Julian H. Lombard

In Dahl salt-sensitive (SS) rats, impaired vascular relaxation can be restored by: (1) minipump infusion of a low (sub-pressor) dose of angiotensin II (ANG II) to restore physiological levels of plasma ANG II, (2) inhibition of 20-HETE production, and (3) introgression of a normally functioning renin allele from the Brown Norway rat (SS-13^BN consomic rat). Unlike SS rats, SS-13^BN rats have normal levels of ANG II on a normal-salt diet and suppressed ANG II on a high-salt (HS) diet. This study tested whether chronically low ANG II levels in SS rats upregulate cytochrome P450-4A (CYP4A) increasing the production of the vasoconstrictor 20-HETE. Although salt-induced suppression of ANG II levels increased reactive oxygen species (ROS) in basilar arteries from SS-13^BN rats in previous studies, this study showed no change in vascular 20-HETE levels in response to ANGII suppression. CYP4A inhibition significantly reduced vascular ROS levels and restored endothelium-dependent relaxation in response to acetylcholine in the middle cerebral artery (MCA) of SS rats and HS-fed SS-13^BN rats. These data demonstrate that both the renin–angiotensin system and the CYP4A/20-HETE pathway play a direct role in the vascular dysfunction of the Dahl SS rat but are independent of each other, even though they may both contribute to vascular dysfunction through ROS production.

When investigating the body's mechanisms for regulating cerebral blood flow, a relative measurement of microcirculatory blood flow can be obtained using laser Doppler flowmetry (LDF). This paper demonstrates a closed skull preparation that allows cerebral blood flow to be assessed without penetrating the skull or installing a chamber or cerebral window. To evaluate autoregulatory mechanisms, a model of controlled blood pressure reduction via graded hemorrhage can be utilized while simultaneously employing LDF. This enables the real time tracking of the relative changes in the blood flow in response to reductions in arterial blood pressure produced by the withdrawal of circulating blood volume. This paradigm is a valuable approach to study cerebral blood flow autoregulation during reductions in arterial blood pressure and, with minor modifications in the protocol, is also valuable as an experimental model of hemorrhagic shock. In addition to evaluating autoregulatory responses, LDF can be used to monitor the cortical blood flow when investigating metabolic, myogenic, endothelial, humoral, or neural mechanisms that regulate cerebral blood flow and the impact of various experimental interventions and pathological conditions on cerebral blood flow.

The present study assessed the effect of nearby construction activity on the responses of rat middle cerebral arteries (MCA) to the endothelium-dependent vasodilator acetylcholine and the NO donor sodium nitroprusside (SNP) and the activity of MaxiK potassium channels in MCA smooth muscle cells from male Sprague–Dawley rats. Two monitoring systems were used to assess vibrations in the animal rooms during and immediately after construction activities near the research building where the animal facility is located. One was a commercially available system; the other was a Raspberry-Pi (RPi)–based vibration monitoring system designed in our laboratory that included a small computing unit attached to a rolling sensor (low sensitivity) and a piezoelectric film sensor (high sensitivity). Both systems recorded increased levels of vibration during construction activity outside the building. During the construction period, vasodilator responses to acetylcholine and SNP were abolished, and MaxiK single-channel current opening frequency and open-state probability in cell-attached patches of isolated MCA myocytes were dramatically decreased. Recovery of acetylcholine- and SNP-induced dilation was minimal in MCA from rats studied after completion of construction but housed in the animal facility during construction, whereas responses to acetylcholine and SNP were intact in rats purchased, housed, and studied after construction. Baseline levels of vibration returned after the completion of construction, concomitant with the recovery of normal endothelium-dependent vasodilation to acetylcholine and of NO sensitivity assessed by using SNP in MCA from animals obtained after construction. The results of this study indicate that the vibration associated with nearby construction can have highly disruptive effects on crucial physiologic phenotypes.

