Sciatic Nerves Of Lewis Rats Research Articles

A Photosealed Cap Prevents Disorganized Axonal Regeneration and Neuroma following Nerve Transection in Rats

Background:Neuroma is a common sequela of traumatic peripheral nerve injury that can result in pain and decreased quality of life for patients. Neuromas result from axonal outgrowth in an attempt to reestablish continuity with the disrupted distal nerve end. Photosealing is a light-activated technique whereby tissues can be securely isolated in a strong and secure manner. This study investigated whether photosealing of autologous vein and crosslinked human amniotic membrane (xHAM) to cap the proximal stump of transected sciatic nerve would prevent disorganized axonal regeneration and neuroma in a rat model.Methods:The right sciatic nerve of Lewis rats (n = 27, 300–350 g) was transected 1 cm proximal to the trifurcation. Animals were randomized to one of three groups (n = 9): no further intervention (Group 1), photosealing with xHAM (Group 2), or photosealing with vein (Group 3). After 60 days, rats were euthanized and their right hindlimbs were re-explored for evidence of disorganized axonal regeneration and/or bulbous neuroma.Results:All untreated control animals were found to have protruding nerve fibers, often invading the adjacent muscle, and 33% of these control animals exhibited a bulbous neuroma. Photosealing with xHAM successfully capped 100% of nerves, with no observable axonal outgrowth. Photosealing with vein prevented axonal outgrowth in eight of nine nerves. No bulbous neuroma was found in any photosealed nerves.Conclusion:Nerve capping with photosealed xHAM or autologous vein can prevent axonal outgrowth in transected nerves, therefore decreasing the likelihood of symptomatic neuroma formation following nerve transection injury or surgical intervention.

Nerve conductions studies in experimental models of autoimmune neuritis: A meta-analysis and guideline

Nerve conduction studies (NCS) are essential to assess peripheral nerve fiber function in research models of immune-mediated neuritis. However, the current lack of standard protocols and reference values impedes data comparability across models and studies. We performed a systematic review and subsequent meta-analysis of the last 30 years of NCS of immune-mediated neuritis in Lewis-rats. Twenty-six papers met the inclusion criteria for meta-analysis. Extracted data showed considerable heterogeneity of recorded nerve conduction velocity (NCV) and compound muscle action potential (CMAP). Studies also significantly differed in terms of technical, methodical, and data reporting issues. The heterogeneity of the underlying studies emphasizes the need for standardization when conducting and reporting NCS in rats. We provide normative values for NCS of the sciatic nerve of Lewis rats and propose seven items that should be addressed when NCS are performed when studying immune paradigms in Lewis rats.

Handcrafted multilayer PDMS microchannel scaffolds for peripheral nerve regeneration.

Injuries that result in the loss of limb functionality may be caused by the severing of the peripheral nerves within the affected limb. Several bioengineered peripheral nerve scaffolds have been developed in order to provide the physical support and topographical guidance necessary for the naturally disorganized axon outgrowth to reattach to distal nerve stumps as an alternative to other procedures, like nerve grafting. PDMS has been chosen for the base material of the scaffolds due to its biocompatibility, flexibility, transparency, and well-developed fabrication techniques. The process of observing the axon outgrowth across the nerve gaps with PDMS scaffolds has been challenging due to the limited number and fineness of longitudinal sections that can be extracted from harvested nerve tissue samples after implantation. To address this, multilayer microchannel scaffolds were developed with the object of providing more refined longitudinal observation of axon outgrowth by longitudinally 'sectioning' the device during fabrication, removing the need for much of the sample preparation process. This device was then implanted into the sciatic nerves of Lewis rats, and then harvested after two and four weeks to analyze the difference in nerve regeneration between two different time periods. The present layer by layer structure, which is separable after nerve regeneration and is treated as an individual layer during the histology process, provides the details of biological events during axonal regeneration. Confocal microscopic imaging showed the details of peripheral nerve regeneration including nerve branches and growth cones observable from within the microchannels of the multilayer PDMS microchannel scaffolds.

