이 작품은 PC에 NIH 부여 DK080351에 의해 그리고 테리와 크리스틴 고어 가족의 관대한 선물에 의해 지원되었다.

실험 프로토콜 뇌하수체 immunogen가 (동반자 문서를 참조) 준비되면, 그것은 SJL 마우스 (잭슨 연구소, 주식 000,686)으로 subcutaneously 주입니다. 두 번째 동일한 주사는 7 일 후에 반복됩니다. 생쥐는 다음 생체내 및 사후 결과에 수집하는 데 사용됩니다. 1 단계. SJL 마우스로 뇌하수체 immunogen의 피하 주입 마우스는 20 MG / ML 2,2,2 - Tribromoethanol 0.5 ML (Avertin, TCI 미국, T - 1420) intraperitoneally를 주사하여 첫번째 anesthetized 있습니다. 생쥐는 다음 2181 문서를 조브로 동반자에 표시된, 뇌하수체 유제 100 μl와 subcutaneously 주입 뇌하수체 추출하고 완전한 프로 인드의 도움이되는의 0.25 MG 1 MG를 포함하고 있습니다. 에멀전은 왼쪽 지느러미 뒷부분 다리 지역 ((50 μL)과 오른쪽 사타구니 지역 (50 μL)에 주입합니다. 뇌하수체 유제는 반대 사이트 (50 오른쪽 지느러미 뒷부분 다리 지역에서 μl과에 일 7 일에 다시 주입 왼쪽 사타구니 지역에 50 μl). 마우스는 처음 3 일 동안 매일 모니터링하고 처음으로 예방 접종 후 일반적으로 28일는 희생의 날 때까지 매주 수 있습니다. 2 단계. 생체내의 결과에의 수집 : 블러드 assays 생쥐가 살아있는 동안 가장 일반적으로 실험 결과를 평가하는 데 사용되는 조직은 혈액이다. 혈액은 일상적 십사일 뇌하수체 항체 또는 크린 시토킨 및 chemokines 같은 다른 마커의 혈청 수준을 측정하는 최초의 예방 접종 후에 그려진 것입니다. 혈액은 다음과 같이 라이브 생쥐에서 수집됩니다. 생쥐는 목의 아저씨에 의해 안전하게 개최되고 턱뼈의 뒤쪽이 있습니다. 턱뼈의 뒤쪽이있는되면, 4mm의 lancets은 은빛의 턱밑 혈관 번들을 사용하고 있습니다. 혈액을 수집 후 지역에 온 장소 압력은 혈종을 피하기 위해 알코올 면봉을 사용합니다. 수집된 혈액은 20 분 동안 2,000g에 centrifuged 및 사용 ° C까지 -80에 저장됩니다. 3 단계. 사후 결과의 수집 : 뇌하수체 조직 병리학 마우스는 일반적으로 28일 첫 예방 접종 후 희생하고 있습니다. 사나의 진단을 수립하는 데 필요한 추기경 기능은 조혈 mononuclear 세포에 의해 뇌하수체의 침투이다. 생쥐는 CO 2 perfused되는 밀폐된 플라스틱 챔버에서 그들을 배치하여 euthanized 있습니다. 마우스는 8 분 총 챔버에 보관됩니다. 안락사 후, 뇌하수체는 동반자 문서에 설명된대로 수집됩니다. 병에 걸린 뇌하수체가 부풀어 오르고 주변 두라 메이터 더 확고 자기편 나타납니다. 샤프 포셉은 뇌하수체를 둘러싼 meninges을 느슨하게하는 데 사용됩니다. 일단 쐐기 꼴의 뼈에서 자유롭게 이동 뇌하수체, 뇌하수체의 한쪽 끝을은 집게로 쥐었다이며, 뇌하수체는 신중하게 해제됩니다. 선 그러면 벡스 테드의 정착액 5 포함된 microcentrifuge 관에 배치됩니다. 뇌하수체는 야간 고정 렌즈 종이에 싼, 그리고 카세트에 배치됩니다. 이 선들은 다음 프로토콜을 사용하여 처리됩니다 : <table border="1"><tbody><tr><td> 역 번호 </td><td> 시약 </td><td> 분 시간 </td><td> 온도 (섭씨) </td></tr><tr><td> 1 </td><td> 70 % 알콜 </td><td> 5 </td><td> 25 </td></tr><tr><td> 2 </td><td> 70 % 알콜 </td><td> 15 </td><td> 25 </td></tr><tr><td> 3 </td><td> 95 % 알콜 </td><td> 15 </td><td> 25 </td></tr><tr><td> 4 </td><td> 95 % 알콜 </td><td> 15 </td><td> 25 </td></tr><tr><td> 5 </td><td> 100 % 알코올 </td><td> 15 </td><td> 25 </td></tr><tr><td> 6 </td><td> 100 % 알코올 </td><td> 7.5 </td><td> 25 </td></tr><tr><td> 7 </td><td> 100 % 알코올 </td><td> 7.5 </td><td> 25 </td></tr><tr><td> 8 </td><td> 크실렌 </td><td> 20 </td><td> 25 </td></tr><tr><td> 9 </td><td> 크실렌 </td><td> 20 </td><td> 25 </td></tr><tr><td> 10 </td><td> 크실렌 </td><tD&gt; 20 </td><td> 25 </td></tr><tr><td> 11 </td><td> 파라핀 </td><td> 30 </td><td> 58 </td></tr><tr><td> 12 </td><td> 파라핀 </td><td> 5 </td><td> 58 </td></tr><tr><td> 13 </td><td> 파라핀 </td><td> 5 </td><td> 58 </td></tr></tbody></table> 처리 후, 뇌하수체 분비는 파라핀에 포함된하고 적어도 5 nonconsecutive 섹션 (5 μm의 두께)가 각각의 선 (腺)에서 절단됩니다. sectioning 후,이 선들은 hematoxylin과 eosin 물들일 및 mononuclear 침투를 정할 분석하고 있습니다. 가장 심각한 침투있는 부분은 점수를 위해 선택됩니다. 심각도는 면역 세포에 의해 앞쪽에 뇌하수체 교체 또는 파괴의 범위의 주관적 평가에 따라 점수입니다 등급 0, 아니 질병, 1 학년, 2% -20 % 참여, 학년이, 20 % -30 %, 3 학년, 30% -50 %; 학년 4, 50 % -90 %; 5 학년, 큰 90 % 이상. 모든 섹션은 섹션의 원본 멀게 두 명으로 득점하고 있습니다.

