Astrocytosis, microglia activation, oligodendrocyte degeneration, and pyknosis following acute spinal cord injury

Glial activation and degeneration are important outcomes in the pathophysiology of acute brain and spinal cord injury (SCI). Our main goal was to investigate the pattern of glial activation and degeneration during secondary degeneration in both gray matter (GM) and white matter (WM) following SCI. Adult rats were deeply anesthetized and injected with 20 nmol of N-methyl-D-aspartate (NMDA) into the ventral horn of rat spinal cord (SC) on T7. Animals were perfused after survival times of 1, 3, and 7 days. Ten-micrometer sections were submitted to immunocytochemistry for activated macrophages/microglia, astrocytes, oligodendrocytes, and myelin. Astrocyte activation was more intense in the vacuolated white matter than in gray matter and was first noticed in this former region. Microglial activation was more intense in the gray matter and was clear by 24 h following NMDA injection. Both astrocytosis and microglial activation were more intense in the later survival times. Conspicuous WM vacuolation was present mainly at the 3-day survival time and decreased by 7 days after the primary damage. Quantitative analysis revealed an increase in the number of pyknotic bodies mainly at the 7-day survival time in both ventral and lateral white matter. These pyknotic bodies were frequently found inside white matter vacuoles like for degenerating oligodendrocytes. These results suggest a differential pattern of astrocytosis and microglia activation for white and gray matter following SCI. This phenomenon can be related to the different pathological outcomes for this two SC regions following acute injury.     

Apoptosis of oligodendrocytes occurs for long distances away from the primary injury after compression trauma to rat spinal cord

We evaluated by in situ nick end labeling the presence of apoptotic glial cells in the spinal cord of rats which have sustained a moderate and severe compression injury at the level of T8–9, resulting in a severe but reversible paraparesis and irreversible paraplegia, respectively. In a previous investigation we found apoptotic glial cells (oligodendrocytes) in the immediate vicinity of the primary lesion (T7 and T10). The present study was designed to evaluate the extent of such cells in the spinal cord even at long distances away from the primary injury. Rats sustaining a moderate and severe compression injury and surviving 4 and 9 days showed a significant increase in the number of apoptotic glial cells at the T1, T5, T7, T12 and L2 levels. At the T10 level the elevation was significant only after day 9. There was no significant increase in the number of these cells at 4 h and 1 day after moderate and severe compression. In general, the apoptotic cells were most often seen in segments adjacent to the compression. They were randomly located in the ventral, lateral and dorsal tracts but were rarely present in the gray matter of the cord. In conclusion, compression trauma to rat spinal cord induces signs of apoptosis in glial cells, presumably oligodendrocytes of the long tracts. This newly discovered type of secondary injury is widely distributed in the damaged spinal cord and occurs even at long distances remote from the initial compression injury. Apoptotic cell death of oligodendrocytes will induce myelin degeneration and cause additional disturbances of axonal function. This cell damage may be a target for future therapy since it occurs after a delay and chemical compounds are now available by which apoptotic cell death can be modified. Received: 26 November 1998 / Revised, accepted: 22 April 1999     

Progesterone attenuates astro- and microgliosis and enhances oligodendrocyte differentiation following spinal cord injury

Reactive gliosis, demyelination and proliferation of NG2+ oligodendrocyte precursor cells (OPC) are common responses to spinal cord injury (SCI). We previously reported that short-term progesterone treatment stimulates OPC proliferation whereas chronic treatment enhances OPC differentiation after SCI. Presently, we further studied the proliferation/differentiation of glial cells involved in inflammation and remyelination in male rats with SCI subjected to acute (3 days) or chronic (21 days) progesterone administration. Rats received several pulses of bromodeoyuridine (BrdU) 48 and 72 h post-SCI, and sacrificed 3 or 21 days post-SCI. Double colocalization of BrdU and specific cell markers showed that 3 days of SCI induced a strong proliferation of S100β+ astrocytes, OX-42+ microglia/macrophages and NG2+ cells. At this stage, the intense GFAP+ astrogliosis was BrdU negative. Twenty one days of SCI enhanced maturation of S100β+ cells into GFAP+ astrocytes, but decreased the number of CC1+ oligodendrocytes. Progesterone treatment inhibited astrocyte and microglia /macrophage proliferation and activation in the 3-day SCI group, and inhibited activation in the 21-day SCI group. BrdU/NG2 double labeled cells were increased by progesterone at 3 days, indicating a proliferation stimulus, but decreased them at 21 days. However, progesterone-enhancement of CC1+/BrdU+ oligodendrocyte density, suggest differentiation of OPC into mature oligondendrocytes. We conclude that progesterone effects after SCI involves: a) inhibition of astrocyte proliferation and activation; b) anti-inflammatory effects by preventing microglial activation and proliferation, and c) early proliferation of NG2+ progenitors and late remyelination. Thus, progesterone behaves as a glioactive factor favoring remyelination and inhibiting reactive gliosis.     