Glial cell line‐derived neurotrophic factor is expressed by inflammatory cells in the sciatic nerves of Lewis rats with experimental autoimmune neuritis

Neurotrophic factors, including glial cell line-derived neurotrophic factor (GDNF), have been known to play a role in neuroprotection in the injured peripheral nervous system (PNS). To evaluate the involvement of GDNF in experimental autoimmune neuritis (EAN) pathogenesis, the expression of GDNF in rat sciatic nerves with EAN was studied. Western blot analysis showed that the level of GDNF protein significantly increased 1.8-fold at the paralytic stage of EAN at day 12 post-immunization (PI) (p < 0.01), and its level further increased approximately 2.5-fold at day 21 PI (p < 0.001) in the sciatic nerves of EAN-affected rats compared with those of control rats, and then declined thereafter at day 28 PI. Immunohistochemical analysis showed that axons and Schwann cells constitutively contained GDNF in normal controls. In sciatic nerves with EAN at day 12 PI, GDNF was immunostained in infiltrating inflammatory cells including macrophages and T cells. Collectively, we postulate that GDNF plays a regulatory role in EAN paralysis. A paradoxical role of inflammatory cells to ameliorate PNS inflammation remains to be further studied in EAN, an animal model of human demyelinating disease.

Distribution of Inducible Nitric Oxide Synthase and Tumor Necrosis Factor-α in the Peripheral Nervous System of Lewis Rats during Ascending Paresis and Spontaneous Recovery from Experimental Autoimmune Neuritis

Background: Inducible nitric oxide synthase (iNOS) and tumor necrosis factor-α (TNF-α) are pleiotropic molecules with widespread action in autoimmune diseases. Objective: This study characterizes the distribution of iNOS and TNF-α in the spinal nerve roots, dorsal root ganglia and sciatic nerve of Lewis rats during experimental autoimmune neuritis (EAN). Methods: Macrophages and neutrophils were identified by immunofluorescence as cellular sources of iNOS and TNF-α at various stages of EAN induced by synthetic peptide 26. Results: As the disease progressed, iNOS- and TNF-α-bearing cells gradually infiltrated the cauda equina, dorsal root ganglia, Th12–L3 spinal roots, and the sciatic nerve. A severer EAN profile developed when more iNOS- and TNF-α-bearing cells were present, and the recovery from EAN was related to the disappearance of these cells and the regeneration of nerve fibers. Conclusions: This is the first report to show iNOS- and TNF-α-immunoreactive cells in dorsal root ganglia during EAN, suggesting an underlying pathology for the neuropathic pain behavior in EAN. Our results suggest that the cells bearing iNOS and TNF-α in the different parts of the peripheral nervous system are involved in the development of the clinical signs observed at each stage of EAN.

Supplementation of acellular nerve grafts with skin derived precursor cells promotes peripheral nerve regeneration

Introduction of autologous stem cells into the site of a nerve injury presents a promising therapy to promote axonal regeneration and remyelination following peripheral nerve damage. Given their documented ability to differentiate into Schwann cells (SCs) in vitro, we hypothesized that skin-derived precursor cells (SKPs) could represent a clinically-relevant source of transplantable cells that would enhance nerve regeneration following peripheral nerve injury. In this study, we examined the potential for SKP-derived Schwann cells (SKP–SCs) or nerve-derived SCs to improve nerve regeneration across a 12 mm gap created in the sciatic nerve of Lewis rats bridged by a freeze-thawed nerve graft. Immunohistology after 4 weeks showed survival of both cell types and early regeneration in SKP seeded grafts was comparable to those seeded with SCs. Histomorphometrical and electrophysiological measurements of cell-treated nerve segments after 8 weeks survival all showed significant improvement as compared to diluent controls. A possible mechanistic explanation for the observed results of improved regenerative outcomes lies in SKP–SCs' ability to secrete bioactive neurotrophins. We therefore conclude that SKPs represent an easily accessible, autologous source of stem cells for transplantation therapies which act as functional Schwann cells and show great promise in improving regeneration following nerve injury.

