Proof-of-concept validation of the mechanism of action of Src tyrosine kinase inhibitors in dystrophic mdx mouse muscle: in vivo and in vitro studies.

Src tyrosine kinase (TK), a redox-sensitive protein overexpressed in dystrophin-deficient muscles, can contribute to damaging signaling by phosphorylation and degradation of ß-dystroglycan (ß-DG). We performed a proof-of-concept preclinical study to validate this hypothesis and the benefit-safety ratio of a pharmacological inhibition of Src-TK in Duchenne muscular dystrophy (DMD). Src-TK inhibitors PP2 and dasatinib were administered for 5 weeks to treadmill-exercised mdx mice. The outcome was evaluated in vivo and ex vivo on functional, histological and biochemical disease-related parameters. Considering the importance to maintain a proper myogenic program, the potential cytotoxic effects of both compounds, as well as their cytoprotection against oxidative stress-induced damage, was also assessed in C2C12 cells. In line with the hypothesis, both compounds restored the level of ß-DG and reduced its phosphorylated form without changing basal expression of genes of interest, corroborating a mechanism at post-translational level. The histological profile of gastrocnemius muscle was slightly improved as well as the level of plasma biomarkers. However, amelioration of in vivo and ex vivo functional parameters was modest, with PP2 being more effective than dasatinib. Both compounds reached appreciable levels in skeletal muscle and liver, supporting proper animal exposure. Dasatinib exerted a greater concentration-dependent cytotoxic effect on C2C12 cells than the more selective PP2, while being less protective against H2O2 cytotoxicity, even though at concentrations higher than those experienced during in vivo treatments. Our results support the interest of Src-TK as drug target in dystrophinopathies, although further studies are necessary to assess the therapeutic potential of inhibitors in DMD.

Alcohol use and abuse: a cross-sectional study among Italian adolescents.

INTRODUCTION: Alcohol is recognized as one of four major risk factors for non-communicable diseases. Exposure to alcoholic beverages during the adolescence has been linked to increased heavier drinking habits: obviously, the age of alcohol initiation resulted an important determinant of alcohol dependence. The aim of this study is to analyze knowledge, attitudes and practices in alcohol habit of adolescent population. METHODS: 943 students from 13 schools (middle and upper secondary schools) of the Bari district were enrolled in the study: in each school one class for each age was randomly selected. The research was carried out by an anonymous, self-administered questionnaire which investigated alcohol consumption, knowledge of alcohol consumption of parents and knowledge of the law regulating alcohol consumption. RESULTS: 34.8% (328) have never consumed alcoholic drinks while 65.2% (615) declare the use of alcohol; the average age of alcohol initiation was 12.2 years. 35.7% (329/921) of mothers and 36.6% (335/915) of fathers drink alcohol only on special occasions. 17.9% (168/939) considered that alcohol could be free sale at all while 16.4% (154/939) reported that sale is forbidden for children under 14. CONCLUSIONS: The higher prevalence of alcohol habits and the poor knowledge on alcohol law seemed to indicated the need of improving public health efforts in the prevention of alcohol consumption among Italian adolescents.

Acute exposure to methylmercury at two developmental windows: focus on neurobehavioral and neurochemical effects in rat offspring.

The neurobehavioral and neurochemical effects produced by prenatal methylmercury exposure (8 mg/kg, gestational-days 8 or 15), were investigated in rats. On postnatal day 40, animals exposed to methylmercury and tested in the open field arena, showed a reduction in the number of rearings, whereas the number of crossings and resting time was not altered with respect to the age-matched control rats. The methylmercury-exposed groups showed a lower level of exploratory behavior as well as an impairment in habituation and working memory when subjected to the novel object exploration task. The neophobia displayed by methylmercury-exposed rats is unlikely to be attributed to a higher degree of anxiety. Prenatal methylmercury exposure did not affect motor coordination or motor learning in 40-day-old rats subjected to the balance task on a rotating rod, and it did not impair the onset of reflexive behavior in pups screened for righting reflex, cliff aversion and negative geotaxis. In cortical cell cultures from pups exposed to methylmercury during gestation, basal extracellular glutamate levels were higher, whereas the KCl-evoked extracellular glutamate levels were lower than that measured in cultures from rats born to control mothers. In addition, a higher responsiveness of glutamate release to N-methyl-D-aspartic acid receptor activation was evident in cortical cell cultures from pups born from methylmercury-treated dams than in cultures obtained from control rats. The present results suggest that acute maternal methylmercury exposure induces, in rat offspring, subtle changes in short-term memory as well as in exploratory behavior. These impairments seem to be associated to alterations of cortical glutamatergic signaling.

