Calcitonin gene-related peptide staining intensity is reduced in rat lumbar motoneurons after spinal cord transection: a quantitative immunocytochemical study

Summary The expression of Calcitonin gene-related peptide (CGRP) has been demonstrated in motoneurons of several species. We have investigated in adult rats the influence of transection of the spinal cord on CGRP immunoreactivity of motoneurons located below the section. Quantative analysis has been performed with computer-assisted image analysis. As early as 48 h after the section, CGRP immunoreactivity is modified, and the reduction is maximal after one month. Then, both the number of immunoreactive cells and the intensity of staining increase until the 5th month. It is concluded that the expression of CGRP is under the influence of supraspinal afferents to the motoneuron.     

Basal lamina at the site of spinal cord transection in the rat: An ultrastructural study

This electron microscopic study confirms that basal lamina (BL) begins to cap the cut end of the spinal cord 15 days after spinal cord transection. BL is first seen immediately adjacent to reactive glial cells but only when there is collagen in the nearby interstitial space. This finding suggests that collagen may provide a trigger to initiate the production of BL by reactive glia. We found no direct evidence that BL in this injury area impeded the outgrowth of regenerating neurites.     

Optical imaging of vascular and metabolic responses in the lumbar spinal cord after T10 transection in rats

Neuronal and vascular reorganization after spinal cord injury (SCI) is scarcely known although its characterization has major implications in understanding the functioning of the altered spinal cord. Several electrophysiological and anatomical lines of evidence support plasticity caudal to the lesion site, but do not provide sufficient clues about neuronal and vascular reorganization after SCI. The aim of the present study was to compare neuronal activation in the lumbar spinal cord between uninjured and SCI rats with novel optical imaging technology. The results showed significant haemodynamic response differences after sciatic nerve stimulation in uninjured controls, in comparison to SCI rats. Both timing and shape of the response were modified. In uninjured rats, blood flow presented an initial dip but was rapidly drained from the activation site through the venous system. In comparison, the blood transfer rate in SCI rats was much slower. Damaged blood vessels at the lesion site after thoracic SCI impacted the vascular response upon neuronal activation in the lumbar spinal cord. This observation is important in the study of spinal cord function after SCI by imaging techniques based on haemodynamics (blood oxygenation level-dependent using functional magnetic resonance imaging (BOLD fMRI) and optical imaging). In conclusion, our results indicate that new avenues quantifying the influence of vascular plumbing will have to be developed to explore the efficacy of rehabilitation and pharmacological therapies by haemodynamic imaging.     

iNOS expression in rat aorta is increased after spinal cord transection: a possible cause of orthostatic hypotension in man

Orthostatic hypotension commonly occurs in persons with spinal cord injury (SCI), limiting rehabilitation and independence. Findings of increased production of nitric oxide (NO) by inducible nitric oxide synthase (iNOS) after exposure to simulated microgravity suggest that increased iNOS expression contributes to OH in persons with SCI. To test this possibility, male Wistar rats underwent surgical transection of the spinal cord (T10) or sham-SCI surgery followed by euthanasia 3, 7 or 14 days later. Expression in thoracic aortic of inducible (iNOS), endothelial (eNOS) and neuronal (nNOS) NOS was then determined. In SCI rats, expression of iNOS mRNA was decreased at 3 days, had returned to normal levels of expression at 7 days and was increased at 14 days post-SCI (1.8-fold). In contrast, levels of eNOS mRNA were increased at 3 days (1.4-fold), then declined over time reaching levels by day 14 that were reduced compared to sham-SCI (0.23-fold). There were no significant effects of SCI on nNOS expression. These findings suggest a possible role for increased iNOS expression in the pathogenesis of OH in persons with SCI.     

