Experimental Allergic Encephalomyelitis In Rats Research Articles

The role of vitamin D through SphK1/S1P in the regulation of MS progression

Sphingosine-1-phosphate (S1P) is biologically active lipid, leading to neuroinflammation and macrophage invasion in central nervous system, plays an important role in the development of multiple sclerosis (MS) model in experimental allergic encephalomyelitis (EAE) rats. Vitamin D is observed to be a key factor in regulating cell S1P levels. We detected vitamin D can alleviate the symptoms of EAE rats, but the exact mechanism is unclear. In PC12 cells, vitamin D can reverse S1P-induced cell death, but the signaling pathway unclear. This study was aimed to investigate S1P regulation mechanism or signaling pathway mediated by vitamin D in EAE and PC12 model. In our experiments, S1P and Sphingosine kinase type 1 (SphK1) mRNA and protein expression in EAE rats group, control group, vitamin D feeding group were detected by HPLC, ELISA, RT-PCR and western blot. PC12 cell death was detected by Propidium (PI) staining. VDR plasmid overexpression and RNA interference, immunofluorescence, real-time cell analysis, protein immunoblotting was used to detect SphK1 transcriptional regulation, cell-substrate attachment quality, the signaling pathway of cell apoptosis and inflammation related gene expression (Bax/Bcl-2, Casepase-3, Il-6, TGF-β, TNF-α). Our study showed vitamin D can reverse the elevation of S1P level in EAE rats, reduce the severity and shorten the course of EAE. 1,25-(OH) 2D3 coupled with vitamin D receptor (VDR) inhibited SphK1 transcription. 1,25-(OH)2D3 significantly reduced PC12 cell death rate induced by S1P, in addition improved the cell substrate attachment quality. 1,25-(OH) 2D3 can block S1P-induced p-ERK activation and PI3K /Akt signaling pathway reduced Il-6, TGF-β, TNF-α cytokine release and Bax/Bcl-2, Casepase-3 apoptosis protein expression. On the other hand, immunofluorescence staining showed 1,25-(OH) 2D3 can increase the expression of neuronal perinuclear protein MAP2 in PC12 cells probably protect nerve cells further. In summary, the ameliorative effect of vitamin D was derived from its ability to reduce S1P levels, provides an idea for vitamin D as a combination therapy for disease.

Memantine Improves the Disturbed Glutamine and γ-Amino Butyric Acid Homeostasis in the Brain of Rats Subjected to Experimental Autoimmune Encephalomyelitis.

Glutamine (Gln), glutamate (Glu), and γ-amino butyric acid (GABA) are essential amino acids for brain metabolism and function. Astrocyte-derived Gln is the precursor for the two most important neurotransmitters in the central nervous system (CNS), which are the excitatory neurotransmitter Glu and the inhibitory neurotransmitter GABA. In addition to their roles in neurotransmission, these amino acids can be used as alternative substrates in brain metabolism that enable metabolic coupling between astrocytes and neurons in the glutamate-glutamine cycle (GGC). The disturbed homeostasis of these amino acids within the tripartite synapse may be involved in the pathogenesis of various neurological diseases. Interactions between astrocytes and neurons in terms of Gln, Glu, and GABA homeostasis were studied in different phases of experimental allergic encephalomyelitis (EAE) in Lewis rats. The results of the study showed a decrease in the transport (uptake and release) of Gln and GABA in both neuronal and astrocyte-derived fractions. These effects were fully or partially reversed when the EAE rats were treated with memantine, a NMDA receptor antagonist. Changes in the expression and activity of selected glutamine/glutamate metabolizing enzymes, such as glutamine synthase (GS) and phosphate-activated glutaminase (PAG), which were affected by memantine, were observed in different phases of EAE. The results suggested perturbed homeostasis of Gln, Glu, and GABA during EAE, which may indicate alterations in neuron-astrocyte coupling and dysfunction of the tripartite synapse. Memantine appears to partially regulate the disturbed relationships between Gln, Glu, and GABA.

