The authors gratefully acknowledge the data storage service SDS@hd supported by the Ministry of Science, Research and the Arts Baden-W&#252;rttemberg (MWK) and the German Research Foundation (DFG) through grant INST 35/1314-1 FUGG and INST 35/1503-1 FUGG. Furthermore, the authors gratefully acknowledge the support from the NCT (National Center for Tumor Diseases in Heidelberg, Germany) through its structured postdoc program and the Surgical Oncology program. We also acknowledge the support through state funds approved by the State Parliament of Baden-W&#252;rttemberg for the Innovation Campus Health + Life Science Alliance Heidelberg Mannheim from the structured postdoc program for Alexander Studier-Fischer: Artificial Intelligence in Health (AIH) - A collaboration of DKFZ, EMBL, Heidelberg University, Heidelberg University Hospital, University Hospital Mannheim, Central Institute of Mental Health, and the Max Planck Institute for Medical Research. Furthermore, we acknowledge the support through the DKFZ Hector Cancer Institute at the University Medical Center Mannheim. For the publication fee, we acknowledge financial support from Deutsche Forschungsgemeinschaft within the funding program &#34;Open Access Publikationskosten&#34; as well as from Heidelberg University.

All animal activities described here were conducted in accredited facilities and approved by the Institutional Animal Care and Use Committee (IACUC) of the Baden-W&#252;rttemberg Regional Council in Karlsruhe, Germany (35-9185.81/G-62/23). Experimental animals were managed according to institutional standards, German laws for animal use and care, the directives of the European Community Council (2010/63/EU), and the ARRIVE guidelines. Male Sprague Dawley rats with an order weight of 400 grams were used after being acclimatized for one week. The details of the reagents and the equipment used in this study are listed in the Table of Materials.
1. Anesthesia and analgesia
<ol>
	<li>Anesthetize the rat with the medication following the institutional protocols. Isoflurane is recommended for induction of sedation followed by an i.p. injection of 100 mg/kg body weight ketamine and 4 mg/kg body weight xylazine. Additional analgesia can be achieved with a s.c. injection of 5 mg/kg body weight carprofen. For details, please refer to Studier-Fischer et al.13.</li>
	<li>Apply ophthalmic ointment to the eyes to prevent dryness.</li>
	<li>Guarantee proper analgetic depth using the toe pinch test with surgical forceps.</li>
	<li>Regularly reassess and adjust the depth of anesthesia during surgery.</li>
</ol>
2. Procedure preparation
<ol>
	<li>Prepare the scrub table with all the required materials and instruments, including releasable microvascular clamps and the applicator (Figure 2A-C), blunt overholt clamp, fine preparation scissor, and forceps.
	<ol>
		<li>Prepare the surgical preparation hooks by folding cannulas at an angle of 135&#176; at 1 cm to the tip and connect them to plastic perfusion tubes via Luer-lock ready to apply tension using a surgical mosquito clamp (Figure 2D,E).</li>
	</ol>
	</li>
	<li>Prepare a rodent surgical exposure apparatus, including Y-shaped fixation rods and a heating pad, as specified in Studier-Fischer et al.13.</li>
	<li>Ensure proper oxygenation is achieved via the inhalation of 100% oxygen using a neonatal face mask (Figure 2F).</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/67171/67171fig02.jpg" /> 
Figure 2: Experimental and animal setup. (A) Surgical instruments and materials required. (B,C) Releasable microvascular clamp and applicator. (D,E) A folded cannula connected to a perfusion tube is used as a surgical preparation hook. (F,G) Rat model oxygenated with a face mask and shaven. (H) Cutaneous incision over complete abdominal length. (I-M) resection of the xiphoid and hemostasis. (N-Q) Hepatic mobilization and dissection of the falciform ligament (arrow 1). (R) Application of preparation hooks and metal stands for exposure of organs after laparotomy. (S) Full visceral exposure of abdominal major vessels using blunt hooks (arrow 2), silicone vessel loops (arrow 3), and surgical compress (arrow 4). (T,U) Abdominal aorta and caval vein. (V) Atraumatic preparation instruments. (W) Humidified cotton swap (arrow 5). (X) Humidified compress in forceps (arrow 6) and blunt overholt clamps (arrow 7). <a href="https://www.jove.com/files/ftp_upload/67171/67171fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Surgical preparation
<ol>
	<li>Shave the surgical access site for a midline laparotomy (Figure 2G). Perform a cutaneous incision over the desired abdominal length, i.e., about 7 cm (Figure 2H), advance with the laparotomy by dissecting the fascia, and stitch the surgical preparation hooks with attached plastic tubes and surgical mosquito clamps through the skin. Expose the surgical situs using the surgical preparation hooks, applying tension to the tissue.</li>
	<li>Ensure that the peritoneum is dissected cranially only until a few millimeters below the beginning of the sternum, leaving some of the peritoneum intact. Place a part of a surgical compress below the sternum (Figure 2I) and resect the xiphoid with strong material scissors. Apply pressure to the resection area using the surgical compress to achieve sufficient hemostasis of this well-perfused region (Figure 2I-M).</li>
