We would like to acknowledge Dennis Mathias for his illustrative work. This work was supported by NIH T32AI 106704-01A1 and the T. Flesch Fund for Organ Transplantation, Perfusion, Engineering and Regeneration at The Ohio State University.

All procedures were performed according to the guidelines of the Institutional Animal Care and the National Research Council&#39;s Guide for the Humane Care and Use of Laboratory Animals (IACUC) and has undergone approval by the Ohio State University IACUC committee.
1. Initial Set-up
<ol>
	<li>Set-up the surgical microscope and the operating theater (Figure 3, Figure 4). Turn on all equipment including that for maintaining anesthesia and monitoring vital signs. Turn on the electrosurgical unit and warming pad. Position the infusion pump near the operating table.
	<ol>
		<li>Draw up 10 mL of liquid isoflurane for inhalation (molecular weight 184.5) in the anesthesia syringe and place it in the anesthesia unit.</li>
		<li>Set-up a 200 mL container of liquid nitrogen near the operating table and another near the centrifuge where blood samples will be processed.</li>
		<li>Position the surgical instruments, 4-0 and 7-0 braided silk suture, sterile cotton swabs, 4x4 non-woven sponges, 5 mL syringes, and 27 gauge insulin syringes near the operating table.</li>
	</ol>
	</li>
	<li>Prepare the isoflurane chamber and ensure that sufficient isoflurane is instilled in the anesthesia induction delivery system.</li>
</ol>
2. Induction of Anesthesia
<ol>
	<li>Before handling the rat put on the following personal protective equipment (PPE): surgical mask, surgical gloves, and disposable gown.</li>
	<li>Weigh the rat and record the weight. 
	NOTE: Sprague Dawley rats should be used.</li>
	<li>Place the rat in the anesthesia chamber and turn on the isoflurane and the oxygen. Induce anesthesia using the isoflurane chamber.</li>
	<li>Clip the animal&#39;s abdominal hair using an electric hair clipper to allow for cleaner exposure (Figure 5).</li>
	<li>Place the animal back in the isoflurane chamber for an additional one minute. Perform a toe pinch to verify depth of anesthesia.</li>
</ol>
3. Procedure
<ol>
	<li>Position the rat with the animal&#39;s nose in the nose cone and four extremities immobilized with restraints or tape on the warming pad.</li>
	<li>Continue anesthesia using the anesthesia delivery system, nose cone and isoflurane with anesthesia at 3.6% for animals weighing between 200 and 250 g and 4% for animals weighing greater than 250 g. Confirm depth of anesthesia by performing a toe pinch and a skin pinch.</li>
	<li>Make a midline abdominal incision from pubis to xiphoid through the skin using sharp scissors (Figure 6).</li>
	<li>Make an incision in the peritoneum along the linea alba from pubis to xiphoid and enter the abdomen taking care not to damage the bladder or bowel. As the liver also sticks to the peritoneum anteriorly near the xiphoid process, ensure that it releases prior to incising the abdominal wall in this area.</li>
	<li>Make a transverse incision through the skin and the peritoneum at the level of the inferior border of the right lobe of the liver.</li>
	<li>Turn the anesthesia down to 1.6% for animals weighing between 200 and 250 g and 2% for animals weighing greater than 250 g.</li>
	<li>Retract the xiphoid process using a curved mosquito clamp.</li>
	<li>Place rib retractors pulling the ribs as far apart as possible from the midline (Figure 7). Cut the falciform, phrenic and gastric ligaments. Flip the liver up using moistened sterile cotton swabs.</li>
	<li>Cut additional ligaments as necessary to gain access to the porta. Perform visceral rotation with saline moistened gauze (Figure 7).</li>
	<li>Remove the loose connective tissue overlying the portal hilum using sharp or blunt dissection. Remove the loose connective tissue overlying the length of the portal vein.</li>
	<li>Use forceps to push through the loose connective tissue posterior to the left portal vein, artery and bile duct making a window and place 4-0 Potts suture but do not cinch down (Figure 8).</li>
	<li>Clear off the loose connective tissue overlying the posterior branch to the portal vein that comes in at approximately the level of the right kidney. This vein will be used for cannulation.</li>
	<li>Draw 0.5 mL of blood out of the inferior vena cava (IVC) with an insulin syringe (Figure 9). Place the 0.5 mL of blood in a small vial, centrifuge at 135 x g for 12 min. Attempt to draw off serum.
	<ol>
		<li>If a distinct line cannot be appreciated between red blood cells and serum, try to centrifuge for an additional 2 - 3 min at 135 x g. Draw off serum and place in a vial for alanine-aminotransferase (ALT). Snap freeze this specimen by placing it directly into liquid nitrogen.</li>
	</ol>
	</li>
	<li>Cut two pieces of 7-0 suture and place near the vein that will be used for cannulation. Place the first 7-0 loop around this vein as far medial as possible. Tie this loop and use it to retract using a curved mosquito clamp (Figure 10). Place a second 7-0 loop on the vein that will be used for cannulation near its intersection with the portal vein and place one tie, but do not cinch down.</li>
	<li>Prepare the infusion pump with a 5 mL syringe with 3 mL of reagent. Prime the tubing.</li>
	<li>Clamp distal portal vein using a microsurgical clamp. 
	NOTE: This will reduce bleeding when the vein is incised for cannulation.</li>
	<li>Cut a 0.5 mm hole in the vein in between the 7-0 stay suture and its intersection with the portal vein using small microsurgical scissors. Use 27-0 catheter to cannulate the left portal venous system (Figure 11, Figure 12). Insert the catheter past the bifurcation of the left and right portal veins.</li>
	<li>Check placement of the cannula by infusing 1 mL of normal saline and watch for the left lateral and median lobes of the liver to blanch. Manually confirm that catheter is past the take-off of the right portal vein, but not beyond the take-off of the portal vein feeding the median lobe.</li>
	<li>Cinch down the Potts suture and start ischemia time. Tighten 7-0 suture around vein and 27-0 catheter to hold it in place and remove the clamp from the distal portal vein.</li>
	<li>Start the infusion of polyethylene glycol-superoxide dismutase (PEG-SOD, 0.00067 g/mL) using the infusion pump. Start the infusion as close as possible to the start of the ischemic time.</li>
</ol>
4. Monitoring
<ol>
	<li>Continue to monitor the animal&#39;s vital signs throughout the infusion. Deliver 2 mL of 0.9% normal saline or 2 mL of PEG-SOD (0.00067 g/mL) dissolved in 0.9% normal saline over a period of 15 min.</li>
</ol>
5. Reperfusion
<ol>
	<li>Allow one hour to pass from the beginning of the ischemic time. This is 1-h of warm ischemia time.</li>
	<li>Remove the Potts suture. Remove the 27-0 catheter. Cinch down the 7-0 suture around the vein. Note the time. This marks the time of reperfusion.</li>
</ol>
6. Continued Sampling
<ol>
	<li>Draw 0.5 mL of blood out of the IVC at 120 min post-reperfusion. Draw the blood slowly to avoid lysing red blood cells. Slowly drip the blood into a vial. Ensure that bleeding from the IVC is controlled after each blood draw.
	<ol>
		<li>If there is continued bleeding apply gentle pressure with a sterile cotton swab or a small 1 cm by 1 cm section cut from gauze.</li>
	</ol>
	</li>
	<li>Centrifuge at 135 x g for 12 min. If sufficient separation is not achieved, try an additional 2 - 3 min at 135 x g.</li>
	<li>Place half of the serum in a vial for later processing for ALT. Snap freeze these specimens.</li>
</ol>
7. Euthanasia
<ol>
	<li>While the rat is still under anesthesia cut the IVC and superior vena cava (SVC) and monitor until blood flow, respiration and heart beat cease.</li>
	<li>Incise the diaphragm and perform a brief hepatectomy by incising the diaphragm in a circle and incising additional connective tissue that remains connecting the liver to the peritoneal cavity. Remove the liver from the peritoneal cavity.</li>
	<li>Take four samples from the left and median lobes of the liver and four samples from the right lobe of the liver. The samples should be as large as possible and their size will be limited only by the amount of available liver tissue. Place these in small, labeled vials, and snap freeze in the liquid nitrogen. Use these for later processing for tissue adenosine triphosphate (ADP), malondialdehyde (MDA) and glutathione (GSH).</li>
</ol>
8. Post-experiment Analysis
<ol>
	<li>Determine glutathione (GSH), malondialdehyde (MDA) and alanine aminotransferase (ALT) activities in liver tissue and serum samples using diagnostic kits according to the manufacturer&#39;s instructions.</li>
	<li>Homogenize the liver tissue with lysis buffer and quantify using a Bradford assay. Analyze tissue lysate by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot using antibodies against cleaved capase-3 and actin. Quantify western blots performed with publicly available software.</li>
</ol>

