Michał Bulc

Immunohistochemical distribution of cocaine and amphetamine regulatory peptide-like immunoreactive (CART-LI) nerve fibers in the circular muscle layer and their relationship to other peptides in the human caecum.

Motor activity of the gastrointestinal tract is extensively controlled by the enteric nervous system (ENS). Numerous neurotransmitters and neuromodulators are responsible for this regulation. One of them is cocaine- and amphetamine-regulated transcript peptide (CART). So far, there are few reports available concerning the distribution, functions, and co-localization of CART in the human gastrointestinal tract. The aim of the present investigation was to study the distribution and degree of co-localization of CART with substances taking part in conducting sensory stimuli, such as: substance P (SP), neurokinin A (NKA), calcitonin gene related peptide (CGRP) and Leu 5 enkephalin (L-ENK) in the circular muscle layer of the human caecum. CART-like immunoreactive (CART-LI) nerve fibers formed a very dense meshwork in the circular muscle layer of the caecum in all patients studied. Moreover, all neuronal substances tested during the present investigation were observed in CART-LI processes, but the degree of co-localization depended on the type of substance. The highest number of CART-positive nerves also contained L-ENK. A slightly lower level of co-localization was observed in the case of CART and SP or NKA, while only single nerve fibers were simultaneously CART- and CGRP-positive.

Long term estradiol-17β administration changes population of the dorsal root ganglia neurons innervating the ovary in the sexually mature gilts.

The influence of estradiol-17β (E₂) overdose on the number and distribution of neurons in the dorsal root ganglia (DRGs) supplying the ovary of adult pigs was investigated. The numbers of ovarian substance P (SP)-, calcitonin gene-related peptide (CGRP)-, galanin (GAL)-, pituitary adenylate cyclase-activating polypeptide (PACAP)-, neuronal isoform of nitric oxide synthase (nNOS)- and estrogen receptors (ERs)-immunoreactive perikarya were also determined. On day 3 of the estrous cycle, the ovaries of both the control and experimental gilts were injected with retrograde tracer Fast Blue. From day 4 of the estrous cycle to the expected day 20 of the second studied cycle, the experimental gilts were injected with E₂, while the control gilts received oil. The DRGs Th16-L5 were then collected and processed for double-labelling immunofluorescence. Injections of E₂ increased the E₂ level in the peripheral blood ∼4-5-fold and reduced the following in the DRGs: the total number of Fast Blue-positive perikarya, the number of large perikarya, the population of perikarya in the L2 and L3 ganglia, the numbers of SP- and/or CGRP-, PACAP-, nNOS-immunoreactive perikarya and the number of large perikarya expressing ERs subtype α and β. These results show that long-term E₂ treatment of adult gilts affects both the spatial and neurochemical organization pattern of ovary sensory innervation. Our findings suggest that elevated E₂ levels occurring during pathological states may regulate the transmission of sensory modalities from the ovary to the spinal cord.

Immunohistochemical characterization of the porcine nodose ganglion.

This study reports for the first time the presence of several biologically active substances in the nodose ganglion of immature female pigs. The expression and distribution pattern of the studied substances was examined using a double-labeling immunofluorescence technique. In order to visualize the entire population of the ganglionic cell bodies, PGP 9.5, the pan-neuronal marker was used. The distribution and relative proportion of immunolocalized substance P, calcitonin gene related peptide, neuronal isoform of nitric oxide synthase and galanin in perikarya were evaluated. Quantitative analysis of the nodose ganglion neurons revealed that 16.187% (±1.22) of the immunoreactive PGP 9.5 was localized in the perikarya of the left-side ganglion whereas 14.234% (±3.63) of the right-side ganglion neurons expressed substance P, respectively. Accordingly, the proportion of the perikarya with calcitonin gene related peptide ranged from 12.667% (±2.66) for the left ganglion compared with 14.875% (±2.33) for the right one. With regard to the neuronal isoform of the nitric oxide synthase expression, our study revealed a population of 18.703% (±2.50) in the left and 13.336% (±1.25) in the right ganglion, respectively. The immunoreactivity for galanin was found in a relatively small population of neurons of the left nodose ganglion, where galanin-immunoreactive perikarya constituted 1.163% (±1.26), while in the contralateral ganglion 0.865% (±0.32) of total perikarya. No nerve fibers immunopositive for the above studied substances were encountered.

