Neuritogenic activity of peripheral nerve myelin proteins in Lewis rats

The neuritogenic activity of human peripheral nerve myelin proteins in Lewis rats was examined. Experimental allergic neuritis (EAN) was induced by human myelin fraction in all Lewis rats examined. Purified human P2 protein induced mild but definite EAN. None of purified P0 protein, total myelin lipids, gangliosides and cerebrosides induced EAN. Addition of total myelin lipids or gangliosides to the P2 protein did not enhance the neuritogenicity of the P2 protein. Bovine P2 protein, which was inactive in rabbits and guinea pigs, showed considerable neuritogenicity in Lewis rats. We postulate that induction of EAN depends both on the species of host animal and the source of antigen.     

Evolution of the cellular response in P2-induced experimental allergic neuritis.

We examined the development of the inflammatory cellular response and demyelination in P2 protein-induced experimental allergic neuritis (EAN). Collections of inflammatory cells were first identified in nerve roots 14 days after immunization. Ia+ cells predominated in the evolving lesions and T-helper cells were the dominant T-cell type with T-suppressor/cytotoxic cells appearing later in the course of the disease. Vesiculation, the earliest change seen in the myelin sheath, appeared before the wave of cellular infiltration. These results indicate that myelin injury precedes inflammation in P2 protein-induced EAN, and provide further evidence that this disorder is indistinguishable from EAN induced with whole peripheral nerve myelin.     

Intranasal administration of recombinant mouse interleukin-12 increases inflammation and demyelination in chronic experimental autoimmune neuritis in Lewis rats

To examine whether interleukin (IL)-12 modulates ongoing chronic experimental autoimmune neuritis (EAN), we evaluated the effects of recombinant mouse IL-12 (rmIL-12) in Lewis rats with chronic EAN, induced by immunization with P0 peptide (180-199) plus complete Freund's adjuvant. Rats were treated intranasally with either 0.1 or 1 microg/rat/day rmIL-12 for 6 days from the onset of clinical chronic EAN, on days 5-10 postimmunization (p.i.). Only high-dose rmIL-12 exacerbated chronic EAN. This clinical effect was associated with higher numbers of inflammatory cells and more severe demyelination in sciatic nerve sections on days 15 and 80 p.i. compared with low-dose rmIL-12-treated rats and phosphate-buffered saline (PBS)-treated control rats. High-dose rmIL-12 increased significantly the lymph node mononuclear cell proliferation in response to P0 peptide 180-199 and IFN-gamma production in the sciatic nerves. These data indicate that intranasally administered IL-12 acts as a proinflammatory cytokine in chronic EAN. Effective inhibition of IL-12 in vivo could be considered for therapeutic use in chronic inflammatory demyelinating polyradiculoneuropathy.     

Oral Administration of Type I Interferon Modulates the Course of Experimental Allergic Neuritis

We investigated the effect of oral administration of type I interferon (IFN) in experimental allergic neuritis (EAN) in Lewis rats immunized with bovine peripheral nerve myelin. Starting at 7 days preceding immunization, rats were fed daily until sacrifice either with 5000 U rat IFN-α/β or mock-IFN. The clinical severity of EAN was significantly reduced in IFN-α/β fed animals compared to mock-IFN fed controls. Demyelination, but not inflammation, was decreased in IFN-α/β fed compared to mock-IFN fed rats at day 20 after immunization. In situ IFN-γ production and inflammation were reduced when evaluated by immunocytochemistry at day 13 after immunization. Spleen cells from IFN-α/β fed compared to mock-IFN fed EAN rats showed significantly reduced proliferation to stimulation with Con A or peripheral nerve myelin. IFN-γ production in draining lymph node cells was significantly reduced after stimulation with bovine peripheral nerve myelin. Our data suggest that oral administration of IFN-α/β reduces the severity of EAN, possibly by a reduction in production.     

