The present studies were supported by United States National Institutes of Health grant R01-HL092188, R01-DK083385 and R01HL098294, and Foundation for Anesthesia Education and Research (FAER) Grants to HKE, a FAER grant to JYD and a DFG (Deutsche Forschungsgemeinschaft) Research Fellowship (GR2121/1-1) to AG.

General remarks. All operations should be performed under an upright dissecting microscope (Leica) and by using a surgical coagulator. The mice in the experimental groups should be matched in age and weight to ensure comparability of the results. Temperature, blood pressure, anesthesia and fluid administration should be stable and monitored during the experiment.
1. Anesthesia and surgery
<ol>
	<li>Renal ischemia experiments can either be performed with simultaneously applying ischemia and reperfusion to both kidneys, or after single nephrectomy in combination with ischemia treatment of the remaining kidney. As bilateral ischemia treatment of both kidneys is associated with increased technical difficulties, a more complex experimental setting and longer surgery, we will perform a right nephrectomy followed by ischemia treatment of the remaining left kidney by utilizing a hanging weight system. We utilized this hanging weight system also for coronary artery occlusion in the heart. However, the procedure is different since the renal artery can be isolated from the surrounding tissue whereas the coronary artery lies within the heart muscle.</li>
 <li>Mice (8-14 weeks of age, either sex) will be anesthetized with pentobarbital at a dose of 70mg/kg. We will maintain anesthesia with approx. 10 mg/kg/h sodium pentobarbital. Overdosing can significantly lower blood pressure and thus can alter the results. In our initial studies we also used Ketamine and xylazine. However, these compounds induced a strong bradycardia (150 /min). Beside the observed bradycardia there is also evidence that both compounds are involved in protective mechanisms during conditions of ischemia. Volatile anesthetics cannot be used either due to their capability to induce also renal protection and low blood pressure. Therefore our final choice for anesthesia was pentobarbital. In addition, ketamine and xylazine are primarily excreted by the kidneys. Nephrectomy and ischemia may prolong ketamine anesthesia.</li>
 <li>Once pain reflexes are absent (tested by squeezing the toes with tweezers) mice will be placed on a temperature-controlled heating table (RT, Effenberg, Munich, Germany to maintain body temperature at 37.0&deg;C) in a left lateral decubitus position. </li>
 <li>Attach the lower and upper extremities to the table by using tape.</li>
 <li>For non-survival surgeries, such as shown in the video demonstration, cover the incision area with mineral oil to prevent inhalation of mouse hair by cutting the skin and thus prevent
	the risk of mouse hair allergies. In case of survival experiments proper aseptic techniques including clipping of hair, disinfection of
	skin with iodophors (e.g. Betadine), sterile handling of autoclaved instruments and using masks a sterile gloves for the surgeon are
	required. Mineral oil is not used in survival surgeries. For survival surgeries sterile NaCl is used</li>
 <li>Right flank incision will be performed with a scissor and a coagulation electrode (Erbe, ICC50, Germany) to prevent bleeding of muscle and skin vessels. In contrast to accessing the kidneys via midline laparotomy, this technique does not require access to the peritoneal cavity.</li>
 <li>The renal pedicle including renal artery and renal vein are ligated using a 4/0 silk suture and the right kidney will be cut out with a scissor without interfering with the adrenal vessels. </li>
 <li>The surgical wound is closed using continuous suture (two stitches) of the muscle wall and skin with a 4/0 suture. </li>
 <li>Animals are then placed in a right lateral decubitus position and a left flank incision will be performed. </li>
 <li>The left kidney is carefully removed from the connective tissues, avoiding the adrenal gland and vessels. </li>
 <li>Next, the kidney is turned with its ventral side down into a lucite cup. The lucite cup should be sterilized for
	survival surgeries. The pole of the kidney is carefully pushed into the lucite cup by holding the surrounding tissue with a forceps.
	Thereafter the pole is carefully pushed into the lucite cup with a forceps. The kidney will be kept wet and warm with a wet swab soaked
	with water at 37.0&deg;C.</li>
 <li>Following exposure of the left kidney and placement into a lucite cup the kidney holder is restrained ventrally to have tension on the renal vessels. A sufficient restraining is important for a successful placement of the suture under the renal artery. Thereafter the kidney holder is fixed and the renal artery can be easily identified as it runs on top of the renal vein. </li>
 <li>The renal artery is dissected from adjacent tissues, close to its take-off from the abdominal aorta with fine forceps. The artery is dissected from the underlying vein with a curved forceps at the very top. </li>
 <li>An 8/0 silk suture (Ethicon, Norderstedt, Germany) is placed under the renal artery by using one forceps as a leading guide. This technique allows interruption of only the arterial blood flow to the kidney without compression of the renal vein, as compared to clamping of the renal pedicle.</li>
 <li>The suture is placed over a small pole (can be a prepared safety pin) and a weight of approx. 1g is attached to each end (eppendorf tube). The weight can be adapted to the kidney size by taking out or adding water into the eppendorf tubes. However, the artery should be slightly pulled upwards, so that no blood is running through the artery but without major stretch on the artery. Major stretch stress on the artery can cause vessel wall damage with thickening of the arterial wall and reduced reperfusion.</li>
 <li>While the weights are unsupported, the renal artery is immediately occluded. In contrast, when the weights are supported, blood flow is immediately restored to the organ. Successful occlusion is confirmed by a change of color from red to white. </li>
 <li>Different ischemia times can be performed as required by the experiment. A complete reperfusion will take place depending on the length of the ischemia time. Ischemia times up to 45 minutes allow recoverable kidney injury. In our experimental settings we used 20 up to 30 minutes of ischemia with a medium renal injury which allows us to see improvement as well as impairment of kidney damage depending on the mouse strain and the treatment. </li>
 <li>After complete reperfusion the kidney can be replaced out of the lucite cup. Therefore the kidney holder is unfixed and the tension on the kidney is released. The cranial pole is gently removed out of the cup with a bland forceps and the caudal pole follows immediately. </li>
 <li>The fixation (tapes) of the mouse can be released and the mouse can be placed in a supine position for e.g. FITC-inulin labeled clearance.</li>
 <li>In subsets of experiments, blood pressure and heart rate can be recorded throughout the surgical procedure. Blood pressure measurements are obtained continuously via a carotid artery catheter. In brief, the carotid artery is exposed via blunt dissection of the paratracheal muscles. Following further exposure and careful avoidance of tissue trauma (particular of the vagal nerve), a catheter is inserted into the vessel using two sutures and a small clamp. Blood pressure is measured with a statham element (WK 280, WKK, Kaltbrunn, Switzerland).</li>
 <li>For FITC-labeled inulin clearance a catheter has to be placed into the left jugular vein for infusion of sodium chloride and FITC-labeled inulin. Furthermore a bladder catheter has to be inserted for urine collection in a time dependent fashion as explained below in detail.</li> 
</ol>
2. Measurement of kidney injury
<ol>
	<li>In general: The following outcome parameters are recommended to determine kidney injury following renal ischemia: Perform FITC-labeled inulin clearance to assess the glomerular filtration rate (GFR). As an alternative determine creatinine clearance and serum creatinine via metabolic cage investigation 24 hours following renal injury. Perform renal H&amp;E staining and a myeloperoxidase (MPO) ELISA to determine renal damage and inflammation 24 hours following renal ischemia. </li>
 <li>Metabolic cage investigations: 
 	<ol>
 <li>Following ischemia the left kidney is turned back into the retroperitoneal position and the wound is closed as described above.</li>
 <li>At the end of surgery mice receive 0.3 ml normal saline IP and recover for 2 hours under a heating lamp. </li>
 <li>Thereafter mice will be placed into metabolic cages (Tecniplast Deutschland, Hohenpeissenberg, Germany) for determination of renal functional parameters for 2 times of 24 hours. </li>
 <li>The water intake of each mouse has to be determined by weighing the empty and filled bottle with water before supply to the mouse and than 24 hours later by weighing the full bottle again. The mouse weight has to be determined as well.</li>
 <li>After 24 hours approximately 75&mu;l blood are taken from the venous plexus behind the eye via a capillary tube under short isoflorane anesthesia.</li>
 <li>Renal function is assessed by measuring plasma and urine creatinine 24 hours after renal ischemia using a commercially available colorimtric method according to the manufacturer`s protocol (LT-SYS, Labor + Technik, Berlin, Germany). Plasma and urine concentrations of Na+, K+ are determined with a flame-emission photometer (ELEX 6361, Eppendorf AG, Hamburg, Germany). Renal excretory and hemodynamic values are calculated using standard formulas15-17. </li>
 
