Experimental and computational evidence for C=O π-bonding in [CH2OH]+ and related oxonium ions.

The question of whether [CH2OH]+ should be described as the hydroxymethyl cation, +CH2OH, or protonated formaldehyde, CH2=OH+, is reconsidered in the light of experimental information and new computational evidence. Previous arguments that the charge distribution in [CH2OH]+ may be probed by considering the incremental stabilisation of [CH2OH]+ induced by homologation on carbon (to give [CH3CHOH]+) or oxygen (to produce [CH2OCH3]+) are critically examined. Cation stabilisation energies are shown to be better indicators of the nature of these oxonium ions. Further insight into the structure of larger CnH2n+1O+ oxonium ions is obtained by considering the site of protonation of enol ethers and related species. Computational information, including AIM (Atoms and Molecules) and NBA (Natural Bond Analysis) charges on the carbon and oxygen atoms in [CH2OH]+ and related species, is considered critically. Particular attention is focused on the calculated bond lengths and barriers to rotation about the C-O bond(s) in [CH2OH]+, [CH3CHOH]+, [(CH3)2COH]+, CH3OH and [CH2OCH3]+ and the C-N bond in [CH2NH2]+. Trends in these data are consistent with appreciable π-bonding only in the C-O connections which correspond to the C=O bond in the parent aldehyde or ketone from which the oxonium ion may be considered to be derived by protonation or alkyl cationation.

Calcium/calmodulin dependent kinase II contributes to persistent central neuropathic pain following spinal cord injury.

Chronic central neuropathic pain after central nervous system injuries remains refractory to therapeutic interventions. A novel approach would be to target key intracellular signaling proteins that are known to contribute to continued activation by phosphorylation of kinases, transcription factors, and/or receptors that contribute to changes in membrane excitability. We demonstrate that one signaling kinase, calcium/calmodulin-dependent kinase II (CaMKII), is critical in maintaining aberrant dorsal horn neuron hyperexcitability in the neuropathic pain condition after spinal cord injury (SCI). After contusion SCI at spinal level T10, activated CaMKII (phosphorylated, pCaMKII) expression is significantly upregulated in the T7/8 spinal dorsal horn in neurons, but not glial cells, and in oligodendrocytes in the dorsal column in the same rats that displayed at-level mechanical allodynia. Furthermore, identified spinothalamic neurons demonstrated significant increases of pCaMKII after SCI compared to sham-treated control animals. However, neither astrocytes nor microglia showed pCaMKII expression in either sham-treated or SCI rats. To demonstrate causality, treatment of SCI rats with KN-93, which prevents CaMKII activation, significantly attenuated at-level mechanical allodynia and aberrant wide dynamic range neuronal activity evoked by brush, pressure, and pinch stimuli and a graded series of von Frey stimuli, respectively. Persistent CaMKII activation contributes to chronic central neuropathic pain by mechanisms that involve maintained hyperexcitability of wide dynamic range dorsal horn neurons. Furthermore, targeting key signaling proteins is a novel, useful therapeutic strategy for treating chronic central neuropathic pain.

Regulation of interleukin-1beta by the interleukin-1 receptor antagonist in the glutamate-injured spinal cord: endogenous neuroprotection.

Elevation of extracellular glutamate contributes to cell death and functional impairments generated by spinal cord injury (SCI), in part through the activation of the neurotoxic cytokine interleukin-1beta (IL-1beta). This study examines the participation of IL-1beta and its regulation by the endogenous interleukin-1 receptor antagonist (IL-1ra) in glutamate toxicity following SCI. Glutamate, glutamatergic agonists and SCI had similar effects on levels of IL-1beta and IL-1ra. Following spinal cord contusion or exposure to elevated glutamate, concentrations of IL-1beta first increased as IL-1ra decreased, and both then changed in the opposite directions. Applying the glutamate agonists NMDA and S-AMPA to the spinal cord caused changes in IL-1beta and IL-1ra levels very similar to those produced by contusion and glutamate. The glutamate antagonists MK801 and NBQX blocked the glutamate-induced changes in IL-1beta and IL-1ra levels. Administering IL-1beta elevated IL-1ra, and administering IL-1ra depressed IL-1beta levels. Infusing IL-beta into the spinal cord impaired locomotion, and infusing IL-1ra improved recovery from glutamate-induced motor impairments. We hypothesize that elevating IL-1ra opposes the damage caused by IL-1beta in SCI by reducing IL-1beta levels as well as by blocking binding of IL-1beta to its receptor. Our results demonstrate that IL-1beta contributes to glutamate damage following SCI; blocking IL-1beta may usefully counteract glutamate toxicity.

