Pharmacology and toxicology of xylazine: quid novum?

The current opioid overdose crisis is characterized by the presence of unknown psychoactive adulterants. Xylazine is an alpha-2 receptor agonist that is not approved for human use but is commonly used in veterinary medicine due to its sedative and muscle-relaxant properties. Cases of human intoxication due to accidental or voluntary use have been reported since the 1980s. However, reports of adulteration of illicit opioids (heroin and illicit fentanyl) with xylazine have been increasing all over Western countries. In humans, xylazine causes respiratory depression, bradycardia, and hypotension-posing individuals, using xylazine-adulterated opioids. We present a narrative review of the latest intoxication cases related to xylazine, to bring awareness to readers and also to help pathologists to detect and deal with xylazine cases.

Fatal acute lung injury after balloon valvuloplasty in a dog with pulmonary stenosis.

A one-year-old French Bulldog was referred for the management of a severe form of pulmonary valve stenosis (PS) complicated by right-sided congestive heart failure. Echocardiography showed severe valvular PS with right ventricular concentric hypertrophy, dilatation and severe right atrial enlargement. A pulmonary balloon valvuloplasty (PBV) was performed with a balloon-to-pulmonary annulus ratio of 1.36. Echocardiography immediately after PBV showed a significant reduction in right atrial and ventricular size, improved opening and mobility of the pulmonary valve leaflets, and a 75% reduction in the pulmonary pressure gradient from 158 mmHg pre-operative to 40 mmHg post-operative. The dog recovered well from anesthesia, but 2 h later, it suddenly showed severe respiratory distress. Focus cardiac ultrasound showed increased left cardiac size with echocardiographic signs of high left ventricular filling pressure. Bedside lung ultrasound showed diffuse numerous-to-confluent B lines, compatible with a severe alveolar-interstitial syndrome. The dog was treated with furosemide, helmet continuous positive airway pressure, and then mechanical ventilation but without success. At post-mortem evaluation, histological examination of the lung showed diffuse, severe broncho-alveolar edema with mixed leukocyte, fibrin, and red blood cell infiltrate. Moreover, severe congestion and multifocal alveolar hemorrhages were evident. All findings were compatible with fatal acute lung injury after PBV secondary to pulmonary reperfusion-ischemia injury and increased pulmonary capillary hydrostatic pressure. Based on the present case, acute lung injury should be considered as a rare but serious complication of PBV.

Closure of an unusual morphology patent ductus arteriosus with a covered stent in a dog.

Patent ductus arteriosus (PDA) with unusual morphology was diagnosed in a 3-year-old German shepherd presented for exercise intolerance. Two interventional PDA closure procedures with various Amplatzer devices and one surgical ligation were attempted to close the PDA. However, PDA closure failed with both methods due to the unusual morphology and the severe enlarged ductus ampulla. Therefore, the patient underwent an aortic covered stent placement, and successful closure of the PDA was achieved. Eight months after the procedure, the dog was free of clinical signs and no residual flow was identified through the PDA on transthoracic echocardiography. Moreover, computed tomography angiography demonstrated correct stent positioning, with no evidence of thrombus formation, damage to the stent, nor any aortic wall abnormalities. Aortic covered stent placement is a viable option in dogs with unusual PDA morphology.

A global cloud free pixel- based image composite from Sentinel-2 data.

Large-scale land cover classification from satellite imagery is still a challenge due to the big volume of data to be processed, to persistent cloud-cover in cloud-prone areas as well as seasonal artefacts that affect spatial homogeneity. Sentinel-2 times series from Copernicus Earth Observation program offer a great potential for fine scale land cover mapping thanks to high spatial and temporal resolutions, with a decametric resolution and five-day repeat time. However, the selection of best available scenes, their download together with the requirements in terms of storage and computing resources pose restrictions for large-scale land cover mapping. The dataset presented in this paper corresponds to global cloud-free pixel based composite created from the Sentinel-2 data archive (Level L1C) available in Google Earth Engine for the period January 2017- December 2018. The methodology used for generating the image composite is described and the metadata associated with the 10 m resolution dataset is presented. The data with a total volume of 15 TB is stored on the Big Data platform of the Joint Research Centre. It can be downloaded per UTM grid zone, loaded into GIS clients and displayed easily thanks to pre-computed overviews.

