Trabecular Bone Parameters, TIMP-2, MMP-8, MMP-13, VEGF Expression and Immunolocalization in Bone and Cartilage in Newborn Offspring Prenatally Exposed to Fumonisins.

Fumonisins are protein serine/threonine phosphatase inhibitors and potent inhibitors of sphingosine N-acyltransferase (ceramide synthase) disrupting de novo sphingolipid biosynthesis. The experiment was conducted to evaluate the effects of fumonisins (FB) exposure from the 7th day of pregnancy to parturition on offspring bone development. The rats were randomly allocated to either a control group (n = 6), not treated with FBs, or to one of the two groups intoxicated with FBs (either at 60 mg FB/kg b.w. or at 90 mg FB/kg b.w. Numerous negative, offspring sex-dependent effects of maternal FB exposure were observed with regards to the histomorphometry of trabecular bone. These effects were due to FB-inducted alterations in bone metabolism, as indicated by changes in the expression of selected proteins involved in bone development: tissue inhibitor of metalloproteinases 2 (TIMP-2), matrix metalloproteinase 8 (MMP-8), matrix metalloproteinase 13 (MMP-13), and vascular endothelial growth factor (VEGF). The immunolocalization of MMPs and TIMP-2 was performed in trabecular and compact bone, as well as articular and growth plate cartilages. Based on the results, it can be concluded that the exposure of pregnant dams to FB negatively affected the expression of certain proteins responsible for bone matrix degradation in newborns prenatally exposed to FB in a dose- and sex-dependent manner.

Immunolocalization of NOS, VIP, galanin and SP in the small intestine of suckling pigs treated with red kidney bean (Phaseolus vulgaris) lectin.

Lectins belong to a family of glycoproteins that can act both beneficially and detrimentally on the morphology of the small intestine. The aim of the study was to determine whether experimental treatment with red kidney bean (Phaseolus vulgaris) lectin influences the chemical code of the small intestine nervous system of suckling pigs. The immunolocalization sites of vasoactive intestinal polypeptide (VIP), nitric oxide synthase (NOS), substance P (SP) and galanin were determined in control and lectin-treated animals. In all segments of the small intestine (duodenum, jejunum, ileum), the subpopulations of VIP-, NOS-, SP- and galanin-immunoreactive (IR) myenteric neurons were unchanged. After lectin stimulation, increased proportions of NOS-IR and decreased numbers of VIP-IR submucous neurons/mucosa innervating nerve fibers were observed in the duodenum, jejunum and ileum. In lectin-treated animals down-regulation of submucous neurons expressing SP and up-regulation of galanin-IR submucous neurons were seen in the duodenum and jejunum (but not in the ileum). The distribution patterns of NOS-IR, galanin-IR and SP-IR nerve fibers supplying the duodenum, jejunum and ileum of the lectin-treated animals showed no substantial differences in relation to control piglets. We conclude that exposure to red kidney bean (P. vulgaris) lectin substantially changes the chemical content of VIP, NOS, SP and galanin in submucous neurons of the small intestine. These results are in line with previous findings outlining the key role(s) of these substances in enteric neuroplasticity processes and may constitute the basis for further functional studies on maturation of the gut.

Aquaporin 1 water channel is expressed on submucosal but not myenteric neurons from the ovine duodenum.

Aquaporins are a large family of small integral membrane proteins that function as molecular water channels. Increasing evidence indicates that an aquaporin 1 (AQP1) water channel is present on the surface of discrete neuronal classes of the central as well peripheral nervous systems. The aim of the present study has been to immunohistochemically localize AQP1 in the enteric nervous system (ENS) of the sheep duodenum. Specific antibodies to vasoactive intestinal polypeptide (VIP) and substance P (SP) were also used to biochemically determine possible function(s) of AQP1-positive enteric neurons. The expression of AQP1 in neuronal cell cultures has been also studied. Under normal conditions, approximately 30% of submucosal neurons exhibit the presence of AQP1 water channels. Neither myenteric neurons nor enteric nerve fibres showed immunoreactivity to AQP1. The vast majority of AQP1-bearing submucosal neurons were immunoreactive (IR) to SP (but not to VIP). Moderate numbers of SP-IR as well as VIP-IR nerve fibres run in close vicinity to AQP1-positive small blood/lymphatic vessels. SP-positive as well as VIP-positive nerve fibres were regularly observed to be in close contact with AQP1-positive submucosal neurons. After 3, 6 and 9 days of in vitro culturing, respectively, myenteric neurons still exhibited no presence of AQP1 channels. The obtained results indicated that in ENS of the ovine duodenum the expression of AQP1 is species-related and predominantly seen in a significant subpopulation of probably sensory submucosal neurons. Since we show no upregulation of AQP1 channels in cultured myenteric neurons we suggest that AQP1 is not a significant factor involved in environmental adaptation of myenteric neurons to the artificial conditions.

Cocaine- and amphetamine-regulated transcript (CART) is expressed in the ovine pancreas.

