Machine intelligence identifies soluble TNFa as a therapeutic target for spinal cord injury.

Traumatic spinal cord injury (SCI) produces a complex syndrome that is expressed across multiple endpoints ranging from molecular and cellular changes to functional behavioral deficits. Effective therapeutic strategies for CNS injury are therefore likely to manifest multi-factorial effects across a broad range of biological and functional outcome measures. Thus, multivariate analytic approaches are needed to capture the linkage between biological and neurobehavioral outcomes. Injury-induced neuroinflammation (NI) presents a particularly challenging therapeutic target, since NI is involved in both degeneration and repair. Here, we used big-data integration and large-scale analytics to examine a large dataset of preclinical efficacy tests combining five different blinded, fully counter-balanced treatment trials for different acute anti-inflammatory treatments for cervical spinal cord injury in rats. Multi-dimensional discovery, using topological data analysis (TDA) and principal components analysis (PCA) revealed that only one showed consistent multidimensional syndromic benefit: intrathecal application of recombinant soluble TNFα receptor 1 (sTNFR1), which showed an inverse-U dose response efficacy. Using the optimal acute dose, we showed that clinically-relevant 90 min delayed treatment profoundly affected multiple biological indices of NI in the first 48 h after injury, including reduction in pro-inflammatory cytokines and gene expression of a coherent complex of acute inflammatory mediators and receptors. Further, a 90 min delayed bolus dose of sTNFR1 reduced the expression of NI markers in the chronic perilesional spinal cord, and consistently improved neurological function over 6 weeks post SCI. These results provide validation of a novel strategy for precision preclinical drug discovery that is likely to improve translation in the difficult landscape of CNS trauma, and confirm the importance of TNFα signaling as a therapeutic target.

Convolutional Neural Network-Based Automated Segmentation of the Spinal Cord and Contusion Injury: Deep Learning Biomarker Correlates of Motor Impairment in Acute Spinal Cord Injury.

BACKGROUND AND PURPOSE: Our aim was to use 2D convolutional neural networks for automatic segmentation of the spinal cord and traumatic contusion injury from axial T2-weighted MR imaging in a cohort of patients with acute spinal cord injury. MATERIALS AND METHODS: Forty-seven patients who underwent 3T MR imaging within 24 hours of spinal cord injury were included. We developed an image-analysis pipeline integrating 2D convolutional neural networks for whole spinal cord and intramedullary spinal cord lesion segmentation. Linear mixed modeling was used to compare test segmentation results between our spinal cord injury convolutional neural network (Brain and Spinal Cord Injury Center segmentation) and current state-of-the-art methods. Volumes of segmented lesions were then used in a linear regression analysis to determine associations with motor scores. RESULTS: Compared with manual labeling, the average test set Dice coefficient for the Brain and Spinal Cord Injury Center segmentation model was 0.93 for spinal cord segmentation versus 0.80 for PropSeg and 0.90 for DeepSeg (both components of the Spinal Cord Toolbox). Linear mixed modeling showed a significant difference between Brain and Spinal Cord Injury Center segmentation compared with PropSeg (P < .001) and DeepSeg (P < .05). Brain and Spinal Cord Injury Center segmentation showed significantly better adaptability to damaged areas compared with PropSeg (P < .001) and DeepSeg (P < .02). The contusion injury volumes based on automated segmentation were significantly associated with motor scores at admission (P = .002) and discharge (P = .009). CONCLUSIONS: Brain and Spinal Cord Injury Center segmentation of the spinal cord compares favorably with available segmentation tools in a population with acute spinal cord injury. Volumes of injury derived from automated lesion segmentation with Brain and Spinal Cord Injury Center segmentation correlate with measures of motor impairment in the acute phase. Targeted convolutional neural network training in acute spinal cord injury enhances algorithm performance for this patient population and provides clinically relevant metrics of cord injury.

Multivariate Analysis of MRI Biomarkers for Predicting Neurologic Impairment in Cervical Spinal Cord Injury.

