Name: establishment of deep hypothermic circulatory arrest in rats
Uploaded: 2022-12-16T12:30:00
Description: Watch this Scientific Journal Video about Establishment of Deep Hypothermic Circulatory Arrest in Rats at JoVE.com

Forfatterne takker Liang Zhang for å bidra til å samle inn videodataene under eksperimentet. Denne studien ble støttet av National Natural Science Foundation of China (Grant nummer: 82070479) og Fundamental Research Funds for de sentrale universitetene (Grant nummer: 3332022128).

Protokollene gjennomgikk en institusjonell gjennomgang og mottok godkjenning fra Institutional Animal Care and Use Committee, Fuwai Hospital, Chinese Academy of Medical Sciences (FW-2021-0005). Alle eksperimentelle prosedyrer ble utført i samsvar med Veiledning for pleie og bruk av forsøksdyr utgitt av National Institutes of Health.
MERK: Hannrotter fra Sprague-Dawley (vekt: 500-600 g, alder: 12-14 uker) ble holdt under standard laboratorieforhold med fri tilgang til mat og vann. Rottene ble...

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C., Cao, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beneficial+effects+of+chlorogenic+acid+treatment+on+neuroinflammation+after+deep+hypothermic+circulatory+arrest+may+be+mediated+through+CYLD/NF-&#954;B+signaling.">Beneficial effects of chlorogenic acid treatment on neuroinflammation after deep hypothermic circulatory arrest may be mediated through CYLD/NF-&#954;B signaling.</a> Brain Research. 1767, 147572 (2021).</li><li>Li, Y. 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=Differential+expression+profiles+of+circular+RNAs+in+the+rat+hippocampus+after+deep+hypothermic+circulatory+arrest.">Differential expression profiles of circular RNAs in the rat hippocampus after deep hypothermic circulatory arrest.</a> Artificial Organs. 45 (8), 866-880 (2021).</li><li>Linardi, D., 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=Slow+versus+fast+rewarming+after+hypothermic+circulatory+arrest:+effects+on+neuroinflammation+and+cerebral+oedema.">Slow versus fast rewarming after hypothermic circulatory arrest: effects on neuroinflammation and cerebral oedema.</a> European Journal of Cardiothoracic Surgery. 58 (4), 792-780 (2020).</li><li>Engelman, 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=The+Society+of+Thoracic+Surgeons,+The+Society+of+Cardiovascular+Anesthesiologists,+and+The+American+Society+of+ExtraCorporeal+Technology:+Clinical+practice+guidelines+for+cardiopulmonary+bypass--Temperature+management+during+cardiopulmonary+bypass.">The Society of Thoracic Surgeons, The Society of Cardiovascular Anesthesiologists, and The American Society of ExtraCorporeal Technology: Clinical practice guidelines for cardiopulmonary bypass--Temperature management during cardiopulmonary bypass.</a> Annals of Thoracic Surgery. 100 (2), 748-757 (2015).</li><li>Jenke, 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=AdipoRon+attenuates+inflammation+and+impairment+of+cardiac+function+associated+with+cardiopulmonary+bypass-induced+systemic+inflammatory+response+syndrome.">AdipoRon attenuates inflammation and impairment of cardiac function associated with cardiopulmonary bypass-induced systemic inflammatory response syndrome.</a> Journal of the American Heart Association. 10 (6), 018097 (2021).</li></ol>

Kanylering er den mest grunnleggende prosedyren for å etablere DHCA hos rotter. Før kanylering vil bløtlegging av arterien med 0,5 ml 2% lidokain gjøre det lettere å kanylere. Etter kanylering er heparinisering med 500 IE/kg heparin via yttervena jugularis nødvendig for å unngå mikrotrombedannelse17. Vi har gjentatte ganger funnet ut at denne dosen heparin kan oppnå målet om en aktivert koagulasjonstid (ACT) >480 s. Gjenoppvarmingsperioden er den vanskeligste delen. Det tok mer ...

