Large-field-of-view scanning electron microscopy of the paraventricular nucleus of the hypothalamus during diet-induced obesity.

Dysregulation in the paraventricular nucleus of the hypothalamus (PVN) is associated with a variety of diseases including those related to obesity. Although most investigations have focused on molecular changes, structural alterations in PVN neurons can reveal underlying functional disruptions. Although electron microscopy (EM) can provide nanometer resolution of brain structures, an inherent limitation of traditional transmission EM is the single field of view nature of data collection. To overcome this, we used large-field-of-view high-resolution backscatter scanning electron microscopy (bSEM) of the PVN. By stitching high-resolution bSEM images, taken from normal chow and high-fat diet mice, we achieved interactive, zoomable maps that allow for low-magnification screening of the entire PVN and high-resolution analyses of ultrastructure at the level of the smallest cellular organelle. Using this approach, quantitative analysis across the PVN revealed marked electron-dense regions within neuronal nucleoplasm following high-fat diet feeding, with an increase in kurtosis, indicative of a shift away from a normal distribution. Furthermore, measures of skewness indicated a shift toward darker clustered electron-dense regions, potentially indicative of heterochromatin clusters. We further demonstrate the utility to map out healthy and altered neurons throughout the PVN and the ability to remotely perform bSEM imaging in situations that require social distancing, such as the COVID-19 pandemic. Collectively, these findings present an approach that allows for the precise placement of PVN cells within an overall structural and functional map of the PVN. Moreover, they suggest that obesity may disrupt PVN neuronal chromatin structure.NEW & NOTEWORTHY Paraventricular nucleus of the hypothalamus (PVN) alterations are linked to obesity-related conditions, but limited knowledge exists about neuroanatomical changes in this region. A large-field-of-view backscatter scanning electron microscopy (bSEM) method was used, which allowed the identification of up to 40 PVN neurons in individual samples. During obesity in mice, bSEM revealed changes in PVN neuronal nucleoplasm, possibly indicating chromatin clustering. This microscopy advancement offers valuable insights into neuroanatomy in both healthy and disease conditions.

Subfornical organ insulin receptors tonically modulate cardiovascular and metabolic function.

Insulin acts within the central nervous system through the insulin receptor to influence both metabolic and cardiovascular physiology. While a major focus has been placed on hypothalamic regions, participation of extrahypothalamic insulin receptors in cardiometabolic regulation remains largely unknown. We hypothesized that insulin receptors in the subfornical organ (SFO), a forebrain circumventricular region devoid of a blood-brain barrier, are involved in metabolic and cardiovascular regulation. Immunohistochemistry in mice revealed widespread insulin receptor-positive cells throughout the rostral to caudal extent of the SFO. SFO-targeted adenoviral delivery of Cre-recombinase in insulin receptorlox/lox mice resulted in sufficient ablation of insulin receptors in the SFO. Interestingly, when mice were maintained on a normal chow diet, deletion of SFO insulin receptors resulted in greater weight gain and adiposity, relative to controls, independently of changes in food intake. In line with this, ablation of insulin receptors in the SFO was associated with marked hepatic steatosis and hypertriglyceridemia. Selective removal of SFO insulin receptors also resulted in a lower mean arterial blood pressure, which was primarily due to a reduction in diastolic blood pressure, whereas systolic blood pressure remained unchanged. Cre-mediated targeting of SFO insulin receptors did not influence heart rate. These data demonstrate multidirectional roles for insulin receptor signaling in the SFO, with ablation of SFO insulin receptors resulting in an overall deleterious metabolic state while at the same time maintaining blood pressure at low levels. These novel findings further suggest that alterations in insulin receptor signaling in the SFO could contribute to metabolic syndrome phenotypes.

Liver sympathetic denervation reverses obesity-induced hepatic steatosis.

