Rat Spinal Cord Tissue Research Articles

Expression of EphB3 receptor protein in injured spinal cord tissues of rats

Expression of EphB3 receptor protein in injured spinal cord tissues of rats

Mechanical Characterization of the Injured Spinal Cord after Lateral Spinal Hemisection Injury in the Rat

The glial scar formed at the site of traumatic spinal cord injury (SCI) has been classically hypothesized to be a potent physical and biochemical barrier to nerve regeneration. One longstanding hypothesis is that the scar acts as a physical barrier due to its increased stiffness in comparison to uninjured spinal cord tissue. However, the information regarding the mechanical properties of the glial scar in the current literature is mostly anecdotal and not well quantified. We monitored the mechanical relaxation behavior of injured rat spinal cord tissue at the site of mid-thoracic spinal hemisection 2 weeks and 8 weeks post-injury using a microindentation test method. Elastic moduli were calculated and a modified standard linear model (mSLM) was fit to the data to estimate the relaxation time constant and viscosity. The SLM was modified to account for a spectrum of relaxation times, a phenomenon common to biological tissues, by incorporating a stretched exponential term. Injured tissue exhibited significantly lower stiffness and elastic modulus in comparison to uninjured control tissue, and the results from the model parameters indicated that the relaxation time constant and viscosity of injured tissue were significantly higher than controls. This study presents direct micromechanical measurements of injured spinal cord tissue post-injury. The results of this study show that the injured spinal tissue displays complex viscoelastic behavior, likely indicating changes in tissue permeability and diffusivity.

Mapping Lipid Alterations in Traumatically Injured Rat Spinal Cord by Desorption Electrospray Ionization Imaging Mass Spectrometry

Desorption electrospray ionization (DESI) mass spectrometry (MS) is used in an imaging mode to interrogate the lipid profiles of 15 μm thin tissue cross sections of injured rat spinal cord and normal healthy tissue. Increased relative intensities of fatty acids, diacylglycerols, and lysolipids (between +120% and +240%) as well as a small decrease in intensities of lipids (-30%) were visualized in the lesion epicenter and adjacent areas after spinal cord injury. This indicates the hydrolysis of lipids during the demyelination process due to activation of phospholipase A(2) enzyme. In addition, signals corresponding to oxidative degradation products, such as prostaglandin and hydroxyeicosatetraenoic acid, exhibited increased signal intensity by a factor of 2 in the negative ion mode in lesions relative to the normal healthy tissue. Analysis of malondialdehyde, a product of lipid peroxidation and marker of oxidative stress, was accomplished in the ambient environment using reactive DESI mass spectrometry imaging. This was achieved by electrospraying reagent solution containing dinitrophenylhydrazine as high-velocity charged droplets onto the tissue section. The hydrazine reacts selectively and rapidly with the carbonyl groups of malondialdehyde, and signal intensity of twice the intensity was detected in the lesions compared to healthy spinal cord. With a small amount of tissue sample, DESI-MS imaging provides information on the composition and distribution of specific compounds (limited by the occurrence of isomeric lipids with very similar fragmentation patterns) in lesions after spinal cord injury in comparison with normal healthy tissue allowing identification of the extent of the lesion and its repair.

Iron is a potential key mediator of glutamate excitotoxicity in spinal cord motor neurons

Threohydroxyaspartate (THA)-induced glutamate excitotoxicity in organotypic culture of rat spinal cord is a well-known model of motor neuron degeneration. THA causes accumulation of synaptic glutamate and over stimulation of the postsynaptic receptor by inhibiting glutamate uptake. This model has also been used to identify agents that inhibit glutamate excitotoxicity by increasing the expression of glutamate transporter. We now show that THA also increases iron level in rat spinal cord tissue, with concomitant modulation of key iron transport and storage proteins, including transferrin receptor, divalent metal-ion transporter 1 and ferritin. More significantly, iron chelator deferoxamine (DFO) was able to completely prevent THA-induced motor neuron degeneration. The protective effect of DFO did not involve enhancing glutamate uptake. These data provide new mechanistic insight into THA-induced glutamate excitotoxicity and suggest that blocking THA-induced iron rise alone may be sufficient for prevention of glutamate excitotoxicity.

