Distribution, organization and innervation of gastric MALT in conventional piglet.

Mucosa-associated lymphoid tissue (MALT) is the initial inductive site for mucosal immunity. It is present in the different layers of the mucosal wall and consists of organized lymphoid tissue which may occur as isolated or aggregated lymphoid follicles (LFs) and interfollicular areas. It is present in many organs, including the pig stomach. Gastric MALT has been intensely studied in experimentally infected pigs but few data are available in healthy, non-gnotobiotic or germ-free animals. In the present study we described the gastric MALT in conventional piglets in the cardiac mucosa of the gastric diverticulum, in the pyloric mucosa, and in the sites of transition from cardiac to oxyntic and from cardiac to pyloric mucosa by means of histological and immunohistochemical stains. The majority of LFs were located in the cardiac mucosa and in the transition from the cardiac to the oxyntic mucosa. Here the LFs were mainly located in the submucosa and reached the mucosa; we called these submucosal lymphoid follicles (SLFs). In the pyloric mucosa and in the transition sites from the cardiac to the pyloric mucosa, LFs were located in the mucosa; we called these mucosal lymphoid follicles (MLFs). In SLFs, a compartmental organization of T and B lymphocytes was present; by contrast, in the MLFs, the T and B cells were intermingled, suggesting the possibility of different roles for the two types of follicles. In the epithelium overlying the lymphoid tissue, numerous T lymphocytes and some cells immunoreactive to cytokeratin-18 were observed. Following the application of the fluorescent tracer DiI into the SLFs of the diverticulum, enteric neurones located in the submucosal plexus were labelled, confirming the interplay between the immune and the enteric nervous system.

An immunohistochemical study of the distribution of nitric oxide synthase-immunoreactive neurons and fibers in the reticular groove of suckling lambs.

The reticular groove (RG) is a specialized region of ruminant forestomach which, in suckling animals, via a vagovagal reflex, transforms itself into a tube to ensure the direct transport of milk from the esophagus to the abomasum. The nervous mechanism controlling the RG movement is not fully understood; however, at this level, the enteric nervous system (ENS) shows the highest neuronal density when compared with other forestomach compartments. Because nitric oxide is considered the putative major mediator of non-adrenergic non-cholinergic smooth muscle relaxation, the aim of the present study was to investigate the ENS of the RG of suckling lambs, both in the floor and in the lip, with particular regard to nitric oxide synthase (NOS)-immunoreactivity (-IR), by means of double immunohistochemical staining. NOS antiserum was used in association with some neurochemical markers which have been utilized by many authors in ENS. A rich innervation of fibers extended along the entire length of the RG. Proceeding distally, the number of neurons stained with a pan-neuronal marker increased; they were more numerous in the lips and lip-floor junction than in the floor itself. However, the percentage of NOS-IR neurons was the same in the proximal and distal parts. Many NOS-IR neurons often co-expressed galanin and dopamine ß-hydroxylase. Neurochemical markers, such as calbindin, calcitonin gene-related peptide, IB4 and neurofilament 200 kDa, usually used to identify primary sensory neurons were not expressed in RG neurons, and the co-localization of NOS with tyrosine hydroxylase and substance P was rarely found. When compared with other districts, the RG showed some peculiar aspects, such as the lack of both neurons in the submucosal plexus and the lack of typical sensory neurons.

Immunohistochemical characterization of TH13-L2 spinal ganglia neurons in sheep (Ovis aries).

Spinal ganglia (SG) neurons are commonly classified according to various specific features. The most widespread classification based on morphological and ultrastructural features subdivides SG neurons into light and small dark neurons. Using immunohistochemical, histochemical and lectin methods, it is possible to further subdivide the small dark neurons into two subpopulations: peptidergic and nonpeptidergic neurons. The majority of studies on SG neurons were carried out on mice and rats; there are few or no studies on large mammals. In this study, some of the widely used neuronal markers, neurofilament 200 kDa (NF200), substance P (SP), calcitonin gene-related peptide (CGRP) and isolectin B4 (IB4), were employed to characterize neuronal nitric oxide synthase (nNOS)-immunoreactivity (-IR) in sheep (Ovis aries) SG (Th13-L2) neurons. The majority of the SG neurons were IB4-labeled (79 +/- 10%), followed by NF200- (45 +/- 4%), NOS- (44 +/- 10%), SP- (42 +/- 5%) and CGRP-IR (35 +/- 7%) neurons. The triple staining experiments showed that a higher percentage (75 +/- 16%) of NOS-IR neurons bound both IB4 and CGRP, or both IB4 and SP (49 +/- 6%). The IB4 marker showed an unexpected staining pattern; in fact, IB4-labeled neurons largely colocalized with NF200, usually considered a marker of light SG neurons, and with CGRP and SP. For this reason, IB4 cannot be employed in sheep to differentiate between light and dark neurons, or between peptidergic and nonpeptidergic neurons. These results suggest the importance of being cautious when comparing data among different species.

