Transient Receptor Potential Melastatin 2 Thermosensitive Neurons in the Preoptic Area Involved in Menopausal Hot Flashes in Ovariectomized Mice.

INTRODUCTION: Menopausal hot flashes are related to hypothalamic preoptic area (POA) dysfunction. Thermosensitive transient receptor potential channels (ThermoTRPs) are involved in temperature sensing and regulation of thermosensitive neurons (TSNs) in the POA. Whether ThermoTRP-TSNs in the POA, particularly the non-noxious thermoreceptor, transient receptor potential melastatin 2 (TRPM2), are involved in the occurrence of hot flashes is still unclear. METHODS: Twenty wild-type and 50 Trpm2-Cre adult female mice were randomly divided into sham (SHAM) and ovariectomy (OVX) groups. In the POA, ERα, ERß, GPR30, TRPA1, TRPM8, TRPM2, and TRPV1 expression was detected by Western blot or/and quantitative real-time polymerase chain reaction and the number of TSNs expressing TRPM2 (TRPM2-TSNs) by immunofluorescence. Before and after TRPM2-TSN activation/inhibition, back (BST) and tail skin temperature (TST) and the proportion of glutamatergic and GABAergic neurons among TRPM2-TSNs were recorded. RESULTS: Compared with SHAM, the expression of ERα, ERß, TRPM2, and TRPM8 in the POA of the OVX group decreased, with a significantly larger change range for TRPM2 than TRPM8. In addition, the number of TRPM2-TSNs showing TRPA1, TRPM8, and TRPV1 expression in the OVX group decreased, and the proportion of glutamatergic and GABAergic neurons in TRPM2-TSNs decreased and increased, respectively. Meanwhile, BST and TST increased. After activating or inhibiting TRPM2-TSNs, the proportions of glutamatergic and GABAergic neurons in TRPM2-TSNs changed, along with the BST and TST. CONCLUSION: In menopause, the abnormal quantity and function of TRPM2-TSNs in the POA is key for the development of hot flashes, characterized by an imbalance in heat dissipation and production due to the corresponding imbalance in glutamatergic and GABAergic neurons.

Pyrotinib in vitro metabolite profiling via rat, dog and human hepatocytes using liquid chromatography-quadrupole/orbitrap mass spectrometry.

RATIONALE: Pyrotinib is an irreversible EGFR/HER2 inhibitor that has shown antitumor activity and tolerance in the treatment of breast cancer. Studies focused on its metabolic pathways and major metabolites are insufficient. In the evaluation of drug safety and therapeutic use, metabolite characterization is critical. The metabolism of pyrotinib in vitro was studied utilizing rat, dog and human hepatocytes in this study. METHODS: Pyrotinib (10 µM) was incubated with hepatocytes in Williams' E medium. The metabolites were examined and profiled using ultrahigh-performance liquid chromatography coupled with quadrupole/orbitrap high-resolution mass spectrometry. The metabolite structures were deduced by comparing their precise molecular weights, fragment ions and retention times with those of the parent drug. RESULTS: A total of 16 metabolites, including 6 novel ones, were discovered and structurally described under the present conditions. Oxidation, demethylation, dehydrogenation, O-dealkylation and glutathione (GSH) conjugation were all involved in the metabolism of pyrotinib in hepatocytes. The most predominant metabolic route was identified as GSH conjugation (M5). CONCLUSIONS: This study generated valuable metabolite profiles of pyrotinib in several species, which will aid in the understanding of the drug's disposition in various species and in evaluating the contribution of metabolites to overall effectiveness and toxicity of pyrotinib.

The imbalance in the aortic ceramide/sphingosine-1-phosphate rheostat in ovariectomized rats and the preventive effect of estrogen.

