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Fiziologia- Physiology 2013.23.2(78)
Fiziologia‑ Physiology 2010supplement

CONTENTS
1. REVIEW: Stem Cells and the Placental Stem Cells....................................................................................................................................................................................................4
Anastasiu D, Bojin F, Gluhovschi A, Stoian D, Balu S, Vizitiu A, Bold T, Craina M, Paunescu V
2. The Taurine Effect on Electrical Cerebral Signal in Old Rats....................................................................................................................................................................................10
Giurgiu Monica - Cristina, Ghita Mihai Aurelian, Zagrean Leon
3. Hydatid Cyst of the Kidney: Preclinical and Clinical Particularities ........................................................................................................................................................................15
Radu Tudor, Istrate Bogdan, Jinga Viorel, Steriu Dan
4. Subclinical Atherosclerois in Rheumatoid Polyarthritis .........................................................................................................................................................................................18
Cristina Florea, Elena Ardeleanu, Alexandru Caraba
5. Identification of the Antibiotic Resistance Phenotypes of Escherichia Coli Strains Isolated in the Urine Specimens of Diabetic Patients...........................................................23
Teodora Chiţă, Delia Muntean, Luminița Badițoiu, Bogdan Timar, Roxana Moldovan, Romulus Timar, Monica Licker
6. Techniques for Endotracheal Intubation in Rats......................................................................................................................................................................................................27
Daniela Chiru, Narcis Ţepeneu, Caius Burian, Adrian Craciun, Ilie Constantin, Mirabela Dima, Valentin Ordodi
7. Correlation between Pulse Wave Velocity and Inflammation Markers in Patients with Cardiovascular Risk Factors ...........................................................................................30
Ioana Suceava, Daniel Lighezan, Corina Serban, Anca Tudor, Roxana Buzas, Simona Dragan
8. Taurine Effect on Working Memory in Healthy Elderly...........................................................................................................................................................................................34
Monica-Cristina Giurgiu, Ana Capisizu, Leon Zagrean

CUPRINS
1. REVIEW: Celulele stem si celulele stem placentare..................................................................................................................................................................................................4
Anastasiu D, Bojin F, Gluhovschi A, Stoian C, Balu S, Vizitiu A, Bold T, Craina M, Paunescu V
2. Efectele taurinei asupra activitatii electrice cerebrale la sobolanii batrani............................................................................................................................................................10
Giurgiu Monica - Cristina, Ghita Mihai Aurelian, Zagrean Leon
3. Chistul hidatic renal: particularitati preclinice si clinice..........................................................................................................................................................................................15
Radu Tudor, Istrate Bogdan, Jinga Viorel, Steriu Dan
4. Ateroscleroza subclinică în poliartrita reumatoidă.................................................................................................................................................................................................18
Cristina Florea, Elena Ardeleanu, Alexandru Caraba
5. Identificarea fenotipurilor de rezistenta la antibiotice a tulpinilor de Escherichia Coli izolate din probele de urina ale pacientilor diabetici.....................................................23
Teodora Chiţă, Delia Muntean, Luminița Badițoiu, Bogdan Timar, Roxana Moldovan, Romulus Timar, Monica Licker
6. Tehnici utilizate pentru intubaţia endotraheală la şobolan....................................................................................................................................................................................27
Daniela Chiru, Narcis Ţepeneu, Caius Burian, Adrian Craciun, Ilie Constantin, Mirabela Dima, Valentin Ordodi
7. Corelaţia dintre viteza de propagare a undei de puls şi markerii inflamatori la pacienţi cu factori de risc cardiovascular...................................................................................30
Ioana Suceava, Daniel Lighezan, Corina Serban, Anca Tudor, Roxana Buzas, Simona Dragan
8. Efectul taurinei asupra memoriei de lucru la varstinicii sanatosi...........................................................................................................................................................................34
Monica-Cristina Giurgiu, Ana Capisizu, Leon Zagrean
2013.23.2 (78)  Fiziologia - Physiology

3

REVIEWSTEM CELLS AND THE PLACENTAL STEM CELLSANASTASIU D1, BOJIN F1, GLUHOVSCHI A2, STOIAN D2, BALU S2, VIZITIU A2, BOLD T2,
CRAINA M2, PAUNESCU V1
1
Department of Functional Sciences, “Victor Babes” University of Medicine and Pharmacy Timisoara
2
Obstetrics-Gynecology Department, “Victor Babes” University of Medicine and Pharmacy Timisoara

ABSTRACTStem cells are cells that divide to produce one daughter cell that is a stem cell and another daughter cell that produces differentiateddescendants. The definition fits a newly fertilized egg but begins to unravel as we move along the developmental pathway. Totipotent embryonic stem (ES) cells can readily be shown to differentiate into essentially all cell phenotypes, whereas most isolates ofadult stem cells (multipotent stem cells) from sources as bone marrow stroma, fat, muscle, and nervous tissue have a more limited
potential for differentiation. Most of the adult stem cells, but not all isolates, have a more limited life span in culture than the ES cells
have. Moreover, the progression from a fertilized egg to an ES cell to an adult stem cell to a differentiated cell may be a continuum
in which few if any steps are irreversible. Placental stem cells are at the crossroads of adult stem cells and ES cells, so that theirincreased potential should be explored in more detailed for further therapeutic strategies.
Key words: embryonic stem cells, adult stem cells, totipotent, multipotent

OVERVIEW OF STEM CELLS
Stem cells differ in their differentiation plasticity or developmental potential but they all have the ability to self-renew.
Depending on the developmental potential, there are more typesof stem cells, for instance pluripotent or multipotent stem cells,
the unipotent and induced pluripotent stem cells. Pluripotent stem
cells can be cultured and isolated from the inner cell mass of theblastocyst or morula stage, and have the ability to differentiate
into cell types from all three embryonic germ layers. These cells
are also referred to as embryonic stem cells (ESCs). Furthermore, multipotent stem cells can derive cell types from lineages
other than their germ layer of origin such an example are/represent the mesenchymal stem cells. They can differentiate into
multiple cell types that can span the ectodermal and mesodermal
lineages. Other stem cells isolated from the child or adult have alimited potential in the differentiation process. The unipotent stem
cells are proliferative stem cells that are committed to a single
lineage and can only differentiate into cells of a related lineage,
except for neural progenitor cells which can develop into bothneuronal and glial cell types. Likewise, primordial germ cells areunipotent and develop into germ cells in the adult. In contrast to
stem cells, nullipotent cells are committed somatic cells. Thesetypes of cells that are reprogramed in vitro to a less differentiatedstate are called induced pluripotent stem cells (iPSCs).
EMBRYONIC-LIKE STEM CELLS
Embryonic stem cells (ESCs) are very important cells
that can be used in the treatment of different diseases such as:
spinal cord injury, heart disease, diabetes etc. because of theirpotential to develop into many cell types, including chondrocytes,
cells of the neural lineage, retinal cells and cardiomyocytes.
Human ESCs are obtained from poor quality embryos donatedfrom in vitro fertilization programs that would have otherwise
been discarded, but, nonetheless, there are ethical and politicalchallenges to be taken into account. The cells are isolated fromthe inner cell mass of five-to-seven day old blastocysts and arecultured with a fibroblast feeder layer. The tumorigenicity of the
ESCs has yet to be resolved (1).
Embryonic germ cells (EGCs) have been used to repair
bladder defects, cartilage degeneration and motor neuron injuries
in rat models. However, research is trying to define a culture
condition that facilitates the best derivation of EGSs. These cells
are derived from PGSs, primordial germ cells who are difficult
to obtain, considering that they are present in the fetal genitalridge by the fifth week of human development. In the female, themitotic expansion begins at the 5th week and in the male at the 8th
week so PGSs in both sexes arrest their expansion at week 10.
Germ-line stem cells (GSCs) and their potential in clinical applications is still ongoing. These cells are associated withfewer ethical concerns because they are derived from testicularbiopsies in men, and have a potential adult source of pluripotent
stem cells. The spermatogonia stem cells have the ability to selfrenew and differentiate into sperm. However, the use of GSCs islimited by the possible restriction for use only in male patients,
the uniparental epigenetic imprint (1).
Induced pluripotent stem cells (iPSCs) are derived fromsomatic, differentiated adult cells such as fibroblasts, keratinocytes, hepatocytes, neural stem cells and gastric epithelial cells.
These iPSC cells are useful not only for in vitro human models
in order to study the molecular mechanisms involved in humanReceived 14th of April 2013. Accepted 24th of May 2013. Adress for correspondence: Diana Anastasiu, MD, PhD student, “Victor Babes” University of Medicine andPharmacy Timisoara, Eftimie Murgu Square No. 2A, RO-3000041, Timisoara, phone/fax: +40256220479; e-mail: diana_anastasiu@yahoo.com

4

Fiziologia - Physiology  2013.23.2 (78)disease, but also for patient specific cell based treatments, aswell as studying a multitude of other afflictions. Current studies
have developed strategies for transdifferentiation, in which a cellof specific lineage is converted to another lineage and then intoanother cell type without reverting to a common progenitor state.
Complications, however, do occur because of the use of genetic
integrated viruses, as vectors in the reprogramming strategies.
These complications include: inflammatory response, replication,
virulence, tumorigenicity, immunogenicity, random integration,and reactivation. Future research focuses on establishing safeand effective methods for reprogramming cells (1).
PLACENTAL COMPARTMENTS
The human placenta is a feto-maternal organ. At term, it has
a discoid shape with a diameter of 15-20 cm and a thicknessof 2-3 cm (3). The placenta has a maternal and a fetal side.
The maternal side has 16-20 cotyledons that are divided by theplacental septa. The fetal adnexa are composed of the placenta,
fetal membranes and umbilical cord. The fetal membranes
amnion and chorion, extend from the margins of the chorionicdisc which enclose the fetus in the amniotic cavity, and the endometrial decidua. The chorionic plate faces the amniotic cavity
and consists of two different structures: the amniotic membrane
and the chorion. The villi originate from the chorionic plate andanchor the placenta through the trophoblast of the basal plateand maternal endometrium. Some villi terminate freely in theintervillous space, while others anchor the placenta to the basal
plate. In the term placenta, the stem villi show an inner core of
fetal vessels with a distinct muscular wall and connective tissue.
Mature intermediate villi and term villi consist of capillary vessels
and thin mesenchyme. The stromal core is separated from thesyncytiotrophoblast by the basement membrane. Between thesyncytiotrophoblast, which is an uninterrupted multinucleatedlayer, and its basement membrane are single aggregated Langhans cytotrophoblastic cells also called cytotrophoblast cells. The
amnion is a thin avascular membrane and is contiguous over theumbilical cord with the fetal skin. Microscopically, the amnion iscomposed of an epithelial layer of columnar and cuboidal cells,
which lie on top of the mesodermal layer consisting of an upper
acellular compact layer and a deeper layer containing dispersed
fibroblasts. The chorionic membrane consists of a mesodermal
layer and a layer of extravillous trophoblast cells (2,4,7).
Embryological development of the placenta
In humans, the placenta starts to develop during theimplantation window, by days 6-7 after fertilization, when theblastocyst implanted. The blastocyst consists of the outer wall,
blastocystic cavity and inner cell mass. The outer wall surrounds
the blastocystic cavity while the inner cell mass, is represented
by a small group of larger cell apposed to the inner surface ofthe trophoblastic vesicle. By days 8-9 the bilayered trophoblastis build. The inner layer becomes the cytotrophoblast, the inner
cells remain temporarily unfused. The outer layer is the syncyti2013.23.2 (78)  Fiziologia - Physiology

otrofoblast, formed by the fusion of neighboring trophoblast cells.
Small vacuoles appear in the syncytiotrophoblastic mass, thesevacuoles grow, become confluent and form a system of lacunae.
The syncytiotrophoblast and cytotrophoblast form the trabeculae,
which intermingle with the hematic lacunae. The primordial villi
are generated by these trabeculae. Those which are in contact
with the decidua basalis give rise to the chorion frondosum whilevilli in contact with the decidua capsularis build/form the chorion
leave. The epiblast and hypoblast are differentiated from theinner cell mass at day 8-9 after fertilization. Small cells from theepiblast appear between the trophoblast, and the embryonicdisc becomes the amniotic cavity. The exocoelomic membrane
and its cavity modify to form the yolk sac, between the hypoblastand cytotrophoblast. The amniotic and chorionic mesoderm isbuilt by the extra embryonic mesoderm that surrounds the yolk
sac and amniotic cavity. The gastrulation process takes place(the bilaminar disc differentiates into ectoderm, mesoderm andendoderm) and, during the 3rd week after fertilization, a definedform develops with a right-left, cranio-caudal and dorsal-ventral
body axes (2,5,6).
PLACENTAL TISSUE CELL POPULATION
Mesenchymal stem cells are defined as stem cells that candifferentiate into multiple cell lineages and have the potential
to self-renew (8,9). Moreover, they secret antiapoptotic andproangiogenic (10) cytokines and possess immunosuppressive
properties (11). Caplan coined the term mesenchymal stem cells(MSCs) in 1991 (12). Bone marrow MSCs represent the primarysource of MCSs and are most commonly used (13). In the 1860s
Cohnheim was the first to hypothesize the existence of MCSs in
the bone marrow (14). Maximov (1920) assumed the importantrole of the marrow stromal tissue in supporting the development
and maintenance of blood and hematopoietic organs (15).
Friedenstein demonstrated for the first time in 1960s that stromalcells could be isolated from whole bone marrow aspirate, basedon differentiation adhesion to tissue culture plastic dishes (16).
Mesenchymal stem cells can also be grouped into: adult MSCs
and fetal MSCs. The adult MSCs are one of the most studied
populations of multipotent ASCs (adult stem cells), they are
isolated from bone marrow, peripheral blood. However MSCs
from the bone marrow have to be obtained through invasive
procedure and stem cell numbers decrease significantly with theage of the individual (3,4,17-19). This brought research to lookfor easily accessible sources of multipotent stem cells, in othertissues for example: peripheral blood (3,20), umbilical cord blood(3, 21-24) and more recently, deciduous tooth (25) and umbilicalcord mesenchyme (26). There is still a debate if in some sources
stem cells do exist (27, 28) and cell volume from these tissues
can be limited (3,22, 25).
The fetal MSCs are isolated from placenta, amniotic fluid,
umbilical cord and umbilical cord blood (2,29). The human placenta is a fetomaternal organ which has both maternal and fetal
tissue. After birth, the term placenta is discarded which meansthat no invasive procedure is necessary. Furthermore, there are5

no ethical conflicts and the tissues can be effectively used forclinical application, as well as research. Although they do nothave the same proliferative and differentiation potentials of ESCs,
the placenta-derived multipotent cells (PDMCs) may be superior
to Adult stem cells and are of fetal origin (2,3).
MCS of maternal origin
Vikram Sabapathy and colleagues isolated placental
mesenchymal stem cells from cotyledons of the maternal side
while the placental membrane from the maternal side was cut
open. About 80 g of cotyledon was exercised and washed with
PBS, cut off into small pieces and blood clots were removed.
The minced placenta was washed with physiological saline andsubjected to digestion with trypsin and collagenase I, and then
the tissues were incubated with 0.25% trypsin for 1hour at 37 oC.
After the trypsinization, the sample was filtered through 250µm
metal sieve. The supernatant was put up for a second digestion
with 12.5 U/mL collagenase I for 1 hour at 37 oC. Collagenase I
digested tissue was passed first through 100µm metal sieve andfiltrate collected was then passed through 100µm cell strainer.
The filtrate with the cell suspension was subjected to centrifugation at 300g for 10min after dual filtration stages. The cell pellet
was resuspended in RBC lysis buffer and centrifuged at 300g
for 10 minutes. At the end the cell pellet was resuspended in
Mesenchymal expansion medium (alphaMEM + 10%FBS+ 50µg/
mL streptomycin + 1Mm L-glutamine) and plated into two 75 cm2
flasks (2). The isolated MSCs formed a homogenous monolayerof adherent spindle-shaped fibroblast-like cells, which are generally term placenta-derived multipotent mesenchymal stem cells.
The colonies became visible after 7 days. By day 14 the MSC
colonies started to proliferate, the flask was almost 60-70%
confluent and ready for splitting. Flow analysis established MCSs
phenotypic characteristics staining positively for mesenchymal
lineage surface markers CD29+, CD73+, CD90+, CD105+,
and negative for hematopoietic marker CD34-, CD45-, CD14-,
HLA-DR-. The expression profile confirms the criteria generally
defined for multipotent mesenchymal stem cells. By visualizing
large lipid vacuoles, mineralized bone with calcium deposits
and Saffranin O positive collagen matrix results the ability ofMSCs to differentiate into adipocyte, osteocytes, chondrocytes,
consequently, mesodermal cell lineage but also translineage
differentiated towards pancreatic progenitor cells, neural cells
and retinal cells displaying plasticity. Furthermore these cells
maintained a normal karyotype while cell cycle or apoptosis
pattern did not alter significantly, are Naïve for stimulatory factors
CD80 and CD86 and have limited expression of MHC-II antigens.
Moreover the soft agar assays showed that placental MSC do
not have the ability to form invasive colonies (2).
Linju B and collaborators concluded that after the plating, theinitial growth consisted of cells with two different morphologies:
a non-fibroblastoid, an epitheliod, polygonal morphology anda fibroblastoid, spindle-shaped morphology. The fibroblastoid
population continued to proliferate after numerous passages,
while the non-fibroblastoid population disappeared after passing
6