Long-term testosterone administration affects the number of paracervical ganglion ovary-projecting neurons in sexually mature gilts.

The influence of testosterone (T) overdose on the number and distribution of ovarian neurons in the paracervical ganglion (PCG) in pigs was examined. To identify the ovarian neurons, on day 3 of the estrous cycle, the ovaries of both the control and experimental gilts were injected with retrograde neuronal tracer Fast Blue. From next day to the expected day 20 of the second studied cycle, experimental gilts were injected with T, while control gilts received oil. The PCG was then collected and processed for double-labeling immunofluorescence. T injections increased the T (∼3.5-fold) and estradiol-17β (∼1.6-fold) levels in the peripheral blood, and reduced the following in the PCG: the total number of Fast Blue-positive neurons, the number of perikarya in the lateral part of the PCG, the numbers of VAChT(+)/SOM(+), VAChT(+)/VIP(+), VAChT(+)/nNOS(+), VAChT(+)/VIP(-), VAChT(+)/DβH(-), VAChT(-)/SOM(-), VAChT(-)/VIP(-), VAChT(-)/nNOS(-) and VAChT(-)/DβH(-) perikarya, In the T-affected PCG, the populations of ovarian perikarya coded VAChT(-)/SOM(+), VAChT(-)/VIP(+) and VAChT(-)/DβH(+), and expressing androgen receptor were increased. After T treatment within the PCG dropped the density of nerve fibers expressing VAChT and/or SOM, VIP, DβH. Obtained data suggest that elevated androgen levels occurring during pathological processes may regulate ovary function(s) by affecting the PCG gonad-supplying neurons.

Changes in expression of inhibitory substances in the intramural neurons of the stomach following streptozotocin- induced diabetes in the pig

AIM Influence of chronic hyperglycemia on chemical coding of enteric neurons in stomach using pig as a model for human diabetic complications. METHODS Ten pigs were divided into two groups: diabetic (D group, n = 5) and control (C group, n = 5). Pigs constituting the experimental group were given streptozotocin (150 mg/kg). Animals were euthanized six weeks after the induction of diabetes. The samples of stomach were collected from animals of both groups. The cryostat sections were processed for double immunofluorescence staining using primary antisera directed towards pan-neuronal marker (Hu C/D) proteins and/or neuronal isoform of nitric oxide synthase (nNOS), vasoactive intestinal peptide (VIP) and galanin (GAL). RESULTS In the control group in the myenteric ganglia (MG) of the corpus we have noted 22.28% ± 1.19% of nNOS positive neurons, while in diabetic group we have found 40.74% ± 2.22% of nNOS immunoreactive perikarya (increase by 82.85 %). In turn in the pylorus we have observed 15.91% ± 0.58% nNOS containing neurons in control animals and 35.38% ± 1.54% in the diabetes group (increase by 122.37%). In the MG of the antrum and submucosal ganglion (SG) in the corpus hyperglycemia did not cause statistically significant changes. With regard to VIP-positive cell bodies in the antrum MG in the control animals we have noted 18.38 ± 1.39% and 40.74% ± 1.77% in the experimental group (increase by 121.65%). While in the corpus we have observed 23.20% ± 0.23% in the control and 30.93% ± 0.86% in the diabetes group (increase by 33.31%). In turn in the pylorus VIP positive cells bodies constituted 23.64% ± 1.56% in the control group and 31.20% ± 1.10% in the experimental group (increase by 31.97%). In the submucosal ganglion in the corpus we have noted 43.61% ± 1.06% in the control animals and 37.00% ± 1.77% in the experimental group (decrease by 15.15%). Expression of GAL-positive perikarya showed statistically significant changes only in the MG of the antrum and pylorus. In the antrum GAL positive perykarya constituted 26.53% ± 1.52% in the control and 36.67% ± 1.02% in the experimental animals (increase by 38.22%). While in the pylorus GAL positive neurons in the control group constituted 16.32% ± 0.92% and 17.99% ± 0.38% in the experimental animals (increase by 10.23%). CONCLUSION Our results support the hypothesis that in the course of diabetes, long term episodes of high glucose serum level may influence the chemical phenotyping of enteric neurons.

