Name: Technical Refinement of a Bilateral Renal Ischemia-Reperfusion Mouse Model for Acute Kidney Injury Research (Video) | JoVE Journal
Uploaded: 2023-11-03T14:20:00.000+00:00
Duration: 3 min 13 s
Description: Learn a refined bilateral renal ischemia-reperfusion protocol in C57BL/6 mice for acute kidney injury research. This method enables consistent induction of renal injury to study therapeutic interventions.

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5 minutes

This model was developed with the financial support from Ministry of Science and Technology, Taiwan (MOST 109-2320-B-030-006-MY3). This manuscript was edited by Wallace Academic Editing.

All animal experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals, published by the US National Institutes of Health (NIH publication no. 85-23, revised 1996). The study protocol was approved by and in accordance with the guidelines of the Institutional Animal Care and Use Committee at Fu-Jen Catholic University. See the Table of Materials for details about all materials and instruments used in this protocol.
1. Preparing the mice
<ol>
	<li>Select 8-week-old C57BL/6 male mice with a weight of 21-23 g.</li>
	<li>House and maintain the mice under a 12 h light and dark cycle at a controlled temperature (21 &#177; 2 &#176;C) with free access to food, standard mouse food pellets, and tap water.</li>
</ol>
2. Anesthesia
<ol>
	<li>Put on a surgical mask and sterile gloves.</li>
	<li>Put the mice under anesthesia with 2% isoflurane mixed with oxygen at 1 L/min in the induction chamber.</li>
	<li>Assess the level of anesthesia by pedal reflex. 
	&#8203;NOTE: Pedal reflex is a retraction of the hind paw in response to a firm toe pinch. The anesthetization is complete when the pedal reflex disappears.</li>
	<li>Move and place each mouse in prone position on a surgery platform with an electric blanket to maintain their body temperature once the anesthetization is complete. Stabilize the body temperature before surgery and monitor with rectal temperature probes.&#160;Apply ophthalmic ointment to both eyes to prevent dryness.</li>
	<li>Tape the paws of the mice to the board.</li>
	<li>Attach a mask to the face of the mice to provide a constant supply of 1% isoflurane and 1 L/min oxygen</li>
	<li>Assess the level of anesthesia by pedal reflex regularly and adjust the anesthetic delivery accordingly during the surgery.</li>
</ol>
3. Bilateral renal IR-AKI surgery
<ol>
	<li>Touch the back and find the lumbar spine of the mice manually. Move along the spine cephalically and look for costovertebral angles that are below both sides of the last rib of the mice.</li>
	<li>Apply hair removal lotion to both sides of the costovertebral angle region for approximately 30 s and then remove the fur with saline.</li>
	<li>Disinfect the shaved skin&#160;with three rounds of&#160;betadine solution and 75% alcohol using cotton balls. 
	NOTE: Maintaining a sterile field for surgery throughout the procedure is key. Apply a surgical drape and use sterile instruments.</li>
	<li>Use fine-tip forceps to gently lift the skin below the left costovertebral angle, and then use scissors&#160;to create a 1 cm oblique dorsolateral incision along the skin tension lines from the lumbar midline on the left flank. Transect the muscle wall of the left flank using scissors to visualize the left kidney.</li>
	<li>Repeat the aforementioned surgical procedures to visualize the right kidney. Remove the small amounts of blood produced during the procedure with sterile cotton swabs.</li>
	<li>Push and separate the left kidney carefully from the surrounding tissue with forceps. Identify the renal pedicle after the left kidney is exposed. 
	NOTE: Be careful not to injure the adrenal gland and the surrounding blood vessels.</li>
	<li>Clamp over the left renal pedicle with a microvascular clip for 25 min. Confirm ischemia by a visible change in kidney color from pink to dark red.</li>
	<li>Cover the clamped kidney with sterile saline wet cotton balls to avoid desiccation during left renal pedicle clamping.</li>
	<li>Repeat the aforementioned surgical procedures to clamp the right renal pedicle with a microvascular clip for 25 min.</li>
	<li>Cover the clamped kidney with sterile saline wet cotton balls to avoid desiccation during right renal pedicle clamping.</li>
	<li>Monitor anesthesia depth and humidity of the sterile saline wet cotton balls periodically.</li>
	<li>Open the left microvascular clip to start reperfusion of the left kidney. Confirm reperfusion by a visible color change of the left kidney from dark red to pink.</li>
	<li>Open the right microvascular clip to start reperfusion of the right kidney.</li>
	<li>After the kidney color change is verified, return the kidney to the abdominal cavity.</li>
	<li>Close the abdominal cavity and skin with 6-0 absorbable suture materials.</li>
	<li>Scrub to disinfect the wound with a betadine solution and 75% alcohol using cotton balls.</li>
	<li>Observe the animal carefully until it begins to move freely and feed. 
	NOTE: Pay close attention to the animals until they have regained sufficient consciousness to maintain sternal recumbency.</li>
	<li>Administer carprofen (5 mg/kg in 0.2 mL, administered subcutaneously) for 2-3 days to prevent postsurgical pain.</li>
</ol>