with trypsin. The tissue that was harvested from the placentas,
were washed several times in PBS, mechanically minced andenzymatically digested with 0.25% trypsin- EDTA for approximately 10 minutes at 37 oC. The homogenate was then centrifuged and suspended in Dulbecco’s modified Eagle’s medium,supplemented by 10% fetal bovine serum. Cell cultures weremaintained at 37 oC with water-saturated atmosphere and 5%
CO2. One to two times every week the medium was replaced.
The cells were recovered with 0.25% trypsin / EDTA and replated
at dilution of 1:3, when they were confluence more than 80%.
Immunologically, PDMCs were negative for HLA-DR and positivefor HLA-ABC, ESC-associated cell surface markers of SSEA-4,
TRA-1-60, and TRA-1-81 (30-32). Furthermore the immunophenotyping of the PDMCs cells were positive for: SH-2/CD105,
SH-3, SH-4, CD29, CD44, CD90/Thy-1, and CD166. These
markers are common to MSCs (6, 21). In addition they are negative for hematopoietic surface markers of CD14, CD34, CD45,
CD117/c-kit, AC133/2, endothelial cell markers of von Willebrandfactor and Flk-1 and glycophorin A. Also the expression of thecostimulatory molecules CD40L, CD80, and CD86 are negativeand not only, there were no markers found in trophoblastic cells
like HLA-G and cytokeratin 7 but the cells were positive for the
nontrophoblastic markers of CD9 and vimentin.
The β-human chorionic gonadotropin (β-hCG) was measured by immunometric assay and after the second passage;
β-hCG levels ranged from 2.16 IU/L to undetectable and in
the culture media of JEG-3 cells, levels of up to 260 IU/L weredetected (3).
MCSs of fetal origin
From the fetal placenta, four regions can be distinguished:amniotic epithelial, amniotic mesenchymal, chorionic mesenchymal and chorionic trophoblastic from which cell populations
are isolated: human amniotic epithelial cells (hAEC), humanamniotic mesenchymal stromal cells (hAMSC), human chorionicmesenchymal stromal cells (hCMSC), and human chorionic
trophoblastic cells (hCTC). Human Amniotic Epithelial Cells havethe ability to differentiate toward all three germ layers, demonstrated in vitro and express stem cell markers (33-36). They haveunique characteristics and, currently, there is strong in vitro andin vivo evidence of neural, pancreatic and hepatic differentiation of hAEC. The mesenchymal stromal cells from the amnionand chorion are thought to be derived from the extraembrionicmesoderm (37). The distinct morphology of cultured hAMSC,hCMSC and hAEC is shown by phase micrographs, hAMSC
is distinguished from hAEC by the expression of CD49d (α4integrin). Also, hAMSC has a hybrid phenotype; the cells havemesenchymal and epithelial characteristics (shown by electron
microscopy). This feature is interpreted as a sign of multipotentiality and is not found in hCMSC which are metabolically quiescentand more primitive (38). Human amniotic and chorionic cells
successfully persistently engraft in multiple organs and tissues
in vivo after either intravenous or intraperitoneal transplantation
into neonatal swine and rats (39).
Fiziologia - Physiology  2013.23.2 (78)Table I. Isolation protocols, phenotype and in vitro differentiation
potential of each cell type
Human amniotic epithe- Human amniotic mesenchy- Human chorioniclial cells
mal stromal cells
mesenchymal stromal
cells
Isolation Mechanical peeling of Mechanical peeling of amnion Mechanical removal of
protocols amnion from the underly- membrane from the underlying surrounding layers aftering chorion followed by chorion followed by digestion treatment with 2.4 U/
digestion with the follow- with various concentrations of ml dispase II at 37C,
ing: dispase II (2,4 U/ml) collagenase (0.75-2mg/MI) AND followed by 1-3 hours
for 1 hour or trypsin EDTA dnASE (20-75µg/ml) for 30-120 treatment with Worthing(different concentrations minutes at 37C
ton collagenase II (270
and incubation times).
U/ml) or collagenaseA
Centrifugation (200g for(0.83 mg/ml)
10 minutes) of solution
containing released cells,
discarding the remaining
membraneP h e n o - Mesenchymal and hemat- Mesenchymal and hematopoietic M e s e n c h y m a l a n dtype at opoietic markers: CD105+, markers:
hematopoietic markpassages CD90+, CD73+,CD44+, CD105+, CD90+, CD73+,CD44+, ers: CD105+, CD90+,
2-4
CD29+, HLA-A,B,C+, C D 2 9 + , H L A - A , B , C + , CD73+,CD44+, CD29+,
CD13+, CD10+, CD166+, CD13+, CD10+, CD166+, HLA-A,B,C+, CD13+,
CD49d-, CD49e+, CD117 CD49c+, CD49d+, CD49e+, CD166+, CD49e+, CD(+/- very weak signal), CD54+,CD166+, CD271 low, 271low, CD10+,CD117-,
CD14-, CD34-,
CD14-, CD34-,
CD14-, CD34-,
CD45-, HLA-DRCD45-, CD31- HLA-DR-, CD133- CD45-,CD133-,HLA,CD3DRAdipogenic:
D i f f e r - Adipogenic:
Adipogenic:
entiation DMEM high glucose (or DMEM, 10% FCS, 0.5mM DMEM high glucose,
potential DMEM/Ham’s F-12 me- isobutylmethylxanthine,200µM 1 0 % F B S , 0 . 5 m Mdium), 10% FBS, 0.5mM indomethacin, 10-6M( 10 la -6) isobutylmethylxanthine,
isobutylmethylxanthine, dexamethasone, 10µg/ml insulin 1µM dexamethasone,
1µM dexamethasone, 10 Bullet Kit Adipogenic Differentia- 10 µM insulin, 200µMµM insulin, 200µM indo- tion Media (Cambrex, Walkers- indomethacin
methacinville, MD, http://www.cam- Bullet Kit Adipogenic
brex.com)
Differetiation Media
Chondrogenic:(Cambrex)
DMEM high glucose, 1%FBS,
6.25µg/ml insulin,10ng/ml Chondrogenic:
Chondrogenic:
DMEM high glucose, 1% TGF-β1, 50ng/ml fresh ascor- DMEM high glucose, 1%
FBS, 6.25µg/ml insulin,
FBS, 6.25µg/ml insulin, bic acid10ng/ml TGF-β1, 50ng/ml DMEM, 6.25 µg/ml insulin, 10ng/ml TGF-β1, 50ng/
6.25µg/ml transferrin, 6.25µg/ml ml fresh ascorbic acidfresh ascorbic acidselenous acid, 5.33µg/ml linolenic acid, 1.25mg/ml BSA,0.35Mm DMEM, 100 nM, dexaproline, 1mM sodium pyruvate, methasone, 50µg/ml
10-7M dexamethasone, 0.1mM L-ascorbic acid-2phosL-ascorbic acid-2-phosphate phate, 1mM sodium
supplemented with 10ng/ml pyruvate, 40µg/ml protransforming growth-β-3line, ITS (5 µg/ml insulin,
DMEM, 100nM dexamethasone, 5µg/ml transferrin, 5 ng/
50µg/ml L-ascorbic acid 2-phos- ml selenous acid), 5ng/
phate, 1mM sodium pyruvate, ml TGF-β1
40 µg/ml proline, ITS ( 5µg/ml
insulin, 5µg/ml transferrin, 5ng/ml
selenous acid), 5ng/ml TGF-β1
Osteogenic:
DMEM high glucose, 10% FBS,
10mM β-glycerophosphate,
Osteogenic:
DMEM high glucose (or 50-200µg/ml ascorbic acid,
DMEM/Ham’s F-12 me- 10-8 M dexamethasone, 1,α25Osteogenic:dium), 10% FBS, 10µM dihydroxyvitamin-D3
dexamethasone, 10 Nm Commercial media: MesenCult 10µM dexamethasone,
1-α,25-dehydroxyvitamin Human Osteogenic Stimulatory 10Nm 1α, 25-dihydroxyD3, 50 µg/ml ascorbic acid, Kit (StemCell Technologies); Bul- vitamin D3, 50 µg/ml
10 Mm β-glycerophosphate let Kit Osteogenic Differentiation ascorbic acid, 10Mm
β-glycerophosphate
MesenCult Human Os- Media (Cambrex)
teogenic Stimulatory Kit(StemCell Technologies,
Vancouver, BC, Canada,
http://www.stemcell.com

Pieternella S. IN ‘T Anker and collaborators indicate thatboth fetal and maternal MSCs can be isolated from the hu2013.23.2 (78)  Fiziologia - Physiology

man placenta, and that the amnion is also a good source for
MSCs. Placenta and amniotic fluid were derived from humansecond-trimester and third-trimester pregnancies. Term thirdtrimester placenta tissue and amniotic fluid was harvested fromwomen who underwent elective cesarean section for breech
presentation and the amniotic fluid was collected by puncture
through the membranes after opening the uterine wall during theoperation. Decidua parietalis was collected by scraping it fom
the chorion and term amnion was obtained by removing it fromthe membranes. From the central region of the maternal-facing
surface of the placenta, decidua basalis tissue was dissected
macroscopically. Second trimester amniotic fluid and placenta
were obtained from women undergoing socially indicated termination of pregnancy, while decidua tissue was collected byultrasound-guided transcervical biopsy with a uterine curette;amniotic fluid was collected by ultrasound transabdominal
puncture using a 22-gauge spinal needle and amnion was collected after the abortion. Tissue specimen of decidua basalis
and decidua parietalis from approximately 1 cm 3 an amnion
from approximately 1cm2 were washed in PBS. Single –cellsuspension of decidua basalis, decidua parietalis and amnion
were made by mincing and flushing the tissue parts through a
100µm nylon filter (Falcon, Becton, Dickinson, San Jose, CA)with washing medium. The amniotic fluid was centrifuged for 10
minutes at 1,283 rpm and pellets were resuspended in Iscove’s
modified Dulbecco’s medium containing 20 U/ml penicillin and20µg streptomycin (P/S) and 2% heat-inactivated fetal calf serumthat is washing medium. All single-cell suspensions were culturedin M199 supplemented with 10% heat-inactivated FCS, P/S,
endothelial cell growth factor and heparin (8 U/ml). The maternal
and fetal origin of the cells was determined by molecular humanleukocyte antigen typing. There has been no difference detectedamong the expression of MSCs markers from different sources.
The second trimester AF and amnion is a rich source of MCSs,
and the growth curves of the both were very similar. Although thesecond trimester AF is an abundant source of MSCs, comparedto the third trimester where proportion of viable cells decreased
with GA, the risk of amniocentesis prohibits the use for routine
clinical use (4).
CELL AND TISSUE BANKING
The European Community directive 2003/94/EC lays down
the principles and guidelines of good manufacturing practice(GMP) for medical products for human use, but also other institutions such as The European Association of Tissue Banks andthe Joint Accreditation Committee-ISCT& EBMT also publish
standards for cell and tissue banking and interpreting laws (4042). Amniotic membranes are widely used in skin transplantation(43), treatment of burned and ulcerated skin (44,45), connective
tissue defects (46), ophthalmology, surgery and wound healing(47,48). Its use has a history of almost 100 years. They can be
freeze-dried, gamma-sterilized, decellularized, glycerol-preserved and cryopreserved. A comparison of the cryopreservationof the membranes with the storage in glycerol at 4 oC showed a7

retained cell viability and release of angiogenic growth factors
and cytokines in the first method while in the glycerol method
resulted immediate cell death (49). It has been confirmed that
other methods like irradiation influences the growth factor content
of amniotic membrane (50). The most experience in preservation of placental tissue derived cells has been gained with cord
blood. Because of the efficiency of cord blood transplantation
many banks were established offering collection and banking for
public and private use. Cord blood contains both mesenchymal
stem cells and hematopoietic stem cells and it can be procured
from natural births or caesarean section (51). It has been proventhat placentae collected from natural birth are more likely to
be contaminated with aerobic or anaerobic bacteria then theplacentae collected from caesarean sections. Therefore it may
be preferential to collect placentae from caesarian sections (2).
Cord blood products containing cryoprotectans are frozen at acontrolled-rate and can be stored in liquid nitrogen for at least
15 years without the loss of their engraftment potential in vivo
(51,52) while fetal membrane stem cells are presently preserved
mainly for research.
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21. Erices A, Conget P, Minguell JJ. Mesenchymal progenitor cells inhuman umbilical cord blood. Br J Haematol 2000; 109: 235-242.
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30. Thomson JA, Itskovitz- Eldor, Shapiro SS, et al. Embryonic stem
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36. Miki T, Lehmann T, Cai H, et al. Stem cell characteristics of amniotic
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38. Wolbank S, Peterbauer A, Fahmer M, et al. Dose dependent immunomodulary effect of human stem cells from amniotic membrane: A
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44. Stern W. The grafting of preserved amniotic membrane to burned

and ulcerated skin. JAMA 2013; 13: 973-974.
45. Sabella W. Use of fetal membranes in skin grafting. Med Rec NY
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46. de Rotth A. Plastic repair of conjunctival defects with fetal membranes. Arch Ophtalmol 1940; 23: 522-525.
47. Kim JC, Tseng SC. Transplantation of preserved human amniotic
membrane for surface reconstruction in severely damaged rabbit corneas. Cornea 1995; 14: 473-484.
48. Tosi GM, Massaro-Giordano M, Caporossi A, et al. Amniotic
membrane transplantation in ocular surface disorders. J Cell Physiol.
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49. Hennebrichler S, Reichl B, Pleiner D, et al. The influence of various
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50. Branski L, Kulp G, Jeschke M, et al. Amniotic membrane as woundcoverage: The effects of irradiation and different processing methods
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51. Chrysler GR, McKenna DH, et al. Cord Blood Banking In: Broxmeyer
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52. Skoric D, Balint B, Petakov M, et al. Collection strategies andcryopreservation of umbilical cord blood. Transfus Med 2007:17;
107-113.

CELULELE STEM SI CELULELE STEM PLACENTAREREZUMAT

Celulele stem sunt celule care se divid pentru a genera o celula progenitoare, care este o celula stem, si o alta celula progenitoare,care produce descendenti diferentiati. Definitia se poate aplica embrionului fecundat, dar nu mai are aplicabilitate pe masura ce neindreptam spre caile de dezvoltare celulara. Celulele embrionare (ES) totipotente se pot diferentia cu usurinta spre toate fenotipurile
celulare esentiale, in timp ce celulele stem adulte (multipotente) izolate din diverse surse tisulare – stroma maduvei osoase hematogene, tesut adipos, muschi, tesut nervos – prezinta un potential de diferentiere limitat. Majoritatea celulelor stem adulte, prezinta
un ciclu de viata redus comparativ cu celulele ES. Mai mult, progresia de la embrionul fecundat, la ES si apoi la celulele stem adulte
si celule diferentiate ar putea fi un proces continuu, in care doar unele etape sa fie ireversibile. Celulele stem placentare se afla laintersectia dintre celulele stem adulte si celulele ES, astfel incat potentialul crescut al acestora ar trebui explorat in detaliu, pentru
utilizarea lor in viitoare terapii celulare.
Cuvinte cheie: celule stem embrionare, celule stem adulte, totipotent, multipotent

2013.23.2 (78)  Fiziologia - Physiology

9THE TAURINE EFFECT ON ELECTRICAL CEREBRAL SIGNALIN OLD RATS
GIURGIU MONICA - CRISTINA, GHITA MIHAI AURELIAN, ZAGREAN LEON
Department of Functional Sciences – Division of Physiology and Fundamental Neurosciences, “Carol Davila” University of Medicine
and Pharmacy, Bucharest, Romania

ABSTRACTIn the aging process, there are a series of structural and functional brain changes that cause modifications in electrical activity.
Taurine is an amino acid which has a osmoregulator, neuroprotector and antioxidant actions in the central nervous system. The aimof the experiment was to test the effects of taurine on brain electrical activity in old rats. Two groups of Wistar rats, 26 months old
were formed.They were chronically cortical implanted. Taurine was administrated on a period of two months in drinking water (1g/kgc). The electroencephalographic recordings were made at the beginning of the experiment, at one month and at two months. The
results indicated the increase in spectral power density in the delta and theta bands and the improvement of the correlation coefficient
between the two cerebral hemispheres.
Keywords: taurine, oxidative stress, cerebral electrical signal, old rats

INTRODUCTION
Aging is accompanied by multiple structural and functional
cortical changes. Among these, may be mentioned the decreasein the number of neurons (1, 2), the loss and modification ofsynapses (3). It was also noticed a reduction in neurons receptor
surface due to decreased number of dendrites and shortening
process (4). Changes in the brain electrical activity can becomputed through quantitative electroencephalography analysis.
Taurine is a widespread amino acid in the body, including
the central nervous system. At this level has osmoregulator,
neuroprotective and antioxidant actions. In the brain, taurine
is found both in neurons and astrocytes.The main pathway for
taurine biosynthesis is that of cisteinsulfuric acid decarboxylase(5). Neurons lacks this enzyme and base on taurine acquisition(6). Cisteinsulfuric acid decarboxylase is primarily found in glial
cells (7). Taurine content decreases in the aging brain (8).Thisaminoacid is taken from plasma through a transport system
based on Na+ and Cl- gradient, which is located in luminal andbasal membranes of endothelials cells from hematoencephalicbarrier (9).The purpose of this article is to present the results
obtained by quantitative electroencephalographic analysis ofchanges in brain electrical signals, induced by taurine in old rats.
Electroencephalographic analysis
Electroencephalography is the recording method of theelectrical activity of the brain. In 1875, Caton R. published thefirst paper about registering spontaneous brain electrical activity in experimental animals (10). In 1924, Berger H. made thefirst electroencephalographic recording in humans, which was

published in 1929 (11).
Brain signals are complex electrical signals. Frequencies
spectrum of electroencephalographic waves was divided into
five bands: delta (0.5 - 4 Hz), theta (4 - 8 Hz), alpha (8 - 13Hz), beta (13 - 30 Hz), gamma (30 - 70 Hz). Delta bandwaves are generated in the cortex and thalamus (12). They
occur in deep sleep, stages 3 and 4 (13) and in brain lesions.
Theta rhythm is seen in superficial sleep, stages 1 and 2 andREM sleep. In adult humans, in wakefulness, there are two
types of theta rhythms: the first is associated with decreased
sensory perception (drowsiness and impaired information
processing) and the second occurs in the frontal midline area
and is associated with attention and mental effort (14). In rats,
hippocampal theta rhythm prevails. It occurs when the animal isinvolved in a motor activity and during REM sleep (15). Alpharhythm is recorded in wakefulness, relaxing state, with closed
eyes. It has the largest amplitude in the occipital region and
it is blocked by the eyes opening and mental concentration.
Beta waves present mainly a fronto - central distribution andare associated with activity, with thinking and concentration.
Gamma rhythm is associated with intense cortical activationand object recognition.
Quantitative electroencephalography analysis deals withmathematical processing of brain electrical signals, the mostfrequently used being the spectral analysis and coherence
analysis.
Fourier Transform is used for data analysis, because it
allows signal decomposition into sinusoid constituents of different frequencies. For a given sample of data, Fourier analysis
is performed using Fast Fourier Transform.
A parameter that can be calculated using Fast Fourier

Received 2nd of February 2013. Accepted 16th of March 2013. Address for correspondence: Giurgiu Monica - Cristina, MD, PhD student, Division of Physiology andFundamental Neurosciences, “Carol Davila” University of Medicine and Pharmacy, Bd. Eroii Sanitari, No. 8, Bucharest, RO-050474, phone: +40722527878; e-mail:monicagiurgiu@gmail.com

10

Fiziologia - Physiology  2013.23.2 (78)Transform is spectral power density. Spectral power density
indicates the distribution of the power at different frequencies. It
represents the Fourier Transform of the autocorrelation function
and has the following formula:∞Sx ( f ) = ∫ Rx (τ )e − 2πif τ dτ−∞The autocorrelation function describes how a signal changes over time and what is the signal correlation in two different
moments in time. It has the following equation:

Rx (τ ) = E[ x (t ) x (t + τ )]To avoid convergence problems, it is considered a signal
recorded on a finite interval of time, T, where T = x (t ) wT (t )x, where 1 for 0 ≤ t ≤ T
2wT = 
T
for
t
>
0

2

Fourier Transform is applied toxT

The result obtained is
T /2

XT ( f ) = ∫ x (t )e − 2πift dt−T /2

The variables are replaced in the equation of the autocorrelation function and the spectral power density formula is obtained.
Studies on analysis methods of quantitative electroencephalography showed that this is highly reliable. Salinsky et al., 1991,
demonstrated that the reliability for 20 seconds epochs is 82%
and for 60 seconds epochs is 92% (16). Gasser et al., 1985,
indicated that in electroencephalographic analysis 20 seconds
epochs are enough to reduce signal variability (17).
MATERIALS AND METHODSThe experiment consisted of recording cortical electricalactivity in studied animals before and after administration oftaurine. All procedures respected the directive 2010/63/EU of
the European Parliament and of the Council on the protectionof animals used for scientific purposes.
The experiment was conducted on two groups, one test andone control, of ten Wistar male rats each. Rats, 26 months old,
were bred in standard conditions in the Biobase of the Universityof Medicine and Pharmacy „Carol Davila”. The animals were anesthetized with chloral hydrate, 400 mg / kgc (Novachim Trading
S.R.L.) and were chronically implanted under stereotaxic control.
The electrodes were placed after the model introduced by Zagrean L., as follows: two electrodes at 5 mm anterior to bregma,
at 3 mm lateral right and left of the midline and two electrodes at
6 mm posterior to bregma, at 4 mm lateral right and left of the2013.23.2 (78)  Fiziologia - Physiology

median line (18). The internal part of the electrodes was placedon the cerebral cortex and the external part was connected toa pin array. The system was secured to the skull with dental
acrylic cement. The scalp was sutured and the animals received
antibiotic. They were placed individually in transparent cages andwere allowed a period of a week to recover from operator stress.
The rats were exposed to a cycle of 12h light, 12 hours dark, withfree access to food and water at a temperature of 20 Celsius
degrees. Taurine (Novachim Trading S.R.L.) was administeredover a period of two months in drinking water in a volume of
20 ml, which contained the daily dose of 1g/kgc (19). For the
rest of the day there was access to taurine - free water. Taurine
administration was started after the first electroencephalographic
recording, which was performed at one week after implant. The
other two recording sessions were performed at one and twomonths from taurine first administration. To exclude the effectsof circadian tests were done at the same time. The recording
system was composed from MP150 units and Acqknowledge
4.2.0 software (BIOPAC System, Inc.).
RESULTSElectrocorticographic recordings were made in anterior- posterior axis, using bipolar derivations. The quantitative
processing of the electrical signal was made with aid of the
Acqknowledge 4.2.0 software. Statistical analysis of the data
was made using Kingsoft Spreadsheets software (Kingsoft Office
Software Corporation Limited).
The average magnitudes of the waves for the five frequency
bands were computed using Fast Fourier Transformation.The
results obtained indicated statistically significant increases inwaves magnitudes in delta and theta bands for the test group.
For the control goup there were no changes in time. The p wascomputed using the values obtained from recordings at control
and at two months moments. The p value for the right hemisphere
was 0.0017 for the delta band and 0.002 for the theta band. For
the left hemisphere the p value was 0.002 for the delta band and0.0023 for the theta band. The changes obtained were represented as percentage in Figures 1 and 2 for each hemisphere. Each
percentage was calculated compared to the control. It should
be noted that the most significant increases from baseline wererecorded at one month.

