Outflow Pressure Research Articles

Elevated intraocular pressure induces Rho GTPase mediated contractile signaling in the trabecular meshwork

Rho GTPase regulated contractile signaling in the trabecular meshwork (TM) has been shown to modulate aqueous humor (AH) outflow and intraocular pressure (IOP). To explore whether elevated IOP, a major risk factor for primary open angle glaucoma (POAG) influences Rho GTPase signaling in the TM, we recorded AH outflow in enucleated contralateral porcine eyes perfused for 4–5 h at either 15 mm or 50 mm Hg pressure. After perfusion, TM tissue extracted from perfused eyes was evaluated for the activation status of Rho GTPase, myosin light chain (MLC), myosin phosphatase target substrate 1 (MYPT1), myristoylated alanine-rich C-kinase substrate (MARCKS) and paxillin. Eyes perfused at 50 mm Hg exhibited a significant decrease in AH outflow facility compared with those perfused at 15 mm Hg. Additionally, TM tissue from eyes perfused at 50 mm Hg revealed significantly increased levels of activated RhoA and phosphorylated MLC, MYPT1, MARCKS and paxillin compared to TM tissue derived from eyes perfused at 15 mm Hg. Taken together, these observations indicate that elevated IOP-induced activation of Rho GTPase-dependent contractile signaling in the TM is associated with increased resistance to AH outflow through the trabecular pathway, and demonstrate the sensitivity of Rho GTPase signaling to mechanical force in the AH outflow pathway.

Sympathetic vasomotor outflow and blood pressure increase during exercise with expiratory resistance.

The purpose of the present study was to elucidate the effect of increasing expiratory muscle work on sympathetic vasoconstrictor outflow and arterial blood pressure (BP) during dynamic exercise. We hypothesized that expiratory muscle fatigue would elicit increases in sympathetic vasomotor outflow and BP during submaximal exercise. The subjects performed four submaximal exercise tests; two were maximal expiratory pressure (PEmax) tests and two were muscle sympathetic nerve activity (MSNA) tests. In each test, the subjects performed two 10-min exercises at 40% peak oxygen uptake using a cycle ergometer in a semirecumbent position [spontaneous breathing for 5 min and voluntary hyperpnoea with and without expiratory resistive breathing for 5 min (breathing frequency: 60 breaths/min, inspiratory and expiratory times were set at 0.5 sec)]. PEmax was estimated before and immediately after exercises. MSNA was recorded via microneurography of the right median nerve at the elbow. PEmax decreased following exercise with expiratory resistive breathing, while no change was found without resistance. A progressive increase in MSNA burst frequency (BF) appeared during exercise with expiratory resistance (MSNA BF, without resistance: +22 ± 5%, with resistance: +44 ± 8%, P < 0.05), accompanied by an augmentation of BP (mean BP, without resistance: +5 ± 2%, with resistance: +29 ± 5%, P < 0.05). These results suggest that an enhancement of expiratory muscle activity leads to increases in sympathetic vasomotor outflow and BP during dynamic leg exercise.

Pulmonary vascular and airway responses to systemic vasoconstrictors in anesthetized BALB/c mice.

There is no systematic study in which the effects of vasoactive substances were investigated on pulmonary vascular resistance (PVR) in in vivo mouse by directly measuring cardiac output and the inflow and outflow pressures in the pulmonary circulation. We determined the responses of PVR, total peripheral resistance (TPR), and airway pressure (AWP) to angiotensin II, endothelin-1, vasopressin, phenylephrine, and thromboxane A2 analog U46619 in anesthetized BALB/c mice. Pulmonary arterial pressure, left atrial pressure (LAP), and aortic blood flow were measured. TPR increased dose-dependently in response to consecutive administration of all vasoconstrictors except vasopressin which reduced TPR at the highest dose of 100 nmol/kg. At high doses of vasoconstrictors, pulmonary arterial pressure and AWP increased due to increased LAP, as demonstrated by the separate LAP elevation experiments. When LAP transiently increased at high doses, PVR did not increase but decreased. Nonetheless, enodothelin-1, angiotensin II, and U46619 increased PVR. Vasopressin at 100 nmol/kg increased AWP without LAP elevation. In conclusion, the high doses of the vasoconstrictors studied here exert indirectly a transient pulmonary vasodilatory and AWP increasing actions due to pulmonary congestion evoked by strong systemic vasoconstriction. Nevertheless, enodothelin-1, angiotensin II, and U46619 cause pulmonary vasoconstriction, and vasopressin constricts airway in anesthetized BALB/c mice.

