Plexus-specific effect of flicker-light stimulation on the retinal microvasculature assessed with optical coherence tomography angiography.

In neural tissues, the coupling between neural activity and blood flow is a physiological key principle in blood flow regulation. We used optical coherence tomography angiography to investigate stimulus-evoked hemodynamic responses in different microvascular layers of the human retina. Twenty-two healthy subjects were included. Vessel density before and during light stimulation was measured using optical coherence tomography angiography and assessed for the superficial, intermediate, and deep capillary plexus of the retinal circulation. Volumetric blood flow was measured using a custom-built Doppler optical coherence tomography system. Our results show that flicker stimulation induced a significant increase in the vessel density of +9.9 ± 6.7% in the superficial capillary plexus, +6.6 ± 1.7% in the intermediate capillary plexus, and +4.9 ± 2.3% in the deep capillary plexus. The hyperemic response of the superficial capillary plexus was significantly higher compared to the intermediate capillary plexus (P = 0.02) and deep capillary plexus (P = 0.002). Volumetric retinal blood flow increased by +39.9 ± 34.9% in arteries and by +29.8 ± 16.8% in veins. In conclusion, we showed a strong increase in the retinal microvascular density in response to light stimulation, with the most pronounced effect in the superficial capillary plexus. This is compatible with the hypothesis that the microvasculature exerts an important function in mediating functional hyperemia in humans.NEW & NOTEWORTHY We present vessel density alterations in response to flicker stimulation using optical coherence tomography angiography and identified the superficial capillary plexus as the layer with the most pronounced effect. This points out the physiological importance of the microvasculature in mediating functional hyperemia and suggests a fine-tuned plexus-specific mechanism to meet cellular metabolic demands.

Maintenance of Tight Junction Integrity in the Absence of Vascular Dilation in the Brain of Mice Exposed to Ultra-High-Dose-Rate FLASH Irradiation.

Persistent vasculature abnormalities contribute to an altered CNS microenvironment that further compromises the integrity of the blood-brain barrier and exposes the brain to a host of neurotoxic conditions. Standard radiation therapy at conventional (CONV) dose rate elicits short-term damage to the blood-brain barrier by disrupting supportive cells, vasculature volume and tight junction proteins. While current clinical applications of cranial radiotherapy use dose fractionation to reduce normal tissue damage, these treatments still cause significant complications. While dose escalation enhances treatment of radiation-resistant tumors, methods to subvert normal tissue damage are clearly needed. In this regard, we have recently developed a new modality of irradiation based on the use of ultra-high-dose-rate FLASH that does not induce the classical pathogenic patterns caused by CONV irradiation. In previous work, we optimized the physical parameters required to minimize normal brain toxicity (i.e., FLASH, instantaneous intra-pulse dose rate, 6.9 · 106 Gy/s, at a mean dose rate of 2,500 Gy/s), which we then used in the current study to determine the effect of FLASH on the integrity of the vasculature and the blood-brain barrier. Both early (24 h, one week) and late (one month) timepoints postirradiation were investigated using C57Bl/6J female mice exposed to whole-brain irradiation delivered in single doses of 25 Gy and 10 Gy, respectively, using CONV (0.09 Gy/s) or FLASH (>106 Gy/s). While the majority of changes found one day postirradiation were minimal, FLASH was found to reduce levels of apoptosis in the neurogenic regions of the brain at this time. At one week and one month postirradiation, CONV was found to induce vascular dilation, a well described sign of vascular alteration, while FLASH minimized these effects. These results were positively correlated with and temporally coincident to changes in the immunostaining of the vasodilator eNOS colocalized to the vasculature, suggestive of possible dysregulation in blood flow at these latter times. Overall expression of the tight junction proteins, occludin and claudin-5, which was significantly reduced after CONV irradiation, remained unchanged in the FLASH-irradiated brains at one and four weeks postirradiation. Our data further confirm that, compared to isodoses of CONV irradiation known to elicit detrimental effects, FLASH does not damage the normal vasculature. These data now provide the first evidence that FLASH preserves microvasculature integrity in the brain, which may prove beneficial to cognition while allowing for better tumor control in the clinic.

