This work was supported by grants from the National Institutes of Health, National Institute of General Medical Science (R01GM063075) and the National Center of Complementary and Alternative Medicine (R01AT05076).

1. Establishment of Animal Model of Sepsis
<ol>
	<li>Mice are anesthetized with ketamine (75 mg/kg, intramuscular, i.m.) and xylazine (10 mg/kg, i.m.), and placed in supine position.</li>
	<li>Fix the feet of the mouse with tape to ensure a stable position.</li>
	<li>Clean the abdomen with 3 alternating scrubs of betadine or other skin disinfectant and alcohol. Then, make a 15 mm midline incision to expose the cecum.</li>
	<li>Ligate the cecum with a 4-0 silk suture at approximately 5.0 mm from the cecal tip, and then puncture the ligated cecal stump once with a 22-gauge needle to allow extrusion of stool.</li>
	<li>The cecum is immediately replaced back to its normal intra-abdominal position.</li>
	<li>Close the incision site in two layers, by first closing the musculature of the abdomen with absorbable sutures, and then closing the skin with wound clips or nonabsorbable sutures. </li>
	<li>Resuscitate mouse with 0.5 ml normal saline solution and a single dose of imipenem (0.5 mg/mouse, subcutaneously) immediately after the completion of the surgical procedure.</li>
	<li>Return the mouse back to a clean cage with free access to food and water.</li>
	<li>At various time points post CLP, herbal extract or component is administered intraperitoneally.</li>
	<li>Animal survival is monitored for more than two weeks. Moribund animals exhibiting difficulties to stand, agonal breathing, severe muscular atrophy and uncontrolled bleeding should be euthanized by overdose carbon dioxide inhalation. </li>
	<li>It is important to note that circulating cytokine levels are important parameters in these studies. Various analgesics have been shown to affect cytokine release and activities, and therefore have been intentionally avoided in postoperative care. </li>
</ol>
2. Preparation of Herbal Extract
<ol>
	<li>Extract herbs in hot water (85 &deg;C) for 1-4 hr (1 hr for leaves, and 4 hr for roots).</li>
	<li>Centrifuge the water-soluble fraction (3300 g, 20 min, 4 &deg;C) to remove insoluble particles.</li>
	<li>Filtrate the supernatant fraction through a 0.2 &mu;m filter.</li>
	<li>The clear water-soluble filtrate fraction is then fractionated by ultrafiltration using Centriprep YM-10 Centrifugal Filter (Catalog no. 4305, Millipore).</li>
	<li>The resulted low (&lt;10 kDa) and high (&gt; 10 kDa) molecular weight fractions (MWF) are screened for HMGB1-inhibiting activities using macrophage cultures.</li>
	<li>Intraperitoneal administration of HMGB1-inhibiting herbal extract or components at 24 hr post CLP to evaluate their therapeutic efficacy.</li>
</ol>
3. Peritoneal Macrophage Isolation
<ol>
	<li>Thioglycollate broth (4%, 2.0 ml) is administered intraperitoneally into normal mice.</li>
	<li>Primary peritoneal macrophages are harvested at 3 days as previously described10.</li>
	<li>Macrophages are pre-cultured in DMEM medium (Gibco BRL, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS), 2 mmol/L glutamine, and 1% penicillin.</li>
	<li>Adherent macrophages are gently washed with, and cultured in, serum-free OPTI-MEM I medium two hours before stimulation with bacterial endotoxin (lipopolysaccharide, LPS, E. coli 0111:B4, Sigma-Aldrich).</li>
	<li>At 16 hours after LPS stimulation, levels of HMGB1 in the culture medium are determined by Western blotting analysis11.</li>
	<li>The relative band intensity was quantified by using the NIH image 1.59 software to determine HMGB1 levels with reference to standard curves generated with purified HMGB1.</li>
</ol>
4. Representative Results
1. CLP induces systemic and local inflammation.
Within a few hours of CLP surgery, animals exhibit clinical signs of sepsis that include piloerection, lethargy, huddling, and decrease in food and water intake. Animals developing a severe peritonitis with consecutive systemic infection normally die within 48 - 96 hr. However, even age-, sex-, and genetic background-matched animals can respond to CLP surgery in a distinguishable manner in the course of experimental sepsis. For instance, at 48 hr post CLP, while some animals may have approached the moribund state (as defined in Figure 1), others may remain at a non-moribund state.