Paraventricular Nucleus in Acupuncture's Inhibition of the Von Bezold Jarsich Reflex

Electroacupuncture (EA) stimulates specific somatic sensory nerves underlying P5‐6 acupoints and modulates parasympathoexcitatory reflex responses through actions in the brainstem. Although Bezold Jarisch‐induced decreases in blood pressure and heart rate reflex responses are modulated by acupuncture through actions in nucleus tractus solitarius and nucleus ambiguus, the role of the hypothalamus is unclear. The paraventricular nucleus (PVN) regulates sympathetic outflow and blood pressure. Vagal afferent stimulation activates neurons in the PVN but little is known about cardiopulmonary physiological actions in this nucleus. We therefore hypothesized that the PVN participates in the Bezold Jarisch responses and EA modulation of parasympathoexcitatory cardiovascular responses. Rats were anesthetized, ventilated, and heart rate and mean blood pressures were monitored. Application of phenylbiguanide (PBG) every 10 min close to the right atrium induced consistent depressor and bradycardia reflex responses. Unilateral microinjection of kainic acid or kynurenic acid in the PVN reduced the reflex responses. Thirty min of bilateral EA at P5‐6 acupoints reduced the depressor and bradycardia responses lasted for &gt;60 min. Unilateral inhibition of the PVN with 50 nl of naloxone reversed the EA‐inhibition during repeated PBG‐induced responses. EA‐related c‐Fos PVN neurons were juxtaposed to endorphinergic PVN fibers. Thus, cardiopulmonary PVN responses are important in processing acupuncture modulation of cardiovascular inhibitory reflexes.Funded by HL‐63313, HL‐72125.

Cystathionine-β-Synthase Gene Transfer Into Rostral Ventrolateral Medulla Exacerbates Hypertension via Nitric Oxide in Spontaneously Hypertensive Rats.

Rostral ventrolateral medulla (RVLM) plays a crucial role in the central regulation of cardiovascular functions. Cystathionine-β-synthase (CBS) is a major hydrogen sulfide (H2S)-generating enzyme that has been identified mainly in the brain. The present study was designed to examine CBS expression and determine its roles and mechanisms of regulating sympathetic outflow and blood pressure (BP) in the RVLM in spontaneously hypertensive rats (SHR). CBS expression was decreased in the RVLM in SHR compared to Wistar-Kyoto (WKY) rats. Accumulating evidences suggest that H2S interacts with nitric oxide (NO) to regulate cardiovascular function. Therefore, we hypothesize that the decrease in CBS expression in the RVLM may be involved in the disorder of l-arginine/NO pathway, which subsequently affects BP in SHR. Overexpression of CBS in the RVLM caused significant increases in BP, heart rate, and urinary norepinephrine excretion in SHR but not in WKY. Acute experiments were carried out at day 7 after gene transfer. NO metabolite levels, neuronal NO synthase, and γ-amino butyric acid were decreased in SHR after CBS gene transfer. Furthermore, pressor responses to microinjection of NG-monomethyl-l-arginine into RVLM were blunt in SHR transfected with AdCBS compared to SHR transfected with AdEGFP. Overexpression of CBS in the RVLM elicits enhanced pressor responses in SHR, but not in WKY, and the NO system is involved in these effects. The results suggest that alterations of H2S signaling in the brain may be associated with the development of hypertension.

Gαi2-protein-mediated signal transduction: central nervous system molecular mechanism countering the development of sodium-dependent hypertension.