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All authors report they have no disclosures.

This series of experiments demonstrated that injection of PEG-SOD into the left and median lobes led to significant decreases in the release of ALT, lipid peroxidation of cell membranes (MDA), and maintenance of glutathione (GSH) when compared with controls (Normal Saline). Liver tissue transaminases including Alanine Aminotransferase (ALT) are established markers of hepatocellular injury. The decrease in ALT when the left lobe is injected with PEG-SOD suggests a protective effect of PEG-SOD. Increased tissue MDA indicates increased lipid peroxidation and is considered a marker of oxidative stress and tissue injury. Overproduction of reactive oxygen species causes an increase in production of MDA26. The significant reduction in tissue MDA in the animal&#39;s left and median lobes when injected with PEG-SOD demonstrates a protective effect of PEG-SOD. This is consistent with the current understanding that PEG-SOD protects cells from damage caused by partially reduced reactive oxygen species27. Additionally, in the presence of reactive oxygen species, glutathione disulfide is reduced to glutathione (GSH)28. The maintenance in GSH in the left and median lobes of the liver injected with PEG-SOD further reinforces the protective effect of PEG-SOD. Additionally it is demonstrated that there is increased cleaved caspase-3, a product of apoptosis, in tissue exposed to ischemia-reperfusion injury. The decrease in cleaved caspase-3 in the left lobe when treated with PEG-SOD suggests that PEG-SOD leads to a decrease in apoptosis.
Superoxide dismutase (SOD) is a critical enzyme in the detoxication of reactive oxygen species. The enzyme catalyzes the conversion of two superoxide anions into hydrogen peroxide and water. The enzyme catalase then converts hydrogen peroxide to water and oxygen, completing the process25. The half-life of native SOD limited its use in experimental models until the development of conjugated polyethylene glycol-superoxide dismutase (PEG-SOD). Conjugation of SOD to polyethylene glycol increases its half-life from 6 min to 14 h. Nguyen et al. demonstrated its ability to mitigate lipid peroxidation in hepatic ischemia in a rat model, using systemic delivery29.
There are a variety of potential modifications of the technique detailed here and some have previously been described in the literature. Depending on the model used molecules have been delivered using inhalation11, epidural infusion12, intraperitoneal injection17,18,21,22, intravenous administration10,14,15,19,23,24 or injection into the peripheral superior mesenteric vein8.
There are several critical steps in this protocol. The most important is the cannulation of the portal vein. Care must be taken that the hole cut in the vein is not too large. The tissue is very elastic and the hole will enlarge on its own. We recommend starting by cutting a hole that is 0.5 mm with the microsurgical scissors. The cannula can be fed through the hole using an instrument, which allows for greater agility than if trying to perform this portion of the procedure by hand. Additionally, while initially feeding the cannula, it should be aimed directly towards the bifurcation of the left and right portal veins to avoid poking a hole through the back wall of the vein. When the cannula tip reaches the bifurcation, it can then be fed into the left vein specifically. Once the cannula is fed into the left portal vein, which supplies both the left and median lobes, its position can be confirmed manually by feeling it inside the vein. Its position can also be confirmed by injecting a small amount of cold saline and seeing the blanching effect on the supplied segments of the liver.
The liver hilar clamp model in the rat provides a reproducible and stable platform for demonstrating hepatic ischemic-reperfusion injury. Variable hilar clamp models have been used by researchers to study the protective effects of anti-oxidants and other small molecules6,7,8,9,10,11,12,13,14. Points of variation include which vessels are clamped, which segment are made ischemic, whether or not the bile duct is included and the length of the period of reperfusion6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21. Additionally, when this model is used to study the impact of administration of a molecule the route of administration is also heterogeneous8,10,11,12,14,15,17,18,19,21,22,23,24. There are several advantages to the described approach. First, direct cannulation of the portal supply to the ischemic segment allows for direct segmental hepatic delivery of the pharmacological substance under study. This allows utilization of the other lobes of the livers as an internal control. Second, segmental hepatic cannulation allows for a reduced volume of distribution for the molecule being studied. This approach thereby reduces the risk of systemic side effects as the substance is injected directly into the liver segment of interest. Direct cannulation of the hepatic segment allows for the substance to be delivered pre-ischemia, intra-ischemia or post-ischemia. This allows for study of the molecule&#39;s effect at any point in the ischemia-reperfusion injury cycle. With increased length of ischemic time and increased level of injury additional opportunity to study liver regeneration would be available.
There are also some limitations of this approach. The first is start-up cost. The purchase of a surgical microscope could be a significant start-up cost for a lab that does not already possess one. This technique may be difficult or impossible without a microscope. The second is learning curve time. Although this procedure is relatively simple it does require some practice and it is likely that a novice will require a significant number of procedures to become an expert.
In summary, this model allows for a reproducible, simple, and cost-effective platform to study hepatic ischemia-reperfusion injury. Although in the protocol described here polyethylene glycol-superoxide dismutase, a free radical scavenger25, was infused, this model could be used to infuse a variety of different pharmacologic substances in order to evaluate their impact on I/R injury in the liver.

