The aging-related changes of NADPH-diaphorase (NADPH-d) in the spinal cord were studied in aged dogs. At all levels of the spinal cord examined, NADPH-d activities were present in neurons and fibers in the superficial dorsal horn, dorsal commissure and in neurons around the central canal. In addition, the sympathetic autonomic nucleus in the thoracic and rostral lumbar segments exhibited prominent NADPH-d cellular staining whereas the sacral parasympathetic nucleus (SPN) in the sacral segments was not well stained. Interestingly, we found abundant NADPH-d positive enlarged-diameter fibers termed megaloneurite, which characteristically occurred in the aged sacral segments, distributed in the dorsal gray commissure (DGC), lateral collateral pathway (LCP) the lateral fasciculi and the central canal compared with the cervical, thoracic and lumbar segments. The dense, abnormal NADPH-d megaloneurites occurred in extending from dorsal entry zone through lamina I along with the lateral boundary of the dorsal horn to the region of the SPN. These fibers were prominent in the S1-S3 segments but not in adjacent segments L5-L7 and Cx1 or in thoracolumbar segments and cervical segments. Double staining with GFAP, NeuN, CGRP, MAP2 and Iba1, NADPH-d megaloneurite colocalized with vasoactive intestinal peptide. Presumably, the megaloneurites may represent, in part, visceral afferent projections to the SPN and/or DGC. The NADPH-d megaloneurites in the aged sacral spinal cord indicated some anomalous changes in the neurites, which might account for a disturbance in the aging pathway of the autonomic and sensory nerve in the pelvic visceral organs.

NADPH diaphorase (N-d) positive neurons has been examined in many animals. N-d neurodegenerative neurites were detected in some animal models. However, detailed information of N-d positivity and aging related changes was still lack in the spinal cord and medulla oblongata of pigeons. In this study, we evaluated the N-d positivity and aging alterations in the spinal cord and medullary oblongata of the pigeon compared with rat and mouse. In pigeons, N-d neurons were more numerous in the dorsal horn, around the central canal and in the column of Terni in the thoracic and lumbar segments and scattered neurons occurred in the ventral horn of spinal segments. N-d neurons also occurred in the white matter of spinal cord. Morphometrical analysis demonstrated in the lumbosacral, cervical and thoracic regions. Compared with young pigeons, the size of N-d soma was significantly altered in aged pigeons. Meanwhile, the dramatic morphological changes occurred in the lumbar to sacral segments. The most important findings of this study were aging-related N-d positive bodies (ANB) in aged pigeons, mainly in the nucleus cuneatus externus (CuE), occasionally in the nuclei gracilis et cuneatus. ANBs were identified in the gracile nuclei in spinal cord in the aged rats and mice. ANBs also detected in the CuE spinal nucleus in the aged rats. Immunohistochemistry also showed that the aging changes occurred in the cell types and neuropeptides in aged animals. The results suggested the weak inflammation and neuronal dysfunction in the spinal cord in aged pigeons. Our results suggested that the ANB could be considered as aging marker in the central nervous system.

NADPH diaphorase (N-d) positive neurons has been examined in many animals. N-d neurodegenerative neurites were detected in some animal models. However, detailed information of N-d positivity and aging related changes was still lack in the spinal cord and medulla oblongata of pigeons. In this study, we evaluated the N-d positivity and aging alterations in the spinal cord and medullary oblongata of the pigeon compared with rat and mouse. In pigeons, N-d neurons were more numerous in the dorsal horn, around the central canal and in the column of Terni in the thoracic and lumbar segments and scattered neurons occurred in the ventral horn of spinal segments. N-d neurons also occurred in the white matter of spinal cord. Morphometrical analysis demonstrated in the lumbosacral, cervical and thoracic regions. Compared with young pigeons, the size of N-d soma was significantly altered in aged pigeons. Meanwhile, the dramatic morphological changes occurred in the lumbar to sacral segments. The most important findings of this study were aging-related N-d positive bodies (ANB) in aged pigeons, mainly in the nucleus cuneatus externus (CuE), occasionally in the nuclei gracilis et cuneatus. ANBs were identified in the gracile nuclei in spinal cord in the aged rats and mice. ANBs were also detected in the CuE spinal nucleus in the aged rats. Immunohistochemistry also showed that the aging changes occurred in the cell types and neuropeptides in aged animals. The results suggested the weak inflammation and neuronal dysfunction in the spinal cord in aged pigeons. Our results suggested that the ANB could be considered as aging marker in the central nervous system.

