Distribution of mitochondria along small-diameter myelinated central nervous system axons

Small-diameter myelinated CNS axons are preferentially affected in multiple sclerosis (MS) and in the hereditary spastic paraplegias (HSP), in which the distal axon degenerates. Mitochondrial dysfunction has been implicated in the pathogenesis of these and other disorders involving axonal degeneration. The aim of this study was to determine whether the frequency of axonal mitochondria changes along the length of small-diameter fibers and whether there is a preferential localization to the region of the node of Ranvier. We find that mitochondrial numbers do not change along the length of a myelinated small-diameter fiber, and, in contrast to the peripheral nervous system, there is no tendency for mitochondrial numbers to increase at the node.     

Quantitative autoradiographic mapping of delta-opioid receptors in the rat central nervous system using [125I][D.Ala2]deltorphin-I

The distribution of β-opioid binding sites was studied by quantitative autoradiography in rat brain and spinal cord using the highly selective ligand [¹²⁵I][D. Ala²]deltorphin-I. The binding properties of [¹²⁵I][D. Ala²]deltorphin-I were investigated by microdensitometry of autoradiographic films with the aid of a computerassisted image-analysis system. [¹²⁵I][D. Ala²]deltorphin-I appeared to interact with a single class of sites in all brain areas (K_D = 0.9 nM). In 23 regions tested, whatever the δ site concentration, DTLET, a δ agonist, appears to be 2 orders of magnitude more effective than DAGO, a μ agonist, in inhibiting specific [¹²⁵I][D. Ala²]deltorphin-I binding. The distribution of [¹²⁵I][D. Ala²]deltorphin-I sites is globally consistent with that of other δ ligands and does not support the existence of a δ-receptor subtype recognized by [D. Ala²]deltorphin-I. [¹²⁵I][D. Ala²]deltorphin-I binding sites were highly confined, exhibiting selective localization in the neocortex and a diffuse pattern in the striatum, accumbens nucleus, claustrum, layer of bulb, amygdaloid nucleus, pontine nuclei, and inferior colliculus. In several areas a rostro-caudal gradient of site concentration was indicated. [D. Ala²]deltorphin-I binding sites were also present in the substantia gelatinosa at all levels of the spinal cord and, unexpectedly, in deeper laminae and the ventral horn. These results demonstrate the ability of [¹²⁵I][D. Ala²]deltorphin-I to characterize low concentrations of binding sites and to reveal new localizations of δ receptors. © 1993 Wiley-Liss, Inc.     

Immunohistochemical localization of the neuropeptide Y Y1 receptor in rat central nervous system

The diverse effects of neuropeptide Y (NPY) are mediated through interaction with G-protein coupled receptors. Pharmacological analysis suggests the Y1 receptor mediates several of NPY's central and peripheral actions. We sought to determine the distribution of Y1 protein throughout the rat central nervous system by means of indirect immunofluorescence using the tyramide signal amplification method and a novel, amino terminally-directed Y1 antisera. This antisera was verified as specific for Y1 by solution-phase competition ELISA, Western blot and in situ blocking experiments. High concentrations of Y1 immunoreactivity were found in the claustrum, piriform cortex (superficial layer), arcuate hypothalamic nucleus, interpeduncular nucleus, paratrigeminal nucleus, and lamina II of the spinal trigeminal nucleus and entire spinal cord. Moderate levels of Y1 immunoreactivity were found the in the main olfactory bulb, dorsomedial part of suprachiasmatic nucleus, paraventricular hypothalamic nucleus, ventral nucleus of lateral lemniscus, pontine nuclei, mesencephalic trigeminal nucleus, external cuneate nucleus, area postrema, and nucleus tractus solitarius. Low levels of Y1 immunostaining were distributed widely throughout layers II-III of the cerebral cortex (i.e., orbital, cingulate, frontal, parietal, insular, and temporal regions), nucleus accumbens core, amygdalohippocampal and amygdalopiriform areas, dentate gyrus, CA1 and CA2 fields of hippocampus, principal and oral divisions of the spinal trigeminal nucleus, islands of Calleja and presubiculum. These findings are discussed with reference to previously reported receptor autoradiography, immunohistochemistry and mRNA analyses to further support the role of Y1 in NPY-mediated biology.     

