Tumour status prediction by means of carbon-ion beam irradiation: comparison of washout rates between in-beam PET and DCE-MRI in rats.

Objective.Tumour response to radiation therapy appears as changes in tumour vascular condition. There are several methods for analysing tumour blood circulatory changes one of which is dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI), but there is no method that can observe the tumour vascular condition and physiological changes at the site of radiation therapy. Positron emission tomography (PET) has been applied for treatment verification in charged particle therapy, which is based on the detection of positron emitters produced through nuclear fragmentation reactions in a patient's body. However, the produced positron emitters are washed out biologically depending on the tumour vascular condition. This means that measuring the biological washout rate may allow evaluation of the tumour radiation response, in a similar manner to DCE-MRI. Therefore, this study compared the washout rates in rats between in-beam PET during12C ion beam irradiation and DCE-MRI.Approach.Different vascular conditions of the tumour model were prepared for six nude rats. The tumour of each nude rat was irradiated by a12C ion beam with simultaneous in-beam PET measurement. In 10-12 h, the DCE-MRI experiment was performed for the same six nude rats. The biological washout rate of the produced positron emitters (k2,1st) and the MRI contrast agent (k2a) were derived using the single tissue compartment model.Main results.A linear correlation was observed betweenk2,1standk2a, and they were inversely related to fractional necrotic volume.Significance.This is the first animal study which confirmed the biological washout rate of in-beam PET correlates closely with tumour vascular condition measured with the MRI contrast agent administrated intravenously.

Acceleration of the Development of Microcirculation Embolism in the Brain due to Capillary Narrowing.

BACKGROUND: Cerebral microvascular obstruction is critically involved in recurrent stroke and decreased cerebral blood flow with age. The obstruction must occur in the capillary with a greater resistance to perfusion pressure through the microvascular networks. However, little is known about the relationship between capillary size and embolism formation. This study aimed to determine whether the capillary lumen space contributes to the development of microcirculation embolism. METHODS: To spatiotemporally manipulate capillary diameters in vivo, transgenic mice expressing the light-gated cation channel protein ChR2 (channelrhodopsin-2) in mural cells were used. The spatiotemporal changes in the regional cerebral blood flow in response to the photoactivation of ChR2 mural cells were first characterized using laser speckle flowgraphy. Capillary responses to optimized photostimulation were then examined in vivo using 2-photon microscopy. Finally, microcirculation embolism due to intravenously injected fluorescent microbeads was compared under conditions with or without photoactivation of ChR2 mural cells. RESULTS: Following transcranial photostimulation, the stimulation intensity-dependent decrease in cerebral blood flow centered at the irradiation was observed (14%-49% decreases relative to the baseline). The cerebrovascular response to photostimulation showed significant constriction of the cerebral arteries and capillaries but not of the veins. As a result of vasoconstriction, a temporal stall of red blood cell flow occurred in the capillaries of the venous sides. The 2-photon excitation of a single ChR2 pericyte demonstrated the partial shrinkage of capillaries (7% relative to the baseline) around the stimulated cell. With the intravenous injection of microbeads, the occurrence of microcirculation embolism was significantly enhanced (11% increases compared to the control) with photostimulation. CONCLUSIONS: Capillary narrowing increases the risk of developing microcirculation embolism in the venous sides of the cerebral capillaries.

Capillary responses to functional and pathological activations rely on the capillary states at rest.

Brain capillaries play a crucial role in maintaining cellular viability and thus preventing neurodegeneration. The aim of this study was to characterize the brain capillary morphology at rest and during neural activation based on a big data analysis from three-dimensional microangiography. Neurovascular responses were measured using a genetic calcium sensor expressed in neurons and microangiography with two-photon microscopy, while neural acivity was modulated by stimulation of contralateral whiskers or by a seizure evoked by kainic acid. For whisker stimulation, 84% of the capillary sites showed no detectable diameter change. The remaining 10% and 6% were dilated and constricted, respectively. Significant differences were observed for capillaries in the diameter at rest between the locations of dilation and constriction. Even the seizures resulted in 44% of the capillaries having no detectable change in diameter, while 56% of the capillaries dilated. The extent of dilation was dependent on the diameter at rest. In conclusion, big data analysis on brain capillary morphology has identified at least two types of capillary states: capillaries with diameters that are relatively large at rest and stable over time regardless of neural activity and capillaries whose diameters are relatively small at rest and vary according to neural activity.

