Hymenolepis diminuta: Effect of infection on ion transport in colon and blood picture of rats

Experimental Parasitology 124 (2010) 285–294

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Hymenolepis diminuta: Effect of infection on ion transport in colon and blood picture of rats Danuta I. Kosik-Bogacka a,*, Irena Baranowska-Bosiacka b, Rusłan Salamatin c a

Department of Biology and Medical Parasitology, Pomeranian Medical University, Powstancow Wielkopolskich Av. 72, 70-111 Szczecin, Poland Department of Biochemistry and Human Nutrition, Pomeranian Medical University, Powstancow Wielkopolskich Av. 72, 70-111 Szczecin, Poland c Department of General Biology and Parasitology, Medical University of Warsaw, 61 Zwirki i Wigury St., 02-091 Warsaw, Poland b

a r t i c l e

i n f o

Article history: Received 15 June 2009 Received in revised form 26 October 2009 Accepted 29 October 2009 Available online 4 November 2009 Keywords: Hymenolepis diminuta Cestode Hymenolepiosis Rat Colon Ion transport Ussing chamber

a b s t r a c t The aim of this study was to examine the effect of an infection with Hymenolepis diminuta on ion transport in an isolated colon and blood picture of rats. Fifty rats were orally infected with five cysticercoids of H. diminuta. The experimental groups of rats were assigned to four groups: group I – 8 days post-infection (dpi), group II – 16 dpi, group III – 40 dpi and group IV– 60 dpi. The control group comprised non-infected rats. The experiments consisted of measuring the transepithelial electrical potential difference (PD) and the transepithelial electrical resistance (R) of the rat colon under controlled conditions as well as during mechanical stimulation (MS) using a modified Ussing chamber. Ion transport was modified using inhibitors of the epithelial sodium channel (amiloride – AMI) and the epithelial chloride channel (bumetanide – BUME), and also using capsaicin (CAPSA), a substance which activates C-fibres. The experimental data presented in this study indicates that experimental hymenolepidosis inhibits sodium and chloride ion transport in the epithelium of the rat colon, with preserved tight junction continuity (except at 40 dpi) and a decreased mechanical sensitivity. The effect of capsaicin on ion transport in the rat colon was varied. In control rats it increased ionic current, and in H. diminuta-infected rats it did not cause any changes in PD. Blood picture in this study showed a statistically significantly lower red blood cells (RBC) count and haemoglobin (HGB) concentration in infected rats in comparison to non-infected. Red cell distribution width (RDW) values and platelet (PLT) count were negatively correlated with the duration of infection, whereas mean corpuscular volume (MCV) value was positively correlated. We did not observe leukocytosis during infection, and amongst the differential leukocyte counts eosinophils and basophils showed statistically significant lower values in infected rats in comparison to non-infected. Our results indicate that hymenolepidosis is associated with the activation of inflammatory mediators and stimulation of nervous fibres, which significantly affects the function of ion channels in the epithelium of the colon in the host. At the same time, a significant decrease in eosinophil count during infection suggests that such an infection did not trigger a strong immunological reaction in rats. Ó 2009 Elsevier Inc. All rights reserved.

1. Introduction Abbreviations: MS, mechanical stimulation; AMI, amiloride; BUME, bumetanide; CAPSA, capsaicine; PD, transepithelial electrical potential difference; dPD, the difference between the maximum stimulated value and control value of PD; R, transepithelial electrical resistance; SP, substance P; NANC, non-adreneric noncholinergic; dpi, days post-infection; WBC, white blood cell count; NEU, number and percentage of neutrophils; LYM, number and percentage of lymphocytes; MONO, number and percentage of monocytes; EOS, number and percentage of eosinophils; BASO, number and percentage of basophils; RBC, red blood cell count; HGB, haemoglobin; HCT, haematocrit; MCV, mean corpuscular volume; MCHC, mean corpuscular haemoglobin; RDW, red cell distribution width; PLT, platelet count; MPV, mean platelet volume; PCT, plateletocrit; PDW, platelet distribution width; C, control group. * Corresponding author. Fax: +48 91 4661671. E-mail address: [email protected] (D.I. Kosik-Bogacka). 0014-4894/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2009.10.014

Infections by gastrointestinal parasites cause immunological responses and pathophysiological changes in their hosts. Due to a close relationship between the local immune system and epithelial cells in the gastrointestinal tract, local immune reactions can directly change the epithelial ion transport, causing an increased secretion, decreased ion absorption or both. Hymenolepis diminuta is a parasite of the small intestine in rodents (mostly mice and rats), and accidentally in humans (Ishih and Uchikawa, 2000; McKay et al., 1991; Reardon et al., 2001). It has no hooks that could mechanically damage host tissues and as such is nontissue invasive tapeworm. However, metabolites produced by H. diminuta have been proved to disrupt the action of

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the gastrointestinal tract with increased secretion of saliva, inhibition of stomach secretion and increased activity of trypsin in the chyme in the duodenum (Fal and Czaplicka, 1991). Infected rats have also been observed to suffer from pathomorphological changes in the small intestine – inflammatory infiltrations in the acute phase of hymenolepidosis, and muscle-deep erosion by the 40th day post-infection (40 dpi). The chronic phase of infection by H. diminuta (60 dpi) shows the disappearance of intestinal villi, which leads to the disruption of the final stages of the digestive process and malabsorption (Fal and Czaplicka, 1991). It has been observed that in rats infected with H. diminuta, the entire small intestine is affected due to the large size of the cestode and circadian migrations associated with the presence of food in the gut (Podesta and Mettrick, 1974). However, the effect on ion transport in the colon has not yet been determined. Studies of ion transport in the epithelium of various organs started in 1958 by investigating the transport of ions in the skin of a frog (Koefoed-Johnsen and Ussing, 1958). At present, a method commonly known as the Ussing method is used in studies on ion transport, for example in the respiratory and gastrointestinal tracts. Most researchers use a voltage clamp and examine the short-circuit current (Riegler et al., 1999). In this work we used an open mode, which preserved functions of current-dependent channels (Cooke, 1994). The basic indicator of a quantifiable electrogenic ion transport is a transepithelial electrical potential difference, resulting, among other things, from the transport of sodium and chloride ions through ion channels situated in epithelial cells (Cooke, 1994). Although no diarrhoea has been reported in hymenolepidosis, it is possible that the invasion of H. diminuta affects the ion transport in the epithelium of both the large and small intestines. Therefore, the aim of this study was to examine the effect of an infection with H. diminuta on ion transport in the colon of the host, rat Rattus norvegicus. Maintenance of constant haematological values within certain limits is indispensable for the performance of the normal physiological function of the organism. Any alteration in such values may impair their metabolic activities. Because many reports on intestinal cestodes infection showed changes in blood pictures (Gill et al., 2007) we also examined the influence of hymenolepidosis on blood parameters in rats.

