FIZIOLOGIA
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Fiziologia - Physiology • 2016.26.1(89)FIZIOLOGIA

physiology
CONTENTS
1. Vitex doniana has Restorative Effect on Sperm Parameters and Hormonal Production in
Male Wistar Rats following Testicular Torsion ................................................................................................................................. 4
Adelodun ST, Adewole OS, Olatunji SY, Odukoya SA, Adekomi AD, Adalumo OA, Adeyeye OB
2. CYP2D6 Enzymatic Deficiency and Extrapyramidal Sides Effects in an Autistic Patient Treated with Risperidone .................. 9
Gradinaru R, Andreescu N, Puiu M
3. Effects of Chronic Copper Sulphate Administration on Feeding Pattern and Markers of Renal
and Liver Functions of Wistar Rats ................................................................................................................................................. 13
Akomolafe RO, Olukiran OS, Imafidon CE, Ayannuga OA, Oyekunle JA, Oladele AA
4. Contribution of Self-Perceived Stress to Postsurgical Life Quality Change of Patients Undergoing Catharact Surgery ........ 20
Ciobotea D, Șerban C, Putnoky S, Fira Mladinescu C, Tuță Sas I, Băcean Miloicov C, Vlaicu B
5. Biocomposite Processing Technology and Medical Applications. A Review ................................................................................ 25
Voisan R, Gingu O
6. Assessment of Obsessive-Compulsive Behavior in Patients with Haemophilia ........................................................................... 31
Hogea L, Nussbaum L, Dumache R
7. Characterization of the Effects of SarcKATP And MitoKATP Modulators on Reperfusion-Induced
Arrhythmias in Isolated Rat Hearts ................................................................................................................................................. 35
Petruș A, Sturza A, Duicu O, Jost N, Muntean D, Baczko I
8. Stem cells from amniotic fluid, an essential tool for regenerative medicine ................................................................................. 40
Honcea A

CUPRINS

1. Vitex doniana are efect restaurator asupra parametrilor spermici și producției hormonale lașobolanii Wistar după torsiunea testiculară ..................................................................................................................................... 4
Adelodun ST, Adewole OS, Olatunji SY, Odukoya SA, Adekomi AD, Adalumo OA, Adeyeye OB
2. Deficitul enzimatic de CYP2D6 şi efectele adverse extrapiramidale la un pacient cu autism tratat cu risperidonă .................... 9
Gradinaru R, Andreescu N, Puiu M
3. Efectele administrării cronice de sulfat de cupru aupra modalităților de hrănire și a markerilor funcționali
renali și hepatici la șobolanii Wistar ............................................................................................................................................... 13
Akomolafe RO, Olukiran OS, Imafidon CE, Ayannuga OA, Oyekunle JA, Oladele AA
4. Contribuția stresului autoperceput la modificarea postoperatorie a calității vieții la pacienți supuși operației de cataractă ......... 20
Ciobotea D, Șerban C, Putnoky S, Fira Mladinescu C, Tuță Sas I, Băcean Miloicov C, Vlaicu B
5. Tehnologia procesării materialelor biocompozite și aplicabilitatea în domeniul medical ............................................................ 25
Voisan R, Gingu O
6. Evaluarea comportamentului obsesiv-compulsiv la pacienții cu hemofilie .................................................................................. 31
Hogea L, Nussbaum L, Dumache R
7. Caracterizarea efectelor modulatorilor sarcKATP și mitoKATP asupra aritmiilor induse de reperfuzie
pe inimi izolate de șobolan ............................................................................................................................................................... 35
Petruș A, Sturza A, Duicu O, Jost N, Muntean D, Baczko I
8. Celulele stem din lichidul amniotic – un instrument esențial pentru medicina regenerativă ...................................................... 40
Honcea A

Fiziologia - Physiology • 2016.26.1(89)3VITEX DONIANA HAS RESTORATIVE EFFECT ON SPERM
PARAMETERS AND HORMONAL PRODUCTION IN MALEWISTAR RATS FOLLOWING TESTICULAR TORSIONSTEPHEN TAIYE ADELODUN*1, OLARINDE STEPHEN ADEWOLE1, SUNDAY YINKA
OLATUNJI2, SAMSON AYODEJI ODUKOYA1, ADEDAYO DAMILARE ADEKOMI3,
OLUSOJI ADEOLA ADALUMO4 AND OPEYEMI BLESSING ADEYEYE4
1Department of Anatomy and Cell Biology, Obafemi Awolowo University, Ile-Ife, Nigeria.
2Department of Anatomy, Ben Carson (Snr.) School of Medicine, Babcock University, Ilisan-Remo, Nigeria.
3Department of Anatomy, Osun State University, Osogbo, Nigeria.
4Department of Physiological Sciences, Obafemi Awolowo University, Ile-Ife, Nigeria.

ABSTRACT
Objective: This study investigated the effects of Vitex doniana leaf extract on testicular torsion of Wistar rats with a view todetermining its effects on sperm motility, sperm count and serum testosterone level.
Methods: Thirty adult male Wistar rats weighing 120 - 160 g, obtained from the Animal House of the College of Health
Sciences, Obafemi Awolowo University, Ile-Ife were used for this study. The rats were randomly assigned into six groups (n =5). All surgical procedures were performed under ketamine anaesthesia (5 mg/kg i.m). All animals in each group had their lefttestes twisted at 720°, left for one hour, sutured back and were then treated for 7 days except group A that had their lefttestes exposed but not twisted for one hour (sham surgery) and then received distilled water for 7 days. Group B rats were left
untreated, groups C, D and E received 50 mg/kg, 100 mg/kg and 200 mg/kg of Vitex doniana extract respectively while group
F received 100 mg/kg of vitamin C. At the end of the experiment, the animals were sacrificed using lethal dose of ketamine(100 mg/kg) and blood samples were collected through cardiac puncture into plain bottles. Serum testosterone, sperm countand motility were measured. Data collected were analyzed using One-way ANOVA and Student Newman- Keuls post- hoctest for multiple comparisons.
Results: There was a significant reduction in the sperm motility in group B (44 ± 2.45%) when compared with group A (93 ±1.23%) and the treated groups: C, D, E and F (55 ± 4.64%; 65 ± 1.58%; 85 ± 2.00% and 79 ± 1.87% respectively) (F =44.03; p = 0.0001). There was a significant reduction in sperm count in group B (88.2 ± 4.63%) when compared with A (130.2± 4.84%) and the treated groups: C, D, E and F (97.8 ± 2.31%; 109.8 ± 4.14%; 117.4 ± 6.62% and 126.8 ± 8.71%
respectively) (F = 8.66; p = 0.0001). Testosterone level in group B was significantly lowered (0.50 ± 0.14 ng/dl) whencompared with group A (1.85 ± 0.52 ng/dl) and the treated groups: C, D, E and F (0.62 ± 0.02 ng/dl; 0.62 ± 0.24 ng/dl; 0.74 ±0.18 ng/dl and 0.88 ± 0.50 ng/dl) (F = 2.20; p = 0.0004).
Conclusion: The study concluded that aqueous extract of Vitex doniana leaves had ameliorative effect on the sperm quality
and testosterone concentration in the torsioned rat model.
Keywords: Vitex doniana, restorative, torsion, sperm, rats

INTRODUCTION
Testicular torsion is a urological emergency that usually
results from rotation of testis around the axis of the spermaticcord. It is frequently observed in newly borns, children andadolescents [1]. Rapid diagnosis and immediate surgicaltreatment are essential to avoid permanent testiculardamage. Delay or misdiagnosis and inappropriate treatment
usually lead to male infertility [2]. The main pathophysiologyof testicular torsion/detorsion of spermatic cord is ischemia/
reperfusion (I/R) injury of the testis [3]. Under normal
conditions, free radicals are produced and their effects arecounterbalanced by the endogenous antioxidant system.
When ROS generation exceeds the defense mechanisms
capacity, oxidative stress is generated and contributes toreversible and irreversible cell injury [4]. Vitex doniana is asavanna species in wooded grassland and can also be foundalong forest edges. It can be found throughout tropical Africa.
Vitex doniana belongs to the family Verbenaceae. This large
genus is distributed throughout the tropics and subtropics [5].
It is commonly known as Mfuru, Mgwobe (Tanzania),
Munyamazi, Muhomozi (Uganda) (USDA,), Dinya, Tinya,
Tunci (Fulani), Ori nla (Yoruba) [6]. In Nigeria, frominformation available from the indigenous traditional healers,Received January 5th, 2016. Accepted February 12th, 2016. Address for correspondence: Stephen Taiye Adelodun,
Department of Anatomy and Cell Biology, Obafemi Awolowo University, Ile-Ife, Nigeria; tel: +2348069823009,
e-mail adeloduntaiye@gmail.com.

4

Fiziologia - Physiology • 2016.26.1(89)a decoction of the chopped stem bark part of V. doniana isprepared and taken orally for treatment of gastroenteritis. It isadministered for ailments including diarrhea and dysentery. It
is also taken to improve fertility and the juice may besqueezed into the eyes to treat eye troubles. It is also used in
the treatment of liver disease.

MATERIALS AND METHODSExtraction of Vitex doniana Leaves
Vitex doniana leaves were collected from the lawnbehind Moremi Hall, Obafemi Awolowo University, Ile-Ife andwere taken to a taxonomist at the Department of Botany,
Obafemi Awolowo University, Ile-Ife for authentication after
which a voucher specimen was deposited at Ife Herbarium
for reference with a reference number IFE-17377433. The
leaves were cleaned and air-dried at room temperature. The
dried leaves were pulverized using an electric blender. The
extraction was done using percolation method which
involved continuous washing of the pulverized leaves withwater at 80 °C and 30 extraction cycles for 3 hours using
Soxhlet extractor. The extract was concentrated in vacuo at
65 °C using a vacuum rotary evaporator and freeze-dried ina lyophilizer. The extract was stored in a desiccator until
used.
Animal Care and Management
Thirty young male Wistar rats weighing between 120 160 g were obtained from the Animal House of the College of
Health Sciences Obafemi Awolowo University Ile-Ife, andused for this research. The rats were randomly assigned into
six groups of five rats per group (Groups A, B, C, D, E and F).
Animals were housed in clean plastic cages under naturallight and dark cycles and at room temperature. Animals in allgroups were fed on normal laboratory chow, and had accessto water ad libitum.
Surgical Procedure and Experimental Protocol
Animals in group A had their testes exposed for one hour
without twisting the spermatic cords, sutured back (shamsurgery) and then were given distilled water for 7 days.
Group B had their spermatic cords twisted at 720°, left for
one hour, sutured back and then received distilled water for 7
days. Group C, D and E also had their spermatic cords
twisted at 720°, left for one hour, sutured back and then wereorally administered with V. doniana extract (50 mg/kg, 100
mg/kg and 200 mg/kg per body weight) respectively for 7
days. Group F had their spermatic cords twisted at 720°, left
for one hour, sutured back and then were orally administeredwith vitamin C (100 mg/kg per day) for 7 days. All surgicalprocedures were performed under ketamine anaesthesia (5mg/kg i.m). V. doniana extract and vitamin C dissolved in
distilled water were administered orally on a daily basis for
Fiziologia - Physiology • 2016.26.1(89)the period of administration using suitable oral cannula.
Vitamin C was obtained from KUNIMED PHARMACHEM
LTD Nigeria.
Sperm Counts and Motility Analysis
The sperm concentration was determined using thehaematocytometer method. 1:20 dilution from each well
mixed sample was prepared by diluting 50 µl of liquefied
semen with 950 µl diluents. The diluents were prepared byadding 50 g of sodium hydrogen trioxocarbonate (NaHCO3),
10 ml of 35% (v/v) formalin and 0.25 g of trypan blue todistilled water and making up the solution to final volume of1000 ml. A fixed volume of the sample was withdrawn withmicro-pipette and delivered onto the edges of Neubauer
chamber of haematocytometer and covered with 22x22 mmcover slip. The weight of the cover slip spread the sample
which made the semen to move to center of Neubauer bycapillary action and standardized so that the analyses werecarried out in a preparation with fixed depth. Both chambersof haematocytometer were scored and the average count
was calculated.
A fixed volume of semen was collected from harvested
epididymis and put in normal saline. Not more than 10 µl of
the semen was withdrawn with micro-pipette and delivered
onto clean glass slide covered by 22 x 22 mm cover slip andstandardized so that the analyses were carried out in a
preparation with depth (i.e., 20 µl). This depth allowed full
expression of the rotating movement of normal spermatozoa.
The weight of the cover slip spread the sample for optimal
viewing. The freshly made, wet preparation was left tostabilize for approximately one minute and the procedure
was carried out at a room temperature between 18 and 24oC
in the laboratory. The microscopic field was scannedsystematically and the motility of each spermatozoon was
graded as being motile or non- motile. Spermatozoa gradedmotile displayed rapid progressive motility along a linear
track, covering a distance of at least half the length of the
spermatozoon per second.
Testosterone Assay
Blood obtained from the left ventricular through cardiac
puncture was assayed for serum testosterone. The level of
testosterone was estimated using Bio-Inteco KIT by Inteco
Diagnostics UK Ltd.
Desired numbers of coated wells were secured in theholder after which 10 µL of standards, specimens andcontrols were dispensed into appropriate wells. Then 50 µL
of rabbit anti-Testosterone reagent was dispensed into eachwell. The mixtures were thoroughly mixed for 30 minutes.
100 µL of Testosterone-HRP Conjugate Reagent wasdispensed into each well and it was incubated at 37oC for 90
minutes. The microwells were rinsed and flicked 5 times withwashing Buffer (1x). 100 µL of TMB Substrate wasdispensed into each well and gently mixed for 10 seconds,
incubated at room temperature (18–22 oC) for 20 minutes.
5

The reaction was stopped by adding 100µL of Stop Solution
to each well. It was gently mixed for 30 seconds and it wasmade sure that all the blue color changed to yellow color
completely. Absorbance was read at 450nm with a microliterwell reader within 15 minutes.
Statistical Analysis
Results were expressed as mean ± standard deviation.
One-way ANOVA was used to analyze data, followed byStudent Newman-Keuls (SNK) test for multiple comparisons.
GraphPad Prism5 (Version 5.03, GraphPad Inc USA.) was
the statistical package used for data analysis. The results
were considered significant when p < 0.05.

RESULTS
Testicular torsion was accompanied by weight loss evenwith the administration of Vitex doniana and vitamin C for the
first 2-3 days in the experimental animals. Weight loss
continued in the control (sham surgery) and torted untreatedgroup (Group A and B) respectively. There was a slightweight gain in the treated groups C, D, E and F towards theend of the experimental period (Table I).

Fig. 1. Effects of Vitex doniana on Sperm Motility of Male Wistar
Rats following Testicular Torsion* significantly different from control at p < 0.05.
# significantly different from B at p < 0.05.

The mean sperm count in group B (torted untreated) is(88.2 ± 4.63 ×100/ml) which is significantly (p < 0.05)
different when compared with groups D, E and F (109.8 ±
4.14, 117.4 ± 6.62 and 126.8 ± 8.71 ×100/ml) (Figure 2).

Table I. Absolute Organ Weight and Difference in Body Weight of Male
Wistar Rats Treated with Vitex donina following Testicular Torsion
TreatmentGroups

InitialBody
Weight
(g)

Final
Body
Weight(g)

Absolute
Testicular
Weight (g)Relative
Testicular
Weight(%)

A

176.00 ±7.97

161.60 ±7.97

1.25 ± 0.07

0.78 ± 0.04

B

225.00 ±5.00*

175.30 ±21.59

0.79 ±
0.14*

0.44 ±
0.05*

C

149.00 ±8.86

148.20 ±9.90*

0.97 ±
0.04*

0.67 ± 0.04

D

156.30 ±8.75

152.60 ±8.41

0.90 ±
0.05*

0.59 ± 0.05

E

173.80 ±11.43

159.60 ±9.46

1.41 ± 0.10

0.89 ± 0.08

F

153.00 ±9.03

143.90 ±9.59*

1.08 ± 0.07

0.78 ± 0.09

Results are presented as Mean ± SEM (n =5).
* Significantly different from Control at p < 0.05.

The mean sperm motility in the torted untreated group B
is (44.0 ± 4.47%) which is significantly (p < 0.05) lower whencompared with the control (93.0 ± 1.23 %) and the torted andtreated groups C, D, E and F (58.0 ± 4.64, 59.0 ± 2.45, 49.0
± 5.57 and 56.0 ± 6.00 %) (Figure 1).

6

Fig. 2. Showing the Effects of Vitex doniana on Sperm Countof Male Wistar Rats following Testicular Torsion* significantly different from control at p < 0.05.
# significantly different from B at p < 0.05

The mean serum testosterone concentration for group B(torted untreated) is (0.50 ± 0.14 ng/dl) which shows asignificant (p < 0.05) decrease when compared with thecontrol (1.85 ± 0.52 ng/dl) and the other torted and treatedgroups; C, D, E and F (0.62 ± 0.02, 0.62 ± 0.24, 0.74 ± 0.18
and 0.88 ± 0.50 ng/dl). Serum testosterone for group F(torted and treated with 100 mg/kg Vitamin C) also shows asignificant (p < 0.05) difference when compared with groups
treated with the extract; C, D and E (0.62 ± 0.02, 0.62 ± 0.24
and 0.74 ± 0.18 ng/dl) (Figure 3).

Fiziologia - Physiology • 2016.26.1(89)Fig. 3. Showing the Effects of Vitex doniana on Testosterone
Level of Male Wistar Rats Following Testicular Torsion* significantly different from control at p < 0.05.
# significantly different from C and D at p < 0.05.

