A part of the work shown in this article was founded by the Center of Integrative Mammalian Research of Ross University School of Veterinary Medicine (RUSVM), Saint Kitts and Nevis. The financial aid provided by the Department of Veterinary Biomedical Sciences of Long Island University College of Veterinary Medicine is greatly appreciated.

The following protocol describes a survival surgery. Therefore, the highest aseptic and surgical practice is applied. All animal experiments were performed in compliance with institutional guidelines and approved by the Institutional Animal Care and Use Committee. To eliminate gender&#160;and strain-based differences in IRI effects, only male C57BL6 mice were used in the study. All animals were matched in age and weight to produce comparable results.
1. Preparation
NOTE: A timeline of the different experimental phases and interventions is shown in Figure 1A.
<ol>
	<li>Clean and disinfect the surgical table before each procedure. Prepare and place all required materials (sterilized instruments and cotton swabs, sterile gauze and drapes, pre-diluted anesthetics, heating pad, sterilized vascular clamp, sterile saline solution, and skin antiseptics and suture) on the surgical table (see the Table of Materials).</li>
	<li>Anesthetize male C57BL6 mice (age range 11-13 weeks) by intraperitoneal injection of ketamine/xylazine (100 mg/kg and 20 mg/kg of body weight, respectively, previously diluted in sterile saline). 
	NOTE: Skilled animal handling is essential to minimize stress for the animal, as stress responses can negatively affect the action of anesthetics.</li>
	<li>After ketamine/xylazine administration, shave the surgical area on the right flank using a razor blade and soap. 
	NOTE: Shaving the skin improves wound healing as well as the general outcomes of survival surgeries.</li>
	<li>Disinfect the skin in the surgical area with 70% alcohol first and then with povidone iodine solution using a cotton swab.</li>
	<li>After the skin preparation, place the mouse on a heating table in a ventral decubitus position and stabilize the body temperature at 37 &#176;C (monitored through rectal and pad sensor probes). 
	NOTE: Kidneys are more easily accessed and surgically exposed when placed in ventral decubitus rather than lateral.</li>
	<li>While the body temperature is stabilized, apply eye ointment to the eyes of the mouse. 
	NOTE: Dissociative anesthetics, such as ketamine, cause the animal&#39;s eyes to remain open while anesthetized.</li>
</ol>
2. Surgery
<ol>
	<li>Once the pain reflexes are absent (toe pinching with tweezers), perform an approximately 1 cm dorso-lateral surgical incision on the right flank using a scalpel blade. Start the incision behind the last rib and continue caudally approximately 1 cm parallel to the lumbar midline.</li>
	<li>Transect the abdominal musculature using scissors to visualize the retroperitoneal space. Remove the small amounts of blood produced during the sectioning of the muscles using sterile cotton swabs. 
	NOTE: Because the dorso-lateral approach is used, the retroperitoneum, and not the peritoneal cavity, is accessed with this procedure.</li>
	<li>Push the right kidney out from the abdominal cavity. Use Graefe forceps to expose the kidney carefully. 
	NOTE: Always keep forceps closed to avoid traumatic injury to the kidney when placed on the abdomen and use it only to carefully push and guide the kidney towards the surgical incision and out of it.</li>
	<li>Slowly expose the right kidney and identify the renal pedicle. Carefully remove the adipose tissue around the pedicle.</li>
	<li>To induce ischemia, place the vascular clamp over the renal artery and vein present in the renal pedicle, avoiding clamping the adjacent ureter. Use a Halsted-Mosquito hemostat for manipulating the vascular clamp. 
	NOTE: Ischemia is confirmed by the visualization of a change in color of the kidney from red-pink to dark purple (Figure 1B).</li>
	<li>Cover the clamped kidney with sterile gauze soaked in saline to avoid desiccation and leave it for 30 min.</li>
	<li>Monitor anesthesia depth and humidity of the gauze periodically during this time. 
	NOTE: The induction dose of anesthesia is sufficient to provide analgesia until the end of the ischemic event; hence, no additional anesthetic injections are required.</li>
	<li>Shortly before the end of the ischemia period, remove the gauze and uncover the kidney. Hold the Halsted-Mosquito hemostat, ready for clamp removal.</li>
	<li>At minute 30, open the vascular clamp with the hemostat and remove it from the renal pedicle to allow reperfusion of the kidney. 
	NOTE: Reperfusion is confirmed by the visualization of a change in the color of the kidney from dark purple to red-pink (Figure 1C).</li>
	<li>Perform the same procedures described above for sham animals without clamping of the renal pedicle.</li>
	<li>After verification of the kidney color change, return the kidney to the abdominal cavity. Close the abdominal muscles with absorbable suture 5-0 using a cruciate pattern. 
	NOTE: A second injection of anesthetics may be required to maintain analgesia during the suturing of the muscles and the skin. Half of the initial dose has proved effective in providing analgesia until the conclusion of the surgery.</li>
	<li>Close the skin with absorbable suture 5-0 using a horizontal mattress pattern. Clean the wound with a povidone iodine solution using a cotton swab.</li>
</ol>
3. Recovery and post-surgery
NOTE: As post-surgical time is the actual reperfusion time, proper post-surgical care is ethically mandatory and scientifically relevant. Reperfusion times can be selected as required by the researcher. Reperfusion times of 4 h, 8 h, 16 h, 1 day, 2 days, 4 days, and 7 days are compared to obtain a sequential overview of pathological changes induced by renal IRI.
<ol>
	<li>Keep the mouse on the heating pad until it starts recovering from anesthesia. 
	NOTE: It is recommended to wait until the mouse begins moving its legs and attempts to move around. In cases when additional anesthetic injections are required during the surgery, the recovery time is longer. Atipamezole, an alpha-2 receptor antagonist, can be administered at a dose of 0.5 mg/kg of body weight intraperitoneally to reverse the xylazine effects and shorten the recovery phase. For pain management, buprenorphine (0.1 mg/kg of body weight, intraperitoneally) is administered pre-operatively and every 6 hours during the recovery and post-surgical phase. The use of non-steroidal anti-inflammatory drugs is discouraged as several drugs in this family induce nephrotoxicity and can, therefore, alter the results.</li>
	<li>After recovery from anesthesia, place the mouse back in its cage with free access to water and food. 
	NOTE: Mashed food can be provided in a Petri dish as well as material for hiding and playing (e.g., paper sheets, paper towel tubes).</li>
	<li>Monitor the mouse daily to assess wound healing, food and water intake, body weight, and behavior. 
	NOTE: Wound healing status was assessed using the following scale: 1, dry; 2, wet; 3, partially opened; 4, opened. Fast wound healing was documented in this study, with more than 90% of dry wounds after day 2.</li>
</ol>
4. Euthanasia and sample collection
<ol>
	<li>Euthanize the mice with sodium pentobarbital administered intraperitoneally at a dose that is twice the anesthetic dose for mice (100 mg/kg).</li>
	<li>Collect fluid and tissue samples as required. 
	NOTE: Both kidneys, whole blood (for blood cell count), serum (for blood biochemistry), urine, the heart, and the lungs were collected. A few microliters of serum are needed for blood biochemistry analysis (blood urea nitrogen (BUN), creatinine, electrolytes). If required, 24 h before euthanasia, mice can be placed into metabolic cages to collect a higher urine volume that allows the determination of renal function parameters.</li>
</ol>