The authors declare that there is no conflict of interest regarding the publication of this article.

The proposed bilateral IR-AKI protocol is suitable for investigating the mechanism of hypoperfusion and reperfusion injury of both kidneys. The protocol suggests that lightweight mice, general anesthesia with isoflurane, a dorsolateral approach to the surgery, and body temperature maintenance during the operation mitigate the associated technical difficulties, shorten the duration of the surgery, and increase the consistency of the procedure for acute bilateral renal IR-AKI research.
Technical...

Cardiac arrest occurs more than 80,000 times annually in the United States1,2. The mortality rate of cardiac arrest is extremely high3,4,5,6. AKI is a major risk factor associated with high mortality and poor neurological outcomes in patients with cardiac arrest after ROSC7,8,9,10,11,12,13. Recovery from AKI is a good predictor of favorable neurological outcomes and discharge from the hospital14,15,16. However, effective therapies for IR-AKI are still lacking15,16,17,18,19. Additional therapeutic strategies are required to further improve the clinical outcomes of the disease.
IR-AKI with bilateral renal ischemia approach is one of the animal models used for AKI research20,21,22,23,24,25,26. Renal IR-AKI animal models are less complicated than a whole-body IR injury model for the study of AKI in patients with sudden cardiac arrest following ROSC6,27,28,29,30. This implies that consistent results from a renal IR-AKI animal model are easier to achieve because of the presence of fewer confounding factors in experiments. Moreover, renal IR-AKI protocols commonly involve a unilateral or bilateral renal pedicle occlusion. Conditions in experiments on bilateral renal IR-AKI are comparable to clinical conditions for AKI following ROSC in patients with sudden cardiac arrest after successful cardiopulmonary resuscitation. Although the pathological characteristics of the kidneys in both models reflect the pathological characteristics of human renal IR injury31,32,33, a bilateral renal ischemia approach is more relevant to AKI under human pathological conditions, such as cardiac failure, vasoconstriction, and septic shock35. Bilateral renal IR-AKI animal models are suitable for studies focusing on renal IR injuries in cardiac arrest following ROSC.
Bilateral renal IR-AKI models are associated with technical difficulties, experimental complexity, and long surgery duration23,26,32,33,35,36. To overcome these technical difficulties, the present study established a reliable bilateral IR-AKI research protocol in mice by making some technical modifications. The proposed protocol resulted in fewer surgical complications, less tissue damage, and a lower likelihood of mortality during surgery. Therefore, it can be used to investigate the pathophysiological processes of AKI after ROSC to develop new therapeutic strategies against renal hypoperfusion and reperfusion damage37,38,39.

<table><tbody><tr><td>Absorbable Suture, 6-0</td><td>Ethicon</td><td>J510G-BX</td><td></td></tr><tr><td>Betadine solution</td><td>Shineteh Istrument</td><td></td><td></td></tr><tr><td>Carprofen</td><td>Sigma</td><td>PHR1452</td><td></td></tr><tr><td>Cotton balls</td><td>Shineteh Istrument</td><td></td><td></td></tr><tr><td>Graefe Forceps</td><td>Fine Science Tools</td><td>11051-10</td><td></td></tr><tr><td>Heating pad</td><td>Shineteh Istrument</td><td></td><td></td></tr><tr><td>Isoflurane</td><td>Piramal Critical Care Inc.</td><td>26675-46-7</td><td></td></tr><tr><td>Moria Vessel Clamp</td><td>Fine Science Tools</td><td>18320-11</td><td></td></tr><tr><td>Olsen-Hegar needle holder</td><td>Fine Science Tools</td><td>12002 - 12</td><td></td></tr><tr><td>Saline</td><td>Shineteh Istrument</td><td></td><td></td></tr><tr><td>Scalpel blades</td><td>Shinva</td><td>s2646</td><td></td></tr><tr><td>Small Animal Anesthesia Machine</td><td>Sheng-Cing Instruments Co.</td><td>STEP AS-01</td><td></td></tr><tr><td>Tissue scissors</td><td>Fine Science Tools</td><td>14072 - 10</td><td></td></tr></tbody></table>

technical refinement of a bilateral renal ischemia-reperfusion mouse model for acute kidney injury research