Spatiotemporal analysis of blood plasma and blood cell flow fluctuations of cerebral microcirculation in anesthetized rats.

Cerebral hemodynamics fluctuates spontaneously over broad frequency ranges. However, its spatiotemporal coherence of flow oscillations in cerebral microcirculation remains incompletely understood. The objective of this study was to characterize the spatiotemporal fluctuations of red blood cells (RBCs) and plasma flow in the rat cerebral microcirculation by simultaneously imaging their dynamic behaviors. Comparisons of changes in cross-section diameters between RBC and plasma flow showed dissociations in penetrating arterioles. The results indicate that vasomotion has the least effect on the lateral movement of circulating RBCs, resulting in variable changes in plasma layer thickness. Parenchymal capillaries exhibited slow fluctuations in RBC velocity (0.1 to 0.3 Hz), regardless of capillary diameter fluctuations (<0.1 Hz). Temporal fluctuations and the velocity of RBCs decreased significantly at divergent capillary bifurcations. The results indicate that a transit of RBCs generates flow resistance in the capillaries and that slow velocity fluctuations of the RBCs are subject to a number of bifurcations. In conclusion, the high-frequency oscillation of the blood flow is filtered at the bifurcation through the capillary networks. Therefore, a number of bifurcations in the cerebral microcirculation may contribute to the power of low-frequency oscillations.

Close association between spreading depolarization and development of infarction under experimental ischemia in anesthetized male mice.

Clinical and experimental evidence suggests that spreading depolarizations (SD) usually occur in patients with ischemic or hemorrhagic stroke when the gray matter of the brain is affected. In this study, we evaluated spatiotemporal changes of cerebral blood flow (CBF) during middle cerebral artery (MCA) occlusion and examined the relationship between SD occurrence and cerebral infarct development. In male isoflurane-anesthetized C57BL/6J mice, CBF changes over the ipsilateral parietal bone were recorded by laser speckle flowgraphy during and after transient (45 min, n = 22) or permanent occlusion (n = 22) of the distal MCA. Infarct volume was evaluated 24 hr after the operation. Upon MCA occlusion, CBF decreased by -55.6 ± 8.5 % in the lowest CBF and linearly recovered with increasing distance from the region. At 1-10 min after onset of occlusion, SD occurred and concentrically propagated from the core region, showing a decrease of CBF in the whole observed area along with a transient hyperemia and oligemia in the normal region. SD spontaneously re-occurred and propagated around the ischemic area in 37 % of mice, accompanied with a marked decrease of CBF in the core or a marked increase of CBF in the normal region. The CBF response to SDs gradually changed from the core to the normal area, depending upon the distance from the core region. Infarction was not observed in transiently (n = 2) or permanently (n = 4) occluded mice without SD. The infarct area tended to be larger with increasing number of SDs in transiently occluded mice. In conclusion, our findings suggest that the occurrence of SD during ischemia might elicit infarct formation and/or influence infarct development.

Measurement of biological washout rates depending on tumor vascular status in15O in-beam rat-PET.

