الفهرس 
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Abstract
The current study was undertaken to explore the possible therapeutic role of single intravenous dose of bone marrow or adipose tissue-derived mesenchymal stem cells in management of Alzheimer’s and Parkinson’s diseases in experimental rat models. Also, this study was extended to compare between the roles of stem cells therapy with the conventional therapies of these neurodegenerative diseases.
For mesenchymal stem cells isolation and preparation; BM-MSCs were harvested from bone marrow of femoral bones of male Sprague-Dawley rats, while, ADMSCs were excised from adipose tissue of both the omentum and the inguinal fat pad of male Sprague-Dawley rats. Then, these cells were grown and propagated in culture. To affirm that the cells in culture are MSCs, they were characterized morphologically by inverted microscope examination. Also, detection of CD14, CD29, CD34, CD44, CD45 and CD106 gene expressions was done by PCR technique. Additionally, multilineage potential was confirmed via in vitro differentiation to adipocytes, chondrocytes and osteocytes.
The biological experimentation was carried out on two models of neurodegenaerative diseases. The first one was Alzheimer’s disease model and the second was Parkinson’s disease model. For AD model, 48 adult female Sprague-Dawley rats were classified into 6 groups (8 rats/group). The first group was normal control group (Negative control group). The groups from second to sixth were orally administered with AlCl3 in a dose of 17 mg/kg b. wt., daily for 75 days for induction of AD disease. Thereafter, the second group was left untreated for 4 months; the third group was treated orally with rivastigmine in a dose of 0.3 mg/kg b. wt. daily for 4 months. While, the fourth group was treated intraperitoneally with cerebrolysin in a dose of 1.08 ml/kg b. wt. that is equivalent to the recommended human dose; 60 mL, 5 days/week for one month and thereafter two times per week for 3 months. The fifth and sixth groups were infused intravenously with a single dose (3 x 106 cells/rat) of BM-MSCs and ADMSCs respectively.
To evaluate the therapeutic efficacy of MSCs either derived from bone marrow or adipose tissue against AD, the ability of the injected MSCs to migrate to the injured female brains was confirmed by the PCR detection of brain Y-chromosome (SRY) gene. Serum TGF-β1, MCP-1 and BDNF levels were assayed by ELISA technique. While, brain ChAT and survivin expressions were determined by immunohistochemical procedure. Brain nestin and SELADIN-1 gene expressions were detected by sqRT-PCR. Moreover, histopathological investigation of brain tissues was done.
The data of the current study revealed that Y-chromosome gene (SRY) was present in the brain tissues of the female AD rats treated with BM-MSCs or ADMSCs indicating that the delivered undifferentiated MSCs were able to home at the injured female brains. It was clearly observed from the biochemical analyses that aluminum chloride administration caused significant increase in serum TGF-β1 and MCP-1 levels associated with significant decrease in serum BDNF level. Also, AlCl3 produced significant decrease in the number of positive cells for ChAT and survivin expressions as well as gene expression levels of brain nestin and SELADIN-1. Moreover, the brain sections of untreated AD group showed various sizes of amyloid plaques formation in the hippocampus associated with oedema, hypoplasia, congested blood capillaries and neuronal degeneration. In addition to, focal gliosis in the cerebrum. Also, there were focal hemorrhage and congestion in the blood capillaries, as well as encephalomalacia of the matrix in the medulla oblongata. Moreover, there were focal astrocytosis, neuronal degeneration with satellatosis and congestion in the blood vessels and capillaries in the striatum.
On the other hand, all treatments produced significant decrease in serum TGF-β1 and MCP-1 levels in concomitant with significant increase in serum BDNF level. Additionally, they caused significant increase in the number of positive cells for ChAT and survivin expressions as well as nestin and SELADIN-1 gene expression levels. Moreover, the brain sections of AD group treated with rivastigmine for 4 months showed intact histological structure of the hippocampus and cerebellum. Meanwhile, the brain sections of AD group treated with cerebrolysin for 4 months showed intact histological structure of the hippocampus. Also, the brain sections of AD group treated with BM-MSCs showed after four months from treatment intact histological structure of the hippocampus. Finally, the brain sections of AD group treated with ADMSCs showed after four months from treatment intact histological structure of the hippocampus and congestion in the cerebral blood vessels.
For PD model, 48 adult female Sprague-Dawley rats were surgically ovariectomized and after one month from ovariectomy they were classified into 6 groups (8 rats/group). The first group was untreated ovariectomized control group. The groups from second to sixth were subcutaneously injected with rotenone in a dose of 12 mg/kg b. wt. daily for 14 days for induction of PD. Then, the second group was left untreated for 4 months; while the third group was treated orally with sinemet in a dose of 20 mg/kg b. wt. daily for 4 months. The fourth group was treated intraperitoneally with cerebrolysin in a dose of 1.08 ml/kg b. wt. that is equivalent to the recommended human dose; 60 mL, 5 days/week for one month and thereafter two times per week for 3 months. The fifth and sixth groups were intravenously infused with a single dose (3 x 106 cells/rat) of BM-MSCs and ADMSCs respectively.
To evaluate the therapeutic efficacy of BM-MSCs and ADMSCs against PD, the capability of the injected MSCs to home to the injured female brains was confirmed via PCR detection of brain SRY gene. Serum TGF-β1, MCP-1 and BDNF levels were assayed by ELISA technique. Brain dopamine level was assayed fluorometrically while, brain TH and nestin genes expression were detected by sqRT-PCR. Brain survivin expression was determined by immunohistochemical procedure. Moreover, histopathological investigation of brain tissues was done.
The results of the present study demonstrated that SRY gene was present in the brain tissues of female PD rats treated with BM-MSCs and ADMSCs revealing that the transplanted undifferentiated MSCs were able to home at the injured female brains. Moreover, the current data indicated that rotenone administration produced significant increase in serum TGF-β1 and MCP-1 levels associated with significant decrease in serum BDNF, brain dopamine and brain TH as well as nestin gene expression levels. Also, it caused insignificant decrease in the number of positive cells for survivin expression. Moreover, the brain sections of untreated PD group showed congestion in the blood vessels and capillaries in the striatum. Hyalinization and plaques formation in the matrix of the striatum were observed indicating the presence of neurodegeneration. Also, there were focal areas of encephalomalacia with gliosis associated with focal neuronal degeneration in the cerebrum. In addition to, diffuse gliosis in between the degenerated cells of the hippocampus. Also, oedema and congestion were noticed in the hippocampus. Meanwhile, the medulla oblongata had focal haemorrhage and neuronal degeneration with lose of Nissl granules of the neuron.
In contrast, all treatments produced significant decrease in serum TGF-β1 and MCP-1 levels in concomitant with significant increase in serum BDNF, brain dopamine and brain TH as well as nestin gene expression levels. Also, these treatments caused insignificant increase in the number of positive cells for brain survivin expression. The brain sections of the PD group treated with sinemet showed oedema in the hippocampus and congestion in the blood vessels in the striatum. Meanwhile, the PD group treated with cerebrolysin showed congestion in the hippocampus and striatum. The brain sections of PD group treated with BM-MSCs showed intact histological structure of the hippocampus and striatum. Finally, the PD group treated with ADMSCs showed intact histological structure of hippocampus with mild congestion in the blood vessels in the striatum.