Patricia Castelucci

Immunohistochemical analysis of neuron types in the mouse small intestine

The definition of the nerve cell types of the myenteric plexus of the mouse small intestine has become important, as more researchers turn to the use of mice with genetic mutations to analyze roles of specific genes and their products in enteric nervous system function and to investigate animal models of disease. We have used a suite of antibodies to define neurons by their shapes, sizes, and neurochemistry in the myenteric plexus. Anti-Hu antibodies were used to reveal all nerve cells, and the major subpopulations were defined in relation to the Hu-positive neurons. Morphological Type II neurons, revealed by anti-neurofilament and anti-calcitonin gene-related peptide antibodies, represented 26% of neurons. The axons of the Type II neurons projected through the circular muscle and submucosa to the mucosa. The cell bodies were immunoreactive for choline acetyltransferase (ChAT), and their terminals were immunoreactive for vesicular acetylcholine transporter (VAChT). Nitric oxide synthase (NOS) occurred in 29% of nerve cells. Most were also immunoreactive for vasoactive intestinal peptide, but they were not tachykinin (TK)-immunoreactive, and only 10% were ChAT-immunoreactive. Numerous NOS terminals occurred in the circular muscle. We deduced that 90% of NOS neurons were inhibitory motor neurons to the muscle (26% of all neurons) and 10% (3% of all neurons) were interneurons. Calretinin immunoreactivity was found in a high proportion of neurons (52%). Many of these had TK immunoreactivity. Small calretinin neurons were identified as excitatory neurons to the longitudinal muscle (about 20% of neurons, with ChAT/calretinin/± TK chemical coding). Excitatory neurons to the circular muscle (about 10% of neurons) had the same coding. Calretinin immunoreactivity also occurred in a proportion of Type II neurons. Thus, over 90% of neurons in the myenteric plexus of the mouse small intestine can be currently identified by their neurochemistry and shape.

P2X2 purine receptor immunoreactivity of intraganglionic laminar endings in the mouse gastrointestinal tract

The distribution of P2X2 purine receptor subunit immunoreactivity has been investigated in the mouse gastrointestinal tract. Immunoreactivity occurred in intraganglionic laminar endings (IGLEs) associated with myenteric ganglia throughout the gastrointestinal tract. In the esophagus, IGLEs supplied every myenteric ganglion. The proportion of ganglia supplied decreased from 85% in the stomach to 10% in the ileum, and from 50% in the caecum to 15% in the distal colon. There was substantial loss of IGLEs from myenteric ganglia of all abdominal regions after bilateral subdiaphragmatic section of the vagus nerves. IGLEs in the esophagus consisted of dense clusters of punctate immunoreactive varicosities. In the stomach and duodenum they had prominent lamellar processes and irregular, but smaller, lamellae were found in other regions. Rare immunoreactive IGLEs occurred in the submucosa of the distal colon. P2X2 receptor immunoreactivity was on the surfaces and in the cytoplasm of a minority of nerve cells in myenteric ganglia. It is concluded that P2X2 purine receptor immunoreactivity is a feature of IGLEs in the mouse, and that P2X receptor agonists may modulate sensitivity of the IGLEs.

Effects of pre- and postnatal protein deprivation and postnatal refeeding on myenteric neurons of the rat small intestine: A quantitative morphological study

We investigated weight gain, the size of the small intestine and numbers and sizes of enteric neurons in rats whose mothers had been deprived of protein during pregnancy and who themselves were deprived postpartum. Postnatally, protein deprivation was for 42 days, or for 21 days with refeeding for a further 21 days. Control animals received normal nourishment. Neurons were located by nicotinamide adenine dinucleotide (NADH) diaphorase staining, by acetylcholinesterase (AChE) activity and immunoreactivity for choline acetyltransferase (ChAT). The collagen and elastic fibers in the myenteric ganglia were evaluated histologically. The myenteric ganglia were regular and uniform in the nourished and refed groups. In the undernourished group, the myenteric ganglia were irregularly arranged and the cytoplasm of most of the neurons showed less intense staining for NADH diaphorase, AChE and ChAT. AChE activity and ChAT immunoreactivity showed that most ganglionic neurons were stained in nourished and refed groups, but the neurons of undernourished rats were unstained or moderately stained. The distribution of the connective tissue of the ganglionic capsule was similar in the three groups. There was a decrease in weight of undernourished rats, which was restored in refed rats. The size of the small intestine of the undernourished group was smaller than in the normally fed group, by about 45%, but it was similar in nourished and refed rats. After 42 days of protein deprivation the numbers of neurons that were revealed by NADH diaphorase were fewer than in well nourished rats, but numbers were not different between nourished and refed rats. These observations indicate that protein deprivation alters histological features and acetylcholinesterase activity of neurons and also reduces body weight but these were restored by refeeding.

