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Fiziologia - Physiology • 2016.26.3(91)FIZIOLOGIA

physiology
CONTENTS
1. Understanding the Blood-Brain Barrier ........................................................................................................................ 3
Scheau C, Scheau AE, Papacocea RI, Ciornei CM, Badarau IA

2. Biochemical Studies of Aqueous Extract of Garlic on the Myocardium of Left Ventricleof High Salt Fed Adult Wistar Rats .............................................................................................................................. 9
Olusola Stephen Saka, Omobola Aderibigbe Komolafe, Oluwadare Ogunlade, Ahmed Afolabi Olayode,
Alice Ayomide Akinjisola, Samson Ayodeji Odukoya, Ronald Ayodele Bejide

3. Age- and Dose-Dependent Haematological Changes in Lactational Lead-Intoxicated Rats ................................. 13
Samson Oludare Ayodeji Odukoya, Oluwole Busayo Akinola

4. Integrating Physiology in the “Urinary System” Module .......................................................................................... 19
Tatu C, Pitic C, Panaitescu C, Cotarca M, Bojin F, Tanasie G, Minca D, Puscasiu D

5. Interaction between Alexithymia and Obsessive - Compulsive Behavior to Hemophilic Patients ....................... 25
Hogea L, Nussbaum L

6. The Utility of Ring Finger Median-Ulnar Sensory Latency Difference in the Early Diagnosis
of Carpal Tunnel Syndrome ....................................................................................................................................... 30
Zahid M. Kadhim, Safaa Mohammed Fadhel Abood, Ali J. Al-Sultany, Ban J. Edan

7. Rh Isoimmunisation in Neonates: Risk Factors, Incidence, Complications and Treatment .................................. 35
Iacob D, Angelescu-Coptil C, Nyiredi A, Dima M, Hocan ME, Nassar M, Iacob RE

CUPRINS
1. Înțelegerea barierei hemato-encefalice ......................................................................................................................... 3
Scheau C, Scheau AE, Papacocea RI, Ciornei CM, Badarau IA

2. Studii biochimice ale extractului apos de usturoi asupra miocardului ventriculului stângla șobolanii Wistar adulți cu dietă hipersodată .......................................................................................................... 9
Olusola Stephen Saka, Omobola Aderibigbe Komolafe, Oluwadare Ogunlade, Ahmed Afolabi Olayode,
Alice Ayomide Akinjisola, Samson Ayodeji Odukoya, Ronald Ayodele Bejide

3. Modificări hematologice induse de vârstă și doză la șobolanii intoxicați lactațional cu plumb ........................... 13
Samson Oludare Ayodeji Odukoya, Oluwole Busayo Akinola

4. Integrarea Fiziologiei în cursul modular „Sistemul Urinar” ...................................................................................... 19
Tatu C, Pitic C, Panaitescu C, Cotarca M, Bojin F, Tanasie G, Minca D, Puscasiu D

5. Interacțiunea dintre alexitimie și comportamentul obsesiv-compulsiv la pacienții cu hemofilie ......................... 25
Hogea L, Nussbaum L

6. Utilitatea detectării diferenței de latență a nervului senzitiv median-ulnar al degetului patru
în diagnosticul precoce al sindromului de tunel carpian ........................................................................................ 30
Zahid M. Kadhim, Safaa Mohammed Fadhel Abood, Ali J. Al-Sultany, Ban J. Edan

7. Izoimunizarea Rh la nou-născut: fiziopatologie, factori de risc, incidență, complicații și tratament ................... 35
Iacob D, Angelescu-Coptil C, Nyiredi A, Dima M, Hocan ME, Nassar M, Iacob RE

Fiziologia - Physiology • 2016.26.3(91)3UNDERSTANDING THE BLOOD-BRAIN BARRIER
CRISTIAN SCHEAU, ANDREEA ELENA SCHEAU, RALUCA IOANA PAPACOCEA,
CĂTĂLINA MARIANA CIORNEI, IOANA ANCA BĂDĂRĂU
“Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania

ABSTRACTThe blood-brain barrier is a mostly impermeable structure separating the blood from the central nervous system. Its existence
has been deduced by scientists in the 19th century, but technological progress allowed viewing the constituency of theblood-brain barrier only in the mid-20th century. The study of the blood-brain barrier continues even today, because all theroles of its components are not fully understood, as new information is uncovered by exploring it in both physiological andpathological situations, in-vivo and in-vitro. The endothelial cells with their tight junctions, the basal lamina, the astrocyte feet,the pericytes and other structural parts of the blood-brain barrier all contribute towards its complex degree of impermeability
due to mechanical, electrical, chemical and immune factors.
Keywords: blood-brain barrier, ultrastructure, neurophysiology

INTRODUCTION
The Blood-Brain Barrier (BBB) is an important structure
which functions as an interface between the blood and thecentral nervous system, restricting the diffusion of mostblood compounds towards the brain, but facilitatingcomplex signaling and regulation mechanisms involved in
the body’s homeostasis. It has a complex morphology withdifferent types of constituents, building towards intensively
studied but not yet completely understood selective
gateway between the central nervous system and thecirculating blood.

THE FORETELLING
OF THE BLOOD-BRAIN BARRIER
The first observations that indirectly pointed towards
the existence of a barrier between the blood and thecentral nervous system belong to Paul Enrlich, which in1885 noticed that with the injection of a dye in the
peripheral venous system of an animal, all the organs were
stained except the brain and spinal cord [1]. He initiallyattributed this particular finding to the difference in binding
affinities of the dye.
The hinting towards the presence of an actual barrier
was done by Max Lewandowsky in 1900, when he noticed
that the peripheral injection cholic acids and sodium
ferrocyanide had no effect on the CNS, while theintraventricular injection of the same compounds exhibits
the specific pharmacological effects [2]. Therefore heconcluded that the blood vessels in proximity to the brain

selectively block certain substances, and he introduced theterm “blood-brain barrier” to describe this mechanism.
Continued research by one of Ehrlich’s students, Edwin
Goldmann, unveiled a more complex grasp on thephysiology of the BBB, published in several steps, mostimportantly in 1913 [3]. Staining studies with trypan blue
showed a coloration of the brain when injected
intraventricularly, but with intravenous injection no staining
was observed except for the choroid plexuses. This
demonstrated a separation between the CNS and the restof the body and also that the choroid plexuses were
gateways towards the brain.
Further observations were obtained by Lina Stern,which noted that an anti-tetanus drug administeredintravenously did not cure tetanus once it affected the brain,thus concluded there is mechanism that protects the CNS
from infectious and toxical compounds in the blood [4]. She
introduced the term “hematoencephalic barrier” to describe
an interface that completely blocks any molecular transfer
towards the brain, a hypothesis proven improper years
later.

THE DEMONSTRATION
OF THE BLOOD-BRAIN BARRIER
The introduction of electron microscopy made it
possible to directly visualize the structural composition of
the BBB. The first studies by Edward Dempsey and his
team, in 1955, demonstrated that the BBB should consistof one or more structures that separate the blood in the

Received August 12th 2016. Accepted September 2nd 2016. Address for correspondence: Assistent Teaching Scheau Cristian,Discipline of Physiology, „Carol Davila” University of Medicine and Pharmacy Bucharest, B-dul Eroilor Sanitari, no. 8, sector 5,
Bucharest, Romania, phone: +4021.318.07.60, e-mail: cristianscheau@gmail.com

4

Fiziologia - Physiology • 2016.26.3(91)capillaries from the neurons, recte the capillary wall, theastrocytic feet and the basal lamina [5].
Subsequent studies published by Van Harrefeld in
1965-1967 used an improved method of fixation by rapid
freezing on a metal plate and substitution fixation at low
temperature shortly after acquiring the biological sample
from the living organism. This procedure permitted theidentification of the extracellular space in the CNS, whichby conventional methods was destroyed and impossible toobjectify [6].
Additional progress was recorded by Thomas Reeseand his colleagues, in 1967-1969 when they demonstrated
the actual role of interendothelial tight junctions, whichsurpassed the astrocytic feet and basal lamina in terms ofsignificance [7].
Following studies using freeze fracture, performed by
Nagy and his team and published in 1984, showed thattight junctions form a complex network restricting thepassage of molecules up to 10-15Å [8].

Fig. 1. The anatomical structure of the blood-brain barrier.
1. Endothelial cell. 2. Basal lamina. 3. Tight junction. 4. Pericyte.
5. Astrocyte foot. 6. Regulatory neuron. 7. Microglia.
8. Perivascular macrophage.

THE SPECIFIC ROLE OF INDIVIDUAL
ANATOMICAL CONSTITUENTS
OF THE BLOOD-BRAIN BARRIER
Endothelial cells
Studies have demonstrated that the most importantcomponent of the BBB is represented by the endothelial
cells, which line the blood vessels and have morphologicalparticularities that differentiate them from those that form
somatic capillaries [9].
The endothelial cells in the brain capillaries have nofenestrations, hold more mitochondria compared to otherendothelia in the body, and possess a continuous basal
lamina to which they are attached through focal adhesions
consisting of transmembrane proteins [10].
Fiziologia - Physiology • 2016.26.3(91)The fundamental role of the endothelial cells of the
BBB is to block the access of hydrophobic intravascularcomponents to the brain, and in the meantime allowing theregulated passage of key metabolites to the extracellular
fluid and astrocyte feet while maintaining local
hydroelectrolytic homeostasis [11].
Another important function of endothelial cells is tosecrete the endothelial growth factor, brain-derived
neurotrophic factor, insulin-like growth factor 1, substance
P and other regulating factors [12-14].
Basal lamina
A crucial part of the BBB that surrounds and binds
endothelial cells, the basal lamina is composed by multiple
types of molecules such as collagen, elastin, proteoglycans,laminin, nidogen, osteonectin, perlecan and fibronectin[15].
The basal lamina plays a supportive role due to its
composition rich in cell adhesion molecules that bind to theendothelial cells and surrounding structures such as the
pericytes, astrocyte feet, and also contribute to the integrityof the tight junctions [16].
Tight junctions
One of the most important factors that reduce thepermeability of various polar solutes and ions in theirpassage between the endothelial cells from the blood
towards the brain [17].
Ultrastructurally, the tight junctions are composed ofproteins and junction adhesion molecules developed in
between two endothelial cells, or for the distal capillary,
between the two extremities of the single endothelial cellcomposing the vessel wall [18]. The proteins that block theaccess of molecules through the tight junction are claudins
and occludins, and not only their presence but their three
dimensional organization are key factors to producing animpassable barrier [19].
Several other cytoplasmic proteins of the endothelial
cells are indirectly involved in the composition of the tightjunctions, which interact with the aforementioned
membrane proteins, signaling them and regulating theirexpression [20].
The tight junctions are also the cause for the highelectrical resistance of the blood-brain barrier causing a
restriction of ion free motion [21].
Pericytes
Enveloping the capillaries of the BBB, the pericytes
play a key role by regulating the vessel caliber, dynamic ofcontraction and structural stability, as well as secretingseveral angiogenic and differentiation factors [22].
The pericytes interact with the basement membrane,
attaching themselves and dynamically interacting
especially through the secretion by the pericytes of the5tissue inhibitor of metalloproteinase 3 and other proteaseinhibitors and enzymes involved in vascular modeling [23].
Endothelial cells secrete pericyte proliferation factorsthat favor their migration and development, and after theirmaturation they bind to the endothelial cells by means oftransforming growth factor-β [24].
Several experiments demonstrated the increased
resistance to apoptosis demonstrated by endothelial cells
bound to pericytes, when compared to isolated endothelial
cells, thus underlining yet another role of pericytes in therobustness of the BBB [25].
Astrocytes
The structural role provided by astrocytes resides
mainly in nutritional support and the clearance of ions andmetabolites. The astrocyte feet cover a significant
circumference of the capillary surface and interact withadjoining neuronal bodies, endothelial cells and basal
lamina, regulating their functions.
Animal experimental studies showed that astrocytes
tend to develop around the endothelial cells and tighten thecapillaries when transplanted into areas with leaky vessels,
thus demonstrating an important role towards the induction
and maintenance of the BBB [26].
In vivo studies evidenced the critical role of theastrocytes in the proper functioning of the BBB, studying
the effect of selective destruction of astrocytes with hypoxic
stress, which leads to the loss of endothelial tight junction
protein expression, destroying the BBB. Restoration of theastrocytes in the affected area reestablished the normal
parameters of the BBB [27].
Various signaling factors secreted by the astrocytes
were identified, such as calcium, TGF-β, IL-6, and Glial
cell-derived neurotropic growth factor (GDNF), some ofthem appearing to play a role in the modulation of the BBB
phenotype, without actually being a factor in its structuralresistance [28].
Regulatory neurons
Neurons do not play a direct role in the structure of the
BBB, but act as modulators by releasing some enzymes
regulating the endothelial cells, as well as conventional
neurotransmitters such as norepinephrine, acetylcholine,
GABA, serotonin and others [29].
Recent studies observed the disruption of the BBB
through an increase of vascular endothelial growth factor
release from the astrocytes activated by ischemic neurons,
findings which were accompanied also by a decrease of
occludin and claudin proteins that further diminished BBB
integrity [30].
Neurons seem to have a signaling feedback with theendothelial cells, including secretion of proteins that
promote synaptogenesis and the overall regulation of thecerebral blood flow, in response to fine changes in theneuronal activity [31].
6

Microglia
Microglia represent the effectors of the immunity system inthe central nervous system, and can be subjected to varioustransformations in their activation process. The neuroinflammation that appears is a common pathogenic pathway
for numerous neurodegenerative diseases [32].
Several cytotoxic mediators are released by microglia
during inflammation, including tumor necrosis factor α thateasily destroys the BBB [33]. On the other hand, the close
proximity of the microglia to the endothelial cells indicate
their contribution to the properties of the BBB, yet the exact
role they are playing is yet to be demonstrated.
Perivascular macrophages
Showing similar structure and immune phenotype as theirblood-circulating counterparts, perivascular macrophages area particular population of cells residing near the astrocytic feetthat compose the BBB [34].
Perivascular macrophages can extend some branchingprocesses that can help engulfing the vessels thus adding
to the physical impermeability of the BBB, and some caneven be found beneath the basement membrane, between
the vessel and the astrocyte feet [35].
In certain infectious or inflammatory conditions,perivascular macrophages can accumulate in the interstitialspaces and either clear along with the acute process, or
remain for a long and unspecific period of time [36].
There are several immunological roles of theperivascular macrophages that have been identified andthey have also been implicated in the response toapoptosis, as well as in the phagocytosis and pinocytosisof various molecules [37].

THE REGULATION
OF THE BLOOD-BRAIN BARRIER
More and more studies point out that there is a certain
dynamics in the response of the BBB to various stimuli inphysiologic and pathological states of the body. Regulating
the strict permeability of the BBB is essential in the
response to factors that can involve the central nervous
system, and thus on one hand protecting the CNS fromharmful molecules, and on the other allowing specificsubstances and cells to cross the barrier and facilitate thebrain’s response to aggression.

