Name: a preclinical model of exertional heat stroke in mice
Uploaded: 2021-07-01T14:30:00
Description: Watch this Scientific Journal Video about A Preclinical Model of Exertional Heat Stroke in Mice at JoVE.com

Dette arbejde blev finansieret af Department of Defense W81XWH-15-2-0038 (TLC) og BA180078 (TLC) og BK og Betty Stevens Endowment (TLC). JMA blev støttet af finansiel støtte fra Kongeriget Saudi-Arabien. Michelle King var fra University of Florida på det tidspunkt, hvor denne undersøgelse blev gennemført. Hun er i øjeblikket ansat ved Gatorade Sports Science Institute, en afdeling af PepsiCo R &D.

Alle procedurer er blevet gennemgået og godkendt af University of Florida IACUC. C57BL/6J han- eller hunmus, ~4 måneder gamle, der vejer inden for henholdsvis 27-34 g og 20-25 g, anvendes til undersøgelsen.
1. Kirurgisk implantation af det telemetriske temperaturovervågningssystem
<ol><li>Ved ankomsten fra sælgeren, lad dyrene hvile i vivarium i mindst 1 uge før operationen for at minimere stress af transport.</li>
	<li>Gruppe hus musene (højst 5 pr bur under lokale IACUC retningslinj...

