Sciatic Nerves Of Experimental Autoimmune Neuritis Rats Research Articles

Lithium alleviates paralysis in experimental autoimmune neuritis in Lewis rats by modulating glycogen synthase kinase-3β activity.

Guillain-Barré syndrome (GBS)-like neuropathy mimics the leading cause of sporadic acute nontraumatic limb paralysis in individuals from developed countries. Experimental autoimmune neuritis (EAN) is an animal model of GBS and of syndromes such as acute canine polyradiculoneuritis, seen in dogs and cats. The involvement of glycogen synthase kinase (GSK)-3β, a pro-inflammatory molecule, in rat EAN is not fully understood. This study evaluated the potential role of GSK-3β in EAN through its inhibition by lithium. Lewis rats were injected with SP26 antigen to induce EAN. Lithium was administered from 1 day before immunization to day 14 post-immunization (PI). Then the rats were euthanized and their neural tissues were prepared for histological and Western blotting analyses. Lithium, an inhibitor of GSK-3, significantly ameliorated EAN paralysis in rats, when administered from day 1 to day 14 PI. This corresponded with reduced inflammation in the sciatic nerves of EAN rats, where phosphorylation of GSK-3β was also upregulated, indicating suppression of GSK-3. These findings suggest that lithium, an inhibitor of GSK-3β, plays a significant role in ameliorating rat EAN paralysis, by suppressing GSK-3β and its related signals in EAN-affected sciatic nerves.

Dimethyl fumarate attenuates experimental autoimmune neuritis through the nuclear factor erythroid-derived 2-related factor 2/hemoxygenase-1 pathway by altering the balance of M1/M2 macrophages.

BackgroundGuillain–Barré syndrome (GBS) is an acute, post-infectious, immune-mediated, demyelinating disease of peripheral nerves and nerve roots. Dimethyl fumarate (DMF), a fumaric acid ester, exhibits various biological activities, including multiple immunomodulatory and neuroprotective effects. However, the potential mechanism underlying the effect of DMF in GBS animal model experimental autoimmune neuritis (EAN) is unclear.MethodsUsing EAN, an established GBS model, we investigated the effect of DMF by assessing clinical score, histological staining and electrophysiological studies. Then, we further explored the potential mechanism by Western blot analysis, flow cytometry, fluorescence immunohistochemistry, PCR, and ELISA analysis. The Mann–Whitney U test was used to compare differences between control group and treatment groups where appropriate.ResultsDMF treatment reduced the neurological deficits by ameliorating inflammatory cell infiltration and demyelination of sciatic nerves. In addition, DMF treatment decreased the level of pro-inflammatory M1 macrophages while increasing the number of anti-inflammatory M2 macrophages in the spleens and sciatic nerves of EAN rats. In RAW 264.7, a shift in macrophage polarization from M1 to M2 phenotype was demonstrated to be depended on DMF application. In sciatic nerves, DMF treatment elevated the level of the antioxidant transcription factor nuclear factor erythroid-derived 2-related factor 2 (Nrf2) and its target gene hemoxygenase-1 (HO-1) which could facilitate macrophage polarization toward M2 type. Moreover, DMF improved the inflammatory milieu in spleens of EAN rats, characterized by downregulation of messenger RNA (mRNA) of IFN-γ, TNF-α, IL-6, and IL-17 and upregulation of mRNA level of IL-4 and IL-10.ConclusionsTaken together, our data demonstrate that DMF can effectively suppress EAN, and the mechanism involves altering the balance of M1/M2 macrophages and attenuating inflammation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-016-0559-x) contains supplementary material, which is available to authorized users.

Lesional accumulation of CD8(+) cells in sciatic nerves of experimental autoimmune neuritis rats.

Experimental autoimmune neuritis (EAN) is a well-known animal model of human demyelinating polyneuropathies. Macrophages are the major immune cells in peripheral nerves and may exert tissue-damage or tissue-protective activity during EAN. While considered to define a subpopulation of T lymphocytes, CD8 expression has been found on certain macrophages that show cytotoxic effects. Here we have studied the spatiotemporal accumulation of CD8(+) cells in sciatic nerves of EAN rats. A robust accumulation of CD8(+) cells was observed in the sciatic nerves of EAN rats, which was positively correlated with the severity of neurological signs in EAN. Moreover, double-labelling experiments showed that the major cellular sources of CD8 were reactive macrophages. Therefore, our data here suggest a pathological role of CD8(+) macrophages in EAN, which makes CD8(+) macrophage a potential therapeutic target for EAN.

Administration of SB203580, a p38 MAPK Inhibitor, Reduced the Expression of MMP9, and Relieved Neurologic Severity in the Experimental Autoimmune Neuritis (EAN) in Rats.

