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Biomedical subjects

A S Greene

Publications and source records attributed to A S Greene.

At least 19 recordsLinked to original sources
PubMed2000

Regional cerebral blood flow responses to variable frequency whisker stimulation: an autoradiographic analysis.

Activation of the rat primary somatosensory barrel field (S1BF) is a commonly used model to study the mechanisms of evoked coupled cortical blood flow changes. However, the relationship between these blood flow changes and variable whisker movement has not been completely characterized. We have previously shown that in urethane anesthetized rats, the magnitude of laser-Doppler measured cortical blood flow changes increase linearly with the frequency of full pad whisker movement over the physiological range of 1.5 to 10.5 s. To further test the hypothesis that local cortical blood flow increases with frequency of whisker movement and underlying neuronal activity, regional cerebral blood flow (rCBF) was determined autoradiographically in seven urethane anesthetized SD rats. Selected rows of whiskers (rows C, D, E) were stimulated at 3 s on the right side of the rat's face and simultaneously at 10 s on the left side for 2 min prior to radioactive tracer administration. Subregions of somatosensory cortex were identified with the aid of thionin and cytochrome oxidase stained sections. Mean rCBF (ml/100 g/min) for S1BF were: S1BF [0 s] left cortex, 146+/-13; S1BF [0 s] right cortex, 158+/-15; S1BF[3 s], 160+/-13; S1BF [10 s] 178+/-14. In both stimulated and nonstimulated regions, the profile of blood flow increased across cortex laminae, peaking in layer IV and decreasing through deeper layers. Maximal blood flow increases elicited by whisker movement occurred in cortical layers I-IV. These data support the hypothesis that whisker movement elicited rCBF changes are input frequency dependent and are most pronounced in cortical layers I though IV. These data provide a strong framework in which to study the mechanisms of neuronal activity-blood flow coupling.

Animals↗
PubMed2000

Genetically defined risk of salt sensitivity in an intercross of Brown Norway and Dahl S rats.

A genetic segregation analysis was performed to identify genes that cosegregate with arterial blood pressure traits reflective of salt sensitivity. A population of 113 F2 male rats was derived from an intercross of inbred SS/JrHsd/Mcw (Dahl salt-sensitive) and BN/SsN/Mcw (Brown Norway) rats. Rats were maintained on an 8% salt diet from the age of 9 to 13 wk, and arterial pressure was measured for 3 h daily during the 4th wk of high salt intake in unanesthetized rats using implanted arterial catheters. At the end of the 3rd day of high-salt pressure recordings, the arterial pressure response to salt depletion was determined 1.5 days following treatment with Lasix and a low-sodium (0. 4%) diet. A genome-wide scan using 265 polymorphic simple sequence length polymorphism (SSLP) markers found that seven arterial pressure phenotypes determined at different times and circumstances, and representing two distinct indexes of salt sensitivity, mapped to the same region of rat chromosome 18. The trait of salt sensitivity was strongly influenced by the presence of SS alleles in this region of chromosome 18, and those rats which were homozygote SS/SS exhibited a significantly greater reduction of mean arterial pressure following sodium depletion (29 +/- 2 mmHg) than homozygote BN/BN (17 +/- 3 mmHg) or heterozygotic (22 +/- 2 mmHg) rats. This region of rat chromosome 18 corresponds to the long arm of human chromosome 5 and a region of human chromosome 18 that has been linked to hypertension in humans. Given the unlikely chance of these different blood pressure traits mapping to the same region, we believe these data provide evidence that this region of rat chromosome 18 plays an important role in salt-induced hypertension.

Alleles↗
PubMed2000

Quantification of the contribution of type 1 and type 2 angiotensin II receptors to the net tissue specific effect of angiotensin II.

Numerous studies have demonstrated changes in receptor number, protein concentration, or mRNA levels and have proposed that these subcellular changes produce physiologic effects. To date, no adequate mathematical analysis has been available to provide a framework for interpretation of such data. In the present study we have combined measurements of angiotensin receptor protein levels with the development of a mathematical model that includes two receptors with opposing actions for a single ligand. This model was used to quantify the net, physiologic response of each receptor population to ANG II stimulation and the effect of altering the expression of receptor populations by a physiologic stimulus. Altered sodium intake was used as the physiologic stimulus and quantification of Western blot analysis and revealed that high sodium diet significantly suppressed AT1 receptor protein in the adrenal gland and aorta and augmented AT2 receptor protein in the aorta. A high sodium diet did not significantly alter AT2 receptor protein in the adrenal gland. Modeling the measured sodium-induced changes in receptor concentration demonstrated that small, subcellular changes in receptor concentration can have a large impact on the net physiologic effect. This model for dual receptor-single ligand interactions should be amenable for other systems.

