Absorptive activities of the efferent ducts evaluated by the immunolocalization of aquaporin water channels and lectin histochemistry in adult cats.

Ultrastructural and biochemical features of efferent ducts (EDs) are indicative of an intense absorptive activity towards the luminal fluid. This function was investigated by 1) the immunohistochemical localization of different aquaporins, integral membrane water channels that facilitate rapid passive movement of water, and 2) the histochemical localization of lectins, known to have specific affinity for glycoconjugate residues. AQP1 was mostly revealed at the apical surface and adluminal cytoplasm of non-ciliated cells and to a minor extent in their lateral plasma membrane, whereas it was absent in ciliated cells. Blood vessels showed AQP1-immunoreactivity, which was present in endothelial cells of venous vessels and capillaries and around the muscular sheath of arteries. AQP9 was expressed in the apical zone of ciliated and non-ciliated cells and in the lateral cell membrane. AQP2 and AQP5 were undetectable. Lectin histochemistry showed that non-ciliated cells contain glycans with terminal Neu5Acalpha2,3Galbeta1,3GalNAc, Neu5Acalpha2,3Galbeta1,4GlcNAc, Galbeta1,4GlcNAc, GalNAc (s-PNA, MAL II, RCA120, SBA reactivity) and with internal/terminal alphaMan (Con A affinity) at the luminal surface and the apical region. In addition, non-ciliated cells expressed oligosaccharides terminating with GalNAc and Neu5Acalpha2,6Gal/GalNAc (SNA reactivity) in the luminal surface and the apical zone, respectively. Ciliated cells revealed glycoconjugates only on cilia, which showed terminal Neu5Acalpha2,3Galbeta1,4GlcNAc (s-RCA120 staining) and GalNAc, as well as internal/terminal alphaMan and GlcNAc (s-WGA, GSA II staining). Data provide evidence for the involvement of different pathways in the bulk reabsorption of water and low molecular weight solutes by the non-ciliated cell of the cat EDs. AQP-mediated trans-cellular route can be hypothesized, together with fluid phase endocytosis mediated by the glycocalix and a well-developed endocytotic apparatus. Epithelial ciliated cells, whose main function is the movement of luminal content, might also participate in absorptive processes to a lesser extent.

Lectin-binding sites on ejaculated stallion sperm during breeding and non-breeding periods.

Stallion sperm from semen collected in Southern Italy during the breeding (June-July) and non-breeding (December-January) periods were analyzed by means of twelve lectins to evaluate the glycoconjugate pattern and to verify whether there are any seasonal differences in the glycosylation pattern of the sperm glycocalyx. The acrosomal cap showed reactivity for Maackia amurensis (MAL II), Sambucus nigra (SNA), Arachis hypogaea (PNA), Glycine max (SBA), Helix pomatia (HPA), Canavalia ensiformis (Con A) Triticum vulgaris (WGA), and Griffonia simplicifolia isolectin II (GSA II) in breeding and non-breeding ejaculated sperm, suggesting the presence of oligosaccharides terminating with Neu5Ac alpha 2,3Gal beta 1,4GlcNAc, Neu5Ac alpha 2,6Gal/GalNAc, with Gal beta 1,3GalNAc, alpha/beta GalNAc and glycans with terminal/internal alpha Man and GlcNAc. During the non-breeding period, the acrosomal cap expressed oligosaccharides terminating with Gal beta 1,4GlcNAc (Ricinus communis(120) affinity) (RCA(120)) and L-Fuc alpha 1,2Gal beta 1,4GlcNAc beta (Ulex europaeus affinity) (UEA I). The equatorial segment placed between the acrosomal cap and post-acrosomal region did not display glycans terminating with GalNAc, GlcNAc, and alpha L-Fuc. The post-acrosomal region of sperm collected in the breeding and non-breeding periods bound Con A, MAL II, SNA, and SBA, thus showing the presence of N-linked oligosaccharides from high-Man content, terminating with Neu5Ac alpha 2,3Gal beta 1,4GlcNAc, Neu5Ac alpha 2,6Gal/GalNAc and GalNAc. In winter, the post-acrosomal region also expressed oligosaccharides terminating with alpha GalNAc, GlcNAc, and L-Fuc alpha 1,2Gal beta 1,4GlcNAc beta (HPA, GSA II, and UEA I staining). The tail of sperm from semen collected during the breeding and non-breeding periods showed a lectin binding pattern similar to the post-acrosomal region, except for the absence of HPA staining in sperm collected during the winter season. These results indicate that the surface of stallion sperm contains different glycocalyx domains and that the glycosylation pattern undergoes changes during the breeding and non-breeding periods.

