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The immunohistochemical profile of neurotrophins and their receptors in the human cranial dura mater was studied by examining certain dural zones in specimens harvested from different regions (frontal, temporal, parietal and occipital). Dural specimens were obtained during neurosurgical operations performed in ten patients for surgical treatment of intracranial lesions (meningiomas, traumas, gliomas, vascular malformations). The dural fragments were taken from the area of the craniotomy at least 8 cm from the lesion as well as from the area in which the meningioma had its dural attachment. Immunohistochemical characterization and distribution of neurotrophins, with their receptors, were analyzed. The concrete role played by these neurotrophic factors in general regulation, vascular permeability, algic responsivity and release of locally active substances in the human dura mater is still controversial. Our study revealed a general structural alteration of dural tissue due to the invasivity of meningiomatous lesions, together with an improved expression of brain derived neurotrophic factor (BDNF) in highly proliferating neoplastic cells and an evident production of nerve growth factor (NGF) in inflammatory cells, suggesting that BDNF has a role in supporting the proliferation rate of neoplastic cells, while NGF is involved in the activation of a chronic inflammatory response in neoplastic areas.

Lymphoid organs are supplied by many nerve endings associated with different kinds of cells and macrophages. The role of this innervation on the release of locally active molecules is still unclear. Lingual tonsils belong to Waldeyer's Ring, in close association with palatine tonsils and nasopharyngeal (adenoids) tonsils, thus constituting part of NALT (nasal-associated lymphoid tissue) together with the tubal tonsils and lateral pharyngeal bands. In this study, we focused our attention on the expression of some neurotrophins (NTs) and their high- and low-affinity receptors in human lingual tonsils. Light immunohistochemistry showed that human tonsillar samples were generally positive for all the NTs investigated (NGF, BDNF, NT-3, NT-4) and their receptors (TrKA, TrKB, TrKC and p75) with some different expression levels. NGF and TrKC were strongly expressed in macrophages, but weakly in lymphocytes. However, BDNF and TrKB was highly expressed in lymphocytes and weaker in macrophages. The low-affinity receptor for NGF, p75, was mainly moderately expressed in the analysed samples. These results suggest the presence of a pattern of neurotrophin innervation in the human lingual tonsil which may play a role in sustaining inflammatory conditions and in modulating a close interaction between the nervous system and the different immune cellular subtypes.

Compared to the normal epidermal epithelium, cholesteatomas have altered growth properties characterized by the excessive growth of keratinocytes leading to mucosal destruction. Either congenital or acquired, these lesions, which grow in the middle ear space, the petrous apex or the mastoid of temporal bones, are mostly considered benign skin tumoral lesions. However, many questions remain concerning their pathophysiology. Numerous studies have been proposed to identify those cholesteatoma lesions at risk of recurrence, a possible event that may cause hearing loss. We examined patients with petrous apex or mastoid cholesteatoma in order to analyze the expression of various neurotransmitters, neurotrophins and their receptors and the Ki-67 antigen for identification of a possible relationship between clinical outcome and histopathological behaviour in terms of the proliferative activity of cholesteatomas. Expression of the analyzed molecules was studied using immunohistochemical methods in seven adult patients with petrous apex cholesteatoma who underwent surgical removal of the lesion. Our results, in accordance with published data, confirm that Molecular Immunology Borstel-1 (MIB-1) and certain neurotransmitters could be useful in the prognostic evaluation of the risk of recurrence of aggressive forms of cholesteatoma.

Background and Aim of the Study: Idiopathic interstitial pneumoniae (IIPs) are characterized by fibroblast proliferation, extracellular matrix deposition and progressive lung function impairment. Because effective therapeutic strategies still remain limited, research has been directed toward the identification of novel targets for additional therapeutic options. The neurotrophins (NTs) nerve growth factor (NGF), brain derived neurotrophic factor (BDNF) and NT-3, beside their importance in nervous, endocrine and immune system activities, participate in chronic inflammatory disorders and in repair processes., Methods: We have investigated NT and high and low affinity NT receptor expression in IIPs using immunoblots and immunohistochemistry. Fourteen idiopatic pulmonary fibrosis/usual interstitial pneumoniae (IPF/UIP), eight non specific pneumoniae (NSIP) and eight respiratory bronchiolitis-associated interstitial lung disease (RB-ILD) were analyzed., Results: Immunoblots revealed that NT and high affinity NT receptor proteins were more abundantly expressed in IPF/UIP than NSIP and RB-ILD patients. In RB-ILD, a faint expression of NT-3 and NT receptors were detected. NT and NT receptor immunostaining was detected in interstitial cells from IPF/UIP, NSIP and RB-ILD patients by immunohistochemistry. Fibroblastic foci in IPF/UIP strongly stained for BDNF and its high affinity receptor TrkB and in lesser amount for NGF, NT-3 and their respective high affinity receptors TrkA and TrkC. Furthermore, in fibroblast culture derived from IPF/UIP patients, the proliferation rate of primary culture and clones derived from primary lines was stimulated by BDNF but down regulated by NT-3. In contrast, NGF did not influence IPF/UIP fibroblasts proliferation., Conclusions: Our data suggest that that NTs may exert differential activities on lung fibroblasts and may be considered as potential regulatory molecules influencing fibroblast behavior in IPF/UIP patients. Therefore, NTs may play a role in IIPs patho-physiology representing novel potential therapeutic targets.

Lonidamine (LND) or [1-(2,4-dichlorobenzyl)-1H-indazole-3-carboxylic acid] is an anticancer and antispermatogenic drug that exerts a large number of effects on tumor cells and germ cells. Sexually mature male Sprague-Dawley rats were housed at 22 degrees C on a 12-h light/12-h dark cycle 1 week before the experiments, with free access to food and water. LND was suspended in 0.5% methylcellulose at a concentration of 10 mg/mL and administered orally at the dose of 10 mL/kg (b.w.) as a single dose. Control rats received an equal amount of vehicle. Testes were removed, fixed for 24 h in 2% glutaraldehyde and 2% paraformaldehyde in 0.1 M sodium phosphate (pH 7.2 at 22 degrees C), rinsed with the same buffer, and stored at room temperature. From each sample, a block of tissue was removed by sectioning through the organ. After dehydration in ethanol at increasing concentrations (70-100%), each block was embedded in paraffin and serial 5 mm thick sections were cut using a rotatory microtome. The immunoreactivity for NTs has been observed in spermatogonia of untreated rats, while the rats treated with LND showed an immunohistochemical localization in all the stages of germinal cells. The generally well-expressed immunoreactivity for the neurotrophins receptors in treated rats observed in our study is presumably attributable to alterations of the receptors' structure and/or expression leading to changes of the activity, affinity, localization or protein interactions that may depend on sensitization of ion channels (induced by LND). Neurotrophins (NTs) appear to be interesting proteins for the modulation of sperm maturation and motility with a prominent role for the nerve growth factor (NGF), that may exert an autocrine or paracrine role. We therefore investigated the location and distribution of immunoreactivity for some neurotransmitters (SP, VIP, CGRP, nNOS, Chat), neurotrophins (NGF, BDNF, NT-3) and their own receptors (TrKA, TrKB, TrKC, p75) in the seminiferous tubules of male rats treated by LND in the light of the literature on this topic.

