This study was supported by a Biomedical Research Institute Grant from Pusan National University Hospital.

This study was approved by the administration of Pusan National University of Medicine and was conducted in accordance with ethical guidelines for the use and care of animals. (certificate no. 2017-119). Prior to any animal studies, institutional approval should be obtained. 
NOTE: All surgical and anesthetic instruments/equipment and reagents recommended for successful surgical presentation and imaging of abdominal organs are detailed in Table 1.
1. Preparation procedures for harvesting of rat kidneys
<ol>
	<li>In preparation for surgery, place 8-week-old Sprague-Dawley rats (weighing 200&#8211;250 g) on a warming pad. Place a rectal thermometer probe in the rectum to monitor core temperature.</li>
	<li>Anesthetize the rat with a 5% mixture of isoflurane gas (induction: 5%, maintenance: 3%).</li>
	<li>To start the operation, place the rat in a supine position after administration of anesthesia. Mount the four limbs of the rat on the operation table with tape.</li>
	<li>Shave and clean the abdomen of the donor rat with germicidal soap. Apply 2% betadine for at least 1&#8211;2 min, and wipe with a 70% ethanol solution. Repeat this sequence three times.</li>
	<li>Cover the operative field with a sterile fenestrated drape.</li>
	<li>Make a vertical abdominal incision and expose the left kidney, ureter, abdominal aorta, and inferior vena cava.</li>
	<li>Visualize and dissect the left kidney, ureter, abdominal aorta, and inferior vena cava just before cutting the pedicle.</li>
</ol>
2. Transcardial perfusion
<ol>
	<li>Before surgery, prepare the perfusion solution.
	<ol>
		<li>Make 50 mL of perfusion solution per rat.</li>
		<li>Mix 1x PBS with approximately 10 U/mL heparin (1 25 kU vial will make 2.5 L of PBS+Hep).</li>
		<li>Mix equal volumes of 8% paraformaldehyde with 1x PBS to make the 4% PFA/1xPBS solution. 
		NOTE: 8% PFA made in water can be stored at 4 &#176;C for up to 2 months. However, 4% PFA diluted in PBS is only stable for 1 week at 4 &#176;C. Make the dilution fresh.</li>
	</ol>
	</li>
	<li>Extend the vertical abdominal incision cranially. Be sure to draw the scissors away from the organs when cutting to avoid damaging the internal organs.</li>
	<li>Continue the incision through the rib cage, and then cut through the diaphragm by lifting the sternum.</li>
	<li>Pin the loose flap of skin out of the way, and free the heart by tearing any connective tissue with the forceps. 
	CAUTION: Do not use the scissors to free the heart and this could result in unwanted bleeding.</li>
	<li>Open the phosphate-buffered saline (PBS) line and ensure that the line is flowing before placing the needle into the left ventricle. Hold the heart gently with blunt forceps, and use a hemostat to control the needle. The needle should be inserted no more than 1/4 inch. 
	NOTE: Insertion greater than 1/4 inch may result in perforation to the other side of the tissue.</li>
	<li>While supporting the heart with the needle and hemostat, locate the right atrium and snip through it with iridectomy scissors. Rest the hemostat on the rat&#8217;s body, and make sure the needle is still positioned inside the heart. 
	NOTE: If the cut is sufficient, there should be blood in the body cavity as the pressure from the PBS flowing into the rat is relieved.</li>
	<li>Carefully unpin the front feet and skin flap.</li>
	<li>Continue perfusing PBS for 4 min or longer if there is still blood visible in the kidney and liver.</li>
</ol>
3. Kidney harvesting and decellularization
<ol>
	<li>Harvest the left kidney with the abdominal aorta and inferior vena cava.</li>
	<li>Ligate the ureter, thoracic aorta, superior vena cava, and branches of the abdominal aorta.</li>
	<li>Keep the organ hydrated in Dulbecco&#8217;s PBS (DPBS) in a 10 cm Petri dish.</li>
	<li>Cannulate the abdominal aorta and inferior vena cava with a 23 Gauge catheter. To remove residual blood, connect the cannula with a peristaltic pump, and wash with DPBS (500 mL) and 16 U/mL heparin for 90 min at a rate of 5 rpm at 37 &#176;C.</li>
	<li>To decellularize the kidney, perfuse the kidney with 1% Triton X-100 (1 L) for 3 h and then with 0.75% sodium dodecyl sulfate (SDS) solution (2 L) for 6 h at a constant pressure of 40 mmHg. 
	NOTE: The kidney will become transparent after 8 h.</li>
	<li>To remove residual SDS, perfuse the sample with 1% penicillin in distilled water (6 L) for 18 h (overnight) and then with sterile DPBS (500 mL) and 16 U/mL heparin for 90 min.</li>
</ol>