NADPH- Diaphorase positive cardiac neurons in the atria of mice. A morphoquantitative study

BackgroundThe present study was conducted to determine the location, the morphology and distribution of NADPH-diaphorase positive neurons in the cardiac nerve plexus of the atria of mice (ASn). This plexus lies over the muscular layer of the atria, dorsal to the muscle itself, in the connective tissue of the subepicardium. NADPH- diaphorase staining was performed on whole-mount preparations of the atria mice. For descriptive purposes, all data are presented as means ± SEM.ResultsThe majority of the NADPH-diaphorase positive neurons were observed in the ganglia of the plexus. A few single neurons were also observed. The number of NADPH-d positive neurons was 57 ± 4 (ranging from 39 to 79 neurons). The ganglion neurons were located in 3 distinct groups: (1) in the region situated cranial to the pulmonary veins, (2) caudally to the pulmonary veins, and (3) in the atrial groove. The largest group of neurons was located cranially to the pulmonary veins (66.7%). Three morphological types of NADPH-diaphorase neurons could be distinguished on the basis of their shape: unipolar cells, bipolar cells and cells with three processes (multipolar cells). The unipolar neurons predominated (78.9%), whereas the multipolar were encountered less frequently (5,3%). The sizes (area of maximal cell profile) of the neurons ranged from about 90 μm2to about 220 μm2. Morphometrically, the three types of neurons were similar and there were no significant differences in their sizes. The total number of cardiac neurons (obtained by staining the neurons with NADH-diaphorase method) was 530 ± 23. Therefore, the NADPH-diaphorase positive neurons of the heart represent 10% of the number of cardiac neurons stained by NADH.ConclusionThe obtained data have shown that the NADPH-d positive neurons in the cardiac plexus of the atria of mice are morphologically different, and therefore, it is possible that the function of the neurons may also be different.

Age-related changes in urinary bladder intramural neurons

A quantitative morphometric evaluation of the intramural plexus of the urinary bladder of adult and aged guinea‐pigs was performed by histological analysis, scanning electron microscopy, and hystochemical methods, such as NADH‐diaphorase and acetylcholinesterase (AChE). The round or oval shaped intramural neurons were revealed among the bundles of the smooth detrusor muscle in clusters containing a variable number of cells in the groups. In both adult control and aged animals, the ganglia were enveloped by a ganglionar capsule of connective tissue mainly composed of type I collagen fibers. The number of neurons NADH‐diaphorase positives estimated in the intramural plexus was 1433 ± 187.71 and 1107 ± 120.67 in the adult control and aged groups, respectively. The perikaryon areas of the NADH‐diaphorase neurons reactives ranged from 216.40 to 1809.30 μm2 in adult control group and from 198.20 to 2096.25 μm2 in aged group. The nuclear area showed an increase in aged animals. The number of AChE‐positive neurons estimated in the intramural plexus was 3294.67 ± 415 μm2 in the adult control group and 1960.33 ± 526 μm2 in the aged group, showing a significant decrease in the latter group. This age‐related morphological change in intramural neurons may contribute to changes in urinary bladder activities in the elderly.

Differential effects of undernourishment on the differentiation and maturation of rat enteric neurons

The colocalization, number, and size of various classes of enteric neurons immunoreactive (IR) for the purinergic P2X2 and P2X7 receptors (P2X2R, P2X7R) were analyzed in the myenteric and submucosal plexuses of control, undernourished, and re-fed rats. Pregnant rats were exposed to undernourishment (protein-deprivation) or fed a control diet, and their offspring comprised the following experimental groups: rats exposed to a normal diet throughout gestation until postnatal day (P)42, rats protein-deprived throughout gestation and until P42, and rats protein-deprived throughout gestation until P21 and then given a normal diet until P42. Immunohistochemistry was performed on the myenteric and submucosal plexuses to evaluate immunoreactivity for P2X2R, P2X7R, nitric oxide synthase (NOS), choline acetyltransferase (ChAT), calbindin, and calretinin. Double-immunohistochemistry of the myenteric and submucosal plexuses demonstrated that 100% of NOS-IR, calbindin-IR, calretinin-IR, and ChAT-IR neurons in all groups also expressed P2X2R and P2X7R. Neuronal density increased in the myenteric and submucosal plexuses of undernourished rats compared with controls. The average size (profile area) of some types of neurons in the myenteric and submucosal plexuses was smaller in the undernourished than in the control animals. These changes appeared to be reversible, as animals initially undernourished but then fed a normal diet at P21 (re-feeding) were similar to controls. Thus, P2X2R and P2X7R are present in NOS-positive inhibitory neurons, calbindin- and calretinin-positive intrinsic primary afferent neurons, cholinergic secretomotor neurons, and vasomotor neurons in rats. Alterations in these neurons during undernourishment are reversible following re-feeding.