BACKGROUND: The prevalence of hypertension in young women is lower than that in age-matched men while the prevalence of hypertension in women is significantly increased after the age of 50 (menopause) and is greater than that in men. It is already known that sphingosine-1-phosphate (S1P) and ceramide regulate vascular tone with opposing effects. This study aimed to explore the effects of ovariectomy and estrogen supplementation on the ceramide/S1P rheostat of the aorta in rats, and to explore a potential mechanism for perimenopausal hypertension and a brand-new target for menopausal hormone therapy to protect vessels. METHODS: In total, 30 female adult SD rats were randomly divided into three groups: The sham operation group (SHAM), ovariectomy group (OVX) and ovariectomy plus estrogen group (OVX + E). After 4 weeks of treatment, the blood pressure (BP) of the rats was monitored by a noninvasive system; the sphingolipid content (e.g., ceramide and S1P) was detected by liquid chromatography-mass spectrometry (LC-MS); the expression of the key enzymes involved in ceramide anabolism and catabolism was measured by real-time fluorescence quantitative polymerase chain reaction (qPCR); and the expression of key enzymes and proteins in the sphingosine kinase 1/2 (SphK1/2)-S1P-S1P receptor 1/2/3 (S1P1/2/3) signaling pathway was detected by qPCR and western blotting. RESULTS: In the OVX group compared with the SHAM group, the systolic BP (SBP), diastolic BP (DBP) and pulse pressure (PP) increased significantly, especially the SBP and PP (P < 0.001). For aortic ceramide metabolism, the mRNA level of key enzymes involved in anabolism and catabolism decreased in parallel 2-3 times, while the contents of total ceramide and certain long-chain subtypes increased significantly (P < 0.05). As for the S1P signaling pathway, SphK1/2, the key enzymes involved in S1P synthesis, decreased significantly, and the content of S1P decreased accordingly (P < 0.01). The S1P receptors showed various trends: S1P1 was significantly down-regulated, S1P2 was significantly up-regulated, and S1P3 showed no significant difference. No significant difference existed between the SHAM and OVX + E groups for most of the above parameters (P > 0.05). CONCLUSIONS: Ovariectomy resulted in the imbalance of the aortic ceramide/S1P rheostat in rats, which may be a potential mechanism underlying the increase in SBP and PP among perimenopausal women. Besides, the ceramide/S1P rheostat may be a novel mechanism by which estrogen protects vessels.

Gender-dependent pharmacokinetics of olaparib in rats determined by ultra-high performance liquid chromatography/electrospray ionization tandem mass spectrometry.

The aim of the present study was to develop a liquid chromatography/electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method for the determination of olaparib in rat plasma. The plasma samples were processed using one-step protein precipitation with acetonitrile and then separated on Waters Acquity BEH C18 column (50 × 2.1 mm, particle size 1.7 µm) using water containing 0.1% formic acid and acetonitrile as mobile phase with optimized gradient elution. Mass spectrometric detection was carried out by selective reaction monitoring mode via positive ESI mode with precursor-to-product transitions of m/z 435.3 > 367.1 and m/z 443.1 > 375.2 for olaparib and 2 H8 -olaparib (internal standard). The method was linear over the concentration range 0.1-2000 ng/ml with correlation coefficient >0.9987. The lower limit of quantitation was 0.1 ng/ml. The method showed excellent accuracy and precision, negligible matrix effect and high extraction recovery. The validated method was subsequently utilized to determine the concentration of olaparib in rat plasma and further applied to the pharmacokinetic study of olaparib in rat plasma. Our results demonstrated that olaparib showed gender-dependent pharmacokinetics in rats. Compared with that in males, olaparib showed high plasma exposure, long half-life, low clearance and high bioavailability in females.

Effects of oestrogen and Cimicifuga racemosa on the cardiac noradrenaline pathway of ovariectomized rats.

