The present study was supported by grants from Kidney Research UK (ST4/2011), the Cunningham Trust (CT11/14) and the Mrs EA Hogg's Charitable Trust.

NOTE: Animal experiments were performed in accordance with the guidelines and regulations imposed by the Animals (Scientific Procedures) Act 1986. Procedures were conducted using sterile (autoclaved) surgical instruments and consumables. Whilst the murine model of renal IRI presented here was performed on an 8-week-old male Balb/c mouse it can be reproducibly performed on a variety of murine strains of either gender typically aged between 7 &#8211; 15 weeks, with the optimum age being 8 weeks. The data presented in the representative results section was obtained from both Balb/c and FVB mice. The application of warmed saline is used to keep the intestines and surgical area moist but it should be carefully monitored and kept to a minimum as the excessive application of fluids can lead to an artifactual lowering of the serum or plasma creatinine levels, which is an important experimental readout.
1. Animal Preparation and Laparotomy
<ol>
	<li>Anesthetize the mouse with ketamine hydrochloride (70 mg/kg) and medetomidine hydrochloride (1 mg/kg) intraperitoneally and confirm the depth of anesthesia by loss of reflexes e.g. toe pinch. The duration of the resulting anesthetic plane is 4 hours. This is sufficient to perform the entire surgical procedure and no supplemental anesthesia is required.</li>
	<li>Remove the hair surrounding the incision area, ensure that the area is free of loose hair, and sanitize the abdominal skin using a dilute chlorhexidine solution.</li>
	<li>Place the mouse on a heated surgical pad in a supine position and fix the upper and lower limbs to the pad using low-tack adhesive tape. Ensure that the upper extremities are maintained in a normal position to prevent lung compression. Throughout the procedure monitor the mouse for thermal burns. If possible it is recommended that a non-electric heat source be used.</li>
	<li>Administer the analgesic buprenorphine hydrochloride (0.06 mg/kg) subcutaneously and apply eye lubricant to the eyes to prevent corneal drying. Analgesics are administered pre-operatively in order to aid the post-surgical recovery.</li>
	<li>Using tissue separating scissors or a scalpel blade make a midline laparotomy incision and bluntly separate the skin from the peritoneum. This allows the skin and peritoneum to be sutured separately during wound closure. An incision of the avascular linea alba is made giving access to the peritoneal cavity.</li>
	<li>To create a clear view of the surgical area insert a retractor and drape the mouse.</li>
</ol>
2. Ureter Division and Right Nephrectomy
<ol>
	<li>Gently push the intestines towards the left side of the abdominal cavity using autoclaved sterilized cotton swabs moistened with saline to expose the right kidney and ureter. Cover the intestines with moistened drapes to prevent drying.</li>
	<li>Lift the right ureter with angled forceps. Ligate the right ureter twice using 6/0 silk-braided suture. For long term experiments, use absorbable sutures for all abdominal surgeries.</li>
	<li>Divide the ureter between the sutures. Whilst the vesico-ureteric junction should prevent urine from leaking into the peritoneum from the bladder the ureter is ligated as a safeguard against any leakage post-operatively during long-term experiments.</li>
	<li>To allow easier access when performing the right nephrectomy, gently push the liver up and to the right using a moistened cotton swab and hold in place using moistened gauze to expose the right renal artery and vein.</li>
	<li>Bluntly dissect the connective tissue and fat along the medial aspect of the right kidney as far as the right renal artery and vein.</li>
	<li>Create a channel underneath the renal artery and vein by carefully sliding the angled forceps underneath the blood vessels. Guide the forceps underneath with the tips closed and gently remove with the tips open in order to facilitate the formation of the channel.</li>
	<li>Repeat step 2.6 until the tips of the forceps are visible through the connective tissues just above the renal artery and vein. Once visible gently rub the tips of the forceps against the tips of another set of angled forceps to gently break the connective tissue.</li>
	<li>With the renal artery and vein clearly accessible they can now be occluded. Carefully slide angled forceps underneath the renal artery and vein with the tips closed. Once in place open the tips of the forceps and guide the haemostatic clip applicator between the tips and firmly apply a haemostatic clip onto the renal artery and vein close to the kidney. Alternatively the renal artery and vein can be ligated using 6/0 silk braided suture.</li>
	<li>Divide the occluded renal artery and vein close to the kidney, which can now be removed with any remaining adherent connective tissue.</li>
	<li>Remove any gauze used to hold the liver and replace the intestines with cotton swabs.</li>
</ol>
3. Left Kidney Ischaemia and Reperfusion
<ol>
	<li>Gently push the intestines towards the right side of the abdominal cavity with cotton swabs to expose the left kidney and ureter and cover with moistened drapes. If necessary the pancreas can be deflected with moistened gauze to allow easier access.</li>