Serotonergic neuron system in the spinal cord of the gar Lepisosteus oculatus (Lepisosteiformes,Osteichthyes) with special regard to the juxtameningeal serotonergic plexus as a paracrine site

Immunohistochemical and electron microscopic studies were carried out to elucidate the structure of the serotonergic neuron system in the spinal cord of the spotted gar, Lepisosteus oculatus, a nonteleost actinopterygian. Serotonin-immunoreactive (5HT-IR) cell bodies and fibers were widely distributed in the spinal cord, constituting an intrinsic neuron system. This system comprised three anatomical cell groups in different portions of the spinal cord, i.e., the rostromedial cell group, the paired ventrolateral cell groups, and the ventral superficial cell group. The rostromedial cell group included cerebrospinal fluid-contacting neurons with intraventricular processes. The immunostained fibers projecting from all three of these cell groups ran in various directions, mainly ventrally and ventrolaterally, and partly gave rise to a dense plexus at the ventrolateral surface of the spinal cord. Immunoelectron microscopy of the relevant portion demonstrated many varicose fibers containing 5HT-immunopositive vesicles. Conventional electron microscopy of the plexus showed that the constituent varicose fibers were unmyelinated and frequently made a direct contact with the basement membrane contiguous to the leptomeniges (meninx primitiva). There, exocytotic figures of cytoplasmic vesicles were demonstrated, suggesting that 5HT may be secreted, in a paracrine way, into the extraspinal space. This specialized area in the gar spinal cord may be referred to as the juxtameningeal serotonergic plexus.     

A mechanical microconnector system for restoration of tissue continuity and long-term drug application into the injured spinal cord

Complete transection of the spinal cord leaves a gap of several mm which fills with fibrous scar tissue. Several approaches in rodent models have used tubes, foams, matrices or tissue implants to bridge this gap. Here, we describe a mechanical microconnector system (mMS) to re-adjust the retracted spinal cord stumps. The mMS is a multi-channel system of polymethylmethacrylate (PMMA), designed to fit into the spinal cord tissue gap after transection, with an outlet tubing system to apply negative pressure to the mMS thus sucking the spinal cord stumps into the honeycomb-structured holes. The stumps adhere to the microstructure of the mMS walls and remain in the mMS after removal of the vacuum. We show that the mMS preserves tissue integrity and allows axonal regrowth at 2, 5 and 19 weeks post lesion with no adverse tissue effects like in-bleeding or cyst formation. Preliminary assessment of locomotor function in the open field suggested beneficial effects of the mMS. Additional inner micro-channels enable local substance delivery into the lesion center via an attached osmotic minipump. We suggest that the mMS is a suitable device to adapt and stabilize the injured spinal cord after surgical resection of scar tissue (e.g., for chronic patients) or traumatic injuries with large tissue and bone damages.     

Pain reactivity of monkeys after lesions to the dorsal and lateral columns of the spinal cord

Summary Macaca speciosa monkeys were trained to escape electrical stimulation of either hindlimb, and they were given the alternative of witholding the response for 10 sec to receive food reward. The force, latency and threshold of escape responses were measured as a function of stimulation intensity before and after lesions of the spinal cord. The stimulus range was set so that pain was certainly evoked by some portion of the intensities above escape threshold, and it was inferred that pain reactivity was altered if the force or latency curves or both were shifted by a lesion. Ipsilateral dorsal column lesions decreased pain reactivity as revealed by higher latencies and lower forces of escape responses. Thresholds were generally unaffected. Ipsilateral lesions of the dorsolateral column produced hyperesthesia, or reduced latencies and increased forces. Thresholds were again unaffected. Contralateral lesions of the lateral column elevated thresholds, and this effect was magnified by inclusion of the dorsal columns in the lesion.Supported by Grant NS07261 from the National Institute of Neurological Diseases and Stroke, U.S. Department of Health, Education and Welfare, Bethesda, Maryland, and the Veterans Administration Hospital, Gainesville, Florida. The research described in this report involved animals maintained in animal care facilities fully accredited by the American Association for Accreditation of Laboratory Animal Care. The animal care was provided in part by N.I.H. grant FR00421. We thank Dorothy Robinson for technical assistance.During this investigation, Dr. Hamilton was supported by a postdoctoral fellowship from the Center for Neurobiological Sciences (grant MH10320 from the National Institutes of Mental Health).     

