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In mice, the number of intestinal villous columnar epithelium cells that incorporate abnormal prion protein (PrPSc) decreases significantly after weaning. In this study, the dynamics of PrPSc uptake during the growth of hamsters were investigated by inoculating scrapie 263K agent orally into suckling and weanling Syrian hamsters and estimating the number of PrPSc-positive villous epithelium cells immunohistochemically. The number of PrPSc-positive cells declined significantly as the hamsters aged. The present results suggest that a tendency toward decline of PrPSc-positive cells with increasing age might be a common phenomenon among the superfamily Muridae.
Transmissible spongiform encephalopathies (or prion diseases) are a group of fatal neurodegenerative diseases characterized by the accumulation of PrPSc, which constitutes the major component of the infectious agents of TSEs, in the CNS (1). Although exposure to TSE infectious agents can come from a variety of sources, such as in the diet, from the environment, and iatrogenically, the main route of infection is thought to be oral. For example, BSE in cattle is spread by consumption of meat bone meal contaminated by BSE agents (2), and variant Creutzfeldt-Jakob disease is thought to be transmitted to humans by BSE agents in the diet (3). Moreover, scrapie in sheep and chronic wasting disease in cervids are suspected to occur because of environmental exposure to the excreta of affected animals (4, 5). After entry by the oral route, TSE agents invade through the intestinal epithelial cell barrier. They accumulate and propagate in the follicular dendritic cells of GALT and then spread to non-GALT–related lymphoid tissues before they reach the CNS through the peripheral nerve system (6).
A correlation between the risks of infection by TSEs and the age at which the host is exposed to the TSE agents has been proposed. Epidemiological studies of BSE have shown that most cattle affected by BSE were exposed to the infectious agents in the first 6 months of life, and that the risk of infection is likely to be higher in younger cattle (7). Moreover, experimental studies of transmission of scrapie have shown that orally inoculated neonatal sheep and intracerebrally or intraperitoneally inoculated hamsters exhibit shorter incubation periods than older animals (8, 9). Although some hypotheses have been proposed to account for the shorter incubation period in neonatal animals (7–9), the details of the mechanisms remain poorly understood.
There are no previous reports on the mechanism of PrPSc invasion in the early stages of infection in young animals. However, in a previous study, our group identified the entry sites of PrPSc from the small intestines of suckling and weanling mice that had been fed orally with the scrapie agent (10). That report suggested that far fewer intestinal villous columnar epithelium cells incorporated PrPSc in weanling mice than in suckling mice, and that the number of PrPSc-positive villous epithelium cells decreased significantly as the mice grew. In TSE research, rodents including mice and hamsters (11), nonhuman primates (12), and small ruminants including sheep (13) and goats (14) have mainly been used as in vivo experimental models. In particular, Syrian hamsters with scrapie are a useful model because of the very short incubation periods associated with some experimental routes of infection, such as intracerebral, intraperitoneal, and oral inoculation (15, 16). However, the uptake dynamics of PrPSc in the intestinal villous epithelium at different growth stages of Syrian hamsters have not been reported. In the present study, the uptake dynamics of PrPSc in the intestinal villous epithelium of Syrian hamsters were explored by feeding suckling and weanling hamsters orally with the scrapie agent and performing immunohistochemistry analysis.