<ol>
	<li>Tzou, S. C., Lupi, I., Landek, M., Gutenberg, A., Tzou, Y. M., Kimura, H., Pinna, G., Rose, N. R., Caturegli, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoimmune+Hypophysitis+of+SJL+mice:+Clinical+Insights+from+a+New+Animal+Model.">Autoimmune Hypophysitis of SJL mice: Clinical Insights from a New Animal Model.</a> Endocrinology. , (2008).</li><li>Twarog, F. J., Rose, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+production+of+thyroid+autoantibodies+in+mice.">The production of thyroid autoantibodies in mice.</a> J Immunol. 101, 242-2450 (1968).</li><li>Patrick, J., Lindstrom, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoimmune+response+to+acetylcholine+receptor.">Autoimmune response to acetylcholine receptor.</a> Science. 180, 871-872 (1973).</li><li>Caturegli, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoimmune+hypophysitis:+an+underestimated+disease+in+search+of+its+autoantigen(s).">Autoimmune hypophysitis: an underestimated disease in search of its autoantigen(s).</a> J Clin Endocrinol Metab. 92, 2038-2040 (2007).</li><li>Beckstead, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+technique+for+preservation+of+fixation-sensitive+antigens+in+paraffin-embedded+tissues:+addendum.">A simple technique for preservation of fixation-sensitive antigens in paraffin-embedded tissues: addendum.</a> J Histochem Cytochem. 43, 345-345 (1995).</li><li>Levine, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allergic+adenophypophysitis:+new+experimental+disease+of+the+pituitary+gland.">Allergic adenophypophysitis: new experimental disease of the pituitary gland.</a> Science. 158, 1190-1191 (1967).</li><li>Yoon, J. W., Choi, D. S., Liang, H. C., Baek, H. S., Ko, I. Y., Jun, H. S., Gillam, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+an+organ-specific+autoimmune+disease,+lymphocytic+hypophysitis,+in+hamsters+by+recombinant+rubella+virus+glycoprotein+and+prevention+of+disease+by+neonatal+thymectomy.">Induction of an organ-specific autoimmune disease, lymphocytic hypophysitis, in hamsters by recombinant rubella virus glycoprotein and prevention of disease by neonatal thymectomy.</a> Journal of Virology. 66, 1210-1214 (1992).</li><li>Davis, J. R., McMahon, R. F., Lowenstein, P. R., Castro, M. G., Lincoln, G. A., McNeilly, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus-mediated+gene+transfer+in+the+ovine+pituitary+gland+is+associated+with+hypophysitis.">Adenovirus-mediated gene transfer in the ovine pituitary gland is associated with hypophysitis.</a> J Endocrinol. 173, 265-271 (2002).</li><li>Rotondo, F., Quintanar-Stephano, A., Asa, S. L., Lombardero, M., Berczi, I., Scheithauer, B. W., Horvath, E., Kovacs, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenohypophysitis+in+rat+pituitary+allografts.">Adenohypophysitis in rat pituitary allografts.</a> Int J Exp Pathol. , (2010).</li></ol>

