Injecting NMDA and Ro 25-6981 in insular cortex induce neuroplastic changes and neuropathic pain-like behaviour.

BACKGROUND: Neuropathic pain is associated with abnormal sensitivity of the central nervous system. Although the mechanism underlying the development of sensitization remains to be fully elucidated, recent studies have reported that neuroplastic changes in the pain circuitry may be involved in hypersensitivity associated with neuropathic pain. However, it is difficult to investigate such phenomena in existing animal pain model. Therefore, in this study, we developed a novel animal model - the circuit plasticity reconstruction (CPR) model - to mimic central sensitization associated with neuroplastic changes. METHOD: NMDA and Ro 25-6981 were injected into the right insular cortex of Sprague-Dawley rats, while electrical stimulation was delivered to the contralateral hind paw. Mechanical allodynia was tested by von Frey test with up-down method, and neuroplastic changes were confirmed by PSA-NCAM-positive immunostaining. RESULT: The mechanical withdrawal threshold of the left hind paw decreased beginning 1 day after CPR modelling and persisted until day 21 comparing to the modified CPR 1 (mod-CPR 1) group (CPR: 91.68 ± 1.8%, mod-CPR 1: 42.71 ± 3.4%, p < 0.001). In contrast, mod-CPR 2 surgery without electrical stimulation did not induce mechanical allodynia. Immunostaining for PSA-NCAM also revealed that neuroplastic changes had occurred in the CPR group. CONCLUSION: Our results demonstrated that CPR modelling induced neuroplasticity within the insular cortex, leading to alterations in the neural circuitry and central sensitization. SIGNIFICANCE: This article represents that the CPR model can mimic the neuropathic pain derived by neuroplastic changes. Our findings indicate that the CPR model may aid the development of novel therapeutic strategies for neuropathic pain and in elucidating the mechanisms underlying pain induced by central sensitization and neuroplastic changes.

An intelligent tutoring system for trauma management (Trauma-Teach): a preliminary report.

Trauma-Teach is an interactive software for tutoring surgical trainees on medical trauma management procedures. Users of the system interact with a virtual patient suffering from trauma injuries. The task of the user is to stabilise the virtual patient, discover the underlying injuries and decide on an appropriate management plan. Artificial intelligence techniques are used to simulate the patient's pulmonary and cardiovascular systems in real time, determine the responses and results of treatments and diagnostics accordingly, model the patient deterioration if wrong actions are taken, and give a measure of reality to the system by selecting actual trauma cases from the hospital's database.

The mastectomy clinical pathway: what has it achieved?

INTRODUCTION: The mastectomy clinical pathway was developed to optimise the clinical care and cost management of breast cancer patients. The aim of this study was to prospectively assess the effect of the mastectomy pathway at the National University Hospital, Singapore over a 7-month period. MATERIALS AND METHODS: A prospective study was carried out on all breast cancer patients admitted for elective mastectomy between March and October 2001. As surrogates of optimised cost and care management, the length of stay and hospital costs, as well as the incidence of complications and unscheduled readmissions, were analysed. Non-pathway mastectomy patients treated from March to October 2000 were used as controls. A total of 83 patients who underwent mastectomy formed the pathway group, while 69 non-pathway patients acted as controls. RESULTS: Compared to controls, the mean length of hospital stay decreased significantly from 4.91 days to 4.10 days (P = 0.018) and the mean cost per case decreased significantly from $5,050 to $4,406 (P = 0.014) for those in the pathway group. There were no significant differences in the complications and unscheduled hospital readmission rates between the two groups (P > 0.05). CONCLUSION: The implementation of mastectomy clinical pathway has improved consistency in patient's treatment, the quality of patient outcome, and has reduced the costs of care and length of hospital stay. In addition, variance analysis of the mastectomy pathway has shown to be valuable for problem identification to improve patient care.

Brain-derived gangliosides suppress the chronic relapsing-remitting experimental autoimmune encephalomyelitis in NOD mice induced with myelin oligodendrocyte glycoprotein peptide.