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	<li>Ray, S. C., Mason, J., O'Connor, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ischemic+renal+injury:+can+renal+anatomy+and+associated+vascular+congestion+explain+why+the+medulla+and+not+the+cortex+is+where+the+trouble+starts.">Ischemic renal injury: can renal anatomy and associated vascular congestion explain why the medulla and not the cortex is where the trouble starts.</a> Seminars in Nephrology. 39 (6), 520-529 (2019).</li><li>Weight, S. C., Bell, P. R., Nicholson, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Renal+ischaemia--reperfusion+injury.">Renal ischaemia--reperfusion injury.</a> The British Journal of Surgery. 83 (2), 162-170 (1996).</li><li>Ratliff, B. B., Abdulmahdi, W., Pawar, R., Wolin, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidant+mechanisms+in+renal+injury+and+disease.">Oxidant mechanisms in renal injury and disease.</a> Antioxidants &amp; Redox Signaling. 25 (3), 119-146 (2016).</li><li>Schrier, R. W., Wang, W., Poole, B., Mitra, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+renal+failure:+definitions,+diagnosis,+pathogenesis,+and+therapy.">Acute renal failure: definitions, diagnosis, pathogenesis, and therapy.</a> The Journal of Clinical Investigation. 114 (1), 5-14 (2004).</li><li>Fern&#225;ndez, A. R., S&#225;nchez-Tarjuelo, R., Cravedi, P., Ochando, J., L&#243;pez-Hoyos, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review:+Ischemia+reperfusion+injury-a+translational+perspective+in+organ+transplantation.">Review: Ischemia reperfusion injury-a translational perspective in organ transplantation.</a> International Journal of Molecular Sciences. 21 (22), 8549 (2020).</li><li>Wu, C. -. L., 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=Tubular+peroxiredoxin+3+as+a+predictor+of+renal+recovery+from+acute+tubular+necrosis+in+patients+with+chronic+kidney+disease.">Tubular peroxiredoxin 3 as a predictor of renal recovery from acute tubular necrosis in patients with chronic kidney disease.</a> Scientific Reports. 7 (1), 43589 (2017).</li><li>Nishida, 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=Systemic+and+sustained+thioredoxin+analogue+prevents+acute+kidney+injury+and+its-associated+distant+organ+damage+in+renal+ischemia+reperfusion+injury+mice.">Systemic and sustained thioredoxin analogue prevents acute kidney injury and its-associated distant organ damage in renal ischemia reperfusion injury mice.</a> Scientific Reports. 10 (1), 20635 (2020).</li><li>Mishra, 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=Neutrophil+gelatinase-associated+lipocalin+(NGAL)+as+a+biomarker+for+acute+renal+injury+after+cardiac+surgery.">Neutrophil gelatinase-associated lipocalin (NGAL) as a biomarker for acute renal injury after cardiac surgery.</a> Lancet. 365 (9466), 1231-1238 (2005).</li><li>Han, W. 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R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Segment-specific+overexpression+of+redoxins+after+renal+ischemia+and+reperfusion:+protective+roles+of+glutaredoxin+2,+peroxiredoxin+3,+and+peroxiredoxin+6.">Segment-specific overexpression of redoxins after renal ischemia and reperfusion: protective roles of glutaredoxin 2, peroxiredoxin 3, and peroxiredoxin 6.</a> Free Radical Biology &amp; Medicine. 51 (2), 552-561 (2011).</li><li>Wei, Q., Dong, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+model+of+ischemic+acute+kidney+injury:+technical+notes+and+tricks.">Mouse model of ischemic acute kidney injury: technical notes and tricks.</a> American Journal of Physiology - Renal Physiology. 303 (11), 1487-1494 (2012).</li><li>Gaut, J. 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(88), e51816 (2014).</li><li>Wei, 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=New+mouse+model+of+chronic+kidney+disease+transitioned+from+ischemic+acute+kidney+injury.">New mouse model of chronic kidney disease transitioned from ischemic acute kidney injury.</a> American Journal of Physiology. Renal Physiology. 317 (2), 286-295 (2019).</li><li>Basile, D. P., Leonard, E. C., Tonade, D., Friedrich, J. 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The authors declare that there are no conflicts of interest regarding this article.