DISCUSSION
Result from this study showed reduced quantity of
testosterone in untreated torted group (group B) and groups
that received lower dose of the extract (group C and D, 50
mg/kg and 100 mg/kg). The decrease in testosterone is inagreement with the findings of Prillaman and Turner [7] who
reported that testicular torsion is the most common genital
trauma of the adolescent boy and has been implicated in
testicular injury, altered hormone production, subfertility andinfertility. Baker and Turner [8] and [9] Turner et al., 2005
reported that endocrine function of the rat testis after repair of
1-hour, 720o torsion is significantly reduced; however,testosterone levels do return to normal values days after the
repair of torsion. This report also agrees with the result fromthe findings of this study.
However, the result from this study also showed a
progressive slight increase in testosterone level of groups E
and F that received higher dose of extract and vitamin C (200mg/kg extract and 100 mg/kg Vitamin C) respectively. This is
an indication of the restorative effect of Vitex doniana extract
at a higher dose and vitamin C on the endocrine function oftestes following testicular torsion.
Ascorbic acid (vitamin C) is a component of the extractand it is an effective scavenger of superoxide radical anion,hydroxide radical, singlet oxygen and reactive nitrogen oxide[10,11]. Vitamin C also contributes to the support ofspermatogenesis, at least in part through its capacity to
reduce vitamin E (α- tocopherol) and maintain thisantioxidant in an active state. Vitamin E is a powerful
lipophilic antioxidant that is absolutely vital for the
maintenance of mammalian spermatogenesis [12]. Vitamin
C is itself maintained in a reduced state by GSH-dependent
dehydroascorbate reductase, which is abundant in the testes[13].
Fiziologia - Physiology • 2016.26.1(89)Johnson [12] has reported that deficiency in vitamin C
leads to oxidative stress in the testes, which disrupt bothspermatogenesis and the production of testosterone. This is inagreement with the result of this study as there was asignificant reduction in the testosterone level in the untreatedtorted group when compared with the extract and vitamin C
treated groups. Conversely, ascorbate administration tonormal animals stimulates both sperm production andtestosterone secretion [14]. This vitamin as also been found tocounteract the testicular oxidative stress induced by exposure
to pro-oxidants such as arsenic, cadmium, endosulfan andalcohol [15,16]. However, no toxicological study has been
done on Vitex doniana to determine any toxic component of it
which can label it as a toxicant, rather it has been found thatthe plant is useful in treatment of ulcers, wounds and malesterility as reported by [17-20].
The outcome of this study revealed that torsion-induced
testicular damage is associated with increased oxidative
stress in the testes. The increased oxidative stress disrupted
the steroidogenic capacity of the Leydig cells to produce
testosterone and the capacity of the germinal epithelium todifferentiate normal spermatozoa. This is in tandem withwhat [21-23] reported that spermatogenesis and Leydig cellsteroidogenesis- the two vital functions of the testes, areimpaired by oxidative stress. This validates oxidative stressas a major factor in the aetiology of male infertility.
Result from this study showed that the extract has
significant effect on the sperm count and motility as there is a
progressive increase in sperm count and motility across thegroups that were treated with the extract and vitamin C whencompared with the torted untreated group. However, it has
been stated above that Baker and Turner [8] and Turner etal., [9] reported that endocrine function of the rat testis after
repair of 1-hour, 720o torsion is significantly reduced;
however, testosterone levels do return to normal values days
after the repair of torsion. This endocrine restoration after
detorsion might be responsible for the significant increase in
the sperm count and motility of the treated group knowing
fully that testosterone is a hormone that preserves the sperm
quality.

CONCLUSION
In conclusion, discovering efficient therapies for
improving a torted testis is the most important goal ofmanaging testicular torsion in humans. This study showed anameliorative effect on the semen quality in the torted animalmodel by treating with Vitex doniana leaf aqueous extract.
Therefore, Vitex doniana extract would be of potential benefit
in the management of some forms of male infertility due totesticular torsion.
In view of the observations from this study, we
recommend a further study into the antioxidant properties ofVitex doniana aqueous leaf extract in torsion-induced

7

testicular damage as induction of oxidative stress is the main
mechanism of damage in this urological challenge.
Conflicts of Interest
The authors declare that they have no conflict of interest.
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2. Atawodi SE, Bulus T, Ibrahim S, Ameh DA, Nok AJ, Mamman
M and Galadima M. In vitro trypanocidal effect of methanolic
extract of some Nigerian savannah plants. Afr. J. Biotech
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8

13. Paolicchi A, Pezzini A and Saviozzi M. Localization of a
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Fiziologia - Physiology • 2016.26.1(89)CYP2D6 ENZYMATIC DEFICIENCY AND EXTRAPYRAMIDALSIDES EFFECTS IN AN AUTISTIC PATIENT TREATED WITH
RISPERIDONERALUCA GRADINARU*, NICOLETA ANDREESCU*, MARIA PUIU
Genetics Discipline, Department of Microscopic Morphology, “Victor Babes” University of Medicine and Pharmacy, Timisoara,
Romania

ABSTRACT
It is known today that genetic variability can have a major impact in the occurrence of adverse effects of antipsychotic
medications such as movement disorders. Unfortunately, in clinical practice, testing the genetic profile of patients is notfrequently performed in order to identify such risks. It is our hope, that in the near future, for a more predictive andpersonalized treatment, identifying the specific genotype of patients and the way this could contribute to the antipsychotic
adverse effects, will become a usual practice.
In this paper we provide a summary of one case report on a 10-year-old autistic patient who suffered from movement
disorders while being treated with risperidone at 0.75mg/day. It was discovered that he has a cytochrome P450 2D6
deficiency, being phenotypically a CYP2D6 intermediate metabolizer, which may explain his susceptibility to develop
extrapyramidal side effects. Although we expected that the active moiety of risperidone would be elevated, this was not
the case. This is an example of the important role that pharmacogenetics can have, to identify the risk of side effects prior
to treatment initiation, and to better guide the clinicians in choosing a personalized therapeutic regime for each patient,with less adverse reactions.
Keywords: Risperidone, CYP2D6 genotype, extrapyramidal syndrome

INTRODUCTION
Autism is a behavioral disorder diagnosed in childhood,characterized by difficulties in communication, stereotypical,
repetitive behavior and decrease of patient's ability tointeract socially. Although the specific causes are not yet
understood, scientific evidence indicates involvement of bothabnormalities in brain development and important genetic
factors. Besides educational and behavioral specializedinterventions, pharmacological management can play apositive role in the treatment of autism related disorders suchas depression, anxiety, hyperactivity, obsessive-compulsive
behavior, auto and hetero aggression.
Risperidone is an atypical antipsychotic whose
pharmacodynamic action is due to the dual dopaminergic(D2) and serotoninergic (5HT2) antagonism in the central
nervous system. In October 2006, risperidone was the first
drug approved by the Food and Drug Administration (FDA)
for the treatment of irritability in children and adolescents with
autism, between 5 and 16 years of age. Clinical efficacy ofrisperidone versus placebo was demonstrated in two clinical
trials conducted over a period of eight weeks, which included

159 pediatric patients with autism. The instruments
employed to assess reduction of irritability were Aberrant
Behavior Checklist (ABC) and Clinical Global Impression Change (CGI-C) scale [1]. Off-label risperidone isadministered also in other behavioral disturbances
associated with autism such as aggression, self-flagellationand spontaneous mood changes [2].
Regarding pharmacokinetic properties, risperidone ismetabolized in the liver by the CYP2D6 enzyme system. The
CYP2D6 gene has several polymorphisms. The identified
allelic variants can be classified as being: functional (e.g. CYP2D6*1 and *2)
 with reduced activity (e.g. CYP2D6*9, *10, *29, *36, *41)
 nonfunctional (e.g. CYP2D6*3-8, *18, *21, *44) [3].
The adverse effects occurring with a frequency greaterthan 5% suffered by pediatric patients with autism treatedwith risperidone, may include dizziness, constipation, drymouth, sedation, fatigue, headache, weight gain andparkinsonian syndrome. Also neurological and other adverse
effects have been reported, such as dyskinesia, neuroleptic
malignant syndrome, impaired motor coordination,arrhythmias, hyperglycemia etc. [4].

Received January 10th 2016. Accepted February 2nd 2016. Address for correspondence: Nicoleta Andreescu, Discipline of
Genetics, University of Medicine and Pharmacy “Victor Babes”, 2 E. Murgu Square, 300041 Timisoara, Romania, tel.:
0040256204476, e-mail: nicollandreescu@yahoo.com*Authors have equal contribution

Fiziologia - Physiology • 2016.26.1(89)9CASE DESCRIPTION
A male patient aged 10, 31 kg, diagnosed with autism,
was prescribed risperidone to improve aggressive behavior,
0.25mg/day for 7 days, then 0.5mg/day for 12 months.
Subsequently, to better control the symptoms, the dose of
risperidone was increased to 1mg/day. Because there was anincrease in the patient’s irritability, it was decided to increase
the dose to 2 mg/day divided in two doses, considering thatthese manifestations are due to disease decompensation as aconsequence of the pre-pubertal stage of the child. 14 days
after increasing the dose, the patient developed akathisia, withextreme psychomotor agitation, stiffness and inability tocontrol the motor function. It was decided to gradually reducethe dose of risperidone to discontinuation.
In order to ameliorate akathisia, treatment with propranolol
was initiated for 30 days. Initiation and discontinuation of
propranolol treatment was made by progressively increasing
respectively decreasing doses. It was noticed thatadministration of propranolol caused a distinct reduction in
intestinal transit with occurrence of constipation.
Subsequently, reintroduction of risperidone therapy wasdecided, in progressive increased doses of 0.25 mg/day for
14 days, then 0.5 mg/day followed by 0.75 mg/day. At adosage of 0.75 mg/day extrapyramidal adverse effects
occurred, manifested by choreiform movements, especiallyof the hands. Analysis of plasma concentrations, ofrisperidone and of the active metabolite, for the three doses,
was conducted (Table I).
Table I. Risperidone dosage vs plasma concentration
Test

Risperidone
dosage

Test

(mg)
1.

2.

3.

0.25

0.50

0.75

Plasmaconcentrations

Ratio
R:9-HO-R(µg/l)
Risperidone< 1.0 *

9-HO-Risperidone

1.1 **

Risperidone

2.3 *9-HO-Risperidone

3.1 **

Risperidone

2.4 *9-HO-Risperidone

3.4 **

0.90

0.74

0.70

At present, after taking various other atypicalantipsychotics, the patient is under treatment withrisperidone, because it was the only drug that proved to beeffective for the treatment of anxiety and aggressive
manifestations associated with autism. Maintenance dosage
which proved to be tolerated by the patient, was set at 0.5
mg/day in two divided doses.
According to the drug maker’s specifications, for thetreatment of irritability associated with autism, the target
dose of risperidone for patients > 20 kg is 1 mg/day. There
are also clinical trials where the maximum daily dose was 2.5
mg for children > 20 kg, but at this dose the therapeutic effect10

has reached a plateau [5]. Although the dose administered tothe patient fit in the therapeutic range, the occurrence ofadverse effects imposed considerable reduction in therisperidone dosage.
Nowadays we know the importance of the allelicconstitution of a certain patient for setting the correct dosage of
medication so as to avoid the occurrence of adverse drug
reactions. Thus, detection of genetic variations in the enzymes
involved in the metabolism of risperidone was pursued, inorder to investigate the patient's general ability to metabolize
drugs and to find out if the patient has an increased risk ofadverse drug reactions to therapeutic doses.
Since risperidone is metabolized by the CYP2D6 enzyme
system, CYP2D6 genotyping was decided and carried out.
Analyses revealed heterozygosis for CYP2D6*4 allele(1846G>A) and CYP2D6*41 (2988G> A) – genotype *4*41.
The presence of the inactive allele and of the low activity
allele is associated with reduced enzymatic activity of
CYP2D6, which means metabolizing capacity of substrates
less than normal [6]. Phenotypically, like 10% of the
Caucasian population, the patient falls into the group of“intermediate metabolizers”. The result of molecularanalyses also indicated a decrease of the CYP2C19
enzymatic function.

DISCUSSION
The first generation of antipsychotic (FGA) drugs was
associated with different severity of extrapyramidal
manifestation in an important percent of patients. The
second generation of antipsychotics (SGA), especially thosewith lower dopamine receptors affinity, was thought to haveless adverse effects, but the majority of the reports showedthat extrapyramidal manifestations can appear even after theuse of SGA [7].
Akathisia, reported in this paper, is one of the clinicalmanifestations of the extrapyramidal syndrome that wasreported after administration of FGA but also SGA [8].
The main metabolic pathway of risperidone ishydroxylation to the active metabolite 9-HO-risperidone
through the CY2D6 liver enzyme system. After administrationof the antipsychotic, the clinical effect occurs due to thesumming up of plasma concentrations of risperidone and its
active metabolite 9-hydroxyrisperidone (active moiety). Initially
it was thought that polymorphism of the CYP2D6 gene, with
reduction of metabolic capacity, has no clinical implications,since decreasing the concentration of the active metabolite
9-HO-risperidone is compensated by increase of theconcentration of risperidone. Subsequently, clinical studies
have found a correlation between CYP2D6 poor metabolizers and the increased incidence of extrapyramidal
adverse effects following administration of risperidone, which
would be in agreement with what we have noticed [9].
Specialized literature uses the R:9-HO-R ratio to assess
the level of activity of CYP2D6. It was suggested that a
Fiziologia - Physiology • 2016.26.1(89)plasma ratio R:9-HO-R between 0.1-0.3 indicates a normal
enzymatic activity, this patients being classified as
extensive metabolizers, while a value >1 is associated withlack of enzymatic activity, this patients being classified aspoor metabolizers [10]. For our patient, CYP2D6
intermediate metabolizer, the average ratio R:9-HO-R is0.78, which is in trend with the things presented above.
The occurrence of these adverse effects in patients, whoreport high risperidone/9-HO-risperidone plasmatic ratio,
characteristic to CYP2D6 deficient metabolizers, may be
explained by the different pharmacodynamic profile of thetwo compounds. In a review, Alamo et al. analyze pharmacological differences between risperidone and the active
metabolite administered as a drug under the name of
paliperidone [11]. Compared to ripseridone, 9-HO-risperidone
has a higher rate of dissociation from the D2 receptors and alower affinity to adrenergic, muscarinic and 5HT2A receptors,
hence the possible differences existing in therapeutic efficacy
and the profile of adverse effects between the two. Also thetwo compounds differ in terms of profile of the second
messenger systems that they regulate [12].
Since the result of the genetic testing performed, places
our patient among intermediate metabolizers, we would
expected that there is an accumulation of risperidone and theactive metabolite, which could explain the occurrence ofadverse effects. But after determination of plasma
concentrations following administration of different doses ofrisperidone, it appeared under-dosed, in spite of the adverse
effects occurred. The analysis laboratory quotes Regenthal
R. for the reference values of these analyses [13]. As far as
we are concerned, we do not know about the existence inliterature of data indicating exactly the optimal plasma
concentration for risperidone for pediatric patients, overwhich the risk of adverse effects exceeds the benefits
provided by the treatment.
The importance of establishing the CYP2D6 genotype inpatients treated with risperidone was suggested by several
studies that included cohorts of children and adolescents
under treatment with risperidone [14, 15].

CONCLUSION
Thus, to implement a personalized therapy withrisperidone, we sustain that CYP2D6 genotyping prior torisperidone treatment can be a useful tools in choosing thecorrect dosage regimen, so as to avoid the occurrence ofadverse drug reactions.

Fiziologia - Physiology • 2016.26.1(89)Acknowledgments
This work was supported by the POSDRU grant no.
159/1.5/S/136893 titled “Strategic partnership for the increase of
the scientific research quality in medical universities through theaward of doctoral and postdoctoral fellowships – DocMed.Net_2.0”,
awarded to Dr. Andreescu Nicoleta.
REFERENCES
1. United States Food and Drug Administration. (October 2006). FDA
approves the first drug to treat irritability associated with autism,
risperdal. http://www.fda.gov/NewsEvents/Newsroom/.
2. Sharma A, Shaw SR. Efficacy of risperidone in managing
maladaptive behaviors for children with autistic spectrum disorder:
a meta-analysis. J Pediatr Health Care 2012; 26(4):291-9.
3. Ingelman-Sundberg M, Sim SC, Gomez A, et al. Influence of
cytochrome P450 polymorphisms on drug therapies:
Pharmacogenetic, pharmacoepigenetic and clinical aspects.
Pharmacol Ther 2007; 116:496-526.
4. Rxlist.com. (2009). Risperdal indications & dosage,
http://www.rxlist.com/risperdal-drug.htm
5. Risperdal(risperidone).
http://www.fda.gov/Safety/
MedWatch/Safety Information/ucm175826.htm.
6. www.snpedia.com/index.php/CYPED6
7. Divac N, Prostran M, Jakovcevski I, et al. Second-Generation
Antipsychotics and Extrapyramidal Adverse Effects. Biomed Res
Int. 2014; 2014: 656370.
8. Shirzadi AA, Ghaemi SN. Side effects of atypical antipsychotics:
extrapyramidal symptoms and the metabolic syndrome. Harv Rev
Psychiatry 2006; 14(3):152-164.
9. de Leon J, Susce MT, Pan RM, et al. The CYP2D6 poor
metabolizer phenotype may be associated with risperidone
adverse drug reactions and discontinuation. J Clin Psychiatry
2005; 66:15-27.
10. de Leon J, Greenlee B, Barber J, et al. Practical guidelines for theuse of new generation antipsychotic drugs (except clozapine) in
adult individuals with intellectual disabilities. Res Dev Disabil 2009;
30:613-669.
11. Álamo C, López-Muñoz F. The Pharmacological Role and ClinicalApplications of Antipsychotics’ Active Metabolites: Paliperidone
versus Risperidone. Clin Exp Pharmacol 2013; 3:117.
12. Clarke WP, Chavera TA, Silva M, et al. Signalling profile
differences: paliperidone versus risperidone, Pharmacol. 2013;
170(3): 532-545.
13. Regenthal R, Koppel M, Preis R. Anestheologie undIntensivmedizin 1999; 3: 129-144.
14. Youngster I, Zachor DA, Gabis LV et al. CYP2D6 genotyping in
paediatric patients with autism treated with risperidone: a preliminarycohort study. Dev Med Child Neurol 2014; 56(10): 990-4.
15. Sherwin CMT, Saldaña SN, Bies RR, et al. Population
pharmacokinetic modeling of risperidone and 9-hydroxyrisperidone
to estimate CYP2D6 subpopulations in children and adolescents.
Ther Drug Monit 2012; 34(5): 535–544.

