We sincerely thank Shin-Han Tseng for the critical discussion and partial execution of the study. This study was supported by EDAHP110003 and NCKUEDA110002 from the Research Foundation of E-DA Hospital and the National Cheng Kung University, Taiwan.

All mouse experiments were approved by the Laboratory Animal Center of the E-DA Hospital/ I-Shou University and were handled according to the &#34;Guide for the Care and Use of Laboratory Animals&#34; (NRC, USA 2011). Male C57BL/6 mice, 7-8 weeks old, were used for the present study.
1. Chemical preparation
<ol>
	<li>Prepare the chemical irritant by diluting 0.1% chlorhexidine gluconate (CG, see Table of Materials) in 15% ethanol.</li>
</ol>
<p class="jove_t...

<ol>
	<li>Han, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+outcome+of+CAPD:+twenty-five+years'+experience+in+a+single+Korean+center.">Improving outcome of CAPD: twenty-five years' experience in a single Korean center.</a> Peritoneal Dialysis International. 27 (4), 432-440 (2007).</li><li>Kawaguchi, Y., Hasegawa, T., Nakayama, M., Kubo, H., Shigematu, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Issues+affecting+the+longevity+of+the+continuous+peritoneal+dialysis+therapy.">Issues affecting the longevity of the continuous peritoneal dialysis therapy.</a> Kidney International Supplements. 62, 105-107 (1997).</li><li>Lee, Y. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vitamin+D+can+ameliorate+chlorhexidine+gluconate-induced+peritoneal+fibrosis+and+functional+deterioration+through+the+inhibition+of+epithelial-to-mesenchymal+transition+of+mesothelial+cells.">Vitamin D can ameliorate chlorhexidine gluconate-induced peritoneal fibrosis and functional deterioration through the inhibition of epithelial-to-mesenchymal transition of mesothelial cells.</a> BioMed Research International. 2015, 595030 (2015).</li><li>Nakamoto, H., Kawaguchi, Y., Suzuki, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+technique+survival+on+peritoneal+dialysis+better+in+Japan.">Is technique survival on peritoneal dialysis better in Japan.</a> Peritoneal Dialysis International. 26 (2), 136-143 (2006).</li><li>Schaefer, F., Klaus, G., Muller-Wiefel, D. 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A., Garosi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+peritoneum:+from+histological+studies+to+mesothelial+transplant+through+animal+experimentation.">The peritoneum: from histological studies to mesothelial transplant through animal experimentation.</a> Peritoneal Dialysis International. 28, 5-9 (2008).</li><li>Fusshoeller, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histomorphological+and+functional+changes+of+the+peritoneal+membrane+during+long-term+peritoneal+dialysis.">Histomorphological and functional changes of the peritoneal membrane during long-term peritoneal dialysis.</a> Pediatric Nephrology. 23 (1), 19-25 (2008).</li><li>Goffin, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peritoneal+membrane+structural+and+functional+changes+during+peritoneal+dialysis.">Peritoneal membrane structural and functional changes during peritoneal dialysis.</a> Seminars in Dialysis. 21 (3), 258-265 (2008).</li><li>Ito, T., Yorioka, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peritoneal+damage+by+peritoneal+dialysis+solutions.">Peritoneal damage by peritoneal dialysis solutions.</a> Clinical and Experimental Nephrology. 12 (4), 243-249 (2008).</li><li>Io, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SAHA+suppresses+peritoneal+fibrosis+in+mice.">SAHA suppresses peritoneal fibrosis in mice.</a> Peritoneal Dialysis International. 35 (3), 246-258 (2015).</li><li>Yoh, K., Ojima, M., Takahashi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Th2-biased+GATA-3+transgenic+mice+developed+severe+experimental+peritoneal+fibrosis+compared+with+Th1-biased+T-bet+and+Th17-biased+RORgammat+transgenic+mice.">Th2-biased GATA-3 transgenic mice developed severe experimental peritoneal fibrosis compared with Th1-biased T-bet and Th17-biased RORgammat transgenic mice.</a> Experimental Animals. 64 (4), 353-362 (2015).</li><li>Karl, Z. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modification+of+the+masson+trichrome+technique+for+routine+laboratory+purposes.">Modification of the masson trichrome technique for routine laboratory purposes.</a> The American Journal of Pathology. 14 (2), 237-243 (1938).</li><li>Cheng, F. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+application+of+magnetite+nanoparticle-mediated+vitamin+D3+delivery+for+peritoneal+dialysis-related+peritoneal+damage.">Novel application of magnetite nanoparticle-mediated vitamin D3 delivery for peritoneal dialysis-related peritoneal damage.</a> International Journal of Nanomedicine. 16, 2137-2146 (2021).</li><li>Ross, A., Willson, V. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Basic and Advanced Statistical Tests: Writing Results Sections and Creating Tables and Figures. , 13-16 (2017).</li><li>Suga, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preventive+effect+of+pirfenidone+against+experimental+sclerosing+peritonitis+in+rats.">Preventive effect of pirfenidone against experimental sclerosing peritonitis in rats.</a> Experimental and Toxicologic Pathology. 47 (4), 287-291 (1995).</li><li>Ishii, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+experimental+sclerosing+encapsulating+peritonitis+model+in+mice.">An experimental sclerosing encapsulating peritonitis model in mice.</a> Nephrology Dialysis Transplantation. 16 (6), 1262-1266 (2001).</li><li>Nishino, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antisense+oligonucleotides+against+collagen-binding+stress+protein+HSP47+suppress+peritoneal+fibrosis+in+rats.">Antisense oligonucleotides against collagen-binding stress protein HSP47 suppress peritoneal fibrosis in rats.</a> Kidney International. 64 (3), 887-896 (2003).</li><li>Mishima, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+expression+of+heat+shock+protein+47+in+rat+model+of+peritoneal+fibrosis.">Enhanced expression of heat shock protein 47 in rat model of peritoneal fibrosis.</a> Peritoneal Dialysis International. 23 (1), 14-22 (2003).</li><li>Kushiyama, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+liposome-encapsulated+clodronate+on+chlorhexidine+gluconate-induced+peritoneal+fibrosis+in+rats.">Effects of liposome-encapsulated clodronate on chlorhexidine gluconate-induced peritoneal fibrosis in rats.</a> Nephrology Dialysis Transplantation. 26 (10), 3143-3154 (2011).</li><li>Nishino, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+lymphocyte+infiltration+in+the+progression+of+mouse+peritoneal+fibrosis+model.">Involvement of lymphocyte infiltration in the progression of mouse peritoneal fibrosis model.</a> Renal Failure. 34 (6), 760-766 (2012).</li><li>Lua, I., Li, Y., Pappoe, L. 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In this study, a mouse PD model is presented by i.p. injection of CG, and the results showed peritoneal fibrosis and functional deterioration in this model, which mimicked the PD patient&#39;s condition.
There are several critical steps in the protocol. First, for performing an i.p. injection of CG or NS, the abdominal wall skin of the mouse must be picked up using forceps to prevent puncture-induced intraperitoneal organ damage. Second, while collecting the peritoneum of the abdominal wall fo...