Absence of connections between spinal nerves and the spinal cord in a twin fetus: a very rare malformation occasionally evidenced at autopsy

We describe a rare case of spinal cord malformation in a dichorionic diamniotic twin fetus aborted at 20 weeks' gestation due to acute chorioamnionitis with placental dysmaturity probably caused by a maternal viral infection. At autopsy, there were no connections between the spinal nerves and the spinal cord. The spinal cord lacked the posterior median fissure and gray matter; only a few neurons were present in the anterior and lateral gray columns. No chromosome anomalies were found. Although we cannot reconstruct the pathogenetic chain of events leading to this malformation, we believe that it is correlated with a maternal viral infection. We hypothesize that this viral infection altered the delicate balance between the factors inducing and those inhibiting fetal spinal neural differentiation.     

脊髓损伤后5-HT2A受体的变化与鼠尾痉挛关系的分析

目的　通过损伤大鼠脊髓腰1节段来探讨损伤下段5-HT2A受体免疫反应变化与鼠尾痉挛状态的关系,为治疗患者脊髓损伤后肢体痉挛提供理论依据。 方法　60只雄性Wistar大鼠随机分为空白组、打击组和横断组（n=20）,用免疫组化法研究5-HT2A受体免疫反应的变化,并分析与鼠尾痉挛状态的关系。所得数据统计学处理。 结果　(1)打击组和横断组5-HT2A受体免疫反应的密度比空白组增高,横断组增高更显著,差异有统计学意义。(2)脊髓损伤后鼠尾痉挛状态与5-HT2A受体的密度和脊髓损伤的程度呈正相关。 结论　脊髓损伤后鼠尾痉挛状态与5-HT2A受体密度的变化呈正相关,应用5-HT2A受体阻滞剂治疗患者的肢体痉挛提供了理论依据。     

Brain derived neurotrophic factor and trk B mRNA expression in the brain of a brain stem-spinal cord regenerating model,the European eel,after spinal cord injury

Evidence from mammalian studies suggests that brain derived neurotrophic factor (BDNF) and its receptor, trk B, are upregulated in neuronal cell bodies after injury. Although fish possess neurotrophins and display rapid functional and morphological recovery after central nervous system (CNS) injury, to date few studies have examined neurotrophin expression during CNS regeneration. In this study, RT-PCR was used to investigate the effect of complete spinal cord transection on the mRNA expression of BDNF and its receptor, trk B, in the eel brain at a range of timepoints after injury. The spatial expression pattern of BDNF mRNA in the brain was also assessed before and after injury using in situ hybridization. Marked changes in BDNF and trk B mRNA levels in the eel brain were not detected during the recovery period after cord transection. In addition, the spatial expression pattern of BDNF mRNA in the eel brain appeared unchanged after injury. Our results are in contrast with the increase reported in mammals but are in line with studies examining neurotrophin expression during CNS regeneration in other anamniotic vertebrates.     

An analysis of response properties of spinal cord dorsal horn neurones to nonnoxious and noxious stimuli in the spinal rat

Summary Electrophysiological properties of neurones in the spinal cord dorsal horn were studied in decerebrated, immobilized spinal rats. Extracellular recordings were performed at the thoraco-lumbar junction level. Each track was systematically located by extracellular injection of pontamine sky blue. According to their responses to mechanical peripheral stimuli, cells were classified in four classes: Class 1 cells: Cells activated only by nonnoxious stimuli. They were divided into — 1A: hair movement and/or touch and 1B: hair movement and/or touch and pressure or pressure only. Class 2 cells: Cells driven by both nonnoxious and noxious stimuli, divided into — 2A: hair movement and/or touch, pressure, pinch and/or pin-prick, and 2B: pressure, pinch and/or pin-prick. Class 3 cells: Cells only activated by noxious stimuli (pinch and/or pin-prick). Class 4 cells: Cells responding to joint movement or pressure on deep tissues.Peripheral transcutaneous or sural nerve stimulation clearly showed that class 1 cells were activated only by A fiber input while 68% of classes 2 and 3 cells received A and C input. Histological examination indicated that cells driven only by noxious input were located either in the deepest part or in the marginal zone (lamina I) of the dorsal horn. Nevertheless, some lamina I cells were also driven by both nonnoxious and noxious stimuli. In addition, there is a great deal of overlap between class 1 and class 2 cells. This fact was confirmed by considering the wide distribution in the dorsal horn of cells receiving A and C input. However, spinal organization of the different classes of cells consists of a preferential distribution rather than a strict lamination. This study indicates that properties of dorsal horn interneurones in the rat have a high degree of similarity with those previously described in other species (cat and monkey).This work was supported by the C.N.R.S. (E.R.A. 237).     