11

DEFICITUL ENZIMATIC DE CYP2D6 ŞI EFECTELE
ADVERSE EXTRAPIRAMIDALE LA UN PACIENT CU
AUTISM TRATAT CU RISPERIDONĂREZUMAT
Este cunoscut astăzi că variabilitatea genetică poate avea un impact major în apariția efectelor adverse ale
medicamentelor antipsihotice, cum ar fi efectele adverse extrapiramidale. Din păcate, în practica clinică, testarea profilul
genetic al pacienților nu se determină în mod frecvent, în vederea identificării unor astfel de riscuri. Este speranța
noastră, ca în viitorul apropiat, pentru personalizarea şi buna predictibilitate a tratamentului medicamentos, identificarea
genotipului pacienților și modul în care acesta ar putea contribui la apariţia efectelor adverse antipsihotice, să devenă opractică obișnuită. Această lucrare a dorit să prezinte un studiu de caz al unui pacient diagnosticat cu autism, în vârstă
de 10 de ani, care ȋn urma tratamentului cu risperidonă la 0,75 mg / zi a dezvoltat efecte adverse extrapiramidale. S-a
descoperit că pacientul prezintă un deficit al citocromul P450 2D6, fiind fentotipic ȋncadrat ca metabolizor intermediar
CYP2D6, ceea ce poate explica susceptibilitatea de apariţie a efectelor secundare extrapiramidale. Deși ne-am așteptatca şi concentraţia activă totală de rispeidonă să fie crescută, acest lucru nu a fost observat. Acest caz este un exemplu alrolului important pe care farmacogenetica ȋl poate avea, pentru a identifica riscul de a dezvolta reacții adverse
medicamentoase înainte de inițierea tratamentului, și pentru a ghida mai bine clinicienii ȋn alegerea unor doze terapeuticepersonalizate pentru fiecare pacient ȋn parte, cu un minim de reacții adverse.
Cuvinte cheie: Risperidona, genotip CYP2D6, sindrom extrapiramidal

12

Fiziologia - Physiology • 2016.26.1(89)EFFECTS OF CHRONIC COPPER SULPHATEADMINISTRATION ON FEEDING PATTERN AND MARKERSOF RENAL AND LIVER FUNCTIONS OF WISTAR RATS
RUFUS O. AKOMOLAFE1, OLAOLUWA S. OLUKIRAN1, CHRISTIAN E.
IMAFIDON1, OLUGBENGBA A. AYANNUGA2, JOHN A. OYEKUNLE3,AYOWOLE A. OLADELE4
1Department of Physiological Sciences, Faculty of Basic Medical Sciences, Obafemi Awolowo University, Ile-Ife, Nigeria
2Department of Anatomy and Cell Biology, Faculty of Basic Medical Sciences, Obafemi Awolowo University, Ile-Ife, Nigeria
3Department of Chemistry, Faculty of Science, Obafemi Awolowo University, Ile-Ife, Nigeria

4Department of Medical Laboratory Science, College of Medicine, Afe Babalola University, Ado-Ekiti, Ekiti State

ABSTRACTThis study was carried out to determine the changes in feeding pattern, plasma and urine concentrations of someorganic constituents which are often used in the assessment of renal function following chroni c administration ofcopper sulphate to Wistar rats. The changes in plasma activities of some liver enzymes were also investigated.
Twenty (20) adult male Wistar rats were divided into four groups of five rats each. The control group received distilledwater orally. Groups II, III and IV were administered orally with 100, 200 and 300 mg/kg/day of copper sulphate,respectively, for 35 days. Results showed significant reductions in body weight, food consumption and water intake inthe experimental groups when compared with the control rats. The plasma creatinine levels of the experimentalgroups increased significantly when compared with the control rats. A significant reduction in urine creatinine wasobserved in the experimental groups when compared with the control rats. This was accompanied by significant
decrease in creatinine clearance. The activities of plasma AST and ALT were significantly elevated in the experimentalgroups when compared with the control rats. Photomicrographs of the kidney of the experimental rats revealed dose
dependent tissue degeneration, while their liver did not show any visible evidence of degeneration.
It is concluded that chronic administration of copper sulphate is toxic to the kidney of rats. Copper sulphate induced liver
necrosis could require a longer period of exposure to develop in rats than the time needed to cause kidney damage.
Keywords: Copper sulphate, kidney, creatinine, urea, rats.

INTRODUCTION
Copper (Cu) is an essential trace element and one of themost important heavy metals capable of producing toxic
effects in man and animals when ingested acutely or
chronically in excess. It is used chiefly for agricultural
purposes as a pesticide and in leather industry [1]. Copper isneeded by the body for a number of functions, predominantly
as a cofactor for a number of enzymes such as
ceruloplasmin,
cytochromeoxidase,dopamine
β-hydroxylase, superoxide dismutase and tyrosinase. It ispresent in several haematinic and its salts are also usedtherapeutically because of their astringent and antiseptic
properties but sometimes copper salts are poisonous forhuman organ system. Exposure of humans to copper occurs
primarily from the consumption of food and drinking water [2].

Chronic copper exposure is increasingly recognized as apublic health issue; its early effects remain largely unknown[3]. Ingestion of significant quantities of copper carries a riskof multi organ failure and death [1]. Initially, copper
accumulates in the liver and disrupts the liver’s ability todetoxify elevated copper concentration in the body, thus itadversely affects the nervous system, reproductive system,
adrenal function, connective tissue and learning ability.
Copper toxicity has also been associated indirectly with anumber of neurological disorders, including Alzheimer’s
disease and prion diseases, such as bovine spongiform
encephalopathy [4].
In a preliminary study of a two-week oral administrationof 100 to 200 mg/kg of copper sulphate, we observedchanges in some of the renal functions and feeding
patterns of Wistar rats. We reported a pattern of anorexia

Received January 12th 2016. Accepted March 16th 2016. Address for correspondence: Olukiran Olaoluwa Sesan,
Department of Physiological Sciences, Obafemi Awolowo University, Ile-Ife, Nigeria; tel: +234 8123872009;e- mail: oolaoluwasesan@gmail.com

Fiziologia - Physiology • 2016.26.1(89)13which manifested as a significant reduction in the food andwater intake of all the experimental rats. The effects of thesame doses of copper on the plasma and urine levels ofsome markers for the assessment of kidney functions in
Wistar rats revealed that their concentrations in urine
reduced and increased marginally in the plasma. This was
accompanied by decrease in creatinine clearance. The
decrease in creatinine clearance is an indication of tissue
damage, which was supposed to have been accompaniedwith a significant increase in plasma concentration ofcreatinine. The fact that the plasma level of creatinine did
not rise significantly in the treated rats could be due to theacute nature of this study [5].
Most of the reported studies on the effect of copper
sulphate on renal function of rats measured the plasma
creatinine and urea concentrations and indicated elevationof these indices as an indication of renal tissue damage.
Elevated plasma creatinine level could not be attributed torenal dysfunction only, it might be elevated as a result ofother pathological conditions like muscle wasting anddamage to other tissues apart from the kidney. A measureof creatinine clearance is a better assessment of renal
handling of this important metabolite as well as blood flow
to the kidneys. We have also reported that short term
administration of Cu did not significantly alter the histologyof the renal tissue of rats. We therefore decided toinvestigate the effects of chronic ingestion of copper
sulphate on the feeding pattern and creatinine clearance
and some markers for the assessment of kidney and liverfunctions in Wistar rats.

MATERIALS AND METHODS
Twenty (20) adult male Wistar rats weighing 120 g - 150
g obtained from the Animal House of the College of Health
Sciences, Obafemi Awolowo University, Ile-Ife, wereallowed to acclimatize in the laboratory for one week before
the commencement of the study. They were kept undernormal environmental conditions with a natural light/darkcycle and were fed on standard rodent pellet diet (CapsFeed PLC, Osogbo, Nigeria) and water ad libitum. Each rat
was housed in a separate metabolic cage (Ohaus R Model;
Ohaus, Pine Brook, NJ, USA) during the experiment toobtain a 24 hr urine sample. All experimental procedures
carried out were in strict compliance with the principles forthe care and use of laboratory animals in Biomedical
Research, College of Health Sciences, Obafemi Awolowo
University, Ile-Ife.

14

Experimental procedure
The rats were divided into four groups of five rats each.
The control group received distilled water orally. Groups II, III
and IV were administered orally with 100, 200 and 300
mg/kg/day of copper sulphate, respectively, for 35 days. On
the 36th day, rats were sacrificed by cervical dislocation.
Blood was collected into separate heparinized bottles forhaematological analysis using an auto-analyzer machine(SFRI Blood Cell Counter, H18 Light, France) and thencentrifuged at 4000 rpm for 15minutes at 4ºC, using a cold
centrifuge (Centurium Scientific, Model 8881). The obtained
plasma was collected into separate plain bottles for theassessment of organic constituents that are routinely used in
the assessment of kidney function. The kidney of each rat
was carefully excised and fixed inside 10% formo-saline forhistopathological studies.
During the experiment, the body weight of the animals
was measured once a week using a weighing balance(Camry; Zhongshan Guangdong, China) to determine theweight gain or loss in each group. Water intake and foodconsumption were measured with the aid of a measuringcylinder and weighing balance respectively. The daily foodconsumption and water intake of the rats was determined bysubtracting the previous day volume of water and weight offood from the left-over.
Biochemical assays
Creatinine, urea, aspartate aminotransferase, alanine
aminotransferase and total bilirubin concentrations in theplasma were assayed with the use of commercially available
biochemical kits (Randox Laboratories Limited, Antrim, UK).
The urine concentrations of urea and creatinine were
estimated in the last samples of urine collected from the rats,
using the same methods that were used in the analysis ofplasma. Creatinine clearance was then calculated.
Creatinine was determined by alkaline picrate method [6].
Urea assay was carried out according to the method ofBerthelot [7]. Aspartate aminotransferase and alanine
aminotransferase were measured by the method of Reitman
and Frankel [8]. Total bilirubin was estimated using themethod of Sherlock [9].
Histological examination
The fixed kidney samples were dehydrated in gradedalcohol, cleared by xylene and embedded in paraffin wax.
The tissues were then cut into 7-8 μm thick sections by amicrotome, fixed on the slides and stained withhaematoxylin-eosin. The slides were examined under a lightmicroscope (Olympus CH; Olympus, Tokyo, Japan) andphotomicrographs were taken with a Leica DM 750 Camera
at x100 and 1000 magnifications.

Fiziologia - Physiology • 2016.26.1(89)Statistical analysis
Statistical analysis was carried out using one-way
analysis of variance (ANOVA) followed by Tukey’s post-hoc
test for multiple comparison using GraphPad 5.03
(GraphPad Software Inc., CA, USA). Differences withprobability values of p < 0.05 were considered significant.

RESULTS
Food consumption and body weight
A significant decrease (p < 0.05) in food consumption
and body weight was observed in the experimental groups
when compared with control group (Table I).

Table I. Effects of graded doses of copper sulphate on food consumption and body weight of rats
I Control

II (100 mg/kg)

III( 200 mg/kg)

IV(300 mg/kg)

Food consumption (g)20.51 ± 1.39

15.93 ± 1.06*

15.93 ± 1.61*

15.20 ± 0.58*

Body weight (g)195.4 ± 8.98

166.3 ± 0.87*

163.4 ± 4.72*

169.5 ± 3.53*

Values are given as mean ± SEM. * = Significantly different from control.
Table II. Effects of graded doses of copper sulphate on water intake and urine volume of rats
I Control

II(100 mg/kg)

III(200 mg/kg)

IV(300 mg/kg)

Water intake (ml)43.60 ± 1.50

32.80 ± 1.39*

30.33 ± 2.95*

35.20 ± 0.58*

Urine volume (ml)4.44 ± 0.12

3.47 ± 0.28§

3.75 ± 0.50§

6.21 ± 0.04*

Values are given as mean ± SEM. * = Significantly different from control. § = Significantly different from group IV.
Table III. Effects of graded doses of copper sulphate on total bilirubin and liver enzymes of rats
TB

I Control
12.12± 0.57

II(100 mg/kg)
13.38± 1.03

III(200 mg/kg)
37.00± 7.21*§

IV(300 mg/kg)
50.64± 10.74*§

AST

51.56± 3.09

107.8± 16.22*

111.2± 5.99*

109.1± 6.85*

ALT

23.54± 1.38

48.43± 11.40*

43.18± 5.86*

47.68± 7.59*

Values are given as mean ± SEM. * = Significantly different from control. § = Significantly different from group II.
AST = Aspartate aminotransferase, ALT = Alanine aminotransferase, TB = Total bilirubin
Table IV. Effects of graded doses of copper sulphate on haematological indices of Wistar rats
WBC Count
LYM%
MON%
GRAN%
LYM Count
MON Count
GRAN CountRBC Count
HGBg Count
MCHpg
MCHCg/dl
PLT*10*3/UL
MPVfl
HCT%
MCVfl

I Control
4.38± 0.44
65.08± 3.26
14.80± 0.72
20.12± 2.66
2.86± 0.34
0.66± 0.07
0.86± 0.18
6.96± 0.16
12.98± 0.29
18.60± 0.05
36.40± 0.24
557.0± 18.14
6.46± 0.15
35.60± 0.72
51.24± 0.43

II(100 mg/kg)
3.76± 0.41
76.22± 2.46*
10.78± 0.87*
13.00± 1.80*§
2.90± 0.38
0.38± 0.04*
0.48± 0.06*
7.38± 0.21
13.82± 0.49
18.68± 0.25
33.16± 0.77*
428.4± 36.16
6.70± 0.09
41.72± 1.92*
56.56± 1.36*

III(200 mg/kg)
3.54± 0.40
78.34± 1.86*
8.46± 0.40*
13.20± 1.69*§
2.80± 0.35
0.30± 0.03*
0.44± 0.05*
7.43± 0.12
13.82± 0.33
18.54± 0.37
33.30± 0.38*
449.2± 59.80
6.80± 0.16
41.42± 0.85*
55.84± 0.78*

IV(300 mg/kg)
3.50± 0.18
85.96± 1.39*#
7.46± 0.50*#
6.58± 0.91*
3.00± 0.16
0.26± 0.02*
0.24± 0.04*
7.14± 0.12
13.20± 0.33
18.44± 0.29
32.14± 0.06*
343.2± 37.34*
6.44± 0.15
41.00± 1.03*
57.48± 0.91*

Values are given as mean ± SEM. * = Significantly different from control. # = Significantly different from group II.
§ = Significantly different from group IV. WBC = White blood cells, LYM = Lymphocyte, MON = Monocyte, GRAN = Granulocyte,

HGBg = haemoglobin, MCH = Mean corpuscular haemoglobin, MCHC = Mean corpuscular haemoglobin concentration, HCT = haematocrit,
MCV = Mean corpuscular volume, MPV = Mean platelet volume, PLT = Platelet count, PCT = Platelet crit.

Fiziologia - Physiology • 2016.26.1(89)15Water intake and urine volume
The water intake of the experimental groups decreased
significantly (p < 0.05) when compared with the control rats
(Table II). This was accompanied by a significant increase (p< 0.05) in urine volume in group IV when compared with thecontrol and groups II and III. However, the urine volume ofgroups II and III was not significantly different (p > 0.05) fromthat of the control group (Table II).
Total bilirubin, aspartate aminotransferase
and alanine aminotransferase
The activities of plasma aspartate aminotransferase(AST) and alanine aminotransferase (ALT) was significantly
elevated (p < 0.05) in the experimental groups whencompared with the control rats (Table III). There was no
significant difference (p > 0.05) in total bilirubin level ofgroup I when compared with the control rats. However, asignificant increase (p < 0.05) in total bilirubin level wasobserved in groups II and III (Table III). Also, the total
bilirubin level of groups II and III was significantly higher
than that of group I.
Haematological indices
There was no significant difference (p > 0.05) in red
blood cell (RBC) and haemoglobin concentrations of theexperimental groups when compared with the control rats
(Table IV). The percentage haematocrit (HCT%), mean
corpuscular volume (MCV) of the experimental groups
increased significantly (p < 0.05), but the mean corpuscularhaemoglobin concentration (MCHC) dropped significantly (p< 0.05) in the experimental groups when compared with thecontrol (Table IV).
The white blood cell (WBC) counts of the treatedgroups was not significantly different (p > 0.05) from that of
the control rats (Table IV). On the other hand, a significant
increase (p < 0.05) in percentage lymphocyte wasobserved in the experimental groups. The percentage
granulocyte and monocyte dropped significantly (p < 0.05)
in the treated groups when compared with the control rats.
There was a significant increase (p < 0.05) in percentage
lymphocyte of group IV when compared with group II,
whilst the percentage monocyte dropped significantly (p <0.05) in group IV when compared with group II.
The percentage granulocyte of group IV reduced
significantly (p < 0.05) when compared with groups II and
III (Table IV).
A significant reduction (p < 0.05) in platelet count andpercentage platelet was seen in group IV when comparedwith the control rats (Table IV).

urea concentration of the experimental groups decreased
significantly (p < 0.05) when compared with the control rats
(Figure 2).

Fig. 1. Effects of copper sulphate on urine urea concentration ofrats. Values are given as mean ± SEM. No significant difference
was observed between groups.

Fig. 2. Effects of copper sulphate on plasma urea concentrationof rats. Values are given as mean ± SEM. * = Significantly
different from control.

Plasma creatinine, urine creatinine and creatinine clearance
A significant reduction (p < 0.05) in urine creatinine wasobserved in the experimental groups when compared withthe control rats (Figure 3). This goes together with asignificant decrease (p < 0.05) in creatinine clearance(Figure 5). The plasma creatinine concentration of theexperimental groups was significantly higher (p < 0.05) thanthat of the control group (Figure 4).

Urine urea and plasma urea
There was no significant difference (p > 0.05) in theconcentration of urea in the urine of the experimental groups
when with the control rats (Figure 1). In contrast, the plasma
16

Fiziologia - Physiology • 2016.26.1(89)Fig. 3. Effects of copper sulphate on urine creatinine
concentration of rats. Values are given as mean
± SEM. * = Significantly different from control.

Fig. 5. Effects of copper sulphate on creatinine clearance of rats.
Values are given as mean
± SEM. *= Significantly different from control.

Histopathological observations
The photomicrographs of the kidneys of theexperimental rats show degenerating glomeruli andobliterated glomerular spaces when compared with thecontrol rats which show normal glomeruli and glomerularspaces (Figure 6).