 </ol>
 </li>
 <li>Inulin clearance: FITC-inulin clearance experiments will be performed following renal ischemia.
 	<ol>
 	<li>The left jugular vein is cannulated for continuous infusion. After dissecting the vein from adjacent tissue with a preparation scissor (Dumont, WPI) place two sutures (4/0) around the vein. Place a clamp on the vein before the proximale suture to stop blood flow. Thereafter close the distal knot and place a weight on one of the distal suture ends to strain tension on the vein. This is very important to get the catheter in the vein. Use a micro-scissor to cut a small opening into the vein. Hold the opening with a fine forceps (Dumont, WPI) and place the catheter into the vein. To advance the catheter take the clamp off. Make sure that there is no tension on the catheter so that you can place it down without holding it. Thereafter close the second proximal suture over the vein and the catheter.</li>
 <li>A catheter is placed in the urinary bladder for timed urine collection. Therefore you cut a short incision in the skin over the bladder. Carefully remove the bladder through the skin hole. Fix the bladder with a wet tissue. Place a prepared suture over the bladder (4/0). </li>
 <li>Cut a short piece of a catheter (1.5cm) and heat on end to get a ring around the catheter which makes it easier to fix it in the bladder. </li>
 <li>Cut a hole into the bladder at its very top. Make the cut between the vessels to prevent bleeding. Insert the big end of the catheter into the bladder and close the suture over the catheter and the bladder.</li>
 <li>All mice receive a bolus of 0.85% sodium chloride solution in an amount equal to 20% of body weight. Continuous infusion is maintained at a rate of 800ul/h/25g body weight and FITC-inulin is added to the infusion for evaluation of whole kidney glomerular filtration rate (GFR). After stabilization of the animals for 20 min, 20 min timed urine collections are performed (four times) for determination of urinary flow rate and FITC-inulin. Blood is obtained in the middle of every period for measurement of FITC-inulin.</li>
 <li>When starting the collection periods for 20 minutes use a timer. Collect the urine by placing the end of the bladder catheter into a small eppendorf tube. Measure the tube weight before and after urine collection so that you can determine the urine amount. Blood samples are taken via retroorbital blood collection form the venous plexus via capillary tubes in the middle of each collection period.</li>