관심 없음 충돌 선언하지 않습니다.

네 기술은 실험 동물에 hypophysitis을 유발하는 것으로보고되었습니다. 프로 인드의 도움이되는 섞인 뇌하수체 단백질의 주입이이 문서에서 설명하는 기술은, 1967 년 6 거슬러 올라가는 가장 오래된 하나이며, 최근에는 한 개선되었습니다. 기타 기술 1992 7 용해 풍진 바이러스 glycoproteins의 분사 2002 년 8 뇌하수체에 직접 아데노 바이러스의 stereotactic 주입, 2010 9</su...

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		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>SJL mice (The Jackson Laboratories)</td>
<td>&nbsp;</td>
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<td>stock 000686</td>
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<td>2,2,2-Tribromoethanol (Avertin)</td>
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<td>TCI America</td>
<td>T-1420</td>
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<td>Beckstead's fixative</td>
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induction of experimental autoimmune hypophysitis in sjl mice

면역 hypophysitis는 민감한 생쥐 한에 도움이되는 섞인 뇌하수체 단백질의 주입에 의해 실험적으로 복제하실 수 있습니다. 마우스 모델은 종종 인간에서 발생하는 동일한 프로세스의 좋은 복제를 제공하고, 우리가 질병이 전개 방법을 학습하실 수 있습니다. 유형 1A 당뇨병 같은자가 면역 질환의 경우 저절로 인간의 대응과 유사한 질병을 개발하는 모델 (NOD 마우스)가 있습니다. 많은 다른 면역 질환, 그러나, 모델은 실험적으로 유도해야합니다. 이 점에서 일반적인 접근 방법은 연구되고있는 기관에서 파생된 지배적인 항원으로 마우스를 주입하는 것입니다. 예를 들어, 면역 thyroiditis에 관심이 조사는 thyroglobulin 2 마우스를 투입하고, myasthenia gravis에 관심있는 사람은 아세틸콜린 수용체 3을 주입. 특정 면역 질환에 대한 autoantigen가 알려져 있지 않을 경우, 수사는 면역 반응의 표적 기관에서 원유 단백질 추출물을 주입. 면역 hypophysitis 들어, 병원성 autoantigen (들)은 4 식별을 유지하고, 따라서 원유 뇌하수체 단백질 준비가 사용됩니다. 이 동영상이 문서에서 우리는 SJL 생쥐의 면역 실험 hypophysitis를 유도하는 방법을 보여줍니다.