Chronic relapsing-remitting experimental autoimmune encephalomyelitis (CREAE) induced with myelin oligodendrocyte glycoprotein peptides 35-55 (MOG(35-55)) in NOD mice was successfully treated with brain-derived gangliosides (GA). The GA treatment suppressed the development and severity of CREAE, both clinically and histologically. Spleen cells from the GA-treated mice displayed markedly inhibited levels of MOG(35-55) specific proliferation and interferon-gamma production. Delayed-type hypersensitivity reactions to MOG(35-55) were suppressed by the GA treatment. GA modulate various T cell effector functions in CREAE and may be an effective therapeutic agent for autoimmune demyelinating diseases such as multiple sclerosis.

Amelioration of experimental autoimmune encephalomyelitis with anti-OX40 ligand monoclonal antibody: a critical role for OX40 ligand in migration, but not development, of pathogenic T cells.

OX40 (CD134) and its ligand (OX40L) have been implicated in T cell activation and migration. In this study, we examined the contribution of these molecules to the pathogenesis of experimental autoimmune encephalomyelitis (EAE) by administering a neutralizing mAb against murine OX40L (RM134L) to proteolipid protein (139-151) peptide-induced EAE in SJL mice. Administration of RM134L effectively ameliorated the disease in both actively induced and adoptively transferred EAE models. Histological examination showed that the RM134L treatment greatly reduced mononuclear cell infiltration into the spinal cord. The RM134L treatment did not inhibit the development of pathogenic T cells, given that proliferative response and IFN-gamma production by draining lymph node cells were not reduced or rather enhanced upon restimulation with proteolipid protein (139-151) in vitro, and these cells effectively transferred EAE to naive SJL mice. Flow cytometric analyses showed that the RM134L treatment inhibited the accumulation of OX40-expressing CD4(+) T cells and the migration of adoptively transferred CD4(+) T cells in the spinal cord. Immunohistochemical staining showed that OX40L was most prominently expressed on endothelial cells in the inflamed spinal cord. These results suggest that the OX40/OX40L interaction plays a critical role for the migration of pathogenic T cells into the CNS in the pathogenesis of EAE.

[A study of free radical defense and oxidative stress in the sera of patients with neuroimmunological disorders].

Free radicals are molecules that contain at least one unpaired electron and by nature are highly reactive and potentially destructive. Free radical damage can play an important role of demyelination. Glutathione peroxidase, which plays a role in free radical defenses, and myeloperoxidase and lactoferrin, which are considered to reflect the strength of oxidative stress, were examined by monoclonal antibody-based enzyme immunoassay on serum samples taken from patients with neuroimmunological disorders, namely, 35 multiple sclerosis(MS), and 2 Baló disease, 10 Guillain-Barré syndrome(GBS), and 25 human T-lymphotropic virus type-1 associated myelopathy (HAM). The levels of glutathione peroxidase in active phase of MS (8.37 +/- 5.59 micrograms/ml: p < 0.05) were increased rather than in inactive phase (5.05 +/- 2.44 micrograms/ml) and control (5.41 +/- 1.40 micrograms/ml), the levels of myeloperoxidase in HAM (95.5 +/- 89.1 ng/ml: p < 0.05) were increased rather than in controls (21.5 +/- 4.1 ng/ml), and the levels of lactoferrin were not significantly increased than in other disease and control. Moreover the levels of myeloperoxidase and lactoferrin are increased in Baló disease (myeloperoxidase 487, 762 ng/ml; not significant, lactoferrin 2.58, 2.77 ng/ml; not significant) than in control (myeloperoxidase 21.5 +/- 4.1 ng/ml, lactoferrin 0.69 +/- 0.32 ng/ml). In conclusion, we have here first demonstrated that the levels of these enzyme were not paralleled in MS and Baló diseases. In GBS the levels of all these enzyme were not increased. Thus, these findings suggest that these enzyme may play an important role of the disease activity of Baló, and may reflect the activity of the defense of MS.

Anti-IL-12 antibody prevents the development and progression of multiple sclerosis-like relapsing--remitting demyelinating disease in NOD mice induced with myelin oligodendrocyte glycoprotein peptide.

