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Alpha-synuclein (α-syn) is a presynaptic neuronal protein and its structural alterations play an important role in the pathogenesis of neurodegenerative diseases, such as Parkinson's disease (PD). It has been originally described in the brain and aggregated α-syn has also been found in the peripheral nerves including the enteric nervous system (ENS) of PD patients. ENS is a network of neurons and glia found in the gut wall which controls gastrointestinal function independently from the central nervous system. Moreover, two types of epithelial cells are crucial in the creation of an interface between the lumen and the ENS: they are the tuft cells and the enteroendocrine cells (EECs). In addition, the abundant enteric glial cells (EGCs) in the intestinal mucosa play a key role in controlling the intestinal epithelial barrier. Our aim was to localize and characterize the presence of α-syn in the normal human jejunal wall. Surgical specimens of proximal jejunum were collected from patients submitted to pancreaticoduodenectomy and intestinal sections underwent immunohistochemical procedure. Alpha-syn has been found both at the level of ENS and the epithelial cells. To characterize α-syn immunoreactive epithelial cells, we used markers such as choline acetyltransferase (ChAT), useful for the identification of tuft cells. Then we evaluated the co-presence of α-syn with serotonin (5-HT), expressed in EECs. Finally, we used the low-affinity nerve growth factor receptor (p75NTR), to detect peripheral EGCs. The presence of α-syn has been demonstrated in EECs, but not in the tuft cells. Additionally, p75NTR has been highlighted in EECs of the mucosal layer and co-localized with α-syn in EECs but not with ChAT-positive cells. These findings suggest that α-syn could play a possible role in synaptic transmission of the ENS and may contribute to maintain the integrity of the epithelial barrier of the small intestine through EECs.

Chemoprevention is referred to as a strategy to inhibit, suppress, or reverse tumor development and progression in healthy people along with high-risk subjects and oncologic patients through using pharmacological or natural substances. Numerous phytochemicals have been widely described in the literature to possess chemopreventive properties, although their clinical usefulness remains to be defined. Among them, caryophyllane sesquiterpenes are natural compounds widely occurring in nature kingdoms, especially in plants, fungi, and marine environments. Several structures, characterized by a common caryophyllane skeleton with further rearrangements, have been identified, but those isolated from plant essential oils, including β-caryophyllene, β-caryophyllene oxide, α-humulene, and isocaryophyllene, have attracted the greatest pharmacological attention. Emerging evidence has outlined a complex polypharmacological profile of caryophyllane sesquiterpenes characterized by blocking, suppressing, chemosensitizing, and cytoprotective properties, which suggests a possible usefulness of these natural substances in cancer chemoprevention for both preventive and adjuvant purposes. In the present review, the scientific knowledge about the chemopreventive properties of caryophyllane sesquiterpenes and the mechanisms involved have been collected and discussed; moreover, possible structure-activity relationships have been highlighted. Although further high-quality studies are required, the promising preclinical findings and the safe pharmacological profile encourage further studies to define a clinical usefulness of caryophyllane sesquiterpenes in primary, secondary, or tertiary chemoprevention.

Cholangiocarcinoma (CCA) is an aggressive group of biliary tract cancers, characterized by late diagnosis, low effective chemotherapies, multidrug resistance, and poor outcomes. In the attempt to identify new therapeutic strategies for CCA, we studied the antiproliferative activity of a combination between doxorubicin and the natural sesquiterpene β-caryophyllene in cholangiocarcinoma Mz-ChA-1 cells and nonmalignant H69 cholangiocytes, under both long-term and metronomic schedules. The modulation of STAT3 signaling, oxidative stress, DNA damage response, cell cycle progression and apoptosis was investigated as possible mechanisms of action. β-caryophyllene was able to synergize the cytotoxicity of low dose doxorubicin in Mz-ChA-1 cells, while producing cytoprotective effects in H69 cholangiocytes, mainly after a long-term exposure of 24 h. The mechanistic analysis highlighted that the sesquiterpene induced a cell cycle arrest in G2/M phase along with the doxorubicin-induced accumulation in S phase, reduced the γH2AX and GSH levels without affecting GSSG. ROS amount was partly lowered by the combination in Mz-ChA-1 cells, while increased in H69 cells. A lowered expression of doxorubicin-induced STAT3 activation was found in the presence of β-caryophyllene in both cancer and normal cholangiocytes. These networking effects resulted in an increased apoptosis rate in Mz-ChA-1 cells, despite a lowering in H69 cholangiocytes. This evidence highlighted a possible role of STAT3 as a final effector of a complex network regulated by β-caryophyllene, which leads to an enhanced doxorubicin-sensitivity of cholangiocarcinoma cells and a lowered chemotherapy toxicity in nonmalignant cholangiocytes, thus strengthening the interest for this natural sesquiterpene as a dual-acting chemosensitizing and chemopreventive agent.