SARS-CoV-2 identification in lungs, heart and kidney specimens by transmission and scanning electron microscopy.

From two COVID-19-related deaths, samples of lung, heart and kidney were collected and processed for Transmission and Scanning Electron Microscopy (TEM and SEM) with the aim of identifying the virus. Virions of SARS-CoV-2 were found in all tissues by TEM and SEM, corroborating the hypothesis that the virus enters the cells of different organs. This is the first report identifying SARS-CoV-2 in different human tissues by TEM and SEM.

Built-up area and population density: Two Essential Societal Variables to address climate hazard impact.

Scientists use Essential Climate Variables to understand and model the Earth's climate. Complementary to the Climate Variables this paper introduces global built-up area and population density, referred to as Essential Societal Variables, that can be used to model human activities and the impact of climate induced hazards on society. Climate impact scenarios inform policy makers on current and future risk and on the cost for mitigation and adaptation measures. The global built-up area and global population densities are generated from Earth observation image archives and from national population census data in the framework of the Global Human Settlement Layer (GHSL) project. The layers are produced with fine granularity for four epochs: 1975, 1990, 2000 and 2015, and will be updated on a regular basis with open satellite imagery. The paper discusses the relevance of global built-up area and population density for a number of policy areas, in particular to understand regional and global urbanization processes and for use in operational crisis management and risk assessment. The paper also provides examples of global statistics on exposure to natural hazards based on the two ESVs and their use in policy making. Finally, the paper discusses the potential of using population and built-up area for developing indicators to monitor the progress in Agenda 2030 including the Sustainable Development Goals (SDGs).

Study of inelastic nuclear interactions of 400 GeV/c protons in bent silicon crystals for beam steering purposes.

Inelastic nuclear interaction probability of 400 GeV/c protons interacting with bent silicon crystals was investigated, in particular for both types of crystals installed at the CERN Large Hadron Collider for beam collimation purposes. In comparison to amorphous scattering interaction, in planar channeling this probability is ∼ 36 % for the quasi-mosaic type (planes (111)), and ∼ 27 % for the strip type (planes (110)). Moreover, the absolute inelastic nuclear interaction probability in the axial channeling orientation, along the ⟨ 110 ⟩ axis, was estimated for the first time, finding a value of 0.6 % for a crystal 2 mm long along the beam direction, with a bending angle of 55 µ rad. This value is more than two times lower with respect to the planar channeling orientation of the same crystal, and increases with the vertical angular misalignment. Finally, the correlation between the inelastic nuclear interaction probability in the planar channeling and the silicon crystal curvature is reported.

Neuroactive steroids and diabetic complications in the nervous system.

Important complications of diabetes mellitus in the nervous system are represented by diabetic peripheral neuropathy and diabetic encephalopathy. In this context, an important link is represented by neuroactive steroids (i.e., steroids coming from peripheral glands and affecting nervous functionality as well as directly synthesized in the nervous system). Indeed, diabetes does not only affect the reproductive axis and consequently the levels of sex steroid hormones, but also those of neuroactive steroids. Indeed, as will be here summarized, the levels of these neuromodulators present in the central and peripheral nervous system are affected by the pathology in a sex-dimorphic way. In addition, some of these neuroactive steroids, such as the metabolites of progesterone or testosterone, as well as pharmacological tools able to increase their levels have been demonstrated, in experimental models, to be promising protective agents against diabetic peripheral neuropathy and diabetic encephalopathy.

Use of an implantable pump for controlled subcutaneous insulin delivery in healthy cats.

