Morphoquantitative study of Rattus norvegicus submucosal plexus by different neuronal evidentiation histochemical techniques / Estudio morfocuantitativo del plexo submucoso de Rattus norvegicus por medio de diferentes técnicas histoquímicas para verificar características neuronales

Enteric nervous plexuses have been the object of several studies, specially the myenteric plexus whose studies describe its organization, functions and alterations. On the other hand, the submucosal plexus has been less studied and still needs descriptive studies. To analyze morphologically and quantitatively submucosal neurons of the jejunum of 90-day-old healthy rats using different techniques for neuronal staining as a way to provide normality data to compare with future experimental studies. Whole mount preparations of the jejunum were submitted to Giemsa, NADH-diaphorase and NADPH-diaphorase techniques to stain the total neuronal population, more metabolically active subpopulation and subpopulation of nitrergic neurons, respectively. Neurons of the submucosal plexus of adult rats are mainly organized in ganglia with varied sized and shapes. Giemsa technique stained 243.93 ± 7.68 neurons per mm2. Regarding the total population stained by Giemsa, NADH- diaphorase positive (139.09 ± 11.14/mm2) neurons represented 57 % and NADPH-diaphorase positive (18.17 ± 0.28/mm2) represented 7.5 %. The area of the cell body was bigger in nitrergic neurons (412.29 ± 150.22) than in the ones stained by Giemsa (254.71 ± 63.32) and NADH-diaphorase positive (243.98 ± 123.82).

El plexo nervioso entérico ha sido objeto de varios estudios, especialmente el plexo mientérico, cuyos estudios consisten en describir su organización, funciones y alteraciones. Por otro lado, el plexo submucoso ha sido menos investigado y todavía necesita estudios descriptivos. Para analizar morfológica y cuantitativamente las neuronas de la submucosa del yeyuno de ratas de 90 días de edad, se realizaron diferentes técnicas de tinción neuronales, en animales sanos, como una forma de proporcionar datos de normalidad y compararlo con futuros estudios experimentales. Se realizaron montajes con preparados enteros del yeyuno que fueron sometidos a las técnicas de Giemsa, de NADPH-diaforasa y NADH-diaforasa para teñir la población total neuronal, subpoblación más activa metabólicamente y subpoblación de neuronas nitrérgicas, respectivamente. Las neuronas del plexo submucoso de ratas adultas se organizan principalmente en los ganglios con variaciones de tamaño y formas. Con la técnica de Giemsa se tiñeron 243.93±7.68 neuronas por mm2. Con respecto a la población total teñida con Giemsa, fueron positivas para NADH- diaforasa en 139.09 ±11.14 / mm2 neuronas, representando el 57% y fueron positivas para NADPH-diaforasa en 18,17 ± 0,28 / mm2 neuronas, lo que representó el 7,5%. El área del cuerpo celular fue mayor en neuronas nitrérgicas (412,29 ± 150.22) que en las teñidas con Giemsa (254,71 ± 63,32) y NADH-diaforasa positivas (243,98 ± 123,82).

Fetal intraluminal portion of the sphincter of Oddi: a histological study using human fetuses

The sphincter of Oddi surrounds a common duct after joining of the bile and pancreatic ducts in the adult ampulla of Vater, but the fetal development of the submucosal portion of the sphincter is still obscure possibly because previous studies used horizontal or frontal sections. We examined serial sagittal histological sections of 12 human fetuses with 36-65 mm crown rump length or CRL (approximately 9-11 weeks) and semi-serial sections of the other 3 fetuses with 210-250 mm (25-30 weeks). Except for 1 fetus (36 mm CRL), fourteen fetuses carried the "intraluminal portion" protruding and floating in the duodenal lumen. Twelve of them had the sphincter extending to the anal side in the duodenal lumen, whereas two extended to the stomach side. The distal end of the sphincter seemed to detach from the duodenal mucosa at and around 9 weeks, and subsequently the common duct seemed to elongate freely without mucosal attachment in mid-term fetuses and, finally, become embedded again in the duodenal mucosa in the postnatal life. A possible discrepancy in growth rate between the sphincter muscle and duodenal mucosa was likely to allow the specific intermediate morphology, i.e., the intraluminal common duct. The fetal accessory papilla did not show such morphology. A minority of cases whose common duct extended to the stomach side might connect with abnormal union of the bile and pancreatic ducts

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Architecture and Chemical Coding of the Inner and Outer Submucous Plexus in the Colon of Piglets.