BACKGROUND AND PURPOSE: Acute markers of spinal cord injury are essential for both diagnostic and prognostic purposes. The goal of this study was to assess the relationship between early MR imaging biomarkers after acute cervical spinal cord injury and to evaluate their predictive validity of neurologic impairment. MATERIALS AND METHODS: We performed a retrospective cohort study of 95 patients with acute spinal cord injury and preoperative MR imaging within 24 hours of injury. The American Spinal Injury Association Impairment Scale was used as our primary outcome measure to define neurologic impairment. We assessed several MR imaging features of injury, including axial grade (Brain and Spinal Injury Center score), sagittal grade, length of injury, maximum canal compromise, and maximum spinal cord compression. Data-driven nonlinear principal component analysis was followed by correlation and optimal-scaled multiple variable regression to predict neurologic impairment. RESULTS: Nonlinear principal component analysis identified 2 clusters of MR imaging variables related to 1) measures of intrinsic cord signal abnormality and 2) measures of extrinsic cord compression. Neurologic impairment was best accounted for by MR imaging measures of intrinsic cord signal abnormality, with axial grade representing the most accurate predictor of short-term impairment, even when correcting for surgical decompression and degree of cord compression. CONCLUSIONS: This study demonstrates the utility of applying nonlinear principal component analysis for defining the relationship between MR imaging biomarkers in a complex clinical syndrome of cervical spinal cord injury. Of the assessed imaging biomarkers, the intrinsic measures of cord signal abnormality were most predictive of neurologic impairment in acute spinal cord injury, highlighting the value of axial T2 MR imaging.

SOD1 overexpression improves features of the oligodendrocyte precursor response in vitro.

Spinal cord injury (SCI) produces a significant loss of oligodendrocytes (OL) and demyelination. The oligodendrocyte precursor cells (OPCs) response includes a group of cellular changes in OPCs that are directed to replenish OL loss from the injury. However, this adaptive response is hampered and OPCs eventually die or fail to differentiate to mature and functional OL. In this study, we wanted to evaluate if overexpression of human superoxide dismutase 1 (hSOD1) in OPCs from the SOD1 transgenic rat could improve some of the features of the OPC response in vitro. We found that hSOD1 overexpression increases the proliferation of OPCs and accelerates their differentiation to mature OL in vitro. Furthermore, hSOD1 overexpression reduces oxidative stress-mediated death in OPCs. These results suggest hSOD1 as a therapeutic target to increase OPC response success and potentially, OL replacement and remyelination after SCI.

Dissociation of the effects of nucleus raphe obscurus or rostral ventrolateral medulla lesions on eliminatory and sexual reflexes.

Rat preparations were used to investigate long-term changes in external anal sphincter (EAS) contractions and reflexive penile erection following electrolytic lesions of the nucleus raphe obscurus (nRO) or the rostral ventrolateral medulla. EAS contractions were measured electromyographically (EAS EMG) following distention of the EAS with a 5-mm probe. Penile erections were measured using a standard ex copula reflex testing paradigm. At 48 h postlesion, 100% of nRO-lesioned animals displayed reflexive erections and the magnitude of EAS EMG was significantly greater in lesioned animals than in sham controls. These results suggested EAS hyperreflexia following destruction of the nRO. By 14 days postlesion, EAS responsiveness in nRO-lesioned animals had returned to levels comparable to nonlesioned animals. No measures of penile erection were affected by nRO lesions. In animals with nucleus gigantocellularis (Gi) and lateral nucleus paragigantocellularis (Gi-lPGi) lesions, no significant changes to EAS reflexes were observed at any time point. At 48 h postoperative, Gi-lPGi lesions significantly reduced the latency to first erection and increased the number of erections elicited relative to controls. Similar facilitation of erection latency was observed at 14 days postlesion, while erection number and flip total were no longer significantly different from controls. These and previous studies suggest that the nRO regulates defecatory reflexes in the rat. These data further suggest that the comingled EAS and bulbospongiosus (BS) motoneurons are controlled by discrete and separate brainstem circuits and that increases in EAS and penile reflexes after spinal cord lesions are mediated by loss of different descending inputs.