Objective.The biological washout of positron emitters should be modeled and corrected in order to achieve quantitative dose range verification in charged particle therapy based on positron emission tomography (PET). This biological washout effect is affected by physiological environmental conditions such as blood perfusion and metabolism, but the correlation to tumour pathology has not been studied yet.Approach.The aim of this study was to investigate the dependence of the biological washout rate on tumour vascular status in rat irradiation. Two types of tumour vascularity conditions, perfused and hypoxic, were modelled with nude rats. The rats were irradiated by a radioactive15O ion beam and time activity curves were acquired by dynamic in-beam PET measurement. Tumour tissue sections were obtained to observe the histology as well. The biological washout rate was derived using a single-compartment model with two decay components (medium decay,k2mand slow decay,k2s).Main results.Allk2mvalues in the vascular perfused tumour tissue were higher than the values of the normal tissue. Allk2mvalues in the hypoxic tumour tissue were much lower than the values of the vascular perfused tumour tissue and slightly lower than the values of the normal tissue.Significance.The dependency of the biological washout on the tumour vasculature conditions was experimentally shown.

Error Evaluation for Automated Diameter Measurements of Cerebral Capillaries Captured with Two-Photon Laser Scanning Fluorescence Microscopy.

Cerebral capillaries respond to changes in neural activity to maintain regional balances between energy demand and supply. However, the quantitative aspects of the capillary diameter responses and their contribution to oxygen supply to tissue remain incompletely understood. The purpose of the present study is to check if the diameters measured from large-scale angiographic image data of two-photon laser scanning fluorescent microscopy (2PLSM) are correctly determined with a custom-written MATLAB software and to investigate how the measurement errors can be reduced, such as at the junction areas of capillaries. As a result, nearly 17% of the measured locations appeared to be outliers of the automated diameter measurements, in particular arising from the junction areas where three capillary segments merged. We observed that about two-thirds of the outliers originated from the measured locations within 6 µm from the branching point. The results indicate that the capillary locations in the junction areas cause non-negligible errors in the automated diameter measurements. Considering the common site of the outliers, the present study identified that the areas within 6 µm from the branch point could be separately measured from the diameter analysis, and careful manual inspection with reference to the original images for these transition areas around the branch point is further recommended.

Time Series Tracking of Cerebral Microvascular Adaptation to Hypoxia and Hyperoxia Imaged with Repeated In Vivo Two-Photon Microscopy.

The present study describes methodological aspects of image analysis for angiographic image data with long-term two-photon microscopy acquired for the investigation of dynamic changes in the three-dimensional (3D) network structure of the capillaries (less than 8 µm in diameter) in the mouse cerebral cortex. Volume images of the identical capillaries over different periods of days up to 32 days were compared for adaptation under either chronic hypoxia (8-9% O2) or hyperoxia (40-50% O2). We observed that the median diameters of measured capillaries were 5.8, 8.4, 9.0, and 8.4 µm at 0, 1, 2, and 3 weeks during exposure to hypoxia, respectively (N = 1, n = 2193 pairs at day 0), and 5.4, 5.7, 5.4, 6.0, and 6.1 µm measured weekly up to 32 days under hyperoxia (N = 1, n = 1025 pairs at day 0). In accordance with these changes in capillary diameters, tissue space was also observed to change in a depth-dependent manner under hypoxia, but not hyperoxia. The present methods provide us with a method to quantitatively determine three-dimensional vascular and tissue morphology with the aid of a computer-assisted graphical user interface, which facilitates morphometric analysis of the cerebral microvasculature and its correlation with the adaptation of brain cells imaged simultaneously with the microvasculature.

Differential pial and penetrating arterial responses examined by optogenetic activation of astrocytes and neurons.

A variety of brain cells participates in neurovascular coupling by transmitting and modulating vasoactive signals. The present study aimed to probe cell type-dependent cerebrovascular (i.e., pial and penetrating arterial) responses with optogenetics in the cortex of anesthetized mice. Two lines of the transgenic mice expressing a step function type of light-gated cation channel (channelrhodopsine-2; ChR2) in either cortical neurons (muscarinic acetylcholine receptors) or astrocytes (Mlc1-positive) were used in the experiments. Photo-activation of ChR2-expressing astrocytes resulted in a widespread increase in cerebral blood flow (CBF), extending to the nonstimulated periphery. In contrast, photo-activation of ChR2-expressing neurons led to a relatively localized increase in CBF. The differences in the spatial extent of the CBF responses are potentially explained by differences in the involvement of the vascular compartments. In vivo imaging of the cerebrovascular responses revealed that ChR2-expressing astrocyte activation led to the dilation of both pial and penetrating arteries, whereas ChR2-expressing neuron activation predominantly caused dilation of the penetrating arterioles. Pharmacological studies showed that cell type-specific signaling mechanisms participate in the optogenetically induced cerebrovascular responses. In conclusion, pial and penetrating arterial vasodilation were differentially evoked by ChR2-expressing astrocytes and neurons.

