Name: generation of a mouse spontaneous autoimmune thyroiditis model
Uploaded: 2023-03-17T14:05:00
Description: Watch this Scientific Journal Video about Generation of a Mouse Spontaneous Autoimmune Thyroiditis Model at JoVE.com

Monoklonale Antikörper der Maus gegen humanes TPO (als Positivkontrollen) wurden von Dr. P. Carayon und Dr. J. Ruf (Marseille, Frankreich) zur Verfügung gestellt. Die Autoren danken allen Teilnehmern dieser Studie und den Mitgliedern unseres Forschungsteams. Diese Arbeit wurde teilweise durch Zuschüsse des Postdoctoral Sustentation Fund des West China Hospital, Sichuan University, China (2020HXBH057) und des Sichuan Province Science and Technology Support Program (Projektnummer 2021YFS0166) unterstützt

Das unten beschriebene Protokoll wurde von den Pflege- und Verwendungsrichtlinien genehmigt, die vom Institutional Animal Care and Use Committee der Universität Sichuan festgelegt wurden.
1. Vorbereitung
<ol><li>Unterbringung aller Mäuse unter bestimmten erregerfreien Bedingungen unter 12-stündigen Hell-Dunkel-Zyklen (Beginn um 07:00 Uhr bzw. 19:00 Uhr). Halten Sie die Raumtemperatur bei 22 °C. Wechseln Sie die Einstreumaterialien jede Woche. Stellen Sie ausreichende Me...

