Spinal cord contusion in the rat: behavioral analysis of functional neurologic impairment

A graded spinal cord injury in rats was produced by dropping a 10-g weight from 2.5, 5.0, 10,0, or 17.5 cm onto the exposed dura at the T8 vertebral level. Groups of rats (N = 10) for each of these weight drop (WD) levels as well as unoperated and WD controls (0 cm) were subjected to behavioral analysis that included evaluation of simple and complex reflexes as well as spontaneous and evoked motor patterns. On the basis of this analysis, we developed a protocol for evaluating functional deficits that follow spinal cord injury in the rat. The resulting combined behavioral score, a measure of functional deficit, closely correlated with the magnitude of the mechanical injury. The protocol used for neurologic assessment was administered routinely by personnel who were easily and rapidly trained. It should therefore prove useful in detecting the effects of treatment on recovery of function in a rat model of spinal cord injury.     

Spinal cord contusion in the rat: morphometric analyses of alterations in the spinal cord

Morphometric analyses were carried out on rat spinal cords which were injured by a weight drop technique. A 10-g weight was dropped 0.0, 2.5, 5.0, 7.5, 10.0, or 17.5 cm onto the dura which was exposed at the T8 vertebral level. Four weeks after injury, lesion volume, lesion length, and the dimensions of the tissue at the epicenter (lesion area, area of gray matter, and area of white matter) were measured and correlated with the height from which the weight was dropped and the results from tests of motor and sensory functional deficit. The results, based on linear regression analyses, indicated significant correlations between certain morphologic parameters (lesion volume, lesion length, and the area of gray and white matter at the epicenter) and both the height from which the weight was dropped and behavioral scores. Because the area of white matter at the epicenter is a very simple measurement which correlates well (r = 0.91) with behavioral outcome, this morphologic feature is a useful quantitative measure of the histopathologic consequences of spinal cord injury.     

Histological and functional evaluation of experimental spinal cord injury: evidence of a stepwise response to graded compression

Most experimental spinal cord injury studies described to date have relied on a limited number of injury gradations, and have tacitly assumed that outcome (functional, histological, and/or neurophysiological) is a monotonically graded function of injury severity. In contrast, the present study provides evidence that functional and morphological outcome after spinal cord compression injury may occur in a discontinuous, non-graded manner in response to linearly graded injury levels. The thoracic spinal cord of adult rats was transiently compressed to thicknesses from 1.8 to 0.8 mm in 0.2 mm steps, or sham injury was administered. Open field motor behavior and segmental reflexes were evaluated up to 21 days post injury and correlated with histological measures and injury level. The highest correlation was between histological outcome and open field motor scores. Among the six injury groups, only three significantly different outcomes were apparent in the open field, reflex, and histological measures, consisting of the injury group pairs 1.8/ 1.6, 1.4/1.2, and 1.0/0.8 mm. At day 21, the 1.8/1.6 mm injury groups were also indistinguishable from the sham injury group. The implications of these findings in terms of therapeutic studies are discussed. Comparison of the temporal outcome patterns among contusion and compression injuries in rats and other species also revealed a significant species difference: a period of delayed or secondary functional loss reported in theguinea pig was not present in the rat.     

An inexpensive apparatus for producing graded spinal cord contusive injury in the rat

We recently described an experimental model of spinal cord contusive injury in the rat in which a modified weight-drop apparatus is used to generate reproducible mild, moderate, or severe final functional deficit. We now report several inexpensive alternatives for portions of the original apparatus which allow the production of contusive injuries without the necessity of an expensive commercial stereotaxic frame or custom-machined clamp holders.     

Graded unilateral cervical spinal cord injury in the rat: evaluation of forelimb recovery and histological effects

The purpose of this study was to develop a model of unilateral cervical (C4-C5) spinal cord contusion injury in the rat and to characterize the functional and histological consequences following three injury levels using a new weight-drop spinal cord injury device. We evaluated forepaw/forelimb and hindlimb functions by: (1) a horizontal ladder beam measuring paw misplacements and slips; and (2) the forelimb preference test which measures the forelimb used for pushing off to rear, for support, and to land on after rearing. Rats with a mild spinal cord injury displayed primarily a forepaw deficit (forepaw misplacements) for 8 weeks after injury. Paw preference also improved after injury, but failed to reach control levels even after 12 weeks. These rats had damage primarily to the rubrospinal, spinocervicothalamic, and the uncrossed lateral corticospinal tracts in the dorsolateral funiculus a well as some loss of the lateral spinothalamic tracts in the lateral funiculus. Rats with a moderate injury had a prominent forepaw deficit still evident at 12 weeks after injury as well as a mild but not significant hindlimb deficit. Paw preference improved slightly 12 weeks. There was a larger lesion in the dorsolateral and lateral funiculi than in mildly injured rats which extended into the ventrolateral funiculi. There was a significant loss of gray matter compared to rats with a mild injury. Rats with a severe injury displayed significant forelimb and hindlimb deficits throughout the 12 week testing period compared to rats with a mild or moderate injury, and also had a more severe paw preference bias (90%). The lesion encompassed the entire dorsolateral, lateral and ventrolateral funiculi with some disruption of the ventral funiculus. There was more significant gray matter necrosis compared to rats with either a mild or moderate injury. Thus, the spinal cord injury device we used may be useful for studying graded cervical spinal cord injury in rats and potential treatments or interventions, because both the behavioral and histological effects are reproducible and consistent.     

