עבודה זו נתמכת על ידי אוניברסיטת גתה בפרנקפורט החולים מענק של קרן המחקר הגרמנית UC

<ol>
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החוקרים אין לי מה לחשוף.

במחקרים קודמים, הפטיטיס ניסיוני דווח לעתים קרובות להיות רק המודלים הנוכחיים רגעיות רבים עבור מחלת כבד אוטואימונית תלוי אינדוקציה כמה פרוטוקולים מורכבים ולא המחלה (עבור ביקורות לראות 3,5). לדוגמה, מספר דגמים להשתמש העכברים הטרנסגניים ביטוי אנטיגנים יעד ספציפיים ולהעביר adoptively היעד אנטיגן ספציפי, בעיקר TCR-מהונדס, ותאי T 14,15. לעתים קרובות זיהום נוסף עם וירוסים, חיידקים או טפילים livertropic יש צורך לגרום למחלות 16-18. לחילופין, ה-DNA חיסון עם פלסמידים קידוד אנטיגנים היעד, כולל CYP2D6 19, משמש כדי לגרום דלקת הכבד. עם זאת, חיסון נוסף עם פלסמידים קידוד Pro ציטוקינים דלקתיים, כגון IL-12, נדרש 20. למרבה הצער, עם כמה יוצאים מן הכלל 15,20 הפטיטיס הוא זמני בלבד. המודל CYP2D6 6,21 משתמש לא שיטה פשוטההו לגרום דלקת כבד אוטואימונית פשוט על ידי הדבקה של עכברים עם adenovirus להביע hCYP2D6, autoantigen מרכזי AIH-2 7,8. משלוח של CYP2D6 על ידי מבנה adenovirus מבטיח גם מיקוד ישיר של הכבד, כמו גם דלקת מקומית המקדמת פירוט של סובלנות. חשוב לציין עם זאת כי הן כייל הנגיף, כמו גם את המסלול של הממשל היא קריטית דגם זה. מצד אחד, Ad-2D6 הוא וירוס שכפול לקויה, ובכך, כייל גבוה מספיק של וירוס נדרש לייצר כמות קריטית של האנטיגן בתוך הכבד. מצד שני, גבוה מדי כייל עלולה לגרום לאי ספיקת כבד חריפה קטלנית. בנוסף, הוכח בעבר כי זיהום של עכברים על ידי titers גבוהות של adenovirus מוביל תשישות תפקודית של התגובה החיסונית 22. במודל זה, השתמשנו בשילוב של זיהום תוך ורידי ו intraperitoneal על מנת לקבל גם חדירת הסלולר כרונית, כמו גם שלוחהensive פיברוזיס. ראינו כי זיהום תוך ורידי בלבד עדיין גורם חדירת הסלולר מסיבי, אבל לא סיסטיק האזור subcapsular, המציין כי דלקת הצפק עקב הזרקת intraperitoneal יכול להיות מעורב הצטברות subcapsular רציפה של קולגן (Hintermann &amp; להטביל, כתב היד בהכנה). עם זאת, חשוב לציין כי סיסטיק הכבד הנצפה הוא אנטיגן ספציפי, שכן לא גילינו הפעלת stellate תאים בכבד וכן בתצהיר קולגן לאחר ההדבקה עם שוויון titers וירוס של ה-GFP, מודעות או מודעות-Control 23. המודל CYP2D6 משקף היבטים רבים של 1,2 AIH האדם, כגון דלקת כבד כרונית המאופיינת על ידי חדירת הסלולר מתמשך לייפת הכבד 6. בנוסף, נוכחות גבוהה כייל נוגדנים נגד CYP2D6 עם ייחוד epitope דומה והפצת 13 ל LKM-1 נוגדנים הנמצאים AIH-2 בחולים מציין מראשהיגיון של התגובה הנפוצה אוטואימונית כרונית. CYP2D6 הוא אנטיגן מרכזי AIH-2, אבל זה לא מוכר על ידי חולים שאובחנו עם AIH תכופים יותר מסוג 1. בנוסף, חולים הסובלים ממחלות כבד אחרות עם אטיולוגיה אוטואימונית, כגון שחמת המרה הראשי (ות&quot;ת) או העיקרי sclerosing cholangitis (PSC), לייצר נוגדנים עצמיים סימן ההיכר עם סגוליות כדי autoantigens כבד שונות ואת הכבדים שלהם להראות תכונות פתולוגיים שונים שבמרכזו קטן (ות&quot;ת ) או גדולים (PSC) צינורות המרה. עם זאת, CYP2D6 מודלים מציעה את ההזדמנות לנתח את מנגנוני immunopathogenic המעורבים בתהליכים דלקתיים כרוניים בכבד כמו המתרחשים במהלך מחלות כבד אוטואימוניות, לזהות את שחקני המפתח המניעים את ההרס אוטואימונית של hepatocytes ולהעריך התערבויות טיפוליות ניתן לרפא את המחלה.