	<li>Use blunt dissection to gently break the connective tissues anterior and posterior to the left renal artery and vein, and then create a channel underneath the vessels in a similar manner to that described in Ureter Division and Right Nephrectomy step 2.4.</li>
	<li>Induce ischaemia of the left kidney by applying a micro serrafine clip onto the renal artery and vein. Successful ischaemia can be visually confirmed by a gradual uniform darkening of the kidney. Blood vessels in addition to the left renal artery and vein may occasionally supply the kidney. If present, these additional blood vessels will also need to be occluded using micro serrafine clips in order to successfully induce ischaemia of the entire kidney.</li>
	<li>Replace the intestines and carefully ensure that there are no abrupt twists that may lead to intestinal ischaemia compromising results. Temporarily close the peritoneum with a single suture.</li>
	<li>Following ischaemia induction immediately place the mouse on a homeothermic blanket with a rectal thermistor attached to a control unit that will maintain the body temperature at 37 &#176;C for the required duration of ischaemia. To define the appropriate length of ischaemia necessary to induce the desired degree of renal injury and kidney failure it is recommended that a titration be performed for each strain and surgical operator.</li>
	<li>Shortly before the end of the ischaemic period re-open the peritoneum and carefully position the intestines to allow access to the clamp and view the kidney. Insertion of the retractor, as shown in the video, is not necessary and was solely performed for presentational purposes.</li>
	<li>Remove the clamp after the period of ischaemia has concluded. Immediately after removal the kidney will rapidly change color from a dark maroon to a healthy dark pink indicating successful reperfusion.</li>
	<li>Again ensure that the intestines are not twisted prior to closing the peritoneum with a blanket stitch using 6/0 braided silk suture. Then close the skin using metallic skin clips.</li>
	<li>To minimize the risk of post-operative infection, apply an antiseptic such as iodine/alcohol solution to the surgical area.</li>
</ol>
4. Post-operative Recovery and Care
<ol>
	<li>Partially reverse anesthesia with atipamezole hydrochloride (2 mg/kg) subcutaneously and administer fluids with a subcutaneous injection of 1&#160;ml&#160;warmed saline to prevent dehydration following surgery.</li>
	<li>Carefully monitor mice until they have recovered consciousness, appear alert and are able to right themselves.</li>
	<li>Allow mice to recover in a heated box kept at 29 &#176;C, located in a quiet environment, for 24 hr. Mice will have an impaired ability to thermoregulate due to the anesthesia and it is therefore crucial that they are housed at an elevated temperature to enable effective recovery. Moistened food can also be provided to encourage fluid and nutrition intake.</li>
	<li>For long-term recovery experiments provide ongoing analgesics and remove skin clips 7 days following surgery.</li>
</ol>
5. Assessing Functional and Structural Kidney Injury and Regeneration
<ol>
	<li>Blood may be collected from the tail vein during experiments or by cardiac puncture at the time of euthanasia at the end of the experiment. Measure renal function by serum creatinine, using a creatinase based method, and blood urea nitrogen (BUN) on a biochemical centrifugal analyzer as described previously7. The renal function of normal healthy mice needs to be determined before establishing a model of renal failure as this can vary markedly depending on the analysis method used e.g. the Jaffe reaction and creatinase based methods for measuring creatinine give different results.</li>
	<li>Examine renal structure by histopathology on paraffin embedded kidney tissue sections stained with Haematoxylin and Eosin (H&#38;E) or Periodic Acid Schiff (PAS). Capture between 5 &#8211; 10 images at 200x magnification within the outer stripe of the outer medulla (OSOM) for each mouse. Assess the level of injury in the OSOM as this region is injured in this model because it is highly vulnerable to hypoxia.</li>
	<li>Use either of the two scoring systems detailed below to measure acute tubular necrosis (ATN) or regeneration. Representative morphology of the classifications used in these systems is illustrated in Figure 1.</li>
	<li>Use a simple binary system to score ATN. On the basis of cellular integrity and morphology mark and count tubules as either viable (intact cell morphology) or necrotic (compromised cell integrity, abnormal cell morphology or loss of cells).
	<ol>
		<li>Express the number of necrotic tubules as a percentage of the total number of tubules (necrotic tubules %).</li>
	</ol>
	</li>
	<li>Classify regeneration using another system based on cell integrity, morphology and nuclei number. Mark and count tubules as healthy, necrotic, injured or recovering according to the criteria listed in the following steps.
	<ol>
		<li>As with the ATN scoring system (detailed in step 5.4) mark tubules with intact normal cells as healthy whilst tubules showing compromised cell integrity, abnormal cell morphology or overt cell loss should be scored as necrotic.</li>
		<li>Classify tubules as injured if they have a thinned cytoplasm containing few nuclei. In contrast, designate tubules containing more nuclei with more normal cell morphology as recovering.</li>
