Appreciation is extended to Dr. T. Taki and Dr. M. Funamoto for their technical support.

Prior to experiment, all rats should be given one week to acclimate. All surgical procedures on animals should be carried out in accordance with the Guide for the Care and Use of Laboratory Animals (www.nap.edu/catalog/5140.html) or other appropriate ethical guidelines. Protocols should be approved by the animal welfare committee at the appropriate institution before proceeding. All subsequent procedures must be performed under aseptic conditions.&#160;
1. Preparing CPB Circuit
Note: Wear personal protective equipment including gloves, eyewear, and a clean coat or disposable gown.
<ol>
	<li>Set-up of CPB circuit
	<ol>
		<li>Connect polyvinyl chloride tubes with a venous reservoir, predesigned for CPB circuit, and a modified neonatal membrane oxygenator as shown in Figure 1. Ensure all connections are tight and do not leak water.</li>
		<li>Set a CPB circuit to the roller pump device according to the manufacturer&#39;s protocols.</li>
		<li>Keep the roller pump on a height-adjustable table and adjust the table&#39;s height to 10 cm below the experimental desk.</li>
	</ol>
	</li>
	<li>Priming of CPB circuit
	<ol>
		<li>Mix 12 mL of hydroxyethyl starch solution with 0.1 mL of heparin and 0.5 mL of 7% sodium bicarbonate solution for priming CPB circuit.</li>
		<li>Prime the circuit with 11 mL of the priming solution, with the pump roller gently rotating. Put an 18-gauge venting needle into the reservoir for venting air.</li>
		<li>Hit the membrane oxygenator several times to deair, with tilting the oxygenator. Air must be obscured completely to avoid air embolism and insufficient oxygenation. During the circuit priming, heat the circuit by an electric heat lamp set on the reservoir.</li>
	</ol>
	</li>
</ol>
2. Procedure Before CPB
NOTE: The surgical field and devices should be disinfected by 70% alcohol or a quaternary ammonium compound before use.
<ol>
	<li>Anesthesia and animal setting
	<ol>
		<li>Anesthetize a rat with 3.0% isoflurane-mixed air inhalation in a vaporizer. Set the rat on a work-stand and intubate a 16-gauge cannula into the trachea. Follow your local animal care guidelines regarding analgesia dosage and frequency (e.g. 
		buprenorphine 0.005 mg/kg s.c.) 
		&#8203;NOTE: Rats must be at deep anesthesia and lose reflexes. The respiration should be rhythmic but not be arrested.</li>
		<li>Transfer the rat to an operating table equipped with an electric heating pad. Start the mechanical ventilation with 8 mL/kg of tidal volume, a respiratory rate of 70 cycles/min, and 30% of inspired oxygen fraction monitored by the oxygen sensor.</li>
		<li>Maintain the anesthesia with 1.5-2.0% isoflurane, and with an additional administration of ketamine/xylazine at CPB initiation.</li>
		<li>Monitor the rectal temperature by using the rectal probe. Maintain normothermic body temperature of 37 &#176;C by adjusting the temperature of the heat pad and placing the circuit over the heat lamp.</li>
		<li>Set the rat in supine position and stretch the four limbs by fixing with needles. Monitor the heart rate by setting the ECG electrode needle to the bilateral shoulder and left abdomen. Place a moist gauze or apply ophthalmic ointment to the eyes to prevent dryness.</li>
	</ol>
	</li>
	<li>Cannulation
	<ol>
		<li>After disinfecting the surface of the whole body by sprayingby spraying 70 % ethanol or another antiseptic solution, shave the hair by a razor on the bilateral inguinal region and right cervical region. Local anesthetics (such as lidocaine) should be used before making the skin incision. NOTE: As an alternative, a surgical scrub of the incision site can be used instead of the whole body spray of 70% ethanol to avoid a drop in body temperature.</li>