Involvement of Acidic Fibroblast Growth Factor in Spinal Cord Injury Repair Processes Revealed by a Proteomics Approach

Acidic fibroblast growth factor (aFGF; also known as FGF-1) is a potent neurotrophic factor that affects neuronal survival in the injured spinal cord. However, the pathological changes that occur with spinal cord injury (SCI) and the attribution to aFGF of a neuroprotective effect during SCI are still elusive. In this study, we demonstrated that rat SCI, when treated with aFGF, showed significant functional recovery as indicated by the Basso, Beattie, and Bresnahan locomotor rating scale and the combined behavior score (p < 0.01-0.001). Furthermore proteomics and bioinformatics approaches were adapted to investigate changes in the global protein profile of the damaged spinal cord tissue when experimental rats were treated either with or without aFGF at 24 h after injury. We found that 51 protein spots, resolvable by two-dimensional PAGE, had significant differential expression. Using hierarchical clustering analysis, these proteins were categorized into five major expression patterns. Noticeably proteins involved in the process of secondary injury, such as astrocyte activation (glial fibrillary acidic protein), inflammation (S100B), and scar formation (keratan sulfate proteoglycan lumican), which lead to the blocking of injured spinal cord regeneration, were down-regulated in the contusive spinal cord after treatment with aFGF. We propose that aFGF might initiate a series of biological processes to prevent or attenuate secondary injury and that this, in turn, leads to an improvement in functional recovery. Moreover the quantitative expression level of these proteins was verified by quantitative real time PCR. Furthermore we identified various potential neuroprotective protein factors that are induced by aFGF and may be involved in the spinal cord repair processes of SCI rats. Thus, our results could have a remarkable impact on clinical developments in the area of spinal cord injury therapy.

A versatile mesoindentation system to evaluate the micromechanical properties of soft, hydrated substrates on a cellular scale

It has become increasingly important to study mechanical properties of substrates on the cellular scale since cells sense and respond to changes in the microenvironment in which they are grown. To study the effects of mechanical substrate properties on the cellular scale, an existing microindentation system has been modified to perform indentation tests on highly hydrated polymeric substrates and tissues. The highly sensitive, modified indentation system, labeled as a mesoindenter, is versatile and can be customized to perform a variety of tests useful for studying tissue mechanics, stress relaxation in polymers, and interfacial adhesion phenomena. To validate the efficacy and accuracy of the system, soft, hydrated hydrogels made from agarose (1-5 wt %), poly(2-hydroxyethyl methacrylate) (p(HEMA)) (60-90% water), and unfixed, saline-perfused rat spinal cord tissue were tested. The results demonstrate that moduli vary with water content and are in line with previously published studies. We also demonstrate that the modulus of hydrogels is sensitive to the preload applied, with modulus increasing with preload. Stress relaxation indentation testing of p(HEMA) showed relaxation behavior that can be modeled using a heredity integral and standard linear model. The mesoindenter is versatile, capable of scanning and testing immersed samples, and easily customized to ascertain mechanical properties of substrates ranging from the kPa to GPa range.

Selective gene transfer into neurons via Na,K‐ATPase β1. Targeting gene transfer with monoclonal antibody and adenovirus vector