Treatment with monoclonal anti-IL-12 antibody injected on day 0, 7 and 10 after immunization with myelin oligodendrocyte glycoprotein (MOG) peptide 35-55 in NOD mice resulted in significant suppression of the development and the severity of the chronic relapsing-remitting experimental autoimmune encephalomyelitis (EAE) both clinically and histologically. The spleen cells from anti-IL-12 antibody treated mice displayed markedly inhibited MOG35-55 specific proliferation and IFN-gamma production. MOG35-55 specific antibody production was enhanced by anti-IL-12 antibody treatment. These results suggest that IL-12 is critically involved in the pathogenesis of MOG-induced EAE and that antibody to IL-12 could be an effective therapeutic agent in the clinical treatment of autoimmune demyelinating diseases such as multiple sclerosis (MS).

Pathogenic immunity in Theiler's virus-induced demyelinating disease: a viral model for multiple sclerosis.

Multiple sclerosis involves inflammatory immune responses in the central nervous system (CNS) and is considered as an autoimmune disease potentially associated with viral infection. The majority of experimental models rely heavily on the autoimmune components since similar diseases can be induced following immunization with various myelin antigens. A very attractive alternative model is the Theiler's murine encephalomyelitis virus-induced demyelinating disease. This disease is primarily a CD4+ T cell-mediated, inflammatory demyelinating disease induced following viral infection. Virus-specific inflammatory Th1 cell responses, rather than cytotoxic T lymphocyte response, play a critical role in the pathogenic immune responses. The major pathogenic epitopes have been identified and these are correlated with a Th1 type response to the epitopes following viral infection. In addition, the initial virus-specific immune response is followed by the autoimmune responses to myelin antigens. Assessment of cytokines produced locally in the CNS during the course of disease suggests involvement of inflammatory cytokines in the disease. Furthermore, the manipulation of inflammatory cytokine levels by administration of either recombinant cytokines or antibodies to the cytokines strongly influences the induction and/or progression of disease, supporting the importance of these inflammatory cytokines in this virus-induced demyelinating disease.

Effect of anti-B7-1 and anti-B7-2 mAb on Theiler's murine encephalomyelitis virus-induced demyelinating disease.

We examined the role of B7-1 and B7-2, costimulatory molecules critical to full activation of T cells, in the development of Theiler's murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD). Treatment with mAbs to B7-1 resulted in significant suppression of the development of this disease both clinically and histologically. In mice treated with these mAbs, the production of TNF-alpha and IFN-gamma in the spleen cells was decreased. The delayed-type hypersensitivity and T cell proliferative response specific for TMEV were decreased by this treatment. In contrast, treatment with Abs to B7-2, resulted in no effect on TMEV-IDD. These data suggest that B7-1 is critically involved in the pathogenesis of TMEV-IDD and that Abs to B7-1 could be a novel therapeutic approach in the clinical treatment of demyelinating diseases such as human multiple sclerosis.

Serum levels of apoptosis-related molecules in patients with multiple sclerosis and human T-lymphotropic virus Type I-associated myelopathy.

We evaluated the presence of soluble Fas (sFas), Fas ligand (sFasL), and Bcl-2 in the sera of patients with multiple sclerosis (MS) or human T-lymphotropic virus type I (HTLV-1)-associated myelopathy (HAM) using an enzyme-linked immunosorbent assay (ELISA). Patients with MS in the active phase had higher sFas and Bcl-2 levels than had controls (sFas, p < 0.005; Bcl-2, p < 0.05) or patients in the inactive phase (p < 0.05). In addition, significantly increased serum levels of sFas were found in patients with HAM (p < 0.005). Interestingly, levels of sFasL in sera from patients with HAM and MS in the active stage were higher than those from controls or from patients with MS in the inactive stage or from other inflammatory neurologic diseases (OIND), although this was not statistically significant. These results suggest that serum sFas, sFasL, and Bcl-2 may play an important role in the pathogenesis of MS and HAM.

Expression and potential role of inducible nitric oxide synthase in the central nervous system of Theiler's murine encephalomyelitis virus-induced demyelinating disease.