<ol>
	<li>Leon, L. R., Bouchama, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heat+stroke.">Heat stroke.</a> Comprehensive Physiology. 5 (2), 611-647 (2015).</li><li>Laitano, O., Leon, L. R., Roberts, W. O., Sawka, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controversies+in+exertional+heat+stroke+diagnosis,+prevention,+and+treatment.">Controversies in exertional heat stroke diagnosis, prevention, and treatment.</a> Journal of Applied Physiology. 127 (5), 1338-1348 (2019).</li><li>King, M. A., 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=Influence+of+prior+illness+on+exertional+heat+stroke+presentation+and+outcome.">Influence of prior illness on exertional heat stroke presentation and outcome.</a> PLOS One. 14 (8), 0221329 (2019).</li><li>Carter, 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=Epidemiology+of+hospitalizations+and+deaths+from+heat+illness+in+soldiers.">Epidemiology of hospitalizations and deaths from heat illness in soldiers.</a> Medicine and Science in Sports and Exercise. 37 (8), 1338-1344 (2005).</li><li>Howe, A. S., Boden, B. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heat-related+illness+in+athletes.">Heat-related illness in athletes.</a> The American Journal of Sports Medicine. 35 (8), 1384-1395 (2007).</li><li>Wallace, R. F., Kriebel, D., Punnett, L., Wegman, D. H., Amoroso, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prior+heat+illness+hospitalization+and+risk+of+early+death.">Prior heat illness hospitalization and risk of early death.</a> Environmental Research. 104 (2), 290-295 (2007).</li><li>Wang, 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=The+association+between+heat+stroke+and+subsequent+cardiovascular+diseases.">The association between heat stroke and subsequent cardiovascular diseases.</a> PLOS One. 14 (2), 0211386 (2019).</li><li>Leon, L. R., Blaha, M. D., DuBose, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time+course+of+cytokine,+corticosterone,+and+tissue+injury+responses+in+mice+during+heat+strain+recovery.">Time course of cytokine, corticosterone, and tissue injury responses in mice during heat strain recovery.</a> Journal of Applied Physiology. 100 (4), 1400-1409 (2006).</li><li>Leon, L. R., DuBose, D. A., Mason, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heat+stress+induces+a+biphasic+thermoregulatory+response+in+mice.">Heat stress induces a biphasic thermoregulatory response in mice.</a> American Journal of Physiology. Regulatory, Integrative and Comparative Physiology. 288 (1), 197-204 (2005).</li><li>Leon, L. R., Gordon, C. J., Helwig, B. G., Rufolo, D. M., Blaha, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermoregulatory,+behavioral,+and+metabolic+responses+to+heatstroke+in+a+conscious+mouse+model.">Thermoregulatory, behavioral, and metabolic responses to heatstroke in a conscious mouse model.</a> American Journal of Physiology. Regulatory, Integrative and Comparative Physiology. 299 (1), 241-248 (2010).</li><li>King, M. A., Leon, L. R., Morse, D. A., Clanton, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unique+cytokine+and+chemokine+responses+to+exertional+heat+stroke+in+mice.">Unique cytokine and chemokine responses to exertional heat stroke in mice.</a> Journal of Applied Physiology. 122 (2), 296-306 (2016).</li><li>Costa, K. A., 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=l-Arginine+supplementation+prevents+increases+in+intestinal+permeability+and+bacterial+translocation+in+Male+Swiss+mice+subjected+to+physical+exercise+under+environmental+heat+stress.">l-Arginine supplementation prevents increases in intestinal permeability and bacterial translocation in Male Swiss mice subjected to physical exercise under environmental heat stress.</a> The Journal of Nutrition. 144 (2), 218-223 (2014).</li><li>Hubbard, 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=Effects+of+exercise+in+the+heat+on+predisposition+to+heatstroke.">Effects of exercise in the heat on predisposition to heatstroke.</a> Medicine and Science in Sports. 11 (1), 66-71 (1979).</li><li>Hubbard, R. W., 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=Rat+model+of+acute+heatstroke+mortality.">Rat model of acute heatstroke mortality.</a> Journal of Applied Physiology: Respiratory, Environmental and Exercise Physiology. 42 (6), 809-816 (1977).</li><li>Hubbard, R. W., 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=Diagnostic+significance+of+selected+serum+enzymes+in+a+rat+heatstroke+model.">Diagnostic significance of selected serum enzymes in a rat heatstroke model.</a> Journal of Applied Physiology: Respiratory, Environmental and Exercise Physiology. 46 (2), 334-339 (1979).</li><li>Hubbard, R. W., 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=Role+of+physical+effort+in+the+etiology+of+rat+heatstroke+injury+and+mortality.">Role of physical effort in the etiology of rat heatstroke injury and mortality.</a> Journal of Applied Physiology: Respiratory, Environmental and Exercise Physiology. 45 (3), 463-468 (1978).</li><li>Garcia, C. K., 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=Sex-dependent+responses+to+exertional+heat+stroke+in+mice.">Sex-dependent responses to exertional heat stroke in mice.</a> Journal of Applied Physiology. 125 (3), 841-849 (2018).</li><li>Garcia, C. K., 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=Effects+of+Ibuprofen+during+Exertional+Heat+Stroke+in+Mice.">Effects of Ibuprofen during Exertional Heat Stroke in Mice.</a> Medicine and Science in Sports and Exercise. 52 (9), 1870-1878 (2020).</li><li>King, M. A., Leon, L. R., Mustico, D. L., Haines, J. M., Clanton, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomarkers+of+multi-organ+injury+in+a+pre-clinical+model+of+exertional+heat+stroke.">Biomarkers of multi-organ injury in a pre-clinical model of exertional heat stroke.</a> Journal of Applied Physiology. 118 (10), (2015).</li><li>Murray, K. O., 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=Exertional+heat+stroke+leads+to+concurrent+long-term+epigenetic+memory,+immunosuppression+and+altered+heat+shock+response+in+female+mice.">Exertional heat stroke leads to concurrent long-term epigenetic memory, immunosuppression and altered heat shock response in female mice.</a> The Journal of Physiology. 599 (1), 119-141 (2021).</li><li>Leon, L. R., DuBose, D. A., Mason, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heat+stress+induces+a+biphasic+thermoregulatory+response+in+mice.">Heat stress induces a biphasic thermoregulatory response in mice.</a> American Journal of Physiology-Regulatory, Integrative and Comparative Physiology. 288, 197-204 (2005).</li><li>Laitano, O., 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=Delayed+metabolic+dysfunction+in+myocardium+following+exertional+heat+stroke+in+mice.">Delayed metabolic dysfunction in myocardium following exertional heat stroke in mice.</a> The Journal of Physiology. 598 (5), 967-985 (2020).</li><li>Iwaniec, 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=Acute+phase+response+to+exertional+heat+stroke+in+mice.">Acute phase response to exertional heat stroke in mice.</a> Experimental Physiology. 106 (1), 222-232 (2020).</li><li>He, S. -. 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=Optimization+of+a+rhabdomyolysis+model+in+mice+with+exertional+heat+stroke+mouse+model+of+EHS-rhabdomyolysis.">Optimization of a rhabdomyolysis model in mice with exertional heat stroke mouse model of EHS-rhabdomyolysis.</a> Frontiers in Physiology. 11, (2020).</li><li>Lopez, J. R., Kaura, V., Diggle, C. P., Hopkins, P. M., Allen, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Malignant+hyperthermia,+environmental+heat+stress,+and+intracellular+calcium+dysregulation+in+a+mouse+model+expressing+the+p.G2435R+variant+of+RYR1.">Malignant hyperthermia, environmental heat stress, and intracellular calcium dysregulation in a mouse model expressing the p.G2435R variant of RYR1.</a> British Journal of Anaesthesia. 121 (4), 953-961 (2018).</li><li>Laitano, O., Murray, K. O., Leon, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overlapping+mechanisms+of+exertional+heat+stroke+and+malignant+hyperthermia:+evidence+vs.+conjecture.">Overlapping mechanisms of exertional heat stroke and malignant hyperthermia: evidence vs. conjecture.</a> Sports Medicine. 50 (9), 115-123 (2020).</li><li>Casa, D. J., Armstrong, L. E., Kenny, G. P., O'Connor, F. G., Huggins, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exertional+heat+stroke:+new+concepts+regarding+cause+and+care.">Exertional heat stroke: new concepts regarding cause and care.</a> Current Sports Medicine Reports. 11 (3), 115-123 (2012).</li></ol>