Excess dietary salt intake is an established cause of hypertension. At present, our understanding of the neuropathophysiology of salt-sensitive hypertension is limited by a lack of identification of the central nervous system mechanisms that modulate sympathetic outflow and blood pressure in response to dietary salt intake. We hypothesized that impairment of brain Gαi2-protein-gated signal transduction pathways would result in increased sympathetically mediated renal sodium retention, thus promoting the development of salt-sensitive hypertension. To test this hypothesis, naive or renal denervated Dahl salt-resistant and Dahl salt-sensitive (DSS) rats were assigned to receive a continuous intracerebroventricular control scrambled or a targeted Gαi2-oligodeoxynucleotide infusion, and naive Brown Norway and 8-congenic DSS rats were fed a 21-day normal or high-salt diet. High salt intake did not alter blood pressure, suppressed plasma norepinephrine, and evoked a site-specific increase in hypothalamic paraventricular nucleus Gαi2-protein levels in naive Brown Norway, Dahl salt-resistant, and scrambled oligodeoxynucleotide-infused Dahl salt-resistant but not DSS rats. In Dahl salt-resistant rats, Gαi2 downregulation evoked rapid renal nerve-dependent hypertension, sodium retention, and sympathoexcitation. In DSS rats, Gαi2 downregulation exacerbated salt-sensitive hypertension via a renal nerve-dependent mechanism. Congenic-8 DSS rats exhibited sodium-evoked paraventricular nucleus-specific Gαi2-protein upregulation and attenuated hypertension, sodium retention, and global sympathoexcitation compared with DSS rats. These data demonstrate that paraventricular nucleus Gαi2-protein-gated pathways represent a conserved central molecular pathway mediating sympathoinhibitory renal nerve-dependent responses evoked to maintain sodium homeostasis and a salt-resistant phenotype. Impairment of this mechanism contributes to the development of salt-sensitive hypertension.

A novel method to study cerebrospinal fluid dynamics in rats

Cerebrospinal fluid (CSF) flow dynamics play critical roles in both the immature and adult brain, with implications for neurodevelopment and disease processes such as hydrocephalus and neurodegeneration. Remarkably, the only reported method to date for measuring CSF formation in laboratory rats is the indirect tracer dilution method (a.k.a., ventriculocisternal perfusion), which has limitations. Anesthetized rats were mounted in a stereotaxic apparatus, both lateral ventricles were cannulated, and the Sylvian aqueduct was occluded. Fluid exited one ventricle at a rate equal to the rate of CSF formation plus the rate of infusion (if any) into the contralateral ventricle. Pharmacological agents infused at a constant known rate into the contralateral ventricle were tested for their effect on CSF formation in real-time. The measured rate of CSF formation was increased by blockade of the Sylvian aqueduct but was not changed by increasing the outflow pressure (0-3cm of H2O). In male Wistar rats, CSF formation was age-dependent: 0.39±0.06, 0.74±0.05, 1.02±0.04 and 1.40±0.06μL/min at 8, 9, 10 and 12 weeks, respectively. CSF formation was reduced 57% by intraventricular infusion of the carbonic anhydrase inhibitor, acetazolamide. Tracer dilution methods do not permit ongoing real-time determination of the rate of CSF formation, are not readily amenable to pharmacological manipulations, and require critical assumptions. Direct measurement of CSF formation overcomes these limitations. Direct measurement of CSF formation in rats is feasible. Our method should prove useful for studying CSF dynamics in normal physiology and disease models.

Reporter mouse strain provides a novel look at angiotensin type-2 receptor distribution in the central nervous system.

Angiotensin-II acts at its type-1 receptor (AT1R) in the brain to regulate body fluid homeostasis, sympathetic outflow and blood pressure. However, the role of the angiotensin type-2 receptor (AT2R) in the neural control of these processes has received far less attention, largely because of limited ability to effectively localize these receptors at a cellular level in the brain. The present studies combine the use of a bacterial artificial chromosome transgenic AT2R-enhanced green fluorescent protein (eGFP) reporter mouse with recent advances in in situ hybridization (ISH) to circumvent this obstacle. Dual immunohistochemistry (IHC)/ISH studies conducted in AT2R-eGFP reporter mice found that eGFP and AT2R mRNA were highly co-localized within the brain. Qualitative analysis of eGFP immunoreactivity in the brain then revealed localization to neurons within nuclei that regulate blood pressure, metabolism, and fluid balance (e.g., NTS and median preoptic nucleus [MnPO]), as well as limbic and cortical areas known to impact stress responding and mood. Subsequently, dual IHC/ISH studies uncovered the phenotype of specific populations of AT2R-eGFP cells. For example, within the NTS, AT2R-eGFP neurons primarily express glutamic acid decarboxylase-1 (80.3±2.8%), while a smaller subset express vesicular glutamate transporter-2 (18.2±2.9%) or AT1R (8.7±1.0%). No co-localization was observed with tyrosine hydroxylase in the NTS. Although AT2R-eGFP neurons were not observed within the paraventricular nucleus (PVN) of the hypothalamus, eGFP immunoreactivity is localized to efferents terminating in the PVN and within GABAergic neurons surrounding this nucleus. These studies demonstrate that central AT2R are positioned to regulate blood pressure, metabolism, and stress responses.

