Glutamate-induced losses of oligodendrocytes and neurons and activation of caspase-3 in the rat spinal cord.

The toxicity of released glutamate contributes substantially to secondary cell death following spinal cord injury (SCI). In this work, the extent and time courses of glutamate-induced losses of neurons and oligodendrocytes are established. Glutamate was administered into the spinal cords of anesthetized rats at approximately the concentration and duration of its release following SCI. Cells in normal tissue, in tissue exposed to artificial cerebrospinal fluid and in tissue exposed to glutamate were counted on a confocal system in control animals and from 6 h to 28 days after treatment to assess cell losses. Oligodendrocytes were identified by staining with antibody CC-1 and neurons by immunostaining for Neuronal Nuclei (NeuN) or Neurofilament H. The density of oligodendrocytes declined precipitously in the first 6 h after exposure to glutamate, and then relatively little from 24 h to 28 days post-exposure. Similarly, neuron densities first declined rapidly, but at a decreasing rate, from 0 h to 72 h post-glutamate exposure and did not change significantly from 72 h to 28 days thereafter. The nuclei of many cells strongly and specifically stained for activated caspase-3, an indicator of apoptosis, in response to exposure to glutamate. Caspase-3 was localized to the nucleus and may participate in apoptotic cell death. However, persistence of caspase-3 staining for at least a week after exposure to glutamate during little to no loss of oligodendrocytes and neurons demonstrates that elevation of caspase-3 does not necessarily lead to rapid cell death. Beyond about 48 h after exposure to glutamate, locomotor function began to recover while cell numbers stabilized or declined slowly, demonstrating that functional recovery in the experiments presented involves processes other than replacement of oligodendrocytes and/or neurons.

Preemptive analgesia with lidocaine prevents Failed Back Surgery Syndrome.

Failed Back Surgery Syndrome (FBSS) is commonly encountered in pain-treatment settings in the United States. We tested whether potential key factors in this syndrome, such as extracellular concentrations of excitatory amino acids (EAAs), are increased in the dorsal horn by synaptic release due to unintentional stretch and/or deformation/compression/transection of dorsal spinal structures during surgery. We hypothesized that pharmacological nerve block as a form of preemptive analgesia prior to any insult to dorsal root neurons will prevent an abnormally high increase in extracellular concentrations of EAAs in the dorsal horn and ultimately the establishment of central sensitization during back surgery. The L4 and L5 dorsal roots were cut bilaterally near the spinal cord to provide an adequate model to test for preemptive analgesia. Amino acid concentrations were measured by dorsal horn microdialysis sampling; EAAs aspartate and glutamate were significantly increased by 80% and 65% respectively, as were other amino acids compared to sham control values. Topical application of 1% Lidocaine, a voltage-gated Na(+) channel blocker, for 10 min prior to L4 and L5 bilateral dorsal rhizotomy (BDR) significantly attenuated the increase in EAA concentrations such that their values were not different from sham controls. Behavioral tests demonstrated significant hindlimb mechanical allodynia after BDRs that was significantly attenuated by Lidocaine pretreatment. Thus, Lidocaine pretreatment could offer a safe measure for prevention of chronic pain for back surgical procedures if given by intramuscular injection, topical administration onto spinal nerves and/or the dorsal spinal surface during surgical procedures that include nerve entrapment release, intervertebral disc modification and laminectomies.

Concentrations of extracellular free zinc (pZn)e in the central nervous system during simple anesthetization, ischemia and reperfusion.

"Free Zn2+" (rapidly exchangeable Zn2+) is stored along with glutamate in the presynaptic terminals of specific specialized (gluzinergic) cerebrocortical neurons. This synaptically releasable Zn2+ has been recognized as a potent modulator of glutamatergic transmission and as a key toxin in excitotoxic neuronal injury. Surprisingly (despite abundant work on bound zinc), neither the baseline concentration of free Zn2+ in the brain nor the presumed co-release of free Zn2+ and glutamate has ever been directly observed in the intact brain in vivo. Here, we show for the first time in dialysates of rat and rabbit brain and human CSF samples from lumbar punctures that: (i) the resting or "tonic" level of free Zn2+ signal in the extracellular fluid of the rat, rabbit and human being is approximately 19 nM (95% range: 5-25 nM). This concentration is 15,000-fold lower than the "300 microM" concentration which is often used as the "physiological" concentration of free zinc for stimulating neural tissue. (ii) During ischemia and reperfusion in the rabbit, free zinc and glutamate are (as has often been presumed) released together into the extracellular fluid. (iii) Unexpectedly, Zn2+ is also released alone (without glutamate) at a variable concentration for several hours during the reperfusion aftermath following ischemia. The source(s) of this latter prolonged release of Zn2+ is/are presumed to be non-synaptic and is/are now under investigation. We conclude that both Zn2+ and glutamate signaling occur in excitotoxicity, perhaps by two (or more) different release mechanisms.

