Novel morphological alteration and neural pathway of NADPH diaphorase positivity in the spinal cord with disc herniation of aged dog: A case report

Neuronal lesion or injury is a traditional approach to investigate neural circuit. Is any new neural pathway or new neurodegeneration related central nerve system injury? Spinal disc herniation can cause the spinal cord injury. However, the histological examination is still lack. It happened that a case of spinal disc herniation of a 10-year old dog was examined with NADPH diaphorase (N-d) histology. We did not find the N-d neurodegenerative aberrant in the tissue of the mid-rostral lumber segment besides the metamorphoses by the compression of the disc herniation. However, the severe neuropathological changes majorly occurred in the lumbosacral spinal cord. We found more diverse neurodegenerative alterations: the aging-related N-d body (ANB), megaloneurite and N-d homogeneous formazan globule in the lumbosacral spinal cord. We also found that a new circuit pathway (intermedial collateral pathway) showed by a megaloneurite between the lateral collateral pathway and the medial collateral pathway. The enormous notch caused by spinal disc herniation located at the mid-rostral lumber segments. The aging-related neurodegeneration occurred the specific lumbosacral segments. The homogeneous formazan globule was round or oval homogeneous N-d positivity which distributed in the gray matter and dorsal column. In the medulla oblongata, ANBs were revealed in the gracile nucleus, nucleus reticularis lateralis (ventrolateral spinal trigeminal nucleus) and middle of the spinal


INTRODUCTION
Thoracolumbar intervertebral disc herniations are a common disease that cause spinal cord injury (SCI) [1][2][3][4][5]. Cerebrospinal fluid (CSF) tau protein is found to be used as a biomarker in severity of SCI in dogs with intervertebral disc herniation [6]. Spinal cord swelling is used as indication for prognosis in intervertebral disc disease of dogs [7]. An experimental model of epidural balloon compression is found to cause the spinal cord occlusion and severe hemorrhages as well as vacuolar formations [8]. However, the neuropathological changes of intervertebral disc herniations are scarcely reported. We have found a megaloneurites of NADPH diaphorase (N-d) in the sacral spinal cord of the aged dogs [9] and aged monkey [10]. Recently, a case of 10-year old dog of lumber disc herniations happened to be investigated with N-d staining. Besides the occurrence of N-d megaloneurites in the sacral spinal cord, we further found the aging-related N-d body (ANB) and round or oval homogeneous N-d positivity in the spinal cord with disc herniations. Homogeneous formazan globule was a new term that we will discuses in the discussion section.

MATERIALS AND METHODS
The tissue preparation Adult female dog (crossbreed dog Pekingese, 7.2kg) was legally obtained from Jinzhou Animal Facility for Department of Surgery (Jinzhou, China) and was breeding maintained in a temperature and humiditycontrolled room as well as fed libitum. This study and experimental protocol were approved by the Ethics Committee on the Use of Animals at Jinzhou Medical University.
The animal was deeply anesthetized by intravenous injection of sodium pentobarbital (60 mg/kg body weight) and perfused through the aorta with physiological saline, followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.4) briefly according to the previous experiments [11]. The tissues of the brain and spinal cord were immersed in the same fixative as in the perfusion for 6 hrs. at 4°C, and transferred to 30% sucrose in 0.1 M PB (pH 7.4). Frozen sections were cut coronally at 40 µm on a cryostat. NADPH diaphorase histochemistry N-d enzyme histochemistry was performed in free-floating approach. In this procedure, Sections were incubated for 5 min in 100 mM sodium phosphate buffer(PBS, pH 7.4) followed by incubation in phosphate buffer (PB, pH 7.4) with 1 mM beta-NADPH, 0.5 mM NBT and 0.3% Triton X-100 at 37℃ for up to 3h. Sections were rinsed with PB, distilled water, dehydrated in a graded ethanol series, and were coverslipped with mounting medium.

