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>
2. Animal treatment
<ol>
	<li>Assign three mice as the control group. Perform intraperitoneal injection (i.p.) of 1 mL/kg of 0.9% normal saline (NS) every other day for 3 weeks, for a total of nine times.</li>
	<li>Assign three mice to the peritoneal fibrosis group. Induce peritoneal fibrosis using chlorhexidine gluconate (CG) by administering i.p. injections of 0.1% of CG in 15% ethanol (step 1.1) at a dose of 12.5 &#181;L/g body weight. Perform this every other day for 3 weeks, for a total of nine times.</li>
</ol>
3. Peritoneal function tests (modified peritoneal equilibration test)
<ol>
	<li>Prepare a dialysis solution containing 4.25% glucose. Draw 0.5 mL of dialysate sample with a syringe, and then check the glucose concentration in the dialysate sample. 
	NOTE: The glucose concentration is determined according to the hexokinase/G6PD method. Dialysate samples were accessed to L-type Glu 2 assay and investigated with a biochemical analyzer (see Table of Materials). This is the initial dialysate glucose concentration.</li>
	<li>Anesthetize the mice by intramuscular injection of Zoletil and Xylazine (prepared in a ratio of 1:2 by volume, see Table of Materials) at a dose of 20 &#181;L/20 gw. Additionally, use vet ointment on the eyes to prevent dryness under anesthesia.</li>
	<li>Perform i.p. instillation of the dialysis solution (2 mL/20 g bodyweight).</li>
	<li>After 30 min, assess and verify the depth of anesthesia with lack of toe pinch reflex. Then, perform a vertical incision in the midline of the abdominal (beneath the xiphoid process), then open the mice&#39;s abdomen and collect the intraperitoneal fluid with a syringe (defined as &#34;volume 1&#34;). Then, measure the weight of a clean and dry cotton and put the cotton into the mice&#39;s abdominal cavity to absorb the residual intraperitoneal fluid. Finally, measure the cotton weight again. 
	NOTE: The weight gain of cotton is equal to the weight of the residual intraperitoneal fluid. Then, convert to the obtained volume (specific gravity: 1 g/cm3; defined as &#34;volume 2&#34;). The final dialysate volume is volume 1 plus volume 2.</li>
	<li>Use 0.5 mL of dialysate sample (final dialysate) to measure the glucose concentration. This is the final dialysate glucose concentration.</li>
	<li>Calculate the net ultrafiltration using the formula15: 
	<img alt="Equation 1" src="/files/ftp_upload/63903/63903eq01.jpg" style="margin: auto;" /></li>
	<li>Calculate the peritoneal permeability using the following formula15: 
	<img alt="Equation 2" src="/files/ftp_upload/63903/63903eq02.jpg" style="margin: auto;" /></li>
</ol>
4. Tissue preparation of the abdominal wall muscle and liver and histological analysis
<ol>
	<li>Sacrifice the mice via cardiac puncture (phlebotomy)3,16.</li>
	<li>Cut the abdominal wall (1 cm x 1 cm) and total hepatectomy. Fix the mice&#39;s abdominal wall and liver tissues overnight in 10% neutral buffered formalin.</li>
	<li>Prepare 3 &#181;m thick paraffin sections of the abdominal wall muscle and liver, and perform histological analysis following previously published report17.</li>
	<li>Evaluate the parietal peritoneum of the abdominal wall and visceral peritoneum of the liver surfaces of the mice using morphometry18.</li>
	<li>Perform statistical analyses using statistics and graphing software (see Table of Materials). Express all data as mean &#177; SD and analyze for statistical significance using a t-test19. Define values with P &#60; 0.05 as significant results.</li>
</ol>

