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 at hjælpe med at indsamle videodataene under eksperimentet. Denne undersøgelse blev støttet af National Natural Science Foundation of China (Grant nummer: 82070479) og Fundamental Research Funds for Central Universities (Grant nummer: 3332022128).

Protokollerne gennemgik en institutionel gennemgang og modtog godkendelse fra Institutional Animal Care and Use Committee, Fuwai Hospital, Chinese Academy of Medical Sciences (FW-2021-0005). Alle forsøgsprocedurer blev udført i overensstemmelse med vejledningen til pleje og brug af forsøgsdyr udgivet af National Institutes of Health.
BEMÆRK: Mandlige Sprague-Dawley rotter (vægt: 500-600 g, alder: 12-14 uger) blev holdt under standard laboratorieforhold med fri adgang til mad og vand. Rott...

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(133), e56986 (2018).</li><li>Ha, J. Y., Kim, J. S., Kim, S. E., Son, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+activation+of+mitophagy+and+autophagy+by+staurosporine+protects+against+dopaminergic+neuronal+cell+death.">Simultaneous activation of mitophagy and autophagy by staurosporine protects against dopaminergic neuronal cell death.</a> Neuroscience Letters. 561, 101-106 (2014).</li><li>Yamamoto, A., Yue, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+and+its+normal+and+pathogenic+states+in+the+brain.">Autophagy and its normal and pathogenic states in the brain.</a> Annual Review of Neuroscience. 37, 55-78 (2014).</li><li>You, X. 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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>

Cannulation er den mest grundlæggende procedure for etablering af DHCA hos rotter. Før kannulation vil blødning af arterien med 0,5 ml 2% lidokain gøre det lettere at kannulate. Efter kannikering er heparinisering med 500 IE / kg heparin via den ydre jugularvene nødvendig for at undgå dannelse af mikrotrombose17. Vi har gentagne gange fundet ud af, at denne dosis heparin kan nå målet om en aktiveret koagulationstid (ACT) >480 s. Genopvarmningsperioden er den sværeste del. Det tog...

Dyb hypotermisk kredsløbsstop (DHCA) er blevet brugt i hjertekirurgi siden 19531. DHCA indebærer at reducere patientens kernetemperatur til dybt hypotermiske niveauer (15-22 ° C), før den globalt afbryder blodgennemstrømningen til kroppen2. Kredsløbssikringen kan give et relativt ublodigt operationsfelt. Dyb hypotermi nedsætter metabolismen, især i hjernen og myokardiet, hvilket er en effektiv metode til beskyttelse mod iskæmi3. DHCA er almindeligt anvendt under operationer for komplekse medfødte hjertesygdomme, aorta bue sygdom, og endda nyre- eller binyretumorer med en vena cava thrombus 4,5. Derfor giver etablering af DHCA-dyremodeller en vigtig reference til forbedring af proceduren og forebyggelse af komplikationer i kliniske omgivelser.
Selvom modeller kan etableres med hjørnetænder6, kaniner7 og andre dyr, foretrækkes det at bruge rotter på grund af deres funktionsdygtighed og lave omkostninger. DHCA-rottemodellen blev beskrevet for første gang i 2006 af Jungwirth et al.8. Det blev konstateret, at varigheden af kredsløbsstop havde indflydelse på de neurologiske resultater. Siden da er DHCA-rottemodeller blevet undersøgt bredt. Det er blevet præciseret, at DHCA kunne fremkalde systemisk inflammatorisk responssyndrom (SIRS)9. I efterfølgende undersøgelser fandt farmakologer, at DHCA-relateret neuroinflammation induceret af SIRS kunne dæmpes af resveratrol10 og triptolid11. Vores team fandt også, at DHCA-relateret neuroinflammation kunne dæmpes ved at hæmme det koldt inducerbare RNA-bindende protein12. I det kardiovaskulære system har superoxiddismutase en kardiobeskyttende virkning på iskæmi / reperfusion (I / R) skader under DHCA13. Disse resultater udvidede forståelsen af DHCA-relaterede patofysiologiske processer og tilbød nye retninger til forbedring af resultaterne af DHCA. Men, resultaterne vedrørende endotoxæmi, oxidativ stress, og autofagi efter DHCA er ufattelige. DHCA bruger den samme operationelle teknologi som kardiopulmonal bypass (CPB)14, men dens ledelsesstrategi er anderledes, og trinene til at generere DHCA varierer på tværs af forskellige hold 8,9,10,11. Denne undersøgelse har til formål at tilvejebringe en metode til at fastslå DHCA-proceduren hos rotter.