1,2-Eliminations from (CH3)2NH+CH2CH3 and (CH3)2NH2+: guided dissociations.

1,2-Eliminations are a varied and extensive set of dissociations of ions in the gas phase. To understand better such dissociations, elimination of CH(2)=CH(2) and CH(3)CH(3) from (CH(3))(2)NH(+)CH(2)CH(3) (1) and of CH(4) from (CH(3))(2)NH(2)(+) are characterized by quantum chemical calculations. Stretching of the CN bond to ethyl is followed by shift of an H from methyl to the bridging position in ethyl and then to N to reach (CH(3))(2)NH(2)(+) + CH(2)=CH(2) from 1. CH(3)CH(3) elimination by H-transfer to C(2)H(5)(+) to form CH(3)NH(+)=CH(2) + CH(3)CH(3) also takes place. (CH(3))(2)NH(2)(+) eliminates methane by CN bond extension followed by beta-H-transfer to give CH(2)=NH(+) + CH(4). Low-energy reactions resembling complex-mediated 1,2-eliminations occur and constitute a hitherto largely unrecognized type of reaction. As in many complex-mediated reactions, these reactions transfer H between incipient fragments. They are distinguished from complex-mediated processes by the fragments not being able to rotate freely relative to each other near the transition state for reaction, as they do in complexes. Most 1,2-eliminations are ion-neutral complex-mediated, occur by the just described lower energy reactions, have 1,1-like transition states, or utilize highly asynchronous 1,2 transition states. All of these avoid synchronized 1,2-transition states that would violate conservation of orbital symmetry.

Microdialysis in central nervous system disorders and their treatment.

Central nervous system (CNS) insults elevate endogenous toxins and alter levels of indicators of metabolic disorder. These contribute to neurotrauma, neurodegenerative diseases and chronic pain and are possible targets for pharmaceutical treatment. Microdialysis samples substances in the extracellular space for chemical analysis. It has demonstrated that toxic levels of glutamate are released and that toxic levels of the reactive species O(2)(-), H(2)O(2), HO. NO and HOONO are generated upon CNS injury. Agent administration by microdialysis can also help elucidate mechanisms of damage and protection, and to identify targets for clinical application. Microdialysis sampling indicates that circuits descending from the brain to the spinal cord transmit and modulate pain signals by releasing neurotransmitter amines and amino acids. Efforts are under way to develop microdialysis into a technique for intensive care monitoring and predicting outcomes of brain insults. Finally, microdialysis sampling has demonstrated in vivo elevation of glial cell line-derived neurotrophic factor following grafting of primed fetal human neural stem cells into brain-injured rats, the first in vivo demonstration of the release of a neurotrophic factor by grafted stem cells. This increased release correlated with significantly improved spatial learning and memory.

Caffeinated coffee blunts the myocardial protective effects of statins against ischemia-reperfusion injury in the rat.

PURPOSE: We asked whether caffeinated coffee (CC) blunts the infarct size (IS)-limiting effects of atorvastatin (ATV). BACKGROUND: Adenosine receptor activation is essential for mediating the IS-limiting effects of statins. Caffeine is a nonspecific adenosine receptor blocker, and thus drinking CC may block the myocardial protective effects of statins. METHODS: Rat received 3-day ATV (10 mg/kg/day) or water by oral gavage once daily. Drinking water was replaced by water + sugar (7.5 g/100 ml), CC with sugar, or decaffeinated coffee (DC) with sugar. On the 4th day, rats were anesthetized and underwent 30 min of coronary artery occlusion and 4 h reperfusion. Area at risk was assessed by blue dye and infarct size by TTC. RESULTS: Body weight and area at risk was comparable among groups. IS was 25.1 +/- 3.9% of the area at risk in the control group. In rats not receiving ATV, CC (25.5 +/- 3.1%) and DC (34.0 +/- 2.8%) did not affect IS. IS was significantly reduced by ATV in the water + sugar (11.7 +/- 0.7%, p = 0.015) and DC (11.5 +/- 1.0%; p < 0.001) groups, but not in the CC group (32.3 +/- 3.0%; p = 0.719). ATV increased myocardial levels of Ser-473 phosphorylated Akt in the water + sugar and DC groups, but not in the CC group. CONCLUSIONS: CC, but not DC, abrogated the IS-limiting effects of ATV by blocking the adenosine receptors and preventing the phosphorylation of Akt. CC did not affect IS in rats not receiving ATV.