	<li>Mobilize the liver dorso-caudally to expose the falciform ligament (Figure 2N) and dissect the ligament (Figure 2O-Q).</li>
	<li>For hemostatic control, depending on where the clamping of the major vessels should be performed, perform a full visceral exposure of abdominal major vessels (Figure 2R) and the vessels slung with silicone vessel loops (Figure 2S-U). Only atraumatic preparation instruments should be used (Figure 2V), such as humidified cotton swaps (Figure 2W), humidified compress in forceps, and blunt overholt clamps (Figure 2X).</li>
</ol>
4. Preparation and clamping of abdominal aorta for arterial ischemia
<ol>
	<li>Perform a left medialization of the upper abdominal organs using atraumatic preparation instruments to gain access to the left adrenal artery (Figure 3A-D).</li>
	<li>Identify the pulsating site typically medial to the cranial extension of the left adrenal artery, indicating the course of the aorta (Figure 3E). Advance through the soft tissue using overholt clamps for blunt dissection in order to access the abdominal aorta (Figure 3F,G).</li>
	<li>Tunnel the abdominal aorta at the most cranial end using blunt overholt clamps (Figure 3H-L) and sling the aorta using a silicone vessel loop (Figure 3M-T).</li>
	<li>Apply a suitable aneurysm microvascular clamp using the silicone loop to slightly luxate the aorta ventrally and guide the aneurysm microvascular clamp along the silicone loop to guarantee isolated aortic clamping (Figure 3U). Depending on the research question, the microvascular clamp can be released again.</li>
</ol>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/67171/67171fig03.jpg" /> 
Figure 3: Preparation and clamping of the abdominal aorta. (A) Exposure of visceral organs. (B-E) Left medialization of upper abdominal organs using atraumatic preparation instruments to gain access to the left adrenal artery. (F,G) Blunt dissection medial of the left adrenal artery at the pulsating site (gray arrow) in order to access the abdominal aorta. (H-L) Tunneling of the abdominal aorta using blunt overholt clamps. (M-T) Slinging the aorta using a silicone vessel loop. (U) Application of a releasable aneurysm microvascular clamp using the silicone loop as guidance. (V-Z) Visualization of the celiac artery (orange) in reference to the aorta (red) and the silicone vessel loop. <a href="https://www.jove.com/files/ftp_upload/67171/67171fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
5. Preparation and clamping of suprahepatic abdominal caval vein for venous congestion
<ol>
	<li>Mobilize the liver to the right using atraumatic preparation instruments, sharply dissect the hepatic ligaments, and further lateralize the liver (Figure 4A-C).</li>
	<li>Open the retrohepatic space at the left crus of the diaphragm using blunt overholt clamps (Figure 4D-G).</li>
	<li>Tunnel the caval vein using blunt overholt clamps (Figure 4H-K) and sling the caval vein using silicone vessel loops (Figure 4L-O).</li>
	<li>Apply a suitable aneurysm microvascular clamp using the silicone loop to slightly luxate the caval vein ventrally and guide the aneurysm microvascular clamp along the silicone loop to guarantee isolated clamping of the caval vein (Figure 4R).</li>
</ol>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/67171/67171fig04.jpg" /> 
Figure 4: Preparation and clamping of suprahepatic abdominal caval vein. (A) Exposure of cranial visceral organs. (B) Tissue-protective mobilization of the liver and sharp dissection of hepatic ligaments using atraumatic preparation instruments. (C) Lateralization of the liver. (D-G) The opening of the retrohepatic space and preparation at the left crus of the diaphragm. (H-K) Tunneling of the caval vein (blue) using blunt overholt clamps. (L-O) Slinging the caval vein using silicone vessel loops. (P,Q) Exertion of tension to tentatively restrict caval blood flow. (R) Application of a releasable aneurysm microvascular clamp using the silicone loop as guidance. <a href="https://www.jove.com/files/ftp_upload/67171/67171fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
6. Clamping of the abdominal aorta and suprahepatic abdominal caval vein for combined malperfusion
<ol>
	<li>Perform the steps above until both the aorta and caval vein are slung with silicone vessel loops. Advance with the application of the aneurysm microvascular clamp for both vessels again using the silicone loop for guidance. Clamping the aorta first and minimizing the time required until subsequent caval clamping to only a few seconds is recommended. 
	NOTE: Depending on the desired scenario and research purpose, malperfusion can be continued over or released after a defined timespan, and animals can either be euthanized by sharp cardiectomy (following institutionally approved protocols) for non-survival applications, or receive stepwise abdominal closure using surgical sutures in case of planned follow-ups and survival experiments. For the present study, the animals were euthanized.</li>
</ol>