		<li>Incise the skin (approximately 5 mm) at the bilateral inguinal regions and right cervical region by scissors and bluntly dissect the tissues to expose the right main femoral artery. Separate the artery carefully from the vein and nerve nearby. Ligate at the end of common femoral artery by 4-0 silk and exposure by tension.</li>
		<li>Cut the arterial wall (approximately 1 mm) of right common femoral artery by micro scissors in perpendicular direction to the artery, and carefully cannulate a 24-gauge intravenous catheter from the incision to a depth of 1 cm for monitoring systemic arterial pressure and analyzing gas partial pressure in arterial blood.</li>
		<li>Administrate heparin sodium (500 IU/kg) from the catheter.</li>
		<li>Follow steps 2.2.2 and 2.2.3 to cannulate a 24-gauge intravenous catheter into the left common femoral artery as an arterial infusion line for the CPB circuit.</li>
		<li>Insert a 17-gauge multi-orifice angiocatheter into the right internal jugular vein and advance it into the right atrium and inferior vena cava (IVC). Do not push the catheter roughly as the vessel can easily break. Connect the catheter to the CPB circuit for venous drainage.</li>
		<li>Cover each cannulated region with a moist gauze to avoid contamination.</li>
	</ol>
	</li>
</ol>
3. Procedure During CPB
<ol>
	<li>Deliver 100% oxygen gas to the oxygenator at 0.8 L/min during CPB and decrease the respiratory rate to 30 cycles/min. The arterial oxygen partial pressure is required from 200 to 400 mmHg.</li>
	<li>At the beginning of CPB, increase the temperature setting of the heat pad to the maximum, 42 &#176;C, to mitigate the immediate drop of the body temperature after CPB initiation. Adjust the setting temperature to 37 &#176;C when the body temperature returns to 36 &#176;C.</li>
	<li>Carefully start the CPB flow and keep an eye on the volume of the blood in the reservoir. An empty reservoir may cause air embolism. If the volume of the blood in the reservoir decreases, lower the pump flow by adjusting the table height, or changing the position of the drainage catheter. Do not re-position the venous drainage catheter, which might easily cause perforation of the right atrium and/or arrhythmia.</li>
	<li>Increase and keep the pump flow at 100 mL/kg/min, while the mean blood pressure is maintained at 70 mmHg. When the appropriate blood pressure is maintained, a small volume of at least 1 mL in the reservoir is acceptable. If there is less than 1 mL of the blood in the reservoir, it may cause an air embolism in organs.</li>
	<li>If the blood pressure is unstable, add 2-3 mL of the priming solution to the circuit (it may cause anemia after the CPB).</li>
</ol>
4. Procedure After CPB
<ol>
	<li>Clamp the venous drainage tube and remove it from the circuit. Infuse the remaining blood in the circuit gradually to the artery to maintain the blood pressure.</li>
	<li>Increase the respiratory rate to 70 cycles/min.</li>
	<li>Remove the venous drainage catheter and the left arterial catheter, then ligate the vessel in the proximal and distal site.</li>
	<li>Remove the arterial line from the right femoral artery 60 min after the end of CPB.</li>
	<li>Clean each wound with saline and close the wound with sutures.</li>
	<li>End the anesthesia and extubate the intratracheal tube after checking the spontaneous breathing of the animal.</li>
	<li>Administer warmed sterile isotonic fluids, and use a heat mat and electrical heat lamp to keep the animals warm. Check the conditions of the animal frequently until recovery from anesthesia. Provide respiratory support when required. NOTE: Once the animal begins moving around, the heat source should be removed from part of the cage to allow the animal to choose the warm or cool side.</li>
	<li>Keep the animal apart from the company of other animals until the respiration fully regains. Do not return the animal to the company until full recovery.</li>
	<li>Check the food and water intake after the recovery from anesthesia and provide appropriate nutritional support. Administer analgesics and check for signs of discomfort or pain.</li>
</ol>