Elimination of H2 from CH3CH=N+HCH3: a synchronous, concerted 1,4-H2 elimination.

Most H2 eliminations from cations in the gas phase are formally 1,1- or 1,2- processes. Larger ring size H2 eliminations are rare and little studied. Thus, whether the 6-center, 1,4- elimination CH3CH=N+HCH3-->CH2=CHN+H=CH2+H2 is concerted and synchronous, as indicated by isotope effects and predicted by conservation of orbital symmetry, is a significant question. This reaction is characterized here by application of QCI and B3LYP theories. CH bond-breaking and H-H bond-making in this reaction are found by theory to be highly synchronized, consistent with previously established isotope effects and in contrast to "forbidden" 1,2-eliminations from organic cations in the gas phase. This reaction is made feasible by its conservation of orbital symmetry, the energy supplied by formation of the H-H bond, and a favorable geometry of the ion for eliminating H2.

The cardioprotective effect of a statin and cilostazol combination: relationship to Akt and endothelial nitric oxide synthase activation.

BACKGROUND: Atorvastatin (ATV) protects against ischemia-reperfusion by upregulating Akt and subsequently, endothelial nitric oxide synthase (eNOS) phosphorylation at Ser-1177. However, when given orally, high doses of ATV (10 mg/kg/d) are needed to achieve maximal protective effect in the rat. Protein kinase A (PKA) also phosphorylates eNOS at Ser-1177. As PKA activity depends on cAMP, cilostazol (CIL), a phosphodiesterase type III inhibitor, may stimulate NO production by activating PKA. HYPOTHESIS: CIL and ATV may have synergistic effects on eNOS phosphorylation and myocardial infarct size (IS) reduction. METHODS: Sprague-Dawley rats received 3-day oral pretreatment with: (1) water; (2) low dose ATV (2 mg/kg/d); (3) CIL (20 mg/kg/d): (4) ATV+CIL. Rats underwent 30 min coronary artery occlusion and 4 h reperfusion, or hearts explanted for immunoblotting without being subjected to ischemia. Area at risk (AR) was assessed by blue dye and IS by triphenyl-tetrazolium-chloride. RESULTS: Body weight and the size of AR were comparable among groups. There were no significant differences among groups in mean blood pressure and heart rate. CIL, but not ATV, reduced IS. IS in the ATV+CIL group was significantly smaller than the other three groups (P < 0.001 for each comparison). ATV, CIL and their combination did not affect total eNOS expression. ATV at 2 mg/kg/d did not affect Ser-1177 P-eNOS levels, whereas CIL increased it (258 +/- 15%). The level of myocardial P-eNOS levels was highest in the ATV+CIL group (406 +/- 7%). CONCLUSIONS: ATV and CIL have synergistic effect on eNOS phosphorylation and IS reduction. By increased activation of eNOS, CIL may augment the pleiotropic effects of statins.

Enhanced cardioprotection against ischemia-reperfusion injury with a dipyridamole and low-dose atorvastatin combination.