Dyp hypoterm sirkulasjonsstans (DHCA) har vært brukt i hjertekirurgi siden 19531. DHCA innebærer å redusere pasientens kjernetemperatur til dypt hypoterme nivåer (15-22 ° C) før det globalt avbryter blodstrømmen til kroppen2. Sirkulasjonsstansen kan gi et relativt blodløst operasjonsfelt. Dyp hypotermi reduserer stoffskiftet, spesielt i hjernen og myokardiet, som er en effektiv metode for beskyttelse mot iskemi3. DHCA brukes ofte under operasjoner for kompleks medfødt hjertesykdom, aortabuesykdom, og til og med nyre- eller binyretumorer med vena cava trombus 4,5. Derfor gir etablering av DHCA-dyremodeller en viktig referanse for forbedring av prosedyren og forebygging av komplikasjoner i kliniske omgivelser.
Selv om modeller kan etableres med hjørnetenner6, kaniner7 og andre dyr, er det å foretrekke å bruke rotter på grunn av deres brukbarhet og lave kostnader. DHCA-rottemodellen ble beskrevet for første gang i 2006 av Jungwirth et al.8. Det ble funnet at varigheten av sirkulasjonsstansen hadde innvirkning på de nevrologiske utfallene. Siden den gang har DHCA-rottemodeller blitt undersøkt bredt. Det er avklart at DHCA kan fremprovosere systemisk inflammatorisk responssyndrom (SIRS)9. I påfølgende studier fant farmakologer at DHCA-relatert nevroinflammasjon indusert av SIRS kunne dempes av resveratrol10 og triptolide11. Vårt team fant også at DHCA-relatert nevroinflammasjon kunne dempes ved å hemme det kaldinduserbare RNA-bindende proteinet12. I kardiovaskulærsystemet har superoksiddismutase en kardioprotektiv effekt på iskemi / reperfusjon (I / R) skader under DHCA13. Disse resultatene utvidet forståelsen av DHCA-relaterte patofysiologiske prosesser og tilbød nye retninger for å forbedre resultatene av DHCA. Resultatene angående endotoksemi, oksidativt stress og autofagi etter DHCA er imidlertid ufullstendige. DHCA bruker samme operasjonelle teknologi som kardiopulmonal bypass (CPB)14, men ledelsesstrategien er forskjellig, og trinnene for å generere DHCA varierer på tvers av ulike team 8,9,10,11. Denne studien tar sikte på å gi en metode for å etablere DHCA-prosedyren hos rotter.

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			<td>Heat Exchanger</td>
			<td>Xi&#8217;an Xijing Medical Appliance Co., Ltd</td>
			<td>Animal-M</td>
			<td></td>
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			<td>Membrane Oxygenator</td>
			<td>Dongguan Kewei Medical Instrument Co., Ltd.</td>
			<td>Micro-M</td>
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			<td>Chengdu Techman Co., Ltd</td>
			<td>BL-420s</td>
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			<td>Changzhou Prefluid Technology Co.,Ltd</td>
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			<td>HFK Bioscience Co.,Ltd.</td>
			<td>/</td>
			<td></td>
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			<td>Sevoflurane</td>
			<td>Maruishi Pharmaceutical Co. Ltd</td>
			<td>H20150020</td>
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			<td>Shaver</td>
			<td>Hangzhou Huayuan Pet Products Co.,Ltd.</td>
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			<td>SPACECABS</td>
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			<td>Ventilator</td>
			<td>Shanghai Alcott Biotech Co., Ltd</td>
			<td>ALC-V8S</td>
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			<td>Water Tank</td>
			<td>Maquet Critical Care AB</td>
			<td>Jostra HCU20-600</td>
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establishment of deep hypothermic circulatory arrest in rats

