The present studies were supported by United States National Institutes of Health grant R01-HL092188, R01-DK083385 and R01HL098294, and Foundation for Anesthesia Education and Research (FAER) Grants to HKE and a DFG (Deutsche Forschungsgemeinschaft) Research Fellowship (GR2121/1-1) and American Heart Association Grant to AG.

General remarks. All operations should be performed under an upright dissecting microscope (Leica) and by using a surgical coagulator. The mice in the experimental and sham groups should be matched in age and weight to ensure comparability of the results. Temperature, blood pressure, anesthesia and fluid administration should be stable and monitored during the entire experiment.
1. Anesthesia and Surgery
<ol>
 <li>C57BL/6 mice should be within an age range of 10 to 16 weeks and they should be gender-matched in the respective groups.</li>
 <li>Anesthetize mice with sodium pentobarbital (70 mg/kg body weight i.p.) and maintain anesthesia with approx. 10 mg/kg/h sodium pentobarbital. Overdosing can significantly lower blood pressure and thus can alter the results.</li>
 <li>Place mice on a temperature-controlled heated table (RT, Effenberg, Munich, Germany) with a rectal thermometer probe attached to a thermal feedback controller to maintain body temperature at 37 &deg;C.</li>
 <li>Place mice in a supine position on the surgery table, with the upper and lower extremities attached to the table with removable tape.</li>
 <li>In case of survival experiments proper aseptic techniques including clipping of hair, disinfection of skin with iodophors (e.g. Betadine), sterile handling of autoclaved instruments, and using masks a sterile gloves for the surgeon are required.</li>
</ol>
2. Portal Triad Occlusion
<ol>
 <li>Cover the incision area with mineral oil to prevent inhalation of mouse hair by cutting the skin and thus preventing the risk of mouse hair allergies. Perform midline laparotomy and incision of the linea alba up to the upper end so that you can easily expose the liver. If possible perform the incision with a coagulation electrode (Erbe, ICC50, Tuebingen, Germany) to prevent bleeding.</li>
 <li>Place the heating table with the mouse in a position that the tail is directed to the surgeon. That position provides you with the best setting to localize the triad and to place the suture.</li>
 <li>Displace the intestine on the right side by using a wet cotton tip swab to expose the portal triad.</li>
 <li>Slightly shift the median and left lobe towards the diaphragm and the right lobe towards the intestine. That will give you a clear view of the portal triad above the bifurcation of right, median/left lobes.</li>
 <li>Carefully inspect the portal triad and hold the right lobe gently down with bland forceps. That will give you a nice view on the left portal triad.</li>
 <li>Once visually identified place the needle with the suture (7/0 nylon suture; Ethicon, Norderstedt, Germany) via a needle holder under the left portal triad, including the hepatic artery, hepatic vein, and bile duct from the left to the right side of the mouse.</li>
 <li>The stitch has to be closely under the triad. A deeper stitch can injure the vena vaca inferior which is located under the triad. Therefore hold the right liver lobes down with the forceps to identify the vena cava before placing the stitch.</li>
 <li>Take the tip of the needle on the right side with a forceps and pull it gently out of the liver area without injuring the liver.</li>
 <li>Attach to each end of the suture an Eppendorf tube (approximately 3 g, filled with water) and place both suture ends over suture holders. Place the suture holder distally of the stitch so that the suture does not interfere with the right triad.</li>
 <li>By applying the weights the triad will be immediately occluded, causing interruption of blood supply to the left and median lobes of the liver. Successful occlusion can be easily confirmed by visual inspection of pale blanching of the ischemic lobes. In contrast, the change of color immediately disappears when the hanging weights are released from the poles and the liver is reperfused. This partial hepatic ischemia model avoids mesenteric congestion by preserving blood flow to the right liver lobes.</li>
 <li>Replace the intestine into the abdominal cavity. Keep the liver and intestine warm with a wet swab soaked with water at 37.0 &deg;C. In addition to keep body temperature stable cover the mice with commercially available food wrap after finishing surgery.</li>
 <li>Perform ischemia as required per experimental protocol. After a defined ischemia time release the weights and reperfusion of the median and left liver lobes will start immediately. This technique allows producing reversible ischemia in mice. If no further ischemia times will be performed carefully remove the suture.</li>
 <li>Add approximately 400 &mu;l sodium chloride into the abdominal cavity 15 minutes before and directly after reperfusion. Continue to substitute fluid every 30 minutes in an amount of 100 &mu;l to compensate fluid loss during the reperfusion time.</li>
 <li>For survival experiments the surgical wound is closed using continuous suture of the muscle wall and skin with a 4/0 suture.</li>
</ol>
3. Changes in Liver Perfusion
The following outcome parameters are recommended to determine liver injury following hepatic ischemia:
Determine liver enzymes (e.g. ALT, AST) and liver histology (H&amp;E staining) following liver ischemia and reperfusion3,9.
<img alt="Figure 1" src="/files/ftp_upload/2550/2550fig1.jpg" /> 
Figure 1. Model of a hanging-weight system for liver ischemia: A hanging weight system has been established to induce liver ischemia of the left and median lobe3. (A) Therefore the caudate and right lobe were gently separated from the left lobe. The right lobe was then slightly shifted to clearly view the portal triad above the bifurcation of right, median and left lobes. An 8.0 nylon suture was placed under the left hepatic triad. (B) At the end of each suture a small weight (3 g Eppendorf tubes filled with water) was attached and applied. Hepatic ischemia is visible by a color change of the left and median lobe from red to pale. This experimental design allows precise occlusion and reperfusion of the left hepatic triad by applying and releasing the weight load.
<img alt="Figure 2" src="/files/ftp_upload/2550/2550fig2.jpg" /> 
Figure 2. Effect of different liver ischemia times on alanine (ALT) and aspartate (AST) aminotransferase in mice. Partial portal triad ischemia was induced as indicated (0 - 60 min ischemia). After 2 hr of reperfusion, ALT (A) and AST (B) were measured. Results are expressed as means &plusmn; SE of 6 - 7 mice/group. *P &lt; 0.05 compared with 0 min ischemia (sham control).

