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 arbeidet ble finansiert av Forsvarsdepartementet W81XWH-15-2-0038 (TLC) og BA180078 (TLC) og BK og Betty Stevens Endowment (TLC). JMA ble støttet av økonomisk støtte fra Kongeriket Saudi-Arabia. Michelle King var ved University of Florida da denne studien ble gjennomført. Hun er for tiden ansatt ved Gatorade Sports Science Institute, en divisjon av PepsiCo R&D.

Alle prosedyrer er gjennomgått og godkjent av University of Florida IACUC. C57BL/6J hann- eller hunnmus, ~ 4 måneder gamle, veier innenfor et område på henholdsvis 27-34 g og 20-25 g, brukes til studien.
1. Kirurgisk implantasjon av det telemetriske temperaturovervåkingssystemet
<ol><li>Ved ankomst fra leverandøren, la dyrene hvile i vivariumet i minst 1 uke før operasjonen for å minimere transportstresset.</li>
	<li>Gruppehus musene (maksimalt 5 per bur under lokale IACUC-retningsli...

<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 gjennomgangen tar sikte på å gi retningslinjer for utførelsen av en preklinisk modell av EHS hos mus. Detaljerte trinn og materialer som kreves for å utføre en reproduserbar EHS-episode med variable alvorlighetsgrader, er gitt. Viktigst, modellen etterligner i stor grad tegn, symptomer og multiorgan dysfunksjon observert hos menneskelige EHS-ofre11,19. Videre tillater denne modellen undersøkelse av mekanismen som ligger til grunn for kort- og...

Varmeslag er preget av sentralnervesystemet dysfunksjon og påfølgende organskader hos hypertermiske personer1. Det er to manifestasjoner av heteslag. Klassisk heteslag (CHS) påvirker for det meste eldre populasjoner under hetebølger eller barn som er igjen i solutsatte kjøretøy i varme sommerdager1. Anstrengelsesvarmeslag (EHS) oppstår når det er manglende evne til å termoregulering tilstrekkelig under fysisk anstrengelse, vanligvis, men ikke alltid, under høye omgivelsestemperaturer som resulterer i nevrologiske symptomer, hypertermi og påfølgende multiorgan dysfunksjon og skade2. EHS forekommer i rekreasjons- og toppidrettsutøvere samt militært personell og i arbeidere med og uten samtidig dehydrering3,4. Faktisk er EHS den tredje ledende årsaken til dødelighet hos idrettsutøvere under fysisk aktivitet5. Det er ekstremt utfordrende å studere EHS hos mennesker, da episoden kan være dødelig eller føre til langsiktige negative helseutfall6,7. Derfor kan en pålitelig preklinisk modell av EHS tjene som et verdifullt verktøy for å overvinne begrensningene for retrospektive og assosiative kliniske observasjoner hos menneskelige EHS-ofre. Prekliniske modeller av CHS hos gnagere og griser har blitt godt karakterisert8,9,10. Prekliniske modeller av CHS oversettes imidlertid ikke direkte til EHS patofysiologi på grunn av de unike effektene av fysisk trening på termoregulatorisk profil og medfødt immunrespons11. I tillegg utgjorde tidligere forsøk på å utvikle prekliniske EHS-modeller hos gnagere betydelige restriksjoner, inkludert overliggende stressstimuli indusert av elektrisk støt, innsetting av en rektal sonde og forhåndsdefinerte maksimale kroppstemperaturer med høydødelighet 12,13,14,15,16 som ikke samsvarer med gjeldende epidemiologiske data. Disse representerer betydelige begrensninger som kan forvirre datatolkning og gi upålitelige biomarkørindekser. Derfor tar protokollen sikte på å karakterisere og beskrive trinnene i en standardisert, svært repeterbar og overførbar preklinisk modell av EHS hos mus som i stor grad er fri for begrensningene nevnt ovenfor. Justeringer av modellen som kan resultere i graderte fysiologiske utfall fra moderat til dødelig varmeslag er beskrevet. Til forfatternes kunnskap er dette den eneste prekliniske modellen av EHS med slike egenskaper, noe som gjør det mulig å forfølge relevant EHS-forskning på en hypotesedrevet måte11,17,18.