Dyp hypoterm sirkulasjonsstans (DHCA) brukes rutinemessig under operasjoner for kompleks medfødt hjertesykdom og aortabuesykdom. Denne studien tar sikte på å gi en metode for å etablere DHCA hos rotter. For å evaluere virkningen av DHCA-prosessen på vitale tegn, ble en normal temperatur kardiopulmonal bypass (CPB) rottemodell uten sirkulasjonsstans brukt som en kontroll. Som forventet førte DHCA til en signifikant reduksjon i kroppstemperatur og gjennomsnittlig arterielt blodtrykk. Blodgassanalysen indikerte at DHCA økte melkesyrenivået, men ikke påvirket blodets pH og konsentrasjonene av hemoglobin, hematokrit, Na +, Cl-, K + og glukose. Videre, sammenlignet med CPB-rotter med normal temperatur, viste resultatene av transmisjonselektronmikroskopi en mild økning i hippocampale autofagosomer hos DHCA-rotter.

Som kontrollgruppe viste rotter med normal temperatur CPB (NtCPB) uten sirkulasjonsstans stabilt gjennomsnittlig arterielt blodtrykk (MAP) og kroppstemperatur under hele prosedyren, mens MAP for DHCA-rottene gikk ned under hjertestansen (p < 0,01, figur 3A). Temperaturen til DHCA-rottene falt raskt i kjølefasen og gjenvunnet gradvis under oppvarmingsfasen. Ved avvenning av rottene fra DHCA-kretsene gikk temperaturen til DHCA-rottene tilbake til normal (figur 3B...

Watch this Scientific Journal Video about Establishment of Deep Hypothermic Circulatory Arrest in Rats at JoVE.com

Establishment of Deep Hypothermic Circulatory Arrest in Rats

Denne protokollen presenterer etablering av dyp hypoterm sirkulasjonsstans hos rotter, som kan brukes til å undersøke systemisk inflammatorisk responssyndrom, iskemi / reperfusjonsskade, oksidativt stress, nevroinflammasjon, etc.

Etablering av dyp hypoterm sirkulasjonsarrest hos rotter

Deep hypothermic circulatory arrest (DHCA) is routinely applied during surgeries for complex congenital heart disease and aortic arch disease. The present ...

