Association of transforming growth factor-beta 1 gene polymorphism with genetic susceptibility to ossification of the posterior longitudinal ligament in Korean patients.

Ossification of the posterior longitudinal ligaments (OPLL) has been considered to be associated with abnormalities of bone metabolism, and transforming growth factor-ß1 (TGF-ß1) has been demonstrated to affect the bone remodeling process. We investigated two SNPs of the TGF-ß1 promoter (-509C>T; rs1800469) and exon 1 (869T>C; rs1982073) in 298 Koreans (98 patients with OPLL and 200 control subjects). The promoter SNP -509C>T was determined by PCR and RFLP, and the TaqMan probe assay was used to determine 869T>C polymorphism genotypes. The subjects were divided into OPLL continuous group (continuous type plus mixed type) and OPLL segmental group (segmental and localized type). We also separately analyzed this association according to gender difference. There was no significant difference in genotype distributions of -509C>T and 869T>C polymorphisms of the TGF-ß1 gene between OPLL patients and controls. A combined analysis of TGF-ß1 -509C>T and 869T>C polymorphisms showed no significant association with OPLL, and a subgroup analysis did not show any significant correlation between the SNP -509C>T or SNP 869T>C and OPLL subgroups. Stratification by gender demonstrated no significant effect. We conclude that promoter region (-509C>T) and exon 1 (869T>C) polymorphisms are not associated with OPLL in the Korean population.

Association between VEGF and eNOS gene polymorphisms and lumbar disc degeneration in a young Korean population.

Disturbances in blood flow to intervertebral discs (IVD) play an important role in IVD degeneration. Vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS) are extremely important angiogenic factors for vasodilation and neovascularization. We investigated the relationship between single nucleotide polymorphisms (SNPs) of the VEGF and eNOS genes and genetic susceptibility to lumbar IVD degeneration in a young adult Korean population. Two hundred and forty-one participants (aged 18 to 30 years), with or without low back pain, were selected for the study. Magnetic resonance imaging was made of the lumbar spine in all participants. The patient group (N = 102) had low back pain clinically and lumbar IVD degeneration radiographically. The control group (N = 139) included subjects with and without low back pain; all were negative radiographically for lumbar IVD degeneration. Using PCR-RFLP analysis, we analyzed VEGF (-2578C>A, -1154G>A, -634G>C, and 936C>T) and eNOS (-786T>C, 4a4b and 894G>T) SNPs. We made combined analyses of the genes and performed haplotype analyses. There were no significant differences in the genotype distribution of polymorphisms of VEGF and eNOS genes among patients and controls. However, the frequency of VEGF -2578CA +AA/-634CC combined genotypes was significantly higher in patients when compared with controls [odds ratio (OR) = 21.00; 95% confidence interval (CI) = 2.590- 170.240]. The frequencies of the -2578A/-1154A/-634C/936C (OR = 3.831; 95%CI = 1.068-13.742), -2578A/-1154A/-634C (OR = 3.356; 95%CI = 1.198-9.400), and -2578A/-634C/936C (OR = 10.820; 95%CI = 2.811-41.656) haplotypes were also significantly higher in patients than in controls. We conclude that the combined genotype VEGF -2578CA+AA/-634CC is a possible risk factor for IVD degeneration and the VEGF -2578A/-1154A/-634C/936C haplotype may increase the risk for development of IVD degeneration. Furthermore, the VEGF -634C allele appears to be associated with susceptibility to IVD degeneration.

Effects of the sodium channel blocker tetrodotoxin on acute white matter pathology after experimental contusive spinal cord injury.

Focal microinjection of tetrodotoxin (TTX), a potent voltage-gated sodium channel blocker, reduces neurological deficits and tissue loss after spinal cord injury (SCI). Significant sparing of white matter (WM) is seen at 8 weeks after injury and is correlated to a reduction in functional deficits. To determine whether TTX exerts an acute effect on WM pathology, Sprague Dawley rats were subjected to a standardized weight-drop contusion at T8 (10 gm x 2.5 cm). TTX (0. 15 nmol) or vehicle solution was injected into the injury site 5 or 15 min later. At 4 and 24 hr, ventromedial WM from the injury epicenter was compared by light and electron microscopy and immunohistochemistry. By 4 hr after SCI, axonal counts revealed reduced numbers of axons and significant loss of large (>/=5 micrometer)-diameter axons. TTX treatment significantly reduced the loss of large-diameter axons. In addition, TTX significantly attenuated axoplasmic pathology at both 4 and 24 hr after injury. In particular, the development of extensive periaxonal spaces in the large-diameter axons was reduced with TTX treatment. In contrast, there was no significant effect of TTX on the loss of WM glia after SCI. Thus, the long-term effects of TTX in reducing WM loss after spinal cord injury appear to be caused by the reduction of acute axonal pathology. These results support the hypothesis that TTX-sensitive sodium channels at axonal nodes of Ranvier play a significant role in the secondary injury of WM after SCI.