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			<td>Xi&#8217;an Xijing Medical Appliance Co., Ltd</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>HFK Bioscience Co.,Ltd.</td>
			<td>/</td>
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			<td>Sevoflurane</td>
			<td>Maruishi Pharmaceutical Co. Ltd</td>
			<td>H20150020</td>
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			<td>Ventilator</td>
			<td>Shanghai Alcott Biotech Co., Ltd</td>
			<td>ALC-V8S</td>
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establishment of deep hypothermic circulatory arrest in rats

Dyb hypotermisk kredsløbsstop (DHCA) anvendes rutinemæssigt under operationer for kompleks medfødt hjertesygdom og aortabuesygdom. Denne undersøgelse har til formål at tilvejebringe en metode til etablering af DHCA hos rotter. For at evaluere virkningen af DHCA-processen på vitale tegn blev en normal temperatur kardiopulmonal bypass (CPB) rottemodel uden kredsløbsstop brugt som kontrol. Som forventet førte DHCA til et signifikant fald i kropstemperaturen og det gennemsnitlige arterielle blodtryk. Blodgasanalysen viste, at DHCA øgede mælkesyreniveauet, men ikke påvirkede blodets pH og koncentrationerne af hæmoglobin, hæmatokrit, Na +, Cl-, K + og glucose. Sammenlignet med CPB-rotter ved normal temperatur viste resultaterne af transmissionselektronmikroskopien desuden en mild stigning i hippocampale autofagosomer hos DHCA-rotterne.

Som kontrolgruppe viste den normale temperatur CPB (NtCPB) rotter uden kredsløbsstop et stabilt gennemsnitligt arterielt blodtryk (MAP) og kropstemperatur under hele proceduren, mens MAP for DHCA-rotterne faldt under hjertestoppet (p < 0,01, figur 3A). Temperaturen på DHCA-rotterne faldt hurtigt i kølefasen og genvandt gradvist under genopvarmningsfasen. Ved fravænning af rotterne fra DHCA-kredsløbene vendte temperaturen på DHCA-rotterne tilbage til normal (<strong class="xfig...

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 protokol præsenterer etableringen af dyb hypotermisk kredsløbsstop hos rotter, som kan anvendes til at undersøge systemisk inflammatorisk responssyndrom, iskæmi / reperfusionsskade, oxidativ stress, neuroinflammation osv.

