Thyrotropin-Releasing Hormone Enhances Choline Acetyltransferase and Creatine Kinase in Cultured Spinal Ventral Horn Neurons

The effect of 0.1 mM thyrotropin-releasing hormone (TRH) on ventral horn neurons was investigated in eight experimental sets of tissue cultures established from ventral and dorsal portions of spinal cords of 13-15-day rat embryos. Cultures were treated with TRH from day 1 for 2-5 weeks. TRH-treated ventral spinal cord cultures (VSCC), compared with control VSCC, had more numerous and more healthy-appearing neurons and thicker bundles of long cell processes. In TRH-treated VSCC, choline acetyltransferase (ChAT) activity was greater than 16 times (p less than 0.005) and creatine kinase greater than 3 times (p less than 0.005) that of control VSCC. Morphologic and biochemical parameters of dorsal spinal cord cultures remained unchanged by TRH treatment. Since lower motor neurons are numerous in the ventral spinal cord (and not present in the dorsal cord) and since lower motor neurons are the major ChAT-containing spinal cord cells, our data demonstrating a beneficial effect of TRH on VSCC suggest a tropic effect of TRH on lower motor neurons.     

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.     

Immunohistochemical examination of intraspinal serotonin neurons and fibers in the chicken lumbar spinal cord and coexistence with Leu-enkephalin

Intraspinal serotonin-positive cells and fibers were examined in the chicken lumbar spinal cord following removal of descending serotonin fibers by spinal transection. Co-localization of Leu-enkephalin immunoreactivity in intraspinal serotonin cells was also examined using a double immunofluorescence labeling technique. By one or two weeks after spinal transection, virtually all supraspinal serotonin fibers were eliminated. Intraspinal serotonin cells were located ventral or ventrolateral to the central canal corresponding to laminae VII, VIII, and IX, and the anterior funiculus. Intraspinal serotonin cells sent fibers to (1) the pia mater on the ventral or ventrolateral surface of the spinal cord; (2) vessels in the spinal cord; (3) sympathetic preganglionic column of Terni; (4) other intraspinal serotonin neurons; (5) the central canal. Some 30%–50% of the intraspinal serotonin cells co-localized with Leu-enkephalin. Intraspinal serotonin fibers co-containing Leu-enkephalin were observed in the pia mater located on the most lateral surface of the spinal cord. Permanent address: This study was supported by Grant-in-Aid for Scientific Research on Priority Area from the Ministry of Education, Science and Culture, Japan.     

Mesenchymal stem cell conditioned medium increases glial reactivity and decreases neuronal survival in spinal cord slice cultures

Ex vivo spinal cord slice cultures (SCSC) allow study of spinal cord circuitry, maintaining stimuli responses comparable to live animals. Previously, we have shown that mesenchymal stem/stromal cell (MSC) transplantation in vivo reduced inflammation and increased nerve regeneration but MSC survival was short-lived, highlighting that beneficial action may derive from the secretome. Previous in vitro studies of MSC conditioned medium (CM) have also shown increased neuronal growth. In this study, murine SCSC were cultured in canine MSC CM (harvested from the adipose tissue of excised inguinal fat) and cell phenotypes analysed via immunohistochemistry and confocal microscopy. SCSC in MSC CM displayed enhanced viability after propidium iodide staining. GFAP immunoreactivity was significantly increased in SCSC in MSC CM compared to controls, but with no change in proteoglycan (NG2) immunoreactivity. In contrast, culture in MSC CM significantly decreased the prevalence of βIII-tubulin immunoreactive neurites, whilst Ca²⁺ transients per cell were significantly increased. These ex vivo results contradict previous in vitro and in vivo reports of how MSC and their secretome may affect the microenvironment of the spinal cord after injury and highlight the importance of a careful comparison of the different experimental conditions used to assess the potential of cell therapies for the treatment of spinal cord injury.     