mie GmbH, Austria), administered in 100 mg/kg body weight (b.w.) intraperitoneally (i.p.). The rats were weighed, and then their blood and colon were taken for analysis. 2.2. Infection Cysticercoids of H. diminuta (strain WMS) were obtained from the culture of cestode infected Tribolium destructor. The cysticercoids taken from the bodies of the insects were then washed with saline solution and administered to the rats via stomach tube (5 cysticercoids in 1 ml of 0.9% NaCl solution). In rats infected with 5 cysticercoids of H. diminuta, from 3 to 5 tapeworms were recorded, the average number was 4.5 which equates to 90% of tapeworms in relation to the number of cysticercoids administered. 2.3. Isolation and incubation of colon The colons were cleared of adjacent connective tissue, cut along the mesentery, cleared of chyme and divided into fragments of about 2 cm2. The tissues were placed in an incubation liquid at 37 °C which was aerated by an external peristaltic pump. After 60 min incubation, the tissues were mounted in the Ussing chamber. The incubation and stimulation were performed using the following chemicals (concentrations in mM): Ringer solution (RH) (Na+ 147.2, K+ 4.0, Ca+ 4.4, Cl 155.6, Hepes 10.0) and RH with the additions: amiloride – AMI (0.1 mmol/l) and bumetanide – BUME (0.1 mmol/l) (all supplied by Aldrich–Sigma). The mechanical stimulation was a stream of fluid from the chamber of the using apparatus flushing the mucosa surface of the colon. The stream was ejected for 30 s from a nozzle 1.5 mm in diameter situated 12 mm from the surface of the intestine, and driven by the action of a peristaltic pump. Standard stimulation comprised eight to nine washes of liquid, total volume 2.45 ml. Besides the mechanical stimulation, a combined mechanical–chemical stimulation was also used (administration of AMI and BUME using a peristaltic pump). In the experiment, we also used a chemical stimulation of C-fibres – capsaicin (20 ml at a concentration of 0.0001%) delivered with a pipette (Aldrich–Sigma). The concentrations of the afore-

2. Materials and methods 2.1. Animals The study was carried out on 60 male Wistar rats, aged about 4 months at the beginning of the experiment. The experiment was carried out at the same time daily at about 9 am. The rats were housed singly, kept on a 12-h-light–dark cycle and were given standard feed and water ad libitum. The study was approved by the Local Ethic Committee for Scientific Experiments on Animals in Szczecin (Poland). The rats were divided into five groups:     

control group – not infected with H. diminuta group I – 8 days post-infection (8 dpi) group II – 16 days post-infection (16 dpi) group III – 40 days post-infection (40 dpi) group IV – 60 days post-infection (60 dpi).

Before each experiment, a coproscopic examination of the rat faeces was performed to ascertain the presence of parasites. After the experiment, rats from both the control and experimental groups were sacrificed with Tiopental anaesthesia (Bioche-

Fig. 1. Diagram of electrical circuit for evaluation of electrical parameters describing isolated epithelial organs according to Ussing’s method. Abbreviations use are: V1 and V2, Ag/AgCl electrodes for measuring transepithelial electrical potential, connecting voltmeter with measuring chamber through electrolyte agar bridges; I1 and I2, Ag/AgCl electrodes for measuring electrical current, connecting the voltage compensation device with measuring chamber through electrolyte agar bridges; mA, ammeter; mV, voltmeter.

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mentioned substances were based on available literature and our own experiments in which these substances were used on mammalian organs (trachea and colon) (Kosik-Bogacka and Kolodziejczyk, 2004; Kosik-Bogacka et al., 2005; Tyrakowski et al., 1998). 2.4. Measuring instruments The experiments used a modified Ussing apparatus. The modification of a conventional Ussing chamber consisted in placing the tissue horizontally. The chamber was connected with the equipment by agar bridges and Ag/AgCl electrodes (Fig. 1). In the experiments, we used an EVC 4000 voltage/current clamp apparatus (manufactured by WPI, USA) to measure voltage and resistance, connected to a BD 111 recorder (Kipp & Zonen, Holland).

WBC, RBC, HGB and PLT were measured directly, but other parameters (HTC, MCV, MCH, MCHC, RDW and PDW) are calculated automatically from the measured parameter data. Haemoglobin was measured by Perkin-Elmer, Lambda 40 UV/VIS spectrophotometer according to cyanmethemoglobin method at a wavelength of 540 nm. The principle of the five-part differential white blood count is based on the erythrocyte membrane lysis done by haemolysing reagents and followed by the differentiation of leukocyte types (NEU, LYM, MONO, EOS and BASO) according to the size of the cell or nucleus. These leukocyte types are detected by peaks and size of histograms (Operator’s, 2000). Literature results confirms that the precision of the analyser fulfils the reproducibility of testing parameters: WBC, RBC, HGB, MCV, MCH, MCHC and PLT (Suljevic et al., 2003). 2.7. Statistical analysis

Measurements in our experiment concerned transepithelial electrical potential difference – PD and transepithelial electrical resistance – R. PD was determined when the amperage of the compensatory current from the outer battery 0 mA and was recorded continuously. The transepithelial electrical potential difference was measured against ground from the serosal side (it was equal to zero), and hence the value on the display reflected the potential of the mucous membrane. R was calculated using Ohm’s law from voltage deflections after the 10 lA current. Each measurement was preceded by a control of the measuring instruments using a synthetic cellophane membrane placed in the Ussing Apparatus in Ringer’s solution (blind experiments).