Intracerebral inoculation of susceptible strains of mice with Theiler's murine encephalomyelitis virus (TMEV) results in immune-mediated demyelinating disease. We examined the pathogenic roles of nitric oxide (NO) and inducible NO synthase (iNOS) in TMEV-induced demyelinating disease (TMEV-IDD). The presence of iNOS was confirmed in the spinal cords of TMEV-infected mice using immunohistochemical staining with anti-iNOS antibody on day 0 (control) and days 15, 30, 60, and 120. Aminoguanidine (AG), a specific inhibitor of iNOS, was injected intraperitoneally (ip) on 1, 3, 5, 8, 10, and 12 days post-TMEV inoculation as induction phase or 15, 17, 19, 22, 24, and 26 days as effector phase. Control animals in each experiment received phosphate-buffered saline (PBS) ip at similar time intervals. Few iNOS-positive cells were observed in the spinal cords of naive SJL/J mice. In the early phase (day 15) of TMEV-IDD, an increase of iNOS-positive cells was detected in the leptomeninges and perivascular space of the spinal cords. The number of iNOS-positive cells was increased and reached its peak on day 60, when histology of the animals showed peak infiltration with inflammatory cells. The clinical course of TMEV-IDD on each day postintracerebral infection was significantly reduced in mice treated with AG in the effector phase, and there was no significant difference between mice treated with AG in induction phase versus those administered PBS. Thus, NO production via iNOS appears to be a pathogenic factor in the effector phase of TMEV-IDD.

IgG subclass switching is associated with the severity of experimental autoimmune encephalomyelitis induced with myelin oligodendrocyte glycoprotein peptide in NOD mice.

We have recently shown that a single dose of the myelin oligodendrocyte glycoprotein (MOG) peptide 35-55 produces a relapsing-remitting demyelinating disease similar to multiple sclerosis (MS) in Lewis rats. In this study we have assessed the possibility that a subclass of anti-MOG35-55 antibodies influences the clinical outcome of these diseases by examining the classes and isotypes of anti-MOG35-55 antibody produced during the course of MOG35-55-induced demyelinating disease in NOD mice. Following immunization, 7 of the 21 injected mice had only mild diseases, while the 14 others had severe progressive and/or relapsing-remitting diseases. There were no differences in anti-MOG35-55 IgG, IgA, IgM, IgG1, IgG2a, and IgG3 antibody titers between the severe and mild symptoms groups. High levels of IgG2b antibody to MOG35-55 were detected in all mice with severe symptoms. In contrast, none of the mice which contracted a mild disease produced anti-MOG35-55 IgG2b. These results suggest that in NOD mice, the IgG2b antibody response to MOG35-55 is associated with the severity of this MS-like demyelinating disease.

Suppression of experimental autoimmune encephalomyelitis by dermatan sulfate.

The effect of dermatan sulfate (DS) on the treatment of Lewis rats with experimental autoimmune encephalomyelitis (EAE) was examined. DS, a sulfated glycosaminoglycan, has been reported to exhibit anticoagulant and fibrinolytic activities. DS treatment (50 mg/kg/day) facilitates recovery from the clinical manifestations of EAE. In this study, the fibrinolytic activity was higher in DS-treated rats than in saline-treated rats. Although the degree of perivascular mononuclear cell infiltration in the spinal cord was not suppressed in DS-treated rats compared to that in saline-treated rats, perivascular fibrin deposition was markedly suppressed in DS-treated rats. These findings suggest that DS would act as an effective therapeutic agent for EAE by preventing fibrin deposition.

Suppressive effect on Theiler's murine encephalomyelitis virus-induced demyelinating disease by the administration of anti-IL-12 antibody.

We examined the role of IL-12, a cytokine critical to the evolution of cellular responses, in the development of Theiler's murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD). Treatment with mAbs to IL-12, especially during the effector phase, resulted in significant suppression of the development of this disease both clinically and histologically. In mice treated with these mAbs, the production of inflammatory and Th1-derived cytokines such as TNF-alpha and IFN-gamma in the spleen cells was decreased, and that of Th2-derived cytokines such as IL-4 and IL-10 was increased. The delayed type hypersensitivity and T cell proliferative response specific for TMEV were decreased by this treatment. These data suggest that IL-12 is critically involved in the pathogenesis of TMEV-IDD and that Abs to IL-12 could be a novel therapeutic approach in the clinical treatment of demyelinating diseases such as human multiple sclerosis.

[A study of soluble form of selectin family in the sera of patients with neuroimmunological disorders].