Watch this Scientific Journal Video about Induction of Experimental Autoimmune Hypophysitis in SJL Mice at JoVE.com

Induction of Experimental Autoimmune Hypophysitis in SJL Mice

이 동영상은 어떻게 조직 병리학하여 심각도를 평가하기 위해 SJL 마우스 및 면역 hypophysitis를 유도하는 방법을 보여줍니다.

SJL 마우스의 실험적 면역 Hypophysitis의 유도

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse ...

induction-of-experimental-autoimmune-hypophysitis-in-sjl-mice

Research

JoVE Journal

Immunology and Infection

11.4K 조회수.  The Johns Hopkins University. 이 동영상은 어떻게 조직 병리학하여 심각도를 평가하기 위해 SJL 마우스 및 면역 hypophysitis를 유도하는 방법을 보여줍니다.

비디오: Induction of Experimental Autoimmune Hypophysitis in SJL Mice

Preparation of Mouse Pituitary Immunogen for the Induction of Experimental Autoimmune Hypophysitis

Autoimmune hypophysitis is a chronic inflammation of the pituitary gland caused or accompanied by autoimmunity1. It has traditionally been considered a rare disease but reporting has increased markedly in recent years. Hypophysitis, in fact, develops not uncommonly as a "side effect" in cancer patients treated with antibodies that block inhibitory receptors expressed on T lymphocytes, such as CTLA-42 and PD-1 receptors. Autoimmune hypophysitis can be induced experimentally by injecting mice with pituitary proteins mixed with an adjuvant3. In this video article we demonstrate how to extract proteins from mouse pituitary glands and how to prepare them in a form suitable for inducing autoimmune hypophysitis in SJL mice.

Autoimmune hypophysitis can be reproduced in mice by injecting an extract of mouse pituitary proteins.

실험 면역 Hypophysitis의 유도를위한 마우스 뇌하수체 Immunogen의 준비

Autoimmune hypophysitis is a chronic inflammation of the pituitary gland caused or accompanied by autoimmunity1.  It has traditionally been considered a ...

연구

면역학 및 감염병학

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 MOG35-55 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.

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

Experimental autoimmune encephalomyelitis (EAE) is an established animal model of multiple sclerosis. C57BL/6 mice are immunized with myelin oligodendrocyte glycoprotein (MOG) peptide 35-55 (MOG35-55), resulting in an ascending flaccid paralysis caused by autoreactive immune cells in the central nervous system. Protocols for disease induction and monitoring will be discussed.

수초 희소 돌기 아교 세포 당 단백질 (MOG<sub&gt; 35-55</sub&gt;) C57BL / 6 쥐에 실험자가 면역 뇌척수염 (EAE) 유도

Multiple sclerosis is a chronic neuroinflammatory demyelinating disorder of the central nervous system with a strong neurodegenerative component. While ...

Induction of Experimental Autoimmune Encephalomyelitis in Mice and Evaluation of the Disease-dependent Distribution of Immune Cells in Various Tissues

