Re-expression of pro-fibrotic, embryonic preserved mediators in irradiated arterial vessels of the head and neck region.

PURPOSE: Surgical treatment of head and neck malignancies frequently includes microvascular free tissue transfer. Preoperative radiotherapy increases postoperative fibrosis-related complications up to transplant loss. Fibrogenesis is associated with re-expression of embryonic preserved tissue developmental mediators: osteopontin (OPN), regulated by sex-determining region Y­box 9 (Sox9), and homeobox A9 (HoxA9) play important roles in pathologic tissue remodeling and are upregulated in atherosclerotic vascular lesions; dickkopf-1 (DKK1) inhibits pro-fibrotic and atherogenic Wnt signaling. We evaluated the influence of irradiation on expression of these mediators in arteries of the head and neck region. MATERIALS AND METHODS: DKK1, HoxA9, OPN, and Sox9 expression was examined immunohistochemically in 24 irradiated and 24 nonirradiated arteries of the lower head and neck region. The ratio of positive cells to total cell number (labeling index) in the investigated vessel walls was assessed semiquantitatively. RESULTS: DKK1 expression was significantly decreased, whereas HoxA9, OPN, and Sox9 expression were significantly increased in irradiated compared to nonirradiated arterial vessels. CONCLUSION: Preoperative radiotherapy induces re-expression of embryonic preserved mediators in arterial vessels and may thus contribute to enhanced activation of pro-fibrotic downstream signaling leading to media hypertrophy and intima degeneration comparable to fibrotic development steps in atherosclerosis. These histopathological changes may be promoted by HoxA9-, OPN-, and Sox9-related inflammation and vascular remodeling, supported by downregulation of anti-fibrotic DKK1. Future pharmaceutical strategies targeting these vessel alterations, e. g., bisphosphonates, might reduce postoperative complications in free tissue transfer.

Radiation-induced afferent arteriolar endothelial-dependent dysfunction involves decreased epoxygenase metabolites.

Chronic kidney disease is a known complication of hematopoietic stem cell transplant (HSCT) and can be caused by irradiation at the time of the HSCT. In our rat model there is a 6- to 8-wk latent period after irradiation that leads to the development of proteinuria, azotemia, and hypertension. The current study tested the hypothesis that decreased endothelial-derived factors contribute to impaired afferent arteriolar function in rats exposed to total body irradiation (TBI). WAG/RijCmcr rats underwent 11 Gy TBI, and afferent arteriolar responses to acetylcholine were determined at 1, 3, and 6 wk. Blood pressure and blood urea nitrogen were not different between control and irradiated rats. Afferent arteriolar diameters were not altered in irradiated rats. Impaired endothelial-dependent responses to acetylcholine were evident at 3 and 6 wk following TBI. Nitric oxide synthase (NOS), cyclooxygenase (COX), and epoxygenase (EPOX) contribution to acetylcholine dilator responses were evaluated. NOS inhibition with N(G)-nitro-l-arginine methyl ester (l-NAME) reduced acetylcholine responses by 50% in controls and 90% in 3-wk TBI rats. COX inhibition with indomethacin did not significantly alter the acetylcholine response in the presence or absence of l-NAME. EPOX inhibition with N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide significantly decreased acetylcholine responses (35%) in controls but did not significantly alter acetylcholine responses (4%) in TBI rats. Biochemical analysis revealed decreased urinary EPOX metabolites but no change in COX, NOS, or reactive oxygen species at 3 wk TBI. Taken together, these results indicate that afferent arteriolar endothelial dysfunction involves a decrease in EPOX metabolites that precedes the development of proteinuria, azotemia, and hypertension in irradiated rats.

Flicker-induced retinal arteriole dilation is reduced by ambient lighting.

