الفهرس 
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Abstract
This study was performed to investigate and compare the neuroprotective effect of Acetyl-L-Carnitine and Caloric Restriction on Aluminum Chloride induce cognitive impairment and to elucidate its possible underlying mechanisms.
This study was carried out on 67 adult male local strain albino rats, initially weighing 200-290 g and were randomly allocated into 4 groups:
I- Control group (n=18): Rats in this group were fed ad libitum and received 1ml distilled water (D.W.)/100g body weight (B.W.) twice daily by gavage, one hour apart for 30 days.
II- Aluminum Chloride induced cognitive deficit group (AlCl3 group, n=16): Rats in this group were fed ad libitum and daily received 1ml D.W./100g B.W., then after 1 hour received 10mg/1ml/100g. B.W. AlCl3 by gavage for 30 days (Prakash and Kumr, 2013).
III- Aluminum Chloride induced cognitive deficit- Acetyl-L-Carnitine pretreated group (AlCl3-Acetyl-L-Carnitine group, n=17): Rats in this group were fed ad libitum and daily received Acetyl-L-Carnitine in a dose of 10mg/1ml/100g. B.W. orally by gavage (Goo et al., 2012), then after one hour, received AlCl3 as AlCl3 group, for 30 days.
IV- Aluminum Chloride induced cognitive deficit- Caloric Restriction subjected group (AlCl3-Caloric Restriction group, n=16): Rats in this group were fed 70% of the daily average caloric intake of the control group (Plata et al., 2019), and received D.W. and AlCl3 as AlCl3 group for 30 days.
All rats were subjected to Behavioral assessment using Open field test (OFT), Y maze spontaneous alternation test, Novel object recognition test (NORT) and Passive avoidance test (PAT). Biochemical assay of serum phosphorylated tau protein (p-Tau), Hippocampal tissue level of Phosphorylated adenosine monophosphate activated protein kinase (pAMPK), Beclin-1, apoptosis regulator Bax and B cell lymphoma (Bcl2). Histological study of the hippocampal tissue by Hematoxylin and Eosin (H&E), Silver stain and GFAP & Ki67 immunohistochemistry.
The results of the present study can be summarized as follows:
All the initial values of the 3 mazes were non-significant among the 4 studied groups, except for the initial stretch attend posture number, an anxiety-related emotional behavior, in AlCl3- Caloric restriction group that showed significant increase as compared to both control and AlCl3- Acetyl-L-Carnitine groups.
Comparing the final values with its corresponding initial values of OFT, Y maze and NOR in control rats, all values showed non-significant differences except for final defecation number in OFT which was significantly decreased as compared to its corresponding initial value. Regarding the Y maze, the final values of total arm entries (TAE), spontaneous alternation performance (SAP), number of possible alternations showed significant decrease as compared with its corresponding initial values, however the final percentage alternation (% alternation) and errors tested by number and percentage of same arm entries (SAE, % SAE), number and percentage of alternative arm return (AAR, %AAR) were comparable to its corresponding initial values. For the NOR only the final discrimination index (DI) is significantly reduced as compared to its corresponding initial value. In passive avoidance test (PAT), the control group showed significant increase in time of 24 hours retention (post-shock latency) as compared to the time of acquisition (pre-shock cross latency) being 300 sec, the maximum test time. In addition to the normal histological picture of the hippocampus proper and dentate gyrus.
Aluminum Chloride induced cognitive deficit group:
Regarding the AlCl3-group, induction of the model was confirmed by the significant reduction in the OFT final ambulation, center square entry number, center square frequency/duration, rearing number, rearing duration, score, grooming number and grooming duration, with significant prolongation in freezing duration when compared with its corresponding initial values, as well as significant decrease in final rearing duration and significant increase in final freezing duration as compared with its corresponding final control values. In addition, Y maze showed significant reduction in final TAE, SAP, number of possible alternation as well as reduced % alternation when compared with its corresponding initial values, with reduction in SAP when compared with its corresponding value in control rats. However, the NOR showed non-significant changes between its final and initial parameters, except for 24 h retention time spent exploring novel object, that was significantly decreased, or between the final values of AlCl3-group and control rats. In the PAT, the time of 24 h retention was non-significant from time of acquisition but significantly reduced from control group time of 24 h retention.