Mouse renal IRI models are popular in biomedical research due to their relatively low operational costs and the availability of diverse transgenic models12. The unilateral renal IRI model presented here mimics characteristic pathological changes observed in human renal IRI such as tubular dilatation, necrosis, and fibrosis13. These results are based on varying reperfusion times.
Critical steps of this protocol include the maintenance of constant body temperature and correct placement of the vascular clamp in the renal pedicle. Body temperature influences the animal&#39;s metabolism14, altering the experimental results both at the physiological and cellular levels15. In this model, the body temperature was stabilized before surgery using rectal and pad sensor probes. In addition, continuous monitoring of the body temperature during the whole surgical procedure is highly recommended, especially before placing the vascular clamp to induce ischemia.
The exposure of the kidney and the proper placement of the vascular clamp are also critical for the success of the experiment. Damage to the renal capsule by improper handling of the forceps during exposure of the kidney through the surgical incision will result in perirenal hemorrhage and inflammation. The vascular clamp should be placed on the renal pedicle occluding the renal artery and the renal vein without affecting the ureter and the suprarenal arteries. Critical for this step is the careful dissection of the adipose tissue surrounding the renal hilum14,16.
This model is cost- and time-effective. Anesthetic consumption per mouse was 156.47 &#177; 37.88 &#181;L (mean &#177; SD, n = 17) of a prediluted ketamine/xylazine cocktail (1:10 ketamine, 1:50 xylazine, in saline; stock solution concentration, 100 mg/mL both). Surgery can be performed in a relatively short period. The total surgery time per mouse was 53 &#177; 5.23 min (mean &#177; SD, n = 17). With trained personnel, several surgeries can be performed at the same time. In our group, one experienced researcher performed the surgery until the clamp was released from the renal pedicle, while a second one took over from wound closing until the recovery of the mouse. With this approach, we were able to perform a high number of surgeries on a single day. In this model, we used the dorsolateral approach, which results in less trauma and reduced fluid and heat loss from the abdominal cavity as compared with the midline approach16.
Previously published protocols have described the renal pedicle clamping technique to induce acute kidney injury in mice17,18,19. However, in those studies, a contralateral nephrectomy was performed in addition to the unilateral IRI with ischemic times ranging from 15 to 26 min. In this protocol, we induced unilateral ischemia for 30 min while preserving the contralateral kidney. This resulted in a survival rate of 100%. However, this model is not suitable to induce azotemic renal damage due partly to the compensatory effect exerted by the non-surgically intervened contralateral kidney. However, keeping one kidney unaffected in the same animal offers the advantage of using longer ischemia times with a higher survival rate. In addition to this, the contralateral kidney can be utilized to assess possible side effects of test drugs or treatments applied during the experimental procedure and to study kidney-kidney crosstalk effects20,21. For example, this model has been useful in showing reactive oxygen species-induced alterations at the cellular level both in the IRI and contralateral, non-surgically intervened kidney11.
This model has a potential application in studies aiming to identify and characterize markers of unilateral renal damage, renal crosstalk effects, post-renal IRI-induced hemodynamic changes, and potential nephrotoxic effects of drug candidates to be used in renal IRI. This detailed description of the main pathological changes serves as a valuable tool to select the most suitable time to study specific cellular processes, from inflammation and necrosis (4 h to 2 days) to regeneration (4 days) and fibrosis (7 days and later).