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ischemia/reperfusion (I/R) injury1. The consequences of I/R injury in the liver have been detailed extensively1,2,3. Consequences of I/R injury detailed in the literature include: generation of reactive oxygen species, initiation of the inflammatory cascade including activation of neutrophils, Kupffer cells, and endothelial cells, activation of the heme oxygenase system and activation of toll-like receptors, an imbalance between endothelin and nitric oxide, activation of nuclear factor-&#954;B, and promotion of proinflammatory cytokine and adhesion molecule synthesis1,2,3. These proinflammatory events may lead to apoptosis, necrosis, organ dysfunction and eventual organ failure3.
I/R injury in organs destined for liver transplantation can lead to early graft loss and contributes to the current donor shortage as marginal organs are more susceptible to injury3. There are currently 15,226 potential recipients on the waiting list for liver transplantation in the United States4 and only 5,950 liver transplants were performed in 20155. Due to this extreme limitation in organ availability, research exploring hepatic I/R injury is needed in order to optimize graft function and organ utilization.
Animal models used to study hepatic I/R injury include rat hilar clamp models and rat liver transplantation models. There are a variety of rat hilar clamp models currently in use. The most common is one in which the portal vein, hepatic artery and bile duct supplying the left lateral and median lobes are clamped using microsurgical clips6,7,8,9,10,11,12 for 30 to 60 min6,7,10,13,14, and then a period of reperfusion from 60 min to 24 h7,9,10,13,14 is allowed. The left lateral and median lobes of the rat liver comprise about 70% of the hepatic parenchyma9. Some protocols designed to study ischemic preconditioning include intermittent clamping of the hilar vessels or the hind-limb prior to a longer period of ischemia induced by clamping the hilar vessels9,13. There are also several modifications described in the literature. The first is to clamp the portal vein and hepatic artery supplying the left lateral and median lobes, but exclude the bile duct15. A second modification is to induce total hepatic ischemia by clamping the portal vein, hepatic artery and bile duct prior to their division16,17,18,19,20. A third modification includes clamping of the hilar vessels to the right lobe for 30 to 60 min8. An additional modification involves clamping the vascular bundle in one hind limb in order to induce injury in the liver13,21. Various approaches to the hilar clamp procedure are illustrated in Figure 1A-D.
Rat liver hilar clamp models have been used to study the impact of different molecules and compounds on hepatic I/R. Depending on the model used these molecules have been delivered using inhalation11, epidural infusion12, intraperitoneal injection17,18,21,22, intravenous administration10,14,15,19,23,24 or injection into the peripheral superior mesenteric vein8.
The model for rat liver hilar clamp detailed in this report includes direct cannulation of the portal supply to the ischemic segment via a side branch of the portal vein (Figure 2), which allows for direct segmental hepatic delivery of the pharmacological substance under study. Our approach is to induce ischemia in the left lateral and median lobes for 60 min, during which time an infusion of the substance under study, in this case, pegylated-superoxide dismutase, a free radical scavenger25, is infused directly into the ischemic segment. Blood samples are taken prior to induction of ischemia and at 120 min post-reperfusion. At this point, the rat is sacrificed and samples are taken from the left and median lobes. Additionally, samples are taken from the right lobe to serve as an internal control.
There are numerous advantages to this approach. First and foremost, when the pharmacologic substance under study can be directly injected into the ischemic segment the volume of distribution is quite low in comparison to the volume of distribution of injection into the systemic circulation or the peritoneal cavity. Additionally, this approach reduces, although does not eliminate, the possibility of systemic side effects.