Nerve Regeneration Promoted in a Tube with Vascularity Containing Bone Marrow-Derived Cells

Bone marrow-derived cells (BMCs) are multipotent cells that have the potential to differentiate into bone, cartilage, fat, muscle, or neuronal lineages such as neurons and glial cells. A silicone tube model containing reverse-pedicled sural vessels was created in the sciatic nerves of Lewis rats. About 1 x 10(7) BMCs, removed from the bone marrow of synergetic rat femurs and cultured in vitro, were transplanted into the 15-mm-long chambers of the silicone tubes. Nerve regeneration in vessel-containing tubes that had received BMCs was significantly greater at 12 and 24 weeks after surgery than in tubes that did not receive cells. Transplantation of fibroblasts instead of BMCs into the vessel-containing tube resulted in reduced axonal regeneration, which was inferior to regeneration in the vessel-containing tube that did not receive cells. Polymerase chain reaction (PCR) studies revealed that in vessel-containing tubes containing transplanted BMCs, about 29% of cells in the regenerated nerve originated from BMCs. Cells identified by in situ hybridization and PKH26 prelabeling as being of BMC origin stained positively for S100 and GFAP. Transplanted BMCs differentiated into cells with phenotypes similar to those of Schwann cells under the influence of neurochemical factors and survived by obtaining nutrients from vessels that had been preinserted into the tube. They thus functioned similarly to Schwann cells, promoting nerve regeneration.

Increased phosphorylation of caveolin-1 in the sciatic nerves of Lewis rats with experimental autoimmune neuritis

The levels of phosphorylated caveolin-1 (p-caveolin-1) were analyzed in the sciatic nerves of Lewis rats with experimental autoimmune neuritis (EAN). Western blot analysis showed that the phosphorylation of caveolin-1 increased significantly in the sciatic nerves of EAN-affected rats at the paralytic stage of EAN on day 14 post-immunization (PI) ( P < 0.05) and declined slightly thereafter during the recovery stage. Immunohistochemistry showed intense p-caveolin-1 immunostaining in some inflammatory macrophages, as well as in T-cells in individual nerve fascicles at the peak stage of EAN, while p-caveolin-1 was weakly expressed in some of the vascular endothelial cells and Schwann cells of normal sciatic nerves. The inflammatory cells with intense p-caveolin-1 expression in the EAN-affected individual nerve fascicles were not positive for terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL), while the TUNEL-positive apoptotic cells in the perineurium, where infiltration initially occurred, were weakly positive for p-caveolin-1. Based on these findings, we postulate that caveolin-1 is phosphorylated in inflammatory cells soon after they infiltrate the sciatic nerve, as well as in the perineurium, and that p-caveolin-1 activates intracellular signaling in inflammatory cells, leading to cell death, which ultimately eliminates the infiltrating inflammatory cells from the sciatic nerves of animals with EAN.

Immunohistochemical study of caveolin-1 in the sciatic nerves of Lewis rats with experimental autoimmune neuritis

The expression of caveolin-1 and the related molecule endothelial nitric oxide synthase (eNOS) was analyzed in the sciatic nerves of Lewis rats with experimental autoimmune neuritis (EAN). Western blot analysis showed that caveolin-1 significantly increased in the sciatic nerves with EAN upon initiation of cell infiltration during the early and peak stages (days 10 and 14 post-immunization, p.i.) and declined thereafter. The pattern of eNOS expression over the course of EAN largely matched that of caveolin-1. Immunohistochemistry showed that in EAN lesions, intense caveolin-1 immunostaining occurred in ED1-positive macrophages as well as in vessels, while the caveolin-1 immunoreaction was reduced in Schwann cells in the inflammatory lesions. Consequently, we postulated that caveolin-1 expression increased in the sciatic nerves with EAN; this possibly mediated either molecular trafficking or nitric oxide generation partly through the activation of eNOS in vascular endothelial cells, as well as in inflammatory macrophages in EAN and/or cellular apoptosis of inflammatory cells.