Rat Model for Visceral Organ Malperfusion Studies

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While peripheral arterial occlusive disease (PAOD) alone, as the most prominent representative of tissue malperfusion conditions, already has a prevalence of around 7%, affecting an estimated 8.5 million adults in the United States alone18, tissue malperfusion in general is a relevant pathomechanism in a majority of surgical and medical conditions. Consequently, appropriate and reproducible animal models are absolutely necessary to address novel research questions in this field.
The three vascular dynamic situations that require distinctive investigation are arterial ischemia, venous congestion, and combined malperfusion. By inducing isolated arterial ischemia, scientists can precisely delineate the temporal and spatial progression of tissue hypoxia, investigating the molecular cascades implicated in ischemia-reperfusion injury, cellular apoptosis, and inflammatory responses. By compromising venous drainage, an oftentimes overlooked facet of vascular pathology, scientists can investigate the interplay between arterial inflow and venous outflow discrepancies, shedding light on the pathophysiology of venous thrombosis, congestion-related tissue edema, and microcirculatory dysfunction. When combining both of these pathological situations, one can investigate the dys-synergistic milieu of combined malperfusion, mirroring the complex pathophysiology encountered in clinical syndromes like acute mesenteric ischemia and ischemic colitis.
Beyond elucidating fundamental pathophysiological mechanisms, the ability to induce visceral arterial ischemia, venous congestion, and combined malperfusion in rats serves as an indispensable platform for evaluating the efficacy of pharmacological interventions, surgical techniques, new therapeutic strategies, and innovative imaging modalities, especially such as HSI14,19,20,21,22. This model is, therefore, a key component in providing the required biological tissue ground truth needed to harness the full potential of HSI in tissue evaluation and identification of perfusion states. By leveraging this experimental setup, researchers can expedite the translation of preclinical findings into clinically viable strategies, ultimately reducing the morbidity and mortality associated with diverse vascular and perfusion disorders.
Illustratively, researchers can employ this model to investigate the efficacy of pharmacological agents targeting ischemia-reperfusion injury pathways, such as antioxidants, anti-inflammatory agents, and vasodilators, thereby delineating their potential utility in clinical practice23,24. Additionally, this model facilitates the assessment of novel surgical approaches, such as mesenteric revascularization techniques and venous decompressive procedures, providing invaluable insights into their feasibility, safety, and long-term efficacy25,26.
Furthermore, this experimental framework enables researchers to explore the intricate interplay between vascular dysfunction and systemic comorbidities, such as diabetes, hypertension, and atherosclerosis, thereby illustrating the intricate web of interconnected pathophysiological pathways orchestrating vascular disease progression27,28.
While there are several publications on selective malperfusion of single organs, such as the liver29,30,31or kidney, in rats32,33, there is a lack of scientific literature addressing malperfusion of the complete viscera in rats, and there is explicitly no methodical protocol. This is, therefore, the claim of this manuscript. Limitations of the presented technique mainly include the invasiveness of the procedure and, depending on the duration of malperfusion, consecutive organ thrombosis, and dysfunction, possibly leading to postoperative suffering through multiorgan failure or abdominal compartment syndrome34,35,36. Careful planning and design, depending on the research question, can help balance the required duration of malperfusion and its pathophysiological consequences.
When troubleshooting common challenges encountered during the procedure, attention should be drawn to the following points and recommendations: (1) Ensure thorough preparation of equipment and medication beforehand to minimize interruptions during the procedure; (2) Perform hemostatic control meticulously by carefully preparing and dissecting avascular planes. Consider using bipolar hemostatic forceps for electric hemostasis, if available; (3) Minimize trauma to tissues by using non-traumatic instruments such as humidified cotton swabs or humidified surgical compress with forceps when making contact with the liver parenchyma; (4) Approximately 20% of animals experienced diffuse superficial liver parenchyma bleeding due to delicate tissue conditions. However, the bleeding stopped in all cases with light compression and patience. These recommendations aim to enhance procedural efficiency and minimize complications during the induction of malperfusion in rat models.
When resecting the xiphoid for improved access to the caval vein, ensure that the peritoneum dorsal to the transition between the xiphoid and sternum is left intact over a few millimeters. The resection site of the xiphoid will be hard and sharp, potentially causing trauma to the superficial liver parenchyma. Therefore, it is recommended that the retrosternal peritoneum caudally be mobilized using forceps and wrapped around the bone stump effectively, covering it and supporting hemostasis. The surgical preparation hooks should be stitched through the cranial ventral abdominal wall with cranial tension so that the peritoneal coverage of the xiphoid stump will remain in place.
When dissecting the falciform ligament, care should be taken to avoid accidentally causing iatrogenic injury to the hepatic vein, as this could be fatal to the animal. Due to the high risk of bleeding during vascular preparation, it is recommended that the majority of surgical preparation be done by spreading with blunt overholt clamps, rather than using sharp dissection instruments. Additionally, silicone vessel loops should be moistened prior to usage to reduce surface friction and minimize the risk of tissue trauma.
When applying the aneurysm microvascular clamp, it is crucial to visualize the exact vascular anatomy. For instance, the celiac artery originates very cranially from the abdominal aorta. If celiac occlusion is desired, such as to investigate hepatic malperfusion, the celiac artery should be visualized in reference to the aorta and the silicone vessel loop. This ensures that the celiac artery is included in the clamped vascular tissue (Figure 2V-Z). There was one case in which the clamp was initially placed caudal to the celiac artery by accident. However, this was promptly recognized due to the missing drop in StO2 liver values, and proper reclamping was successfully performed.
The most hazardous preparation step is tunneling the caval vein. This step requires gentle movements and patience, and overholt clamps should only be spread when certain there is no contact with the caval vein. It can be challenging to judge this, as the caval vein will appear as thin avascular connective tissue when slight compression is applied, causing the contained blood to disappear in both directions. There is also a risk of accidental pleural opening and creating a pneumothorax when tunneling the caval vein too cranially. This can be a serious and life-threatening complication, especially since the animal is spontaneously breathing, and no invasive respiratory measures can be taken. It has been found helpful to slightly retract the preparation instruments and continue more caudally to avoid this complication. In cases of hemodynamically relevant and visible pneumothorax with bulging of the hepatic diaphragm, a trans-diaphragmatic one-time puncture and aspiration of the trapped air using a 30 G needle and a small syringe can be recommended as a rescue strategy. This technique was successfully employed in one animal to save it intraoperatively.
Finally, special care should be taken when applying the microvascular clamps to avoid including surrounding connective tissue, which could lead to insufficient occlusion of the desired vessel.
While this protocol is intended as a step-by-step guide for global visceral malperfusion, the clamping site can be adjusted according to the specific research question due to the extensive vascular preparation and mobilization depicted in Figure 1T-V. Therefore, selected malperfusion of organ groups or single organs is also an option when choosing the clamping site further distally along the vascular tree, such as selectively clamping the celiac trunk for hepatic ischemia. By offering a detailed and reproducible methodology, this protocol facilitates a standardized approach for controlled reversible arterial ischemia, venous congestion, and combined malperfusion in rat models, leading to improved data reliability, robustness, researcher independence, and comparability across future animal studies. Consequently, it represents an indispensable tool within the biomedical research armamentarium, offering insights into the complex interplay between vascular compromise, tissue injury, and therapeutic interventions. By harnessing the versatility of this experimental setup, researchers can investigate the mysteries around vascular pathophysiology, forging new frontiers in translational medicine, and ultimately enhancing patient outcomes in the realm of vascular health.

