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

Författarna tackar Liang Zhang för att ha hjälpt till att samla in videodata under experimentet. Denna studie stöddes av National Natural Science Foundation of China (bidragsnummer: 82070479) och grundforskningsfonderna för de centrala universiteten (bidragsnummer: 3332022128).

Protokollen genomgick en institutionell granskning och fick godkännande från Institutional Animal Care and Use Committee, Fuwai Hospital, Chinese Academy of Medical Sciences (FW-2021-0005). Alla experimentella procedurer utfördes i enlighet med Guide for the Care and Use of Laboratory Animals som publicerades av National Institutes of Health.
OBS: Manliga Sprague-Dawley-råttor (vikt: 500-600 g, ålder: 12-14 veckor) hölls under normala laboratorieförhållanden med fri tillgång till mat ...

<ol>
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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=Triptolide+improves+neurobehavioral+functions,+inflammation,+and+oxidative+stress+in+rats+under+deep+hypothermic+circulatory+arrest.">Triptolide improves neurobehavioral functions, inflammation, and oxidative stress in rats under deep hypothermic circulatory arrest.</a> Aging. 13 (2), 3031-3044 (2021).</li><li>Liu, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+target+to+reduce+microglial+inflammation+and+neuronal+damage+after+deep+hypothermic+circulatory+arrest.">A novel target to reduce microglial inflammation and neuronal damage after deep hypothermic circulatory arrest.</a> Journal of Thoracic and Cardiovascular Surgery. 159 (6), 2431-2444 (2020).</li><li>Pinto, 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=The+extracellular+isoform+of+superoxide+dismutase+has+a+significant+impact+on+cardiovascular+ischaemia+and+reperfusion+injury+during+cardiopulmonary+bypass.">The extracellular isoform of superoxide dismutase has a significant impact on cardiovascular ischaemia and reperfusion injury during cardiopulmonary bypass.</a> European Journal of Cardio-Thoracic Surgery. 50 (6), 1035-1044 (2016).</li><li>Hirao, S., Masumoto, H., Itonaga, T., Minatoya, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+recovery+cardiopulmonary+bypass+model+without+transfusion+or+inotropic+agents+in+rats.">A recovery cardiopulmonary bypass model without transfusion or inotropic agents in rats.</a> Journal of Visualized Experiments. (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. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+cardiopulmonary+bypass+model:+Application+of+a+miniature+extracorporeal+circuit+composed+of+asanguinous+prime.">Rat cardiopulmonary bypass model: Application of a miniature extracorporeal circuit composed of asanguinous prime.</a> Journal of Extra-Corporeal Technology. 37 (1), 60-65 (2005).</li><li>Chen, Q., Lei, Y. Q., Liu, J. F., Wang, Z. 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 är det mest grundläggande förfarandet för att etablera DHCA hos råttor. Före cannulation kommer blötläggning av artären med 0,5 ml 2% lidokain att göra det lättare att kannulatera. Efter cannulation är heparinisering med 500 IE/kg heparin via den yttre halsvenen nödvändig för att undvika mikrotrombbildning17. Vi har upprepade gånger funnit att denna dos heparin kan uppnå målet om en aktiverad koagulationstid (ACT) >480 s. Omvärmningsperioden är den svårast...

