الفهرس 
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Abstract
Doxorubicin (DOX) is a prominent mainstay member of the anthracycline antineoplastic agents. It is one of the most potent FDA-approved agents for treatment of solid tumors and hematological malignancies. The mechanism underlying DOX-induced chemobrain is thought to be cytokine-induced oxidative and nitrosative damage to brain tissues. Astaxanthin (AST), a naturally occurring carotenoid, is reputable for its outstanding antioxidant, anti-inflammatory and antiapoptotic activities. Therefore, the aim of the current study was to experiment DOX-induced cognitive deficits rodent model, determine the neuroprotective dose of AST against DOX-induced behavioral and neurobiological abnormalities and elucidate the underlying neuroprotective effect in terms of oxidative, inflammatory and apoptotic arbiters.
The following parameters were investigated:
➢ Step-through passive avoidance
➢ Locomotor activity test
➢ Assessment of neurodegeneration: hematoxylin and eosin (H&E) and toluidine blue staining
➢ Acetylcholinesterase (AChE) activity in hippocampal tissues
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➢ Oxidative stress markers: reduced glutathione (GSH), lipid peroxidation product (MDA) and, catalase activity in hippocampal tissues
➢ Inflammatory markers: tumor necrosis factor alpha (TNF-α), prostaglandin E-2 (PGE-2), cyclooxygenase-2 (COX-2)
➢ Astrocytes activation marker: glial fibrillary acidic protein (GFAP) levels in hippocampal tissues
➢ Apoptotic markers: active-cytochrome c and caspase-3 activity in hippocampal tissues.
The findings of the present investigation can be summarized as follows:
I) Experimenting different DOX-induced cognitive deficits rodent models
➢ Within the step-through passive avoidance paradigm, a single dose of 8 mg/kg of DOX induced memory impairment as indicated by the shortening in the step-through latency by 50% as compared to the control group, while the administration of DOX at a dose of 6 mg/kg/w for two weeks considerably shortened the escape latency by 82%. Moreover, administration of 3 mg/kg/w and 2 mg/kg/w for four consecutive weeks significantly shortened
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the escape latency by 92% and 93% respectively compared to the control group.
➢ DOX did not affect locomotor activity.
➢ Histopathological examination revealed that a single dose of 8 mg/kg of DOX resulted in nuclear pyknosis and degeneration in some of the neuronal cells of the hippocampus, while the administration of DOX at a dose of 6 mg/kg/w for two weeks resulted in nuclear pyknosis and degeneration in numerous hippocampal neuronal cells. On the other hand, administration of either 3 mg/kg/w or 2 mg/kg/w for four consecutive weeks resulted in nuclear pyknosis and degeneration in most of the hippocampal neuronal cells.
Based on the results of behavioral tests and histopathological examination, model D (2 mg/kg/w, for 4 weeks, i.p.) was chosen as the most robust model for inducing cognitive dysfunction.
II) Determining the neuroprotective dose of AST against DOX-induced behavioral and neurobiological abnormalities
➢ In the step-through passive avoidance paradigm, the lower dose of AST (25 mg/kg/d, 5d/week, for 4 weeks, P.O) was solely able to completely reverse DOX-induced amnesia as evident by
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restoration of the normal step-through latency while the medium (50 mg/kg/d, 5d/week, for 4 weeks, P.O) and high dose (100 mg/kg/d 5d/week, for 4 weeks, P.O) restored 52% and 33% of the normal escape latency respectively.
➢ Different doses of AST did not affect the locomotor activity.
➢ Treatment with low dose of AST completely restored the hippocampal histological structure while the medium and the high doses showed minimal and moderate hippocampal neurodegeneration respectively.
➢ Administration of 25 mg/kg of AST restored the normal GSH and MDA levels. On the other hand, co-treatment with 50 mg/kg elevated GSH and reduced MDA levels by 97% and 21% respectively as compared to DOX-treated group. Meanwhile, co-administration of 100 mg/kg of AST reduced MDA level by 17% compared to DOX treated group however, it failed to elicit any significant upsurge of GSH level.
Based on the results of neurobehavioral tests, histopathological examination and oxidative stress parameters, the lower dose of AST (25 mg/kg/d, 5d/week, for 4 weeks, P.O) was the most effective in halting the damage imposed by DOX therefore, it was chosen to complete further neurochemical and immunohistochemical assessments.
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III) Elucidating the mechanisms underlying AST neuroprotective effect in terms of oxidative, inflammatory and apoptotic arbiters
➢ In the step-through passive avoidance paradigm, AST (25 mg/kg) reversed DOX-induced amnesia as evident by restoration of the normal step-through latency.
➢ AST and/or DOX did not affect the locomotor activity of the treated rats
➢ Treatment with 25 mg/kg of AST completely reversed DOX-induced hippocampal neurodegenerative changes. AST alone group was analogous to the control group.
➢ Hippocampal neurodegeneration was further confirmed using toluidine blue staining. DOX-treated group complete loss of the hippocampal histological architecture. Conversely, AST (25 mg/kg) treatment restored the normal neuronal features manifested by the rounded neuronal build, euchromatic cytoplasm and defined nuclei.
➢ DOX administration significantly increased AChE activity. On the other hand, AST treatment precluded any alteration in the enzyme activity.
➢ AST reverted DOX-induced hippocampal oxidative stress as evidenced by the normalization of GSH and MDA levels and catalase activity.
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➢ AST halted DOX-induced hippocampal inflammatory insult as shown by the statistically significant downsurge of TNF-α, PGE-2, COX-2 and GFAP levels.
➢ AST demonstrated efficient antiapoptotic activity where it dramatically reverted DOX-induced activation of the executioner caspase 3 along with reduction of expression of hippocampal-active cytochrome c.
In conclusion, the present study provides evidence that systemic administration of DOX at a dose of 2 mg/kg/w for four consecutive weeks effectively recapitulated clinically reported DOX-induced chemobrain reports. Moreover, the naturally occurring carotenoid, AST could present a promising anti-amnestic and neuroprotective agent which could guard against DOX-induced cognitive deficits. Furthermore, we highlighted that the underlying molecular mechanisms beyond the cognitive boosting effects of AST could be at least partly attributed to a myriad of actions including its antioxidant, anti-inflammatory and antiapoptotic activities. Accordingly, the current study serves as an impetus supporting clinical investigation of the neuroprotective impact of AST supplementation in DOX-treated cancer patients.