Brain homogenates (25%) derived from terminally ill hamsters infected with scrapie strain 263K (15) were prepared in order to administer the scrapie agent to uninfected animals. Doses of 200 μL of the homogenates were administrated orally to Syrian hamsters at 10, 15, 20, and 25 days of age (n= 6 for each age group). Age-matched control hamsters (n= 2 for each age group) were administered the same volume of 25% homogenates of normal hamster brain. The same dose was repeated 3 hrs after the first administration, the hamsters were killed, and the small intestine harvested 1 hr after the second administration. All animals were treated in accordance with procedures authorized by the Animal Experiment Committee of the Nihon University College of Bioresource Sciences. The samples were fixed in PBS containing 4% paraformaldehyde (Wako Pure Chemical Industries, Osaka, Japan) for 2 hrs before being washed in PBS containing 6.8% sucrose (Wako Pure Chemical Industries). After dehydration in 100% acetone (Wako Pure Chemical Industries) for 1 hr, the tissue samples were embedded in resin (Technovit 8100; Heraeus Kulzer, Wehrheim, Germany) (10) in accordance with the manufacturer's instructions and then sectioned at a thickness of 5 μm. The sections were pretreated with 0.1% CaCl2 (Wako Pure Chemical Industries) at pH 7.8 containing 0.01% trypsin (Wako Pure Chemical Industries) for 15 min at 37°C, and immersed in 0.3% hydrogen peroxide in methanol (Wako Pure Chemical Industries) for 30 min to inactivate endogenous peroxidase. After incubation with mouse anti-PrP monoclonal antibody 3F4 (2 mg/mL; diluted 1:200 with PBS; purchased from Funakoshi, Tokyo, Japan) at 4°C overnight and secondary horseradish peroxidase-coupled goat anti-mouse IgG antibody (4 μg/mL; Nichirei, Tokyo, Japan) at room temperature for 30 min, diaminobenzidine (Wako Pure Chemical Industries) was applied and incubated for 10 min. The sections were counterstained by hematoxylin for 45 sec, as described previously (10). The number of PrPSc positive cells in each visual field was counted at six random points at 200× magnification in a total of 54 specimens per group (nine from each hamster) and expressed as the mean ± standard deviation. The observer assessing the intestinal sections to determine the intensity of infection did not know from which animals the sections had been prepared. Student's t test was used for statistical analysis (P < 0.01) of the differences between the groups.
Abnormal prion proteins were observed in the villous epithelium in all groups of hamsters that had received the scrapie agent by the oral route (Fig. 1), but no PrPSc were observed in any of the groups fed normal hamster brain homogenate (Fig. 1e–h). In the 10- and 15-day-old hamsters, PrPSc-positive cells were observed relatively easily in the intestinal villous epithelium (Fig. 1a, b). However, fewer villous epithelial cells with incorporated PrPSc were observed in the 20- and 25-day-old hamsters, despite an increased number of PrPSc that had not been incorporated but had attached to the surface of the villous epithelium (Fig. 1c, d). Statistical analysis revealed that the number of PrPSc-positive cells decreased significantly with increasing age: the most PrPSc were observed in the 10-day-old hamsters, followed by the 15-day-old and 20-day-old animals. The fewest PrPSc-positive cells were found in the 25-day-old hamsters (Fig. 2). This age-related decline in PrPSc-positive cells is similar to that observed in mice (10), suggesting that it might be a common phenomenon in the superfamily Muridae.
Figure 1. Intestinal villi from Syrian hamsters fed with (a−d) 263K-infected or (e−h) normal hamster brain homogenate. PrPSc-positive cells (arrows) were easily observed in (a) 10 and (b) 15 day old Syrian hamsters, but fewer were observed at (c) 20 and (d) 25 days, despite an increase in the amount of PrPSc attached to the surface of the villous epithelium (arrowheads). No incorporated PrPSc were observed in any of the groups fed with normal hamster brain: (e) 10 days, (f) 15 days, (g) 20 days and (h) 25 days. Scale bars represent 20 μm.
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Figure 2. Age-related incorporation of PrPSc into intestinal villous epithelium cells of Syrian hamsters at 10, 15, 20, and 25 days of age fed with 263K-infected hamster brain homogenate. The number of PrPSc positive cells in each visual field was counted at six random points at 200× magnification in a total of 54 specimens per group (nine from each hamster). The number of PrPSc-positive cells decreased significantly (**P < 0.01) with increasing age. Data are expressed as mean ± standard deviation, and statistical differences were determined by Student's t-test.