Denne tekniske gennemgang har til formål at give retningslinjer for udførelsen af en præklinisk model af EHS hos mus. Detaljerede trin og materialer, der kræves til udførelse af en reproducerbar EHS episode af varierende sværhedsgrader er fastsat. Vigtigere, modellen i vid udstrækning efterligner tegn, symptomer, og multi-organ dysfunktion observeret i menneskelige EHS ofre11,19. Desuden giver denne model mulighed for undersøgelse af den mekanisme, der li...

Hedeslag er karakteriseret ved dysfunktion i centralnervesystemet og efterfølgende organskader hos hypertermiske emner1. Der er to manifestationer af hedeslag. Klassisk hedeslag (CHS) påvirker for det meste ældre befolkninger under hedebølger eller børn tilbage i solbeskinnede køretøjer i varme sommerdage1. Anstrengende hedeslag (EHS) opstår, når der er en manglende evne til at termoregulere tilstrækkeligt under fysisk anstrengelse, typisk, men ikke altid, under høje omgivelsestemperaturer resulterer i neurologiske symptomer, hypertermi, og efterfølgende multi-organ dysfunktion og skader2. EHS forekommer i rekreative og elite atleter samt militært personel og i arbejdere med og uden samtidig dehydrering3,4. Ehs er faktisk den tredje hyppigste årsag til dødelighed hos atleter under fysisk aktivitet5. Det er yderst udfordrende at studere EHS hos mennesker, da episoden kan være dødelig eller føre til langsigtede negative sundhedsmæssige resultater6,7. Derfor kan en pålidelig præklinisk model af EHS tjene som et værdifuldt redskab til at overvinde begrænsningerne af retrospektive og associative kliniske observationer hos menneskelige EHS-ofre. Prækliniske modeller af CHS hos gnavere og svin har været godt karakteriseret8,9,10. Prækliniske modeller af CHS kan dog ikke direkte oversættes til EHS patofysiologi på grund af de unikke virkninger af motion på den termoregulerende profil og medfødte immunrespons11. Derudover udgjorde tidligere forsøg på at udvikle prækliniske EHS-modeller hos gnavere betydelige begrænsninger, herunder overlejrede stressstimuli forårsaget af elektrisk stød, indsættelse af en rektal sonde og foruddefinerede maksimale kernekropstemperaturer med høje dødelighedsrater12,13,14,15,16 der ikke svarer til aktuelle epidemiologiske data. Disse udgør betydelige begrænsninger, der kan forvirre datafortolkning og give upålidelige biomarkørindekser. Derfor har protokollen til formål at karakterisere og beskrive trinene i en standardiseret, meget gentagelig og oversættelig præklinisk model af EHS hos mus, der stort set er fri for ovennævnte begrænsninger. Justeringer af modellen, der kan resultere i graduerede fysiologiske resultater fra moderat til dødelig hedeslag er beskrevet. Så vidt forfatterne ved, er dette den eneste prækliniske model af EHS med sådanne egenskaber, hvilket gør det muligt at forfølge relevant EHS-forskning på en hypotesedrevet måde11,17,18.

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a preclinical model of exertional heat stroke in mice

Hedeslag er den mest alvorlige manifestation af varmerelaterede sygdomme. Klassisk hedeslag (CHS), også kendt som passiv hedeslag, forekommer i hvile, mens anstrengende hedeslag (EHS) forekommer under fysisk aktivitet. EHS adskiller sig fra CHS i ætiologi, klinisk præsentation, og følgesygdomme af multi-organ dysfunktion. Indtil for nylig har kun modeller af CHS været veletablerede. Denne protokol har til formål at give retningslinjer for en raffineret præklinisk musemodel af EHS, der er fri for vigtige begrænsende faktorer såsom brug af anæstesi, tilbageholdenhed, rektalsonder eller elektrisk stød. Mandlige og kvindelige C57Bl/6 mus, instrumenteret med kernetemperatur (Tc) telemetriske sonder blev udnyttet i denne model. For at gøre køretilstanden bekendt gennemgår mus 3 ugers træning ved hjælp af både frivillige og tvungne løbehjul. Derefter kører mus på et tvunget hjul inde i et klimakammer, der er indstillet til 37,5 °C og 40%-50% relativ luftfugtighed (RH), indtil de udviser symptombegrænsning (f.eks. bevidsthedstab) ved Tc på 42,1-42,5 °C, selv om der kan opnås passende resultater ved kammertemperaturer mellem 34,5-39,5 °C og fugtighed mellem 30%-90%. Afhængigt af den ønskede sværhedsgrad fjernes mus straks fra kammeret for at komme sig i omgivelsestemperaturen eller forblive i det opvarmede kammer i længere tid, hvilket fremkalder en mere alvorlig eksponering og en højere forekomst af dødelighed. Resultaterne sammenlignes med sham-matchede øvelse kontrol (EXC) og / eller naive kontroller (NC). Modellen afspejler mange af de patofysiologiske resultater observeret i human EHS, herunder tab af bevidsthed, svær hypertermi, multi-organ skader samt inflammatorisk cytokin frigivelse, og akut fase reaktioner i immunsystemet. Denne model er ideel til hypotese-drevet forskning til at teste forebyggende og terapeutiske strategier, der kan forsinke starten af EHS eller reducere multi-organ skader, der karakteriserer denne manifestation.