<ol>
	<li>Rook, M., Rand, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predictors+of+long-term+outcome+after+liver+transplant.">Predictors of long-term outcome after liver transplant.</a> Curr. Opin. Organ. Transplant. 16, 499-504 (2011).</li><li>Henrion, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ischemia/reperfusion+injury+of+the+liver:+pathophysiologic+hypotheses+and+potential+relevance+to+human+hypoxic+hepatitis.">Ischemia/reperfusion injury of the liver: pathophysiologic hypotheses and potential relevance to human hypoxic hepatitis.</a> Acta. Gastroenterol. Belg. 63, 336-347 (2000).</li><li>Hart, M. L., Much, C., Kohler, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+hanging-weight+system+for+liver+ischemic+preconditioning+in+mice.">Use of a hanging-weight system for liver ischemic preconditioning in mice.</a> Am. J. Physiol. Gastrointest. Liver Physiol. 294, 1431-1440 (2008).</li><li>Schneider, M., Geyte, K., Fraisl, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+or+silencing+of+the+PHD1+prolyl+hydroxylase+protects+livers+of+mice+against+ischemia/reperfusion+injury.">Loss or silencing of the PHD1 prolyl hydroxylase protects livers of mice against ischemia/reperfusion injury.</a> Gastroenterology. 138, 1143-1154 (2010).</li><li>Coriat, R., Leconte, M., Kavian, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mangafodipir+Protects+against+Hepatic+Ischemia-Reperfusion+Injury+in+Mice.">Mangafodipir Protects against Hepatic Ischemia-Reperfusion Injury in Mice.</a> PLoS One. 6, e27005 (2011).</li><li>Sakai, N., Van Sweringen, H. L., Schuster, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receptor+activator+of+NF-kappaB+ligand+(RANKL)+protects+against+hepatic+ischemia/reperfusion+injury+in+mice.">Receptor activator of NF-kappaB ligand (RANKL) protects against hepatic ischemia/reperfusion injury in mice.</a> Hepatology. , (2011).</li><li>Zhang, J., Hu, W., Xing, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Protective+Role+of+CD59+and+Pathogenic+Role+of+Complement+in+Hepatic+Ischemia+and+Reperfusion+Injury.">The Protective Role of CD59 and Pathogenic Role of Complement in Hepatic Ischemia and Reperfusion Injury.</a> Am. J. Pathol. 179, 2876-2884 (2011).</li><li>Beldi, G., Banz, Y., Kroemer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deletion+of+CD39+on+natural+killer+cells+attenuates+hepatic+ischemia/reperfusion+injury+in+mice.">Deletion of CD39 on natural killer cells attenuates hepatic ischemia/reperfusion injury in mice.</a> Hepatology. 51, 1702-1711 (2010).</li><li>Hart, M. L., Much, C., Gorzolla, I. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+adenosine+production+by+ecto-5'-nucleotidase+protects+during+murine+hepatic+ischemic+preconditioning.">Extracellular adenosine production by ecto-5'-nucleotidase protects during murine hepatic ischemic preconditioning.</a> Gastroenterology. 135, 1739-1750 (2008).</li><li>Beraza, N., Ludde, T., Assmus, U., Roskams, T., Vander Borght, S., Trautwein, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatocyte-specific+IKK+gamma/NEMO+expression+determines+the+degree+of+liver+injury.">Hepatocyte-specific IKK gamma/NEMO expression determines the degree of liver injury.</a> Gastroenterology. 132, 2504-2517 (2007).</li><li>Lentsch, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+and+function+of+hepatocyte+NF-kappaB+in+postischemic+liver+injury.">Activation and function of hepatocyte NF-kappaB in postischemic liver injury.</a> Hepatology. 42, 216-218 (2005).</li><li>Luedde, T., Assmus, U., Wustefeld, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deletion+of+IKK2+in+hepatocytes+does+not+sensitize+these+cells+to+TNF-induced+apoptosis+but+protects+from+ischemia/reperfusion+injury.">Deletion of IKK2 in hepatocytes does not sensitize these cells to TNF-induced apoptosis but protects from ischemia/reperfusion injury.</a> J. Clin. Invest. 115, 849-859 (2005).</li><li>Kapitsinou, P. P., Liu, Q., Unger, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatic+HIF-2+regulates+erythropoietic+responses+to+hypoxia+in+renal+anemia.">Hepatic HIF-2 regulates erythropoietic responses to hypoxia in renal anemia.</a> Blood. , (2010).</li></ol>

