Research On Aging-related NADPH-diaphorase Positive Megaloneurites In The Lumbosacral Spinal Cord Of The Aged Dogs And Monkeys

ObjectiveTo investigate the nicotinamide adenine dinucleotide phosphatediaphorase(NADPH-d)positivity and further determine whether NADPH-d abnormality in the lumbosacral spinal cord of aged rats appeared in the dogs and non-human primates.MethodsThe experiment used young(less than 2-year-old,n=8)and aged(more than 10-year-old,n=8)dogs(Canis lupus familiaris)of both sexes,as well as young(less than 5-year-old,n=8)and aged(more than 15-year-old,n=8)cynomolgus monkeys of both sexes.These animals did not show any neurological deficits before experiments.In the dog’s experiment,NADPH-d histochemical staining,NADPH-d histochemistry combined with NeuN,CGRP,VIP or GFAP immunofluorescence double-staining,and VIP immunohistochemistry of sacral sections were performed in the spinal cord of dogs.In the monkey’s experiment,the spinal cords were processed with NADPH-d histochemical staining,and NADPH-d histochemical combined with CGRP,VIP,and NPY immunofluorescence double-labeling.ResultsNADPH-d histochemical staining results indicated that,in the aged dogs and monkeys,a new pattern of fiber-like NADPH-d activity in the dorsal horn(DH),dorsal root entry zone(DREZ)and dorsal gray commissure(DGC)of the sacral spinal cord was found to be extremely different from regular NADPH-d positive fibers,especially in the lateral collateral pathway(LCP)of S1-S3 segments.The swelling giant NADPH-d positive alteration was named as megaloneurite.The dense,abnormal NADPH-d megaloneurites occurred in extending from dorsal root entry zone(DREZ)through Lissauer’s tract(LT)along with the lateral boundary of the dorsal horn to the region of the sacral parasympathetic nucleus(SPN)and/or reached the DGC through the medial collateral pathway(MCP).In the transverse sections of sacral and coccygeal spinal cord of aged dogs and monkeys,clearly-expressed dense punctate NADPH-d activity was noted in the DREZ and the lateral white matter of the LCP,which was extremely different from normal nerve cells and neuroglia cells.These abnormal NADPH-d positive alterations exhibited the analogous morphology with the NADPH-d positive megaloneurites on the horizontal sections.One another alternative tumid NADPH-d positive abnormality was observed around the central canal(CC)of the sacral-coccygeal sections of aged dogs.The irregular intensely-stained NADPH-d positive abnormalities,unlike normal neurons,appeared in a mass-like or strand-like manner around the CC.We termed this NADPH-d abnormality as a variation of megaloneurites.In addition,the dense spherical NADPH-d positive degenerative structures were also detected in the gracile nucleus of aged monkeys.The results of NADPH-d histochemistry combined with NeuN,CGRP,VIP,GFAP or NPY immunofluorescence double-staining showed that,the NADPHd positive megaloneurites colocalized with VIP in the DREZ,LCP,DGC as well as around the CC of aged lumbosacral spinal cord,but not colocalized with NeuN,CGRP,GFAP or NPY immunofluorescence.VIP immunohistochemistry in sacral spinal cord of aged dogs showed that just a few of megaloneurites appeared in the MCP.ConclusionsThe abundant NADPH-d positive megaloneurites occurred in the sacral spinal cord of aged dogs and monkeys,which characteristically distributed in the DREZ,DGC,LCP and around the CC compared with the cervical,thoracic and lumbar segments.NADPH-d positive megaloneurites colocalized with VIP in the aged lumbosacral spinal cord,but not in young animals.The megaloneurites might be a swelling of the transganglionic fibers in the sacral spinal cord.The results of this experiment suggested that the megaloneurites might be considered as a specific aging marker,which were associated with pelvic organ dysfunction and disease in the elderly.