Modification of the Marmarou and Foda model of diffuse axonal injury (DAI) improves percentage survival of rats at 24 h and increases the amount of DAI identified.

More than two decades ago, Marmarou published a valid model for producing diffuse axonal injury (DAI) in rats. Since then, both mild and severe injuries have been obtained by researchers using the original method and a weight of 450 g. However, the diffuse brain injuries produced in rats were only similar to those seen in humans when the rats sustained severe brain injuries. In these cases, rat mortality in the original article was around 50%, and the cause of death was prolonged apnea post-impact. Rat survival after impact is critical for studying the progression of DAI. In order to explain the cause of death in human victims with cranial trauma who do not show gross brain injury, testing for the presence of DAI is essential. Thus, in order to minimize local and cervical injuries to increase rat survival, attention should be paid to the following aspects: a wider head protector disc should be used, the head of the rat should be elevated at the time of impact, and the foam bed should be soft enough to allow the movement caused by acceleration. With our modified method, rat survival increased by 30% compared to the original model (80% versus 50%). Moreover, 85.7% of rats demonstrated DAI after 24 h of survival. With these modifications, injuries appear in the same locations as in humans; thus, the method is suitable for the study of traumatic DAI in humans.

Histological and histomorphometric study of human palatal mucosa: Implications for connective tissue graft harvesting.

AIMS: To analyse the histological structure and histomorphometric characteristics of human hard palatal mucosa in order to determine the donor site of choice for connective tissue grafts from a histological point of view. MATERIALS AND METHODS: Palatal mucosa samples from six cadaver heads were harvested at four sites: incisal, premolar, molar and tuberosity. Histological and immunohistochemical techniques were performed, as was histomorphometric analysis. RESULTS: In the current study, we found that the density and size of cells were higher in the superficial papillary layer, whereas the thickness of the collagen bundles increased in the reticular layer. Excluding the epithelium, the mean percentage of lamina propria (LP) and submucosa (SM) was 37% and 63%, respectively (p < .001). LP thickness showed similar values in the incisal, premolar and molar regions, and a significantly greater thickness in tuberosity (p < .001). The thickness of SM increased from incisal to premolar and molar, disappearing in the tuberosity (p < .001). CONCLUSIONS: As dense connective tissue of LP is the tissue of choice for connective tissue grafts, the best donor site from a histological point of view is tuberosity because it is composed only of a thick LP without the presence of a loose submucosal layer.

The cold-sensing ion channel TRPM8 regulates central and peripheral clockwork and the circadian oscillations of body temperature.

AIM: Physiological functions in mammals show circadian oscillations, synchronized by daily cycles of light and temperature. Central and peripheral clocks participate in this regulation. Since the ion channel TRPM8 is a critical cold sensor, we investigated its role in circadian function. METHODS: We used TRPM8 reporter mouse lines and TRPM8-deficient mice. mRNA levels were determined by in situ hybridization or RT-qPCR and protein levels by immunofluorescence. A telemetry system was used to measure core body temperature (Tc). RESULTS: TRPM8 is expressed in the retina, specifically in cholinergic amacrine interneurons and in a subset of melanopsin-positive ganglion cells which project to the central pacemaker, the suprachiasmatic nucleus (SCN) of the hypothalamus. TRPM8-positive fibres were also found innervating choroid and ciliary body vasculature, with a putative function in intraocular temperature, as shown in TRPM8-deficient mice. Interestingly, Trpm8-/- animals displayed increased expression of the clock gene Per2 and vasopressin (AVP) in the SCN, suggesting a regulatory role of TRPM8 on the central oscillator. Since SCN AVP neurons control body temperature, we studied Tc in driven and free-running conditions. TRPM8-deficiency increased the amplitude of Tc oscillations and, under dim constant light, induced a greater phase delay and instability of Tc rhythmicity. Finally, TRPM8-positive fibres innervate peripheral organs, like liver and white adipose tissue. Notably, Trpm8-/- mice displayed a dysregulated expression of Per2 mRNA in these metabolic tissues. CONCLUSION: Our findings support a function of TRPM8 as a temperature sensor involved in the regulation of central and peripheral clocks and the circadian control of Tc.