Lymphoid organs are supplied by many nerve endings associated with different kinds of cells and macrophages. The role of these neuromediators on the release of locally active molecules is still unknown. Here we focused our attention on the expression of some neurotrophins (NTs), their high- and low-affinity receptors and several neurotransmitters in human palatine tonsils. Light and electron microscopy immunohistochemistry showed that human tonsillar samples were positive for all analyzed neurotrophins (NGF, BDNF and NT-3) and their high-affinity receptors (TrkA, TrkB and TrkC, respectively). All of these molecules were strongly expressed in macrophages whereas, in some patients, a weaker specific staining of lymphocytes and blood vessels was also found. The low-affinity receptor for NGF (p75) was always absent in the analysed samples. RT-PCR confirmed the occurrence of specific transcripts for NTs and their high-affinity receptors as well as the absence of mRNA for p75 protein. Also, specific immunoreactivity for neurotransmitters SP, VIP, CGRP, ChAT and nNOS was mainly expressed by macrophagic cells. These results suggest the presence of an extensive network of innervation in the human palatine tonsils which may play a role in the regulation of some immune functions as well as in the modulation of a possible functional scenario of interactions among different immune cellular subtypes.

Neurotrophin (NT) and NT receptor expression was assessed in 12 typical (TC) and 8 atypical (AC) human pulmonary carcinoids by Western blot and immunohistochemistry. TC and AC carcinoid express to different extent NT and NT receptor proteins. Nerve growth factor (NGF) was expressed by 83% of the TC but not by the AC carcinoids. Brain derived neurotrophic factor (BDNF) was expressed by 33 and 100% of TC and AC carcinoids, respectively. NT-3 was expressed by 58% of the TC and 38% of AC carcinoids. TC carcinoids express high affinity NT receptors while 50% of the AC carcinoids express the TrkB receptor. Our results demonstrate that NGF/TrkA and BDNF/TrkB signaling need to be considered as regulatory pathways that may address survival, differentiation and/or aggressiveness of human pulmonary carcinoids. Contrarily to the BDNF/TrkB, expression of the NGF/TrkA signaling may overcome aggressiveness of carcinoid cells. NTs may be useful as markers in the clinic.

Mammalian peribronchial tissue is supplied by several peptide-containing nerve fibers. Although it is well established that different neuropeptides exert significant effects on bronchial and vascular tone in the lungs, the role played by some neuromediators on the general regulation, differentiation and release of locally active substances is still controversial. We studied the innervation of rat peribronchial tissue by immunohistochemical techniques. The immunoperoxidase method with nickel amplification was applied to detect the distribution of nerve fibers using antibodies against the general neuronal marker PGP 9.5 (neuron-specific cytoplasmic protein), while the cholinacetyltransferase immunoreactivity was studied by immunohistochemistry. A slight immunoreactivity for NT receptors is observed in lung bronchial epithelium. There is increasing evidence that NTs may act with a paracrine mechanism regulating functional activity of neuronal and non-neuronal structures. A specific immunoreactivity for NTs and NT receptors was also demonstrated within different layers of large, medium and small sized intrapulmonary arteries and veins, according to a recent study of our group. Moreover our data describe the expression of NTs and NT receptors in lymphoid aggregates of the lung (BALT) in which both lymphocytes and macrophages express TrkA receptor and synthesize NTs. Our results show the presence of an extensive network of innervation in the rat peribronchial tissue, confirming a morphological basis for a possible neural modulation of the respiratory mucosa and the physiological/pathophysiological mechanisms of the lung.

Spontaneously hypertensive rats represent a widely used animal model of hypertension. Altered neurotrophin regulation has been related to systemic hypertension. Lung cells synthesize and release neurotrophins(NT) which are involved in several pulmonary disturbances. Our study explored the expression of NT and their receptors using molecular biology and immunohistochemistry. Spontaneously 6-, 20- and 29- weeks hypertensive (SHR) and Wistar Kyoto rat (WKY) lungs were used. SHR and WKY lungs expressed neurotrophin and neurotrophin receptor mRNAs and protein transcripts. Age and hypertension dependent increase was clearly noticeable. Furthermore, immunohistochemistry revealed that their increase was mainly noticeable within bronchial and vascular components of the lung. Although the role of NT in sympathetic hyperinnervation and progression of systemic hypertension is a well established phenomenon, their increase in SHR lungs, strongly suggest that NT may be operative during pulmonary hypertension and in subsequent airway and pulmonary vascular remodeling.

Neurotrophins (NTs) promote survival and differentiation of central and peripheral neurons, and display several activities also in non-neuronal cells. Human lungs synthesize and release NTs, which are probably involved in the pathophysiology of pulmonary disturbances. In this article the expression and anatomic localization of nerve growth factor, brain-derived neurotrophic factor, and NT-3 and of corresponding high-affinity receptors TrkA, TrkB (full-length and truncated [TR-] isoforms), TrkC, and of the low-affinity p75 receptor, were assessed in surgical samples from adult human lung by reverse transcriptase-polymerase chain reaction, Western blot, and immunohistochemistry. NTs and their cognate receptor mRNA and protein transcripts were detected by reverse transcriptase-polymerase chain reaction and immunoblotting, respectively, nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) mRNA and corresponding protein transcripts being the most expressed. High levels of TrkB-[TR-] mRNA and of its protein transcript were also demonstrated, whereas a low expression of p75 mRNA and of corresponding protein transcript were found. Microanatomic analysis of immunohistochemical study revealed that bronchial epithelial cells were immunoreactive for different NTs, with a higher intensity of BDNF immune staining compared with other NTs, but did not express NT receptor immunoreactivity. Alveolar cells were immunoreactive for TrkA and TrkC receptor protein, but did not display immunoreactivity for NTs or other receptors investigated. Gland cells expressed NT and high-affinity NT receptor immunoreactivity, but not p75 receptor immunoreactivity. NT and low-affinity receptor immunoreactivity was observed within neurons and satellite cells of parasympathetic ganglia as well as in nerve fiber-like structures supplying the bronchopulmonary tree. An obvious immunoreactivity for NTs and NT receptor protein was also observed in intrapulmonary branches of pulmonary artery. Pulmonary lymphocytes and macrophages express nerve growth factor and high-affinity NT receptor immunoreactivity. The role of NTs in non-neuronal tissue including lung has not been clarified yet. The widespread expression of NTs and their receptors in different components of the lung suggests that these factors may contribute to regulate cell function in human lung.

The anatomy of the gonadal vein has been the subject of several studies relating particularly to the aetiology and therapy of varicocele and left ovarian vein syndrome. Venography shows the presence of valves, the collateral branches, the anastomoses between the left gonadal vein and the retroperitoneal venous networks and the effective pathways of venous reflux. The authors observed a particular congenital anomaly of the left gonadal vein in the dissection of a female cadaver, and studied the venographic pattern of a male patient with left idiopathic varicocele. The aim of this study was to investigate, with the aid of a review of the literature, the embryo-pathogenetic basis of congenital abnormalities of the left gonadic vein, stressing those factors most conducive to errors in the diagnosis and therapy of varicocele and left ovarian vein syndrome, particularly in the scleroembolisation therapy of idiopathic varicocele.