KEY POINTS: Non-alcoholic fatty liver disease, characterized in part by elevated liver triglycerides (i.e. hepatic steatosis), is a growing health problem. In this study, we found that hepatic steatosis is associated with robust hepatic sympathetic overactivity. Removal of hepatic sympathetic nerves reduced obesity-induced hepatic steatosis. Liver sympathetic innervation modulated hepatic lipid acquisition pathways during obesity. ABSTRACT: Non-alcoholic fatty liver disease (NAFLD) affects 1 in 3 Americans and is a significant risk factor for type II diabetes mellitus, insulin resistance and hepatic carcinoma. Characterized in part by excessive hepatic triglyceride accumulation (i.e. hepatic steatosis), the incidence of NAFLD is increasing - in line with the growing obesity epidemic. The role of the autonomic nervous system in NAFLD remains unclear. Here, we show that chronic hepatic sympathetic overactivity mediates hepatic steatosis. Direct multiunit recordings of hepatic sympathetic nerve activity were obtained in high fat diet and normal chow fed male C57BL/6J mice. To reduce hepatic sympathetic nerve activity we utilized two approaches including pharmacological ablation of the sympathetic nerves and phenol-based hepatic sympathetic nerve denervation. Diet-induced NAFLD was associated with a nearly doubled firing rate of the hepatic sympathetic nerves, which was largely due to an increase in efferent nerve traffic. Furthermore, established high fat diet-induced hepatic steatosis was effectively reduced with pharmacological or phenol-based removal of the hepatic sympathetic nerves, independent of changes in body weight, caloric intake or adiposity. Ablation of liver sympathetic nerves was also associated with improvements in liver triglyceride accumulation pathways including free fatty acid uptake and de novo lipogenesis. These findings highlight an unrecognized pathogenic link between liver sympathetic outflow and hepatic steatosis and suggest that manipulation of the liver sympathetic nerves may represent a novel therapeutic strategy for NAFLD.

Insulin increases ventilation during euglycemia in humans.

Evidence from animal studies indicates that hyperinsulinemia, without changes in glucose, increases ventilation via a carotid body-mediated mechanism. However, whether insulin elevates ventilation in humans independently of changes in glucose remains unclear. Therefore, we tested the hypothesis that insulin increases ventilation in humans during a hyperinsulinemic-euglycemic clamp in which insulin was elevated to postprandial concentrations while glucose was maintained at fasting concentrations. First, in 16 healthy young men ( protocol 1), we retrospectively analyzed respiration rate and estimated tidal volume from a pneumobelt to calculate minute ventilation during a hyperinsulinemic-euglycemic clamp. In addition, for a direct assessment of minute ventilation during a hyperinsulinemic-euglycemic clamp, we retrospectively analyzed breath-by-breath respiration rate and tidal volume from inspired/expired gasses in an additional 23 healthy young subjects ( protocol 2). Clamp infusion elevated minute ventilation from baseline in both protocols ( protocol 1: +11.9 ± 4.6% baseline, P = 0.001; protocol 2: +9.5 ± 3.8% baseline, P = 0.020). In protocol 1, peak changes in both respiration rate (+13.9 ± 3.0% baseline, P < 0.001) and estimated tidal volume (+16.9 ± 4.1% baseline, P = 0.001) were higher than baseline during the clamp. In protocol 2, tidal volume primarily increased during the clamp (+9.7 ± 3.7% baseline, P = 0.016), as respiration rate did not change significantly (+0.2 ± 1.8% baseline, P = 0.889). Collectively, we demonstrate for the first time in humans that elevated plasma insulin increases minute ventilation independent of changes in glucose.

Endoplasmic reticulum stress in the pathogenesis of hypertension.

NEW FINDINGS: What is the topic of this review? This review highlights the emerging role of disruptions in endoplasmic reticulum (ER) function, namely ER stress, as a contributor to hypertension. What advances does it highlight? This review presents an integrative view of ER stress in cardiovascular control systems, including systems within the brain, kidney and peripheral vasculature, as related to development of hypertension. The endoplasmic reticulum (ER) is a cellular organelle specialized in the synthesis, folding, assembly and modification of proteins. In situations of increased protein demand, complex signalling pathways, termed the unfolded protein response, influence a series of cellular feedback loops to control ER function strictly. Although this is initially a compensatory attempt to maintain cellular homeostasis, chronic activation of the unfolded protein response, known as ER stress, leads to sustained changes in cellular function. A growing body of literature points to ER stress in diverse cardioregulatory systems, including the brain, kidney and vasculature, as central to the development of hypertension. Here, these recent findings from essential and obesity-related forms of hypertension are highlighted in an integrative manner, with discussion of the potential upstream causes and downstream consequences of ER stress. Given that hypertension is a leading medical and socio-economic global challenge, emerging findings suggest that targeting ER stress might represent a viable strategy for the treatment of hypertensive disease.

NMDA Receptor Plasticity in the Hypothalamic Paraventricular Nucleus Contributes to the Elevated Blood Pressure Produced by Angiotensin II.