Spatiotemporal Patterns of SSeCKS Expression After Rat Spinal Cord Injury

Src suppressed C kinase substrate (SSeCKS) was identified as a PKC substrate/PKC-binding protein, which plays a role in mitogenic regulatory activity and has a function in the control of cell signaling and cytoskeletal arrangement. However its distribution and function in the central nervous system (CNS) lesion remain unclear. In this study, we mainly investigated the mRNA and protein expression and cellular localization of SSeCKS during spinal cord injury (SCI). Real-time PCR and Western blot analysis revealed that SSeCKS was present in normal whole spinal cord. It gradually increased, reached a peak at 3 days for its mRNA level and 5 days for its protein level after SCI, and then declined during the following days. In ventral horn, the expression of SSeCKS underwent a temporal pattern that was similar with the whole spinal cord in both mRNA and protein level. However, in dorsal horn, the mRNA and protein for SSeCKS expression were significantly increased at 1 day for its mRNA level and 3 days for its protein level, and then gradually declined to the baseline level, ultimately up-regulated again from 7 to 14 days. The protein expression of SSeCKS was further analysed by immunohistochemistry. The positively stained areas for SSeCKS changed with the similar pattern to that of protein expression detected by immunoblotting analysis. Double immunofluorescence staining showed that SSeCKS immunoreactivity (IR) was found in neurons, astrocytes, oligodendrocytes of spinal cord tissues within 5 mm from the lesion site. Importantly, injury-induced expression of SSeCKS was co-labeled by active caspase-3 (apoptotic marker), Tau-1 (the marker for pathological oligodendrocyte) and β-1,4-galactosyltransferase 1 (GalT). All the results suggested that SSeCKS might play important roles in spinal cord pathophysiology and further research is needed to have a good understanding of its function and mechanism. Feng Xiao and Min Fei contributed equally to this work.     

The development of an improved physical surrogate model of the human spinal cord—Tension and transverse compression

To prevent spinal cord injury, optimize treatments for it, and better understand spinal cord pathologies such as spondylotic myelopathy, the interaction between the spinal column and the spinal cord during injury and pathology must be understood. The spinal cord is a complex and very soft tissue that changes properties rapidly after death and is difficult to model. Our objective was to develop a physical surrogate spinal cord with material properties closely corresponding to the in vivo human spinal cord that would be suitable for studying spinal cord injury under a variety of injurious conditions. Appropriate target material properties were identified from published studies and several candidate surrogate materials were screened, under uniaxial tension, in a materials testing machine. QM Skin 30, a silicone elastomer, was identified as the most appropriate material. Spinal cords manufactured from QM Skin 30 were tested under uniaxial tension and transverse compression. Rectangular specimens of QM Skin 30 were also tested under uniform compression. QM Skin 30 produced surrogate cords with a Young's modulus in tension and compression approximately matching values reported for in vivo animal spinal cords (0.25 and 0.20 MPa, respectively). The tensile and compressive Young's modulus and the behavior of the surrogate cord simulated the nonlinear behavior of the in vivo spinal cord.     

Effects of Methylprednisolone and Hyperbaric Oxygen on Oxidative Status after Experimental Spinal Cord Injury: A Comparative Study in Rats

The effects of hyperbaric oxygen (HBO) therapy or methylprednisolone on the oxidative status were evaluated in experimental spinal cord injury. Clip compression method was used to produce acute spinal cord injury rats. Hyperbaric oxygen was administered twice daily for a total of eight 90 min-sessions at 2.8 atmospheres. Methylprednisolone was first injected with a bolus of 30 mg/kg followed with an infusion rate of 5.4 mg/kg/h for 24 h. Five days after clip application animals were sacrificed and their traumatized spinal cord segment were excised. Tissue levels of thiobarbituric acid reactive substances (TBARS), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were evaluated to reflect oxidant/antioxidant status. Non-treated clip-operated animals reflected significantly higher SOD, GSH-Px and TBARS levels that were found to be significantly higher than the sham-operated. Methylprednisolone was not able to lower these levels. HBO administration diminished all measured parameters significantly; however, their levels appeared already to be high when compared with sham animals. According to these results obtained on the 5th day after induction, HBO, but not methylprednisolone, seems to procure prevention against oxidative spinal cord injury.     