Adjunctive mesenchymal stem/stromal cells augment microvascular function in poststenotic kidneys treated with low-energy shockwave therapy.

Effective therapeutic strategies are needed to preserve renal function in patients with atherosclerotic renal artery stenosis (ARAS). Low-energy shockwave therapy (SW) and adipose tissue-derived mesenchymal stem/stromal cells (MSCs) both stimulate angiogenesis repair of stenotic kidney injury. This study tested the hypothesis that intrarenal delivery of adipose tissue-derived MSCs would enhance the capability of SW to preserve stenotic kidney function and structure. Twenty-two pigs were studied after 16 weeks of ARAS, ARAS treated with a SW regimen (bi-weekly for 3 weeks) with or without subsequent intrarenal delivery of adipose tissue-derived MSCs and controls. Four weeks after treatment, single-kidney renal blood flow (RBF) before and after infusion of acetylcholine, glomerular filtration rate (GFR), and oxygenation were assessed in vivo and the renal microcirculation, fibrosis, and oxidative stress ex vivo. Mean arterial pressure remained higher in ARAS, ARAS + SW, and ARAS + SW + MSC compared with normal. Both SW and SW + MSC similarly elevated the decreased stenotic kidney GFR and RBF observed in ARAS to normal levels. Yet, SW + MSC significantly improved RBF response to acetylcholine in ARAS, and attenuated capillary loss and oxidative stress more than SW alone. Density of larger microvessels was similarly increased by both interventions. Therefore, although significant changes in functional outcomes were not observed in a short period of time, adjunct MSCs enhanced pro-angiogenic effect of SW to improve renal microvascular outcomes, suggesting this as an effective stratege for long-term management of renovascular disease.

Effect of X-rays on transcript expression of rat brain microvascular endothelial cells: role of calcium signaling in X-ray-induced endothelium damage.

Radiation-induced brain edema is a serious adverse effect of radiotherapy. Although there are many causes of radiation-induced brain edema, the pathogenesis is not clear and clinical treatment is not ideal. Therefore, knowing the differential expression of the brain microvascular endothelial cell (BMEC) transcriptome after brain radiotherapy may shed light on the pathogenesis of radiation-induced brain edema. The present study used RNA-Seq technique to identify 383 BMEC transcripts differentially expressed (many 2-fold or higher; P < 0.05) between control and X-ray-treated primary cultured rat BMECs. Compared with controls, X-ray-treated BMECs had 183 significantly up-regulated transcripts and 200 significantly down-regulated transcripts. The differentially expressed genes were associated with the biological processes of the cell cycle, apoptosis, vascular permeability, and extracellular junctions. The functional changes identified in the X-ray-treated BMECs included Ca2+ signaling, phosphoinositide 3-kinase-Akt signaling, and methionine degradation. These results indicated that transcript expression was substantially affected by radiation exposure and the proteins encoded by these differentially expressed genes may play a significant role in radiotherapy-induced brain edema. Our findings provide additional insight into the molecular mechanisms of radiation-induced brain edema and may be helpful in the development of clinical treatment of this adverse reaction to radiotherapy.

Contrast-Enhanced Ultrasound to Detect Early Microvascular Changes in Skeletal Muscle after High-Dose Radiation Treatment.