Inducing inflammation following subacute spinal cord injury in female rats: A double-edged sword to promote motor recovery

The inflammatory response following spinal cord injury is associated with increased tissue damage and impaired functional recovery. However, inflammation can also promote plasticity and the secretion of growth-promoting substances. Previously we have shown that inducing inflammation with a systemic injection of lipopolysaccharide in the chronic (8 weeks) stage of spinal cord injury enhances neuronal sprouting and the efficacy of rehabilitative training in rats. Here, we tested whether administration of lipopolysaccharide in female rats in the subacute (10 days) stage of spinal cord injury would have a similar effect. Since the lesioned environment is already in a pro-inflammatory state at this earlier time after injury, we hypothesized that triggering a second immune response may not be beneficial for recovery. Contrary to our hypothesis, we found that eliciting an inflammatory response 10 days after spinal cord injury enhanced the recovery of the ipsilesional forelimb in rehabilitative training. Compared to rats that received rehabilitative training without treatment, rats that received systemic lipopolysaccharide showed restored motor function without the use of compensatory strategies that translated beyond the trained task. Furthermore, lipopolysaccharide treatment paradoxically promoted the resolution of chronic neuroinflammation around the lesion site. Unfortunately, re-triggering a systemic immune response after spinal cord injury also resulted in a long-term increase in anxiety-like behaviour.     

Peroxynitrite-induced oligodendrocyte toxicity is not dependent on poly(ADP-ribose) polymerase activation

Oligodendrocyte loss is a characteristic feature of several CNS disorders, including multiple sclerosis (MS) and spinal cord injury. However, the mechanisms responsible for oligodendrocyte destruction remain undefined. As recent studies have implicated peroxynitrite in the pathogenesis of both spinal cord injury and MS, we have examined whether peroxynitrite may mediate at least some of the oligodendrocyte damage and demyelination observed in these conditions. Primary rat oligodendrocytes were exposed to authentic peroxynitrite in vitro and assessed for cytotoxicity. Mitochondrial function, measured by the reduction of MTT to formazan, and mitochondrial membrane potential were used as indicators of cell viability. Cell death was quantitated by measuring either the release of lactate dehydrogenase from, or the uptake of propidium iodide into, damaged and dying cells. Peroxynitrite dose-dependently reduced the viability of primary oligodendrocytes and induced cell death. Furthermore, peroxynitrite significantly increased DNA strand breakage and the activity of poly(ADP-ribose) polymerase (PARP) in oligodendrocyte cultures. To identify whether PARP activation plays a role in peroxynitrite-induced oligodendrocyte toxicity, we examined the effects of the PARP inhibitors 3-aminobenzamide (3AB) and 5-iodo-6-amino-1,2-benzopyrone (INH(2)BP) on mitochondrial function and cell death in oligodendrocytes. The presence of 3AB and INH(2)BP did not protect oligodendrocytes from peroxynitrite-induced cytotoxicity. However, both compounds significantly reduced PARP activity in these cells. Primary oligodendrocytes generated from PARP-deficient mice were also highly susceptible to peroxynitrite-induced cell death. Therefore, our results show that peroxynitrite exerts cytotoxic effects on oligodendrocytes in vitro independently of PARP activation.     

Effects of BW755C, a mixed cyclo-oxygenase-lipoxygenase inhibitor, following traumatic spinal cord injury in rats

BW755C is an inhibitor of both cyclo-oxygenase and lipoxygenase, which has been found to have protective effects after myocardial ischemia in dogs. Impact injury to the spinal cord is associated with tissue ischemia as well as with the accumulation of eicosanoids. In the present studies we evaluated the effects of BW755C after traumatic spinal cord injury in rats. Drug treatment reduced thromboxane B2 levels and improved neurological recovery as compared to treatment with equal-volume physiological saline. The findings suggest that this drug or related compounds may be useful for the treatment of clinical spinal cord injury.     

