<meta charset="utf8"></head><body>تطوير زراعة الرئة مع التروية القائمة على PolyhHb في نماذج EVLP للفئران

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
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			<td>10 cc insulin syringe 29 G x 1/2&#34; needle</td>
			<td>B-D</td>
			<td>309301</td>
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			<td>Calcium Chloride dihydrate (CaCl2.2H2O)</td>
			<td>Sigma Aldrich</td>
			<td>10035-04-8</td>
			<td>For modified Ringer&#39;s lactate</td>
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			<td>&#160;VK 73-3763</td>
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			<td>11253-25</td>
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			<td>Lauda</td>
			<td>LCK 1879</td>
			<td></td>
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			<td>Expired human leukoreduced, packed RBC units</td>
			<td>Wexner Medical Center 
			Canadian Blood Services 
			Zen-Bio Inc</td>
			<td></td>
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			<td>Fiberoxygenator D150</td>
			<td>Hugo Sachs Elektronik</td>
			<td>PY2 73-3762</td>
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			<td>Forceps</td>
			<td>Fine Science tools</td>
			<td>11027-12</td>
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			<td>Glutaraldehyde (C5H8O2 70 wt%)</td>
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			<td>111-30-8 (G7776)</td>
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			<td>Halsted-Mosquito Hemostat</td>
			<td>Roboz Surgical</td>
			<td>RS-7112</td>
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			<td>Heparin 30,000 units per 30 ml</td>
			<td>APP Pharmaceuticals</td>
			<td></td>
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			<td>Perfusate additive</td>
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			<td>IL2 Tube set for perfusate</td>
			<td>Harvard Apparatus</td>
			<td>733842</td>
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			<td>IPL-2 Basic Lung Perfusion System</td>
			<td>Harvard Apparatus</td>
			<td></td>
			<td></td>
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			<td></td>
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			<td>Left Atrium cannula</td>
			<td>Harvard Apparatus</td>
			<td>730712</td>
			<td></td>
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			<td>G420</td>
			<td>gas contactor</td>
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			<td>low potassium dextran glucose solution (perfadex)</td>
			<td>XVIVO</td>
			<td></td>
			<td>solution flushing the lung</td>
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			<td>Masterflex Platinum Coated Tubing(Size: 73,17,16,24)</td>
			<td>Cole-Palmer</td>
			<td></td>
			<td></td>
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			<td>N-Acetyl-L-cysteine (NALC, C5H9NO3S)</td>
			<td>Sigma Aldrich</td>
			<td>616-91-1 (A7250)</td>
			<td>For modified Ringer&#39;s lactate</td>
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			<td>Nalgene Vessels (10L, 20L)</td>
			<td>Nalgene</td>
			<td></td>
			<td>Filtration vessels</td>
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			<td>Peristaltic Pump</td>
			<td>&#160;Ismatec</td>
			<td>&#160;ISM 827B</td>
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			<td>PES, 0.65 &#181;m TFF module</td>
			<td>Repligen</td>
			<td>N02-E65U-07-N</td>
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			<td>Kent Scientific Corporation</td>
			<td>PS-MSTAT-RT</td>
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			<td>polyethersulfone (PES), 0.2 &#181;m TFF module</td>
			<td>Repligen</td>
			<td>N02-S20U-05-N</td>
			<td></td>
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			<td>Polysulfone (PS), 500 kDa TFF module</td>
			<td>Repligen</td>
			<td>N02-P500-05-N</td>
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			<td>Potassium Chloride (KCl)</td>
			<td>Fisher Scientific</td>
			<td>7447-40-7</td>
			<td>For PBS</td>
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			<td>&#160;ADInstruments</td>
			<td>730045</td>
			<td></td>
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			<td>Pulmonary Artery cannula</td>
			<td>Harvard Apparatus</td>
			<td>730710</td>
