Official
Official Journal
Journal ofof thethe Romanian
Romanian Society
Society ofof Physiological
Physiological Sciences
Sciences

CHIEF EDITOR
CO-CHIEF EDITORSASSOCIATE EDITORSEXECUTIVE EDITORSFRANCISC SCHNEIDERIOANA SISKACARMEN TATUMIHAI NECHIFORSORIN RIGA
FLORINA bOjIN
GAbRIELA TANASIEDACIANA NISTORCALIN MUNTEANEDITORIAL BOARD
ARDELEAN
ARDELEAN AUREL
AUREL
BADIU
BADIU GHEORGHE
GHEORGHE
BĂDĂRĂU
BĂDĂRĂU ANCA
ANCA
BENEDEKBENEDEK GYÖRGYGYÖRGYBENGA
BENGA GHEORGHE
GHEORGHE
BUNU
BUNU CARMENCARMEN
COJOCARU
COJOCARU MANOLEMANOLE
CUPARENCU
CUPARENCU BARBUBARBUCONSTANTINCONSTANTIN NICOLAE
NICOLAEHAULICĂHAULICĂ ION
ION
IANCĂUIANCĂU MARIAMARIAMIHALAŞ
MIHALAŞ GEORGETAGEORGETAMUNTEANMUNTEAN DANINADANINAMUREŞANMUREŞAN ADRIANAADRIANANESTIANUNESTIANU VALERIU
VALERIU
OPREA
OPREA TUDOR
TUDOR(Arad)(Arad)(Constanţa)(Constanţa)(Bucureşti)(Bucureşti)(Szeged)(Szeged)(Cluj)(Cluj)(Timişoara)(Timişoara)(Bucureşti)(Bucureşti)(Oradea)(Oradea)(Bucureşti)(Bucureşti)(Iaşi)(Iaşi)(Craiova)(Craiova)(Timişoara)(Timişoara)(Timişoara)(Timişoara)(Cluj)(Cluj)(Craiova)(Craiova)(New(New Mexico)
Mexico)PĂUNESCU
PĂUNESCU VIRGIL
VIRGILPETROIUPETROIU ANA
ANAPOPESCU
POPESCU LAURENŢIU
LAURENŢIU
RÁCZRÁCZ OLIVER
OLIVERRIGA
RIGA DAN
DANSABĂUSABĂU MARIUS
MARIUSSIMIONESCUSIMIONESCU MAIA
MAIA
SIMON
SIMON ZENOZENOSAULEASAULEA I.I. AUREL
AUREL
SWYNGHEDAUWSWYNGHEDAUW BERNARDBERNARDTANGUAYTANGUAY M.
M. ROBERTROBERTTATUTATU FABIANFABIAN ROMULUS
ROMULUSVLAD
VLAD AURELIANAURELIAN
VOICU
VOICU VICTOR
VICTORZĂGREANZĂGREAN LEON
LEON

(Timişoara)(Timişoara)(Timişoara)(Timişoara)(Bucureşti)(Bucureşti)(Košice)(Košice)(Bucureşti)(Bucureşti)(Tg.
(Tg. Mureş)Mureş)(Bucureşti)(Bucureşti)(Timişoara)(Timişoara)(Chişinău)(Chişinău)(Paris)(Paris)(Canada)(Canada)(Timişoara)(Timişoara)(Timişoara)(Timişoara)(Bucureşti)(Bucureşti)(Bucureşti)(Bucureşti)AccREDITED
AccREDITED By
By cNcSIS
cNcSIS -- B
B+cATEgORycATEgORy -- cODE
cODE 240
240
http://journals.indexcopernicus.com/karta.php?action=masterlist&id=4929
http://journals.indexcopernicus.com/karta.php?action=masterlist&id=4929
http://www.ebscohost.com/titleLists/a9h-journals.pdf
http://www.ebscohost.com/titleLists/a9h-journals.pdf
Publication
Publication data:
data: Fiziologia
Fiziologia (Physiology)(Physiology) isis issued
issued quarterly
quarterly
Subscription
Subscription rates:
rates: Subscriptions
Subscriptions run
run aa full
full calendar
calendar year.
year. Prices
Pricesareare give
give perper volume,
volume, surface
surface postage
postage included.
included.
Personal
Personal subscription:
subscription: Romania
Romania -- 100
100 RON,
RON, Outside
OutsideRomania
Romania -- 35$
35$ (must(must bebe inin thethe namename of,of, billed
billed to,to, andand paid
paid byby anan
individual.
individual. Order
Order must
must bebe marked
marked “personal“personal subscription”)
subscription”)
Institutional
Institutional subscription:
subscription: 50$
50$ (regular(regular rate)
rate)
SingleSingle issues
issues andand backback volumes:
volumes: Information
Information on
on availability
availability
andand prices
prices cancan bebe obtained
obtained throughthrough thethe Publisher.
Publisher.
ChangeChange ofof address:
address: Both
Both old
old andand new
new address
address should
should be
be statedstatedandand send
send toto thethe subscription
subscription source.
source.
Bibliographic
Bibliographicindices:
indices:We
Wehopehopethis
thisjournal
journalwill
willbeberegularly
regularlylisted
listedinin bibliographic
bibliographic services,
services, including
including “Current“Current Contents”
Contents”..
Book
Book Reviews:
Reviews: Books
Books areare accepted
accepted forfor review
review byby special
specialagreement.
agreement.
Advertising:Advertising: Correspondence
Correspondence andand rate
rate requests
requests should
should bebe
addressed
addressed toto thethe Publisher.
Publisher.

Fiziologia‑‑ Physiologysupplement
Fiziologia
Physiology 20102010
supplement
2012.23.3(75)
 Fiziologia- Physiology

1.
1. FORFOR SUBSCRIPTIONSUBSCRIPTION ADDRESSADDRESSHVB
HVB Bank
Bank TIMISOARA
TIMISOARA
RO
RO 21
21 BACXBACX 0000000218508250
0000000218508250
TIMISOARATIMISOARA –– ROMANIA
ROMANIAPENTRUPENTRU REVISTAREVISTA„FIZIOLOGIA„FIZIOLOGIA –– PHYSIOLOGY”
PHYSIOLOGY”
2.
2. CORRESPONDENCE
CORRESPONDENCE SHOULDSHOULD BE
BE
ADDRESSEDADDRESSED TO
TO THETHE CHIEF
CHIEF EDITOREDITOR
PROF.
PROF. DR.
DR. FRANCISC
FRANCISC SCHNEIDER
SCHNEIDERPO
PO BOX
BOX 135135
300024300024 –– TIMISOARA
TIMISOARA –– ROMANIA
ROMANIAe-mail:e-mail: carmen.tatu@umft.rocarmen.tatu@umft.ro
EdituraEditura EUROSTAMPAEUROSTAMPA
Tel./fax:
Tel./fax: 0256-204816
0256-204816
ISSN
ISSN 1223
1223 –– 2076
2076

111

Official Journal of the Romanian Society of Physiological Sciences

Instructions to Authors
Submission: Only original papers in English areconsidered and should be sent to:Prof. dr. Francisc Schneider
Chief Editor of “Fiziologia”
PO Box 135
300024, TIMISOARA, ROMANIA
Tel./Fax: 40-256/490507
Manuscripts should be submitted in triplicate setsof illustrations (of which one is an original), typewritten
doublespaced on one side of the paper, with a wide margin.
Conditions: All manuscripts are subject to editorial review.
Manuscripts are received with the explicit understanding thatthey are not under simultaneous consideration by any otherpublication. Submission of an article for publication implies thetransfer of the copyright from the author to the publisher upon
acceptance. Accepted papers become the permanent propertyof “Fiziologia” (Physiology) and may not be reproduced by anymeans, in whole or in part, without the written consent of the
publisher. It is the author’s responsibility to obtain permission to
reproduce illustrations, tables, etc. from other publications.
Arrangement:
Title page: The first of each paper should indicate the title(main title underlined), the authors’ names, and the institutewhere the work was conducted. A short title for use as runninghead is also required.
Keywords: for indexing purposes, a list of 3-10 keywords
in English and Romanian is essential.
Abstract: Each paper needs abstract and title in Romanianand English language, fonts size 9, Arial Narrow.
Bady text: fonts size 10, Arial Narrow.
Small type: Paragraphs which can or must be set in smaller
type (case histories, test methods, etc.) should be indicated witha „p” (petit) in the margin on the left-hand side.
Footnotes: Avoid footnotes. When essential, they are
numbered consecutively and typed at the foot of the appropriate
page, fonts size 8, Arial Narrow.
Tables and illustrations: Tables (numbered in Roman
numerals) and illustrations (numbered in Arabic numerals)
should be prepared on separate sheets, fonts size 9, Arial Narrow.
Tables require a heading, and figures a legend, also prepared ona separate sheet. For the reproduction of illustrations, only good
drawings and original photographs can be accepted; negatives
or photocopies cannot be used. When possible, group several
illustrations on one block for reproduction (max. size 140x188mm) or provide crop marks. On the back of each illustration
indicate its number, the author’s name, and article title. Colour

2

illustration are reproduced at the author’s expense.
References: In the text identify references by Arabic
figures, (in brackets), fonts size 9, Arial Narrow. Material
submitted for publication but not yet accepted should be
noted as “unpublished data” and not be included in thereference list. The list of references should include only thosepublications which are cited in the text. The references should
be numbered and arranged alphabetically by the authors’names. The surnames of the authors followed by initials
should be given. There should be no punctuation signs otherthan a comma to separate the authors. When there are morethan 3 authors, the names of the 3 only are used, followedby “et al”. abbreviate journal names according to the Index
Medicus system. (also see International Committee of Medical
Journal Editors: Uniform Requirements for manuscripts
submitted to biomedical journals. Ann Intern Med 1982; 96:766 – 771).

Examples:(a) Papers published in periodicals: Kauffman HF, van der
Heide S, Beaumont F, et al: Class-apecific antibody determination
against Aspergillus fumigatus by mean of the enzyme-linked
immunosorbent assay. III. Comparative study: IgG, IgA, IgM, ELISA
titers, precipitating antibodies and IGE biding after fractionationof the antigen. Int Arch Allergy Appl Immunol 1986; 80: 300 –306.

(b) Monographs; Matthews DE, Farewell VT: Using andUnderstanding Medical Statistics. Basel, Karger, 1985.
(c) Edited books: Hardy WD Jr, Essex M: FeLV-inducted felineacquired immune deficiency syndrome: A model for human
AIDS; in Klein E(ed): Acquired Immunodeficiency Syndrome. Prog Allergy,
Busel, Karger, 1986, vol 37, 353 – 376.
Full address: The exact postal address complete withpostal code of the senior author must be given; if correspondenceis handled by someone else, indicate this accordingly. Add theE-mail address if possible.
Page charges: There is no page charge for papers of
4 or fewer printed pages (including tables, illustrations andreferences).
Galley proofs: unless indicated otherwise, galley proofs
are sent to the first-named author and should be returned withthe least possible delay. Alternations made in galley proofs, otherthan the corrections of printer’s errors, are charged to the author.
No page proofs are supplied.
Reprints: Order forms and a price list are sent with thegalley proofs. Orders submitted after the issue is printed are
subject to considerably higher prices. Allow five weeks fromdate of publication for delivery of reprints.

Fiziologia- Physiology 2012.23.3(75)
Fiziologia‑ Physiology 2010supplement

CONTENTS

1. Diagnostic Aspects in Adult Patients with Thyroid Disease and Type 2 Diabetes ....................................................................................................................................................4
Adriana Gherbon, Lavinia Noveanu, Georgeta Mihalaş
2. The Morphology of Colonies from Breast Cancer SK-BR-3 Cells in Semisolid Media Depends on the Media Composition ................................................................................9
Cean Ada, Istratoaie Bogdan, Cristea Mirabela, Ivan Alexandra, Anghel Simona, Gavriliuc Oana, Bojin Florina, Tanasie Gabriela, Tatu Carmen, Panaitescu Carmen, Paunescu Virgil
3. A Simple ECG Recording Hardware for Langendorff Isolated Heart Experiments ................................................................................................................................................15
Marcel Perian, Dan Dobreanu, Carmen Caldararu, Marius Sabau
4. Analysis of the Evolution in Time of the Effects of Monotonous Auditory Stimulation on Cortical Biopotentials ...............................................................................................18
Marius Georgescu, Daniel Georgescu, Dragoş Alexandru, Daniela Puscasiu, Carmen Tatu, Maria Iancău
5. Physiology and Physiopathology of Hepatic Encephalopathy ..............................................................................................................................................................................23
Scheau Cristian, Lazăr Mihai, Bunea Magda, Ciornei Cătălina, Papacocea Raluca, Ion Daniela Adriana, Bădărău Anca Ioana
6. The Effects of Adenosine Amine Congener (ADAC) on the Sound Stress in Wistar Rats .....................................................................................................................................26Goanţă CM, Sandu S, Pîslaru A, Zăgrean L
7. Prevalence of Refractive Errors in Schoolchildren in Urban Environment .............................................................................................................................................................30
Luminita Turcin
8. Changes of Interleukin-12 Serum Levels in Patients with Chronic Hepatitis C Virus Infection before Interferon Alpha Treatment .....................................................................34
Manole Cojocaru, Simona Alexandra Iacob, Dorina Banică, Eugenia Panaitescu, Diana Gabriela Iacob
9. Etiology and Pathophysiology of Seizures, Epilepsy and Status Epilepticus after Stroke .....................................................................................................................................36
Vintilescu Raluca, Balseanu Adrian, Mitu Cristina, Dina Ciprian, Georgescu Marius, Iancau Maria
10. The Survey of Nosocomial Infections from a University Clinic Institute with Cardiovascular Profile ..................................................................................................................41
Livia Stângă, Luminiţa Mirela Bădiţoiu, Matilda Rădulescu, Delia Berceanu-Văduva, Elena Hogea, Delia Muntean, Piluţ C., Monica Licker, Roxana Moldovan

CUPRINS
1. Caracteristici diagnostice ale pacientilor adulti cu afectiune tiroidiana si Diabet Zaharat tip 2 .............................................................................................................................4
Adriana Gherbon, Lavinia Noveanu, Georgeta Mihalaş
2. Aspectele morfologice ale coloniilor celulare formate de linia tumorala mamara SK-BR-3 sunt corelate cu compozitia mediului de cultura semisolid ..................................9
Cean Ada, Istratoaie Bogdan, Cristea Mirabela, Ivan Alexandra, Anghel Simona, Gavriliuc Oana, Bojin Florina, Tanasie Gabriela, Tatu Carmen, Panaitescu Carmen, Paunescu Virgil
3. Dispozitiv simplu pentru înregistrarea ECG în experimente pe inima izolată în sistem Langendorff ..................................................................................................................15
Marcel Perian, Dan Dobreanu, Carmen Caldararu, Marius Sabau
4. Analiza evolutiei in timp a efectelor stimularii auditive monotonale asupra biopotentialelor corticale .............................................................................................................18
Marius Georgescu, Daniel Georgescu, Dragoş Alexandru, Daniela Puscasiu, Carmen Tatu, Maria Iancău
5. Fiziologia şi fiziopatologia encefalopatiei hepatice ...............................................................................................................................................................................................23
Scheau Cristian, Lazăr Mihai, Bunea Magda, Ciornei Cătălina, Papacocea Raluca, Ion Daniela Adriana, Bădărău Anca Ioana
6. Adenosine amine congener (ADAC) şi efectele sale asupra comportamentului şobolanilor Wistar supusi stresului sonor ...............................................................................26Goanţă CM, Sandu S, Pîslaru A, Zăgrean L
7. Prevalenta erorilor de refractie la scolarii din mediul urban .................................................................................................................................................................................30
Luminita Turcin
8. Variaţii ale concentraţiilor de interleukină-12 în serul pacienţilor cu infecţie cronică cu virusul hepatitei C înaintea tratamentului cu interferon alpha .................................34
Manole Cojocaru, Simona Alexandra Iacob, Dorina Banică, Eugenia Panaitescu, Diana Gabriela Iacob
9. Epilepsia post accident vascular cerebral ...............................................................................................................................................................................................................36
Vintilescu Raluca, Balseanu Adrian, Mitu Cristina, Dina Ciprian, Georgescu Marius, Iancau Maria
10. Monitorizarea infectiilor nosocomiale dintr-un Institut Clinic Universitar cu profil cardiovascular ...................................................................................................................41
Livia Stângă, Luminiţa Mirela Bădiţoiu, Matilda Rădulescu, Delia Berceanu-Văduva, Elena Hogea, Delia Muntean, Piluţ C., Monica Licker, Roxana Moldovan2012.23.3 (75)  Fiziologia - Physiology

3

DIAGNOSTIC ASPECTS IN ADULT PATIENTS WITH THYROIDDISEASE AND TYPE 2 DIABETESADRIANA GHERBON, LAVINIA NOVEANU, GEORGETA MIHALAŞ
Department III-Functional Sciences, Physiology, “Victor Babes” University of Medicine and Pharmacy Timisoara

ABSTRACT

Patients with diabetes have an increased prevalence of thyroid disease compared with non-diabetic population. In the case of type2 diabetes mellitus, hypothyroidism is the most common disorder seen. Type 2 diabetes is often associated with hyperthyroidism(Basedow-Graves’ disease and multinodular toxic goiter).
The purpose of this study is to determine the main diagnosis aspects of thyroid diseases in adult patients with type 2 diabetes. The
adults group studied was represented by 290 cases, aged 18-80 years. Gender distribution was net in favor of women being represented by 252 women and 38 men. They used clinical, imaging, biochemical, hormonal and immunological parameters.
Keywords: type 2 diabetes, thyroid disease, diagnosis, thyroid ultrasound, thyroid hormones, antithyroid antibodies

INTRODUCTION
The thyroid disorders are common in the general population;
their prevalence increases with age. The screening for thyroid
disorders is indicated in certain high-risk groups as new bornand elderly people (8).
The hypothyroidism is the most common thyroid disorderin adults and older women. Its origin is usually autoimmune,
presenting as atrophic primary hypothyroidism or Hashimoto
thyroiditis. It may be also secondary to radioactive iodine
treatment or thyroid surgery. In rare cases, occurs secondary
hypothyroidism to hypothalamic or pituitary disease (8).
By contrast, the hyperthyroidism is less common, with aratio female/male 9/1. The Graves’s disease is most common
and usually affects young adults. The toxic multinodular goiterusually occurs in older people (8).
The patients with diabetes mellitus (DM) have an increased prevalence of thyroid disease compared with non-diabetic
population (8).
The most common association is between thyroid disease
and type 1 diabetes, but a number of studies also indicate ahigh prevalence of thyroid disorders in patients with type 2
diabetes, the hypothyroidism is the most common disorderencountered (1).
In the case of association of IGT and IFG with thyroid disorders, they usually occur as a result of excess thyroid hormones. It
was found that these associations are more common in women.
In a U.S. study shows that at patients with thyroid disease,glucose intolerance was present in 38% of cases, and incidenceof clinical diabetes was 2-3% (6, 7).
Other authors have obtained a prevalence of thyroid disorders in patients with type 2 diabetes of 2.5%, the most common
endocrine disorder were sub clinical hypothyroidism (4.1%) (5).
DM type 2 is frequently associated with hyperthyroidism(Graves-Basedow disease and toxic multinodular goiter). DM
type 2 is present in 11% of patients with Graves-Basedow

disease and in 5% of those with toxic multinodular goiter. The
glucose intolerance is also frequently associated with hyperthyroidism; its prevalence is much higher compared to type 2
diabetes (72.3%). In the case of the toxic multinodular goiterincidence of impaired glucose tolerance was much higher,
namely 85%, and in the case of the Graves-Basedow diseasearound 54% (4).
Also, in the case of euthyroid diffuse goiter, its prevalencein the people with DM varies between 3.4% and 17%; being
discussed the treatment with sulfonylurea involvement in its
appearance (2).
The diagnosis of thyroid dysfunction in patients with diabetes, based only on clinical manifestations, can be difficult. An
unsatisfactory glycemic control can cause similar symptoms withthe hyperthyroidism, such as weight loss despite increased appetite, and fatigue. On other hand, the severe diabetic nephropathy
can be omitted in patients with hypothyroidism because thesepatients may present edemas, fatigue, and pallor and weightgain (8).
To further complicate the diagnostic process, unbalance DM,
with or without complications, can cause alterations of thyroid
function tests, which can occur in patients without associatedthyroid disease. The typical changes are represented by thedecrease of T3, determined by the decrease of T4 conversion
in T3, decreased serum T4, due to lower fixing on protein andlower serum TSH (8).
The most sensitive test to detect the thyroid dysfunction isthe determination of TSH, which allows the certain diagnosisof the hypo-and hyperthyroidism. It also allows the diagnosis
of sub clinical thyroid dysfunction; characterized only throughadjusting the level of TSH, with T3 or T4 in the normal range,
the patients with this type of dysfunction is usually asymptomatic (8).
The thyroid dysfunction can produce significant biochemical and clinical changes. Thus, the sub clinical hypothyroidism

Received 10th of July 2012. Accepted 20th of August 2012. Address for correspondence: Adriana Gherbon, MD, PhD, Department of Physiology, “Victor Babes” Universityof Medicine and Pharmacy Timisoara, Romania, Eftimie Murgu Square No. 2A, 300041, Timisoara, phone/fax: +40256220479; e-mail: adrianagherbon@yahoo.com

4

Fiziologia - Physiology  2012.23.3 (75)

may increase LDLc and aggravating pre-existing dyslipidemia,
increasing the risk of arteriosclerosis. On other hand, the sub
clinical hyperthyroidism may increase the risk of cardiac arrhythmias and can exacerbate a pre-existing coronary heartdisease. Because the patients with diabetes are an increased
risk for cardiovascular disease, the diagnosis and treatment ofassociated thyroid disease is important (8).
The presence on antiperoxidase thyroid antibodies (antiTPO
AB) has a role in predicting occurrence of autoimmune thyroid
disease, particularly of the hypothyroidism. Auto antibodies may
occur long before obvious clinical manifestations of autoimmune
thyroid disease and predict the appearance of clinical disease.
The patients with polyglandular autoimmune syndrome may
present auto antibodies against the same antigen. At the patientswith present antiTPO AB must make their annual screening forearly detection of thyroid dysfunction and establishment of an
appropriate treatment (3).
Also, at all the patients with diabetes should be performed ascreen for adrenal auto antibodies, celiac disease, steroid, gastricparietal and thyroid even at diagnosis. Also, at the patients who
have one of the diseases listed above must be confirmed existence of diabetes by determining the ICA, GAD, IA2, in absenceof characteristic clinical manifestations (3).
At the family members of patients with diabetes must also
determine auto antibodies, especially if the diabetic patient
has thyroid auto antibodies. Identifying of the auto antibodies
presence should be followed by hormonal assessment and thetreatment of thyroid disease (3).
MATERIAL AND METHOD
Investigated population
The study included adult subjects with diabetes mellitus,
which in time present thyroid disease, or adult subjects withthyroid disease that subsequently present diabetes mellitus.
The study group comprised 290 cases, aged 18-80 years.
Gender distribution was net in favor of women being represented
by 252 women and 38 men.
Methods of investigation
Methods of investigation were the clinical data - history,
present status, and imaging - thyroid ultrasound, biochemistry- carbohydrate metabolism parameters: fasting blood glucose,urine glucose, glycosylated hemoglobin and thyroid hormones
investigations and some immunological parameters
Glucose determination was performed by enzymatic techniques with glucose oxidase. Were considered normal fastingblood glucose between 70-110 mg%, diabetes mellitus - fastingblood glucose values above 126 mg%, impaired glucose tolerance - fasting blood glucose values between 110-126 mg% andthe oral glucose tolerance test (OGTT) at 2 h between 140-200
mg% and fasting impaired glucose tolerance - fasting blood
glucose values between 110-126 mg% and OGTT at 2 h under
2012.23.3 (75)  Fiziologia - Physiology

140 mg%.
Determination of glycosylated hemoglobin (HbA1c) was
achieved through the DiaStat program for glycosylated hemoglobin HbA1c that measures the ratio of glycated hemoglobin
to total HbA.
Determination of serum levels of TSH, free fraction of serum
level of triiodothyronine (FT3), free fraction of thyroxin (FT4) were
ARCHITECT quantitative method, which is an immunologic
determination by chemiluminescence’s with small Chemilumnescent Micro particle Immunoassay (CMIA). The following values
were considered normal: TSH = 0.465 - 4.68 mIU/ml, FT3 = 3.69
-10.4 pmol/l, FT4 = 10 - 28.2 pmol/l.
Immunological parameters were represented by somemarkers of thyroid autoimmunity – antiperoxidase (antiTPO)
and antithyroglobulin (antiTG) antibodies (AB). To determine
the serum titers of antiTPO AB, AxSYM antiTPO kit was used,
the method is enzyme immunoassay with micro particles, Meia
(Micro particle Enzyme Immunoassay). It was considered
normal: antiTPO AB <35 IU / ml. To determine the serum
titers of antiTG AB, AxSYM antiTG kit was used, the method
is enzyme immunoassay with micro particles, Meia (Microparticle Enzyme Immunoassay). It was considered normal:
antiTG AB <55 IU/ ml.
Thyroid ultrasound performed in all cases is a non-invasive
method of exploration that allows measurement of thyroid volume, thyroid study report with cervical anatomical structures andthyroid parenchyma changes.
RESULTS AND DISCUSSION
In the case of the type 2 diabetes group, family history
was present in 1.96% cases. In terms of the clinical symptoms, the patients with Graves-Basedow disease presented
the specific thyreotoxic disease syndrome. In the case of
ACT, some patients were asymptomatic, and some presented
typical symptoms of hypothyroidism. In the case of the nodular goiter, they were discovered incidentally at ultrasoundexamination (incidentalomas) or by clinical symptoms due
to compression. In the case of the malignant tumors, thesymptoms were very different: firm goiter, loco-regional
adenopathy, quick growth of the formation spontaneous orby thyroxin suppression therapy. In the case of the euthyroid
diffuse goiter, was no symptoms, the diagnosis was basedon clinical examination and ultrasound. In the case of the
sub acute thyroiditis, the symptoms occurred after an upper
respiratory tract infection, framing local pain, inflammatory
syndrome, functional disorders (transient hyperthyroidism,
followed by transient hypothyroidism).
To assess the goiter was used inspection and palpation andwere taken into account the WHO goiter staging criteria. Basedon these criteria, at the studied group with type 2 diabetes wehave obtained the results given in Table I.
A percentage of 18.62% cases presented the goiter stage 1
a, 8.96% had goiter stage 1 b, 7.24% stage 2 and 4.13% stage
3, with the relatively firm consistency. Most patients (61.03%)
had no goiter on clinical examination (Figure 1).
5and ACT moderate hypoechogenity (+ + and ++/+++) (53.32%
cases for Graves-Basedow disease and 48.04% for ACT) andintense (+ + + + + + and above) (16.66% for Graves-Basedow
disease, and 24.68% for TCA) prevailed (Figure 1).

