Neonatal Mouse Intestine Research Articles

Oral administration of bone marrow-derived mesenchymal stem cells attenuates intestinal injury in necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is a major cause of morbidity in premature infants. However, effective treatment options for NEC are currently lacking. This study aimed to determine the optimal dose of intraperitoneally administered bone marrow-derived mesenchymal stem cells (BM-MSCs) and investigate the therapeutic potential of orally administered BM-MSCs in NEC. Neonatal mice were fed maternal breast milk for the first 2 days of life. On day 3, the neonatal mice were randomly divided into control, negative control, and BM-MSC-treated groups. Lipopolysaccharide (LPS) was administered for 3 days, and cold stress (4°C, 10 minutes) was applied 3 times a day to induce NEC. High-dose (1×106 cells) or low-dose (1×105 cells) BM-MSCs were administered intraperitoneally 1 or 3 times between days 6 and 8 to treat the NEC. The orally administered group received a low dose of BM-MSCs on day 6. Furthermore, except for the control group, intraepithelial cells (IECs) of the small intestine of neonatal mice were treated with LPS and exposed to 5% O2/95% N2 hypoxic stress for 2 hours. Thereafter, each was treated with BM-MSCs. Tissue injury, apoptosis, and inflammatory marker levels were significantly reduced after BM-MSC administration. Oral administration was as effective as intraperitoneal administration, even at a low dose (1×105 cells) of BM-MSCs. The efficacy of high (1×106 cells) or multiple divided doses of BM-MSCs did not differ from that of low-dose treatment. Significantly improved wound healing was observed after BM-MSC administration to injured IECs. The oral administration of BM-MSCs is a promising treatment option for NEC in infants. Further human studies of BM-MSCs are necessary to determine the optimal dose required to achieve safe and effective outcomes.

Live Intravital Intestine with Blood Flow Visualization in Neonatal Mice Using Two-photon Laser Scanning Microscopy

This protocol describes a novel technique to investigate the microcirculation dynamics underlying the pathology in the small intestine of neonatal mice using two-photon laser-scanning microscopy (TPLSM). Recent technological advances in multi-photon microscopy allow intravital analysis of different organs such as the liver, brain and intestine. Despite these advances, live visualization and analysis of the small intestine in neonatal rodents remain technically challenging. We herein provide a detailed description of a novel method to capture high resolution and stable images of the small intestine in neonatal mice as early as postnatal day 0. This imaging technique allows a comprehensive understanding of the development and blood flow dynamics in small intestine microcirculation.

Histopathological evaluation of crypt fission during intestinal development in neonatal mice.

Pathological evaluation of juvenile toxicity studies requires the understanding of normal tissue development at different ages. Here, we report the morphological features of the neonatal mouse intestine, focusing on crypt fission. Postnatal day (PND) 7 and 14 mice showed fewer crypts and less mature epithelial morphology compared to PND 21 and 28. Crypt fission occurred in three stages: 1) flattening of the crypt base into a skirt shape, 2) penetration of myofibroblasts into the crypt base center, and 3) complete separation of a single crypt into two daughter crypts. The ratio of crypt fission to total number of crypts was the highest at PND 14 and 7 in the jejunum and colon, respectively. Crypt fission, a key phenomenon for balance or imbalance in epithelial cell hierarchy, including stem and differentiated cells, is related to tissue injury repair and tumorigenesis. Therefore, examining crypt fission can provide valuable insights into current conditions of intestine.