The expression of muscarinic cholinergic receptor subtypes was investigated in peripheral blood lymphocytes (PBL) of bronchial asthma patients by a combined kinetic and equilibrium labeling technique for radioligand binding assay of muscarinic cholinergic receptor subtypes and by receptor immunochemistry and immunocytochemistry. An increased expression of M2 and to a lesser extent of M5 receptors and no changes of M4 receptor were observed in PBL of asthmatics compared to control individuals. The increase was related to bronchial hyperresponsiveness detected by methacholine challenge test. Analysis of M3 receptor expression revealed biphasic changes, with a decreased receptor density in patients with normal, mild and moderate responses to methacholine test and a recovery to levels similar to those found in healthy individuals in severe responders to methacholine test. The demonstration of a different expression of lymphocyte muscarinic receptors in asthma suggests that cholinergic system may participate to a molecular framework influencing immune functions in asthma.

Dopamine D4 receptors mediate inhibition of vasopressin-dependent sodium reabsorption by dopamine in collecting tubules. At present, the distribution of D4 receptors in other renal districts remains an open issue. The renal distribution of D4 receptor was assessed in normally innervated and denervated male Sprague-Dawley rats by quantitative immunohistochemistry using an anti-dopamine D4 receptor rabbit polyclonal antibody. D4 receptor protein immunoreactivity was observed perivascularly in the adventitia and the adventitia-media border. The density of perivascular dopamine D4 receptor was higher in afferent and efferent arterioles than in other segments of the renal vascular tree. Renal denervation abolished perivascular dopamine D4 receptor protein immunoreactivity. In renal tubules, the epithelium of collecting tubules showed the highest dopamine D4 receptor protein immunoreactivity, followed by the epithelium of proximal and distal tubules. No dopamine D4 receptor protein immunoreactivity was observed in the epithelium of the loop of Henle. Denervation did not change dopamine D4 receptor protein immunoreactivity in renal tubules. These results indicate that rat kidney expresses dopamine D4 receptors located both prejunctionally and nonprejunctionally in collecting, proximal, and distal tubules. This suggests that the dopamine D4 receptor may be involved in the control of neurotransmitter release and in renal hemodynamic and tubule function.

Postnatal development in the expression of dopamine D1-like and D2-like receptors was investigated in peripheral blood lymphocytes of male Wistar rats aged 1, 3, 4, 8, 12 and 16 weeks of age by radioligand binding assay techniques. Sample of frontal cortex, striatum and hippocampus were also investigated as reference tissues. The dopamine D1-like receptor antagonist [3H]SCH 23390 and the dopamine D2-like receptor agonist [3H]7-OH-DPAT were used as radioligands. The affinity (K(d)) of [3H]SCH 23390 or of [3H]7-OH-DPAT binding was unchanged in lymphocytes of rats of different age groups. The density (B(max)) of [3H]SCH 23390 binding sites increased from the 1st to the 3rd week of age, remained constant from the 3rd to the 8th week of age, and then increased slightly at 12 and 16 weeks of age. The B(max) value of [3H]7-OH-DPAT binding to lymphocytes increased from the 1st to the 3rd week of age, remained constant from the 3rd to the 4th week, increased again until the 12th week and then plateaued. Dopamine D1-like and D2-like receptor maturation in frontal cortex, hippocampus and striatum revealed an increased receptor density until the 4th week of age and a relative stabilization of receptor density values between the 4th to the 12th week depending on the area considered. Comparatively postnatal maturation of lymphocyte dopamine D1-like receptors displayed a pattern different from that of brain areas investigated, whereas maturation of D2-like receptors displayed a pattern similar to that of striatum. The quantitative and/or qualitative dissimilarities between development of lymphocyte and brain dopamine receptors suggest that from a developmental point of view lymphocyte dopamine receptors probably cannot be considered as a marker of homologous brain receptors.

Dopamine D1-D5 receptor protein immunoreactivity and tyrosine hydroxylase (TH) immunoreactivity were investigated on the mesenteric arterial tree by immunohistochemistry. The density of various dopamine receptors and TH immunoreactivity was compared between young (6-month-old), adult (15-month-old) and senescent (24-month-old) Fischer 344 rats by computer-assisted microdensitometry. The dopamine D1-like (D1 and D5) receptors were localized on the tunica media of different sized mesenteric artery branches. The D2-like (D2, D3 and D4) receptors as well as TH immunoreactivity were localized only on the adventitia-media transitional zone of mesenteric arterial tree. Expression of the D1 and D5 receptors was decreased in both adult and senescent rats compared to the young rats, suggesting an age-related decline in these receptors. Of the D2-like receptors, the expression of the D2 receptor was decreased as a function of age, while the D3 receptor was unchanged in the senescent rats compared to the young rats. Expression of the D4 receptor was increased in adult, but was unchanged in the senescent rats compared to young animals. TH immunoreactivity was increased as a function of age. The above data suggest that reduction in the D1, D2 and D5 receptor expression may contribute to the deficiency in the dopamine-mediated vasorelaxation and hence blood flow in the mesenteric vascular tree in aging. The different sensitivity to aging of sympathetic neuroeffector junctions labeled by TH and of dopamine D2-like receptors that are known to be prejunctional, suggests that age-related changes of dopamine receptor expression in the mesenteric vasculature reflect more complicated mechanisms than simple up- or down-regulation phenomena.

Objective: The expression of alpha1-adrenergic receptor subtypes in peripheral blood lymphocytes was investigated in 28 essential hypertensive patients as well as in the peripheral blood lymphocytes and aorta of spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats., Methods: Alpha1-adrenergic receptors were quantified by radioligand binding assays, employing [3H]-prazosin as the radioligand in association with compounds displaying different degrees of selectivity for alpha1A-, alpha1B- and alpha1D-adrenergic receptor subtypes., Results: The affinity of [3H]-prazosin binding was similar in peripheral blood lymphocytes of different stage essential hypertensive and normotensive subjects or of SHR and age-matched normotensive WKY rats as well as in the aortas of SHR and WKY rats. The radioligand binding assay revealed no change in the expression of alpha1-adrenergic receptors in peripheral blood lymphocytes of essential hypertensives compared with normotensive subjects; a moderate decrease of alpha1B-adrenergic receptors and an increase of alpha1D-adrenergic receptors. The relative densities of the alpha1-adrenergic receptor subtypes were similar in the three groups of essential hypertensives. In peripheral blood lymphocytes and in aorta of SHR, [3H]-prazosin binding was significantly reduced compared with normotensive WKY rats. The expression of alpha1-adrenergic receptor subtypes in peripheral blood lymphocytes of SHR was similar to that found in peripheral blood lymphocytes of essential hypertensives., Conclusions: Changes of lymphocyte alpha1-adrenergic receptor subtypes in essential hypertensives are similar to those observed in lymphocytes and vascular tissues of animal models of hypertension. This suggests that assays of lymphocyte alpha1-adrenergic receptors may represent an indirect marker of their involvement in essential hypertension.

