A genomic-systems biology map for cardiovascular function.

With the draft sequence of the human genome available, there is a need to better define gene function in the context of systems biology. We studied 239 cardiovascular and renal phenotypes in 113 male rats derived from an F2 intercross and mapped 81 of these traits onto the genome. Aggregates of traits were identified on chromosomes 1, 2, 7, and 18. Systems biology was assessed by examining patterns of correlations ("physiological profiles") that can be used for gene hunting, mechanism-based physiological studies, and, with comparative genomics, translating these data to the human genome.

Characterization of a local renin-angiotensin system in rat gingival tissue.

BACKGROUND: The systemic renin-angiotensin system (RAS) promotes the plasmatic production of angiotensin (Ang) II, which acts through interaction with specific receptors. There is growing evidence that local systems in various tissues and organs are capable of generating angiotensins independently of circulating RAS. The aims of this study were to investigate the expression and localization of RAS components in rat gingival tissue and evaluate the in vitro production of Ang II and other peptides catalyzed by rat gingival tissue homogenates incubated with different Ang II precursors. METHODS: Reverse transcription-polymerase chain reaction assessed mRNA expression. Immunohistochemical analysis aimed to detect and localize renin. A standardized fluorimetric method with tripeptide hippuryl-histidyl-leucine was used to measure tissue angiotensin-converting enzyme (ACE) activity, whereas high performance liquid chromatography showed products formed after the incubation of tissue homogenates with Ang I or tetradecapeptide renin substrate (TDP). RESULTS: mRNA for renin, angiotensinogen, ACE, and Ang II receptors (AT(1a), AT(1b), and AT(2)) was detected in gingival tissue; cultured gingival fibroblasts expressed renin, angiotensinogen, and AT(1a) receptor. Renin was present in the vascular endothelium and was intensely expressed in the epithelial basal layer of periodontally affected gingival tissue. ACE activity was detected (4.95 +/- 0.89 nmol histidyl-leucine/g/minute). When Ang I was used as substrate, Ang 1-9 (0.576 +/- 0.128 nmol/mg/minute), Ang II (0.066 +/- 0.008 nmol/mg/minute), and Ang 1-7 (0.111 +/- 0.017 nmol/mg/minute) were formed, whereas these same peptides (0.139 +/- 0.031, 0.206 +/- 0.046, and 0.039 +/- 0.007 nmol/mg/minute, respectively) and Ang I (0.973 +/- 0.139 nmol/mg/minute) were formed when TDP was the substrate. CONCLUSION: Local RAS exists in rat gingival tissue and is capable of generating Ang II and other vasoactive peptides in vitro.

Substitution of Brown Norway chromosome 16 preserves cardiac function with aging in a salt-sensitive Dahl consomic rat.

Determination of the genetic factors that control the progression of left ventricular hypertrophy (LVH) to heart failure has been difficult despite extensive study in animal models. Here we have characterized a consomic rat model of LVH resulting from the introgression of chromosome 16 from the normotensive Brown Norway (BN) rat onto the genetic background of the Dahl salt-sensitive (SS/Mcwi) rat by marker assisted breeding. The SS-16BN/Mcwi consomic rats are normotensive but display LVH equivalent to the hypertensive SS/Mcwi rats at early ages. In this study we tracked the development of LVH by echocardiography and analyzed changes in cardiac function and morphology with aging in the SS-16BN/Mcwi, SS/Mcwi, and BN to determine if the consomic SS-16BN/Mcwi was a model of hypertrophic cardiomyopathy (HCM). Aging SS-16BN/Mcwi rats showed no evidence of heart failure or impaired cardiac function upon extensive analysis of left ventricle function by echocardiography and pressure-volume relationships, while their parental SS/Mcwi experienced deterioration in function between 18 and 36 wk of age. In addition aging SS-16BN/Mcwi did not exhibit tissue remodeling common to pathological hypertrophy and HCM such as increased fibrosis and reduced capillary density in the myocardium. In fact, SS-16BN/Mcwi were better protected from developing LV fibrosis with age than either the hypertensive SS/Mcwi or normotensive BN parental strains. This suggests that a gene or genes on chromosome 16 may be involved with both blood pressure regulation and preservation of cardiac function with aging.

Mapping baroreceptor function to genome: a mathematical modeling approach.