Upregulation of the HLH Id gene family in neural progenitors and glial cells of the rat spinal cord following contusion injury.

Spinal cord injury (SCI) leads to a complex sequence of cellular responses, including astrocyte activation, oligodendrocyte death, and ependymal cell proliferation. Inhibitors of DNA binding (Id1, Id2, Id3) belong to a helix-loop-helix (HLH) gene family. Id genes have been implicated in playing a vital role in the proliferation of many cell types, including astrocytes and myoblasts. In the present study, the expression of Id family members in spinal cord after contusion injury was investigated by in situ hybridization. Id1, Id2, and Id3 mRNA expression was upregulated 5 mm rostral and caudal to the lesion center, and reached maximal levels 3 days after SCI. In addition, cell populations expressing Id1, Id2, and Id3 mRNA were maximally increased 3 days after SCI. The increase in Id2 and Id3 mRNA expression and Id2 and Id3 mRNA+ cells was still observed at 8 days. The Id mRNA expressing cells were phenotyped by combining immunostaining of cell-specific markers with in situ hybridization. Glial fibrillary acidic protein (GFAP)+ astrocytes were found to express all three Id mRNA, whereas S-100alpha+ astrocytes only expressed high levels of Id2 and Id3 mRNA. Cells having a neural progenitor morphology and the marker nestin appeared after SCI and they expressed Id1, Id2, and Id3 mRNA. Interestingly, some Rip+ oligodendrocytes located in the areas close to the central canal expressed Id3 mRNA after injury. In conclusion, Id genes are upregulated in a time-dependent manner in astrocytes, oligodendrocytes, and neural progenitor subpopulations after SCI, suggesting that they play major roles in cellular responses following SCI.

Inhibition of pudendal reflexes in spinal rats. Reassessing the role of serotonin.

The effects of serotonin (5-HT) and thyrotropin-releasing hormone (TRH) on penile reflexes were investigated in intact and spinally transected male rats. Doses of intrathecal 5-HT (0.0, 1.13, 2.26, 11.3, 22.6, and 113.0 nmol), in a range previously shown to inhibit pudendal reflexes in anesthetized spinal preparations, prolonged the latency to the first penile erection in awake intact rats. However, these doses also provoked hyperreactivity and vocalization. Doses of intrathecal TRH (100 and 500 pmol) that effectively inhibited penile erection in intact animals were less effective in spinalized animals. Finally, a combination of subthreshold doses of TRH (100 pmol) and 5-HT (4.0 nmol) at a ratio known to affect other TRH/5-HT-mediated circuits significantly extended erection latency in animals with spinal transections. These data suggest that 5-HT and TRH are both involved in the inhibitory circuits regulating penile erection, either through corelease onto the same population of cells or through independent release onto different populations of neurons.

Tumor necrosis factor-alpha induces cFOS and strongly potentiates glutamate-mediated cell death in the rat spinal cord.

Excitotoxic cell death due to glutamate release is important in the secondary injury following CNS trauma or ischemia. Proinflammatory cytokines also play a role. Both glutamate and tumor necrosis factor-alpha (TNF(alpha)) are released immediately after spinal cord injury. Neurophysiological studies show that TNF(alpha) can potentiate the effects of glutamatergic afferent input to produce hyperactivation of brain-stem sensory neurons. Therefore, we hypothesized that TNF(alpha) might act cooperatively with glutamate to affect cell death in the spinal cord as well. Nanoinjections of either TNF(alpha) (60 pg) or kainate (KA; 32 ng) alone into the thoracic gray resulted in almost no tissue damage or cell death 90 min after injection. However, the combination of TNF(alpha) plus KA at these same doses produced a large area of tissue necrosis and neuronal cell death, an effect which was blocked by the AMPA receptor antagonist CNQX (17 ng). These results suggest that secondary injury may involve potentiation of AMPA receptor-mediated excitatory cell death by TNF(alpha).