Neuron-selective gene transfer is an attractive therapeutic strategy for neurological disorders. However, optimal targets and gene delivery systems remain to be determined. Following immunization of mice with PC12 cells, hybridomas were screened by beta-Gal reporter gene assay using FZ33 fiber-modified adenovirus vectors. Subsequently, the efficacy and specificity of monoclonal antibody (mAb)-mediated gene transfer via FZ33 and FdZ adenovirus vectors were evaluated by flow cytometry, chemiluminescent beta-Gal reporter gene assay, and immunocytochemistry. Finally, the antigen recognized by the mAb was identified by mass spectrometry and transfection analysis. A hybridoma clone 6E3 producing monoclonal antibody, mAb6E3, was screened. Flow cytometry, chemiluminescent beta-Gal reporter gene assay, and immunocytochemistry with mAb6E3 and the fiber mutant adenovirus demonstrated efficient gene transfer into the PC12 cells. Treatment of neuron-glia cocultures with mAb6E3 and FdZ adenovirus resulted in neuron-selective gene transfer. Immunohistochemical images of rat spinal cord tissue showed that mAb6E3 reacts specifically with neurons. Finally, Na,K-ATPase beta1 was identified as the antigen of mAb6E3. Hybridoma screening using FZ33 fiber-modified adenovirus vectors serves as an efficient approach to detect antigens in mAb-targeted gene transfer. Neuronal tropism in the central nervous system through mAb6E3 represents an important initial step towards neuron-selective gene transfer in the treatment of local neurological disorders, such as spinal cord injury.

Tissue spinal cord response in rats after implants of polypyrrole and polyethylene glycol obtained by plasma

Most of the biomaterials used nowadays for the reconstruction of the spinal cord (SC) tissue after an injury, tested in animals, have obtained modest results. This work presents a study about the compatibility of two novel, non-biodegradable, semi-conductive materials, obtained by plasma polymerization: iodine-doped pyrrole (PPy/I) and pyrrole-polyethylene glycol (PPy/PEG). Both polymers, separately, were implanted in the SC tissue of rats after a transection. Prior to implantation, the elemental composition and the physico-chemical properties of polymers were studied by electron scanning microscopy, IR Spectroscopy and thermogravimetric analysis. We used adult female Long Evans rats, subjected to SC transection. Animals were randomized to be allocated in one of the treatment groups and were killed four weeks after the lesion for histology study. Results showed that both implants were integrated to the SC tissue, as inflammatory and gliotic responses, similar to those observed in the control group, and rejection of the implant, were not evident. Moreover, the immediate effect of PPy/I or PPy/PEG in the injured SC prevented secondary tissue destruction, as compared to non-implanted control animals. In conclusion, implants of semi-conductive polymers were well-tolerated and integrated favorably to SC tissue after transection.

Expression change of stem cell-derived neural stem/progenitor cell supporting factor gene in injured spinal cord of rats

To explore the expression change of stem cell-derived neural stem/progenitor cell supporting factor (SDNSF) gene in the injuried spinal cord tissues of rats, and the relation between the expressions of SDNSF and nestin. The spinal cord contusion model of rat was established according to Allen's falling strike method. The expression of SDNSF was studied by RT-PCR and in situ hybridization (ISH), and the expression of nestin was detected by immunochemistry. RT-PCR revealed that SDNSF mRNA was upregulated on day 4 after injury, peaked on day 8-12, and decreased to the sham operation level on day 16. ISH revealed that SDNSF mRNA was mainly expressed in the gray matter cells, probably neurons, of spinal cord. The immunohistochemistry showed that accompanied with SDNSF mRNA upregulation, the nestin-positive cells showed erupted roots, migrated peripherad and proliferation on the 8-day slice. However, the distribution pattern of these new cells was different from that of SDNSF-positive cells. (1) SDNSF is expressed in the gray matter of spinal cord. The expression of SDNSF mRNA in the spinal cord varies with injured time. (2) The nestin-positive cells proliferate accompanied with spinal cord injury repair, but do not secrete SDNSF.

Increase in bFGF-responsive neural progenitor population following contusion injury of the adult rodent spinal cord

