Cellular Neuroinflammation in a Lateral Forceps Compression Model of Spinal Cord Injury

Postinjury inflammation has been implicated in secondary degeneration following injury to the spinal cord. The cellular inflammatory response to injury has not been described in the lateral compression injury model, although various types of compression injuries account for ～20% of human spinal cord injuries (SCI). Here, we used forceps to induce a moderate compression injury to the thoracic spinal cord of female Sprague‐Dawley rats. We evaluated innate and adaptive components of the inflammatory response at various times postinjury using immunohistochemical techniques. We show that components of innate immunity (e.g., macrophages and dendritic cells) peak between 1 and 2 weeks postinjury but persist through 42 days postinjury (dpi). CD163 and CD206 expression, associated with an anti‐inflammatory, reparative phenotype, was upregulated on activated macrophages in the injury site, as were MHC class II antigens. The expression of MHC class II antigens is necessary for the initiation of adaptive immunity and was accompanied by an influx of T cells. T cells were initially restricted to gray matter at the injury epicenter but were later observed throughout the lesioned parenchyma. In summary, we demonstrate that lateral forceps compression of the spinal cord produces a neuroinflammatory response similar to that described in human spinal cord trauma and in other experimental models of spinal cord trauma, thus is an appropriate model to study secondary neurodegeneration in SCI. Anat Rec, 2013. © 2013 Wiley Periodicals, Inc.     

Developmental changes in leg coordination of the chick at embryonic days 9, 11, and 13: uncoupling of ankle movements

Acute repair of crushed guinea pig spinal cord by polyethylene glycol. We have studied the responses of adult guinea pig spinal cord white matter to a standardized compression within a sucrose gap recording chamber. This injury eliminated compound action potential (CAP) conduction through the lesion, followed by little or no recovery of conduction by 1 h postinjury. We tested the ability of polyethylene glycol (PEG) to repair the injured axons and restore physiological function. Local application of PEG (1,800 MW, 50% by weight in water) for approximately 2 min restored CAP conduction through the injury as early as 1 min post PEG application. The recovery of the CAP 相似文献   

Control of membrane sealing in injured mammalian spinal cord axons

The process of sealing of damaged axons was examined in isolated strips of white matter from guinea pig spinal cord by recording the "compound membrane potential," using a sucrose-gap technique, and by examining uptake of horseradish peroxidase (HRP). Following axonal transection, exponential recovery of membrane potential occurred with a time constant of 20 +/- 5 min, at 37 degrees C, and extracellular calcium activity ([Ca(2+)](o)) of 2 mM. Most axons excluded HRP by 30 min following transection. The rate of sealing was reduced by lowering calcium and was effectively blocked at [Ca(2+)](o) 相似文献   

Changes in axonal physiology and morphology after chronic compressive injury of the rat thoracic spinal cord

The spinal cord is rarely transected after spinal cord injury. Dysfunction of surviving axons, which traverse the site of spinal cord injury, appears to contribute to post-traumatic neurological deficits, although the underlying mechanisms remain unclear. The subpial rim frequently contains thinly myelinated axons which appear to conduct signals abnormally, although it is uncertain whether this truly reflects maladaptive alterations in conduction properties of injured axons during the chronic phase of spinal cord injury or whether this is merely the result of the selective survival of a subpopulation of axons. In the present study, we examined the changes in axonal conduction properties after chronic clip compression injury of the rat thoracic spinal cord, using the sucrose gap technique and quantitatively examined changes in the morphological and ultrastructural features of injured axonal fibers in order to clarify these issues. Chronically injured dorsal columns had a markedly reduced compound action potential amplitude (8.3% of control) and exhibited significantly reduced excitability. Other dysfunctional conduction properties of injured axons included a slower population conduction velocity, a longer refractory period and a greater degree of high-frequency conduction block at 200 Hz. Light microscopic and ultrastructural analysis showed numerous axons with abnormally thin myelin sheaths as well as unmyelinated axons in the injured spinal cord. The ventral column showed a reduced median axonal diameter and the lateral and dorsal columns showed increased median diameters, with evidence of abnormally large swollen axons. Plots of axonal diameter versus myelination ratio showed that post-injury, dorsal column axons of all diameters had thinner myelin sheaths. Noninjured dorsal column axons had a median myelination ratio (1.56) which was within the optimal range (1.43-1.67) for axonal conduction, whereas injured dorsal column axons had a median myelination ratio (1.33) below the optimal value. These data suggest that maladaptive alterations occur postinjury to myelin sheath thickness which reduce the efficiency of axonal signal transmission.In conclusion, chronically injured dorsal column axons show physiological evidence of dysfunction and morphological changes in axonal diameter and reduced myelination ratio. These maladaptive alterations to injured axons, including decrease in myelin thickness and the appearance of axonal swellings, contribute to the decreased excitability of chronically injured axons. These results further clarify the mechanisms underlying neurological dysfunction after chronic neurotrauma and have significant implications regarding approaches to augment neural repair and regeneration.     