Table I. The goiter stages distribution in patients with type 2 diabetes

Goiter stages
0nTotal (n=290)

%

n

1a

1b

2

%

n % n

%

3
n

%

177 61.03 54 18.62 26 8.96 21 7.24 12 4.13

Graves-Basedow 32 53.33 4 6.66 12 20 7 11.66 5 8.33
disease(n = 60)
Autonomous
3
thyroid adenoma(n = 3)100

Hyperthyroidism induced by
Amiodarone(n= 4)

100

4

-

-

-

-

-

-

-

-

Thyroid disease TV type
type
normal higher
n

-

-

-

-

-

-

-

-

Euthyroid diffuse 51 40.8 43 34.4 12 9.6 12 9.6 7
goiter (n= 125)

5.6

Nodular euthy3 42.85 2 28.57 roid goiter (n = 7)--

2 28.57 -Differentiatedthyroid carcinoma(n= 10)9

ACT (n = 77)

71 92.2 4 5.19 1 1.3 1 1.3--

Sub acute thyroiditis (n = 4)

3

-

-

90

75

-

1

-

25

1 10 -

-

-

-

-

-

-

-

The thyroid ultrasound allows the accurate assessmentof thyroid volume and thyroid parenchyma appearance appreciation. As consequence of iodine intake study in the Banat
plain, especially in the Timisoara, the normal thyroid volumes
were within the following values: women - 10.27 ± 2.09 ml andin males - 12.18 ± 2.52 ml. In the case of our study, the goiter
has been diagnosed with thyroid volume exceeded 3 standard
deviation (SD) (females > 16.54 ml and men > 19.74 ml).
In the case of the type 2 diabetes group, the biggest thyroid
volume (TV) was found in patients with Graves-Basedow disease(34.33 ± 32.2 ml) and at those with euthyroid diffuse goiter(25.27 ± 19.17 ml). The smallest TV was found in patients with
ACT 10.65 ± 5.23 ml. All the thyroid carcinoma cases received
surgery, the TV being evaluated also intraoperatory.
The results about the TV value and the hypoechogenity
intensity of thyroid parenchyma, according to sex and type ofthyroid disease are shown in Table II.
In the case of type 2 diabetes groups most cases presented
hypoechogenity (86.9%). Normoechogenity was present in
13.1% cases. Discrete hypoechogenity was present in 36%
cases; moderate hypoechogenity was present in 28.57% cases.
Intense hypoechogenity was present only in 11.9% cases. It isnoted that for thyroid carcinoma (100% cases), nodular euthyroid goiter (100%), sub acute thyroiditis (100%) and euthyroid
diffuse goiter (76% cases) discrete hypoechogenity (+ and + / ++) prevailed, while in the case of the Graves-Basedow disease
6

Table II. The classification of cases according to the TV and the type ofthyroid parenchyma hypoechogenity

%

n

%

Hypoechogenity type
mild
n

%

moder- intense
ate
n

%

n

%

Graves-Basedow 19 31.66 41 68.33 17 28.32 32 53.32 10 16.66
disease (n=60)
Autonomous
1 33.33 2 66.67 3 100
thyroid adenoma
(n=3)

-

-

-

-

Hyperthyroidism 2
induced by Amiodarone (n=4)

4 100

-

-

-

-

Euthyroid diffuse 44 35.2 81 64.8 95 76
goiter (n=125)

-

30 24

-

Nodular euthyroid 4 57.14 3 42.86 7 100
goiter (n=7)

-

-

-

-

Differentiated thy- 8
roid carcinoma(n=10)

-

-

-

-

50

80

2

50

2

20 10 100

ACT (n=77)

77 100 --16 20.8 37 48.04 19 24.68

Sub acute
thyroiditis (n=4)

2

50

4 100

+++/++++

1%

Hypoecogenity type

Thyroid disease
type

50

2

+++

10%

++/+++

10%

-

-

-

-

25%

++
9%

+/++
+

32%

0%

10%

20%

30%

40%

Cases prevalenceFig. 1. The hypoechogenity intensity of the thyroid parenchyma in adults
group with type 2 diabetes

The homogeneity of the thyroid parenchyma echogenity(96.7% cases) was superior thyroid parenchyma inhomogeneity(3.1% of cases) divided by the 290 members of the group. The
thyroid functionality evaluation was performed by determining
serum levels of TSH and FT4. Depending on the thyroid disease
and the type of the changes in glycemic balance, we obtained
the following results:
Fiziologia - Physiology  2012.23.3 (75)Table III. Evaluation of thyroid functionality

Parameters
Graves-Basedow
diseaseAutonomous thyroid
adenomaHyperthyroidism induced by Amiodarone
Euthyroid diffuse goiter
Nodular euthyroid goiter
Differentiated thyroid
carcinoma
ACT
Sub acute thyroiditis

TSH (mIU/ml)
0.11 ± 0.14

FT4 (pmol/l)
41.07 ± 19.44

2.19 ± 2.12

19.66 ± 12.79

0.16 ± 0.15

4.45 ± 14.98

1.82 ± 1.14
1.76 ± 0.85
1.9 ± 1.07

14.96 ± 4.49
16.86 ± 6.63
21.04 ± 6.99

10.77 ± 20.07
2.25 ± 1.76

13.24 ± 5.49
19.42 ± 11.72

The Graves-Basedow disease was encountered in 60
patients with type 2 diabetes. Depending on the TSH value was
performed functional classification of the cases. All cases with
Graves-Basedow disease were accompanied by thyrotoxicosis.
The correlation between type 2 diabetes and Graves-Basedow
disease was not significant (r = -0.10, p <0.001). The GravesBasedow disease complications were represented by cardiothyreosis, the most common complications that occur as aconsequence of cardiac overload and metabolic abnormalities
in myocardium. This takes the form of heart failure secondary
tachyarrhythmia’s, with increased flow and with resistance totonicardiac treatment.
Autonomous thyroid adenomas were represented by the“warm” or “hot” nodules. The latter have evolved with less severe
thyrotoxicosis than Basedow Graves’ disease. Autonomous
thyroid adenoma was found in 3 patients with type 2 diabetes.
Depending on the TSH value was performed functional classification of cases.
In the case of type 2 diabetes, 66.67% of autonomous
thyroid adenoma cases were euthyroid and 33.34% presented
sub clinical hyperthyroidism.
Iodine-induced hyperthyroidism was found in only 4 patientswith type 2 diabetes. All cases were induced by administrationof amiodarone. In all these TSH was low and FT4 increased.
Depending on the TSH value was performed functional classification of cases. All the above cases functionally were presented
with hyperthyroidism.
Euthyroid diffuse goiter was encountered in 125 patientswith type 2 diabetes. In all these the hormones functionalparameters were normal. Depending on the TSH value was
performed functional classification of cases. All cases withdiffuse goiter functionally were euthyroid. Nodular euthyroid
goiter was represented by adenomas and thyroid cysts.
Nodular euthyroid goiter was encountered in 7 cases with
type 2 diabetes. Depending on the TSH value was performed
functional classification of cases. All cases with nodular goiter
functionally were euthyroid.
Thyroid carcinoma was encountered in 10 patients with
type 2 diabetes. Depending on the TSH value was performed
2012.23.3 (75)  Fiziologia - Physiology

functional classification of cases. All cases with thyroid carcinoma functionally were initially euthyroid. All cases received required treatment (total thyroidectomy, treatment with I131, thyroid
suppressive therapy). ACT was found in 77 patients with type2 diabetes. Depending on the TSH value was performed functional classification of cases. In the case of the type 2 diabetes
group, 70.13% (n = 54) cases presented normal values, and29.87% (n = 23) cases increased values of TSH. In this groupprevailed ACT with euthyroidism (70.13%), followed by ACTwith clinical hypothyroidism (25.97%) and then ACT with sub
clinical hypothyroidism (3.89%). Also, the correlation coefficient
between serum concentration of TSH and HbA1c in all caseswith type 2 diabetes and ACT was insignificant (r = - 0.046, p =0.30). It is generally accepted that evolution of ACT is stages.
The risk of hypothyroidism is higher in women over 45 years,
presenting slightly increased serum TSH concentrations, andelevated titers of antiTPO AB. The thyroxin therapy induces adecrease in goiter volume at 50-90% cases, corrects the thyroid
function and after some authors decreased antibodies titers.
The sub acute thyroiditis was encountered only at 4 patientswith type 2 diabetes. In these entire hormones functionalparameters were normal. Depending on the TSH value was
performed functional classification of cases. All cases with subacute thyroiditis functionally were euthyroid. The sub acutethyroiditis may evolve initially with transient hyperthyroidism,
followed then by hypothyroidism.
Humoral immunity was assessed by determining thyroid
serum antibodies titers: antiTPO AB and antiTG AB at 58/77
patients with type 2 diabetes. In the case of the type 2 diabetes
group with antiTPO AB, they were present in high titers in 79.31%
cases (n = 46) and in normal titers in 20.69% cases (n = 12). It
was found elevated values of antiTPO AB in 79.31% ACT cases.
AntiTPO AB was normal in 20.69% of cases with ACT. At every
group we appreciated the onset age of diabetes, current age,diabetes duration, BMI, the values of blood glucose, HbA1c, TSH
and FT4. (Tab. IV). It was found significant differences between
the 2 groups regarding HbA1c value and insignificant for the restof the parameters studied.
Table IV. Comparative data (p) between cases with positive antiTPO AB andnegative antiTPO AB in adults with type 2 diabetes studied group

Parameters

Negative
antiTPO AB
43
55.25 ± 8.8
53.83 ± 9.19

Positive
antiTPO AB
46
54.67 ± 10.56
51.65 ± 10.99

Cases number (%)
Age (years)
Onset age of diabetes(years)DM duration (years)
1.41 ± 1.5
3.02 ± 6.38
BMI (kg/m2)
34.81 ± 6.01
32.12 ± 6.17
HbA1c (%)
7.25 ± 1.56
8.59 ± 2.69
Blood glucose (mg %) 115.58 ± 22.37 132.43 ± 42.55
TSH (mIU/ml)
6.69 ± 9.91
7.75 ± 18.15
FT4 (pmol/l)
15.84 ± 7.79
13.9 ± 6

p
0.65
0.84
0.49
0.12
0.18
0.03
0.06
0.78
0.43

7

CONCLUSIONS

Fig. 2. Diagnostic algorithm at thyroid diseases
in patients with type 2 diabetes

In conclusion, thyroid damage is common in the patientswith diabetes and may cause significant metabolic changes. The
main thyroid disease associated with type 2 diabetes and sub
clinical changes in glycemic balance was diffuse and nodular
euthyroid goiter. Therefore, thyroid function regular screening
is indicated in all the patients with diabetes for establishment ofearly treatment of sub clinical thyroid dysfunction (3). The bestscreening test is the TSH determination. At the patients with

type 2 diabetes, the TSH determination is performed at diabetes
diagnosis and then at a minimum 5 years interval (3).
In the case of the patients with thyroid disease is obligatory
annual determination of glycemia. In the case of elevated blood
glucose levels, according to the values obtained, it is necessary
employment in one of the category: type 1 diabetes, type 2
diabetes or IGT (3).
In conclusion, we propose the following algorithm to diagnose the thyroid disease at patients with type 2 diabetes:
REFERENCES
1. Cooper GS, Stroehla BC. The epidemiology of autoimmune diseases. Autoimmune Rev, 2003; 2(3): 119 – 25
2. Guney E, Efe B, Akalin A, Kebapci N, Erenoglu E. Thyroid Diseasein Diabetes Mellitus. Turkish Journal of Endocrinology and Metabolism,
1999; 3: 119 – 122
3. Kukreja A, Maclaren N. Autoimmune Endocrine Disorders.In PediatricEndocrinology (electronic book), 2002, cap. 6
4. Paul DT, Mollah FH, Alam MK, Fariduddin M, Azad K, Arslan MI.
Glycemic status in hyperthyroid subjects. Mymensingh Med J, 2004;
13 (1): 71-5.
5. Radaideh AR, Nusier MK, Amari FL, Bateiha AE, El-Khateeb MS,
Naser AS, Ajlouni KM. Thyroid dysfunction in patients with type 2
diabetes mellitus in Jordan. Saudi Med J, 2004; 25 (8): 1046-1050.
6. Wartofsky L. Myxedema coma. In: Werner SC, Ingbar SH, Braverman LE, Utiger RD, eds. Werner & Ingbar’s the Thyroid: A Fundamental
and Clinical Text. 8th ed. Philadelphia, Pa: Lipincott Williams & Wilkins,
2000; 843-847.
7. Werner SC, Ingbar SH, Braverman LE. Organ system manifestationsof hypothyroidism (Section C). In: Werner and Ingbar’s the Thyroid: A
Fundamental and Clinical Text. 8th ed. Philadelphia, Pa: Lipincott Williams & Wilkins, 2000; 774-847.
8. Wu P. Thyroid disease and diabetes. Clinical Diabetes, 2000; 18: 1.

CARACTERISTICI DIAGNOSTICE ALE PACIENTILOR ADULTI CU AFECTIUNE TIROIDIANA SI DIABET ZAHARAT TIP 2
REZUMAT

Pacienţii cu diabet zaharat prezintă o prevalenţă crescută a afecţiunilor tiroidiene comparativ cu populaţia nediabetică. În cazul
diabetului zaharat tip 2, hipotiroidismul este cea mai frecventă tulburare întâlnită. DZ tip 2 se asociază frecvent şi cu hipertiroidismul(boala Graves-Basedow şi guşa multionodulară toxică). Scopul acestui studiu este de a determina principalele aspecte de diagnostic
ale afec ţiunilor tiroidiene la pacienţii adulţi cu DZ tip 2. Lotul de adulţi studiat a fost reprezentat de 290 cazuri, cu vîrste cuprinseîntre 18-80 ani. Distribuţia pe sexe a fost netă în favoarea femeilor, fiind reprezentată de 252 femei, şi de 38 bărbaţi. Au fost utilizaţi
parametrii clinici, imagistici, biochimici, hormonali şi imunologici.
Cuvinte cheie: diabet zaharat tip 2, afecţiuni tiroidiene, diagnostic, ecografie tiroidiană, hormoni tiroidieni, anticorpi antitiroidieni

8

Fiziologia - Physiology  2012.23.3 (75)THE MORPHOLOGY OF COLONIES FROM BREAST CANCER
SK-BR-3 CELLS IN SEMISOLID MEDIA DEPENDS ON THE
MEDIA COMPOSITION
CEAN ADA1, ISTRATOAIE BOGDAN1, CRISTEA MIRABELA2, IVAN ALEXANDRA1, ANGHELSIMONA2, BOJIN FLORINA1, TANASIE GABRIELA1,2, TATU CARMEN1,2, PANAITESCU CARMEN1,2, PAUNESCU VIRGIL1,2
1
Department of Functional Sciences, “Victor Babes” University of Medicine and Pharmacy Timisoara, Romania
2
Immunophysiology and Biotechnologies Center, Clinical County Emergency Hospital Timisoara, Romania

ABSTRACTThe 3D culture systems allows rich tissue-specific architecture and specific cell morphology, multiple interactions between cells, astrong cell adhesion, polarization of the cells, restricted cell mobility, cell growth in all directions and offers an extracellular matrix
that mimics the native one. The aim of this study was to investigate the morphology of cancer cells colonies, from SK-BR-3 line,
when cultured in three semisolid media (3D) and to investigate if surface markers expression and proliferation is influenced by the
3D culture conditions. For our experiments we used a commercial cell line SK-BR-3, human breast adenocarcinoma isolated from apleural effusion. Confluent monolayer cultures were obtained after thawing SK-BR-3 cells frozen at passage 4. The confluent cultures
were detached and four experimental groups were created: cells cultured in MethoCult, cells cultured in Human Methylcellulose BaseMedia, cells cultured in Matrigel and cells cultured in monolayer (control). After a culture period of 10 days cells were recovered from
3D matrixes and part of them was analyzed, while part of them were replated for subsequent analysis. Our experiments showed thatthe morphology of cell colonies from SK-BR-3 is highly dependent of the semisolid media in which the cells are cultured. The protein
based semisolid media are better for supporting cellular proliferation compared with methylcellulose based media. The flowcytometry
analysis of the surface markers showed no significant difference in expression, except for CD44. Cells cultured in Matrigel seem tohave a low expression of CD44 compared with cells cultured in monolayer, MethoCult and MethylCellulose. Inclusion of cancer cells
in semisolid media had no effect in reducing cell proliferation as shown by high Ki67 marker expression.
Key words: SK-BR-3 cell line, 3D culture systems, phenotypic markers, cellular proliferation

INTRODUCTION
Monolayer cell culture is the most common technique usedin laboratories all over the world, but recently the limitation ofthis technique has directed the research into finding techniquesthat allows culturing the cells in vitro but with morphology andinteractions resembling more with in vivo conditions. Cells inmonolayer culture have poor tissue-specific architecture, are flatand extended, have limited interactions, weak cell adhesion, fastor free mobility, high proliferation, a directional growth, partiallypolarization and absent or poor extracellular matrix remodeling.
The 3D culture systems allows rich tissue-specific architectureand specific cell morphology, multiple interactions between cells,
a strong cell adhesion, polarization of the cells, restricted cell
mobility, cell growth in all directions and offers an extracellularmatrix that mimics the native one (5).
The extracellular matrix (ECM) is a mixture of substances(proteoglycans, proteins, signaling molecules, etc.) that, in thepast, was considered to have only a structural role in supporting
cells and an environment for cell migration. Recent advancementin science showed that ECM plays an active role in cell adhesion(1), signaling (12) and cell differentiation (9).

Mammary gland cells are in tight contact with each otherand with ECM, and trough cell-mediated contraction they sense
the stiffness of their microenvironment and respond with appropriate signaling regulating gene expression and differentiation.
Mechanical regulation is playing an important role in breast
carcinoma progression, since the alteration of ECM depositions, composition, and organization results in stiffer matricesthat activate signaling pathways that induce cell proliferation,
facilitating tumor cell invasion, and promote progression ofcancer. Schedin P. (11) underlined the important role of mechanic forces in the mammary gland in maintaining physiologic
function of cells (11). The aim of this study was to investigate
the morphology of cancer cells colonies, from SK-BR-3 cell line,
when cultured in three semisolid media (3D) and to investigate
if surface markers expression and proliferation is influenced by
the 3D culture conditions.
MATERIALS AND METHODS
Cell line
For our experiments we used a commercial cancerous cell

Received 20th of August 2012. Accepted 10th of September 2012. Address for correspondence: Biotech Eng Ada Cean, MS, PhD, Department of of Functional Sciences,“Victor Babes” University of Medicine and Pharmacy Timisoara, Romania, Immunophysiology and Biotechnologies Center, Eftimie Murgu Square No. 2A, RO-30041,
e-mail: telea_ada@animalsci-tm.ro2012.23.3 (75)  Fiziologia - Physiology

9line SK-BR-3, human breast adenocarcinoma. This cell line wasderived by G. Trempe and L.J. Old in 1970 from a pleural effusionfrom a 43 years old female patient, diagnosed with mammary
gland adenocarcinoma. This is a hypertriploid human cell linewith the modal chromosome number of 84, occurring in 34% of
cells.Cells having 80 chromosomes also occurring in 28% of thecells. The SK-BR-3 cell line overexpresses the HER2/c-erb-2
gene product, but has no expression for estrogen or progesterone receptors. Ultrastructural features include microvilli anddesmosomes, glycogen granules, large lysosomes, bundles ofcytoplasmic fibrils.
Maintaining this cell culture requires the following conditions: culture medium McCoy’s 5a Medium Modified (ATCC,
Manassas, USA), supplemented with 10% Fetal Calf Serum(PromoCell, Heidelberg, Germany) and 2% Pen/Strep (10,000
IU/mL, PromoCell) and incubation at 37°C, 5% CO2.
Experimental design
Confluent monolayer cultures were obtained after thawing
SK-BR-3 cells frozen at passage 4. The confluent cultures weredetached by standard trypsin method, live cells counted and four
experimental groups were created: cells cultured in MethoCult,cells cultured in Human Methylcellulose Base Media, cells
cultured in Matrigel and cells cultured in monolayer (control).
After a culture period of 10 days cells were recovered from 3D
matrixes and part of them was analyzed and part of them were
replated for subsequent analysis.
Cell evaluation
Cells were evaluated during the culture in semisolid matrix,
immediately after recovering them from matrix and after removal
of matrix after a short 2D culture (4 days).
During the 3D culture period the evaluation was performedby monitoring each day, at inverted microscope (Olympus IX70),
the development of the cells.
At the end of the 3D culture period the total number of live
cells was determined by Trypan Blue exclusion method and thenumber was determined using Neubauer hemacytometer. Cells
were analyzed using flowcytometric method for evaluation of
surface receptors and adhesion proteins expression. Protein
expression pattern was also evaluated and confirmed by immunofluorescence analysis.
After recovery from matrix cells were replated in 2 cm2 glasschamber culture dishes (104 cells /cm2) and cultured for 4 days
in monolayer system.
Semisolid media cell culture
For our experiments we used three semisolid media,
MethoCult (Stem Cell, Vancouver, Canada), Human Methylcellulose Base Media (R&D Systems, Minneapolis, SUA), based on
methylcellulose, and Matrigel Basement Membrane Matrix (BDBiosciences, San Jose, CA, USA), based on proteins. For theculture of cells in semisolid media a 10X cell suspension was
prepared in McCoy’s Medium (Gibco, BRL, Invitrogen, Carlsbad,
CA, USA) with 50% FCS (Fetal Calf Serum, PromoCell).
10

MethoCult and Methylcellulose media were warmed at
37°C, shortly before usage. The cell suspension and methylcellulose based media were mixed and dispensed, with a syringewith 18G, in 10 cm2 culture dishes. The thickness of the cell layer
was approximately 1mm. The culture dishes were introduced ina 100 cm 2 dish, without lid and with additional 10cm2 with water,in order to prevent drying of the matrix. The 100 cm2 dish wascovered with the lid and the cells were cultured in this system for
10 days. Cells were recovered from these matrixes by diluting,up to 10 times.
Matrigel matrix was thawed overnight at 4°C. The cellsuspension was mixed with the matrix, and dispensed, with aprecooled (-20°C, at least 2 hours) syringe, in 10 cm2 culture
dish. We used 1-1,5 mL of Marigel for each culture dish of 10cm2,
which resulted in a thickness of the cell layer of 1mm. The dishwas introduced at incubator in humid atmosphere, at 37°C, for
30 minutes than 2 mL of culture media (McCoy’s, with 10% FCS)
was laid on top on the stabilized matrix. Cells were cultured for
10 days, with the media refreshed at every 3 days. Cells wererecovered from Matrigel using Dispase (BD Biosciences), 100
UI/10cm2 (aprox. 2mL).
Flowcytometry
After 10 days of culture, and recovery of the cells frommatrix, the cells were resuspended in PBS (Phosphate Buffer
Saline, Sigma, St. Louis, MO, USA), for flowcytometeric analysis. 105 cells were placed in each analysis tube, and mouseanti-human fluorochrome-conjugated antibodies were added
at a dilution specified in the manufacturer’s protocol. The cells
were incubated with the elected antibodies in the dark, at room
temperature for 30 min. After the incubation period, cells were
washed twice with 2 mL Cell Wash Solution (BD Biosciences,
San Jose, CA, USA) and resuspended in 500 μL of the same
solution, for further analysis.
The antibodies used for the flowcytometric analysis, purchased from BD Biosciences were the following: PE-conjugated,
CD29 (Integrin beta-1), EpCAM (Epithelial cell adhesion molecule), VEGF-R2 (Vascular Endothelial Growth Factor Receptor2), CXCR4 (Chemokine Receptor Type 4), and FITC-conjugatedCD90 (Thy-1), HER2 (Human Epidermal Growth Factor Receptor2), CD24 (Heat Stable Antigen Homologue), CD44 (Receptor
for hyaluronic acid).
The analysis was performed on a four-color capable FACS
Calibur (Becton-Dickinson) flowcytometer. Data analysis was
performed with Windows Multiple Document Interface Flow
Cytometry Application software (WinMDI version 2.9).
Immunofluorescence analysis
By immunofluorescence assay the expression of CD29
(β1 integrin, Abcam, Cambridge, UK), Her2 (Human Epidermal
Grow Factor Receptor 2, DakoCytomation, Glostrup, Denmark)and Ki67 (cellular marker for proliferation, Dako) was by immunofluorescence assay.
Cells that were analyzed immediately after recovery frommatrix were transferred on a microscope glass by spinning using
Fiziologia - Physiology  2012.23.3 (75)

a cytocentrifuge (Thermo Scientific Cytospin 4). For each sample
were used 5 104 cells. After spinning cells were fixed using
methanol and placed at 4°C in PBS until staining was performed.
The immunofluorescence staining was performed as follows: cells were fixed with methanol for 10 minutes at -20 oC,
washed 2 times in PBS, and then the primary antibody was added(diluted according to manufacturer specifications). The samples
were incubated for 24 h at 4°C, in a humidified chamber, washed
2 times with PBS, and then the secondary antibody was added,
followed by 1 h incubation period, in the dark. The final step was
nuclear staining using DAPI (Sigma).
The expression of CD29 was analyzed on cells fixed immediately after recovering from the semisolid matrix, using as
primary antibody Mouse monoclonal to Integrin beta 1 (Abcam)
and as secondary antibody Alexa Fluor 594 goat anti-mouse(Invitrogen, Carlsbad, CA, USA).
HER2 expression was analyzed on cells fixed immediately
after recovery from matrix and also on cells fixed after 5 daysof culture after recovery from semisolid matrix, using Polyclonal
Rabbit Anti-Human c-erbB-2 Oncoprotein (Dako) as primary
antibody, and Alexa Fluor 488 donkey anti-rabbit as secondary
antibody.
Cell proliferation was analyzed on fixed cells, cultured in 2D for
4 days after recovery from semisolid matrix using as primary
antibody Monoclonal Mouse Anti-Human – Ki67 Antigen Clone
MIB-1 (Dako) and as secondary antibody Alexa Fluor 594 goatanti-mouse (Invitrogen).
RNA extraction and RT-PCR
RNA extraction was performed with GenElute Mammalian Total RNA (Sigma), and the extraction protocol was
performed according to manufacturer protocol. RNA concentration was measured with Nandodrop ND-100 (Wilmington,
DE, USA) spectrophotometer. For RT-PCR we used 200 ng/
reaction and fragments of 113 bp from HER 2 (ERBB2 gene)mRNA were amplified. The primers used were the following: Forward CTG-GTG-ACA-CAG-CTT-ATC-CCC-T and
Reverse ATC-CCC-TTG-GCA-ATC-TGC-A. The PCR was
performed with Thermal Cycler 2720 (Applied Biosystems)
and the program used was: 50°C - 31’, 95°C-15, (94°C-1’,
60°C-1’, 72°C-1’) x 35 cycles, 72°C-10’, 4°C-∞. Samples
were migrated in 2% agarose gel (Sigma) with 1% ethidium
bromide for nucleic acids staining, at 75V, 30’. Gels wereanalyzed in UV light with Flor-S TM MultiImager (BioRad,
BioRad, California, USA).
RESULTS AND DISCUSSIONS
During the culture period cell development was monitored
daily. In figure 1 the microscopic aspect of the cells, cultured
in semisolid matrix, is represented, at the start of the culture
period (day 1), in the middle (day 5) and at the end of the 3D
culture (day 10).
During the culture period we observed that cells in Matrigel
had a different morphological aspect when compared with cells
2012.23.3 (75)  Fiziologia - Physiology

in MethylCellulose or MethoCult. Cells in Matrigel developed
into large invasive colonies with numerous cellular extensions
and protrusions that connected with each other at the end of thecultured period (fig. 1. C, F and I). Cells in MethoCult formed
small, round, colonies that did not connect with each other during
the culture period (fig.1. A, D and G). Cells that were cultured
in MethylCellulose had an intermediate developmental patternmost of the colonies had no cellular extensions, but through theend of the culture period more cells started to exhibit extensions,the colonies were smaller in diameter that the cells in Matrigel but
bigger than the one in MethoCult, some of them even connect
with others (fig. 1. B, E and H).