 </ol>
 
 
 </li>
 <li>Blood and the kidney can be collected after each procedure for further analysis e.g. MPO ELISA, cytokine EISA, histology.</li>
 
</ol>
3. Representative Results:


<img src="/files/ftp_upload/2549/2549fig1.jpg" alt="Figure 1"/> 
Figure 1. Model of a hanging-weight system for renal ischemia: A hanging weight system has been established to induce renal ischemia of the left kidney. Therefore an 8.0 nylon suture was placed under the renal artery and at the end of each suture a small weight (1g) was attached. This experimental design allows reversible occlusion of the renal artery by applying and releasing the weight load.

<ol>
	<li>Abuelo, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normotensive+ischemic+acute+renal+failure.">Normotensive ischemic acute renal failure.</a> N Engl J Med. 357, 797-805 (2007).</li><li>Thadhani, R., Pascual, M., Bonventre, J. V. <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.">Acute renal failure.</a> N Engl J Med. 334, 448-460 (1996).</li><li>Lameire, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pathophysiology+of+acute+renal+failure.">The pathophysiology of acute renal failure.</a> Crit Care Clin. 21, 197-210 (2005).</li><li>Gelman, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pathophysiology+of+aortic+cross-clamping+and+unclamping.">The pathophysiology of aortic cross-clamping and unclamping.</a> Anesthesiology. 82, 1026-1060 (1995).</li><li>Yalavarthy, R., Edelstein, C. L., Teitelbaum, I. <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+and+chronic+kidney+disease+following+liver+transplantation.">Acute renal failure and chronic kidney disease following liver transplantation.</a> Hemodial Int. 11, S7-S12 (2007).</li><li>Schrier, R. W., Wang, W. <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+and+sepsis.">Acute renal failure and sepsis.</a> N Engl J Med. 351, 159-169 (2004).</li><li>Grenz, A., Osswald, H., Eckle, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Reno-Vascular+A2B+Adenosine+Receptor+Protects+the+Kidney+from+Ischemia.">The Reno-Vascular A2B Adenosine Receptor Protects the Kidney from Ischemia.</a> PLoS Medicine. 5, e137-e137 (2008).</li><li>Grenz, A., Zhang, H., Hermes, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+E-NTPDase1+(CD39)+to+renal+protection+from+ischemia-reperfusion+injury.">Contribution of E-NTPDase1 (CD39) to renal protection from ischemia-reperfusion injury.</a> FASEB J. 21, 2863-2873 (2007).</li><li>Grenz, A., Zhang, H., Eckle, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+role+of+ecto-5'-nucleotidase+(CD73)+in+renal+ischemia.">Protective role of ecto-5'-nucleotidase (CD73) in renal ischemia.</a> J Am Soc Nephrol. 18, 833-845 (2007).</li><li>Akcay, A., Nguyen, Q., Edelstein, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mediators+of+inflammation+in+acute+kidney+injury.">Mediators of inflammation in acute kidney injury.</a> Mediators Inflamm. , 137072-137072 (2009).</li><li>Faubel, S., Ljubanovic, D., Poole, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+CD4+T-cell+depletion+is+not+sufficient+to+prevent+ischemic+acute+renal+failure.">Peripheral CD4 T-cell depletion is not sufficient to prevent ischemic acute renal failure.</a> Transplantation. 80, 643-649 (2005).</li><li>Wei, Q., Bhatt, K., He, H. Z., Mi, Q. S., Haase, V. H., 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=Targeted+deletion+of+Dicer+from+proximal+tubules+protects+against+renal+ischemia-reperfusion+injury.">Targeted deletion of Dicer from proximal tubules protects against renal ischemia-reperfusion injury.</a> J Am Soc Nephrol. 21, 756-761 (2010).</li><li>Joo, J. D., Kim, M., Horst, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+and+delayed+renal+protection+against+renal+ischemia+and+reperfusion+injury+with+A1+adenosine+receptors.">Acute and delayed renal protection against renal ischemia and reperfusion injury with A1 adenosine receptors.</a> Am J Physiol Renal Physiol. 293, F1847-F1857 (2007).</li><li>Grenz, A., Eckle, T., Zhang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+hanging-weight+system+for+isolated+renal+artery+occlusion+during+ischemic+preconditioning+in+mice.">Use of a hanging-weight system for isolated renal artery occlusion during ischemic preconditioning in mice.</a> Am J Physiol Renal Physiol. 292, F475-F485 (2007).</li><li>Qi, Z., Whitt, I., Mehta, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serial+determination+of+glomerular+filtration+rate+in+conscious+mice+using+FITC-inulin+clearance.">Serial determination of glomerular filtration rate in conscious mice using FITC-inulin clearance.</a> Am J Physiol Renal Physiol. 286, 590-596 (2004).</li><li>Lorenz, J. N., Gruenstein, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple,+nonradioactive+method+for+evaluating+single-nephron+filtration+rate+using+FITC-inulin.">A simple, nonradioactive method for evaluating single-nephron filtration rate using FITC-inulin.</a> Am J Physiol. 276, 172-177 (1999).</li><li>Lorenz, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+practical+guide+to+evaluating+cardiovascular,+renal,+and+pulmonary+function+in+mice.">A practical guide to evaluating cardiovascular, renal, and pulmonary function in mice.</a> Am J Physiol Regul Integr Comp Physiol. 282, 1565-1582 (2002).</li></ol>