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	<li>Ralli, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hashimoto's+thyroiditis:+An+update+on+pathogenic+mechanisms,+diagnostic+protocols,+therapeutic+strategies,+and+potential+malignant+transformation.">Hashimoto's thyroiditis: An update on pathogenic mechanisms, diagnostic protocols, therapeutic strategies, and potential malignant transformation.</a> Autoimmunity Reviews. 19 (10), 102649 (2020).</li><li>Zhang, Q. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphocyte+infiltration+and+thyrocyte+destruction+are+driven+by+stromal+and+immune+cell+components+in+Hashimoto's+thyroiditis.">Lymphocyte infiltration and thyrocyte destruction are driven by stromal and immune cell components in Hashimoto's thyroiditis.</a> Nature Communications. 13 (1), 775 (2022).</li><li>Ruggeri, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoimmune+comorbidities+in+Hashimoto's+thyroiditis:+different+patterns+of+association+in+adulthood+and+childhood/adolescence.">Autoimmune comorbidities in Hashimoto's thyroiditis: different patterns of association in adulthood and childhood/adolescence.</a> European Journal of Endocrinology. 176 (2), 133-141 (2017).</li><li>Resende de Paiva, C., Gr&#248;nh&#248;j, C., Feldt-Rasmussen, U., von Buchwald, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+between+Hashimoto's+thyroiditis+and+thyroid+cancer+in+64,628+patients.">Association between Hashimoto's thyroiditis and thyroid cancer in 64,628 patients.</a> Frontiers in Oncology. 7, 53 (2017).</li><li>Ehlers, M., Schott, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hashimoto's+thyroiditis+and+papillary+thyroid+cancer:+are+they+immunologically+linked.">Hashimoto's thyroiditis and papillary thyroid cancer: are they immunologically linked.</a> Trends in Endocrinology and Metabolism. 25 (12), 656-664 (2014).</li><li>Medenica, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+cell+and+gene+therapies+in+the+treatment+of+infertility+in+patients+with+thyroid+autoimmunity.">The role of cell and gene therapies in the treatment of infertility in patients with thyroid autoimmunity.</a> International Journal of Endocrinology. 2022, 4842316 (2022).</li><li>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+genetics+of+autoimmune+thyroiditis:+the+first+decade.">The genetics of autoimmune thyroiditis: the first decade.</a> Journal of Autoimmunity. 37 (2), 88-94 (2011).</li><li>Kolypetri, P., King, J., Larijani, M., Carayanniotis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genes+and+environment+as+predisposing+factors+in+autoimmunity:+acceleration+of+spontaneous+thyroiditis+by+dietary+iodide+in+NOD.H2(h4)+mice.">Genes and environment as predisposing factors in autoimmunity: acceleration of spontaneous thyroiditis by dietary iodide in NOD.H2(h4) mice.</a> International Reviews of Immunology. 34 (6), 542-556 (2015).</li><li>Terplan, K. L., Witebsky, E., Rose, N. R., Paine, J. R., Egan, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+thyroiditis+in+rabbits,+guinea+pigs+and+dogs,+following+immunization+with+thyroid+extracts+of+their+own+and+of+heterologous+species.">Experimental thyroiditis in rabbits, guinea pigs and dogs, following immunization with thyroid extracts of their own and of heterologous species.</a> The American Journal of Pathology. 36 (2), 213-239 (1960).</li><li>Alexopoulos, H., Dalakas, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+immunobiology+of+autoimmune+encephalitides.">The immunobiology of autoimmune encephalitides.</a> Journal of Autoimmunity. 104, 102339 (2019).</li><li>Noviello, C. M., Kreye, J., Teng, J., Pr&#252;ss, H., Hibbs, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+mechanisms+of+GABA+receptor+autoimmune+encephalitis.">Structural mechanisms of GABA receptor autoimmune encephalitis.</a> Cell. 185 (14), 2469-2477 (2022).</li><li>Pudifin, D. J., Duursma, J., Brain, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+autoimmune+thyroiditis+in+the+vervet+monkey.">Experimental autoimmune thyroiditis in the vervet monkey.</a> Clinical and Experimental Immunology. 29 (2), 256-260 (1977).</li><li>Esquivel, P. S., Rose, N. R., Kong, Y. C. <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+autoimmunity+in+good+and+poor+responder+mice+with+mouse+thyroglobulin+and+lipopolysaccharide.">Induction of autoimmunity in good and poor responder mice with mouse thyroglobulin and lipopolysaccharide.</a> The Journal of Experimental Medicine. 145 (5), 1250-1263 (1977).</li><li>Kong, Y. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HLA-DRB1+polymorphism+determines+susceptibility+to+autoimmune+thyroiditis+in+transgenic+mice:+definitive+association+with+HLA-DRB1*0301+(DR3)+gene.">HLA-DRB1 polymorphism determines susceptibility to autoimmune thyroiditis in transgenic mice: definitive association with HLA-DRB1*0301 (DR3) gene.</a> The Journal of Experimental Medicine. 184 (3), 1167-1172 (1996).</li><li>Kotani, T., Umeki, K., Hirai, K., Ohtakia, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+murine+thyroiditis+induced+by+porcine+thyroid+peroxidase+and+its+transfer+by+the+antigen-specific+T+cell+line.">Experimental murine thyroiditis induced by porcine thyroid peroxidase and its transfer by the antigen-specific T cell line.</a> Clinical and Experimental Immunology. 80 (1), 11-18 (1990).</li><li>Ng, H. P., Banga, J. P., Kung, A. W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+murine+model+of+autoimmune+thyroiditis+induced+with+homologous+mouse+thyroid+peroxidase.">Development of a murine model of autoimmune thyroiditis induced with homologous mouse thyroid peroxidase.</a> Endocrinology. 145 (2), 809-816 (2004).</li><li>Ng, H. P., Kung, A. W. C. <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+autoimmune+thyroiditis+and+hypothyroidism+by+immunization+of+immunoactive+T+cell+epitope+of+thyroid+peroxidase.">Induction of autoimmune thyroiditis and hypothyroidism by immunization of immunoactive T cell epitope of thyroid peroxidase.</a> Endocrinology. 147 (6), 3085-3092 (2006).</li><li>Ellis, J. S., Braley-Mullen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+by+which+B+cells+and+regulatory+T+Cells+influence+development+of+murine+organ-specific+autoimmune+diseases.">Mechanisms by which B cells and regulatory T Cells influence development of murine organ-specific autoimmune diseases.</a> Journal of Clinical Medicine. 6 (2), 13 (2017).</li><li>Fang, Y., Yu, S., Braley-Mullen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contrasting+roles+of+IFN-gamma+in+murine+models+of+autoimmune+thyroid+diseases.">Contrasting roles of IFN-gamma in murine models of autoimmune thyroid diseases.</a> Thyroid. 17 (10), 989-994 (2007).</li><li>Fang, Y., Zhao, L., Yan, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemokines+as+novel+therapeutic+targets+in+autoimmune+thyroiditis.">Chemokines as novel therapeutic targets in autoimmune thyroiditis.</a> Recent Patents on DNA &amp; Gene Sequences. 4 (1), 52-57 (2010).</li><li>Chen, C. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibodies+to+thyroid+peroxidase+arise+spontaneously+with+age+in+NOD.H-2h4+mice+and+appear+after+thyroglobulin+antibodies.">Antibodies to thyroid peroxidase arise spontaneously with age in NOD.H-2h4 mice and appear after thyroglobulin antibodies.</a> Endocrinology. 151 (9), 4583-4593 (2010).</li><li>Ruf, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+between+immunological+structure+and+biochemical+properties+of+human+thyroid+peroxidase.">Relationship between immunological structure and biochemical properties of human thyroid peroxidase.</a> Endocrinology. 125 (3), 1211-1218 (1989).</li><li>McLachlan, S. M., Aliesky, H. A., Chen, C. R., Chong, G., Rapoport, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breaking+tolerance+in+transgenic+mice+expressing+the+human+TSH+receptor+A-subunit:+thyroiditis,+epitope+spreading+and+adjuvant+as+a+'double+edged+sword'.">Breaking tolerance in transgenic mice expressing the human TSH receptor A-subunit: thyroiditis, epitope spreading and adjuvant as a 'double edged sword'.</a> PLoS One. 7 (9), e43517 (2012).</li><li>McLachlan, S. M., Aliesky, H. A., Chen, C. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breaking+tolerance+in+transgenic+mice+expressing+the+human+TSH+receptor+A-subunit:+thyroiditis,+epitope+spreading+and+adjuvant+as+a+'double+edged+sword’[J].">Breaking tolerance in transgenic mice expressing the human TSH receptor A-subunit: thyroiditis, epitope spreading and adjuvant as a 'double edged sword’[J].</a> PLoS One. 7 (9), e43517 (2012).</li><li>Hutchings, P. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Both+CD4(+)+T+cells+and+CD8(+)+T+cells+are+required+for+iodine+accelerated+thyroiditis+in+NOD+mice.">Both CD4(+) T cells and CD8(+) T cells are required for iodine accelerated thyroiditis in NOD mice.</a> Cellular Immunology. 192 (2), 113-121 (1999).</li><li>Xue, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+changes+of+CD4+CD25+++regulatory+T+cells+in+NOD.H-2h4+mice+with+iodine-induced+autoimmune+thyroiditis.">Dynamic changes of CD4+CD25 + regulatory T cells in NOD.H-2h4 mice with iodine-induced autoimmune thyroiditis.</a> Biological Trace Element Research. 143 (1), 292-301 (2011).</li><li>Hou, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+halofuginone+on+the+pathogenesis+of+autoimmune+thyroid+disease+in+different+mice+models.">Effect of halofuginone on the pathogenesis of autoimmune thyroid disease in different mice models.</a> Endocrine, Metabolic &amp; Immune Disorders Drug Targets. 17 (2), 141-148 (2017).</li><li>McLachlan, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissociation+between+iodide-induced+thyroiditis+and+antibody-mediated+hyperthyroidism+in+NOD.H-2h4+mice.">Dissociation between iodide-induced thyroiditis and antibody-mediated hyperthyroidism in NOD.H-2h4 mice.</a> Endocrinology. 146 (1), 294-300 (2005).</li><li>Danailova, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nutritional+management+of+thyroiditis+of+hashimoto.">Nutritional management of thyroiditis of hashimoto.</a> International Journal of Molecular Sciences. 23 (9), 5144 (2022).</li><li>Carayanniotis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+parameters+linking+thyroglobulin+iodination+with+autoimmune+thyroiditis.">Molecular parameters linking thyroglobulin iodination with autoimmune thyroiditis.</a> Hormones. 10 (1), 27-35 (2011).</li><li>Verginis, P., Li, H. S., Carayanniotis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tolerogenic+semimature+dendritic+cells+suppress+experimental+autoimmune+thyroiditis+by+activation+of+thyroglobulin-specific+CD4+CD25++T+cells.">Tolerogenic semimature dendritic cells suppress experimental autoimmune thyroiditis by activation of thyroglobulin-specific CD4+CD25+ T cells.</a> Journal of Immunology. 174 (11), 7433-7439 (2005).</li><li>Flynn, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superiority+of+thyroid+peroxidase+DNA+over+protein+immunization+in+replicating+human+thyroid+autoimmunity+in+HLA-DRB1*0301+(DR3)+transgenic+mice.">Superiority of thyroid peroxidase DNA over protein immunization in replicating human thyroid autoimmunity in HLA-DRB1*0301 (DR3) transgenic mice.</a> Clinical and Experimental Immunology. 137 (3), 503-512 (2004).</li><li>Akeno, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IFN-&#945;+mediates+the+development+of+autoimmunity+both+by+direct+tissue+toxicity+and+through+immune+cell+recruitment+mechanisms.">IFN-&#945; mediates the development of autoimmunity both by direct tissue toxicity and through immune cell recruitment mechanisms.</a> Journal of Immunology. 186 (8), 4693-4706 (2011).</li></ol>