NEW FINDINGS: What is the central question of this study? We investigated the effects of oestrogen and Cimicifuga racemosa on the stellate ganglion, cardiac noradrenaline pathway and Ca2+ -calmodulin-dependent protein kinase II in ovariectomized rats. What is the main finding and its importance? The right stellate ganglion, but not the left, may be associated with decreased left ventricular noradrenaline content in ovariectomized rats. Oestrogen can reverse all changes caused by ovariectomy. Cimicifuga racemosa did not affect left ventricular noradrenaline, but decreased protein expression of ß1 -adrenergic receptor and Ca2+ -calmodulin-dependent protein kinase II. The results might explain potential effects of C. racemosa on the cardiovascular system and provide new insights into cardiovascular protection. The aim of this study was to investigate the effects of oestrogen and Cimicifuga racemosa on the stellate ganglion, cardiac noradrenaline (NA) pathway and Ca2+ -calmodulin-dependent protein kinase II (CaMK II). Forty adult female Sprague-Dawley rats were randomly divided into the following four groups: sham operated (SHAM); ovariectomized (OVX); ovariectomized with oestrogen treatment (E2); and ovariectomized with C. racemosa treatment (iCR). After 4 weeks of treatment, the NA content was determined by high-performance liquid chromatography, and dopamine ß-hydroxylase (DBH) and noradrenaline transporter (NET) expression were detected by immunohistochemistry. Western blotting was used to determine NET, ß1 -adrenergic receptor (ß1 -AR) and CaMK II expression. Compared with the SHAM group, body weights, DBH and NET expression in the right stellate ganglia, and NET, ß1 -AR and CaMK II expression in the left ventricles of the OVX group were increased, whereas left ventricular NA content was decreased; DBH and NET expression in the left stellate ganglion was not significantly different. The indexes of the E2 group were similar to those of the SHAM group. Moreover, in the iCR group, DBH, NET, ß1 -AR and CaMK II expression was decreased; NET expression and NA content of the left ventricle remained unchanged. Our conclusions are as follows. First, the right stellate ganglion, but not the left, may be associated with decreased left ventricular NA content in OVX rats. Second, oestrogen increases the left ventricular NA content and adjusts the expression of DBH and NET in the right stellate ganglion and restores ß1 -AR and CaMK II protein expression to normal levels. Third, C. racemosa does not affect left ventricular NA, but decreases the protein expression of ß1 -AR and CaMK II.

Does Cimicifuga racemosa have the effects like estrogen on the sublingual gland in ovariectomized rats?

BACKGROUND: Cimicifuga racemosa is one of the herbs used for the treatment of climacteric syndrome, and it has been cited as an alternative therapy to estrogen. Apart from hectic fevers, dyspareunia and so on, dry mouth also increase significantly after menopause. It has not yet been reported whether C. racemosa has any impact on the sublingual gland, which may relate to dry mouth. In an attempt to determine this, we have compared the effects of estrogen and C. racemosa on the sublingual gland of ovariectomized rats. RESULTS: HE staining showed that the acinar cell area had contracted and that the intercellular spaces were broadened in the OVX (ovariectomized rats) group, while treatment with estradiol (E2) and iCR (isopropanolic extract of C. racemosa) improved these lesions. Transmission electron microscopy showed that rough endoplasmic reticulum expansion in mucous and serous acinar epithelial cells and apoptotic cells was more commonly seen in the OVX group than in the SHAM (sham-operated rats) group. Mitochondria and plasma membrane infolding lesions in the striated ducts were also observed. These lesions were alleviated by both treatments. It is of note that, in the OVX + iCR group, the volume of mitochondria in the striated duct was larger than in other groups. Immunohistochemical staining showed that the ratio of caspase-3 positive cells was significantly increased in the acinar cells of the OVX group compared with the SHAM group (p < 0.05); and the MA (mean absorbance) of caspase-3 in the striated ducts also increased (p < 0.05). Estradiol decreased the ratio of caspase-3 positive cells and the MA of caspase-3 in striated ducts significantly (p < 0.05). ICR also reduced the ratio of caspase-3 positive cells and the MA in the striated ducts (p < 0.05), but the reduction of the MA in striated ducts was inferior to that of the OVX + E2 group (p < 0.05). CONCLUSION: Both estradiol and iCR can inhibit subcellular structural damage, and down-regulate the expression of caspase-3 caused by ovariectomy, but their effects were not identical, suggesting that both drugs confer a protective effect on the sublingual gland of ovariectomized rats, but that the specific location and mechanism of action producing these effects were different.