Abstract 602: RAS-induced Hypertension Is Primarily Mediated By (Pro)renin Receptor Activation At The Paraventricular Nucleus Of The Hypothalamus.

Studies have shown that renin-angiotensin-system (RAS)-induced inflammation at the paraventricular nucleus of the hypothalamus (PVN) contributes to hypertension development by increasing sympathetic outflow and elevating blood pressure. Recent data suggest that PRR activation increases pro-inflammatory cytokine (PIC) production in an Ang II-independent manner helping sustain kidney inflammation, retinal disease and diabetic neuropathy. Astrocytes house all the components of the RAS and help regulate the immune response within the brain. However, the role of (pro)renin (PRO) receptor (PRR) activation at the PVN on blood pressure regulation and astrocyte activation has not been evaluated. We show that at the PVN, 12 weeks old spontaneously hypertensive rats (SHRs) have significantly (p &lt; 0.05) higher PRR mRNA levels than normotensive Wistar Kyoto (WKY) and Sprague Dawley (SD) rats (1.6 ± 0.3 vs. 0.96 ± 0.4 &amp; 1.0 ± 0.2, SHR, WKY and SD respectively). In situ-hybridization data using RNA Scope, confirmed these results as qualitative analysis from coronal sections containing the PVN from SD and WKY rats showed lower PRR signal when compared to those from SHRs. Our data shows that bilateral downregulation of the PRR, using a short-hairpin RNA (shRNA) vector construct, at the PVN of SHRs (n = 5) reduces blood pressure compared to control animals (n = 5) with PRR levels intact (146.1 ± 6.6 vs. 133.3 ± 9.0 mmHG; p &lt; 0.05). Similarly, PRR downregulation at the PVN can also reduce the pressor response triggered by sub-cutaneous Ang II infusion (200ng/kg/min) in SD rats (143 ± 13.2 vs. 114.8 ± 13.2 mmHG, p &lt; 0.05; control vs. shRNA animals, respectively). In both experimental designs, blood pressure decreases were not accompanied by changes in heart rate. Our results also show lower glial fibrillary acidic protein (GFAP) and interleukin (IL)-1β mRNA levels at the PVN in shRNA-injected animals and using the Image J tool we show a trend for astroglial area fraction in the PVN to decrease. These changes are in concert with our in vitro data which show that PRO increases PICs levels and astrogliosis in cultured astrocytes. Our findings show that at the PVN, PRR activation is key in RAS-induced hypertension and potential mechanisms may include astrocyte- and neuroimmune activation.

Abstract 081: Reporter Mouse Strain Provides a Novel Look at Angiotensin Type-2 Receptor Distribution in Central Nervous System Cardiovascular Control Centers