Regeneration of the rat spinal cord after thoracic segmentectomy: Growth and restoration of nerve conductors

The results presented in our previous report (Morfologiya, 127, No. 2 (2005)) provided evidence that consolidation of the spinal cord (SC) after thoracic segmentectomy in rats occurs as a result of the formation of a connective tissue scar, which is quicker when the defect is filled with collagen gel. The present report describes analysis of semithin sections and transmission electron microscopy studies demonstrating that regenerating nerve conductors traverse connective tissue in structures whose organization is identical to that of peripheral nerves. In the zone of SC rarefaction caudal to the trauma site, myelination of growing axons is mediated by glial cells without the formation of nerve trunks. Large numbers of fine regenerating conductors were seen at the sites of degenerated myelin fibers in the fasciculi of the white matter in the lumbar segments of the SC. __________ Translated from Morfologiya, Vol. 129, No. 1, pp. 30–38, January–February, 2006.     

The use of state-dependent modulation of spinal reflexes as a tool to investigate the organization of spinal interneurons

This review examines the proposition that state-dependent modulation of transmission through spinal reflex pathways can be used as an investigative tool to reveal details about the organization of spinal interneurons into functional circuits. The first set of examples includes the use of spinal and supraspinal lesions, as well as the administration of the drug l-dihydroxyphenylalanine (l-DOPA), to produce different, relatively stable ”states” of the central nervous system (CNS), revealing previously unsuspected spinal pathways activated by the flexor reflex afferents (FRA). The second set of examples deals with the use of fictive locomotion and scratching to investigate the organization of oligosynaptic excitatory and inhibitory reflex pathways from cutaneous and muscle afferents. As in the first set of examples, several hitherto unknown reflex pathways have been found only during the flexion or extension phases of rhythmic locomotion, which are regarded as different CNS states. Differences in the patterns of control can be used to infer the existence of distinct sets of reflex pathway interneurons that have remarkably precise input/output relations. Received: 4 February 1999 / Accepted: 19 April 1999     

稳定内皮和周细胞微管改善脊髓损伤中微循环障碍的作用及其机制

目的 探讨稳定内皮细胞和周细胞微管改善脊髓损伤(SCI)中微循环障碍的作用及其机制.方法 培养大鼠脑微血管内皮细胞和周细胞,建立糖氧剥夺(OGD)模型,采用CCK-8法检测细胞活力,采用免疫荧光和Western blotting分别检测α-微管蛋白(α-tubulin)表达;建立脊髓横断损伤模型(n=36),采用免疫荧...     

Immunocytochemical localization of amino acid neurotransmitter candidates in the ventral horn of the cat spinal cord: a light microscopic study

