Cauda equina syndrome and nitric oxide synthase immunoreactivity in the spinal cord of the dog

The development of the cauda equina syndrome in the dog and the involvement of spinal nitric oxide synthase immunoreactivity (NOS-IR) and catalytic nitric oxide synthase (cNOS) activity were studied in a pain model caused by multiple cauda equina constrictions. Increased NOS-IR was found two days post-constriction in neurons of the deep dorsal horn and in large, mostly bipolar neurons located in the internal basal nucleus of Cajal seen along the medial border of the dorsal horn. Concomitantly, NOS-IR was detected in small neurons close to the medioventral border of the ventral horn. High NOS-IR appeared in a dense sacral vascular body close to the Lissauer tract in S1-S3 segments. Somatic and fiber-like NOS-IR appeared at five days post-constriction in the Lissauer tract and in the lateral and medial collateral pathways arising from the Lissauer tract. Both pathways were accompanied by a dense punctate NOS immunopositive staining. Simultaneously, the internal basal nucleus of Cajal and neuropil of this nucleus exhibited high NOS-IR. A significant decrease in the number of small NOS immunoreactive somata was noted in laminae I-II of L6-S2 segments at five days post-constriction while, at the same time, the number of NOS immunoreactive neurons located in laminae VIII and IX was significantly increased. Moreover, high immunopositivity in the sacral vascular body persisted along with a highly expressed NOS-IR staining of vessels supplying the dorsal sacral gray commissure and dorsal horn in S1-S3 segments. cNOS activity, based on a radioassay of compartmentalized gray and white matter regions of lower lumbar segments and non-compartmentalized gray and white matter of S1-S3 segments, proved to be highly variable for both post-constriction periods.     

Experimental cauda equina compression induces HSP70 synthesis in dog

The heat shock protein 70 (HSP70) is a key component of the stress response induced by various noxious conditions such as heat, oxygen stress, trauma and infection. In present study we have assessed the consequences of the compression of lower lumbar and sacral nerve roots caused by a multiple cauda equina constrictions (MCEC) on HSP70 immunoreactivity (HSP70-IR) in the dog. Our data indicate that constriction of central processes evokes HSP70 up-regulation in the spinal cord (L7, S1-Co3) as well as in the corresponding dorsal root ganglion cells (DRGs) (L7-S1) two days following injury. A limited number of bipolar or triangular HSP-IR neurons were found in the lateral collateral pathway (LCP) as well as in the pericentral region (lamina X) of the spinal cord. In contrast, a high number of HSP70 exhibiting motoneurons with fine processes appeared in the ventral horn (laminae VIII-IX) of lumbosacral segments. Concomitantly, close to them a few lightly HSP70-positive neuronal somata or cell bodies lacking the HSP70-IR occurred. In the DRGs, HSP70 expression was mildly up-regulated in small and medium-sized neurons and in satellite cells. On the contrary, DRGs from intact or sham-operated dogs did not reveal HSP70 specific neuronal staining. In conclusion, we have demonstrated that the MCEC in dogs mimicking the cauda equina syndrome in clinical settings evokes expression of HSP70 synthesis in specific neurons of the lumbo-sacro-coccygeal spinal cord segments and in small and medium sized neurons of corresponding DRGs. This suggests that HSP70 may play an active role in neuroprotective processes partly by maintaining intracellular protein integrity and preventing the neuronal degeneration in this experimental paradigm.     

Glucocorticoid Receptors and Enzyme Induction in the Spinal Cord of Rats: Effects of Acute Transection