The kidneys are among the highest perfused organs in the body and are extremely susceptible to changes in blood perfusion1. Renal ischemia-reperfusion injury (IRI) remains the leading cause of acute renal failure2,3 and is associated with high morbidity and high mortality in hospitalized patients4. With limited therapeutic options available,4,5 renal IRI is currently the focus of several research efforts in biomedicine6,7 aiming for the development of novel therapeutic targets and the characterization of early and sensitive markers of renal injury8,9,10. Identifying a reliable, time&#160;and cost-effective animal model is considered essential to meet these needs. This paper presents a simple and effective mouse model of unilateral renal IRI. Ischemia is induced by clamping of the right renal pedicle for 30 min11,12. A crucial part of this model is choosing the most suitable reperfusion time that will reproduce the pathological events of interest, such as tubular necrosis, polymorphonuclear inflammatory cell infiltration, or fibrosis. Therefore, researchers are provided with this sequential overview of representative pathological changes expected in the IRI kidney.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Atipamezole hydrochloride</td>
			<td>Penn Veterinary Supply, Inc., PA, USA</td>
			<td>PVS8700</td>
			<td>5 mg/mL</td>
		</tr>
		<tr>
			<td>Buprenorphine</td>
			<td>Penn Veterinary Supply, Inc., PA, USA</td>
			<td>PRMBURPEN22</td>
			<td>0.3 mg/mL</td>
		</tr>
		<tr>
			<td>Commercial euthanasia solution</td>
			<td>various suppliers</td>
			<td>na</td>
			<td>e.g., Euthasol Virbac (sodium pentobarbital 390 mg/mL + sodium phenytoin 50 mg/mL)</td>
		</tr>
		<tr>
			<td>Eye ointment Puralube</td>
			<td>Dechra Veterinary Products, KS, USA</td>
			<td>na</td>
			<td>3.5 g (1/8 oz)</td>
		</tr>
		<tr>
			<td>Heating pad RightTempJr</td>
			<td>Kent Scientific, CT, USA</td>
			<td>&#160;RT-JR-20</td>
			<td>Consider the one with two temperature probes</td>
		</tr>
		<tr>
			<td>Ketamine hydrochloride</td>
			<td>Penn Veterinary Supply, Inc., PA, USA</td>
			<td>VED1220</td>
			<td>100 mg/ml</td>
		</tr>
		<tr>
			<td>S&#38;T Vascular clamp</td>
			<td>Fine Science Tools, Inc., Germany</td>
			<td>00398-02</td>
			<td>Jaw dimensions: 5.5 x 1.5 mm; length: 11 mm</td>
		</tr>
		<tr>
			<td>Sterile Disposable Towel Drapes
</td>
			<td>Kent Scientific, CT, USA 
</td>
			<td>SURGI-5023-3
</td>
			<td>Disposable, individualy packed
</td>
		</tr>
		<tr>
			<td>Surgical instruments (Graefe forceps, Halsted-Mosquito hemostat, scissors, etc)</td>
			<td>Fine Science Tools, Inc., Germany</td>
			<td>Various</td>
			<td>Consider the extra fine straight scissor and the angled Graefe forceps</td>
		</tr>
		<tr>
			<td>Vicryl suture</td>
			<td>Ethicon US, LLC</td>
			<td>J493G</td>
			<td>Size 5-0</td>
		</tr>
		<tr>
			<td>Xylazine hydrochloride</td>
			<td>Penn Veterinary Supply, Inc., PA, USA</td>
			<td>VAM4821</td>
			<td>100 mg/mL</td>
		</tr>
	</tbody>
</table>

an effective mouse model of unilateral renal ischemia-reperfusion injury

Ischemia-reperfusion injury (IRI) is the leading cause of acute renal failure and is a significant contributor to delayed graft function. Animal models are the only available resources that mimic the complexities of the IRI-associated damage encountered in vivo. This paper describes an effective mouse model of unilateral renal IRI that delivers highly reproducible data. Ischemia is induced by occluding the right renal pedicle for 30 min followed by reperfusion. In addition to the surgical procedure, a sequential overview of the expected physiological and histopathological changes following renal IRI will be provided by comparing data from seven different reperfusion times (4 h, 8 h, 16 h, 1 day, 2 days, 4 days, and 7 days). Critical data for planning experiments ahead, such as mean surgical time, average anesthetic consumption, and body weight changes over time, will be shared. This work will help researchers implement a reliable renal IRI model and select the appropriate reperfusion time that aligns with their intended investigative goals.