Members of the selectin family mediate the first step of leukocyte-endothelial cell interaction in inflammatory lesions. Soluble(s) L-selectin, sE-selectin and sP-selectin were examined by monoclonal antibody-based enzyme linked immunosorbent assay on serum samples taken from patients with neuroimmunological disorders, namely, 35 cases of multiple sclerosis (MS), 18 of Guillain-Barré syndrome (GBS), 7 of Miller-Fisher syndrome (MFS), 8 of chronic inflammatory demyelinating polyneuropathy (CIDP), and 25 of human T-lymphotropic virus type-1 associated myelopathy (HAM). The levels of sL-selectin in active phase of MS (2.20 +/- 0.6 mg/ml: p < 0.05) were increased rather than in inactive phase (0.6 +/- 0.25 mg/ml) and control (1.47 +/- 0.24 mg/ml), the levels of sE-selectin in HAM (37.6 +/- 25.7 ng/ml: p < 0.05) were increased, and the levels of sP-selectin were increased in active phase of MS (179.5 +/- 103.8 ng/ml), GBS (151.2 +/- 81.6 ng/ml), CIDP (198.6 +/- 81.9 ng/ml), and HAM (115.3 +/- 73.5 ng/ml). Moreover, the levels of all soluble selectin family are more increased in active phase of MS (sL-selectin 2.20 +/- 0.6 mg/ml: p < 0.05, sE-selectin 44.2 +/- 32.8 ng/ml: p < 0.05, sP-selectin 179.5 +/- 103.8 ng/ml: p < 0.05) than in inactive phase of MS(sL-selectin 0.6 +/- 0.25 mg/ml, sE-selectin 9.8 +/- 2.6 ng/ml, sP-selectin 63.7 +/- 26.6 ng/ml). In conclusion, we have here first demonstrated that levels of all soluble selectin family were increased in the bloods of patients with MS in active phase. In GBS only the levels of sP-selectin were increased. Thus, these findings suggest that soluble selectin family may reflect the disease activity of multiple sclerosis.

The effect of pentoxifylline (PTX) on Theiler's murine encephalomyelitis (TMEV)-induced demyelinating disease.

Pentoxifylline (PTX) has been recently shown to have a variety of immunomodulatory effects. PTX suppresses the production of tumor necrosis factor-alpha (TNF-alpha) and T helper type 1 (Th1) cytokine, interferon-gamma (IFN-gamma), whereas it increases the production of Th2 cytokines, such as interleukin-4 (IL-4) and IL-10. In the pathogenesis of Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease (TMEV-IDD), encephalitogenic Th1 cells may play a major role. We examined the effect of PTX treatment on TMEV-IDD. We treated SJL/J mice, inoculated TMEV intracerebrally, with either PTX or saline from days -2 to 12 and days 14 to 27 postintracerebral infection. In the group of mice treated with PTX from days -2 to 12, the onset of TMEV-IDD was suppressed. On the other hand, in the group of mice treated with PTX from days 14 to 27 or saline, the onset of TMEV-IDD was not inhibited. The results of enzyme-linked immunospot (ELISPOT) assay of spleen cells of mice showed that the production of TNF-alpha and IFN-gamma was significantly inhibited (TNF-alpha and IFN-gamma, p < 0.001) and IL-4 and IL-10 production was significantly increased (IL-4, P < 0.001; and IL-10, P < 0.05, respectively) in the group of mice treated with PTX from days -2 to 12. These findings suggest that PTX suppresses the onset of TMEV-IDD by suppressing the production of TNF-alpha and modulating Th1-dominant immune responses into Th2-dominant ones.

Elevated serum levels of IFN-gamma, IL-4 and TNF-alpha/unelevated serum levels of IL-10 in patients with demyelinating diseases during the acute stage.