<ol>
	<li>Kawai, 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=Brief+report:+HLA-mismatched+renal+transplantation+without+maintenance+immunosuppression.">Brief report: HLA-mismatched renal transplantation without maintenance immunosuppression.</a> New England Journal of Medicine. 358 (4), 353-361 (2008).</li><li>Rana, D., Zreiqat, H., Benkirane-Jessel, N., Ramakrishna, S., Ramalingam, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+decellularized+scaffolds+for+stem+cell-driven+tissue+engineering.">Development of decellularized scaffolds for stem cell-driven tissue engineering.</a> Journal of Tissue Engineering and Regenerative Medicine. 11 (4), 942-965 (2017).</li><li>Fu, R. H., 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=Decellularization+and+recellularization+technologies+in+tissue+engineering.">Decellularization and recellularization technologies in tissue engineering.</a> Cell Transplantation. 23 (4-5), 621-630 (2014).</li><li>Hopkinson, 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=Optimization+of+amniotic+membrane+(AM)+denuding+for+tissue+engineering.">Optimization of amniotic membrane (AM) denuding for tissue engineering.</a> Tissue Engineering. Part C, Methods. 14 (4), 371-381 (2008).</li><li>Shupe, T., Williams, M., Brown, A., Willenberg, B., Petersen, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+the+decellularization+of+intact+rat+liver.">Method for the decellularization of intact rat liver.</a> Organogenesis. 6 (2), 134-136 (2010).</li><li>Petersen, T. H., 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=Tissue-engineered+lungs+for+in+vivo+implantation.">Tissue-engineered lungs for in vivo implantation.</a> Science. 329 (5991), 538-541 (2010).</li><li>Fernandez-Perez, J., Ahearne, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+decellularization+methods+on+extracellular+matrix+derived+hydrogels.">The impact of decellularization methods on extracellular matrix derived hydrogels.</a> Scientific Reports. 9 (1), 14933 (2019).</li><li>Naik, A., Griffin, M., Szarko, M., Butler, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+the+decellularization+process+of+an+upper+limb+skeletal+muscle;+implications+for+muscle+tissue+engineering.">Optimizing the decellularization process of an upper limb skeletal muscle; implications for muscle tissue engineering.</a> Artificial Organs. 44 (2), 178-183 (2020).</li><li>Crapo, P. M., Gilbert, T. W., Badylak, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+overview+of+tissue+and+whole+organ+decellularization+processes.">An overview of tissue and whole organ decellularization processes.</a> Biomaterials. 32 (12), 3233-3243 (2011).</li><li>Mason, C., Dunnill, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+brief+definition+of+regenerative+medicine.">A brief definition of regenerative medicine.</a> Regenerative Medicine. 3 (1), 1-5 (2008).</li><li>Guyette, J. P., 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=Perfusion+decellularization+of+whole+organs.">Perfusion decellularization of whole organs.</a> Nature Protocols. 9 (6), 1451-1468 (2014).</li><li>Song, J. J., 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=Regeneration+and+experimental+orthotopic+transplantation+of+a+bioengineered+kidney.">Regeneration and experimental orthotopic transplantation of a bioengineered kidney.</a> Nature Medicine. 19 (5), 646-651 (2013).</li></ol>

The authors have no conflicts of interest to disclose.

