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. 2001 Jul;49(1):56–65. doi: 10.1136/gut.49.1.56

Involvement of nerves and calcium channels in the intestinal response to Clostridium difficile toxin A: an experimental study in rats in vivo

J Sorensson 1, M Jodal 1, O Lundgren 1
PMCID: PMC1728359  PMID: 11413111

Abstract

BACKGROUND—The involvement of nerves and calcium channels in the intestinal response to Clostridium difficile toxin A (luminal concentration 1 or 15 µg/ml) was studied in the small intestine of rats in vivo.
METHODS—Inflammation was quantified by estimating myeloperoxidase (MPO) activity in the intestinal lumen, extravascular accumulation of Evan's blue (EB) in the intestine, and number of red blood cells (RBCs) in veins in histological sections. Intestinal damage was estimated using a histological grading system. In some experiments net fluid transport was recorded using a gravimetric technique.
RESULTS—In acutely denervated intestines, toxin A caused marked destruction of the villi, increased luminal release of MPO activity, and augmentation of intestinal content of EB and venous RBCs. Denervating the intestine 3-4 weeks prior to the actual experiment prevented the development of villus damage and significantly decreased the number of RBCs in intestinal veins in experiments with a low toxin concentration, whereas no effect was demonstrated on luminal MPO activity. Using a high toxin concentration, chronic denervation decreased only the number of RBCs. Pretreatment with hexamethonium (low toxin concentration; acute denervation) attenuated the effect of toxin A on morphology, luminal MPO activity, and number of RBCs. Pretreatment with nifedipine (low toxin concentration; acute denervation) significantly decreased intestinal MPO activity and number of RBCs. Tissue accumulation of EB was not influenced by experimental manipulation. Net fluid transport was measured in experiments exposing the intestinal mucosa to a high toxin concentration. Fluid secretion caused by the toxin was significantly attenuated by intravenous hexamethonium whereas no effect was observed after administration of nifedipine or granisetron.
CONCLUSIONS—At a low toxin concentration, intramural reflexes are involved in the inflammatory response whereas axon reflexes contribute to tissue damage. At a high toxin concentration no nervous involvement in the toxin A response was demonstrated except for fluid secretion evoked by the toxin.


Keywords: cholera toxin; enteric nervous system; 5-hydroxytryptamine; myeloperoxidase; red blood cells

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Figure 1  .

Figure 1  

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Effect of Clostridium difficile toxin A (1 µg/ml physiological saline) on myeloperoxidase (MPO) activity in the intestinal lumen at various times after exposing one segment to the toxin. A control segment was only exposed to physiological saline. (A) Experiments (n=6) in which the intestines were acutely denervated by cutting the periarterial nerves. (B) Results from the experiments (n=5) in which the intestines were denervated 4-6 weeks prior to the experiments ("chronic denervation"). Values are given as mean (SEM).

Figure 2  .

Figure 2  

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Effect of Clostridium difficile toxin A (15 µg/ml physiological saline) on myeoloperoxidase activity (MPO) in the intestinal lumen at various times after exposing one segment to the toxin. A control segment was only exposed to physiological saline. (A) Experiments (n=6) in which the intestines were acutely denervated by cutting the periarterial nerves. (B) Results from experiments (n=5) in which the intestines were denervated 4-6 weeks prior to the experiments ("chronic denervation"). Values are given as mean (SEM).

Figure 3  .

Figure 3  

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Effect of Clostridium difficile toxin A (1 µg/ml physiological saline) on myeoloperoxidase activity (MPO) in the lumen of acutely denervated intestinal segments of animals pretreated with hexamethonium, a nicotinic receptor blocker. MPO activity at various times after exposing one segment to the toxin is shown. A control segment was only exposed to physiological saline. MPO activity response to the toxin was markedly attenuated by the drug (compare figs 1 and 3). Values are mean (SEM); n=5.

Figure 4  .

Figure 4  

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Effect of Clostridium difficile toxin A (1 µg/ml physiological saline) on myeoloperoxidase activity (MPO) in the intestinal lumen in animals pretreated with nifedipine, a blocker of calcium channels of the L-type. Intestinal segments were acutely denervated. MPO activity at various times after exposing one segment to the toxin is shown. A control segment was only exposed to physiological saline. MPO activity response to the toxin was markedly attenuated by the drug (compare figs 1 and 4). Values are mean (SEM); n=6.

Figure 5  .

Figure 5  

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Hypothetical model based on the findings of the present study. Nerves participate both in the development of the inflammatory response and in the events leading to tissue damage, at least when toxin concentrations are not too high. The results of the present study suggest that an intramural reflex(es) is involved in the toxin response leading to inflammation whereas an axon reflex(es) participates in the development of tissue damage.

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