Cell death and plasticity after experimental spinal cord injury.
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Biomedical subjects
M S Beattie
Publications and source records attributed to M S Beattie.
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.
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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.
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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.
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Apoptosis of microglia and oligodendrocytes after spinal cord contusion in rats.
Following spinal cord contusion in the rat, apoptosis has been observed in the white matter for long distances remote from the center of the lesion and is primarily associated with degenerating fiber tracts. We have previously reported that many of the apoptotic cells are oligodendrocytes. Here we show that the oligodendrocyte death is maximal at 8 days postinjury and suggest that loss of oligodendrocytes may result in demyelination of axons that have survived the initial trauma. There are two mechanisms that may account for the observed oligodendrocyte apoptosis. The apoptotic cell death may result from the loss of trophic support after axonal degeneration or it may be the consequence of microglial activation. The hypothesis that oligodendrocyte apoptosis is secondary to microglial activation is supported by our observations of microglia with an activated morphology in the same regions as apoptosis and apparent contact between some of the apoptotic oligodendrocytes and microglial processes. In addition to oligodendrocyte apoptosis, a subpopulation of microglia appears to be susceptible to apoptotic cell death as well, as evidenced by the presence of apoptotic bodies in OX42 immunopositive profiles. Thus, the population of apoptotic cells following spinal cord contusion is comprised of oligodendrocytes and putative phagocytic microglia or macrophages. Given the delayed time course of oligodendrocyte death, the apoptotic death of oligodendrocytes may be amenable to pharmacological intervention with subsequent improvement in functional recovery.
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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.
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Endogenous repair after spinal cord contusion injuries in the rat.
Contusion injuries of the rat thoracic spinal cord were made using a standardized device developed for the Multicenter Animal Spinal Cord Injury Study (MASCIS). Lesions of different severity were studied for signs of endogenous repair at times up to 6 weeks following injury. Contusion injuries produced a typical picture of secondary damage resulting in the destruction of the cord center and the chronic sparing of a peripheral rim of fibers which varied in amount depending upon the injury magnitude. It was noted that the cavities often developed a dense cellular matrix that became partially filled with nerve fibers and associated Schwann cells. The amount of fiber and Schwann cell ingrowth was inversely related to the severity of injury and amount of peripheral fiber sparing. The source of the ingrowing fibers was not determined, but many of them clearly originated in the dorsal roots. In addition to signs of regeneration, we noted evidence for the proliferation of cells located in the ependymal zone surrounding the central canal at early times following contusion injuries. These cells may contribute to the development of cellular trabeculae that provide a scaffolding within the lesion cavity that provides the substrates for cellular infiltration and regeneration of axons. Together, these observations suggest that the endogenous reparative response to spinal contusion injury is substantial. Understanding the regulation and restrictions on the repair processes might lead to better ways in which to encourage spontaneous recovery after CNS injury.
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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.
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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.
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Graded histological and locomotor outcomes after spinal cord contusion using the NYU weight-drop device versus transection.
Injury reproducibility is an important characteristic of experimental models of spinal cord injuries (SCI) because it limits the variability in locomotor and anatomical outcome measures. Recently, a more sensitive locomotor rating scale, the Basso, Beattie, and Bresnahan scale (BBB), was developed but had not been tested on rats with severe SCI complete transection. Rats had a 10-g rod dropped from heights of 6.25, 12.5, 25, and 50 mm onto the exposed cord at Tl 0 using the NYU device. A subset of rats with 25 and 50 mm SCI had subsequent spinal cord transection (SCI + TX) and were compared to rats with transection only (TX) in order to ascertain the dependence of recovery on descending systems. After 7-9 weeks of locomotor testing, the percentage of white matter measured from myelin-stained cross sections through the lesion center was significantly different between all the groups with the exception of 12.5 vs 25 mm and 25 vs 50 mm groups. Locomotor recovery was greatest for the 6.25-mm group and least for the 50-mm group and was correlated positively to the amount of tissue sparing at the lesion center (p < 0.0001). BBB scale sensitivity was sufficient to discriminate significant locomotor differences between the most severe SCI (50 mm) and complete TX (p < 0.01). Transection following SCI resulted in a drop in locomotor scores and rats were unable to step or support weight with their hindlimbs (p < 0.01), suggesting that locomotor recovery depends on spared descending systems. The SCI + TX group had a significantly greater frequency of HL movements during open field testing than the TX group (p < 0.005). There was also a trend for the SCI + TX group to have higher locomotor scores than the TX group (p > 0.05). Thus, spared descending systems appear to modify segmental systems which produce greater behavioral improvements than isolated cord systems.