			<td></td>
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			<td>Pump Head tubing (Size: 73,17,16,24)</td>
			<td>PharMed BPT</td>
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			<td>Puralube&#160;Ophthalmic Ointment</td>
			<td>Dechra</td>
			<td>NA</td>
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			<td>Scissors</td>
			<td>Fine Science tools</td>
			<td>14090-11</td>
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			<td>SCP Servo controller for perfusion type 704</td>
			<td>Harvard Apparatus</td>
			<td>732806</td>
			<td></td>
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			<td>Small Animal Ventilator model 683</td>
			<td>Harvard Apparatus</td>
			<td>55-000</td>
			<td></td>
		</tr>
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			<td>Sodium Chloride (NaCl)</td>
			<td>Fisher Scientific</td>
			<td>7647-14-5 (S271-10)</td>
			<td>For PBS and saline</td>
		</tr>
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			<td>Sodium cyanoborohydride (NaCNBH3)</td>
			<td>Sigma Aldrich</td>
			<td>25895-60-7</td>
			<td></td>
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			<td>Sodium Dithionite (Na2S2O4)</td>
			<td>Sigma Aldrich</td>
			<td>7775-14-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide (NaOH)</td>
			<td>Fisher Scientific</td>
			<td>1310-73-2</td>
			<td>For modified Ringer&#39;s lactate</td>
		</tr>
		<tr>
			<td>Sodium Lactate (NaC3H5O3)</td>
			<td>Sigma Aldrich</td>
			<td>867-56-1</td>
			<td>For modified Ringer&#39;s lactate</td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic (Na2HPO4)</td>
			<td>Fisher Scientific</td>
			<td>7558-79-4</td>
			<td>For PBS</td>
		</tr>
		<tr>
			<td>Sodium phosphate monobasic (NaH2PO4)</td>
			<td>Fisher Scientific</td>
			<td>7558-80-7</td>
			<td>For PBS</td>
		</tr>
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			<td>SomnoSuite Small Animal Anesthesia System</td>
			<td>Kent Scientific Corporation</td>
			<td>SS-MVG-Module</td>
			<td></td>
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			<td>Sprague-Dawley rats</td>
			<td>Envigo</td>
			<td></td>
			<td></td>
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			<td>TAM-A transducer amplifier module type 705/1</td>
			<td>Harvard Apparatus</td>
			<td>73-0065</td>
			<td></td>
		</tr>
		<tr>
			<td>TAM-D transducer amplifier type 705/2</td>
			<td>Harvard Apparatus</td>
			<td>&#160;73-1793</td>
			<td></td>
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			<td>TCM time control module type 686</td>
			<td>Harvard Apparatus</td>
			<td>731750</td>
			<td></td>
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			<td>Tracheal cannula</td>
			<td>Harvard Apparatus</td>
			<td>733557</td>
			<td></td>
		</tr>
		<tr>
			<td>Tube set for moist chamber</td>
			<td>Harvard Apparatus</td>
			<td>&#160;73V83157</td>
			<td></td>
		</tr>
		<tr>
			<td>Tubing Cassette</td>
			<td>Cole-Parmer</td>
			<td>IS 0649</td>
			<td></td>
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			<td>Tweezer #5 Dumostar</td>
			<td>Kent Scientific Corporation</td>
			<td>&#160;INS500085-A</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezer #5 stainless steel, curved</td>
			<td>Kent Scientific Corporation</td>
			<td>IND500232</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezer #7 Titanium</td>
			<td>Kent Scientific Corporation</td>
			<td>&#160;INS600187</td>
			<td></td>
		</tr>
		<tr>
			<td>Tygon E-3603 Tubing 2.4 mm ID</td>
			<td>Harvard Apparatus</td>
			<td>721017</td>
			<td>perfusate line entering lung</td>
		</tr>
		<tr>
			<td>Tygon E-3603 Tubing 3.2 mm ID</td>
			<td>Harvard Apparatus</td>
			<td>721019</td>
			<td>perfusate line leaving lung</td>
		</tr>
		<tr>
			<td>Vannas-Tubingen Spring Scissors</td>
			<td>Fine Science Tools</td>
			<td>15008-08</td>
			<td></td>
		</tr>
		<tr>
			<td>VCM ventilator control module type 681</td>
			<td>Harvard Apparatus</td>
			<td>731741</td>
			<td></td>
		</tr>
		<tr>
			<td>William&#39;s E Media</td>
			<td>Gibco, ThermoFisher Scientific</td>
			<td>A12176-01</td>
			<td>Perfusate additive</td>
		</tr>
		<tr>
			<td>Xylazine 100 mg per 1 ml</td>
			<td>Akorn</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