PURPOSE: To investigate the impact of ambient room lighting on the magnitude of flicker light-induced retinal vasodilations in healthy individuals. METHODS: Twenty healthy nonsmokers participated in a balanced 2 × 2 crossover study. Retinal vascular imaging was performed with the dynamic vessel analyzer under reduced or normal ambient lighting, then again after 20 minutes under the alternate condition. Baseline calibers of selected arteriole and venule segments were recorded in measurement units. Maximum percentage dilations from baseline during 20 seconds of luminance flicker were calculated from the mean of three measurement cycles. Within-subject differences were assessed by repeated measures analysis of variance with the assumption of no carryover effects and pairwise comparisons from the fitted model. RESULTS: Mean (SD) maximum arteriole dilations during flicker stimulation under reduced and normal ambient lighting were 4.8% (2.3%) and 4.1% (1.9%), respectively (P = 0.019). Maximum arteriole dilations were (mean ± 95% confidence interval) 0.7% ± 0.6% lower under normal ambient lighting compared with reduced lighting. Ambient lighting had no significant effect on maximum venular dilations during flicker stimulation or on the baseline calibers of arterioles or venules. CONCLUSIONS: Retinal arteriole dilation in response to luminance flicker stimulation is reduced under higher ambient lighting conditions. Reduced responses with higher ambient lighting may reflect reduced contrast between the ON and OFF flicker phases. Although it may not always be feasible to conduct studies under reduced lighting conditions, ambient lighting levels should be consistent to ensure that comparisons are valid.

Dynamic responses in retinal vessel caliber with flicker light stimulation in eyes with diabetic retinopathy.

PURPOSE: This study investigated the responses of retinal vessels to flickering light in diabetic patients with various stages of diabetic retinopathy (DR). METHODS: This cross-sectional observational study evaluated adult subjects with diabetes mellitus. The Dynamic Vessel Analyzer (DVA) was used to measure retinal vascular dilatation in response to diffuse illuminance flicker. Diabetic retinopathy was graded from retinal photography. RESULTS: There were 279 subjects in total, with a mean age of 59.9 ± 9.2 years. The majority were male (73%) and the mean HbA1c level and mean duration of diabetes were 7.7% ± 1.4% and 13.9 ± 10.4 years, respectively. After adjustments for age, sex, smoking, duration of diabetes, HbA1c, hypertension, and hyperlipidemia, the responses of both retinal arterioles and venules to flicker stimulation decreased continuously with increasing stages of diabetic retinopathy (P = 0.008 and <0.001, respectively). Subjects with reduced arteriolar dilation responses were more likely to have any DR (odds ratio, OR, 1.20, [95% confidence interval (CI), 1.01-1.45], P = 0.045, per SD decrease). Subjects with reduced venular dilation responses were more likely to have any DR, moderate DR, or vision-threatening DR (OR: 1.27 [1.04-1.53], P = 0.02; OR: 1.27 (1.06-1.49), P = 0.007; and OR: 1.51 (1.14-1.50), P = 0.002; per SD decrease, respectively). CONCLUSIONS: The responses of retinal arterioles and venules to flickering light are reduced in subjects with DR, and decrease progressively with more severe stages of DR.

Role of nitric oxide in responses of pial arterial vessels to low-intensity red laser irradiation.

The responses of rat pial vessels to red laser irradiation can be mediated by NO. NO mainly affects major arteries and did not contribute to reactivity of small pial arteries and precortical arterioles.

External beam radiotherapy for head and neck cancers is associated with increased variability in retinal vascular oxygenation.

BACKGROUND: Radiation retinopathy is a possible post-treatment complication of radiation therapy. The pathophysiologic mechanism is hypothesized to be microvascular in origin, but evidence is limited. In an effort to study retinal oxygenation in these patients, we herein evaluate the repeatability and variability of retinal oximetry measurements in subjects who had previously received radiation and make comparisons to a cohort of unirradiated subjects. METHODS: Using retinal oximetry, a non-invasive imaging modality, we performed in vivo measurements of arteriole (SaO2) and venule SO2 (SvO2) in subjects (n = 9, 18 retinas) who had received incidental radiation to their retinas (≥ 45 Gy to one retina) and in healthy subjects (n = 20, 40 retinas). A total of 1367 SO2 observations on 593 vessels in 29 persons were analyzed to assess three sources of variance in vessel SO2: 1) variance in repeated measurements of the same vessel ("repeatability"), 2) variance in different vessels within the same subject ("within-subject variability"), and 3) variance between subjects ("between-subject variability"). RESULTS: Retinal oximetry measurements were highly repeatable in both irradiated patients and unirradiated subjects. The within-subject variability of SvO2 and SaO2 measurements constituted the highest component of variance in both groups and was significantly higher in venules vs. arterioles (relative effect size 1.8, p<0.001) and in irradiated subjects vs. unirradiated subjects (relative effect size 1.6, p<0.001). CONCLUSIONS: Retinal oximetry is a highly repeatable technology and can be reliably used to study vascular oxygenation in irradiated subjects. Different vessels within the same subject exhibit a high degree of variability, suggesting that pooled analyses of multiple vessels are most likely to be informative of regional retinal oxygenation. Finally, irradiated subjects exhibited significantly higher within-subject variability in SO2 measurements, suggesting that radiation may cause regional alterations in retinal oxygen delivery and/or metabolism.