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MASCIS evaluation of open field locomotor scores: effects of experience and teamwork on reliability. Multicenter Animal Spinal Cord Injury Study.
The Multicenter Animal Spinal Cord Injury Study (MASCIS) adopted a modified 21-point open field locomotor scale developed by Basso, Beattie, and Bresnahan (BBB) at Ohio State University (OSU) to measure motor recovery in spinal-injured rats. BBB scores categorize combinations of rat hindlimb movements, trunk position and stability, stepping, coordination, paw placement, toe clearance, and tail position, representing sequential recovery stages that rats attain after spinal cord injury. A total of 22 observers from 8 participating centers assessed 18 hindlimbs of 9 rats at 2-6 weeks after graded spinal cord injury. The observers were segregated into 10 teams. The teams were grouped into 3 cohorts (A, B, and C), consisting of one experienced team from OSU and two non-OSU teams. The cohorts evaluated the rats in three concurrent and sequential sessions. After viewing a rat for 4 min, individual observers first assigned scores without discussion. Members of each team then discussed and assigned a team score. Experience (OSU vs. non-OSU) and teamwork (individual vs. team) had no significant effect on mean scores although the mean scores of one cohort differed significantly from the others (p = 0.0002, ANOVA). However, experience and teamwork significantly influenced reliability of scoring. OSU team scores had a mean standard deviation or discordance of 0.59 points, significantly less than 1.31 points for non-OSU team scores (p = 0.003, ANOVA) and 1.30 points for non-OSU individual scores (p = 0.001, ANOVA). Discordances were greater at the upper and lower ends of the scale, exceeding 2.0 in the lower (< 5) and upper (> 15) ends of the scale but were < 1.0 for scores between 4 and 16. Comparisons of non-OSU and OSU team scores indicated a high reliability coefficient of 0.892 and a correlation index (r2) of 0.894. These results indicate that inexperienced observers can learn quickly to assign consistent BBB scores that approach those given by experienced teams, that the scores are most consistent between 4 and 16, and that experience improves consistency of team scores.
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Thyrotropin-releasing hormone (TRH) and CNS regulation of anorectal motility in the rat.
The effect of thyrotropin-releasing hormone (TRH) upon anorectal motility was investigated in acute male rat preparations. Micromolar doses of TRH were intrathecally (i.t.) infused at the L6 spinal level at a rate of 1 microliter/min over 8 min. TRH infusions in 1.0-1000 microM concentrations elicited biphasic, dose-dependent anorectal contractions as measured by a rectal manometer. The 100 microM dose yielded the most significant increase in contractions over the greatest period of time. Atropine, administered as a pretreatment (100 micrograms s.c.), blocked contractions normally produced by i.t. infusion of TRH (1000 microM). Intravenous infusions of atropine (10 micrograms) through a jugular catheter immediately blocked anorectal contractions produced by i.t. infusion of 100 microM TRH. Sectioning of the hypogastric nerve, which supplies sympathetic innervation to the colon and internal anal sphincter, did not significantly affect contractions induced by 100 microM TRH applied intrathecally. Disruption of the major pelvic ganglion fibers, however, completely abolished the contractions induced by 100 microM TRH, either through the interruption of preganglionic parasympathetic fibers in the pelvic nerve, or by disrupting postganglionic fibers. These findings extend the role of TRH in the regulation of defecatory behaviors.
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Distribution of NADPH-diaphorase reactivity in the spinal cord of metamorphosing and adult Xenopus laevis.
