Activation of coeruleospinal noradrenergic inhibitory controls during withdrawal from morphine in the rat

We previously reported that withdrawal from morphine induces the expression of Fos, a marker of neuronal activity, in spinal cord neurons, particularly in laminae I and II of the superficial dorsal horn, and that the magnitude of Fos expression is increased in rats with a midthoracic spinal transection. We suggested that loss of withdrawal-associated increases in descending inhibitory controls that arise in the brainstem underlie the increased Fos expression after spinal transection. Here, we addressed the origin of the supraspinal inhibition. We injected rats intracerebroventricularly with saline or anti-dopamine-beta-hydroxylase-saporin, a toxin that destroys noradrenergic neurons of the locus coeruleus. Eleven days later, we implanted rats with morphine or placebo pellets, and after 4 d, we precipitated withdrawal with naltrexone. One hour later, the rats were killed, their brains and spinal cords were removed, and transverse sections of the brains and spinal cords were immunoreacted with an antibody to Fos. In placebo-pelleted rats, the toxin injection did not alter behavior and did not induce expression of the Fos protein. However, compared with saline-injected withdrawing rats, the toxin-treated rats that underwent withdrawal demonstrated an intense withdrawal behavior rarely seen in the absence of toxin, namely forepaw fluttering. The rats also had significantly increased Fos-like immunoreactivity in all laminae of the cervical cord and in laminae I and II and the ventral horn of the lumbar cord. No differences were recorded in the sacral cord. We conclude that the effects of spinal transection in rats that withdraw from morphine in part reflect a loss of coeruleospinal noradrenergic inhibitory controls.     

Neuroimaging and pathology of the spinal cord in compressive cervical myelopathy

Magnetic resonance imaging (MRI) has enabled us to see the spinal intramedullary pathology as differences in signal intensity. Intramedullary high intensity lesions were observed on T2-weighted MRI in patients with cervical spondylotic myelopathy (20.0%) and ossification of the posterior longitudinal ligament (OPLL) of the cervical spine (25.7%). The frequency of this findings was proportional to the clinical severity of myelopathy and degree of spinal cord compression. The pathophysiological basis of such signal abnormality was presumed to vary from acute edema to chronic myelomalacia. The intramedullary lesion on MRI is considered to be the main site of lesion responsible for the neurological symptom because of a good correlation between the neurological level and high intensity level. We found from nine autopsy cases of OPLL that there are distinct differences in severity and extent of pathological changes between the spinal cord with a boomerang-shaped cross-section and that with a triangular-shaped cross-section. In the boomerang-shaped cases, major pathological changes were restricted to the gray matter and the white matter was relatively well preserved. Secondary wallerian degeneration was restricted to the fasciclus cuneatus the fibers of which were derived from the affected segments. In the cases of a triangular shape, pathological changes were more severe, both white and gray matter were involved. There were severe pathological changes over more than one segment, and both descending degeneration of the lateral pyramidal tracts and ascending degeneration of the posterior column, including the fasciclus gracilis, were observed. In conclusion, it is clinically very important to understand the pathological basis of the compressed spinal cord on neuroimages.     

MRI characterization of diffusion coefficients in a rat spinal cord injury model

Apparent diffusion coefficients (ADC) were measured in a rat spinal cord weight-drop injury model. After sacrifice, the spinal cords were fixed in situ and excised for MR imaging and ADC measurement. Diffusion is anisotropic in normal gray and white matter. There were significant decreases in ADCs measured along the longitudinal axis of the injured cord and increases in ADCs measured transverse to the cord. Injured segments demonstrated reductions in diffusion anisotropy in the white matter. Diffusion was completely isotropic at the epicenter of the weight-drop injury. Significant decreases in longitudinal ADC and increases in transverse ADC were observed in portions of the cord which appeared normal on conventional spin-echo and calculated T2 images. Thus ADC measurement may complement routine imaging for evaluation of spinal cord injury.     