<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. E., Mehls, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+practice+of+peritoneal+dialysis+in+children:+results+of+a+longitudinal+survey.+Mid+European+Pediatric+Peritoneal+Dialysis+Study+Group+(MEPPS).">Current practice of peritoneal dialysis in children: results of a longitudinal survey. Mid European Pediatric Peritoneal Dialysis Study Group (MEPPS).</a> Peritoneal Dialysis International. 19, 445-449 (1999).</li><li>Woodrow, G., Turney, J. H., Brownjohn, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technique+failure+in+peritoneal+dialysis+and+its+impact+on+patient+survival.">Technique failure in peritoneal dialysis and its impact on patient survival.</a> Peritoneal Dialysis International. 17 (4), 360-364 (1997).</li><li>Schmidt, D. W., Flessner, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+and+treatment+of+encapsulating+peritoneal+sclerosis:+basic+and+translational+research.">Pathogenesis and treatment of encapsulating peritoneal sclerosis: basic and translational research.</a> Peritoneal Dialysis International. 28, 10-15 (2008).</li><li>Augustine, T., Brown, P. W., Davies, S. D., Summers, A. M., Wilkie, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Encapsulating+peritoneal+sclerosis:+clinical+significance+and+implications.">Encapsulating peritoneal sclerosis: clinical significance and implications.</a> Nephron Clinical Practice. 111 (2), 149-154 (2009).</li><li>Di Paolo, N., Nicolai, G. 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. J. 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=Peritoneal+Equilibration+Test.">Peritoneal Equilibration Test.</a> Peritoneal Dialysis International. 7 (3), 138-148 (1987).</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=The+clinical+implication+of+vitamin+D+nanomedicine+for+peritoneal+dialysis-related+peritoneal+damage.">The clinical implication of vitamin D nanomedicine for peritoneal dialysis-related peritoneal damage.</a> International Journal of Nanomedicine. 14, 9665-9675 (2019).</li><li>Goldner, J. 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. S., Asahina, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myofibroblastic+conversion+and+regeneration+of+mesothelial+cells+in+peritoneal+and+liver+fibrosis.">Myofibroblastic conversion and regeneration of mesothelial cells in peritoneal and liver fibrosis.</a> The American Journal of Pathology. 185 (12), 3258-3273 (2015).</li><li>Kitamura, M., 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=Epigallocatechin+gallate+suppresses+peritoneal+fibrosis+in+mice.">Epigallocatechin gallate suppresses peritoneal fibrosis in mice.</a> Chemico-Biological Interactions. 195 (1), 95-104 (2012).</li></ol>

The authors have nothing to disclose.

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 for histologic analyses, the area damaged by i.p. injections must be avoided.
Among the several experimental animal models of peritoneal fibrosis, the most common is the CG model because of its ease of use and adaptability. Suga et al.20 were the first to report a CG-induced peritoneal fibrosis rat model in 1995. i.p. injections of 0.1% CG and 15% ethanol dissolved in 2 mL of saline were used daily for 26 days. IshiI et al.21 used C57BL/6 mice and administered 0.3 mL of 0.1% CG with 15% ethanol dissolved in saline i.p. injection for a total of 56 days, where an experimental sclerosing encapsulating peritonitis was induced in mice. Nishino et al.22 used Wistar rats that received i.p. injections of 0.1% CG daily in 15% ethanol dissolved in 2 mL of saline for 28 days. Mishima et al.23 used a similar method to induce peritoneal fibrosis in Sprague-Dawley (SD) rats in the same year. Kushiyama et al.24 used SD rats and administered 0.1% CG in 15% ethanol dissolved in saline (1.5 mL/100 g body weight) i.p. injections three times a week for 21 days. Nishino et al.25 used mice daily, administering an injection of 0.1% CG in 15% ethanol intraperitoneally, dissolved in 0.2 mL of saline for 7 days and 15 days. Lua et al.26 used tamoxifen emulsified in sesame oil at 12.5 mg/mL, dissolved in ethanol, and i.p. injected into mice at 100 mg/g body weight during a 3-day interval. After 2 weeks, 0.1% CG in 15% ethanol/phosphate-buffered saline (1.5 mL/100 g) was injected to the mice every other day for a total of 10 doses. Yoh et al.14 used SD rats and administered 1.5 mL/100 g body weight of 0.1% CG in 15% ethanol dissolved in saline i.p. injections three times a week for 21 days. Yoh et al. used 10-week-old mice and administered 0.1% CG (0.01 ml/g body weight) in 15% ethanol i.p. injections three times a week for a total of 21 days. In the same year, lo et al.13 also used a similar method.
The present model has some limitations. First, in this animal model, CG was used as a chemical stimulant to induce functional deterioration due to peritoneal fibrosis instead of dialysate. CG is a chemical irritant, and its repeated administration can lead to degeneration of mesothelial cells and inflammatory responses, thus causing excessive fibrosis. Inflammation and neovascularization were observed, and these findings were similar to those observed in patients with PD. Although CG injections result in significant peritoneal thickening, a previous study showed that the fibrin deposition was relatively weaker27. Second, the mice used in the present study did not have kidney disease; consequently, the effect of uremic toxins on the peritoneum could not be assessed. Third, we did not evaluate inflammation, angiogenesis, and extracellular matrix deposition in the peritoneum. However, according to a previous study13, the same animal model has already shown that the numbers of both F4/80-positive cells and CD31-positive vessels increased after CG exposure. Therefore, it must be noted that the results obtained in this animal model cannot fully represent the condition of PD in peritoneal dialysis patients. In patients with PD, the mechanism of peritoneal damage is complex and may follow different patterns.
Despite all these limitations, the present model is simple, easy to use, and feasible compared to other animal models of PD, according to the previous studies3,13,14,16,18,25. This method represents a PD-related peritoneal fibrosis model that can be applied to PD field research.

