Cortical microvascular blood flow velocity mapping by combining dynamic light scattering optical coherence tomography and two-photon microscopy.

Significance: The accurate large-scale mapping of cerebral microvascular blood flow velocity is crucial for a better understanding of cerebral blood flow (CBF) regulation. Although optical imaging techniques enable both high-resolution microvascular angiography and fast absolute CBF velocity measurements in the mouse cortex, they usually require different imaging techniques with independent system configurations to maximize their performances. Consequently, it is still a challenge to accurately combine functional and morphological measurements to co-register CBF speed distribution from hundreds of microvessels with high-resolution microvascular angiograms. Aim: We propose a data acquisition and processing framework to co-register a large set of microvascular blood flow velocity measurements from dynamic light scattering optical coherence tomography (DLS-OCT) with the corresponding microvascular angiogram obtained using two-photon microscopy (2PM). Approach: We used DLS-OCT to first rapidly acquire a large set of microvascular velocities through a sealed cranial window in mice and then to acquire high-resolution microvascular angiograms using 2PM. The acquired data were processed in three steps: (i) 2PM angiogram coregistration with the DLS-OCT angiogram, (ii) 2PM angiogram segmentation and graphing, and (iii) mapping of the CBF velocities to the graph representation of the 2PM angiogram. Results: We implemented the developed framework on the three datasets acquired from the mice cortices to facilitate the coregistration of the large sets of DLS-OCT flow velocity measurements with 2PM angiograms. We retrieved the distributions of red blood cell velocities in arterioles, venules, and capillaries as a function of the branching order from precapillary arterioles and postcapillary venules from more than 1000 microvascular segments. Conclusions: The proposed framework may serve as a useful tool for quantitative analysis of large microvascular datasets obtained by OCT and 2PM in studies involving normal brain functioning, progression of various diseases, and numerical modeling of the oxygen advection and diffusion in the realistic microvascular networks.

Aerobic exercise reverses aging-induced depth-dependent decline in cerebral microcirculation.

Aging is a major risk factor for cognitive impairment. Aerobic exercise benefits brain function and may promote cognitive health in older adults. However, underlying biological mechanisms across cerebral gray and white matter are poorly understood. Selective vulnerability of the white matter to small vessel disease and a link between white matter health and cognitive function suggests a potential role for responses in deep cerebral microcirculation. Here, we tested whether aerobic exercise modulates cerebral microcirculatory changes induced by aging. To this end, we carried out a comprehensive quantitative examination of changes in cerebral microvascular physiology in cortical gray and subcortical white matter in mice (3-6 vs. 19-21 months old), and asked whether and how exercise may rescue age-induced deficits. In the sedentary group, aging caused a more severe decline in cerebral microvascular perfusion and oxygenation in deep (infragranular) cortical layers and subcortical white matter compared with superficial (supragranular) cortical layers. Five months of voluntary aerobic exercise partly renormalized microvascular perfusion and oxygenation in aged mice in a depth-dependent manner, and brought these spatial distributions closer to those of young adult sedentary mice. These microcirculatory effects were accompanied by an improvement in cognitive function. Our work demonstrates the selective vulnerability of the deep cortex and subcortical white matter to aging-induced decline in microcirculation, as well as the responsiveness of these regions to aerobic exercise.

Differential reductions in the capillary red-blood-cell flux between retina and brain under chronic global hypoperfusion.

Significance: It has been hypothesized that abnormal microcirculation in the retina might predict the risk of ischemic damages in the brain. Direct comparison between the retinal and the cerebral microcirculation using similar animal preparation and under similar experimental conditions would help test this hypothesis. Aim: We investigated capillary red-blood-cell (RBC) flux changes under controlled conditions and bilateral-carotid-artery-stenosis (BCAS)-induced hypoperfusion, and then compared them with our previous measurements performed in the brain. Approach: We measured capillary RBC flux in mouse retina with two-photon microscopy using a fluorescence-labeled RBC-passage approach. Key physiological parameters were monitored during experiments to ensure stable physiology. Results: We found that under the controlled conditions, capillary RBC flux in the retina was much higher than in the brain (i.e., cerebral cortical gray matter and subcortical white matter), and that BCAS induced a much larger decrease in capillary RBC flux in the retina than in the brain. Conclusions: We demonstrated a two-photon microscopy-based technique to efficiently measure capillary RBC flux in the retina. Since cerebral subcortical white matter often exhibits early pathological developments due to global hypoperfusion, our results suggest that retinal microcirculation may be utilized as an early marker of brain diseases involving global hypoperfusion.