Effects of estradiol valerate and remifemin on norepinephrine signaling in the brain of ovariectomized rats.

AIMS: We investigated the norepinephrine pathway changes from the locus coeruleus (LC) to the preoptic area of the hypothalamus (POAH) in the brain of ovariectomized rats under low estrogen levels and explored the therapeutic effects of estradiol valerate (E2) and Remifemin (ICR) on these changes. METHODS: 40 female Sprague-Dawley rats were randomly divided into the following groups: surgery with vehicle (SHAM), ovariectomy surgery with vehicle (OVX), ovariectomy with E2 treatment (OVX + E2), and ovariectomy with Remifemin (OVX + ICR). After 4 weeks of treatment, we observed the changes by immunohistochemistry. RESULTS: (1) The average optical density of DBH-ir fibers and the number of α1-adrenoreceptor- and estrogen receptor (ER)α-positive neurons in the main nuclei of POAH were all reduced in OVX rats compared with the SHAM group. The above changes were normalized in all nuclei of the POAH in the E2 group, while they were normalized in some nuclei in the ICR group. Coexpression of ERα and α1-adrenoreceptor was observed in the POAH. (2) The number of DBH- and ERα-positive neurons in the LC decreased in the OVX group compared with the SHAM group and increased after treatment with E2 and ICR. Coexpression of ERα and DBH was observed in the LC. CONCLUSION: Low estrogen (OVX) altered norepinephrine synthesis in the LC, the projection of norepinephrine fibers and α1-adrenoreceptor expression in the POAH. Both E2 and ICR normalized the norepinephrine pathway, but E2 achieved greater effects than ICR. ICR had different effects in different nuclei in the POAH and its therapeutic effect was better in the LC.

Potential diagnostic biomarkers for immunogenic cell death in elderly female patients with ischemic stroke: identification and analysis.

Ischemic stroke (IS) is of increasing concern given the aging population and prevalence of unhealthy lifestyles, with older females exhibiting higher susceptibility. This study aimed to identify practical diagnostic markers, develop a diagnostic model for immunogenic cell death (ICD)-associated IS, and investigate alterations in the immune environment caused by hub genes. Differentially expressed genes associated with ICD in IS were identified based on weighted gene co-expression network analysis and the identification of significant modules. Subsequently, machine learning algorithms were employed to screened hub genes, which were further assessed using Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and Gene Set Enrichment Analysis. A nomogram mode lwas then constructed for IS diagnosis, and its diagnostic value was assessed using a receiver operating characteristic curve. Finally, alterations in immune cell infiltration were assessed within patients with IS, and the pan-cancer expression patterns of hub genes were evaluated. Three hub genes associated with ICD (PDK4, CCL20, and FBL) were identified. The corresponding nomogram model for IS diagnosis could effectively identify older female patients with IS (area under the curve (AUC) = 0.9555). Overall, the three hub genes exhibit good diagnostic value (AUC > 0.8). CCL20 and FBL are significantly associated with the extent of immune cells infiltration. Moreover, a strong link exists between hub gene expression and pan-cancer prognosis. Cumulatively, these results indicate that ICD-related hub genes critically influence IS progression in older females, presenting novel diagnostic and therapeutic targets for personalized treatment.

Oxidative stress plays an important role in the central regulatory mechanism of orofacial hyperalgesia under low estrogen conditions.

Hyperalgesia occurs in the orofacial region of rats when estrogen levels are low, although the specific mechanism needs to be investigated further. Furthermore, oxidative stress plays an important role in the transmission of pain signals. This study aimed to explore the role of oxidative stress in orofacial hyperalgesia under low estrogen conditions. We firstly found an imbalance between oxidative and antioxidant capacity within the spinal trigeminal subnucleus caudalis (SP5C) of rats after ovariectomy (OVX), resulting in oxidative stress and then a decrease in the orofacial pain threshold. To investigate the mechanism by which oxidative stress occurs, we used virus as a tool to silence or overexpress the excitatory amino acid transporter 3 (EAAT3) gene. Further investigation revealed that the regulation of glutathione (GSH) and reactive oxygen species (ROS) can be achieved by regulating EAAT3, which in turn impacts the occurrence of oxidative stress. In summary, our findings suggest that reduced expression of EAAT3 within the SP5C of rats in the low estrogen state may decrease GSH content and increase ROS levels, resulting in oxidative stress and ultimately lead to orofacial hyperalgesia. This suggests that antioxidants could be a potential therapeutic direction for orofacial hyperalgesia under low estrogen conditions, though more research is needed to understand its mechanism.