Immunohistological study of the density and distribution of human penile neural tissue: gradient hypothesis.

Immunohistological patterns of density and distribution of neural tissue in the human penis, including the prepuce, are not fully characterized, and effects of circumcision (partial or total removal of the penile prepuce) on penile sexual sensation are controversial. This study analyzed extra- and intracavernosal innervation patterns on the main penile axes using formalin-fixed, paraffin-embedded human adult and fetal penile tissues, single- and double-staining immunohistochemistry and a variety of neural and non-neural markers, with a special emphasis on the prepuce and potential sexual effects of circumcision. Immunohistochemical profiles of neural structures were determined and the most detailed immunohistological characterizations to date of preputial nerve supply are provided. The penile prepuce has a highly organized, dense, afferent innervation pattern that is manifest early in fetal development. Autonomically, it receives noradrenergic sympathetic and nitrergic parasympathetic innervation. Cholinergic nerves are also present. We observed cutaneous and subcutaneous neural density distribution biases across our specimens towards the ventral prepuce, including a region corresponding in the adult anatomical position (penis erect) to the distal third of the ventral penile aspect. We also describe a concept of innervation gradients across the longitudinal and transverse penile axes. Results are discussed in relation to the specialized literature. An argument is made that neuroanatomic substrates underlying unusual permanent penile sensory disturbances post-circumcision are related to heightened neural levels in the distal third of the ventral penile aspect, which could potentially be compromised by deep incisions during circumcision.

Activation of Hypothalamic AMP-Activated Protein Kinase Ameliorates Metabolic Complications of Experimental Arthritis.

OBJECTIVE: To investigate whether thermogenesis and the hypothalamus may be involved in the physiopathology of experimental arthritis (EA). METHODS: EA was induced in male Lewis rats by intradermal injection of Freund's complete adjuvant (CFA). Food intake, body weight, plasma cytokines, thermographic analysis, gene and protein expression of thermogenic markers in brown adipose tissue (BAT) and white adipose tissue (WAT), and hypothalamic AMP-activated protein kinase (AMPK) were analyzed. Virogenetic activation of hypothalamic AMPK was performed. RESULTS: We first demonstrated that EA was associated with increased BAT thermogenesis and browning of subcutaneous WAT leading to elevated energy expenditure. Moreover, rats experiencing EA showed inhibition of hypothalamic AMPK, a canonical energy sensor modulating energy homeostasis at the central level. Notably, specific genetic activation of AMPK in the ventromedial nucleus of the hypothalamus (a key site modulating energy metabolism) reversed the effect of EA on energy balance, brown fat, and browning, as well as promoting amelioration of synovial inflammation in experimental arthritis. CONCLUSION: Overall, these data indicate that EA promotes a central catabolic state that can be targeted and reversed by the activation of hypothalamic AMPK. This might provide new therapeutic alternatives to treat rheumatoid arthritis (RA)-associated metabolic comorbidities, improving the overall prognosis in patients with RA.

Is there a correlation between HER2 gene amplification level and response to neoadjuvant treatment with trastuzumab and chemotherapy in HER2-positive breast cancer?

There are contradictory data regarding the correlation between HER2 amplification level determined by in situ hybridization and evolution after treatment with anti-HER2 therapies. The aim of this study was to correlate quantitative results of FISH (ratio HER2/CEP17 and number of HER2 signals/nucleus) with pathological response achieved after neoadjuvant treatment with trastuzumab and chemotherapy. For this purpose, we analysed 100 consecutive HER2-positive cases of breast carcinoma treated with neoadjuvant therapy. HER2 amplification determined by FISH was found in 92 of the 100 cases studied. pCR was obtained in 58% of the patients whose tumours presented amplification. In contrast, no pCR was obtained in the 8 patients with non-amplified tumours. A significant direct correlation between HER2 high amplification (HER2/CEP17 ratio > 5 or HER2 signals/nucleus > 10) and pCR was found. In conclusion, HER2 amplification levels are clinically relevant because they provide oncologists with valuable information on the possibilities of achieving pCR after neoadjuvant treatment.