Multiple sclerosis is presumed to be an inflammatory autoimmune disease, which is characterized by lesion formation in the central nervous system (CNS) resulting in cognitive and motor impairment. Experimental autoimmune encephalomyelitis (EAE) is a useful animal model of MS, because it is also characterized by lesion formation in the CNS, motor impairment and is also driven by autoimmune and inflammatory reactions. One of the EAE models is induced with a peptide derived from the myelin oligodendrocyte protein (MOG)35-55 in mice. The EAE mice develop a progressive disease course. This course is divided into three phases: the preclinical phase (day 0 - 9), the disease onset (day 10 - 11) and the acute phase (day 12 - 14). MS and EAE are induced by autoreactive T cells that infiltrate the CNS. These T cells secrete chemokines and cytokines which lead to the recruitment of further immune cells. Therefore, the immune cell distribution in the spinal cord during the three disease phases was investigated. To highlight the time point of the disease at which the activation/proliferation/accumulation of T cells, B cells and monocytes starts, the immune cell distribution in lymph nodes, spleen and blood was also assessed. Furthermore, the levels of several cytokines (IL-1&#946;, IL-6, IL-23, TNF&#945;, IFN&#947;) in the three disease phases were determined, to gain insight into the inflammatory processes of the disease. In conclusion, the data provide an overview of the functional profile of immune cells during EAE pathology.

This manuscript describes the methods for induction and scoring of the experimental autoimmune encephalomyelitis (EAE) model, together with the assessment of immune cell distribution and mRNA cytokine levels in lymph nodes, spleen, blood and spinal cord using flow cytometry and quantitative PCR, respectively, at various disease phases.

다양한 조직에서 면역 세포의 질병에 의존하는 유통의 마우스 및 평가에 실험자가 면역 뇌척수염의 유도

Multiple sclerosis is presumed to be an inflammatory autoimmune disease, which is characterized by lesion formation in the central nervous system (CNS) ...

A Non-invasive and Technically Non-intensive Method for Induction and Phenotyping of Experimental Bacterial Pneumonia in Mice

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant preclinical advances in our understanding of the underlying cellular and molecular pathogenesis. In vivo mouse models capture the integrated physiology and resilience of the host defense response in a manner not revealed by alternative, simplified ex vivo approaches. Several methods have been described in the literature for intrapulmonary inoculation of bacteria in mice, including aerosolization, intranasal delivery, peroral endotracheal cannulation under &#39;blind&#39; and visualized conditions, and transcutaneous endotracheal cannulation. All methods have relative merits and limitations. Herein, we describe in detail a non-invasive, technically non-intensive, inexpensive, and rapid method for intratracheal delivery of bacteria that involves aspiration (i.e., inhalation) by the mouse of an infectious inoculum pipetted into the oropharynx while under general anesthesia. This method can be used for pulmonary delivery of a wide variety of non-caustic biological and chemical agents, and is relatively easy to learn, even for laboratories with minimal prior experience with pulmonary procedures. In addition to describing the aspiration pneumonia method, we also provide step-by-step procedures for assaying the subsequent in vivo pulmonary innate immune response of the mouse, in particular, methods for quantifying bacterial clearance and the cellular immune response of the infected airway. This integrated and simple approach to pneumonia assessment allows for rapid and robust evaluation of the effect of genetic and environmental manipulations upon pulmonary innate immunity.

Several methods have been described in the literature for modeling bacterial pneumonia in mice. Herein, we describe a non-invasive, inexpensive, rapid method for inducing pneumonia via aspiration (i.e., inhalation) of a bacterial inoculum pipetted into the oropharynx. Downstream methods for assessment of the pulmonary innate immune response are also detailed.

마우스의 실험 세균성 폐렴의 유도 및 표현형에 대한 비 침습적 및 기술적 비 집약적 인 방법

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant ...

Induction of Drug-Induced, Autoimmune Hepatitis in BALB/c Mice for the Study of Its Pathogenic Mechanisms