It is established that angiotensin-II acts at its type-1 receptor (AT1R) in the brain to increase sympathetic outflow and blood pressure, and modulate fluid balance. However, the role of the angiotensin type-2 receptor (AT2R) in the neural control of these processes has received far less attention, largely because of an inability to effectively localize these receptors at a cellular level in the brain. The present studies combine the use of a bacterial artificial chromosome transgenic AT2R-eGFP reporter mouse with recent advances in in situ hybridization (ISH) to circumvent this obstacle. Dual IHC/ ISH studies validated the AT2R-eGFP reporter mice by determining that eGFP and AT2R mRNA were highly co-localized within the nucleus of the solitary tract (NTS; 98.0 ± 0.18 %; 125 ± 3.6 of 127 ± 3.9 cells; n = 4). Analysis of eGFP immunoreactivity in the brain revealed localization to neurons within nuclei that regulate blood pressure and fluid balance (e.g., NTS and median preoptic nucleus [MnPO]). Additional IHC/ISH studies uncovered the phenotype of specific AT2R-eGFP cells. For example, within the NTS, AT2R-eGFP neurons primarily express glutamic acid decarboxylase-67 (GABAergic; 80 ± 2.8 %; 225 ± 12.5 of 280 ± 8.4 cells; n = 4), while only a subset express vesicular glutamate transporter-2 (glutamatergic; 18.2 ± 2.9 %; 50.8 ± 7.7 of 280 ± 8.4 cells) or AT1R (8.7 ± 1.0 %; 22 ± 2.2 of 256 ± 11.7 cells). No co-localization was observed with tyrosine hydroxylase in the NTS. Although AT2R-eGFP neurons were not observed within the paraventricular hypothalamic nucleus (PVN), eGFP was localized to efferents terminating in the PVN and to GABAergic neurons surrounding this nucleus. Retrograde neuronal tract tracing studies revealed that many eGFP-positive efferents to the PVN arise from neurons in the MnPO. Based on these neuroanatomical results, we hypothesized that activation of central AT2R would decrease blood pressure. Consistent with this hypothesis, chronic administration of the selective AT2R agonist, compound 21 (7.5 ng/h into the lateral cerebral ventricle) reduced baseline mean arterial blood pressure relative to control mice (103 ± 1.65 v. 110 ± 1.70 mmHg; n = 16; p = 0.02). These studies demonstrate that central AT2R are positioned to regulate blood pressure.

Biomechanical strain as a trigger for pore formation in Schlemm's canal endothelial cells

The bulk of aqueous humor passing through the conventional outflow pathway must cross the inner wall endothelium of Schlemm's canal (SC), likely through micron-sized transendothelial pores. SC pore density is reduced in glaucoma, possibly contributing to obstructed aqueous humor outflow and elevated intraocular pressure (IOP). Little is known about the mechanisms of pore formation; however, pores are often observed near dome-like cellular outpouchings known as giant vacuoles (GVs) where significant biomechanical strain acts on SC cells. We hypothesize that biomechanical strain triggers pore formation in SC cells. To test this hypothesis, primary human SC cells were isolated from three non-glaucomatous donors (aged 34, 44 and 68), and seeded on collagen-coated elastic membranes held within a membrane stretching device. Membranes were then exposed to 0%, 10% or 20% equibiaxial strain, and the cells were aldehyde-fixed 5 min after the onset of strain. Each membrane contained 3-4 separate monolayers of SC cells as replicates (N = 34 total monolayers), and pores were assessed by scanning electron microscopy in 12 randomly selected regions (∼65,000 μm2 per monolayer). Pores were identified and counted by four independent masked observers. Pore density increased with strain in all three cell lines (p < 0.010), increasing from 87 ± 36 pores/mm2 at 0% strain to 342 ± 71 at 10% strain; two of the three cell lines showed no additional increase in pore density beyond 10% strain. Transcellular “I-pores” and paracellular “B-pores” both increased with strain (p < 0.038), however B-pores represented the majority (76%) of pores. Pore diameter, in contrast, appeared unaffected by strain (p = 0.25), having a mean diameter of 0.40 μm for I-pores (N = 79 pores) and 0.67 μm for B-pores (N = 350 pores). Pore formation appears to be a mechanosensitive process that is triggered by biomechanical strain, suggesting that SC cells have the ability to modulate local pore density and filtration characteristics of the inner wall endothelium based on local biomechanical cues. The molecular mechanisms of pore formation and how they become altered in glaucoma may be studied in vitro using stretched SC cells.

Optimising the Windkessel model for cardiac output monitoring during changes in vascular tone.

Algorithms for estimating cardiac output (CO) from the arterial blood pressure wave have been observed to be inaccurate during changes in vascular tone. Many such algorithms are based on the Windkessel model of the circulation. We investigated the optimal analytical approaches and assumptions that make up each algorithm during changes in vascular tone. Several analytical approaches and assumptions were evaluated on data from 15 critically ill patients by comparison with thermodilution measurements. We found that the most accurate algorithms assumed a constant compliance for the duration of the beat. They produced a percentage error of ±31% by maintaining the compliance and outflow terms in the Windkessel model. For any algorithm, the following assumptions gave highest accuracy: (i) outflow pressure into the microcirculation is zero; (ii) end of systole is identified using the second derivative of pressure. None of the tested algorithms reached the clinically acceptable accuracy of ±30%.