Role of TTX-sensitive and TTX-resistant sodium channels in Adelta- and C-fiber conduction and synaptic transmission

Thin afferent axons conduct nociceptive signals from the periphery to the spinal cord. Their somata express two classes of Na+ channels, TTX-sensitive (TTX-S) and TTX-resistant (TTX-R), but their relative contribution to axonal conduction and synaptic transmission is not well understood. We studied this contribution by comparing effects of nanomolar TTX concentrations on currents associated with compound action potentials in the peripheral and central branches of Adelta- and C-fiber axons as well as on the Adelta- and C-fiber-mediated excitatory postsynaptic currents (EPSCs) in spinal dorsal horn neurons of rat. At room temperature, TTX completely blocked Adelta-fibers (IC50, 5-7 nM) in dorsal roots (central branch) and spinal, sciatic, and sural nerves (peripheral branch). The C-fiber responses were blocked by 85-89% in the peripheral branch and by 65-66% in dorsal roots (IC50, 14-33 nM) with simultaneous threefold reduction in their conduction velocity. At physiological temperature, the degree of TTX block in dorsal roots increased to 93%. The Adelta- and C-fiber-mediated EPSCs in dorsal horn neurons were also sensitive to TTX. At room temperature, 30 nM blocked completely Adelta-input and 84% of the C-fiber input, which was completely suppressed at 300 nM TTX. We conclude that in mammals, the TTX-S Na+ channels dominate conduction in all thin primary afferents. It is the only type of functional Na+ channel in Adelta-fibers. In C-fibers, the TTX-S Na+ channels determine the physiological conduction velocity and control synaptic transmission. TTX-R Na+ channels could not provide propagation of full-amplitude spikes able to trigger synaptic release in the spinal cord.     

Role of Na(v)1.9 in activity-dependent axon growth in motoneurons

Spontaneous neural activity promotes axon growth in many types of developing neurons, including motoneurons. In motoneurons from a mouse model of spinal muscular atrophy (SMA), defects in axonal growth and presynaptic function correlate with a reduced frequency of spontaneous Ca(2+) transients in axons which are mediated by N-type Ca(2+) channels. To characterize the mechanisms that initiate spontaneous Ca(2+) transients, we investigated the role of voltage-gated sodium channels (VGSCs). We found that low concentrations of the VGSC inhibitors tetrodotoxin (TTX) and saxitoxin (STX) reduce the rate of axon growth in cultured embryonic mouse motoneurons without affecting their survival. STX was 5- to 10-fold more potent than TTX and Ca(2+) imaging confirmed that low concentrations of STX strongly reduce the frequency of spontaneous Ca(2+) transients in somatic and axonal regions. These findings suggest that the Na(V)1.9, a VGSC that opens at low thresholds, could act upstream of spontaneous Ca(2+) transients. qPCR from cultured and laser-microdissected spinal cord motoneurons revealed abundant expression of Na(V)1.9. Na(V)1.9 protein is preferentially localized in axons and growth cones. Suppression of Na(V)1.9 expression reduced axon elongation. Motoneurons from Na(V)1.9(-/-) mice showed the reduced axon growth in combination with reduced spontaneous Ca(2+) transients in the soma and axon terminals. Thus, Na(V)1.9 function appears to be essential for activity-dependent axon growth, acting upstream of spontaneous Ca(2+) elevation through voltage-gated calcium channels (VGCCs). Na(V)1.9 activation could therefore serve as a target for modulating axonal regeneration in motoneuron diseases such as SMA in which presynaptic activity of VGCCs is reduced.     

