Name: technical refinement of a bilateral renal ischemia-reperfusion mouse model for acute kidney injury research
Uploaded: 2023-11-03T14:20:00
Description: Watch this Scientific Journal Video about Technical Refinement of a Bilateral Renal Ischemia-Reperfusion Mouse Model for Acute Kidney Injury Research at JoVE.com

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. Prepar...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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

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...

Watch this Scientific Journal Video about Technical Refinement of a Bilateral Renal Ischemia-Reperfusion Mouse Model for Acute Kidney Injury Research at JoVE.com

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

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 ...

Acute kidney injury is a major risk factor associated with high mortality in patients with cardiac arrest after return of circulation. This procedure can be performed to induce acute kidney injury. This protocol is to induce renal ischemia reperfusion accurately, securely, and consistently.Demonstrating the procedure will be Huang Cheng-Wei, an assistant from my laboratory. To begin, place an anesthetized eight-week-old C57BL/6 male mouse in the prone position on a surgery platform with an electric blanket to maintain body temperature. Next, touch the back, find the lumbar spine, and look for costovertebral angles below both sides of the last rib.Apply hair removal lotion to shave and scrub the hair near both sides of the costovertebral angle region. Disinfect the surgical area using a Betadine solution and 75%alcohol using cotton balls. Use fine tip forceps to gently lift the skin below the left costovertebral angle.Then, using scissors, create a one-centimeter oblique dorsolateral incision along the skin tension lines from the lumbar midline on the left flank. Next, transect the muscle wall of the left flank to visualize the left kidney. Push and separate the left kidney carefully from the surrounding tissue with forceps.Then, identify the renal pedicle. Clamp the left renal pedicle with a microvascular clip. Confirm the ischemia by a visible change in kidney color from pink to dark red.To avoid desiccation during clamping, cover the kidney with sterile saline-wet cotton balls. After 25 minutes, open the left microvascular clip to start reperfusion of the left kidney. Return the kidney to the abdominal cavity.Close the abdominal cavity with 6/0 absorbable suture materials. Observe the mouse until it begins to move freely and feed. Renal ischemia after renal pedicle clamping is shown here.A kidney color change from pink to dark red reveals that the kidney perfusion has become inadequate. Compared to the controls, serum levels of blood urea nitrogen and creatinine increased two days after renal reperfusion. This technique paves a way for researchers to explore novel therapeutic approaches against acute renal injury.

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

Research

JoVE Journal

Medicine

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

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms at work during MI and developing effective therapeutic approaches requires methodology to reproducibly simulate the clinical incidence and reflect the pathophysiological changes associated with MI. Here, we describe a surgical method to induce MI in mouse models that can be used for short-term ischemia-reperfusion (I/R) injury as well as permanent ligation. The major advantage of this method is to facilitate location of the left anterior descending artery (LAD) to allow for accurate ligation of this artery to induce ischemia in the left ventricle of the mouse heart. Accurate positioning of the ligature on the LAD increases reproducibility of infarct size and thus produces more reliable results. Greater precision in placement of the ligature will improve the standard surgical approaches to simulate MI in mice, thus reducing the number of experimental animals necessary for statistically relevant studies and improving our understanding of the mechanisms producing cardiac dysfunction following MI. This mouse model of MI is also useful for the preclinical testing of treatments targeting myocardial damage following MI.

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.

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms ...

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

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

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

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

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, ...

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

Ischemia-reperfusion injury (IRI) occurs when there is transient hypoxia due to the obstruction of blood flow (ischemia) followed by a subsequent re-oxygenation of the tissues (reperfusion). In the skin, ischemia-reperfusion (IR) is the main contributing factor to the pathophysiology of pressure ulcers. While the cascade of events leading up to the inflammatory response has been well studied, the spatial and temporal responses of the different subsets of immune cells to an IR injury are not well understood. Existing models of IR using the clamping technique on the skin flank are highly invasive and unsuitable for studying immune responses to injury, while similar non-invasive magnet clamping studies in the skin flank are less-than-ideal for intravital imaging studies. In this protocol, we describe a robust model of non-invasive IR developed on mouse ear skin, where we aim to visualize in real-time the cellular response of immune cells after reperfusion via multiphoton intravital imaging (MP-IVM).

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.

Ischemia-reperfusion injury (IRI) occurs when there is transient hypoxia due to the obstruction of blood flow (ischemia) followed by a subsequent ...

Two-vessel Occlusion Mouse Model of Cerebral Ischemia-reperfusion

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

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

In this study, a middle cerebral artery (MCA) occlusion mouse model is employed to study cerebral ischemia-reperfusion. A reproducible and reliable mouse ...

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 ...