Long-distance axonal regeneration in the transected adult rat spinal cord is promoted by olfactory ensheathing glia transplants

The lack of axonal regeneration in the injured adult mammalian spinal cord leads to permanent functional impairment. To induce axonal regeneration in the transected adult rat spinal cord, we have used the axonal growth-promoting properties of adult olfactory bulb ensheathing glia (EG). Schwann cell (SC)-filled guidance channels were grafted to bridge both cord stumps, and suspensions of pure (98%) Hoechst-labeled EG were stereotaxically injected into the midline of both stumps, 1 mm from the edges of the channel. In EG-transplanted animals, numerous neurofilament-, GAP-43-, anti-calcitonin gene-related peptide (CGRP)-, and serotonin-immunoreactive fibers traversed the glial scars formed at both cord-graft interfaces. Supraspinal serotonergic axons crossed the transection gap through connective tissue bridges formed on the exterior of the channels, avoiding the channel interior. Strikingly, after crossing the distal glial scar, these fibers elongated in white and periaqueductal gray matter, reaching the farthest distance analyzed (1.5 cm). Tracer-labeled axons present in SC grafts were found to extend across the distal interface and up to 800 microm beyond in the distal cord. Long-distance regeneration (at least 2.5 cm) of injured ascending propriospinal axons was observed in the rostral spinal cord. Transplanted EG migrated longitudinally and laterally from the injection sites, reaching the farthest distance analyzed (1.5 cm). They moved through white matter tracts, gray matter, and glial scars, overcoming the inhibitory nature of the CNS environment, and invaded SC and connective tissue bridges and the dorsal and ventral roots adjacent to the transection site. Transplanted EG and regenerating axons were found in the same locations. Because EG seem to provide injured spinal axons with appropriate factors for long-distance elongation, these cells offer new possibilities for treatment of CNS conditions that require axonal regeneration.     

Roles of ascending inhibition during two rhythmic motor patterns in Xenopus tadpoles

We have investigated the effects of ascending inhibitory pathways on two centrally generated rhythmic motor patterns in a simple vertebrate model, the young Xenopus tadpole. Tadpoles swim when touched, but when grasped respond with slower, stronger struggling movements during which the longitudinal pattern of motor activity is reversed. Surgical spinal cord transection to remove all ascending connections originating caudal to the transection (in tadpoles immobilized in alpha-bungarotoxin) did not affect "fictive" swimming generated more rostrally. In contrast, cycle period and burst duration both significantly increased during fictive struggling. Increases were progressively larger with more rostral transection. Blocking caudal activity with the anesthetic MS222 (pharmacological transection) produced equivalent but reversible effects. Reducing crossed-ascending inhibition selectively, either by midsagittal spinal cord division or rostral cord hemisection (1-sided transection) mimicked the effects of transection. Like transection, both operations increased cycle period and burst duration during struggling but did not affect swimming. The changes during struggling were larger with more rostral hemisection. Reducing crossed-ascending inhibition by spinal hemisection also increased the rostrocaudal longitudinal delay during swimming, and the caudorostral delay during struggling. Weakening inhibition globally with low concentrations of the glycine antagonist strychnine (10-100 nM) did not alter swimming cycle period, burst duration, or longitudinal delay. However, strychnine at 10-60 nM decreased cycle period during struggling. It also increased burst duration in some cases, although burst duration increased as a proportion of cycle period in all cases. Strychnine reduced longitudinal delay during struggling, making rostral and caudal activity more synchronous. At 100 nM, struggling was totally disrupted. By combining our results with a detailed knowledge of tadpole spinal cord anatomy, we conclude that inhibition mediated by the crossed-ascending axons of characterized, glycinergic, commissural interneurons has a major influence on the struggling motor pattern compared with swimming. We suggest that this difference is a consequence of the larger, reversed longitudinal delay and the extended burst duration during struggling compared with swimming.     