Physiological parameters 
Mice recovered from this unilateral renal IRI surgery uneventfully; appeared active and alert; and showed normal eating, drinking, and behavior by the following day. Some mice may have post-IRI body weight loss, although it is usually less than 10% of the initial body weight (Figure 2). Greater body weight losses (&#707;10%) can be detrimental, and those animals should be removed from the study. Sham-operated mice did not show body weight changes post-surgery (measured 24 h after surgery). Most mice recovered their initial body weight between days 4 and 7 post-surgery (see IRI 7-day group, Figure 2). Renal function can be assessed using traditional markers such as blood urea nitrogen (BUN) and creatinine. Additionally, electrolyte levels in serum (sodium, potassium, and chloride) and an automated differential blood count were included in the analysis.
Histopathological changes 
Assessment of histopathological findings was done using 4% paraformaldehyde-fixed, paraffin-embedded whole mid-sagittal sections of the kidney stained with hematoxylin/eosin (HE), periodic acid Schiff, and Masson&#39;s trichrome stains. The most evident changes produced by this unilateral renal IRI model can be seen at the cortico-medullary junction, specifically in the proximal tubules, thick ascending limbs of Henle&#39;s loop, and distal convoluted tubules, as well as in the tubular interstitium (see the legend for Figure 3). Microscopic images showing the most characteristic lesions following IRI in the kidney can be seen in Figure 3. A list of the sequential histopathological findings is provided in Table 1.
A tubular injury scoring system was developed to categorize the damage over time (Figure 4). In this, five defined alterations were assessed by three different evaluators: 1) tubular epithelial attenuation; 2) brush border loss; 3) tubular necrosis; 4) luminal obstruction; and 5) presence of proteinaceous cast. An assignment of &#34;1&#34; indicates that the alteration is present, &#34;0&#34; that it is absent.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62749/62749fig01.jpg" /> 
Figure 1: Experimental renal IRI model in mouse. (A) Phases of experiments and interventions (anesthesia induction, ischemia, and reperfusion) are shown. Please note the changes in the color of the right kidney to dark red during ischemia (B) to pink during reperfusion (C). (D) Macroscopic appearance of the IRI right kidney (red arrow) compared to the contralateral non-IRI kidney of the same animal 24 h after surgery. Red arrow in (B) shows the position of the hemostatic clamp. Abbreviation: IRI = Ischemia-reperfusion injury. <a href="https://www.jove.com/files/ftp_upload/62749/62749fig01large.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/62749/62749fig02.jpg" /> 
Figure 2:&#160;Body weight of mice before and after renal IRI. Individual data are shown. Abbreviations: IRI = Ischemia-reperfusion injury; h = hours; d = days. <a href="https://www.jove.com/files/ftp_upload/62749/62749fig02large.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/62749/62749fig03.jpg" /> 
Figure 3: Typical microscopic lesions observed in the cortex and the cortico-medullary junction of IR-operated mice. Sham and different reperfusion times are shown (indicated above each picture). (A) Intact structures are shown in sham (magnification 40x; scale bar = 20 &#181;m). Arrows in IRI 4 h indicate the presence of proteinaceous cast in the tubular lumen (magnification 40x; scale bar = 20 &#181;m). Arrows in IRI 8 h show tubular dilatation (magnification 40x; scale bar = 50 &#181;m). Black arrow in IRI 16 h shows tubular cast in medullary segments; white arrows show areas of cellular necrosis (magnification 40x; scale bar = 50 &#181;m). Black arrows in IRI 1 d indicate tubular dilatation (magnification 10x; scale bar = 100 &#181;m). Black arrow in IRI 2 d shows enlarged cell nuclei; white arrowheads show areas of lymphocyte and macrophage infiltration (magnification 40x; scale bar = 50 &#181;m). White arrowheads in IRI 4 d indicate mitotic tubular cells (magnification 40x; scale bar = 50 &#181;m). Black arrow in IRI 7 d shows an area of focal fibrosis; white arrowhead shows an area of regeneration (magnification 20x; scale bar = 100 &#181;m). (B) PAS staining showing the renal cortex of mice during early reperfusion (4 h, 8 h, and 16 h). Note the progressive attenuation of the brush border (arrows). Magnifications 40x; scale bars = 50 &#181;m (C) Masson trichrome staining of sham and IRI 7 d mice showing areas of interstitial fibrosis (white arrows). Magnification 40x; scale bars = 50 &#181;m. Abbreviations: IRI = Ischemia-reperfusion injury; Glo = glomerulus; PCT = proximal convoluted tubule; DCT = distal convoluted tubule; CD = collecting duct; PAS = periodic acid Schiff; d = day. <a href="https://www.jove.com/files/ftp_upload/62749/62749fig03large.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/62749/62749fig04.jpg" /> 
Figure 4: Tubular injury score of sham- and IRI-operated mice. Scoring system scale 1 to 5 for tubular epithelial attenuation; brush border loss; tubular necrosis; luminal obstruction; and presence of proteinaceous cast. An assignment of &#34;1&#34; indicates that the alteration is present, &#34;0&#34; that it is absent. Individual values are shown. Bars represent mean &#177; SD (n = 4). Abbreviation: IRI = Ischemia-reperfusion injury. <a href="https://www.jove.com/files/ftp_upload/62749/62749fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Time after IRI</td>
			<td>Most significant pathological changes</td>
		</tr>
		<tr>
			<td>4 h</td>
			<td>Tubular obstruction</td>
		</tr>
		<tr>
			<td></td>
			<td>Protein cast in lumen</td>
		</tr>
		<tr>
			<td>8 h</td>
			<td>Tubular dilatation</td>
		</tr>
		<tr>
			<td></td>
			<td>Incipient necrosis</td>
		</tr>
		<tr>
			<td></td>
			<td>Attenuation of epithelium</td>
		</tr>
		<tr>
			<td>16 h</td>
			<td>Cellular necrosis</td>
		</tr>
		<tr>
			<td></td>
			<td>Tubular cast</td>
		</tr>
		<tr>
			<td></td>
			<td>Neutrophil infiltration</td>
		</tr>
		<tr>
			<td>1 day</td>
			<td>Necrosis</td>
		</tr>
		<tr>
			<td></td>
			<td>Tubular dilatation</td>
		</tr>
		<tr>
			<td></td>
			<td>Neutrophil infiltration</td>
		</tr>
		<tr>
			<td>2 days</td>
			<td>Tubular dilatation</td>
		</tr>
		<tr>
			<td></td>
			<td>Lymphocyte and macrophage infiltration</td>
		</tr>
		<tr>
			<td></td>
			<td>Enlarged cell nuclei</td>
		</tr>
		<tr>
			<td>4 days</td>
			<td>Prominent mitotic activity in tubule cells</td>
		</tr>
		<tr>
			<td>7 days</td>
			<td>Focal fibrosis</td>
		</tr>
		<tr>
			<td></td>
			<td>Areas of regeneration</td>
		</tr>
	</tbody>
</table>
Table 1: Most significant pathological changes over time. Diagnosed based on microscopic examination of 4-6 animals per group.