Post Transplantation Cyclophosphamide Improves Outcome of Autologous Hematopoietic Stem Cell Transplantation in Animal Model of Multiple Sclerosis

Experimental allergic encephalomyelitis (EAE) is the animal model of multiple sclerosis (MS). Autologous hematopoietic stem cell transplantation (AHSCT) has recently been recognized as the standard treatment for MS. The aim of our experiment was to investigate the effect of AHSCT with the addition of low-dose post-transplantation cyclophosphamide (Cy) on EAE in rats. Low dose post-transplantation Cy is used in haploidentical HSCT to reduce the risk of graft versus host disease. We hypothesized that it could bring additional benefit in autologous HSCT in autoimmune diseases. Rats with evoked EAE were treated with high dose (125 mg/kg) Cy, followed by AHSCT or high dose (125 mg/kg) Cy followed by AHSCT followed by low dose (20 mg/kg) Cy in two-time schedules—with the therapy applied during the pre-symptomatic or symptomatic phase of the disease. Both AHSCT and AHSCT with post-transplantation Cy in accordance with both time schedules reduce the intensity of the inflammatory response in the CNS, in comparison with non-treated EAE rats. The reduction of clinical symptoms was present in all AHSCT treatment protocols, however, it was significantly stronger when post-transplantation Cy was given during the symptomatic phase of the disease. AHSCT with the addition of post HSCT low dose Cy improved the results of AHSCT by not only reducing the intensity of inflammation in the CNS but also by significantly reducing the clinical symptoms in treated animals when compared to AHSCT alone. We provide an experimental rationale that the addition of post-transplantation Cy may improve the outcome of HSCT in MS.Graphic

Ultrastructural changes in the spinal cord of rats with experimental allergic encephalomyelitis under the influence of human umbilical cord-derived multipotent mesenchymal stromal cells cryopreserved according to different protocols

The transplantation of multipotent mesenchymal stromal cells (MMSCs) is considered to be a possible therapy of multiple sclerosis. For the clinical application of human umbilical cord-derived MMSCs (UC-MMSCs) it is necessary to develop a method of their cryopreservation taking into account the type of cryoprotective media and to investigate the possibility of using these cells for therapeutic purposes in vivo. The purpose of the study was to investigate the effect of UC-MMSCs, cryopreserved in solutions of different composition, on the processes of demyelination and remyelination of the spinal cord of rats with experimental allergic encephalomyelitis (EAE) as a model of multiple sclerosis. Materials and methods. The EAE was modeled by subcutaneous administration of homogenized spinal cord of adult rats with complete Freund's adjuvant. On the 18th day rats with moderate relapsing-remitting form of EAE were suboccipitally injected 1•106 UC-MMSCs, cryopreserved in cryoprotective media containing dimethyl sulfoxide (DMSO), fetal bovine serum (FBS), ethylene glycol, trehalose and sucrose at different composition. On the 35th and 60th days, the studies of ultrastructural changes of the lumbar spinal cord (L3-L5) were performed, assessing the degree of demyelination of nerve fibers by the ratio of myelin sheath (MS) thickness to the diameter of the axis cylinder (AC) of axons. Results. In rats with moderate EAE from the 35th to the 60th day after the modelling of the disorder, destructive changes and signs of demyelination in the spinal cord increased; the MS/AC index corresponded to the average degree of axon demyelination. Suboccipitally administered cryopreserved UC-MMSCs to EAE rats, depending on the used cryopreservation solution, slowed or stopped the demyelination, decreased the MS/AC index to a low degree of axonal demyelination. Reducing the concentration of DMSO in the cryopreservation medium from 10 % to 4 % and adding 6 % trehalose provided a better effectiveness of UC-MMSCs in decreasing the degree of demyelination in EAE. At the same time, the standard solution (10 % DMSO, 90 % FBS) provided these effect, but to a lesser extent. The use of a multicomponent cryopreservation medium containing 15 % ethylene glycol, 3 % DMSO, 10 % sucrose, 12 % trehalose and 60 % FBS did not achieve the goal of maintaining the effects of UC-MMSCs to reduce the degree of demyelination in EAE. Conclusions. To maintain the therapeutic properties of UC-MMSCs, it is advisable to add a reduced concentration of DMSO (4 %) and 6 % trehalose to the cryopreservation medium, supplemented with 90 % fetal bovine serum.