CONCLUSIONSThe blood-brain barrier is a very highly selective
permeability barrier that uses different morphological andphysiological mechanisms to separate the circulating bloodfrom the central nervous system. The blood-brain barrier’s
constituents which are very heterogeneous in nature,
ranging from nervous endings and nerve cells, vascular
Fiziologia - Physiology • 2016.26.3(91)cells and their connections, immune-response cells andothers, help create a barrier that opposes its transition bymechanical, electrical, chemical and immune forces, allwhile being strictly regulated by the central nervous system.
The understanding of the blood-brain barrier is critical inorder to control its functions in pathological conditions,
intentionally modifying the permeability for specificsubstances.
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increasing VEGF expression. J Neurochem. 2014;
129(1):120-9.
31. Abbott NJ, Ronnback L, Hansson E. Astrocyteendothelialinteractions at the blood-brain barrier. Nat. Rev. Neurosci.
2006; 7: 41-53.
32. Dickstein DL, Biron KE, Ujiie M, Pfeifer CG, Jeffries AR,
Jefferies WA. Abeta peptide immunization restores
blood-brain barrier integrity in Alzheimer disease. FASEB J.
2006; 20: 426-433.
33. Da Fonseca ACC, Matias D, Garcia C, et al. The impact of
microglial activation on blood-brain barrier in brain diseases.
Frontiers in Cellular Neuroscience. 2014; 8: 362.
34. Guillemin GJ, Brew BJ. Microglia, macrophages, perivascularmacrophages, and pericytes: a review of function andidentification. J Leukoc Biol. 2004; 75(3): 388-97.
35. Nacer A, Movila A, Baer K, Mikolajczak SA, Kappe SHI,
Frevert U. Neuroimmunological Blood Brain Barrier Opening
in Experimental Cerebral Malaria. PLoS Pathogens. 2012;8(10): e1002982.

736. Owens T, Bechmann I, Engelhardt B. Perivascular spaces
and the two steps to neuroinflammation. J Neuropathol Exp
Neurol. 2008; 67(12): 1113-21.

37. Man S, Ubogu EE, Ransohoff RM. Inflammatory cell
migration into the central nervous system: a few new twists
on an old tale. Brain Pathol. 2007; 17(2): 243-50.

ÎNȚELEGEREA BARIEREI HEMATO-ENCEFALICEREZUMAT
Bariera hemato-encefalică este o structura cvasi-impenetrabilă ce separă sângele de sistemul nervos central. Existența
ei a fost dedusă de oameni de știință încă din secolul 19, însă, abia la mijlocul secolului al 20-lea, progresul tehnologic
a permis vizualizarea directă a constituenților acesteia. Studiul barierei hemato-encefalice continuă și în prezent, dincauză că nu toate rolurile componenților acesteia au fost înțelese pe deplin, noi informații fiind descoperite prin
explorări în situații atât fiziologice cât și patologice, in-vivo și in-vitro. Celulele endoteliale și joncțiunile lor strânse,lamina bazală, terminațiile astrocitare, pericitele și celelalte porțiuni structurale ale barierei hemato-encefalice
contribuie la gradul acesteia complex de impermeabilitate prin factori mecanici, electrici, chimici și imuni.
Cuvinte cheie: bariera hemato-encefalică, ultrastructură, neurofiziologie

8

Fiziologia - Physiology • 2016.26.3(91)BIOCHEMICAL STUDIES OF AQUEOUS EXTRACTOF GARLIC ON THE MYOCARDIUM OF LEFT
VENTRICLE OF HIGH SALT FED ADULT WISTAR RATS
OLUSOLA STEPHEN SAKA*1, OMOBOLA ADERIBIGBE KOMOLAFE 1,
OLUWADARE OGUNLADE2, AHMED AFOLABI OLAYODE1, ALICE AYOMIDEAKINJISOLA3, SAMSON AYODEJI ODUKOYA1, RONALD AYODELE BEJIDE1
1Department of Anatomy and Cell Biology, Obafemi Awolowo University, Ile-Ife, Osun State, Nigeria
2Department of Physiological Sciences, Obafemi Awolowo University, Ile-Ife, Osun State, Nigeria
3Department of Human Anatomy, Ladoke Akintola University of Technology, Ogbomoso, Oyo State, Nigeria

ABSTRACT
Background: This study was designed to demonstrate the restorative effect of garlic extract on the biochemical activities ofthe myocardium of left ventricle of high salt fed adult Wistar rats.
Methods: Twenty-five healthy female Wistar rats weighing 130-180 g were randomly assigned into 5 groups of 5 rats each
(Groups A, B, C, D and E). Rats in group A were fed with standard laboratory pellets, while groups B, C, D and E were fed on thehigh-salt diet for five weeks. Thereafter, daily administration of 50 mg kg-1, 100 mg kg-1 and 150 mg kg-1 of the garlic extract were
giving orally to groups C, D and E respectively for 3 weeks while rats in groups A and B were administered distilled water. The rats
were sacrificed under ketamine anesthesia (30mg/kg i.m). Blood samples were collected for the measurement of serum
electrolytes and part of the heart ventricle was homogenized for the determination of lactate dehydrogenase activity. One-way
ANOVA was used to analyze data, followed by Student Newman-keuls (SNK) test for multiple comparison.
Result: Result showed that the LDH activity, plasma level of sodium and potassium of group B rats were significantly higher(P < 0.05) when compared with the control group.
Conclusion: The study concluded that aqueous garlic administered altered the above-mentioned biochemical parameters
toward normal value; this indicated that it contains the restorative effect on myocardium disorder.
Keywords: biochemical, high salt fed, myocardium, garlic

INTRODUCTION
Salt intake observed more than a half century ago adose-dependent decrease in survival of rats fed a standard
diet containing increasing amounts of salt [1]. Researchshowed that, it increased mortality by acceleratingarteriosclerosis, renal parenchymal damage andcardiovascular disorders. These outcomes wereassociated with changes in blood pressure. This dramatic
illustration of the effect of excess salt intake on mammaliancardiovascular and renal physiology has not been stressed,
but recent developments have revitalized these original
findings, with emphasis on the role of the renin-angiotensin
aldosterone system (RAAS) in cardiovascular disease [2].
Garlic had been reported to have prophylactic,
restorative and curative properties in various conditions
such as microbial infection, thrombosis, hypertension,
hyperglycemia, hyperlipidemia, cancer, and thrombosis[3,4]. The contents of the extract include sulfur active

principles mainly in the form of cysteine derivatives such ass-alkyl cysteine and sulfaxides, which decompose into avariety of thiosulfinates and polysulfides by the action of an
enzyme allinase on extraction [5]. Evidence from numerous
studies has pointed out that garlic can bring about plasma
lipid normalization, enhance fibrinolytic activity and inhibit
platelet aggregation and thromboxane formation [4]. Several
reports have suggested that garlic has protective effectagainst stroke and atherosclerosis [6]. Fresh garlic extracts
have been found to lower blood pressure in spontaneously
hypertensive rats and in anesthetized dogs [7].
High prevalence of cardiovascular disease, side effectof available antihypertensive drugs, recent trend in any
development from medicinal plant, this study investigatedalternative therapy that are likely to be more effective andcheaper than the current therapies. The goal of this study
was to show that garlic extract still possess the restorative
effect on the biochemical activities on the myocardium ofleft ventricle of high salt fed (HSF) adult Wistar rats.

Received August 15th 2016. Accepted September 22nd 2016. Address for correspondence: SAKA, Olusola Stephen, M.Sc.
Department of Anatomy and Cell Biology, Obafemi Awolowo University, Ile-Ife, Osun State, Nigeria; phone: +2348067646211;
e-mail: olusolasaka1@gmail.com

Fiziologia - Physiology • 2016.26.3(91)9MATERIALS AND METHOD
Animal Care and Management
Twenty-five healthy female Wistar rats weighing
130-180 g obtained from the Animal Holding of College of
Health Sciences, Obafemi Awolowo University Ile-Ife wereused for this research. The rats were randomly assigned
into 5 groups of 5 rats each (Groups A, B, C, D and E).
They were maintained on standard laboratory rat pellets
before the commencement of the experiment and water
was provided ad libitum.
Plant Material and Preparation of Extract
Cloves of garlic bulb were procured from Sabo market
in Ile-Ife and identified by a taxonomist in the Departmentof Botany, Obafemi Awolowo University, Ile-Ife. They were
weighed and then blended with water. After which they
were filtered into a conical flask using a filter paper and afunnel to produce a clear juice. Then, the filtrate wasfreeze-dried using lyophilizer and stored in a desiccator. An
aliquot portion of the crude extract residue was dissolved in
distilled water which was been used on each day of theexperiment [8].
Preparation of High Salt Diet (HSD)
HSD containing 8% sodium chloride was preparedspecially by replacing 0.3% sodium chloride-containing
standard diet with 8% sodium chloride [9,10].
Animal Treatment
Group A was the control, group B was negative control,
while groups C, D and E were the test groups. Rats ingroup A were fed with standard laboratory pellets, whilegroups B, C, D and E were fed on the high-salt diet for five
weeks. Thereafter, daily administration of 50 mg kg-1, 100
mg kg-1 and 150 mg kg-1 of the garlic extract were giving
orally to groups C, D and E respectively for 3 weeks, whilerats in group B was left untreated for the same period. The
extract solution was administered orally, using oral cannulaand duration of the experiment was 8 weeks.
Sacrifice of Animals
At the end of the experiment, the rats were sacrificed
under ketamine anesthesia (30mg/kg i.m). Blood samples
were collected for the measurement of serum electrolytes
and part of the heart ventricle was homogenized for thedetermination of lactate dehydrogenase (LDH) activity.
Homogenization of Myocardiumof Left Ventricle of the HeartThe tissues were excised, weighed and were chopped
into small pieces with a scalpel holder and blade. Each of
the tissues was washed three times in ice-cold 0.9% KCl.
All subsequent manipulations were done at 4oC. The
10tissues were then homogenized in Teflon glass
homogenizer within ten fold volumes excess of 5 mm TRIS
homogenization buffer (PH 7.7 at 2oC). The
homogenization buffer contained 3.338 mm TRIS HCL,
1.669 mm TRIS base, 250 mm sucrose and 2 mm EGTA,
The homogenates were centrifuged at 5000 rpm for 5
minutes to obtain clear supernatant. The supernatant wasthen assayed for the levels of activity of lactate
dehydrogenase enzymes using Randox kit for LDH(Randox Laboratory, Northern Ireland).
Measurement of Serum Electrolytes
Blood sample from each rats was collected separately
into clean capped plain tubes and allowed to stand for 30
minutes for clotting to occur. These were then centrifuged at2500 revolution per minutes for 15 minutes. The serum was
extracted into clean test tube for the analysis of sodium andpotassium. This was measured using Flame photometry
method at wavelength of 590 nm for sodium and 770 nm forpotassium.
Photomicrography
Stained sections were viewed under a LEICA research
microscope (LEICA DM750, Switzerland) with digital camera
attached (LEICA ICC50) and digital photomicrographs weretaken at various magnifications.

RESULTS
One way ANOVA revealed that there was significant
difference (F4, 20 = 4.217; p = 0.0174) in activity of LDH
across all experimental groups. Post-hoc analysis showedthat LDH activity of HSF group was significantly higher (p =0.0174) than the control group. The LDH activity which was
induced by HSD was significantly reversed by garlic extract.
The 100 mg/kg of garlic extract produced the highest level ofreversal, which was not significantly different whencompared with control (Figure 1). Post-hoc analysis showedthat serum sodium ion concentration of group B was
significantly higher than the control group (p<0.05). The
increased serum sodium ion concentration which was
induced by the HSD was reversed by garlic extract in groups
C, D and E (Table I). There was significant difference inserum potassium ion concentration across all
experimental groups (F4, 20 = 28.54; p<0.05). Serum
potassium ion concentration of group B was significantly
lower than the control group (p<0.05). The serum
potassium ion concentration which was lowered by the
HSD was significantly and dose-dependently reversed byextract in groups C, D and E. However, the highest dose ofgarlic used in this study (150 mg/kg) did not produce acomplete reversal of HSD-induced hypokatrimia to such alevel that was significantly higher than the vehicle group(p<0.05) (Table II).
Fiziologia - Physiology • 2016.26.3(91)Fig. 1. Effect of Aqueous Extract Garlic Activity on LDH of
Myocardium of Left Ventricle of Rats Fed with HSD; n = 5, values
are expressed as LDH Activity (u/l) ± SEM, *relative to control at
p<0.05; # relative to group B (HSF) at p<0.05.
Table I. Effect of Aqueous Garlic Extract on Serum Sodium Ion ofRats Fed with HSD; n = 5, values are expressed as serum
sodium (mmol/L) ± SEM, *relative to control; # relative to group B(HSF) at p<0.05.

GroupsA
B

Serum sodium ion(mmol/L) ±SEM
137.30±0.42144.90±0.06*

CDE

141.80±0.19*#
141.10±0.27*#
139.10±0.02#

Table II. Effect of aqueous garlic extract on serum potassium ion ofrats fed with HSD; n = 5, values are expressed as serum potassium(mmol/L) ± SEM, * relative to control; # relative to group B (HSF); a
relative to group C (HSD + 50 mg kg-1 of garlic extract); b relative togroup D (HSD + 100 mg kg-1 of garlic extract); p<0.05.

GroupsA
B
C
D
E

Serum potassium ion(mmol/L) ±SEM
4.49±0.18
3.95±0.02*
4.03±0.02*
4.67±0.04#a
4.99±0.02*#ab

DISCUSSION
Biochemical studies of aqueous extract of garlic on the
myocardium of left ventricle of HSF adult wistar rats werecarried out in this study. It was observed that the lactate
dehydrogenase (LDH) was significantly higher in the HSF
group than the control group (p<0.05). Similarly, theconcentration of plasma sodium and potassium ion was
significantly higher in HSF group than the control group
Fiziologia - Physiology • 2016.26.3(91)(p<0.05). This suggests that HSD resulted in hyperkaliemia,
hypernatrimia and cardiovascular disorder in myocardiumof left ventricle of the heart. High salt intake promotes theelevation of blood pressure, cardiac hypertrophy, theimpairment of left ventricular relaxation, endothelial
dysfunction and kidney injury [8], while some studies havelinked the HSD–induced cardiovascular disorder tosalt-induced hypertension [9]. Increasing evidence frommultiple clinical studies showed that excess salt intake wasrelated to cardiovascular organ damage, independent ofblood pressure [10]. The pathophysiological mechanisms
responsible for high salt diet–induced remains debatable.
It is well known that lactate dehydrogenase (LDH) is
one of the diagnostic marker enzymes of myocardialdamage. Presence of this biomarker in heart tissue
homogenate (HTH) is indicative of myocardial integrity andtheir release in serum signifies myocardial injury. The
results of this study showed that LDH activity increased in
the HSD. However, garlic extract has restorative effects onhigh salt induced cardiac damage as revealed by reductionin the concentration of LDH of treated groups especiallythose with 100 mg kg-1 and 150 mg kg-1 of garlic extract. It
was reported that garlic consumption had significant
restorative effect on both animals and human [11]. It wasalso reported that aqueous garlic extract was beneficial inameliorating LDH activity in Lead-induced oxidative
damage in the rat heart [12].
The active ingredient in garlic extract is known as
Allicin (diallyl thiosulfinate) which is a mainly organosulfur[13] had been shown to reverse the effect of high salt inthis research. Garlic had been reported to exhibit potent
angiotensin converting enzyme (ACE) inhibitory activity[14]. The reduction in ACE activity results to reduction inplasma level of angiotensin-II. Angiotensin II is also known
to cause cardiovascular remodeling [15]. Garlic
extract-induced inhibition of ACE probably leads toreduction in angiotensin-II, leading to decrease adrenal
production of aldosterone [16]. The Garlic extract-induced
reduction of plasma level of angiotensin-II followed by natriand diuresis may be responsible for the reversal of the highsalt-induced cardiovascular disorder.

