Met-enkephalin-like immunoreactivity in rat forebrain and spinal cord using hydrogen peroxide and Triton X-100. Ultrastructural study

Using two immunocytochemical methods, we have shown in light microscopy that the met-enkephalin-like immunoreactivity within striatum and spinal cord of the rat is differentially distributed in either perikarya or nerve terminals according to the technical conditions used [1]. The present electron microscopic study has been undertaken in order to elucidate the subcellular localization of immunoprecipitates according to the same technical conditions. In the neostriatum, numerous met-enkephalin-containing perikarya were stained (principally at the level of rough endoplasmic reticulum) when tissue sections were treated with hydrogen peroxide (H2O2) only, prior to the immunocytochemical procedure. However, injections of colchicine were required to demonstrate perikarya in the dorsal horn of the spinal cord. At variance with previous results, numerous dendritic profiles and nerve terminals were also reactive in this condition. Neurotubules, mitochondria, large granular vesicles (LGVs) and small synaptic vesicles were stained within these structures. The addition of a low concentration of Triton-X-100 (0.02%) in the first incubation medium often resulted in the disappearance of most perikarya and in the staining of only LGVs in nerve terminals. The addition of a higher concentration of Triton-X-100 (0.1%) produced diffusion of immunoprecipitates at the level of nerve terminals, which was probably responsible for the increased intensity of staining and, subsequently, for the better demonstration of fibre varicosities in light microscopy. On the contrary, the disappearance of reactive perikarya seemed to result from the diffusion of the non-protected peptide out of the cytoplasm. The diverse ultrastructural localizations of met-enkephalin-like immunoreactivity in striatum and spinal cord are finally discussed in light of intrinsic connections or afferents described in the literature.     

Differential expression of proteoglycans at central and peripheral nodes of Ranvier

The nodes of Ranvier are regularly spaced gaps between myelin sheaths that are markedly enriched in voltage-gated sodium channels and associated proteins. Myelinating glia play a key role in promoting node formation, although the requisite glial signals remain poorly understood. In this study, we have examined the expression of glial proteoglycans in the peripheral and central nodes. We report that the heparan sulfate proteoglycan, syndecan-3, becomes highly enriched with PNS node formation; its ligand, collagen V, is also concentrated at the PNS nodes and at lower levels along the abaxonal membrane. The V1 isoform of versican, a chondroitin sulfate proteoglycan, is also present in the nodal gap. By contrast, CNS nodes are enriched in versican isoform V2, but not syndecan-3. We have examined the molecular composition of the PNS nodes in syndecan-3 knockout mice. Nodal components are normally expressed in mice deficient in syndecan-3, suggesting that it has a nonessential role in the organization of nodes in the adult. These results indicate that the molecular composition and extracellular environment of the PNS and CNS nodes of Ranvier are significantly distinct.     

Postnatal differentiation of rat optic nerve fibers: Electron microscopic observations on the development of nodes of Ranvier and axoglial relations

The postnatal differentiation of rat optic nerve fibres was examined by transmission electron microscopy. The results show that many early developing axons contain clusters of vesiculotubular profiles prior to Myelination. At places vesicular elements appear to fuse with the axolemma, and, in addition, some axons exhibit deep axolemmal invaginations and axoplasmic lamellated bodies. It is suggested that these feature might reflect axolemmal remodeling, possibly involving axoglial signalling and/or functional differentiation of the axolemma. The size distribution of unmyelinated optic nerve axons changes little during development. Ensheathment of larger axons commences 6 days postnatally. The subsequent formation of compact sheaths are a few microns long and separated by long bare axon segments. In optic nerves from 10–12-day-old rat pups, a few sheaths consisting of about five layers border primitive asymmetric nodes with a patchy axolemmal undercoating. Extensions from one of the terminating sheaths are often associated with undercoated patches of axolemma. Relatively differentiated nodes of Ranvier first appear 14–16 days after birth. The continued nodal maturation involves establishment of a regular nodal geometry, increasing distinctness of the axolemmal undercoating, and formation of perinodal astrocytic processes embedded in an extracellular node gap substance. The results are compared with available data on the conduction properties of rat optic nerve fibres during development.     

