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

De auteurs bedanken Liang Zhang voor het helpen verzamelen van de videogegevens tijdens het experiment. Deze studie werd ondersteund door de National Natural Science Foundation of China (Grant nummer: 82070479) en de Fundamental Research Funds for the Central Universities (Grant number: 3332022128).

De protocollen ondergingen een institutionele beoordeling en kregen goedkeuring van de Institutional Animal Care and Use Committee, Fuwai Hospital, Chinese Academy of Medical Sciences (FW-2021-0005). Alle experimentele procedures werden uitgevoerd in overeenstemming met de Gids voor de verzorging en het gebruik van proefdieren gepubliceerd door de National Institutes of Health.
OPMERKING: Mannelijke Sprague-Dawley-ratten (gewicht: 500-600 g, leeftijd: 12-14 weken) werden gehouden onder standaa...

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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>

Cannulatie is de meest fundamentele procedure voor het vaststellen van DHCA bij ratten. Vóór cannulatie zal het weken van de slagader met 0,5 ml 2% lidocaïne het gemakkelijker maken om te cannuleren. Na cannulatie is heparinisatie met 500 IE/kg heparine via de uitwendige halsader noodzakelijk om microtrombusvorming te voorkomen17. We hebben herhaaldelijk ontdekt dat deze dosis heparine het doel van een geactiveerde stollingstijd (ACT) >480 s kan bereiken. De opwarmperiode is het moeili...

Diepe onderkoelde circulatoire arrestatie (DHCA) wordt sinds 1953 gebruikt bij hartchirurgie1. DHCA omvat het verlagen van de kerntemperatuur van de patiënt tot diep onderkoelde niveaus (15-22 ° C) voordat de bloedtoevoer naar het lichaam wereldwijd wordt onderbroken2. De circulatiestilstand kan zorgen voor een relatief bloedeloos operatieveld. Diepe onderkoeling vermindert het metabolisme, vooral in de hersenen en myocardium, wat een effectieve methode is om te beschermen tegen ischemie3. DHCA wordt vaak toegepast tijdens operaties voor complexe aangeboren hartaandoeningen, aortaboogaandoeningen en zelfs nier- of bijniertumoren met een vena cava-trombus 4,5. Daarom biedt het opstellen van DHCA-diermodellen een belangrijke referentie voor de verfijning van de procedure en de preventie van complicaties in klinische omgevingen.
Hoewel modellen kunnen worden vastgesteld met hoektanden6, konijnen7 en andere dieren, heeft het de voorkeur om ratten te gebruiken vanwege hun bruikbaarheid en lage kosten. Het DHCA-rattenmodel werd in 2006 voor het eerst beschreven door Jungwirth et al.8. Het bleek dat de duur van de circulatiestilstand een impact had op de neurologische uitkomsten. Sindsdien zijn DHCA-rattenmodellen breed onderzocht. Er is verduidelijkt dat DHCA systemisch ontstekingsreactiesyndroom (SIRS)9 kan veroorzaken. In latere studies ontdekten farmacologen dat de DHCA-gerelateerde neuro-inflammatie geïnduceerd door SIRS kon worden verzwakt door resveratrol10 en triptolide11. Ons team ontdekte ook dat DHCA-gerelateerde neuro-inflammatie kon worden verzwakt door het koud-induceerbare RNA-bindende eiwit12 te remmen. In het cardiovasculaire systeem heeft superoxide dismutase een cardioprotectief effect op ischemie / reperfusie (I / R) -verwondingen tijdens DHCA13. Deze resultaten breidden het begrip van DHCA-gerelateerde pathofysiologische processen uit en boden nieuwe richtingen voor het verbeteren van de resultaten van DHCA. De resultaten met betrekking tot endotoxemie, oxidatieve stress en autofagie na DHCA zijn echter niet overtuigend. DHCA gebruikt dezelfde operationele technologie als de cardiopulmonale bypass (CPB)14, maar de managementstrategie is anders en de stappen om DHCA te genereren verschillen tussen verschillende teams 8,9,10,11. De huidige studie heeft tot doel een methode te bieden voor het vaststellen van de DHCA-procedure bij ratten.