Blocking NF-κB Activation in Ly6c+ Monocytes Attenuates Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) is a devastating disease affecting premature infants with intestinal inflammation and necrosis. The neonatal intestinal inflammatory response is rich in macrophages, and blood monocyte count is low in human NEC. We previously found that NF-κB mediates the intestinal injury in experimental NEC. However, the role of NF-κB in myeloid cells during NEC remains unclear. Herein, inhibitor of kappaB kinase β (IKKβ), a critical kinase mediating NF-κB activation, was deleted in lysozyme M (Lysm)-expressing cells, which were found to be Cd11b+Ly6c+ monocytes but not Cd11b+Ly6c- macrophages in the dam-fed neonatal mouse intestine. NEC induced differentiation of monocytes into intestinal macrophages and up-regulation of monocyte recruitment genes (eg, L-selectin) in the macrophage compartment in wild-type mice, but not in pups with IKKβ deletion in Lysm+ cells. Thus, NF-κB is required for NEC-induced monocyte activation, recruitment, and differentiation in neonatal intestines. Furthermore, pups with Lysm-IKKβ deletion had improved survival and decreased incidence of severe NEC compared with littermate controls. Decreased NEC severity was not associated with an improved intestinal barrier. In contrast, NEC was unabated in mice with IKKβ deletion in intestinal epithelial cells. Together, these data suggest that recruitment of Ly6c+ monocytes into the intestine, NF-κB activation in these cells, and differentiation of Ly6c+ monocytes into macrophages are critical cellular and molecular events in NEC development to promote disease.

Enteric Neural Stem Cells Expressing Insulin-Like Growth Factor 1: A Novel Cellular Therapy for Hirschsprung's Disease in Mouse Model.

Transplanting enteric neural stem cells (ENSCs) is an innovative approach for replacing enteric neurons in Hirschsprung's disease (HSCR). However, posttransplantation cell survival and differentiation limit efficacy. We aimed to investigate whether transplantation of ENSCs engineered with insulin-like growth factor 1 (IGF-1) could improve survival and differentiation of the engrafted cells and promote functional recovery of the aganglionic colon. ENSCs were isolated from the intestine of neonatal mice and genetically modified to express IGF-1. After implantation into the mice aganglionic colon induced by benzalkonium chloride, survival and differentiation of engrafted cells were assessed by immunohistochemistry for GFP, neuronal, and glial cell markers. Colonic motility was quantified by colonic bead expulsion time and response to electrical field stimulation (EFS). Expression of the neural marker nNOS and the glial cell marker (glial fibrillary acidic protein [GFAP]) was increased in IGF-1-ENSCs. IGF-1 had also improved neuroglial differentiation of ENSCs following transplantation in vivo. Colonic motility was significantly improved after IGF-1-ENSC transplantation, as demonstrated by reduction of colonic bead expulsion time and recovery of EFS-induced relaxation. IGF-1 has enhanced neurogenesis and function of ENSCs upon transplantation for enteric nervous system replacement, which may provide a potential novel therapy for the treatment of HSCR.

Dextran sodium sulfate (DSS) induces necrotizing enterocolitis-like lesions in neonatal mice.

BackgroundNecrotizing enterocolitis (NEC) is an inflammatory bowel disease of preterm human newborns with yet unresolved etiology. An established neonatal murine model for NEC employs oral administration of lipopolysaccharides (LPS) combined with hypoxia/hypothermia. In adult mice, feeding dextran sodium sulfate (DSS) represents a well-established model for experimental inflammatory bowel disease. Here we investigated the effect of DSS administration on the neonatal murine intestine in comparison with the established NEC model.Methods3-day-old C57BL/6J mice were either fed formula containing DSS or LPS. LPS treated animals were additionally stressed by hypoxia/hypothermia twice daily. After 72 h, mice were euthanized, their intestinal tissue harvested and analyzed by histology, qRT-PCR and flow cytometry. For comparison, adult C57BL/6J mice were fed with DSS for 8 days and examined likewise. Untreated, age matched animals served as controls.ResultsAdult mice treated with DSS exhibited colonic inflammation with significantly increased Cxcl2 mRNA expression. In contrast, tissue inflammation in neonatal mice treated with DSS or LPS plus hypoxia/hypothermia was present in colon and small intestine as well. Comparative analysis of neonatal mice revealed a significantly increased lesion size and intestinal Cxcl2 mRNA expression after DSS exposure. Whereas LPS administration mainly induced local neutrophil recruitment, DSS treated animals displayed increased monocytes/macrophages infiltration.ConclusionsOur study demonstrates the potential of DSS to induce NEC-like lesions accompanied by a significant humoral and cellular immune response in the small and large intestine of neonatal mice. The new model therefore represents a good alternative to LPS plus hypoxia/hypothermia administration requiring no additional physical stress.