De typiske termoreguleringsprofiler under hele EHS-protokollen og tidlig genfinding af en mus er illustreret i figur 1A. Denne profil omfatter fire forskellige faser, der kan defineres som kammerets opvarmningsfase, trinvis øvelse, øvelsesfase med konstant tilstand og et restitutionsstadium ved enten en hurtig afkøling (R) eller alvorlig (S) metode17. De vigtigste termoreguleringsresultater omfatter maksimalt opnåede Tc (Tc,max) og den tid, det tager at nå Tc, ma...

Watch this Scientific Journal Video about A Preclinical Model of Exertional Heat Stroke in Mice at JoVE.com

A Preclinical Model of Exertional Heat Stroke in Mice

Protokollen beskriver udviklingen af en standardiseret, gentagelig, præklinisk model af anstrengende hedeslag (EHS) hos mus fri for negative eksterne stimuli såsom elektrisk stød. Modellen giver en platform for mekanistiske, forebyggende og terapeutiske undersøgelser.

En præklinisk model af anstrengende hedeslag hos mus

Heat stroke is the most severe manifestation of heat-related illnesses. Classic heat stroke (CHS), also known as passive heat stroke, occurs at rest, ...

Tidligere har kun passive modeller af hedeslag været veletablerede. Denne model af anstrengende hedeslag er fri for store begrænsninger og mere nøje efterligner den menneskelige patofysiologi. Det er ikke muligt effektivt at studere hedeslag hos mennesker, fordi det er en livstruende tilstand.Denne prækliniske model bevarer de kliniske egenskaber ved menneskelig anstrengende hedeslag. Denne teknik hjælper med at teste virkningerne af afkøling sats på inddrivelse fra anstrengende hedeslag, comorbiditeter fra narkotika indtagelse, samt langsigtede konsekvenser af hedeslag eksponering. Demonstrere proceduren vil være Jamal Alzahrani, en ledende ph.d.-studerende fra Dr.Clanton's lab.Efter bedøvelse af musen skal du bruge øjensmøre som Dyrlægens salve til at beskytte dyrets øjne mod skader eller skader under operationen. For at forberede det kirurgiske sted skal du barbere underlivet med små dyrehårklippere eller en kommercielt tilgængelig hårfjerner. Giv den første dosis af subkutane 0,1 milligram pr. kg buprenorphin.Skrub området med tre vasker povidone-jod eller chlorhexidin, efterfulgt af 70% isopropylalkohol skyl, og overfør derefter musen til det kirurgiske område, brug en klæbende drapering til at isolere det kirurgiske sted på musen. Ved hjælp af sterile instrumenter i aseptisk teknik, lav et ca. en centimeter snit på midterlinjen langs linea alba ca. 0,5 centimeter fra omkostningsmarginen, fjern derefter huden fra muskellaget og lav et lidt mindre snit på linea alba, der pas på ikke at beskadige tarmene eller indre organer. Når muskellaget er åbent, skal du placere det sterile telemeter i intraperitoneal hulrum foran de kauudale arterier og vener og ryghul til fordøjelsesorganerne, så det kan flyde frit.Luk abdominal åbning med en steril 5-O absorberes sutur og lukke huden ved hjælp af en simpel afbrudt søm med 5-O proline sutur. Placer musen i sit rene bur med en tæt mikrobølge varmepude under buret. Overvåg musen hvert 15. minut i løbet af den første time af genopretning fra anæstesi og vend derefter tilbage til dyrehuset.Natten før den anstrengende hedeslag protokol, placere musen i miljøkammeret ved stuetemperatur til akklimatisering til kammeret. Brug dataindsamlingssystemet til at indsamle kontinuerlig kernetemperatur eller TC i gennemsnit over 30-sekunders intervaller natten over. Om morgenen af den anstrengende hedeslag protokol, sørg for, at musen er på eller under det normale interval af døgntemperatur på 36 til 37,5 grader Celsius, før du øger kammertemperaturen for at sikre, at musen ikke har feber eller har oplevet unødig stress i denne periode.Når musen er stabil og inden for en række normale hvilende kernetemperatur, skal du fjerne mad og vand og veje dyret. Luk kammerdøren og øg kammertemperaturen til et mål på 37,5 grader Celsius og 40 til 50% relativ luftfugtighed eller den ønskede miljøtemperatur og fugtighed. Kontroller kammertemperaturen og fugtigheden med en kalibreret temperatur- og fugtighedsmåler.Omgiv kammeret med et mørklægningsgardin for at holde lyset og forstyrrelserne minimale under protokollen. Overvåg musen