F. van Meir

NITRIC OXIDE SYNTHASE EXPRESSION IN ENTERIC NEURONS DURING DEVELOPMENT IN THE PIG DUODENUM

Abstract The expression of the constitutive neural isoform of nitric oxide synthase (bNOS) is dynamic and thus forms an ideal parameter to evaluate whether development and region affect the enteric nervous system. By applying NADPH-diaphorase histochemistry on whole-mount preparations of the myenteric and submucosal plexuses and by using the ’unbiased counting frame’, a qualitative and quantitative description of bNOS-expression in enteric neurons in the pig duodenum in various developmental stage and region was obtained. Examinations were carried out on the oral and aboral duodenum of fetal pigs from the second half of gestation, of 1–2-day-old pigs and of 6–8-week-old pigs. In the pig duodenum, three enteric plexuses were readily distinguished: the inner submucous, the outer submucous and the myenteric plexuses. All three plexuses already harboured, to different degrees, bNOS-expressing neurons at midgestation. Although the enteric nervous system was present at midgestation, the enteric neurons had not yet reached their adult phenotype and morphology. During gestation, the number of inner submucous bNOS-expressing neurons increased approximately 50-fold, whereas after birth that number fell to about 10% of the prenatal value. During further postnatal development it returned to prenatal values. In addition, the number of bNOS-expressing myenteric neurons doubled postnatally. These changes favour a role for NO in mediating the development of enteric neurons and point to a greater necessity for inhibitory innervation in the adult pig as compared with the fetal pig. Furthermore, the number of bNOS-expressing outer submucosal and myenteric neurons was significantly higher in the oral duodenal segment compared with the aboral duodenal segment. This regional difference suggests that the oral duodenal segment is more prominently involved in the regulation of NO-mediated gastrointestinal processes than the aboral one. The developmentally and regionally dependent bNOS-expression can be explained by shifts and differences in the balanced system of hormones, presynaptic input and target-derived signals that affects neurotransmitter expression.

Stereologic Characteristics of Pig Small Intestine During Normal Development

Stereologic methods were used to study the behavior of the pigs intestinal wall during periods that are characterized by a high incidence of gastrointestinal disorders. For this purpose conventionally stained transverse and vertical paraffin sections were made of the small intestine (duodenum, jejunum, and ileum) of fetal, neonatal, and weaned pigs. The volumes of the intestinal walls were estimated using Cavalieris method. Subsequently, the surface density (Sv) of the tunica mucosa and the volume densities (Vv) of the different small intestinal elements were estimated. Finally, the surface and volumes per serosal surface area (Ss and Vs) were calculated. The decrease of Sv can be attributed to the finding that the mucosal surface increases to a lesser extent compared with the volume of the intestinal wall. The Vs of the various layers increased postnatally, illustrating that the intestinal wall thickens. Despite an increasing total mucosal surface, this postnatal thickening causes Ss to decline. Each of these changes is temporally related to dietary changes, an increased antigen load, and an increased need for protection. Additionally, the regional differences of the various parameters match the qualitative descriptions of the small intestine of the pig and relate to region-specific functions.

Selective visualisation of neuroepithelial bodies in vibratome slices of living lung by 4-Di-2-ASP in various animal species.

Pulmonary neuroepithelial bodies (NEBs) are extensively innervated organoid groups of neuroendocrine cells that lie in the epithelium of intrapulmonary airways. Our present understanding of the morphology of NEBs is comprehensive, but direct physiological studies have so far been challenging because the extremely diffuse distribution of NEBs makes them inaccessible in vivo and because a reliable in vitro model is lacking. Our aim has been to optimise an in vitro method based on vibratome slices of living lungs, a model that includes NEBs, the surrounding tissues and at least part of their complex innervation. This in vitro model offers satisfactory access to pulmonary NEBs, provided that they can be differentiated from other tissue elements. The model was first optimised for living rat lung slices. Neutral red staining, reported to stain rabbit NEBs, proved unsuccessful in rat slices. On the other hand, the styryl pyridinium dye, 4-(4-diethylaminostyryl)-N-methylpyridinium iodide (4-Di-2-ASP), showed brightly fluorescent cell groups, reminiscent of NEBs, in the airway epithelium of living lung slices from rat. In addition, nerve fibres innervating the NEBs were labelled. The reliable and specific labelling of pulmonary NEBs by 4-Di-2-ASP was corroborated by immunostaining for protein gene-product 9.5. Live cell imaging and propidium iodide staining further established the acceptable viability of 4-Di-2-ASP-labelled NEB cells in lung slices, even over long periods. Importantly, the in vitro model and 4-Di-2-ASP staining procedure for pulmonary NEBs appeared to be equally reproducible in mouse, hamster and rabbit lungs. Diverse immunocytochemical procedures could be applied to the lung slices providing an opportunity to combine physiological and functional morphological studies. Such an integrated approach offers additional possibilities for elucidating the function(s) of pulmonary NEBs in health and disease.

