Article Text


Calpain inhibitor I and colonic inflammation induced by DNBS in the rat
  1. A Ballinger1,
  2. O Azooz1
  1. 1Department of Adult and Paediatric Gastroenterology, St Bartholomew's and the Royal London School of Medicine and Dentistry, London E1 2AT, UK;
  1. a.b.ballinger{at}
  1. C Thiemermann2,
  2. S Cuzzocrea3
  1. 2William Harvey Research Institute, St Bartholomew's and the Royal London School of Medicine and Dentistry, London, UK
  2. 3Department of Pharmacology, University of Messina, Italy

Statistics from

We read with interest the paper by Cuzzocrea et al in which they demonstrated the marked beneficial effects of calpain inhibitor I on colonic inflammation induced by dinitrobenzene sulphonic acid (DNBS) in the rat. On the basis of their findings, they suggest that calpain inhibitor I treatment may be useful in inflammatory bowel disease (Gut 2001;48:478–88).

It is debatable whether a model that is associated with a mortality of 40% and 80% at two and six days, respectively, after induction of colitis has any relevance to human disease. The different numbers given in the text (seven animals per group) and figure 4 (10 rats in each group) further confuse interpretation of the mortality data. The authors have apparently taken their methods for induction of colitis from previously published work although the paper that they quote described induction of ileitis (not colitis) in guinea pigs.1 We also use a rat model of colitis and would suggest that intrarectal administration of 20 mg trinitrobenzene sulphonic acid in 40% ethanol is associated with a mortality of less than 8% at seven days.

In a separate experiment, the authors assessed the effect of calpain I inhibitor on the histological severity of colitis, weight loss, and expression of adhesion molecules in the colon. It is not clear from either the methods or the results as to how many animals were in each group. In the text the authors state that seven animals were in the sham group. However, in figures 1, 3, 5, 6, and 9, values are given for the mean of 10 rats in each group. The numbers of animals in the colitis group is not given in the text but the figures states 10 rats/group. Was this 10 rats at four days after induction of colitis or 10 rats at the beginning of the experiment (day 0)? If 10 rats remained at four days, what was the starting number; presumably many more with a predicted mortality of about 60%?


Authors' reply

We very much appreciate the interest of Drs Ballinger and Azooz in our work.

In 1997, we discovered that calpain inhibitor I exerted potent anti-inflammatory effects in rats with endotoxin shock.1 We therefore speculated that calpain inhibitor I may reduce the degree of inflammation and tissue injury associated with other models of inflammation, including colitis. The model of hapten induced colitis used by us is similar, but not identical, to the one introduced by John Wallace and colleagues in 1989 (Gut 2001;48:478–8) in which instillation of a solution containing a “barrier breaker” (0.25 ml of 50% ethanol) and the hapten 2,4,6-trinitrobenzene sulphonic acid (TNBS 5–30 mg) are used to cause colonic ulceration and inflammation in the rat. Clearly, the severity of colitis caused and the mortality of any given model may vary with the experimental design used. We would like to propose that the high mortality associated with our model of hapten induced colitis is a reflection of the fact that our animals were exposed to the hapten for a relatively long period. As stated in the methods section of our article, adult male Wistar rats were anaesthetised with isoflurane and received intrarectal administration of 25 mg of dinitrobenzene sulphonic acid (DNBS) for a period of 15 minutes.2 Exposure of the colon of the mouse to 1 mg of TNBS even for very short periods (30 seconds) also results in a substantial degree of mortality3 but mortality may vary between studies even if the same experimental protocol is used by the same investigator.4 When investigating the effects of insulin-like growth factor 1 on the linear growth retardation associated with colitis,5 Ballinger et al exposed prepuberal rats (26 days old, anaesthetised with Hypnorm) of both sexes to 8 mg of TNBS in 40% ethanol for an unspecified period of time. When investigating the role of neuropeptide Y on the weight loss associated with experimental colitis, investigators from this group used intrarectal administration of 20 mg of TNBS (for an unspecified period of time) to cause colitis in male Wistar rats.6 Unfortunately, neither study provides any histological evidence of the severity of the colitis or any mortality data.5, 6 We understand from Dr Ballinger that the mortality of 8% cited in her letter is a summary of experiments carried out by her group over the last 4–5 years. These mortality values have not been published.

We apologise if the number of animals studied was less clear than we thought. All of the data outlined in figures 1, 3, 5, 6, and 9 were, indeed, based on results obtained from 10 animals (survivors). The n numbers of the individual groups at the beginning of the experiments were the following: sham operated treated with vehicle, n=10; sham operated animals treated with calpain inhibitor I, n=10; DNBS control, n=18; DNBS rats treated with calpain inhibitor I, n=13.

In the final paragraph of our article (Gut 2001;48:478–88), we stated that our findings suggest that calpain inhibitor I may be useful in conditions associated with local or systemic inflammation, including inflammatory bowel disease. In the last few months, we have reported that calpain inhibitor I also attenuates the multiple organ injury and dysfunction associated with haemorrhagic shock7 as well as the tissue injury associated with pleuritis (induced with carrageenan) and arthritis (induced by collagen).8 All of these findings support the view1 that calpain inhibitor I exerts potent anti-inflammatory effects in vivo.


View Abstract

Request permissions

If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.

Linked Articles