Normal distribution of VGLUT1 synapses on spinal motoneuron dendrites and their reorganization after nerve injury.

Peripheral nerve injury induces permanent alterations in spinal cord circuitries that are not reversed by regeneration. Nerve injury provokes the loss of many proprioceptive IA afferent synapses (VGLUT1-IR boutons) from motoneurons, the reduction of IA EPSPs in motoneurons, and the disappearance of stretch reflexes. After motor and sensory axons successfully reinnervate muscle, lost IA VGLUT1 synapses are not re-established and the stretch reflex does not recover; however, electrically evoked EPSPs do recover. The reasons why remaining IA synapses can evoke EPSPs on motoneurons, but fail to transmit useful stretch signals are unknown. To better understand changes in the organization of VGLUT1 IA synapses that might influence their input strength, we analyzed their distribution over the entire dendritic arbor of motoneurons before and after nerve injury. Adult rats underwent complete tibial nerve transection followed by microsurgical reattachment and 1 year later motoneurons were intracellularly recorded and filled with neurobiotin to map the distribution of VGLUT1 synapses along their dendrites. We found in control motoneurons an average of 911 VGLUT1 synapses; ~62% of them were lost after injury. In controls, VGLUT1 synapses were focused to proximal dendrites where they were grouped in tight clusters. After injury, most synaptic loses occurred in the proximal dendrites and remaining synapses were declustered, smaller, and uniformly distributed throughout the dendritic arbor. We conclude that this loss and reorganization renders IA afferent synapses incompetent for efficient motoneuron synaptic depolarization in response to natural stretch, while still capable of eliciting EPSPs when synchronously fired by electrical volleys.

[Expression and distribution of NGF and p75 during rabbit tibial nerve repair induced by human hair keratin conduits].

OBJECTIVE: To study the expression and distribution of nerve growth factor (NGF) and low-affinity neurophin receptor p75 during human hair keratin conduit-induced repair of rabbit tibial nerves. METHODS: Rabbit tibial nerves were transected and connected by either routine suture or by conduits made of human hair keratin (HHK), and after different time periods, paraffin-embedded sections of the nerve tissue at the damaged sites and the adjacent tissues, with normal rabbit tibial nerve sections as control, were prepared for immunohistochemistry. RESULTS: No positive NGF staining was observed in normal tibial nerve tissues, but 76 days after the surgery, strong NGF positivity was detected in the newly generated nerve tissue around the HHK implants until 100 days after the surgery, which was absent in the tissues around the suture. As for p75, there was no positive staining observed in normal tibial nerve tissue. Light positive p75 staining was found in the mature nerve tissues around the HHK implants, where the newly generated tissues were strongly p75-positive during the period between 76-and loo-days after surgery. CONCLUSIONS: HHK and its degenerative product, but not routine suture, can induce the production of NGF and p75 to create a favorable micro-environment for nerve regeneration. More NGF and p75 are produced in newly generated neurons than in mature ones.

The effect of pre and postnatal cadmium exposure on somatosensory evoked potentials: relation to lipid peroxidation.

Pregnant swiss albino rats were divided into three groups: control (C), gestational exposure of cadmium (G-Cd) and gestational/postnatal exposure of cadmium (GP-Cd) groups. Control animals received tap water and the rats of GP-Cd group received Cd as CdCl2 in their drinking water during the experimental period. G-Cd group was given Cd during pregnancy, but given tap water after birth. Twenty-two days after birth, 15 rats (for each group) were taken from their mothers and continued to be treated with Cd (GP-Cd group) or tap water (C and G-Cd groups) for an additional 38 days. On postnatal day (PND) 60, somatosensory evoked potentials (SEPS) of three groups were recorded following left posterior tibial nerve (PTN) stimulation. The mean latencies of N1, P1, and N2, components were significantly prolonged in both Cd groups compared with control group. The mean latency of N1 in the GP-Cd group was longer than control and the G-Cd groups. There was no significant amplitude differences among groups. On the other hand, thiobarbituric acid reactive substances (TBARS), an indicator of lipid peroxidation, were increased in the sciatic nerves of both groups compared with control group. A significant increase in the TBARS level of the brain was found only in GP-Cd group due to significant accumulation of Cd.

Intraneural angiosarcoma and angiosarcoma arising in benign and malignant peripheral nerve sheath tumours: clinicopathological and immunohistochemical analysis of four cases.