促红细胞生成素对实验性自身免疫性神经炎大鼠的治疗作用及机制

目的研究促红细胞生成素(Erythropoietin,EPO)对实验性自身免疫性神经炎(Experimental autoimmune neuritis,EAN)的治疗作用及机制。方法将Lewis大鼠共12只随机分的为EPO治疗组6只和PBS对照组6只,使用大鼠外周神经P257-81肽段诱发急性EAN模型,每天测量体重和神经功能评分至实验结束,免疫后第7天开始进行EPO治疗。第14天(高峰期)取大鼠坐骨神经,用免疫组化染色观察T细胞炎症浸润。第21天(恢复期)取大鼠淋巴结,用荧光定量PCR方法检测IFN-γ、IL-17、IL-4和Foxp3的相对表达。使用放射性3H-TdR掺入法检测淋巴细胞增殖实验中EPO的抑制作用。结果 EPO治疗后大鼠EAN严重度显著降低;免疫组化显示治疗组外周神经T细胞浸润减少;荧光定量PCR结果显示治疗组淋巴结中IFN-γ和IL-17表达降低,而IL-4和Foxp3表达增加;淋巴细胞增殖实验表明EPO剂量依赖地抑制淋巴细胞增殖。结论 EPO对EAN有明显治疗作用,这种治疗作用可能与EPO抑制T细胞的炎症浸润和增殖反应,抑制Th1/Th17类细胞因子炎症损伤,促进Th2/Treg类细胞因子免疫抑制作用有关。     

Cellular mRNA expression of interferon-gamma, IL-4 and transforming growth factor-beta (TGF-beta) by rat mononuclear cells stimulated with peripheral nerve myelin antigens in experimental allergic neuritis.

Experimental allergic neuritis (EAN) serves as a useful model for inflammation in the peripheral nervous system. To study the potential role of important immunoregulatory and effector cytokines in EAN, we examined the expression of mRNA for interferon-gamma (IFN-gamma), IL-4 and TGF-beta by in situ hybridization in lymph node and splenic cells cultured with bovine peripheral nerve myelin (BPM), P2 and P0 during the course of EAN in Lewis rats. Levels of IFN-gamma mRNA-expressing mononuclear cells (MNC) from lymph nodes and spleens roughly correlated with clinical status, consistent with a disease-promoting role for IFN-gamma. BPM, P0 and P2-reactive IFN-gamma mRNA-expressing T cells appeared in lymph nodes and spleen before onset of the disease, whereas a significant TGF-beta response to BPM, P2 and P0 was observed at lower levels than the IFN-gamma response and at onset of recovery, consistent with a disease down-regulating role of TGF-beta. IL-4 mRNA-expressing cells were found at levels similar to TGF-beta mRNA-expressing cells, and with the latest peak of the three cytokines examined. This result suggests that IL-4 may also suppress IFN-gamma expression at late recovery phase of EAN.     

Elimination of CD8+ T Cells in Vivo Does Not Break Induced Immunospecific Tolerance to Experimental Allergic Neuritis in Rats

The role of CD8+ T 'cytotoxic/suppressor' T cells in induced immunospecific tolerance and during recovery after actively induced disease was examined by means of elimination of CD8+ cells from Lewis rats using in vivo treatment by Ox8 monoclonal antibodies, in experimental allergic neuritis (EAN). Animals depleted of CD8+ T cells after recovery from EAN did not show any clinical signs of relapse. Other animals were pretreated with the peripheral nerve basic protein P2 and thereby rendered resistant to disease induction with a potentially neuritogenic emulsion. The elimination of CD8+ T cells did not result in EAN here either. Thus, the CD8+ T-cell population does not seem to participate in the suppression of this autoimmune disease under these experimental conditions.     

