Shear stress-induced vasodilation in porcine coronary conduit arteries is independent of nitric oxide release

The present study was performed to determine the importance of nitric oxide in eliciting epicardial coronary artery dilation during sustained increases in shear stress in the absence of pulsatile flow. Isolated first-order porcine epicardial coronary conduit arteries (approximately 500 microm) were preconstricted (U-46619) and subjected to steady-state changes in flow in vitro. Nonpulsatile flow (shear stress range from 0 to approximately 100 dyn/cm2) produced a graded dilation of epicardial arteries. Inhibiting nitric oxide synthase with 10(-5) M N(omega)-nitro-L-arginine methyl ester (L-NAME) blocked bradykinin-induced vasodilation but did not affect the flow-diameter relation or the maximum change in diameter from static conditions (67 +/- 10 microm in control vs. 71 +/- 8 microm after L-NAME, P = not significant). The addition of indomethacin (10(-5) M) had no effect on flow-mediated vasodilation. Depolarizing vascular smooth muscle with KCl (60 mM) or removing the endothelium blocked bradykinin vasodilation and completely abolished flow-mediated responses. The K+ channel blocker tetraethylammonium chloride (TEA; 10(-4)M) attenuated flow-mediated vasodilation (maximum diameter change was 110 +/- 18 microm under control conditions vs. 58 +/- 10 microm after TEA, P < 0.001). These data indicate that epicardial coronary arteries dilate to steady-state changes in nonpulsatile flow via a mechanism that is independent of nitric oxide production. The ability to completely block this with KCl and attenuate it with TEA supports the hypothesis that epicardial coronary arteries dilate to steady levels of shear stress through hyperpolarization of vascular smooth muscle. This may be secondary to the release of an endothelium-dependent hyperpolarizing factor.     

Impaired NO-mediated vasodilation with increased superoxide but robust EDHF function in right ventricular arterial microvessels of pulmonary hypertensive rats

Pulmonary hypertension (PH) causes right ventricular (RV) hypertrophy and, according to the extent of pressure overload, eventual heart failure. We tested the hypothesis that the mechanical stress in PH-RV impairs the vasoreactivity of the RV coronary microvessels of different sizes with increased superoxide levels. Five-week-old male Sprague-Dawley rats were injected with monocrotaline (n=126) to induce PH or with saline as controls (n=114). After 3 wk, coronary arterioles (diameter = 30-100 microm) and small arteries (diameter = 100-200 microm) in the RV were visualized using intravital videomicroscopy. We evaluated ACh-induced vasodilation alone, in the presence of N(omega)-nitro-L-arginine methyl ester (L-NAME), in the presence of tetraethylammonium (TEA) or catalase with or without L-NAME, and in the presence of SOD. The degree of suppression in vasodilation by L-NAME and TEA was used as indexes of the contributions of endothelial nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF), respectively. In PH rats, ACh-induced vasodilation was significantly attenuated in both arterioles and small arteries, especially in arterioles. This decreased vasodilation was largely attributable to reduced NO-mediated vasoreactivity, whereas the EDHF-mediated vasodilation was relatively robust. The suppressive effect on arteriolar vasodilation by catalase was similar to TEA in both groups. Superoxide, as measured by lucigenin chemiluminescence, was significantly elevated in the RV tissues in PH. SOD significantly ameliorated the impairment of ACh-induced vasodilation in PH. Robust EDHF function will play a protective role in preserving coronary microvascular homeostasis in the event of NO dysfunction with increased superoxide levels.     

Acute local subcutaneous VEGF165 injection for augmentation of skin flap viability: efficacy and mechanism