		<li>Express each tubule classification as a percentage of the total tubule number.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Kinsey, G. R., Li, L., Okusa, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation+in+acute+kidney+injury.">Inflammation in acute kidney injury.</a> Nephron Exp Nephrol. 109, (2008).</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> Am J Physiol Renal Physiol. 303, (2012).</li><li>Ferenbach, D. A., Kluth, D. C., Hughes, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemeoxygenase-1+and+renal+ischaemia-reperfusion+injury.">Hemeoxygenase-1 and renal ischaemia-reperfusion injury.</a> Nephron Exp Nephrol. 115, (2010).</li><li>Ferenbach, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+induction+of+macrophage+hemeoxygenase-1+is+protective+during+acute+kidney+injury+in+aging+mice.">The induction of macrophage hemeoxygenase-1 is protective during acute kidney injury in aging mice.</a> Kidney Int. 79, 966-976 (2011).</li><li>Ferenbach, D. 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=Macrophage/monocyte+depletion+by+clodronate,+but+not+diphtheria+toxin,+improves+renal+ischemia/reperfusion+injury+in+mice.">Macrophage/monocyte depletion by clodronate, but not diphtheria toxin, improves renal ischemia/reperfusion injury in mice.</a> Kidney Int. 82, 928-933 (2012).</li><li>Ferenbach, D. 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=Macrophages+expressing+heme+oxygenase-1+improve+renal+function+in+ischemia/reperfusion+injury.">Macrophages expressing heme oxygenase-1 improve renal function in ischemia/reperfusion injury.</a> Mol Ther. 18, 1706-1713 (2010).</li><li>Keppler, 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=Plasma+creatinine+determination+in+mice+and+rats:+an+enzymatic+method+compares+favorably+with+a+high-performance+liquid+chromatography+assay.">Plasma creatinine determination in mice and rats: an enzymatic method compares favorably with a high-performance liquid chromatography assay.</a> Kidney Int. 71, 74-78 (2007).</li><li>Vinuesa, E., 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=Macrophage+involvement+in+the+kidney+repair+phase+after+ischaemia/reperfusion+injury.">Macrophage involvement in the kidney repair phase after ischaemia/reperfusion injury.</a> J Pathol. 214, 104-113 (2008).</li><li>Friedewald, J. J., Rabb, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+cells+in+ischemic+acute+renal+failure.">Inflammatory cells in ischemic acute renal failure.</a> Kidney Int. 66, 486-491 (2004).</li><li>Gandolfo, M. 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=Foxp3++regulatory+T+cells+participate+in+repair+of+ischemic+acute+kidney+injury.">Foxp3+ regulatory T cells participate in repair of ischemic acute kidney injury.</a> Kidney Int. 76, 717-729 (2009).</li><li>Burne, M. 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=Identification+of+the+CD4++T+cell+as+a+major+pathogenic+factor+in+ischemic+acute+renal+failure.">Identification of the CD4+ T cell as a major pathogenic factor in ischemic acute renal failure.</a> J Clin Invest. , 1283-1290 (2001).</li><li>Burne-Taney, M. 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=B+cell+deficiency+confers+protection+from+renal+ischemia+reperfusion+injury.">B cell deficiency confers protection from renal ischemia reperfusion injury.</a> J Immunol. 171, 3210-3215 (2003).</li><li>Kinsey, G. 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=Regulatory+T+cells+suppress+innate+immunity+in+kidney+ischemia-reperfusion+injury.">Regulatory T cells suppress innate immunity in kidney ischemia-reperfusion injury.</a> J Am Soc Nephrol. 20, 1744-1753 (2009).</li><li>Li, L., Okusa, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophages,+dendritic+cells,+and+kidney+ischemia-reperfusion+injury.">Macrophages, dendritic cells, and kidney ischemia-reperfusion injury.</a> Semin Nephrol. 30, 268-277 (2010).</li><li>Lin, S. 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=Macrophage+Wnt7b+is+critical+for+kidney+repair+and+regeneration.">Macrophage Wnt7b is critical for kidney repair and regeneration.</a> Proc Natl Acad Sci U S A. 107, 4194-4199 (2010).</li><li>Lee, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+macrophage+phenotypes+contribute+to+kidney+injury+and+repair.">Distinct macrophage phenotypes contribute to kidney injury and repair.</a> J Am Soc Nephrol. 22, 317-326 (2011).</li><li>Kumar, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dexamethasone+ameliorates+renal+ischemia-reperfusion+injury.">Dexamethasone ameliorates renal ischemia-reperfusion injury.</a> J Am Soc Nephrol. 20, 2412-2425 (2009).</li><li>Sharples, E. 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=Erythropoietin+protects+the+kidney+against+the+injury+and+dysfunction+caused+by+ischemia-reperfusion.">Erythropoietin protects the kidney against the injury and dysfunction caused by ischemia-reperfusion.</a> J Am Soc Nephrol. 15, 2115-2124 (2004).</li><li>Gueler, F., 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=Statins+attenuate+ischemia-reperfusion+injury+by+inducing+heme+oxygenase-1+in+infiltrating+macrophages.">Statins attenuate ischemia-reperfusion injury by inducing heme oxygenase-1 in infiltrating macrophages.</a> Am J Pathol. 170, 1192-1199 (2007).</li><li>Delbridge, M. S., Shrestha, B. M., Raftery, A. T., ElNahas, A. M., Haylor, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+body+temperature+in+a+rat+model+of+renal+ischemia-reperfusion+injury.">The effect of body temperature in a rat model of renal ischemia-reperfusion injury.</a> Transplant Proc. 39, 2983-2985 (2007).</li></ol>