Morpho-functional parameters of neurons in the sensorimotor cortex and neuroapoptosis under conditions of an induced experimental allergic encephalomyelitis in rats and a course of intranasal gel administration containing n-phenylacetyl-l-prolylglycine (N

Purpose. The aim of the research is to evaluate a neuroprotective effect of the nasal dosage form containing Noopept (n-phenylacetyl-l-prolylglycine ethyl ester) by the degree of influence on morphofunctional parameters of neurons in the sensorimotor cortex of animals with experimental allergic encephalomyelitis.Methods. The experiments were conducted on 260 outbred albino rats weighing 190-220 g which were obtained from the nursery of the State Institution «Institute ofPharmacology and Toxicology of the Academy of Medical Sciences of Ukraine». The quarantine (acclimatization period) for all animals lasted 14 days. An animal model of experimental allergic encephalomyelitis (EAE) which has clinical manifestations and pathogenic mechanisms similar to multiple sclerosis was used for the study. There were five groups of animals in the experiment: 1) the intact group (10 rats); 2) the control group – non-treated subjects with EAE which received saline solution (20 rats); 3) the animals with EAE which received basic treatment – 3,4 mg / kg of Methylprednisolone (MP) intraperitoneally, administered slowly in saline solution with a volume of not more than 1/10 of the blood volume of a rat (20 rats); 4) the animals with EAE which received MP and a nasal gel containing n-phenylacetyl-l-prolylglycine at a dose of 10 mg / kg (20 rats); 5) the animals with EAE which received MP and Citicolinum (Ceraxon, «Ferrer Internacional S.A.», Spain) series D003U1, 500 mg / kg, intragastrically (20 rats). Morphometric studies were carried out with the help of Axioskop microscope (Ziess,Germany), a magnification of x40. The image of neurons in the area of the CA-1 zone of the hippocampus, obtained from the microscope using a highly sensitive video camera COHU-4922 (COCHU Inc.,USA).Results. It has been shown that the combination therapy with Methylprednisolone and an intranasal gel containing n-phenylacetyl-l-prolylglycine intensified neuroprotectivity in the animals with EAE. In particular, a neuronal density in rats with the experimental pathology, which received a combination of Methylprednisolone and a gel containing n-phenylacetyl-l-prolylglycine, increased by 14,7 (р <0,05) and their area equaled in values with animals from the intact group; at the same time, the amount of RNA increased by 9,2%. In addition, in conditions of this experiment n-phenylacetyl-l-prolylglycine decreased density of apoptotic and destructive cells by 32,4% (р <0,05) in relation to the control and the MP groups, and the amount of apoptotic neurons – by 44,6% (р <0,05). Judging by effect on the neuronal density and the density of apoptotic and destructively altered neurons in the sensorimotor cortex of outbred albino rats with EAE, a combination of Methylprednisolone and a gel containing n-phenylacetyl-l-prolylglycine reliably exceeds the combined therapy of Methylprednisolone and Citicolinum and the MP monotherapy. A higher, according to some indicators, neuroprotective, nootropic, anxiolytic activity of a gel containing n-phenylacetyl-l-prolylglycine is due to its intranasal route of administration. Conclusions: For the first time, we have obtained reliable data on the neuroprotective effect of the new dosage form containing n-phenylacetyl-l-prolylglycine (Noopept) – an intranasal gel – under conditions of an induced experimental multiple sclerosis. It has been shown that a course of intranasal administration of a gel containing n-phenylacetyl-l-prolylglycine (Noopept) at a dose of 10 mg / kg in combination with Methylprednisolone leads to a decrease in the death of neurons in the sensorimotor cortex of rats with EAE and an increase in the amount of RNA in them. It has been shown that a course of intranasal administration of a gel containing n-phenylacetyl-l-prolylglycine (Noopept) at a dose of 10 mg / kg in combination with Methylprednisolone leads to neuroapoptosis inhibition – a decrease in the density of apoptotic and destructively altered neurons in the sensorimotor cortex of outbred albino rats with EAE. It has been found that judging by effect on the neuronal density and the density of apoptotic and destructively altered neurons in the sensorimotor cortex of the outbred albino rats with EAE, a combination of Methylprednisolone and a gel containing n-phenylacetyl-l-prolylglycine reliably exceeds the combined therapy of Methylprednisolone and Citicolinum and the MP monotherapy.