The obtained results were analysed statistically using Statistica 6.1 software. Arithmetical mean and standard deviation (SD) were calculated for each of the studied parameters. As most of the distributions deviated from the normal distribution (Shapiro–Wilk test), non-parametric tests were used for the analyses. Correlations between the parameters were examined with Spearman’s rank correlation coefficient (Rs). To assess the differences between the parameters studied, Kruskal–Wallis ANOVA and Mann–Whitney U-tests were used. The level of significance was p < 0.05. All electrophysiological parameters are expressed as arithmetical mean SD. The Student’s t-test was used to determine the statistical significance of differences between means. The value of p < 0.05 was considered as the significance level. The analyses were performed using the Statgraphics software package.

2.6. Blood sample

3. Results

Whole blood from the heart of rats was sampled by 3.0 ml vacutainer blood tubes (Becton Dickinson Vacutainer Systems) containing K3EDTA as an anticoagulant. By means of a laser haematology analyser Cell-Dyn 3700 SL (Abbott-USA), 17 parameters were measured at the same time: white blood cell count (WBC), number and percentage of neutrophils (NEU), number and percentage of lymphocytes (LYM), number and percentage of monocytes (MONO), number and percentage of eosinophils (EOS), number and percentage of basophils (BASO), red blood cell count (RBC), haemoglobin (HGB), haematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCHC), red cell distribution width (RDW), platelet count (PLT), mean platelet volume (MPV), plateletocrit (PCT) and platelet distribution width (PDW).

3.1. Basic electrophysiological parameters in the colon epithelium of non-infected and tapeworm-infected rats

2.5. Measurement of electrophysiological parameters

In the colon of control rats (non-infected with H. diminuta, 0 dpi), PD was about 1.5 mV, and R was about 146 X cm2 (Table 1). Mechanical stimulation (MS) of the mucosal side of the isolated colon wall caused a hyperpolarization of tissues (dPD) as presented in Fig. 2A. An increase in PD was observed during MS, peaking at the end of stimulation. After 4–5 min, PD returned to the initial control value. The infection of rats with H. diminuta significantly affected the values of electrophysiological parameters. At 8 dpi, PD and dPD decreased by about 59% and 79%, respectively (Fig. 3A) and R increased by 23% compared to the control group. At 16 and 40 dpi,

Table 1 Basic electrophysiological parameters of the colon for control and Hymenolepis diminuta-infected rats. Parameters

Days post-infection (dpi) 0 (n = 10)

PD (mV) R (X cm2) MS1 (mV) AMI (mV) MS2 (mV) BUME (mV) MS3 (mV)

1.5 ± 0.01 146 ± 6.1 0.7 ± 0.2 0.1 ± 0.1 0.3 ± 0.0 0.0 ± 0.1 0.1 ± 0.1

8 (n = 12)

16 (n = 14)

0.62 ± 0.2* 180.22 ± 35.48 0.15 ± 0.04* 0.1 ± 0.03 0.9 ± 0.05* 0.01 ± 0.04 0.06 ± 0.05

0.43 ± 0.06* 132.78 ± 11.12 0.14 ± 0.03* 0.04 ± 0.02 0.05 ± 0.02* 0.11 ± 0.03 0.04 ± 0.05

40 (n = 12) 0.49 ± 0.08* 85 ± 8.1* 0.17 ± 0.2* 0.04 ± 0.04 0.05 ± 0.04* 0.09 ± 0.04* 0.19 ± 0.04

60 (n = 12) 0.1 ± 0.0* 136.4 ± 7.3 0.0 ± 0.0* 0.0 ± 0.0 0.1 ± 0.0* 0.0 ± 0.0 0.0 ± 0.0

Mean values and ±standard error of the mean are given. PD is a value of the transepithelial electrical potential difference before mechanical stimulation, and dPD is a difference between the maximum stimulated value and control value of PD. R is the value of the transepithelial electrical resistance of the colon in rats, n is the size of groups, MS – mechanical stimulation, AMI – amiloride, BUME – bumetanide. * Significantly different at p < 0.05 compared to control group (0 dpi).

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Fig. 2. The effect of mechanical and chemical stimulation on the transepithelial electrical potential difference of the isolated colon of control rats. Fragments of the colon were incubated in Ringer solution. Stimulation was achieved through rinsing the mucous surface with Ringer solution (A), Ringer solution with the addition of amiloride (B), Ringer solution after the administration of amiloride (C), Ringer solution with the addition of bumetanide (D) and Ringer solution after the administration of bumetanide (E). Arrows signify the beginning and end of stimulation. A single experiment is shown.

PD and dPD decreased by about 70% compared to control values. R was different in both groups. At 16 dpi, R in the colon was lower by 90% compared with control, and at 40 dpi, R was 40% lower than in the control group. At 60 dpi, PD of the colon was only 0.1 mV, and mechanical stimulation did not cause any changes in PD. Only R remained similar to control values.

3.2. The effect of ion transport inhibitors (amiloride and bumetanide) on the parameters of the colon in non-infected and tapeworm-infected rats In order to identify the ion transport pathway that influences electrophysiological parameters of the isolated colon of non-infected and infected rats, we applied two ion transport inhibitors to the stimulation fluid: AMI and BUME (which efficiently block transepithelial pathways for Na+ and Cl , respectively). In order to determine the effect of the inhibitors on mechanical sensitivity, we used MS with Ringer’s solution, both before and after the administration of the inhibitors. In control rats (0 dpi), AMI inhibited hyperpolarization (Fig. 2B) and decreased reaction by 57% during MS (Table 1 and Fig. 2C). At 8 dpi, AMI inhibited hyperpolarization (Fig. 3B), and MS using AMI resulted in depolarization (Fig. 3C). Similarly, at 16, 40 and 60 dpi,

Fig. 3. The effect of infection by Hymenolepis diminuta on the electrophysiological parameters of the colon. The fragments of the colon of rats (8 dpi) were incubated in Ringer solution. The stimulation involved rinsing the mucous surface with Ringer solution (A), Ringer solution with the addition of amiloride (B), Ringer solution after the administration of amiloride (C), Ringer solution with the addition of bumetanide (D) and Ringer solution after the administration of bumetanide (E). Arrows signify the beginning and end of stimulation. A single experiment is shown.