Three-dimensional microvascular network reconstruction from in vivo images with adaptation of the regional inhomogeneity in the signal-to-noise ratio.

OBJECTIVE: Quantification of angiographic images with two-photon laser scanning fluorescence microscopy (2PLSM) relies on proper segmentation of the vascular images. However, the images contain inhomogeneities in the signal-to-noise ratio (SNR) arising from regional effects of light scattering and absorption. The present study developed a semiautomated quantification method for volume images of 2PLSM angiography by adjusting the binarization threshold according to local SNR along the vessel centerlines. METHODS: A phantom model made with fluorescent microbeads was used to incorporate a region-dependent binarization threshold. RESULTS: The recommended SNR for imaging was found to be 4.2-10.6 that provide the true size of imaged objects if the binarization threshold was fixed at 50% of SNR. However, angiographic images in the mouse cortex showed variable SNR up to 45 over the depths. To minimize the errors caused by variable SNR and a spatial extent of the imaged objects in an axial direction, the microvascular networks were three-dimensionally reconstructed based on the cross-sectional diameters measured along the vessel centerline from the XY-plane images with adapted binarization threshold. The arterial volume was relatively constant over depths of 0-500 µm, and the capillary volume (1.7% relative to the scanned volume) showed the larger volumes than the artery (0.8%) and vein (0.6%). CONCLUSIONS: The present methods allow consistent segmentation of microvasculature by adapting the local inhomogeneity in the SNR, which will be useful for quantitative comparison of the microvascular networks, such as under disease conditions where SNR in the 2PLSM images varies over space and time.

Biological washout modelling for in-beam PET: rabbit brain irradiation by 11C and 15O ion beams.

Positron emission tomography (PET) has been used for dose verification in charged particle therapy. The causes of washout of positron emitters by physiological functions should be clarified for accurate dose verification. In this study, we visualized the distribution of irradiated radioactive beams, 11C and 15O beams, in the rabbit whole-body using our original depth-of-interaction (DOI)-PET prototype to add basic data for biological washout effect correction. Time activity curves of the irradiated field and organs were measured immediately after the irradiations. All data were corrected for physical decay before further analysis. We also collected expired gas of the rabbit during beam irradiation and the energy spectrum was measured with a germanium detector. Irradiated radioactive beams into the brain were distributed to the whole body due to the biological washout process, and the implanted 11C and 15O ions were concentrated in the regions which had high blood volume. The 11C-labelled 11CO2 was detected in expired gas under the 11C beam irradiation, while no significant signal was detected under the 15O beam irradiation as a form of C15O2. Results suggested that the implanted 11C ions form molecules that diffuse out to the whole body by undergoing perfusion, then, they are incorporated into the blood-gas exchange in the respiratory system. This study provides basic data for modelling of the biological washout effect.

Spatiotemporal dynamics of red blood cells in capillaries in layer I of the cerebral cortex and changes in arterial diameter during cortical spreading depression and response to hypercapnia in anesthetized mice.