HT tritt aufgrund einer Störung des Autoimmunsystems auf, die durch Lymphozyten verursacht wird, die in die Schilddrüse eindringen, die Schilddrüsenfunktion weiter beeinträchtigen und gleichzeitig schilddrüsenspezifische Antikörper produzieren. Die Serum-TSH-, TgAb- und TPOAb-Spiegel bei HT-Patienten sind signifikant erhöht27. Gegenwärtig werden zwei Haupttypen von Mausmodellen verwendet, um die Ätiologie der Autoimmunthyreoiditis zu untersuchen: EAT und SAT29. EAT...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/64609">Generation of a Mouse Spontaneous Autoimmune Thyroiditis Model</a>. The Protocol section was updated.
Step 3.1.1 of the Protocol was updated from:
After the induction, anesthetize the mice with a volume of 0.01 mL/g anesthetic by intraperitoneal injection. Prepare the anesthetic by mixing midazolam (40 &#181;g/100 &#181;L for sedation), medetomidine (7.5 &#181;g/100 &#181;L for sedation), and butorphanol tartrate (50 &#181;g/100 &#181;L for analgesia) in phosphate-buffered saline (PBS).
to
After the induction, anesthetize the mice with a volume of 0.01 mL/g anesthetic by intraperitoneal injection. Prepare the anesthetic by mixing midazolam (40 &#181;g/100 &#181;L for sedation), medetomidine (7.5 &#181;g/100 &#181;L for sedation), and butorphanol tartrate (50 &#181;g/100 &#181;L for analgesia) in phosphate-buffered saline (PBS). 
NOTE: The specific concentrations of each component in the anesthesia mixture are: midazolam 13.33&#181;g/100&#181;L, medetomidine 2.5&#181;g/100&#181;L, and butorphanol 16.7&#181;g/100&#181;L. For specific dosages used in mice, the doses are: midazolam 4&#181;g/g, medetomidine 0.75&#181;g/g, and butorphanol 1.67&#181;g/g.&#160;Anesthesia depth was confirmed when the mouse&#39;s limb muscles relaxed, the whiskers had no touch response, and there was loss of&#160; pedal reflex.
Step 3.1.2 of the Protocol was updated from:
After the mice are anesthetized, cut off their whiskers with ophthalmic scissors to prevent blood from flowing down the whiskers and causing hemolysis. Fix the mouse with one hand and press the skin of the eye to make the eyeball protrude. Quickly remove the eyeball and draw 1 mL of blood into the microcentrifuge tube via a capillary tube.
to
After the mice are anesthetized, prepare the peripheral blood samples, by fixing the mouse with one hand and pressing the eye skin to protrude the eyeball. Then, insert the capillary tube into the inner corner of the eye and penetrate at a 30-45 degree angle to the plane of the nostril. Apply pressure while gently rotating the capillary tube. Blood will flow into the tube via capillary action.
Step 3.2.1 of the Protocol was updated from:
Dissect the chest wall to expose the heart, cut open the right atrium, and infuse saline into the left ventricle by an intravenous infusion needle attached to a 20 mL syringe until the tissue turns white.
to
Humanely euthanize the animal according to the institutional policies. Then, dissect the chest wall to expose the heart, cut open the right atrium, and infuse saline into the left ventricle by an intravenous infusion needle attached to a 20 mL syringe until the tissue turns white.

An erratum was issued for:&#160;<a href="https://www.jove.com/t/64609">Generation of a Mouse Spontaneous Autoimmune Thyroiditis Model</a>. The Protocol section was updated.

