J. Virol.-2013-Istrate-JVI.02927-13

8/10/2019 J. Virol.-2013-Istrate-JVI.02927-13

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Rotavirus infection increases intestinal motility but not permeability at the onset of1

diarrhoea2Running title: Intestinal motility and permeability in early rotavirus infection3

4

Claudia Istratea,b,z

, Marie Hagbomb,z

, Elena Vikstrmc, Karl-Eric Magnusson

c and Lennart5

Svenssonb#

6

7

Grupo de Virologia, Unidade da Microbiologia Mdica, Centro de Malria e outras Doenas8

Trpicas, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Lisboa,9

Portugala; Division of Molecular Virology, Department of Clinical and Experimental Medicine,10

University of Linkping, Linkping, Swedenb

; Division of Medical Microbiology, Department11

of Clinical and Experimental Medicine, University of Linkping, Linkping, Swedenc

12

13

ZM.H and C.I contributed equally to this work14

15

Abstract word count: 24216

Text word count: 508217

18

# Corresponding Author:19

Lennart Svensson20

Division of Molecular Virology, Department of Clinical and Experimental Medicine21

University of Linkping, 581 85 Linkping, Sweden22

Email: [email protected]

Phone: +46 (0)10 103 88 03; Fax: +46 (0)10 103 13 7524

JVI Accepts, published online ahead of print on 26 December 2013

J. Virol. doi:10.1128/JVI.02927-13

Copyright 2013, American Society for Microbiology. All Rights Reserved.


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ABSTRACT25

The disease mechanisms associated with onset and secondary effects of rotavirus (RV) diarrhoea26

remain to be determined and may not be identical. In this study we investigated whether onset of27

RV diarrhoea is associated with increased intestinal permeability and/or motility. To study the28

transit time, fluorescent FITC-dextran was given to RV-infected adult and infant mice. Intestinal29

motility was also studied with an opioid receptor agonist (loperamide) and a muscarinic receptor30

antagonist (atropine). To investigate whether RV increases permeability at onset of diarrhoea,31

fluorescent 4- and 10-kDa dextran were given to infected and non-infected mice and fluorescence32

intensity measured subsequently in serum. RV increased transit time in adult and infant mice.33

Increased motility was detected as early as 6 hours post infection (h p.i) in adults and 24 h p.i. in34

pups, and persisted up to 72 h p.i in both models. Both loperamide and atropine decreased35

intestinal motility and attenuated diarrhoea. Analysis of passage of fluorescence dextran from the36

intestine into serum indicated unaffected intestinal permeability at onset of diarrhoea (24-48 h37

p.i.). We show that RV-induced diarrhoea is associated with increased intestinal motility viaan38

activation of the myenteric nerve plexus, which in turn stimulates muscarinic receptors on39

intestinal smooth muscles.40

41

Important section. We show that RV-infected mice have increased intestinal motility at the42

onset of diarrhoea and that this is not associated with increased intestinal permeability. These43

new observations will contribute to a better understanding of the mechanisms involved in RV44

diarrhoea.45

46

47


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INTRODUCTION48

While rotavirus (RV) is well established as a major cause of severe acute gastroenteritis in49

young children all over the world, the knowledge of the mechanisms behind diarrhoea and50

vomiting is still largely unknown. The fluid loss due to diarrhoea may be caused by several51

mechanisms [1, 2]. High concentrations of poorly absorbable compounds may create an osmotic52

force causing a loss of fluid across the intestinal epithelium (osmotic diarrhoea). Secretory53

diarrhoea is characterized by an overstimulation of the intestinal secretory capacity, not coupled54

to an inhibition of fluid absorptive mechanisms. Furthermore, if the barrier function of the55

epithelium is compromised by loss of epithelial cells or disruption of tight junction, hydrostatic56

pressure in blood- and lymphatic vessels may cause water and electrolytes to accumulate in57

lumen (exudative diarrhoea) [1]. In most types of diarrhoea more than one of these58

pathophysiological mechanisms are involved.59

Several hypotheses have been forwarded to explain RV-induced diarrhoea, including a60

functional role of the virus-encoded enterotoxin NSP4 [3-5], malabsorption secondary to failing61

transport of electrolytes and/or glucose/amino acids [6], villus ischemia [7-10], stimulation of the62

enteric nervous system (ENS) [11] and of enterochromaffin (EC) cells [12]. At the cellular and63

tissue level, a new understanding of the disease mechanisms is beginning to emerge [2].64