<ol>
	<li>Gibbon, 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=Application+of+a+mechanical+heart+and+lung+apparatus+to+cardiac+surgery.">Application of a mechanical heart and lung apparatus to cardiac surgery.</a> Minn Med. 37 (3), 171-185 (1954).</li><li>Apostolakis, E., Filos, K. S., Koletsis, E., Dougenis, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lung+dysfunction+following+cardiopulmonary+bypass.">Lung dysfunction following cardiopulmonary bypass.</a> J Cardiac Surg. 25 (1), 47-55 (2010).</li><li>Popovic, P., Horecky, J., Popovic, V. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Instrumental+responses+in+rats+after+hypothermic+cardiopulmonary+by-pass.">Instrumental responses in rats after hypothermic cardiopulmonary by-pass.</a> P Soc Exp Biol Med. 126 (1), 225-228 (1967).</li><li>Fabre, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+recovery+model+of+partial+cardiopulmonary+bypass+in+the+rat.">A recovery model of partial cardiopulmonary bypass in the rat.</a> Perfusion. 16 (3), 215-220 (2001).</li><li>Hirao, S., Masumoto, H., 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=Rat+cardiopulmonary+bypass+models+to+Investigate+multi-organ+injury.">Rat cardiopulmonary bypass models to Investigate multi-organ injury.</a> Clin Surg. 2, 1-6 (2017).</li><li>Jungwirth, B., 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=Neurologic+outcome+after+cardiopulmonary+bypass+with+deep+hypothermic+circulatory+arrest+in+rats:+description+of+a+new+model.">Neurologic outcome after cardiopulmonary bypass with deep hypothermic circulatory arrest in rats: description of a new model.</a> J Thorac Cardiov Sur. 131 (4), 805-812 (2006).</li><li>de Lange, F., Yoshitani, K., Podgoreanu, M. V., Grocott, H. P., Mackensen, G. B. <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+survival+model+of+cardioplegic+arrest+and+cardiopulmonary+bypass+in+rats:+a+methodology+paper.">A novel survival model of cardioplegic arrest and cardiopulmonary bypass in rats: a methodology paper.</a> J Cardiothorac Surg. 3, 51 (2008).</li><li>Peters, S., 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=An+experimental+model+of+myocardial+infarction+and+controlled+reperfusion+using+a+miniaturized+cardiopulmonary+bypass+in+rats.">An experimental model of myocardial infarction and controlled reperfusion using a miniaturized cardiopulmonary bypass in rats.</a> Interact Cardiovasc Th. 19 (4), 561-564 (2014).</li><li>Engels, 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+cardiopulmonary+bypass+with+deep+hypothermic+circulatory+arrest+rat+model+for+the+investigation+of+the+systemic+inflammation+response+and+induced+organ+damage.">A cardiopulmonary bypass with deep hypothermic circulatory arrest rat model for the investigation of the systemic inflammation response and induced organ damage.</a> J Inflamm. 11 (26), (2014).</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> Eur J Cardio-Thorac. 50 (6), 1035-1044 (2016).</li><li>Zhang, Z., Wu, Y., Zhao, Y., Xiao, X., Liu, J., Zhou, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+changes+in+HMGB1+levels+correlate+with+inflammatory+responses+during+cardiopulmonary+bypass.">Dynamic changes in HMGB1 levels correlate with inflammatory responses during cardiopulmonary bypass.</a> Exp Ther Med. 5 (5), 1523-1527 (2013).</li><li>Kohno, T., 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=Impact+of+serum+high-mobility+group+box+1+protein+elevation+on+oxygenation+impairment+after+thoracic+aortic+aneurysm+repair.">Impact of serum high-mobility group box 1 protein elevation on oxygenation impairment after thoracic aortic aneurysm repair.</a> Heart Vessels. 26 (3), 306-312 (2011).</li><li>Tseng, C. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+serum+biomarkers+and+clinical+factors+on+intensive+care+unit+mortality+and+6-month+outcome+in+relatively+healthy+patients+with+severe+pneumonia+and+acute+respiratory+distress+syndrome.">Impact of serum biomarkers and clinical factors on intensive care unit mortality and 6-month outcome in relatively healthy patients with severe pneumonia and acute respiratory distress syndrome.</a> Dis Markers. 2014, (2014).</li><li>Paparella, D., Yau, T. M., Young, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiopulmonary+bypass+induced+inflammation:+pathophysiology+and+treatment.+An+update.">Cardiopulmonary bypass induced inflammation: pathophysiology and treatment. An update.</a> Eur J Cardio-Thorac. 21 (2), 232-244 (2002).</li><li>Hirao, S., 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=Recombinant+human+soluble+thrombomodulin+prevents+acute+lung+injury+in+a+rat+cardiopulmonary+bypass+model.">Recombinant human soluble thrombomodulin prevents acute lung injury in a rat cardiopulmonary bypass model.</a> J Thorac Cardiov Sur. , (2017).</li><li>Yamazaki, S., Inamori, S., Nakatani, T., Suga, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activated+protein+C+attenuates+cardiopulmonary+bypass-induced+acute+lung+injury+through+the+regulation+of+neutrophil+activation.">Activated protein C attenuates cardiopulmonary bypass-induced acute lung injury through the regulation of neutrophil activation.</a> J Thorac Cardiov Sur. 141 (5), 1246-1252 (2011).</li><li>Wang, C. T., Zhang, L., Wu, H. W., Wei, L., Xu, B., Li, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Doxycycline+attenuates+acute+lung+injury+following+cardiopulmonary+bypass:+involvement+of+matrix+metalloproteinases.">Doxycycline attenuates acute lung injury following cardiopulmonary bypass: involvement of matrix metalloproteinases.</a> Int J Clin Exp Patho. 7 (11), 7460-7468 (2014).</li><li>Liu, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Curcumin+attenuates+cardiopulmonary+bypass-induced+lung+oxidative+damage+in+rats.">Curcumin attenuates cardiopulmonary bypass-induced lung oxidative damage in rats.</a> J Cardiovasc Pharm T. 17 (4), 395-402 (2012).</li><li>Taki, T., 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=Fetal+mesenchymal+stem+cells+ameliorate+acute+lung+injury+in+a+rat+cardiopulmonary+bypass+model.">Fetal mesenchymal stem cells ameliorate acute lung injury in a rat cardiopulmonary bypass model.</a> J Thorac Cardiov S. 153 (3), 726-734 (2017).</li><li>Zhu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+a+novel+rat+model+without+blood+priming+during+normothermic+cardiopulmonary+bypass.">Establishment of a novel rat model without blood priming during normothermic cardiopulmonary bypass.</a> Perfusion. 29, 63-69 (2014).</li><li>Inoue, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+anesthesia+worsens+outcome+of+rats+with+inflammatory+responses.">Deep anesthesia worsens outcome of rats with inflammatory responses.</a> Inflamm Res. 65 (7), 563-571 (2016).</li><li>Bradfield, J. F., Schachtman, T. R., McLaughlin, R. M., Steffen, E. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+and+physiologic+effects+of+inapparent+wound+infection+in+rats.">Behavioral and physiologic effects of inapparent wound infection in rats.</a> Lab Anim Sci. 42 (6), 572-578 (1992).</li></ol>

