Painful nerve injury upregulates thrombospondin‐4 expression in dorsal root ganglia

Thrombospondin‐4 (TSP4) belongs to a family of large, oligomeric extracellular matrix glycoproteins that mediate interactions between cells and interactions of cells with underlying matrix components. Recent evidence shows that TSP4 might contribute to the generation of neuropathic pain. However, there has been no systematic examination of TSP4 expression in the dorsal root ganglia (DRG) after injury. This study, therefore, investigates whether TSP4 protein level is changed in DRG after injury following spinal nerve ligation (SNL) and spared nerve injury in rats by performing Western blotting, immunohistochemistry, and immunocytochemistry. After nerve ligation, TSP4 protein level is upregulated in the axotomized somata of the fifth lumbar (L5) DRG. There is substantial additional TSP4 in the nonneuronal compartment of the L5 DRG that does not costain for markers of satellite glia, microglia, or Schwann cells and appears to be in the interstitial space. Evidence of intracellular overexpression of TSP4 persists in neurons dissociated from the L5 DRG after SNL. These findings indicate that, following peripheral nerve injury, TSP4 protein expression is elevated in the cytoplasm of axotomized sensory neurons and in the surrounding interstitial space. © 2014 Wiley Periodicals, Inc.     

trkB-like immunoreactivity in rat dorsal root ganglia following sciatic nerve injury

The expression of full-length trkB protein, the functional high affinity receptor for BDNF and NT-4, was examined by immunohistochemistry in adult rat L4–L5 dorsal root ganglia after different types of sciatic nerve lesions. In normal ganglia, 52.5% of the neurons showed trkB-like immunoreactivity. Size measurements demonstrated that trkB-like immunoreactivity was seen predominantly in small- and medium-sized cells. This was confirmed by the finding that 28% of all trkB-positive neurons showed affinity to RT97, an antibody which lanels a neurofilament epitope specific for medium-sized and large primary afferent neurons. After crush, section or neuroma formation of the sciatic nerve, the proportion of trkB-positive cells was 64.5%, 58% and 61.9%, respectively. Since trkB-receptors are present in regenerating primary afferent neurons, these data could indicate that BDNF and/or NT-4 are involved in sensory nerve fiber regeneration after adult injury.     

Fibrous meningeal tumours with extensive non-calcifying collagenous whorls and glial fibrillary acidic protein expression: the whorling-sclerosing variant of meningioma

Meningiomas comprise a wide range of morphological patterns. We describe unusual fibrous meningeal tumours in two patients, composed of extensive non-calcifying collagenous whorls of varying size, resembling non-calcified psammoma bodies, while interposed tumour cells are sparse. Immunohistochemistry showed expression of S-100, vimentin and glial fibrillary acidic protein, whereas only single tumour cells stained for epithelial membrane antigen. Electron microscopy detected desmosomes or desmosome-like structures in both specimens. We conclude that these tumours represent a peculiar whorling-sclerosing variant of fibrous meningioma. Recognition of this meningioma variant is important in the differential diagnosis of meningioma versus other fibrous tumours of the meninges, including solitary fibrous tumours of the meninges, unusual forms of desmoplastic gliomas or chondroid tumours.     

周围神经损伤后脊神经节感觉神经元胞体形态学的变化

目的 研究周围神经损伤后脊神经节感觉神经元胞体形态学的变化以探讨其主要死广性质。方法 切断并原位吻合大鼠右侧坐骨神经，左侧不作任何处理，作为对照；于术后不同时间取L4-L6脊神经节作光镜和电镜观察，观察脊神经节感觉神经元胞体形态的变化。结果 光镜下，损伤的脊神经节感觉神经元胞体染色质浓染；电镜下，细胞膜内陷，分割细胞内容物成凋亡小体；而对侧脊神经节感觉神经元胞体均一、无变化。结论 大鼠坐骨神经损伤后，脊神经节感觉神经元有死亡，其胞体的形态学变化符合细胞凋亡特征。     