Fig. 4. Effects of copper sulphate on plasma creatinine
concentration of rats. Values are given as mean
± SEM. * = Significantly different from control.

Fig. 6. Photomicrographs of the renal cortex (A; Control, B; 100 mg/kg of CuS04, C; 200 mg/kg of CuS04, D; 300 mg/kg of CuS04) showing
Normal (Yellow arrow) and degenerating (Black arrow) glomeruli. Normal (Blue arrow) and obliterated (Green arrow) glomerular spaces
were noted. Macula densa (Red arrow) and epithelial cells (Brown arrow) appear normal across the groups. White arrow points toglomeruli with reduced glomerular spaces. Stain H&E. Mag. X100 (Upper panel) and X1000 (Middle and Lower panels).

Fiziologia - Physiology • 2016.26.1(89)17DISCUSSION
The present study demonstrated that copper sulphate,
in a dose dependent manner, altered the plasma and urine
concentrations of some markers for the assessment ofkidney function in the experimental rats. This indicated that
this salt can induce renal toxicity. A significant decrease infood consumption was observed in the experimentalgroups with a corresponding decrease in body weight
when compared with the control rats. These observedchanges are in accordance with the findings of Haywood[10], who reported that one of the most consistent clinicalsigns indicative of toxicity in animals administered withcopper is a reduced growth rate which is accompanied bya decrease in body weight. The water intake of theexperimental groups reduced significantly when comparedwith the control group, but the urinary volume of groups II
and III was not significantly different from the control rats.
However, the urinary volume of group IV increased
significantly when compared with the control rats andgroups II and III. The significant increase in urine output
without a corresponding increase in the water intake thatwas observed in group IV may have resulted from theeffect of copper sulphate on the production of antidiuretic
hormone (ADH) from the supraoptic nuclei of the
hypothalamus or decrease in responsiveness ofvasopressin (V2) receptors in the tubular epithelial
membrane to ADH [11], thereby leading to decrease
reabsorption of water from the distal convoluted tubule andcollecting duct of the kidneys. However, this still requires
further studies. The degeneration of the renal tissue as
revealed by the photomicrographs could be responsible forthe loss of ability of the renal tubules to concentrate urine.
This could lead to dehydration and a severe depletion of
the major electrolytes of their body fluid with the attendantconsequences on the cardiovascular system.
In this study, the plasma creatinine levels of theexperimental groups were significantly higher than that of
the control rats. The creatinine concentrations in the urine
reduced significantly in the experimental groups whencompared with the control rats. These observed changes
are in accordance with the results of Sinkovic et al andGalhardi et al [2,12]. However, this is in contrast with thefinding of Abou-Seif et al [13] who reported thatadministration of copper (II) complexes in rats caused asignificant increase in superoxide dismutase activity
without alteration in blood creatinine and urea levels whencompared with the control rats. This could be to due acutenature of their study.
Creatinine clearance is important in assessing theexcretory function as well as blood flow to the kidneys. A
decrease in creatinine clearance is an indication of amarked reduction in glomerular filtration rate and renal
blood flow, resulting from a rise in renal vascular resistanceor damage to the glomerular capillary endothelium. The
decrease in renal blood flow may cause renal ischemia
18

resulting in impaired tubular reabsorption and secretion ofsubstances. The significant decrease in creatinine
clearance that was observed in the experimental groups isan indication of reduced blood flow to the kidneys as wellas significant renal tissue damage, a fact that was also
corroborated by the photomicrographs of the renal tissue
and the elevated level of creatinine in their plasma. The
decrease in urine creatinine is a further evidence of
reduced ability of the renal tubules to extract and remove
creatinine from the plasma of the experimental rats.
The liver is particularly rich in aspartate
aminotransferase (AST) and alanine aminotransferase(ALT) and damage to this organ which involves necrosis ofcells or increased cell permeability can be expected toraise the activities of the enzymes [14,15]. The activities ofplasma AST, ALT and total bilirubin level increased
significantly in the experimental groups when comparedwith the control group, but the photomicrographs of theliver of these groups revealed no significant alteration in
the histology of their hepatic tissue. Similarly, in a different
study, chronic exposure to 750 μg/g copper sulfate in rats
did not result in a development of substantial hepatic
damage [16]. This suggests that copper sulphate may haveinduced the release of the transaminases from otherorgans such as the kidney, heart, skeletal muscles [17,18].
Furthermore, the photomicrographs of the experimentalgroups showed appreciable degeneration of their renal
tissues.
The red blood cell count and haemoglobin
concentration of the experimental groups was not
significantly different from the control rats. This is incontrast with the findings of previous researchers whoreported that one of the major haematological manifestationsof copper sulphate poisoning is intravascular haemolysis andmethaemoglobinaemia [19-24]. However, the haematocrits
and mean corpuscular volume of the treated groups
increased significantly when compared with the control rats.
This may be an indication of haemoconcentration due toplasma volume reduction caused by dehydration [25]. A
significant decrease in granulocyte and monocyte counts
was observed in the experimental groups when comparedwith the control rats. This shows that the experimental rats
may be under stress which resulted in the release of
epinephrine. The initial response to epinephrine releasebegins in the amygdala, which triggers a neural responsein the hypothalamus. This is followed by activation of thepituitary gland and secretion of the adrenocorticotropic
hormone (ACTH) [26]. The adrenal gland is activatedalmost simultaneously and releases epinephrine. The
release of chemical messengers results in the productionof the cortisol, which increases blood pressure, blood
glucose, and suppresses the immune system [27].
Platelets or thrombocytes are the blood cell fragmentsthat aid the formation of blood clot. The significant
decrease in platelet count that was observed in group IV
could be attributed to the inhibitory effect of copper
Fiziologia - Physiology • 2016.26.1(89)sulphate on the production of thrombopoietin, a circulating
protein factor which facilitates megakaryocyte maturation,is produced constitutively by the kidneys [11].

CONCLUSION
From the results of this study, it is concluded thatchronic administration of copper sulphate to rats induced
nephrotoxicity which manifested as renal tissue damage,decreased creatinine clearance, and elevated levels ofplasma and urine creatinine resulting from impaired renal
function and elevation of the activities of aminotransferases
in the blood.
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19

CONTRIBUTION OF SELF-PERCEIVED STRESS TOPOSTSURGICAL LIFE QUALITY CHANGE OF PATIENTSUNDERGOING CATHARACT SURGERYCIOBOTEA DANA1, ȘERBAN COSTELA2, PUTNOKY SALOMEIA1,
FIRA MLADINESCU CORNELUȚA1, TUȚĂ SAS IOANA1,
BĂCEAN MILOICOV CODRUȚA1, VLAICU BRIGITHA1
1Departament 14 - Microbiology, Discipline of Hygiene, ”Victor Babeș” University of Medicine and Pharmacy Timișoara
2Departament 3 - Functional science, Discipline of Informatics and Biostatistics, ”Victor Babeș” University of Medicine andPharmacy Timișoara

ABSTRACTThe research aimed to investigate pre- and postsurgical self-perceived stress and life quality of patients programmed toreceive crystalline implants in relation to demographics and clinical status, but also to assess the contribution ofmodified stress to the improvement of life quality in between assessments. The study included 53 patients withcatharact in whom the procedure of phacoemulsification was used for crystalline replacement. Presurgically,
demographic and clinical variables do not influence the size of the two measures, which have 20% common variance.
Postsurgically, the stres level decreased and life quality improved. The reduction of stress level and the increase of
sleep duration explain 38% of life quality variance.

INTRODUCTION

MATERIAL AND METHOD

Anxiety and stress in patients before surgicalinterventions represent a global problem which meay leadto profound effects. Among these effects the risk ofincreased postsurgical pain, the risk of infection, and thedecreased healing rate may be mentioned. There are
multiple factors affecting the level of presurgical anxiety,including the need for surgery, the perceived loss of control,
the fear of postsurgical pain and the alteration of body
image [1].
Numerous studies demonstrated that the improvementin visual acuity induced by catharact surgery leads toconsiderable gain in real life activities, in emotional andsocial life components [2].
In the present study we intended to investigate theself-perceived stress and life quality in patients scheduled
to receive crystalline implants, the size of changes in thetwo measurements as a consequence of catharact surgery
in relation with the demographic and clinical situations andthe contribution of changed stress levels to a changed life
quality in between assessments.

Material
The study included 53 catharact patients who adressed
the Clinic of Ophthalmology in Timisoara between January
and April 2015, with the following demographic
characteristics: 49.1% of patients belong to the masculine
gender, with an age median value of 70 years and age
interval between 50 and 84 years.
Presurgically, for the eye with the best results there
were 69.8% patients without visual deficiency or with minor
visual deficiencies, 20.8% with moderate visual deficiency
and 9.4% with severe deficiencies or cecity. For the eyewith the worst results, we found 20.8% patients withoutvisual deficiencies or with minor deficiencies, 24.5% withmoderate visual deficiencies and 54.7% with severe
deficiencies or with cecity. One month after the surgicalintervention, visual acuity was classified as no visual deficit
in all patients, with 83% being recorded with maximum
values.

Received February 5th 2016. Accepted March 20th 2016. Address for correspondence: Costela Serban, Department of
Functional Sciences, “Victor Babes” University of Medicine and Pharmacy Timisoara, 2A Eftimie Murgu Square, Timisoara,
RO-300041; phone: +0256490507, e-mail: costela.serban@gmail.com

20

Fiziologia - Physiology • 2016.26.1(89)MethodPre- and postsurgically, patients were clinicallyassessed and responded the Visual Functioning
Questionnaire (VFQ) and a question regarding theself-assessed stress level. Testing for corrected anduncorrected visual acuity (VA) was performed using theSnellen chart and an autorefractometer.
The VFQ questionnaire was developed and validated
by RAND, starting from the 51 questions variant developedby the National Eye Institute [3]. VFQ-25 is a public
document, freely available for all researchers provided they
identify the measure as such in all publications [4,5]. The
questionnaire measures life quality in patients with eye
pathologies and intermediate results are coupled into 12
sub-scales. Each sub-scale was calculated according to
the methods described by the developers of NEI-VFQ andmay vary from 0 to 100, where 0 is the worst score and 100
indicates there is no sight-related disability. The generalscore of life quality is calculated as an average between the
12 sub-scales. For interpretations in this paper we worked
with the general score for life quality. The association
between questionnaire scores and objective measurements
of VA to establish visual deficits proved to be significantly
related in the group of elderly adults as well [6].
For the self-assessment of stress perceived during thelast month we used a 10 points Likert scale, where 0
represented a reduced level and 10 represented theextreme level.
The surgical intervention consisted of crystalline implant
replacement by a procedure called phacoemulsification.
Data processing and interpretation used modern
methods of advanced medical statistics. Data were
electronically filed using the Microsoft Excel, version 2007
and processed with PASW 18 (SPSS 18) 2010. The
threshold for statistical significance was set at p<0.05. For
comparing parametrical data we used the t and ANOVA
tests. For pre- / postsurgical comparisons we used thelinear general model for repeated measurements, and forpredicting changes in life quality multiple liniar regression
was used.

RESULTSStressPresurgically, patients self-assessed their stress during
the recent period on a scale with 1 representing a
stressless situation and 10 representing maximum stress.
For the initial situation, the stress level has an averagevalue of 6.00, with SD of 2.38 points, with values between
a minimum 1 and a maximumn 10. Gender (p=0.493) andage (p=0.559) of the patients did not influence theperceived level of stress. We intended to find if thepresurgical stress level differs depending on the visual
deficit class of the worst eye, but differences are notsignificant, p=0.808.
Thre increased stress level correlates to the low
number of sleeping hours per night, both during the week, τ=-0.331, p<0.001, as well as during weekends, τ=-0.303,
p<0.001, the size of the association being small in bothsituations.
Postsurgically, patients self-assessed their stress levelat an average of 3.09 points, SD 1.65 points, with values
situated at a minimum of 1 and a maximum of 8. A mixed
between-within subject analysis of variance was conducted
to assess the impact of initial categories of VA deficit on
participants perceived level of stress during the last month,evaluated pre- and postsurgically. We found no significant
interaction between the evolution in time of theself-assessed stress level depending on VA deficit
categories, Wilks Lambda=0.999, F(2.50)=0.14, p=0.986,
but we found that the pre- postsurgical evolution of theself-perceived stress is statistically significant, Wilks
Lambda=0.432, F(1.50)=65.8, p<0.001, partial eta squared=0.568, representing a large size effect. (Table I, Figure 1).

Table I. Mean values and standard deviation of the stress level and life quality score
Classification of initial visual deficiency - Worst eye
No visual deficiency

Visual deficiency

Preop

1 monthpostopPreop

Stress level

5.6 +/- 2.62

2.8 +/- 2.09

5.9 +/- 2.25

Life quality

41.1 +/- 20.58

73.6 +/-13.68

28.3 +/- 20.20

The size of the difference between the presurgical andthe one month-postsurgical situations regarding the

Fiziologia - Physiology • 2016.26.1(89)1 month postop

CecityPreop

1 monthpostop

3.1 +/- 1.48

6.2 +/-2.45

3.2 +/-1.61

70.8 +/-13.49

33.8 +/-21.09

71.5 +/-14.95

perceived stress level is not statistically significant between
VA deificit classes, p=0.986 (Table II).

21

Table II. Mean values and standard deviation of changes in stress levels and life quality scores between measurements
Classification of initial visual deficiency - Worst eye
No visualdeficiency(N=25)(N=11)

Visualdeficiency(N=17)

CecitySig

Total

Stress (postop-preop) (Mean+/- SD)-2.8 +/-2.82-2.9 +/-2.00-3.0 +/-2.56

0.986-2.9 +/-2.40

Life quality (postop-preop)(Mean +/- SD)

32.9 +/- 13.90

43.4 +/-12.33

39.1 +/-16.40

0.258

39.2 +/-14.91(N=53)

p=0.258, but we did find that the pre- / postsurgicalevolution of the life quality score is statsitically significant,Wilks Lambda=0.136, F(1.42)=267.2, p<0.001, partial eta
squared =0.864, representing a large size effect. (Table I,
Figure 2).

Fig. 1. Distribution of the evolution of average stress levels
between the initial and postsurgical situations depending
on VA deficit classes in the worst eyeLife quality
Presurgically patients slef-assess their life quality at amean value 33.5 +/- 20.75 points. The general VFQ score
correlates neither to demographic variables of gender(p=0.06), and age (p=0.362) nor to the VA deficitclassification in the worst eye (p=0.765), but we found asignificant association to the reported number of sleeping
hours per night, τ=0.383, p=0.003, r=0.15, which issignificantly lower in patients who admit night awakenings,
t(45)=2.16, p=0.036, r=0.31, with a medium sized
difference (Table I, Figure 2).
Presurgically, life quality and reported stress record
20% common variance, τ=-0.444, p<0.001, r=0.20.
Postsurgically, the global life quality score VFQ
records a mean value of 71.7 +/- 14.0 points, with scores
between a minumum of 39.6 and a maximum of 96.3. A
mixed between-within subject analysis of variance wasconducted to assess the impact of initial categories of VA
deficit on life quality scores, evaluated pre- andpostsurgically. We found no significant interaction between
the time evolution of the life quality score depending on VA
deficit categories, Wilks Lambda=0.937, F(2.42)=1.4,

22

Fig. 2. Distribution of mean scores of life quality between
initial and postsurgical situations, depending
on VA deficit in the worst eye

The size of difference between the pre- and one month
postsurgical situations regarding life quality scores does
not differ statistically between VA deficit classes, p=0.258(Table II).
We intended to test the contribution of stress levelchanges as well as of changed sleeping hours to life
quality changes measured as difference between the final
situation, one month after surgery and the initial,
presurgical situation. Multiple regression was used toestablish the ability of changed stress levels and modified
number of sleeping hours in predicting changes in life
quality scores. Preliminary analyses were conducted toensure that normality, linearity, multiple colinearity andhomoscedanscity premises were not failed. The total
variance explained by the model was 38.2%, F(2.44)=12.9,
p<0.001. Both variables had a significant contribution to
the model, stress recording the highest beta value

Fiziologia - Physiology • 2016.26.1(89)(beta=0.418, p=0.003), and for additional sleeping hours
beta=0.326, p=0.016. The changed stress level has thehighest unique contribution of 15% to the model, theunique contribution of additional sleeping hours being
9.1%, the remaining 14.1% variance being common to thetwo measures (Table III).

Table III. Coefficients for predicting changed life quality scores in between measurements
Model

Unstandardized
Coefficients
B

Std. Error(Constant)

20.672

4.212

dif_hours_week

5.004

2.002

dif_stress

2.855

.893

Standardized
Coefficients

t

Sig.

Beta

95.0% Confidence Interval
for B
Lower
Bound

Upper
Bound

4.908

.000

12.17129.173

.326

2.500

.016

.964

9.043

.418

3.197

.003

1.053

4.658

a. Dependent Variable: dif_VFQ

DISCUSSIONS
Demographic and clinical variables did not influence
the perceived stress level or life quality, as assessed forthe month prior to catharact surgery. Other authors [7]found in patients with eye pathology inducing decreasedsight that the state of mind and anxiety were associatedwith objective measures of the visual function. Kroll [8]found that specific situation coping was a mediator
between the patient's personality type and adaptation
criteria to the stress level as measured before catharact
surgery. Pruessner et al., using functional magnetic
resonance (fMRI) showed that stress exposure and theactivation of the hypothalamus-hypophysis-adrenal axis
are associated with reduced activities in some major
portions of the limbic system allowing them to speculate on
the effects of stress upon cognitive and emotional brain
regulation.
We found that increased stress levels correlate to thedecreased number of sleeping hours per night and to
reduced life quality scores. Within the controledenvironment of the somnology laboratory, in a case-control
study, Mikel et al [9] showed that sleep deprivation
decreases the psychological threshold of stress perception
manipulated by cognitive tests, time pressure andperformance feedback.
In our study we showed that the postsurgical reductionof the stress level and the increase in life quality levels are
significant, the modification size being similar in all 3 VA
classes.
A recent study [10] demonstrated that among catharact
patients assessed by IDATE-T (Anxiety Scale) and
WHOQOL (WHO Quality of Life Questionnaire) there is ahigh psychological burden. The authors showed thatpatients' life quality is inversely related to the anxiety level.
This finding confirms previous studies which demonstrated
reduced life quality scores in catharact patients [11] and a
Fiziologia - Physiology • 2016.26.1(89)negative impact of anxiety disorders on the life quality ofpatients who underwent catharact surgery [12].
The increase in life quality may be explained in over
38% of cases by the decrease in stress levels and increasein the number of hours of sleep. These results support
our recommendation to patients who postpone catharact
surgery or who present counterindications for catharact
surgery to undergo stress reduction measures by specificpsychotherapy and blue light therapy for decreasing
anxiety and depression and improve sleep duration [13,14].