Various protocols have been used for decellularization of organs and other tissues. The optimal decellularization protocol should preserve the three-dimensional architecture of the extracellular matrix (ECM). In general, such protocols consist of lysing the cell membrane by physical processing or ionic solutions, dissociating the cytoplasm and nucleus from the ECM by enzymatic processing or detergents, and then removing cellular debris from the tissue3. Physical processes include scraping, solutio...

Cell culture techniques are applied for regeneration of functional tissues and organs to replace diseased or damaged organs. Allogenic organ transplantation is currently the most common treatment for irreversible organ damage; however, this approach requires the use of immunosuppression to prevent rejection of the transplanted organ. Moreover, despite advances in transplant immunology, 20% of transplant recipients may experience acute rejection within 5 years, and within 10 years after transplantation, 40% of recipients may lose their transplanted graft or die1.
Advances in tissue engineering technologies have yielded in a new paradigm for transplantation of new organs without immune rejection using differentiated stem cells. After stem cell differentiation, a scaffold, called a synthetic extracellular matrix, is needed to facilitate the generation of three-dimensional organs and enable the new tissue to thrive within the recipient. Scaffolds from decellularized native organs have advantages, including a more effective environment for establishment of cells and enhancement of stem cell proliferation, although these mechanisms have not been fully elucidated2. In particular, the kidney is a suitable organ for scaffold generation because it has abundant circulation and a niche for stem cell establishment. Additionally, because of the complex structure of the kidney, it is difficult to artificially regenerate kidneys for organ transplantation.
In this report, we introduce a method of developing vascularized scaffolds using decellularized organs in a rat model to facilitate future animal studies for tissue engineering purposes.

<table>
	<tbody>
		<tr>
			<td>1 cc syringe (inject probes and vehicle solutions</td>
			<td>Becton Dickinson</td>
			<td>305217</td>
			<td></td>
		</tr>
		<tr>
			<td>10-0 ethilon for vessel anastomosis</td>
			<td>Ethicon</td>
			<td>9032G</td>
			<td></td>
		</tr>
		<tr>
			<td>25 gauge inch guide needle(for vascular catheters)</td>
			<td>Becton Dickinson</td>
			<td>305145</td>
			<td></td>
		</tr>
		<tr>
			<td>3-0 PDS incision closure rat</td>
			<td>Ethicon</td>
			<td>Z316H</td>
			<td></td>
		</tr>
		<tr>
			<td>3-0 Prolene incision closure rat</td>
			<td>Ethicon</td>
			<td>8832H</td>
			<td></td>
		</tr>
		<tr>
			<td>3-0 silk spool vascular access/ligation in rat</td>
			<td>Braintree Scientific</td>
			<td>SUT-S 110</td>
			<td></td>
		</tr>
		<tr>
			<td>4-0 PDS incision closure mouse</td>
			<td>Ethicon</td>
			<td>Z773D</td>
			<td></td>
		</tr>
		<tr>
			<td>4-0 Prolene incision closure mouse</td>
			<td>Ethicon</td>
			<td>8831H</td>
			<td></td>
		</tr>
		<tr>
			<td>5-0 silk spool vascular access/ligation in mouse</td>
			<td>Braintree Scientific</td>
			<td>SUT-S 106</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Scissors to cut fascia/connective tissue</td>
			<td>Fine Science Tools</td>
			<td>14058-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Halsey needle holder</td>
			<td>Fine Science Tools</td>
			<td>12001-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Kelly Hemostat for rats: muscle clamp to minimize bleeding when cut</td>
			<td>Fine Science Tools</td>
			<td>13018-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethelyne 50 tubing, catheter tubing 100 ft</td>
			<td>Braintree Scientific</td>
			<td>.023&#34; &#215; .038&#8221;</td>
			<td></td>
		</tr>
		<tr>
			<td>Schwartz microserrefine vascular clamps</td>
			<td>Fine Science Tools</td>
			<td>18052-01 (straight)</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>18052-03 (curved)</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Scissors to cut skin</td>
			<td>Fine Science Tools</td>
			<td>14002-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Vannas-Tubingen Spring scissors for arteriotomy</td>
			<td>Fine Science Tools</td>
			<td>15003-08</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of decellularized kidney scaffolds in rats