Adrenal Glands↗
PubMed2000

Development of an implantable muscle stimulator: measurement of stimulated angiogenesis and poststimulus vessel regression.

OBJECTIVE: We developed a lightweight, totally implantable electrical stimulator designed to elicit contraction of skeletal muscle. The stimulator can be programmed to run for different on-off intervals in a given time period in a fully automatic mode. Using the stimulator, angiogenesis was promoted in order to study the rate at which vessel growth and subsequent regression occurs after stimulus removal. METHODS: A fully implanted digital stimulator was designed and fabricated. The stimulator was embedded subcutaneously in the thoracolumbar region of male Sprague-Dawley rats and the electrodes were tunneled under the skin to the common peroneal nerve of the right hind limb. The stimulator elicited muscle contraction in the hind limb at 10 s-1 using square-wave pulses 0.3 ms in duration, evoking contraction of specific muscles for 8 hours/day for 7 days. RESULTS: Chronic stimulation of the skeletal muscles innervated by the common peroneal nerve led to significant increases in blood vessel density in the tibialis anterior (TA; 26%) and the extensor digitorum longus (EDL; 19%) within 7 days. The vessel density remained elevated at 3 days and 7 days poststimulation, but subsequently decreased to control levels by 14 days poststimulation. CONCLUSION: The new stimulator can promote significant increases in vessel density within 7 days, allowing study of both stimulated vessel growth and poststimulus rarefaction. Because of its small size and reliable timing cycles, the stimulator should prove to be a valuable tool in studying these phenomena.

Animals↗
PubMed1998

Blood flow increases linearly in rat somatosensory cortex with increased whisker movement frequency.

It has long been known that the level of neuronal activity is correlated to the level of localized blood flow. Despite the importance of functional hyperemia in the brain, the relationship between blood flow and electrical activity has not been clearly demonstrated parametrically in a single region of cerebral cortex. We investigated both the magnitude and temporal characteristics of the blood flow response in somatosensory cortex while varying the frequencies of whisker movement. The full whisker pad on one side of the rat's face was repeatedly moved for 13 s at frequencies of 1.5, 2, 3, 4, 6, 8, and 10.5 Hz, and the resulting changes in blood flow were quantified using Laser-Doppler flowmetry (LDF). The magnitude of the blood flow response increased linearly with increasing frequency while the temporal parameters of time to half maximal value and time to return halfway to baseline after stimulus termination did not vary. Baseline blood flow levels were elevated by breathing rats on a 5% CO2 mixture. No significant alteration in the LDF plateau response to whisker movement was observed compared to normal air, suggesting sustained vasodilation reserve capacity remained after CO2-induced vasodilation. These data demonstrate linear blood flow responses to presumptive linear increases in neuronal activity with sufficient vascular reserve capacity to overcome moderate CO2-induced dilation, and support the use of blood flow changes in neuroimaging studies. They provide a framework to study the neurobiological signal transduction mechanisms coupling neuronal electrical activity with regional alterations in blood flow.

Animals↗
PubMed1998

Gender-specific protection from microvessel rarefaction in female hypertensive rats.

