Introduction

The lumbosacral spinal cord is unique among spinal segments due to its autonomic centers that regulate the pelvic visceral organs1. Within this region, the sacral parasympathetic nucleus (SPN)2 and the dorsal gray commissure (DGC) are critical for receiving projections from primary visceral and somatic afferent fibers originating from the colon3, bladder4,5,6, external urethral sphincter5,7, urethra8 and the pudendal nerve9,10. Somatic and visceral afferent fibers extend from the dorsal root entry zone (DREZ) through Lissauer’s tract (LT) to the region of the SPN and/or reach the DGC through the lateral collateral pathway (LCP) and medial collateral pathway (MCP)10,11,12,13,14. In addition, the gracile nucleus in the medulla oblongata plays a critical function in transmitting and processing visceral and somatosensory information15,16. Visceral and somatosensory signals ascend along the posterior funiculus of the sacral spinal cord to the gracile nucleus, which then emanates fibers that continue to ascend to higher parts of the brain15.

The nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) has been used as a histochemical marker in both central and peripheral nervous systems17. The NADPH-d activity has been used to study neuritic dystrophy in the aging condition18 as well as neurodegenerative changes19,20,21. The NADPH-d activity is widespread in visceral and somatosensory pathways and is associated with neuronal senility changes in the sacral spinal cord and gracile nucleus22,23,24. The large number of NADPH-d positive neurons in the spinal cord appears to be involved in visceral regulation, innervating most of the pelvic visceral organ25,26,27,28. Notably, NADPH-d positive reactivity in the sacral spinal cord is distributed in the visceral afferent pathway29,30,31, which is very similar to the central projections of afferent neurons innervating the pelvic viscera30,32.

Our previous studies have demonstrated that the aging-related NADPH-d positive bodies (ANBs) specifically appear in the sacral spinal cord of aged rats33. ANBs are clearly distinct from normal neural structures, with distinctive morphological and distributional characteristics, and are a distinctive age-related neuroanatomical change. ANBs present spherical or multi-angular shapes existed in the dorsal region of the aged sacral spinal cord. With increasing age, there is a gradual increase in the incidence and size of ANBs. The major distribution of ANBs indicates some anomalous neuronal changes that may account for the disturbance of autonomic and sensory pathways in the pelvic visceral organs in the aging condition.

Recently, we also found NADPH-d positive megaloneurites in the sacral spinal cord of aged dogs34. Megaloneurites are an aging-related aberrant structures characterized by abnormally enlarged diameter and length that are distinct from normal neural structures and are not found in young dogs. Megaloneurites are selectively present on dorsal sensory afferent pathways in the sacral spinal cord and are usually co-localized with vasoactive intestinal peptide (VIP) immunoreactivity, suggesting a potential role in the autonomic nervous system. Megaloneurites may disrupt sensory afferent pathways in the autonomic nervous system, leading to pelvic visceral dysfunction with aging, and serve as a biomarker for age-related neurodegenerative changes in the spinal cord.

ANBs are clearly distinguished from megaloneurites, but both forms of abnormal alterations may be associated with aging and potential neurodegenerative changes. These findings have prompted us to investigate the prevalence of aging-related neurodegenerations across different species, particularly in non-human primates, which are evolutionarily closer to humans. In the present study, we aimed to determine the occurrence and characterization of megaloneurites and/or ANBs in the sacral spinal cord of non-human primates. These degenerative changes, if present, may act as markers of aging in the pelvic visceral organs and could provide valuable information for understanding the neuroanatomical changes that accompany aging and potentially provide targets for therapeutic interventions in aging-related neurodegenerative diseases.