大鼠脊髓损伤后轴突病理变化与胶质瘢痕的关系

目的 观察脊髓损伤(SCI)后轴突变化及其与胶质瘢痕的关系.方法 应用Allen's法建立大鼠脊髓损伤模型,通过行为学评分、免疫荧光及神经束路示踪等观察SCI后轴突的病理变化,及其与胶质瘢痕的关系,并测量胶质瘢痕的厚度.结果 SCI后损伤处的轴突呈断裂、扭曲状,SCI后1 周损伤轴突呈再生趋势,2周时再生明显,与此相应动物运动功能逐渐恢复,4周时胶质瘢痕形成,再生的轴突被瘢痕阻挡.头尾侧胶质瘢痕厚度(107.00±20.12)μm大于两侧边厚度(69.92±24.37)μm.结论 SCI后轴突仍具有再生能力,但被胶质瘢痕所阻挡,瘢痕厚度的测量为将来去除胶质瘢痕提供了实验依据.     

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     

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.     

Electrical stimulation of intact peripheral sensory axons in rats promotes outgrowth of their central projections

A lesion of a peripheral nerve before a second injury (conditioning lesion, CL), enhances peripheral and central regeneration of dorsal root ganglion (DRG) axons. This effect is mediated by elevated neuronal cAMP. Here we wanted to investigate whether electrical stimulation (ES) of an intact nerve, which has been shown to accelerate peripheral axon outgrowth, is also effective in promoting axon regeneration of injured DRG axons in vitro and of the central DRG axons in vivo and, whether this effect is mediated by elevation of cAMP. For the in vitro assay, the intact sciatic nerve of adult rats was stimulated at 20 Hz for 1 h, 7 days before harvest and primary culture of DRG neurons on a growth permissive substrate. In the in vivo study, the central axons of the lumbosacral DRGs were cut in the Th8 dorsal column, and the sciatic nerve was either cut or left intact, and subjected to 1 h ES at 20 Hz or 200 Hz. In vitro, ES increased neurite outgrowth 4-fold as compared to non-stimulated DRG neurons. In vivo, ES at 20 Hz significantly increased axon outgrowth into the central lesion site as compared to the Sham control. The 20 Hz ES was as effective as the CL in increasing axon outgrowth into the lesion site but not in promoting axonal elongation even though 20 Hz ES increased intracellular cAMP levels in DRG neurons as effectively as the CL. Thus elevation of cAMP may account for the central axonal outgrowth after ES and a CL.     

Sprouting and regeneration of sensory axons after destruction of ensheathing glial cells in the leech central nervous system

To test the importance of glia during regeneration of mechanosensory neuron axons in the leech central nervous system, individual glial cells that ensheathed the axons were destroyed by intracellular protease injection. Recordings with intracellular microelectrodes showed that glial-desheathed axons re-established synaptic connections with their appropriate target cell, a motor neuron, as frequently and as selectively as control, glial-ensheathed axons. Intracellular staining with horseradish peroxidase showed that desheathed regenerating axons sprouted more than controls, and loss of the glial cell in some cases caused uninjured (intact) axons to sprout. Successful, accurate regeneration could occur whether axons grew along normal or along aberrant pathways. The distal stumps of some sensory axons severed from cell bodies and maintained in organ culture survived without their glial sheath for up to 3 weeks. These experiments show that, although loss of the glial cell affects sensory axon growth, the glial cell is not required for accurate axonal regeneration in the leech central nervous system.     

Role of glial cells in the differentiation and function of myelinated axons

Myelinated axons are highly differentiated in the vicinity of the node of Ranvier, both structurally and with respect to ion channel distribution. Evidence is reviewed showing that axonal differentiation depends upon two distinct types of interaction between glial cells and the axolemma, one at the node itself, with astrocyte processes, and the second, more extensive one, in the paranodal region, with oligodendrocyte processes. In the peripheral nervous system, Schwann cells fulfill both roles. Glial or Schwann cell abnormalities, due to genetic deficiencies, diseases or experimental procedures, result in corresponding abnormalities in the axolemma and can have devastating effects on nerve fiber function. An example, the myelin-deficient mutant rat, is presented, and the defects underlying the profound and ultimately lethal neurological abnormalities seen in this mutant are discussed in relation to abnormalities in its axoglial interactions.     

Hypomyelination in the neonatal rat central and peripheral nervous systems following tellurium intoxication

Summary Neonatal rats were exposed to Tellurium (Te), via the mother's milk, from the day of birth until sacrifice at 7, 14, 21, and 28 days of age. Light and electron microscopy revealed Schwann cell and myelin degeneration in the sciatic nerves at each age studied. These changes were similar to those described in weanling rats as a result of Te intoxication. In the CNS, hypomyelination of the optic nerves was convincingly demonstrated at 14, 21, and 28 days of age, accompanied by some evidence of myelin degeneration. These changes were also seen in the ventral columns of the cervical spinal cords, although less markedly, and were confirmed by quantitative methods. There was little evidence of oligodendrocyte pathology in the CNS, and it appears that degeneration of these cells is not the primary cause of the CNS hypomyelination, in contrast to the PNS where Schwann cell degeneration has been shown to precede the myelin pathology.Supported by grants from the NIH (NS-23214) and the NMSS (RG 1791-9-1)     