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	<tbody>
		<tr>
			<td>Heat Exchanger</td>
			<td>Xi&#8217;an Xijing Medical Appliance Co., Ltd</td>
			<td>Animal-M</td>
			<td></td>
		</tr>
		<tr>
			<td>Membrane Oxygenator</td>
			<td>Dongguan Kewei Medical Instrument Co., Ltd.</td>
			<td>Micro-M</td>
			<td></td>
		</tr>
		<tr>
			<td>Monitor</td>
			<td>Chengdu Techman Co., Ltd</td>
			<td>BL-420s</td>
			<td></td>
		</tr>
		<tr>
			<td>Roller Pump</td>
			<td>Changzhou Prefluid Technology Co.,Ltd</td>
			<td>BL100</td>
			<td></td>
		</tr>
		<tr>
			<td>SD Rat</td>
			<td>HFK Bioscience Co.,Ltd.</td>
			<td>/</td>
			<td></td>
		</tr>
		<tr>
			<td>Sevoflurane</td>
			<td>Maruishi Pharmaceutical Co. Ltd</td>
			<td>H20150020</td>
			<td></td>
		</tr>
		<tr>
			<td>Shaver</td>
			<td>Hangzhou Huayuan Pet Products Co.,Ltd.</td>
			<td>/</td>
			<td></td>
		</tr>
		<tr>
			<td>Vaporizer</td>
			<td>SPACECABS</td>
			<td>/</td>
			<td></td>
		</tr>
		<tr>
			<td>Ventilator</td>
			<td>Shanghai Alcott Biotech Co., Ltd</td>
			<td>ALC-V8S</td>
			<td></td>
		</tr>
		<tr>
			<td>Water Tank</td>
			<td>Maquet Critical Care AB</td>
			<td>Jostra HCU20-600</td>
			<td></td>
		</tr>
	</tbody>
</table>

establishment of deep hypothermic circulatory arrest in rats

Diepe hypothermische circulatoire arrestatie (DHCA) wordt routinematig toegepast tijdens operaties voor complexe aangeboren hartaandoeningen en aortaboogaandoeningen. De huidige studie heeft tot doel een methode te bieden voor het vaststellen van DHCA bij ratten. Om de impact van het DHCA-proces op vitale functies te evalueren, werd een ratmodel met een normale temperatuur cardiopulmonale bypass (CPB) zonder circulatoire arrestatie gebruikt als controle. Zoals verwacht leidde DHCA tot een significante daling van de lichaamstemperatuur en de gemiddelde arteriële bloeddruk. De bloedgasanalyse gaf aan dat DHCA de melkzuurspiegels verhoogde, maar de pH van het bloed en de concentraties van hemoglobine, hematocriet, Na +, Cl−, K + en glucose niet beïnvloedde. Bovendien toonden de resultaten van de transmissie-elektronenmicroscopie, vergeleken met de CPB-ratten met een normale temperatuur, een lichte toename van hippocampale autofagosomen bij de DHCA-ratten.

Als controlegroep vertoonden de cpb-ratten met normale temperatuur (NtCPB) zonder circulatoire arrestatie een stabiele gemiddelde arteriële bloeddruk (MAP) en lichaamstemperatuur gedurende de hele procedure, terwijl de MAP van de DHCA-ratten afnam tijdens de hartstilstand (p < 0,01, figuur 3A). De temperatuur van de DHCA-ratten daalde snel tijdens de koelfase en herstelde zich geleidelijk tijdens de opwarmfase. Bij het spenen van de ratten van de DHCA-circuits keerde de temperatuur...

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

Dit protocol presenteert de vaststelling van diepe hypothermische circulatiestilstand bij ratten, die kan worden toegepast om systemisch ontstekingsreactiesyndroom, ischemie / reperfusieletsel, oxidatieve stress, neuro-inflammatie, enz. Te onderzoeken.