Expression of hypothalamic feeding-related peptide genes and neuroendocrine responses in an experimental allergic encephalomyelitis rat model

Experimental allergic encephalomyelitis (EAE) is considered to be a useful animal model of human multiple sclerosis (MS). However, among the various symptoms of MS, the mechanisms contributing to inflammatory anorexia remain unclear. In the present study, we used an EAE rat model to examine changes in expression levels of hypothalamic feeding-related peptide genes and neuroendocrine responses such as the hypothalamo-neurohypophysial system and the hypothalamo-pituitary-adrenal (HPA) axis. The weight gain and cumulative food intake in EAE rats in the early days after immunization was significantly lower than that of the control group. The expression of orexigenic peptide genes Npy and Agrp were significantly increased, whereas the levels of anorectic peptide genes (Pomc and Cart) were significantly decreased in the hypothalamus of EAE rats. There was also a significant increase in the mRNA and plasma oxytocin (OXT) but not of arginine vasopressin (AVP) in the supraoptic and paraventricular nuclei (PVN) of EAE rats at days 12 and 18 after immunization. The expression of corticotropin-releasing hormone (Crh) and Avp was downregulated and upregulated, respectively, in the parvocellular division of the PVN at day 12 after immunization. The expression level of Pomc in the anterior pituitary significantly increased, accompanied by increased plasma corticosterone levels, at days 6, 12, and 18 after immunization. These results suggest that inflammatory anorexia in rat EAE may be caused by activation of the OXT-ergic pathway and HPA axis via changes in the expression of hypothalamic feeding-related peptides, including Avp but not Crh.

Autologous Haematopoietic Stem Cell Transplantation Followed by Low‐dose Cyclophosphamide Ameliorates the Disease Course and Influences the Microglia Phenotype in Rats With Experimental Allergic Encephalomyelitis

Experimental allergic encephalomyelitis (EAE) is the animal model of multiple sclerosis (MS), the human autoimmune disease, which leads to neurodegeneration. Immune response is developed by autoreactive lymphocytes infiltrating the CNS, but further maintained by resident immune cells, such as microglia. Microglia, until recently was recognized as pro‐inflammatory cells. Lately, it was discovered, that microglia can also differentiate into an anti‐inflammatory phenotype involved in the regression of inflammatory reaction and regenerative processes. Therapy based on the autologous haematopoietic stem cell transplantation (HSCT) was recently approved for treatment of highly‐active or progressive MS. In some regimens the additional conditioning is used after the HSCT to eliminate the autoreactive cells, which may persist after the first dose of chemotherapy.In our experiment, we aimed to investigate the effect of HSCT preceded by high‐dose of cyclophosphamide (CP) on the disease course in rats with evoked EAE, and if the additional, low‐dose of CP following the high‐dose of CP and HSCT may improve the observed effect.Rats with evoked EAE (day 0) were treated with (i) high‐dose (125mg/kg) CP (day +6), followed by HSCT (day +7), or (ii) high‐dose (125mg/kg) CP, followed by HSCT, followed by low‐dose (20mg/kg) CP (day +10, +11). Control animals were (iii) healthy non‐treated and (iv) EAE non‐treated. Clinical symptoms of EAE (0–5 scale) were observed during the disease course and after the resolution of symptoms rats were euthanized. Spinal cords were collected for immunofluorohistochemical analysis to exmine the microglia phenotype in the CNS.Results showed that the applied therapy in the EAE rats, including only the high‐dose CP followed by HSCT did neither improve clinical symptoms observed in the rats nor influenced the microglia phenotype in comparison to the EAE non‐treated animals. However, the additional low‐dose CP after HSCT resulted in the significant reduction of clinical symptoms and increase in the number of anti‐inflammatory microglia in the CNS. There was no effect observed of applied therapies on the numer of pro‐inflammatory microglia.Our results demonstrated, that the appliction of additional low‐dose CP after HSCT led to the increase in the number of anti‐inflammatory microglia in the CNS. This shift into the anti‐inflammatory phenotype might be engaged in the reduction of clinical symptoms in the EAE rats and accentuates the role of microglia in MS pathomechanism.Support or Funding InformationSupported by grant no. 2018/02/X/NZ5/01487 financed by the National Science Centre

Comparative Study of the Influence of Iem-1556 and Glatiramer Acetate (Copaxon) on the Severity of Neurologic Disorders and the Duration of Experimental Allergic Encephalomielitis in the Rats

The effect of N-decyltropine chloride (IEM-1556) and the reference drug glatiramer acetate (GA) on the severity of neurological disorders and the duration of the experimental allergic encephalomyelitis (EAE), modeling the processes of neural inflammation, demyelination and neurodegeneration characteristic for multiple sclerosis were studied. EAE in female Wistar rats was induced by a single subcutaneous (SC) inoculation of the homologous spinal cord homogenate in complete Freund’s adjuvant. The test preparations were administered from 2 to 16 days after induction of EAE. The severity of the disease was assessed in scores (from 0 to 6) by the presence in animals of persistent paresis and paralysis. The course systemic administration of IEM-1556 in a dose of 3 mg/kg reduced the severity and duration of EAE in rats, comparable to GA. Advantage of IEM-1556 before GA is the possibility of non-invasive application, as well as the presence of analgesic, antiparkinsonian and antidepressant action. It is assumed that the therapeutic effect of IEM-1556 is related to its ability to release endogenous adenosine, which causes neuroprotective, analgesic, antiparkinsonian and antidepressant effects of the drug.