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><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 H&lt;sub&gt;2&lt;/sub&gt;O</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, peritoneal fibrosis is more severe than in the control saline group (NS). In Figure 3A, the ultrafiltration rate decreased in the CG group, and glucose mass transport indicated that the peritoneal permeability increased in the CG-induced group (Figure 3B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63903/63903fig01.jpg" /> 
Figure 1: Fibrosis of the parietal peritoneum of the abdominal wall in peritoneal dialysis (PD) mouse model. (A) For the group exposed to CG, peritoneal fibrosis is more severe than in the control group (NS) under Masson&#39;s trichrome staining. (B) Quantified data of (A) represented as the mean &#177; standard deviation, n &#8805; 3; P &#60; 0.01. For (A), scale bar = 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63903/63903fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63903/63903fig02.jpg" /> 
Figure 2: Fibrosis of the visceral peritoneum of liver surfaces in peritoneal dialysis (PD) mouse model. (A) For the group exposed to CG, peritoneal fibrosis is more severe than in the control group (NS) under Masson&#39;s trichrome staining. (B) Quantified data of (A) represented as the mean &#177; standard deviation, n &#8805; 3; P &#60; 0.005. For (A), scale bar = 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63903/63903fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63903/63903fig03.jpg" /> 
Figure 3: Deterioration of peritoneal function in the PD mouse model. (A) In the chlorhexidine gluconate-exposed group (CG), the ultrafiltration rate was significantly lower than in the control saline group (NS). (B) The glucose mass transport also indicated that CG-induced an increase in peritoneal permeability. Data are represented as the mean &#177; standard deviation, n &#8805; 3; P &#60; 0.005. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63903/63903fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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Nanyang Technological University

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1.7K Views.  I-Shou University. Het vaststellen van een diermodel voor peritoneale dialyse, PD, is erg belangrijk. We hopen dat het de technologie en de werkzaamheid van PD-therapie kan verbeteren en uiteindelijk de overleving van de patiënt kan verbeteren. Dit peritoneale dialysemuismodel is eenvoudig, gemakkelijk te gebruiken en zichtbaar in vergelijking met andere peritoneale dialysediermodellen.Een geschikt peritoneaal dialysediermodel kan onderzoekers helpen om de peritoneale, fysiologische en pathologische ver...

