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 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.
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'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.
All mouse experiments were approved by the Laboratory Animal Center of the E-DA Hospital/ I-Shou University and were handled according to the "Guide for the Care and Use of Laboratory Animals" (NRC, USA 2011). Male C57BL/6 mice, 7-8 weeks old, were used for the present study.
1. Chemical preparation
2. Animal treatment
3. Peritoneal function tests (modified peritoneal equilibration test)
4. Tissue preparation of the abdominal wall muscle and liver and histological analysis
In Figure 1A,B, the parietal peritoneum of the abdominal wall was markedly thicker and more fibrotic under Masson'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).
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's trichrome staining. (B) Quantified data of (A) represented as the mean ± standard deviation, n ≥ 3; P < 0.01. For (A), scale bar = 100 µm. Please click here to view a larger version of this figure.
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's trichrome staining. (B) Quantified data of (A) represented as the mean ± standard deviation, n ≥ 3; P < 0.005. For (A), scale bar = 100 µm. Please click here to view a larger version of this figure.
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 ± standard deviation, n ≥ 3; P < 0.005. Please click here to view a larger version of this figure.
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'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.
The authors have nothing to disclose.
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.
0.9% Normal Saline | Y F CHEMICAL CORP., New Taipei City, Taiwan | – | |
10% neutral buffered formalin | Taiwan Burnett International Co., Ltd., Taipei City, Taiwan | 00002A | |
Automatic biochemical analyzer | Hitachi Ltd., Tokyo, Japan | Labospect Series 008 | for determining glucose concentration |
Chlorhexidine digluconate solution, 20% in H2O | Sigma-Aldrich, MO, USA | C9394 | diluted to 0.1% with 15% ethanol for injection |
Ethanol | Avantor Performance Materials, LLC, PA, USA | BAKR8006-05 | diluted to 15% with normal saline for working concentration |
Glucose (Dianeal) | Baxter International, Inc., IL, USA | FNB9896 | Commercial dialysis solution (4.25%) |
GraphPad Prism 8.0 | GraphPad Software, Inc., CA, US | ||
L-type Glu 2 assay | FUJIFILM Wako, Japan | 461-32403 | |
Xylazine 20 | Juily Pharmaceutical Co., Ltd., New Taipei City, Taiwan | – | |
Zoletil 50 | Virbac Laboratories, Carros, France | – |