Hypertension induced by angiotensin II (Ang II) is associated with glutamate-dependent dysregulation of the hypothalamic paraventricular nucleus (PVN). Many forms of glutamate-dependent plasticity are mediated by NMDA receptor GluN1 subunit expression and the distribution of functional receptor to the plasma membrane of dendrites. Here, we use a combined ultrastructural and functional analysis to examine the relationship between PVN NMDA receptors and the blood pressure increase induced by chronic infusion of a low dose of Ang II. We report that the increase in blood pressure produced by a 2 week administration of a subpressor dose of Ang II results in an elevation in plasma membrane GluN1 in dendrites of PVN neurons in adult male mice. The functional implications of these observations are further demonstrated by the finding that GluN1 deletion in PVN neurons attenuated the Ang II-induced increases in blood pressure. These results indicate that NMDA receptor plasticity in PVN neurons significantly contributes to the elevated blood pressure mediated by Ang II.

A synthetic luciferin improves in vivo bioluminescence imaging of gene expression in cardiovascular brain regions.

Bioluminescence imaging is an effective tool for in vivo investigation of molecular processes. We have demonstrated the applicability of bioluminescence imaging to spatiotemporally monitor gene expression in cardioregulatory brain nuclei during the development of cardiovascular disease, via incorporation of firefly luciferase into living animals, combined with exogenous d-luciferin substrate administration. Nevertheless, d-luciferin uptake into the brain tissue is low, which decreases the sensitivity of bioluminescence detection, particularly when considering small changes in gene expression in tiny central areas. Here, we tested the hypothesis that a synthetic luciferin, cyclic alkylaminoluciferin (CycLuc1), would be superior to d-luciferin for in vivo bioluminescence imaging in cardiovascular brain regions. Male C57B1/6 mice underwent targeted delivery of an adenovirus encoding the luciferase gene downstream of the CMV promoter to the subfornical organ (SFO) or paraventricular nucleus of hypothalamus (PVN), two crucial cardioregulatory neural regions. While bioluminescent signals could be obtained following d-luciferin injection (150 mg/kg), CycLuc1 administration resulted in a three- to fourfold greater bioluminescent emission from the SFO and PVN, at 10- to 20-fold lower substrate concentrations (7.5-15 mg/kg). This CycLuc1-mediated enhancement in bioluminescent emission was evident early following substrate administration (i.e., 6-10 min) and persisted for up to 1 h. When the exposure time was reduced from 60 s to 1,500 ms, minimal signal in the PVN was detectable with d-luciferin, whereas bioluminescent images could be reliably captured with CycLuc1. These findings demonstrate that bioluminescent imaging with the synthetic luciferin CycLuc1 provides an improved physiological genomics tool to investigate molecular events in discrete cardioregulatory brain nuclei.

Arterial baroreflex control of sympathetic nerve activity and heart rate in patients with type 2 diabetes.

Despite greater blood pressure reactivity to acute cardiovascular stressors and a higher prevalence of hypertension in type 2 diabetes (T2D) patients, limited information is available regarding arterial baroreflex (ABR) control in T2D. We hypothesized that ABR control of muscle sympathetic nerve activity (MSNA) and heart rate (HR) are attenuated in T2D patients. Seventeen T2D patients (50 ± 2 yr; 31 ± 1 kg/m2), 9 weight-matched controls (WM-CON, 46 ± 2 yr; 32 ± 2 kg/m2) and 10 lean controls (Lean-CON, 49 ± 3 yr; 23 ± 1 kg/m2), underwent bolus infusions of sodium nitroprusside (100 µg) followed 60 s later by phenylephrine (150 µg) and weighted linear regression performed. No group differences in overall sympathetic baroreflex gain were observed (T2D: -2.5 ± 0.3 vs. WM-CON: -2.6 ± 0.2 vs. Lean-CON: -2.7 ± 0.4 arbitrary units·beat·mmHg-1, P > 0.05) or in sympathetic baroreflex gain when derived separately during blood pressure (BP) falls (nitroprusside) and BP rises (phenylephrine). In contrast, overall cardiac baroreflex gain was reduced in T2D patients compared with Lean-CON (T2D: 8.2 ± 1.5 vs. Lean-CON: 15.6 ± 2.9 ms·mmHg-1, P < 0.05) and also tended to be reduced in WM-CON (9.3 ± 1.9 ms·mmHg-1) compared with Lean-CON (P = 0.059). Likewise, during BP rises, cardiac baroreflex gain was reduced in T2D patients and weight-matched controls compared with lean controls (P < 0.05), whereas no group differences were found during BP falls (P > 0.05). Sympathetic and cardiac ABR gains were comparable between normotensive and hypertensive T2D patients (P > 0.05). These findings suggest preserved ABR control of MSNA in T2D patients compared with both obese and lean age-matched counterparts, with a selective impairment in ABR HR control in T2D that may be related to obesity.