Oprichting van diepe onderkoelde circulatoire arrestatie bij ratten

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

Oprichting van diepe onderkoelde circulatoire arrestatie bij ratten. Diepe onderkoelde circulatoire arrestatie, DHCA wordt routinematig toegepast tijdens operaties van complexe aangeboren hartaandoeningen en aortaboogaandoeningen. De huidige studie heeft tot doel een methode te bieden voor het vaststellen van DHCA bij ratten.De protocollen kregen een institutionele beoordeling en kregen goedkeuring van de Institutional Animal Care and Use Committee, Fuwai Hospital, Chinese Academy of Medical Sciences. Voorbereidende werkzaamheden. Zorg ervoor dat CPB-circuits een reservoir bevatten dat is gewijzigd door morfroomdruppel, rolpomp, warmtewisselaar, membraanoxygenator, verbindingsbuis en een watertank die het circuit heeft aangesloten en 12 milliliter hydroxyzetmeel maakt met één milliliter heparinenatrium en één milliliter zoutoplossing prime het circuit met 40 milliliter primingoplossing met de rolpomp voorzichtig draaiend.Twee, anesthesie en cannulatie. Sluit de buis aan op de ventilator en zijparameters. Leg een elektrische verwarmingsdeken en rechts en kijk met tape naar rechts.Breng oogheelkundige zalf aan op de ogen om uitdroging te voorkomen. Scheer het haar op het rechter liesgebied, het rechter cervicale gebied en de staart met het scheerapparaat. Desinfecteer vervolgens de huid met aldehyde en alcohol.Snijd de huid en het linker liesgebied en ontleed de spier en het zachte weefsel om de linker femorale ader en slagader bloot te leggen. Scheid de slagader zorgvuldig. Cannulaat 22 gauge intraveneuze katheter in de linker femorale slagader.Ligated slagader en katheter met zijde. Gebruik zoutoplossing met heparine om de canule te spoelen om stolling te voorkomen. Sluit de katheter aan op de perssensor om de bloeddruk te controleren.Snijd de huid van de staart en gebruik vervolgens een scalpel om de oppervlakkige specia van de staartslagader te snijden om de staartslagader bloot te leggen die zich in het midden van het chirurgische veld bevindt. Cannuleer de staartslagader met de 22 gauge intraveneuze katheter. Ligaat slagader en katheter met zijde.Gebruik zoutoplossing met heparine om de katheter te spoelen om stolling te voorkomen. Snijd de huid van de rechter jugular van de ader en scheid vervolgens spier en weefsel om de ader bloot te leggen. Breng een 16 gauge zelfgemaakte multiage intraveneuze katheter in de rechter uitwendige jugular van de ader en plaats deze voorzichtig in de rechter inferieure vena cava of het rechter atrium.Dien heparinenatrium toe waarbij de rechter externe ader elk gecannuleerd gebied bedekt met vochtige ballen om besmetting te voorkomen. Drie, verbind het circuit met de katheter in de staartslagader en houd de pomp laag met één tot twee milliliter per minuut. Verbind vervolgens het reservoir met de katheter in de rechter externe halsader.Zorg ervoor dat er altijd een bloedspiegel van ongeveer een centimeter in het reservoir zit. Zet het waterreservoir aan en stel eerst de watertemperatuur in op 37 graden Celsius. Nadat de bloeddruk stabiel is, verhoogt u voorzichtig de stroompomp tot 80 tot 100 milliliter per minuut.Vier, afkoelen. Stel de kamertemperatuur in op ongeveer 20 graden Celsius. Doe ijsblokjes in deze wegwerphandschoenen en leg ze vervolgens op het juiste hoofd en de juiste zijkanten.De temperatuur van de tank werd voorgesteld om real-time te zijn volgens de rectale temperatuur van de rijst. Verander de relevante parameters van de ventilator correct op basis van de resultaten van bloedgasanalyse. Vijf, diepe onderkoeling circulatoire arrestatie.Dan daalt de rectale temperatuur naar 15 tot 20e eeuw celsius. Verander het ijspak en gooi de handschoenen weg om ervoor te zorgen dat diepe onderkoeling tijdens het stoppen van de bloedsomloop wordt gehandhaafd. Stop de rolpomp.Houd het reservoir in contact met de omgeving en laat het bloed langzaam van de externe halsader naar het reservoir lopen. Besteed aandacht aan de bloeddrukhervorming. Nadat er geen bloeddruk en hartslag is, stopt u de drainage en houdt u het reservoir gesloten.Zet de ventilator uit. Zes, warming-up en reperfusie. Verwijder alle wegwerphandschoenen en verhoog de kamertemperatuur tot 25 graden Celsius.Herstel de geheugenzuurstof tussen ventilatie terwijl de veneuze afvoerbuis wordt geknipt. Zet de rollerpomp aan om ervoor te zorgen dat het bloed in het reservoir langzaam terug is naar het juiste lichaam. Zet