Analysis of the Gadolinium retention in the Experimental Autoimmune Encephalomyelitis (EAE) murine model of Multiple Sclerosis

ObjectivesThe aim of this study is to quantitatively investigate, at the preclinical level, the extent of Gd retention in the CNS, and peripheral organs, of immune-mediated murine models (Experimental Autoimmune Encephalomyelitis –EAE) of Multiple Sclerosis, compared to control animals, upon the injection of gadodiamide. The influence of the Gadolinium Based Contrast Agent administration timing during the course of EAE development is also monitored.MethodsEAE mice were injected with three doses (1.2 mmol/kg each) of gadodiamide at three different time points during the EAE development and sacrificed after 21 or 39 days. Organs were collected and the amount of Gd was quantified through Inductively Coupled Plasma-Mass Spectrometry. Transmission electron microscopy (TEM) and MRI techniques were applied to add spatial and qualitative information to the obtained results.ResultsIn the spinal cord of EAE group, 21 days after gadodiamide administration, a significantly higher accumulation of Gd occurred. Conversely, in the encephalon, a lower amount of Gd retention was reached, even if differences emerged between EAE and controls mice. After 39 days, the amounts of retained Gd markedly decreased. TEM validated the presence of Gd in CNS. MRI of the encephalon at 7.1T did not highlight any hyper intense region.ConclusionIn the spinal cord of EAE mice, which is the mostly damaged region in this specific animal model, a preferential but transient accumulation of Gd is observed. In the encephalon, the Gd retention could be mostly related to inflammation occurring upon immunization rather than to demyelination.     

Spasticity Treatment Ameliorates the Efficacy of Melatonin Therapy in Experimental Autoimmune Encephalomyelitis (EAE) Mouse Model of Multiple Sclerosis

Multiple sclerosis (MS) is a progressive inflammatory demyelinating disease in the central nervous system (CNS). Melatonin is an effective treatment in MS patients and experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. Melatonin secretion peaks at 2 AM, concomitant with the time at which the muscles are resting and the body is exerting its antioxidant activity. The current study was designed to investigate combination treatment of baclofen, a muscle relaxant drug, and melatonin in EAE mice. Results showed that melatonin (Mel) alone or in combination with baclofen (Bac + Mel) reduced clinical scores and demyelination by significantly increasing myelin oligodendrocyte glycoprotein (MOG) levels, a marker for mature oligodendrocytes, compared to EAE mice. Moreover, Mel or Bac + Mel therapy caused a significant increase in IL-4 serum levels, an anti-inflammatory cytokine, whereas IFN-γ serum levels, a pro-inflammatory cytokine, were significantly reduced. On the other hand, Mel or Bac + Mel caused a significant reduction in malondialdehyde (MDA) levels, a marker of oxidative stress, in comparison to EAE mice. In contrast, the activity of antioxidant enzymes catalase (CAT) and superoxide dismutase (SOD) was significantly increased in Mel and Bac + Mel groups. In summary, combination therapy improved clinical scores and tend to enhance the efficiency of melatonin treatment by further promoting remyelination, decreasing inflammation, and stimulating the activity of antioxidant enzymes, which suggests that prior spasticity treatment increases the efficacy of melatonin therapy in EAE mouse model of MS. Further experimental and clinical studies are needed to ensure the beneficial role of this combination strategy.     

A CB2 Receptor Agonist Reduces the Production of Inflammatory Mediators and Improves Locomotor Activity in Experimental Autoimmune Encephalomyelitis

Background:Cannabinoids (CBs) have been found to regulate the immune system, affect innate and adaptive immune responses, and reduce inflammatory reactions. This study assessed the therapeutic effects of GW-405833 synthetic CB2 agonist on inflammatory factors as well as locomotor activity in experimental autoimmune encephalomyelitis (EAE).Methods:In this experimental study, 48 adult male C57BL/6 mice were randomly and equally assigned to eight groups. By injecting 250 mg of MOG35-55 peptide, EAE was induced. Every other day for 17 days after EAE onset, EAE-afflicted mice in groups 1–3 received an intraperitoneal injection of GW-405833 at a dose of 3, 10, and 30 mg/kg, respectively. Clinical status and locomotor activity, measured using the beam walking assay, were assessed every other day during the first 17 days after EAE onset. Mice were euthanized in day 17th of treatment and the serum levels of the IL-1β, IL-12, CRP, and TNF-α proinflammatory cytokines as well as IL-4 and TGF-β anti-inflammatory cytokines were measured by ELISA method.Results:Clinical manifestations of EAE in groups 2 and 3 were significantly milder than group 4 and locomotor activity in groups 1–3 was significantly better than group 4 in days 5–17 (p< 0.05). GW-405833 also significantly decreased the levels of IL-12, TNF-α, and CRP and significantly increased the levels of IL-4 and TGF-β but had no significant effects on the level of IL-1β. GW-405833 was not associated with significant side effects.Conclusion:The CB2 receptor agonist GW-405833, improves clinical conditions and reduces inflammation in mice with EAE.Key Words: Clinical evaluation, Experimental autoimmune encephalomyelitis, GW-405833, Locomotor activity, Multiple sclerosis, Proinflammatory cytokines     