Watch this Scientific Journal Video about An Effective Mouse Model of Unilateral Renal Ischemia-Reperfusion Injury at JoVE.com

An Effective Mouse Model of Unilateral Renal Ischemia-Reperfusion Injury

Renal ischemia-reperfusion injury is associated with high morbidity and mortality in hospitalized patients. Here, we present a simple and effective mouse model of unilateral renal ischemia-reperfusion injury and provide a sequential overview of representative pathological changes observed in the kidney.

Ischemia-reperfusion injury (IRI) is the leading cause of acute renal failure and is a significant contributor to delayed graft function. Animal models ...

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6.1K Views.  Long Island University College of Veterinary Medicine. Renal ischemia-reperfusion injury is associated with high morbidity and mortality in hospitalized patients. Here, we present a simple and effective mouse model of unilateral renal ischemia-reperfusion injury and provide a sequential overview of representative pathological changes observed in the kidney.

Video: An Effective Mouse Model of Unilateral Renal Ischemia-Reperfusion Injury

Normothermic Cardiac Arrest and Cardiopulmonary Resuscitation: A Mouse Model of Ischemia-Reperfusion Injury

Acute Kidney Injury (AKI) is a common, highly lethal, complication of critical illness which has a high mortality1-4 and which is most 
frequently caused by whole-body hypoperfusion.5,6 Successful reproduction of whole-body hypoperfusion in rodent models has been fraught with 
difficulty.7-9,9,10 Models which employ focal ischemia have repeatedly demonstrated results which do not translate to the clinical setting, and larger 
animal models which allow for whole body hypoperfusion lack access to the full toolset of genetic manipulation possible in the mouse.11,12 However, in recent 
years a mouse model of cardiac arrest and cardiopulmonary resuscitation has emerged which can be adapted to model AKI.13 This model reliably reproduces 
physiologic, functional, anatomic, and histologic outcomes seen in clinical AKI, is rapidly repeatable, and offers all of the significant advantages of a murine surgical 
model, including access to genetic manipulative techniques, low cost relative to large animals, and ease of use. Our group has developed extensive experience 
with use of this model to assess a number of organ-specific outcomes in AKI.14,15

A powerful model for perioperative and critical care related acute kidney injury is presented. Using whole body hypoperfusion induced by cardiac arrest it is possible to nearly replicate the histologic and functional changes of clinical AKI.

Acute Kidney Injury (AKI) is a common, highly lethal, complication of critical illness which has a high mortality1-4 and which is most 
frequently caused ...

Ischemia-reperfusion Model of Acute Kidney Injury and Post Injury Fibrosis in Mice

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often unreported mortality rates that may confound analyses. Bilateral renal pedicle clamping is commonly used to induce IR-AKI, but differences between effective clamp pressures and/or renal responses to ischemia between kidneys often lead to more variable results. In addition, shorter clamp times are known to induce more variable tubular injury, and while mice undergoing bilateral injury with longer clamp times develop more consistent tubular injury, they often die within the first 3 days after injury due to severe renal insufficiency. To improve post-injury survival and obtain more consistent and predictable results, we have developed two models of unilateral ischemia-reperfusion injury followed by contralateral nephrectomy. Both surgeries are performed using a dorsal approach, reducing surgical stress resulting from ventral laparotomy, commonly used for mouse IR-AKI surgeries. For induction of moderate injury BALB/c mice undergo unilateral clamping of the renal pedicle for 26 min and also undergo simultaneous contralateral nephrectomy. Using this approach, 50-60% of mice develop moderate AKI 24 hr after injury but 90-100% of mice survive. To induce more severe AKI, BALB/c mice undergo renal pedicle clamping for 30 min followed by contralateral nephrectomy 8 days after injury. This allows functional assessment of renal recovery after injury with 90-100% survival. Early post-injury tubular damage as well as post injury fibrosis are highly consistent using this model.

We describe models of moderate and severe ischemia-reperfusion-induced kidney injury in which the mice undergo unilateral renal pedicle clamping followed by simultaneous or delayed contralateral nephrectomy, respectively. These models consistently give rise to renal dysfunction and post-injury fibrosis, but injury severity and survival are dependent on mouse background, age and surgical equipment.

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often ...

Renal Ischaemia Reperfusion Injury: A Mouse Model of Injury and Regeneration

Renal ischaemia reperfusion injury (IRI) is a common cause of acute kidney injury (AKI) in patients and occlusion of renal blood flow is unavoidable during renal transplantation. Experimental models that accurately and reproducibly recapitulate renal IRI are crucial in dissecting the pathophysiology of AKI and the development of novel therapeutic agents. Presented here is a mouse model of renal IRI that results in reproducible AKI. This is achieved by a midline laparotomy approach for the surgery with one incision allowing both a right nephrectomy that provides control tissue and clamping of the left renal pedicle to induce ischaemia of the left kidney. By careful monitoring of the clamp position and body temperature during the period of ischaemia this model achieves reproducible functional and structural injury. Mice sacrificed 24 hr&#160;following surgery demonstrate loss of renal function with elevation of the serum or plasma creatinine level as well as structural kidney damage with acute tubular necrosis evident. Renal function improves and the acute tissue injury resolves during the course of 7 days following renal IRI such that this model may be used to study renal regeneration. This model of renal IRI has been utilized to study the molecular and cellular pathophysiology of AKI as well as analysis of the subsequent renal regeneration.

The mouse model of renal ischaemia reperfusion injury described here comprises of a right nephrectomy that provides control tissue and clamping of the left renal pedicle to induce ischaemia that results in acute kidney injury. This model uses a midline laparotomy approach with all steps performed via one incision.