Fig.1. Percentage changes of waves magnitudes in right cerebral hemisphere. It was noticed the increased magnitudes of cerebral waves of the delta andtheta bands at 1 month and at to 2 months.

11and it can be applied to signal analysis. The absolute value of r
ranges between 0 and 1. The degree of correlation is proportionalwith the r absolute value. The correlation coefficient between thetwo hemispheres was computed with the aid of Acqknowledge
4.2.0 software. Its value increased at one month and two months
compared to control. The results are shown in Figure 5.

Fig.2. Percentage changes of waves magnitudes in left cerebral hemisphere.
It was noticed the increased magnitudes of the two bands at the two registration moments.

Another analyzed parameter was the spectral power density(PSD). This was computed with aid of the Acqknowledge 4.2.0.
software witch used a Welsh approximation algorithm to average
epochs of the signal and reduce noise effect and a Hamming
window tapering. It was calculated the average power spectral
density for each frequency band. Statistically significant increases were observed for the density of delta and theta bands inthe test group. For the control goup there were no changes intime. The results are shown in Figures 3 and 4.

Fig.3. Spectral power density in right cerebral hemisphere (MicroV^2/Hz). It
can be observed the progressive increase of PSD values in the test groupcompared to the control group.The most important change was observed forthe delta band. The p value for delta band was 0.0004 and for the theta band
was 0.0013.

Fig.4. Spectral power density in left cerebral hemisphere (MicroV^2/Hz).
It can be observed the increased values of PSD for delta and theta bands,
indicating that at 1 month the increase was the most important. The p value
for the delta band was 0.0006 and for the theta band was 0.0014.

Correlation coefficient (r) is an important statistical index andshows the degree of correlation between the two phenomena
12

Fig.5. Correlation coefficient between the two hemispheres. It can be noticed
that the value of correlation coefficient increased gradually.

DISCUSSION
The aging process is accompanied by a series of brain changes such as loss of neurons in various structuresand loss of synapses. From functional point of view, it wasnoticed a decreases in the discharge rate of cortical neurons(20). Electroencephalographic recordings shows a decreasein the magnitude of electric signal from the delta and theta
bands (21).
Brain aging is associated with increased levels of reactive
oxygen species (22), that react with nucleic acids, proteins andmembrane lipids generating gene mutations, impairments or lossof enzyme activity, abnormal protein clearance and altered cell
membrane permeability (23, 24, 25).
Protein modifications include carbonyl formation and covalent modification of cysteine, lysine, and histidine residues by thelipid peroxidation product 4-hydroxynonenal. In aged brain havebeen detected high levels of lipid peroxidation products (26).
Taurine is one of the most abundant amino acids in theorganism, being found in the brain and spinal cord. Taurine is anosmoregulator, a neuroprotector and antioxidant agent.
Endoplasmic reticulum stress due to the accumulation of
misfolded proteins interferes with neuronal signaling. In PC12cell cultures and primary neuronal cultures, in oxidative stressand excitotoxicity conditions, taurine reduces the endoplasmic
reticulum stress by decreasing the levels of certain proteinssuch as Grp 78, CHOP/GADD153, p-IRE and p-eI0046–2
alpha protein (27, 28). Taurine acts as antioxidant also by
increasing the levels of protective enzymes against reactive
oxygen species, as glutathione peroxidase and superoxide
dismutase (29).
Administration of taurine improved alterations in electricalactivity. Recordings made at one month and at two months
from the start of taurine administration indicated an increasein waves magnitude from delta and theta bands. Anotherresult was an increase in the power spectral density of thetwo bands for the group test. Improving the synchronizationof electrical activity between the two hemispheres has been
Fiziologia - Physiology  2013.23.2 (78)

demonstrated by the progressively higher values of thecorrelation coefficient.
CONCLUSION
Taurine has benefic effects over cortical electrical activity.
The changes induces by taurine administration over a period of
two months in analyzed parameters, may due to its antioxidant
activity in central nervous system.
ACKNOWLEDGEMENT
The experiment presented, including procedures, were partof TD grant, number 449/2007, which was approved by the Boardof University of Medicine and Pharmacy „Carol Davila” and by
the National Council of Scientific Research in Higher Educationfrom Romania.
DISCLOSURE STATEMENT
There are no conflicts of interest between authors. The
article is not submitted to other journal for publication.
REFERENCES
1. Henderson G, Tomlinson BE, Gibson PH. Cell counts in human cerebral cortex in normal adults throughout life using an image analysing
computer. Journal of the Neurological Sciences 1980; 46: 113-136.
2. Terry RD, De Teresa R, Hansen LA. Neocortical cell counts in normal
human adult aging. Annals of Neurology 1987; 21: 530 - 539.
3. Adams I, Jones DG. Quantitative ultrastructural changes in rat cortical synapses during early-, mid- and late-adulthood. Brain Research
1982; 239: 349 - 363.
4. Scheibel ME, Lindsay RD, Tomiyasu U. Progressive dendritic
changes in aging human cortex. Experimental Neurology 1975; 47:
392 - 403.
5. Wu J-Y, Moss LG, Chen MS. Tissue and regional distribution ofcysteic acid decarboxylase in bovine brain. A new assay method.
Neurochem Res 1979; 4: 201 - 212.
6. Tappaz M, Almarghini K, Do K. Cysteine sulfinate decarboxylase inbrain: identification, characterization and immunocytochemical locationin astrocytes. AdV Exp Med Biol 1994; 359: 257 - 268.
7. Almarghini K, Remy A, Tappaz M. Immunocytochemistry of thetaurine biosynthesis enzyme, cysteine sulfinate decarboxylase, inthe cerebellum: evidence for a glial localization. Neuroscience 1991;
43: 111 – 119.
8. Benedetti MS, Russo A, Marrari P, et al. Effects of ageing on thecontent in sulfur-containing amino acids in rat brain. Journal of Neural
Transmission / General Section JNT 1991; 86(3): 191 - 203.
9. Tamai I, Senmaru M, Terasaki T, et al. Na(+)- and Cl(-)-dependenttransport of taurine at the blood-brain barrier. Biochem Pharmacol
1995; 50(11): 1783 - 1793.
10. Caton R. The electric currents of the brain. Br Med J 1875; 2: 278.
11. Berger H. Über das Elektrenkephalogramm des Menschen. Archive
Fuer Psychiatrie Und Nervenkrankheiten 1929; 87: 527 - 570.
12. Brigo F. Intermittent rhythmic delta activity patterns. Epilepsy Behav.
2011; 20: 254 - 256.
13. Niedermeyer E. Historical Aspects; in Niedermeyer E & Lopesda Silva F(ed): Electroencephalography: Basic Principles, ClinicalApplications, and Related Fields, Williams & Wilkins, 1993, 1 - 14.
14. Schacter D. EEG theta waves and psychological phenomena: A
review and analysis. Biological Psychology 1977; 5: 47 - 82.
15. Vanderwolf CH. Hippocampal electrical activity and voluntary
movement in the rat. Electroencephalogr Clin Neurophysiol 1969;
26(4): 407 - 418.
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16. Salinsky MC, Oken BS, Morehead L. Test-retest reliability in EEG
frequency analysis. Electroencephalogr Clin Neurophysiol 1991; 79(5):382 - 392.
17. Gasser T, Bächer P, Steinberg H. Test-retest reliability of spectral
parameters of the EEG. Electroencephalogr Clin Neurophysiol 1985;
60: 312 - 319.
18. Zagrean L, Munteanu A-M, Coculescu M. Preliminary EEG studyof protective effects of Tebonin in transient global cerebral ischemia
in rats. Rom J Physiol 1998; 35:161 - 168.
19. Pierno S, de Luca AM, Camerino C. Chronic Administration of
Taurine to Aged Rats Improves the Electrical and Contractile Properties of Skeletal Muscle Fibers1, The Journal of Pharmacology andExperimental Therapeutics 1998; 286(3): 1183 - 1190.
20. Roy D, Singh R. Age-related change in the multiple unit activity of
the rat brain parietal cortex and the effect of centrophenoxine. Experimental Gerontology 1988; 23: 161 - 174.
21. John ER, Ahn H, Prichep L. Developmental equations for theelectroencephalogram. Science 1980; 210: 1255 - 1258.
22. Driver AS, Kodavanti PRS, Mundy WR. Age-related changes inreactive oxygen species production in rat brain homogenates. Neurotoxicol Teratol 2000; 22: 175 - 181.
23. Finkel T. Oxidant signals and oxidative stress. Curr Opin Cell Biol
2003; 15: 247 - 254.
24. Harman D. Aging: A theory based on free radical and radiation
chemistry. J Gerontol 1956; 11: 298 - 300.
25. Dei R, Takeda A, Niwa H. Lipid peroxidation and advanced glycation
endproducts in the brain in normal aging and in Alzheimer’s disease
Acta Neuropathol 2002; 104: 113 - 122.
26. Poon HF, Calabrese V, Scapagnini G. Free radicals and brain aging.
Clin Geriatr Med 2004; 20: 329 - 359.
27. Pan C, Giraldo GS, Prentice H, et al. Taurine protection of PC12
cells against endoplasmic
reticulum stress induced by oxidative stress. J Biomed Sci 17 2010;
Suppl 1 :S1 - S17.
28. Pan C, Prentice H, Price AL. Beneficial effect of taurine on hypoxiaand glutamate-induced endoplasmic reticulum stress pathways inprimary neuronal culture. Amino Acids 2012; 43: 845 - 855.
29. Vohra BP, Hui X. Taurine protects against carbon tetrachloride
toxicity in the cultured neurons and in vivo. Arch Physiol Biochem
2001; 109: 90 - 94.

13

EFECTELE TAURINEI ASUPRA ACTIVITATII ELECTRICE CEREBRALE
LA SOBOLANII BATRANIREZUMATIn procesul de imbatranire apar o serie de modificari cerebrale structurale si functionale care determina modificarea activitatii electrice. Taurina este un aminoacid, care la nivelul sistemului nervos central are rol osmoreglator, neuroprotector si antioxidant. Scopul
experimentului a fost testarea efectelor taurinei asupra activitatii electrice cerebrale la sobolanii batrani. Au fost constituite doua loturi,
unul control si unul test, de sobolani Wistar in varsta 26 luni. Acestia au fost implantati cronic cortical. Administrarea taurinei s - a facutpe o perioada de doua luni in apa de baut (1g/kgc). Inregistrarile electroencefalografice s-au realizat la inceputul experimentului, lao luna si la doua luni. Rezultatele au indicat cresterea densitatii puterii spectrale in benzile delta si theta si imbunatatirea gradului decorelatie intre cele doua emisfere cerebrale la lotul test.
Cuvinte cheie: taurina, stres oxidativ, semnale electrice cerebrale, sobolani batrani

14

Fiziologia - Physiology  2013.23.2 (78)

HYDATID CYST OF THE KIDNEY: PRECLINICAL AND CLINICALPARTICULARITIESRADU TUDOR1, ISTRATE BOGDAN2, JINGA VIOREL1, STERIU DAN3
1
Urology Clinical Hospital “Prof. Dr. Th. Burghele” Bucharest, Romania
2
Center of Neurobiology and Molecular Physiology, University of Bucharest, Romania
3
University of Medicine and Pharmacy “Carol Davila” Bucharest, Romania

ABSTRACT

Renal echinococcosis is rarely occurring in comparison with liver and lung localization. Kidney involvement reveals a percentage of
6% in confirmed cases of hydatid disease. In this paper we point the elements of physiology, preclinical and clinical aspects of hydatid
cyst of the kidney. In the same manner are presented the diagnostic steps with the differential connotations. Since the availability of
nuclear medicine and clinical laboratory brings new insights in the diagnostic accuracy, medical and surgical treatment remains themost important benefit for the patient.
Key words: kidney, echinococcosis, immunology, diagnostic, surgery, considerations

INTRODUCTION
A renal hydatid cyst is a parasitic infection (benign) causedby larval growth of the cestode Echinococcus granulosus.
Synonym: Taenia echinococcus
Disease: Hydatiosis, Hydatid disease, Echinococcosis.
Elements of morphology
Adult parasite, specially occurred in dogs, is the lowest
cestode of medical importance. Its measure is from 2.5 to 9 mm,
consisting from a scolex, a neck and only three attached segments(proglottids). The first proglottid is short and contains immature
genital organs. In the second proglotte, a bit elongated, thegenital organs are completely developed. The scolex is granular,
measuring 0.3 mm in diameter equipped with 4 suckers and28-40 hooks arranged in two rows (1). The uterus reaches the
maximum development in the last segment being composed fromshort diverticles which as filled with eggs become wider, taking
the aspect of a bag full of eggs. After the proglotte’s rupture, theeggs are eliminated in feces. The definitive host can accommodate
thousands of adult parasites, their lifespan is between 6 months
and 2 years. The eggs are typical for most taeniid species, small
and round 34-41µm in diameter, thick-shelled, containing ahexacanth (6-hooked) embryo (oncosphere) (Figure 1).

Fig.1. Invaginated and exvaginated ptotosleces (after Steriu D)

The evolutive cycle
Definitive hosts are carnivores such as dogs, wolfs, foxes,
hyena. Gravid proglottids or released eggs are shed in faeces
and after their ingestion by the host; a oncosphere larva isreleased and penetrates the intestinal epithelium into lamina
propria. The passively pass through blood or lymph into thetarget organs, where it develop into hydatid cyst. The hydatide
is compose of a thick cuticle coated inside by a proligere
membrane and inside by a adventia membrane. Inside are foundthe hydatic liquid and scoleces. At the contact zone between
parasite and infected organ there is a dense coating which canreach a thickness of 1-2 mm, produced by the condensation andinfiltration of the organ that develop hydatid cyst. The growth ofhydatid cyst is concentric with a rate of 1-5cm/year (Figure 2).

Fig.2. The evolutive cycle (after D.Steriu)
Received 2nd of February 2013. Accepted 5th of March 2013. Address for correspondence: Marius Tudor Radu, MD urologist, Urology Clinical Hospital “Prof.Dr.Th.
Burghele” Bucharest 5, 050659 Romania, e-mail: tudor.radu@yahoo.com

2013.23.2 (78)  Fiziologia - Physiology

15

In humans the cysts can reach a volume of several liters
and they can contain hundreds of thousands protoscoleces.
The infectious potential of hydtid cyst is enormous, one single
proligere vesicle contains between 10-120 scoleces, 1cm3 ofsand contains 400.000 scoleces, and a hydatid cyst cam contain3-6 cm3 of hydatid sand.

can be recognized macroscopically, being as well the only
pathognomonic evidence of a hydatid cyst that has ruptured in
the urinary tract.
Other: renal colic, fever, allergic reactions.

Patogenesis
Hydatid cyst can develop in humans in two situations: byingestion of eliminated embriosphere from dog, it forms a primary
hydatida, located in different organs, depending on blood stream
passive migration of the parasite (1). Most embryos are retainedat the level of hepatic capillary network (60-70%) especially inthe right lobe; 20-30 % retired in the pulmonary filter and only
10% passes in the circulation, being disseminated in muscles(4%), spleen (2%), brain (1%), kidney (1%), orbit (0.5%), bones(1-2%). Usually they are located in one single organ but there areclinical cases described with plurivisceral localization. It depends
presumably by the intensity of the contamination. Other modality
in production of hydatiosis is the rupture of the primary hydatid
cyst followed by the protoscoleces release.
The protoscoleces are converted in new hydatids,
generating local secondary echinococcosis or if disseminate
at a distance of the primitive focus, it produces systemic
secondary echinococcosis (2). The parasited organ will influence
its further development determining the shape and latent
period, when becomes symptomatic. The rhythm of hydatid
cyst development is very different and may vary depending on
the host, the parasitic tissue being as well conditioned by thecyst fertility and organ tolerance. The hydatid cyst can damage
profoundly the infested organ through the action of parenchyma
destruction accompanied by the hypertrophy of free territory.
The hydatid cyst once constituted can describe three evolutive
stages: spontaneous involution, rupture, infection. Initially thecyst is univesicular and after 6-8 months the vesicles becomes
fertile (proligere vesicles and protoscoleces). The thickness ofthe cuticle grows gradually by adding new lamellar structures
increasing the liquid quantity till 5 to 800 cm3. After a variable
number of years the hydatide may die, replaced by a formation
well tolerated by the body. The hydatid cavity will be surrounded
by a fibrous capsule charged with limestone salts. The cyst
rupture can be caused by a trauma or an increased renalpressure.

Immunoserologic studies
Immunoserological diagnosis is helpful in determinationof parasitic etiology, if the factor that influences the result isknown. Firstly, a negative result does not exclude the diagnosisof hydatiosis because some cyst carriers have no detectableantibodies. The presence of a detectable immune response isconditioned by the localization, integrity and vitality of the cyst.
The immunological diagnosis consists in detection of anti-E.
Granulosus antibodies can be detected from the patient’s sera,
via indirect hemagglutination, immunoelectrophoresis, ELISA,
Casoni’s test including immunofluorescence. The highest
specificity is done by the test based on the detection of antibodies
against 5 specific antigens.

Clinical manifestations
Most of the infections progresses asymptomatic. In
other cases, the clinical manifestations are determined by the
localization of the cyst, by dimension and it state: intact, cracked,
infected. Renal hydatid cyst can remain asymptomatic for many
years and can be discovered accidentally (3).
Cardinal symptoms: flank pain (lumbar and abdominal),
perirenal reactions suggests the appearance of “fixed tumor”
indicate a solid tumor actually. Digestive symptoms: subcostal
pain, abdominal distension and vomiting.
Hydatiuria (the presence of daughter vesicles in urine)
16

DIAGNOSTICRadiologic studies
These studies are the most helpful tool. During the 1960’s
and 1980’s retrograde pyelography and less often aretiography
were used. Plain abdominal radiography can be sometimes
useful. The classification of the hydatid cysts was done,
according with Gharbi studies (Table I) (3):Table I. Garby ultrasonographic classification

Type
I
II
III
IV
V

Criterion
Well limited liquid cyst, with parietal echo backing
Cyst with a detached membrane or a floating
membranePartitioned cyst with a honeycomb picture
Heterogeneous echo structure formation,
sometimes with liquid predominance andsometimes with solid predominanceDense reflecting pictures with a posterior shadecone, corresponding to the calcified cyst

Computed tomography usually after ultrasonography is
mandatory in most of the cases. Renal echography should be
performed as well.
Surgical terminology and procedures
Treatment of cyst → cystectomy
Resection of the preeminent dome → partial pericystectomy
Total cystectomy → Total pericystectomy
Epidemiology
Humans are occasionally infected: infection is caused byoccupational factors, the more exposed people are hunters
Fiziologia - Physiology  2013.23.2 (78)and the persons which process the foxes furs (1). The dogs are
infected by capturing and eating rodents which can carry thelarval form of the parasite, becoming sources of infection forhumans trough the eggs eliminated via feces.
Methods of prevention
Infection can be avoided by removing the eggs from thefruits, vegetables or other foods. It is proved that the eggs aredestroyed after exposure for three hours at 45°C or -80°C (1). In
endemic areal the measures of hygiene recommends’, avoiding
the contact with foxes and their excrements, in order to protect
the people having a high risk of contamination. It is indicatedfor hunters to wear rubber gloves when processing the fox’s fur
and for other people is indicated to wash the fruits and boil thevegetables before consuming/prepare them.
CONCLUSIONS
Renal hydatiosis due to Echinococcus is an endemicparasitic zoonosis characterized by worldwide distribution. The
most commonly involved anatomical locations are the liver andthe lung. The renal cases are rare.
The immunoserological diagnosis may contribute in orderto monitor the patient after the surgical intervention.
CT is recommended in the pre-operatory phase, suggesting
to surgeon a indispensable vascular cartography, if it is decided
a nephrectomy, especially for the retroperitoneal procedures,
giving information on the benign nature of the process.
The exploratory puncture is formally contraindicated due tothe risk of secondary seeding.
Imagistic techniques are important in the diagnosis of renal
hydatiosis and should be corroborated with laboratory data.
ACKNOWLEDGEMENTS
We would like to acknowledge to Prof. Univ. Dr. Lotus
Elena Mester for the important contribution in scientifical support
with regards to biology of the parasites. We also would like toacknowledge to Mihaela Radu.
REFERENCES
1. Radulescu S. Medical Parasitology, ALL Medical, Bucharest, 2000.
2. Zmerli S, Mohsen A, Chami I, Onakdi EM, Slama MRB. Hydatid cystof the kidney, Diagnosis and treatment. World J. Surg. 2001; 25: 68-74.
3. Gharbi HA, Hassine W, Brauner HW, Dupuch K. Ultrasoundexamination of the hydatid liver. Radiology 1991; 139:453.
4. Gillespie S, Pearson RD. Clinical Parasitology. Principles andPractice. Ed. John Wiley& Sons Ltd., 2010.

