Developmental changes in tibia and humerus of goose: morphometric, densitometric, and mechanical analysis.

The skeletal system of young animals undergoes a series of intensive and rapid changes. In this study, we aimed to verify the hypothesis that geese exhibit a distinct pattern of bone growth compared to gallinaceous species. Specifically, we hypothesised that geese would experience an accelerated growth rate in the humerus bone, which can be attributed to the increased wing mobility facilitated by their rearing in free-range systems. This need for access to both ground and water environments contributes to the unique demands placed on their skeletal development. We focused on evaluating the mechanical properties and geometry of the humerus as the forelimb bone, and the tibia as the hindlimb bone. The 320 geese used in this study were divided into 12 groups according to sex (females and males) and age (0-,1-,3-,6-,8-,12-,14-week-old). To assess bone mechanical properties, a three-point bending test was performed, along with densitometry and morphological measurements. The tibiae of the geese showed the most intensive growth until 6 weeks of age and then stabilised. The wing bones (humerus) showed only slight changes in the first weeks after hatching, and then a rapid growth between the third and sixth week, both in terms of mechanical and morphological properties. This is most likely due to a change in the geese's living environment during this period, i.e., allowing them to leave their enclosures and enter open space, which gives them the opportunity to use their wings, resulting in the rapid growth of the wing bones to support increasing muscle mass in this area. This study increases our understanding of bone growth and development in domesticated birds, specifically waterfowl species, and highlights the importance of rearing methods on the proper bone development and functionality of the entire skeletal system, and thus, on their welfare.

Prenatal acrylamide exposure results in time-dependent changes in liver function and basal hematological, and oxidative parameters in weaned Wistar rats.

Acrylamide (ACR) is a toxic compound commonly found in fried, baked and heat-processed starchy foods. The current study investigated the time-dependent effects of maternal exposure to non-toxic ACR doses on the oxidative stress, liver function, and basal blood morphology of the rat offspring. Pregnant, Wistar rats were randomly divided into the control group or the groups administrated with ACR (3 mg/kg b.w./day): long exposure for 15 days, medium exposure for 10 days and short exposure for 5 days during pregnancy. Body mass, blood morphology and hematology, serum concentrations of growth hormone, IGF-1, TNF-α, IL-1ß, IL-6 and insulin, liver histomorphometry, liver activity of beclin1, LC2B and caspase3, markers of oxidative stress and the activity of antioxidative enzymes in blood serum and the liver were measured in offspring at weaning (postnatal day 21). Even short prenatal exposure to ACR led to oxidative stress and resulted in changes in liver histomorphometry and upregulation of autophagy/apoptosis. However, the most significant changes were observed following the long period of ACR exposure. This study has shown for the first time that ACR is responsible for changes in body mass in a time-dependent manner, which could lead to more serious illnesses like overweight and diabetes later in life.

Immunoreactivity of acetylcholinesterase and M1 muscarinic receptors in the hippocampus and striatum of rats treated with Rebaudioside A.

Rebaudioside A (RebA) is a steviol glycoside used for production of sweeteners. It was shown that the glycosides affect memory and learning processes. The aim of the study was to investigate neurons immunoreactive for acetylcholinesterase (AChE) and M1 muscarinic receptors (mAChRs-M1) of the hippocampal CA1 and CA3 fields and striatal caudateputamen (CP) and globus pallidus (GP) in rats receiving RebA. RebA was administrated to adult rats for 45 days in dilutions of 1 mg and 2 mg RebA/ml water. Indirect peroxidase-antiperoxidase immunohistochemical reaction was conducted on frontal sections containing the hippocampus and striatum with use of antibodies against AChE and mAChRs-M1. Immunoreactive for the studied proteins neurons were morphologically and morphometrically assessed in hippocampal CA1 and CA3 fields and in CP and GP. Microscopic observations did not reveal significant changes in morphology of immunoreactive neurons, which suggests no neurotoxic effect of the studied glycoside on these cells. Morphometric analyses revealed an increase in the density of AChE and mAChRs-M1 immunoreactive neurons. A decrease in reaction intensity of AChE-positive neurons was also demonstrated in the hippocampal CA1 field and in GP. In contrast, an increase in reaction intensity of mAChRs-M1-positive neurons was found in CA1, CA3 fields and in CP and GP. The results of our preliminary studies indicate that RebA administrated to rats has an impact on cholinergic neurons in the studied area. The results suggest a possible increase in the activity of the cholinergic system, responsible for memory and learning processes, after administration of RebA.