AMI inhibited the reaction, and MS to a small extent unblocked the reaction. The application of BUME in the stimulation solution for control rats (0 dpi) inhibited hyperpolarization (Fig. 2D) and reaction during MS (Table 1 and Fig. 2E). Similarly, at 8, 16 and 60 dpi, the administration of BUME inhibited hyperpolarization (Fig. 3D), which was not unblocked by MS (Fig. 3E). Only at 40 dpi, did both BUME and MS result in slight hyperpolarization. 3.3. The effect of capsaicin (a C-fibre stimulator) on ion transport In a separate experimental series, we checked the influence of capsaicin (CAPSA) on electrophysiological parameters of colon of control rats and rats infected with H. diminuta. The effect of CAPSA on a colon incubated in Ringer’s solution is presented in Table 2 and Fig. 4. In control rats, it caused hyperpolarization that was twice that caused by MS, but did not have any effect on reaction during MS – both before and after the administration of CAPSA the value was similar. In rats infected with H. diminuta (at 8, 16, 40 and 60 dpi) the administration of CAPSA neither significantly affected PD nor reaction during MS. 3.4. Blood picture 3.4.1. Erythrocyte indices during cestode infection Erythrocyte indices determined during H. diminuta infection of rats are presented in Table 3. Red blood cell count (RBC) in infected

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Table 2 The effect of H. diminuta infection on the electrophysiological parameters of the colon of rats during mechanical stimulation before and after the administration of capsaicin. Parameters (mV)

Days post-infection (dpi) 0 (n = 10)

MS1 CAPSA MS2

8 (n = 12)

0.5 ± 0.01 1.0 ± 0.1 0.5 ± 0.2

16 (n = 14)

0.06 ± 0.05 0.0 ± 0.0* 0.03 ± 0.03

40 (n = 12)

0.04 ± 0.04 0.01 ± 0.02* 0.06 ± 0.04

60 (n = 12)

0.07 ± 0.03 0.05 ± 0.06* 0.08 ± 0.03*

0.04 ± 0.04 0.1 ± 0.05* 0.02 ± 0.02

Mean values and ±standard error of the mean are given; n is the number of the examined fragments, PD – transepithelial electrical potential difference, dPD – difference between the maximum stimulated value and control value of PD. MS – mechanical stimulation, CAPSA – 20 ml of capsaicin at a concentration of 0.0001 was injected on the mucous surface of the colon of rats. * Significantly different at p < 0.05 compared to control group (0 dpi).

rats was statistically significantly lower than in the control group (0 dpi) as early as 16 dpi (p = 0.049). That significant decrease remained until 60 dpi. Reduction in RBC count at 60 dpi was 5.8% in comparison to control group. A significant decrease in haemoglobin concentration (HGB) in infected rats was observed at 16 dpi (p = 0.012). The decrease in comparison to control (by 3%) remained until 60 dpi, but was then already statistically insignificant. A significantly lower red cell distribution width (RDW), was observed at 40 dpi (p = 0.029), which remained significantly lower in comparison to control group at 60 dpi (by 8.0%, at p = 0.029). RDW values were quite strongly negatively correlated (Rs = 0.50, p = 0.00062) with the duration of infection. A lower MPV in comparison to the control group was observed as early as 8 dpi (p = 0.003). Although MPV had increased by 60 dpi, it was statistically insignificant. MPV values were moderately correlated (Rs = 0.42, p = 0.0082) with the infection duration. A moderate negative correlation (Rs = 0.35, p = 0.03) was also observed between the number of platelets (PLT) and the duration of infection. A decrease in PLT at 60 dpi in comparison to control group was statistically insignificant. Fig. 4. The effect of capsaicin (CAPSA) on ion transport of the isolated colon of control rats (A) and rats infected by H. diminuta (8 dpi) (B). The colon was incubated in Ringer solution. Thirty seconds-long mechanical stimulation by a jet of Ringer fluid was applied before and after injection of capsaicin (CAPSA) on the mucous membrane with 20 ll of the solution at a concentration of 0.0001%.

3.4.2. Leukocyte counts and WBC relative counts (%) during cestode infection Leukocyte counts during H. diminuta infection of rats are presented in Table 3. Amongst the leukocyte counts, only eosinophils showed statistically significant lower values in rats at 8 dpi com-

Table 3 Blood morphology parameters, during of H. diminuta infection in intestine of rats. Parameters

WBC NEU LYM MONO EOS BASO RBC HGB HCT MCV MCH MCHC RDW PLT MPV

Days post-infection (dpi) 0 (n = 5)

8 (n = 12)

16 (n = 9)

40 (n = 8)

60 (n = 9)

9.09 ± 3.31 1.48 ± 0.30 6.45 ± 2.71 0.54 ± 0.29 0.37 ± 0.09 0.25 ± 0.08 9.09 ± 0.34 9.71 ± 0.29 0.70 ± 0.02 77.46 ± 2.99 1.07 ± 0.04 13.76 ± 0.14 18.64 ± 1.24 904.80 ± 142.22 8.84 ± 0.69

7.40 ± 1.33 1.55 ± 0.53 5.00 ± 1.02 0.36 ± 0.16 0.27 ± 0.08* 0.18 ± 0.16 8.65 ± 0.56 9.34 ± 0.52 0.69 ± 0.03 79.36 ± 2.95 1.08 ± 0.04 13.62 ± 0.16 17.96 ± 0.91 1015.5 ± 61.78 7.34 ± 0.33*