Differential innervation of the goldfish tonic red muscles and twitch white muscles by neuropeptide-immunoreactive motoneurons

Neuropeptides in the motor nerves innervating the red and white muscles of the goldfish Carassius auratus were examined. In the tonic red muscles, varicose nerve endings immunoreactive for both calcitonin gene-related peptide and substance P were found spread over the surface of the muscle fibers, but in the twitch white muscles only scattered nerve endings immunoreactive for calcitonin gene-related peptide were found. At the electron microscopic observation, dense electron products immunoreactive for calcitonin gene-related peptide and for substance P (SP) were detected in the motor nerve endings making synapses on the muscle fibers of the red muscles. In the spinal cord, all of the motor neurons showed immunoreactivity to calcitonin gene-related peptide, but the motor neurons immunoreactive for substance P were restricted to the ventrolateral group that has been shown to project predominantly to the red muscles. These results suggest that the motor neurons innervating the red and white muscles of the goldfish are distinct in their neuropeptide content. The present study also raises the possibility that SP might be related to the unique physiological properties of the tonic type red muscles, probably by direct binding to the acetylcholine receptors.     

Olfactory ensheathing cells induce less host astrocyte response and chondroitin sulphate proteoglycan expression than Schwann cells following transplantation into adult CNS white matter

Both Schwann cells and olfactory ensheathing cells (OECs) create an environment favorable to axon regeneration when transplanted into the damaged CNS. However, transplanted cells can also exert an effect on the host tissue that will influence the extent to which regenerating axons can grow beyond the transplanted area and reenter the host environment. In this study equivalent numbers of Lac-Z-labeled Schwann cells and OECs have been separately transplanted into normal white matter of adult rat spinal cord and the host astrocyte response to each compared. Schwann cell transplantation resulted in a greater area of increased glial fibrillary acidic protein (GFAP) expression compared to that associated with OEC transplantation. This was accompanied by a greater increase in the expression of axon growth inhibitory chrondroitin sulfate proteoglycans (CSPGs) following Schwann cell transplantation compared to OEC transplantation. However, no differences were detected in the increased expression of the specific CSPG neurocan following transplantation of the two cell types. These results mirror differences in the interactions between astrocytes and either Schwann cells or OECs observed in tissue culture models and reveal one aspect of the complex biology of creating regeneration-promoting environments by cell transplantation where transplanted OECs have favorable properties compared to transplanted Schwann cells.     

Perinodal astrocytic processes at nodes of ranvier in developing normal and glial cell deficient rat spinal cord

This study examined, during normal development and during the development of a glial cell deficient axon population, the nature of astrocyte involvement at the central nodes of Ranvier on spinal cord axons. One condition examined was the ventral funiculus of normal 7-day-old rats. At this age, the lumbar spinal cord underwent an active phase of gliogenesis, and axons were seen in various stages of myelination. Perinodal astrocytic processes were routinely observed at nodes of axons on which myelin sheaths exceeded 8 compact lamellae. Perinodal astrocytic processes were also seen in close proximity to axolemma at most developing nodes. This study also examined the lumbar spinal cords of rats which were X-irradiated on the third postnatal day. This procedure caused a profound reduction in the astrocyte and oligodendrocyte population in 13- and 18-day-old rats, while sparing the neuronal elements. Thus, axo-glial relationships observed in this tissue are unlikely to be random occurrences. Despite the reduction in glial cells, some oligodendrocyte-myelinated axons were observed in the irradiated spinal cords. Perinodal astrocytes were seen at all oligodendrocyte-derived nodes observed in the irradiated cord and appeared to have a specific relationship to the node of Ranvier. The presence of astrocytic processes at the normal, developing node and at the nodes in glial cell deficient spinal cords suggests that astrocytes may be necessary to the function of nodal axolemma. In irradiated spinal cords, where the glial cells are markedly reduced, apposition between astrocytic and oligodendrocytic membrane at the paranode and internode was also seen and was so common that it is highly unlikely to be due to random occurrences. These observations further suggest that in addition to the presumptive role at the nodes, astrocytes may play an inductive or supportive role in the development and maintenance of central myelin.     

Cholecystokinin-like immunoreactivity in the rat spinal cord: Effects of thoracic transection

A study of cholecystokinin-like immunoreactivity in the lumbar (L1-L5) spinal cord segments of rats was realised 24-48 hours after complete thoracic transection (T6-T8). A comparison was made with corresponding spinal cord segments from control and sham-operated animals. The immunocytochemical study with light microscopy showed cholecystokinin-like immunoreactive cell bodies in laminae VII and X at L1-L5, caudal to the transection. In addition, the immunoreactivity was greatly enhanced in bundles of the dorsolateral funiculus compared to sham-operated animals. Our results suggest that part of cholecystokinin-like cell bodies of laminae VII and X send projections to supraspinal sites. Some of these supraspinal projections would go through the dorsolateral funiculus. In the lumbar dorsal horn of operated animals, the immunoreactivity was greatly enhanced in lamina I, while it was slightly decreased in lamina II, compared to control animals. Using electron microscopy, in lamina I, the immunoreactivity localized in different neurites was generally very intense. Moreover, axon terminals showed swelling: their mean size was 0.8-1.8 microns (0.5-1.2 in control animals). This result suggests that some cholecystokinin-like neurons also project to lamina I of rostral cervical segments. In lamina II, numerous degenerating axons were observed (24 hours after thoracic spinal transection). This would suggest that part of descending cholecystokinin-like projections terminate in lamina II.     