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In regard to the uptake of PrPSc in hamsters, one study of Syrian hamsters fed 263K scrapie agent orally focused on Peyer's patches at 1, 2, 4, and 8 days after inoculation (17). In this study, deposition of PrPSc was observed in the Peyer's patches of hamsters at 2, 4 and 8 days after inoculation but not at 1 day after inoculation, suggesting that orally inoculated PrPSc accumulates in Peyer's patches two or more days after inoculation (17). However, no reports on PrPSc uptake by the intestinal villous epithelium in suckling and weanling hamsters immediately after oral exposure to TSE agents have been published.
There are two main entry sites in the intestine for PrPSc: GALT, including M cells of the follicle associated epithelium and Peyer's patches, and non-GALT structures such as villous epithelium. Findings from study of a cell culture model suggest that M cell dependant transcytosis plays an important role in uptake of PrPSc from the mucosal surface of the intestine (18). In addition, mice lacking Peyer's patches and mesenteric lymph nodes are resistant to oral infection byTSE (19, 20). Thus, GALT is thought to be an obligatory site for infection by TSE agents via the oral route. In regard to non-GALT structures, one report has suggested that PrPSc is transported through the enterocytes and lamina propria to the villous lacteal glands (21). Recent studies have shown that the 37 kDa/67 kDa laminin receptors expressed on the apical brush border of enterocytes (22) and ferritin (23) also play a role in the entry of PrPSc from intestine. Notably, treatment of scrapie infected mice with anti-37 kDa/67 kDa laminin receptor antibody prolongs the length of time between onset of disease and the death of all infected animals (24). These reports also found that non-GALT structures have an important role as primary entry sites for oral infection by TSE agents and TSE pathology (21–24).
In our previous study, most of the PrPSc was incorporated into the villous epithelium of suckling and weanling mice (10); the present study showed a similar phenomenon. In addition, our other study showed that PrPSc incorporation into the villous epithelium is inhibited by the Fc receptor blocker. Therefore the neonatal Fc receptor, which expresses on columnar epithelial cells, is associated in some way with the uptake of PrPSc from the intestine in the suckling and weanling periods (25). Furthermore, other researchers have reported that uptake of PrPSc into the intestinal villous epithelium can also be observed in 1 day old neonatal mice inoculated with brain homogenate from diseased sheep (26). Therefore, it appears that the villous epithelium may act as the main entry site for PrPSc invasion in immature rodents because of incomplete development of Peyer's patches, whereas GALT is an important entry site for PrPSc in mature animals.
An association between risk of infection with TSEs and host age has been described previously (7–9). In regard to the difference between young and old animals in pathogenesis of TSEs, some reports have suggested that aspects of the status of follicular dendritic cells, such as their maturation, degree of expression of PrPc and function, influence the onset of scrapie in mice with peripheral scrapie infection (27, 28). With respect to the results of our previous (10) and present studies, fewer PrPSc-positive villous epithelium cells were found in weanling than in suckling mice and hamsters. If the number of PrPSc-positive villous epithelium cells is decreased, the chance of PrPSc invasion from the intestine may also decrease. Therefore, the dynamics and number of PrPSc-positive villous epithelium cells may have some effect on scrapie pathogenesis. However, our data does not allow us to ascertain whether this tendency results in a decrease in susceptibility to prion infection. In order to clarify associations between the dynamics and number of PrPSc-positive villous epithelium cells and infection with TSEs in immature rodents, further studies are needed to determine the fate of PrPSc after uptake from the intestine and the effect on TSEs pathogenesis of oral exposure to TSE agents in juvenile animals.
In conclusion, the previously reported age-dependent decline in the number of PrPSc incorporated into villous epithelium cells in mice has now also been observed in Syrian hamsters. Syrian hamsters are known to have a shorter incubation period for TSEs infection than other experimental animals; hence, they might be more useful models for evaluating the correlation between the numbers of intestinal villous epithelium cells with incorporated PrPSc and age-related risks of TSEs.