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العنوان
Comparative Study between Bone Marrow Mesenchymal
Stem Cells and their Derived Exosomes on Neurological
Dysfunction in Experimentally Induced Alzheimer
Disease in Rat Model /
المؤلف
Elnahrawy, Esraa Ahmed Baioumy.
هيئة الاعداد
باحث / إسراء أحمد بيومى النحراوي
مشرف / جيهان محمود حامد
مشرف / وسام عزت مرسى
مشرف / منال سعيد عبد الحميد
تاريخ النشر
2023.
عدد الصفحات
370 P. :
اللغة
الإنجليزية
الدرجة
الدكتوراه
التخصص
علم وظائف الأعضاء (الطبية)
تاريخ الإجازة
1/1/2023
مكان الإجازة
جامعة عين شمس - كلية الطب - قسم الفسيولوجيا الطبية
الفهرس
Only 14 pages are availabe for public view

from 370

from 370

Abstract

The present study was planned to compare the effect of Bone Marrow mesenchymal stem cells and Bone Marrow Mesenchymal stem cells derived exosomes on cognitive functions (learning and memory) and locomotor function in lipopolysaccharide (LPS)-induced Alzheimer rat model.
This study was performed on 40 adult albino male rats over 5 weeks and were randomly allocated into four equal groups, 10 rats each:
group I: Control rat group (C): Rats in this group received intraperitoneal injection of 1ml 0.9% saline (the solvent for lipopolysaccharide {LPS})/rat for five consecutive days, two hours before the daily performance of the behavioral tests. On the 5th day, and after performing behavioral tests, rats received a single intravenous (I.V) injection of 1ml phosphate-buffered saline (PBS)/rat, the media for stem cells and exosomes transplantation.
group II: Experimentally induced Alzheimer group (Alz.): Rats in this group received intraperitoneal injection of 250 µg/kg LPS for five consecutive days according to Zhu et al., (2014) two hours before the daily performance of the behavioral tests. On the 5th day, and after performing behavioral tests, rats received a single intravenous injection of 1ml phosphate-buffered saline (PBS)/rat, the media for stem cells and exosomes transplantation.
group III: Mesenchymal stem cell transplanted Alzheimer group (Alz./MSCs): Rats in this group received intraperitoneal injection of 250 µg/kg LPS for five consecutive days according to Zhu et al., (2014), two hours before the daily performance of the behavioral tests. On the 5th day, and after performing behavioral tests, rats received single intravenous transplantation with PKH26 dye-labelled bone marrow mesenchymal stem cells in a dose of 1 x 106 cells/1ml PBS per rat, over 5 minutes, according to Bae et al., (2007); Park et al., (2013)a & Nasiri et al., (2019).
group IV: Mesenchymal stem cells derived exosomes transplanted Alzheimer group (Alz./exosomes): Rats in this group received intraperitoneal injection of 250 µg/kg LPS for five consecutive days according to Zhu et al., (2014) two hours before the daily performance of the behavioral tests. On the 5th day, and after performing behavioral tests, rats received single intravenous transplantation with 100 μg total protein of PKH26 dye-labeled mesenchymal stem cells derived exosomes precipitated in 1 ml PBS per rat, over 5 minutes, according to Zhang et al., (2015); Dabrowska et al., (2019) & Zhao et al., (2019)a.
Behavioral assessment (Morris Water Maze test, Passive Avoidance test, Pole test and RotaRod test) was done twice:
• The first assessment was done on the first 5 days of the experiment, concomitant to LPS injection.
• The second assessment was done in the last 5 days of the experiment, during the 4th week that followed PBS/ MSCs/exosomes injection.
Initial and final body weights were recorded, and percentage changes were calculated. On the day of sacrifice, retro-orbital blood samples and hippocampal tissues were collected for assessment of:
Æ Serum malondialdehyde (MDA)
Æ Serum superoxide dismutase (SOD).
Æ Hippocampal Neurotransmitter as acetylcholine (Ach).
Æ Hippocampal Amyloid-β (Aβ) peptides expression levels.
Æ Hippocampal tumor necrosis Factor-α (TNF-α).
Æ Hippocampal apoptotic markers as BAX and Bcl2.
Later, a histopathological examination of brain tissue sections was performed.
The present study revealed that LPS-induced Alzheimer group (Alz.) showed deterioration in spatial learning and memory denoted by the significant prolongation of the mean latency time in Morris Water Maze test. Also, the short retention latency time of Passive Avoidance test reflected decline in contextual memory. LPS- injected rats were unable to maintain balance on RotaRod and consumed longer time in Pole test reflecting motor impairment.
These behavioral changes were positively correlated with serum MDA, hippocampal TNF-α, BAX, amyloid beta and were negatively correlated with serum SOD, hippocampal Bcl-2 and Ach.