Distal skin ischemic necrosis is a common complication in skin flap surgery. The pathogenesis of skin flap ischemic necrosis is unclear, and there is no clinical treatment available. Here, we used the 4 x 10 cm rat dorsal skin flap model to test our hypothesis that subcutaneous injection of vascular endothelial growth factor 165 (VEGF165) in skin flaps at the time of surgery is effective in augmentation of skin flap viability, which is associated with an increase in nitric oxide (NO) production, and the mechanism involves 1) an increase in skin flap blood flow in the early stage after surgery and 2) enhanced angiogenesis subsequently to sustain increased skin flap blood flow and viability. We observed that subcutaneous injection of VEGF165 in skin flaps at the time of surgery increased skin flap viability in a dose-dependent manner. Subcutaneous injection of VEGF165 at the dose of 2 microg/flap increased skin flap viability by 28% (P < 0.05; n = 8). Over 80% of this effect was blocked by intramuscular injection of the NO synthase (NOS) inhibitor Nomega-nitro-L-arginine (13 mg/kg) 45 min before surgery (P < 0.05; n = 8). The VEGF165 treatment also increased skin flap blood flow (2.68 +/- 0.63 ml x min(-1) x 100 g(-1)) compared with the control (1.26 +/- 0.10 ml x min(-1) x 100 g(-1); P < 0.05, n = 6) assessed 6 h postoperatively. There was no change in skin flap capillary density at this time point. VEGF165-induced increase in capillary density (32.2 +/- 1.1 capillaries/mm2; P < 0.05, n = 7) compared with control (24.6 +/- 1.4 capillaries/mm2) was seen 7 days postoperatively. There was also evidence to indicate that VEGF165-induced NO production in skin flaps was stimulated by activation of NOS activity followed by upregulation of NOS protein expression. These observations support our hypothesis and for the first time provide an important insight into the mechanism of acute local VEGF165 protein therapy in mitigation of skin flap ischemic necrosis.     

Direct effect of ethanol on human vascular function

Epidemiological studies indicate that moderate ethanol consumption reduces cardiovascular mortality. Cellular and animal data suggest that ethanol confers beneficial effects on the vascular endothelium and increases the bioavailability of nitric oxide. The purpose of this study was to assess the effect of ethanol on endothelium-dependent, nitric oxide-mediated vasodilation in healthy human subjects. Forearm blood flow (FBF) was determined by venous occlusion plethysmography in healthy human subjects during intra-arterial infusions of either methacholine (0.3, 1.0, 3.0, and 10.0 mcg/min, n = 9), nitroprusside (0.3, 1.0, 3.0, and 10.0 mcg/min, n = 9), or verapamil (10, 30, 100, and 300 mcg/min, n = 8) before and during the concomitant intra-arterial infusions of ethanol (10% ethanol in 5% dextrose). Additionally, a time control experiment was conducted, during which the methacholine dose-response curve was measured twice during vehicle infusions (n = 5). During ethanol infusion, mean forearm and systemic alcohol levels were 227 +/- 30 and 6 +/- 0 mg/dl, respectively. Ethanol infusion alone reduced FBF (2.5 +/- 0.1 to 1.9 +/- 0.1 ml.dl(-1).min(-1), P < 0.05). Despite initial vasoconstriction, ethanol augmented the FBF dose-response curves to methacholine, nitroprusside, and verapamil (P < 0.01 by ANOVA for each). To determine whether this augmented FBF response was related to shear-stress-induced release of nitric oxide, FBF was measured during the coinfusion of ethanol and N(G)-nitro-L-arginine (L-NAME; n = 8) at rest and during verapamil-induced vasodilation. The addition of L-NAME did not block the ability of ethanol to augment verapamil-induced vasodilation. Ethanol has complex direct vascular effects, which include basal vasoconstriction as well as potentiation of both endothelium-dependent and -independent vasodilation. None of these effects appear to be mediated by an increase in nitric oxide bioavailability, thus disputing findings from preclinical models.     

Prostanoids contribute to cutaneous active vasodilation in humans

The specific mechanisms by which skin blood flow increases in response to a rise in core body temperature via cutaneous active vasodilation are poorly understood. The primary purpose of this study was to determine whether the cyclooxygenase (COX) pathway contributes to active vasodilation during whole body heat stress (protocol 1; n = 9). A secondary goal was to verify that the COX pathway does not contribute to the cutaneous hyperemic response during local heating (protocol 2; n = 4). For both protocols, four microdialysis fibers were placed in forearm skin. Sites were randomly assigned and perfused with 1) Ringer solution (control site); 2) ketorolac (KETO), a COX-1/COX-2 pathway inhibitor; 3) NG-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor; and 4) a combination of KETO and L-NAME. During the first protocol, active vasodilation was induced using whole body heating with water-perfused suits. The second protocol used local heaters to induce a local hyperemic response. Red blood cell flux (RBC flux) was indexed at all sites using laser-Doppler flowmetry, and cutaneous vascular conductance (CVC; RBC flux/mean arterial pressure) was normalized to maximal vasodilation at each site. During whole body heating, CVC values at sites perfused with KETO (43 +/- 9% CVCmax), L-NAME (35 +/- 9% CVCmax), and combined KETO/L-NAME (22 +/- 8% CVCmax) were significantly decreased with respect to the control site (59 +/- 7% CVCmax) (P < 0.05). Additionally, CVC at the combined KETO/L-NAME site was significantly decreased compared with sites infused with KETO or L-NAME alone (P < 0.05). In the second protocol, the hyperemic response to local heating did not differ between the control site and KETO site or between the L-NAME and KETO/L-NAME site. These data suggest that prostanoids contribute to active vasodilation, but do not play a role during local thermal hyperemia.     