Neural Control of Non-vasomotor Organs in Hypertension.

Hypertension affects over 25 % of the population with the incidence continuing to rise, due in part to the growing obesity epidemic. Chronic elevations in sympathetic nerve activity (SNA) are a hallmark of the disease and contribute to elevations in blood pressure through influences on the vasculature, kidney, and heart (i.e., neurogenic hypertension). In this regard, a number of central nervous system mechanisms and neural pathways have emerged as crucial in chronically elevating SNA. However, it is important to consider that "sympathetic signatures" are present, with differential increases in SNA to regional organs that are dependent upon the disease progression. Here, we discuss recent findings on the central nervous system mechanisms and autonomic regulatory networks involved in neurogenic hypertension, in both non-obesity- and obesity-associated hypertension, with an emphasis on angiotensin-II, salt, oxidative and endoplasmic reticulum stress, inflammation, and the adipokine leptin.

Endoplasmic reticulum and oxidant stress mediate nuclear factor-κB activation in the subfornical organ during angiotensin II hypertension.

Endoplasmic reticulum (ER) stress and reactive oxygen species (ROS) generation in the brain circumventricular subfornical organ (SFO) mediate the central hypertensive actions of Angiotensin II (ANG II). However, the downstream signaling events remain unclear. Here we tested the hypothesis that angiotensin type 1a receptors (AT1aR), ER stress, and ROS induce activation of the transcription factor nuclear factor-κB (NF-κB) during ANG II-dependent hypertension. To spatiotemporally track NF-κB activity in the SFO throughout the development of ANG II-dependent hypertension, we used SFO-targeted adenoviral delivery and longitudinal bioluminescence imaging in mice. During low-dose infusion of ANG II, bioluminescence imaging revealed a prehypertensive surge in NF-κB activity in the SFO at a time point prior to a significant rise in arterial blood pressure. SFO-targeted ablation of AT1aR, inhibition of ER stress, or adenoviral scavenging of ROS in the SFO prevented the ANG II-induced increase in SFO NF-κB. These findings highlight the utility of bioluminescence imaging to longitudinally track transcription factor activation during the development of ANG II-dependent hypertension and reveal an AT1aR-, ER stress-, and ROS-dependent prehypertensive surge in NF-κB activity in the SFO. Furthermore, the increase in NF-κB activity before a rise in arterial blood pressure suggests a causal role for SFO NF-κB in the development of ANG II-dependent hypertension.

Circumventricular organ-hypothalamic circuit endoplasmic reticulum stress drives hepatic steatosis during obesity.

OBJECTIVE: Nonalcoholic fatty liver disease (NAFLD), characterized by excess liver triglyceride accumulation (hepatic steatosis), leads to an increased risk for cardiometabolic diseases and obesity-related mortality. Emerging evidence points to endoplasmic reticulum (ER) stress in the central nervous system as critical in NAFLD pathogenesis. Here, we tested the contribution of ER stress in a circumventricular organ-hypothalamic circuit in NAFLD development during obesity. METHODS: C57BL/6J male mice were fed a high-fat diet (HFD) or normal chow. A combination of histological, viral tracing, intersectional viral targeting, and in vivo integrative physiological approaches were used to examine the role of ER stress in subfornical organ to hypothalamic paraventricular nucleus projecting neurons (SFO➔PVN) in NAFLD during diet-induced obesity. RESULTS: Immunohistochemical analysis revealed marked unfolded protein response activation in the SFO, particularly in excitatory SFO➔PVN neurons of HFD-fed animals. Moreover, intersectional viral inhibition of ER stress in SFO➔PVN neurons resulted in a reduction in hepatomegaly, hepatic steatosis, and a blunted increase in body weight gain during diet-induced obesity, independent of changes in food intake, substrate partitioning, energy expenditure, and ambulatory activity. CONCLUSIONS: These results indicate that ER stress in an SFO➔PVN neural circuit contributes to hepatic steatosis during obesity.