Mapping the 5-HTergic neural pathways in perimenopausal mice and elucidating the role of oestrogen receptors in 5-HT neurotransmission.

Perimenopausal syndrome (PMS) encompasses neuropsychiatric symptoms, such as hot flashes and depression, which are associated with alterations in the 5-HTergic neural pathway in the brain. However, the specific changes and mechanisms underlying these alterations remain unclear. In this study, ovariectomized mice were used to successfully establish a perimenopause model, and the changes in the expression of 5-HT and its receptors (5-HT1AR and 5-HT2AR) across 72 brain regions in these ovariectomized mice were assessed by immunohistochemistry. Although both 5-HT and 5-HT1AR were widely expressed throughout the brain, only a limited number of regions expressed 5-HT2AR. Notably, decreased expression of 5-HT was observed across almost all brain regions in the ovariectomy (OVX) group compared with the Sham group. Altered expression of both receptors was found within areas related to hot flashes (the preoptic area) or mood disorders (the amygdala). Additionally, reduced oestrogen receptor (ER)α/ß expression was detected in cells in the raphe nucleus (RN), an area known to regulate body temperature. Results showed that ERα/ß positively regulate the transcriptional activity of the enzymes TPH2/MAOA, which are involved in serotonin metabolism during perimenopause. This study revealed the changes in 5-HT neuropathways (5-HT, 5-HT1AR and 5-HT2AR) in perimenopausal mice, mainly in brain regions related to regulation of the body temperature, mood, sleep and memory. This study clarified that the expression of oestrogen receptor decreased in perimenopause, which regulated the transcription levels of TPH2 and MAOA, and ultimately led to the reduction of 5-HT content, providing a new target for clinical diagnosis and treatment of perimenopausal diseases.

Effect of Low-Temperature Plasma Surface Treatment on Bonding Properties of Single-Lap Joint of Thermosetting Composites.

Bonding is one of the main forms of composite bonding. In order to investigate the effect of low-temperature plasma surface treatment on the bonding properties of carbon fiber-reinforced epoxy resin composites (CF/EP), a single-lap joint of CF/EP was prepared. The surface of the CF/EP was treated with atmospheric pressure "low-temperature plasma spray" equipment, and the tensile shear strength, surface morphology, surface contact angle and surface chemical composition of the CF/EP before and after plasma treatment were characterized. Finally, the samples were treated with traditional sandblasting, compared and analyzed. The results show that the effect of low-temperature plasma surface treatment on CF/EP joints is better than that of traditional sandblasting treatment. After low-temperature plasma surface treatment, the tensile shear strength of the CF/EP single-lap joint increased by 119.59% at most, and the failure form of the joint changed from untreated interface failure to mixed failure dominated by cohesion failure. Plasma can etch the surface of composite materials, the mechanical interlock between the carbon fiber and glue is enhanced and the bonding performance of the composite is improved. In addition, after low-temperature plasma surface treatment, the introduction of a large number of oxygen-containing active groups such as C-O and C=O can increase the surface free energy, reduce the contact angle and improve the surface activity and wettability of the composites. However, too long a treatment time will lead to excessive plasma etching of carbon fibers, thus weakening the active effect of the oxygen-containing active groups on the surface of the composites, and the surface wettability is no longer improved, but the adhesive properties of CF/EP are reduced. This paper plays a guiding role in the bonding technology of composite materials.

Transient receptor potential M2 channel in the hypothalamic preoptic area and its impact on thermoregulation during menopause.