Fig.1. Microscopic aspect of the SK-BR-3 cells during the culture in semisolid
matrix, MethoCult A (day 1), D (day 5) and G (day 10), MethylCellulose B(day 1), E (day 5) and H (day 10), Matrigel C (day 1), F (day 5) and I (day 10)

After removing the cells form matrix as described above
the total number of viable cells was: 1.32 x 106 for MethoCult,
1.44 x 106 for cells in MethylCellulose, and 7.8 x 106 forMatrigel. Our observations are consisted with the specialty
literature, in which is mentioned the fact that cells in 3D matrix
have different morphologies compared with cells in 2D (8). In
specialty literature the colonies formed by the cells in 3D media
are classified into four distinct morphological groups: round,mass, grape-like, stellate, and determined that SK-BR-3 cells
fall into grape-like category (6, 8). Our observations showed
that, according to Praic A. Kenney, (8) colonies classification,
the cells from SK-BR-3 line cultured in MethoCult and MethylCellulose can be classified as mass rather than grape-likecolonies. Towards the end of the culture period, the cells in
MethylCellulose developed into stellate colonies. Our studies
revealed that colonies of cells in MethoCult and MethylCellulose are not uniform in morphology, some of them were stellate,mass or grape-like, but we noticed no round colonies. On the
opposite, cells cultured in Matrigel formed uniform morphology
colonies, and all of them were stellate. Our findings shows thatthe morphology of SK-BR-3 colonies depends greatly of themedia in which they are cultured, thus explaining the difference
between the morphology of the SK-BR-3 colonies formed withindifferent media used.
11Regardless of experimental group, SK-BR-3 cells arestrongly expressing CD29 adhesion molecule on cellular surface,as provided by flowcytometric analysis (Figure 3 A, B, C, and
D). These results are in accordance with the findings of otherresearch groups (Park C. Catherine, et. al. 2006) which reveledaberrant expression of CD29 (β1 integrin) in human breast
carcinoma.

Fig.2. Flowcytometry histograms, showing negative cells for CD90 (A),
EpCam (B), Her2 (C), VGF-R2 (D), CD24 (E) and positive for CD29 (F), regardless of the media the cells were cultured in. Interestingly is that for CD44
(G) and CXCR4 (H) cells cultured in Matrigel were negative while cells fromcontrol were indicating a slightly more positive population for CD44. Cells
cultured in MethoCult and MethylCellulose seem to be two populations one
CD44+ weak and one CD44-, but the overall expression of CD44 was week.
For CXCR4 (G) the situation is similar with CD44 meaning that the control
cells seem positive meanwhile cells cultured in Matrigel are negative.

Fig.4. The expression of HER2 analyzed immediately after removal fromsemisolid media, revealed by immunofluorescence; A - Control, B - MethoCult,
C - MethylCellulose, D - Matrigel

SK-BR-3 cells from Control and MethylCelullose groupare strongly expressing HER2 immediately after recovery from
culture, unlike cells cultured in MethoCult and Matrigel thatshowed a low expression for HER2, immediately after removalof the cells from culture.

CD44 are cell adhesion molecules, glycoprotein membersof hyaluronate receptor family, whose major function is to bindto the ligands from extracellular matrix (ECM) (7). The CD44characteristic of SK-BR-3 cells is consistent with data in specialty
literature in which SK-BR-3 cells are mentioned as 84% CD44-/
CD24+ (13). After flowcytometric analysis of the cells cultured
in 3D media we can say that no significant differences wereobserved between the three semisolid media and control, withthe respect of the surface molecules expression.

Fig.5. The expression of HER2 analyzed by immunofluorescence, after 5days of culture in 2D from recovery of the cells from semisolid media, AControl, B- MethoCult, C- MethylCellulose, D- Matrigel

Fig.3. The expression of CD29 analyzed immediately after removal fromsemisolid media by immunofluorescence; A - Control, B - MethoCult, C MethylCellulose, D - Matrigel

12

In all experimental variants, HER2 expression was high(Figure 5), this being an usual feature of breast cancer cells.
HER2/ErbB has an important role in cell differentiation, proliferation, and survival. Human cancer cells display an aberrant
signaling through the HER2 pathway (3). Overexpression of
HER2, is noticed in 15 to 30% of human breast tumors, this isprognostic for poor outcome and predictive of a response to
trastuzumab treatment (2). Our results obtained by flowcitometry
Fiziologia - Physiology  2012.23.3 (75)

analysis showed that cells were negative for HER2 (Figure 2 C),
also the results obtained when cells were analyzed immediately
after recovery from semisolid media showed a low expressionfor HER2 in cells from MethoCult and Matrigel (Figure 4 B and
D). When these data were compared with the results obtained
after 5 days of culture we noticed a shift in HER2 expression
from weak to strong (Figure 5). For evaluation of HER2 expression in all experimental groups, we performed RT-PCR, and theresults are presented in Figure 6. The PCR analysis showed that
HER2 mARN was present in all the RNA samples extracted at
the moment of recovery from semisolid matrix, and also showedthat there were no difference between 2D cultured cells and3D cultured cells. Our findings suggest that maybe during theprocess of extracting the cells from semisolid matrix HER2 epitopes were damaged, and the antibody could not bound to it toemit a fluorescent signal. During the culture period the epitope
was repaired and the positive signal for HER2 was registered.

Fig.6. RT-PCR for HER2 expression; 1. Control, 2. MethoCult, 3. MethylCellulose, 4. Matrigel, 5. Negativ control, 6. Ledder 50 bp,(Gene Ruler: Fermentas
SM 0331)

Fig.7. The expression of Ki-67 analyzed by immunofluorescence, after5 daysof culture in 2D from recovery of the cells from semisolid media, A- Control,
B- MethoCult, C- MethylCellulose, D- Matrigel

The results obtained so far suggest that the inclusion ofcancer cells in semisolid media had no effect in reducing cellproliferation as shown by Ki 67 expression (figure 7. A, B, C,
and D). Ki67 is a nuclear non-histone protein, and its absencein quiescent cells and its universal expression in proliferative
cells make it an excellent marker for cell proliferation (14). Ki67
along with ER and Her2 are markers used in deciding the course
2012.23.3 (75)  Fiziologia - Physiologyof treatment for patients with breast cancer, patients with highKi67 values are treated by chemotherapy, high ER indicatesto endocrine therapy and, recently, Her2 positive patients aretreated by anti-Her2 therapy (trastuzumab). Recent studies
shown that Ki67 can be used in the prognosis of the patient, a
low level of expression indicate to a favorable prognosis whilea high expression of Ki67 suggest a highly proliferative tumor(10). Recent studies showed that Ki67 can be a useful tool inindicating the recurrence in ER-positive, Her2-negative breast
cancer patients. The main problem with fully acceptance ofKi67 is that this prognostic/predictive parameter has a highdegree of variability, such as dependency of factors like humanerror or sampling the right part of the tissue, and the specificantibody used, therefore it is important that the procedures tobe standardized (4).
CONCLUSION
Our experiments showed that the morphology of SK-BR-3cell colonies is highly dependent of the semisolid media in
which the cells are cultured. In MethoCult colonies have mass
morphology, in MethylCellulose for the first part of the culture
period they have Mass morphology and towards the end someof the colonies develop stellate morphology. Cells from SKBR-3 cultured in Matrigel, in our laboratory conditions, form
large invasive colonies with numerous cellular extensions andprotrusions, having stellate morphology.
The protein based semisolid media are better for supporting cellular proliferation compared with methylcellulose based
media. From the three semisolid media tested, cell developed
better into Matrigel, the number of viable cells recovered at
the end of 3D culture was greater in Matrigel (7.8 x 106 cells)
compared with MethoCult (1.32 x 106 cells) and MethylCellulose(1.44 x 106 cells).
The flowcytometric analysis of the surface markers showedno significant difference in expression, except CD44. Cells
cultured in Matrigel seem to have a low expression in CD44
compared with cells cultured in monolayer, MethoCult and MethylCellulose. The process of extracting the cells from matrix andfrom monolayer culture may damage the HER2 epitopes since
we had no, or very low expression for HER 2 immediately after
recovery (by flowcytometry and immunofluorescence), but after
5 days of culture the expression was exacerbated and RT-PCR
showed that HER2 mARN expression was present at the timethe 3D culture period was finished.
Inclusion of cancer cells in semisolid media had no effectin reducing cell proliferation as shown by high Ki67 expression.
Acknowledgements
This work was supported by CNCSIS – UEFISCSU, project
number PNII – IDEI 319/2011, and POSDRU 89/1.5/S/60746.
REFERENCES

1. Cukierman E, Pankov R, Stevens DR, Yamada KM. Taking cell–matrix adhesions to the third dimension. Science, 2001, 294: 1708-12.

13

2. DeFazio-EL, Strommen K, Dao-Pick T, Parry G, Goodman L,
Winslow J. Quantitative assays for the measurement of HER1-HER2
heterodimerization and phosphorylation in cell lines and breast tumors:applications for diagnostics and targeted drug mechanism of action.
Breast Cancer Research 2011, 13:R44.
3. Dua R, Zhang J, Nhonthachit P, Penuel E, Petropoulos C, Parry
G. EGFR over-expression and activation in high HER2, ER negative
breast cancer cell line induces trastuzumab resistance. Breast Cancer
Research and Treatment, 2010; 122(3): 685-697.
4. Fasanella S, Leonardi E, Cantaloni C, Eccher C, Bazzanella I, Aldovini
D, et.al. Proliferative activity in human breast cancer: Ki-67 automatedevaluation and the influence of different Ki-67 equivalent antibodies.
Diagnostic Pathology, 2011, 6 (Suppl 1): S7.
5. Geckil H, Feng Xu, Xiaohui Z, SangJun M, Utkan D. Engineeringhydrogels as extracellular matrix mimics. Nanomedicine, 2010, 5(3);469-484.
6. Genee Y Lee, Kenny PA, Lee EH, and Bissell MJ. Three-dimensional
culture models of normal and malignant breast epithelial cells. Nat
Methods, 2007; 4(4): 359-365.
7. Goodison SV, Urquidi DT. CD44 cell adhesion molecules. J Clin
Pathol: Mol Pathol, 1999; 52:189-196.
8. Kenny PA, Genee YL, Myers CA, Neve RM, Semeiks JR, Spellman

PT, Lorenz K, et.al. The morphologies of breast cancer cell lines inthree-dimensional assays correlate with their profiles of gene expression. Molecular Oncology, 2007; 1: 84-96.
9. Lund AW, Yener B, Stegemann JP, Plopper GE. The Natural andEngineered 3D Microenvironment as a Regulatory Cue During Stem
Cell Fate Determination. Tissue Eng Part B Rev., 2009; 15(3): 371-380.
10. Nishimura R, Tomofumi O, Yasuhiro O, Rumiko T, Yasuo T, Nobuyuki
A. Changes in the ER, PgR, HER2, p53 and Ki-67, biological markers
between primary and recurrent, breast cancer: discordance rates andprognosis. World Journal of Surgical Oncology, 2011; 9: 131.
11. Schedin P. and Keely P. J., Mammary Gland ECM Remodeling,
Stiffness, and Mechanosignaling in Normal Development and Tumor
Progression, Cold Spring Harb Perspect Biol January 2011, 1;3(1).
12. Shawn C. O., Shoichet M. S., Design of three-dimensional biomimetic scaffolds, Journal of Biomedical Materials Research Part A,
Volume 94A, Issue 4, pages 1321–1331
13. Sheridan C, Kishimoto H, Fuchs RK, Mehrotra S, Bhat- Nakshatri
P, Turner CH, et. al. CD44+/CD24- breast cancer cells exhibit enhanced
invasive properties: an early step necessary for metastasis. Breast
Cancer Research, 2006; 8: R59.
14. Urruticoechea A, Smith IE, Dowsett M. Proliferation Marker Ki-67 in
Early Breast Cancer. Journal Of Clinical Oncology, Biology of Neoplasia,
2005; 23: 7212-20.

ASPECTELE MORFOLOGICE ALE COLONIILOR CELULARE FORMATE
DE LINIA TUMORALA MAMARA SK-BR-3 SUNT CORELATE CU COMPOZITIA MEDIULUI DE CULTURA SEMISOLIDREZUMAT

Sistemele 3D utilizate pentru culturile celulare permit dezvoltarea in vitro a arhitecturii specific tisulare, a unei morfologii celulare specific,
a multiple interactiuni intercelulare, adeziune celulara ferma, polarizare, mobilitate celulara redusa, crestere celulara multidirectionala,oferind totodata o matrice extracelulara care mimeaza tesutul nativ. Scopul acestui studiu a fost de a investiga morfologia coloniilor
celulelor tumorale ale liniei SK-BR-3, cultivate in 3 tipuri de medii semisolide (3D) si de a analiza expresia markerilor de suprafata si
proliferarea sub influenta conditiilor de cultura 3D. Pentru experimentele desfasurate am utilizat linia celulara SK-BR-3 de adenocarcionm mamar uman, izolata din efuzia pleurala. Culturile celulare monostrat confluente au fost obtinute dupa dezghetarea celulelor
SK-BR-3 aflate la pasajul 4. Celulele aflate la confluenta au fost detasate enzimatic si au fost impartite in 4 grupuri experimentale:celule cultivate in MethoCult, celule cultivate in Human Methylcellulose Base Media, celule cultivate in Matrigel si celule cultivate inmonostrat (control). Dupa o perioada de 10 de la initierea culturii, celulele au fost izolate din matricile 3D, iar o parte dintre acestea
au fost analizate, in timp ce o alta parte au fost re-cultivate in vederea analizelor ulterioare. Rezultatele obtinute in urma experimentelor au aratat ca morfologia coloniilor celulare ale liniei SK-BR-3 este dependenta intr-o foarte mare masura de tipul mediului
semisolid in care au fost cultivate. Mediile semisolide imbogatite in proteine asigura un suport nutritiv superior, inducand o cresterea proliferarii celulare, comparativ cu mediile pe baza de metilceluloza. Analiza flowcitometrica a markerilor de suprafata nu a relevat
diferente semnificative in expresia acestora, comparativ cu grupul de control, cu exceptia CD44. Celulele cultivate in Matrigel auprezentat expresie scazuta a CD44, comparativ cu celulele cultivate in monostrat, MethoCult si MethylCellulose. Cultivarea celulelor
tumorale in medii semisolide nu a avut efecte semnificate asupra proliferarii celulare, evaluata prin expresia markerului nuclear Ki67.
Cuvinte cheie: linia celulara SK-BR-3, sisteme de cultura 3D, markeri fenotipici, proliferare celulara

14

Fiziologia - Physiology  2012.23.3 (75)

A SIMPLE ECG RECORDING HARDWARE FOR LANGENDORFFISOLATED HEART EXPERIMENTS
MARCEL PERIAN, DAN DOBREANU, CARMEN CALDARARU, MARIUS SABAU
Târgu Mureş University of Medicine and Pharmacy, Physiology Department

ABSTRACTThe isolated perfused small mammalian heart probably represents the optimal compromise in the conflict between the quantityand quality of data that can be acquired from an experimental model versus its clinical relevance. The ECG recording is done via
circumferential electrode arrays around the heart. The amplifier needs to amplify useful heart-generated potentials and to reject
as much as possible the parasitic signals picked up from environment. The amplifier designed by our team is based on AD623
integrated circuit produced by Analog Devices. This circuit have a very high CMRR (110 dB) over a large frequency range. The
use of an optocoupler at the output of the amplifier ensures galvanic separation (as high as 3KV), but also allows the amplifier tooperate in a very large range of output voltages (between 650mV and 24V). We had no need for filtering, but if it is necessary anda hardware filter is not available, the filtering can be done after the recording, using the analysis software features. The quality of
the recorded trace is very good and we consider this amplifier a reliable alternative to the high-cost amplifiers offered for several
Langendorff systems on the market.
Key words: ECG, amplifier, CMRR, Langendorff, isolated heart, AD623, optocoupler

INTRODUCTION
The isolated perfused small mammalian heart probably represents the optimal compromise in the conflict between the quantityand quality of data that can be acquired from an experimental
model versus its clinical relevance (1). The Langendorff perfusion system allows “normal” functioning of a small animal heart
during several hours with continuous monitoring of electrical andmechanical activity. The ECG recording is done via circumferential
electrode arrays around the heart and one or more ECG leads
can be obtained by switching adequate electrode pairs. During theexperiments the heart is immersed into nutritive and oxygenatedliquid, acting as a shunt for the electrical potential generated bymyocardium. This nutritive column liquid, approximately 5 m long,acts as “parasite antenna” for the surrounding electric parasites.
The amplitude of electrical signals at the electrodes leads isaround 1 mV, and a very high gain amplifier is needed for thosesignals. This kind of amplifier requires also a complete electricalinsulation between recording/display setup (usually a PC withanalog-digital converter) and the experimental liquid medium.
Our team developed such an amplifier using dedicated integrated
circuits assembled in an original topology.
MATERIAL AND METHODSThe ECG amplifier needs to amplify useful heart-generated
potentials and to reject as much as possible the parasitic signals
picked up from environment. This task can be achieved using
differential amplifiers, which have in fact two inputs respective tothe ground. This kind of amplifier is able to suppress the signalsof same amplitude and phase from the inputs and will amplify
only the voltage variations between the two inputs. Any parasitic

signal received from the environment will have approximately
the same amplitude on the both leads and the same phase(supposing that inputs cables have equal lengths), so it will bestrongly attenuated. Amplifier capacity to reject those parasitic
signals is measured by common-mode rejection ratio (CMRR).
The CMRR is defined as the ratio of the powers of the differential
gain over the common-mode gain, measured in positive decibels(dB). To be appropriate for ECG recording, such amplifier needs
a CMRR higher than 60 dB (2, 3). For performance reasons,
we have chosen for this task an integrated circuit produced by
Analog Devices, the AD623 (4). This differential amplifier, also
called instrumentation amplifier, has a very high CMRR (110 dB)
over a large frequency range. Another very important parameterof a differential amplifier is its own noise, which can affect thequality of recorded signals. The AD623 has a very low noise(35 nV/√Hz) and also a very low power consumption (1.3 mW),
allowing him to operate several days by using a single 9V battery.
A very simple ECG module can be found in the official AD623
datasheet from Analog Devices (Figure 1).

Fig. 1. A simple ECG amplifier

Received 15th of August 2012. Accepted 1st of September 2012. Address for correspondence: Dr. Marcel Perian, Târgu Mureş University of Medicine and Pharmacy,
Physiology Department, Gh. Marinescu Street No. 38, 540000 Targu Mures, Mures, Romania, e-mail: marcel_perian@yahoo.com

2012.23.3 (75)  Fiziologia - Physiology

15

This kind of ECG amplifier has several limitations. In realmeasurement conditions the input electrode impedance mayvary many KΩ, which will greatly alter the CMRR (measured byproducer with maximum 1KΩ imbalance between electrodes), soa very high impedance buffer at the input is a must. This kind of
buffer can be done using MOSFET operational amplifiers, with
input impedances of several MΩ, so that imbalances of several
thousands KΩ are insignificant. The TL074 (5) quadruple low-noise
operational amplifier from Texas Instruments is appropriate for thistask, as it can offer an input impedance of 1012 Ohms, making all
usual imbalances insignificants. It is also a low noise – low powerdevice (6mW), able to operate long time from a simple 9V battery.
One of the most important sources of parasitic electricity
on ECG systems is the electric potential developed between theperfusing solution of the Langendorff system and the ground,despite correct grounding of the system. To prevent interferences between this potential and the ECG signals, the perfusion
liquid must be “driven” with an opposite signal, having the same
amplitude as the “parasitic” signal, canceling each other (6).
This can be done using one of the four operational amplifiers
contained in the TL074.
Frequently during Langendorff experiments we need to pace
the heart using electric stimuli higher than the maximum input
voltage of TL074 (15V) or even we need to defibrillate the heart
using electric shocks. To be able to withstand to those voltages,
the input of the amplifier needs to be “clamped” to safe levels.
We obtained this effect using fast silicone diodes in anti-parallel,allowing maximum amplitude of 0.7V at the input of the amplifier.
The last available operational amplifier in the TL074 package
will be used as output buffer for AD623 and variable-gain amplifier.
The AD623 itself allows presettable gain using a resistor between
pins 1 and 8, but we preferred using a gain =1 to minimize thenoise. This buffer will drive a PNP transistor connected to an optocoupler. The CNY17-2 optocoupler is used to ensure complete
galvanic separation between recording system (PC) and theLangendorff system. CNY17-2 ensures 3.3 KV separations, muchsuperior to any voltage used during our experiments. The TL074
integrated circuit works with dual power supply and AD623 can also
work with dual power supplies, so we designed a simple voltage
divider using two usual transistors. This divider will “convert” asingle 9V power supply into a symmetrical + and – 4.5V.
The final schematic is shown in Figure 2.

RESULTS
The amplifier is 6 X 9 cm large, easy to handle during theexperiments. The recordings we have done with this amplifier
are of high quality, as it can be seen on Figure 3.

Fig. 3. Biphasic shock during fast VT post-cardioplegic arrest on whistar rat
isolated heart. Sinusal rhythm after the shock

The amplifier is injecting the signal into almost any type of
analog-digital converter capable to handle signals in a frequency
range of 1Hz and 250Hz (The Poly system, the DT 300 series
from Data Translation Inc., etc). The technical performancerequirements for ECG digital conversion are not very high, soeven self-constructed analog-digital converters can be used.
Almost every producer of AD converters is offering ready-to-use
software or platforms to develop specific software for biologicaluse (Data Translation by ex.). There are also many free software
applications which can be used (WinScope or FreeVIEW by ex.).
Our system is allowing us to measure almost every parameter
we need during Langendorff experiments. To be able to precisely
quantify the amplitude of ECG signals, the system requires
calibration before start recording. This can be done by applying
a 1mV signal at the input and tuning the gain knob to measureexactly 10 div. on the screen (Figure 4).

Fig. 4. Calibrating the gain and measuring cardiac frequency

Fig. 2. Complete schematic of the ECG module

16

The heart rate can be very easy determined knowing the time
duration between ECG elements (R-R interval by ex.) and using
formula: HR=1000/t (msec) X 60. In Fig. 4 we can see an RR
interval of 380 msec (the red circle), so the heart was runningat 158 beats/min. The ventricular electromechanical delay canalso be measured, if intraventricular pressure information is alsoavailable (Figure 5).
Fiziologia - Physiology  2012.23.3 (75)Fig.5. Electromechanical delay in post-cardioplegic whistar rat

Even if not very often used in ECG analysis, the setup is able
to perform frequency and spectral analysis (Figure 6).