Prenatal Stress Impairs Spinal Cord Oligodendrocyte Maturation via BDNF Signaling in the Experimental Autoimmune Encephalomyelitis Model of Multiple Sclerosis

One of the most substantial and established environmental risk factors for neurological and psychiatric disorders is stress exposure, whose detrimental consequences hinge on several variables including time. In this regard the gestational period is known to present an intrinsic vulnerability to environmental insults and thus stressful events during pregnancy can lead to severe consequences on the offspring’s brain development with long-term repercussions throughout adulthood. On this basis, we investigated the long-lasting impact of prenatal stress exposure on the susceptibility to the experimental autoimmune encephalomyelitis (EAE), a well-established murine model of multiple sclerosis. Although stress is considered a triggering factor for this chronic, progressive, autoimmune disease, little is known about the underlying mechanisms. To this end, EAE was induced by immunization with MOG35-55/CFA and pertussis toxin administration in adult female C57BL/6 mice born from control or stressed dams exposed to restraint stress during the last days of gestation. Our results demonstrate that gestational stress induces a marked increase in the severity of EAE symptoms in adulthood. Further, we highlight an altered maturation of oligodendrocytes in the spinal cord of prenatally stressed EAE mice, as indicated by the higher levels of GPR17, a marker of immature oligodendrocyte precursor cells. These behavioral and molecular alterations are paralleled by changes in the expression and signaling of the neurotrophin BDNF, an important mediator of neural plasticity that may contribute to stress-induced impaired remyelination. Since several already marketed drugs are able to modulate BDNF levels, these results pave the way to the possibility of repositioning these drugs in multiple sclerosis.

     

Detecting Deoxyhemoglobin in Spinal Cord Vasculature of the Experimental Autoimmune Encephalomyelitis Mouse Model of Multiple Sclerosis Using Susceptibility MRI and Hyperoxygenation

Susceptibility-weighted imaging (SWI) detects hypointensities due to iron deposition and deoxyhemoglobin. Previously it was shown that SWI detects hypointensities in the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis (MS), most of which are due to intravascular deoxyhemoglobin, with a small proportion being due to iron deposition in the central nervous system parenchyma and demyelination. However, animals had to be sacrificed to differentiate these two types of lesions which is impractical for time course studies or for human application. Here, we proposed altering the inspired oxygen concentration during imaging to identify deoxyhemoglobin-based hypointensities in vivo. SWI was performed on lumbar spinal cords of naive control and EAE mice using 30% O₂ then 100% O2. Some mice were imaged using 30% O₂, 100% O₂ and after perfusion. Most SWI-visible hypointensities seen with 30% O₂ changed in appearance upon administration of 100% O₂, and were not visible after perfusion. That hypointensities changed with hyperoxygenation indicates that they were caused by deoxyhemoglobin. We show that increasing the inspired oxygen concentration identifies deoxyhemoglobin-based hypointensities in vivo. This could be applied in future studies to investigate the contribution of vascular-based hypointensities with SWI in EAE and MS over time.     

Myelin Oligodendrocyte Glycoprotein (MOG35-55) Induced Experimental Autoimmune Encephalomyelitis (EAE) in C57BL/6 Mice

Multiple sclerosis is a chronic neuroinflammatory demyelinating disorder of the central nervous system with a strong neurodegenerative component. While the exact etiology of the disease is yet unclear, autoreactive T lymphocytes are thought to play a central role in its pathophysiology. MS therapy is only partially effective so far and research efforts continue to expand our knowledge on the pathophysiology of the disease and to develop novel treatment strategies. Experimental autoimmune encephalomyelitis (EAE) is the most common animal model for MS sharing many clinical and pathophysiological features. There is a broad diversity of EAE models which reflect different clinical, immunological and histological aspects of human MS. Actively-induced EAE in mice is the easiest inducible model with robust and replicable results. It is especially suited for investigating the effects of drugs or of particular genes by using transgenic mice challenged by autoimmune neuroinflammation. Therefore, mice are immunized with CNS homogenates or peptides of myelin proteins. Due to the low immunogenic potential of these peptides, strong adjuvants are used. EAE susceptibility and phenotype depends on the chosen antigen and rodent strain. C57BL/6 mice are the commonly used strain for transgenic mouse construction and respond among others to myelin oligodendrocyte glycoprotein (MOG). The immunogenic epitope MOG_35-55 is suspended in complete Freund''s adjuvant (CFA) prior to immunization and pertussis toxin is applied on the day of immunization and two days later. Mice develop a "classic" self-limited monophasic EAE with ascending flaccid paralysis within 9-14 days after immunization. Mice are evaluated daily using a clinical scoring system for 25-50 days. Special considerations for care taking of animals with EAE as well as potential applications and limitations of this model are discussed.     