Intestinal permeability and motility are two physiological mechanisms most rarely investigated,65

but currently being implicated in diarrheal syndromes [2, 13]. Intestinal permeability has only66

rarely been investigated in humans infected with RV [14] . In the early 1980s, Stintzing and co-67

workers [15] was assessed the intestinal permeability in young children with RV infection using68

polyethylene glycol (PEG) of different molecular weights, and a significantly low urinary69

recovery of PEG was noted. A similar observation was made in adults by Serrander and co-70

workers [16]. Moreover, Johansen et al [17] found that children with acute RV infection excreted71


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less PEG in their urine than healthy children and children with enteropathogenicEscherichia coli72

(EPEC) infections. In vitro studies, on the other hand, have shown that RV can increase the73

permeability of polarized human epithelial Caco-2 cells [18], probably due to a reorganization of74

the tight junction proteins claudin-1, occludin and ZO-1 [19]. Furthermore, the RV enterotoxin75

NSP4, induces paracellular leakage in polarized epithelial cells and prevents lateral targeting of76

ZO-1 [20]. In line with this, it has also been demonstrated in Ussing chamber experiments that77

the electrical tissue conductance is increased in RV-infected intestines [11, 21].78

Intestinal motility has only occasionally been studied during RV infection. It is well79

established that stimulating the vagal efferent nerves to the gut increases intestinal motility via a80

direct effect on the intestinal musculature and/or via an excitation of the intestinal cells of Cayal81

[22]. Atropine, the classical muscarinic receptor antagonist, can block these effects. It is also well82

known that naturally occurring opioids can be used as antidiarrheal agents, but their adverse83

central nervous effects make them undesirable as remedies. Loperamide, an opioid receptor84

agonist, differs from other similar drugs by binding predominantly to intestinal tissue and when85

given orally it gains almost no access to the central nervous system (CNS). Its antidiarrheal86

action is thought to result from diminished intestinal motility by interfering with the myenteric87

plexa [22, 23]. Furthermore, loperamide can inhibit intestinal secretion caused by bacterial88

enterotoxins [24, 25]. Regarding RV infections, Yamashiro and co-workers [26] found a89

significant effect of the stool score on day 3-5 of treatment. Meta-analyses confirmed that90

patients treated with loperamide are less likely to have diarrhoea 24 h after treatment and present91

with a shorter duration of diarrhoea and fewer stools compared to patients in the placebo group92

[27].93

Previous human permeability and motility studies have been performed at a time when94

children already have documented RV diarrhoea. Thus, it has remained unresolved whether the95


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effects on motility and permeability are early or late host responses following diarrhoea, rather96

than the actual cause to diarrhoea onset.97

The objective of this study was to investigate whether and how intestinal permeability and98

motility are altered at the onset of RV diarrhoea. To address this issue, different-sized fluorescent99

dextran was given to RV-infected and non-infected adult and infant mice, to determine both100

permeability and transit kinetics. Intestinal motility was also studied after administration of the101

opioid receptor agonist loperamide or the muscarinic receptor antagonist atropine. Our study102

gives new important understanding of the mechanisms triggering diarrhoea.103

104

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107

108

109

110

111

112

113

114

115

116

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119


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MATERIAL AND METHODS120

Animals. RV nave BALB/c, 8 weeks old adults and 5-7 days old infant mice (B&K121

Laboratories, Sollentuna, Sweden) were used. They were housed in standard cages with free122

access to food and water. Pregnant females were transferred to individual cages 1 week before123

the expected day of birth and off spring remained with their mother during the experimental124

period. The animal ethics committee in Stockholm approved the experimental protocol125

(N291/010).126

127

Rotavirus infection and criteria for diarrhoea. Mice (adults and infants) were orally infected128

with 10 L/animal (100 DD50 diarrhoea doses) of wild-type murine RV (EDIM strain) as129

previously described [28]. Only infant mice were examined for diarrhoea since adult mice have130

previously been shown to not develop diarrhoea [29] . Adult mice were infected to investigate131

whether any changes in motility and permeability occur in the absence of diarrhoea. Diarrhoea132

was defined as a liquid yellow stool revealed by gentle abdominal palpation as described133

elsewhere [11, 30]. The percentage of mice with diarrhoea was calculated as the number of pups134

with typical diarrhoeal stools (loose, yellow, liquid faeces) divided by the total number of pups in135

the same group. Mice were confirmed to have diarrhoea before being included in the treatment136

studies with loperamide and atropine.137

138

Motility studies. In the motility experiments, adult (n=6 to 13, at each time-point) and infant139