Measurements of cerebral microvascular blood flow, oxygenation, and morphology in a mouse model of whole-brain irradiation-induced cognitive impairment by two-photon microscopy and optical coherence tomography: evidence for microvascular injury in the cerebral white matter.

Whole-brain irradiation (WBI, also known as whole-brain radiation therapy) is a mainstay treatment modality for patients with multiple brain metastases. It is also used as a prophylactic treatment for microscopic tumors that cannot be detected by magnetic resonance imaging. WBI induces a progressive cognitive decline in ~ 50% of the patients surviving over 6 months, significantly compromising the quality of life. There is increasing preclinical evidence that radiation-induced injury to the cerebral microvasculature and accelerated neurovascular senescence plays a central role in this side effect of WBI. To better understand this side effect, male C57BL/6 mice were first subjected to a clinically relevant protocol of fractionated WBI (5 Gy, two doses per week, for 4 weeks). Nine months post the WBI treatment, we applied two-photon microscopy and Doppler optical coherence tomography to measure capillary red-blood-cell (RBC) flux, capillary morphology, and microvascular oxygen partial pressure (PO2) in the cerebral somatosensory cortex in the awake, head-restrained, WPI-treated mice and their age-matched controls, through a cover-glass-sealed chronic cranial window. Thanks to the extended penetration depth with the fluorophore - Alexa680, measurements of capillary blood flow properties (e.g., RBC flux, speed, and linear density) in the cerebral subcortical white matter were enabled. We found that the WBI-treated mice exhibited a significantly decreased capillary RBC flux in the white matter. WBI also caused a significant reduction in capillary diameter, as well as a large (although insignificant) reduction in segment density at the deeper cortical layers (e.g., 600-700 µm), while the other morphological properties (e.g., segment length and tortuosity) were not obviously affected. In addition, we found that PO2 measured in the arterioles and venules, as well as the calculated oxygen saturation and oxygen extraction fraction, were not obviously affected by WBI. Lastly, WBI was associated with a significant increase in the erythrocyte-associated transients of PO2, while the changes of other cerebral capillary PO2 properties (e.g., capillary mean-PO2, RBC-PO2, and InterRBC-PO2) were not significant. Collectively, our findings support the notion that WBI results in persistent cerebral white matter microvascular impairment, which likely contributes to the WBI-induced brain injury and cognitive decline. Further studies are warranted to assess the WBI-induced changes in brain tissue oxygenation and malfunction of the white matter microvasculature as well.

Optical measurement of microvascular oxygenation and blood flow responses in awake mouse cortex during functional activation.

The cerebral cortex has a number of conserved morphological and functional characteristics across brain regions and species. Among them, the laminar differences in microvascular density and mitochondrial cytochrome c oxidase staining suggest potential laminar variability in the baseline O2 metabolism and/or laminar variability in both O2 demand and hemodynamic response. Here, we investigate the laminar profile of stimulus-induced intravascular partial pressure of O2 (pO2) transients to stimulus-induced neuronal activation in fully awake mice using two-photon phosphorescence lifetime microscopy. Our results demonstrate that stimulus-induced changes in intravascular pO2 are conserved across cortical layers I-IV, suggesting a tightly controlled neurovascular response to provide adequate O2 supply across cortical depth. In addition, we observed a larger change in venular O2 saturation (ΔsO2) compared to arterioles, a gradual increase in venular ΔsO2 response towards the cortical surface, and absence of the intravascular "initial dip" previously reported under anesthesia. This study paves the way for quantification of layer-specific cerebral O2 metabolic responses, facilitating investigation of brain energetics in health and disease and informed interpretation of laminar blood oxygen level dependent functional magnetic resonance imaging signals.

Intracranial pressure spikes trigger spreading depolarizations.