In the porcine colon, the submucous plexus is divided into an inner submucous plexus (ISP) on the epithelial side and an outer submucous plexus (OSP) on the circular muscle side. Although both plexuses are probably involved in the regulation of epithelial functions, they might differ in function and neurochemical coding according to their localization. Therefore, we examined expression and co-localization of different neurotransmitters and neuronal markers in both plexuses as well as in neuronal fibres. Immunohistochemical staining was performed on wholemount preparations of ISP and OSP and on cryostat sections. Antibodies against choline acetyltransferase (ChAT), substance P (SP), somatostatin (SOM), neuropeptide Y (NPY), vasoactive intestinal peptide (VIP), neuronal nitric oxide synthase (nNOS) and the pan-neuronal markers Hu C/D and neuron specific enolase (NSE) were used. The ISP contained 1,380 ± 131 ganglia per cm2 and 122 ± 12 neurons per ganglion. In contrast, the OSP showed a wider meshwork (215 ± 33 ganglia per cm2) and smaller ganglia (57 ± 3 neurons per ganglion). In the ISP, 42% of all neurons expressed ChAT. About 66% of ChAT-positive neurons co-localized SP. A small number of ISP neurons expressed SOM. Chemical coding in the OSP was more complex. Besides the ChAT/±SP subpopulation (32% of all neurons), a nNOS-immunoreactive population (31%) was detected. Most nitrergic neurons were only immunoreactive for nNOS; 10% co-localized with VIP. A small subpopulation of OSP neurons was immunoreactive for ChAT/nNOS/±VIP. All types of neurotransmitters found in the ISP or OSP were also detected in neuronal fibres within the mucosa. We suppose that the cholinergic population in the ISP is involved in the control of epithelial functions. Regarding neurochemical coding, the OSP shares some similarities with the myenteric plexus. Because of its location and neurochemical characteristics, the OSP may be involved in controlling both the mucosa and circular muscle.

Organization of the innervation of the oesophagus and stomach in chinchilla (Chinchilla laniger, Molina).

Using the histochemical and histological techniques the morphology and topography of the autonomic plexuses of the oesophagus and stomach in chinchilla (Chinchilla laniger) was investigated. AChE-positive and adrenergic structures in the myenteric and submucosal plexus were observed. According to the results the cholinergic structures were formed by the different in shape network meshes, various size of the neurons agglomerations and nerve fibres. Adrenergic fibres were found. Density of these structures varied in different parts of the investigated organs. This study is the first attempt to localize the intramural autonomic plexuses in this species.

Morphoquantitative study of the submucous plexus (of Meissner) of the jejunum-ileum of young and old guinea pigs.

OBJECTIVE: To study the aging of submucous plexus of the small intestine (jejunum-ileum) of the guinea pigs from the quantitative, structural and ultrastructural perspective. METHOD: Chemical preparations of membrane of the jejunum-ileum of old and young animals with the use of light and electronic microscope. RESULTS: The ganglia of young animals presented between 1 and 56 neurons and the old animals presented from 1 to 30 neurons. The mean density of the ganglia by cm(2) in the young jejunum-ileum was of 551±36.89 and in the old one 413±11.86. The density of the neurons was 5011±291.11 neurons/cm(2) average in young animals and 2918±120.70 neurons/cm(2) in the old ones. The size of the neurons varied in both age groups. The collagen fibers in the ganglia of old animals they were condensed. Degenerated mitochondrias in the interior of the cell were frequent in the old animals. CONCLUSION: In submucous plexus of the jejunum-ileum there is a loss of 38% of the neurons with aging.