While the implications of tissue perfusion deprivation in the form of local or systemic tissue malperfusion conditions have long been recognized, they persist as one of the primary causes of morbidity and mortality in both the United States and Europe1. These malperfusion conditions are the third leading cause of tissue degradation, following malignancy and septic inflammation, but have a far wider spectrum of origins compared to the latter two2.
This spectrum ranges from local mechanisms such as atrial fibrillation with thromboembolic occlusion, vasoconstriction, and iatrogenic or traumatic dissection, to systemic mechanisms such as cardiac insufficiency or shock, sepsis, hypovolemia, and steal phenomena. These diverse mechanisms underlie a variety of medical and surgical conditions. The significant morbidity and mortality associated with these conditions have directed medical attention toward procedures for reinstating blood flow to malperfused tissues in order to forestall necrosis and restore organ function over the decades3.
This research effort has resulted in a variety of pharmaceutical, medical, and surgical solutions to restore physiological organ perfusion, including advances in bypass surgery4, endovascular procedures5, extracorporeal membrane oxygenation6,7, organ machine perfusion during transplantation8, and artificial blood substitutes9.
Yet, despite these significant developments, malperfusion, especially of visceral organs, remains a pressing issue in patient care, and the demand for further research on biomedical processes and rescue strategies is high. Valid biological models are of utmost importance in enabling this kind of research. Due to the multifactorial aspects of tissue perfusion research, which include not only cell biology but also vascular microanatomy and rheology, an appropriate model requires a degree of biological complexity that only a complete model organism can provide, making rodents the obvious model of choice.
Tissue malperfusion can be differentiated into three distinct conditions: isolated arterial ischemia, isolated venous congestion, and combined malperfusion10. Clinically relevant scenarios for these conditions include (1) Arterial ischemia: Atrial fibrillation with thromboembolic occlusion, septic emboli, vasoconstriction, iatrogenic or traumatic vascular dissection or clamping, cardiac insufficiency or shock, aortic dissection, sepsis and hypovolemia, extreme arterial obstruction due to external constriction, pulmonary artery embolism, chronic arterial vascular occlusive diseases, or steal phenomena; (2) Venous congestion: Iatrogenic or traumatic vascular dissection or clamping, heart failure, liver fibrosis or cirrhosis, venous obstruction due to external constriction, venous thrombosis, venous insufficiency, and Budd-Chiari syndrome; (3) Combined malperfusion: Combinations of the above conditions and advanced stages of the aforementioned conditions, such as secondary venous congestion due to ischemia-induced fibrosis or secondary arterial ischemia due to congestion-induced retention, as well as specific organ conditions like ischemic inflammation (e.g., ischemic colitis)11,12.
This article, therefore, aims to provide a step-by-step model to induce controlled, reversible visceral arterial ischemia, venous congestion, and combined malperfusion via midline laparotomy in rats for both survival and non-survival applications, as depicted in Figure 1. The experimental model offers a controlled environment for studying the multifaceted dynamics of arterial ischemia, venous congestion, and their combined sequelae, emulating clinically relevant scenarios encountered in various conditions.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67171/67171fig01.jpg" /> 
Figure 1: Overview of the protocol. Schematic depiction of vascular rat anatomy and depiction of the clamping site (gray arrow). (A) Physiological perfusion. (B) Arterial ischemia. (C) venous congestion. (D) Combined malperfusion. <a href="https://www.jove.com/files/ftp_upload/67171/67171fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Atraumatic preparation forceps</td>
			<td>Aesculap</td>
			<td>FB395R</td>
			<td>DE BAKEY ATRAUMATA atraumatic forceps, straight</td>
		</tr>
		<tr>
			<td>Blunt overholt clamp</td>
			<td>Aesculap</td>
			<td>BJ012R</td>
			<td>BABY-MIXTER preparation and ligature clamp, bent, 180 mm</td>
		</tr>
		<tr>
			<td>Cannula</td>
			<td>BD (Beckton, Dickinson)</td>
			<td>301300</td>
			<td>BD Microlance 3 cannula 20 G</td>
		</tr>
		<tr>
			<td>Fixation rods</td>
			<td>legefirm</td>
			<td>500343896</td>
			<td>tuning forks used as y-shaped metal fixation rods</td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>Royal Gardineer</td>
			<td>IP67</td>
			<td>Royal Gardineer Heating Pad Size S, 20 Watt</td>
		</tr>
		<tr>
			<td>Plastic perfusor tube</td>
			<td>M. Schilling GmbH</td>
			<td>S702NC150</td>
			<td>connecting tube COEX 150 cm</td>
		</tr>
		<tr>
			<td>Preparation scissors</td>
			<td>Aesculap</td>
			<td>BC177R</td>
			<td>JAMESON preparation scissors, bent, fine model, blunt/blunt, 150 mm (6&#34;)</td>
		</tr>
		<tr>
			<td>Silicone vessel loop tie</td>
			<td>SERAG WIESSNER</td>
			<td>SL26</td>
			<td>silicone vessel loop tie 2,5 mm red</td>
		</tr>
		<tr>
			<td>Spraque Dawley rat</td>
			<td>Janvier Labs</td>
			<td>RN-SD-M</td>
			<td>Spraque Dawley rat</td>
		</tr>
		<tr>
			<td>Steel plate</td>
			<td>Maschinenbau Feld GmbH</td>
			<td>C010206</td>
			<td>Galvanized sheet plate, 40 x 50 cm, thickness 4.0 mm</td>
		</tr>
		<tr>
			<td>Yasargil clip</td>
			<td>Aesculap</td>
			<td>FE795K</td>
			<td>YASARGIL Aneurysm Clip System 
			Phynox Temporary (Standard) Clip</td>
		</tr>
		<tr>
			<td>Yasargil clip applicator</td>
			<td>Aesculap</td>
			<td>FE558K</td>
			<td>YASARGIL Aneurysm Clip Applicator 
			Phynox (Standard)</td>
		</tr>
	</tbody>
</table>