CONCLUSION
The result of this study indicated that high salt diet causes
significant histomorphological changes on myocardium of left
ventricle of rats as evidenced by significant increase of LDH,
serum sodium and potassium. Garlic extract has restorative
and ameliorative properties on these high salt diet-inducedchanges in cardiovascular system.
CONFLICT OF INTEREST
There was no conflict of interest among the authors andevery necessary detail was agreed upon during the
preparation of the work.

11

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12

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Fiziologia - Physiology • 2016.26.3(91)AGE- AND DOSE-DEPENDENT HAEMATOLOGICALCHANGES IN LACTATIONAL LEAD-INTOXICATED RATS
SAMSON OLUDARE AYODEJI ODUKOYA*1, OLUWOLE BUSAYO AKINOLA2
1Department of Anatomy and Cell Biology, Obafemi Awolowo University, Ile-Ife, Nigeria;
2Department of Anatomy, University of Ilorin, Ilorin, Nigeria.

ABSTRACT
Objective: High to low lead (Pb) concentrations in breast milk has been found to perturb some biological events in the
postnatal life. While postnatal Pb exposure has been reported to impair some haematological parameters in mammals,
the age-dependent haematological signature of lactational Pb poisoning is not clear. This study investigated the effectsof Pb exposure during lactating period on the haematological profile of rats at certain post -lactational ages using varying
doses of Pb.
Methods: Lactating mothers and their pups were randomly divided into 4 groups comprising 24 pups each. The treatmentgroups received 10 mg/dL, 30 mg/dL, and 70 mg/dL of lead acetate in their drinking water from postnatal day one (P1) to P21 of
the lactating period. The control rats received distilled water. At P22, P60, P90 and P120, the pups from each group wereeuthanized and blood was collected and analyzed for blood Pb levels, packed cell volume (PCV) and total white blood cell(WBC) count.
Results: Lactational lead poisoning resulted in higher blood lead levels among the exposed pups (p < 0.01 compared with
controls); the PCV did not show statistically significant differences from the controls until P90 and P120. Moreover, WBC
count was significantly higher (P>0.05) from the control as an initial response to Pb intoxication except in the group treatedwith the highest Pb dose, where WBC count was significantly reduced.
Conclusion: We concluded that lactational Pb intoxication is a means of Pb poisoning in rat pups, and that high concentrationof Pb in maternal breast milk is associated with reductions in WBC counts in the pups. High lactational Pb is also associated with
post-lactational impairment of the immune system and anemic responses in rat pups.
Keywords: lead poisoning, lactational, postnatal, haematological, RBC, WBC.

INTRODUCTION
Lead poisoning (also known as plumbism,
colicapictorum, saturnism, Devon colic, or painter's colic) isa medical condition in humans and other vertebrates
caused by increased levels of the heavy metal lead (Pb) in
the body, that is, an intoxication resulting from absorption of
hazardous levels of lead into body tissues [1,2]. Pb iswell-known as a major global environmental hazard. The
Centre for Disease Control (CDC) [4,5] currently considers
lead poisoning the leading environmental health threat tochildren in the US [3]. No safe threshold for lead exposure
has been discovered; that is, there is no known sufficiently
small amount of lead that will not cause harm to the body.
The Centers for Disease Control [4,5] (US) has set thestandard of elevated blood lead level for adults to be 10µg/dl of the whole blood. For children the number is set
much lower at 5 µg/dl of blood as of 2012, down from aprevious 10 µg/dl [4,5].
The alterations in hematological changes serve as
the earliest indicator of toxic effects on tissue [6]. Anemia

may result when the cell membranes of RBCs become
more fragile as a result of damage to their membrane [7].
According to Harkness and Wagner [8], meanhemoglobin levels for rodents vary from 10 to 17 g/dL.
High levels of lead exposure during gestation andlactation can severely damage heme synthesis [9].
Hallen et al., [10] established that lactational lead
exposure results in far higher (6 times) intoxication thanplacental intoxication. Maternal Pb can also betransferred to infants during breastfeeding. The placentaand breast ensure that the developing fetus and young
child are exposed early [11].
While studies on the impact of lactational Pb intoxicationon haematological indices remain very scanty, mostprevious studies were limited to only one dose, and studies
limited to a particular period post weaning. This study was
therefore designed to investigate the effects of lactational
lead intoxication on pups’ packed cells volume (PCV) andtotal white blood cells count at different ages and doses
following weaning.

Received September 12th 2016. Accepted October 2nd 2016. Address for correspondence: Odukoya SOA, PhD, Department of
Anatomy and Cell Biology, Obafemi Awolowo University, Ile-Ife, Nigeria; phone: +2348036153842; +2348076959940; e-mail:goshevnigeria@yahoo.com

Fiziologia - Physiology • 2016.26.3(91)13MATERIALS AND METHODS
Chemicals
Lead II Acetate trihydrate (Pb(CH3COO2).3H2O) was a
product of Guangzhou Jinhuada Chemical Reagent Co.,
Ltd, China. Other chemicals and reagents were of analyticalgrade and were procured locally.
Animals and lead acetate treatment
Wistar rats bred in the animal holdings of the Departmentof Anatomy, Bowen University Iwo, Osun state, Nigeria wereused for this study. Sexually mature rats (120-150 days old)
were mated under standard laboratory conditions at aconstant light/dark cycle. The pregnant rats were isolated into
separate cages and fed with standard rat chow from Ladoke
Feeds, Ibadan and water was supplied ad libitum. The day
each dam was littered was marked day 0.5.
Throughout the lactating period (3 weeks postnatal), 3
groups of nursing dams were exposed to varying doses oflead acetate in their drinking water [lower dose: 10 mg/dL[12]; moderate dose: 30 mg/dL [13]; and higher dose: 70
mg/dL [14]. We expected that the pups were indirectly
exposed to Pb via the dams’ breastmilk [11]. Control dams
were administered distilled water, and so their pups had nolactational Pb exposure.
Collection of biological samples
At postnatal day 22 (P22), i.e. one-day post-weaning,
P60, P90 and P120, pups (n = 6) from each group wereeuthanized using 40 mg/Kg body weight sodium pentobarbital(IP). The total body length and whole body weight for eachanimal were determined. The thorax was exposed to gain
access to the heart; cardiac puncture was performed tocollect blood into heparinized specimen bottles.
White blood cell countThis was done with Hawskley haemocytometer (MarlBorough business park, lancing, UK).). The white beadpipette is filled with blood up 0.5 marking. It is then diluted
with Turk’s solution (1:20) to the 11th gradation on thepipette. The diluted fluid was then released unto thecounting chamber and covered with cover slip. The counting
chamber was then placed under the microscope andexamined with scanning (X4), low power (X10), and highpower (X40) objective lens through which the counting wasdone. Results were expressed as counts per mm3.
Packed cell volume (PCV)
In the determination of PCV, the heparinized capillary
tube was filled with blood through one end with the otherend secured with plasticine. The capillary tubes were thenplaced in the micro hematocrit centrifuge, well counterbalanced and spun for 15 minutes at 3000 revolutions per
minute (rpm). Results were expressed in percentages.

14

Quantification of blood lead
This procedure was carried out to establish theconcentration of lead in the blood of the pups at the point ofsacrifice. For each of the rats, a sample solution of whole
blood and 2M concentrated H2SO4 was prepared by diluting
into a specimen bottle, 1ml of whole blood with 3ml of 2M
concentrated H2SO4 (ratio 1:3). The acid was used to digest
the red blood cells to release lead within the blood cells. The
solutions were centrifuged for 15 minutes at 4000 rpm. Eachsupernatant was decanted into a new heparinized specimen
bottle and the centrifugation was repeated again to ensurethat the supernatant was clear. The clear supernatants were
decanted into specimen bottles and each sample wasanalyzed using the atomic absorption spectrometer (AAS).
The AAS was set at the standard wavelength for lead
analysis; 283.3nm. Five standard lead analysis solutions (0,2.5, 5, 10 & 20 ppm) were run through the AAS followed byeach sample. The lead concentration in the serum wasrecorded by the AAS.

RESULTS
There was statistically significant difference between
the control (0.237 ± 0.036; 0.541 ± 0.123; 0.633 ± 0.314
and 1.547 ± 0.567, Mean ± SEM) and the treated groups
10mg/dl (7.330 ±1.965; 5.928±1.013; 5.092±0.962 and4.568±0.009 Mean ± SEM); 30mg/dl (20.722 ±1.631;
15.416±1.844; 7.356±1.356 and 5.120±0.267, Mean ±SEM) and 70mg/dl (26.686 ±1.548; 20.656±1.712;
15.782±0.478 and 12.187±0.115, Mean ± SEM) at thedifferent stages of investigation (Postnatal days 22,60,90
and 120), P<0.01. Although almost negligible whencompared with the treated animals, the control animals still
showed some levels of Pb in their blood; this shows thepersistence of Pb presence in the environment. * represents
statistical significance at P>0.01 compared with the control(Figures 1 and 2).

Fig. 1. Mean blood lead levels (µg/dl)

Fiziologia - Physiology • 2016.26.3(91)groups showed statistically significant low PCV at P120
compared with the control (52.830 ±0.833); 10mg/dl(50.830 ± 1.376); 30mg/dl (47.830 ± 0.872); 70mg/dl(45.670 ±0.667). * represents statistical significance atp>0.05 compared with the control (Figures 4 and 5).
While the control group showed initial increase from P21
to P60 and subsequent stability, 10 mg/dl group was
unstable while 30 mg/dl and 70 mg/dl groups showed initial
increases from P21 to P60 with subsequent statisticallysignificant decreases in PCV as presented above. p>0.05

Fig. 2. Mean packed cell volume (%)

Fig. 4. Control vs. BLL

Fig.3. Mean white blood cell count (mm3)The percentage reduction in BLL within each group at
P60, P90 and P120 from P21are:10mg/dl (68%; 42% and20%); 30mg/dl (57%; 25% and 10%) and 70mg/dl (64%; 37%
and 16%) respectively. This shows closely related but variant
rates of body Pb elimination across the groups (Figure 3).
Effects of Lactational Lead Exposure
on Packed Cell Volume in Rats
The PCV in the control and the treated groups initiallyshowed some level of constancy that is not significantly
different at postnatal days 22 and 60, until postnatal day 90
where 30mg/dl and 70mg/dl showed some statisticallysignificant low levels of PCV, and postnatal day 120 when
all treated groups expressed some statistically significant
low levels of PCV compared with the control.
No statistically significant difference was recorded
among the treated groups and the control at P22 and P60(p>0.05). Compared with the control (52.830 ± 0.749),
30mg/dl (49.830 ± 1.222) and 70mg/dl (47.830 ± 0.654)
groups were significantly lower at P90, while all treatedFiziologia - Physiology • 2016.26.3(91)Fig. 5. Mean blood lead level (µg/dl)

Effects of Lactational Pb Exposure
on Total White Blood Cell Counts in Rats
The initial reaction of white blood cells to lead wasobserved as significant level of leukocytosis in 30mg/dl
group and slightly seen in 10mg/dl group (not statisticallysignificant), by postnatal day 60, the two groups had shownstatistically significant increase in total white blood count. By
postnatal day 90, the 10mg/dl group had fully recovered15

while the 30mg/dl group was still slightly higher than thecontrol group but none was statistically significant. Up till
postnatal day 90, the 70mg/dl group was consistently
leukopenic at a statistically significant level when comparedwith the control. By postnatal day 120, all the treated groups
exhibited no significant difference from the control.
Only the 30mg/dl group (6408.33 ± 386.095) showedsignificantly higher total WBC count at postnatal day 21
compared with the control (4666.67 ± 180.123). At P60, the10mg/dl group (7247.17 ± 155.996) and 30mg/dl group(7238.33 ± 201.501) showed statistically significant higher
levels of total WBC count compared with the control (5858.33± 166.542), whereas these groups showed no statisticallysignificant difference from the control at P90 and P120. At
P21, P60 and P90, the 70 mg/dl group (3666.67 ± 299.692;
2541.67 ± 159.904 and 2883.00 ± 231.541, respectively)
showed statistically significant lower total WBC comparedwith the control, but not significantly different from control at
P120. * represents statistically significant difference at p>0.05
compared with the control (Figures 6 and 7).

Fig. 6. Mean packed cell volume

Fig. 7. Mean total WBC / mm3

16

DISCUSSION
This study evaluated age-dependent haematologicalcharacteristics in lactational Pb-exposed rats at certain
postnatal time points. We have shown that while PCV
remained largely undisturbed at 21 and 60 postnatal daysin rats exposed to lead via the breast milk, total white blood
cell counts were significantly reduced in the early postnatal
period in these pups, especially at the highest Pb dose of
70 mg/dl. Lower doses of Pb showed significantly higher
total WBC count, a probable initial response to Pb induced
inflammation.
Hallen et al., [10] reported that lactational lead
exposure results in far higher (6 times) intoxication thanplacental intoxication. Hua-Wang [15] also showed that
there is only a difference of 3% between blood lead levels
and breast milk lead levels in rats [16,17]. It thus implies
that lead concentrations in breast milk are almost as highas lead concentrations in blood. Hence, in our study, we
anticipated that the high blood lead levels recorded bymeans of atomic absorption spectrometer was suggestiveof proportionally similar lead levels in the exposed dams’breast milk, and by extension, in the pups fed from sucked
Pb-contaminated milk.
Several hematological perturbations have beenrecorded sequel to lead intoxication. Kales et al [18]showed that lead poisoning was associated with basophilicstippling, low hemoglobin and higher protoporphyrin.
Findings from studies by Yu [7] also showed that the initial
impact of lead poisoning is to alter the cell membrane of
red blood cells, and it was subsequently demonstrated byPatrick [19] that 99 % of the circulating lead in blood isfound in red blood cells and only 1% in the plasma.
Therefore, the absence of significant differences in PCV
between the control and lead-treated groups in the present
study further suggest that the initial impact of leadpoisoning on RBC is more of qualitative (morphologic)
perturbations rather than quantitative impact, as initiallyreported by Sachdev and Veena [9].
On the other part, statistically significant differences in
the PCV were observed at the longer duration
post-exposure (90 and 120 days). This might be anindication of hematopoietic perturbation. It has been
established that the half-life of lead in the bones may be as
long as 20-30 years in man, but only weeks to months insoft tissues and about 30 days in the blood [20]. Lead also
inhibits ferrochelatase (haemsynthetase) which catalyses
the introduction of ferrous iron into the porphyrin ring toform heme, a process in haematopoiesis [21].
Lead poisoning has been implicated in anemia,
reticulocytosis, and basophilic stippling since thebeginning of the century. Lead binds copiously to the redcells [22] with about 50 times as much found within thebone marrow [23]. These abnormalities are more
pronounced in the erythroid series, especially ineffective
erythroid hyperplasia [18].
Fiziologia - Physiology • 2016.26.3(91)When lead enters into a biological system either
through skin absorption, inhalation and/or ingestion, thefirst place of call is the blood which would circulate it roundthe body and it is first deposited in soft tissues and thenhard tissues e.g. bones, teeth [6,24]. The bones eventuallyharbor the highest level of lead. This means that high levelof lead in the bones would potentially disrupt the normal
process of hematopoiesis in the bone marrow, and thismay proceed for a long time due to slow deposition of leadin bones [19,25,14].
On the other hand, Peris and Salibian [26] reported
significant increase in reticulocytes as an early response
to Pb intoxication. Noori Mughai et al., [27] associated thisincrease in total leukocyte count mainly to increase innuetrophil and monocyte counts, and an insignificant
increase in lymphocytes count. This early response to Pb
intoxication was explained by Yagminas et al., [28] aschanges due to Pb induced inflammation. These findings
are only consistent with our findings in the 10 mg/dl and 30
mg/dl groups.
Noori Mughai et al. [27] reported initialthrombocytopenia after intoxication followed bythrombocytosis. Sudakova et al. [29] reported leucopenia(low WBC count) as an initial response in acute plumbism
which was consistent with our findings in the 70 mg/dl
treated group, they reported a disappearance of this low
WBC count after acute plumbism was resolved which also
was consistent with our findings in this group.