BALB/c mice (n=5 to 13, at each time-point) received orally 10 l of 4-kDa FITC-dextran probe140

(Sigma; cat.no: FD-4) at 6, 12, 24, 48, 72 and 96 h p.i.. The probe was and also given to non-141

infected mice. The adult mouse dose was 0.5 mg/mice and infant mouse dose was 0.25 mg/mice.142

FITC-dextran was dissolved in Milli-Q water and the solutions were freshly prepared before each143


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experiment. For time points of 24 h p.i and later, diarrhoea was confirmed in all infected infant144

mice that received the probe. After 30 min for adults and 15 min for infant mice, the animals145

were sacrificed and the whole intestines, from stomach to rectum, were used for determination of146

transit time by UV light measurement, taking photos, using a FITC specific filter (Bio-Rad Filter147

520DF30) in a ChemiDoc System (BioRad, ChemiDoc XRS). The front part of the main148

accumulating FITC-dextran was defined from the photo and the software program Illustrator CS6149

was used to most exactly measure the intestinal length and migration of the FITC-dextran probe.150

Intestinal transit was calculated at different time-points, on how far the FITC-dextran probe has151

passed in per cent of the entire length of the intestine, from the pylorus to the rectum. Motility152

experiments were performed at two different occations.153

154

Anti-diarrhoeal drug experiments. Loperamide hydrochloride and atropine sulphate salt155

monohydrate was purchased from Sigma (Sigma; loperamide cat.no: 34014; atropine cat.no:156

A0257) and dissolved in Milli-Q water. Loperamide (10 mg/kg) or atropine (6 mg/kg) was157

administered orally to adult and infant mice at 40 and 44 h p.i.. Four hours after the second dose,158

at 48 h p.i., mice received 4-kDa FITC dextran and were then sacrificed after 15 (infant) and 30159

(adult) min, for determination of transit time. In infant mice the anti-diarrhoeal effect of160

loperamide and atropine was checked at 4 h after the first dose and 4 h after the second dose (48161

h p.i.) of treatment, a time-point associated with severe diarrhoea of almost all mice. In the162

loperamide experiment, mice were checked for diarrhoea also on the following day and received163

a final dose of loperamide at 64 h p.i., the effect of which was investigated 4 h later.164

165

Intestinal permeability studies. The transmural passage into the blood of fluorescently 10- and166

4-kDa dextran was used to assess the barrier characteristics of infant and adult mice infected with167


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RV from 6 up to 96 h in comparison to non-infected animals. The barrier properties were168

described in two ways: (i) by a permeability index equal to the ratio between the relative uptake169

of fluorescent 10- and 4-kDa dextran, and (ii) by the fold-increase (Fi) in either marker compared170

to the basal value for non-infected animals. The values are given as fluorescence intensity (FI).171

The relative permeation of the 10- and 4-kDa probes was used to calculate a permeability index172

(R) through their relative fluorescence intensities, F10and F4in blood/serum, i.e. R=F10/F4173

Briefly, RV-nave BALB/c mice, non-infected and RV-infected, received orally a mixture of 4-174

kDa FITC-dextran (adult mouse dose of 2.5 mg/mice, infant mouse dose of 0.7 mg/mice) and 10-175

kDa Rhodamine-dextran (adult dose 5.0 mg/mice, infant mouse dose 1.4 mg/mice). The176

fluorescently conjugated dextran (Sigma Aldrich, cat.no: FD-4 (4-kDa FITC-dextran) and R881177

(10-kDa Rhodamine-daxtran)) was freshly prepared in Milli-Q water before use. Blood was178

collected in serum tubes (BD Microtainer Tubes, cat.no: 365951) 3 h post tracer-administration179

and centrifuged for 5 min at 1677xg. The serum was diluted 1/50 in Milli-Q water and the180

fluorescence intensities of FITC (494/518 nm) and Rhodamine (565/580 nm) were measured with181

a fluorescence spectrophotometer (Perkin-Elmer,LS-3B,Waltham,182

Massachusetts, USA).183

184

Histology. Specimens (0.5 cm) were taken from the duodenum, the middle part of the jejunum,185

and the lowest part of ileum, from adult and infant mice, non-infected and infected mice at 48 h186

p.i.. The specimens were fixed in 4% phosphate-buffered formaldehyde (Histolab, Sweden,187

cat.no: 02176) and then embedded in paraffin. From each specimen sections of 5-m-thickness188

were cut and placed onto glass slides (SuperFrost Plus Menzel, Histolab, Sweden, cat.no: 06400).189

