Changes In Arteriolar Diameter Research Articles

Leukocyte adherence inhibits adenosine-dependent venular control of arteriolar diameter and nitric oxide

Venular control of arteriolar perfusion has been the focus of several investigations in recent years. This study investigated 1) whether endogenous adenosine helps control venule-dependent arteriolar dilation and 2) whether venular leukocyte adherence limits this response via an oxidant-dependent mechanism in which nitric oxide (NO) levels are decreased. Intravital microscopy was used to assess changes in arteriolar diameters and NO levels in rat mesentery. The average resting diameter of arterioles (27.5 +/- 1.0 microm) paired with venules with minimal leukocyte adherence (2.1 +/- 0.3 per 100-microm length) was significantly larger than that of unpaired arterioles (24.5 +/- 0.8 microm) and arterioles (23.3 +/- 1.3 microm) paired with venules with higher leukocyte adherence (9.0 +/- 0.5 per 100-microm length). Local superfusion of adenosine deaminase (ADA) induced significant decreases in diameter and perivascular NO concentration in arterioles closely paired to venules with minimal leukocyte adherence. However, ADA had little effect on arterioles closely paired to venules with high leukocyte adherence or on unpaired arterioles. To determine whether the attenuated response to ADA for the high-adherence group was oxidant dependent, the responses were also observed in arterioles treated with 10(-4) M Tempol. In the high-adherence group, Tempol fully restored NO levels to those of the low-adherence group; however, the ADA-induced constriction remained attenuated, suggesting a possible role for an oxidant-independent vasoconstrictor released from the inflamed venules. These findings suggest that adenosine- and venule-dependent dilation of paired arterioles may be mediated, in part, by NO and inhibited by venular leukocyte adherence.

Differential roles for potassium and acetylcholine in producing the dilations resulting from a single tetanic skeletal muscle contraction

The rapid onset of the dilation within 2 seconds of a single tetanic contraction suggests that the dilators involved may be products of muscle activation (e.g. acetylcholine (ACh) and potassium (K+)). To investigate the role of ACh and K+ in these rapid dilations we stimulated 4–5 skeletal muscle fibres in the anaesthetized hamster cremaster preparation in situ and measured the change in diameter of arterioles at the site of overlap with the stimulated muscle fibres before and after a single contraction (250ms train duration) over a range of stimulus frequencies (4, 10, 20, 30, 40 and 60Hz). Muscle fibres were stimulated in the absence and presence of a muscarinic antagonist atropine (10−5M), Na+K+ATPase inhibitor ouabain (10−4M), KIR channel inhibitor barium chloride (5x10−5M), or Kv channel inhibitor 3,4-diaminopyridine (10−3M). We observed significant inhibitions of the rapid dilation at all stimulus frequencies with inhibitors of Na+K+ATPase, KIR and Kv channels; across all stimulus frequencies, we observed an average decrease in dilation of 40.5+/−3.9%, 49.8+/−4.4% and 43.0+/−4.7% respectively. Atropine did not inhibit the rapid dilation (average decrease of 7.1+/−5.9%) but did attenuate a secondary dilation that occurs within 20 seconds of a single contraction at specific stimulus frequencies (20, 30 and 40Hz) as well as the dilation 10 seconds following a 60Hz contraction. Thus K+ appears to be involved in the rapid dilation in response to a single muscle contraction at all stimulus frequencies while the contribution of ACh is delayed and is stimulus frequency dependent. This work was supported by NSERC.

Comparison of hypercapnia‐ and acetazolamide‐induced cerebrovascular responses in piglets

Acetazolamide (AZD) produces cerebral vasodilation. The underlying mechanism is unclear, but it is assumed to be largely due to CO2 retention and acidosis. We tested if cerebrovascular effects of AZD were similar to hypercapnia in the newborn pig. We simultaneously used the closed cranial window/intravital microscopy technique to determine pial arteriolar diameters and laser-Doppler flowmetry (LDF). Anesthetized (Na-thiopenthal+α-chloralose), ventilated, 1-day old instrumented piglets (n=26) were divided into 4 experimental groups: time control (n=8), indomethacin or ibuprofen treatments (1 or 30 mg/kg, iv, n=6-6, respectively), and global ischemia/reperfusion (I/R, 10 min induced by elevated intracranial pressure, n=6). Responses to 5–10 % inhaled CO2 were recorded before and after the treatments, and then to AZD (10–20 mg/kg, iv). Consistent with previous studies, hypercapnia produced dose-dependent pial arteriolar vasodilation and similar changes in LDF data that was abolished by indomethacin, unaltered by ibuprofen and significantly attenuated after I/R. AZD elicited similar pial vasodilation (33±7%, change from baseline, 10 mg/kg) and increase in the LDF signal (31±9%). AZD-induced vasodilation was also sensitive to indomethacin and I/R but was unaltered by ibuprofen. Thus, the mechanism of AZD-induced vasodilation appears to be similar to hypercapnia, and pial arteriolar diameter changes reflect changes in cortical perfusion. Supported by OTKA F 043101.