The distribution of immunoreactivities to six amino acids, possibly related to synaptic function, was investigated in the motor nucleus of the cat L7 spinal cord (laminae VII and IX) using a postembedding peroxidase-antiperoxidase technique. Consecutive 0.5 m transverse sections of plastic-embedded tissue were incubated with antisera raised against protein-glutaraldehyde conjugates of -aminobutyric acid (GABA), glycine, aspartate, glutamate, homocysteate, and taurine. This method allowed localization of the different immunoreactivities in individual cell profiles. The results showed that all these amino acids, except homocysteate, could be clearly detected in either neuronal or glial elements in the ventral horn. In cell bodies of neurons in lamina VII, immunoreactivity was observed for aspartate, glutamate, GABA, and glycine. Adjacent section analysis revealed that combinations of immunoreactivity for glycine/glutamate/aspartate, GABA/glycine/glutamate/aspartate and glutamate/aspartate, respectively, may occur in one and the same cell. In the motor nuclei (lamina IX), immunoreactivity to amino acids was observed in two types of neuron. Large cells, probably representing -motoneurons, were harboring immunoreactivity to both glutamate and aspartate, while a few small neurons in this area displayed a colocalization of glycine, glutamate, and aspartate. Dendrites and axons in the motor nuclei cocontained glycine/glutamate/aspartate, GABA/glycine/glutamate/aspartate, and glutamate/aspartate immunoreactivities. In both laminae VII and IX, taurine-like immunoreactivity was absent in neuronal cell bodies, but highly concentrated in perivascular cells and small cells with a morphology resembling that of glial cells. A punctate immunolabeling, in all probability representing labeling of nerve terminals, could be demonstrated in the ventral horn for GABA, glycine, and glutamate, but not with certainty for aspartate or taurine. A quantitative estimate of the covering of cell bodies of -motoneuron size by immunoreactive puncta revealed that glycine immunoreactive terminal-like structures were most abundant (covering 26–42% of the somatic membrane), while glutamate immunoreactive terminals were seen least frequently (5–9% covering). GABA-immunoreactive terminals covered from 10 to 24% of the soma surface. A colocalization of GABA and glycine immunoreactivities in putative nerve terminals could be shown both in the neuropil and in close relation to cell bodies of motoneurons. These results suggest that among the studied amino acids probably only three, namely GABA, glycine, and glutamate, can be considered to be neurotransmitter candidates in the ventral horn of the cat spinal cord.     

Curcumin improves the recovery of motor function and reduces spinal cord edema in a rat acute spinal cord injury model by inhibiting the JAK/STAT signaling pathway

Curcumin, a yellow pigment extracted from Carcuma longa, has been demonstrated to have extensive pharmacological activity in various studies, and it exhibits protective effects on injuries involving a number of human organs. The present study was designed to evaluate the potential effect and underlying mechanism of curcumin on the motor function and spinal cord edema in a rat acute spinal cord injury (SCI) model. The SCI model was induced by a heavy object falling. At 30 min after the SCI was successfully induced, the animals were intraperitoneally given 40 mg/kg curcumin. The Basso, Beattie and Bresnahan scores showed that curcumin moderately improved the recovery of the motor function in the injured rats, and hematoxylin–eosin staining demonstrated the role of this compound in reducing the hemorrhage, edema and neutrophil infiltration of the traumatic spinal cord. Furthermore, curcumin also inhibited the SCI-associated aquaporin – 4 (AQP4) overexpression and glial fibrillary acidic protein (GFAP) and repressed the unusual activation of the JAK/STAT signaling pathway. In conclusion, our data demonstrate that curcumin exhibits a moderately protective effect on spinal cord injury, and this effect might be related to the inhibition of overexpressed AQP4 and GFAP and the activated JAK/STAT signaling pathway. Curcumin may have potential for use as a therapeutic option for spinal cord injuries.     

Reorganization of the ependyma during axolotl spinal cord regeneration: changes in intermediate filament and fibronectin expression.

Changes in intermediate filament content and extracellular matrix material showed that the injury response of ependymal cells in lesioned axolotl spinal cord involves an epithelial-to-mesenchymal transformation, and that fibrous astrocytes are excluded from the remodeling lesion site. Antibody localization was used to visualize cytokeratin-, vimentin-, and glial fibrillary acidic protein- (GFAP-) containing intermediate filaments, as well as the adhesive glycoprotein fibronectin. In normal axolotl spinal cord cytokeratins were found near the apical surface of the ependymal cells. Transmission electron microscopic examination suggested that these cytokeratins were in tonofilaments. Cytokeratin expression was lost and vimentin production was initiated in ependymal cells 2-3 weeks following spinal cord injury. There was a period of approximately 1-2 weeks when cytokeratins and vimentin were co-expressed in vivo. This co-expression was maintained in vitro by culture on a fibronectin-coated substratum. As the central canal reformed, vimentin expression was lost. Ependymal cells lacked GFAP intermediate filaments, but GFAP was present in fibrous astrocytes of the neuropil and white matter. Following injury, GFAP localization showed that fibrous astrocytes disappeared from the remodeling lesion site and reappeared only after the ependymal epithelium reformed and newly myelinated axons were found. Fibronectin expression closely followed the expression of vimentin during mesenchymal ependymal cell outgrowth. These results suggest that the ependymal cell outgrowth requires changes in cell shape followed by changes in production of extracellular matrix.     