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם הציוד </td><td> חברה </td><td> מספר קטלוגי </td><td> תגובות </td></tr><tr><td> 23G מחט </td><td> BD Biosciences </td><td> 300800 </td><td></td></tr><tr><td> 27 ½ G מחט </td><td> VWR </td><td> 612-0151 </td><td></td></tr><tr><td> 30 ½ מחט G </td><td> BD Biosciences </td><td> 304000 </td><td></td></tr><tr><td> אלאנין הבדיקה רצועות aminotransferase (GPT / ALT) </td><td> Roche Diagnostics </td><td> 10 745 138 202 </td><td></td></tr><tr><td> Aspartate מקלוני הבדיקה aminotransferase (יש / AST) </td><td> Roche Diagnostics </td><td> 10 745 120 202 </td><td></td></tr><tr><td> אלחוש יחידת Univentor 400 </td><td> <a href="http://www.agnthos.se" target="_blank" >AgnTho של</a> </td><td></td><td></td></tr><tr><td> בסיס תבניות, 37x24x10 מ&quot;מ חד פעמיות </td><td> VWR </td><td> 720-0208 </td><td></td></tr><tr><td> תא מסננת 70mm </td><td> VWR </td><td> 734-0003 </td><td></td></tr><tr><td> Cryostat </td><td> Leica </td><td> CM1850 UV </td><td></td></tr><tr><td> צינורות נימי Heparinized </td><td> דיג </td><td> 3123987 </td><td></td></tr><tr><td> מיקרוסקופ מחליק Superfrost פלוס </td><td> מנצל גלזר </td><td> J1800AMNZ </td><td></td></tr><tr><td> Microtainer SST צינורות </td><td> BD Biosciences </td><td> 365951 </td><td></td></tr><tr><td> Microtiter צלחות, V-התחתונות </td><td> VWR </td><td> 391-1924 </td><td></td></tr><tr><td> Reflotron פלוס </td><td> Roche Diagnostics </td><td></td><td> Determiniation של סרום AST / ALT </td></tr><tr><td colspan="4"></td></tr><tr><td> שם OF מגיב </td><td> חברה </td><td> מספר קטלוגי </td><td> תגובות </td></tr><tr><td> הארנב נגד העכבר נגד קולגן מסוג IgG אני, </td><td> Chemicon </td><td> AB765P </td><td></td></tr><tr><td> Biotinylated נגד ארנב עז IgG </td><td> וקטור (AXXORA) </td><td> VC-BA-1000-MC15 </td><td></td></tr><tr><td> FITC-מצומדות נוגדנים נגד CD8a העכבר </td><td> דרום ביוטק </td><td> 1550-1502 </td><td></td></tr><tr><td> PE-מצומדות העכבר אנטי IFNγ נוגדן </td><td> BD Biosciences </td><td> 554412 </td><td></td></tr><tr><td> PE-מצומדות נגד העכבר CD16/CD32 נוגדנים (FCR בלוק) </td><td> BD Biosciences </td><td> 553141 </td><td></td></tr><tr><td> Aquatex </td><td> VWR </td><td> 1.08562.0050 </td><td></td></tr><tr><td> Avidin / ביוטין חסימת ערכת </td><td&gt; וקטור (AXXORA) </td><td> VC-SP-2001-KI01 </td><td></td></tr><tr><td> Brefeldin </td><td> סיגמא </td><td> B6542 </td><td></td></tr><tr><td> Collagenase IV </td><td> סיגמא </td><td> C5138-100 מ&quot;ג </td><td></td></tr><tr><td> DAB המצע ערכת 3&#39;3&#39;diaminobenzidine </td><td> וקטור (AXXORA) </td><td> VC-SK-4100-KI01 </td><td></td></tr><tr><td> DNase </td><td> סיגמא </td><td> DN-25 </td><td></td></tr><tr><td> עלית מגיב ABC </td><td> וקטור (AXXORA) </td><td> VC-PK-7100-L050 </td><td></td></tr><tr><td> Eosin G / Y פתרון </td><td> רוט </td><td> X883.1 </td><td></td></tr><tr><td> עוברי עגלים בסרום (FCS) </td><td> Biochrom AG </td><td> S 0115 </td><td></td></tr><tr><td> Isofluran </td><td> Forene </td><td> B506 </td><td></td></tr><tr><td> מאייר Hematoxylin פתרון </td><td> AppliChem </td><td> A4840, 1000 </td><td></td></tr><tr><td> אוקטובר מתחם </td><td> סאקורה Finetek אירופה </td><td> 4583 </td><td></td></tr><tr><td> PBS שנאגרו פורמלדהיד 10% </td><td> רוט </td><td> A146.3 </td><td></td></tr><tr><td> Percoll </td><td> Amersham </td><td> 17-0891-01 </td><td></td></tr><tr><td> רוטי, Histokit </td><td> רוט </td><td> 6638.1 </td><td></td></tr><tr><td> RPMI </td><td> Invitrogen </td><td> 61870 </td><td></td></tr><tr><td> Saponin מקליפת quillaja </td><td> סיגמא </td><td> S7900 </td><td></td></tr></tbody></table>