Doxorubicin represents a valuable choice for different cancers, although the severe side effects occurring at the high effective dose limits its clinical use. In the present study, potential strategies to potentiate low-dose doxorubicin efficacy, including a metronomic schedule, characterized by a short and repeated exposure to the anticancer drug, and the combination with the natural chemosensitizing sesquiterpenes β -caryophyllene and β -caryophyllene oxide, were assessed in human hepatoma HepG2 cells. The involvement of P-glycoprotein (P-gp) in the HepG2-chemosensitization to doxorubicin was evaluated. Also, the direct interaction of caryophyllene sesquiterpenes with P-gp was characterized by molecular docking and dynamic simulation studies. A metronomic schedule allowed us to enhance the low-dose doxorubicin cytotoxicity and the combination with caryophyllane sesquiterpenes further potentiated this effect. Also, an increased intracellular accumulation of doxorubicin and rhodamine 123 induced by caryophyllane sesquiterpenes was found, thus suggesting their interference with P-gp function. A lowered expression of P-gp induced by the combinations, with respect to doxorubicin alone, was observed too. Docking studies found that the binding site of caryophyllane sesquiterpene was next to the ATP binding domain of P-gp and that β -caryophyllene possessed the stronger binding affinity and higher inhibition potential calculated by MM-PBSA. Present findings strengthen our hypothesis about the potential chemosensitizing power of caryophyllane sesquiterpenes and suggest that combining a chemosensitizer and a metronomic schedule can represent a suitable strategy to overcome drawbacks of doxorubicin chemotherapy while exploiting its powerful activity., Competing Interests: The authors declare no conflict of interest.

Cholangiocarcinoma (CCA) is an aggressive biliary tract malignancy with limited treatment options and low survival rates. The intrahepatic subtype comprises two forms: mucin-iCCA and mixed-iCCA. Epidermal growth factor-like domain multiple (EGFL7) is overexpressed in less differentiated liver tumors. The aim of this study was to assess the presence of EGFL7 due to its possible role in the growth of CCA. Hematoxylin and Eosin and periodic acid-Schiff staining were used to evaluate the morphological aspects and glycogen deposition. Immunohistochemistry and immunofluorescence were performed to identify the presence of EGFL7 both in tumor sections ex vivo and in appropriate cell lines in culture. We found that EGFL7 is expressed in malignant cholangiocytes of mixed-iCCA and absent in mucin-iCCA. In conclusion the expression of EGFL7 might be useful in the classification of CCA subtypes.

Influenza virus replicates intracellularly exploiting several pathways involved in the regulation of host responses. The outcome and the severity of the infection are thus strongly conditioned by multiple host factors, including age, sex, metabolic, and redox conditions of the target cells. Hormones are also important determinants of host immune responses to influenza and are recently proposed in the prophylaxis and treatment. This study shows that female mice are less susceptible than males to mouse-adapted influenza virus (A/PR8/H1N1). Compared with males, PR8-infected females display higher survival rate (+36%), milder clinical disease, and less weight loss. They also have milder histopathological signs, especially free alveolar area is higher than that in males, even if pro-inflammatory cytokine production shows slight differences between sexes; hormone levels, moreover, do not vary significantly with infection in our model. Importantly, viral loads (both in terms of viral M1 RNA copies and tissue culture infectious dose 50%) are lower in PR8-infected females. An analysis of the mechanisms contributing to sex disparities observed during infection reveals that the female animals have higher total antioxidant power in serum and their lungs are characterized by increase in (i) the content and biosynthesis of glutathione, (ii) the expression and activity of antioxidant enzymes (peroxiredoxin 1, catalase, and glutathione peroxidase), and (iii) the expression of the anti-apoptotic protein Bcl-2. By contrast, infected males are characterized by high expression of NADPH oxidase 4 oxidase and phosphorylation of p38 MAPK, both enzymes promoting viral replication. All these factors are critical for cell homeostasis and susceptibility to infection. Reappraisal of the importance of the host cell redox state and sex-related effects may be useful in the attempt to develop more tailored therapeutic interventions in the fight against influenza.