The biological response of normal tissue to high-dose radiation treatment remains poorly understood. Alterations to the microenvironment, specifically the microvasculature, have been implicated as a significant contributor to tumoral cytotoxicity. We used contrast-enhanced ultrasound (CEU) perfusion imaging, which is uniquely suited to assess functional status of the microcirculation, to measure microvascular blood flow after high-dose irradiation to normal skeletal muscle tissue in a murine model. Proximal hindlimbs of wild-type C57Bl/6 mice were irradiated with a single fraction using 6 MV photons, 1 cm bolus and a dynamic wedge. Quantitative perfusion CEU imaging of the skeletal muscle was performed at days 1 and 8 postirradiation in three different regions of interest (ROIs): 1. 15 Gy external-beam irradiated leg; 2. 12 Gy irradiated 5 mm proximal area; 3. single ROI in the nonirradiated contralateral (CL) hindlimb. Perfusion imaging was also performed in the hindlimb of nonirradiated mice. CEU time-intensity data were analyzed to measure microvascular blood flow (MBF, also referred to as perfusion), and its parametric components of microvascular flux rate and functional microvascular blood volume (MBV). Plasma measurements of two potent vasoconstrictors, endothelin-1 and angiotensin II, were also performed to assess systemic response. CEU perfusion imaging values for the 12 and 15 Gy irradiated limb regions were pooled. At day 1, MBF in the irradiated limb was significantly lower than in the CL limb (P = 0.016) but quite similar to the nonirradiated mice. At day 8, both limbs of irradiated mice exhibited a trend towards lower MBF than the limbs of nonirradiated mice (28% decrease in mean MBF, P = 0.149 for CL; 39% decrease, P = 0.065 for irradiated limb). Compared to nonirradiated animals, the reduction in perfusion in irradiated limbs at day 8 may have been more influenced by the microvascular flux rate (25% decrease in the mean, P = 0.079) than the MBV (12% decrease in the mean, P = 0.328). Examination of vasoactive compounds revealed that the average plasma concentration for endothelin-1 at day 8 postirradiation was significantly higher in 14 irradiated animals than in 4 nonirradiated animals (3.07 pg/ ml vs. 2.51 pg/ml; P = 0.011). Up to day 8 after high-dose irradiation, flow deficits in irradiated muscle appear to be a consequence of increased vascular resistance more so than loss or functional de-recruitment of microvascular units.

Impact of Single Dose Photons and Carbon Ions on Perfusion and Vascular Permeability: A Dynamic Contrast-Enhanced MRI Pilot Study in the Anaplastic Rat Prostate Tumor R3327-AT1.

We collected initial quantitative information on the effects of high-dose carbon (12C) ions compared to photons on vascular damage in anaplastic rat prostate tumors, with the goal of elucidating differences in response to high-LET radiation, using dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). Syngeneic R3327-AT1 rat prostate tumors received a single dose of either 16 or 37 Gy 12C ions or 37 or 85 Gy 6 MV photons (iso-absorbed and iso-effective doses, respectively). The animals underwent DCE-MRI prior to, and on days 3, 7, 14 and 21 postirradiation. The extended Tofts model was used for pharmacokinetic analysis. At day 21, tumors were dissected and histologically examined. The results of this work showed the following: 1. 12C ions led to stronger vascular changes compared to photons, independent of dose; 2. Tumor growth was comparable for all radiation doses and modalities until day 21; 3. Nonirradiated, rapidly growing control tumors showed a decrease in all pharmacokinetic parameters (area under the curve, Ktrans, ve, vp) over time; 4. 12C-ion-irradiated tumors showed an earlier increase in area under the curve and Ktrans than photon-irradiated tumors; 5. 12C-ion irradiation resulted in more homogeneous parameter maps and histology compared to photons; and 6. 12C-ion irradiation led to an increased microvascular density and decreased proliferation activity in a largely dose-independent manner compared to photons. Postirradiation changes related to 12C ions and photons were detected using DCE-MRI, and correlated with histological parameters in an anaplastic experimental prostate tumor. In summary, this pilot study demonstrated that exposure to 12C ions increased the perfusion and/or permeability faster and led to larger changes in DCE-MRI parameters resulting in increased vessel density and presumably less hypoxia at the end of the observation period when compared to photons. Within this study no differences were found between curative and sub-curative doses in either modality.

Calreticulin stabilizes F-actin by acetylating actin and protects microvascular endothelial cells against microwave radiation.