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a preclinical model of exertional heat stroke in mice

Varmeslag er den mest alvorlige manifestasjonen av varmerelaterte sykdommer. Klassisk varmeslag (CHS), også kjent som passivt varmeslag, oppstår i ro, mens anstrusjonelt varmeslag (EHS) oppstår under fysisk aktivitet. EHS skiller seg fra CHS i etiologi, klinisk presentasjon og oppfølger av multiorgan dysfunksjon. Inntil nylig har bare modeller av CHS blitt godt etablert. Denne protokollen tar sikte på å gi retningslinjer for en raffinert preklinisk musemodell av EHS som er fri for store begrensende faktorer som bruk av anestesi, selvbeherskelse, rektal sonder eller elektrisk støt. Mannlige og kvinnelige C57Bl/6 mus, instrumentert med kjernetemperatur (Tc) telemetriske sonder ble brukt i denne modellen. For å bli kjent med løpemodus gjennomgår mus 3 ukers trening ved hjelp av både frivillige og tvungne løpehjul. Deretter løper mus på et tvungent hjul inne i et klimakammer satt til 37,5 °C og 40%-50% relativ fuktighet (RH) til de viser symptombegrensning (f.eks. bevissthetstap) ved Tc på 42,1-42,5 °C, selv om egnede resultater kan oppnås ved kammertemperaturer mellom 34,5-39,5 °C og fuktighet mellom 30%-90%. Avhengig av ønsket alvorlighetsgrad fjernes mus fra kammeret umiddelbart for utvinning i omgivelsestemperatur eller forblir i det oppvarmede kammeret i lengre tid, noe som indikerer en mer alvorlig eksponering og en høyere forekomst av dødelighet. Resultatene sammenlignes med sham-matchede treningskontroller (EXC) og/eller naive kontroller (NC). Modellen speiler mange av de patofysiologiske resultatene som observeres i human EHS, inkludert tap av bevissthet, alvorlig hypertermi, multiorganskade samt inflammatorisk cytokinfrigjøring og akutte faseresponser i immunsystemet. Denne modellen er ideell for hypotesedrevet forskning for å teste forebyggende og terapeutiske strategier som kan forsinke utbruddet av EHS eller redusere multiorganskaden som karakteriserer denne manifestasjonen.

De typiske termoregulatoriske profilene under hele EHS-protokollen og tidlig gjenoppretting av en mus er illustrert i figur 1A. Denne profilen består av fire forskjellige faser som kan defineres som kammervarmestadiet, inkrementell treningstrinn, steady-state treningsstadium og et gjenopprettingsstadium med enten en rask kjøling (R) eller alvorlig (S) metode17. De viktigste termoregulatoriske resultatene inkluderer maksimal Tc oppnådd (Tc, maks) og tiden som kreves...

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 utviklingen av en standardisert, repeterbar, preklinisk modell av anstrengelsesvarmeslag (EHS) hos mus fri for ugunstige ytre stimuli som elektrisk støt. Modellen gir en plattform for mekanistiske, forebyggende og terapeutiske studier.