Supplemental sodium butyrate stimulates different gastric cells in weaned pigs.

Sodium butyrate (SB) is used as an acidifier in animal feed. We hypothesized that supplemental SB impacts gastric morphology and function, depending on the period of SB provision. The effect of SB on the oxyntic and pyloric mucosa was studied in 4 groups of 8 pigs, each supplemented with SB either during the suckling period (d 4-28 of age), after weaning (d 29 to 39-40 of age) or both, or never. We assessed the number of parietal cells immunostained for H+/K+-ATPase, gastric endocrine cells immunostained for chromogranin A and somatostatin (SST) in the oxyntic mucosa, and gastrin-secreting cells in the pyloric mucosa. Gastric muscularis and mucosa thickness were measured. Expressions of the H+/K+-ATPase and SST type 2 receptor (SSTR2) genes in the oxyntic mucosa and of the gastrin gene in the pyloric mucosa were evaluated by real-time RT-PCR. SB increased the number of parietal cells per gland regardless of the period of administration (P < 0.05). SB addition after, but not before, weaning increased the number of enteroendocrine and SST-positive cells (P < 0.01) and tended to increase gastrin mRNA (P = 0.09). There was an interaction between the 2 periods of SB treatment for the expression of H/K-ATPase and SSTR2 genes (P < 0.05). Butyrate intake after weaning increased gastric mucosa thickness (P < 0.05) but not muscularis. SB used orally at a low dose affected gastric morphology and function, presumably in relationship with its action on mucosal maturation and differentiation.

Anatomical evidence for ileal Peyer's patches innervation by enteric nervous system: a potential route for prion neuroinvasion?

We have examined the innervation of the gut-associated lymphoid system of the sheep ileum, with a view to identifying potential sites for neuroinvasion by pathogens, such as prions (PrP(Sc)). Special attention has been paid to the follicles of Peyer's patches (PPs), which are major sites of PrP(Sc) accumulation during infection. Evidence exists that the enteric nervous system, together with the parasympathetic and sympathetic pathways projecting to the intestine, are important for PrP(Sc) entry into the central nervous system. Thus, PrP(Sc) might move from PPs to the neurons and nerve fibres that innervate them. We investigated, by immunohistochemistry and retrograde tracing (DiI) from the follicles, the distribution and phenotype of enteric neurons innervating the follicles. Antibodies against protein gene product 9.5, tyrosine hydroxylase, dopamine beta hydroxylase, choline acetyltransferase, calbindin (CALB), calcitonin gene-related peptide (CGRP), and nitric oxide synthase were used to characterise the neurons. Immunoreactivity for each of these was observed in fibres around and inside PP follicles. CGRP-immunoreactive fibres were mainly seen at the follicular dome. Retrograde tracing revealed submucosal neurons that contributed to the innervation of PPs, including Dogiel type II neurons and neurons immunoreactive for CALB and CGRP. The major source of the adrenergic fibres are the sympathetic ganglia. Our results thus suggest that enteric and sympathetic neurons are involved during the first stage of neuroinvasion, with neurons connecting to them acting as potential carriers of PrP(Sc) to the central nervous system.

Morphology and neurochemistry of descending and ascending myenteric plexus neurons of sheep ileum.

The specific patterns of gastrointestinal motility in large herbivores may relate to differences in the organization of enteric nerve circuits, compared with other mammals. To investigate this possibility, we characterized the morphologies, chemical phenotypes, and projections of myenteric plexus (MP) neurons of the sheep ileum. Morphologies and projections were investigated after application of the carbocyanine dye (1,1', di-octadecyl-3,3,3',3',-tetramethylindo-carbocyanine perchlorate, DiI) to fixed tissues. To study chemical phenotypes, the fluorescent tracer Fast Blue (FB) was injected into the wall of the ileum, in vivo, 12-14 cm oral to the ileo-caecal junction. Over 80% of the descending and ascending DiI-labeled neurons had typical Dogiel type I morphology, whereas only a few Dogiel type II neurons were observed. Nevertheless, there were long projections (up to 10 cm) of Dogiel type II neurons in both directions. Both type II and type I neurons were neurofilament immunoreactive (IR). We observed long projections of descending (up to 18 cm) and ascending (up to 12-14 cm) FB-labeled MP neurons. Nitric oxide synthase (NOS)-IR, peripheral choline acetyltransferase (pChAT)-IR, and substance P (SP)-IR occurred in both descending and ascending myenteric neurons. NOS-IR was in approximately 60% of FB-labeled descending and ascending neurons, whereas those expressing pChAT-IR were 67 +/- 15% and 60 +/- 14%, respectively. Descending neurons expressing SP-IR were 48 +/- 15% and ascending were 56 +/- 12%. NOS-IR and pChAT-IR, and SP-IR and pChAT-IR were commonly colocalized in both ascending and descending pathways. In descending pathways, almost all SP-IR neurons were also pChAT-IR (98 +/- 3%) and NOS-IR (99 +/- 2 NOS(+)/SP(+)/pChAT(-)). Many FB-labeled descending neurons showed both NOS- and pChAT-IR. Descending neurons may represent inhibitory motor neurons (NOS(+)/SP(+)/pChAT(-)) and two classes of interneurons (pChAT(+)/NOS(-), and pChAT(+)/NOS(+)/SP(+)). In ascending pathways, most neurons are pChAT(+)/NOS(+)/SP(+). Thus, in sheep, ascending interneurons and ascending excitatory motor neurons both have the same phenotype, and other markers are needed to distinguish them.

