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     

Morphology of dendrite bundles in the cervical spinal cord of the rat: a light microscopic study

Histological staining techniques and Golgi-Cox impregnation revealed three discrete dendrite bundles in the ventral horn of the rat cervical spinal cord. A midline dendrite bundle (MDB) traversed the ventromedial gray matter (C3-6), a central dendrite bundle (CDB) coursed the medial aspect of the ventral horn (C3-5), and a lateral dendrite bundle (LDB) traveled in the ventrolateral gray matter (C2-4). At the light microscopic level, the three dendrite bundles were composed of longitudinally oriented intertwined dendrites that coursed in close apposition among motoneuron perikarya, neuroglia, and capillaries. A gradient of packing density of dendrites in the bundles existed, the MDB displaying the greatest packing density and the LDB forming the most loosely interwoven dendritic plexus. Dendrites contributing to the bundles originated from several different motoneuron pools. Smaller transverse dendrite bundles radiated from the longitudinal dendrite bundles at right angles and appeared to interconnect the MDB, CDB, and LDB. Transverse dendrite bundles also exited the MDB and LDB to course into the anterior and lateral funiculi, respectively. The presence of dendrite bundles among fields of motoneurons suggests that dendrite bundles may provide an anatomical substrate for the synchronization of neuronal activity for coordination of muscle groups involved in particular movements. Dendrite bundles also would provide a means whereby functionally similar motoneurons can receive and integrate similar synaptic inputs, and thus allow these inputs to modulate and coordinate groups of neurons that act as a functional unit. The presence of transverse dendrite bundles interconnecting the longitudinal bundles may permit the fine tuning of motoneuron activity for better coordination of movements involving synergistic and antagonistic muscle groups.     

Fluororuby as a marker for detection of acute axonal injury in rat spinal cord

Axonal damage is a common pathological consequence of spinal cord injury. Previous studies have detected axonal injury with silver stains for degeneration or immunohistochemistry for alterations in components such as beta-amyloid precursor protein, neurofilament or ubiquitin. Fluororuby has recently been introduced as a neuronal tracer in studies of spinal cord injury and regeneration. Our study was carried out to determine whether Fluororuby can be used to identify injured axons and monitor the time course of axonal damage. Adult rats underwent needle puncture injury to the white matter in the midline and lateral spinal cord at T11. At the same time, 0.05 microl of Fluororuby was injected into the cord at the same sites. After survival times ranging from 6 h to 3 weeks, spinal cords were cut into longitudinal frozen sections and examined with confocal microscopy. Fluororuby was found to label key features of axonal injury including axonal swelling, retraction balls and disrupted axons. Damaged axons close to the injury site were consistently labeled within 6 h, with indications of swollen and disconnected axons spreading further from the site during the first week. Fewer injured axons were labeled after 1 week survival, but the marker revealed longer distances of degenerating axons both distal and rostral to the injury site. Our findings indicate that Fluororuby is a quick, sensitive, reliable and technically simple fluorescent marker for early stages of acute axonal injury and degeneration.     

Heterogeneous astroglial response in the rat spinal cord to long-term portacaval shunt: an immunohistochemical study.

Glial fibrillary acidic protein (GFAP) immunoreactivity has been used to study the astroglial response in the rat spinal cord to long-term portacaval shunt (PCS). The astroglial response in PCS rats is heterogeneous. In general, astrocytes show a loss of GFAP immunoreactivity, as well as shrinking and pyknosis in their nuclei; however, while GFAP reactivity was unchanged in the periependymal region, it was strongly increased in the dorsolateral region of the spinal cord (lateral spinal nucleus, dorsal root entry zone, and the most dorsal region of the dorsal horn). Three possibilities are postulated to explain how astrocytes, in the periependymal and dorsolateral regions, can support the effects of PCS: a) astrocytes related to glutamatergic pathways ought to possess a more efficient ammonia uptake and detoxification system, b) long-term PCS can activate nociceptive pathways (substancePergic fibers), and c) astrocytes located in periependymal and dorsolateral regions can be exposed to lower concentrations of ammonia because of its diffusion into the cerebro-spinal fluid close to these regions.     