The paraffin was removed through 2 steps of 5-min incubations in tissue clear solution (Sakura,190

Tokyo, Japan, Code: 1466,) and the tissues were hydrated through a series of ethanol (EtOH)191


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baths (99.5% EtOH 2x5 min, 95% EtOH 1x5 min, 70% EtOH 1x5 min) followed by a final step192

in PBS. Slides were then stained with hematoxylin and eosin for 5 min, rinsed in tap-water and193

mounted with Pertexmounting medium (Histolab, Sweden, cat.no: 00814) and a cover glass.194

195

Statistical analysis. Data was analysed with non-parametric two-tailed Mann-Whitney test and196

Kruskal-Wallis test, using the Prism Software 5.0 (GraphPad, San Diego, CA) or Fishers exact197

test. Values were considered significant at p 0.05.198

199


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RESULTS200

Rotavirus increases intestinal motility in adult and infant mice. Alterations in intestinal201

transit/motility have been associated with several forms of gastrointestinal diseases, promoting202

the development of pharmaceutical drugs that attenuate intestinal motility. For RV infection it203

still remains unclear whether RV could increase intestinal transit and thereby contribute to204

diarrhoea.205

Therefor, to obtain an all-over view of the possible involvement of increased motility in RV206

diarrhoea we infected infant and adult mice with murine RV, 10 L/animal (100 x DD50207

diarrhoea doses) and characterized the kinetics of intestinal transit of a 4-kDa FITC-dextran208

probe. We observed that the probe have passed along the entire intestine within 15 min in209

infected infant mice at 48 h p.i., more rapidly compared to non-infected mice (p0,001) (Fig.1A210

and B). Interestingly, the peak of transit 48 h p.i. coincided with the time of most severe211

diarrhoea symptoms in pups, as 12 out of 13 mice had diarrhoea (Table 1). RV infection in mice212

was confirmed by eosin and haematoxylin staining of tissue sections of different parts of the213

small intestine (Fig.2). The characteristic vacuolisation of the epithelial cells was seen on villi but214

not in the crypts.215

We next investigated whether adult mice, in spite of not responding with diarrhoea216

following RV infection, might have altered intestinal motility. Theoretically, RV infection could217

stimulate the myenteric plexus and thus motility in absence of diarrhoea [2]. Indeed, we found218

that the infection in adult mice did not resulted in increased intestinal motility, as determined by219

the transit of FITC-dextran (Fig.1C). However, a slight effect was found at 24 h p.i., a time point220

typically associated with onset of diarrhoea in pups.221

222

223


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Atropine, a muscarinic receptor antagonist, attenuates rotavirus motility and diarrhoea.224

The motility studies with atropine treatment orally, using a dose of 6 mg/kg, were performed as225

described in material and methods. We observed a significant decrease of intestinal motility in226

infected infant and adult mice treated with atropine as compared to non-treated mice at 48 h p.i.227

(Fig.3A and 3B). In non-infected infant mice atropine had a modest effect on motility (Fig.3A).228

Since the effect of atropine was also observed in non-infected mice (Fig.3A and 3B), it seemed to229

be likely not virus specific.230

We also investigated whether atropine could attenuate RV-induced diarrhoea in infant mice. Only231

mice with confirmed diarrhoea were included and atropine was given at 40 h and 44 p.i.. The232

effect of the 2 doses of atropine was evaluated at 48 h p.i.. In the mock-treated group 6/7 still had233

diarrhoea, in contrast to only 3/11 in the atropine-treated group (p


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the untreated infected mice (Fig.4A, line 4) but was delayed in loperamide treated and infected248

mice (Fig.4A, line 5). Giving loperamide to non-infected infant mice had a modest effect on249

motility (Fig.4B). No significant effect on intestinal motility was observed in infected adult mice250

treated with loperamide as compared to infected non-treated adults at 48 h p.i. (Fig.4C). Again251

the drug had only modest effect on non-infected adult mice (Fig.4C). We conclude that opioid252

receptor agonists can attenuate the increased intestinal motility in RV-infected mice.253

Next we investigated whether loperamide could attenuate RV-induced diarrhoea using the254

same experimental protocol as in the transit studies, i.e. infant mice was given doses of255

loperamide at 40 h and 44 h p.i.. In the mock-treated group 12/13 had diarrhoea at 40 h p.i.,256

which was maintained during the course of experiment until termination at 68 h p.i. (Table 1),257

and among infected mice 8/9 had diarrhoea at 40 h p.i before administration of loperamide (Table258