Etablering af dyb hypotermisk kredsløbsstop hos rotter

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

Etablering af dyb hypotermisk kredsløbsstop hos rotter. Dyb hypotermisk kredsløbsstop, DHCA anvendes rutinemæssigt under operationer af kompleks medfødt hjertesygdom og aortabusygdom. Denne undersøgelse har til formål at tilvejebringe en metode til etablering af DHCA hos rotter.Protokollerne modtog en institutionel gennemgang og fik godkendelse fra Institutional Animal Care and Use Committee, Fuwai Hospital, Chinese Academy of Medical Sciences. Forberedende arbejde. Sørg for, at CPB-kredsløb indeholder et reservoir, der er modificeret fra morphs-dropper, rullepumpe, varmeveksler, membranoxygenator, forbindelsesrør og en vandtank, der er forbundet kredsløbet og gør 12 milliliter hydroxystivelse med en milliliter heparinnatrium og en milliliter saltvand prime kredsløbet med 40 milliliter primingopløsning med rullepumpen forsigtigt roterende.To, anæstesi og kannulation. Tilslut røret med ventilatoren og sideparametrene. Sæt et elektrisk varmetæppe og det højre og vend til højre med tape.Påfør oftalmisk salve på øjnene for at forhindre tørhed. Barber håret på højre lyskeområde, højre cervikale region og halen med barbermaskinen. Desinficer derefter huden med aldehyd og alkohol.Skær huden og den venstre inguinale region og disseker muskel- og vævets bløde knogle for at udsætte venstre lårbensvene og arterie. Adskil arterien omhyggeligt. Kanylat 22 gauge intravenøst kateter i venstre lårbensarterie.Ligated arterie og kateter med silke. Brug saltvand indeholdende heparin til at skylle kanylen for at undgå koagulering. Tilslut kateteret med pressesensoren for at overvåge blodtrykket.Skær halehuden og brug derefter en skalpel til at skære halearteriens overfladiske specia for at udsætte halearterien, som er midt i det kirurgiske felt. Kanylat halearterien med 22 gauge intravenøst kateter. Ligate arterie og kateter med silke.Brug saltvand indeholdende heparin til at skylle kateteret for at undgå koagulering. Skær huden på den højre halsvene og adskil derefter muskel og væv for at udsætte venen. Indsæt et 16 gauge hjemmelavet multiage intravenøst kateter i den højre ydre vene og læg det forsigtigt i den højre ringere vena cava eller det højre atrium.Administrer heparinnatrium, hvor den højre ydre vene dækker hvert kanyleret område med fugtige kugler for at undgå forurening. Tre, tilslut kredsløbet med kateteret i halearterien og hold pumpen lav hastighed en til to milliliter pr. Minut. Tilslut derefter reservoiret med kateteret i højre ydre jugular vene.Sørg for, at der altid er et blodniveau på cirka en centimeter i reservoiret. Tænd for vandtanken og indstil vandtemperaturen til 37 celsius først. Når blodtrykket er stabilt, skal du forsigtigt øge strømningspumpen op til 80 til 100 ml pr. Minut.Fire, køling. Indstil stuetemperaturen til omkring 20 celsius. Sæt isterninger i disse engangshandsker og læg dem derefter på højre hoved og sider.Tankens temperatur blev foreslået til realtid i henhold til risens rektale temperatur. Skift de relevante parametre for ventilatoren korrekt i henhold til resultaterne af blodgasanalysen. Fem, dyb hypotermi kredsløbssikring.Derefter falder den rektale temperatur til 15 til 20. celsius. Skift ispakken og bortskaf handskerne for at sikre opretholdelse af dyb hypotermi under kredsløbsstop. Stop rullepumpen.Hold reservoiret i kontakt med miljøet og dræn blodet langsomt fra den ydre jugular vene til reservoiret. Vær opmærksom på blodtryksreformen. Når der ikke er blodtryk og hjerteslag, skal du stoppe dræningen og holde reservoiret lukket.Sluk for respiratoren. Seks, opvarmning og reperfusion. Fjern alle engangshandsker og øg stuetemperaturen til 25 celsius.Gendan hukommelsens iltinterventilation, mens du holder det venøse drænrør klippet. Tænd for rullepumpen for at sikre, at blodet i reservoiret langsomt er tilbage til den rigtige krop. Tænd ventilatoren, efter at blodniveauet i reservoiret forbliver en centimeter.Brug drænrøret og dræn blodet langsomt fra højre atrium til reservoiret. Tænd for varmelampen, varmepuden og vandtanken. Indstil temperaturen på vandtanken til 25 celsius.Juster den derefter til venstre temperatur rettidigt. Fjern varmelampen, når den rektale temperatur vender tilbage til 34 celsius. Syv, fravænning af CPB.Reducer langsomt og gradvist pumpens strømningshastighed og juster vena-slægtens hastighed i strømningshastigheden reduceres til en milliliter pr. Minut. Hold reservoiret i kontakt med miljøet. Tilsæt det resterende blod i et kredsløb med en strømningshastighed på en milliliter pr. Minut.Stop hukommelsesoxygeneringen og rullepumpen. Aflivet efter en periode med mekanisk ventilation og en dyb anæstesi. Repræsentative resultater.Som kontrolgruppe, den normale temperatur CPB, NtCPB, rotte. Uden kredsløbsstop viste en stabil gennemsnitlig arterieblodtryk MAP og kropstemperatur under hele proceduren. Mens MAP af DHCA rotter faldt under hjertestop.Temperaturen på DHCA-rotter faldt hurtigt i kølefasen og genvandt gradvist i den roterende fase. Fravænning af DHCA kredsløb temperaturen på DHCA rotter vendte tilbage til normal. Ovenstående data viste, at vores DHCA-procedure efterlignede de faktiske temperatur- og MAP-ændringer hos patienter i klinikker.Effekten af DHCA-processor og stigning blev undersøgt ved blodgasanalyse. Efter fuldblodskontakt med den primære opløsning var koncentrationen af hæmoglobin højere end seks gram pr. Decilitter i begge grupper. Fravænningen af DHCA-overfladen steg til ni gram pr. decitlitter på grund af geninfusion af resterende blod i CPB-kredsløbet til højre.Hæmatokrit, HCT, viste den samme tendens til HB. Efter DHCA reperfusion steg lactatsyre hurtigt, hvilket var mere udtalt i DHCA-gruppen. pH faldt efter DHCA-proceduren, hvilket sandsynligvis var resultatet af akkumulering af mælkesyre. Under hele eksperimentet viste koncentrationen af natrium, chlorid, kaliumioner og glucose ikke signifikante forskelle ved hvert 10 punkt.Dette resultat tyder på, at DHCA kun inducerede den øgede til laktatsyre, men ikke påvirkede blodets pH og koncentrationen af hæmoglobin, hæmatokrit, natriumioner, chloridioner, kaliumioner og glucose. For at evaluere virkningen af DHCA på neuronal autofagi brugte vi transmissionselektromikroskopet og overraskende fandt og øgede antallet af autofagosomer i hippocampus hos DHCA-rotter. Ovenstående data viste, at vores DHCA-model efterlignede faktiske temperatur- og MAP-ændringer hos patienter i klinikkerne.Denne model kan anvendes til at undersøge systemisk inflammatorisk responssyndrom, neuroinflammation osv.

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 af Epstein-Barr virus Vækst-transformerede lymfoblastoide cellelinjer

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.

Komplet thymektomi i voksne rotter med Non-invasiv endotrakeal intubation

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.

Forebyggelse af varmestress bivirkninger i rotter ved<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 af den ringere Vena Cava: en Murine Model for dyb venøs trombose

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.

Dyb venøs trombose induceret af Stasis i mus overvåges af høj frekvens ultralyd

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.

Dyb Dermal injektion som en Model af Candida albicans hudinfektion histologisk 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 af Viral infektion og analyse af Host-Virus interaktion 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 ...