Stress-induced Hypertension Rats Research Articles

Co-expression of interleukin (IL)-17RA and IL-36γ in the hypothalamic paraventricular nucleus of the stress-induced hypertension rats

Objectives: The up-regulation of proinflammatory cytokines in the hypothalamic paraventricular nucleus (PVN) is well demonstrated to be involved in the development of neurogenic hypertension, including stress-induced hypertension (SIH). IL-17A has been found to be increased in the PVN of several hypertensive animal models and has been shown to play a key role in the development of hypertension. Although IL-36γ was found to be expressed in spinal neurons, its role in hypertension remains elusive. Here, we investigated the co-expression of IL-17 receptor A (IL-17RA) and IL-36γ in the PVN cells of SIH rats. Methods: The electric foot shock combined with buzzer noise stressors were used to make hypertensive rat model. The immunochemical staining or immunofluorescence staining was used to reveal cells as requested. The Western blot was used to detect the related protein levels. Results and Conclusion: In the PVN of the SIH rats, the number of CD3+CD4+ T cells was significantly increased by the immunochemical staining. Additionally, the protein levels of RORγt and IL-17A were significantly upregulated by Western blot, confirming the infiltration of CD4+ T cells and differentiation into Th17 cells in the PVN of SIH rats. Immunofluorescence staining revealed abundant expression of IL-17RA in PVN neurons, with relatively less expression in astrocytes or microglia. Furthermore, IL-36γ positive cells and protein expression of IL-36R were significantly increased. Notably, this study demonstrates for the first time that most IL-36γ cells were strongly colocalized with IL-17RA positive cells in the PVN of SIH rats, and the colocalized cells were significantly higher in SIH rats. This suggests that IL-17A secreted by infiltrated Th17 cells may stimulate PVN neurons to produce IL-36γ via IL-17RA, indicating that the combination of IL-17 and IL-36γ might produce strong pro-inflammatory effects in the PVN of SIH rats.

CircRNA Galntl6 sponges miR-335 to ameliorate stress-induced hypertension through upregulating Lig3 in rostral ventrolateral medulla

Rostral ventrolateral medulla (RVLM) is thought to serve as a major vasomotor center that participates in controlling the progression of stress-induced hypertension (SIH). Circular RNAs (circRNAs) perform important functions in the regulation of diverse physiological and pathological processes. However, information concerning the functions of RVLM circRNAs on SIH remains limited. RNA sequencing was performed to profile circRNA expression in RVLMs from SIH rats, which were induced by electric foot shocks and noises. The functions of circRNA Galntl6 in reducing blood pressure (BP) and its potential molecular mechanisms on SIH were investigated via various experiments, such as Western blot and intra-RVLM microinjection. A total of 12,242 circRNA transcripts were identified, among which circRNA Galntl6 was dramatically downregulated in SIH rats. The upregulation of circRNA Galntl6 in RVLM effectively decreased the BP, sympathetic outflow, and neuronal excitability in SIH rats. Mechanistically, circRNA Galntl6 directly sponged microRNA-335 (miR-335) and restrained it to reduce oxidative stress. Reintroduction of miR-335 observably reversed the circRNA Galntl6-induced attenuation of oxidative stress. Furthermore, Lig3 can be a direct target of miR-335. MiR-335 inhibition substantially increased the expression of Lig3 and suppressed oxidative stress, and these favorable effects were blocked by Lig3 knockdown. CircRNA Galntl6 is a novel factor that impedes SIH development, and the circRNA Galntl6/miR-335/Lig3 axis represents one of the possible mechanisms. These findings demonstrated circRNA Galntl6 as a possibly useful target for the prevention of SIH.

Microglia-derived TNF-α contributes to RVLM neuronal mitochondrial dysfunction via blocking the AMPK–Sirt3 pathway in stress-induced hypertension