1). Only the 8 mice with confirmed diarrhoea were included in the loperamide-treated group.259

Already after the first dose (40 h.p.i), none of them displayed diarrhoea at 44 h p.i. (Table 1), and260

they showed no sign of diarrhoea (48 h.p.i) following administration of a second dose. The effect261

of Loperamide was highly significant (p


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multifactorial and may in the case of RV infection include disturbances in intestinal permeability.272

Previous human studies investigating intestinal barrier functions [15, 17] were all conducted at a273

time-point at which subjects already had diarrhoea. Thus, it remains unclear whether permeability274

changes are a late host response following diarrhoea and/or the result of extensive lesions of the275

epithelia, rather than the actual cause of diarrhoea onset.276

To address this issue, we administered orally 4-kDa FITC-dextran and 10-kDa Rhodamine-277

dextran to infant and adult mice at 6, 12, 24, 48, 72 and 96 h p.i.. Adult and infant animals were278

bled at 3 h post tracer administration to assess the transmural passage of the probes. The use of279

two different-sized probes allowed us to investigate in more detail whether the intestinal barrier280

was influenced by the RV infection. The plasma concentration of the two probes as reflected in281

their optical densities was estimated during the course of infection as a measure of their intestinal282

permeability. A ratiobetween the permeability of the two probes was also determined (Table 3),283

as well as the change of permeability to either compound (fold increase) as compared to the284

corresponding non-infected animal.285

We found that infected infant in contrast to adult mice, responded with a significantly286

increase in permeability for both the 4- and 10-kDa probes at 6 and 12 h p.i. (p


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pathological changes were not sufficient to induce increased permeability for 4- and 10-kDa296

dextran probes.297

A reduced surface area should principally affect the permeability of the 10- and 4-kDa298

probes to the same extent and yield parallel decline of either marker in the blood. However, if299

there is a perturbation of the junctional integrity, the paracellular uptake of the larger 10-kDa300

probe might be enhanced relatively to the smaller 4-kDa probe [31]. In the early phase of301

infection the 10/4 kDa ratio was balanced, based on a 20-fold increased for the 4-kDa probe and a302

18-fold increased for the 10-kDa probe. A pronounced 11-fold increase in 10/4 kDa ratio was303

however observed at 24 h p.i and a 17.9 fold increase at 72 h p.i., caused by the significant304

tightening of the epithelium for the 4-kDa probe (Table 3). Interestingly, at 48 h p.i., when the305

clinical signs of diarrhea are obvious, the 10/4 kDa ratio was 4.5 caused by a decreased306

permeability of the 10-kDa probe (Table 3).307

To get further insight into the mechanisms associated with onset of diarrhoea, we308

investigated whether adult infected mice not responding with diarrhoea from the small intestine309

[28] had a perturbed intestinal barrier. Accordingly, a set of experiments identical to the infant310

mice permeability studies was performed in adult mice. The permeability of the 4-kDa probe was311

decreased at time-points 6, 12 and 24 h p.i. (Fig.6A). A limited permeability increase for the 4-312

kDa probe was observed at 48 and 72 h p.i., but did not reach statistical significance. Rather there313

was a significantly reduced permeability at 24 and 96 h p.i. (p


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DISCUSSION320

321

Several disease mechanisms such as malabsorption, altered permeability, motility disturbances,322

stimulation of enterochromaffin cells, activation of the ENS and the RV NSP4 toxin have been323

associated with the fluid loss in RV diarrhoea [2, 3, 5-8, 12, 13]. Some of these mechanisms may324

indeed occur after the onset of diarrhoea, rather than being the actual cause. In this study we325

have investigated how intestinal transit (motility) and epithelial permeability are related to the326

onset of RV diarrhoea. These two important disease-associated mechanisms have not been327

studied together in the early phase of RV diarrhoea.328

Motility of the small intestine, as in all parts of the digestive tract, is controlled329

predominantly by excitatory and inhibitory signals from the ENS, however, modulated by inputs330

from the CNS, whereas intestinal secretion is controlled mainly by intrinsic nerves and in331

particular by the submucosal plexus [22]. The autonomic nervous system control of motility is332

exerted mainly via the release of acetylcholine and effect on muscarinic receptors. Hence, it is333

possible to block this motor response by atropine as also demonstrated in this study. Considering334

in vivo studies in several experimental models of acute diarrhoea, atropine has failed to attenuate335

the rate of fluid secretion in the small intestine.336

In the present study gut motility was determined by studying the intestinal transit of the 4-337

kDa FITC-dextran probe in infant and adult mice. Interestingly, in infant mice, the onset of338

diarrhoea occurred concomitantly with a high intestinal transit of FITC-dextran at 48 h p.i.339

