Arabinogalactan as active compound in the management of corneal wounds: in vitro toxicity and in vivo investigations on rabbits.

PURPOSE: Aims of the present investigation were to prove that natural polysaccharide arabinogalactan (AG) is well tolerated after ocular administration and exerts a high restoring effect on corneal epithelium abrasions. MATERIALS AND METHODS: AG interactions with corneal cells, as well as its effect on their proliferation, were evaluated employing rabbit corneal epithelial cell cultures. The effects due to the presence of benzalkonium chloride (BAK) were also studied on cell cultures, ex vivo on rabbit isolated corneas, evaluating the hydration level, and on the healing rate of experimental corneal wounds in rabbits. Furthermore, the healing process of corneal lesions treated with an experimental 5.0% AG solution was studied and compared with those obtained applying solutions of hyaluronic acid and tamarind seed polysaccharide, both chosen as a reference by virtue of their well-known adjuvant properties on corneal trophism; the study was carried out by light and transmission electron microscopy. RESULTS: BAK showed toxic effects on corneal epithelium in all experiments. AG proved to stimulate the growth of the corneal epithelial cells by interacting at the level of the cell plasma membrane. The microscopy observations of the epithelial surface of AG-treated damaged corneas revealed a well-restored and histologically organized ultrastructure characterized by fully formed microvilli and glycocalyx; the healing process resulted faster with respect to spontaneously recovered untreated corneas. CONCLUSION: Our results suggest that AG can interact with corneal epithelial cells without any toxic side effect; moreover, it proved to stimulate cell proliferation, thus promoting tissue re-epithelialization and reorganization just 48 hr post-wounding.

Short-term effects of 3,4-methylen-dioxy-metamphetamine (MDMA) on 5-HT(1A) receptors in the rat hippocampus.

The first effects of 3,4-methylen-dioxy-metamphetamine (MDMA, "ecstasy"), on serotonin 1A (5-HT(1A)) receptors in rat hippocampus were determined by means of [(3)H]-8-hydroxy-dipropylamino-tetralin ([(3)H]-8-OH-DPAT) and 5'guanosine-(gamma-[(35)S]-thio)triphosphate ([(35)S]-GTPgammaS) binding as well as inhibition of forskolin (FK)-stimulated adenylyl cyclase (AC) activity. The study was completed by [(35)S]-GTPgammaS functional autoradiography experiments carried out in frontal sections of rat brain, including the hippocampal region. Results showed that MDMA was either able to displace [(3)H]-8-OH-DPAT binding (K(i) congruent with 500 nM) or to reduce the number of specific sites (B(max)) without affecting K(d). The drug also failed to change the [(35)S]-GTPgammaS binding or to inhibit AC velocity, underlying its behavior as a non-competitive 5-HT(1A) receptor antagonist. Further, MDMA (1 or 100 microM), partially antagonized either [(35)S]-GTPgammaS binding stimulation of the agonists 5CT and 8-OH-DPAT or the AC inhibition induced by 5CT and DP-5CT. However, in contrast to binding studies, in AC assays the amphetamine displayed an effect also on EC(50), always being less potent than the reference antagonist WAY100,635. In functional autoradiography, MDMA behaved either as a partial 5-HT(1A) antagonist in limbic areas or, added alone, as an agonist, increasing the coupling signal presumably through 5-HT release from synapses. Interestingly, the selective 5-HT re-uptake inhibitor (SSRI) fluoxetine had no effect on MDMA [(35)S]-GTPgammaS binding activation. This latter finding indicates that the amphetamine can release 5-HT via alternative mechanisms to 5-HT transporter binding, probably via membrane synaptic receptors or vesicular transporters. The release of other transmitters is not excluded. Therefore, our results encourage at extending the study of MDMA biochemical profiles, in the attempt to elucidate those amphetamine-induced pathways with a potential for neurotoxicity or psycho-stimulant activity.

[3H] muscimol receptors sites in the carp (Cyprinus carpio L.) brain: binding assay and autoradiographic distribution.

Autoradiographic and binding techniques were used to study the presence of [(3)H]muscimol receptors sites in the carp brain. The radioligand was distributed with an high degree of anatomical selectivity. We found abundant labelling in the cerebellum, in the nucleus diffusus lobi inferioris, and in the torus longitudinalis. No labelling was detected within the epithalamus, thalamus and hypothalamus, while the telencephalon and the rhombencephalon displayed a low density of [(3)H]muscimol receptors sites. Binding assay showed the highest concentration of receptor sites in the nucleus diffusus lobi inferioris and the lowest in the medulla oblongata. Presence of [(3)H]muscimol binding sites within the visceral sensory area was noted. The rank order of displacement efficacy of unlabelled ligands observed, suggested that in brain membranes of carp the receptor binding of [(3)H]muscimol has the same pharmacological specificity previously reported in a large number of experiments with tissue homogenates. A general agreement in binding and autoradiography was observed. The present findings suggested that muscimol receptor could be involved in neuronal pathway controlling basic central actions like gustatory signal processing or spatial learning acquisition and retention.