Microvascular Diameter Responses to the Controlled Release of Platelet Derived Growth Factor‐BB (PDGF‐BB) and Transforming Growth Factor‐β1 (TGF‐β1)

PDGF-BB, a smooth muscle cell (SMC) chemoattractant and mitogen, and TGF-β1, a SMC differentiation factor, may have utility as therapeutic arteriogenesis factors. However, few studies have examined whether the controlled release of PDGF-BB or TGF-β1 can amplify arteriogenesis. To this end, we utilized the mouse dorsal skinfold window chamber model, which allows for repeated vessel measurements over time and naturally exhibits arteriolar and venular structural lumen expansion. An 85:15 poly(DL-lactide-co-glycolide) microsphere containing either 0.2μg/mg PDGF-BB, 0.008μg/mg TGF-β1 or 4μg/mg BSA was inserted on Day 7. Microvascular diameter measurements were made at Days 7, 10 and 14 on individual vessels within the release profile of the microsphere (4mm) during topical superfusion of adenosine (10−4 M). Interestingly, PDGF-BB treatment elicited no change in arteriolar structural diameter [PDGF-BB: =25.0μm (48.0%) vs. BSA: =23.1μm (44.2%)], but a significant increase in venular structural diameter [PDGF-BB: =48.9μm (61.9%) vs. BSA: =14.2μm (18.2%)]. TGF-β1 treatment significantly attenuated normal arteriolar [TGFβ1: =7.8μm (17.1%) vs. BSA: =23.1μm (44.2%)] and venular [TGF-β1: =−14.4μm (−1.2%) vs. BSA: =14.2μm (18.2%)] structural lumen expansion. Because PDGF-BB elicited no further increase in arteriolar diameter and TGF-β1 caused vessel constriction, our results do not support the therapeutic potential of these factors. However, the mechanisms of these diameter responses remain unknown, and alterations in growth factor concentration and/or release kinetics may be needed to show efficacy. Supported by NIH HL65958.

Perturbation of intraparenchymal arterioles during K+-induced cortical spreading depression in cats

During K+-induced cortical spreading depression (CSD) in the rat cerebral cortex, we previously observed repetitive wave-ring spread of oligemia/hypoperfusion followed by hyperemia/hyperperfusion. The oligemia/hypoperfusion occurred before upstream feeding arteriolar changes, suggesting that the capillary changes of the oligemia/hypoperfusion was of neuronal origin (Tomita M et al (2005) J CBF & M, in press). In this communication, we examined intraparenchymal arteriolar changes in the early phase of tissue oligemia. Six cats were used. Under -chloralose-urethan anesthesia, a skull window was opened in the parieto-temporal region. The dura was removed and the cerebral cortex was exposed. An optical fiber of 150 m in diameter was inserted into the brain tissue obliquely from 3 mm behind the skull window, so that the light source was positioned at the center of the ROI 1 mm below the pia. Intraparenchymal 20–40 m arterioles were both trans-illuminated in silhouette from below and epi-illuminated from above. They were monitored with a Canon video camera, and images were taken into a computer through a Scion frame grabber card (LG-3). When K+ was microinjected into the cortex at a site near the ROI, oligemic wave-ring spread was reproduced as reported previously (Tomita Y et al (2002) Neurosci Lett:322,157). We found that capillary flow in the oligemic area became very slow, with clustered RBCs passing through small vessels like ants marching. During oligemia/hypoperfusion, we noticed in all cases segmental constriction/dilatation of arteriole(s), forming a sausage-string shape (Fig. 1 and Fig. 2a) which had never been seen in other series of experiments. Chronological changes of arteriolar diameter were seen; for example, in 2 cases, a part of the arteriole suddenly contracted by 50% for about 1 to 1.5 sec and then dilated to 250% at 10 sec and remained dilated for more than 1 min (Fig. 2b). In the remaining cases, the time courses of constriction/dilatation of arteriole(s) were rather slow. In one case, the vascular dilatation occurred at a branching site and moved peripherally along the arteriole, like the movement of an egg swallowed by a snake. Despite these events, subtraction pictures revealed that the majority of the epi-cortical pial arteries did not change their diameter in this early phase. Based on these observations, we speculate that arterioles were perturbed by discrepant controls of high K+ concentration in the extracellular fluid, unknown feedback signals from oligemic tissue, and autonomic ganglionic regulation.