8.21 ± 3.93 1.70 ± 0.63 5.60 ± 3.49 0.42 ± 0.21 0.28 ± 0.11* 0.17 ± 0.09 8.54 ± 0.58* 9.06 ± 0.45* 0.67 ± 0.04 79.36 ± 4.04 1.06 ± 0.08 13.44 ± 0.65 17.91 ± 1.89 1018.4 ± 280.7 8.69 ± 1.07

8.26 ± 1.89 1.60 ± 0.46 5.88 ± 1.51 0.71 ± 0.90 0.24 ± 0.16* 0.19 ± 0.14 8.54 ± 0.67* 9.35 ± 0.57 0.68 ± 0.04 79.50 ± 2.58 1.10 ± 0.03 13.80 ± 0.27 17.09 ± 1.11** 881.63 ± 80.75 8.71 ± 0.62

6.30 ± 2.08 1.33 ± 0.32 4.24 ± 1.74 0.36 ± 0.19 0.17 ± 0.09** 0.20 ± 0.10 8.56 ± 0.48* 9.40 ± 0.51 0.68 ± 0.04 79.98 ± 1.76 1.10 ± 0.02 13.74 ± 0.17 16.98 ± 0.97** 847.56 ± 122.83 9.34 ± 1.25

Change (%) vs 0 30 10 34 33 53 21.5 5.8 3.0 2.7 3.3 3.0 0.11 8.9 6.3 5.7

Values expressed as arithmetic mean (± SD). WBC, white blood cells; NEU, neutrophils; LYM, lymphocytes; MONO, monocytes; EOS, eosinophils; BASO, basophils; HGB, haemoglobin; HCT, packet cell volume; MCV, mean corpuscular volume; MCH, mean corpuscular haemoglobin; MCHC, mean corpuscular haemoglobin concentration; RDW, red cells distribution width; PLT, platelets; MPV, mean plate volume. * p < 0.05 vs control group (0 dpi) (Mann–Whitney test). ** p < 0.001 vs control group (0 dpi) (Mann–Whitney test).

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Table 4 The percent of leukocytes during H. diminuta infection in intestine of rats. Parameters

NEU LYM MONO EOS BASO

Days post-infection (dpi) 0 (n = 5)

8 (n = 12)

16 (n = 9)

40 (n = 8)

60 (n = 9)

17.46 ± 3.93 69.34 ± 5.68 5.77 ± 1.19 4.61 ± 2.7 2.84 ± 0.29

21.35 ± 5.05 67.73 ± 8.05 4.76 ± 1.75 3.73 ± 0.93 2.16 ± 1.69*

22.28 ± 6.85 66.26 ± 8.71 5.16 ± 1.15 4.14 ± 2.14 2.13 ± 1.04*

19.58 ± 4.05 70.83 ± 4.96 4.45 ± 2.63 2.92 ± 1.67 2.25 ± 1.53

22.36 ± 5.87 65.96 ± 6.99 5.78 ± 2.44 2.84 ± 1.33* 3.04 ± 0.70

Change (%) vs 0 28 4.9 0.1 38 7

Values expressed as arithmetic mean (± SD). NEU, neutrophils; LYM, lymphocytes; MONO, monocytes; EOS, eosinophils; BASO, basophils. * p < 0.05 vs control group (0 dpi) (Mann–Whitney test).

pared with control group (p = 0.043). That significantly lower value was maintained until 60 dpi (p = 0.0039). A 53% reduction in EOS counts at 60 dpi was seen in comparison to control group. With the increasing duration of infection, the number of eosinophils decreased (Rs = 0.48, p = 0.001). Also the percentage of EOS in the total number of leukocytes decreased significantly over the time of infection (Rs = 0.31, p = 0.042) (Table 4). Lower, but statistically insignificant, was the basophils count in infected rats in comparison to control group. The percentage of basophils in leukocytes decreased statistically significantly on 8 and 20 dpi in comparison to control (p = 0.036; p = 0.041, respectively), but over the next days of infection this decrease was not statistically significant in comparison to control group (Table 4). No statistically significant differences were found in total white blood cell counts in infected or control rats. 4. Discussion Our examination of the colon of rats experimentally infected with H. diminuta indicates that the infection affected ion transport in the colon and changed the blood picture. 4.1. Ion transport in control conditions In the epithelium of the colon, the processes of secretion of chloride ions and absorption of sodium ions take place simultaneously, but with different intensity (Cooke, 1994). It leads to the segregation of electrical charges. Anions accumulates in the layer of mucus covering the colon, and cations accumulates in the interstitial space. In our previous publications we described two basic components of transepithelial electrical potential difference (Kosik-Bogacka and Kolodziejczyk, 2004; Kosik-Bogacka et al., 2005; Tyrakowski et al., 1998). The stable component, usually marked as PD, is a parameter dependent on the ion transport processes, regulated over longer periods of time. The variable component, usually marked as dPD, depends on mechanical stimulation, and is associated with short-lived hyperpolarization caused by an increased transport of sodium and chloride ionic currents. The transepithelial electrical potential difference depends on the species and organ. In mice, the PD of the colon is about 2.3 mV (Davies et al., 1998), in rabbits 3.2 mV (Kosik-Bogacka et al., 2005). In this study, the value of the transepithelial electrical potential difference in the rat colon was about 1.5 mV. PD of the rat colon was in our study half that obtained by Greenwood-Van Meerveld et al. (1999), which may be associated with the fact that a different breed of rats was used; namely Wistar rats where Greenwood-Van Meerveld et al. experimented on Sprague–Dawley rats. Transport through the epithelium of the gastrointestinal tract may take place not only via the transcellular route but also via a paracellular one. Contrary to the directed transcellular transport, depending upon the transporting systems in cellular membranes

