الفهرس 
Aim of the workOnly 14 pages are availabe for public view
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Abstract
Psychosis affects multiple brain structures including the hippocampal
formation. Studies of the hippocampal regions indicate that CA1 is the most
affected region by psychosis.
Clozapine is an atypical anti-psychotic drug well known as the treatment of
choice in TRS. However, it is associated with several side effects such as
metabolic syndrome and agranulocytosis.
Many studies indicate that AFA extract has a neuroprotective effect due to
its vitamin B12 content and antioxidant anti-inflammatory components including
phycocyanins, carotenoids and polyunsaturated fatty acids which inhibit neuronal
toxicity and protect against neurodegeneration.
The present study was performed to compare the therapeutic effects of AFA
extract with that of clozapine on CA1 region of the hippocampus of adult male
albino rats treated with ketamine to induce psychosis; clinically, histologically and
biochemically.
7.1. Materials and Methods:
Sixty adult male albino rats weighing 200-250 g were used in this study.
They were classified into five groups, each group consisted of 12 rats.
group I: (Control group): was subdivided into two subgroups:
Subgroup Ia: (Plain control): consisted of 6 rats and was kept without any
treatment throughout the experimental period.
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Subgroup Ib: (Sham control): consisted of 6 rats and received 0.4 ml
intraperitoneal saline once daily for 14 consecutive days.
group II: (AFA extract group): received AFA extract (200 mg/kg/d) from the 8th
day to the 21st day. AFA extract was dissolved in distilled water and given by
gastric tube.
group III: (Psychosis group): received ketamine HCl (25 mg/kg/d) by
intraperitoneal injections once daily for 14 consecutive days. Ketamine HCL was
available as 500 mg/ 10 ml vials. Each 0.1 ml of ketamine HCL was diluted in 0.3
ml of saline.
group IV: (Psychosis group treated with clozapine): received intraperitoneal
ketamine HCl (25 mg/kg/d) for 14 consecutive days. from the 8th day and till the
21st day, this group received intraperitoneal clozapine (5 mg/kg/d) dissolved in
saline.
group V: (Psychosis group treated with AFA extract): received intraperitoneal
ketamine HCl (25 mg/kg/d) for 14 consecutive days. from the 8th day and till the
21st day, this group received AFA extract (200 mg/kg/d).
OFT was done on the 7th and 21st days of the experiment to test positive
symptoms. Each rat was placed in the center of a square box with a floor divided
into 25 equal squares and allowed to explore freely for 5 minutes. Number of
crossed squares was counted.
SPT was conducted on the 5th and 19th days of the experiment to test
negative symptoms. The SPT consisted of adaptation and test phases. The
adaptation phase lasted for two days. After 18 hours of food and water deprivation,
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the test phase was performed. The test phase was conducted on the 7th and 21st days
of the experiment. In the test phase, two identical water bottles were used, one
containing 100 ml of 1.5% sucrose solution while the second containing 100 ml of
water. Sucrose solution and water consumption were recorded after one hour. The
sucrose preference was calculated. Sucrose preference index (%) = sucrose
consumption [g] / (sucrose consumption [g] + water consumption [g]) × 100%.
NORT was done on the 7th and 21st days of the experiment to test cognitive
symptoms. The test consisted of two sessions. During the first session
(familiarization), two identical objects were placed in opposite corners and the rat
was given three minutes to explore the objects. After the first session, the rat was
returned to its home cage for 60 minutes. During the second session (test), one of
the original objects was replaced by a new different object and the exploration was
allowed for two minutes. The memory index was then calculated for the test
session: Memory index = N/(N+F). N= time spent by the rat exploring the novel
object, F= time spent exploring the familiar object.
Body weight measurements were recorded for all rats on days 1 and 21.
Percentage change of body weight = (Weight at day 21 - Weight at day 1)/ Weight
at day 1 × 100.
Animals from the above-mentioned groups were anaesthetized by diethyl
ether inhalation then sacrificed. Blood samples were collected for biochemical
assay of serum total cholesterol, triglycerides, blood glucose and WBCs. Brain
weight was measured then the right cerebral hemispheres were used to measure
brain AChE activity, MDA levels, SOD and CAT activity levels. The left
hemispheres were dissected to reach the hippocampus. The hippocampus of each
animal was carefully dissected for histological paraffin sections to undergo light
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microscopic study; histologically by hematoxylin and eosin for routine study and
toluidine blue stain for detection of Nissl’s granules. Moreover,
immunohistochemical study was performed to detect GFAP, a marker of
astrocytes, P53, a marker of apoptosis, and MBP, a marker of myelination.