No conflicts of interest declared.

In summary, the present study describes a novel technique for performing renal ischemia in a murine model using a hanging-weight system for renal artery occlusion. In fact, this study demonstrates highly reproducible injury, thus minimizing the variability associated with clamping of the renal pedicle.

<table border="1">
	<tbody>
 <tr>
 	<th>Name of the reagent</th>
 <th>Company</th>
 <th>Catalogue number</th>
 <th>Comments</th>
 </tr>
 <tr>
 	<td>Sodium Pentobarbital</td>
 <td>Vortech Pharmaceutical Ls, Ltd</td>
 <td>V.P.L. 9372

		</td>
			<td>4mg/mL in saline</td>
 </tr>
 <tr>
 	<td>Suture, silk 4.0</td>
 <td>Harvard Apparatus</td>
 <td>517698</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Suture, Prolene 8.0</td>
 <td>Ethicon, USA</td>
 <td>M8739</td>
 <td>reusable</td>
 </tr>
 <tr>
 	<td>dissecting microscope </td>
 <td>Leica</td>
 <td>n/a</td>
 <td>consider generous working distance</td>
 </tr>
 <tr>
 	<td>Heating Table</td>
 <td>Rt, Effenberger, Germany</td>
 <td>n/a</td>
 <td>only and single provider</td>
 </tr>
 <tr>
 	<td>Surgical instruments</td>
 <td>WPI, Dumont</td>
 <td>&nbsp;</td>
 <td>a fine microvessel scissor</td>
 </tr>
 
 </tbody>
</table>

use of a hanging-weight system for isolated renal artery occlusion

In hospitalized patients, over 50% of cases of acute kidney injury (AKI) are caused by renal ischemia 1-3. A recent study of hospitalized patients revealed that only a mild increase in serum creatinine levels (0.3 to 0.4 mg/dl) is associated with a 70% greater risk of death than in persons without any increase 1. Along these lines, surgical procedures requiring cross-clamping of the aorta and renal vessels are associated with a renal failure rates of up to 30% 4. Similarly, AKI after cardiac surgery occurs in over 10% of patients under normal circumstances and is associated with dramatic increases in mortality. AKI are also common complications after liver transplantation. At least 8-17% of patients end up requiring renal replacement therapy 5. Moreover, delayed graft function due to tubule cell injury during kidney transplantation is frequently related to ischemia-associated AKI 6. Moreover, AKI occurs in approximately 20% of patients suffering from sepsis 6.The occurrence of AKI is associated with dramatic increases of morbidity and mortality 1. Therapeutic approaches are very limited and the majority of interventional trials in AKI have failed in humans. Therefore, additional therapeutic modalities to prevent renal injury from ischemia are urgently needed 3, 7-9. 
To elucidate mechanisms of renal injury due to ischemia and possible therapeutic strategies murine models are intensively required 7-13. Mouse models provide the possibility of utilizing different genetic models including gene-targeted mice and tissue specific gene-targeted mice (cre-flox system). However, murine renal ischemia is technically challenging and experimental details significantly influence results. We performed a systematic evaluation of a novel model for isolated renal artery occlusion in mice, which specifically avoids the use of clamping or suturing the renal pedicle 14. This model requires a nephrectomy of the right kidney since ischemia can be only performed in one kidney due to the experimental setting. In fact, by using a hanging-weight system, the renal artery is only instrumented once throughout the surgical procedure. In addition, no venous or urethral obstruction occurs with this technique. We could demonstrate time-dose-dependent and highly reproducible renal injury with ischemia by measuring serum creatinine. Moreover, when comparing this new model with conventional clamping of the whole pedicle, renal protection by ischemic preconditioning is more profound and more reliable. Therefore his new technique might be useful for other researchers who are working in the field of acute kidney injury.