The expression of neurotrophins (NTs) and related high- and low-affinity receptors was studied in surgical samples of histologically diagnosed human tumors of the lower respiratory tract. The experiment was conducted with 30 non-small cell lung cancer specimens and in eight small cell lung cancer specimens by Western blot analysis and immunohistochemistry to assess expression and distribution of NT and NT receptor proteins in tissues examined. Immunoblots of homogenates from human tumors displayed binding of anti-nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and NT-3 antibodies as well as of anti-tyrosine-specific protein kinase (Trk) A, TrkB, and TrkC receptor antibodies, with similar migration characteristics than those displayed by human beta-NGF and proteins from rat brain. A specific immunoreactivity for NTs and NT receptors was demonstrated in vessel walls, stromal fibroblasts, immune cells, and sometimes within neoplastic cell bodies. Approximately 33% of bronchioloalveolar carcinomas exhibited a strong membrane NGF and TrkA immunoreactivity, whereas 46% adenocarcinomas expressed an intense TrkA immunoreactivity but a weak immunostaining for NGF within tumor cells. Moreover, squamous cell carcinomas developed an intense TrkA immunoreactivity only within stroma surrounding neoplastic cells. A faint BDNF and TrkB immunoreactivity was documented in adenocarcinomas, squamous cell carcinomas, and small cell lung cancers. NT-3 and its corresponding TrkC receptor were found in a small number of squamous cell carcinomas within large-size tumor cells. No expression of low-affinity p75 receptor protein was found in tumor cells. The detection of NTs and NT receptor proteins in tumors of the lower respiratory tract suggests that NTs may be involved in controlling growth and differentiation of human lung cancer and/or influencing tumor behavior.

The Authors present a case of inflammatory pseudotumour (IPT) of the urinary bladder occurring in a 57-year-old female patient, who was referred to our department with haematuria, stranguria and hypogastric pain. Ultrasonographic, radiological and endoscopic examinations showed a sessile, ulcerated, easily bleeding bladder formation; urinary cytology revealed no atypical transitional cells. Abdomino-pelvic computed tomography analysis showed thickening of the bladder walls and infiltration of the perivesical fat. Histopathologically, the formation was indicated as an inflammatory pseudotumour (IPT) of the bladder. The patient underwent TURB (transurethral resection of the bladder) and was discharged clinically healed on postoperative day 4. A one-year follow up revealed no evidence of recurrence. On the basis of their experience and a thorough review of the literature review, the Authors discuss the clinico-pathological features of IPT of the bladder and the possible factors involved in the malignant transformation of IPT. In conclusion, the benign nature of the lesion is stressed.

The expression of dopamine receptors by human platelets was investigated by Western blot analysis and immunocytochemical techniques using antibodies raised against dopamine D1-D5 receptor protein. The influence of dopamine D1-like and D2-like receptor agonists on adrenaline-induced platelet aggregation was also investigated. Western blot analysis revealed that platelet membranes bind anti-dopamine D3 or D5 receptor protein antibodies, but not anti-D1, D2 or D4 receptor protein antibodies. Cytospin centrifuged human platelets exposed to anti-dopamine D3 or D5 receptor protein antibodies developed a specific immune staining, whereas no positive staining was noticeable in platelets exposed to other antibodies tested. Both the D1-like receptor agonist 1-phenyl2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol hydrochloride (SKF 38393) and the D2-like receptor agonist 7-hydroxy-N,N-di-n-propyl-2-aminotetralin (7-OH-DPAT) dose-dependently inhibited adrenaline-induced platelet aggregation. These effects were decreased respectively by the D-like and D2-like receptor antagonists R(+)-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepin-7-ol hydrochloride (SCH 23390) and (-)sulpiride. The above findings indicate that human platelets express dopamine D3 and D5 receptors probably involved in the regulation of platelet function.

Left testicular vein anatomy has received more attention due to the presence of competent or incompetent venous valves and bypassing anastomoses, which are involved in venographic diagnosis and embolisation of varicocele. The left gonadal vein develops, in both males and females, between the 5th and 7th intrauterine weeks, being derived from the distal or postrenal portion of the left subcardinal vein. The varicocele aetiologic hypothesis leads to ontogenetic disturbances in the development of the secondary venous system. Retrograde testicular venography shows the precise anatomy of the left pampiniform plexus, while anterograde testicular venography identifies the presence of the valve and possible continence. In the present case sclerotherapy could not be achieved due to testicular vein anomalies. Sclerotherapy versus surgical high ligature of the left testicular vein in cases of left idiopathic varicocele with testicular vein anomalies is discussed.

The localization of neurotrophins (NTs) and NT receptors was analyzed in sections of human extra- and intrapulmonary arteries by Western blot analysis and immunohistochemistry. In extrapulmonary branches of human pulmonary artery, NT and NT receptor immunoreactivity was located in the tunica intima, within endothelium, in the tunica media, within smooth muscle and in the tunica adventitia. In different sized intrapulmonary arteries, NT and NT receptor immunoreactivity was observed primarily in the tunica adventitia. A faint NT and NT receptor immunoreactivity was observed in the tunica media of large-sized branches of intrapulmonary arteries, but not within medium- or small-sized intrapulmonary vessels or in tunica intima of different sized intrapulmonary arteries. These findings suggest that NTs may have a role in the control of vascular responses in the pulmonary system acting as local paracrine or autocrine mediators. The possible relevance of the NT system in human pulmonary vasculature identified in this study is discussed., (Copyright 2000 S. Karger AG, Basel)

Dopamine D1-D5 receptor protein immunoreactivity was investigated in different sized pial, renal and mesenteric artery branches using immunohistochemical techniques and anti-dopamine D1-D5 receptor protein antibodies. Faint dopamine D1 receptor protein immunoreactivity was observed in smooth muscle of tunica media of pial, renal and mesenteric artery branches. Dopamine D2 receptor protein immunoreactivity was located in the adventitia and adventitia-media border of pial and renal artery branches and to a lesser extent of mesenteric artery branches. No dopamine D3 receptor protein immunoreactivity was observed in pial and mesenteric arteries. In renal arteries a moderate dopamine D3 receptor immunoreactivity was detectable in the adventitia and adventitia-media border. A strong dopamine D4 receptor protein immunoreactivity displaying the same localization of dopamine D2 receptor protein was observed in pial and mesenteric arteries, but not in renal artery branches. Moderate dopamine D5 receptor protein immunoreactivity was observed in smooth muscle of the tunica media of pial, renal and mesenteric artery branches. Bilateral removal of superior cervical ganglia, from which sympathetic supply to cerebral circulation originate abolished dopamine D2 and D4 receptor protein immunoreactivity in pial arteries but was without effect on dopamine D1 and D5 receptor protein immunoreactivity. These findings indicate that systemic arteries express dopamine D1-like (D1 and D5) and D2-like (D2, D3 and D4) receptor subtypes displaying respectively a muscular (postjunctional) and prejunctional localization. The specific distribution of dopamine D2-like receptor subtypes in systemic arteries suggests that they may have a different role in regulating blood flow through the vascular beds investigated.

A case of myofibroblastic pseudosarcomatous tumor of the bladder occurred in a patient previously submitted to TURB is reported. Aim of this investigation is to demonstrate, on the basis of the literature, the importance of histopathology in the diagnosis of bladder pseudosarcoma, since endoscopical and radiological methods can erroneously show a neoplasm. A 65-year-old male patient with hematuria, submitted to ultrasonography and cystoscopy, revealed a bladder superficial papillary formation. A TURB was performed and the histological study showed a superficial bladder cancer (T1-G1-2). After a second hematuria episode with stranguria a new bladder formation was diagnosed and resected; the histological study revealed a bladder pseudosarcoma. The absolute benignity of the lesion and the slight difference in the symptomatology between urothelial carcinoma and pseudosarcoma are underlined. Furthermore, the importance of histopathology to obtain an accurate diagnosis is stressed. The difficulty of a preoperative diagnosis without histopathological examination is pointed out.