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			<td height="21" style="height:21px;width:288px;">Sprague-Dawley Rat&#160;</td>
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			<td>Isotec</td>
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			<td>INS600240</td>
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			<td>Sigma Aldrich&#160;</td>
			<td>S9549 SIGMA</td>
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			<td>Kent Scientific</td>
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			<td>Baxter</td>
			<td>NDC 0338-0048-04</td>
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			<td>104-2411</td>
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			<td>Henry Schein Animal Health</td>
			<td>1009175</td>
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			<td>Terumo</td>
			<td>22-272328</td>
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			<td>BD&#160;</td>
			<td>REF 309603</td>
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			<td>Deknatel, Inc.</td>
			<td>198737LP</td>
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			<td>REF 103-S</td>
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			<td>USA Scientific&#160;</td>
			<td>1418-7410</td>
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			<td>Roboz&#160;</td>
			<td>RS-5610</td>
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			<td>S&#38;T&#160;</td>
			<td>SAA-15</td>
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			<td>S&#38;T&#160;</td>
			<td>JFCL-7</td>
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			<td>S&#38;T</td>
			<td>FRAS-15 RM-8</td>
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			<td>ROBOZ</td>
			<td>RS-5440</td>
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			<td>FST 14958-11</td>
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			<td>S&#38;T&#160;</td>
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			<td>Fine Scientific Tools</td>
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			<td>Fine Scientific Tools</td>
			<td>18055-01</td>
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			<td>Fine Scientific Tools</td>
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			<td>Fine Scientific Tools</td>
			<td>18055-03</td>
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			<td height="21" style="height:21px;">Name</td>
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			<td>Biovision</td>
			<td>K752-100</td>
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			<td>Abcam&#160;</td>
			<td>ab118970</td>
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			<td height="21" style="height:21px;">Glutathione (GSH) assay</td>
			<td>Cayman Chemical</td>
			<td>7030002</td>
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			<td>Cell Signaling Tecnology</td>
			<td>Antibody 9661</td>
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			<td height="21" style="height:21px;">ImageJ Software&#160;</td>
			<td>National Institutes of Health&#160;</td>
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			<td height="21" style="height:21px;">RIPA Lysis and Extraction Buffer&#160;</td>
			<td>Millipore</td>
			<td>10-188</td>
			<td></td>
		</tr>
	</tbody>
</table>

method of direct segmental intra-hepatic delivery using a rat liver hilar clamp model

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ischemia/reperfusion (I/R) injury with myriad negative consequences. Potential I/R injury in marginal organs destined for liver transplantation contributes to the current donor shortage secondary to a decreased organ utilization rate. A significant need exists to explore hepatic I/R injury in order to mediate its impact on graft function in transplantation. Rat liver hilar clamp models are used to investigate the impact of different molecules on hepatic I/R injury. Depending on the model, these molecules have been delivered using inhalation, epidural infusion, intraperitoneal injection, intravenous administration or injection into the peripheral superior mesenteric vein. A rat liver hilar clamp model has been developed for use in studying the impact of pharmacologic molecules in ameliorating I/R injury. The described model for rat liver hilar clamp includes direct cannulation of the portal supply to the ischemic hepatic segment via a side branch of the portal vein, allowing for direct segmental hepatic delivery. Our approach is to induce ischemia in the left lateral and median lobes for 60 min, during which time the substance under study is infused. In this case, pegylated-superoxide dismutase (PEG-SOD), a free radical scavenger, is infused directly into the ischemic segment. This series of experiments demonstrates that infusion of PEG-SOD is protective against hepatic I/R injury. Advantages of this approach include direct injection of the molecule into the ischemic segment with consequent decrease in volume of distribution and reduction in systemic side effects.