Tissue engineering is a cutting-edge discipline in biomedicine. Cell culture techniques can be applied for regeneration of functional tissues and organs to replace diseased or damaged organs. Scaffolds are needed to facilitate the generation of three-dimensional organs or tissues using differentiated stem cells in vivo. In this report, we describe a novel method for developing vascularized scaffolds using decellularized rat kidneys. Eight-week-old Sprague-Dawley rats were used in this study, and heparin was injected into the heart to facilitate flow into the renal vessels, allowing heparin to perfuse into the renal vessels. The abdominal cavity was opened, and the left kidney was collected. The collected kidneys were perfused for 9 h using detergents, such as Triton X-100 and sodium dodecyl sulfate, to decellularize the tissue. Decellularized kidney scaffolds were then gently washed with 1% penicillin/streptomycin and heparin to remove cellular debris and chemical residues. Transplantation of stem cells with the decellularized vascular scaffolds is expected to facilitate the generation of new organs. Thus, the vascularized scaffolds may provide a foundation for tissue engineering of organ grafts in the future.

The gross morphology of rat kidneys was dark red (Figure 1A). After decellularization, the kidney became pale and translucent (Figure 1D). Residual genomic DNA was assessed with a commercial kit according to the manufacturer&#8217;s instructions, in decellularized kidney scaffolds and compared with that in native kidneys (control). Quantitative analysis confirmed that tissue genomic DNA was almost eliminated after decellularization. From 14 cases, the average DN...

Watch this Scientific Journal Video about Preparation of Decellularized Kidney Scaffolds in Rats at JoVE.com

Preparation of Decellularized Kidney Scaffolds in Rats

This protocol introduces a method to develop a scaffold using decellularized rat kidneys. The protocol includes decellularization and recellularization processes to confirm bioavailability. Decellularization is performed using Triton X-100 and sodium dodecyl sulfate.

Tissue engineering is a cutting-edge discipline in biomedicine. Cell culture techniques can be applied for regeneration of functional tissues and organs ...

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4.0K Views.  Pusan National University Hospital. This protocol introduces a method to develop a scaffold using decellularized rat kidneys. The protocol includes decellularization and recellularization processes to confirm bioavailability. Decellularization is performed using Triton X-100 and sodium dodecyl sulfate.