BackgroundNeuroinflammation in the rostral ventrolateral medulla (RVLM) has been associated with the pathogenesis of stress-induced hypertension (SIH). Neuronal mitochondrial dysfunction is involved in many pathological and physiological processes. However, the impact of neuroinflammation on neuronal mitochondrial homeostasis and the involved signaling pathway in the RVLM during SIH are largely unknown.MethodsThe morphology and phenotype of microglia and the neuronal mitochondrial injury in vivo were analyzed by immunofluorescence, Western blot, RT-qPCR, transmission electron microscopy, and kit detection. The underlying mechanisms of microglia-derived tumor necrosis factor‐α (TNF-α) on neuronal mitochondrial function were investigated through in vitro and in vivo experiments such as immunofluorescence and Western blot. The effect of TNF-α on blood pressure (BP) regulation was determined in vivo via intra-RVLM microinjection of TNF-α receptor antagonist R7050.ResultsThe results demonstrated that BP, heart rate (HR), renal sympathetic nerve activity (RSNA), plasma norepinephrine (NE), and electroencephalogram (EEG) power increased in SIH rats. Furthermore, the branching complexity of microglia in the RVLM of SIH rats decreased and polarized into M1 phenotype, accompanied by upregulation of TNF‐α. Increased neuronal mitochondria injury was observed in the RVLM of SIH rats. Mechanistically, Sirtuin 3 (Sirt3) and p-AMPK expression were markedly downregulated in both SIH rats and TNF-α–treated N2a cells. AMPK activator A769662 upregulated AMPK–Sirt3 signaling pathway and consequently reversed TNF-α–induced mitochondrial dysfunction. Microinjection of TNF-α receptor antagonist R7050 into the RVLM of SIH rats significantly inhibited the biological activities of TNF-α, increased p‐AMPK and Sirt3 levels, and alleviated neuronal mitochondrial injury, thereby reducing c-FOS expression, RSNA, plasma NE, and BP.ConclusionsThis study revealed that microglia-derived TNF-α in the RVLM impairs neuronal mitochondrial function in SIH possibly through inhibiting the AMPK–Sirt3 pathway. Therefore, microglia-derived TNF-α in the RVLM may be a possible therapeutic target for the intervention of SIH.

PDZD8-mediated endoplasmic reticulum–mitochondria associations regulate sympathetic drive and blood pressure through the intervention of neuronal mitochondrial homeostasis in stress-induced hypertension

Neuronal hyperexcitation in the rostral ventrolateral medulla (RVLM) drives heightened sympathetic nerve activity and contributes to the etiology of stress-induced hypertension (SIH). Maintenance of mitochondrial functions is central to neuronal homeostasis. PDZD8, an endoplasmic reticulum (ER) transmembrane protein, tethers ER to mitochondria. However, the mechanisms of PDZD8-mediated ER–mitochondria associations regulating neuronal mitochondrial functions and thereby mediating blood pressure (BP) in the RVLM of SIH were largely unknown. SIH rats were subjected to intermittent electric foot shocks plus noise for 2 h twice daily for 15 consecutive days. The underlying mechanisms of PDZD8 were investigated through in vitro experiments by using small interfering RNA and through in vivo experiments, such as intra-RVLM microinjection and Western blot analysis. The function of PDZD8 on BP regulation in the RVLM was determined in vivo via the intra-RVLM microinjection of adeno-associated virus (AAV)2-r-Pdzd8. We found that the c-Fos-positive RVLM tyrosine hydroxylase (TH) neurons, renal sympathetic nerve activity (RSNA), plasma norepinephrine (NE) level, BP, and heart rate (HR) were elevated in SIH rats. ER–mitochondria associations in RVLM neurons were significantly reduced in SIH rats. PDZD8 was mainly expressed in RVLM neurons, and mRNA and protein levels were markedly decreased in SIH rats. In N2a cells, PDZD8 knockdown disrupted ER–mitochondria associations and mitochondrial structure, decreased mitochondrial membrane potential (MMP) and respiratory metabolism, enhanced ROS levels, and reduced catalase (CAT) activity. These effects suggested that PDZD8 dysregulation induced mitochondrial malfunction. By contrast, PDZD8 upregulation in the RVLM of SIH rats could rescue neuronal mitochondrial function, thereby suppressing c-Fos expression in TH neurons and decreasing RSNA, plasma NE, BP, and HR. Our results indicated that the dysregulation of PDZD8-mediated ER–mitochondria associations led to the loss of the activity homeostasis of RVLM neurons by disrupting mitochondrial functions, thereby participating in the regulation of SIH pathology.

IFN-γ deficiency in the rostral ventrolateral medulla contributes to stress-induced hypertension by impairing microglial synaptic engulfment.