The aim of this study was to examine the safety and reliability of a research-grade implantable pump for controlled delivery of insulin glargine in cats. For this purpose, a small telemetrically controlled drug delivery pump with a refillable reservoir was implanted into the subcutaneous tissues of the dorsal neck in 10 clinically healthy cats. The reservoir was filled with insulin glargine, and the pump was programmed to deliver four boluses of 0.25 IU/kg, 2-3 weeks apart. As a control, insulin glargine (0.25 IU/kg) was injected SC. Blood glucose and plasma insulin glargine concentrations were measured before each bolus and SC injection and for 8 h afterward. Cats were monitored for signs of discomfort. Pumps were easily implanted and well tolerated by all cats. The experiment was completed in five of 10 cats. In four, the pump failed because of technical reasons; another cat developed severe hypoglycaemia attributable to insulin leakage. Overall, plasma insulin glargine increased after six of eight (75%) initial boluses and after one of 16 (6%) successive boluses. Glucose decreased after seven of eight (88%) initial boluses and after four of 16 (25%) successive boluses. Only the first bolus significantly increased plasma insulin glargine (P = 0.008) and decreased glucose (P = 0.008). Of 20 SC injections, 10 (50%) increased plasma insulin glargine (P <0.001) and 12 (60%) decreased glucose (P <0.001). The pump did not cause discomfort in cats, but life-threatening hypoglycaemia occurred in one. Frequent device problems suggest that the pump needs improvements. Because successive boluses did not increase plasma insulin glargine, this type of insulin may not be appropriate with the pump.

Axon diameter and axonal transport: In vivo and in vitro effects of androgens.

Testosterone is a sex hormone involved in brain maturation via multiple molecular mechanisms. Previous human studies described age-related changes in the overall volume and structural properties of white matter during male puberty. Based on this work, we have proposed that testosterone may induce a radial growth of the axon and, possibly, modulate axonal transport. In order to determine whether this is the case we have used two different experimental approaches. With electron microscopy, we have evaluated sex differences in the structural properties of axons in the corpus callosum (splenium) of young rats, and tested consequences of castration carried out after weaning. Then we examined in vitro the effect of the non-aromatizable androgen Mibolerone on the structure and bidirectional transport of wheat-germ agglutinin vesicles in the axons of cultured sympathetic neurons. With electron microscopy, we found robust sex differences in axonal diameter (males>females) and g ratio (males>females). Removal of endogenous testosterone by castration was associated with lower axon diameter and lower g ratio in castrated (vs. intact) males. In vitro, Mibolerone influenced the axonal transport in a time- and dose-dependent manner, and increased the axon caliber as compared with vehicle-treated neurons. These findings are consistent with the role of testosterone in shaping the axon by regulating its radial growth, as predicted by the initial human studies.

White matter as a transport system.

There are two ways to picture white matter: as a grid of electrical wires or a network of roads. The first metaphor captures the classical function of an axon as conductor of action potentials (and information) from one brain region to another. The second one points to the important role of axons in a bi-directional transport of biological molecules and organelles between the cell body and synapse. Given the wide variety of such cargoes, a well-functioning axonal transport is critical for a number of processes, including neurotransmission, metabolism and viability of neurons. This selective review will emphasize the need for considering axonal transport when interpreting functional consequences of inter-individual variations in the structural properties of white matter. We start by describing the space occupied by white matter and techniques used in vivo for its characterization. We then provide examples of key features of maturation and aging of white matter, as well as some of the common abnormalities observed in neurodevelopmental and neurodegenerative disorders. Next, we review work that motivated our focus on axonal diameter, and explain the relationships between transport and cytoskeleton within the axon. We will conclude by describing molecular machinery of axonal transport and genes that may contribute to inter-individual variations in axonal diameter and axonal transport.

Neuroprotective effects of progesterone in chronic experimental autoimmune encephalomyelitis.