Immunohistochemical localization of connective tissue growth factor in the rat central nervous system

Connective tissue growth factor (CTGF) is an immediate early growth-responsive gene but its distribution and significance in the central nervous system (CNS) are unknown. We investigated the distribution of CTGF-like immunoreactivity (CTGF-IR) in the rat CNS using a specific antiserum against CTGF oligopeptide. The majority of CTGF-IR was observed in astrocytes. Ependymal cells lining the wall of the cerebral ventricle and tanycytes lining the central canal of the spinal cord showed the strongest CTGF-IR, while there was a diffuse but weak signal in the gray matter of the spinal cord. CTGF-IR was also detected in the cytoplasm of a subpopulation of pyramidal neurons in the cerebral cortex. Our results showed that CTGF-IR is widely distributed in the CNS at both regional and cellular levels, suggesting a complex functional role in the CNS.     

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     

Neurofilaments assume a less random architecture at nodes and in other regions of axonal compression

Neurofilament distributions were mathematically characterized in four chicken somatic motor axons at each of four histologically distinct regions: compact myelinated regions, compact myelinated regions associated with Schwann cell nuclei, Schmidt-Lanterman clefts, and nodes of Ranvier. Compact myelinated regions had the largest cross-sectional areas, the lowest neurofilament densities, and the most random neurofilament organizations — nodes of Ranvier had the smallest cross-sectional areas, the highest neurofilament densities, and the most ordered architectures. In these myelinated axons, the closest natural neurofilament spacing was 25 nm. Mathematical analyses of serial sections suggested that neurofilament interactions are sufficiently weak and transient to permit a full range of variation from random to ordered cytoskeletal architectures as the neurofilaments move longitudinally through the few micron span of the paranodal-nodal region of a single axon.     

Increased expression of growth-associated protein 43 immunoreactivity in axons following compression trauma to rat spinal cord

Growth-associated protein 43 (GAP43) is one compound used to indicate growth of axonal endings during development and regeneration, particularly of peripheral neurons. Using immunohistochemistry, we have studied the expression of GAP43 in the spinal cord of rats subjected to mild, moderate or severe compression injury and used neurofilament immunostaining to demonstrate axonal injuries. Samples removed from the compressed T8–9, the cranial T7 and the caudal T10 segments were studied at 4 h, 24 h, 4 days and 9 days after injury. Control rats showed a moderate immunostaining of neurons in dorsal root ganglia, weak staining of ventral motor neurons and, with the exception of the corticospinal tracts, a weak staining in some axons of the longitudinal tracts of the cord. Injury in the compressed region led to increased GAP43 immunoreactivity in axons of normal and expanded size. This occurred particularly 1–4 days after injury and normalized 9 days thereafter. More marked immunostaining was present in the cranial and caudal segments. The corticospinal tracts never showed such staining. The increase of GAP43 immunostaining is presumably caused by disturbed axonal transport from neurons with the capacity to synthesize and transport the GAP43 antigen. Transported material may thus be available for regeneration of axons, but this source of material may vary between different classes of axons within the cord. Received: 11 December 1995 / Revised, accepted: 19 January 1996     

Retrogradely transported fluorogold accumulates in lysosomes of neurons and is detectable ultrastructurally using post-embedding immuno-gold methods

For ultrastructural studies, it is of great interest to be able to combine anatomical tracer techniques with sensitive immunohistochemical methods. Fluorogold (FG) is a fluorescent and retrogradely transported anatomical tracer, which is commonly used to label neurons in the brain and spinal cord for light microscopic studies. We here describe a method for detecting FG-labeled somata in the electron microscope using a high resolution post-embedding immuno-gold method. For this purpose, spinal motoneurons were retrogradely labeled by an intraperitoneal injection of FG in the adult rat. The rats were intravascularly perfused with a fixative solution containing 2% paraformaldehyde and 1–2% glutaraldehyde. Vibratome sections of spinal cord tissues were cryo-protected in glycerol, freeze substituted in methanol containing uranyl acetate, and embedded in the Lowicryl HM20 resin at low temperatures. Electron microscopic analysis demonstrated atypical lysosome-like structures in the cytoplasm of FG-labeled motoneurons. Subsequent post-embedding immuno-gold labeling demonstrated prominent accumulation of FG in numerous lysosomes but not in other organelles or cytoplasmic compartments of the labeled neurons. The protocol is versatile and allows for combining anatomical tracing of neurons with, e.g., neuro-transmitter studies in the electron microscope. We suggest that the described method for sensitive detection of FG in the spinal cord may also have broad applicability to other areas of the central nervous system.     