Metformin treatment restores the altered microvascular reactivity in neonatal streptozotocin-induced diabetic rats increasing NOS activity, but not NOS expression

Abnormalities in vascular function are well recognized in diabetes. Hyperglycemia may be central to the pathogenesis of vascular dysfunction but is not certain whether improvements in glycaemic control will improve vascular function. The effects of metformin, an antidiabetic agent that improves insulin sensitivity and glycaemic control, on the microvascular reactivity have not been reported in neonatal streptozotocin-induced (n-STZ) diabetes. Diabetes was induced by STZ injection (160 mg/kg, ip) in neonates (2-day-old) Wistar rats. n-STZ diabetic rats were treated with metformin (300 mg/kg, 15 d, by gavage). Using intravital microscopy the changes in mesenteric arteriolar and venular diameters were determined in anesthetized control and n-STZ diabetic rats, before and after topical application of endothelium-dependent vasodilator agents, mediators or not of the inflammatory response, and endothelium-independent vasodilator agent. We also determined the total nitric oxide synthase (NOS) activity (conversion of L-arginine to citrulline) and endothelial(e), inducible(i), and neuronal(n) NOS expression (using polymerase chain reaction after reverse transcription of the mRNAs into cDNAs) in the mesentery of metformin-treated n-STZ diabetic and vehicle-treated n-STZ diabetic and control rats. Although metformin treatment did not correct the high glycaemic levels and the impaired glucose tolerance, the reduced vasodilator responses and total NOS activity in n-STZ diabetic rats were corrected by the treatment. Neither diabetes nor metformin treatment altered the expression of the three NOS isoforms. We concluded that metformin restores the reduced response to vasodilator agents, independently of the correction of the metabolic alterations. Improvement of total NOS activity might be in part responsible for the correction.

Selective Inhibitors Differentially Affect Cyclooxygenase-Dependent Pial Arteriolar Responses in Newborn Pigs

Cyclooxygenase (COX)-derived prostanoids play an important role in the cerebrovascular control of newborns. In humans and in the widely accepted model of piglets, both the COX-1 and the COX-2 isoforms are expressed in cerebral arteries. However, the involvement of these isoforms in cerebrovascular control is unknown. Therefore we tested if specific inhibitors of COX-1 and/or COX-2 would differentially affect pial arteriolar responses to COX-dependent stimuli in piglets. Anesthetized, ventilated piglets (n = 35) were equipped with a closed cranial window, and changes in pial arteriolar diameters (baseline approximately 100 microm) to hypercapnia (ventilation with 5-10% CO(2), 21% O(2), balance N(2)), arterial hypotension (40 mm Hg MABP achieved by blood withdrawal), and Ach (Ach, 10-100 microM) were determined via intravital microscopy. Arteriolar responses were repeatedly tested 15 min after IV administration of selective COX-1 and COX-2 inhibitors SC-560 and NS-398 (1-1 mg/kg), and nonselective inhibitors indomethacin (0.3-1 mg/kg), acetaminophen (30 mg/kg), and ibuprofen (30 mg/kg). Hypercapnia resulted in concentration-dependent, reversible, (approximately 20-40%) increases in pial arteriolar diameters that were unaffected by NS-398, SC-560, acetaminophen and ibuprofen. In contrast, 0.3 mg/kg indomethacin significantly reduced, 1 mg/kg virtually abolished the vasodilation. Arterial hypotension elicited (approximately 15-20%) vasodilation that was similarly reduced by NS-398 and indomethacin but was unaltered by SC-560. Ach dose-dependently constricted pial arterioles. This response was similarly attenuated by NS-398, indomethacin, and ibuprofen, but left intact by SC-560. We conclude that the assessed COX-dependent vascular reactions appear to depend largely on COX-2 activity. However, hypercapnia-induced vasodilation was found indomethacin-sensitive instead of a COX-dependent response in the piglet.