The morphometric measurements were done by the Image J analyzer for
calculation of the thickness of the pyramidal cell layer, the number of pyramidal
cells, color intensity of Nissl’s granules, the number of GFAP positive cells, the
number of P53 positive cells and the area percentage of MBP positive expression.
Statistical analysis was done for all calculated parameters.
7.2. Results:
Non-significant difference between group I and II in all examined
parameters.
Concerning behavioral tests, locomotor activity was significantly increased
in group III, decreased in group IV and V while sucrose preference index and
memory index were significantly decreased in group III, increased in group IV and
V. The increase in sucrose preference index and memory index in group V was
more significant than the increase in group IV.
Percentage change of body weight was significantly decreased in group III.
There was a significant increase in percentage change of body weight in group IV
and V as compared with group III. However, the increase in group IV was more
significant than the increase in group V.
Biochemical results revealed that serum total cholesterol, triglycerides and
blood glucose levels were significantly increased while WBCs count was
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significantly decreased in group IV. Brain AChE activity and MDA levels were
significantly increased in group III, decreased in group IV and V. Brain SOD and
CAT activity levels were significantly decreased in group III, increased in group
IV and V. The decrease in brain MDA and increase in brain SOD and CAT activity
in group V was significant from group IV.
Brain weight was significantly decreased in group III, increased in group IV
and V.
Histological study of hippocampal sections from group III (Psychosis group)
showed loss of hippocampal tissue integrity with marked decrease in the thickness
and the number of pyramidal cells. Pyramidal cells were degenerated with
shrunken hyperchromatic nuclei and perinuclear halos. The neuropil of both
molecular and polymorphic layers showed many vacuolations, dilated congested
blood vessels and increased astrocyte number. There was a significant decrease of
Nissl’s granules content of the cytoplasm of the pyramidal cells as detected in
toluidine blue stained sections.
Immunohistochemical study of these animals revealed significant increase in
the number of GFAP immunoreactive astrocytes, P53 positive cells and decrease in
area percentage of MBP positive expression, as confirmed statistically.
Animals from group IV (Psychosis group treated with clozapine) showed
significant amelioration of the pathological changes of the hippocampus.
Compared with the psychosis group, the pyramidal layer showed apparent increase
in the thickness and the number of cells. Most pyramidal cells appeared normal
with basophilic cytoplasm, large vesicular nuclei and prominent nucleoli but few
cells appeared degenerated with hyperchromatic nuclei and perinuclear halos.
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Other cells were swollen and lost their nuclei. Few congested blood vessels and
few astrocytes were observed in the neuropil of both molecular and polymorphic
layers. There was a significant increase of Nissl’s granules content of the
pyramidal cells as detected in toluidine blue stained sections.
Immunohistochemical study of these animals revealed a significant decrease
in the number of GFAP immunoreactive astrocytes, P53 positive cells and increase
in area percentage of MBP positive expression as compared with the psychosis
group.
Animals from group V (Psychosis group treated with AFA extract) showed
significant amelioration of the pathological changes of the hippocampus. The
pyramidal layer showed apparent increase in the thickness and the number of cells.
Many pyramidal cells appeared normal with basophilic cytoplasm, large vesicular
nuclei and prominent nucleoli but some cells were degenerated with
hyperchromatic nuclei and perinuclear halos. Few swollen cells losing their nuclei
were observed. The neuropil of both molecular and polymorphic layers contained
few vacuolations, congested blood vessels and astrocytes. There was a significant
increase of Nissl’s granules content of the pyramidal cells as detected in toluidine
blue stained sections.
Immunohistochemical study of these animals revealed significant decrease
in the number of GFAP immunoreactive astrocytes, P53 positive cells and increase
in area percentage of MBP positive expression as compared with the psychosis
group.
7.3. Conclusion:
from the present study, it is concluded that:
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1- Subanesthetic ketamine administration induces psychosis with degenerative
changes in the hippocampus due to oxidative stress, cholinergic dysfunction,
gliosis, upregulation of P53 protein and decreased myelination.
2- Clozapine administration as a treatment of psychosis improves behavior and
degenerative changes of the hippocampus but induces major side effects including
metabolic syndrome and agranulocytosis.
3- AFA extract improves psychotic behavior, ameliorates degenerative changes in
the hippocampus through decreasing oxidative stress, reducing
acetylcholinesterase activity, decreasing gliosis, downregulating P53 protein and
enhancing remyelination without causing the major side effects of clozapine.
7.4. Recommendations:
• It is useful to prescribe AFA extract for the treatment of psychosis.
• Furthermore, studies are needed to evaluate the role of AFA extract on
psychosis in different doses and periods of treatment.
• A combination of clozapine and AFA extract could be studied to gain all
benefits of both modalities in the treatment of resistant cases.