We investigated IFN-gamma, IL-4, IL-10 and TNF-alpha in the sera of 42 patients with demyelinating diseases: multiple sclerosis (MS) (21), Guillain-Barré syndrome (GBS) (14), Miller-Fisher syndrome (MFS) (7) during the acute or active stage and 12 normal controls using an originally devised sensitive sandwich enzyme-linked immunosorbent assay (ELISA). We found elevated serum levels of IFN-gamma derived from T helper 1 (Th1) cells and of IL-4 derived from Th2 cells in MS, GBS and MFS. It has been reported in several studies that IL-4 production is increased in MS; however, there have been no reports of elevated serum IL-4 levels in MS. We here describe elevated serum levels of IL-4 in MS, GBS and MFS for the first time. Serum TNF-alpha levels were significantly elevated in patients with MS (p < 0.01). Serum TNF-alpha levels also tended to be elevated in patients with GBS and MFS, but not significantly elevated compared with those of controls. Serum IL-10 levels were not elevated significantly in these diseases, although several patients had higher levels of serum IL-10 than controls. These findings suggest that simultaneous activation of both Th1 and Th2 cells may occur during the acute stage, indicating systemic involvement of the immune system in patients with MS, GBS and MFS. Levels of serum IFN-gamma were two- to three-fold higher than those of serum IL-4 in all three demyelinating diseases. We may then conclude that Th1 response may be dominant in GBS and MFS during the acute stage.

Role of individual T-cell epitopes of Theiler's virus in the pathogenesis of demyelination correlates with the ability to induce a Th1 response.

Intracerebral inoculation of susceptible strains of mice with Theiler's murine encephalomyelitis virus (TMEV) results in immune-mediated demyelination. Three major T-cell epitopes have previously been identified within the VP1 (VP1233-250), VP2 (VP274-86), and VP3 (VP324-37) capsid proteins in virus-infected SJL/J mice. These epitopes appear to account for the majority ( approximately 90%) of major histocompatibility complex class II-restricted T-cell responses to TMEV. Interestingly, the effect of immunization with synthetic peptides bearing the predominant T-cell epitopes on the course of TMEV-induced demyelination indicates that T cells reactive to the VP1 and VP2 epitopes, but not VP3, accelerate the pathogenesis of demyelination. The predominant pathogenic role of the T cells is verified by similar immunization with the fusion proteins containing the entire individual capsid proteins. The order of appearance and level of T cells specific for the individual epitopes during the course of demyelination are similar to each other. However, cytokine profiles of T cells from virus-infected mice indicate that T cells specific for the VP1 (and perhaps the VP2) epitope are Th1, whereas T cells reactive to VP3 are primarily Th2. These results suggest that Th1-type cells specific for VP1 and VP2 are involved in the pathogenesis of viral demyelination induced by TMEV. Thus, a predominance of Th1-inducing viral epitopes is likely critical for the pathogenesis of demyelination.

A new point mutation (3426, A to G) in mitochondrial NADH dehydrogenase gene in Korean diabetic patients which mimics 3243 mutation by restriction fragment length polymorphism pattern.

Mitochondrial tRNA(Leu)(UUR) gene mutation is one of the candidates in the pathogenesis of NIDDM. Especially the 3243 (A-->G) mutation is associated with the maternally-inherited diabetes and deafness. To evaluate the prevalence and characteristics of the 3243 point mutation in Koreans, we screened 433 Korean diabetic patients (220 men and 213 women). Genomic DNA was extracted from peripheral white blood cells and PCR was carried out with mitochondrial DNA primers (3130-3149, 3558-3539) encompassing the 3243 position. After digestion with Apa-1, five subjects showed polymorphism suggesting 3243 point mutation but when we directly sequenced the amplified DNA with an automatic sequencer, only 2 of the 5 patients were shown to have 3243 (A-->G) mutation and the other 3 subjects had 3426 (A-->G) mutation rather than 3243 mutation. Two diabetic patients with 3243 mutation were lean (BMI = 14.4, 17.0 kg/m2), had relatively lower fasting C-peptide concentrations (0.9 ng/ml each), and required insulin for management. In contrast, those with 3426 point mutation were not lean (BMI = 22.6-28.0 kg/m2), had relatively higher C-peptide levels (3.9-5.4 ng/ml), and could be managed with oral hypoglycemic agents. None of the 5 patients had deafness. In conclusion, the prevalence of 3243 point mutation in Korean diabetic patients was approximately 0.5% and we found a new mutation mimicking 3243 mutation by PCR-RFLP (restriction fragment length polymorphism) pattern. We suggest that sequencing of the PCR product or designing smaller PCR fragment size to enhance the specificity may help to identify the exact location of the point mutation.