Basic fibroblast growth factor increases long-term survival of spinal motor neurons and improves respiratory function after experimental spinal cord injury.

Acute focal injection of basic fibroblast growth factor (FGF2) protects ventral horn (VH) neurons from death after experimental contusive spinal cord injury (SCI) at T8. Because these neurons innervate respiratory muscles, we hypothesized that respiratory deficits resulting from SCI would be attenuated by FGF2 treatment. To test this hypothesis we used a head-out plethysmograph system to evaluate respiratory parameters in conscious rats before and at 24 hr and 7, 28, and 35 d after SCI. Two groups of rats (n = 8 per group) received either FGF2 (3 microg) beginning 5 min after injury or vehicle (VEH) solution alone. We found significantly increased respiratory rate and decreased tidal volume at 24 hr and 7 d after SCI in the VEH-treated group. Ventilatory response to breathing 5 or 7% CO(2) was also significantly reduced. Recovery took place over time. Respiration remained normal in the FGF2-treated group. At 35 d after injury, histological analyses were used to compare long-term neuron survival. FGF2 treatment doubled the survival of VH neurons adjacent to the injury site. Because the number of surviving VH neurons rostral to the injury epicenter was significantly correlated to the ventilatory response to CO(2), it is likely that the absence of respiratory deficits in FGF2-treated rats was caused by its neuroprotective effect. Our results demonstrate that FGF2 treatment prevents the respiratory deficits produced by thoracic SCI. Because FGF2 also reduced the loss of preganglionic sympathetic motoneurons after injury, this neurotrophic factor may have broad therapeutic potential for SCI.

Neural stem cells are uniquely suited for cell replacement and gene therapy in the CNS.

In recent years, it has become evident that the developing and even the adult mammalian CNS contain a population of undifferentiated, multipotent cell precursors, neural stem cells, the plastic properties of which might be of advantage for the design of more effective therapies for many neurological diseases. This article reviews the recent progress in establishing rodent and human clonal neural stem cell lines, their biological properties, and how these cells can be utilized to correct a variety of defects, with prospects for the near future to harness their behaviour for neural stem cell-based treatment of diseases in humans.

Evidence that local non-NMDA receptors contribute to functional deficits in contusive spinal cord injury.

To investigate the role of non-N-methyl-D-aspartate (non-NMDA) types of excitatory amino acid (EAA) receptors in traumatic spinal cord injury, we administered 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)-quinoxaline (NBQX), a potent and specific antagonist of non-NMDA receptors, to rats with a standardized contusive spinal cord injury. Focal infusion of NBQX into the injury site significantly reduced long-term hindlimb functional deficits as well as decreasing the time required for the rats to establish a reflex bladder. The results suggest that non-NMDA receptors at or near the injury site are involved in producing a portion of the functional deficits that result from contusive spinal cord injury.

Evaluation of cardiorespiratory parameters in rats after spinal cord trauma and treatment with NBQX, an antagonist of excitatory amino acid receptors.

2,3-Dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxaline (NBQX), a selective antagonist of amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/kainate receptors, ameliorates functional deficits and tissue loss after experimental contusive spinal cord injury (SCI). Data suggest that NBQX acts via local receptors at the injury site. However, potential systemic effects of NBQX could also modify consequences of SCI. We therefore examined effects of therapeutic doses of NBQX on cardiorespiratory parameters (CRP) including: mean arterial pressure, heart rate, respiratory rate and arterial blood gas. We found no significant effect on these CRP of either focal microinjection or intravenous administration of NBQX at doses that are therapeutic for SCI, in either uninjured rats, or rats after SCI. The results support the hypothesis that NBQX affects SCI by acting locally rather than through systemic effects and demonstrate NBQX treatment paradigms without adverse effects on CRP.

Translational spinal cord injury research: preclinical guidelines and challenges.