CONCLUSION
This study has been able to demonstrate that Pb
exposure during lactation period is a means of Pb
poisoning in rat pups, and that high concentration of Pb inmaternal breast milk is associated with reductions in WBC
counts in the pups at each of the days studied. Thesefindings suggest that high lactational Pb is associated with
post-lactational impairment of the immune system andanemic responses in rat pups.
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Effects of Pollutants. CRC Press, 2005.
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10. Hallén IP, Jorhem L, Oskarsson A. Placental and lactationaltransfer of lead in rats: a study on the lactational process andeffects on offspring. Arch Toxicol. 1995; 69 (9): 596-602.
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12. Dietrich KN, Krafft KM, Bornschein RL, Hammond PP,
Berger O, Succop PA. Low-level fetal lead exposure effecton neurobehavioural development in early infancy.
Pediatrics. 1987; 80: 721-30.
13. Sokol RZ. Hormonal effects of lead acetate in the male rat.
Biol Repro. 1987; 37(5): 1135-8.
14. Avalos Javier. Public Health Goal for Lead in Drinking Water.
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Protection Agency; Office of Environmental Health Harzard
Assessment, Pesticide and Environmental Toxicology
Branch, California State, U.S.A. 2009; 1-45.
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Yu T, Zhang Y, Meng XH, Xu DX. Maternal lead exposure
during lactation persistently impairs testicular development
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KorschMJ and Cameron MA. Mobilization of lead from theskeleton during the postnatal period is larger than during
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Jacobson SW, et al. Higher infant blood lead levels withlonger duration of breastfeeding. J Pediatrics. 2009; 155(5):663-667.
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Saper. Hematopoietic toxicity from lead-containing
Ayurvedic medications. Med Sci Monit. 2007; 13(7):CR295-CR298.
19. Patrick L. Lead toxicity: A review of the literature. Part 1:
Exposure, evaluation, and treatment. Altern Med Rev. 2006;
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20. Verstraeten SV, Aimo L, Oteiza PI. Aluminium and lead:molecular mechanisms of brain toxicity. Arch Toxicol. 2008;
82(11): 789-802.
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22. White JM, Selhi HS. Lead and the red cell. Br J Haematol.
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25. Juberg R. Lead and Human Health: an update.
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1726. Peris Fink NE, Salibian, A. Hematological parameter in
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Sagheb, Mohammad Barbarestani. Effects of chronic lead
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18

28. Yagminas AP, Franklin CA, Villeneuve DC, Gilman AP, Little
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exposure. Toxicol. Genet. 1983; 17: 3-7.

Fiziologia - Physiology • 2016.26.3(91)INTEGRATING PHYSIOLOGYIN THE “URINARY SYSTEM” MODULECARMEN TATU, CARMEN PITIC, CARMEN PANAITESCU, MONICA COTARCĂ,FLORINA BOJIN, GABRIELA TĂNASIE, DELIA MINCĂ, DANIELA PUŞCAŞIU
University of Medicine and Pharmacy “Victor Babes” Timisoara

ABSTRACT
Victor Babes University of Medicine and Pharmacy Timisoara ran a project which included a series of extracurricular
modular courses to evaluate the impact of integrated medical curricula. “The Ur inary System” was part of this effort.
The modular course ensured vertical and horizontal integration in order to provide students with thoroughunderstanding of kidney structure and functions, as well as selected features of various kidney pathologies and wasoffered as two summer school sessions (in 2013 and 2015 years). The course consisting of lectures and practicalapplications was scheduled for a week, also including workshops and small group discussions in a problem -basedlearning setting. A total number of 66 students were selected based on letters of intent and interviews. At the end of
the summer school, students’ knowledge was assessed by a multiple choice question (MCQ) test, while the modular
course itself was evaluated by students through a questionnaire. MCQ results were above average, which was
expected since this was a free chosen activity by students who were among the first 10% of their respective year of
study. At the end of the course, the students were able to discriminate between normal and pathological results and tocorrelate clinical presentation with results, and acquired a better understanding of the newest developments in thefield, while the teachers gained experience in working together. Overall, the summer school experience was highly
appreciated, as seen from the analysis of answers to the questionnaires for students and teachers. Conclusions: (1)Small group teaching enhances the quality of teaching and learning processes. (2) Close interaction with students
enables appropriate feedback for improvement of teaching process. (3) An integrated approach to teaching andlearning allows students to better integrate pre-clinical knowledge into clinical context.
Key words: medical education, integrated modular course, small group discussions

INTRODUCTION
Medical education programmes worldwide differ
remarkably, with a wide range of approaches on suchaspects as quality standards, curricula or methods ofdelivering education. World Health Organisation
acknowledged that the great differences in the contents ofmedical curricula, leading to a high variation in thecompetences of medical graduates, should be reduced by
setting “Global Minimum Essential Requirements” formedical professionals across the world [1]. There has beenmuch debate about the quality of medical education during
the last decades, centred on the idea of connecting
education with society needs by tailoring appropriate
medical curricula [2] and promoting competence-based
education [3].
An integrated medical curriculum would serve theultimate goal of preparing medical graduates able to copewith current healthcare demands. Integration of medical
curriculum, either horizontal or vertical, or both has been

already implemented or is in progress in many medical
schools around the world, although large differences
between countries exist, with the highest percentages ofschools delivering integrated curricula found in thedeveloped countries [4]. The aim of integration is to bring
together basic and clinical sciences across the entire
curriculum in order to enable the medical student toconcurrently develop theoretical knowledge and practicalskills [5].
Horizontal integration, i.e. between disciplines in thesame phase of the traditional curriculum (either pre-clinicalor clinical) and vertical integration of disciplines in different
phases (pre-clinical and clinical) [6] are both required toachieve a fully integrated medical curriculum. Designingand implementing a modular curriculum is a way toaccomplish integration at all levels, as several disciplines
are grouped under a single module. Nevertheless, it
requires careful planning of modules in the curriculum inorder to reach the desired educational outcomes [7].

Received August 2nd 2016. Accepted September 20th 2016. Address for correspondence: Associate Professor Carmen Tatu,
MD, PhD, Department of Functional Sciences, Discipline of Physiology, “Victor Babes” University of Medicine and Pharmacy
Timisoara, Eftimie Murgu Square No. 2A, RO-300041, Timisoara, Romania; phone/fax: +40256490507; e-mail:carmen.tatu@umft.roFiziologia - Physiology • 2016.26.3(91)19

DESIGN
Such an approach was attempted by the Victor Babes
University of Medicine and Pharmacy Timisoara (UMFVBT)
within the eMediqual (European Quality and Professional
Competence in Medical University Education andManagement’, Sectorial Operational Programme Human
Resources Development 2007 – 2013, ref. POSDRU
86/1.2/S/63815) project (2011 – 2013) in which
curriculum development was defined as a generalobjective. Several extracurricular modular courses were
designed to evaluate how an integrated medical
curriculum would improve the quality of both teaching andlearning. One of these was “The Urinary System”, asummer schools offered by UMFVBT to students enrolled
in the 2nd to 5th year of study. It was first organised in2013 as a task in the project and then again in 2015 due
to its success among students.
The modular course was designed to ensure bothvertical and horizontal integration in order to provide
students with a thorough understanding of kidney structureand functions, as well as selected features of various
kidney pathologies. Horizontal integration was achieved byan interdisciplinary approach, i.e. bringing together
pre-clinical disciplines (Anatomy, Histology, Physiology,
Pathophysiology and Microbiology) on the one hand, andclinical ones on the other hand (Nephrology and Urology),
with Semiotics linking the two levels. Vertical integration
between pre-clinical and clinical disciplines was further
supported by adding paraclinical disciplines (ClinicalLaboratory and Medical Imaging). Case scenarios provided
within workshops and small group discussions served theaim of applying integrated knowledge acquired frompre-clinical, paraclinical and clinical disciplines. Even
immunology basics were integrated with urology andnephrology in the context of kidney transplantation.
Six months before the first session of the actual course(July, 15-20, 2013), a team of 18 professors started todevelop the summer school programme during a series ofmeetings in which they formulated the scope of integration,
learning objectives and strategies, themes, teaching
methods, clinical cases and assessment method. This
team work facilitated integration between pre-clinical,
paraclinical and clinical knowledge, ensuring adequate andrelevant information was provided to students in order tohelp them acquire a comprehensive view on the normal
and pathological kidney function, as well as to develop
their problem-solving skills and clinical abilities.
Ten major themes, starting with the study of nephronand ending with the management of renal patient, werebuilt around several case reports presented to students
as a premise of teaching and ground of discussion.
Seven hours of lectures and practical applications werescheduled each day, with lectures followed by workshops
and small group discussions or practical works. The
entire module was conceived so as to allow the use of20

both expository and interactive teaching methods, withproblem-based learning at the core of the educational
approach. At the very beginning of the modular course,
the students were confronted with a case scenario (Box 1)that needed to be solved lately, after they would haveacquired enough knowledge to attempt a diagnosis. Thisset the stage for the theoretical information delivered
during the week in the form of lectures. Physiological andpathophysiological mechanisms described in the first partof the module were then used to build knowledge on
various renal pathologies. The goals were to facilitate
student understanding of the urinary system and normal
vs. altered functions, and to provide theoreticalbackground and practical tools that would allow them torecognise pathological changes involved in various
kidney diseases.
Box 1.
Case scenario – Diabetes-associated chronic
kidney disease
A 42-year-old male patient presents with a complaint ofpolyuria and xerostomia aggravated during the last 6months. He is overweight (BMI 27 kg/m2), his bloodpressure is 170/100 mmHg, heart rate 80 bpm, sinus
rhythm.
He has irregular eating habits with frequent consumptionof fast-food meals and carbonated drinks, a sedentary
lifestyle and he is a smoker.
One month before presentation his health status wasevaluated at work: blood pressure 145/70 mmHg, heart
rate 80 bpm, sinus rhythm, fasting glucose 105 mg/dl.
Current laboratory data: blood glucose 225 mg/dl,
HbA1c 11.1%, cholesterol 241 mg/dl, HDL-C 37 mg/dl;
triglycerides 369 mg/dl, LDL-C 130 mg/dl, TGO/AST 19
U/L, TGP/ALP 30 U/L, creatinine 1.40 mg/dl, urea 60
mg/dl, eGFR/MDRD 56 ml/min/1.73m2; ACR 300 mg/g
urinary creatinine; ionogram within normal limits.
Workshops and small group discussions
Workshops were focused on specific topics and newcase scenarios were introduced to students which wereafterwards solved during the small group discussions
part. For example, the Physiology workshop “Functional
Exploration of the Kidney” was designed around four
themes: Fluid and Electrolyte Balance, Acid-BaseBalance, Urinalysis and Renal Excretion of Protein
Catabolic By-Products. Theoretical information,
including normal values for each of the parameters were
provided and multiple-choice questions were asked at
key points to help students summarize and self-assess
their learning progress (see Box 2 for an example).
Students were encouraged to collectively find thecorrect answer by analysing and correlating case dataas compared with normal parameters and had to explain
their choice.
Fiziologia - Physiology • 2016.26.3(91)Box 2.
Acid-base balance(decompensated metabolic acidosis)
Evaluate the acid-base balance given the following
values:pH 7.52;[HCO3-] 40 mEq/l;
PCO2 48 mm Hg.
The second part of workshops was devoted to small
group discussions (5 - 6 students/group), which wereintended to enhance critical thinking, effective
communication and collaborative team-work. Within the
Physiology workshop each group was assigned a casescenario (Box 3) on one of the four previously discussed
themes and a few questions were formulated to help themestablish a direction for discussions. The groups had 20 25 minutes to analyse the case data and prepare ananswer, then each group introduced its case to the classand discussions were encouraged by the facilitator. Aftereach group’s presentation, the students were expected to
discuss, ask questions, debate, compare and argue theirconclusions in such a way that they would have reached aconsensus. A major aim of these sessions was to provide aframework for student-centred learning, with the teacher
assuming merely the role of guiding the process. Teachers
also set the rules and expectations at the beginning of
small group discussions, clarified concepts, encouraged
active participation of all group members and summarized
the conclusions at the end of sessions.
Box 3.
Case scenario – Kidney function
Evaluate functional status of kidney in a 65-year-old
female patient suffering of diabetes mellitus for 15 years
who complaints of leg edema, nocturia and hematuria.
Her blood pressure is 180/100 mm Hg.
Blood tests: urea 80 mg/dl, creatinine 2.5 mg/dl,
glucose 240 mg/dl.
Urinalysis: diuresis 3000 ml/day, density 1010 g/cm3,
pH 4.5, proteins (+++), blood glucose (++), ketones (+),
bilirubin (-), urobilinogen (+).
Urine sediment test: 6-8 WBC/per field, 18-20 RBC/perfield, 8-10 RBC casts/per field.
Points to consider: Urinalysis parameters indicative for kidney
function.
 What suggest the values of diuresis, urine densityand pH in the case?
 Correlation of urinalysis with other data.
In the last day of the module, students were asked torecall the first case scenario (Box 1) and to make adiagnosis using and integrating knowledge acquired
throughout the course. The module ended with a sessionof joint teaching, intended to correlate knowledge across all
Fiziologia - Physiology • 2016.26.3(91)disciplines involved. The multidisciplinary approach of renal
patient has incorporated content from basic, paraclinicaland clinical disciplines focused on the process of
integrating data to achieve a high level of confidence when
making a diagnosis.
Student selection
A total number of 66 students participated, 35 in 2013
and31 in 2015. Students were selected based on letters ofintent and interviews. In their letters of intent, the students
were asked to explain why they consider useful to attend
the course. Not surprisingly, most of the students declared
their main aim was to acquire new medical information in
general or specifically regarding the urinary system,
although some 30% of them also mentioned the integrative
perspective would help them become better doctors, withsome also stressing the importance of the modular course
for their personal and professional development. Theinterviews were based on a set of five questions, with eachanswer being awarded 1 to 5 points by two interviewers
individually and the overall score was used to establish ahierarchy at the end of the interviews. It was considered
this approach will help ensure both transparency andequity. Although these questions were previously prepared
and asked, the interviewers focused on the discussions
raised and not merely on the simple answers of students.