Comprehensive surveys of circulating cytokines revealed dramatic differences in levels of several cytokines (e.g., IL-6, KC, MIP-2, and sTNFR1) between mice in the moribund and non-moribund states (Figure 1)12. Notably, these inflammatory mediators have been classified as surrogate markers of sepsis because their circulating levels are reliable predictors of lethal outcome in experimental12-14 or clinical sepsis15. In addition, CLP also induced local release of various pro- and anti-inflammatory cytokines and chemokines. For instance, at 48 hr post CLP, significant amounts of cytokines (e.g., IL-6) and chemokines (KC and MIP-2) can still be measured not only systemically in the blood, but also locally in the peritoneal lavage fluid (Figure 2).
2. Screening for HMGB1-inhibiting herbal extract or components.
Using macrophage cultures, we were able to evaluate the capacities of various herbal extract or components in inhibiting endotoxin-induced HMGB1 release (Figure 3). In light of their capacity in inhibiting HMGB1 release, we further explored their efficacy in animal models sepsis. Given the late and prolonged kinetics of HMGB1 accumulation in experimental sepsis8, the first dose of HMGB1 inhibitors was given 24 hr after the onset of sepsis - a time point when mice developed clear signs of sepsis including lethargy, diarrhea, and piloerection. As shown in Figure 4, delayed and repeated administration of a major green tea component, epigallocatechin-3- gallate (EGCG), beginning at 24 h after onset of sepsis, significantly rescued mice from lethal sepsis12. Even when given orally, EGCG still rescued mice from lethal sepsis, significantly increasing animal survival rates from 16% to 44%16. It would be important to determine whether combinational therapies with several herbal components could achieve a significant better protection in animal models of sepsis. Taken together, these experimental data validated our approach to screen novel therapeutic agents using an animal model of CLP-induced sepsis.
5. Representative Results
<img alt="Figure 1" src="/files/ftp_upload/3926/3926fig1.jpg" /> 
Figure 1. Circulating levels of surrogate markers are dramatically higher in septic animals approaching moribund state. Balb/C mice were subjected to sepsis by CLP and monitored for the development of signs of sickness. At a late stage of sepsis (52 hr post CLP), blood was collected from 3 normal mice (-CLP), 3 septic mice approaching the moribund state, and 3 septic mice in the non-moribund state. Serum was pooled from each group, and assayed for the cytokine profile by antibody arrays. Note the dramatic difference in the relative levels of several surrogate markers between different groups. Adapted from doi:10.1371/journal.pone.0001153.g006 with granted permission from the publisher.
<img alt="Figure 2" src="/files/ftp_upload/3926/3926fig2.jpg" /> 
Figure 2. Detection of local and systemic cytokines and chemokines at 48 hr post CLP. Balb/C mice were subjected to sepsis by CLP, and blood or peritoneal fluid were harvested at 48 h post CLP. Relative levels of several cytokines and chemokines in pooled serum or peritoneal lavage fluid were measured by cytokine antibody arrays, and expressed as arbitrary units (AU). As controls, blood and peritoneal fluid samples were obtained from normal healthy animals (-CLP) without laparotomy.
<img alt="Figure 3" src="/files/ftp_upload/3926/3926fig3.jpg" /> 
Figure 3. Herbal component dose-dependently inhibited endotoxin-induced HMGB1 release in primary macrophage cultures. Primary murine peritoneal macrophages were stimulated with LPS in the absence, or presence of herbal component (e.g., EGCG, 15 &mu;M). At 16 hours after LPS stimulation, levels of HMGB1 in the culture medium were determined by Western blotting analysis. Adapted from doi:10.1371/journal.pone.0001153.g006 with granted permission from the publisher.