Alveolar macrophages play a crucial role in regulating lung immune responses and in maintaining the integrity of the respiratory tract. Neurotrophins (NTs), besides to their neurotrophic activities, exhibit physiological effects in the immune system. In this study, nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), NT-3 and low- (p75) and high affinity (Trks) NT receptors were investigated by immunocytochemistry in cytospin centrifuged preparations of human alveolar macrophages. Approximately 2.5% alveolar macrophages were immunoreactive for NGF, whereas no macrophages displaying immunoreactivity for BDNF or NT-3 were observed. A 3.5% macrophages displayed immunoreactivity for TrkA-receptor protein, 10% for TrkB-receptor protein (full length isoform), and 2% for TrkC-receptor protein. No low-affinity p75 NT and TrkB[-] truncated isoform receptor immunoreactive macrophages were found. These findings support the hypothesis that NTs and the corresponding receptors may play a role in regulating immunological and functional activity of alveolar macrophages via paracrine/autocrine mechanisms.

Molecular biology studies have shown that human peripheral blood lymphocytes express a dopamine D5 receptor, whereas no information is available on dopamine D receptor, the other dopamine D1-like receptor subtype. Radioligand binding assay investigations with the nonsubtype selective dopamine D1-like receptor antagonist [3H]SCH 23390 as radioligand have suggested the presence of a dopamine D5 receptor in human peripheral blood lymphocytes. However, so far no evidence was provided as whether or not human peripheral blood lymphocytes express a dopamine D1 receptor. In this study, we have investigated dopamine D1 and D5 receptor mRNA and the influence of antibodies against dopamine D1 and D5 receptors on [3H]SCH 23390 binding to intact human peripheral blood lymphocytes. The two receptors were also analyzed by immunocytochemistry. Dopamine D5 receptor, but not D1 mRNA, was detected in human peripheral blood lymphocytes. Anti-dopamine D5 receptor antibodies, but not anti-dopamine D1 receptor antibodies, significantly decreased [3H]SCH 23390 binding to human peripheral blood lymphocytes. A dark-brown immunoreactivity was visualized in cytospin centrifuged human peripheral blood lymphocytes exposed to anti-dopamine D5, but not to anti-dopamine D1 receptor antibodies. These data collectively indicate that dopamine D5 receptor is the only dopamine D1-like receptor subtype expressed by human peripheral blood lymphocytes.

Age-related changes of glial fibrillary acidic protein (GFAP) immunoreactivity were investigated in the cerebellar cortex of young (3 months), adult (12 months) and old (24 months) rats using immunohistochemical techniques associated with image analysis. In young rats, cell bodies of GFAP-immunoreactive astrocytes were found in the white matter and in the granular layer of cerebellar cortex. Radially-oriented branches of astrocytes which are sited in the granular layer were also observed in the molecular layer. The number of GFAP-immunoreactivity astrocytes of white matter was decreased in adult and old rats in comparison with young cohorts, whereas their size increased progressively from 3 to 24 months old. The number and the size of GFAP-immunoreactive astrocytes of the granular layer was similar in young and adult rats. An increased number and size of GFAP-immunoreactive astrocytes was noticeable in old rats in comparison with younger cohorts. The number of radially oriented branches of the molecular layer was the same in the three age groups investigated. The above results indicate that GFAP-immunoreactive astrocytes of rat cerebellar cortex undergo age-related changes. The not homogeneous sensitivity to aging of cerebellar astrocytes suggests that evaluation of changes of different cell populations of cerebellar cortex should represent an important step of research on aging cerebellum.

We investigated the expression of alpha1-adrenergic receptor subtypes in intact human peripheral blood lymphocytes using reverse transcription-polymerase chain reaction (RT-PCR) and radioligand binding assay techniques combined with antibodies against the three subtypes of alpha1-adrenergic receptors (alpha1A, alpha1B, and alpha1D). RT-PCR amplified in peripheral blood lymphocytes a 348-bp alpha1A-adrenergic receptor fragment, a 689-bp alpha1B-adrenergic receptor fragment, and a 540-bp alpha1D-adrenergic receptor fragment. Radioligand binding assay with [3H]prazosin as radioligand revealed a high-affinity binding with a dissociation constant value of 0. 65+/-0.05 nmol/L and a maximum density of binding sites of 175. 3+/-20.5 fmol/10(6) cells. The pharmacological profile of [3H]prazosin binding to human peripheral blood lymphocytes was consistent with the labeling of alpha1-adrenergic receptors. Antibodies against alpha1A-, alpha1B-, and alpha1D-receptor subtypes decreased [3H]prazosin binding to a different extent. This indicates that human peripheral blood lymphocytes express the three alpha1-adrenergic receptor subtypes. Of the three different alpha1-adrenergic receptor subtypes, the alpha1B is the most represented and the alpha1D, the least. Future studies should clarify the functional relevance of alpha1-adrenergic receptors expressed by peripheral blood lymphocytes. The identification of these sites may represent a step for evaluating whether they represent a marker of alpha1-adrenergic receptors in cardiovascular disorders or for assessing responses to drug treatment on these receptors.

Ca2+ channels of the L-type were assayed in human peripheral blood lymphocytes of normotensive control subjects and of essential hypertensives using radioligand binding assay techniques. The dihydropyridine Ca2+ channel blocker [3H](+)-PN 200-110 [isopropyll-4-(2,1,3-benzoxadiazol-4-yl)1,4-dihydro-5-methox ycarbonyl-2,6-dimethyl-3-pyridine carboxylate] was used as a ligand. [3H](+)-PN 200 110 was bound specifically to human peripheral blood lymphocytes in a manner consistent with the labeling of Ca2+ channels of the L-type. No significant differences in the dissociation constant (Kd), in the maximum density of binding sites (Bmax) or in the pharmacological profile of [3H](+)-PN 200 110 binding were found between normotensive subjects and different degree essential hypertensives. Analysis of the intralymphocytic free Ca2+ concentration did not reveal differences between normotensive subjects and essential hypertensives. Although hypertension is associated with altered membrane handling of Ca2+, no changes in the expression of peripheral blood lymphocyte Ca2+ channels of the L-type or in intralymphocytic Ca2+ concentrations were found in essential hypertensives. Human peripheral blood lymphocytes therefore cannot represent a peripheral marker of altered Ca2+ handling in hypertension.

The microanatomical localization of dopamine D1A and D1B receptor subtypes was investigated in sections of rat kidney using immunohistochemicals techniques with antidopamine D1A and D1B receptor antibodies. Microanatomical analysis was limited to the various components of nephron. Dopamine D1A receptor immunoreactivity was found primarily in the epithelium of loop of nephron (loop of Henle) and of collecting tubules. A less intense immunoreactivity was observed within proximal and distal convoluted tubules as well as in juxtaglomerular complex. Dopamine D1B receptor immunoreactivity was found primarily in proximal and distal convoluted tubules and within the juxtaglomerular complex. A less intense immunoreactivity was observed in the epithelium of collecting tubules followed by the loop of nephron. The demonstration of the localization of dopamine D1A and D1B, receptor subtypes along the nephron may contribute to better define their significance in physiological and pathological conditions.