Blocking weight-induced spinal cord injury in rats: effects of TRH or naloxone on motor function recovery and spinal cord blood flow

The ability of thyrotropin releasing hormone (TRH) or naloxone to reduce the motor function deficit and to improve the spinal cord blood flow (SCBF) was investigated in a rat spinal cord compression injury model. Spinal cord injury was induced by compression for 5 min with a load of 35 g on a 2.2 x 5.0 mm sized compression plate causing a transient paraparesis. One group of animals was given TRH, one group naloxone and one group saline alone. Each drug was administered intravenously as a bolus dose of 2 mg/kg 60 min after injury followed by a continuous infusion of 2 mg/kg/h for 4 h. The motor performance was assessed daily on the inclined plane until Day 4, when SCBF was measured with the 14C-iodoantipyrine autoradiographic method. It was found that neither TRH nor naloxone had promoted motor function recovery or affected SCBF 4 days after spinal cord 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.     

Increase in nerve growth factor-like immunoreactivity and decrease in choline acetyltransferase following contusive spinal cord injury

We have previously described a graded spinal cord injury model in the rat. Mild contusive injury results in an initially severe functional deficit that is attenuated over time to reveal the mild chronic deficits that characterize this injury. In this study, we have shown that mild contusive injury also results in a significant decrease in choline acetyltransferase (ChAT) activity during the first week after injury. At 1 week ChAT activity is maximally reduced at the site of the contusion and is also significantly lowered throughout the spinal cord. ChAT activity then rebounds during the following 3 weeks, partially at the injury site where there is considerable loss of gray and white matter, and completely in rostral and caudal cord segments. The rebound in ChAT activity is temporally associated with the partial recovery of function. Further, the changes in ChAT activity after injury are mirrored by changes in nerve growth factor-like immunoreactivity (NGF-LI) as determined by a specific two-site ELISA. NGF-LI increases significantly after injury, reaching a maximum at 7 days after contusion and at the injury site. However, levels of NGF-LI are also significantly increased throughout the spinal cord. NGF-LI then decreases at 2 and 4 weeks as ChAT activity rebounds. Further experiments will be needed to examine the possibility of a role for NGF in promoting the recovery of function after spinal cord injury.     

Effect of a single huge dose of methylprednisolone on blood flow,evoked potentials,and histology after acute spinal cord injury in the rat

Abstract

Effects ofa single, huge dose of methylprednisolone on post-traumatic spinal cord blood flow (SCBF), evoked potentials and histological changes were studied in a rat model ofspinal cord injury. The purpose of this study was to assess the optimal dose of methylprednisolone for the treatment of rat spinal cord injury. Twenty-five male Wistar rats were subjected to an acute clip compression injury at 51 g for 1 min at CB-T1, and then received an intravenous bolus injection of one of the following 30 min after injury: vehicle, 30, 60, 120 or 240 mg kg ^-1 methylprednisolone. SCBF was measured at the injury site and an adjacent area with the' hydrogen clearance technique. Sensory evoked potentials following sciatic stimulation were recorded from the somatosensory and cerebellar cortices. Descending volleys were recorded from T9-10 spinal cord following cerebellar stimulation. SCBF and evoked potential recordings were repeated until perfusion-fixation at 4 h after injury. After injury, SCBF at both levels significantly dropped, and all evoked potentials disappeared in all animals. None of the doses of methylprednisolone improved post-traumatic SCBF, or evoked potentials. Qua;ntitative histological assessment ,of the injured cords revealed no significant differences in hemorrhages or cavitation in the spinal cord among the treatment groups. This study showed that a single huge dose of methylprednisolone from 30 to 240 mg kg- ¹ had no beneficial effects on the traumatized rat spinal cord in the acute stage. [Neural Res 1997; 19: 289–299]     

Behavioral assessment of functional deficit in rats with contusive spinal cord injury