Neurotraumatology: Ultrasonic evaluation of the central nervous system

Abstract

In this study we describe the technique of intraoperative ultrasound imaging of brain and spinal cord in trauma patients. The images are shown and their interpretation is discussed. This intraoperative imaging allows for localization of hematomas, bone fragments and indriven foreign bodies (j.e., pieces of plastic, glass, metal, etc.). DISC matenal and bone fragments deep to the spinal cord can be localized with this technique. Real-time ultrasound can be used to guide instruments within the brain and, thereby, provide dynamic guidance for removal of bone fragments and foreign bodies dynamically. In summary, Intraoperative real-time ultrasonic Imaging is of use to the neurosurgeon in the treatment of the neurotrauma patient. [Neural Res 1997; 19: 317–322]

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Myelin-induced experimental allergic encephalomyelitis in Lewis rats: tumor necrosis factor α levels in serum and cerebrospinal fluid Immunohistochemical expression in glial cells and macrophages of optic nerve and spinal cord

Tumor necrosis factor a (TNFa) activity was measured in serum and cerebrospinal fluid (CSF) of Lewis rats after experimental allergic encephalomyelitis (EAE) induction and during the clinical course of acute disease. TNFa bioactivity expression preceded the clinical symptoms and paralleled the severity of disease. We further investigated the identity of the central nervous system (CNS) cells involved in TNFa expression and their regional localization during EAE. Tissue sections of brain, cerebellum, dorsal spinal cord and optic nerve were studied by indirect double labelling immunofluorescence. Spinal cord white matter and optic nerve showed a widespread TNFa immunoreactivity at critical stages of EAE in macrophages/microglia and astrocytes. We have shown changes in CSF/serum albumin ratio and immunoglobulin G index during EAE. Our results confirm the very important role of TNFa in EAE.     

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     

Characterization of glial subpopulations in cultures of the ovine central nervous system

The cellular composition and in vitro development of glial cultures derived from the rat CNS has been well studied. However, less information is available on similar cultures from other species, particularly higher mammals. To study ovine glial development in vitro, cultures from 50-day fetal to adult animals were characterized with various immunocytochemical markers, which are frequently used to define neural cell subsets in rat cultures. As in rats, both A2B5+ and A2B5- astrocytes can be identified in ovine cultures. However, ovine A2B5+ and A2B5- could not be reliably differentiated by their morphology, which was more influenced by whether the cells were in serum-free or serum-containing media than by their A2B5-positive or -negative status. In addition, ovine A2B5+ astrocytes were present in cultures from early fetal brain before the development of identifiable oligodendrocytes, unlike rat type II astrocytes, which develop only after the appearance of oligodendrocytes. An A2B5+ cell, morphologically similar to the rat 02-A cell, can be found in cultures from fetal ovine cerebrum or cerebellum. A2B5+/glial fibrillary acidic protein (GFAP)- cells in cultures from 100- to 115-day ovine cerebellum appeared to differentiate into A2B5+ astrocytes in serum-containing media. However, in serum-free media, although the A2B5+ cells assumed a more "oligodendroglial-like" morphology, they did not express galactocerebroside or myelin basic protein, suggesting that these cells may not be bipotential as is the rat 02-A cell. Oligodendroglial differentiation was not induced by treatment with dibutyryl cyclic AMP or insulin-like growth factor I. Many cells in cultures from a variety of fetal ages did not label with any of the immunocytochemical markers used, suggesting the need for more cell-type-specific markers to identify neural cell subsets in higher mammals.     

大鼠脊髓源性神经干细胞的培养分化及其特异性研究

目的 研究大鼠脊髓源性神经干细胞培养和分化的特异性。方法 从孕17d的SD大鼠胚胎脊髓中分离，培养神经干细胞并用血清诱导其分化，通过免疫荧光化学方法研究其特性。结果 在血清的诱导下，脊髓源性神经干细胞大多数分化成GFAP阳性的星形胶质细胞，少数分化为tubulin-β阳性的神经细胞；与脑源性神经干细胞分化的神经细胞相比较，其分化出的神经细胞的突起长度明显延长。结论 脊髓源性神经干细胞在体外具有多向分化潜能，但与脑源性神经干细胞有明显差别。     

Asymmetrical regional changes in energy metabolism of the central nervous system during walking