kontinuerligt under protokollen via eksterne IR-belyste kameraer. Fokuser det andet kamera på temperatur- og fugtighedsskærmen, der er placeret tæt på løbehjulet.Foretag justeringer af controlleren for miljøkammersætpunktet for at sikre nøjagtige temperaturaflæsninger i nærheden af dyret. Når kammeret har nået sin måltemperatur målt ved det andet kamera på temperaturmåleren, skal du hurtigt åbne kammerdøren og placere musen i det tvungne løbehjul. Start den tvungne løbehjul protokol med en hastighed på 2,5 meter i minuttet, og øge hastigheden med 0,3 meter i minuttet hvert 10. minut, indtil musen når en kernetemperatur på 41 grader Celsius.Når kernetemperaturen er opnået, skal du tillade hastigheden at forblive konstant, indtil symptombegrænsningen opstår, karakteriseret ved et tilsyneladende tab af bevidsthed, et tilbagefald eller besvimelse og manglende evne til at fortsætte med at køre eller holde fast i hjulet. Bekræft det tidspunkt, hvor musen har tre tilbagevendt på hjulet uden tegn på fysisk respons. Du kan også identificere humane slutpunkter efter lokale IACUC-regler for at afgøre, hvornår protokollen skal stoppes.Dette endepunkt er lidt over symptombegrænsningen hos stort set alle mus. For at udføre den hurtige køleprotokol skal du stoppe hjulet, når musen når symptombegrænsning og straks fjerne musen fra det tvungne løbehjul. Afvej musen og læg den tilbage i sit hjemmebur for at komme sig ved stuetemperatur.I løbet af denne tid skal du lade kammerdøren stå åben og returnere inkubatorens sætpunkt til stuetemperatur, så kammeret kan køle hurtigt, hvilket resulterer i større end 99% langsigtet overlevelse. For at udføre en mere alvorlig anstrengende hedeslag eksponering, holde dyrets bur inden for 37,5 grader Celsius kammer under den anstrengende hedeslag protokol. Når dyret når symptombegrænsning, skal de forblive i løbehjulet, indtil de vender tilbage til bevidsthed som observeret af fjernkameraet, og fjern derefter hurtigt musen fra løbehjulet og returnere den direkte til dets forvarmede bur for at resultere i en meget langsommere køleprofil, hvilket i det væsentlige eliminerer den anstrengende hedeslaghytermisk fase.Fjern filtertoppen fra buret i løbet af denne tid for at forbedre ekvilibreringen med kammeret. Et genvindingsbur, der er forkølet til stuetemperatur, kan bruges til at udføre en mindre alvorlig alternativ procedure for at resultere i en undertrykt hypotermisk fase med en overlevelsesrate på 100% For den alvorlige anstrengende hedeslagprotokol skal du omhyggeligt overvåge musene under genopretning og løbende kontrollere humane endepunkter, herunder normale bevægelser under genopretning, såsom grooming, normal vejrtrækning og slikke. Overvåg kernetemperaturen i løbet af denne tid.Musene er usandsynligt, at inddrive, hvis deres kernetemperatur vender retning i løbet af inddrivelse fase, i sidste ende over 40 grader Celsius. I et sådant tilfælde skal du afslutte eksperimentet og fortsætte med at evaluere musen for standard humane slutpunkter. De typiske termoreguleringsprofiler under hele den anstrengende hedeslag protokol i tidlig inddrivelse af en mus omfatter af fire forskellige faser, der kan defineres som kammeret opvarmning fase, trinvis øvelse fase, stabil tilstand øvelse fase, og en nyttiggørelse fase ved enten en hurtig eller langsom afkøling metode.For den alvorlige model indikerede genopretningstemperaturprofilen, at kernetemperaturen forblev over 37 grader Celsius i hele den to timers genopretningsperiode. Hunmus var mere modstandsdygtige over for hedeslag i denne model og løb næsten dobbelt længere afstande end hanmus. Ingen forskel blev observeret i den tid, der kræves for at komme sig til 39,5 grader Celsius i de to modeller.Men tiden til at køle til miljøtemperaturen på 37,5 grader Celsius blev stærkt forlænget. Det vigtigste er den omhyggelige overvågning af dyrene til enhver tid efter operationen og post-anstrengende hedeslag. Denne metode har hjulpet med at studere de langsigtede virkninger af hedeslag eksponering op til flere måneder, og har også givet os mulighed for at måle virkningerne af yderligere faktorer i hedeslag sygdom, såsom indtagelse af nonsteroidal antiinflammatoriske lægemidler og epigenetik.