Functional changes in genetically dysmyelinated spinal cord axons of shiverer mice: role of juxtaparanodal Kv1 family K+ channels

Axonal dysfunction after spinal cord injury (SCI) and other types of neurotrauma is associated with demyelination and exposure of juxtaparanodal K+ channels. In this study, sucrose gap electrophysiology using selective and nonselective K+ channel blockers, confocal immunohistochemistry, and Western blotting were used to study the role of Kv1.1 and Kv1.2 K+ channel subunits in dysmyelination-induced spinal cord axonal dysfunction in shiverer mice, which lack the gene encoding myelin basic protein (MBP) and exhibit incomplete myelin sheath formation on CNS axons. The shiverer spinal cord axons exhibited smaller amplitude of compound action potentials (CAPs), reduced conduction velocity, reduced excitability, and greater degree of high-frequency conduction failure. The "fast" K+ channel blocker 4-aminopyridine, the toxin DTX-I, which targets the Kv1.1 and Kv1.2, but not DTX- K, which has higher selectivity for Kv1.1, increased the amplitude and area of CAPs of shiverer mice spinal cord axons but had insignificant effects in wild-type mice. Confocal immunohistochemistry showed that, unlike wild-type mice, which have a precise juxtaparanodal localization of the Kv1.l and Kv1.2 K+ channel subunits, shiverer mouse axons displayed a dispersed distribution of these subunits along the internodes. In contrast, the Kv1.l and Kv1.2 subunits, Na+ channels remained highly localized to the nodal regions. Western blotting showed an increased expression of Kv 1.1 and 1.2 in the shiverer mouse spinal cord. These results provide evidence that the neurological deficits associated with myelin deficiency reflect the altered distribution and expression of the K+ channel subunits Kv1.l and Kv1.2 along the internodes of spinal cord axons associated with the biophysical consequences caused by alterations in the myelin sheaths.     

大鼠脊髓损伤后神经细胞凋亡及相关调控蛋白Caspase-3表达的时空分布

目的 观察大鼠脊髓损伤（SCI）后神经元细胞凋亡和相关调控蛋白Caspase-3表达的时间、空间分布规律。方法 SD大鼠50只,分为假手术组和SCI组,钳夹法建立SCI模型,用结晶紫（CV）染色法、TUNEL法和免疫组织化学方法观察SCI后6h、12h、24h、3d和7d损伤中心到头端0～5.00mm空间范围内脊髓神经细胞存活、凋亡以及相关调控蛋白Caspase-3的表达状况。 结果 CV染色发现,SCI后6h～7d在0～0.50mm空间范围内均未发现存活的神经细胞,在1.00～3.50mm距离内,存活的神经细胞数逐渐增加,4.00mm处达峰值;TUNEL结果发现,SCI后6h～7d在1.05～4.55mm范围内,神经细胞出现凋亡,3d时在2.55mm处,细胞凋亡达高峰,7d时显著减少;免疫组织化学结果发现,SCI后6h～7d在1.10～4.60mm范围内,神经元细胞出现Caspase-3阳性表达,3d在3.10mm处Caspase-3表达达到高峰,7d显著减少。 结论 SCI后,凋亡的神经细胞及其相关调控蛋白Caspase-3的表达有一定的时空分布规律。     

The ionic mechanisms underlying N-methyl-D-aspartate receptor-induced, tetrodotoxin-resistant membrane potential oscillations in lamprey neurons active during locomotion

Activation of N-methyl-D-aspartate (NMDA) receptors can induce tetrodotoxin (TTX)-resistant membrane potential oscillations as well as fictive locomotion in the in vitro preparation of the lamprey spinal cord. The ionic basis of these oscillations were investigated in the presence of N-methyl-D,L-aspartate and TTX. Addition of blocking agents (2-amino-5-phosphonovalerate and tetraethylammonium (TEA)) and selective removal or substitution of certain ions (Mg2+, Ca2+, Na+, Ba2+) were used in the analysis of the oscillations. The depolarizing phase of the oscillation requires Na+ ions but not Ca2+ ions. The depolarization becomes larger if TEA is administered in the bath, which presumably is due to a blockade of potassium (K+) channels activated during the depolarizing phase. The repolarization appears to depend on a Ca2+ entry, which presumably acts indirectly by an activation of Ca2+-dependent K+ channels. Together with the NMDA-induced voltage dependence, this will bring the membrane potential back down to a hyperpolarized level.     

