Name: method of direct segmental intra-hepatic delivery using a rat liver hilar clamp model
Uploaded: 2017-04-02T13:00:00.000+00:00
Duration: 9 min 22 s
Description: Watch this Scientific Journal Video about Method of Direct Segmental Intra-hepatic Delivery Using a Rat Liver Hilar Clamp Model at JoVE.com

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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Superoxide Dismutase (PEG-SOD)</td><td>Sigma Aldrich&amp;nbsp;</td><td>S9549 SIGMA</td><td></td></tr><tr><td>GenieTouch</td><td>Kent Scientific</td><td></td><td></td></tr><tr><td>Normal Saline</td><td>Baxter</td><td>NDC 0338-0048-04</td><td></td></tr><tr><td>4 x 4 Non-Woven Sponges&amp;nbsp;</td><td>Criterion</td><td>104-2411</td><td></td></tr><tr><td>Sterile Q-Tips</td><td>Henry Schein Animal Health</td><td>1009175</td><td></td></tr><tr><td>U-100 27 Gauge Insulin Syringe</td><td>Terumo</td><td>22-272328</td><td></td></tr><tr><td>5 mL Syringe</td><td>BD&amp;nbsp;</td><td>REF 309603</td><td></td></tr><tr><td>4-0 Braided Silk Suture</td><td>Deknatel, Inc.</td><td>198737LP</td><td></td></tr><tr><td>7-0 Braided Silk Suture</td><td>Teleflex Medical&amp;nbsp;</td><td>REF 103-S</td><td></td></tr><tr><td>1.8 mL Arcticle Cryogenic Tube</td><td>USA Scientific&amp;nbsp;</td><td>1418-7410</td><td></td></tr><tr><td>&lt;strong&gt;Microsurgical Instruments&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&amp;nbsp;&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>Small Scissors&amp;nbsp;</td><td>Roboz&amp;nbsp;</td><td>RS-5610</td><td></td></tr><tr><td>Large Scissors</td><td>S&amp;amp;T&amp;nbsp;</td><td>SAA-15</td><td></td></tr><tr><td>Forceps - Large Angled</td><td>S&amp;amp;T&amp;nbsp;</td><td>JFCL-7</td><td></td></tr><tr><td>Forceps - Small Angled&amp;nbsp;</td><td>S&amp;amp;T</td><td>FRAS-15 RM-8</td><td></td></tr><tr><td>Clip Applier</td><td>ROBOZ</td><td>RS-5440</td><td></td></tr><tr><td>Scissors - non micro</td><td>FST 14958-11</td><td>14958-11</td><td></td></tr><tr><td>Forceps - Straight Tip&amp;nbsp;</td><td>S&amp;amp;T&amp;nbsp;</td><td>FRS-15 RM8TC</td><td></td></tr><tr><td>Large Microsurgical Clip</td><td>Fine Scientific Tools</td><td>18055-01</td><td></td></tr><tr><td>Small Microsurgical Clip</td><td>Fine Scientific Tools</td><td>18055-01</td><td></td></tr><tr><td>Small Microsurgical Clip</td><td>Fine Scientific Tools</td><td>18055-02</td><td></td></tr><tr><td>Small Microsurgical Clip</td><td>Fine Scientific Tools</td><td>18055-03</td><td></td></tr><tr><td>&lt;strong&gt;Other Instruments&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&amp;nbsp;&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>Small Mosquito Clamps</td><td>Generic&amp;nbsp;</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Analysis&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&amp;nbsp;&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>Alannine aminotransferase (ALT) assay&amp;nbsp;</td><td>Biovision</td><td>K752-100</td><td></td></tr><tr><td>Malondialdehye (MDA) assay&amp;nbsp;</td><td>Abcam&amp;nbsp;</td><td>ab118970</td><td></td></tr><tr><td>Glutathione (GSH) assay</td><td>Cayman Chemical</td><td>7030002</td><td></td></tr><tr><td>Antibodies - Cleaved Caspase-3 and Actin&amp;nbsp;</td><td>Cell Signaling Tecnology</td><td>Antibody 9661</td><td></td></tr><tr><td>ImageJ Software&amp;nbsp;</td><td>National Institutes of Health&amp;nbsp;</td><td></td><td></td></tr><tr><td>RIPA Lysis and Extraction Buffer&amp;nbsp;</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="https://cloudflare-jove-com.bdigitaluss.remotexs.co/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="https://cloudflare-jove-com.bdigitaluss.remotexs.co/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="https://cloudflare-jove-com.bdigitaluss.remotexs.co/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="https://cloudflare-jove-com.bdigitaluss.remotexs.co/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="https://cloudflare-jove-com.bdigitaluss.remotexs.co/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="https://cloudflare-jove-com.bdigitaluss.remotexs.co/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="https://cloudflare-jove-com.bdigitaluss.remotexs.co/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="https://cloudflare-jove-com.bdigitaluss.remotexs.co/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="https://cloudflare-jove-com.bdigitaluss.remotexs.co/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

Universidad San Sebastian

Research

JoVE Journal

Medicine

14.0K Views.  The Ohio State University Wexner Medical Center. 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.