Caralluma fimbriata Wall. is currently used as a "natural slimming" food supplement, likely due to its content in pregnane glycosides. In the present study, a commercially available Caralluma fimbriata extract (Slimaluma ® ; CFE, 100 mg/kg) has been evaluated for its ability to affect the ingestive behaviour in female rats, also with reference to the modulation of the brain neuropeptides NPY and ORX.The interference of CFE with α-amylase and lipase enzymes has been investigated in vitro, as possible peripheral mechanism of action. Also, the chemical composition of CFE has been assessed by NMR and spectrophotometric analysis. Results from in vivo study showed that CFE induced effects neither on blood parameters, nor on liver and gut histomorphology. Interestingly, a reduction in body weight gain with an increase in water intake and hypothalamic levels of NPY and ORX peptides were found. Phytochemical analysis, showed CFE contained about 12% of pregnane glycosides and 1.3% of polyphenols. Present results suggest possible effects of C. fimbriata on ingestive behaviour, likely mediated by central and peripheral mechanisms., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

GSK-3 is a ubiquitously expressed serine/threonine kinase existing as GSK-3 α and GSK-3 β isoforms, both active under basal conditions and inactivated upon phosphorylation by different upstream kinases. Initially discovered as a regulator of glycogen synthesis, GSK-3 is also involved in several signaling pathways controlling many different key functions. Here, we discuss recent advances regarding (i) GSK-3 structure, function, regulation, and involvement in several cancers, including hepatocarcinoma, cholangiocarcinoma, breast cancer, prostate cancer, leukemia, and melanoma (active GSK-3 has been shown to induce apoptosis in some cases or inhibit apoptosis in other cases and to induce cancer progression or inhibit tumor cell proliferation, suggesting that different GSK-3 modulators may address different specific targets); (ii) GSK-3 involvement in autophagy modulation, reviewing signaling pathways involved in neurodegenerative and liver diseases; (iii) GSK-3 role in oxidative stress and autophagic cell death, focusing on liver injury; (iv) GSK-3 as a possible therapeutic target of natural substances and synthetic inhibitors in many diseases; and (v) GSK-3 role as modulator of mammalian aging, related to metabolic alterations characterizing senescent cells and age-related diseases. Studies summarized here underline the GSK-3 multifaceted role and indicate such kinase as a molecular target in different pathologies, including diseases associated with autophagy dysregulation.

The maternal separation protocol in rodents is a widely recognized model of early life stress allowing acute and chronic physiological consequences to be studied. An (1)H NMR-based metabolomic approach was applied to urines to evaluate the systemic metabolic consequences of maternal separation stress in female rats after the beginning of weaning and 4 weeks later when the rats were reaching adulthood. Furthermore, because maternal separation is considered as a model mimicking the inflammatory bowel syndrome, the lactulose/mannitol test was used to evaluate the influence of postnatal maternal separation on gut permeability and mucosal barrier function by (1)H NMR spectroscopy analysis of urine. The results showed no statistical differences in gut permeability due to maternal separation. The application of ANOVA simultaneous component analysis allowed the contributions of physiological adaptations to the animal's development to be separated from the metabolic consequences due to postnatal stress. Systemic metabolic differences in the maternally separated pups were mainly due to the tryptophan/NAD pathway intermediate levels and to the methyladenosine level. Urinary NMR-based metabolic profiling allowed us to disentangle the metabolic adaptive response of the rats to postnatal stress during the animal's growth, highlighting the metabolic changes induced by weaning, gut closure, and maturity.