Lectin-binding sites in isolated equine cumulus-oocyte complexes: differential expression of glycosidic residues in complexes recovered with compact or expanded cumulus.

Equine cumulus-oocyte complexes (COCs) were analyzed by means of 13 lectins to evaluate their glycoconjugate patterns and to verify differences between COCs recovered with compact (Cp) and expanded (Exp) cumulus. Cumulus cells showed a similar staining pattern in both Cp and Exp COCs with all lectins used, except for a higher reactivity with SNA and GSA II in Cp COCs and SBA in Exp COCs. The zona pellucida (ZP) showed (1) uniform staining with MAL II, RCA(120), and SBA in both Cp and Exp COCs, (2) trilaminar binding pattern with WGA as well as higher Con A reactivity in the outer region of both types of COCs, (3) uniform staining with PNA only in Exp COCs, (4) uniform and trilaminar binding pattern with SNA in Cp and Exp COCs, respectively, and (5) major reactivity with GSA II in Exp COCs. Ooplasm showed similar staining intensity with Con A, HPA, GSA I-B(4), and WGA in both Cp and Exp COCs, with stronger reactivity to GSA II in Exp COCs, whereas SNA, UEA I, and LTA binding sites were present only in Cp COCs. Oocyte cortical granules of both Cp and Exp COCs reacted with Con A and WGA. These results suggest that, in the mare, viable (Cp) and atretic (Exp) COCs display different glycoconjugate staining pattern, which may account for the different maturation and developmental competence of COCs.

Brain morphology and immunohistochemical localization of the gonadotropin-releasing hormone in the bluefin tuna, Thunnus thynnus.

The present study was focused on the morphology of the diencephalic nuclei (likely involved in reproductive functions) as well as on the distribution of GnRH (gonadotropin-releasing hormone) in the rhinencephalon, telencephalon and the diencephalon of the brain of bluefin tuna (Thunnus thynnus) by means of immunohistochemistry. Bluefin tuna has an encephalization quotient (QE) similar to that of other large pelagic fish. Its brain exhibits well-developed optic tecta and corpus cerebelli. The diencephalic neuron cell bodies involved in reproductive functions are grouped in two main nuclei: the nucleus preopticus-periventricularis and the nucleus lateralis tuberis. The nucleus preopticus-periventricularis consists of the nucleus periventricularis and the nucleus preopticus consisting of a few sparse multipolar neurons in the rostral part and numerous cells closely packed and arranged in several layers in its aboral part. The nucleus lateralis tuberis is located in the ventral-lateral area of the diencephalon and is made up of a number of large multipolar neurones. Four different polyclonal primary antibodies against salmon (s)GnRH, chicken (c)GnRH-II (cGnRH-II 675, cGnRH-II 6) and sea bream (sb)GnRH were employed in the immunohistochemical experiments. No immunoreactive structures were found with anti sbGnRH serum. sGnRH and cGnRH-II antisera revealed immunoreactivity in the perikarya of the olfactory bulbs, preopticus-periventricular nucleus, oculomotor nucleus and midbrain tegmentum. The nucleus lateralis tuberis showed immunostaining only with anti-sGnRH serum. Nerve fibres immunoreactive to cGnRH and sGnRH sera were found in the olfactory bulbs, olfactory nerve and neurohypophysis. The significance of the distribution of the GnRH-immunoreactive neuronal structures is discussed.