Advances in the neurobiology of spinal cord injury (SCI) have prompted increasing attention to opportunities for moving experimental strategies towards clinical applications. Preclinical studies are the centerpiece of the translational process. A major challenge is to establish strategies for achieving optimal translational progression while minimizing potential repetition of previous disappointments associated with clinical trials. This chapter reviews and expands upon views pertaining to preclinical design reported in recently published opinion surveys. Subsequent discussion addresses other preclinical considerations more specifically related to current and potentially imminent cellular and pharmacological approaches to acute/subacute and chronic SCI. Lastly, a retrospective and prospective analysis examines how guidelines currently under discussion relate to select examples of past, current, and future clinical translations. Although achieving definition of the "perfect" preclinical scenario is difficult to envision, this review identifies therapeutic robustness and independent replication of promising experimental findings as absolutely critical prerequisites for clinical translation. Unfortunately, neither has been fully embraced thus far. Accordingly, this review challenges the notion "everything works in animals and nothing in humans", since more rigor must first be incorporated into the bench-to-bedside translational process by all concerned, whether in academia, clinical medicine, or corporate circles.

Neural and anatomical abnormalities of the gastrointestinal system resulting from contusion spinal cord injury.

Gastrointestinal (GI) abnormalities resulting from spinal cord injury (SCI) are challenging disorders that have not been examined experimentally using clinically relevant models. In this study, female Sprague-Dawley rats (n=5/groupx4: T10-T11 contusion, laminectomy, or naïve) were fasted for 24 h before being submitted to dye recovery assays (Phenol Red solution, 1.5 ml/rat; per oral) on GI emptying/transiting at 48 h or 4 weeks postinjury (p.i.). Compared with controls, SCI significantly increased dye recovery rate (DRR, determined by spectrophotometry) in the duodenum (+84.6%) and stomach (+32.6%), but decreased it in the jejunum (-64.1% and -49.5%) and ileum (-73.6% and -70.1%) at 48 h and 4 weeks p.i., respectively (P

Spinal cord injury causes rapid osteoclastic resorption and growth plate abnormalities in growing rats (SCI-induced bone loss in growing rats).

UNLABELLED: Spinal cord injury causes severe bone loss. We report osteoclast resorption with severe trabecular and cortical bone loss, decreased bone mineral apposition, and growth plate abnormalities in a rodent model of contusion spinal cord injury. These findings will help elucidate the mechanisms of osteoporosis following neurological trauma. INTRODUCTION: Limited understanding of the mechanism(s) that underlie spinal cord injury (SCI)-induced bone loss has led to few treatment options. As SCI-induced osteoporosis carries significant morbidity and can worsen already profound disability, there is an urgency to advance knowledge regarding this pathophysiology. METHODS: A clinically relevant contusion model of experimental spinal cord injury was used to generate severe lower thoracic SCI by weight-drop (10 g x 50 mm) in adolescent male Sprague-Dawley rats. Body weight and gender-matched naïve (no surgery) rats served as controls. Bone microarchitecture was determined by micro-computed tomographic imaging. Mature osteoclasts were identified by TRAP staining and bone apposition rate was determined by dynamic histomorphometry. RESULTS: At 10 days post-injury we detected a marked 48% decrease in trabecular bone and a 35% decrease in cortical bone at the distal femoral metaphysis by micro-CT. A 330% increase in the number of mature osteoclasts was detected at the growth plate in the injured animals that corresponded with cellular disorganization at the chondro-osseous junction. Appositional growth studies demonstrated decreased new bone formation with a mineralization defect indicative of osteoblast dysfunction. CONCLUSIONS: Contusion SCI results in a rapid bone loss that is the result of increased bone resorption and decreased bone formation.

Local blockade of sodium channels by tetrodotoxin ameliorates tissue loss and long-term functional deficits resulting from experimental spinal cord injury.

Although relatively little is known of the mechanisms involved in secondary axonal loss after spinal cord injury (SCI), recent data from in vitro models of white matter (WM) injury have implicated abnormal sodium influx as a key event. We hypothesized that blockade of sodium channels after SCI would reduce WM loss and long-term functional deficits. To test this hypothesis, a sufficient and safe dose (0.15 nmol) of the potent Na+ channel blocker tetrodotoxin (TTX) was determined through a dose-response study. We microinjected TTX or vehicle (VEH) into the injury site at 15 min after a standardized contusive SCI in the rat. Behavioral tests were performed 1 d after injury and weekly thereafter. Quantitative histopathology at 8 weeks postinjury showed that TTX treatment significantly reduced tissue loss at the injury site, with greater effect on sparing of WM than gray matter. TTX did not change the pattern of chronic histopathology typical of this SCI model, but restricted its extent, tripled the area of residual WM at the epicenter, and reduced the average length of the lesions. Serotonin immunoreactivity caudal to the epicenter, a marker for descending motor control axons, was nearly threefold that of VEH controls. The increase in WM at the epicenter was significantly correlated with the decrease in functional deficits. The TTX group exhibited a significantly enhanced recovery of coordinated hindlimb functions, more normal hindlimb reflexes, and earlier establishment of a reflex bladder. The results demonstrate that Na+ channels play a critical role in WM loss in vivo after SCI.