<img alt="Figure 4" src="/files/ftp_upload/3926/3926fig4.jpg" /> 
Figure 4. Herbal component rescued mice from lethal sepsis. Balb/C mice were subjected to lethal sepsis by CLP, and herbal component (EGCG) was intraperitoneally administered at +24, +48, +72 hours post the onset of sepsis. Animal survival was monitored for up to two weeks. The Kaplan-Meier method was used to compare the differences in mortality rates between groups. *, p &lt; 0.05 versus saline. Adapted from doi:10.1371/journal.pone.0001153.g006 with granted permission from the publisher.

<ol>
	<li>Wichterman, K. A., Baue, A. E., Chaudry, I. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sepsis+and+septic+shock--a+review+of+laboratory+models+and+a+proposal.">Sepsis and septic shock--a review of laboratory models and a proposal.</a> J. Surg. Res. 29, 189-201 (1980).</li><li>Baker, C. C., Chaudry, I. H., Gaines, H. O., Baue, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+factors+affecting+mortality+rate+after+sepsis+in+a+murine+cecal+ligation+and+puncture+model.">Evaluation of factors affecting mortality rate after sepsis in a murine cecal ligation and puncture model.</a> Surgery. 94, 331-335 (1983).</li><li>Hubbard, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cecal+ligation+and+puncture.">Cecal ligation and puncture.</a> Shock. 24, 52-57 (2005).</li><li>Akira, S., Takeda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toll-like+receptor+signalling.">Toll-like receptor signalling.</a> Nat. Rev. Immunol. 4, 499-511 (2004).</li><li>Baggiolini, M., Loetscher, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemokines+in+inflammation+and+immunity.">Chemokines in inflammation and immunity.</a> Immunol. Today. 21, 418-420 (2000).</li><li>Balkwill, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokines--soluble+factors+in+immune+responses.">Cytokines--soluble factors in immune responses.</a> Curr. Opin. Immunol. 1, 241-249 (1988).</li><li>Wang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HMG-1+as+a+late+mediator+of+endotoxin+lethality+in+mice.">HMG-1 as a late mediator of endotoxin lethality in mice.</a> Science. 285, 248-251 (1999).</li><li>Yang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversing+established+sepsis+with+antagonists+of+endogenous+high-mobility+group+box+1.">Reversing established sepsis with antagonists of endogenous high-mobility group box 1.</a> Proc. Natl. Acad. Sci. U.S.A. 101, 296-301 (2004).</li><li>Qin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+HMGB1+in+apoptosis-mediated+sepsis+lethality.">Role of HMGB1 in apoptosis-mediated sepsis lethality.</a> J. Exp. Med. 203, 1637-1642 (2006).</li><li>Ray, A., Dittel, B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+Mouse+Peritoneal+Cavity+Cells.">Isolation of Mouse Peritoneal Cavity Cells.</a> J. Vis. Exp. (35), e1488 (2010).</li><li>Rendon-Mitchell, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IFN-gamma+Induces+High+Mobility+Group+Box+1+Protein+Release+Partly+Through+a+TNF-Dependent+Mechanism.">IFN-gamma Induces High Mobility Group Box 1 Protein Release Partly Through a TNF-Dependent Mechanism.</a> J. Immunol. 170, 3890-3897 (2003).</li><li>Li, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Major+Ingredient+of+Green+Tea+Rescues+Mice+from+Lethal+Sepsis+Partly+by+Inhibiting+HMGB1.">A Major Ingredient of Green Tea Rescues Mice from Lethal Sepsis Partly by Inhibiting HMGB1.</a> PLoS ONE. 2, e1153 (2007).</li><li>Osuchowski, M. F., Welch, K., Siddiqui, J., Remick, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+cytokine/inhibitor+profiles+reshape+the+understanding+of+the+SIRS/CARS+continuum+in+sepsis+and+predict+mortality.">Circulating cytokine/inhibitor profiles reshape the understanding of the SIRS/CARS continuum in sepsis and predict mortality.