This experiment was performed with 2 groups of n = 3 rats each. Three rat livers were injected with 2 mL of normal saline (NS) with the infusion pump over a period of 15 min. Three rat livers were injected with 2 mL of normal saline (NS) mixed with pegylated-superoxide dismutase (PEG-SOD, 0.00067 g/mL) with the infusion pump over a period of 15 min. As described in the above protocol, blood samples were taken pre-hilar clamp and at 120-min post-reperfusion. Additionally, after completion of 120-min of reperfusion four liver tissue samples were taken from the left and median lobes and four liver samples were taken from the right lobe of the rat liver.
Serum Alanine Aminotransferase (ALT) was measured pre-hilar clamp and at 120-min post-reperfusion in control (NS) and experimental (PEG-SOD) animals. There was a significant difference between the ALT level of control (NS) animals pre-hilar clamp and at 120-min post-reperfusion. There was a significant difference between ALT level of control (NS) and experimental animals (PEG-SOD) at 120-min (Figure 13A). Tissue malonaldehyde (MDA) was measured for control (NS) and experimental (PEG-SOD) animals in both right and left lobes of the liver. Tissue MDA in right lobe (non-hilar clamp) with control injection (NS) and experimental injection (PEG-SOD) demonstrate no significant difference. Left lobe (post-hilar clamp and reperfusion) tissue MDA with control injection (NS) is significantly different than right lobe (non-hilar clamp) p &#60; 0.001. Left lobe (post-hilar clamp and reperfusion) has significantly different levels of tissue MDA with control injection (NS) versus experimental injection (PEG-SOD) p &#60; 0.005 (Figure 13B). Tissue glutathione (GSH) was measured and tissue glutathione in right lobe (non-hilar clamp) with control injection (NS) and experimental injection (PEG-SOD) demonstrate no significant difference. Left lobe (post-hilar clamp and reperfusion) tissue GSH with control injection (NS) is significantly different than right lobe (non-hilar clamp) with control injection (NS) p &#60; 0.05. Left lobe (post-hilar clamp and reperfusion) has significantly different levels of tissue glutathione with control injection (NS) versus experimental injection (PEG-SOD) p &#60; 0.005 (Figure 13C). Western blot was performed comparing right and left lobe of control animals and demonstrates increased cleaved caspase-3 in the left lobe after hilar clamp and reperfusion (Figure 13D). A second western blot was performed comparing the left lobes of animals treated with control and with PEG-SOD (Figure 13E). This demonstrates decreased cleaved caspase-3 in the liver tissue of animals treated with PEG-SOD. Densitometry was also performed demonstrating that the level of cleaved caspase-3 in liver tissue is significantly increased in the left versus right lobe of control animals (Figure 13F). In comparing the left lobe liver tissue of experimental animals, infused with PEG-SOD, and left lobe liver tissue of control animals, infused with Normal Saline, densitometry demonstrates significantly decreased cleaved caspase-3 in animals treated with PEG-SOD in comparison to animals treated with control (Figure 13G).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/54729/54729fig1.jpg" /> 
Figure 1: Anatomical Illustrations. A. Anatomical illustration of the rat liver. B. Anatomical illustration of the rat liver. The portal pedicle to the left and median lobes of the liver is clamped. The left and median lobes are ischemic. C. Anatomical illustration of the rat liver. The portal pedicle to the left lobe is clamped. The left lobe is ischemic. D. Anatomical illustration of the rat liver. The portal pedicle to the right lobe is clamped and the right lobe is ischemic.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/54729/54729fig2.jpg" /> 
Figure 2: Anatomical Illustrations. Anatomical illustration of the rat liver with portal vein cannulated via a side branch. The portal pedicle to the left and median lobes of the liver is surrounded by a suture and a microvessel clamp has been used to tighten around the vascular bundle. The left and median lobes are ischemic.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/54729/54729fig3.jpg" /> 
Figure 3: Instrument Set-up. This figure demonstrates the instrument set-up.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/54729/54729fig4.jpg" /> 
Figure 4: Operating Room Set-up. This figure demonstrates the operating room set-up. <a href="http://cloudflare.jove.com/files/ftp_upload/54729/54729fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/54729/54729fig5.jpg" /> 
Figure 5: Trimming of Abdominal Hair. This figure demonstrates the trimming of the abdominal hair. <a href="http://cloudflare.jove.com/files/ftp_upload/54729/54729fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/54729/54729fig6.jpg" /> 
Figure 6: Immobilization and Skin Incision. This figure demonstrates the immobilization of the rat and the skin incision. <a href="http://cloudflare.jove.com/files/ftp_upload/54729/54729fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/54729/54729fig7.jpg" /> 
Figure 7: Rib Retractor Placement and Evisceration. This figure demonstrates the rib retractor placement and evisceration. <a href="http://cloudflare.jove.com/files/ftp_upload/54729/54729fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/54729/54729fig8.jpg" /> 
Figure 8: Placement of Suture. This figure demonstrates the placement of the suture. <a href="http://cloudflare.jove.com/files/ftp_upload/54729/54729fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/54729/54729fig9.jpg" /> 
Figure 9: Blood Draw from the Inferior Vena Cava. This figure demonstrates blood draw from the inferior vena cava. <a href="http://cloudflare.jove.com/files/ftp_upload/54729/54729fig9large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 10" class="xfigimg" src="/files/ftp_upload/54729/54729fig10.jpg" /> 
Figure 10: Vein Branch Tied Off and Retracted. This figure demonstrates vein branch tied off and retracted. <a href="http://cloudflare.jove.com/files/ftp_upload/54729/54729fig10large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 11" class="xfigimg" src="/files/ftp_upload/54729/54729fig11.jpg" /> 
Figure 11: Process of Cannulation. This figure demonstrates the process of cannulation. <a href="http://cloudflare.jove.com/files/ftp_upload/54729/54729fig11large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 12" class="xfigimg" src="/files/ftp_upload/54729/54729fig12.jpg" /> 
Figure 12: Cannulation. This figure demonstrates the cannulation. <a href="http://cloudflare.jove.com/files/ftp_upload/54729/54729fig12large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 13" class="xfigimg" src="/files/ftp_upload/54729/54729fig13.jpg" /> 
Figure 13: Representative Results: Direct Segmental Intrahepatic Delivery of Pegylated-Superoxide Dismutase Using a Rat Hilar Clamp Model. NS = normal saline. PEG-SOD = pegylated-superoxide dismutase, ALT = alanine aminotransferase, MDA = malondialdehyde. A. Serum Alanine Aminotransferase (ALT, mU/mL) compared between pre-hilar clamp and 120-min post-reperfusion. There is a significant difference between control (NS) pre-hilar clamp and control (NS) at 120-min post-reperfusion (p &#60; 0.001). There is also a significant difference between control (NS) and experimental groups (PEG-SOD) at 120-min post-reperfusion (p &#60; 0.05). A student&#39;s T-test was used. Error bars represent standard deviation. B. Tissue malondialdehyde in right lobe (non-hilar clamp) with control injection (NS) and experimental injection (PEG-SOD) demonstrate no significant difference. Left lobe (post-hilar clamp and reperfusion) tissue malondialdehyde with control injection (NS) is significantly different than right lobe (non-hilar clamp) p &#60; 0.001. Left lobe (post-hilar clamp and reperfusion) has significantly different levels of tissue malonaldehyde with control injection (NS) versus experimental injection (PEG-SOD) p &#60; 0.005. A student&#39;s T-test was used. Error bars represent standard deviation. C. Tissue glutathione in right lobe (non-hilar clamp) with control injection (NS) and experimental injection (PEG-SOD) demonstrate no significant difference. Left lobe (post-hilar clamp and reperfusion) tissue glutathione with control injection (NS) is significantly different than right lobe (non-hilar clamp) with control injection (NS) p &#60; 0.05. Left lobe (post-hilar clamp and reperfusion) has significantly different levels of tissue glutathione with control injection (NS) versus experimental injection (PEG-SOD) p &#60; 0.005. A student&#39;s T-test was used. Error bars represent standard deviation. D. Western blot demonstrating increased cleaved caspase-3 in liver tissue of the left lobe (post-hilar clamp and reperfusion) versus the right lobe (non-hilar clamp) of control animals (Normal Saline). E. Western blot demonstrating decreased cleaved caspase-3 in liver tissue of animals treated with PEG-SOD in comparison to animals treated with control (Normal Saline). F. Level of cleaved caspase-3 in liver tissue is significantly increased in post-hilar clamp and reperfusion animals (p &#60; 0.05). A student&#39;s T-test was used. Error bars represent standard deviation. G. In comparing left lobe liver tissue of experimental animals (infused with PEG-SOD) and left lobe liver tissue of control animals (infused with Normal Saline), there is significantly decreased cleaved caspase-3 in animals treated with PEG-SOD in comparison to animals treated with control (Normal Saline). A student&#39;s T-test was used. Error bars represent standard deviation.