En preklinisk modell av anstrengelsesvarmeslag 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 bare passive modeller av heteslag vært veletablerte. Denne modellen av anspresjonell varmeslag er fri for store begrensninger og etterligner nærmere den menneskelige patofysiologien. Det er ikke mulig å effektivt studere varmeslag hos mennesker fordi det er en livstruende tilstand.Denne prekliniske modellen beholder de kliniske egenskapene til menneskelig anstrengelsesvarmeslag. Denne teknikken bidrar til å teste effekten av kjølehastighet på utvinning fra anstrusjonelt varmeslag, komorbiditeter fra legemiddelinntak, samt langsiktige konsekvenser av varmeslageksponering. Å demonstrere prosedyren vil være Jamal Alzahrani, en senior ph.d.-student fra Dr.Clantons laboratorium.Etter bedøvelse av musen, bruk øyesmøre som Vet Salve for å beskytte dyrets øyne mot skade eller skade under operasjonen. For å forberede det kirurgiske stedet, barber underlivet med små dyrehårklippere eller en kommersielt tilgjengelig hårfjerner. Administrer den første dosen av subkutane 0,1 milligram per kilo buprenorfin.Skrubb området med tre vasker av povidon-jod eller klorhexidin, etterfulgt av 70% isopropylalkoholskylling, og overfør deretter musen til det kirurgiske området, bruk en klebende gardin for å isolere det kirurgiske stedet på musen. Ved hjelp av sterile instrumenter i aseptisk teknikk, gjør en omtrent en centimeter snitt på midtlinjen langs linea alba ca 0,5 centimeter fra kostnadsmarginen, deretter skille huden fra muskellaget og gjøre et litt mindre snitt på linea alba være forsiktig så du ikke skader tarmene eller indre organer. Når muskellaget er åpent, plasserer du det sterile telemeteret i intraperitonealhulen foran kaudale arterier og årer og dorsale til fordøyelsesorganene slik at det kan flyte fritt.Lukk bukåpningen med en steril 5-O absorberbar sutur og lukk huden ved hjelp av en enkel avbrutt søm med 5-O prolin sutur. Plasser musen i sitt rene bur med en tettsittende mikrobølgevarmepute under buret. Overvåk musen hvert 15.Kvelden før den anspsjonelle varmeslagprotokollen, plasser musen i miljøkammeret ved romtemperatur for å akklimatisere seg til kammeret. Bruk datainnsamlingssystemet til å samle kontinuerlig kjernetemperatur eller TC i gjennomsnitt over 30-sekunders intervaller over natten. På morgenen av anstrengelsesmessig varmeslagprotokoll må du sørge for at musen er på eller under det normale området av diurnal temperatur på 36 til 37,5 grader Celsius før du øker kammertemperaturen for å sikre at musen ikke har feber eller har opplevd unødig stress i denne perioden.Når musen er stabil og innenfor en rekke normale hvilekjernetemperatur, fjern maten og vannet og vei dyret. Lukk kammerdøren og øk kammertemperaturen til et mål på 37,5 grader Celsius og 40 til 50% relativ fuktighet eller ønsket miljøtemperatur og fuktighet. Kontroller kammertemperaturen og fuktigheten med en kalibrert temperatur- og fuktighetsmonitor.Omgi kammeret med en blendingsgardin for å holde lys og forstyrrelser minimale under protokollen. Overvåk musen kontinuerlig under protokollen via eksterne IR-opplyste kameraer. Fokuser det andre kameraet på temperatur- og fuktighetsmonitoren plassert nær løpehjulet.Gjør justeringer på kontrolleren for miljøkammerets settpunkt for å sikre nøyaktige temperaturavlesninger nær dyret. Når kammeret har nådd måltemperaturen målt ved det andre kameraet på temperaturmonitoren, åpner du raskt kammerdøren og plasserer musen i det tvungne løpehjulet. Start tvungen kjørehjulprotokoll med en hastighet på 2,5 meter per minutt, og øk hastigheten med 0,3 meter per minutt hvert 10.Når kjernetemperaturen er oppnådd, la hastigheten forbli konstant til symptombegrensning oppstår, preget av et tilsynelatende bevissthetstap, et bakoverfall eller besvimelse, og manglende evne til å fortsette å løpe eller holde fast på hjulet. Bekreft tidspunktet når musen har tre bakoverrotasjoner på hjulet uten tegn på fysisk respons. Du kan også identifisere humant endepunkt etter lokale