CHISTUL HIDATIC RENAL: PARTICULARITATI PRECLINICE SI
CLINICEREZUMAT

Echinococoza renala este rar intalnita in comparatie cu cea hepatica si cea pulmonara. Implicarea renala arata un procentaj de 6%
din totalul cazurilor confirmate. In acest articol subliniem elemtele de fiziologie, aspectele preclinice si clinice ale chistului hidatic renal.
In aceeasi maniera sunt prezentate etapele diagnosticului cu conotatii diferentiale. Intrucat disponibilitatile moderne ale medicinei
nucleare si laboratorului clinic aduc noi perspective in acuratetea diagnosticului, tratamentul medical si chirurgical raman beneficiul
primordial pentru pacient.
Cuvinte cheie: rinichi, echinococoza, imunologie, diagnostic, chirurgie, consideratii

2013.23.2 (78)  Fiziologia - Physiology

17

SUBCLINICAL ATHEROSCLEROIS IN RHEUMATOIDPOLYARTHRITISCRISTINA FLOREA, ELENA ARDELEANU, ALEXANDRU CARABA
University of Medicine and Pharmacy ”Victor Babes” Timisoara

ABSTRACT

RP is considered an autoimmune disease due to the presence of autoantibodies, such as anti-cyclic citrullinated peptide antibodies(tested as anti-cyclic citrullinated peptide), that may be present many years before clinical manifestation of RP. Atherosclerosis hascommon features with autoimmune diseases, as showed in the study in which β2-glycoprotein I-reactive transfer lymphocytes increase
atherosclerosis in diseased mice. Cardiovascular diseases (CVD) are a major cause of comorbidity and mortality, reportedly related
to approximately 60% of the mortality risk in RP.
Aim: The aim of this paper is to assess atherosclerotic lesions by carotid ultrasound and to characterise the factors responsible for
their onset in PR patients. Material and methods: This study has been conducted on a group of based on the criteria set by American
College of Rheumatology-ACR/European League Against Rheumatism-ELA revised in 2010. Medication used by patients consistedof methotrexate and sulfasalazine. The control group was composed of 17 healthy women of age similar to RP patients. Both groups
underwent carotid ultrasound by ALOKA ProSound 4000 echograph, using 10 MHz linear probe. Biochemical and immunologicalinvestigation consisted in determination of: total cholesterol (Abbott photometry), C reactive protein (CRP - Turbidimetry) triglycerides(Abbott reagent), anti-cyclic citrullinated peptide antibodies (Microparticle enzyme immunoassay), rheumatoid factor (photometry)and platelet sedimentation rate (PSR - Electro Optical System Technologies).RP activity has been appreciated by the means of
DAS28 (Disease Activity Score) index.
Results: Carotid IMT in RA patients has been strongly correlated with: DAS28 index (r = 0.869, p < 0.01), disease evolution time (r= 0.944, p < 0.01), systolic blood pressure value (r = 0.946, p < 0.01). Medium correlations were recorded between this parameterand diastolic blood pressure value (r = 0.568, p < 0.05), number of relapses (r = 0.669, p < 0.01), and value of total cholesterol (r =0.678, p < 0.01), respectively. Carotid IMT has been weakly correlated with the values of triglycerides, anti-cyclic citrullinated peptide
antibodies, PSR and CRP.
Conclusion: Subclinical atherosclerosis has high incidence among patients with RP. Besides the traditional cardiovascular risk factors,
other factors related to the main disease, as well as to its treatment occur in these patients. Vascular ultrasound of common carotid
artery is a non-invasive mean to identify and quantify subclinical atherosclerosis in order to apply therapies to prevent the worseningof disease and the onset of clinically manifest atherosclerosis.
Keywords: subclinical atherosclerosis, rheumatoid arthritis

INTRODUCTION
Rheumatoid polyarthritis (RP) is a chronic systemic
inflammatory disease marked by joint inflammation andsensitivity, as well as by synovial joint destruction, leading tosevere disability and premature mortality (1). RP is considered
an autoimmune disease due to the presence of autoantibodies,such as anti-cyclic citrullinated peptide antibodies (tested asanti-cyclic citrullinated peptide), that may be present many years
before clinical manifestation of RP (2).
Austrian pathologist Carol von Rokitansky (1840) and Rudolf
Virchow were the first to describe the nature of inflammation inatherosclerosis. Although Rokitansky believed the inflammation issecondary to other processes, Virchow promoted atherosclerosis
as a primary inflammatory disease (2).
Atherosclerosis is an inflammatory process in the large
and medium arteries, characterized by infiltration of monocytes/
macrophages and activated T cells into intima. Atherosclerosis
has common features with autoimmune diseases, as showed in

the study in which β2-glycoprotein I-reactive transfer lymphocytes
increase atherosclerosis in diseased mice (2). Cardiovasculardiseases (CVD) are a major cause of comorbidity and mortality,
reportedly related to approximately 60% of the mortality risk in
RP. Traditional risk factors for CVD and inflammation-related
factors appear to play a critical role in the increase of CVD riskin RP patients.
Higher CVD risk has been reported in RP (3). Dyslipidemia
is often positive in RP, but seems to be somewhat different.
An increased prevalence of small dense atherogenic LDL
particles has been determined in RP, this being considered anearly atherogenesis stage. Role of corticosteroids and othertreatments needs to be considered. Methotrexate treatment
appears to be associated with an increase in CVD risk.
Substitution with folate on the other hand seems beneficial (4).
The mechanisms inducing increased mortality by CVD in RP
are present early in the natural evolution of this disease (I).
Several types of cardiac involvement may occur in RP (e.g.,ischemic disease secondary to atherosclerosis), which are the

Received 22nd of March 2013. Accepted 26th of May 2013. Address for correspondence: Cristina Florea, MD, University of Medicine and Pharmacy ”Victor Babeș”
Timisoara, Eftimie Murgu Square No. 2A, RO-300041, Timisoara, phone/fax: +40256220479; e-mail: cristina_demendy@yahoo.com

18

Fiziologia - Physiology  2013.23.2 (78)

main CVD death causes in RP patients (1,2). Other risk factors
for subclinical atherosclerosis onset are: smoking, functional
endothelial abnormalities chronic inflammation (1,2).
Ultrasound evaluation of common carotid artery (intimamedia thickness, presence of atheromatous plaques) is a noninvasive method to early detect atherosclerosis. The aim of thispaper is to assess atherosclerotic lesions by carotid ultrasoundand to characterize the factors responsible for their onset in
PR patients.

Results of immunological explorations and DAS28 values
are presented in Table II.

MATERIAL AND METHODSThis study has been conducted on a group of based on
the criteria set by American College of Rheumatology-ACR/
European League Against Rheumatism-ELA revised in 2010(5). Medication used by patients consisted of methotrexate andsulfasalazine. The control group was composed of 17 healthy
women of age similar to RP patients.
Both groups underwent carotid ultrasound by ALOKA
ProSound 4000 echograph, using 10 MHz linear probe.
Evaluation of carotid intima-media thickness (IMT) was done at
20 mm before common carotid bifurcation (normal value < 0.5mm). Atheromatous plaque has been defined as focal thickeningof vascular wall, with or without calcifications.
Biochemical and immunological investigation consisted indetermination of: total cholesterol (Abbott photometry), C reactive
protein (CRP - Turbidimetry) triglycerides (Abbott reagent),
anti-cyclic citrullinated peptide antibodies (Microparticle enzyme
immunoassay), rheumatoid factor (photometry) and platelet
sedimentation rate (PSR - Electro Optical System Technologies).
RP activity has been appreciated by the means of DAS28(Disease Activity Score) index.
Data were presented as mean ± standard deviation. Statistic
analysis was performed by the means of Pearson (for correlation)and t-Student (for comparison) tests, p < 0.01 being considered
statistically significant.

Higher carotid IMT and higher incidence of atheromatous plaques
were recorded in RP patients, as compared to the control group(Table III, Figures 1, 2, and 3).

Table II. Results of immunological exploration, DAS28 index
Parameter
Anti-cyclic citrullinated peptide antibodies
Rheumatoid Factor
DAS28 index
Number of relapses

Value
123U/ml
232U/ml
4.635±0.4808
2.71±1.359

Table III. Carotid parameters of two groups
Parameter
IMT (mm)
Atheromatous plaques (%)

Group
RA
0.706±0.2015
47.05%

p
Control
0.583±0.126
29.41%

P<0.01
P<0.01

Fig.1.

RESULTS
Both groups (RP and control) were similar as regards theage, while some of the traditional cardiovascular risk factors
were absent, such as: diabetes mellitus, chronic kidney disease.
Average disease evolution time was 2.58±1.46 years (Table I)

Fig.2.

Table I. Compared characteristics of study groups
Parameter
Age (years)
Average disease evolution(years)Total Cholesterol (mg/dl)
Triglycerides (mg/dl)
Smoke (%)
Blood pressure (%)
Blood pressure (mmHg)

GroupsRP
Control
42.6 ±4.14 years
42.6 ±4.14 years
2.58±1.46 years
0201.24±23.965
161±26.7
23.52
41.17
159.12±25.44/
79.71±7.389

2013.23.2 (78)  Fiziologia - Physiology

196.2±29.02
153±46.26
17.64
29.41
137.083±19.59/
82.916±9.87

Fig.3.

Carotid IMT in RA patients has been strongly correlated with:
DAS28 index (r = 0.869, p < 0.01) (Figure 4), disease evolution
19

time (r = 0.944, p < 0.01) (Figure 5), systolic blood pressure
value (r = 0.946, p < 0.01) (Figure 6). Medium correlations wererecorded between this parameter and diastolic blood pressure
value (r = 0.568, p < 0.05), number of relapses (r = 0.669, p< 0.01), and value of total cholesterol (r = 0.678, p < 0.01),
respectively. Carotid IMT has been weakly correlated with thevalues of triglycerides, anti-cyclic citrullinated peptide antibodies,
PSR and CRP.
Fig.8. Correlation between IMT and number of relapses

Fig.4. Correlation between IMT and DAS

Fig.9. Correlation between IMT and total cholesterol

Carotid IMT, as well as atheromatous plaques incidence hadhigher values in smokers than in non-smokers (Table IV).
Table IV. Carotid parameters at smokers vs. non-smokers

Fig.5. Correlation between IMT and disease evolution time

Smokers

Non-smokerspNumber of patients

7

27

<0.01

IMT (mm)0.8429±0.345

0.9077±0.281<0.01

Atheromatous plaque (%)

85.71%

29.62%<0.01

Patients with carotid atheromatous plaques had a longer disease
evolution time, a higher DAS28 index compared to those withoutatheromatous plaques (Table V).
Table V. Clinical and biological parameters at patients withatheromatous plaquesFig.6. Correlation between IMT and systolic blood pressure

Fig.7. Correlation between IMT and diastolic blood pressure

20

Parameter

Atheromatous plaques

p

With

Without

Total Cholesterol (mg/dl)

209.66±20.25

208.71±27.08<0.01

SBP (mmHg)

170.43±20.50

137.50±13.56<0.01

DBP (mmHg)

80.65±6.79

79.16±7.33

<0.01

DAS28

4.65±0.50

4.26±0.23<0.01

Disease evolution time (years)

3.46±1.68

2.16±0.93<0.01

Number of relapses

3.17±1.33

2.00±1.20<0.01

Patients on corticotherapy had greater intima-media thickness
and higher atheromatous plaques incidence (Table VI).
Fiziologia - Physiology  2013.23.2 (78)Table VI. Carotid parameters according to treatment used
Parameter

Type of treatmentpCorticotherapy

Another therapy

Number of patients

23

11

<0.01

IMT (mm)

0.81±0.16

0.60±0.15<0.01

Atheromatous plaques (%)

73.26%

67.8%

<0.01

DISCUSSION
RP is a chronic inflammatory disease mainly affecting
women up to 45 years of age and declining up to 75 years.
Although therapeutic schemes have been significantly improved,
thus increasing the survival time, both morbidity and mortality
among these patients remained high, chiefly because of CVD.
RP has been suggested to be comparable with type 2 diabetes
mellitus as an independent risk factor for CVD (2,4). Traditionalrisk factors for cardiovascular diseases and inflammation-related
factors seem to play a major role in explaining the increase of
CVD risk in RP patients (2,4). Increased prevalence of CVD isthe consequence of an early extensive atherogenesis process.
Non-invasive evaluation of subclinical atherosclerosis
patients by vascular ultrasound of common carotid arteryquantifies two parameters: IMT and atheromatous plaquespresence. In a meta-analysis, Tyrell et al showed thatatherosclerosis, ascertained by measurement of IMT, is indeed
higher in rheumatic diseases (2). RP appears to be an importantrisk factor for early atherosclerosis and CVD development.
Under these conditions, a number of epidemiological, clinicaland laboratory investigations suggest that chronic inflammationand immune irregularities specific to RP play an essential
role in atherosclerosis acceleration [1]. Permanent endothelial
dysfunction predisposes to organic damage of the vascular wall,
which can be detected before the disease onset by ultrasoundmeasurements of IMT. Many investigations showed evidencesof carotid IMT increase in PR (I). This cannot be explained bycorticosteroid treatments, but seems instead to be associatedwith the markers of systemic inflammation and disease duration,
thus emphasising the importance of RP as atherosclerosis risk
factor (1). The role of rheumatic factors in this context is unknown,
although the rheumatoid factor is often positive in smokers(4). Dyslipidemia is frequently positive in RP, with low density
lipoproteins and triglycerides increased similarly to inflammatory
and infectious diseases (4,7). It is possible that patients with
autoimmune diseases, such as RP, to have a lower tolerance toatherosclerosis as compared to healthy individuals (7).
Carotid IMT recorded higher values in RP patients comparedto the control group, the difference being statistically significant.
Strong and medium correlations, respectively, have beenrecorded between studied carotid parameters and traditionalrisk factors for atherosclerosis: hypertension, smoking, totalcholesterol. Medium correlation (r = 0.678, p < 0.01) between
IMT and total cholesterol value has also been reported by otherresearchers. Lipid profile in RP is characterised by lower LDL
and HDL and higher triglycerides. LDL of RP patients had also
an increased capacity to bind proteoglycans, which is deemed an
2013.23.2 (78)  Fiziologia - Physiology

early step in atherosclerosis development (7). Lipid peroxidation
might play a role in RP and LDL oxidised LDL holds foamy cells
that have been described in synovial RP. Moreover, the oxidative
stress is higher in RP, being associated with atherosclerosis (4,8).
Hypertension is one of the most studied traditional risk
factors of atherosclerosis, carotid abnormalities being correlatedwith the increase in blood pressure values. A strong correlation
between IMT and blood pressure value (Figure 6) was foundin this study. Hypertension action in atherogenesis process iscomplex, increased blood pressure values leading to endothelial
dysfunction, changes in the biology of certain active substancesin the vascular wall, LDL-cholesterol transport to the vascularwall. RA treatment and life style of RP patients may favour
physical inactivity, hypertension being involved in accelerated
atherosclerosis in RP (1).
IMT and atheromatous plaques prevalence were higher insmokers as compared to non-smokers. Smoking contributes tothe onset of atherosclerosis by lipid profile changes (2,4).
Study of RP dependent parameters revealed strong,medium and weak with IMT and presence of atheromatous
plaques, respectively.
The strongest correlation has been recorded between
IMT and DAS28 index (Figure 4). The mean value of thisindex has been significantly higher in patients with carotid
atheromatosis. DAS28 index evaluates the number ofswollen, sensitive joints, while the general assessment ofdisease activity is performed using an analogue visual scaleof 100mm and PSR or CRP (9). Atherogenesis process isgradual, the risk factors persistence inducing and worsening
the existing lesions.
Strong correlation has been established between IMT and
RP evolution time (Figure 5), which can be explained by thefact that a longer evolution means: older age, persistence of
inflammation and risk of more evolutive attacks. Atheromatous
plaques have been found in patients with prolonged evolutionof RP.
Emergence of an evolutive attack of RP means severe
inflammation, important immunity disturbances and higher
doses of corticoids for its control. As atherosclerosis is strongly
associated with inflammation, the number of attacks is higher,
setting the stage for the onset of atherosclerosis (6).
As the immunological and inflammation markers varyover time, which is a consequence of post-therapy relapses
and remissions, their value at a certain evolutive time does notcorrelate with IMT and the presence of carotid atheromatous
plaques, respectively (4,6).
The role of corticosteroids and other treatments should
be considered. Methotrexate treatment is associated with anincrease of CVD risk. Folate substitutes were used in the past
years, which are beneficial (4,7). Higher doses corticotherapy
may interfere with atherogenesis by its effect on the traditional
cardiovascular risk factors (dyslipidemia, hypertension, diabetes
mellitus), which can be induced or worsened because thistherapeutic approach involves also the existence of an active
severe form of RP (4).
21CONCLUSION
Subclinical atherosclerosis has high incidence among
patients with RP. Besides the traditional cardiovascular risk
factors, other factors related to the main disease, as well as
to its treatment occur in these patients. Vascular ultrasound ofcommon carotid artery is a non-invasive mean to identify andquantify subclinical atherosclerosis in order to apply therapies
to prevent the worsening of disease and the onset of clinicallymanifest atherosclerosis.
REFERENCES
1. Shoenfeld Y, Gerli R, Doria A, Matsuura E, Cerinic MM, Ronda
N, Jara LJ, Abu-Shakra M, Meroni PL, Sherer Y. Accelerated
Atherosclerosis in Autoimmune Rheumatic Diseases. Circulation.
2005; 112: 3337-47.
2. Frostegard J. Rheumatic Diseases – Insights Into Inflammation andAtherosclerosis. Arterioscler Thromb Vasc Biol 2010; 30: 892-893.

3. Handbook of Systemic Autoimmune Diseases,Volume 1: The Heartin Systemic Autoimmune Diseases, Ed.by: Andrea Doria and Paolo
Pauletto University of Padova, Padova, Italy Series Editor Ronald A.
Asherson, 2004.
4. Frostegard J. Cardiovascular co-morbidity in patients with rheumatic
diseases. Arthritis Research & Therapy 2011, 13:225.
5. Aletaha D, Neogi T, Silman AJ, Funovits J, Felson DT, Binghman
CO, Birnbaum NS, et al. Rheumatoid arthritis classification criteria:an American College of Rheumatology/European League Against
Rheumatism collaborative initiative Ann Rheum Dis 2010;69:1580-1588.
6. Sherer Y, Shoenfeld Y. Atherosclerosis. Annals of the Rheumatic
Diseases, 2002; 61: 97-99.
7. Frostegard J. Atherosclerosis in patients with autoimmune disorders
Arterioscler Thromb Vasc Biol., 2005; 25: 1776-85.
8. Ionescu R. Esentialul in reumatologie, Ed 2, Bucharest, Amaltea
Ed, 2007.
9. Hakim A, Clunie G, Haq I. Oxford handbook of rheumatology, ed. 3,
Oxford University Press, 2011.