AIMS: Angiosarcomatous differentiation represents the least common form of heterologous differentiation in malignant peripheral nerve sheath tumours (MPNST), and is seen most frequently in patients with neurofibromatosis type 1. More rarely, it has been reported in patients without stigmata of neurofibromatosis, or in benign nerve sheath tumours and peripheral nerves. This study was undertaken to confirm this rare association. METHODS AND RESULTS: Four cases of angiosarcoma arising in a peripheral nerve, in a long-standing schwannoma and in two MPNST are described. Immunohistochemical studies were performed on paraffin sections with the alkaline phosphatase-antialkaline phosphatase method. An intraneural high-grade epithelioid angiosarcoma arose in the left posterior tibial nerve of a 78-year-old man, a well to moderately differentiated angiosarcoma was seen in an ancient schwannoma of the lateral neck in a 73-year-old women, and an angiosarcoma of varying grades of differentiation developed in a recurrent MPNST in the thigh of 38-year-old man. In addition a high-grade MPNST in the axillary region of a 30-year-old man showed foci of heterologous high-grade angiosarcomatous differentiation. The neural and endothelial lines of differentiation were confirmed in each case by positive immunohistochemical staining for neural and endothelial markers, respectively. In all cases tested, the neural differentiated cells stained immunohistochemically positive for antibodies against vascular endothelial growth factor. CONCLUSIONS: This study confirms the rare association of angiosarcoma arising in peripheral nerves, as well as in benign and malignant peripheral nerve sheath tumours.

Neural architecture in transected rabbit sciatic nerve after prolonged nonreinnervation.

Observations have been made on the rabbit sciatic nerve distal to a transection, with survival periods of up to 26 mo and prevention of reinnervation. It was confirmed that the nerve becomes compartmented by fibroblast processes and that a zone of fine collagen fibrils develops around the Schwann cell columns that constitute the Büngner bands. The Schwann cells become progressively more atrophic but after 6 mo of denervation still expressed low affinity p75 nerve growth factor receptor (NGFR), the latest stage at which this was examined. NGFR was also expressed by the processes of the fibroblasts producing the endoneurial compartmentation. By 26 mo after transection the site of previous nerve fibres was indicated by sharply demarcated domains of approximately circular outline in transverse section consisting of densely packed longitudinally oriented collagen fibrils. Some of these domains still possessed centrally situated Schwann cells or residual basal lamina but many were acellular. The central collagen fibrils in these domains were of smaller diameter than those situated peripherally but were of larger size than those that form around the Büngner bands during wallerian degeneration. The peripherally located fibrils in the domains were of the same calibre as for normal endoneurial collagen. The collagen domains were encircled by fibroblast processes or at times enclosed in a perineurial cell ensheathment. Long-standing axonal loss therefore leads to a striking reorganisation of the internal architecture of peripheral nerve trunks. The findings may be relevant for the interpretation of the appearances in chronic peripheral neuropathies in man.

Complex regional pain syndrome type I (RSD): pathology of skeletal muscle and peripheral nerve.

BACKGROUND: Reflex sympathetic dystrophy (RSD) (recently reclassified as complex regional pain syndrome type I) is a syndrome occurring in extremities and, when chronic, results in severe disability and untractable pain. RSD may be accompanied by neurologic symptoms even when there is no previous neurologic lesion. There is no consensus as to the pathogenic mechanism involved in RSD. To gain insight into the pathophysiology of RSD, we studied histopathology of skeletal muscle and peripheral nerve from patients with chronic RSD in a lower extremity. METHODS: In eight patients with chronic RSD, an above-the-knee amputation was performed because of a nonfunctional limb. Specimens of sural nerves, tibial nerves, common peroneal nerves, gastrocnemius muscles, and soleus muscles were obtained from the amputated legs and analyzed by light and electron microscopy. RESULTS: In all patients, the affected leg showed similar neurologic symptoms such as spontaneous pain, hyperpathy, allodynia, paresis, and anesthesia dolorosa. The nerves showed no consistent abnormalities of myelinated fibers. In four patients, the C-fibers showed electron microscopic pathology. In all patients, the gastrocnemius and soleus muscle specimens showed a decrease of type I fibers, an increase of lipofuscin pigment, atrophic fibers, and severely thickened basal membrane layers of the capillaries. CONCLUSION: In chronic RSD, efferent nerve fibers were histologically unaffected; from afferent fibers, only C-fibers showed histopathologic abnormalities. Skeletal muscle showed a variety of histopathologic findings, which are similar to the histologic abnormalities found in muscles of patients with diabetes.

Pathogenesis of the neurotoxicity caused by anti-GD2 antibody therapy.