Intrathecal Neostigmine Prevents Intrathecal Clonidine from Attenuating Hypercapnic Cerebral Vasodilation in Rabbits

We previously demonstrated that lumbar intrathecal alpha(2) agonists attenuate hypercapnia-induced cerebral vasodilation. The combination of intrathecal clonidine and neostigmine is being investigated as pain therapy. The effects of their combination on cerebrovascular reactivity are unknown. We allocated rabbits anesthetized with pentobarbital to two groups: (a) clonidine (normal saline followed 30 min later by clonidine 2 microg/kg, both into the lumbar intrathecal space; n = 6), and (b) neostigmine-pretreatment (neostigmine 2 microg/kg followed 30 min later by clonidine 2 microg/kg, both into the lumbar intrathecal space; n = 6). We then evaluated the hypercapnia-induced changes in pial arteriolar diameter in these two groups using the closed cranial window preparation. The pial arteriolar dilator response to hypercapnia was significantly attenuated in the clonidine group (14% +/- 4%, 4% +/- 4%, 6% +/- 6%, and 5% +/- 7% for before and 30, 60, and 90 min, respectively). Neither normal saline nor neostigmine alone induced any change in the cerebral reactivity to hypercapnia. Pretreatment with neostigmine completely prevented the clonidine-induced attenuation of the hypercapnic cerebral vasodilation attenuated by intrathecal clonidine (16% +/- 7%, 15% +/- 6%, 12% +/- 6%, and 16% +/- 8%, respectively).

Influence of insulin on the microvascular response to inflammatory mediators in neonatal streptozotocin diabetic rats

To investigate the effect of insulin on microvascular responses to inflammatory mediators in a model of type 2 diabetes mellitus. We used the neonatal streptozotocin (n-STZ)-induced diabetes model. Diabetes was induced in male newborn (2-day-old) Wistar rats through STZ administration. Experiments were performed 10-12 weeks later. Rats were divided into control (sham-injected) and study (n-STZ) groups. Using a closed-circuit video camera coupled to a microscope, changes in mesenteric arteriolar and venular diameters induced by topical application of the inflammatory mediators histamine, bradykinin and platelet-activating factor were assessed in chloral hydrate-anesthetized rats. The n-STZ rats received NPH insulin s.c. for either 4 h or 12 days. Impaired arteriole and venule responses to the inflammatory mediators tested were observed in n-STZ rats. Both acute and chronic insulin treatment corrected the alterations. We conclude that insulin is beneficial, restoring microvascular reactivity to inflammatory mediators in type 2 diabetes.

Retinal Artery and Vein Diameters during Pregnancy in Diabetic Women

To study systemic and retinal circulatory variables and vasoactive hormones during pregnancy in women with and without diabetes. Prospective study of 45 women with type 1 diabetes and 6 healthy pregnant women, from the first trimester of pregnancy to half a year after delivery, by using a fundus photographic method of determining retinal vessel diameters. In diabetic women, the mean arterial blood pressure increased from 90.7 +/- 10.9 mm Hg (mean +/- SD) in the first trimester to 102.4 +/- 16.4 mm Hg in the third trimester, accompanied by retinal arteriolar constriction, from a mean diameter of 95.5 +/- 11.3 to 92.2 +/- 12.9 microm (P = 0.007), arteriolar constriction being prominent in nonsmokers, from 96.6 +/- 11.1 to 92.3 +/- 13.0 microm (P < 0.001; n = 38), but absent in smokers, from 89.2 +/- 11.5 to 92.0 +/- 13.6 microm (P = 0.28). Healthy nonsmoking women demonstrated an increase in blood pressure during pregnancy comparable to that in diabetic women, but the change in mean retinal arteriolar diameter, from 88.5 +/- 10.9 microm in the first to 91.6 +/- 10.2 microm in the third trimester, did not reach statistical significance (P = 0.38). Diabetic retinopathy levels increased during pregnancy, but the change in arteriolar diameter from the first to the third trimester did not correlate with retinopathy, arterial blood pressure, HbA(1c), or atrial natriuretic peptide. Plasma angiotensin II correlated positively with the change in arteriolar diameter in women who did not smoke (P < 0.05). After delivery, retinal vessel diameters returned to the first trimester range in all subgroups. From the first to the third trimester of pregnancy, blood pressure increased and retinal arteriolar diameter decreased in diabetic women, but the arteriolar constriction associated with pregnancy and systemic arterial blood pressure increase was remarkably absent in diabetic women who smoked tobacco before and during pregnancy. It is unknown whether smoking inhibits this vasoconstrictive response by deactivating mechanisms of physiological adaptation or by activating these mechanisms before the first trimester of pregnancy.