Taurine in rat posterior pituitary: Localization in astrocytes and selective release by hypoosmotic stimulation

Taurine, a γ-aminobutyric acid (GABA)-like acidic amino acid, has previously been shown to be prominently localized to astrocytes in the supraoptic nucleus, the neurons of which contain only small amounts, and to have inhibitory actions on supraoptic neuronal activity. In the present study, taurine distribution in the neurohypophysis was determined by using a well-characterized monoclonal antibody against taurine itself. Preembedding immunohistochemistry was performed at light and electron microscopic levels by using diaminobenzidine and gold-substituted silver-intensified peroxidase (GSSP) methods. At the light microscopic level, the distribution pattern and cellular localization of taurine immunoreactivity corresponded to that of glial fibrillary acidic protein. Pituicyte cell bodies and processes displayed dense taurine immunoreactivity. Electron microscopic observations revealed strong taurine GSSP reactions in these neural lobe astrocytes, but weak taurine reactivity was seen within only some neurosecretory axons. High-performance liquid chromatography analyses demonstrated that in vitro hypoosmotic stimulation (reduction of 40 mOsm/kg) of isolated posterior pituitaries resulted in preferential increases in taurine release into the bathing medium without increased release of other amino acids. Conversely, tissue concentrations of taurine significantly decreased with hypoosmotic perfusion, while glutamate, glutamine, and GABA concentrations were not reduced. These results indicate that taurine is mainly concentrated in neurohypophysial astrocytes, which are known to engulf the neurosecretory axonal processes and terminals. Taurine released from pituicytes under basal and hypoosmotic conditions may act to suppress axon terminal depolarization and thereby depress release of neurohypophysial peptides.J.Comp. Neurol. 381:513-523, 1997. © 1997 Wiley-Liss, Inc.     

Chronic nerve compression injury induces a phenotypic switch of neurons within the dorsal root ganglia

Chronic nerve compression (CNC) injury initiates a series of pathological changes within the peripheral nerve at the site of injury. However, to date, little work has been performed to explore neuronal cell body responses to CNC injury. Here we show a preferential upregulation of growth-associated protein-43 (GAP-43) and enhanced Fluoro Ruby uptake by the small-diameter calcitonin gene-related protein (CGRP) and isolectin B4 (IB4)-positive neurons in the L4 and L5 ipsilateral dorsal root ganglion (DRG) 2 weeks and 1 month post injury. Furthermore, L4 and L5 DRGs ipsilateral to CNC injury also demonstrated a marked reduction in neurofilament 200 (NF-200) neurons and an increase in CGRP and IB4 neurons at early time points. All numbers normalized to values comparable to those of control when the DRG was evaluated 6 months post injury. Quantification of glial-derived neurotrophic factor (GDNF) protein revealed an upregulation in L4 and L5 DRG followed by a return to baseline values at later stages following injury. Upregulation of GDNF expression by Schwann cells was also readily apparent with both immunohistochemistry and Western blot analysis of 1 month compressed sciatic nerve specimens. Thus, CNC induces a phenotypic change in the DRG that appears to be temporally associated with increases in GDNF protein expression at and near the site of the compression injury in the nerve.     

Evidence of substance P immunoreactive neurons in dorsal root ganglia and vagal ganglia projecting to the guinea pig pylorus

The origin of extrinsic substance P fibers in the guinea pig pyloric wall was investigated by combining retrograde axonal tracing and indirect immunofluorescence techniques. After injection of Fast Blue into the pyloric wall labeled cells were found in the T7-T9 dorsal root ganglia and the nodose and jugular ganglia. About 60% of the labeled cells in the dorsal root ganglia contained substance P-like immunoreactivity. After local application of colchicine, a few substance P positive cells were observed in the nodose and jugular ganglia, some of which also contained Fast Blue.     