Video: Preparation of Decellularized Kidney Scaffolds in Rats

Acute Myocardial Infarction in Rats

With heart failure leading the cause of death in the USA (Hunt), biomedical research is fundamental to advance medical treatments for cardiovascular diseases. Animal models that mimic human cardiac disease, such as myocardial infarction (MI) and ischemia-reperfusion (IR) that induces heart failure as well as pressure-overload (transverse aortic constriction) that induces cardiac hypertrophy and heart failure (Goldman and Tarnavski), are useful models to study cardiovascular disease. In particular, myocardial ischemia (MI) is a leading cause for cardiovascular morbidity and mortality despite controlling certain risk factors such as arteriosclerosis and treatments via surgical intervention (Thygesen). Furthermore, an acute loss of the myocardium following myocardial ischemia (MI) results in increased loading conditions that induces ventricular remodeling of the infarcted border zone and the remote non-infarcted myocardium. Myocyte apoptosis, necrosis and the resultant increased hemodynamic load activate multiple biochemical intracellular signaling that initiates LV dilatation, hypertrophy, ventricular shape distortion, and collagen scar formation. This pathological remodeling and failure to normalize the increased wall stresses results in progressive dilatation, recruitment of the border zone myocardium into the scar, and eventually deterioration in myocardial contractile function (i.e. heart failure). The progression of LV dysfunction and heart failure in rats is similar to that observed in patients who sustain a large myocardial infarction, survive and subsequently develops heart failure (Goldman). The acute myocardial infarction (AMI) model in rats has been used to mimic human cardiovascular disease; specifically used to study cardiac signaling mechanisms associated with heart failure as well as to assess the contribution of therapeutic strategies for the treatment of heart failure. The method described in this report is the rat model of acute myocardial infarction (AMI). This model is also referred to as an acute ischemic cardiomyopathy or ischemia followed by reperfusion (IR); which is induced by an acute 30-minute period of ischemia by ligation of the left anterior descending artery (LAD) followed by reperfusion of the tissue by releasing the LAD ligation (Vasilyev and McConnell). This protocol will focus on assessment of the infarct size and the area-at-risk (AAR) by Evan's blue dye and triphenyl tetrazolium chloride (TTC) following 4-hours of reperfusion; additional comments toward the evaluation of cardiac function and remodeling by modifying the duration of reperfusion, is also presented. Overall, this AMI rat animal model is useful for studying the consequence of a myocardial infarction on cardiac pathophysiological and physiological function.

The rat model of acute myocardial infarction (AMI) is useful to study the consequence of a MI on cardiac pathophysiological and physiological function.

With heart failure leading the cause of death in the USA (Hunt), biomedical research is fundamental to advance medical treatments for cardiovascular ...

Roux-en-Y Gastric Bypass Operation in Rats

Currently, the most effective therapy for the treatment of morbid obesity to induce significant and maintained body weight loss with a proven mortality benefit is bariatric surgery1,2. Consequently, there has been a steady rise in the number of bariatric operations done worldwide in recent years with the Roux-en-Y gastric bypass (gastric bypass) being the most commonly performed operation3. Against this background, it is important to understand the physiological mechanisms by which gastric bypass induces and maintains body weight loss. These mechanisms are yet not fully understood, but may include reduced hunger and increased satiation4,5, increased energy expenditure6,7, altered preference for food high in fat and sugar8,9, altered salt and water handling of the kidney10 as well as alterations in gut microbiota11. Such changes seen after gastric bypass may at least partly stem from how the surgery alters the hormonal milieu because gastric bypass increases the postprandial release of peptide-YY (PYY) and glucagon-like-peptide-1 (GLP-1), hormones that are released by the gut in the presence of nutrients and that reduce eating12.
During the last two decades numerous studies using rats have been carried out to further investigate physiological changes after gastric bypass. The gastric bypass rat model has proven to be a valuable experimental tool not least as it closely mimics the time profile and magnitude of human weight loss, but also allows researchers to control and manipulate critical anatomic and physiologic factors including the use of appropriate controls. Consequently, there is a wide array of rat gastric bypass models available in the literature reviewed elsewhere in more detail 13-15. The description of the exact surgical technique of these models varies widely and differs e.g. in terms of pouch size, limb lengths, and the preservation of the vagal nerve. If reported, mortality rates seem to range from 0 to 35%15. Furthermore, surgery has been carried out almost exclusively in male rats of different strains and ages. Pre- and postoperative diets also varied significantly.
Technical and experimental variations in published gastric bypass rat models complicate the comparison and identification of potential physiological mechanisms involved in gastric bypass. There is no clear evidence that any of these models is superior, but there is an emerging need for standardization of the procedure to achieve consistent and comparable data. This article therefore aims to summarize and discuss technical and experimental details of our previously validated and published gastric bypass rat model.

Numerous studies using gastric bypass rat models have been recently conducted to uncover the underlying physiological mechanisms of Roux-en-Y gastric bypass operations. This article aims to demonstrate and discuss the technical and experimental details of our published gastric bypass rat model to understand advantages and limitations of this experimental tool.