Erratum: Generation of a Mouse Spontaneous Autoimmune Thyroiditis Model

Die Hashimoto-Thyreoiditis (HT), auch bekannt als chronische lymphatische Thyreoiditis oder Autoimmunthyreoiditis, wurde erstmals 1912 berichtet1. HT ist gekennzeichnet durch Lymphozyteninfiltration und Schädigung des Schilddrüsenfollikelgewebes. Labortests manifestieren sich hauptsächlich in einem Anstieg schilddrüsenspezifischer Antikörper, einschließlich Anti-Thyreoglobulin-Antikörper (TgAb) und Anti-Schilddrüsenperoxidase-Antikörper (TPOAb)2. Die Inzidenz von HT liegt im Bereich von 0,4 % bis 1,5 % und macht 20 % bis 25 % aller Schilddrüsenerkrankungen aus, und dieser Wert ist in den letzten Jahren gestiegen3. Darüber hinaus wurde in einer Vielzahl von Studien berichtet, dass HT mit der Onkogenese und dem Rezidiv des papillären Schilddrüsenkarzinoms (PTC) assoziiert ist4,5; Die möglichen Mechanismen sind noch umstritten. Die Autoimmunthyreoiditis ist auch ein wichtiger Faktor bei der weiblichen Unfruchtbarkeit6. Daher muss die Pathogenese der HT klar sein, wofür ein stabiles und einfaches Tiermodell unerlässlich ist.
Um die Ätiologie der HT zu untersuchen, wurden in den vorliegenden Studien zwei Haupttypen von Mausmodellen verwendet, darunter die experimentelle Autoimmunthyreoiditis (EAT) und die spontane Autoimmunthyreoiditis (SAT) 7,8. Empfängliche Mäuse wurden mit spezifischen Schilddrüsenantigenen (einschließlich der rohen Schilddrüse, des gereinigten Thyreoglobulins [TG], der Schilddrüsenperoxidase [TPO], der rekombinanten TPO-Ektodomäne und ausgewählter TPO-Peptide) immunisiert, um das EAT-Mausmodell zu etablieren. Darüber hinaus werden die Adjuvantien, darunter Lipopolysaccharid (LPS), vollständiges Freund-Adjuvans (CFA) und andere ungewöhnliche Adjuvantien, auch während der Immunisierung verwendet, um die Immuntoleranz 9,10,11,12,13,14,15,16,17 abzubauen.
Das SAT-Modell ist ein wichtiges Modell, um die spontane Entwicklung einer Autoimmunthyreoiditis zu untersuchen, die auf NOD basiert. H-2h4 Mäuse. Das NOD. Die H-2h4-Maus ist ein neuer Stamm, der aus der Kreuzung von NOD und B10 gewonnen wurde. A(4R)-Mäuse, gefolgt von mehrfachen Rückkreuzungen auf NOD, mit dem Autoimmunthyreoiditis-Suszeptibilitätsgen IAk18,19. NICKEN. H-2h4-Mäuse entwickeln keinen Diabetes, haben aber eine hohe Inzidenz von Autoimmunthyreoiditis und Sjögren-Syndrom (SS)19. Studien haben gezeigt, dass das intrazelluläre Adhäsionsmolekül-1 (ICAM-1) im Schilddrüsengewebe von NOD stark exprimiert wird. H-2h4-Mäuse im Alter von 3-4 Wochen. Darüber hinaus wird mit zunehmender Jodzufuhr die Immunogenität des Thyreoglobulinmoleküls erhöht, was die Expression von ICAM-1, das eine wichtige Rolle bei der Infiltration von Monozyten spielt, weiter hochreguliert21. Dieses Modell simuliert den Autoimmunprozess und verifiziert gleichzeitig den Zusammenhang zwischen Joddosis und Schwere der Erkrankung. Die etablierte Methode ist stabil und hat eine hohe Erfolgswahrscheinlichkeit. Das SAT-Modell wird seit vielen Jahren zur Induktion einer Autoimmunthyreoiditis eingesetzt und ist nach wie vor eine effektive Methode, um die Pathogenese der Autoimmunthyreoiditis zu untersuchen. Die derzeitige Bauweise des EAT-Modells ist jedoch komplizierter und teurer; Verschiedene Labore verwenden unterschiedliche Immunisierungsmethoden und Injektionsstellen. Darüber hinaus haben Mäuse mit unterschiedlichem genetischem Hintergrund unterschiedliche Induktionsraten, die weitere Studien erfordern, um den potenten Mechanismus aufzudecken.
Die Entwicklung einer Thyreoiditis im SAT-Modell ist jedoch mit Natriumjodid, Sexualdimorphismus und den Aufzuchtbedingungen verbunden. Um das geeignete Verfahren der Autoimmunthyreoiditis im SAT-Modell aufzuzeigen, wurde in diesem Artikel die Methode zur Induktion der Autoimmunthyreoiditis unter verschiedenen Bedingungen beschrieben. Darüber hinaus ermöglicht es die Untersuchung der Pathogenese und des immunologischen Verlaufs der Autoimmunthyreoiditis in verschiedenen Stadien dieser Erkrankung.

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	<tbody>
		<tr>
			<td>Butorphanol tartrate</td>
			<td>Supelco</td>
			<td>L-044&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Dexmedetomidine hydrochloride&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>145108-58-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Enzyme-linked immunosorbent assay (ELISA) well</td>
			<td>Sigma-Aldrich</td>
			<td>M9410-1CS</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>macklin</td>
			<td>64-17-5&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Freund&#8217;s Adjuvant, Complete&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>F5881&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Freund&#8217;s Adjuvant, Incomplete&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>F5506</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Mouse IgG&#160;</td>
			<td>invitrogen</td>
			<td>SA5-10275&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Midazolam solution&#160;</td>
			<td>Supelco</td>
			<td>M-908&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse/rat thyroxine (T4) ELISA</td>
			<td>Calbiotech</td>
			<td>DKO045</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>macklin</td>
			<td>30525-89-4&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium iodide</td>
			<td>Sigma-Aldrich</td>
			<td>P4864</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Iodine</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>7681-82-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Thyroglobulin</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>T1126</td>
			<td></td>
		</tr>
		<tr>
			<td>Thyroglobulin&#160; ELISA Kit</td>
			<td>Thermo Scientific</td>
			<td>EHTGX5</td>
			<td></td>
		</tr>
		<tr>
			<td>TSH ELISA</td>
			<td>Calbiotech</td>
			<td>DKO200</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>macklin</td>
			<td>1330-20-7</td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of a mouse spontaneous autoimmune thyroiditis model