Amelioration of functional deficits from spinal cord trauma with systemically administered NBQX, an antagonist of non-N-methyl-D-aspartate receptors.

Excitatory amino acid (EAA) receptors play a significant role in delayed neuronal death after ischemic and traumatic injury to the CNS. Recent data based on focal microinjection experiments have demonstrated that 2,3-dihydro-6-nitro-7-sulfamoyl-benzo(f)quinoxaline (NBQX), a highly selective and potent antagonist of non-N-methyl-D-aspartate ionotropic EAA receptors, i.e., those preferring alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) or kainate, can reduce histopathology and functional deficits after traumatic spinal cord injury (SCI). Thus, non-NMDA receptors at or near the injury site appear to be important in secondary injury processes that contribute significantly to the consequences of SCI. We have now examined the effects of systemic NBQX, using intravenous infusion, the most commonly used and temporally efficient clinical mode of drug administration. Standardized contusive SCI was produced at the T8 vertebral level in Sprague-Dawley rats. Beginning at 15 min postin-jury, NBQX was administered intravenously at 1 mg/kg/min for 30 min. Behavioral tests of hindlimb functional deficits were performed at 1 day and weekly for 1 month after SCI. Spinal cord tissue was then examined morphometrically to compare lesion size and amount of spared tissue. We found that intravenous administration of NBQX significantly reduced functional impairment after SCI. The effects included more rapid and extensive recovery of hindlimb reflexes, more rapid establishment of a reflex bladder, and a more rapid and greater degree of recovery of coordinated use of hindlimbs in open field locomotion, swimming, and maintaining position on an inclined plane. The profile of effects was similar to that seen with focal microinjection of NBQX, suggesting that even with systemic administration, the drug acts mainly at the injury site. Further, the results support a therapeutic potential for NBQX, or similar drugs that antagonize non-NMDA receptors and inhibit secondary injury processes after SCI.

Dose-dependent reduction of tissue loss and functional impairment after spinal cord trauma with the AMPA/kainate antagonist NBQX.

Initial studies on the role of glutamate receptors in traumatic spinal cord injury (SCI) implicated the NMDA subclass of ionotropic glutamate receptors in contributing to functional deficits. Recently we obtained evidence suggesting that non-NMDA ionotropic receptors may participate in producing a portion of the behavioral impairment after SCI. To test this hypothesis we have conducted a dose-response experiment, focally injecting 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxaline (NBQX; 1.5, 5, or 15 nmol), a highly selective antagonist of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/kainate receptors, or vehicle alone, into the injury site beginning at 15 min after a standardized contusive SCI. Behavioral tests of hindlimb reflex and coordinated sensorimotor function were performed 1 d after injury and weekly thereafter. At 4 weeks, spinal cord tissue was examined using quantitative histopathological and immunocytochemical techniques. We found a dose-dependent reduction in tissue loss at the thoracic injury site, with greater residual amounts of both gray matter and myelinated white matter. The maximum dose (15 nmol) significantly reduced the average length of the lesions and doubled the area of residual white matter at the epicenter. Serotonin immunoreactivity caudal to the lesion, used as a marker for descending motor control axons, was also increased in a dose-related manner and nearly tripled with the highest dose of NBQX as compared to controls. Most importantly, the reduced tissue loss in NBQX-treated groups was correlated with reduced functional deficits. There was a dose-dependent enhancement of speed and degree of recovery of both reflex and coordinated hindlimb motor activity, and reduction in the time required for establishing a reflex bladder. The long-term functional deficits at 4 weeks after SCI were reduced in a dose-related manner. Further, regression analyses demonstrated a significant correlation between the increase in amount of residual tissue and improvement in hindlimb function. Our results suggest that in this type of incomplete contusive SCI, a large and functionally important proportion of the tissue loss appears to be due to secondary injury mediated by local AMPA/kainate receptors.

Delayed antagonism of AMPA/kainate receptors reduces long-term functional deficits resulting from spinal cord trauma.