Immunohistochemical study on the expression of biologically active substances in the endocrine pancreas of the European bison.

Previous morphological studies of mammalian pancreatic islets have been performed mainly in domestic and laboratory animals. Therefore, the present immunohistochemical investigation was conducted in a wild species, the European bison, using antibodies against glucagon-like peptide-1 (GLP1), glucagon, insulin and somatostatin. Morphological analyses revealed that the mean area of the endocrine pancreas constituted 2.1±0.1% of the whole area of the pancreas, while the mean area of a single pancreatic islet was 13301.5±686.5 µm2. Glucagon-immunoreac- tive cells accounted for 22.4±1.1% and occupied 19.4±0.4% of the average islet area. As many as 14.3±1.4% of pancreatic islet cells were shown to express GLP1, which constituted 12.6±0.8% of the mean area of the islet. Insulin expression was confirmed in 67.6±0.7% of pancreatic islet cells, which represented 62.3±4.9% of the mean total area of the pancreatic islet. As many as 8.5±1.3% of cells stained for somatostatin. The somatostatin-immunoreactive cell area was 4.9±0.3% of the mean pancreatic islet area. In summary, we have determined in detail for the first time the morphometry and islet composition of the European bison pancreas. The distri- bution patterns of immunoreactivities to the substances studied in the European bison show many similarities to those described in other ruminant species.

Colocalization pattern of cocaine- and amphetamine-regulated transcript peptide and parvalbumin immunoreactivity in the hippocampus proper of the chinchilla.

This study set out to investigate, for the first time, the distribution and colocalization pattern of cocaine-and amphetamine-regulated transcript (CART) and one of the calcium binding- -proteins: parvalbumin (PV) in the chinchilla's hippocampus proper (HP). HP, consisting of Ammon's horn (CA) and the dentate gyrus (DG), is an important component of the limbic system, involved in learning and memory processes. CA showed a higher immunoreactivity of CART (-IR) compared to DG. CART-IR neurons were mainly observed in the molecular layer of DG and in the pyramidal layer of CA. CART-IR fibers were present in the granular layer; in the hilus numerous mossy fibers were detected, while in the molecular layer CART-IR fibers were not found. In all CA fields (CA1-CA3), CART-IR fibers were only present in the lacuno- sum-molecular layer. Immunofluorescence with double- labeling showed that only CART-IR cells stained positive for PV, whereas in CART-IR fibers there was no PV-positive reaction. Our research supplements missing knowledge about the distribution and colocalization pattern of CART with PV in the chinchilla's hippocampus, and also provides a better understanding of the similarities and differences among individuals of the same species and also with other mammals.

Histomorphometrical changes in intestine structure and innervation following experimental fumonisins intoxication in male Wistar rats.

Fumonisins are highly toxic metabolites produced by Fusarium proliferatum and Fusarium verticillioides. Little is known about the effects of a chronic low level of fumonisins on intestinal structure and innervation in monogastric animals, even though the intestine is the first organ exposed to fumonisins. The influence of the most prevalent strains of fumonisins, FB1 and FB2, on intestinal and liver morphology, the enteric nervous system and intestinal epithelial cell prolif- eration was investigated in an experimental rat model of fumonisin intoxication. Adolescent (5-weeks-old), male Wistar rats were randomly divided into a control group (C group) not treated with fumonisins or intoxicated with fumonisins (FB group). FB1 together with FB2 were daily administered intragastrically at a dose of 90 mg/kg body weight for 21 days. The damaging effect was assessed by determination of the activity of ALAT and AspAT. Samples from the small intes- tine and liver were taken and blood samples were collected to determine the activity of gamma- -glutamyl transferase (GGT) and amylase. The exposure to FBs resulted in histopathological degenerative alterations in hepatocytes, including mild vacuolar degeneration and ballooning. FB exposure was also toxic in the duodenum and jejunum, where significant changes in morphology, cell proliferation, collagen wall fibres and innervation were observed. Taken together, the results obtained strengthen the hypothesis that chronic exposure to FBs could induce intestinal damage, including damage to the enteric nervous system and may have consequences for general health.