Watch this Scientific Journal Video about Use of a Hanging-weight System for Isolated Renal Artery Occlusion at JoVE.com

Use of a Hanging-weight System for Isolated Renal Artery Occlusion

A precise murine model for acute kidney injury (AKI) due to ischemia is an important tool to investigate acute kidney injury and possibly find therapeutic tools to treat renal injury. The hanging weight system offers a tool for immediate and reliable renal artery occlusion and reperfusion without causing renal congestion.

In hospitalized patients, over 50% of cases of acute kidney injury (AKI) are caused by renal ischemia 1-3. A recent study of hospitalized patients ...

use-of-a-hanging-weight-system-for-isolated-renal-artery-occlusion

Research

JoVE Journal

Medicine

15.8K Views.  University of Colorado. A precise murine model for acute kidney injury (AKI) due to ischemia is an important tool to investigate acute kidney injury and possibly find therapeutic tools to treat renal injury. The hanging weight system offers a tool for immediate and reliable renal artery occlusion and reperfusion without causing renal congestion. 

Video: Use of a Hanging-weight System for Isolated Renal Artery Occlusion

Modeling Stroke in Mice - Middle Cerebral Artery Occlusion with the Filament Model

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are very limited. To meet the need for novel therapeutic approaches, experimental stroke research frequently employs rodent models of focal cerebral ischaemia. Most researchers use permanent or transient occlusion of the middle cerebral artery (MCA) in mice or rats.
Proximal occlusion of the middle cerebral artery (MCA) via the intraluminal suture technique (so called filament or suture model) is probably the most frequently used model in experimental stroke research. The intraluminal MCAO model offers the advantage of inducing reproducible transient or permanent ischaemia of the MCA territory in a relatively non-invasive manner. Intraluminal approaches interrupt the blood flow of the entire territory of this artery. Filament occlusion thus arrests flow proximal to the lenticulo-striate arteries, which supply the basal ganglia. Filament occlusion of the MCA results in reproducible lesions in the cortex and striatum and can be either permanent or transient. In contrast, models inducing distal (to the branching of the lenticulo-striate arteries) MCA occlusion typically spare the striatum and primarily involve the neocortex. In addition these models do require craniectomy. In the model demonstrated in this article, a silicon coated filament is introduced into the common carotid artery and advanced along the internal carotid artery into the Circle of Willis, where it blocks the origin of the middle cerebral artery. In patients, occlusions of the middle cerebral artery are among the most common causes of ischaemic stroke. Since varying ischemic intervals can be chosen freely in this model depending on the time point of reperfusion, ischaemic lesions with varying degrees of severity can be produced. Reperfusion by removal of the occluding filament at least partially models the restoration of blood flow after spontaneous or therapeutic (tPA) lysis of a thromboembolic clot in humans.
In this video we will present the basic technique as well as the major pitfalls and confounders which may limit the predictive value of this model.

Filamentous occlusion of the Middle cerebral artery is a common model for studying ischemic stroke in mice.

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are ...