Observations so far obtained in experimental autoimmune encephalomyelitis (EAE) have revealed the promising neuroprotective effects exerted by progesterone (PROG). The findings suggest that this neuroactive steroid may potentially represent a therapeutic tool for multiple sclerosis (MS). However, up to now, the efficacy of PROG has been only tested in the acute phase of the disease, whereas it is well known that MS expresses different features depending on the phase of the disease. Accordingly, we have evaluated the effect of PROG treatment in EAE induced in Dark Agouti rats (i.e. an experimental model showing a protracted relapsing EAE). Data obtained 45 days after EAE induction show that PROG treatment exerts a beneficial effect on clinical score, confirming surrogate parameters of spinal cord damage in chronic EAE (i.e. reactive microglia, cytokine levels, activity of the Na(+) ,K(+) -ATPase pump and myelin basic protein expression). An increase of the levels of dihydroprogesterone and isopregnanolone (i.e. two PROG metabolites) was also observed in the spinal cord after PROG treatment. Taken together, these results indicate that PROG is effective in reducing the severity of chronic EAE and, consequently, may have potential with respect to MS treatment.

Histopathological markers for the diagnosis of anaphylactic death.

The postmortem diagnosis of anaphylactic death may be frustrating when victims are not hospital patients, even more so when they are recovered dead. The frequent lack of specific morphological findings in such cases means that diagnosis by the forensic pathologist must rely solely on exclusion criteria or circumstantial evidence. However, a diagnostic approach based on case history, analysis of circumstances, available clinical and necropsy findings, as well as toxicology, histopathology and biohumoral data, often allows demonstration of the cause of death. Some useful reflections on microscopic morphological data have come from two recent cases, where thorough data collection provided a reasonably certain diagnosis of anaphylactic death and systemic inflammatory response syndrome-related cardiac arrest, respectively. In both cases tissue histopathology proved crucial, since histochemical (GIEMSA) and immunohistochemical analysis (CD117 and tryptase) documented a large number of mast cells in tissues, particularly the laryngeal wall, and a discrepancy between cells positive for GIEMSA and tryptase and those positive for CD117. Staining for CD117 was also detected in cells with dendrite morphology and in a subpopulation of small lymphocytes with incised nuclei. The morphological findings of these cases are discussed, especially those obtained with immunohistochemistry, and the need for the latter data to be interpreted by experienced medical staff in the framework of a thorough analysis of all the data collected is highlighted.

Sex-dimorphic effects of dehydroepiandrosterone in diabetic neuropathy.

Our recent observations have demonstrated that gonadectomy in female, but not in male diabetic animals, exert protection in the peripheral nervous system and that these effects were associated with an increase in the levels of dehydroepiandrosterone (DHEA) in the female sciatic nerve [Pesaresi M, Giatti S, Cavaletti G, Abbiati F, Calabrese D, Bianchi R, Caruso D, Garcia-Segura LM, Melcangi RC (2011) Exp Neurol 228:215-221]. That is interesting because the neuroprotective effects of this neuroactive steroid have so far only been analyzed in male diabetic animals. Using the experimental model of streptozotocin-induced diabetic neuropathy, we have here compared the effect of DHEA treatment in male and in female animals. Data obtained indicate that DHEA treatment is able to counteract the decrease in nerve conduction velocity (NCV) induced by diabetes in both sexes. However, it was only in females that this neuroactive steroid was able to reestablish NCV to control levels. In addition, it was only in females that DHEA exerted neuroprotective actions on functional (i.e., thermal sensitivity) or molecular parameters, such as gene expression of myelin proteins. Sex-depending neuroprotective effects of DHEA were also confirmed by the finding that it was only in females that this neuroactive steroid fully restored the intra-epidermal nerve fiber density, which was decreased by diabetes. Interestingly, the metabolic fate of DHEA is also different in males and females. Thus, analysis of the neuroactive steroid levels after the treatment with DHEA indicates that in the sciatic nerve of male diabetic animals 17α-estradiol levels decrease in association with an increase of its isomer 17ß-estradiol and with a decrease in the levels of α-androstane-3α, 17ß-diol. These changes were not observed in the sciatic nerve of females. Altogether, these results suggest that DHEA could be considered as a candidate for a sex-specific therapy based on neuroactive steroids.