The number of neural progenitor cells, especially nestin + cells or BrdU-uptake cells is sparse in the normal adult rodent spinal cord. However, in the present study, we show that after spinal cord injury (SCI), many ordinarily quiescent cells were activated to become nestin + and undergo mitosis (BrdU +) in the ependymal layer as well as in the parenchyma of the spinal cord. Nestin + cells and BrdU + cells were in most cases immunohistochemically GFAP +, some of which displayed radial glial cell morphology and partly participated in the border formation of the lesion. The culturing of injured rat spinal cord tissues generated more neurospheres earlier than did the culturing of intact tissues, and these neurosphere cells were multipotent and bFGF-responsive. Immunohistochemical analysis showed that there existed many bFGF + cells after SCI, the number of which were almost 15 times greater than that in an intact spinal cord. Increased bFGF production after SCI might activate quiescent progenitor cells, and thus initiate their cell proliferation. Finally, SCI to the nestin-promoter green fluorescent protein (GFP) transgenic mice showed broad proliferation of progenitor cells that were induced in the injured spinal cord. The culturing of injured spinal cord tissues from these transgenic mice provides direct evidence that neurospheres can be generated by SCI-activated nestin + cells. Thus, the activation of bFGF-responsive progenitor cells and the concomitant increase in the population of bFGF + cells following SCI might be beneficial for spinal cord repair if these progenitor cells are properly manipulated.

Disruption of the hyaluronan‐based extracellular matrix in spinal cord promotes astrocyte proliferation

Astrocyte proliferation is tightly controlled during development and in the adult nervous system. In the present study, we find that a high-molecular-weight (MW) form of the glycosaminoglycan hyaluronan (HA) is found in rat spinal cord tissue and becomes degraded soon after traumatic spinal cord injury. Newly synthesized HA accumulates in injured spinal cord as gliosis proceeds, such that high-MW HA becomes overabundant in the extracellular matrix surrounding glial scars after 1 month. Injection of hyaluronidase, which degrades HA, into normal spinal cord tissue results in increased numbers of glial fibrillary acidic protein (GFAP)-positive cells that also express the nuclear proliferation marker Ki-67, suggesting that HA degradation promotes astrocyte proliferation. In agreement with this observation, adding high- but not low-MW HA to proliferating astrocytes in vitro inhibits cell growth, while treating confluent, quiescent astrocyte cultures with hyaluronidase induces astrocyte proliferation. Collectively, these data indicate that high-MW HA maintains astrocytes in a state of quiescence, and that degradation of HA following CNS injury relieves growth inhibition, resulting in increased astrocyte proliferation.

Effects of Antithrombin III on Myeloperoxidase Activity, Superoxide Dismutase Activity, and Malondialdehyde Levels and Histopathological Findings after Spinal Cord Injury in the Rat

Among the many possible mechanisms of the secondary spinal cord injury (SCI), microcirculatory disturbances as a result of activated leukocyte-induced endothelial cell injury is important because it is potentially treatable and reversible. Currently, clinically available pharmacological agents for treatment of acute SCI do not inhibit neutrophil activation. The effect of antithrombin III (AT-III) on neutrophil activation was studied in rats with SCI produced with an aneurysm clip on the T2-T7 segments. Forty rats were randomly allocated to four groups. Group I (10 rats) was killed to provide normal spinal cord tissue for testing. Group II (10 rats) underwent a six-segment laminectomy for the effects of total laminectomy to be determined. In Group III, 10 rats underwent a six-segment laminectomy and SCI was produced by extradural compression of the exposed cord. The same procedures were performed in 10 rats in Group IV, but they also received one (250 IU/kg) intraperitoneal injection of AT-III immediately after the injury and a second dose 24 hours later. The animals from Groups II through IV were killed 48 hours after the trauma. The effect of AT-III on the myeloperoxidase activity, superoxide dismutase activity, and malondialdehyde levels and histopathological findings were studied. Myeloperoxidase activity, superoxide dismutase activity, and malondialdehyde levels were significantly lower and there was less histopathological damage in the AT-III treatment group than in the trauma group. The results demonstrate that AT-III treatment may reduce secondary structural changes in damaged rat spinal cord tissue by inhibiting leukocyte activation.