Etablering av dyp hypoterm sirkulasjonsstans hos rotte. Dyp hypoterm sirkulasjonsstans, DHCA brukes rutinemessig under operasjoner av kompleks medfødt hjertesykdom og aortabuesykdom. Denne studien tar sikte på å gi en metode for å etablere DHCA hos rotter.Protokollene mottok en institusjonell gjennomgang og fikk godkjenning fra Institutional Animal Care and Use Committee, Fuwai Hospital, Chinese Academy of Medical Sciences. Forberedende arbeid. Sørg for at CPB-kretser inneholder et reservoar modifisert fra morphs dropper, rullepumpe, varmeveksler, membranoksygenator, tilkoblingsrør og en vanntank koblet kretsen og gjør 12 milliliter hydroksystivelse med en milliliter heparinnatrium og en milliliter saltvann prime kretsen med 40 milliliter grunningsløsning med rullepumpen forsiktig roterende.To, anestesi og kanylering. Koble røret med ventilatoren og sideparametrene. Sett et varmeteppe og høyre og vend mot høyre med tape.Påfør oftalmisk salve på øynene for å forhindre tørrhet. Barber håret på høyre inngangsregion, høyre livmorhalsregion og halen med barbermaskinen. Desinfiser deretter huden med aldehyd og alkohol.Klipp huden og venstre inngangsregion og disseker muskel- og vevsbløtbenet for å eksponere venstre lårben og arterie. Separat arterien forsiktig. Kanylert 22 gauge intravenøst kateter inn i venstre lårarterie.Ligert arterie og kateter med silke. Bruk saltvann som inneholder heparin til å skylle kanylen for å unngå koagulering. Koble kateteret til trykksensoren for å overvåke blodtrykket.Klipp huden på halen og bruk deretter en skalpell for å kutte den overfladiske arten av halearterien for å eksponere halearterien som ligger midt i det kirurgiske feltet. Kanylere halearterien med 22 gauge intravenøst kateter. Ligate arterie og kateter med silke.Bruk saltvann som inneholder heparin til å skylle kateteret for å unngå koagulering. Klipp huden på høyre jugular av venen, og skill deretter muskel og vev for å eksponere venen. Sett inn et 16 gauge hjemmelaget multiage intravenøst kateter i høyre ytre jugular av venen og legg den forsiktig inn i riktig dårligere vena cava eller høyre atrium.Administrer heparinnatrium hvor høyre ytre vene dekker hvert kanylert område med fuktige baller for å unngå forurensning. Tre, koble kretsen med kateteret i halearterien og hold pumpen lav hastighet en til to milliliter per minutt. Koble deretter reservoaret med kateteret i høyre ytre jugularvene.Pass på at det alltid er et blodnivå på omtrent en centimeter i reservoaret. Slå på vanntanken og sett vanntemperaturen på 37 celsius først. Etter at blodtrykket er stabilt, øk forsiktig strømningspumpen opp til 80 til 100 milliliter per minutt.Fire, kjøling. Sett romtemperaturen til rundt 20 celsius. Legg isbiter i disse engangshanskene og legg dem deretter på høyre hode og sider.Temperaturen på tanken ble foreslått til sanntid i henhold til rektaltemperaturen til risen. Endre de relevante parametrene til ventilatoren riktig i henhold til resultatene av blodgassanalysen. Fem, dyp hypotermi sirkulasjon arrestasjon.Deretter faller rektaltemperaturen til 15 til 20 celsius. Bytt ispose og kast hanskene for å sikre opprettholdelse av dyp hypotermi under sirkulasjonsstans. Stopp rullepumpen.Hold reservoaret i kontakt med miljøet og tøm blodet sakte fra den ytre jugulære venen til reservoaret. Vær oppmerksom på blodtrykksreformen. Etter at det ikke er blodtrykk og hjerteslag, stopp dreneringen og hold reservoaret lukket.Slå av respiratoren. Seks, oppvarming og reperfusjon. Fjern alle engangshanskene og øk romtemperaturen til 25 celsius.Gjenopprett minnet oksygen inter ventilasjon mens du holder venøse dreneringsrør klipping. Slå på rullepumpen for å sikre at blodet i reservoaret sakte er tilbake til riktig kropp. Slå på ventilatoren etter at blodnivået i reservoaret forblir en centimeter.Bruk dreneringsrøret og tøm blodet sakte fra høyre atrium til reservoaret. Slå på varmelampen, varmeputen og vanntanken. Sett temperaturen på vanntanken til 25 celsius.Juster den deretter til venstre temperatur i tide. Fjern varmelampen etter at rektaltemperaturen har gått tilbake til 34 celsius. Syv, avvenning av CPB.Sakte og gradvis redusere rullen av pumpens strømningshastighet og juster vena-slektens hastighet i strømningshastigheten reduseres til en milliliter per minutt. Hold reservoaret i kontakt med miljøet. Tilfør det gjenværende blodet i en krets med en strømningshastighet på en milliliter per minutt.Stopp minneoksygenering og rullepumpen. Avlivet etter en periode med mekanisk ventilasjon og dyp anestesi. Representative resultater.Som kontrollgruppe, normal temperatur CPB, NtCPB, rotte. Uten sirkulasjonsstansen viste man stabilt gjennomsnittlig blodtrykksblodtrykk MAP og kroppstemperatur under hele inngrepet. Mens MAP av DHCA-rotter ble redusert under hjertestansen.Temperaturen på DHCA-rotter falt raskt i kjølefasen og kom seg gradvis i løpet av den roterende fasen. Avvenning av DHCA-krets temperaturen på DHCA-rotter gikk tilbake til normal. Ovennevnte data indikerte at vår DHCA-prosedyre imiterte den faktiske temperaturen og MAP-endringene til pasienter i klinikker.Effekten av DHCA-prosessor og stigning ble undersøkt ved blodgassanalyse. Etter fullblodskontakten med den primære løsningen var konsentrasjonen av hemoglobin høyere enn seks gram per desilitter i begge grupper. Avvenningen fra DHCA-overflaten økte den til ni gram per desilitter på grunn av reinfundering av gjenværende blod i CPB-kretsen til høyre.Hematokriten, HCT, viste lignende tendens til HB. Etter DHCA-reperfusjon økte laktatsyren raskt, noe som var mer uttalt i DHCA-gruppen. pH ble redusert etter DHCA-prosedyren, som mest sannsynlig var et resultat av laktatsyreakkumulering. Under hele forsøket viste konsentrasjonen av natrium, klorid, kaliumioner og glukose ikke signifikante forskjeller ved hvert 10-punkt.Dette resultatet antyder at DHCA bare induserte økningen til laktatsyre, men påvirket ikke blodets pH og konsentrasjonen av hemoglobin, hematokrit, natriumioner, kloridioner, kaliumioner og glukose. For å evaluere virkningen av DHCA på nevronal autofagi brukte vi overføringselektromikroskopet og overraskende fant og økte antall autofagosomer i hippocampus av DHCA-rotter. Ovennevnte data indikerte at vår DHCA-modell imiterte faktiske temperatur- og MAP-endringer hos pasienter i klinikkene.Denne modellen kan brukes til å undersøke systemisk inflammatorisk responssyndrom, nevroinflammasjon, et cetera.