To gain information about the genetic basis of a complex disease such as hypertension, blood pressure averages are often obtained and used as phenotypes in genetic mapping studies. In contrast, direct measurements of physiological regulatory mechanisms are not often obtained, due in large part to the time and expense required. As a result, little information about the genetic basis of physiological controlling mechanisms is available. Such information is important for disease diagnosis and treatment. In this article, we use a mathematical model of blood pressure to derive phenotypes related to the baroreceptor reflex, a short-term controller of blood pressure. The phenotypes are then used in a quantitative trait loci (QTL) mapping study to identify a potential genetic basis of this controller.

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.

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.

Autoregulation of the systemic circulation in conscious rats.

Autoregulation of blood flow in various organ systems is a well-documented phenomenon. However, the net effect of these regional autoregulatory responses on the systemic circulation has not been studied in conscious rats despite the now extensive use of rats in cardiovascular research. The ability of the systemic circulation to autoregulate cardiac output has been proposed to play an important role in the development of increased vascular resistance in volume-dependent forms of hypertension. To better understand these events, we characterized responses to acute increases and decreases in blood volume in conscious areflexic rats that were chronically instrumented with arterial and venous catheters and an electromagnetic flow probe around the ascending aorta. Neurohumoral blockade was achieved with chlorisondamine (10 mg/kg), methscopolamine (0.5 mg/kg), captopril (1.0 mg/kg), and d(CH2)5Tyr(Me)arginine vasopressin (10 micrograms/kg). Mean arterial pressure was restored to normal levels with a constant i.v. norepinephrine infusion, which resulted in normal values of cardiac output, total peripheral resistance, and blood gases. Blood volume expansion (0.9 ml i.v. blood infusion for 6 minutes) increased cardiac output 9 +/- 1% and mean arterial pressure 30 +/- 3% and caused a 22 +/- 2% increase in total peripheral resistance (n = 7). Blood volume contraction (6-minute withdrawal of 0.9 ml of blood) decreased cardiac output 12 +/- 1% and mean arterial pressure 26 +/- 4%, which resulted in a 16 +/- 4% decrease in total peripheral resistance (n = 8). The slopes of the pressure-flow relationships during volume expansion were 0.24 and 0.41 during volume contraction, as compared with a nonautoregulating system (slope = 1) and a completely autoregulating system (slope = 0).(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Insulin does not reduce forearm alpha-vasoreactivity in obese hypertensive or lean normotensive men.

Evidence supports the hypothesis that an impaired capacity of insulin to antagonize norepinephrine-induced vasoconstriction increases alpha-adrenergic tone in overweight young men with insulin resistance and mild hypertension. Therefore, the effects of regionally infused insulin at 100 microU/mL on forearm blood flow (milliliters per deciliter per minute) and responses to norepinephrine were measured in seven obese hypertensive and eight lean normotensive men younger than 45 years old. The obese hypertensive men were hyperinsulinemic and insulin resistant compared with the normotensive men, as evidenced by abnormal values for fasting insulin (15.5 +/- 1.6 versus 7.2 +/- 0.8 microU/mL, P < .001), the insulin area under the curve in response to a 2-hour oral glucose tolerance test (12.0 +/- 1.5 versus 6.7 +/- 1.1 mU x min/dL, P < .01), and the disappearance rate of glucose during a 15-minute insulin tolerance test (2.7 +/- 0.3 versus 4.1 +/- 0.3 mg%/min, P < .05). The logarithm of the norepinephrine EC50 was not significantly different in obese hypertensive men (mean, 95% confidence interval: -8.15, -8.42 to -7.87) versus lean normotensive men (-7.91, -8.23 to -7.59). The 2-hour regional insulin infusion at 100 microU/mL did not significantly alter the EC50 for norepinephrine in either group. Insulin at this concentration induced significant and similar increases of forearm blood flow in the hypertensive and normotensive groups (1.7 +/- 0.4 versus 1.7 +/- 0.6 mL/100 mL per minute, P = NS). At approximately 100 microU/mL, insulin does not antagonize norepinephrine-induced vasoconstriction in the forearm circulation of either obese hypertensive or lean normotensive men.(ABSTRACT TRUNCATED AT 250 WORDS)

Microvessel changes in hypertension measured by Griffonia simplicifolia I lectin.