controlled reversible visceral arterial ischemia, venous congestion and combined malperfusion via midline laparotomy in rats

Besides sepsis and malignancy, malperfusion is the third leading cause of tissue degradation and a major pathomechanism for various medical and surgical conditions. Despite significant developments such as bypass surgery, endovascular procedures, extracorporeal membrane oxygenation, and artificial blood substitutes, tissue malperfusion, especially of visceral organs, remains a pressing issue in patient care. The demand for further research on biomedical processes and possible interventions is high. Valid biological models are of utmost importance in enabling this kind of research. Due to the multifactorial aspects of tissue perfusion research, which include not only cell biology but also vascular microanatomy and rheology, an appropriate model requires a degree of biological complexity that only an animal model can provide, rendering rodents the obvious model of choice. Tissue malperfusion can be differentiated into three distinct conditions: (1) isolated arterial ischemia, (2) isolated venous congestion, and (3) combined malperfusion. This article presents a detailed step-by-step protocol for the controlled and reversible induction of these three types of visceral malperfusion via midline laparotomy and clamping of the abdominal aorta and caval vein in rats, underscoring the significance of precise surgical methodology to guarantee uniform and dependable results. Prime examples of possible applications of this model include the development and validation of innovative intraoperative imaging modalities, such as Hyperspectral Imaging (HSI), to objectively visualize and differentiate malperfusion of gastrointestinal, gynecological, and urological organs.

This protocol was performed in 10 male rats (mean weight 403 g &#177; 26 g) in a non-survival setting. The success rate was defined by survival over 20 min after arterial clamping, venous clamping, and combined clamping for 5 min with 10 min of reperfusion, each of which was 100%. The mean duration of the preparation from skin incision until both vessels were slung with silicone loops was 11 min 45 s &#177; 3 min 23 s.
For validation of the 4 different malperfusion states, index parameters for oxygenation (StO2) and perfusion (NIR) were measured using hyperspectral imaging (HSI) across 5 visceral organs (Figure 5).
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/67171/67171fig05.jpg" /> 
Figure 5: Validation of the malperfusion model. (A,B) Quantification of HSI oxygenation and perfusion values across four different perfusion states and five different visceral organs with n = 10 animals. (C-F) RGB and color-coded index pictures of HSI recordings containing visceral organs across 4 different perfusion states. Error bars indicate standard deviation. The scale bar depicts 5 cm. <a href="https://www.jove.com/files/ftp_upload/67171/67171fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Values were provided in arbitrary units and showed a significant decrease in the malperfusion states compared to the physiological organ state (Table 1). The hyperspectral results were in line with recent publications indicating that viability and perfusion of tissue can be evaluated using organ-specific HSI StO2 cut-off values that matched the values seen in this study14,15. Exemplarily for the stomach, these were 64.1% (&#177;9.4%) for physiological perfusion,43.1% (&#177;7.4%) for arterial ischemia, 40.5% (&#177;5.4%) for venous congestion and 39.3% (&#177;4.5%) for combined malperfusion.
Since these were non-survival experiments, there is no experimental data on the long-term outcomes of the animals. However, other studies report 100% and 57% survival over 24 h for rats that underwent 30 min and 60 min of superior mesenteric artery clamping16,17 and successfully correlate it with serum levels of Heat Shock Protein 70. Consequently, this might be a possible method to assess outcomes in future survival studies according to different clamping times.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>parameter</td>
			<td>organ</td>
			<td>baseline</td>
			<td>arterial ischemia</td>
			<td>venous congestion</td>
			<td>combined malperfusion</td>
		</tr>
		<tr>
			<td rowspan="5">StO2</td>
			<td>stomach</td>
			<td>64.1% (&#177;9.4%)</td>
			<td>43.1% (&#177;7.4%)</td>
			<td>40.5% (&#177;5.4%)</td>
			<td>39.3% (&#177;4.5%)</td>
		</tr>
		<tr>
			<td>small bowel</td>
			<td>78.4% (&#177;5.1%)</td>
			<td>44.8% (&#177;5.5%)</td>
			<td>38.0% (&#177;7.9%)</td>
			<td>41.9% (&#177;6.9%)</td>
		</tr>
		<tr>
			<td>colon</td>
			<td>74.6% (&#177;5.0%)</td>
			<td>56.0% (&#177;6.3%)</td>
			<td>51.3% (&#177;4.1%)</td>
			<td>51.8% (&#177;2.9%)</td>
		</tr>
		<tr>
			<td>liver</td>
			<td>39.5% (&#177;9.7%)</td>
			<td>16.9% (&#177;2.6%)</td>
			<td>9.5% (&#177;0.8%)</td>
			<td>9.3% (&#177;1.1%)</td>
		</tr>
		<tr>
			<td>kidney</td>
			<td>71.0% (&#177;3.8%)</td>
			<td>26.3% (&#177;3.0%)</td>
			<td>18.6% (&#177;2.5%)</td>
			<td>21.2% (&#177;2.6%)</td>
		</tr>
		<tr>
			<td rowspan="5">NIR</td>
			<td>stomach</td>
			<td>20.0% (&#177;9.3%)</td>
			<td>8.3% (&#177;6.7%)</td>
			<td>6.8% (&#177;5.1%)</td>
			<td>7.5% (&#177;8.1%)</td>
		</tr>
		<tr>
			<td>small bowel</td>
			<td>38.6% (&#177;17.4%)</td>
			<td>12.9% (&#177;11.0%)</td>
			<td>6.3% (&#177;6.5%)</td>
			<td>5.7% (&#177;5.9%)</td>
		</tr>
		<tr>
			<td>colon</td>
			<td>12.6% (&#177;13.7%)</td>
			<td>5.3% (&#177;8.7%)</td>
			<td>3.8% (&#177;7.5%)</td>
			<td>2.6% (&#177;4.7%)</td>
		</tr>
		<tr>
			<td>liver</td>
			<td>40.4% (&#177;13.1%)</td>
			<td>0.3% (&#177;0.7%)</td>
			<td>0.0% (&#177;0.1%)</td>
			<td>0.0% (&#177;0.0%)</td>
		</tr>
		<tr>
			<td>kidney</td>
			<td>10.4% (&#177;5.2%)</td>
			<td>0.0% (&#177;0.0%)</td>
			<td>0.0% (&#177;0.1%)</td>
			<td>0.0% (&#177;0.0%)</td>
		</tr>
	</tbody>
</table>
Table 1: Tissue parameters. HIS StO2 oxygenation and NIR perfusion values in arbitrary units across 5 visceral organs and 4 different perfusion states.