ATEROSCLEROZA SUBCLINICĂ ÎN POLIARTRITA REUMATOIDĂREZUMATDată fiind prezența autoanticorpilor, cum ar fi anticorpii anti-peptid ciclic citrulinați proteici (testați ca anti-peptid ciclic citrulinat), carepot precede manifestări clinice a PR cu mulți ani, PR este considerat o boală autoimună. Ateroscleroza are caracteristici comune
cu bolile autoimune, după cum s-a indicat în studiul în care limfocitele de transfer β2-glicoproteice I-reactive cresc ateroscleroza lașoarecii cu această boală. BCV sunt o cauză majoră a co-morbidității și mortalității, fiind raportate a fi relaționate cu aproximativ
60% din riscul mortalității în PR.
Scop: Scopul lucrării este reprezentat de evaluarea leziunilor aterosclerotice, prin intermediul ultrasonografiei carotidiene și
caracteristicile factorilor responsabili de apariția acestora la pacienții cu PR. Material si metode: Studiul a fost efectuat pe un grup
de 17 paciente cu PR. Diagnosticul PR a fost stabilit pe baza criteriilor Colegiului American de Reumatologie (American College of
Rheumatology-ACR)/ Liga Europeană Împotriva Reumatismului (European League Against Rheumatism-ELA), revizuite în 2010.
Grupul martor a fost constituit din 17 persoane de sex feminin, sănătoase, cu vârstă asemănătoare cu cea a pacientelor cu PR.
Ambelor grupuri li s-a efectuat ultrasonografia carotidiană cu ajutorul ecografului ALOKA ProSound 4000, utilizând sonda liniară de
10MHz. Aprecierea grosimii intimă-medie (GIM) carotidiană s-a efectuat la 10mm înaintea bifurcației arterei carotide comune (normal< 0,5mm). Placa de aterom a fost definită drept o îngroșare focală a peretelui vascular, cu sau fără calcificări. Investigațiile biochimiceși imunologice au constat în determinarea: colesterolului total (fotometrie Abbot), proteina C reactivă (PCR) (Turbidimetry), trigliceride(reactiv Abbott), anticorpi anti-peptid ciclic citrulinat (Microparticle enzyme immunoassay), factor reumatoid (fotometrie), VSH (Electro
Optical System Technologies). Activitatea PR a fost apreciată cu ajutorul indicelui DAS28 (Disease Activity Score-Scorul activității bolii).
Rezultate: GIM carotidian la pacientele cu PR s-a corelat puternic cu: indicele DAS28 (r = 0,869, p < 0,01), durata de evoluție a bolii(r = 0,944, p < 0,01), valoarea tensiunii arteriale sistolice (r = 0,946, p < 0,01). Corelații medii au fost înregistrate între acest parametru
și valoarea tensiunii arteriale diastolice (r = 0,568, p < 0,01), numărul recăderilor (r = 0,669, p < 0,01), respectiv valoarea colesterolului
total (r = 0,678, p < 0,01). GIM carotidiană s-a corelat slab cu valoarea trigliceridelor, anticorpilor anti-peptid ciclic citrulinat, VSH, PCR.
Concluzie: Ateroscleroza subclinică are o incidență crescută în rândul pacietelor cu PR. Pe lângă factorii de risc cardiovasculari
tradiționali, la acești pacienți intervin și alți factori legați de boala de bază și tratamentul acesteia. Ultrasonografia vasculară la nivelul
arterei carotide comune reprezintă un mijloc neinvaziv pentru identificarea și cuantificarea aterosclerozei subclinice în vederea aplicării
mijloacelor terapeutice de prevenire a agravării și apariției bolii aterosclerotice și cardiovasculare clinic manifeste.
Cuvinte-cheie: ateroscleroza subclinică, artrita reumatoidă

22

Fiziologia - Physiology  2013.23.2 (78)IDENTIFICATION OF THE ANTIBIOTIC RESISTANCE
PHENOTYPES OF ESCHERICHIA COLI STRAINS ISOLATEDIN THE URINE SPECIMENS OF DIABETIC PATIENTSTEODORA CHIŢĂ1,2, DELIA MUNTEAN3, LUMINIŢA BADIŢOIU4, BOGDAN TIMAR1,2, ROXANAMOLDOVAN3, ROMULUS TIMAR1,2, MONICA LICKER3
1“Victor Babeş” University of Medicine and Pharmacy Timişoara, Romania
2
Emergency Clinical County Hospital Timişoara - Clinic of Diabetes, Nutrition and Metabolic Diseases
3
Department of Microbiology, “Victor Babeş” University of Medicine and Pharmacy Timişoara, Romania
4
Department of Epidemiology, “Victor Babeş” University of Medicine and Pharmacy Timişoara, Romania

ABSTRACTThe survey aimed to determine the antimicrobial resistance of the Escherichia coli strains involved in producing urinary tract infections
in diabetic patients, hospitalised in the Diabetes Clinic, from January to December 2012. The study included 109 Escherichia coli
isolates from 570 urine specimens collected from 1470 patients. Germs identification relied on cultural and biochemical characters.
Final identification and antimicrobial testing were performed using the Vitek 2 (Bio Merieux France) automatic analyzer. Out of the
570 urine specimens, 158 samples (27.71%) were positive. From these, 109 were E. coli. The framing of the resistance phenotypes
showed a low percentage of Extended-Spectrum Beta-lactamase (ESBL) producing strains. Conclusions: The most frequently
isolated germ from the total number of collected urine specimens was E. coli. Fortunately, we isolated only a small percentage of
ESBL multidrug resistant type. 30.27% from our E. coli strains were included in the wild phenotype, while 47.71% were includedin the high level penicillinase producing phenotype. The highest percentage of antimicrobial resistance was recorded in β-lactam
antibiotics, fluoroquinolones and trimethoprim/sulfamethoxazole.
Key words: urinary tract infection, E. coli, antibiotic resistance, diabetes mellitus

INTRODUCTION
The urinary tract is one of the most common sites of infection. Urinary tract infections (UTIs) are usually acquired by theascending route from the urethra to the bladder and may proceed
to the kidney. Ascending infections of the urinary tract are mostcommonly caused by Escherichia coli. (1, 2).
Diabetes mellitus appears to be a risk factor for developing
UTI (3). Diabetic patients may suffer more severe clinical forms
of infection and when diabetic neuropathy interferes with normal
bladder emptying, persistent UTI commonly occur (2). Immunologic factors are also implicated in developing an UTI: functional
disorders of polymorphonuclear (PMN) granulocytes, reduced
chemotaxy, impaired phagocytosis, decreased killing activity of
PMN granulocytes, low levels of serum immunoglobulins, etc. (4).
In some studies, diabetic women have been found to havea higher prevalence of bacteriuria, while, in others, no difference
was found in its prevalence between patients with and without
diabetes. The higher prevalence of UTIs in diabetic patients insome series may be attributed to more frequent catheterization(1).
The aim of this study was to determine the resistance toantiinfectious chemotherapeutic agents of the Escherichia coli
strains involved in the etiology of UTIs diagnosed in diabetic

patients.
MATERIAL AND METHOD
The study was conducted in the Diabetes Clinic of theEmergency Clinical County Hospital Timişoara and included 109
Escherichia coli strains isolated from 570 urine specimens. The
urine specimens were collected from 1470 diabetic patients (847
women and 623 men), both with type 1 and type 2 diabetes mellitus, admitted to the Clinic in 2012 (from January to December).
Urine specimens were collected according to sample specific collection protocols.
Quantitative bacterial culture of the urine specimens were
performed by inoculating culture media (Columbia agar supplemented with 5% sheep blood and Mac Conkey agar) with ameasured amount of urine with calibrated loop designed to deliver
a known volume.
Germs identification was generally done according tomorphological, cultural, biochemical characters and pathogenicity tests.
All the strains involved in the etiology of urinary tract infections were identified using the Vitek 2 Compact automatedsystem (bioMerieux).
The antimicrobial resistance of the germs isolated form

Received April 4th 2013. Accepted May 20th 2013. Adress for correspondence: Teodora Chita, MD, UMF Victor Babes Timisoara, Emergency Hospital Timisoara County;
10 Iosif Bulbuca Street, Timisoara, RO-300736; phone/fax: +40256220479; e-mail: teodora.chita@gmail.com

2013.23.2 (78)  Fiziologia - Physiology

23the collected samples was assessed by performing the MIC(Minimum Inhibitory Concentration) test, with automated reading
and classification into resistance phenotypes by use of the Vitek
2 Compact analyzer, according to the CLSI (Clinical Laboratory
and Standards Institute Inc.).
Quality control strains used in testing were E. coli ATCC
35218 and E. coli ATCC 25922.
RESULTSOut of the 570 urine specimens, 158 samples (27.71%)
were positive. Gram negative bacilli from the Enterobacteriaceae
family were predominant. In 109 samples it was E. coli.
The antimicrobial resistance patterns for the Escherichia
coli isolates are showed in Tables I, II and the resistance phenotypes in Table III.
Table I. Resistance to ß-lactam antibiotics in Escherichia coli isolates.
ß-lactam antibiotic

Susceptible

Intermediate

Resistant

No.

%

No.

%

No.

%

Ampicillin

33

30.27

1

0.92

75

68.81

Amoxicillin/
clavulanic acid

41

37.61

11

10.09

57

52.30

Piperacillin

39

35.78

30

27.52

40

36.70

Piperacillin/tazobactam

87

79.82

16

14.68

6

5.50

38

34.87

28

25.69

43

39.44

Cefoxitin

105

96.33

1

0.92

3

2.75

Cefotaxime

99

90.82

1

0.92

9

8.26

Ceftazidime

99

90.82

1

0.92

9

8.26

Cefepime

101

92.67

-

-

8

7.33

Imipenem

109

100

-

-

-

-

Table II. Resistance to other antibiotics in Escherichia coli isolates.
Susceptible

Intermediate

No.

%

No.

%

No.

%

Amikacin

108

99.08

-

-

1

0.92

Gentamicin

100

91.75

2

1.83

7

6.42

Ciprofloxacin

78

71.56

5

4.59

26

23.85

Norfloxacin

78

71.56

-

-

31

28.44

Tetracycline

106

97.25

-

-

3

2.75

Nitrofurantoin

106

97.25

1

0.92

2

1.83

Trimethoprim/sulfamethoxazole

68

62.39

-

-

41

37.61

24

No.
33
32
3
4
1
10
1
1
1
2
1
4
2
3
3
1
1
4
1
1
109

%
30.27
29.35
2.75
3.67
0.92
9.18
0.92
0.92
0.92
1.83
0.92
3.67
1.83
2.75
2.75
0.92
0.92
3.67
0.92
0.92
100

Legend: ESBL- extended spectrum beta-lactamase, Low PASE- lowlevel penicillinase, High PASE- high level penicillinase, CASE- cephalosporinase, CHN- high level cephalosporinase, GM- gentamycin,
AG- aminoglycosides, Q- fluoroquinolones, SXT-trimethoprim-sulphamethoxazole, FT- nitrofurantoin, Te- tetracycline.
DISCUSSION

Cefazolin

Antibiotic

Table III. Resistance phenotypes in Escherichia coli isolates.
Phenotypes
Wild typeHigh PASE
Low PASE+SXT
Low PASE+Q+SXT
Low PASE+GM+Q+FT
High PASE+Q+SXTHigh PASE+GM+Q+SXTHigh PASE+GM+Q+FT+SXTHigh PASE+AG+Q+SXTHigh PASE+TE+SXTHigh PASE+Q+TE
High PASE+SXT
CASE+SXT
CASE+Q+SXT
CHN+Q+SXT
ESBL+SXT
ESBL+GM+Q
ESBL+Q+SXT
ESBL+GM+SXT
ESBL+GM+Q+SXT
Total

ResistantIn our study, the most frequently isolated germ in the collected urine specimens was E. coli (109 strains, representing
68.98%). These data are similar to those found in various studies,in which E. coli was the most frequently isolated germ in diabetic
and non-diabetic patients diagnosed with UTIs (5, 6).
Studying the antimicrobial resistance and resistance phenotypes in the 109 E. coli strains revealed that 8 of 109 strains(7.33%) belonged to the ESBL phenotype. All these strainspresented also resistance to other classes of antibiotics.
The ESBLs are beta-lactamases capable of conferring
bacterial resistance to the penicillins, first-, second, and thirdgeneration cephalosporins, and aztreonam (but not the cephamycins or carbapenems) by hydrolysis of these antibiotics, andwhich are inhibited by beta-lactamase inhibitors such as clavulanic acid or tazobactam. The most common extended-spectrum
phenotypes arise from point mutations in the blaTEM, blaSHV, or
blaCTX genes resulting in alterations of the primary aminoacid
sequence of the enzyme (7). Since these genes are generally
found on plasmids, many of the organisms that harbor ESBLs
also are resistant to other classes of antibiotics, such as aminoglycosides, fluoroquinolones, tetracyclines, chloramphenicol, andsulfonamides (8). Many strains are multiresistant, being sensitive
only to imipenem (9).
There is a considerable geographical difference in theoccurrence of ESBLs in the European countries. In a SENTRY
worldwide surveillance program report, ESBL phenotypes were
Fiziologia - Physiology  2013.23.2 (78)

detected in 1-8% of E. coli strains (8).
In the United States, multidrug resistance (≥3 antimicrobial
drug classes) in E. coli increased from 7.2% during the 1950s to
63.6% during the 2000s (10).
The greatest resistance increase, explored in US outpatients, occured from 2000 to 2010, for ciprofloxacin (3% to 17.1%),
trimethoprim-sulfamethoxazole (17.9% to 24.2%), nitrofurantoin(0.8% to 1.6%) and ceftriaxone (0.2% to 2.3%) (11).
The proportion of E. coli isolates resistant to ciprofloxacin
also increased significantly between 2007 (20.0%) and 2011(29.2%), in the Canadian hospitals (12).
In 2007, the EARSS (European Antimicrobial Resistance
Surveillance System) reported 2.5% E. coli strains resistant to 4classes of antibiotics. Also, in 2011, EARSS reported an increasein the number of XDR (extensively drug-resistant) E. coli strains– resistant to all antibiotics, except carbapenems (13).
Of the 109 E. coli strains, isolated by us, 31 (28.44%)
presented a high level of resistance to quinolones, 41 (37.61%)
presented resistance to trimethoprim-sulfamethoxazole, 2(1.83%) were resistant to nitrofurantoin and 3 (2.75%) wereresistant to tetracycline. We did not isolate any strain withimipenem resistance.
CONCLUSIONS
The most frequently isolated germ in the urine specimensof diabetic patients was E. coli. 30.27% of our total E. coli
strains were included in the wild phenotype, while 47.71% were
included in the high level penicillinase producing phenotype. The
highest percentage of antimicrobial resistance was recorded in
β-lactam antibiotics (ampicillin – 68.81%, amoxicillin/clavulanic
acid – 52.30%, piperacillin 36.70% and cefazolin 39.44%), fluoroquinolones (ciprofloxacin – 23.85%, norfloxacin – 28.44%)
and trimethoprim/sulfamethoxazole (37.61%). Fortunately, onlya small percentage of ESBL producing E.coli isolates havebeen recorded.

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13. European Centre for Disease Prevention and Control: Annual
epidemiological report 2011. Reporting on 2009 surveillance data and2010 epidemic intelligence data. Stockholm: ECDC; 2011(http://www.
ecdc.europa.eu/en/publications/Publications/1111_SUR_Annual_Epidemiological_Report_on_Communicable_Diseases_in_Europe.pdf)

IDENTIFICAREA FENOTIPURILOR DE REZISTENTA LA ANTIBIOTICEA TULPINILOR DE ESCHERICHIA COLI IZOLATE DIN PROBELE DE
URINA ALE PACIENTILOR DIABETICIREZUMAT

Obiective: Scopul acestui studiu este de a determina rezistenta la preparate antimicrobiene a tulpinilor de Escherichia
coli implicate in etiologia infectiilor de tract urinar la pacientii diabetici internati intr-o sectie de diabet, in perioada ianuarie - decembrie 2012. Material si metoda: In studiu au fost incluse un numar de 109 tulpini de Escherichia coli izolate
din 570 uroculturi, recoltate de la 1470 pacienti. Identificarea germenilor s-a realizat pe baza caracterelor culturale sibiochimice ale germenilor. Identificarea finala si testele de sensibilitate au fost realizate cu ajutorul analizorului Vitek 2(Bio Merieux France). Rezultate: Din cele 570 uroculturi, 158 probe (27.71%) au fost pozitive. Dintre ele, 109 tulpini au
fost cele de Escherichia coli. Incadrarea in fenotipuri de rezistenta a evidentiat un procent scazut de tulpini secretoarede BLSE. Concluzii: Germenul cel mai frecvent izolat din totalul uroculturilor a fost E. coli. Un procent de 30.27 % au
fost incadrate in fenotipul salbatic, in timp ce 41.47% au apartinut fenotipului producator de penicilinaza de nivel crescut.
Cel mai mare procent de rezistenta s-a evidentiat la β-lactamine, fluoroquinolone si trimethoprim/sulfamethoxazole. Din
fericire, procentul tulpinilor multirezistente de tip BLSE a fost scazut.
Cuvinte cheie: infectii de tract urinar, E. coli, rezistenta la antibiotice, diabet zaharat
2013.23.2 (78)  Fiziologia - Physiology

25

TECHNIQUES FOR ENDOTRACHEAL INTUBATION IN RATSDANIELA CHIRU1, NARCIS ŢEPENEU2, CAIUS BURIAN3, ADRIAN CRACIUN1, ILIE CONSTANTIN4,MIRABELA DIMA4, VALENTIN ORDODI3
1
Department of Pediatrics, “Victor Babeş” University of Medicine and Pharmacy, Timişoara
2
Department of Pediatric Surgery, “Victor Babeş” University of Medicine and Pharmacy, Timişoara
3
Department of Physiology, “Victor Babeş” University of Medicine and Pharmacy, Timişoara
4
Department of Neonaltology, “Victor Babeş” University of Medicine and Pharmacy, Timişoara

ABSTRACT

Tracheal intubation is often necessary for experiments involving anesthetized animals. In rats, endotracheal intubation is challenging
and more difficult due to the small size of anatomical landmarks and also due to no standard intubation equipment available. This
review aims to highlight the techniques available for endotracheal intubation in rats.
Key words: endotracheal intubation, rat, intubation techniques

INTRODUCTION
Airways access is important for ventilation, administration of
inhaled anesthetics or instillation of drugs, and maintenance of
airway patency is often necessary for survival experiments. On
most medium sized experimental animals, endotracheal intubation can easily be performed under general anesthesia using
a pediatric laryngoscope and small commercial endotracheal
tubes. Instead, on small animals, such as rodents, in which
oropharyngeal access is limited, endotracheal intubation is challenging. In rats, endotracheal intubation is more difficult due to thesmall size of anatomical landmarks (e.g. small palate, larynx andepiglottis). No visualization of epiglottis and vocal cords results
in repeated intubation attempts which can cause trauma to theoral cavity and death of the animal due to respiratory failure.
Several methods are available for intubation of the rat;
however, not all techniques are easy to perform. In addition, no
standard intubation equipment is available for rats.
In rats, tracheal intubation can be achieved with speciallydesigned tubes for this purpose or with an intravenous plastic
catheter for human use. Most commonly used for this purpose is14- or 16-gauge catheter, with length from 45 to 60 mm. Unlike
metal tubes, these catheters have the advantage of less injury
of the upper airways. A modified Foley catheter having an inflatable cuff near the tip to minimize the leak can be also used as a
tracheal tube (1). From vocal cords to bifurcation, the trachea inrats weighing 300–530 g is 3.58 mm on average (range 3.0-4.5mm) (2). The catheter can be secured to the maxilla with adhesive
tape or can be left free, without fixation.
The procedure is performed after the animal is previously
prepared. Inserting the catheter into the trachea requires adequate general anesthesia of the rat and local anesthesia of
tongue, epiglottis and vocal cords with a solution of 4% lidocaine.
Local anesthesia is not mandatory; some authors perform intubation without it (3,4). Others found that local anesthesia greatly
reduces the difficulty of intubation by reducing glottis spasm if
general anesthesia is more superficial (5,6).
Several techniques have been developed for endotracheal
intubation in rats:- Blind intubation technique;- Transillumination of the neck;- Direct illumination of the trachea;- Direct laryngoscopy using an otoscope for human use;
- Direct laryngoscopy using a laryngoscope specially designed for rats; and- Video-assisted endotracheal intubation.
Blind intubation technique
Stark et al. (7) presented a technique for blind oral tracheal intubation using a modified 16-gauge intravenous catheter
adapted to the anatomy of the rat’s upper airways. This method
can be performed much more rapidly than conventional methods,
which requires considerable time and skill. The disadvantages forthis method are that several attempts were necessary to place
the tube correctly. Cheong et al. (8) used a blind placement of a
16-gauge intravenous catheter and a modified 18-gauge epidural
needle, and the correct placement of the catheter was verified
using a rodent ventilator. By this method the authors tried toreduce the number of intubation attempts.
Transillumination of the neck
Tracheal intubation using percutaneous transillumination
technique is relatively simple, and requires a strong external lightsource that penetrates the skin to illuminate the larynx (4,9-13).
The light source is placed oblique from caudal to cranial. After immobilization of the upper incisors and tongue retraction, epiglottis
and glottis are visualized as a bright red spot. Brown et al. (4) used
a metal laryngoscope to lift the lower jaw of the mouse and keep
the mouth open. The catheter is inserted into the trachea after
cleaning pharyngeal secretions with a cotton swab (Figure 1).