We have previously reported a method for assessing functional deficits in rats after contusive spinal cord injury in which a Combined Behavioral Score (CBS) is calculated that is indicative of the overall percent deficit (Exp. Neurol., 88: 123-134, 1985). The test battery used includes several neurologic tests as well as the Motor Score in which use of the hindlimbs in locomotion is graded. In this report we present correction criteria in order to reduce potential interlaboratory variability in assessing functional deficit by the CBS. Groups of rats were subjected to contusive injury using a weight drop technique. The calculated CBS at 4 weeks was compared to that obtained if the Motor Score was over- or underestimated by 1 grade. The results indicated a considerable effect on the calculated CBS, especially when the Motor Score was underestimated in mildly injured animals. Behavioral test data were examined in terms of the distribution of the responses to the various other behavioral tests in relation to the Motor Score. The results were used to develop a set of correction criteria that minimized the effects on the CBS of subjective errors in the Motor Score.     

大鼠脊髓冲击伤模型制作方法的改良探索

目的:探索一种制作稳定性强、重复性好的脊髓损伤模型的简易方法。方法:应用简易重物坠落打击装置建立大鼠脊髓中度损伤组(5g×8cm)、重度损伤组(5g×16cm)模型和假手术组,每组大鼠均n=10。通过运动功能评分法(BBB)评分、苏木精-伊红染色观察脊髓损伤后两组大鼠功能及病理的变化特点,以评价该制作方法的可靠性。结果:BBB评分显示两组大鼠后肢运动功能均有不同程度的恢复,但中度损伤大鼠的恢复明显优于重度损伤大鼠(P<0.05)。组织病理学观察显示脊髓损伤后脊髓结构紊乱,有胶质瘢痕及空洞形成,重度损伤组脊髓空洞面积明显大于中度组(P<0.05)。结论:该方法制作的大鼠脊髓模型能将不同打击力度造成的损伤区分开,并且模型的行为学与病理学结果相吻合,说明此模型具有良好的稳定性、重复性和一致性,适合脊髓损伤实验研究的应用。     

Experimental acute traumatic injury of the adult rat spinal cord by a subdural inflatable balloon: methodology, behavioral analysis, and histopathology.

We describe an experimental model to produce closed traumatic injuries to the spinal cord of adult rats. This model uses an inflatable balloon that is introduced in the dorsal subdural space and moved to a location rostral to the laminectomy site. The spinal cord trauma can be graded by varying either the duration of compression or the volume of saline used to inflate the balloon. The locomotor deficit of animals with various degrees of injury has been assessed at increasing delays after trauma. The parameters generating transient or definitive deficits of varying intensity were defined. Some injured animals underwent nuclear magnetic resonance imaging. Detailed histopathological studies demonstrated that the extent of the spinal lesion was significantly correlated with the physical parameters of compression and with the severity of the behavioral deficit.     

Spinal cord injury in rats: inability of nimodipine or anti-neutrophil serum to improve spinal cord blood flow or neurologic status

The role of a calcium-mediated increase in vascular resistance and of vascular damage caused by polymorphonuclear leukocytes (PMNLs) in the development of neurologic deficit and disturbance of spinal cord circulation following spinal cord compression was studied in the rat. Spinal cord injury was induced by 5 min of compression with a load of 35 g on a 2.2 X 5.0 mm compression plate. This caused transient paraparesis. The rats received either the calcium receptor antagonist nimodipine or an anti-rat neutrophil serum (ANS). Nimodipine was infused i.v. for 4 h in an amount of 1.5 micrograms/kg/min starting 60 min after trauma. The number of circulating PMNLs was depleted by intraperitoneal injection of an ANS raised in sheep given 12 h before trauma. This caused a reduction to about 2% of the pre-ANS value. Controls received saline or normal sheep serum. The motor performance was assessed daily on the inclined plane. On day one, the day after injury, the capacity angle had decreased from about 63 degrees preoperatively to close to 32 degrees in the experimental groups. There was then a slow improvement in both the control and experimental groups and on day 4 the capacity angle was close to 43 degrees in all 3 groups. Spinal cord blood flow, as measured with the 14C-iodoantipyrine autoradiography method, was similar in all groups on day 4. As neither the neurologic dysfunction nor the spinal cord blood flow was affected by post-trauma treatment with nimodipine or pretreatment with ANS, the possibility that calcium-mediated vasoconstriction or PMNLs play a role in the development of posttraumatic neurologic disability was not supported by this study.     

Therapeutic efficacy of Ac-DMQD-CHO, a caspase 3 inhibitor, for rat spinal cord injury.

