histological interfaces of liver, kidney and cerebrum in ... · cv s . hamad, s. et al. /zjpas:...

126

ZANCO Journal of Pure and Applied Sciences

The official scientific journal of Salahaddin University-Erbil

ZJPAS (2016), 28 (5); 621-135

Histological Interfaces of Liver, Kidney and Cerebrum in Male Rats

exposed to Fluoxetine

Sarkawt H. Hamad1, Dlshad Hussein Hassan

1, Khder H. Rasul

2, Nazar Mohammed Shareef

Mahmood3

6- Biology Department, Faculty of Science, Soran University, Soran, Erbil, Kurdistan Region, Iraq.

2- Department of Biology, College of Science, Salahaddin University, Erbil, Kurdistan Region, Iraq.

3- Bjeel Preparatory school, Directorate of Education - Akre, General Directorate of Education – Duhok, Ministry of

Education- KRG-Iraq.

1. INTRODUCTION

Depression is a condition of unfavorable

mood and dislikes to do activities which lead to

abnormal behavior and aggressive feeling.

Depressed mood is a normal occurrence in

response to adversity in all individuals and it is

very common among persons who had a

trouble and disease (Johnstone et al., 2007).

The diagnoses such as Beck Depression

Inventory and Children’s Depression Inventory

have been used for assessment of depression

and severity of its symptoms (Biros et al.,

2008, Jang et al., 2016). In general, depressed

mood may not need a professional treatment,

but in most cases, the patients take treatments,

so that the production and using of

antidepressant such as selective serotonin

reuptake inhibitors are the most frequently

prescribed drugs to treat moody disease among

A R T I C L E I N F O

A B S T R A C T

Article History:

Received: 21/07/2016

Accepted: 61/08/2016

Published: 28/11/2016

Fluoxetine is an antidepressant of selective serotonin reuptake inhibitor

drug. It is used in treatment of depression, panic and anxiety. Also, it may

decrease the risk of suicide in those over the age of sixty-five years. The

current study was planned to investigate the histological effect of oral

administration of fluoxetine (10 mg/kg body weight/day) on liver, kidney and

cerebrum of male rats. Rats were divided randomly and equally into two

groups; control group (n=8) and fluoxetine-treated group (n=8). After one

month of administration; liver, kidney and cerebrum tissues would be taken to

make histological slides. Histological alterations were observed in liver such

as presence of few dead hepatocytes, congested blood vessels and

inflammatory cells. Kidney also showed histological changes; in the cortex

region include shrunken glomeruli tuft, hemorrhage and degenerated cells

while in the medullary region included thickened wall of tubules and some of

the epithelial tubule cells were degenerated. Alterations in the cerebrum were

the presence of dead neuronal cells in the second and third layers of grey

matter were due to fluoxetine. In conclusion, fluoxetine had a minute effect on

the histological structure of liver, kidney and cerebrum tissues.

Keywords:

Fluoxetine

Depression

Liver

Kidney

Cerebrum.

*Corresponding Author:

Sarkawt H. Hamad

[email protected]

Hamad, S. et al. /ZJPAS: 2016, 28(5): 126-135

127

them fluoxetine (Anderson, 2000, Inkielewicz-

Stepniak, 2011).

Fluoxetine is a selective serotonin reuptake

inhibitor and as a first line drug which is used

as a treatment of depression and many

neuropsychiatric disorders (Cipriani et al.,

2005, Wernicke, 2004). Serotonin in the

central nervous system act as neurotransmitter

included in variety of physiological and

behavioral functions and considered to have

an important role in the control of pain (Bardin,

2011).

Depression and drug using such as

fluoxetine gradually increases in all ages

especially among adults and teenagers in

Kurdistan region-Iraq. Fluoxetine is easily

available by the patients; it metabolizes in the

liver and excreted in the urine. This study was

planned to determine the histological effects of

fluoxetine on liver, kidney and cerebrum.

2. MATERIALS AND METHODS

2.1. Experimental Animals

Sixteen adult Wister male albino rats (8-10

weeks old) and weighing (200-270 gm) were

conducted in this study. They were housed in

plastic rat cages (56 x 39 x 19 cm) in groups

of eight rats per cage in a room with controlled

temperature of (22 ± 1 oC), 12 hours light and

12 hours dark by using an automated light-

switching device, in the animal house of

Biology department, Faculty of Science, Soran

University; under supervision and approval of

local scientific committee and animal care

rules. Rats were fed with standard laboratory

chow and allowed drinking water ad libitum.