Enteroglial and neuronal involvement without apparent neuron loss in ileal enteric nervous system plexuses from scrapie-affected sheep.

The enteric nervous system (ENS) probably plays a dominant role in sheep scrapie pathogenesis, but little is known about the cell types involved. We investigated the ileal myenteric and submucosal plexuses of four naturally and four orally experimentally scrapie-affected ARQ/ARQ Sarda sheep, as well as those of 12 healthy-control Sarda sheep carrying different PrP genotypes. All scrapie-affected animals, euthanized at clinical-disease end stage, showed PrPd deposition within enteric glial cells (EGCs) and calbindin-immunoreactive (CALB-IR) and neuronal nitric oxide synthase (nNOS)-IR neurons. Whole-mount investigations revealed no significant differences between the densities of total, CALB-IR and nNOS-IR neurons in scrapie-affected versus healthy sheep, irrespective of PrP genotype. Our results suggest that EGCs and CALB-IR and nNOS-IR neurons are probably involved in the pathogenesis of natural and oral experimental sheep scrapie. Furthermore, the infectious agent may be less pathogenic towards ENS neurons than it is towards central nervous system neurons.

Nitric oxide synthase immunoreactivity and NADPH-d histochemistry in the enteric nervous system of Sarda breed sheep with different PrP genotypes in whole-mount and cryostat preparations.

Until now, significant differences in the neurochemical pattern of enteric neurons have been demonstrated in all species studied; however, some strong similarities also occur across species, such as the occurrence of nitric oxide synthase immunoreactivity (NOS-IR) in inhibitory motor neurons to muscle. In consideration of the insufficient data regarding the enteric nervous system (ENS) of sheep, we investigated the myenteric plexus and submucosal plexus of the ovine ileum. Since the pivotal role of the ENS in the early pathogenesis of sheep scrapie, the "prototype" of prion diseases, has been suggested, we have focused our observations also on the host's PrP genotype. We have studied the morphology and distribution of NOS-IR neurons and their relationships with the enteric glia in whole-mount preparations and in cryostat sections. NOS-IR neurons, always encircled by glial processes, were located in both plexuses. Many NOS-IR fibers were seen in the circular muscle layer, in the submucosa, and in the mucosa. In the submucosa they were close to the lymphoid tissue. No differences in the distribution and percentage of NOS-IR fibers and neurons were observed among sheep carrying different PrP genotype, thus making unlikely their contribution in the determinism of susceptibility/resistance to scrapie infection.

A continuous dietary supply of free calcium formate negatively affects the parietal cell population and gastric RNA expression for H+/K+-ATPase in weaning pigs.

Baby formula acidification can be used to reduce diarrhea. Calcium formate is a dietary acidifier frequently used in animal weaning diets; it is also a source of available calcium. Gastric acidification reduces gastrin release and hydrochloric acid (HCl) secretion. To study the medium-term effects on fundic gastric mucosa, we fed weaning pigs control diets or diets supplemented with free or fat-protected calcium formate. We evaluated the following: 1) the number of HCl-secreting parietal cells, by immunohistochemistry using an antibody against H(+)/K(+)-ATPase; 2) the number of enteroendocrine cells immunohistochemically stained with chromogranin A (CGA), somatostatin, and histamine (HIS); and 3) the expression of the H(+)/K(+)-ATPase gene, by real-time RT-PCR in the oxyntic mucosa. Cells co-staining for CGA and HIS were defined as enterochromaffin-like (ECL) cells. Pigs fed calcium formate had fewer parietal cells and a lower expression of the H(+)/K(+)-ATPase gene than the controls (P < 0.05). This reduction did not occur in pigs fed fat-protected calcium formate. Somatostatin immune-reactive cells were also more numerous in pigs fed free calcium formate than in controls (P < 0.05). The number of ECL cells was not affected. Using covariance analysis, the number of parietal cells explained part of the differences in the expression of H(+)/K(+)-ATPase gene (positive correlation, r = 0.385, P < 0.01), and excluded the statistical significance of the diet. In the future, the effects on the oxyntic mucosa should be checked when the diet supplemented with calcium formate is discontinued. Furthermore, a reduction in the number of parietal cells could impair the absorption of vitamin B-12 due to a reduced secretion of the intrinsic factor by these cells.