Possible Involvement of Interleukin-1 in Cyclooxygenase-2Induction After Spinal Cord Injury in Rats

Abstract : A standardized compression injury of rat spinal cord brought about a time-dependent biphasic production of thromboxane A₂ (detected as thromboxane B₂) and prostaglandin I₂ (detected as 6-ketoprostaglandin F_1α. Thromboxane B₂ was predominant during the first 1 h, whereas the 6-ketoprostaglandin F_1α level exceeded that of thromboxane B₂ at 8 h postinjury. As examined by inhibitor experiments and northern blotting, cyclooxygenase-1 was responsible for the first phase, and cyclooxygenase-2 was involved in the second phase. On compression injury the levels of interleukin-1α and -1β detected as mRNA and protein increased and peaked at 2-4 h. Injection of exogenous interleukin-1 α into the spinal cord resulted in an increase of cyclooxygenase-2 mRNA content and a predominant production of 6-ketoprostaglandin F_1α resembling the second phase of eicosanoid production. Concomitantly, extravascular migration of polymorphonuclear leukocytes was enhanced after the interleukin-1α injection. These cells together with vascular endothelial cells and glial cells were stained positively with an anti-cyclooxygenase-2 antibody. The results suggest that the immediate eicosanoid synthesis after spinal cord injury was due to the constitutive cyclooxygenase-1 and the delayed synthesis of eicosanoids was attributable to the induction of cyclooxygenase-2 mediated by interleukin-1 α.     

Protective effect of vitamin E on spinal cord injury by compression and concurrent lipid peroxidation

Studies were made on the influence of vitamin E on the effects of compression injury of the spinal cord associated with ischemia in rats. The motor disturbance induced by spinal cord injury was greatly reduced by vitamin E supplementation. After injury, the spinal cord evoked potentials showed greater recovery of both amplitude and latency in the vitamin E-supplemented group than in the control group. Spinal cord blood flow was promptly restored and remained normal after injury in the vitamin E-supplemented group, but was significantly decreased from 3 h after injury in the control group. Thiobarbituric acid (TBA)—reactive substances in the spinal cord was immediately increased by compression injury in both groups, and after injury it persisted at a high value for 24 h in the control group, but decreased within 1 h in the vitamin E-supplemented group. Pathological examination of the spinal cord showed less damage, such as bleeding and edema, in the vitamin E-supplemented group than in the control group. Vitamin E may have protective effects on the spinal cord by inhibiting damage induced by lipid peroxidation and/or by sustaining the blood flow by maintaining the normal metabolism of arachidonic acid.     

Functional recovery after transplantation of bone marrow-derived human mesenchymal stromal cells in a rat model of spinal cord injury

Background aimsSpinal cord injury (SCI) is a medically untreatable condition for which stem cells have created hope. Pre-clinical and clinical studies have established that these cells are safe for transplantation. The dose dependency, survivability, route of administration, cell migration to injury site and effect on sensory and motor behavior in an SCI-induced paraplegic model were studied.MethodsA spinal cord contusion injury model was established in rats. Bone marrow (BM) mesenchymal stromal cells (MSC) were tagged to facilitate tracing in vivo. Two different doses (2 and 5 million cells/kg body weight) and two different routes of infusion (site of injury and lumbar puncture) were tested during and after the spinal shock period. The animals were tested post-transplantation for locomotor capacity, motor control, sensory reflex, posture and body position. Stem cell migration was observed 1 month post-transplantation in spinal cord sections.ResultsThe overall results demonstrated that transplantation of BM MSC significantly improved the locomotor and sensory behavior score in the experimental group compared with the sham control group, and these results were dose dependent. All the infused stem cells could be visualized at the site of injury and none was visualized at the injected site. This indicated that the cells had survived in vivo, were probably chemoattracted and had migrated to the lesion site.ConclusionsMSC transplanted with a lumbar puncture method migrate to the site of injury and are the most suitable for SCI healing. These cells demonstrate a dose-dependent effect and promote functional recovery when injected during or after the spinal shock period.     