All authors have nothing to disclose regarding commercial support.

In this rat CPB model, the serum and lung expression levels of inflammatory cytokines and HMGB-1, a key transcription factor regulating the inflammatory responses, dramatically increased after CPB. Previous clinical studies showed that the serum secretion of HMGB-1 level is elevated in patients undergoing cardiovascular surgery11, and the peak serum HMGB-1 level during CPB was associated with more severe systemic inflammatory response syndrome and lung oxygenation impairment after CPB12. Additionally, the serum HMGB-1 level is an independent biomarker to predict intensive care unit mortality in patients with severe pneumonia and acute respiratory distress syndrome (ARDS)13. Severe CPB-related ARDS associated with inflammatory cytokines and HMGB-1 activation is successfully simulated by this rat CPB model.
Numerous studies on CPB-related systemic inflammation have been conducted in rat models because of their usefulness for investigating the systemic inflammatory responses, including proinflammatory cytokine expression14, nuclear kappa B activation15, neutrophil activation by adhesion molecules16, and matrix metalloproteinase-9 activities17. Oxidative stress such as the activation of the HO-1 protein during CPB has been also investigated by using rat CPB models18.
The rat CPB model has recently been developed in various procedures to simulate the clinical situations of complications from CPB. The enzymatic, genetic, and histologic effects for myocardium can be investigated by a cardiac arrest model7. Moreover, a DHCA model is reported to reveal the mechanisms associated with adverse cerebral outcome after thoracic aortic surgery, and to investigate potential neuroprotective strategies6. These models, based on the conventional rat CPB model, are valuable to evaluate the various effects of CPB, closely mimicking clinical situations in cardiovascular surgeries.
The survival rate of this model depends on the investigator&#39;s technical improvement. The most critical factors related to morbidity are bleeding during the separation of vessels, cardiac rupture, and retroperitoneum hematoma, which generally occurs from technical failure. Good exposure, appropriate tension to the vessels, and gentle cannulation are surgical requirements for success. Keeping the mean blood pressure of at least 60 mmHg by controlling the reserved blood and additional solution is important for avoiding extreme anemia and lower limb deficiency. Air embolism to the intestine and/or other abdominal organs is also critical to avoid; once this occurs, the procedure cannot be completed.
In early practice of these procedure, completion of CPB is approximately 20% because of the difficulty in cannulation, and up to 10% survival 4 h after CPB. After experience with 20 models, the survival rate dramatically improved to nearly 80% because of the stable hemodynamics during model preparation.
At the beginning of establishing this model, total volume of the CPB circuit was 3 mL which is higher than that of the present one. Therefore, the excessive colloidal volume induced critical anemia during CPB (mean hematocrit: 14.7%)19. By decreasing the CPB circuit volume and surgical bleeding, the hematocrit during CPB dramatically increased to 21.3%, which allowed maintaining stable hemodynamics.
The 17-gauge multi-orifice is very suitable for the cannulation from the internal jugular vein to IVC through the right atrium, because of its suitable length and softness, which prevents wearing out of the venous wall. Moreover, the multi-orifice and sufficient inner diameter allows sufficient venous return and a high success rate. Introducing the catheter into IVC is technically difficult because of its blind manner and would easily perforate the venous or atrial wall.
Arterial cannulation for the femoral artery of rats tends to cause limb ischemia compared to tail artery or carotid artery20. In our experience, instability of hemodynamics (mean BP less than 60 mmHg), ligation of the deep femoral artery, and critical anemia may cause severe leg damage. Maintaining blood pressure, cannulating a higher region of the common femoral artery, and keeping a higher hematocrit are all solutions to prevent limb ischemia caused by the innate rich collateral perfusion of rats.
Avoidance of catecholamine and blood transfusion is necessary to accurately investigate hemodynamic, biochemical and/or physiological responses of testing materials and interventions. There is no need for vasopressor and transfusion at all during the procedure in our model. Deep anesthesia augments the release of inflammatory cytokines and subsequently deteriorates organ function21. Appropriate anesthesia depth, smooth and gentle cannulation of the animal trachea for intubation, and femoral artery cannulation for blood pressure monitoring without bleeding are important before initiation of CPB. If there is hemodynamic instability before initiation of CPB, success rate will be remarkably decreased. During CPB, the flow rate of CPB must be maintained by adjusting the volume in the reservoir. At 60 min after initiation of CPB, the careful addition of volume (1-3 mL saline) is required because of the hemodynamic instability by inflammatory processes. However, the addition of excessive colloidal volume causes anemia which may lead to unstable conditions. After CPB, the blood in the CPB circuit should be returned to the rat gently in 30 min. Instability after CPB means that there is potential morbidity such as ischemia in limbs or intestine.
The suitable anesthesia depth is one of tips in this model to decrease mortality and morbidity. Inhalant anesthetics provide adequate control of depth and duration of anesthesia. When we intubate a cannula to the trachea, rats must be at deep anesthesia enough to lose reflexes. The respiration should be rhythmic but not be arrested. Respiratory arrest indicates extremely deep anesthesia, hypotension, and/or imminent death, which causes systemic inflammation. Contrarily, insufficient anesthesia causes more stress to the animal. During CPB, body movement of the animal is critical to reduce because of possible accidental removal of the cannulas. Controlling the isoflurane vaporizer by monitoring heart rate, arterial pressure, and chest wall movement is required during the procedure. At the initiation of CPB, the blood concentration of isoflurane may decrease from dilatation by primed volume. Additional intraperitoneal pentobarbital may allow stable depth of anesthesia.
Post-operative infections in rats will cause numerous changes in physiologic parameters22. A long survival model requires strict maintenance of sterile conditions during the procedure. A separate room and a clean working area for aseptic procedures are desirable. The surgical field and devices should be disinfected by 70% alcohol or a quaternary ammonium compound before use. Every material which likely affects contamination, such as cannulas, solutions, and suture materials, must be in a sterile pack. During CPB, covering the surgical field with moist small gauze prevents the sites from potential sources of contamination. Before closing the wounds, removing blood from the wound and rinsing it with sterile water are necessary to avoid wound infection. In our experience, no wound infection has occurred 24 h after the operation.
There are limitations of this CPB model in that is cannot completely mimic clinical situations. Firstly, this heart beating model exhibits pulsatile flow which is different from clinical cardiac surgery with arrested heart. Secondly, this model can be conducted without thoracotomy, and this would not fully reproduce clinical CPB with median thoracotomy, which leads to massive bleeding during the surgical procedure.
In conclusion, this recovery CPB model allows studies of early- and long-term multiple organ damage associated with CPB. Major advantages of this model include the reduction of possible biases by blood transfusions, vasopressors, or inotropic agents. This model is suitable to investigate therapeutic strategies aimed at preventing multi-organ injury during CPB.