Die Hashimoto-Thyreoiditis (HT) hat sich in den letzten Jahren zur häufigsten Autoimmunerkrankung der Schilddrüse entwickelt. Sie zeichnet sich durch eine Infiltration von Lymphozyten und den Nachweis spezifischer Serum-Autoantikörper aus. Obwohl der mögliche Mechanismus noch nicht geklärt ist, hängt das Risiko einer Hashimoto-Thyreoiditis mit genetischen und umweltbedingten Faktoren zusammen. Derzeit gibt es verschiedene Arten von Modellen der Autoimmunthyreoiditis, darunter die experimentelle Autoimmunthyreoiditis (EAT) und die spontane Autoimmunthyreoiditis (SAT).
EAT bei Mäusen ist ein häufiges Modell für HT, das mit Lipopolysaccharid (LPS) in Kombination mit Thyreoglobulin (Tg) immunisiert oder mit einem vollständigen Freund-Adjuvans (CFA) ergänzt wird. Das EAT-Mausmodell ist bei vielen Mäusearten weit verbreitet. Der Krankheitsverlauf ist jedoch eher mit der Tg-Antikörperantwort assoziiert, die in verschiedenen Experimenten variieren kann.
SAT wird auch häufig bei der Untersuchung von HT im NOD verwendet. H-2h4 Maus. Das NOD. Die H2h4-Maus ist ein neuer Stamm, der aus der Kreuzung der nicht-adipösen diabetischen Maus (NOD) mit der B10 gewonnen wurde. A(4R), das für HT mit oder ohne Jodzufuhr signifikant induziert ist. Während der Induktion wird die NOD. Die H-2h4-Maus weist einen hohen TgAb-Spiegel auf, der von einer Lymphozyteninfiltration im Schilddrüsenfollikelgewebe begleitet wird. Für diese Art von Mausmodell gibt es jedoch nur wenige Studien, um den pathologischen Prozess bei der Induktion von Jod umfassend zu bewerten.
In dieser Studie wird ein SAT-Mausmodell für die HT-Forschung etabliert und der pathologische Veränderungsprozess nach einer langen Zeit der Jodinduktion evaluiert. Durch dieses Modell können Forscher die pathologische Entwicklung von HT besser verstehen und neue Behandlungsmethoden für HT untersuchen.

Die histologischen Veränderungen unterschieden sich auffallend bei Frauen und Männern, in der Dauer der Jodaufnahme und in der Lösung von NaI. Wie in Abbildung 1 dargestellt, ~10 % von NOD. H-2h4-Mäuse entwickelten SAT auch ohne Jodinduktion im Alter von 24 Wochen, und alle Mäuse entwickelten schließlich eine Thyreoiditis. Bei regelmäßiger Wässerung gab es keinen signifikanten Unterschied in den histologischen Veränderungen zwischen Männchen und Weibchen. Die Zugabe von NaI zum Tr...

Watch this Scientific Journal Video about Generation of a Mouse Spontaneous Autoimmune Thyroiditis Model at JoVE.com

Generation of a Mouse Spontaneous Autoimmune Thyroiditis Model

Es wurden mehrere Arten von Tiermodellen der Hashimoto-Thyreoiditis etabliert, ebenso wie die spontane Autoimmunthyreoiditis bei der NOD-Maus. H-2h4-Mäuse sind ein einfaches und zuverlässiges Modell für die HT-Induktion. Dieser Artikel beschreibt diesen Ansatz und bewertet den pathologischen Prozess für ein besseres Verständnis des SAT-Mausmodells.

Generierung eines Mausmodells für spontane Autoimmunthyreoiditis

In recent years, Hashimoto's thyroiditis (HT) has become the most common autoimmune thyroid disease. It is characterized by lymphocyte infiltration and ...