Neurofunctional effects of developmental alcohol exposure in alcohol-preferring and alcohol-nonpreferring rats.

The neurofunctional effects of developmental alcohol exposure (3% v/v solution from day 15 of gestation to day 7 after parturition) have been investigated in Sardinian alcohol-preferring (sP) and alcohol-nonpreferring (sNP) rat lines, selectively bred for opposite alcohol preference and consumption. Alcohol exposure significantly decreased the rate of ultrasonic emission in sP male pups; whereas, it did not affect this indicator of emotional reactivity in sNP animals. Perinatal alcohol intake did not influence either learning of an active avoidance task or hippocampal long-term potentiation in both offspring lines. Significant differences in time spent exploring novel objects were observed between control sP and sNP rats subjected to the novel exploration object test. Alcohol exposed sP rats, but not alcohol exposed sNP rats, apparently lost the capacity to discriminate between the novel and the familiar object, even though this difference is difficult to interpret because of the large differences in the respective responses to the novel objects. Neurochemical experiments have shown that basal levels of dopamine (DA) and homovanillic acid (HVA) were significantly higher in the nucleus accumbens (NAC) of sP rats with respect to sNP animals. Perinatal alcohol did not affect basal DA and HVA concentrations or amphetamine-induced DA increase and HVA decrease in the NAC of either sP or sNP offspring. These results suggest that subtle behavioral alterations induced by developmental exposure to low doses of alcohol, which do not cause malformations and/or overt neurotoxicity, may be associated with genetic factors, although not necessarily those responsible for differences in alcohol preference.

Prenatal exposure to low concentrations of carbon monoxide alters habituation and non-spatial working memory in rat offspring.

Inhalation of low concentrations (75 and 150 ppm) of carbon monoxide (CO) by pregnant rats from days 0 to 20 of gestation leads to alterations in habituation and working memory in young adult male offspring subjected to the novel exploration object test. In particular, lack of habituation upon the second presentation of the objects and failure in the ability to discriminate between the novel and the familiar object were found in CO (75 and 150 ppm)-exposed offspring. These alterations were not accompanied by changes in spontaneous motor activity (open field test). The subtle behavioral deficits observed in the present study have been produced by prenatal exposure to CO levels resulting in maternal blood carboxyhaemoglobin (HbCO) concentrations equivalent to those observed in human cigarette smokers.

Maternal cocaine exposure alters mesolimbic dopaminergic function in rat offspring.

Hooded Lister female rats were treated with either saline or cocaine (20 mg/kg s.c.) from gestational day 10 every other day until weaning (postnatal day 25). In vivo microdialysis has shown that maternal cocaine exposure significantly decreases basal extracellular concentrations of dopamine in the nucleus accumbens of young-adult offspring (4 weeks after cessation of cocaine treatment). Moreover, the increase in extracellular dopamine levels induced by a challenge dose of K+ (intracerebral 60 mM K+ artificial cerebrospinal fluid (aCSF) infusion) or cocaine (15 mg/kg i.p.) was significantly attenuated in rats exposed to cocaine during perinatal life with respect to controls. The alterations in mesolimbic dopamine transmission observed in these experiments might underlie behavioral abnormalities induced in rat offspring by maternal exposure to cocaine at dose levels which do not produce gross malformations and/or overt neurotoxic effects.

Perinatal cocaine reduces responsiveness to cocaine and causes alterations in exploratory behavior and visual discrimination in young-adult rats.

Lister hooded female rats were exposed to either saline or cocaine (20 mg/kg s.c.) from gestational day 10 every other day until weaning (postnatal day 25). The effects of maternal cocaine exposure on novelty-induced exploration and on spontaneous and cocaine-induced motor activity were evaluated in young-adult male offspring (4 weeks after weaning). Rats exposed to cocaine during development spent less time exploring two novel objects. Lack of habituation upon the second presentation of the objects and failure in the ability to discriminate between the novel and familiar object were also found in cocaine exposed offspring. Moreover, maternal cocaine treatment did not affect spontaneous motor activity (active time, average speed and rearing) in rats subjected to an open field test. Furthermore, perinatal exposure to cocaine significantly attenuated acute cocaine (15 mg/kg i.p.)-induced hyperactivity. These data indicate that developmental exposure to cocaine, at dose levels below those producing gross malformations and/or overt signs of neurotoxicity, causes behavioral changes characterized by an altered responsiveness to environmental and pharmacological challenges.