Epidemiologic studies reveal that women have a significantly lower age-adjusted morbidity and mortality from cardiovascular disease than men, suggesting that gender is a cardiovascular disease risk factor. The mechanism of the "gender protection" is unknown. In this study, we investigated the microvascular remodeling in reduced renal mass plus a high salt (4.0% NaCl) diet model of hypertension (RRM + HS). We hypothesized that women would be protected from the increase in blood pressure and from the microvascular rarefaction associated with RRM + HS hypertension. Studies were designed to determine whether female rats were less susceptible to changes in microvessel density during RRM + HS. Microvessel density was measured in male and female low salt (0.4% LS) sham-operated controls (Sham + LS) and after 3 days or 4 weeks of RRM + HS hypertension. The microcirculation of hind limb (medial and lateral gastrocnemius, plantaris, soleus) muscles was visualized using rhodamine-labeled Griffonia simplicifolia I lectin. Tissue sections were examined by videomicroscopy and microvessel density was determined by quantitative stereology. As shown previously, mean arterial pressure increased to 160 +/- 8 mm Hg and microvessel density decreased (>30% decrease in all beds) in male RRM + HS. In contrast, mean arterial pressure of female RRM + HS rats was modestly increased from 101 +/- 2 to 118 +/- 4 mm Hg. Despite previous results showing a reduction in microvessel density of both normotensive and hypertensive male rats on a high salt diet, microvessel density of female RRM + HS rats was not reduced at either time. These results suggest that gender protection in the RRM rat extends beyond an attenuation of the increase in pressure to an immunity from microvascular rarefaction.

Animals↗
PubMed1998

Laser-Doppler flowmetry utilizing a thinned skull cranial window preparation and automated stimulation.

For several decades, cranial windows have been used to investigate questions relating to cerebral blood flow and its regulation. In general, these techniques have utilized either 'open' cranial windows for the direct observation of the intracranial vasculature, or 'closed' cranial windows in which the skull and dura are removed and replaced with a clear seal, such as a coverslip. Here we describe a method of studying blood flow responses elicited by the physiological stimulus of whisker movement while using a 'thinned skull' cranial window created over the rat whisker-barrel cortex. This method employing an automated whisker stimulator coupled with laser-Doppler flowmetry focused through the thinned skull cranial window, is less invasive than other cranial window techniques, and allows for the study of the effects of stimulation parameters and systemically administered compounds on whisker movement elicited blood flow responses. Automated whisker stimulation and data collection also allow for precise temporal averaging of laser-Doppler measured responses, leading to increased precision in determining the true shape of the evoked blood flow response pattern.

Animals↗
PubMed1998

Localization of the ANG II type 2 receptor in the microcirculation of skeletal muscle.

Only functional studies have suggested the presence of the ANG II type 2 (AT2) receptor in the microcirculation. To determine the distribution of this receptor in the rat skeletal muscle microcirculation, a polyclonal rabbit anti-rat antiserum was developed and used for immunohistochemistry and Western blot analysis. The antiserum was prepared against a highly specific and antigenic AT2-receptor synthetic peptide and was validated by competition and sensitivity assays. Western blot analysis demonstrated a prominent, single band at approximately 40 kDa in cremaster and soleus muscle. Immunohistochemical analysis revealed a wide distribution of AT2 receptors throughout the skeletal muscle microcirculation in large and small microvessels. Microanatomic studies displayed an endothelial localization of the AT2 receptor, whereas dual labeling with smooth muscle alpha-actin also showed colocalization of the AT2 receptor with vascular smooth muscle cells. Other cells associated with the microvessels also stained positive for AT2 receptors. Briefly, this study confirms previous functional data and localizes the AT2 receptor to the microcirculation. These studies demonstrate that the AT2 receptor is present on a variety of vascular cell types and that it is situated in a fashion that would allow it to directly oppose ANG II type 1 receptor actions.

Adrenal Glands↗
PubMed1998

Documentation of angiotensin II receptors in glomerular epithelial cells.

Angiotensin II decreases glomerular filtration rate, renal plasma flow, and glomerular capillary hydraulic conductivity. Although angiotensin II receptors have been demonstrated in mesangial cells and proximal tubule cells, the presence of angiotensin II receptors in glomerular epithelial cells has not previously been shown. Previously, we have reported that angiotensin II caused an accumulation of cAMP and a reorganization of the actin cytoskeleton in cultured glomerular epithelial cells. Current studies were conducted to verify the presence of angiotensin II receptors by immunological and non-peptide receptor ligand binding techniques and to ascertain the activation of intracellular signal transduction in glomerular epithelial cells in response to angiotensin II. Confluent monolayer cultures of glomerular epithelial cells were incubated with angiotensin II, with or without losartan and/or PD-123,319 in the medium. Membrane vesicle preparations were obtained by homogenization of washed cells followed by centrifugation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membrane proteins followed by multiscreen immunoblotting was used to determine the presence of angiotensin II receptor type 1 (AT1) or type 2 (AT2). Angiotensin II-mediated signal transduction in glomerular epithelial cells was studied by measuring the levels of cAMP, using radioimmunoassay. Results obtained in these experiments showed the presence of both AT1 and AT2 receptor types in glomerular epithelial cells. Angiotensin II was found to cause an accumulation of cAMP in glomerular epithelial cells, which could be prevented only by simultaneous use of losartan and PD-123,319, antagonists for AT1 and AT2, respectively. The presence of both AT1 and AT2 receptors and an increase in cAMP indicate that glomerular epithelial cells respond to angiotensin II in a manner distinct from that of mesangial cells or proximal tubular epithelial cells. Our results suggest that glomerular epithelial cells participate in angiotensin II-mediated control of the glomerular filtration barrier.