Three-dimensional analysis of the vascular system in the rat spinal cord with scanning electron microscopy of vascular corrosion casts. Part 1: Normal spinal cord

Vascular corrosion casts of polyester resin in the normal spinal cord at C4-C6 and C7-T1 were inspected three-dimensionally by scanning electron microscopy in 13 rats. Arteries and veins were easily differentiated by the impression pattern of endothelial nuclei on the casts. The centrifugal arterial system from the sulcal arteries supplied most of the gray and white matter in the ventral and lateral spinal cord. Each sulcal artery supplied only one side of the cord. The average number of sulcal arteries was 2.6 per mm. The centripetal arterial system from the posterior spinal arteries fed the posterior gray and white matter. In contrast with classical concepts, there was no pial arterial plexus on the ventral and ventrolateral surface except for infrequent transverse branches from the anterior spinal artery. In the posterior columns, two types of large veins were identified: the posterior medial septal veins and the posterior oblique veins that drained the posterior columns, medial posterior gray matter, and posterior gray commissure. The remainder of the gray and white matter was drained by the sulcal veins and the radial veins. This method clearly demonstrates the three-dimensional structure of both the arterial and venous system in the rat spinal cord.     

Brain-derived neurotrophic factor stimulates hindlimb stepping and sprouting of cholinergic fibers after spinal cord injury

Neurotrophic factors have been proposed as a therapeutic treatment for traumatic brain and spinal cord injury. The present study determined whether exogenous administration of one such factor, brain-derived neurotrophic factor (BDNF), could effect behavioral recovery and/or histopathological changes after spinal cord injury. Adult rats received a mild or moderate contusion injury or complete transection of the mid-thoracic spinal cord. Immediately thereafter, they were infused intrathecally with vehicle or BDNF for 28 days. Behavioral recovery was evaluated for 6 weeks after injury, at which time the rats were sacrificed and the spinal cord tissue was examined histologically. The infusion of BDNF resulted in acute stimulation of hindlimb activity. These effects included activation of alternating airstepping in injured rats when the hindlimbs were unloaded as well as slight improvements in the rate of recovery in open field locomotion score. BDNF infusion was also associated with enhanced growth of cholinergic fibers at the injury epicenter, but did not affect white matter sparing or density of serotonergic axons at or below the injury site. Based on immunohistochemical detection of BDNF protein distribution, these described effects are likely to be mediated by the activation of cells and axons within the central injury region and the along the peripheral rim of the spinal cord. Together, these findings demonstrate that the exogenous infusion of BDNF after spinal trauma can influence postinjury outcome through mechanisms that include acute stimulation of hindlimb activity and neuritogenesis at the injury site.     

Recent advances in the large-scale production of antibody fragments using lower eukaryotic microorganisms

BACKGROUND AND OBJECTIVES: Magnetic resonance microscopy (MRM) is a technique that is worthwhile for anesthesiologists because it allows spinal cord and plexus anatomy to be visualized three dimensionally and followed over time in the same animal. For example, the long-term effect of indwelling intrathecal or plexus catheters can be studied in situ, and convective and diffusive forces within intrathecal, epidural, or nerve sheath spaces can be investigated. Further, diffusion-weighted MRM, which measures an "apparent diffusion coefficient" (ADC), can be used to track the presence of ischemia, hypoperfusion, or cytotoxic edema. This study investigates problems associated with the use of in vivo MRM for spinal cord and peripheral nerve studies in the rat. METHODS: Twenty-one anesthetized female Fisher CDF rats were used. Group 1 (n=7) was used for anatomic three-dimensional studies. Groups 2 (n=4), 3 (n=4), and 4 (n=6) were used for measurements of the ADC. Group 2 served as controls, group 3 received lumbar intrathecal catheters, and group 4 received cervical intrathecal catheters. RESULTS: Cervical spine, lumbar spine, and spinal nerves and ganglia were accurately visualized with MRM. As a rule, spinal cord gray and white matter were better demonstrated using diffusion-weighted proton stains. By contrast, T2-weighted proton staining superiorly demonstrated structures surrounding the spinal cord. In groups 3 and 4, indwelling intrathecal catheters did not affect the spinal cord ADC, indicating normal blood flow and no cytotoxic edema. Contrast studies revealed nonhomogeneous distribution of contrast predominately in the lateral and ventral intrathecal space. CONCLUSION: Three-dimensional diffusion-weighted MRM displays cervical and lumbar spine anatomy accurately in vivo. Apparent diffusion coefficients measurements are feasible in rat cervical spinal cord with intrathecal catheters. Spinal cord ADCs are unaffected by intrathecal catheters, indicating normal spinal cord perfusion.     