We investigated the therapeutic efficacy of Ac-DMQD-CHO, a caspase-3 inhibitor, and functional recovery in spinal cord injury in a rat model. Thirty rats were randomized into three groups of 10 each. In groups 2 and 3, spinal cord trauma was produced in the thoracic region. Group 3 rats were treated with Ac-DMQD-CHO. Treatment responses were evaluated based on histopathological and TUNEL staining findings at 24 h and 5 days post-injury. Neurologic performance was assessed during and following treatment. Twenty-four hours after injury, light microscopy examination revealed diffuse hemorrhagic necrosis, edema, vascular thrombi, and polymorphonuclear leukocyte infiltration in group 2 and 3 rats, but cavitation and demyelinization were less prominent in group 3. At this time point, treatment of the rats with Ac-DMQD-CHO significantly reduced the number of apoptotic cells. Traumatic injury to the spinal cord causes apoptosis and administration of Ac-DMQD-CHO decreases apoptosis and improves functional outcome.     

Gene transfer of glial cell line-derived neurotrophic factor promotes functional recovery following spinal cord contusion

Neuronal cell death and the failure of axonal regeneration cause a permanent functional deficit following spinal cord injury (SCI). Administration of recombinant glial cell line-derived neurotrophic factor (GDNF) has previously been reported to rescue neurons following severe SCI, resulting in improved hindlimb locomotion in rats. In this study, thus, GDNF gene therapy using an adenoviral vector (rAd-GDNF) was examined in rats following SCI induced by dropping the NYU weight-drop impactor from a height of 25 mm onto spinal segment T9-T10. To evaluate the efficacy of intraspinal injection of recombinant adenovirus into the injured spinal cord, we observed green fluorescent protein (GFP) gene transfer in the contused spinal cord. GFP was effectively expressed in the injured spinal cord, and the most prominently transduced cells were astrocytes. The expression of GDNF was detected only in rats receiving rAd-GDNF, not the controls, and remained detectable around the injured site for at least 8 days. Open-field locomotion analysis revealed that rats receiving rAd-GDNF exhibited improved locomotor function and hindlimb weight support compared to the control groups. Immunohistochemical examination for the neuronal marker, calcitonin gene-related peptide (CGRP), showed an increase in CGRP+ neuronal fibers in the injured spinal cord in rats receiving rAd-GDNF treatment. Collectively, the results suggest that adenoviral gene transfer of GDNF can preserve neuronal fibers and promote hindlimb locomotor recovery from spinal cord contusion. This research should provide information for developing a clinical strategy for GDNF gene therapy.     

Acute transplantation of olfactory ensheathing cells or Schwann cells promotes recovery after spinal cord injury in the rat

We compared the neurological and electrophysiological outcome, glial reactivity, and spared spinal cord connectivity promoted by acute transplantation of olfactory ensheathing cells (group OEC) or Schwann cells (group SC) after a mild injury to the rat spinal cord. Animals were subjected to a photochemical injury of 2.5 min irradiation at the T8 spinal cord segment. After lesion, a suspension containing 180,000 OECs or SCs was injected. A control group (group DM) received the vehicle alone. During 3 months postsurgery, behavioral skills were assessed with open field-BBB scale, inclined plane, and thermal algesimetry tests. Motor (MEPs) and somatosensory evoked potentials (SSEPs) were performed to evaluate the integrity of spinal cord pathways, whereas lumbar spinal reflexes were evaluated by the H reflex responses. Glial fibrillary acidic protein and proteoglycan expressions were quantified immunohistochemically at the injured spinal segments, and the preservation of corticospinal and raphespinal tracts caudal to the lesion was evaluated. Both OEC- and SC-transplanted groups showed significantly better results in all the behavioral tests than the DM group. Furthermore, the OEC group had higher MEP amplitudes and lower H responses than the other two groups. At the injury site, the area of spared parenchyma was greater in transplanted than in control injured rats. OEC-transplanted animals had reduced astrocytic reactivity and proteoglycan expression in comparison with SC-transplanted and DM rats. Taken together, these results indicate that transplantation of both OEC and SC has potential for restoration of injured spinal cords. OEC grafts showed superior ability to reduce glial reactivity and to improve functional recovery.     