Immunohistochemical evidence of the co-localisation of cocaine and amphetamine regulatory peptide with neuronal isoform of nitric oxide synthase, vasoactive intestinal peptide and galanin within the circular muscle layer of the human caecum

The enteric nervous system consists of about one hundred million of neurons. In big mammals (including humans) intestinal enteric neuronal cells are grouped into three types of intramural ganglia located within myenteric, as well as outer and inner submucosal plexuses, which are connected by numerous nerve fibres. Both nerve fibres and cell bodies located in the gastrointestinal tract utilise a broad spectrum of active substances. One of them is cocaine- and amphetamine-regulated transcript peptide (CART). The goal of the current study was to determinate the distribution and degree of co-localisation of CART with substances taking part in intestinal motor activity by double labelling immunofluorescence technique. During the study CART-, neuronal isoform of nitric oxide synthase (nNOS)-, vasoactive intestinal peptide (VIP)- and/or galanin (GAL) - like immunoreactive (LI) nerve fibres in the circular muscle layer of the human caecum were observed in all patients studied. The degree of co-localisation of particular substances with CART depended on their type. The majority of CART-LI fibres contained simultaneously nNOS, slightly lower degree of co-localisation was observed in the case of the VIP, while simultaneously CART- and GAL-positive nerve fibres were observed less often.

The foetal pig pineal gland is richly innervated by nerve fibres containing catecholamine-synthesizing enzymes, neuropeptide Y (NPY) and C-terminal flanking peptide of NPY, but it does not secrete melatonin

Innervation of the mammalian pineal gland during prenatal development is poorly recognized. Therefore, immunofluorescence studies of the pineals of 70- and 90-day-old foetuses of the domestic pig were performed using antibodies against tyrosine hydroxylase (TH), dopamine-β-hydroxylase (DβH), neuropeptide Y (NPY) and C-terminal flanking peptide of NPY (CPON). The investigated glands were supplied by numerous nerve fibres containing TH and DβH. The density of these fibres was higher in the distal and middle parts of the gland than in the proximal one. NPY and CPON were identified in the majority of DβH-positive fibres as well as in a small population of DβH-negative fibres localized mainly in the proximal part of the pineal. The immunoreactive fibres were more numerous in 90-day-old foetuses than in 70-day-old ones. The effect of norepinephrine on melatonin secretion by the foetal pineals in the short-term organ culture was studied to determine the role of DβH-positive fibres during prenatal life. For the same purpose melatonin was measured in the blood in the umbilical cords and in the jugular vein of the mother. The pineals of both groups of foetuses did not secrete melatonin in the organ culture, independently of the presence or absence of norepinephrine in the medium. Melatonin concentrations in the blood in the umbilical cords of foetuses from the same litter and in the jugular vein of their mother were similar. The presence of adrenergic nerve fibres in the pig pineal during gestation does not seem to be associated with the control of melatonin secretion.