The influence of bilberry fruit on memory and the expression of parvalbumin in the rat hippocampus.

Therefore, the aim of the present study was to evaluate the possible effect of bilberry fruit (Vaccinium myrtillus L.) supplement in a daily diet on the cognitive behaviour of the rats and the expression of paravalbumin (PV) in populations of hippocampal neurons. It has been postulated that the antioxidants present in bilberry fruit may act as neuroprotective factors playing also a significant role as memory enhancements. Forty Wistar rats with a similar average body weight (460 ± 0.4 g) were divided into four groups (n=10 per group). The control group received standard feed (210 g/week), whereas animals of experimental groups received standard feed supplemented with bilberry (per os) at consumed doses of 2 g (group I), 5 g (group II), and 10 g/kg b.w./ /day (group III). After three months of feeding with bilberry, the modified elevated plus-maze test (mEPM) was performed. After 32 weeks of feeding, brains were collected and PV-immunoreactive (ir) neurons were immunohistochemically visualized. In the modified elevated plus-maze test, transfer latency examined 2 h and 24 h after the acquisition session was significantly shorter (p⟨0.05) in the group II in comparison with the control group. In CA1 and CA2/CA3 hippocampal fields as well as dentate gyrus of all experimental groups, a significant (p⟨0.05) decrease in number of PV-ir neurons were found. In relation to the control group, the mean subpopulation of PV-ir neurons found in groups II and III were significantly reduced. The subpopulations of PV-ir neurons found in DG of all experimental groups were significantly reduced in comparison to the control. In conclusion the in the present paper we demonstrated a relationship between the diet rich in a bilberry fruit and process of memory as well as numbers of calcium- binding protein-expressing hippocampal neurons. Our results may be source of basic knowledge for further research aiming at neuroprotective role of the bilberry fruit.

Analysis of mechanical properties of bones and tendons shows that modern hybrid rye can be introduced to corn-wheat based diet in broiler chickens as an alternative energy source irrespective of xylanase supplementation.

This study focused on analyzing the effects of inclusion of modern hybrid rye to corn-wheat diet on mechanical properties of bones and tendons. A total of 224 broiler chickens were fed a diet without rye inclusion or a diet containing 15% of hybrid rye cv. Brasetto. The diets were either unsupplemented or supplemented with xylanase (minimum activity 1000 FXU/g, dose 200 mg/kg of feed). Each dietary group consisted of 56 birds. On day 42, selected chickens (n = 7 from each group) were slaughtered. Tibia were analyzed for mineralization, geometry, and biomechanical characteristics of bone mid-diaphysis. The mechanical properties of digital flexor III tendon were also assessed. Bone mineral density and bone ash percentage did not differ when both diets were given without xylanase. Enzyme supplementation increased bone mineral density (P < 0.01) in both dietary groups, whereas bone ash percentage (P < 0.01) increased only for corn-wheat diet. Rye inclusion had no effect on bone mid-shaft geometrical traits related to tibia weight-bearing capacity (cross-sectional area, cortical index, and mean relative wall thickness). Performed bending test showed no effect of hybrid rye inclusion on bone mechanical endurance. When xylanase was supplemented, bone length (P < 0.01) and weight (P < 0.05) decreased, whereas yield load (P < 0.01), stiffness (P < 0.05), Young modulus (P < 0.05), elastics stress (P < 0.01), and ultimate stress (P < 0.01) increased, irrespective of rye presence. The tendon tensile strain test showed that in corn-wheat diet enzyme supplementation positively influenced rupture force (P < 0.05) and tendon stiffness (P < 0.01). Xylanase supplementation increased the value of energy required to tendon rupture, irrespective of rye inclusion (P < 0.05). Study showed that modern hybrid rye varieties can be introduced to corn-wheat diets of broiler chickens in the aspect of animal welfare related to the development and homeostasis of musculoskeletal system, irrespective of xylanase supplementation. The enzyme addition positively affected biomechanical properties of bones and tendons.