Video: A Mice Model of Chlorhexidine Gluconate-Induced Peritoneal Damage

An Intravital Microscopy-Based Approach to Assess Intestinal Permeability and Epithelial Cell Shedding Performance

Intravital microscopy of the gut using confocal imaging allows real time observation of epithelial cell shedding and barrier leakage in living animals. Therefore, the intestinal mucosa of anesthetized mice is topically stained with unspecific staining (acriflavine) and a fluorescent tracer (rhodamine-B dextran), mounted on a saline solution-rinsed plate and directly imaged using a confocal microscope. This technique can complement other non-invasive techniques to identify leakage of intestinal permeability, such as transmucosal passage of orally administered tracers. Besides this, the approach presented here allows the direct observation of cell shedding events at real-time. In combination with appropriate fluorescent reporter mice, this approach is suitable for shedding light into cellular and molecular mechanisms controlling intestinal epithelial cell extrusion, as well as to other biological processes. In the last decades, interesting studies using intravital microscopy have contributed to knowledge on endothelial permeability, immune cell gut homing, immune-epithelial communication and invasion of luminal components, among others. Together, the protocol presented here would not only help increase the understanding of mechanisms controlling epithelial cell extrusion, but could also be the basis for the developmental of other approaches to be used as instruments to visualize other highly dynamic cellular process, even in other tissues. Among technical limitations, optical properties of the specific tissue, as well as the selected imaging technology and microscope configuration, would in turn, determine the imaging working distance, and resolution of acquired images.

Taking advantage of intravital microscopy, the method presented here enables real-time visualization of intestinal epithelial cell shedding in living animals. Therefore, topically stained intestinal mucosa (acriflavine and rhodamineB-dextran) of anesthetized mice is imaged up to single-cell resolution using confocal microscopy.

Intravital microscopy of the gut using confocal imaging allows real time observation of epithelial cell shedding and barrier leakage in living animals. ...

Immunology and Infection

Protocol to Create Chronic Wounds in Diabetic Mice

Chronic wounds develop as a result of defective regulation in one or more complex cellular and molecular processes involved in proper healing. They impact ~6.5M people and cost ~$40B/year in the US alone. Although a significant effort has been invested in understanding how chronic wounds develop in humans, fundamental questions remain unanswered. Recently, we developed a novel mouse model for diabetic chronic wounds that have many characteristics of human chronic wounds. Using db/db-/- mice, we can generate chronic wounds by inducing high levels of oxidative stress (OS) in the wound tissue immediately after wounding, using a one-time treatment with inhibitors specific to the antioxidant enzymes catalase and glutathione peroxidase. These wounds have high levels of OS, develop biofilm naturally, become fully chronic within 20 days after treatment and can remain open more for more than 60 days. This novel model has many features of diabetic chronic wounds in humans and therefore can contribute significantly to advancing fundamental understanding of how wounds become chronic. This is a major breakthrough because chronic wounds in humans cause significant pain and distress to patients and result in amputation if unresolved. Moreover, these wounds are very expensive and time-consuming to treat, and lead to significant loss of personal income to patients. Advancements in this field of study through the use of our chronic wound model can significantly improve health care for millions who suffer under this debilitating condition. In this protocol, we describe in great detail the procedure to cause acute wounds to become chronic, which has not been done before.

Chronic wounds are developed from acute wounds on a diabetic mouse model by inducing high levels of oxidative stress after a full-thickness cutaneous wound. The wound is treated with inhibitors for catalase and glutathione peroxidase, resulting in impaired healing and biofilm development by bacteria present in the skin microbiome.

Chronic wounds develop as a result of defective regulation in one or more complex cellular and molecular processes involved in proper healing. They impact ...

A Clinically Relevant Murine Model of Peritoneal Fibrosis by Dialysate and Catheters

Peritoneal fibrosis can occur in patients undergoing peritoneal dialysis (PD), and patients with severe peritoneal fibrosis have high morbidity and mortality. Peritonitis, high-glucose peritoneal dialysis fluid, and a long period of PD precipitate the onset of peritoneal fibrosis. An animal study of peritoneal fibrosis is needed due to the limitations of human and in vitro studies. However, most animal models do not mimic clinical conditions. To study peritoneal fibrosis, we developed a clinically relevant murine model by implanting a peritoneal catheter and injecting 2.5% high-glucose PD fluid plus 20 mM methylglyoxal (MGO) into the peritoneal cavity daily for 21 days. Implantation of the peritoneal catheter avoids peritoneal injury by needles and mimics clinical PD patients. Immunofluorescence staining showed that myofibroblasts accumulated in the fibrotic peritoneum. The experimental group had lower ultrafiltration volume and peritoneal membrane transport function (peritoneal equilibration test). In this article, we provide a detailed protocol of the model.