In 1953, Dr. John H. Gibbon Jr. successfully performed the first cardiac surgery using CPB1, and it subsequently became an essential modality in cardiovascular surgery. While the techniques and devices have been dramatically refined, multi-organ complications related to CPB still compromise the outcome of cardiovascular surgeries, and may affect postoperative morbidity and mortality2. CPB-related organ damage is caused by immune system activation and synthesis of proinflammatory cytokines, compliment activation, and production of oxygen free radicals2. Its pathophysiology, however, has not been fully elucidated.
Animal models recapitulating the clinical usage of CPB enable the clarification of the pathophysiological processes during and after CPB; this can facilitate pre-clinical studies in developing strategies to avoid these complications. Since Popovic et al. first reported a rat CPB model in 19673, rat CPB models have been refined, and have gradually taken the place of large-animal models due to greater cost-effectiveness, convenient experimental processes, and a plethora of testing methods in genetic and protein levels. Additionally, inbred rats can be genetically identical, reducing possible biological biases.
Fabre et al. first established a recovery model that allowed the study of the long-term multiple organ sequelae of CPB4. The advantages of this simple survival model are the flexibility (CPB flow and duration), stable vital condition, and reproducibility in systemic inflammation. Rat CPB models have become crucial for the investigation of therapeutic strategies that aim to prevent multi-organ injury during CPB5, and various models for simulating the clinical situations during CPB have recently been developed. De Lange et al. developed a cardiac arrest model, which can be used to characterize the enzymatic, genetic, and histological responses related to myocardial injury7. Peters et al. arranged myocardial infarction and controlled reperfusion using a miniaturized CPB model to analyze heart disfunction through the focal ischemia and reperfusion injury8. Jungwirth et al. first established a deep hypothermic circulatory arrest (DHCA) model, which can elucidate the global ischemia and reperfusion injury by DHCA and supports potential neuroprotective strategies6. Studies using DHCA investigate the influence of hypothermia, reperfusion, and/or hemolysis-triggered signaling events9. Deep hypothermia may affect the activation and inactivation of various enzymes and pathways and the mechanisms remain unknown10. On the other hand, cardiac arrest models or heart ischemia models must be used to investigate ischemia and reperfusion heart injury. These various rat CPB models that highly recapitulate human CPB may reveal pathological processes related to CPB and help mitigate CPB-related complications.
This protocol demonstrates a simple CPB model without transfusion or inotropic agents in a rat. This model allows for the study of long-term multiple organ sequelae of CPB.

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	<tbody>
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			<td height="21" style="height:21px;width:216px;">Rodent Ventilator 7025</td>
			<td style="width:140px;">Ugo Basile</td>
			<td align="right" style="width:145px;">7025</td>
			<td style="width:167px;">Ventilator</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">OxiQuant B</td>
			<td style="width:140px;">ENVITEC</td>
			<td style="width:145px;">46-00-0023</td>
			<td>Oxygen Sensor</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">CMA 450 Temperature Controller</td>
			<td style="width:140px;">CMA</td>
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			<td>Temperature Controller</td>
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			<td height="21" style="height:21px;width:216px;">CMA 450 Heating Pad</td>
			<td style="width:140px;">CMA</td>
			<td align="right" style="width:145px;">8003763</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">CMA 450 Rectal Probe</td>
			<td style="width:140px;">CMA</td>
			<td align="right" style="width:145px;">8003761</td>
			<td></td>
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			<td height="42" style="height:42px;width:216px;">DIN(8) to Disposable BP Transducer</td>
			<td style="width:140px;">ADInstruments</td>
			<td style="width:145px;">MLAC06</td>
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			<td height="21" style="height:21px;width:216px;">Disposable BP Transducer</td>
			<td style="width:140px;">ADInstruments</td>
			<td style="width:145px;">MLT0670</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">IX-214 Data Recorder</td>
			<td style="width:140px;">iWorx Systems</td>
			<td style="width:145px;">IWX-214</td>
			<td>amplifier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">LabScribe software</td>
			<td style="width:140px;">iWorx Systems</td>
			<td style="width:145px;"></td>
			<td>software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Roller pump</td>
			<td style="width:140px;">Furue Science</td>
			<td style="width:145px;">Model RP-VT</td>
			<td>pump</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Happy Cath</td>
			<td style="width:140px;">Medikit</td>
			<td style="width:145px;">EB 19G 4HCLs PP</td>
			<td>17-gauge multiorifice angiocatheter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SURFLO ETFE I.V. Catheter</td>
			<td style="width:140px;">Terumo</td>
			<td style="width:145px;">SR-OX2419CA</td>
			<td>24-gauge angiocatheter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Oxygenator</td>
			<td style="width:140px;">Mera</td>
			<td style="width:145px;">HPO-002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CPB circuit</td>
			<td style="width:140px;">Mera</td>
			<td style="width:145px;">custom-made</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hespander fluid solution</td>
			<td style="width:140px;">Fresenius Kabi</td>
			<td style="width:145px;">3319547A4035</td>
			<td>Hydroxyethyl starch</td>
		</tr>
	</tbody>
</table>

a recovery cardiopulmonary bypass model without transfusion or inotropic agents in rats

Cardiopulmonary bypass (CPB) is indispensable in cardiovascular surgery. Despite the dramatic refinement of CPB technique and devices, multi-organ complications related to prolonged CPB still compromise the outcome of cardiovascular surgeries, and may worsen postoperative morbidity and mortality. Animal models recapitulating the clinical usage of CPB enable the clarification of the pathophysiological processes that occur during CPB, and facilitate pre-clinical studies to develop strategies protecting against these complications. Rat CPB models are advantageous because of their greater cost-effectiveness, convenient experimental processes, abundant testing methods at the genetic or protein levels, and genetic consistency. They can be used for investigating the immune system activation and synthesis of proinflammatory cytokines, compliment activation, and production of oxygen free radicals. The rat models have been refined and have gradually taken the place of large-animal models. Here, we describe a simple CPB model without transfusion and/or inotropic agents in a rat. This recovery model allows the study of the long-term multiple organ sequelae of CPB.