The dynamics of axolemmal disruption in guinea pig spinal cord following compression

Membrane damage has been postulated as a critical factor in mediating axonal degeneration in brain and spinal cord trauma. Despite compelling evidence of membrane disruption as a result of physical insults in both in vivo and in vitro studies, the dynamics of such damage over the time post injury in in vivo studies has not been well documented. Using a well-characterized in vivo guinea pig spinal cord compression model and horseradish peroxidase exclusion assay, we have documented significant membrane disruption at 1 hr, 3 days, and 7 days following injury. Furthermore, the membrane damage was found to spread laterally 10 mm beyond the center of original compression site in both rostral and caudal directions. A second-degree polynomial fit of the measured data predicts a bilateral spread of approximately 20–21 mm of membrane disruption from the epicenter of injury over a period of about 20 days. Thus, this study shows that membrane damage exists days, and possibly weeks, after spinal cord trauma in live guinea pigs. This provides the evidence necessary to investigate the role of membrane damage in triggering axonal deterioration in the future. Furthermore, this study has also revealed a long therapeutical window for membrane repair and functional enhancement following traumatic injury in the central nervous system.     

Effect of bone fragment impact velocity on biomechanical parameters related to spinal cord injury: A finite element study

Several experimental and computational studies have investigated the effect of bone fragment impact on the spinal cord during trauma. However, the effect of the impact velocity of a fragment generated by a burst fracture on the stress and strain inside the spinal cord has not been computationally investigated, even though spinal canal occlusion and peak pressure at various impact velocities were provided in experimental studies. These stresses and strains are known factors related to clinical symptoms or injuries. In this study, a fluid-structure interaction model of the spinal cord, dura mater, and cerebrospinal fluid was developed and validated. The von-Mises stress distribution in the cord, the longitudinal strain, the cord compression and cross-sectional area at the impact center, and the obliteration of the cerebrospinal fluid layer were analyzed for three pellet sizes at impact velocities ranging from 1.5 m/s to 7.5 m/s. The results indicate that stress in the cord was substantially elevated when the initial impact velocity of the pellet exceeded a threshold of 4.5 m/s. Cord compression, reduction in cross-sectional area, and obliteration of the cerebrospinal fluid increased gradually as the velocity of the pellet increased, regardless of the size of the pellet. The present study provides insight into the mechanisms underlying spinal cord injury.     

Finite Element Study of the Mechanical Response in Spinal Cord during the Thoracolumbar Burst Fracture

Background

The mechanical response of the spinal cord during burst fracture was seldom quantitatively addressed and only few studies look into the internal strain of the white and grey matters within the spinal cord during thoracolumbar burst fracture (TLBF). The aim of the study is to investigate the mechanical response of the spinal cord during TLBF and correlate the percent canal compromise (PCC) with the strain in the spinal cord.

Methodology/Principal Findings

A three-dimensional (3D) finite element (FE) model of human T12-L1 spinal cord with visco-elastic property was generated based on the transverse sections images of spinal cord, and the model was validated against published literatures under static uniaxial tension and compression. With the validated model, a TLBF simulation was performed to compute the mechanical strain in the spinal cord with the PCC. Linear regressions between PCC and strain in the spinal cord show that at the initial stage, with the PCC at 20%, and 45%, the corresponding mechanical strains in ventral grey, dorsal grey, ventral white, dorsal white matters were 0.06, 0.04, 0.12, 0.06, and increased to 0.14, 0.12, 0.23, and 0.13, respectively. At the recoiled stage, when the PCC was decreased from 45% to 20%, the corresponding strains were reduced to 0.03, 0.02, 0.04 and 0.03. The strain was correlated well with PCC.