AIMS: Calreticulin (CRT) is a multifunctional protein that protects endothelial cells by alleviating actin cytoskeleton injury, but the underlying mechanism remains unclear. CRT was recently identified as a novel acyltransferase; acetylation at the N-terminus of actin monomers strengthens actin polymerization. This study was undertaken to determine whether CRT protects human microvascular endothelial cells (HMECs) against microwave radiation through actin acetylation. MATERIALS AND METHODS: We prepared a eukaryotic-derived recombinant CRT and incubated the HMECs with it prior to microwave exposure. We then assessed cell injury and endothelial function, detected actin polymerization and acetylation after HMECs exposure to S-band high-power microwaves. Coimmunoprecipitation, pull-down, and ex vitro acetylation reaction were performed to determine whether actin is a novel substrate of CRT acyltransferase. Finally, we employed the mutant experiments to demonstrate the acetylation sites contributing to CRT acetyltransferase activity. KEY FINDINGS: Microwave radiation induced severe cell injury and endothelial contact dysfunction, reduced the polymerization of actin filaments, and destroyed the actin arrangement, ultimately reducing acetylated actin expression. CRT treatment upregulated actin acetylation levels, promoted polymerization, and facilitated thicker and longer F-actin stress fibre formation. Pre-incubation with CRT rescued microwave-induced cell injury, decreased actin acetylation, and rendered the actin cytoskeleton radiation-retardant. The level of acetyl-actin was positively correlated with actin polymerization. Actin was identified as a novel substrate of CRT, being acetylated mainly through the CRT P-domain at lys-206 and -207. SIGNIFICANCE: This work provides a better understanding of the underlying mechanism of CRT-induced cytoprotection, and suggests a novel therapeutic target for microwave radiation-related diseases with endothelial dysfunction.

Assessment of chronic radiation proctopathy and radiofrequency ablation treatment follow-up with optical coherence tomography angiography: A pilot study.

BACKGROUND: Chronic radiation proctopathy (CRP) occurs as a result of pelvic radiation therapy and is associated with formation of abnormal vasculature that may lead to persistent rectal bleeding. While incidence is declining due to refinement of radiation delivery techniques, CRP remains one of the major complications of pelvic radiation therapy and significantly affects patient quality of life. Radiofrequency ablation (RFA) is an emerging treatment modality for eradicating abnormal vasculature associated with CRP. However, questions remain regarding CRP pathophysiology and optimal disease management. AIM: To study feasibility of optical coherence tomography angiography (OCTA) for investigating subsurface vascular alterations in CRP and response to RFA treatment. METHODS: Two patients with normal rectum and 8 patients referred for, or undergoing endoscopic RFA treatment for CRP were imaged with a prototype ultrahigh-speed optical coherence tomography (OCT) system over 15 OCT/colonoscopy visits (2 normal patients, 5 RFA-naïve patients, 8 RFA-follow-up visits). OCT and OCTA was performed by placing the OCT catheter onto the dentate line and rectum without endoscopic guidance. OCTA enabled depth-resolved microvasculature imaging using motion contrast from flowing blood, without requiring injected dyes. OCTA features of normal and abnormal microvasculature were assessed in the mucosa and submucosa. Blinded reading of OCTA images was performed to assess the association of abnormal rectal microvasculature with CRP and RFA treatment, and rectal telangiectasia density endoscopic scoring. RESULTS: OCTA/OCT images are intrinsically co-registered and enabled depth-resolved visualization of microvasculature in the mucosa and submucosa. OCTA visualized normal vascular patterns with regular honeycomb patterns vs abnormal vasculature with distorted honeycomb patterns and ectatic/tortuous microvasculature in the rectal mucosa. Normal arterioles and venules < 200 µm in diameter versus abnormal heterogenous enlarged arterioles and venules > 200 µm in diameter were visualized in the rectal submucosa. Abnormal mucosal vasculature occurred in 0 of 2 normal patients and 3 of 5 RFA-naïve patients, while abnormal submucosal vasculature occurred more often, in 1 of 2 normal patients and 5 of 5 RFA-naïve patients. After RFA treatment, vascular abnormalities decreased, with abnormal mucosal vasculature observed in 0 of 8 RFA-follow-up visits and abnormal submucosal vasculature observed in only and 2 of 8 RFA-follow-up visits. CONCLUSION: OCTA visualizes depth-resolved microvascular abnormalities in CRP, allowing assessment of superficial features which are endoscopically visible as well as deeper vasculature which cannot be seen endoscopically. OCTA/OCT of the rectum can be performed in conjunction with, or independently from endoscopy. Further studies are warranted to investigate if OCTA/OCT can elucidate pathophysiology of CRP or improve management.