CCL20 Displays Antimicrobial Activity Against Cryptosporidium parvum, but Its Expression Is Reduced During Infection in the Intestine of Neonatal Mice.

CCL20 is a chemokine with antimicrobial activity. We investigated its expression and role during neonatal cryptosporidiosis, a worldwide protozoan enteric disease leading to severe diarrhea. Surprisingly, during infection by Cryptosporidium parvum, CCL20 production by the intestine of neonatal mice is reduced by a mechanism independent both of the enteric flora and of interferon γ, a key cytokine for the resolution of this infection. However, oral administration of recombinant CCL20 to neonatal mice significantly reduced the parasite load by a mechanism that was independent of immune cell recruitment and occurred instead by direct cytolytic activity on free stages of the parasite. MiR21 functionally targets CCL20 and is upregulated during the infection, thus contributing to the downregulation of the chemokine. Our findings demonstrate for the first time the direct antiparasitic activity of CCL20 against an enteric protozoan and its downregulation during C. parvum infection, which is detrimental to parasite clearance.

Probiotics stimulate enterocyte migration and microbial diversity in the neonatal mouse intestine.

Beneficial microbes and probiotics show promise for the treatment of pediatric gastrointestinal diseases. However, basic mechanisms of probiosis are not well understood, and most investigations have been performed in germ-free or microbiome-depleted animals. We sought to functionally characterize probiotic-host interactions in the context of normal early development. Outbred CD1 neonatal mice were orally gavaged with one of two strains of human-derived Lactobacillus reuteri or an equal volume of vehicle. Transcriptome analysis was performed on enterocyte RNA isolated by laser-capture microdissection. Enterocyte migration and proliferation were assessed by labeling cells with 5-bromo-2'-deoxyuridine, and fecal microbial community composition was determined by 16S metagenomic sequencing. Probiotic ingestion altered gene expression in multiple canonical pathways involving cell motility. L. reuteri strain DSM 17938 dramatically increased enterocyte migration (3-fold), proliferation (34%), and crypt height (29%) compared to vehicle-treated mice, whereas strain ATCC PTA 6475 increased cell migration (2-fold) without affecting crypt proliferative activity. In addition, both probiotic strains increased the phylogenetic diversity and evenness between taxa of the fecal microbiome 24 h after a single probiotic gavage. These experiments identify two targets of probiosis in early development, the intestinal epithelium and the gut microbiome, and suggest novel mechanisms for probiotic strain-specific effects.

The potential effects of Lactobacillus acidophilus on the prevention and treatment of neonatal mice infected with human rotavirus

Objective To investigate the potential effect of Lactobacillus acidophilus (L. acidophilus) on prevention and treatment of neonatal mice infected with human rotavirus (HRV). Methods Sixty 4-day-old kunming mice were randomly divided into control group. HRV infected group, L. acidophilus pretreated group (treated before HRV infection ) and L. acidophilus treated group(treated after HRV infection). The manifestation and pathological changes in small intestine of neonatal mice were observed. The HRV antigen in the feces and intestines were measured by ELISA and fluorescent-focus assay, respectively. Results The severity and duration of diarrhea as well as mortality in L. acidophilus pretreated group and treated group were lower than those in HRV infected group. The duration of HRV-antigens shedding following infection was considerably prolonged in HRV infected group compared to that in L. acidophilus pretreated group and treated group. Furthermore, decreased expression of HRV antigen and little pathological changes in intestinal mucosa were found in L. acidophilus pretreated group and treated group when compared with HRV infected group. Conclusion L. acidophilus may be used as an alternative approach for the prevention and treatment of neonatal mice infected with HRV. Key words: Rotavirus; Lactobacillus acidophilus ; Neonatal mice; Diarrhea; Intestinal mucosa