Degeneration and sprouting of identified descending supraspinal axons after contusive spinal cord injury in the rat.

Contusive spinal cord injury (SCI) results in the formation of a chronic lesion cavity surrounded by a rim of spared fibers. Tissue bridges containing axons extend from the spared rim into the cavity dividing it into chambers. Whether descending axons can grow into these trabeculae or whether fibers within the trabeculae are spared fibers remains unclear. The purposes of the present study were (1) to describe the initial axonal response to contusion injury in an identified axonal population, (2) to determine whether and when sprouts grow in the face of the expanding contusion cavity, and (3) in the long term, to see whether any of these sprouts might contribute to the axonal bundles that have been seen within the chronic contusion lesion cavity. The design of the experiment also allowed us to further characterize the development of the lesion cavity after injury. The corticospinal tract (CST) underwent extensive dieback after contusive SCI, with retraction bulbs present from 1 day to 8 months postinjury. CST sprouting occurred between 3 weeks and 3 months, with penetration of CST axons into the lesion matrix occurring over an even longer time course. Collateralization and penetration of reticulospinal fibers were observed at 3 months and were more extensive at later time points. This suggests that these two descending systems show a delayed regenerative response and do extend axons into the lesion cavity and that the endogenous repair can continue for a very long time after SCI.

Intrathecal methiothepin and thyrotropin-releasing hormone effects on penile erection.

Intrathecal thyrotropin-releasing hormone (TRH) potently inhibits penile erection at all doses (100, 500, 1000 or 5000 pmol) tested so far. Since the serotonin receptor antagonist methiothepin (MT) inhibits TRH responses in other systems, this study tested the hypothesis that MT-sensitive receptors mediate the effect of TRH on penile erection in rats. When compared to controls, the highest doses of IT TRH (0, 10 or 500 pmol) or MT (5 or 50 nmol) significantly altered penile reflex latency. When coadministered (50 nmol MT/500 pmol TRH), the effect of TRH was reversed, suggesting that the high dose of MT antagonized the inhibitory actions of TRH. The low dose of MT (5 nmol) did not block the 500 pmol TRH inhibition of reflex latency. These data further suggest that MT sensitive receptors are important in (1) mediating normal penile reflexes and (2) mediating the inhibitory response to TRH.

Boron neutron capture therapy of brain tumors: functional and neuropathologic effects of blood-brain barrier disruption and intracarotid injection of sodium borocaptate and boronophenylalanine.

Sodium borocaptate (BSH) and boronophenylalanine (BPA) are two drugs that have been used clinically for boron neutron capture therapy (BNCT) of brain tumors. We previously have reported that hyperosmotic mannitol-induced disruption of the blood-brain barrier (BBB-D), followed by intracarotid (i.c.) administration of BPA or BSH, either individually or in combination, significantly enhanced tumor boron delivery and the efficacy of BNCT in F98 glioma bearing rats. The purpose of the present study was to determine the short-term neuropathologic consequences of this treatment and the long-term effects on motor and cognitive function, as well as the neuropathologic sequelae 1 year following neutron capture irradiation. BBB-D was carried out in non-tumor bearing Fischer rats by infusing a 25% solution of mannitol i.c. followed by i.c. injection of BPA or BSH, either individually or in combination, immediately thereafter. Animals were euthanized 2 days after compound administration, and their brains were processed for neuropathologic examination, which revealed sporadic, mild, focal neuronal degeneration, hemorrhage, and necrosis. To assess the long-term effects of such treatment followed by neutron capture irradiation, non-tumor bearing rats were subjected to BBB-D after which they were injected i.c. with BPA (25 mg B/kg body weight (b.w)) or BSH (30 mg B/kg b.w.) either individually or in combination (BPA 12.5 mg and BSH 14 mg B/kg b.w.). Two and a half hours later they were irradiated at the Medical Research Reactor, Brookhaven National Laboratory, Upton, NY, with the same physical radiation doses (5.79, 8.10 or 10.06 Gy), delivered to the brain, as those that previously had been used for our therapy experiments. The animals tolerated this procedure well, after which they were returned to Columbus, Ohio where their clinical status was monitored weekly. After 1 year, motor function was assessed using a sensitive and reliable locomotor rating scale for open field testing in rats and cognitive function was evaluated by their performance in the Morris water maze, the results of which were similar to those obtained with age matched controls. After functional evaluation, the rats were euthanized, their brains were removed, and then processed for neuropathologic examination. Subtle histopathologic changes were seen in the choroid plexuses of irradiated animals that had received BPA, BSH or saline. Radiation related ocular changes consisting of keratitis, blepharitis, conjunctivitis and cataract formation were seen with similar frequency in most rats in each treatment group. Based on these observations, and the previously reported significant therapeutic gain associated with BBB-D and i.c. injection of BSH and BPA, the present observations establish its safety in rats and suggest that further studies in large animals and humans are warranted.