Dysfunctional expression of matrix metalloproteinase-9 (MMP-9) has been found to be closely associated with the experimental autoimmune neuritis (EAN). In this study, we explored the role of p38 mitogen-activated protein kinases (p38 MAPK) in the regulation of MMP-9 in sciatic nerves of EAN rats. We analyzed the expression changes of MMP-2, MMP-3, MMP-9, and mitogen-activated protein kinases (MAP kinases) in sciatic nerves of EAN rats and investigated the effect of p38 MAPK inhibitor (SB203580) on MMP-9 expression and pathological changes in EAN. Real-time PCR and western blot analyses showed that the expression of MMP-2 exhibited no significant changes throughout the course of EAN, while MMP-3 and MMP-9 presented the relatively increased expression compared with that in control group. MAP kinases, including p38 MAPK, ERK1/2, and JNK1/2, were activated in the sciatic nerve of EAN rats, and phosphorylated p38 MAPK showed similar patterns of expression to MMP-9. The expression of MMP-9 and phosphorylated p38 MAPK in sciatic nerves were in positive correlation with disease severity. In addition, SB203580 treatment significantly reduced the mRNA and protein level of MMP-9 in sciatic nerves on the peak phase of EAN. Inhibition of p38 MAPK also relieved neurologic severity and ameliorated pathological changes in EAN. In conclusion, SB203580 may ameliorate clinical deficit and pathological changes in EAN by reducing the MMP-9 expression in sciatic nerves, which suggest that p38 MAPK inhibitor such as SB203580 could be considered as a therapeutic candidate in autoimmune neuropathies.

Lesional infiltration of receptor activator of nuclear factor-κB ligand+ cells in experimental autoimmune neuritis rats

Experimental autoimmune neuritis (EAN) is a T cell-mediated autoimmune demyelinating inflammatory disease of the peripheral nervous system. Receptor activator of NF-κB ligand (RANKL), a member of the tumor necrosis factor family, regulates proliferation of mature T cells. Here, we have studied the expression of RANKL in sciatic nerves of EAN rats. EAN was induced in male Lewis rats. The spatiotemporal expression of RANKL in sciatic nerves of EAN rats was investigated using immunohistochemistry. In sciatic nerves of normal rats RANKL(+) cells were rarely seen. EAN induced a significant accumulation of RANKL(+) cells in sciatic nerves and there was a significant positive correlation of the time course of RANKL(+) cell accumulations with neurological scores of EAN rats. The major cellular resources of RANKL in sciatic nerves were T cells and macrophages. The positive association of RANKL(+) cell accumulations with neurological scores of EAN rats together with the known functions of RANKL indicated that RANKL might play a role in pathologic development of EAN and need further investigation.

Distinct expression of Tim-3 during different stages of rat experimental autoimmune neuritis

Experimental autoimmune neuritis (EAN) is a well-known animal model of human demyelinating polyneuropathies and is characterized by inflammation and demyelination in the peripheral nervous system. Tim-3 had been identified as a Th1-specific marker negatively regulating autoimmunity or inflammatory diseases. Here we have studied by immunohistochemistry the spatiotemporal accumulation of Tim-3+ cells in sciatic nerves of EAN rats, particularly focusing on its association with alternatively activated macrophages. Our results showed that time course of Tim-3+ cell accumulation correlated positively with disease progression of EAN; but distinct major cellular resources of Tim-3 were observed at different disease stages of EAN: during the early phase of EAN, the main cellular resource were T cells, but at the peak and during recovery phase of EAN, Tim-3 was mostly expressed on CD68+ macrophages or CD163+ cells. Further investigation suggested that accumulation of CD163+ cells, particularly their relative abundances to activated macrophages at different time points, were in accordance with the recovery from EAN. Therefore, Tim3+ cells might include a distinct macrophage population, which may be involved in anti-inflammatory effect and recovery from EAN.

Doxycycline Attenuates Peripheral Inflammation in Rat Experimental Autoimmune Neuritis

Experimental autoimmune neuritis (EAN) is a T cell-mediated autoimmune inflammatory demyelinating disease of the peripheral nervous system and widely-used animal model of human inflammatory demyelinating polyradiculoneuropathies. Doxycycline is a well-known antibiotic and has been reported to have neuroprotective and anti-inflammatory effects. Here we investigated the effects of doxycycline on rat EAN. Therapeutic treatment with doxycycline (40 mg/kg body weight daily from the Day 9 to Day 14 post immunization) significantly attenuated the severity of EAN, decreased inflammatory infiltration of macrophages, B- and T-cells and demyelination in sciatic nerves of EAN rats. Pro-inflammatory molecules including matrixmetalloproteinase-9, inducible nitric oxide synthase and interleukin-17 were greatly decreased in sciatic nerves by administration of doxycycline as well. Taken together, our data showed that doxycycline could effectively suppress the peripheral inflammation to improve outcome of EAN, which suggests that doxycycline may be considered as a potential candidate of pharmacological treatment for neuropathies.