Effect of vascular delay on viability, vasculature, and perfusion of muscle flaps in the rabbit

The delay procedure is known to augment pedicled skin or muscle flap survival. In this study, we set out to investigate the effectiveness of vascular delay in two rabbit muscle flap models. In each of the muscle flap models, a delay procedure was carried out on one side of each rabbit (n = 20), and the contralateral muscle was the control. In the latissimus dorsi flap model, two perforators of the posterior intercostal vessels were ligated. In the biceps femoris flap model, a dominant vascular pedicle from the popliteal artery was ligated. After the 7-day delay period, the bilateral latissimus dorsi flaps (based on the thoracodorsal vessels) and the bilateral biceps femoris flaps (based on the sciatic vessels) were elevated. Animals were divided into three groups: part A, assessment of muscle flap viability at 7 days using the tetrazolium dye staining technique (n = 7); part B, assessment of vascular anatomy using lead oxide injection technique (n = 7); and part C, assessment of total and regional capillary blood flow using the radioactive microsphere technique (n = 6). The results in part A show that the average viable area of the latissimus dorsi flap was 96 +/- 0.4 percent (mean +/- SEM) in the delayed group and 84 +/- 0.7 percent (mean +/- SEM) in the control group (p < 0.05, n = 7), and the mean viable area of the biceps femoris flap was 95 +/- 2 percent in the delayed group and 78 +/- 5 percent in the control group (p < 0.05, n = 7). In part B, it was found that the line of necrosis in the latissimus dorsi flap usually appeared at the junction between the second and third vascular territory in the flap. Necrosis of the biceps femoris flap usually occurred in the third territory, and occasionally in both the second and the third territories. In Part C, total capillary blood flow in delayed flaps (both the latissimus dorsi and biceps femoris) was significantly higher than that in the control flaps (p < 0.05). Increased regional capillary blood flow was found in the middle and distal regions, compared with the control (p < 0.05, n = 6). In conclusion, ligation of either the dominant vascular pedicle in the biceps femoris muscle flap or the nondominant pedicle in the latissimus dorsi muscle flap in a delay procedure 1 week before flap elevation improves capillary blood flow and muscle viability. Vascular delay prevents distal flap necrosis in two rabbit muscle flap models.     

Dilation of rat diaphragmatic arterioles by flow and hypoxia: roles of nitric oxide and prostaglandins.

The in vitro responses to ACh, flow, and hypoxia were studied in arterioles isolated from the diaphragms of rats. The endothelium was removed in some vessels by low-pressure air perfusion. In endothelium-intact arterioles, pressurized to 70 mmHg in the absence of luminal flow, ACh (10(-5) M) elicited dilation (from 103 +/- 10 to 156 +/- 13 microm). The response to ACh was eliminated by endothelial ablation and by the nitric oxide synthase antagonists NG-nitro-L-arginine (L-NNA; 10(-5) M) and NG-nitro-L-arginine methyl ester (L-NAME, 10(-5) M) but not by indomethacin (10(-5) M). Increases in luminal flow (5-35 microl/min in 5 microl/min steps) at constant distending pressure (70 mmHg) elicited dilation (from 98 +/- 8 to 159 +/- 12 microm) in endothelium-intact arterioles. The response to flow was partially inhibited by L-NNA, L-NAME, and indomethacin and eliminated by endothelial ablation and by concurrent treatment with L-NAME and indomethacin. The response to hypoxia was determined by reducing the periarteriolar PO2 from 100 to 25-30 Torr by changing the composition of the gas used to bubble the superfusing solution. Hypoxia elicited dilation (from 110 +/- 9 to 165 +/- 12 microm) in endothelium-intact arterioles but not in arterioles from which the endothelium had been removed. Hypoxic vasodilation was eliminated by treatment with indomethacin and was not affected by L-NAME or L-NNA. In rat diaphragmatic arterioles, the response to ACh is dependent on endothelial nitric oxide release, whereas the response to hypoxia is mediated by endothelium-derived prostaglandins. Flow-dilation requires that both nitric oxide and cyclooxygenase pathways be intact.     