The spinal cord is a glucocorticoid-responsive tissue, as demonstrated by hormonal effects on enzyme induction and by the presence of type II and type I glucocorticoid receptors in cytoplasmic extracts of this CNS region. Using microdissection techniques, we have found in the present investigation that glucocorticoid type II receptors are the most abundant class detected in gray (ventral and dorsal horns) and white (lateral funiculus) matter and that the distribution of type II sites among these regions was quantitatively similar. Type I sites were also quantified, with a slight prevalence in gray matter as opposed to white matter. Furthermore, stimulation of an inducible enzyme, ornithine decarboxylase (ODC), was found in ventral horn and lateral funiculus but not in dorsal horn after administration of dexamethasone (DEX), a type II receptor ligand. We also found that surgical transection of the spinal cord, while markedly increasing ODC activity per se, did not prevent the stimulatory effect of DEX administration on ODC activity measured in the lumbar enlargement of the spinal cord located below the surgical lesion. Taken together, the results suggest a direct effect of glucocorticoids on ODC activity in the spinal cord of rats, probably mediated by glucocorticoid receptors (type II) found in target cells of the ventral horn and lateral funiculus. The results also indicate that glucocorticoid receptors of the dorsal horn were not involved in ODC induction, and a function for these receptors awaits the results of further experimentation.     

Fos Protein Expression in Sacral Spinal Cord in Relation to Early Phase of Cauda Equina Syndrome in Dogs

1. The aim of the present study is to map the incipient phase of Fos expression in the sacral spinal cord neuronal pools of multiple cauda equina constrictions canine model.2. Fos-positive neurons were found bilaterally in the lateral portion of superficial dorsal horn layers (Laminae I–III) and along the lateral edge of the dorsal horn accompanied by the lateral collateral pathway, fibers of Lissauer's tract, terminating at the sacral parasympathetic nucleus. Similarly, high Fos expression was detected in the ventral portion of the dorsal sacral commissure and in the dorsomedial portion of the anterior horns at S₁–S₃ segment level. Finally, a clearly expressed Fos-positivity was disclosed bilaterally in the neuropil of the nucleus Y in the anterior horn.3. Data from the present study show that continuous stimulation of the central fibers of sacral dorsal root ganglia neurons, i.e., fibers of sacral primary afferents, unlike those using various stimulations of the peripheral fibres offers an unusual pattern of Fos-like immunoreactivity.     

The regional changes of the catalytic NOS activity in the spinal cord of the rabbit after repeated sublethal ischemia

The regional distribution of catalytic NOS activity was studied in the lumbosacral segments of the spinal cord of the rabbit during single (8-min), twice (8-, 8-min) and thrice repeated (8-, 8-, 9-min) sublethal ischemia followed each time by 1h of reperfusion. Single ischemia/reperfusion induced a significant increase of cNOS activity in almost all spinal cord regions, with the exception of non-significant increase in the dorsal horn. Sublethal ischemia repeated twice produced a significant decrease of enzyme activity in the intermediate zone and ventral horn and an increase in the white matter columns. Within thrice repeated ischemia, the activity of cNOS in the gray matter regions was similar to that found after a single ischemia/reperfusion. For all the animals subjected to single and twice repeated sublethal ischemic insults, there was no neurological impairment. Following thrice repeated ischemic insults, four out of five of the experimental animals recovered only partially and one was completely paraplegic. Our results do not indicate a cumulative effect of repeated sublethal ischemia on cNOS activity and, consequently, on NO production. The NO generated during thrice repeated ischemia/reperfusion appears to have a detrimental effect on the neurological outcome.     

AMINO ACID AND SUBSTANCE P CONTENTS IN SPINAL CORD OF CATS WITH EXPERIMENTAL HIND-LIMB RIGIDITY PRODUCED BY OCCLUSION OF SPINAL CORD BLOOD SUPPLY

Abstract— Experimental hind-limb rigidity of spinal origin was produced in cats by temporary occlusion of thoracic aorta and internal mammary arteries. In the lumbar segments (L6- S1) of these rigid cats, the monosynaptic reflex recorded from ventral roots was enhanced whereas the polysynaptic reflexes as well as the dorsal root reflexes were almost abolished. On morphological examination of the lumbar spinal cord, the number of interneurons was greatly reduced, whereas the small sized cells, presumably glial cells, were increased by about two times. Ventral horn motoneurons were also reduced. The lumbar spinal cords of the rigid cats were analysed for amino acid and substance P contents. Four major amino acids, aspartate, glutamate, glycine and GABA, were definitely reduced in both grey and white matter except that the glutamate level in the dorsal white was within the normal range. Content and distribution pattern of substance P were not altered in the lumbar cord of the rigid cats. These results are consistent with the notions that GABA occurs in the dorsal horn interneurons subserving primary afferent depolarisation, and that substance P is concentrated in primary afferent fibre terminals. The implications of the decrease of aspartate, glutamate and glycine in the spinal cord of rigid cats are discussed.     