RESULTSAt the end of the course, students’ knowledge wasassessed by a multiple choice question (MCQ) test, while
the modular course itself was evaluated by students throughquestionnaires. The overall results showed students wereable to discriminate between normal and pathological results
and to correlate clinical presentation with results. They alsoacquired a better understanding of the newest developmentsin the field. By developing and implementing the module,
teachers gained experience in working together, as they had
to harmonize course contents in order to avoid repetition or
resolve discrepancies.
Student assessment
Each teacher provided 10 MCQs, out of which 50
were selected for the final test, with 20 discipline-basedquestions and 30 integrated questions. Overall, MCQ
results were above average, which was expected since thiswas a free chosen activity by students who were among
the first 10% of their respective year of study. All thestudents scored over 65%.
Module evaluation
A questionnaire for students (Box 4) with 8 closed and2 open questions was formulated to evaluate the modular
course and to get their feedback. A five-level Likert scale21was used (where 1= strongly disagree and 5=strongly
agree) for the close-end questions. Analysis of student
questionnaires (Figures 1 and 2) showed a high level ofsatisfaction (average score range: 4.31 – 4.73 in 2013session and 4.5 – 4.96 in 2015).
Box 4.
Questionnaire for students
1. Lectures answered my needs and expectations.
2. Workshops helped me understand theoreticalconcepts taught during lectures.
3. Teaching methods were interactive.
4. Lecture materials were well structured.
5. Labs were adequately equipped.
6. This module increased my interest in the subject.
7. This module increased my knowledge of urinary
system.
8. Adequate methods were used for student
assessment, which mirrors my knowledge.
The two open questions were intended to give students
the opportunity to comment on the strengths andweaknesses of the module as perceived by them. They
were instructed to address the modular course as a whole,as well as any other particular aspects they might
considered relevant, including but not limiting to thoseevaluated in the closed questions. Most students
appreciated the better level of interaction with teachers,
particularly during the small group discussions; theintegration of basic sciences (including Physiology) withclinical scenarios is facilitating learning and comprehension;
the integrated approach of medical education is better than
the traditional one. Among the weaknesses of the module,
students pointed out that too much and sometimes
unnecessary theoretical information was delivered. They
found the integrated approach more challenging, although
hard to pursue at times.

Fig. 1. Module evaluation. Urinary System - 2013

22

Fig. 2. Module evaluation. Urinary System - 2015

DISCUSSION AND CONCLUSIONS
During the last decades, medical education
throughout the world is undergoing major changes, since
the need for new, fresh paradigms was recognised. A
shifting point in the views on medical education camewith the adoption of Edinburgh Declaration [8] during theWorld Conference on Medical Education in 1988. The setof proposed actions included building the curriculum toachieve professional competence and social values,
ensuring the national health priorities are reflected in the
curriculum content and integrating education in practicethrough a problem-based approach. The declaration
triggered an extensive reform of medical education
systems around the world, as it was agreed by medical
educators in all the regions of the world [9].
The Bologna Declaration [10] in 1999 has furthered thereform within the European Union, this time encompassing
the educational process as a whole, regardless of the fieldof study. It was aimed to increase the competitiveness of
the EU higher education, while making national systems
more comparable and compatible. The developments since
are commonly referred to as the Bologna Process. The
principle of harmonization involved, among others, thecreation of a two-cycle structure, first degree –undergraduate level (three or four years) and master’s
degree (five years from entry), with the third cycle, doctorallevel (seven or eight years since entry) [11], added later.
However, medical educators have been concerned that the
Bologna process would not be suitable to medicine,especially when it comes to the two-cycle framework,
which was understood as a pre-clinical (first cycle)/clinical(second cycle) model [12]. Two projects, MEDINE(2004-2007) and MEDINE2 (2009-2013) involving all the
EU member states, promoted by universities and fundedby the European Commission, have addressed various
aspects of medical education in connection with the
Bologna process [13]. A report [14] on common learning
outcomes/competences for the Bachelor of Medicine inEurope was issued following the surveys conducted within
Fiziologia - Physiology • 2016.26.3(91)the second project, concluding that a fully integrated
curriculum with early clinical experience is possible anddesired. A European learning outcomes framework basedon integrated curricula would facilitate the application of
Bologna process to medical education [15] and this clearlyshows its principles are able to drive quality improvement
and harmonization of medical education throughout
Europe.
Romania, as a signatory of the Bologna Declaration,
has started a top-down reform of its higher education
system, which, however, remains rather technical in nature[16], with implementation of the two-cycle model, theEuropean Credit Transfer System or the quality assurance
system. Moreover, as in the case of other European
countries, modular study programmes are merely
attempted, with less than 25% of elective courses, while
the medical sciences programmes are not designed
according to the Bologna two-cycle model [17].
Shifting from a traditional to an integrated curriculum
requires much effort in order to correlate subjects
previously taught in different, often unconnected
departments, but there are several advantages that neednot to be neglected: lower fragmentation of medical
courses, motivation and shaping attitudes of students,
higher effectiveness of teaching, higher level objectives,
improved staff communication and collaboration andrationalization of teaching resources [6].
This module has shown the efforts to integrate
pre-clinical, paraclinical and clinical knowledge using
student-centred teaching strategies with problem-basedlearning as central approach are very useful for fine tuning
both teaching and learning. Three major conclusions canbe drawn, as follows: (1) Small group teaching enhances
the quality of teaching and learning processes. (2) Close
interaction with students enables appropriate feedback for
improvement of teaching process. (3) An integrated
approach to teaching and learning allows students to better
integrate pre-clinical knowledge into clinical context.
ACKNOWLEDGEMENT
European Quality and Professional Competence in
Medical University Education and Management’, Sectorial
Operational Programme Human Resources Development
2007 – 2013, ref. POSDRU 86/1.2/S/63815

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6. Harden RM, Sowden S, Dunn WR. Educational strategies incurriculum development: the SPICES model. Medical
education. 1984;18(4):284-97.
7. Modo M, Kinchin I. A Conceptual Framework forInterdisciplinary Curriculum Design: A Case Study in
Neuroscience. Journal of Undergraduate Neuroscience
Education. 2011;10(1):A71.
8. http://wfme.org/projects/wfme-publications/99-the-edinburghdeclaration/file
9. http://wfme.org/documents/about-wfme/79-wfme-history-of-th
e-first-forty-years-1972-2012/file
10. http://www.magna-charta.org/resources/files/text-of-the-bolog
na-declaration
11. Van der Wende MC. The Bologna Declaration: Enhancing thetransparency and competitiveness of European higher
education. Higher education in Europe. 2000;25(3):305-10.
12. Patrício M, Harden RM.The Bologna Process–A global vision
for the future of medical education. Medical Teacher.
2010;32(4):305-15.
13. http://tuningacademy.org/medine-medicine/?lang=en
14. http://medine2.com/Public/docs/outputs/wp4/DV4.14.1_Sum
mary%20Brochure%20-%20Tuning%201st%20Cycle%20De
grees%20in%20Medicine.pdf
15. Cumming A. The Bologna process, medical education andintegrated learning. Medical teacher. 2010;32(4):316-8.
16. Szolár É. The Bologna Process in Romania. InThe Bologna
Process in Central and Eastern Europe. SpringerFachmedien
Wiesbaden. 2014: 183-222
17. Westerheijden DF, Beerkens E, Cremonini L, Huisman J,
Kehm B, Kovac A, Lazetic P, McCoshan A, Mozuraityte N,
Souto-Otero M, Weert E. Bologna process independent
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Higher Education Area. Final report to the European
Commission.

23

INTEGRAREA FIZIOLOGIEI ÎN CURSUL MODULAR„SISTEMUL URINAR”
REZUMAT
Universitatea de Medicină şi Farmacie Victor Babeş Timişoara a derulat un proiect care a inclus o serie de cursuri
modulare extracuriculare în vederea evaluării impactului generat de o curriculă medical integrată. “Sistemul urinar” afăcut parte din acest demers. Cursul modular a asigurat integrarea verticală şi orizontală, pentru a oferi studenţilor
înţelegerea aprofundată a structurii şi funcţiilor renale, precum şi anumite caracteristici ale diferitelor patologii renale şia fost organizat sub forma a două şcoli de vară (în anii 2013 şi 2015). Modulul, constând în cursuri şi aplicaţii practice,a fost programat pe perioada unei săptămâni şi a inclus workshopuri şi discuţii pe grupuri mici în format PBL(problem-based learning). Au fost selectaţi 66 de studenţi în total, pe baza scrisorilor de intenţie şi a interviurilor. Lasfârşitul şcolii de vară, cunoştinţele acumulate de studenţi au fost evaluate printr-un test tip MCQ, iar cursul modular afost evaluat de studenţi printr-un chestionar. Rezultatele studenţilor au fost peste medie, ceea ce era de aşteptat,
deoarece a fost vorba de o activitate liber aleasă de studenţi plasaţi între primii 10% din anii lor de studii. La sfârşitul
cursului studenţii au fost capabili să facă diferenţa între rezultatele normale şi patologice, să coreleze cazurile clinice cu
rezultatele şi au dobândit cunoştinţe mai aprofundate în domeniu. În acelaşi timp profesorii au câştigat experienţă în cepriveşte munca în echipă. Experienţa acumulată în cadrul şcolii de vară a fost unanim apreciată, aşa cum a reieşit dinanaliza răspunsurilor la chestionarele date studenţilor şi profesorilor. Concluzii: (1) Metoda predării/învățării în grupuri
mici îmbunătățește atât calitatea procesului de predare, cât și a celui de învățare. (2) Interacțiunea strânsă cu studenții
facilitează obținerea feedback-ului necesar pentru perfecționarea procesului de predare. (3) Abordarea integrată apredării şi învăţării permite studenţilor o mai bună integrare a cunoştinţelor preclinice în contextul clinic.
Cuvinte cheie: educaţie medicală, curs modular integrat, discuţii pe grupuri mici

24

Fiziologia - Physiology • 2016.26.3(91)INTERACTION BETWEEN ALEXITHYMIAAND OBSESSIVE - COMPULSIVE BEHAVIORTO HEMOPHILIAC PATIENTSLAVINIA HOGEA*1, LAURA NUSSBAUM*2
1Psychology Discipline, Neurosciences Department, University of Medicine and Pharmacy “V. Babeș” Timișoara
2Pedopsychiatry Discipline, Neurosciences Department, University of Medicine and Pharmacy “V. Babeș” Timișoara, Seniorspecialist Pediatric Psychiatry, Head of the Clinic of Child and Adolescent Psychiatry, Timisoara*The authors have equal rights - first authors

ABSTRACTIntroduction. Hemophilia is "a genetic transmitted disorder", it belongs to the category of rare diseases, diseases that facedevastating and crippling developments, affecting each person beyond physical problems.
Material and methods. The present study is descriptive and analytical and took place within the Medical Center forevaluation and rehabilitation "Cristian Serban" Buziaş and Pediatric Clinic Timisoara, 100 patients that have presented
themselves for evaluation of comprehensive treatment and recovery were monitored.
Results. Our results show that the prevalence and levels of alexithymia were higher in patients with low health perspective,
with poor social relations and with obsessive-compulsive behaviors.
Conclusions. Psychological consequences of patients experiencing a chronic condition and with complications are numerous:
the possibility of disability, dependency of the basic family, emotional and behavioral disorders, the inability to sustain
themselves, difficulties of integration into social life.
Key words: haemophilia, alexithymia, obsessive-compulsive behavior

INTRODUCTION
The presence of the malady in the patient's life
compels him to deal with the changes imposed on his bodyfor all his life. This event can be devastating both physicaland psychical and loads the patient with a series of losses
that customize this pathology: loss of independence;
lifestyle changes; uncertainties regarding the future; thefeeling of helplessness; friends separation; changes inphysical health; changes in body image; emotional andbehavioral disorders [1].
Aim: Hemophilia form, obsessive-compulsive behaviors
level, general health, physical functioning, social relations
domain may be predictors of the alexithymia level inhemophiliac patients.

MATERIAL AND METHODS
The batch for clinical study is made up of 100 (50%)
patients with hemophilia A (35.5%) and B (14.5%).
By the form of severity there can be found 83 subjects
diagnosed with severe hemophilia (41.5%), 13 subjects

diagnosed with medium hemophilia (6.5%) and 4
diagnosed with mild hemophilia (2%) (Figure 1).

Fig. 1. Distribution by type of hemophilia patients

To verify this hypothesis we have used the results
obtained at SECOC (scale for evaluation ofobsessive-compulsive Behaviors-Yale-Brown Scale) tomeasure the level of obsessive-compulsive behaviors andfor the measurement of alexithymia results from TAS(Toronto Alexithymia Scale) have been used. For the

Received September 10th 2016. Accepted October 2nd 2016. Address for correspondence: As. Professor Lavinia Hogea, PhD,
Department of Psychology, “Victor Babes” University of Medicine and Pharmacy, no. 2 Eftimie Murgu Square,
RO-300041Timisoara, Romania; phone: +40745042035; e-mail: laviniahogea@yahoo.com

Fiziologia - Physiology • 2016.26.3(91)25measurements of the dimensions of general health andphysical functioning the results of the Questionnaire forhealth and quality of life (MOS- SF–36) have been used,
and to measure the domain social relationships resultsfrom World Health Organization Quality of Life (WHOQOL)
have been used.

were included in the study. According to the data from
Table I, in which the matrix of correlations between
predictors in the model is presented, a situation ofmulticolinearity has not been identified. Analyzing Table II,
we noted that between hemophilia form, alexithymia, pain,
general health, environment, there are not very strong
correlations, which indicates that the possibility of amulticolinearity is reduced [2].

RESULTS AND DISCUSSION
To test the hypothesis a multi-hierarchical linear
regression in two steps was made. Given that both thedependent variable and criterion (alexithymia level) as wellas predictors are measured by means of a numerical scale,it means that the conditions have been met for the use of
linear regression [2]. In step one the criterion form
hemophilia was introduced (medium, severe) because thisvariable to be kept in check, and in step two predictors
subjected for analysis were introduced: the form ofhemophilia, obsessive-compulsive behaviors level, general
health, physical functioning, social relations.
Table. I. Values obtained in the form of haemophilia dimensions,the obsessive compulsive behavior, general health, physicalfunctioning, the social relations and alexithymia

Table III. The value of R² and R² adjusted
Regresion
model

R

R²

R²
Adjusted

1

.002

.000

-.010

2

.549

.302

.264

Predictors: the form of hemophilia, the level ofobsessive-compulsive behaviors, physical functioning,
general health, social relations.
Table IV. The value of significance test F
Regresionmodel 1

Sum ofsquares

Df

Mean ofsquares

F

Sig

.083
15316.027
15316.110

1
98
99

0.83
156.289

.001

.982

6.93

Regresion
Waste
Total

42.43

17.13

Regresionmodel 2Sum ofsquares

Df

Mean ofsquares

F

Sig

100

54.75

22.11

Social relationships

100

11.49

1.13

.000

69.83

12.43

5
94
99

8.116

100

4618.473
10697.637
15316.110

923.695
113.805

Alexithymia

Regresion
Waste
Total

Lot

Mean

Standard
deviation

Form of haemophilia

100

2.79

0.49

Obsesive-compulsive
behavior

100

9.67

General health100

Physical functioning

Table II. The correlation matrix of variables in subject
Form ofhaemophilia

Obsesivecompulsive
behavior

GeneralhealthPhysicalfunctioningForm ofhaemophilia-

Obsesivecompulsive
behavior.076-

Generalhealth

-.16

-.03

-

Physicalfunctioning

-.27

-.18

.52*

-

Socialrelationships

-.03

.13

.13

.25*

Socialrelationships-

To identify any instances of collinearity, the matrix ofcorrelations have been installed between predictors that26

As a result of data analysis it is observed that the firstmodel does not explain better than those based on mean
the data dispersion, F(1.98)= .982, p> .05. Model number
2, which comprises in addition to the form of hemophilia,
obsessive-compulsive behaviors, physical functioning,
general health, social relationships domain, issignificantly statistically better than the one which was
obtained on the basis of the mean of the values F(5.94)=
8.116, p< .01. Of the five predictors, three of them (thelevel of obsessive compulsive behaviors, overall health,and social relations) is presented statistically significant
as it can be seen in table 4. This model is able to explain
in a ratio of adjusted form 26,4% (R² adjusted = .264) of
the evolution of dispersal obsessive-compulsive
behaviors level, while the remaining 73.6% remains
unexplained, because it is due to other factors notentered in the regression model.