Results

NADPH diaphorase activity in the sacral spinal cord

The NADPH-d positive reactivities were extensively distributed in the dorsal regions of the monkey’s spinal cord, and in the sacral spinal cord, where we focused our attention, a substantial amount of NADPH-d positive reactivities could be detected in the regions of the DREZ, LCP, SPN and DGC (Fig. 1A). Notably, we found abnormal megaloneurites and ANBs in these regions of the sacral spinal cord in aged monkeys, which were not present in young monkeys. The NADPH-d staining revealed a moderately dense fiber network of dendrites and axon terminals as well as moderately stained neurons in the dorsal horn, DREZ, LCP, and DGC of the sacral spinal cord of young monkeys (Fig. 1B, C). In the sacral spinal cord of aged monkeys, NADPH-d positive megaloneurites (Fig. 1D-G) were predominantly distributed in the DGC and LCP, and ANBs (black arrowheads in Fig. 1D, H, and I) were found largely in the SPN and superficial dorsal horn. Megaloneurites and ANBs were selectively detected in the sacral spinal cord but not in the lumbar or thoracic and cervical segments (Fig. 1J-L). The general location and selective segmental distribution of the NADPH-d positive megaloneurites and ANBs were related to the central projection of the primary pelvic visceral sensation, mostly located dorsal of the sacral spinal cord.

Figure 1
Figure 1
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Microphotographs of NADPH-d positive reactivity in young and aged monkeys at the sacral spinal cord. All of the transverse sections are taken at the same levels. The illustration represents the magnification of the corresponding rectangular frame. A demonstrates the schematic diagram of the distribution NADPH-d staining in transverse sections at different spinal cord segments in monkeys. Black circles in the schematic diagram represent NADPH-d positive neurons, blue shapes represent megaloneurites, and green asterisks represent ANBs. NADPH-d positive reactivity in the dorsal horn and DGC of sacral spinal cord of young (B, C) and aged (D, E) monkeys. The megaloneurites and ANBs in the sacral spinal cord of aged monkeys are completely different from the surrounding normal fibers and neurons (F-I). The NADPH-d positive activity in the dorsal horn of cervical (J), thoracic (K) and lumbar (L) segments of aged monkeys. Frequency distribution of diameters of NADPH-positive fibers in the sacral spinal cord of aged (M) and young (N) monkeys. Comparison of the diameter of NADPH diphosphatase positive fibers in young and aged monkeys (O). The asterisks indicate vascular structures. Open arrowheads: NADPH-d positive neurons, black arrowheads: ANBs, open arrows: normal NADPH-d positive neurites, white arrowheads: megaloneurites. Scale bar in B, D, J-L = 100 μm, in C, E, F-I = 50 μm, ***P < 0.001.

Statistical data showed that in the sacral spinal cord of aged monkeys, the diameter of the majority of the NADPH-d megaloneurites ranged from 2 to 6 μm, with the thickest ones reaching 9 μm, whereas the diameter of normal fibers was typically less than 1 μm (Fig. 1M). In the sacral spinal cord of young monkeys, the diameter of most fibers was less than 1 μm, and no megaloneurites were detected (Fig. 1N). The average diameter of megaloneurites (3.718 ± 0.093 μm) was significantly thicker than the diameter of normal fibers (1.124 ± 0.017 μm) in aged monkeys; the diameter of normal fibers in aged monkeys was also significantly thicker than that of normal fibers in young (0.827 ± 0.013 μm) (Fig. 1O).

NADPH-d activity in the white matter and the DREZ of the sacral spinal cord

In transverse sections of the sacral spinal cord of aged monkeys, massive punctate NADPH-d aberrant reactivities (Fig. 2A) were detected in the lateral white matter of the LCP compared with that of young monkeys (Fig. 2B), and these abnormal alterations were dramatically different from normal nerve cells. It was confirmed to be similar to megaloneurites, which may be in close association with megaloneurites penetrating deeply into the white matter. Longitudinal sections of the sacral spinal cord indicated that the punctate NADPH-d alterations were longitudinally-arranged fibrous extending rostro-caudally and were greatly different from normal fiber bundles and vascular structures in the white matter (Fig. 2C). Numerous NADPH-d positive fibers and varicosities were evident in the lateral white matter of the LCP in young monkeys, and in some instances, these fibers could be traced along the whole length of the section (Fig. 2D). Given the same morphology and characteristics, we still refer to the aged and segmental related alterations in the white matter as megaloneurites.