Hypothesis: This study tested the hypothesis that dietary activation of the master antioxidant and cell protective transcription factor nuclear factor, erythroid ‐2‐like 2 (NRF2), protects against salt‐induced vascular dysfunction by restoring redox homeostasis in the vasculature.Methods: Male Sprague‐Dawley rats and Syrian hamsters were fed a HS (4.0% NaCl) diet containing ~60 mg/kg/day Protandim supplement for 2 weeks and compared to controls fed HS diet alone.Results: Protandim supplementation restoredendothelium‐dependent vasodilation in response to acetylcholine (ACh) in middle cerebral arteries (MCA)of HS‐fed rats and hamster cheek pouch arterioles, and increased microvessel density in the cremastermuscle of HS‐fed rats. The restored dilation to ACh in MCA of Protandim‐treated rats was prevented by inhibiting nitric oxide synthase (NOS) with L‐NAME [100 μM] and was absent in MCA from Nrf2^(−/−) knockout rats fed HS diet. Basilar arteries from HS‐fed rats treated with Protandim exhibited significantly lower staining for mitochondrial oxidizing species than untreated animals fed HS diet alone; and Protandim treatment increased MnSOD (SOD2) protein expression in mesenteric arteries of HS‐fed rats.Conclusions: These results suggest that dietary activation of NRF2 protects against salt‐induced vascular dysfunction, vascular oxidative stress, and microvascular rarefaction by upregulating antioxidant defenses and reducing mitochondrial ROS levels.

Objectives: This study sought to determine whether salt‐induced ANG II suppression contributes to impaired CBF autoregulation.Methods: Cerebral autoregulation was evaluated with LDF during graded reductions of blood pressure. Autoregulatory responses in rats fed HS (4% NaCl) diet vs LS (0.4% NaCl) diet were analyzed using linear regression analysis, model‐free analysis, and a mechanistic theoretical model of blood flow through cerebral arterioles.Results: Autoregulation was intact in LS‐fed animals as MAP was reduced via graded hemorrhage to approximately 50 mm Hg. Short‐term (3 days) and chronic (4 weeks) HS diet impaired CBF autoregulation, as evidenced by progressive reductions of laser Doppler flux with arterial pressure reduction. Chronic low dose ANG II infusion (5 mg/kg/min, i.v.) restored CBF autoregulation between the pre‐hemorrhage MAP and 50 mm Hg in rats fed short‐term HS diet. Mechanistic‐based model analysis showed a reduced myogenic response and reduced baseline VSM tone with short‐term HS diet, which was restored by ANG II infusion.Conclusions: Short‐term and chronic HS diet lead to impaired autoregulation in the cerebral circulation, with salt‐induced ANG II suppression as a major factor in the initiation of impaired CBF regulation.

This protocol describes the use of in vitro television microscopy to evaluate vascular function in isolated cerebral resistance arteries (and other vessels), and describes techniques for evaluating tissue perfusion using Laser Doppler Flowmetry (LDF) and microvessel density utilizing fluorescently labeled Griffonia simplicifolia (GS1) lectin. Current methods for studying isolated resistance arteries at transmural pressures encountered in vivo and in the absence of parenchymal cell influences provide a critical link between in vivo studies and information gained from molecular reductionist approaches that provide limited insight into integrative responses at the whole animal level. LDF and techniques to selectively identify arterioles and capillaries with fluorescently-labeled GS1 lectin provide practical solutions to enable investigators to extend the knowledge gained from studies of isolated resistance arteries. This paper describes the application of these techniques to gain fundamental knowledge of vascular physiology and pathology in the rat as a general experimental model, and in a variety of specialized genetically engineered "designer" rat strains that can provide important insight into the influence of specific genes on important vascular phenotypes. Utilizing these valuable experimental approaches in rat strains developed by selective breeding strategies and new technologies for producing gene knockout models in the rat, will expand the rigor of scientific premises developed in knockout mouse models and extend that knowledge to a more relevant animal model, with a well understood physiological background and suitability for physiological studies because of its larger size.