Renal ischaemia reperfusion injury (IRI) is a common cause of acute kidney injury (AKI) in patients and occlusion of renal blood flow is unavoidable ...

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic cerebrovascular accidents account for 80% of all strokes. A common cause of IR injury is the rapid inflow of fluids following an acute/chronic occlusion of blood, nutrients, oxygen to the tissue triggering the formation of free radicals.
Ischemic stroke is followed by blood-brain barrier (BBB) dysfunction and vasogenic brain edema. Structurally, tight junctions (TJs) between the endothelial cells play an important role in maintaining the integrity of the blood-brain barrier (BBB). IR injury is an early secondary injury leading to a non-specific, inflammatory response. Oxidative and metabolic stress following inflammation triggers secondary brain damage including BBB permeability and disruption of tight junction (TJ) integrity.
Our protocol presents an in vitro example of oxygen-glucose deprivation and reoxygenation (OGD-R) on rat brain endothelial cell TJ integrity and stress fiber formation. Currently, several experimental in vivo models are used to study the effects of IR injury; however they have several limitations, such as the technical challenges in performing surgeries, gene dependent molecular influences and difficulty in studying mechanistic relationships. However, in vitro models may aid in overcoming many of those limitations. The presented protocol can be used to study the various molecular mechanisms and mechanistic relationships to provide potential therapeutic strategies. However, the results of in vitro studies may differ from standard in vivo studies and should be interpreted with caution.

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is associated with a high rate of morbidity and mortality. The goal of the in vitro model of oxygen-glucose deprivation and reoxygenation (OGD-R) described here is to assess the effects of ischemia reperfusion injury on a variety of cells, particularly in blood-brain barrier (BBB) endothelial cells.

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic ...

Murine Model of Intestinal Ischemia-reperfusion Injury

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, hypotension, necrotizing enterocolitis, bowel transplantation, trauma and chronic inflammation. Intestinal ischemia-reperfusion (IR) injury is a consequence of acute mesenteric ischemia, caused by inadequate blood flow through the mesenteric vessels, resulting in intestinal damage. Reperfusion following ischemia can further exacerbate damage of the intestine. The mechanisms of IR injury are complex and poorly understood. Therefore, experimental small animal models are critical for understanding the pathophysiology of IR injury and the development of novel therapies.
Here we describe a mouse model of acute intestinal IR injury that provides reproducible injury of the small intestine without mortality. This is achieved by inducing ischemia in the region of the distal ileum by temporally occluding the peripheral and terminal collateral branches of the superior mesenteric artery for 60 min using microvascular clips. Reperfusion for 1 hr, or 2 hr after injury results in reproducible injury of the intestine examined by histological analysis. Proper position of the microvascular clips is critical for the procedure. Therefore the video clip provides a detailed visual step-by-step description of this technique. This model of intestinal IR injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Here we describe the detailed procedure of intestinal ischemia-reperfusion in mice which results in reproducible injury without mortality to encourage the standardization of this technique across the field. This model of intestinal ischemia-reperfusion injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, ...

A Mouse Model of Retinal Ischemia-Reperfusion Injury Through Elevation of Intraocular Pressure

Retinal ischemia-reperfusion (I/R) is a pathophysiological process contributing to cellular damage in multiple ocular conditions, including glaucoma, diabetic retinopathy, and retinal vascular occlusions. Rodent models of I/R injury are providing significant insights into mechanisms and treatment strategies for human I/R injury, especially with regard to neurodegenerative damage in the retinal neurovascular unit. Presented here is a protocol for inducing retinal I/R injury in mice through elevation of intraocular pressure (IOP). In this protocol, the ocular anterior chamber is cannulated with a needle, through which flows the drip of an elevated saline reservoir. Using this drip to raise IOP above systolic arterial blood pressure, a practitioner temporarily halts inner retinal blood flow (ischemia). When circulation is reinstated (reperfusion) by removal of the cannula, severe cellular damage ensues, resulting ultimately in retinal neurodegeneration. Recent studies demonstrate inflammation, vascular permeability, and capillary degeneration as additional elements of this model. Compared to alternative retinal I/R methodologies, such as retinal arterial ligation, retinal I/R injury by elevated IOP offers advantages in its&#160;anatomical specificity, experimental tractability, and technical accessibility, presenting itself as a valuable tool for examining neuronal pathogenesis and therapy in the retinal neurovascular unit.

This article describes a procedure for inducing retinal ischemia-reperfusion injury by elevated intraocular pressure in mice. Retinal ischemia-reperfusion injury by elevated intraocular pressure serves to model human pathologies characterized by compromised oxygen and nutrient delivery in the retina, enabling researchers to examine potential cellular mechanisms and treatments for human diseases of the retinal neurovascular unit.

Retinal ischemia-reperfusion (I/R) is a pathophysiological process contributing to cellular damage in multiple ocular conditions, including glaucoma, ...