Lectin binding pattern of Schwann cells and macrophages in 2,5-hexanedione-induced axonal degeneration in rats.

The lectin binding pattern of both Schwann cells and macrophages has been studied during axonal degeneration induced in the rat sciatic nerve by chronic administration of 2,5-hexanedione (0.8 ml/kg per day i.p. for 20 days). In particular, the present study aimed to establish a possible relationship between macrophage activation and expression of lectin binding sites. To identify and distinguish between Schwann cells and macrophages, electron microscopy was combined with the lectin staining method. On 2,5-hexanedione injury, a drastic disorganization of both axon and myelin sheath occurred and nerve fibers were replaced by a chain of ovoids. Besides the well-established concept that Schwann cells and macrophages cooperate in the removal of the myelin debris during axonal degeneration, evidence is presented that expression of binding sites to lectins is closely related to macrophage activation. Monocytes occasionally present in control nerves were labelled only by Con A and sialidase-peanut sequence; in 2,5-hexanedione degeneration monocytes, prephagocytes (macrophages with minute bubbles) and phagocytes (macrophages with large bubbles) were labelled also by peanut, wheat germ and BSA I-B4; moreover, phagocytes were labelled by soybean as well, thus showing a clearly differentiation-dependent binding pattern. Since changes in lectin binding pattern may reflect changes in complex carbohydrate structures, the results show that the expression of certain glycoproteins may be closely related to activation of macrophages in response to toxic injuries.

Alterations in the ontogeny of rat pup ultrasonic vocalization produced by prenatal exposure to nitrogen dioxide.

Wistar female rats were exposed to low concentrations of nitrogen dioxide, NO2 (1.5 and 3 ppm) from day 0 to day 20 of pregnancy. The results show that prenatal exposure to this oxidant gas produced significant changes in the duration pattern of ultrasonic vocalizations emitted by male pups removed from their nest. In particular, a significant decrease in the length of ultrasonic calls was found in both 10- and 15-day-old rats exposed to NO2 (3 ppm) during gestation. These alterations were found at dose levels which did not significantly affect reproduction parameters, body weight gain and motor activity development. These findings suggest that gestational exposure to NO2, at concentrations below those associated with overt signs of toxicity, induces in rat offspring subtle behavioral changes characterized by altered ontogeny of ultrasonic emission.

Changes in the frequency of splenic immunocompetent cells in rats exposed to carbon monoxide during gestation.

The aim of the present study was to evaluate whether prenatal exposure to relatively low concentrations of carbon monoxide (CO) may alter the frequency of splenic cells either in young (15-21 days) or in aged rats (18 months). Wistar female rats were exposed to 75 and 150 ppm of CO from day 0 to day 20 of pregnancy, respectively. The results show that prenatal exposure to 150 ppm of CO significantly decreases the number of leucocyte common antigen (LCA+) cells in 21 day old male rats, whereas other cellular populations, such as macrophages, Major Histocompatibility (MHC) II cells, T and B lymphocytes display only a trend towards a reduction without achieving statistical significance. The alterations in LCA+ cell frequency produced by gestational exposure to CO were reversible. These data further extend previous findings showing that rats prenatally exposed to moderate concentrations of CO exhibit subtle immunological changes in the absence of overt signs of toxicity.

Immunological changes produced in rats by prenatal exposure to carbon monoxide.

Wistar female rats were exposed to relatively mild concentrations of carbon monoxide (CO) (75 and 150 p.p.m.) from day 0 to day 20 of pregnancy. The results show that splenic macrophage phagocytosis of Candida albicans was significantly decreased in 15 and 21 day old male rats exposed to CO (150 p.p.m.) during pregnancy. Moreover, splenic macrophage killing was significantly reduced in 15 day old male pups prenatally exposed to 75 and 150 ppm of CO. Prenatal CO (150 p.p.m.) significantly decreased splenic macrophage O2- release in both 15 and 21 day old pups. CO-induced alterations in the immune system were not observed in 60 day old rats. These findings indicate that gestational exposure to relatively mild concentrations of CO induces in rat offspring reversible immunological changes characterized by an altered splenic macrophage function.