Effects of extreme hemodilution with hemoglobin-based O2 carriers on microvascular pressure

A surface-modified polyethylene glycol-conjugated human hemoglobin (MP4) and alpha alpha-cross-linked human hemoglobin (alpha alpha Hb) were used to restore oxygen carrying capacity in conditions of extreme hemodilution (hematocrit 11%) in the hamster window model preparation. Changes in microvascular function were analyzed in terms of effects on capillary pressure and functional capillary density (FCD). MP4, at 1.0 +/- 0.2 g/dl blood concentration, significantly lowered mean arterial pressure (MAP) below baseline (99.6 +/- 7.6 mmHg) to 82.4 +/- 6.9 mmHg (P < 0.05) and decreased of FCD to 70 +/- 9%. alpha alpha Hb caused a greater recovery in MAP to 94.4 +/- 6.2 mmHg and lowered FCD to 62 +/- 8%. However, differences between alpha alpha Hb and MP4 in FCD were not statistically significant. Capillary pressures were in the ranges of 17-21 mmHg for MP4 and 15-19 mmHg for alpha alpha Hb, with both significantly lower than baseline (P < 0.05). Pressure in 80-microm-diameter arterioles was significantly increased with alpha alpha Hb relative to MP4 (P < 0.05). These results were compared with previous findings on the relation between capillary pressure and FCD; they supported the concept of a relationship between FCD and capillary pressure. Measurement of changes in arteriolar diameter, microvascular blood flow, and FCD show that there was no statistical difference between using alpha alpha Hb and MP4 in extreme hemodilution. Microvascular resistance in arterioles with a diameter range of 70-80 microm showed an increase relative to control with alpha alpha Hb, whereas MP4 caused a decrease.

Chronic prenatal exposure to cocaine alters cerebrovascular responses in newborn pigs.

Maternal cocaine abuse may increase the incidence of perinatal asphyxia. In nonexposed asphyxiated neonates, decreased cerebrospinal fluid (CSF) cAMP concentrations are associated with poor neurological outcome. On the other hand, cocaine increases central nervous system (CNS) cAMP. Therefore, we hypothesized that in utero cocaine exposure may increase brain cAMP and thereby preserve cerebrovascular responses to cAMP-dependent stimuli following asphyxia. Pregnant pigs received either cocaine (1 mg/kg, i.v.) twice weekly during the last trimester or normal saline vehicle (sham-control) and were allowed to deliver vaginally at term. Cranial windows were implanted in the newborn pigs within the first week of life and used to collect CSF for cAMP determinations and to assess changes in pial arteriolar diameters (PAD). In the first part of the study, pial arteriolar responses to different vasodilator and vasoconstrictor stimuli were evaluated in piglets prior to asphyxia (n = 20). In newborn pigs exposed to cocaine, cerebrovascular responses to hypercapnia and norepinephrine were significantly exaggerated compared to controls. Then, piglets were randomly selected for the second part of the study that involved prolonged asphyxia (n = 12). In cocaine-exposed but not sham-control piglets, CSF cAMP increased markedly during asphyxia. In the sham piglets, but not the cocaine-exposed piglets, CSF cAMP fell progressively below the baseline during recovery. Cerebrovascular reactivity to cAMP-dependent stimuli (hypercapnia and isoproterenol) was preserved during recovery from asphyxia in the cocaine-exposed piglets but significantly attenuated in the sham controls. We conclude that piglets with chronic prenatal exposure to cocaine show exaggerated cerebrovascular responses to vasogenic stimuli and preserved cAMP-dependent cerebral vasoreactivity following asphyxia.

CAMP modulates cGMP-mediated cerebral arteriolar relaxation in vivo.

No studies have specifically addressed whether cAMP can influence nitric oxide (NO)/cGMP-induced cerebral vasodilation. In this study, we examined whether cAMP can enhance or reduce NO-induced cerebral vasodilation in vivo via interfering with cGMP efflux or through potentiating phosphodiesterase 5 (PDE5)-mediated cGMP breakdown, respectively, in cerebral vascular smooth muscle cells (CVSMCs). To that end, we evaluated, in male rats, the effects of knockdown [via antisense oligodeoxynucleotide (ODN) applications] of the cGMP efflux protein multidrug resistance protein 5 (MRP5) and PDE5 inhibition on pial arteriolar NO donor [S-nitroso-N-acetyl penicillamine (SNAP)]-induced dilations in the absence and presence of cAMP elevations via forskolin. Pial arteriolar diameter changes were measured using well-established protocols in anesthetized rats. In control (missense ODN treated) rats, forskolin elicited a leftward shift in the SNAP dose-response curves (approximately 50% reduction in SNAP EC50). However, in MRP5 knockdown rats, cAMP increases were associated with a substantial reduction in SNAP-induced vasodilations (reflected as a significant 35-50% lower maximal response). In the presence of the PDE5 inhibitor MY-5445, the repression of the NO donor response accompanying forskolin was prevented. These findings suggest that cAMP has opposing effects on NO-stimulated cGMP increases. On the one hand, cAMP limits CVSMC cGMP loss by restricting cGMP efflux. On the other, cAMP appears to enhance PDE5-mediated cGMP breakdown. However, because increased endogenous cAMP seems to potentiate NO/cGMP-induced arteriolar relaxation when MRP5 expression is normal, the effect of cAMP to reduce cGMP efflux appears to predominate over cAMP stimulation of cGMP hydrolysis.