Functional analysis of the mouse Fcgrt 5′ proximal promoter

The Brambell receptor (FcRB, FcRn) mediates transmission of immunity from mother to young perinatally and plays a central role in IgG protection and homeostasis throughout life. Developmental and tissue specific expression is via transcriptional regulation of the gene for the receptor heavy chain, Fcgrt. In neonatal mouse intestine, expression is high to absorb IgG from mother's milk, but then nearly absent in adult intestine and in most other tissues. In this initial functional characterization, reporter gene assays revealed at least two upstream promoter regions (−372/−140 and −105/−1), each with repressor and activator functions, and a downstream activator domain (+1/+78). The gene carries no upstream TATA element or Inr sequence, and an apparent downstream promoter feature (DPE) lacks typical context for an active element, rendering uncertain the nature of the organizing feature for the transcription initiation complex. Electrophoretic mobility shift analysis of the proximal upstream region identified transcription factor (TF) binding to motifs for NF1, Sp1 (GT box) and Ets. Binding to the GT box and Ets motif was observed only with mature cell sources, and not with neonatal enterocyte extracts. Site-directed mutagenesis confirmed that TF binding to the GT box up-regulates promoter activity in adult (NIH3T3) cells, whereas binding to the Ets motif represses activity. Binding of NF1 protein to the Fcgrt promoter was confirmed in nuclear extracts of NIH3T3 cells and in adult mouse enterocytes, whereas an apparently different TF bound uniquely to the NF1 site in neonatal enterocyte extracts as the sole identified candidate for an expected developmental-specific high-level activator in this tissue. These data indicate regions of potential importance in the Fcgrt proximal promoter and additionally suggest that the selective temporal and spatial availability of specific TFs may contribute to the developmental and tissue-specific regulation of Fcgrt expression.

Experimental transmission of abnormal prion protein (PrPsc) in the small intestinal epithelial cells of neonatal mice.

Using an immunohistochemical method, we attempted to detect the transmission of abnormal prion protein (PrPsc) to the enterocytes of the small intestine of neonatal mice by oral exposure with sheep brain affected by scrapie. Five 1-day-old neonatal mice were exposed by oral inoculation to the homogenized brain of a scrapie-affected sheep. In the small intestine of all mice 1 hour after inoculation, immunoreactivity with antinormal prion protein (PrPc) antibody was seen in the cytoplasm of villus enterocytes. This finding suggests transmission of abnormal PrPsc into the cytoplasm of enterocytes. In control mice treated with normal sheep brain, no PrPc signal was seen in enterocytes of the small intestine. Immunopositivity for neurofilament protein and glial fibrillary acidic protein was seen in the cytoplasm of enterocytes of mice inoculated with scrapie and normal sheep brain. This suggests that the enterocytes of neonatal mice can absorb PrPsc and other macromolecular proteins of the sheep brain affected by scrapie and may be more important than previously thought as a pathway for PrPsc transmission in neonatal animals.

The neonatal IgG receptor FcRn, is expressed in the human fetal intestine and the non-malignant fetal intestinal epithelial cell line

The neonatal Fc receptor FcRn functions in rodents to transfer maternal gammaglobulins (IgG) from mother to young via the neonatal intestine. In humans most of this transfer is presumed to occur in utero via the placenta. The fetus swallows amniotic fluid that contains immunogiobulins but it is unknown whether this transfer also occurs via the fetal intestine. Human FcRn has been identified in the human syncitiotrophoblast and plays a role in the materno-fetal transfer of antibody. It is also expressed in adult intestine and may be important in IgG catabolism. Objective: We hypothesize that the FcRn receptor is expressed in the human fetal intestine and may play a role in IgG transport from the amniotic fluid/colostrum into the fetal circulation. We wish to demonstrate expression of the FcRn 1) along different segments of the human fetal intestine and 2) in a human non-malignant fetal intestinal epithelial cell line (H4). Methods: Lysates were prepared from human fetal stomach, small intestine and colon from a 22 week gestation fetus and from H4 cells. Caco-2 cells and T-84 cells were used as positive controls. Prepared lysates were run on Western blots and were probed with anti-FcRn antibodies. Bound antibody was detected by HRP-conjugated secondary antibody and enhanced chemiluminescence. FcRn expression was normalized against GAPDH and quantitated by densitometry. Immunostaining of human fetal intestine, neonatal mouse intestine (+ contro[), H4 cells and T-B4 ceils (+ control), was done with a rabbit anti-human FcRn antibody and detected with the appropriate conjugated secondary antibody for microscopy. Results: The FcRn heavy chain was detected from the fetal intestine and H4 cells on Western blot analysis. On immunostaining, a subapical staining pattern for FcRn was seen on human fetal intestine. The H4 cells also stained positive for FcRn. Conclusion: FcRn expression is demonstrated in the human fetal intestine and in the human non-malignant fetal intestinal epithelial cell line (H4) suggesting that FcRn may play a role in the uptake and transport of IgG in the fetus.

