Potential Neuroprotective Effect of Melatonin in the Hippocampus of Male BTBR Mice.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder identified by impairments in common social interactions and repetitive behaviors. In ASD patients, substantial morphological alterations have been observed in the hippocampus, which represents an important region for the development of social skills. Melatonin, commonly found in many foods and plants, is also produced by the pineal gland. This indolamine, known to regulate the circadian rhythm, shows antioxidant and anti-inflammatory properties. We therefore hypothesized that melatonin may reduce oxidative stress and inflammation in the hippocampus of ASD patients. We explored our hypothesis using the BTBR mouse, a well-regarded murine transgenic model for ASD. Immediately after weaning, male BTBR and C57BL/6 mice underwent an 8-week treatment with melatonin or vehicle. Later, through immunohistochemistry and the immunoblotting analysis of the hippocampus, we evaluated the overall expression and cellular localization of Nrf2 and SOD1, two enzymes involved in the oxidative stress response. Similarly, we evaluated NLRP3 and NFkB, two mediators of inflammation, and GAD67, an enzyme responsible for the synthesis of GABA. Ultimately, we addressed melatonin's potential to regulate iron metabolism through a DAB-enhanced Perls reaction assay. Results showed melatonin's potential for modulating the analyzed markers in BTBR mice, suggesting a potential neuroprotective effect in ASD patients.

LRRK2 Kinase Inhibition Attenuates Neuroinflammation and Cytotoxicity in Animal Models of Alzheimer's and Parkinson's Disease-Related Neuroinflammation.

Chronic neuroinflammation plays a crucial role in the progression of several neurodegenerative diseases (NDDs), including Parkinson's disease (PD) and Alzheimer's disease (AD). Intriguingly, in the last decade, leucine-rich repeat kinase-2 (LRRK2), a gene mutated in familial and sporadic PD, was revealed as a key mediator of neuroinflammation. Therefore, the anti-inflammatory properties of LRRK2 inhibitors have started to be considered as a disease-modifying treatment for PD; however, to date, there is little evidence on the beneficial effects of targeting LRRK2-related neuroinflammation in preclinical models. In this study, we further validated LRRK2 kinase modulation as a pharmacological intervention in preclinical models of AD- and PD-related neuroinflammation. Specifically, we reported that LRRK2 kinase inhibition with MLi2 and PF-06447475 (PF) molecules attenuated neuroinflammation, gliosis and cytotoxicity in mice with intracerebral injection of Aß1-42 fibrils or α-syn preformed fibrils (pffs). Moreover, for the first time in vivo, we showed that LRRK2 kinase activity participates in AD-related neuroinflammation and therefore might contribute to AD pathogenesis. Overall, our findings added evidence on the anti-inflammatory effects of LRRK2 kinase inhibition in preclinical models and indicate that targeting LRRK2 activity could be a disease-modifying treatment for NDDs with an inflammatory component.

Anatomical basis of retrograde thoracic veins flow and its implications in complex thoracic wall reconstructive surgery.

PURPOSE: Internal thoracic veins are increasingly used as recipient's vessels in chest wall reconstructive surgery due to their predictable anatomy and to the possibility to make a double venous anastomosis, exploiting the retrograde flow within them. Over the years, retrograde flow had been explained by the absence of valves in internal thoracic veins, which have been found recently instead. Therefore, our aim is to analyze the retrograde flow and its relationship with valves in the internal thoracic veins. METHODS: We evaluated 32 internal thoracic veins of 16 fresh-frozen specimens with undamaged thoracic cages by dynamic analysis focused on retrograde flow assessment through a partial external circulation system obtained cannulating the subclavian veins. Gross anatomical and morphological evaluations about the presence of valves and their pattern were then made. RESULTS: Efficient, partial, and absent retrograde flow was, respectively, found in 17/30, 8/30 and ITVs and 5/30 internal thoracic veins. Following Arnez's classification, 20/32 Type I and 12/32 Type II internal thoracic veins were identified. Valves were observed in 10/16 specimens (62.50%) corresponding to 36.67% of examined veins (11/30). Three valves were found between the 2nd intercostal space and 12 valves in the 3rd intercostal space. 13/15 valves were bicuspid, 2/15 tricuspid. A significant correlation (p < 0.001) between the retrograde flow and the presence of valves in internal thoracic veins was observed. CONCLUSION: Our study suggests a possible influence of the presence and the number of valves in the efficient retrograde flow of the internal thoracic veins, suggesting that, especially for more complex cases, a preoperative or intraoperative evaluation of the chest wall drainage should be recommended.