Regarding the biochemical results, there was elevated hippocampal phosphorylated tau protein (p TAU). Also, there was significant increase of phosphorylated adenosine monophosphate kinase (pAMPK). On the other hand, there was a reduction in the level of hippocampal Beclin-1and the associated enhanced apoptosis represented by the elevated hippocampal proapoptotic Bax, Bax/Bcl2 ratio and reduced hippocampal antiapoptotic Bcl-2.
Regarding the histological studies, there was excess accumulation of intracellular neurofibrillary tangles and extracellular amyloid plaques in silver-stained sections of area CA1 of rat hippocampus proper and dentate gyrus. The histopathological picture showed shrunken pyramidal and granule cells with pyknotic nuclei, in addition to decreased thickness and number of surviving cells of pyramidal layer of CA1, CA2 and CA3 areas of hippocampus proper and suprapyramidal blade of granule cell layer of dentate gyrus in the H&E sections and negative immunoreactivity of nuclear protein Ki 67 in the granule cell layer and in the subgranular zone of dentate gyrus, associated with astroglial injury indicated by reduced positive immunoreactivity of glial fibrillary acidic protein (GFAP).
Aluminum Chloride induced cognitive deficit- Acetyl-L-Carnitine pretreated group:
AlCl3-Acetyl-L-Carnitine group showed non-significant differences in the final values of all parameters of OFT, Y maze and NORT as compared with its corresponding initial values except for the significantly decreased final ambulation, rearing number, score and significantly increased final freezing. In addition to a significantly increased time of 24 h retention as compared to time of acquisition, being 300 msec indicating no entry to the dark compartment containing the electric shock.
Compared to control group, rats received Acetyl-L-Carnitine showed non-significant changes except for a significant increase in the final freezing duration of OFT. Compared to AlCl3-group, only significant prolongation in the time of 24 h retention was observed being comparable to controls.
Regarding the biochemical assay, there was significant reduction in serum p TAU when compared with AlCl3 group, being comparable to controls. Also, there was the significant reduction in p AMPK when compared with AlCl3 group, being comparable to controls. On the other hand, there was significant elevation in hippocampal Beclin-1when compared with AlCl3 group, being non-significant from controls. In addition to significantly associated decline apoptosis, signified by the reduction in proapoptotic hippocampal Bax and Bax/Bcl2, as well as elevation in hippocampal Bcl2 compared with AlCl3 group being comparable to control rats.
Regarding the histological finding, the neurofibrillary tangles were still detected in affected pyramidal cells of area CA1 of rat hippocampus proper with amyloid plaques in area CA1 of hippocampus proper and dentate gyrus as shown in silver-stained sections. Also, there were few cells with karyolitic nuclei, others were shrunken with pyknotic nuclei, many other cells contain apparently healthy vesicular nuclei in area CA1 of hippocampus proper and dentate gyrus. However, the thickness and the number of surviving cells of pyramidal layer of CA1, CA2 and CA3 areas of hippocampus proper and suprapyramidal blade of granule cell layer of dentate gyrus were significantly decreased as compared to control, being non-significant from AlCl3 group except for thickness of suprapyramidal blade of granule cell layer of dentate gyrus that was significantly thicker as compared to AlCl3 group as shown in H&E-stained sections. Moreover, positive Ki-67 immunoreactive nuclei in the suprapyramidal blade and subgranular zone of dentate gyrus was detected indicating neural cells survival and proliferation. Also, astrocytes showed positive immunoreactivity of GFAP and appeared as large cells with multiple thin processes, however, the number and area percentage of GFAP were significantly decreased as compared to controls.