Technical Refinement of a Bilateral Renal Ischemia-Reperfusion Mouse Model for Acute Kidney Injury Research

Cardiac arrest poses a large public health burden. Acute kidney injury (AKI) is an adverse marker in survivors of cardiac arrest following the return of spontaneous circulation (ROSC) after successful cardiopulmonary resuscitation. Conversely, recovery of kidney function from AKI is a predictor of favorable neurological outcomes and hospital discharge. However, an effective intervention to prevent kidney damage caused by cardiac arrest after ROSC is lacking, suggesting that additional therapeutic strategies are required. Renal hypoperfusion and reperfusion are two pathophysiological mechanisms that cause AKI after cardiac arrest. Animal models of ischemia-reperfusion-induced AKI (IR-AKI) of both kidneys are comparable with patients with AKI following ROSC in a clinical setting. However, IR-AKI of both kidneys is technically challenging to analyze because the model is associated with high mortality and wide variation in kidney damage, which may affect the analysis. Lightweight mice were chosen, placed under general anesthesia with isoflurane, subjected to surgery with a dorsolateral approach, and their body temperature maintained during operation, thereby reducing tissue damage and establishing a reproducible acute renal IR-AKI research protocol.

The quality of the bilateral renal IR-AKI surgery should be assessed before further microscopic or molecular analysis. During surgery, renal ischemia should be confirmed by seeing whether the kidney has changed color from pink to dark red soon after the renal pedicle is clamped with a microvascular clip (Figure 1). After surgery, kidney damage caused by IR-AKI surgery can be further validated with a few microliters of serum through submandibular blood collection for biochemical analysis wher...

This study established a protocol focusing on the technical refinement of a mouse model of bilateral renal ischemia-reperfusion for acute kidney injury research.

Cardiac arrest poses a large public health burden. Acute kidney injury (AKI) is an adverse marker in survivors of cardiac arrest following the return of ...

technical-refinement-bilateral-renal-ischemia-reperfusion-mouse-model

Research

JoVE Journal

Medicine

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 Model of Acute Kidney Injury and Post Injury Fibrosis in Mice

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: A Mouse Model of Injury and Regeneration

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

An Effective Mouse Model of Unilateral Renal Ischemia-Reperfusion Injury

This article presents an ischemic CKD model using unilateral renal IRI with contralateral nephrectomy performed one day prior to sample collection; this protocol enables serum-based functional assessment, high survival, and reproducible CKD pathology at 28 and 42 days, providing a versatile platform for mechanistic and interventional studies.

Mouse Model of Acute to Chronic Kidney Disease Transition Induced by Renal Ischemia/Reperfusion Injury

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.

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

We introduce a surgical method to induce experimental ischemia/reperfusion (I/R) injury to simulate myocardial infarction (MI) in mouse models that allows for more clarity in positioning of the ligation on the left anterior descending artery (LAD) to increase the reproducibility of MI experiments in mice.

A Murine Model of Myocardial Ischemia-reperfusion Injury through Ligation of the Left Anterior Descending Artery

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.

Murine Model of Intestinal Ischemia-reperfusion Injury

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.

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

This protocol describes the induction of an ischemia-reperfusion (IR) model on mouse ear skin using magnet clamping. Using a custom-built intravital imaging model, we study in vivo inflammatory responses post-reperfusion. The rationale behind the development of this technique is to extend the understanding of how leukocytes respond to skin IR injury.

Inducing Ischemia-reperfusion Injury in the Mouse Ear Skin for Intravital Multiphoton Imaging of Immune Responses

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

Two-vessel Occlusion Mouse Model of Cerebral Ischemia-reperfusion

Learn a refined bilateral renal ischemia-reperfusion protocol in C57BL/6 mice for acute kidney injury research. This method enables consistent induction of renal injury to study therapeutic interventions.

Technical Refinement of a Bilateral Renal Ischemia-Reperfusion Mouse Model for Acute Kidney Injury Research

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