Use of a Hanging Weight System for Coronary Artery Occlusion in Mice

Murine studies of acute injury are an area of intense investigation, as knockout mice for different genes are becoming increasingly available 1-38. Cardioprotection by ischemic preconditioning (IP) remains an area of intense investigation. To further elucidate its molecular basis, the use of knockout mouse studies is particularly important 7, 14, 30, 39. Despite the fact that previous studies have already successfully performed cardiac ischemia and reperfusion in mice, this model is technically very challenging. Particularly, visual identification of the coronary artery, placement of the suture around the vessel and coronary occlusion by tying off the vessel with a supported knot is technically difficult. In addition, re-opening the knot for intermittent reperfusion of the coronary artery during IP without causing surgical trauma adds additional challenge. Moreover, if the knot is not tied down strong enough, inadvertent reperfusion due to imperfect occlusion of the coronary may affect the results. In fact, this can easily occur due to the movement of the beating heart.
Based on potential problems associated with using a knotted coronary occlusion system, we adopted a previously published model of chronic cardiomyopathy based on a hanging weight system for intermittent coronary artery occlusion during IP 39. In fact, coronary artery occlusion can thus be achieved without having to occlude the coronary by a knot. Moreover, reperfusion of the vessel can be easily achieved by supporting the hanging weights which are in a remote localization from cardiac tissues.
We tested this system systematically, including variation of ischemia and reperfusion times, preconditioning regiments, body temperature and genetic backgrounds39. In addition to infarct staining, we tested cardiac troponin I (cTnI)
as a marker of myocardial infarction in this model. In fact, plasma levels of cTnI correlated with infarct sizes (R2=0.8). Finally, we could show in several studies that this technique yields highly reproducible infarct sizes during murine IP and myocardial infarction6, 8, 30, 40, 41. Therefore, this technique may be helpful for researchers who pursue molecular mechanisms involved in cardioprotection by IP using a genetic approach in mice with targeted gene deletion. Further studies on cardiac IP using transgenic mice may consider this technique.

A murine model for myocardial ischemia and ischemic preconditioning is an important tool study cardioprotective mechanisms in vivo. Here, we report an easy applicable in situ model for cardiac IP using a hanging-weight system for coronary artery occlusion.

Murine studies of acute injury are an area of intense investigation, as knockout mice for different genes are becoming increasingly available 1-38. ...

Use of a Hanging-weight System for Liver Ischemia in Mice

Acute liver injury due to ischemia can occur during several clinical procedures e.g. liver transplantation, hepatic tumor resection or trauma repair and can result in liver failure which has a high mortality rate1-2. Therefore murine studies of hepatic ischemia have become an important field of research by providing the opportunity to utilize pharmacological and genetic studies3-9. Specifically, conditional mice with tissue specific deletion of a gene (cre, flox system) provide insights into the role of proteins in particular tissues10-13 . Because of the technical difficulty associated with manually clamping the portal triad in mice, we performed a systematic evaluation using a hanging-weight system for portal triad occlusion which has been previously described3. By using a hanging-weight system we place a suture around the left branch of the portal triad without causing any damage to the hepatic lobes, since also the finest clamps available can cause hepatic tissue damage because of the close location of liver tissue to the vessels. Furthermore, the right branch of the hepatic triad is still perfused thus no intestinal congestion occurs with this technique as blood flow to the right hepatic lobes is preserved. Furthermore, the portal triad is only manipulated once throughout the entire surgical procedure. As a result, procedures like pre-conditioning, with short times of ischemia and reperfusion, can be easily performed. Systematic evaluation of this model by performing different ischemia and reperfusion times revealed a close correlation of hepatic ischemia time with liver damage as measured by alanine (ALT) and aspartate (AST) aminotransferase serum levels3,9. Taken together, these studies confirm highly reproducible liver injury when using the hanging-weight system for hepatic ischemia and intermittent reperfusion. Thus, this technique might be useful for other investigators interested in liver ischemia studies in mice. Therefore the video clip provides a detailed step-by-step description of this technique.

We established a novel murine model of a hanging weight system for portal triad occlusion. This technique may be useful for future investigations of ischemia in murine hepatic models.

Acute liver injury due to ischemia can occur during several clinical procedures e.g. liver transplantation, hepatic tumor resection or trauma repair and ...

Mouse Model of Middle Cerebral Artery Occlusion

Stroke is the most common fatal neurological disease in the United States 1. The majority of strokes (88%) result from blockage of blood vessels in the brain (ischemic stroke) 2. Since most ischemic strokes (~80%) occur in the territory of middle cerebral artery (MCA) 3, many animal stroke models that have been developed have focused on this artery. The intraluminal monofilament model of middle cerebral artery occlusion (MCAO) involves the insertion of a surgical filament into the external carotid artery and threading it forward into the internal carotid artery (ICA) until the tip occludes the origin of the MCA, resulting in a cessation of blood flow and subsequent brain infarction in the MCA territory 4. The technique can be used to model permanent or transient occlusion 5. If the suture is removed after a certain interval (30 min, 1 h, or 2 h), reperfusion is achieved (transient MCAO); if the filament is left in place (24 h) the procedure is suitable as a model of permanent MCAO. This technique does not require craniectomy, a neurosurgical procedure to remove a portion of skull, which may affect intracranial pressure and temperature 6. It has become the most frequently used method to mimic permanent and transient focal cerebral ischemia in rats and mice 7,8. To evaluate the extent of cerebral infarction, we stain brain slices with 2,3,5-triphenyltetrazolium chloride (TTC) to identify ischemic brain tissue 9. In this video, we demonstrate the MCAO method and the determination of infarct size by TTC staining.

