Intrinsic Optical Signals Research Articles

Enhanced auditory responses in visual cortex of blind rats using intrinsic optical signal imaging

Functional maturation of the visual cortex is induced by visual experiences during critical periods. Blind animals and humans exhibit improved auditory abilities after losing their vision. Here we investigated the response of the visual cortex to white noise stimuli during the progression of photoreceptor degeneration in a rat model of blindness (Royal College of Surgeons [RCS] (rdy/rdy) rats). Optical coherence tomography of RCS (+/+) rats with normal visual function revealed normal photoreceptor cells, whereas 3-month-old RCS (rdy/rdy) rats demonstrated photoreceptor cell degeneration. Visual cortex responses (VCRs) to a single flash stimulus were negligible in 3-month-old photoreceptor-degenerated rats. However, VCRs with white noise stimuli were significantly increased in blind versus RCS rats (+/+). Slight changes in the intrinsic optical signals of the control rats were observed on the ventral side of the visual cortex. In contrast, responses were markedly increased throughout the visual cortex of RCS (rdy/rdy) rats. These results indicate that the visual cortex rapidly acquires auditory system function over the first 3 months of life and that the entire visual cortex, rather than just the portion close to the auditory cortex, responds to white noise.

ChiSCAT: Unsupervised Learning of Recurrent Cellular Micromotion Patterns from a Chaotic Speckle Pattern.

There is considerable evidence that action potentials are accompanied by "intrinsic optical signals", such as a nanometer-scale motion of the cell membrane. Here we present ChiSCAT, a technically simple imaging scheme that detects such signals with interferometric sensitivity. ChiSCAT combines illumination by a chaotic speckle pattern and interferometric scattering microscopy (iSCAT) to sensitively detect motion in any direction. The technique features reflective high-NA illumination, common-path suppression of vibrations, and a large field of view. This approach maximizes sensitivity to motion, but does not produce a visually interpretable image. We show that unsupervised learning based on matched filtering and motif discovery can recover underlying motion patterns and detect action potentials. We demonstrate these claims in an experiment on blebbistatin-paralyzed cardiomyocytes. ChiSCAT opens the door to action potential measurement in scattering tissue, including a living brain.

Nimodipine inhibits spreading depolarization, ischemic injury, and neuroinflammation in mouse live brain slice preparations

Nimodipine is used to prevent delayed ischemic deficit in patients with aneurysmal subarachnoid hemorrhage (aSAH). Spreading depolarization (SD) is recognized as a factor in the pathomechanism of aSAH and other acute brain injuries. Although nimodipine is primarily known as a cerebral vasodilator, it may have a more complex mechanism of action due to the expression of its target, the L-type voltage-gated calcium channels (LVGCCs) in various cells in neural tissue. This study was designed to investigate the direct effect of nimodipine on SD, ischemic tissue injury, and neuroinflammation. SD in control or nimodipine-treated live mouse brain slices was induced under physiological conditions using electrical stimulation, or by subjecting the slices to hypo-osmotic stress or mild oxygen-glucose deprivation (mOGD). SD was recorded applying local field potential recording or intrinsic optical signal imaging. Histological analysis was used to estimate tissue injury, the number of reactive astrocytes, and the degree of microglia activation. Nimodipine did not prevent SD occurrence in mOGD, but it did reduce the rate of SD propagation and the cortical area affected by SD. In contrast, nimodipine blocked SD occurrence in hypo-osmotic stress, but had no effect on SD propagation. Furthermore, nimodipine prevented ischemic injury associated with SD in mOGD. Nimodipine also exhibited anti-inflammatory effects in mOGD by reducing reactive astrogliosis and microglial activation. The results demonstrate that nimodipine directly inhibits SD, independent of nimodipine's vascular effects. Therefore, the use of nimodipine may be extended to treat acute brain injuries where SD plays a central role in injury progression.

The Impact of Photopigment Bleaching on the Human Rod Photoreceptor Subretinal Space Measured Via Optical Coherence Tomography.

