Awake chronic mouse model of targeted pial vessel occlusion via photothrombosis

Animal models of stroke are used extensively to study the mechanisms involved in the acute and chronic phases of recovery following stroke. A translatable animal model that closely mimics the mechanisms of a human stroke is essential in understanding recovery processes as well as developing therapies that improve functional outcomes. We describe a photothrombosis stroke model that is capable of targeting a single distal pial branch of the middle cerebral artery with minimal damage to the surrounding parenchyma in awake head-fixed mice. Mice are implanted with chronic cranial windows above one hemisphere of the brain that allow optical access to study recovery mechanisms for over a month following occlusion. Additionally, we study the effect of laser spot size used for occlusion and demonstrate that a spot size with small axial and lateral resolution has the advantage of minimizing unwanted photodamage while still monitoring macroscopic changes to cerebral blood flow during photothrombosis. We show that temporally guiding illumination using real-time feedback of blood flow dynamics also minimized unwanted photodamage to the vascular network. Finally, through quantifiable behavior deficits and chronic imaging we show that this model can be used to study recovery mechanisms or the effects of therapeutics longitudinally.R01 EB021018 - NIBIB NIH HHS; R01 MH111359 - NIMH NIH HHS; R01 NS108472 - NINDS NIH HHSPublished versio

Chronic cranial windows for long term multimodal neurovascular imaging in mice

Chronic cranial windows allow for longitudinal brain imaging experiments in awake, behaving mice. Different imaging technologies have their unique advantages and combining multiple imaging modalities offers measurements of a wide spectrum of neuronal, glial, vascular, and metabolic parameters needed for comprehensive investigation of physiological and pathophysiological mechanisms. Here, we detail a suite of surgical techniques for installation of different cranial windows targeted for specific imaging technologies and their combination. Following these techniques and practices will yield higher experimental success and reproducibility of results.R21 EY030727 - NEI NIH HHS; R01 NS108472 - NINDS NIH HHS; R01 NS057198 - NINDS NIH HHS; K99 AG063762 - NIA NIH HHS; R01 DA050159 - NIDA NIH HHS; R01 EB021018 - NIBIB NIH HHS; R01 MH111359 - NIMH NIH HHSPublished versio

The Role of Toll-Like Receptor 2 and 4 Innate Immunity Pathways in Intracortical Microelectrode-Induced Neuroinflammation

We have recently demonstrated that partial inhibition of the cluster of differentiation 14 (CD14) innate immunity co-receptor pathway improves the long-term performance of intracortical microelectrodes better than complete inhibition. We hypothesized that partial activation of the CD14 pathway was critical to a neuroprotective response to the injury associated with initial and sustained device implantation. Therefore, here we investigated the role of two innate immunity receptors that closely interact with CD14 in inflammatory activation. We implanted silicon planar non-recording neural probes into knockout mice lacking Toll-like receptor 2 (Tlr2−/−), knockout mice lacking Toll-like receptor 4 (Tlr4−/−), and wildtype (WT) control mice, and evaluated endpoint histology at 2 and 16 weeks after implantation. Tlr4−/− mice exhibited significantly lower BBB permeability at acute and chronic time points, but also demonstrated significantly lower neuronal survival at the chronic time point. Inhibition of the Toll-like receptor 2 (TLR2) pathway had no significant effect compared to control animals. Additionally, when investigating the maturation of the neuroinflammatory response from 2 to 16 weeks, transgenic knockout mice exhibited similar histological trends to WT controls, except that knockout mice did not exhibit changes in microglia and macrophage activation over time. Together, our results indicate that complete genetic removal of Toll-like receptor 4 (TLR4) was detrimental to the integration of intracortical neural probes, while inhibition of TLR2 had no impact within the tests performed in this study. Therefore, approaches focusing on incomplete or acute inhibition of TLR4 may still improve intracortical microelectrode integration and long term recording performance

