A Mice Model of Chlorhexidine Gluconate-Induced Peritoneal Damage

Summary

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.

Abstract

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.

Introduction

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 treatment^1,2,3,4,5,6. Peritoneal fibrosis is one of the most serious complications^7,8. Peritoneal fibrosis is characterized by the deposition and accumulation of extracellular matrix within the interstitium, and neo-angiogenesis and vasculopathy of the peritoneum^9,10.

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 accumulation^11,12. 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 previously^13,14. This PD mouse model is simple, easy to use, and feasible compared to other PD animal models.

Protocol

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

1. Prepare the chemical irritant by diluting 0.1% chlorhexidine gluconate (CG, see Table of Materials) in 15% ethanol.

2. Animal treatment

1. 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.
2. 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 µL/g body weight. Perform this every other day for 3 weeks, for a total of nine times.

3. Peritoneal function tests (modified peritoneal equilibration test)

1. 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.
2. 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 µL/20 gw. Additionally, use vet ointment on the eyes to prevent dryness under anesthesia.
3. Perform i.p. instillation of the dialysis solution (2 mL/20 g bodyweight).
4. 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's abdomen and collect the intraperitoneal fluid with a syringe (defined as "volume 1"). Then, measure the weight of a clean and dry cotton and put the cotton into the mice'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 "volume 2"). The final dialysate volume is volume 1 plus volume 2.
5. Use 0.5 mL of dialysate sample (final dialysate) to measure the glucose concentration. This is the final dialysate glucose concentration.
6. Calculate the net ultrafiltration using the formula¹⁵:
   
7. Calculate the peritoneal permeability using the following formula¹⁵:
   

4. Tissue preparation of the abdominal wall muscle and liver and histological analysis

1. Sacrifice the mice via cardiac puncture (phlebotomy)^3,16.
2. Cut the abdominal wall (1 cm x 1 cm) and total hepatectomy. Fix the mice's abdominal wall and liver tissues overnight in 10% neutral buffered formalin.
3. Prepare 3 µm thick paraffin sections of the abdominal wall muscle and liver, and perform histological analysis following previously published report¹⁷.
4. Evaluate the parietal peritoneum of the abdominal wall and visceral peritoneum of the liver surfaces of the mice using morphometry¹⁸.
5. Perform statistical analyses using statistics and graphing software (see Table of Materials). Express all data as mean ± SD and analyze for statistical significance using a t-test¹⁹. Define values with P < 0.05 as significant results.

Representative Results

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.

Discussion

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 studies^{3,13,14,16,18,25}. This method represents a PD-related peritoneal fibrosis model that can be applied to PD field research.

Materials

Name	Company	Catalog Number	Comments
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	-