Effects of baclofen on spinal reflexes and persistent inward currents in motoneurons of chronic spinal rats with spasticity

In the months after spinal cord injury, motoneurons develop large voltage-dependent persistent inward currents (PICs) that cause sustained reflexes and associated muscle spasms. These muscle spasms are triggered by any excitatory postsynaptic potential (EPSP) that is long enough to activate the PICs, which take > 100 ms to activate. The PICs are composed of a persistent sodium current (Na PIC) and a persistent calcium current (Ca PIC). Considering that Ca PICs have been shown in other neurons to be inhibited by baclofen, we tested whether part of the antispastic action of baclofen was to reduce the motoneuron PICs as opposed to EPSPs. The whole sacrocaudal spinal cord from acute spinal rats and spastic chronic spinal rats (with sacral spinal transection 2 mo previously) was studied in vitro. Ventral root reflexes were recorded in response to dorsal root stimulation. Intracellular recordings were made from motoneurons, and slow voltage ramps were used to measure PICs. Chronic spinal rats exhibited large monosynaptic and long-lasting polysynaptic ventral root reflexes, and motoneurons had associated large EPSPs and PICs. Baclofen inhibited these reflexes at very low doses with a 50% inhibition (EC50) of the mono- and polysynaptic reflexes at 0.26 +/- 0.07 and 0.25 +/- 0.09 (SD) microM, respectively. Baclofen inhibited the monosynaptic reflex in acute spinal rats at even lower doses (EC50 = 0.18 +/- 0.02 microM). In chronic (and acute) spinal rats, all reflexes and EPSPs were eliminated with 1 microM baclofen with little change in motoneuron properties (PICs, input resistance, etc), suggesting that baclofen's antispastic action is presynaptic to the motoneuron. Unexpectedly, in chronic spinal rats higher doses of baclofen (20-30 microM) significantly increased the total motoneuron PIC by 31.6 +/- 12.4%. However, the Ca PIC component (measured in TTX to block the Na PIC) was significantly reduced by baclofen. Thus baclofen increased the Na PIC and decreased the Ca PIC with a net increase in total PIC. By contrast, when a PIC was induced by 5-HT (10-30 microM) in motoneurons of acute spinal rats, baclofen (20-30 microM) significantly decreased the PIC by 38.8 +/- 25.8%, primarily due to a reduction in the Ca PIC (measured in TTX), which dominated the total PIC in these acute spinal neurons. In summary, baclofen does not exert its antispastic action postsynaptically at clinically achievable doses (< 1 microM), and at higher doses (10-30 microM), baclofen unexpectedly increases motoneuron excitability (Na PIC) in chronic spinal rats.     

Pathological changes of isolated spinal cord axons in response to mechanical stretch

White matter strips extracted from adult guinea-pig spinal cords were maintained in vitro and studied physiologically using a double sucrose gap technique and anatomically using a horseradish peroxidase assay. The amplitude of compound action potentials was monitored continuously before, during, and after elongation. Three types of conduction blocks resulting from stretch injury were identified: an immediate, spontaneously reversible component, which may result from a transient increase in membrane permeability and consequent disturbance of ionic distribution; a second component that was irreversible within 30–60 min of recording, perhaps resulting from profound axolemmal disruption; and a third component, which may be due to perturbation of the myelin sheath, that was reversible with application of 100 μM of the potassium channel blocker, 4-aminopyridine. The intensity of the conduction deficits correlated with the extent of initial stretch over a full range of severity. Stimulus–response data indicate that mechanical damage to axons in stretch was evenly distributed across the caliber spectrum. Morphological examinations revealed that a small portion of axons exhibited membrane damage at 2 min following stretch and appeared to be largely sealed at 30 min after injury. Further, in the entire length of the cord strip subjected to stretch, axons closer to the surface were found to be more likely to suffer membrane damage, which distinguished stretch injury from compression injury.