The survival of transplanted mesenchymal stem cells from humans Wharton’s jelly of the umbilical cord in the central nervous system of rats with experimental allergic encephalomyelitis after their suboccipital injection

Human Wharton’s Jelly of mesenchymal stem cells (hWJ-MSCs) have a considerable advantage in comparison with the cells from other sources; their therapeutic potential in treating the central nervous system (CNS) diseases is higher than that of other MSCs. That’s why hWJ-MSCs can be a new alternative treatment of CNS demyelinization damages including multiple sclerosis (MS).Purpose. To study the persistence and distribution of hWJ-MSCs in different CNS segments following suboccipital transplantation into cerebrospinal fluid (CSF) of rats with experimental allergic encephalomyelitis (EAE).Methods. Isolation and cultivation of hWJ-MSCs in vitro. Immunological phenotyping by flow cytometry. EAE induction. Suboccipital injection of MSCs into EAE rats cerebrospinal fluid. Persistence of hWJ-MSCs in the CNS of EAE rats was assayed by PCR in tissue samples at days 2, 3, 4 and 5 using primers for amplifying nucleic alpha satellite sequences of human 17th chromosome.Results. PCR-assays for alpha-satellite sequences revealed human DNA to be detected in the treated rats within 5 days after suboccipital injection at the peak of disease. The human DNA was traced in CSF and various segments of the spinal cord.Conclusions. The data obtained suggest that suboccipitally delivered hWJ-MSCs, survive and can migrate through the CSF from the injection site (cisterna magna) to various segments of CNS.

Effects of Probiotic Enterococci and Glatiramer Acetate on the Severity of Experimental Allergic Encephalomyelitis in Rats

The intestinal microbiota is currently regarded as a potential target for treatments in many pathologies underlying the genesis of inflammation, autoimmune reactions, and neurodegeneration. Multiple sclerosis (MS) is a disease whose pathogenesis combines all these processes. MS also involves impairment to the balance between the components of the intestinal microbiota, with development of dysbiosis. Various probiotics are widely used to correct dysbiotic conditions – bacteria with proven useful properties. We report here the use of a model of multiple sclerosis – experimental allergic encephalomyelitis (EAE) – to study the ability of the probiotic Enterococcus faecium strain L-3 to decrease disease severity in rats when used alone and in combination with glatiramer acetate (GA). Administration of E. faecium L-3 was found to decrease the severity of EAE in rats to essentially the same extent as GA. However, simultaneous use of probiotic enterococci with GA produced no protective action. It is suggested that these agents stimulate different components of the immune system, as their actions produce increases in different populations of immune cells circulating in the blood. The study results demonstrate the ability of E. faecium L-3 to produce significant direct and indirect (via correction of dysbacteriosis) influences on the immune system in MS, which allows these bacteria to be regarded as a potential agent for immunocorrection in autoimmune, inflammatory, and neurodegenerative disease.

Effect of dimethyl fumarate on heme oxygenase-1 expression in experimental allergic encephalomyelitis in rats.

<i>Multiple sclerosis (MS) is an autoimmunological disease leading to neurodegeneration. The etiology of the disease remains unknown, which strongly impedes the development of effective therapy. Most MS treatments focus on modulating the activity of the immune system. Dimethyl fumarate (DMF) exerts a broad spectrum of action, such as modulating immune cell differentiation towards anti-inflammatory subtypes, influencing cytokine production, regulating immune cell migration into the central nervous system, and activating intracellular antioxidant mechanisms. It is well established that activation of the nuclear factor E2 (Nrf2)-dependent pathway, leading to expression of the second-phase antioxidant enzymes, is influenced by DMF. In our experiments we used female Lewis rats in an animal model of MS - experimental allergic encephalomyelitis (EAE). The rats were fed with dimethyl fumarate to test the expression of heme oxygenase-1 (HO-1), one of the second-phase antioxidant enzymes, at specific time points of the symptomatic phases of the disease: on the first day of the occurrence of clinical symptoms (10th day post immunization, DPI); at the peak of clinical symptoms (14th DPI); and at the end of the relapse (21st DPI). The results showed that HO-1 expression, at both the mRNA and protein level, is influenced by DMF administration only at the very beginning of the symptomatic phase of EAE, and not at the peak of clinical symptoms, nor at the end of the relapse. This indicates that the regulation of the Nrf2-dependent antioxidant pathway by DMF occurs at a certain time interval (early EAE/MS) and strongly underlines the importance of the earliest introduction of the therapy to the patient. </i>.