Dysfunctional neurons and microglia in the rostral ventrolateral medulla (RVLM) have been implicated in the pathogenesis of stress-induced hypertension (SIH). Functional perturbation of microglial synaptic engulfment can induce aberrant brain circuit activity. IFN-γ is a pleiotropic cytokine that plays a role in regulating neuronal activity. However, existing research on the exploration of the effects of microglia on synapses in the RVLM is lacking, particularly on the function of IFN-γ in microglial synaptic engulfment involved in SIH. A SIH rat model was established by electric foot shocks combined with noise stimulation. The underlying mechanism of IFN-γ on synaptic density and microglial synaptic engulfment was investigated through in-vivo and in-vitro experiments involving gain of function, immunofluorescence, quantitative real-time PCR, western blot, and morphometric analysis. Furthermore, the function of IFN-γ in neuronal activity, renal sympathetic nerve activity (RSNA), and blood pressure (BP) regulation was determined through in-vivo and in-vitro experiments involving Ca2+ imaging, immunofluorescence, platinum-iridium electrode recording, ELISA, the femoral artery cannulation test, and the tail-cuff method. The BP, heart rate, RSNA, plasma norepinephrine, and the number of c-Fos-positive neurons in SIH rats increased compared with those in control rats. Pre and postsynaptic densities in the RVLM also increased in SIH rats. IFN-γ and CCL2 expression levels were significantly reduced in the RVLM of the SIH group, whose microglia also exhibited an impaired capacity for synapse engulfment. IFN-γ elevation increased CCL2 expression and microglial synaptic engulfment and decreased synaptic density in vivo and in vitro. However, CCL2 inhibition reversed these effects. Moreover, the reduction of neuronal excitability, RSNA, plasma norepinephrine, and BP by IFN-γ was abrogated through CCL2 expression. IFN-γ deficiency in the RVLM impaired the microglial engulfment of synapses by inhibiting CCL2 expression and increasing synaptic density and neuronal excitability, thereby contributing to SIH progression. Targeting IFN-γ may be considered a potential strategy to combat SIH.Graphical Abstract, http://links.lww.com/HJH/C203.

LncRNA INPP5F ameliorates stress-induced hypertension via the miR-335/Cttn axis in rostral ventrolateral medulla.

The rostral ventrolateral medulla (RVLM) is an essential vasomotor center responsible for regulating the development of stress-induced hypertension (SIH). Long non-coding RNAs (lncRNAs) play critical roles in various physiopathology processes, but existing research on the functions of RVLM lncRNAs on SIH has been lacking. In this study, we investigated the roles of RVLM lncRNAs in SIH. Genome-wide lncRNA profiles in RVLM were determined by RNA sequencing in a SIH rat model established using electric foot shocks plus noises. The hypotensive effect of lncRNA INPP5F and the underlying mechanisms of lncRNA INPP5F on SIH were explored through in vivo and in vitro experiments, such as intra-RVLM microinjection and immunofluorescence. We discovered 10,179 lncRNA transcripts, among which the lncRNA INPP5F expression level was significantly decreased in SIH rats. Overexpression of lncRNA INPP5F in RVLM dramatically reduced the blood pressure, sympathetic nerve activity, and neuronal excitability of SIH rats. LncRNA INPP5F overexpression markedly increased Cttn expression and reduced neural apoptosis by activating the PI3K-AKT pathway, and its inhibition had opposite effects. Mechanistically, lncRNA INPP5F acted as a sponge of miR-335, which further regulated the Cttn expression. LncRNA INPP5F was a key factor that inhibited SIH progression, and the identified lncRNA INPP5F/miR-335/Cttn/PI3K-AKT/apoptosis axis represented one of the possible mechanisms. LncRNA INPP5F could serve as a therapeutic target for SIH.

Up-regulation of miR-335 and miR-674-3p in the rostral ventrolateral medulla contributes to stress-induced hypertension.

The rostral ventrolateral medulla (RVLM) is known as the vasomotor center that plays a crucial role in mediating the development of stress-induced hypertension (SIH). MicroRNAs (miRNAs) are involved in many different biological processes and diseases. However, studies that evaluated the roles of miRNAs in the RVLM during SIH do not exist. Here, we performed RNA sequencing to explore the genome-wide miRNA profiles in RVLM in an SIH rat model established by administering electric foot-shocks and noises. The function of miRNAs in blood pressure regulation was determined in vivo via the intra-RVLM microinjection of the agomir or antagomir. Furthermore, the underlying mechanisms of miRNAs on SIH were investigated through in vitro and in vivo experiments, like gain-of-function. We discovered 786 miRNA transcripts among which 4 were differentially expressed. The over-expression of miR-335 and miR-674-3p in RVLM dramatically increased the heart rate (HR), arterial blood pressure (ABP), systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean arterial pressure (MAP) levels of normotensive rats, whereas the knockdown of miR-335 and miR-674-3p in RVLM markedly reduced the HR, ABP, SBP, DBP, and MAP levels of SIH rats. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation revealed that miR-335 and miR-674-3p participated in regulating the development of SIH from different aspects, like apoptosis-multiple species pathway. Sphk1, whose expression was markedly decreased in SIH, was identified as a novel target of miR-335. MiR-335 over-expression substantially reduced the expression of Sphk1 and promoted neural apoptosis, and its inhibition had opposite effects. Re-introduction of Sphk1 dramatically abrogated the apoptosis induced by miR-335. This study provides the first systematic dissection of the RVLM miRNA landscape in SIH. MiR-335 and miR-674-3p act as SIH promoters, and the identified miR-335/Sphk1/apoptosis axis represents one of the possible mechanisms. These miRNAs can be exploited as potential targets for the molecular-based therapy of SIH.

