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.

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.

Stereologic characteristics of pig small intestine during normal development.

Stereologic methods were used to study the behavior of the pig's 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 Cavalieri's 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.

A stereologic evaluation of glucagon-like peptide-1 (GLP-1) mucosal cells in the small intestine of the developing pig.

Studies that investigate possible developmental changes in gastrointestinal hormones in the pig are sparse and contradictory. Therefore, a quantitative morphological study using stereologic methods on paraffin sections of the different small intestinal regions (cranial and caudal duodenum, jejunum and ileum) of the developing pig (second half of the gestation, neonatal and weaning period) has been conducted. The sections were processed for GLP-1-immunohistochemistry. During the investigated time span, the volume of GLP-1-IR cells increased approximately 40-fold in both the jejunum and ileum, notwithstanding a decrease of their volume density. The ileal small intestinal segment contained the highest volume density of GLP-1-IR cells. In contrast, GLP-1-IR cells were only occasionally encountered in the duodenum. The development-related changes of the investigated parameters coincide with dietary changes and the regional differences are in accordance with functional reports that point to GLP-1 as a gastrointestinal hormone that ediates the 'ileal brake' and stimulates glucose-dependent pancreatic insulin secretion.

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.

Stereologic description of the changing expression of constitutive nitric oxide synthase and heme oxygenase in the enteric plexuses of the pig small intestine during development.

The similarities between heme oxygenase-2 (HO-2) and nitric oxide synthase (nNOS) and the transient expression of nNOS during development led us to investigate whether both systems are similarly affected by changes that occur during development and by regional differences along the small intestine. By combining 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 small intestine of fetal, 1-2-day and 5-6-week-old pigs. In all age groups, three enteric plexuses were distinguished. The presence of HO-2-immunoreactive (HO-2-IR) and NADPH diaphorase-positive neurons corresponded to earlier morphological and physiological reports. Nevertheless, the total number of nitrergic neurons remained constant or decreased in the enteric plexuses, whereas the total number of HO-2-IR neurons displayed an overall increase. Changing concentrations of glucocorticoids, target-derived signals, presynaptic input, and an effect of HO-2 activity on nNOS synthesis are likely to play roles in the observed developmental changes. The numerical density of HO-2-IR neurons remained relatively constant along the intestinal tract; in contrast, the nitrergic neurons were most numerous in the inner submucous and myenteric plexus in the duodenum and ileum, respectively. It is believed that the duodenal nitrergic neurons in the inner submucous plexus could be involved in the regulation of duodenal secretion processes, whereas the region-dependent density in the myenteric plexus possibly forms the morphological basis for a regionally different participation of NO in the relaxation of the small intestine.

Microwave staining of enteric neurons using cuprolinic blue (Quinolinic phthalocyanin) combined with enzyme histochemistry and peroxidase immunohistochemistry.

Methods that visualize subsets as well as the entire enteric neuron population are not readily available or have proved to be unreliable. Therefore, we attempted to combine NADPH-d histochemistry, AChE histochemistry, and CGRP immunohistochemistry, techniques that mark subsets of enteric neurons, with a technique that appeared to visualize the entire enteric neuron population, the cuprolinic blue staining method. To guarantee representative staining results, the individual staining methods were modified by using microwaves. In addition, this preserved the characteristics of each of the individual techniques. The distribution of NADPH-d, AChE, and CGRP corresponded well with previous morphological and physiological reports. Consequently, the different combinations gave rise to rapid, useful, and ready-to-use double labeling techniques. Their main advantage is that they simultaneously visualize the total population as well as subsets of enteric neurons.

Nitric oxide synthase expression in enteric neurons during development in the pig duodenum.

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.

Microwave staining of intestinal whole-mount preparations with Cuprolinic blue.

Encouraged by the knowledge that microwaves have a beneficial effect on immunohistochemical reactions, the present study aimed to find out whether microwaves could improve the Cuprolinic Blue staining of enteric neurons as well as the actual method that has been developed for gastrointestinal whole-mount preparations. In addition to incorporating a microwave application in the method described by Holst and Powley (1995), some other modifications were made: two incubations before incubation in the staining solution and free-floating incubations. In the whole-mount preparations, most, if not all, enteric neurons were stained by Cuprolinic Blue. These neurons appeared as blue-green cells with non-reacting nuclei and neuronal processes. At higher magnification, the cytoplasm was characterized by a fine blue-green granulation, and the nucleolus in the nucleus appeared as a blue iridescent structure. Non-specific staining occurred in fibrocytes and epithelial cells but, because of their location and appearance, they could easily be distinguished from neurons. The modified incubations and the incorporation of a microwave application into the conventional Cuprolinic Blue staining method turn the method into an easy-to-use one that seems to visualize most, if not all, enteric neurons in whole-mount preparations of the pig jejunum.

