The rise in cytoplasmic ubiquitin levels is an early step in the response of parasympathetic ganglionic neurons to axonal injury followed by regeneration.

We investigated the involvement of ubiquitin in the neuronal response to axonal injury in the quail parasympathetic ciliary ganglion by immuno-light and electron microscopy. Image analysis of immunoreacted cryosections shows that ubiquitin-immunoreactivity in the ciliary neurons increases significantly 6 hours after postganglionic nerve crush. The immunolabeling reaches a peak 1 day after injury and begins to decrease between days 3 and 6 when, in contrast to the cytoplasm, numerous highly eccentric nuclei are strongly immunolabeled. Electron microscopy shows ubiquitin-immunoreactivity associated with cytoplasmic organelles and with several postsynaptic densities of the numerous synapses established by the preganglionic boutons on the soma of the ciliary neurons. The number of immunopositive postsynaptic densities increases significantly 1 day after axonal damage, followed by temporary detachment of the preganglionic boutons from the injured neurons between days 3 and 6. The early increase in cytoplasmic ubiquitin-immunoreactivity suggests a prompt ubiquitination of damaged proteins addressed to degradation, while the nuclear immunolabeling may reflect high histone ubiquitination, a process involved in keeping chromatin transcriptionally active. The possible ubiquitin-mediated removal of postsynaptic apparatus constituents such as ACh receptors, proteins involved in their clustering and stabilization, and/or adhesion molecules may be a crucial step for the detachment of the preganglionic boutons, thus favoring injury-induced synaptic plasticity.

Effects of axotomy on the expression and ultrastructural localization of N-cadherin and neural cell adhesion molecule in the quail ciliary ganglion: an in vivo model of neuroplasticity.

Postganglionic nerve crush of the avian ciliary ganglion induces detachment of preganglionic terminals from the soma of the injured ciliary neurons, followed by reattachment at about the same time that the postganglionic axons regenerate to their targets. In order to determine the role played by cell adhesion molecules in this response, we have studied injury-induced changes in the amount and distribution of N-cadherin and neural cell adhesion molecule, together with modifications in the expression of their messenger RNAs. Both N-cadherin and neural cell adhesion molecule immunoreactivities associated with postsynaptic specializations decreased between one and three days following postganglionic nerve crush, preceding the detachment of the preganglionic boutons. Immunoreactivities subsequently increased between 13 and 20 days, in parallel with restoration of synaptic contacts on the ganglion cells and the progressive reinnervation of the peripheral targets. In contrast to the rapid decrease in immunoreactivity, the messenger RNA levels of N-cadherin and neural cell adhesion molecule both increased after crush, and remained elevated throughout the 20-day period of the experiment. These results are consistent with roles for N-cadherin and neural cell adhesion molecule in the maintenance of synaptic contacts. The rapid regulation of these proteins in injury-induced synaptic plasticity occurs at the post-transcriptional level, whereas longer term regulation associated with the re-establishment of synapses may be promoted by the increased levels of gene expression.

Degeneration and regeneration of neuromuscular junctions in chicken iris muscle after crush of the ciliary nerves: a study of ultrastructural changes and of cholinergic enzymes.

Degeneration of neuromuscular junctions in the iris muscle was observed to have occurred 4 days after ciliary nerve crush. By day 10 reinnervation had commenced and by day 30 the maturation of neuromuscular junctions was nearly complete. The loss and recovery of acetylcholinesterase activity in the iris muscle paralleled the denervation-reinnervation process, with recovery being completed by day 30, whereas the loss in the activity of choline acetyltransferase had not yet completely recovered at this time. The acetylcholinesterase activity localized cytochemically at synaptic sites followed the same trend as the total activity in the iris muscle, whereas acetylcholinesterase localized at myo-muscular junctions showed only slight changes. The acetylcholinesterase molecular forms displayed changes in their relative proportions, which could be related to the time course of the denervation-reinnervation process and to the cytochemical localization of the activity.

Ultrastructural alterations induced in quail ciliary neurons by postganglionic nerve crush and by Ricinus toxin administration, separately and in combination.