Review of current evidence for apoptosis after spinal cord injury.

The initial mechanical tissue disruption of spinal cord injury (SCI) is followed by a period of secondary injury that increases the size of the lesion. The secondary injury has long been thought to be due to the continuation of cellular destruction through necrotic (or passive) cell death. Recent evidence from brain injury and ischemia suggested that cellular apoptosis, an active form of programmed cell death seen during development, could play a role in CNS injury in adulthood. Here, we review the evidence that apoptosis may be important in the pathophysiology of SCI. There is now strong morphological and biochemical evidence from a number of laboratories demonstrating the presence of apoptosis after SCI. Apoptosis occurs in populations of neurons, oligodendrocytes, microglia, and, perhaps, astrocytes. The death of oligodendrocytes in white matter tracts continues for many weeks after injury and may contribute to post-injury demyelination. The mediators of apoptosis after SCI are not well understood, but there is a close relationship between microglia and dying oligodendrocytes, suggesting that microglial activation may be involved. There is also evidence for the activation of important intracellular pathways known to be involved in apoptosis in other cells and systems. For example, some members of the caspase family of cysteine proteases are activated after SCI. It appears that the evolution of the lesion after SCI involves both necrosis and apoptosis. It is likely that better understanding of apoptosis after SCI will lead to novel strategies for therapeutic interventions that can diminish secondary injury.

An analysis of changes in sensory thresholds to mild tactile and cold stimuli after experimental spinal cord injury in the rat.

Changes in sensory function including chronic pain and allodynia are common sequelae of spinal cord injury (SCI) in humans. The present study documents the extent and time course of mechanical allodynia and cold hyperalgesia after contusion SCI in the rat using stimulation with graded von Frey filaments (4.97-50.45 g force) and ice probes. Fore- and hind-paw withdrawal thresholds to plantar skin stimulation were determined in rats with a range of SCI severities (10-g weight dropped from 6.25, 12.5, or 25 mm using the MASCIS injury device); animals with 25-mm injuries most consistently showed decreased hind-paw withdrawal thresholds to touch and cold, which developed over several weeks after surgery. Stimulation of the torso with graded von Frey hairs was performed at specified locations on the back and sides from the neck to the haunch. Suprasegmental responses (orientation, vocalization, or escape) to mechanical stimulation of these sites were elicited infrequently in the laminectomy control rats and only during the first 3 weeks after surgery, whereas in 25-mm SCI rats, such responses were obtained for the entire 10 weeks of the study. These data suggest that rats with contusion SCI may exhibit sensory alterations relevant to human spinal cord injuries.

Descending projections from the nucleus raphe obscurus to pudendal motoneurons in the male rat.