Conclusion: The results of the present study reject the hypothesis that epithelial inclusions into the ossicles could cause cholesteatoma recurrences, but strongly suggest the performance of a safe cleaning procedure for ossicular remnants to make them usable in ossiculoplasty in patients with partially or non-encapsulated cholesteatoma., Objective: The aim of the study was to define, before any sort of cleaning procedure, if there is any epithelial inclusion inside the ossicles of patients with cholesteatoma and if the findings could be correlated with surgical aspect of cholesteatoma., Methods: The specimens used for this study comprised 19 mallei and 15 incudes, which were obtained intraoperatively from 24 patients. Each ossicle was grouped on the basis of the intraoperative aspect of the cholesteatoma as follows. Grade 1: 10 ossicles obtained from encapsulated cholestatoma, non-invasive, easily cleavable. Grade 2: 14 ossicles obtained from partially encapsulated cholesteatoma, non-invasive, not easily cleavable. Grade 3: 10 ossicles obtained from non-encapsulated cholesteatoma, invasive, not cleavable. Two stapes and one malleus were taken from patients who underwent middle ear surgery for conductive hearing loss and were used as controls. The ossicles were examined histopathologically after removal., Results: Our results do not show any epithelial inclusion inside the ossicles independently from the macroscopic aspect or growing aggressiveness of cholesteatoma. In addition there was no infiltration of inflammatory cells in grade 1, but it was present in one incus (7.1%) of grade 2 and in five ossicles (50%) of grade 3. In ossicles of grade 3 up to four layers of epithelial cells were found on the surface of the ossicles.

Aim: The present study assessed the ability of Chelidonium majus to potentiate the hepatic effect of a sub-toxic dose of acetaminophen, in rats., Results: C. majus, when administered alone, did not alter the liver function parameters in male, whereas an increase in fibrinogen level was found in female rats. Moreover, it did not affect the hepatic histomorphology in both male and female rats. The sub-toxic dose of acetaminophen induced: a significant increase in activated partial thromboplastin time in both genders, a focal hepatocellular necrosis with minor lymphocytes infiltrate and a slight but significant increase in total bilirubin, AST, and ALT in male rats, and in prothrombin time in female rats. The co-administration of C. majus did not increase the effects induced by acetaminophen, in both genders., Conclusions: C. majus does not modify the hepatic effects of acetaminophen in our in vivo experimental model., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

There is a general consensus that liver fibrosis in humans is potentially reversible, while scepticism prevails on the concept that cirrhosis can be truly reversed. The availability of suitable experimental models is fundamental for disease research. The experimental murine model of liver cirrhosis induced by carbon tetrachloride (CCl(4)) reproduces both the histological picture of the postnecrotic cirrhosis and its biochemical and clinical parameters. Normal hepatic structure is modified by formation of regeneration nodules. Fibrosis represents a morphological element of disease and an effect of hepatocyte necrosis. However, the relevance for research of this well-established model of liver cirrhosis is hampered by some spontaneous cirrhosis regression reported in mice and rats. It has been reported that CCl(4) also induces experimental liver cirrhosis in rabbits, but it is not known whether the process is reversible in this species. The aim of our study was to investigate this question. Male New Zealand White rabbits were treated intragastrically with CCl(4) or the vehicle only for 19 weeks and groups were sacrificed three and five months after treatment interruption. Cirrhotic and control livers were processed for routine light microscopy and for morphometric study of fibrosis by semiquantitative evaluation. The degree of fibrosis was based on the Knodell's scoring system.

Aim: The aim of this study was to determine whether GSH-C4, a hydrophobic glutathione derivative, affects in vitro and in vivo influenza virus infection by interfering with redox-sensitive intracellular pathways involved in the maturation of viral hemagglutinin (HA)., Results: GSH-C4 strongly inhibited influenza A virus replication in cultured cells and in lethally infected mice, where it also reduced lung damage and mortality. In cell-culture studies, GSH-C4 arrested viral HA folding; the disulfide-rich glycoprotein remained in the endoplasmic reticulum as a reduced monomer instead of undergoing oligomerization and cell plasma-membrane insertion. HA maturation depends on the host-cell oxidoreductase, protein disulfide isomerase (PDI), whose activity in infected cells is probably facilitated by virus-induced glutathione depletion. By correcting this deficit, GSH-C4 increased levels of reduced PDI and inhibited essential disulfide bond formation in HA. Host-cell glycoprotein expression in uninfected cells was unaffected by glutathione, which thus appears to act exclusively on glutathione-depleted cells., Innovation: All currently approved anti-influenza drugs target essential viral structures, and their efficacy is limited by toxicity and by the almost inevitable selection of drug-resistant viral mutants. GSH-C4 inhibits influenza virus replication by modulating redox-sensitive pathways in infected cells, without producing toxicity in uninfected cells or animals. Novel anti-influenza drugs that target intracellular pathways essential for viral replication ("cell-based approach") offer two important potential advantages: they are more difficult for the virus to adapt to and their efficacy should not be dependent on virus type, strain, or antigenic properties., Conclusion: Redox-sensitive host-cell pathways exploited for viral replication are promising targets for effective anti-influenza strategies.