Sympathoexcitation and pressor responses induced by ethanol in the central nucleus of amygdala involves activation of NMDA receptors in rats.

The central nervous system plays an important role in regulating sympathetic outflow and arterial pressure in response to ethanol exposure. However, the underlying neural mechanisms have not been fully understood. In the present study, we tested the hypothesis that injection of ethanol in the central nucleus of the amygdala (CeA) increases sympathetic outflow, which may require the activation of local ionotropic excitatory amino acid receptors. In anesthetized rats, CeA injection of ethanol (0, 0.17, and 1.7 μmol) increased splanchnic sympathetic nerve activity (SSNA), lumbar sympathetic nerve activity (LSNA), and mean arterial pressure (MAP) in a dose-dependent manner. A cocktail containing ethanol (1.7 μmol) and kynurenate (KYN), an ionotropic excitatory amino acid receptor blocker, showed significantly blunted sympathoexcitatory and pressor responses compared with those elicited by CeA-injected ethanol alone (P < 0.01). A cocktail containing ethanol and d-2-amino-5-phosphonovalerate, an N-methyl-d-aspartate (NMDA) receptor antagonist, elicited attenuated sympathoexcitatory and pressor responses that were significantly less than ethanol alone (P < 0.01). In addition, CeA injection of acetate (0.20 μmol, n = 7), an ethanol metabolite, consistently elicited sympathoexcitatory and pressor responses, which were effectively blocked by d-2-amino-5-phosphonovalerate (n = 9, P < 0.05). Inhibition of neuronal activity of the rostral ventrolateral medulla (RVLM) with KYN significantly (P < 0.01) attenuated sympathoexcitatory responses elicited by CeA-injected ethanol. Double labeling of immune fluorescence showed NMDA NR1 receptor expression in CeA neurons projecting to the RVLM. We conclude that ethanol and acetate increase sympathetic outflow and arterial pressure, which may involve the activation of NMDA receptors in CeA neurons projecting to the RVLM.

Yes! Sex matters: sex, the brain and blood pressure.

The role of the brain in hypertension between the sexes is known to be important especially with regards to the effects of circulating sex hormones. A number of different brain regions important for regulation of sympathetic outflow and blood pressure express estrogen receptors (ERα and ERβ). Estradiol, acting predominantly via the ERα, inhibits angiotensin II activation of the area postrema and subfornical organ neurons and inhibits reactive oxygen generation that is required for the development of Angiotensin II-induced neurogenic hypertension. Estradiol activation of ERβ within the paraventricular nucleus and the rostral ventral lateral medulla inhibits these neurons and inhibits angiotensin II, or aldosterone induced increases in sympathetic outflow and hypertension. Understanding the cellular and molecular mechanisms underlying ERα and ERβ actions within key brain regions regulating blood pressure will be essential for the development of "next generation" selective estrogen receptor modulators (SERMS) that can be used clinically for the treatment of neurogenic hypertension.

Salusin-β in rostral ventrolateral medulla increases sympathetic outflow and blood pressure via superoxide anions in hypertensive rats

Rostral ventrolateral medulla (RVLM) plays crucial roles in central cardiovascular regulation. Salusin-β is a multifunctional bioactive peptide and is expressed in the brain and various peripheral tissues. The present study was designed to identify the expression of salusin-β in the RVLM, and to determine its roles and mechanisms in regulating sympathetic outflow and blood pressure in hypertension. Renovascular hypertension was induced with two-kidney, one-clip (2K1C) method in rats. Acute experiments were carried out 4 weeks after the 2K1C or sham operation under urethane and alpha-chloralose anesthesia. Salusin-β level and the number of salusin-β-like immunopositive neurons in the RVLM were greatly increased in 2K1C rats. Microinjection of salusin-β into the RVLM increased renal sympathetic nerve activity, mean arterial pressure and heart rate, whereas antisalusin-β IgG (SIgG) reduced renal sympathetic nerve activity and mean arterial pressure in 2K1C rats, but not in sham-operated rats. The effects of salusin-β were abolished by the RVLM pretreatment with superoxide anion scavenger tempol or polyethylene glycol superoxide dismutase, or NAD(P)H oxidase inhibitor apocynin, but not attenuated by the RVLM microinjection or intravenous injection of dTyr(CH2)5(Me)AVP, an antagonist of V1 receptors of arginine vasopressin. Salusin-β increased NAD(P)H oxidase activity and superoxide anion level in the RVLM, which were prevented by the pretreatment with SIgG. Microinjection of salusin-β or SIgG into the RVLM had no significant effect on the arginine vasopressin level in the RVLM and plasma. Salusin-β in the RVLM increases sympathetic outflow, blood pressure and heart rate via NAD(P)H oxidase-derived superoxide anions in renovascular hypertensive rats.