A forebrain-hypothalamic ER stress driven circuit mediates hepatic steatosis during obesity.

OBJECTIVE: Non-alcoholic fatty liver disease (NAFLD) affects 1 in 3 adults and contributes to advanced liver injury and cardiometabolic disease. While recent evidence points to involvement of the brain in NAFLD, the downstream neural circuits and neuronal molecular mechanisms involved in this response, remain unclear. Here, we investigated the role of a unique forebrain-hypothalamic circuit in NAFLD. METHODS: Chemogenetic activation and inhibition of circumventricular subfornical organ (SFO) neurons that project to the paraventricular nucleus of the hypothalamus (PVN; SFO→PVN) in mice were used to study the role of SFO→PVN signaling in NAFLD. Novel scanning electron microscopy techniques, histological approaches, molecular biology techniques, and viral methodologies were further used to delineate the role of endoplasmic reticulum (ER) stress within this circuit in driving NAFLD. RESULTS: In lean animals, acute chemogenetic activation of SFO→PVN neurons was sufficient to cause hepatic steatosis in a liver sympathetic nerve dependent manner. Conversely, inhibition of this forebrain-hypothalamic circuit rescued obesity-associated NAFLD. Furthermore, dietary NAFLD is associated with marked ER ultrastructural alterations and ER stress in the PVN, which was blunted following reductions in excitatory signaling from the SFO. Finally, selective inhibition of PVN ER stress reduced hepatic steatosis during obesity. CONCLUSIONS: Collectively, these findings characterize a previously unrecognized forebrain-hypothalamic-ER stress circuit that is involved in hepatic steatosis, which may point to future therapeutic strategies for NAFLD.

Antibodies elicited by Plasmodium falciparum circumsporozoite proteins lacking sequentially deleted C-terminal amino acids reveal mouse strain and epitopes specific differences.

Malaria affects ∼ » billion people globally and requires the development of additional tools to aid in elimination efforts. The recently approved RTS,S/AS01 vaccine represents a positive step, however, the moderate efficacy necessitates the development of more efficacious vaccines. PfCSP is a key target antigen for pre-erythrocytic vaccines aimed at preventing Plasmodium falciparum malaria infections. Epitopes within the central repeat region and at the junction of the repeat and N-terminal domain are well documented as major protective B cell epitopes. On the other hand, a majority of antibodies against the epitopes in the C-terminal domain, have been shown to be non-protective against sporozoite challenge. The C-terminal domain, however, contains CD4+ and CD8+ T cell epitopes previously shown to be important for regulating immune responses. The present study was designed to further explore the immunomodulatory potential of the C-terminal domain using DNA vaccines encoding PfCSP with sequential C-terminal truncations following known T cell epitopes. Five DNA vaccines encoding different truncations of PfCSP within the C-terminal domain were administered via intramuscular route and in vivo electroporation for effective immunogenicity. Protection in mice was evaluated by challenge with transgenic P. berghei expressing PfCSP. In Balb/c mice, antibody responses and protective efficacy were both affected progressively with sequential deletion of C-terminal amino acid residues. Similar studies in C57Bl/6 mice revealed that immunizations with plasmids encoding truncated PfCSP showed partial protection from sporozoite challenge with no significant differences in antibody titers observed compared to full-length PfCSP DNA immunized mice. Further analysis revealed murine strain-specific differences in the recognition of specific epitopes.

Central Feminization of Obese Male Mice Reduces Metabolic Syndrome.

Metabolic syndrome encompasses a spectrum of conditions that increases the risk for cardiovascular and metabolic diseases. It is widely accepted that the sex hormone estrogen plays a protective metabolic role in premenopausal women, in part through central nervous system (CNS) mechanisms. However, most work to date has focused on the loss of estrogen in females (e.g., menopause). Interestingly, transgender individuals receiving feminizing gender affirming therapy (i.e., estrogen) are relatively protected from metabolic syndrome conditions, pointing to a role for CNS estrogen in the development of metabolic syndrome in men. Here, we show that estrogen signaling in the brain protects males from metabolic syndrome and obesity related complications. First, short-term CNS specific supplementation of low-dose 17-ß-estradiol in diet-induced obese male mice resulted in a significant reduction in body weight in parallel with a decrease in food intake without alterations in energy expenditure. In conjunction, central supplementation of estrogen reduced visceral adiposity, including epididymal and abdominal regions, with slighter decreases in subcutaneous inguinal and thermogenic brown adipose tissue. Furthermore, central estrogen administration reduced the liver manifestation of metabolic syndrome including hepatomegaly and hepatic steatosis. Collectively, these findings indicate that a lack of estrogen action in the brain may predispose males to metabolic syndrome pathogenesis.