Upregulation of the phosphorylated form of CREB in spinothalamic tract cells following spinal cord injury: relation to central neuropathic pain.

Spinal cord injury (SCI) often leads to the generation of chronic intractable neuropathic pain. The mechanisms that lead to chronic central neuropathic pain (CNP) following SCI are not well understood, resulting in ineffective treatments for pain relief. Studies have demonstrated persistent hyperexcitability of dorsal horn neurons which may provide a substrate for CNP. We propose a number of similarities between CNP mechanisms and mechanisms that occur in long-term potentiation, in which hippocampal neurons are hyperexcitable. One biochemical similarity may be activation of the transcription factor, cyclic AMP response element-binding protein (CREB), via phosphorylation (pCREB). The current study was designed to examine whether tactile allodynia that develops in segments rostral to SCI (at-level pain) correlates with an increase in CREB phosphorylation in specific neurons known to be involved in allodynia, the spinothalamic tract (STT) cells. This study determined that, in animals experiencing at-level allodynia 35 days after SCI, pCREB was upregulated in the spinal cord segment rostral to the injury. In addition, pCREB was found to be upregulated specifically in STT cells in the rostral segment 35 days after SCI. These findings suggest one mechanism of maintained central neuropathic pain following SCI involves persistent upregulation of pCREB expression within STT cells.

Exogenous Bcl-xL fusion protein spares neurons after spinal cord injury.

Spinal cord injury (SCI) induces neuronal death, including apoptosis, which is completed within 24 hr at and around the impact site. We identified early proapoptotic transcriptional changes, including upregulation of proapoptotic Bax and downregulation of antiapoptotic Bcl-xL, Bcl-2, and Bcl-w, using Affymetrix DNA microarrays. Because Bcl-xL is the most robustly expressed antiapoptotic Bcl-2 molecule in adult central nervous system, we decided to characterize better the effect of SCI on Bcl-xL expression. We found Bcl-xL expressed robustly throughout uninjured spinal cord in both neurons and glia cells. We also found Bcl-xL localized in different cellular compartments: cytoplasmic, mitochondrial, and nuclear. Bcl-xL protein levels decreased in the cytoplasm and mitochondria 2 hr after SCI and persisted for 24 hr. To test the contribution of proapoptotic decreases in Bcl-xL to neuronal death, we augmented endogenous Bcl-xL levels by administering Bcl-xL fusion protein (Bcl-xL FP) into injured spinal cords. Bcl-xL FP significantly increased neuronal survival, suggesting that SCI-induced changes in Bcl-xL contribute considerably to neuronal death. Because Bcl-xL FP increases survival of dorsal horn neurons and ventral horn motoneurons, it could become clinically relevant in preserving sensory and motor functions after SCI.

Evidence that infiltrating neutrophils do not release reactive oxygen species in the site of spinal cord injury.

The release of reactive oxygen species (ROS) by neutrophils, which infiltrate the region of damage following spinal cord injury (SCI), was investigated to determine if such release is significant following spinal cord injury. The relationship of extracellular levels of hydroxyl radicals and hydrogen peroxide obtained by microdialysis sampling and oxidized protein levels in tissue to neutrophil infiltration following spinal cord injury was examined. Neither of the reactive oxygen species were elevated in the site of spinal cord injury relative to their concentrations in normal tissue at a time (24 h) when the numbers of neutrophils were maximum in the site of injury. Surprisingly, ablation with a neutrophil antiserum actually increased the level of oxidized proteins in Western blots. Thus, our findings are (1) that neutrophils, which infiltrate the site of damage following a spinal cord injury, do not release detectable quantities of reactive oxygen species; and (2) that the presence of neutrophils reduces the concentrations of oxidized proteins in the site of spinal cord injury. Therefore, release of reactive oxygen species by neutrophils does not contribute significantly to secondary damage following spinal cord injury. Reduced levels of oxidized proteins in the presence of neutrophils may reflect removal of damaged tissue by neutrophils.