The effect of obesity on the bone morphometry and histomorphometry in male and female Wistar rats.

The study was undertaken to determine the effect of continuation or changes of the diet on the morphometry and histomorphometry of bone in male and female Wistar rats with experimen- tally induced obesity by high energetic diet. Sixty-four 90-day-old Wistar rats obtained from obese parents (16 male, 16 female) and control parents (16 male, 16 female) were used in this study. After 21 days of the baby period, rats were divided into four groups: obese rats fed with high energy feed (F/F), control rats fed with a standard diet (C/C), obese rats with changed diet from high energy diet to control diet (F/C) and control rats with changed diet from control diet to high energy diet (C/F). After 90 days of experimental feeding, the rats were sacrificed. Thereafter, body weight and the isolated humerus were measured and next, the histological stainings and counts were done. Our results revealed that change in the parent's diet from F to C in the female leads to increased bone growth length and reduction of body weight in female and male. Reverse diet changes (from C to F) lead to decreased bone length only in the female. Moreover, the con- tinuation by offspring of both sexes with a high-energy diet contributes to a reduction in osteo- cytes, reduction in bone marrow cavity and cortical expansion, but a change in nutrition from parents' standard diet to high-energy diet leads to increase in osteocytes dimensions. The contin- uation of feeding with F diet promotes the accumulation of adipocytes in the bone marrow in female and male, and correction of nutrition from F to standard diet leads to a reduction in their number in the bone marrow compared to groups continuing feeding with high-energy diet.

Effect of feeding with bilberry fruit on the expression pattern of αCaMKII in hippocampal neurons in normal and diabetic rats.

αCaMKII, widely occurring in the central nervous system, plays a significant role in cognitive processes. It is well known that diabetes is a risk factor that may trigger brain atrophy, cognitive dysfunction and finally lead to memory loss. Antioxidants richly present in bilberry fruits are believed to have significant effects on diabetes-related brain dysfunctions mainly due to their abilities to modulate neurotransmitter release that lead to reduction of the negative impact of free radicals on cognitive processes. The aim of the present research was to immunohistochemically investigate the expression patterns of αCaMKII in hippocampal neurons from non-diabetic, diabetic and diabetic rats fed with an extract of bilberry fruit. The obtained results show that in comparison to the control group, in diabetic rats hippocampal neurons immunoreactive (ir) to αCaMKII were swollen and the lengths of the neuronal fibres were reduced. Further study shows that in diabetic rats fed with bilberry fruit, αCaMKII-positive nerve fibres were significantly longer when compared to the groups of diabetic and control rats. Additionally, we observed statistically significant changes in the average larger diameter of αCaMKII-ir hippocampal neurons between groups of diabetic rats (with vs. without supplement of bilberry fruit). The results of the present work suggest that antioxidants present in bilberry fruits influence the morphology of and possibly exhibit beneficial and neuroprotective effects on hippocampal neurons during diabetes. It is likely that changes in the appearance of αCaMKII-expressed hippocampal neurons may reflect the diabetes-evoked rise in Ca2+ level in the cerebral nerve terminals. The present research extends our knowledge of preventive mechanisms for cognitive dysfunctions occurring in the brain during diabetes.

Distribution and immunohistochemical characteristics of cocaine- and amphetamineregulated transcript-positive nerve elements in the pelvic ganglia of the female pig.