Cellular localization and enzymatic activity of cathepsin B after spinal cord injury in the rat

Mechanical spinal cord injury (SCI) initiates a cascade of pathochemical and pathophysiological events, collectively known as the secondary injury. There has been a long-standing interest in understanding the activation and involvement of proteases in this secondary injury process. Several proteases including the calpains, caspases and matrix metalloproteinases are activated by perturbations to the spinal cord and have been linked to cell death following SCI and in other models of CNS disease and insult. Cathepsin B (Cath B), a potent lysosomal protease, has also been implicated in the pathology of CNS diseases including brain tumors, Alzheimer's disease, amyotrophic lateral sclerosis and stroke. Previously, we reported significant increases in Cath B mRNA and protein expression following contusion-SCI. This characterization of Cath B continues with the experiments reported herein, which were designed to examine Cath B enzymatic activity and cellular localization following contusion-SCI in the rat. Cath B enzymatic activity was significantly increased in the injury epicenter at 5 and 7 days post-injury and was highly correlated with increases in the active forms of the Cath B protein reported earlier. Furthermore, the immunohistochemical analyses revealed that the post-injury increases in expression and enzymatic activity at the injury epicenter were due to the presence of a large and diverse population of inflammatory cells. However, in areas adjacent to the injury epicenter, it appears that parenchymal neurons may also contribute to these increases. Our findings coupled with the documented role of Cath B in other CNS pathologies make this potent protease an attractive candidate for involvement in the tissue destruction associated with the secondary injury cascade following SCI.     

Intact aggrecan and chondroitin sulfate-depleted aggrecan core glycoprotein inhibit axon growth in the adult rat spinal cord

Aggrecan is a chondroitin sulfate (CS)/keratan sulfate (KS)-substituted proteoglycan (PG) abundant in cartilage which is also present within the mammalian embryonic, adult, and injured adult central nervous system (CNS). Although its role within the CNS is not clear, cell culture studies show that when substituted with CS, aggrecan inhibits neurite extension. To better understand the inhibitory effect of aggrecan on injured adult axons in vivo, we developed a model to independently test intact aggrecan and CS-depleted aggrecan core glycoprotein. Acute rat spinal cord hemisection cavities were filled with a growth-promoting matrix, Matrigel, and severed dorsal rootlets were placed into this matrix. This created an assay in which axons readily grew. The extent of ingrowth in this baseline assay was compared to the ingrowth in Matrigel loaded with intact aggrecan or the purified core glycoprotein of aggrecan. Our results show that both intact aggrecan and equivalent concentrations of the core glycoprotein component significantly inhibit axonal growth in this model system. These results confirm that aggrecan can inhibit the growth of adult axons in vivo and suggest that the inhibitory effects of aggrecan may be mediated, at least in part, by structures located on the core glycoprotein in the absence of the bulk of the CS chains.     

Expression of endothelial barrier antigen immunoreactivity in blood vessels following compression trauma to rat spinal cord

The endothelial barrier antigen (EBA) recognised by a monoclonal antibody is expressed in rat cerebral microvessels possessing blood-brain barrier properties but only weakly by fenestrated vessels. We have studied the expression of this marker in the spinal cord of control rats and compared the findings with those seen in rats subjected to compression injury at the T8–9 level with a survival period of 4 h, 24 h, 4 days and 9 days. To that end, formalin-fixed paraffin-embedded material was immunostained by the avidin-biotin-peroxidase complex method. Sections from control rats presented a distinct immunostaining at the site of the endothelial cells of almost all microvessels in the grey and white matter of the cord. The anterior and posterior spinal arteries did not show such staining. Neurons and glial cells were unstained. Rats which had survived 4 h after a moderate or severe compression trauma still showed immunoreactivity in intramedullary microvessels at the site of injury. There was a moderate reduction of vascular immunoreactivity at 24 h and a pronounced loss of such reactivity at 4 days after trauma. At 9 days after compression the expression of the endothelial barrier antigen had almost been normalised in the microvessels of the cord. In conclusion, using immunohistochemistry, EBA can be demonstrated in noninjured rat spinal cord microvessels, while the staining disappears at the site of compression trauma to the cord. The EBA marker can be used to indicate sites of vascular injury in spinal cord compression injury. The factors causing the disappearance and restitution of the antigen are unknown. Received: 1 October 1997 / Revised, accepted: 9 January 1998     