Overexpression of NaV1.6 in the rostral ventrolateral medulla in rats mediates stress-induced hypertension via glutamate regulation

ABSTRACT Background The rostral ventrolateral medulla (RVLM) plays a key role in mediating the development of stress-induced hypertension (SIH). Furthermore, enhanced glutamate transport within glutamatergic neurons in the RVLM mediates pressor responses. Data from our previous studies suggest that the voltage-gated sodium channel NaV1.6 is overexpressed in neurons in the RVLM in SIH model rats and participates in the resulting elevation of blood pressure. However, previous studies have not investigated the relationship between NaV1.6 expression and glutamatergic neurons. Methods Here, we constructed an SIH rat model by knocking down NaV1.6 via microinjection of clustered regularly interspaced short palindromic repeats (CRISPR) guide RNA into the RVLM. Glutamate-related markers were quantified by Western blotting and immunofluorescence, and blood pressure was measured in the rats. Results Our findings showed that vesicular glutamate transporter 1 (VGluT1) protein expression in the RVLM was higher in SIH rats than in Control rats, and GAD67 protein expression in SIH rats was lower than that in Control rats. Therefore, the number of VGluT1-positive neurons increased, while the number of GAD67-labeled neurons decreased after stress. After knocking down NaV1.6 expression in the RVLM, VGluT1 expression and the number of VGluT1-positive neurons decreased relative to those in SIH rats, while GAD67 protein expression and the number of GAD67-labeled neurons increased relative to those in SIH rats. Conclusions These results indicate that overexpression of NaV1.6 in the RVLM may mediate the transport and transformation of glutamate in neurons, and NaV1.6 may participate in SIH.

Sigma-1 Receptor Activation Suppresses Microglia M1 Polarization via Regulating Endoplasmic Reticulum-Mitochondria Contact and Mitochondrial Functions in Stress-Induced Hypertension Rats.

Exposure to stress plays a detrimental role in the pathogenesis of hypertension via neuroinflammation pathways. Microglial neuroinflammation in the rostral ventrolateral medulla (RVLM) exacerbates stress-induced hypertension (SIH) by increasing sympathetic hyperactivity. Mitochondria of microglia are the regulators of innate immune response. Sigma-1R (σ-1R) localizes to the mitochondria-associated membranes (MAMs) and regulates endoplasmic reticulum (ER) and mitochondria communication, in part through its chaperone activity. The present study aims to investigate the protective role of σ-1R on microglial-mediated neuroinflammation. Stress-induced hypertension (SIH) was induced in rats using electric foot shocks and intermittent noise. Arterial blood pressure (ABP), heart rate (HR), and renal sympathetic nerve activity (RSNA) were measured to evaluate the sympathetic nervous system (SNS) activities. SKF10047 (100µM), an agonist of σ-1R, was administrated to rats, then σ-1R localization and MAM alterations were detected by immuno-electron microscopy. Mitochondrial calcium homeostasis was examined in primary microglia and/or BV-2 microglia cells. The effect of SKF10047 treatment on the mitochondrial respiratory function of cultured microglia was measured using a Seahorse Extracellular Flux Analyzer. Confocal microscopic images were performed to indicate mitochondrial dynamics. Stress reduces σ-1R's localization at the MAMs, leading to decreased ER-mitochondria contact and IP3R-GRP75-VDAC calcium transport complexes expression in the RVLM of rats. SKF10047 promotes the length and coverage of MAMs in the prorenin-treated microglia. Prorenin treatment increases mitoROS levels, and inhibits Ca2+ signalling between the two organelles, therefore negatively affects ATP production in BV2 cells, and these effects are reversed by SKF10047 treatment. We found mitochondrial hyperfusion and microglial M1 polarization in prorenin-treated microglia. SKF10047 suppresses microglial M1 polarization and RVLM neuroinflammation, subsequently ameliorates sympathetic hyperactivity in stress-induced hypertensive rats. Sigma-1 receptor activation suppresses microglia M1 polarization and neuroinflammation via regulating endoplasmic reticulum-mitochondria contact and mitochondrial functions in stress-induced hypertension rats.

Upregulation of Chemokines in the Paraventricular Nucleus of the Hypothalamus in Rats with Stress-Induced Hypertension.