Drug-induced autoimmune hepatitis (DIH) is the most common hepatic drug-induced hypersensitization process observed in approximately 9 to 12% of patients with autoimmune hepatitis. The overwhelming majority of patients with DIH are women. The underlying mechanisms of these sex differences in prevalence are unclear because of the paucity of animal models that mimic human disease. Even so, underlying mechanisms are widely believed to be associated with human leukocyte antigen haplotypes and sex hormones. In contrast, using a DIH mouse model, we have uncovered that IL-4 initiated&#160;CD4+ T cells directed against an epitope of cytochrome P450 2E1 induces influx of neutrophils, macrophages and mast cells into the livers of female BALB/c mice. Using this model, we have also shown that IL-33-induced FoxP3+regulatory T cells confer protection against DIH in female and male mice. This DIH model is induced by immunizing mice with an epitope of CYP2E1 that has been covalently altered with a drug metabolite that has been associated with DIH. This epitope is recognized by patients with DIH. Our method induces robust and reproducible hepatitis and autoantibodies that can be utilized to study the pathogenesis of DIH. While in vivo studies can cause undue pain and distress in mice when done improperly, the advantage of an in vivo model is the ability to evaluate the pathogenesis of disease in a large number of mice. Additionally, biological effects of the altered liver proteins can be studied using invasive procedures. The addition of in vitro studies to the experimental design allows rapid repetition and mechanistic analysis at a cellular level. Thus, we will demonstrate our model protocol and how it can be utilized to study in vivo and in vitro mechanisms of DIH.

We describe an in vivo immunization, translational hepatitis model in BALB/c mice that can be utilized to study the pathogenesis of drug-induced autoimmune hepatitis including sex differences seen in this disease. We will describe how this model demonstrates reproducible analyses using in vivo and in vitro experimental techniques.

병원성 메커니즘 연구를위한 BALB / c 마우스에서 약물 유도,자가 면역 간염의 유도

Drug-induced autoimmune hepatitis (DIH) is the most common hepatic drug-induced hypersensitization process observed in approximately 9 to 12% of patients ...

Visualizing Impairment of the Endothelial and Glial Barriers of the Neurovascular Unit during Experimental Autoimmune Encephalomyelitis In Vivo

The neurovascular unit (NVU) is composed of microvascular endothelial cells forming the blood-brain barrier (BBB), an endothelial basement membrane with embedded pericytes, and the glia limitans composed by the parenchymal basement membrane and astrocytic end-feed embracing the abluminal aspect of central nervous system (CNS) microvessels. In addition to maintaining CNS homeostasis the NVU controls immune cell trafficking into the CNS. During immunosurveillance of the CNS low numbers of activated lymphocytes can cross the endothelial barrier without causing BBB dysfunction or clinical disease. In contrast, during neuroinflammation such as in multiple sclerosis or its animal model experimental autoimmune encephalomyelitis (EAE) a large number of immune cells can cross the BBB and subsequently the glia limitans eventually reaching the CNS parenchyma leading to clinical disease. 
Immune cell migration into the CNS parenchyma is thus a two-step process that involves a sequential migration across the endothelial and glial barrier of the NVU employing distinct molecular mechanisms. If following their passage across the endothelial barrier, T cells encounter their cognate antigen on perivascular antigen-presenting cells their local reactivation will initiate subsequent mechanisms leading to the focal activation of gelatinases, which will enable the T cells to cross the glial barrier and enter the CNS parenchyma. Thus, assessing both, BBB permeability and MMP activity in spatial correlation to immune cell accumulation in the CNS during EAE allows to specify loss of integrity of the endothelial and glial barriers of the NVU. 
We here show how to induce EAE in C57BL/6 mice by active immunization and how to subsequently analyze BBB permeability in vivo using a combination of exogenous fluorescent tracers. We further show, how to visualize and localize gelatinase activity in EAE brains by in situ zymogaphy coupled to immunofluorescent stainings of BBB basement membranes and CD45+ invading immune cells.

Here, we present protocols to investigate impairment of the neurovascular unit during experimental autoimmune encephalomyelitis in vivo. We specifically address how to determine blood-brain barrier permeability and gelatinase activity involved in leukocyte migration across the glia limitans.&#160;

Vivo에서 실험적인 자기 면역 뇌 중 Neurovascular 단위의 내 피와 폐해 장벽의 장애 시각화

The neurovascular unit (NVU) is composed of microvascular endothelial cells forming the blood-brain barrier (BBB), an endothelial basement membrane with ...

Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase (G6PI)-Induced RA Mice

Rheumatoid arthritis (RA) is a complex chronic inflammatory autoimmune disease. The pathogenesis of the disease is related to invariant natural killer T (iNKT) cells. Patients with active RA present fewer iNKT cells, defective cell function, and excessive polarization of Th1. In this study, an RA animal model was established using a mixture of hGPI325-339 and hGPI469-483 peptides. The iNKT cells were obtained by in vivo induction and in vitro purification, followed by infusion into RA mice for adoptive immunotherapy. The in vivo imaging system (IVIS) tracking revealed that iNKT cells were mainly distributed in the spleen and liver. On day 12 after cell therapy, the disease progression slowed down significantly, the clinical symptoms were alleviated, the abundance of iNKT cells in the thymus increased, the proportion of iNKT1 in the thymus decreased, and the levels of TNF-&#945;, IFN-&#947;, and IL-6 in the serum decreased. Adoptive immunotherapy of iNKT cells restored the balance of immune cells and corrected the excessive inflammation of the body.

Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase G6PI-Induced RA Mice

This protocol uses G6PI mixed peptides to construct rheumatoid arthritis models that are closer to that of human rheumatoid arthritis in CD4+ T cells and cytokines. High purity invariant natural killer T cells (mainly iNKT2) with specific phenotypes and functions were obtained by in vivo induction and in vitro purification for adoptive immunotherapy.

포도당-6-인산염 이사머라제(G6PI)-유도 RA 마우스에서 iNKT 세포의 입양 면역 요법

Rheumatoid arthritis (RA) is a complex chronic inflammatory autoimmune disease. The pathogenesis of the disease is related to invariant natural killer T ...

Flow Cytometric Analysis of Lymphocyte Infiltration in Central Nervous System during Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible genetic background. Experimental autoimmune encephalomyelitis (EAE) is a typical disease model of MS widely used for investigating the pathogenesis in which T lymphocytes specific for myelin antigens initiate an inflammatory reaction in CNS. It is very important to assess how lymphocytes in the CNS regulate the development of disease. However, the approach for measuring the quantity and quality of infiltrated lymphocytes in the CNS is very limited due to the difficulties in isolating and detecting infiltrated lymphocytes from the brain. This manuscript presents a protocol for that is useful for the isolation, identification, and characterization of infiltrated lymphocytes from the CNS and will be helpful for our understanding of how lymphocytes are involved in the development of the CNS autoimmune disease.

This manuscript presents a protocol to induce active experimental autoimmune encephalomyelitis (EAE) in mice. A method for the isolation and characterization of the infiltrated lymphocytes in the central nervous system (CNS) is also presented to show how lymphocytes are involved in the development of CNS autoimmune disease.

실험적인 자가면역 뇌척수염 중 중추신경계에서 림프구 침투의 흐름 세포 분석

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible ...

Experimental Autoimmune Uveitis: An Intraocular Inflammatory Mouse Model

Experimental Autoimmune Uveitis (EAU) is driven by immune cells responding to self-antigens. Many features of this non-infectious, intraocular inflammatory disease model recapitulate the clinical phenotype of posterior uveitis affecting humans. EAU has been used reliably to study the efficacy of novel inflammatory therapeutics, their mode of action and to further investigate the mechanisms that underpin disease progression of intraocular disorders. Here, we provide a detailed protocol on EAU induction in the C57BL/6J mouse - the most widely used model organism with susceptibility to this disease. Clinical assessment of disease severity and progression will be demonstrated using fundoscopy, histological examination and fluorescein angiography. The induction procedure involves subcutaneous&#160;injection of an emulsion containing a peptide (IRBP1-20) from the ocular protein interphotoreceptor retinoid binding protein (also known as retinol binding protein 3), Complete Freund&#39;s Adjuvant (CFA) and supplemented with killed Mycobacterium tuberculosis. Injection of this viscous emulsion on the back of the neck is followed by a single intraperitoneal injection of Bordetella pertussis toxin. At the onset of symptoms (day 12-14) and under general anesthesia, fundoscopic images are taken to assess disease progression through clinical examination. These data can be directly compared with those at later timepoints and peak disease (day 20-22) with differences analyzed. At the same time, this protocol allows the investigator to assess potential differences in vessel permeability and damage using fluorescein angiography. EAU can be induced in other mouse strains - both wildtype or genetically modified - and combined with novel therapies offering flexibility for studying drug efficacy and/or disease mechanisms.