Increased phosphorylation of c-Jun NH (2)-terminal protein kinase in the sciatic nerves of Lewis rats with experimental autoimmune neuritis

The phosphorylation of c-Jun NH (2)-terminal protein kinase (JNK), one of the mitogen-activated protein kinases, was analyzed in the sciatic nerves of Lewis rats with experimental autoimmune neuritis (EAN). Western blot analysis showed that the expression levels of both phosphorylated JNK1 (p-JNK1, approximately 46 kDa) and phosphorylated JNK2 (p-JNK2, approximately 54 kDa) in the sciatic nerves of rats with EAN increased significantly (p < 0.05) at day 14 post-immunization (PI) and remained at this level at days 24 and 30 PI, with a slight decrease. In EAN-affected sciatic nerves, there was intense immunostaining for p-JNK in the infiltrating inflammatory cells (especially ED1-positive macrophages) and Schwann cells on days 14-24 PI, compared with those of controls. Some macrophages with increased p-JNK immunoreactivity was shown to be apoptotic, while some Schwann cells remained survived in this rat EAN model, suggesting that JNK is differentially involved in the EAN-affected sciatic nerves. These findings suggest that JNK phosphorylation is closely associated with the clearance of inflammatory cells as well as the activation of Schwann cells in the EAN affected sciatic nerves.

Enhanced expression of netrin-1 protein in the sciatic nerves of Lewis rats with experimental autoimmune neuritis: Possible role of the netrin-1/DCC binding pathway in an autoimmune PNS disorder

Netrin-1 is a chemotropic factor that plays an important role as a survival factor in the adult nervous system. To investigate whether netrin-1 is involved in autoimmune injury of the peripheral nervous system (PNS), the temporal expression of netrin-1 protein was analyzed in the sciatic nerves of Lewis rats with experimental autoimmune neuritis (EAN). Western blot analysis revealed a significant increase in the level of netrin-1 protein in the sciatic nerves of rats on days 11 to 24 post-immunization (p.i.) compared to controls; netrin-1 expression declined by day 30 p.i. Immunohistochemistry revealed that netrin-1 protein was expressed weakly in Schwann cells and vessels in the sciatic nerves of normal and CFA-immunized control rats. In the sciatic nerves of EAN-affected rats, netrin-1 immunoreactivity was increased mainly in the cell membrane and extracellular matrix of OX42-positive macrophages and S100-positive Schwann cells at the peak and recovery phases of EAN. Moreover, the putative netrin-1 receptor, deleted in colorectal cancer (DCC), was expressed mainly in axons, some macrophages, and Schwann cells in EAN-affected sciatic nerves, although the level of protein expression did not change significantly over the course of EAN. We suggest that a significant increase in netrin-1 expression contributes to host cell survival and axon regeneration to counter autoimmune injury and inflammation, which may play a role in recovery from EAN-induced paralysis.

Activation of p38 mitogen-activated protein kinase in the early and peak phases of autoimmune neuritis in rat sciatic nerves

To examine the involvement of p38 mitogen-activated protein kinase (MAPK) in autoimmune disorders of the peripheral nerve system, we analyzed the phosphorylation of p38 MAPK protein in the sciatic nerves of Lewis rats with experimental autoimmune neuritis (EAN). Western blot analysis showed that phosphorylated p38 (p-p38) MAPK protein was significantly increased in the sciatic nerves of rats in the early and peak phases of EAN, and declined gradually thereafter. Immunohistochemistry showed that p-p38 MAPK levels were increased in the infiltrating inflammatory cells, including T cells and macrophages, as well as in blood vessels and some Schwann cells in EAN-affected sciatic nerves, as compared to the sciatic nerves of controls. Some inflammatory cells and a few Schwann cells were also positive for TUNEL reaction at the peak and recovery phases of EAN. In conclusion, we postulate that the phosphorylation of p38 MAPK is involved in the elimination of infiltrating inflammatory cells during the course of EAN and may possibly modulate recovery in autoimmune disorders of the peripheral nervous system.

Upregulation of osteopontin in Schwann cells of the sciatic nerves of Lewis rats with experimental autoimmune neuritis

We examined the expression of osteopontin (OPN) in the sciatic nerves of rats with experimental autoimmune neuritis (EAN), using immunohistochemistry and immunoblotting, to study its involvement in the pathogenesis of autoimmune peripheral nervous system diseases. Constitutive OPN expression was detected in some Schwann cells; expression was increased after immunization with adjuvant alone. At day 14 after induction of EAN, many Schwann cells had a granular pattern of immunoreactivity, whereas very few inflammatory cells were OPN-positive. Even after recovery from hindlimb paralysis, at 24 days post-immunization, OPN expression remained elevated in the Schwann cells. The results suggest that OPN expression in Schwann cells is easily induced by immunostimulation, and further enhanced by the inflammatory reaction in EAN. Continued elevation of OPN after recovery may represent a functional recovery after a transient inflammatory insult.