BackgroundThe neuroinflammation of paraventricular nucleus (PVN) of the hypothalamus has been implicated in the development of hypertension. The promoted invasion of peripheral immune cells into PVN may be attributed to the upregulation of chemokines, then exacerbating neuroinflammation. We studied the expressions of chemokines, activation of microglial cells, and inflammatory mediators in PVN of rats with stress-induced hypertension (SIH).Material/MethodsSIH was induced by electrical foot shock combined with noise for 2 h twice a day, at an interval of 4 h for 14 consecutive days. At the end of the 14th day, fresh PVN tissues were collected to measure the expressions of chemokines using the RayBiotech antibody array.ResultsWe are the first to report that the expression of CXCL7 was extremely high in PVN of control rats, and was significantly lower in SIH rats. The expressions of CCL2 and CX3CL1 in PVN of SIH rats significantly exceeded those of control rats. The numbers of CX3CR1 (receptor of CX3CL1)-immunostained cells and oxycocin-42 (OX-42, marker of microglia)-positive cells increased in PVN of the SIH rats. The stress enhanced the protein expressions of proinflammatory cytokines IL-6 and IL-17 and reduced those of anti-inflammatory cytokines TGF-β and IL-10 in PVN.ConclusionsIn PVN of SIH rats, chronic stress induced neuroinflammation characterized by the activated microglia and upregulated proinflammatory cytokines. Expressions of chemokines CXCL7, CX3CL1, and CCL2 were altered. The causal link of chemokines to PVN neuroinflammation and hypertension remain to be determined.

TLR4/MyD88/NF-κB Signaling in the Rostral Ventrolateral Medulla Is Involved in the Depressor Effect of Candesartan in Stress-Induced Hypertensive Rats.

This study aimed to investigate whether the proinflammatory and pressor effects of endogenous angiotensin II (AngII) are mediated by binding to the AngII type 1 receptor (AT1R) and subsequently activating central Toll-like receptor 4 (TLR4) in the rostral ventrolateral medulla (RVLM) of stress-induced hypertensive rats (SIHR). The stress-induced hypertension (SIH) model was established by random electric foot shocks combined with noise stimulation. Mean arterial pressure, heart rate, plasma norepinephrine, and RVLM AngII and TLR4 increased in a time-dependent manner in SIHR. Pro-inflammatory cytokines (tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β)), myeloid differentiation factor 88 (MyD88), and nuclear factor (NF)-κB also increased, while anti-inflammatory cytokine IL-10 decreased in the RVLM of SIHR. These changes were attenuated by 14-day intracerebroventricular (ICV) infusion of VIPER (a TLR4 inhibitor) or candesartan (an AT1R antagonist). Both TLR4 and AT1R were expressed in the neurons and microglia in the RVLM of SIHR. Candesartan attenuated the expression of TLR4 in the RVLM of SIHR. This study demonstrated that endogenous AngII may activate AT1R to upregulate TLR4/MyD88/NF-κB signaling and subsequently trigger an inflammatory response in the RVLM of SIHR, which in turn enhanced sympathetic activity and increased blood pressure.

Gut microbiota modulates stress-induced hypertension through the HPA axis

Stress is associated with an increased risk of hypertension, and the incidence of stress-related hypertension has risen rapidly in recent years; however, the underlying mechanisms remain elusive. Gut dysbiosis has been demonstrated to contribute to hypertension and hyperactivation of the hypothalamus-pituitary-adrenal (HPA) axis. Based on our previous findings showing the altered gut microbiota in the rats of stress-induced hypertension (SIH), the present study aims to investigate whether the stress-induced alteration in gut microbiota can lead to the dysfunction of the HPA axis which contributes to the development of SIH. SIH was developed in rats subjected to electric foot-shock combined with buzzer noise stressors. The gut microbiota of rats were deleted by administering an antibiotic cocktail containing ampicillin (1 g/L), vancomycin (500 mg/L), neomycin (1 g/L), and metronidazole (1 g/L) in drinking water. The serum levels of adrenocorticotropic hormone (ACTH) and corticosterone (CORT) were tested using enzyme-linked immunosorbent assay (ELISA). The mRNA expression of glucocorticoid receptor (GR) and corticotropin-releasing factor (CRF), CRFR1 and CRFR2 was detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR). The cellular protein expressions of corticotropin-releasing hormone (CRH), c-fos, and GR were examined by immunohistochemical staining. In the present study, SIH rats showed a hyperactive HPA axis as indicated by the increased CRH expression in the paraventricular nucleus (PVN) of the hypothalamus, the elevated serum ACTH or CORT concentrations, and increased adrenal gland index. The decreased GR expression and increased CRFR1 in the hypothalamus might underlie the hyperactivation of the HPA axis. The microbial deletion by antibiotics mitigated the hyperactivation of the HPA axis and attenuated the stress-induced elevation of blood pressure, indicating that the causal link of gut microbiota to SIH is mediated, at least in part, by the HPA axis activity. Our findings shed new light on the mechanisms underlying SIH.