Topical hexylaminolevulinate and aminolevulinic acid photodynamic therapy: complete arteriole vasoconstriction occurs frequently and depends on protoporphyrin IX concentration in vessel wall.

Vascular responses to photodynamic therapy (PDT) may influence the availability of oxygen during PDT and the extent of tumor destruction after PDT. However, for topical PDT vascular effects are largely unknown. Arteriole and venule diameters were measured before and after hexylaminolevulinate (HAL) and aminolevulinic acid (ALA) PDT and related to the protoporphyrin IX (PpIX) concentration in the vessel wall. A mouse skin fold chamber model and an intravital confocal microscope allowed direct imaging of the subcutaneous vessels underlying the treated area. In both HAL and ALA groups over 60% of arterioles constricted completely, while venules generally did not respond, except for two larger veins that constricted partially. Arteriole vasoconstriction strongly correlated with PpIX fluorescence intensity in the arteriole wall. Total PpIX fluorescence intensity was significantly higher for HAL than ALA for the whole area that was imaged but not for the arteriole walls. In conclusion, complete arteriole vasoconstriction occurs frequently in both HAL and ALA based topical PDT, especially when relatively high PpIX concentrations in arteriole walls are reached. Vasoconstriction will likely influence PDT effect and should be considered in studies on topical HAL and ALA-PDT. Also, our results may redefine the vasculature as a potential secondary target for topical PDT.

Novel versus traditional risk markers for diabetic retinopathy.

AIMS/HYPOTHESIS: To explore the relative contribution of novel and traditional risk markers for diabetic retinopathy (DR). METHODS: A clinic-based study of 224 diabetic patients (85 type 1, 139 type 2) from a diabetes clinic was performed. DR was graded from fundus photographs according to the Airlie House Classification system and classified as absent or present (at least ETDRS level 14). Novel risk markers assessed included serum apolipoprotein (Apo) AI and B, skin microvascular responses to acetylcholine (endothelium-dependent) and sodium nitroprusside (endothelium-independent) iontophoresis, flicker-light-induced retinal vasodilation and retinal vascular tortuosity. Relative contribution was determined by semi-partial correlation coefficient generated from a logistic regression model containing all traditional and novel risk markers simultaneously. RESULTS: There were 144 (64.3%) participants with DR. Of the novel markers, ApoAI, flicker-light-induced vasodilation and retinal arteriolar tortuosity were significantly associated with DR, independently of traditional measures (all p < 0.03). Diabetes duration contributed most (51%) to the risk of DR, followed by ApoAI (16%), systolic blood pressure (13%), retinal arteriolar tortuosity (8%) and flicker-light-induced venular and arteriolar dilation (3% and 0.5%, respectively). CONCLUSIONS/INTERPRETATION: ApoAI and retinal arteriolar tortuosity made considerable contributions to DR risk, independently of traditional risk markers. Findings from this study suggest that serum ApoAI and retinal arteriolar tortuosity may be novel and independent risk markers of DR.

Microbeam radiation-induced tissue damage depends on the stage of vascular maturation.

PURPOSE: To explore the effects of microbeam radiation (MR) on vascular biology, we used the chick chorioallantoic membrane (CAM) model of an almost pure vascular system with immature vessels (lacking periendothelial coverage) at Day 8 and mature vessels (with coverage) at Day 12 of development. METHODS AND MATERIALS: CAMs were irradiated with microplanar beams (width, ∼25 µm; interbeam spacing, ∼200 µm) at entrance doses of 200 or 300 Gy and, for comparison, with a broad beam (seamless radiation [SLR]), with entrance doses of 5 to 40 Gy. RESULTS: In vivo monitoring of Day-8 CAM vasculature 6 h after 200 Gy MR revealed a near total destruction of the immature capillary plexus. Conversely, 200 Gy MR barely affected Day-12 CAM mature microvasculature. Morphological evaluation of Day-12 CAMs after the dose was increased to 300 Gy revealed opened interendothelial junctions, which could explain the transient mesenchymal edema immediately after irradiation. Electron micrographs revealed cytoplasmic vacuolization of endothelial cells in the beam path, with disrupted luminal surfaces; often the lumen was engorged with erythrocytes and leukocytes. After 30 min, the capillary plexus adopted a striated metronomic pattern, with alternating destroyed and intact zones, corresponding to the beam and the interbeam paths within the array. SLR at a dose of 10 Gy caused growth retardation, resulting in a remarkable reduction in the vascular endpoint density 24 h postirradiation. A dose of 40 Gy damaged the entire CAM vasculature. CONCLUSIONS: The effects of MR are mediated by capillary damage, with tissue injury caused by insufficient blood supply. Vascular toxicity and physiological effects of MR depend on the stage of capillary maturation and appear in the first 15 to 60 min after irradiation. Conversely, the effects of SLR, due to the arrest of cell proliferation, persist for a longer time.