2.2. Experimental design

Rats were divided randomly into two

groups; group 1: control group (n=8), group 2:

fluoxetine-exposed rats in which rats were

orally administrated with 10 mg/kg body

weight/day (n=8).

2.3. Anesthesia, dissection and removal of

the organs

At the end of the experiment, all rats were

anesthetized with intraperitoneal injection of a

mixture of Ketamine Hydrochloride (80 mg /

Kg) and Xylazine (12 mg / Kg), then liver,

kidneys and cerebrum were removed, cut into

smaller pieces (approximately 0.5cm in

thickness) in petri dish which contained a

fixative (Bouin’s fluid) and then transferred

into Bouin’s fluid for fixation.

2.4. Histological preparation (Paraffin

method)

Fixed tissues (liver, kidney and cerebrum)

were removed from Bouin’s fluid and

dehydrated by a serial concentration of ethanol

in ascending manner, infiltrated with paraffin

wax after the clearing. Paraffin wax also used

for making tissue blocks. The tissue blocks

were cut into four to six micrometer thick

paraffin sections by using rotary microtome

(Bright, MIC) and stained with hematoxylin

and eosin (H&E) (Bancroft et al., 1977).

Finally, light microscope (digital binocular

compound microscope 40x-2000x, built-in

3MP USB camera) was used to examine and

photo taken.

3. RESULTS AND DISCUSSION

Daily oral administration of fluoxetine for

one month caused several histological

alterations in liver, kidney and cerebrum of

rats.

3.1. Effect of fluoxetine on liver

Histological sections of liver control rats

showed normal hepatic architecture in which


128

many healthy hepatocytes were polyhedral in

shape appeared, exhibited centrally round or

ovoid shape nucleus and well- known plasma

membrane. Blood sinusoids appeared normally

and located between hepatocytes. Central vein

were normal (Fig. 3.1 and Fig. 3.2). The liver

sections of fluoxetine treated rats showed that

most of hepatocytes would be appeared as

normal and there is no obvious architecture

change except that a few of the hepatocytes

degenerated, a little inflammatory infiltration

of leukocytes and congested blood vessel

would be observed (Fig. 3.3 and Fig. 3.4).

Histopathological examination of the liver

revealed hepatic injury after fluoxetine

treatment (Yilmaz et al., 2016). Furthermore,

hepatocellular hydropic vacuolar degeneration,

portal area and lobular inflammation were

observed as a result of rat exposure to

fluoxetine which agree with our results (Özden

et al., 2005).

3.2 Effect of fluoxetine on kidney

The paraffin sections of the cortical region

showed well-designated glomeruli and kidney

tubules both proximal convoluted tubules and

distal convoluted tubules in the kidney of

control group rats (Fig. 3.5). The medullary

region of the control kidney rats also showed a

normal appearance of tubules as well as their

epithelial cells (Fig. 3.6). In contrast, fluoxetine

caused alterations in kidney rats of treated

group, the cortex region showed shrinking of

glomeruli, hemorrhage and dead cells (Fig. 3.7)

while in the medullary region, thickened the

wall of kidney tubules and degeneration of

some kidney tubule epithelial cells were

observed (Fig. 3.8). At doses of 3 and 10

mg/kg, the significant reductions in renal nerve

activity were observed especially 15 minutes

after the intravenous injection of fluoxetine

(Tiradentes et al., 2014). The hyponatremia

arose during fluoxetine antidepressant therapy

(ten Holt et al., 1996). Also, the sodium ions

level decreased, Potassium unchanged and

antidiuretic hormone remained unchanged,

whereas the AQP2 protein abundance and

water absorption in the inner medullary

collecting duct were increased (Moyses et al.,

2008).