Microglia-Derived NLRP3 Activation Mediates the Pressor Effect of Prorenin in the Rostral Ventrolateral Medulla of Stress-Induced Hypertensive Rats

Increased microglial activation and neuroinflammation within autonomic brain regions such as the rostral ventrolateral medulla (RVLM) have been implicated in stress-induced hypertension (SIH). Prorenin, a member of the brain renin-angiotensin system (RAS), can directly activate microglia. The present study aimed to investigate the effects of prorenin on microglial activation in the RVLM of SIH rats. Rats were subjected to intermittent electric foot-shocks plus noise, this stress was administered for 2 h twice daily for 15 consecutive days, and mean arterial pressure (MAP) and renal sympathetic nerve activity (RSNA) were monitored. The results showed that MAP and RSNA were augmented, and this paralleled increased pro-inflammatory phenotype (M1) switching. Prorenin and its receptor (PRR) expression and the NLR family pyrin domain containing 3 (NLRP3) activation were increased in RVLM of SIH rats. In addition, PLX5622 (a microglial depletion agent), MCC950 (a NLRP3 inhibitor), and/or PRO20 (a (Pro)renin receptor antagonist) had antihypertensive effects in the rats. The NLRP3 expression in the RVLM was decreased in SIH rats treated with PLX5622. Mito-tracker staining showed translocation of NLRP3 from mitochondria to the cytoplasm in prorenin-stimulated microglia. Prorenin increased the ROS-triggering M1 phenotype-switching and NLRP3 activation, while MCC950 decreased the M1 polarization. In conclusion, upregulated prorenin in the RVLM may be involved in the pathogenesis of SIH, mediated by activation of the microglia-derived NLRP3 inflammasome. The link between prorenin and NLRP3 in microglia provides insights for the treatment of stress-related hypertension.

Central changes in the Kv10.2 potassium channel in stress-induced hypertension rats.

Ion channels play as a pivotal role in hypertension in the processes of maintenance of vascular tone and sympathetic excitement of hypertension. The Kv10.2 channel (encoded by the Kcnh5 gene) belongs to the EAG voltage-gated superfamily. It is distributed widely in the brain, such as the hippocampus, the cortex, and the olfactory bulb. To date, the expression of Kv10.2 in central nervous system nuclei that regulates cardiovascular function and its inter-relationship with hypertension are still unclear. Here, electric foot-shock stressors with noise were used to establish the stress-induced hypertensive (SIH) rat model. The expression of Kv10.2 in the rostral ventrolateral medulla, the nucleus tractus solitarius, and the paraventricular nucleus (PVN) was examined by immunohistochemical staining and western blots. The following results were obtained: (a) the expression level of Kv10.2 was increased obviously in the paraventricular nucleus of SIH rats, whereas no significant difference was found in the rostral ventrolateral medulla and the nucleus tractus solitarius. (b) Kv10.2 was located in neurons. (c) Vesicular glutamate transporter 1 as a protein mark of glutamate neurons was increased in the paraventricular nucleus of the SIH group. (d) The expression of vesicular glutamate transporter 1 protein in neurons was significantly decreased when the Kcnh5 gene was knocked down by small interfering RNA in vitro. These findings indicate that the changes in Kv10.2 may be related to SIH, which may provide a potential avenue for further investigation of SIH.

Infiltrating T helper 17 cells in the paraventricular nucleus are pathogenic for stress-induced hypertension

Central neuroinflammation produced by both innate and adaptive immunities plays a major role in the development of stress-induced hypertension (SIH), but successful T cell immunoregulation for SIH requires that the T cells can access brain tissues. So far, both the effects of T helper 17 (Th17) cells on SIH and the pathway for T cells entry into the brain were unknown. Here we show that the blood pressure (BP), heart rate (HR) and the norepinephrine(NE) of the SIH rats were considerably higher, the numbers of Th17 cells and IL-17 were higher, relative to control. Anti-IL-17 attenuated the elevation of BP and HR of the SIH rats when microinjected into the paraventricular nucleus (PVN).Alb-FITC, after infusion into the carotid artery, were found in the brain parenchyma of the PVN in the SIH rats. We concluded that Th17 cells infiltrated the parenchyma of the paraventricular nucleus (PVN) via a compromised blood brain barrier (BBB) in response to stress and Th17 cells and IL-17 play an important role in the pathophysiology of SIH.