Functional and clinical changes in upper limb spastic patients treated with botulinum toxin (BTX).

Spasticity is a motor disorder characterized by a velocity-dependent increase in tonic stretch reflexes (muscle tone) with exaggerated tendon jerks. In order to study the usefulness of botulinum toxin type A (BTX) as a therapy for spasticity, we studied 15 patients affected by spasticity secondary to stroke. Tests included: clinical evaluation of tone (Ashworth scale); active angles of extension and flexion at elbow and wrist; Hmax/Mmax ratio from flexor carpi radialis (FCR); Hreflex presynaptic inhibition from FCR during vibration; Task score; and video recording. Patients were injected with BTX into one or more muscles with total doses not exceeding 200 International Units (IU). The tests were performed immediately prior to injection and repeated 2 weeks afterwards. Furthermore, in eight patients, testing was also performed one month after BTX injection. Between two weeks and one month after BTX there were no statistically significant differences. A statistically significant difference in the Task and Ashworth scores before and after treatment emerged (p < 0.0014), but only 6 patients showed a clear improvement in motor performance. Overall, we observed an improvement in the angle of active extension and flexion at the wrist and elbow. There were no significant changes in the Hmax/Mmax ratio and the Hreflex presynaptic inhibition during vibration. All the patients reported a subjective improvement. The results suggest that subjective benefits can be gained from the use of BTX in patients affected by spasticity, and that the degree of motor improvement seems to depend on the motor recovery obtained before treatment.

Mechanism of binocular interaction in refraction errors: study using pattern-reversal visual evoked potentials.

In this study we sought to determine whether a natural condition involving fine discrimination, for example moderately severe myopia, might yield interesting information regarding the binocular interaction expressed by visual evoked potentials (VEPs). We studied ten normal subjects with a mild refraction deficits. Transient VEPs were elicited by monocular and binocular stimulation under conditions of natural and lens-corrected vision. The visual stimulus was a pattern-reversal checkerboard consisting of 15' and 40' checks. VEPs in response to binocular stimulation were compared with monocular VEPs. We plotted the monocular 'better-VEP' and 'worse-VEP' response, since significant differences between individual eye stimulations were present. We found no significant difference between the mean N75 and P100 latencies of the binocular VEP and the better monocular VEP, regardless of the check size used and of natural or corrected vision. Under all stimulus conditions, the mean amplitude of the N75-P100 of the binocular VEPs was also larger than the better monocular VEP response. The difference proved more significant when we stimulated our subjects with smaller squares and left vision uncorrected. The mean P100-N145 amplitude obtained with binocular stimulation was larger than the better monocular VEP response only when using small checks (15') and uncorrected vision. Overlapping latencies are consistent with an earlier hypothesis that monocular and binocular VEPs originate postsynaptically from the binocular neurons in the primary visual cortex. The gain in amplitude achieved by binocular stimulation may depend upon the removal of 'tonic interocular inhibition' and/or on a cortical modulatory mechanism.

Reversible changes of motor cortical outputs following immobilization of the upper limb.

We mapped the cortical representations of the abductor pollicis brevis, flexor carpi radialis, biceps and deltoid muscles in six subjects with unilateral wrist fractures, immediately after the removal of the splint. This was repeated 1 month later in three out of the six subjects. Duration of immobilization was 1 month. Muscle maps were obtained by delivering four focal magnetic pulses for each scalp position (1 cm apart with reference to Cz) over the contralateral hemisphere. Motor evoked potentials (MEPs) were averaged off-line and expressed as a percentage of the motor action potential evoked by supramaximal peripheral nerve stimulation. Volume, area and threshold of the motor maps showed no significant hemispheric differences within each muscle in 10 control subjects. In the first recording session the volume of each immobilized muscle was distinctly higher when compared to that of controls in terms of absolute value and side-to-side ratio. This finding disappeared 1 month later. Moreover, MEP amplitude difference recorded from hand muscle could be reversed during a small tonic voluntary contraction. Immobilization had no significant effect on the threshold for activation of the target muscles and on the area of the motor map. The increase in MEP amplitudes occurred without changes in spinal excitability as tested by the F wave. These findings suggest that immobilization of the upper limb induces a reversible enhancement of the excitability of structures along the corticomotoneuronal pathway. Sustained restriction of volitional movements and reduction in somatic sensory inputs might promote this functional modulation of the motor system.