establishment-of-deep-hypothermic-circulatory-arrest-in-rats

Research

JoVE Journal

Immunology and Infection

Orthotopic Hind-Limb Transplantation in Rats

Composite tissue allotransplantation (CTA) now represents a valid therapeutic option after the loss of a hand, forearm or digits and has become a novel therapeutic entity in reconstructive surgery. However, long term high-dose multi-drug immunosuppressive therapy is required to ensure graft survival, bearing the risk of serious side effects which halters broader application. Further progression in this field may depend on better understanding of basic immunology and ischemia reperfusion injury in composite tissue grafts.
To date, orthotopic hind limb transplantation in rats has been the preferred rodent model for reconstructive transplantation (RT), however, it is an extremely demanding procedure that requires extraordinary microsurgical skills for reattachment of vasculature, bones, muscles and nerves.
We have introduced the vascular cuff anastomosis technique to this model, providing a rapid and reliable approach to rat hind limb transplantation. This technique simplifies and shortens the surgical procedure and enables surgeons with basic microsurgical experience to successfully perform the operation with high survival and low complication rates. The technique seems to be well suited for immunological as well as ischemia reperfusion injury (IRI) studies.

Here we describe the orthotopic rat hind-limb transplantation procedure, which seems to be the gold standard in vivo model for composite tissue allotransplantation research.

Composite tissue allotransplantation (CTA) now represents a valid therapeutic option after the loss of a hand, forearm or digits and has become a novel ...

Diagnosis of Ecto- and Endoparasites in Laboratory Rats and Mice

Internal and external parasites remain a significant concern in laboratory rodent facilities, and many research facilities harbor some parasitized animals. Before embarking on an examination of animals for parasites, two things should be considered. One: what use will be made of the information collected, and two: which test is the most appropriate. Knowing that animals are parasitized may be something that the facility accepts, but there is often a need to treat animals and then to determine the efficacy of treatment. Parasites may be detected in animals through various techniques, including samples taken from live or euthanized animals. Historically, the tests with the greatest diagnostic sensitivity required euthanasia of the animal, although PCR has allowed high-sensitivity testing for several types of parasite. This article demonstrates procedures for the detection of endo- and ectoparasites in mice and rats. The same procedures are applicable to other rodents, although the species of parasites found will differ.

This article describes various procedures for screening rats and mice to detect endo- or ectoparasitism. Several diagnostic assays will be demonstrated, both those suitable for use on live animals and those used after euthanasia of the animal. Photographs to aid in identification of rat and mouse parasites will be included.

Internal and external parasites remain a significant concern in laboratory rodent facilities, and many research facilities harbor some parasitized ...