Cocaine- and amphetamine-regulated transcript (CART) peptides are widely expressed not only in the brain but also in numerous endocrine/neuroendocrine cells as well as in neurons of the peripheral nervous system. The present study investigated the distribution patterns of CART-like immunoreactivity in the pelvic plexus (PP) of the female pig. The co-expression of CART with principal neurotransmitter markers: choline acetyltransferase (ChAT), tyrosine hydroxylase (TH), serotonin (5-HT) or biologically active neuropeptides: pituitary adenylate cyclase-activating polypeptide (PACAP), substance P (SP), calbindin was analyzed using double immunohistochemical stainings. Amongst neurons immunopositive to Hu C/D panneuronal marker as many as 4.1 ± 1.2% in right and 4.4 ± 1.6% in left pelvic ganglia were found to express CART. The vast majority of CART-IR ganglionic neurons were predominantly small in size and were evenly scattered throughout particular ganglia. Immunoreactivity to CART was also detected in numerous nerve terminals (which frequently formed pericellular formations around CART-negative perikarya) as well as in numerous nerve fibres within nerve branches interconnecting the unilateral pelvic ganglia. Immunohistochemistry revealed that virtually all CART-IR neurons were cholinergic in nature and CART-IR basket-like formations frequently encircled TH-positive/CART-negative perikarya. None of CART-IR ganglionic neurons showed immunoreactivity to SP, PACAP, 5-HT or calbindin. CART-IR nerve fibres ran in a close vicinity to serotonin-containing cells or faintly labelled SP-expressing neurons. On the other hand, PACAP-IR, SP-IR (but not 5-HT-positive) nerve terminals were found to run in close proximity to CART-IR neurons. Our results indicate that: 1) CART present in PP may influence the activity of pelvic ganglionic neurons/SIF cells, 2) PP should be considered as a potential source of CART-like supply to pelvic viscera and 3) functional interactions between CART and SP or PACAP are possible at the periphery.

Review: Exogenous butyrate: implications for the functional development of ruminal epithelium and calf performance.

The importance of the use of exogenous butyrate in calves' diets is due to its role as a factor stimulating the functional development of ruminal epithelium and improving calf performance during the transition from preruminant to ruminant status. Our review will first present results related to effects of the administration of butyrate in calves' diets on the development of ruminal epithelium toward a more effective absorption and metabolism of fermentation products from the rumen. The introduction of sodium butyrate at a level of about 0.3% of diet dry matter is accompanied by an increase to 35% in butyrate concentration in the rumen of 33-day-old calves. Mutual reliance between an enhanced ruminal concentration of butyrate and the activities of transcription factors, genes and proteins involved in cell proliferation, ketogenesis and the maintenance of cell pH homeostasis in the ruminal epithelial cells has been clearly confirmed in many experiments. Second, the review presents results related to the effects of the introduction of butyrate salts in the diet on calf performance. Of 11 studies a positive effect was found in six; five of these were obtained from the calves that started receiving butyrate supplement at a level of about 0.3% diet dry matter from the age of 3 to 5 days. Results indicate that when a supplement is given to calves soon after birth the functional development of ruminal epithelium in cooperation with the endocrine and digestion systems is transferred into improving the efficiency of rearing. There have been no studies on the effects of greater amounts of butyrate salts in milk replacer; butyrate constitutes about 1.2% of the whole cow's milk dry matter. In older calves, when butyrate administration is provided as a component of a starter concentrate at the increasing inclusion rate from 0.3% to 3.0%, the practical effect in calf performance relates to the risk of depression of rumen pH below 5.5 and accompanying disruption of the organization of the ruminal epithelial tissue. The higher risk is noted in calves received starter with substantial content of a rapidly degradable starch. At present, the insufficient number of positive results confirming the beneficial effect of butyrate supplements in terms of an improvement in performance does not allow their recommendation for use in the practical feeding of calves.

Galanin is Co-Expressed with Substance P, Calbindin and Corticotropin-Releasing Factor (CRF) in The Enteric Nervous System of the Wild Boar (Sus scrofa) Small Intestine.