Author Spotlight: Advancing Spectral Characterization of Physiological and Malperfused Tissues

This article introduces a standardized procedure for controlled, reversible malperfusion of visceral organs in rat models. The aim is to induce these malperfusion states with a high degree of methodological certainty and control while maintaining technical simplicity and error resilience.

Controlled Reversible Visceral Arterial Ischemia, Venous Congestion and Combined Malperfusion via Midline Laparotomy in Rats

Besides sepsis and malignancy, malperfusion is the third leading cause of tissue degradation and a major pathomechanism for various medical and surgical ...

controlled-reversible-visceral-arterial-ischemia-venous-congestion

Research

JoVE Journal

Medicine

250 Views.  Heidelberg University Hospital. This article introduces a standardized procedure for controlled, reversible malperfusion of visceral organs in rat models. The aim is to induce these malperfusion states with a high degree of methodological certainty and control while maintaining technical simplicity and error resilience.

Video: Author Spotlight: Advancing Spectral Characterization of Physiological and Malperfused Tissues

Microsurgical Venous Pouch Arterial-Bifurcation Aneurysms in the Rabbit Model: Technical Aspects

For ruptured human cerebral aneurysms endovascular embolization has become an equivalent alternative to aneurysm clipping.1 However, large clinical trials have shown disappointing long-term results with unacceptable high rates of aneurysm recanalization and delayed aneurysm rupture.2 To overcome these problems, animal experimental studies are crucial for the development of better endovascular devices.3-5
Several animal models in rats, rabbits, canines and swine are available.6-8 Comparisons of the different animal models showed the superiority of the rabbit model with regard to hemodynamics and comparability of the coagulation system and cost-effectiveness.9-11 
The venous pouch arterial bifurcation model in rabbits is formed by a venous pouch sutured into an artificially created true bifurcation of both common carotid arteries (CCA). The main advantage of this model are true bifurcational hemodynamics.12 The major drawbacks are the sofar high microsurgical technical demands and high morbidity and mortality rates of up to 50%.13 These limitations have resulted in less frequent use of this aneurysm model in the recent years. These shortcomings could be overcome with improved surgical procedures and modified peri- and postoperative analgetic management and anticoagulation.14-16 Our techniques reported in this paper demonstrate this optimized technique for microsurgical creation of arterial bifurcation aneurysms.

An optimized technique for the microsurgical creation of arterial bifurcation aneurysms mimicking bifurcation human cerebral aneurysms is described. A venous pouch is sutured into an artificially created true bifurcation of both common carotid arteries. Facilitated microsurgical techniques and aggressive postoperative anticoagulation and analgesia lead to minimized morbidity rates and high aneurysm patency rates.

For ruptured human cerebral aneurysms endovascular embolization has become an equivalent alternative to aneurysm clipping.1 However, large clinical trials ...