Upregulation of type I interleukin−1 receptor after traumatic spinal cord injury in adult rats

Post-traumatic inflammation response has been implicated in secondary injury mechanisms after spinal cord injury (SCI). Interleukin-1 (IL-1) is a key inflammatory mediator that is increasingly expressed after SCI. The action of IL-1 is mediated through its functional receptor, type I interleukin-1 receptor (IL-1RI). However, whether this receptor is expressed after SCI remains to be elucidated. In the present study, the temporospatial expression of IL-1RI was detected in rats that received a moderate contusive SCI (a 10 g rod dropped at a height of 12.5 mm) at the ninth to tenth thoracic vertebral level using a widely used New York University impact device. Our study demonstrated that IL-1RI was slightly increased at 4 h post-injury compared to the normal or sham-operated controls, reached the peak at 8 h at mRNA level (4.44-fold, P<0.01) and 1 d at protein level (2.62-fold, P<0.01). IL-1RI remained at its elevated levels for a relatively long duration (4 h–7 days). Spatially, IL-1RI was observed throughout the entire length of a 10 mm-long cord segment containing the injury epicenter. Colocalization of IL-1RI was found in neurons, oligodendrocytes, astrocytes, and activated microglia. Our results suggest that the elevated expression of IL-1RI after SCI may contribute to posttraumatic inflammation responses of IL-1.     

Apoptotic death following Fas activation in human oligodendrocyte hybrid cultures

The purpose of this study was to determine how oligodendrocytes die following Fas receptor activation. An immortalized human oligodendrocyte hybrid line (MO3.13) was challenged with Fas ligand (FasL), and cell death was assessed by flow cytometry and DNA gel electrophoresis. Caspase activation was determined by either Western immunoblotting on cell extracts or by whole-cell flow cytometry. FasL challenge clearly induced substantial apoptotic cell death in the oligodendrocyte hybrid cell line, as judged by flow cytometry and by the presence of prominent low molecular weight DNA banding patterns after gel electrophoresis. Western immunoblots showed marked increases in cleaved caspase-1, 8, and 3, indicating that the extrinsic Fas death receptor-induced pathway was activated. The intrinsic mitochondrial pathway was also activated, but only at a minimal level. These findings demonstrate that there are several independent molecular sites within the extrinsic caspase cascade in oligodendrocytes where inhibitory compounds may be capable of blocking cell death in vivo.     

miR-138对脊髓全横断大鼠行为学的影响

目的探讨miR-138在脊髓全横断大鼠运动功能恢复过程中是否发挥调控作用。方法打开SD大鼠T9-T11椎板,脊髓内分别注射miR-138、DEPC处理水后制备脊髓全横断模型实验组与对照组。术后3d、65d取材,观察脊髓红肿范围、瘢痕长度;期间进行BBB运动功能评分;术后30d应用体感诱发电位(SEP)检测神经功能恢复情况。结果第5～9周miR-138组大鼠BBB评分明显低于对照组(P<0.05)。对照组后肢有运动的大鼠所占比例逐渐增多,而miR-138组该比例未见增加(27.03±16.2,6.5±11.72)(P<0.05)。SEP显示miR-138组大鼠左后肢均未测出潜伏期和波幅,右后肢潜伏期和波幅检出率均为16.7%,明显低于对照组(P<0.05)。术后3dmiR-138组脊髓横断处头端红肿长度较对照组增加(P<0.05)。术后65d脊髓横断处瘢痕明显,与周围软组织及椎骨粘连。miR-138组与对照组比较,瘢痕长度(1.07±0.21,1.03±0.12)cm及周长(1.00±0.17,1.05±0.09)cm均无显著性差异(P>0.05)。结论脊髓全横断大鼠局部应用miR-138后运动功能恢复较对照组...     