Two-vessel Occlusion Mouse Model of Cerebral Ischemia-reperfusion

In this study, a middle cerebral artery (MCA) occlusion mouse model is employed to study cerebral ischemia-reperfusion. A reproducible and reliable mouse model is useful for investigating the pathophysiology of cerebral ischemia-reperfusion and determining potential therapeutic strategies for patients with stroke. Variations in the anatomy of the circle of Willis of C57BL/6 mice affects their infarct volume after cerebral-ischemia-induced injury. Studies have indicated that distal MCA occlusion (MCAO) can overcome this problem and result in a stable infarct size. In this study, we establish a two-vessel occlusion mouse model of cerebral ischemia-reperfusion through the interruption of the blood flow to the right MCA. We distally ligate the right MCA and right common carotid artery (CCA) and restore blood flow after a certain period of ischemia. This ischemia-reperfusion injury induces an infarct of stable size and a behavioral deficit. Peripheral immune cells infiltrate the ischemic brain within the 24 h infiltration period. Additionally, the neuronal loss in the cortical area is less for a longer reperfusion duration. Therefore, this two-vessel occlusion model is suitable for investigating the immune response and neuronal recovery during the reperfusion period after cerebral ischemia.

A mouse model of cerebral ischemia-reperfusion is established to investigate the pathophysiology of stroke. We distally ligate the right middle cerebral artery and right common carotid artery and restore blood flow after 10 or 40 min of ischemia.

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Evaluation of Coronary Flow Reserve After Myocardial Ischemia Reperfusion in Rats

Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via recanalization of the coronary artery is the most effective strategy for reducing the size of ischemic myocardium. The coronary microvasculature cannot be visualized and imaged&#160;in vivo, but there are several invasive and noninvasive techniques that can be used to assess parameters which depend directly on coronary microvascular function. The endothelial function after ischemia reperfusion can be assessed also at the level of the coronary circulation via the coronary flow reserve (CFR).&#160;In this study, peak velocity of left anterior descending (LAD) coronary arteries was measured in rats in vivo via Transthoracic Doppler Echocardiography during resting and stress challenge (induced by Dobutamine). A normal heart can increase its coronary blood flow up to four times above the resting values during stress induction. Following ischemia reperfusion, we found a significantly diminished CFR, which can be used as a marker of coronary microvascular dysfunction. CFR has opened a window on the importance of microvascular dysfunction and has been shown to predict cardiovascular risk independent of whether the severe obstructive disease is present.

Coronary flow reserve (CFR), is defined as the ratio of maximal coronary blood flow to the resting coronary blood flow. We present a protocol for evaluating CFR in rats via ultrasound, which offers the opportunity to predict cardiovascular risk factors in the absence of obstructive coronary disease.

Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via ...

A Pre-clinical Rat Model for the Study of Ischemia-reperfusion Injury in Reconstructive Microsurgery

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many areas of biomedical research due to its cost-effectiveness and its translation to humans. This protocol describes a method to create a preclinical free skin flap model in rats with ischemia-reperfusion injury. The described 3 cm x 6 cm rat free skin flap model is easily obtained after the placement of several vascular ligatures and the section of the vascular pedicle. Then, 8 h after the ischemic insult and completion of the microsurgical anastomosis, the free skin flap develops the tissue damage. These ischemia-reperfusion injury-related damages can be studied in this model, making it a suitable model for evaluating therapeutic agents to address this pathophysiological process. Furthermore, two main monitoring techniques are described in the protocol for the assessment of this animal model: transit-time ultrasound technology and laser speckle contrast analysis.

Here, we describe a pre-clinical animal model for studying the pathophysiology of ischemia-reperfusion injury in reconstructive microsurgery. This free skin flap model based on the superficial caudal epigastric vessels in the rat may also allow for the evaluation of different therapies and compounds to counteract ischemia-reperfusion injury-related damage.

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many ...

Delayed Intramyocardial Delivery of Stem Cells after Ischemia Reperfusion Injury in a Murine Model

There is significant interest in the use of stem cells (SCs) for the recovery of cardiac function in individuals with myocardial injuries. Most commonly, cardiac stem cell therapy is studied by delivering SCs concurrently with the induction of myocardial injury. However, this approach presents two significant limitations: the early hostile pro-inflammatory ischemic environment may affect the survival of transplanted SCs, and it does not represent the subacute infarction scenario where SCs will likely be used. Here we describe a two-part series of surgical procedures for the induction of ischemia-reperfusion injury and delivery of mesenchymal stem cells (MSCs). This method of stem cell administration may allow for the longer viability and retention around damaged tissue by circumventing the initial immune response. A model of ischemia reperfusion injury was induced in mice accompanied by the delivery of mesenchymal stem cells (3.0 x 105), stably expressing the reporter gene firefly luciferase under the constitutively expressed CMV promoter, intramyocardially 7 days later. The animals were imaged via ultrasound and bioluminescent imaging for confirmation of injury and injection of cells, respectively. Importantly, there was no added complication rate when performing this two-procedure approach for SC delivery. This method of stem cell administration, collectively with the utilization of state-of-the-art reporter genes, may allow for the in vivo study of viability and retention of transplanted SCs in a situation of chronic ischemia commonly seen clinically, while also circumventing the initial pro-inflammatory response. In summary, we established a protocol for the delayed delivery of stem cells into the myocardium, which can be used as a potential new approach in promoting regeneration of the damaged tissue.

Stems cells are continuously investigated as potential treatments for individuals with myocardial damage, however, their decreased viability and retention within injured tissue can impact their long-term efficacy. In this manuscript we describe an alternative method for stem cell delivery in a murine model of ischemia reperfusion injury.

There is significant interest in the use of stem cells (SCs) for the recovery of cardiac function in individuals with myocardial injuries. Most commonly, ...