No conflicts of interest declared.

The present study describes a technique of performing ischemia in a murine model by using a hanging-weight system for occlusion of the left hepatic portal triad. Although it is an easy method to learn the following pitfalls should be taken into account. A very common mistake in the beginning is a deep stitch which includes the portal triad and the vena cava. The breathing and heart rate will slow down within 15 minutes due to the increased resistance and the decrease in returning blood to the heart resulting in an early death of the mice. The best way to prevent the including of the vena cava is to localize the vena cava before the stitch by holding the right lobes down with a forceps. Another important issue is that the weights are not to heavy which can result in too much stress on the left and right portal trial which also stops blood flow to the right lobes. Therefore the amount of water in the Eppendorf vials should be adjusted to the mouse weight and the suture holders should be placed distally from the stitch location. On the opposite if the weights are to low blood pressure is not completely stopped. Both mistakes are easily detectable by an additional color change to pale of the right lobe or insufficient color change of the median and left lobes, respectively. Therefore the hepatic color change following applying the weights should be controlled every 10 minutes. Another common surgical complication is the injuring of the liver with the needle. Therefore the right lobes should be held down with a bland forceps so that the tip of the needle can be easily seen and taken with a needle holder. If these pitfalls in the beginning are taken into account the hanging-weight system for hepatic ischemia provides a highly reproducible injury due to ischemia by minimizing the variability of liver damage associated with clamping of the portal triad. By using a hanging-weight system, the portal triad is only manipulated once throughout the entire surgical procedure, causing significantly less damage to the hepatic lobes. In addition, no hepatic or intestinal congestion occurs with this technique. More tissue trauma occurs by removing and replacing vessel clamps, especially with reapplication of the clamp during multiple ischemia cycles like during ischemic preconditioning. The use of hanging weights that are in a remote location from the liver tissue, according to our observations, provides the advantage of reliable occlusion while preventing tissue trauma due to manipulation of the liver lobes by reapplication of a clamp. Taken together, the present study provides feasibility of the hanging-weight system for portal triad occlusion during ischemia, minimizing the variability and limitations associated with clamping. Thus this technique might be of interest for other investigators who consider studying hepatic ischemia.