Expression of the type 1 and type 2 receptors for tumor necrosis factor after traumatic spinal cord injury in adult rats

Posttraumatic inflammation has been implicated in secondary tissue damage after spinal cord injury (SCI). Tumor necrosis factor-alpha (TNF-alpha) is a key inflammatory mediator that is increasingly expressed after SCI. The effect of TNF-alpha is mediated through its receptors TNFR1 (p55) and TNFR2 (p75). However, whether these two receptors are expressed after SCI has not been demonstrated. In the present study, the temporo-spatial expression of TNFR1 and TNFR2 was examined in rats that had received a 10 g impact injury dropped at a height of 12.5 mm using the New York University impact device. In sham operates, no detectable TNFR1 or TNFR2 immunoreactivity (IR) was observed. In contused spinal cord, TNFR1 protein expression and immunoreactivity (IR) were detected as early as 15 min postinjury, reached its peak at 8 h, and declined markedly after 1 and 3 days postinjury. The temporal pattern of TNFR2 expression was similar to that of TNFR1 but its expression peaked at 4 h postinjury. During peak expression, TNFR1- and TNFR2-IR were most intense at the site of injury and decreased gradually from the injury epicenter. TNFR1- and TNFR2-positive cells included neurons, astrocytes, and oligodendrocytes. Methylprednisolone (MP), a synthetic glucocorticoid, partially inhibited the injury-induced expression of TNFR1 and TNFR2, an effect which could be reversed by RU486, an antagonist of glucocorticoid receptors. We suggest that the expression of TNFR1 and TNFR2 after SCI may contribute to posttraumatic inflammatory responses of TNF-alpha.     

Systemic hypothermia following spinal cord compression injury in the rat: an immunohistochemical study on MAP 2 with special reference to dendrite changes

Systemic hypothermia has been shown to exert neuroprotective effects in experimental ischemic CNS models caused by vascular occlusions. The present study addresses the question as to whether systemic hypothermia has similar neuroprotective qualities following severe spinal cord compression trauma using microtubule-associated protein 2 (MAP2) immunohistochemistry combined with the avidin-biotin-peroxidase complex method as marker to identify neuronal and dendritic lesions. Fifteen rats were randomized into three equally sized groups. One group sustained thoracic laminectomy, the others severe spinal cord compression trauma of the T8-9 segment. The control group contained laminectomized animals submitted to a hypothermic procedure in which the esophageal temperature was reduced from 38 °C to 30 °C. The two trauma groups were either submitted to the same hypothermic procedure or kept normothermic during the corresponding time. All animals were sacrificed 24 h following the surgical procedure. The MAP2 immunostaining in the normothermic trauma group indicated marked reductions in MAP2 antigen in the cranial and caudal peri-injury zones (T7 and T10, respectively). This reduction was much less pronounced in the hypothermic trauma group. In fact, the MAP2 antigen was present in almost equally sized areas in both the hypothermic groups independent of previous laminectomy alone or the addition of trauma. Our study thus indicates that hypothermia has a neuroprotective effect on dendrites of rat spinal cords subjected to compression trauma. Received: 8 November 1999 / Revised: 7 January 2000 / Accepted: 11 January 2000     

Spinal versus brain microglial and macrophage activation traits determine the differential neuroinflammatory responses and analgesic effect of minocycline in chronic neuropathic pain

Substantial evidence indicates involvement of microglia/macrophages in chronic neuropathic pain. However, the temporal-spatial features of microglial/macrophage activation and their pain-bound roles remain elusive. Here, we evaluated microglia/macrophages and the subtypes in the lumbar spinal cord (SC) and prefrontal cortex (PFC), and analgesic-anxiolytic effect of minocycline at different stages following spared nerve injury (SNI) in rats. While SNI enhanced the number of spinal microglia/macrophages since post-operative day (POD)3, pro-inflammatory MHCII⁺ spinal microglia/macrophages were unexpectedly less abundant in SNI rats than shams on POD21. By contrast, less abundant anti-inflammatory CD172a (SIRPα)⁺ microglia/macrophages were found in the PFC of SNI rats. Interestingly in naïve rats, microglial/macrophage expression of CD11b/c, MHCII and MHCII⁺/CD172a⁺ ratio were higher in the SC than the cortex. Consistently, multiple immune genes involved in anti-inflammation, phagocytosis, complement activation and M2 microglial/macrophage polarization were upregulated in the spinal dorsal horn and dorsal root ganglia but downregulated in the PFC of SNI rats. Furthermore, daily intrathecal minocycline treatment starting from POD0 for two weeks alleviated mechanical allodynia most robustly before POD3 and attenuated anxiety on POD9. Although minocycline dampened spinal MHCII⁺ microglia/macrophages until POD13, it failed to do so on cortical microglia/macrophages, indicating that dampening only spinal inflammation may not be enough to alleviate centralized pain at the chronic stage. Taken together, our data provide the first evidence that basal microglial/macrophage traits underlie differential region-specific responses to SNI and minocycline treatment, and suggest that drug treatment efficiently targeting not only spinal but also brain inflammation may be more effective in treating chronic neuropathic pain.     

