الفهرس 
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Abstract
A window of hope is opened for schizophrenic patients through the utilization and emersion of omega 3 fatty acids in prevention and treatment protocols. These fatty acids are major structural components of the phospholipid membrane. Interestingly, in humans, omega-3 fatty acids are required to maintain under normal conditions, cell membranes, brain function and the transmission of nerve impulses. Moreover, these fatty acids facilitate the transfer of atmospheric oxygen to the blood plasma, favor hemoglobin synthesis and contribute to cell division processes. Owing to variety of functions performed by omega-3 FA, it is suggested that several abnormalities of phospholipid structure of neuronal membranes affect mental health. As a result of previously mentioned facts, many inquiries about using omega-3 supplementation in schizophrenic patients can produce significant improvement in symptoms of schizophrenia. The pathophysiology of schizophrenia can partially clarify the use of omega-3 FA in prevention and treatment of schizophrenia. It is stated that stress due to the accumulation of reactive oxygen species (ROS) is incriminated in pathophysiology of the disease. The presence of oxidative stress documented by increased levels of ROS, reactive nitrogen species (RNS) or by an imbalance in the activity of endogenous antioxidant systems (antioxidant enzymes). So as a consequence to the increase in ROS and the failure of the endogenous antioxidant system, a damage to DNA, proteins and membrane lipids can occur.
In the present study the cytosar and ketamine rat models of schizophrenia were used to examine the prophylactic as well as the therapeutic effect of omega-3 when used alone or in combination with haloperidol and quetiapine on neurodevelopmental schizophrenia (cytosar induced schizophrenia) and pharmacological induced schizophrenia (ketamine induced schizophrenia) in rats.
The study was carried out on approximately 20 nulliparous albino Wistar female rats, 8- to 12 weeks old 250-280 g. Pregnant dams had been assigned into ﴾A﴿ and ﴾B﴿ groups. group (A) consisted of 7 pregnant rats that constituted the animal pool for neurodevelopmental model of schizophrenia. Whereas, group (B) consisted of 6 pregnant rats that represented the animal pool for ketamine induced schizophrenia model.
group (A): (Cytosar induced schizophrenia)
It consisted of six pregnant rats that were injected with cytosar (30 mg ⁄ kg, intarperitoneal (i.p.) in phosphate buffered saline, PBS,) on the morning of embryonic day 19.5 (E 19.5) and E20.5. Pups were weaned at postnatal day (PND) 21 and male offspring were grouped, housed. The seventh pregnant rat received nothing until completion of gestation and her male offspring served as control group. (200)
Male off springs 0f group A were divided into:
1. group Ι (control group):
• Consisted of 10 male rats which were off-springs the 7thpregnant rat:
• Animals received 0.2 ml saline by oral gavage for 7 months.
2. group ΙI (Omega-3 FA prophylactic group):
• Consisted of 10 male rats which were off-springs of one of cytosar treated pregnant dams:
• This dam received omega-3 FA at a dose of 0.8 g/kg diluted in sesame oil which was given by orogastric gavage (oral route) once daily, started on day one of pregnancy till term i.e. from 21-24 days/.
3. group ΙΙI (cytosar induced schizophrenia non treated group):
• Consisted of 10 male rats which were off-springs of one of cytosar treated pregnant dams:
• Animals received 0.2 ml saline by oral gavage for 7 months.
• This group sereved as a control for group ΙV, group V, group VI and group VII.
4. group ΙV (Cytosar induced schizophrenia treated with haloperidol) :
• Consisted of 10 male rats which were off-springs of one of cytosar treated pregnant dams:
• These rats received intramuscular (i.m.) injection of 38 mg/kg/month of haloperidol-depo diluted in saline for about 6 months. Rats were lightly anesthetized with isoflurane and half of the calculated drug volume was injected in each hind leg. Dosages were given once per month (between one and 4 pm) for 6 months, started on post natal day (PND) 41.
5. group V (Cytosar induced schizophrenia treated with quetiapine) :
• Consisted of 10 male rats which were off-springs of one of cytosar treated pregnant rats:
• These rats received quetiapine 10 mg/kg diluted in 2% gum acacia by orogastric gavage once a day for 21 days, started on post natal day (PND) 41.