Galanin is a neuropeptide widely present in the enteric nervous system of numerous animal species and exhibiting neurotransmittery/neuromodulatory roles. Colocalization patterns of galanin with substance P (SP), corticotropin-releasing factor (CRF) and calbindin were studied in the small intestine of the wild boar using immunofluorescence technique. We demonstrated the presence of SP in substantial populations of galanin-immunoreactive (IR) submucous neurons. Additionally, different amounts of nerve fibres exhibiting simultaneous presence of galanin and SP were noted in the small intestinal smooth musculature, submucous ganglia, lamina muscularis mucosae and mucosa. In the wild boar duodenum, jejunum and ileum, the co-expression of galanin and calbindin was limited to minor populations of submucous neurons only. Single galanin-/CRF-IR nerve fibres were exclusively present in the duodenal and jejunal (but not ileal) mucosa. These results strongly suggest that galanin participates in neuronal control of the wild boar small intestine also by functional co-operation with other biologically active neuropeptides.

Pituitary adenylate cyclase-activating peptide-27 (PACAP-27) is co-stored with galanin, substance P and corticotropin releasing factor (CRF) in intrapancreatic ganglia of the sheep.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptide existing in two variant forms (of either 27 or 38 residues), widely present in numerous organs and evoking multiple effects both in the central and peripheral nervous systems. The present study was undertaken to evaluate the distribution pattern of PACAP-27 expression in the ovine pancreas. Using double immunohistochemical stainings co-localizations of PACAP-27 with galanin, SP or CRF were studied in intrapancreatic neurons. In intrapancreatic ganglia, immunoreactivty to PACAP-27 was found in 87.6 ± 5.4% of PGP 9.5-positive intrapancreatic neurons but not in intraganglionic nerve fibres. Numerous PACAP-27-immunoreactive nerve terminals were also observed between pancreatic acini and around small arterioles. No immunoreactivity to PACAP-27 was found in the endocrine pancreas. In 42.9 ± 6.2% of PACAP-27-immunoreactive intrapancreatic neurons the expression of galanin was also found. Statistically lower subpopulation (12.4 ± 4.0%) of intrapancreatic neurons exhibited simultaneously the immunoreactivity to PACAP-27 and SP. The expression of CRF was detected in the relatively smallest group (3.2 ± 1.4%) of PACAP-27-positive intrapancreatic neurons. The present results suggest that in the ovine pancreas PACAP-27 may play an important role as mediator of pancreatic functions. In PACAP-related pancreatic activities, a modulatory role of galanin, SP and to a lower extend of CRF is also likely.

Immunocytochemical detection of calretinin in the claustrum and endopiriform nucleus of the chinchilla.

Calretinin (CR) as a buffer and sensor protein plays an important role in regulatory processes of Ca²âº and anty-apoptotic cellular protection. In the present study, immunohistochemical peroxidase-antiperoxidase (PAP) method was used in order to determine the numbers, morphology, morphometry and distribution pattern of CR in neurons of the chinchilla's claustrum (Cl) and endopiriform nucleus (EN). In Cl and EN the presence of several classes of neurons with different immunoreactivity to CR was found. In Cl, CR-immunoreactive (IR) neurons were predominantly found in close vicinity to insular border while CR-IR neurons were evenly scattered throughout EN. In general, immunoreaction to CR was observed in neuronal cytoplasm, nucleus and in fibres-like nerve extensions. Statistical analysis showed the differences between average large diameter as well as cross-sectional area of CR-IR neurons present in Cl and EN. It is suggested, that CR acting as a calcium binding protein may play a role in neuronal network. Further co-localization studies are necessary to fully elucidate neurophysiology and neuropathology of the chinchilla's Cl and EN neurons.

Immunoreactivity to cocaine- and amphetamine-regulated transcript in the enteric nervous system of the pig and wild boar stomach.