Reducing age-dependent monocyte-derived macrophage activation contributes to the therapeutic efficacy of NADPH oxidase inhibition in spinal cord injury

ObjectiveThe average age at the time of spinal cord injury (SCI) has increased to 43 years old. Middle-aged mice (14 months old, MO) exhibit impaired recovery after SCI with age-dependent increases in reactive oxygen species (ROS) production through NADPH oxidase (NOX) along with pro-inflammatory macrophage activation. Despite these aging differences, clinical therapies are being examined in individuals regardless of age based upon preclinical data generated primarily using young animals (∼4 MO). Our objective is to test the extent to which age affects SCI treatment efficacy. Specifically, we hypothesize that the effectiveness of apocynin, a NOX inhibitor, is age-dependent in SCI.MethodsApocynin treatment (5 mg/kg) or vehicle was administered 1 and 6 h after moderate T9 contusion SCI (50kdyn IH) and then daily for 1 week to 4 and 14 MO mice. Locomotor and anatomical recovery was evaluated for 28 days. Monocyte-derived macrophage (MDM) and microglial activation and ROS production were evaluated at 3 and 28 days post-injury.ResultsApocynin improved functional and anatomical recovery in 14 but not 4 MO SCI mice. Apocynin-mediated recovery was coincident with significant reductions in MDM infiltration and MDM-ROS production in 14 MO SCI mice. Importantly, microglial activation was unaffected by treatment.ConclusionThese results indicate that apocynin exhibits age-dependent neuroprotective effects by blocking excessive neuroinflammation through NOX-mediated ROS production in MDMs. Further, these data identify age as a critical regulator for SCI treatment efficacy and indicate that pharmacologically reduced macrophage, but not microglia, activation and ROS production reverses age-associated neurological impairments.     

At-level neuropathic pain is induced by lumbosacral ventral root avulsion injury and ameliorated by root reimplantation into the spinal cord

Neuropathic pain is common after traumatic injuries to the cauda equina/conus medullaris and brachial plexus. Clinically, this pain is difficult to treat and its mechanisms are not well understood. Lesions to the ventral roots are common in these injuries, but are rarely considered as potential contributors to pain. We examined whether a unilateral L6-S1 ventral root avulsion (VRA) injury in adult female rats might elicit pain within the dermatome projecting to the adjacent, uninjured L5 spinal segment. Additionally, a subset of subjects had the avulsed L6-S1 ventral roots reimplanted (VRA+Imp) into the lateral funiculus post-avulsion to determine whether this neural repair strategy elicits or ameliorates pain. Behavioral tests for mechanical allodynia and hyperalgesia were performed weekly over 7 weeks post-injury at the hindpaw plantar surface. Allodynia developed early and persisted post-VRA, whereas VRA+Imp rats exhibited allodynia only at 1 week post-operatively. Hyperalgesia was not observed at any time in any experimental group. Quantitative immunohistochemistry showed increased levels of inflammatory markers in laminae III-V and in the dorsal funiculus of the L5 spinal cord of VRA, but not VRA+Imp rats, specific to areas that receive projections from mechanoreceptive, but not nociceptive, primary afferents. These data suggest that sustained at-level neuropathic pain can develop following a pure motor lesion, whereas the pain may be ameliorated by acute root reimplantation. We believe that our findings are of translational research interest, as root implantation surgery is emerging as a potentially useful strategy for the repair of cauda equina/conus medullaris injuries.     