Atorvastatin (ATV) limits infarct size (IS) by activating Akt and ecto-5-nucleotidase, which generates adenosine. Activated Akt and adenosine activate endothelial nitric oxide synthase (eNOS). When given orally, high doses (10 mg/kg) are needed to achieve full protection. We determined whether dipyridamole (DIP), by preventing the reuptake of adenosine, has a synergistic effect with ATV in reducing myocardial IS. In this study, rats received 3-days of the following: water, ATV (2 mg.kg(-1).day(-1)), DIP (6 mg.kg(-1).day(-1)), or ATV + DIP. In addition, rats received 3-days of the following: aminophylline (Ami; 10 mg.kg(-1).day(-1)) or Ami + ATV + DIP. Rats underwent 30 min of myocardial ischemia followed by 4 h of reperfusion (IS protocol), or hearts were explanted for immunoblotting. As a result, IS in the controls was 34.0 +/- 2.8% of the area at risk. ATV (33.1 +/- 2.1%) and DIP (30.5 +/- 1.5%) did not affect IS, whereas ATV + DIP reduced IS (12.2 +/- 0.5%; P < 0.001 vs. each of the other groups). There was no difference in IS between the Ami alone (48.1 +/- 0.8%) and the Ami + ATV + DIP (45.8 +/- 2.9%) group (P = 0.422), suggesting that Ami completely blocked the protective effect. Myocardial adenosine level in the controls was 30.6 +/- 3.6 pg/microl. ATV (51.0 +/- 4.9 pg/microl) and DIP (51.5 +/- 6.8 pg/microl) caused a small increase in adenosine levels, whereas ATV + DIP caused a greater increase in adenosine levels (66.4 +/- 3.1 pg/microl). ATV and DIP alone did not affect myocardial Ser473 phosphorylated-Akt and Ser1177 phosphorylated-eNOS levels, whereas ATV + DIP significantly increased them. In conclusion, low-dose ATV and DIP had synergistic effects in reducing myocardial IS and activation of Akt and eNOS. This combination may have a potential benefit in augmenting the eNOS-mediated pleiotropic effects of statins.

Serum albumin improves recovery from spinal cord injury.

A neuroprotective factor is shown to be present in mammalian serum. This factor is identified by Western blotting to be serum albumin. The serum factor and albumin both protected cultured spinal cord neurons against the toxicity of glutamate. The inability of K252a, a blocker of the high affinity tyrosine kinase receptor for members of the nerve growth factor family, to block the neuroprotective effect of the serum factor established that the serum factor is not a member of the nerve growth factor family. Post-injury injection of albumin intravenously or into the site of injury immediately after injury both improved significantly locomotor function according to Basso-Beattie-Bresnahan assessment and spontaneous locomotor activity recorded with a photobeam activity system. Albumin has multiple mechanisms whereby it may be neuroprotective, and it is a potentially useful agent for treating neurotraumas.

Mediating delta-opioid-initiated heart protection via the beta2-adrenergic receptor: role of the intrinsic cardiac adrenergic cell.

Stimulation of cardiac beta(2)-adrenergic receptor (beta(2)-AR) or delta-opioid receptor (DOR) exerts a similar degree of cardioprotection against myocardial ischemia in experimental models. We hypothesized that delta-opioid-initiated cardioprotection is mediated by the intrinsic cardiac adrenergic (ICA) cell via enhanced epinephrine release. Using immunohistochemical and in situ hybridization methods, we detected in situ tyrosine hydroxylase (TH) mRNA and TH immunoreactivity that was colocalized with DOR immunoreactivity in ICA cells in human and rat hearts. Western blot analysis detected DOR protein in ICA cells isolated from rat ventricular myocytes. The physiology of DOR expression was examined by determining changes of cytosolic Ca(2+) concentration ([Ca(2+)](i)) transients in isolated rat ICA cells using fluorescence spectrophotometry. Exposing the selective delta-opioid agonist D-[Pen(2,5)]enkephalin (DPDPE) to ICA cells increased [Ca(2+)](i) transients in a concentration-dependent manner. Such an effect was abolished by the Ca(2+) channel blocker nifedipine. HPLC-electrochemical detection demonstrated a 2.4-fold increase in epinephrine release from ICA cells following DPDPE application. The significance of the ICA cell and its epinephrine release in delta-opioid-initiated cardioprotection was demonstrated in the rat myocardial infarction model and ICA cell-ventricular myocyte coculture. DPDPE administered before coronary artery occlusion or simulated ischemia-reperfusion reduced left ventricular infarct size by 54 +/- 15% or myocyte death by 26 +/- 4%, respectively. beta(2)-AR blockade markedly attenuated delta-opioid-initiated infarct size-limiting effect and abolished delta-opioid-initiated myocyte survival protection in rat ICA cell-myocyte coculture. Furthermore, delta-opioid agonist exerted no myocyte survival protection in the absence of cocultured ICA cells during ischemia-reperfusion. We conclude that delta-opioid-initiated myocardial infarct size reduction is primarily mediated via endogenous epinephrine/beta(2)-AR signaling pathway as a result of ICA cell activation.