鞘内注射胶质细胞抑制剂对背根神经节压迫大鼠脊髓星形胶质细胞的影响

BACKGROUND： Astrocytes are considered to provide nutritional support in the central nervous system. However, recent studies have confirmed that astrocytes also play an important role in chronic pain.
OBJECTIVE： To investigate the effects of intrathecal injection of fluorocitrate, minocycline or both on astrocyte activation and proliferation in the spinal dorsal horn of compressed dorsal root ganglion in rats.
DESIGN, TIME AND SETTING： The neurology randomized controlled animal study was performed at the Jiangsu Institute of Anesthesia Medicine, from September 2006 to April 2007. MATERIALS： A total of 96 male Sprague Dawley rats, aged 6-8 weeks, were selected for this study. Following intrathecal catheterization, 80 rats underwent steel bar insertion into the L4-5 intervertebral foramina to make a stable compression on the L4-5 posterior root ganglion. Thus rat models of ganglion compression were established. Minocycline and fluorocitrate were purchased from Sigma, USA.
METHODS： A total of 96 rats were randomly and equally divided into six groups. Rat L4, L5 transverse process and intervertebral foramina were exposed in the sham operation group, but without steel bar insertion. The model group did not receive any manipulations. Rats in the phosphate buffered saline （PBS） group were intrathecally injected with 0.01 mmol/L PBS （20 μL）. Rats in the fluorocitrate group were subjected to 1 μmol/L fluorocitrate （20 μL）. Rats in the minocycline group were intrathecally injected with 5 g/L minocycline （20 μL）. Rats in the minocycline and fluorocitrate group received a mixture （20 μL） of 5 g/L minocycline and 1 μmol/L fluorocitrate. Following model establishment, drugs were administered once a day.
MAIN OUTCOME MEASURES： At 7 and 14 days following model induction, glial fibrillary acidic protein expression in the spinal dorsal horn was measured by immunofluorescence microscopy. Six sections with significant glial fibrillary acidic protein -positive expression were obtained to count astrocytes under an inverted microscope.
RESULTS： No significant differences in astrocyte count were detected between the fluorocitrate and model groups. Cell bodies were small with a few processes in the fluorocitrate group, compared with the model group. The astrocyte count decreased significantly in the minocycline group and the minocycline and fluorocitrate group compared with the sham operation, model, PBS and fluorocitrate groups （P 〈 0.01）. The decrease in astrocyte count was mainly found in layers Ⅲ–Ⅳ of the spinal dorsal horn. Cell body volume was smaller and process numbers were fewer in the minocycline group and the minocycline and fluorocitrate group, compared with the model and PBS groups.
CONCLUSION： Fluorocitrate can inhibit astrocyte activation, but does not affect astrocyte proliferation. However, minocycline can inhibit the activation and proliferation of astrocytes.     

Immunocytochemical localization of glial fibrillary acidic protein (GFAP) in the area postrema of the cat. Light and electron microscopic study

Glial fibrillary acidic protein (GFAP) was demonstrated in the cytoplasm and processes of ependymal cells and astroglial components of the area postrema of the cat. These observations differ from the findings in the ependyma of the ventricular cavities which are consistently negative for the protein. Since some studies have suggested sensory functions of the glial cells in this emetic chemoreceptor trigger zone, a careful consideration of morphological and biochemical attributes of these cells seems appropriate.     

BDNF is involved in sympathetic sprouting in the dorsal root ganglia following peripheral nerve injury in rats