Suitability of allogeneic sertoli cells for ex vivo gene delivery in the injured spinal cord

Cell-based gene delivery for gene therapy offers the advantages of long-term stable expression of proteins without the safety concerns associated with viral vectors. However, issues of immune rejection prevent the widespread use of allogeneic cell implants. In this study, we determine if Sertoli cells, known for their immune privileged status, are suitable vehicles for allogeneic cell-based gene delivery into the injured spinal cord. As proof of concept, Sertoli cells were modified with recombinant adenovirus expressing enhanced green fluorescent protein (eGFP) or a human trophic factor, neurotrophin-3 (hNT-3), and eGFP. Genetically modified Sertoli cells retained their immunosuppressive ability in vitro, based upon lymphocyte proliferation assays, and were capable of generating biologically relevant levels of NT-3. Similarly, modified, allogeneic cells, implanted into the acutely injured spinal cord, reduced the early inflammatory response while producing significant levels of hNT-3 for at least 3 days after grafting. Moreover, these cells survived for at least 42 days after implantation in the injured cord. Together, these results demonstrate that Sertoli cells function in immunomodulation, can be engineered to produce bioactive molecules, and show long-term survival after implantation into the hostile environment of the acutely injured spinal cord. Such long-term survival represents an important first step toward developing an optimal cell-based delivery system that generates sustained expression of a therapeutic molecule.     

Evidence that local non-NMDA receptors contribute to functional deficits in contusive spinal cord injury

To investigate the role of (non-NMDA) types of excitatory amino acid (EAA) receptors in traumatic spinal cord injury, we administered 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)-quinoxaline (NBQX), a potent and specific antagonist of non-NMDA receptors, to rats with a standardized contusive spinal cord injury. Focal infusion of NBQX into the injury site significantly reduced long-term hindlimb functional deficits as well as decreasing the time required for the rats to establish a reflex bladder. The results suggest that non-NMDA receptors at or near the injury site are involved in producing a portion of the functional deficits that result from contusive spinal cord injury.     

Immune activation is required for NT-3-induced axonal plasticity in chronic spinal cord injury

After an unilateral lesion of the corticospinal tract (CST) at the level of the medulla over-expression of Neurotrophin-3 (NT-3) in lumbar spinal cord motoneurons induced axonal sprouting of the intact CST in the acutely injured but not uninjured or chronically injured spinal cord in rats. This suggested that processes associated with immune-mediated wound healing may act with NT-3 to induce neuroplasticity. To test whether immune processes were involved we measured NT-3-induced axonal sprouting in immunosuppressed compared to immunocompetent rats. Rats were immunosuppressed with anti-leukocyte antibodies 1 day before receiving a CST lesion and then 2 weeks later NT-3 was over-expressed in the lumbar spinal motoneurons with an adenoviral vector carrying the NT-3 gene targeted to the motoneurons by retrograde transport. At 35 days post-lesion no axonal sprouting was measured in immunosuppressed rats whereas axonal sprouting was measured in the immunocompetent rats. We then tested whether re-evoking an immune response in chronically lesioned rats would induce neuroplasticity. Rats received CST lesions and then 4 months later were treated with systemic injections of lipopolysaccharide (LPS) 7 days before NT-3 was over-expressed in the lumbar spinal motoneurons. Axonal sprouting was observed in the LPS treated rats but not in control animals that were not treated with LPS. Further studies showed that lesioning the CST activated and LPS reactivated microglia and CD4(+) T-cells in the acutely lesioned and chronically lesioned rats, respectively. However, immunosuppression only decreased the number of activated CD4(+) T-cells suggesting they were responsible for the support of axonal growth. These observations demonstrate that processes associated with immune-mediated wound healing play a role in NT-3-induced neuroplasticity after injury.     

Structure-activity relationships of TRH analogs in rat spinal cord injury

Effects of thyrotropin-releasing hormone (TRH) analogs were compared in rats to evaluate the structure-activity relationships of such compounds in the treatment of traumatic spinal cord injury. CG3703, a TRH analog having a modified amino-terminus, significantly improved motor recovery and somatosensory-evoked responses after trauma; in contrast, RX77368, which has a modified carboxy-terminus, was without effect, even at doses up to 10 mg/kg. These findings confirm and extend findings in cats, using other TRH analogs in a different model of spinal trauma. Together, data from rat and cat studies are consistent with the hypothesis that the integrity of the C-terminal amino acid may be critical for the beneficial effects of treatment with TRH and TRH analogs in experimental spinal injury, and suggest that a variety of other TRH analogs having substitutions of the pyroglutamyl or histidyl moieties of the tripeptide may also prove to be effective in the treatment of such injury.