The purpose of this study was to investigate rod photopigment bleaching-driven intrinsic optical signals (IOS) in the human outer retina and its measurement repeatability based on a commercial optical coherence tomography (OCT) platform. The optical path length of the rod photoreceptor subretinal space (SRS), that is, the distance between signal bands of rod outer segment tips and retinal pigment epithelium, was measured in 15 healthy subjects in ambient light and during a long-duration bleaching white-light exposure. On 2 identical study days (day 1 and day 2 [D1 and D2]), light stimulation resulted in a significant decrease in rod SRS by 21.3 ± 7.6% and 19.8 ± 8.5% (both P<0.001), respectively. The test-retest reliability of the SRS maximum change of an individual subject was moderate for single measures (intraclass correlation coefficient [ICC] = 0.730, 95% confidence interval [CI] = 0.376, 0.900, P<0.001) and good for average measures (ICC = 0.844, 95% CI = 0.546, 0.947, P<0.001). The mean area under the stimulus response curve with values of 14.8 ± 9.4 and 15.5 ± 7.5µm × minutes (P=0.782) showed excellent agreement between the stimulus response on D1 and D2. Intermittent dark adaptation of the retina led to an initial increase of the SRS by 6.1% (P=0.018) and thereafter showed a decrease toward baseline, despite continued dark adaptation. The data indicate the potential of commercial OCT in measuring slow IOS in the outer retina suggesting that the rod SRS could serve as a biomarker for photoreceptor function. The presented approach could provide an easily implementable clinical tool for the early detection of diseases affecting photoreceptor health.

Intrinsic signal optoretinography of dark adaptation abnormality due to rod photoreceptor degeneration.

This research aims to investigate the potential of using intrinsic optical signal (IOS) optoretinography (ORG) to objectively detect dark adaptation (DA) abnormalities related to rod photoreceptor degeneration. Functional optical coherence tomography (OCT) was employed in both wild-type (WT) and retinal degeneration 10 (rd10) mice to conduct this assessment. Dynamic OCT measurements captured the changes in retinal thickness and reflectance from light-to-dark transition. Comparative analysis revealed significant IOS alterations within the outer retina. Specifically, a reduction in thickness from external limiting membrane (ELM) peak to retinal pigment epithelium (RPE) peak was observed (WT: 1.13 ± 0.69µm, 30min DA; rd10: 2.64 ± 0.86µm, 30min DA), as well as a decrease in the intensity of the inner segment ellipsoid zone (EZ) in 30min DA compared to light adaptation (LA). The reduction of relative EZ intensity was notable in rd10 after 5min DA and in WT after 15min DA, with a distinguishable difference between rd10 and WT after 10min DA. Furthermore, our findings indicated a significant decrease in the relative intensity of the hypo-reflective band between EZ and RPE in rd10 retinas during DA, which primarily corresponds to the outer segment (OS) region. In conclusion, the observed DA-IOS abnormalities, including changes in ELM-RPE thickness, EZ, and OS intensity, hold promise as differentiators between WT and rd10 mice before noticeable morphological abnormalities occur. These findings suggest the potential of this non-invasive imaging technique for the early detection of dysfunction in retinal photoreceptors.

Synaptic Zn2+ contributes to deleterious consequences of spreading depolarizations

Spreading depolarizations (SDs) are profound waves of neuroglial depolarization that can propagate repetitively through injured brain. Recent clinical work has established SD as an important contributor to expansion of acute brain injuries and have begun to extend SD studies into other neurological disorders. A critical challenge is to determine how to selectively prevent deleterious consequences of SD. In the present study, we determined whether a wave of profound Zn2+ release is a key contributor to deleterious consequences of SD, and whether this can be targeted pharmacologically. Focal KCl microinjection was used to initiate SD in the CA1 region of the hippocampus in murine brain slices. An extracellular Zn2+ chelator with rapid kinetics (ZX1) increased SD propagation rates and improved recovery of extracellular DC potential shifts. Under conditions of metabolic compromise, tissues showed sustained impairment of functional and structural recovery following a single SD. ZX1 effectively improved recovery of synaptic potentials and intrinsic optical signals in these vulnerable conditions. Fluorescence imaging and genetic deletion of a presynaptic Zn2+ transporter confirmed synaptic release as the primary contributor to extracellular accumulation and deleterious consequences of Zn2+ during SD. These results demonstrate a role for synaptic Zn2+ release in deleterious consequences of SD and show that targeted extracellular chelation could be useful for disorders where repetitive SD enlarges infarcts in injured tissues.

Research on the classification of early-stage brain edema by combining intrinsic optical signal imaging and laser speckle contrast imaging.