DNA microarray analysis of the contused spinal cord: effect of NMDA receptor inhibition.

Spinal cord injury (SCI)-induced neurodegeneration leads to irreversible and devastating motor and sensory dysfunction. Post-traumatic outcomes are determined by events occurring during the first 24 hours after SCI. An increase in extracellular glutamate concentration to neurotoxic levels is one of the earliest events after SCI. We used Affymetrix DNA oligonucleotide microarrays (with 1,322 DNA probes) analysis to measure gene expression in order to test the hypothesis that SCI-induced N-methyl-D-aspartate (NMDA) receptor activation triggers significant postinjury transcriptional changes. Here we report that SCI, 1 hour after trauma, induced change in mRNA levels of 165 genes and expression sequence tags (ESTs). SCI affected mRNA levels of those genes that regulate predominantly transcription factors, inflammation, cell survival, and membrane excitability. We also report that NMDA receptor inhibition (with -(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine hydrogen maleate [MK-801]) reversed the effect of SCI on about 50% of the SCI-affected mRNAs. Especially interesting is the finding that NMDA receptor activation participates in the up-regulation of inflammatory factors. Therefore, SCI-induced NMDA receptor activation is one of the dominant, early signals after trauma that leads to changes in mRNA levels of a number of genes relevant to recovery processes. The majority of MK-801 effects on the SCI-induced mRNA changes reported here are novel. Additionally, we found that the MK-801 treatment also changed the mRNA levels of 168 genes and ESTs that had not been affected by SCI alone, and that some of their gene products could have harmful effects on SCI outcome.

Involvement of metabotropic glutamate receptors in excitatory amino acid and GABA release following spinal cord injury in rat.

Spinal cord injury (SCI) leads to an increase in extracellular excitatory amino acid (EAA) concentrations resulting in glutamate receptor-mediated excitotoxic events. The glutamate receptors include ionotropic (iGluRs) and metabotropic (mGluR) receptors. Of the three groups of mGluRs, group-I activation can initiate intracellular pathways that lead to further transmitter release. Groups II and III mGluRs function mainly as autoreceptors to regulate neurotransmitter release. In an effort to examine the role of mGluRs in the increase in EAAs following SCI, we administered AIDA, a potent group-I mGluR antagonist immediately after injury. To determine subtype specific roles of the group-I mGluRs, we evaluated EAA release following LY 367385 (mGluR1 antagonist) and MPEP (mGluR5 antagonist) administration. To evaluate group-II and -III mGluRs we administered APDC (group-II agonist) and L-AP4 (group-III agonist) immediately following injury; additionally, we initiated treatment with CPPG (group-II/-III antagonist) and LY 341495 (group-II antagonist) 5 min prior to injury. Subjects were adult male Sprague-Dawley rats (225-250 g), impact injured at T10 with an NYU impactor (12.5 mm drop). Agents were injected into the epicenter of injury, amino acids where collected by microdialysis fibers inserted 0.5 mm caudal from the edge of the impact region and quantified by HPLC. Treatment with AIDA significantly decreased extracellular EAA and GABA concentrations. MPEP reduced EAA concentrations without affecting GABA. Combining LY 367385 and MPEP resulted in a decrease in EAA and GABA concentrations greater than either agent alone. L-AP4 decreased EAA levels, while treatment with LY 341495 increased EAA levels. These results suggest that mGluRs play an important role in EAA toxicity following SCI.

Bcl-xL expression after contusion to the rat spinal cord.