Morphoquantitative study of the submucous plexus (of Meissner) of the jejunum-ileum of young and old guinea pigs / Estudo morfoquantitativo do plexo submucoso (de Meissner) do jejuno-íleo de cobaias jovens e velhas

OBJECTIVE: To study the aging of submucous plexus of the small intestine (jejunum-ileum) of the guinea pigs from the quantitative, structural and ultrastructural perspective. METHOD: Chemical preparations of membrane of the jejunum-ileum of old and young animals with the use of light and electronic microscope. RESULTS: The ganglia of young animals presented between 1 and 56 neurons and the old animals presented from 1 to 30 neurons. The mean density of the ganglia by cm² in the young jejunum-ileum was of 551±36.89 and in the old one 413±11.86. The density of the neurons was 5011±291.11 neurons/cm² average in young animals and 2918±120.70 neurons/cm² in the old ones. The size of the neurons varied in both age groups. The collagen fibers in the ganglia of old animals they were condensed. Degenerated mitochondrias in the interior of the cell were frequent in the old animals. CONCLUSION: In submucous plexus of the jejunum-ileum there is a loss of 38 percent of the neurons with aging.

OBJETIVO: Estudar o envelhecimento do plexo submucoso do intestino delgado (jejuno-íleo) das cobaias do ponto de vista quantitativo, estrutural e ultra-estrutural. MÉTODO: Preparados de membrana do jejuno-íleo de animais jovens e velhos com a utilização de microscopia de luz e eletrônica. RESULTADOS: Os gânglios de animais jovens apresentaram entre 1 e 56 neurônios e os animais velhos apresentaram de 1 a 30 neurônios. A densidade média dos gânglios por cm² no jejuno-íleo jovem foi de 551±36,89 e no velho foi de 413±11,86. A densidade dos neurônios foi de 5011±291,11 neurônios/cm² em média nos animais jovens e 2918±120,70 neurônios/cm² nos velhos. O tamanho dos neurônios variou em ambos os grupos etários. As fibras colágenas nos gânglios de animais velhos estavam mais condensadas. Mitocôndrias degeneradas no interior da célula foram freqüentes nos animais velhos. CONCLUSÃO: No plexo submucoso do jejuno-íleo há uma perda de 38 por cento dos neurônios com o envelhecimento.

Synaptic transmission from the submucosal plexus to the myenteric plexus in Guinea-pig ileum.

Stimulation of the myenteric plexus results in activation of submucosal neurons and dilation of arterioles, one way that motility and secretion can be coupled together. The present study aimed to examine the converse, whether myenteric neurons receive synaptic input from the submucosal plexus (SMP). Intracellular recordings were made from guinea-pig ileal myenteric neurons while the SMP was electrically stimulated. Of the 29 neurons studied (13 S and 16 AH neurons), stimulation of the SMP evoked a synaptic potential in only seven cells, or 24% of neurons. When the SMP was situated oral to the myenteric plexus, 4 of 13 (31%) myenteric neurons had synaptic input. When it was situated circumferential, 2 of 8 (25%) had input, and when the SMP was situated anal 1 of 8 (13%) had input. Overall, 5 of the 13 (38%) S neurons responded with fast excitatory post-synaptic potentials (EPSPs), one of which also showed a slow EPSP, while 2 of the 16 (13%) AH neurons responded with a slow EPSP. This study indicates that the synaptic input from the SMP to myenteric neurons is relatively sparse. Whether this input is less important than the myenteric to submucosal input or simply represents a more selective form of control is unknown.

Woodruff's plexus.

OBJECTIVE: To identify the nature of Woodruff's plexus, which has been frequently mentioned in the rhinological literature but has never been properly characterised. STUDY DESIGN AND SETTING: A study using 21 cadaveric specimens, combining microdissection of the mucosa of the posterior part of the inferior meatus, Spalteholz 'clearing' of specimens injected with latex ink, and histological analysis of sections of the inferior meatal mucosa. RESULTS: Microdissection revealed a superficial plexus of thin walled vessels in the inferior meatus, which were also seen in cleared, injected specimens. Histological sections showed these vessels to be large, thin walled veins with very little muscle or fibrous tissue, within a thin mucosa relatively devoid of other structures. CONCLUSION: Woodruff's plexus is a venous plexus in the posterior part of the inferior meatus. SIGNIFICANCE: This is the first time Woodruff's plexus has been properly characterised. Whilst the significance of the plexus itself is uncertain, its long overdue identification as a venous plexus provides a platform for further study and discussion.