Vascular endothelial growth factor stimulates differential signaling pathways in in vivo microcirculation.

Vascular endothelial growth factor (VEGF) induces mild vasodilation and strong increases in microvascular permeability. Using intravital microscopy and digital integrated optical intensity image analysis, we tested, in the hamster cheek pouch microcirculation, the hypothesis that differential signaling pathways in arterioles and venules represent an in vivo regulatory mechanism in the control of vascular diameter and permeability. The experimental design involved blocking specific signaling molecules and simultaneously assessing VEGF-induced changes in arteriolar diameter and microvascular transport of FITC-Dextran 150. Inhibition of Akt [indirectly via phosphatidylinositol 3-kinase with LY-294002 or wortmannin] or PKC (with bisindolylmaleimide) reduced VEGF-induced hyperpermeability. However, phosphatidylinositol 3-kinase/Akt inhibition enhanced the early phase and attenuated the late phase of VEGF-induced vasodilation, whereas blocking PKC had no effect. Inhibition of extracellular signal-regulated kinase (ERK)-1/2 (with PD-98059 or AG-126) also reduced VEGF-induced hyperpermeability but did not block VEGF-induced vasodilation. Blockade of endothelial nitric oxide synthase (with N(omega)-monomethyl-l-arginine) inhibited VEGF-induced changes in both permeability and diameter. Furthermore, immunofluorescence studies with human umbilical vein endothelial cells revealed that bisindolylmaleimide, PD-98059, and l-NMMA attenuate VEGF-induced reorganization of vascular endothelial cadherin. Our data demonstrate that 1) endothelial nitric oxide synthase is a common convergence pathway for VEGF-induced changes in arteriolar diameter and microvascular permeability; 2) PKC and ERK-1/2 do not play a major role in VEGF-induced vasodilation in the hamster cheek pouch microcirculation; and 3) Akt, PKC, and ERK-1/2 are elements of the signaling cascade that regulates VEGF-stimulated microvascular hyperpermeability. Our data provide evidence for differential signaling as a regulatory step in VEGF-stimulated microvascular dynamics.

Microcirculatory effects of pulsed electromagnetic fields

Purpose: Pulsed electromagnetic fields (PEMF) are used clinically to expedite healing of fracture non-unions, however, the mechanism of action by which PEMF stimulation is effective is unknown. The current study examined the acute effects of PEMF stimulation on arteriolar microvessel diameters in the rat cremaster muscle. The study hypothesis was that PEMF would increase arteriolar diameters, a potential mechanism involved in the healing process. Methods: Local PEMF stimulation/sham stimulation of 2 or 60 min duration was delivered to the cremaster muscle of anesthetized rats. Arteriolar diameters were measured before and after stimulation/sham stimulation using intravital microscopy. Systemic hemodynamics also were monitored during PEMF stimulation. Results: Local PEMF stimulation produced significant ( p<0.001) vasodilation, compared to pre-stimulation values, in cremasteric arterioles in anesthetized rats ( n=24). This dilation occurred after 2 min of stimulation (9% diameter increase) and after 1 h of stimulation (8.7% diameter increase). Rats receiving “sham” stimulation ( n=15) demonstrated no statistically significant change in arteriolar diameter following either “sham” stimulation period. PEMF stimulation of the cremaster ( n=4 rats) did not affect systemic arterial pressure or heart rate, nor was it associated with a change in tissue environmental temperature. Conclusions: These results support the hypothesis that local application of a specific PEMF waveform can elicit significant arteriolar vasodilation. Systemic hemodynamics and environmental temperature could not account for the observed microvascular responses.