3.3. Effect of fluoxetine on cerebrum

As shown in figure 3.9, the histological

slides showed that the cerebrum of control rats

have normal appearance of their structure such

as layers of grey matter and normal neuronal

cells, while dead pyramidal cells in second and

third layers of grey matter were observed in

the sections through the cerebrum of

fluoxetine-exposed rats (Fig. 8.10).It has been

suggested that fluoxetine cause increasing the

concentration of serotonin in synaptic cleft and

vasodilation of small cerebral arteries such as

branches of the anterior cerebral arteries which

induced by calcium channel openers (Ungvari

et al., 2000). In addition, the

electrophysiological studies have further

demonstrated that fluoxetine inhibits different

types of calcium channels in the neurons (Deak

et al., 2000). Fluoxetine at 0.03 mM enhanced

nicotine- and choline-induced relaxations in

which nicotine induced norepinephrine release

from cerebral perivascular sympathetic nerves

but vasorelaxation was blocked by higher

concentration of fluoxetine (>0.3 mM) that is

mean, the high concentration of fluoxetine

cause decrease neurogenic vasodilation while

low concentration of fluoxetine cause increase

neurogenic vasodilation (Chen et al., 2012).

Furthermore, fluoxetine alters the levels and

composition of brain GABA(A) receptors and

reduces the responsiveness of GABA(A)-R to

GABA-mimetic drugs such as pentobarbital

(Matsumoto et al., 2007).


129

Figure 3.1: Photomicrograph from liver a control rat showing the normal central vein (CV), normal hepatocytes and blood

sinusoids. H&E. 100X.

Figure 3.2: A magnified photomicrograph from liver control rat section showing normal appearance of hepatocytes (black

arrow) which polyhedral in shape, round or ovoid shape nucleus and well-defined plasma membrane. Blood sinusoids (S)

and central vein (CV) are normal. H&E. 400X.

CV S

CV


130

Figure 3.3: Photomicrograph from liver treated rat with fluoxetine showing that the hepatocytes will appear as normal and

there is no architecture change. A little inflammatory infiltration of leukocytes (black arrow) and congested blood vessel

(white arrow) will present due to fluoxetine drug. H&E. 100X.

Figure 3.4: A magnified histological section of liver treated rat with fluoxetine showing congested blood vessels (white

arrow) and leukocyte infiltration inflammation (black arrow). Most of hepatocytes show normal architecture and a few of

them degenerated. H&E. 400X.


131

Figure 3.1: Section through the cortex of the kidney of control rat showing normal glomeruli (black arrow) and kidney

tubules (white arrow). H&E. 400X.

Figure 3.1: Medullary region in the kidney of control rat having a normal appearance of tubules (T) H&E. 400X.

T

T


132

Figure 3.7: Photomicrograph from kidney cortex of treated rat with fluoxetine showing shrunken tuft of glomeruli (black

arrow), hemorrhagic area (H) and degenerated cell area (D). H&E. 400X.

Figure 3.8: Section through the kidney of fluoxetine treated rat showing the kidney tubules in the medullary region which

they had thickened wall (black arrow) and some of their cells were degenerated (white arrow). H&E. 400X.

H

D


133

Figure 3.9: Sections through the cerebrum of control rats. A) Grey matter of cerebrum shows different layers with normal

structure. H&E. 100X. B) Normal appearance of grey matter in which normal pyramidal cells (black arrow) and glial cells

(white arrow) were observed. H&E. 400X. C) Oil immersion magnification power showing normal pyramidal cells (black

arrow) and glial cells (white arrow) in cerebral cortex layer. H&E. 1000X.

A

A

C

B

A


134

Figure 3.10: Photomicrographs from cerebrum treated rats with fluoxetine. A) Grey matter of cerebrum which shows

layers. H&E. 100X. B) Dead cells (black arrow) were observed due to fluoxetine in second and third layers of grey matter.

H&E. 400X. C) High power magnification shows clearly dead cells (black arrow). H&E. 1000X.

4. CONCLUSIONS

The present work was designed to

determine the histological effect of fluoxetine

on liver, kidney and cerebrum of male rats. The

light microscopic examination showed that

orally administration of fluoxetine for a period

of one month had a bantam histological effect

of liver, kidney and cerebrum of male rats.

A

A

B

C


135

Conflict of Interest

There is no conflict of interest.

REFERENCES

ANDERSON, I. M. 2000. Selective serotonin reuptake

inhibitors versus tricyclic antidepressants: a meta-

analysis of efficacy and tolerability. J Affect Disord,

58, 19-36.

BANCROFT, J. D., STEVENS, A. & DAWSON, I. M.

S. 1977. Theory and Practices of Histological

Techniques. Edinburg, London, New York.

ChurchillLivingstone.

BARDIN, L. 2011. The complex role of serotonin and 5-

HT receptors in chronic pain. Behav Pharmacol, 22,

390-404.

BIROS, M. H., HICK, K., CEN, Y. Y., MANN, J.,

GAETZ, A., HANSEN, R. & SCHIMING, R. 2008.