Structure of enteric neurons.

The ENS contains numerous different neuron populations which belong to three main groups, primary afferent neurons, interneurons and effector neurons. The most extensive knowledge on the different enteric neuron types is derived from studies in the guinea pig. A significant obstacle for the transfer of this knowledge to putative equivalent enteric neurons of other species, including human, is species differences as to their morphological, chemical, physiological etc. phenotypes. Modern morphological classifications are based on the work of the Russian histologist Dogiel. Since the late 1970s, refined morphological classifications of enteric neurons beyond Dogiel have been attempted mainly in two species, the pig and the guinea pig. These reflect the immunohistochemical diversity of enteric neurons more precisely but are far from being complete. In this paper, we follow two aims. First, we have presented an overview on the chemical coding of the morphological neuron types described by Stach in the pig intestine. In doing so, we have pointed out the difference between the definitions of type I neurons given by Dogiel and Stach. Second, we have attempted to provide a basis for the morpho-chemical classification of human enteric neurons as revealed by their immunoreactivity for NFs and several neuroactive substances or related markers. According to results from guinea pig, where there is functional evidence, human morphological type II neurons (non-dendritic, multiaxonal; co-reactive for NF, CAR, SOM, SP) seem to be the intrinsic primary afferent neurons. This conclusion is based primarily on structural equivalence. Human ENK-positive, stubby (type I) neurons maybe ascending interor motorneurons. In contrast, nitrergic, VIP-reactive spiny (type I) neurons maybe descending inter- or motor neurons. Further, morphologically defined human neuron types, i.e. type III, type V and dendritic type II neurons, are non-nitrergic but could not be chemically defined as yet. Future investigations of morpho-chemical characteristics of human enteric neurons including also other cytoskeletal markers will provide a broader basis for neurohistopathological diagnostics of gut diseases.

Topographic peculiarities of the submucous plexus in the human anorectum--consequences for histopathologic evaluation of rectal biopsies.

Chronic colorectal motility disorders are commonly encountered in the pediatric population. While most cases can be managed successfully by conservative therapy, a subgroup of patients suffers from severe constipation and requires further diagnostic procedures to identify the underlying pathologies, such as aganglionosis, hypoganglionosis or intestinal neuronal dysplasia (IND). The present study provides reference data about the quantitative distribution of nerve cells and ganglia within the submucosal plexus of the human anorectum from healthy subjects. Anorectal specimens (n = 15) obtained postmortem were divided into 6 segments beginning from the dentate line (S1 = 0-2 cm, S 2 = 2-4 cm, S3 = 4-6 cm, S4 = 6-8 cm, S5 = 8-10 cm, S6 = 10-12 cm). From each segment sections (6 microm thickness) were immunostained with a pan-neuronal marker (Protein Gene Product 9.5) to visualize the enteric nervous system. A morphometric analysis was carried out for each segment recording the number of ganglia and nerve cells of the submucous plexus. Neither ganglia nor nerve cells showed a uniform distribution pattern, but decreased continuously towards the anus. However, even the lowest segments (S1, S2) contained nerve cells and were not aganglionic. In the remaining segments ganglia with 7 or more nerve cells could be detected. The findings demonstrate segment-specific quantitative differences of the anorectal submucous plexus which should be taken into consideration for the histopathologic evaluation of rectal biopsies. Moreover, the data support the concept of a physiologic hypoganglionosis of the anal canal.

Quantitative morphometric analysis of the submucous plexus in age-related control groups.