Neurobehavioral changes produced in rats by prenatal exposure to carbon monoxide.

Wistar female rats were exposed to relatively mild concentrations of carbon monoxide (75 and 150 ppm) from day 0 to day 20 of pregnancy. The results show that prenatal exposure to CO (150 ppm) produced a significant reduction in the minimum frequency of ultrasonic calls emitted by rat pups removed from their nest. Moreover, a significant decrease in the responsiveness (rate of calling) to a challenge dose of diazepam (0.25 mg/kg) was found in male pups exposed to CO (150 ppm) during gestation. Prenatal CO (75 and 150 ppm) did not significantly affect locomotor activity or D-amphetamine-induced hyperactivity in both 14- and 21-day-old animals. Furthermore, adult male rats exposed to this chemical (150 ppm) during gestation exhibited significant alterations in the acquisition of an active avoidance task. CO-induced learning disruption does not seem to be linked to changes in the emotionality of animals. These findings suggest that gestational exposure to CO induces in rat offspring both short- and long-term behavioral changes characterized by altered ontogeny of emotional responsiveness to environmental challenges and by learning impairment.

Behavioral and neurochemical changes produced in rats by developmental treatments with psychotropic drugs.

The timing of developmental administration of psychotropic drugs affecting dopaminergic and GABAergic neurotransmission is crucial for the induction of specific neurobehavioral and neurochemical changes in rodents. Compensatory mechanisms occurring in response to a prolonged treatment with some neuroleptic and anxiolytic agents during development seem to be markedly different from those occurring in response to a prolonged administration in adult animals.

Behavioral changes produced in rats by developmental exposure to flumazenil, a benzodiazepine receptor antagonist.

1. Prolonged administration of a benzodiazepine receptor antagonist, such as flumazenil (given to the mother at a dose of 3 mg/kg s.c. from day 14 to day 20 of gestation), produced subtle behavioral changes in rat pups. 2. Flumazenil treatment decreased the rate of ultrasonic vocalization in 15-day old male pups removed from their nest. 3. No significant changes in the locomotor activity of the flumazenil-treated group with respect to controls was found at the end of the second and fourth postnatal week. 4. These results suggest that late prenatal exposure to flumazenil induces in rat offspring behavioral changes characterized by decreased emotional responsiveness to environmental challenges.

Changes in peripheral nervous system activity produced in rats by prenatal exposure to carbon monoxide.

The present experiments were designed to investigate whether alterations of peripheral nervous system activity may be produced in male Wistar rats by prenatal exposure (from day 0 to day 20 of pregnancy) to relatively low levels of CO (75 and 150 ppm). The voltage clamp analysis of ionic currents recorded from sciatic nerve fibres showed that prenatal exposure to CO produced modifications of sodium current properties. In particular, in 40-day-old rats exposed to CO (75 and 150 ppm) during gestation, the inactivation kinetics of transient sodium current were significantly slowed. Analysis of the potential dependence of steady-state Na inactivation, h infinity (V), showed that the percentage of the maximum number of activable Na channels at the normal resting potential (-80 mV) was increased to approximately 85% in CO-exposed rats. Moreover, the voltage-current relationship showed a negative shift of sodium equilibrium potential in CO treated animals. In 270-day-old CO-exposed rats, parameters of sodium inactivation were not significantly modified; the reversal potential was still lower with respect to controls. The results indicate that prenatal exposure to mild CO concentrations produces reversible changes in sodium inactivation kinetics and on irreversible change in sodium equilibrium potential. These alterations could reflect CO influence on the rate of ion channel development.

[Experimental studies on the role of suture materials in the healing of anastomoses of the colon. Mechanical and histological study]. / Ricerche sperimentali sul ruolo del materiale di sutura nella guarigione delle anastomosi coliche. Studio meccanico ed istologico.

The relation between suture material and consolidation and healing in operations on the colon was investigated by using silk and catgut in one group of disinfected stray dogs subjected to right or left hemicolectomy and resection of the transverse portion, and a synthetic reabsorbable material (polyglycolic acid) in another. The following parameters were assessed following removal of the segment containing the anastomosis after various periods of time: resistance to biomechanical tests, stenosis of the lumen, extent and duration of inflammation and regeneration at the edge. Reabsorbable material was associated with less overall inflammation and healing was more rapid. Consolidation of the suture was not significantly different in the two groups.