Occult depressive symptoms in adolescent emergency

department patients. Arch Pediatr Adolesc Med, 162,

769-73.

CHEN, M. F., HUANG, Y. C., LONG, C., YANG, H. I.,

LEE, H. C., CHEN, P. Y., HOFFER, B. J. & LEE, T.

J. 2012. Bimodal effects of fluoxetine on cerebral

nitrergic neurogenic vasodilation in porcine large

cerebral arteries. Neuropharmacology, 62, 1651-8.

CIPRIANI, A., BRAMBILLA, P., FURUKAWA, T.,

GEDDES, J., GREGIS, M., HOTOPF, M.,

MALVINI, L. & BARBUI, C. 2005. Fluoxetine

versus other types of pharmacotherapy for

depression. Cochrane Database Syst Rev, Cd004185.

DEAK, F., LASZTOCZI, B., PACHER, P., PETHEO,

G. L., VALERIA, K. & SPAT, A. 2000. Inhibition of

voltage-gated calcium channels by fluoxetine in rat

hippocampal pyramidal cells. Neuropharmacology,

39, 1029-36.

INKIELEWICZ-STEPNIAK, I. 2011. Impact of

fluoxetine on liver damage in rats. Pharmacol Rep,

63, 441-7.

JANG, C. H., JOO, M. C., NOH, S. E., LEE, S. Y., LEE,

D. B., LEE, S. H., KIM, H. K. & PARK, H. I. 2016.

Effects of Hippotherapy on Psychosocial Aspects in

Children With Cerebral Palsy and Their Caregivers:

A Pilot Study. Ann Rehabil Med, 40, 230-6.

JOHNSTONE, T., VAN REEKUM, C. M., URRY, H.

L., KALIN, N. H. & DAVIDSON, R. J. 2007. Failure

to regulate: counterproductive recruitment of top-

down prefrontal-subcortical circuitry in major

depression. J Neurosci, 27, 8877-84.

MATSUMOTO, K., PUIA, G., DONG, E. & PINNA, G.

2007. GABA(A) receptor neurotransmission

dysfunction in a mouse model of social isolation-

induced stress: possible insights into a non-

serotonergic mechanism of action of SSRIs in mood

and anxiety disorders. Stress, 10, 3-12.

MOYSES, Z. P., NAKANDAKARI, F. K. &

MAGALDI, A. J. 2008. Fluoxetine effect on kidney

water reabsorption. Nephrol Dial Transplant, 23,

1173-8.

ÖZDEN, H., BILDIRICI, K. ı., ÜSTÜNER, D.,

ÜSTÜNER, C., CENGIZ, B. P., TÜLAY, A. &

YıLMAZ, V. 2005. Histopathologic examination of

rat liver after experimental application of fluoxetine.

Türkiye Ekopatoloji Dergisi, 11, 9-15.

TEN HOLT, W. L., VAN IPEREN, C. E., SCHRIJVER,

G. & BARTELINK, A. K. 1996. Severe

hyponatremia during therapy with fluoxetine. Arch

Intern Med, 156, 681-2.

TIRADENTES, R. V., PIRES, J. G., SILVA, N. F.,

RAMAGE, A. G., SANTUZZI, C. H. & FUTURO

NETO, H. A. 2014. Effects of acute administration of

selective serotonin reuptake inhibitors on sympathetic

nerve activity. Braz J Med Biol Res, 47, 554-9.

UNGVARI, Z., PACHER, P. & KOLLER, A. 2000.

Serotonin reuptake inhibitor fluoxetine decreases

arteriolar myogenic tone by reducing smooth muscle

[Ca2+]i. J Cardiovasc Pharmacol, 35, 849-54.

WERNICKE, J. F. 2004. Safety and side effect profile of

fluoxetine. Expert Opin Drug Saf, 3, 495-504.

YILMAZ, A., ELBEY, B., YAZGAN, U. C., DONDER,

A., ARSLAN, N., ARSLAN, S., ALABALIK, U. &

ASLANHAN, H. 2016. Protective Effects of Caffeic

Acid Phenethyl Ester on Fluoxetine-Induced

Hepatotoxicity: An Experimental Study. Biomed Res

Int, 2016, 1247191.

histological interfaces of liver, kidney and cerebrum in ... · cv s . hamad, s. et al. /zjpas:...

Documents