Protective effect of Rolipram in experimental autoimmune neuritis: protection is associated with down-regulation of IFN-gamma and inflammatory chemokines as well as up-regulation of IL-4 in peripheral nervous system

Rolipram, a phosphodiesterase type 4 inhibitor, is reported to have anti-inflammatory effects. It can markedly downregulate antigen-driven T cell proliferation and suppress TNF-(alpha and TNF-beta production in vitro and in vivo, which have led to its use in the treatment of a number of autoimmune disorders including experimental autoimmune encephalomyelitis (EAE) and experimental autoimmune neuritis (EAN). EAN is a CD4+ T cell-mediated demyelinating autoimmune disease of peripheral nervous system (PNS) that represents an animal model for the study of the immunopathogenesis and immunotherapy of Guillain-Barré syndrome (GBS) in human. In the previous study, we reported that suppression of EAN by Rolipram was associated with down-regulated myelin antigen-induced T cell responses as well as downregulated IFN-gamma and TNF-alpha production. Here we report that EAN induced in Lewis rats by inoculation with the PNS P2 protein peptide 57-81 and Freund's complete adjuvant (FCA), was strongly suppressed by Rolipram administered twice daily intraperitoneally from day 9 post immunization (p.i.), i.e. after onset of clinical EAN to day 18 p.i. This clinical effect was associated with dose-dependent down-regulated production of IFN-gamma and the chemokines macrophage inflammatory protein-1 alpha (MIP-1 alpha, MIP-2 and monocyte chemotactic protein-1(MCP-1) as well as up-regulated IL-4 production in sciatic nerve sections from Rolipram-treated EAN rats at maximum of clinical EAN, i.e. on day 14 p.i.. These findings suggest that Rolipram may be useful in certain T cell-dependent autoimmune diseases and inflammatory neuropathies. These observations call for further studies on the potential role of Rolipram in the treatment of autoimmune diseases.     

Mechanical allodynia and spinal up-regulation of P2X4 receptor in experimental autoimmune neuritis rats

Experimental autoimmune neuritis (EAN) is the animal model of acute inflammatory demyelinating polyradiculoneuropathy (AIDP) that is the most common subtype of Guillain-Barre syndrome (GBS). While neuropathic pain is a common symptom of GBS, its underlying mechanisms remain elusive. Central sensitization, particularly spinal glia (microglia and astrocytes) activation, is important for the initiation and maintenance of neuropathic pain. P2X(4) receptor (P2X(4)R) is an ATP-gated ion channel and its spinal up-regulation has been found to be crucial for the development of neuropathic pain following peripheral nerve injury. The initiation of mechanical allodynia in rat EAN was observed at day 9 before the onset of neurological signs. Maximal level of mechanical allodynia was observed from days 17-19 and then a slow recovery, long after the cessation of typical neurological signs of EAN, until day 37 was observed. Expression of P2X(4)R in lumbar spinal cords was studied by immunohistochemistry. P2X(4)R immunoreactivity (IR) was mainly seen in gray matter, particularly in the dorsal horn. Accumulation of P2X(4)R(+) cells in the lumbar dorsal horn was observed at day 9, reached the maximal level at day 17 and remained elevated until day 37 after immunization. Furthermore, a negative correlation between the density of P2X(4)R(+) cells in the lumbar dorsal horn with mean hind-paw withdrawal threshold in EAN rats was seen, indicating that P2X(4)R might contribute to EAN mechanical allodynia. Double staining revealed that almost all P2X(4)R(+) cells co-expressed CD68, a marker for reactive microglia, but not the astrocyte marker, glial fibrillary acidic protein (GFAP). Our data demonstrate that EAN induces mechanical allodynia and P2X(4)R expression in spinal microglia, suggesting that EAN is a good animal model for neuropathic pain in polyneuropathy and spinal microglia activation might participate in EAN-induced neuropathic pain.     