Establishment of Epstein-Barr Virus Growth-transformed Lymphoblastoid Cell Lines

Infection of B cells with Epstein-Barr virus (EBV) leads to proliferation and subsequent immortalization, resulting in establishment of lymphoblastoid cell lines (LCL) in vitro. Since LCL are latently infected with EBV, they provide a model system to investigate EBV latency and virus-driven B cell proliferation and tumorigenesis1. LCL have been used to present antigens in a variety of immunologic assays2, 3. In addition, LCL can be used to generate human monoclonal antibodies4, 5 and provide a potentially unlimited source when access to primary biologic materials is limited6, 7.
A variety of methods have been described to generate LCL. Earlier methods have included the use of mitogens such as phytohemagglutinin, lipopolysaccharide8, and pokeweed mitogen9 to increase the efficiency of EBV-mediated immortalization. More recently, others have used immunosuppressive agents such as cyclosporin A to inhibit T cell-mediated killing of infected B cells7, 10-12.
The considerable length of time from EBV infection to establishment of cell lines drives the requirement for quicker and more reliable methods for EBV-driven B cell growth transformation. Using a combination of high titer EBV and an immunosuppressive agent, we are able to consistently infect, transform, and generate LCL from B cells in peripheral blood. This method uses a small amount of peripheral blood mononuclear cells that are infected in vitroclusters of cells can be demonstrated. The presence of CD23 with EBV in the presence of FK506, a T cell immunosuppressant. Traditionally, outgrowth of proliferating B cells is monitored by visualization of microscopic clusters of cells about a week after infection with EBV. Clumps of LCL can be seen by the naked eye after several weeks. We describe an assay to determine early if EBV-mediated growth transformation is successful even before microscopic clusters of cells can be demonstrated. The presence of CD23hiCD58+ cells observed as early as three days post-infection indicates a successful outcome.

We describe a method for generating transformed B cell lines using Epstein-Barr virus. We also illustrate a novel assay that can identify B cells destined to undergo transformation as early as three days after infection.

Etablering av Epstein-Barr Virus Vekst-forvandlet lymfoblastoid Cell Lines

Infection of B cells with Epstein-Barr virus (EBV) leads to proliferation and subsequent immortalization, resulting in establishment of lymphoblastoid ...

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

The current article outlines a protocol to establish an in vitro cell culture model system to study the interaction of a facultative intracellular human fungal pathogen Candida glabrata with human macrophages which will be a useful tool to advance our knowledge of fungal virulence mechanisms.

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

Complete Thymectomy in Adult Rats with Non-invasive Endotracheal Intubation

Thymectomy in neonatal rodents is an established and reliable procedure for immunological studies. However, in adult rats, complications of hemorrhage and pneumothorax from pleural disruption can result in a significant mortality rate. This protocol is a simple method of rat thymectomy that utilizes a mini-sternotomy and endotracheal intubation. Intubation is accomplished with a non-invasive and easily reproducible method and allows for positive pressure ventilation to prevent pneumothorax and a controlled airway that allows sufficient time for careful thymus dissection to minimize pleural disruption. A 1.5 cm sternal incision decreases contact with mediastinal vessels and pleura, while still providing full visualization of the thymus. Following exposure of the mediastinum, the thymus is removed by blunt dissection under magnification. The pleural space is then sealed by suture closure of the pre-tracheal muscles followed by the application of surgical glue. The thorax is then closed by suture closure of the sternum, followed by suture closure of the skin. All thymectomies were complete as evidenced by immunohistochemical (IHC) staining of mediastinal tissue, and absence of na&#239;ve T-cells by flow cytometry, and the procedure had a 96% survival rate. This method is suitable when complete thymectomy with minimal complications is desired for further immunological studies in athymic adult rats.

Rodent thymectomy is a valuable technique in immunological research. Here, a protocol for complete thymectomy in adult rats using a mini-sternotomy along with non-invasive intubation and positive pressure ventilation to minimize perioperative morbidity and mortality is described.

