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

Los anticuerpos monoclonales de ratón contra TPO humano (utilizados como controles positivos) fueron proporcionados por el Dr. P. Carayon y el Dr. J. Ruf (Marsella, Francia). Los autores agradecen a todos los participantes en este estudio y a los miembros de nuestro equipo de investigación. Este trabajo fue apoyado en parte por subvenciones del Fondo de Sustentación Postdoctoral del Hospital de China Occidental, Universidad de Sichuan, China (2020HXBH057) y el Programa de Apoyo a la Ciencia y Tecnología de la Provincia de Sichuan (Proyecto No. 2021YFS0166)

El protocolo que se describe a continuación fue aprobado por las pautas de cuidado y uso establecidas por el Comité Institucional de Cuidado y Uso de Animales de la Universidad de Sichuan.
1. Preparación
<ol><li>Aloje a todos los ratones en condiciones específicas libres de patógenos en ciclos de luz-oscuridad de 12 h (a partir de las 07:00 a.m. y 07:00 p.m., respectivamente). Mantener la temperatura ambiente a 22 °C. Cambie los materiales de la ropa de cama cada sema...

<ol>
	<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. 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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>

La TH ocurre debido a un trastorno del sistema autoinmune causado por linfocitos que se infiltran en la glándula tiroides, lo que afecta aún más la función tiroidea, al tiempo que produce anticuerpos específicos para la tiroides. Los niveles séricos de TSH, TgaB y TPOAb en pacientes con HTA están significativamente elevados27. En la actualidad, dos tipos principales de modelos murinos son ampliamente utilizados para estudiar la etiología de la tiroiditis autoinmune: EAT y SAT<sup class="xr...

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

La tiroiditis de Hashimoto (TH), también conocida como tiroiditis linfocítica crónica o tiroiditis autoinmune, se informó por primera vez en 19121. La TH se caracteriza por infiltración de linfocitos y daño al tejido folicular tiroideo. Las pruebas de laboratorio se manifiestan principalmente como un aumento de los anticuerpos específicos de la tiroides, incluidos los anticuerpos antitiroglobulina (TgAb) y los anticuerpos antioxidasa antitiroidea (TPOAb)2. La incidencia de HTA está en el rango de 0,4%-1,5%, representando 20%-25% de todas las enfermedades tiroideas, y este valor ha aumentado en los últimos años3. Además, un gran número de estudios han reportado que la TH se asocia con la oncogénesis y recurrencia del carcinoma papilar de tiroides (PTC)4,5; Los posibles mecanismos siguen siendo controvertidos. La tiroiditis autoinmune también es un factor importante en la infertilidad femenina6. Por lo tanto, la patogénesis de la HTA debe ser clara, para lo cual es esencial un modelo animal estable y simple.
Para estudiar la etiología de la HTA, se han empleado dos tipos principales de modelos murinos, incluyendo la tiroiditis autoinmune experimental (EAT) y la tiroiditis autoinmune espontánea (SAT) en los presentes estudios 7,8. Los ratones susceptibles fueron inmunizados con antígenos tiroideos específicos (incluyendo la tiroides cruda, tiroglobulina purificada [TG], peroxidasa tiroidea [TPO], ectodominio TPO recombinante y péptidos TPO seleccionados) para establecer el modelo murino EAT. Además, los adyuvantes, incluyendo lipopolisacárido (LPS), adyuvante completo de Freund (CFA) y otros adyuvantes inusuales, también se utilizan durante la inmunización para descomponer la tolerancia inmune 9,10,11,12,13,14,15,16,17.
El modelo SAT es un modelo importante para estudiar el desarrollo espontáneo de tiroiditis autoinmune, que se basa en NOD. Ratones H-2H4. El NOD. El ratón H-2h4 es una nueva cepa obtenida del cruce de NOD y B10. Ratones A(4R), seguidos de múltiples retrocruces a NOD, con el gen de susceptibilidad a la tiroiditis autoinmune IAk18,19. CABECEO. Los ratones H-2h4 no desarrollan diabetes, pero tienen una alta incidencia de tiroiditis autoinmune y síndrome de Sjogren (SS)19. Los estudios han encontrado que la molécula de adhesión intracelular-1 (ICAM-1) está altamente expresada en el tejido tiroideo de NOD. Ratones H-2h4 a las 3-4 semanas de edad. Además, con el aumento de la ingesta de yodo, se mejora la inmunogenicidad de la molécula de tiroglobulina, lo que regula aún más la expresión de ICAM-1, que desempeña un papel importante en el proceso de infiltración de monocitos21. Este modelo simula el proceso autoinmune mientras verifica la relación entre la dosis de yodo y la gravedad de la enfermedad. El método establecido es estable, con una alta probabilidad de éxito. El modelo SAT se ha aplicado para inducir tiroiditis autoinmune durante muchos años y sigue siendo un método eficaz para estudiar la patogénesis de la tiroiditis autoinmune. Sin embargo, el método de construcción actual del modelo EAT es más complicado y costoso; Diferentes laboratorios utilizan diferentes métodos de inmunización y sitios de inyección. Además, los ratones con diferentes antecedentes genéticos tienen diferentes tasas de inducción, que necesitan más estudios para revelar el potente mecanismo.
Sin embargo, el desarrollo de tiroiditis en el modelo SAT se asocia con yoduro de sodio, dimorfismo sexual y condiciones de crianza. Para revelar el procedimiento apropiado de tiroiditis autoinmune en el modelo SAT, este artículo describió el método de inducción de tiroiditis autoinmune en diferentes condiciones. Además, permite el estudio de la patogénesis y el progreso inmunológico de la tiroiditis autoinmune en diferentes etapas de esta enfermedad.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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