The authors have no competing or conflicting interests to disclose.

Renal IRI is an important cause of AKI with no specific treatment available. The experimental study of renal IRI has been highly informative with previous work demonstrating the role of macrophages, dendritic cells, lymphocytes, regulatory T-cells as well as other cells and mediators in the induction of both the acute injury and healing phase5,8-16. In addition, experimental renal IRI has been used to assess the effect of various therapeutic agents4,17-19.
The model of re...

Ischaemia reperfusion injury (IRI) is a common mode of injury for multiple organs including the kidney, heart and brain. Renal IRI may lead to acute kidney injury (AKI) in patients and no specific treatment is available. AKI as a result of IRI has a complicated pathogenesis involving both the innate and adaptive immune response1. An experimental model of renal IRI offers the potential to dissect the key cells and mediators involved in the pathogenesis of AKI as well as the subsequent renal regeneration that ensues over subsequent days. Furthermore the effects of novel therapeutic agents upon disease processes can be assessed.
The overall goal of the experimental model of renal IRI described here is to induce both acute functional and structural kidney injury. Some investigators have utilized a model that involves the induction of bilateral IRI2. Although the bilateral renal IRI model is of use, the unilateral renal IRI model has the advantage of a right nephrectomy being undertaken at the time of surgery. The right nephrectomy tissue serves as valuable control tissue in studies involving a pretreatment step that either induces or suppresses the expression of a gene or protein. For example, we have used this model to assess the preconditioning effects of heme arginate (HA) injection 24 hours before renal IRI3. The successful induction of the cytoprotective enzyme heme-oxygenase-1 (HO-1) by HA before IRI was confirmed in the right nephrectomy control tissue4. HA reduced renal IRI in aged mice in part via an HO-1 dependent mechanism. Similarly, we have used the model in macrophage depletion studies to examine the role of macrophages in renal IRI5. Immunohistochemical analysis of the right nephrectomy tissue can be used to confirm the efficacy of the ablation methodology. The right nephrectomy tissue can therefore be used to both confirm and quantify the level of induction or inhibition of the molecule of interest in each individual experimental animal. This model will be of interest to researchers who are using drugs or other compounds to modulate the expression of genes or proteins etc. prior to the induction of renal IRI.
Other studies have used flank incisions to access the kidneys. The model described here uses a single midline abdominal surgery to perform both the right nephrectomy and induce ischaemia reperfusion of the left kidney. This surgical approach provides excellent visualization of the surgical field including the renal pedicles and color changes that follow renal pedicle clamping. Our published experience with the model4-6 indicates that mice quickly recover from the surgery with a near 100% survival rate.
Lastly, kinetic analysis of the model over a period of 7 days indicates that this model exhibits restoration of both renal function and tubular integrity, with significant tubular cell proliferation.