Komplett tymektomi hos voksne rotter med Non-invasiv endotrakeal intubering

Thymectomy in neonatal rodents is an established and reliable procedure for immunological studies. However, in adult rats, complications of hemorrhage and ...

Prevention of Heat Stress Adverse Effects in Rats by Bacillus subtilis Strain

This study was designed to evaluate the protective effect of the Bacillus subtilis strain against complications related to heat stress. Thirty-two Sprague-Dawley rats were used in this study. Animals were orally treated twice a day for two days with B. subtilis BSB3 strain or PBS. The next day after the last treatment, each group was divided and two experimental groups (one treated with PBS and one treated with B. subtilis) were placed at 45 oC for 25 min. Two control groups stayed for 25 min at room temperature. All rats were euthanized and different parameters were analyzed in all groups. Adverse effects of heat stress are registered by the decrease of villi height and total mucosal thickness in the intestinal epithelium; translocation of bacteria from the lumen; increased vesiculation of erythrocytes and elevation of the lipopolysaccharides (LPS) level in the blood. The protective efficacy of treatment is evaluated by prevention of these side effects. The protocol was set up for the oral treatment of rats with bacteria for prevention of heat stress complications, but this protocol can be modified and used for other routes of administration and for analysis of different compounds.

Prevention of Heat Stress Adverse Effects in Rats by Bacillus subtilis Strain

We described a protocol for prevention of heat stress effects in rats by oral pre-treatment with beneficial bacteria. This protocol can be modified and used for various routes of administration and for analysis of different compounds.

Forebygging av varmestress bivirkninger hos rotter etter<em&gt; Bacillus subtilis</em&gt; Strain

This study was designed to evaluate the protective effect of the Bacillus subtilis strain against complications related to heat stress. Thirty-two ...

Stenosis of the Inferior Vena Cava: A Murine Model of Deep Vein Thrombosis

Deep vein thrombosis (DVT) and its devastating complication, pulmonary embolism, are a severe health problem with high mortality. Mechanisms of thrombus formation in veins remain obscure. Lack of mobility (e.g., after surgery or long-haul flights) is one of the main factors leading to DVT. The pathophysiological consequence of the lack of mobility is blood flow stagnation in venous valves. Here, a model is described that mimics such flow disturbance as a thrombosis-driving factor. In this model, partial flow restriction (stenosis) in the inferior vena cava (IVC) is created. Closure of about 90% of the IVC lumen for 48 h results in development of thrombi structurally similar to those in humans. The similarities are: i) most of the thrombus volume is red, i.e., consists of red blood cells and fibrin, ii) presence of a white part (lines of Zahn), iii) non-denuded endothelial monolayer, iv) elevated plasma D-Dimer levels, and v) possibility to prevent thrombosis by low molecular weight heparin. Limitations include variable size of thrombi and the fact that a certain percentage of wild-type mice (0 - 35%) may not produce a thrombus. In addition to visual observation and measurement, thrombi may be visualized by non-invasive technologies, such as ultrasonography, which allows for monitoring the dynamics of thrombus development. At shorter time points (1 - 6 h), intravital microscopy may be applied to directly observe events (e.g., recruitment of cells to the vessel wall) preceding thrombus formation. Use of this method by several teams around the world has made it possible to uncover basic mechanisms of DVT initiation and identify potential targets that might be beneficial for its prevention.

We describe here stenosis in the inferior vena cava as a murine model of deep vein thrombosis. This model recapitulates blood flow restriction, one of the major triggers of venous thrombosis in humans.

Stenose dårligere Vena cava: en Murine modell av dyp venetrombose

Deep vein thrombosis (DVT) and its devastating complication, pulmonary embolism, are a severe health problem with high mortality. Mechanisms of thrombus ...

