The Role Of Apoptosis In The Neurodegenerative Disease By Subchronic Induction Of Acrylamide

Objective Occupational exposure of humans and experimental intoxication of laboratory animals with acrylamide monor (ACR) produces a neuropathy characterized by nerve degeneration. In order to study the role of apoptosis in the neurodegenerative disease by subchronic induction of acrylamide, Neuronal morphologic changes, DNA cleavage and the expression of bcl-2, bax and caspase-3 in the central nervous system(CNS) and the peripheral nervous system(PNS) was assessed following the administration of ACR. Methods1. Male Wistar rats weighing 180~230g were divided into three groups, each 10 animals. The rats in group 1 served as control, and received normal saline. The animals in group 2 and 3 were given ACR dissolved in physiological saline(20, 40 mg/kg ip 3 days/week) for 8 weeks.2. Neurobehavioral indices were determined per week. Special tests included observations of spontaneous locomotion in an open field, measurements of hindlimb landingfoot splay, nervous sensory to pain and heat and the rotator test.3. The research effort has been made to decipher the mechanism of apoptosis in ACR-induced neurotoxicity: we used a combination of morphologic criteria (Haematoxylin and Eosin staining, HE staing) and the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) technique together with DNA agarose gel electrophoresis for DNA fragmentation, immunohistochemistry for Bcl-2 and Bax proteins and Western blotting analyze of bcl-2, bax and caspase-3 expression in the CNS and PNS tissue.Results1. Compared to the control, weight loss following the administration of ACR, theconduction of sensory nerve to heat changed (the high dose group increased by 55% and the low dose decreased by 39%), the conduction of pain fasted (on the third week the high dose group decreased by 45%, on the seventh week the low dose group decreased by 45%), the hindlimb landingfoort splay of high dose group increased by 109% and low dose group increased by 59%, the time staying on the rotating cylinder decreased (the high dose group decreased by 98% and the low dose group decreased by 81 %).2. HE staining showed darkly stained degenerating neurons in the cerebral cortex and the lost of purkinje cell in the cerebellar cortex following the administration of acrylamide, the change being most severe in the high dose group; Compared to control, HE staining did not reveal overt neuronal loss in the cerebral cortex and the spinal cord after ACR administration.3. Immunoreactivity of Bcl-2 and Bax was localized in the cytoplasm of neurons in control and ACR-treated rats. The change of Bcl-2 and Bax immunoreactivity was observed in the neurons of the CNS following ACR administration:1) Cerebral cortex: Compared to control, the immunoreactivity of Bcl-2 was markedly decreased(14%, P <0.05) in the low dose group, but the high dose group with a up-pattern of Bcl-2 immunoreactivity (20%, P <0.05); No significant change was observed in the immunoreactivity of Bax in control and ACR-treated rats.2) Cerebellum: compared to control, neuronal Bcl-2 immunoreactivity was markedly decreased(22%, P<0.01) in the low dose group, this decrease was accompanied by a concomitant increase of staining for Bax protein(22%, P< 0.05); increased Bcl-2 immunoreactivity(62%, P<0.0l) was observed in the high dose group, Bax immuneoreactivity was decreased(30%, P<0.01).3) Spinal cord: Compared to control, the immunoreactivity of Bcl-2 protein was markedly decreased(20%, P <0.05) in the low dose group, but the high dose group Bcl-2 immunoreactivity was elevated (12%, P <0.05); No significant change was observed in the immunoreactivity of Bax in control and ACR-treated rats.4. Western blotting disclosed modification in the intensity of the bands corresponding to Bcl-2, Bax and Procaspase-3 between control and ACR-treated rats:1) Cerebral cortex: Compared to control, densitometric analysis showed that the relative amount of bcl-2 expression was markedly decreased(22%, P <0.05) in the low dose group, but the high dose group with a up-expression of Bcl-2 (26%, P <0.05); there was no significant change in the expression of bax and caspase-3 in control and ACR-treated rats.2) Cerebellum: compared to control, the relative amount of neuronal Bcl-2 was markedly decreased(25%, p<0.05) in the low dose group, this decrease was accompanied by a concomitant increase of Bax protein(14%, P<0.05); increased Bcl-2 expression(13%, P<0.05) was observed in the high dose group, however there was a significant decrease in Bax expression(17%, P< 0.05); there was no significant change in the expression of caspase-3 in control and ACR-treated rats.3) Spinal cord: Compared to control, densitometric analysis showed that the relative amount of bcl-2 expression was markedly decreased(33%, P <0.01) in the low dose group, but the high dose group with a up-expression of Bcl-2(25%, P <0.05); there was no significant change in the expression of bax in control and ACR-treated rats; expression of the inactive 32 kDa caspase-3 precursor showed an increase following ACR administration, the low dose group increased by 41% and the high dose increased by 132%( P<0.01).4) Sciatic nerve: Compared to control, increased Bcl-2 expression(104%, P<0.01) was observed in the low dose group, there was a concomitant increase in Bax expression(49%, P<0.05); there was no significant change in the expression of caspase-3 in control and the low dose group. In the high dose group, densitometric analysis showed that bcl-2(196%, P<0.01), bax(94%, P<0.01) and caspase-3(66%, P<0.01) expression was all markedly increased following ACR administration.5. TUNEL showed abundant positive neurons in the cerebral cortex, cerebellar cortex and spinal cord after DNase I treat; TUNEL-positive neurons were completely absent in the ACR-treated rats and controls (negative and experimental control), which indicated there was no DNA fragmentation.6. Agarose gel electrophoresis of DNA extracted from the cerebral cortex of the control rats did not show any fragmentation, DNA extracted from the cerebralcortex of the ACR-treated rats exhibited a smear of DNA; Nuclear DNA extracted from cerebellum of the low dose ACR-treated rats exhibited the non-typical 180~200bp banding pattern characteristic of apoptotic cell death, these fragments were superimposed over a smear of DNA extracted from the high dose group, suggestive of the occurrence of non-specific DNA fragmentation associated with necrosis. No DNA fragmentation was observed in DNA extracted from the spinal cord of either control or ACR-treated rats. Conclusion1. There was obvious neurotoxicity after subchronic intraperitioneal administration of ACR to the adult rats:1) ACR changed the feeling of heat, increased the sensitivity of pain, and damaged the coordination ability of muscles.2) ACR induced a massive degeneration of neurons in the cerebral cortex identified as shrunken cell nuclei with chromatin condensation; ACR also induced the loss of the cerebellar purkinje cells.3) The Bcl-2/ Bax ratio was altered in cerebral cortex, cerebellum and spinal cord of ACR-treated rats. The ratio was dropped in the low dose rats compared to the controls, in contrast, it showed a increase in the high dose rats.4) Expression of caspase-3 in the spinal cord and the sciatic nerve showed an increase following ACR administration.5) bcl-2 and bax expression was markedly increased in the sciatic nerve following ACR administration.2. Thus, subchronic administration of ACR affected the expression of death related proteins in the CNS and PNS, which indicate there is the early molecular regulatory mechanism of apoptosis in the neuropathy induced by ACR; The change of the Bcl-2/Bax ratio in the cerebellum suggest the potential role of apoptosis in the purkinje cellar death induced by ACR; The increase of Bcl-2 and Bax protein expression also indicate that the axonal degeneration is associated with the apoptotic molecular regulatory mechanism.