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			<td height="21" style="height:21px;width:265px;">Name of Material/ Equipment</td>
			<td style="width:125px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
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			<td></td>
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			<td height="42" style="height:42px;">Tissue scissors</td>
			<td style="width:125px;">Fine Science Tools</td>
			<td>14072 - 10</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Micro-Adson forceps (Rat toothed)</td>
			<td style="width:125px;">Fine Science Tools</td>
			<td style="width:119px;">11019 - 12</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;">S&#38;T JFA-5bTC Forceps - SuperGrip Angled</td>
			<td style="width:125px;">Fine Science Tools</td>
			<td>00649-11</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Colibri retractor</td>
			<td style="width:125px;">Fine Science Tools</td>
			<td>17000 - 04</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Micro clip applicator</td>
			<td style="width:125px;">Fine Science Tools</td>
			<td>18057-14</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Micro serrafines&#160;</td>
			<td style="width:125px;">Fine Science Tools</td>
			<td>18055-04</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Olsen-Hegar needle holder</td>
			<td style="width:125px;">Fine Science Tools</td>
			<td>12002 - 12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;"></td>
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			<td></td>
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			<td height="21" style="height:21px;width:265px;">Hemoclip Plus Ligating Clips Small</td>
			<td style="width:125px;">Weck</td>
			<td style="width:119px;">533837</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:265px;">Autoclip Wound Clip System, 9mm</td>
			<td style="width:125px;">Harvard Apparatus</td>
			<td style="width:119px;">PY2 52-3748</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:265px;">Silk Black Braided Suture, Size 6-0</td>
			<td style="width:125px;">Harvard Apparatus</td>
			<td style="width:119px;">723288</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;width:265px;">Standard Heat Matt</td>
			<td style="width:125px;"></td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:265px;">Homeothermic Blanket &#38; Control Unit</td>
			<td style="width:125px;">Harvard Apparatus</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;"></td>
			<td style="width:125px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Lacri-Lube</td>
			<td style="width:125px;">Allergan</td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Vetasept Chlorhexidine&#160;&#160;</td>
			<td style="width:125px;">AnimalCare</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vetalar : Ketamine hydrochloride</td>
			<td style="width:125px;"></td>
			<td style="width:119px;"></td>
			<td>100mg/ml solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Domitor : medetomidine hydrochloride&#160;</td>
			<td style="width:125px;"></td>
			<td style="width:119px;"></td>
			<td>1mg/ml</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;">Vetergesic : Buprenorphine hydrochloride&#160;</td>
			<td style="width:119px;"></td>
			<td>0.3mg/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Antisedan : Atipamezole hydrochoride</td>
			<td style="width:125px;"></td>
			<td style="width:119px;"></td>
			<td>5mg/ml</td>
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</table>

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.

Tubular injury and recovery may be assessed by H&#38;E or PAS staining of tissue sections following renal IRI. Tubules located within the OSOM are classified as healthy, injured, necrotic or recovering according to cell morphology, integrity and nuclei number (Figure 1). The functional and structural injury in this model is dependent upon the duration of ischaemia. A progressive increase in the severity of renal dysfunction, assessed by plasma creatinine and BUN, is evident as the duration of ischaemia i...

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

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49.3K Views.  University of Edinburgh. 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.

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

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 ...