The early detection and pathological classification of brain edema are very important for symptomatic treatment. The dual-optical imaging system (DOIS) consists of intrinsic optical signal imaging (IOSI) and laser speckle contrast imaging (LSCI), which can acquire cerebral hemodynamic parameters of mice in real-time, including changes of oxygenated hemoglobin concentration ( ), deoxyhemoglobin concentration (ΔCHbR) and relative cerebral blood flow (rCBF) within the field of view. The slope sum of , ΔCHbR and rCBF was proposed to classify vasogenic edema (VE) and cytotoxic edema (CE). The slope sum values in the VE and CE group remain statistically different and the classification results provide higher accuracy of more than 93% for early brain edema detection. In conclusion, the differences of hemodynamic parameters between VE and CE in the early stage were revealed and the method helps in the classification of early brain edema.

BLEscope: A Bluetooth Low Energy (BLE) Microscope for Wireless Multicontrast Functional Imaging.

Recent advances in low-power wireless-capable system-on-chips (SoCs) have accelerated diverse Internet of Things (IoT) applications, encompassing wearables, asset monitoring, and more. Concurrently, the field of neuroimaging has experienced escalating demand for lightweight, untethered, low-power systems capable of imaging in small animals. This article explores the feasibility of using a low-power asset monitoring system as the basis of a new architecture for fluorescence and hemodynamic contrast-based wireless functional imaging. The core system architecture hinges on the fusion of a Bluetooth Low Energy (BLE) 5.2 SoC and a low-power 560×560, 8-bit monochrome CMOS image sensor module. Successful integration of a multicontrast optical front-end consisting of a fluorescence channel (FL) and an intrinsic optical signal (IOS) channel resulted in the creation of a wireless microscope called 'BLEscope'. Next, we developed a wireless (i.e. BLE) protocol to remotely operate the BLEscope via a laptop and acquire in vivo images at 1 frame per second (fps). We then conducted a comprehensive characterization of the BLEscope to assess its optical capabilities and power consumption. We report a new benchmark for continuous wireless imaging of ∼1.5 hours with a 100 mAh battery. Via the FL channel of the BLEscope, we successfully tracked the kinetics of an intravenously injected fluorescent tracer and acquired images of fluorescent brain tumor cells in vivo. Via the IOS channel, we characterized the differential response of normal and tumor-associated blood vessels to a carbogen gas inhalation challenge. When miniaturized, the BLEscope will result in a new class of low-power, implantable or wireless microscopes that could transform preclinical and clinical neuroimaging applications.

An optical fiber chlorogenic acid sensor using a Chitosan membrane coated bent optical fiber probe

Hydrogel polymers have hydrophilic function groups bonded on the polymer's backbone network. Water molecules and compounds soluble in aqueous solution can permeate into hydrogel's network. This property was employed in this work in developing an optical fiber chemical sensor for detecting chlorogenic acid (CGA). A Chitosan membrane was coated on a bent optical fiber probe (BOFP) by simply dipping the BOFP into a Chitosan solution, which was made by dissolving solid Chitosan in a 2 % acetic acid solution, and pulling out. When such a Chitosan-coated BOFP was exposed to CGA in an aqueous sample solution, CGA molecules permeate into the Chitosan membrane, and were detected through monitoring the compound's intrinsic optical absorption signal at 400 nm. Chitosan has one amine group on each of its glucose rings, which helps the membrane concentrating CGA (a weak acid) from aqueous sample solution. Therefore, the sensor shows high sensitivity in detecting CGA with a detection limit of 0.018 μg/mL. The sensor's response to CGA is reversible, because CGA permeation into/out of the polymer network is a reversible process. The effectiveness of the developed sensor for detecting CGA was verified though analyzing CGA in green coffee extract products. The analytical results obtained with the developed sensor agree well with results obtained with a traditional UV/Vis optical absorption spectrometric method. The effectiveness of the sensor for analyzing CGA in green coffee extract samples was also verified through standard addition and recovery experiment with obtained recovery rate ranging from 97 % to 100 %.