Background: Cassia angustifolia L. (senna) is traditionally used as a laxative. Its major components are sennosides that are responsible for the laxative effect. Senna is recommended for the short-term treatment of acute constipation. Nevertheless people use its preparations as self-medication, often for long periods, to treat chronic constipation thus exposing themselves to adverse reactions. Most reactions were associated with hepatotoxicity., Aims: The present study was aimed to evaluate the toxicity of a C. angustifolia leafextract (standardized at 60% of sennosides) on rat liver cells and the long-term effects on liver functions, in Wistar rats., Methods: Cytotoxicity was assessed in a buffalo normal rat liver cell line (BRL-3A) by the trypan blue assay and the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide reduction test. In vivo effects were observed after oral administration of the extract for 4 or 8 weeks at doses of 12 and 58 mg/kg/day. At the end of treatment, animals were sacrificed, the postmortem examination was performed and serum was used for biochemical analysis. Liver samples were used for histomorphological and immunohistochemical examination along with the determination of oxidative stress parameters., Results and Conclusion: In BRL-3A cells, the extract was cytotoxic at concentrations that appear largely higher than those attainable in humans. In Wistar rats, the extract did not induce any significant change in all of the parameters tested. In summary, the present study indicates a lack of hepatotoxicity of senna at doses higher than those generally used in humans., (Copyright © 2011 S. Karger AG, Basel.)

Aim of the Study: Aerial parts of Chelidonium majus L. (Papaveraceae family) are traditionally used in the treatment of gallstones and dyspepsia, however several cases of hepatotoxicity are reported. In this work we evaluated the effects on liver function of a C. majus extract, obtained from the herbal material responsible for one case of hepatotoxicity., Materials and Methods: Experiments were performed in Wistar rats, after oral administration of doses corresponding to 1.5 and 3g/(kg day) of herbal drug, for 2 or 4 weeks. Blood samples were collected to perform biochemical analysis, whereas liver samples were used for histomorphological and immunohistochemical examination along with the determination of oxidative stress parameters., Results: No significant modification in animal body weight, food consumption, enzyme activities, hepatic histomorphology and MDA formation, at either time or dosage level. Conversely, C. majus induced a slight but significant decrease of GSH levels and SOD activity, especially at the high dose., Conclusions: Our study suggests that C. majus, at doses about 50 and 100 times higher than those generally used in humans, does not alter hepatic function. However, the reduction in GSH levels and SOD activity suggests particular attention in use of C. majus or its preparations in situations (pharmacological treatments, physio-pathological conditions, etc.) that can compromise liver function.

Unlabelled: Cimicifuga racemosa (black cohosh) is a herbaceous perennial plant, that has been traditionally used for a variety of ailments (dyspepsia, climacteric complaints, muscular rheumatisms, menstrual cramps). From laboratory and clinical studies, black cohosh seems to have a relatively good safety profile, even if a number of case reports of hepatotoxicity were a matter of recent concern., Aim: A number of case reports indicated that C. racemosa could induce hepatotoxicity. We evaluated the effects of black cohosh extract on liver morphology, and on levels of various hepatic function indices in rats., Methods: Wistar rats received 300mg/kg/day of C. racemosa extract by gavage, for 30 days. Biochemical analysis of serum was conducted by an automated, random-access clinical chemistry analyzer. Liver samples were used for hystomorphological and immunohistochemical examination, for the detection of apoptosis (TUNEL assay), and for the determination of GSH level (spectrophotometrical analysis)., Results: C. racemosa extract does not affect liver morphology and hepatic function indices, in rats., Conclusions: On the basis of experimental data, the use of 300mg/kg/day of black cohosh appears quite safe in rats. Nevertheless, in humans the safety of C. racemosa should be further monitored, in terms of patient-related factors.