Enterococcus faecium strain L-3 and glatiramer acetate ameliorate experimental allergic encephalomyelitis in rats by affecting different populations of immune cells.

The effect of probiotic Enterococcus faecium strain L-3 was studied in rats with experimental allergic encephalomyelitis (EAE). Glatiramer acetate (GA) was used as control drug. E. faecium strain L-3 and GA both were able to reduce the severity of EAE in a similar fashion. Both approaches increased the proportion of EAE resistant rats and rats with mild disease, prolonged the inductive phase of EAE and reduced the disease duration. Study of the phenotypes of immune cells in blood revealed the differences in immunoregulatory pathways that mediate the protective action of probiotic or GA treatment of EAE. The presence of pronounced protective and immunomodulating effects of the probiotic E. faecium strain L-3 opens an opportunity of its application for the treatment of multiple sclerosis.

Inflammation severely alters thyroid hormone signaling in the central nervous system during experimental allergic encephalomyelitis in rat: Direct impact on OPCs differentiation failure.

Differentiation of oligodendrocyte precursor cells (OPCs) into myelinating oligodendrocytes is severely impaired by inflammatory cytokines and this could lead to remyelination failure in inflammatory/demyelinating diseases. Due to the role of thyroid hormone in the maturation of OPCs and developmental myelination, in this study we investigated (i) the possible occurrence of dysregulation of thyroid hormone signaling in the CNS tissue during experimental neuroinflammation; (ii) the possible impact of inflammatory cytokines on thyroid hormone signaling and OPCs differentiation in vitro. The disease model is the experimental allergic encephalomyelitis in female Dark-Agouti rats, whereas in vitro experiments were carried out in OPCs derived from neural stem cells. The main results are the following: (i) a strong upregulation of cytokine mRNA expression level was found in the spinal cord during experimental allergic encephalomyelitis; (ii) thyroid hormone signaling in the spinal cord (thyroid hormone receptors; deiodinase; thyroid hormone membrane transporter) is substantially downregulated, due to the upregulation of the thyroid hormone inactivating enzyme deiodinase 3 and the downregulation of thyroid hormone receptors, as investigated at mRNA expression level; (iii) when exposed to inflammatory cytokines, deiodinase 3 is upregulated in OPCs as well, and OPCs differentiation is blocked; (iv) deiodinase 3 inhibition by iopanoic acid recovers OPCs differentiation in the presence on inflammatory cytokines. These data suggest that cellular hypothyroidism occurs during experimental allergic encephalomyelitis, possibly impacting on thyroid hormone-dependent cellular processes, including maturation of OPCs into myelinating oligodendrocytes. GLIA 2016;64:1573-1589.

GDNF Enhances Therapeutic Efficiency of Neural Stem Cells-Based Therapy in Chronic Experimental Allergic Encephalomyelitis in Rat.

Multiple sclerosis (MS) is an autoimmune disease in the CNS. The current immunomodulating drugs for MS do not effectively prevent the progressive neurological decline. Neural stem cells (NSCs) transplantation has been proven to promote repair and functional recovery of experimental allergic encephalomyelitis (EAE) animal model for MS, and glial cell line-derived neurotrophic factor (GDNF) has also been found to have capability of promoting axonal regeneration and remyelination of regenerating axons. In the present study, to assess whether GDNF would enhance therapeutic effect of NSCs for EAE, GDNF gene-modified NSCs (GDNF/NSCs) and native NSCs were transplanted into each lateral ventricle of rats at 10 days and rats were sacrificed at 60 days after EAE immunization. We found that NSCs significantly reduced the clinical signs, and GDNF gene-modification further promoted functional recovery. GDNF/NSCs more profoundly suppressed brain inflammation and improved density of myelin compared with NSCs. The survival of GDNF/NSCs was significantly higher than that of transplanted NSCs. Transplanted GDNF/NSCs, in contrast to NSCs, differentiated into more neurons and oligodendrocytes. Moreover, the mRNA expression of oligodendrocyte lineage cells in rats with GDNF/NSCs was significantly increased compared to rats with NSCs. These results suggest that GDNF enhances therapeutic efficiency of NSCs-based therapy for EAE.