and in the active one depending upon energy, the intercellular transport is bidirectional and passive. It takes place through tight junctions between epithelial cells in the apical part, and in the basal–lateral part through stoma (Kunzelmann and Mall, 2002). The integrity of tight junctions between cells and the degree of permeability of a tissue for ions was shown by R (Gitter et al., 2000). Many factors may change a resistance (for example proinflammatory factors) (Berkers et al., 1990). The epithelium of the colon are highly resistant (tight) tissues (Wills, 1981). In our study, R was about 146 X cm2. These results are consistent with those obtained by other authors studying the colon of rats (Knauf et al., 1984). It has been observed that various mechanical and chemical stimulations affecting the epithelial tissue cause local changes in the ionic currents, motor activity and the permeability of vascular vessels. Signals, mostly nervous ones triggered by the stimulus, may also be sent to other sections of the gastrointestinal tracts, thus forming functional units (Cooke, 1994). In this study, hyperpolarization after MS of the colon wall by jets from a peristaltic pump, caused changes in the transepithelial ion transport. It induced the movement of mucus lining on the surface of the colon epithelium. Literature shows that the reaction to mechanical stimulation involves both ion channels, which are sensitive to MS, and the endings of C-fibres situated in the intestine epithelium (Bevans and Geppetti, 1994). Mechanical stimulation induces secretion of non-adrenergic non-cholinergic (NANC) neuropeptides from afferent neural endings, which changes the transport of sodium and chloride ions in the colon epithelium. 4.2. The influence of infection with H. diminuta on ion transport in the colon Mucous membranes of the gastrointestinal tract are the site of contact with gastrointestinal parasites. The main role is played by the mucosal-associated lymphoid tissue (MALT), present in the mucous and submucous membranes of the gastrointestinal tract (Furness et al., 1999). In the intestine one may find a number of lines of defence against harmful factors in line the intestinal microenvironment, namely the intact epithelium, mucosa, peristaltic movements, physiological intestinal microflora of mucus lining, enteric nervous system (ENS) and the mucosal immune system (Wallace, 2001). It has been observed that mediators released from fibroblasts, mastocytes, neutrophils, macrophages and eosinophils, directly or indirectly affect the ion transport and motor activity of intestines. In this study we observed that infection with H. diminuta, a tapeworm living in the small intestine, causes changes in ion transport in the colon, an organ situated some distance from the site of infection. The changes in ion transport in organs other than the biotope of the parasite have also been reported after application of an antigen Trichinella spiralis in the colon of guinea pigs (Wang et al., 1991) and Fasciola hepatica on the rat colon (Kosik-Bogacka and Kolodziejczyk, 2004; O’Malley et al., 1993). These results and

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other literature data confirm the hypothesis of a common mucosal immune system (McDermott and Bienenstock, 1979). The aforementioned changes in the colon may be supplemented by doming of sensitized immunocytes originating in the small intestine (Broaddus and Castro, 1994). It could also be associated with the dissemination of blood-born antigens to extra-intestinal sites, binding homocytropic antibodies to mucosal mast cells (Broaddus and Castro, 1994). Histamine, secreted for example by mast cells, affects ion transport. It has been confirmed that histamine stimulates the electrogenic secretion of Cl and inhibits the electro-neutral absorption of NaCl in the rat colon (Schultheiss et al., 2006). Helminthiasis in rats is connected with an increased number of mastocytes (Fal and Czaplicka, 1991) who reported that the greatest number of mastocytes can be found in the jejunum of the infected rats, increasing from 2 to 25 dpi when it peaked. Substances released from the granulation of degranulated mastocytes have a positive effect on the expulsion of the parasite, the presence of mastocytes and the increase in eosinophils is a response of the host to the inflammatory state. In this study, infected rats were observed to have a significant decrease in PD and dPD in the colon, especially at 60 dpi (Table 1). It could be associated with increased changes in the small intestine: inflammatory swellings, erosion and the extension of the lymphatic system at 60 dpi (Fal and Czaplicka, 1991). These facts indicate that H. diminuta inhibits ion transport in the epithelium of the host’s colon. It suggests that hymenolepidosis in rats causes an activation of inflammatory mediators and the stimulation of nervous fibres, which significantly affects the function of ion channels in the epithelium of the colon. Perhaps it is associated with the decrease in the sensitivity of sensory receptors and a decrease in the amount of secreted neuropeptides that stimulate ion transport in epithelial cells. Similarly, decreased intestinal permeability and decreased absorption of liquids, glucose and electrolytes have been reported in the small intestine of rats infected with H. diminuta (Podesta and Mettrick, 1974). However, the colon of mice infected with H. diminuta, between 10 and 14 dpi, did not experience any changes in the passive and stimulated ion transport (short-circuit current and ion conductance) (Reardon et al., 2001). It may have been caused by the fact the parasite in mice is expulsion before it reaches maturity (between 10 and 14 dpi) and does not achieve a considerable biomass, as in rats. In our study on the infected colon we did not observe stimulation of chloride secretion, reported in helminthic infections and described as an immediate (type I) hypersensitivity reaction (Jarrett and Miller, 1982). Such a reaction was described in isolated colonic mucosae from sensitized rats in response to antigen Nippostrongylus brasiliensis (Baird et al., 1985) and F. hepatica (O’Malley et al., 1993). During experimental hymenolepidosis, the transepithelial electrical resistance (R) did not change until 40 dpi. As R depends on the permeability of tight cellular junctions, one may conclude that the presented changes in development during MS of the isolated colon resulted from changes in the active ion transport at a stable level of extracellular passive ion transport. Our results are confirmed by the lack of diarrhoea in hymenolepidosis, usually caused by increased permeability of the tissue due to the destruction of tight junctions among cells. Only in the chronic phase (40 dpi) did the value of R decrease, which could have been caused by the increased permeability of tight junctions and could have been associated with the enhanced inflammatory processes in the small intestine.