Femoral Arterial and Venous Catheterization for Blood Sampling, Drug Administration and Conscious Blood Pressure and Heart Rate Measurements

In multiple fields of study, access to the circulatory system in laboratory studies is necessary. Pharmacological studies in rats using chronically implanted catheters permit a researcher to effectively and humanely administer substances, perform repeated blood sampling and assists in conscious direct measurements of blood pressure and heart rate. Once the catheter is implanted long-term sampling is possible. Patency and catheter life depends on multiple factors including the lock solution used, flushing regimen and catheter material. This video will demonstrate the methodology of femoral artery and venous catheterization of the rat. In addition the video will demonstrate the use of the femoral venous and arterial catheters for blood sampling, drug administration and use of the arterial catheter in taking measurements of blood pressure and heart rate in a conscious freely-moving rat. A tether and harness attached to a swivel system will allow the animal to be housed and have samples taken by the researcher with minimal disruption to the animal. To maintain patency of the catheter, careful daily maintenance of the catheter is required using lock solution (100 U/ml heparinized saline), machine-ground blunt tip syringe needles and the use of syringe filters to minimize potential contamination. With careful aseptic surgical techniques, proper catheter materials and careful catheter maintenance techniques, it is possible to sustain patent catheters and healthy animals for long periods of time (several weeks).

Chronic catheterization of blood vessels in the rat is often required for administration of substances, obtain blood sample over a period of time or for direct conscious blood pressure measurements. Femoral arterial catheterization of the rat and corresponding measurements of blood pressure in the conscious animal will be demonstrated.

In multiple fields of study, access to the circulatory system in laboratory studies is necessary. Pharmacological studies in rats using chronically ...

Porcine Liver Transplantation Without Veno-Venous Bypass As an Extended Criteria Donor Model

Liver transplantation is regarded as the gold standard for the treatment of a variety of fatal hepatic diseases. However, unsolved issues of chronic graft failure, ongoing organ donor shortages, and the increased use of marginal grafts call for the improvement of current concepts, such as the implementation of organ machine perfusion. In order to evaluate new methods of graft reconditioning and modulation, translational models are required. With respect to anatomical and physiological similarities to humans and recent progress in the field of xenotransplantation, pigs have become the main large animal species used in transplantation models. After the initial introduction of a porcine orthotopic liver transplant model by Garnier et al. in 1965, several modifications have been published over the past 60 years.
Due to specifies-specific anatomical traits, a veno-venous bypass during the anhepatic phase is regarded as a necessity to reduce intestinal congestion and ischemia resulting in hemodynamic instability and perioperative mortality. However, the implementation of a bypass increases the technical and logistical complexity of the procedure. Furthermore, associated complications such as air embolism, hemorrhage, and the need for a simultaneous splenectomy have been reported previously.
In this protocol, we describe a model of porcine orthotopic liver transplantation without the use of a veno-venous bypass. The engraftment of donor livers after static cold storage of 20 h - simulating extended criteria donor conditions - demonstrates that this simplified approach can be performed without significant hemodynamic alterations or intraoperative mortality and with regular uptake of liver function (as defined by bile production and liver-specific CYP1A2 metabolism). The success of this approach is ensured by an optimized surgical technique and a sophisticated anesthesiologic volume and vasopressor management.
This model should be of special interest for workgroups focusing on the immediate postoperative course, ischemia-reperfusion injury, associated immunological mechanisms, and the reconditioning of extended criteria donor organs.

In this protocol, a model of porcine orthotopic liver transplantation after static cold storage of donor organs for 20 h without the use of a veno-venous bypass during engraftment is described. The approach uses a simplified surgical technique with minimization of the anhepatic phase and sophisticated volume and vasopressor management.

Liver transplantation is regarded as the gold standard for the treatment of a variety of fatal hepatic diseases. However, unsolved issues of chronic graft ...

Continuous Venous-Arterial Doppler Ultrasound During a Preload Challenge

A preload challenge (PC) is a clinical maneuver that, first, increases the cardiac filling (i.e., preload) and, second, calculates the change in cardiac output. Fundamentally, a PC is a bedside approach for testing the Frank-Starling-Sarnoff (i.e., "cardiac function") curve. Normally, this curve has a steep slope such that a small change in the cardiac preload generates a large change in the stroke volume (SV) or cardiac output. However, in various disease states, the slope of this relationship flattens such that increasing the volume into the heart leads to little rise in the SV. In this pathological scenario, additional cardiac preload (e.g., intravenous fluid) is unlikely to be physiologically effective and could lead to harm if organ congestion evolves. Therefore, inferring both the cardiac preload and output is clinically useful as it may guide intravenous (IV) fluid resuscitation. Accordingly, the goal of this protocol is to describe a method for contemporaneously tracking the surrogates of cardiac preload and output using a novel, wireless, wearable ultrasound during a well-validated preload challenge.

The Frank-Starling-Sarnoff curve is clinically important and describes the relationship between cardiac preload and output. This report illustrates a novel method of simultaneous jugular venous and carotid arterial Doppler velocimetry as transient surrogates of cardiac preload and output, respectively; this approach is enabled by wireless, wearable Doppler ultrasound.

A preload challenge (PC) is a clinical maneuver that, first, increases the cardiac filling (i.e., preload) and, second, calculates the change in cardiac ...

Surgical Approach, Challenges, and Resolutions for Uterus Transplantation in Rats

Uterine transplantation (UTx) is a new approach for treating women with absolute uterine factor infertility (AUFI). An estimated 3%-5% of women suffer from AUFI. These women were deprived of the option to have children until the advent of UTx. The clinical application of UTx was driven by experimental studies in animals, and the first successful UTx was achieved in rats. Given their physiological, immunological, genetic, and reproductive characteristics, rats are a suitable model system for such transplants. In particular, their short gestation period is a clear advantage, as the usual endpoint of experimental UTx is successful pregnancy with live birth. The biggest challenge for rat models remains the small anatomy, which requires advanced microsurgical skills and experience. Although UTx has led to pregnancy in the clinic, the procedure is not established and requires continuous experimental optimization. Here, a detailed protocol is presented, including essential troubleshooting for rat UTx, which is expected to make the entire procedure easier to grasp for those without experience in this type of microsurgery.