IACUC-regler for å finne ut når protokollen skal stoppes.Dette endepunktet er litt over symptombegrensning i stort sett alle mus. For å utføre den raske kjøleprotokollen, stopp hjulet når musen når symptombegrensning og fjern musen umiddelbart fra det tvungne løpehjulet. Vei musen og plasser den tilbake i hjemmeburet for å gjenopprette ved romtemperatur.I løpet av denne tiden, la kammerdøren stå åpen og returner inkubatorens settpunkt til romtemperatur slik at kammeret avkjøles raskt, noe som resulterer i større enn 99% langsiktig overlevelse. For å utføre en mer alvorlig anstrengelsesmessig varmeslageksponering, hold dyrets bur innenfor 37,5 grader Celsius-kammeret under anstrengelsesmessig varmeslagprotokoll. Når dyret når symptombegrensning, la dem forbli i løpehjulet til de kommer tilbake til bevisstheten som observert av fjernkameraet, fjern musen deretter raskt fra løpehjulet og returner den direkte til det forvarmede buret for å resultere i en mye langsommere kjøleprofil, og eliminerer i hovedsak den anspresjonelle varmeslag hypotermiske fasen.Fjern filtertoppen fra buret i løpet av denne tiden for å forbedre likevekten med kammeret. Et gjenopprettingsbur forhåndskjølt til romtemperatur kan brukes til å utføre en mindre alvorlig alternativ prosedyre for å resultere i en undertrykt hypotermisk fase med en overlevelsesrate på 100%For den alvorlige anstrengelsesmessige varmeslagprotokollen, må du nøye overvåke musene under gjenoppretting og sjekke kontinuerlig for humane endepunkter, inkludert normale bevegelser under gjenoppretting, for eksempel grooming, normal pusting og slikking. Overvåk kjernetemperaturen i denne perioden.Musene er usannsynlig å gjenopprette hvis kjernetemperaturen reverserer retningen under utvinningsfasen, til slutt overstiger 40 grader Celsius. I et slikt tilfelle avslutter du eksperimentet og fortsetter å evaluere musen for standard humane endepunkter. De typiske termoregulatoriske profilene under hele anstrengelsesmessig varmeslagprotokoll i tidlig gjenoppretting av en mus består av fire forskjellige faser som kan defineres som kammervarmestadiet, inkrementell treningsstadium, steady state-treningsstadium og et gjenopprettingsstadium ved enten en rask eller langsom kjølemetode.For den alvorlige modellen indikerte utvinningstemperaturprofilen at kjernetemperaturen holdt seg over 37 grader Celsius gjennom den to timer lange gjenopprettingsperioden. Kvinnelige mus var mer motstandsdyktige mot varmeslag i denne modellen og kjørte nesten to ganger lengre avstander enn mannlige mus. Ingen forskjell ble observert i tiden som kreves for å gjenopprette til 39,5 grader Celsius i de to modellene.Imidlertid var tiden for å avkjøle til miljøtemperaturen på 37,5 grader Celsius sterkt forlenget. Det viktigste er nøye overvåking av dyrene til enhver tid etter operasjonen og post-anstrusjonell varmeslag. Denne metoden har bidratt til å studere de langsiktige effektene av varmeslageksponering opptil flere måneder, og har også gjort det mulig for oss å måle effekten av ytterligere faktorer i varmeslagssykdom, for eksempel inntak av ikke-steroide antiinflammatoriske legemidler og epigenetikk.

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 murine modell av subaraknoidalblødning

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.

Eksitotoksiske Stimulering av Hjerne Microslices som en<em&gt; In vitro</em&gt; Modell av 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.

En Preklinisk Murine Modell av 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 av 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 ...

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 hjerneslag modell basert på Transient Cerebral Hypoksi-iskemi

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 tilnærming for Middle Cerebral arterien okklusjon og reperfusjon Induced 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 avbildning av Neuroinflammation bruke [11C] DPA-713 i en musemodell av iskemiske hjerneslag

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