ANIMALS AND T SPIRALIS INFECTION

Male Sprague-Dawley rats (200–250 g) (purchased from Charles River Laboratories) were kept in filtered cages under controlled ambient temperature and light-dark cycle (14:10 hours). Rats were infected by administering 1.0 ml of phosphate buffered saline containing 7500 T spiralis larvae by gavage. The larvae were obtained from male CD1 mice infected 30–90 days previously byT spiralis using a previously described method.11 Animals had unrestrained access to water but were fasted for 18 hours before experiments. Rats were studied six days after infection and age matched non-infected animals served as controls. For immunohistochemistry, animals were killed by cervical dislocation. For functional studies, rats were anaesthetised with a ketamine hydrochloride (90 mg/kg) and xylazine (20 mg/kg) mixture given intramuscularly and were placed on a heating pad to maintain the animal's temperature at about 36°C throughout the duration of the study. Supplemental anaesthesia was given throughout the study as required. At the end of the pseudoaffective response experiment (see below), animals were killed by cervical dislocation. All experimental procedures were approved by the Animal Care Committee at McMaster University and were in accordance with guidelines of the Canadian Council on Animal Care.

PSEUDOAFFECTIVE CARDIOAUTONOMIC RESPONSE TO GASTRIC DISTENSION

The approach has been described in detail previously, including the afferent and efferent pathways involved.2 Briefly, the cardioautonomic reflex response to noxious gastric distension obtained in control animals was compared with the response observed in age, sex, and weight matched T spiralis infected rats (n=6 in both groups). Following laparotomy and duodenotomy, a 2×2 cm balloon was inserted into the duodenum and subsequently positioned into the stomach. The gastric volume-cardiac response relationship was then examined using volumes ranging from 3 to 15 ml, which were applied for a 60 second period. The response to each volume was studied on three separate occasions in each animal. Continuous electrocardiographic recordings were performed through three electrodes applied on the right and left shoulders and the left leg of the animal.

IMMUNOHISTOCHEMISTRY

Specimens of jejunum, DRG (T1-L1), and spinal cord obtained fromT spiralis infected and control rats (n=6 each group) were rapidly removed and processed for either routine histology or immunohistochemistry. For the latter, tissues were fixed by immersion in 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, for 6–8 hours at 4°C and subsequently placed in 25% sucrose in 0.1 M phosphate buffer for cryoprotection until sectioning. Specimens were cut with a cryostat at 10–12 μm, mounted onto chrome-alum gelatin coated slides, and processed with the immunofluorescence method, as previously described.12 The antibodies used included a rabbit polyclonal antibody anti-SP, directed against the C terminal portion of the tachykinin amino acid sequence (antibody code 8701, diluted 1:500),12 ,13 and a rabbit polyclonal anti-NK-1, directed against the C terminus of the intracellular loop of the receptor sequence (diluted 1:1000).14

Specificity studies included control experiments commonly applied in immunohistochemical protocols.12-14

Sections were analysed by a Leitz Orthoplan microscope equipped with a Ploem epi-illumination system with “I2” or “L2” filter cubes to detect fluorescence.

IMMUNOREACTIVE PATTERNS AND QUANTITATIVE ANALYSIS OF SP IMMUNOLABELLED GANGLION CELL BODIES

Tissue sections from both infected and controls rats were processed for immunohistochemistry simultaneously. Comparison between infected and control sections was performed in parallel by two examiners (RDeG and GB) in a blinded fashion.

The number (mean (SEM)) of immunolabelled SP neural cell bodies per section in each DRG (from T1 to L1) was evaluated by counting the labelled perikarya by means of direct microscopic analysis. This analysis was performed on 12 consecutive sections per each DRG of infected and control rats.

STATISTICAL ANALYSIS

All studies involved at least four rats. For quantitative analysis of SP labelled DRG neural cells, the Mann-Whitney test for unpaired data was used. Change in heart rate was expressed as per cent change in heart rate compared with the immediately preceding 60 second control period before distension. The Student's ttest and ANOVA (one and two way, and repeated measures) were used as appropriate. Statistical significance was determined at p<0.05.

PSEUDOAFFECTIVE CARDIAC RESPONSE TO GASTRIC DISTENSION

In control animals, as previously documented, gastric distension elicited a volume dependent pseudoaffective decrease in heart rate which was first present with volumes of 6 ml and maximal with volumes ranging from 9 to 15 ml (p<0.05 v resting heart rate). T spiralis infection did not change resting intraballoon pressures. In T spiralis infected animals (fig 1), there also was a similar volume dependent pseudoaffective cardioautonomic reflex response but while the magnitude of the response was comparable, there was a significant rightward shift in T spiralisinfected animals.

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Figure 1

Volume dependent pseudoaffective increase in heart rate associated with gastric distension in control and Trichinella spiralis infected rats. Data are presented as mean (SD) per cent decrease in heart rate compared with resting heart rate immediately before distension. *p<0.05 versus resting heart rate; †p<0.05 versus control animals.

JEJUNUM

Specificity was demonstrated by lack of immunoreaction in sections in which the primary antisera preadsorbed with the appropriate peptides.