Effect of Naloxone After Spinal Cord Injury in the Rat

Endorphins have been implicated in the pathophysiology of spinal cord injury (SCI). Although some possible mechanisms for the therapeutic actions of naloxone have been postulated, the exact mechanism of these favorable actions is not yet known with certainty. The benefit of naloxone in the recovery of neurologic function has generally been attributed to its action at the opiate receptors. The effect of naloxone on the inducible nitric oxide synthase (iNOS) immunoreactivity, superoxide dismutase (SOD) level, and ultrastructural findings were studied in rats at the early and late stages of SCI, produced with an aneurysm clip on the T2 to T7 segments. Seventy rats were randomly allocated to 4 groups. The animals in group 1 (10 rats) were killed to provide normal spinal cord tissue for testing. Group 2 (20 rats) underwent 6-segment laminectomy so that the effects of total laminectomy could be determined. Group 3 (20 rats) underwent 6-segment T2 to T7 laminectomy, and SCI was produced by extradural compression of the exposed cord. The same procedures were performed in the 20 rats in group 4, but these rats also received 1 (2 mg/kg) intraperitoneal injection of naloxone immediately after the injury, a second dose 24 hours after trauma, and a third dose 48 hours after trauma. Half of the animals from groups 3 and 4 were killed 2 hours after trauma, and the other half were killed 48 hours after trauma. The exposed cord segments were immediately removed and processed for analysis. The results showed that naloxone treatment reduces secondary structural changes in damaged rat spinal cord tissue by affecting iNOS and SOD activity.

Pharmacological and functional biochemical properties of d-Ala 2- d-Nle 5-enkephalin-Arg-Phe

Tyr- d-Ala-Gly-Phe- d-Nle-Arg-Phe (DADN) a synthetic analogue of the endogenous Met-enkephalin-Arg-Phe (Tyr-Gly-Gly-Phe-Met-Arg-Phe; MERF), was investigated in radioligand binding assays, [ 35S]GTPγS stimulation experiments as well as in in vivo algesiometric tests. Binding properties of [ 3H]DADN were measured in crude membrane fractions of rat spinal cord tissues and in homogenates of Chinese hamster ovary (CHO) cells selectively expressing δ-, κ-or μ-opioid receptors. The highest affinity for [ 3H]DADN binding was observed in membranes from CHO cells transfected with μ-opioid receptors confirming the μ-selectivity of the peptide. Unlabeled DADN was also investigated in functional biochemical experiments by measuring opioid receptor-mediated G-protein activation in rat brain membrane fractions. The peptide stimulated the activity of the regulatory G-proteins in a concentration dependent manner, and the stimulation was efficiently inhibited in the presence of μ-receptor specific antagonist ligands further supporting the selectivity profile of DADN. Intrathecally administered DADN produced a dose-related, naloxone-reversible antinociception in rat hot water tail-flick tests. Among the selective opioid antagonists tested, the δ-selective naltrindole (NTI) and the κ-specific norbinaltorphimine (norBNI) showed only slight blocking effects compared with naloxone. The results obtained in the in vitro agonist-stimulated [ 35S]GTPγS binding assays are in good agreement with the opioid agonist effect seen in the in vivo pain test.

NAD(P)H oxidase, superoxide dismutase, catalase, glutathione peroxidase and nitric oxide synthase expression in subacute spinal cord injury

Primary trauma to the spinal cord triggers a cascade of cellular and molecular events that promote continued tissue damage and expansion of the lesion for extended periods following the initial injury. Oxidative and nitrosative stresses play an important role in progression of spinal cord injury (SCI). In an attempt to explore the biochemical origin of oxidative/nitrosative stress associated with secondary SCI, we studied expression of the superoxide (O 2· −)-generating enzyme, NAD(P)H oxidase, antioxidant enzymes [superoxide dismutase (CuZn SOD, Mn SOD), catalase, glutathione peroxidase (GPX)], nitric oxide synthases (NOS) and a byproduct of NO–O 2· − interaction (nitrotyrosine) in the spinal cord tissues of rats 16 h and 14 days after surgical resections of a 5-mm segment of the cord below T8 or sham-operation. Immunodetectable NAD(P)H oxidase subunits (gp91 phox and P67 phox), Mn SOD, inducible NOS (iNOS), endothelial NOS (eNOS), and nitrotyrosine were elevated in the transected cords on day 1 and day 14. Neuronal NOS (nNOS) was unchanged on day 1 and significantly depressed on day 14. GPX was unchanged on day 1 and significantly elevated on day 14. Catalase was unchanged in the cord tissue surrounding the transection site at both points. Thus, concurrent upregulations of NAD(P)H oxidase, eNOS and iNOS (but not nNOS), work in concert to maintain oxidative and nitrosative stress in the injured cord tissue.