Catalytic nitric oxide synthase activity in the white and gray matter regions of the spinal cord of rabbits

The latest research reveals that nitric oxide as a gas messenger may diffuse into the surrounding extracellular fluid and act locally upon neighboring target cells. However, several observations raise the possibility that nitric oxide may also be released at a greater distance from the neuronal cell body. The catalytic nitric oxide synthase (cNOS) activity was therefore studied in the cervicothoracic and lumbosacral segments of the spinal cord of rabbits, including the white matter of dorsal columns (DC), lateral columns (LC) and ventral columns (VC), as well as the gray matter of dorsal horns (DH), intermediate zone (IZ) and ventral horns (VH). Lower cNOS activity was found in the white matter of both cervicothoracic (47%) and lumbosacral (30%) regions, whereas that detected in the gray matter of the lumbosacral part of the spinal cord was considerably higher (70%). Enzyme activity varied from 43.4 to 77.2 dpm/microg protein in the cervicothoracic segments of the gray matter in the descending order: DH>VH>IZ. Similar cNOS activity was found in the white matter of the cervicothoracic segments (42.1-62.8 dpm/microg protein). When the activity of cNOS was compared in the lumbosacral segments, the highest enzyme activity was found in DH of the gray matter (198.7 dpm/microg protein) and the lowest cNOS in DC (45.8 dpm/microg protein) of the white matter. It was concluded that the white matter of the spinal cord contains similar cNOS activity in comparison to the gray matter.     

Distribution of Calcitonin Gene-Related Peptide in Rat and Rabbit Spinal Cords: Effect of Intrathecal 5,7-Dihydroxytryptamine

Calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) was measured in selected regions of the cervical, thoracic, and lumbar spinal cord of untreated rabbits and, following intrathecal injection of the serotonergic neurotoxin 5,7-dihydroxytryptamine (5,7-DHT), in the thoracolumbar cord in rats using a sheep antiserum raised against tyrosine0 calcitonin gene-related peptide28-37. In the cervical, thoracic, and lumbar segments of the rabbit spinal cord, CGRP-LI levels were 15-50-fold higher in the dorsal than in the ventral grey region in the same segment. The only segmental variation in CGRP-LI levels was in the dorsal white region, where levels in the thoracic cord were lower than those in cervical or lumbar segments. Within individual spinal segments, the pattern of distribution of CGRP-LI in the rabbit spinal cord was analogous to that in other species previously examined, including rat, human, and cat spinal cord. Intrathecal injection of 5,7-DHT, which caused 85-91% depletion of 5-hydroxytryptamine and 5-hydroxyindoleacetic acid from the thoracolumbar ventral spinal cord, did not affect choline acetyltransferase activity, which is colocalized with CGRP in motoneurones in this spinal cord region. In contrast, intrathecal 5,7-DHT produced a threefold increase in CGRP-LI in the ventral thoracolumbar cord, suggesting that spinal motoneurones selectively increase production of CGRP 10 days after neurotoxin-induced denervation of bulbospinal raphe neuronal input.     

Spatiotemporal Changes of NGF,BDNF and NT-3 in the Developing Spinal Cords of Embryonic Chicken

Spatiotemporal changes of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) in the spinal cords of chick embryonic stage day 7 (E7) and day 14 (E14) were examined by using immunohistochemistry and Western blot. Intensive NGF immunoreaction (IR) was detected in the white matter of the spinal cords, while BDNF-IR in perikaryon and neurite, and NT-3-IR in the nucleus and cytoplasm were seen in the neurons of the ventral horn in the gray matter. Comparatively, the expressions for three growth factors have expanded largely into the dorsal horn at E14, and the level of proteins for these growth factors increased significantly in the spinal cords from E7 to E14. Morphological observation showed that the lumbar spinal cords of E7 appeared rectangular, whereas it gave a butterfly shape in the gray matter consisting of the typical ventral horn, dorsal horn and intermediate zone at E14. The present findings indicated that the spatiotemporal changes of NGF, BDNF and NT-3 could be associated to the morphological changes of developing spinal cords, suggesting the possible roles of three growth factors in the development of spinal cords.     