Figure 1&#160;shows the entire CPB circuit. The physiological variables in this model are shown in Figure 2, and include rectal temperature, mean arterial blood pressure, and heart rate. Figure 3 shows the arterial blood gas analyses during CPB, including partial pressure of arterial oxygen, partial pressure of arterial carbon dioxide, hematocrit, base excess, serum expression of potassium, and potential of hydrogen. Throughout the procedure, the heart rate and mean arterial pressure were stable. The rectal temperature and hematocrit decreased at the beginning of CPB because of hemodilution caused by the priming volume. The partial pressure of arterial oxygen significantly increased during CPB due to membrane oxygenation. Representative hematoxylin and eosin staining images of lungs are shown in Figure 4, which includes the images of the CPB group (Figure 4B, D) and sham operation group (Figure 4A, C), and the ratios of PaO2/FiO2 after CPB compared with the baseline (Figure 4E). For interstitial edema, inflammatory cell infiltration, and hemorrhage, a significant difference was observed in the CPB group. Figure 5 shows serum concentration of tumor necrosis factor-&#945; (TNF-&#945;), interleukin-6 (IL-6), and high mobility group box 1 (HMGB1) levels after CPB.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56986/56986fig1.jpg" /> 
Figure 1: The CPB circuit. The entire CPB circuit consists of a reservoir (8 mL), a membrane oxygenator with a priming volume of 3.3 mL, and a roller pump. All parts are connected via polyvinyl chloride tubes, including a venous drainage line (outer diameter (OD), 3.3 mm; inner diameter (ID), 2 mm; about 1.4 mL), an arterial line (OD, 2 mm; ID, 1.2 mm; about 1.2 mL) and a tube attaching the pump roller (OD, 6.6 mm; ID, 4.5 mm; about 2.8 mL). Total priming volume is 11 mL including approximately 3 mL in the reservoir. <a href="https://cloudflare.jove.com/files/ftp_upload/56986/56986fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56986/56986fig2.jpg" /> 
Figure 2: Physiological variables. Physiological variables during and after CPB, including (A) rectal temperature, (B) mean arterial blood pressure, and (C) heart rate (n = 6). The points and error bars are presented as mean and standard deviation. CPB, cardiopulmonary bypass; Pre, before CPB; CPB X, X min after the initiation of CPB; Post X, X min after the end of CPB. <a href="https://cloudflare.jove.com/files/ftp_upload/56986/56986fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56986/56986fig3.jpg" /> 
Figure 3: Arterial blood gas analyses. Arterial blood gas analyses during and after CPB. (A) partial pressure of arterial oxygen, (B) partial pressure of arterial carbon dioxide, (C) hematocrit, (D) base excess, (E) serum expression of potassium, and (F) potential of hydrogen (n = 6). The points and error bars are presented as mean and standard deviation. CPB, cardiopulmonary bypass; Pre, before CPB; CPB X, X min after the initiation of CPB; Post X, X min after the end of CPB. <a href="https://cloudflare.jove.com/files/ftp_upload/56986/56986fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56986/56986fig4.jpg" /> 
Figure 4: Histological analysis for lungs. Representative hematoxylin and eosin staining images for the sham operation group (A, C) and the cardiopulmonary bypass performed group (B, D).&#160;4 h after CPB. The severity of respiratory distress was correlated to the extent of pathological findings in edema and hemorrhage. Scale bars; 50 &#181;m (A, B), 1 mm (C, D). (E) The ratio of PaO2/FiO2 after CPB compared with the baseline in sham group and control group (n = 6 in each group). The points and error bars are presented as mean and standard deviation. *p &#60; 0.01 vs. Sham group. CPB, cardiopulmonary bypass; PaO2, partial pressure of arterial oxygen; FiO2, fraction of inspired oxygen; Post X, X min after the end of CPB. <a href="https://cloudflare.jove.com/files/ftp_upload/56986/56986fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56986/56986fig5.jpg" /> 
Figure 5: Evaluation of inflammatory responses in serum. Serum concentration of (A) tumor necrosis factor-&#945; (TNF-&#945;), (B) interleukin-6 (IL-6), and (C) high mobility group box 1 (HMGB1) levels after CPB. n = 5 for each group: Sham group and CPB group, rats undergoing CPB. The points and error bars are presented as mean and standard deviation. *p &#60; 0.05, &#8224;p &#60; 0.001 vs. Sham group. CPB, cardiopulmonary bypass; Post X, X min after the end of CPB. <a href="https://cloudflare.jove.com/files/ftp_upload/56986/56986fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Recovery Cardiopulmonary Bypass Model Without Transfusion or Inotropic Agents in Rats at JoVE.com

A Recovery Cardiopulmonary Bypass Model Without Transfusion or Inotropic Agents in Rats

Here, we present a protocol to describe a simple recovery cardiopulmonary bypass model without transfusion or inotropic agents in a rat. This model allows the study of the long-term multiple organ sequelae of cardiopulmonary bypass.

Cardiopulmonary bypass (CPB) is indispensable in cardiovascular surgery. Despite the dramatic refinement of CPB technique and devices, multi-organ ...

a-recovery-cardiopulmonary-bypass-model-without-transfusion-or

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

7.8K Views.  Kyoto University. Here, we present a protocol to describe a simple recovery cardiopulmonary bypass model without transfusion or inotropic agents in a rat. This model allows the study of the long-term multiple organ sequelae of cardiopulmonary bypass.

Video: A Recovery Cardiopulmonary Bypass Model Without Transfusion or Inotropic Agents in Rats

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 ...