Sunscreen or simulated sweat minimizes the impact of acute ultraviolet radiation on cutaneous microvascular function in healthy humans.

NEW FINDINGS: What is the central question of this study? Are ultraviolet radiation (UVR)-induced increases in skin blood flow independent of skin erythema? Does broad-spectrum UVR exposure attenuate NO-mediated cutaneous vasodilatation, and does sunscreen or sweat modulate this response? What are the main findings and their importance? Erythema and vascular responses to UVR are temporally distinct, and sunscreen prevents both responses. Exposure to UVR attenuates NO-mediated vasodilatation in the cutaneous microvasculature; sunscreen or simulated sweat on the skin attenuates this response. Sun over-exposure may elicit deleterious effects on human skin that are separate from sunburn, and sunscreen or sweat on the skin may provide protection. ABSTRACT: Exposure to ultraviolet radiation (UVR) may result in cutaneous vascular dysfunction independent of erythema (skin reddening). Two studies were designed to differentiate changes in erythema from skin vasodilatation throughout the 8 h after acute broad-spectrum UVR exposure with (+SS) or without SPF-50 sunscreen (study 1) and to examine NO-mediated cutaneous vasodilatation after acute broad-spectrum UVR exposure with or without +SS or simulated sweat (+SW) on the skin (study 2). In both studies, laser-Doppler flowmetry was used to measure red cell flux, and cutaneous vascular conductance (CVC) was calculated (CVC = flux/mean arterial pressure). In study 1, in 14 healthy adults (24 ± 4 years old; seven men and seven women), the skin erythema index and CVC were measured over two forearm sites (UVR only and UVR+SS) before, immediately after and every 2 h for 8 h post-exposure (750 mJ cm-2 ). The erythema index began to increase immediately post-UVR (P < 0.05 at 4, 6 and 8 h), but CVC did not increase above baseline for the first 4-6 h (P ≤ 0.01 at 6 and 8 h); +SS prevented both responses. In study 2, in 13 healthy adults (24 ± 4 years old; six men and seven women), three intradermal microdialysis fibres were placed in the ventral skin of the forearm [randomly assigned to UVR (450 mJ cm-2 ), UVR+SS or UVR+SW], and one fibre (non-exposed control; CON) was placed in the contralateral forearm. After UVR, a standardized local heating (42°C) protocol quantified the percentage of NO-mediated vasodilatation (%NO). The UVR attenuated %NO compared with CON (P = 0.01). The diminished %NO was prevented by +SS (P < 0.01) and +SW (P < 0.01). Acute broad-spectrum UVR attenuates NO-dependent dilatation in the cutaneous microvasculature, independent of erythema. Sunscreen protects against both inflammatory and heating-induced endothelial dysfunction, and sweat might prevent UVR-induced reductions in NO-dependent dilatation.

Radiation-Induced Skin Changes after Postmastectomy Radiation Therapy: A Pilot Study on Indicators for Timing of Delayed Breast Reconstruction.

BACKGROUND: Delayed autologous breast reconstruction is commonly recommended in patients requiring postmastectomy radiation. This study examines gross and histologic changes in the breast skin of patients who have undergone postmastectomy radiation to help determine when radiation-induced skin changes begin to stabilize. METHODS: A prospective pilot study was conducted on eight patients with invasive breast cancer who required mastectomy and radiotherapy. At the time of mastectomy and 2, 4, 6, 8, and 12 months after completion of radiotherapy, a punch biopsy was taken from the radiated mastectomy skin of each patient. Serial standardized photographs were taken before and after radiotherapy to evaluate the degree of hyperpigmentation and graded by three blinded plastic surgeons. Skin biopsies were processed for histologic assessment of inflammation, elastin organization, and vascularity. RESULTS: Grading of patient photographs revealed an increase in hyperpigmentation after radiotherapy compared with baseline with a gradual improvement over time. SMAD3 immunostaining demonstrated a trend toward an increase in inflammation over 12 months. The elastin distribution within samples showed an increase in fiber disorganization, thickening, and clumping, with no improvement throughout the study period. The average number of vessels per high powered field decreased steadily through the duration of the study. CONCLUSION: Histologic changes in dermal inflammation, elastin organization, and vascularity do not appear to correspond with the gradual improvement of hyperpigmentation, resulting from postmastectomy radiation. These histologic changes persist beyond the 12 month observation period and will require clinical correlation to determine the potential impact on postoperative outcomes.