Fig. 6. Spectral analysis of a QRS complex

DISCUSSIONSThe quality of the recorded trace is very good if correct
grounding of the entire setup is provided. Amplifiers developed byother authors (3) showed similar performances. The electric noise
induced by the amplifier itself is insignificant. The use of regular
50Hz current should be avoided in the proximity of the Langendorff
setup because of the induced noise. If the use of 50Hz current
cannot be avoided in the proximity of the setup, supplemental
filtering with a notch filter on 50Hz can be added. Even a simple

50 Hz low-pass filter can be used (7), since the ECG signals havea frequency spectrum usually below 40Hz. If a hardware filter is
not available, the filtering can be done after the recording, using
the analysis software features. Another reason to use 9V batteries
for this amplifier is to avoid 50Hz parasitical signals.
By switching the electrode pairs in the peri-cardiac array,different leads can be recorded, with different amplitudes. The
morphologic analysis of the recorded trace can be done easily,
although amplitude measurements are a little bit “complicated”
because of the variable gain. There are two solutions for this (ifnecessary): the use of a calibrated potentiometer with gain markson the dial, or, using short calibration pulses with 1mV amplitude
as marker at the beginning of a record.
The use of an optocoupler at the output of the amplifier ensures galvanic separation (as high as 3KV), but also allows the amplifier to operate in a very large range of output voltages (between
650mV and 24V), the range of the output signal depends only on
the voltage applied to the CE output junction of the optocoupler.
We consider this amplifier a reliable alternative to thehigh-cost amplifiers offered for several Langendorff systems on
the market and it is in use in our laboratory from over 3 years
without faults.
REFERENCES
1. Sutherland FJ, Hearse DJ. The isolated blood and perfusion fluid perfusedheart. Pharmacological research: the official journal of Italian Pharmacological Society, 2000; 41(6): 613-627.
2. Levkov C, Mihov G, Ivanov R, et al. Removal of power-line interference
from ECG: a review of the subtraction procedure. Biomedical engineering
online, 2005; 4: 50.
3. Dobrev D. Two-electrode low supply voltage electrocardiogram signal
amplifier. Medical &biological engineering & computing, 2004; 42(2): 272-76.
XXXX. www.analog.com/static/imported-files/data_sheets/AD623.pdf.2008.
XXXX. http://www.ti.com/product/tl074.2009.
Metting van Rijn AC, Peper A, Grimberg CA. High-quality recording of bioelectric events. Part 1. Interference reduction, theory and practice. Medical& biological engineering & computing, 1990; 28(5): 389-97.
Yoo SK, Kim NH, Song JS, et al. Simple self-tuned notch filter in a biopotential amplifier. Medical & biological engineering & computing, 1997;
35(2): 151-54.

DISPOZITIV SIMPLU PENTRU ÎNREGISTRAREA ECG ÎN EXPERIMENTEPE INIMA IZOLATĂ ÎN SISTEM LANGENDORFFREZUMAT

Metoda Langendorff de perfuzie a cordului izolat de mamifere mici reprezintă un instrument extrem de util în activitatea de studiu a
diverşilor mediatori sau substanţe medicamentoase cardioactive. Una dintre cele mai importante părţi ale unui sistem Langendorff
este amplificatorul pentru semnale ECG, care trebuie să îndeplinească condiţii deosebite din punct de vedere al rejecţiei paraziţilor
electrici din mediu şi al zgomotului propriu. Amplificatorul proiectat şi realizat de noi este realizat în jurul un circuit integrat AD623
produs de Analog Devices Inc., cu un CMRR de 110dB într-o plajă de frecvenţe foarte largă. Pe parcursul majorităţii înregistrărilor
realizate de noi, nu am avut nevoie de filtrare a semnalelor, dar la nevoie amplificatorului i se pot adăuga şi etaje de filtrare a semnalului, sau aceasta poate fi realizată la nivel software, în momentul prelucrării datelor. Amplificatorul propus de noi asigură în acelaşi
timp separaţia galvanică necesară între sistemul Langendorff şi sistemul de achiziţie şi înregistrare computerizată.
Cuvinte-cheie : ECG, amplificator, Langendorff, cord izolat, AD623, optocuplor
2012.23.3 (75)  Fiziologia - Physiology

17

ANALYSIS OF THE EVOLUTION IN TIME OF THE EFFECTS
OF MONOTONOUS AUDITORY STIMULATION ON CORTICAL
BIOPOTENTIALSMARIUS GEORGESCU1, DANIEL GEORGESCU1*, DRAGOŞ ALEXANDRU1, DANIELA PUSCASIU2, CARMEN TATU3, MARIA IANCĂU1
1
Department of Functional Disciplines, University of Medicine and Pharmacy of Craiova, Romania
2
Department of Microscopic Morphology, University of Medicine and Pharmacy “Victor Babes” Timisoara, Romania
3
Department of Functional Sciences, University of Medicine and Pharmacy “Victor Babes” Timisoara, Romania
ABSTRACTMonotonous stimulation can be accomplished by using continuous, predictive or repetitive stimuli and results in a functional state of thecentral nervous system characterized by lowered cerebral activity. The present study investigates the effects of several monotonous
auditory input on cerebral biopotentials. This was done by means of recording the electroencephalography (EEG) under stimulationby three auditory stimuli different in regards to frequencies, amplitudes, tonality and the induced psychoemotional state (S1, S2, S3),
on 11 volunteers (average age 23 years). Data were acquisitioned during 20 minutes of auditory stimulation, minute per minute, foreach of the 3 stimulation signals. By analysing the obtained EEG patterns, specific changes in cortical biopotentials were observed:
the subjectively pleasant sounds, S2 and S3 exert a similar influence on cortical micropotentials, by increasing all frequency bands,
except Alpha2, while the subjectively unpleasant one, S1 induces decrease of all EEG bands except low frequency ones (Delta and
Theta). The obtained results can be partially explained by the nature of the sound stimulus and each induced psychoemotional state
could be associated with a characteristic EEG pattern.
Keywords: EEG, auditory stimulation, cortical biopotentials, music, brain rhythms

INTRODUCTION
Monotony is definable as a result of constant stimulations
that are predictive or repetitive. The environment in which thosestimulations occur may also have an influence on performancein a degree that is equivalent to the monotony of the action itself(1). Monotony is characterized by decreased cerebral activation,accompanied by decreased vigilance, sleepiness, and a lower
degree of attention (1,2).
Repetitive stimuli are generally recognized as being monotonous. They are associated with decreased performance and lackof attention (1). However Wertheim (3), suggests that monotony
may be determined by the predictability of stimuli in the environment rather than by repetitivity. The degree of complexity in a task
is also deemed an important factor, and its effects are explainedby two theories: the theory of excitement and the theory of accommodation (4). Thus, tasks that have decreased requirements
lead to cognitive underload, lack of vigilance, lowered attention
and monotony, while tasks that have increased requirements
and cognitive overload lead to tiredness (5).
There are numerous studies that focus on the effect ofexternal stimulation on cortical biopotentials, especially in caseof responses to visual stimulation (6,7). The focus on using
rhythmic auditory and visual stimulation as a way of inducing
relaxation and hypnosis emerged towards the end of the XXthcentury (8). Varying repetitive visual stimulation in psychophysiologic and neurophysiologic research are frequent, withrepetitive visual stimulation patterns generally presented in

an on-off mode (for example flickering light) (9), or throughinverted pattern changes (for example checkerboard pattern)(10). Leaving aside visual stimulation, auditory stimulation
through repetitive acoustic stimuli, be they artificial or naturally
produced, either solely or in association with different activities
can provide useful new data regarding psychic sensorial andsuperior cognitive processes (11-13). Music has proved to bean essential instrument in the understanding of human emotion
and knowledge, and the cerebral mechanisms that underline
them. Some researchers maintain that human musical abilities
achieved a key phylogenetic role in the evolution of language,
and that musical behavior promoted and supported important
evolutionary functions such as cooperation, communication, andsocial cohesion (14). From the dawn of society, music was used
to stimulate emotions and treat a variety of diseases: epilepsy,
migraine, arrhythmia, schizophrenia, dementia, anxiety, neurosis
asthenia, stress, depression, autism, palpitations, cardiac insufficiency, arterial hypertension, insomnia (15-20). Despite all this,EEG investigations of cerebral activity under acoustic stimulationin general, and in particular by music, are not abundant.
The present study focuses on investigating the cerebral
electrical activity under the influence of three different types ofmonotonous sounds, both artificial and recorded from nature, different in regards to their tonality, amplitudes, frequencies and theinduced psychoemotional state. The effects of the three different
auditory stimulations was tracked on all EEG frequency bands
in time, minute per minute, during the whole stimulation period.

Received 20th of August 2012. Accepted 10th of September 2012. Address for correspondence: Daniel Georgescu, Department of Functional Disciplines, UMF Craiova,
Petru Rareş Street No. 2-4, RO-200349, Craiova, Romania, e-mail: d_g_home@yahoo.com

18

Fiziologia - Physiology  2012.23.3 (75)

RESULTS AND DISCUSSIONS
Brain rhythms were highlighted through spectral analysis
obtained on the basis of the Fourier Transform in its FFT variant(Fast Fourier Transform).
The three sounds utilized were considered representative
to achieving a monotonous unpleasant auditory stimulation incase of S1, a monotonous soothing stimulation in case of S2,
and a pleasant but tensing stimulation in case of S3, respectively.
2012.23.3 (75)  Fiziologia - Physiology

To study evolution in time choice values were chosen fromthe auditory projection area (the area covered by the P3, P4,
O1 and O2 electrodes). For each of the four electrodes, thefrequency spectrum was obtained with the help of FFT and the
median value was chosen. Through averaging, a single synthetic
value was obtained, capable of characterizing the projection area
for auditory stimulation.
Table I contains mean values for the entire spectrum, recorded during the 20 minutes of stimulation (S period), minute perminute, for each of the 3 stimulation signals. From these values
graphic 1a was compiled, which shows a decrease of values
for S1 stimulation and a slight increase in case of S2 and S3.
We further analysed the evolution in time for each EEG
band of frequency, in order to understand what gives the observed tendencies for the total frequencies. Mean values for each
EEG frequency bands were obtained during the 20 minutes ofstimulation, minute per minute, for each of the 3 auditory stimuli(data not shown) and a characteristic graphic presentation of theobtained values and a regression line for each freaquency band
were made (Figures 1.b-g).

Stimulation Signal
S1

S2

S3

Stimulation
MinuteTable I. Mean values for the total spectrum of EEG frequencies under stimulation with S1, S2 and S3
Stimulation
MinuteMATERIALS AND METHODS
The investigated group was made up of 11 men, averageage 23, all right handed, and homogenous regarding professionaland extraprofessional activity. The subjects were chosen to
perceive the band of frequencies between 45 - 16500 Hz at thesame sound level of 40 dB. After medical examination, subjects
suffering from neurological disturbances or with a history of
drug or ethanol abuse were excluded from the study. Beginningwith 12 hours prior to the EEG recording, none of the following
substances - alcohol, tea, caffeine, chocolate, hormones, B
group vitamins, sleeping pills, hypotensive drugs, tranquilizers,
sedatives - were allowed. Permission for experiments with humansubjects for scientific purposes was obtained from The EthicalCommission of the University of Craiova, Romania.
Three sounds were used: S1 (recorded in a car moving over arough surface), S2 (recorded in a rainforest) and S3 (recorded
after Mozart’s K448 Sonata). The EEG was recorded undercontinuous auditory stimulation, for 20 minutes, using a pair of“hook-on-ear” headphones connected to a netbook computer
which was running Windows Media Player 11 under Windows
XP Service Pack 3, powered by batteries to avoid parasitic
currents. The level of the sound’s intensity was measured witha NM102 Noise Meter.
The acquisition of cortical biopotentials was achieved byan industrially produced electroencephalograph, Nihon-Kohden
EEG-9200. The electrodes were placed according to the classic
10-20 system, bipolar acquisition (21), with references the 2 ears,
and an extra EKG derivation (left and right hands, the right foot)with the principal role of signal quality control. Recordings weremade in identical experimental conditions: subjects with the same
degree of psychical and physical tiredness, eyes closed, sitting
still, absent ambient sound and lighting, no disruptive ambient
electrical fields.
The procedure was carried out as following - 3 valid recordings per each subject, each one made using a different soundfrom the three ones (S1, S2 and S3), with the following steps:
1. subjects close the eyes at the operator’s command;
2. after 5 minutes of silence (L1 period) the operator begins
the auditory stimulation;
3. after 20 minutes of stimulation (S period) the operator
stops the auditory stimulation;
4. after 5 minutes of silence (L2 period) the operator stops
the recording.
To avoid inducing a modulation of the rhythm subjects were
not instructed on any particular mental activity and were given
complete freedom.

Stimulation Signal
S1

S2

S3

1

0.938656 0.962212 0.891253

11

0.861442 0.908226 0.988340

20.932399 0.905647 0.815672

120.911615 0.762348 0.900762

3

1.031920 0.915591 1.041086

13

1.089471 0.790584 0.990134

40.954790 0.881337 0.838366

14

0.842747 0.953101 0.871593

50.998890 0.911405 0.902208

150.794623 0.995684 0.906115

60.914366 0.813502 0.988902

160.752672 1.147037 0.849638

7
8
9
10

0.952155 0.872317 1.080548
0.826402 0.893476 1.046803
0.879170 0.894339 0.935941
0.756029 0.842600 0.875139

17
18
19
20

0.750711 1.059012 0.980915
0.834362 0.981376 1.134763
0.786714 0.979071 1.402991
0.773682 0.991940 0.923973

In Figures 1.b and 1.c, the mean values are graphically
represented and regression lines are drawn to represent tendencies of increase or decrease over time. By analyzing thetwo figures we see that, unlike bands Delta and Theta, Alphabands have a tendency to decrease in time under S1 stimulation(which conforms to the behavior of the entire EEG spectrum). A
general characteristic of the response to the 3 stimulations is arelatively higher spread, as well as a tendency of increase in time
for this spread. By further analysis a different behavior for bands
Alpha1 and Alpha2 is noticeable under S2 and S3 stimulation:
Alfa1 tends to increase, while Alfa2 tends to decrease.
The results for the Beta1 and Beta2 frequencies are
depicted in Figure 1d and 1e. By analyzing the graphics it isobserved that Beta EEG bands respect the overall behavior of
the EEG total spectrum.
The last investigated bands are Delta and Teta. By analyzing
graphic 1.f. we notice that the Delta frequency band does not19respect the trend of the entire frequency spectrum, a slighttendency of growth on all 3 stimulations being noticeable, with asignificantly higher rate than in case of S2 stimulation. The Teta
frequecy band behavies somewhat similarly to Delta without anoticeable reponse to S2 stimulation (Figure 1g).
Table II centralizes both the equations of the regression lines
obtained for all EEG frequency bands for each stimulation with
S1, S2, S3, as well as the tendencies to increase or decrease.
Thus, S1 decreases the mean value of EEG frequency bands
in time, with the exception of Delta and Theta, confirming its
subjectively unpleasant nature. S2 and S3 signals, subjectively
pleasant to listen to (with the exception of Alpha2) increase themean values of EEG bands. If for S2 stimulation, which is discreet
and has no rhythm interruptions we would have expected sucha result, for S3 which presents multiple changes in rhythm andvolume (Mozart) we would have expected a behavior that issimilar to S1.

d.

e.

a.

f.
b.

g.
c.

20

Fig. 1. Graphic presentation of the values and regression lines when stimulated by S1, S2, S3 signals for: a) the Total EEG Frequency Spectrum; b) theAlpha1 band; c) the Alpha2 band; d) the Beta1 band; e) the Beta2 band; f) the
Delta band; g) the Theta band

Fiziologia - Physiology  2012.23.3 (75)Table II. The equations of regression lines and tendencies for EEG bands
under stimulation by S1, S2, S3

StimuFrequency
lationBand
SignalAlpha1 (A1)
Alpha 2 (A2)
Beta1 (B1)
S1
Beta2 (B2)
Delta (D)Theta (T)Total
Alpha 1 (A1)
Alpha 2 (A2)
Beta1 (B1)
S2
Beta2 (B2)
Delta (D)Theta (T)Total
Alpha 1 (A1)
Alpha 2 (A2)
Beta1 (B1)
S3
Beta2 (B2)
Delta (D)Theta (T)Total

Regression Line

Tendencyy = -0.0047x + 0.7761
y = -0.0206x + 1.1059
y = -0.0077x + 0.9666
y = -0.0067x + 1.0363
y = 0.0059x + 0.8558
y = 0.0100x + 0.9180
y = -0.0107x + 0.9917
y = 0.0072x + 0.6880
y = -0.005x + 0.9676
y = 0.0094x + 0.8270
y = 0.0038x + 0.8833
y = 0.0607x + 0.8072
y = 0.0186x + 0.9622
y = 0.0068x + 0.8515
y = 0.0206x + 0.6852
y = -0.0131x + 1.1653
y = 0.0160x + 0.8047
y = 0.0071x + 0.8882
y = 0.0210x + 1.1777
y = 0.0297x + 0.8714
y = 0.0081x + 0.8830

Decreasing
Decreasing
Decreasing
Decreasing
Increasing
Increasing
Decreasing
Increasing
Decreasing
Increasing
Increasing
Increasing
Increasing
Increasing
Increasing
Decreasing
Increasing
Increasing
Increasing
Increasing
Increasing

Literature data regarding the influence of music on the human brain’s electrical activity is conflicting. Some authors (22, 23)
reported increases in power of the Alpha rhythm while listening
to music, while other researchers (24) observed an increasein power of Theta activity compared to the background of adecrease in total power of the Alpha rhythm. Exposure to otherclassical music pieces (fragments from Mozart’s Concertos for
Piano and Orchestra nos. 20 and 21) of a comparable intensity
level, around 40 dB, resulted in virtually no wide generalizedchanges of EEG power in the Alfa and Delta bands, while thepower increase of the high-frequency EEG bands (Alfa2, Beta1,
Beta2, Gamma) embraced almost the entire cortex (25). The
observed differences in the obtained results between our analysis
and (25) can be partially explained by the difference in lengthof stimulation and partially by the difference in the stimuli parameters – solo instrument piece versus multiple instruments, inorchestra. The discrepancies observed in literature data may berelated to the fact that most works don’t distinct between musicalstyles, and often the duration and intensity of the musical piece
was not taken into account, all of which makes the comparison
with literature data hard to achieve.
CONCLUSIONSThe obtained results indicate that each auditory stimulus
produces specific changes in cortical biopotentials. While thesubjectively pleasant sounds, S2 and S3 exert a similar influence on the EEG frequency bands, the subjectively unpleasant2012.23.3 (75)  Fiziologia - Physiology

one, S1 has a somewhat different effect. Thus, except Alpha2,
all the other frequency bands tend to increase under both S2and S3 stimulation, whereas S1 induces decrease of all EEG
bands except low frequency ones (Delta and Theta). This canbe partially explained by the nature of sound stimulus and eachinduced psychoemotional state could be associated with acharacteristic EEG pattern. Full consideration is given to further
investigations with other type of acoustic stimuli and different
stimulation length in order to provide new insights in the areaof knowledge regarding the effects of auditory stimulation on
cerebral activity and to assist in accumulating new data regarding
psychic sensorial and superior cognitive processes.
Acknowledgments
This work was partially supported by the strategic grant
POSDRU/88/1.5/S/52826, Project ID52826 (2009), co-financedby the European Social Fund-Investing in People, within the
Sectorial Operational Programme Human Resources Development 2007-2013.
REFERENCES
1. Thiffault P, Bergeron J. Monotony of Road Environment and Driver
fatigue: A Simulator Study. Accident Analysis and Prevention 2003;
35: 381-391.
2. Lal SKL, Craig A. A Critical Review of the Psychophysiology of Driver
Fatigue. Biological Psychology, 2001; 55: 173-194.
3. Wertheim AH. Highway Hypnosis: A theoretical Analysis; in: Gale AG,
Brown ID, Haslegrave CM, Moorhead I & Taylor S (Eds.): Vision in vehicles - III Amsterdam: Elsevier Science Publishers B.V, 1991, 467-472.
4. Smit AS, Eling PA, Coenen AML. Mental Effort Causes Vigilance
Decrease Due to Resource Depletion. Acta Psychologica, 2004; 115:
35-42.
5. Wilde GJ, Stinson JF. The Monitoring of Vigilane in Locomotive
Engineers. Accident Analysis and Prevention, 1983; 15: 87-93.
6. Martini N, Menicucci D, Sebastiani L, Bedini R, Pingitore A, Vanello
N, Milanesi M, Landini L, Gemignani A. The Dynamics of EEG Gamma
Responses to Unpleasant Visual Stimuli: From Local Activity to Functional Connectivity. Neuroimage, 2012; 60: 922-932.
7. Tekin E, Engin M, Dalbasti T, Engin EZ. The Evaluation of EEG
Response to Photic Stimulation in Normal and Diseased Subjects.
Computers in Biology and Medicine, 2009; 39: 53-60.
8. Teplan M, Krakovská A, Stolc S. Direct Effects of Audio-Visual
Stimulation on EEG. Computer Methods and Programs in Biomedicine,2011; 102: 17-24.
9. Soeta Y, Uetani S, Ando Y. Propagation of Repetitive Alpha Waves
Over the Scalp in Relation to Subjective Preferences for a Flickering
Light. International Journal of Psychophysiology, 2002; 46: 41-52.
10. Bai X, Liu Z, Zhang N, Chen W, He B. Three-Dimensional Source
Imaging from Simultaneously Recorded ERP and BOLD-fMRI. IEEE
Transactions on Neural Systems and Rehabilitation Engineering, 2009;
17: 101-106.
11. Jaušovec N, Jaušovec K. Differences in Induced Gamma and Upper
Alpha Oscillations in the Human Brain Related to Verbal/Performanceand Emotional Intelligence. International Journal of Psychophysiology,
2005; 56: 223–235.
12. Corral M J, Escera C. Effects of Sound Location on Visual Task
Performance and Electrophysiological Measures of Distraction. Neu-21

roreport, 2008; 19: 1535-1539.
13. Jaušovec N, Jaušovec K, Gerlic I. The Influence of Mozart’s Musicon Brain Activity in the Process of Learning. Clinical Neurophysiology,
2006; 117: 2703-2714.
14. Koelsch S, Offermanns K, Franzke P. Music in the Treatment ofAffective Disorders: an Exploratory Investigation of a New Method forMusic-Therapeutic Research. Music Perception, 2010; 27: 307-316.
15. Bodner E, Iancu J, Gilboa A, Sarel A, Mazor A, Amir D. Finding
Words for Emotions: the Reactions of Patients with Major Depressive
Disorder Towards Various Musical Excerpts. Arts in Psychotherapy,
2007; 34: 142-150.
16. Peng S M, Koo M, Kuo JC. Effect of Group Music Activity as an
Adjunctive Therapy on Psychotic Symptoms in Patients with Acute
Schizophrenia. Archives of Psychiatric Nursing, 2010; 24: 429-434.
17. Sherratt K, Thornton A, Hatton C. Music Interventions for People
with Dementia: a Review of the Literature. Aging & Mental Health,
2004; 8: 3-12.
18. Rimland B, Edelson SM. A Pilot Study of Auditory Integration
Training in Autism. Journal of Autism and Developmental Disorders,
1995; 25: 61-70.

19. Schneider S, Schönle PW, Altenmüller E, Münte TF. Using MusicalInstruments to Improve Motor Skill Recovery Following a Stroke. Journalof Neurology, 2007; 254: 1339-46.
20. Hughes JR. The Mozart Effect: Additional Data. Epilepsy & Behavior,
2002; 3: 182-184.
21. Niedermeyer E, da Silva FL. Electroencephalography: Basic
Principles, Clinical Applications, and Related Fields, Lippincott Williams& Wilkins, 2011.
22. Sulimov AV, Lyubimova Yu V, Pavlygina RA, Davydov VI. Spectral
Analysis of Human EEG during Listening to Music. Zhurnal vyssheĭ
nervnoĭ deyatelnosti imeni I P Pavlova, 2000; 50: 62-67.
23. Ikeda T. Concentration-Effect and Underestimation of Time by
Acoustic Stimuli. Shinrigaku Kenkyu, 1992; 63: 157-162.
24. Yuan Q, Liu XH, Li DC, Wang HL, Liu YS. Effects of Noise andMusic on EEG Power Spectrum. Space Medicine & Medical Engineering, 2000; 19: 401-404.
25. Pavlygina RA, Sakharov DS, Davydov VI. Spectral Analysis of
the Human EEG during Listening to Musical Compositions. Human
Physiology, 2004; 30: 54-60.