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harmful effect of parasites in the gastrointestinal tract, predominantly C-fibres. Elements of the nervous system and sensory receptors in the intestinal wall integrate the motoric and secretory function of intestines through synaptic effects, nervous endings and the NANC neurotransmitters (substance P – SP, nerokinin A – NKA, calcitonin gene-related peptide – CGRP and others), secreted from sensory endings (Cooke, 1994; Kosik-Bogacka et al., 2005; Riegler et al., 1999). The role of these neuropeptides is the preservation of integrity of tissues and damage repair (Cooke, 1994). Changes in the level of NANC neuropeptides, for example SP, have been described in cestode and nematode infections (McKay et al., 1991; Swain et al., 1992). Substance P is a pro-inflammatory neuropeptide, stimulating mastocytes to secrete inflammatory mediators, for example histamine. Substance P elevates the short-circuit current, decreases ileal Na+ absorption and stimulates Cl secretion (Walling et al., 1977). It has been observed that SP may be a non-cholinergic neurotransmitter regulating ion secretion in parasite infections. An increase in intestinal immunoreactive-substance P has been described in the jejunum of rats infected with N. brasiliensis (Masson et al., 1996) and T. spiralis (Kataeva et al., 1994). An increase in SP has also been described in rats infected with H. diminuta (McKay et al., 1991). An excess amount of released NANC neuropeptides, including SP from nervous endings of C-fibres, leads to neurogenic inflammation (Ciancio and Chang, 1992). A local hyperaemia is observed, along with an increase in permeability of vascular vessels and epithelium, and the release of other typical inflammatory mediators. In consequence, it leads to muscle contractions, stimulation of mucosa secretion and ion transport (Cooke, 1994). There are many substances that trigger the stimulation of C-fibres. These are inflammatory mediators such as histamine, prostaglandins PGF-2a, PGE-2, PGI-2 and irritating agents, such as capsaicin (Bevan and Geppetti, 1994). In this study capsaicin caused hyperpolarization in non-infected rats – it was twice the value before mechanical stimulation and did not affect the reaction during MS. Before and after CAPSA administration, the value of reaction was the same. A capsaicin-induced increase in hyperpolarization could have been associated with the stimulated secretion of neuropeptides from the endings of C-fibres. Many publications show that the stimulation of C-fibres by capsaicin triggers the release of NANC neuropeptides and also with afferent impulses (Bevan and Geppetti, 1994). In rats infected with H. diminuta, the administration of CAPSA did not have any significant effect on ion transport or reaction during mechanical stimulation. Lack of hyperpolarization in the colon of infected rats in the presence of CAPSA could have been caused by an excess stimulation of C-fibres by H. diminuta. Probably it is associated with the decreased release of SP or an altered sensitivity to SP during H. diminuta infection. This can be confirmed by the 80–90% reduction of mucosal neuropeptide content in rats infected with T. spiralis which were treated with capsaicin as neonates or as adults before infection (Swain et al., 1992). It could have also had some connection with the change in distribution of nerves or changes in neuronal function. Varilek et al. (1991) observed granuloma in mice which were a consequence of infection with Schistosoma mansoni, a local destruction of neurons in the distal part of the ileum and the proximal section of the colon. Long et al. (1980) observed the reduction of SP and VIP nerves in people infected with Trypanosoma cruzi.

4.3. The influence of infection with H. diminuta in rats on the function of C-fibres in the colon

4.4. The effect of amiloride and bumetanide on ion transport

Besides the immune system, the enteric nervous system (ENS), predominantly C-fibres, provides protective functions against the

In the epithelium of the colon of mammals, an electro-neutral sodium transport exists that depends on the absorption of chloride

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(Kosik-Bogacka et al., 2005). Therefore, in order to identify the routes of ion transport responsible for the transepithelial electrical potential in the colon of rats, we used two ion transport inhibitors: AMI and BUME. AMI is a known selective inhibitor of Na+ in epithelial tissues that blocks channels in a fast and reversible manner (Benos et al., 1995). In the epithelium of the colon of mammals, sodium channels are blocked at AMI concentrations from 0.1 to 1 mmol/l (Benos et al., 1995). The application of AMI may inhibit the phase of absorption of sodium ions and obtain the advantage of chloride ion secretion. The administration of amiloride, using a peristaltic pump, on the mucous surface of the colon of rats was used to determine the participation of epithelial sodium pumps in the formation and changes of PD. The immediate effect of AMI on the colon in control rats was visible in the inhibition of the reaction during stimulation and the decreased reaction during MS (Table 1). That type of reaction in the colon of rats to amiloride was caused by the closure of apical sodium channels by a stimulation solution that contained AMI. Similarly, in rats infected with H. diminuta, the application of amiloride inhibited hyperpolarization; mechanical stimulation after AMI resulted in depolarization but only slightly unblocked the reaction. Another inhibitor that was applied in our study was bumetanide, an inhibitor of the transepithelial chloride ion transport. BUME blocks the cotransporting basal–lateral mechanism Na+K+2Cl (Moore et al., 1995). In the presence of BUME, the reaction of dPD depends solely on the transport of sodium ions. Such experimental conditions may be described as the pharmacological isolation of the chloride current. The application of BUME, used for the stimulation of the colon of control rats and rats infected with H. diminuta, inhibited both hyperpolarization and reaction during MS after BUME (Table 1). It shows that not only the transport of sodium ions but also of chloride ions can be responsible for hyperpolarization. One may suppose that either chloride ions or sodium ions are responsible for hyperpolarization after MS, and also that during infection of intestine parasites the transport of sodium and chloride ions is inhibited. 4.5. Blood picture In our study we observed a significantly lower RBC count and HGB concentration, with no significant changes in MCHC in infected rats when compared with non-infected ones. The reduction in RBC count, HGB concentration and MCHC values was also noted by Gill et al. (2007) in R. norvegicus, naturally infected with H. diminuta and H. nana. Marthur and Johnson (1989) investigated blood profiles of Rattus rattus naturally infected with Vampirolepis fraterna (Cestoda) and observed a lower RBC count but significantly enhanced level of HGB. In our study RDW values, an indicator of anisocytosis (variability of RBC size), were significantly lower than the control. RDW was negatively correlated with the duration of infection. There was a positive correlation between the duration of infection and MCV values, but the increase in MCV values at 60 dpi in comparison to control group did not reach the level of significance. In the aforementioned work by Gill et al. (2007) a significantly enhanced value of MCV was noticed. The fall in RBC count recorded in the infected rats may be attributed to blood loss (various types of anaemia and haemorrhage), erythropoietin deficiency, haemolysis (erythrocyte destruction), malnutrition (nutritional deficiency of iron, folic acid, vit. B12 or B6) or some other factors (Gill et al., 2007). Since the erythrocytes are rich in haemoglobin, the reduction in RBC recorded in our study may be one of the reasons for the decrease in HGB concentration during cestode infection in rats. The decreased HCT values in the infected rats may be also due to anaemia (Gill et al., 2007), which is also supported by the fact that low values of RBC and HGB concentra-