After contusion-derived spinal cord injury, (SCI) there is localized tissue disruption and energy failure that results in early necrosis and delayed apoptosis, events that contribute to chronic central pain in a majority of patients. We assessed the extent of contusion-induced apoptosis of neurons in a known central pain-signaling pathway, the spinothalamic tract (STT), which may be a contributor to SCI-induced pain. We observed the loss of STT cells and localized increase of DNA fragmentation and cytoplasmic histone-DNA complexes, which suggested potential apoptotic changes among STT neurons after SCI. We also showed SCI-associated changes in the expression of the antiapoptotic protein Bcl-xL, especially among STT cells, consistent with the hypothesis that Bcl-xL regulates the extent of apoptosis after SCI. Apoptosis in the injured spinal cord correlated well with prompt decreases in Bcl-xL protein levels and Bcl-xL/Bax protein ratios at the contusion site. We interpret these results as evidence that regulation of Bcl-xL may play a role in neural sparing after spinal injury and pain-signaling function.

Engraftment of serotonergic precursors enhances locomotor function and attenuates chronic central pain behavior following spinal hemisection injury in the rat.

Spinal cord injury (SCI) results in abnormal locomotor and pain syndromes in humans. T13 spinal hemisection in the rat results in development of permanent mechanical allodynia and thermal hyperalgesia partially due to interruption of descending inhibitory modulators such as serotonin (5-HT). We hypothesize that lumbar transplantation of nonmitotic cells that tonically secrete antinociceptive and trophic compounds will reduce the pain-like behavior and enhance locomotor recovery after SCI. We used RN46A-B14 cells, a conditionally immortalized (SV40tsTag) rat neuronal cell line derived from E13 raphe bioengineered to secrete both 5-HT and BDNF in vitro at both permissive (33 degrees C) and nonpermissive (39 degrees C) temperatures. Three groups (n = 72) of 30-day-old male Sprague-Dawley rats were spinally hemisected at T13 and allowed 4 weeks for adequate recovery of locomotor function and development of allodynia and hyperalgesia. Immunosuppressed animals received either lumbar RN46A-B14 (n = 24) or control RN46A-V1 (n = 24) empty-vector transplants or no cell (n = 24) transplant. HPLC analysis of media and CSF demonstrated increases of both in vitro and in vivo 5-HT levels at 28 days in RN46A-B14 animals. ELISA demonstrated BDNF secretion in vitro and in vivo by RNA46A-B14 cells. Locomotor function (BBB scale) and nociceptive behaviors measured by paw withdrawals to von Frey filaments, radiant heat, and noxious pin stimuli were tested for 4 weeks posttransplant. Animals receiving RN46A-B14 cells demonstrated significantly improved locomotor function and reductions in both fore- and hindlimb mechanical allodynia and thermal hyperalgesia compared to controls receiving RN46A-V1 or no transplants. These effects were modulated by the 5-HT antagonist methysergide and reuptake inhibitor fluvoxamine. Bromodeoxyuridine and 5-HT immunoreactivity confirmed cell survival and graft location 4 weeks posttransplantation. These results support the therapeutic potential of bioengineered serotonin-secreting cell lines in reducing chronic central pain following spinal cord injury.

IL-1 receptor antagonist prevents apoptosis and caspase-3 activation after spinal cord injury.

One of the consequences of cytokine-orchestrated inflammation after CNS trauma is apoptosis. Our hypothesis is that cell death in the spinal cord after injury results in part from increased synthesis and release of IL-1beta. Using a ribonuclease protection assay, we demonstrated that there is increased transient expression of IL-1beta mRNA and, by using IL-1beta protein ELISA assay, that there are increased IL-1beta protein levels in the contused rat spinal cord, initially localized to the impact region of the spinal cord (segment T8). Using an ELISA cell death assay, we showed that there is apoptosis in the spinal cord 72 h after injury, a finding that was confirmed by measuring caspase-3 activity, which also significantly increased at the site of injury 72 h after trauma. Treatment of the contused spinal cord at the site of injury with the IL-1 receptor antagonist (rmIL-lra, 750 ng/mL) for 72 h using an osmotic minipump completely abolished the increases in contusion-induced apoptosis and caspase-3 activity.

Metalloproteinase increases in the injured rat spinal cord.

Up-regulation of matrix metalloproteinases MMP-9 and MMP-2 after injury to the spinal cord (SCI) is demonstrated. MMP-9 activity maximized at 12-24 h, and MMP-2 rose at 5 days post-injury. MMP-3 was not detectable by zymographic analysis, so its level of expression was, at most, very low. The level of tissue inhibitor of metalloproteinases in the spinal cord was not altered by injury, perhaps permitting increased MMP-9 and MMP-2 activities in situ. Ablating them with an antibody demonstrated that infiltrating neutrophils were the principal source of MMP-9 activity after spinal cord injury, suggesting that neutrophils utilize that proteinase in responding to spinal cord injury. MMP-9 and MMP-2 probably contribute to breakdown of the extracellular matrix following SCI.