We demonstrate in the video a method for producing a middle cerebral artery occlusion in adult mice using an intraluminal monofilament. We also show how to evaluate the extent of cerebral infarction by 2,3,5-triphenyltetrazolium chloride (TTC) staining.

Stroke is the most common fatal neurological disease in the United States 1. The majority of strokes (88%) result from blockage of blood vessels in the ...

Utilizing a Cranial Window to Visualize the Middle Cerebral Artery During Endothelin-1 Induced Middle Cerebral Artery Occlusion

Creation of a cranial window is a method that allows direct visualization of structures on the cortical surface of the brain1-3. This technique can be performed in many locations overlying the rat cerebrum, but is most easily carried out by creating a craniectomy over the readily accessible frontal or parietal bones. Most frequently, we have used this technique in combination with the endothelin-1 middle cerebral artery occlusion model of ischemic stroke to quantify the changes in middle cerebral artery vessel diameter that occur with injection of endothelin-1 into the brain parenchyma adjacent to the proximal MCA4, 5. In order to visualize the proximal portion of the MCA during endothelin -1 induced MCAO, we use a technique to create a cranial window through the temporal bone on the lateral aspect of the rat skull (Figure 1). Cerebral arteries can be visualized either with the dura intact or with the dura incised and retracted. Most commonly, we leave the dura intact during visualization since endothelin-1 induced MCAO involves delivery of the vasoconstricting peptide into the brain parenchyma. This bypasses the need to incise the dura directly over the visualized vessels for drug delivery. This protocol will describe how to create a cranial window to visualize cerebral arteries in a step-wise fashion, as well as how to avoid many of the potential pitfalls pertaining to this method.

This article describes a method for visualizing rat cerebral arteries through a cranial window using temporal craniectomy in order to view proximal portions of the middle cerebral artery (Figure 1). This versatile method can be combined with various techniques of drug delivery to measure cerebral artery reactivity in vivo.

Creation of a cranial window is a method that allows direct visualization of structures on the cortical surface of the brain1-3. This technique can be ...

Optimized System for Cerebral Perfusion Monitoring in the Rat Stroke Model of Intraluminal Middle Cerebral Artery Occlusion

The translational potential of pre-clinical stroke research depends on the accuracy of experimental modeling. Cerebral perfusion monitoring in animal models of acute ischemic stroke allows to confirm successful arterial occlusion and exclude subarachnoid hemorrhage. Cerebral perfusion monitoring can also be used to study intracranial collateral circulation, which is emerging as a powerful determinant of stroke outcome and a possible therapeutic target. Despite a recognized role of Laser Doppler perfusion monitoring as part of the current guidelines for experimental cerebral ischemia, a number of technical difficulties exist that limit its widespread use. One of the major issues is obtaining a secure and prolonged attachment of a deep-penetration Laser Doppler probe to the animal skull. In this video, we show our optimized system for cerebral perfusion monitoring during transient middle cerebral artery occlusion by intraluminal filament in the rat. We developed in-house a simple method to obtain a custom made holder for twin-fibre (deep-penetration) Laser Doppler probes, which allow multi-site monitoring if needed. A continuous and prolonged monitoring of cerebral perfusion could easily be obtained over the intact skull.

Cerebral perfusion monitoring has been demonstrated to improve accuracy in ischemic stroke models. Technical difficulties often limit the use of this essential tool for cerebrovascular research. In this video, an optimized system is shown to obtain a single or multi-site hemodynamic monitoring during intraluminal middle cerebral artery occlusion in rats.

The translational potential of pre-clinical stroke research depends on the accuracy of experimental modeling. Cerebral perfusion monitoring in animal ...

An Isolated Working Heart System for Large Animal Models

Since its introduction in the late 19th century, the Langendorff isolated heart perfusion apparatus, and the subsequent development of the working heart model, have been invaluable tools for studying cardiovascular function and disease1-15. Although the Langendorff heart preparation can be used for any mammalian heart, most studies involving this apparatus use small animal models (e.g., mouse, rat, and rabbit) due to the increased complexity of systems for larger mammals1,3,11. One major difficulty is ensuring a constant coronary perfusion pressure over a range of different heart sizes &#8211; a key component of any experiment utilizing this device1,11. By replacing the classic hydrostatic afterload column with a centrifugal pump, the Langendorff working heart apparatus described below allows for easy adjustment and tight regulation of perfusion pressures, meaning the same set-up can be used for various species or heart sizes. Furthermore, this configuration can also seamlessly switch between constant pressure or constant flow during reperfusion, depending on the user&#8217;s preferences. The open nature of this setup, despite making temperature regulation more difficult than other designs, allows for easy collection of effluent and ventricular pressure-volume data.

Most studies involving the Langendorff apparatus use small animal models due to the increased complexity of systems for larger mammals. We describe a Langendorff system for large animal models that allows for use across a range of species, including humans, and relatively easy data acquisition.