Dynamic retinal vessel response to flicker in obesity: A methodological approach.

Obesity and related metabolic disorders affect vascular endothelial function. The use of the Dynamic Vessel Analyzer (DVA) represents a modern methodological approach to analyze vascular function in the retinal microcirculation. Whether the dynamic reaction to flicker stimulation in retinal vessels is altered in obese subjects is investigated. Retinal vessel reactions to flicker stimulation were examined by DVA in 46 obese individuals (49.6±10.0years) and 46 age- and gender-matched healthy controls. The clinical examination included anthropometry, blood pressure measurements and blood sampling. Mean maximal arteriolar dilation in response to flicker was reduced in the obese group (3.2±1.8%) compared to controls (4.1±2.0%, p<0.05) and the time to maximal arteriolar dilation was prolonged (18.0±9.4s vs. 14.6±3.8s, p=0.03). In addition, mean maximal venular dilation was reduced in obese subjects (3.9±1.7% vs. 4.7±1.8%, p<0.05). Among the microvascular parameters, the most significant correlation with waist circumference was found for the "area under the reaction curve 50-80s after stimulation" in arterioles (r=-0.40; p<0.001). Functional retinal arteriolar reactivity to flicker stimulation differs between obese and healthy lean subjects. Time course analysis of retinal vessel response and its quantitative parameters can comprehensively characterize alterations of retinal vessel reactivity in metabolic disease.

Flicker light-induced retinal vasodilation in diabetes and diabetic retinopathy.

OBJECTIVE: Flicker light-induced retinal vasodilation may reflect endothelial function in the retinal circulation. We investigated flicker light-induced vasodilation in individuals with diabetes and diabetic retinopathy. RESEARCH DESIGN AND METHODS: Participants consisted of 224 individuals with diabetes and 103 nondiabetic control subjects. Flicker light-induced retinal vasodilation (percentage increase over baseline diameter) was measured using the Dynamic Vessel Analyzer. Diabetic retinopathy was graded from retinal photographs. RESULTS: Mean +/- SD age was 56.5 +/- 11.8 years for those with diabetes and 48.0 +/- 16.3 years for control subjects. Mean arteriolar and venular dilation after flicker light stimulation were reduced in participants with diabetes compared with those in control subjects (1.43 +/- 2.10 vs. 3.46 +/- 2.36%, P < 0.001 for arteriolar and 2.83 +/- 2.10 vs. 3.98 +/- 1.84%, P < 0.001 for venular dilation). After adjustment for age, sex, diabetes duration, fasting glucose, cholesterol and triglyceride levels, current smoking status, systolic blood pressure, and use of antihypertensive and lipid-lowering medications, participants with reduced flicker light-induced vasodilation were more likely to have diabetes (odds ratio 19.7 [95% CI 6.5-59.1], P < 0.001 and 8.14 [3.1-21.4], P < 0.001, comparing lowest vs. highest tertile of arteriolar and venular dilation, respectively). Diabetic participants with reduced flicker light-induced vasodilation were more likely to have diabetic retinopathy (2.2 [1.2-4.0], P = 0.01 for arteriolar dilation and 2.5 [1.3-4.5], P = 0.004 for venular dilation). CONCLUSIONS: Reduced retinal vasodilation after flicker light stimulation is independently associated with diabetes status and, in individuals with diabetes, with diabetic retinopathy. Our findings may therefore support endothelial dysfunction as a pathophysiological mechanism underlying diabetes and its microvascular manifestations.