Dynamics of functional impairments during focal transient ischemia in three-dimensional cortical space

Ischemic injury in the cerebral cortex results in decreased electrical activity across all frequency bands and the emergence of abnormal electrophysiological patterns, including spreading depolarization (SD) and negative ultraslow potential (NUP) [1, 2]. Despite this, the specific dynamics of these changes in electrical activity within the three-dimensional cortical space during ischemia remain incompletely described and poorly understood. To simultaneously investigate changes in electrical activity across the layers of the cerebral cortex and in the horizontal cortical space, we used two linear 16-channel silicone probes (Neuronexus, USA) in combination with a flexible transparent 60-channel matrix of subdural electrodes (MIPT, Russia) and intrinsic optical signal imaging (IOS, 665 nm, transillumination mode) during focal ischemia induced by intracortical injection of the potent vasoconstrictor endothelin-1 (ET-1, 1 μL, 1 μM). These experiments were carried out in head-restrained rats under urethane anesthesia (1.5 g/kg). Formation of the ischemic lesion following ET-1 administration was correlated with clusters of SDs that demonstrated considerable variability in their propagation patterns within both vertical and horizontal planes. The initial SDs originated from the injection site of ET-1 and diffused across all cortical layers. Subsequently, the initiation point of the ensuing SDs gradually shifted towards the deeper layers while the electrical activity showed inadequate recovery between SDs within the injection site. SDs originating in the surrounding cortex did not invade the area near the injection point. Instead, they tended to spread around, often compartmentalizing in the superficial layers of the cortex. Some SDs were observed deep in the cortex, while others were detected on the surface via superficial electrodes and IOS, without leaving typical intracortical electrode traces. Electrographic activity was significantly depressed, especially in the superficial layers around the injection site, three hours after ET-1 administration. However, it returned to pre-ET-1 levels at the remote site, with a spatial gradient observed in subdural electrodes. Functional impairments corresponded to the histological lesion observed in coronal brain sections. Recently discovered NUPs were initiated by SD and were most prominent in the electrodes closest to the ET-1 injection site. These NUPs reached their maximal amplitude at one hour and subsided three hours after ET-1 injection. Our research indicates intricate dynamics in the creation of an ischemic focal point. The data gathered suggest that the emergence of cerebral harm during focal ischemia is associated with the growth of a focus extending both horizontally and vertically across cortical dimensions. This growth is fueled by the generation of SDs within the ischemic penumbra.

Multimodality imaging reveals angiogenic evolution in vivo during calvarial bone defect healing.

The healing of calvarial bone defects is a pressing clinical problem that involves the dynamic interplay between angiogenesis and osteogenesis within the osteogenic niche. Although structural and functional vascular remodeling (i.e., angiogenic evolution) in the osteogenic niche is a crucial modulator of oxygenation, inflammatory and bone precursor cells, most clinical and pre-clinical investigations have been limited to characterizing structural changes in the vasculature and bone. Therefore, we developed a new multimodality imaging approach that for the first time enabled the longitudinal (i.e., over four weeks) and dynamic characterization of multiple in vivo functional parameters in the remodeled vasculature and its effects on de novo osteogenesis, in a preclinical calvarial defect model. We employed multi-wavelength intrinsic optical signal (IOS) imaging to assess microvascular remodeling, intravascular oxygenation (SO2), and osteogenesis; laser speckle contrast (LSC) imaging to assess concomitant changes in blood flow and vascular maturity; and micro-computed tomography (μCT) to validate volumetric changes in calvarial bone. We found that angiogenic evolution was tightly coupled with calvarial bone regeneration and corresponded to distinct phases of bone healing, such as injury, hematoma formation, revascularization, and remodeling. The first three phases occurred during the initial two weeks of bone healing and were characterized by significant in vivo changes in vascular morphology, blood flow, oxygenation, and maturity. Overall, angiogenic evolution preceded osteogenesis, which only plateaued toward the end of bone healing (i.e., four weeks). Collectively, these data indicate the crucial role of angiogenic evolution in osteogenesis. We believe that such multimodality imaging approaches have the potential to inform the design of more efficacious tissue-engineering calvarial defect treatments.

Quantitative hemodynamic imaging: a method to correct the effects of optical properties on laser speckle imaging.