OBJECTIVE: Control of red blood cell velocity in capillaries is essential to meet local neuronal metabolic requirements, although changes of capillary diameter are limited. To further understand the microcirculatory response during cortical spreading depression, we analyzed the spatiotemporal changes of red blood cell velocity in intraparenchymal capillaries. METHODS: In urethane-anesthetized Tie2-green fluorescent protein transgenic mice, the velocity of fluorescence-labeled red blood cells flowing in capillaries in layer I of the cerebral cortex was automatically measured with our Matlab domain software (KEIO-IS2) in sequential images obtained with a high-speed camera laser-scanning confocal fluorescence microscope system. RESULTS: Cortical spreading depression repeatedly increased the red blood cell velocity prior to arterial constriction/dilation. During the first cortical spreading depression, red blood cell velocity significantly decreased, and sluggishly moving or retrograde-moving red blood cells were observed, concomitantly with marked arterial constriction. The velocity subsequently returned to around the basal level, while oligemia after cortical spreading depression with slight vasoconstriction remained. After several passages of cortical spreading depression, hypercapnia-induced increase of red blood cell velocity, regional cerebral blood flow and arterial diameter were all significantly reduced, and the correlations among them became extremely weak. CONCLUSIONS: Taken together with our previous findings, these simultaneous measurements of red blood cell velocity in multiple capillaries, arterial diameter and regional cerebral blood flow support the idea that red blood cell flow might be altered independently, at least in part, from arterial regulation, that neuro-capillary coupling plays a role in rapidly meeting local neural demand.

Positron emission tomography of cerebral angiogenesis and TSPO expression in a mouse model of chronic hypoxia.

The present study aimed to examine whether positron emission tomography (PET) could evaluate cerebral angiogenesis. Mice were housed in a hypoxic chamber with 8-9% oxygen for 4, 7, and 14 days, and the angiogenic responses were evaluated with a radiotracer, 64Cu-cyclam-RAFT-c(-RGDfK-)4, which targeted αVß3 integrin and was imaged with PET. The PET imaging results showed little uptake during all of the hypoxic periods. Immunofluorescence staining of the ß3 integrin, CD61, revealed weak expression, while the microvessel density assessed by CD31 staining increased with the hypoxic duration. These observations suggest that the increased vascular density originated from other types of vascular remodeling, unlike angiogenic sprouting. We then searched for any signs of vascular remodeling that could be detected using PET. PET imaging of 11C-PK11195, a marker of the 18-kDa translocator protein (TSPO), revealed a transient increase at day 4 of hypoxia. Because the immunofluorescence of glial markers showed unchanged staining over the early phase of hypoxia, the observed upregulation of TSPO expression probably originated from non-glial cells (e.g. vascular cells). In conclusion, a transient increase in TSPO probe uptake was detected with PET at only the early phase of hypoxia, which indicates an early sign of vascular remodeling induced by hypoxia.

Visual evaluation of kinetic characteristics of PET probe for neuroreceptors using a two-phase graphic plot analysis.

Objectives In PET studies for neuroreceptors, tracer kinetics are described by the two-tissue compartment model (2-TCM), and binding parameters, including the total distribution volume (V T), non-displaceable distribution volume (V ND), and binding potential (BPND), can be determined from model parameters estimated by kinetic analysis. The stability of binding parameter estimates depends on the kinetic characteristics of radioligands. To describe these kinetic characteristics, we previously developed a two-phase graphic plot analysis in which V ND and V T can be estimated from the x-intercept of regression lines for early and delayed phases, respectively. In this study, we applied this graphic plot analysis to visual evaluation of the kinetic characteristics of radioligands for neuroreceptors, and investigated a relationship between the shape of these graphic plots and the stability of binding parameters estimated by the kinetic analysis with 2-TCM in simulated brain tissue time-activity curves (TACs) with various binding parameters. Methods 90-min TACs were generated with the arterial input function and assumed kinetic parameters according to 2-TCM. Graphic plot analysis was applied to these simulated TACs, and the curvature of the plot for each TAC was evaluated visually. TACs with several noise levels were also generated with various kinetic parameters, and the bias and variation of binding parameters estimated by kinetic analysis were calculated in each TAC. These bias and variation were compared with the shape of graphic plots. Results The graphic plots showed larger curvature for TACs with higher specific binding and slower dissociation of specific binding. The quartile deviations of V ND and BPND determined by kinetic analysis were smaller for radioligands with slow dissociation. Conclusions The larger curvature of graphic plots for radioligands with slow dissociation might indicate a stable determination of V ND and BPND by kinetic analysis. For investigation of the kinetics of radioligands, such kinetic characteristics should be considered.