Figure 2
Figure 2
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The NADPH-d positive alterations in the white matter and DREZ of the sacral spinal cord. The location and morphology of the megaloneurites and ANBs in the white matter and LCP of the sacral spinal cord of aged monkeys (A). NADPH-d positive activity in the sacral spinal cord of young monkeys (B). In the longitudinal section, NADPH-d staining fibers in the white matter of the lateral fasciculus of the aged monkeys (C) and young monkeys (D). The distribution of NADPH-d activities in the DREZ of the sacral spinal cord of aged monkeys (E) and young monkeys (F). Longitudinal sections of aged monkeys (G) at the sacral segment showed aberrant NADPH-d positive megaloneurites in the DREZ, transitionally continuous with LT. The illustration represents the magnification of the corresponding rectangular frame. Asterisks indicate vascular structures in C and D. Open arrowheads: NADPH-d positive neurons, black arrowheads: ANBs, open arrows: normal NADPH-d positive neurites, white arrowheads: megaloneurites. Scale bar = 50 μm.

In the transverse sections of the sacral spinal cord of aged monkeys (Fig. 2E), showed numerous punctate intensely NADPH-d activities in the DREZ accompanied with the megaloneurites. These punctate NADPH-d abnormalities occurred specifically in the caudal spinal cord of the aged and were not present in young animals (Fig. 2F). In longitudinal sections, these punctate NADPH-d alterations had the same morphology as megaloneurites (Fig. 2G). The properties of the punctate anomalous structures in the longitudinal sections were that the afferent fibers from the dorsal rootlets underwent some pathological changes in the DREZ, forming NADPH-d megaloneurites that were continuous with Lissauer’s tract (Fig. 2G). Compared to the other segments of the spinal cords, the megaloneurites mainly occurred in the DREZ of the sacral segment in aged monkeys.

Double staining with three neuropeptides

The double-staining of NADPH-d histochemistry combined with CGRP, Neuropeptide Y and VIP immunofluorescence were used to characterize megaloneurites and ANBs. In the DGC, no structures corresponding to NADPH-d positive megaloneurites were detected by Neuropeptide Y (Fig. 3A-C), CGRP immunofluorescence (Fig. 3D-F). For VIP immunoreactivity, VIP and NADPH-d megaloneurites co-localized positively in DGC (white arrowheads in Fig. 3G-I). Similarly, in the white matter, LCP and DREZ of the sacral spinal cord of aged monkeys, NADPH-d positive megaloneurites were not co-localized with Neuropeptide Y (Fig. 4A-C, J-L) or CGRP (Fig. 4D-F, M-O) immunofluorescence, whereas co-localized with VIP immunofluorescence (Fig. 4G-I, P-R). However, the ANBs (black arrowheads) distributed in the superficial dorsal horn and SPN were not co-localized with the VIP immunofluorescence, indicating that the composition of the ANB was different from the NADPH-d positive megaloneurites and they were two different degenerative structures appearing in the lumbosacral spinal cord.

Figure 3
Figure 3
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The double-staining of NADPH-d histochemistry combined with Neuropeptide Y, CGRP and VIP immunofluorescence in the DGC of the sacral spinal cord from aged monkeys (n = 3). Neuropeptide Y (A-C) and CGRP (D-F) immunofluorescence failed to label the megaloneurites, whereas VIP immunofluorescence co-localized with the megaloneurites (G-I). Asterisks indicate vascular structures. Black/white arrows: megaloneurites, open arrowheads: normal NADPH-d positive neurons. Scale bar = 50 μm.

Figure 4
Figure 4
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The double-staining of NADPH-d histochemistry combined with Neuropeptide Y, CGRP and VIP immunofluorescence in the white matter, LCP and DREZ of the sacral spinal cord from aged monkeys (n = 3). The megaloneurites were not co-localized with Neuropeptide Y (A-C, J-L) or CGRP (D-F, M-O) immunofluorescence in the white matter, LCP and DREZ but were labelled by VIP immunofluorescence (G-I, P-R). The ANBs (black arrows) distributed in the superficial dorsal horn and SPN were not co-localized with the VIP immunofluorescence. Black arrowheads: ANBs, black/white arrows: megaloneurites. Scale bar = 50 μm.

NADPH-d activity in the medulla oblongata

In the medulla oblongata of aged monkeys, primarily in the gracile nucleus and cuneate nucleus (Fig. 5A-D), a large number of spheroidal ANBs were detected compared with young monkeys (Fig. 5E-H). These results coincide with those obtained from previous studies in the gracile nucleus of aged rats35. ANBs in the gracile nucleus and cuneate nucleus were not co-labelled with VIP immunoreactivity (data not shown here).