Angiotensin Receptor Antagonists↗
PubMed1998

Life and death in the microcirculation: a role for angiotensin II.

OBJECTIVE: Angiotensin II (ANGII) plays a critical role in the maintenance of the microcirculation and in the anatomical loss of microvessels (rarefaction) that occurs in low renin forms of hypertension and in animals fed a high-salt diet. Elevations in sodium intake can trigger a series of hemodynamic and hormonal responses culminating in a substantial rarefaction of small arterioles and capillaries in both normal and reduced renal mass hypertensive rats. METHODS: Immunohistochemistry, Northern blot, and reverse transcription-polymerase chain reaction (RT-PCR) analysis of microdissected blood vessels were used to localize ANGII receptors in the microcirculation. Chronic infusion of ANGII and other physiologic and pharmacologic manipulations of the reninangiotensin system in rats was combined with morphologic and mathematical analysis of the network architecture. RESULTS: We have shown that rarefaction of the microcirculation can cause an increase in total peripheral resistance, reduced tissue perfusion, decreased oxygen delivery, and impaired organ function. Although the mechanisms by which this occurs are not well understood, a number of key observations point to a role for the renin-angiotensin system in this effect. First, ANGII infused systemically at subpressor levels, or locally into the skeletal muscle interstitium, can induce significant microvessel growth. Second, localization of ANGII receptor proteins by immunohistochemistry and Western blotting and RNA localization by RT-PCR confirm the presence of AT1 receptors, which are growth-stimulatory, and AT2 receptors, which are growth-inhibitory in the microcirculation. Third, maintenance of ANGII at normal levels during periods of hypertension or high-salt diet completely eliminates rarefaction. CONCLUSIONS: Taken together, these results support the hypothesis that ANGII acting through AT1- and AT2-receptor mechanisms modulate vessel density during high-salt diet and hypertension.

Angiotensin II↗
PubMed1997

Reversal of microvascular rarefaction and reduced renal mass hypertension.

This study examined the microcirculatory and renin-angiotensin system changes following the reversal of hypertension in reduced renal mass rats. Nine-week-old Sprague-Dawley reduced renal mass rats were placed on a low or high sodium diet for 4 or 8 weeks or a combination of 4 weeks of high sodium followed by 4 weeks of low sodium. Blood pressure was directly measured during the development of hypertension and its reversal. Plasma renin activity, angiotensin-converting enzyme activity, and angiotensin II concentrations were measured throughout the experiment. The cremaster and hindlimb muscles were removed, and microvascular density was determined by quantitative stereology. Four weeks of high sodium increased blood pressure (152+/-7 mm Hg) and reduced microvessel density (13.7%). Reduced renal mass hypertension was rapidly reversed after the rats were returned to a low sodium diet (124+/-7 mm Hg after 3 days), and microvascular density returned to control levels. After 4 weeks of high sodium, circulating plasma renin activity and angiotensin II fell by 94% and 82%, respectively. Plasma angiotensin-converting enzyme activity was increased after 2 weeks of high sodium but returned to control levels after 4 weeks of high sodium. This study demonstrates that microvascular density is reduced in reduced renal mass hypertensive rats following exposure to high sodium diet and this is associated with a fall in circulating plasma renin activity and angiotensin II levels. Microvascular density can return to normal levels after a reactivation of the circulating renin-angiotensin system. This study provides further evidence for the hypothesis that modulation of the renin-angiotensin system is important in the regulation of microvascular structure.

Analysis of Variance↗
PubMed1997

Suppression of angiotensin-converting enzyme expression and activity by shear stress.