Evaluation of the mechanism of vascular endothelial growth factor improvement of ischemic flap survival in rats

This study evaluated the effects of exogenous vascular endothelial growth factor (VEGF) on the regulation of cytokines in a rat dorsal ischemic skin flap model. Exogenous VEGF (1 microg/ml) was injected subdermally into the flaps of 12 rats before the flaps were sutured back in place. Another 12 rats with flaps received saline injections, as a control group. Biopsy specimens were obtained from the flaps treated with VEGF or saline solution, at positions 2.5, 5.5, and 8.5 cm from the distal edge of the flaps, at 12 hours (n = 6 for each group) and 24 hours (n = 6 for each group) after suturing of the flaps. Expression of cytokine, growth factor, and inducible nitric oxide synthase was measured. The results demonstrated that expression of tumor necrosis factor-alpha and nitric oxide synthase in the distal part of the VEGF-treated flaps was significantly decreased, compared with the control values, at 12 and 24 hours postoperatively. It was concluded that administration of exogenous VEGF could protect flaps from ischemia-reperfusion injury through the regulation of proinflammatory cytokines and the inhibition of cytotoxic nitric oxide production.     

Role of nitric oxide in the vascular effects of local warming of the skin in humans.

Local warming of skin induces vasodilation by unknown mechanisms. To test whether nitric oxide (NO) is involved, we examined effects of NO synthase (NOS) inhibition with NG-nitro-L-arginine methyl ester (L-NAME) on vasodilation induced by local warming of skin in six subjects. Two adjacent sites on the forearm were instrumented with intradermal microdialysis probes for delivery of L-NAME and sodium nitroprusside. Skin blood flow was monitored by laser-Doppler flowmetry (LDF) at microdialysis sites. Local temperature (Tloc) of the skin at both sites was controlled with special LDF probe holders. Mean arterial pressure (MAP; Finapres) was measured and cutaneous vascular conductance calculated (CVC = LDF/MAP = mV/mmHg). Data collection began with a control period (Tloc at both sites = 34 degrees C). One site was then warmed to 41 degrees C while the second was maintained at 34 degrees C. Local warming increased CVC from 1.44 +/- 0.41 to 4.28 +/- 0.60 mV/mmHg (P < 0.05). Subsequent L-NAME administration reduced CVC to 2.28 +/- 0.47 mV/mmHg (P < 0.05 vs. heating), despite the continued elevation of Tloc. At a Tloc of 34 degrees C, L-NAME reduced CVC from 1.17 +/- 0.23 to 0.75 +/- 0.11 mV/mmHg (P < 0.05). Administration of sodium nitroprusside increased CVC to levels no different from those induced by local warming. Thus NOS inhibition attenuated, and sodium nitroprusside restored, the cutaneous vasodilation induced by elevation of Tloc; therefore, the mechanism of cutaneous vasodilation by local warming requires NOS generation of NO.     

Central nitric oxide modulates hindquarter vasodilation elicited by AMPA receptor stimulation in the NTS of conscious rats

Microinjection of S-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) in the nucleus of the solitary tract (NTS) of conscious rats causes hypertension, bradycardia, and vasoconstriction in the renal, mesenteric, and hindquarter vascular beds. In the hindquarter, the initial vasoconstriction is followed by vasodilation with AMPA doses >5 pmol/100 nl. To test the hypothesis that this vasodilation is caused by activation of a nitroxidergic pathway in the NTS, we examined the effect of pretreatment with the nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME, 10 nmol/100 nl, microinjected into the NTS) on changes in mean arterial pressure, heart rate, and regional vascular conductance (VC) induced by microinjection of AMPA (10 pmol/100 nl in the NTS) in conscious rats. AMPA increased hindquarter VC by 18 +/- 4%, but after pretreatment with L-NAME, AMPA reduced hindquarter VC by 16 +/- 7% and 17 +/- 9% (5 and 15 min after pretreatment, P < 0.05 compared with before pretreatment). Pretreatment with L-NAME reduced AMPA-induced bradycardia from 122 +/- 40 to 92 +/- 32 beats/min but did not alter the hypertension induced by AMPA (35 +/- 5 mmHg before pretreatment, 43 +/- 6 mmHg after pretreatment). Control injections with D-NAME did not affect resting values or the response to AMPA. The present study shows that stimulation of AMPA receptors in the NTS activates both vasodilatatory and vasoconstrictor mechanisms and that the vasodilatatory mechanism depends on production of nitric oxide in the NTS.     

Differing flow patterns between ischemically challenged flap skin and flap skeletal muscle: implications for salvage regimens.