a-preclinical-model-of-exertional-heat-stroke-in-mice

Research

JoVE Journal

Medicine

Modeling Stroke in Mice - Middle Cerebral Artery Occlusion with the Filament Model

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are very limited. To meet the need for novel therapeutic approaches, experimental stroke research frequently employs rodent models of focal cerebral ischaemia. Most researchers use permanent or transient occlusion of the middle cerebral artery (MCA) in mice or rats.
Proximal occlusion of the middle cerebral artery (MCA) via the intraluminal suture technique (so called filament or suture model) is probably the most frequently used model in experimental stroke research. The intraluminal MCAO model offers the advantage of inducing reproducible transient or permanent ischaemia of the MCA territory in a relatively non-invasive manner. Intraluminal approaches interrupt the blood flow of the entire territory of this artery. Filament occlusion thus arrests flow proximal to the lenticulo-striate arteries, which supply the basal ganglia. Filament occlusion of the MCA results in reproducible lesions in the cortex and striatum and can be either permanent or transient. In contrast, models inducing distal (to the branching of the lenticulo-striate arteries) MCA occlusion typically spare the striatum and primarily involve the neocortex. In addition these models do require craniectomy. In the model demonstrated in this article, a silicon coated filament is introduced into the common carotid artery and advanced along the internal carotid artery into the Circle of Willis, where it blocks the origin of the middle cerebral artery. In patients, occlusions of the middle cerebral artery are among the most common causes of ischaemic stroke. Since varying ischemic intervals can be chosen freely in this model depending on the time point of reperfusion, ischaemic lesions with varying degrees of severity can be produced. Reperfusion by removal of the occluding filament at least partially models the restoration of blood flow after spontaneous or therapeutic (tPA) lysis of a thromboembolic clot in humans.
In this video we will present the basic technique as well as the major pitfalls and confounders which may limit the predictive value of this model.

Filamentous occlusion of the Middle cerebral artery is a common model for studying ischemic stroke in mice.

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are ...

Gene Transfer for Ischemic Heart Failure in a Preclinical Model

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine their efficacy and safety.
Gene therapy using viral vectors is one of the most promising approaches for treating cardiac diseases. Viral delivery of various different genes by changing the carrier gene has immeasurable therapeutic potential.
In this video, the full process of an animal model of heart failure creation followed by gene transfer is presented using a swine model. First, myocardial infarction is created by occluding the proximal left anterior descending coronary artery. Heart remodeling results in chronic heart failure. Unique to our model is a fairly large scar which truly reflects patients with severe heart failure who require aggressive therapy for positive outcomes. After myocardial infarct creation and development of scar tissue, an intracoronary injection of virus is demonstrated with simultaneous nitroglycerine infusion. Our injection method provides simple and efficient gene transfer with enhanced gene expression. This combination of a myocardial infarct swine model with intracoronary virus delivery has proven to be a consistent and reproducible methodology, which helps not only to test the effect of individual gene, but also compare the efficacy of many genes as therapeutic candidates.

A method of gene transfer for the treatment of ischemic heart failure is described using a swine model of myocardial infarction. Our simple and reproducible method enables us to readily evaluate the efficacy of various gene transfers with a very simple and reproducible way.

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine ...

Intraluminal Middle Cerebral Artery Occlusion (MCAO) Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Mice

Stroke is the third leading cause of death and the leading cause of disability in the world, with an estimated cost of near $70 billion 
in the United States in 20091,2. The intraluminal middle cerebral artery occlusion (MCAO) model was developed by Koizumi4 in 1986 to simulate this 
impactful human pathology in the rat. A modification of the MCAO method was later presented by Longa3. Both techniques have been widely used to identify 
molecular mechanisms of brain injury resulting from ischemic stroke and potential therapeutic modalities5. This relatively noninvasive method in rats has been 
extended to use in mice to take advantage of transgenic and knockout strains6,7. To model focal cerebral ischemia, an intraluminal suture is advanced via 
the internal carotid artery to occlude the base of the MCA. Retracting the suture after a specified period of time mimics spontaneous reperfusion, but the 
suture can also be permanently retained. This video will be demonstrating the two major approaches for performing intraluminal MCAO procedure in mice in a 
stepwise fashion, as well as providing insights for potential drawbacks and pitfalls. The ischemic brain tissue will subsequently be stained by 
2,3,5-triphenyltetrazolium chloride (TTC) to evaluate the extent of cerebral infarction8.