Gait analysis of adult paraplegic rats after spinal cord repair

This study presents a novel detailed method of analysis of rat gait and uses this method to demonstrate recovery of forward locomotion patterns in adult rats made paraplegic by surgical spinal cord transection and subjected to a novel strategy for spinal cord repair. Six normal rats were compared to five animals in which the cord was transected at T8-T9, and a 5-mm segment of the spinal cord removed, and to seven animals in which, following spinal cord transection and removal of a spinal cord segment, multiple intercostal peripheral nerve bridges were implanted, rerouting pathways from white to gray matter in both directions. The implanted area was filled with fibrin glue containing acidic fibroblast growth factor. Details of the repair strategy have been published (H. Cheng, Y. Cao, and L. Olson, 1996, Science 273: 510-513). Gait analysis was carried out 3 and 4 months after surgery and once in the normal animals. Animals were allowed to walk across a runway with a transparent floor. Each test consisted of five trials, and each trial was videorecorded from underneath. Using frame-by-frame playback, individual footprints were then recorded regarding location and order of limb use, as well as step quality (degree of weight bearing, etc.). These data allowed measuring runway transit time, five different measures of step numbers, all possible temporal patterns of limb use, stride length, and base of support. Transected controls remained paralyzed in the hindlimbs with only occasional reflex hindlimb movements without weight bearing. Animals subjected to the full repair procedure were significantly faster than the controls, used their hindlimbs for 25-30% of the movements, and regained several of the specific limb recruitment patterns used by normal rats. Taken together, the gait analysis data demonstrate remarkable recovery of coordinated gait in the repaired animals, which was significantly better than controls for all relevant parameters, while at the same time clearly inferior to normal rats for most of the examined parameters. We conclude that normal rats use a multitude of interchangeable step sequence patterns, and that our spinal cord repair strategy leads to recovery of some of these patterns following complete spinal cord transection. These data suggest functionally relevant neuronal communication across the lesion.     

Effect of spinal cord transection on N-methyl-D-aspartate receptors in the cord

Spinal cord injury can lead to an exaggeration of transmission through spinal pathways, resulting in muscle spasticity, chronic pain, and abnormal control of blood pressure and bladder function. These conditions are mediated, in part, by N-methyl-D-aspartate (NMDA) receptors on spinal neurons, but the effects of cord injury on the expression or function of these receptors is unknown. Therefore, antibodies to the NMDA-R1 receptor subunit and binding of [3H]MK-801 were used to assess NMDA receptors in the spinal cord. Receptor density in rats with intact spinal cords was compared to that in rats 1 and 2 weeks after spinal cord transection (SCT) at the mid-thoracic level. At 1 and 2 weeks after SCT, [3H]MK-801 binding was reduced in most laminae in cord segments caudal to the injury, whereas no decrease in amount of R1 subunit immunoreactivity was observed. No significant changes in [3H]MK-801 binding and NMDA-R1 immunoreactivity could be seen rostral to the transection. Since [3H]MK-801 binding requires an open ion channel, the discrepancy between [3H]MK-801 binding and immunocytochemistry may indicate a loss of functional receptors without a consistent change in their total number. Therefore, the exaggerated reflexes that are well established in rats 2 weeks after cord injury must be mediated by a mechanism that withstands attenuation of NMDA receptor function.     

A histochemical investigation of catecholamines in spinal cord injury

The catecholamine hypothesis of progressive spinal cord necrosis following mechanical trauma was investigated with the histofluorometric method. Forty-four adult mongrel dog were examined as control, L1 crush-injured, and crush-injured with prior T1 total transection groups. In crush injured dogs, catecholamines were present in a 1 cm length of white matter at the crush site, with the greatest accumulation in the deep lateral and ventral funiculi. Gray matter fluorescence was not enhanced. Prior transection did not abolish the intense accumulation of catecholamines at the site of the cord injury. We propose that the catecholamines accumulating at the cord injured site are not central in origin, but represent an uptake mechanism into white matter as a reflection of cord microperfusion.     