Characterization by theory of H-transfers and onium reactions of CH3CH2CH2N+H=CH2.

H-transfers by 4-, 5-, and 6-membered ring transition states to the pi-bonded methylene of CH3CH2CH2NH+=CH2 (1) are characterized by theory and compared with the corresponding transfers in cation radicals. Four-membered ring H-transfers converting 1 to CH3CH2CH=N+HCH3 (2) and CH3N+H=CH2 to CH2=NH+CH3 are high-energy processes involving rotation of the source and destination RHC= groups (R = H or C2H5) to near bisection by skeletal planes; migrating hydrogens move near these planes. The H-transfer 1 --> CH3C+HCH2NHCH3 (3) has a higher energy transition-state than 1 --> 2, in marked contrast to the corresponding relative energies of 4- and 5-membered ring H-transfers in cation-radicals. Six-membered ring H-transfer-dissociation (1 --> CH2=CH2 + CH2=N+HCH3) is a closed shell analog of the McLafferty rearrangement. It has a lower energy transition-state than either 1 --> 2 or 1 --> 3, but is still a much higher energy process than 6-membered ring H-transfers in aliphatic cation radicals. In contrast to the stepwise McLafferty rearrangement in cation radicals, H-transfer and CC bond breaking are highly synchronous in 1 --> CH3N+H=CH2 + CH2=CH2. H-transfers in propene elimination from 1 are ion-neutral complex-mediated: 1--> [CH3CH2CH2+ ---NH=CH2] --> [CH3C+HCH3 NH=CH2] --> CH3CH = CH2 + CH2=NH2+. Intrinsic reaction coordinate tracing demonstrated that a slight preference for H-transfer from the methyl containing the carbon from which CH2=NH is cleaved is due to CH2=NH passing nearer this methyl than the other on its way to abstracting H, i.e., some memory of the initial orientation of the partners accompanies this reaction.

Human fetal neural stem cells grafted into contusion-injured rat spinal cords improve behavior.

Grafted human neural stem cells (hNSCs) may help to alleviate functional deficits resulting from spinal cord injury by bridging gaps, replacing lost neurons or oligodendrocytes, and providing neurotrophic factors. Previously, we showed that primed hNSCs differentiated into cholinergic neurons in an intact spinal cord. In this study, we tested the fate of hNSCs transplanted into a spinal cord T10 contusion injury model. When grafted into injured spinal cords of adult male rats on either the same day or 3 or 9 days after a moderate contusion injury, both primed and unprimed hNSCs survived for 3 months postengraftment only in animals that received grafts at 9 days postinjury. Histological analyses revealed that primed hNSCs tended to survive better and differentiated at higher rates into neurons and oligodendrocytes than did unprimed counterparts. Furthermore, only primed cells gave rise to cholinergic neurons. Animals receiving primed hNSC grafts on the ninth day postcontusion improved trunk stability, as determined by rearing activity measurements 3 months after grafting. This study indicates that human neural stem cell fate determination in vivo is influenced by the predifferentiation stage of stem cells prior to grafting. Furthermore, stem cell-mediated facilitation of functional improvement depends on the timing of transplantation after injury, the grafting sites, and the survival of newly differentiated neurons and oligodendrocytes.

Intradermal injection of capsaicin induces acute substance P release from rat spinal cord dorsal horn.

Increased release of substance P (SP) from the dorsal horn following noxious stimuli, such as spinal administration of capsaicin, has been demonstrated in previous studies. However, changes in the release of SP in response to intradermal injection of capsaicin still remain unknown. This study was designed to demonstrate in vivo spinal SP release following intradermal injection of capsaicin (3%, 50 microl), using polyimide tubing with a single hole introduced into the rat dorsal horn. The changes in the content of SP in the rat dorsal horn tissues before and after capsaicin (3%, 50 microl) injection were also investigated. The SP concentration in the samples was analyzed using an enzyme-linked immunosorbent assay (ELISA). We found that intradermal injection of capsaicin induced a quick SP release within the dorsal horn. The peak of the release appeared around 10 min after the injection. In contrast, intradermal injection of capsaicin had no significant effect on the SP content in the dorsal horn. This study has provided direct evidence of the effect of intradermal injection of capsaicin on SP release within the dorsal horn, with the major source being from the central terminals of primary afferents.