Spreading depolarizations are highly prevalent and spatiotemporally punctuated events worsening the outcome of brain injury. Trigger factors are poorly understood but may be linked to sudden worsening in supply-demand mismatch in compromised tissue. Sustained or transient elevations in intracranial pressure are also prevalent in the injured brain. Here, using a mouse model of large hemispheric ischaemic stroke, we show that mild and brief intracranial pressure elevations (20 or 30 mmHg for just 3 min) potently trigger spreading depolarizations in ischaemic penumbra (4-fold increase in spreading depolarization occurrence). We also show that 30 mmHg intracranial pressure spikes as brief as 30 s are equally effective. In contrast, sustained intracranial pressure elevations to the same level for 30 min do not significantly increase the spreading depolarization rate, suggesting that an abrupt disturbance in the steady state equilibrium is required to trigger a spreading depolarization. Laser speckle flowmetry consistently showed a reduction in tissue perfusion, and two-photon pO2 microscopy revealed a drop in venous pO2 during the intracranial pressure spikes suggesting increased oxygen extraction fraction, and therefore, worsening supply-demand mismatch. These haemodynamic changes during intracranial pressure spikes were associated with highly reproducible increases in extracellular potassium levels in penumbra. Consistent with the experimental data, a higher rate of intracranial pressure spikes was associated with spreading depolarization clusters in a retrospective series of patients with aneurysmal subarachnoid haemorrhage with strong temporal correspondence. Altogether, our data show that intracranial pressure spikes, even when mild and brief, are capable of triggering spreading depolarizations. Aggressive prevention of intracranial pressure spikes may help reduce spreading depolarization occurrence and improve outcomes after brain injury.

Quantitation of cerebral oxygen tension using phasor analysis and phosphorescence lifetime imaging microscopy (PLIM).

Time-domain measurements for fluorescence lifetime imaging microscopy (FLIM) and phosphorescence lifetime imaging microscopy (PLIM) are conventionally computed by nonlinear curve fitting techniques to model the time-resolved profiles as mono- or multi-exponential decays. However, these techniques are computationally intensive and prone to fitting errors. The phasor or "polar plot" analysis method has recently gained attention as a simple method to characterize fluorescence lifetime. Here, we adapted the phasor analysis method for absolute quantitation of phosphorescence lifetimes of oxygen-sensitive phosphors and used the phasor-derived lifetime values to quantify oxygen partial pressure (pO2) in cortical microvessels of awake mice. Our results, both experimental and simulated, demonstrate that oxygen measurements obtained from computationally simpler phasor analysis agree well with traditional curve fitting calculations. To our knowledge, the current study constitutes the first application of the technique for characterizing microsecond-length, time-domain phosphorescence measurements and absolute, in vivo quantitation of a vital physiological parameter. The method shows promise for monitoring cerebral metabolism and pathological changes in preclinical rodent models.

Two-photon microscopic imaging of capillary red blood cell flux in mouse brain reveals vulnerability of cerebral white matter to hypoperfusion.

Despite the importance of understanding the regulation of microvascular blood flow in white matter, no data on subcortical capillary blood flow parameters are available, largely due to the lack of appropriate imaging methods. To address this knowledge gap, we employed two-photon microscopy using a far-red fluorophore Alexa680 and photon-counting detection to measure capillary red blood cell (RBC) flux in both cerebral gray and white matter, in isoflurane-anesthetized mice. We have found that in control animals, baseline capillary RBC flux in the white matter was significantly higher than in the adjacent cerebral gray matter. In response to mild hypercapnia, RBC flux in the white matter exhibited significantly smaller fractional increase than in the gray matter. Finally, during global cerebral hypoperfusion, RBC flux in the white matter was reduced significantly in comparison to the controls, while RBC flux in the gray matter was preserved. Our results suggest that blood flow in the white matter may be less efficiently regulated when challenged by physiological perturbations as compared to the gray matter. Importantly, the blood flow in the white matter may be more susceptible to hypoperfusion than in the gray matter, potentially exacerbating the white matter deterioration in brain conditions involving global cerebral hypoperfusion.

More homogeneous capillary flow and oxygenation in deeper cortical layers correlate with increased oxygen extraction.