(Fig.1A and B). To get further insight into the pathophysiological mechanisms responsible for340

the increased motility, we treated non-infected and RV-infected mice with the -opioid receptor341

agonist loperamide, attenuating the activity of the myenteric plexus [23] and with atropine,342

blocking muscarinic receptors on smooth muscles. Loperamide differs from other opioid343

analogues as it binds predominantly to intestinal tissue. Thus, when given orally it has virtually344


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no access to the CNS. Loperamide is a widely used anti-diarrhoeal agent, its effects being345

attributed to an inhibitory action on smooth muscle tone and peristalsis attenuating both346

cholinergic and non-cholinergic influences on intestinal smooth muscles [32, 33]. The effect of347

loperamide was not restricted to infant mice as adult non-infected and infected responded with348

attenuated intestinal motility, strongly suggesting that the effect is not virus-specific. The efficacy349

was very pronounced, since the diarrhoea in infant mice was completely abolished in 8/8 mice 4350

h after a single dose. When giving a second dose, it appeared that the effect was time-dependent351

since 6/8 mice exhibited diarrhoea 20 h after the second dose. In humans, maximal plasma352

loperamide concentration is achieved within 3-5 h in humans [34], which corresponds with the353

significant effect observed within 4 h in the mouse model. It has also been proposed, that354

loperamide apart from its inhibitory action on smooth muscle tone, also can stimulate absorption355

of fluid, electrolytes and glucose and moreover reverse the prostaglandin PGE2 and cholera toxin-356

induced secretion to absorption [24]. In previous studies loperamide has been administered to357

individuals with on-going diarrhoea, therefore the question whether increased gut motility could358

be associated with onset of diarrhoea or a systemic host response following diarrhoea is still359

unresolved.360

The effect of atropine on infected infant mice after 2 doses (6 mg/kg) at 40 and 44 h p.i.,361

determined a clear reduction in motility as well as reduced diarrhoea, where only 3/11 mice had362

diarrhoea after atropine treatment compared of 6/7 in the mock-treated group.363

To conclude, our results obtained with atropine, the classical muscarinic receptor364

antagonist, and loperamide, a -opioid receptor agonist, clearly suggest that ENS is involved in365

the motility response to rotavirus infection. We have earlier found that the fluid loss in RV366

diarrhoea is at least partly induced by an activation of the ENS [11].367


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Adult mice were explored to determine whether motility changes were associated with368

water and electrolyte loss (i.e. diarrhoea) from the small intestine or whether motility changes369

occurred independently of diarrhoea. Previous studies have shown that the small intestine of370

infant mice [11, 35] but not adult mice [28] respond with electrolyte and water secretion371

(diarrhoea) following infection with murine RV. In diarrhoea-resistant adult mice there was a372

most modest increase of intestinal motility at 24 h p.i. (Fig.1C), but no over all increased motility373

during the course of infection.374

The participation of prostaglandins (PGs) in RV diarrhoea may be another factor explaining375

the different time courses for secretion and motility. It has been shown that PGs are produced376

under the influence of microorganisms and have immune-modulatory, anti-inflammatory as well377

as pro-inflammatory actions [36]. Moreover, they can stimulate water secretion [37-40], an effect378

that can be blocked by drugs attenuating nerve activity, such as hexamethonium, a nicotinic379

receptor blocker or lidocaine, a local anaesthetic [41], thus indicating that nerves are involved.380

Loperamide is also a potent inhibitor of intestinal fluid secretion induced by PGE2and cholera381

toxin. In children with RV gastroenteritis elevated levels of PGE2and PGF2in both plasma and382

stools have been found and treatment with the COX inhibitor aspirin reduced the duration of383

diarrhoea [26]. Moreover, intravenous infusion of PG causes abdominal cramps [42], inhibit384

intestinal motility [43, 44] and stimulates water secretion [24, 38, 45]. Moreover RV induces385