In summary, we have developed an in vitro model of axonal stretch that provides the ability to monitor changes in the properties of central myelinated axons following stretch injury in the absence of pathological variables related to vascular damage. This initial investigation found no evidence of secondary deterioration of axons in the first 30 min after stretch in vitro, although there was evidence of both transient and lasting physiological and anatomical damage to axons and their myelin sheaths.     

Serotonin facilitates a persistent calcium current in motoneurons of rats with and without chronic spinal cord injury

In the months after spinal cord transection, motoneurons in the rat spinal cord develop large persistent inward currents (PICs) that are responsible for muscle spasticity. These PICs are mediated by low-threshold TTX-sensitive sodium currents (Na PIC) and L-type calcium currents (Ca PIC). Recently, the Na PIC was shown to become supersensitive to serotonin (5-HT) after chronic injury. In the present paper, a similar change in the sensitivity of the Ca PIC to 5-HT was investigated after injury. The whole sacrocaudal spinal cord from acute spinal rats and spastic chronic spinal rats (S2 level transection 2 mo previously) was studied in vitro. Intracellular recordings were made from motoneurons and slow voltages ramps were applied to measure PICs. TTX was used to block the Na PIC. For motoneurons of chronic spinal rats, a low dose of 5-HT (1 microM) significantly lowered the threshold of the Ca PIC from -56.7 +/- 6.0 to -63.1 +/- 7.1 mV and increased the amplitude of the Ca PIC from 2.4 +/- 1.0 to 3.0 +/- 0.73 nA. Higher doses of 5-HT acted similarly. For motoneurons of acute spinal rats, low doses of 5-HT had no significant effects, whereas a high dose (about 30 microM) significantly lowered the threshold of the L-Ca PIC from -58.5 +/- 14.8 to -62.5 +/- 3.6 mV and increased the amplitude of the Ca PIC from 0.69 +/- 1.05 to 1.27 +/- 1.1 nA. Thus Ca PICs in motoneurons are about 30-fold supersensitive to 5-HT in chronic spinal rats. The 5-HT-induced facilitation of the Ca PIC was blocked by nimodipine, not by the I(h) current blocker Cs(+) (3 mM) or the SK current blocker apamin (0.15 microM), and it lasted for hours after the removal of 5-HT from the nCSF, even increasing initially after removing 5-HT. The effects of 5-HT make motoneurons more excitable and ultimately lead to larger, more easily activated plateaus and self-sustained firing. The supersensitivity to 5-HT suggests the small amounts of endogenous 5-HT below the injury in a chronic spinal rat may act on supersensitive receptors to produce large Ca PICs and ultimately enable muscle spasms.     

Phenytoin protects spinal cord axons and preserves axonal conduction and neurological function in a model of neuroinflammation in vivo

Axonal degeneration within the spinal cord contributes substantially to neurological disability in multiple sclerosis (MS). Thus neuroprotective therapies that preserve axons, so that they maintain their integrity and continue to function, might be expected to result in improved neurological outcome. Sodium channels are known to provide a route for sodium influx that can drive calcium influx, via reverse operation of the Na+/Ca2+ exchanger, after injury to axons within the CNS, and sodium channel blockers have been shown to protect CNS axons from degeneration after experimental anoxic, traumatic, and nitric oxide (NO)-induced injury. In this study, we asked whether phenytoin, which is known to block sodium channels, can protect spinal cord axons from degeneration in mice with experimental allergic encephalomyelitis (EAE), which display substantial axonal degeneration and clinical paralysis. We demonstrate that the loss of dorsal corticospinal tract (63%) and dorsal column (cuneate fasciculus; 43%) axons in EAE is significantly ameliorated (corticospinal tract: 28%; cuneate fasciculus: 17%) by treatment with phenytoin. Spinal cord compound action potentials (CAP) were significantly attenuated in untreated EAE, whereas spinal cords from phenytoin-treated EAE had robust CAPs, similar to those from phenytoin-treated control mice. Clinical scores in phenytoin-treated EAE at 28 days were significantly improved (1.5, i.e., minor righting reflex abnormalities) compared with untreated EAE (3.8, i.e., near-complete hindlimb paralysis). Our results demonstrate that phenytoin has a protective effect in vivo on spinal cord axons, preventing their degeneration, maintaining their ability to conduct action potentials, and improving clinical status in a model of neuroinflammation.     