In this study, the authors tested the hypothesis that there is a significant difference in spatial patterns of reflow in skin as opposed to skeletal muscle after an ischemic insult. The authors believe that this pathophysiologic difference between the two flap types has significant implications for flap salvage strategies. Bilateral buttock skin flaps (10 x 18 cm) and latissimus dorsi myocutaneous flaps (10 x 20 cm) were elevated in Landrace pigs (n = 7). Flaps on one side of the animal were randomly assigned to 6 hours of arterial occlusion, with the contralateral side acting as control. At 15 minutes, 1 hour, and 4 hours after reflow, radioactive microspheres (15 microm) were injected into the left ventricle. After 18 hours of reperfusion, skin and muscle viability were estimated by intravenous fluorescein and soaking in nitroblue tetrazolium, respectively. Flow rates in the skin with an ischemia-reperfusion injury were significantly reduced (30 to 53 percent), at all time intervals, compared with controls. The flow rate in the fluorescent skin with ischemia-reperfusion injury of the latissimus dorsi flaps (0.037 ml/min/g at 15 min) was greater than in that of the buttock flaps (0.018 ml/min/g). The muscle flaps with ischemia-reperfusion injury had significantly higher flow rates than control muscle flaps at all time intervals studied (at 1 hour, 0.32 ml/min/g compared with 0.16 ml/min/g, respectively). In flap skeletal muscle, an early hyperemic phase during reperfusion maintains a significant blood flow to all regions, including the area of the flap that is destined for necrosis. In flap skin, however, there is a marked decrease in flow rates. These differences have important implications for the intravascular delivery of therapeutic agents to the damaged portions of the flap. Transdermal drug delivery systems should be explored as an alternative to intravascular regimens for the salvage of flap skin with ischemia-reperfusion injury.     

Role of nitric oxide in methacholine-induced sweating and vasodilation in human skin.

The purpose of this study was to determine whether the nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) demonstrates significant muscarinic-receptor antagonism during methacholine (MCh)-stimulated sweating in human forearm skin. Three intradermal microdialysis probes were placed in the skin of eight healthy adults (4 men and 4 women). MCh in the range of 0.033-243 mM in nine steps was perfused through a microdialysis probe with and without the presence of the nitric oxide synthase inhibitor L-NAME (10 mM) or the L-arginine analog NG-monomethyl-L-arginine (L-NMMA; 10 mM). Local sweat rate (sweat rate) and skin blood flow (laser-Doppler velocimetry) were measured directly over each microdialysis probe. We observed similar resting sweat rates at MCh only, MCh and L-NAME, and MCh and L-NMMA sites averaging 0.175 +/- 0.029, 0.186 +/- 0.034, and 0.139 +/- 0.027 mg x min(-1) x cm(-2), respectively. Peak sweat rate (0.46 +/- 0.11, 0.56 +/- 0.16, and 0.53 +/- 0.16. mg x min(-1) x cm(-2)) was also similar among all three sites. MCh produced a sigmoid-shape dose-response curve and 50% of the maximal attainable response (0.42 +/- 0.14 mM for MCh only) was shifted rightward shift in the presence of L-NAME or L-NMMA (2.88 +/- 0.79 and 3.91 +/- 1.14 mM, respectively; P < 0.05). These results indicate that nitric oxide acts to augment MCh-stimulated sweat gland function in human skin. In addition, L-NAME consistently blunted the MCh-induced vasodilation, whereas L-NMMA did not. These data support the hypothesis that muscarinic-induced dilation in cutaneous blood vessels is not mediated by nitric oxide production and that the role of L-NAME in attenuating acetylcholine-induced vasodilation may be due to its potential to act as a muscarinic-receptor antagonist.     

Acetylcholine-induced vasodilation is mediated by nitric oxide and prostaglandins in human skin.