Currently, the most effective therapy for the treatment of morbid obesity to induce significant and maintained body weight loss with a proven mortality ...

Orthotopic Small Bowel Transplantation in Rats

Small bowel transplantation has become an accepted clinical option for patients with short gut syndrome and failure of parenteral nutrition (irreversible intestinal failure). In specialized centers improved operative and managing strategies have led to excellent short- and intermediate term patient and graft survival while providing high quality of life 1,3. Unlike in the more common transplantation of other solid organs (i.e. heart, liver) many underlying mechanisms of graft function and immunologic alterations induced by intestinal transplantation are not entirely known6,7. Episodes of acute rejection, sepsis and chronic graft failure are the main obstacles still contributing to less favorable long term outcome and hindering a more widespread employment of the procedure despite a growing number of patients on home parenteral nutrition who would potentially benefit from such a transplant. The small intestine contains a large number of passenger leucocytes commonly referred to as part of the gut associated lymphoid system (GALT) this being part of the reason for the high immunogenity of the intestinal graft. The presence and close proximity of many commensals and pathogens in the gut explains the severity of sepsis episodes once graft mucosal integrity is compromised (for example by rejection). To advance the field of intestinal- and multiorgan transplantation more data generated from reliable and feasible animal models is needed. The model provided herein combines both reliability and feasibility once established in a standardized manner and can provide valuable insight in the underlying complex molecular, cellular and functional mechanisms that are triggered by intestinal transplantation. We have successfully used and refined the described procedure over more than 5 years in our laboratory 8-11. The JoVE video-based format is especially useful to demonstrate the complex procedure and avoid initial pitfalls for groups planning to establish an orthotopic rodent model investigating intestinal transplantation.

Small bowel transplantation has become an accepted treatment option for patients with irreversible intestinal failure. Our experimental model of orthotopic small bowel transplantation in rats serves as a reliable tool to address underlying immunologic and inflammatory processes that complicate intestinal transplantation.

Small  bowel transplantation has become an accepted clinical option for patients with  short gut syndrome and failure of parenteral nutrition ...

Orthotopic Liver Transplantation in Rats

Clinical progress in the field of liver transplantation has been largely supported by animal models1,2. Since the publication of the first orthotopic rat liver transplantation in 1979 by Kamada et al.3, this model has remained the gold standard despite various proposed alternative techniques4. Nevertheless, its broader use is limited by its steep learning curve5.
In this video paper, we show a simple and easy-to-establish revision of Kamada's two-cuff technique. The suprahepatic vena cava anastomosis is performed manually with a running suture, and the vena porta and infrahepatic vena cava anastomoses are performed utilizing a quick-linker cuff system6. Manufacturing the quick-linker kit is shown in a separate video paper.

We present an easy-to-establish revision of the classical two-cuff technique for orthotopic liver transplantation in rat.

Clinical progress in the field of liver transplantation has been largely supported by animal models1,2. Since the publication of the first orthotopic rat ...

Assessment of Vascular Function in Patients With Chronic Kidney Disease

Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this is only partially explained by traditional CVD risk factors. Vascular dysfunction is an important non-traditional risk factor, characterized by vascular endothelial dysfunction (most commonly assessed as impaired endothelium-dependent dilation [EDD]) and stiffening of the large elastic arteries. While various techniques exist to assess EDD and large elastic artery stiffness, the most commonly used are brachial artery flow-mediated dilation (FMDBA) and aortic pulse-wave velocity (aPWV), respectively. Both of these noninvasive measures of vascular dysfunction are independent predictors of future cardiovascular events in patients with and without kidney disease. Patients with CKD demonstrate both impaired FMDBA, and increased aPWV. While the exact mechanisms by which vascular dysfunction develops in CKD are incompletely understood, increased oxidative stress and a subsequent reduction in nitric oxide (NO) bioavailability are important contributors. Cellular changes in oxidative stress can be assessed by collecting vascular endothelial cells from the antecubital vein and measuring protein expression of markers of oxidative stress using immunofluorescence. We provide here a discussion of these methods to measure FMDBA, aPWV, and vascular endothelial cell protein expression.