Central blockade of the AT1 receptor attenuates pressor effects via reduction of glutamate release and downregulation of NMDA/AMPA receptors in the rostral ventrolateral medulla of rats with stress-induced hypertension.

Glutamatergic activity in the rostral ventrolateral medulla (RVLM), which is an important brain area where angiotensin II (Ang II) elicits its pressor effects, contributes to the onset of hypertension. The present study aimed to explore the effect of central Ang II type 1 receptor (AT1R) blockade on glutamatergic actions in the RVLM of stress-induced hypertensive rats (SIHR). The stress-induced hypertension (SIH) model was established by electric foot shocks combined with noises. Normotensive Sprague-Dawley rats (control) and SIHR were intracerebroventricularly infused with the AT1R antagonist candesartan or artificial cerebrospinal fluid for 14 days. Mean arterial pressure (MAP), heart rate (HR), plasma norepinephrine (NE), glutamate, and the expression of N-methyl-D-aspartic acid (NMDA) receptor subunit NR1, and α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors in the RVLM increased in the SIH group. These increases were blunted by candesartan. Bilateral microinjection of the ionotropic glutamate receptor antagonist kynurenic acid, the NMDA receptor antagonist D-2-amino-5-phosphonopentanoate, or the AMPA/kainate receptors antagonist 6-cyano-7-nitroquinoxaline-2,3-dione into the RVLM caused a depressor response in the SIH group, but not in other groups. NR1 and AMPA receptors expressed in the glutamatergic neurons of the RVLM, and glutamate levels, increased in the intermediolateral column of the spinal cord of SIHR. Central Ang II elicits release of glutamate, which binds to the enhanced ionotropic NMDA and AMPA receptors via AT1R, resulting in activation of glutamatergic neurons in the RVLM, increasing sympathetic excitation in SIHR.

Upregulation of AT1 Receptor Mediates a Pressor Effect Through ROS-SAPK/JNK Signaling in Glutamatergic Neurons of Rostral Ventrolateral Medulla in Rats With Stress-Induced Hypertension.

The present study examined whether angiotensin II (Ang II) mediates the pressor effect through nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-derived reactive oxygen species (ROS)-mitogen-activated protein kinase (MAPK) signaling in the glutamatergic neurons of the rostral ventrolateral medulla (RVLM) in stress-induced hypertensive rats (SIHR). The SIHR model was established using electric foot-shocks combined with noises for 15 days. We observed that Ang II type 1 receptor (AT1R) and the glutamatergic neurons co-localized in the RVLM of SIHR. Furthermore, glutamate levels in the intermediolateral column of the spinal cord were higher in SIHR than in controls. Microinjection of Ang II into the RVLM of SIHR activated stress-activated protein kinase/Jun N-terminal kinase (SAPK/JNK), extracellular signal-regulated protein kinase (ERK) 1/2, and p38MAPK. Compared with controls, the activation of SAPK/JNK, ERK1/2, p38MAPK, and ROS in the RVLM were higher in SIHR, an effect that was blocked by an NADPH oxidase inhibitor (apocynin) and an AT1R antagonist (candesartan). RVLM microinjection of apocynin or a SAPK/JNK inhibitor (SP600125), but not an ERK1/2 inhibitor (U0126) or a p38MAPK inhibitor (SB203580), decreased AT1R mRNA and mean arterial blood pressure (MABP) in SIHR. The increase of AT1R protein expression and MABP was inhibited by intracerebroventricular infusion (ICV), for 14 days, of SP600125, but not U0126 or SB203580 in SIHR. We conclude that Ang II modulates the pressor effect through AT1R-dependent ROS-SAPK/JNK signaling in glutamatergic neurons in the RVLM of SIHR.

Upregulation of Nav1.6 expression in the rostral ventrolateral medulla of stress-induced hypertensive rats.

The rostral ventrolateral medulla (RVLM) plays a key role in mediating the development of stress-induced hypertension (SIH) by excitation and/or inhibition of sympathetic preganglionic neurons. The voltage-gated sodium channel Nav1.6 has been found to contribute to neuronal hyperexcitability. To examine the expression of Nav1.6 in the RVLM during SIH, a rat model was established by administering electric foot-shocks and noises. We found that Nav1.6 protein expression in the RVLM of SIH rats was higher than that of control rats, peaking at the tenth day of stress. Furthermore, we observed changes in blood pressure correlating with days of stress, with systolic blood pressure (SBP) found to reach a similarly timed peak at the tenth day of stress. Percentages of cells exhibiting colocalization of Nav1.6 with NeuN, a molecular marker of neurons, indicated a strong correlation between upregulation of Nav1.6 expression in NeuN-positive cells and SBP. The level of RSNA was significantly increased after 10 days of stress induction than control group. Compared with the SIHR, knockdown of Nav1.6 in RVLM of the SIHR decreased the level of SBP, heart rate (HR) and renal sympathetic nerve activity (RSNA). These results suggest that upregulated Nav1.6 expression within neurons in the RVLM of SIH rats may contribute to overactivation of the sympathetic system in response to SIH development.