Shear stress caused by the frictional forces of a fluid moving over a cell monolayer is an important regulator of gene expression. In this study, we investigated the effect of shear stress on angiotensin-converting enzyme (ACE) expression and promoter activity in vitro and on local vascular ACE activity in vivo. ACE activity measured in bovine pulmonary artery endothelial (BPAE) cells was reduced by 49.5% after exposure to a shear stress of 20 dyne/cm2 for 18 hours. Short-term shearing (2 hours) elevated ACE activity in BPAE cells, whereas long-term shearing produced a time-dependent reduction in ACE activity by 23.3%, 33.5%, and 48.9% at 8, 12, and 18 hours, respectively. Northern blot analysis revealed that shear stress (20 dyne/cm2 for 18 hours) significantly reduced ACE mRNA expression by 82%. To determine the mechanism of ACE activity and message reduction, the effect of shear on transcriptionally related events was determined in a rabbit aortic endothelial cell line (W3LUC) stably transfected with 1.3 kb of a rat ACE promoter/luciferase construct. Different shear stress magnitudes (5 to 20 dyne/cm2) caused suppression of luciferase activity by an average of 40.7%. ACE promoter activity was suppressed by 2 hours of shear stress (24.7%) and was further inhibited at time periods > 8 hours. In vivo elevations in shear stress were created by placing a stainless steel clip over a 12-mm region of the rat abdominal aorta. Restriction of vessel diameter increased blood flow velocity and caused reduction in vascular ACE activity by 40%. These studies suggest that elevations in the level of shear stress alter endothelial cell function by suppressing ACE gene and protein expression in vitro and in vivo.

Acetylcholine↗
PubMed1996

Decreases in steady-state muscle performance and vessel density in reduced renal mass hypertensive rats.

In this study we hypothesized that reduced renal mass (RRM) hypertension, which is associated with a chronic reduction in vessel density, could reduce steady-state muscle performance. Vessel density and isometric tetanic force from the in situ gastrocnemius-plantaris-soleus muscle group were studied in age-matched normotensive sham-operated control (HSS) and hypertensive RRM rats on a high-salt (4.0% NaCl) diet (HSRRM) and a low-salt (0.4% NaCl) diet (LSS and LSRRM, respectively). The Achilles tendon was isolated and connected to a force transducer. Peak isometric tension elicited by sciatic nerve stimulation (1/s for 10 min, 50 impulses/s for 330 ms) was not different between the groups. In the LSS, HSS, and LSRRM groups, tension decreased similarly at 3 min to 375 +/- 22, 447 +/- 26, and 424 +/- 21 g/g, respectively. Tension was significantly reduced in the HSRRM group (203 +/- 45 g/g) relative to the LSS, HSS, and LSRRM groups by 3 min. These differences in steady-state tension persisted throughout the remainder of the experiment. Microvessel density, measured by the lectin fluorescence technique, was reduced significantly only in the soleus, plantaris, and medial gastrocnemius of the HSRRM rats. We conclude that RRM hypertension results in reductions in vessel density (rarefaction) that are associated with decreased steady-state muscle performance.

Animals↗
PubMed1996

Opposing actions of angiotensin II on microvascular growth and arterial blood pressure.

We performed studies to further elucidate the mechanisms of angiotensin II (Ang II)-induced angiogenesis of the microvasculature. Rats were placed on a high salt diet (4% NaCl), and Ang II was infused at a subpressor rate (5 ng/kg per minute) for 3 days. Blood pressure was measured daily for 2 control and 3 infusion days. Microvessel density in the cremaster muscle was measured at the end of the infusion. Vessel density in rats that received subpressor Ang II infusion increased by 12.6% compared with rats that received vehicle infusion. When the angiotensin type 2 (AT2) receptor antagonist PD 123319 was coinfused with Ang II, blood pressure was elevated and vessel density increased above that observed with Ang II infusion alone (23% increase). When the AT1 receptor antagonist losartan was coinfused with Ang II, blood pressure was lower than control and vessel density was reduced compared with the Ang II group but was still greater than control (7.8% increase). In this study, Ang II stimulated angiogenesis in the rat cremaster muscle; this effect was enhanced by AT2 antagonism and inhibited by AT1 antagonism. Ang II infusion at a subpressor dose resulted in a pressor response with AT2 antagonism and a depressor response with AT1 antagonism. This suggests that in the microvasculature, the AT1 receptor mediates angiogenesis and vasoconstriction, and the AT2 receptor mediates an inhibition of angiogenesis and vasodilation.