Increased protein oxidation and decreased creatine kinase BB expression and activity after spinal cord contusion injury.

Traumatic injury to the spinal cord triggers several secondary effects, including oxidative stress and compromised energy metabolism, which play a major role in biochemical and pathological changes in spinal cord tissue. Free radical generation and lipid peroxidation have been shown to be early events subsequent to spinal cord injury. In the present study, we demonstrated that protein oxidation increases in rat spinal cord tissue after experimental injury. As early as h after injury, the level of protein carbonyls at the injury epicenter was significantly higher than in control (169%, p < 0.05) and increased gradually over the next 4 weeks to 1260% of control level. Both caudal and rostral parts of the injured spinal cord demonstrated a mild increase of protein carbonyls by 4 weeks postinjury (135-138%, p < 0.05). Immunocytochemical analysis of protein carbonyls in the spinal cord cross-sections showed increased protein carbonyl immunoreactivity in the epicenter section compared to rostral and caudal sections of the same animal or control laminectomy animals. Increased protein carbonyl formation in damaged spinal cord tissue was associated with changes in activity and expression of an oxidative sensitive enzyme, creatine kinase BB, which plays an important role in the maintenance of ATP level in the CNS tissue. Damage to CK function in the CNS may severely aggravate the impairment of energy metabolism. The results of our study indicate that events associated with oxidative damage are triggered immediately after spinal cord trauma but continue to occur over the subsequent 4 weeks. These results suggest that antioxidant therapeutic strategies may be beneficial to lessen the consequences of the injury and potentially improve the restoration of neurological function.

Effect of anti-rat interleukin-6 antibody after spinal cord injury in the rat: inducible nitric oxide synthase expression, sodium- and potassium-activated, magnesium-dependent adenosine-5'-triphosphatase and superoxide dismutase activation, and ultrastructural changes.

The inflammatory cells that accumulate at the damaged site after spinal cord injury (SCI) may secrete interleukin-6 (IL-6), a mediator known to induce the expression of inducible nitric oxide synthase (iNOS). Any increased production of NO by iNOS activity would aggravate the primary neurological damage in SCI. If this mechanism does occur, the direct or indirect effects of IL-6 antagonists on iNOS activity should modulate this secondary injury. In this study, the authors produced spinal cord damage in rats and applied anti-rat IL-6 antibody to neutralize IL-6 bioactivity and to reduce iNOS. They determined the spinal cord tissue activities of Na+-K+/Mg++ adenosine-5'-triphosphatase (ATPase) and superoxide dismutase, evaluated iNOS immunoreactivity, and examined ultrastructural findings to assess the results of this treatment. Seventy rats were randomly allocated to four groups. Group I (10 rats) were killed to provide normal spinal cord tissue for testing. In Group II 20 rats underwent six-level laminectomy for the effects of total laminectomy alone to be determined. In Group III 20 rats underwent six-level T2-7 laminectomy and SCI was produced by extradural compression of the exposed cord. The same procedures were performed in the 20 Group IV rats, but these rats also received one (2 microg) intraperitoneal injection of anti-rat IL-6 antibody immediately after the injury and a second dose 24 hours posttrauma. Half of the rats from each of Groups II through IV were killed at 2 hours and the other half at 48 hours posttrauma. The exposed cord segments were immediately removed and processed for analysis. The results showed that neutralizing IL-6 bioactivity with anti-rat IL-6 antibody significantly attenuates iNOS activity and reduces secondary structural changes in damaged rat spinal cord tissue.