BACKGROUND: Decreased estrogen levels can cause abnormal thermosensitivity of the preoptic area (POA) in the hypothalamus during menopause, which may cause hot flashes. Thermosensitive transient receptors (ThermoTRPs) affect the thermosensitivity of neurons. It is worth exploring whether ThermoTRPs change under low estrogen state and participate in the abnormal thermoregulation of POA. METHODS: Adult female Sprague-Dawley rats were randomly divided into sham operation (SHAM), ovariectomy (OVX) and estrogen treatment after ovariectomy (OVX+E) groups. Under 10 â„ƒ, 18 â„ƒ, 25 â„ƒ, 37 â„ƒ and 45 â„ƒ incubations, their skin temperature was monitored and the expression of TRPA1, TRPM8, TRPM2, and TRPV1 in POA were investigated. RESULTS: The skin temperature of ovariectomized rats changed faster and more dramatically under different incubation temperatures. The results at mRNA level show that only the expression of TRPM2 decreased in POA of OVX group compared with the other two groups at 25 â„ƒ, TRPA1 expression in POA of the three groups increased at 10 â„ƒ, TRPM8 increased at 10 â„ƒ and 18 â„ƒ, TRPV1 increased at 10 â„ƒ and 45 â„ƒ, while the expression of TRPM2 decreased at 10 â„ƒ and 18 â„ƒ and increased at 37 â„ƒ and 45 â„ƒ. In all these cases, the magnitudes of the changes were less in the OVX group relative to the other two groups. The further immunohistochemical and Western blot results of TRPM2 and the activated TRPM2 positive cells labeled by c-Fos were consistent with the results of mRNA level. CONCLUSIONS: The expression and thermosensitivity of TRPM2 in POA changed greatly under different incubation temperatures, but the changes in ovariectomized rats were less. This may be the key factor triggering thermoregulation dysfunction under low estrogen and may cause hot flashes.

The lymphatic drainage systems in the brain: a novel target for ischemic stroke?

Recent studies have proposed three lymphatic drainage systems in the brain, that is, the glymphatic system, the intramural periarterial drainage pathway, and meningeal lymphatic vessels, whose roles in various neurological diseases have been widely explored. The glymphatic system is a fluid drainage and waste clearance pathway that utilizes perivascular space and aquaporin-4 protein located in the astrocyte endfeet to provide a space for exchange of cerebrospinal fluid and interstitial fluid. The intramural periarterial drainage pathway drives the flow of interstitial fluid through the capillary basement membrane and the arterial tunica media. Meningeal lymphatic vessels within the dura mater are involved in the removal of cerebral macromolecules and immune responses. After ischemic stroke, impairment of these systems could lead to cerebral edema, accumulation of toxic factors, and activation of neuroinflammation, while restoration of their normal functions can improve neurological outcomes. In this review, we summarize the basic concepts of these drainage systems, including drainage routes, physiological functions, regulatory mechanisms, and detection technologies. We also focus on the roles of lymphatic drainage systems in brain injury after ischemic stroke, as well as recent advances in therapeutic strategies targeting these drainage systems. These findings provide information for potential novel strategies for treatment of stroke.

RIIß-PKA in GABAergic Neurons of Dorsal Median Hypothalamus Governs White Adipose Browning.

The RIIß subunit of  cAMP-dependent protein kinase A (PKA) is expressed in the brain and adipose tissue. RIIß-knockout mice show leanness and increased UCP1 in brown adipose tissue. The authors have previously reported that RIIß reexpression in hypothalamic GABAergic neurons rescues the leanness. However, whether white adipose tissue (WAT) browning contributes to the leanness and whether RIIß-PKA in these neurons governs WAT browning are unknown. Here, this work reports that RIIß-KO mice exhibit a robust WAT browning. RIIß reexpression in dorsal median hypothalamic GABAergic neurons (DMH GABAergic neurons) abrogates WAT browning. Single-cell sequencing, transcriptome sequencing, and electrophysiological studies show increased GABAergic activity in DMH GABAergic neurons of RIIß-KO mice. Activation of DMH GABAergic neurons or inhibition of PKA in these neurons elicits WAT browning and thus lowers body weight. These findings reveal that RIIß-PKA in DMH GABAergic neurons regulates WAT browning. Targeting RIIß-PKA in DMH GABAergic neurons may offer a clinically useful way to promote WAT browning for treating obesity and other metabolic disorders.