Excitatory amino acid (EAA) receptors play a significant role in delayed neuronal death after ischemic and traumatic injury to the CNS. Focal microinjection experiments have demonstrated that 2,3-dihydro-6-nitro-7-sulfamoyl-benzo(f)quinoxaline (NBQX), a highly selective and potent antagonist of non-N-methyl-D-aspartate ionotropic EAA receptors, i.e., those preferring alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) or kainate, can reduce histopathology and functional deficits when administered at 15 min after traumatic spinal cord injury (SCI). Similarly, intravenous infusion of NBQX, beginning at 15 min postinjury (p.i.), results in a significant amelioration of the functional deficits produced by experimental SCI. However, if antagonists of AMPA/kainate receptors were to be used therapeutically for patients with SCI, administration would likely be delayed for several hours after injury. We therefore examined the effects of NBQX administered at 4 h after SCI on functional deficits and histopathology in a standardized rat model of contusive SCI. An incomplete SCI was produced in Sprague-Dawley rats at T8 with a weight-drop device (10 g x 2.5 cm). NBQX (15 nmol), or vehicle alone, was microinjected into the injury site 4 h later. Recovery of hind limb reflexes, postural control, and locomotor function was determined by a battery of behavioral tests performed for 8 weeks. Spinal cord tissue was then fixed by perfusion and used for morphometric and immunocytochemical analyses. Previous studies with acute NBQX treatment showed significant functional improvement by 1 week; the effects of delayed NBQX treatment on functional deficits were not discernible until 3-4 weeks after SCI. Thereafter, significant reductions in hindlimb deficits were demonstrated in two independent studies. The nature and magnitude of the reductions in chronic deficits were similar to those observed previously when NBQX was administered acutely at 15 min after SCI. Morphometric analyses showed that delayed treatment with NBQX resulted in sparing of gray matter adjacent to the injury site but no significant effect on the area of white matter at the epicenter. However, serotonin immunoreactivity below the lesion, used as a marker for preservation of one supraspinal pathway, was significantly higher in the NBQX-treated group. These results support a therapeutic potential for NBQX, and presumably other AMPA antagonists, in SCI by demonstrating effectiveness in a clinically relevant time frame. They indicate the importance of assessing chronic functional deficits in evaluating the therapeutic potential of a treatment paradigm. Further, they suggest the intriguing hypothesis that mechanisms underlying early functional recovery after SCI are, at least in part, distinct those from those involved in reducing chronic functional deficits.

2,3-Dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxaline reduces glial loss and acute white matter pathology after experimental spinal cord contusion.

Focal microinjection of 2, 3-dihyro-6-nitro-7-sulfamoyl-benzo(f)quinoxaline (NBQX), an antagonist of the AMPA/kainate subclass of glutamate receptors, reduces neurological deficits and tissue loss after spinal cord injury. Dose-dependent sparing of white matter is seen at 1 month after injury that is correlated to the dose-related reduction in chronic functional deficits. To determine whether NBQX exerts an acute effect on white matter pathology, female, adult Spague Dawley rats were subjected to a standardized weight drop contusion at T-8 (10 gm x 2.5 cm) and NBQX (15 nmol) or vehicle (VEH) solution focally injected into the injury site 15 min later. At 4 and 24 hr, tissue from the injury epicenter was processed for light and electron microscopy, and the histopathology of ventromedial white matter was compared. The axonal injury index, a quantitative representation of axoplasmic and myelinic pathologies, was significantly lower in the NBQX group at 4 hr (2.7 +/- 0.24, mean +/- SE) and 24 hr (1.4 +/- 0.19) than in VEH controls (3.8 +/- 0.33 and 2.1 +/- 0.20, respectively). Counts of glial cell nuclei indicated a loss of at least 60% at 4 and 24 hr after injury in the VEH group compared with uninjured controls. NBQX treatment reduced this glial loss by half. Immunohistochemistry revealed that the spared glia were primarily oligodendrocytes. Thus, the chronic effects of NBQX in reducing white matter loss after spinal cord injury appear to be attributable to the reduction of acute pathology and may be mediated through the protection of glia, particularly oligodendrocytes.

Basic and acidic fibroblast growth factors protect spinal motor neurones in vivo after experimental spinal cord injury.

We studied the effect of a single focal injection of recombinant basic (FGF2) or acidic (FGF1) fibroblast growth factor on the survival of spinal motor neurones at 24 h after a standardized spinal cord contusion injury (SCI) in the rat. Both FGF2 and FGF1 (3 microg), microinjected into the injury site at 5 min post-injury (p.i.), protected at least two functionally important classes of spinal motor neurones, autonomic preganglionic neurones in the intermediolateral (IML) column and somatic motor neurones in the ventral horn (VH). Moreover, there was enhanced choline acetyltransferase (ChAT) immunoreactivity in surviving VH and IML neurones, suggesting an improved functional status. Thus, neurotrophic factors such as FGF2 and FGF1 may contribute to an overall strategy to treat acute SCI and improve recovery of function.