Intraluminal Middle Cerebral Artery Occlusion MCAO Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Mice

The intraluminal middle cerebral artery occlusion (MCAO) model is the most frequent used model among experimental ischemic stroke models. Here we will demonstrate the entire model in detail with the guide of Laser Doppler flowmetry, and its representative results.

Stroke is the third leading cause of death and the leading cause of disability in the world, with an estimated cost of near $70 billion 
in the United ...

A Murine Model of Subarachnoid Hemorrhage

In this video publication a standardized mouse model of subarachnoid hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of Willis perforation (CWp) and proven by intracranial pressure (ICP) monitoring. Thereby a homogenous blood distribution in subarachnoid spaces surrounding the arterial circulation and cerebellar fissures is achieved. Animal physiology is maintained by intubation, mechanical ventilation, and continuous on-line monitoring of various physiological and cardiovascular parameters: body temperature, systemic blood pressure, heart rate, and hemoglobin saturation. Thereby the cerebral perfusion pressure can be tightly monitored resulting in a less variable volume of extravasated blood. This allows a better standardization of endovascular filament perforation in mice and makes the whole model highly reproducible. Thus it is readily available for pharmacological and pathophysiological studies in wild type and genetically altered mice.

A standardized mouse model of subarachnoid hemorrhage by intraluminal Circle of Willis perforation is described. Vessel perforation and subarachnoid bleeding are monitored by intracranial pressure monitoring. In addition various vital parameters are recorded and controlled to maintain physiologic conditions.

En murin model af subarachnoid blødning

In this video publication a standardized mouse model of subarachnoid  hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of  Willis ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

Excitotoksiske Stimulering af Brain Microslices som en<em&gt; In vitro</em&gt; Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

A Preclinical Murine Model of Hepatic Metastases

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, murine pancreatic tumor model of hepatic metastases via a hemispleen injection of syngeneic murine pancreatic tumor cells is described. This model mimics many of the clinical conditions in patients with metastatic disease to the liver. Mice consistently develop metastases in the liver allowing for investigation of the metastatic process, experimental therapy testing, and tumor immunology research.

A preclinical, murine model of hepatic metastases performed via a hemispleen injection technique.

Et præklinisk musemodel af levermetastaser

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, ...

Modeling Stroke in Mice: Permanent Coagulation of the Distal Middle Cerebral Artery

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical options are available for a small proportion of stroke patients in the acute phase. Current research is intensively searching for novel therapeutic strategies and is increasingly focusing on the sub-acute and chronic phase after stroke because more patients might be eligible for therapeutic interventions in a prolonged time window. These delayed mechanisms include important pathophysiological pathways such as post-stroke inflammation, angiogenesis, neuronal plasticity and regeneration. In order to analyze these mechanisms and to subsequently evaluate novel drug targets, experimental stroke models with clinical relevance, low mortality and high reproducibility are sought after. Moreover, mice are the smallest mammals in which a focal stroke lesion can be induced and for which a broad spectrum of transgenic models are available. Therefore, we describe here the mouse model of transcranial, permanent coagulation of the middle cerebral artery via electrocoagulation distal of the lenticulostriatal arteries, the so-called &#8220;coagulation model&#8221;. The resulting infarct in this model is located mainly in the cortex; the relative infarct volume in relation to brain size corresponds to the majority of human strokes. Moreover, the model fulfills the above-mentioned criteria of reproducibility and low mortality. In this video we demonstrate the surgical methods of stroke induction in the &#8220;coagulation model&#8221; and report histological and functional analysis tools.

Various murine models of middle cerebral artery occlusion (MCAO) are widely used in experimental brain research. Here, we demonstrate the model of transcranial permanent distal MCAO which produces consistent cortical infarction of a size corresponding to damage imposed by the majority of human ischemic strokes.

Modellering Stroke i mus: Permanent Koagulering af den distale Middle Cerebral Artery

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical ...

A Thrombotic Stroke Model Based On Transient Cerebral Hypoxia-ischemia

Stroke research has endured many setbacks in translating neuroprotective therapies into clinical practice. In contrast, the real-world therapy (tPA thrombolysis) rarely produces benefits in mechanical occlusion-based experimental models, which dominate preclinical stroke research. This split between the bench and bedside suggests the need to employ tPA-responsive models in preclinical stroke research. To this end, a simple and tPA-reactive thrombotic stroke model is invented and described here. This model consists of transient occlusion of the unilateral common carotid artery and delivery of 7.5% oxygen through a face mask in adult mice for 30 min, while maintaining the animal rectal temperature at 37.5 &#177; 0.5 &#176;C. Although reversible ligation of the unilateral carotid artery or hypoxia each suppressed cerebral blood flow only transiently, the combination of both insults caused lasting reperfusion deficits, fibrin and platelet deposition, and large infarct in the middle cerebral artery-supplied territory. Importantly, tail-vein injection of recombinant tPA at 0.5, 1, or 4 hr post-tHI (10 mg/kg) provided time-dependent reduction of the mortality rate and infarct size. This new stroke model is simple and can be standardized across laboratories to compare experimental results. Further, it induces thrombosis without craniectomy or introducing pre-formed emboli. Given these unique merits, the tHI model is a useful addition to the repertoire of preclinical stroke research.