Aluminum Chloride induced cognitive deficit- Caloric Restriction subjected group:
AlCl3-Caloric restriction group showed non-significant changes in OFT parameters except for significant prolongation in final freezing duration when compared with its corresponding initial value and its final value of control group. Compared to AlCl3 group, AlCl3-Caloric restriction group showed significant increase in rearing number and rearing duration as well as significant increase in score. But there was a significant increase in initial and final stretch attend posture as compared with AlCl3-Acetyl-L-Carnitine group. Regarding the Y maze, the final TAE, SAP, number of possible alternations were significantly decreased when compared with its corresponding initials, however, the final SAP showed significant increase when compared with its final value of AlCl3 group. However, the NORT showed non-significant changes between its final and initial parameters, except for time spent exploring object A, that was significantly decreased, as well as between its final values and those of control, AlCl3 and Acetyl-L-Carnitine groups. PAT showed significantly increased time of 24 h retention as compared with its time of acquisition and with time of 24 h retention of AlCl3 group, being 300 msec.
Regarding the biochemical assay, there was significant reduction in serum p TAU when compared with AlCl3 group, being comparable to controls. Also, there was significantly decreased p AMPK as compared to AlCl3 group, being comparable to controls. Unlike the Acetyl-L-Carnitine supplemented group, apoptosis was still activated in the group subjected to Caloric restriction as compared to control rats, being non significantly less than AlCl3 group but not reaching the control values. On the other hand, there was elevated hippocampal level of Beclin-1 when compared with AlCl3 group, being non-significant from control group, without inhibition of apoptosis as denoted by the significantly increased hippocampal Bax, Bax/Bcl2 ratio and significantly decreased hippocampal Bcl2 as compared to control rats being non significantly changed from AlCl3 group.
Regarding the histological results, the silver-stained section of this group showed affected pyramidal cells with deep brown neurofibrillary tangles and amyloid plaques in neuropil in between in area CA1 of hippocampus proper and the molecular layer of dentate gyrus. In addition to the H&E sections that showed some shrunken pyramidal cells with pyknotic nuclei and deeply stained homogenous basophilic cytoplasm, others show karyolitic nuclei, some cells appear relatively healthy with central, round vesicular nuclei and prominent nucleoli in area CA1 of hippocampus proper, also, the dentate gyrus showed many granule cells with deeply stained pyknotic nuclei, others display an irregular clumping of nuclear chromatin (karyorrhexis) and some are vesicular with prominent nucleolus, with significant increase in the thickness and number of surviving cells of pyramidal layer as compared to rats of AlCl3 group, being comparable to control rats only for the thickness. And significant decrease in the thickness and number of surviving cells of suprapyramidal blade of granule cell layer of dentate gyrus compared to controls, but significantly increased as compared to AlCl3 and AlCl3-Acetyl-L-Carnitine groups. Also, showed few positive Ki-67 immunoreactive nuclei in the dentate gyrus. Moreover, astrocytes expressed numerous long cytoplasmic processes and showed an apparent increase in positive immunoreactivity of GFAP as compared to of AlCl3 group, with significant increase in the mean number of GFAP positive astrocytes compared to AlCl3 and AlCl3-Acetyl-L-Carnitine group, but significant decrease in the area percentage of GFAP immunoreactive astrocytes as compared with control group.
Conclusions and Recommendation:
It could be concluded that each of Acetyl-L-Carnitine and Caloric Restriction partially improved the AlCl3 induced behavioral, cognitive, biochemical and histological changes, with more ameliorative effect of Acetyl-L-Carnitine on hippocampal apoptotic markers, with more obvious behavioral and histological improvement with Caloric Restriction. So, we recommend implementation of a low-caloric diet (20-40%) fortified with Acetyl-L-Carnitine for the elderly subjects as a promising strategy to prevent or delay the pathogenesis of AD.