Peripheral nerve injury results in sympathetic sprouting around large diameter sensory neurons in the dorsal root ganglia (DRG). The mechanism underlying this pathological phenomenon is not known. Brain-derived neurotrophic factor (BDNF) is up-regulated in large sensory neurons and ensheathing satellite cells following a sciatic nerve injury. In the present study, we investigated the effects of BDNF on the sympathetic sprouting in the DRG, by delivering BDNF antibody or antisense oligodeoxynucleotide to injured DRGs, or by delivering exogenous BDNF to intact DRGs. The sheep antibody to BDNF, characterized by bioassays and dot blots, specifically reacted with BDNF but not other neurotrophins. Noradrenergic fibers were visualized by immunostaining of tyrosine hydroxylase (TH) and quantified by an NIH Imaging program. Two weeks following L5 spinal nerve lesion, a dramatic increase in TH-immunoreactive (-ir) fibres was observed in both ipsi- and contralateral DRGs in normal sheep IgG treated rats. BDNF antibody significantly reduced the sprouting of sympathetic nerves in both ipsi- and contra-lateral DRGs by 67% and 42% respectively. BDNF antisense oligodeoxynucleotide, by inhibiting BDNF synthesis in DRGs, also significantly suppressed the sprouting by 67% and 60% respectively in the ipsi- and contra-lateral DRGs. Delivery of exogenous BDNF into an intact L5 DRGs resulted in an increase in the sprouting by 4.2-fold. Our results clearly indicate that BDNF, synthesized in and secreted from the DRGs, is involved in the sympathetic sprouting in the DRG following the peripheral nerve injury.     

BDNF is involved in sympathetic sprouting in the dorsal root ganglia following peripheral nerve injury in rats

Peripheral nerve injury results in sympathetic sprouting around large diameter sensory neurons in the dorsal root ganglia (DRG). The mechanism underlying this pathological phenomenon is not known. Brain-derived neurotrophic factor (BDNF) is up-regulated in large sensory neurons and ensheathing satellite cells following a sciatic nerve injury. In the present study, we investigated the effects of BDNF on the sympathetic sprouting in the DRG, by delivering BDNF antibody or antisense oligodeoxynucleotide to injured DRGs, or by delivering exogenous BDNF to intact DRGs. The sheep antibody to BDNF, characterized by bioassays and dot blots, specifically reacted with BDNF but not other neurotrophins. Noradrenergic fibres were visualized by immunostaining of tyrosine hydroxylase (TH) and quantified by an NIH Imaging program. Two weeks following L5 spinal nerve lesion, a dramatic increase in TH-immunoreac-tive (-ir) fibres was observed in both ipsi- and contralateral DRGs in normal sheep IgG treated rats. BDNF antibody significantly reduced the sprouting of sympathetic nerves in both ipsi- and contra-lateral DRGs by 67% and 42% respectively. BDNF antisense oligodeoxynucleotide, by inhibiting BDNF synthesis in DRGs, also significantly suppressed the sprouting by 67% and 60% respectively in the ipsi- and contralateral DRGs. Delivery of exogenous BDNF into an intact L5 DRGs resulted in an increase in the sprouting by 4.2-fold. Our results clearly indicate that BDNF, synthesized in and secreted from the DRGs, is involved in the sympathetic sprouting in the DRG following the peripheral nerve injury.     

Axotomy-induced neuronal death and reactive astrogliosis in the lateral geniculate nucleus following a lesion of the visual cortex in the rat

Following a unilateral lesion of the visual cortex (cortical areas 17, 18, and 18a) in adult rats, neurons in the ipsilateral dorsal lateral geniculate nucleus (LGN) are axotomized, which leads to their atrophy and death. The time course of this neuronal degeneration was studied quantitatively, and the astroglial response was examined with glial fibrillary acidic protein immunohistochemistry. More than 95% of the neurons in the ipsilateral LGN survive during the first 3 days following a lesion of the visual cortex. However, in the next 4 days, massive neuronal death ensues, reducing the number of surviving neurons to approximately 33% of normal by the end of the first postoperative week. Between 2 weeks and 24 weeks postoperatively, the number of neurons present in the LGN declines very gradually from 34% to 17% of normal. Three days after a lesion of the visual cortex, the mean cross-sectional areas of ipsilateral LGN neurons are 13% smaller than normal (87%). By 1 week after the operation, surviving LGN neurons have atrophied to 66% of their normal area. Subsequently, the size of surviving neurons declines slowly to approximately 50% of normal at 24 weeks after the cortical lesion. Astrocytes in the ipsilateral LGN also react to cortical damage. At 1 day after a lesion of the visual cortex, glial fibrillary acidic protein immunoreactivity in the LGN is almost undetectable, but a distinct increase in immunoreactivity is seen at 3 days. Immunoreactivity peaks between 1 week and 2 weeks postoperatively and, thereafter, remains intense for at least 24 weeks. Thus, following a lesion of the visual cortex, the somata of neurons in the LGN remain essentially normal morphologically for about 3 days before the onset of rapid atrophy and death. Moreover, most of the neural cell death that occurs in the LGN after axotomy takes place in the last half of the first postoperative week. J. Comp. Neurol. 392:252–263, 1998. © 1998 Wiley-Liss, Inc.     