Abnormal Microvasculature in Laryngectomy Mucosal Margins may be Associated with Increased Risk of Fistula.

Pharyngocutaneous fistula after laryngectomy is common and significantly increases the morbidity of the procedure. Intraoperative, objective variables that can reliably predict fistula formation would be useful to surgeons deciding how to reconstruct the laryngectomy defect. Retrospective chart review of 50 radiated patients and 10 non-radiated patients who underwent total laryngectomy at a single tertiary care institution. Patients with pharyngocutaneous fistula were selected to ensure a representative sample were available for comparison. All patients had pathology slides available for re-review by a single, blinded pathologist. Margins of both radiated (n = 50) and non-radiated (n = 10) larynges were examined for 7 histologic features, and odds ratios were calculated to assess whether these features were associated with fistula. When evaluating all 60 patients, both telangiectatic capillaries and hyalinized arterioles were associated with fistula (OR 3.72 and 9.21, respectively). Collinearity between the variables was evaluated; findings indicated a high likelihood of having hyalinized arterioles if telangiectatic capillaries were also present (OR 31.67 [3.13, 320.06]). Microvascular changes in radiated tissue have previously been described in other anatomic subsites, but the larynx and pharynx have not been specifically evaluated. Laryngectomy mucosal margins appear to display similar changes, and evidence of this damage may be associated with fistula formation. These features could potentially guide the surgeon to alter the reconstructive technique.

Microvascular abnormalities secondary to radiation therapy in neovascular age-related macular degeneration: findings from the INTREPID clinical trial.

PURPOSE: To report the incidence and features of retinal microvascular abnormalities (MVAs) occurring secondary to stereotactic radiotherapy (SRT) in a randomised double-masked sham-controlled clinical trial at 21 European sites. METHODS: Two hundred and thirty participants with neovascular age-related macular degeneration (AMD) treated with at least three intravitreal antivascular endothelial growth factor (anti-VEGF) injections prior to enrolment, and demonstrating a continuing need for re-treatment. INTERVENTIONS: 16 Gy, 24 Gy or sham SRT. All three groups received pro re nata anti-VEGF injections if the lesion was judged to be active at review visits. Colour fundus images from baseline and 6 months and fluorescein angiograms from baseline and annual visits were graded for measures of morphological outcome and safety using a prespecified protocol with accompanying definitions to distinguish RT-related MVA from non-specific retinal vessel abnormalities that are known to occur in neovascular AMD. The main outcome measure was MVA detected by months 12, 24 and 36 after enrolment. RESULTS: The frequency of MVAs in the combined SRT arms was 0% in year 1, 13.1% in year 2 and 30.3% in year 3. The area of MVA was small and the mean change in visual acuity in year 2 was similar in a subset of SRT eyes with MVAs, versus those without MVAs. MVA was considered to have possibly contributed to vision loss in 2 of 18 cases with MVA in year 2, and 5 of 37 cases in year 3. CONCLUSION: Treatment with SRT is associated with development of subtle MVAs that have little or no impact on visual outcome. These findings can help clinicians recognise the retinal MVAs that occur in response to SRT.

Impairment of lymphatic endothelial barrier function by X-ray irradiation.

PURPOSE: Although the microvascular system is a significant target for radiation-induced effects, the lymphatic response to radiation has not been extensively investigated. This is one of the first investigations characterizing the lymphatic endothelial response to ionizing radiation. MATERIALS AND METHODS: Rat mesenteric lymphatic endothelial cells (RMLECs) were exposed to X-ray doses of 0, 0.5, 1, 1.5, and 2 Gy. RMLEC cellular response was assessed 24 and 72-h post-irradiation via measures of cellular morphometry and junctional adhesion markers. RMLEC functional response was characterized through permeability experiments. RESULTS: Cell morphometry showed radiation sensitivity at all doses. Notably, there was a loss of cell-to-cell adhesion with irradiated cells increasing in size and cellular roundness. This was coupled with decreased ß-catenin and VE-cadherin intensity and altered F-actin anisotropy, leading to a loss of intercellular contact. RMLEC monolayers demonstrated increased permeability at all doses 24 h post-irradiation and at 2-Gy 72 h post-irradiation. CONCLUSIONS: In summary, lymphatics show radiation sensitivity in the context of these cell culture experiments. Our results may have functional implications of lymphatics in tissue, with endothelial barrier dysfunction due to loss of cell-cell adhesion leading to leaky vessels and lymphedema. These preliminary experiments will build the framework for future investigations towards lymphatic radiation exposure response.