The histochemical NADPH-diaphorase reaction has identified distinct neuronal populations in the nervous system of several species. Considerable evidence suggests that NADPH-d is a neuronal nitric oxide synthase (NOS). We examined spinal cords of adult and metamorphosing Xenopus laevis (XL) for developmental differences in NADPH-d reactivity. In adult XL, labeling was found in all dorsal root ganglia (DRGs) and in their termination sites within the dorsal horn (cutaneous afferent field) and intermediate gray (muscle afferent field). Cell bodies in the intermediate gray regions containing the autonomic preganglionic neurons were labeled in thoracic and sacral sections. Neurons located in the medial (MMC) and lateral motor columns (LMC) of the ventral horn were also stained. In metamorphosing XL, reactivity was detected in neurons in the intermediate gray, in the MMC and in the LMC as in the adult. Additionally, primary motoneurons including those innervating tail musculature were labeled. Neurons in the DRGs were stained at all stages; in the dorsal horn, the density of staining reflected the development of the sensory afferent fields. The conservation of NADPH-d reactivity in adult and metamorphosing XL spinal neurons suggests that NOS may be involved in processes independent of developmental changes occurring in XL spinal cord.
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A sensitive and reliable locomotor rating scale for open field testing in rats.
Behavioral assessment after spinal cord contusion has long focused on open field locomotion using modifications of a rating scale developed by Tarlov and Klinger (1954). However, on-going modifications by several groups have made interlaboratory comparison of locomotor outcome measures difficult. The purpose of the present study was to develop an efficient, expanded, and unambiguous locomotor rating scale to standardize locomotor outcome measures across laboratories. Adult rats (n = 85) were contused at T7-9 cord level with an electromagnetic or weight drop device. Locomotor behavior was evaluated before injury, on the first or second postoperative day, and then for up to 10 weeks. Scoring categories and attributes were identified, operationally defined, and ranked based on the observed sequence of locomotor recovery patterns. These categories formed the Basso, Beattie, Bresnahan (BBB) Locomotor Rating Scale. The data indicate that the BBB scale is a valid and predictive measure of locomotor recovery able to distinguish behavioral outcomes due to different injuries and to predict anatomical alterations at the lesion center. Interrater reliability tests indicate that examiners with widely varying behavioral testing experience can apply the scale consistently and obtain similar scores. The BBB Locomotor Rating Scale offers investigators a more discriminating measure of behavioral outcome to evaluate treatments after spinal cord injury.
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Distribution and morphology of sacral spinal cord neurons innervating pelvic structures in Xenopus laevis.
Relatively little is known about the organization of neural input to pelvic viscera in amphibia. In this study, sacral spinal efferent neurons were labeled in Xenopus laevis frogs by application of horseradish peroxidase (HRP) to the tenth spinal nerve, to pelvic musculature, or to the pelvic nerve. DiI was applied to the pelvic nerve with similar results. Labeled spinal neurons were located in the intermediate gray or in the ventral horn. Neurons in the tenth dorsal root ganglion, but not in the spinal cord, were labeled after application of HRP or DiI to the pudendal nerve. The labeled neurons in the spinal cord intermediate gray were in a position comparable to that of the mammalian sacral parasympathetic nucleus (SPN). Two apparent subdivisions included 1) a medial cluster of cells with mediolaterally oriented dendrites and 2) a lateral group with dorsoventrally oriented dendrites. An intermediate group, not clearly classed with the other two, was also identifiable. In some cases, labeled tenth nerve primary afferents were seen in contact with efferent neurons of the intermediate gray. Labeled neurons in the ventral horn medial to the lateral motor column were small, with dendrites oriented mediolaterally, in a position comparable to that of the mammalian Onuf's nucleus. The peripheral targets of DiI-labeled pelvic nerve axons were the compressor cloaca muscle, cloaca, and bladder. DiI-labeled pudendal nerve axons distributed peripherally to cloacal lip and medial thigh integument. These data suggest that the pudendal nerve in amphibians is purely sensory and that both somatic and autonomic motor axons traverse the pelvic nerve.
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Modeling of acute spinal cord injury in the rat: neuroprotection and enhanced recovery with methylprednisolone, U-74006F and YM-14673.