Microtubule-associated protein 2 appears in axons of cultured dorsal root ganglia and spinal cord neurons after rotavirus infection

The immunohistochemical distribution of microtubule-associated protein 2 (MAP2), being normally restricted to nerve cell bodies and dendrites, became altered in rat dorsal root ganglia and spinal cord neurons in cultures infected with rhesus rotavirus. MAP2 appeared in axons of both sources of neurons as displayed with monoclonal antibodies to MAP2a+b and MAP2a+b+c at 48 hr post-infection (p.i.). Other cytoskeletal elements, i.e., tau, MAP1, MAP5, neurofilament, actin, and tubulin, did not reveal any alterations in the rotavirus-infected neurons. One of the rotavirus cytosolic proteins, the inner capsid protein vp6, was expressed in axons at 48 hr p.i. simultaneously with the appearance of MAP2, while two other viral proteins, vp4 and NS28, remained in the nerve cell bodies. By quantitative enzyme-linked immunosorbent assay (ELISA) a binding of single-shelled rotaviruses, which express vp6 on their surfaces, to purified MAP2 was found. There was no binding of these viral particles to tau or tubulin proteins. This study indicates that a selective interaction between certain viral and neuronal cytoskeletal proteins can occur and that a non-cytolytic viral infection can cause alterations in the polarized sorting of neuronal proteins. © 1993 Wiley-Liss, Inc.     

Termination patterns of serotoninergic medullary raphespinal fibers in the rat lumbar spinal cord: an anterograde immunohistochemical study

Electrical and chemical stimulation given in the ventral medullary raphe nuclei inhibits spinal nociceptive reflexes and spinal nociceptive transmission; serotoninergic receptors have been demonstrated to partially mediate that inhibition. In the present study, the termination patterns of raphespinal fibers in the rat lumbar spinal cord demonstrating serotonin-like immunoreactivity were examined by using the anterograde tracer Phaseolus vulgaris leucoagglutinin (PHA-L) in combination with immunohistochemistry. Fibers and terminations from the ventral medullary raphe nuclei (raphe magnus and raphe pallidus) demonstrating both PHA-L- and serotonin-like immunoreactivity were identified in all laminae of the dorsal horn and the ventral horn. Networks of large fibers, characterized by large boutons, and which did not demonstrate serotonin-like immunoreactivity, were identified in deeper laminae of the dorsal horn. The heterogeneous morphology of raphespinal fibers identified in the dorsal horn suggests that these fibers also may be heterogeneous in neurochemistry and function. Medial medullary sites outside the raphe nuclei were found to innervate the ventral horn and all laminae of the dorsal horn, with the exception of lamina I. Descending fibers and terminations also demonstrating serotonin-like immunoreactivity were identified in deep laminae (III, IV, V, VI) of the dorsal horn and in the ventral horn. Similarly, large fiber networks were identified which did not demonstrate serotonin-like immunoreactivity.     

Development of serotonin immunoreactivity in the rat spinal cord and its plasticity after neonatal spinal cord lesions

The postnatal maturation of spinal pathways may account for the gradual time course of postnatal development of behavior and also account for the greater anatomical reorganization which often follows damage to the developing CNS compared to the mature CNS. The purpose of the current study was to examine (1) the prenatal and postnatal development of the descending serotonergic (5-HT) projection to the spinal cord and (2) the effects of a neonatal spinal cord lesion on this development. In addition, we wished to determine (3) whether transplants of fetal spinal cord tissue placed into the neonatal lesion site alter the plasticity of the 5-HT projection to the cord. Peroxidase-antiperoxidase immunocytochemical techniques were used. At embryonic day 14 (E14), no 5-HT immunoreactive fibers could be identified at any spinal cord level. By E18 the first axons were identified in the white matter only at all spinal cord levels. At birth, 5-HT immunoreactive fibers were present both in the white matter and in the gray matter at all cord levels. The projection within the gray matter was diffuse and considerably less dense than in the adult. The postnatal maturation of the 5-HT projection within the gray matter of the spinal cord followed rostral to caudal and ventral to dorsal gradients. During the first weeks postnatal, the 5-HT immunoreactivity within the cord increased to attain an adult pattern and density by 14 days in the cervical cord and 21 days in the thoracic and lumbar cord. The effect of a spinal cord hemisection at birth on the anatomical reorganization of the descending serotonergic innervation of the cord was compared with the effect of the same lesion in the adult. In the adult animal, mid-thoracic hemisection decreased the 5-HT content of the ventral horn of the lumbar spinal cord caudal and ipsilateral to the lesion to 8% of that on the intact side. When this same lesion was made in the newborn animal, the innervation was 43% of that on the intact side. When a transplant of fetal spinal cord tissue was inserted into the lesion site in the newborn animals, there was even greater 5-HT innervation caudal to the lesion, 83% of that on the intact side. These results indicate that there is considerable postnatal development and plasticity of the descending serotonergic projection to the spinal cord, and this plasticity is enhanced by the presence of a spinal cord transplant at the site of the lesion.     