A Neonatal Mouse Spinal Cord Compression Injury Model

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal cord. A major current area of research is focused on the mechanisms of adaptive plasticity that underlie spontaneous or induced functional recovery following SCI. Spontaneous functional recovery is reported to be greater early in life, raising interesting questions about how adaptive plasticity changes as the spinal cord develops. To facilitate investigation of this dynamic, we have developed a SCI model in the neonatal mouse. The model has relevance for pediatric SCI, which is too little studied. Because neural plasticity in the adult involves some of the same mechanisms as neural plasticity in early life1, this model may potentially have some relevance also for adult SCI. Here we describe the entire procedure for generating a reproducible spinal cord compression (SCC) injury in the neonatal mouse as early as postnatal (P) day 1. SCC is achieved by performing a laminectomy at a given spinal level (here described at thoracic levels 9-11) and then using a modified Yasargil aneurysm mini-clip to rapidly compress and decompress the spinal cord. As previously described, the injured neonatal mice can be tested for behavioral deficits or sacrificed for ex vivo physiological analysis of synaptic connectivity using electrophysiological and high-throughput optical recording techniques1. Earlier and ongoing studies using behavioral and physiological assessment have demonstrated a dramatic, acute impairment of hindlimb motility followed by a complete functional recovery within 2 weeks, and the first evidence of changes in functional circuitry at the level of identified descending synaptic connections1.

This article describes a method for generating a reproducible spinal cord compression injury (SCI) in the neonatal mouse. The model provides an advantageous platform for studying mechanisms of adaptive plasticity that underlie spontaneous functional recovery.

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal ...

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, ...

A Mouse Model of Single and Repetitive Mild Traumatic Brain Injury

Mild Traumatic Brain Injury (mTBI) can result in the acute loss of brain function, including a period of confusion, a loss of consciousness (LOC), focal neurological deficits and even amnesia. Athletes participating in contact sports are at high risk of exposure to large number of mTBIs. In terms of the level of injury in a sporting athlete, a mTBI is defined as a mild injury that does not cause gross pathological changes, but does cause short-term neurological deficits that are spontaneously resolved. Despite previous attempts to model mTBI in mice and rats, many have reported gross adverse effects including skull fractures, intracerebral bleeding, axonal injury and neuronal cell death. Herein, we describe our highly reproducible animal model of mTBI that reproduces clinically relevant symptoms. This model uses a custom made pneumatic impactor device to deliver a closed-head trauma. This impact is made under precise velocity and deformation parameters, creating a reliable and reproducible model to examine the mechanisms that contribute to effects of single or repetitive concussive mTBI.

Athletes absorb several hundred mild traumatic brain injuries (mTBI)/concussions every year; however, the consequence of these on the brain is poorly understood. Therefore, an animal model of single and repetitive mTBI that consistently replicates clinically relevant symptoms provides the means to advance the study of mTBI and concussion.

Mild Traumatic Brain Injury (mTBI) can result in the acute loss of brain function, including a period of confusion, a loss of consciousness (LOC), focal ...

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, ...

A Rat Lung Transplantation Model of Warm Ischemia/Reperfusion Injury: Optimizations to Improve Outcomes

From our experience with rat lung transplantation, we have found several areas for improvement. Information in the existing literature regarding methods for choosing appropriate cuff sizes for the pulmonary vein (PV), pulmonary artery (PA), or bronchus (Br) are varied, thus making the determination of proper cuff size during rat lung transplantation an exercise of trial and error. By standardizing the cuffing technique to use the smallest effective cuff appropriate for the size of the vessel or bronchus, one can make the transplantation procedure safer, faster, and more successful. Since diameters of the PV, PA, and Br are related to the body weight of the rat, we present a strategy to choosing an appropriate size using a weight-based guide. Since lung volume is also related to body weight, we recommend that this relationship should also be considered when choosing the proper volume of air for donor lung inflation during warm ischemia as well as for the proper volume of PBS to be instilled during bronchoalveolar lavage (BAL) fluid collection. We also describe methods for 4th intercostal space dissection, wound closure, and sample collection from both the native and transplanted lobes.

Here, we present optimizations to a rat lung transplantation model that serve to improve outcomes. We provide a size guide for cuffs based on body weight, a measurement strategy to ascertain the 4th intercostal space, and methods of wound closure and BAL (bronchoalveolar lavage) fluid and tissue collection.

From our experience with rat lung transplantation, we have found several areas for improvement. Information in the existing literature regarding methods ...