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

Manufacturing Devices and Instruments for Easier Rat Liver Transplantation

Orthotopic rat liver transplantation is a popular model, which has been shown in a recent JoVE paper with the use of the "quick-linker" device. This technique allows for easier venous cuff-anatomoses after a reasonable learning curve. The device is composed of two handles, which are carved out from scalpel blades, one approximator, which is obtained by modifying Kocher's forceps, and cuffs designed from fine-bore polyethylene tubing. The whole process can be performed at a low-cost using common laboratory material. The present report provides a step-by-step protocol for the design of the required pieces and includes stencils.

We describe the design of the &#x201C;quick-linker&#x201D; device for easier orthotopic rat liver transplantation.

Orthotopic rat liver transplantation is a popular model, which has been shown in a recent JoVE paper with the use of the "quick-linker" device. This ...

Bioengineering

Orthotopic Liver Transplantation in Rats

Clinical progress in the field of liver transplantation has been largely supported by animal models1,2. Since the publication of the first orthotopic rat liver transplantation in 1979 by Kamada et al.3, this model has remained the gold standard despite various proposed alternative techniques4. Nevertheless, its broader use is limited by its steep learning curve5.
In this video paper, we show a simple and easy-to-establish revision of Kamada's two-cuff technique. The suprahepatic vena cava anastomosis is performed manually with a running suture, and the vena porta and infrahepatic vena cava anastomoses are performed utilizing a quick-linker cuff system6. Manufacturing the quick-linker kit is shown in a separate video paper.

We present an easy-to-establish revision of the classical two-cuff technique for orthotopic liver transplantation in rat.

Clinical progress in the field of liver transplantation has been largely supported by animal models1,2. Since the publication of the first orthotopic rat ...

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 ...

Transarterial Administration of Oncolytic Viruses for Locoregional Therapy of Orthotopic HCC in Rats

Hepatocellular carcinoma (HCC) is a disease with limited treatment options and poor prognosis. In recent years, oncolytic virotherapies have proven themselves to be potentially powerful tools to fight malignancy. Due to the unique dual blood supply in the liver, it is possible to apply therapies locally to orthotopic liver tumors, which are predominantly fed by arterial blood flow. We have previously demonstrated that hepatic arterial delivery of oncolytic viruses results in safe and efficient transduction efficiency of multifocal HCC lesions, resulting in significant prolongation of survival in immune competent rats. This procedure closely mimics the application of transarterial embolization in patients, which is the standard palliative care provided to many HCC patients. The ability to administer tumor therapies through the hepatic artery in rats allows for a highly sophisticated preclinical model for evaluating novel viral vectors under development. Here we describe the detailed protocol for microdissection of the hepatic artery for infusion of oncolytic virus vectors to treat orthotopic HCC.

Oncolytic virotherapies are under development as novel therapeutics for the treatment of hepatocellular carcinoma (HCC). Here we describe a method for locoregional therapy of HCC via hepatic arterial administration of oncolytic virus.

Hepatocellular carcinoma (HCC) is a disease with limited treatment options and poor prognosis.  In recent years, oncolytic virotherapies have proven ...

Rat Model of the Associating Liver Partition and Portal Vein Ligation for Staged Hepatectomy (ALPPS) Procedure

Recent clinical data support an aggressive surgical approach to both primary and metastatic liver tumors. For some indications, like colorectal liver metastases, the amount of liver tissue left behind after liver resection has become the main limiting factor of resectability of large or multiple liver tumors. A minimal amount of functional tissue is required to avoid the severe complication of post-hepatectomy liver failure, which has high morbidity and mortality. Inducing liver growth of the prospective remnant prior to resection has become more established in liver surgery, either in the form of portal vein embolization by interventional radiologists or in the form of portal vein ligation several weeks prior to resection. Recently, it was shown that liver regeneration is more extensive and rapid, when the parenchymal transection is added to portal vein ligation in a first stage and then, after only one week of waiting, resection performed in a second stage (Associating Liver Partition and Portal vein ligation for Staged hepatectomy = ALPPS). ALPPS has rapidly become popular across the world, but has been criticized for its high perioperative mortality. The mechanism of accelerated and extensive growth induced by this procedure has not been well understood. Animal models have been developed to explore both the physiological and molecular mechanisms of accelerated liver regeneration in ALPPS. This protocol presents a rat model that allows mechanistic exploration of accelerated regeneration.

Rat Model of the Associating Liver Partition and Portal Vein Ligation for Staged Hepatectomy ALPPS Procedure

Inducing rapid liver hypertrophy using Associating Liver Partition and Portal vein ligation for a Staged hepatectomy (ALPPS) has been proposed for resection of borderline resectable liver tumors. This model may elucidate mechanisms involved in rapid hypertrophy and allows testing of drugs that promote or block the acceleration of regeneration.