Expression of constitutive endothelial and inducible nitric oxide synthase in the sciatic nerve of Lewis rats with experimental autoimmune neuritis

This study examined the expression of constitutive endothelial nitric oxide synthase (eNOS) and inducible NOS (iNOS) in the sciatic nerve of Lewis rats with experimental autoimmune neuritis (EAN). Western blot analysis showed that both eNOS and iNOS expressions in the sciatic nerves of rats increased significantly during the peak stage of EAN, but declined thereafter. Only minimal amounts of these enzymes were identified in normal rat sciatic nerves. Immunohistochemical studies showed that eNOS was increased in vascular endothelial cells and Schwann cells, but not in inflammatory cells, during the peak stage of EAN. However, iNOS was found mainly in inflammatory macrophages in sciatic nerve EAN lesions. These findings suggest that, depending on the stage of peripheral nervous system autoimmune disease, the increased expressions of both eNOS and iNOS might be involved in either the production of detrimental effects during the induction stage of EAN or in the recovery from EAN paralysis.

Increased lipocortin-1 (annexin-1) expression in the sciatic nerve of Lewis rats with experimental autoimmune neuritis.

Lipocortin-1 exerts a potent immunosuppressive effect on pathogenic T cells. In multiple sclerosis and experimental autoimmune encephalomyelitis levels of lipocortins are raised, suggesting their involvement in the recovery from an immunological insult or in neural regeneration. To further understand the role of lipocortins in the peripheral nervous system we have characterized lipocortin-1 levels and cellular distribution of lipocortin-1 immunoreactivity in sciatic nerves of rats with experimental autoimmune neuritis (EAN), a model of human Guillain-Barré syndrome. EAN was induced actively by immunization with bovine peripheral myelin (active EAN) or by adoptive-transfer (AT-EAN) of P2-specific T cells. Cellular infiltrates in serial and semithin cryosections were characterized by immunohistochemistry. In parallel, lipocortin-1 levels in tissue extracts were quantified by a sandwich-ELISA. Only weak lipocortin-1 immunoreactivity was found in nerves of control animals injected with non-pathogenic T cells. The majority of macrophages and lymphocytes in EAN lesions exhibited lipocortin-1 immunoreactivity. Some very heavily stained cells showed a distribution and morphology similar to ED-2-positive macrophages which were abundant during early stages of EAN. Lipocortin-1 expression in T cells and macrophages was proven by immunocytochemical studies in semithin serial sections. In tissue extracts, lipocortin-1 levels increased from 0.24 +/- 0.14 micrograms/mg protein in controls receiving non-pathogenic T cells to a maximum of 0.55 +/- 0.1 micrograms/mg protein in AT-EAN at the peak of disease, and then slowly decreased during clinical recovery but still remained elevated. In dose-response studies in AT-EAN, highest values of lipocortin-1 (0. 71 +/- 0.23 micrograms/mg protein) were recorded after transfer of 2 x 10(7) T cells. Increased levels of lipocortin-1 were also measured in active EAN but occurred during the recovery phase (0.65 +/- 0.27 micrograms/mg protein). By analogy with other immune-mediated disorders, increased lipocortin-1 expression in the inflamed sciatic nerve in EAN may exert immunoregulatory functions in-situ and contribute to the termination of the autoimmune response.

Intraneural activated T cells cause focal breakdown of the blood-nerve barrier.