The principal aims of this study have been to elucidate the nature of glycoconjugates produced by the two distinct parts of bovine Brunner glands, peripheral and central areas of lobules, and to investigate the presence of sialyl acid residues. Bovine duodenal tissues, embedded in paraffin wax, were investigated by means of both conventional histochemical methods (PAS, AB, HID) and biotinylated lectins (Con A, DBA, SBA, GS-I-B4, PNA, sWGA, GS-II, UEA-I, LPA, LFA). Conventional histochemical methods allowed us to accurately define two different areas: a central and a peripheral area. The central area, composed of secretory tubular tracts and the excretory duct, contained neutral glycoconjugates. The peripheral area was formed by both terminal alveolar and tubular secretory tracts and contained both neutral and acidic glycoconjugates, the latter partly carboxylated and partly sulfated. Lectin histochemistry confirmed differences highlighted by conventional histochemical methods and allowed us to characterise glycoprotein profiles of the preterminal and terminal tracts. The preterminal tracts and the excretory duct contained glycoconjugates with terminal D-Gal beta(1-3)GalNAc, alpha-D-Gal, alpha/beta-D-GalNAc, alpha/beta-D-GlcNAc, and internal beta(1-4) D-GlcNAc and alpha-Man residues. The terminal tracts were characterised by terminal alpha-L fucose, beta-D-GalNac, alpha/betaD-GlcNAc, alpha-D-Gal, alpha-D-GalNAc, and sialic acid residues. Internal beta(1-4) D-GlcNAc and alpha-Man residues were also identified. Finally, secretion of bovine Brunner glands is characterised by both O-linked and N-linked glycoproteins: cells located in the preterminal tracts and in the excretory duct produce mainly O-linked glycoproteins while cells located in the terminal tracts produce N-linked glycoproteins.

The occurrence of S100 proteins in neurons of the mammalian peripheral nervous system is still controversial. This study was designed to investigate this topic in dorsal root ganglia (DRG) and the enteric nervous system (ENS) of several mammalian species (horse, buffalo, cow, sheep, pig, dog, rabbit and rat), as well as in DRG, paravertebral sympathetic ganglia (SG) and ENS of the adult man. Rat embryos of E17 and E19 were also examined. The material was fixed in Bouin's fixative, paraffin-embedded and processed for immunohistochemistry, combined with image analysis, using a panel of mono and polyclonal antibodies against S100alpha, S100beta or S100alpha + beta (referred to here as S100) proteins. In all species examined, strong S100 protein immunoreactivity (IR) was found in satellite glial cells and Schwann cells, which also showed S100alpha and S100beta IR in humans. Furthermore, faint S100 protein IR was observed in a subpopulation of DRG intermediate- and large-sized sensory neurons in humans, buffalo, sheep, and pig. The rat was the only species showing clear S100 and S100beta in neurons, labelling in about 30-35% in adults (small, intermediate and large in size), and about 88% at E17 and 42% at E19, respectively. Weak S100alpha protein IR was observed in most of human SG neurons. In ENS, S100 protein IR was restricted to enteric glial and Schwann cells, with the exception of cow and goat in which a subset of neurons in both the myenteric and submucous plexuses displayed strong S100 protein IR. Neuronal S100alpha IR and glial S100beta IR was found in the human ENS. The present results demonstrate intra- and inter-specific differences in the expression of S100 proteins by neurons of the peripheral nervous system among mammalian species. Furthermore, they also suggest that neuronal S100 protein, at least in humans, consists of both S100alpha and S100beta.

Some morphometrical parameters of the axons making up the contralateral sciatic nerves, both in the male and female rabbits were calculated and compared by means of a Zeiss Vidas image analyser (Ober Kochen, Germany). The results show that the fibres constituting the left nerve have a greater mean diameter but a lower mean density that those constitution the right nerve. This suggests that the diameter of the myelinated fibres and the density of both the myelinated and unmyelinated fibres do not vary from male to female. On the other hand, the G ratio and the diameter of the unmyelinated axons do, since the nerves on the right side (in both sexes) have higher morphometric values, on average, than the contralateral ones.