QUANTITATIVE HISTOCHEMISTRY OF γ-AMINOBUTYRIC ACID IN CAT SPINAL CORD WITH SPECIAL REFERENCE TO PRESYNAPTIC INHIBITION

Abstract— A method is described for quantifying the GABA distribution in cat spinal cord at 200–500 μn resolution. Isolated spinal cord (L5–S1) was frozen and sectioned at about 150 μm thickness. The frozen tissue section was cut into 200 or 500 μm square blocks. The GABA content of each square tissue block was determined by enzymic micromethods and GABA distribution was mapped quantitatively. Average GABA concentrations were: 0·4 mmol/l. in white matter, 1·2 mmol/l. in ventral horn and 1·7 mmol/l. in dorsal horn. The highest concentrations of GABA (2–3 mmol/l.) were found in the dorsolateral part of dorsal horn. In order to destroy the interneurons of dorsal horn, the blood vessels supplying the dorsal horn of the lumbar enlargement were unilaterally cauterized. Seven to 30 days after operation, both the size of dorsal root potential and the GABA level in the dorsal horn were markedly reduced on the cauterized side. These results suggest that GABA is highly concentrated in the interneurons of dorsal horn and functions as a transmitter of presynaptic inhibition.     

Depletion of vesicular zinc in dorsal horn of spinal cord causes increased neuropathic pain in mice

Zinc enriched (ZEN) neurons and terminals are abundant in the rodent spinal cord. Zinc ions have been suggested to modulate the excitability of primary afferent fibers believed to be important in nociceptive transmission. To test the hypothesis that vesicular zinc concentration is related to neuropathic pain we applied Chung’s rodent pain model on BALB/c mice, and traced zinc transporter 3 (ZnT3) proteins and zinc ions with immunohistochemistry and autometallography (AMG), respectively. Under anesthesia the left fifth lumbar spinal nerve was ligated in male mice in order to produced neuropathic pain. The animals were then sacrificed 5 days later. The ZnT3 immunoreactivity was found to have decreased significantly in dorsal horn of fourth, fifth, and sixth lumbar segments. In parallel with the depressed ZnT3 immunoreactivity the amount of vesicular zinc decreased perceptibly in superficial gray matters of especially layer I-IV of the same segments. The transection-induced reduction of vesicular zinc in ZEN terminals of the dorsal horn was synchronic to reduced pain threshold, as measured by von Frey method. In a separate study, we observed intensive zinc selenite precipitation in somata of the smaller spinal ganglion cell, but 5 days after spinal nerve transection zinc precipitation was also found in the lager ganglion cells. The present results indicate that zinc may be involved in pain mechanism in the spinal ganglion level. These results support the hypothesis that vesicular zinc might have a modulatory role for neuropathic pain. Thus, increased pain sensitivity might be related to reduce vesicular zinc level in the dorsal spinal gray matter.     

Hrp-labeled sources of descending propriospinal pathways in cats

Unilateral injections of horseradish peroxidase into the cat spinal cord at different segmental levels revealed a laminar distribution of spinal interneurons that are sources of ipsilateral and contralateral propriospinal pathways of different lengths. The majority of the long pathways connecting cervical and lumbar segments are formed by neurons located in the central quadrants (laminae VII and VIII) bilaterally; a few such neurons also are present in the marginal layer and in lateral zones at the base of the dorsal horn (ipsilaterally). The zones containing numerous propriospinal neurons forming short (extending over a few segments) connections were more extensive. In the lumbar portion neurons which were sources of short uncrossed pathways tended to be concentrated in the lateral areas of the base of the dorsal horn, intermediate zone, and ventral horn, whereas sources of crossed pathways were concentrated in the ventromedial zones of gray matter. In the cervical portion "short" propriospinal neurons forming both ipsilateral and contralateral projections were concentrated in the lateral zones of gray matter. Neurons of the marginal layer and substantia gelatinosa and neurons of intermediolateral sympathetic nuclei also were sources of descending propriospinal pathways. Some propriospinal axons were intermediate in length. The distribution of neurons with axons of this kind largely coincided with the distribution of neurons that were sources of long propriospinal pathways. The connection between the spatial distribution of different groups of propriospinal neurons and the organization of the synaptic inputs into them, and also correlation between the morphological and functional characteristics of these neurons are discussed.     