</a> J. Immunol. 177, 1967-1974 (2006).</li><li>Heuer, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+protein+C+and+other+biomarkers+as+predictors+of+mortality+in+a+rat+cecal+ligation+and+puncture+model+of+sepsis.">Evaluation of protein C and other biomarkers as predictors of mortality in a rat cecal ligation and puncture model of sepsis.</a> Crit. Care. Med. 32, 1570-1578 (2004).</li><li>Bozza, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokine+profiles+as+markers+of+disease+severity+in+sepsis:+a+multiplex+analysis.">Cytokine profiles as markers of disease severity in sepsis: a multiplex analysis.</a> Crit. Care. 11, R49 (2007).</li><li>Li, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EGCG+stimulates+autophagy+and+reduces+cytoplasmic+HMGB1+levels+in+endotoxin-stimulated+macrophages.">EGCG stimulates autophagy and reduces cytoplasmic HMGB1 levels in endotoxin-stimulated macrophages.</a> Biochem. Pharmacol. 81, 1152-1163 (2011).</li><li>Beutler, B., Milsark, I. W., Cerami, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Passive+immunization+against+cachectin/tumor+necrosis+factor+protects+mice+from+lethal+effect+of+endotoxin.">Passive immunization against cachectin/tumor necrosis factor protects mice from lethal effect of endotoxin.</a> Science. 229, 869-871 (1985).</li><li>Tracey, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-cachectin/TNF+monoclonal+antibodies+prevent+septic+shock+during+lethal+bacteraemia.">Anti-cachectin/TNF monoclonal antibodies prevent septic shock during lethal bacteraemia.</a> Nature. 330, 662-664 (1987).</li><li>Eskandari, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-tumor+necrosis+factor+antibody+therapy+fails+to+prevent+lethality+after+cecal+ligation+and+puncture+or+endotoxemia.">Anti-tumor necrosis factor antibody therapy fails to prevent lethality after cecal ligation and puncture or endotoxemia.</a> J. Immunol. 148, 2724-2730 (1992).</li><li>Ziegler, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+of+gram-negative+bacteremia+and+septic+shock+with+HA-1A+human+monoclonal+antibody+against+endotoxin.+A+randomized,+double-blind,+placebo-controlled+trial.+The+HA-1A+Sepsis+Study+Group.">Treatment of gram-negative bacteremia and septic shock with HA-1A human monoclonal antibody against endotoxin. A randomized, double-blind, placebo-controlled trial. The HA-1A Sepsis Study Group.</a> N. Engl. J. Med. 324, 429-436 (1991).</li><li>Ziegler, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+of+gram-negative+bacteremia+and+shock+with+human+antiserum+to+a+mutant+Escherichia+coli.">Treatment of gram-negative bacteremia and shock with human antiserum to a mutant Escherichia coli.</a> N. Engl. J. Med. 307, 1225-1230 (1982).</li><li>Abraham, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+and+safety+of+monoclonal+antibody+to+human+tumor+necrosis+factor+alpha+in+patients+with+sepsis+syndrome.+A+randomized,+controlled,+double-blind,+multicenter+clinical+trial.+TNF-alpha+MAb+Sepsis+Study+Group.">Efficacy and safety of monoclonal antibody to human tumor necrosis factor alpha in patients with sepsis syndrome. A randomized, controlled, double-blind, multicenter clinical trial. TNF-alpha MAb Sepsis Study Group.</a> JAMA. 273, 934-941 (1995).</li><li>Cohen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adjunctive+therapy+in+sepsis:+a+critical+analysis+of+the+clinical+trial+programme.">Adjunctive therapy in sepsis: a critical analysis of the clinical trial programme.