Figure 5
Figure 5
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Microphotographs of NADPH-d positive reactivity in the gracile nucleus and cuneate nucleus of age and young monkeys. Note abnormal spherical ANBs (black arrowheads) in the gracile nucleus (A-B) and cuneate nucleus (C-D) of aged monkeys. The regular NADPH-d positive neurons were observed in the gracile nucleus (E-F) and cuneate nucleus (G-H) of young monkeys. Open arrowheads: NADPH-d positive neurons, black arrowheads: ANBs. Scale bar in A, C, E, G = 100 μm, in B, D, F, H = 50 μm.

Discussion

Many studies have proven that NADPH-d reactivity occurs extensively in the neurons and sensory pathways in the spinal cord29,36,37,38,39,40. Our previous study demonstrated that ANBs occurred in the lumbosacral spinal cord of aged rats33. Recent studies have also shown that NADPH-d positive megaloneurites, a novel neurodegeneration, were found in the sacral spinal cord of aged dogs34. Although the normal and some experimental NADPH-d positivity were studied in the spinal cord of monkeys, few reports were available on the aging-related NADPH-d alterations in the sacral spinal cord41.

The major finding of this study was the identification of two forms of neurodegeneration, ANBs and megaloneurites, in the sacral spinal cord of aged monkeys, and the description of their basic morphological properties, segmental and laminar distribution. Aging neuropathological studies of NADPH-d positivity could be used to reveal the neuronal terminal-pathy of aged conditions23 and neurodegenerative animal models42. In our previous study, ANBs were detected in the gracile nucleus and lumbosacral spinal cord of aged rats36. We similarly detected spherical aberrant ANBs in the gracile nucleus and cuneate nucleus as well as in the dorsal horn of the sacral spinal cord of aged monkeys. The central-projecting axons of the first-order sensory neurons in DRG could configure bifurcation in the spinal cord. The bifurcating branches could terminate in the corresponding spinal segments and ascend to dorsal column nuclei respectively43. Besides somatic sensory inputs, dorsal column nuclei also receive visceral sensory inputs16. We considered that the ANBs in aged rats and monkeys were postulated as dying back in the lumbosacral spinal cord and gracile nucleus accompanied by aging33,35,44,45.

The NADPH-d positive megaloneurites with morphology and organization were first discovered in the sacral spinal cord in aged dogs34. The megaloneurites were different from the previously reported meganeurites, which occurred proximal to axonal initial segments of the somas in younger organisms46. The anatomical locations of the two structures are also strikingly different. The megaloneurites were considered to be swollen neuronal processes, which were enlarged-diameter formations much longer than meganeurites46. Normal NADPH-d positive fibers stained with clear punctate and considerable varicosities. Megaloneurites were intensively stained with less varicosities, and traceable for substantial distances in sacral segments, especially in the longitudinal sections.

Similar to the experimental results in the sacral spinal cord of aged dogs34, the dense hypertrophic megaloneurites extend from DREZ through LT along the lateral edge of the dorsal horn to the SPN and DGC in the sacral spinal cord of aged monkeys, and the fibers in this pathway might play a role in visceral reflex pathways11,14, as the DGC and the LCP receive terminations from the somatic and visceral afferents11,12,13,47. Functionally, the sacral spinal cord was known to be associated with bowel bladder, and sexual dysfunction48,49,50. Previous tracing experiments in rats have also demonstrated that a large percentage of afferent neurons projecting to pelvic visceral organs and specifically to the bladder and urethra exhibit NADPH-d activity6,30,51. The distribution of the megaloneurites overlapped both the efferent and afferent pathways of the autonomic system, which regulates the pelvic organs. Segmental specialized aged-related changes may be also considered for the segmental distribution of NADPH-d neurons in the spinal cord52. The neuron disorder of aging may be caused by environmental factors. For example, NADPH-d neurons in the white matter are also vulnerable to chronic heavy metal poisoning53.