Development of an astrocytic response to lesions of the spinal cord in the north american opossum: An immunohistochemical study using anti-glial fibrillary acidic protein

We have shown previously that rubral axons grow around a lesion of their pathway in developing opossums and that a critical period exists for that plasticity. The critical period begins when rubral axons first reach the level of the lesion and ends sometime between postnatal days (PD) 26 and 30. The aim of the present study was to examine the development of an astrocytic response to lesioning the spinal cord to determine if there is a temporal correlation between the development of such a response and the end of the critical period. The astrocytic response was examined immunohistochemically, 2 and 4 weeks after hemisecting the thoracic spinal cord, using an antibody to glial fibrillary acidic protein (GFAP). A response was first seen at PD21 in the 2-week series. The response was relatively mild, however, and limited to the white matter. When the lesion was made at PD26. the response was still restricted to the white matter, but hypertrophied astrocytes were found at the gray/white matter junction and cystic cavities were present. When the lesion was made at PD41, the response had spread to the gray matter and it occupied a larger area rostral and caudal to the lesion than at earlier ages. The animals allowed to survive 4 weeks after lesioning were subjected to a second operation 4-5 days before sacrifice so that Fast Blue could be injected bilaterally two to three segments caudal to the lesion. When the hemisection was made at PD15, a response was present in the ventral and ventrolateral funiculi, but not in that part of the lateral funiculus that contains rubrospinal axons. As expected from previous studies, rubral neurons were labeled contralateral to the lesion, suggesting that their axons had grown around it. When the lesion was made at PD21, the glial response was still limited to the white matter, but it extended into that part of the lateral funiculus that contains rubral axons. In spite of the presence of a glial response, rubral neurons were still labeled contralateral to the lesion. When the lesion was made at PD26, hypertrophied astrocytes were present at the gray/white matter junction and small cavities were noted at the lesion site. In such cases, there was no evidence for rubrospinal plasticity. An astrocytic response was not observed in the gray matter of the dorsal horn, an area used by rubral axons to grow around a lesion of their pathway, until well after the end of the critical period. We conclude that the initial development of a glial scar in the white matter after lesioning does not determine the end of the critical period for rubrospinal plasticity. Loss of rubrospinal plasticity correlates most closely with the appearance of hypertrophied astrocytes at the gray/white matter junction and the formation of cystic cavities.     

Serotoninergic medullary raphespinal projection to the lumbar spinal cord in the rat: a retrograde immunohistochemical study.

Classically the raphespinal system has been regarded as a serotoninergic system; inhibition of spinal nociceptive transmission produced by stimulation of the medullary raphe nuclei is mediated partially by spinal serotoninergic receptors. However, recent evidence suggests that the raphe nuclei are not homogeneous populations of serotoninergic cells. The objective of the present study was to re-examine, in the rat, the serotoninergic raphespinal projection to the lumbar spinal cord, and to determine the relative contribution of serotoninergic raphespinal neurons to the total population of raphespinal neurons. Microinjections of wheat-germ agglutinin horseradish peroxidase conjugate coupled to colloidal gold into the lumbar spinal cord resulted in the retrograde labeling of 53% and 59% of the serotoninergic neurons in the raphe nuclei and in the para-raphe zone, respectively. Conversely, 47% and 28% of the retrogradely labeled neurons in the raphe and para-raphe zone, respectively, demonstrated serotonin-like immunoreactivity. Thus, contrary to previous reports, the present results suggest 1) that only about half of the serotoninergic neurons in the raphe nuclei and in the surrounding para-raphe zone project to the lumbar spinal cord, and 2) that a large proportion of the neurons in the raphe nuclei (53%) and in the surrounding para-raphe zone (72%) that project to the lumbar spinal cord are not serotoninergic.     