Studying cerebral hemodynamics may provide diagnostic information on neurological conditions. Wide-field imaging techniques, such as laser speckle imaging (LSI) and optical intrinsic signal imaging, are commonly used to study cerebral hemodynamics. However, they often do not account appropriately for the optical properties of the brain that can vary among subjects and even during a single measurement. Here, we describe the combination of LSI and spatial-frequency domain imaging (SFDI) into a wide-field quantitative hemodynamic imaging (QHI) system that can correct the effects of optical properties on LSI measurements to achieve a quantitative measurement of cerebral blood flow (CBF). We describe the design, fabrication, and testing of QHI. The QHI hardware combines LSI and SFDI with spatial and temporal synchronization. We characterized system sensitivity, accuracy, and precision with tissue-mimicking phantoms. With SFDI optical property measurements, we describe a method derived from dynamic light scattering to obtain absolute CBF values from LSI and SFDI measurements. We illustrate the potential benefits of absolute CBF measurements in resting-state and dynamic experiments. QHI achieved a 50-Hz raw acquisition frame rate with a field of view and flow sensitivity up to . The extracted SFDI optical properties agreed well with a commercial system (). The system showed high stability with low coefficients of variations over multiple sessions within the same day () and over multiple days (). When optical properties were considered, the in-vivo hypercapnia gas challenge showed a slight difference in CBF ( to 0.5% difference). The in-vivo resting-state experiment showed a change in CBF ranking for nine out of 13 animals when the correction method was applied to LSI CBF measurements. We developed a wide-field QHI system to account for the confounding effects of optical properties on CBF LSI measurements using the information obtained from SFDI.

Inhibiting metabotropic glutamate receptor 5 after stroke restores brain function and connectivity.

Stroke results in local neural disconnection and brain-wide neuronal network dysfunction leading to neurological deficits. Beyond the hyper-acute phase of ischaemic stroke, there is no clinically-approved pharmacological treatment that alleviates sensorimotor impairments. Functional recovery after stroke involves the formation of new or alternative neuronal circuits including existing neural connections. The type-5 metabotropic glutamate receptor (mGluR5) has been shown to modulate brain plasticity and function and is a therapeutic target in neurological diseases outside of stroke. We investigated whether mGluR5 influences functional recovery and network reorganization rodent models of focal ischaemia. Using multiple behavioural tests, we observed that treatment with negative allosteric modulators (NAMs) of mGluR5 (MTEP, fenobam and AFQ056) for 12 days, starting 2 or 10 days after stroke, restored lost sensorimotor functions, without diminishing infarct size. Recovery was evident within hours after initiation of treatment and progressed over the subsequent 12 days. Recovery was prevented by activation of mGluR5 with the positive allosteric modulator VU0360172 and accelerated in mGluR5 knock-out mice compared with wild-type mice. After stroke, multisensory stimulation by enriched environments enhanced recovery, a result prevented by VU0360172, implying a role of mGluR5 in enriched environment-mediated recovery. Additionally, MTEP treatment in conjunction with enriched environment housing provided an additive recovery enhancement compared to either MTEP or enriched environment alone. Using optical intrinsic signal imaging, we observed brain-wide disruptions in resting-state functional connectivity after stroke that were prevented by mGluR5 inhibition in distinct areas of contralesional sensorimotor and bilateral visual cortices. The levels of mGluR5 protein in mice and in tissue samples of stroke patients were unchanged after stroke. We conclude that neuronal circuitry subserving sensorimotor function after stroke is depressed by a mGluR5-dependent maladaptive plasticity mechanism that can be restored by mGluR5 inhibition. Post-acute stroke treatment with mGluR5 NAMs combined with rehabilitative training may represent a novel post-acute stroke therapy.

Spreading Depolarizations Suppress Hematoma Growth in Hyperacute Intracerebral Hemorrhage in Mice.

Spreading depolarizations (SDs) occur in all types of brain injury and may be associated with detrimental effects in ischemic stroke and subarachnoid hemorrhage. While rapid hematoma growth during intracerebral hemorrhage triggers SDs, their role in intracerebral hemorrhage is unknown. We used intrinsic optical signal and laser speckle imaging, combined with electrocorticography, to investigate the effects of SD on hematoma growth during the hyperacute phase (0-4 hours) after intracortical collagenase injection in mice. Hematoma expansion, SDs, and cerebral blood flow were simultaneously monitored under normotensive and hypertensive conditions. Spontaneous SDs erupted from the vicinity of the hematoma during rapid hematoma growth. We found that hematoma growth slowed down by >60% immediately after an SD. This effect was even stronger in hypertensive animals with faster hematoma growth. To establish causation, we exogenously induced SDs (every 30 minutes) at a remote site by topical potassium chloride application and found reduced hematoma growth rate and final hemorrhage volume (18.2±5.8 versus 10.7±4.1 mm3). Analysis of cerebral blood flow using laser speckle flowmetry revealed that suppression of hematoma growth by spontaneous or induced SDs coincided and correlated with the characteristic oligemia in the wake of SD, implicating the vasoconstrictive effect of SD as one potential mechanism of action. Our findings reveal that SDs limit hematoma growth during the early hours of intracerebral hemorrhage and decrease final hematoma volume.