Fig. 1. Transillumination technique (5)Received 12th of February 2013. Accepted 5th of March 2013. Address for correspondence: Narcis Tepeneu, MD, Emergency Hospital for Children “Louis Ţurcanu”, Iosif
Nemoianu Street, No. 2, Timişoara, Romania; phone/fax: +40256220479; e-mail: nftepeneu@yahoo.com

26

Fiziologia - Physiology  2013.23.2 (78)Direct illumination of the trachea
This method can be performed using a fiber-optic light guidefor illumination (14,15) or a surgical operating microscope formagnification and illumination in order to facilitate the procedure(6,16). Jou et al. (2) used an intubation wedge made from an
obliquely sectioned standard 3-ml plastic syringe and a ordinary
lamp for external illumination to facilitate the introduction of thecatheter into the trachea under direct vision.
Direct laryngoscopy using an otoscope for human use
Tracheal intubation using an otoscope allows direct visualization of the glottis (17), but is more laborious. It involves theexecution of several maneuvers and so the time needed for
intubation increase. This fact can have negative consequencesin an animal who is insufficient preoxygenated (5).
The otoscope is introduced into the oropharynx and by direct
vision, a guide wire is inserted into the trachea and a 16-gauge
catheter is glided over the wire. After that, the guide is removed
and the rat is connected to the rodent ventilatory. Kastl et al.
(18) uses a special positioning of the rat in dorsal recumbency
and a semisuspended position on an inclined metal plate. This
makes the access to the upper airways easier and can facilitate
the technique (Figure 2).

Fig. 2. Direct laryngoscopy with an otoscope (18)

Direct laryngoscopy using a laryngoscope specially designed for rats
This method for intubation in rats allows direct visualization of the glottis and intubation in one time. The technique inrats is the same like in adults. Several devices for this purpose
were invented. Direct laryngoscopy with a head-mounted lightsource was proposed by Alpert et al. (19). A laryngoscope,
fashioned from a weighing spatula and a glassfibre cable whichis connected to a cold light source, was used by Boersma etal. (20). The Schaefer’s et al. (21) intubation method requires
no special manufacture of equipment, because it employs thehuman laryngoscope equipped with an infant blade (size 0). In
1986, a fiberoptic laryngoscope has been developed by Costa
et al. (22). A rat laryngoscope was also invented by Linden et al.
(23) and Molthen et al. (24). Mothen fashioned a light-carrying
laryngoscopic blade (laryngoscope) from readily available acrylicpolymethyl methacrylate tubing.
Ordodi et al. (25) developed a simple device for intubation ofrats, consisting of the barrel of a standard 2-ml syringe welded toa 6-cm metal spout, and inside the barrel of the syringe, is a lightbulb from a flashlight (Figure 3). The intubation can be perform
rapidly and relatively simple. The anesthetized rat, positioned in
dorsal recumbency, has its head held in position by a flexible plastic
tube hooked around the upper incisors. The tong is put aside, after
cleaning the oral secretion with a cotton swab. The laryngoscope
blade is inserted, and after the visualization of the glottis, the catheter is inserted thru the metal spout into the trachea (4).
2013.23.2 (78)  Fiziologia - Physiology

Fig. 3. Direct laryngoscopy using a rat-laryngoscope (25)Video-assisted endotracheal intubation
This technique was proposed for small animals intubation by
Vergardi et al. (26), Clary et al. (27) and Fuentes et al. (2), using
equipment commonly found in the endosurgical research setting.
This method of direct vision of the trachea uses either a straight,
small arthroscope (26,27), or a rigid endoscope (2), connected to alight source and a videocamera. Videocamera is carefully inserted
to the left of the incisors and inserted at the base of the tongue toallow visualization of the glottis and the rhythmic opening of the
vocal cords (Figure 4). Secretions were removed by a cannula connected to a syringe (26) or using a cotton-tip applicator (27). Under
direct videoendoscopic guidance, the catheter is inserted into thetrachea directly (26) (Figure 5) or using a guide represented by astiff wire (27) or a blunted and bent metal needle (2). The catheter
is advanced over the guide, and then the guide is removed andthe rat is connected to the rodent ventilator.

Fig. 4. The rat’s larynx on videointubation (26)Fig. 5. Correct position of the endotracheal catheter (white arrow)(27); C - endotracheal catheter, E - epiglottis, P - soft palate

27

DISCUSSION
A variety of methods have been described for endotracheal
intubation of the rat. In the majority of studies intubation techniques are very complicated or require expensive equipment.
Blind intubation sometimes requires 3 or 4 attempts to position
the endotracheal tube correctly in the trachea and may lead tocomplications or death to the animal. Other non-invasive rat
tracheal intubations under direct view often require elaborated
animal position and special equipment including a laryngoscope,
an otoscope with an incorporated light source, a surgical microscope, a head-mounted mirrorreflected adjustable-focus light,
an endoscope, or some other special devices. However, oneof these devices may be not available in every laboratory. The
laryngeal opening in rats is very small, measuring 1.5 mm indiameter in 200 g specimens (7), and therefore visualization withan untrained eye can be difficult, even with proper illumination.
Clearly, the intubation of the trachea under direct vision remains
the standard for a safe intubation in rats.
CONCLUSIONSTechniques for endotracheal intubation in rats vary according to the experience of authors and to possibilities of laboratory
equipment. With the exception of laryngoscopes especiallydesigns for rat intubation, all other techniques uses equipmentfrom human use, adapted to the small size of the animal. The
fact that so many different methods for rodent intubation havebeen described probably indicates that there is no ideal method.
ACKNOWLEDGEMENT
This paper is supported by the Sectorial Operational
Programme Human Resources Development (SOP HRD)
2007-2013, financed from the European Social Fund andby the Romanian Government under the contract number
POSDRU/107/1.5/S/82839.
REFERENCES
1. Fox MS, Welch I, Hobson D, Santyr GE. A novel intubation technique for minimally invasive longitudinal studies of rat lungs using
hyperpolarized 3He magnetic resonance imaging. Lab Anim. 2012
Oct; 46(4): 311-7.
2. Jou IM, Tsai YT, Tsai CL, Wu MH, Chang HY, Wang NS. Simplified
rat intubation using a new oropharyngeal intubation wedge. J. Appl.
Physiol. 2000 Nov; 89(5): 1766-70.
3. Fuentes JM, Hanly EJ, Bachman SL, Alexander AR, Marohn MR,
Talamini MA. Videoendoscopic endotracheal intubation in the rat: A
comprehensive rodent model of laparoscopic surgery. Journal of Surgical Research 2004; 122(2), 240-8.
4. Brown RH, Walters DM, Greenberg RS, Mitzer W. A method for endotracheal intubation and pulmonary functional assessment for repeatedstudies in mice. J Appl Physiol 1999; 87: 2362-5.
5. Ordodi VL. Implicaţii ale ingineriei biomedicale în medicina
experimentală. PhD Thesis 2006.
6. Kramer K, Grimbergen JA, van Iperen DJ, van Altena K, Voss HP,
Bast A. Oral endotracheal intubation of guineapigs. Laboratory Animals
1998; 32: 162-4.
7. Stark RA, Nahrwold ML, Cohen PJ. Blind oral tracheal intubation ofrats. J Appl Physiol. 1981 Nov; 51(5): 1355-6.
8. Cheong SH, Lee KM, Yang YI, Seo JY, Choi MY, Yoon YC. Blindoral endotracheal intubation of rats using a ventilator to verify correct
placement. Lab Anim. 2010 Jul; 44(3): 278-80.
9. Yasaki S, Dyck PJ. A simple method for rat endotracheal intubation.
Lab Anim Sci. 1991 Dec; 41(6): 620-2.

28

10. Cambron H, Latulippe JF, Nguyen T, Cartier R. Orotracheal intubation of rats by transillumination. Lab. Anim. Sci. 1995; 45(3): 303-4.
11. Lizio R, Westhof A, Lehr CM, Klenner T. Oral endotracheal intubationof rats for intratracheal instillation and aerosol drug delivery. Lab Anim.
2001 Jul; 35(3): 257-60.
12. Rivard AL, Simura KJ, Mohammed S, Magembe AJ, Pearson HM,
Hallman MR, et al. Rat intubation and ventilation for surgical research.
J Invest Surg. 2006 Jul-Aug;19(4): 267-74.
13. Na N, Zhao DQ, Huang ZY, Hong LQ. An improved method for rat
intubation and thymectomy. Chin Med J (Engl). 2011 Sep; 124(17):2723-7.
14. Thet LA. A simple method of intubating rats under direct vision. Lab.
Anim. Sci. 1983; 33(4): 368-9.
15. Rivera B, Miller S, Brown E, Price R. A novel method for endotracheal
intubation of mice and rats used in imaging studies. Contemp Top Lab
Anim Sci 2005; 44: 52-55.
16. Pena H, Cabrera C. Improved endotracheal intubation technique inthe rat. Lab Anim Sci. 1980 Aug; 30(4 Pt 1): 712-3.
17. Weksler B, Ng B, Lenert J, Burt M. A simplified method for endotracheal intubation in the rat. J. Appl. Physiol. 1994; 76(4): 1823-5.
18. Kastl S, Kotschenreuther U, Hille B, Schmidt J, Gepp H, Hohenberger W. Simplification of rat intubation on inclined metal plate. Adv
Physiol Educ. 2004 Dec; 28(1-4): 29-32.
19. Alpert M, Goldstein D, Triner L. Technique of endotracheal intubationin rats. Lab. Anim. Sci. 1982; 32(1): 78-9.
20. Boersma FP, Wieringa RA. Rat intubation. A method for safe andfast intubation of the rat trachea under direct vision. Anaesthesist. 1982
May; 31(5): 239-40.
21. Schaefer CF, Brackett DJ, Downs P, Tompkins P, Wilson MF. Laryngoscopic endotracheal intubation of rats for inhalation anesthesia.
J Appl Physiol. 1984 Feb; 56(2): 533-5.
22. Costa DL, Lehmann JR, Harold WM, Drew RT. Transoral tracheal
intubation of rodents using a fiberoptic laryngoscope. Lab Anim Sci.
1986 Jun; 36(3): 256-61.
23. Linden RD, Shields CB, Zhang YP, Edmonds HL, Hunt MA. A laryngoscope designed for intubation of the rat. Contemp Top Lab Anim
Sci. 2000 Mar; 39(2): 40-2.
24. Molthen RC. A simple, inexpensive, and effective light- carrying
laryngoscopic blade for orotracheal intubation of rats. J Am Assoc Lab
Anim Sci. 2006 Jan; 45(1): 88-93.
25. Ordodi VL, Mic FA, Mic AA, Sandesc D, Paunescu V. A simple device
for intubation of rats. Lab Anim (NY). 2005 Sep; 34(8): 37-9.
26. Vergari A, Polito A, Musumeci M, Palazzesi S, Marano G. Videoassisted orotracheal intubation in mice. Lab Anim 2003; 37: 204-7.
27. Clary EM, O’Halloran EK, de la Fuente SG, Eubanks S. Videoendoscopic
endotracheal intubation of the rat. Lab Anim. 2004 Apr; 38(2): 158-61.

Fiziologia - Physiology  2013.23.2 (78)

TEHNICI UTILIZATE PENTRU INTUBAŢIA ENDOTRAHEALĂ LA ŞOBOLAN
REZUMATIntubaţia traheală este deseori necesară pentru experimente pe animale anesteziate. La şobolani, intubaţia endotraheală este
provocatoare şi totodată dificilă datorită pe de o parte a mărimii mici a reperelor anatomice, iar pe de altă parte lipsei de standardizarea echipamentului disponibil pentru intubaţie. Acest review işi propune să evidenţieze tehnicile disponibile de intubaţie endotraheală
la şobolani.
Cuvinte cheie: intubaţie endotraheală, şobolan, tehnici de intubaţie

2013.23.2 (78)  Fiziologia - Physiology

29

CORRELATION BETWEEN PULSE WAVE VELOCITY
AND INFLAMMATION MARKERS IN PATIENTS WITHCARDIOVASCULAR RISK FACTORSIOANA SUCEAVA, DANIEL LIGHEZAN, CORINA SERBAN, ANCA TUDOR, ROXANA BUZAS,SIMONA DRAGAN
University of Medicine and Pharmacy “Victor Babes”, Timisoara, Romania

ABSTRACTIn recent years, arterial stiffness expressed by pulse wave velocity and circulating inflammatory markers such as fibrinogen and highsensitive C-reactive protein (hsCRP) were recognized as risk factors for cardiovascular disease (CVD).The purpose of this study
was to evaluate the association between pulse wave velocity and inflammatory markers in patients with cardiovascular risk factors.
The study included 223 patients with at least 2 risk factors for atherosclerosis, divided considering the presence of coronary arterydisease (CAD): 140 patients with angiographically confirmed CAD and 83 patients without CAD. The patients were compared with acontrol group of 74 healthy age subjects (CON). The following parameters were determined: blood pressure, total cholesterol (TC),triglycerides (TG), LDL-cholesterol, HDL-cholesterol, fasting serum glucose, fibrinogen and hsCRP. Arterial stiffness was quantified
by pulse wave velocity (PWV), using Arteriograph device (Tensiomed Ltd., Budapest, Hungary). CAD patients had significantly
increased values of inflammatory markers as compared with patients without CAD and controls (all p<0.001). The patients with CAD
had significant increased values of PWV compared with patients without CAD and CON subjects (12.5±0.7 vs 10.9±0.6 vs 8.5±0.07
m/s, all p<0.001). A significant medium correlation was found between PWV and fibrinogen (r=0.641, p<0.001) and between PWV
and hsCRP in all subjects (r= 0.551, p<0.001). In this study inflammatory markers were significantly correlated with PVW, therefore,
inflammation may influence arterial stiffness in patients with cardiovascular risk factors.
Keywords: pulse wave velocity, hsCRP, fibrinogen

INTRODUCTION
Recent investigations of atherosclerosis have focused onarterial stiffness and inflammation, providing new insight intomechanisms of disease (1). An increase in peripheral vascular
resistance associated with increased stiffness of central elastic
arteries represents hallmarks of this ageing effect on the vasculature, referred to as early vascular ageing (2). Many cardiovascular risk factors such as age, smoking, arterial hypertension,
diabetes mellitus, end stage renal disease are associated withincreased arterial stiffness (3, 4). Besides these traditional risk
factors, chronic inflammation of the cardiovascular system may
also affect PWV (5). High-sensitivity C reactive protein (hsCRP)
seems to have an important relation to the risk of cardiovasculardiseases in a variety of clinical settings including healthy subjects
and patients with cardiovascular diseases (6).
Furthermore, the Hisayama Study suggested that brachialankle pulse wave velocity is a significant predictive factor for
cardiovascular disease in the general Japanese population andsubstantially improves cardiovascular risk assessment, being
included within the framework of large-scale clinical studies (7, 8).
Its application as a routine tool for clinical patient evaluation has
been hampered until now by the absence of reference values (8).
The aim of this study was to evaluate the association between pulse wave velocity and inflammatory markers in patientswith cardiovascular risk factors.

MATERIAL AND METHODS
The study included 223 patients with at least 2 risk factors for atherosclerosis, divided considering the presence ofcoronary artery disease (CAD): 140 patients with angiographically confirmed CAD and 83 patients without CAD. The patientswere compared with a control group of 74 healthy age subjects(CON). We recorded age, sex, smoking habits, hypertension,
atherogenic dyslipidemia, and diabetes mellitus as cardiovascular risk factors. We excluded from the study the patients with
unavailable data for fibrinogen, hsCRP and PWV measurement
and the patients with identified genetic cause of hypertension orsecondary hypertension.
Clinical evaluation included blood pressure measurement,
physical examination, chest radiograph, 12-lead electrocardiogram. While on their usual diet, a venous blood sample wasdrawn from an antecubital vein in all subjects after an overnight
fast to determine TC, TG and using standard enzymatic method
on a COBAS Integra 400 plus analyzer.
Hypertension was diagnosed according to 2013 European
Guidelines for the Management of Arterial Hypertension (9).
Diabetes was defined by the current intake of oral hypoglycemictreatment or use of insulin.
Assessment of arterial stiffness
Measurement of arterial stiffness indices was done earlyReceived May 1st 2013. Accepted June 2nd 2013. Address for correspondence: Corina Serban, MD, PhD, Department of Functional Sciences, University of Medicine
and Pharmacy “Victor Babes” Timisoara, Eftimie Murgu Square No. 2A, RO-3000041, Timisoara, phone/fax: +40256220479; e-mail: dr.corinaserban@yahoo.com

30

Fiziologia - Physiology  2013.23.2 (78)in the morning 1 day before coronary angiography with patients
fasted and before the administration of the medications. The
assessment of arterial stiffness was noninvasively performed
with the Arteriograph (Tensiomed Ltd., Budapest, Hungary) by anoperator who was blinded to the results of coronary angiography
and other findings. All measurements were taken in the supine
position in a quiet, temperature-controlled room (approximately
22oC) after a brief period of rest in the morning.
Informed consent was obtained for each man who agreed
to participate in our study. The study protocol was approved by
the local ethics committee. The study was conducted according
to the Declaration of Helsinki, and the written informed consent
was obtained from each subject.
Statistical analysis
Database and processing were performed using statistical software SPSS Statistical Software Package, version
15.0 (SPSS Inc, Chicago, Illinois, USA). Continuous data arepresented as mean ± standard deviation for continuous variables
and number of subjects (n) and percentage (%), respectively, forcategorical variables. The independent sample t test was used
to reveal significant differences in parameters between patientswith and without CAD and controls. The statistically analysis
was carried out with Pearson’s test for correlation. p < 0.05 wasconsidered statistically significant.

Serum creatinine
0.9±0.3(mg/dL)
Blood urea nitro37±11.4
gen (mg/dl)
Uric acid levels
6.3±1.8(mg/dl)Fibrinogen (mg/dl) 513.2±110.3

0.8±0.1

0.7±0.08<0.00138.3±6.930.1±70.001

5.7±1.7

4.7±1.1<0.001357.6±57.2

301.8±21.5

hsCRP (mg/l)<0.001

3.6±1.1

3.2±1.4

1.06±1.2

PVW (m/s)<0.00112.5±0.7

10.9±0.6

8.5±0.07<0.001

SBP: systolic blood pressure, DBP: diastolic blood pressure, PVW:
pulse wave velocity; high sensitive C-reactive protein: hsCRP

Table I shows that the parameters of lipid profile (TC, TG,
LDL-C and HDL-C), fibrinogen and hsCRP were significantly
higher in patients than in controls (all p < 0.001). Mean levelsof fibrinogen in serum were significantly higher in CAD patients
than in those without CAD and than in CON group (513.2 ±110.3 mg/dl versus 357.6 ± 57.2 mg/dl versus 301.8 ± 21.5,
all p < 0.001).
The patients with CAD had significantly increased PWVvalues compared with patients without CAD ( p < 0.001) andCON group (p < 0.001) (Figure 1).
The correlation between PWV and fibrinogen was moderateand significant and is showed in Figure 2 (r = 0.641, p < 0.001).