Since its introduction in the late 19th century, the Langendorff isolated heart perfusion apparatus, and the subsequent development of the working heart ...

Surgical Approach for Middle Cerebral Artery Occlusion and Reperfusion Induced Stroke in Mice

Stroke is a leading cause of death worldwide and continues to be one of the major causes of long-term adult disabilities. About 87% of strokes are ischemic in origin and occur in the territory of the middle cerebral artery (MCA). Currently the only Food and Drug Administration (FDA) approved drug for the treatment of this devastating disease is tissue plasminogen activator (tPA). However, tPA has a small therapeutic window for administration (3 - 6 hr), and is only effective in 4% of the patients who actually receive it. Current research focuses on understanding the pathophysiology of stroke in order to find potential therapeutic targets. Thus, reliable models are crucial, and the MCA occlusion (MCAo) model (also termed the intraluminal filament or suture model) is deemed to be the most clinically relevant surgical model of ischemic stroke, and is fairly non-invasive and easily reproducible. Typically the MCAo model is used with rodents, especially with mice due to all the genetic variations available for this species. Here we describe (and present in the video) how to successfully perform the MCAo model (with reperfusion) in mice to generate reliable and reproducible data.

In order to understand the pathophysiology of stroke, it is important to use reliable models. This paper will describe one of the most frequently used stroke models in mice, termed the middle cerebral artery occlusion (MCAo) model (also termed the intraluminal filament or suture model) with reperfusion.

Stroke is a leading cause of death worldwide and continues to be one of the major causes of long-term adult disabilities. About 87% of strokes are ...

Middle Cerebral Artery Occlusion Allowing Reperfusion via Common Carotid Artery Repair in Mice

The ischemic stroke is a major cause of adult long-term disability and death worldwide. The current treatments available are limited, with only tissue plasminogen activator (tPA) as an approved drug treatment to target ischemic strokes. Current research in the field of ischemic stroke focuses on better understanding the pathophysiology of stroke, to develop and investigate novel pharmaceutical targets. Reliable experimental stroke models are crucial for the progression of potential treatments. The middle cerebral artery occlusion (MCAO) model is clinically relevant and the most frequently used surgical model of ischemic stroke in rodents. However, the outcomes of this model, such as lesion volume, are associated with high levels of variability, particularly in mice. The alternative MCAO model described here allows the reperfusion of the common carotid artery (CCA) and the increased perfusion of the middle cerebral artery (MCA) territory, using a tissue pad with fibrinogen-based sealant to repair the vessel, and the improved welfare of the mice by avoiding external carotid artery (ECA) ligation. This reduces the reliance on the Circle of Willis, which is known to be highly anatomically variable in mice. Representative data show that using this alternative surgical approach decreases the variability in lesion volumes between the traditional MCAO approach and the alternative approach described here.

Intraluminal filament occlusion of the middle cerebral artery is the most frequently used in vivo model of experimental stroke in rodents. An alternative surgical approach to allow common carotid artery repair is performed here, which allows the reperfusion of the common carotid artery and a full reperfusion to the middle cerebral artery territory.

The ischemic stroke is a major cause of adult long-term disability and death worldwide. The current treatments available are limited, with only tissue ...

A Modified Two Kidney One Clip Mouse Model of Renin Regulation in Renal Artery Stenosis

Renal artery stenosis is a common condition in patients with coronary or peripheral vascular disease where the renin angiotensin aldosterone system (RAAS) is overactivated. In this context, there is a narrowing of the renal arteries that stimulate an increase in the expression and release of renin, the rate-limiting protease in RAAS. The resulting rise in renin expression is a known driver of renovascular hypertension, frequently associated with kidney injury and end organ damage. Thus, there is a great interest in developing novel treatments for this condition. The molecular and cellular mechanism of renin control in renal artery stenosis is not fully understood and warrants further investigation. To induce renal artery stenosis in mice, a modified 2 kidney 1 clip (2K1C) Goldblatt mouse model was developed. The right kidney was stenosed in wild type mice and sham operated mice were used as control. After renal artery stenosis, we determined renin expression and kidney injury. Kidneys were harvested, and fresh cortices were used to determine protein and mRNA expression of renin. This animal model is reproducible and can be used to study pathophysiological responses, molecular and cellular pathways involved in renovascular hypertension and kidney injury.

A modified 2 kidney 1 clip (2K1C) Goldblatt mouse model was developed using polyurethane tubing to initiate renal artery stenosis, inducing an increase in renin expression and kidney injury. Here, we describe a detailed procedure of preparing and placing the cuff onto the renal artery to generate a reproducible and consistent 2K1C mouse model.

Renal artery stenosis is a common condition in patients with coronary or peripheral vascular disease where the renin angiotensin aldosterone system (RAAS) ...