An increased number and density of the so-called "giant ganglia" (seven or greater ganglion cells per ganglion) serve as histopathological criteria for a bowel motility disorder called intestinal neuronal dysplasia of the submucous plexus (IND B). However, because these morphological criteria have been defined based upon observations in constipated patients, the diagnostic value of previous studies is open to controversy. Moreover, no age-related reference data from unaffected controls are available. This study reports on data from unaffected controls on the variability of size and distribution of ganglia in the submucous plexus during development. Therefore, for the first time, the normal status has been defined. Four age groups have been defined: (a) premature births, gestational age less than 35 weeks; (b) 1-365 days; (c) 1-14 years and (d) 15 years to greater than 70 years). All of these groups revealed giant ganglia in the submucous plexus. With advancing age, there was a decrease in the number of giant ganglia (from 32.7% in group a to 11.2% in group d) accompanied by an inverse increase in the mean distance between all ganglia (from 0.52 mm in group a to 1.17 mm in group d). The data presented permit the conclusion that the criteria mentioned above are not apt to define IND B as an entity, since they do not allow a sufficient demarcation from the age-correlated normal values presented here.

Topography and neurochemistry of the enteric ganglia in the proventriculus of the duck (Anas platyrhynchos).

The topographical distribution of the enteric ganglia has been investigated in the proventriculus of the duck using protein gene product 9.5 (PGP 9.5) immunohistochemistry. Myenteric ganglia were usually located between the outer longitudinal and the inner circular muscle layer. Submucous ganglia were sparsely distributed and seemed to be substituted by ganglia located in the tunica mucosa. The neurochemical profile of proventricular ganglion cells was also investigated using nicotinamide adenine dinucleotide phosphate reduced-diaphorase (NADPH-d)-histochemistry and pituitary adenylate cyclase activating peptide (PACAP)/galanin (Gal) double-labelling immunohistochemistry. The majority of mucosal ganglion cells were shown to contain the NADPH-d enzyme and both the investigated peptides. These findings provide evidence for the presence of a mucosal ganglionated plexus in the glandular stomach of birds. Moreover, the neurochemical characteristics of this plexus suggest that it plays an important role in regulating several mucosal functions and, in particular, the production and the composition of the gastric juice.

Morphometric quantification of normal submucous plexus in the distal rectum of adult healthy volunteers.

OBJECTIVE: Inadequate morphometric characterization of the normal adult submucous plexus has precluded the diagnosis of colonic dysganglionoses associated with constipation, such as intestinal neuronal dysplasia type B (IND B). The internal submucous plexus (Meissner plexus) was morphometrically quantified in adult healthy volunteers. DESIGN: Open, prospective morphometric study in balanced groups of female and male volunteers. PARTICIPANTS: Thirty-seven adult healthy male and female volunteers with normal bowel function and no history of gastrointestinal disease. METHODS: Four jumbo rectal biopsies (3-5 mm3) were taken 5 and 10 cm above the pectinate line. Two expert gastrointestinal pathologists assessed biopsy sections after specific nerve cell staining for lactic dehydrogenase, nitric oxide synthase and acetylcholinesterase, mainly for characteristics of ganglia and nerve cells in the submucous plexus. RESULTS: No healthy individual demonstrated over 20% of submucosal ganglia as giant ganglia or more than four giant ganglia per 30 sections (the morphometric criteria for IND B). Single submucosal nerve cells and ganglion numbers halved between 10 and 5 cm above the pectinate line, but there were no age or gender differences. The biological variability of nerve cell and ganglion density in the submucous plexus was large. CONCLUSIONS: Healthy adults show less than 20% of submucosal ganglia as giant ganglia and no more than four giant ganglia per 30 rectal biopsy sections. There is therefore no overlap with the histomorphological criteria of IND B. These data therefore support the specificity of the previously defined criteria for IND B in adults.

An immunohistochemical study of the organization of ganglia and nerve fibres in the mucosa of the porcine intestine.