Evoked activity of spinal neurons in the early period after sciatic nerve division

The character of dorsal horn motoneurons and interneurons evoked by stimulation of the dorsal root, and activity of Renshaw cells in response to stimulation of the ventral root were studied in albino rats in the lower lumbar segments of the spinal cord 5 days after sciatic nerve division. A significant increase in the mean amplitude of excitatory postsynaptic potentials of motoneurons was observed on the side of division of the nerve. No significant change in membrane potential and in the threshold of appearance of the action potential of these motoneurons took place. The mean number of action potentials and the duration of discharge of the Renshaw cells and dorsal horn interneurons likewise were not significantly changed.Dnepropetrovsk Medical Institute, Ukrainian Ministry of Health. Translated from Neirofiziologiya, Vol. 24, No. 3, pp. 306–314, May–June, 1992.     

Short-term changes of NADPH diaphorase-exhibiting neuronal pools in the spinal cord of rabbit after repeated sublethal ischemia

The aim of this study was the histochemical characterization of NADPH diaphorase-positive neuronal pools in the rabbit lumbosacral segments using a model of single, repeated and multiple sublethal spinal cord ischemia. Following a single 8-min sublethal spinal cord ischemia and 1-hour reperfusion, the staining of NADPH diaphorase-exhibiting neurons in the dorsal horn, pericentral region, dorsal gray commissure and sacral parasympathetic nucleus was comparable with the control sections. In contrast to the foregoing sublethal ischemia, a regionally different somatic NADPH diaphorase (NADPHd) staining was found after multiple sublethal spinal cord ischemia. Whereas an almost complete loss of the staining of large NADPHd-exhibiting somata in the pericentral region was detected, the staining of the NADPHd-exhibiting neuronal pools in the deep dorsal horn and sacral parasympathetic nucleus was fully preserved. Concomitantly, a prominent reduction of small NADPH diaphorase-positive neurons was noted in the superficial dorsal horn layers of lower lumbar and sacral segments.     

扬子鳄颈髓一氧化氮合酶(NOS)和乙酰胆碱酯酶(AChE)阳性神经元的分布

本实验分别应用还原型尼克酰胺嘌呤二核苷酸脱氢酶（ＮＡＤＰＨ－ｄ）和乙酰胆碱酯酶（ＡＣｈＥ）方法,对扬子鳄颈髓ＮＯＳ和ＡＣｈＥ阳性神经元的分布进行了研究。结果表明：颈髓前角、中央灰质均含有ＮＯＳ和ＡＣｈＥ阳性神经元,颈髓后角有较为丰富的ＮＯＳ和ＡＣｈＥ阳性纤维和终末以及显色淡的ＮＯＳ阳性神经元。     

Sciatic nerve transection decrease substance P immunoreactivity in the lumbosacral spinal cord of the frog (Rana catesbeiana)

Using immunohistochemistry and optical densitometry, substance P (SP) was investigated in the lumbar spinal cord of the frog Rana catesbeiana after sciatic nerve transection. In control animals, there was a high density of SP fibers in the Lissauer's tract and in the mediolateral band of the dorsal gray matter. Other SP immunoreactive fibers were observed in the dorsal part of the lateral funiculus and in the ventral horn. No SP label was found in any cell bodies. After axotomy, SP immunoreactive fibers decreased in the Lissauer's tract on the same side of the lesion. The other regions remained labeled. The changes were observed at 3 days following axonal injury and persisted at 5, 8 and 15 days. At 20 days, there was no significant difference between the axotomized side and the control one, thus indicating a recovery of the SP expression. These results indicate that the frog may be used as a model to study the effects of peripheral axotomy, contributing to elucidate the SP actions in the pain neuropath.     