Selective degeneration of dorsal root ganglia and dorsal nerve roots in methyl mercury-intoxicated rats: a stereological study

The components of the nervous system of rats that are most critically affected by methyl mercury are still a matter of debate. A recent stereological study of rats with typical symptoms resulting from methyl mercury intoxication demonstrated that the morphology of cerebellar granule cells and Purkinje cells were unchanged at the light microscopic level, even though there was pronounced degeneration of myelinated axons in dorsal nerve root nerves. In the present study, unbiased stereological methods were used to quantify morphological changes in the dorsal root ganglion, and dorsal and ventral nerve roots of the rats used in the previous study. The rats were treated with methyl mercury (2 mg daily/kg, per os) for a 19-day period that was followed by a 32-day period without treatment. The means of the total numbers of A-cell and B-cell perikarya in the dorsal root ganglion of the intoxicated rats were reduced by 60% and 24%, respectively. The mean volume of A-cell perikarya in rats of the experimental group was reduced by 22%, whereas the mean volume of B-cell perikarya was the same in the two groups. In the experimental group, the total number of myelinated axons in the dorsal nerve roots was reduced by 60%, whereas no difference was found in the ventral nerve roots. The areas of axon and myelin sheath, dorsal and ventral nerve roots were not affected. This study demonstrates that extensive loss of dorsal root ganglion cells and myelinated axons in dorsal nerve roots precedes light microscopical changes in the ventral nerve roots and the cerebellum of rats intoxicated with methyl mercury. Received: 16 January 1998 / Revised, accepted: 23 February 1998     

Neuropathology and blood flow of nerve, spinal roots and dorsal root ganglia in longstanding diabetic rats

Vascular perfusion and neuropathologic evaluation of the lumbar spinal roots and dorsal root ganglia (DRG) were studied in rats with longstanding (duration 12–15 months) streptozotocin-induced diabetes and age- and sex-matched control rats. We also undertook nerve conduction studies including F-wave recordings and measured blood flow in sciatic nerve, DRG, and superior cervical ganglion (SCG). Light microscopically, changes of the myelin sheath in the dorsal and ventral roots and vacuolated cells in the DRG were the major findings, being significantly higher in diabetic rats than in control rats. The effects of the diabetic state on myelin splitting were greater in the dorsal than ventral roots. Electron microscopic studies revealed a gradation of changes in myelin from mild separation to severe ballooning of myelin with relative axonal sparing. DRG cells showed vacuoles of all sizes with cristae-like residues, suggestive of mitochondria. These findings suggest that diabetes mellitus has a dual effect; it accelerates the normal age-related degenerative changes in the spinal roots and DRG, and it also has a selective effect on the sensory neuron. Nerve conduction studies showed markedly reduced conduction velocities in the distal nerve segments and prolonged F-wave latency and proximal conduction time despite the shorter conduction pathway in diabetic rats. Blood flow, which was measured using iodo[¹⁴C]antipyrine autoradiography, was significantly reduced in the sciatic nerves, DRG, and SCG of diabetic rats. We suggest that the combination of hyperglycemia and ischemia results in oxidative stress and a predominantly sensory neuropathy. Received: 26 February 1996 / Revised: 12 June 1996 / Revised, accepted: 13 August 1996     