The response to postganglionic nerve crush and Ricinus toxin administration by the ciliary neurons of the quail ciliary ganglion was investigated at the ultrastructural level. The toxin was either applied at the crush site on the postganglionic nerves or injected into the anterior eye chamber without any other operative intervention. Crush of postganglionic nerves without toxin administration and saline injection into the anterior eye chamber served as controls for the two toxin administration procedures. Postganglionic nerve crush caused a distinct chromatolytic reaction, accompanied by massive detachment of the preganglionic axon terminals from the ciliary neurons and loss of most of the synapses, both chemical and electrical. This process does not induce cell death and is reversible. Saline injection in the anterior eye chamber caused a moderate retrograde reaction in some of the ciliary neurons, presumably as a consequence of paracentesis. The changes consisted mainly of an increase of perikaryal neurofilaments with, at most, a minor detachment of the preganglionic boutons from a small portion of the cell body at the nuclear pole. Ricinus toxin administration induced neuronal degeneration following a pattern common to both delivery modes. The degenerative process consisted of disruption and detachment of polyribosomes from the rough endoplasmic reticulum, an increase of smooth cisterns and tubules, a dramatic increase of neurofilament bundles, compartmentalization of the cytoplasmic organelles and, finally, karyorrhexis and cell lysis. The final stages of Ricinus toxin degeneration involve a progressive accumulation of extracellular flocculo-filamentous material and cell lysis. After administration of Ricinus toxin to the crush site, ricin-affected neurons showed withdrawal of the preganglionic boutons from a portion of the ciliary neuron, especially at the nuclear pole. After Ricinus toxin injection into the anterior eye chamber, however, the bouton shell surrounding the affected ciliary neurons remained intact in the early stages of degeneration. Detachment of the preganglionic terminals and disruption of the cell junctions, therefore, is the consequence of nerve crush and not of the toxin itself. This study demonstrated that quail ciliary neurons are a suitable model for experimental neuropathology and neurotoxicology.

Quantitative study of neuronal degeneration induced by Ricinus toxin and crush of postganglionic nerves in the ciliary ganglion of quail.

The effects of Ricinus toxin on the neurons of the ciliary ganglia were investigated in the quail. The neuronal death and the morphological alterations of the ganglionic cells were assessed following injection of the toxin in the anterior chamber of the eye or after application of the toxin on the postganglionic nerves at a crush site. A 45% loss of choroid neurons without loss of ciliary neurons was observed after postganglionic nerve crush alone. Injection of the toxin in the anterior chamber of the eye led to a selective loss of ciliary neurons (38%). Application of the toxin to the crushed postganglionic nerves led to a loss from both neuronal populations (40% of total neurons). This work indicates that different procedures result in selective lesion of the different neuronal populations in the ciliary ganglion.

Activities of 3':5' cyclic nucleotide phosphodiesterases in the superior cervical ganglion of rat: characterization, compartmentalization and observations in young and old animals.

We investigated the presence and features of "low Km" 3'-5' cyclic nucleotide phosphodiesterase activity in the homogenates and extracts of rat superior cervical ganglion. The DEAE chromatographic elution profile of a Triton X-100 extract showed two peaks of cAMP phosphodiesterase activity eluted at 280 and 600 mM sodium acetate and two peaks of cGMP phosphodiesterase activity eluted at 300 and at 500 mM sodium acetate. The activity was poorly stimulated by calcium-calmodulin and neither stimulated or inhibited by cGMP. Both cGMP PDE peaks were inhibited by zaprinast, with IC50's of 1.4 microM and 0.28 microM: their Km values were 4.4 and 3.8 microM, respectively. These features, together with cGMP binding activity, indicate that both enzymes belong to the phosphodiesterase V family. The Km values of the first and second cAMP phosphodiesterase peaks were 1.7 and 3.8 microM. Although both peaks displayed a cAMP specific hydrolysis, only the second peak was inhibited by RO 20-1724, with an IC50 of 8 microM. Preganglionic denervation indicated that the bulk of phosphodiesterase activity is localized in ganglion cells. In order to investigate possible effects of aging on the ganglionic function, phosphodiesterase activity was assayed in the ganglia of young (3 months) and old (25 months) male Fisher rats. The chromatographic profiles and kinetic features revealed no significant differences between young and old rats.

Neurotoxic action of 2,5-hexanedione on the autonomic nervous system: ultrastructural and functional alterations in the rat sympathetic superior cervical ganglion.

In rats treated for 14 days with 2,5-hexanedione, the efficiency of ganglionic transmission was markedly reduced whereas only faint ultrastructural changes occurred in a few preganglionic fibers; evident signs of axonal pathology were observed on the 30th day of treatment. Choline acetyltransferase activity and acetylcholine formation showed no alteration at any time. The autonomic system is affected early during 2,5-hexanedione neuropathy, functional changes being more marked than morphological lesions.