The critical role of IL-12p40 in initiating,enhancing, and perpetuating pathogenic events in murine experimental autoimmune neuritis

Interleukin 12 (IL-12) is a proinflammatory cytokine with important immunoregulatory activities and is critical in determining the differentiation and generation of Th1 cells. For the present study, we investigated the role of endogenous IL-12 in the pathogenesis of experimental autoimmune neuritis (EAN), which is a CD4+ T-cell mediated autoimmune inflammatory disease of the peripheral nervous system. EAN is used as an animal model for Guillain-Barré syndrome of humans. Here, EAN was established in IL-12 p40 deficient mutant (IL-12-/-) C57BL/6 mice by immunization with P0 peptide 180-199, a purified component of peripheral nerve myelin, and Freund's complete adjuvant. In these IL-12-/- mice the onset of clinical disease was delayed, and the incidence and severity of EAN were significantly reduced compared to that in wild-type mice.The former group's clinical manifestations were associated with less P0-peptide 180-199 induced secretion of interferon-gamma (IFN-gamma) by splenocytes in vitro and low production of anti-P0-peptide 180-199 IgG2b antibodies in serum. Fewer IFN-gamma and TNF-alpha producing cells, but more cells secreting IL-4, were found in sciatic nerve sections from IL-12-/- mice, consistent with impaired Th1 functions and response. However, the IL-12 deficiency appeared not to affect P0 peptide 180-199-specific T-cell proliferation. These results indicate that IL-12 has a major role in the initiation, enhancement and perpetuation of pathogenic events in EAN by promoting a Th1 cell-mediated immune response and suppressing the Th2 response. This information augments consideration of IL-12 as a therapeutic target in Guillain-Barré syndrome and other T-cell-mediated autoimmune diseases.     

Involvement of cyclooxygenase-1 and -2 in the sciatic nerve of rats with experimental autoimmune neuritis

The expression of both cyclooxygenase (COX)-1 and COX-2, which are representative enzymes in prostaglandin synthesis, was evaluated in the sciatic nerve of rats with experimental autoimmune neuritis (EAN). Western blot analysis showed that both COX-1 and COX-2 were significantly increased in the sciatic nerve at the peak stage of EAN and declined during the recovery stage. Vascular endothelial cells in normal sciatic nerves immunostained for both COX-1 and COX-2. COX-1 was mainly detected in macrophages, and not in other cell types, while COX-2 was detected in Schwann cells and axons as well as inflammatory macrophages in EAN lesions. This suggests that COXs are involved in the pathogenesis of peripheral demyelinating disease, including EAN, and the major cellular source of both COXs in EAN lesions is inflammatory macrophages. Furthermore, COX-2 is enhanced in some Schwann cells and neural elements, possibly mediating peripheral nervous system inflammation.     

雷公腾多甙对实验性变态反应性神经炎周围神经的影响

目的:了解雷公腾多甙(Tripterygium polyglucoside,TPG)对实验性变态反应性神经炎(experimental allergic neuritis,EAN)周围神经的影响。方法:在模型大鼠出现EAN临床症状后连续给予TPG14d,观察EAN临床症状、周围神经电生理与组织病理的变化。结果:免疫后23d EAN坐骨神经的运动神经传导速度和复合肌肉动作电位(compound muscle action potential,CMAP)的波幅明显降低,潜伏期和时限显著延长(P0.05,与对照组比较)。大量炎性细胞侵入坐骨神经,多数神经纤维呈现严重的髓鞘脱失和相当程度的轴索变性;位于郎飞结间隙和近结旁段的轴膜钠、钾离子通道基本检测不到(P0.05,与对照组比较)。TPG干预显著减轻了CMAP波幅的降低和潜伏期与时限的延长,坐骨神经炎性反应和脱髓鞘减轻,部分轴膜钠、钾离子通道得以保留(P0.05与EAN组比较)。结论:TPG作为一种可能减少格林-巴利综合征持久性神经功能损害的药物值得进一步研究。     

In Vivo Demyelination Induced by Intraneural Injection of Anti-Galactocerebroside Serum: A Morphologic Study