Patients with severe peritoneal fibrosis have high morbidity and mortality. The mechanism of peritoneal fibrosis is unclear. In this study, we describe a simple experimental murine model of peritoneal fibrosis induced by the injection of peritoneal dialysis fluid.

Peritoneal fibrosis can occur in patients undergoing peritoneal dialysis (PD), and patients with severe peritoneal fibrosis have high morbidity and ...

Establishing a Mouse Model for Candida albicans-Associated Catheter-Related Infection

A Catheter-Related Candida albicans Infection Model in Mouse

Catheter-related infection (CRI) is a common nosocomial infection caused by candida albicans during catheter implantation. Typically, biofilms are formed on the outer surface of the catheter and lead to disseminated infections, which are fatal to patients. There are no effective prevention and treatment management in clinics. Therefore, it is urgent to establish an animal model of CRI for the preclinical screening of new strategies for its prevention and treatment. In this study, a polyethylene catheter, a widely used medical catheter, was inserted into the back of the BALB/c mice after hair removal. Candida albicans ATCC MYA-2876 (SC5314) expressing enhanced green fluorescent protein was subsequently inoculated on the skin's surface along the catheter. Intense fluorescence was observed on the surface of the catheter under a fluorescent microscope 3 days later. Mature and thick biofilms were found on the surface of the catheter via scanning electron microscopy. These results indicated the adhesion, colonization, and biofilm formation of candida albicans on the surface of the catheter. The hyperplasia of the epidermis and the infiltration of inflammatory cells in the skin specimens indicated the histopathological changes of the CRI-associated skin. To sum up, a mouse CRI model was successfully established. This model is expected to be helpful in the research and development of therapeutic management for candida albicans associated CRI.

Author Spotlight: Enhancing Candida albicans Detection in Catheter Infections Using Fluorescent Protein Tagging 

We establish a mouse model of C.albicans-associated catheter-related infection (CRI), in which biofilm forms on the catheter, and the interaction between C.albicans and host correlates well with the clinical CRI. This model helps screen therapies for C.albicans biofilm-associated CRI, laying a foundation for clinical transformation.

Catheter-related infection (CRI) is a common nosocomial infection caused by candida albicans during catheter implantation. Typically, biofilms are formed ...

Inducing Pancreatic Injury in Mice Through Intraperitoneal Drug Injection

Preparing a Mice Model of Severe Acute Pancreatitis via a Combination of Caerulein and Lipopolysaccharide Intraperitoneal Injection

The treatment of severe acute pancreatitis (SAP), with high mortality rates, poses a significant clinical challenge. Investigating the pathological changes associated with SAP using animal models can aid in identifying potential therapeutic targets and exploring novel treatment approaches. Previous studies primarily induced pancreatic injury through retrograde bile duct injection of sodium taviaurocholate, but the impact of surgical damage on the quality of the animal model remains unclear. In this study, we employed various frequencies of intraperitoneal Caerulein injections combined with different doses of LPS to induce pancreatic injury in C57BL/6J mice and compared the extent of injury across five intraperitoneal injection protocols. Regarding inducing acute pancreatitis in mice, an intraperitoneal injection protocol is proposed that results in a mortality rate as high as 80% within 5 days. Specifically, mice received ten daily intraperitoneal injections of Caerulein (50 &#181;g/kg), followed by an injection of LPS (15 mg/kg) one hour after the last Caerulein administration. By adjusting the frequency and dosage of injected medications, one can manipulate the severity of pancreatic injury effectively. This model exhibits strong controllability and has a short replication cycle, making it feasible for completion by a single researcher without requiring expensive equipment. It conveniently and accurately simulates key disease characteristics observed in human SAP while demonstrating a high degree of reproducibility.