Transplantation of primed human fetal neural stem cells improves cognitive function in rats after traumatic brain injury.

Traumatic brain injury (TBI) often produces cognitive impairments by primary or secondary neuronal loss. Stem cells are a potential tool to treat TBI. However, most previous studies using rodent stem or progenitor cells failed to correlate cell grafting and cognitive improvement. Furthermore, the efficacy of fetal human neural stem cells (hNSCs) for ameliorating TBI cognitive dysfunction is undetermined. This study therefore characterized phenotypic differentiation, neurotrophic factor expression and release and functional outcome of grafting hNSCs into TBI rat brains. Adult Sprague-Dawley rats underwent a moderate parasagittal fluid percussion TBI followed by ipsilateral hippocampal transplantation of hNSCs or vehicle 1 day post-injury. Prior to grafting, hNSCs were treated in vitro for 7 days with our previously developed priming procedure. Significant spatial learning and memory improvements were detected by the Morris water maze (MWM) test in rats 10 days after receiving hNSC grafts. Morphological analyses revealed that hNSCs survived and differentiated mainly into neurons in the injured hippocampus at 2 weeks after grafting. Furthermore, hNSCs expressed and released glial-cell-line-derived neurotrophic factor (GDNF) in vitro and when grafted in vivo, as detected by RT-PCR, immunostaining, microdialysis and ELISA. This is the first direct demonstration of the release of a neurotrophic factor in conjunction with stem cell grafting. In conclusion, human fetal neural stem cell grafts improved cognitive function of rats with acute TBI. Grafted cells survived and differentiated into neurons and expressed and released GNDF in vivo, which may help protect host cells from secondary damage and aid host regeneration.

Increases in the activated forms of ERK 1/2, p38 MAPK, and CREB are correlated with the expression of at-level mechanical allodynia following spinal cord injury.

Rats given moderate spinal cord injury (SCI) display increases in the expression of the activated form of the transcription factor cyclic AMP responsive element binding protein (CREB) in spinal segments of dermatomes corresponding to permanent mechanical allodynia, a model of chronic central neuropathic pain (CNP; (Crown, E.D., Ye, Z., Johnson, K.M., Xu, G.Y., McAdoo, D.J., Westlund, K.N., Hulsebosch, C.E., 2005. Upregulation of the phosphorylated form of CREB in spinothalamic tract cells following spinal cord injury: relation to central neuropathic pain. Neurosci. Lett. 384, 139-144)). Given that not all rats that receive moderate SCI develop CNP, the current study was designed to further analyze changes in persistent CREB activation and in the activation state of upstream intracellular signaling cascades (e.g., mitogen-activated protein kinases [MAPKs]) in populations of rats that receive SCI and weeks later develop CNP and rats that receive SCI but do not develop CNP. The results indicate that activated kinases such as pERK 1/2, p-p38 MAPK, but not pJNK, are upregulated in injured rats that develop CNP as compared to injured rats that fail to develop CNP. In addition, the current results replicated our previous finding that activated CREB is upregulated following SCI, however, only in SCI rats that developed CNP. Taken together, these results indicate that activation of intracellular signaling cascades traditionally associated with long-term potentiation and memory is associated with the expression of chronic CNP following SCI.

Transcriptional profiling of spinal cord injury-induced central neuropathic pain.