Commonly used methods for assessing reductions in microvascular density (rarefaction) in hypertension detect only perfused microvessels. In the present study, samples of cremaster and spinotrapezius muscles were taken from rats with chronic (4-week) reduced renal mass hypertension and normotensive sham-operated control rats, as well as from 12-week-old spontaneously hypertensive rats and their normotensive Wistar-Kyoto control strain. Mean arterial pressure was 149 +/- 8 mm Hg in the rats with reduced renal mass hypertension, 114 +/- 7 mm Hg in sham-operated rats, 177 +/- 9 mm Hg in spontaneously hypertensive rats, and 95 +/- 4 mm Hg in Wistar-Kyoto rats. Muscle samples were incubated with rhodamine-labeled Griffonia simplicifolia I lectin, which identifies both perfused and nonperfused microvessels. Microvascular density was assessed by counting intersections with a 20-microns grid. Microvessel density was significantly reduced in cremaster muscles of both spontaneously hypertensive and reduced renal mass hypertensive rats, and in the spinotrapezius muscle of spontaneously hypertensive rats, compared with their respective normotensive controls. Further studies in the reduced renal mass rats on low salt diets indicated that lectin binding was also decreased as salt intake was increased, independent of blood pressure. This change was not due to an alteration in lectin-binding affinity. These studies indicate that lectin binding can be a useful tool for assessing microvessel density that does not depend on the perfusion state of the vessels and that rarefaction due to hypertension is not evenly distributed in all vascular beds. These results also provide evidence that dietary salt intake alone can influence microvessel density, as measured by the lectin technique.

Structural changes during microvascular rarefaction in chronic hypertension.

Previous physiological studies have suggested that loss of microvessels (anatomic rarefaction) occurs in the skeletal muscle microcirculation of rats with chronic hypertension. However, little is known of the exact structural changes that occur during the process of anatomic rarefaction. The purpose of this study was to examine the muscle at the ultrastructural level to search for evidence of microvessel degeneration that would correlate with the concept of anatomic rarefaction in chronic hypertension. Cremaster muscles were removed from normal rats and from rats with chronic reduced renal mass hypertension, which was produced by a 75% reduction in kidney mass followed by salt loading (4% NaCl chow with water ad libitum) for 4 weeks. The muscles were fixed and prepared for histological examination by light and electron microscopy. Atrophy and degeneration of both endothelial cells and vascular smooth muscle cells were observed in many arterioles of the hypertensive rats. Some arterioles of hypertensive rats were degenerated to such an extent that the original identity of the cells could not be determined. The hypertensive rats also exhibited degeneration of capillaries and extravasation and uptake of red blood cells into lymphatic vessels. In contrast, age-matched control rats exhibited normal histology. The results of this study support previous physiological evidence for anatomic rarefaction in the cremaster muscle of chronically hypertensive rats.

Structural alterations of microvascular smooth muscle cells in reduced renal mass hypertension.

Loss of microvessels (anatomic rarefaction) occurs in chronic reduced renal mass (RRM) hypertension and is mediated via structural degeneration of vascular smooth muscle (VSM) and endothelial cells. The purpose of the present study was to determine if structural changes occur in VSM cells of the microvessels that remain in the tissue of rats with chronic RRM hypertension. Samples of cremaster muscles were taken from normotensive control rats and rats with acute (3-7 days) and chronic (14-28 days) RRM hypertension (75% reduction in kidney mass with 4% NaCl loading). The samples were fixed in situ and processed for light and electron microscopy. Ultrastructural morphology of VSM cells in terminal arterioles of control animals was normal. Although VSM morphology in many microvessels of RRM hypertensive rats was also normal, some vessels exhibited structural changes that were not present in arterioles of the normotensive animals. The most striking change was the appearance of more extensive dense bodies anchoring the contractile filaments around the outer membrane of the cells. Extreme vasoconstriction was observed in some arterioles of RRM rats as long as 2 weeks after salt loading. Focal areas of VSM cell proliferation were evident. Many of the changes occurring in RRM were detected as early as 1 week after the onset of hypertension. These observations suggest that renal mass reduction-salt loading hypertension is associated with early structural and functional changes in the VSM cells.

Measurement of peripheral blood flow by thermodilution techniques.