After treatment of melanomas with anti-GD2 monoclonal antibody (MAb) (14G2a), some patients develop sensorimotor demyelinating polyneuropathy with and without the syndrome of inappropriate antidiuretic hormone (SIADH). To clarify what causes the neurotoxicity of anti-GD2 MAb, we investigated the immunohistochemical localization of GD2 in the human nervous system. Anti-GD2 MAb (14G2a) reacted with the myelin sheaths in the peripheral nerves as well as with the pituicyte cytoplasm in the posterior lobe of the pituitary gland. We assume that the binding of anti-GD2 MAb to peripheral nerve myelin and the pituicytes in the posterior pituitary causes sensorimotor demyelinating neuropathy and SIADH.

L-carnitine and acetyl-L-carnitine in human nerves from normal and diabetic subjects.

Marked reduction in the contents of L-carnitine and acetyl-L-carnitine has been reported in peripheral nerves of rats with experimental diabetes. Since these substances have been claimed to improve a number of signs and symptoms of peripheral neuropathy in controlled clinical trials, this study was aimed at assessing whether nerves from diabetic subjects would also reveal similar decrease in the concentration of L-carnitine and acetyl-L-caritine. To this end, these substances were measured in nerves obtained from 11 patients with diabetic neuropathy, 13 patients with ischemic non-diabetic neuropathy, and 12 normal controls. Nerves from patients with either diabetic neuropathy and ischemic non-diabetic neuropathy showed levels of both carnitines lower than those from normal controls. However, differences among the three groups were not statistically significant, indicating that a reduction in these amino acids probably represents only a co-factor in the development of the variegated clinical picture of human diabetic neuropathy.

Acrylamide disrupts elemental composition and water content of rat tibial nerve. III. Recovery.

We have previously demonstrated that subacute and subchronic acrylamide (ACR) intoxication are associated with a loss of subcellular elemental regulation in myelinated axons and Schwann cells of rat tibial nerve (LoPachin et al., Toxicol. Appl. Pharmacol. 115, 21-34, 1992; LoPachin et al., Toxicol. Appl. Pharmacol. 115, 35-43, 1992). In the present study, rats were allowed to recover partially from subchronic oral ACR intoxication (2.8 mM in drinking water for approximately 30 days). Elemental composition and water content of tibial nerve myelinated axons and Schwann cells were measured by electron probe X-ray microanalysis. Results show that K and Cl concentrations in larger tibial nerve axons were shifted toward normal values or above. For the most part, small axons also exhibited elemental changes that reflected recovery from ACR intoxication. Mitochondria displayed elemental changes that were similar to corresponding axoplasm. Schwann cells in tibial nerve of recovering animals had altered Na, P, Cl, K, and Mg concentrations that were similar in magnitude and extent to those occurring during ACR intoxication. In contrast, myelin displayed few changes. These results suggest that the recovery process following ACR intoxication is associated with characteristic changes in subaxonal elemental composition that might be related to repair mechanisms. That recovery-related elemental changes differ from those associated with intoxication provides additional support for the hypothesis (LoPachin et al., Toxicol. Appl. Pharmacol. 115, 21-34, 1992) that perturbation of elemental regulation is a specific component of ACR neurotoxicity. The observation of persistent Schwann cell disruption during recovery might reflect either long-term secondary consequences or delayed recovery from direct injury. Further studies are necessary to resolve this issue.

Identification of G protein subtypes in peripheral nerve and cultured Schwann cells.

In this study, we investigated the expression of various G proteins in whole sciatic nerves, in myelin and nonmyelin fractions from these nerves, and in membranes of immortalized Schwann cells. In myelin, nonmyelin, and Schwann cell membranes we detected two 39-40-kDa pertussis toxin substrates that were resolved on separation on urea-gradient gels. Two cholera toxin substrates with apparent molecular masses of 42 and 47 kDa were present in nerve and brain myelin and in Schwann cell membranes. In these membranes, a third 45-kDa cholera toxin substrate, which displayed the highest labeling, was also present. Immunoblotting with specific antisera allowed the identification of G(o) alpha, Gi1 alpha, Gi2 alpha, Gi3 alpha, Gq/G11 alpha, and the two isoforms of Gs alpha in nerve homogenates, nerve, and brain myelin fractions. In Schwann cell membranes we identified G(o) alpha, Gi2 alpha, Gi3 alpha, and proteins from the Gq family, but no immunoreactivity toward anti-Gi1 alpha antiserum was detected. In these membranes, anti-Gs alpha antibody recognized the three cholera toxin substrates mentioned above, with the 45-kDa band displaying the highest immunoreactivity. Relative to sciatic nerve myelin, the Schwann cell membranes revealed a significantly higher expression of Gi3 alpha and the absence of Gi1 alpha. The different distribution of G proteins among the different nerve compartments might reflect the very specialized function of Schwann cells and myelin within the nerve.