En los últimos años, la tiroiditis de Hashimoto (TH) se ha convertido en la enfermedad tiroidea autoinmune más común. Se caracteriza por la infiltración de linfocitos y la detección de autoanticuerpos séricos específicos. Aunque el mecanismo potencial aún no está claro, el riesgo de tiroiditis de Hashimoto está relacionado con factores genéticos y ambientales. En la actualidad, existen varios tipos de modelos de tiroiditis autoinmune, incluida la tiroiditis autoinmune experimental (EAT) y la tiroiditis autoinmune espontánea (SAT).
La EAT en ratones es un modelo común para la HTA, que se inmuniza con lipopolisacárido (LPS) combinado con tiroglobulina (Tg) o se complementa con adyuvante de Freund completo (CFA). El modelo de ratón EAT está ampliamente establecido en muchos tipos de ratones. Sin embargo, es más probable que la progresión de la enfermedad esté asociada con la respuesta de anticuerpos Tg, que puede variar en diferentes experimentos.
SAT también es ampliamente utilizado en el estudio de HTA en la NOD. Ratón H-2H4. El NOD. El ratón H2h4 es una nueva cepa obtenida del cruce del ratón diabético no obeso (NOD) con el B10. A(4R), que se induce significativamente para TH con o sin yodo de alimentación. Durante la inducción, el NOD. El ratón H-2h4 tiene un alto nivel de TgAb acompañado de infiltración de linfocitos en el tejido folicular tiroideo. Sin embargo, para este tipo de modelo de ratón, hay pocos estudios para evaluar exhaustivamente el proceso patológico durante la inducción de yodo.
En este estudio se establece un modelo de ratón SAT para la investigación de HT, y el proceso de cambio patológico se evalúa después de un largo período de inducción de yodo. A través de este modelo, los investigadores pueden comprender mejor el desarrollo patológico de la TH y detectar nuevos métodos de tratamiento para la HTA.

Los cambios histológicos fueron sorprendentemente diferentes en mujeres y hombres, la duración de la ingesta de yodo y la solución de NaI. Como se muestra en la Figura 1, ~10% de NOD. Los ratones H-2h4 desarrollaron SAT incluso sin inducción de yodo a la edad de 24 semanas, y todos los ratones finalmente desarrollaron tiroiditis. Cuando se les administró agua regular, no hubo diferencias significativas en los cambios histológicos entre hombres y mujeres. La adición de NaI al agua pota...

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

Se han establecido varios tipos de modelos animales de tiroiditis de Hashimoto, al igual que la tiroiditis autoinmune espontánea en el ratón NOD. Los ratones H-2h4 son un modelo simple y confiable para la inducción de HT. Este artículo describe este enfoque y evalúa el proceso patológico para una mejor comprensión del modelo murino SAT.