the cyp2d6 animal model: how to induce autoimmune hepatitis in mice

הפטיטיס אוטואימונית היא מחלה אוטואימונית נדירה אך סכנת חיים של הכבד של 1,2 שהגורם לה אינו ידוע. במהלך ניסיונות רבים בעבר נעשו כדי לייצר מודל בעל חיים המשקף את המאפיינים של המחלה האנושית 3-5. עם זאת, מודלים שונים אינדוקציה של המחלה היה מורכב למדי, לעתים קרובות הפטיטיס היה חולף רק 3-5. לכן, פיתחנו מודל עכבר פשוטה המשתמשת autoantigen אדם מרכזי מסוג 2 הפטיטיס אוטואימונית (AIH-2), כלומר hCYP2D6, כמו ההדק 6. 1 סוג כבד כליות נוגדנים microsomal (LKM-1) נוגדנים הכרה hCYP2D6 הם סימן ההיכר של AIH-2 7,8. מסירת hCYP2D6 אל wildtype FVB או C57BL / 6 עכברים היתה לבנות adenovirus (Ad-2D6), אשר מבטיחה אספקה ​​ישירה של האנטיגן מפעילה אל הכבד. לכן, דלקת מקומית שלאחר מכן יוצר שדה פורה 9 לפיתוח הבאות של אוטואימוניות. גombination של הזרקה תוך ורידית ו intraperitoneal של Ad-2D6 היא הדרך היעילה ביותר כדי לגרום נזק לטווח ארוך אוטואימונית אל הכבד (סעיף 1). כאן אנו מספקים פרוטוקול מפורט על איך אוטואימונית מחלות כבד מושרה במודל CYP2D6 וכיצד היבטים שונים של נזק לכבד ניתן להעריך. ראשית, רמות בסרום של סמנים המעידים על הרס hepatocyte, כגון aminotransferases, כמו גם את titers של hCYP2D6 נוגדנים נקבעים באמצעות דגימת דם retroorbitaly (סעיף 2). שנית, התגובה hCYP2D6 ספציפי תא T מאופיינת באיסוף לימפוציטים של הטחול והכבד. על מנת לקבל לימפוציטים כבד טהור, הכבדים הם perfused ידי PBS דרך וריד השער (סעיף 3), מתעכל על קולגן מטוהרים על שיפוע Percoll (סעיף 4). התדירות של hCYP2D6 ספציפיים בתאי T מנותח על ידי גירוי עם hCYP2D6 פפטידים וזיהוי של תאים מייצרי IFNγ ידי cytometry זרימה (סעיף 5). שלישית,חדירת הסלולר סיסטיק והוא נקבע על ידי אימונוהיסטוכימיה של סעיפים כבד (סעיף 6). משטר ניתוח כזה חייב להתנהל על פי כמה לאחר תחילת המחלה, כדי להוכיח את האופי הכרוני של המודל. עוצמת התגובה החיסונית מתאפיינת בתדירות הפעילות של hCYP2D6-T הספציפיים ו / או תאים מסוג B ואת מידת הנזק הכבד סיסטיק צריך להעריך להערכה בדיעבד של הטיפולים האפשריים כדי למנוע, לעכב או לבטל את autodestructive תהליך של הכבד.