Watch this Scientific Journal Video about Method of Direct Segmental Intra-hepatic Delivery Using a Rat Liver Hilar Clamp Model at JoVE.com

Method of Direct Segmental Intra-hepatic Delivery Using a Rat Liver Hilar Clamp Model

A unique rat liver hilar clamp model was developed for studying the impact of pharmacologic molecules in ameliorating ischemia-reperfusion injury. This model includes direct cannulation of the portal supply to the ischemic liver segment via a branch of the portal vein, allowing for direct hepatic delivery.

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ...

method-direct-segmental-intra-hepatic-delivery-using-rat-liver-hilar

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JoVE Journal

Medicine

14.0K Views.  The Ohio State University Wexner Medical Center.  Een unieke rattenlever hilaire clamp model ontwikkeld voor het bestuderen van de effecten van farmacologische moleculen bij het verbeteren van ischemie-reperfusieletsel. Dit model omvat directe canulatie van het portaal toevoer naar het segment ischemische lever via een tak van de poortader, zodat hij direct hepatische afgifte. 

Video: Method of Direct Segmental Intra-hepatic Delivery Using a Rat Liver Hilar Clamp Model

Hyperinsulinemic-Euglycemic Clamp in the Conscious Rat

Type 2 diabetes (T2D) is rapidly rising in prevalence. Characterized by either inadequate insulin production or the inability to utilize insulin produced, T2D results in elevated blood glucose levels. The "gold-standard" in assessing insulin sensitivity is a hyperinsulinemic-euglycemic clamp or insulin clamp. In this procedure, insulin is infused at a constant rate resulting in a drop in blood glucose. To maintain blood glucose at a constant level, exogenous glucose (D50) is infused into the venous circulation. The amount of glucose infused to maintain homeostasis is indicative of insulin sensitivity. Here, we show the basic clamp procedure in the chronically catheterized, unrestrained, conscious rat. This model allows blood to be collected with minimal stress to the animal. Following the induction of anesthesia, a midline incision is made and the left common carotid artery and right jugular vein are catheterized. Inserted catheters are flushed with heparinized saline, then exteriorized and secured. Animals are allowed to recover for 4-5 days prior to experiments, with weight gain monitored daily. Only those animals who regain weight to pre-surgery levels are used for experiments. On the day of the experiment, rats are fasted and connected to pumps containing insulin and D50. Baseline glucose is assessed from the arterial line and used a benchmark throughout the experiment (euglycemia). Following this, insulin is infused at a constant rate into the venous circulation. To match the drop in blood glucose, D50 is infused. If the rate of D50 infusion is greater than the rate of uptake, a rise in glucose will occur. Similarly, if the rate is insufficient to match whole body glucose uptake, a drop will occur. Titration of glucose continues until stable glucose readings are achieved. Glucose levels and glucose infusion rates during this stable period are recorded and reported. Results provide an index of whole body insulin sensitivity. The technique can be refined to meet specific experimental requirements. It is further enhanced by the use of radioactive tracers that can determine tissue specific insulin-stimulated glucose uptake as well as whole body glucose turnover.

The hyperinsulinemic-euglycemic clamp is the "gold standard" for the assessment of insulin action. Insulin is infused at a constant rate stimulating glucose uptake. The amount of exogenous glucose infused to counter this drop is indicative of insulin sensitivity. Here the procedure is performed on a conscious, unrestrained rat.

Type 2 diabetes (T2D) is rapidly rising in prevalence. Characterized by either inadequate insulin production or the inability to utilize insulin produced, ...