Previous physiological and behavioral studies have shown that the nucleus raphe obscurus (nRO) modulates pelvic floor reflex function (Yamanouchi and Kakeyama [1992] Physiol. Behav. 51:575-579; Beattie et al. [1996] Soc. Neurosci. Abstr. 22:722.4; Holmes et al. [1997] Brain Res. 759:197-204). In the present study, small injections of fluorescent tracers were used to investigate direct descending projections from the rostral and caudal portions of the brainstem nRO to retrogradely labeled pudendal motoneurons (MN) in the male rat. The caudal nRO projects into the ventral and lateral funiculi of the spinal cord, with arborizations in the thoracic intermediolateral cell column; in laminae VII, IX, and X of the lumbosacral cord; and in the sacral parasympathetic nucleus (SPN). Many identified external anal sphincter and ischiocavernosus MNs appeared to be in direct apposition with fibers originating from the caudal nRO; and more than half of the bulbospongiosus MNs that were identified appeared to receive such descending input. In addition to the nRO spinal autonomic and pudendal motoneuronal targets, projections were observed to regions of the intermediate gray that contain interneurons organizing the pelvic floor reflexes and to MN pools that are involved in functionally related somatic activities. Finally, several neurons in the lumbar enlargement were labeled retrogradely with FluoroRuby after injections into the nRO and the immediately adjacent reticular formation. Thus, the nRO may be in a position to modulate the coordinated actions of autonomic preganglionic and functionally related skeletal MN activity involved in sexual and eliminative reflex functions.

Adult opossums (Didelphis virginiana) demonstrate near normal locomotion after spinal cord transection as neonates.

When the thoracic spinal cord of the North American opossum (Didelphis virginiana) is transected on postnatal day (PD) 5, the site of injury becomes bridged by histologically recognizable spinal cord and axons which form major long tracts grow through the lesion. In the present study we asked whether opossums lesioned on PD5 have normal use of the hindlimbs as adults and, if so, whether that use is dependent upon axons which grow through the lesion site. The thoracic spinal cord was transected on PD5 and 6 months later, hindlimb function was evaluated using the Basso, Beattie, and Bresnahan (BBB) locomotor scale. All animals supported their weight with the hindlimbs and used their hindlimbs normally during overground locomotion. In some cases, the spinal cord was retransected at the original lesion site or just caudal to it 6 months after the original transection and paralysis of the hindlimbs ensued. Surprisingly, however, these animals gradually recovered some ability to support their weight and to step with the hindlimbs. Similar recovery was not seen in animals transected only as adults. In order to verify that descending axons which grew through the lesion during development were still present in the adult animal, opossums subjected to transection of the thoracic cord on PD5 were reoperated and Fast blue was injected several segments caudal to the lesion. In all cases, neurons were labeled rostral to the lesion in each of the spinal and supraspinal nuclei labeled by comparable injections in unlesioned, age-matched controls. The results of orthograde tracing studies indicated that axons which grew through the lesion innervated areas that were appropriate for them.

External anal sphincter hyperreflexia following spinal transection in the rat.

In the present study, long-term and short-term rat preparations were used to develop a model for investigating external anal sphincter (EAS) reflexes in intact and spinal cord-injured (SCI) rats. In this model, EAS distension with an external probe elicits reflex contractions of the EAS in intact, unanesthetized animals. At 2 h after spinal cord transection, none of the lesioned animals displayed EAS EMG activity. In fact, once distended, the EAS was incapable of maintaining closure of the anal orifice. Over a period of 4 days, spinalized animals developed a hyperreflexia of the EAS response. By 48 h, the rectified, integrated EAS EMG was significantly elevated in comparison with nonlesioned controls (EAS hyperreflexia). In addition, the duration of the EAS EMG bursts in response to sphincter distension had significantly increased. At 6 weeks after injury, the EAS was significantly hyperreflexic as measured by EMG burst duration and burst area. As with intact animals, posttransection EAS reflexes were highly anesthesia sensitive. These studies indicate that (1) brief distension of the anal orifice is sufficient to evoke a physiologically relevant reflexive activation of the EAS in the rat, (2) the 2- to 24-h postinjury areflexia observed in these experiments may be a suitable model for the study of spinal shock, and (3) the observed EAS hyperreflexia after chronic SCI may represent the permanent effects of removing descending inhibitory circuits and segmental plasticity, making this reflex an appropriate measure of defecatory dysfunction after spinal cord injury.