Acrylamide disrupts elemental composition and water content of rat tibial nerve. I. Myelinated axons.

The mechanism by which acrylamide (ACR) produces distal axonopathy in humans and laboratory animals is unknown. The possibility that this neuropathy involves deregulation of elements and water in rat peripheral nerve has been investigated. Electron probe X-ray microanalysis was used to measure percentages of water and concentrations (mmol element/kg dry or wet wt) of Na, P, S, Cl, K, Ca, and Mg in axoplasm and mitochondrial areas of tibial nerve axons. Results show that when rats were intoxicated with ACR by either the oral (2.8 mM in drinking water, up to 60 days) or the intraperitoneal (ip, 50 mg/kg/day x 5 or 10 days) route, a progressive loss of internodal axoplasmic K, Cl, and Na regulation was observed in subpopulations of myelinated fibers. Elemental deregulation was manifest as a shift in mean elemental content, widening of the corresponding concentration range, and a statistically significant increase in data variance. In internodal axonal regions, elemental composition of mitochondrial areas was not altered by ip ACR intoxication, whereas oral exposure was associated with delayed changes in Na, K, Cl, Ca, and Mg. In swollen axons, axoplasm and mitochondrial areas exhibited complete loss of element and water compartmentalization. This global decompartmentalization of swollen axons was quantitatively similar regardless of the route or length of ACR exposure. The results of this study suggest that a progressive loss of elemental regulation in axoplasm of myelinated tibial nerve fibers might be mechanistically related to ACR neurotoxicity.

Acrylamide disrupts elemental composition and water content of rat tibial nerve. II. Schwann cells and myelin.

The effects of subchronic and subacute acrylamide (ACR) intoxication on elemental composition (Na, P, S, Cl, K, Ca, Mg) and water content of Schwann cell body cytoplasm and myelin were assessed in rat tibial nerve. Electron probe X-ray microanalysis demonstrated that, in control rats, peripheral nerve glia and myelin exhibited highly characteristic distributions of elements and water and that ACR intoxication was associated with disruption of this normal subcellular distribution. When rats were intoxicated with ACR by either the oral (2.8 mM in drinking water for 15, 22, 30, and 60 days) or the intraperitoneal (50 mg/kg/day x5 and 10 days) route, an exposure-dependent loss of cytoplasmic Na, K, P, Cl, Mg, and water regulation was detected in Schwann cell cytoplasm. Maximum development of elemental deregulation occurred after 30 days of oral ACR exposure and 10 days of ip treatment. The cytoplasmic elements involved and their corresponding quantitative changes were similar regardless of the route of ACR intoxication. Analysis of myelin revealed that both oral and parenteral ACR exposure caused early, persistent increases in dry weight Na, P, and water content. However, Cl dry weight concentrations were increased by oral exposure and decreased by ip ACR injection. Results of this study indicate that ACR intoxication is associated with a significant disturbance of subcellular element and water distribution in tibial nerve Schwann cells and myelin. The pattern of elemental disruption is typical of reversible cell damage and, therefore, Schwann cell injury might play a role in the expression of ACR neurotoxicity.

Distribution of elements and water in peripheral nerve of streptozocin-induced diabetic rats.

Accumulating evidence suggests that alterations in Na, Ca, K, and other biologically relevant elements play a role in the mechanism of cell injury. The pathogenesis of experimental diabetic neuropathy is unknown but might include changes in the distribution of these elements in morphological compartments. In this study, this possibility was examined via electron-probe X-ray microanalysis to measure both concentrations of elements (millimoles of element per kilogram dry or wet weight) and cell water content (percent water) in frozen, unfixed, unstained sections of peripheral nerve from control and streptozocin-induced diabetic rats. Our results indicate that after 20 wk of experimental diabetes, mitochondria and axoplasm from myelinated axons of proximal sciatic nerve displayed diminished K and Cl content, whereas in tibial nerve, the intraaxonal levels of these elements increased. In distal sciatic nerve, mitochondrial and axoplasmic levels of Ca were increased, whereas other elemental alterations were not observed. These regional changes resulted in a reversal of the decreasing proximodistal concentration gradients for K and Cl, which exist in nondiabetic rat sciatic nerve. Our results cannot be explained on the basis of altered water. Highly distinctive changes in elemental distribution observed might be a critical component of the neurotoxic mechanism underlying diabetic neuropathy.