</a> Br. Med. Bull. 55, 212-225 (1999).</li><li>Dellinger, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surviving+Sepsis+Campaign:+international+guidelines+for+management+of+severe+sepsis+and+septic+shock:+2008.">Surviving Sepsis Campaign: international guidelines for management of severe sepsis and septic shock: 2008.</a> Crit. Care Med. 36, 296-327 (2008).</li><li>Wang, H., Zhu, S., Zhou, R., Li, W., Sama, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+potential+of+HMGB1-targeting+agents+in+sepsis.">Therapeutic potential of HMGB1-targeting agents in sepsis.</a> Expert. Rev. Mol. Med. 10, e32 (2008).</li><li>Wang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+aqueous+extract+of+a+popular+herbal+nutrient+supplement,+Angelica+sinensis,+protects+mice+against+lethal+endotoxemia+and+sepsis.">The aqueous extract of a popular herbal nutrient supplement, Angelica sinensis, protects mice against lethal endotoxemia and sepsis.</a> J. Nutr. 136, 360-365 (2006).</li><li>Li, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cardiovascular+drug+rescues+mice+from+lethal+sepsis+by+selectively+attenuating+a+late-acting+proinflammatory+mediator,+high+mobility+group+box+1.">A cardiovascular drug rescues mice from lethal sepsis by selectively attenuating a late-acting proinflammatory mediator, high mobility group box 1.</a> J. Immunol. 178, 3856-3864 (2007).</li><li>Fukuyama, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mixed+bacterial+infection+model+of+sepsis+in+rabbits+and+its+application+to+evaluate+superantigen-adsorbing+device.">Mixed bacterial infection model of sepsis in rabbits and its application to evaluate superantigen-adsorbing device.</a> Blood Purif. 23, 119-127 (2005).</li></ol>

A.E.S. and H.W. are co-inventors of patent applications related to HMGB1 inhibitors (tanshinones) as potential therapeutic agents for sepsis.

In the laboratory, several animal models of sepsis have been employed to understand the pathogenesis of sepsis in order to develop potential novel therapies. Their clinical relevance remains a subject of debate before the successful translation of animal studies into clinical applications for sepsis. Although neutralizing antibodies against early cytokines (e.g., TNF) were protective in animal models of bacteremia/endotoxemia17,18, they actually worsen survival in animal model of sepsis19. Similarly...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>Betadine </td>
 <td>Purdue Products L.P.</td>
 <td>25655-41-8</td>
 </tr>
 <tr>
 <td>imipenem</td>
 <td>Merck &amp; Co., Inc.</td>
 <td>9882821</td>
 </tr>
 <tr>
 <td>Ketamine HCl</td>
 <td>Hospira Inc.</td>
 <td>RL-0065</td>
 </tr>
 <tr>
 <td>Xylazine</td>
 <td>Lloyd Laboratories</td>
 <td>4821</td>
 </tr>
 <tr>
 <td>Autoclip </td>
 <td>Becton Dickinson</td>
 <td>427631</td>
 </tr>
 <tr>
 <td>4-0 silk suture</td>
 <td>Roboz</td>
 <td>SUT-15-2</td>
 </tr>
 <tr>
 <td>Surflo I.V. Catheter</td>
 <td>Terumo</td>
 <td>SR*OX2419CA</td>
 </tr>
 <tr>
 <td>RayBio mouse cytokine antibody array</td>
 <td>RayBiotech, Inc.</td>
 <td>AAM-CYT-3</td>
 </tr>
 <tr>
 <td>Thioglycollate </td>
 <td>Becton Dickinson</td>
 <td>211716</td>
 </tr>
 </tbody>
</table>

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.

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Use of Animal Model of Sepsis to Evaluate Novel Herbal 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 ...

use-of-animal-model-of-sepsis-to-evaluate-novel-herbal-therapies

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19.3K Views.  North Shore – LIJ Health System. 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.