Watch this Scientific Journal Video about The CYP2D6 Animal Model: How to Induce Autoimmune Hepatitis in Mice at JoVE.com

The CYP2D6 Animal Model: How to Induce Autoimmune Hepatitis in Mice

זיהום של עכברים עם adenovirus להביע autoantigen אדם גדול ציטוכרום P450 2D6 (hCYP2D6) המוכרים על ידי סרה של חולים הסובלים מסוג 2 תוצאות אוטואימוניות הפטיטיס בצורה מתמשכת של מחלה אוטואימונית בתיווך הכבד המאופיינת סיסטיק נרחב צהבת, ואת הדור של CYP2D6 ספציפית התגובה החיסונית.

CYP2D6 מודל החיה: כיצד לגרום דלקת כבד אוטואימונית עכברים

Autoimmune hepatitis is a rare but life threatening autoimmune disease of the liver of unknown etiology1,2. In the past many attempts have been made to ...

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18.9K Views.  Goethe University Hospital Frankfurt. זיהום של עכברים עם adenovirus להביע autoantigen אדם גדול ציטוכרום P450 2D6 (hCYP2D6) המוכרים על ידי סרה של חולים הסובלים מסוג 2 תוצאות אוטואימוניות הפטיטיס בצורה מתמשכת של מחלה אוטואימונית בתיווך הכבד המאופיינת סיסטיק נרחב צהבת, ואת הדור של CYP2D6 ספציפית התגובה החיסונית.

Video: The CYP2D6 Animal Model: How to Induce Autoimmune Hepatitis in Mice

Pressure Controlled Ventilation to Induce Acute Lung Injury in Mice

Murine models are extensively used to investigate acute injuries of different organs systems (1-34). Acute lung injury (ALI), which occurs with prolonged mechanical ventilation, contributes to morbidity and mortality of critical illness, and studies on novel genetic or pharmacological targets are areas of intense investigation (1-3, 5, 8, 26, 30, 33-36). ALI is defined by the acute onset of the disease, which leads to non-cardiac pulmonary edema and subsequent impairment of pulmonary gas exchange (36). We have developed a murine model of ALI by using a pressure-controlled ventilation to induce ventilator-induced lung injury (2). For this purpose, C57BL/6 mice are anesthetized and a tracheotomy is performed followed by induction of ALI via mechanical ventilation. Mice are ventilated in a pressure-controlled setting with an inspiratory peak pressure of 45 mbar over 1 - 3 hours. As outcome parameters, pulmonary edema (wet-to-dry ratio), bronchoalveolar fluid albumin content, bronchoalveolar fluid and pulmonary tissue myeloperoxidase content and pulmonary gas exchange are assessed (2). Using this technique we could show that it sufficiently induces acute lung inflammation and can distinguish between different treatment groups or genotypes (1-3, 5). Therefore this technique may be helpful for researchers who pursue molecular mechanisms involved in ALI using a genetic approach in mice with gene-targeted deletion.

A murine model for ventilator induced lung injury is an important tool to study an acute lung injury in vivo. Here, we report an easy applicable in situ model for acute lung injury using high-pressure mechanical ventilation to induce acute failure of the lung.