Role of melatonin in autism spectrum disorders in a male murine transgenic model: Study in the prefrontal cortex.

Autism spectrum disorders (ASDs) are a group of clinically heterogeneous neurodevelopmental disorders sharing common features related to impaired social and communication abilities in addition to stereotyped behaviors. ASD patients present encephalic morphological, physiological, and biomolecular alterations with low levels of melatonin due to alterations in its pathways. Therefore, even if ASDs have traditionally been framed as behavioral disorders, several lines of evidence are accumulating that ASDs are characterized by certain anatomical and physiological abnormalities, including oxidative stress and inflammation in peripheral biomarkers, but likewise present in human brain tissue also characterized by alterations in synaptic remodeling and neuromodulation. Melatonin has also protective and antioxidant properties, so we can therefore hypothesize that alterations in melatonin's pathways may be one of the causes of the symptomatology of autism. The aim of the present study was to analyze the beneficial effect induced by melatonin administration and its possible mechanism of action in a transgenic mouse model of autism, immediately after weaning. The male mice were daily treated per os with melatonin (10 mg/Kg/day) or vehicle for 8 weeks starting from the sixth week of life. The antioxidant modulation, the GABAergic/glutamatergic impairment, and the synaptic remodeling in the prefrontal cortex have been evaluated. Social and repetitive behaviors were also evaluated. The behavioral results showed no statistical evidences, instead the immunohistochemical results indicated the ability of melatonin to promote the activity of antioxidant system, the GABAergic/glutamatergic equilibrium, and the synaptic remodeling. The results show that melatonin may be a possible adjuvant therapeutic strategy in ASDs.

Melatonin Efficacy in Obese Leptin-Deficient Mice Heart.

Cardiomyocytes are particularly sensitive to oxidative damage due to the link between mitochondria and sarcoplasmic reticulum necessary for calcium flux and contraction. Melatonin, important indoleamine secreted by the pineal gland during darkness, also has important cardioprotective properties. We designed the present study to define morphological and ultrastructural changes in cardiomyocytes and mainly in mitochondria of an animal model of obesity (ob/ob mice), when treated orally or not with melatonin at 100 mg/kg/day for 8 weeks (from 5 up to 13 week of life). We observed that ob/ob mice mitochondria in sub-sarcolemmal and inter-myofibrillar compartments are often devoid of cristae with an abnormally large size, which are called mega-mitochondria. Moreover, in ob/ob mice the hypertrophic cardiomyocytes expressed high level of 4hydroxy-2-nonenal (4HNE), a marker of lipid peroxidation but scarce degree of mitofusin2, indicative of mitochondrial sufferance. Melatonin oral supplementation in ob/ob mice restores mitochondrial cristae, enhances mitofusin2 expression and minimizes 4HNE and p62/SQSTM1, an index of aberrant autophagic flux. At pericardial fat level, adipose tissue depot strictly associated with myocardium infarction, melatonin reduces adipocyte hypertrophy and inversely regulates 4HNE and adiponectin expressions. In summary, melatonin might represent a safe dietary adjuvant to hamper cardiac mitochondria remodeling and the hypoxic status that occur in pre-diabetic obese mice at 13 weeks of life.

Acute mercury exposition of virgin, pregnant, and lactating rats: Histopathological kidney and liver evaluations.