Fiziologia - Physiology • 2016.26.3(91)Table V. The predictors value included in the regression model
Predictors

Non-standardized
coefficients

Standardized
coefficie
nts

BStandard
error

Beta

Intercept

109.161

12.904Form ofhemophilia-1.675

2.240

-.067

Obsessive-

.736

.161

Generalhealth-.241

Physicalfunctioning
Socialrelationships

Model 2

t

Sig.

8.459

.000

-.748

.457

.410

4.579

.000

.074

-.331

-3.262

.002

.050

.061

.090

.822

.413

-2.987

.999

-.271

-2.990.004

compulsive
behavior

Furthermore, Table V gives us important information
about those predictors that contribute to the effectivenessof the model as well as about their share within the
regression model. On the basis of the significance test t
shown in Table V, it is found that from the five predictors
included in model three are contributing statisticallysignificant to the evolution of the hemophiliac patients’alexithymia level, i.e. the level of obsessive-compulsive
behaviors, overall health, social relations.
The level of obsessive-compulsive behaviors has apositive value (b =.736) therefore establishes a direct
relationship between alexithymia and obsessive-compulsive
behaviors level. Thus, when the level ofobsessive-compulsive behaviors increase a unit, alexithymia
tends to increase by one unit (specifically with 0.73) underconditions in which the constant influence of other predictors
is kept constant.
Overall health has a negative value (b=.241) therefore
sets an inverse relationship between overall health andalexithymia level. Thus, when it increases by one unit,
alexithymia level tends to decrease with one unit (moreprecisely -0.24) under conditions in which the influence ofother predictors is kept constant.
The field of social relationships has a negative value(b=-2.987) therefore sets an inverse relationship between
the field of social relationships and the alexithymia level.
Thus, when it increases by one unit, the alexithymia leveltends to decrease with one unit (more precisely-2.98) inthe terms on which the influence of other predictors is kept
constant.
Other predictors, contrary to initial expectations
according to statistical results do not significantly influence
the increase or decrease of the level of alexithymia to ailinghemophilia patients.
Fiziologia - Physiology • 2016.26.3(91)

Our results show that the prevalence and level ofalexithymia were higher in patients with low perspective on
health, with low social relations and with obsessivecompulsive behaviors.
In regards to the study of the relationship between
alexithymia and obsessive-compulsive behaviors, it isfound that it is consistent with the results found in otherstudies, which noted that the percentage of patients with aweak or absent perspective of alexithymia is relevant topatients with obsessive compulsive disorder [3, 4].
In the scientific literature, Matsunaga et al. havereported significant correlations in patients withobsessive-compulsive disorder and severe alexithymia[5,6]. The results of the study conducted by Rachman et al.
[7] are consistent with our hypothesis that obsessivecompulsive behavior is predictor for alexithymia, meaningthat the greater the severity of obsessions andcompulsions increase, the higher is the level ofalexithymia.
In another study, a number of subjects 5418 aged
between 30 and 97 years were investigated with the
TAS-20 regarding the level of alexithymia and theperception of quality of life with the HRQOL. The results
were in line with the present study, which included
significant correlations between the scales of quality of life
and alexithymia [8]. Quality of life depends largely on theability of hemophiliacs to adapt to chronic disease and to
the difficult circumstances which may arise.
Health is a fundamental resource for individuals,
communities and societies overall. For the individual, toenjoy a good health is of paramount importance [9].
Lumley et al. examined pairing alexithymia withrelationships and social skills, with physical and mental
health problems. Relations between alexithymia, measured
with the TAS-20, social variables, physical health anddepression were examined both in young healthy adults
and patients.
Alexithymia was associated statistically significant witha deficit of social skills and social relationships. It has been
noted the association between alexithymia, somatizations,depression and physical health, and the social support was
not a significant one [10]. Terry has conducted a survey ona sample of 158 subjects and they were questioned about
the relationship between alexithymia and satisfaction in
intimate relationships: the general satisfaction of therelationship and sexual satisfaction in the relationship. The
analysis showed a negative relationship between themoderate level of alexithymia and both of the measured
variables. These results assist other previous studies
showing a strong connection between alexithymia and arange of interpersonal problems [11].
Alexithymia, defined as the inability to identify or
describe emotions, has been shown to be associated witha large number of the patient's chronic pain. A number of
129 patients with muscular dystrophy and chronic pain
were administered TAS-20, SNR (pain intensity), the27interference of pain (Short Pain Inventory), the SF-36(scales of mental health and vitality). Significantly elevated
TAS scores were significantly associated with the intensity
and the interference of pain, and, in the case of vitality andmental health, associations have been reduced [12]. To
investigate the relationship between alexithymia and pain
in a sample of patients with chronic pain, 100 (67 women,
33 men) patients with chronic musculoskeletal pain werequestioned in this regard by completing the TAS-20,
PANAS, and VAS. For instance, it was observed that there
is a direct relationship between the two, so the painincreases the effect of alexithymia [13].
Roghaye et al. investigated, within a research, the role ofmoderator of self-efficacy in the relationship between
alexithymia and pain (pain severity and disability) in patientswith chronic pain. 100 patients with chronic musculoskeletal
pain, both women and men, participated in this research. All
participants were asked to complete the measurement scaleof alexithymia (TAS-20), self-efficacy questionnaire (CPSEQ)
scale for severity of pain (VAS), and disability questionnaire(PDQ). The results showed that the self-efficacy has lowered
the effect of alexithymia in pain and disability severity level.
So that it can be concluded that the relationship between
alexithymia and pain in patients with chronic pain was
influenced by self-efficacy [14].

CONCLUSIONS
Between the level of obsessive compulsive behaviors
and overall health it is established a direct relationship, inthe sense that the more careful, more precautions by
avoiding knocks and falls, the protection related to the
choice of clothing, the more they assess their overall healthstatus as being better (adjusted R ² = 264).
- The level of obsessive-compulsive behaviors has apositive value (b =.736) therefore it is established adirect relationship between alexithymia and the levelof obsessive-compulsive behaviors.
- Overall health has a negative value (b = -.241), andthus, an inverse relationship exists between the levelof overall health and alexithymia.
- The field of social relationships has a negative value(b = -2.987), obtaining an inverse relation between
alexithymia and social relations level.
- The higher the level of obsessive-compulsive
behaviors increases, the more their need for social
support is greater. Social support explores how theperson felt, engagement, encouragement support
from family and friends.
The relation between the functional physical scale and28

obsessive-compulsive behaviors is a reversed one, whichmeans that daily activity significantly lowers while the levelof protective, reassurance behaviors is increased.
Other predictors, contrary to initial expectations, andaccording to statistical results do not significantly influence
the increase or decrease in the level of alexithymia inpatients suffering from hemophilia.
REFERENCES
1. Cosma A. Hemofilicul îndrumător practic pentru bolnavi,familiile lor şi educatori. Ed. Medicală, 1983, Bucharest.
2. Sava FA. Analiza datelor in cercetarea psihologica. Metodestatistice complementare. ASCR, Cluj-Napoca, 2004.
3. Insel TR, Akiskal HS. Obsessive-compulsive disorder withpsychotic features: a phenomenological analysis. Am J
Psychiatr. 1986; 143:1527-1533.
4. Catapano F, et al. Insight e resistenza nei pazienti ossessiv,
1996; 126-132.
5. Matsunaga H, Kiriike N, Matsui T, Oya K, Iwasaki Y,
Koshimune K, Miyata A, Stein DJ. Obsessive-compulsive
disorder with poor insight. Compr Psychiatry. 2002;
43:150-157.
6. Palomo NAL, Alcoverro FO, Hurtado RG, Guerrero PA.
Thought disorders in obsessive-compulsive disorder. Actas
Esp Psiquiatr. 2003; 31:100-102.
7. Rachman S, Hodgson R. Obsession and compulsions.
Prentice-Hall: Englewood Cliffs, 1980.
8. Aino KM, Samuli I, et al. Alexithymia and Health-Related
Quality of Life in a General Population. Psychosomatics.
2009; 50(1): 59-68.
9. Rapport pour le SPF, 2004
10. Lumley MA, Neely LC, Burger AJ. The assessment ofalexithymia in medical settings: implications forunderstanding and treating health problems. J Pers Assess,
2007; 89: 230-46.
11. Terry P, Humphreys LM, Wood JDA. Alexithymia andsatisfaction in intimate relationships. Personality andIndividual Differences, 2009; 46(1): 43-47.
12. Hosoi M, Molton IR, Jensen MP, Ehde DM, Amtmann S,
O’Brien S, Arimura T, Kubo C. Relationships among
alexithymia and pain intensity, pain interference, and vitality
in persons with neuromuscular disease: Considering theeffect of negative affectivity. Pain, 2010; 149(2): 273-277.
13. Tooyserkani MA, Besharat MA, Koochi S. The ModeratingRole of Positive and Negative Affects on the Relationshipbetween Alexithymia and Experience of Pain in Chronic Pain
Patients. Procedia - Social and Behavioral Sciences, 2011;
30: 154-158.
14. Mirjalili RA, Besharat MA, Koochi S. The Moderating Role of
Self-Efficacy on the Relationship between Alexithymia andSeverity of Pain in Chronic Pain Patients. Procedia - Social
and Behavioral Sciences, 2011; 30: 149-153.

Fiziologia - Physiology • 2016.26.3(91)INTERACȚIUNEA DINTRE ALEXITIMIEȘI COMPORTAMENTUL OBSESIV-COMPULSIVLA PACIENȚII CU HEMOFILIEREZUMAT
Introducere. Hemofilia este „o dezordine genetică transmisă”, face parte din categoria bolilor rare, boli care se
confruntă cu evoluţii devastatoare şi handicapante, afectand fiecare persoană dincolo de problemele fizice.
Material și metoda. Studiul de faţă este unul descriptiv şi analitic şi s-a desfăşurat în cadrul Centrului Medical de
Evaluare şi Recuperare „Cristian Şerban” Buziaş şi a Clinicii de Pediatrie Timişoara, au fost urmăriţi 100 de pacienţi ces-au prezentat în vederea evaluării comprehensive, a tratamentului şi recuperării fizice.
Rezultate. Rezultatele noastre arată că prevalenţa şi nivelul alexitimiei au fost mai mari la pacienţii cu perspectivă
scăzută asupra stării de sănătate, cu relaţii sociale scăzute şi cu comportamente obsesiv-compulsive.
Concluzii. Consecinţele psihologice ale pacienţiilor care se confruntă cu o afecţiune cronică şi cu complicaţii suntnumeroase: posibilitatea apriţiei invalidităţii, dependenţa de familia de bază, tulburari emotionale si de comportament,
incapacitatea de a se întreţine singuri, dificultăţi de integrare în viaţa socială.
Cuvinte cheie: hemofilie, alexitimie, comportament obsesiv-compulsiv

Fiziologia - Physiology • 2016.26.3(91)29THE UTILITY OF RING FINGER MEDIAN-ULNARSENSORY LATENCY DIFFERENCE IN THE EARLYDIAGNOSIS OF CARPAL TUNNEL SYNDROMEZAHID M. KADHIM1, SAFAA MOHAMMED FADHEL ABOOD2,
ALI J. AL-SULTANY2, BAN J. EDAN1
1Physiology Department, College of medicine, Babylon University, Iraq
2Al-Hilla General Hospital, Babylon, Iraq
3Internal Medicine Department, College of medicine, Babylon University, Iraq

ABSTRACT
Study highlights: Ring finger media-ulnar sensory latency difference is useful in the early diagnosis of carpal tunnel syndrome;
Ring finger media-ulnar sensory latency difference can be used as a screening test for carpal tunnel syndrome; Ring finger
median-ulnar sensory latency difference can be used as complimentary test for doubtful cases of carpal tunnel syndrome
Objectives: to study the utility of ring finger median-ulnar sensory latency difference in the early diagnosis and assessment ofcarpal tunnel syndrome.
Methods: 180 patients with 300 hands matched to 100 healthy volunteers (200 hands). Patients where suffering from signs andsymptoms of carpal tunnel syndrome and were tested by ordinary sensory & motor NCS of median & ulnar nerves. Patients withnegative results were evaluated by ring finger median-ulnar sensory latency difference.
Results: 137 patients (220 hands) showed a difference between median & ulnar nerves sensory responses recording ring finger
greater than 0.4 micro second and were categorized to have early mild carpal tunnel syndrome while the remaining patients andcontrol show less or no difference.
Conclusion: the use of ring finger median-ulnar sensory latency difference is helpful in the assessment and early diagnosis of
median nerve entrapment in the carpal tunnel because it increase the diagnostic sensitivity and should be used ascomplementary part of routine electrodiagnostic assessment of carpal tunnel syndrome.
Significance: ring finger median-ulnar sensory latency difference is easy to perform and need no additional equipment,
accessories and experience and so it can be useful for screening and aid in the early diagnosis of carpal tunnel syndrome.
Keywords: carpal tunnel syndrome, ring finger median-ulnar sensory latency difference, electrodiagnosis of carpal tunnel
syndrome.

INTRODUCTION
Carpal tunnel syndrome (CTS) is a common peripheral
entrapment neuropathy which has been related tooccupational factors including deviated wrist postures,
especially when combined with repetitive and forceful useof the hand [1]. Patients complain of nocturnal numbness
and pain in the hand and lateral three digits, andsometimes weakness and disuse of the thumb due tochronic median nerve compression at the wrist [2].
Nerve conduction studies (NCS) are commonly used toconfirm the clinical diagnosis of CTS and it has a highdegree of sensitivity (95%) and specificity (98%) [3].
However, it had shown that there is no relationshipbetween nerve conduction results and clinical outcome
measures after carpal tunnel release [4]. Also correlation ofelectrodiagnostic severity and clinical severity does notalways reveal an exact match although they are highly
correlated for diagnosis [5]. In spite of this, NCS provides
the most objective, quantitative & non-invasive assessmentof myelinated nerve fibre dysfunction, and has an importantcomplimentary function in cases of atypical clinicalpresentation, or when other underlying causes such asneuropathy are suspected [2].