6. group VI: (Cytosar induced schizophrenia treated with haloperidol and omega 3 FA):
• Consisted of 10 male rats which were off-springs of one of cytosar treated pregnant rats:
• These rats received i.m. injection of 38 mg/kg/month of haloperidol-depo diluted in normal saline for about 6 months together with omega-3 FA 0.4 g/kg/day orally diluted in sesame oil by gastric gavage once daily for 6 months.
7. group VII: (Cytosar induced schizophrenia treated with quetiapine and omega- 3 FA):
• Consisted of 10 male rats which were off-springs of one of cytosar treated pregnant dams:
• These rats received quetiapine 10 mg/kg diluted in 2% gum acacia by orogastric gavage once a day for 21 days, together with omega-3 FA 0.4 g/kg/day diluted in sesame oil orally by gastric gavage once daily for 21 days. The treatment started on PND 41.
group (B): (Ketamine induced schizophrenia) was divided into 2 subgroups:
1. group (B-1) Omega-3 FA prophylactic group:
• This group consisted of a pregnant dam that was left to complete pregnancy. 10 male off springs received at PND 30 omega-3 FA at a dose of 0.8 g/kg diluted in sesame oil, given by orogastric gavage (oral route) once daily for 21 days. Then Ketamine was injected at a dose of 25 mg/kg i.p. diluted in normal saline for 7 days.
2. group (B-2) kitamine induced model of schizophrenia:
• This group consisted of five pregnant dams that were left to complete pregnancy. Fifty male off springs received ketamine at a dose of 30 mg/kg i.p. diluted in normal saline for five consecutive days. The ketamine was injected on PND 11.
• Animals were divided into:
1. group (B-2a) kitamine induced model of schizophrenia untreated:
• Consisted of 10 male rats with ketamine induced schizophrenia.
• Animals received 0.2 ml saline by oral gavage.
• This group served as a control for group B-2b, B-2c, group B-2d and group B-2e.
2. group (B-2b) kitamine induced model of schizophrenia treated with haloperidol:
• Consisted of 10 male rats with ketamine induced schizophrenia.
• These rats received i.m. injection 38 mg/kg/month of haloperidol-depo diluted in normal saline for about 6 months.
3. group (B-2c) kitamine induced model of schizophrenia treated with quetiapine:
• Consisted of 10 male rats with ketamine induced schizophrenia.
• These rats received quetiapine 10 mg/kg diluted in 2% gum acacia by orogastric gavage once a day for 21 days.
4. group (B-2d) kitamine induced model of schizophrenia treated with haloperidol and omega-3 FA:
• Consisted of 10 male rats with ketamine induced schizophrenia.
• These rats received i.m. injection 38 mg/kg/month of haloperidol-depodiluted in normal saline for about 6 months together with omega-3 FA 0.4 g/kg/day diluted in sesame oil orally by gastric gavage once daily for 6 months.
5. group (B-2e) kitamine induced model of schizophrenia treated with quetiapine and omega-3 FA:
• Consisted of 10 male rats: with ketamine induced schizophrenia.
• These rats received quetiapine 10 mg/kg diluted in 2% gum acacia by orogastric gavage once a day for 21 days, together with omega-3 FA 0.4 g/kg/day diluted in sesame oil orally by gastric gavage once daily for 21 days. The treatment started on PND 41.
Base-line behavioral tests were performed for all animals after they were numbered. These tests included the following:
1. Social (play) behavior. (Demonstrated the deficits of social functioning) on PND 22.
2. Spontaneous activity in an open field (OFT). (Demonstrated the hyperlocomotion) on PND 35. 3.
Morris Water Maze (MWM). (Demonstrated spatial learning and memory which were features of cognition) on PND 40.