Cats were injected with 2-deoxy-[¹⁴C]glucose (2-DG) while walking on a moving treadmill (experimental group), or sitting down on a stationary one (controls). After a 45-min equilibration period they were anesthetized, and their central nervous system (CNS) was removed rapidly and frozen. The tissue blocks were sectioned serially, and X-ray film exposed to the sections was used for quantitative densitometric analysis by Sokoloff's method. The utilization of glucose in a CNS region (LCMRg) was regarded as a measure of that region's energy metabolic activity and — indirectly — of its functional status. The walking cats exhibited significantly higher LCMRg in many but not all places of the neuraxis, compared to the control group. Also, LCMRg was symmetrical (side to side) in the control group but significantly asymmetrical in certain regions of the CNS in the experimental group. In all but one of these cats the LCMRg was greater in the right side of the gray matter of the cervical spinal cord and in the left visual and motor cortices and caudate nucleus. The finding that the motor cortex and other supraspinal regions become more active during walking suggests they may contribute to the control of locomotion and/or processing of related sensory data. The side to side assymetry in the spinal cord and hemispheres during walking may be related to the phenomenon of lateral dominance.     

Neuroinflammation in the central nervous system: Symphony of glial cells

Neuroinflammation in the central nervous system (CNS) is an important subject of neuroimmunological research. Emerging evidence suggests that neuroinflammation is a key player in various neurological disorders, including neurodegenerative diseases and CNS injury. Neuroinflammation is a complex and well-orchestrated process by various groups of glial cells in CNS and peripheral immune cells. The cross-talks between various groups of glial cells in CNS neuroinflammation is an extremely complex and dynamic process which resembles a well-orchestrated symphony. However, the understanding of how glial cells interact with each other to shape the distinctive immune responses of the CNS remains limited. In this review, we will discuss the joint actions of glial cells in three phases of neuroinflammation, including initiation, progression, and prognosis, the three movements of the symphony, as the role of each type of glial cells in neuroinflammation depends on the nature of inflammatory cues and specific course of diseases. This perspective of glial cells in neuroinflammation might provide helpful clues to the development of the early diagnosis and therapeutic intervention of the various CNS diseases.     

False resurrections: distinguishing regenerated from spared axons in the injured central nervous system

Several recent studies report that axon regeneration can be induced in the mature mammalian nervous system by novel treatments or genetic manipulations. In assessing these reports, it is important to be mindful of the history of regeneration research, which is littered with the corpses of studies that reported regeneration that later proved incorrect. One important reason is the "spared axon conundrum," in which axons that survive a lesion are mistakenly identified as having regenerated. Here, we illustrate the problem and propose criteria that may be used to identify regenerated vs. spared axons, focusing on the injured spinal cord.     

Projection of nodose ganglion cells to the upper cervical spinal cord in the rat

Afferent fibers mediating pain from myocardial ischemia classically are believed to travel in sympathetic nerves to enter the thoracic spinal cord. After sympathectomies, angina pectoris still may radiate to the neck and inferior jaw. Sensory fibers from those regions are thought to enter the central nervous system through upper spinal cord segments. We postulated that axons from nodose ganglion cells might project to cervical cord segments. The purpose of this study was to determine the density and pathway of vagal afferent innervation to the upper cervical spinal cord. Following an injection of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) into the upper cervical spinal cord, approximately 5.8% of cells in the nodose ganglion contained reaction product. Cervical vagotomy did not diminish the density of WGA-HRP labeled cells in the nodose ganglion. However, a spinal cord hemisection cranial to the injection site eliminated labeling of nodose cells. These data indicate that a portion of vagal afferent neurons project from the nodose ganglion to the upper cervical spinal cord. In addition, vagal afferent fibers reach the spinal cord via a central route rather than through dorsal root ganglia.     

Growing and regenerating axons in the visual system of teleosts are recognized with the antibody RT97

We have analyzed the immunolabeling with the antibody RT97, a good marker for ganglion cell axons in several species, in the normal and regenerating visual pathways of teleosts. We have demonstrated that RT97 antibody recognizes several proteins in the tench visual system tissues (105, 115, 160, 200, 325 and 335 kDa approximately). By using immunoprecipitation and Western blot we have found that after crushing the optic nerve the immunoreactivity to anti RT97 increased markedly in the optic nerve. In immunohistochemical analysis we also found a different pattern of labeling in normal and regenerating visual pathways. In normal tench RT97 is a good marker for the horizontal cells in the retina, for growing ganglion cell axons which run along the optic nerve from the retina to the optic tectum and of the axon terminals in the stratum opticum and stratum fibrosum and griseum superficiale in the optic tectum. After optic nerve crush, no immunohistochemistry modifications were observed in the retina. However, in accordance with Western blot experiments, in the optic nerve intensely stained groups of regenerating axons appeared progressively throughout the optic nerve as far as the optic tectum. We conclude that the antibody RT97 is an excellent marker of growing and regenerating axons of the optic nerve of fish.