MATERIALS AND METHODSThis case control study was conducted at the period ofJuly 2012 to May 2015. It included 180 patients and 100
control subjects. Control subjects have the same age andgender parameters as those of patients.
Diagnosis of CTS was based on the American
Academy of Neurology clinical diagnostic criteria [6]Received October 2nd 2016. Accepted October 23rd 2016. Address for correspondence: Dr. Zahid M. Kadhim MBChB, MSc, PhD
Neurophysiology, MD, Lecturer Physiology Department, College of Medicine, Babylon University, Iraq; phone:009647801853810; e-mail: zahid2ali@yahoo.com

30

Fiziologia - Physiology • 2016.26.3(91)summarized here: paresthesia, pain, swelling, weakness orclumsiness of the hand provoked or worsened by sleep,sustained hand or arm position, repetitive action of thehand or wrist that is mitigated by changing posture or byshaking of the hand; sensory deficits in the medianinnervated region of the hand and motor deficit or
hypotrophy of the median innervated thenar muscle;
symptoms elicited by Phalen test (1 min passive forced
flexion of the wrist), performed on each patient.
Exclusion criteria
1. positive ordinary NCS
2. clinical or electrodiagnostic features of polyneuropathy or radiculopathy
3. paste history of disease that affect nerve function
like diabetes
4. drug or toxic exposure that damage nerves likechemotherapeutic agentsEvery patient undergoes sensory NCS of median nerve
recording at index finger and ulnar nerve recording at the
fifth finger. Also motor NCS was done for median and ulnar
nerves with F-wave studies.
Patients with negative results on these tests were
included in the study. They underwent sensory NCS for
median and ulnar nerves recording ring finger andstimulating at the wrist at equal distances.
An onset latency difference of 0.4 µsec was considered
as abnormal and supports the diagnosis of CTS.
Statistics
The collected data were revised and introduced to acomputer using Statistical package for Social Science(SPSS 15.0.1 for windows; SPSS Inc, Chicago, IL, 2001).
Data were presented and suitable analysis was done
according to the type of data obtained for each parameter.
Data were expressed as Mean ± SD for quantitative
measures and both number and percentage for categorized
data. The comparison between two groups was assessedusing the unpaired t-test for numeric variables. The
comparison between three groups was assessed using
ANOVA test for parametric data. ROC curve was used to findsensitivity and specificity of the test (7).

RESULTS

Table II. Symptom profile of the patients.
Symptom
Parasthesia
Pain
Nocturnal symptoms
Weakness

Table I. Demographic data of study group.
Patient36±10162/185

Fiziologia - Physiology • 2016.26.3(91)Control
30±8
91/9
0

No. of patients (percentage)
173 (95%)
126 (79%)73 (27%)13 (2.5%)

Nerve conduction study findings
The results of nerve conduction study of both medianand ulnar nerves both motor and sensory findings areshown in Tables III, IV, and V.
Table III. NCS findings of median nerve motor study for bothpatients and control.
Variables
Latency (ms)
Amplitude (mv)CV (m/s)Patient
Control
Patient
Control
Patient
Control

3.3±0.4
2.8±0.1
9.7±2.1
7.7±1.458±5
58±6

P-value
0.003
0.004
0.8

Table IV. NCS results of ulnar nerve motor study for both patientsand control.
Variables
Latency (ms)
Amplitude(mv)
CV (m/s)Patient
Control
Patient
Control
Patient
Control

2±0.3
2±0.2
10.7±1.8
9.9±1.661±561±6P-value
0.9
0.3
0.8

Table V. NCS findings of median nerve sensory study for bothpatients and control.
Variables
Latency (µs)
Amplitude (µv)

Demographic data
The demographic data of patients and controls isshown on Table I.

VariableAge (years)
Sex (female/male)
Duration of symptoms (months)

Symptom profile
The study found that most patients were presented
with parasthesia of hand at median nerve distribution andpain, while control group was symptom free. The symptom
profile of patients is shown in Table II.

Patient
Control
Patient
Control

2±0.3
2±0.2
10.7±1.8
9.9±1.6

P-value
0.9
0.3

Table VI. NCS findings of ulnar nerve sensory part for both patientsand control.
Variables
Latency (µs)
Amplitude (µv)

Patient
Control
Patient
Control

1.8±0.2
1.7±0.1
42±20
37±12

P-value
0.5
0.5

31

Table VII. The results ring finger median-ulnar sensory latency
difference in both patients and control.
Latencydifference(0.4µs)Patient

Control

P-value

Sensitivity

Specificity

0.7±0.5

0.2±0.1

0.002

70%

100%

Fig. 1. ROC curve showing sensitivity and specificity of ring finger
media-ulnar sensory latency difference.

DISCUSSION
The most commonly reported symptom is parasthesia
and this is rational because most of patients presented
in the early phase of disease when sensory fibers onlyor more severely affected by compression than motor
fibers. Pain is the second most common presenting
symptom and it might reflect both motor and sensory
fiber lesion [8].
Motor weakness was the last commonly reported
symptom indicting that motor fibers are less severely
injured.
The diagnosis of carpal tunnel syndrome is difficultand complicated by the lack of agreement on a "goldstandard" diagnostic method for ensuring its presence orabsence [9], however a rigorous diagnosis of CTS isneeded because it forms the basis of accurate treatment.
Poor response or outcome of surgical treatment is often
related to poor initial diagnosis [8].
There is a diversity of electrophysiological techniquesthat are used to assess median nerve conduction across
carpal tunnel. These tests are the routine motor andsensory conduction studies of the median nerve which
has a sensitivity of 49-84%. Unfortunately, theseelectrophysiological tests can be normal in a32

considerable number of patients with CTS. The use ofother more sensitive comparative techniques thatcompare the median sensory or motor conduction across
carpal tunnel with an adjacent nerve in the same handwhich does not pass through the carpal tunnel andpresumed to be normal (direct internal comparison) [10].
These comparative tests increase the sensitivity ofelectrodiagnosis of CTS to 95% [11]. In addition to that,comparative tests help to eliminate the effects oftemperature, age and even of superimposed diseases on
the results. In this circumstance, the presence of at least
two abnormal sensitive electrophysiological techniques
for the diagnosis of CTS is required [12].
The commonest reference nerve is the ulnar nerve. It
is well known that ring finger has dual sensory
innervation with its lateral aspect supplied by median
nerve while the medial aspect supplied by ulnar nerve.
The median sensory innervation to ring finger isconsidered to be highly susceptible to impairment due totransverse ligament compression in the wrist. Monga andLaidlow (1982) [13] found that the amplitude of thesensory nerve action potentials of the ring finger always
were lower than those of the index and middle finger,even in healthy subjects. This fact indicates that sensory
innervation for the ring finger is provided by a lesser
number of fibers from the median nerve than is the casefor the index finger and the middle finger; hence its
threshold for injury is lower. Monga and Laidlow (1982)[13] and Uncini et al., 1989 [14] reported that the sensory
median fibers of the ring finger are damaged earlier thanthose of the index finger. The reason is possibly the
anatomic location of the sensory fibers in the median
nerve below the transverse ligament, so that the sections
supplying the ring finger are exposed to compression
especially early.
Because of all that, the median versus ulnar sensory
latency difference recording ring finger is one of thecomparative tests with high sensitivity and specificity inthe diagnosis of CTS [15].
Uncini et al. [14] reported that median versus ulnar
digit four sensory latency difference test had the highest
sensitivity in comparison to other comparative tests. Also
Celik and Guven reported that median sensory CV of thedigit two had sensitivity of 81.5% while median versus
ulnar digit four sensory latency difference had asensitivity of 92% [16].
Other comparative tests possibly have lower
sensitivity like 2nd lumberical interosseus latency
difference motor study (83%) and median-radial sensory
study (82%) [17].
The current study found that the sensitivity of this test
was 70% and the specificity was 100%. Report from otherstudies give widely variable results like Andary (42%) [18],Jackson & Clifford (44%) [19], Uncini et al. (78%) [20],Pease et al. (88.6%) [21], Monga et al. (93%) [22], Cioni
et al. (99.2%) [23], Charles et al. (100%) [24] andFiziologia - Physiology • 2016.26.3(91)Johnson et al. (100%) [25]. The possible explanation for
the variability between our results and the above results
is our patients have mild CTS and showed normal routine
NCS of median nerve. So if we select patients with CTS
with variable severities, the sensitivity would be higher
and might reach up to 95-100%. Other causes ofcontroversy in the results could be due to selecting
patients: is there difference in age group, are thesymptomatic hands due to CTS? Is there nerve
compression with structural lesion or just ischemic
reversible initial abnormalities? Are there other causes?
Is the "routine" median sensory nerve conduction normal
or near normal [26]?
The study has few limitations like the absence of gold
standard test for the diagnosis of CTS. Also having a larger
sample size of patients with mild CTS in whom the routine
tests is normal will help to confirm the results.

CONCLUSION
Ring finger median-ulnar sensory latency difference isa reliable method for the screening and early diagnosis of
CTS. It is performed easily and quickly without additional
equipment or accessories and give high sensitivity andspecificity in the diagnosis of CTS.
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Fiziologia - Physiology • 2016.26.3(91)RH ISOIMMUNISATION IN NEONATES:RISK FACTORS, INCIDENCE, COMPLICATIONSAND TREATMENTDANIELA IACOB1*, CLAUDIU ANGELESCU-COPTIL1, ALEXANDRA NYIREDI2,MIRABELA DIMA1, HOCAN MARIA-ELENA3, MUSTAFA NASSAR4, RADU EMIL IACOB2
1University of Medicine and Pharmacy “Victor Babes” Timisoara – Department of Neonatology
2University of Medicine and Pharmacy “Victor Babes” Timisoara – Department of Pediatric Surgery
3Emergency Children Hospital “Louis Turcanu” Timisoara
4University of Medicine and Pharmacy “Victor Babes” Timisoara – Student

ABSTRACT
Rh incompatibility is a hemolytic disease of neonates, which is the leading cause for moderate and severe Jaundice andAnemia in fetus and newborn. During pregnancy Rh negative phenotype women may develop antibodies against their Rh
positive fetuses RBCs. Rh Disease remains significant problem in obstetric and neonatology, which may lead to severe
hemolysis and even hydrops fetalis in fetus and newborn without proper and timely prevention and treatment.
Keywords: Rh isoimmunisation, neonates, risk factors, jaundice.

INTRODUCTION
Hemolytic Disease of the fetus and newborn can result
from many blood groups incompatibility, but the Rh
Isoimmunisation is responsible for the most of moderateand severe cases despite the widespread use of anti D
immunoglobulin as prevention method [6]. The hemolytic
disease in infants and newborns is an immune mediateddestruction of fetal and newborn RBCs. This is usually due
to antibodies made by the mother directed against the
fetus red blood cells. It is typically caused by Rh
incompatibility, which occurs when there is difference
between the Rh blood group of the mother and the fetus.
Typically happens when the fetal red blood cells express apaternally inherited red blood cell antigen (Rh positive)
which is not present on maternal red blood cells (Rhnegative) [9].
The objectives of this paper is to study the prevalence ofRh negative phenotype, incidence of Rh disease, It’s risk
factors ,and the effectiveness of protective measures andtreatment.

METHODS
A prospective study of neonates that have beendelivered in BEGA hospital in Timisoara city, Romania for
one year period. The period of study was from the first ofJanuary 2015 until the end of December 2015. The Data
has been collected regarding The Blood Rh and ABO grouptypes for both the mother and newborn, Gestational Age,
Gravidity and Parity numbers, abortion history, type ofdelivery, family history of Jaundice, pathologies during
pregnancy and the method of treatment.
If the Newborns have developed clinical findings
suggesting newborn hemolytic disease, the total serum
bilirubin and hemoglobin levels were measured.
If Plasma Bilirubin >4mg/dl and/or hemoglobin level lessthan 15mg/dl for full term neonate and less than 13 forpreterm neonates, then the newborns were admitted and fullinvestigations and treatment started.
If the clinical findings has not been suggestive forhemolytic disease, and the Bilirubin blood levels were below
4 mg/dl and Hemoglobin levels were above 15 for full term
and above 13 for preterm newborns then the newborns
were discharged home and the family has been informed tobring the newborn after 2-3 days for follow up.
Investigations done for neonates:
1) Blood group and Rh typing2) Total Serum Bilirubin (TSB)
3) Hemoglobin4) Hematocrit5) APGAR score

Received August 27th 2016. Accepted September 12th 2016. Address for correspondence: Daniela Iacob, MD, PhD University of
Medicine and Pharmacy “Victor Babes” Timisoara, Department of Neonatology, Eftimie Murgu Square No. 2, Timisoara 300041,
Romania; phone: +40742558574; e-mail: danielariacob@yahoo.com

Fiziologia - Physiology • 2016.26.3(91)356) Gestational Age7) Other Pathologies

study, from first of January 2015 till the end of December
2015 is 24 out of 2456 (1%).

Investigations done for mothers:
1) Blood group and Rh typing
2) Gravidity and Parity numbers
3) Abortion History4) Pathologies during pregnancy
5) Type of Delivery

RESULTS2456 Pregnant women delivered during this period, allof them have been examined for their Rh type, 186 of them
have Rh negative. From those 186 pregnant women 167
delivered neonates whom Rh is positive, and 19 whom Rh is
negative. Those 19 Rh negative newborns were excluded
from the study (because only Rh positive neonates candevelop Rh disease).
Rh phenotype prevalence: the prevalence ofpreganant women whom Rh phenotype is negative in BEGA
hospital during one year study has been determined fromthe data the have been collected.
Out of 2456 pregnant women there are only 186 whom
Rh phenotype is negative [7.5%] .
The National Center for Biotechnology Information(US) has conducted a study in 2005 about the Rh negative
phenotype prevalence regarding the races. They havefound the Rh negative phenotype most commonly present in
the Basques race (30-35%) then Caucasians (15%), lesscommon in Blacks (8%), and rare in Asians (1%) [5].

Fig. 2. The incidence of Rh disease in BEGA Hospital

The incidence of Rh disease in population depends on
the prevalence of Rh negative phenotype and on theprenatal care. The incidence of Rh disease markedly
reduced since blood Rh typing and the administration ofanti-D immunoglobulin became a routine prophylaxis
measure. In the US, the Centers for Disease Control andPrevention reported in 2003 the incidence of Rh hemolytic
disease is 6.8 cases per 1000 live births [10].

Fig. 1. The prevalence of Rh (-) phenotype
around the world comparing to our study

Fig. 3. The incidence of Rh disease in USA
comparable to BEGA Hospital

Incidence: the incidence of Rh disease in BEGA
hospital has been deducted from the data in our study, outof 2456 pregnant women, there are 186 women with Rh
negative, 167 whom newborns have Rh positive phenotype,and only 24 newborns developed the Rh disease. Thus theincidence of Rh disease in BEGA hospital during one year

Jaundice etiology: From the 186 pregnant women with
Rh negative phenotype, there are 167 neonates having Rh
positive phenotype, and only 24 out of those 167 developed
hemolytic disease due to Rh incompatibility, the other 143
either have not developed jaundice or developed jaundice
due to other causes than Rh incompatibility.

36

Fiziologia - Physiology • 2016.26.3(91)Out of those 143 , 105 have not developed jaundice,and 38 developed jaundice due to variety of casues [18 dueto physilogical jaundice, 12 due to obstetrical trauma, 2 due
to ABO incompatibility, 6 due to maternal fetal infection].

The concurrence of mothers and newborns with thesame ABO type has been calculated from the data as thefollow: 98 of cases the mothers and neonates have thesame blood ABO type and 69 have different bloodgroups.

Fig. 6. The concurrence of mothers and newborns
with the same ABO

Fig. 4. The distribution of jaundice causes in newborns with Rh+
that have born to Rh negative mother.

Rh Coexistent with ABO Incompatibility: Theprevalence of newborns with ABO incompatibility
coexistent Rh incompatibility has been calculated from thedata of our study, there are 12 cases out of 167 Rh
negative mothers (7%), where the mothers have blood
type O and the neonates with either blood type A or B.

Mothers ABO Blood group Distribution: The distributionof ABO blood group type of the mothers with Rh negative
phenotype whom newborns have Rh positive phenotype asfollow, out of 167 women there are [83 have type A, 42 havetype O, 28 have type B and 14 have type AB].