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>Sodium Pentobarbital (Fatal Plus)</td>
 <td>Vortech Pharmaceutical Ls, Ltd </td>
 <td>V.P.L. 9372 </td>
 <td>4 mg/mL in saline</td>
 </tr>
 <tr>
 <td>Suture, Prolene 8.0</td>
 <td>Ethicon, USA </td>
 <td>M8739 </td>
 <td>reusable</td>
 </tr>
 <tr>
 <td>dissecting microscope </td>
 <td>Leica</td>
 <td>n/a</td>
 <td>consider generous working distance</td>
 </tr>
 <tr>
 <td>Heating Table</td>
 <td>Rt, Effenberger, Germany</td>
 <td>n/a</td>
 <td>only and single provider</td>
 </tr>
 <tr>
 <td>Surgical instruments</td>
 <td>WPI, Dumont</td>
 <td>&nbsp;</td>
 <td>Forceps, needle holder</td>
 </tr>
 <tr>
 <td>Coagulator</td>
 <td>Erbe, ICC50, Tuebingen, Germany</td>
 <td>n/a</td>
 <td>Different providers of coagulators</td>
 </tr>
 </tbody>
</table>
Table 1. Specific reagents and equipment.

use of a hanging-weight system for liver ischemia in mice

Acute liver injury due to ischemia can occur during several clinical procedures e.g. liver transplantation, hepatic tumor resection or trauma repair and can result in liver failure which has a high mortality rate1-2. Therefore murine studies of hepatic ischemia have become an important field of research by providing the opportunity to utilize pharmacological and genetic studies3-9. Specifically, conditional mice with tissue specific deletion of a gene (cre, flox system) provide insights into the role of proteins in particular tissues10-13 . Because of the technical difficulty associated with manually clamping the portal triad in mice, we performed a systematic evaluation using a hanging-weight system for portal triad occlusion which has been previously described3. By using a hanging-weight system we place a suture around the left branch of the portal triad without causing any damage to the hepatic lobes, since also the finest clamps available can cause hepatic tissue damage because of the close location of liver tissue to the vessels. Furthermore, the right branch of the hepatic triad is still perfused thus no intestinal congestion occurs with this technique as blood flow to the right hepatic lobes is preserved. Furthermore, the portal triad is only manipulated once throughout the entire surgical procedure. As a result, procedures like pre-conditioning, with short times of ischemia and reperfusion, can be easily performed. Systematic evaluation of this model by performing different ischemia and reperfusion times revealed a close correlation of hepatic ischemia time with liver damage as measured by alanine (ALT) and aspartate (AST) aminotransferase serum levels3,9. Taken together, these studies confirm highly reproducible liver injury when using the hanging-weight system for hepatic ischemia and intermittent reperfusion. Thus, this technique might be useful for other investigators interested in liver ischemia studies in mice. Therefore the video clip provides a detailed step-by-step description of this technique.