Atrophy and neuron loss: effects of a protein-deficient diet on sympathetic neurons.

Protein deficiency is one of the biggest public health problems in the world, accounting for about 30–40% of hospital admissions in developing countries. Nutritional deficiencies lead to alterations in the peripheral nervous system and in the digestive system. Most studies have focused on the effects of protein‐deficient diets on the enteric neurons, but not on sympathetic ganglia, which supply extrinsic sympathetic input to the digestive system. Hence, in this study, we investigated whether a protein‐restricted diet would affect the quantitative structure of rat coeliac ganglion neurons. Five male Wistar rats (undernourished group) were given a pre‐ and postnatal hypoproteinic diet receiving 5% casein, whereas the nourished group (n = 5) was fed with 20% casein (normoproteinic diet). Blood tests were carried out on the animals, e.g., glucose, leptin, and triglyceride plasma concentrations. The main structural findings in this study were that a protein‐deficient diet (5% casein) caused coeliac ganglion (78%) and coeliac ganglion neurons (24%) to atrophy and led to neuron loss (63%). Therefore, the fall in the total number of coeliac ganglion neurons in protein‐restricted rats contrasts strongly with no neuron losses previously described for the enteric neurons of animals subjected to similar protein‐restriction diets. Discrepancies between our figures and the data for enteric neurons (using very similar protein‐restriction protocols) may be attributable to the counting method used. In light of this, further systematic investigations comparing 2‐D and 3‐D quantitative methods are warranted to provide even more advanced data on the effects that a protein‐deficient diet may exert on sympathetic neurons.

Differential effects of intestinal ischemia and reperfusion in rat enteric neurons and glial cells expressing P2X2 receptors.

BACKGROUND Intestinal ischemia followed by reperfusion (I/R) may occur following intestinal obstruction. In rats, I/R in the small intestine leads to structural changes accompanied by neuronal death. AIM The objective was to analyze the impact of I/R injury on different neuronal populations in the myenteric plexus of the rat ileum after different periods of reperfusion. METHODS The superior mesentery artery was occluded for 45 minutes, and animals were euthanized after 24 hours and 1 week of reperfusion. Immunohistochemical analyses were performed with antibodies against the P2X2 receptor in combination with antibodies against nitric oxide synthase (NOS), choline acetyltransferase (ChAT), calbindin, calretinin, the pan-neuronal marker anti-HuC/D, or S100β (glial marker). RESULTS Dual immunolabeling demonstrated that approximately 100% of NOS-, ChAT-, calbindin-, and calretinin-immunoreactive neurons in all groups expressed the P2X2 receptor. Following I/R, the neuronal density decreased in the P2X2 receptor-, ChAT-, calretinin-, and HuC/D-immunoreactive neurons at 24 hours and 1 week following injury compared to the densities in the control and sham groups. The calbindin-immunoreactive neuron density was not reduced in any of the groups. The density of enteric glial cells increased by 40% in the I/R group compared to the density in the sham groups. We also observed increases of 12%, 16%, and 23% in the neuronal cell body profile areas of the NOS-, ChAT-, and calbindin-immunoreactive neurons, respectively, at 1 week following I/R. However, the average size of the calretinin-immunoreactive neurons was reduced by 12% in the I/R group at 24 hours. CONCLUSIONS This work demonstrates that I/R is associated with a significant loss of different classes of neurons in the myenteric plexus accompanied by morphological changes and an increased density of enteric glial cells; all of these effects may underlie conditions related to intestinal motility disorder.

Differential regulation of FGF-2 in neurons and reactive astrocytes of axotomized rat hypoglossal nucleus. A possible therapeutic target for neuroprotection in peripheral nerve pathology.