Molecular biology studies have demonstrated that human peripheral blood lymphocytes express dopamine D2-like receptors belonging to the D3 and D4 receptor subtypes, whereas the characterization of these receptors using radioligand binding assay techniques provided conflicting results. The preferential dopamine D3 receptor agonist [3H]7-hydroxy-N, N-di-n-propyl-2-aminotetralin ([3H]7-OH-DPAT) was used recently for labeling lymphocyte dopamine D3 receptor. However, the selectivity of this compound for the D3 receptor was questioned. In this study we have investigated human peripheral blood lymphocyte dopamine receptor subtypes labeled by [3H]7-OH-DPAT using a conventional radioligand binding assay technique and antibodies against dopamine D2-like receptor subtypes. [3H]7-OH-DPAT was specifically bound to intact human peripheral blood lymphocytes with a dissociation constant (Kd) value of 0.32 + 0.03 nM and a maximum density of binding sites (Bmax) of 18.2 + 0.8 fmol/2 x 10(6) cells. [3H]7-OH-DPAT binding was unaffected by antibodies against dopamine D2 and D2S receptors. Anti-dopamine D3 and D4 receptor antibodies reduced [3H]7-OH-DPAT binding by about 53% and 32% respectively. Combination of anti D3 and D4 receptor antibodies reduced remarkably [3H]7-OH-DPAT binding. The above results suggest that the dopamine receptor agonist [3H]7-OH-DPAT labels dopamine D3 and D4 receptor subtypes in human peripheral blood lymphocytes. The use of antibodies raised against dopamine receptor subtypes in combination with radioligand binding assay may contribute to define receptor subtypes expressed by human peripheral blood lymphocytes in health and disease.

Glial cells are sensitive to aging, but the real extent of age-related quantitative and qualitative changes of these brain cellular elements has not yet been clarified. Brain volume undergoes age-related changes, but several studies on the number of glial cells have not taken this important variable into account. In this study we quantitatively evaluated the number and morphology of glial fibrillary acidic protein (GFAP)-immunoreactive astroglia in the frontal cortex and in the CA1 subfield of the hippocampus of male Sprague-Dawley rats of aged 12 and 24 months, considered adult and aged, respectively. The volume of frontal cortex was unchanged in the two age groups investigated, whereas the volume of hippocampus was higher in aged rats. An increase in the number and size of GFAP-immunoreactive astrocytes was observed in the frontal cortex and in the CA1 subfield of the hippocampus of aged rats. The numeric increase in astrocytes was more pronounced in the hippocampus than in the frontal cortex, whereas age-related hypertrophy of astroglia was more accentuated in the frontal cortex. The possible significance of hyperplasia and hypertrophy of GFAP-immunoreactive astrocytes in the frontal cortex and in the CA1 subfield of the hippocampus of aged rats is discussed.

The pharmacological profile and the anatomical localization of Ca2+ channels of the L-type were investigated in the human pulmonary artery to identify possible mechanisms involved in the regulation of the pulmonary vascular tone. Analysis was performed on slide-mounted frozen sections of human pulmonary artery using radioligand binding assay techniques associated with light microscope autoradiography. [3H]-Nicardipine was used as ligand. Human renal and right coronary arteries also were used as systemic reference arteries. Binding of [3H]-nicardipine to sections of human pulmonary artery was time-, temperature- and concentration-dependent, saturable and reversible. In the human pulmonary artery, the apparent equilibrium dissociation constant (Kd) was 0.12+/-0.02 nM and the maximum density of binding sites (Bmax) was 38.15+/-2.25 fmol/mg tissue. Kd values were 0.3+/-0.01 nM and 0.5+/-0.02 in the human renal artery and right coronary artery respectively. Bmax values were 248+/-16 fmol/mg tissue and 173+/-9.5 fmol/mg tissue in the human renal artery and right coronary artery respectively. The pharmacological profile of [3H]-nicardipine binding to sections of human pulmonary artery was consistent with the labeling of Ca2+ channels of the L-type. It was similar in the pulmonary artery and in the human renal and right coronary arteries. Light microscope autoradiography revealed a high density of [3H]-nicardipine binding sites within smooth muscle of the tunica media of human pulmonary artery as well as of human renal and right coronary arteries. A lower accumulation of the radioligand occurred in the tunica adventitia. No specific binding was noticeable in the tunica intima. Our data suggest that human pulmonary artery expresses Ca2+ channels of the L-type sensitive to dihydropyridines. These sites have similar affinity and lower density than those expressed by systemic arteries. The presence of Ca2+ channels of the L-type in human pulmonary artery suggests that their pharmacological manipulation may be considered in the treatment of pulmonary hypertension.

To study the short and long term effects of myocardial injury on sympathetic nerve fibers, a necrotizing lesion was performed on the diaphragmatic side of rat myocardium by freeze-thawing. Animals were sacrificed at 2, 6, 18, 28 and 105 days after the surgical procedure and paraffin-embedded hearts were subjected to peroxidase immunohistochemistry. According to previous studies cardiac nerves were visualized by staining their surrounding Schwann cells with an anti-S100 protein antibody. Catecholaminergic axons were specifically identified by an anti-tyrosine hydroxylase antibody. No S-100 positive structures were found in 2-day lesions (denervation). Starting from day 6,S-100 positive structures became progressively more evident (reinnervation) and persisted up to day 105. Many of these newly formed nerve fibers were positive for tyrosine hydroxylase, indicating that a necrotic injury of rat myocardium causes a disappearance of sympathetic innervation which is followed by a phase of sympathetic reinnervation.

Herbst corpuscles are the avian equivalent to the mammalian Pacinian corpuscles. In this study we used indirect peroxidase-anti peroxidase (PAP) immunohistochemistry to analyze the distribution in the pigeon cutaneous Herbst corpuscles, of several markers which are known to specifically label the axon, the Schwann-related and perineurial-related cells in Pacinian corpuscles. The distribution of the assessed antigens on Herbst corpuscles was as follows: i) the central axon displayed positive immunoreactivity for neurofilament proteins and neuron specific enolase; ii) the lamellar cells forming the inner-core were positive for S-100 protein and vimentin, whereas fibroblast surrounding them were vimentin and epithelial membrane antigen positive; iii) the capsule was focally immunolabelled for vimentin, and regularly for epithelial membrane antigen. No immunoreactivity was found neither for cytokeratins nor for glial fibrillary acidic protein. These observations demonstrate that the immunohistochemical profile of cutaneous (beak skin and rictus) Herbst corpuscles in pigeon is similar to that of the mammalian Pacinian corpuscles.

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 target organs of neurotrophin-dependent sympathetic and sensory neurons, including the skin, synthesize and release neurotrophins, primarily NGF. Neurotrophins undergo retrograde axonal transport, and exert specific function in the perikarya of the responsive neurons. Moreover, evidence exists for an autocrine and/or paracrine function of neurotrophins in the skin. This study analyses the immunohistochemical localization of low (gp75) and high-affinity (gp140 trkA, gp145trkB and gp145trkC) neurotrophin receptor proteins in the human glabrous skin. We consider that the expression of neurotrophin receptors may be indicative of neurotrophin activity. Specific gp75 and gp140trkA-like immunoreactivity (IR) were observed highly co-localized in (1) epidermis, primarily in the basal keratinocytes; 2) sweat glands; (3) blood vessel walls, mainly in the muscular layer; (4) Schwann and perineurial cells of nerve trunks; (5) periaxonic cells forming sensory nerve formations (Meissner's and Pacini's corpuscles); (6) large axons of nerve bundles and of sensory corpuscles; gp145trkB-like and gp145trkC-like were found labelling nerve fibers and sensory nerve formations, as well as blood vessels and sweat glands, but not epidermic cells. The results suggest that, in addition to the well known neurotrophic functions, neurotrophins may also regulate unknown functions in non-nervous cutaneous cells, which are targets for neurotrophin-dependent sympathetic and sensory neurons.