Autoimmunthyreoiditis, die Folge einer Krankheit, die die menschliche Gesundheit ernsthaft beeinträchtigt. Obwohl es derzeit keine wirksame Behandlung gibt, bieten die Tiermodelle eine Plattform für die Erforschung dieser Krankheit. Dieses Modell ist mit einfachen Schritten und einer hohen Erfolgsquote leicht zu konstruieren.Es stimuliert den Autoimmunprozess, indem es den Zusammenhang zwischen unserem Sterben und der Schwere der Krankheit überprüft. Befestigen Sie zunächst die Maus mit Stecknadeln auf dem Seziertisch. Um die Schilddrüsengewebeprobe vorzubereiten, präparieren Sie die Brustwand und legen Sie das Herz frei.Schneiden Sie den rechten Vorhof auf und injizieren Sie mit einer 20-Milliliter-Spritze Kochsalzlösung intravenös in die linke Herzkammer, bis sich das Gewebe weiß verfärbt. Sterilisieren Sie den Hals mit 75%igem Ethanol. Schneiden Sie mit einer Gewebeschere die Maushaut entlang des Halses und der Mittellinie von der Oberseite des Brustbeins bis zum Unterkiefer ab, um das Halsgewebe vollständig freizulegen.Lokalisieren Sie die Unterkieferdrüsen und den vorderen Luftröhrenmuskel und trennen Sie sie mit einer Augenschere und einer Pinzette. Sobald die Luftröhre und der Schilddrüsenknorpel freigelegt sind, entfernen Sie Gewebe unter dem Knorpel mit einer gebogenen Pinzette. Nachdem Sie den Knorpel und die Luftröhre entnommen haben, schneiden Sie die distale Linse des Knorpels und die proximale Linse der Luftröhre durch.Entfernen Sie dann die Schilddrüse und die Luftröhre. Die HE-Färbung zeigte pathologische Veränderungen in der Schilddrüse unter Jodaufnahme. Alle Mäuse entwickelten eine Thyreoiditis, auch ohne Jodinduktion.Es wurde jedoch eine beschleunigte Entwicklung einer Thyreoiditis mit steigender Natriumiodidkonzentration beobachtet. Weibliche Mäuse neigen dazu, eine schwerere Thyreoiditis zu entwickeln als männliche Mäuse unter der gleichen Bedingung, aber die Unterschiede in der Schwere der lymphozytären Infiltration waren vernachlässigbar. Die Thyreoglobulin-Antikörperspiegel waren im Alter von 16 Wochen in klarem Wasser nicht signifikant erhöht.Die Supplementierung mit Natriumjodid hingegen zeigte bei männlichen und weiblichen Mäusen einen vergleichbaren Anstieg der Antikörperspiegel. Die Schilddrüsenperoxidase-Antikörper wurden nach 24 Wochen in normalem Wasser gefunden, und weibliche Mäuse zeigten nach 72 Wochen höhere Werte als männliche Mäuse. Die 0,05%ige Natriumiodid-Edition zeigte jedoch nach 64 Wochen die höchsten Antikörper.Darüber hinaus waren die TSH-Spiegel bei männlichen NODH2-H4-Mäusen höher als bei weiblichen NODH2-H4-Mäusen, mit oder ohne Jodid-Supplementierung. Des Weiteren zeigten die T4-Spiegel eine abnehmende Tendenz zur Induktionszeit im Jodwasser. Es sollte beachtet werden, dass 0,05 % Natrium in der am besten geeigneten Konzentration zur Reduzierung des Salzmodells.Außerdem ist beim Isolieren von Proben mehr Vorsicht geboten. Denn die Mauszelle ist relativ klein und leicht zu beschädigen. Die Etablierung des Tiermodells ist hilfreich für die Untersuchung des Pfades des Genozids und die Behandlung des Autoimmunzellstatus.Und bietet eine Grundlage für die Entwicklung neuer Behandlungsmethoden.

generation-of-a-mouse-spontaneous-autoimmune-thyroiditis-model

Research

JoVE Journal

Immunology and Infection

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.

Vorbereitung der Maus Pituitary Immunogen für die Induktion der experimentellen autoimmunen Hypophysitis

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

Forschung

Immunologie und Infektion

Induction of Experimental Autoimmune Hypophysitis in SJL Mice

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse models allow us to study how diseases unfold, often providing a good replica of the same processes occurring in humans. For some autoimmune diseases, like type 1A diabetes, there are models (the NOD mouse) that spontaneously develop a disease similar to the human counterpart. For many other autoimmune diseases, however, the model needs to be induced experimentally. A common approach in this regard is to inject the mouse with a dominant antigen derived from the organ being studied. For example, investigators interested in autoimmune thyroiditis inject mice with thyroglobulin2, and those interested in myasthenia gravis inject them with the acetylcholine receptor3. If the autoantigen for a particular autoimmune disease is not known, investigators inject a crude protein extract from the organ targeted by the autoimmune reaction. For autoimmune hypophysitis, the pathogenic autoantigen(s) remain to be identified4, and thus a crude pituitary protein preparation is used. In this video article we demonstrate how to induce experimental autoimmune hypophysitis in SJL mice.

This video shows how to induce autoimmune hypophysitis in SJL mice and how to assess its severity by histopathology.

Induktion der experimentellen autoimmunen Hypophysitis in SJL Mäuse

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

Parasite Induced Genetically Driven Autoimmune Chagas Heart Disease in the Chicken Model

The Trypanosoma cruzi acute infections acquired in infancy and childhood seem asymptomatic, but approximately one third of the chronically infected cases show Chagas disease up to three decades or later. Autoimmunity and parasite persistence are competing theories to explain the pathogenesis of Chagas disease 1, 2. To separate roles played by parasite persistence and autoimmunity in Chagas disease we inoculate the T. cruzi in the air chamber of fertilized eggs. The mature chicken immune system is a tight biological barrier against T. cruzi and the infection is eradicated upon development of its immune system by the end of the first week of growth 3. The chicks are parasite-free at hatching, but they retain integrated parasite mitochondrial kinetoplast DNA (kDNA) minicircle within their genome that are transferred to their progeny. Documentation of the kDNA minicircle integration in the chicken genome was obtained by a targeted prime TAIL-PCR, Southern hybridizations, cloning, and sequencing 3, 4. The kDNA minicircle integrations rupture open reading frames for transcription and immune system factors, phosphatase (GTPase), adenylate cyclase and phosphorylases (PKC, NF-Kappa B activator, PI-3K) associated with cell physiology, growth, and differentiation 3, 5-7, and other gene functions. Severe myocarditis due to rejection of target heart fibers by effectors cytotoxic lymphocytes is seen in the kDNA mutated chickens, showing an inflammatory cardiomyopathy similar to that seen in human Chagas disease. Notably, heart failure and skeletal muscle weakness are present in adult chickens with kDNA rupture of the dystrophin gene in chromosome 1 8. Similar genotipic alterations are associated with tissue destruction carried out by effectors CD45+, CD8&gamma;&delta;+, CD8&alpha; lymphocytes. Thus this protozoan infection can induce genetically driven autoimmune disease.