In spinal cord, white matter is distinguished from grey matter in that it contains ascending and descending axonal tracts. While grey matter gets concentrated with neuronal cell bodies. Notable cell bodies and sensory modality of cerebral spinal fluid (CSF) in white matter are still elusive in certain segment of the spinal cord. Monkey Spinal cord was examined by NADPH diaphorase (NADPH-d) histochemistry. We found that NADPH-d positive neurons clustered and featured flat plane in mediolateral funiculus in caudal thoracic and rostral lumber spinal cord, especially evident in the horizontal sections. Majority of NADPH-d funicular neurons were relatively large size and moderately-or lightly-stained neurons. In horizontal section, the multipolar processes of the neurons were thicker than that of regular other neurons. The processes oriented laterally or obliquely in the lateral funiculus. Some of neuronal cell bodies and proximal processes attached NADPH-d positive buttons or puncta. The neuronal processes interlaced network medially linked to lateral horn (intermediolateral nucleus, IML) and laterally to subpial region, in which formed subpial plexus with subpial NADPH-d neurons. Subpial plexus appeared to contacting externally with CSF. The subpial plexus patterned like round brackets located in lateromarginal pial surface. Compared with sympathetic IML in rostral thoracic segments and sacral parasympathetic IML, the funicular plexus configurated a specialized neuro-texture in caudal thoracic segments. The dendritic arbor of funicular neuron featured variety geometric plane shapes. The funicular plexus oriented exclusive layered flat-plane organization between lateral horn and subpial region in caudal thoracic and rostral lumber spinal cord. The subpial plexus may work as CSF sensor outside of spinal cord. The cluster of funicular neurons may function as locomotion sensor, besides visceral regulation. Different to periventricular CSF contacting or pericentral canal structures, NADPH-d funicular neurons and subpial plexus that located in the pial surface. With advantage of NADPH-d, we found funicular neurons which termed academically as funicular plexus and specialized localization for subpial structure we termed subpial plexus. The funicular texture was regarded as neuronal bridge between the interior CSF in the central canal and external CSF out of the pial surface.

Megaloneurite of NADPH diaphorase (NADPH-d) positivity is a new kind of aging-related neurodegeneration and also co-localized with vasoactive intestinal peptide (VIP) in the sacral spinal cord of aged dog and monkey. However, no immunocytochemistry of VIP was exclusively tested in the aged dog and no evidence has been reported in the aged human spinal cord. Aged dogs were used to examine the distribution of VIP immunopositivity in the sacral spinal cord. Immunocytochemistry of VIP and alpha-synuclein were also examined in the aged human spinal cord. The VIP immunopositivity in aged dog was reconfirmed our previous finding illustrated by immunofluorescent study. Megalogneurite was also identified by nitric oxide synthase (NOS) immunoreaction in aged dog. The VIP positive megaloneurites both in age dog and human were detected in dorsal root entry zoon, Lissauer's tract, dorsal commissural nucleus and anterior commissural gray as well as in the lateral funiculus of the sacral spinal cord exclusive of other segments of spinal cord. Alpha-synuclein positivity was present mini-aggregation and Lewy body in the sacral spinal cord of aged human, that also occurred in the lumber, thoracic and cervical spinal cord. It was firstly tested that VIP megaloneurites occurred in the aged human sacral spinal cord, especially in the white matter. Megaloneurites identified by NADPH-d-VIP-NOS immunoreaction could implicate for the dysfunction of pelvic organs in the aged human being. Vasoactive intestinal peptide (VIP) acts on neuronal cells to support physiological condition and protect neurons survival in pathological condition[1-3]. Different from other neuropeptides, the distribution of VIP is restricted to the sacral cord[4]. Or it is majorly distributed in the sacral spinal cord and a few of VIP positivity is detected in the other segment of the spinal cord in cat[5].VIP neurons are small-size neurons and central original source for peripheral innervation[6]. VIP-ergic afferent neurons projected to the pelvic organs are bigger proportion that the other segments, while the lower lumbosacral segments of the neuropeptide innervation was also more priority to the upper lumber segment[7]. VIP fibers are majorly found in the Lissauer's tract (LT), superficial lamina,