This study investigated the acute effects of angiotensin-(1-7) and AVE0991 on active tone and vasodilator responses to bradykinin and acetylcholine in isolated mesenteric arteries from Sprague-Dawley rats fed a high-salt (HS; 4% NaCl) versus a normal salt (NS; 0.4% NaCl) diet. Angiotensin-(1-7) and AVE0991 elicited relaxation, and angiotensin-(1-7) unmasked vasodilator responses to bradykinin in arteries from HS-fed rats. These effects of angiotensin-(1-7) and AVE0991 were inhibited by endothelium removal, A779, PD123319, HOE140 and L-NAME. Angiotensin-(1-7) also restored the acetylcholine-induced relaxation that was suppressed by the HS diet. Vasodilator responses to bradykinin and acetylcholine in the presence of angiotensin-(1-7) were mimicked by captopril and the AT₂ receptor agonist CGP42112 in arteries from HS-fed rats. Thus, in contrast to salt-induced impairment of vascular relaxation in response to vasodilator stimuli, angiotensin-(1-7) induces endothelium-dependent and NO-mediated relaxation, unmasks bradykinin responses via activation of mas and AT₂ receptors, and restores acetylcholine-induced vasodilation in HS-fed rats. AT₂ receptor activation and angiotensin-converting enzyme (ACE) inhibition shared the ability of angiotensin-(1-7) to enhance bradykinin and acetylcholine responses in HS-fed rats. These findings suggest a therapeutic potential for mas and/or AT₂ receptor activation and ACE inhibition in restoring endothelial function impaired by elevated dietary salt intake or other pathological conditions.

Objective: The potential contribution of CYP4A enzymes to endothelial dysfunction in Dahl salt‐sensitive rats was determined by comparison to SS‐5^BN consomic rats having chromosome 5 carrying CYP4A alleles from the BN rat introgressed into the SS genetic background.Methods: The following experiments were performed in cerebral arteries from HS‐fed SS and SS‐5^BN rats ± the SOD inhibitor DETC and/or the superoxide scavenger Tempol: (i) endothelial function was determined via video microscopy ± acute addition of the CYP4A inhibitor DDMS or Tempol; (ii) vascular oxidative stress was assessed with DHE fluorescence ± acute addition of DDMS, l‐NAME, or PEG‐SOD; and (iii) CYP4A protein levels were compared by western blotting.Results: In DETC‐treated SS‐5^BN and HS‐fed SS rats, (i) DDMS or Tempol ameliorated vascular dysfunction, (ii) DDMS reduced vascular oxidative stress to control levels, (iii) chronic Tempol treatment reduced vascular CYP4A protein expression, and (iv) combined treatment with Tempol and l‐NAME prevented the reduction in CYP4A protein expression in MCA of HS‐fed SS rats.Conclusion: The CYP4A pathway plays a role in vascular dysfunction in SS rats and there appears to be a direct role of reduced NO availability due to salt‐induced oxidant stress in upregulating CYP4A enzyme expression.

Nuclear factor (erythroid-derived 2)-like-2 (NRF2) is a master antioxidant and cell protective transcription factor that upregulates antioxidant defenses. In this study we developed a strain of Nrf2 null mutant rats to evaluate the role of reduced NRF2-regulated antioxidant defenses in contributing to endothelial dysfunction and impaired angiogenic responses during salt-induced ANG II suppression. Nrf2^−/− mutant rats were developed using transcription activator-like effector nuclease technology in the Sprague-Dawley genetic background, and exhibited a 41-bp deletion that included the start codon for Nrf2 and an absence of immunohistochemically detectable NRF2 protein. Expression of mRNA for the NRF2-regulated indicator enzymes heme oxygenase-1, catalase, superoxide dismutase 1, superoxide dismutase 2, and glutathione reductase was significantly lower in livers of Nrf2^−/− mutant rats fed high salt (HS; 4% NaCl) for 2 wk compared with wild-type controls. Endothelium-dependent dilation to acetylcholine was similar in isolated middle cerebral arteries (MCA) of Nrf2^−/− mutant rats and wild-type littermates fed low-salt (0.4% NaCl) diet, and was eliminated by short-term (3 days) HS diet in both strains. Low-dose ANG II infusion (100 ng/kg sc) reversed salt-induced endothelial dysfunction in MCA and prevented microvessel rarefaction in wild-type rats fed HS diet, but not in Nrf2^−/− mutant rats. The results of this study indicate that suppression of NRF2 antioxidant defenses plays an essential role in the development of salt-induced oxidant stress, endothelial dysfunction, and microvessel rarefaction in normotensive rats and emphasize the potential therapeutic benefits of directly upregulating NRF2-mediated antioxidant defenses to ameliorate vascular oxidant stress in humans.