Nucleus raphe obscurus (nRO) regulation of anorectal motility in rats.

Previous research has demonstrated that anorectal contractions in the rat are modulated by activation of spinal autonomic circuits. In the present study, anterograde tracing of descending pathways originating from the caudal nucleus raphe obscurus (nRO) revealed that this nucleus projects to cells within the intermediolateral (IML) cell column of the thoracic cord and the sacral parasympathetic nucleus (SPN). These anatomical studies suggested that the nRO may influence the regulation of spinal reflexes of the pelvic floor. In a second set of experiments, acute rat preparations were used to investigate changes in anorectal motility during electrical stimulation of the nRO. Anorectal contractions were measured by a fluid-filled manometer. Electrical stimulation of the nRO significantly reduced spontaneous anorectal activity when compared to baseline contractions recorded for 1 min prior to stimulation. Stimulation sites outside the nRO did not affect anorectal contractions when compared to either (a) the 1-min pre-stimulation baseline for that site or (b) the 1-min stimulation period for sites within the nRO. Stimulation of caudal portions of the nRO were more likely than the rostral nRO to reduce anorectal contractions. Given that the SPN contains preganglionic neurons which may be involved in control of anorectal contractions (mediated via the pelvic nerve), the studies presented here suggest a functional role for nRO regulation of preganglionic motoneurons innervating the distal gut of the rat.

Differential effects of intrathecal thyrotropin-releasing hormone (TRH) on perineal reflexes in male rats.

The effects of thyrotropin-releasing hormone (TRH) on the sexual and defecatory reflexes regulated by pudendal motoneurons were investigated. Intrathecal TRH (10 microliters volume; 0.0, 0.01, 1.0 or 100 microM concentration) at lumbosacral spinal segments (L4-S1) in acute preparations produced a dose-dependent increase in external anal sphincter (EAS), but not bulbospongiosus (BS), electromyographic (EMG) activity. Intraspinal (L6) injection of 100 microM TRH (1 microliter/micropipette), significantly increased EAS EMG activity in acute preparations. Electromyographic activity of the BS muscle was unchanged. All doses of intrathecal TRH (10 microliters volume; 0, 10, 50, 100, or 500 microM concentration) in awake animals significantly reduced the proportion of responders to a penile reflex test. Subsequently, all measures of penile reflexes were significantly reduced. Glans tumescence and defecation bouts before or during penile reflex testing were unaffected by intrathecal TRH as were indices of behavioral and motor hyper-reactivity analogous to that produced by serotonin. These data indicate that pudendal motoneurons, in the dorsomedial nucleus, are differentially regulated by neuropeptides present in the lumbosacral spinal cord.

Apoptosis and delayed degeneration after spinal cord injury in rats and monkeys.

Apoptosis is a morphologically defined form of programmed cell death seen in a variety of circumstances, including immune cell selection, carcinogenesis and development. Apoptosis has very recently been seen after ischemic or traumatic injury to the central nervous system (CNS), suggesting that active cell death as well as passive necrosis may mediate damage after CNS injury. After spinal cord injury (SCI) in the rat, typical post-traumatic necrosis occurred, but in addition, apoptotic cells were found from 6 hours to 3 weeks after injury, especially in the spinal white matter. Apoptotic cells were positive for oligodendrocyte markers. After SCI in monkeys, apoptotic cells were found within remote degenerating fiber tracts. Both secondary degeneration at the site of SCI and the chronic demyelination of tracts away from the injury appear to be due in part to apoptosis. As cytokines have been shown to mediate oligodendrocyte death in vitro, it seems likely that chronic demyelination after CNS injury shares features with chronic degenerative disorders like multiple sclerosis.