Effects of litter size on protein, choline acetyltransferase (cat), and dopamine-beta-hydroxylase (DBH) of a mouse sympathetic ganglion.

Litters of 5, 10 and 17 pups were formed at birth. At 7, 14, 21, 28 and 60 days after birth mice were weighed and the cervical superior ganglia were examined for total protein and for CAT and DBH activities. At each age, the parameters examined were fitted into regression lines on the reciprocal of the litter size (L.S.R.); a highly positive correlation was found at each age between body weight and L.S.R., with maximum differences at weaning (day 21) followed by partial recovery after ad libitum feeding until day 60. A positive correlation was also found for total ganglion protein at days 21 and 28, with recovery practically completed at day 60. For CAT activity a positive correlation with L.S.R. appeared at day 14, with increasing coefficient and significance in later periods, until day 60. The temporal pattern of DBH activity for the 5-pup litter showed a peak at day 21, followed by a 35% decrease at day 28, with a steady level until day 60. A similar trend was observed for the 10- and 17-pup litters, but the peak occurred at day 28. Significant differences were found as early as day 7 between the 5- and the 17-pup litters, the maximum differences in the 3 experimental conditions being found at weaning, with a high and significant correlation coefficient, which persisted at day 60. Thus, the biochemical development and maturation of both presynaptic cholinergic nerve terminals and postsynaptic adrenergic neurones was evidently affected by the itter size during the suckling period, with persistent effects.

Neurotoxic effects of 2,5-hexanedione in rats: early morphological and functional changes in nerve fibres and neuromuscular junctions.

The study was directed at detecting changes occurring in the early stages of the neurotoxic process induced by an intensive treatment with 2,5-hexanedione. Rats were injected intraperitoneally each day with 450 mg/kg of body weight for the first 14 days and then with 300 mg/kg for an additional 20 days. After 34 days of treatment typical axonal lesions were observed in the sciatic branches, together with electrophysiological signs of denervation in several fibres of the leg muscles. Morphological changes were also found in axons of the spinal cord and optic tracts. At an earlier stage (days 13 to 18), when clinical signs appeared, in the absence of morphological changes in peripheral nerves and of denervated muscle fibres, significant alterations in the functioning of several neuromuscular junctions were observed: increase in frequency and amplitude of miniature end-plate potentials, reduction of the mean quantal content of the evoked end-plate potential (epp) and absence of epp's in some fibres. These changes indicate a progressive functional impairment of nerve terminals which could possibly represent: (a) a primary alteration of the membrane resting and action potentials; (b) a secondary effect of changes in axonal transport. Both these hypothetical events could result from the inhibition of glycolitic enzymes of nervous tissue by 2,5-hexanedione.

[Alpha-interferon therapy: an echographic study of left ventricular function and a clinical assessment of cardiovascular complications]. / Terapia con alfa interferone: studio ecocardiografico della funzione ventricolare sinistra e valutazione clinica delle complicanze cardiovascolari.

Many side-effects of alpha interferon (alpha-I) therapy have occurred as a result of its widespread clinical applications. The hypothesis of alpha-I related cardiomyopathy is particularly interesting. Our study involved echocardiographic evaluation of left ventricular function and cardiovascular complications in 35 patients with chronic hepatitis treated with alpha-I at 20 MU/week for an average of 10.8 months. The results were compared with those of a control group. Of the values studied, only Max E V and the E/A ratio were statistically significant. No cardiovascular side-effects were found.

[Prevention of paroxysmal atrial fibrillation with propafenone after withdrawal of amiodarone because of side effects]. / Profilassi della fibrillazione atriale parossistica con propafenone dopo sospensione di amiodarone per effetti collaterali.

The authors evaluated the efficacy of medium term prophylaxis of atrial fibrillation (AF) with propafenon (P) in 33 symptomatic patients. Consecutive patients were treated with P replacing previous therapy with amiodarone (A) withdrawn on account of adverse side effects occurring on average after 1.8 years' treatment. Quantification of AF episodes was based on symptoms reported by patients, ECG, and dynamic Holter-ECG performed every 3-6 months. During A treatment (average daily dosage 216 mg) 32% of patients had reported more than two episodes of AF, 52% one or two episodes, and 16 none during the last 6 months. During 6 months of P treatment (average daily dosage 586 mg) 28% had more than two episodes, 64% had one or two, and 8% had none. The difference of incidence of AF episodes between the two treatments was not statistically significant. Side effects requiring withdrawal of the drug were not observed with P. The results obtained confirm P as a valid therapeutic resource for treatment of recurrent paroxysmall AF. In addition, the drug was also well tolerated during medium term application.