Our understanding of how capillary blood flow and oxygen distribute across cortical layers to meet the local metabolic demand is incomplete. We addressed this question by using two-photon imaging of resting-state microvascular oxygen partial pressure (PO2) and flow in the whisker barrel cortex in awake mice. Our measurements in layers I-V show that the capillary red-blood-cell flux and oxygenation heterogeneity, and the intracapillary resistance to oxygen delivery, all decrease with depth, reaching a minimum around layer IV, while the depth-dependent oxygen extraction fraction is increased in layer IV, where oxygen demand is presumably the highest. Our findings suggest that more homogeneous distribution of the physiological observables relevant to oxygen transport to tissue is an important part of the microvascular network adaptation to local brain metabolism. These results will inform the biophysical models of layer-specific cerebral oxygen delivery and consumption and improve our understanding of the diseases that affect cerebral microcirculation.

Two-photon phosphorescence lifetime microscopy of retinal capillary plexus oxygenation in mice.

Impaired oxygen delivery and/or consumption in the retinal tissue underlies the pathophysiology of many retinal diseases. However, the essential tools for measuring oxygen concentration in retinal capillaries and studying oxygen transport to retinal tissue are still lacking. We show that two-photon phosphorescence lifetime microscopy can be used to map absolute partial pressures of oxygen (pO2) in the retinal capillary plexus. Measurements were performed at various retinal depths in anesthetized mice under systemic normoxic and hyperoxic conditions. We used a newly developed two-photon phosphorescent oxygen probe, based on a two-photon absorbing platinum tetraphthalimidoporphyrin, and commercially available optics without correction for optical aberrations of the eye. The transverse and axial distances within the tissue volume were calibrated using a model of the eye's optical system. We believe this is the first demonstration of in vivo depth-resolved imaging of pO2 in retinal capillaries. Application of this method has the potential to advance our understanding of oxygen delivery on the microvascular scale and help elucidate mechanisms underlying various retinal diseases.

Phasor analysis of NADH FLIM identifies pharmacological disruptions to mitochondrial metabolic processes in the rodent cerebral cortex.

Investigating cerebral metabolism in vivo at a microscopic level is essential for understanding brain function and its pathological alterations. The intricate signaling and metabolic dynamics between neurons, glia, and microvasculature requires much more detailed understanding to better comprehend the mechanisms governing brain function and its disease-related changes. We recently demonstrated that pharmacologically-induced alterations to different steps of cerebral metabolism can be distinguished utilizing 2-photon fluorescence lifetime imaging of endogenous reduced nicotinamide adenine dinucleotide (NADH) fluorescence in vivo. Here, we evaluate the ability of the phasor analysis method to identify these pharmacological metabolic alterations and compare the method's performance with more conventional nonlinear curve-fitting analysis. Visualization of phasor data, both at the fundamental laser repetition frequency and its second harmonic, enables resolution of pharmacologically-induced alterations to mitochondrial metabolic processes from baseline cerebral metabolism. Compared to our previous classification models based on nonlinear curve-fitting, phasor-based models required fewer parameters and yielded comparable or improved classification accuracy. Fluorescence lifetime imaging of NADH and phasor analysis shows utility for detecting metabolic alterations and will lead to a deeper understanding of cerebral energetics and its pathological changes.

Intrinsic optical signal imaging of the blood volume changes is sufficient for mapping the resting state functional connectivity in the rodent cortex.

OBJECTIVE: Resting state functional connectivity (RSFC) allows the study of functional organization in normal and diseased brain by measuring the spontaneous brain activity generated under resting conditions. Intrinsic optical signal imaging (IOSI) based on multiple illumination wavelengths has been used successfully to compute RSFC maps in animal studies. The IOSI setup complexity would be greatly reduced if only a single wavelength can be used to obtain comparable RSFC maps. APPROACH: We used anesthetized mice and performed various comparisons between the RSFC maps based on single wavelength as well as oxy-, deoxy- and total hemoglobin concentration changes. MAIN RESULTS: The RSFC maps based on IOSI at a single wavelength selected for sensitivity to the blood volume changes are quantitatively comparable to the RSFC maps based on oxy- and total hemoglobin concentration changes obtained by the more complex IOSI setups. Moreover, RSFC maps do not require CCD cameras with very high frame acquisition rates, since our results demonstrate that they can be computed from the data obtained at frame rates as low as 5 Hz. SIGNIFICANCE: Our results will have general utility for guiding future RSFC studies based on IOSI and making decisions about the IOSI system designs.