Myogenic responses of isolated adipose tissue arterioles

Previous in vivo studies indicate that vascular autoregulation does take place in adipose tissue. We tested the hypothesis that adipose tissue arterioles can develop a myogenic response to increases in transmural pressure. Arterioles, isolated from the inguinal fat pad of male Wistar rats, were placed in a microvessel chamber containing a Kreb’s bicarbonate-buffered solution (pH 7.4) gassed with 10% O 2 (5% CO 2; 85% N 2). Vessels were cannulated and pressurized to 100 mm Hg and studied under no-flow conditions. Control diameters were obtained at 100 mm Hg. Changes in arteriolar diameter were observed and measured by television microscopy and videocaliper. Diameters, in response to 20 mm Hg step increases in transmural pressure, were measured before and after removal either of extracellular calcium or of the endothelium, and administration of indomethacin (10 −5 M) or L-NAME (3 × 10 −4 M). Removal of calcium resulted in an increase in control diameter of 81% and completely eliminated the myogenic response. In contrast, administration of indomethacin increased control diameter by 13%. L-NAME significantly enhanced the myogenic response; however, neither endothelium removal nor indomethacin had any significant effect. These results indicate that adipose tissue arterioles are capable of eliciting a myogenic response that could contribute to the regulation of blood flow in vivo. Furthermore, it appears that calcium is essential for the myogenic response and that nitric oxide significantly contributes to the modulation of baseline myogenic tone, as well as the myogenic response.

ADP-induced pial arteriolar dilation in ovariectomized rats involves gap junctional communication.

It was previously shown that, despite the loss of nitric oxide (NO) dependence, ADP-induced pial arteriolar dilation was not attenuated in estrogen-depleted [i.e., ovariectomized (Ovx)] rats. Additional evidence suggested that the NO was replaced by an endothelium-dependent hyperpolarizing factor (EDHF)-like mechanism. To further characterize the nascent EDHF role in Ovx females, the current study was undertaken to test whether, in Ovx rats, ADP-induced pial arteriolar dilation retained its endothelial dependence and whether gap junctions are involved in that response. A closed cranial window and intravital microscopy system was used to monitor pial arteriolar diameter changes in anesthetized rats. The endothelial portion of the ADP-induced dilation was evaluated using light dye endothelial injury (L/D). The study was organized around three experimental approaches. First, the responses of pial arterioles to ADP before and after L/D exposure in intact and Ovx female rats were tested. L/D reduced the ADP response by 50-70% in both groups, thereby indicating that the endothelium dependence of ADP-induced vasodilation is not altered by chronic estrogen depletion. Second, the NO synthase inhibitor N(omega)-nitro-L-arginine (L-NNA) and the prostanoid synthesis inhibitor indomethacin (Indo) were coapplied. In intact females, L-NNA-Indo attenuated the response to ADP by 50%, with no further changes upon the addition of L/D. On the other hand, L-NNA-Indo did not affect ADP reactivity in Ovx rats, but subsequent L/D exposure reduced the ADP response by >50%. The NO-prostanoid-independent, but endothelium-dependent, nature of the response in Ovx females is a hallmark of EDHF participation. Third, gap junctional inhibition strategies were applied. A selective inhibitor of gap junctional function, Gap 27, did not affect ADP reactivity in intact females but reduced the the ADP response by 50% in Ovx females. A similar result was obtained following application of a connexin43 antisense oligonucleotide. These findings suggest that the nascent EDHF dependency of ADP-induced pial arteriolar dilation in Ovx females involves connexin43-related gap junctional communication.

Lipids and nitric oxide in porcine retinal and choroidal blood vessels.

Lipid profiles of porcine retina, and retinal and choroidal vessels were analyzed using the gas chromatography/mass spectrometry (GC/MS) technique. The retina and both isolated retinal and choroidal vessels contained saturated fatty acid stearic acid and polyunsaturated fatty acids, including arachidonic (C20:4, AA), a precursor for vasoactive prostaglandin (PG-2) series, and W-6 docosahexaenoic acids (C22:6, DHA). However, eicosapentaenoic acid (C20:5, EPA), a precursor for PG-3 series, was not detected in these vessels. When stearic acid was used for normalization of tissue sample, the retina contained relatively higher amounts of DHA than AA, retinal vessels had equal amounts of AA and DHA, while choroidal vessels contained higher amounts of AA than DHA. We also examined the endogenous synthesis of vasoactive endothelial-derived factors like PGs and nitric oxide (NO). Since vasoactive angiotensin II (Ang II) releases these products from blood vessels, this polypeptide was used in a porcine retinal circulation model. The porcine retinal central artery was perfused with oxygenated/heparinized physiological salt solution at 37 degrees C. Changes in A1 and A2 arteriolar diameters induced by Ang II were determined in the absence and presence of the nitric oxide synthase inhibitor, l-NO Arginine (LNOA), and cyclooxygenase inhibitor, flurbiprofen (FB). The central retinal artery designated as the first order arteriole A1 and subsequent branch was defined as A2. Luminal diameters of Al and A2 arterioles were 35 +/- 2 am and 10 +/- 1 microm, respectively. Topical Ang II (10(-10) M-10(-6) M) caused small vasoconstrictions in a dose-dependent manner. This response was enhanced after inhibition of PG synthesis by (10-6 M) FB. Ang II induced-constrictions were further enhanced in the presence of NO synthase inhibitor, LNOA (10(-7) M). There was slightly more increase in Ang II-induced vasoconstrictions in the presence of both NO and PG inhibitors, suggesting that NO may cause release of PGs from these vessels. This study demonstrates that the porcine retinal arterioles have the ability to regulate vasoconstriction responses induced by Ang II by synthesis and release of endogenous vasodilating PGs and NO (especially NO); and these substances may play a vital role in porcine retinal circulation.