Binocular interaction in normal vision studied by pattern-reversal visual evoked potential (PR-VEPS).

Monocular and binocular visual evoked potentials (VEPs) in response to different check size (15-21-38-84 minutes or arc) were studied in 14 subjects with normal visual acuity and stereopsis. The binocular VEP amplitude is slightly higher than the VEP amplitude on stimulation of the "better eye" and significantly higher than the VEP amplitude on stimulation of the "worse eye"; this effect is observed using small checks and almost exclusively involved N75-P100. Both the N75 and P100 peaks occur earlier after binocular than monocular stimulation. The shortening of the N75 mean latency is significantly greater than that of the P100 mean latency when larger check sizes are used. The mean latency of the N145 potential is not significantly different in monocular and binocular stimulus conditions. The slight summation effect and latency shortening in the binocular VEPs are not consistent with the hypothesis that it is the sum of separate monocular signals originating from the visual cortex that gives rise to the response. The early components of both monocular and binocular VEPs are thought to be of post-synaptic origin (outside layer 4c of area 17), where the inputs become mixed so that most cells receive information from both eyes. The amplitude enhancement of binocular VEPs, which mainly occurs when using small checks, may be related to the increase in the total amount of cortical activity representing the macular region; this may account for binocular superiority in fine spatial resolution. The latency shortening in binocular conditions can be explained by considering that the critical determinant of the latency is the fundamental spatial frequency of the pattern. When coarse patterns are used, their effectiveness in parafoveal stimulation may affect the VEPs, with a significant contribution coming from the more peripheral retina. The enlargement of the visual field when the eyes see simultaneously may therefore further reduce the latency of the response when using the larger checks suitable for eccentric stimulation.

Effect of pharmacologically induced mydriasis on the normal variability of retinal oscillatory potentials in man.

In this study we describe certain aspects of normal variability of oscillatory potentials, with particular reference to interaction between pupil size and stimulus intensity. The mean latencies of the earlier oscillatory potentials, O1 and O2, were significantly shorter when adopting the dilated pupil condition than when stimulating with a normal pupil. This occurred at various light intensities, although the difference was more significant at the highest intensity. O1 and O2 latencies became significantly longer as the stimulus intensity was progressively reduced. The latencies of the later components O3 and O4 were unaffected. The O1 and O2 amplitudes were not influenced by pupil size, but a significant reduction occurred with the progressive decrease in stimulus intensity. The later components O3 and O4 are greatly reduced in amplitude after pupil dilatation using higher stimulus intensities; the O4 potential may even disappear in 10% of the cases. These data provide further support for the existence of two distinct behavior patterns for earlier and later oscillatory potentials. Although light adaptation may affect oscillatory potentials, we suggest that in our experimental conditions, oscillatory potential changes may occur as a result of the activity of the neural modulating system, not only as a result of photoreceptor interaction.

GABA concentrations in different cerebral areas of bass (dicentrarchus labrax).

In these investigations GABA concentrations in different cerebral areas (cortex, diencephalon, optic lobes and mesencephalon) of bass (Dicentrarchus labrax) were evaluated. According to the results obtained, the GABA, like other chemical mediators (noradrenaline, dopamine, serotonin), investigated in different animal species, shows a variable distribution in the CNS of bass. The highest levels of GABA were observed in the cerebral cortex and the lowest ones in the mesencephalon. Mean values were observed in the diencephalon and the optic lobes. The study of the GABA-ergic system of the CNS of fish, could provide a valid contribution to the comprehension of several biochemical and physiopathological mechanisms responsible for the behavioural activity and/or the appearance of neurotoxic phenomena.