In the jejunum of control rats, SP-IR (fig 2) was identified in varicose nerve endings targeting the different layers of the gut wall as well as in myenteric and submucosal ganglion cell bodies. In particular, nerve processes positive for SP-IR supplied themuscularis externa, the submucosal vasculature, and the mucosa (fig 2A). A rather dense network of SP labelled processes was found in the deep muscular plexus, the lining situated between the inner part of the muscle layer, and the submucosa of the jejunum. SP containing nerve fibres were also identified around stained and unstained ganglion cells of the submucosal and, to a lower extent, myenteric plexus (fig 2C). NK-1-IR (fig 3) was identified mainly in myenteric and submucosal neurones of the rat jejunum as well as in nerve fibres occasionally identified either within the myenteric plexus or distributed in the muscular layer. No NK-1 immunolabelling was identified around blood vessels in the submucosa or in the mucosa of the jejunum. However, in the deep muscular plexus of the jejunum, NK-1 staining was localised to non-neuronal cells whose appearance was that of the interstitial cells of Cajal (fig3A).

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Figure 2

Representative photomicrographs showing substance P (SP) immunoreactivity in the control (A, C) and Trichinella spiralis infected (B, D) jejunum. Note the marked increase in the density and staining intensity of the SP-immunoreactive (IR) nerve processes running throughout the villi in the inflamed jejunum (B) in comparison with controls (A). An increase in SP-IR was also found in myenteric neurones of the inflamed jejunum (D) in comparison with controls (C). cm, circular muscle; lm, longitudinal muscle; mp, myenteric plexus. Calibration bar, 25 μm.

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Figure 3

Neurokinin 1-immunoreactivity (NK-1-IR) in the jejunal deep muscular plexus of Trichinella spiralis infected (B) and control (A) rats. NK-1 receptor immunolabelling was identified in non-neuronal elements with the appearance of interstitial cells of Cajal (arrows in A). In inflamed jejunum (B), NK-1 immunostaining localised to the interstitial cells of Cajal (arrows) was markedly decreased in comparison with controls. cm, circular muscle; sm, submucosa. Calibration bar, 25 μm.

The distribution pattern of both SP and NK-1-IRs was not modified inT spiralis infected rats. However, in infected rats, SP containing nerves showed an increased number and staining intensity in the muscularis externaand mucosa (fig 2B) compared with controls (fig 2A). Also, SP-IR was markedly increased in submucosal and myenteric neurones (fig 2D) in the small intestine of infected animals in comparison with controls (fig2C). In contrast, NK-1-IR displayed a reduction in staining intensity both in enteric neurones (that is, myenteric and submucosal plexus) and particularly in the interstitial cells of Cajal (fig 3B) compared with controls (fig 3A).

DRG

SP immunolabelling was localised to small and medium sized neuronal cell bodies and nerves from T1 to L1 of control animals (fig4A). In infected animals, SP-IR cell bodies showed increased density and staining intensity (fig 4B). As shown in fig 5, these findings were confirmed by quantitative analysis which demonstrated a significantly increased number of SP containing neurones in infected compared with control rats (57 (8) v 23 (7) positive cell bodies, respectively; p<0.05). No NK-1-IR ganglion cell bodies were observed in any infected or control rats.

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Figure 4

Photomicrographs showing substance P-immunoreactive (SP-IR) neurones of the dorsal root ganglia (DRG) in Trichinella spiralis infected (B) and control (A) rats. Note the increased SP-IR in the small-medium sized DRG neurones of T spiralis infected (B) in comparison with controls (A). Calibration bar, 25 μm.

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Figure 5

Mean (SEM) number of substance P-immunoreactivity (SP-IR) dorsal root ganglia (DRG) neurones in the Trichinella spiralis infected and control rats. Neurokinin 1 (NK-1) is also shown, although this tachykinin receptor was not detected in any ganglion cells of either infected or control rats. *p<0.05 versus controls.

SPINAL CORD

In the spinal cord of control rats, SP (fig 6A, B) and to a minor extent NK-1 (fig 6C, D) labelled nerve processes were seen predominantly in the outermost laminae of the dorsal horn. Conversely, in infected rats, there was a marked increase in the density and staining intensity of SP containing nerves in the dorsal horn (fig 6B) whereas NK-1-IR was undetectable (fig 6D).

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Figure 6

Representative photomicrographs showing substance P-immunoreactivity (SP-IR) (A, B) and neurokinin 1-immunoreactivity (NK-1-IR) (C, D) in the spinal cord of control (A, C) and Trichinella spiralis infected (B, D) rats. In the spinal cord of control animals, SP immunostaining was detectable in nerve fibres (A) whereas NK-1-IR was visualised in neurones and nerve fibres (C) mainly identified in the outermost laminae of the dorsal horns. In T spiralis infected rats, there was a striking increase in the density and staining intensity of SP neural network (B) compared with controls (A). In relation to the SP increase, we found a marked decrease in NK-1-IR in the dorsal horn of T spiralis infected rats (D) compared with controls (C). Calibration bar, 25 μm.