الفهرس 
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Abstract
Alzheimer’s disease (AD) is the most common cause of age-related neurodegeneration and cognitive decline. AD more commonly occurs in females than in males, so it is necessary to consider new treatments specifically targeting this population. Alzheimer disease pathology starts early before symptoms appear so that the study aims to protect next generation against pathological hallmarks induced by AD. The work was designed to use bone marrow derived mesenchymal stem cells and gamma secretase inhibitor GSI-953 (begacestat) against AD induced by single intracerebroventricular injection of amyloid beta 25-35 to protect the newborns from cognitive impairment, neurodegenerative cytokines, histopathological degenerations.
The study was carried out on 50 mature female rats and 30 adult mature male used for mating. The female rats were divided into five groups as following
Group1: Ten adult female rats subjected to intacerebroventricular injection with 10µl of isotonic saline before pregnancy.
Group2: before pregnancy 30 adult female rats subjected to intracerebroventricular injection with amyloid beta 25-35
Group3: adult female rats intravenously injected with DMEM starting from pregnancy
Adult female rats subjected to i.c.v injection with Aβ were divided into
Group4: include ten adult female rats treated with mesenchymal stem cells during pregnancy one time /week
Group5: Which treated with gamma secretase inhibitor (begacestat) 2.5mg/kg.b.w.
The remaining ten animals acted as AD group (negative control). These groups were subjected to behavioral tests (novel object recognition and Y-maze test) before pregnancy for Alzheimer disease establishment.
The mature adult female rats and their newborns were sacrificed at postnatal day 7, 14 and 21. Blood samples were collected and allowed to clot then centrifuge to separate serum which used for biochemical analysis. Brain samples (cortex and hippocampus) were isolated to be used in gene quantification, western blot analysis, histological analysis and immunohistochemistry analysis.
Behavioral tests including NOR and Y-maze test that were carried out on AD group before pregnancy showed marked short memory impairment in the Y-maze test as the time spent in the new arm was decreased compared with that of rats receiving saline and novel object recognition test showed defect in reference memory.
Histological study of newborns cerebral cortex showed marked reduction in outer molecular layer thickness and increase in number of degenerated neurons alongside all the tested ages in AD group while MSCs and GSI-953 treatment restored the thickness of outer molecular layer and degenerative cells to the normal range. In addition newborns hippocampus dams subjected to Aβ 25-35 injection showed reduction in thickness and number of pyramidal cells forming cornu ammonis (CA1 and CA2) and showed shrunked cells with pyknotic nuclei, moreover the dentate gryus showed distortion of subgranular zone and separation of this layer from granular layer, also granular layer showed degenerative granular cells. On the other hand newborns hippocampus of AD dams treated with MSCs and GSI-953 showed normal histological structure.
Congo red staining of dams’ hippocampus and their newborns showed Aβ deposition in AD group, while saline injected dams treated with MSCs and GSI-953 and their newborns showed no amyloid deposition.
Microglial cells detection using iba-1 antibody in newborns cortex and hippocampus of AD group were activated and indicated by marked increase in cell soma size, decrease in processes length and decrease in number of processes. On the other side MSCs and GSI-953 treatment for AD showed normal microglial cells.
Concerning neuroinflammatory cytokines including IL-1β and TNF-α measurements, Aβ25-35 injection into dams caused significant elevation in newborns serum level of IL-1β and TNF-α, while ad treatment using MSCs and GSI-953 reduced cytokines elevation to reach to normal levels. Serum level of brain derived neurotrophic factor and glycogen-s-kinase-3β in AD group was significantly reduced BDNF concentration but significantly elevated GSK-3β in comparing with saline injected group. MSCs and GSI-953 treatment for AD markedly increased BDNF and reduced GSK-3β relative to AD group.
Quantitative real time polymerase chain reaction (qRTPCR) was used to quantify gene expression of BDNF, caspase-3, NF-κB, TGF-3β and TNFR in cerebral cortex and hippocampus of newborns illustrating up regulation of expression of caspase-3, NF-κB, TGF-3β and TNFR while showed significant down regulation of BDNF in AD group, while using MSCs and GSI-953 treatment significantly reversed gene expression of all tested genes.
Protein concentration of amyloid precursor protein (APP) and hyperphosphorylated tau protein (p-tau) was markedly increased in cortex and hippocampus of newborns of AD group while MSCs and GSI-953 treatment significant reduced concentration of APP and P-tau.
In conclusion, the cognitive deficits induced by the i.c.v. injections of Aβ25–35 was improved by treating adult female rats with i.v. injections of BM-MSCs or the oral administration of the γ-secretase inhibitor GSI-953 (Begacestat). Additionally, prenatal BM-MSC and GSI-953 treatments protected against hippocampus and cerebral cortex maldevelopment in neonates. These protective effects in the neonatal rat hippocampus were linked to a reduced expression of APP and hyperphosphorylated tau protein, the suppression of microglial activation, the decreased expression of proinflammatory cytokines (IL-1β and TNF-α), and the down regulation of brain levels of caspase-3, NF- κB, and TGF-β. Moreover, the down-regulation of BDNF expression and increased GSK-3β activity induced by Aβ 25–35 were reversed by BM-MSC and GSI-953 treatments. Therefore, these therapies seem to prevent Aβ25–35-induced neurodegeneration and cognitive impairments by preventing the formation of Aβ plaque and NFTs as well as the resulting neuroinflammation. However, additional investigations are needed to determine the precise mechanism responsible for the anti-inflammatory effect of GSI-953 and BM-MSCs.