The effects of monolateral lesioning of the nucleus basalis magnocellularis (NBM) and of choline alphoscerate treatment on histochemically reactive vesicular zinc stores were assessed in the rat brain using the sulphide-silver histochemical technique. Histochemically reactive zinc stores are located primarily within association fibres of the neuropil of the cerebral cortex as well as in the mossy fibres of the hippocampus. The density of cortical and hippocampal sulphide-silver positive fibres, which might have a role in cognitive and mnemonic processes, parallels the density of zinc-containing presynaptic buttons. Unilateral lesions of NBM caused a remarkable decrease of sulphide-silver positive fibres from the 4th week after lesioning in the neuropil of the ipsilateral fronto-parietal cortex and from the 3rd week in the mossy fibres of the ipsilateral hippocampus. Treatment with choline alphoscerate, which is a precursor in the biosynthesis of brain phospholipids that increases the bioavailability of acetylcholine in the nervous tissue, restored, in part, the density and pattern of sulphide-silver positive fibres in the fronto-parietal cortex and in the hippocampus. The data suggest that, analogously to reports from Alzheimer's disease patients, lesions of the NBM cause a decrease of zinc stores in the rat brain. Choline alphoscerate treatment is able to counter the expression of this phenomenon which accompanies experimental lesions of the NBM.

The study was designed to assess whether Ca2+ channel blockers of the dihydropyridine family (felodipine, nicardipine, nifedipine, nimodipine, nitrendipine and nisoldipine), and of the phenylalkylamine family (verapamil) have any effect on alpha-adrenoceptor binding to sections of the human right coronary and mammary arteries, measured using [3H]-dihydroergocryptine (DHT) as a ligand. Increasing concentrations of nicardipine, verapamil and nitrendipine competed with [3H]-DHT binding to sections of the human coronary and mammary arteries. The other compounds tested were without effect. Among the competitors of [3H]-DHT binding, nicardipine was the most powerful, with a 50% inhibition (IC50) value of approximately 10 nM. The pharmacological profile of competition with [3H]-DHT binding by nicardipine, in the presence or in the absence of guanosine triphosphate and NaCl, is consistent with antagonist activity of the dihydropyridine derivative at the alpha-adrenoceptor. This property may account for the lower sympathetic stimulatory activity elicited by nicardipine, in comparison with other Ca2+ channel blockers used in cardiovascular therapy.

Muscarinic cholinoceptor subtypes (M1 and M2) were studied in membrane particles of the rat frontoparietal cortex 72 h and 1, 2, 3, and 4 weeks after ipsilateral lesioning of the nucleus basalis magnocellularis (NBM). The affinity of the ligand used to characterize muscarinic cholinoceptors, 3H-quinuclidinyl benzilate did not significantly change in lesioned compared with sham-operated rats as well as the density of high affinity (M1) sites. Low affinity muscarinic cholinoceptors (M2 sites) were significantly decreased in NBM-lesioned rats 72 h and 1 week after lesioning. The density of M2 sites did not significantly differ in lesioned rats 2 or 3 weeks after NBM lesioning, but increased, in comparison with sham-operation 4 weeks after NBM lesioning. These findings suggest that frontoparietal M2 muscarinic cholinoceptors, which probably have a presynaptic localization, are sensitive to NBM lesions. Their changes at different times after NBM lesioning suggest the occurrence of loss, compensation and upregulation of cholinergic projections arising to the neocortex from the NBM.

We have recently demonstrated that monolateral lesions of the Nucleus Basalis Magnocellularis (NBM), which is a nucleus sending cholinergic projections to the fronto-parietal cortex, cause a loss in the intensity of Timm staining in the intrahippocampal pathway of mossy fibres (MF). Moreover, these lesions induce ultrastructural changes consistent with the occurrence of degeneration of presynaptic buttons of MF. The present study was designed to quantify the effects of NBM lesioning on the morphology of the presynaptic buttons of MF. Moreover the effects of 4-week choline alfoscerate (alphaGFC) treatment on the density of Timm staining and on the ultrastructure of presynaptic buttons of MF were assessed, alphaGFC, which was given at an oral daily dose of 100 mg/kg, is a precursor in the biosynthesis of several brain phospholipids which increases the availability of choline in the nervous tissue. Monolateral lesions of NBM cause, 4 weeks after lesioning, a significant decrease in the intensity of Timm staining in the MF area accompanied by a loss of about 23% of presynaptic buttons of MF. Moreover about 40% of presynaptic buttons of MF show an impaired morphology. alphaGFC administration restored the intensity of Timm staining in the MF area. In alphaGFC-treated rats, the loss of presynaptic buttons and the number of impaired buttons were reduced to about 12% and 27%, respectively in comparison with non-treated animals. These results confirm and extend our previous observations indicative of the occurrence of transneuronal degenerations in the MF of the hippocampus after monolateral NBM lesioning. Moreover these findings show that alphaGFC treatment is able to counter in part these degenerative changes.

Assessment was made of the effects of intracerebroventricular (i.c.v.) administration of ethylcholine mustard aziridinium (AF64A) and of the monoamine oxidase (MAO)-B inhibitor L-deprenyl on the acetylcholine (ACh) biosynthetic enzyme choline acetyltransferase (ChAT), on the ACh catabolic enzyme acetylcholinesterase (AChE) and on the density of ACh muscarinic M-1 and M-2 receptor sites. In addition, the effect of AF64A and of L-deprenyl treatment on the localization of AChE activity in the CA-1 and CA-3 fields of the hippocampus was evaluated by combined enzyme histochemistry and microdensitometry techniques. I.c.v. injection of AF64A induced, 4 weeks after administration of the neurotoxin, a remarkable increase of MAO-B activity, and a significant reduction of ChAT and AChE activities in the hippocampus but not in the neostriatum which was used as a reference tissue. Hippocampal muscarinic M-1 receptors were unaffected by AF64A administration, whereas M-2 sites were reduced after neurotoxin injection. Enzyme histochemistry analysis showed that the loss of AChE induced by AF64A was more pronounced in the CA-3 than in the CA-1 field of the hippocampus. Treatment with L-deprenyl induced, from a dose of 11.17 microM/kg/day, a significant reduction of MAO-B activity in the hippocampus. The expression of ChAT and AChE, as well as the density of M-2 receptors, was increased after L-deprenyl administration in the hippocampus but not in the neostriatum. An increase in AChE reactivity was noticeable in the CA-1 and CA-3 fields of the hippocampus of AF64A-injected rats treated with L-deprenyl.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of intracerebroventricular (ICV) administration of ethylcholine mustard aziridinium (AF64A) and of the monoamine oxidase (MAO)-B inhibitor L-deprenyl on MAO-A and MAO-B activities and on the morphology of the rat neostriatum and hippocampus were studied. The ICV administration of AF64A was without effect on MAO-A and MAO-B in the neostriatum and caused an increase of MAO-B but not of MAO-A in the hippocampus. No changes in neostriatal micro-anatomy were noticeable in AF64A-injected rats, whereas the neurotoxin caused an impairment in hippocampal micro-anatomy consisting in the loss of nerve cells and of silver-gold impregnated fibres in the CA-1--CA-3 fields. The treatment of AF64A-injected animals with doses of L-deprenyl from 11.17 microM/kg/day significantly reduced MAO-B activity in the hippocampus and improved the morphology of the hippocampus formation. L-deprenyl was without effect on MAO-A activity both in the neostriatum and in the hippocampus, as well as on neostriatal MAO-B activity and morphology. The possibility that MAO-B inhibition may represent a principle for the treatment of age-related physiological and pathological changes characterized by increased MAO-B activity is discussed.