Recent advances in the diagnosis and treatment of cardiovascular pathology, including balloon angioplasty of atherosclerotic lesions in peripheral vascular disease, have led to an increased need for in vivo quantitation of blood flow. This study has three purposes: (1) to validate thermodilution techniques as a viable method for measuring low blood flow rates, (2) to calibrate accurately thermodilution catheters at these low flows, and (3) to develop an animal model that can be used to quantitate and compare many different flow measuring techniques. Modified commercially available 6F thermodilution catheters were used with a standard cardiac output computer to measure flows between 200 and 700 ml/minute. Eight anesthetized dogs were surgically interfaced with a variable flow, pressure, and compliance carotid-carotid/jugular bypass perfusion system. Three milliliters of normal saline at room temperature were injected through the catheters intra-arterially to measure different flows below, at, and above physiologic pressures and compliances. Results of this study indicate that with proper calibration, thermodilution techniques of measuring arterial and venous flows between 200 and 700 ml/minute are simple, accurate, and reliable. Using the designed system to generate known flows in vivo at various physiologic conditions allowed easy calibration of catheters and should facilitate calibration and comparison of other measurement techniques.

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.

Breathing periodicity in intact and carotid body-denervated ponies during normoxia and chronic hypoxia.

Periodic oscillations in pulmonary ventilation (VI), tidal volume (VT), and inspiratory and expiratory times (TI and TE) were studied during normoxia (arterial PO2 = 95 Torr) and 48 h of hypoxia (arterial PO2 = 40-50 Torr) in awake intact (n = 8) and carotid body-denervated (CBD; n = 8) ponies. Periodic oscillations were identified by fast-Fourier transformation of breath-by-breath data and quantitated by determining the power ratio of significant periodic oscillations to total power of data sequence. Periodic oscillations of 0.063-0.500 cycles/breath were observed in all parameters during both normoxia and hypoxia. During normoxia, CBD accentuated periodicity of VT (P < 0.02) and VI (P < 0.01) but did not change TI or TE periodicity (P > 0.05). These findings suggest that carotid chemoreceptors serve to stabilize breathing (i.e., decrease periodicity) during normoxia, conceivably because of their shorter response time compared with that of central chemoreceptors. During certain periods of hypoxia, periodicity of VT and VI was significantly (P < 0.05) increased in intact ponies. The response to hypoxia in CBD ponies was variable, with VI periodicity significantly (P < 0.05) increasing, decreasing, or unchanging. Because some CBD ponies significantly changed their periodicity during hypoxia compared with normoxia, we conclude that carotid chemoreceptors are not requisite for hypoxia-induced changes in periodic breathing. In addition, our observations in both groups of ponies during normoxia and hypoxia suggest that multiple mechanisms may lead to periodic oscillations in breathing.

Treatment of imipramine overdose in children.

Enuresis is a common problem often treated effectively with imipramine hydrochloride. The usefulness of this therapy carries with it, however, the risk of accidental overdose by younger siblings of these enuretic patients. Traditional support measures are effective in the treatment of the mild to moderate overdose, while separate symptomatic treatment of seizures and cardiac arrhythmias is possible as outlined herein. Physostigmine offers a single alternate treatment which is effective in the full panorama of life-threatening manifestations of an imipramine overdose.

Anesthesia alters NO-mediated functional hyperemia.

Many properties of nitric oxide, NO, (localization, diffusiveness, half-life, vasodilatory affects) have supported its potential role in mediating the link between local cerebral activity and blood flow. However, evidence that both supports and refutes a role for NO in functional hyperemia have been presented. The present study employed multiple nitric oxide synthase inhibitors, two anesthetic regimes and laser-Doppler flowmetry to test the hypothesis that NO is critically involved in mediating the functional hyperemic response within rodent whisker-barrel cortex (WBC). In urethane anesthetized animals, functional hyperemic responses were obtained both before and after 1 mg/kg atropine infusion, 30 mg/kg i.v. L-NAME (N-Nitro-L-arginine methylester) infusion, 30 mg/kg L-NA (N-Nitro-L-arginine) infusion or 25 mg/kg 7-NI (7-nitroindazole). L-NAME was also tested in a group of animals pretreated with halothane before urethane anesthesia. Neither the magnitude of the blood flow response nor its time course was altered by NO blockade or atropine administration when compared to pre-infusion controls in urethane anesthetized rats. In contrast, animals that were pretreated with halothane exhibited a 33% inhibition of functional hyperemia after L-NAME administration. Taken together, these data do not support a primary role for NO in rat WBC functional hyperemia and suggest that previous reports of inhibition may have been secondary to the anesthesia employed.

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.

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.

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.