Then the animals were sacrificed and blood was collected from the aortic trunk for separation of serum. The serum was separated by centrifugation of the blood samples. Then sera were aliquotted into clean eppendorf tubes and stored at ≤ -200C. The frontal cortex was removed on ice, weighed and homogenized. The supernatants were separated by medical centrifuge , aliquotted and stored in two eppendorf at -20ºC waiting for the following parameters to be measured:
a. Dopamine level by ELISA
b. EGF level by ELISA
Results:
The results of the present study were evaluated in two phases:
Phase I
Paired-t-test was used in current study to analyze the results obtained from control group I (at beginning vs. at end of experiment), omega-3 FA prophylactic group II (at beginning vs. at end of experiment), omega-3 FA prophylactic group B-1 (at beginning vs. at end of experiment), cytosar induced schizophrenia non-treated group III (at beginning vs. at end of experiment) and kitamine induced schizophrenia non-treated group B-2a (at beginning vs. at end of experiment).
The following results were obtained:
• The control group I showed non significant change in social play test parameters (pinning, mounting/crawling, grooming, approaching/ following being alone with distance between pair≥10cm and time spent in the same place), number of crossings, immobility time and frequency of rearing in OFT and time to reach the platform in MWM test.
• Omega-3 FA prophylactic group II showed non significant change in social play test parameters, number of crossings, immobility time and frequency of rearing in OFT and time to reach the platform in MWM test.
• Omega-3 FA prophylactic group B-1 showed non significant change in social play test parameters, number of crossings, immobility time and frequency of rearing in OFT and time to reach the platform in MWM test.
• Cytosar induced schizophrenia non-treated group III showed significant decrease in frequency of pinning, mounting/ crawling, grooming, approaching/ following, significant increase in time rat being alone with distance between pair≥10 cm and time spent in the same place in social play test. This group showed significant increase in number of crossings, significant decrease in immobility time and significant increase in frequency of rearing in OFT. Also this group showed significant increase in time to reach the platform in MWM test.
• Ketamine induced schizophrenia non-treated group B-2a showed significant decrease in frequency of pinning, mounting/ crawling, grooming, approaching/ following, significant increase in time rat being alone with distance between pair≥10 cm and time spent in the same place in social play test. This group showed significant increase in number of crossings, significant decrease in immobility time and significant increase in frequency of rearing in OFT. In addition, this group showed significant increase in time to reach the platform in MWM test.
Phase II.
Anova test was used to evaluate results of behavioral tests, brain weigh, serum and frontal cortex dopamine levels and serum and frontal cortex EGF levels.
The following results were obtained:
I. Social play test:
• Frequency of pinning: revealed a significant decrease frequency of pinning in rats that were subjected to induction of schizophrenia compared to the control group.
• Mounting/ crawling: revealed significant decrease in frequency of mounting and crawling in rats that were subjected to induction of schizophrenia compared to control group.
• Grooming: revealed significant decrease in frequency of grooming in rats that were subjected to induction of schizophrenia compared to control group.
• Approaching/ following: revealed significant decrease in frequency of approaching and following in rats that were subjected to induction of schizophrenia compared to control group.
• Time rat being alone with distance between pair≥10 cm: revealed significant increase time rat being alone with distance between pair≥10cm in rats that were subjected to induction of schizophrenia compared to control group.
• Time spent in the same place: revealed significant increase in time spent in the same place in rats that were subjected to induction of schizophrenia compared to control group.
II. OFT:
• Number of crossings: revealed significant increase in number of crossingsin rats that were subjected to induction of schizophrenia compared to control group.
• Immobility time: revealed significant decrease in immobility time in rats that were subjected to induction of schizophrenia compared to control group.
• Frequency of rearing: revealed significant increase in frequency of rearing in rats that were subjected to induction of schizophrenia compared to control group.
III. MWM test:
• Time to reach the platform: revealed significant increase in time to reach the platform in rats that were subjected to induction of schizophrenia compared to control group.
VI. Brain weight: revealed insignificant change in weight of brain in rats that were subjected to induction of schizophrenia compared to control group.
V. Dopamine level:
• Frontal cortex dopamine level: revealed significant increase in frontal cortex dopamine level in rats that were subjected to induction of schizophrenia compared to control group.
• Serum dopamine level: revealed significant increase in serum dopamine level in rats that were subjected to induction of schizophrenia compared to control group.