de ventilator aan nadat de bloedspiegel in het reservoir een centimeter blijft.Gebruik de drainagebuis en laat het bloed van het rechteratrium langzaam naar het reservoir lopen. Zet de verwarmingslamp, het verwarmingskussen en de watertank aan. Stel de temperatuur van het waterreservoir in op 25 graden Celsius.Stel het vervolgens tijdig in op de linkertemperatuur. Verwijder de verwarmingslamp nadat de rectale temperatuur is teruggekeerd naar 34 graden Celsius. Zeven, spenend van het CPB.Verlaag langzaam en geleidelijk het roldebiet van de pomp en pas de vena genus snelheid aan in het debiet verminderen tot één milliliter per minuut. Houd het reservoir in contact met de omgeving. Infundeer het resterende bloed in een circuit met een stroomsnelheid van één milliliter per minuut.Stop de geheugenoxygenatie en de rolpomp. Geëuthanaseerd na een periode van mechanische beademing en een diepe anesthesie. Representatieve resultaten.Als controlegroep, de normale temperatuur CPB, NtCPB, rat. Zonder de circulatoire arrestatie toonde een stabiele gemiddelde arterie bloeddruk MAP en lichaamstemperatuur gedurende de hele procedure. Terwijl de MAP van DHCA-ratten afnam tijdens de hartstilstand.De temperatuur van DHCA-ratten daalde snel tijdens de koelfase en herstelde zich geleidelijk tijdens de draaiende fase. Het spenen van DHCA-circuit de temperatuur van DHCA-ratten keerde terug naar normaal. De bovenstaande gegevens gaven aan dat onze DHCA-procedure de werkelijke temperatuur- en MAP-veranderingen van patiënten in klinieken imiteerde.Het effect van DHCA-processor en stijging werd onderzocht door bloedgasanalyse. Na het volbloedcontact met de primaire oplossing was de concentratie hemoglobine in beide groepen hoger dan zes gram per decilitter. Het spenen van het DHCA-oppervlak nam toe tot negen gram per decilitter vanwege het opnieuw inbrengen van achtergebleven bloed in het CPB-circuit naar rechts.De hematocriet, HCT, vertoonde dezelfde neiging als HB. Na DHCA-reperfusie nam lactaatzuur snel toe, wat meer uitgesproken was in de DHCA-groep. de pH daalde na de DHCA-procedure, wat hoogstwaarschijnlijk het gevolg was van lactaatzuuraccumulatie. Tijdens het hele experiment vertoonden de concentratie van natrium, chloride, kaliumionen en de glucose geen significante verschillen op elk 10 punt.Dit resultaat suggereert dat DHCA alleen de verhoogde lactaatzuur induceerde, maar de pH van het bloed en de concentratie van hemoglobine, hematocriet, natriumionen, chloride-ionen, kaliumionen en glucose niet beïnvloedde. Om de impact van DHCA op neuronale autofagie te evalueren, gebruikten we de transmissie-elektromicroscoop en vonden en verhoogden verrassend het aantal autofagosomen in de hippocampus van DHCA-ratten. De bovenstaande gegevens gaven aan dat ons DHCA-model de werkelijke temperatuur- en MAP-veranderingen van patiënten in de klinieken imiteerde.Dit model kan worden toegepast om systemisch ontstekingsreactiesyndroom, neuro-inflammatie, et cetera te onderzoeken.

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.

Orthotope Hind-Limb Transplantatie bij ratten

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.

Diagnose van Ecto-en endoparasieten bij laboratoriumratten en muizen

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.

Oprichting van Epstein-Barr Virus Growth-getransformeerde lymfoblastoïde cellijnen

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.

Oprichting van een<em&gt; In vitro</em&gt; Systeem om Intracellular Gedrag van Studie<em&gt; Candida glabrata</em&gt; In Human THP-1 macrofagen

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.

Compleet thymectomie bij volwassen ratten met niet-invasieve Luchtpijpintubatie

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.

Preventie van hittestress schadelijke effecten bij ratten door<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 van de Vena Cava Inferior: een lymfkliertest Model van diep-veneuze 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.

Diep-veneuze trombose geïnduceerd door Stasis in muizen gecontroleerd door hoge frequentie Ultrasonografie

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.

Diepe dermale injectie als de een Model van Candida albicans infectie van de huid voor histologisch 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 ...

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.

Oprichting van virale infectie en analyse van Host-Virus interactie bij 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 ...