ANALIZA EVOLUTIEI IN TIMP A EFECTELOR STIMULARII AUDITIVEMONOTONALE ASUPRA BIOPOTENTIALELOR CORTICALEREZUMAT

Stimularea monotonala poate fi realizata prin utilizarea stimulilor continui, predictivi sau repetitivi si are ca rezultat un status functionalal sistemului nervos central caracterizat prin scaderea activitatii cerebrale. Studiul prezent investigheaza efectele diversilor stimuli
monotonali asupra biopotentialelor cerebrale. Evaluarea a fost realizata prin inregistrarea electroencefalografica (EEG) in urma
stimularii cu trei tipuri diferite de stimuli, in ceea ce priveste frecventele, amplitudinile, tonalitatea si statusul psihoemotional indus(S1, S2, S3), la 11 voluntari (varsta medie de 23 ani). Achizitia datelor a fost realizata timp de 20 de minute de stimulare auditiva, in
fiecare minut, pentru fiecare dintre cele 3 semnale de stimulare. Prin analiza modelelor EEG obtinute, au fost observate modificari
specifice ale biopotentialelor corticale: sunetele subiectiv placute, S2 si S3, exercita o influenta similara asupra micropotentialelor
corticale, prin cresterea tuturor benzilor de frecventa, cu exceptia alfa2, in timp ce sunetele subiectiv neplacute, S1, induc o scaderea tuturor benzilor EEG, cu exceptia celor cu frecventa scazuta (delta si teta). Rezultatele obtinute pot fi explicate partial prin natura
stimului auditiv si fiecare status psihoemotional indus poate fi asociat cu un model EEG caracteristic.
Cuvinte cheie: EEG, stimulare auditiva, biopotentiale corticale, muzica, ritmuri cerebrale

22

Fiziologia - Physiology  2012.23.3 (75)PHYSIOLOGY AND PHYSIOPATHOLOGY OF HEPATIC ENCEPHALOPATHYSCHEAU CRISTIAN1, LAZĂR MIHAI2, BUNEA MAGDA1, CIORNEI CĂTĂLINA1, PAPACOCEARALUCA1, ION DANIELA ADRIANA2, BĂDĂRĂU ANCA IOANA2
1
Department of Physiology I, “Carol Davila” University of Medicine and Pharmacy, Bucharest
2
Department of Physiopathology, “Carol Davila” University of Medicine and Pharmacy, Bucharest

ABSTRACT

A good knowledge of the physiological and physiopathological processes of hepatic encephalopathy (HE) can lead to a better
understanding of this syndrome and to an improved approach on its treatment. A large number of mechanisms are involved in thepathogenesis of HE, some of which are tightly inter-connected. This paper summarizes the most important aspects implicated in
the development of HE in the attempt to uncover future research directions, as well as provide useful information, with potential
clinical impact.
Keywords: Hepatic encephalopathy, physiology, physiopathology, mechanisms, theories, animal research, MRI, spectroscopy

INTRODUCTION
Hepatic Encephalopathy (HE) is a syndrome characterized
by a series of potentially reversible neurological deficits which
occur in a patient with chronic or acute significant liver disease
and/or porto-systemic blood shunting. The neurological andneuropsychiatric alterations are summarized in Table I. Thechanges in consciousness, attention span, and orientation may
vary, and so, the staging of the extent of neurological impairment
is performed using the West Haven Criteria for Semi-quantitative
Grading of Mental Status (1). Minimal HE is found in the majorityof patients with liver cirrhosis, and can be detected by a seriesof neuropsychological tests, with variable sensitivity.
Table I. Neurological alterations in hepatic encephalopathy

Cognitive findings
Disorientation
Altered consciousness
Short attention span
Memory impairment
Affective disorder
Emotional disturbances

Motor findings
Hyperreflexia
Asterixis
Extensor plantar reflexes
Rigidity
TremorHemiplegia (rare)

In the physiopathology of Hepatic Encephalopathy, a numberof hypothesis have been suggested, some of which are listed
below:
Ammonia as a direct neurotoxin
The suspicion that ammonium is involved in the development of HE was first materialized by Eck in 1877, when hedescribed the effect of nitrogenous substances in dogs withporto-systemic anastomosis (2). The healthy dogs with Eckfistula developed severe neurologic symptoms when they werefed meat. Later, in 1952, Gabuzda (3) observed reversible

neuropsychiatric changes in cirrhotic patients treated for asciteswith ion-exchange resins that absorb sodium and release ammonium ions.
High blood concentrations of ammonia may not correlate withthe presence of significant HE in at least 10% of cases (4).
Furthermore, several tests showed no success in inducing HE
in patients with chronic liver failure by administering ammonia.
These facts suggest that there are other mechanisms involved.
Ammonia as an indirect neurotoxin
This theory states that ammonia in high amounts in thecentral nervous system (CNS) induces astrocyte cells swelling.
Ammonium crosses the blood-brain barrier (BBB) and in order
to be cleared, it is used by the astrocytes in the conversion of
glutamate to glutamine, which in excess causes cellular edema.
Increased γ-aminobutyric acid (GABA) activity produces aninhibition of the energy usage by the other neuronal cells (5).
Current methods, such as Magnetic Resonance Spectroscopy,
allow a non-invasive quantification of these metabolites in thebrain, with very good sensitivity and specificity.
This theory also provides an explanation for the patients with HE
and low ammonium blood levels – in this case, ammonia may
have already crossed the BBB and started being metabolized.
Mercaptans as additional neurotoxins
Mercaptans have been implicated as a pathogenic factor
in HE. High blood concentrations of methanethiol have been
isolated in patients with hepatic dysfunction, and even more
specific in patients with HE, but they did not correlate with thedegree of the symptoms (6). Mercaptans are produced in thesmall intestine by bacteria through metabolizing amino-acids
which contain sulphur. The neurotoxic effect of mercaptans is
considered to be synergic and competing with ammonia (7). It
has been proven that foetor hepaticus is produced by the pres-

Received 12th of July 2012. Accepted 20th of August 2012. Address for correspondence: Scheau Cristian, MD, PhD student, “Carol Davila” University of Medicine andPharmacy, Bucharest, 8 Eroilor Sanitari Boulevard, 5th district, 050474, Romania; telephone: 0723.77.88.40; e-mail: cristianscheau@gmail.com

2012.23.3 (75)  Fiziologia - Physiology

23

ence of mercaptans in high concentrations.

of HE have been found (12).

False neurotransmitters theory
Tyramine, octopamine, synephrine and beta-phenylethanolamines are neurotransmitters obtained as secondary productsof hepatic insufficiency. Usually they have reduced or no effect,with the sole result of blocking the normal neurotransmission.
High plasmatic levels correlate with the presence of liver cirrhosis
and severe coma. The theory is well documented and has been
introduced in the 1970’s (8).

Endogenous opioids theory
Studies on rats with induced HE have demonstrated a hypersensitivity to morphine, as well as increased pain sensitivity,
findings which are specific to the endogenous opioid system(13). The increased activation of the latter was also correlatedwith behavioral alterations, such as increased ethanol intake
in cirrhotic patients (14). These findings suggest that the endogenous opioid system has a role in the physiopathology ofneuropsychiatric symptoms in patients with liver failure.

Imbalance between aromatic amino-acids (AAA) andbranched-chain amino-acids (BCAA)First proposed by Fisher et al in 1974 (9), he stated that inpatients with liver insufficiency there is an abnormally increased
ratio between AAA and BCAA. This occurs mainly becauseof the decreased AAA hepatic metabolization doubled by anincreased catabolism of BCAA in the skeletal muscles. The
main AAA which accumulate in excess are tyrosine, tryptophan
and phenylalanine, which pass the BBB and can lead to alteredneurotransmission (through synthesis of false neurotransmitters,such as octapamine) (8).
The altered ratio also connects with the levels of ammonia
in the plasma: in the hyperammonemia that occurs in hepatic
insufficiency, increased levels of ammonia are normalized by
combining with glutamate and resulting synthesis of glutamine;
this process occurs in the brain and skeletal muscle, in the latter BCAA metabolization results in forming glutamate, which isthen converted to glutamine. The excess glutamine in the central
nervous system facilitates AAA influx in the brain.
Gamma aminobutyric acid theory
Introduced in the early 80’s, this theory relies on the factthat intestinal gamma aminobutyric acid (GABA) crosses anabnormally permeable BBB to find excessive amounts of receptors, combined effect which induce an increased CNS sensitivity
to GABA inhibition. In addition, the high amount of receptors
allow binding of barbiturics and benzodiazepines, leading to anincreased effect of the two (10). The experiments used a rabbit
with liver failure induced by administering galactosamine, a verystrong hepatotoxin. One of the most important features of GABA
is that it can induce coma in high amounts, playing an importantrole in HE from this point of view.
Cerebral metals deposit theory
Metals such as manganese have been identified in thebrain of patients with HE, in the globus pallidus bilaterally, andcorrelate with Parkinsonian manifestations. The deposits canbe easily identified by Magnetic Resonance Imaging and they
appear as typical hyperintensities on T1 spin echo sequences.
Manganese is implicated in the normal functioning of superoxide
dismutase, synthetases and peroxidases, and in excess it is neurotoxic inducing astrocyte swelling (11). Variations in serum andcerebrospinal fluid levels of copper have also been speculated,but no correlation between them and the presence and degree
24

Nitric oxide theory
Nitric oxide is a gas molecule produced in the context ofhyperammonia leading to memory deficits and cerebral edema,
through increased oxidative stress and decreased antioxidant
mechanisms (15). In patients with liver insufficiency there is also
an over expression of nitric oxide synthase (NOS) in the neuronalcells, which is responsible for the excessive production of nitricoxide. The mechanism was studied on rats with porto-caval
anastomosis (16). The substrate for NOS is L-arginine, and its
cerebral uptake and availability in hyperammonemia increases
in parallel with that of NOS. Arginine can also function as a
substrate for glutamate, thus explaining the replenishment ofglutamate in the astrocytes in the case of increased glutamine
production (17).
CONCLUSION
Numerous hypotheses have been introduced over time, in
the attempt to thoroughly and completely explain the pathogenesis of hepatic encephalopathy. A series of in-vitro studies, as wellas animal experiments have documented clear and statisticallycoherent correlations, and thus fundament new theories. Clinicalevidence and new biological and imagistic investigations try to
uncover the last less-known physiopathological processes, in theattempt to fully understand this syndrome. The large number of
mechanisms involved suggests that the process of HE onset anddevelopment is a complex one, with multiple causes, some ofwhich are intricate. A good knowledge and understanding of thephysiopathology of HE can lead to a quick and correct diagnosis,
and to a good insight in the management of this syndrome.
ACKNOWLEDGEMENT: This paper is supported by the Sectoral
Operational Programme Human Resources Development (SOP
HRD) 2007-2013, financed from the European Social Fundand by the Romanian Government under the contract number
POSDRU/107/1.5/S/82839
REFERENCES
1. Ferenci P, Lockwood A, Mullen K, et al. Hepatic encephalopathy –Definition, nomenclature, diagnosis and quantification: Final report of
the working party at the 11th World Congresses of Gastroenterology,
Vienna, 1998. Hepatology, 2002; 35: 716-721.
2. Eck NV. Ligature of the portal vein. Voen Med J St. Petersburg(Russ), 1877; 130:1-2.

Fiziologia - Physiology  2012.23.3 (75)

3. Gabuzda D Jr, Philips GB, Davidson CS. Reversible toxic manifestations in patients with cirrhosis of the liver given cation-exchange resins.
N Engl J Med, 1952; 246: 124-130.
4. Abou Assi S, Vlahcevic ZR. Hepatic Encephalopathy. Metabolic
consequence of cirrhosis often is reversible, Postgrad Med 2001, 102:
52-60, 63.
5. Ryan JM, Shawcross DL. Hepatic encephalopathy. Medicine, 2011;
39 (10): 617-620.
6. H Al Mardini, K Bartlett. CO Record, Blood and brain concentrationsof mercaptans in hepatic and methanethiol induced coma. Gut., 1984;
25(3): 284-290.
7. The Merck Manual, February 2010
8. Fischer JE, Baldessarini RJ. False neurotransmitters and hepatic
failure. Lancet., 1971; 10(2):75.80.
9. Fischer JE, Yoshimura N, Aguirre A, James JH, Cummings MG,
Abel RM, Deindoerfer F. Plasma amino acids in patients with hepatic
encephalopathy. Effects of amino acid infusions. Am J Surg., 1974;
127: 40-7.
10. Schafer DF, Jones EA. Hepatic encephalopathy and the -y-aminobutyric acid neurotransmitter system. Lancet, 1982; i:18-19.
11. Rose C, Butterworth RF, Zayed J, et al. Manganese deposition inbasal ganglia structures results fromboth portal-systemic shunting andliver dysfunction. Gastroenterology 1999; 117:640-44.
12. Singh V, Bhattacharya SK, Kacchawaha JS. Cerebrospinalfluid
copper in acute hepatic encephalopathy Indian Journal of Clinical
Biochemistry 1992; 7(1): 31-32.
13. Salomon F, Beaubernard C, Thangapregassam MJ, Grange D,
Bismuth H. Encéphalopathie hépatique expérimentale. II–Étude dela réaction à la douleur chez le rat avec anastomose porto-cave. Biol
Gastroenterol, 1976; 9: 105-108.
14. De Waele JP, Audet RM, Rose C, Butterworth RF. The portacavalshunted rat: A new model for the study of the mechanisms controllingvoluntary ethanol consumption and ethanol dependence. Alcohol Clin
Exp Res 1997; 21: 305-310.
15. Rao VL. Nitric oxide in hepatic encephalopathy and hyperammonemia. Neurochem Int., 2002; 41(2-3): 161-70.
16. Rao VL, Audet RM, Butterworth RF. Portacaval shunting andhyperammonemia stimulate the uptake of L-3H-arginine but not L-3Hnitroarginine into rat brain synaptosomes. J Neurochem, 1997; 68:
337-343.
17. Mans AM, DeJoseph MR, Hawkins RA. Metabolic abnormalities
and grade of encephalopathy in acute hepatic failure. J Neurochem,
1994; 63:1829-38.

FIZIOLOGIA ŞI FIZIOPATOLOGIA ENCEFALOPATIEI HEPATICEREZUMATO bună cunoaştere a proceselor fiziologice şi fiziopatologice ale encefalopatiei hepatice (EH) poate conduce la o mai bună înţelegere
a acestui sindrom, şi la o abordare terapeutică adecvată. În patogeneza EH sunt implicate un număr mare de mecanisme, unele dintre
ele fiind strâns inter-conectate. Această lucrare sumarizează cele mai importante aspecte implicate în dezvoltarea EH, cu intenţia dea deschide noi direcţii de cercetare, şi de asemenea, de a aduce informaţii utile, cu potenţial impact clinic.
Cuvinte cheie: Encefalopatie hepatică, fiziologie, fiziopatologie, mecanisme, teorii, studii pe animale, IRM, spectroscopie.

2012.23.3 (75)  Fiziologia - Physiology

25

THE EFFECTS OF ADENOSINE AMINE CONGENER (ADAC)
ON THE SOUND STRESS IN WISTAR RATS
GOANŢĂ CM, SANDU S, PÎSLARU A, ZĂGREAN L
Division of Physiology and Neuroscience, University of Medicine and Pharmacy „Carol Davila”, Bucharest, Romania

ABSTRACT

Noise is a stress factor that cannot be avoided in everyday life with significant implications in morbidity. Its effects are not limited
to the inner ear but are reflected back to the entire body, psyche, and behavior. ADAC is a selective A1 adenosine receptor agonist
which was used before to provide neuroprotection in experimental models of cerebral ischemia and Huntington’s disease.
This study included 30 Wistar rats divided into 3 groups: one group was injected with ADAC for 7 days, a second group with saline
for 7 days, and the third group was the control group. Rats injected with ADAC and saline were exposed to a white noise with anintensity of 100 dB, for 2 hours per day during 14 days and were subject to behavioral tests (Open Field Test, Elevated Plus Maze
Test) before exposure and subsequently after 7 and 14 days respectively. Their weight was monitored daily during the first 7 days
and then on day 14. We have noticed a slight weight gain in the noise exposed rats. Animals injected with ADAC have performed
better on behavioral tests.
Keywords: noise stress, ADAC, anxiety, depression, Open field test, Elevated plus maze.

INTRODUCTION
The World Health Organization informs that 275 million people
worldwide are suffering from hearing loss (1). These worrying
figures are a manifestation of growing exposure to increased
noise. Noise pollution is due to the most basic things: cars inthe streets, iPods, appliances, music in restaurants or clubs,
background noise inside supermarkets. In large cities, it is estimated that the noise level from the traffic areas is over 65 dB
and 20% of Europe’s population is subject to this noise stress (2).
The effects of noise on the human body have been extensively
studied and historically it was concluded that acoustic stressaffects not only the human ear but in an even more severe way
it affects the nervous, endocrine, and cardiovascular systems.
Furthermore these effects became apparent at a pollution levelof 50 dB (3,4).
Prolonged exposure to noise and continuous changes ofmetabolic disorders due to endogenous factors associated tonoise stress can lead to chronic diseases such as atherosclerosis, hypertension, and cardiac ischemia (5).
Studies to date on human subjects show that exposure tonoise affects the sympathetic nervous and endocrine systems
and leads to nonspecific physiological reactions such as increased blood pressure, increased heart rate (6), vasoconstriction, increased levels of stress hormones, and sometimes evenchanges visible on the electrocardiogram (7).
These reactions of the body to noise stress were recorded
at moderate levels of noise, especially when the noise wassuperimposed to individual activities (learning, concentration,

attention) (8). Another interesting aspect is the reactions above
do not occur only when people are awake but also during sleep(9). At the psychological level, people working or living in polluted
noise have to deal with acute anxiety or depression (10,11).
Adenosine receptor agonists have been used successfullyin treating cerebral and heart ischemia and their protective function at the cellular level was successfully demonstrated (12).
The same types of receptors have been located in the cochlea(13) so it would be possible these compounds are also effective at this level. Adenosine amine congener may be a better
option than other type A1 agonists because it has a reduced
number of peripheral side effects (14). The most common side
effects noticed in the administration of A1 receptor agonists arebradycardia, hypotension, and hypothermia. The lack of theseside effects and the high affinity to receptors in the brain is due
to its chemical structure which has been modified and its ability
to cross the blood-brain barrier (15).
MATERIAL AND METHODS
Animals. For this study we used 30 Wistar rats, 12
weeks of age weighing 200-210 grams. Rats were divided
into 3 groups: a control group, a group which was administered ADAC, and another group which was administereda vehicle solution prepared similarly to the ADAC solution.
Animals were housed 4 in each cage in a room with a constanttemperature of 25 degrees with food and water ad libitum.
Treatment. ADAC was obtained from Sigma Aldrich, and was
prepared as follows: 6 mg of ADAC were dissolved in 200 µl of

Received 1th of April 2012. Accepted 21th of May 2012. Address for correspondence: Dr. Goanta CM, Division of Physiology and Neuroscience, University of Medicine
and Pharmacy “Carol Davila”, Bucharest, Romania, phone: +40213180762, e-mail: cristina.goanta@gmail.coma26

Fiziologia - Physiology  2012.23.3 (75)

1N HCl and then 120 ml of PBS 0.1 M and pH = 7 were added.
The final solution had a concentration of 50 µg / ml. The vehicle
solution was prepared using 200μl of 1N HCl and 120 ml of PBS
0.1 M, pH = 7.4. The pH of the final solutions was 7.3 - 7.4 inboth cases. The solutions thus obtained were stored in microcontainers at a temperature of -20° C. Before each injection, thesolutions were brought to 37° C. We administered the substances
every day for 7 days at an interval of 24 hours apart, starting 6
hours after the first exposure to noise. The amount was injected
intraperitoneally and was adjusted according to the rat weight(200μl/100 g body weight). We chose this dose as it is describedin the literature to be effective in protecting the brain against
ischemia without any adverse cardiovascular effects. Body
weight and intra-rectal temperature were monitored to highlightpotential side effects such as weight gain or loss or hypo/hyperthermia. The rectal temperature was monitored before and after
the ADAC administration at 30 and 60 minutes. The body weight
was monitored daily for the first 7 days and then on day 14.

in all three groups of rats, but with no significant difference
between them (Figure 1.a). We did not observe any hypo/
hyperthermia when analyzing rectal temperature (Figure 1.b).

Noise stress. Rats were exposed to white noise at an
intensity of 100 dB for 2 hours a day during 14 days. The
sound intensity was measured in the vicinity of cages using an
EMC-DT 85 A sound level meter and intensity deviations were
kept to a minimum (100 ± 1 dB).
Behavioral analysis. We performed two tests: The OpenField Test (OFT) and the Elevated Plus Maze (EPM). The OFT
takes place in a square arena with the side of 60 cm, divided into
two zones: a peripheral zone about 10 cm wide, located near thewall, and a central zone. The animal behavior was recorded with acamera suspended above the arena and connected to a computer.
The program used to record the animals as well as for data extraction was Ethovision 3.0. Each recording lasted for 10 minutes.
Between testing of each rat the arena was cleaned with alcohol
10%. The measured parameters were the distance that the animals
moved, the time they spent in each of the two areas. Stressed andanxious animals will spend a longer period of time standing near
the arena walls, and will record lower values ​​of distance traveled
and time spent in the center of the arena. EPM is a test which also
analyzes anxiety and depression and is performed using a wooden
maze with 4 perpendicular arms of equal size (50x10cm), located
50 cm from the floor. Two arms have 40 cm high walls and arecalled closed arms and the other two do not have walls and arecalled open arms. Rats were placed in the center of the maze facing
a closed arm and were registered for 5 minutes with a suspended
camera connected to a computer. Data acquisition was made with
Ethovision 3.0. The parameters that were followed were the distance traveled and the time spent in each of the open and closed
arms. Between tests the maze was cleaned with 10% alcohol.
Statistical analysis was performed in Excel, using AVERAGE,
STANDEV, TTest tests.
RESULTS
Temperature and Weight. We noticed an increase in weight2012.23.3 (75)  Fiziologia - Physiology

Fig.1. Weight and temperature. 1.a. Weight was measured before noise
stress and then at 7 and 14 days of noise exposure.1.b.Temperature wasbmeasured before ADAC treatment
and at 30 and 60 minutes after administration.

Fig.2. The distance rats traveled through the OFT arena. After 7 days ofnoise stress p=0.039333 and after 14 days p=0.0012

In the OFT we noticed a significant difference between thethree groups in what concerns the distance traveled, both groups
exposed to noise traveling shorter distances than the control
group and the group injected with vehicle traveling a shorter
distance than the one injected with ADAC (Figure 2).
27

DISCUSSION

Fig.3. The time rats spent in the central and in the peripheral zones of the
OFT arena. At 7 days of sound stress p=0.00886 and at 14 days p= 0.006543

Subjects from the sound stressed groups spent a shorter
period of time in the central zone than the ones in the control
group, those in the group injected with ADAC spending a longer
period of time in the central zone when compared with the groupinjected with the vehicle solution (Figure 3).

Fig. 4. The distance rats traveled through the EPM labyrinth. At 7 days after
noise stress p=0.017497 and at 14 days p= 0.017035

The EPM results confirmed the results that were obtained
from the OFT data: sound stressed rats spent longer periods oftime in the closed arms when compared to the time spent in the
open arms (Figure 4), and traveled shorter distances than thecontrol group rats (Figure 5).

In order for sensory systems to be functioning properly,
people need a certain sound comfort. Noise can break thisbalance affecting various organs and systems ranging from thealteration of the immune response to impaired social behavior
leading to anxiety and depression (16). This study is the firststep in our attempt to prove ADAC’s protective role against
noise pollution (17). Anxiety and depression are conditions that
occur frequently in people living in sound polluted environments.
Anxiety and depression in rats can be studied with the two tests
listed above, the OFT and the EPM. Results are interpreted using
two parameters: the distance rats travel through the maze andthe time they spend in the light and in the dark zones. Anxious
animals will travel shorter distances, and eventually will be immobile and avoid open spaces, preferring to stay in dark areas(in the closed arms of the maze or in the peripheral area near
the walls) (18). Some studies have shown that there are somespecific periods during life in which each rat is more sensitive tonoise exposure (19), namely during early life and then at an older
age, the period between childhood and young adult being themost severely affected by exposure to noise (20). We performed
behavioral tests and the results showed that the noise stressed
rats became more anxious than those which were not exposed
to noise, and those in the group injected with ADAC were lessanxious than those in the group injected with a vehicle solution.
Another interesting observation is that in some of the measurements made at 14 days after the start of the noise exposure, rats
performed better than in those made at 7 days after exposure,which leads us to think that animals can somehow adapt to thenoise polluted environment (21). With regard to weight changes
our results show some variation in weight, the group injected with
ADAC with a slightly higher increase than the other two, however
without being statistically significant. The literature confirms aweight gain of rats exposed to white noise compared to thosewhich were not exposed to white noise (22).
CONCLUSIONADAC administration had a beneficial effect, rats that were
injected with ADAC performing better in the tests to which they
have been subjected. We can say that the administration of
ADAC in Wistar rats has a protective role against the depression
and anxiety induced by noise stress. Another important aspectis that we did not notice any side effects such as hypothermia or
bradycardia as opposed to other A1 adenosine receptor agonists.

Fig.5. The amount of time rats spent inside the open arms and inside theclosed arms of the EPM. After 7 days of noise stress p=0.026433 and after 14days p=0.0379994

28

REFERENCES
1. Prevention of noise induced hearing loss-report of the WHO-PDH
Informal consultation, Geneva,28-30 October 1997
2. Uran SL, Caceres LG, Guelman LR. Effects of loud noise on hippocampal and cerebellar-related behaviors. Role of oxidative state.
Brain Reaserche; 1382: 102-114.
3. Lenzi P, Frenzilli G, Gesi M, Ferrucci M, Lazzeri G, Fornai F, Nigro

Fiziologia - Physiology  2012.23.3 (75)

M. DNA damage associated with ultrastructural alterations in rat myocardium after loud noise exposure. Environ. Health Perspect., 2003;
111: 467-471.
4. Rabat A. Extra-auditory effects of noise in laboratory animals: therelationship between noise and sleep. J. Am.Assoc. Lab. Anim. Sci.
2007; 46(1): 35-41.
5. Babisch W. Cardiovascular effects of noise. Noise and Health, 2011:201-204.
6. Wolk R, Gami AS, Garcia-Touchard A, Somers VK. Sleep and cardiovascular disease. Curr Probl Cardiol 2005; 30: 625-62.
7. McEwen BS. Stress, adaption, and disease. Allostasis and allostaticload. Ann N Y Acad Sci 1998;840:33-44.
8. Maschke C, Harder J, Ising H, Hecht K, Thierfelder W. Stresshormone changes in persons exposed to simulated night noise. Noise
Health 2002; 5: 35-45.
9. Muzet A. Environmental noise, sleep and health. Sleep Med Rev
2007; 11: 135-42.
10. Lee KW, Kim SJ, Park JB, Min KB, Kil HG, Lee C, Lee KJ. Relationship Between Aircraft Noise Exposure and Depression, Anxiety, Stressin Korea. Epidemiology, 22: S258-S258.
11. Rylander R. Physiological aspects of noise-induced stress andannoyance. Journal of Sound and Vibration 2004; 227: 471-478.
12. Jacobson KA, Xie R, Young L, Chang L, Liang BT. A novel pharmacological approach to treating cardiac ischemia:binary conjugates of A1
and A3 adenosine receptor agonists. J Biol Chem 2000; 275: 30272-79.
13. Maggirwar MS, Rybak SB, Whitworth LP, Ramkumar C. Expression
and function of adenosine receptors in the chinchilla cochlea. Hear Res
1997; 105: 130-140.