Radiation-induced astrogliosis and blood-brain barrier damage can be abrogated using anti-TNF treatment.

PURPOSE: In this article, we investigate the role of tumor necrosis factor-alpha (TNF) in the initiation of acute damage to the blood-brain barrier (BBB) and brain tissue following radiotherapy (RT) for CNS tumors. METHODS AND MATERIALS: Intravital microscopy and a closed cranial window technique were used to measure quantitatively BBB permeability to FITC-dextran 4.4-kDa molecules, leukocyte adhesion (Rhodamine-6G) and vessel diameters before and after 20-Gy cranial radiation with and without treatment with anti-TNF. Immunohistochemistry was used to quantify astrogliosis post-RT and immunofluorescence was used to visualize protein expression of TNF and ICAM-1 post-RT. Recombinant TNF (rTNF) was used to elucidate the role of TNF in leukocyte adhesion and vessel diameter. RESULTS: Mice treated with anti-TNF showed significantly lower permeability and leukocyte adhesion at 24 and 48 h post-RT vs. RT-only animals. We observed a significant decrease in arteriole diameters at 48 h post-RT that was inhibited in TNF-treated animals. We also saw a significant increase in activated astrocytes following RT that was significantly lower in the anti-TNF-treated group. In addition, immunofluorescence showed protein expression of TNF and ICAM-1 in the cerebral cortex that was inhibited with anti-TNF treatment. Finally, administration of rTNF induced a decrease in arteriole diameter and a significant increase in leukocyte adhesion in venules and arterioles. CONCLUSIONS: TNF plays a significant role in acute changes in BBB permeability, leukocyte adhesion, arteriole diameter, and astrocyte activation following cranial radiation. Treatment with anti-TNF protects the brain's microvascular network from the acute damage following RT.

Development of a rat model of photothrombotic ischemia and infarction within the caudoputamen.

BACKGROUND AND PURPOSE: Basal ganglia infarction is typically caused by the occlusion of deep arteries and the formation of relatively small lesions called lacunes. In the present study, a rat model of lacunar infarction was induced by photothrombotic occlusion of the small vessels within the caudate-putamen and subsequently characterized. METHODS: Male Sprague-Dawley rats (n=143) were anesthetized, and Rose Bengal dye (20 mg/kg) was intravenously injected. The left caudoputamen was exposed to cold white light for 5 to 10 minutes via a stereotaxically implanted polymethylmethacrylate optic fiber (0.5-0.75 mm diameter). Neurological and morphological changes were assessed at various times during the following 6 weeks. Local cerebral blood flow was measured 90 minutes after photothrombosis by [(14)C]-N-isopropyl-p-iodoamphetamine quantitative autoradiography. The time course of blood-brain barrier opening and ischemic brain edema as well as the effects of aspirin and tissue plasminogen activator treatment were also determined. RESULTS: A virtually round infarct with thrombosed parenchymal vessels surrounded by a layer of selective neuronal death was formed within the caudoputamen; it turned into a cystic cavity (lacune) over 6 weeks. A central zone of markedly reduced blood flow and surrounding oligemic zone were observed 90 minutes after light exposure. Lesion size was proportional to light exposure, and the severity and duration of neurological deficits paralleled infarct size. Early blood-brain barrier opening with edema peaked at day 1. After tissue plasminogen activator treatment, infarction volume and neurological deficits were reduced. CONCLUSIONS: This study describes a new rat model of lacunar infarction by photothrombotic occlusion of the microvessels within the caudoputamen. With this model, infarct size correlates with the severity and duration of the neuropathology and can be varied by altering light exposure.

Static magnetic fields affect capillary flow of red blood cells in striated skin muscle.

Blood flowing in microvessels is one possible site of action of static magnetic fields (SMFs). We evaluated SMF effects on capillary flow of red blood cells (RBCs) in unanesthetized hamsters, using a skinfold chamber technique for intravital fluorescence microscopy. By this approach, capillary RBC velocities (v(RBC)), capillary diameters (D), arteriolar diameters (D(art)), and functional vessel densities (FVD) were measured in striated skin muscle at different magnetic flux densities. Exposure above a threshold level of about 500 mT resulted in a significant (P < 0.001) reduction of v(RBC) in capillaries as compared to the baseline value. At the maximum field strength of 587 mT, v(RBC) was reduced by more than 40%. Flow reduction was reversible when the field strength was decreased below the threshold level. In contrast, mean values determined at different exposure levels for the parameters D, D(art), and FVD did not vary by more than 5%. Blood flow through capillary networks is affected by strong SMFs directed perpendicular to the vessels. Since the influence of SMFs on blood flow in microvessels directed parallel to the field as well as on collateral blood supply could not be studied, our findings should be carefully interpreted with respect to the setting of safety guidelines.