Cocaine- and amphetamine-regulated transcript (CART) is a recently discovered peptide inducing strong anxiogenic-like effect. CART distribution and its role(s) at periphery are not well understood. Immunohistochemisty was utilized to investigate the distribution patterns of CART in the stomach of the pig and wild boar. Double immunohistochemisty was applied to elucidate whether CART-immunoreactive (IR) neuronal elements coexpress galanin, substance P (SP) and neuropeptide Y (NPY). In the pig stomach, different proportions of CART-IR myenteric neurons were found in the antrum (42.3 ± 3.5%), corpus (18.0 ± 1.9%) and pylorus (33.2 ± 3.0%). CART-IR myeneric neurons were also found in the antrum, corpus and pylorus of the wild boar stomach (41.7 ± 3.2, 36.0 ± 2.2 and 35.8 ± 3.5%; respectively). In both species, none of gastric submucous neurons were CART-IR; however, CART-IR nerve fibres encircled submucous perikarya. In all portions of the pig and wild boar stomach, CART-IR nerve fibres were frequently found in the smooth muscle layer as well as in the lamina muscularis mucosae. In all regions of the pig and wild boar stomach, the expression of galanin and SP was found in CART-IR myenteric neurons and smooth muscle-supplying nerve fibres. CART/NPY coexpression was not found in the porcine stomach; however, in different regions of the wild boar stomach, subpopulations of CART-IR/NPY-IR myenteric neurons were noted. In conclusion, in this study, the existence and distribution patterns of CART in discrete regions of the pig and wild boar stomach were described in details. Colocalization studies revealed that in both animal species, a functional cooperation of CART with several neuropeptides is likely.

Changes in expression of calbindin 28 kDa in the small intestine of red kidney bean (Phaseolus vulgaris) lectin-treated suckling piglets.

Calbindin (CB) is a calcium binding protein playing a role in calcium uptake and anti-apoptotic cellular protection. The expression of CB was immunohistochemically studied in the small intestine of normal and red bean kidney lectin-treated suckling piglets. In the duodenum and jejunum (but not ileum) of lectin-treated animals overexpression of CB was noted in chromogranin A-immunoreactive (CgA-IR) neuroendocrine (NE) cells. In both control and experimental group a small population of CB-IR NE cells exhibited the presence of somatostatin (but not serotonin, histamine or CRF). After the lectin treatment, an increased (however not statistically significant) immunoreactivity to CB was found in a small subpopulation of neurons of outer submucous (but not inner submucous and myenteric) plexus. It is suggested that there is a functional interaction between lectin administration and CB-expression in the porcine small intestine. Future studies will be needed to clarify this processes.

Neurochemical properties of aquaporin 1-expressing sensory neurons from the ovine trigeminal ganglion.

Aims of the present study were to investigate the distribution and morphology of aquaporin 1-immunoreactive (AQP1-IR) neurons in the sensory ganglia of the sheep. Double immunohistochemical staining was applied to figure out whether substance P (SP), calcitonin gene-related peptide (CGRP) and galanin are present in AQP1-bearing primary afferent neurons. The expression of AQP1 was present only in trigeminal ganglion, whereas in nodose ganglion, jugular ganglion as well as C(1) -C(7) dorsal root ganglia no presence of AQP1 was found. In trigeminal ganglion, 15.4 ± 2.3% of Hu C/D-IR neurons (pan-neuronal marker) showed the presence of AQP1. The vast majority of AQP1-IR trigeminal sensory neurons (approximately 69.6 ± 3.3%, n = 5) were classified as middle in size, 28.6 ± 3.0% of AQP1-IR neurons were small and only 1.8 ± 0.6% of AQP1-positive neurons were large in size. Amongst the population of AQP1-IR trigeminal neurons as many as 58.5 ± 3.9% were immunopositive to SP, 30.7 ± 2.3% showed the presence of CGRP and 10.9 ± 0.2% coexpressed galanin. In trigeminal ganglion, SP-IR as well as CGRP-IR (but not galanin-IR) nerve fibres were found in close neighbourhood of AQP1-IR neurons. It is concluded that AQP1 is present in certain neuronal subsets of the ovine trigeminal ganglion; however, the exact role of this water channel has to be elucidated.