Fig. 7. The distribution of Rh incompatibility Coexistent
with ABO incompatibility.
Fig. 5. Distribution of mother’s ABO blood group in Rh- mothers

Fiziologia - Physiology • 2016.26.3(91)37Rh coexistent ABO incompatibility for either the A or B
blood group antigen will reduce the risk of maternal Rh
sensitization. The rapid immune clearance of the fetal
erythrocytes before Rh sensitization can proceed to asignificant extent due to ABO incompatibility. Fortunately,
hemolytic disease due ABO incompatibility is less severe
than that of Rh isoimmunisation, but it confers no protection
once sensitization has occurred.
In the 12 cases with Rh coexistent with ABO
incompatibility only 2 cases have developed mild jaundice
and anemia, and none of them developed Rh sensitization.
The results emphasizes what we have discussed in the firstchapter that coexistent ABO incompatibility is a protection
factor and reduces the Rh sensitization occurrence.
We classified the newborns into two groups, the firstgroup is the ABO compatible newborns, and the second
group is the ABO incompatible newborns.
In the first group, out of 155 compatible newborns 24
developed Rh disease (15.4%), and in the second groupzero newborns developed Rh disease (0%).

Fig. 9. The distribution of Newborns related
to their gestation age

As shown in Figure 10, from 167 newborns, only 19
newborns are premature and 148 are mature. Assumingthat newborns who born before 37 weeks are consideredpremature. The global incidence of preterm delivery wasestimated by world health organization to be 9.6% [19]. In
our study we can conclude that Rh incompatibility doesn’t
increase the incidence of preterm delivery.

Fig. 8. The distribution of Rh disease in ABO compatible
comparing to ABO Incompatible fetuses.

The distribution of Newborns with Rh
Incompatibility according to Gestational Age: in ourstudy the gestational age data of 167 neonates has beencollected and shown the following number of neonates bornin each specific gestational age (41 weeks: 21 neonates, 40
weeks: 49 neonates, 39 weeks: 42 neonates, 38 weeks: 28
neonate, 37 weeks: 8 neonates, 36 weeks: 19 neonates).

38

Fig. 10. Distribution of newborns according to maturity.

Distribution of newborns according to Gender: outof 167 newborns, there are 105 male newborns and 62
female newborns. Thus the ratio between male and female
is 1.7:1. As shown in Figure 11 there are 63% males whichis by far more than female patients.
Fiziologia - Physiology • 2016.26.3(91)Fig. 11. Distribution of newborns according to gender

We found out that from those who developed Rh
disease 20 are males and only 4 females from all the 167 Rh
incompatible newborns. As we mentioned in the first part,male infants are reported to have an increased risk todevelop Rh disease than females, although the basis for thisobservation is unclear.
The incidence of Rh disease sensitization in Rh
incompatible newborns as follow: 20 patients out of the 105
males (19%), and 4 out of the 62 females (6.4%).

Fig. 13. Prevalence of Newborns that developed anemia
in the first day

Distribution of Newborns according to type ofdelivery: in our study 90 (53.8%) women had delivered by
Caesarean section and 77 (46.2%) women delivered naturaldelivery. From the 24 neonates that developed jaundice due
to Rh disease, 16 of them have been delivered by C-section
and 8 have been delivered naturally.

Fig. 12. Number of cases that developed Rh
disease according to gender.

Fig. 14. Development of Rh disease according
to type of delivery

The prevalence of newborns who developed
anemia in the first day: considering that each newborn
who has hemoglobin below 15 mg/dl is anemic weclassified the newborns into normal and anemic neonates.
We found that 42 out of 169 newborns had anemia in thefirst day, as it is shown in Figure 13 (25% of the newborns
were anemic). Anemia is a common complication of Rh
disease which occurs due to hypo-regenerative andhemolytic mechanisms [1,2].

We observed that the incidence of Rh disease
increased in newborns that have been delivered by
C-section.

Fiziologia - Physiology • 2016.26.3(91)Distribution of women according to number ofpregnancies: in our study out of 167 there are 70 womenwith I pregnancies, 41 with II pregnancies, 14 with III
pregnancies, 7 with IV pregnancies, 21 with V pregnancies,
and 14 with VI pregnancies.

39

Fig. 15. Distribution of women according to number of pregnancies

We noticed from Figure 15 that there are 70 out of 167
newborns have been delivered in the first pregnancy(Primi-gravida), and 97 are considered multigravida(delivered after more than one pregnancy).
The distribution of Rh disease according to numberof gravidity: From the 24 newborns that developed Rh
disease there are 4 newborns that are considered gravida I,
6 newborns are gravida II, 3 newborns are gravida III, 2
newborns are gravida IV, 5 newborns are gravida V and 4
newborns are gravida VI.

Fig. 17. Comparison between the incidence of Rh disease inprimigravida and multigravida.

This result confirms what we have mentioned in the firstpart, that birth order is significant risk factor, thus the first
newborn has minimal risk to develop the disease, unlesssensitization has occurred previously. Once sensitization
has occurred, subsequent pregnancies are at a progressive
risk for fetal and newborn hemolytic disease.
Distribution of cases according to peak Bilirubinlevels: In our study we classified the patients in 3 groups
according to peak bilirubin levels, first group has bilirubinlevel below 13 mg/dl, second group between 13 and 16
mg/dl and the third group above 16 mg/dl. In our study wemeasured the newborns bilirubin levels every day until clear
improvement and declining in bilirubin level noticed.

Fig. 16. Shows the number of newborns who developed Rh
disease related to the number of gravida.

There are only 4 newborns out of 70 (5%) ofprimi-gravida mothers developed Rh disease, while there
are 20 out of 96 (20.8%) of multigravida mothers developed
Rh disease.
Fig. 18. The distribution of patients according
to peak bilirubin level.

40

Fiziologia - Physiology • 2016.26.3(91)We noticed in Figure 18 that most newborns thatdeveloped jaundice due to Rh disease had peak plasma
Bilirubin Values between 13 and 16 mg/dl.
Treatment: all the 24 newborns with Rh diseasedeveloped jaundice in the first 24 hours, 18 of themneeded phototherapy alone, 4 patients needed
phototherapy and albumin, and 2 patients needed
phototherapy, albumin and blood exchange transfusion.

Fig. 20. Show the distribution of patients according
to duration of phototherapy

DISCUSSION

Fig. 19. The distribution of our study group according to themethod of treatment

Phototherapy is non-invasive treatment method
which decreases bilirubin levels and reduces the numberof total exchange transfusions required, thus avoiding
neurological and other possible complications ofhyper-bilirubin concentration [15,16]. Phototherapy isstarted if bilirubin rises 0.5 mg/dL/h or if total bilirubin
exceeds 10, 12, or 14 mg/dL at 12, 18, or 24 h of life,
respectively.
Distribution of cases according to the duration ofphototherapy treatment needed: In each day after birth
we were measuring bilirubin levels, whenever there isindication for phototherapy we give one episode ofphototherapy per day, which is about 6-8 hours duration.
In our study from the 24 patient, 7 patients needed 2 daysof phototherapy, 11 patient needed 3 days ofphototherapy and 6 patients needed 4 days ofphototherapy.

Fiziologia - Physiology • 2016.26.3(91)Since blood Rh typing and the administration of anti-D
immunoglobulins for Rh negative mothers becomes aroutine prophylaxis measure, the Incidence of Rh diseasehas been dramatically reduced.
Despite these advances to prevent the development of
Rh disease, the disease still poses major health problem tothe mothers and newborns.
The incidence of Rh disease in population depends on
the prevalence of Rh negative phenotype, which is variedamong the races. The Rh negative phenotype is commonly
present in the Basques race about 35% and less common
in Asians about 1% [4,7]. Thus it is very important to findthe prevalence of Rh negative phenotype in our study in
BEGA hospital.
Rh disease still major cause of anemia and fetalhydrops, which resulted from poor prenatal care andprevention, thus in our study we focused on the incidenceof Rh disease in BEGA hospital and to compare to the
Global results to evaluate the efficacy of the prevention
measures and prenatal care.
There are many causes which may lead to jaundiceduring the neonatal period, thus the differential diagnosis
is important to exclude other possible causes than Rh
disease. Thus in our study we classified the patients
according to their jaundice causes. Many studies andtheories claim that ABO incompatibility offers a significant
degree of protection against Rh sensitization. The mainfeature for production of Rh antibodies is the survival of
fetal RBCs in the maternal circulation for long enoughperiod to stimulate the anti-D antibodies production. If the
fetal RBCs are Incompatible with maternal anti A or B they
are rapidly hemolyzed so that the Rh antigens are notavailable to produce antigenic stimulus [13,14,17].
41

Therefore in our study we classified the newborns into
ABO compatible and incompatible to compare theincidence difference between them. We noticed in somestudies that Male infants are reported to have an increased
risk of developing the Rh disease, although the basis forthis observation is unclear. Thus we aimed to compare theratio between male and female in our study to observe if
there are similar results.
Anemia is considered as common complication of the
Rh isoimmunisation, which is caused by the hemolytic
mechanism. Thus we obtained the hemoglobin values in
the first day of newborns to evaluate the percentage ofanemic newborns.
In 90% of cases, sensitization occurs during delivery.
Therefore, most first born infants with Rh-positive blood
type are not affected because the short period from first
exposure of Rh-positive fetal erythrocytes to the birth,which is insufficient to produce a significant maternal IgG
antibody response.
The risk and severity of sensitization response
increases with each subsequent pregnancy involving a
fetus with Rh-positive blood. In women who are prone to
Rh incompatibility, the second pregnancy with an
Rh-positive fetus often produces a mildly anemic infant,whereas succeeding pregnancies produce more seriously
affected infants who ultimately may die in utero frommassive antibody-induced hemolytic anemia [11,12,18].
In our study we classified the newborns related to theirmother number of gravidity, to compare the incidence ofRh disease in each group to prove that the incidence
increased by each subsequent pregnancy as written intheory.
Depending on the severity of the disease, eachnewborn will develop different degree of jaundice andanemia and so different treatment method will be needed.
Some patients needs only phototherapy, others may needalbumin and even blood exchange transfusion if severe
anemia developed [3,8].
In our study we classified the patients depending on
their method if treatment needed, to detect the percentage
for each treatment needed and so the severity of thedisease.

RESULTS
The prevalence of preganant women whom Rh
phenotype is negative in BEGA hospital during one year is
Out of 2456 pregnant women there are only 186 whom Rh
phenotype is negative [7.5%].
The incidence of Rh disease in BEGA hospital has
been deducted from the data in our study, out of 2456
pregnant women, there are 186 women with Rh negative,
167 whom newborns have Rh positive phenotype, andonly 24 newborns developed the Rh disease. Thus theincidence of Rh disease in BEGA hospital during one year

42

study, from first of January 2015 till the end of December
2015 is 24 out of 2456 (1%).
From the 186 pregnant women with Rh negative
phenotype, 167 neonates has Rh positive phenotype, only
24 out of those 167 had developed hemolytic disease due
to Rh Incompatibility (14%), the other 143 either have notdeveloped jaundice or developed jaundice due to othercauses than Rh incompatibility. Out of those 143, 105 havenot developed Jaundice (63%), and 38 developed jaundice
due to variety of casues [18 due to physilogical jaundice(11%), 12 due to obstetrical trauma (7%), 2 due to ABO
incompatibility (1%), 6 due to Maternal Fetal infection(4%)].
The Prevalence of Newborns who have Rh Coexistent
with ABO Incompatibility has been concluded from thedata of our study, there are 12 cases out of 167 Rh
negative mothers (7%), where the mothers have blood
type O and the neonates with either blood type A or B. We
classified the newborns into two groups, the first group is
the ABO compatible newborns, and the second group is
the ABO incompatible newborns. In the ABO compatiblegroup, out of 155 compatible newborns 24 developed Rh
disease (15.4%), and in the ABO Incompatible group zero
newborns developed Rh disease (0%).
The incidence of Rh disease sensitization in Rh
incompatible newborns as follow: 20 patients out of 105
males (19%), and 4 out of 62 females (6.4%).
The prevalence of newborns who developed anemia in
the first day: considering that each newborn who hashemoglobin below 15 mg/dl is anemic we classified thenewborns into normal and anemic neonates.
We found that 42 out of 169 newborns had anemia in
the first day, as it is shown in figure 13 (25% of thenewborns were anemic).
The distribution of newborns that developed Rh
disease according to number of gravidity: from the 24
newborns that developed Rh disease there are 4
newborns that are considered gravida I, 6 newborns are
gravida II, 3 newborns are gravida III, 2 newborns are
gravida IV, 5 newborns are gravida V and 4 newborns are
gravida VI.
There are only 4 newborns out of 70 (5%) ofprimigravida mothers developed Rh disease, while there
are 20 out of 96 (20.8%) of multigravida mothers
developed Rh disease.
All the 24 newborns with Rh disease developed
jaundice in the first 24 hours, 18 (75%) of them needed
phototherapy alone, 4 patients (17%) needed
phototherapy and albumin and 2 patients (8%) needed
phototherapy, albumin and blood exchange transfusion.

CONCLUSION
1. The prevalence of Rh negative phenotype in BEGA
hospital is 7.5% which is considered less common thanFiziologia - Physiology • 2016.26.3(91)

2.

3.

4.

5.

6.
7.

Basques (35%) and Caucasians (15%) but more than
Asians (1%). This means that they are in moderate riskof developing Rh disease comparable to other races.
Only 14% of Rh negative women whom fetus has Rh
positive antigens develop jaundice due to Rh disease.
While 86% either do not develop Rh disease ordevelop jaundice due to other disease. Thus wededucted that not all Rh negative mother whom fetus is
Rh positive develop Rh disease, only 14% of newborns
develop the disease.
The incidence of Rh disease in BEGA Hospital is 9.9
per 1000 which is similar to the incidence conducted in
USA by the US, the Centers for Disease Control andPrevention reported in 2003, 6,8 per 1000. This
incidence is much less than two decades ago, and thisimprovement is due to the blood Rh typing and theadministration of anti-D immunoglobulins becomes aroutine prophylaxis measure.
We concluded from our study that ABO incompatibility
offers a significant degree of protection against Rh
sensitization since the results showed decreased
incidence of Rh disease in ABO Incompatible
newborns compared to ABO Compatible newborns.
In our study being Male newborns were consideredsignificant risk factor to develop Rh disease, because
20 out of 105 males (19%), and 4 out of 62 females(6.4%) developed Rh disease.
Anemia is common complication in Rh disease, since
25% of patients had anemia in the first day of life.
Birth order is considered important factor, since there
are only 4 newborns out of 70 (5%) of Primi-gravida
mothers developed Rh disease, while there are 20 outof 96 (20.8%) of multigravida mothers developed Rh
disease. Thus we concluded that multigravida isconsidered significant risk factor.

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43

IZOIMUNIZAREA RH LA NOU-NASCUT:FIZIOPATOLOGIE, FACTORI DE RISC,
INCIDENTA, COMPLICATII SI TRATAMENTREZUMAT
Incompatibilitatea Rh este o boala hemolitica la nou-nascut care reprezinta cauza principala de anemie si icter
moderat si sever la fetusi si nou-nascuti. In timpul sarcinii femeile cu fenotipul Rh negativ pot dezvolta anticorpi
impotriva fetusilor cu Rh pozitiv. Incompatibilitatea Rh ramane o problema semnificativa in obstretica si
neonatologie, care poate duce la hemoliza severa si chiar hidrops fetal in viata intrauterina si la nou-nascut farapreventie si tratament adecvat si la timp.
Cuvinte cheie: izoimunizarea Rh, nou-nascut, factori de risc, icter.

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Fiziologia - Physiology • 2016.26.3(91)