AIDA reduces glutamate release and attenuates mechanical allodynia after spinal cord injury.

Spinal cord injury (SCI) leads to an increase in extracellular excitatory amino acid (EAA) concentrations, resulting in glutamate receptor-mediated excitotoxicity and central sensitization. To test contributions of group I metabotropic glutamate receptors (mGluRs) in SCI induced release of glutamate and in behavioral outcomes of central sensitization following injury, we administered 1-aminoindan-1,5-dicarboxylic acid (AIDA; 0.1 nmol intraspinally), a potent group I mGluR antagonist, to rats immediately after spinal cord contusion injury. EAAs were collected by microdialysis and quantified using HPLC. AIDA significantly decreased extracellular glutamate but not aspartate concentrations and significantly attenuated the development of mechanical but not thermal allodynia. These results suggest mGluRs play an important role in injury-induced EAA release and in central sensitization following SCI.

Transplants of adrenal medullary chromaffin cells reduce forelimb and hindlimb allodynia in a rodent model of chronic central pain after spinal cord hemisection injury.

In the majority of patients, spinal cord injury (SCI) results in abnormal pain syndromes in which non-noxious stimuli become noxious (allodynia). To reduce allodynia, it would be desirable to implant a permanent biological pump such as adrenal medullary chromaffin cells (AM), which secrete catecholamines and opioid peptides, both antinociceptive substances, near the spinal cord. We tested this approach using a recently developed a mammalian SCI model of chronic central pain, which results in development of mechanical and thermal allodynia. Thirty day-old male Sprague-Dawley rats were spinally hemisected at T13 and allowed 4 weeks for recovery of locomotor function and development of allodynia. Nonimmunosuppressed injured animals received either control-striated muscle (n = 7) or AM (n = 10) transplants. Nociceptive behavior was tested for 4 weeks posttransplant as measured by paw withdrawals to von Frey filaments, radiant heat, and pin prick stimuli. Hemisected animals receiving AM demonstrated statistically significant reductions in both fore- and hindlimb mechanical and thermal allodynia, but not analgesia, when compared to hemisected animals receiving striated muscle transplants (P < 0.05). Tyrosine hydroxylase immunoreactivity indicated prolonged transplant survival and production of catecholamines. HPLC analysis of cerebrospinal fluid samples from animals receiving AM transplants demonstrated statistically significant increases in levels of dopamine (sevenfold), norepinephrine (twofold), and epinephrine (threefold), compared to control values several weeks following transplant (P < 0.05). By 28 days posttransplant, however, antinociceptive effects were diminished. These results support the therapeutic potential of transplanted AM in reducing chronic central pain following spinal cord injury.

Evidence that reversed glutamate uptake contributes significantly to glutamate release following experimental injury to the rat spinal cord.

Released excitatory amino acids contribute significantly to secondary damage following spinal cord injury. Reversal of normal transport due to cell membrane depolarization may contribute to this release. We tested this by administering dihydrokainic acid (DHK), a non-transported glutamate uptake blocker, into the rat spinal cord by microdialysis in association with contusion spinal cord injury. Glutamate release in response to injury was reduced by 34% (P<0.05) when 3 mM DHK was administered within the microdialysis fiber, suggesting that reversed transport is an important contributor to glutamate release upon spinal cord injury.

Adenosine release upon spinal cord injury.

The hypothesis that release of adenosine following spinal cord injury (SCI) may provide neuroprotective feedback is explored. Consistent with this hypothesis, substantial release of adenosine, estimated to reach 100 microM in the extracellular space, was detected by microdialysis sampling immediately following contusion SCI. There is also considerable release of excitatory amino acids following SCI. The latter was not affected by administration of the general adenosine receptor antagonist theophylline and the A1 antagonist 8-cyclopentyl-1,3-dipropylxanthine, implying that the adenosine released following SCI does not significantly influence the release of neurotoxic amino acids. Administration of the concentration of glutamate released upon SCI into the spinal cord caused only about 1% as much release of adenosine as did injury, evidence that elevated excitatory amino acids do not elicit an appreciable fraction of the release of adenosine that follows SCI. Results obtained suggest that release of endogenous adenosine is not neuroprotective by blocking release of excitatory amino acids following SCI.