AUY954, a selective S1P 1 modulator, prevents experimental autoimmune neuritis

Experimental autoimmune neuritis (EAN) is a T cell-mediated autoimmune inflammatory demyelinating disease of the peripheral nervous system and an animal model of human inflammatory demyelinating polyradiculoneuropathy. AUY954, which targets selectively the sphingosine-1-phosphate receptor 1 (S1P 1), is known to sequester lymphocytes into secondary lymphoid tissues. In EAN rats, AUY954 greatly prevented paraparesis if administrated from the day of immunization. T cell, B cell, and macrophage infiltration, inflammatory demyelination, and local expression of interleukine-17 and matrix metalloproteinase-9 in sciatic nerves of EAN rats were significantly decreased by AUY954 treatment. Therefore, S1P 1 modulation might be a potential treatment option for inflammatory neuropathies.

FTY720 attenuates lesional interleukin‐17+ cell accumulation in rat experimental autoimmune neuritis

Experimental autoimmune neuritis (EAN) is a well-known animal model of human demyelinating polyneuropathies. Here we have studied the spatiotemporal accumulation of interleukin (IL)-17(+) cells in sciatic nerves of EAN rats and effects of FTY720, an agonist of sphingosine-1-phosphate (S1P) receptors. In this study, we examined the spatiotemporal expression of IL-17 using immunohistochemistry and RT-PCR, and analysed the IL-17(+) cell proportion in blood and lymph nodes using flow cytometry. In sciatic nerves of EAN rats, IL-17(+) cells were mainly found to concentrate around blood vessels and IL-17(+) cell accumulation was temporally correlated with severity of neurological signs. FTY720, which has been shown to reduce severity of EAN, attenuated accumulation of IL-17(+) cells in sciatic nerves, decreased IL-17(+) cell proportion in peripheral blood, but increased IL-17(+) cell proportion in lymph nodes, suggesting the involvement of S1P signal pathway in regulating IL-17(+) cell trafficking. our data are consistent with the possibility that IL-17(+) cells might contribute to the pathogenesis of EAN and the S1P signal pathway may be involved in the in vivo trafficking of IL-17(+) cells.

Improved outcome of EAN, an animal model of GBS, through amelioration of peripheral and central inflammation by minocycline

Experimental autoimmune neuritis (EAN) is a widely used animal model of the human acute inflammatory demyelinating polyradiculoneuropathy, which is the most common subtype of Guillain-Barré Syndrome. EAN is pathologically characterized by breakdown of the blood-nerve barrier, infiltration of reactive immune cells, local inflammation, demyelination in the peripheral nervous system and mechanical allodynia. Minocycline is known to have neuroprotective and anti-inflammatory effects. Furthermore, relieve of neuropathic pain following minocycline administration was observed in a variety of animal models. Here, we investigated the effects of minocycline on rat EAN. Suppressive treatment with minocycline (50 mg/kg body weight daily immediately after immunization) significantly attenuated the severity and duration of EAN. Macrophage and T-cell infiltration and demyelination in sciatic nerves of EAN rats treated with minocycline were significantly reduced compared to phosphate-buffered saline (PBS)-treated EAN rats. mRNA expressions of matrix metallopeptidase-9, inducible nitric oxide synthase and pro-inflammatory cytokines interleukin-1 β and tumour necrosis factor-α in EAN sciatic nerves were greatly decreased by administration of minocycline as well. Furthermore, minocycline attenuated mechanical allodynia in EAN rats and greatly suppressed spinal microglial activation. All together, our data showed that minocycline could effectively suppress the peripheral and spinal inflammation (immune activation) to improve outcome in EAN rats, which suggests that minocycline may be considered as a potential candidate of pharmacological treatment for autoimmune-mediated neuropathies.

Toll-like receptor-2, CD14 and heat-shock protein 70 in inflammatory lesions of rat experimental autoimmune neuritis

Experimental autoimmune neuritis (EAN) is a well-known animal model of Guillain-Barré syndrome (GBS) characterized by inflammation and demyelination in the peripheral nervous system. Toll-like receptors (TLRs) together with their co-receptors form the first line of the self-defense, and play important roles in innate immune responses and inflammation. TLRs can be activated by endogenous ligands, like heat shock protein 70 (HSP70). In this study, we examined the spatiotemporal expressions of TLR2, CD14 and Hsp70 in EAN rats using immunohistochemistry and RT-PCR. A significant up-regulation of TLR2, CD14 and Hsp70 was seen in sciatic nerves of EAN rats and correlated with disease severity. Furthermore, activated macrophages were the main cellular resource of TLR2, CD14 and Hsp70 in EAN. Our results suggest that TLR2-, CD14- or Hsp70-based immunomodulation might have potential in the control of unwanted innate immune system activation in inflammatory neuropathies.