Cerebral metabolism in a mouse model of Alzheimer's disease characterized by two-photon fluorescence lifetime microscopy of intrinsic NADH.

Disruptions and alterations to cerebral energy metabolism play a vital role in the onset and progression of many neurodegenerative disorders and cerebral pathologies. In order to precisely understand the complex alterations underlying Alzheimer's disease (AD) progression, in vivo imaging at the microscopic level is required in preclinical animal models. Utilizing two-photon fluorescence lifetime imaging microscopy and the phasor analysis method, we have observed AD-related variations of endogenous fluorescence of reduced nicotinamide adenine dinucleotide (NADH) in vivo. We collected NADH FLIM images from the cerebral cortices of both APPswe:PS1dE9 mice to model amyloid ß plaque accumulation and corresponding age-matched wildtype controls. Distinct variations in NADH fluorescence lifetime between wildtype and AD mice, as well as variations related to proximity from amyloid plaques, are obvervable via the phasor method. The combination of NADH FLIM and phasor analysis allows for a minimally invasive, high-resolution technique to characterize the adverse effects of amyloid ß accumulation on mitochondrial energy metabolism and could guide our understanding of preclinical AD pathology.

Shear-induced diffusion of red blood cells measured with dynamic light scattering-optical coherence tomography.

Quantitative measurements of intravascular microscopic dynamics, such as absolute blood flow velocity, shear stress and the diffusion coefficient of red blood cells (RBCs), are fundamental in understanding the blood flow behavior within the microcirculation, and for understanding why diffuse correlation spectroscopy (DCS) measurements of blood flow are dominantly sensitive to the diffusive motion of RBCs. Dynamic light scattering-optical coherence tomography (DLS-OCT) takes the advantages of using DLS to measure particle flow and diffusion within an OCT resolution-constrained three-dimensional volume, enabling the simultaneous measurements of absolute RBC velocity and diffusion coefficient with high spatial resolution. In this work, we applied DLS-OCT to measure both RBC velocity and the shear-induced diffusion coefficient within penetrating venules of the somatosensory cortex of anesthetized mice. Blood flow laminar profile measurements indicate a blunted laminar flow profile and the degree of blunting decreases with increasing vessel diameter. The measured shear-induced diffusion coefficient was proportional to the flow shear rate with a magnitude of ~0.1 to 0.5 × 10-6 mm2 . These results provide important experimental support for the recent theoretical explanation for why DCS is dominantly sensitive to RBC diffusive motion.

Capillary red blood cell velocimetry by phase-resolved optical coherence tomography.

We present a phase-resolved optical coherence tomography (OCT) method to extend Doppler OCT for the accurate measurement of the red blood cell (RBC) velocity in cerebral capillaries. OCT data were acquired with an M-mode scanning strategy (repeated A-scans) to account for the single-file passage of RBCs in a capillary, which were then high-pass filtered to remove the stationary component of the signal to ensure an accurate measurement of phase shift of flowing RBCs. The angular frequency of the signal from flowing RBCs was then quantified from the dynamic component of the signal and used to calculate the axial speed of flowing RBCs in capillaries. We validated our measurement by RBC passage velocimetry using the signal magnitude of the same OCT time series data.

Fluorescence lifetime microscopy of NADH distinguishes alterations in cerebral metabolism in vivo.

Evaluating cerebral energy metabolism at microscopic resolution is important for comprehensively understanding healthy brain function and its pathological alterations. Here, we resolve specific alterations in cerebral metabolism in vivo in Sprague Dawley rats utilizing minimally-invasive 2-photon fluorescence lifetime imaging (2P-FLIM) measurements of reduced nicotinamide adenine dinucleotide (NADH) fluorescence. Time-resolved fluorescence lifetime measurements enable distinction of different components contributing to NADH autofluorescence. Ostensibly, these components indicate different enzyme-bound formulations of NADH. We observed distinct variations in the relative proportions of these components before and after pharmacological-induced impairments to several reactions involved in glycolytic and oxidative metabolism. Classification models were developed with the experimental data and used to predict the metabolic impairments induced during separate experiments involving bicuculline-induced seizures. The models consistently predicted that prolonged focal seizure activity results in impaired activity in the electron transport chain, likely the consequence of inadequate oxygen supply. 2P-FLIM observations of cerebral NADH will help advance our understanding of cerebral energetics at a microscopic scale. Such knowledge will aid in our evaluation of healthy and diseased cerebral physiology and guide diagnostic and therapeutic strategies that target cerebral energetics.