Generación de un modelo de tiroiditis autoinmune espontánea en ratones

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

Tiroiditis autoinmune, el resultado de una enfermedad que afecta seriamente la salud humana. Aunque actualmente no existe un tratamiento efectivo, los modelos animales proporcionan una plataforma para estudiar esta enfermedad. Este modelo es fácil de construir con pasos simples y una alta tasa de éxito.Estimula el proceso autoinmune con la verificación de la relación entre nuestra muerte y la gravedad de la enfermedad. Para comenzar, fije el mouse en la tabla de disección con pines. Para preparar la muestra de tejido tiroideo, diseccione la pared torácica y exponga el corazón.Corte la aurícula derecha e inyecte solución salina por vía intravenosa en el ventrículo izquierdo, usando una jeringa de 20 mililitros hasta que el tejido se vuelva blanco. Esterilizar el cuello con etanol al 75%. Usando tijeras de tejido, corte la piel del ratón a lo largo del cuello, línea media desde la parte superior del esternón hasta la mandíbula, para exponer completamente el tejido del cuello.Localice las glándulas submandibulares y el músculo de la tráquea anterior y sepárelos con tijeras oftálmicas y fórceps. Una vez que la tráquea y el cartílago tiroides están expuestos, retire el tejido debajo del cartílago con pinzas curvas. Después de recoger el cartílago y la tráquea, corte la lente distal del cartílago y el cristalino proximal de la tráquea.Luego extirpe la glándula tiroides y la tráquea. La tinción HE mostró cambios patológicos en la glándula tiroides, bajo ingesta de yodo. Todos los ratones desarrollaron tiroiditis, incluso sin inducción de yodo.Sin embargo, se observó un desarrollo acelerado de tiroiditis con el aumento de la concentración de yoduro de sodio. Los ratones hembra tienden a desarrollar tiroiditis más grave que los machos bajo la misma condición, pero las diferencias en la gravedad de la infiltración linfocítica fueron insignificantes. Los niveles de anticuerpos contra la tiroglobulina no aumentaron significativamente en agua corriente a las 16 semanas de edad.Mientras que, la suplementación con yoduro de sodio mostró un aumento comparable en los niveles de anticuerpos en ratones machos y hembras. Los anticuerpos contra la peroxidasa tiroidea se encontraron a las 24 semanas en agua regular; y los ratones hembra mostraron niveles más altos que los machos a las 72 semanas. Sin embargo, la edición de yoduro de sodio al 0,05% mostró los anticuerpos más altos a las 64 semanas.Además, los niveles de TSH fueron más altos en ratones NODH2 H4 machos que en hembras; con o sin suplementación con yoduro. Además, los niveles de T4 mostraron una tendencia decreciente al tiempo de inducción en agua con yodo. Cabe señalar que 0.05% de sodio a la concentración más adecuada para reducir el modelo de sal.Además, se debe tener más cuidado al aislar las muestras. Porque la célula del ratón es relativamente pequeña y se daña fácilmente. El establecimiento del modelo animal es útil para el estudio del genocidio del camino y el tratamiento del estado celular autoinmune.Y proporciona una base para el desarrollo de un nuevo método de tratamiento.

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.

Preparación de inmunógeno ratón hipófisis para la inducción de la hipofisitis autoinmune experimental

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

Investigación

Inmunología e Infección

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.

La inducción de la hipofisitis autoinmune experimental en ratones SJL

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.

Parásito inducido genéticamente Impulsado enfermedad autoinmune de Chagas en el modelo de Pollo

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.

Aislamiento de leucocitos infiltrantes de ratón Piel Usando enzimática Digest y Separación de degradado

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.

Generación e identificación de GM-CSF Derivado alveolares-como los macrófagos y las células dendríticas A partir de médula ósea de ratones

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.

Producción simple y eficiente y purificación de ratón mielina de oligodendrocitos de la glucoproteína de la encefalomielitis autoinmune experimental Estudios

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.

Cefoperazona tratados modelo de ratón clínicamente relevante<em&gt; Clostridium difficile</em&gt; R20291 Strain

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.

Un modelo de rata ocular seca autoinmune crónica con aumento de células T de memoria efector en tejido de globo ocular

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.

Estudios de Virus de influenza A en un modelo murino de infección

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.

Un modelo de ratón controlado para la Sepsis Neonatal polimicrobiana

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