Reverse Genetics Mediated Recovery of Infectious Murine Norovirus

Human noroviruses are responsible for most cases of human gastroenteritis (GE) worldwide and are recurrent problem in environments where close person-to-person contact cannot be avoided 1, 2. During the last few years an increase in the incidence of outbreaks in hospitals has been reported, causing significant disruptions to their operational capacity as well as large economic losses. The identification of new antiviral approaches has been limited due to the inability of human noroviruses to complete a productive infection in cell culture 3. The recent isolation of a murine norovirus (MNV), closely related to human norovirus 4 but which can be propagated in cells 5 has opened new avenues for the investigation of these pathogens 6, 7. 
MNV replication results in the synthesis of new positive sense genomic and subgenomic RNA molecules, the latter of which corresponds to the last third of the viral genome (Figure 1). MNV contains four different open reading frames (ORFs), of which ORF1 occupies most of the genome and encodes seven non-structural proteins (NS1-7) released from a polyprotein precursor. ORF2 and ORF3 are contained within the subgenomic RNA region and encode the capsid proteins (VP1 and VP2, respectively) (Figure 1). Recently, we have identified that additional ORF4 overlapping ORF2 but in a different reading frame is functional and encodes for a mitochondrial localised virulence factor (VF1) 8.
Replication for positive sense RNA viruses, including noroviruses, takes place in the cytoplasm resulting in the synthesis of new uncapped RNA genomes. To promote viral translation, viruses exploit different strategies aimed at recruiting the cellular protein synthesis machinery 9-11. Interestingly, norovirus translation is driven by the multifunctional viral protein-primer VPg covalently linked to the 5' end of both genomic and subgenomic RNAs 12-14. This sophisticated mechanism of translation is likely to be a major factor in the limited efficiency of viral recovery by conventional reverse genetics approaches. 
Here we report two different strategies based on the generation of murine norovirus-1 (referred to as MNV herewith) transcripts capped at the 5' end. One of the methods involves both in vitro synthesis and capping of viral RNA, whereas the second approach entails the transcription of MNV cDNA in cells expressing T7 RNA polymerase. The availability of these reverse genetics systems for the study of MNV and a small animal model has provided an unprecedented ability to dissect the role of viral sequences in replication and pathogenesis 15-17.

Noroviruses are a major cause of gastroenteritis yet molecular techniques for their characterisation are still relatively new. Here we report two different reverse genetics approaches for the efficient recovery of murine norovirus (MNV), the only member of this genus which can be propagated in cell culture.

Human noroviruses are responsible for most cases of human gastroenteritis (GE) worldwide and are recurrent problem in environments where close ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or A. baumannii

Efficacy of candidate antibacterial treatments must be demonstrated in animal models of infection as part of the discovery and development process, preferably in models which mimic the intended clinical indication. A method for inducing robust lung infections in immunocompetent rats and mice is described which allows for the assessment of treatments in a model of serious pneumonia caused by S. pneumoniae, H. influenzae, P. aeruginosa, K. pneumoniae or A. baumannii. Animals are anesthetized, and an agar-based inoculum is deposited deep into the lung via nonsurgical intratracheal intubation. The resulting infection is consistent, reproducible, and stable for at least 48 h and up to 96 h for most isolates. Studies with marketed antibacterials have demonstrated good correlation between in vivo efficacy and in vitro susceptibility, and concordance between pharmacokinetic/pharmacodynamic targets determined in this model and clinically accepted targets has been observed. Although there is an initial time investment when learning the technique, it can be performed quickly and efficiently once proficiency is achieved. Benefits of the model include elimination of the neutropenic requirement, increased robustness and reproducibility, ability to study more pathogens and isolates, improved flexibility in study design and establishment of a challenging infection in an immunocompetent host.

A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or  A. baumannii

A method for establishing lung infections in immunocompetent rodents is described. With proficiency, this method can be performed quickly and easily to induce stable infection with many isolates of S. pneumoniae, H. influenzae, P. aeruginosa, K. pneumoniae and A. baumannii.

Efficacy of candidate antibacterial treatments must be demonstrated in animal models of infection as part of the discovery and development process, ...

Optimized Interferon-gamma ELISpot Assay to Measure T Cell Responses in the Guinea Pig Model after Vaccination

The guinea pig has played a pivotal role as a relevant small animal model in the development of vaccines for infectious diseases such as tuberculosis, influenza, diphtheria, and viral hemorrhagic fevers. We have demonstrated that plasmid-DNA (pDNA) vaccine delivery into the skin elicits robust humoral responses in the guinea pig. However, the use of this animal to model immune responses was somewhat limited in the past due to the lack of available reagents and protocols to study T cell responses. T cells play a pivotal role in both immunoprophylactic and immunotherapeutic mechanisms. Understanding T cell responses is crucial for the development of infectious disease and oncology vaccines and accommodating delivery devices. Here we describe an interferon-gamma (IFN-&#947;) enzyme-linked immunospot (ELISpot) assay for guinea pig peripheral blood mononuclear cells (PBMCs). The assay enables researchers to characterize vaccine-specific T-cell responses in this important rodent model. The ability to assay cells isolated from the peripheral blood provides the opportunity to track immunogenicity in individual animals.

The development of the interferon-gamma ELISpot assay for guinea pig PBMCs allows the characterization of T-cell responses in this highly relevant model for studying infectious diseases. We have applied the assay to measure T cell responses associated with intradermal delivery of DNA vaccines.

The guinea pig has played a pivotal role as a relevant small animal model in the development of vaccines for infectious diseases such as tuberculosis, ...