Merkel cells of human oral mucosa express the pluripotent stem cell transcription factor Sox2.

Merkel cells are neuroendocrine cells associated to a neural sensitive ending and localized primarily in the epidermis, although they are also found in oral mucosa. Sox2 or SRY-box2 is a key transcription factor important in the maintenance of embryonic neural crest stem cell pluripotency. Sox2 has been described in Merkel cells of skin and in Merkel cell carcinomas, but not specifically in oral Merkel cells. The aims of the present study were to analyze the density of Merkel cells in human oral mucosa and to study the expression of Sox2 in these cells. For these purposes, immunohistochemical analyses for Sox2 and CK20 (the best marker for Merkel cells) were automatically performed on sections of normal human oral mucosa. Double immunofluorescence for Sox2 and CK20 was also performed. To analyze the density of Merkel cells, CK20 positive cells were counted in each sample and the length of the epithelial apical edge was measured (cells/mm). Merkel cells, demonstrated by CK20 immunoreactivity, were found in 95% of oral mucosa specimens studied (n=21). Mean density of Merkel cells in oral mucosa was 1.71±2.34 cells/mm. Sox2 immunoreactivity was found in the nuclei of scattered cells located at the basal layer. Serial sections immunostained for Sox2 and CK20 showed that Sox2-positive cells of oral mucosa coexpressed CK20, confirming that they were Merkel cells. Immunofluorescence for Sox2 and CK20 showed colocalization of both markers, demonstrating that virtually all oral Merkel cells expressed Sox2. This transcription factor could play a role in Merkel cell maturation and maintenance.

Hypothalamic dopamine signaling regulates brown fat thermogenesis.

Dopamine signaling is a crucial part of the brain reward system and can affect feeding behavior. Dopamine receptors are also expressed in the hypothalamus, which is known to control energy metabolism in peripheral tissues. Here we show that pharmacological or chemogenetic stimulation of dopamine receptor 2 (D2R) expressing cells in the lateral hypothalamic area (LHA) and the zona incerta (ZI) decreases body weight and stimulates brown fat activity in rodents in a feeding-independent manner. LHA/ZI D2R stimulation requires an intact sympathetic nervous system and orexin system to exert its action and involves inhibition of PI3K in the LHA/ZI. We further demonstrate that, as early as 3 months after onset of treatment, patients treated with the D2R agonist cabergoline experience an increase in energy expenditure that persists for one year, leading to total body weight and fat loss through a prolactin-independent mechanism. Our results may provide a mechanistic explanation for how clinically used D2R agonists act in the CNS to regulate energy balance.

MCH Regulates SIRT1/FoxO1 and Reduces POMC Neuronal Activity to Induce Hyperphagia, Adiposity, and Glucose Intolerance.

Melanin-concentrating hormone (MCH) is an important regulator of food intake, glucose metabolism, and adiposity. However, the mechanisms mediating these actions remain largely unknown. We used pharmacological and genetic approaches to show that the sirtuin 1 (SIRT1)/FoxO1 signaling pathway in the hypothalamic arcuate nucleus (ARC) mediates MCH-induced feeding, adiposity, and glucose intolerance. MCH reduces proopiomelanocortin (POMC) neuronal activity, and the SIRT1/FoxO1 pathway regulates the inhibitory effect of MCH on POMC expression. Remarkably, the metabolic actions of MCH are compromised in mice lacking SIRT1 specifically in POMC neurons. Of note, the actions of MCH are independent of agouti-related peptide (AgRP) neurons because inhibition of γ-aminobutyric acid receptor in the ARC did not prevent the orexigenic action of MCH, and the hypophagic effect of MCH silencing was maintained after chemogenetic stimulation of AgRP neurons. Central SIRT1 is required for MCH-induced weight gain through its actions on the sympathetic nervous system. The central MCH knockdown causes hypophagia and weight loss in diet-induced obese wild-type mice; however, these effects were abolished in mice overexpressing SIRT1 fed a high-fat diet. These data reveal the neuronal basis for the effects of MCH on food intake, body weight, and glucose metabolism and highlight the relevance of SIRT1/FoxO1 pathway in obesity.