Peritoneal dialysis (PD) is a type of renal replacement therapy. However, PD has problems that cannot be solved. For example, long-term PD treatment can cause peritoneal damage, eventually leading to ultrafiltration failure and withdrawal of treatment1,2,3,4,5,6. Peritoneal fibrosis is one of the most serious complications7,8. Peritoneal fibrosis is characterized by the deposition and accumulation of extracellular matrix within the interstitium, and neo-angiogenesis and vasculopathy of the peritoneum9,10.
The main causes of these peritoneal changes are recurrent peritonitis and non-biocompatibility of the dialysate, which are hyperosmotic, high glucose, low pH, and glucose degradation product accumulation11,12. Therefore, suitable animal experimental models can help researchers better study the peritoneum&#39;s physiological and pathological changes during PD therapy. Therefore, establishing an animal PD model is important for improving PD technology and dialysis efficacy and prolonging patient survival. This study aimed to generate a PD mouse model by intraperitoneal (i.p.) injection of chlorhexidine gluconate (CG), as described previously13,14. This PD mouse model is simple, easy to use, and feasible compared to other PD animal models.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.9% Normal Saline</td>
			<td>Y F CHEMICAL CORP., New Taipei City, Taiwan</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>10% neutral buffered formalin</td>
			<td>Taiwan Burnett International Co., Ltd., Taipei City, Taiwan</td>
			<td>00002A</td>
			<td></td>
		</tr>
		<tr>
			<td>Automatic biochemical analyzer</td>
			<td>Hitachi Ltd., Tokyo, Japan</td>
			<td>Labospect Series 008</td>
			<td>for determining glucose concentration</td>
		</tr>
		<tr>
			<td>Chlorhexidine digluconate solution, 20% in H2O</td>
			<td>Sigma-Aldrich, MO, USA</td>
			<td>C9394</td>
			<td>diluted to 0.1% with 15% ethanol for injection</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Avantor Performance Materials, LLC, PA, USA</td>
			<td>BAKR8006-05</td>
			<td>diluted to 15% with normal saline for working concentration</td>
		</tr>
		<tr>
			<td>Glucose (Dianeal)</td>
			<td>Baxter International, Inc., IL, USA</td>
			<td>FNB9896</td>
			<td>Commercial dialysis solution (4.25%)</td>
		</tr>
		<tr>
			<td>GraphPad Prism 8.0</td>
			<td>GraphPad Software, Inc., CA, US</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>L-type Glu 2 assay</td>
			<td>FUJIFILM Wako, Japan</td>
			<td>461-32403</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine 20</td>
			<td>Juily Pharmaceutical Co., Ltd., New Taipei City, Taiwan</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Zoletil 50</td>
			<td>Virbac Laboratories, Carros, France</td>
			<td>-</td>
			<td></td>
		</tr>
	</tbody>
</table>

