Palmitoylethanolamide reduces pain-related behaviors and restores glutamatergic synapses homeostasis in the medial prefrontal cortex of neuropathic mice.

BACKGROUND: Enhanced supraspinal glutamate levels following nerve injury are associated with pathophysiological mechanisms responsible for neuropathic pain. Chronic pain can interfere with specific brain areas involved in glutamate-dependent neuropsychological processes, such as cognition, memory, and decision-making. The medial prefrontal cortex (mPFC) is thought to play a critical role in pain-related depression and anxiety, which are frequent co-morbidities of chronic pain. Using an animal model of spared nerve injury (SNI) of the sciatic nerve, we assess bio-molecular modifications in glutamatergic synapses in the mPFC that underlie neuropathic pain-induced plastic changes at 30 days post-surgery. Moreover, we examine the effects of palmitoylethanolamide (PEA) administration on pain-related behaviours, as well as the cortical biochemical and morphological changes that occur in SNI animals. RESULTS: At 1 month, SNI was associated with mechanical and thermal hypersensitivity, as well as depression-like behaviour, cognitive impairments, and obsessive-compulsive activities. Moreover, we observed an overall glutamate synapse modification in the mPFC, characterized by changes in synaptic density proteins and amino acid levels. Finally, with regard to the resolution of pain and depressive-like syndrome in SNI mice, PEA restored the glutamatergic synapse proteins and changes in amino acid release. CONCLUSIONS: Given the potential role of the mPFC in pain mechanisms, our findings may provide novel insights into neuropathic pain forebrain processes and indicate PEA as a new pharmacological tool to treat neuropathic pain and the related negative affective states. Graphical Abstract Palmitoylethanolamide: a new pharmacological tool to treat neuropathic pain and the related negative affective states.

Apoptosis during chick inner ear development: some observations by TEM and TUNEL techniques.

In order to clarify the occurrence, distribution and possible role of apoptosis during inner ear development, the ultrastructural aspects (by TEM) (at 9-19 incubation day and 1 day after hatching) and the distribution of the apoptotic phenomenon (by the TdT-mediated dUTP nick end-labeling technique), were studied in the crista ampullaris of chick embryo at 5-19 days of incubation to hatching and of postnatal 1-day old chick. We found, in the sensorial epithelium, dark supporting cells in chick embryos and mainly dark hair cells in postnatal chicks, both with ultrastructural features consistent with those of apoptosis. The presence of apoptotic phenomena was confirmed by the TUNEL technique. According to our findings, it is hypothesized that apoptosis in the inner ear may be involved: 1) at first, in macroscopic remodelling of the membranous labyrinth in early developmental stages, 2) later, in the correct differentiation of the hair and of the supporting cells, leading to characteristic cellular pattern formation and 3) finally, in physiological cell turnover of the postnatal chicken sensorial epithelium of the crista.

Calcification processes in the chick otoconia and calcium binding proteins: patterns of tetracycline incorporation and calbindin-D28K distribution.

In order to clarify the otoconia formation and turnover, tetracycline, an antibiotic that precipitates at calcifying fronts and serves as a fluorescent marker, was injected into eggs at different stages of chick embryonic development, as well as into postnatal chicken and into adult animals. The changes in the intensity, location patterns and time course of fluorescent labelling in each examined stage in the otolithic organs was studied. The presence and distribution of calbindin (CB)-D28K, one of the calcium-binding proteins constantly found in the mammalian and chicken cochlea and also in otolithic membrane of some adult mammals, was studied. Results in embryonal stages, postnatal and adult animals allow us to postulate that otoliths are mainly produced during the embryonal phase, but they may also be produced throughout the whole life span. Results also indicate that otoconia are dynamic structures which undergo turnover. The correspondence between the patterns of CB-D28K immunoreactivity and tetracycline fluorescence may indicate that CB-D28K participates in the formation of otoconia.

Immunocytochemistry of carbonic anhydrase and non-radioactive detection of its RNA by in situ hydridization in the chick embryo membranous labyrinth.