Experiments were conducted to investigate the effect of activated T cells on the blood-nerve barrier (BNB) in experimental allergic neuritis (EAN). T cells reactive to the P2 component of myelin (P2 T cells) and known to cause EAN were injected into the sciatic nerve of Lewis rats. Animals were then given daily intraperitoneal (i.p.) injections of serum with known demyelinating activity (rabbit EAN serum) or control serum. Serial nerve conduction studies across the injected segment were performed and nerves were removed at various stages for histology. Focal conduction block and perivascular demyelination were evidence in T cell injected nerves of animals treated with EAN serum. In animals treated with control serum no conduction block was seen and only perivascular infiltrates without demyelination were present. Similar results were obtained with T cells reactive to non-neural antigens, although the effect was less marked. Systemically administered rabbit immunoglobulin (Ig) was demonstrated within the endoneurium of P2 T cell injected nerves by immunofluorescence and the endoneurial blood vessels showed increased permeability to circulating horseradish peroxidase (HRP). These findings demonstrate that activated T cells cause focal breakdown of the BNB, allowing circulating antimyelin antibody to enter the endoneurium with consequent focal demyelination. P2 reactive EAN producing T cells do not cause significant demyelination when injected intraneurally (i.n.) in the absence of circulating antimyelin antibody. Intraneural injection of tumour necrosis factor alpha (TNF-alpha) yielded similar results, causing conduction block and perivascular demyelination in the presence of circulating antimyelin antibody but not in control serum treated animals.

Comparative Histology of Experimental Allergic Neuritis Induced with Minimum Length Neuritogenic Peptides by Adoptive Transfer with Sensitized Cells or Direct Sensitization

Using synthetic peptides representing specific regions of the bovine myelin protein P2, the minimum peptide length requirement for the T-cell epitope necessary for successful production of experimental allergic neuritis (EAN) has been shown to involve residues 61-70 of the P2 protein. In this study, morphometric analysis was used to compare the histologic changes in sciatic nerves of Lewis rats after disease was induced by P2 specific neuritogenic T-cell lines (P(2)3) or, alternatively, by direct inoculation of synthetic peptides representing residues 60-70 or 61-70 of the P2 protein sequence. Inoculation with cell line P(2)3 stimulated with residue 61-70 failed to elicit demyelination in sciatic nerves. However, cells stimulated with residue 60-70 produced inflammation, endoneurial edema, mild demyelination and axonal degeneration within seven days. In contrast, disease induced with either peptide by direct sensitization was more severe. Morphometric analysis revealed that inflammation was most severe in animals sensitized to the decapeptide. In the sciatic nerve, axonal degeneration was proportional in frequency to the extent of inflammation. Inflammation was especially intense in spinal roots with extensive destruction of axons including unmyelinated fibers. Spinal root changes were associated with Wallerian degeneration in the posterior white matter tracts of the spinal cord and were apparently secondary to endoneurial inflammation. Disruption of the blood-nerve-barrier (BNB), evident as physical separation of endothelial cells in association with severe perivascular inflammation, was observed.

Role of nonspecific myelin destruction by delayed type hypersensitivity in primary demyelination

To clarify the role of nonspecific myelin destruction mediated by delayed type hypersensitivity (DTH) in primary demyelination, DTH to tuberculin was induced within the endoneurium by intraneural injection of purified protein derivative (PPD) or sonicated Mycobacterium tuberculosis into the sciatic nerves of Lewis rats and guinea pigs which had previously been sensitized to tuberculin. The morphological features of the nerves proximal to the site of needle insertion were assessed 5 days after injection. By changing the PPD concentration of solution for intraneural injection, various degrees of DTH reaction could be produced in the nerve. Infiltration of mononuclear cells including macrophages was observed around the vessels and in the vicinity of the myelin sheaths. Although nonspecific damage of axons, myelin sheaths and Schwann cells was observed in areas heavily infiltrated with inflammatory cells, primary demyelination was hardly recognized. Another group of Lewis rats previously immunized with galactocerebroside (GC), the major glycolipid hapten of myelin, in Freund's complete adjuvant received intraneural injection of PPD or GC liposomes. Neither cellular nor humoral immunity to GC was detected in these rats. The nerves injected with GC liposomes showed no inflammatory cell infiltration except for a few macrophages containing liposomes and those injected with PPD showed infiltration of mononuclear cells without primary demyelination. Our findings reveal that nonspecific myelin destruction induced by DTH does not play an important role in immune-mediated demyelination.