Functional recovery of stepping in rats after a complete neonatal spinal cord transection is not due to regrowth across the lesion site

Rats receiving a complete spinal cord transection (ST) at a neonatal stage spontaneously can recover significant stepping ability, whereas minimal recovery is attained in rats transected as adults. In addition, neonatally spinal cord transected rats trained to step more readily improve their locomotor ability. We hypothesized that recovery of stepping in rats receiving a complete spinal cord transection at postnatal day 5 (P5) is attributable to changes in the lumbosacral neural circuitry and not to regeneration of axons across the lesion. As expected, stepping performance measured by several kinematics parameters was significantly better in ST (at P5) trained (treadmill stepping for 8 weeks) than age-matched non-trained spinal rats. Anterograde tracing with biotinylated dextran amine showed an absence of labeling of corticospinal or rubrospinal tract axons below the transection. Retrograde tracing with Fast Blue from the spinal cord below the transection showed no labeled neurons in the somatosensory motor cortex of the hindlimb area, red nucleus, spinal vestibular nucleus, and medullary reticular nucleus. Retrograde labeling transsynaptically via injection of pseudorabies virus (Bartha) into the soleus and tibialis anterior muscles showed no labeling in the same brain nuclei. Furthermore, re-transection of the spinal cord at or rostral to the original transection did not affect stepping ability. Combined, these results clearly indicate that there was no regeneration across the lesion after a complete spinal cord transection in neonatal rats and suggest that this is an important model to understand the higher level of locomotor recovery in rats attributable to lumbosacral mechanisms after receiving a complete ST at a neonatal compared to an adult stage.     

Phosphinic acid derivatives as baclofen agonists and antagonists in the mammalian spinal cord: an in vivo study

The actions of a series of derivatives of 3-aminopropyl-phosphinic acid as baclofen agonists and antagonists have been examined on the synaptic excitation of neurones by impulses in primary afferent fibres in the lumbar spinal cords of pentobarbitone-anaesthetised cats and rats. Both the pre-and postsynaptic inhibitory actions of microelectrophoretic (-)-baclofen were reduced by similarly administered CGP 35 348, 36 742, 46 381, 52 432, 54 626 and 55 845, the latter being the most potent antagonist. None of these antagonists either decreased or increased the excitability of spinal neurones, and the inhibitory action of GABA was reduced only by local concentrations of antagonists which also reduced the action of piperidine-4-sulphonic acid, a GABA_A agonist. Although the weak inhibitory effect of 3-aminopropylphosphinic acid in both the rat and the cat was not reduced by these baclofen antagonists, the pre-and postsynaptic inhibitory effects of 3-aminopropyl-methyl-osphinic acid (CGP 35 024), which was more potent than (-)-baclofen, were reduced by the antagonists. Like (-)-baclofen, CGP 35 024 was relatively ineffective in reducing transmitter release in the cord from the terminals of excitatory spinal interneurones, the terminals of excitatory tracts in the dorsolateral funiculus and the cholinergic terminals of motor axon collaterals. In both rat and cat cords, receptors for (-)-baclofen could not be demonstrated to be activated by microelectrophoretic GABA, possibly because of the predominantly dendritic location of GABA_B receptors. Spinal pre-and postsynaptic baclofen receptors appeared to be pharmacologically similar but differed from those in the higher central nervous system of the rat, where 3-aminopropylphosphinic acid has been reported to be an effective baclofen agonist. The compounds tested, particularly CGP 55 845 and 46 381, will be of use in further investigations of the physiological relevance of baclofen receptors at central synapses where GABA may be the transmitter.