This work investigated the effects of mercury chloride (HgCl2 ) acute exposure on virgin, pregnant and lactating rats by determination of renal and hepatic morphological and ultrastructural parameters and the expression of oxidative stress and stress tolerance markers, due to kidney and liver are the organs that more accumulate inorganic mercury. Adult Wistar rats virgin (90 days old), pregnant (18th gestation day) and lactating (7th lactation day) were injected once with HgCl2 (5 mg/kg) or saline (controls). We observed that HgCl2 exposure of virgin rats caused significant inflammatory infiltration and severe morphological variations, like glomeruli atrophy, dilatation of Bowman's capsule, tubular degeneration and hepatocytes alteration. Moreover, virgin rats presented mitochondrial modification, important oxidative stress and increase in stress tolerance proteins at both kidney and liver level, compared with virgin controls. In detail, virgin rats exposed to HgCl2 presented significantly elevated level of inducible nitric oxide synthase, heat shock protein 27 and glucose regulated proteins 75 expressions at both renal tubular and hepatocytes level, respect untreated virgin rats. Interestingly, pregnant and lactating rats exposed to HgCl2 presented weak renal and liver morphological alterations, showing weak inflammatory infiltration and no significant difference in structural mitochondrial transmembrane protein, oxidative stress markers and stress tolerance proteins expressions respect controls (virgin, pregnant and lactating rats). Although, both control and HgCl2 -exposed pregnant and lactating rats showed renal glomeruli greater in diameter respect virgin rats. In conclusion, we believe that virgin rats are more sensitive to HgCl2 toxicity respect pregnant and lactating rats. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1500-1512, 2017.

A comparison of melatonin and α-lipoic acid in the induction of antioxidant defences in L6 rat skeletal muscle cells.

Aging is characterized by a progressive deterioration in physiological functions and metabolic processes. The loss of cells during aging in vital tissues and organs is related to several factors including oxidative stress and inflammation. Skeletal muscle degeneration is common in elderly people; in fact, this tissue is particularly vulnerable to oxidative stress since it requires large amounts of oxygen, and thus, oxidative damage is abundant and accumulates with increasing age. Melatonin (N-acetyl-5-methoxytryptamine) is a highly efficient scavenger of reactive oxygen species and it also exhibits beneficial anti-inflammatory and anti-aging effects. This study investigated the susceptibility of rat L6 skeletal muscle cells to an induced oxidative stress following their exposure to hydrogen peroxide (50 µM) and evaluating the potential protective effects of pre-treatment with melatonin (10 nM) compared to the known beneficial effect of alpha-lipoic acid (300 µM). Hydrogen peroxide-induced obvious oxidative stress; it increased the expression of tumour necrosis factor-alpha and in turn promoted nuclear factor kappa-B and overrode the endogenous defence mechanisms. Conversely, pre-treatment of the hydrogen peroxide-exposed cells to melatonin or alpha-lipoic acid increased endogenous antioxidant enzymes, including superoxide dismutase-2 and heme oxygenase-1; moreover, they ameliorated significantly oxidative stress damage and partially reduced alterations in the muscle cells, which are typical of aging. In conclusion, melatonin was equally effective as alpha-lipoic acid; it exhibited marked antioxidant and anti-aging effects at the level of skeletal muscle in vitro even when it was given in a much lower dose than alpha-lipoic acid.

Mitochondrial and metabolic dysfunction in renal convoluted tubules of obese mice: protective role of melatonin.

Obesity is a common and complex health problem, which impacts crucial organs; it is also considered an independent risk factor for chronic kidney disease. Few studies have analyzed the consequence of obesity in the renal proximal convoluted tubules, which are the major tubules involved in reabsorptive processes. For optimal performance of the kidney, energy is primarily provided by mitochondria. Melatonin, an indoleamine and antioxidant, has been identified in mitochondria, and there is considerable evidence regarding its essential role in the prevention of oxidative mitochondrial damage. In this study we evaluated the mechanism(s) of mitochondrial alterations in an animal model of obesity (ob/ob mice) and describe the beneficial effects of melatonin treatment on mitochondrial morphology and dynamics as influenced by mitofusin-2 and the intrinsic apoptotic cascade. Melatonin dissolved in 1% ethanol was added to the drinking water from postnatal week 5-13; the calculated dose of melatonin intake was 100 mg/kg body weight/day. Compared to control mice, obesity-related morphological alterations were apparent in the proximal tubules which contained round mitochondria with irregular, short cristae and cells with elevated apoptotic index. Melatonin supplementation in obese mice changed mitochondria shape and cristae organization of proximal tubules, enhanced mitofusin-2 expression, which in turn modulated the progression of the mitochondria-driven intrinsic apoptotic pathway. These changes possibly aid in reducing renal failure. The melatonin-mediated changes indicate its potential protective use against renal morphological damage and dysfunction associated with obesity and metabolic disease.