The megaloneurites were characterized as longitudinally extending fiber bundles in both transverse and longitudinal sections of the sacral spinal cord. The morphology of megaloneurites was clearly distinguished from the vascular structures found under light microscopy. The megaloneurites occurred in the DREZ, which is the transitional location between the peripheral nervous system and CNS in the spinal cord and brain stem54. The pelvic visceral sensory information from the urogenital organs is routed to the sacral spinal cord through centrally-projecting axons that pass into the CNS via the DREZ and continue with LT. DREZ plays an important role in neurodegeneration55 and axon routing during development56. All these segmental selective deteriorations demonstrated that the sacral segments exited relevant adverse conditions more vulnerable to aging degenerations.

The double-staining experiment demonstrated that VIP immunoreactivity and NADPH-d megaloneurites positively co-localized in the DREZ, DGC, LCP and white matter of the sacral spinal cord of aged monkeys. VIP is involved in the information transmission and physiological regulation of many functions of the organism and is released in the central and peripheral nervous system to support neuronal survival under physiological and pathological conditions57,58. Studies have shown that VIP can be considered as a marker for parasympathetic nerves59 and that VIP-containing fibers and terminals are well distributed in the lumbosacral spinal cord and play an important role in the efferent and afferent pathways of the autonomic nervous system that innervate the pelvic organs60,61,62,63,64. In our previous study, megaloneurites in the sacral spinal cord of aged dogs similarly overlapped with the distribution of VIP immunoreactivity, suggesting that megaloneurites may affect sensory afferent pathways in the autonomic nervous system in the aging condition, leading to pelvic visceral dysfunction.

Conclusion

In summary, this study revealed a special occurrence of the megaloneurites and ANBs in the sacral spinal cord of aged monkeys. These results confirm our previous discovery in aged rats and dogs, respectively. Furthermore, NADPH-d megaloneurites could be co-labelled by VIP immunofluorescence, whereas ANBs were not, indicating that megaloneurites and ANBs were two distinct forms of neurodegeneration. Our results suggested that megaloneurites and ANBs are recognized as specific aging markers associated with progressive aging deterioration and aberrant structures.

Materials and methods

Animal and tissue preparation

Young (3.8 ± 0.7 years old, n = 4) and aged (23.8 ± 4.3 years old, n = 5) female cynomolgus monkeys, weighing 3–5 kg, were used in our experiments. All animals were housed under a 12:12-h light/dark cycle in the animal care facilities. The facilities are certified by the Council on Accreditation of the Association for Assessment and Accreditation of Laboratory Animals Care (International) and China National Accreditation Service for Conformity Assessment. The ambient temperature was maintained at 24 ± 2 ℃ and relative humidity at 65 ± 4%. Reverse osmosis water was available ad libitum. Normal food, fresh fruit and vegetables were supplied twice daily. Animals were purchased from Beijing Xierxin Biological Resources Research Institute (Permit Number: 11805300010425, Beijing, China), Wincon TheraCells Biotechnologies Co., Ltd. (Permit Number: WD-0312010, Nanning, China) and Hainan Jingang Biotech Co., Ltd (Permit Number: 46001200000114, Hainan, China). These animals did not show any neurological deficits before experiments and were humanely euthanized. The experimentations have been conducted in accordance with the ARRIVE guidelines (https://arriveguidelines.org). All experimental procedures followed the International Guiding Principles for Biomedical Research Involving Animals and the Laboratory Animal-Guideline for ethical review of animal welfare (GB/T 35892 − 2018) and were approved by the Ethics Committee in Animal and Human Experimentation of the Jinzhou Medical University (IACUC Number: 2014JCATBH0E-NNSF-0004).

The animals were anesthetized with sodium pentobarbital (Laboratory Animal-Guidelines for euthanasia, GB/T 39760 − 2018, 80 mg/kg i.v.) and perfused transcardially with saline followed by 4% paraformaldehyde in a 0.01 M phosphate buffer saline (PBS, pH 7.4). Following perfusion fixation, the spinal cords and brain were obtained and placed in 25% sucrose for 48 h. Following dehydration and fixation, the spinal cord and medulla oblongata tissues were divided into cervical, thoracic, lumbar, and sacral segments, as well as the gracile nucleus of the medulla. A subset of these tissues (from three aged and two young monkeys) was sectioned transversely into 40 μm slices using a cryostat. To observe the longitudinal morphology and distribution of NADPH-diaphorase positivity, additional monkeys (two aged and two young) were selected, and longitudinal sections of 40 μm in thickness were prepared from each spinal segment. To facilitate the comparison between transverse and longitudinal sections from the same tissue, a 5 mm length of each spinal segment was sectioned transversely during the preparation of longitudinal slices. Sections from each segment were divided equally into three sets and collected into well plates containing 0.3% Triton X-100 in PBS.