לחץ אוורור מבוקר כדי לגרום פגיעה ריאות חריפה על עכברים

Murine models are extensively used to investigate acute injuries of different organs systems (1-34). Acute lung injury (ALI), which occurs with prolonged ...

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

סמנים ביולוגיים במודל חיה לחשיפה קושרת עצבית, המטולוגית, והתנהגות של PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

Protocol for Long Duration Whole Body Hyperthermia in Mice

Hyperthermia is a general term used to define the increase in core body temperature above normal. It is often used to describe the increased core body temperature that is observed during fever. The use of hyperthermia as an adjuvant has emerged as a promising procedure for tumor regression in the field of cancer biology. For this purpose, the most important requirement is to have reliable and uniform heating protocols. We have developed a protocol for hyperthermia (whole body) in mice. In this protocol, animals are exposed to cycles of hyperthermia for 90 min followed by a rest period of 15 min. During this period mice have easy access to food and water. High body temperature spikes in the mice during first few hyperthermia exposure cycles are prevented by immobilizing the animal. Additionally, normal saline is administered in first few cycles to minimize the effects of dehydration. This protocol can simulate fever like conditions in mice up to 12-24 hr. We have used 8-12 weeks old BALB/Cj female mice to demonstrate the protocol.

This paper describes a protocol for whole body hyperthermia in mice that can stimulate fever like conditions up to 12-24 hr.

פרוטוקול ליפרתרמיה הארוכה משך כל גוף בעכברים

Hyperthermia is a general term used to define the increase in core body temperature above normal. It is often used to describe the increased core body ...

Use of Animal Model of Sepsis to Evaluate Novel Herbal Therapies

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several techniques, including infusion of exogenous bacterial toxin (endotoxemia) or bacteria (bacteremia), as well as surgical perforation of the cecum by cecal ligation and puncture (CLP)1-3. CLP allows bacteria spillage and fecal contamination of the peritoneal cavity, mimicking the human clinical disease of perforated appendicitis or diverticulitis. The severity of sepsis, as reflected by the eventual mortality rates, can be controlled surgically by varying the size of the needle used for cecal puncture2. In animals, CLP induces similar, biphasic hemodynamic cardiovascular, metabolic, and immunological responses as observed during the clinical course of human sepsis3. Thus, the CLP model is considered as one of the most clinically relevant models for experimental sepsis1-3.
Various animal models have been used to elucidate the intricate mechanisms underlying the pathogenesis of experimental sepsis. The lethal consequence of sepsis is attributable partly to an excessive accumulation of early cytokines (such as TNF, IL-1 and IFN-&gamma;)4-6 and late proinflammatory mediators (e.g., HMGB1)7. Compared with early proinflammatory cytokines, late-acting mediators have a wider therapeutic window for clinical applications. For instance, delayed administration of HMGB1-neutralizing antibodies beginning 24 hours after CLP, still rescued mice from lethality8,9, establishing HMGB1 as a late mediator of lethal sepsis. The discovery of HMGB1 as a late-acting mediator has initiated a new field of investigation for the development of sepsis therapies using Traditional Chinese Herbal Medicine. In this paper, we describe a procedure of CLP-induced sepsis, and its usage in screening herbal medicine for HMGB1-targeting therapies.

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection, and can be simulated by a surgical technique termed cecal ligation and puncture (CLP). Here we describe a method to use CLP-induced animal model to screen medicinal herbs for therapeutic agents.

שימוש במודל של בעלי חיים אלח דם להערכת טיפולים צמחים רומן

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several ...

Visualization and Analysis of Blood Flow and Oxygen Consumption in Hepatic Microcirculation: Application to an Acute Hepatitis Model