Surgical Procedures for a Rat Model of Partial Orthotopic Liver Transplantation with Hepatic Arterial Reconstruction

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as studies on graft preservation and ischemia-reperfusion injury 1,2, immunological responses 3,4, hemodynamics 5,6, and small-for-size syndrome 7. The rat OLT is among the most difficult animal models in experimental surgery and demands advanced microsurgical skills that take a long time to learn. Consequently, the use of this model has been limited. Since the reliability and reproducibility of results are key components of the experiments in which such complex animal models are used, it is essential for surgeons who are involved in rat OLT to be trained in well-standardized and sophisticated procedures for this model.
While various techniques and modifications of OLT in rats have been reported 8 since the first model was described by Lee et al. 9 in 1973, the elimination of the hepatic arterial reconstruction 10 and the introduction of the cuff anastomosis technique by Kamada et al. 11 were a major advancement in this model, because they simplified the reconstruction procedures to a great degree. In the model by Kamada et al., the hepatic rearterialization was also eliminated. Since rats could survive without hepatic arterial flow after liver transplantation, there was considerable controversy over the value of hepatic arterialization. However, the physiological superiority of the arterialized model has been increasingly acknowledged, especially in terms of preserving the bile duct system 8,12 and the liver integrity 8,13,14.
In this article, we present detailed surgical procedures for a rat model of OLT with hepatic arterial reconstruction using a 50% partial graft after ex vivo liver resection. The reconstruction procedures for each vessel and the bile duct are performed by the following methods: a 7-0 polypropylene continuous suture for the supra- and infrahepatic vena cava; a cuff technique for the portal vein; and a stent technique for the hepatic artery and the bile duct.

Orthotopic liver transplantation in rats is an indispensable experimental model for biomedical research. Here we present our surgical procedures for orthotopic rat liver transplantation with hepatic arterial reconstruction using a 50% partial graft.

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as ...

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. As this technology expands and improves, the ability to potentially evaluate and improve the quality of substandard lungs prior to transplant is a critical need. In order to more rigorously evaluate these approaches, a reproducible animal model needs to be established that would allow for testing of improved techniques and management of the donated lungs as well as to the lung-transplant recipient. In addition, an EVLP animal model of associated pathologies, e.g., ventilation induced lung injury (VILI), would provide a novel method to evaluate treatments for these pathologies. Here, we describe the development of a rat EVLP lung program and refinements to this method that allow for a reproducible model for future expansion. We also describe the application of this EVLP system to model VILI in rat lungs. The goal is to provide the research community with key information and &#8220;pearls of wisdom&#8221;/techniques that arose from trial and error and are critical to establishing an EVLP system that is robust and reproducible.

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. Here, we describe the development of a rat EVLP program and refinements that allow for a reproducible model for future expansion.

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed ...

The Dimethylnitrosamine Induced Liver Fibrosis Model in the Rat

Four to six week old, male Wistar rats were used to produce animal models of liver fibrosis. The process requires four weeks of administration of 10 mg/kg dimethylnitrosamine (DMN), given intraperitoneally for three consecutive days per week. Intraperitoneal injections were performed in the fume hood as DMN is a known hepatoxin and carcinogen. The model has several advantages. Firstly, liver changes can be studied sequentially or at particular stages of interest. Secondly, the stage of liver disease can be monitored by measurement of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) enzymes. Thirdly, the severity of liver damage at different stages can be confirmed by sacrifice of animals at designated time points, followed by histological examination of Masson&#39;s Trichome stained liver tissues. After four weeks of DMN dosing, the typical fibrosis score is 5 to 6 on the Ishak scale. The model can be reproduced consistently and has been widely used to assess the efficacy of potential anti-fibrotic agents.

We describe a method to produce an animal model of liver fibrosis in the rat, and assess the degree of fibrosis by histological examination of the liver. The model can be used to study the development of liver disease as well as to test the efficacy of potential anti-fibrotic agents.

Four to six week old, male Wistar rats were used to produce animal models of liver fibrosis. The process requires four weeks of administration of 10 mg/kg ...

Intravascular Delivery of Biologics to the Rat Kidney

The renal microvascular compartment plays an important role in the progression of kidney disease and hypertension, leading to the development of End Stage Renal Disease with high risk of death for cardiovascular events. Moreover, recent clinical studies have shown that renovascular structure and function may have a great impact on functional renal recovery after surgery. Here, we describe a protocol for the delivery of drugs into the renal artery of rats. This procedure offers significant advantages over the frequently used systemic administration as it may allow a more localized therapeutic effect. In addition, the use of rodents in pharmacodynamic analysis of preclinical studies may be cost effective, paving the way for the design of translational experiments in larger animal models. Using this technique, infusion of rat recombinant Vascular Endothelial Growth Factor (VEGF) protein in rats has induced activation of VEGF signaling as shown by increased expression of FLK1, pAKT/AKT, pERK/ERK. In summary, we established a protocol for the intrarenal delivery of drugs in rats, which is simple and highly reproducible.

The administration of drugs for recovery of kidney function requires control of the localization and distribution of the therapeutic compound. Here, we describe in detail a simple technique for intrarenal delivery of drugs in rats. This procedure may be easily performed with no mortality and high reproducibility.

The renal microvascular compartment plays an important role in the progression of kidney disease and hypertension, leading to the development of End Stage ...

A Rat Model of Orthotopic Liver Transplantation Using a Novel Magnetic Anastomosis Technique for Suprahepatic Vena Cava Reconstruction

The rat model of orthotopic liver transplantation (OLT) is essential for transplant research. It is a very sophisticated animal model and requires a steep learning curve. The introduction of the cuff technique for anastomosis of the portal vein (PV) and infrahepatic vena cava (IHVC) has significantly simplified the transplant procedure in rats. However, due to the short anterior wall of the recipients' suprahepatic vena cava (SHVC), the cuff technique is very difficult to use for the reconstruction of the SHVC. Most researchers in this field still use the hand-suture technique for SHVC reconstruction, which makes it the bottleneck step in rat orthotopic liver transplantation. The magnetic anastomosis technique (i.e., magnamosis) is a method of connecting two vessels using the attractive force between two magnets. Our recent study has shown that the magnetic anastomosis technique is superior to the hand-suture technique for SHVC reconstruction in rats. In this article, we show a step-by-step protocol for SHVC reconstruction in rats using the novel magnetic anastomosis technique. In this model, the reconstruction of the PV and IHVC was performed by the standard cuff technique, while the reconstruction of the bile duct (BD) was performed by a stent technique. The hepatic re-arterialization was not performed. The magnetic anastomosis technique made SHVC reconstruction much easier and significantly shortened the anphepatic phase. After a reasonable learning curve, even researchers without advanced microsurgical skills can produce reliable and reproducible results using this rat model of OLT.