Acetylcholine (ACh) can effect vasodilation by several mechanisms, including activation of endothelial nitric oxide (NO) synthase and prostaglandin (PG) production. In human skin, exogenous ACh increases both skin blood flow (SkBF) and bioavailable NO levels, but the relative increase is much greater in SkBF than NO. This led us to speculate ACh may dilate cutaneous blood vessels through PGs, as well as NO. To test this hypothesis, we performed a study in 11 healthy people. We measured SkBF by laser-Doppler flowmetry (LDF) at four skin sites instrumented for intradermal microdialysis. One site was treated with ketorolac (Keto), a nonselective cyclooxygenase antagonist. A second site was treated with NG-nitro-L-arginine methyl ester (L-NAME) to inhibit NO synthase. A third site was treated with a combination of Keto and L-NAME. The fourth site was an untreated control site. After the three treated sites received the different inhibiting agents, ACh was administered to all four sites by intradermal microdialysis. Finally, sodium nitroprusside (SNP) was administered to all four sites. Mean arterial pressure (MAP) was monitored by Finapres, and cutaneous vascular conductance (CVC) was calculated (CVC = LDF/MAP). For data analysis, CVC values for each site were normalized to their respective maxima as effected by SNP. The results showed that both Keto and L-NAME each attenuated the vasodilation induced by exogenous ACh (ACh control = 79 +/- 4% maximal CVC, Keto = 55 +/- 7% maximal CVC, L-NAME = 46 +/- 6% maximal CVC; P < 0.05, ACh vs. Keto or L-NAME). The combination of the two agents produced an even greater attenuation of ACh-induced vasodilation (31 +/- 5% maximal CVC; P < 0.05 vs. all other sites). We conclude that a portion of the vasodilation effected by exogenous ACh in skin is due to NO; however, a significant portion is also mediated by PGs.     

Time course of flow-induced vasodilation in skeletal muscle: contributions of dilator and constrictor mechanisms

The purpose of this study was to determine the time course of flow-induced vasodilation in soleus and gastrocnemius muscle arterioles and the mechanisms that underlie vasodilatory responses to an increase in intraluminal flow. Vasodilation was assessed during 20 min of continuous exposure to intraluminal flow. Both soleus and gastrocnemius muscle arterioles dilated in response to flow, although the magnitude of vasodilation was greater in arterioles from the gastrocnemius muscle. Neither blockade of nitric oxide synthase with N(G)-nitro-L-arginine methyl ester (L-NAME) nor blockade of cyclooxygenase with indomethacin inhibited the initial vasodilation (0-2 min) in arterioles from either muscle. In contrast, vasodilation to sustained exposure to flow (2-20 min) was eliminated by treatment with L-NAME in arterioles from both muscles. Both depolarization with 40 mM KCl and blockade of Ca(2+)-activated K(+) channels inhibited the initial flow-induced dilation, and the inhibition was greater in gastrocnemius muscle arterioles than soleus muscle arterioles. In the presence of L-NAME, prolonged exposure to flow resulted in constriction in soleus and gastrocnemius muscle arterioles. This constriction was abolished by endothelin receptor blockade. These results indicate that the time course and magnitude of flow-induced vasodilation differs between arterioles from soleus and gastrocnemius muscles. The immediate response to increased flow is greater in gastrocnemius muscle arterioles and involves activation of K(+) channels. In arterioles from both soleus and gastrocnemius muscles, vasodilation to sustained flow exposure occurs primarily through production of nitric oxide. In the absence of nitric oxide, sustained exposure to flow results in pronounced constriction that is mediated by endothelin.     

Effects of sodium pentobarbital anesthesia on blood flow in skin, myocutaneous, and fasciocutaneous flaps in swine.

Drug effect on flap blood flow is most commonly determined in anesthetized animals, yet the effect of the anesthetic is often poorly understood. Halothane and nitrous oxide cause profound changes in skin blood flow and thus provide an unsuitable anesthetic technique for use in measuring drug effects on skin and myocutaneous flaps in swine. The goal of this study was to determine the effects of sodium pentobarbital anesthesia on cardiovascular parameters and blood flow in skin, myocutaneous, and fasciocutaneous flaps in pigs. In seven pigs, 7 forelimb skin flaps, 7 forelimb fasciocutaneous flaps, 14 arterial buttock flaps, and 14 latissimus dorsi flaps were created. Blood flow was measured at 2-cm intervals along each flap while the animal was awake and anesthetized. A cardiac depressant effect of pentobarbital was observed, but no change in blood flow could be demonstrated in control skin or control muscle. However, pentobarbital did significantly increase blood flow in all viable portions of arterial and random skin flaps, fasciocutaneous flaps, and the cutaneous segments of the latissimus dorsi flap. These demonstrated effects of pentobarbital should be taken into consideration in designing and analyzing studies of flap blood flow in the acute postoperative phase.     