Central neuropathic pain (CNP) is an important problem following spinal cord injury (SCI), because it severely affects the quality of life of SCI patients. As in the patient population, the majority of rats develop significant allodynia (CNP rats) after moderate SCI. However, about 10% of SCI rats do not develop allodynia, or develop significantly less allodynia than CNP rats (non-CNP rats). To identify transcriptional changes underlying CNP development after SCI, we used Affymetrix DNA microarrays and RNAs extracted from the spinal cords of CNP and non-CNP rats. DNA microarry analysis showed significantly increased expression of a number of genes associated with inflammation and astrocytic activation in the spinal cords of rats that developed CNP. For example, mRNA levels of glial fibrilary acidic protein (GFAP) and Aquaporin 4 (AQP4) significantly increased in CNP rats. We also found that GFAP, S100beta and AQP4 protein elevation persisted for at least 9 months throughout contused spinal cords, consistent with the chronic nature of CNP. Thus, we hypothesize that CNP development results, in part, from dysfunctional, chronically "over-activated" astrocytes. Although, it has been shown that activated astrocytes are associated with peripheral neuropathic pain, this has not previously been demonstrated in CNP after SCI.

Administration of glutamate into the spinal cord at extracellular concentrations reached post-injury causes functional impairments.

In vivo experiments addressing the role of released glutamate in damage caused by neurotrauma seldom administer glutamate itself because it usually produces relatively little damage when administered into central nervous system (CNS) tissue in vivo. However, because of recent observations that glutamate administered into the spinal cord at the levels attained following spinal cord injury (SCI) kills neurons and oligodendrocytes, we tested the effects of administering glutamate at those concentrations on locomotor function. The Basso-Beattie-Bresnahan (BBB) test and activity box measures demonstrated that those glutamate concentrations produce lasting functional impairments. Several parameters provided by the activity box provided sensitive measures of the degree of post-SCI impairment, demonstrating their substantial potential for evaluating outcomes of SCI. Results obtained also enhance evidence that glutamate toxicity contributes to secondary damage following SCI and suggest that damage to white matter is an important contributor to such damage.

The effect of glutamate receptor blockers on glutamate release following spinal cord injury. Lack of evidence for an ongoing feedback cascade of damage --> glutamate release --> damage --> glutamate release --> etc.

It is widely hypothesized that excitotoxicity of released glutamate following a CNS insult is propagated by the cyclic cascade: glutamate release --> damage --> glutamate release --> further damage --> etc. We tested this hypothesis by determining the effects of attempting to interrupt the loop by administering glutamate receptor antagonists and Na(+)-channel blockers on glutamate release following spinal cord injury (SCI). The effects of administering the NMDA receptor blockers MK-801 and memantine, the AMPA/kainate receptor blockers NBQX and GYKI 52466, the AMPA receptor desensitization blocker cyclothiazide and the sodium channel blockers riluzole, mexiletine and QX-314 on post-SCI were determined. Agents were administered into the site of injury by direct injection, by microdialysis or systemically. None of these agents had an appreciable effect on glutamate release following SCI. Thus, it is unlikely that the above cascade produces significant secondary glutamate release and ongoing damage following SCI, although such cascades may worsen other CNS insults. We attribute our results to overwhelming effects of much greater release by direct mechanical damage and reversal of transport following SCI.

Circumvention of orbital symmetry restraints by 1,3-H-shifts of enolic radical cations.

The reaction coordinates of 1,3-H-shifts across double bonds are traced by theory for three reactions, CH(3)C(OH)CH(2)(+*) (1) --> CH(3)C(O(+*))CH(3) (2), CH(2)C(OH)(2)(+*) (3) --> CH(3)CO(2)H(+*) (4) and CH(3)C(OH)CH(2)(+*) (1) --> CH(2)C(OH)CH(3)(+*) (1'), to explore how the need to conserve orbital symmetry influences the pathways for these reactions. In the first and second reactions, prior to the start of the H-transfer the methylene rotates from being in the skeletal plane to being bisected by it. Thus these reactions are neither antarafacial nor suprafacial, but precisely between those possibilities. This stems from a counterbalancing between the need to conserve orbital symmetry and the large distorting forces required to attain an allowed antarafacial transition state. In contrast to the first two reactions, 1 --> 1' follows a suprafacial pathway. However, this pathway does not violate conservation of orbital symmetry, as it utilizes lower lying orbitals of appropriate symmetry rather than the antisymmetric uppermost occupied allyl-type orbital. Changes in geometry which presumably produce asymmetric vibrational excitation and the unequal losses of methyl that follow 1 --> 2, i.e., nonergodic behavior, are also characterized.