The reconstruction of the suprahepatic vena cava (SHVC) remains a difficult step in rat orthotopic liver transplantation. In this article, we show a step-by-step protocol for SHVC reconstruction in rats using a novel magnetic anastomosis technique.

The rat model of orthotopic liver transplantation (OLT) is essential for transplant research. It is a very sophisticated animal model and requires a steep ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Generation of a Rat Model of Acute Liver Failure by Combining 70% Partial Hepatectomy and Acetaminophen

Acute liver failure (ALF) is a clinical condition caused by various etiologies resulting in the loss of metabolic, biochemical, synthesizing, and detoxifying functions of the liver. In most irreversible liver damage cases, orthotropic liver transplant (OLT) remains the only available treatment. To study the therapeutic potential of a treatment for ALF, its prior testing in an animal model of ALF is essential. In the current study, an ALF model in rats was developed by combining 70% partial hepatectomy (PHx) and injections of acetaminophen (APAP) that provides a therapeutic window of 48 h. The median and left lateral lobes of the liver were removed to excise 70% of the liver mass and APAP was given 24 h postsurgically for 2 days. Survival in ALF-induced animals was found to be severely decreased. The development of ALF was confirmed by altered serum levels of the enzymes alanine amino transferase (ALT), aspartate amino transferase (AST), alkaline phosphatase (ALP); changes in prothrombin time (PT); and assessment of the international normalized ratio (INR). Study of the gene expression profile by qPCR revealed an increase in expression levels of genes involved in apoptosis, inflammation, and in the progression of liver injury. Diffused degeneration of hepatocytes and infiltration of immune cells was observed by histological evaluation. The reversibility of ALF was confirmed by the restoration of survival and serum levels of ALT, AST, and ALP after intrasplenic transplantation of syngeneic healthy rat hepatocytes. This model presents a reliable alternative to the available ALF animal models to study the pathophysiology of ALF as well as to evaluate the potential of a novel therapy for ALF. The use of two different approaches also makes it possible to study the combined effect of physical and drug-induced liver injury. The reproducibility and feasibility of current procedure is an added benefit of the model.

The acute liver failure animal model developed in the current study presents a feasible alternative for the study of potential therapies. The current model employs the combined effect of physical and drug-induced hepatic injury and provides a suitable time window to study the potential of novel therapies.

Acute liver failure (ALF) is a clinical condition caused by various etiologies resulting in the loss of metabolic, biochemical, synthesizing, and ...

A Rat Carotid Artery Pressure-Controlled Segmental Balloon Injury with Periadventitial Therapeutic Application

Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the luminal surface area in arteries thereby reducing adequate blood flow to organs and distal tissues. Clinically, revascularization procedures such as balloon angioplasty with or without stent placement aim to restore blood flow. Although these procedures reestablish blood flow by reducing plaque burden, they damage the vessel wall, which initiates the arterial healing response. The prolonged healing response causes arterial restenosis, or re-narrowing, ultimately limiting the long-term success of these revascularization procedures. Therefore, preclinical animal models are integral for analyzing the pathophysiological mechanisms driving restenosis, and provide the opportunity to test novel therapeutic strategies. Murine models are cheaper and easier to operate on than large animal models. Balloon or wire injury are the two commonly accepted injury modalities used in murine models. Balloon injury models in particular mimic the clinical angioplasty procedure and cause adequate damage to the artery for the development of restenosis. Herein we describe the surgical details for performing and histologically analyzing the modified, pressure-controlled rat carotid artery balloon injury model. Additionally, this protocol highlights how local periadventitial application of therapeutics can be used to inhibit neointimal hyperplasia. Lastly, we present light sheet fluorescence microscopy as a novel approach for imaging and visualizing the arterial injury in three-dimensions.

The rat carotid artery balloon injury mimics the clinical angioplasty procedure performed to restore blood flow in atherosclerotic vessels. This model induces the arterial injury response by distending the arterial wall, and denuding the intimal layer of endothelial cells, ultimately causing remodeling and an intimal hyperplastic response.

Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the ...

Reduced Complications after Arterial Reconnection in a Rat Model of Orthotopic Liver Transplantation

The rat orthotopic liver transplantation (OLT) model is a powerful tool to study acute and chronic rejection. However, it is not a complete representation of human liver transplantation due to the absence of arterial reconnection. Described here is a modified transplantation procedure that includes the incorporation of hepatic artery (HA) reconnection, leading to a marked improvement in transplant outcomes. With a mean anhepatic time of 12 min and 14 s, HA reconnection results in improved perfusion of the transplanted liver and an increase in long-term recipient survival from 37.5% to 88.2%. This protocol includes the use of 3D-printed cuffs and holders to connect the portal vein and infrahepatic inferior vena cava. It can be implemented for studying multiple aspects of liver transplantation, from immune response and infection to technical aspects of the procedure. By incorporating a simple and practical method for arterial reconnection using a microvascular technique, this modified rat OLT protocol closely mimics aspects of human liver transplantation and will serve as a valuable and clinically relevant research model.

The goal of this study is to modify the rat orthotopic liver transplant model to better represent human liver transplantation and improve recipient survival. The presented method reestablishes hepatic arterial inflow by connecting the donor liver&#39;s common hepatic artery to the recipient liver&#39;s proper hepatic artery.

The rat orthotopic liver transplantation (OLT) model is a powerful tool to study acute and chronic rejection. However, it is not a complete representation ...

Method of Direct Segmental Intra-hepatic Delivery Using a Rat Liver Hilar Clamp Model

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