Watch this Scientific Journal Video about Use of a Hanging-weight System for Liver Ischemia in Mice at JoVE.com

Use of a Hanging-weight System for Liver Ischemia in Mice

We established a novel murine model of a hanging weight system for portal triad occlusion. This technique may be useful for future investigations of ischemia in murine hepatic models.

Acute liver injury due to ischemia can occur during several clinical procedures e.g. liver transplantation, hepatic tumor resection or trauma repair and ...

use-of-a-hanging-weight-system-for-liver-ischemia-in-mice

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15.7K vues.  University of Colorado, Denver. Nous avons établi un modèle murin roman d'un système de poids suspendu pour le portail triade d'occlusion. Cette technique peut être utile pour des enquêtes futures de l'ischémie dans les modèles murins hépatiques.

Vidéo: Use of a Hanging-weight System for Liver Ischemia in Mice

Use of a Hanging Weight System for Coronary Artery Occlusion in Mice

Murine studies of acute injury are an area of intense investigation, as knockout mice for different genes are becoming increasingly available 1-38. Cardioprotection by ischemic preconditioning (IP) remains an area of intense investigation. To further elucidate its molecular basis, the use of knockout mouse studies is particularly important 7, 14, 30, 39. Despite the fact that previous studies have already successfully performed cardiac ischemia and reperfusion in mice, this model is technically very challenging. Particularly, visual identification of the coronary artery, placement of the suture around the vessel and coronary occlusion by tying off the vessel with a supported knot is technically difficult. In addition, re-opening the knot for intermittent reperfusion of the coronary artery during IP without causing surgical trauma adds additional challenge. Moreover, if the knot is not tied down strong enough, inadvertent reperfusion due to imperfect occlusion of the coronary may affect the results. In fact, this can easily occur due to the movement of the beating heart.
Based on potential problems associated with using a knotted coronary occlusion system, we adopted a previously published model of chronic cardiomyopathy based on a hanging weight system for intermittent coronary artery occlusion during IP 39. In fact, coronary artery occlusion can thus be achieved without having to occlude the coronary by a knot. Moreover, reperfusion of the vessel can be easily achieved by supporting the hanging weights which are in a remote localization from cardiac tissues.
We tested this system systematically, including variation of ischemia and reperfusion times, preconditioning regiments, body temperature and genetic backgrounds39. In addition to infarct staining, we tested cardiac troponin I (cTnI)
as a marker of myocardial infarction in this model. In fact, plasma levels of cTnI correlated with infarct sizes (R2=0.8). Finally, we could show in several studies that this technique yields highly reproducible infarct sizes during murine IP and myocardial infarction6, 8, 30, 40, 41. Therefore, this technique may be helpful for researchers who pursue molecular mechanisms involved in cardioprotection by IP using a genetic approach in mice with targeted gene deletion. Further studies on cardiac IP using transgenic mice may consider this technique.

A murine model for myocardial ischemia and ischemic preconditioning is an important tool study cardioprotective mechanisms in vivo. Here, we report an easy applicable in situ model for cardiac IP using a hanging-weight system for coronary artery occlusion.

Murine studies of acute injury are an area of intense investigation, as knockout mice for different genes are becoming increasingly available 1-38. ...

