الفهرس 
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Abstract
This study will be designed to evaluate and compare the possible therapeutic effect of both single and multiple injections of adipose-derived mesenchymal stem cells (AMSCs) and ascorbic acid on the cerebral cortex in induced type1DM in adult male albino rats.
Drugs and chemicals used included streptozotocin (STZ), which was obtained from Sigma Corporation in the form of powder (1g vial). The required dose was weighed using a digital scale and dissolved in citrate buffer. Ascorbic Acid (AA): was supplied by Development of Chemical Industries (Giza, Egypt) in a 1g Vitacid C effervescent tablet form. The required doses were weighed using a digital scale and dissolved in water.
Forty four adult male albino rats each weighing 200g were used in the present study. The animals were housed in the Animal House of Physiology Department, Faculty of Medicine, Cairo University under good hygienic conditions of air, temperature, fed ad libitum and allowed for free water supply. The animals were treated according to the ethical guidelines of Cairo University.
The animals were divided into the following groups, kept in separate cages.
Donor Group: 4 rats from which AMSCs were isolated
group I (Control group): Five rats subdivided into:
The first rat received a single IP injection of 0.5 ml citrate buffer and left for 8 weeks without therapy.
The second rat received a single IP injection of 0.5 ml citrate buffer. 4 weeks later, it was given 1ml of PBS IV via the tail vein once (on start of 5th week) and left for another 4 weeks without therapy.
The third rat received citrate buffer and PBS as for the second rat together with daily oral AA dissolved in 0.5 ml water using a syringe without a needle for 4 weeks.
The fourth rat received citrate buffer and PBS as for the 2nd and 3rd rats, via the tail vein but four times (on start of weeks 5, 6, 7, 8).
The fifth rat received citrate buffer and PBS as for the fourth rat together with daily oral AA dissolved in 0.5 ml water using a syringe without a needle for 4 weeks.
All control rats are sacrificed 8 weeks from the start of the experiment.
group II (Diabetic Group): It included 7 rats. Diabetes was induced by a single IP injection of STZ at a dose of 50 mg/kg body weight dissolved in 0.5 ml citrate buffer for each rat. Three days following STZ injection, diabetes was confirmed by measuring the blood glucose level. The animals were considered diabetic if their blood glucose level was higher than 200 mg/dl. The rats were left for 8 weeks without therapy.
group III (Diabetic treated with AMSCs single injection Group): It included 7 rats, diabetes was induced and confirmed as in group II. Four weeks following STZ injection (on start of 5th week), 1x106 of cultured and labeled rat AMSCs, suspended in 1ml PBS, were injected once into the tail vein. Stem cell isolation, culture, labeling and phenotyping were performed. AMSCs isolation was performed from the rat abdomen. The rats were left for another 4 weeks without therapy.
group IV (Diabetic treated with AMSCs single injection and AA Group): 7 rats, diabetes was induced and confirmed as in groups II and III. Four weeks following STZ injection, 1ml of cultured and labeled AMSCs were isolated, suspended and injected as in group III. Combined daily oral administration of 500mg/Kg of AA dissolved in 0.5 ml of water was performed for each of the 7 rats for another 4 weeks using a syringe without a needle.
group V (Diabetic treated with AMSCs multiple injection Group): 7 rats, diabetes was induced and confirmed as in group II, III and IV. Four weeks following STZ injection, 1ml of cultured and labeled rat AMSCs, were isolated and injected into the tail vein as in group III and IV, but four times (on start of weeks 5, 6, 7, 8).
group VI ((Diabetic treated with AMSCs multiple injection and AA Group): 7 rats diabetes was induced and confirmed as in groups II, III, IV and V. Four weeks following STZ injection, 1ml of cultured and labeled AMSCs were isolated and injected as in group V. Combined daily oral administration of AA was performed for each of the 7 rats for another 4 weeks using a syringe without a needle.
Animals of groups II, III, IV,V and VI were sacrificed 8 weeks from the start of the experiment. Before sacrifice tail vein blood samples were collected at the end of the 8th week for blood glucose level estimation. The animals belonging to control and corresponding experimental groups were sacrificed by cervical dislocation under anaesthesia at the end of the experiment. The skull was opened by bone cutter at the temporal regions. The brain was exposed and immediately dissected out and placed in 10 % formol saline for 48 hours at RT. Specimens were embedded into paraffin wax. Coronal 5μm thick sections were prepared and sections were subjected to the following studies:
A) Serological Study: Measurement of fasting blood glucose level.