exploring alternative perfusion solutions using next-generation polymerized hemoglobin-based oxygen carriers in a model of rat ex vivo lung perfusion

Like any field in solid organ transplantation, lung transplantation suffers from a shortage of donor organs. In order to increase the donor pool, significant research has been dedicated to investigating the potential of allografts that were once thought to be unsuitable for transplantation, i.e., extended criteria donors (ECD). These allografts can be considered ECD for a milieu of reasons, including questionable quality, poor function, infection, trauma, prolonged warm or cold ischemic times, and advanced age1,2. In certain cases, where these lungs are suitable for immediate transplant3, it is often advantageous to providers and recipients alike to evaluate these lungs for an additional time to determine their suitability for transplantation. Ex vivo lung perfusion (EVLP) is such a technology that allows for extended assessment of potential lung allografts in a closed circuit outside the donor2,4,5,6,7, affording the transplant provider the ability to determine transplantation suitability. EVLP has shown the ability to adequately assess donor organs8,9,10,11, decrease the effects of ischemic reperfusion injury (IRI)12,13 and increase the donor pool14,15 thus making lung transplantation a more accessible treatment for all.
In general, an EVLP system is a closed system with a ventilatory circuit (achieved by connecting a ventilator to the trachea to introduce air into the system) and a vascular circuit (achieved by connecting the left atrium (LA) to the pulmonary artery (PA) with tubing)7. The vascular circuit has perfusate running through the tubing to give the lung vital nutrients and oxygen while limiting the cold ischemic time (CIT)5,8,16,17. This solution is either blood-based (i.e., via the addition of packed red blood cells (PRBCs))16,17 or acellular-based (i.e., no PRBCs)4,5. However, there are several notable disadvantages to using PRBCs. If using PRBCs from donors who died from trauma or brain-dead donors (BDD), these fluids often contain large amounts of inflammatory cytokines, which may increase cellular damage during EVLP as well as increase levels of cell-free hemoglobin (Hb), heme, iron, and cell fragments which deliver additional damage to cells18,19. Furthermore, as these donors are often multi-organ, the collection of PRBCs prior to procurement could lead to decreasing blood volume in the donor and subsequently increasing ischemia to all organs. If using PRBCs from another source, providers could face blood shortages as this is a scarce material in and of itself20,21. Finally, PRBCs are prone to mechanical lysis on the EVLP circuit regardless of their source, releasing Hb and other components that contribute to cellular damage.
Thus, for many reasons, it could be advantageous to use an artificial red blood cell substitute, i.e., hemoglobin-based oxygen carriers (HBOCs), as a perfusate supplement. One particularly promising HBOC is polymerized human hemoglobin (PolyhHb). PolyhHb is synthesized from Hb purified from expired PRBCs that were deemed unsuitable for immediate transfusion22. They have been shown to be viable blood substitutes in hemorrhagic shock23 and transplantation24 and can be produced in large quantities22. However, large-scale adoption of PolyhHb has been unsuccessful due to unforeseen complications such as vasoconstriction, increasing blood pressure, and cardiac arrest23,25. The reasons behind these findings were likely due to the presence of cell-free Hb or low molecular weight Hb polymers (&#60; 500 kDa) in the PolyhHb solution, as they have a propensity to extravasate into the tissue space, which resulted in decreased nitric oxide availability, subsequent vasoconstriction, systemic hypertension, and ultimately oxidative tissue injury26,27. To improve upon these issues, the Palmer Laboratory has worked to develop a next-generation PolyhHb that contains minimal low MW species and cell-free Hb, which has demonstrated improved biophysical characteristics and in vivo responses22,28,29,30. Several transfusion studies in animals have shown that if low molecular weight Hb polymers are eliminated from the HBOC, vasoconstriction, systemic hypertension, and oxidative damage can be mitigated28,29,31,32,33,34,35. Therefore, making this next-generation PolyhHb a promising perfusate candidate.
Here, we describe the application of a next-generation PolyhHb to be used in a perfusate and the protocol by which this perfusion solution can be tested in a model of rat EVLP. The goal of this study is to provide the lung transplant community with key information in designing and developing novel perfusion solutions, as well as provide protocols to test them in clinically relevant translational transplant models.

<meta charset="utf8"></head><body>تسليط الضوء على المؤلف: استخدام الجيل التالي من الهيموغلوبين البشري المبلمر لتحسين تقييم الرئة المانحة والحفاظ عليها في الفئران

استكشاف حلول التروية البديلة باستخدام الجيل التالي من ناقلات الأكسجين المبلمرة القائمة على الهيموجلوبين في نموذج لنضح الرئة خارج الجسم الحي للفئران

Ex vivo lung perfusion or EVLP enables extended evaluation of donor lungs. There are two perfusion solutions, an acellular and cellular. This study details using a polymerized human hemoglobin or a poly HB-based oxygen carrier as a perfusate in the protocol for testing this solution in a rat EVLP model.The recent advances in ex vivo organ perfusion have enabled expanding the donor organ pool with improved function. Working to extend normothermic profusion times with tailored perfusates will allow for further advances in this space. Current perfusion techniques use cellular or acellular perfusates, each with pros and cons.Perfusion composition is crucial for metabolic support, reducing inflammation and maintaining oncotic pressure. However, existing solutions can cause pulmonary edema and damage. Thus, it is necessary to develop novel perfusion solutions that prevent excessive damage while maintaining proper cellular homeostasis.A promising hemoglobin-based oxygen carrier or HBOC is polymerized human hemoglobin synthesized from expired packed red blood cells. Our laboratory developed a next generation poly HB with minimal low molecular weight species and cell-free hemoglobin. This has shown improved biophysical characteristics and has reduced vasoconstriction, hypertension, and oxidative damage in animal studies.These characteristics make it a promising perfusate candidate.

Lung transplantation is hampered by the lack of suitable donors. Previously, donors that were thought to be marginal or inadequate were discarded. ...

exploring-alternative-perfusion-solutions-using-next-generation

Research

JoVE Journal

Medicine

Exploring Alternative Perfusion Solutions Using Next-Generation Polymerized Hemoglobin-Based Oxygen Carriers in a Model of Rat Ex Vivo Lung Perfusion

286 Views.  The Ohio State University Wexner Medical Center. Ex vivo lung perfusion or EVLP enables extended evaluation of donor lungs. There are two perfusion solutions, an acellular and cellular. This study details using a polymerized human hemoglobin or a poly HB-based oxygen carrier as a perfusate in the protocol for testing this solution in a rat EVLP model.The recent advances in ex vivo organ perfusion have enabled expanding the donor organ pool with improved function. Working to extend normothermic profusion times with tailored perfusates...