Role of L- and N-type calcium channels in the pathophysiology of traumatic spinal cord white matter injury

Recent work has suggested a potential role for voltage-gated Ca(2+) channels in the pathophysiology of anoxic central nervous system white matter injury. To examine the relevance of these findings to neurotrauma, we conducted electrophysiological studies with inorganic Ca(2+) channels blockers and L- and N-subtype-specific calcium channel antagonists in an in vitro model of spinal cord injury. Confocal immunohistochemistry was used to examine for localization of L- and N-type calcium channels in spinal cord white matter tracts. A 30-mm length of dorsal column was isolated from the spinal cord of adult rats, pinned in an in vitro recording chamber and injured with a modified clip (2g closing force) for 15s. The functional integrity of the dorsal column was monitored electrophysiologically by quantitatively measuring the compound action potential at two points with glass microelectrodes. The compound action potential decreased to 71.4+/-2.0% of control (P<0. 05) after spinal cord injury. Removal of extracellular Ca(2+) promoted significantly greater recovery of compound action potential amplitude (86.3+/-7.6% of control; P< 0.05) after injury. Partial blockade of voltage-gated Ca(2+) channels with cobalt (20 microM) or cadmium (200 microM) conferred improvement in compound action potential amplitude. Application of the L-type Ca(2+) channel blockers diltiazem (50 microM) or verapamil (90 microM), and the N-type antagonist omega-conotoxin GVIA (1 microM), significantly enhanced the recovery of compound action potential amplitude postinjury. Co-application of the L-type antagonist diltiazem with the N-type blocker omega-conotoxin GVIA showed significantly greater (P<0.05) improvement in compound action potential amplitude than application of either drug alone. Confocal immunohistochemistry with double labelling for glial fibrillary acidic protein, GalC and NF200 demonstrated L- and N-type Ca(2+) channels on astrocytes and oligodendrocytes, but not axons, in spinal cord white matter.In conclusion, the injurious effects of Ca(2+) in traumatic central nervous system white matter injury appear to be partially mediated by voltage-gated Ca(2+) channels. The presence of L- and N-type Ca(2+) channels on periaxonal astrocytes and oligodendrocytes suggests a role for these cells in post-traumatic axonal conduction failure.     

Effects of ion channel blockade on the distribution of Na, K, Ca and other elements in oxygen-glucose deprived CA1 hippocampal neurons