CONCLUSIONS
‐
‐

‐
‐

Presurgically, the stress level and life quality do notdepend on demographic variables or on the objective
level of visual deficit
Postsurgically, one month after phacoemulsification,
the stress level and life quality improved in a liniary
relation, correlated neither to the initial clinical status
nor to the clinical level of improvement in theoperated eye
The size of life quality change may be 38.2% justified
by changes in stress levels and sleep duration, as
recorded before and after catharact surgery
We think that presurgical adjuvant methods such as
blue light exposure to reduce anxiety and to improve
sleep, but also psychological therapy are beneficial in
coping with stress and anxiety, depending on thetype of patient personality.

REFERENCES
1. Bailey L. Continuing Education Strategies for Decreasing
Patient Anxiety in the Perioperative Setting. AORN Journal.
2010;92(4):445–460

23

2. Lamoureux E, Fenwick E, Pesudovs K, Tan D. The impact of
cataract surgery on quality of life. Curr Opin Ophthalmol.
2011; 22 (1): 19-27.
3. http://www.rand.org/health/surveys_tools/vfq.html
4. Mangione CM, Lee PP, Gutierrez PR, et al. Development of
the 25-item National Eye Institute Visual Function
Questionnaire (VFQ-25). Arch Ophthalmol. 2001;119:1050-1058.
5. World Health Organization International Statistical
Classification of Diseases and Related Health Problems 10th
revision Current version Version for 2003 Chapter VII H54
Blindnessandlowvision.
http://www.who.int/classifications/icd/en/
6. Owen CG, Rudnicka AR, Smeeth L, Evans JR, Wormald RP,
Fletcher AE. Is the NEI-VFQ-25 a useful tool in identifying
visual impairment in an elderly population? BMC Ophthalmol.
2006, 9;6:24.
7. Eramudugolla R, Wood J, Anstey KJ. Co-morbidity ofdepression and anxiety in common age-related eye diseases:
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2013; 5: 56.
8. Knoll N, Rieckmann N, Schwarzer R. Coping as a mediator
between personality and stress outcomes: a longitudinal
study with cataract surgery patients. Eur J Pers. 2005, 19:229-247.

9. Minkel JD, Banks S, Htaik O, Moreta MC, Jones CW,
McGlinchey EL, Simpson NS, Dinges DF. Sleep deprivationand stressors: evidence for elevated negative affect in
response to mild stressors when sleep deprived. Emotion,
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10. Fazzi L, Dobrianskyj FM, Abreu Reggi JR, et al. The
influence of anxiety on quality of life among patients with anindication for cataract surgery. Sao Paulo Med. J. 2015;
133(2)
11. Skiadaresi E, McAlinden C, Pesudovs K, et al. Subjective
Quality of Vision Before and After Cataract Surgery. Arch
Ophthalmol. 2012;130(11):1377-1382.
12. Yokoi T, Moriyama M, Hayashi K, et al. Predictive factors forcomorbid psychiatric disorders and their impact on
vision-related quality of life in patients with high myopia. Int
Ophthalmol. 2014; 34(2): 171-83.
13. Lieverse R, Nielen MM, Veltman DJ, et al. Bright light inelderly subjects with nonseasonal major depressive disorder:
a double blind randomised clinical trial using early morning
bright blue light comparing dim red light treatment. Trials
2008, 9: 48.
14. Lieverse R, Van Someren EJ, Nielen MM, et al. Bright lighttreatment in elderly patients with nonseasonal major
depressive disorder: a randomized placebo-controlled trial.
Arch Gen Psychiatry 2011, 68: 61-70.

CONTRIBUȚIA STRESULUI AUTOPERCEPUT LA
MODIFICAREA POSTOPERATORIE A CALITĂȚII VIEȚII
LA PACIENȚI SUPUȘI OPERAȚIEI DE CATARACTĂREZUMAT
Cercetarea și-a propus investigarea pre și postoperatorie a stresului autoperceput și a calității vieții la pacienți programați pentru
implant de cataractă în relație cu statusul demografic și clinic, dar și contribuția modificării stresului la ameliorarea calității vieții între
evaluări. Au fost incluși 53 pacienți cu diagnostic de cataractă, la care s-a folosit procedeul de facoemulsificare pentru înlocuirea
cristalinului. Preoperator, variabilele demografice și clinice nu influențează mărimea celor două măsuri, ele având 20% varianță
comună. Postoperator nivelul de stres s-a redus, iar calitatea vieții s-a îmbunătățit. Reducerea nivelului de stres și creșterea duratei
somnului explică 38% din varianța calității vieții.

24

Fiziologia - Physiology • 2016.26.1(89)BIOCOMPOSITE PROCESSING TECHNOLOGYAND MEDICAL APPLICATIONSROXANA VOISAN1, OANA GINGU2
1UPU-SMURD SCJU Timisoara
2Faculty of Mechanics, University of Craiova

ABSTRACT
Porous biocomposite materials - hydroxyapatite reinforced by titanium -made by powder metallurgy technology, arequalitatively assessed from the point of view of their behavior along the wear tests against metallic and ceramic
counterparts. This assessment is monitored through some representative parameters of wear tests: friction coefficient,
wear rate and wear track area. The experimental results point out the distinction of the wear behavior between the
biocomposite materials which are elaborated by two different processes: spark plasma sintering and two step sintering.
The wear results, related to the structural properties of the biocomposite materials, could recommend each of theprocessing technologies for specific bone grafts manufacturing, namely for the cortical or trabecular hard tissue.
Because of some disadvantages reported up to now (mainly due to the difference between the mechanicalcharacteristics of the human bone and the metallic implant), the biocomposite become an interesting solution to
improve the hard tissue implants for the human body.
Keywords: biomaterials, hydroxyapatite, titanium, bone

INTRODUCTION
One of the most important targets of the science is todevelop new materials for bone implants. Titanium andTi-based alloys have been usually used as artificial bonematerials because of their great mechanical properties andthey show non-toxic, i.e., when these materials are utilized
for bone implants they are considered bioinert [1].
Titanium alloys present the greatest biocompatibility
among metallic materials for biomedical applications
because of its high chemical stability in the human body,relative low module of elasticity, low density, high strengthand corrosion resistance. However, even if they are
considered into bioinert materials and its biocompatibility is
inferior to that of hydroxyapatite (HAP: Ca(PO4)3OH),
which is grouped into bioactive materials [2]. Due to thereported geometrical instability, its reinforcing by someform of stable osteosynthesis, preferably with an angularly
stable plate is recommended [3]. Much better biomaterials
for hard tissue replacement implants may be acquired by
the preparation of titanium composites. The mostcommonly used materials are ceramics such ashydroxyapatites, bioglasses or silicas.
Therefore, the connection of excellent biocompatibility
and bioactivity of ceramics with good mechanicalproperties of titanium is considered to be a promising
approach for fabrication of more perfect hard tissue
replacement implants. There are two creative ways to meetthis idea: use of ceramics as a coating or as a reinforcing
phase in metallic-ceramic composites [4].
Direct HAP deposition methods on titanium substrate
are, for example: ion plating [5], plasma spray method [6],pulse laser deposition method [7,8], RF magnetron
sputtering [9], super plastic joining method [10], ion beam
dynamic mixing method [11], processing of nanostructured
biomaterials [12, 13], obtaining of an intermediate layer atcoating/implant interface [14, 15].
The most approached processing technologies for the
HAP/Ti advanced biomaterials are: powder metallurgy [16],pulsed laser deposition [17,18], microarc oxidation [19],microwave sintering [20], radio-frequency thermal plasma
spraying (RF-TPS) technology [21], electrochemicaltreatment [22, 23].
In order to obtain nanostructured sintered biocomposites,
two steps sintering (TSS) and spark plasma sintering (SPS)
are developed at different technological parameters in thisresearch.
One of the challenges of tissue engineering field is tocontrol the friction respectively wear phenomena occurring
during the implants / grafts long life. The medical studies

Received February 12th 2016. Accepted March 6th 2016. Address for correspondence: Roxana Voisan, UPU-SMURD, “Pius
Brinzeu” Emergency Clinical County Hospital Timisoara, Liviu Rebreanu 156 Street, Timisoara, RO-300723; phone:+40356433111, e-mail: ciolacu.roxana@yahoo.com

Fiziologia - Physiology • 2016.26.1(89)25and research report clinical cases regarding wear
processes going on the adjacent surfaces of the bone andimplant in direct contact.
The wear phenomenon is not an intrinsic material
property, but is a system property. Its evolution andmonitoring instruments depend on the entire tribologicalsystem comprising the following elements: the contact
surfaces, the lubricant presence (or not), the relative
speed/movement of the counterparts in contact, thesurface roughness and temperature.
The wear mechanisms developing in the human body,
between the implants / grafts and bone tissue are as follow:- the adhesive wear is a result of micro-junctions
caused by welding between the opposing asperities
on the rubbing surfaces of the counterparts;- the fatigue wear is caused by the normal cycling
loading during friction;- the fretting wear is the derivative of the fatigue wear,
when the tangential loading acts between the twocounterparts, Figure 1. This is the most reported
wear mechanism at the interface between the bone
and implants / grafts [3].
- the abrasive wear is acting in all above mentioned
cases when wear particles (debris) are released by
one of the contact surfaces.

Fig.1 Schematic representation of the fretting wears mechanism

CRITERIA FOR BIOMATERIALS APPLICATIONS
The main ideea, is that a biomaterial represents any
kind of material, natural or synthetic that is used toestablish a medical device, in order to treat, augment, or
replace any tissue, organ, and/or any body function. The
purpose is that this biomaterial remains there for asignificant period of time.
When we decide to use an implantable biomaterial,
there are some criteria anyone needs to know about:‐ Toxicity (citotoxicity, genotoxicity, etc.) [29,31]‐ Natural tissue growth‐ Rough surface‐ Porous structure and stress shielding

26

One of those criteria, and we are talking here about
toxicity, needs a lot more of our attention. For example,
when we say cytotoxicity, it refers to chemicals, materials
our even mediator cells, able to destroy living cells. The
most important consequences will be undergo necrosis(accidental cell death) and apoptosis (programmed cell
death).
We also have to measure cytotoxicity, and thatinvolves the cell membrane integrity, evaluated by using
trypan blue, or propidium iodide, also protease biomarkers(MTT or MTS redox potential assay).
Genotoxicity represents the property of a chemical
agent, able to damage the genetic information within a cell,causing mutations.
Genotoxicity is confused with mutagenicity, because all
mutagens are genotoxic but not all genotoxic substances
are mutagenic.
A substance or a material that have the property ofgenotoxicity, are known as genotoxins. Some genotoxins
can lead to cancer. They can cause mutations in cells andthe cells can divide or grow uncontrollably.
Some mutations can be teratogenic, meaning they cancause birth defects, affecting the genetic information byforming strong chemical bounds between genotoxins andDNA or RNA.
Immediately after implantation, when the biologicaltissue or the biological components meet the biomaterial,
the protein adsorption occurs which lead to various
consequences.
Coagulation system activation, platelet adhesion andactivation determinate thrombosis. Bacterial adhesion leadto infection. Leukocyte adhesion and activation, plus
complement system activation generate inflammation.
Morphologically speaking the device must be dense,
nonporous and nearly inert. It will not form a bond with thetissue and the response from the tissue is dependent on fit,rather than chemistry. The porous structure, in order togrow is a very important aspect. The pores must be larger
than 100μm diameter so that capillaries can provide blood
supply to ingrown connective tissue porous inert implants(example- Hydroxyapatite coated porous implants) [30].
The effects of the implant on the host, due to theconsequences we talked before, are very important,because we talk about toxicity, modification of normal
healing (encapsulation, foreign body reaction), infection,
tumorigenesis, embolization, hypersensitivity, elevation of
implant elements in the blood, etc. There are also physicaland mechanical effects such as, abrasive wear, fatigue,
stress corrosion and also biological effects, enzymatic
degradation and calcification.
Also, a very important issue we have to take inconsideration, regards the status of the host. Age andhealth, immunological and metabolic status, tissue
damage, contamination, are demanding for a proper
implantation. A healthy patient will respond better and will
have good prognosis. Instead, a patient with renal
Fiziologia - Physiology • 2016.26.1(89)diseases, cardiovascular diseases, metabolic bonediseases, radiation bone therapy, patient on steroid
medication, or pregnant women, are the most often cases
for ineffective implantation [30].
Also, there are some major contraindications, such as:
patients with uncontrolled diabetes, degenerative diseases,
hypercalcemia, osteomyelitis and malignant tumors.

MATERIALS AND EXPERIMENTAL PROCEDURE
The studied biocomposite materials are based on
nanostructured HAP reinforced by micrometric TiH2
particles.

Fig. 2. SEM image of composite mixture

For the research HAP is used as powder particles(<200 nm, type Aldrich), as matrix. The preparation of HAP
powders before sintering is presented in other research[24]. The reinforcement is micrometric TiH2 powder
particles (<100 µm, water atomised).
The weight ratio between the components is mentionedin [25] as well as the preparation method for the mixture
before compaction and sintering. In Figure 2 the SEM
image of composite mixture is presented.
Spark Plasma Sintering (SPS) represents anadvanced sintering route dedicated to ceramic parts aswell as metallic and composite products. Plasma heating
system provides much lower sintering temperature (TSPS)
and time (about minutes) than the classic sintering route,Figure 3. Because of these great advantages, SPS allows
nanostructures elaboration, too.
Due to the advantages offered by lower sintering
temperatures and dwell times as compared to the classic
sintering parameters (Figure 3), SPS is also recommended
for nanostructured materials processing [26] when
nanopowders / nanostructured powders are used as raw
materials.

Fiziologia - Physiology • 2016.26.1(89)Fig. 3. Schematic representation of the thermal cycles
for the SPS vs. the classic sintering (CS)The SPS treatment was made on National Institute for
Research and Development for Technical Physics, Iasi,
Romania, uses (SPS)-FCT-(FAST) HPD5 equipment,
working under the following terms: max. current intensity
Imax. = 20 kA; max. sintering temperature 2400oC (working
temperature 2200oC); sintering atmosphere: vacuum; max.
load: 50kN, for fundamental and applicative research inadvanced materials elaboration.
For this research the SPS treatment has beendeveloped in vacuum at 10000C, respectively 11000C, thedwell times for each temperature being 10 min., 15 min.
and 20 min. The heating rate was for 100C/min. and thepunches load was 7kN.
In the Table I the parameters of the SPS treatment arepresented.
Table I.Technological parameters for HAP/Tibiocomposites processing by SPS
Temperature, 0C

Dwell time, min

1000

10

15

20

1100

10

15

20

Another sintering technique used in this study is TSS.
After cold compaction stage at 120 MPA, the TSS
technique consists in a first heating of the sample to alower temperature (T1) than the classic sintering
temperature (TCS), followed by the cooling down to a lower
temperature (T2<T1) for suppressing the grain boundary
migration. The sample is held at this temperature T2, until
full densification (Figure 4). Based on our previous
thermogravimetric analysis [27], the technologicalparameters regarding both the time and sintering
temperature were established (Table II).
The biocomposite cylindrical billets of 12 mm diameter
have been processed by unilateral cold compaction in a
metallic
die,
at
120
MPa,
onA009electromechanical-computerized 100kN testing machine,
equipped with TCSoft2004Plus software.

27

After cold compaction stage at 120 MPa, TSS
technique, fig.3, consists in a first heating of the sample toa lower temperature (T1) than the classic sintering
temperature, followed by the quick cooling down to a lower
temperature (T2<T1) for suppressing the grain boundary
migration. The sample is held for hours at this temperature
T2, until full densification.
For TSS route a laboratory Nabertherm chamber
furnace was used, type L5/12, max. temperature 12000C,
with protective gas connection on the rear wall of thefurnace and temperature controller on the front panel of thefurnace. Argon atmosphere (99.98% purity) was delivered
along the entire sintering treatment.

temperature than CS route respectively T1 just for structuraldensification, in such conditions in order to avoid thenanoparticles growth beyond the nanometric scale. Thus,
TSS is recommended for sintered nanostructure processing.
Cylindrical specimens with 12 mm diameter using cold
compaction pressure of 90-150 MPa were obtained.
In this research the effects of SPS technique on the
microstructural changes for HAP/Ti biocomposites has
been studied. Also, the effects of the TSS route, in fact,second sintering temperature (T2) and dwell time on theporosity and microstructure of biocomposites HAP/Ti were
the objects of this particular study.
The friction tests were described by Gingu et al. [28].
The friction couple is ball-on-disc and this choice issupported by the analogy with the friction systems that
could occur inside the human body between bones andcorresponding implants/grafts as well as to the fretting
wear mechanism described above. Figure 5 schematically
represents the ball-on-disc friction couple.

Fig. 4. Thermal cycle for TSS:
Tcs = temperature for the classic sintering;T1: first step temperature of TSS;
T2: second step temperature of TSS

The sintering was developed by TSS route, the firststep at 9000C, for 1-5 min, followed by the second step atthree different temperatures from 7000C to 8000C and for
different sintering times: 300/600/900/1200 min [28].
Table II. Technological parameters for HAP/Ti biocomposites
processing by TSS
1st step parameters

2nd step parameters

T1 [0C]T2 [0C]

Dwell time τ1 [min.]Dwell time τ2 [min.]