Consequences of maternal cocaine on cerebral microvascular functions in piglets

Maternal cocaine abuse is associated with fetal and neonatal neurological abnormalities. Prolonged exposure to cocaine can induce blood flow disorders, growth restriction, and hypoxia in the newborn. We investigated the impact of chronic fetal cocaine exposure on cerebral microvascular reactivity and autonomic function in the piglets. Pregnant pigs received cocaine (1 mg/kg i.v.; twice weekly) or saline throughout the last trimester. Prenatal exposure to cocaine did not have any significant effect on the birth weight of the piglets as compared to the control. Following delivery, effects of recurrent prenatal cocaine exposure on cerebral microvascular functions were examined in piglets (3–6 days old). Pial arteriolar responses to applications of 5-hydroxytryptamine (5-HT), endothelin-1 (ET-1), and clonidine were examined using closed cranial windows. Functional effects of prenatal cocaine exposure on changes in mean arterial pressure (MAP) and pial arteriolar diameter induced by intracisternal injection (i.c.) of clonidine (1 μg/kg) were also determined. Topical applications of 5-HT, ET-1, and clonidine dose-dependently decreased pial arteriolar diameter in the control and these constrictions were significantly enhanced in the in utero cocaine-exposed piglets. Prenatal cocaine exposure did not have any significant effects on the resting MAP and heart rate as there were no differences between the groups. IC clonidine caused sustained decrease in MAP in both groups but the decrease was more pronounced in the cocaine than the control group. IC clonidine causes cerebral microvascular dilation coincident with the development of hypotension. Such dilation was severely attenuated in the cocaine group, even though the hypotension was much more pronounced than in the control. In conclusion, prenatal cocaine exposure resulted in attenuated autoregulatory vasodilation and potentiated responses to vasoconstrictor agents. The mechanisms behind the effects of in utero cocaine exposure on alteration of newborn cerebral functions need further investigation. Such actions may be important in development of cerebral pathologies associated with recurrent prenatal cocaine exposure.

Cerebrovasodilatory Contribution of Endogenous Carbon Monoxide During Seizures in Newborn Pigs

Carbon monoxide (CO) and the excitatory amino acid glutamate both dilate cerebral arterioles in newborn pigs. The key enzyme in CO synthesis is heme oxygenase, which is highly expressed in neurons with glutamatergic receptor activity as well as cerebral microvessels. During seizures the extracellular level of glutamate is increased, which results in excessive depolarization of neurons. We hypothesized that CO is a mediator of excitatory amino acid–induced dilation of the cerebral microvasculature during seizures. Three groups of piglets were examined:1) i.v. normal saline (sham control), 2) topical chromium mesoporphyrin (Cr-MP, 15 × 10−6 M), and 3) i.v. tin-protoporphyrin (Sn-PP, 4 mg/kg). Synthetic metalloporphyrins (Cr-MP and Sn-PP) are heme oxygenase inhibitors, thereby reducing CO synthesis. Implanted closed cranial windows were used to monitor changes in pial arteriolar diameters. Seizures were induced by administration of i.v. bicuculline. Changes in pial arteriolar diameters were monitored during 30 min of status epilepticus. The percent increase in pial arteriolar dilation in the saline group during seizures was 68 ± 3%. In the metalloporphyrin groups, the pial arteriolar dilation was markedly reduced (35 ± 3% and 13 ± 1%, for Cr-MP and Sn-PP, respectively;p < 0.05, compared with the saline group). We conclude that metalloporphyrins by inhibition of heme oxygenase and prevention of CO synthesis attenuate pial arteriolar dilation during seizures. Therefore, CO appears to be involved in cerebral vasodilation caused by glutamatergic seizures.