14. Lubitz DKJE, Lin RCS, Paul IA, Beenhakker M, Boyd M, Bischofberger N, Jacobson KA. Postischemic administration of adenosine
amine congener (ADAC): analysis of recoveryin gerbils. Eur J Pharmacol 1996; 316: 171-9.
15. Jacobson KA, Kirk KL, Padgett WL, Daly JW. Functionalized
congeners of adenosine: preparation of analogues with high affinity for
A1-adenosine receptors. J Med Chem 1985; 28: 1341-46.
16. Turner JG, Parrish JL, Hughes LF, Toth LA, Caspary DM. Hearing inlaboratory animals: strain differences and nonauditory effects of noise.
Comp. Med. 2005; 55: 12-23.
17. Uran SL, Caceres LG, Guelman LR. Effects of loud noise on hippocampal and cerebellar-related behaviors. Role of oxidative state.
Brain Reaserche; 1382: 102-114.
18. Brenes JC, Padilla M, Fornaguera J. A detailed analysis of openfield habituation and behavioral and neurochemical antidepressant-like
effects in postweaning enriched rats. Behav. Brain Res. 2009; 197:
125-137.
19. Ohlemiller KK. Contributions of mouse models to understanding ofage - and noise-related hearing loss. Brain Res. 2006; 1091: 89-102.
20. Henry KR. Lifelong susceptibility to acoustic trauma: changing patterns of cochlear damage over the life span of the mouse. Audiology
1983; 22(4): 372-383.
21. McEwen BS, Gianaros PJ. Stress- and allostasis-induced brain
plasticity. Annu. Rev. Med. 2011; 62: 5.1-5.15.
22. Sembulingam K, Sembulingam P, Namasivayam A. Effect ofchronic noise stress on some selected stress indices in albino rats. J
Environ Biol; 19: 63-66.   

ADENOSINE AMINE CONGENER (ADAC) ŞI EFECTELE SALE ASUPRA COMPORTAMENTULUI ŞOBOLANILOR WISTAR SUPUSI STRESULUI SONORREZUMAT

Zgomotul este un factor stresant in crestere direct proportionala cu dezvotarea tehnologica si cu implicatii semnificative in ceea cepriveste morbiditatea. Efectele pe care le are asupra corpului uman nu se limitează la nivelul urechii interne ci se răsfrâng asupraîntregului organism,asupra psihicului şi comportamentului. În studiul de fata vrem sa arătăm efectele protectoare pe care le poate
avea administrarea Adenosine Amine Congenerului asupra comportamentului şobolanilor Wistar .ADAC-ul este un agonist selectiv
al receptorilor adenozinici de tip A1. Printre cele mai importante efecte ale sale se numara protectia impotriva ischemiei cerebrale.
În acest studiu au fost incluşi 30 de şobolani Wistar, împărţiţi în 3 loturi: 1-injectat cu Adenosine Amine Congener timp de 7 zile,
2-cu soluţie salină timp de 7 zile şi 3-a fost lotul control. Şobolanii injectaţi cu Adenosine amine congener şi soluţie vehicul au fostexpuşi unui sunet alb cu o intensitate de 100dB, 2 ore pe zi timp de 14 zile şi le-au fost efectuate teste comportamentale (Open
Field Test, Elevated Plus Maze test) înainte de expunere şi apoi la 7 şi 14 zile. Le-a fost monitorizată zilnic greutatea în primele 7
zile si apoi la 14 zile. Am observat o uşoară creştere în greutate a şobolanilor, precum şi o creştere a apetitului la toate loturile deşobolani stresaţi fonic. Animalele injectate cu Adenosine Amine Congener au avut performanţe mai bune la testele comportamentale.
Cuvinte cheie: stres fonic, ADAC, open field test, elevated plus maze, anxietate, depresie

2012.23.3 (75)  Fiziologia - Physiology

29

PREVALENCE OF REFRACTIVE ERRORS IN SCHOOLCHILDREN IN URBAN ENVIRONMENTLUMINITA TURCIN
Turcin Clinic“Vasile Goldis” University, Arad, Romania

ABSTRACTThe main aim of this study is to establish the refractive errors prevalence in urban schoolchildren from Arad County.
Materials and Methods: We have examined 509 pupils aged 6 to 11 years enrolled in elementary classes of 5 schools in Arad cityduring September - December 2011. We have investigated the following parameters: age, sex, objective refraction and visual acuity.
Refraction was measured with Potec 5000 autorefractokeratometer under cycloplegia, which was obtained with cyclopentolate, 4times in one hour. Myopia was defined as refractive errors of at least -1.0 SD, hyperopia + 1.5SD and astigmatism 1.0CD.
Results and discussions: We found 130 pupils with refractive errors out of which 17 cases of myopia, 43 of hyperopia, and 70 casesof astigmatism. Fifty pupils were diagnosed de novo with refractive pathology. Thirty-three subjects didn’t wear any correction tool
despite their pathology.
Conclusion: Astigmatism is the refractive error with the highest prevalence, followed by hyperopia and myopia. The prevalence ofastigmatism was higher in female (64.29%) than male subjects (35.71%) [p < 0.001] and the prevalence of hyperopia was higher inmales (65.12%) than females (34.88%) [p < 0.005]. Elementary schoolchildren are a high risk group for developing refractive errors.
Key words: refractive errors, children, urban environment, amblyopia, myopia, hyperopia, astigmatism

INTRODUCTION
Childhood visual impairment due to refractive errors is oneof the most common problems in school children and second
leading cause of treatable blindness (6). Most of the children
with uncorrected refractive errors are asymptomatic and hence
screening helps in early detection and timely interventions. In
countries with high school attendance (like Romania), integration of vision screening within screening for other health issues
is recommended (1). However, differences in the availability ofaccess to eye care services and even the magnitude of refractive
errors between rural and urban students are not considered (2).
In this study we focus on the urban environment. Future studies
will discuss rural ophthalmic pathology in preschool- and schoolchildren and the differences between the two environments.
Refractive error is one of the most common causes of visual
impairment around the world and the second leading cause oftreatable blindness (6). In a study, Bucsa D and collaboratorsconcluded than the most common disorders in preschool- andschoolchildren are refractive errors (5).
Reliable data on prevalence and distribution of refractive errors
from population-based surveys are needed to plan cost-effective programs devoted to the reduction of visual impairment and blindness.
Undiscovered and untreated refractive errors are an important cause of low visual acuity or amblyopia. We found little recent
data in the literature regarding the prevalence of myopia, hypermetropia and astigmatism at schoolchildren in urban Romania.
Therefore, our objective is to determine the prevalence of thispathology in urban children population. We must underline theimportance of the screening of refractive errors because of thenegative consequences that result from the early misdiagnoseof these health problems.
Amblyopia or lazy eye and its risk factors is a decrease invisual acuity resulting from abnormal visual development in children, and is a major public health problem. Amblyopia is the mostcommon cause of monocular or in some cases binocular vision loss
in infants and young adults (7). Amblyopia affects approximately
2-4% of the population (8). Most cases are associated with eye
misalignment, usually esotropia in infancy or early childhood(9-10). Less frequently, anisometropia [difference in refractive
error between the two eyes] or a combination of strabismus andanisometropia are causally associated with amblyopia. In children
under 7 years, amblyopia was associated with strabismus in
38% of cases, with anisometropia in 37% of cases and with both
strabismus and anisometropia in 24% of cases (11).
In the present work, prevalence and pattern of refractive
errors (myopia, hyperopia, astigmatism) among school children
in Arad has been studied for planning appropriate eyecare programs to reduce the burden of visual impairment among younger
population in this area.
MATERIALS AND METHODS
The study was conducted between September and December 2011. Verbal consents of school director, teachers andparents were obtained for screening the children. The research
protocol adhered to the provisions of the Declaration of Helsinki
for research involving human beings.
Target group size was calculated by the means of Kish
and Leslie’s formula for an expected prevalence of 30% with

Received 20th of August 2012. Accepted 10th of September 2012. Address for correspondence: Dr. Luminita Turcin, MD, PhD student, “Vasile Goldis” University, Arad, Romania

30

Fiziologia - Physiology  2012.23.3 (75)

confidence limit of 5% and confidence level 99%.
We have examined 509 pupils aged 6 to 11 years enrolled
in elementary classes of 5 schools in Arad city. There was noacute pathology that would have influenced refraction. The distantvision of a child was tested utilizing Snellen’s Illiterate ‘E’ chart.
The visual acuity was tested with the chart at 6 meters. If uncorrected vision was <0.6 in either eye, the child was declared tohave defective vision. A cover-uncover test was then performed toconfirm the diagnosis of strabismus. If eyes moved after removalof the cover, the child was considered to have a “phoria”; and if
the degree of deviation did not change on cover and uncover, thechild was considered to have a “tropia” [> 5 degree / 10Δ diopter(D)]. The eye movements were tested in 6 cardinal directions torule out paralytic or restrictive strabismus. Anterior segment was
examined with flashlight to detect cataract; congenital anomalies
like anophthalmos, microphthalmos, large corneas; and evidenceof previous eye surgery. Objective refraction was measured with
Potec 5000 autorefractokeratometer under cycloplegia which was
obtained with cyclopentolate 1% 4 times in 1 hour. This procedure
was applied to all children, regardless of visual acuity.
Statistic analysis was conducted with Epi Info 7.
Emmetropia was defined as a spherical equivalent between-1.00 and +1.00. Myopia was considered when the measured
objective refraction was more than or equal to –1.0 sphericalequivalent diopters in one or both eyes. Hyperopia was consideredwhen the measured objective refraction was greater than +1.50
spherical equivalent diopters in one or both eyes provided no eye
was myopic. Astigmatism was considered to be visually significant
if ≥1.00 D. Results are presented in tables and charts below.
All children with uncorrected refractive error were given
low cost spectacles. . Children with eye diseases were further
examined and managed at the base clinic free of charge. The
study results were shared with the scientific fraternity and policies
for improving eye care of children were proposed.
RESULTSWe found the following results: out of the total of 509 children
examined, 379 (74.46%) were emmetropic and 130 (25.54%)
were found with refractive errors (ammetropic). There were 17(3.34%) cases of myopia, 43 (8.45%) of hypermetropia and 70(13.75%) cases of astigmatism. Results are shown in Table I.
Table I. Number of children and distribution of refractive errors over classes

Class No. pupils Total refrac- Myopia Hyperopia Astigmatismtive errors
I.
139
32
5
9
18
II.
149
40
5
12
23
III.
112
29
3
10
16
IV.
109
29
4
12
13
Total
509
130
17
43
70

This means that 25.54% of children from urban environment included in the study have ophthalmic refractive pathology.
3.34% have myopia, 8.45% have hypermetropia and 13.75%
2012.23.3 (75)  Fiziologia - Physiology

have astigmatism.
There were no statistical differences in prevalence of refractive errors between genders.
Chart 1 shows the proportion of the different types of refractive errors. Myopia represents 13.08%, hyperopia 33.08% andastigmatism 53.85% of discovered refractive errors.

MiopiaHyperopia
Astigm atism
Chart 1. Proportion of the three types of refractive errors

In this study we examined 230 (45.18%) male and 279(54.82%) female children. We discovered 7 (41.17%) cases of boys
and 10 (58.83%) cases of girls with myopia. There is no statisticallysignificant difference between sexes in myopia. We discovered
28 (65.11%) cases of boys and 15 (34.89%) cases of girls withhyperopia. We found 25 (35.71%) cases of boys and 45 (64.29%)
cases of girls with astigmatism. Prevalence of astigmatism washigher in girls (64.29%) than in boys (35.71%) [p < 0.001], while
hypermetropia was more prevalent in boys (65.11%) than in girls(34.89%) [p < 0.005]. Data are shown in Table II.
Table II. Distribution of refractive errors over sexes

Pupils Myopia Hyperopia Astigmatism
Males 230
7
28
25
Females 279
10
15
45
Total
509
17
43
70

Refractive
60
70
130

Out of the total number of 509 children examined, 130(25.54%) were found with refractive errors. Fifty (38.46%) children
were newly discovered with ophthalmic refractive pathology and33 (25.38%) didn’t wear optical correction although they knew
about their condition. Hence 83 pupils overall didn’t wear opticalcorrection for their ophthalmologic pathology, because either they
didn’t know about it or they were not compliant with the treatment.
Forty-seven (9.23%) of the 509 children examined wore
optical correction at the time the study was conducted.
Table III. Newly and neglected refractive errors distribution

No. c Percent (%) Myo- Hyper- Astigpia opia matism
No. of pupils
509
100
Total refractive errors
130
25.54
17 43
70
Newly discovered refractive 50
9.82
5
18
27
errorsKnown and uncorrected
33
6.48
4
14
15
refractive errors

31

We found 5 (3.84%) new myopia cases, 18 (13.86%) new
hyperopia cases and 27 (20.76%) new astigmatism cases. The
number of new cases, expressed as percentage of total numberof children examined, was as follows: 0.98% myopia cases,
3.53% hyperopia cases and 5.39% astigmatism cases.
We found 4 (3.07%) known and neglected myopia cases, 14(10.76%) known and neglected hyperopia cases and 15 (11.53%)
known and neglected astigmatism cases. The number of known,
yet neglected cases, expressed as percentage of total number ofchildren examined, was: 0.78% myopia cases, 2.75% hyperopia
cases and 2.94% astigmatism cases. Forty-seven (9.23%) fromthe 509 children examined wore optical correction at the time thestudy was conducted. Results are shown in Table III.
Considering each type of refractive error, we have 21.41%
new myopia cases, 23.52% known and neglected myopia cases,
41.86% new hyperopia cases, 32.55% known and neglected
hyperopia cases 38.54% new astigmatism cases and 21.42%
known and neglected astigmatism cases.
52.94% of the children with myopia, 74.41% of the children
with hyperopia and 60% of the children with astigmatism didn’t
wear optical correction at the time of examination because either
they didn’t know about their pathology or they have shown low
compliance with the treatment.
Related to the total number of children examined, we found
1.77% untreated myopia cases, 6.17% untreated hypermetropia
cases and 8.25% untreated astigmatism cases. Statisticallysignificant differences can be seen between myopic and hypermetropic cases (1.77% and 6.17% p < 0.001) and between
myopic and astigmatism cases (1.77% and 8.25% p<0.0001).
There was no statistically significant difference between hypermetropic and astigmatism cases.
Table IV. Age groups distribution of refractive errors

Age No % Myo- % Hyper- % Astig- %
pupia
opia
matism
pils
cases
cases
cases
6 23 4.52
2 11.76
3
6.98
5
7.14
7 111 21.81 3 17.65
7 16.28 13 18.57
8 87 17.09 4 23.53 11 25.58 17 24.29
9 99 19.45 4 23.53
8
18.6
9 12.86
10 109 21.41 2 11.76
6 13.95 11 15.71
11 80 15.72 2 11.76
8
18.6
15 21.43
total 509 100 17
100
43
100
70
100
The prevalence of myopia, hyperopia and astigmatism overage groups after one year is shown in Table IV. There was no
statistically significant difference in the prevalence evolution overthese age groups in neither of the studied refractive pathologies.
DISCUSSION
In 2003 Budau M. and collaborators conducted a screening
assessment of refractive errors of children investigated at “Luis32

Turcanu” Hospital’s Ambulatory. They concluded that out of the
646 children, 407--63% (CI95 = 59.1-66.7) had refraction errors,
out of which 1.5% (CI95 = 0.8-2.9) were myopic, whereas 49.8%(CI95 = 45.9-53.8) were hyperopic. Astigmatism was found in
11.8% (CI95 = 9.4-14.6) and the mean age was 10.7 years (4).
Compared to our study myopia cases have similar prevalence(3.54% in our study and 1.5% in the cited study [p < 0.02]). The
results are different in the case of astigmatism, which we found tobe the most frequent; while in the above mentioned study hyperopia had the highest prevalence. We believe the differences arise
from the different definitions of the studied pathology. Also, thetarget group was different than ours. Patients who went to “Luis
Turcanu” Hospital’s Ambulatory already had some symptoms andpossibly a current disease. This could be a good explanation forthe high percentage of refractive errors found (63%).
In a study Bucsa D. and collaborators concluded than themost common disorders in preschool - and schoolchildren are
refractive errors (5). We found that 74.46% of children from thestudy were emmetropic and 25.54% ammetropic. This could
mean that about one quarter of elementary schoolchildren haverefractive errors.
In 2001, Hendrickson K and Bleything W conducted ascreening of Romanian children and adults. They found thefollowing data: 45% of the children were emmetropic, 27%
were myopic, and 28% were hyperopic; 42% of the adults were
emmetropic, 16% were myopic, and 42% were hyperopic. When
compared with other nations the prevalence of myopia washigher in the Romanian children, whereas in adults the hyperopia
was higher. With-the-rule astigmatism had the highest occurrence when compared to other axis orientations, yet the overalloccurrence of astigmatism was less than that found in othernations for both children and adults. Incidence of astigmatism
was lower compared to other nations in both children and adults.
The prevalence of strabismus and other ocular diseases was
lower in the Romanian children as compared to other nations (3).
CONCLUSIONS
The most prevalent ophthalmic pathology in urban schoolchildren is astigmatism, followed by hyperopia and myopia.
Astigmatism is more prevalent in girls and myopia is moreprevalent in boys. There is no statistically significant differencein the prevalence of myopia cases in the two sexes.
We didn’t find any variation of prevalence between age
groups from 6 to 11 in any of the studied refractive pathology.
The most prevalent newly discovered ophthalmic pathology
in urban schoolchildren is astigmatism, followed by hypermetropia and myopia. Elementary schoolchildren are a high risk
group for developing refractive errors. From those hyperopia andastigmatism are the main risk factors of developing amblyopia.
The most prevalent known and uncorrected (neglected, poor
treatment compliance) ophthalmic pathology in urban schoolchildren is astigmatism, followed by hypermetropia and myopia.
The screening of school and preschool children should
be carried out periodically. Most children are unaware of their
Fiziologia - Physiology  2012.23.3 (75)

refractive errors.
Children aged 6 to 11 and their parents should be educatedabout signs and symptoms of refractive errors, ocular hygiene
and the risk factors involved in the development of these errors,
especially amblyopia and other ocular pathological problems.
We believe that the newly discovered refractive errors canbe addressed by screenings like this one. It is a good way to
discover and treat children ophthalmic pathology and prevent
amblyopia. Effective VA screening strategies and cycloplegic
refraction measurements are needed to eliminate this easily
treatable cause of visual impairment.
REFERENCES
1. World Health Organization. Elimination of avoidable visual disability
due to refractive error Report of an informal planning meeting. pp. 6-10.
WHO/PBL/00.79.
2. Amruta SP, Khandekar R, Dharmadhikari S, Dole K, Gogate K,
Deshpande M. Prevalence of Uncorrected Refractive Error and Other
Eye Problems Among Urban and Rural School Children. Middle East
Afr J Ophthalmol 2009; 16(2): 69-74.
3. Hendrickson K, Bleything W. The visual profile of Romanian children

and adults assessed through vision screenings. Pacific University,
College of Optometry, Forest Grove, Oregon, USA. Optometry 2001;
72(6): 388-96.
4. Budău M, Gamaniuc E, Moise C, Korbuly B. Study of refractive
changes in Timiş County. Oftalmologia 2003; 58(3): 64-9.
5. Bucşă D, Gavăt V, Albu A. Aspects of child and youth health revealedby medical check-up in Botosani County, Romania. Rev Med Chir Soc
Med Nat Iasi 2011; 115(4): 1225-30.
6. Dandona R, Dandona L. Refractive error blindness. Bull World HealthOrgan. 2001; 79: 237-43.
7. Doshi NR, Rodriguez MLF. Amblyopia. Am Fam Physician 2007;
75: 361-7.
8. Chang CH, Tsai RK, Sheu MM. Screening amblyopia of preschool
children with uncorrected vision and stereopsis tests in Eastern Taiwan.
Eye 2007; 21: 1482-8.
9. Woodruff G, Hiscox F, Thompson JR, Smith LK. Factors affecting
the outcome of children treated for amblyopia. Eye 1994; 8: 627-631.
10. The Pediatric Eye Disease Investigator Group. The Clinical Profile ofModerate Amblyopia in Children Younger Than 7 Years. Arch Ophthalmol
2002; 120: 281-287.
11. Shaw DE, Fielder AR, Minshull C, Rosenthal AR. Amblyopia: factors
influencing age of presentation. Lancet 1988; 2: 207-209.
12. Prevalence of Refractive Errors in Primary School Children [7-15Years] of Qazvin City. Eur J Sci Res 2009; 28: 174-185.

PREVALENTA ERORILOR DE REFRACTIE LA SCOLARII DIN MEDIULURBANREZUMAT

Scopul principal al acestui studiu a fost de a stabili prevalenta erorilor de refractie la scolarii din mediul urban din Judetul Arad.
Materiale si metode: Au fost examinati 509 scolari cu varste cuprinse intre 6 si 11 ani, inscrisi in clasele elementare in 5 scoli din
Arad, in perioada Septembrie-Decembrie 2011. Au fost investigati urmatorii parametri: varsta, sexul, refractia obiectiva si acuitatea
vizuala. Refractia a fost determinata utilizand autorefractokeratomerul Potec 5000, pe ochiul cicloplegic, care a fost indus cu ciclopentolat, de 4 ori in decurs de 1 ora. Miopia a fost definita ca eroare de refractie de cel putin -1.0 SD, hypermetropia ca + 1.5 SD,
iar astigmatismul ca 1.0 CD.
Rezultate si discutii: Au fost determinate erori de refractie la 130 elevi, dintre care 17 au prezentat miopie, 43 hipermetropie si 70
au fost cazuri de astigmatism. 50 de scolari au fost diagnosticati de novo cu patologie refractiva. 33 de subiecti nu beneficiau detratament corector in momentul investigatiei, indiferent de patologia determinata.
Concluzii: Astigmatismul este eroarea de refractie cu cea mai crescuta prevalenta, fiind urmata de hipermetropie si miopie. Prevalenta
astigmatismului a fost mai mare la scolarii de sex feminin (64.29%) comparativ cu subiectii de sex masculin (35.71%) [p < 0.001],
iar prevalenta hipermetropiei a fost crescuta la elevii de sex masculin (65.12%), comparativ cu sexul feminin (34.88%) [p < 0.005].
Elevii aflati in scoala elementara reprezinta un grup cu risc inalt de a dezvolta erori de refractie.
Cuvinte cheie: erori de refractie, copii, mediul urban, ambliopie, miopie, hipermetropie, astigmatism2012.23.3 (75)  Fiziologia - Physiology

33

CHANGES OF INTERLEUKIN-12 SERUM LEVELS IN PATIENTSWITH CHRONIC HEPATITIS C VIRUS INFECTION BEFORE
INTERFERON ALPHA TREATMENTMANOLE COJOCARU1, SIMONA ALEXANDRA IACOB2, DORINA BANICĂ3, EUGENIA PANAITESCU2, DIANA GABRIELA IACOB2
1“Titu Maiorescu” University, Faculty of Medicine, Bucharest
2“Carol Davila” University of Medicine and Pharmacy, Bucharest
3“Marius Nasta” Institute of Pneumology, Bucharest

ABSTRACTInterleukin (IL)-12 is a cytokine secreted by antigen-presenting cells (APC) such as activated macrophages and dendritic cells (DC).
IL-12 might play a crucial role in the course of HCV infection. In this study, we aimed to investigate the serum levels of IL-12 in patientswith chronic hepatitis C virus (HCV) infection, its roles in the pathogenesis of chronic HCV infection and to analyze how IL-12 related
to histologic findings and laboratory parameters. Serum IL-12 levels were measured by ELISA. Serum IL-12 levels of the patientswith chronic HCV infection were found to be elevated when compared to those of healthy controls (274.3 pg/mL (107.5-469.7 pg/mL)
vs. 43.6 pg/mL (38.3-76.2 pg/mL); p<0.001). There was significant correlation between IL-12 and ALT levels (r=0.512; p<0.001). The
serum IL-12 was correlated with the histological activity score (p<0.001). In conclusion, since serum levels of IL-12 were higher inpatients with chronic HCV infection, this citokine might play an important role in the inflammatory process in chronic HCV infection.
Key words: interleukin-12, chronic hepatitis C virus infection