Transneuronal tracing of central autonomic regions involved in cardiac sympathetic afferent reflex in rats

Stimulation of cardiac afferents (CA) increased sympathetic outflow and blood pressure. The goal of the current study is to determine the central autonomic nuclei involved in the regulation of cardiac sympathetic afferent reflex (CSAR) which has been proved in previously functional studies. Neuroanatomical method and pseudorabies virus (PRV) transynaptic retrograde trace technique will be performed to investigate the relationship between kidney and heart and the temporal order of the most PRV-labeled neurons in the central nervous system. Recombinant PRV expressing enhanced green fluorescence protein (EGFP) was injected into the left kidney of rats as a specific trans-synaptic retrograde tracer in neurons. After 2, 3, 4, 5, 6, 7, 8 or 9days, brain, spinal cord and heart were collected for immunofluorescence staining. The temporal order of PRV labeled neurons was found in the ipsilateral intermediolateral nucleus (IML) of T8–T12 spinal segments on day 3; bilateral rostroventrolateral medulla (RVLM), paraventricular nucleus (PVN) and nucleus of the solitary tract (NTS) on day 4; and left and right ventricular walls and ventricular septum of the heart on day 9. In rats with renal denervation, no PRV-infected neurons or cardiomyocytes were found after PRV injection. In conclusion, PRV trans-synaptic retrograde trace confirms that CA, NTS, PVN, RVLM, IML and renal nerves do exist to be involved in the regulation of CSAR and there is a close relationship between heart and kidney. CA is mainly located in the left ventricular wall, right ventricular wall and ventricular septum.

Pharmacological Regulation of SPARC by Lovastatin in Human Trabecular Meshwork Cells

Statins have been shown to increase aqueous outflow facility. The matricellular protein SPARC (secreted protein acidic and rich in cysteine) is a critical mediator of aqueous outflow and intraocular pressure (IOP). Here, we examine the effects of lovastatin on SPARC expression in trabecular meshwork (TM) cells, exploring the molecular mechanisms involved. Primary cultured human TM cells were incubated for 24, 48, and 72 hours with 10 μM lovastatin. In separate cultures, media was supplemented with either farnesyl pyrophosphate (FPP) or geranylgeranyl pyrophosphate (GGPP) for the duration of the 72-hour time point experiment. Trabecular meshwork cells were also pretreated for 24 hours with lovastatin followed by 24-hour stimulation with 3 ng/mL TGF-β2. Cell lysates and media were harvested and relative mRNA and protein level changes were determined. Krüppel-like factor 4 (KLF4) localization in normal human anterior segments was examined by immunofluorescence. Adenovirus expressing human KLF4 was used and relative changes in SPARC mRNA and protein levels were assessed. Incubating TM cells with lovastatin suppressed SPARC mRNA and protein levels. This effect was reversed upon media supplementation with GGPP but not FPP. Pretreating cells with lovastatin inhibited TGF-β2 induction of SPARC. The KLF4 transcription factor was expressed throughout the TM and the inner and outer walls of Schlemm's canal. Lovastatin treatment upregulated KLF4 mRNA and protein levels. Overexpression of KLF4 downregulated SPARC expression. Collectively, our data identify lovastatin as an important pharmacological suppressor of SPARC expression in TM cells, and provide further insight into the molecular mechanisms mediating statin enhancement of aqueous outflow facility.