The inoculation of Trypanosoma cruzi in fertile eggs prior to incubation renders the parasite kDNA minicircle integration in embryo cells genome. Crossbreeding reveals the vertical transfer of the mutations to progeny. The kDNA integrates into coding regions at several chromosomes and the chickens die with an inflammatory autoimmune heart disease.

Parasite Induced Genetisch Angetrieben Autoimmune Chagas-Herz-Krankheit in der Hähnchen-Modell

The Trypanosoma cruzi acute infections acquired in infancy and childhood seem asymptomatic, but approximately one third of the chronically infected cases ...

Isolation of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune cells in rodent models of skin inflammation. Here, we describe a protocol for the digestion of mouse skin in a nutrient-rich solution with collagenase D, followed by separation of hematopoietic cells using a discontinuous density gradient. Cells thus obtained can be used for in vitro studies, in vivo transfer, and other downstream cellular and molecular analyses including flow cytometry. This protocol is an effective and economical alternative to expensive mechanical dissociators, specialized separation columns, and harsher trypsin- and dispase-based digestion methods, which may compromise cellular viability or density of surface proteins relevant for phenotypic characterization or cellular function. As shown here in our representative data, this protocol produced highly viable cells, contained specific immune cell subsets, and had no effect on integrity of common surface marker proteins used in flow cytometric analysis.

This protocol describes enzymatic digestion of mouse skin in nutrient-rich medium followed by gradient separation to isolate leukocytes. Cells thus derived can be used for diverse downstream applications. This is an effective, economical, and improved alternative to tissue dissociation machines and harsher trypsin and dispase-based tissue digestion protocols.

Isolation der infiltrierenden Leukozyten aus Mäusehaut unter Verwendung enzymatischer Digest und Gradiententrennung

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune ...

Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. However, they are difficult to isolate in sufficient numbers to study them in detail, therefore, in vitro models have been developed. In vitro cultures of bone marrow-derived macrophages and dendritic cells are well-established and valuable methods for immunological studies. Here, a method for culturing and identifying both DCs and macrophages from a single culture of primary mouse bone marrow cells using the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) is described. This protocol is based on the established procedure first developed by Lutz et al. in 1999 for bone marrow-derived DCs. The culture is heterogeneous, and MHCII and fluoresceinated hyaluronan (FL-HA) are used to distinguish macrophages from immature and mature DCs. These GM-CSF derived macrophages provide a convenient source of in vitro derived macrophages that closely resemble alveolar macrophages in both phenotype and function.

Bone marrow cells cultured with granulocyte macrophage colony stimulating factor (GM-CSF) generate a heterogeneous culture containing macrophages and dendritic cells (DCs). This method highlights using MHCII and hyaluronan (HA) binding to differentiate macrophages from the DCs in the GM-CSF culture. Macrophages in this culture have many similarities to alveolar macrophages.

Erzeugung und Identifizierung von GM-CSF Abgeleitet Alveolare ähnlichen Makrophagen und dendritische Zellen aus Knochenmark der Maus

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. ...

Simple and Efficient Production and Purification of Mouse Myelin Oligodendrocyte Glycoprotein for Experimental Autoimmune Encephalomyelitis Studies

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS), thought to occur as a result of autoimmune responses targeting myelin. Experimental autoimmune encephalomyelitis (EAE) is the most common animal model of CNS autoimmune disease, and is typically induced via immunization with short peptides representing immunodominant CD4+ T cell epitopes of myelin proteins. However, B cells recognize unprocessed protein directly, and immunization with short peptide does not activate B cells that recognize the native protein. As recent clinical trials of B cell-depleting therapies in MS have suggested a role for B cells in driving disease in humans, there is an urgent need for animal models that incorporate B cell-recognition of autoantigen. To this end, we have generated a new fusion protein containing the extracellular domain of the mouse version of myelin oligodendrocyte glycoprotein (MOG) as well as N-terminal fusions of a His-tag for purification purposes and the thioredoxin protein to improve solubility (MOGtag). A tobacco etch virus (TEV) protease cleavage site was incorporated to allow the removal of all tag sequences, leaving only the pure MOG1-125 extracellular domain. Here, we describe a simple protocol using only standard laboratory equipment to produce large quantities of pure MOGtag or MOG1-125. This protocol consistently generates over 200 mg of MOGtag protein. Immunization with either MOGtag or MOG1-125 generates an autoimmune response that includes pathogenic B cells that recognize the native mouse MOG.

We describe a simple protocol using only basic lab equipment to generate and purify large quantities of a fusion protein that contains mouse Myelin Oligodendrocyte Glycoprotein. This protein can be used to induce experimental autoimmune encephalomyelitis driven by both T and B cells.

Einfache und effiziente Herstellung und Reinigung von Maus-Myelin-Oligodendrozyten-Glykoprotein für experimentellen autoimmunen Enzephalomyelitis Studies

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS), thought to occur as a result of autoimmune responses ...

Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291

Clostridium difficile is an anaerobic, gram-positive, spore-forming enteric pathogen that is associated with increasing morbidity and mortality and consequently poses an urgent threat to public health. Recurrence of a C. difficile infection (CDI) after successful treatment with antibiotics is high, occurring in 20-30% of patients, thus necessitating the discovery of novel therapeutics against this pathogen. Current animal models of CDI result in high mortality rates and thus do not approximate the chronic, insidious disease manifestations seen in humans with CDI. To evaluate therapeutics against C. difficile, a mouse model approximating human disease utilizing a clinically-relevant strain is needed. This protocol outlines the cefoperazone mouse model of CDI using a clinically-relevant and genetically-tractable strain, R20291. Techniques for clinical disease monitoring, C. difficile bacterial enumeration, toxin cytotoxicity, and histopathological changes throughout CDI in a mouse model are detailed in the protocol. Compared to other mouse models of CDI, this model is not uniformly lethal at the dose administered, allowing for the observation of a prolonged clinical course of infection concordant with the human disease. Therefore, this cefoperazone mouse model of CDI proves a valuable experimental platform to assess the effects of novel therapeutics on the amelioration of clinical disease and on the restoration of colonization resistance against C. difficile.

Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291

This protocol outlines the cefoperazone mouse model of Clostridium difficile infection (CDI) using a clinically-relevant and genetically-tractable strain, R20291. Emphasis on clinical disease monitoring, C. difficile bacterial enumeration, toxin cytotoxicity, and histopathological changes throughout CDI in a mouse model are detailed in the protocol.

Cefoperazon behandelten Maus-Modell der klinisch relevanten<em&gt; Clostridium difficile</em&gt; Dehnungs R20291

Clostridium difficile is an anaerobic, gram-positive, spore-forming enteric pathogen that is associated with increasing morbidity and mortality and ...

A Chronic Autoimmune Dry Eye Rat Model with Increase in Effector Memory T Cells in Eyeball Tissue

Dry eye disease is a very common condition that causes morbidity and healthcare burden and decreases the quality of life. There is a need for a suitable dry eye animal model to test novel therapeutics to treat autoimmune dry eye conditions. This protocol describes a chronic autoimmune dry eye rat model. Lewis rats were immunized with an emulsion containing lacrimal gland extract, ovalbumin, and complete Freund&#39;s adjuvant. A second immunization with the same antigens in incomplete Freund&#39;s adjuvant was administered two weeks later. These immunizations were administered subcutaneously at the base of the tail. To boost the immune response at the ocular surface and lacrimal glands, lacrimal gland extract and ovalbumin were injected into the forniceal subconjunctiva and lacrimal glands 6 weeks after the first immunization. The rats developed dry eye features, including reduced tear production, decreased tear stability, and increased corneal damage. Immune profiling by flow cytometry showed a preponderance of CD3+ effector memory T cells in the eyeball.

This report describes a method to induce chronic experimental autoimmune dry eye in Lewis rats through immunization with an emulsion of rat lacrimal gland extract, ovalbumin, and complete Freund's adjuvant, followed by the injection of lacrimal gland extract and ovalbumin into the forniceal subconjunctiva and lacrimal glands six weeks later.

Ein chronisches Autoimmun-Trockenaugen-Rattenmodell mit Zunahme im Effekt-Speicher T-Zellen in Augapfel-Gewebe

Dry eye disease is a very common condition that causes morbidity and healthcare burden and decreases the quality of life. There is a need for a suitable ...

Influenza A Virus Studies in a Mouse Model of Infection

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone considering direct medical and hospitalization expenses and work absenteeism2. Animal models are crucial in Influenza A virus (IAV) studies to evaluate viral pathogenesis, host-pathogen interactions, immune responses, and the efficacy of current and/or novel vaccine approaches as well as antivirals. Mice are an advantageous small animal model because their immune system is evolutionarily similar to that found in humans, they are available from commercial vendors as genetically identical subjects, there are multiple strains that can be exploited to evaluate the genetic basis of infections, and they are relatively inexpensive and easy to manipulate. To recapitulate IAV infection in humans via the airways, mice are first anesthetized prior to intranasal inoculation with infectious IAVs under proper biosafety containment. After infection, the pathogenesis of IAVs is determined by monitoring daily the morbidity (body weight loss) and mortality (survival) rate. In addition, viral pathogenesis can also be evaluated by assessing virus replication in the upper (nasal mucosa) or lower (lungs) respiratory tract of infected mice. Humoral responses upon IAV infection can be rapidly evaluated by non-invasive bleeding and secondary antibody detection assays aimed at detecting the presence of total or neutralizing antibodies. Here, we describe the common methods used to infect mice intranasally (i.n) with IAV and evaluate pathogenesis, humoral immune responses and protection efficacy.

Influenza A viruses (IAVs) are important human respiratory pathogens. To understand the pathogenicity of IAVs and to perform preclinical testing of novel vaccine approaches, animal models mimicking human physiology are required. Here, we describe techniques to evaluate IAV pathogenesis, humoral responses and vaccine efficacy using a mouse model of infection.

Influenza-A-Virus-Studien in einem Mausmodell der Infektion

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone ...

A Controlled Mouse Model for Neonatal Polymicrobial Sepsis

Neonatal sepsis remains a global burden. A preclinical model to screen effective prophylactic or therapeutic interventions is needed. Neonatal mouse polymicrobial sepsis can be induced by injecting cecal slurry intraperitoneally into day of life 7 mice and monitoring them for the following week. Presented here are the detailed steps necessary for the implementation of this neonatal sepsis model. This includes making a homogeneous cecal slurry stock, diluting it to a weight- and litter-adjusted dose, an outline of the monitoring schedule, and a definition of observed health categories used to define humane endpoints. The generation of a homogeneous cecal slurry stock from pooled donors allows for the administration into many litters over time, reducing the variation between donors, and preventing the use of potentially toxic glycerol. The monitoring strategy used allows for the anticipation of survival outcome and the identification of mice that would later progress to death, allowing for an earlier identification of the humane endpoint. Two main behavioral features are used to define the health scores, namely, the ability of the neonatal mice to right themselves when placed on their back and their level of mobility. These criteria could potentially be applied to address humane endpoints in other studies of neonatal disease in mice, as long as a pilot study is performed to confirm accuracy. In conclusion, this approach provides a standardized method to model newborn sepsis in mice, while providing resources to assess animal welfare used to define early humane endpoints for challenged animals.

This protocol provides the necessary steps to establish and evaluate neonatal sepsis in 7-day-old mice.

Eine kontrollierte Mausmodell für neonatale Sepsis Biofilm

Neonatal sepsis remains a global burden. A preclinical model to screen effective prophylactic or therapeutic interventions is needed. Neonatal mouse ...