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 pig's 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 non-specific-reacting VIP-IR glandular cells were noticed in the basal parts of the fundic, antral and pyloric gastric glands. The distribution pattern of VIP in the fetal pig resembles that of the adult pig. This suggests a possible functional role for VIP during fetal life and/or puts forward the suggestion that the stomach of a fetal pig from the second half of the gestation period is prepared, from then on, for postnatal function. High similarities with regard to the general distribution pattern of VIP in the stomach have also been noted between the fetal pig and humans, proving once more that the fetal pig can serve as a good animal model in several research areas. Finally, the morphological data provided here may, combined with the physiological significance of VIP, contribute to a better insight into the physiopathology of economically important gastro-intestinal disorders in the pig, such as gastric ulceration.

Planimetry of bronchoalveolar macrophages. Importance of preparation and staining techniques.

Bronchoalveolar lavage seems a well-established, valuable research tool in the study of alveolar macrophages. The influence of fixation, cytocentrifugation and staining procedures on the cellular and nuclear size has been investigated by planimetry. As a reference, mean profile areas of 109 and 39 microns 2 for cell and nucleus, respectively, were measured for alveolar macrophages suspended in the hemocytometer. For comparison, stained Cytospin preparations were measured. Unfixed cells were compressed during cytocentrifugation. The cellular profile areas for Cytospin preparations increased about 15% and 70% after May-Grünwald-Giemsa and Feulgen staining, respectively. The nuclear area was approximately 25% larger for both staining procedures as compared to the hemocytometer values. When the cells had been fixed prior to cytocentrifugation, these differences were less conspicuous. No significant differences were observed after May-Grünwald-Giemsa staining, showing a cellular area of 114 microns 2 and a nuclear area of 45 microns 2. Depending on the staining procedure, low nucleus:cell ratios (31%) were observed after Feulgen staining, while higher values (about 43%) were measured after May-Grünwald-Giemsa staining, regardless of which fixation or centrifugation procedure had been followed. In conclusion, these findings indicate that fixation should be carried out in order to prevent cell changes resulting from cytocentrifugation. Moreover, different staining procedures considerably influence the measurement of cellular and nuclear profile areas and the determination of nucleus:cell ratios.

The alveolar pores of Kohn in young postnatal rat lungs and their relation with type II pneumocytes.

In order to obtain more information on the development, morphology and function of the pores of Kohn, the lungs of Wistar rats are studied during their early postnatal period, up to 3 weeks of age, by scanning and transmission electron microscopy. The substantial development of the interalveolar pores on days 14 and 21 coincides with the period of septal rearrangement when secondary interalveolar septa become lengthened and thinner. The high frequency of transseptal type II pneumocytes from day 7 onwards, and their typical localization near the pores of Kohn at this period of lung development especially suggests that type II pneumocytes are engaged in the formation of the pores of Kohn. During early lung development, the pores of Kohn seem to serve as passageways for alveolar macrophages.

Microperoxisomes in type II pneumocytes and interstitial cells of postnatal rat lungs.

Microperoxisomes in alveolar type II pneumocytes and lipid interstitial cells are visualized using the diaminobenzidine method for catalase. These organelles, with a diameter ranging from 100-800 nm, respectively 200-500 nm, and with no crystalline cores or densities, are in close relation with the endoplasmic reticulum. No luminal continuity between microperoxisomes and the endoplasmic reticulum is observed. Postfixation with unbuffered ferrocyanide-reduced osmiumtetroxide contributes to a better localization of microperoxisomes in both celltypes.

Development of alveolar septa and formation of alveolar pores during the early postnatal period in the rat lung.

In order to investigate the formation of alveolar pores, lungs of rats, after intratracheal perfusion of glutaraldehyde, are processed at postnatal days 1, 7, 14, 16 and 21 for light and transmission electron microscopy and at days 7 and 16 for scanning electron microscopy. The initial low secondary crests of day 1 rapidly elongate to pleats subdividing the primary saccules. The ledges of some pleats partly grow toward each other as ring like diaphragms, leaving openings whose boundary is composed of alveolar epithelium separated by a basal lamina from a connective tissue sheath with capillaries. At day 7, in scanning electron microscopy the lumina of some rudimentary alveoli communicate by apertures of different sizes, as a result of the outgrowth of curved alveolar pleats which narrow to a ring-like aperture. The interalveolar openings observed in scanning electron microscopy resemble those investigated by light and transmission electron microscopy. The number of interalveolar pores increases from day 7 on; they become more and more frequent at days 14, 16 and 21, respectively. It appears that alveolar multiplication in newborn rats proceeds not only by segmentation of terminal respiratory units but also by compoundment of septa. The difference between genuine pores and transsections of folds in transmission electron microscopy will be given closer attention in this study. Also, the incidence and location of type II pneumocytes during rapid enlargement of the alveolar surface area is discussed.