Paradoxical hypotension following increased hematocrit and blood viscosity

Hematocrit (Hct) of awake hamsters and CD-1 mice was acutely increased by isovolemic exchange transfusion of packed red blood cells (RBCs) to assess the relation between Hct and blood pressure. Increasing Hct 7-13% of baseline decreased mean arterial blood pressure (MAP) by 13 mmHg. Increasing Hct above 19% reversed this trend and caused MAP to rise above baseline. This relationship is described by a parabolic function (R2 = 0.57 and P < 0.05). Hamsters pretreated with the nitric oxide (NO) synthase (NOS) inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) and endothelial NOS-deficient mice showed no change in MAP when Hct was increased by <19%. Nitrate/nitrite plasma levels of Hct-augmented hamsters increased relative to control and L-NAME treated animals. The blood pressure effect was stable 2 h after exchange transfusion. These findings suggest that increasing Hct increases blood viscosity, shear stress, and NO production, leading to vasodilation and mild hypotension. This was corroborated by measuring A1 arteriolar diameters (55.0 +/- 21.5 microm) and blood flow in the hamster window chamber preparation, which showed statistically significant increased vessel diameter (1.04 +/- 0.1 relative to baseline) and microcirculatory blood flow (1.39 +/- 0.68 relative to baseline) after exchange transfusion with packed RBCs. Larger increases of Hct (>19% of baseline) led blood viscosity to increase >50%, overwhelming the NO effect through a significant viscosity-dependent increase in vascular resistance, causing MAP to rise above baseline values.     

Improvement of survival of skin flaps by combined gene transfer of hepatocyte growth factor and prostacyclin synthase

BACKGROUND: Increasing the local blood flow is a critical factor for long-term survival of skin flaps. Thus, a molecular therapy to increase the blood flow by means of an angiogenic factor is considered to be a useful strategy to improve skin flap survival. We focused on a combined strategy to stimulate not only angiogenesis, but also vasodilation of local microvessels, using co-transfection of the hepatocyte growth factor (HGF) and prostacyclin synthase (PGIS) genes to enhance the survival of random-pattern skin flaps. METHODS AND RESULTS: A 2 x 8 cm full thickness cranial pedicled random-pattern flap was made on the back of each 12-week-old male rat. At 3 days before operation, 400 microg of human HGF and PGIS naked plasmid DNA or control plasmid was transfected into the flaps by needle-less injection using a Shima Jet, resulting in successful expression of human HGF and PGIS in the skin flaps. Transfection of both genes into the distal half of skin flaps at 3 days prior to operation significantly increased the survival rate of skin flaps, while transfection all over the flaps did not. In addition, transfection prior to operation was more effective than simultaneous treatment. Moreover, co-transfection of these genes improved the survival area of skin flaps, accompanied by an increase in blood flow of skin flaps, even in a diabetic model. CONCLUSIONS: Overall, these results indicate that combination treatment with HGF and PGIS genes by Shima Jet could be an effective strategy to improve skin flap survival.     

Effect of capsulectomy on the hemodynamics and viability of random-pattern skin flaps raised on expanded skin in the pig

Skin flaps constructed on expanded skin usually include the underlying capsular tissue. It has been hypothesized that capsulectomy may jeopardize the viability of the expanded skin flap. The experiments reported herein were designed to test this hypothesis. Specifically, we studied the hemodynamics and viability of random-pattern skin flaps (8 X 20 cm) raised on delayed bipedicle flaps (group A) and on expanded skin pockets with capsulectomy at the time of flap elevation (group B) or with intact underlying capsular tissue (group C). Each group was randomly assigned to each flank in 16 pigs. Skin pockets were expanded by inflation of subcutaneous silicone tissue expanders with sterile saline (299 +/- 7 ml; X +/- SEM) over a period of 3 weeks. At the end of this period, the bipedicle flaps were constructed. Eight days later, random-pattern skin flaps were raised on bipedicle flaps and skin pockets. The length and area of skin flap viability, judged by the fluorescein dye test performed 1 day postoperatively, were not significantly different (p greater than 0.05) among groups A, B, and C (n = 31 to 32). There also were no significant differences (p greater than 0.05) in total skin capillary blood flow measured 1 day postoperatively (A = 2.6 +/- 0.4, B = 2.4 +/- 0.4, and C = 2.7 +/- 0.6 ml/min per flap; n = 15 to 16) and in skin viability assessed 7 days postoperatively (A = 74 +/- 2, B = 75 +/- 2, and C = 76 +/- 2 percent; n = 16) among delayed skin flaps and skin flaps raised on expanded skin pockets with or without capsulectomy. The results of this flap viability study were confirmed in 5 minipigs in a separate experiment. We conclude that capsulectomy did not have a detrimental effect on the hemodynamics and viability of random-pattern skin flaps raised on expanded skin. Furthermore, we hypothesize that skin flaps raised on expanded skin are similar to delayed skin flaps in that the skin blood flow is optimally augmented; therefore, the capsular tissue does not add significant blood supply to the overlying skin.     