Autoradiographic localization and binding study of benzodiazepines receptor sites in carp brain (Cyprinus carpio L.).

This study demonstrates, for the first time, by both autoradiography and binding assay that [3H]Ro 15-1788 binds to carp brain with a high degree of anatomical selectivity. Saturation binding of the radioligand was determined in seven anatomically defined regions and suggested the presence of one class of binding sites (Type I-lke). In general, there was a good correlation between the autoradiographic and the binding data. By far, the optic tectum and the vagal, facial, and glossopharyngeal lobes showed the majority of [3H]Ro 15-1788 binding sites. Low to negative concentration of binding sites was detected in the cerebellum. The location of [3H]Ro 15-1788 binding sites in particular brain regions, indicates that benzodiazepine receptors could be associated with pathways involved in the control of basic central functions as spatial learning acquisition and retention, and feeding behaviour.

Immunohistochemical distribution of neuropeptide Y in the mesencephalon and rhombencephalon of carp, Cyprinus carpio L. (Cyprinidae: Teleostei).

The localization of neuropeptide Y (NPY)-immunoreactive elements was investigated in the mesencephalon and rhombencephalon of carp, Cyprinus carpio, by using antisera raised against porcine NPY and the immunoperoxidase technique. Concurrently, to identify the distribution of NPY-immunoreactivity, we developed an atlas of the studied areas based on Nissl-stained sections. The NPY-immunoreactive (NPY-ir) elements were located in many zones of the mesencephalon and rhombencephalon. In the mesencephalon, positive fibers were the most abundant elements while neurons were scarce. The rhombencephalon rostral part was characterized by a low to moderate fiber density, distributed in the ventro-medial and ventro-lateral region. Differently the caudal part of the rhombencephalon exhibited several NPY-ir elements. In particular, a high density of immunoreactivity was located in the gustatory area at the level of the nucleus (n.) originis nervi glossopharyngei, in the n. nervi vagi, and in the vagal lobe. The latter can be considered a valid neuroanatomical model for the study of gustatory signal processing in vertebrates. Our results regarding the primary gustatory centers give neuroanatomical support to the view that NPY may act as a neurotransmitter and/or a neuromodulator in a wide neural network for feeding behavior control.

Development of cultured rabbit corneal epithelium for drug permeation studies: a comparison with excised rabbit cornea.

The aim of this study was to prepare and test an artificial corneal epithelium (reconstituted rabbit corneal epithelium, RRCE) exhibiting barrier characteristics and paracellular permeability similar to those of native rabbit cornea. The RRCE was obtained from a rabbit corneal epithelium (RCE) cell line grown for 8 days in submerged culture, then for 7 days in air-interface conditions on Snapwell polyester membranes. Permeation studies on the RRCE were carried out in comparison with rabbit excised corneas in vitro, using timolol maleate (TM) as the test drug, alone and in association with the following ocular permeation enhancers: benzalkonium chloride, ethylene-diaminetetraacetic acid sodium salt, polyethoxylated castor oil, polyoxyethylene stearyl ether, sodium deoxycholate, and escin. The integrity of the RRCE was assessed by measuring the transepithelial electrical resistance (TEER) during culture time and after every permeation experiment. When TM was tested alone, the permeation parameters (apparent permeability coefficient, lag time) obtained with the RRCE were similar to those of excised rabbit corneas. The artificial epithelium, however, was less sensitive than native cornea to the effect of permeation enhancers.

Autoradiographic distribution of neuropeptide Y binding sites in the brain of the carp Cyprinus carpio L. (Cyprinidae, Teleostei).

The present study reports the distribution of neuropeptide Y (NPY)-binding sites in the brain of the adult carp Cyprinus carpio L. Radioiodinated NPY was used as tracer in the autoradiographic procedure. The NPY-binding sites (NPY-bs) were widely distributed in the carp brain. Generally, a good match was observed between the distribution of NPY-bs and the distribution of NPY-immunoreactive (NPY-ir) elements previously reported in the forebrain of the carp. Low to moderate concentration of NPY-bs were found in the telencephalon, this finding indicates that NPY may play a role in the processing of olfactory inputs and in more complex behaviours like spatial learning acquisition and retention, whose importance could correlated with similar results obtained in mammals. Moreover, in the rhombencephalon, the presence of NPY-bs at level of lobus vagus and the lobus facialis suggests that NPY may be implicated in food-seeking behaviour and swallowing reflex.