INTRODUCTION
IL-12, a heterodimer composed of 2 subunits of p40 and
p35 and secreted mainly by antigen-presenting cells (APC) suchas activated macrophages and dendritic cells (DC), is a crucial
mediator between innate and adaptive immune responses. IL-12
induces rapid and efficient production of IFN-γ (1,2). In addition,
IL-12 correlates with virus clearance. Experimental studies havesupported the role of immune response mechanisms in liver
injury of HCV infections. The exact mechanism of hepatocellular
damage in chronic HCV infection remains unclear (3-5).
The aim of this study was to investigate the serum levels of IL-12
in chronic hepatitis C, its roles in the pathogenesis of chronic
HCV infection and to analyze how IL-12 related to histologicalfindings and laboratory parameters.
MATERIAL AND METHODS
Fifteen patients (6 male, 9 female, mean age 47.35±10.78
years) with chronic HCV infection and 14 healthy subjects (6male, 8 female, mean age 35.00±15.45 years) were includedin this study. The diagnosis of the patients with chronic HCV
infection was established on the basis of clinical, laboratory,
ultrasonographic and histopathology findings. The healthy
subjects had negative hepatitis serology, normal liver function
tests and normal ultrasonographic findings. Serum IL-12 levels
were measured by ELISA. Each sample was tested in duplicate.
The levels of the IL-12 were also compared with the traditional
biochemical ALT and viral (HCV RNA) indicator of infection
activity. Histological activity score was evaluated in the chronic

hepatitis C infected patients. The Mann-Whitney U test wasused to compare IL-12 and alanine transaminase (ALT) levels
between the cases and the controls. Discrete variables were
evaluated with Chi-square test. Odds ratios (OR) and 95%
confidence intervals (CI) were calculated for IL-12. Correlation
analysis was done by the Spearman test. A value of p<0.05 was
accepted as significant.
The research was carried out with each patient’s informed
consent and Ethics Committee approval.
RESULTS
All patients had positive HCV RNA with PCR. We found that
HCV-infected patients with greater necro-inflammatory activity ofliver showed greater IL-12 production. A significant increase in IL12 production was observed only in patients with chronic hepatitis
C who cleared the virus (P<0.001). The greater production of
IL-12 associated with greater necro-inflammatory activity of liverin HCV infected patients. Serum IL-12 levels of the patients withchronic HCV infection were found to be elevated when comparedto those of healthy controls (274.3 pg/mL (107.5-469.7 pg/mL) vs.
43.6 pg/mL (38.3-76.2 pg/mL); p<0.001). There was significant
correlation between IL-12 and ALT levels (r=0.512; p<0.001).
The serum IL-12 was correlated with the histologic activity score(p<0.001). There was statistically significant correlation between
serum IL-12 levels and severity of necro-inflammatory changes
in liver biopsy specimens (p<0.001). It may play an importantrole in promoting inflammatory reactions. Since serum levelsof IL-12 were higher in patients with chronic HCV infection thiscytokine might play an important role in the inflammatory process

Received 1th of April 2012. Accepted 21th of May 2012. Address for correspondence: Dr. Manole Cojocaru, Titu Maiorescu University, Faculty of Medicine, Department of
Physiology, Center for Rheumatic Diseases, Bucharest; Thomas Masaryk No. 5 Sector 2 Postal Code 020983, Bucharest, Romania, e-mail: manole.cojocaru@yahoo.com

34

Fiziologia - Physiology  2012.23.3 (75)

in HCV infection.
DISCUSSION
Recent studies have suggested that IL-12 is one of the mostimportant cytokines in the immune defense against intracellularpathogens. Few studies in adult patients report on the role in thecourse of HCV infection. Cytokines are key mediators of inflammation, apoptosis, necrosis and fibrosis and they are actively
involved in the regeneration process of liver tissue after injury.
Massive induction of the pro-inflammatory cytokines IL-12 andIFN-γ in liver specimens is apparently not counterbalanced by
the anti-inflammatory cytokine IL-10. IL-12 has an important role
against intracellular pathogens by promoting Th1 cell development, cell mediated cytotoxicity and IFN-γ production. Our results
revealed higher values in the serum levels of IL-12 in patientswith chronic HCV infection as compared to normal controls andit was correlated with the histological activity score (6-8).
IL-12 stimulating NK cells activity, cytotoxic T lymphocytes proliferation and IFN-γ production, plays an important role in the pathogenesis of HCV infection. IL-12 is the dominant cytokine, which
specifically promotes antigen-dependent Th1 cell differentiation,
suppresses Th2 function and may contribute to the viral clearance.
IL-12 is the dominant cytokine, which specifically promotes Th0cell differentiation to Th1 cells, suppresses Th2, enhances IFN
production and stimulates T and NK cells cytotoxicity (9).
The increase of serum IL-12 always followed the ALT flare,
confirming the function of IL-12 in promoting Th1 cell development, and function of macrophage. In HCV infection massive
hepatocytes are killed by macrophages. This explains why HCV
infected patients with greater necro-inflammatory activity of liver
showed greater IL-12 production, and why Th1 predominancein HCV infection was correlated with the direct cytotoxic effectand the inhibition of viral replication. This means that strategies
to prevent macrophage influx may be beneficial to patients with
hepatitis. The roles of cytokines in the liver damage are complex.
Serum IL-12 levels can inform about the inflammatory activity in the liver. IL-12 serum levels seem to be reliable parameter
informing about the inflammatory activity and extend of fibrosis in

the liver (10). Further research is needed to explore the aspectsof this relationship.
CONCLUSION
Our results suggest that serum levels of IL-12 reflect the
inflammatory activity of hepatitis. IL-12 shows correlation withbiochemical and histopatological markers of hepatitis.
CONFLICT OF INTEREST
The authors have declared no conflicts of interest
REFERENCES
WHO. Hepatitis C. Fact Sheet 164. 2000; http://www.who.int
1. Alter MJ. Epidemiology of hepatitis C virus infection. World J Gastroenterol. 2007; 13: 2436-41.
2. Sun QL, Ran W. Review of cytokine profiles in patients with hepatitis.
World J Gastroenterol. 2004; 10(12): 1709-15
3. Birk RW, Gratchev A, Hakly N, et al. Alternative activation of antigenpresenting cells: concepts and clinical relevance. Hautarzt 2001; 52:
193-200
4. Jacobson Brown PM, Neuman MG. Immunopathogenesis of hepatitis
C viral infection: Th1/Th2 responses and the role of cytokines. Clin
Biochem. 2001; 34: 167-71.
5. Sobue S, Nomura T, Ishikawa T. Th1/Th2 cytokine profiles andtheir relationship to clinical features in patients with chronic hepatitis
C virus infection. J Gastroenterol. 2001; 36: 544-51.
6. Liu M, Chang CW, Mcgilvray I, et al. Fulminant viral hepatitis: molecular and cellular basis; a clinical implications. Expert Rev Mol Med.
2001; 28: 1-9
7. Viso AT R, Duarte MIS, Pagliari C, et al. Tissue and serum immune
response in chronic hepatitis C with mild histological lesions. Mem Inst
Oswaldo Cruz, Rio de Janeiro 2010; 105(1) 25-32.
8. Schvoerer E, Navas MC, Thumann C, et al. Production of interleukine-18 and interleukine-12 in patients suffering from chronic hepatitis
C virus infection before antiviral therapy. J Med Virol. 2003; 70: 588-93.
9. Tilg H, Kaser A, Moschen AR. How to modulate inflammatory cytokines in liver diseases. Liver Int 2006; 26: 1029-39.

Acknowledgement
This paper was supported by CNCSIS-UEFISCSU, PNII, code1165/2009

VARIAŢII ALE CONCENTRAŢIILOR DE INTERLEUKINĂ-12 ÎN SERULPACIENŢILOR CU INFECŢIE CRONICĂ CU VIRUSUL HEPATITEI CÎNAINTEA TRATAMENTULUI CU INTERFERON ALPHAREZUMATInterleukina (IL)-12 este o citokină produsă de celulele prezentatoare de antigen (APC), precum macrofagele activate şi celulele
dendritice (DC). IL-12 s-ar putea să prezinte un rol esenţial în cursul infecţiei cronice cu HCV. Obiectivele acestui studiu a fost să
determinăm nivelurile de IL-12 în serul pacienţilor cu infecţie cronică cu virusul hepatitei C (HCV), importanţa acesteia în patogenia
infecţiei cu HCV şi să analizăm relaţia IL-12 cu aspectele histologice şi cu unul dintre parametrii de laborator. Nivelurile de IL-12
în ser au fost determinate folosind tehnica ELISA. Nivelurile de IL-12 în serul pacienţilor cu infecţie cronică cu HCV au fost găsite
crescute comparativ cu lotul martor (274,3 pg/ml (107,5-469,7 pg/ml) vs. 43,6 pg/ml (38,3-76,2 pg/ml); p<0.001). S-a observat ocorelaţie semnificativă între IL-12 şi nivelurile de ALT (r=0,512; p<0,001). IL-12 s-a corelat cu scorul histologic de gravitate (p<0,001).
În concluzie, deoarece nivelurile de IL-12 în ser au fost mai crescute la pacienţi cu infecţie cronică cu HCV, această citokină s-ar
putea să joace un rol important în procesul inflamator în infecţia cronică cu HCV.
Cuvinte cheie: interleukina-12, infecţia cronică cu virusul hepatitei C
2012.23.3 (75)  Fiziologia - Physiology

35

ETIOLOGY AND PATHOPHYSIOLOGY OF SEIZURES, EPILEPSY AND STATUS EPILEPTICUS AFTER STROKE
VINTILESCU RALUCA, BALSEANU ADRIAN, MITU CRISTINA, DINA CIPRIAN, GEORGESCUMARIUS, IANCAU MARIA
Department of Physiology, University of Medicine and Pharmacy of Craiova, Faculty of Medicine

ABSTRACTOver the age of 60, acute seizures, epilepsy and status epilepticus have the highest incidences. The primary etiological factor forthe new onset seizures is the cerebrovascular disease. The cerebrovascular etiologies include cerebral infarction, cerebrovascularatherosclerosis, and cerebral hemorrhage. Cerebral infarction is the most frequent - 30%; it increases the risk of late-onset seizures
23 times. Epileptic seizures can be early and late-onset. This division is important, because the pathophysiology and prognosis ofboth are different. Early-onset seizures appear one or two weeks after stroke, but most of them occur after 24 hours. Late poststroke seizures occur within several months to years after the injury. The development of epilepsy is accompanied by excessive
glutamatergic activity and damage in the balance between glutamatergic and GABA-ergig synaptic transmission. Some of the risk
factors of post-stroke seizures: ischemic stroke subtype, stroke location and stroke severity. Cortical location is associated withearly-onset seizures after ischemic stroke. Stroke severity determines the outcome in stroke patients. The treatment of epileptic
seizures depends on the clinical context.
Keywords: epilepsy, stroke, risk factors

INTRODUCTION
Cerebrovascular complications are a risk factor in thedevelopment of seizures and epilepsy (1-4). Seizures may occur in 2-20% of all patients with stroke (1-5). Two unprovokedseizures, separated 24 hours apart, are required to diagnose
epilepsy. The latest definition of epilepsy shows that a single
epileptic seizure is enough as long as there is a predisposition
to generate epileptic seizures (6).
Epilepsy is expressed by recurrent, unprovoked, epileptic
seizures, and by the cognitive, psycho-social and social consequences of this condition (7,8). It is very important to make a clinical distinction between provoked and unprovoked etiologies forseizures. Cardiac failure, vasovagal syncope, different metabolic
conditions, different medications, alcohol and drug use are thecauses of provoked etiologies. This category also includes acutehead injuries or acute cerebrovascular events (9).
Etiologically, epilepsies are divided into three categories:
idiopathic, symptomatic, and presumed symptomatic. Idiopathic
epilepsies may have genetic causes, leading to the alteration ofbasic neuronal regulation. Symptomatic epilepsies are caused by
the effects of an epileptic lesion, which can be focal (a tumor), ora metabolic defect, giving rise to widespread injury to the brain.
Cryptogenic epilepsies imply a lesion that is difficult or impossible to discover during evaluation. In approximately 60% of all
epilepsies the etiology is unknown. When the etiology is known,
they include traumatic brain injury, ischemic stroke, intra-cerebral
hemorrhage, infections, tumors, cortical dysplasia, several neurodegenerative diseases, or acute symptomatic seizures suchas febrile seizures or status epilepticus (10).

Epidemiology and etiology of epilepsy in the elderly
Over the age of 60, acute seizures, epilepsy and statusepilepticus have the highest incidences (11-14). The primary
etiological factor for the new onset seizures is the cerebrovascular disease. The cerebrovascular etiologies include cerebral
infarction, cerebrovascular atherosclerosis and cerebral
hemorrhage. Cerebral infarction is the most frequent - 30%; itincreases the risk of late-onset seizures 23 times (15). Other
etiological factors of epilepsy in the elderly include brain tumor,
toxic and metabolic abnormalities, neurodegenerative and (15)neuropsychiatric disorders. Dementia is an important cause fordeveloping seizures. Alzheimer’s disease has a higher rate of
epilepsy. The explanation for the higher rate of epilepsy in Alzheimer’s disease may be the accumulation of amyloid plaques
and neurofibirillary tangles and focal areas of disproportionate
neuronal degeneration. After an ischemic stroke, patients often
express delayed epileptic seizures. Epileptic seizures can beearly and late-onset (16).
Early-onset seizures appear one or two weeks after stroke,
but most of them occur after 24 hours. Late post-stroke seizures
occur within several months to years after the injury. This division is important, because the pathophysiology and prognosisof both are different.
A large number of patients with early seizures do not develop late seizures or recurrent epilepsy, only about one thirdof them will develop these types of complications. The risk ofsubsequent epilepsy is much higher if a late seizure occurs (17).

Received 10th of April 2012. Accepted 25th of May 2012. Address for correspondence: Balseanu Tudor-Adrian, MD, PhD, Department of Physiology, Faculty of Medicine, University of Medicine and Pharmacy of Craiova 2-4 Petru Rares Street, RO-200349 Craiova, Romania, Phone: +40 741015387, e-mail: adibalseanu@yahoo.
com, adibals@umfcv.ro36

Fiziologia - Physiology  2012.23.3 (75)Patients with ischemic stroke onset between 15 and 45
years develop seizures in the first year after the stroke (18).
Status epilepticus is characterized by recurrent epileptic
seizures lasting hours-to-days, without return to baseline mental
status between seizures. Status epilepticus can have convulsive
or non-convulsive forms. In elderly patients status epilepticus has
significant morbidity and mortality (19). Status epilepticus may
be a symptom of acute stroke, about 24.8% (20). Non convulsive
status is associated with early-onset post-stroke seizure patients(85%) and convulsive status (50%) is associated with late-onset
group. The early onset status epilepticus has a higher mortality
rate than the late onset status-epilepticus (21).
Pathophysiology of seizures
Some features of symptomatic epilepsy include the distinction between early and late seizures, the latent period, the clinicalphenomenology and response to treatment and the risk factors.
The epilepsy is a pathological reaction of brain tissue to insult.
Homeostatic or systemic disturbances are the causes of early
seizures. For example, hyperglycemia at the time of ischemia
enhances the risk for seizures (17).
Late-onset seizures may be caused by gliosis, the occurrence of a meningocerebral cicatrix, selective neuronalcell death and apoptosis, changes in membrane properties,
mitochondrial changes, receptor changes, deafferentation andcollateral sprouting (22).
The brain depends on its blood flow which is a supply ofoxygen and glucose. Ischemia induces a delayed selective damage of CA1 pyramidal neurons in the hippocampus, while CA3
and dentate gyrus neurons are resistant (23).
After acute ischemia, extracellular concentrations ofglutamate are increased. The glutamate is an excitatory neurotransmitter, associated with secondary neuronal injury (24).
The initial pathological and pathophysiological damage is influenced by excessive glutamatergic activity. There is no seizure
activity after this initial damage, however pathophysiological andstructural alterations occur in key brain regions, which culminate
in the expression of epilepsy. Epileptogenesis is the process bywhich injured neuron surviving the acute insult together withwell-preserved neurons form epileptic neuronal networks (25).
Overstimulation of glutamate receptors determines glutamate excitotoxicity. Excessive neuronal depolarization is followedby an increase in free intracellular calcium, entering through voltage gated calcium channels and glutamate receptor/channels.
Neuronal dysfunction and alteration in morphology/structure ordeath are determined by activation of the calcium-dependent
signaling pathways (26, 27). An acute increased extracellularglutamate concentration, N-methyl-D-aspartate (NMDA) receptors and calcium-dependent mechanisms lead to neuronal cell
death (28).
The development of epilepsy is accompanied by excessive
glutamatergic activity and damage in the balance between glutamatergic and GABAergig synaptic transmission (29, 30). This
imbalance may be followed by molecular and/or functional altera2012.23.3 (75)  Fiziologia - Physiology

tion in glutamatergic transmission. The damage in the GABAergic
system indirectly increases glutamatergic transmission (25).
An ischemic insult also affects GABAergic inhibitory transmission, in the area adjacent to cortical lesions and in remotebrain regions. An important reduction in the number GABAergic
interneurons is caused by global ischemia in the CA3 area of hippocampus. GABAergic cells form a population of neurons which
are divided into two classes according to their axonal projection:
somatic and dendritic targeting interneurons. They play different
roles in the hippocampus. In the hippocampus there are twomain kinetic classes of GABAA synaptic responses. GABA A fast
synaptic events are mediated by somatic and proximal dendritic
synapses and GABA A slow synaptic events are mediated bydendritic synapses (31, 32). There are three types of GABA
receptors, GABAA, GABAB and GABAC. GABAA and GABAC
are ionotropic receptors, which consist of membrane channels,
whose opening permits the passage of Cl ¯ and HCO3¯. GABAB receptors are located pre- and post-synaptic, and they arecoupled to G-proteins (33).
GABA A receptors consists of the following subunits: α, β,γ, and δ. A subunit subtype is important, because it confers different pharmacological and physiological properties to receptor
isoforms. α2, α3, and/ or α5 subunits are diminished during
development, unlike the α1 subunit, which is up-regulated andis dominant during adulthood. The α1 subunit has an important
decrease in the visual cortex one week after the injury. On thecontrary, an important up-regulation of the α3 subunit occurs in
the contra lateral cortex one month after the ischemia (34-37).
Epileptic activity can be suppressed by the GABAergic
system. Epileptiform discharges are produced by the reductionor blockade of inhibitory transmission and, if repeated, it may
cause epileptogenesis. Epileptogenesis can be prevented ordelayed by enhancing GABAergic inhibition (25).
Studies suggest that in humans idiopathic epilepsies areconnected with mutations at the GABAA receptor level (X H) (38).
The mechanism by which epileptogenesis is induced in the
amygdale is the excessive activation of glutamate receptors.
It is not yet clear which of the glutamate receptor subtypes
should be activated for epileptogenesis. There are three typesof glutamate receptors: AMPA receptor, NMDA receptor andkainat receptor (39).
Out of these receptors, NMDA receptors play an importantrole in inducing epileptogenesis in the amygdale, as in otherbrain regions (25).
Several months after stroke, functional map reorganization
has been observed, leading to a partial recovery of function in
the visual cortex. The consequence of this phenomenon is thatthe neurons that survive the injury suffer delayed morphological changes in the level of expression of proteins involved indendritic and axonal plasticity (MAP-2, GAP 43) (40). Several months after global ischemia, there is a shift of the resting
membrane potential of CA3 pyramidal neurons toward positive
values. The regulation of the cell excitability depends on the Na+/
K+-ATPase. Several weeks after ischemia of the CA3 pyramidalcells, a decrease of the Na+/K+-ATPase has been noticed (23).
37Ischemia, besides producing neuronal degeneration leading to
the damage of brain function, may also lead to important network
recovery (23).
Environmental and pathological conditions can affectthe rate of cell proliferation, migration and differentiation andthis affects the neurogenesis. Seizures are the most common
pathological conditions which have an important influence onneurogenesis (41).
Neurogenesis
Neurogenesis is the development of new functioning neurons, in the adult brain. The dentate gyrus of the hippocampus
and the subventricular zone of the lateral ventricles are the two
major neurogenic regions in the brain (42-46). A stimulation offorebrain subventricular zone cell proliferation and neurogenesis
has been noticed after focal ischemia (47). The first studies
showed that the increase in subventricular zone neurogenesis
after stroke is transient, but later studies demonstrate that it
persists for four months after ischemia (48-50).
The subventricular zone neuroblasts usually migrate to theolfactory bulb, via chain-like formations, in the rostral migratory
stream. However, a large part of them migrate in chains toward theischemic striatum after focal ischemia. Because of this redirectedmigration, fewer neuroblasts reach the ipsilateral bulb. The matrix
metalloproteases and chemokine / chemokine receptor interaction
are the molecular factors that induce this ectopic migration to periinfarct regions. This migration, of subventricular zone neuroblast
to the injured striatum lasts for up to a year after ischemia (51-55).
Seizures increase dentate granule cell neurogenesis andit contributes to aberrant network reorganization in the adult rat
hippocampus (56). Dentate granule cells, generated after seizure
activity, begin sending axons aberrantly into the dentate inner molecular layer, by day 24 after stimulation (57-59). The population of
newly generated migratory neurons in the subgranular zone of thedentate gyrus is enlarged by the periodic stimulation of the brain
by seizure-evoking pharmacological manipulations, performed
within the developmental range of hippocampus neurogenesis.
These neurons can contribute to the formation of new, recurrent
excitatory circuits within the hippocampus formation (60).
Risk factors of post-stroke seizures
Some of the risk factors of post-stroke seizures: ischemic
stroke subtype, stroke location and stroke severity (24). Hypertension raised serum cholesterol and left ventricular hypertrophy
lead to the development of seizures or epilepsy, even if there isno overt stroke (17).
Stroke subtype
Early-onset seizures
The frequency of early-onset seizures is higher in case ofhemorrhagic strokes (3-19%), cerebral vein thrombosis (12-34%)
or subarachnoid hemorrhage (4-18%) than in case of ischemic
stroke (61). Partial seizures with or without secondary generaliza38

tion are frequent in cerebral vein thrombosis (61).
Late-onset seizures
The frequency of late-onset seizures is in case of hemorrhagic strokes 2.6-6%, cerebral vein thrombosis 5-26.6%,
subarachnoid hemorrhage 7-9% and in case of ischemic stroke
2-4% (61).
Seizures are more common with cardioembolic infarction
than other types of ischemic stroke (24). O possible explanationof seizures after cardioembolic stroke, induced by cortical emboli,
is the depolarization within an ischemic penumbra, reperfusion
after the fragmentation and distal migration of the embolus, ora combination of these two factors (62).
The risk of seizures is increased by hemorrhagic transformation of ischemic lesions. The hemorrhagic transformation can be
an independent factor for seizures, in patients with first stroke (17).
The excitability of the cortical ischemic penumbra tissue could be
increased the presence of hemorrhagic transformation in corticalregions, where it is associated with edema. The brain excitability
in the presence of hemorrhagic transformation may be an independent predictor of status epilepticus in acute ischemic stroke.
Middle cerebral artery aneurysms, intra-parenchyma hematoma,
cerebral infarction, hypertension and thickness of the cistern clot
are risk factor for seizures after subarachnoid hemorrhage (62).
Stroke location
Early-onset seizures
Cortical location is associated with early-onset seizures
after ischemic stroke (24). Hemispheric subcortical lesions,determined by small vessel disease, induce seizures with a frequency between 0-3.5%, but this mechanism in not understood(62). Lobar site is the most epileptogenic location in intracerebral
hemorrhage (62). Seizures are predicted by caudate, temporalor parietal involvement within the cortex (62). The risk factorsof early-onset seizures after cerebral vein thrombosis are theexistence of the focal deficits,the cortical vein thrombosis andparenchyma lesions (61).
Late-onset seizures
Cortical location is associated with late-onset seizures after
ischemic stroke (61).
Stroke severity and size of infarct
The stroke severity is predictive of seizures independentlyof the ischemic lesion. There is a relation between the risk ofseizures and the size of the infarct (17). Stroke severity and sizeof infarct are related to status epilepticus (62). Stroke severity
determines the outcome in stroke patients. Increased in hospitalmortality is caused by early post-ischemic seizures.
Seizure type in post-stroke epilepsy
All types of partial seizures can be observed. Early–onFiziologia - Physiology  2012.23.3 (75)

set seizures are most often simple partial (without impaired
consciousness), with motor symptoms or complex partial (withimpaired consciousness). They can be partial evolving into
secondary generalized seizures. Stroke represents one of themost important causes of status epilepticus in adults (22-32%)
Status epilepticus doesn’t seem to be influenced by the stroke
type, ischemic or hemorrhagic, but it would be more frequent incase of severe stroke (61).
Treatment
The treatment of epileptic seizures depends on the clinicalcontext. Status epilepticus requires urgent intravenous therapy.
Early seizures did not require long-term antiepileptic therapy
to prevent recurrence, but they require short-term antiepileptic
drug treatment. Oral antiepileptic drug therapy is required inlate-onset seizures.
t-PA is contraindicated to patients with seizures. Long-term
anticoagulant therapy can’t be given to patients experiencing
seizures with falls. In renal or hepatic impairment patients, antiepileptic drugs should be use prudently and the dosage needs
to be modified (17).
REFERENCES
1. Myint PK, Staufenberg EF, Sabanathan K. Post-stroke seizure andpost-stroke epilepsy. Postgrad Med J 2006; 82: 568-572.
2. Silverman IE, Restrepo L, Mathews GC. Poststroke seizures. Arch
Neurol 2002; 59: 195-201.
3. Hauser WA, Annegers JF, Kurland LT. Incidence of epilepsy andunprovoked seizures in Rochester, Minnesota: 1935-1984. Epilepsia
1993; 34: 453-468.
4. Hauser WA, Annegers JF, Kurland LT. Prevalence of epilepsy in
Rochester, Minnesota: 1940-1980. Epilepsia 1991; 32: 429-445.
5. Burneo JG. Revisiting the well-established subject of stroke andepilepsy. Epilepsy Curr 2007; 7: 42-44.
6. Fisher RS, van Emde Boas W, Blume W, et al. Epileptic seizuresand epilepsy: definitions proposed by the International League Against
Epilepsy (ILAE) and the International Bureau for Epilepsy (IBE). Epilepsia, 46: 470-472.
7. Chang BS, Lowenstein DH. Epilepsy. N Engl J Med 2003; 349:12571266.
8. Engel JJ and Pedley TA Epilepsy: A Comprehensive Textbook 2008;
Lippincott Williams & Wilkins, Philadelphia
9. Banerjee PN, Filippi D, and Allen Hauser W The descriptive epidemiology of epilepsy-a review. Epilepsy Res 2009; 85: 31-45.
10. Hauser WA, Annegers JF, Kuland LT. Incidence of epilepsy andunprovoked seizures in Rochester, Minnesota 1935-1984. Epilepsia,
1993; 34: 453-468.
11. Annegers JF, Hauser WA, Lee JR, et al. Incidence of acute
symptomatic seizures in Rochester, Minnesota, 1935-1984. Epilepsia,
1995; 36: 327-333.
12. DeLorenzo RJ, Hauser WA, Towne AR, et al. A prospective,
population-based epidemiologic study of status epilepticus in Richmond,
Virginia. Neurology 1996; 46: 1029-35.
13. Cloyd J, Hauser V, Towne A, Ramsay R, et al. Epidemiologicaland medical aspects of epilepsy in the elderly. Epilepsy Research
2006; 68: S39-48.
14. Rowan AJ, Ramsay RE, Collins JF et al. VA Cooperative Study 428
Group New onset geriatric epilepsy: a randomized study of gabapentin,
2012.23.3 (75)  Fiziologia - Physiology