Vascular-centric mapping of in vivo blood oxygen saturation in preclinical models.

Assessing intravascular blood oxygen saturation (SO2) is crucial for characterizing in vivo microenvironmental changes in preclinical models of injury and disease. However, most conventional optical imaging techniques for mapping in vivo SO2 assume or compute a single value of the optical path-length in tissue. This is especially detrimental when mapping in vivo SO2 in experimental disease or wound healing models that are characterized by vascular and tissue remodeling. Therefore, to circumvent this limitation we developed an in vivo SO2 mapping technique that utilizes hemoglobin-based intrinsic optical signal (IOS) imaging combined with a vascular-centric estimation of optical path-lengths. In vivo arterial and venous SO2 distributions derived with this approach closely matched those reported in the literature, while those derived using the single path-length (i.e. conventional) approach did not. Moreover, in vivo cerebrovascular SO2 strongly correlated (R2>0.7) with changes in systemic SO2 measured with a pulse oximeter during hypoxia and hyperoxia paradigms. Finally, in a calvarial bone healing model, in vivo SO2 assessed over four weeks was spatiotemporally correlated with angiogenesis and osteogenesis (R2>0.6). During the early stages of bone healing (i.e. day 10), angiogenic vessels surrounding the calvarial defect exhibited mean SO2 that was elevated by10% (p<0.05) relative to that observed at a later stage (i.e., day 26), indicative of their role in osteogenesis. These correlations were not evident with the conventional SO2 mapping approach. The feasibility of our wide field-of-view in vivo SO2 mapping approach illustrates its potential for characterizing the microvascular environment in applications ranging from tissue engineering to cancer.

High-Sensitivity Intrinsic Optical Signal Imaging Through Flexible, Low-Cost Adaptations of an Upright Microscope.

Intrinsic optical signal imaging (IOSI) is a staple technique in modern neuroscience. Pioneered >30 years ago, IOSI allows macroscopic mapping of neuronal activity throughout the cortex. The technique has been used to study sensory processing and experience-dependent plasticity, and is often used as an adjunctive procedure to localize cortical areas for subsequent targeting by other imaging or physiology techniques. Despite the ubiquity of IOSI in neuroscience, there are few commercially available turn-key IOSI systems. As a result, investigators have typically resorted to building their own imaging systems. Over the years, simplified systems built either as dedicated rigs or incorporated into existing microscope platforms have been developed. Here we present a straightforward set of adaptations that can be applied to any standard upright microscope, using readily available, inexpensive, commercial parts for illumination, optics, and signal detection, that enables high-sensitivity IOSI. Using these adaptations, we are able to readily map sensory-evoked signals across the somatosensory and visual cortex, including single-whisker barrel cortical activity maps in mice. We show that these IOSI maps are highly reproducible across animals and can be used to study plasticity mechanisms in the somatosensory cortex. We also provide open-source applications to control illumination and analyze raw data to generate activity maps. We anticipate that these resources will be useful for neuroscience investigators looking to add IOSI capabilities to an existing microscope in the laboratory on a budget.

Intrinsic, widefield optical imaging of hemodynamics in rodent models of Alzheimer's disease and neurological injury.

The complex cerebrovascular network is critical to controlling local cerebral blood flow (CBF) and maintaining brain homeostasis. Alzheimer's disease (AD) and neurological injury can result in impaired CBF regulation, blood-brain barrier breakdown, neurovascular dysregulation, and ultimately impaired brain homeostasis. Measuring cortical hemodynamic changes in rodents can help elucidate the complex physiological dynamics that occur in AD and neurological injury. Widefield optical imaging approaches can measure hemodynamic information, such as CBF and oxygenation. These measurements can be performed over fields of view that range from millimeters to centimeters and probe up to the first few millimeters of rodent brain tissue. We discuss the principles and applications of three widefield optical imaging approaches that can measure cerebral hemodynamics: (1)optical intrinsic signal imaging, (2)laser speckle imaging, and (3)spatial frequency domain imaging. Future work in advancing widefield optical imaging approaches and employing multimodal instrumentation can enrich hemodynamic information content and help elucidate cerebrovascular mechanisms that lead to the development of therapeutic agents for AD and neurological injury.