Differential appearance of T cell subsets in the large and small intestine of neonatal mice.

We examined the appearance of intestinal intraepithelial lymphocytes (IEL) during the first 12 wk of life to gain insight into postnatal factors that contribute to the differences found between IEL in the large and small intestines of adult mice. Intestinal T cells were very infrequent at birth, but increased in number in the large and small intestine during the first 4 wk of life and then stabilized. The small intestinal epithelium at 2 wk of age contained mostly T cell receptor (TCR) alphabeta+, CD2+ T cells, unlike IEL in adult mice, which were composed of nearly equal proportions of CD2-, TCR alphabeta+ and TCR gammadelta+ cells. Between 2 and 3 wk of age, TCR gammadelta+, CD2- IEL increased greatly in the small intestine, whereas TCR alphabeta+ cells expressing CD2 decreased. By contrast, IEL in the large intestine at 2 and 3 wk of age were mostly TCR alphabeta+, CD2+ T cells similar to large intestinal IEL in adult mice. And finally, the expression of CD69 increased earlier and to higher levels on TCR alphabeta+ and TCR gammadelta+ IEL in the small intestine than in the large intestine. Our results demonstrate that IEL in the large and small intestine are phenotypically similar during suckling and that differences between these populations are established after weaning. Furthermore, the earlier accumulation of IEL with an activated adult IEL phenotype in the small intestine suggests that these T cells mature or expand in the gut and contribute to the maturation of immune function during postnatal life in mice.

Enteropathogenicity of Plesiomonas shigelloides and Aeromonas spp. in experimental mono- and coinfection with Cryptosporidium parvum in the intestine of neonatal BALB/c mice

Enteropathogenicity of Plesiomonas shigelloides, Aeromonas hydrophila, A. caviae and A. sobria was studied both in monoinfections and in coinfections with coccidium Cryptosporidium parvum in neonatal BALB/c mice. In monoinfection experiments, neonatal BALB/c mice were orally infected with 7×10 7 or 7×10 8 CFU, respectively, of a strain of P. shigelloides or a strain of an Aeromonas spp. In coinfection experiments, the neonatal mice were, in addition to being orally infected with one of the four bacterial species, orally infected with an inoculum containing 10 5 oocysts of C. parvum. Results from monoinfections with P. shigelloides revealed long-term colonisation of the neonatal mouse intestine by this pathogen, along with associated pathological lesions. The lesions varied in severity from atrophy to necrosis of the mucosal inner surface of the ileum and colon, with predilection to the colon and brush border of colonic enterocytes. The effects of coinfection of P. shigelloides with C. parvum were characterised by bacteremia and heavy colonisation of the intestine by P. shigelloides. In addition, extensive necrotising inflammatory changes in the ileum and colon were accompanied by diarrhoea and deaths of coinfected mice. In contrast, the results from monoinfections of neonatal mice with Aeromonas spp. showed only a short-term colonisation of the intestine by the pathogen. However, when mice were coinfected with A. hydrophila and C. parvum, then the growth of the bacterial species was prolonged, and occurred in both the spleen and intestine. However, no substantial clinical or histopathological changes were observed in mice, whether monoinfected with Aeromonas spp. or coinfected with C. parvum. Our study suggests that experimental monoinfections of neonatal BALB/c mice with P. shigelloides, Aeromonas spp. and C. parvum, together with coinfections (each bacterial species with the protozoan C. parvum), may serve as a useful model to study the initial steps of gastrointestinal colonisation and diarrhoeal disease syndromes caused by enteropathogenic bacteria and protozoa, individually and in combination.