Population-based frequency assessment of HPV-induced lesions in patients with borderline Pap tests in the Emilia-Romagna Region: the PATER study.

BACKGROUND: The PATER study assessed the frequency of high-risk (HR) and low-risk (LR) human papillomavirus (HPV) in HPV-induced lesions in patients with borderline cytology. METHODS: This retrospective observational cohort study was designed to evaluate ASCUS patients detected through a local cervical cancer screening programme and referred to the Department of Gynaecology and Obstetrics at the S. Orsola-Malpighi University Hospital in Bologna, in the period between January 2000 and December 2007. RESULTS: In 1047 patients aged 38.4 ± 9.6 years (range 23-65 years), 34.8% (n = 364) was positive for HR- or LR-HPV DNA. The mean age of women with HPV infection was significantly lower compared with the negative group (36.8 ± 9.4 versus 39.3 ± 9.6 years; p < 0.001). Overall, 357 (34.1%) women had cervical lesions: 279 (26.6%) had CIN1, 18 (1.7%) CIN2, and 60 (5.7%) CIN3+. HR-HPV genotype was detected in 83.3%, and 91.5% of patients with CIN2 and CIN3+ respectively. Among the 124 CIN1 HPV-positive women, 8.9% harboured LR-HPV genotypes, 80.6% HR-HPV and 10.5% a combination of HR- and LR-HPV. HPV-6 and 11 accounted for 19.4% of all HPV-positive CIN1 lesions. CONCLUSION: Our study suggest that: in ASCUS patients over 40 years there is a low risk of positivity for HPV infection; the HPV DNA testing in patients with CIN3+ and a mean age close to 40 years is highly sensitive (98.3%) and acceptably specific (75.5%); the frequency of LR-HPV (alone or in combination with HR) in ASCUS cytology is not negligible. A tetravalent-based HPV vaccination alongside the screening programme would provide considerable clinical, organizational, and economic benefits.

Effect of short-and long-term gonadectomy on neuroactive steroid levels in the central and peripheral nervous system of male and female rats.

Significant levels of neuroactive steroids are still detected in the nervous system of rodents after the removal of peripheral steroidogenic glands. However, the influence of the plasma levels of gonadal steroids on the levels of neuroactive steroids in the nervous system has not so far been clarified in detail. Accordingly, by liquid chromatography tandem mass spectrometry, we have analysed the levels of neuroactive steroids in the sciatic nerve, in three central nervous system (CNS) regions (i.e. cerebellum, cerebral cortex and spinal cord) and in the plasma of male and female animals. The levels present in gonadally intact animals were compared with those present in short- and long-term gonadectomised animals. We observed that: (i) changes in neuroactive steroid levels in the nervous system after gonadectomy do not necessarily reflect the changes in plasma levels; (ii) long-term gonadectomy induces changes in the levels of neuroactive steroids in the peripheral nervous system (PNS) and the CNS that, in some cases, are different to those induced by short-term gonadectomy; (iii) the effect of gonadectomy on neuroactive steroid levels is different between the PNS and the CNS and within different CNS regions; and (iv) the effects of gonadectomy on neuroactive steroid levels in the nervous system show sex differences. Altogether, these observations indicate that the nervous system adapts its local levels of neuroactive steroids in response to changes in gonadal hormones with sex and regional specificity and depending on the duration of the peripheral modifications.

Neuroprotective effects of a ligand of translocator protein-18 kDa (Ro5-4864) in experimental diabetic neuropathy.