Early dendrite development in spinal cord cell cultures: A quantitative study

Neurons in dissociated cell culture provide a favorable system for the quantitative analysis of structural changes and the examination of structure-function relationships during development. Fragment C of tetanus toxin was used to label neurons in murine spinal cord cell cultures and dendrite outgrowth was monitored by a number of measures. The dissociated neurons increased in morphologic complexity from approximate spheres to highly branched structures during the first week in culture. Much of the structural complexity of the dendrite arbor, as quantified by fractal dimension, was established within 48 hr after plating, i.e., prior to the development of interneuronal contacts. During the first few days in culture, dendrite branching complexity increased more rapidly than dendrite size, whereas after 4 days, fractal dimension remained relatively constant while dendrites continued to grow. Fractal analysis has provided data which suggest that the early development of dendrite branching complexity is determined intrinsically. Fractal dimension, as an effective index of morphologic complexity, should be a useful tool for the further study of extrinsic signals which might modify the generation or stabilization of dendrite form. © 1993 Wiley-Liss, Inc.     

Pre- and postsynaptic localization of RC3/neurogranin in the adult rat spinal cord: an immunohistochemical study

RC3 (neurogranin; BICKS) is a neuron-specific calmodulin-binding protein kinase C substrate. Thus far, immunohistochemical studies on the localization of RC3 revealed its presence in all neuronal phenotypes, which were restricted to specific areas in the neostriatum, the neocortex, and the hippocampus. RC3 was mostly found in cell bodies and dendrites, with some infrequent presence in axonal profiles, i.e. in the internal capsule. Until now, RC3 expression was reported to be absent in the adult rat spinal cord. RC3 might, however, act as an intermediate of protein kinase C-mediated signaling pathways during synaptic development and plasticity. We hypothesized a role for this 78-amino-acid protein in dendritic plasticity occurring after spinal cord injury. To our surprise, an immunohistological analysis of the uninjured adult rat spinal cord revealed the presence of RC3-positive cell bodies and dendrites in specific regions in the gray matter. Interestingly, axon-containing structures, such as the dorsal and ventral corticospinal tract, were also found to be RC3-positive. This axonal labeling was confirmed by preembedding electron microscopy. In conclusion, we demonstrate here that RC3 is present in the adult rat spinal cord in pre- and postsynaptic structures.     

Expression of ubiquitin-like immunoreactivity in axons after compression trauma to rat spinal cord

The ubiquitin-mediated proteolytic pathway is an important mode of protein degradation in various tissues. Since breakdown of proteins may occur in axons after injury we evaluated the presence of ubiquitin-like immunoreactive material in rat spinal cord following compression injury of mild, moderate and severe degrees at T8–9 level, resulting in no neurological deficit, reversible paraparesis and paraplegia of the hind limbs, respectively. Rats with mild to severe compression injury surviving 1–4 days showed numerous, intensely immunoreactive expanded axons at the site of compression. The labelled axons were randomly distributed in the longitudinal tracts but they were never found in the corticospinal tracts. No labelling was detected by 9 days after injury. In addition, the presence of labelled axons was investigated in the T7 and the T10 segments from rats with moderate compression. No labelling was seen in T7, but in T10 segments many immunoreactive axons were present. Control rats did not show immunoreactive axons in the spinal cord. Neurons of dorsal root ganglia, trigeminal ganglia and of the grey matter of the spinal cord were immunoreactive. Cerebral cortical neurons did not show ubiquitin expression. Thus, compression of the rat spinal cord causes a transient accumulation of ubiquitin-like immunoreactive material in axonal swellings. Even though the dynamics of ubiquitin conjugates are not fully understood, the observed axonal accumulation presumably reflects arrested anterograde axonal transport of protein chiefly derived from neurons of dorsal root ganglia and the local neurons of the spinal cord. The presence of ubiquitin in damaged axons is one prerequisite for degradation of abnormal proteins by the ubiquitin-mediated proteolytic pathway, which may be activated in reactive axonal swellings. Received: 21 June 1995 / Revised: 11 August 1995 / Accepted: 25 September 1995     

Neuroendocrine secretory protein 55 (NESP55) in the spinal cord of rat: an immunocytochemical study