RNA polymerase II pausing factor NELF in CD8+ T cells promotes antitumor immunity.

T cell factor 1 (TCF1) is required for memory and stem-like CD8+ T cell functions. How TCF1 partners with other transcription factors to regulate transcription remains unclear. Here we show that negative elongation factor (NELF), an RNA polymerase II (Pol II) pausing factor, cooperates with TCF1 in T cell responses to cancer. Deletion of mouse Nelfb, which encodes the NELFB subunit, in mature T lymphocytes impairs immune responses to both primary tumor challenge and tumor antigen-mediated vaccination. Nelfb deletion causes more exhausted and reduced memory T cell populations, whereas its ectopic expression boosts antitumor immunity and efficacy of chimeric antigen receptor T-cell immunotherapy. Mechanistically, NELF is associated with TCF1 and recruited preferentially to the enhancers and promoters of TCF1 target genes. Nelfb ablation reduces Pol II pausing and chromatin accessibility at these TCF1-associated loci. Our findings thus suggest an important and rate-limiting function of NELF in anti-tumor immunity.

Effective Functional Immunogenicity of a DNA Vaccine Combination Delivered via In Vivo Electroporation Targeting Malaria Infection and Transmission.

Plasmodium falciparum circumsporozoite protein (PfCSP) and Pfs25 are leading candidates for the development of pre-erythrocytic and transmission-blocking vaccines (TBV), respectively. Although considerable progress has been made in developing PfCSP- and Pfs25-based vaccines, neither have elicited complete protection or transmission blocking in clinical trials. The combination of antigens targeting various life stages is an alternative strategy to develop a more efficacious malaria vaccine. In this study, female and male mice were immunized with DNA plasmids encoding PfCSP and Pfs25, administered alone or in combination via intramuscular in vivo electroporation (EP). Antigen-specific antibodies were analyzed for antibody titers, avidity and isotype by ELISA. Immune protection against sporozoite challenge, using transgenic P. berghei expressing PfCSP and a GFP-luciferase fusion protein (PbPfCSP-GFP/Luc), was assessed by in vivo bioluminescence imaging and blood-stage parasite growth. Transmission reducing activity (TRA) was evaluated in standard membrane feeding assays (SMFA). High levels of PfCSP- and Pfs25-specific antibodies were induced in mice immunized with either DNA vaccine alone or in combination. No difference in antibody titer and avidity was observed for both PfCSP and Pfs25 between the single DNA and combined DNA immunization groups. When challenged by PbPfCSP-GFP/Luc sporozoites, mice immunized with PfCSP alone or combined with Pfs25 revealed significantly reduced liver-stage parasite loads as compared to mice immunized with Pfs25, used as a control. Furthermore, parasite liver loads were negatively correlated with PfCSP-specific antibody levels. When evaluating TRA, we found that immunization with Pfs25 alone or in combination with PfCSP elicited comparable significant transmission reduction. Our studies reveal that the combination of PfCSP and Pfs25 DNAs into a vaccine delivered by in vivo EP in mice does not compromise immunogenicity, infection protection and transmission reduction when compared to each DNA vaccine individually, and provide support for further evaluation of this DNA combination vaccine approach in larger animals and clinical trials.

In vivo assessment of neurocardiovascular regulation in the mouse: principles, progress, and prospects.

A growing body of evidence indicates that a number of common complex diseases, including hypertension, heart failure, and obesity, are characterized by alterations in central neurocardiovascular regulation. However, our understanding of how changes within the central nervous system contribute to the development and progression of these and other diseases remains unclear. As with many areas of cardiovascular research, the mouse has emerged as a key species for investigations of neuroregulatory processes because of its amenability to highly specific genetic manipulations. In parallel with the development of increasingly sophisticated murine models has come the miniaturization and advancement in methodologies for in vivo assessment of neurocardiovascular end points in the mouse. The following brief review will focus on a number of key direct and indirect experimental approaches currently in use, including measurement of arterial blood pressure, assessment of cardiovascular autonomic control, and evaluation of arterial baroreflex function. The advantages and limitations of each methodology are highlighted to allow for a critical evaluation by the reader when considering these approaches.