Effect of renal denervation on left ventricular mass and function in patients with resistant hypertension: data from a multi-centre cardiovascular magnetic resonance imaging trial

Sympathetic stimulation induces left ventricular hypertrophy and is associated with increased cardiovascular risk. Catheter-based renal denervation (RDN) has been shown to reduce sympathetic outflow and blood pressure (BP). The present multi-centre study aimed to investigate the effect of RDN on anatomic and functional myocardial parameters, assessed by cardiac magnetic resonance (CMR), in patients with resistant hypertension. Cardiac magnetic resonance was performed in 72 patients (mean age 66 ± 10 years) with resistant hypertension (55 patients underwent RDN, 17 served as controls) at baseline and after 6 months. Clinical data and CMR results were analysed blindly. Renal denervation significantly reduced systolic and diastolic BP by 22/8 mm Hg and left ventricular mass index (LVMI) by 7.1% (46.3 ± 13.6 g/m(1.7) vs. 43.0 ± 12.6 g/m(1.7), P < 0.001) without changes in the control group (41.9 ± 10.8 g/m(1.7) vs. 42.0 ± 9.7 g/m(1.7), P = 0.653). Ejection fraction (LVEF) in patients with impaired LVEF at baseline (<50%) significantly increased after RDN (43% vs. 50%, P < 0.001). Left ventricular circumferential strain as a surrogate of diastolic function in the subgroup of patients with reduced strain at baseline increased by 21% only in the RDN group (-14.8 vs. -17.9; P = 0.001) and not in control patients (-15.5 vs. -16.4, P = 0.508). Catheter-based RDN significantly reduced BP and LVMI and improved EF and circumferential strain in patients with resistant hypertension, occurring partly BP independently.

Cerebrospinal fluid absorption block at the vertex in chronic hydrocephalus: obstructed arachnoid granulations or elevated venous pressure?

BackgroundThe lack of absorption of CSF at the vertex in chronic hydrocephalus has been ascribed to an elevation in the arachnoid granulation outflow resistance (Rout). The CSF infusion studies measuring Rout are dependent on venous sinus pressure but little is known about the changes in pressure which occur throughout life or with the development of hydrocephalus.MethodsTwenty patients with chronic hydrocephalus underwent MR venography and MR flow quantification techniques. The venous outflow pressure was estimated from the sinus blood flow and the cross-sectional area of the transverse sinuses. Adult controls as well as a normal young cohort were selected to estimate the change in sinus pressure which occurs throughout life and following the development of hydrocephalus. Significance was tested with a Student’s t-test.ResultsThe size of the transverse sinuses was unchanged from the 1st to the 5th decade of life, indicating a stable outflow resistance. However, the blood flow was reduced by 42%, indicating a likely similar reduction in pressure gradient across the sinuses. The sinuses of hydrocephalus patients were 38% smaller than matched controls, indicating a 2.5 times increase in resistance. Despite the 24% reduction in blood flow, a significant increase in sinus pressure is suggested.ConclusionsThe size of the venous sinuses normally does not change over the age range investigated but sinus pressure is reduced proportional to an age-related blood flow reduction. Hydrocephalus is associated with much smaller sinuses than normal and an elevation in venous pressure may explain the lack of CSF absorption into the arachnoid granulations in chronic hydrocephalus.

Surgical Management of Moderate Sized Spontaneous Cerebellar Hematomas: Role of Intracranial Pressure Monitoring.

Acute onset spontaneous cerebellar hematoma (SCH) is a neurosurgical emergency with unpredictable natural history and outcome. Specific guidelines are available for management of small ( < 2.5 cm) and large sized (4.0 cm < ) SCH. But, literature is sparse for treatment of moderate sized SCH (2.5-4.0 cm). In a retrospective analysis of 32 patients, with moderate sized acute SCH, external ventricular drainage (EVD) tube was inserted for evaluation of ICP and evacuation of cerebrospinal fluid. All patients with ICP > 25 mmHg underwent emergency SCH evacuation. Postoperatively, EVD was continued for 48-72 hours for ICP monitoring. Patients showing no improvement or persistent ventriculomegaly underwent ventriculoperitoneal shunt (VPS) insertion for the management of hydrocephalus. 29/32 patients needed operative intervention either in the form of hematoma evacuation or shunt placement. 10 patients improved to GOS 5 and 7 to GOS 4. Overall mortality was 25% (n=8). Despite SCH evacuation, none of the patients with ICP > 40 mmHg experienced good outcome (p < 0.0001). 47% of patients with moderate sized SCH need surgical evacuation. EVD insertion not only helps in ICP management but also includes the ability to gradually decompress ventricular system by varying the outflow pressure. Apart from radiologic findings, ICP measurement should be a guideline in management decisions.