a mice model of chlorhexidine gluconate-induced peritoneal damage

Peritoneal fibrosis is an important complication of peritoneal dialysis (PD). To investigate and address this problem, an appropriate animal model of PD is required. The present protocol establishes a chlorhexidine gluconate (CG) induced peritoneal fibrosis model that mimics the condition of a patient with PD. Peritoneal fibrosis was induced by intraperitoneal injection of 0.1% of CG in 15% ethanol for 3 weeks (administered every other day), for a total of nine times in male C57BL/6 mice. Peritoneal functional tests were then performed on day 22. After the mice were sacrificed, the parietal peritoneum of the abdominal wall and the visceral peritoneum of the liver were harvested. They were thicker and more fibrotic when analyzed microscopically after Masson's trichrome staining. The ultrafiltration rate decreased, and glucose mass transport indicated a CG-induced increase in peritoneal permeability. The PD model thus established may have applications in improving PD technology, dialysis efficacy, and prolonging patient survival.

In Figure 1A,B, the parietal peritoneum of the abdominal wall was markedly thicker and more fibrotic under Masson&#39;s trichrome staining17, indicating that in the CG-exposed group, peritoneal fibrosis is more severe than in the control saline group (NS). In Figure 2A,B, the visceral peritoneum of the liver surfaces was also markedly thicker and more fibrotic, thus proving that in the CG-exposed group, p...

Watch this Scientific Journal Video about A Mice Model of Chlorhexidine Gluconate-Induced Peritoneal Damage at JoVE.com

A Mice Model of Chlorhexidine Gluconate-Induced Peritoneal Damage

The present protocol establishes a peritoneal dialysis (PD) mouse model of chlorhexidine gluconate (CG)-induced peritoneal fibrosis. The current model is simple and easy to use compared to other PD animal models.

Peritoneal fibrosis is an important complication of peritoneal dialysis (PD). To investigate and address this problem, an appropriate animal model of PD ...

Establishing animal model for peritoneal dialysis, PD, is very important. We hope it can improve the technology and the efficacy of PD therapy and eventually improve the patient's survival. This peritoneal dialysis mouse model is simple, easy to use, and visible compared to other peritoneal dialysis animal models.A suitable peritoneal dialysis animal model can help researchers to study the peritoneal, physiological, and the pathological change during PD therapy. Demonstration of the procedure will be Fang-Ling Liao, a research assistant from my laboratory. To begin, prepare the chemical irritant by diluting 0.1%chlorhexidine gluconate in 15%ethanol.Next, assign three mice as the control group and inject one milliliter per kilogram of 0.9%normal saline intraperitoneally every other day for three weeks for a total of nine times. Then assign three mice to the peritoneal fibrosis group, and induce peritoneal fibrosis by administering an intraperitoneal injection of 0.1%chlorhexidine gluconate solution prepared earlier at a dose of 12.5 microliters per kilogram of the body weight. For peritoneal function tests, prepare a dialysis solution containing 4.25%glucose.Then use a syringe to draw 0.5 milliliters of dialysate sample. Prepare an injection of zoletil and xylazine and anesthetize the mice by administering an intramuscular injection. Perform intraperitoneal installation of the dialysis solution at a dose of two milliliters per 20 grams of body weight.Make a small incision in the midline of the abdomen and collect the major intraperitoneal fluid with a syringe. After 30 minutes, perform a vertical incision in the midline of the abdomen beneath the xiphoid process and open the mouse's abdomen to collect the residual intraperitoneal fluid with a syringe. Next, measure the weight of a clean and dry cotton swab.Put the cotton into the mouse's abdominal cavity to absorb the residual intraperitoneal fluid and then remove it. Measure the cotton weight again. Masson's trichrome staining showed that the parietal peritoneum of the abdominal wall in the chlorhexidine gluconate-exposed group was markedly thicker and fibrotic than the control group.The visceral peritoneum of the liver surfaces was also significantly thicker and fibrotic in the chlorhexidine gluconate-exposed group than in the control saline group, indicating that peritoneal fibrosis is more severe in the chlorhexidine gluconate-exposed group. The chlorhexidine gluconate-exposed group showed decreased ultrafiltration rate and increased glucose mass transport, indicating that the peritoneal permeability increases in the chlorhexidine gluconate exposed mice While performing an intraperitoneal injection procedure, use the forceps and pick the abdominal skin of the mouse to prevent puncture-induced intraperitoneal organ damage.

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1.7K Views.  I-Shou University. Establishing animal model for peritoneal dialysis, PD, is very important. We hope it can improve the technology and the efficacy of PD therapy and eventually improve the patient's survival. This peritoneal dialysis mouse model is simple, easy to use, and visible compared to other peritoneal dialysis animal models.A suitable peritoneal dialysis animal model can help researchers to study the peritoneal, physiological, and the pathological change during PD therapy. Demonstration of the pr...