In this report we present a protocol that allows the investigator to generate a mouse model of intraocular uveitis. More commonly referred to as experimental autoimmune uveitis (EAU), this established model captures many aspects of human disease. Herein, we will describe how to induce and monitor disease progression using several readouts.

실험적 자가면역 포도막염: 안구내 염증성 마우스 모델

Experimental Autoimmune Uveitis (EAU) is driven by immune cells responding to self-antigens. Many features of this non-infectious, intraocular ...

자가면역뇌척수염에서 Mycobacterium paratuberculosis에 의한 면역원성 향상

Adjuvant Activity of Mycobacterium paratuberculosis in Enhancing the Immunogenicity of Autoantigens During Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein (MOG) requires immunization by a MOG peptide emulsified in complete Freund&#39;s adjuvant (CFA) containing inactivated Mycobacterium tuberculosis. The antigenic components of the mycobacterium activate dendritic cells to stimulate T-cells to produce cytokines that promote the Th1 response via toll-like receptors. Therefore, the amount and species of mycobacteria present during the antigenic challenge are directly related to the development of EAE. This methods paper presents an alternative protocol to induce EAE in C57BL/6 mice using a modified incomplete Freund&#39;s adjuvant containing the heat-killed Mycobacterium avium subspecies paratuberculosis strain K-10.
M. paratuberculosis, a member of the Mycobacterium avium complex, is the causative agent of Johne&#39;s disease in ruminants and has been identified as a risk factor for several human T-cell-mediated disorders, including multiple sclerosis. Overall, mice immunized with Mycobacterium paratuberculosis showed earlier onset and greater disease severity than mice immunized with CFA containing the strain of M. tuberculosis H37Ra at the same doses of 4 mg/mL. The antigenic determinants of Mycobacterium avium subspecies paratuberculosis (MAP)&#160;strain K-10 were able to induce a strong Th1 cellular response during the effector phase, characterized by significantly higher numbers of T-lymphocytes (CD4+ CD27+), dendritic cells (CD11c+ I-A/I-E+), and monocytes (CD11b+ CD115+) in the spleen compared to mice immunized with CFA. Furthermore, the proliferative T-cell response to the MOG peptide appeared to be highest in M. paratuberculosis-immunized mice. The use of an encephalitogen (e.g., MOG35-55) emulsified in an adjuvant containing M. paratuberculosis in the formulation may be an alternative and validated method to activate dendritic cells for priming myelin epitope-specific CD4+ T-cells during the induction phase of EAE.

저자 스포트라이트: 실험적 자가면역 뇌척수염 중 자가항원의 면역원성 향상에 대한 Mycobacterium paratuberculosis의 보조 활성  

Here, we present an alternative protocol to actively induce experimental autoimmune encephalomyelitis in C57BL/6 mice, using the immunogenic epitope myelin oligodendrocyte glycoprotein (MOG)35-55 suspended in incomplete Freund's adjuvant containing the heat-killed Mycobacterium avium subspecies paratuberculosis.

실험적 자가면역 뇌척수염 동안 자가항원의 면역원성을 향상시키는 Mycobacterium paratuberculosis 의 보조 활성

Experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein (MOG) requires immunization by a MOG peptide emulsified in ...