Distribution of Foxp3 + T-regulatory cells in experimental autoimmune neuritis rats

T-regulatory cells expressing the forkhead box transcription factor 3 (Foxp3) play essential roles in immune homeostasis. Experimental autoimmune neuritis (EAN) is an autoantigen-specific T-cell-mediated disease model for human demyelinating inflammatory disease of the peripheral nervous system. We investigated the distribution of Foxp3 + cells in sciatic nerves, spleen and lymph nodes of EAN rats, and the influence of FTY720 on the localization of Foxp3 + cells in EAN rats. In sciatic nerves of EAN rats, accumulation of Foxp3 + cells was not seen during the pre-symptomatic phase (until Day 9) or during early or peak disease activity. In contrast, Foxp3 + cell accumulation was regularly seen in the recovery from neurologic disease, suggesting a contribution of Foxp3 + cells to the resolution of EAN. FTY720 was given at onset of EAN (Day 10) until Day 18. Following FTY720 administration, percentages of Foxp3 + cells in EAN rats were increased in circulating blood, but reduced in lymph nodes compared to the PBS control. FTY720 treatment suppressed total numbers but increased percentages of Foxp3 + cells in sciatic nerves of EAN rats. These data not only suggests a protective role of Foxp3 + cells in EAN but also provides a potential way to alter localization of Foxp3 + cells in vivo.

Valproic acid attenuates inflammation in experimental autoimmune neuritis.

Valproic acid (VPA) is a short-chain branched fatty with anti-inflammatory, neuro-protective and axon-remodeling effects. We investigated the effects of VPA in rats in which experimental autoimmune neuritis (EAN) had been induced (EAN rats). VPA (300 mg/kg, intraperitoneally) administration to EAN rats once daily immediately following immunization significantly suppressed mRNA levels of interferon-gamma, tumor necrosis factor-alpha, interleukin (IL)-1beta, IL-4, IL-6 and IL-17 in the lymph nodes of EAN rats. In peripheral blood and sciatic nerves of EAN rats, Foxp3(+) cells were increased but IL-17(+) cells were decreased during VPA treatment. Furthermore, suppressive and therapeutic treatment with VPA greatly attenuated both accumulation of macrophages, T cells and B cells, and demyelination in sciatic nerves, and greatly reduced the severity and duration of EAN. In summary, our data demonstrated that VPA could effectively suppress inflammation in EAN, suggesting that VPA could be a potent candidate for treatment of autoimmune neuropathies.

Dynamics of production of MIP-1α, MCP-1 and MIP-2 and potential role of neutralization of these chemokines in the regulation of immune responses during experimental autoimmune neuritis in Lewis rats

Experimental autoimmune neuritis (EAN) is an inflammatory autoimmune demyelinating disease of the peripheral nervous system (PNS) and represents an animal model of Guillain–Barré syndrome (GBS), which is a major inflammatory demyelinating disease of the PNS in humans. In the present study, the dynamics of the expression of the chemokines macrophage inflammatory protein-1α (MIP-1α), MIP-2 and monocyte chemotactic protein-1 (MCP-1) were determined in the sciatic nerves of EAN rats. Additionally, the effect of neutralizing antibodies against MIP-1α, MIP-2 and MCP-1 on the clinical course of EAN and the chemokine expression was investigated. The maximum of MIP-1α positive cells in the sciatic nerves was seen on day 14 post immunization (p.i.) correlating with the development of severe clinical signs. Administration of an anti-MIP-1α antibody suppressed the clinical signs of EAN and inhibited inflammation and demyelination in the sciatic nerve. Peak numbers of MCP-1 positive cells in the sciatic nerves were detected on day 7 p.i. Administration of an anti-MCP-1 antibody caused a delay of onset of EAN. However, 4 of the 6 EAN rats receiving the anti-MCP-1 antibody showed the same degree of inflammatory cell infiltration and demyelination in the sciatic nerves as sham-treated EAN rats, whereas only 2 EAN rats had less inflammation and demyelination. The numbers of MIP-2 positive cells reached a maximum on day 21 p.i. Anti-MIP-2 antibody failed to suppress the clinical signs of EAN and the inflammation and demyelination in the sciatic nerves. Only administration of the anti-MIP-1α antibody resulted in a significant reduction in the number of chemokine (MIP-1α)-positive cells and ED1-positive macrophages in the sciatic nerves. The present results demonstrate that MIP-1α and MCP-1 may play a role in the immunopathogenesis of EAN, and that MIP-1α induced trafficking of inflammatory cells can be inhibited by immunoneutralization. Further elucidation of the regulation and coordination of MIP-1α and MCP-1 production may lead to new therapeutic approaches to GBS in humans.