Cryosections of pre-irradiated adult rat spinal cord tissue support axonal regeneration in vitro

Neonatal X-irradiation of central nervous system (CNS) tissue markedly reduces the glial population in the irradiated area. Previous in vivo studies have demonstrated regenerative success of adult dorsal root ganglion (DRG) neurons into the neonatally-irradiated spinal cord. The present study was undertaken to determine whether these results could be replicated in an in vitro environment. The lumbosacral spinal cord of anaesthetised Wistar rat pups, aged between 1 and 5 days, was subjected to a single dose (40 Gray) of X-irradiation. A sham-irradiated group acted as controls. Rats were allowed to reach adulthood before being killed. Their lumbosacral spinal cords were dissected out and processed for sectioning in a cryostat. Cryosections (10 μm-thick) of the spinal cord tissue were picked up on sterile glass coverslips and used as substrates for culturing dissociated adult DRG neurons. After an appropriate incubation period, cultures were fixed in 2% paraformaldehyde and immunolabelled to visualise both the spinal cord substrate using anti-glial fibrillary acidic protein (GFAP) and the growing DRG neurons using anti-growth associated protein (GAP-43). Successful growth of DRG neurites was observed on irradiated, but not on non-irradiated, sections of spinal cord. Thus, neonatal X-irradiation of spinal cord tissue appears to alter its environment such that it can later support, rather than inhibit, axonal regeneration. It is suggested that this alteration may be due, at least in part, to depletion in the number of and/or a change in the characteristics of the glial cells.

Formation of 6-nitro-norepinephrine from nitric oxide and norepinephrine in the spinal cord and its role in spinal analgesia

Spinally released norepinephrine is thought to produce analgesia in part by stimulating α 2-adrenergic receptors, which in turn leads to nitric oxide synthesis. Also, nitric oxide is known to react with norepinephrine in vivo in the brain to form 6-nitro-norepinephrine, which inhibits neuronal norepinephrine reuptake. In the present study, we tested the hypothesis that formation of 6-nitro-norepinephrine occurs in the spinal cord and that intrathecal administration of 6-nitro-norepinephrine produces analgesia by stimulating norepinephrine release. 6-Nitro-norepinephrine was present in rat spinal cord tissue and microdialysates of the dorsal horn and intrathecal space. Intrathecal norepinephrine injection increased 6-nitro-norepinephrine. 6-Nitro-norepinephrine also stimulated norepinephrine release in dorsal spinal cord in vitro. Intrathecal injection of 6-nitro-norepinephrine produced antinociception and interacted additively with norepinephrine for antinociception. Spinal noradrenergic nerve destruction increased antinociception from intrathecally injected norepinephrine, but decreased antinociception from 6-nitro-norepinephrine. These results suggest a functional interaction between spinal nitric oxide and norepinephrine in analgesia, mediated in part by formation of 6-nitro-norepinephrine. Stimulation of auto-inhibitory α 2-adrenergic receptors at noradrenergic synapses decreases norepinephrine release. Paradoxically, α 2-adrenergic agonist injection increases and α 2-adrenergic antagonist injection decreases norepinephrine release in the spinal cord. 6-Nitro-norepinephrine may be an important regulator of spinal norepinephrine release and could explain the positive feedback on norepinephrine release after activation of spinal α 2-adrenergic receptors.

Commercial recombinant human beta-nerve growth factor and adult rat dorsal root ganglia contain an identical molecular species of nerve growth factor prohormone.

Examination of commercial recombinant human beta-nerve growth factor (rh-beta-NGF) preparations with polyclonal antibodies specific to 13-kDa NGF and pro-NGF-specific domains revealed the presence of high-molecular-mass immunoreactive proteins, including a 60-kDa NGF prohormone. On incubation with a mixture of N- and O-specific glycosidases, the 60-kDa NGF pro-hormone generated a 32-kDa protein corresponding to the molecular size of NGF precursor predicted by the cloned human NGF cDNA. Highly sensitive chemiluminescence immunoblot analysis of adult rat dorsal root ganglia, spinal cord, and colon tissues with NGF- and pro-NGF domain-specific antibodies also revealed the presence of high-molecular-mass proteins, including the 60-kDa NGF prohormone. Based on the presence of the 60-kDa NGF prohormone in dorsal root ganglia and its efferent tissues, we suggest that proteolytically unprocessed, glycosylated NGF prohormone may mediate interactions between neurons and the tissues they innervate.