Intraperitoneal drug administration is a safe and effective non-invasive approach for inducing pancreatic injury. This study compared five distinct intraperitoneal injection protocols on mice to induce varying degrees of pancreatic injury and established a model of severe pancreatic injury to investigate the pathological changes and treatment strategies for severe acute pancreatitis (SAP).

The treatment of severe acute pancreatitis (SAP), with high mortality rates, poses a significant clinical challenge. Investigating the pathological ...

A Retrograde Implantation Approach for Peritoneal Dialysis Catheter Placement in Mice

Murine models are employed to probe various aspects of peritoneal dialysis (PD), such as peritoneal inflammation and fibrosis. These events drive peritoneal membrane failure in humans, which remains an area of intense investigation due to its profound clinical implications in managing patients with end-stage kidney disease (ESKD). Despite the clinical importance of PD and its related complications, current experimental murine models suffer from key technical challenges that compromise the models&#39; performance. These include PD catheter migration and kinking and usually warrant earlier catheter removal. These limitations also drive the need for a greater number of animals to complete a study. Addressing these drawbacks, this study introduces technical improvements and surgical nuances to prevent commonly observed PD catheter complications in a murine model. Moreover, this modified model is validated by inducing peritoneal inflammation and fibrosis using lipopolysaccharide injections. In essence, this paper describes an improved method to create an experimental model of PD.

This article describes modifications of a procedure to implant a peritoneal dialysis catheter in a murine model to avoid major technical issues observed with the conventional techniques.

Murine models are employed to probe various aspects of peritoneal dialysis (PD), such as peritoneal inflammation and fibrosis. These events drive ...

Rodent Model of Intestinal Ischemia-Reperfusion Injury via Occlusion of the Superior Mesenteric Artery

Intestinal ischemia-reperfusion injury (IRI) is associated with a myriad of conditions in both veterinary and human medicine. Intestinal IRI conditions, such as gastric dilatation volvulus (GDV), mesenteric torsion, and colic, are observed in animals such as dogs and horses. An initial interruption of blood flow causes tissues to become ischemic. Although necessary to salvage viable tissue, subsequent reperfusion can induce further injury. The main mechanism responsible for IRI is free radical formation upon reperfusion and reintroduction of oxygen into damaged tissue, but there are many other components involved. The resulting local and systemic effects often impart a poor prognosis. 
Intestinal IRI has been the subject of extensive research over the past 50 years. An in vivo rodent model in which the base of the superior mesenteric artery (SMA) is temporarily ligated is currently the most common method used to study intestinal IRI. Here, we describe a model of intestinal IRI utilizing isoflurane anesthesia in 21% O2 medical air that yields reproducible injury, as demonstrated by consistent histopathology of the small intestines. Tissue injury was also assessed in the colon, liver, and kidneys.

We describe how to generate a widely used surgical model of intestinal ischemia-reperfusion injury (IRI) in rodents. The procedure involves occlusion of the superior mesenteric artery followed by the restoration of blood flow. This model is useful for studies investigating occlusive causes of intestinal IRI in both veterinary and human medicine.

Intestinal ischemia-reperfusion injury (IRI) is associated with a myriad of conditions in both veterinary and human medicine. Intestinal IRI conditions, ...

Inducing Apical Periodontitis in Mice

The mechanisms involved in local induced inflammation can be studied using several available animal models. One of these is the induction of apical periodontitis (AP). Apical periodontitis is a common pathology of an inflammatory nature in the periodontal tissues surrounding the tooth root. In order to better understand the nature and mechanism of this pathology it is advantageous to perform the procedure in mice. The induction of this odontogenic inflammation is achieved by drilling into the mouse tooth until the dental pulp is exposed. Next, the tooth pulp remains exposed to be contaminated by the natural oral flora over time, causing apical periodontitis. After this time period, the animal is sacrificed, and the tooth and the jaw bone can be analyzed in various ways. Typical analyses include micro-CT imaging (to evaluate bone resorption), histological staining, immunohistochemistry, and RNA expression. This protocol is useful for research in the field of oral biology to better understand this inflammatory process in an in vivo experimental setting with uniform conditions. The procedure requires a careful handling of the mice and the isolated jaw, and a visual demonstration of the technique is useful. All technical aspects of the procedures leading to induced apical periodontitis and its characterization in a mouse model are demonstrated.