Withaferin A Promotes White Adipose Browning and Prevents Obesity Through Sympathetic Nerve-Activated Prdm16-FATP1 Axis.

The increasing prevalence of obesity has resulted in demands for the development of new effective strategies for obesity treatment. Withaferin A (WA) shows a great potential for prevention of obesity by sensitizing leptin signaling in the hypothalamus. However, the mechanism underlying the weight- and adiposity-reducing effects of WA remains to be elucidated. In this study, we report that WA treatment induced white adipose tissue (WAT) browning, elevated energy expenditure, decreased respiratory exchange ratio, and prevented high-fat diet-induced obesity. The sympathetic chemical denervation dampened the WAT browning and also impeded the reduction of adiposity in WA-treated mice. WA markedly upregulated the levels of Prdm16 and FATP1 (Slc27a1) in the inguinal WAT (iWAT), and this was blocked by sympathetic denervation. Prdm16 or FATP1 knockdown in iWAT abrogated the WAT browning-inducing effects of WA and restored the weight gain and adiposity in WA-treated mice. Together, these findings suggest that WA induces WAT browning through the sympathetic nerve-adipose axis, and the adipocytic Prdm16-FATP1 pathway mediates the promotive effects of WA on white adipose browning.

Changes in vesicular glutamate transporter 2 (Vglut2) and vesicular GABA transporter 1 (Vgat1) in the orofacial pain and temperature perception pathway under low estrogen conditions.

OBJECTIVE: To investigate the effects of estrogen on the threshold and temperature of orofacial pain and explore the influence on the function of glutamate and GABA neurons in the orofacial pain temperature perception pathway by observing the expression of vesicular glutamate transporter 2 (Vglut2) and vesicular GABA transporter 1 (Vgat1). METHODS: A total of 24 adult female Sprague-Dawley rats were divided into three groups: sham operation (SHAM), ovariectomized (OVX) and ovariectomized plus estrogen intervention (OVX+E) (n = 8 per group). The threshold of mechanical pain of the orofacial region was assessed with von Frey filaments, and the temperature of the rat orofacial region was monitored by infrared thermography. Changes in the expression of Vglut2 and Vgat1 in glutamatergic and GABAergic neurons in the trigeminal ganglion (TG), spinal trigeminal nucleus (Sp5C), lateral parabrachial nucleus (LPB) and ventral posteromedial nucleus of the thalamus (VPM) were assessed by immunostaining and Western blotting. RESULTS: Under low-estrogen conditions, the mechanical pain threshold of the orofacial region of rats decreased significantly, and the temperature of the orofacial region increased significantly. The expression of Vglut2 and Vgat1 in the TG and Sp5C showed a downward trend, and the decline in Vgat1 was greater than that in Vglut2. Conversely, both proteins were upregulated in the LPB and VPM, and the magnitude of the changes in Vglut2 was greater than that in Vgat1. Estrogen therapy reversed these changes. CONCLUSION: Under low-estrogen conditions, the proportion of glutamate and GABA neurons in the orofacial pain and temperature sensation pathway changes, which leads to the imbalance of neurotransmission function and the enhancement of excitatory transmission of these two kinds of neurons and finally leads to a decrease in the orofacial pain threshold and an increase in temperature.

Glutamatergic and GABAergic neurons in the preoptic area of the hypothalamus play key roles in menopausal hot flashes.