RESULTS
We did not realize that the dog suffered spinal disc herniation. We found the disc herniation when we opened the vertebral lamina and took the spinal cord. The spinal disc herniation revealed at the L3 and S1. Before sacrifice of the dog, the locomotion of the dog seemed no clear dyskinesia when walk the dog. The dog could control excretion and defecation during outdoor walk. We presented example sections of the spinal cord related different segment for the spinal disc herniation as well as the medulla oblongata. The spinal disc herniation located in the ventral of the lumber spinal cord. The compression of the disc herniation caused a signification notch (Figure 1). There was an obvious notch in the ventral of the spinal cord. However, the disc herniation in situ did not cause noticeable neurodegeneration at the level of the notch. Figure 2 caudally located to segment of Figure 1. Vacuolar dendrites of the neurons in vicinity of the central gray matter.
In our previous study, the N-d positive thick neurite termed as megaloneurite is observed in the sacral spinal cord of the aged dog [9]. In the present study, megaloneurites were consistently observed in the dorsal horn of the caudal segments. Figure 3 showed an example of a section that giant megaloneurites occurred in one side dorsal horn while contrast with the same region in the other side. In the Figure 3 B, we pointed out that the megaloneurite (arrowhead) between the lateral collateral pathway [12] [12] and the medial collateral pathway (MCP) could be a new N-d pathway of the autonomic sensory circuit. Temporary, we termed the intermedial collateral pathway with abbreviation ICP. The diameter of the megaloneurite could be as large as 27μm. A tremendous N-d dystrophy and megaloneurites showed in the sacral spinal cord with the notch caused by disc herniation (Figure 4). Besides regular ANBs, we noticed that a massive of N-d spheroids revealed new profile criteria which we termed as homogeneous formazan globule. According to ANBs in aged rat, ANB shows more diversity from size, contour and intensity [13]. The homogeneous formazan globule revealed as round-or ovalshaped non-soma formation and was not found in the normal aged dog [9]. Some of homogeneous formazan globule could also be described like droplet. In this segment, homogeneous formazan globules occurred not only in gray matter but also in the white matter distributed in the dorsal column, lateral funiculus and ventral funiculus. There was also occurrence of the similar variety of neurodystrophy in the intact sacral spinal cord of no herniated compression notch ( Figure 5). In the sacral spinal cord distal to the disc herniation, the homogeneous formazan globules greatly reduced ( Figure 6). The megaloneurite also reduced. The MCP showed in the figure and indicated the sensory input. The diameter of the megaloneurite also reduced compared with that in Figure 3.
The aging-related N-d neurodegeneration occurred normally in the lumbosacral spinal cord of the aged dog [9,14]. In the thoracic segment rostral to the disc herniation, no megaloneurite and homogeneous formazan globules were detected besides occasional small size ANBs (Figure 7). The gracile nucleus receives the primary sensory input bellow the mid thoracic spinal cord [15,16]. N-d dystrophy corelates to the lumbosacral spinal cord [17] and also occurs in the gracile nucleus of dog [18]. Consideration of the ANBs and the homogeneous formazan globules in the dorsal column, we further examined the gracile nucleus. In Figure 8, ANBs and lightly formazan profiles were detected in the gracile nucleus. Next, ANBs were also detected in the other structures of the medulla, such as in the medial lemniscus (ML), the hypoglossal nucleus ( Figure 9) and the ventral lateral of the medulla as well as the spinal trigeminal nucleus ( Figure 10). Taken together, the lumen of the central canal regionally collapsed at or near the spinal disc herniation. Away from the disc herniation, the cavity of the central canal was filling around.