Alteration of serum immunoglobins, C-reactive protein, vitamin D, and electrolyteby atenolol and amlodipine in stress-induced hypertensive rats.

The present study is designed for the assessment of various pathological changes like immunoglobins, C-reactive protein, vitamin D, sodium, potassium, calcium in stress-induced hypertensive rats. Albino Wistar rats of sex male were grouped into six. Each group consists of six animals. Groups were Group I (normal control), Group II (disease control), Group III (amlodipine control), Group IV (atenolol control), Group V (amlodipine treatment), and Group VI (atenolol treatment). Group II, V, and VI animals exposed to regular stress by placing them in cages individually and giving foot electric shocks (1mA, 50ms duration with 0.5-1min of intervals regulated randomly by a computer)along withforced swimming (30 min) in order to induce hypertension in rats. This stress was given two times daily (morning and evening) for regular 15 days. Induction of hypertension was confirmed by measuring the tail arterial pressure of blood and angiotensin II. For next 1 month, Group III and V animals are treated with amlodipine with 1mg/kg, s.c. dose while Group IV and VI animals were given 10mg/kg, s.c. the dose of atenolol once daily. At the end of the experimental work, blood collected, rats sacrificed, and serum separated. Serum sodium, potassium, immunoglobins, C-reactive protein, vitamin D, and calcium were measured by semi-auto-analyzer. Stress-induced hypertension in rats produced altered serum sodium, potassium, immunoglobins, C-reactive protein, vitamin D, and calcium level which is restored by atenolol. Administration of amlodipine in animals without hypertension shows alteration in the level of immunoglobins, calcium, vitamin D, and electrolytes.

Neuroinflammation contributes to autophagy flux blockage in the neurons of rostral ventrolateral medulla in stress-induced hypertension rats

BackgroundNeuroinflammation plays hypertensive roles in the uninjured autonomic nuclei of the central nervous system, while its mechanisms remain unclear. The present study is to investigate the effect of neuroinflammation on autophagy in the neurons of the rostral ventrolateral medulla (RVLM), where sympathetic premotor neurons for the maintenance of vasomotor tone reside.MethodsStress-induced hypertension (SIH) was induced by electric foot-shock stressors with noise interventions in rats. Systolic blood pressure (SBP) and the power density of the low frequency (LF) component of the SAP spectrum were measured to reflect sympathetic vasomotor activity. Microglia activation and pro-inflammatory cytokines (PICs (IL-1β, TNF-α)) expression in the RVLM were measured by immunoblotting and immunostaining. Autophagy and autophagic vacuoles (AVs) were examined by autophagic marker (LC3 and p62) expression and transmission electron microscopy (TEM) image, respectively. Autophagy flux was evaluated by RFP-GFP-tandem fluorescent LC3 (tf-LC3) vectors transfected into the RVLM. Tissue levels of glutamate, gamma aminobutyric acid (GABA), and plasma levels of norepinephrine (NE) were measured by using high-performance liquid chromatography (HPLC) with electrochemical detection. The effects of the cisterna magna infused minocycline, a microglia activation inhibitor, on the abovementioned parameters were analyzed.ResultsSIH rats showed increased SBP, plasma NE accompanied by an increase in LF component of the SBP spectrum. Microglia activation and PICs expression was increased in SIH rats. TEM demonstrated that stress led to the accumulation of AVs in the RVLM of SIH rats. In addition to the Tf-LC3 assay, the concurrent increased level of LC3-II and p62 suggested the impairment of autophagic flux in SIH rats. To the contrary, minocycline facilitated autophagic flux and induced a hypotensive effect with attenuated microglia activation and decreased PICs in the RVLM of SIH rats. Furthermore, SIH rats showed higher levels of glutamate and lower level of GABA in the RVLM, while minocycline attenuated the decrease in GABA and the increase in glutamate of SIH rats.ConclusionsCollectively, we concluded that the neuroinflammation might impair autophagic flux and induced neural excitotoxicity in the RVLM neurons following SIH, which is involved in the development of SIH.