Intraneural injection of rabbit anti-galactocerebroside (anti-GC) serum produced focaldemyelinative lesions in rat sciatic nerves. Recipient rats developed a sensory motordeficit of the toes and feet on the side injected with anti-GC serum. Schwann cellabnormalities in recipient nerves were apparent by 20 minutes, followed by myelinsplitting and vesiculation over the next 8 hours. Macrophages first appeared in moder-ate numbers by 15 hours, and degraded myelin was completely phagocvtized by 5 days.An acute inflammatory reaction consisting of endoneurial edema, polymorphonuclearcell infiltration, and fibrin extravasation also was prominent. In vivo demyelinativeactivity of rabbit anti-GC serums was removed by pre-incubation with GC or central orperipheral nervous system myelin and was also lost when the serums were heated at 56C for 30 minutes and injected into nerves of rats previously injected with cobra venomfactor. Anti-GC antibodies are present in the serum of rabbits with experimentalallergic neuritis (WNV-EAN) and encephalomyelitis (WM-EAE) produced, respectively,by immunization with whole peripheral nerve or brain white matter and may play arole in the pathogenesis of demyelination in GC-induced EAN, WN-EAN, or WM-EAE.     

Suppression of chronic experimental autoimmune neuritis by nasally administered recombinant rat interleukin-6

Experimental autoimmune neuritis (EAN) is a CD4+ T-cell-mediated demyelinating disease of the peripheral nervous system (PNS) and serves as experimental model for human immune-demyelinating neurophathies, especially the Guillain-Barré syndrome. In this study, we examined the effect of recombinant rat interleukin-6 (rrIL-6) on chronic EAN in Lewis rats induced by immunization with P2 peptide 57-81 and Freund's complete adjuvant (FCA). Nasal administration of rat rIL-6 (1 microg/rat/day) beginning in the initial phase of EAN as a therapeutic agent, decreased the severity and the duration of clinical EAN. Low-grade inflammation and suppression of regional demyelination within the sciatic nerves were seen in rrIL-6-treated rats. Hyporesponsiveness of lymph node T cells, down-regulation of serum tumour necrosis factor-alpha (TNF-alpha) and increased levels of P2-specific immunoglobulin G1 (IgG1) antibodies document that nasal administration of rrIL-6 was effective systemically. However, because of the non-specific nature of the treatment and multiple effects of IL-6, more experience and great caution are needed, before nasal administration of IL-6 can be considered as a treatment of human autoimmune demyelinating neurophathies.     

Involvement of Cyclooxygenase‐1 and ‐2 in the Sciatic Nerve of Rats with Experimental Autoimmune Neuritis

The expression of both cyclooxygenase (COX)‐1 and COX‐2, which are representative enzymes in prostaglandin synthesis, was evaluated in the sciatic nerve of rats with experimental autoimmune neuritis (EAN).

Western blot analysis showed that both COX‐1 and COX‐2 were significantly increased in the sciatic nerve at the peak stage of EAN and declined during the recovery stage. Vascular endothelial cells in normal sciatic nerves immunostained for both COX‐1 and COX‐2. COX‐1 was mainly detected in macrophages, and not in other cell types, while COX‐2 was detected in Schwann cells and axons as well as inflammatory macrophages in EAN lesions. This suggests that COXs are involved in the pathogenesis of peripheral demyelinating disease, including EAN, and the major cellular source of both COXs in EAN lesions is inflammatory macrophages. Furthermore, COX‐2 is enhanced in some Schwann cells and neural elements, possibly mediating peripheral nervous system inflammation.     

骨髓基质干细胞治疗实验性自身免疫性神经炎的研究

目的:探讨骨髓基质干细胞(BMSC)移植治疗实验性自身免疫性神经炎(experimentalautoimmuneneuritis,EAN)的疗效以及相应的作用机制。方法:用P0180-199多肽与弗氏完全佐剂的混合液免疫Lewis大鼠,建立EAN动物模型。治疗组在免疫后第10天,尾静脉回输荧光染料PKH26标记的BMSC(2×106个细胞/只),通过临床评估、免疫组化及ELISA等方法,研究了BMSC对EAN的治疗作用。结果:回输的BMSC能向脱髓鞘神经组织周围迁移,减轻脱髓鞘的病理改变和炎性细胞浸润。与对照组比较,治疗组CD4 和CD8 T细胞的浸润显著减少(P<0.05),血清中IFN-γ和TNF-α的水平明显降低(P<0.05),培养上清中IL-4的水平显著增加(P<0.05)。结论:BMSC静脉移植治疗EAN有一定的疗效。BMSC通过调节细胞因子的表达逆转Th1/Th2型细胞之间的失衡而发挥治疗作用,并能够抑制T细胞活化增殖。     