The anatomical localization of gamma-aminobutyric acid type A (GABA-A) receptor sites in the rat superior cervical ganglion was studied using combined radioreceptor binding and autoradiographic techniques. 3H-Muscimol was used as a ligand of GABA-A receptor sites. The binding was consistent with the labelling of GABA-A sites. The dissociation constant value was 6.4 nmol/l, and the maximum density of binding sites was 146 +/- 7.8 fmol/mg tissue. Light microscope autoradiography revealed the accumulation of 3H-muscimol mainly in superior portions of the ganglion. Binding sites are located primarily in the neuropil rather than within ganglionic neurons. It is probable that the sites revealed by autoradiography are involved in the inhibition of acetylcholine release from ganglionic neurons.

Aging-dependent changes of some enzymatic activities related to the glycolytic anaerobic pathway (lactate-dehydrogenase, LDH), to the Krebs's cycle (succinate dehydrogenase, SDH) and to the activity of the respiratory oxidative chain (NADH2 - tetrazolium reductase, NADH-D) were studied in the nephron of 4-(young) and 24- (aged) month-old female Wistar rats. In the renal glomeruli LDH and NADH-D activities were reduced with aging, while SDH did not undergo aging-dependent changes. In both the proximal and the distal convoluted tubules, LDH reactivity slightly increased while NADH-D remarkably decreased in old rats; SDH did not show aging-dependent changes. In the loop of Henle LDH was slightly increased, SDH was unchanged and NADH-D was decreased in aged in comparison to young rats. The three enzyme activities investigated were significantly reduced in the collecting tubules of aged animals. The above results are suggestive of the existence of aging changes in mechanisms related with energy transduction in the rat kidney, affecting at a different extent the cell populations of the nephron.

The age-related anatomical changes in the rat hippocampus were evaluated in male Sprague-Dawley rats of 3 (young), 12 (mature) and 24 (aged) months by counting the number of nerve cells in the CA1 and CA3 fields and in the dentate gyrus and by measuring the density of Nissl bodies in the cytoplasm of the pyramidal and granule neurons of the above areas. Moreover, the effect of 3 months choline alfoscerate treatment on the anatomical parameters examined was evaluated. The number of pyramidal neurons of the CA1 field and of granule neurons of the dentate gyrus was not significantly changed between young and mature animals, but it was decreased in aged rats. The number of pyramidal neurons of the CA3 field showed a progressive age-dependent reduction. The density of Nissl bodies was the highest in the cytoplasm of pyramidal or granule neurons in mature rats followed in descending order by young and aged animals. Choline alfoscerate treatment counteracted the age-related loss of nerve cells in the 3 hippocampal portions examined and slow-drown the decrease of Nissl bodies in the cytoplasm of pyramidal or of granule neurons in the hippocampus. The significance of changes induced by choline alfoscerate in the hippocampus of aged rats and the possible mechanism of action of the compound are discussed.

The sulfide staining technique, known also as neo-Timm staining, predominantly stains associational fibres arising from cortical interneurons located primarily in the neuropil of layers I-III of the rat cerebral cortex. The density of these fibres, considered to have a possible role in cognitive and mnemonic processes, has been demonstrated to be related to the density of zinc-containing presynaptic buttons in the cerebral cortex. The unilateral injection of ibotenic acid into the nucleus basalis magnocellularis (NBM) resulted in no changes in the density of sulfide staining in the first 3 weeks after neurotoxin injection and in a significant loss of sulfide staining in the neuropil of cortical layers I-III 4 weeks after NBM lesion in the fronto-parietal cortex ipsilaterally to the lesion. These data suggest that unilateral lesioning of the NMB may cause changes of chemo-specific zinc-containing intracortical pathways. Moreover, they indicate that, in a manner similar to that described in the brain of Alzheimer's disease patients, NBM-lesioned rats show a decrease of zinc tissue stores in the fronto-parietal cortex.

Lesions of the nucleus basalis magnocellularis (NBM) cause depletion of choline acetyltransferase (ChAT) in the cerebral cortex and behavioral changes consisting of impaired ability to learn avoidance tasks. Since hippocampal mossy fibers (MF) are involved in the elaboration of passive avoidance responses, we analyzed MF by means of Timm's histochemical technique and electron microscopy, to find out whether monolateral lesions of NBM had any effect on MF system. NBM-lesioned rats, 3 weeks after lesioning, showed a significant and progressive decrease in the density of Timm staining as well as significant changes of the morphology of synapic boutons of the MF. These results suggest that, although NBM does not send direct projections to the hippocampus, lesions of this nucleus may have a neurodegenerative effect on the intrahippocampal pathway involved in avoidance responses.

The influence of ageing on the noradrenergic innervation of the ovary was studied in female Wistar rats using high pressure liquid chromatography (HPLC) with electrochemical detection and catecholamine histofluorescence techniques. Old age was accompanied by a significant decrease in ovarian noradrenaline levels. In young animals (3-month-old) noradrenergic nerve fibres were distributed primarily to blood vessels and in lesser amounts to the interstitial glands. In aged animals (24-month-old) perivascular noradrenergic fibres were reduced by more than 40%; interstitial gland nerve fibres were reduced by approximately 20%. The possibility that impaired noradrenergic ovarian innervation occurring in old age may be in some way related with age-dependent failure in reproductive activity is discussed.

Enzyme histochemical techniques associated with microphotometry were used to evaluate the reactivity of glutamate dehydrogenase (GDH) in the hippocampus of young (3 month) adult (12 month) and old (26 month) male Sprague-Dawley rats. In young rats the highest level of enzymatic reactivity was found in the stratum oriens of the CA-1-CA-4 fields followed in descending order by the molecular layer of fascia dentata, and by the layer of mossy fibers. In all the layers investigated GDH reactivity was higher in adult that in young rats. The older animals exhibited lower GDH reactivity than adult rats in almost all of the layers and higher enzymatic reactivity than in young rats in the stratum oriens of the CA-1-CA-4 fields. The hippocampus is known to be involved in the acquisition of two-way avoidance learning and is thought to utilize glutamate as one of its main neurotransmitters. These data suggest a possible relationship between hippocampal glutamatergic transmission and impairment of acquisition of avoidance learning occurring with age. Moreover, the possibility that a decreased glutamate catabolism occurring in old rats may contribute to the neuronal loss described in the hippocampus of aged rats is discussed.

Age-related changes in the density of the noradrenergic perivascular plexus supplying the uterine artery and its intrauterine branches were studied in rats 4, 12 and 24 months old using the glyoxylic acid fluorescence technique. Moreover, uterine noradrenaline levels were determined by means of high pressure liquid chromatography with electrochemical detection. No changes in the density of noradrenergic innervation or in uterine noradrenaline levels were noticeable in 12 month in comparison with 4 month rats. In contrast, a significant decrease in the density of the noradrenergic perivascular plexus of uterine artery and of its intrauterine branches as well as decrease in noradrenaline concentrations of uterine horns and cervix were observed in 24 month rats. The above findings indicate an age-related impairment of uterine noradrenergic innervation.