Expression of myostatin in human hematopoietic cells unveils novel autocrine/paracrine actions for the hormone.

Myostatin is a member of the transforming growth factor ß (TGFß) superfamily that has a well-established role as a mediator of muscle growth and development. However, myostatin is now emerging as a pleiotropic hormone with multiple actions in the regulation of the metabolism as well as several aspects of both cardiac and smooth muscle cells physiology. In addition, myostatin is also expressed in several nonmuscular cells where its physiological role remains to be elucidated in most cases. In this report, we have shown that both myostatin and its receptor system are expressed in blood cells and in hematopoietic cell lines. Furthermore, myostatin treatment promotes differentiation of both HL60 and K562 cells through a mechanism that involves activation of extracellular signal-regulated kinases 1/2 and p38-mitogen-activated protein kinase, thus leading to the possibility that myostatin may be a paracrine/autocrine factor involved in the control of haematopoiesis. In addition, the presence of myostatin expression in immune cells could envisage a novel role for the hormone in the pathogenesis of inflammatory diseases.

Publisher Correction: Obestatin controls skeletal muscle fiber-type determination.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Improvement of Duchenne muscular dystrophy phenotype following obestatin treatment.

BACKGROUND: This study was performed to test the therapeutic potential of obestatin, an autocrine anabolic factor regulating skeletal muscle repair, to ameliorate the Duchenne muscular dystrophy (DMD) phenotype. METHODS AND RESULTS: Using a multidisciplinary approach, we characterized the ageing-related preproghrelin/GPR39 expression patterns in tibialis anterior (TA) muscles of 4-, 8-, and 18-week-old mdx mice (n = 3/group) and established the effects of obestatin administration at this level in 8-week-old mdx mice (n = 5/group). The findings were extended to in vitro effects on human immortalized DMD myotubes. An analysis of TAs revealed an age-related loss of preproghrelin expression, as precursor of obestatin, in mdx mice. Administration of obestatin resulted in a significant increase in tetanic specific force (33.0% ± 1.5%, P < 0.05), compared with control mdx mice. Obestatin-treated TAs were characterized by reduction of fibres with centrally located nuclei (10.0% ± 1.2%, P < 0.05) together with an increase in the number of type I fibres (25.2% ± 1.7%, P < 0.05) associated to histone deacetylases/myocyte enhancer factor-2 and peroxisome proliferator-activated receptor-gamma coactivator 1α axis, and down-regulation of ubiquitin E3-ligases by inactivation of FoxO1/4, indexes of muscle atrophy. Obestatin reduced the level of contractile damage and tissue fibrosis. These observations correlated with decline in serum creatine kinase (58.8 ± 15.2, P < 0.05). Obestatin led to stabilization of the sarcolemma by up-regulation of utrophin, α-syntrophin, ß-dystroglycan, and α7ß1-integrin proteins. These pathways were also operative in human DMD myotubes. CONCLUSIONS: These results highlight the potential of obestatin as a peptide therapeutic for preserving muscle integrity in DMD, thus allowing a better efficiency of gene or cell therapy in a combined therapeutic approach.

p53 in AgRP neurons is required for protection against diet-induced obesity via JNK1.

p53 is a well-known tumor suppressor that has emerged as an important player in energy balance. However, its metabolic role in the hypothalamus remains unknown. Herein, we show that mice lacking p53 in agouti-related peptide (AgRP), but not proopiomelanocortin (POMC) or steroidogenic factor-1 (SF1) neurons, are more prone to develop diet-induced obesity and show reduced brown adipose tissue (BAT) thermogenic activity. AgRP-specific ablation of p53 resulted in increased hypothalamic c-Jun N-terminal kinase (JNK) activity before the mice developed obesity, and central inhibition of JNK reversed the obese phenotype of these mice. The overexpression of p53 in the ARC or specifically in AgRP neurons of obese mice decreased body weight and stimulated BAT thermogenesis, resulting in body weight loss. Finally, p53 in AgRP neurons regulates the ghrelin-induced food intake and body weight. Overall, our findings provide evidence that p53 in AgRP neurons is required for normal adaptations against diet-induced obesity.