The pathophysiology of brain ischemia and reperfusion injury involves perturbation of intraneuronal ion homeostasis. To identify relevant routes of ion flux, rat hippocampal slices were perfused with selective voltage- or ligand-gated ion channel blockers during experimental oxygen-glucose deprivation and subsequent reperfusion. Electron probe X-ray microanalysis was used to quantitate water content and concentrations of Na, K, Ca and other elements in morphological compartments (cytoplasm, mitochondria and nuclei) of individual CA1 pyramidal cell bodies. Blockade of voltage-gated channel-mediated Na+ entry with tetrodotoxin (1 microM) or lidocaine (200 microM) significantly reduced excess intraneuronal Na and Ca accumulation in all compartments and decreased respective K loss. Voltage-gated Ca2+ channel blockade with the L-type antagonist nitrendipine (10 microM) decreased Ca entry and modestly preserved CA1 cell elemental composition and water content. However, a lower concentration of nitrendipine (1 microM) and the N-, P-subtype Ca2+ channel blocker omega-conotoxin MVIIC (3 microM) were ineffective. Glutamate receptor blockade with the N-methyl-D-aspartate (NMDA) receptor-subtype antagonist 3-(2-carboxypiperazin-4-yl) propyl-1-phosphonic acid (CPP; 100 microM) or the alpha-amino-3-hydroxy-5-methyl-4-isoazole propionic acid (AMPA) receptor subtype blocker 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 microM/100 microM glycine) completely prevented Na and Ca accumulation and partially preserved intraneuronal K concentrations. Finally, the increase in neuronal water content normally associated with oxygen-glucose deprivation/reperfusion was prevented by Na+ channel or glutamate receptor blockade.Results of the present study demonstrate that antagonism of either postsynaptic NMDA or AMPA glutaminergic receptor subtypes provided nearly complete protection against ion and water deregulation in nerve cells subjected to experimental ischemia followed by reperfusion. This suggests activation of ionophoric glutaminergic receptors is involved in loss of neuronal osmoregulation and ion homeostasis. Na+ channel blockade also effectively diminished neuronal ion and water derangement during oxygen-glucose deprivation and reperfusion. Prevention of elevated Nai+ levels is likely to provide neuroprotection by decreasing presynaptic glutamate release and by improving cellular osmoregulation, adenosine triphosphate utilization and Ca2+ clearance. Thus, we suggest that voltage-gated tetrodotoxin-sensitive Na+ channels and glutamate-gated ionotropic NMDA or AMPA receptors are important routes of ion flux during nerve cell injury induced by oxygen-glucose deprivation/reperfusion.     

Therapeutic time window for methylprednisolone in spinal cord injured rat.

Recent clinical trials have reported that methylprednisolone sodium succinate administered within 8 hours improves neurological recovery in human spinal cord injury (SCI). Methylprednisolone, however, was ineffective and possibly even deleterious when given more than 8 hours after injury. This finding suggests that a therapeutic time window exists in spinal cord injury. In order to determine the doses, durations and timing of methylprednisolone treatment for optimal neuroprotection, a single or two bolus dose of methylprednisolone (30 mg/kg) was administered at 10, 30, 120, 150 and 240 min. after three graded spinal cord injury. The primary outcome measure was 24-hour spinal cord lesion volumes estimated from spinal cord Na+ and K+ shifts. A single 30 mg/kg dose of methylprednisolone at 10 min. after injury significantly reduced 24-hour lesion volumes in injured rat spinal cords. However, any other methylprednisolone treatment starting 30 min. or more after injury had no effect on 24-hour lesion volumes compared to the vehicle control group. Moreover, delayed treatment increased lesion volumes in some cases. These results suggest that the NYU SCI model has a very short therapeutic window.     

The action potential of chick dorsal root ganglion neurones maintained in cell culture.

1. The directly evoked action potential of dissociated, embryonic, chick, dorsal root ganglion (DRG) neurones maintained in cell culture is prolonged compared to spinal cord cell spikes and the re-polarization phase is marked by a plateau. 2. Evidence was obtained that both Ca2+ and Na+ carry inward current across the active soma membrane. Ca2+ because: overshooting spikes persist in tetrodotoxin (TTX) or Na+-free media; in the presence of TTX (or absence of Na+) spike size varies directly with extracellular Ca2+ and spikes are eliminated by Co2+. Na+ because: spikes persist in the presence of Co2+ or Ca2+-free media; in the presence of Co2+ (or absence of Ca2+) spike varies directly with extracellular Na+ and spikes are blocked by TTX. 3. On the other hand, Ca2+ plays less if any role in action potentials conducted along sensory nerve cell processes. Conducted spikes could not be evoked in TTX containing or Na+-free media. 4. A long-lasting depolarization follows the action potential in some neurones. This depolarization is associated with an increase in membrane conductance and appears to drive the membrane potential to ca. -30mV. It persists when conducted impulses are blocked so it is probably not a recurrent synaptic potential. 5. It is suggested that combined Ca2+-Na+ spikes observed in isolated sensory neurones in vitro reflect the action potential of adult sensory cells but the possibility that they represent an early stage in development is also discussed.     