RESULTSThe baseline characteristics of the subjects, the meanvalues of lipid profile parameters, renal parameters (serum
creatinine, blood urea nitrogen, uric acid levels) inflammatory
markers (fibrinogen and hsCRP) and PVW are summarized inTable I. No significant differences were determined between
groups referring gender, and smoking habits.
Table I. Characteristics of the studied group (mean ± SD values)
Parameters

CAD group(n=140)Age60.5±10.8

Without
CAD group(n=83)
56.63±8.9

CON(n=74)

pvalue55.8±5.2<0.001

Gender females/
males (%)
Family history for
CAD (%)
Smoking (%)

51.4/48.6

51.8/48.2

54.1/45.9

0.93

42.9

67.5

44.6

0.001

37.1

47

35.1

0.23

SBP (mmHg)

154.9±20.9123.7± 6.3<0.001

DBP (mmHg)

91.1±12.3

157.8 ±
13.5
93.67±7.4

72.6±5.3<0.001

Glucose (mg/dL)

117.1±4498.7±12.1

90.9±8.7<0.001

Total cholesterol(mg/dL)
Triglycerides (mg/dL)
LDL-C (mg/dL)

262±39.5

268.1±35.8 181.27±13.044 <0.001

168.3±79.2

147.3±49.6

111.9±27.8<0.001145±26126.3±23.8

115.6±18.5<0.001

HDL - C (mg/dL)

35.9±4.3

40.5±7.5

46.7±6.4<0.001

2013.23.2 (78)  Fiziologia - Physiology

Fig.1. The PVW values in three studied groups

Fig.2. Correlation between PVW and fibrinogen31

It was found a correlation moderate and significant between
PWV and hsCRP (r=0.551, p<0.001) (Figure 3).

of its simplicity, reproducibility, and inexpensiveness. PVW andassociated inflammatory markers may be of clinical use for early
diagnosis of subclinical atherosclerosis, in the development ofmultimarker strategies to reduce the rate of subsequent cardiovascular events.
CONCLUSIONSIn this study inflammatory markers were significantly correlated with PVW, therefore, inflammation may influence arterial
stiffness in patients with cardiovascular risk factors. PVW andassociated inflammatory markers may be of clinical use for early
diagnosis of subclinical atherosclerosis, in the development ofmultimarker strategies to reduce the rate of subsequent cardiovascular events.

Fig.3. Correlation between PVW and hsCRP

DISCUSSION
It seems that, arterial stiffness measured by carotid-femoral
PWV may reflect the sclerotic rather than atherotic componentof atherosclerosis and it is more closely correlated to age, bloodpressure, plasma glucose and arterial thickness (10, 11). However, findings with regard to its relation with other risk factors
have been inconsistent.
This prospective observational study could reveal risk associations but could not establish causal relationships or explore
potential mechanisms. Similar with the results of others studies(12, 13), in this study was found that PVW was significantly
associated with serum levels of hsCRP and fibrinogen, suggesting that inflammation is involved in arterial stiffening. On
the contrary, a recent study showed that inflammation does not
influence arterial stiffness and pulse-wave velocity in patientswith coronary artery disease (14). Another study observed thattumor necrosis factor-α and C-reactive protein appear to be critical inflammatory cytokines associated with reductions in arterial
elasticity in older women (15). Studies have had conflicting result
until now because techniques based on the pulse wave lack
precision, assume that arterial stiffness is uniform throughout
the path of the pulse wave, and that it is constant throughout
the cardiac cycle (16).
A study that explored the association between PWV and theextent of angiographically proved CAD in patients with angina
and without any previous history of heart disease concluded thatPWV is an independent predictor of significant CAD, but wasneither associated significantly with the extent of CAD nor withthe risk of revascularization (17). In a recent study, a higher dairy
food intake was associated with lower PWV and accompanying
reductions of pulse pressure and SBP (18).
Early diagnosis and prevention of CAD have stimulated asearch for reliable noninvasive methods through detection ofchanges in the endothelium by determining PWV and inflammatory markers as hsCRP and fibrinogen (19). PWV has been
becoming a widely adopted index of arterial stiffness because
32

Competing interests
The authors declare that they have no competing interests.
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2. Nilsson PM, Khalili P, Franklin SS. Blood pressure and pulse wave
velocity as metrics for evaluating pathologic ageing of the cardiovascularsystem. Blood Pressure. 2013. Epub 2013/06/12.
3. Laugesen E, Rossen NB, Hoyem P, Christiansen JS, Knudsen ST,
Hansen KW, et al. Reproducibility of pulse wave analysis and pulse
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Clinical and Laboratory Investigation. 2013. Epub 2013/06/20.
4. Aatola H, Hutri-Kahonen N, Juonala M, Viikari JS, Hulkkonen J,
Laitinen T, et al. Lifetime risk factors and arterial pulse wave velocity inadulthood: the cardiovascular risk in young Finns study. Hypertension.
2010; 55(3): 806-11.
5. Nagano M, Nakamura M, Sato K, Tanaka F, Segawa T, Hiramori K.
Association between serum C-reactive protein levels and pulse wave
velocity: a population-based cross-sectional study in a general population. Atherosclerosis. 2005; 180(1): 189-95.
6. Serban C, Dragan S. The Relationship between Inflammatory andOxidative Stress Biomarkers, Atherosclerosis and Rheumatic Diseases.
Current Pharmaceutical Design. 2013. Epub 2013/04/10.
7. Ninomiya T, Kojima I, Doi Y, Fukuhara M, Hirakawa Y, Hata J, et al.
Brachial-ankle pulse wave velocity predicts the development of cardiovascular disease in a general Japanese population: the Hisayama
Study. Journal of Hypertension. 2013;31(3):477-83.
8. Determinants of pulse wave velocity in healthy people and in the presence of cardiovascular risk factors: ‘establishing normal and reference
values’. European Heart Journal. 2010;31(19):2338-50.
9. Mancia G, Fagard R, Narkiewicz K, Redon J, Zanchetti A, Bohm M, etal. 2013 ESH/ESC Guidelines for the management of arterial hypertension: The Task Force for the management of arterial hypertension of theEuropean Society of Hypertension (ESH) and of the European Societyof Cardiology (ESC). European Heart Journal. 2013;34(28):2159-219.
10. Zhang Y, Agnoletti D, Protogerou AD, Topouchian J, Wang JG, Xu Y,
et al. Characteristics of pulse wave velocity in elastic and muscular arteries: a mismatch beyond age. Journal of Hypertension. 2013;31(3):554-9;

Fiziologia - Physiology  2013.23.2 (78)11. Cecelja M, Chowienczyk P. Dissociation of aortic pulse wave velocity
with risk factors for cardiovascular disease other than hypertension: asystematic review. Hypertension. 2009;54(6):1328-36.
12. Andoh N, Minami J, Ishimitsu T, Ohrui M, Matsuoka H. Relationshipbetween markers of inflammation and brachial-ankle pulse wave velocity in Japanese men. International Heart Journal. 2006;47(3):409-20.
13. Schnabel R, Larson MG, Dupuis J, Lunetta KL, Lipinska I, Meigs
JB, et al. Relations of inflammatory biomarkers and common genetic variants with arterial stiffness and wave reflection. Hypertension.
2008;51(6):1651-7.
14. Blann AD, Kuzniatsova N, Lip GY. Inflammation does not influence
arterial stiffness and pulse-wave velocity in patients with coronary arterydisease. Journal of Human Hypertension. 2013. Epub 2013/03/29.
15. Fisher G, Hunter GR, Glasser SP. Associations between arterial
elasticity and markers of inflammation in healthy older women. The

Journals of Gerontology Series A, Biological Sciences and Medical
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16. Messas E, Pernot M, Couade M. Arterial wall elasticity: state of
the art and future prospects. Diagnostic and Interventional Imaging.
2013;94(5):561-9.
17. Chae MJ, Jung IH, Jang DH, Lee SY, Hyun JY, Jung JH, et al. The
Brachial Ankle Pulse Wave Velocity is Associated with the Presence ofSignificant Coronary Artery Disease but Not the Extent. Korean Circulation Journal. 2013;43(4):239-45.
18. Crichton GE, Elias MF, Dore GA, Abhayaratna WP, Robbins MA.
Relations between dairy food intake and arterial stiffness: pulse wave
velocity and pulse pressure. Hypertension. 2012;59(5):1044-51.
19. Alarhabi AY, Mohamed MS, Ibrahim S, Hun TM, Musa KI, Yusof Z.
Pulse wave velocity as a marker of severity of coronary artery disease.
J Clin Hypertens (Greenwich). 2009;11(1):17-21.

CORELAŢIA DINTRE VITEZA DE PROPAGARE A UNDEI DE PULSŞI MARKERII INFLAMATORI LA PACIENŢI CU FACTORI DE RISC
CARDIOVASCULARREZUMATÎn ultimii ani, rigiditatea arterială exprimată prin viteza de propagare a undei pulsului şi markerii inflamatori precum fibrinogenul şi proteina
C reactivă înalt sensibilă (hsCRP) sunt recunoscute ca factori de risc pentru bolile cardiovasculare (BCV). Scopul acestui studiu a fostevaluarea relaţiei dintre viteza de propagare a undei de puls şi markerii inflamtori la pacienţi cu factori de risc cardiovasculari. Studiul
a inclus 223 de pacienţi cu cel puţin 2 factori de risc CV pentru ateroscleroză, împărţiţi în funcţie de prezenţa bolii coronariene: 140de pacienţi cu boală coronariană confirmată angiografic şi 83 de pacienţi fără boală coronariană. Pacienţii au fost comparaţi cu ungrup de control (CON) de 74 de subiecţi sănătoşi. Au fost determinaţi următorii parametri: tensiunea arterială, colesterolul total (CT),trigliceridele (TG), LDL-colesterolul, HDL-colesterolul, glicemia serică, fibrinogenul şi hsCRP. Rigiditatea arterială a fost cunatificată
prin viteza de propagare a undei de puls (PWV) folsind aparatul Arteriograf (Tensiomed Ltd., Budapest, Hungary). Pacineţii cu boală
coronariană au avut valori semnificativ crescute ale markerilor inflamatori comparativ cu pacienţii fără boală coronariană şi grupul
de control (toate p<0.001). The patients with CAD had significant increased values of PWV compared with patients without CAD
and CON subjects (12.5±0.7 vs 10.9±0.6 vs 8.5±0.07 m/s, all p<0.001). A fost obţinută o corelaţie medie semnificativă între PWV şi
fibrinogen (r= 0.641, p<0.001) şi între PWV şi hsCRP la toţi subiecţii (r= 0.551, p<0.001). În acest studiu markerii inflamatori au fostsemnificativ corelaţi cu PVW, aşadar, inflamaţia ar putea influenţa rigiditatea arterial la pacienţii cu factori de risc cardiovasculari.
Cuvinte cheie: viteza de propagare a undei de puls, fibrinogen, hsCRP

2013.23.2 (78)  Fiziologia - Physiology

33

TAURINE EFFECT ON WORKING MEMORY IN HEALTHY
ELDERLY
MONICA – CRISTINA GIURGIU *, ANA CAPISIZU**, LEON ZAGREAN** Physiology II, “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania** Geriatrics – Gerontology Department, “Sf. Luca” Chronics Pathologies Hospital, Bucharest, Romania

ABSTRACT

Demographic aging is relevant for health field objectives. In normal aging occurs a decline in executive processes, as manipulationof information in working memory, coordination of multiple simultaneous operations. In nervous system, taurine acts as neuroprotective agent. To assess the working memory performance we used the Stroop test. Plasma concentration of homovanillic acid havebeen used as an indicator of dopaminergic activity of the central nervous system. We tested two groups, one test and one control,
each consisting of ten human subjects over sixty five years old, female and male. We recorded reaction times and we determinedthe concentration of the homovanillic acid before the administration of taurine, 1500 mg/ day, at one, at two and at three months oftaurine administration. Working memory activation is related with dopamine release in the prefrontal cortex. Age-related decline in
dopamine system and in working memory is observed in humans. Results obtained indicate the decrease of the reaction times andof the interference index and the increase of homovanillic acid plasma concentration after taurine administration.
Keywords: aging brain, Stroop test, working memory, taurine, dopamine, homovanillic acid

INTRODUCTION
Characteristic for our century is the demographic aging
that determined WHO to include aging problem among the five
health problems of the world. WHO showed that the percentage of people with age equal or greater than 65 is expected toincrease from 14% in 2010 to 25% in 2050. Aging is associatedwith structural and cognitive brain changes that limit functional
capacity. The primary cognitive functions most affected by age
are attention and memory. The purpose of this article is to demonstrate taurine effects to improve working memory, function
whose parameters are altered in aging. To assess the working
memory performance we used the Stroop test.
Brain aging and working memory
With aging, the volume of the brain reduces, especiallyin the frontal cortex. It was found that the volume of the brain
decreases with about 5% per decade, after 40 years (1). The
most obvious changes are gyri atrophy, sulci expansion and increase in ventricles volume. At microscopic level, it was observed
loss of neurons in neocortex, hippocampus and cerebellum, adecrease in size and shape of neurons and a reduced cerebral
vascularization. Nimchinsky et al. reported a loss of 50% in
spines on apical dendrites of pyramidal cells in the prefrontal
cortex of old animals (2). Other changes include decreased levelsof serotonin (3), GABA (4) dopamine, somatostatin (5) and of
metabolites of acetylcholine, dopamine and norepinephrine (6).
There are also, intracellular calcium homeostasis disturbed (7),
mitochondrial dysfunction and increased production of reactive

oxygen species (8). Gene transcription is affected (9), kinases
and phosphatases are altered (10) and protein changes occur
such as formation of carbonyl groups (11) covalent modificationof cysteine residues, lysine and histidine, nitration of tyrosine
residues (12, 13) and glycation (14).
Studies with MRI, and PET MRIf showed that the mostimportant structural changes in aging occur in the prefrontal
cortex, where it produces both white and grey matter atrophy(15) and especially in the lateral prefrontal cortex (16). Thereis evidence that cognitive deficits of older adults correspond toneuropsychological profile associated with lateral frontal lesions(17). Executive functions supported by prefrontal cortex (FPC)
systems can be conceived in terms of control and planningmechanisms that mediate and guide goal - directed behavior.
These processes include cognitive functions such as attention,
working memory, cognitive flexibility (e.g. switch between sets
of attention) and making decisions.
Working memory term was proposed by Baddeley (18, 19)
and indicates a space with limited storage capacity, where theinformation is kept active through repetition for 3 up to 30 s andmanipulated to complete task. Ricks and Wiley (20) suggest thatworking memory is focused on complex cognitive tasks including
reasoning, planning, handling and coordination of precepts andactions. Connection between the frontal lobes and manipulating
information in working memory is supported by numerous neuroimaging studies (21) including the discovery that dorso – lateralprefrontal cortex indicate greater activity when working memory
content is rearranged but it is maintained in its original form (22,23). Maintaining the information in front of interference is theReceived 22nd of March 2013. Accepted 16th of May 2013. Address for correspondence: Giurgiu Monica - Cristina, MD, PhD student, Division of Physiology andFundamental Neurosciences, “Carol Davila” University of Medicine and Pharmacy, Bd. Eroii Sanitari, No. 8, Bucharest, RO-050474, phone: +40722527878; e-mail:monicagiurgiu@gmail.com

34

Fiziologia - Physiology  2013.23.2 (78)

critical function of working memory capacity (24, 25, 26). Working
memory also keeps action plans, representations of goals andrelevant information for task in an accessible form, allowing the
choice of a correct response (27).
In normal aging occurs a decline in executive processes,
as inhibition of irrelevant information, coordination of multiple
simultaneous operations, manipulation of information in working
memory and control of episodic memory operations (28). Hasher and Zacks (24) showed that cognitive deficits of elderly
are related to the inability to ignore the irrelevant informationfrom the environment while focusing on information relevant to
the objective. Inhibition deficits occur in tasks that require theselection of the information from the environment or inhibition ofa strong association or a common response. If elderly distinguishwith difficulty the relevant information from irrelevant one, meansthat they store information irrelevant to the task, reducing storage
capacity available for information relevant to the task.
Stroop test
In 1886 Cattell (29) showed it that lasts longer to nameobjects and properties of objects (colors) than to read the corresponding words. The explanation was that reading words is morepracticed than naming images or words and thus he introduced
the concept of automaticity in psychology (30,31). Almost 50
years later, Stroop (32) combined the words and colors, creating a conflict situation and the test that today bears his name.
Classic Stroop test consists of three types of stimuli: neutral (astring of “XXXX” printed in different colors), congruent or without
interference (the meaning of the word and the printing color are
the same, e.g.: yellow written in yellow) and incongruent or withinterference (the meaning of the word and the printing color
are different, e.g.: yellow written in blue). Solving test requires
naming the printing color of the words and ignoring the meaningof those. It is recorded the reaction time (latency), which is thetime from the appearance of the stimulus until the participants’
answer, given verbally or on computer. Stroop interference effectrefers to increase reaction time to interference stimuli compared
to reaction time for neutral stimuli.
PET and MRIf results showed the strongest activated cerebral areas during color – word Stroop test are anterior cingulate
cortex (33, 34) and lateral prefrontal cortex , especially dorso- lateral prefrontal cortex. Anterior cingulate cortex is consideredsusceptible to conflict and dorso - lateral prefrontal cortex isassociated with the implementation of cognitive control (35, 36).
Interference Stroop effect was explained in terms of response to competition. Greater reaction time and greater interference
are present when irrelevant stimulus attributes (word meaning)are analyzed fastest than relevant attributes (color) and thus
undesirable response is therefore available.
Automatic processing can be defined as implicit or intuitive
thinking, that occurs unconsciously and effortlessly, while controlled processing is conscious, intentional and accompanied
by effort. One explanation is that during a lifetime of practice,reading became automated. In theory developed by Cohen etal. (37), evidence accumulates separately for meaning and color
2013.23.2 (78)  Fiziologia - Physiology

so that the total activation received by output units (meaning,color) will determine who will reach first the threshold in orderto answer. Resolving a conflict created by incongruent tests canbe done either by increasing relevant response activation and/or by increasing irrelevant response inhibition (38, 39, 40, 41).
Inhibition of the response is a function of executive control (42).
The advantage of congruent stimuli versus incongruent ones is
explained by the fact that for the first category both attributes(meaning and color) counts for the correct answer.
Interference Stroop effect is greater with age and could be
explained by decreased efficiency of inhibition processes (43) byslowing that occurs with age (44) and by decreasing in processing
resources (45). These changes are based on morphologicalmodifications from anterior cingulate cortex that is involved inconflict monitoring and from the lateral prefrontal cortex (46),which is involved in maintaining the aim.
Taurine – a short overview
Taurine or 2 - aminoetansulfonic acid was first isolated fromox bile in 1827 by Tiedemann and Gmelin (47). It is a nonessential amino acid in rodents, an essential one in cats and conditional
essential one in humans. Taurine is a neutral amino acid, withboth groups, amino and sulfonic, ionized, dissociation of the last
one giving the biological and functional specificity (48). It is oneof the most abundant intracellular free amino acids.
Methionine and cysteine are the precursors of taurine. Its
average daily rate of synthesis is between 0.4 - 1.0 nmol (50125 mg). Taurine biosynthesis involves the sequential oxidationof cysteine to cysteine - sulfinic acid, catalyzed by cysteine
dioxygenase, decarboxylation by the cysteine - sulfinic aciddecarboxylase (CSAD) to hypotaurine and its transformation
into taurine by hypotaurin dehydrogenase. An alternative pathfor taurine synthesis, involves cysteamine production (49).
Taurine is found in neurons and astrocytes (50, 51) and its
biosynthesis involves metabolic cooperation between astrocytes
and neurons. CSAD is rate limiting enzyme in taurine synthesis
and is found in astrocytes (52, 53). CSAD is activated in phosphorylated state and inhibited in dephosphorylated state. Protein
kinase C is the enzyme responsible for CSAD phosphorylationand phosphatase 2C is responsible for CSAD dephosphorylation.
Neurons do not have CSAD (54, 53) and they are based on
astrocytes deposits of hypotaurine (55) and on taurine acquisition. Cellular content of taurine is mainly determined by thesum of three processes: synthesis from methionine / cysteine,
active uptake by taurine transporters and its release via flow
volume – sensitive pathways. An adult of 70 kg contains about
70g of taurine, with an intracellular concentration of taurine in
the range from 5 to 50 mM, while plasma concentration is about
100 microM. Taurine concentration is different in different tissues.
In the brain, it is between 0.8 - 5.3 mmol / g (48).
Taurine is transported by a specific transporter TAUT, whose
sequence places it in family of transporters Na and Cl dependent(56, 57). In the brain there are two types of transporters, TAUT1
and TAUT2, TAUT2 being located mostly in glial cells (58, 59).
TAUT is regulated by activation of two enzymes Ca dependent:
35