Thalidomide Prolongs Experimental Autoimmune Neuritis in Lewis Rats

Thalidomide is reported to have immunomodulatory and anti-inflammatory effects, which have led to its use in the treatment of a number of immune-mediated disorders, including leprosy, discoid lupus and Behcet's disease, and to prevent immunological rejection phenomena following skin and bone marrow grafts. Experimental autoimmune neuritis (EAN) is a CD4⁺ T-cell-mediated demyelinating autoimmune disease, which represents an animal model for the study of the immunopathogenesis and immunotherapy of Guillain–Barré syndrome (GBS) in humans. We examined the effect of thalidomide in Lewis rats with EAN, which was induced by immunization with bovine peripheral nerve myelin (BPM) and complete Freund's adjuvant (CFA). Thalidomide prolonged clinical EAN when given at a dose of 200 mg/kg/day by gavage. This clinical effect was associated with increased numbers of inflammatory cells in sciatic nerve sections and elevated numbers of interferon-γ (IFN-γ) mRNA-expressing cells among lymph node mononuclear cells from thalidomide-treated EAN rats on day 17 postimmunization, i.e. at the peak of clinical EAN. The finding that thalidomide prolongs clinical EAN is in agreement with the clinical polyneuropathy reported in patients receiving treatment with thalidomide and limits its clinical usefulness.     

Myelin ultrastructure of sciatic nerve in rat experimental autoimmune neuritis model and its correlation with associated protein expression

To explore the relationship of peripheral nerve ultrastructure and its associated protein expression in experimental autoimmune neuritis (EAN). EAN was established in Lewis rats using an emulsified mixture of P0 peptide 180-199, Mycobacterium tuberculosis, and incomplete Freund’s adjuvant. Rats immunized with saline solution were used as a control group. Sciatic nerve ultrastructure and immunofluorescence histopathology were measured at the neuromuscular severity peak on day 18 post-induction. Cell-specific protein markers were used for immunofluorescence histopathology staining to characterize sciatic nerve cells: CD3 (T cell), Iba-1 (microglia), S100 (myelin), and neurofilament 200 (axon). The results showed that swelling of the myelin lamellae, vesicular disorganization, separation of the myelin lamellae, and an attenuation or disappearance of the axon were observed by transmission electron microscopy in the EAN group. CD3 and Iba-1 increased significantly in the structures characterized by separation or swelling of the myelin lamellae, and increased slightly in the structures characterized by vesicular of the myelin lamellae, S100 decreased in the structures characterized by vesicular disorganization or separation of the myelin lamellae. And neurofilament 200 decreased in the structures characterized by separation of the myelin lamellae. Furthermore, we found that Iba1 were positive in the myelin sheath, and overlapped with S100, which significantly indicated that Schwann cells played as macrophage-like cells during the disease progression of ENA. Our findings may be a significant supplement for the knowledge of EAN model, and may offer a novel sight on the treatment of Guillain-Barré syndrome.     

INCREASED EXPRESSION OF PHOSPHOLIPASE D1 IN THE SCIATIC NERVE OF RATS WITH EXPERIMENTAL AUTOIMMUNE NEURITIS

Phospholipase D1 (PLD1) expression in the sciatic nerve was studied in induced experimental autoimmune neuritis (EAN) in Lewis rats. PLD1 immunoreactivity was seen in some Schwann cells in the sciatic nerves of normal rats. In parallel with the progression of EAN, PLD1-positive Schwann cells significantly increased in number and showed intense immunoreactivity. PLD1 was also detected in some ED1+ macrophages in EAN lesions. These results suggest that PLD1 in macrophages and Schwann cells plays an important role in the activation of these cells in the pathogenesis of EAN, an animal model of human peripheral demyelinating disease.