大鼠脊髓压迫损伤后COX-2基因和蛋白的表达

目的观察大鼠脊髓压迫损伤后COX-2mRNA和蛋白在脊髓组织内表达的时间特征,以及COX-2的空间分布特点。方法以压迫装置致脊髓损伤后,在伤后不同时间点(30min、3h、6h、24h、72h、1w)分别应用RT-PCR、Western blotting、免疫组化技术检测COX-2mRNA和蛋白表达和分布。结果RT-PCR和Western blotting显示,COX-2mRNA和蛋白伤后30min表达开始增加,于伤后6h达到峰值,伤后1w表达接近基础水平。免疫组化提示,COX-2蛋白在假手术组表达仅见于脊髓血管内皮细胞,伤后COX-2蛋白表达见于损伤区附近脊髓灰质内的神经元和血管内皮细胞,损伤中心部位COX-2免疫阳性细胞少见。结论脊髓压迫损伤早期可快速诱导COX-2基因的表达上调和蛋白的合成,伤后COX-2蛋白分布发生变化,主要位于脊髓神经元和血管内皮细胞。     

Carbon monoxide-releasing molecule tricarbonyldichlororuthenium (II) dimer induces concentration-dependent alterations in the electrophysiological properties of axons in mammalian spinal cord

Traumatic spinal cord injury (SCI) typically involves intraparenchymal hemorrhage and a cascade of inflammatory and cytotoxic processes leading to tissue necrosis and apoptosis. A consequence of the hemorrhage is the accumulation of deoxygenated heme proximal and distal to the epicenter of the lesion. The heme oxygenase (HO) system is an endogenous heme degradation system and is upregulated following neurotrauma. The breakdown of heme via HO activity yields the byproducts carbon monoxide (CO), biliverdin, and iron. CO has documented neuromodulatory properties; however, the effects of elevated concentrations of CO on axonal conduction in the spinal cord have not previously been studied. The present study tested the hypothesis that CO causes alterations in the electrophysiological properties of axons within the isolated guinea-pig spinal cord. Ex vivo spinal cord preparations were exposed to 100, 500, and 1000 microM concentrations of the carbon monoxide-releasing molecule (CORM) 2 for 30 min in a double sucrose gap electrophysiological recording system and the compound action potential (CAP) and membrane potential (CMP) were recorded continuously during pretreatment, CORM-2 treatment, and washout (30 min) with Krebs' solution. CAP amplitude and area were significantly (P<0.05) reduced following treatment with 500 and 1000 microM CORM-2 and did not recover during washout. No effect on CMP was observed, however, stimulus-peak latency did increase significantly (P<0.05) following CORM-2 treatment at these concentrations, and a decrease in the amplitude of the second CAP elicited by paired-pulse stimulation was also evident at interpulse intervals of 2 and 4 ms. These results are consistent with a CO-induced alteration in axonal conduction, possibly attributable to modified Na+ channel conductance. They also identify a new mechanism by which post-traumatic hemorrhage contributes to the neurological deficits observed following SCI.     

Important role of reverse Na(+)-Ca(2+) exchange in spinal cord white matter injury at physiological temperature

Spinal cord injury is a devastating condition in which most of the clinical disability results from dysfunction of white matter tracts. Excessive cellular Ca(2+) accumulation is a common phenomenon after anoxia/ischemia or mechanical trauma to white matter, leading to irreversible injury because of overactivation of multiple Ca(2+)-dependent biochemical pathways. In the present study, we examined the role of Na(+)-Ca(2+) exchange, a ubiquitous Ca(2+) transport mechanism, in anoxic and traumatic injury to rat spinal dorsal columns in vitro. Excised tissue was maintained in a recording chamber at 37 degrees C and injured by exposure to an anoxic atmosphere for 60 min or locally compressed with a force of 2 g for 15 s. Mean compound action potential amplitude recovered to approximately 25% of control after anoxia and to approximately 30% after trauma. Inhibitors of Na(+)-Ca(2+) exchange (50 microM bepridil or 10 microM KB-R7943) improved functional recovery to approximately 60% after anoxia and approximately 70% after traumatic compression. These inhibitors also prevented the increase in calpain-mediated spectrin breakdown products induced by anoxia. We conclude that, at physiological temperature, reverse Na(+)-Ca(2+) exchange plays an important role in cellular Ca(2+) overload and irreversible damage after anoxic and traumatic injury to dorsal column white matter tracts.