Deep Vein Thrombosis Induced by Stasis in Mice Monitored by High Frequency Ultrasonography

Venous thrombosis is a common condition affecting 1 - 2% of the population, with an annual incidence of 1 in 500. Venous thrombosis can lead to death through pulmonary embolism or results in the post-thrombotic syndrome, characterized by chronic leg pain, swelling, and ulceration, or in chronic pulmonary hypertension resulting in significant chronic respiratory compromise. This is the most common cardiovascular disease after myocardial infarction and ischemic stroke and is a clinical challenge for all medical disciplines, as it can complicate the course of other disorders such as cancer, systemic disease, surgery, and major trauma.
Experimental models are necessary to study these mechanisms. The stasis model induces consistent thrombus size and a quantifiable amount of thrombus. However, it is necessary to systematically ligate side branches of the inferior vena cava to avoid variability in thrombus sizes and any erroneous data interpretation. We have developed a non-invasive technique to measure thrombus size using ultrasonography. Using this technique, we can assess thrombus development and resolution over time in the same animal. This approach limits the number of mice required for quantification of venous thrombosis consistent with the principle of replacement, reduction, and refinement of animals in research. We have demonstrated that thrombus weight and histological analysis of thrombus size correlate with measurement obtained with ultrasonography. Therefore, the current study describes how to induce deep vein thrombosis in mice using the inferior vena cava stasis model and how to monitor it using high frequency ultrasound.

The present protocol describes the steps to obtain venous thrombosis using a stasis model. In addition, we are using a non-invasive method to measure thrombus formation and resolution over time.

Dyp venetrombose indusert av Stasis i mus overvåket av høyfrekvente Ultrasonography

Venous thrombosis is a common condition affecting 1 - 2% of the population, with an annual incidence of 1 in 500. Venous thrombosis can lead to death ...

Deep Dermal Injection As a Model of Candida albicans Skin Infection for Histological Analyses

The skin is an extremely extended organ of the body and, due to this large surface, it is continuously exposed to microorganisms. Skin damage can easily lead to infections in the dermis which can, in turn, result in the dissemination of pathogens into the bloodstream. Understanding how the immune system fights infections at the very early stage and how the host can eliminate the pathogens is an important step to set the base for future therapeutic interventions. Here we describe a model of Candida albicans infection that can visualize the processes that occur early during an infection, including when the pathogen has passed the epithelial barrier, as well as the immune response elicited by the C. albicans invasion. We used this infection model to perform histological analyses that show the immune cells that infiltrate the skin as well as the presence and localization of the pathogen. Samples collected after the infection can be processed for RNA extraction.

Deep Dermal Injection As a Model of Candida albicans Skin Infection for Histological Analyses

Here we describe a protocol that allows histological and molecular analysis of skin samples after Candida albicans intradermal injection. This protocol maintains the structural integrity of the skin and allows for the localization of tissue-resident or newly recruited immune cells as well as the pathogen distribution.

Dyp Dermal injeksjon som en modell av Candida albicans hudinfeksjon for histologiske analyser

The skin is an extremely extended organ of the body and, due to this large surface, it is continuously exposed to microorganisms. Skin damage can easily ...

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our knowledge of prevention and treatment of viral infection. The fruit fly Drosophila melanogaster has proven to be one of the most efficient and productive model organisms to screen for antiviral factors and investigate virus-host interaction, due to powerful genetic tools and highly conserved innate immune signaling pathways. The procedure described here demonstrates a nano-injection method to establish viral infection and induce systemic antiviral responses in adult flies. The precise control of the viral injection dose in this method enables high experimental reproducibility. Protocols described in this study include the preparation of flies and the virus, the injection method, survival rate analysis, the virus load measurement, and an antiviral pathway assessment. The influence effects of viral infection by the flies' background were mentioned here. This infection method is easy to perform and quantitatively repeatable; it can be applied to screen for host/viral factors involved in virus-host interaction and to dissect the crosstalk between innate immune signaling and other biological pathways in response to viral infection.

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

This protocol describes how to establish viral infection in vivo in Drosophila melanogaster using the nano-injection method and basic techniques to analyze virus-host interaction.

Etablering av Viral infeksjon og analyse av Host-Virus samhandling i Drosophila Melanogaster

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our ...