This reflected that inflammatory and oxidative states induced apoptosis and amyloidogenesis. The formation of Aβ plaques in return aggravates oxidative, inflammatory and apoptotic states. So, it is very crucial in AD pathogenesis as it initiates a vicious cycle of neurodegeneration. The significant reduction in hippocampal acetylcholine pointed to the impairment in the cholinergic neurons. This impairment has a direct link with the cognitive and motor dysfunction evidenced by the previously mentioned correlation and based on the importance of hippocampal acetylcholine in learning and memory processing.
This neurodegeneration was confirmed by the presence of degenerated neurons and eosinophilic plaque-like areas in cortex and scattered degeneration in the pyramidal neurons of Cornu Amonis (CA1), (CA2), (CA3) and dentate gyrus. The inability to gain weight owing to the catabolic state induced by LPS might share in motor weakness and dysfunction.
In this work, both lines of treatment, either the intravenous transplantation of bone marrow-derived mesenchymal stem cells (MSCs) or the intravenous transplantation of BM-MSCs-derived exosomes were able to improve cognitive and motor dysfunction. This was evidenced by the shortening of the mean escape latency time Also, in Passive Avoidance test, significant prolongation in retention latency time in the illuminated compartment reflects the improvement in the ability to process psychological learning of the aversive conditioning and emotional memory. Motor function improvement was deduced by the prolonged mean latency time on the RotaRod test and the significant decrease in turn time and descent time in the Pole test. Further, histopathological picture was mitigated with few degenerated neurons in the cortex or in the Cornu Amonis of hippocampus.
The improvement imparted by the transplanted BM-MSCS could be related to their ability to home to the injured tissues evident by the appearance of the PKH26-labelled injected stem cells engrafted in the brain tissue shown with the red fluorescent. Homed BM-MSCs either transdifferentiated into astrocytes and neurons or exerted a paracrine role to enhance neurogenesis and alleviate spatial learning and memory.
BM-MSCs can exert anti-apoptotic, anti-inflammatory and anti-oxidative effects evidenced by the significant increase in hippocampal Bcl-2 and Ach levels together with the significant decrease in BAX, MDA and TNF-α levels. This improved neurodegeneration and restored cholinergic neurons. In addition, it interrupted the vicious cycle of amyloidogenesis and Aβ-induced oxidative stress.
Based on the studies claiming that one of the major mechanisms of MSC therapy depends on their exosome-based paracrine action. This work was designed to inject exosomes directly to the experimental rats, hoping to get the benefit without the risk of tumorigenicity being cell free therapy. One month following I.V exosome transplantation, they were detected and tracked in the brain tissue 4 weeks after intravenous transplantation by the appearance of red florescent in brain tissues proving that exosomes were able to cross blood brain barrier and deliver their cargo to the injured cells.
BM-MSCs-derived exosomes were more effective than homed bone marrow derived-stem cells in mitigating spatial learning and memory, contextual memory and locomotor activity with values comparable to the control.
This improvement could be attributed to their anti-apoptotic, anti-inflammatory and anti-oxidative effects or the ability to decrease brain amyloid beta peptide levels. Restoration of hippocampal neurons was noticed in histopathological examination. Acetylcholine significant increase denotes cholinergic neuron restoration which added to maintenance of body weight could explain locomotor improvement.
Therefore, it is conclouded that exosomes can serve as a preferred promising neuroprotective management to Alzheimer disorder, being a cell free therapy that can reach the brain tissue.

Recommendation
This study recommends the use of mesenchymal stem cells derived exosomes as an alternative to mesenchymal stem cells therapy as they are cell free therapy that can reach brain tissue, added to its ability to induce better improvement in cognitive and locomotor dysfunction, oxidative and inflammatory status.
Further studies should be done to elucidate other possiple mechanisms exerted by exosomes to impart this improvement.
Also, exosomes could be used in other neurodegenerative diseases researches or in clinical trials.