Impact of age on critical closing pressure of the cerebral circulation during dynamic exercise in humans.

Limited information is available regarding cerebral vascular responses to dynamic exercise in older adults. We examined the influence of age on exercise-induced changes in the critical closing pressure (CCP) of the cerebral vasculature. Twelve young and twelve older subjects performed two bouts of steady-state cycling at low and moderate intensities (30 and 50% heart rate reserve). Mean arterial pressure (MAP), middle cerebral artery blood velocity (MCA V) and partial pressure of end-tidal carbon dioxide ( ) were measured. The CCP was estimated by linear extrapolation of pairs of systolic and diastolic blood pressure and MCA V waveforms. Exercise-induced increases in MAP were greater in older subjects (P < 0.01), while mean MCA V (MCA V(mean)) responses to exercise were similar between groups (P = 0.59). The CCP was elevated from rest during low- and moderate-intensity exercise in both groups (moderate exercise: young, +13 ± 2 mmHg and older, +22 ± 2 mmHg; P < 0.01), with the older subjects exhibiting greater increases in CCP during both exercise intensities (moderate exercise: young, +43 ± 9% rest versus older, +153 ± 45% rest; P = 0.04). In contrast, cerebral vascular conductance index (MCA V(mean)/MAP; CVCi) was only decreased during moderate exercise in older subjects (P < 0.01) and CVCi was not altered from rest in young subjects during low- or moderate-intensity cycling. No age-group differences were observed in at rest or during two intensities of exercise (P = 0.40). These data demonstrate that older subjects exhibit larger exercise-induced increases in CCP and decreases in CVCi. Thus, ageing is associated with greater increases in cerebral vascular tone during low- and moderate-intensity dynamic exercise.

Sensory Circumventricular Organs, Neuroendocrine Control, and Metabolic Regulation.

The central nervous system is critical in metabolic regulation, and accumulating evidence points to a distributed network of brain regions involved in energy homeostasis. This is accomplished, in part, by integrating peripheral and central metabolic information and subsequently modulating neuroendocrine outputs through the paraventricular and supraoptic nucleus of the hypothalamus. However, these hypothalamic nuclei are generally protected by a blood-brain-barrier limiting their ability to directly sense circulating metabolic signals-pointing to possible involvement of upstream brain nuclei. In this regard, sensory circumventricular organs (CVOs), brain sites traditionally recognized in thirst/fluid and cardiovascular regulation, are emerging as potential sites through which circulating metabolic substances influence neuroendocrine control. The sensory CVOs, including the subfornical organ, organum vasculosum of the lamina terminalis, and area postrema, are located outside the blood-brain-barrier, possess cellular machinery to sense the metabolic interior milieu, and establish complex neural networks to hypothalamic neuroendocrine nuclei. Here, evidence for a potential role of sensory CVO-hypothalamic neuroendocrine networks in energy homeostasis is presented.

In Vitro and In Vivo Enhancement of Temozolomide Effect in Human Glioblastoma by Non-Invasive Application of Cold Atmospheric Plasma.

Glioblastoma (GBM) is one of the most aggressive forms of adult brain cancers and is highly resistant to treatment, with a median survival of 12-18 months after diagnosis. The poor survival is due to its infiltrative pattern of invasion into the normal brain parenchyma, the diffuse nature of its growth, and its ability to quickly grow, spread, and relapse. Temozolomide is a well-known FDA-approved alkylating chemotherapy agent used for the treatment of high-grade malignant gliomas, and it has been shown to improve overall survival. However, in most cases, the tumor relapses. In recent years, CAP has been used as an emerging technology for cancer therapy. The purpose of this study was to implement a combination therapy of CAP and TMZ to enhance the effect of TMZ and apparently sensitize GBMs. In vitro evaluations in TMZ-sensitive and resistant GBM cell lines established a CAP chemotherapy enhancement and potential sensitization effect across various ranges of CAP jet application. This was further supported with in vivo findings demonstrating that a single CAP jet applied non-invasively through the skull potentially sensitizes GBM to subsequent treatment with TMZ. Gene functional enrichment analysis further demonstrated that co-treatment with CAP and TMZ resulted in a downregulation of cell cycle pathway genes. These observations indicate that CAP can be potentially useful in sensitizing GBM to chemotherapy and for the treatment of glioblastoma as a non-invasive translational therapy.