Thromboembolic stroke models are vital tools for optimizing the recanalization therapy. Here we report a murine thrombotic stroke model based on transient cerebral hypoxic-ischemic (tHI) insult, which triggers thrombosis and infarction, and responds favorably to tissue plasminogen activator (tPA)-mediated fibrinolysis in a therapeutic window similar to those in stroke patients.

En Trombotisk Stroke model baseret på Forbigående Cerebral Hypoxi-iskæmi

Stroke research has endured many setbacks in translating neuroprotective therapies into clinical practice. In contrast, the real-world therapy (tPA ...

Surgical Approach for Middle Cerebral Artery Occlusion and Reperfusion Induced Stroke in Mice

Stroke is a leading cause of death worldwide and continues to be one of the major causes of long-term adult disabilities. About 87% of strokes are ischemic in origin and occur in the territory of the middle cerebral artery (MCA). Currently the only Food and Drug Administration (FDA) approved drug for the treatment of this devastating disease is tissue plasminogen activator (tPA). However, tPA has a small therapeutic window for administration (3 - 6 hr), and is only effective in 4% of the patients who actually receive it. Current research focuses on understanding the pathophysiology of stroke in order to find potential therapeutic targets. Thus, reliable models are crucial, and the MCA occlusion (MCAo) model (also termed the intraluminal filament or suture model) is deemed to be the most clinically relevant surgical model of ischemic stroke, and is fairly non-invasive and easily reproducible. Typically the MCAo model is used with rodents, especially with mice due to all the genetic variations available for this species. Here we describe (and present in the video) how to successfully perform the MCAo model (with reperfusion) in mice to generate reliable and reproducible data.

In order to understand the pathophysiology of stroke, it is important to use reliable models. This paper will describe one of the most frequently used stroke models in mice, termed the middle cerebral artery occlusion (MCAo) model (also termed the intraluminal filament or suture model) with reperfusion.

Kirurgisk Approach for mellem-cerebral arterieokklusion og reperfusion induceret Stroke i mus

Stroke is a leading cause of death worldwide and continues to be one of the major causes of long-term adult disabilities. About 87% of strokes are ...

PET Imaging of Neuroinflammation Using [11C]DPA-713 in a Mouse Model of Ischemic Stroke

Neuroinflammation is central to the pathological cascade following ischemic stroke. Non-invasive molecular imaging methods have the potential to provide critical insights into the temporal dynamics and role of certain neuroimmune interactions in stroke. Specifically, Positron Emission Tomography (PET) imaging of translocator protein 18 kDa (TSPO), a marker of activated microglia and peripheral myeloid-lineage cells, provides a means to detect and track neuroinflammation in vivo. Here, we present a method to accurately quantify neuroinflammation using [11C]N,N-Diethyl-2-[2-(4-methoxyphenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl]acetamide ([11C]DPA-713), a promising second generation TSPO-PET radiotracer, in distal middle cerebral artery occlusion (dMCAO) compared to sham-operated mice. MRI was performed 2 days post-dMCAO surgery to confirm stroke and define the infarct location and volume. PET/Computed Tomography (CT) imaging was carried out 6 days post-dMCAO to capture the peak increase in TSPO levels following stroke. Quantitation of PET images was conducted to assess the uptake of [11C]DPA-713 in the brain and spleen of dMCAO and sham mice to assess central and peripheral levels of inflammation. In vivo [11C]DPA-713 brain uptake was confirmed using ex vivo autoradiography.

PET Imaging of Neuroinflammation Using [11C]DPA-713 in a Mouse Model of Ischemic Stroke

Positron Emission Tomography (PET) imaging of translocator protein 18 kDa (TSPO) provides a non-invasive means to visualize the dynamic role of neuroinflammation in the development and progression of brain diseases. This protocol describes TSPO-PET and ex vivo autoradiography to detect neuroinflammation in a mouse model of ischemic stroke.

PET-billeddannelse af Neuroinflammation ved hjælp af [11C] DPA-713 i en musemodel af iskæmisk slagtilfælde

Neuroinflammation is central to the pathological cascade following ischemic stroke. Non-invasive molecular imaging methods have the potential to provide ...