Here, we present a protocol to locally induce apical periodontitis in mice. We show how to drill a hole in the mouse&#39;s tooth and expose its pulp, in order to cause local inflammation. Analysis methods to investigate the nature of this inflammation, such as micro-CT and histology, are also demonstrated.

The mechanisms involved in local induced inflammation can be studied using several available animal models. One of these is the induction of apical ...

Biology

Investigating the Molecular Mechanisms Underlying Murine Abdominal Aortic Aneurysm

A Mouse Abdominal Aortic Aneurysm Model by Periadventitial Calcium Chloride and Elastase Infiltration

Abdominal aortic aneurysm (AAA) is a life-threatening disease associated with high mortality rates. It is characterized by the permanent dilation of the abdominal aorta with at least a 50% increase in arterial diameter. Various animal models of AAA have been introduced to mimic the pathophysiological changes and study the underlying mechanisms of AAA. Among these models, the calcium chloride (CaCl2)- and elastase-induced AAA models are commonly used in mice. However, these methods have certain limitations. Traditional intraluminal porcine pancreatic elastase (PPE) perfusion is associated with high technical difficulty and a high rupture rate, while periadventitial administration of PPE yields inconsistent results. In addition, the CaCl2-induced AAA model lacks human AAA features, such as atherothrombosis and aneurysm rupture. Therefore, the combined application of CaCl2 and PPE has been proposed as an approach to enhance success rates and induce greater diameter increases in AAA animal models. This manuscript presents a comprehensive protocol for establishing a mouse AAA model through periaortic infiltration of PPE and CaCl2 in the infrarenal segment of the abdominal aorta. By following this protocol, we can achieve an AAA formation rate of approximately 90% with technical simplicity and reproducibility. Further ultrasound and histological experiments confirm that this model effectively replicates the morphological and pathological changes observed in human AAA.

Author Spotlight: Development and Characterization of a Mouse Model for Abdominal Aortic Aneurysm

This manuscript presents a protocol for establishing a mouse abdominal aortic aneurysm model using calcium chloride and elastase, combining the advantages of previous modeling methods. This model can be utilized to investigate the pathophysiological mechanisms underlying abdominal aortic aneurysms.

Abdominal aortic aneurysm (AAA) is a life-threatening disease associated with high mortality rates. It is characterized by the permanent dilation of the ...

Optimized Protocol for Mouse Wound Healing Model with Stem Cell-Derived Exosomes

Mouse Wound Models and Preparation of Single-Cell Suspensions 

The management of acute full-thickness skin injuries presents a considerable challenge in clinical practice. The complexity of wound healing, involving diverse cell populations and the intricate wound microenvironment, complicates the assessment of treatment effects and their interactions using traditional experimental approaches. Single-cell transcriptome sequencing technology has revolutionized the understanding of cellular functions and mechanisms during wound healing. However, the variability in methodologies for creating mouse wound models introduces confounding factors that can impact experimental results. Furthermore, the process of dissociating mouse skin tissues presents significant technical hurdles, highlighting the critical need for high-quality single-cell suspensions for accurate transcriptome sequencing. Therefore, this study aims to optimize the construction of mouse wound models to accurately assess changes in wound closure while eliminating variables that could influence the subsequent sequencing result. Additionally, the preparation of single-cell suspensions was performed using both enzyme preparation protocols and test kit protocols to optimize cost and effectiveness.

Mouse Wound Models and Preparation of Single-Cell Suspensions

This study presents two crucial experimental techniques: the construction of a murine wound model for assessing wound closure and the preparation of single-cell suspensions from murine skin to preserve cellular viability.

The management of acute full-thickness skin injuries presents a considerable challenge in clinical practice. The complexity of wound healing, involving ...

A Mice Model of Chlorhexidine Gluconate-Induced Peritoneal Damage

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