PKC (transporter inhibitor) and calmodulin (transporter stimulator) (60, 61). Taurine increases chloride conductance in the
cerebellum, substantia nigra and olfactory bulb (62). Taurine isexcreted in urine and bile. In kidney, taurine is filtered at the glomerular level and reabsorbed partially in nephron tubes througha Na-dependent transport system specific for alpha amino acids.
The amount of taurine excreted daily varies between 0.22 - 1.85
mmol and is influenced by genetic factors, age, dietary intake,renal function and clinical conditions (63).
Mechanisms of action of taurine are complex, with important cardio - vascular, nervous and digestive effects. In the liveris involved in bile acid conjugation (64). On cardio - vascularsystem taurine has antiarrhythmic, positive chronotropic andinotropic effects (65, 66). It is hypotensive (67), hypocholesterolemiat (68) and antiatherogenic (69). In retina is the mostabundant amino acid and is involved in normal vision. In nervous system taurine controls cell growth and differentiation (70),
acts as trophic factor (71) affects cell migration and modulate
neurotransmission (72, 73). Taurine is involved in maintaining
the structural integrity of the membrane (74) in modulating
protein phosphorylation (75) It acts as osmoregulator (76,77, 78, 79) and neuroprotective agent in ischemia (80) and in
neurotoxicity induced by L - glutamate (81). Taurine interfereswith calcium equilibrium (82). It is known that intracellular free
Ca levels are maintained at low values through its sequestration
in intracellular calcium stores, such as endoplasmic reticulum,
mitochondria and by pumping it through Ca ATP - ase in theextracellular space. In the presence of taurine, glutamateinduced increase in intracellular Ca is reduced by reversing
Na / Ca carrier. At resting membrane potential, Na / Ca carrier
works to remove Ca from the cell. At depolarization conditions
such as stimulation by glutamate, it works in reverse and makes Ca influx. Taurine promotes the Ca efflux through Na / Ca
exchanger by increasing Ca concentration near the transporter(83). Taurine inhibits Ca release from internal deposits andvarious Ca voltage-dependent channels such as L- , N - andP / Q type (84 , 85).
Increased oxidative stress and accumulation of oxidative
modified molecules (proteins, nucleic acids and lipids) promotes dysfunction of various metabolic and signaling pathways(86). Cell toxicity induced by oxidative stress is reduced bytaurine. It does not directly bind superoxide, hydrogen peroxide and hydroxyl radical (87) but couple directly HOCl in
the presence of myeloperoxidase. Neurons may face energy
deficits resulting from alterations in cerebral vascularisationand mitochondrial function (88). Major source of superoxide
generation in mitochondria are complexes I and III. Slowing
the flow of electrons through the respiratory chain determines
electron transfer from complexes I and III to an alternative
receiver, such as oxygen (89). Taurine prevents diversionof electrons to superoxide generation by improving electrontransport function (90).
Taurine concentration decreases in aging rodent brain (91,92). Taurine supplementation in the diet restored the tissue
concentrations of taurine (93, 94).
36

MATERIALS AND METHODS
Stroop test was administered to two groups, one test andone control. The test group was formed of six women and four
men with age between sixty five and seventy years. The control
group was formed of six women and four men with age between
sixty five and seventy years. Test participation was unconstrained, each subject signing an informed consent before being
admitted to the study. Initially, each participant was presented
information on the purpose and progress of the study and about
taurine. Subjects in the study were patients of “Sf. Luca” Chronics
Pathologies Hospital, Bucharest and had the right to withdraw
from the study at any time.
Groups’ inclusion criteria were: age over 65 years, absenceof associated pathology, absence of a current treatment, absenceof a history of psychiatric or neurological disorders or brain trauma with loss conscience. Volunteers completed a Mini Mental
State Examination, the minimum score for participation was 25(95, 96). Subjects were tested for color vision by Ishihara tests.
The Stroop test followed the model proposed by MacLeod (97)in the chapter entitled “The Stroop task in cognitive research”
and consisted of three subsets of stimuli: neutral (a string of“XXXX” printed in colors), incongruent or with interference (themeaning of the word and the printing color were different, e.g.:yellow written in blue) and congruent or without interference (themeaning of the word and the printing color are the same, e.g.:yellow written in yellow). We used the four standard colors: green,
red, yellow and blue. Each subset had a total of 36 slides. The
test was administered on the computer. Subjects were seatedin a chair with monitor (NEC MultiSync FC color monitor, 17inch) placed at 40 cm away from the eye. All tests were printed
in large bold size 24 Helvetica font, in the center of the screen,
on a black background. The order of presentation of the trials
was randomized. Responses were given via a keyboard withkeys in four colors already named. Using the keyboard has theadvantage that no answers were lost. Comparison from literature
between the reaction times obtained from verbal and manual
response mode showed higher reaction times for manual version.
Subjects were advised to respond as fast as they could. Test
slides remained on the screen until each subject responded bypressing a key. Test slides were separated with white slides thatwere displayed 250ms.
We recorded reaction times on Stroop Test before taurine
administration (control), at one month, at two months and at threemonths, after the beginning of taurine administration. Response
times were registered using SuperLab 4.0 software. The mostused performance index for Stroop test is the interference index,
defined as the difference between reaction time to incongruent
stimuli and reaction time to neutral stimuli (97). The results werecompared with the control group.
We determined the concentration of the homovanillic acid,the major metabolite of the dopamine, in the plasma. We collected blood samples from participants at 9.00 am, before administration of the taurine, at one month, at two months and at threemonths from taurine administration. Plasma was separated by
Fiziologia - Physiology  2013.23.2 (78)centrifugation and samples were stored at - 70°C. Homovanillicacid concentration was determined by high performance liquid
chromatography.
We choose this testing period because The European Food
Safety Authority (98) has studied the effects of taurine on lots ofrats for periods of about three months. The Scientific Committee
on Food (99) admitted that oral administration of taurine at doses
between 600 and 1000 mg / kg / day was well tolerated by maleand female rats and did not lead to behavioral changes. Studies
revealed no genotoxic, carcinogenic or teratogenic effect of taurine. Also, theScientific Committee on Food (100) studies showedthat taurine levels return to initial value approximately 8 hours
after the administration. Taurine was administrated at dose of 500
mg x 3/day. Taurine was manufactured by Nutritech, the productbeing notified by the Institute of Food Bioresources, Bucharest.
RESULTSThe recorded times were neuter time, time with interference
and time without interference. Even if the two groups contain
only ten participants each group, the p is below 0.05 for the data
analyzed. Mean reaction times recorded are presented in Table I.
Table I. Mean reaction times before taurine administration(control), at
one month, at two months and at three months
Neuter timeTimes without interference

Times with Interference
interference index

Control
Control group2026.5625

1867.375

2240.62575

214.06325

Test group

1905.51802.725

2159.0483

253.9983

One month
Control group

1940.65

1808.9334

2173.7944

233.1444

Test group

1430.808

1354.175

1684.0602

253.2522

post-hoc analysis.
We have first analyzed the variance of means for the control
group. Our assumption is that, in the case of the group witch had
not received taurine, the means will not decrease significantly,
between the initial moment and the successive recordings at one
month, two months and three months. The one-way ANOVA forthe control group for neuter time is presented in Table II.
Tabel II. One-way ANOVA for control group for neuter time. As
expected, the significance value for the F-test is above 0.05, meaning that there is no significant variation in means between the four
recordings: initially, after one month, after two months, and after threemonths.
Reaction times
Sum of Squares

df

Mean Square

F

Sig.

BetweenGroups162847.876

354282.625

0.359

0.783

WithinGroups2419058.455

16151191.153

Total

2581906.331

19

Reaction times do not vary by themselves over time, only ifthey are influenced by the administration of taurine. For neuter
times, with interference times and without interference times,
for the test group the variation between means has been tested
with ANOVA. For the neuter time, there is a significant variation
between the means, that is, reaction times decrease significantly
between the initial moment, the one month recording, the twomonths recording, and the three months recording. In order tobetter describe the structure of this variance, we have built ameans plot, shown in Figure 1.

Two months
Control group2056.833

1947.333

2275.875

219.0413

Test group1339.95

1298.35

1467.628

127.678

Three months
Control group

1946.583

1824.333

2155.033

208.45

Test group1132.417

1063.75

1228.586

96.16888

We have used one-way ANOVA in order to test whether
there are statistically significant differences between the meansof the values recorded initially, at one month, at two months
and at three months. In other words, we wanted to test if thedecrease of the reaction times along the three-month period isstatistically significant.
Considering that there are more than three groups, ANOVA
is a suitable analysis. Also, in case of violation of the presumptionof normality of the sample, the analysis is robust enough. We
have also used contrasts analysis, comparing more in-depth
the differences between the groups, and also Dunnett test, as2013.23.2 (78)  Fiziologia - Physiology

Fig.1. Means plot for test group for neuter time. It may be seen thatthe most visible decrease in the reaction time is between the initialrecording (control) and the recording at one month. However, between the initial moment and the three-months recording the reduction inreaction time is significant.

In order to better analyze the structure of the differences
between the means of the considered groups, we have designed a two-layer contrast analysis. The contrast coefficients areshown in Table III.
37

Table III. Contrast coefficients for test group for neuter time. In thefirst layer, we have contrasted the first and the second group, while inthe second layer we have contrasted the pair formed by the first andsecond group with the pair formed by the third and forth group.
Contrast

Time intervals
Initial

1
2

1
0.5

At one
month
-1
0.5

At
two
months0-0.5

At threemonths0-0.5

The contrast analysis for test group for neuter time is shownin Table IV.
Table IV. Contrast analysis for test group for neuter time. In the case ofequal variances assumed, we may see that both group 1 (initially recorded
values) differs significantly (p lower than 0.05) from group 2 (values recorded after one month), and groups 1 and 2 together differ from groups 3 and
4 (values recorded after two months and three months, respectively).
Contrast
Neutertime

Assumeequal
variances
Does notassumeequal
variances

Std.
Error

t

df

1
2

Value
of Contrast
474.242425.399167.446118.402

2.832
3.593

36
36

Sig.
(2-tailed)
0.008
0.001

1
2

474.242425.399223.101
118.402

2.126
3.593

10.70613.450

0.058
0.003

At onemonth

Control -474.988 288.500 0.499 -1340.088
At two216.432
168.940 0.737 -296.133
monthsAt three 448.144
159.778 0.086 -49.836
monthsAt twoControl -691.420 258.963 0.112 -1507.578-216.432 168.940 0.737 -728.998
months At onemonth
At three 231.712
96.7400.150 -54.880
monthsAt three Control -923.132* 253.082 0.024 -1733.863-448.144 159.778 0.086 -946.124
months At one
month
At two-231.712 96.740
0.150 -518.304
months*. The mean difference is significant at the 0.05 level.

390.112728.998
946.124124.738
296.133
518.304-112.401
49.83654.880We have analyzed the means plot for reaction times withinterference, presented in Figure 2, in order to better interpretthe differences between the groups.

The ANOVA for test group for reaction times with interference
is presented in Table V.
Table V. ANOVA for test group for reaction times with interference. It
may be seen that the significance is below 0.05, meaning that in thiscase, of interference, the means vary significantly between the four
moments recorded.
Times with interference

df Mean Square F

Between Groups

Sum of Squares
4643039.696

Sig.

3

1547679.899 6.686 0.001

Within Groups

8333227.853

36231478.551

Total

12976267.550 39

We have used the post-hoc Dunnett test to analyze multiple
comparisons Table VI.
Table VI. Multiple comparisons for test group for time with interference. It may be seen that, in the case of interference, the mostsignificant difference appears between the values recorded initiallyand the values recorded at three months.

Dependent Variable: reaction times with interference
Dunnett T3(I) time(J) time Mean
Std.
Sig.
95% Confidence
interinterDifferen- Error
Interval
Lower
Upper
vals
vals
ce (I-J)
Bound
Bound
Control At one
474.988288.500 0.499 -390.112
1340.088
month
At two691.420258.963 0.112 -124.738
1507.578
monthsAt three 923.132* 253.082 0.024 112.401
1733.863
months38

Fig.2. Means plot for test group for the time with interference. It may
be seen that the pattern is similar to the one obtained for the neutertime.

We have further investigated the differences between thegroups by contrast analysis in Table VII.
Table VII. Contrast analysis for test group for the time with interference. With equal variances assumed, differences are significant, at
0.05 level, both between group 1 and group 2, and between groups 1
and 2 and groups 3 and 4. The reaction times decrease more in the
beginning of the experiment and then more smoothly.
ContrastTime
withinterference

Assumeequal
variances
Does
not assume
equalvariances

Std.
Error

t

df

1
2

Value
of Contrast
474.988569.782

215.164
152.144

2.208
3.745

36
36

Sig.
(2-tailed)
0.034
0.001

1
2

474.988569.782288.500
152.144

1.646
3.745

14.82518.137

0.121
0.001

The ANOVA for reaction times without interference is presented in Table VIII.
Fiziologia - Physiology  2013.23.2 (78)Table VIII. ANOVA for the test group for time without interference. It
may be seen that there are significant variances in means between
the four moments when the results were recorded. In other words,
reaction times without interference improve over time.

Reaction times without interference
Sum of Squares df
BetweenGroupsWithinGroupsTotal

2785985,931

3

Mean Square
928661,977

4795868,063

36133218,557

7581853,994

39

F

Sig.

6,971

0,001

The means plot for reaction time without interference isshown in Figure 3.
Fig.4. The evolution of the index of interference. It may be seen that,while for the control group it remains fairly constant over time, for thetest group the interference index decreases gradually.

The comparative graphs, made with Excell software fromMicrosoft Office, present the evolution of the neuter time, timewith interference and time without interference for the two groups,
at the four moments considered.
Fig.3. Means plot for test group for time without interference. It may
be seen for time without interference, there is a obvious decrease
between control and first month and between the second month andthe third month.

The contrast analysis for test group for the time without
interference is presented in Table IX.
Table IX. Contrast analysis for test group for the time without
interference. With equal variances assumed, we notice a significant
difference between group 1 and group 2, as well as between groups
1 and 2 and groups 3 and 4.
Contrast

Reactiontimes
without
interference

Assumeequal variances
Does notassumeequal variances

Std.
Error

t

df

1
2

Value
of Contrast
448.550
391.425163.229
115.420

2.748
3.391

36
36

Sig.
(2-tailed)
0.009
0.002

1
2

448.550
391.425220.792
115.420

2.032
3.391

10.69112.700

0.068
0.005

Fig.5. Evolution of time with interference. It may be seen the decrease of the neuter times in each month with taurine administration in
test group.

A conclusion that may be drawn from this analysis is that,the pattern of evolution is preserved across the three instances –neuter time, time with interference and time without interference.
We analyzed the interference index with the aid of theT – test. For the test group, Tables X, the p is 0,036 and it isincluded in the accepted interval.
Table X. One-Sample T – Test. The values of the interference index
decrease significantly between recordings.
Test Value = 253
t
df
Sig.
(2-tailed)

Intf_index_test-2.591

7

0.036

Mean
Difference

95% Confidence Interval of the Difference
Lower

Upper

-70.228-134.327-6.1292013.23.2 (78)  Fiziologia - Physiology

Fig.6. Evolution of time with interference. The values of the times
with interference reduce every month with taurine administration in
test group.

39

Fig.7. Evolution of time with interference. Times without interference
decrease each month with taurine administration in test group.

It may be seen that the two groups start from approximately
the same point and are gradually set apart by the administrationof the taurine, the gap becoming visible after one month, and also
that, while the control group does not suffer significant changes,
the response times of the test group are modified in time.
The homovanillic analysis indicate progressive increase of
its levels at one month, at two months and at three months forthe test group. For the control group there were not significant
changes in levels of homovanillic acid. For the test group, the pvalue at one month was 0.004. At the three months the p value
was 0.002. We present results of the mean concentration of
homovanillic acid as percentage reported at its value before
taurine administration. The statistical analysis was made with theaid of the Kingsoft Spreadsheets from Kingsoft Office Software
Corporation Limited.

Fig.8. Percentage modifications in the test group for homovanillicacid levels. The most important statistically increases were at one
moth and at three months.

DISCUSSION
There are four dopaminergic pathways in the brain, namely
mesocortical, the mesolimbic, striatal and tubero - infundibular
system (101). Mesocortical dopamine system modulates theactivity of working memory in dorsolateral prefrontal cortex. Alto40

et al demonstrated that during working memory activation there
is dopamine release in the prefrontal cortex (102).
By combining dopamine receptor analysis with cell recordings in monkeys it was demonstrated that D1 receptors
selectively modulate memory fields, in dorso - lateral prefrontal
cortex (103). Improvement of working memory performanceand predominant effects of D1 receptors over D2 ones in thismechanism was demonstrated by simultaneous testes withpergolide (D1 and D2 receptor agonist) and bromocriptine (D2receptor agonist) on working memory tasks. The performance
was improved by pergolide and not by bromocriptine (104).
Dopamine levels decrease by 10% per decade starting in
adulthood and are associated with the decline of cognitive andmotor performance. Age-related decline in dopamine system andin working memory is observed in humans and primates (105,106, 107). Decreased dopaminergic function in aging include
decreased dopamine synthesis, demonstrated with PET studies
in humans (108), reduced dopamine concentration, receptor
density and availability of carrier (109). Diminished dopaminergic
activity in aging is associated with functional changes especiallyin the prefrontal cortex, striatum and medial temporal lobes (110,111, 112, 113). It was shown that decreased dopaminergic projections and dopamine availability in prefrontal cortex with aging
mediates age-related cognitive deficits, including difficulties ofcontextual processing and of working memory (114).
An improvement of working memory performance was observed at low doses of D1 receptor agonist (0.0001 mg / kg) in
old monkeys that have a natural reduction in extracellular levelsof dopamine in prefrontal cortex (115, 105, 116). Extracellular
dopamine levels were significantly incr eased by taurine administration intracerebroventricular (117) or intraperitoneal (118)or directly in the striatum (119). Ericson et al. (120) showed that
local infusion of taurine (1, 10 or 100 mM) increases dopaminelevels in the nucleus accumbens.
Recorded results indicate progressive decrease reaction
times for each subset of the test during the three months oftaurine administration and a decreased in interference.
Homovanillic acid (HVA) is the main product of metabolismof dopamine, resulting from both 3-methoxytyramine deaminationand 3,4-dihydroxyphenylacetic acid methylation (121). Plasma
concentration of homovanillic acid have been used as a peripheric indicator of dopaminergic activity of the central nervous
system (122). This aminoacid levels decrease in the aging brain(123). The determined levels of homovanillic acid indicate thattaurine administration generate progressive increase of homovanillic acid concentration in plasma, in healthy elderly.
CONCLUSIONTaking into account the results obtained by us, the decrease of the reaction times and of the interference index andthe increase of homovanillic acid plasma concentration after
taurine administration, the information from literature that taurine
increase dopamine levels that decreased with aging, that plasma
concentration of homovanillic acid was used as an indicator of
Fiziologia - Physiology  2013.23.2 (78)

dopaminergic activity of the central nervous system, the implication of dopamine in working memory, we can say that taurine
improve the working memory through the increase of dopamine
concentration.
ACKNOWLEDGEMENTS
The presented results are part of the research grant TD
No. 449/2007 offered by the Board of the National Council of
Scientific Research in Higher Education of Romania and wasapproved by the Board of the University of Medicine and Pharmacy „Carol Davila”, Bucharest.
DISCLOSURE STATEMENT
There are no conflicts of interest or financial disclosure. The
manuscript is not considered for publication by any other journal.
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EFECTUL TAURINEI ASUPRA MEMORIEI DE LUCRU LA VARSTNICIISANATOSIREZUMAT

Imbatranirea demografica este importanta pentru obiectivele domeniului sanatatii. In imbatranirea normala se produce un declin alproceselor executive, cum ar fi manipularea informatiei in memoria de lucru, coordonarea de operatii multiple simultan. In sistemul
nervos, taurina actioneaza ca agent neuroprotector. Pentru a evalua performanta memoriei de lucru am folosit testul Stroop. Concentratia plasmatica a acidului homovanilic a fost folosita ca un indicator al activitatii dopaminergice din sistemului nervos central. Am
testat doua grupuri, unul test si unul control, fiecare fiind alcatuit din zece subiecti umani, barbati si femei peste saizeci si cinci deani. Am inregistrat timpii de reactie si am determinat concentratia acidului homovanilic inainte de administrarea taurinei, 1500 mg/
day, la o luna, la doua luni si la trei luni de administrare a taurinei. Activarea memoriei lucru este corelata cu eliberarea dopaminei lanivelul cortexului prefrontal. Declinul legat de varsta in sistemul dopaminergic si la nivelul memoriei de lucru a fost observat la subiectii
umani. Rezultatele obtinute indica scaderea timpilor de reactie si a indexului de interferenta si cresterea concentratiei plasmatice a
acidului homovanilic dupa administrarea taurinei.
Cuvinte cheie: imbatranire cerebrala, test Stroop, memoria de lucru, taurina, dopamina, acidul homovanilic

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