[Comparative anatomo-microscopic observations on the esophagus of teleosts with different feeding habits]. / Osservazioni anatomo-microscopiche comparate sull'esofago di teleostei a diverso regime alimentare.

The authors describe the micro-anatomy of the oesophagus of different diet fish: herbivorous (Boops salpa), omnivorous (Liza aurata) and carnivorous fish (Blennius gattorugine and Gaidropsarus mediterraneus). In all the observed species the oesophagus is formed by a tunica mucosa, tunica muscularis and tunica serosa. The tunica mucosa of the herbivorous fish arises to form thin plicae which protrude into the lumen and are covered with squamous stratified epithelium. The goblet cells do not arrange among the covering cells but take place laterally and on the basis of the plicae. The tunica muscularis is constituted by an external circular layer and an inner longitudinal layer of striated muscle. The oesophagus of omnivorous fish shows thick plicae covered with a higher epithelium than that of the herbivorous fish. Among the epithelial cells a lot of taste buds have been found. The tunica muscularis is like that of the herbivorous fish. In the carnivorous fish Blennius the tunica mucosa presents thin plicae like those of the herbivorous fish but they are covered with a thicker epithelium. The tunica muscularis is formed by an external circular layer and by an inner longitudinal layer which constitutes two diametrically opposed bundles placed dorsally and ventrally. In the other carnivorous fish Gaidropsarus, the tunica mucosa shows very thick plicae covered with a quite high squamous epithelium among which there are many goblet cells. In this fish too numerous taste buds have been found. The tunica muscularis is arranged in the same manner observed in the other carnivorous fish.

"Cyto-assisted", ultrasound-guided biopsy in the diagnosis of focal disease of the abdomen.

From May 1988 to June 1990, 260 patients with abdominal focal disease underwent ultrasonically guided percutaneous fine needle biopsy. The technique of so-called "FNAB-CYT" is described. The procedure entailed very few complications: the mortality rate was 0% and the morbidity rate was 1.5%. The specificity of the technique was 100%, and sensitivity was 93.43%. False-negative diagnoses were made in 8 cases, and in 5 patients the cytologic diagnoses were uncertain. There were no false-positives. The possibility of an immediate control of the collected material by the pathologist avoids the risk of inadequate samples and reduces the number of biopsies for the patient. Comparison among diagnoses on rapid and definitive preparations and histologic ones was carried out to evaluate the diagnostic efficiency of the cytologic procedure. In our experience, a rapid staining of the smears allowed a correct cytologic diagnosis in 87.7% of the cases within approximately 5 min of the biopsy. On the basis of our experience, the authors recommend FNAB-CYT as a routine first-level (less invasive) procedure for diagnosis of abdominal focal disease.

[Comparative macroscopic and microscopic anatomical observations on pyloric ceca in some bony fish]. / Osservazioni anatomo-macro-microscopiche comparate sui ciechi pilorici di alcuni pesci ossei.

The authors report the variable number of pyloric caeca of some species of fish and describe their macroanatomy. They also describe and compare the microanatomy of pyloric caeca in three species; in particular they point out the thickness of tunica muscularis, the shape and length of villi which protrude into the lumen and the variable number of goblet cells. The tunica muscularis is very strong in Uranoscopus and consists of three strata: external and inner longitudinal strata, middle circular stratum. The tunica muscularis is less thick in Diplodus and in Scorpaena. The lamina propria and epithelium form villi which protrude into the lumen. They are covered with columnar epithelium and goblet cells which are more numerous in Uranoscopus and Scorpaena and less numerous in Diplodus. At last the authors describe in pyloric caeca of Scorpaena the presence of a network which engages all the lumen; it consists of connective tissue septa which directly derive from the lamina propria and is covered with the same columnar epithelium and goblet cells.