Rat Models of Acute and/or Chronic Peritoneal Injuries Including Peritoneal Fibrosis and Peritoneal Dialysis Complications

Fig. 1

Histopathological changes in peritonitis induced by mechanical scraping or in peritonitis induced by mechanical scraping following repeated zymosan injections. Frames a, b, e, f, i, j, m, and n are peritoneum with mechanical scraping in rats, and frames c, d, g, h, k, l, o, and p are peritoneum with mechanical scraping and repeated zymosan injections. Left frame of each set shows the macroscopic appearance of parietal peritoneum. Right side of each set shows the microscopic appearance stained with H&E. The original magnifications were 100. The timing of sacrifice is displayed on the left side for each set. Arrows in frame c indicate small plaques. On day 5, fusion of the plaques was observed, arrowed around red swelling (magnified and shown with arrow in right bottom corner). On day 18, the surface of peritoneum was covered with white fibrous tissue and subperitoneal vessels were obscured (arrowheads in frame k). Fibrous tissue formation was accompanied with neoangiogenesis (arrowheads in right bottom corner in frame k). White arrowhead shows vessels obscured under thickened peritoneum on day 36 (frame o). External face of peritoneum. Scale bars are in the upper left corner of frame a for macroscopic appearance and b for microscopic appearance. Reproduced from ref. [6], with permission of the American Association of Immunologists (Copyright 2009. The American Association of Immunologists, Inc.)

1.2 A Rat Model of Peritonitis Induced with Injection of a Zymosan Suspension after Mechanical Scraping of the Peritoneum

This model is a modification of the above peritonitis model caused by mechanical scraping, adding five daily administrations of zymosan [6]. Zymosan is a cellular component of yeast as a fungus and activates the complement system through the alternative pathway [11]. In PD patients, fungal peritonitis is known as a severe complication with poor prognosis. A single episode of fungal infection has been reported as sufficient to cause the development of EPS [1]. This model is ideally suited to studying the pathogenesis of fungal peritonitis in PD patients. In this model, a large accumulation of inflammatory cells in the peritoneum is clearly observed on day 3 with thickening of the subperitoneum. Interestingly, in most animals, an accumulation of inflammatory cells is still apparent on day 36 (Fig. 1).

2 Materials

2.1 Animals

We use 7-week-old Sprague-Dawley rats (Japan SLC, Hamamatsu, Japan), 210–230 g initial body weight. Animals are acclimatized for a few days before any treatment and maintained on a 12-h light/dark cycle under conventional laboratory conditions, with free access to water and food.

2.2 Acute Peritoneal Injury Model

Disinfectant for surgical wounds: Ethanol (70–80 % v/v) to clean the animal skin before the surgical procedure (see Note 1 ).

Anesthetic: Pentobarbital sodium (see Note 2 ).

Surgical instruments: Operating scissors (straight sharp/blunt) and straight iris scissors, thumb forceps, blunt-nose thumb forceps, Kocher forceps, mosquito forceps, and a needle holder. Preoperatively, surgical instruments should be sterilized by dry-heat sterilization (2 h at 160 °C). Surgical procedures are performed under aseptic conditions.

Sterilized cotton gauze.

Ethanol.

Sterile isotonic saline (0.9 % NaCl).

Pre-sterilized or disposable sutures (3-0 nylon) and surgical needles (reverse cutting edge and 1/2 circle by choice).

15-mL sterile polypropylene tubes (BD Falcon Conical Centrifuge tubes; BD Biosciences, Bedford, MA, USA).

2.3 Peritonitis Model

The following additional materials are required for the zymosan A model:

50-mL sterile polypropylene tubes (BD Falcon Conical Centrifuge tubes).

Zymosan A (Sigma-Aldrich, St. Louis, MO, USA).

Dianeal PD-4 4.25 % used as a 4.25 % acidic PD fluid (PDF) (pH ~5.0; Baxter, Tokyo, Japan) [6, 7] or Dianeal-N PD-4 2.5 % used as a 1.5 % neutral PDF (pH ~6.4; Baxter [12] to prepare a zymosan suspension.

2.4 Tissue Processing

Fixative: 10 % buffered formalin to fix peritoneal tissues for light microscopy (LM) study.

Graded alcohols (50, 75, 100 % v/v) for dehydration and clearing solvent.

Paraffin embedding wax and cassettes.

Cryogenic embedding compound: OCT™ compound (Sakura Fine Technical, Tokyo, Japan) for immunofluorescence (IF).

Liquid nitrogen.

3 Methods

The following animal studies have been approved by the Animal Experimentation Committee of Nagoya University Graduate School of Medicine and are performed according to the Animal Experimentation Guidelines of Nagoya University Graduate School of Medicine (Nagoya, Japan).