Peripheral neuropathy represents an important complication of diabetes involving a spectrum of structural, functional and biochemical alterations in peripheral nerves. Recent observations obtained in our laboratory have shown that the levels of neuroactive steroids present in the sciatic nerve of rat raised diabetic by a single injection of streptozotocin (STZ) are reduced and that, in the same experimental model, treatment with neuroactive steroids, such as progesterone, testosterone and their derivatives show neuroprotective effects. On this basis, an interesting therapeutic strategy could be to increase the levels of neuroactive steroids directly in the nervous system. With this perspective, ligands of translocator protein-18 kDa (TSPO) may represent an interesting option. TSPO is mainly present in the mitochondrial outer membrane, where it promotes the translocation of cholesterol to the inner mitochondrial membrane, and, as demonstrated in other cellular systems, it allows the transformation of cholesterol into pregnenolone and the increase of steroid levels. In the diabetic model of STZ rat, we have here assessed whether treatment with Ro5-4864 (i.e., a ligand of TSPO) could increase the low levels of neuroactive steroids in sciatic nerve and consequently to be protective in this experimental model. Data obtained by liquid chromatography-tandem mass spectrometry show that treatment with Ro5-4864 was able to significantly stimulate the low levels of pregnenolone, progesterone and dihydrotestosterone observed in the sciatic nerves of diabetic rats. The treatment with Ro5-4864 also counteracted the impairment of NCV and thermal threshold, restored skin innervation density and P0 mRNA levels, and improved Na+,K+-ATPase activity. In conclusion, data here reported show for the first time that a TSPO ligand, such as Ro5-4864, is effective in reducing the severity of diabetic neuropathy through a local increase of neuroactive steroid levels.

Neuroprotective effects of dihydroprogesterone and progesterone in an experimental model of nerve crush injury.

A satisfactory management to ensure a full restoration of peripheral nerve after trauma is not yet available. Using an experimental protocol, in which crush injury was applied 1 cm above the bifurcation of the rat sciatic nerve for 20 s, we here demonstrate that the levels of neuroactive steroids, such as pregnenolone and progesterone (P) metabolites (i.e. dihydroprogesterone, DHP, and tetrahydroprogesterone, THP) present in injured sciatic nerve were significantly decreased. On this basis, we have focused our attention on DHP and its direct precursor, P, analyzing whether these two neuroactive steroids may have neuroprotective effects on biochemical, functional and morphological alterations occurring during crush-induced degeneration-regeneration. We demonstrate that DHP and/or P counteract biochemical alterations (i.e. myelin proteins and Na(+),K(+)-ATPase pump) and stimulate reelin gene expression. These two neuroactive steroids also counteract nociception impairment, and DHP treatment significantly decreases the up-regulation of myelinated fibers' density occurring in crushed animals. Altogether, these observations suggest that DHP and P (i.e. two neuroactive steroids interacting with progesterone receptor) may be considered protective agents in case of nerve crush injury.

Influence of peroxisome proliferator-activated receptor gamma activation by its endogenous ligand 15-deoxy Delta12,14 prostaglandin J2 on nitric oxide production in term placental tissues from diabetic women.

Diabetes induces alterations which condition placental remodelling. The levels of nitric oxide (NO) (a modulator of placental invasiveness, differentiation and proliferation) were higher in term placental explants from diabetic patients when compared to controls. Peroxisome proliferator-activated receptor gamma (PPARgamma) activation by its endogenous ligand 15-deoxy Delta(12,14)prostaglandin J(2) (15dPGJ(2)), is a differentiating factor of adipocytes and other cell types, such as trophoblasts. 15dPGJ(2) is also able to down-regulate NO production in different cell types. Our study evaluated the levels of 15dPGJ(2) and PPARgamma and the influence of PPARgamma activation by 15dPGJ(2) on the production of NO, in term placental tissues from control, pre-gestational and gestational diabetic patients. Our results showed that 15dPGJ(2) was present in human term placenta, and that its levels were diminished in gestational (P<0.05) and pre-gestational (P<0.002) diabetic women when compared to controls. Exogenous 15dPGJ(2) addition (2 x 10(-6) mol/l) down-regulated NO production in placenta from control (P<0.001) and pre-gestational diabetic (P<0.01) patients, but failed to do so in gestational diabetic women, whose placental PPARgamma expression was diminished in comparison to controls (P<0.001). As the exogenous activation of PPARgamma prevented NO overproduction in placenta from pre-gestational diabetic women, it may have the potential to improve fetal outcome in this pathology.