The degree of vascular dysfunction and contributing physiological mechanisms can be assessed in patients with chronic kidney disease by measuring brachial artery flow-mediated dilation, aortic pulse-wave velocity, and vascular endothelial cell protein expression.

Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this ...

Murine Kidney Transplant Technique

The first mouse kidney transplant technique was published in 19731 by the Russell laboratory. Although it took some years for other labs to become proficient in and utilize this technique, it is now widely used by many laboratories around the world. A significant refinement to the original technique using the donor aorta to form the arterial anastomosis instead of the renal artery was developed and reported in 1993 by Kalina and Mottram 2 with a further advancement coming from the same laboratory in 1999 3. While one can become proficient in this model, a search of the literature reveals that many labs still experience a high proportion of graft loss due to arterial thrombosis. We describe here a technique that was devised in our laboratory that vastly reduces the arterial thrombus reported by others 4,5. This is achieved by forming a heel-and-toe cuff of the donor infra-renal aorta that facilitates a larger anastomosis and straighter blood flow into the kidney.

The goal of this manuscript is to describe the steps required to perform a kidney transplant in a mouse, paying particular attention to the details of the arterial anastomosis.

The first mouse kidney transplant technique was published in 19731 by the Russell laboratory. Although it took some years for other labs to become ...

Normothermic Ex Vivo Kidney Perfusion for the Preservation of Kidney Grafts prior to Transplantation

Kidney transplantation has become a well-established treatment option for patients with end-stage renal failure. The persisting organ shortage remains a serious problem. Therefore, the acceptance criteria for organ donors have been extended leading to the usage of marginal kidney grafts. These marginal organs tolerate cold storage poorly resulting in increased preservation injury and higher rates of delayed graft function. To overcome the limitations of cold storage, extensive research is focused on alternative normothermic preservation methods.
Ex vivo normothermic organ perfusion is an innovative preservation technique. The first experimental and clinical trials for ex vivo lung, liver, and kidney perfusions demonstrated favorable outcomes.
In addition to the reduction of cold ischemic injury, the method of normothermic kidney storage offers the opportunity for organ assessment and repair. This manuscript provides information about kidney retrieval, organ preservation techniques, and isolated ex vivo normothermic kidney perfusion (NEVKP) in a porcine model. Surgical techniques, set up for the perfusion solution and the circuit, potential assessment options, and representative results are demonstrated.

Normothermic Ex Vivo Kidney Perfusion for the Preservation of Kidney Grafts prior to Transplantation

The severe organ shortage has resulted in increased use of marginal kidney grafts for transplantation. This has triggered interest in alternative storage methods, since marginal grafts especially tolerate cold storage poorly. The technique of normothermic ex vivo kidney perfusion (NEVKP) represents a novel preservation method for kidney grafts prior to &#160;transplantation.

Kidney transplantation has become a well-established treatment option for patients with end-stage renal failure. The persisting organ shortage remains a ...

Histological Quantification of Chronic Myocardial Infarct in Rats

Myocardial infarction is defined as cardiomyocyte death due to prolonged ischemia; an inflammatory response and scar formation (fibrosis) follow the ischemic injury. Following the initial acute phase, chronic remodeling of the left ventricle (LV) modifies the structure and function of the heart. Permanent coronary ligation in small animals has been widely used as a reference model for a chronic model of MI. Thinning of the infarcted wall progressively develops to transmural fibrosis. Histological assessment of infarct size is commonly performed; nevertheless, a standardization of the methods for quantification is missing. Indeed, important methodological aspects, such as the number of sections analyzed and the sampling and quantification methods, are usually not described and therefore preclude comparison across investigations. Too often, quantification is performed on a single section obtained at the level of the papillary muscles. Because novel strategies aimed at reducing infarct expansion and remodeling are under investigation, there is an important need for the standardization of accurate heart sampling protocols. We describe an accurate method to quantify the infarct size using a systematic sampling of harvested rat heart and image analyses of trichromatic stained histological sections obtained from base to apex. We also provide evidence that calculating the expansion index (EI) allowed for infarct size assessment, taking into account changes of the left ventricle throughout the remodeling.