VI. EGF level:
• Frontal cortex EGF level: revealed insignificant decrease in frontal cortex EGF level in rats that were subjected to induction of schizophrenia compared to control group.
• Serum EGF level: revealed significant decrease in serum EGF level in rats that were subjected to induction of schizophrenia compared to control group.
When the effects of the studied drugs were assessed on the different studied parameters, it was apparent that:
• Haloperidol produced: an improvement in all the previously mentioned parameters except tissue and serum EGF levels when evaluated after induction of schizophrenia, as compared to schizophrenia induced non-treated group and control group. The improvement in the parameters was in the form of: significant increase in frequency of pinning, mounting and crawling, grooming and approaching and following with decreased time rat being alone with distance between pairs ≥10 cm and time spent in same place in social play test. There was significant decrease in number of crossings, significant increase in immobility time and significant decrease in frequency of rearing in OFT. There was also significant decrease in time to reach the platform in MWM test. Regarding biochemical deficit, haloperidol treatment had no effect on brain weight, haloperidol decreased level of both tissue and serum. Haloperidol was able to decrease tissue dopamine level significantly in ketamine model of schizophrenia and was able to decrease serum dopamine level significantly in cytosar model of schizophrenia. On the other hand, haloperidol had no effect on tissue and serum EGF level.
• Quetiapine produced: an improvement in all the previously mentioned parameters when evaluated after induction of schizophrenia, as compared to schizophrenia induced non-treated group and control group. The improvement in the parameters was in the form of: significant increase in frequency of pinning, mounting and crawling, grooming and approaching and following with decreased time rat being alone with distance between pairs ≥10 cm and time spent in same place in social play test. There was significant decrease in number of crossings, significant increase in immobility time and significant decrease in frequency of rearing in OFT. There was also significant decrease in time to reach the platform in MWM test. Regarding biochemical deficit, quetiapine treatment had no effect on brain weight, quetiapine decreased level of both tissue and serum dopamine and increased both tissue and serum EGF level.
• Haloperidol combined with omega-3 FA produced: an improvement in all the previously mentioned parameters when evaluated after induction induction of schizophrenia, as compared to schizophrenia induced non-treated group and control group. The improvement in the parameters was in the form of: significant increase in frequency of pinning, mounting and crawling, grooming and approaching and following with decreased time rat being alone with distance between pairs ≥10 cm and time spent in same place in social play test. There was significant decrease in number of crossings, significant increase in immobility time and significant decrease in frequency of rearing in OFT. There was also significant decrease in time to reach the platform in MWM test. Regarding biochemical deficit, haloperidol and omega-3 treatment had no effect on brain weight, haloperidol combined with omega-3 decreased level of both tissue and serum dopamine. Interestingly, combining omega-3 with haloperidol led to increase in both tissue and serum EGF level that haloperidol alone had no effect on them.
• Quetiapine combined with omega-3 FA produced: an improvement in all the previously mentioned parameters when evaluated after induction induction of schizophrenia, as compared to schizophrenia induced non-treated group and control group. The improvement in the parameters was in the form of: significant increase in frequency of pinning, mounting and crawling, grooming and approaching and following with decreased time rat being alone with distance between pairs ≥10 cm and time spent in same place in social play test almost with no statistical difference when compared to control group. There was significant decrease in number of crossings, significant increase in immobility time and significant decrease in frequency of rearing in OFT with no statistical difference when compared to control group. There was also significant decrease in time to reach the platform in MWM test to the extent that rats reached the platform during almost the same time as control rats. Regarding biochemical deficit, quetiapine and omega-3 treatment had no effect on brain weight, quetiapine combined with omega-3 caused decrease in level of both tissue and serum dopamine to around same range as control group . Interestingly, combining omega-3 quetiapine led to increase in both tissue and serum EGF level, with tissue level even higher than control group and serum level almost the same range as control group. So this group showed the highest significance when compared to other groups as this combination improved all parameters that were disrupted by induction of schizophrenia.
In summary, the results of the present study indicated that the antipsychotic-like action of omega-3 FA is synergistic with that of first and second generation anyipsychotics. Omega-3 FA add-on therapy may be used as a method to augment the limited therapeutic effects of available antipsychotic drugs.