Deletion of the Cold Thermoreceptor TRPM8 Increases Heat Loss and Food Intake Leading to Reduced Body Temperature and Obesity in Mice.

The coupling of energy homeostasis to thermoregulation is essential to maintain homeothermy in changing external environments. We studied the role of the cold thermoreceptor TRPM8 in this interplay in mice of both sexes. We demonstrate that TRPM8 is required for a precise thermoregulation in response to cold, in fed and fasting. Trpm8-/- mice exhibited a fall of 0.7°C in core body temperature when housed at cold temperatures, and a deep hypothermia (<30°C) during food deprivation. In both situations, TRPM8 deficiency induced an increase in tail heat loss. This, together with the presence of TRPM8-sensory fibers innervating the main tail vessels, unveils a major role of this ion channel in tail vasomotor regulation. Finally, TRPM8 deficiency had a remarkable impact on energy balance. Trpm8-/- mice raised at mild cold temperatures developed late-onset obesity and metabolic dysfunction, with daytime hyperphagia and reduction of fat oxidation as plausible causal factors. In conclusion, TRPM8 fine-tunes eating behavior and fuel utilization during thermoregulatory adjustments to mild cold. Persistent imbalances in these responses result in obesity.SIGNIFICANCE STATEMENT The thermosensitive ion channel TRPM8 is required for a precise thermoregulatory response to cold and fasting, playing an important role in tail vasoconstriction, and therefore heat conservation, as well as in the regulation of ingestive behavior and metabolic fuel selection upon cooling. Indeed, TRPM8-deficient mice, housed in a mild cold environment, displayed an increase in tail heat loss and lower core body temperature, associated with the development of late-onset obesity with glucose and lipid metabolic dysfunction. A persistent diurnal hyperphagia and reduced fat oxidation constitute plausible underlying mechanisms in the background of a deficient thermoregulatory adjustment to mild cold ambient temperatures.

Pharmacological stimulation of p53 with low-dose doxorubicin ameliorates diet-induced nonalcoholic steatosis and steatohepatitis.

OBJECTIVE: Recent reports have implicated the p53 tumor suppressor in the regulation of lipid metabolism. We hypothesized that the pharmacological activation of p53 with low-dose doxorubicin, which is widely used to treat several types of cancer, may have beneficial effects on nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH). METHODS: We used long-term pharmacological activation of p53 by i.p. or oral administration of low-dose doxorubicin in different animal models of NAFLD (high fat diet containing 45% and 60% kcal fat) and NASH (methionine- and choline-deficient diet and choline deficiency combined with high fat diet). We also administered doxorubicin in mice lacking p53 in the liver and in two human hepatic cells lines (HepG2 and THLE2). RESULTS: The attenuation of liver damage was accompanied by the stimulation of fatty acid oxidation and decrease of lipogenesis, inflammation, and ER stress. The effects of doxorubicin were abrogated in mice with liver-specific ablation of p53. Finally, the effects of doxorubicin on lipid metabolism found in animal models were also present in two human hepatic cells lines, in which the drug stimulated fatty acid oxidation and inhibited de novo lipogenesis at doses that did not cause changes in apoptosis or cell viability. CONCLUSION: These data provide new evidence for targeting p53 as a strategy to treat liver disease.

Breast cancer subtype discrimination using standardized 4-IHC and digital image analysis.