There is a considerable discrepancy between oxygen supply and demand in the liver because hepatic oxygen consumption is relatively high but about 70% of the hepatic blood supply is poorly oxygenated portal vein blood derived from the gastrointestinal tract and spleen. Oxygen is delivered to hepatocytes by blood flowing from a terminal branch of the portal vein to a central venule via sinusoids, and this makes an oxygen gradient in hepatic lobules. The oxygen gradient is an important physical parameter that involves the expression of enzymes upstream and downstream in hepatic microcirculation, but the lack of techniques for measuring oxygen consumption in the hepatic microcirculation has delayed the elucidation of mechanisms relating to oxygen metabolism in liver. We therefore used FITC-labeled erythrocytes to visualize the hepatic microcirculation and used laser-assisted phosphorimetry to measure the partial pressure of oxygen in the microvessels there. Noncontact and continuous optical measurement can quantify blood flow velocities, vessel diameters, and oxygen gradients related to oxygen consumption in the liver. In an acute hepatitis model we made by administering acetaminophen to mice we observed increased oxygen pressure in both portal and central venules but a decreased oxygen gradient in the sinusoids, indicating that hepatocyte necrosis in the pericentral zone could shift the oxygen pressure up and affect enzyme expression in the periportal zone. In conclusion, our optical methods for measuring hepatic hemodynamics and oxygen consumption can reveal mechanisms related to hepatic disease.

An optical system was developed to visualize hepatic microcirculation with FITC-labeled erythrocytes and to measure the partial pressure of oxygen in the microvessels with laser-assisted phosphorimetry. This method can be used to investigate physiological and pathological mechanisms by analyzing microvascular structure, diameter, blood flow velocity, and oxygen tension.

הדמיה וניתוח של זרימת הדם ואת צריכת החמצן microcirculation כבד: בקשה דגם הפטיטיס חריפה

There is a considerable discrepancy between oxygen supply and demand in the liver because hepatic oxygen consumption is relatively high but about 70% of ...

Implanting Glass Spinal Cord Windows in Adult Mice with Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) in adult rodents is the standard experimental model for studying autonomic demyelinating diseases such as multiple sclerosis. Here we present a low-cost and reproducible glass window implantation protocol that is suitable for intravital microscopy and studying the dynamics of spinal cord cytoarchitecture with subcellular resolution in live adult mice with EAE. Briefly, we surgically expose the vertebrae T12-L2 and construct a chamber around the exposed vertebrae using a combination of cyanoacrylate and dental cement. A laminectomy is performed from T13 to L1, and a thin layer of transparent silicone elastomer is applied to the dorsal surface of the exposed spinal cord. A modified glass cover slip is implanted over the exposed spinal cord taking care that the glass does not directly contact the spinal cord. To reduce the infiltration of inflammatory cells between the window and spinal cord, anti-inflammatory treatment is administered every 2 days (as recommended by ethics committee) for the first 10 days after implantation. EAE is induced only 2-3 weeks after the cessation of anti-inflammatory treatment.
Using this approach we successfully induced EAE in 87% of animals with implanted windows and, using Thy1-CFP-23 mice (blue axons in dorsal spinal cord), quantified axonal loss throughout EAE progression. Taken together, this protocol may be useful for studying the recruitment of various cell populations as well as their interaction dynamics, with subcellular resolution and for extended periods of time. This intravital imaging modality represents a valuable tool for developing therapeutic strategies to treat autoimmune demyelinating diseases such as EAE.

We describe a method for implanting and maintaining glass windows over the exposed spinal cords of adult mice for studying experimental autoimmune encephalomyelitis. These windows provide chronic optical access to the spinal cord for monitoring cell dynamics at the subcellular level in live animals using two-photon microscopy.

השתלת זכוכית חוט השדרה של Windows בעכברים בוגרים עם ניסיון אוטואימוניות Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) in adult rodents is the standard experimental model for studying autonomic demyelinating diseases such as ...

A Magnetic Microbead Occlusion Model to Induce Ocular Hypertension-Dependent Glaucoma in Mice