Molecular ecological analysis of the succession and diversity of sulfate-reducing bacteria in the mouse gastrointestinal tract.

Intestinal sulfate-reducing bacteria (SRB) growth and resultant hydrogen sulfide production may damage the gastrointestinal epithelium and thereby contribute to chronic intestinal disorders. However, the ecology and phylogenetic diversity of intestinal dissimilatory SRB populations are poorly understood, and endogenous or exogenous sources of available sulfate are not well defined. The succession of intestinal SRB was therefore compared in inbred C57BL/6J mice using a PCR-based metabolic molecular ecology (MME) approach that targets a conserved region of subunit A of the adenosine-5'-phosphosulfate (APS) reductase gene. The APS reductase-based MME strategy revealed intestinal SRB in the stomach and small intestine of 1-, 4-, and 7-day-old mice and throughout the gastrointestinal tract of 14-, 21-, 30-, 60-, and 90-day-old mice. Phylogenetic analysis of APS reductase amplicons obtained from the stomach, middle small intestine, and cecum of neonatal mice revealed that Desulfotomaculum spp. may be a predominant SRB group in the neonatal mouse intestine. Dot blot hybridizations with SRB-specific 16S ribosomal DNA (rDNA) probes demonstrated SRB colonization of the cecum and colon pre- and postweaning and colonization of the stomach and small intestine of mature mice only. The 16S rDNA hybridization data further demonstrated that SRB populations were most numerous in intestinal regions harboring sulfomucin-containing goblet cells, regardless of age. Reverse transcriptase PCR analysis demonstrated APS reductase mRNA expression in all intestinal segments of 30-day-old mice, including the stomach. These results demonstrate for the first time widespread colonization of the mouse intestine by dissimilatory SRB and evidence of spatial-specific SRB populations and sulfomucin patterns along the gastrointestinal tract.

A Study of the Microcirculation in Whole Villi of Neonatal Mice Using a Peroxidase Histochemical Staining Method

The anatomy of the microcirculation of intestinal villi from the upper, middle, and lower small intestine of neonatal mice from 8 to 14 days old was studied using a histochemical peroxidase technique that specifically stained erythrocytes. Over 8-14 days, there was little chronological variation between the same regions of gut; the exception was the lower intestine, which, in younger mice, was noticeably less well perfused with erythrocytes. Vascular beds in the middle and lower intestine comprised a hairpin loop with cross-connections. In the upper intestine, the capillary beds were generally more complex, particularly in apical regions of the villi. Most villi were well perfused with erythrocytes, but a minority (less than 10%) contained considerably fewer red cells, some to the point of being totally ischemic. Other villi (less than 5%) were hyperemic, and the vascular beds packed and engorged with red cells. Usually, the packing density (hematocrit) of red cells within blood vessels increased progressively from villus base to apex. Red-cell deformation was more prevalent at villus apices, with marked crenation in some villi, yet in the basal regions of these same villi, the red cells were of normal discoid shape. The peroxidase staining technique produces a reliable histological picture of red cells circulating through villi. It also reveals differential perfusion of erythrocytes between and within villi, and that blood vessels pass through hypertonic zones in the apical regions of villi.