Wide-field optical imaging of neurovascular coupling during stroke recovery

Functional neuroimaging, which measure vascular responses to brain activity, are invaluable tools for monitoring and treating stroke patients both in the acute and chronic phases of recovery. However, vascular responses after stroke are almost always altered relative to vascular responses in healthy subjects and it is still unclear if these alterations reflect the underlying brain physiology or if the alterations are purely due to vascular injury. In other words, we do not know the effect of stroke on neurovascular coupling and are therefore limited in our ability to use functional neuroimaging to accurately interpret stroke pathophysiology. There is a need for animal models to investigate the effect of stroke on neurovascular coupling to aid in better interpreting the results from functional neuroimaging. To that end, we first implemented a mouse photothrombotic stroke model that mimics the physiology of a human stroke and therefore has high clinical relevance. Mice were implanted with bilateral cranial windows to allow long term multimodal optical access. The occlusion procedure was performed in awake animals while simultaneously monitoring changes to cerebral blood flow. Our optimized photothrombotic stroke to the somatosensory forelimb region produced a sustained behavioral deficit in the contralateral forelimb that could be monitored longitudinally. Next, we implemented simultaneous imaging of neuronal activity, through fluorescent calcium imaging, and hemodynamics, through intrinsic optical signal imaging, to investigate neurovascular coupling during stroke recovery. Additionally, we identified a novel use for spatial frequency domain imaging to quantify the spatial extent of the stroke core. Finally, we combined the mouse stroke model and imaging platforms to investigate the effect of stroke on neurovascular coupling. We found that acute stroke led to the abolishment of both calcium and hemodynamic responses to sensory stimulation. This elimination of response was associated with a loss of correlation between calcium and hemodynamic activity in the acute phase. To quantify neurovascular coupling, we modeled spatiotemporal hemodynamics by convolving neural activity and hemodynamic response functions obtained from deconvolution. Hemodynamic response functions from healthy animals were unable to model hemodynamics in the acute phase, suggesting neurovascular uncoupling. However, hemodynamics could be modeled in the chronic phase, indicating chronic recoupling. Acute stroke also resulted in increased global brain oscillations, which showed distinct patterns in calcium and hemodynamics, and the increase in contralesional calcium activity was associated with increased functional connectivity. We also show that early return of responses, neurovascular recoupling, and global oscillations were predictors of improved behavioral outcomes.2022-09-26T00:00:00

Optimizing the precision of laser speckle contrast imaging

Abstract Laser speckle contrast imaging (LSCI) is a rapidly developing technology broadly applied for the full-field characterization of tissue perfusion. Over the recent years, significant advancements have been made in interpreting LSCI measurements and improving the technique’s accuracy. On the other hand, the method’s precision has yet to be studied in detail, despite being as important as accuracy for many biomedical applications. Here we combine simulation, theory and animal experiments to systematically evaluate and re-analyze the role of key factors defining LSCI precision—speckle-to-pixel size ratio, polarisation, exposure time and camera-related noise. We show that contrary to the established assumptions, smaller speckle size and shorter exposure time can improve the precision, while the camera choice is less critical and does not affect the signal-to-noise ratio significantly

Neurovascular coupling is preserved in chronic stroke recovery after targeted photothrombosis

Functional neuroimaging, which measures hemodynamic responses to brain activity, has great potential for monitoring recovery in stroke patients and guiding rehabilitation during recovery. However, hemodynamic responses after stroke are almost always altered relative to responses in healthy subjects and it is still unclear if these alterations reflect the underlying brain physiology or if the alterations are purely due to vascular injury. In other words, we do not know the effect of stroke on neurovascular coupling and are therefore limited in our ability to use functional neuroimaging to accurately interpret stroke pathophysiology. To address this challenge, we simultaneously captured neural activity, through fluorescence calcium imaging, and hemodynamics, through intrinsic optical signal imaging, during longitudinal stroke recovery. Our data suggest that neurovascular coupling was preserved in the chronic phase of recovery (2 weeks and 4 weeks post-stoke) and resembled pre-stroke neurovascular coupling. This indicates that functional neuroimaging faithfully represents the underlying neural activity in chronic stroke. Further, neurovascular coupling in the sub-acute phase of stroke recovery was predictive of long-term behavioral outcomes. Stroke also resulted in increases in global brain oscillations, which showed distinct patterns between neural activity and hemodynamics. Increased neural excitability in the contralesional hemisphere was associated with increased contralesional intrahemispheric connectivity. Additionally, sub-acute increases in hemodynamic oscillations were associated with improved sensorimotor outcomes. Collectively, these results support the use of hemodynamic measures of brain activity post-stroke for predicting functional and behavioral outcomes

Improving the characterization of ex vivo human brain optical properties using high numerical aperture optical coherence tomography by spatially constraining the confocal parameters

SIGNIFICANCE: The optical properties of biological samples provide information about the structural characteristics of the tissue and any changes arising from pathological conditions. Optical coherence tomography (OCT) has proven to be capable of extracting tissue's optical properties using a model that combines the exponential decay due to tissue scattering and the axial point spread function that arises from the confocal nature of the detection system, particularly for higher numerical aperture (NA) measurements. A weakness in estimating the optical properties is the inter-parameter cross-talk between tissue scattering and the confocal parameters defined by the Rayleigh range and the focus depth. AIM: In this study, we develop a systematic method to improve the characterization of optical properties with high-NA OCT. APPROACH: We developed a method that spatially parameterizes the confocal parameters in a previously established model for estimating the optical properties from the depth profiles of high-NA OCT. RESULTS: The proposed parametrization model was first evaluated on a set of intralipid phantoms and then validated using a low-NA objective in which cross-talk from the confocal parameters is negligible. We then utilize our spatially parameterized model to characterize optical property changes introduced by a tissue index matching process using a simple immersion agent, 2,2'-thiodiethonal. CONCLUSIONS: Our approach improves the confidence of parameter estimation by reducing the degrees of freedom in the non-linear fitting model.Published versio