Neurochemical characteristics of calbindin-like immunoreactive coeliac-cranial mesenteric ganglion complex (CCMG) neurons supplying the pre-pyloric region of the porcine stomach

The present study was designed to determine the distribution, morphology and co-localization of calbindin-D28k (CB) with other neuroactive substances in the coeliac-cranial mesenteric ganglion complex (CCMG) neurons supplying the prepyloric region of the porcine stomach. In all animals, a median laparotomy was performed and the fluorescent retrograde neuronal tracer Fast Blue was injected into the wall of the stomach prepyloric area. On the 28th day, all animals were euthanized and the CCMG complexes were then collected and processed for double-labelling immunofluorescence for CB and tyrosine hydroxylase (TH), galanin (GAL), somatostatin (SOM), leu 5-enkephalin (LENK), vasoactive intestinal peptide (VIP), substance P (SP) and cocaine- and amphetamine-regulated transcript peptide (CART), Immunohistochemistry revealed that 8.27±0.51% of FB-positive neurons expressed CB-like immunoreactivity. Furthermore, CB co-localized with TH, GAL and SOM in retrogradely labelled cell bodies, whereas CART, LENK, VIP and SP were detected only in nerve terminals surrounding FB+/CB+ neurons. The presence of CB in the stomach-projecting neurons may indicate the contribution of CB in the sympathetic regulation of the stomach function. Furthermore, CB-LI neurons had a catecholaminergic character and co-localized with TH, GAL and SOM, which suggests multiple functions of this neuroactive substance in the CCMG neurons supplying the porcine prepyloric area.

Changes in Somatostatin-Like Immunoreactivity in the Sympathetic Neurons Projecting to the Prepyloric Area of the Porcine Stomach Induced by Selected Pathological Conditions

The aim of the present study was to define changes in the expression of somatostatin (SOM) in the sympathetic perikarya innervating the porcine stomach prepyloric area during acetylsalicylic-acid-induced gastritis (ASA) and experimentally induced hyperacidity (HCL) and following partial stomach resection (RES). On day 1, the stomachs were injected with neuronal retrograde tracer Fast Blue (FB). Animals in the ASA group were given acetylsalicylic acid orally for 21 days. On the 22nd day after FB injection, partial stomach resection was performed in RES animals. On day 23, HCL animals were intragastrically given 5 ml/kg of body weight of a 0.25 M aqueous solution of hydrochloric acid. On day 28, all pigs were euthanized. Then, 14-μm thick cryostat sections of the coeliac-superior mesenteric ganglion (CSMG) complexes were processed for routine double-labelling immunofluorescence. All pathological conditions studied resulted in upregulation of SOM-like (SOM-LI) immunoreactivity (from 14.97 ± 1.57% in control group to 33.72 ± 4.39% in the ASA group, to 39.02 ± 3.65% in the RES group, and to 29.63 ± 0.85% in the HCL group). The present studies showed that altered expression of SOM occurs in sympathetic neurons supplying the prepyloric area of the porcine stomach during adaptation to various pathological insults.

Long-term treatment with testosterone alters ovary innervation in adult pigs

BackgroundIntraovarian distribution and density of nerve fibres immunoreactive (IR) to protein gene product 9.5 (PGP 9.5) and containing dopamine-β-hydroxylase (DβH), neuropeptide Y (NPY), somatostatin (SOM), galanin (GAL) were determined.MethodsFrom day 4 of the first oestrous cycle to day 20 of the second studied cycle, experimental gilts (n = 3) were injected with testosterone (T), while control gilts (n = 3) received corn oil.ResultsAfter T administration the numbers of fibres IR to PGP 9.5 and fibres IR to DβH, NPY and SOM were decreased. Fewer PGP 9.5- and DβH-IR terminals were observed within the ground plexus and around arteries and medullar veins, and medium tertiary follicles, and DβH-IR terminals in the vicinity of small tertiary follicles. T decreased the density of NPY-IR fibres in the medullar part of the ground plexus, and SOM-IR in the cortical part of the ground plexus.ConclusionsThe obtained data show that long-term T treatment of gilts decreases the total number of intraovarian fibres, including sympathetic ones. These results suggest that elevated T levels that occur during pathological states may affect the innervation pattern of ovaries, and their function(s).