Neural Stem Cells Transplantation Promotes Brain Repair after Chronic Experimental Allergic Encephalomyelitis in Rat

The recent studies have showed neural stem cells (NSCs) transplantation promoted repair and functional recovery of chronic experimental allergic encephalomyelitis (EAE) animal model for MS based on the concept of cell replacement. In the present study, NSC...

Effect of recombinant Lactococcus lactis producing myelin peptides on neuroimmunological changes in rats with experimental allergic encephalomyelitis.

Multiple sclerosis (MS) is a human autoimmune neurodegenerative disease with an unknown etiology. Despite various therapies, there is no effective cure for MS. Since the mechanism of the disease is based on autoreactive T-cell responses directed against myelin antigens, oral tolerance is a promising approach for the MS treatment. Here, the experiments were performed to assess the impact of oral administration of recombinant Lactococcus lactis producing encephalogenic fragments of three myelin proteins: myelin basic protein, proteolipid protein, and myelin oligodendrocyte glycoprotein, on neuroimmunological changes in rats with experimental allergic encephalomyelitis (EAE) - an animal model of MS. Lactococcus lactis whole-cell lysates were administered intragastrically at two doses (103 and 106 colony forming units) in a twenty-fold feeding regimen to Lewis rats with EAE. Spinal cord slices were subjected to histopathological analysis and morphometric evaluation, and serum levels of cytokines (IL-1b, IL-10, TNF-α and IFN-γ) were measured. Results showed that administration of the L. lactis preparations at the tested doses to rats with EAE, diminished the histopathological changes observed in EAE rats and reduced the levels of serum IL-1b, IL-10 and TNF-α, previously increased by evoking EAE. This suggests that oral delivery of L. lactis producing myelin peptide fragments could be an alternative strategy to induce oral tolerance for the treatment of MS.

Effects of human Wharton’s jelly-derived mesenchymal stem cells and Interleukin-10 on behavioural responses of rats with experimental allergic encephalomyelitis

On the rat model of experimental allergic encephalomyelitis (EAE), as analogue of multiple sclerosis of human, we studied the effect of Wharton’s jelly-derived mesenchymal stem cells and interleukin-10 on the functional parameters of the CNS.Materials and methods. EAE was induced with spinal cord homogenate of rats with complete Freund’s adjuvant. MSCs were isolated by the explants technique from Wharton’s jelly of the human umbilical cord and cultured up to two passages. Recombinant IL-10 was administered intravenously on the day 10 after induction of EAE and subocipitally on the day 17 at a dose of 0.2 mg per animal. Cell transplantation performed subocipitally on the day 17 at a dose of 106 cells per animal. Behavioral reactions studied in the “open field” test three times: on the day 12, 15 and 24 after the induction of EAE.Results. Induced EAE results in significant changes in adaptive behavior of rats on the 12th day as suppression of orienting-exploratory and activation of emotional activity. The application of MSCs and IL-10 has normalized the rates of cognitive activities (orienting-exploratory activity) and emotional sphere (the level of fear, anxiety).Conclusion. Combined treatment of induced EAE in rats with interleukin-10 and human Wharton’s jelly-derived mesenchymal stem cells is effective at correction of behavioral responses of animals.

Effects of beta 2 adrenergic agonists on axonal injury and mitochondrial metabolism in experimental autoimmune encephalomyelitis rats.