Obesity-related dysfunction of the aorta and prevention by melatonin treatment in ob/ob mice.

In this study, we hypothesized that melatonin administration can minimize alterations in aorta morphology in an animal model of obesity (ob/ob mice). The animals were divided into four groups: (i) control lean mice, (ii) control lean mice treated with melatonin, (iii) ob/ob mice and (iv) ob/ob mice treated with melatonin. The synthetic melatonin was dissolved in 1% ethanol and added to the drinking water from postnatal week 5-13 at a final dose of 100 mg/kg body weight/day. Compared with the obese mice, melatonin intake was associated with a significant decrease in body weight and water consumption. Histological analysis showed that the aortic wall of ob/ob mice had a high Tunica media/lumen ratio and that the elastic fibers in the media layer appeared disrupted and degraded. Moreover, the aorta of ob/ob mice displayed a higher degree of collagen accumulation in the Tunica media compared to the normal aorta. The aorta of ob/ob mice treated with melatonin had a lower Tunica media/lumen ratio and collagen accumulation in comparison with untreated ob/ob mice. Our results showed that whereas melatonin had no apparent histological effects on the aorta in lean mice with normal weight, its administration in ob/ob mice can lead to a reduction in body weight and can ameliorate aorta histopathological dysfunction. This experimental study indicates an apparent protective role for melatonin on the aorta in obesity and melatonin could possibly be an effective tool in the management of obesity-related vascular complications.

Provinol prevents CsA-induced nephrotoxicity by reducing reactive oxygen species, iNOS, and NF-kB expression.

Cyclosporine A (CsA) use is associated with several side effects, the most important of which is nephrotoxicity that includes, as we previously showed, tubular injury and interstitial fibrosis. Recently, many researchers have been interested in minimizing these effects by pharmacological interventions. To do this, we tested whether the administration of a red wine polyphenol, Provinol (PV), prevents the development of CsA-induced nephrotoxicity. Rats were treated for 21 days and divided into four groups: control; group treated with PV (40 mg/kg/day by oral administration in tap water); group treated with CsA (15 mg/kg/day by subcutaneous injection); group treated with CsA plus PV. CsA produced a significant increase of systolic blood pressure; it did not affect urinary output, but caused a significant decrease in creatinine clearance. These side effects were associated with an increase in conjugated dienes, which are lipid peroxidation products, inducible NO-synthase (iNOS), and nuclear factor (NF)-kB, which are involved in antioxidant damage. However, PV prevented these negative effects through a protective mechanism that involved reduction of both oxidative stress and increased iNOS and NF-kB expression induced by CsA. These results provide a pharmacological basis for the beneficial effects of plant-derived polyphenols against CsA-induced renal damage associated with CsA.

Cyclosporine A induces vascular fibrosis and heat shock protein expression in rat.

The immunosuppressive drug Cyclosporine A (CsA) has been successfully used in several diseases with immunological basis and in transplant patients. However, the therapeutic treatment induces several side effects, which include the development of severe hypertension, renal failure and cardiotoxicity in the majority of the patients. Since the mechanism by which CsA induces hypertension is not well defined, the aim of this study is to evaluate the morphological changes and the expression of heat shock proteins (HSPs) in the thoracic aorta of CsA-treated rats. The study was carried out on 40 male Wistar rats with an average weight of 200-250 g. The animals were divided into four groups. Groups I and II were injected subcutaneously (sc) daily with castor oil for 15 or 30 days and used as control; group III and IV were injected sc daily with CsA (15 mg/Kg/day) for 15 or 30 days. After the end of the treatment, the thoracic aortae were removed and treated for morphological (Sirius Red) and immunohistochemical evaluation (HSP 25, alphaB-crystallin and HSP 47). The results indicate that CsA induces (1) time-dependent vascular damage visible as fibrosis mainly in intima-media tunica of aorta, and (2) a clear increase in HSP expression. In fact, after 30 days of treatment, HSP and alphaB-crystallin are increased in all tunicas, whereas HSP47 only in tunica media and adventitia. These findings could suggest that these proteins are up-regulated after CsA treatment in order to play a defensive role in vascular damage.