Molecular forms of acetylcholinesterase in the ciliary ganglion and iris of the chick. Developmental changes and effects of axotomy.

Four molecular forms of acetylcholinesterase (AChE) have been observed in extracts from ciliary ganglion and iris by gel electrophoresis followed by Koelle's reaction and densitometry on gels. Forms I, II, and III (order of anodic migration) are present in the 'soluble fraction' (SF) while forms II, III, and IV appear in the 'membranous fraction' (MF). 10 days after postganglionic axotomy in young chickens, the relative proportions of the membranous forms were changed in both the ciliary ganglion and iris, with a marked reduction of form IV occurring. A characteristic evolution of both the soluble and membranous forms of AChE was evident through different developmental stages (10th day of incubation; 2nd, 11th and 56th day posthatching). In the ciliary ganglion the faster components were reduced in favor of form IV. Conversely, the relative proportion of the faster components was increased in the iris. The observed changes in the AChE forms are compared with changes in the structure and function of these organs known to occur in the conditions under study. Evidence is presented which suggests a junctional localization for the form IV in both the ciliary ganglion and iris.

Molecular forms of cholinesterases in cerebrospinal fluid, blood plasma, and brain tissue of the beagle dog.

In the present study we have investigated the activity of AChE and BuChE in the cisternal CSF of the Beagle dog and have outlined the characteristics of the molecular forms of both enzymes. The same enzymes were also investigated in samples from blood plasma and brain tissue. It is concluded that AChE in CSF originates from the spinal cord and brain tissues as a result of some secretory process. A combined origin from both blood plasma and brain tissue appears to be probable for the BuChE of the CSF.

[Cardiac anomalies in systemic lupus erythematosus: their prevalence and relation to duration, disease activity and the presence of antiphospholipid antibodies]. / Anomalie cardiache nel lupus eritematosus sistemico: prevalenza e relazione con durata, attività della malattia e presenza di anticorpi antifosfolipidi.

We conducted an echocardiographic study to determine the incidence and spectrum of morphologic and functional cardiac abnormalities in systemic lupus erythematosus (SLE) and to relate these findings to the disease activity and duration, and the presence of antiphospholipid (APL) antibodies. Thirty consecutive patients with LES (5 male and 25 female, mean age 37 +/- 11 years) were studied with a clinical cardiovascular examination and M-mode, 2-D Doppler echocardiogram. All patients fulfilled the American Rheumatism Association criteria for diagnosis of SLE. Disease activity was scored using the "Lupus Activity Criteria Count". The duration of the disease was less than 1 year in 5 patients (16.7%), between 1 and 5 years in 7 (23.3%), and superior to 5 years in 18 (60%). No patient had a history of rheumatic fever or infective endocarditis. All patients had received steroid therapy. In 26.7% of patients the disease was active, and in 33.3% APL antibodies were present. Patients were matched by number, age and sex with the control group. In 73.3% of the patients the echocardiogram resulted abnormal; valvular disease occurred in 30% and the echocardiographic features were of diffuse thickening, with 4 mitral and 2 aortic regurgitations. No valvular dysfunctions were significant, nor was Libman-Sacks endocarditis present. Pericardial disease, effusion or thickening was detected in 33.3% of the echocardiograms. Furthermore, there was one patient with left ventricular mild hypertrophy; 2 with a mild enlargement of the left ventricle with no segmental abnormalities of wall motion and no systolic disfunction; 8 patients (26.7%) were normal. Compared with the control group, patients with SLE had an increased prevalence of echocardiographic abnormalities, especially pericardial (p < 0.001) and valvular (p < 0.01). No association was found between activity, duration of the disease and prevalence of cardiac abnormalities. On the contrary, an association between the presence of APL antibodies and cardiac abnormalities at the echocardiographic examination was evident (p < 0.05).

Long-term protein deficiency in adult rats : Effects on different proteins of a sympathetic ganglion.

The superior cervical ganglia (SCG) of adult rats were examined for total protein, LDH and TOH activity, and S-100 and tubulin content after 30 days of a proteinfree diet. After the depletion period, significantly lower values in all these parameters, in comparison with those in animals of the same age fed the complete diet, were found. The difference between the resistance of SCG and that of CNS to protein deficiency is discussed.