Localization of NADPH-diaphorase-containing neurons in sensory ganglia of the rat

The presence of reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase activity was studied histochemically in the sensory ganglia of the rat. Supraspinally, the trigeminal ganglion possessed only a few cells positively stained for NADPH-diaphorase, while a large number of positive neurons was found in the nodose ganglion. In the dorsal root ganglia, the distribution of positive cells showed a peculiar pattern in relation to spinal levels. Very minor populations (less than 2% of the total ganglionic cells) exhibited positive reaction in ganglia at levels ranging from the first cervical (C1) to fourth thoracic (T4) and from the second lumber (L2) through the entire sacral levels. In the middle to lower thoracic levels (from T5 to L1), however, abundant diaphorase-positive cells were observed. From these positive neurons it was possible to trace intensely stained nerve fibers. In the lower thoracic level, for example, dense positive fibers were seen in the ramus communicans. Retrograde tracing studies revealed that diaphorase-containing neurons in the lower thoracic level project at least partly to the gastric wall and the celiac ganglion. These results indicate that the diaphorase-positive ganglionic neurons in the thoracicolumbar levels may carry autonomic visceral afferent information. Double staining with NADPH-diaphorase histochemistry and peptide immunohistochemistry revealed that NADPH-diaphorase colocalizes with calcitonin gene-related peptide and substance P in many of these visceral afferent neurons.     

Systemic hypothermia following compression injury of rat spinal cord: An immunohistochemical study on the expression of vimentin and GFAP

The objective of the present study was to determine whether systemic hypothermia up-regulated vimentin and glial fibrillary acidic protein (GFAP) expression in the spinal cord as a result of trauma in normothermic rats. Experimental rats underwent thoracic laminectomy alone or laminectomy plus severe spinal cord compression, with or without reduction of core temperature. In hypothermic rats without trauma, vimentin was expressed in ependymal cells and a few intramedullary vessels but not in capillaries. Normothermic rats with trauma exhibited a marked up-regulation of vimentin in the capillaries and in reactive astrocytes in all thoracic segments of the spinal cord. Expression of vimentin was significantly reduced in hypothermic rats with trauma. Expression of GFAP was reduced in hypothermic rats without trauma. However, trauma induced up-regulation of GFAP in astrocytes in all thoracic segments. Hypothermia reduced this response in rats with trauma but to a lesser extent than vimentin. These results suggest that systemic hypothermia reduces the up-regulation of vimentin in capillaries and reactive astrocytes in injured normothermic rats and support the hypothesis that systemic hypothermia influences microvascular and astrocyte responses in the peri-injury zones following spinal cord compression trauma.     

Griffonia simplicifolia isolectin B4 identifies a specific subpopulation of angiogenic blood vessels following contusive spinal cord injury in the adult mouse

After traumatic spinal cord injury (SCI), disruption and plasticity of the microvasculature within injured spinal tissue contribute to the pathological cascades associated with the evolution of both primary and secondary injury. Conversely, preserved vascular function most likely results in tissue sparing and subsequent functional recovery. It has been difficult to identify subclasses of damaged or regenerating blood vessels at the cellular level. Here, adult mice received a single intravenous injection of the Griffonia simplicifolia isolectin B4 (IB4) at 1-28 days following a moderate thoracic (T9) contusion. Vascular binding of IB4 was maximally observed 7 days following injury, a time associated with multiple pathologic aspects of the intrinsic adaptive angiogenesis, with numbers of IB4 vascular profiles decreasing by 21 days postinjury. Quantitative assessment of IB4 binding shows that it occurs within the evolving lesion epicenter, with affected vessels expressing a temporally specific dysfunctional tight junctional phenotype as assessed by occludin, claudin-5, and ZO-1 immunoreactivities. Taken together, these results demonstrate that intravascular lectin delivery following SCI is a useful approach not only for observing the functional status of neovascular formation but also for definitively identifying specific subpopulations of reactive spinal microvascular elements.