Use of a Hanging-weight System for Isolated Renal Artery Occlusion

In hospitalized patients, over 50% of cases of acute kidney injury (AKI) are caused by renal ischemia 1-3. A recent study of hospitalized patients revealed that only a mild increase in serum creatinine levels (0.3 to 0.4 mg/dl) is associated with a 70% greater risk of death than in persons without any increase 1. Along these lines, surgical procedures requiring cross-clamping of the aorta and renal vessels are associated with a renal failure rates of up to 30% 4. Similarly, AKI after cardiac surgery occurs in over 10% of patients under normal circumstances and is associated with dramatic increases in mortality. AKI are also common complications after liver transplantation. At least 8-17% of patients end up requiring renal replacement therapy 5. Moreover, delayed graft function due to tubule cell injury during kidney transplantation is frequently related to ischemia-associated AKI 6. Moreover, AKI occurs in approximately 20% of patients suffering from sepsis 6.The occurrence of AKI is associated with dramatic increases of morbidity and mortality 1. Therapeutic approaches are very limited and the majority of interventional trials in AKI have failed in humans. Therefore, additional therapeutic modalities to prevent renal injury from ischemia are urgently needed 3, 7-9. 
To elucidate mechanisms of renal injury due to ischemia and possible therapeutic strategies murine models are intensively required 7-13. Mouse models provide the possibility of utilizing different genetic models including gene-targeted mice and tissue specific gene-targeted mice (cre-flox system). However, murine renal ischemia is technically challenging and experimental details significantly influence results. We performed a systematic evaluation of a novel model for isolated renal artery occlusion in mice, which specifically avoids the use of clamping or suturing the renal pedicle 14. This model requires a nephrectomy of the right kidney since ischemia can be only performed in one kidney due to the experimental setting. In fact, by using a hanging-weight system, the renal artery is only instrumented once throughout the surgical procedure. In addition, no venous or urethral obstruction occurs with this technique. We could demonstrate time-dose-dependent and highly reproducible renal injury with ischemia by measuring serum creatinine. Moreover, when comparing this new model with conventional clamping of the whole pedicle, renal protection by ischemic preconditioning is more profound and more reliable. Therefore his new technique might be useful for other researchers who are working in the field of acute kidney injury.

A precise murine model for acute kidney injury (AKI) due to ischemia is an important tool to investigate acute kidney injury and possibly find therapeutic tools to treat renal injury. The hanging weight system offers a tool for immediate and reliable renal artery occlusion and reperfusion without causing renal congestion.

In hospitalized patients, over 50% of cases of acute kidney injury (AKI) are caused by renal ischemia 1-3. A recent study of hospitalized patients ...

Use of Animal Model of Sepsis to Evaluate Novel Herbal Therapies

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several techniques, including infusion of exogenous bacterial toxin (endotoxemia) or bacteria (bacteremia), as well as surgical perforation of the cecum by cecal ligation and puncture (CLP)1-3. CLP allows bacteria spillage and fecal contamination of the peritoneal cavity, mimicking the human clinical disease of perforated appendicitis or diverticulitis. The severity of sepsis, as reflected by the eventual mortality rates, can be controlled surgically by varying the size of the needle used for cecal puncture2. In animals, CLP induces similar, biphasic hemodynamic cardiovascular, metabolic, and immunological responses as observed during the clinical course of human sepsis3. Thus, the CLP model is considered as one of the most clinically relevant models for experimental sepsis1-3.
Various animal models have been used to elucidate the intricate mechanisms underlying the pathogenesis of experimental sepsis. The lethal consequence of sepsis is attributable partly to an excessive accumulation of early cytokines (such as TNF, IL-1 and IFN-&gamma;)4-6 and late proinflammatory mediators (e.g., HMGB1)7. Compared with early proinflammatory cytokines, late-acting mediators have a wider therapeutic window for clinical applications. For instance, delayed administration of HMGB1-neutralizing antibodies beginning 24 hours after CLP, still rescued mice from lethality8,9, establishing HMGB1 as a late mediator of lethal sepsis. The discovery of HMGB1 as a late-acting mediator has initiated a new field of investigation for the development of sepsis therapies using Traditional Chinese Herbal Medicine. In this paper, we describe a procedure of CLP-induced sepsis, and its usage in screening herbal medicine for HMGB1-targeting therapies.

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection, and can be simulated by a surgical technique termed cecal ligation and puncture (CLP). Here we describe a method to use CLP-induced animal model to screen medicinal herbs for therapeutic agents.