A Precise Pathogen Delivery and Recovery System for Murine Models of Secondary Bacterial Pneumonia

Secondary bacterial pneumonias following influenza infections consistently rank within the top ten leading causes of death in the United States. To date, murine models of co-infection have been the primary tool developed to explore the pathologies of both the primary and secondary infections. Despite the prevalence of this model, considerable discrepancies regarding instillation procedures, dose volumes, and efficacies are prevalent among studies. Furthermore, these efforts have been largely incomplete in addressing how the pathogen may be directly influencing disease progression post-infection. Herein we provide a precise method of pathogen delivery, recovery, and analysis to be used in murine models of secondary bacterial pneumonia. We demonstrate that intratracheal instillation enables an efficient and accurate delivery of controlled volumes directly and evenly into the lower respiratory tract. Lungs can be excised to recover and quantify the pathogen burden. Following excision of the infected lungs, we describe a method to extract high quality pathogen RNA for subsequent transcriptional analysis. This procedure benefits from being a non-surgical method of delivery without the use of specialized laboratory equipment and provides a reproducible strategy to investigate pathogen contributions to secondary bacterial pneumonia.

Here, we present methods to improve secondary bacterial pneumonia studies by providing a non-invasive route of instillation into the lower respiratory tract followed by pathogen recovery and transcript analysis. These procedures are reproducible and can be performed without specialized equipment such as cannulas, guide wires, or fiber optic cables.

Secondary bacterial pneumonias following influenza infections consistently rank within the top ten leading causes of death in the United States. To date, ...

Single-cell Screening Method for the Selection and Recovery of Antibodies with Desired Specificities from Enriched Human Memory B Cell Populations

The human antibody repertoire represents a largely untapped source of potential therapeutic antibodies and useful biomarkers. While current computational methods, such as next generation sequencing (NGS), yield enormous sets of data on the antibody repertoire at the sequence level, functional data is required to identify which sequences are relevant for a particular antigen or set of antigens. Here, we describe a method to identify and recover individual antigen-specific antibodies from peripheral blood mononuclear cells (PBMCs) from a human blood donor. This method utilizes an initial enrichment of mature B cells and requires a combination of phenotypic cell markers and fluorescently-labeled protein to isolate IgG memory B cells via flow cytometry. The heavy and light chain variable regions are then cloned and re-screened. Although limited to the memory B cell compartment, this method takes advantage of flow cytometry to interrogate millions of B cells and returns paired heavy and light chain sequences from a single cell in a format ready for expression and confirmation of specificity. Antibodies recovered with this method can be considered for therapeutic potential, but can also link specificity and function with bioinformatic approaches to assess the B cell repertoire within individuals.

The BSelex method to identify and recover individual antigen-specific antibodies from human peripheral blood mononuclear cells combines flow cytometry with single cell PCR and cloning.

The human antibody repertoire represents a largely untapped source of potential therapeutic antibodies and useful biomarkers. While current computational ...

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are cleared from the human lung alveoli by being phagocytosed by innate monocytes and/or neutrophils. This protocol offers a fast and reliable measurement of phagocytosis by flow cytometry using fluorescein isothiocyanate (FITC)-labeled conidia for co-incubation with human leukocytes and subsequent counterstaining with an anti-FITC antibody to allow discrimination of internalized and cell-adherent conidia. Major advantages of this protocol are its rapidness, the possibility to combine the assay with cytometric analysis of other cell markers of interest, the simultaneous analysis of monocytes and neutrophils from a single sample and its applicability to other cell wall-bearing fungi or bacteria. Determination of percentages of phagocytosing leukocytes provides a means to microbiologists for evaluating virulence of a pathogen or for comparing pathogen wildtypes and mutants as well as to immunologists for investigating human leukocyte capabilities to combat pathogens.

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

This protocol provides a fast and reliable method to quantitatively measure phagocytosis of Aspergillus fumigatus conidia by human primary phagocytes using flow cytometry and to discriminate phagocytosis of conidia from mere adhesion to leukocytes.

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are ...

Visualization and Quantification of High-Dimensional Cytometry Data using Cytofast and the Upstream Clustering Methods FlowSOM and Cytosplore

The complexity of data generated by mass cytometry has necessitated new tools to rapidly visualize analytic outcomes. Clustering methods like Cytosplore or FlowSOM are used for the visualization and identification of cell clusters. For downstream analysis, a newly developed R package, Cytofast, can generate a rapid visualization of results from clustering methods. Cytofast takes into account the phenotypic characterization of cell clusters, calculates the cell cluster abundance, then quantitatively compares groups. This protocol explains the applications of Cytofast to the use of mass cytometry data based on modulation of the immune system in the tumor microenvironment (i.e., the natural killer [NK] cell response) upon tumor challenge followed by immunotherapy (PD-L1 blockade). Demonstration of the usefulness of Cytofast with FlowSOM and Cytosplore is shown. Cytofast rapidly generates visual representations of group-related immune cell clusters and correlations with immune system composition. Differences are observed in the clustering analysis, but separation between groups are visible with both clustering methods. Cytofast visually shows the patterns induced by PD-L1 treatment that include a higher abundance of activated NK cell subsets, expressing a higher intensity of activation markers (i.e., CD54 or CD11c).

Cytofast is a visualization tool used to analyze output from clustering. Cytofast can be used to compare two clustering methods: FlowSOM and Cytosplore. Cytofast can rapidly generate a quantitative and qualitative overview of mass cytometry data and highlight the main differences between different clustering algorithms.

The complexity of data generated by mass cytometry has necessitated new tools to rapidly visualize analytic outcomes. Clustering methods like Cytosplore ...