Distribution and chemical coding of calretinin- and calbindin-expressing enteric neurons in the duodenum of the sheep.

Recent decades has brought significant advances in our knowledge of the chemical coding and function of enteric neurons. Calcium ions are important second messenger involved in many aspects of neuron physiology. In the present study, we analyzed immunohistochemically the presence of calcium binding proteins (calretinin and calbindin) in various subpopulations of enteric neurons from the ovine duodenum. Ten percent of submucous neurons were immunoreactive (IR) to calretinin. The presence of calretinin was not detected in myenteric neurons. Calretinin-expressing nerve fibres were found in both myenteric and submucous ganglia, between the circular and longitudinal smooth muscle layers and in the lamina muscularis mucosae. Calretinin-IR submucous neurons did not exhibit the presence of SP, NPY and VIP. Co-localization of calretinin and serotonin was found only in a small number of submucous neurons. Calbindin was expressed in 35% of myenteric neurons and in 60% of submucous neurons. Nerve fibres containing calbindin were localized in myenteric and submucous ganglia where they frequently formed basket-like formations. Calbindin-positive nerve fibres emerging from myenteric ganglia ran between the circular and longitudinal smooth muscle layers. Immunoreactivity to calbindin was also visualized in the lamina muscularis mucosae, around mucosal glands and blood vessels. None of calbindin-IR myenteric neurons revealed immunoreactivity to SP, NPY, VIP and serotonin. Virtually all calbindin-expressing submucous neurons were SP-positive. In moderate numbers of submucous perikarya, co-incidence of calbindin and NPY, calbindin and VIP or calbindin and serotonin was observed. We conclude that in the ovine duodenum, the expression of calretinin and calbindin is species specific. Co-localization studies and distribution patterns indicate that in the duodenum of the sheep, calretinin and calbindin may be present in several functional subclasses of enteric neurons.

Immunodetection of cocaine- and amphetamine-regulated transcript in the rumen, reticulum, omasum and abomasum of the sheep.

Enteric nerves harbour a wide array of neuropeptides playing a key role in the regulation of gastrointestinal tract functions. In this study, the distribution patterns of cocaine- and amphetamine-regulated transcript-immunoreactive (CART-IR) nerve fibres as well as the percentages of CART-positive enteric neurons were immunohistochemically assessed in the rumen, reticulum, omasum and abomasum of the sheep. Double staining were applied, to elucidate whether neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), substance P (SP), somatostatin or serotonin co-exist in CART-IR gastric structures. In the rumen, reticulum, omasum and abomasum, a majority of myenteric neurons identified by immunoreactivity to Hu C/D were CART-positive (47.1 +/- 3.6%, 45.1 +/- 3.0%, 41.6 +/- 2.6% and 40.9 +/- 2.9% respectively). The smooth musculature of the forestomachs as well as abomasum was innervated with numerous CART-IR nerve fibres. Blood vessels-associated CART-positive nerve terminals were identified in the submucosa of the reticulum only. Lamina muscularis mucosae of the omasum and abomasum was moderately innervated with CART-IR nerve terminals. In the abomasum sparse CART-IR nerve fibres were seen between mucosal glands. A small population of endocrine cells of the abomasum also exhibited the presence of CART. All neuronal elements as well as endocrine cells IR to CART were negative to somatostatin and/or serotonin. No immunoreactivity to VIP, NPY and/or SP was found in myenteric ganglia-projecting CART-positive nerve fibres. The co-localization of CART with VIP, NPY and/or SP was regularly observed in myenteric neurons as well as the smooth muscle layer- and lamina muscularis mucosae-projecting nerve fibres. CART-IR nerve terminals located between mucosal glands of the abomasum frequently co-stored VIP, NPY and/or SP. Although the exact function of CART in the ovine forestomachs/stomach has to be elucidated, several potential functions (like enteric nerves protection) have been suggested.