AKP-11 - A Novel S1P1 Agonist with Favorable Safety Profile Attenuates Experimental Autoimmune Encephalomyelitis in Rat Model of Multiple Sclerosis

Sphingosine-1-phosphate receptor 1 (S1P1) mediated regulation of lymphocyte egress from lymphoid organs is recognized as the mechanism of FTY720 (Fingolimod, Gilenya) efficacy in relapsing-remitting forms of multiple sclerosis (RRMS). In this study we describe a novel S1P1 agonist AKP-11, next generation of S1P1 agonist, with immunomodulatory activities in cell culture model and for therapeutic efficacy against an animal model of MS, i.e. experimental autoimmune encephalomyelitis (EAE) but without the adverse effects observed with FTY720. Like FTY720, AKP-11 bound to S1P1 is internalized and activates intracellular AKT and ERKs cellular signaling pathways. In contrast to FTY720, AKP-11 mediated S1P1 downregulation is independent of sphingosine kinase activity indicating it to be a direct agonist of S1P1. The S1P1 loss and inhibition of lymphocyte egress by FTY720 leads to lymphopenia. In comparison with FTY720, oral administration of AKP-11 caused milder and reversible lymphopenia while providing a similar degree of therapeutic efficacy in the EAE animal model. Consistent with the observed reversible lymphopenia with AKP-11, the S1P1 recycled back to cell membrane in AKP-11 treated cells following its withdrawal, but not with withdrawal of FTY720. Accordingly, a smaller degree of ubiquitination and proteolysis of S1P1 was observed in AKP-11 treated cells as compared to FTY720. Consistent with previous observations, FTY720 treatment is associated with adverse effects of bradycardia and lung vascular leaks in rodents, whereas AKP-11 treatment had undetectable effects on bradycardia and reduced lung vascular leaks as compared to FTY720. Taken together, the data documents that AKP-11 treatment cause milder and reversible lymphopenia with milder adverse effects while maintaining therapeutic efficacy similar to that observed with FTY720, thus indicating therapeutic potential of AKP-11 for treatment of MS and related autoimmune disorders.     

两种方法制备豚鼠脊髓匀浆诱导EAE模型的比较

目的建立Wistar大鼠实验性自身免疫性脑脊髓炎模型,并检测可溶性血管细胞黏附分子-1在EAE大鼠中的动态表达。方法两种方法制备豚鼠脊髓匀浆:一种方法不用磷酸盐缓冲液(PBS)灌注,直接将豚鼠麻醉后取豚鼠全脊髓制备匀浆,即非灌注法;另一种方法用磷酸盐缓冲液将豚鼠灌注后,再取豚鼠全脊髓制备匀浆,即灌注法。分别采用上述两种方法制备的豚鼠脊髓匀浆为抗原,免疫Wistar大鼠建立EAE的动物模型,取脑、脊髓组织石腊包埋,病理切片,光镜观察;采用双抗体夹心法酶联免疫吸附实验(ELISA)检测血清中sVCAM-1的表达。结果非灌注组的发病率、临床评分和sVCAM-1的表达显著性升高(P<0.05)。结论采用非灌注法制备的豚鼠全脊髓匀浆作为抗原,能够成功诱发Wistar大鼠的EAE模型,为研究多发性硬化的发病机制及治疗奠定了一定的基础。     

The Beneficial Effects of Resveratrol on Experimental Autoimmune Encephalomyelitis (EAE) in C57BL/6J Mouse Model

Journal of Evolutionary Biochemistry and Physiology - Multiple sclerosis (MS) is a disease of the central nervous system of unknown cause and limited therapeutical treatments. In this study we...     

Identification of a Gene (GPR30) with Homology to the G-Protein-Coupled Receptor Superfamily Associated with Estrogen Receptor Expression in Breast Cancer

Using the technique of differential cDNA library screening, a cDNA clone was isolated from an estrogen receptor (ER)-positive breast carcinoma cell line (MCF7) cDNA library based upon the overexpression of this gene compared to an ER-negative cell line (MDA-MB-231). Sequence analysis of this clone determined that it shared significant homology to G-protein-coupled receptors. This receptor, GPCR-Br, was abundantly expressed in the ER-positive breast carcinoma cell lines MCF7, T-47D, and MDA-MB-361. Expression was absent or minimal in the ER-negative breast carcinoma cell lines BT-20, MDA-MB-231, and HBL-100. GPCR-Br was ubiquitously expressed in human tissues examined but was most abundant in placenta. GPCR-Br expression was examined in 11 primary breast carcinomas. GPCR-Br was detected in all 4 ER-positive tumors and only 1 of 7 ER-negative tumors. Based upon PCR analysis in hybrid cell lines, the gene for GPCR-Br (HGMW-approved symbol GPR30) was mapped to chromosome 7p22. The pattern of expression of GPCR-Br indicates that this receptor may be involved in physiologic responses specific to hormonally responsive tissues.     