Recent clinical data support an aggressive surgical approach to both primary and metastatic liver tumors. For some indications, like colorectal liver ...

Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy

Split liver transplantation and living liver donor liver transplantation were developed in the clinic to utilize liver organs in a more efficient manner. To better understand the mechanism behind these surgical procedures, a rat partial liver transplantation (PLTx) model was established for relevant surgical studies. Because of the complexity of the rat PLTx model, a protocol with detailed descriptions is required. An article published previously reported a protocol in which ex vivo hepatectomy was used to achieve 50% rat PLTx. In contrast to this protocol, we introduced a re-arterialized PLTx with an in vivo 70% hepatectomy. An updated vessel-oriented hepatectomy was incorporated into the rat PLTx to refine the microsurgical procedure. The portal veins and hepatic arteries of the left lateral lobe and the median lobe were individually dissected and ligated before removal of the liver parenchyma, thereby decreasing the probability of bleeding in the remnant liver stump. Furthermore, an end-to-side vessel anastomosis between the common hepatic artery and the enlarged proper hepatic artery was introduced to re-arterialize the hepatic artery. By using this end-to-side vessel anastomosis technique, the diameter of the anastomosis was enlarged, thereby decreasing the difficulty of hand suture and maintaining a high rate of anastomotic patency. Moreover, the cuff anastomosis of the infrahepatic vena cava was slightly modified. A section of circumferential liver parenchyma around the vena cava of a recipient was preserved during cuff anastomosis to maintain the three-dimensional shape of the vascular lumen. This section of liver parenchyma was removed after completing the anastomosis. With this modification, the step involving placement of stay sutures was omitted, thereby further shortening the cuff anastomosis time. By using this protocol of rat PLTx, a low liver enzyme level, an intact liver lobular architecture and a high survival rate were achieved after microsurgery.

Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy

Here, a protocol involving re-arterialized rat partial liver transplantation is presented. Specifically, 70% liver was resected in vivo by using an updated technique of vessel-oriented hepatectomy. The hepatic artery was reconstructed in an end-to-side manner. The cuff technique was modified to shorten the anastomosis time of the infrahepatic vena cava.

Split liver transplantation and living liver donor liver transplantation were developed in the clinic to utilize liver organs in a more efficient manner. ...

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 ...

A Small Animal Model of Ex Vivo Normothermic Liver Perfusion

There is a significant shortage of liver allografts available for transplantation, and in response the donor criteria have been expanded. As a result, normothermic ex vivo liver perfusion (NEVLP) has been introduced as a method to evaluate and modify organ function. NEVLP has many advantages in comparison to hypothermic and subnormothermic perfusion including reduced preservation injury, restoration of normal organ function under physiologic conditions, assessment of organ performance, and as a platform for organ repair, remodeling, and modification. Both murine and porcine NEVLP models have been described. We demonstrate a rat model of NEVLP and use this model to show one of its important applications &#8212; the use of a therapeutic molecule added to liver perfusate. Catalase is an endogenous reactive oxygen species (ROS) scavenger and has been demonstrated to decrease ischemia-reperfusion in the eye, brain, and lung. Pegylation has been shown to target catalase to the endothelium. Here, we added pegylated-catalase (PEG-CAT) to the base perfusate and demonstrated its ability to mitigate liver preservation injury. An advantage of our rodent NEVLP model is that it is inexpensive in comparison to larger animal models. A limitation of this study is that it does not currently include post-perfusion liver transplantation. Therefore, prediction of the function of the organ post-transplantation cannot be made with certainty. However, the rat liver transplant model is well established and certainly could be used in conjunction with this model. In conclusion, we have demonstrated an inexpensive, simple, easily replicable NEVLP model using rats. Applications of this model can include testing novel perfusates and perfusate additives, testing software designed for organ evaluation, and experiments designed to repair organs.

A Small Animal Model of Ex Vivo Normothermic Liver Perfusion

There is a significant liver donor shortage, and criteria for liver donors have been expanded. Normothermic ex vivo liver perfusion (NEVLP) has been developed to evaluate and modify organ function. This study demonstrates a rat model of NEVLP and tests the ability of pegylated-catalase, to mitigate liver preservation injury.

There is a significant shortage of liver allografts available for transplantation, and in response the donor criteria have been expanded. As a result, ...

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 ...

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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Manufacturing Devices and Instruments for Easier Rat Liver Transplantation

Orthotopic Liver Transplantation in Rats

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Transarterial Administration of Oncolytic Viruses for Locoregional Therapy of Orthotopic HCC in Rats

Rat Model of the Associating Liver Partition and Portal Vein Ligation for Staged Hepatectomy (ALPPS) Procedure

Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy

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

A Small Animal Model of Ex Vivo Normothermic Liver Perfusion

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

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