The present protocol describes all the essential steps for successful uterine transplantation (UTx) in rats. The rat model has proven suitable to promote the clinical implementation of UTx; however, rat UTx is a highly complex procedure requiring careful instructions.

Uterine transplantation (UTx) is a new approach for treating women with absolute uterine factor infertility (AUFI). An estimated 3%-5% of women suffer ...

Liver Transplantation in Mouse and Analyzing Allograft Patency

Successful Orthotopic Liver Transplantation in Mice Utilizing Microcomputed Tomography Angiography

Microcomputed tomography (microCT) angiography is an invaluable resource to researchers. New advances in this technology have allowed for high-quality images to be obtained of micro-vasculature and are high-fidelity tools in the field of organ transplantation. In this model of orthotopic liver transplantation (OLT) in mice, microCT affords the opportunity to evaluate allograft anastomosis in real time and has the added benefit of not having to sacrifice study animals. The choice of contrast, as well as image acquisition settings, create a high-definition image, which gives researchers invaluable information. This allows for evaluation of the technical aspects of the procedure as well as potentially evaluating different therapeutics over an extended duration of time. In this protocol, we detail an OLT model in mice in a stepwise fashion and finally describe a microCT protocol that can give high-quality images, which aid researchers in in-depth analysis of solid organ transplantation. We provide a step-by-step guide for liver transplantation in a mouse, as well as briefly discuss a protocol for evaluating the patency of the graft through microCT angiography.

Author Spotlight: Enhancing Transplantation Research Through MicroCT Angiography in Murine Models 

In this protocol, we discuss the implementation of a model of successful orthotopic liver transplantation (OLT) in mice. Additionally, adjuvants to further analyze allograft patency after successful OLT in a mouse are discussed as well, specifically utilizing microcomputed tomography (microCT) scans.

Microcomputed tomography (microCT) angiography is an invaluable resource to researchers. New advances in this technology have allowed for high-quality ...

Tracheotomy-Based Endotracheal Intubation Protocol for Non-Survival Rat Models

Endotracheal Intubation via Tracheotomy and Subsequent Thoracotomy in Rats for Non-Survival Applications

Endotracheal intubation and subsequent ventilation are often basic requirements for translational research in rat models for various interventions that require controlled or high ventilation pressures or access to the thoracic cavity and organs. Conventional endoorotracheal intubation using the anatomically existing route through the mouth is well suited for survival experiments. However, this procedure poses some challenges, including generally higher levels of the required experience and technical skill, more advanced equipment, and greater time effort with relevant intubation failure rates and complications such as tracheal perforation, temporary systemic hypooxygenation, and relevant aerial leakage.
This manuscript, therefore, presents a detailed step-by-step protocol for endotracheal intubation through tracheotomy in non-survival rat models when guaranteed intubation success, constant oxygenation levels, high ventilation pressures, or open thoracotomy are required.
The protocol emphasizes the importance of meticulous surgical technique to ensure consistent and reliable outcomes, especially for researchers who are inexperienced or lack routine in the technique of endoorotracheal intubation via direct laryngoscopy. This procedure is, therefore, expected to minimize animal suffering and unnecessary animal losses.

Here we introduce a standardized procedure for endotracheal intubation via tracheotomy followed by thoracotomy in rats, aimed at enhancing the precision and reproducibility of non-survival applications requiring invasive ventilation and exposure of thoracic organs in in vivo rat models.

Endotracheal intubation and subsequent ventilation are often basic requirements for translational research in rat models for various interventions that ...

Midline Laparotomy Surgery in Donor Mice for Orthotopic Liver Transplantation

Midline Laparotomy Surgery in Recipient Mice for Orthotopic Liver Transplantation

Optimizing Oviductal Embryo Transfer in Cloned Pigs

Embryo Transfer Surgery via Laparotomy in Gilts

This protocol aims to demonstrate the surgical technique for transferring cloned pig embryos to the oviduct, a method widely used in the production of genetically modified pigs for biomedical research. Nine gilts underwent hormonal synchronization and laparotomy for the transfer of cloned embryos produced by somatic cell nuclear transfer (SCNT) at stages of up to 4 cells on day 2 to the oviduct. Gestational diagnosis was conducted via ultrasound examination 30 days post-transfer surgery. Six out of the nine operated gilts exhibited signs of pregnancy on ultrasound examination. However, as there was no progression in fetal development as assessed by echography, the gilts underwent necropsy at 60 days for the collection of biological material and assessment of the reproductive system. Adhesions were observed in the uterine horns, ovaries, and oviducts. From the uterine lumen of two of the euthanized gilts, one and four embryonic structures with gestational ages ranging between 12 to 20 days were obtained. Despite the absence of live piglets, likely attributed to the low-efficiency rate of transferring cloned pig embryos, which is influenced by various factors, including the number and quality of transferred embryos, the presented surgical technique proved to be rapid and safe.

Author Spotlight: Surgical Methods and Outcomes in Oviductal Cloned Pig Embryo Transfers

This protocol describes the application of the surgical technique used to transfer cloned pig embryos via laparotomy in gilts.

This protocol aims to demonstrate the surgical technique for transferring cloned pig embryos to the oviduct, a method widely used in the production of ...