Effects of Schistosoma mansoni infection on somatostatin and somatostatin receptor 2A expression in mouse ileum

Abstract  Intestinal schistosomiasis is accompanied by motility‐related dysfunctions but the underlying mechanisms are not well‐known. Therefore, the presence and effects on intestinal contractility of somatostatin (SOM) and its receptor, SSTR2A, were investigated in the ileum of normal and infected mice. The distribution of SOM and SSTR2A was visualized using immunocytochemistry. Radioimmunoassay combined with oogram studies was performed to determine SOM levels and contractility measurements were determined in organ bath experiments. Schistosomiasis resulted in a significant decrease in somatostatin‐positive endocrine cells, whereas the number of somatostatin‐immunoreactive (IR) neuronal cell bodies did not change. From 8 weeks postinfection onwards, an increase was noted in somatostatin‐IR nerve fibres in both villi and granulomas. The staining intensity for SSTR2A, expressed in somatostatin‐negative myenteric cholinergic neurones, increased during infection suggesting an upregulation of this receptor. SOM levels were negatively correlated with the number of eggs during the acute phase, and were elevated during the chronic phase. Pharmacological experiments revealed that schistosomiasis diminished the inhibitory effect of SOM on neurogenic contractions. We can conclude that schistosomiasis influences the distribution and expression levels of SOM and SSTR2A in the murine ileum, which might explain the changed motility pattern.

Intrinsic Innervation of the Stomach of the Fetal Pig: an Immunohistochemical Study of VIP‐Immunoreactive Nerve Fibres and Cell Bodies

Using an immunohistochemical technique, the presence and distribution of vasoactive intestinal polypeptide (VIP) was investigated in cryostat sections, both tangential and transverse, of the fetal pigs stomach. In all fetuses and in all gastric segments investigated, VIP‐like immunoreactive (IR) nerve‐cell bodies were seen in all intramural ganglia, and VIP‐IR nerve fibres were found in all layers of the gastric wall except the tunica serosa. Consequently, VIP‐IR nerve fibres were found to form a periglandular network, to accompany arterioles, to interconnect the intramural ganglia, to encircle both VIP‐IR‐negative and ‐positive neurons, and were found in all muscle layers. Despite the fact that VIP‐IR seems to be restricted to the intramural nervous elements, some nonspecific‐reacting VIP‐IR glandular cells were noticed in the basal parts of the fundic, antral and pyloric gastric glands.

Developmental changes in heme-oxygenase-2 and bNOS expression in enteric neurons in the pig duodenum.

There exists much parallelism between carbon monoxide- and nitric oxide-generating systems. Therefore, we wondered whether developmental and functional differences along the duodenum similarly affect, part of them, namely, heme oxygenase-2-(HO-2) and neural isoform of nitric oxide synthase- (nNOS) expressing neurons. By applying NADPH diaphorase histochemistry and HO-2 immunohistochemistry on whole-mount preparations and by using stereologic methods, a qualitative and quantitative description of HO-2 and nNOS expression was obtained. Examinations were carried out on the duodenum of fetal, neonatal and weaned pigs. At all ages, three enteric plexuses were readily distinguished. The presence of both enzymes fits in with other morphological and physiological reports. However, the expression of both enzymes significantly changed during development. The number of HO-2-IR neurons increased approximately 20-fold in the inner submucous and almost doubled in the myenteric plexus. In addition, the number of nNOS-expressing neurons displayed a significant decrease in the outer submucous plexus after weaning. High levels of glucocorticoids may cause the perinatally increased HO-2 expression, whereas an influence on nNOS expression is doubtful. Therefore, it seems that notwithstanding the high similarity between both systems, their expression is regulated differently in the pig duodenum.

Anatomy, Embryology, Histology and Physiology of the Spleen

The spleen is the largest lymphoid organ in the body and is interposed within the circulatory system. The spleen has multiple functions in the human body: 1. It clears the circulation of micro-organisms, par- ticulate antigens and other foreign material. 2. It makes the majority of antibodies and enhances cellular immunity to antigens. 3. It removes normal and abnormal blood cells from the circulation. 4. It plays an important role in extramedullary haematopoiesis.