The post-mortem assessment of myocardial infarction (MI) in rodents is based on quantification of the infarct on stained heart sections. We describe an accurate method to quantify the infarct size using systematic sampling of harvested rat hearts from base to apex and image analyses of trichrome-stained histological sections.

Myocardial infarction is defined as cardiomyocyte death due to prolonged ischemia; an inflammatory response and scar formation (fibrosis) follow the ...

Assessment of Kidney Function in Mouse Models of Glomerular Disease

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function in diabetic nephropathy and podocyte-specific VEGF-A knock-out mice; therefore, this protocol describes the full kidney work-up used in our lab to assess these mouse models of glomerular disease, enabling a vast amount of information regarding kidney and glomerular function to be obtained from a single mouse. In comparison to alternative methods presented in the literature to assess glomerular function, the use of the method outlined in this paper enables the glomerular phenotype to be fully evaluated from multiple aspects. By using this method, the researcher can determine the kidney phenotype of the model and assess the mechanism as to why the phenotype develops. This vital information on the mechanism of disease is required when examining potential therapeutic avenues in these models. The methods allow for detailed functional assessment of the glomerular filtration barrier through measurement of the urinary albumin creatinine ratio and individual glomerular water permeability, as well as both structural and ultra-structural examination using the Periodic Acid Schiff stain and electron microscopy. Furthermore, analysis of the genes dysregulated at the mRNA and protein level enables mechanistic analysis of glomerular function. This protocol outlines the generic but adaptable methods that can be applied to all mouse models of glomerular disease.

This protocol describes a full kidney work-up that should be carried out in mouse models of glomerular disease. The methods allow for detailed functional, structural, and mechanistic analysis of glomerular function, which can be applied to all mouse models of glomerular disease.

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function ...

Platelet-based Detection of Nitric Oxide in Blood by Measuring VASP Phosphorylation

Platelets are the blood components responsible for proper blood clotting. Their function is highly regulated by various pathways. One of the most potent vasoactive agents, nitric oxide (NO), can also act as a powerful inhibitor of platelet aggregation. Direct NO detection in blood is very challenging due to its high reactivity with cell-free hemoglobin that limits NO half-life to the millisecond range. Currently, NO changes after interventions are only estimated based on measured changes of nitrite and nitrate (members of the nitrate-nitrite-NO metabolic pathway). However precise, these measurements are rather difficult to interpret vis a vis actual NO changes, due to the naturally high baseline nitrite and nitrate levels that are several orders of magnitude higher than expected changes of NO itself. Therefore, the development of direct and simple methods that would allow one to detect NO directly is long overdue. This protocol addresses a potential use of platelets as a highly sensitive NO sensor in blood. It describes initial platelet rich plasma (PRP) and washed platelet preparations and the use of nitrite and deoxygenated red blood cells as NO generators. Phosphorylation of VASP at serine 239 (P-VASPSer239) is used to detect the presence of NO. The fact that VASP protein is highly expressed in platelets and that it is rapidly phosphorylated when NO is present leads to a unique opportunity to use this pathway to directly detect NO presence in blood.

Here, we present a protocol to address the potential use of platelets as a highly sensitive nitric oxide sensor in blood. It describes initial platelet preparation and the use of nitrite and deoxygenated red blood cells as nitric oxide generators.

Platelets are the blood components responsible for proper blood clotting. Their function is highly regulated by various pathways. One of the most potent ...