COX2 mRNA and secretion of PGE2 already within 8 h p.i [46]. These observations make it386

tempting to speculate that the decreased motility with remaining diarrhoea 72-96 h p.i. might be387

induced by increasing concentrations of PGs. The action of PGs on smooth muscles is, however,388

not yet fully understood, whether they act directly on the muscle cells or modulate of the release389

of neurotransmitters [47].390


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In several in vitro, animal and human studies both increased and decreased permeability391

have been reported. Regarding RV, only a few human studies have been done [15-17], and all392

these studies were however conducted on already RV-infected children and the time course for393

conceivable permeability changes was not studied.394

The use of a combination of two markers of different sizes to measure changes in the small395

intestinal permeability has proved to be sensitive and a specific approach [16, 48]. Interestingly,396

infected infant mice responded with a significant increase in permeability for both probes at 6 and397

12 h p.i, (Fig.5A+B) whereas adults only showed an increase for the 10-kDa probe at 12 h p.i.398

(Fig.6A+B). The increased permeability at 6 and 12 h.p.i was due to the administration of the399

fecal debris but was not associated with diarrhoea nor increased motility. Furthermore oral400

administration of 10% fecal suspension from uninfected animals did not result in diarrhoea. An401

obvious conclusion from this unexpected observation is that increased permeabilityper seis not402

sufficient to cause diarrhoea, since at these early time-points mice do not have diarrhoea. The403

onset of diarrhoea (24-48 h p.i.) was not associated with increased permeability to the two404

dextran probes, despite significant lytic lesions in the small intestine at 48 h p.i. (Fig.2A and B).405

This conclusion is further supported by our observation that even adult mice have similar406

pathological lesions at the same time points yet no diarrhoea [28]. Apparently, RV induced407

pathological lesions per se in the small intestine (Fig.2) are not sufficient to account for408

permeability changes or electrolyte changes during RV infections. A similar observation was409

reported by Heyman and co-workers [49] who studied transepithelial fluxes of horseradish410

peroxidase (HRP; molecular mass about 44 kDa) from mucosa to serosa in EDIM-infected mice411

in an Ussing chamber. During RV diarrhoea (days 2-8) no change in mucosal-to-serosa412

permeability to HRP was observed. On the other hand, during convalescence (days 9-14) when413

virus was no longer present in the mucosa, there was a 10-fold increase in HRP transport. They414


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concluded that RV causes a transient rise in the gut permeability during convalescence [49]. In415

agreement with this observation, we found a significant transient rise in permeability 72 h p.i in416

both infant (p


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571

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FIGURE LEGENDS573

574

FIG 1 Intestinal motility is increased at the time-point of severe diarrhoea, 48 h p.i. (n=5 to 13).575

(A) Illustration of the transit of fluorescent FITC-dextran in the entire intestine of EDIM-infected576

and non-infected infant mice, 15 min post dextran. FITC dextran was visualized by UV-577

spectrophotometer (Chemi-Doc, Bio-Rad, with a FITC specific filter) and was identified as a578

fluorescent-dependent white color, reaching different intestinal segments. (B and C) Transit of579

FITC-dextran in infant (B) and adult (C) mice, 15 respectively 30 min post dextran. Onset of580

diarrhoea in infant mice occurred between 24-48 h p.i. and was most severe at 48 h p.i with581

yellow lose form. Values are based on the transit length of FITC-dextran as percentage of the582

total length of the intestine. Krustal-Wallis; p583

584

FIG 2 Histology of the small intestinal epithelium of EDIM-infected and non-infected infant585

mice. Tissue sections are stained with hematoxylin and eosin. (A and B) Jejenum, 48 h p.i.,586

showing the characteristic vacuolization of cells in the mid and top of villi of a mice with587

diarrhoea and rotavirus in the stool. (C) Non-infected tissue from the duodenum part.588

589

FIG 3Atropine decreases the intestinal motility in EDIM-infected infant and adult mice, 48 h590

p.i.. Transit of FITC-dextran in infant (A) and adult (B) mice, 15 respectively 30 min post591

dextran. FITC dextran was visualized by UV-spectrophotometer (Chemi-Doc, Bio-Rad, with a592

FITC specific filter) and was identified as a fluorescent white color, reaching different intestinal593

segments. Values are based on the transit length of FITC-dextran as percentage of the total length594

of the intestine. Mann-Whitney; p595

596


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FIG 4Loperamide decreases the intestinal motility in EDIM-infected infant and adult mice, 48 h597

p.i.. (A) Transit of fluorescent FITC-dextran in the entire intestine of EDIM-infected and non-598

infected, treated and non-treated infant mice, 15 min post dextran. FITC dextran was visualized599

by UV-spectrophotometer (Chemi-Doc, Bio-Rad, with a FITC specific filter) and was identified600

as a fluorescent white color, reaching different intestinal segments. (B and C) Transit of FITC-601

dextran in infant (B) and adult (C) mice, 15 respectively 30 min post dextran. Values are based602

on the transit length of FITC-dextran as percentage of the total length of the intestine. Mann-603