DISCUSSION
The case study was completed in a 10-year-old dog accompanied by spinal disc herniation. We have already reported the megaloneurite in the sacral spinal cord of aged dog [9,14]. In this study, the N-d positive megaloneurite occurred conformally in the sacral spinal cord. We further found that numerous ANBs and enormous homogeneous formazan globules revealed in the gray matter and dorsal column. The homogeneous formazan globule was a novel type of neuropathological profile according to current morphological criteria. In general, megaloneurite and ANB occurred in the dorsal of the gray matter, for example, dorsal horn, Lissauer's tract, dorsal commissural nucleus and intermediolateral nucleus as well as the mediolateral funiculus. We found that the distribution of homogeneous formazan globules occurred not only in the dorsal of the gray matter but also in the ventral horn. The enormous notch caused by spinal disc herniation located at the mid-rostral lumber segments. We did not find the other N-d neurodegenerative aberrant in the tissue of the mid-rostral lumber segment besides the metamorphoses by the compression of the disc herniation. It seemed that the compression did not cause compression dependent N-d neurodegeneration in situ segment. However, the severe neuropathological changes majorly occurred in the caudal sacral spinal cord. More occurrence of ANB revealed than that of normal aging changes [9]. We supposed that the compression of disc herniation may cause to spread neuropathology from the dorsal part to the ventral area of spinal cord. We should emphasis that the agingrelated changes implicated sacral dependent specific mechanism, because the no substantial N-d injury in situ disc herniation and N-d degeneration occurred in distal to disc herniation. The homogeneous formazan globules featured as decellularized extracellular matrix. In the medulla oblongata, ANBs were revealed in the gracile nucleus, nucleus reticularis lateralis (ventrolateral to spinal trigeminal nucleus) and middle of the spinal trigeminal nucleus. In the other species, the similar irregular profiles of the homogeneous formazan globules are detected in the gracile nucleus of aged rats [17]. In the present study, the homogeneous formazan globules were a relative regular profile of the round and oval shape in the sacral spinal cord. Compared with the normal aging alterations in the sacral spinal cord and or the gracile of rat [13,17], dog [9] and monkey [10] as well as pigeon [19], the homogeneous formazan globules was a novel neurodegenerative formation.
The degenerated intervertebral disk reveal in the spinal disc herniation [20]. Extruded disk herniation is relevant to cell senescence of disc degeneration [21]. The neuropathology of spinal disc herniation is lack in the aging animals. Rat model of lumber disc herniation are used to investigate of the dorsal root ganglia (DRG) and spinal cord [22][23][24][25][26]. Chronic compression of the DRG ipsilaterally induce upregulation of nNOS neurons within the superficial dorsal horn of spinal cord [27]. Basically, nNOS positivity is corelated with N-d expression in the central nervous system [28,29]. However, some previous investigations demonstrate that Nd is not completely identical to nNOS [13,[30][31][32]. In our previous study, megaloneurite still identified by the nNOS immunochemistry [14]. Some of N-d dystrophic spheroids in the gracile nucleus also reveal partially colocalization with nNOS immunochemistry [17]. It demonstrates that the N-d neurodegeneration shows diverse properties. Compared with strong stained ANB of sharp boundary, homogeneous formazan globule may be the swollen axons [33] or the deposit of N-d caused by the axonal leakage [34]. ANB is the abbreviation for aging-related N-d body. We thought that the homogeneous formazan globule was compression-related change.
The dystrophic formation in gracile nucleus is aging dependent and has been termed as neuroaxonal dystrophy (NAD) [35][36][37]. The N-d gracile dystrophy has been reported in the aged rat [36,38]. We find that N-d neuritic dystrophy occurs in both of the gracile nucleus and cuneatus nucleus of the dorsal column nuclei as well as the spinal trigeminal nucleus [17]. The N-d neuritic dystrophy is pathognomonic morphologic change [17]. In the previous study, the N-d dystrophy is not frequently detected in the aged dog. In the present study, the N-d dystrophy distinctly distributed in the gracile nucleus and several other nuclei.
The medial of dorsal horn of the MCP and the lateral dorsal horn of the LCP are function region for a certain condition [39]. In general anatomy of the thoracolumbar and sacral spinal cord in the dog, N-d positive LCP tracks to the dorsal commissural gray and [40]. In our present study, the megaloneurite in Figure 3 occurrent located lamina Ⅲ-Ⅳ between the LCP and the MCP. The positive labeling of the neuronal tracing of LCP and the dorsal lateral funiculus tracks extending into laminae V-VII in the dorsal commissural gray and vicinity of the parasympathetic nucleus [41]. The immunoactivity of substance P and not VIP distributed in the dorsal horn between the LCP and MCP [42] or no notable evidence demonstrates the confirmation about the fiber connection [43]. In this study, substance P immunoactivity does not show megaloneurite-like profile. Megaloneurite is colocalization with VIP [9,10]. We thought that the immunoactivity without evidence of projecting fiber may not make a conclusion because the medial portion of the substance P immunoactivity turns to the dorsal commissural region [44]. The two ends of megaloneurite in figure 3 provided clear orientation between the LCP and the MCP. The LCP and MCP are important for urogenital circuit [45] and also for neuropathological condition [46]. The neurons of medial dorsal horn and lateral dorsal horn can be activated for a physiological function [47]. There was a missing link of notable megaloneurite in the MCP in the Figure 3. Actually, the N-d MCPs were verifiably presented in the Figure 6-1. We also report that megaloneurites locate in the medial dorsal horn of N-d positivity [9] and the VIP positive MCP [14] in aged dog. We postulated that the megaloneurite formed a specific anatomy-oriented functional circuit. As mentioned in results, we already named it a temporary term: intermedial collateral pathway (ICP). We should re-examine the normal spinal cord to verify the neuronal anatomical structure in future.
In summary, the megaloneurite provided a specific anatomy-oriented morphology to reveal a certain circuit in the lumbosacral spinal cord. To our best knowledge, we first time to report the triple morphological alterations of N-d positivity in spinal disc herniation of aged dog. The homogeneous formazan globule was novel neurodegeneration that may implicate the specification of the vulnerable aging deterioration in the sacral spinal cord. We also found that a new circuit pathway: intermedial collateral pathway (ICP) showed by giant megaloneurite between the LCP and the MCP. In the medulla oblongata, ANBs were revealed in the gracile nucleus, nucleus reticularis lateralis (ventrolateral spinal trigeminal nucleus) and middle of the spinal trigeminal nucleus.

CONFLICT OF INTERESTS
The authors have no conflicts of interest to declare.