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

Photothrombosis-induced Focal Ischemia as a Model of Spinal Cord Injury in Mice

Spinal cord injury (SCI) is a devastating clinical condition causing permanent changes in sensorimotor and autonomic functions of the spinal cord (SC) below the site of injury. The secondary ischemia that develops following the initial mechanical insult is a serious complication of the SCI and severely impairs the function and viability of surviving neuronal and non-neuronal cells in the SC. In addition, ischemia is also responsible for the growth of lesion during chronic phase of injury and interferes with the cellular repair and healing processes. Thus there is a need to develop a spinal cord ischemia model for studying the mechanisms of ischemia-induced pathology. Focal ischemia induced by photothrombosis (PT) is a minimally invasive and very well established procedure used to investigate the pathology of ischemia-induced cell death in the brain. Here, we describe the use of PT to induce an ischemic lesion in the spinal cord of mice. Following retro-orbital sinus injection of Rose Bengal, the posterior spinal vein and other capillaries on the dorsal surface of SC were irradiated with a green light resulting in the formation of a thrombus and thus ischemia in the affected region. Results from histology and immunochemistry studies show that PT-induced ischemia caused spinal cord infarction, loss of neurons and reactive gliosis. Using this technique a highly reproducible and relatively easy model of SCI in mice can be achieved that would serve the purpose of scientific investigations into the mechanisms of ischemia induced cell death as well as the efficacy of neuroprotective drugs. This model will also allow exploration of the pathological changes that occur following SCI in live mice like axonal degeneration and regeneration, neuronal and astrocytic Ca2+ signaling using two-photon microscopy.

Photothrombosis is a minimally invasive and highly reproducible procedure to induce focal ischemia in the spinal cord and serves as a model of spinal cord injury in mice.

Spinal cord injury (SCI) is a devastating clinical condition causing permanent changes in sensorimotor and autonomic functions of the spinal cord (SC) ...

Use of a Low-flow Digital Anesthesia System for Mice and Rats

A traditional vaporizer depends on flowing gas and atmospheric pressure for passive anesthetic vaporization. Newly developed direct injection vaporizers utilize a syringe pump to directly administer volatile anesthetics into a gas stream. Unlike a traditional vaporizer, it can be used at very low flow rates, making it ideal for use on mice and rats. 
The equipment's capability to use low flow rates could result in a substantial cost savings due to the reduced need for anesthetic agents, compressed gas, and charcoal scavenging filters1. A lower flow rate means less waste of anesthetic gas and likely reduces the risk of anesthetic exposure to laboratory personnel. Thus, the high levels of precision and safety associated with direct injection vaporizers, along with a reduced need for anesthetic agents, compressed gas, and charcoal filters are beneficial for research requiring small animal anesthesia. 
The goal of this protocol is to demonstrate the use of a syringe-driven direct injection vaporizer as part of a digital, low-flow anesthesia system. The direct injection vaporizer is capable of accurately delivering anesthesia at very low flow rates compared to a traditional vaporizer, making it a promising alternative for controlled gas anesthetic delivery to rodents.

Here, we present a protocol to more safely and efficiently administer anesthetic gas to mice using a digital, low flow anesthesia system utilizing a syringe-driven direct injection vaporizer.

A traditional vaporizer depends on flowing gas and atmospheric pressure for passive anesthetic vaporization. Newly developed direct injection vaporizers ...

Ultrasound-guided Intracardiac Injection of Human Mesenchymal Stem Cells to Increase Homing to the Intestine for Use in Murine Models of Experimental Inflammatory Bowel Diseases