SB209670, a potent endothelin receptor antagonist, prevents or delays axonal degeneration after spinal cord injury

We developed a rat spinal cord transection injury model and investigated whether endogenous endothelin takes part in axonal degeneration after injury, by using a potent nonselective endothelin receptor antagonist, SB209670. Light microscopic analysis showed that axonal degeneration of the spinal cord was clearly observed one week after injury, supported by immunohistochemical study with anti-neurofilament antibody. Electron microscopic observation showed enlargement and shrinking of spinal axons in the injured sites one week after injury. Application of SB209670 to the lesion sites markedly inhibited axonal damage after injury. These results suggest that endogenous endothelin plays a role in axonal degeneration after spinal cord injury and that SB209670 prevents or delays the axonal degeneration after CNS damage.     

An unusual spinal intradural arachnoid cyst

Spinal intradural arachnoid cysts are seen most frequently in the thoracic region, particularly near the midline posteriorly. A thoracic intradural arachnoid cyst in this typical location is reported, with the additional unusual finding of herniation of the spinal cord through an anterior defect in the dura matter. The MRI findings are described.     

Shrinking spinal cord following transverse myelopathy in a patient with systemic lupus erythematosus and the phospholipid antibody syndrome

A 38-year-old woman with systemic lupus erythematosus and the phospholipid antibody syndrome was admitted because of rapidly evolving symptoms consistent with a transverse myelopathy at the TH9/10 level. Magnetic resonance imaging (MRI) showed slight diffuse swelling and increased signal intensity of the spinal cord. She was treated with high dose methylprednisolone plus azathioprine and aspirin. Four months later she had achieved almost complete remission with minimal residual sphincter disturbances. Despite the clinical recovery, repeated MRI at 4 months and 4 years showed diffuse and irreversible atrophy of the entire spinal cord.     

Sensory neurons regenerate more dominantly than motoneurons during the initial stage of the regenerating process after peripheral axotomy

This study was designed to determine whether sensory neurons or motoneurons were dominant during the earlier stage of the regeneration process after peripheral axotomy. After transection of the right sciatic nerves of rats, epineurial end neurorrhaphy was performed. At 5, 7 and 14 days postoperatively, the nerves were re-transected at the positive pinch site, and their proximal stumps were exposed to the retrograde neurotracer, Fluoro-Gold (F-G). Seventy-two hours later, the lumbar spinal cords and the L4 and L5 dorsal root ganglia (DRG) were harvested and evaluated. The incidence and the intensity of F-G labelling in DRG were significantly higher than in anterior horns (AH). These results demonstrated that sensory neurons were more dominant than motoneurons in nerve regeneration.     

Quantitative evaluation of calcitonin gene-related peptide and substance P levels in rat spinal cord following peripheral nerve injury

Levels of calcitonin gene-related peptide immunoreactivity (CGRP-ir) and substance P immunoreactivity (SP-ir) in the lumbar dorsal spinal cord of rats with either sciatic nerve transection or chronic constriction injury (CCI) were measured using radioimmunoassay. Significant decreases in CGRP-ir and SP-ir occurred in the ipsilateral spinal cord at 10 and 31 days after nerve transection. An ipsilateral decrease in SP-ir occurred 60 days after CCI. In addition, contralateral decreases in CGRP-ir and SP-ir occurred 31 days after transection and 60 days after CCI. Transection of the sciatic nerve produced greater decreases in peptide levels than did the CCI. Changes in spinal levels of these peptides may be involved in the appearance of neuropathic signs associated with nerve injury.     