Influence of flickering light on the retinal vessels in diabetic patients.

OBJECTIVE: Stimulation of the retina with flickering light increases retinal vessel diameters in humans. Nitric oxide is a mediator of the retinal vasodilation to flicker. The reduction of vasodilation is considered an endothelial dysfunction. We investigated the response of retinal vessels to flickering light in diabetic patients in different stages of diabetic retinopathy. RESEARCH DESIGN AND METHODS: We studied 53 healthy volunteers, 68 type 1 diabetic patients, and 172 type 2 diabetic patients. The diameter of retinal vessels was measured continuously online with the Dynamic Vessel Analyzer (DVA). Diabetic retinopathy was classified using Early Treatment Diabetic Retinopathy Study criteria. Changes in vasodilation are expressed as percent change over baseline values. RESULTS: After adjustments for age, sex, and antihypertensive treatment, the response of retinal arterioles to diffuse luminance flicker was significantly diminished in patients with type 1 diabetes compared with healthy volunteers. The vasodilation of retinal arterioles and venules decreased continuously with increasing stages of diabetic retinopathy. The retinal arterial diameter change was 3.6 +/- 2.1% in the control group, 2.6 +/- 2.5% in the no diabetic retinopathy group, 2.0 +/- 2.7% in the mild nonproliferative diabetic retinopathy (NPDR) group, 1.6 +/- 2.2% in the moderate NPDR group, 1.8 +/- 1.9% in severe NPDR group, and 0.8 +/- 1.6% in proliferative diabetic retinopathy group. CONCLUSIONS: Flicker responses of retinal vessels are abnormally reduced in diabetic patients. This decreased response deteriorated with increasing stages of retinopathy. The response was already reduced before clinical appearance of retinopathy. The noninvasive testing of retinal autoregulation with DVA might prove to be of value in early detection of diabetic vessel pathological changes.

Glial cells dilate and constrict blood vessels: a mechanism of neurovascular coupling.

Neuronal activity evokes localized changes in blood flow. Although this response, termed neurovascular coupling, is widely used to monitor human brain function and diagnose pathology, the cellular mechanisms that mediate the response remain unclear. We investigated the contribution of glial cells to neurovascular coupling in the acutely isolated mammalian retina. We found that light stimulation and glial cell stimulation can both evoke dilation or constriction of arterioles. Light-evoked and glial-evoked vasodilations were blocked by inhibitors of cytochrome P450 epoxygenase, the synthetic enzyme for epoxyeicosatrienoic acids. Vasoconstrictions, in contrast, were blocked by an inhibitor of omega-hydroxylase, which synthesizes 20-hydroxyeicosatetraenoic acid. Nitric oxide influenced whether vasodilations or vasoconstrictions were produced in response to light and glial stimulation. Light-evoked vasoactivity was blocked when neuron-to-glia signaling was interrupted by a purinergic antagonist. These results indicate that glial cells contribute to neurovascular coupling and suggest that regulation of blood flow may involve both vasodilating and vasoconstricting components.

Radiation induces endothelial dysfunction in murine intestinal arterioles via enhanced production of reactive oxygen species.

OBJECTIVE: Endothelial dysfunction and vascular dysregulation contribute to the pathological effects of radiation on tissues. The objectives of this study were to assess the acute effect of irradiation on acetylcholine (Ach)-induced dilation of gut submucosal microvessels. METHODS AND RESULTS: Rats were exposed in vivo to 1 to 9 cGy in 3 fractions per week on alternate days for 3 successive weeks for a total dose of up to 2250 cGy. Submucosal microvessels were isolated after varying levels of irradiation. Diameters of isolated vessels were measured using videomicroscopy, and the dose-response relationship to Ach was determined. Dihydroethidine and 2', 7'-dichlorodihydrofluorescein diacetate fluorescent probes were used to assess reactive oxygen species (ROS) production. After constriction (30% to 50%) with endothelin, dilation to graded doses of Ach (10(-9)-10(-4) M) was observed in control vessels (maximal dilation [MD] 87+/-3%; n=7). However, Ach-induced dilation was reduced in vessels from irradiated rats (MD=3+/-9%; n=7; P= or <0.05 versus controls). Significant increases in superoxide and peroxides were observed in irradiated microvessels. Irradiated microvessels pretreated with superoxide dismutase-mimetic demonstrated significant improvement in Ach-induced vasodilation compared with irradiation alone, suggesting that superoxide contributes to impaired dilation to Ach after irradiation. CONCLUSIONS: Radiation induces acute microvascular dysfunction in the resistance arterioles of the intestine. Enhanced ROS contribute to this dysfunction and therefore may represent a novel therapeutic target to minimize radiation toxicity in the gut.