Post-synaptic Density-95 (PSD-95) Binding Capacity of G-protein-coupled Receptor 30 (GPR30), an Estrogen Receptor That Can Be Identified in Hippocampal Dendritic Spines

The estrogen 17β-estradiol (E2) modulates dendritic spine plasticity in the cornu ammonis 1 (CA1) region of the hippocampus, and GPR30 (G-protein coupled estrogen receptor 1 (GPER1)) is an estrogen-sensitive G-protein-coupled receptor (GPCR) that is expressed in the mammalian brain and in specific subregions that are responsive to E2, including the hippocampus. The subcellular localization of hippocampal GPR30, however, remains unclear. Here, we demonstrate that GPR30 immunoreactivity is detected in dendritic spines of rat CA1 hippocampal neurons in vivo and that GPR30 protein can be found in rat brain synaptosomes. GPR30 immunoreactivity is identified at the post-synaptic density (PSD) and in the adjacent peri-synaptic zone, and GPR30 can associate with the spine scaffolding protein PSD-95 both in vitro and in vivo. This PSD-95 binding capacity of GPR30 is specific and determined by the receptor C-terminal tail that is both necessary and sufficient for PSD-95 interaction. The interaction with PSD-95 functions to increase GPR30 protein levels residing at the plasma membrane surface. GPR30 associates with the N-terminal tandem pair of PDZ domains in PSD-95, suggesting that PSD-95 may be involved in clustering GPR30 with other receptors in the hippocampus. We demonstrate that GPR30 has the potential to associate with additional post-synaptic GPCRs, including the membrane progestin receptor, the corticotropin releasing hormone receptor, and the 5HT1a serotonin receptor. These data demonstrate that GPR30 is well positioned in the dendritic spine compartment to integrate E2 sensitivity directly onto multiple inputs on synaptic activity and might begin to provide a molecular explanation as to how E2 modulates dendritic spine plasticity.     

The Synergistic Local Immunosuppressive Effects of Neural Stem Cells Expressing Indoleamine 2,3-Dioxygenase (IDO) in an Experimental Autoimmune Encephalomyelitis (EAE) Animal Model

Neurodegenerative diseases provoke robust immunological reactions in the central nervous system (CNS), which further deteriorate the neural tissue damage. We hypothesized that the expression levels of indoleamine 2,3-dioxygenase (IDO), an enzyme that has potent immune suppressive activities, in neural stem cells (NSCs) would have synergistic therapeutic effects against neurodegenerative diseases, since NSCs themselves have low IDO expression. In this study, the synergistic immune suppressive effects of rat fetal NSCs expressing IDO (rfNSCs-IDO) were validated by mixed leukocyte reaction (MLR) in vitro and an experimental autoimmune encephalomyelitis (EAE) animal model in vivo. rfNSCs-IDO showed significantly more suppressive effects on T cell proliferation in the MLR compared to control rfNSCs (rfNSCs-Cont). Importantly, IDO inhibition using 1-methyl-DL-tryptophan (1-MT), an IDO inhibitor, reversed the synergistic effects, confirming IDO-specific effects in rfNSCs-IDO. In the EAE animal model, systemic rfNSCs-IDO injections resulted in significant local immune suppression in the cervical lymph nodes and CNS, evidenced by a reduction in the number of activated T lymphocytes and an increase in regulatory T cell numbers, which induced significantly fewer clinical symptoms and faster recovery. In contrast, rfNSCs-Cont failed to reduce symptoms in the EAE animal models, although they showed local immune suppression, which was significantly less than that in rfNSCs-IDO. Taken together, IDO expression in NSCs synergistically potentiates the immune suppression activities of NSCs and could be applicable for the development of therapeutic modalities against various neurodegenerative diseases.     

猪雌激素受体（ESR）基因对产仔数性状的影响

猪产仔数是重要的经济性状,产仔数的提高将会大大地增加商品肉猪的产量,给现代养猪生产带来巨大的经济效益。ＥＳＲ基因是影响猪产仔数的主效基因,而且与猪的生长发育性状及酮体性状之间不存在负的基因多效性影响。采用ＰＣＲ－ＲＦＬＰｓ的方法,通过对５个不同品种的、特别是我国地方品种的２６２头母猪进行了基因多态性分析,研究表明ＥＳＲ基因在这种种群对产仔数均有极显著影响,ＥＳＲ位点ＢＢ基因型母猪平均比ＡＡ基因型母