Impact of temporal resolution on estimating capillary RBC-flux with optical coherence tomography.

Optical coherence tomography (OCT) has been used to measure capillary red blood cell (RBC) flux. However, one important technical issue is that the accuracy of this method is subject to the temporal resolution ( ? t ) of the repeated RBC-passage B-scans. A ceiling effect arises due to an insufficient ? t limiting the maximum RBC-flux that can be measured. In this letter, we first present simulations demonstrating that ? t = 1.5 ?? ms permits measuring RBC-flux up to 150 ?? RBCs / s with an underestimation of 9%. The simulations further show that measurements with ? t = 3 and 4.5 ms provide relatively less accurate estimates for typical physiological fluxes. We provide experimental data confirming the simulation results showing that reduced temporal resolution (i.e., a longer ? t ) results in an underestimation of mean flux and compresses the distribution of measured fluxes, which potentially confounds physiological interpretation of the results. The results also apply to RBC-passage measurements made with confocal and two-photon microscopy for estimating capillary RBC-flux.

Optical coherence tomography imaging of capillary reperfusion after ischemic stroke.

Although progress has been made for recanalization therapies after ischemic stroke, post-treatment imaging studies show that tissue reperfusion cannot be attained despite satisfactory recanalization in a significant percentage of patients. Hence, investigation of microcirculatory changes in both surface and deep cortical levels after ischemia reperfusion is important for understanding the post-stroke blood flow dynamics. In this study, we applied optical coherence tomography (OCT) imaging of cerebral blood flow for the quantification of the microcirculatory changes. We obtained OCT microangiogram of the brain cortex in a mouse stroke model and analyzed the data to trace changes in the capillary perfusion level (CPL) before, during, and after the stroke. The CPL changes were estimated in 1 and 2 h ischemia groups as well as in a non-ischemic sham-operated group. For the estimation of CPL, a decorrelation amplitude-based algorithm was implemented and used. As a result, the CPL considerably decreased during ischemia but recovered to the baseline when recanalization was performed 1 h after ischemia; however, the CPL was significantly reduced when recanalization was delayed to 2 h after ischemia. These data demonstrate that ischemia causes microcirculation dysfunction, leading to a decreased capillary reperfusion after recanalization. Microcirculatory no-reflow warrants more rigorous assessment in clinical trials, whereas advanced optical imaging techniques may provide mechanistic insight and solutions in experimental studies.

CD40L-Tri, a novel formulation of recombinant human CD40L that effectively activates B cells.

CD40L has a well-established role in enhancing the immunostimulatory capacity of normal and malignant B cells, but a formulation suitable for clinical use has not been widely available. Like other TNF family members, in vivo and in vitro activity of CD40L requires a homotrimeric configuration, and growing evidence suggests that bioactivity depends on higher-order clustering of CD40. We generated a novel formulation of human recombinant CD40L (CD40L-Tri) in which the CD40L extracellular domain and a trimerization motif are connected by a long flexible peptide linker. We demonstrate that CD40L-Tri significantly expands normal CD19+ B cells by over 20- to 30-fold over 14 days and induces B cells to become highly immunostimulatory antigen-presenting cells (APCs). Consistent with these results, CD40L-Tri-activated B cells could effectively stimulate antigen-specific T responses (against the influenza M1 peptide) from normal volunteers. In addition, CD40L-Tri could induce malignant B cells to become effective APCs, such that tumor-directed immune responses could be probed. Together, our studies demonstrate the potent immune-stimulatory effects of CD40L-Tri on B cells that enable their expansion of antigen-specific human T cells. The potent bioactivity of CD40L-Tri is related to its ability to self-multimerize, which may be facilitated by its long peptide linker.