Microglial turnover in the injured CNS: activated microglia undergo delayed DNA fragmentation following peripheral nerve injury

Microglial proliferation and activation are common events in the injured CNS. The mechanisms, however, by which activated microglia are eliminated following a pathological stimulus are still poorly understood. The present study has therefore examined microglial proliferation by 3H-thymidine autoradiography and programmed cell death by terminal transferase-mediated nick end labeling (TUNEL) and in situ end labeling (ISEL) of nuclear DNA fragments in two models of peripheral nerve injury, i.e. sciatic and hypoglossal nerve transection in the rat. In these models, microglial activation and proliferation occur in CNS projection areas, i.e. in the ventral and dorsal gray matter of lumbar spinal cord and in the nucleus gracilis after sciatic nerve transection as well as in the axotomized hypoglossal nucleus. At these sites, microglial proliferation had a relatively sharp peak between days 2 and 3 post-lesion and then rapidly declined. DNA fragmentation was detected in lectin (GSI-B4)-positive microglia from day 6 after axotomy onward, reached an apparent peak at day 21 and was downregulated by day 60, i.e. the latest time point investigated. However, the expression of bcl-2 and c-myc, i.e. genes potentially controlling programmed cell death, was found to be unchanged during this period. Programmed cell death thus appears to be one mechanism by which activated microglia are gradually eliminated following CNS injury and steady state of microglial cell numbers is achieved in vivo. Expression of microglial growth factors may be instrumental in controlling these processes.     

Peripheral nerve insult induces NMDA receptor-mediated, delayed degeneration in spinal neurons

Injury of a peripheral nerve gives rise to adaptive functional and structural alterations in spinal neurons. We report that the rearrangement of the spinal circuitry in response to sciatic nerve transection in adult rats involves a delayed mode of degeneration of lumbar spinal cord neurons. Nuclear fragmentation was detected by the TUNEL technique 7 days after sciatic neurectomy but not after 3 or 14 days. Dying cells were preferentially located in the ipsilateral superficial dorsal horn and expressed the neuronal cytoskeletal marker SMI-31. Degeneration was prevented by continuous systemic treatment with the NMDA receptor-antagonist MK-801. These data are supportive that apoptosis is induced in spinal neurons in a transsynaptic manner by an early signal from injured afferent fibres via activation of spinal NMDA receptors.     

Magnetic resonance imaging study on the results of surgery for cervical compression myelopathy

The morphologic changes and signal intensity of the spinal cord on preoperative magnetic resonance images were correlated with postoperative outcomes in 74 patients undergoing decompressive cervical surgery for compressive myelopathy. The transverse area of the spinal cord on T1-weighted images at the level of maximum compression was closely correlated with the severity of myelopathy, duration of disease, and recovery rate as determined by the Japanese Orthopaedic Association score. In patients with ossification of the posterior longitudinal ligament or cervical spondylotic myelopathy, the increased intramedullary T2-weighted magnetic resonance imaging signal at the site of maximal cord compression and duration of disease significantly influenced the rate of recovery. A multiple regression equation was then developed with these three variables to predict surgical outcomes.     

Spasticity in rats with sacral spinal cord injury

We have investigated sacral spinal cord lesions in rats with the goal of developing a rat model of muscular spasticity that is minimally disruptive, not interfering with bladder, bowel, or hindlimb locomotor function. Spinal transections were made at the S2 sacral level and, thus, only affected the tail musculature. After spinal transection, the muscles of the tail were inactive for 2 weeks. Following this initial period, hypertonia, hyperreflexia, and clonus developed in the tail, and grew more pronounced with time. These changes were assessed in the awake rat, since the tail is readily accessible and easy to manipulate. Muscle stretch or cutaneous stimulation of the tail produced muscle spasms and marked increases in muscle tone, as measured with force and electromyographic recordings. When the tail was unconstrained, spontaneous or reflex induced flexor and extensor spasms coiled the tail. Movement during the spasms often triggered clonus in the end of the tail. The tail hair and skin were extremely hyperreflexive to light touch, withdrawing quickly at contact, and at times clonus could be entrained by repeated contact of the tail on a surface. Segmental tail muscle reflexes, e.g., Hoffman reflexes (H-reflexes), were measured before and after spinalization, and increased significantly 2 weeks after transection. These results suggest that sacral spinal rats develop symptoms of spasticity in tail muscles with similar characteristics to those seen in limb muscles of humans with spinal cord injury, and thus provide a convenient preparation for studying this condition.