Spinal dopaminergic system of the rat: light and electron microscopic study using an antiserum against dopamine, with particular emphasis on synaptic incidence

The mapping of the spinal dopaminergic innervation has been performed in the adult rat using an anti-dopamine antiserum. Immunoreactive fibers were detected with the light microscope in the dorsal horn (mainly in laminae III-IV), in the intermediolateral cell column (IML), in the peri-ependymal region and in the ventral horn. The ultrastructural analysis of dopaminergic innervation showed mainly axodendritic contacts and fewer axosomatic ones. In the ventral horn and the IML, the pattern of dopaminergic innervation exhibited a majority of classical synapses. In the dorsal horn, dopaminergic innervation was partly non-synaptic (at cervical level), whereas numerous axodendritic synapses were observed at thoraco-lumbar level. Previous studies described the non-synaptic organization of serotonergic and noradrenergic projections in the dorsal horn. It is thus hypothesized that the monoaminergic systems, involved in pain modulation within the dorsal horn, act partly through volume transmission. In contrast, these systems would modulate the motor and autonomic functions through classical synapses.     

Extracellular edema and glial response to it in the cerebellum of suckling rats with low-dose lead encephalopathy

Summary Newborn rats were exposed to daily intraperitoneal injections of 10 mg lead nitrate per kg body weight for the first 15 postnatal days. The growth and mortality of the lead-exposed animals did not differ from their control litter-mates, injected with vehicle only. In our previous studies, focal hemorrhages and spongy areas as well as breakdown of blood-brain barrier to plasma proteins were shown by light microscopy in the cerebellar parenchyma of 15-day-old rats exposed to this dose. In spite of these signs of edema, measurements of brain tissue specific gravity did not show increased water content. In the present investigation we examined the ultrastructure of the brain lesions in these rats with low-dose lead encephalopathy, focusing on signs of edema, and evaluated astroglial reaction by immunocytochemical staining for glial fibrillary acidic protein (GFAP). The electron microscopic findings were compatible with extracellular edema in the cerebellum of 15-day-old lead exposed rats. The number of GFAP-positive cell bodies in the gray substance of the cerebellar cortex was increased in the 15-day-old lead-exposed rats as compared with the controls of the same age, a finding which is presumably related to the leakage of plasma proteins. Both these findings were lacking at 20 days of age, suggesting reversibility of the lead-induced changes.Supported by grants from the Swedish Medical Research Council (project nos 12X-03488 and 12X-07123), the Finnish Medical Research Council, the Medical Faculty, University of Göteborg, from the Göteborg Royal Society for Sciences and Arts, and from the Swedish Worker Environment Fund (project no 81-0149)     

Spheroids and altered axons in the spinal gray matter of the normal cat

Summary In our recent ultrastructural studies on synapses of the nucleus dorsalis, central cervical nucleus, and anterior horn of the spinal cord of the normal cat we happened to find spheroids and several types of axonal alterations. These spheroids were up to 39 m in diameter. They were found in myelinated and unmyelinated terminal axons and at the node of Ranvier and showed two different types of internal structure. One type was large and composed of spirally arranged neurofilaments and mitochondria in increased quantity, although the mean population density of mitochondria was not high being 1.4/m² as compared to the normal value 2.0/m². Another type was smaller and consisted of small mitochondria and dense bodies which were increased in number: their mean population densities were 4.5/m² and 1.9/m², respectively.At present, the rare occurrence of spheroids and atypical axonal alterations makes it difficult to determine the origin of affected axons, although some of them presumably arise from primary afferents.     

Co-induction of HSP70 and heme oxygenase-1 in macrophages and glia after spinal cord contusion in the rat

HSP70 and heme oxygenase-1 (HO-1) are thought to be markers of cell injury and oxidative stress, respectively. We have immunolocalized these proteins in the spinal cord at 1-14 days after contusion. HSP70 and HO-1 were co-induced in glia and macrophages within the injured segment at all time points. This co-induction may reflect complementary functions that serve to protect these cells as they respond to the postcontusional environment.