Angiotensin II↗
PubMed1996

Rapid microvessel rarefaction with elevated salt intake and reduced renal mass hypertension in rats.

To identify the sequence of events associated with the development of reduced vessel density (rarefaction) in hypertension, microvessel density and ultrastructure were assessed in the cremaster muscle of rats subjected to a 75% surgical reduction of renal mass and normotensive sham-operated control rats. Rats with reduced renal mass (RRM rats) and sham-operated rats were then maintained on either a high salt (4.0% NaCl) or a low salt (0.4% NaCl) diet for 3 days. Acute exposure to the high salt diet significantly increased mean arterial pressure in RRM rats but did not affect sham-operated control rats. Quantitative fluorescence microscopy of cremaster muscle whole mounts using rhodamine-labeled Griffonia simplicifolia I lectin revealed substantial rarefaction of microvessels in both RRM hypertensive rats and normotensive sham-operated rats on a high salt diet relative to corresponding control rats on a low salt diet. Confocal microscopy revealed a heterogeneous distribution of microvessels in RRM rats on a high salt diet, with some areas largely devoid of vessels. RRM and sham-operated rats on a high salt diet both exhibited changes in arteriolar ultrastructure, which included a loss of basement membranes and a dissociation of the endothelial and smooth muscle components of the vascular wall, resulting in a loss of vessel integrity. These observations demonstrate that a rapid loss of microvessels can occur not only in rats with RRM hypertension but also in normotensive rats on a high salt diet. This loss of microvessels results from structural alterations, which differ from the degenerative processes associated with microvascular rarefaction in rats with chronic RRM hypertension.

Animals↗
PubMed1995

A computerized method for determination of microvascular density.

Currently accepted stereological methods for vascular density measurements involve manual counting of labeled vessels with a grid image overlay and determination of vessel-grid intersections. This method both is tedious and may be prone to error; therefore, our laboratory has developed a method for computer-automated determination of microvascular density using digital image processing techniques. An image of the microvasculature is acquired using computer videomicroscopy. The image is processed in three general steps, involving (1) background correction, (2) thresholding of the gray level image to create a binary image, and (3) processing of the binary image using erosion, dilation, and skeletonization algorithms. Testing of this procedure was performed on 328 typical images of skeletal muscle tissue sections taken from the hindlimb of Sprague-Dawley rats and quantitated by both traditional measures and our new computer method. Results from this comparison reveal that the automated vessel counting is highly correlated (r2 = 0.71) and decreased analysis time from 15 min/image to 30 sec/image when compared with manual counting methods. Our computer-based method also appears to be superior to the traditional method due to the unbiased and nonsubjective nature of determining vessel-grid intersections.

Animals↗
PubMed1995

Hemodynamic and microcirculatory changes during development of renal hypertension.

To study the progression of vessel loss (rarefaction) during the development of hypertension, a plastic window was chronically implanted over the biceps femoralis muscle of reduced renal mass (RRM) and sham-operated control (SOC) rats on a low-salt diet (0.4%). Blood pressure was measured directly via a catheter implanted in the femoral artery, and tissue blood flow was measured by laser-Doppler flowmetry before and after local topical application of sodium nitroprusside. Measurements were made on the control day and after an increase in sodium intake (4.0%) in RRM rats on days 5, 10, 14, 21, and 28. SOC rats on a low-salt diet served as the control group. RRM rats became hypertensive (167 mmHg), and vascular resistance increased after a change in sodium intake. Resting tissue blood flow decreased by 19% by day 28 in RRM but did not change in SOC. Sodium nitroprusside administration reduced vascular resistance to the same level in both RRM and SOC during control and at day 10; however, on all other days, sodium nitroprusside was not able to fully dilate the RRM microcirculation compared with that of SOC. At the same time, microvascular density estimated by computer fluorescence microscopy was decreased by 25% in RRM rats. These studies indicate that the development of RRM hypertension is characterized by a transition from increased tone and vessel closure (functional rarefaction) to anatomic vessel loss (structural rarefaction).

Analysis of Variance↗