An immunocytochemical study was carried out with the aim to localize carbonic anhydrase isozymes (CA I and CA II) in the chick membranous labyrinth. CA I and CA II were localized in the same cells and, during the early developmental stages, were diffusely present in the labyrinthine epithelium. At later stages, the sensorial epithelium of the maculae acusticae, crista ampullaris and papilla basilaris, as well as labyrinthine dark cells and the epithelium of the endolymphatic sac strongly stained by the immunocytochemical procedure. In 1-day and 1-week old chicks, CA was present in the same sites as at advanced embryonal stages. In order to detect mRNA for carbonic anhydrase II, in situ hybridization was also used. Strong hybridization signals were observed in the sensorial epithelium of the saccule, utricle and crista ampullaris, in the tegmentum vasculosum, the endolymphatic sac, the erythrocytes and the choroid plexus in embryos older than 10 days. This positivity persisted until the day after hatching. The authors discuss the significance of labyrinthine CA and, based on in situ hybridization results, suggest that melanocytes do not provide labyrinthine dark cells with enzyme protein.

Bethanechol versus antiacids in the treatment of gastroesophageal reflux.

To compare the efficacy of bethanechol in the treatment of gastroesophageal reflux with that of antiacids, a prospective, cross-over study was undertaken, in which 20 affected infants and children were randomized into two groups on 6-week alternate bethanechol and antiacids oral medication. Patients were evaluated clinically and by esophageal pH-metry before and after each treatment. Clinical score amelioration was achieved earlier than reflux number reduction and with similar incidence in both groups of patients, irrespectively to the initial medication; moreover, the differences in the degree of improvement between the two groups after either treatment were not found to be significant. These results fail to show that bethanechol is more effective than antiacids in controlling gastroesophageal reflux; moreover, bethanechol is more difficult to administer and offers a higher rate of undesired side effects.

Calcite in the statoconia of amphibians: a detailed analysis in the frog Rana esculenta.

The otoconia of Amphibia, especially of the frog Rana esculenta, were studied by X-ray diffraction and scanning electron microscopy (SEM). The SEM studies showed that the membranous labyrinth of Amphibia contains two populations of crystals, which can easily be distinguished by their forms. The X-ray diffraction data indicated that these two populations consist of calcite and aragonite; the endolymphatic sac, the saccule and the lagena contain aragonite, whereas calcite is only found in the otolithic membrane of the utricle. The genetic and functional significance of the existence of two crystalline forms of calcium carbonate in the membranous labyrinth are discussed.

Age-related changes in the prevalence of precipitating antibodies to BK virus in infants and children.

Nearly 1000 sera from children were tested by immunoelectro-osmophoresis against BK virus, and age-specific prevalence rates were estimated from birth until the age of 12 years. Declining rates during the first 12 months showed the waning of passive immunity, which at birth reflects the mother's immune status. The changes of prevalence suggested that the peak incidence of primary infections occurred at about 2 years, with an estimated peak annual rate of 24.6%.

Development, structure and composition of the otoliths in vertebrates.

The vertebrate otoliths are biologically-altered crystals composed of an organic matrix and inorganic constituents. The organic component contains proteins and an acidic mucosubstance with sulphuric and carboxylic groups. The inorganic component of the otoliths in gnathostome vertebrates is almost always composed of polymorphs of calcium carbonate, i.e. calcite, aragonite and rarely, vaterite. This work reviews mainly the morphological aspects (by scanning electron microscopy), and phenomena of seeding and of growth of otolithic biocrystals. Factors essential for seeding and growth of otolithic biocrystals are discussed. Congenital otolithic defects, in fact, can be caused by various environmental factors (carbonic anhydrase inhibitors, manganese and zinc dietary deficiencies) and by genetic factors. The main site of the formation of endogenous otoliths is believed to be the macula and in some cases the endolymphatic sac. Origin of the organic and inorganic constituents of the biocrystals is also discussed. Studies on the process of decalcification of otoliths in some species indicate the endolymphatic sac as a versatile calcium deposit.

Scanning electron microscopic and x-ray diffraction studies of otoconia in the lizard Podarcis s. sicula.

The otoliths of embryos and young animals of the lizard Podarcis s. sicula were studied by X-ray diffraction and scanning electron microscopy. Two types of crystal that give different X-ray diffraction patterns were found in the membranous labyrinth of Podarcis. The crystals consist of calcite or aragonite and are easily distinguished by scanning electron microscopy because of their different morphology. The two calcium carbonate crystal forms are not mixed at random but are present in the embryo from the very beginning in specific sites. The endolymphatic sac contains aragonite crystals while the saccule contains calcite crystals adjacent to the wall, in addition to a preponderance of aragonite crystals. The utricle and lagena contain only calcite crystals. The presence of two crystal forms of calcium carbonate in the membranous labyrinth are discussed in terms of differing genetic and functional significance.