Djup hypotermisk cirkulationsstillestånd (DHCA) har använts vid hjärtkirurgi sedan 19531. DHCA innebär att patientens kärntemperatur sänks till djupt hypotermiska nivåer (15-22 °C) innan blodflödet till kroppen avbryts globalt2. Cirkulationsstoppet kan ge ett relativt oblodigt operationsfält. Djup hypotermi minskar ämnesomsättningen, särskilt i hjärnan och myokardiet, vilket är en effektiv metod för skydd mot ischemi3. DHCA används vanligtvis under operationer för komplex medfödd hjärtsjukdom, aortabågssjukdom och till och med njur- eller binjuretumörer med en vena cava trombus 4,5. Därför ger upprättandet av DHCA-djurmodeller en viktig referens för förfining av proceduren och förebyggande av komplikationer i kliniska miljöer.
Även om modeller kan etableras med hundar6, kaniner7 och andra djur, är det att föredra att använda råttor på grund av deras användbarhet och låga kostnader. DHCA-råttmodellen beskrevs för första gången 2006 av Jungwirth et al.8. Det visade sig att varaktigheten av cirkulationsstoppet hade en inverkan på de neurologiska resultaten. Sedan dess har DHCA-råttmodeller undersökts brett. Det har klargjorts att DHCA kan provocera systemiskt inflammatoriskt responssyndrom (SIRS)9. I efterföljande studier, farmakologer fann att DHCA-relaterade neuroinflammation inducerad av SIRS kunde dämpas av resveratrol10 och triptolid11. Vårt team fann också att DHCA-relaterad neuroinflammation kunde dämpas genom att hämma det kallinducerbara RNA-bindande proteinet12. I hjärt-kärlsystemet har superoxiddismutas en hjärtskyddande effekt på ischemi/reperfusionsskador (I/R) under DHCA13. Dessa resultat utvidgade förståelsen för DHCA-relaterade patofysiologiska processer och erbjöd nya riktningar för att förbättra resultaten av DHCA. Emellertid, resultaten om endotoximi, oxidativ stress, och autofagi efter DHCA är ofullständiga. DHCA använder samma operativa teknik som hjärt-lung-bypass (CPB)14, men dess hanteringsstrategi är annorlunda och stegen för att generera DHCA skiljer sig åt mellan olika team 8,9,10,11. Denna studie syftar till att tillhandahålla en metod för att fastställa DHCA-förfarandet hos råttor.

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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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		<tr>
			<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>
			<td>BL100</td>
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			<td>SD Rat</td>
			<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>Shaver</td>
			<td>Hangzhou Huayuan Pet Products Co.,Ltd.</td>
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			<td>SPACECABS</td>
			<td>/</td>
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			<td>Ventilator</td>
			<td>Shanghai Alcott Biotech Co., Ltd</td>
			<td>ALC-V8S</td>
			<td></td>
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		<tr>
			<td>Water Tank</td>
			<td>Maquet Critical Care AB</td>
			<td>Jostra HCU20-600</td>
			<td></td>
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establishment of deep hypothermic circulatory arrest in rats

Djup hypotermisk cirkulationsstopp (DHCA) appliceras rutinmässigt under operationer för komplex medfödd hjärtsjukdom och aortabågsjukdom. Denna studie syftar till att tillhandahålla en metod för att etablera DHCA hos råttor. För att utvärdera effekten av DHCA-processen på vitala tecken användes en normal temperatur kardiopulmonell bypass (CPB) råttmodell utan cirkulationsstopp som en kontroll. Som förväntat ledde DHCA till en signifikant minskning av kroppstemperaturen och det genomsnittliga arteriella blodtrycket. Blodgasanalysen indikerade att DHCA ökade mjölksyranivåerna men inte påverkade blodets pH och koncentrationerna av hemoglobin, hematokrit, Na+, Cl−, K+ och glukos. Dessutom, jämfört med CPB-råttor med normal temperatur, visade resultaten av transmissionselektronmikroskopin en mild ökning av hippocampus autofagosomer hos DHCA-råttorna.

Som kontrollgrupp visade normaltemperaturen CPB (NtCPB) råttor utan cirkulationsstopp ett stabilt genomsnittligt arteriellt blodtryck (MAP) och kroppstemperatur under hela proceduren, medan MAP för DHCA-råttorna minskade under hjärtstoppet (p < 0,01, figur 3A). Temperaturen hos DHCA-råttorna sjönk snabbt under kylningsfasen och återhämtade sig gradvis under uppvärmningsfasen. Vid avvänjning av råttorna från DHCA-kretsarna återgick temperaturen hos DHCA-råttorna till de...

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

Detta protokoll presenterar upprättandet av djup hypotermisk cirkulationsstopp hos råttor, som kan tillämpas för att undersöka systemiskt inflammatoriskt responssyndrom, ischemi / reperfusionsskada, oxidativ stress, neuroinflammation etc.