Despite the favorable treatment of cranial nerve neuropathology in adulthood, some cases are resistant to therapy leading to permanent functional impairments. In many cases, suitable treatment is problematic as the therapeutic target remains unknown. Basic fibroblast growth factor (bFGF, FGF-2) is involved in neuronal maintenance and wound repair following nervous system lesions. It is one of few neurotrophic molecules acting in autocrine, paracrine and intracrine fashions depending upon specific circumstances. Peripheral cranial somatic motor neurons, i.e. hypoglossal (XII) neurons, may offer a unique opportunity to study cellular FGF-2 mechanisms as the molecule is present in the cytoplasm of neurons and in the nuclei of astrocytes of the central nervous system. FGF-2 may trigger differential actions during development, maintenance and lesion of XII neurons because axotomy of those cells leads to cell death during neonatal ages, but not in adult life. Moreover, the modulatory effects of astroglial FGF-2 and the Ca+2-binding protein S100β have been postulated in paracrine mechanisms after neuronal lesions. In our study, adult Wistar rats received a unilateral crush or transection (with amputation of stumps) of XII nerve, and were sacrificed after 72h or 11 days. Brains were processed for immunohistochemical localization of neurofilaments (NF), with or without counterstaining for Nissl substance, glial fibrillary acidic protein (GFAP, as a marker of astrocytes), S100β and FGF-2. The number of Nissl-positive neurons of axotomized XII nucleus did not differ from controls. The NF immunoreactivity increased in the perikarya and decreased in the neuropil of axotomized XII neurons 11 days after nerve crush or transection. An astrocytic reaction was seen in the ipsilateral XII nucleus of the crushed or transected animals 72h and 11 days after the surgery. The nerve lesions did not change the number of FGF-2 neurons in the ipsilateral XII nucleus; however, the nerve transection increased the number of FGF-2 glial profiles by 72h and 11 days. Microdensitometric image analysis revealed a short lasting decrease in the intensity of FGF-2 immunoreactivity in axotomized XII neurons by 72h after nerve crush or transection and also an elevation of FGF-2 in the ipsilateral of glial nuclei by 72h and 11 days after the two lesions. S100β decreased in astrocytes of 11-day-transected XII nucleus. The two-color immunoperoxidase for the simultaneous detection of the GFAP/FGF-2 indicated FGF-2 upregulation in the nuclei of reactive astrocytes of the lesioned XII nucleus. Astroglial FGF-2 may exert paracrine trophic actions in mature axotomized XII neurons and might represent a therapeutic target for neuroprotection in peripheral nerve pathology.

Distribution of the P2X2 receptor and chemical coding in ileal enteric neurons of obese male mice (ob/ob)

AIM To investigate the colocalization, density and profile of neuronal areas of enteric neurons in the ileum of male obese mice. METHODS The small intestinal samples of male mice in an obese group (OG) (C57BL/6J ob/ob) and a control group (CG) (+/+) were used. The tissues were analyzed using a double immunostaining technique for immunoreactivity (ir) of the P2X2 receptor, nitric oxide synthase (NOS), choline acetyl transferase (ChAT) and calretinin (Calr). Also, we investigated the density and profile of neuronal areas of the NOS-, ChAT- and Calr-ir neurons in the myenteric plexus. Myenteric neurons were labeled using an NADH-diaphorase histochemical staining method. RESULTS The analysis demonstrated that the P2X2 receptor was expressed in the cytoplasm and in the nuclear and cytoplasmic membranes only in the CG. Neuronal density values (neuron/cm(2)) decreased 31% (CG: 6579 ± 837; OG: 4556 ± 407) and 16.5% (CG: 7796 ± 528; OG: 6513 ± 610) in the NOS-ir and calretinin-ir neurons in the OG, respectively (P < 0.05). Density of ChAT-ir (CG: 6200 ± 310; OG: 8125 ± 749) neurons significantly increased 31% in the OG (P < 0.05). Neuron size studies demonstrated that NOS, ChAT, and Calr-ir neurons did not differ significantly between the CG and OG groups. The examination of NADH-diaphorase-positive myenteric neurons revealed an overall similarity between the OG and CG. CONCLUSION Obesity may exert its effects by promoting a decrease in P2X2 receptor expression and modifications in the density of the NOS-ir, ChAT-ir and CalR-ir myenteric neurons.