The use of rodent models of glaucoma has been essential to understand the molecular mechanisms that underlie the pathophysiology of this multifactorial neurodegenerative disease. With the advent of numerous transgenic mouse lines, there is increasing interest in inducible murine models of ocular hypertension. Here, we present an occlusion model of glaucoma based on the injection of magnetic microbeads into the anterior chamber of the eye using a modified microneedle with a facetted bevel. The magnetic microbeads are attracted to the iridocorneal angle using a handheld magnet to block the drainage of aqueous humour from the anterior chamber. This disruption in aqueous dynamics results in a steady elevation of intraocular pressure, which subsequently leads to the loss of retinal ganglion cells, as observed in human glaucoma patients. The microbead occlusion model presented in this manuscript is simple compared to other inducible models of glaucoma and also highly effective and reproducible. Importantly, the modifications presented here minimize common issues that often arise in occlusion models. First, the use of a bevelled glass microneedle prevents backflow of microbeads and ensures that minimal damage occurs to the cornea during the injection, thus reducing injury-related effects. Second, the use of magnetic microbeads ensures the ability to attract most beads to the iridocorneal angle, effectively reducing the number of beads floating in the anterior chamber avoiding contact with other structures (e.g., iris, lens). Lastly, the use of a handheld magnet allows flexibility when handling the small mouse eye to efficiently direct the magnetic microbeads and ensure that there is little reflux of the microbeads from the eye when the microneedle is withdrawn. In summary, the microbead occlusion mouse model presented here is a powerful investigative tool to study neurodegenerative changes that occur during the onset and progression of glaucoma.

Here, we present a protocol to induce ocular hypertension in the murine eye that results in the loss of retinal ganglion cells as observed in glaucoma. Magnetic microbeads are injected into the anterior chamber and attracted to the iridocorneal angle using a magnet to block the outflow of aqueous humour.

לחסימה Microbead מגנטי דגם כדי לגרום Ocular Hypertension-Dependent גלאוקומה עכברים

The use of rodent models of glaucoma has been essential to understand the molecular mechanisms that underlie the pathophysiology of this multifactorial ...

Balloon-based Injury to Induce Myointimal Hyperplasia in the Mouse Abdominal Aorta

The use of animal models is essential for a better understanding of MH, one major cause for arterial stenosis.In this article, we demonstrate a murine balloon denudation model, which is comparable with established vessel injury models in large animals. The aorta denudation model with balloon catheters mimics the clinical setting and leads to comparable pathobiological and physiological changes. Briefly, after performing a horizontal incision in the aorta abdominalis, a balloon catheter will be inserted into the vessel, inflated, and introduced retrogradely. Inflation of the balloon will lead to intima injury and overdistension of the vessel. After removing the catheter, the aortic incision will be closed with single stiches. The model shown in this article is reproducible, easy to perform, and can be established quickly and reliably. It is especially suitable for evaluating expensive experimental therapeutic agents, which can be applied in an economical fashion. By using different knockout-mouse strains, the impact of different genes on MH development can be assessed.

This article demonstrates a murine model to study the development of myointimal hyperplasia (MH) after aortic balloon injury.

מבוסס על בלון פציעה לזירוז היפרפלזיה Myointimal באבי העורקים בטן העכבר

The use of animal models is essential for a better understanding of MH, one major cause for arterial stenosis.In this article, we demonstrate a murine ...

A Closed-chest Model to Induce Transverse Aortic Constriction in Mice

Research on cardiac hypertrophy and heart failure is frequently based on pressure overload mouse models induced by TAC. The standard procedure is to perform a partial thoracotomy to visualize the transverse aortic arch. However, the surgical trauma caused by the thoracotomy in open-chest models changes the respiratory physiology as the ribs are dissected and left unattached after chest closure. To prevent this, we established a minimally invasive, closed chest approach via lateral thoracotomy. Herein we approach the aortic arch via the 2nd intercostal space without entering the chest cavities, leaving the mouse with a less traumatic injury to recover from. We perform this operation using standard laboratory settings for open chest TAC procedures with equal survival rates. Apart from maintaining physiological breathing patterns due to the closed chest approach, the mice seem to benefit by showing rapid recovery, as the less invasive technique appears to facilitate a fast healing process and to reduce immune response after trauma.

Here, we present a protocol of Transverse Aortic constriction (TAC) via a lateral thoracotomy. This technique is a minimally invasive, closed chest surgical procedure aiming to simulate pressure overload and heart failure in mice utilizing standard TAC laboratory settings.

דגם סגור-החזה לזירוז רוחבי. התרחבות אבי העורקים בעכברים

Research on cardiac hypertrophy and heart failure is frequently based on pressure overload mouse models induced by TAC. The standard procedure is to ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

Contractility איזומטרי מדידה של עורק מצע המעי העליון העכבר באמצעות חוט Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...