Conclusions/Significance

The simulation shows that the strain in the spinal cord correlated well with the PCC, and the mechanical strains in the ventral regions are higher than those in the dorsal regions of spinal cord tissue during burst fracture, suggesting that the ventral regions of the spinal cord may susceptible to injury than the dorsal regions.     

Evidence for Ascending and Descending Intraspinal as Well as Primary Sensory Somatostatin Projections in the Rat Spinal Cord

Somatostatin distribution was measured quantitatively in the rat spinal cord by radioimmunoassay. Rostro-caudally, somatostatin content was about 50% higher in lumbar-sacral cord than in cervical or thoracic levels. The dorso-ventral distribution is more uneven: somatostatin is highest in the dorsal horn, where the peptide is 15 times as concentrated as it is in the ventral white matter, the region of lowest concentration. However, measurable amounts of the peptide were found in all regions studied. Dorsal root ganglionectomy decreased somatostatin levels in the dorsal cord, supporting the previously proposed role for this peptide as a primary sensory neurotransmitter or modulator; but somatostatin content also was decreased both rostral and caudal to spinal transection, indicating the presence of ascending and descending somatostatin pathways within the spinal cord. Brain levels did not change. Met-enkephalin and substance P were also measured after the above surgical manipulations. Met-enkephalin content was not altered and substance P content was lowered significantly only after ganglionectomy. Although this study confirms the primary sensory neuron as the origin of a part of spinal cord somatostatin, it further indicates the presence of ascending and descending somatostatin pathways within the rat spinal cord.     

Avian pancreatic polypeptide (APP) immunoreactive neurons in the spinal cord and spinal trigeminal nucleus

Using indirect immunofluorescence technique, avian pancreatic polypeptide (APP) immunoreactive cell bodies and fibres have been observed in the superficial laminae of the dorsal horn of the spinal cord and of the spinal trigeminal nucleus. Fibres were also seen in the ventral horns, in low numbers at the cervical and thoracic levels and in high numbers at the lower lumbar and upper sacral levels. Neither total cord transection, nor dorsal rhizotomy, nor capsaicin treatment seemed to affect the APP systems described above. The present findings suggest that an APP-like peptide may be involved in processing of sensory information at the level of the first relay station.     

Topical application of dynorphin A (1-17) antiserum attenuates trauma induced alterations in spinal cord evoked potentials,microvascular permeability disturbances,edema formation and cell injury: an experimental study in the rat using electrophysiological and morphological approaches

Summary.  Dynorphin is a neuropeptide that is present in high quantities in the dorsal horn of the spinal cord. The peptide is actively involved in pain processing pathways. However, its involvement in spinal cord injury is not well known. Alteration in dynorphin immunoreactivity occurs following a focal trauma to the rat spinal cord. Infusion of dynorphin into the intrathecal space of the cord results in ischemia, cell damage and abnormal motor function. Antibodies to dynorphin when injected into the intrathecal space of the spinal cord following trauma improve motor recovery, reduce edema and cell changes. However, influence of dynorphin on trauma induced alteration in spinal cord bioelectrical activity is still not known. Spinal cord evoked potentials (SCEP) are good indicator of spinal cord pathology following trauma. Therefore, in present investigation, influence of dynorphin antibodies on trauma induced changes in SCEP were examined in our rat model. In addition, spinal cord edema formation, microvascular permeability disturbances and cell injury were also investigated. Our results show that topical application of dynorphin antiserum (1 : 200) two min before injury markedly attenuated the SCEP changes immediately after injury. In the antiserum treated animals, a significant reduction in the microvascular permeability, edema formation and cell injury was observed in the traumatised spinal cord. These observations suggest that (i) dynorphin is involved in the altered bioelectrical activity of the spinal cord following trauma, (ii) the peptide actively participates in the pathophysiological processes of cell injury in the spinal cord trauma, and (iii) the dynorphin antiserum has potential therapeutic value for the treatment of spinal cord injuries. Received July 3, 2001 Accepted August 6, 2001 Published online July 31, 2002     

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.