Rodent model of chronic central pain after spinal cord contusion injury and effects of gabapentin.

Spinal cord injury (SCI) often results in abnormal pain syndromes in patients. We present a recently developed SCI mammalian model of chronic central pain in which the spinal cord is contused at T8 using the NYU impactor device (10-g rod, 2.0-mm diameter, 12.5-mm drop height), an injury which is characterized behaviorally as moderate. Recovery of locomotor function was assessed with an open field test and scored using the open field test scale (BBB scale). Somatosensory tests of paw withdrawal responses accompanied by supraspinal responses to both mechanical punctate (von Frey hairs) and nonpunctate (4 mm diameter blunt probe) as well as thermal (radiant heat) peripheral stimuli were performed. Comparisons at the level of the individual animal between precontusion and postcontusion responses indicated significant increases in reactions to low threshold punctate mechanical stimuli, non-punctate stimuli and thermal stimuli (p < 0.05). To demonstrate the validity of this model as a central pain model, gabapentin, an agent used clinically for central pain, was given i.p. at 10 or 30 mg/kg. Gabapentin treatment significantly and reversibly changed the responses, consistent with the attenuation of the abnormal sensory behavior, and the attenuated responses lasted for the duration of the drug effect (up to 6 h). These results support the use of the spinal contusion model in the study of chronic central pain after SCI.

Changes in amino acid concentrations over time and space around an impact injury and their diffusion through the rat spinal cord.

Release of amino acids, particularly the neurotoxin glutamate, in and around the site of an experimental spinal cord injury was characterized over time by microdialysis. Increases in amino acid concentrations caused by injury decline steeply and then slowly over distance from the impact area, becoming undetectable beyond about 5 mm from the injury epicenter. Diffusion profiles determined in the cord by administering amino acids through one microdialysis fiber and sampling them in a parallel fiber declined steeply with distance. Distant increases coincided temporally with those in the injury epicenter. We conclude that elevated amino acids more than about 1 mm into the periimpact zone are predominantly released in that region rather than diffusing into it from the trauma epicenter. In the outer areas of lesion development, glutamate does not appear to reach concentrations ordinarily toxic, and elevated concentrations do not persist nearly as long as the therapeutic window of NBQX in any part of the lesion. Therefore, the mechanisms whereby excitatory amino acid antagonists reduce the dimensions of injury lesions are unclear. However, sensitization of neurons following impact injury could be important in amino acid neurotoxicity.

Neurotoxicity of glutamate at the concentration released upon spinal cord injury.

Damage caused by administering glutamate into the spinal cord was characterized histologically. Glutamate destroyed neurons for several hundred micrometers around the administering microdialysis fiber. At 24 h after treatment, significant (P = 0.036) loss of neurons was observed (75%) relative to control (47%) near the fiber when glutamate was administered for 1 h at a concentration outside the fiber approximating the maximum glutamate released upon spinal cord injury. Significant loss of neurons (P = 0.006, 0.022) was also caused by administering a combination of glutamate at about its average concentration released upon injury over the 1 h period of administration in combination with the maximum aspartate concentration released upon injury. This work provides a direct demonstration that the concentrations of excitatory amino acids released upon spinal cord injury are neurotoxic. The destruction of neurons by exposure to excitatory amino acids when there is also substantial loss of neurons simply from the presence of the microdialysis fiber may reflect sensitization of neurons to excitotoxicity by stress.

Sampling of low molecular weight iron by microdialysis following spinal cord injury.

We combined the use of desferrioxamine as an iron chelator, microdialysis sampling, and iron analysis by atomic absorption spectroscopy to measure extracellular levels of low molecular weight (LMW) iron in vivo in the spinal cord. Low molecular weight iron is free iron plus iron bound to small molecules. We show that the extracellular LMW-iron concentration is not increased significantly in situ by trauma to the rat spinal cord, suggesting that an extracellular elevation in LMW-iron at the site of injury is not the major initiating factor for lipid peroxidation following spinal cord injury.