Etablering av djup hypotermisk cirkulationsarrest hos råttor

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

Etablering av djup hypotermisk cirkulationsarrest hos råtta. Djup hypotermisk cirkulationsstopp, DHCA appliceras rutinmässigt under operationer av komplex medfödd hjärtsjukdom och aortabågsjukdom. Denna studie syftar till att tillhandahålla en metod för att etablera DHCA hos råttor.Protokollen fick en institutionell granskning och fick godkännande från Institutional Animal Care and Use Committee, Fuwai Hospital, Chinese Academy of Medical Sciences. Förberedelsearbete. Se till att CPB-kretsar innehåller en behållare modifierad från morfdroppare, rullpump, värmeväxlare, membransyresättare, anslutningsrör och en vattentank ansluten kretsen och gör 12 ml hydroxistärkelse med en milliliter heparinnatrium och en milliliter saltlösning primera kretsen med 40 ml grundlösning med rullpumpen försiktigt roterande.Två, anestesi och cannulation. Anslut röret med ventilatorn och sidoparametrarna. Sätt en elektrisk värmefilt och höger och vänd åt höger med tejp.Applicera oftalmisk salva på ögonen för att förhindra torrhet. Raka håret på höger inguinalregion, höger livmoderhalsregion och svansen med rakapparaten. Desinficera sedan huden med aldehyd och alkohol.Skär huden och den vänstra inguinalregionen och dissekera muskel- och vävnadsmjuka benet för att exponera vänster lårbens ven och artären. Separera artären försiktigt. Cannulate 22 gauge intravenös kateter i vänster lårbensartär.Ligerad artär och kateter med siden. Använd saltlösning som innehåller heparin för att spola kanylen för att undvika koagulering. Anslut katetern med presssensorn för att övervaka blodtrycket.Skär svansens hud och använd sedan en skalpell för att skära den ytliga specia i svansartären för att exponera svansartären som ligger mitt i det kirurgiska fältet. Cannulate svansartären med 22 gauge intravenös kateter. Ligatartär och kateter med siden.Använd saltlösning som innehåller heparin för att spola katetern för att undvika koagulering. Skär huden på höger hals i venen och separera sedan muskler och vävnad för att exponera venen. Sätt in en 16 gauge hemlagad multiage intravenös kateter i den högra yttre halsen av venen och lägg den försiktigt i rätt underlägsen vena cava eller höger atrium.Administrera heparinnatrium där den högra yttre venen täcker varje kannulerad region med fuktiga bollar för att undvika kontaminering. Tre, anslut kretsen med katetern i svansartären och håll pumpen låg hastighet en till två milliliter per minut. Anslut sedan behållaren med katetern i den högra yttre halsvenen.Se till att det alltid finns en blodnivå på cirka en centimeter i behållaren. Slå på vattentanken och ställ in vattentemperaturen på 37 celsius först. När blodtrycket är stabilt, öka försiktigt flödespumpen upp till 80 till 100 ml per minut.Fyra, kylning. Ställ in rumstemperaturen på cirka 20 celsius. Lägg isbitar i dessa engångshandskar och lägg dem sedan på höger huvud och sidor.Tankens temperatur föreslogs i realtid enligt risets rektala temperatur. Ändra de relevanta parametrarna för ventilatorn ordentligt enligt resultaten av blodgasanalysen. Fem, djup hypotermi cirkulationsstopp.Sedan sjunker rektaltemperaturen till 15 till 20: e Celsius. Byt ispaketet och kassera handskarna för att säkerställa underhåll av djup hypotermi under cirkulationsstopp. Stoppa rullpumpen.Håll behållaren i kontakt med miljön och dränera blodet långsamt från den yttre halsvenen till behållaren. Var uppmärksam på blodtrycksreformen. När det inte finns något blodtryck och hjärtslag, stoppa dräneringen och håll behållaren stängd.Stäng av ventilatorn. Sex, uppvärmning och reperfusion. Ta bort alla engångshandskar och öka rumstemperaturen till 25 grader.