tion were found in the infected rats. It has been indicated by Moshin et al. (1991) that the MCV values usually rise with a deficiency of vit. B12, folic acids, niacin and cobalt due maybe to parasitic infection (fascioliosis), and attributed to macrocytic anaemia resulting from the deficiency. It has been suggested that low MCH and MCHC indices indicate hypochromic anaemia in cestode infected rats (Gill et al., 2007; Moshin et al., 1991). However, our study did not show a decrease in MCHC and MCH, hence we cannot draw conclusions on hypochromic anaemia during H. diminuta infection. In our study, we did not observe leukocytosis during infection, and amongst the differential leukocyte counts eosinophils showed statistically significant lower values in infected rats in comparison to non-infected. Also the percentage of EOS in the total number of leukocytes was decreasing over the time of infection. In the aforementioned report by Gill et al. (2007) it was observed that the values of WBC and eosinophils were significantly higher during cestode infection (WBC count increased almost three times in infected animals compared with non-infected). Mathur and Johnson (1989) also observed a significantly enhanced level of leukocytes in R. rattus naturally infected with V. fraterna. A significant increase in WBC count was also reported during Fasciola gigantica infection in cattle (Moshin et al., 1991), and with Heterakis gallinae and Ascardia galli in sparrows (Raza et al., 2006). In contrast to these reports, Chaudhry et al. (1991) observed a slight reduction in WBC count during Strongylus infection in equines. Leukocytosis during infection suggests the immune response of the host against infection. Increased monocyte, neutrophil and eosinophil counts have been reported in animals for different types of parasites (Moshin et al., 1991; Sommerfelt et al., 2006). However, a decrease in eosinophil and lymphocyte levels was also reported during H. gallinae and A. galli infection in sparrows (Raza et al., 2006). It has been indicated that eosinophils are used by the organism to protect against allergic reactions and parasites (Abramson and Melton, 2000). Nutman et al. (1987) and Weller (1992) also demonstrated that parasitic disease is the principal cause of eosinophilia in many non-industrialised parts of the word. In established parasitic infections only moderately high blood eosinophil count are found, but in the early stages of infection when parasites are migrating though tissues, high counts were noted (Weller, 1992). The organisms most likely to cause eosinophilia are parasites (helminthes, protozoan), fungi, a few viruses and bacteria (Abramson and Melton, 2000). Starke and Oaks (2001) observed that during H. diminuta in Sprague–Dawley rats, eosinophils were present in the connective tissues of both submucosa and lamina propria in infected and non-infected ilea of rats. Small numbers of eosinophils were also observed in the ileum muscularis externa of infected rats at 15 and 32 dpi, but not in non-infected rats. Our observation of a significant decrease in eosinophil count during cestode infection suggests that such an infection did not trigger a strong immunological reaction in rats. Basophils are hypersensitive effector cells and perform an important role in host defence mechanisms, in innate immunity against pathogenic organisms and allergic reactions, mediated by immunoglobulin E antibodies (Ishih and Uchikawa, 2000). The basophil count can be high in response to a number of causes and factors such as viral infection, haemolytic anaemia, long standing toxic anaemia, myxoedema, hypothyroidism, some conditions that cause inflammation (Abramson and Melton, 2000). In our study we observed a significant decrease in the number of basophils during infection, and then a statistically insignificant increase at 60 dpi in comparison to control group. The percentage of basophils in leukocytes also decreased, although significantly in the initial phase of infection – at 60 dpi that decrease was no longer statistically significant in comparison to control. Gill et al. (2007) also observed significant changes in basophils count as compared with control group. This observation is in agreement with those

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by Mitre and Nutman (2003), who reported no correlation between parasitic infection and peripheral basophilia in humans. Although numerous investigations have demonstrated the existence of cell-mediated immunity, there have been few reports of this type of response associated with intestinal tapeworms. Machnicka and Choromanski (1983) described positive results of macrophage migration inhibition test (MMI) during H. diminuta infection in rats. The negative results of the MMI test observed at later postinfection times are considered a manifestation of immunosupression caused by the presence of H. diminuta. The phenomenon has been described in numerous bacterial, protozoan and nematode infections such as: leprosy, tuberculosis, generalised leishmaniasis and filariasis (Machnicka and Choromanski, 1983). The nature of the immunodepressed state associated with parasitic infection is poorly understood, it seems likely that antigenic overloading is the cause of desensitization in response to H. diminuta (Machnicka and Choromanski, 1983). The results of our studies confirm the results by Machnicka and Choromanski (1983) that the persistence of H. diminuta in rats is associated with the state of specific cellular unresponsiveness. It cannot be ruled out that the adaptation of the rat to H. diminuta is expressed by this phenomenon. Further studies are needed to assess whether or not immune unresponsiveness is restricted to H. diminuta is antigens. 5. Conclusions We have shown that during hymenolepidosis in rats: (i) Ion transport is inhibited in the epithelium of the colon, mainly sodium ions, with the preserved integrity of tight junctions. (ii) The effect of capsaicin on ion transport in rat colon is varied – in control rats it increased ionic current, and in infected rats it did not cause any changes in PD. (iii) Blood picture is changed: RBC count and HGB concentration were lower, but only RDW values and PLT count negatively and MCV values positively correlated with the infection time; leukocytosis did not occur; the numbers of eosinophils and basophils were lower.

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