The primary aims of this study were to investigate mitochondrial metabolism during experimental allergic encephalomyelitis (EAE) animal model axonal injury and to determine the correlation among neurological function scores, pathological changes, and the activities of the BB isoenzyme of creatine kinase (CK-BB), catalase (CAT), and calpain in the brain tissues of EAE rats. Another goal was to preliminarily define the mechanism of mitochondrial metabolism resulting from the effect of beta 2 adrenergic agonists in the process of EAE animal model axonal damage. EAE was induced in specific pathogen free Wistar rats by guinea pig spinal cord homogenate, complete Freund's adjuvant, and pertussis vaccine. We recorded the behavioral change in EAE rats, detected pathological changes in central nervous tissue, and observed the changes of the CK-BB, CAT, and calpain in the EAE rat brain and spinal cord. The results indicated that the average neurologic function score increased in the EAE group compared to that of the controls (P < 0.01). In addition, CAT and CK-BB activities significantly decreased and the calpain activity significantly increased compared with those of the control group (P < 0.05). The decrease of the activity of central nervous CK-BB and CAT content, as well as the increase of calpain activity at the highest time point were considered to be the consequences of EAE. Furthermore, the results revealed that use of salbutamol could alleviate disease symptoms and reduce the recurrence of the EAE disease.

Effect of oral administration of pig spinal cord hydrolysate on clinical and histopathological symptoms of experimental allergic encephalomyelitis in rats.

Oral tolerance is the natural occurring phenomenon of a decreased immune response to previously fed antigens, which prevents induction of a response to dietary antigens. One of the mechanisms is deletion of T lymphocytes reactive to the fed antigen. Knowing that phenomenon, it seems appropriate to engage this mechanism for treatment of autoimmune diseases. Multiple sclerosis (MS) is an autoimmunological disease which causes neurological impairment in humans. Autoreactive T lymphocytes migrate through the open blood-brain barrier into the central nervous system (CNS), where they recognize myelin antigens as foreign, and induce an inflammatory response against the myelin sheath, which causes demyelination and even axonal loss. Experimental allergic encephalomyelitis (EAE), an animal model of MS, resembles the autoimmunological aspect of the disease. We used a broad spectrum of myelin antigens to induce EAE, and also to induce oral tolerance by giving myelin epitopes intragastrically to rats. The aim of our study was to evaluate whether pig spinal cord hydrolysate given intragastrically is able to evoke oral tolerance in rats with an animal model of MS - EAE. In our experiments we fed female Lewis rats with pig spinal cord hydrolysate at doses of 5, 20 and 100 mg per kg of body weight. We observed diminished clinical symptoms of ongoing EAE in rats fed with all doses of pig spinal cord hydrolysate. In the histopathological study, intensity of the inflammatory process in spinal cord was similar in rats not fed with EAE and in rats fed with lower doses of pig spinal cord hydrolysate. In animals fed with the highest dose of pig spinal cord hydrolysate, intensification of the inflammatory response was observed. These results were confirmed by morphometric evaluations. We found that feeding animals with preparations containing myelin antigens can reduce EAE symptoms, which may indicate oral tolerance induction, but the obtained results also underline the importance of dose of the orally given antigens, because of the possibility of enhancement of the inflammatory process in the CNS.

Vitamin D therapy in experimental allergic encephalomyelitis could be limited by opposing effects of sphingosine 1-phosphate and gelsolin dysregulation.

Several studies support a protective effect of vitamin D on multiple sclerosis and experimental allergic encephalomyelitis (EAE), but the mechanisms of these favorable effects are unclear. Our study demonstrates that sphingosine 1-phosphate (S1P) is upregulated in the serum and spinal cords of EAE rats, but that vitamin D reverses the upregulation to alleviate inflammation. Vitamin D, however, cannot prevent the disease process, suggesting that other factors may be involved. To identify additional factors that might limit vitamin D efficacy, we assessed the effects of vitamin D on plasma gelsolin (pGSN), a regulator of S1P that is downregulated in the CSF of MS patients. Our results show that pGSN is downregulated in the serum of EAE rats, whereas its cellular form, cytoplasmic gelsolin (cGSN), is upregulated in the spinal cord of EAE rats. Importantly, vitamin D causes a downregulation of both pGSN and cGSN, which may counteract the positive effects of S1P decrease. Furthermore, 48 and 42 kDa caspase-3 cleavage products of cGSN are detected in EAE spinal cords, suggesting enhanced apoptotic activity, but these cleaved products undergo a similar decrease upon vitamin D treatment. To directly test the role of cGSN in the apoptotic process, we performed RNA interference in PC-12, a rat sympathetic nerve cell line. Results verify that cGSN suppresses apoptosis induced by TNF-α. Collectively, these results support a therapeutic effect of vitamin D that is derived from its ability to reduce S1P, but is limited by its simultaneous effect in reducing pGSN and cGSN. Based on these observations, we postulate that combined therapy with recombinant human pGSN and vitamin D may produce more beneficial effect in treating multiple sclerosis.