Crohn&#39;s disease (CD) is a common chronic inflammatory disease of the small and large intestines. Murine and human mesenchymal stem cells (MSCs) have immunosuppressive potential and have been shown to suppress inflammation in mouse models of intestinal inflammation, even though the route of administration can limit their homing and effectiveness 1,3,4,5. Local application of MSCs to colonic injury models has shown greater efficacy at ameliorating inflammation in the colon. However, there is paucity of data on techniques to enhance the localization of human bone marrow-derived MSCs (hMSCs) to the small intestine, the site of inflammation in the SAMP-1/YitFc (SAMP) model of experimental Crohn&#39;s disease. This work describes a novel technique for the ultrasound-guided intracardiac injection of hMSCs in SAMP mice, a well-characterized spontaneous model of chronic intestinal inflammation. Sex- and age-matched, inflammation-free AKR/J (AKR) mice were used as controls. To analyze the biodistribution and the localization, hMSCs were transduced with a lentivirus containing a triple reporter. The triple reporter consisted of firefly luciferase (fl), for bioluminescent imaging; monomeric red fluorescent protein (mrfp), for cell sorting; and truncated herpes simplex virus thymidine kinase (ttk), for positron emission tomography (PET) imaging. The results of this study show that 24 h after the intracardiac administration, hMSCs localize in the small intestine of SAMP mice as opposed to inflammation-free AKR mice. This novel, ultrasound-guided injection of hMSCs in the left ventricle of SAMP mice ensures a high success rate of cell delivery, allowing for the rapid recovery of mice with minimal morbidity and mortality. This technique could be a useful method for the enhanced localization of MSCs in other models of small-intestinal inflammation, such as TNF&#916;RE6. Future studies will determine if the increased localization of hMSCs by intra-arterial delivery can lead to increased therapeutic efficacy.

Murine studies in models of colonic inflammation have demonstrated that a small percentage (1 - 5%) of mesenchymal stem cells (MSC) injected intravenously or intraperitoneally home to the inflamed colon1,2. This study shows that ultrasound-guided intracardiac injections of MSCs result in increased localization to the intestine.

Crohn&#39;s disease (CD) is a common chronic inflammatory disease of the small and large intestines. Murine and human mesenchymal stem cells (MSCs) have ...

Murine Precision-Cut Liver Slices as an Ex Vivo Model of Liver Biology

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic fatty liver disease, metabolic liver disease, and liver cancer) requires experimental manipulation of animal models and in vitro cell cultures. Both techniques have limitations, such as the requirement of large numbers of animals for in vivo manipulation. However, in vitro cell cultures do not reproduce the structure and function of the multicellular hepatic environment. The use of precision-cut liver slices is a technique in which uniform slices of viable mouse liver are maintained in laboratory tissue culture for experimental manipulation. This technique occupies an experimental niche that exists between animal studies and in vitro cell culture methods. The presented protocol describes a straightforward and reliable method to isolate and culture precision-cut liver slices from mice. As an application of this technique, ex vivo liver slices are treated with bile acids to simulate cholestatic liver injury and ultimately assess the mechanisms of hepatic fibrogenesis.

This protocol provides a simple and reliable method for the production of viable precision-cut liver slices from mice. The ex vivo tissue samples can be maintained under laboratory tissue culture conditions for multiple days, providing a flexible model to examine liver pathobiology.

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic ...

Organ Ischemia-Reperfusion Injury by Simulating Hemodynamic Changes in Rat Liver Transplant Model

Orthotopic liver transplantation (OLT) in rats is a tried and proven animal model used for preoperative, intraoperative, and postoperative studies, including ischemia-reperfusion injury (IRI) of extrahepatic organs. This model requires numerous experiments and devices. The duration of anhepatic phase is closely related to the time to develop IRI after transplantation. In this experiment, we used hemodynamic changes to induce extrahepatic organ damage in rats and determined the maximum tolerance time. The time until the most severe organ injury varied for different organs. This method can easily be replicated and can also be used to study IRI of the extrahepatic organs after liver transplantation.

This paper provides a detailed description of how to build an animal model of the anhepatic phase (liver ischemia) in rats to facilitate basic research into ischemia-reperfusion injury after liver transplantation.

Orthotopic liver transplantation (OLT) in rats is a tried and proven animal model used for preoperative, intraoperative, and postoperative studies, ...

Liver Transplantation in Mouse and Analyzing Allograft Patency

Successful Orthotopic Liver Transplantation in Mice Utilizing Microcomputed Tomography Angiography

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

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

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

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