Dynamic diameter response of intraparenchymal penetrating arteries during cortical spreading depression and elimination of vasoreactivity to hypercapnia in anesthetized mice.

Cortical spreading depression (CSD) induces marked hyperemia with a transient decrease of regional cerebral blood flow (rCBF), followed by sustained oligemia. To further understand the microcirculatory mechanisms associated with CSD, we examined the temporal changes of diameter of intraparenchymal penetrating arteries during CSD. In urethane-anesthetized mice, the diameter of single penetrating arteries at three depths was measured using two-photon microscopy during passage of repeated CSD, with continuous recordings of direct current potential and rCBF. The first CSD elicited marked constriction superimposed on the upstrokes of profound dilation throughout each depth of the penetrating artery, and the vasoreaction temporally corresponded to the change of rCBF. Second or later CSD elicited marked dilation with little or no constriction phase throughout each depth, and the vasodilation also temporally corresponded to the increase of rCBF. Furthermore, the peak dilation showed good negative correlations with basal diameter and increase of rCBF. Vasodilation induced by 5% CO2 inhalation was significantly suppressed after CSD passage at any depth as well as hyperperfusion. These results may indicate that CSD-induced rCBF changes mainly reflect the diametric changes of the intraparenchymal arteries, despite the elimination of responsiveness to hypercapnia.

Changes in effective diffusivity for oxygen during neural activation and deactivation estimated from capillary diameter measured by two-photon laser microscope.

The relation between cerebral blood flow (CBF) and cerebral oxygen extraction fraction (OEF) can be expressed using the effective diffusivity for oxygen in the capillary bed (D) as OEF = 1 - exp(-D/CBF). The D value is proportional to the microvessel blood volume. In this study, changes in D during neural activation and deactivation were estimated from changes in capillary and arteriole diameter measured by two-photon microscopy in awake mice. Capillary and arteriole vessel diameter in the somatosensory cortex and cerebellum were measured under neural activation (sensory stimulation) and neural deactivation [crossed cerebellar diaschisis (CCD)], respectively. Percentage changes in D during sensory stimulation and CCD were 10.3 ± 7.3 and -17.5 ± 5.3 % for capillary diameter of <6 µm, respectively. These values were closest to the percentage changes in D calculated from previously reported human positron emission tomography data. This may indicate that thinner capillaries might play the greatest role in oxygen transport from blood to brain tissue.

Dynamic Flow Velocity Mapping from Fluorescent Dye Transit Times in the Brain Surface Microcirculation of Anesthetized Rats and Mice.

OBJECTIVE: This study aimed to develop a new method for mapping blood flow velocity based on the spatial evolution of fluorescent dye transit times captured with CLSFM in the cerebral microcirculation of anesthetized rodents. METHODS: The animals were anesthetized with isoflurane, and a small amount of fluorescent dye was intravenously injected to label blood plasma. The CLSFM was conducted through a closed cranial window to capture propagation of the dye in the cortical vessels. The transit time of the dye over a certain distance in a single vessel was determined with automated image analyses, and average flow velocity was mapped in each vessel. RESULTS: The average flow velocity measured in the rat pial artery and vein was 4.4 ± 1.2 and 2.4 ± 0.5 mm/sec, respectively. A similar range of flow velocity to those of the rats was observed in the mice; 4.9 ± 1.4 and 2.0 ± 0.9 mm/sec, respectively, although the vessel diameter in the mice was about half of that in the rats. CONCLUSIONS: Flow velocity in the cerebral microcirculation can be mapped based on fluorescent dye transit time measurements with conventional CLSFM in experimental animals.