B) Histological Study:
Hematoxylin and eosin.
C) Histochemical Study:
1. Congo red (CR) stain to demonstrate the amyloid plaques.
2. Prussian blue (Pb) stain to demonstrate the injected ADSCs labeled with iron oxide.
D) Immunohistochemical Study:
1. Synaptophysin immunostaining for neuron structural function organization.
2. CD44 immunostaining, for detecting endogenous and exogenous undifferentiated MSCs.
E) Morphometric Study:
1. Area of glial cells. 2. Area of deformed neurons.
3. Area of CR +ve masses. 4. Count of Pb+ve cells.
5. Area% of synaptophysin +v IE.
6. Count of CD44 +ve cells.
F) Statistical Analysis.
The mean blood glucose values indicated a significant increase in group II compared to control and other experimental groups, in group III compared to control and groups IV, V and VI, in addition to groups IV and V compared to control and group VI.
group II demonstrated multiple large acidophilic masses surrounded by clear areas and exhibiting multiple dark nuclei in the EP layer, some deformed neurons, multiple neurons with acidophilic cytoplasm, rarified neuropil and obvious proliferation of glial cells exhibiting elongated nuclei.
group III recruited small masses with dark nuclei and surrounded by clear areas in the EP layer, congested vessels, some neurons with acidophilic cytoplasm, some deformed neurons and rarified neuropil. Sections of groups IV and V showed few very small masses surrounded by clear areas in the EP layer, few deformed neurons, few neurons with acidophilic cytoplasm surrounded by clear areas and focal rarefaction of neuropil. Sections of group VI revealed focal areas of rarified neuropil and few deformed neurons.
Control group showed dull red staining of the neurons as well as the neuropil in EP layer with CR. Multiple strongly +ve large and small masses were demonstrated among the neurons of EP layer in group II. group III revealed fewer strongly +ve and smaller masses in EP layer. Groups IV and V revealed few strongly +ve small masses in EP layer compared to groups II and III. group VI was comparable to control group. Groups I and II showed –ve Pb staining layer. group III recruited few Pb +ve spindle cells, group IV showed few Pb +ve spindle cells, group V showed multiple Pb +ve spindle cells and group VI showed multiple Pb +ve spindle cells among neurons in EP layer.
group I demonstrated +ve synaptophysin IE among multiple pyramidal neurons, group II showed minimal +ve IE among accidental pyramidal neurons, group III recruited +ve IE among few pyramidal neurons, groups IV and V showed +ve IE among some pyramidal neurons and group VI demonstrated +ve IE among multiple pyramidal neurons in the EP layer. Concerning CD44 IE, group II revealed few CD44 +ve spindle cells, group III showed some +ve cells and group IV demonstrated multiple +ve cells fused with few neurons, in blood vessels and in the neuropil of EP layer. group V demonstrated multiple +ve cells fused with some neurons and in group VI numerous +ve cells were seen fused with multiple neurons and in the neuropil of the EP layer.
The mean area of glial cells and that of deformed neurons showed a significant increase in group II compared to other groups and in group III compared to control, groups IV, V and VI, in groups IV and V compared to control and group VI. The mean area of CR +ve masses showed a significant increase in group II compared to other groups and in group III compared to control, groups IV and V. A significant decrease in the count of Pb +ve cells was recorded in group III compared to groups IV, V and VI. In addition a significant decrease was detected in group IV compared to groups V and VI, while in group V the significant decrease was proved compared to group VI. The mean area of synaptophysin +ve IE showed a significant decrease in group II compared to the other groups, in group III compared to groups I, V and VI, in group IV compared to groups I and VI and a significant decrease was evident in group V compared to groups I and VI. The mean count of CD44 +ve cells indicated a significant decrease in group II compared to the other groups, in group III compared to groups V and VI and in groups IV and V compared to group VI.
In conclusion, T1DM induced cerebral cortical inflammatory and degenerative morphological changes. AMSCs proved a definite therapeutic effect that was more noticeable in response to multiple injections. Combined AA and AMSCs therapy guaranteed the most remarkable effect, that can be related to activated migration and trans-differentiation.