300
900

1-5

700 – 800

600
900
1200

The advantage of TSS vs. CS route is that the first step
(T1-temperature and τ1-dwell time) develops shortly, above
or below the temperature of CS, just to initiate the diffusion
process between the nanoparticles or micronic powders andnanometric ones (in case of such mixture). The second step(T2-temperature and τ2-dwell time) develops at a lower

28

Fig. 5. Schematic representation of the ball-on-disc
friction couple used for the wear behavior assessmentof the biocomposite materials

CONCLUSIONSThe Hap/Ti biocomposite materials are recommended
to be used for bone grafts elaboration by two technologies:
SPS and TSS. Each of these technologies provides
advantages especially from the point of view of the wear
behavior of the biocomposites.
Thus, the SPS technology could be applied in the caseof cortical bone grafts because of high porosity obtained forthe respective biocomposites (5…12%). The wear behaviorof these materials is assessed as high quality response,
comparable with other technologies, but more complex.
As far as the TSS process concerns, it depends on theprocessing parameters in order to assure high wear
behavior of the processed materials that could be used forthe trabecular bone grafts due to their high porosity(20…60)%.
Also, a very important aspect is, a very accurate casehistory, done by the physician and all the criteria must bevery well chosen. Surgeons must be convinced with the longterm durability and reliability of biocomposite materials.

Fiziologia - Physiology • 2016.26.1(89)Cellular, molecular, and genetic level research is
producing exciting new information that has improved
understanding of the host/biomaterials interactions.
That is why the development of new generations ofbiomaterials and implant devices and also the improved
strategies for their evaluation and use should be thenumber one priority.
ACKNOWLEDGMENTS
We
herebyacknowledge
theresearch
project
PN-II-PT-PCCA-2013-4-2094, title: ''Research on the bone
substitution with biocomposite materials processed by powder
metallurgy specific techniques'', acronym BONY, for the financial
support.
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30. Wang X. Overview on Biocompatibilities of Implantable
Biomaterials. In Advances in Biomaterials Science andBiomedical Applications, R. Pignatello, Ed. InTech, 2013.
31. Leggat PA, Smith DR, Kedjarune U. Surgical Applications ofCyanoacrylate Adhesives: A Review of Toxicity. ANZ Journal ofSurgery, 2007; 77(4): 209-213.

29

TEHNOLOGIA PROCESARII MATERIALELOR
BIOCOMPOZITE SI APLICABILITATEAIN DOMENIUL MEDICALREZUMAT
Materiale biocompozite poroase, pe baza de hidroxiapatita ranforsata cu particule de titan, au fost obtinute printehnologia metalurgiei pulberilor. In cadrul acestei lucrari biocompositele elaborate sunt evaluate din punct de vedere
calitativ in ceea ce priveste comportamentul la uzare, avand ca si piesa conjugata produse metalice si ceramice.
Parametrii de evaluare calitativa sunt: coeficientul de frecare, rata uzarii si suprafata urmei uzata. Rezultatele
experimentale evidentiaza diferenta privind comportamentul la uzare al biocompozitelor obtinute prin doua metodediferite: sinterizarea cu descarcare in plasma (SPS) respectiv sinterizarea in doi pasi (TSS). Valorile experimentale
obtinute, coroborate cu proprietatile structurale ale biocompozitelor studiate, pot recomanda fiecare dintre tehnologiile
mentionate pentru fabricarea grafoanelor osoase respectiv SPS pentru tesut osos cortical, iar TSS pentru tesut
trabecular.
Din cauza unor dezavantaje raportate pana in prezent, (mai ales provocate de diferentele din structura osoasa umana
si implantul metalic), biocompositele au inceput sa reprezinte o solutie interesanta in ceea ce priveste cresterea
capacitatii de integrare a implantului in organismul uman.
Cuvinte cheie: biomateriale, biocompozite tip hidroxiapatita /titan, structura osoasa.

30

Fiziologia - Physiology • 2016.26.1(89)ASSESSMENT OF OBSESSIVE-COMPULSIVE BEHAVIORIN PATIENTS WITH HAEMOPHILIALAVINIA HOGEA*1, LAURA NUSSBAUM*2, RALUCA DUMACHE3
1Psychology Discipline, Neurosciences Department, “Victor Babes” University of Medicine and Pharmacy Timișoara, Romania
2Pedopsychiatry Discipline, Neurosciences Department, “Victor Babes” University of Medicine and Pharmacy Timisoara, Romania
3Department of Forensic Medicine, Bioethics, Deontology and Medical Law, Neurosciences Department, “Victor Babes”

University of Medicine and Pharmacy Timisoara, Romania

ABSTRACTIntroduction: Hemophilia, like other rare disease, is a chronic disease, debilitating or crippling threatening life and thequality of life it requires a special effort and support from the community.
Permanent disability to which a hemophiliac body is prone to leads to an alteration of the psyche, with repercussions on
the whole personality of the individual. Intense and permanent headaches with witch the patient is facing are causing an
increase in psychic tension and internalization of one's personality, even more so, the hemorrhagic accidents are more
frequent and more intense.
Material and methods: in the study there were 200 subjects grouped into two equal groups (patients with hemophilia A
and B and healthy population) aged between 16 and 45 years.
Results: statistically significant differences exist between the hemophiliacs and non-hemophiliacs, hemophiliacs have ahigher level of obsessive compulsive behavior, compared to non-hemophiliacs.
Conclusions: Patients with chronic conditions, pose a twice as high risk for the occurrence of psychiatric complications,compared to their healthy peers.
Key words: hemophilia, obsessive-compulsive behavior

INTRODUCTION
Hemophiliac patients must go through periods ofdisability, must suffer lengthy treatments and in themeantime they must deal with various experiences andfeelings, such as fear and uncertainty [1]. The process ofadapting to the disease is a difficult one and can lead overtime to the appearance of psychological dysfunctional
symptoms, which can be associated with psychiatricmorbidity, both to the patient and members of his family [1].
Hemophilia patient's mental disorders are much more
numerous than those presented to the doctor, because
both the patients and their parents often conceal their
existence. Fear, embarrassment, anxiety or mistrust may
be some of the reasons why the patient does notcommunicate the existence of psychological problems.
The type of mental disorders encountered more often
at hemophiliac patients are: mentally retarded, anxiety,nocturnal psychogenic enuresis, retardation in language
acquisitions, anxious-phobious neurosis, affective mooddisorder, Tourette Syndrome, ADHD, conduct disorder,
stuttering, sociopathic behavior disorder, separation anxiety,night pavor, opioid drug abuse, alcohol abuse, depressive
reaction, self-harming, suicidal tendency, focus and attention
disorders, temporary depression moods, headaches,
insomnia.

PURPOSE
There are statistically significant differences regarding
the level of obsessive-compulsive behaviors in hemophiliac
patients compared to the control group.

MATERIAL AND METHODS
The study was conducted on a sample of 200 subjects
grouped into two groups of equal size. The clinical batch
consists of 100 people with hemophilia A and B (50%), withages between 16 and 45 years, while the control group ismade up of 100 people without hemophilia (50%) (Figure 1).
Inclusion criteria in the study are: diagnosis ofhemophilia A or hemophilia B; patients aged between 16
and 45 years; intelligence quotient > 100.

Received February 24th 2016. Accepted March 4th 2016. Address for correspondence: Hogea Lavinia, Psychology
Discipline, Neurosciences Department, “Victor Babes” University of Medicine and Pharmacy Timisoara, Eftimie Murgu SquareNo. 2A, Timisoara, RO-300041, e-mail: laviniahogea@yahoo.com*The first two authors should be considered as first authors due to the equal contribution to this study

Fiziologia - Physiology • 2016.26.1(89)31To collect data, we used the SECOC scale (the scaleof assessment of the Obsessive Compulsive Behaviors-Yale Brown Scale) and anamnestic information.

RESULTS

Fig. 1. The distribution of subjects in the study group

In terms of age, 123 participants aged 16-25 years old(61.5%), 49 are aged between 26-35 years (24.5%), while
28 are between 36 to 45 years (14%) (Figure 2).

To verify this hypothesis we have used the results of
the anamnesis and the SECOC (scale for evaluation ofobsessive-compulsive Behaviors-Yale-Brown Scale) for
measurement of obsessive-compulsive behaviors.
The work hypothesis is one-sided, to test the
hypothesis an univariate ANOVA 2 was made (people
suffering from hemophilia, and people who do not sufferfrom hemophilia) X 3 (age-16-25years, 26-35 years 36-45
years old) to investigate the differences between groups
and the interaction effect. To validate this hypothesis we
invoke the following quantitative arguments:

Fig. 2. Patients distribution by age
Table I. Averages and standard deviations in size behaviors obsessive-compulsive

obsessive-compulsive
behaviors

Haemophilics

Non haemophilics

M

AS

M

AS

M

AS

M

AS

M

AS

9.67

6.93

6.22

6.64

8.34

7.00

6.97

6.62

7.89

7.66

Table I shows the means and standard deviations forobsessive-compulsive behaviors to hemophiliacs andnon-hemophiliacs, respectively, in age groups. At thedimension of the obsessive-compulsive behaviors, total
scores ranged from 0 to 28 (M = 7.94; As = 6.99).

Age
16-25

Age
36-45

Age
26-35

For testing the dispersion homogeneity we used theLevene test. The result is not statistically significant, whichindicates that the dispersions are homogeneous [2], F (5,194) = 1.041, p >. 05.

Table II. 3X2 factorial ANOVA summary table of obsessive compulsive behaviors
Source dispersion

SS

Df

MS

F

P

Group
Age
Group X age
Error
Total

336.244138.28126.3488952.936
22359.00

1
2
2
194
200

336.24469.14013.17446.1497.286*
1.498
.285

.008*
.226
.752

Note: ** p< .001; * p< .05

32

Fiziologia - Physiology • 2016.26.1(89)Fig. 3. X age group interaction and obsessive compulsive
behaviors

Of the three ratios F in Table 2, only one is statisticallysignificant, the main effect F "lot" [F (1, 194) = 7.286 p <.
01 (M = 9.67 versus M = 6.22)]. This means that there arestatisticallysignificant
differences
betweennon-hemophiliacs and hemophiliacs, hemophiliacs
have a higher level of obsessive-compulsive behaviors,
compared with non-hemophiliacs, due to the fact thatthe main effect for batches (hemophiliac versus
non-hemophiliac) is presented as statistically significant.
The effect of the variable age over theobsessive-compulsive behaviors variable is statisticallyinsignificant F(2, 194) = 1.498, p > .05. This means that no
significant differences were found between the three agegroups in hemophiliacs (M = 10.55 on group 16-25 years,
M = 9.11 on group 26-35 years and M = 8.32 on group36-45 years) compared to non-hemophiliacs (M = 6.78 on
group 16-25 year, M = 4.36 on group 26-35 years and M =6.33 on group 36-45 years).
Also, the interaction effect of group, age,obsessive-compulsive behaviors is insignificant statistically,
F (2, 194) = 0.285, p > .05, and neither the analysis of theinteraction graphic does not attest to the existence of aninteraction of the variables contained in the study (Figure 3).
Hemophilia is life-threatening due to the lack of
coagulation factors. Thus the hemophiliac patients sufferfrom various psychological problems. The risk of bleeding
causes stress and difficulty in coping with the situation.
As part of a study made by Fakhari in Shahid Ghazi
Tabatabai Hematology Oncology Center from Tabriz,hemophiliac patients were evaluated with regard to possible
psychiatric disorders. 48 hemophiliac patients were selected
randomly and were evaluated using the questionnaire
SCL-90, and the control group was made of 40 people.
The prevalence of the various psychopathologicalproblems among hemophiliac patients was significant. 67%of them were anxious, 60% were depressed, 60 percent
had the aggressive features, 72% had obsessive

Fiziologia - Physiology • 2016.26.1(89)compulsive traits and 71% had psychiatric manifestations,while 52% had features. The study showed that a highpercentage of patients suffering from mental disorders
hemophiliac and in need of psychological and social
support.
These results confirm the data of this study concerning
the evaluation of obsessive compulsive behavior.
Compulsive behavior obsessive is characterized bypersistent obsessions, intrusive and distressing (persistent
thoughts, impulses, or images) or compulsions (repetitive
behaviors, excessive), with impairments marking thequality of life [3, 4].
On the other hand, in a study from Italy, the results
showed that hemophiliacs have a good psychological
adaptation to their illness except lower self-esteem
compared to healthy subjects.
A study from Australia has investigated thepsychosocial issues faced by young people who are livingwith a chronic medical condition. A number of 35 young
people were interviewed, and it was found that many
young people with chronic illnesses are relatively resistantto the challenges of disease [5].
A document of the Department of psychology in
London reports the results of a cross national survey on
patients with severe hemophilia type A and B. Over one
third of the hemophiliac participants compared to thehealthy population have expressed concerns about the useof drugs, the most common being painkillers they also
have experienced a high rate of various types of mental
disorders.
Most of the time, the mental problems of patients withhemophilia are forgotten, so it is proposed to grant a higher
attention on this issue.

CONCLUSIONSAs a result of this study it has been observed that thehemophiliac patients present an obsessive-compulsive array.
The significant differences between the two groups of thestudy concerning the level of obsessive-compulsive behaviors:
F (1, 194) = 7.286, p < .01 (M = 9.67 versus M = 6.22).
The lack of coagulation factors puts the hemophiliac
patient's life in danger, with consequences on mental
health. The risk of bleeding causes stress and difficulty incoping with the situation. Exaggerated concerns forprotection against injuries can turn in time in obsessive
compulsive behaviors.
The increased frequency of hemarthroses in patientswith severe form of the disease, the emergence andevolution of the hemophilic chronic harthropathy, which
produces chronic disability and changes the body image,the frequent admissions for the administration ofsubstitutionary treatment, are factors that may contribute tothe occurrence of mental disorders to hemophiliacs.

33

REFERENCES
1. Cosma A. Hemofilicul îndrumător practic pentru bolnavi,familiile lor şi educatori. Ed. Medicală, Bucureşti, 1983.
2. Sava FA. Analiza datelor in cercetarea psihologica. Metodestatistice complementare, ASCR. Cluj-Napoca, 2004.
3. Eisen JL, Rasmussen SA. Obsessive-compulsive disorderwith psychotic features. J Clin Psychiatr, 1993; 54: 373-379.
4. Eisen JL, Phillips KA, Coles ME, Rasmussen SA. Insight inobsessive compulsive disorder and body dysmorphic disorder.
Compr Psychiatry, 2004; 45: 10-15.
5. Olsson CA, Bond L, Johnson MW, Forer DL, Boyce MF,
Sawyer SM. Adolescent chronic illness: a qualitative study ofpsychosocial adjustment. Ann Acad Med Singapore, 2003;
32(1): 43-50.
6. Cassis FRMY. Psychosocial Care for People with Hemophilia.
Treatement of hemophilia, Sao Paulo, Brazil, 2007; 44.

EVALUAREA COMPORTAMENTULUIOBSESIV-COMPULSIV LA PACIENȚII CU HEMOFILIEREZUMAT
Introducere: Hemofilia, asemenea celorlalte boli rare, este o boală cronică debilitantă sau handicapantă, ameninţătoarede viaţă şi de calitate a vieţii, necesită efort şi susţinere specială din partea comunităţii. Invaliditatea permanentă lacare este supus un organism tarat hemofilic antrenează o alterare a psihicului, cu repercusiuni asupra întregii
personalităţii individuale. Durerile intense şi permanente cu care se confruntă provoacă o creştere a tensiunii psihice şio interiorizare a personalităţii, cu atât mai mult, cu cât accidentele hemoragice sunt mai frecvente şi mai intense.
Material si metode: Din studiu au făcut parte 200 de subiecţi grupaţi în două loturi egale (pacienți cu hemofilie A și B și
populație sănătoasă), cu vârste cuprinse între 16 şi 45 de ani.
Rezultate: Există diferenţe semnificative statistic între hemofilici şi nonhemofilici, hemofilicii au un nivel mai mare alcomportamentelor obsesiv-compulsive, comparativ cu nonhemofilicii.
Concluzii: Pacienții cu afecţiuni cronice, prezintă un risc dublu în apariţia complicaţiilor psihice faţă de semenii lorsănătoşi.
Cuvinte cheie: hemofilie, comportament obsesiv-compulsiv

34

Fiziologia - Physiology • 2016.26.1(89)CHARACTERIZATION OF THE EFFECTS OF SARCKATP
AND MITOKATP MODULATORS ON REPERFUSION-INDUCEDARRHYTHMIAS IN ISOLATED RAT HEARTSALEXANDRA PETRUȘ1, ADRIAN STURZA1,2, OANA DUICU1,2,
NORBERT JOST1,2, DANINA MUNTEAN1,2, ISTVAN BACZKO1
1Department of Pathophysiology, 2Center for Translational Research and Systems Medicine, “Victor Babeș” University of

Medicine and Pharmacy, Timișoara, Romania

ABSTRACT
Malignant tachyarrhythmias, ventricular fibrillation (VF) and tachycardia (VT), associated with ischemia/reperfusion (I/R)
injury represent a major cause of sudden cardiac death worldwide. Pharmacological modulation of the cardiac
ATP-sensitive potassium channels, either sarcolemmal (sarcKATP) or mitochondrial (mitoKATP), has been reported bymany (but not all) studies to be cardioprotective against the deleterious effects of postischemic reperfusion. The aim of
the present study was to assess the effects of HMR-1098 (10 μΜ), a cardioselective sarcKATP inhibitor and of diazoxide(DZX, 50 μΜ), a mitoKATP opener, given either independently or together, on I/R-related arrhythmias. A brief episodeof regional ischemia was elicited by ligation of the left anterior descending coronary artery (LAD) in
Langendorff-perfused rat hearts (constant flow mode) and the compounds were given prior to the induction of ischemia.
In separate administration, HMR-1098 and DZX significantly reduced the duration of reperfusion-related VF (but not ofVT) as compared to the control group. Also, the incidence of both tachyarrhythmias was diminished (albeit notsignificantly) by the individual administration of these compounds. Intriguingly, neither isolated nor combined
administration of HMR-1098 and DZX were able to significantly decrease the duration of ventricular tachycardia (VT).
Moreover, in the presence of both compounds the beneficial effect on VF duration, recorded with each of the drug
when given solely, was lost. In isolated rat hearts, the independent administration of a sarcKATP inhibitor and a
mitoKATP opener mitigated the duration of reperfusion-induced ventricular fibrillation, whereas the association of thesecompounds did not influence the incidence and duration of malignant tachyarrhythmias.
Keywords: isolated rat heart, sarcolemmal and mitochondrial ATP-sensitive potassium channels, ischemia-related
malignant arrhythmias

INTRODUCTION
Ischemia-related ventricular tachyarrhythmias are
currently the major cause of sudden cardiac deathworldwide [1]. Preventing malignant arrhythmias in thesetting of myocardial ischemia represents an unmet
therapeutic target, ultimately aimed at decreasing mortality
due to coronary artery disease [2]. The efficiency of current
antiarrhythmic therapy is still under debate and a couple ofstudies have shown that several antiarrhythmic drugs canrather increase the risk of lethal arrhythmias in patients
recovering after myocardial infarction [3]. The majormechanism underlying ventricular arrhythmias isrepresented by reentry, classically promoted by theheterogeneity in action potential duration (APD) and tissue
repolarization, respectively. The duration of action potential

can be influenced, among others, by the activity of theATP-sensitive potassium channels (KATP) that couple
membrane excitability with cellular energetics [4]. Thesechannels were firstly described by Noma [5] in thesarcolemmal membrane of cardiomyocytes (sarcKATP). A
decade later, Inoue et al. [6] described a second type of
KATP channels located in the inner mitochondrial membrane(mitoKATP). The ATP-dependent potassium channels areclosed during normoxic conditions, when the cellular levelof ATP is high, and open when ATP concentration drops
during myocardial ischemia, as a result of the accumulationof ischemic metabolites ADP, lactate and H+ [2].
Paradoxically, both activation and inhibition of sarcKATP
channels have been associated with antiarrhythmic effects[7]. In particular, opening the sarcKATP channels has been
considered responsible for a significant potassium efflux

Received 12th of January 2016.Accepted 15th of March 2016. Address for correspondence: Danina Muntean, Departmentof Pathophysiology, “Victor Babeș” University of Medicine and Pharmacy of Timişoara; 14 Tudor Vladimirescu st., RO-300173
Timisoara,Tel/Fax: +40-256-493085; e-mail: daninamuntean@umft.roFiziologia - Physiology • 2016.26.1(89)35with the subsequent shortening of the APD and theincrease in the incidence of re-entrant arrhythmias,
respectively [3,7-9]. Accordingly, several studies suggested
that inhibition of sarcKATP channels can protect the heart
against ischemia/reperfusion-induced arrhythmias, andalso improve the survival rates [10-12].
In the past 2 decades, studies have shifted to theinvestigation of mitoKATP channels as effectors ofcardioprotection [13]. In this regard, opening the mitoKATP
channels was found to preserve mitochondrial structure[14], increase ATP preservation during ischemia and thefunctional recovery at reperfusion [15], and also todecrease infarct size [16].
In the present study we thought to assess the effect of
the combined administration of a cardioselective sarcKATP
inhibitor and a mitoKATP opener on ischemia-induced
arrhythmias in the isolated rat heart model.