In order to elucidate the organization of the enteric nervous system in the mucous plexus, wholemounts from six intestinal regions in six pigs were studied by vasoactive intestinal peptide, substance P, nitric oxide synthase and neurofilament proteins immunohistochemistry. The mucous plexus of both large and small intestine contained ganglia and isolated neurons. They were many and comparably larger in the caecum and colon, few in the ileum, and fewer and smaller in the jejunum. The mucous plexus was subdivided into the lamina muscularis mucosae and lamina proprial subplexuses, and based on location the latter was subdivided further in order to clarify their variations with respect to the amount, sizes and shapes of ganglia and neurons, sizes and orientation of nerve strands and immunoreactivities. Ganglia were situated at different topographical levels in the lamina muscularis mucosae subplexus, outer proprial and interglandular proprial meshworks in the lamina proprial subplexus with the majority of ganglia occurring in the outer proprial meshwork. The mucous plexus in the intestine of the pig is thus a ganglionated plexus showing marked segmental variation in the amount of intramucosal ganglia and isolated nerve cells. These new observations, calls for a re-examination of the mucous plexus to elucidate the regulatory mechanisms of importance in mucosal functions and consideration of the mucous plexus in the intestine of the pig to be one of the major ganglionated plexuses.

The longitudinal smooth muscle layer of the pig small intestine is innervated by both myenteric and submucous neurons.

Originally, intestinal motility was thought to be exclusively regulated by myenteric neurons. Some years ago, however, it was demonstrated in large mammals that submucous neurons also participate in the innervation of the circular smooth muscle layer. To date, no information is available about the submucous innervation of the longitudinal smooth muscle layer (LM). This study provides evidence that in the small intestine of large mammals, the LM is innervated not only by the myenteric plexus, but also by the inner and outer submucous plexuses (ISP and OSP). In the porcine small intestine, the involved neurons can be subdivided into the following neurochemically distinct populations: leu-enkephalin (ENK)- and/or substance P (SP)-IR neurons and nitric oxide synthase (NOS)- and/or vasoactive intestinal polypeptide (VIP)-IR neurons. In the myenteric plexus, the majority of VIP- and/or NOS-IR neurons and ENK(+)/SP(-)-IR neurons exhibit descending projections, whereas ENK(+)/SP(+)-IR neurons preferentially have ascending projections. The ENK(-)/SP(+)-IR neurons do not show a polarized pattern. In the OSP, only ENK(+)/SP(-)- and VIP(+)/NOS(-)-IR neurons display a polarized (descending) projection pattern, whereas no polarization can be noted in the ISP. Morphological analysis of the traced neurons revealed that, in general, myenteric descending LM motor neurons have larger cell bodies than ascending ones and, in addition, myenteric descending VIP- and/or NOS-IR neurons have longer projections than ENK and/or SP-IR neurons. In conclusion, the present study demonstrates the involvement of not only myenteric, but also submucous neurons in the innervation of the LM. The two major populations are descending nitrergic neurons and ascending tachykinergic motor neurons, but also other subpopulations with specific projection patterns and neurochemical features have been identified.

Localization and characterization of neuropeptide receptors in human colon.

Information about the expression of neuropeptide receptors is limited in human peripheral tissues, such as the gastrointestinal tract, as compared to the brain. A detailed evaluation of binding sites for gastrin-releasing peptide (GRP), neuropeptide Y, vasoactive intestinal peptide (VIP)/pituitary adenylate cyclase-activating polypeptide (PACAP), gastrin/cholecystokinin, neurotensin, substance P and somatostatin was therefore undertaken in human colon using in vitro receptor autoradiography and subtype characterization with receptor-selective ligands. GRP receptors, Y2 receptors, PACAP type1-receptors, cholecystokinin-A receptors, neurotensinl and sst2 receptors were abundantly expressed in the myenteric plexus. Y2, neurotensinl and sst2 receptors were also strongly expressed in the submucosal plexus. Furthermore, expression of GRP receptors, neurokinin (NK)1 receptors, VIP type2-receptors and sst2 receptors was found in the mucosa-directed margin of the circular smooth muscle where the interstitial cells of Cajal are located. A variable and complementary expression of GRP receptors, VIP/PACAP receptors, Y2 neurotensinl, NK1 and somatostatin receptors was found in the circular and longitudinal smooth muscle. NK1 and Y1 receptors were often detected in arteries and veins, while VIP/PACAP and sst2 receptors were found in lymphoid follicles. Y2, VIP type, and sst2 receptors were present in the colonic mucosa. Y2 was strongly expressed in the muscularis mucosae. This study shows that neuropeptide receptors are expressed in high amounts and in highly specific patterns in distinct targets in the human colon, suggesting a major physiological role for these peptides. The data represent the molecular basis to investigate the regulation by neuropeptides of colonic functions and to develop neuropeptide drugs aimed at interacting with these receptors in colonic diseases, such as Hirschsprung's and Crohn's diseases.