Postirradiation Necrosis after Slow Microvascular Breakdown in the Adult Rat Spinal Cord is Delayed by Minocycline Treatment.

To better understand the spatiotemporal course of radiation-induced central nervous system (CNS) vascular necrosis and assess the therapeutic potential of approaches for protecting against radiation-induced necrosis, adult female Sprague Dawley rats received 40 Gy surface dose centered on the T9 thoracic spinal cord segment. Locomotor function, blood-spinal cord barrier (BSCB) integrity and histology were evaluated throughout the study. No functional symptoms were observed for several months postirradiation. However, a sudden onset of paralysis was observed at approximately 5.5 months postirradiation. The progression rapidly led to total paralysis and death within less than 48 h of symptom onset. Open-field locomotor scores and rotarod motor coordination testing showed no evidence of neurological impairment prior to the onset of overt paralysis. Histological examination revealed minimal changes to the vasculature prior to symptom onset. However, Evans blue dye (EvB) extravasation revealed a progressive deterioration of BSCB integrity, beginning at one week postirradiation, affecting regions well outside of the irradiated area. Minocycline treatment significantly delayed the onset of paralysis. The results of this study indicate that extensive asymptomatic disruption of the blood-CNS barrier may precede onset of vascular breakdown by several months and suggests that minocycline treatment has a therapeutic effect by delaying radiation-induced necrosis after CNS irradiation.

MRI Study on the Changes of Bone Marrow Microvascular Permeability and Fat Content after Total-Body X-Ray Irradiation.

In this study, we investigated microvascular perfusion status, changes to fat content and fatty acid composition in the bone marrow of rat femurs after total-body irradiation by quantitative permeability parameters of dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and ex vivo high-resolution magic angle spinning (HRMAS) 1H nuclear magnetic resonance spectroscopy (NMRS). Thirty-six Sprague-Dawley rats were randomly assigned to either an irradiated or nonirradiated control group. Permeability imaging using DCE-MRI and HRMAS 1H NMRS was performed before irradiation, as well as at days 4 and 7 postirradiation. The volume transfer constant (Ktrans) values increased to 2.219 ± 0.418/min ( P < 0.01) at day 4 and to 2.760 ± 0.217/min at day 7 ( P < 0.01) postirradiation. The plasma fraction (vp) values gradually decreased. The proportion of (n-6) polyunsaturated fatty acids (PUFA) gradually reached a peak at day 7, the proportion of (n-3) PUFA gradually decreased and the proportion of saturated fatty acids gradually increased. After irradiation, Ktrans at different times showed significant negative correlation with (n-3) PUFA ( r = -0.6393, P < 0.01) and significant positive correlation with (n-6) PUFA ( r = 0.6841, P < 0.05). These findings indicate that bone marrow microcirculation perfusion and vascular permeability correlated with fat content at an early time point after irradiation. A pathophysiological mechanism may exist based on fat-vascular permeability in the case of injury to bone marrow microcirculation.

Heart irradiation reduces microvascular density and accumulation of HSPA1 in mice.