The immunohistochemical expression of a novel chromogranin-like protein, neuroendocrine secretory protein 55 (NESP55), in the rat spinal cord was investigated. NESP55-immunoreactive cells were detected in the ventral horn, intermediate laminae, and deep dorsal horn, comprising motoneurons, autonomic neurons, and interneurons throughout all spinal segments. Within laminae I-II of the dorsal horn, one or two NESP55-positive cells were often seen. Nerve fibers also contained NESP55 immunoreactivity (IR) and were particularly prominent in the ventral horn. No nerve terminals/varicosities appeared to contain NESP55 in any spinal lamina. Double-staining experiments revealed that a high proportion of the NESP55-positive neurons were cholinergic. Moreover, NESP55-IR in the motoneurons was evenly distributed in the whole cytoplasm with a finely granular appearance. In contrast, the fluorescent material in the preganglionic neurons was concentrated in the perinuclear region and largely overlapped with the trans-Golgi network marker TGN38. Our data provide detailed morphological information on the distribution of NESP55-IR in the rat spinal cord. Also, the differential intracellular expression of NESP55-IR in the spinal motoneurons and autonomic neurons suggests that NESP55 may be processed into different secretory granules and may be involved in both constitutive and regulated pathways in these neurons.     

Use of serotonin immunocytochemistry as a marker of injury severity after experimental spinal trauma in rats

In experimental models of spinal cord trauma there is often a relatively poor correlation between light microscopic histological changes and motor recovery. Previously it was shown that spinal cord levels of immunoreactive TRH and substance P, by radioimmunoassay, are significantly reduced caudal to the injury site. Since much of the substance P and TRH in the spinal cord derives from cells within the ventral medulla, many of which also contain serotonin, we examined changes in serotonin immunoreactivity within the spinal cord caudal to the injury site in rats subjected to varying degrees of impact trauma to the thoracic cord. Reductions in immunocytochemical staining of serotonin in ventral gray matter of the lumbar region at two weeks after trauma were significantly correlated with the degree of injury severity as reflected by motor impairment. Changes in the region of the central canal, but not dorsal horn, were also correlated with injury severity. These findings indicate that serotonin immunocytochemical analysis may permit better correlation between anatomical and functional outcome after spinal cord injury than generally utilized light microscopic methods.     

Intramedullary subependymoma of the cervical spinal cord: a case report with immunohistochemical study

Spinal subependymomas, which have a relatively benign nature, are very rare tumors. It is difficult to distinguish spinal subependymomas from other intramedullary spinal tumors based on neuroradiological findings. A case of cervical intramedullary subependymoma in a 63-year-old female is reported. The diffused enlargement of the spinal cord at C2 level involved the lesion with isointensity on a T1-weighted MRI and relatively high intensity on a T2-weighted MRI. Enhancement in the small part of the tumor was observed on a T1-weighted MRI with gadolinium administration. The tumor occupied the left side of the spinal cord, and was totally removed through a laminoplasty of C2. Immunohistochemistry was useful for pathological diagnosis. The clinical feature of this patient is described with the review of literatures.     

大鼠实验性脊髓损伤后胶质瘢痕分布规律研究

目的观察慢性脊髓损伤胶质瘢痕形成的时间与空间分布特征。方法应用Allen′s法建立大鼠脊髓打击伤模型,通过神经功能评分、体感及运动诱发电位、组织病理学及免疫荧光双标记方法确定胶质瘢痕形成时间及分布。结果神经功能评分、诱发电位、组织病理学显示伤后4周进入相对稳定状态,伤后4周损伤区形成空洞,胶质瘢痕位于空洞壁。结论脊髓损伤后4周进入慢性期,研究伤后脊髓空洞及胶质瘢痕形态,为判断切除胶质瘢痕的界限提供形态学依据。     

Tactile discrimination and cortical unit activity after lesions in the dorsal spinal cord of rat

Bilateral lesions in either dorsal columns or dorsal lateral columns of albino rats produced only a mild, transient deficit on a discrimination between rostrally and caudally directed strokes on the flanks. The proportion of single units in somatosensory cortical area I that were driven by gentle mechanical stimuli was not reliably reduced by either lesion.     

An immunohistochemical study of methionine-enkephalin-Arg-Gly-Leu-like immunoreactivity-containing neurons in the parasympathetic preganglionic regions of the rat spinal cord

The localization of the methionine-enkephalin-Arg⁶-Gly⁷-Leu⁸ (Met-Enk-Arg-Gly-Leu)-like immunoreactivity-containing neurons in the rat lumbosacral spinal cord was immunohistochemically examined by an antiserum very specific to Met-Enk-Arg-Gly-Leu. The immunoreactive neurons occupied the positions corresponding to the parasympathetic preganglionic nuclei determined by the previous horseradish peroxidase (HRP)-tracing experiments. The present study suggests that the parasympathetic preganglionic neurons in the rat lumbosacral spinal cord produce preproenkephalin A and its related peptides.