During menopause, when estrogen levels are low, abnormalities in the hypothalamic preoptic area (POA) of the thermoregulatory center can cause hot flashes. However, the involved neural population has not been identified. Proteomics showed that under low estrogen, differentially expressed proteins in the hypothalamus were associated with glutamatergic and GABAergic synapses. RNAscope, Western blotting and qRT-PCR indicated that the number of glutamatergic neurons in the POA was decreased, while the number of GABAergic neurons was increased. Chemogenetics showed that the rat body temperature decreased slowly after glutamatergic neurons were activated and increased quickly after glutamatergic neurons were inhibited, while it increased quickly after GABAergic neurons were activated and decreased slowly after GABAergic neurons were inhibited. RNAscope, immunofluorescence, Western blotting and qRT-PCR further showed that glutamate decarboxylase (GAD) 1 expression in the POA was increased, while GAD2 expression in the POA was decreased; that thermosensitive transient receptor potential protein (ThermoTRP) M (TRPM) 2 expression in glutamatergic neurons was decreased, while TRPM8 expression in GABAergic neurons was increased; and that estrogen receptor (ER) α and ß expression in the POA was decreased, and ERα and ERß expressed in both glutamatergic and GABAergic neurons. Estrogen therapy corrected these abnormalities. In addition, CUT&Tag and Western blot after injection of agonists and inhibitors of ERs showed that ERα and ERß were both transcription factors in glutamatergic and GABAergic synapses. Mechanistically, during menopause, estrogen may regulate the transcription and expression of GADs and ThermoTRPs through ERs, impacting the number and function of glutamatergic and GABAergic neurons, resulting in unbalanced heat dissipation and production in the POA and ultimately triggering hot flashes.

Transcriptomic changes in the hypothalamus of ovariectomized mice: Data from RNA-seq analysis.

BACKGROUND: Menopausal symptoms can affect the physical and mental health of females and are often related to abnormal function of the hypothalamus. In this study, we evaluated changes in the hypothalamus transcriptome in ovariectomized mice to identify key mRNAs, and systematically elucidated the possible molecular mechanisms underlying the menopausal syndrome to provide a theoretical basis for clinical diagnosis and treatment. METHODS: Forty-six adult female C57BL/6 J mice were randomly divided into SHAM and OVX groups, 23 mice per group. Eight weeks after the procedure, differentially expressed genes (DEGs) in the hypothalamus were identified through RNA-sequencing. DEGs were analyzed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genome (KEGG) analyses. Key DEGs were then evaluated using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and immunohistochemical staining. RESULTS: Compared with SHAM group, 7295 genes were upregulated, and 8979 genes were downregulated in the hypothalamus of OVX mice with a fold change of 1.5 (log2 fold change ≥0.585). GO and KEGG analyses suggested these key genes were involved in thermoregulation, food intake, glucose and lipid metabolism, cardiovascular regulation, biological rhythm, and endocrine regulation. CONCLUSIONS: Differential expression of genes in the hypothalamus of OVX mice involved in thermoregulation, eating, sleeping, homeostasis, and endocrine regulation 8 weeks after ovariectomy suggest potential roles in the pathogenesis of climacteric syndrome.

The impairment of intramural periarterial drainage in brain after subarachnoid hemorrhage.

Interstitial fluid (ISF) from brain drains along the basement membranes of capillaries and arteries as Intramural Periarterial Drainage (IPAD); failure of IPAD results in cerebral amyloid angiopathy (CAA). In this study, we test the hypothesis that IPAD fails after subarachnoid haemorrhage (SAH). The rat SAH model was established using endovascular perforation method. Fluorescence dyes with various molecular weights were injected into cisterna magna of rats, and the pattern of IPAD after SAH was detected using immunofluorescence staining, two-photon fluorescent microscope, transmission electron microscope and magnetic resonance imaging tracking techniques. Our results showed that fluorescence dyes entered the brain along a periarterial compartment and were cleared from brain along the basement membranes of the capillaries, with different patterns based on individual molecular weights. After SAH, there was significant impairment in the IPAD system: marked expansion of perivascular spaces, and ISF clearance rate was significantly decreased, associated with the apoptosis of endothelial cells, activation of astrocytes, over-expression of matrix metalloproteinase 9 and loss of collagen type IV. In conclusion, experimental SAH leads to a failure of IPAD, clinically significant for long term complications such as CAA, following SAH.