PURPOSE: Improvement of radiotherapy techniques reduces the exposure of normal tissues to ionizing radiation. However, the risk of radiation-related late effects remains elevated. In the present study, we investigated long-term effects of radiation on heart muscle morphology. MATERIALS AND METHODS: We established a mouse model to study microvascular density (MVD), deposition of collagen fibers, and changes in accumulation of heat shock 70 kDa protein 1 (HSPA1) in irradiated heart tissue. Hearts of C57BL/6 mice received a single dose of X­ray radiation in the range 0.2-16 Gy. Analyses were performed 20, 40, and 60 weeks after irradiation. RESULTS: Reduction in MD was revealed as a long-term effect observed 20-60 weeks after irradiation. Moreover, a significant and dose-dependent increase in accumulation of HSPA1, both cytoplasmic and nuclear, was observed in heart tissues collected 20 weeks after irradiation. We also noticed an increase in collagen deposition in hearts treated with higher doses. CONCLUSIONS: This study shows that some changes induced by radiation in the heart tissue, such as reduction in microvessel density, increase in collagen deposition, and accumulation of HSPA1, are observed as long-term effects which might be associated with late radiation cardiotoxicity.

Noninvasive assessment of cutaneous alterations in mice exposed to whole body gamma irradiation using optical imaging techniques.

We report the results of a study carried out to investigate the potential of optical techniques such as optical coherence tomography, Mueller matrix spectroscopy, and cross-polarization imaging for noninvasive monitoring of the ionizing radiation exposure-induced alterations in cutaneous tissue of mice. Radiation dose-dependent changes were observed in tissue microvasculature and tissue optical parameters like retardance and depolarization as early as 1 h post radiation exposure. Results suggest that these optical techniques may allow early detection of radiation dose-dependent alterations which could help in screening of population exposed to radiation.

Changes in microvascular density differentiate metabolic health outcomes in monkeys with prior radiation exposure and subsequent skeletal muscle ECM remodeling.

Radiation exposure accelerates the onset of age-related diseases such as diabetes, cardiovascular disease, and neoplasia and, thus, lends insight into in vivo mechanisms common to these disorders. Fibrosis and extracellular matrix (ECM) remodeling, which occur with aging and overnutrition and following irradiation, are risk factors for development of type 2 diabetes mellitus. We previously demonstrated an increased incidence of skeletal muscle insulin resistance and type 2 diabetes mellitus in monkeys that had been exposed to whole body irradiation 5-9 yr prior. We hypothesized that irradiation-induced fibrosis alters muscle architecture, predisposing irradiated animals to insulin resistance and overt diabetes. Rhesus macaques (Macaca mulatta, n = 7-8/group) grouped as nonirradiated age-matched controls (Non-Rad-CTL), irradiated nondiabetic monkeys (Rad-CTL), and irradiated monkeys that subsequently developed diabetes (Rad-DM) were compared. Prior radiation exposure resulted in persistent skeletal muscle ECM changes, including a relative overabundance of collagen IV and a trend toward increased transforming growth factor-ß1. Preservation of microvascular markers differentiated the irradiated diabetic and nondiabetic groups. Microvascular density and plasma nitrate and heat shock protein 90 levels were lower in Rad-DM than Rad-CTL. These results are consistent with a protective effect of abundant microvasculature in maintaining glycemic control within radiation-induced fibrotic muscle.

Acute effects of focused ultrasound-induced increases in blood-brain barrier permeability on rat microvascular transcriptome.

Therapeutic treatment options for central nervous system diseases are greatly limited by the blood-brain barrier (BBB). Focused ultrasound (FUS), in conjunction with circulating microbubbles, can be used to induce a targeted and transient increase in BBB permeability, providing a unique approach for the delivery of drugs from the systemic circulation into the brain. While preclinical research has demonstrated the utility of FUS, there remains a large gap in our knowledge regarding the impact of sonication on BBB gene expression. This work is focused on investigating the transcriptional changes in dorsal hippocampal rat microvessels in the acute stages following sonication. Microarray analysis of microvessels was performed at 6 and 24 hrs post-FUS. Expression changes in individual genes and bioinformatic analysis suggests that FUS may induce a transient inflammatory response in microvessels. Increased transcription of proinflammatory cytokine genes appears to be short-lived, largely returning to baseline by 24 hrs. This observation may help to explain some previously observed bioeffects of FUS and may also be a driving force for the angiogenic processes and reduced drug efflux suggested by this work. While further studies are necessary, these results open up intriguing possibilities for novel FUS applications and suggest possible routes for pharmacologically modifying the technique.