Breast cancer is a heterogeneous disease. Surrogate classification of intrinsic subtypes of invasive carcinomas by combined immunohistochemistry for estrogen receptor (ER), progesterone receptor (PR), HER2, and Ki67 (4-IHC) has increased steadily since the 2011 St Gallen symposium, due to its rapid subtyping of tumors at a reasonable cost. An important step in improving 4-IHC reproducibility and reliability will be to provide reference values from the routine use of standardized 4-IHC followed by image analysis. The aims of the current study were (1) to analyze invasive breast carcinomas using standardized 4-IHC and quantitative image analysis and (2) to compare the results obtained in the classification of biological subtypes using current Ki67 and PR threshold values proposed by different authors to sub-classifying the luminal A-like and the luminal B-like (HER2-negative) subtypes. Five hundred twenty-one tumors were analyzed by standardized immunohistochemistry, with automatic image analysis, and HER2 FISH technique. Positivity for ER was found in 82.7% and for PR in 70.1% of cases. Using the Allred scoring system, hormone receptor results showed a bimodal distribution, particularly for ER. HER2 positivity was found in 15.7% of cases, and the mean Ki67 score was 32.3%. Using the most recently proposed surrogate definitions for the classification of luminal breast cancer subtypes, the percentages of different subtypes that we found were similar to those published with genomic platforms: 40.7% luminal A-like, 32.4% luminal B-like/HER2-negative, 9.8% luminal B-like/HER2-positive, 6.0% HER2-positive, and 11.1% triple negative. Standardized 4-IHC with automatic image analysis constitutes a low-cost method for surrogate definitions of biological subtypes of breast cancer that delivers accurate results in a day.

Obestatin Increases the Regenerative Capacity of Human Myoblasts Transplanted Intramuscularly in an Immunodeficient Mouse Model.

Although cell-based therapy is considered a promising method aiming at treating different muscular disorders, little clinical benefit has been reported. One of major hurdles limiting the efficiency of myoblast transfer therapy is the poor survival of the transplanted cells. Any intervention upon the donor cells focused on enhancing in vivo survival, proliferation, and expansion is essential to improve the effectiveness of such therapies in regenerative medicine. In the present work, we investigated the potential role of obestatin, an autocrine peptide factor regulating skeletal muscle growth and repair, to improve the outcome of myoblast-based therapy by xenotransplanting primary human myoblasts into immunodeficient mice. The data proved that short in vivo obestatin treatment of primary human myoblasts not only enhances the efficiency of engraftment, but also facilitates an even distribution of myoblasts in the host muscle. Moreover, this treatment leads to a hypertrophic response of the human-derived regenerating myofibers. Taken together, the activation of the obestatin/GPR39 pathway resulted in an overall improvement of the efficacy of cell engraftment within the host's skeletal muscle. These data suggest considerable potential for future therapeutic applications and highlight the importance of combinatorial therapies.

Obestatin controls skeletal muscle fiber-type determination.

Obestatin/GPR39 signaling stimulates skeletal muscle growth and repair by inducing both G-protein-dependent and -independent mechanisms linking the activated GPR39 receptor with distinct sets of accessory and effector proteins. In this work, we describe a new level of activity where obestatin signaling plays a role in the formation, contractile properties and metabolic profile of skeletal muscle through determination of oxidative fiber type. Our data indicate that obestatin regulates Mef2 activity and PGC-1α expression. Both mechanisms result in a shift in muscle metabolism and function. The increase in Mef2 and PGC-1α signaling activates oxidative capacity, whereas Akt/mTOR signaling positively regulates myofiber growth. Taken together, these data indicate that the obestatin signaling acts on muscle fiber-type program in skeletal muscle.

A Functional Link between AMPK and Orexin Mediates the Effect of BMP8B on Energy Balance.

AMP-activated protein kinase (AMPK) in the ventromedial nucleus of the hypothalamus (VMH) and orexin (OX) in the lateral hypothalamic area (LHA) modulate brown adipose tissue (BAT) thermogenesis. However, whether these two molecular mechanisms act jointly or independently is unclear. Here, we show that the thermogenic effect of bone morphogenetic protein 8B (BMP8B) is mediated by the inhibition of AMPK in the VMH and the subsequent increase in OX signaling via the OX receptor 1 (OX1R). Accordingly, the thermogenic effect of BMP8B is totally absent in ox-null mice. BMP8B also induces browning of white adipose tissue (WAT), its thermogenic effect is sexually dimorphic (only observed in females), and its impact on OX expression and thermogenesis is abolished by the knockdown of glutamate vesicular transporter 2 (VGLUT2), implicating glutamatergic signaling. Overall, our data uncover a central network controlling energy homeostasis that may be of considerable relevance for obesity and metabolic disorders.