The pharmacological properties and the anatomical localization of dopamine (DA) D2-like receptors were studied in the cerebellum of 3 months, 12 months, and 24 months male Wistar rats using combined radioligand binding and autoradiographic techniques with [3H]-spiroperidol as a ligand. The binding was consistent with the labelling of the DA D3 receptor subtype. The affinity for DA D2-like receptors was similar in the cerebellar cortex of the three animal groups investigated, whereas the density of binding sites (Bmax value) assessed using conventional radioligand binding techniques was reduced as a function of aging. Light microscope autoradiography revealed the localization of binding sites primarily in the molecular layer and to a lesser amount in Purkinje neurons layer. A loss of binding sites was noticeable with aging in the grey matter of the cerebellar cortex. It affected primarily the molecular layer. Analysis of radioligand binding data and light microscope autoradiography suggests that age-related changes of DA D2-like receptors depend in part by structural alterations of cerebellar cortex and in part by modifications in receptor expression.

The pharmacological properties and the anatomical localisation of dopamine D3 receptor were assessed in the rat cerebellar cortex using radioligand binding techniques associated with light microscope autoradiography and 7-[3H]hydroxy-N,N-di-n-propyl-2-aminotetralin (7-[3H]OH-DPAT) as a ligand. 7-[3H]OH-DPAT was specifically bound to sections of rat cerebellar cortex with a dissociation constant (Kd) of 0.5 nM and a maximum density of binding sites (Bmax) of 97 +/- 4 fmol/mg tissue. The rank order of potency of competitors of 7-[3H]OH-DPAT binding and the observation that guanosine triphosphate did not affect radioligand binding suggest the labelling of a dopamine D3 receptor. 7-[3H]OH-DPAT binding sites are located mainly in the molecular layer and in lesser amounts in the Purkinje neuron layer, primarily within the cell body of Purkinje neurons. No specific accumulation of silver grains was observed in the granule neuron layer or in the white matter of the cerebellar cortex. The localisation of a putative dopamine D3 receptor within Purkinje neurons suggests that this site may have functional relevance in the cerebellar cortex.

Age-dependent changes in the innervation of the pigeon (Columba livia, L.) bursa of Fabricius, from hatching to 120 days of age, were studied by fluorescence-histochemical and neurochemical methods for demonstrating noradrenergic and acetylcholinesterase (AChE)-reactive nerve fibres respectively. The distribution of both nerve fibre types was largely perivascular. Furthermore, a few isolated nerve fiber profiles were observed beneath the bursal epithelium, in the interfollicular septa and in the follicular cortex. No nerve fibre profiles reaching the medulla of the lymphoid follicles were observed. In addition to nerve fibres, AChE reactive neuron-like cells were encountered within the capsule and interfollicular septa. AChE reactivity was also found in dendritic-like cells localized in the cortical and cortico-medullary border. No changes in the density of perivascular noradrenergic innervation were noticeable during the ages studied, whereas the density of AChE-reactive fibres supplying vessels reached the adult pattern at 30 days, and then remained unvaried. The density of non-perivascular nerve fiber profiles, specially the AChE reactive type, increased until 30 days, remained unchanged until 75 days and then increased with aging (90-120 days). The interrelationship between the autonomic nervous system and the immune system is discussed.

The present study was undertaken to determine, by means of immunohistochemical techniques, image analysis and ultrastructural methods, whether the secretory-dendritic cells (SDC) of the pigeon bursa of Fabricius undergo changes from hatching to the involutive stage (120 days) of the organ. A monoclonal antibody against vimentin (VIM) was used to label SDC. VIM-like immunoreactivity (VIM-L IR) was observed labelling dendritic cell profiles in all age groups. These cells are primarily localized within the medulla and at the cortico-medullary border of the lymphoid follicles. At hatching VIM-L IR was present mainly in the cell bodies, whereas during post-hatching bursal growth (7 to 75 days) it was also present in the cell processes. Conversely, the involutive period examined (90-120 days) was characterized by a progressive decrease of VIM-L IR in the SDC processes. Quantitative studies confirmed the immunohistochemical findings. At the ultrastructural level, there was a progressive increase from 0 to 90 days of age in both the number and size of secretory granules and break-down bodies, as well as in the length of the SDC processes. The involutive stage showed the reverse phenomena. The present results demonstrate that the SDC of the pigeon bursa of Fabricius undergo age-related changes parallel with that of the organ. The possible involvement of SDC in the maintenance of the bursal microenvironment and their role in the maturation of lymphoid line cells is discussed.