Hemorrhagic changes in experimental spinal cord injury models

Early hemorrhagic changes in the spinal cord were compared in three experimental spinal cord injury models in the rat in order to determine the nature and consistency of spinal cord hemorrhage following specific and quantitated forces of injury. The spinal cords were injured by weight-dropping, aneurysm clip and extradural balloon compression techniques. Hemorrhagic changes were assessed quantitatively by the image analyser at 1 and 3 hours after injury. Tissue damage was assessed by determining the percentage of total cross sectional area containing hemorrhage. The extent of hemorrhage at site of injury in the clip and balloon preparations was equal, but several times lower in the weight-drop induced injury. Within each experimental group no appreciable differences were observed at the site of injury between the 1 and 3 hours preparations. The variability of damage within experimental groups was most in the weight-dropping and balloon and least in the clip preparations. Differences were also indicated with respect to the distribution of hemorrhage in grey versus white matter. These findings may be of significance when functional recovery is considered in various experimental acute spinal cord injury models.     

Therapeutic efficacy of SJA6017, a calpain inhibitor, in rat spinal cord injury

Apoptosis is an important element of the secondary processes that occur after spinal cord injury. Calpain and caspases are key proteases in apoptotic cell death. We evaluated the neuroprotective effects of SJA6017 (a calpain inhibitor) and measured functional recovery in a rat spinal cord injury model. Thirty Wistar albino rats were divided into three groups of 10 animals each: sham-operated (group 1), trauma control (group 2) and trauma-plus-SJA6017 treatment (group 3). Spinal cord trauma was produced in the thoracic region of the animals. Rats in group 3 received SJA6017 1 min after trauma. Treatment efficacy was evaluated after injury using light microscopy and TUNEL staining. Neurological performance was assessed using an inclined plane and a modified version of the Tarlov's grading scale. Group 2 rats showed moderate trauma with widespread edema, hemorrhage, vascular thrombi and necrosis 24 h after injury. Group 3 rats had significantly reduced tissue injury and apoptosis. Tarlov scores revealed that group 3 rats also had ameliorated recovery of limb function. Our results demonstrate that treatment with SJA6017 reduces apoptotic cell death, preserves spinal cord tissue and improves functional outcome. Treating calpain-induced apoptosis with this agent may be a feasible therapeutic strategy for patients with spinal cord injury.     

神经干细胞与许旺细胞共移植于大鼠损伤脊髓

目的 观察神经干细胞与许旺细胞共移植于大鼠半横断脊髓损伤处神经干细胞的迁移、存活、分化及对损伤脊髓的修复作用.方法 绿色荧光蛋白(GFP)标记脊髓神经下细胞后与许旺细胞共移植于大鼠半横断脊髓损伤处,免疫荧光染色和电镜技术分别观察神经下细胞的迁移、存活、分化及新生的髓鞘.皮层运动诱发电位(CMEPs)及BBB评分分别检测大鼠运动功能的恢复.结果 在神经干细胞与许旺细胞共移植组,损伤脊髓的头端、尾端及对侧町见明显的GFP阳性细胞及GaLC/GFP、GFAP/GFP、NSE/GFP、SYN/GFP舣阳性细胞,电镜下新生的髓鞘最多,CMEPs恢复百分率和振幅明显高于其他两组,但BBB评分与神经干细胞单移植组差异无统计学意义.结论 神经干细胞和许旺细胞体内共移植可促进神经干细胞的辽移、存活、分化及脊髓运动功能的恢复.     

ApoE mimetic ameliorates motor deficit and tissue damage in rat spinal cord injury

Apolipoprotein E (apoE), a plasma protein responsible for transporting lipid and cholesterol, modulates responses of the central nervous system to injury. Small peptides derived from the receptor‐binding region of apoE can simulate some important bioactivities of apoE holoprotein and offer neuroprotection against brain injury. We tested whether COG1410, an apoE‐mimetic peptide, provides protection in a rat model of spinal cord injury (SCI). Traumatic injury was created at T8 by a cortical impact device. Injured rats were randomized to four treatment groups: vehicle, 0.15, 0.3, or 0.6 mg/kg COG1410; sham surgery rats received vehicle. Basso, Beattie, Bresnahan neurological score was evaluated prior to injury and at 1, 3, 7, and 14 days after injury. Histological changes were evaluated at 14 days. All injured rats lost body weight during the first week following injury. Body weight recovery was significantly improved in rats treated with COG1410. Mechanical impact resulted in severe motor deficit, and most animals had a BBB score of 0–1 at 24 hours postinjury. COG1410‐treated rats showed significantly improved functional recovery and ameliorated motor deficit at 14 days postinjury. Histological analysis showed that COG1410 groups had a significantly reduced lesion size at the site of injury, a larger preserved luxol fast blue‐stained area, and more visible neurons in the surrounding area of injury. Microglial activation was also significantly suppressed. These findings indicate that this apoE mimetic effectively improved neurological and histological outcome following SCI in rats, and the effect was associated with inhibition of microglial activation. © 2014 Wiley Periodicals, Inc.