A Study of the Microcirculation in Whole Villi of Neonatal Mice Using a Peroxidase Histochemical Staining Method

SummaryThe anatomy of the microcirculation of intestinal villi from the upper, middle, and lower small intestine of neonatal mice from 8 to 14 days old was studied using a histochemical peroxidase technique that specifically stained erythrocytes. Over 8–14 days, there was little chronological variation between the same regions of gut; the exception was the lower intestine, which, in younger mice, was noticeably less well perfused with erythrocytes. Vascular beds in the middle and lower intestine comprised a hairpin loop with cross‐connections. In the upper intestine, the capillary beds were generally more complex, particularly in apical regions of the villi. Most villi were well perfused with erythrocytes, but a minority (<10%) contained considerably fewer red cells, some to the point of being totally ischemic. Other villi (<5%) were hyperemic, and the vascular beds packed and engorged with red cells. Usually, the packing density (hematocrit) of red cells within blood vessels increased progressively from villus base to apex. Red‐cell deformation was more prevalent at villus apices, with marked crenation in some villi, yet in the basal regions of these same villi, the red cells were of normal discoid shape. The peroxidase staining technique produces a reliable histological picture of red cells circulating through villi. It also reveals differential perfusion of erythrocytes between and within villi, and that blood vessels pass through hypertonic zones in the apical regions of villi.

An in Vitro Model to Study Aspects of the Pathophysiology of Murine Rotavirus‐Induced Diarrhoea

SummaryAn in vitro system is described and validated for studying transport of solutes and water in both uninfected and rotavirus‐infected neonatal mouse intestine. Control intestine exhibited stable water absorption for periods of up to 40 min. Water absorption was temperature‐dependent, Na‐dependent, and enhanced by glucose‐containing perfusion solutions. Theophylline induced net secretion of water by control intestinal tissue. Water transport by rotavirus‐infected lower small intestine was significantly depressed as compared to control levels, and rotavirus‐infected middle small intestine exhibited net secretion of water. Upper small intestine and colon from infected animals did not differ significantly from control tissues in their ability to transport water. Water secretion by infected middle small intestine was reversed to absorption by glucose‐containing solutions.

An in Vitro Model to Study Aspects of the Pathophysiology of Murine Rotavirus-Induced Diarrhoea

An in vitro system is described and validated for studying transport of solutes and water in both uninfected and rotavirus-infected neonatal mouse intestine. Control intestine exhibited stable water absorption for periods of up to 40 min. Water absorption was temperature-dependent. Na-dependent, and enhanced by glucose-containing perfusion solutions. Theophylline induced net secretion of water by control intestinal tissue. Water transport by rotavirus-infected lower small intestine was significantly depressed as compared to control levels, and rotavirus-infected middle small intestine exhibited net secretion of water. Upper small intestine and colon from infected animals did not differ significantly from control tissues in their ability to transport water. Water secretion by infected middle small intestine was reversed to absorption by glucose-containing solutions.

An electron microscopic investigation of time-related changes in the intestine of neonatal mice infected with murine rotavirus.

Seven-day-old mice were infected orally with murine rotavirus (EDIM) and regions of the gut examined at 24 h intervals up to 7 days by electron microscopy. Structural changes were correlated with data on viral antigen production, thymidine kinase activity, and clinical signs of diarrhea. No pathological changes were detected in the colon. Infection and structural damage were confined to the small intestine, with middle regions showing the most pronounced changes. Constriction of villus bases, edema of the lamina propria, and vacuolation of enterocytes occurred at 24 h postinfection (PI), i.e., before evidence of major virus replication. Transient villus atrophy occurred at 48 h PI. Recovery of villus length was evident by 72 h PI accompanied by evidence of marked enterocyte replication at villus bases. Many enterocytes were damaged with little evidence for the presence of virus particles. By 96 h PI, villi had almost recovered from infection although some enterocytes were still damaged; no virus particles were detected in these cells. A second phase of villus damage and edema of the lamina propria occurred at 120 h PI; the pathology resembled that at 24-48 h PI. By 144 to 168 h PI, recovery of the mucosa from infection was virtually complete. We suggest that many of the pathological features following rotavirus infection result from rotavirus-induced ischemia of villi and that diarrhea results from malabsorption of fluid by damaged villi and hypersecretion of ions released from increased numbers of dividing cells at villus-crypt borders.