Local heat produces a shear-mediated biphasic response in the thermoregulatory microcirculation of the Pallid bat wing

Investigators report that local heat causes an increase in skin blood flow consisting of two phases. The first is solely sensory neural, and the second is nitric oxide mediated. We hypothesize that mechanisms behind these two phases are causally linked by shear stress. Because microvascular blood flow, endothelial shear stress, and vessel diameters cannot be measured in humans, bat wing arterioles (26.6 +/- 0.3, 42.0 +/- 0.4, and 58.7 +/- 2.2 microm) were visualized noninvasively on a transparent heat plate via intravital microscopy. Increasing plate temperature from 25 to 37 degrees C increased flow in all three arterial sizes (137.1 +/- 0.3, 251.9 +/- 0.5, and 184.3 +/- 0.6%) in a biphasic manner. With heat, diameter increased in large arterioles (n = 6) by 8.7 +/- 0.03% within 6 min, medium arterioles (n = 8) by 19.7 +/- 0.5% within 4 min, and small arterioles (n = 8) by 31.6 +/- 2.2% in the first minute. Lidocaine (0.2 ml, 2% wt/vol) and NG-nitro-L-arginine methyl ester (0.2 ml, 1% wt/vol) were applied topically to arterioles (approximately 40 microm) to block sensory nerves, modulate shear stress, and block nitric oxide generation. Local heat caused only a 10.4 +/- 5.5% increase in diameter with neural blockade (n = 8) and only a 7.5 +/- 4.1% increase in diameter when flow was reduced (n = 8), both significantly lower than control (P < 0.001). Diameter and flow increases were significantly reduced with NG-nitro-L-arginine methyl ester application (P < 0.05). Our novel thermoregulatory animal model illustrates 1) regulation of shear stress, 2) a nonneural component of the first phase, and 3) a shear-mediated second phase. The time course of dilation suggests that early dilation of small arterioles increases flow and enhances second-phase dilation of the large arterioles.     

Effects of exercise training on the vascular reactivity of the whole kidney circulation in rabbits.

Exercise training is known to improve vasodilating mechanisms mediated by endothelium-dependent relaxing factors in the cardiac and skeletal muscle vascular beds. However, the effects of exercise training on visceral vascular reactivity, including the renal circulation, are still unclear. We used the experimental model of the isolated perfused rabbit kidney, which involves both the renal macro- and microcirculation, to test the hypothesis that exercise training improves vasodilator mechanisms in the entire renal circulation. New Zealand White rabbits were pen confined (Sed; n = 24) or treadmill trained (0% grade) for 5 days/wk at a speed of 18 m/min during 60 min over a 12-wk period (ExT; n = 24). Kidneys isolated from Sed and ExT rabbits were continuously perfused in a nonrecirculating system under conditions of constant flow and precontracted with norepinephrine (NE). We assessed the effects of exercise training on renal vascular reactivity using endothelial-dependent [acetylcholine (ACh) and bradykinin (BK)] and -independent [sodium nitroprusside (SNP)] vasodilators. ACh induced marked and dose-related vasodilator responses in kidneys from Sed rabbits, the reduction in perfusion pressure reaching 41 +/- 8% (n = 6; P < 0.05). In the kidneys from ExT rabbits, vasodilation induced by ACh was significantly enhanced to 54 +/- 6% (n = 6; P < 0.05). In contrast, BK-induced renal vasodilation was not enhanced by training [19 +/- 8 and 13 +/- 4% reduction in perfusion pressure for Sed and ExT rabbits, respectively (n = 6; P > 0.05)]. Continuous perfusion of isolated kidneys from ExT animals with N(omega)-nitro-L-arginine methyl ester (L-NAME; 300 microM), an inhibitor of nitric oxide (NO) biosynthesis, completely blunted the additional vasodilation elicited by ACh [reduction in perfusion pressure of 54 +/- 6 and 38 +/- 5% for ExT and L-NAME + ExT, respectively (n = 6; P < 0.05)]. On the other hand, L-NAME infusion did not affect ACh-induced vasodilation in Sed animals. Exercise training also increased renal vasodilation induced by SNP [36 +/- 7 and 45 +/- 10% reduction in perfusion pressure for Sed and ExT rabbits, respectively (n = 6; P < 0.05)]. It is concluded that exercise training alters the rabbit kidney vascular reactivity, enhancing endothelium-dependent and -independent renal vasodilation. This effect seems to be related not only to an increased bioavailability of NO but also to the enhanced responsiveness of the renal vascular smooth muscle to NO.