The topography, architecture and structure of the enteric nervous system in the jejunum and ileum of cattle.

To date, there appear to have been no detailed and clear descriptions of the nerve plexuses and their subdivisions in the intestine of cattle. In this study, the enteric nervous system in the jejunum and ileum of 12 1-y-old calves was examined using neurofilament protein and vasoactive intestinal peptide immunohistochemistry in wholemounts and paraffin sections combined with staining of paraffin and historesin sections with haematoxylin and eosin. The main organisation of the plexuses was similar to that of the pig, horse and man with external and internal submucous plexuses being morphologically distinct, with further subdivisions of the internal submucous plexus into the external and internal subplexuses. However, in contrast to pig, horse and man, the submucous layer was firmly attached to the inner circular muscle layer. The myenteric plexus was well developed with large ganglia, and primary and secondary nerve strands. Its main axis was oriented parallel to the outer longitudinal smooth muscle; large ganglia and primary nerve strands fused to form complex ganglia, and 2 types of tertiary nerve strands were observed. Antibodies to neurofilament proteins and vasoactive intestinal peptide revealed adendritic, pseudouniaxonal or multiaxonal type II neurons only in the myenteric and submucous plexuses. This appears to be the first report of the identification of isolated uniaxonal, multidendritic type IV neurons in the mucous pericryptal plexus. The new information presented here provides further evidence for the existence of anatomical and functional differences between the external and internal submucous plexuses and for supporting the nomenclature proposed earlier.

The organisation of the enteric nervous system in the submucous and mucous layers of the small intestine of the pig studied by VIP and neurofilament protein immunohistochemistry.

The arrangement of the enteric ganglia and nerve fibre plexuses was examined in the submucous and mucous layers and around Peyer's patches of the porcine small intestine to clarify their organisation. Immunohistochemistry of vasoactive intestinal peptide (VIP) and neurofilament proteins in wholemounts, chopped or paraffin sections was used to locate the neural elements. The ganglia of the internal and external submucous plexuses were situated at 2 different topographic locations, being clearly demarcated by the submucosal vascular arcades and differing in neuronal composition. The internal submucous plexus was the only contributor to the plexus surrounding the follicles of Peyer's patches as a continuous mesh of 3 ganglionated nerve subplexuses. VIP-immunoreactive fibres from this mesh innervated the dome. The mucosal plexus, which was subdivided into 4 subunits--the outer proprial, inner proprial, pericryptal and villous plexuses--contained a few solitary neuronal perikarya. Labelling for neurofilament proteins revealed Dogiel types II, IV and VI neurons. The observations reveal several new features in the enteric nervous system of the pig and clarify its nomenclature.

In situ visualization of bronchial submucosal glands and their secretory response to acetylcholine.

Airway submucosal glands secrete both macromolecules and liquid, yet the mechanisms by which these substances are secreted are not well understood. In this study, a video microscope was used to directly visualize the submucosal glands in isolated porcine distal bronchi and to observe their responses to acetylcholine (ACh), a glandular secretagogue. Submucosal glands were classified as either "antral," "linear," or "convoluted" glands based on the morphology of their terminal collecting ducts. Because antral duct glands were most easily visualized, the response to ACh was studied in detail in this gland type. Within 5-10 s after addition of 10 microM ACh, the cross-sectional area of the gland duct openings to the airway surface increased severalfold but returned to pre-ACh dimensions within 1 min. Between 30 s and 10 min after ACh addition, spherical particles (1-10 microm) entered the antral ducts from distal acini and exited through the duct openings to the airway surface. Some of the particles were retained within the antral duct where they were kept in constant motion by the action of cilia present within the antral duct. The particles, which are likely to contain the macromolecular secretory products of mucous and/or serous cells, maintained their spherical shape within the gland duct, suggesting that the secretion product was membrane bound. To our knowledge, these studies provide the first description of airway submucosal gland secretion as viewed in situ.