lamotrigine, and carbamazepine. Neurology, 2005; 64: 1868.
15. Varelas PN, Mirski MA. Seizures in the adult intensive care unit. J
Neurosurg Anesthesiol 2001; 13:163-75.
16. Menon B, Shorvon S. Epilepsy Research 2009; 87: 1-11.
17. Naess H, Nyland HI, Thomassen L, et al. Long-term outcome of cerebral infarction in young adults. Acta Neurol. Scand. 2004; 110: 107-112.
18. Cloyd J, Hauser W, Towne A, et al. Epidemiological and medical
aspects of epilepsy in the elderly. Epilepsy Research 2006; 68: S39-48.
19. DeLorenzo RJ, Hauser WA, Towne AR, et al. A prospective,
population-based epidemiologic study of status epilepticus in Richmond,
Virginia. Neurology 1996: 46; 1029-35.
20. Nazire, Afsar, Dilaver, et al. Stroke and status epilepticus: stroke
type, type of status epilepticus and prognosis. Seizure 2003; 12: 23-27.
21. Jennett B. Posttraumatic epilepsy. Adv Neurol. 1979; 22:137-147.
22. Epsztein J, Ben-Ari Y, Represa A, at al. Late-onset epileptogenesis and seizure genesis: lessons from models of cerebral ischemia.
Neuroscientist 2008; 14(1): 78-90.
23. Camilo O, Goldstein L. Seizures and Epilepsy after ischemic stroke.
Stroke 2004; 35: 1769-75.
24. Aroniadou-Anderjaska V, Fritsch B, Qashu F, et al. Pathology andpathophysiology of the amygdale in epilepstogenesis and epilepsy.
Epilepsy Research 2008; 78 (2-3): 102-116.
25. DeLorenzo RJ, Sun DA, Deshpande LS. Cellular mechanisms
underlying acquired epilepsy: the calcium hypothesis of the induction
and maintenance of epilepsy. Pharmacol Ther 2006; 111:288-325.
26. McNamara JO, Huang YZ, Leonard AS. Molecular signallingmechanisms underlying epileptogenesis. Sci STKE 2006; 12.
27. Krantic S, Mechawar N, Reix S, et al. Molecular basis of programmed cell death involved in neurodegeneration. Trends Neurosci
2005; 28: 670-6.
28. Bernard C. Dogma and dreams: experimental lessons for epilepsy
mechanism chasers. Cell Mol. Life Sci 2005; 62: 1177-81.
29. El-Hassar L, Milh M, Wendling F, et al. Cell domain-dependentchanges in the glutamatergic and GABAergic drives during epileptogenesis in the rat CA1 region. J. Physiol 2007; 578: 193-211.
30. Freund TF, Buzsaki G. Interneurons of the hippocampus. Hippocampus 1996; 6: 347-470.
31. Banks MI, White JA, Pearce RA. Interactions between distinct
GABA(A) circuits in hippocampus. Neuron 2000; 25: 449-457.
32. Panayiotopoulus CP. Atlas of Epilepsies 2007; 186.
33. Taketo M, Yoshioka T. Developmental change of GABA(A) receptormediated current in rat hippocampus. Neuroscience 2000; 96: 507-14.
34. Heinen K, Bosman LW, Spijker S, et al. GABAA receptor maturation in relation to eye opening in the rat visual cortex. Neuroscience
2004; 124:161-71.
35. Laurie DJ, Wisden W, Seeburg PH. The distribution of thirteen
GABAA receptor subunit mRNAs in the rat brain: III. Embryonic andpostnatal development. J Neurosci 1992; 12: 4151-172.
36. Pirker S, Schwarzer C, Wieselthaler A, Sieghart W, Sperk G.
GABA(A) receptors: immunocytochemical distribution of 13 subunits
in the adult rat brain. Neuroscience 2000; 101: 815-850.
37. Lerche H, Weber YG, Jurkat-Rott K, et al. Ion channel defects in
idiopathic epilepsies. Curr.Pharm. Des 2005; 11: 2737-52.
38. Kahle W, Frotscher M. Color atlas of human anatomy: Nervous
system and sensory organs 2010.
39. Zepeda A, Sengpiel F, Guagnelli MA, et al. Functional reorganizationof visual cortex maps after ischemic lesions is accompanied by changes
in expression of cytoskeletal proteins and NMDA and GABA(A) receptor
subunits. J Neurosci 2004; 24: 1812-21.
40. Eriksson PS, Perfilieva E, Bjork-Eriksson T, et al. Neurogenesis in
the adult human hippocampus. Nat Med, 1998; 4: 1313-17.

3941. Kaplan MS, Bell DH. Mitotic neuroblasts in the 9-dayold and
11-month-old rodent hippocampus. J Neurosci, 1984; 4: 1429-41.
42. Luskin MB. Restricted proliferation and migration of postnatal
generated neurons derived from the forebrain subventricular zone.
Neuron, 1993; 11: 173-189.
43. Doetsch F, Garcia-Verdugo JM, Alvarez-Buylla A. Cellular composition and three-dimensional organization of the subventricular germinal
zone in the adult mammalian brain. J Neurosci, 1997; 17: 5046-61.
44. Luskin MB, Mc Dermott K. Divergent lineages for oligodendrocytes
and astrocytes originating in the neonatal forebrain subventricular zone.
Glia, 1994; 11: 211-226.
45. Morschead CM, Reynolds BA, Craig CG, et al. Neural stem cells
in the adult mammalian forebrain: a relatively quiescent subpopulationof subependymal cells. Neuron, 1994; 13: 1071-82.
46. Arvidsson A, et al. Neuronal replacement from endogenous precursors in the adult brain after stroke. Nat Med 2002; 8: 963-970.
47. Parent JM, et al. Rat forebrain neurogenesis and striatal neuron
replacement after focal stroke. Ann Neurol 2002; 52: 802-813.
48. Thored P, et al. Persistent production of neurons from adult brain
stem cells during recovery after stroke. Stem Cells 2006; 24: 739-747.
49. Yamashita T, et al. Subventricular zone-derived neuroblasts migrate
and differentiate into mature neurons in the post-stroke adult striatum.
J Neurosci 2006; 26: 6627-36.
50. Lee SR, et al. Involvement of matrix metalloproteinase in neuroblast
cell migration from the subventricular zone after stroke. J Neurosci
2006; 26: 3491-95.
51. Liu XS, et al. Angiopoietin 2 mediates the differentiation and migration of neural progenitor cells in the subventricular zone after stroke. J

Biol Chem 2009; 284: 22680-9.
52. Ohab JJ, et al. A neurovascular niche for neurogenesis after stroke.
J Neurosci 2006; 26: 13007-16.
53. Robin AM, et al. Stromal cell-derived factor 1alpha mediates neuralprogenitor cell motility after focal cerebral ischemia. J Cereb Blood Flow
Metab 2006; 26: 125-134.
54. Yan YP, et al. Monocyte chemo attractant protein-1 plays a criticalrole in neuroblast migration after focal cerebral ischemia. J Cereb Blood
Flow Metab 2007; 27: 1213-24.
55. Parent JM, Yu TW, Leibowitz RT, et al. Dentate granule cell neurogenesis is increased by seizures and contributes to aberrant network
reorganization in the adult rat hippocampus. J. Neurosci. 1997; 17:3727-38.
56. Espósito MS, Piatti VC, Laplagne DA, et al. Neuronal differentiation in the adult hippocampus recapitulates embryonic development. J
Neurosci. 2005; 25(44): 10074-86.
57. Zhao C, Teng EM, Summers RG, et al. Distinct morphological stagesof dentate granule neuron maturation in the adult mouse hippocampus.
J. Neurosci. 2006; 26: 3-11.
58. Kron MM, Zhang H, Parent JM. The developmental stage of dentate
granule cells dictates their contribution to seizure-induced plasticity. J
Neurosci. 2010; 30(6): 2051-59.
59. Buckmaster PS, Zhang GF, Yamawaki R. Axon sprouting in a modelof temporal lobe epilepsy creates a predominantly excitatory feedback
circuit. J. Neurosci. 2002; 22: 6650-58.
60. Leger JM, Mas J. Accidents vasculaires cerebraux 2009, 1071-1085.
61. Silverman I, Restrepo L, Gregory C. Post stroke seizures. Arch.
Neurol. 2002; 59: 195-201.

EPILEPSIA POST ACCIDENT VASCULAR CEREBRALREZUMAT

Crizele epileptice, epilepsia și statusul epileptic au o incidență crescută la pacienții cu vârsta peste 60 ani. Principalul factor etiologic
pentru apariția crizelor epileptice este reprezentat de bolile cerebrovasculare. Etiologia bolilor cerebrovasculare include infarctul
cerebral, ateroscleoroza cerebrală și hemoragia cerebrală. Infarctul cerebral este cel mai frecvent – 30% și crește riscul de apariție alcrizelor epileptice de 23 de ori. Crizele epileptice pot apare precoce sau tardiv, această clasificare este importantă deoarece fiziopatologia și prognosticul este diferit.Crizele epileptice precoce apar în prima sau a doua săptămână post accident vascular cerebral,iar cele tardive pot apare după luni sau ani post accident vascular cerebral. Epilepsia se însoțește de o activitate glutamatergică
excesivă și un dezechilibru al transmisiei sinaptice glutamatergice și GABAergice. Apariția crizelor epileptice post accident vascular
cerebral ischemic depinde de subtipul, localizarea și severitatea accidentului vascular. Localizarea corticală se asociază cu apariția
crizelor epileptice precoce. Severitatea accidentului vascular ischemic determină recuperarea pacințior. Tratamentul crizelor epilepticedepinde de contextul clinic.
Cuvinte cheie: epilepsia, accidentul vascular cerebral, factori de risc

40

Fiziologia - Physiology  2012.23.3 (75)THE SURVEY OF NOSOCOMIAL INFECTIONS FROM A
UNIVERSITY CLINIC INSTITUTE WITH CARDIOVASCULARPROFILELIVIA STÂNGĂ, LUMINIŢA MIRELA BĂDIŢOIU, MATILDA RĂDULESCU, DELIA
BERCEANU-VĂDUVA, ELENA HOGEA, DELIA MUNTEAN, PILUŢ C., MONICA LICKER,ROXANA MOLDOVAN
University of Medicine and Pharmacy „Victor Babeş” Timişoara

ABSTRACT

Objective: The survey aimed at identifying the frequency, characteristics and microbiological risk factors regarding nosocomial
infections from a clinic institute with cardiovascular profile.
Material and method: Germ identification relied on cultural and biochemical characters. Final identification and antibiogram were
performed using the Vitek 2 (Bio Merieux France) automatic analyser.
Results: During 2008-2011, 21 patients were identified and declared to have nosocomial infections, all with surgical profile.
According to the type of nosocomial infections, firstly there came the assisted post-ventilation pneumonia with 7 cases (33.3 %),followed by the postoperative wound deep infection with 6 cases (28.6%) and the septicemia condition - 5 cases (23.8%) andmediastinitis -1 case (4.8%). Six of these patients had an unfavorable evolution ending with exitus. Out of the 49 micorbiologicalsamples, S. aureus strains were mainly identified (10 samples-20.40%), Enterobacter clocae and Pseudomonas aeruginosa ( with
9 samples each – 18.35%), but also other species especially gram-negative bacilli.
Conclusions: Postoperative nosocomial infections of cardiovascular surgery represent a serious problem of the medical system
directly correlated with morbidity and mortality, with intraoperative factors as well as with postoperative condition management.
Key words: nosocomial infection, cardiac surgery, ICU, ventilator-associated pneumonia, deep surgical site infection

INTRODUCTION
Nosocomial infections represent the most important
noncardiac complications from the cardiovascular surgery (1).
They involve substantial morbidity, long-term hospitalization(hospital stay), high mortality and costs that need great efforts
from hospitals (2-3).
Patterns of patient referral, length and extent of surgicalprocedures the utilization of perioperative catheters and the highdependency on the medical staff represent important factors ofsubmitting patients to nosocomial infections (3).
Generally, the epidemiologic process of nosocomial infections, no matter the causal agent, the clinic-epidemiologic formof expression as well as the profile of the unit/hospital wherethese take place, result in the interaction of 3 decisive factors:
the source of infection, the pathway and the organism receptivity,
but also of some favorable factors (natural and socio-economic).
Although the same types of nosocomial pathology can be
met in most surgical units, still, each of these units has its own
peculiarities according to its specificity. Thus, the nosocomial
infections that appear in cardiovascular surgery are: nosocomial
pneumonias, urinary tract infections, blood tissue infections,
surgical wound infections, catheter-related infections, prosthetic
valve infection and vascular grafting, mediastinitis.
The diagnosis of postoperative infections is sometimes
difficult, since clinical and laboratory signs of inflammation may
be caused not only by infection, but also tissue injury and mainlyby the systemic inflammatory response syndrome (SIRS) associated with cardiopulmonary bypass. In addition, surgical patiens
usually receive systemic antibiotics, especially in the ICU, thus
negatively influencing blood culture yield (3).
MATERIAL AND METHOD
From November 2007 to November 2011 a study dealing
with nosocomial infections was performed in a clinic university
cardiovascular institute. The collection of samples from patientswas performed by qualified staff, in sterile containers proper toeach type of sample, strictly observing the general regulations
for the collection of samples for the bacteriological examination.
Special attention was given to the collection of these samples
because a bad collection could damage from the beginning thebacteriologic diagnosis
The identification of these germs relied on the cultural andbiochemical characters. Final identification and antibiogram were
performed using the automatic analyzer Vitek 2 (bio Meriuex
France).
RESULTS AND DISCUSSION
During the study period, 21 patients were identified anddeclared to have nosocomial infections, all of them having had
surgical history (Figure 1).

Received 20th of August 2012. Accepted 10th of September 2012. Address for correspondence: Stanga Livia, MD, PhD student, “Victor Babes” University of Medicine
and Pharmacy Timisoara, Eftimie Murgu Square No. 2A, RO-30041, Romania, e-mail: stangalivia@yahoo.com

2012.23.3 (75)  Fiziologia - Physiology

41condition, as can be seen in Table II.
Table II. Patients distribution according nosocomial infection typeFig. 1. Annually distribution of patients with nosocomial infection

The specific literature reveals high variations in nosocomial
infections rate of cardiovascular surgery. The ESGNI-008 study(European Study Group of Hospital Infection), performed in 42
hospitals of 13 European countries, reported high incidenceof nosocomial infections in patients after having undergone
cardiovascular surgery - 26.8 % (2). Another study performed
by Fowler and co-workers showed a percent of only 3.51 % (4).
The average age of these patients was 65 year old, among
which 18 males (85.71% ), accounting for the higher frequency
of ischemic coronary disease in male persons (Table I).
Table I. Gender distribution of patients with nosocomial infection

Year
2008
2009
2010
2011

Gender
M
F
M
M
F
M

n
4
1
3
7
2
4

%
100.00%25.00%
75.00%
100.00%33.33%
66.67%IC 95%
39.80% 100.00%
0.60%
80.60%
19.40% 99.40%
59.00% 100.00%
4.30%
77.70%
22.30% 95.70%

The cases were declared both by the two surgical units
and by the intensive care unit (Figure 2).

Fig. 2. Surgical management of patients with nosocomial infection in IBCV

According to the type of nosocomial infections, firstly
there came the ventilator-associated pneumonia, followed by
the postoperative wound deep infection and the septicemia
42Year NI type2008 Postoperative wound
deep infection
Medistinitisn %IC 95%
2 50.00% 6.80% 93.20%

SepsisTotal
2009 Ventilator-associatedpneumonia
SepsisTotal
2010 Ventilator-associatedpneumonia
Postoperative wound
deep infection
Sepsis. Ventilatorassociated pneumonia
Total
2011 Ventilator-associatedpneumonia
Postoperative wound
deep infection
SepsisTotal

1 25.00% 0.60% 80.60%
4 100.00%
1 25.00% 0.60% 80.60%

1 25.00% 0.60% 80.60%

3 75.00% 19.40% 99.40%
4 100.00%
4 57.14% 18.40% 90.10%
1 14.28% 0.40% 57.90%
2 28.57% 3.70% 71.00%
7 100.00%
2 33.33% 4.30% 77.70%
3 50.00% 11.80% 88.20%
1 16.67% 0.40% 64.10%
6 100.00%

The ventilator-associated pneumonia was the mostfrequent cause of nosocomial infections, the 7 cases analysed
representing 33.30 % from the whole amount of infections.
Nevertheless significant differences were noticed in the studies
from the specific literature. The ESGNI-008 study (European
Study Group of Hospital Infection) had as result 57% (2) while
Hortal and co-workers report 45.7 % (5) and Ioanna Lola andco-workers obtained 16.7% (6).
The postoperative wound deep infection is the second mostfrequent nosocomial infection, the six cases representing 28.6
% of the total number of infections. Luca Salvatore de Santoand co-workers revealed in one of their studies 27.7 % (7), while
Argyris Michalopoulos and co-workers reported 17.7% (3).
Bacteraemia is responsible for 23.8 % of the nosocomial
infections, which agrees to the studies made by Falagas ME
and co-workers who reported 26.9 %(8) and Trthon A and coworkers 27.6% (9).
A lower rate of frequency along nosocomial infection isrepresented by mediastinitis 4.80, Ioanna L (6) and co-workers
reporting 3.3% and Gualis J. and co-workers between 0.4%
and 2 % (4).
Six of these patients had an unfavorable evolution ending
with exitus, following ventilator-associated pneumonia - 50 %,sepsis - 33.33% and deep infection of sternal wound -16.67 %(Figure 3).
Fiziologia - Physiology  2012.23.3 (75)2009 Central venous
catheterBlood

2 33.33% 4,30%

77.70%

4 66.67% 22.30%

95.70%

Total
2010 Bronchial aspirate
Central venous
catheterWound secretion

6 100.00%
5 29.41% 10.30%
1 5.88% 0.10%

56.00%
28.70%

9 52.94% 27.80%

77.00%

Secretion
tracheostomy
Blood
Total
Bronchial aspirate

1 5.88%0.10%

28.70%

1 5.88% 0.10%
17 100.00%
9 45.00% 23.10%

28.70%

Wound secretion

10 50.00% 27.20%

72.80%

Blood
Total

1 5.00% 0.10%
20 100.00%

24.90%

2011

Fig. 3. The evolution nosocomial infection cases

The burden of these nosocomial cases resides not only inmortality, but also in a raise in hospitalization cost consecutive
to prolonged stay and in antimicrobial and supportively intensive
treatment necessity.
After statistic tests were made, a significant raise in the
total number of hospitalization stay days could not be noticed
and neither in the number of days necessary for the treatmentof the nosocomial infection during the studied period. The
signifying index p value in 2010 vs 2009 was of 0.089 for the
first variable and 0.287 for the second variable (Figure 4).

68.50%Out of the 49 microbiological samples, S. aureus strains
were mainly identified (10 samples - 20.40%), Enterobacterclocae and Pseudomonas aeruginosa ( with 9 samples each– 18.35%), but also other species especially gram-negative
ones (Figure 5).

Fig. 4. The evolution of the average number of hospitalization and treatmentdays with nosocomial infection

49 microbiological samples were collected, most frequently
wound secretions (22-44.89%), bronchial aspirate (15-30.61%)
and hemocultures (6-12.24%) as can be noticed in Table III.
Table III. The distribution of pathological products collected from patients withnosocomial infection

Year

Pathologic product n %IC 95%

2008 Bronchial aspirate
Puncture liquid

1 16.67% 0.40%
1 16.67% 0.40%

64.10%
64.10%

Wound secretion

3 50.00% 11.80%

88.20%Mediastinal lavage

1 16.67% 0.40%

64.10%

Total

6 100.00%

2012.23.3 (75)  Fiziologia - Physiology

Fig. 5. The distribution of samples according to the isolated species

32 strains were analysed in order to determine their
sensitivity to antibiotics, proving highly resistant to Cefoxitin,
SXT and CAZ:
The frequency of nosocomial infections reported to thetotal number of discharges from the cardiovascular surgicalunits, to the number of discharges of the whole institute, ismuch lower than the figures appeared in the specific literature(Figure 6).

43

Fig. 6. The IN prevalence identified during the study period

In a study performed in 2011 in a hospital from Greece,
Ioanna Lola and co-workers report 13.95 % (6) while other study,
also from Greece, made in 2005 by Argyris Michalopoulos andco-workers, reveal 5-21% (3). Luca Salvatore de Santa andco-workers made a study in a hospital from Italy and obtained
9% (7).
CONCLUSIONS
1. Nosocomial infection is and will be an actual publichealth problem in Romania where a subreporting andsubassesment of this issue is noticed.
2. Most frequet diagnosis of nososcomial infection are
ventilator-associated pneumonia, postoperative wound deep
infection and septicemia condition.
3. The etiology of nosocomial infections is determinedmostly by gram-negative bacteria.
4. Old age and chronic immunosuppressive diseases arehigh risk factors for nosocomial infection.

REFERENCES
1. S Welsby IJ, Bennett-Guerrero E, Atwell D, White WD, Newman
MF, Smith PK, Mythen MG. The association of complication
type with mortality and prolonged stay after cardiac surgery withcardiopulmonary bypass. Anesth Analg 2002; 94: 1072-1078.
2. Bouza E, Hortal J, Munoz P, Pascau J, Perez MJ. European Study
Group on Nosocomial Infections, European Workgroup of Cardiothoracic
Intensivists.Postoperative infections after major heart surgery andprevention of ventilator-associated pneumonia: a one-day European
prevalence study (ESGNI-008). J Hosp Infect, 2006; 64: 224-230.
3. Michalopoulosa A, Geroulanosb S, Rosmarakisc ES, Falagasc
ME. Frequency, characteristics, and predictors of microbiologically
documented nosocomial infections after cardiac surgery. Eur J CardioThor Surg, 2006; 29: 456-460.
4. Fowler VG Jr, O’Brien SM, Muhlbaier LH, Corey GR, Ferguson
TB, Peterson ED: Clinical predictors of major infections after cardiac
surgery. Circulation, 2005; 112: I358-65.
5. Hortal J, Giannella M, Perez MJ, Barrio JM, Desco M, Bouza
E, Munoz P. Incidence and risk factors for ventilator-associatedpneumonia after major heart surgery. Intensive Care Med, 2009; 35:
1518-25.
6. Lola I, Levidiotou S, Petrou A, Arnaoutoglou H, Apostolakis E,
Papadopoulos GS. Are there independent predisposing factors for
postoperative infections following open heart surgery? J Cardiothor
Surg, 2011; 6: 151.
7. De Santoa LS, Banconeb C, Santarpinob G, Romanoc
G, De Feob M, Scardonec M, Galdieric N, Cotrufob M.
Microbiologically documented nosocomial infections after
cardiac surgery: an 18-month prospective tertiary care centre
report. Eur J Cardio-Thor Surg, 2008; 33: 666-672.
8. Falagas ME, Rosmarakis ES, Rellos K, Michalopoulos A, Samonis
G, Prapas SN: Microbiologically documented nosocomial infections
after coronary artery bypass surgery without cardiopulmonary bypass.
J Thorac Cardiovasc Surg, 2006, 132: 481-90.
9. Trethon A, Prinz G, Varga A, Kocsis I. Characteristics of nosocomial
bloodstream infections at a Hungarian cardiac surgery centre. Acta
Microbiol Immunol Hung, 2012; 59(2): 271-83.

MONITORIZAREA INFECTIILOR NOSOCOMIALE DINTR-UN INSTITUT
CLINIC UNIVERSITAR CU PROFIL CARDIOVASCULARREZUMAT

Scop: S-a urmărit frecvenţa, caracteristicile şi factorii de risc microbiologici privind infecţiile nosocomiale dintr-un institut clinic cu
profil cardiovascular.
Material şi metodă: Identificarea germenilor s-a bazat pe caracterele culturale şi pe cele biochimice. Identificarea finală şi
antibiograma au fost efectuate utilizând analizorul automat Vitek2 (Bio Merieux France).
Rezultate: Au fost identificaţi şi declaraţi în perioada 2008 - 2011, 21 de pacienţi cu infecţii nosocomiale, toţi cu profil chirurgical.
Din punct de vedere al tipului de infecţie nosocomială pe primul loc s-a situat pneumonia post ventilaţie asistată cu 7 cazuri(33,3%), urmată de infecţia nosocomială profundă de plagă postoperatorie cu 6 cazuri (28,6%), starea septică 5 cazuri (23,8%)
şi mediastinita 1 caz (4,8%). Dintre aceşti pacienţi, 6 au avut evoluţie nefavorabilă soldată cu exitus. Din cele 49 de probe s-au
identificat în special tulpini de S.aureus (10 probe - 20,40%), Enterobacter cloacae şi Pseudomonas aeruginosa cu câte( 9 probe –18,35%), dar şi alte specii, cu precădere de bacili gram negativi.
Concluzii: Infecţiile nosocomiale post chirurgie cardiovasculară reprezintă o problemă serioasă a sistemului medical în corelaţie
directă cu morbiditatea şi mortalitatea, cu factorii intraoperatorii cât şi cu managementul stării post operatorii.
Cuvinte cheie: infecţie nosocomială, chirurgie cardiovasculară, ATI, pneumonie postventilaţie asistată, infecţie nosocomială
profundă de plagă postoperatorie

44

Fiziologia - Physiology  2012.23.3 (75)