NADPH diaphorase histochemistry

NADPH-d staining was performed with transverse and longitudinal sections from one-third-well plates of each young and aged animal, and free-floating sections were used for NADPH-d staining33,34. The tissue sections were stained by NADPH-d histochemistry, with incubation in 0.01 M PBS (PH 7.4), 0.3% Triton X-100 containing 1.0 mM reduced-NADPH (Sigma, St. Louis, MO, USA) and 0.2 mM nitro blue tetrazolium (NBT, Sigma), at 37℃ for 2 to 3 h. The reaction was stopped by washing the sections with 0.01 M PBS.

Double immunofluorescence staining

Following NADPH-d histochemical staining of the sacral spinal cord sections, a selection of 30 slices of adjacent sections with typical megaloneurites from each aged animal (three aged animals included) were subjected to double-staining of NADPH-d with calcitonin gene-related peptide (CGRP), vasoactive intestinal peptide (VIP) or neuropeptide Y (NPY) immunofluorescence, respectively. The sections were collected in 24-well plates containing 0.01 M PBS and washed three times. Then sections were incubated for 1 h at room temperature in blocking solution (0.01 M PBS) at pH 7.4 with 1% BSA and processed for free-floating immunofluorescence using primary polyclonal antibodies that label CGRP (mouse; 1:100, Sigma, C7113, USA), VIP (rabbit, 1:1000, Sigma, HPA017324, USA), NPY (rabbit; 1:2000, Sigma, N9528, USA). The sections were incubated with the primary antibody in PBS containing 1% BSA for 24 h at 4 °C, respectively. For a negative control, a few of sections were incubated without primary antibody in each immunofluorescence test. The sections were then washed three times with PBS. Fluorescent-conjugated secondary antibodies (Goat anti-Rabbit IgG (H + L), Alexa Fluor 594 [1:800, Life] and Goat anti-Mouse IgG, Alexa Fluor 488 [1:800, Life]), were applied to the sections for 1.5 h at 37 °C in the dark. Finally, after several washes with PBS, the sections were counterstained with DAPI for 10 min. The sections were mounted onto slides and cover-slipped. For controls of immunofluorescence staining omitted the primary antibody, no specified immunostaining was detected in the control experiments.

Image acquisition and measurement of fibers

Images were captured with a DP80 camera in an Olympus BX53 microscope (Olympus, Japan). Sections were observed under the light microscope and 20 sections from the sacral spinal cord in each animal were quantitated using Olympus image analysis software (Cellsens Standard, Olympus). The diameter of 500 NADPH-d megaloneurites and normal fibers were also measured with Neurolucida 360 (MBF Bioscience, Inc, USA). Unbiased rating was set by the examiners who were not aware of the grouped labelling in the sections.

Statistics analysis and figure edition

GraphPad Prism 9.0 (GraphPad Software, La Jolla, CA) software was used to analyze the frequency distribution and the differences in the diameters of NADPH-d positive fibers in the sacral spinal cord of young and aged monkeys. To quantify and characterize the distribution of NADPH-d positive fibers, we statistically analyze the frequency distribution of the diameters of megaloneurites and normal fibers in the sacral spinal cord of aged and young monkeys. In addition, to analyze the differences in the diameter of NADPH-d positive normal fibers and megaloneurites within the sacral spinal cord across different age groups, for consistently normally distributed data, we used unpaired t-tests to compare the diameters of NADPH-d positive fibers in young and aged monkeys. Data are expressed as mean ± standard error, and the significance level was set at P < 0.05. Figures were edited with CorelDRAW Graphics Suite 2020 (Ottawa, Canada).