Återställ minnets syreventilation samtidigt som du håller det venösa dräneringsröret klippt. Slå på rullpumpen för att se till att blodet i behållaren långsamt är tillbaka till rätt kropp. Slå på ventilatorn efter att blodnivån i behållaren förblir en centimeter.Använd dräneringsröret och dränera blodet från det högra atriumet till behållaren långsamt. Slå på värmelampan, värmedynan och vattentanken. Ställ in temperaturen på vattentanken till 25 Celsius.Justera den sedan till vänster temperatur i rätt tid. Ta bort värmelampan efter att rektaltemperaturen återgått till 34 celsius. Sju, avvänjning av CPB.Sakta och gradvis minska rullen av pumpflödeshastigheten och justera vena genushastigheten i flödeshastigheten minska till en milliliter per minut. Håll behållaren i kontakt med miljön. Infusera det återstående blodet i en krets med en flödeshastighet på en milliliter per minut.Stoppa minnets syresättning och rullpumpen. Avlivas efter en period av mekanisk ventilation och en djup anestesi. Representativa resultat.Som kontrollgrupp har normaltemperaturen CPB, NtCPB, råtta. Utan cirkulationsstoppet visade ett stabilt medelartärblodtryck MAP och kroppstemperatur under hela proceduren. Medan MAP av DHCA råttor minskade under hjärtstilleståndet.Temperaturen hos DHCA-råttor sjönk snabbt under kylningsfasen och återhämtade sig gradvis under den roterande fasen. Avvänjningen av DHCA-kretsen temperaturen hos DHCA-råttor återgick till det normala. Ovanstående data indikerade att vårt DHCA-förfarande imiterade de faktiska temperatur- och MAP-förändringarna hos patienter på kliniker.Effekten av DHCA-processor och uppgång undersöktes genom blodgasanalys. Efter helblodskontakt med den primära lösningen var koncentrationen av hemoglobin högre än sex gram per decilitter i båda grupperna. Avvänjningen från DHCA-ytan, den ökade till nio gram per decilitter på grund av återingjutning av kvarvarande blod i CPB-kretsen till höger.Hematokriten, HCT, visade liknande tendens till HB. Efter DHCA reperfusion laktat syra ökade snabbt vilket var mer uttalat i DHCA-gruppen. pH minskade efter DHCA-proceduren, vilket troligen var resultatet av laktatsyraackumulering. Under hela experimentet visade koncentrationen av natrium, klorid, kaliumjoner och glukosen inte signifikanta skillnader vid varje 10 punkt.Detta resultat tyder på att DHCA endast inducerade ökningen till laktatsyra men påverkade inte blodets pH och koncentrationen av hemoglobin, hematokrit, natriumjoner, kloridjoner, kaliumjoner och glukos. För att utvärdera effekten av DHCA på neuronal autofagi använde vi transmissionselektromikroskopet och fann överraskande och ökade antalet autofagosomer i hippocampus hos DHCA-råttor. Ovanstående data indikerade att vår DHCA-modell imiterade faktiska temperatur- och MAP-förändringar hos patienter i klinikerna.Denna modell kan tillämpas för att undersöka systemiskt inflammatoriskt responssyndrom, neuroinflammation, 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.

Orthotopic Hind-Limb Transplantation hos råttor

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.

Diagnos av Ecto-och endoparasiter i laboratorium råttor och möss

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.

Upprättande av Epstein-Barr virus Tillväxt-transformerade lymfoblastoida cell-linjer

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.

Inrättande av en<em&gt; In vitro</em&gt; System för att studera intracellulära Beteende<em&gt; Candida glabrata</em&gt; I Human THP-1 makrofager

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 vuxna råttor med icke-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.

Förebyggande av värmestress Biverkningar hos råttor efter<em&gt; Bacillus subtilis</em&gt; Stam

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.

Stenos av Inferior Vena Cava: en murin modell av djup ventrombos

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.

Djup ventrombos som induceras av Stasis hos möss som övervakas av högfrekvent ultraljud

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.

Djup Dermal injektion som en modell av Candida albicans hudinfektion för histologiska 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.

Inrättandet av virusinfektion och analys av värd-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 ...