Whitney; p0,05(*), p604

605

FIG 5Intestinal permeability is not increased at the time for onset of diarrhoea nor at the time of606

most severe diarrhoea in infant mice. Permeability of 4-kDA FITC-dextran (A) and 10-kDa607

Rhodamine-dextran (B) through the intestinal epithelium into serum at different h p.i. was608

measured by spectrophotometry at wave lengths of respectively fluorophore (494/518 and609

565/580 nm), values are given in fluorescence intensity (FI); Mann-Whitney; p0,001(***);610

p0,01(**); p0,05(*).611

612

FIG 6Intestinal permeability is not increased at the most critical time-point of EDIM infection in613

adult mice. Permeability of 4-kDA FITC-dextran (A) and 10-kDa Rhodamine-dextran (B)614

through the intestinal epithelium into serum at different h p.i. was measured by fluorescence at615

wavelengths specific for each fluorophor (494/518 and 565/580 nm), values are given in616

fluorescence intensity (FI); Mann-Whitney; p0,05(*).617

618

619


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25

620

TABLE 1The opioid receptor agonist loperamide attenuate rotavirus diarrhoea621

40 h p.i. 44 h p.i. 48 h p.i. 64 h p.i. 68 h p.i.

EDIM infectedUntreated (n=13)

12/13 12/13 12/13 12/13 12/13

EDIM infectedLoperamide treated (n=9)

8/9 0/8 0/8 6/8 2/8

Non-infectedLoperamide treated (n=6)

0/6 0/6 0/6 0/6 0/6

Non-infectedUntreated (n=2)

0/2 0/2 0/2 0/2 0/2

622

623

624

Loperamide

Loperamide

Loperamide

Diarrhoea in Infant Mice


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26

TABLE 2The muscarinic antagonist atropine attenuate rotavirus diarrhoea625

626

627

628

629

630

40 h p.i. 44 h p.i. 48 h p.i.

EDIM infectedUntreated (n=7)

7/7 7/7 6/7

EDIM infectedAtropine treated (n=11)

11/13 2/11 3/11

Atropine Atropine

Diarrhoea in Infant Mice


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631

TABLE 3Permeability of dextran probes in rotavirus infected infant mice632

Hour

p.i.

Fold Increase

10kDa

FI

10kDa

Fold Increase

4kDa

FI

4kDa

Ratio

10/4kDan=

6 1.8 18,1 (0.9)* 20.1 8,2 (8.6) 0.6

12 1.4 14,5 (1.3) 8.6 2,0 (2.6) 1.2

24 1.0 9,9 (2.1) 0.6 0,9 (0.2) 11.0

48 0.6 5,9 (0.4) 0.9 1,3 (0.3) 4.5

72 1.4 14,3 (1.2) 0.6 0,8 (0.0) 17.9

96 0.5 4,7 (1.0) 1.9 2,6 (0.9) 1.8

Non-

infected

1.0 10,1 (0.3) 1.0 1,4 (0.4) 7.2

633

FI. Fluorescence Intensity, Mean value.634

*=SEM635


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636

TABLE 4Permeability of dextran probes in rotavirus infected adult mice637

Hour

p.i.

Fold Increase

10kDa

FI

10kDa

Fold Increase

4kDa

FI

4kDa

Ratio

10/4kDan=

6 1.1 8.7 (0.3)* 0.4 1.1 (0.2) 7.9

12 1.3 9.9 (0.4) 0.4 1.2 (0.2) 8.3

24 0.9 6.7 (0.6) 0.3 1.0 (0.1) 6.7

48 1.0 7.7 (0.2) 1.2 3.7 (2.5) 2.1

72 1.1 8.6 (0.2) 2.6 7.9 (3.6) 1.1

96 1.1 8.2 (0.4) 0.3 0.8 (0.0) 10.3

Non-

infected

1.0 7.7 (0.6) 1.0 3.0 (1.0) 2.6

638

FI. Fluorescence Intensity, Mean value.639

*=SEM640

641

642

643

644

645

646

647

648

649

650

651

652

653


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