Indirect immunohistochemistry was applied to demonstrate the presence of cocaine- and amphetamine-regulated transcript (CART) peptide expression in the pancreas of the sheep. Using double immunocytochemical staining, the co-incidence of vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY) or substance P (SP) in CART-immunoreactive (IR) nerve fibers and intrapancreatic neurons was analyzed. Immunoreactivity to CART was detected in endocrine cells predominantly localized at the islet periphery. The exocrine pancreas and blood vessels were intensively innervated by CART-IR nerve fibers. Moderate numbers of CART-IR nerve terminals were found in the connective tissue, while the ductal system and islets were poorly supplied with CART-IR nerve endings. No islet penetrating CART-IR nerve fibers were detected. Approximately 53.7+/-1.8% of intrapancreatic neurons displayed immunoreactivity to CART and pancreatic ganglia were moderately supplied with CART-IR nerve fibers. Dependent upon the pancreas region, CART-IR nerve fibers showed a varying degree of co-existence of SP, VIP or NPY. CART-IR intrapancreatic neurons very frequently co-localized with SP, moderately with VIP and rarely with NPY. We conclude that abundant immunoreactivity to CART in the ovine pancreas and the co-existence of CART with other regulatory peptides may reflect a possible involvement of CART in hormone and enzyme secretion as well as regulation of pancreatic blood flow.

A co-localization study on the ovine pancreas innervation.

The expression of DbetaH and several neuropeptides was investigated in neuronal elements of the ovine pancreas using double immunocytochemical stainings. Immunoreactivities to DbetaH, NPY, VIP and SP were seen to various extents in nerve terminals associated with the acini, islets, ducts, blood vessels, interlobular connective tissue as well as in the neurons of intrapancreatic ganglia. The expression of CGRP was limited to nerve fibers lying in the connective tissue septa, amongst the acini and in close vicinity to the pancreatic blood vessels. Single GRP-positive nerve endings were located around the acini, ducts and in the interlobular connective tissue. With the exception of the ductal system in a co-localization of NPY with DbetaH was frequently found in all regions of the pancreas. Moderately numerous blood vessel-associated VIP-positive nerve fibers as well as the vast majority of VIP-containing intrapancreatic neurons were found to co-express DbetaH. Single SP-immunoreactive (IR) nerve fibers of the exocrine pancreas and interlobular connective tissue as well as SP-positive intrapancreatic neurons additionally showed the presence of DbetaH. The co-localization of VIP and NPY was found in nerve terminals located around the blood vessels and acini, in the connective tissue septa as well as in numerous pancreatic neuronal perikarya. Rare nerve terminals located between the acini and around small blood vessels as well as several neurons of intrapancreatic ganglia were VIP-IR/ SP-IR. Simultaneous expression of SP and CGRP was found in nerve fibers supplying large pancreatic arteries and veins, interlobular connective tissue and, occasionally, around the acini. Throughout the pancreas the population of CGRP-positive nerve endings showed lack of VIP and NPY. In a moderate number of GRP-containing nerve fibers, a co-expression of NPY was noted. Nerve terminals containing both GRP and VIP were detected sporadically, whereas none of the GRP-positive nerve terminals showed expression of SP. We conclude that the presented noradrenergic as well as peptidergic innervation patterns of the ovine pancreas are species-dependent. On the basis of the occurrence of DbetaH, NPY, VIP and SP (alone or in combination) in pancreatic neuronal elements we can suggest that these substances presumably act as regulators of the endocrine and/or exocrine pancreas. Involvement of CGRP and GRP in the ovine pancreas physiology seems to be of minor importance. The co-localization study indicated that the pancreas of the sheep is innervated from several sources including intrinsic as well as extrinsic ganglia.

Neurochemical properties of the middle cervical ganglion in the sheep.

The neurochemical properties of the ovine middle cervical ganglion (MCG) were studied using antibodies raised against tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DbetaH), neuropeptide Y (NPY), substance P (SP), calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP) and galanin (GAL). Double-labelling immunocytochemistry revealed that the vast majority (95.5 +/- 0.8%) of postganglionic sympathetic MCG neurons expressed simultaneously both catecholamine-synthesizing enzymes (neurons were TH/DbetaH-positive). A large population of noradrenergic neurons exhibited immunoreactivity (IR) to NPY (62.2 +/- 2.2%), but single NPY-positive perikarya-lacking noradrenergic markers were also observed (2.0 +/- 0.3%). None of the examined MCG neuronal somata contained SP, CGRP, GAL or VIP. A moderate number of noradrenergic nerve fibres located amongst neuronal cell bodies was also found. In small number of these terminals the presence of NPYor GAL (but not CGRP or VIP) was detected. The ovine MCG was numerously innervated with SP-immunoreactive nerve fibres which sometimes formed basket-like formations around postganglionic neurons. The MCG exhibited a sparse CGRP-immunoreactive innervation and lacked VIP-positive nerve terminals. In many aspects the chemical coding of MCG postganglionic neurons and nerve terminals resembles that found in other mammalian cervico-thoracic paravertebral ganglia, but some important species-dependent differences exist. The functional implications of these differences remain to be elucidated.