Inhibitory effects of dorsal horn and excitant effects of ventral horn intraspinal microinjections of norepinephrine and serotonin in the cat

Norepinephrine and serotonin microinjections (10 μg) into the dorsal spinal gray matter depressed C-fiber reflexes induced by electrical stimulation of the superficial peroneal nerve or radiant heat stimulation of the footpad. Microinjection of norepinephrine or serotonin into the ventral gray matter facilitated C-fiber reflexes. These studies lend support to the suggestion that dorsal horn projections of neuronal systems which utilized norepinephrine or serotonin as neurotransmitters inhibit nociceptive spinal reflexes, and ventral horn projections facilitate spinal reflexes.     

A modified peroxidase--antiperoxidase procedure for improved localization of tissue antigens: localization of substance P in rat spinal cord

A procedure is presented which modifies the Sternberger peroxidase--antiperoxidase (PAP) technique in order to visualize additional amounts of immunodeposits representing the antigen substance (SP) in 5-micrometer paraffin tissue sections of rat spinal cord. For increased sensitivity, the new procedure utilizes a "double bridge" and diaminobenzidine in low pH buffer. The modifications have made possible the visualization of immunoreactive beaded processes and punctate bodies, which were then traced to determine patterns of SP circuitry. Using the modified PAP procedure, the greatest number of immunoreactive processes appeared in the dorsal horn, where some punctate bodies and varicose processes could be seen adjacent to the myelinated afferent fiber bundles that penetrate the substantia gelatinosa as dorsal root collaterals. Additional immunoreactive processes and punctate bodies coursed through the myelinated afferent fiber bundles that penetrate the dorsolateral white matter, and extend into the intermediolateral gray region. Substance P was also identified within immunoreactive processes found in Rexed's laminae V and VI, as well as the central canal region, the dorsal gray commissure, and the ventral gray and white commissures. Since the modifications improved the visualization of SP-containing processes in sparsely populated regions of the spinal cord, especially the ventral horn, they may be useful in demonstrating other antigens that normally occur in small quantities within tissues.     

Characterisation of transverse slice culture preparations of postnatal rat spinal cord: preservation of defined neuronal populations

Spinal cord injury induces degenerative and regenerative processes and complex interactions of neurons with non-neuronal cells. In order to develop an in vitro tool for the investigation of such processes, we prepared and characterised spinal cord slice cultures (SCSC) from Wistar rats (p0–12). SCSC were sustained in vitro up to 12 days and characterised by immunohistochemistry. Calbindin⁺ neurons, distributed across the entire gray matter, were visible also after longer culture periods. NeuN⁺ neurons were best preserved in the dorsal horn whereas large NeuN⁺ and choline acetyltransferase⁺ motoneurons in the ventral horn vanished after 3 days in vitro. Nestin immunoreactivity was found in animals of all age groups, either in cells interspersed in the ependymal lining around the central canal or in cells resembling protoplasmic astrocytes. Glial fibrillary acidic protein⁺ astrocytes, initially restricted to the white matter, invaded the gray matter of SCSC early during the culture period. Microglial cells, stained by Griffonia simplicifolia isolectin B₄, were rapidly activated in the dorsal tract and in the gray matter but declined in number with time. SCSC derived from p0 or p3 animals showed a better preservation of the cytoarchitecture than cultures derived from older animals. In summary, SCSC undergo degenerative changes, but they contain defined neuronal populations, the cytoarchitecture is partially preserved and the glial reaction is limited.     

Responses of spontaneously active spinal neurons to temperature stimulation of the skin

Changes in spontaneous unit activity in the dorsal zones of the spinal cord in response to temperature stimulation of the skin of the thigh and leg were investigated in acute experiments on cats lightly anesthetized with pentobarbital. Two groups of neurons were distinguished by the character of their response. The firing rate of the first group of neurons was changed only in response to cold or warmth. The neurons of the second group responded in opposite ways to the two types of stimulation. Frequency characteristics of spontaneous activity of temperature-sensitive units located in both white and gray matter were determined. In all the lumbar segments their depth was not below the 5th layer of gray matter. Mechanisms of the changes in spontaneous activity and the possible pathways of conduction of temperature sensation are discussed.