MATERIAL AND METHODSAll the experiments were performed using aLangendorff retrograde perfusion system in the constantflow mode. The perfusion buffer Krebs-Henseleit (NaCl
118.0 mmol/L; KCl 3.2 mmol/L; MgSO4 1.2 mmol/L;
NaHCO3 25.0 mmol/L; NaH2PO4 1.18 mmol/L; CaCl2 2.5mmol/L; glucoză 11.1 mmol/L) was oxygenated withcarbogen (95% O2, 5% CO2), at least 15 minutes prior to
the experiment and kept at 370C and pH=7.4. All
experimental procedures were conducted in accordancewith the Directive 2010/63/EU and the Romanian Law nr.
43/May 2014 concerning the protection of animals used forscientific purposes. The experimental protocol wasapproved by the Committee for Research Ethics of “Victor
Babes” University for Medicine and Pharmacy of Timişoara,
Romania.
Most reagents were purchased from Sigma Aldrich.
Experimental protocol
Male Sprague-Dawley rats (n= 32) were anesthetizedusing a mixture of xylazine (5 mg/kg) and ketamine (30mg/kg). After the abdominal laparotomy, 500 IU of heparin
was administered via the portal vein to prevent thrombosis.
The heart was rapidly excised, rinsed with cold heparinizedKrebs-Henseleit solution and mounted on the cannula of
the Langendorff apparatus for the retrograde perfusion.
After cannulation, hearts were allowed to stabilize for 3
minutes prior being subjected to 8 minutes of regional
ischemia followed by 10 minutes of reperfusion. Regional
ischemia was elicited by the ligation of left anterior
descending (LAD) coronary artery. A brief episode ofnon-necrotic ischemia was purportedly chosen in order toinvestigate the 'pure' antiarrhythmic effect, i.e unrelated tothe death of the cardiomyocytes. The compounds,
HMR-1098 (10 μΜ) and DZX (50 μΜ) were dissolved in36

dimethyl sulfoxide (DMSO, final concentration less than
0.05%) and added to the perfusate before the stabilization
period. The experimental protocol is presented in Figure 1.

Fig. 1. Experimental protocol.
(A) Control group, (B) HMR-1098, (C) DZX,(D) HMR-1098 + DZX treated groups.

Statistics
Ventricular arrhythmic events were analyzed asincidence and duration expressed as log10 from the total
reperfusion time (600s). Statistical analysis was performed
using one-way ANOVA and Tukey’s test as Post-hoc
comparison among the groups. A value of p<0.05 wasconsidered statistically significant.

RESULTS
The present study was purported to assess the 'pure'
antiarrhythmic properties of two KATP modulators, asarcKATP inhibitor and a mitoKATP opener, and whether
their combined administration might provide an additiveeffect.
The Effects of HMR-1098 and DZX on
Tachyarrhythmias Incidence
The incidence of VT and VF in the study groups isshown in Table 1. The brief 10 min period of postischemic
reperfusion was associated with VF in all animals and VT
in 5 out of the 8 rats of the control group, respectively. A
decreasing tendency in the incidence of both types ofmalignant arrhythmias in the presence of either KATP
modulator when given independently was recorded.
However, when given together, the incidence of botharrhythmias was comparable to the one of the control
group (5 out of 8 animals for VT, and 7 out of 8 animals forVF) - Table I.

Fiziologia - Physiology • 2016.26.1(89)DISCUSSIONS

Table I. The incidence of VF and VT.
Control

HMR-1098

DZX

DZX+ HMR-1098

VT

5/8

3/8

4/8

5/8

VF

8/8

5/8

6/8

7/8

Normalheart
rhythm

2/8

8/8

7/8

6/8

The Effects of HMR-1098 and DZX on
Tachyarrhythmia Duration
Surprisingly, neither the individual nor the combined
administration of HMR-1098 and DZX were able to reducethe duration of VT as compared to controls (Figure 2).

Fig. 2. The isolated and combined effects of HMR-1098
and DZX on VT duration.
(n = 8/group, p = NS)

In contrast, inhibition of sarcKATP channels with theselective compound HMR-1098 and opening of mitoKATP
channels with DZX was each followed by a significant
decrease in the mean duration of VF (Figure 3). However,
the association of both KATP modulators abolished thebeneficial effect of the individual administration on VF
duration (Figure 3).

Fig.3. The isolated and combined effects of HMR-1098
and DZX on VF duration.
(n=8/group, **p<0.01 for HMR-1098 vs.
Control, *p<0.05 for DZX vs. Control)

Fiziologia - Physiology • 2016.26.1(89)The major finding of this paper is that the sarcKATP
inhibitor and the mitoKATP opener, albeit beneficial inindividual administration against VF, were not able todiminish the incidence and duration of reperfusion-related
ventricular arrhythmias when jointly given prior to theischemic episode.
The cardioprotective effects, including theantiarrhythmic ones, of KATP modulators were extensively[for recent comprehensive reviews see refs. 17, 18]. In
particular, glibenclamide, a non-selective sarcKATP inhibitor
belonging to the second generation of sulphonylureas, wassystematically reported to prevent/reduce the incidence ofarrhythmias in several in vitro and in vivo experimental
models and also in the clinical arena [17, 22-23].
HMR-1098 is a sulfonylthiourea, reported as
cardioselective blocker of cardiac sarcKATP channels bysome (but not all) authors [24] that, at variance fromglibenclamide, did not influence the blood glucose levels[25] or the coronary flow under normoxic and hypoxic
conditions [26]. Conflicting results are available in theliterature with respect to the antiarrhythmic action of
HMR-1098. Accordingly, Fischbach et al. demonstrated
that pre-treatment with 3 μΜ HMR-1098 was able toprevent the apparition of VF in isolated rabbit hearts, andalso to block (at 1μΜ) the VF induced by pinacidil, anon-selective KATP agonist [27]. In the canine old
myocardial infarction model, HMR-1098 (but notglibenclamide) improved the scores of programmed
electrical stimulation-induced ventricular arrhythmias, asreported by Zhu et al. [28]. At variance, Gok et al. reported
that HMR-1098 (3 and 30 μmol/l) was not able to prevent
VT and VF in isolated rat hearts subjected to 30 min ofcoronary occlusion followed by 30 min of reperfusion [29].
In our model of short I/R injury, we observed decrease intheincidence
(but
not
the
duration)ofreperfusion-associated VT in the presence of HMR-1098.
Conversely, both incidence and duration of VF weredimished, albeit statistical significance (P < 0.01, vs.
control) was reached only in case of the latter.
DZX is a benzothiadiazine with important
cardioprotective properties that has been reported to exert
antiarrhythmic effects in the rat model of I/R injury, both invivo and in vitro [reviewed in ref. 17]. In our study,
pre-treatment with DZX effects comparable to the ones of
HMR-1098 with respect to the incidence and duration of VT
and VF. Thus, in independent administration, DZX
mitigated (albeit non-significantly) the incidence of both VT
and VF, had no effect on VT duration, and significantly
diminished the VF duration (P < 0.05, vs. control).
Surprisingly, in combined administration, the KATP
modulators had no beneficial effect on either incidence orduration of VT and VF.
In the setting of I/R injury, cardioprotection associatedwith mitoKATP opening has been ascribed to the following

37

mechanisms: mitochondrial swelling, inhibition of ATP
synthesis during ischemia, decreased mitochondrial Ca2+
overload, modulation of ROS production during ischemia
and reperfusion, respiratory inhibition, and mild uncoupling- mechanisms that underlie the infarct size reduction andthe increase in functional recovery at reperfusion [30]. In
the literature there are only a few studies that examined
the antiarrhythmic effect of mitoKATP opening. Thus, inanesthetized rabbits, two selective mitoKATP openers,
nicorandil and minoxidil, when administered prior toregional ischemia of LAD, were able to increase thesurvival rate and decrease arrhythmogenesis, respectively[2]. In the canine model of in vivo I/R injury, DZX protected
the myocardium against ischemia/reperfusion- induced
arrhythmias, effect blocked by the administration of 5-HD,
the classic mitoKATP blocker [31].
Interestingly, the loss of mitochondrial selectivity has
been reported during ischemia for both DZX and nicorandil,when they can activate sarcKATP as result of increased
ADP concentration [32]. In line with this observation we
can speculate that the loss of antiarrhythmic effects whenassociating DZX to HMR-1098 was the consequence ofcounteracting the effect of the latter on sarcKATP; however,
we did not measure the ADP concentration to confirm that
it was decreased. However, other mechanisms can be also
incriminated since DZX has been reported to elicit
KATP-independent effects in cardiomyocytes [33]. Further
experiments aimed at assessing the particular effect ofeach drug on the sarcKATP channel activity are required.

CONCLUSIONSIn isolated rat hearts, the independent administration ofa sarcKATP inhibitor and a mitoKATP opener mitigated theduration of reperfusion-induced ventricular fibrillation,
whereas the association of these compounds did not
influence the incidence and duration of malignant
tachyarrhythmias.
ACKNOWLEDGEMENTS
This work was supported by a grant of the Ministry of
National Education, CNCS – UEFISCDI, project number
PN-II-ID-PCE-2012-4-0512 (N.J.) and by the doctoral fellowship
POSDRU no. 159/1.5/S/136893 – DocMed.Net_2.0. (A.P.) We
would like to thank to Mr. Karoly Acsai from the Department ofPharmacology and Pharmacotherapy from the Faculty of
Medicine, “Victor Babes” University of Medicine and Pharmacy,
Timisoara for his valuable contribution to the study.
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13. Garlid KD, Paucek P, Yarov-Yarovoy V, et al. The
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14. Rousou A, Ericsson M, Federman M, et al. Opening of
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through the modulation of mitochondrial matrix volume,
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15. Garlid KD. Opening mitochondrial KATP in the heart - What
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735-741.

CARACTERIZAREA EFECTELOR MODULATORILORSARCKATP ȘI MITOKATP ASUPRA ARITMIILOR INDUSE
DE REPERFUZIE PE INIMI IZOLATE DE ȘOBOLAN
REZUMAT
Tahiaritmiile maligne, fibrilația și tahicardia ventriculară (FV și TV) asociate ischemiei/reperfuziei (I/R) miocardicereprezintă o cauză majoră de moarte cardiacă subită la nivel global. Modularea farmacologică a canalelor cardiace depotasiu dependente de ATP localizate atât la nivel sarcolemal (sarcKATP) cât și la nivel mitocondrial (mitoKATP) a fostasociată cu cardioprotecție în condițiile ischemiei/reprefuziei miocardice în numeroase studii, fără însă ca această
observație să fie fără echivoc. Scopul prezentului studiu a fost de a evalua efectele administrării izolate și în asociere a
HMR-1098 (10 μΜ), inhibitorului selectiv al canalelor sarcKATP și respectiv, a diazoxidului (DZX, 50 μΜ), deschizătorului
canalelor mitoKATP, asupra tahiaritmiilor induse de reperfuzia postischemică. Ischemia regională a fost indusă prinligatura arterei coronare anterioare descendente stângi la nivelul inimilor izolate de șobolan perfuzate retrograd tip
Langendorff (în modul de lucru cu flux constant), iar compușii au fost administrați anterior inducerii ischemiei regionale.
În administrare independentă, atât HMR-1098 cât și DZX au redus semnificativ durata FV (dar nu și a TV) comparativ
cu lotul martor. De asemenea, aplicarea lor individuală a redus (deși nu semnificativ) incidența ambelor tahiaritmii
maligne. Surprinzător, nici administrarea independentă nici cea în asociere a compușilor nu au condus la o reducere
semnificativă a duratei TV. Mai mult, administrarea combinată a condus la pierderea efectului benefic obținut în cazul
aplicării individuale asupra duratei FV. În concluzie, în cazul inimilor izolate de șobolan, administrarea independentă aunui inhibitor al sarcKATP și respectiv, a unui deschizător al mitoKATP, a redus durata fibrilației ventriculare la reperfuzia
postischemică, în timp ce asocierea acestor compuși nu a influențat incidența și durata tahiaritmiilor maligne.
Cuvinte cheie: inimă izolată de șobolan, canale de poatasiu sarcolemale si mitocondriale dependente de ATP, aritmii
maligne induse de ischemie
Fiziologia - Physiology • 2016.26.1(89)39STEM CELLS FROM AMNIOTIC FLUID,
AN ESSENTIAL TOOL FOR REGENERATIVE MEDICINEHONCEA ADINA
Ovidius University, Constanta

ABSTRACT
Since the existence of stem cells in human amniotic fluid was reported, the knowledge about these cells has increased
dramatically. In recent years it has become increasingly evident the hope of using stem cells and human progenitor cells
to replace dysfunctional tissues. The major problem in this area of research is to identify new sources for isolatingprogenitor cells or stem cells that do not raise ethical issues. As an alternative to these sources of cells, the amniotic fluid
is easily collected by ultrasound guided transabdominal puncture. Compared to embryonic stem cells, amniotic stem cells
can be obtained without affecting human embryos, thus attenuating ethical controversies. The authors also described thehigh proliferative potential, and cultivation possibility for long periods with genomic stability maintained. AFS cells could
be used for stem cell-based therapies; in addition, it is now increasingly accepted as an optimal tool for basic research.
Biological, biochemical, immunological tests and morphological investigations showed that AFS cells are a new andspecific entity, distinct from ES cells or other types of stem cells, such as those that can be isolated from epithelial
sources or trophoblast tissue.
Key words: embryonic stem cells, amniotic stem cells, proliferative potential

INTRODUCTION
In recent years it has become increasingly evident thehope of using stem cells and human progenitor cells to
replace dysfunctional tissues.
The major problem in this area of research is to identify
new sources for isolating progenitor cells or stem cells that
do not raise ethical issues.
Also, because the logistical and ethical problems limit
the use of embryonic stem cells (ESCs), a special attention
was given to recent use of alternative sources of stem cells,such as mesenchymal stem cells (MSCs) in the bonemarrow (BM), amniotic fluid (AF), placenta, and umbilicalcord [1]. BM cells are a common source of MSCs in clinical
trials; however, their use has several limitations. First of all,the collection of BM from a patient is an invasive procedureand secondly, the frequency of MSCs in the BM is low.
Therefore, the search for alternative sources of useful
MSCs for clinical applications is important [2]. Currently,
existing MSC in umbilical cord blood is challenged with
reports showing that the isolation of such cells is difficult,having a low yield and inter-individual variations [3,4)]. The
placenta contains a large number of progenitor cells or stem
cells that can be obtained through minimally invasive
techniques [5]. Cells that were isolated from placenta
include amniotic epithelial cells, stromal chorionic amniotic
cells, MSCs, chorionic trophoblast cells and hematopoietic
stem cells [6].

As an alternative to these sources of cells, the amniotic
fluid is easily collected by ultrasound guided transabdominal
puncture. The potential proliferation of mesenchymal
stromal cells from amniotic fluid (AFMSCs) exceeds that of
MSC derived from BM [2]. AFS cells have a high capacity forrenewal - more than 250 doublings without any detectable
loss of telomere length of chromosomes [7]. The presenceof certain markers of undifferentiated cells shows that
AFMSCs may be less differentiated than most BM-MSCs,
and closer to pluripotent ESCs. AFMSCS’s have phenotypiccharacteristics similar to MSCs from other sources, such asumbilical cord, blood and bone marrow; all negative
AFMSCs for hematopoietic markers, such as CD45, CD34
and CD14 [8] while positive for mesenchymal markers suchas CD90, CD105, CD73, CD166.
Cellular Composition of the Amniotic Fluid
Human amniotic fluid obtained during amniocentesis
process includes a variety of cells derived from embryonicand extra-embryonic tissues [9] including stem cells.
Compared to embryonic stem cells, amniotic stem cells
can be obtained without affecting human embryos, thus
attenuating ethical controversies.
Due to the widespread use of routine prenatal genetic
tests, amniotic cells have become the subject of recent years’
research to enrich current knowledge about their origins andproperties.