We used a new injury device that produces consistent spinal cord contusion injuries (T8) in rats to compare the behavioral and histologic effects of methylprednisolone sodium succinate (MPSS) administration, the clinical standard of therapy after acute spinal cord injury (ASCI), with the 21-aminosteroid, U-74006F (U74), and the TRH analogue, YM-14673 (YM), at different trauma doses. Three sequential experiments were conducted: Experiment 1. U74 (3.0/1.5/1.5 mg/kg; 10/5/5 mg/kg; 30/15/15 mg/kg), MPSS (30/15/15 mg/kg), or vehicle were administered intravenously (i.v.) at 5 min, 2 and 6 h after the injury (n = 8/group). U74 (10/5/5 mg/kg) and MPSS animals scored better than controls (Days 8-43) in open field walking (OFW); no other differences were seen between groups. Experiment 2. Dose-response evaluation of MPSS determined more effective doses. Groups (n = 16) receiving 30/30/30/30 mg/kg and 60/60/60/60 mg/kg i.v. at 5 min and 2, 4, and 6 h after the injury had better OFW scores than controls (Days 8-29; Day 29). Both groups performed better than controls (Days 8-29) on inclined plane (IP); 30 mg/kg animals scored higher on Day 29. Percentage tissue spared (%TS) at the lesion center was greater for 60 mg/kg animals (23.4%) than controls (17.3%). Experiment 3. Compounds were administered as in experiment 2 (n = 15/group); MPSS (60/30/30/30 mg/kg) and YM (1/1/1/1 mg/kg and 1 mg/kg/day ip) were most effective. YM and MPSS combination produced no additive effects. YM animals scored better than MPSS and control animals in OFW (Days 8-29) and better than controls on IP (Days 8-29; Day 29) and grid walking (Day 29). MPSS animals scored better than controls on IP (Days 8-29). YM and MPSS groups had greater %TS than controls. This series of experiments demonstrates the utility of this injury model and simple behavioral measures for preclinical assessment of pharmacologic agents. Under these experimental conditions, U74 demonstrated equivalent efficacy to MPSS, and YM demonstrated greater efficacy than MPSS in the treatment of ASCI.
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A comparison of YM-14673, U-50488H, and nalmefene after spinal cord injury in the rat.
A reproducible spinal cord injury model was used to compare the efficacy of three compounds previously shown to improve neurologic recovery after injury in rats: The thyrotropin releasing hormone (TRH) analogue, YM-14673; the specific kappa-opioid agonist, U-50488H; and the opioid antagonist, nalmefene, which has increased activity at kappa-receptors. A moderate injury in rats that results in recovery of uncoordinated gross locomotion was made at spinal T9 by rapid displacement (1.1. mm) of the cord. Compounds (or vehicle) were given either by intravenous bolus or by continuous mini-osmotic pump over 7 days, beginning 30 min after the injury as follows: controls (saline), YM-14673 (1 mg/kg bolus), U-50488H (10 mg/kg bolus), U-50488H (0.425 mg/kg/h continuous infusion x 7 days); nalmefene (0.1 mg/kg bolus); and nalmefene (0.021 mg/kg/h continuous infusion x 7 days). Neurologic recovery was assessed for 4 weeks by open-field walking, inclined plane, grid walking, and footprint analysis. The percentage of white matter spared was determined at the lesion epicenter. Only those groups given a bolus of YM14673, U-50488H, and nalmefene had open-field performance better than the scores of controls. Animals that received a bolus of YM-14673 also scored better than controls on the inclined plane and were more likely than controls to recover sufficiently to be tested by both grid walking and footprint analysis. Improved behavioral recovery was not found in groups that received chronic drug infusion. Histology demonstrated significant sparing of white matter for the YM-14673-treated group compared with controls; groups given a U-50488H and nalmefene bolus showed a trend for greater sparing of white matter. The results confirm a beneficial effect for these compounds and suggest that they may be useful in treatment of clinical spinal cord injury.
3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗
Evidence for alterations of synaptic inputs to sacral spinal reflex circuits after spinal cord transection in the cat.
Quantitative electron microscopy was used to study potential alterations in the synaptic inputs to HRP-labeled preganglionic neurons (PGNs) in the sacral parasympathetic nucleus (SPN) and to motoneurons (MNs) in Onuf's nucleus (ON) after short (4 days)- or longterm (10-11 weeks) spinal transection as compared to normal controls. Transection resulted in an apparent reorganization and replacement of synaptic input to ON MNs whereas chronic denervation of PGNs in the SPN was observed. These synaptic alterations may play a role in the changes in the eliminative reflexes (e.g., bladder-sphincter dyssynergia, induction of cutaneously elicited elimination) that are observed after spinal cord lesions.
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