Static magnetic fields alter arteriolar tone in vivo.

This study was designed to directly quantify the effect of localized static magnetic field (SMF) exposure on the diameter of microvessels in adult rat skeletal muscle in vivo. Microvascular networks in the exteriorized rat spinotrapezius microvasculature were exposed to a localized, uniform 70 mT SMF for 15 min. Arteriolar vessel diameters were measured; and the extent of vessel contraction, microvascular tone, was calculated before exposure, immediately after exposure, and 15 and 30 min after removal of the field. A calculated value of high tone corresponds to vessels that are vasoconstricted and a calculated value of low tone refers to vessels that are vasodilated. Vessels with initial tone <15% showed an increasing trend in tone and, conversely, vessels with initial tone >15% showed a significant (P < 0.05) decrease in tone 15 and 30 min following application, respectively. Further classification of the data with regards to the initial vessel diameter demonstrated that vessels with initial diameters <30 microm and initial tone <15%, smaller diameter vessels that are initially vasodilated, showed significant (P < 0.05) increase in tone immediately, 15 and 30 min following SMF exposure. Additionally, <30 microm vessels with >15% initial tone, smaller diameter vessels that are initially vasoconstricted, demonstrated a significant (P < 0.05) decrease in tone 30 min after SMF exposure. Vessels with initial diameters >30 microm had no significant response to the SMF. These results imply that SMF exposure influences arteriolar diameters, and therefore microvascular tone, in a restorative fashion acting to normalize the tone to the median tone value of 15% following exposure. Because this response occurs primarily in the resistance arterioles, which significantly influence tissue perfusion, SMF application could be efficacious for the treatment of both ischemic and edematous tissue disorders involving compromised microvascular function.

Connexin expression and conducted vasodilation along arteriolar endothelium in mouse skeletal muscle.

Functional hyperemia requires the coordination of smooth muscle cell relaxation along and between branches of the arteriolar network. Vasodilation is conducted from cell to cell along the arteriolar wall through gap junction channels composed of connexin protein subunits. Within skeletal muscle, it is unclear whether arteriolar endothelium, smooth muscle, or both cell layers provide the cellular pathway for conduction. Furthermore, the constitutive profile of connexin expression within the microcirculation is unknown. We tested the hypothesis that conducted vasodilation and connexin expression are intrinsic to the endothelium of arterioles (17 +/- 1 microm diameter) that supply the skeletal muscle fibers in the cremaster of anesthetized C57BL/6 mice. ACh delivered to an arteriole (500 ms, 1-microA pulse; 1-microm micropipette) produced local dilation of 17 +/- 1 microm; conducted vasodilation observed 1 mm upstream was 9 +/- 1 microm (n = 5). After light-dye treatment to selectively disrupt endothelium (250-microm segment centered 500 microm upstream, confirmed by loss of local response to ACh while constriction to phenylephrine and dilation to sodium nitroprusside remained intact), we found that conducted vasodilation was nearly abolished (2 +/- 1 microm; P < 0.05). Whole-mount immunohistochemistry for connexins revealed punctate labeling at borders of arteriolar endothelial cells, with connexin40 and connexin37 in all branches and connexin43 only in the largest branches. Immunoreactivity for connexins was not apparent in smooth muscle or in capillary or venular endothelium, despite robust immunolabeling for alpha-actin and platelet endothelial cell adhesion molecule-1, respectively. We conclude that vasodilation is conducted along the endothelium of mouse skeletal muscle arterioles and that connexin40 and connexin37 are the primary connexins forming gap junction channels between arteriolar endothelial cells.