Received January 15th 2016. Accepted March 4th 2016. Address for correspondence: Honcea Adina, Ovidius University
Constanta, Romania; 19 Bujorului Street, Constanta; phone: 0762043499, e-mail: adina_honcea@yahoo.com

40

Fiziologia - Physiology • 2016.26.1(89)The types and properties of amniotic fluid cells vary withgestational age. By cultivation, AFS cells can be separated
into adherent cells able to form colonies and non-adherent
cells. Based on morphological and growth characteristics,
amniotic fluid cells can be classified into three types:
amniotic fluid specific cells – type AF, epithelioid cells - type
E and fibroblast cells - F [10]. AF and E type cells appear at
the beginning of cultivation. AF cell type resists during
cultivation, while type E significantly decreases. It is
assumed that E type cells come from fetal skin and urine,
AF type cells from the trophoblast and fetal membranes andF type cells from fibrous connective tissue and dermal
fibroblasts. AF type cells produce human chorionicgonadotropin, estrogen and progesterone, which suggestthat these cells have their origin in the trophoblastic tissue.
Also, F type cells are deemed to originate in themesenchymal tissues. Approximately 1% of cells in cultures
obtained by amniocentesis for prenatal diagnosis of genetic
disorders express the surface antigen c-kit (CD117). This
receptor protein is present in human ES cells, primordial
germ cells, and many of somatic stem cells. In 2004, agroup of researchers managed to isolate monoclonal AFS
cells based on the expression of stem cell markers, c-Kit
and Oct4 [11] using flow cytometry and minimal dilution.
Three years later, another research group isolated
monoclonal AFS cells via flow cytometric selection andminimal dilution, which expressed the stem cell markers
c-Kit and Oct4 [12]. The authors also described the highproliferative potential, and cultivation possibility for long
periods with genomic stability maintained.
Stem cells derived from amniotic fluid (amniotic fluid stem
cells - AFS) express both embryonic and adult stem cellmarkers and unlike ESC proliferate very well without support
cells (feeder). In recent years, increasing evidence
demonstrated AFS ability to differentiate into multiple
lineages including those of the embryonic germ layers[2,9,10,13-16]. All this data allowed classification of thesecells as pluripotent [7,17,18]. Thus, AFS cells no longer needan ectopic induction of pluripotency.
However, AFS cells are different from ESC pluripotent
stem cells and from multipotent adult stem cells, and may
represent a new class of stem cell plasticity whose properties
are somewhere between embryonic and adult stem cells.
Moreover, approximately 90% of AFS cells express thetranscription factor Oct-4, known to be expressed inembryonic carcinoma cells, embryonic germ cells andembryonic stem cell in addition, AFS cells express TERT
marker of pluripotent stem cells [19].
AFS cells are now becoming increasingly accepted as an
optimal tool for fundamental research [20-24] preceding their
possible application in therapy.
It is known that the morphology, the composition and theproperties of AFS cell growth are affected by certain fetal
pathologies such as, for example, a neural tube defect or
gastroschisis [20,25,26].

Fiziologia - Physiology • 2016.26.1(89)Potential Ways of Differentiation Followed by AFS
Stem cells are useful tools for the study of the processesof differentiation in terms of molecular and cellularinvestigations.
AFS cells are genomically stable and do not harborneither the epigenetic memory nor somatic mutations of
already differentiated source cells. Furthermore iPS cells
have been reported to accumulate karyotype abnormalities
and gene mutations during propagation in culture [20,21].
Since AFS discovery, it was tried to be determined thedifferentiation potential of AFS cells by initiating successfully
the generation of various lines from a single stem cell. Quite
often, the researchers only use a mixture of cells fromamniotic fluid obtained by specific procedures of cultivation.
However, as is known, derivatives of amniotic fluid cells
represent mixtures containing a wide variety ofdifferentiated and undifferentiated cells. It is therefore
important that any study for a potential differentiating
specific cell lines to clarify the type of cell from which they
started, highlighting the biological and immunocytochemical
characteristics. Furthermore, evidence that AFS cells haveindeed pluripotent differentiation potential can be obtained
from a single stem cell. Unicellular and practical approaches
are required after minimal dilution experiments [25].
ES cells when cultured in the absence of differentiation
factors can spontaneously form three-dimensional
multicellular organisms called embryoid bodies. At first,
embryoid bodies were generally regarded as a starting point
for highlighting the differentiation of stem cells into different
cell lines. Consequently, the formation of embryoid bodyfollowed by different approaches for inducing differentiation
was an appropriate way to prove pluripotent differentiation
potential of a specific type of stem cell [21, 27]. Therefore, it
was of great interest the research that starting from a single
AFS cell has determined the formation of embryoid bodies.
It was shown that monoclonal human AFS cells can form
organisms when grown without anti-differentiation factors inconditions in which they are not able to attach to the surfaceof culture vessels and without contact with feeder cells. The
formation of such three-dimensional multicellular organisms
is accompanied by a decrease in the expression of stem cellmarkers in different lines and induced differentiation [27].
The study that has shown the potential AFS cells to form
embryoid bodies was the ultimate proof for their pluripotent
character. In addition, now it led to the revision andinvestigation of three-dimensional structures and othertissue structure of many processes of differentiation during
early embryogenesis [27].
ES cells right from the first in vitro culture have been
described as being tumorigenic. Similarly, iPS cells induced
tumor formation when transplanted subcutaneously to themice. However, in AFS cells has not been reported theformation of tumors in immunocompromised mice.
Gene expression profiles suggest that AFS cells havethe potential to differentiate into cardiomyocytes, as well as
in other mesoderm derivatives, in addition to neural cells [7].
41

AFS cells have been shown to express neuronalmarkers (such as CD133, nestin, neurofilament, CNPA,
p75, BDNF, and neurotophin-3) [9]. The presence of several
markers of dopaminergic neurons (TGF-β3, FGF8, SHH,
and β-catenin) in AFS cells demonstrates the existence ofdifferentiating neuronal cells engaged in the path,corresponding to a transcriptional profile similar tomesencephalic dopaminergic neurons [14,28]. These cells
can become nestin positive neural stem cells, and thendopamine and glutamate receptive neurons.
In a suitable environment, AF-derived mesenchymal
stem cells (AF-MSCs) can form functional osteoblasts thatcan produce bone mineralization or may form smooth muscle
cells [19]. Further, liver cells obtained by differentiating
AF-MSCs are capable of secreting high levels of urea and to
express liver proteins, such as α-fetoprotein, albumin, growth
factors and hepatocyte nuclear factor [7,18].
Many groups have confirmed the existence of Oct4+ /c-kit+ AFS cells and reported their potential to differentiate on
the hematopoietic, neurogenic, osteogenic, chondrogenic,
adipogenic, renal, hepatic, and various other lines [2,29-37].
Although, in terms of their biological properties andmarkers expression they appear to be more similar to ES
cells, AFS cells exact origin remains unknown. Biological,
biochemical, immunological tests and morphologicalinvestigations showed that AFS cells are a new and specificentity, distinct from ES cells or other types of stem cells,such as those that can be isolated from epithelial sources or
trophoblast tissue.
AFS and Regenerative Medicine
In 2001, it was suggested that the cells in the amniotic
fluid may be used in tissue engineering approach for thesurgical repair of congenital abnormalities in the perinatal
period.
Initially the idea was to make it function as areconstruction graft for implantation, even before birth or in
the neonatal period. This is of particular interest to children
born with a defect in the wall that are too small for taking a
graft for reconstructive surgery [9,38]. The results obtainedin animal models are indeed encouraging.
Studies done for SE human cell transplantation therapy
prevent possible immunological and tumorigenicity. As waspointed out, in contrast to cells derived from the cell mass of
blastocyst, AFS cells are not tumorigenic in mice after
transplantation [7].
Studies have shown that the AFS cells can be used forregenerative therapy approaches [20-24]. One such study
showed the usefulness of AFS cells in regenerative
mammary gland [11]. Other investigations have shown that
may aid in muscle and cardiac disease [19,39]. Due to theirneurogenic potential there are enough reasons to launchtherapies based on AFS for diseases of the nervous systemsuch as Parkinson's disease, spinal cord injury, multiple
sclerosis or stroke. Neural stem cells, which have been42

studied for this purpose and can be found in the adult central
nervous system and the developing embryo, are not easily
accessible and raise ethical concerns.
Kidney acute and chronic diseases are disorders withhigh rate of morbidity and mortality. Transplantation remains
the most effective treatment option for most patients with
end-stage renal disease. But unfortunately, the lack ofcompatible organs is a real problem. Treatment strategies
are based on conventional kidney dialysis, but the mortality
rate of patients requiring chronic dialysis is quite high.
Therefore, the use of stem cells in tissue repair kidney
becomes increasingly plausible. Several recently publishedstudies for renal cell differentiation AFS cells are tempting
further investigations in this direction. [24,40-44].
A recent report has shown that human AFS cells canintegrate into the kidney tissues when injected into theisolated embryonic murine kidney [41]. It was also provedthat AFS cells have the potential to differentiate tonephrogenic lineage following a pathway regulated by the
mammalian target of rapamycin (mTOR) [42]. Moreover, theinjection of AFS cells into kidneys of mice withrhabdomyolysis associated acute tubular necrosis, could
mediate a protective effect [43].
AFS cells can survive, migrate significant distances, anddifferentiate when transplanted into the brains of adult rats
[7,45]. Also, AF-MSCs after 3 weeks of culture weretransplanted into the brain of both normal and ischemic rats,
and showed that transplanted cells were able to migratefrom the injection site along the corpus callosum in order tohave access to more than one region of the brain. Somepositive effects obtained by MSC transplants can be
assigned to production of trophic factors and cytokines bythese cells, and the increasing levels of neurotrophic factors
in the ischemic region, as a response to administration of
MSCs [46].
Among the limits of using AFS cells is the cost ofobtaining such a cell which is much higher than for othertypes of stem cells.
There are also regarded as a source of
immuno-incompetent graft cells, in large part thanks to theirimmature immunogenic system, immunogenic properties
and phenotypic instability [47]. Studies have also shown thatstem cells derived from the placenta can be an alternative
source of stem cells [48]. This can cause problems during
cell growth, where cells accumulation could lead tospontaneous differentiation. In addition, investigations are
warranted to discover evidence of meiotic divisions withinthese stem cells derived from the placenta. These
unregulated divisions not only can lead to the possibility ofmalignant tumors, but can also lead to cell defects. These
negative side effects should be taken into account to reveal
safety profile of AFS cells, with the resulting parametersveracity.
These interesting options for cell-based treatment
could lead to the development of further appealing therapies
against human and veterinary diseases [49] (Table I).
Fiziologia - Physiology • 2016.26.1(89)Table I. Target disease and references
Target disease

References

Prenatal diagnosis

Cananzi et al., 2009 (17)Peripheral nerve regeneration, sciatic nerve regeneration
following crush

Cheng et al., 2010 (27)

Myocardial infarction (cardiac and smooth muscles) andischemia

Chiavegato et al., 2007 (46); Delo et al., 2010 (38)Tumors and genetic disorders

De Coppi et al., 2007 (7); Yen et al., 2005 (49)

Organ-specific regeneration

De Sacco et al., 2010 (51)

Tissue engineering, cell transplantation, and gene therapy

Fauza, 2004 (13)Diabetes

In’t Anker et al., 2003 (2)Neurodegenerative diseases and nervous system injuries

Tsai et al., 2006 (15)Mammary gland regeneration

Klemmt et al., 2010 (11)Parkinson’s, Alzheimer’s, heart disease, diabetes, stroke,
spinal cord injuries, and burns

Prusa et al., 2002 (10); Yang et al., 2009 (49)

Muscular disease

Mauro et al., 2010 (39)

CONCLUSIONS
Biological, biochemical, immunological, as well asmorphological studies have shown that the AFS cells
represent a new and specific entity of stem cells, distinct fromES cells or other types of stem cells that can be isolated thanamniotic epithelial or trophoblast.

As a result, the amniotic fluid could be a safe and easily
accessible source of AFS cells to be used for therapeutic
purposes, without major ethical concerns, given thatamniocentesis is a universally accepted form of prenatal
diagnostic tests (Table II).

Table II. Research results for amniotic fluid stem cells
Year
2003
2003
2004
2004
2004

2004

2005
2006

Title
Amniotic fluid as a novel source of mesenchymal stem
cells for therapeutic transplantationOct-4-expressing cells in human amniotic fluid: A newsource for stem cell research?
Amniotic fluid and placental stem cells
Isolation of mesenchymal stem cells of fetal or maternal
origin from human placenta
Fetal stem cells

Isolation of human multipotent mesenchymal stem cells
from second-trimester amniotic fluid using a novel
two-stage culture protocol
Isolation of multipotent cells from human term placenta
Stable expression of a neuronal dopaminergic
progenitor phenotype in cell lines derived from humanamniotic fluid cells

Fiziologia - Physiology • 2016.26.1(89)Reserch results
Amniotic fluid is found as a source of MSCs for cotransplantation in
conjuction with UCB-derived hematopoietic stem cells(2)Amniotic fluid may represent a new source for isolation of humanOct-4-positive stem cells(10)Amniotic stem cells have been found to have therapeutic effects intissue engineering, cell transplantation, and gene therapy(13)Amnion is found as a novel source of fetal MSCs; could becontributing to the presence of MSCs in AF(52)Fetal stem cells have been found to be a rich source of hemopoietic
stem cells (HSC), which proliferate more rapidly than those in cord
blood or adult bone marrow [1)Human multipotent MSCs are present in second-trimester amniotic
fluid. Amniotic fluid is found to be an excellent alternative source forinvestigation of human MSCs [16]Placenta-derived multipotent cells (PDMCs) were found to exhibit
many markers common to mesenchymal stem cells(53)Cell lines can be derived from subcultures of amniocentesis [14]

43

2006

Clonal amniotic fluid-derived stem cells express
characteristics of both mesenchymal and neural stem
cells

2006
2007
2007
2007
2007
2008

2008

2009

2010

2010

2010

2010

Stem cells in the umbilical cord
Isolation of amniotic stem cell lines with potential fortherapy
Stem cells. Versatile stem cells without the
ethical baggage?
Human amniotic fluid-derived stem cells havecharacteristics of multipotent stem cells
Tissue engineering from human mesenchymal
amniocytes: A prelude to clinical trials
Deleted in Azoospermia-ike (DAZL) gene-expressing
cells in human amniotic fluid: A new source for germ
cells research?
Isolation and characterization of cells from human term
placenta: Outcome of the First International Workshop
on Placenta Derived Stem
Cells
Stem cells derived from amniotic fluid: New potentials inregenerative medicine
Enhancement of regeneration with glia cell line-derived
neurotrophic factor-transduced human amniotic fluid
mesenchymal stem cells after sciatic nerve crush injury
Human amniotic fluid as a potential new source of organ
specific precursor cells for future regenerative medicine
applications
Murine amniotic fluid stem cells contributemesenchymal but not epithelial components toreconstituted mammary ducts
Isolation, characterization, and in vitro differentiation of
ovine amniotic stem cells

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Amniotic fluid was found to provide a promising source of neuralprogenitor cells that could be used in the future cellular therapies forneurodegenerative disease and nervous system injuries [15]Umbilical cord-derived stem cells are compared with adult bonemarrow-derived mesenchymal stem cells [54]Differentiated cells derived from human AFS cells, which can display
specialized functions including neuronal lineage cells [7]A cell derived from amniotic fluid was found to have characteristics ofembryonic stem (ES) cells [55]
HAFFTs are pluripotent stem cells that are less differentiated [32]
Human mesenchymal amniocytes can retain their progenitor
phenotype [56]
Human Amniotic fluid represents a new source for the isolation ofhuman stem cells [57]Placental tissue is found to be a source of cells for regenerative
medicine [5]Amniotic fluid stem cells have represented as a type of pluriopotent
stem cell with characteristics between embryonic and adult stem cells[17]
The GDNF-modified human AFMSCs appeared to promote nerve
regeneration [28]Progenitor cells in amniotic fluid have been found to provide a use forregenerative medicine purposes [51]Amniotic fluid cell’s 3 germ layers have been found to be valuable forcellular therapy and tissue engineering [11]Stem cell regenerative therapy has been found to treat many humandiseases [19]6. Parolini O, et al. Toward cell therapy using placentaderived cells:
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CELULELE STEM DIN LICHIDUL AMNIOTIC –UN INSTRUMENT ESENȚIAL PENTRU MEDICINAREGENERATIVĂREZUMATÎncă de la raportarea existenţei celulelor stem amniotice, cunoștinţele despre aceste celule au crescut în mod evident. Înultimii ani a devenit din ce în ce mai limpede speranţa de a folosi celule stem şi celule precursoare umane pentru a înlocuițesuturile disfuncționale. Problema majoră în acest domeniu de cercetare este acela de a identifica noi surse pentru
izolarea celulelor precursoare sau celule stem, care să nu ridice probleme etice. Ca o alternativă la alte surse de celule,lichidul amniotic este ușor de colectat prin puncție transabdominală ghidată ultrasonografic. Prin comparaţie cu celulele
stem embrionare, celulele stem amniotice pot fi obținute fără a afecta embrionii umani, atenuând astfel controversele
etice. Autorii au descris, de asemenea, potențialul proliferativ ridicat, precum și posibilitatea de cultivare a celulelor stem
amniotice pentru perioade lungi de timp cu menţinerea stabilităţii genomice. Celulele AFS ar putea fi folosite pentru
terapii pe bază de celule stem; în plus, sunt tot mai uşor acceptate ca un instrument optim pentru cercetarea de bază.
Testele imunologice, biochimice, biologice şi investigațiile morfologice au arătat că celulele AFS sunt o entitate nouă și
specifică, distinctă de celule stem embrionare sau alte tipuri de celule stem, cum ar fi cele care pot fi izolate din surse
epiteliale sau țesutul trofoblastic.
Cuvinte cheie: celule stem embrionare, celule stem amniotice, potențial proliferativ

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Fiziologia - Physiology • 2016.26.1(89)