Nimodipine exerts a protective effect against spreading depolarization and neuroinflammation

Hypothesis, objective: Voltage gated Ca2+ channels (VGCCs) are widely expressed in the central nervous system and implicated in neuronal injury and microglial activation. Nimodipine, a potent cerebrovascular dilator and VGCC antagonist is found to be effective against the vasoconstriction caused by spreading depolarization (SD) in preclinical studies of ischemia. However, it is still unclear whether the beneficial effect of nimodipine is achieved by the improvement of perfusion or a direct action on neuronal or glial cells. Our aim was to explore the direct action of nimodipine on the nervous tissue and microglia. Methods: Brain slices prepared from C57BL/6 mice (n=16) were perfused with artificial cerebrospinal fluid (aCSF). After 30 min nimodipine (nimo; 10 μM) incubation, low glucose aCSF (5 mM) and transient anoxia (1 min) were applied to mimic ischemia and to elicit SD. Intrinsic optical signal imaging was used to analyze SD features, TTC staining was applied to assess tissue injury. Primary mixed and pure microglia cultures prepared from the cortex of neonatal SPRD rats were treated with lipopolysaccharide (LPS;20 ng/ml) and nimodipine (5,10,20 μM), alone or in combination for 24 h. Microglial activation was evaluated by Iba1 immunolabeling (degree of arborization expressed by a transformation index (TI)), Western blot analysis and the visualization of phagocytosis with fluorescent microbeads. Results: In brain slices, nimodipine reduced the focal area of SD (2.37±0.94 vs 3.38±0.88%, nimo vs control), the total cortical area affected by SD (17.12±8.63 vs 39.88±22.42%, nimo vs control) and the propagation velocity of SD (0.19±0.79 vs 1.59±2.29mm/min, nimo vs control). Nimodipine decreased tissue injury, viability expressed as the number of TTC-stained particles (4.48±1.45 vs 3.52±1.52 particle/1000 μm2, nimo vs control). Nimodipine reduced microglial activation in LPS-treated cultures reflected by more ramified cell morphology (3.1±0.91 vs 1.89±0.41 TI, LPS+nimo vs LPS), decreased Iba1 intensity in Western blot analysis (108.3±20.1 and 77.2±8.49 vs 213.4±29.7 integrated optical density, LPS+nimo 10 and 20 μM vs LPS) and attenuated phagocytic activity (6±10 vs 22±20 bead/cell LPS+nimo vs LPS). Conclusion: Nimodipine was protective against SD and successfully reduced neuroinflammation by decreasing microglial activation. Neuroprotection was achieved independent of nimodipine’s vascular action. Our data suggest that nimodipine may be applicable against neuroinflammation and ischemia, in which neurodegeneration is believed to be linked to neurotoxic microglial activation. NKFIH K134377 and K134334, H2020 No. 739593, NAP3.0, TKP2021-EGA-28 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Low-intensity ultrasound stimulation modulates time-frequency patterns of cerebral blood oxygenation and neurovascular coupling of mouse under peripheral sensory stimulation state

Previous studies have demonstrated that transcranial ultrasound stimulation (TUS) not only modulates cerebral hemodynamics, neural activity, and neurovascular coupling characteristics in resting samples but also exerts a significant inhibitory effect on the neural activity in task samples. However, the effect of TUS on cerebral blood oxygenation and neurovascular coupling in task samples remains to be elucidated. To answer this question, we first used forepaw electrical stimulation of the mice to elicit the corresponding cortical excitation, and then stimulated this cortical region using different modes of TUS, and simultaneously recorded the local field potential using electrophysiological acquisition and hemodynamics using optical intrinsic signal imaging. The results indicate that for the mice under peripheral sensory stimulation state, TUS with a duty cycle of 50% can (1) enhance the amplitude of cerebral blood oxygenation signal, (2) reduce the time-frequency characteristics of evoked potential, (3) reduce the strength of neurovascular coupling in time domain, (4) enhance the strength of neurovascular coupling in frequency domain, and (5) reduce the time-frequency cross-coupling of neurovasculature. The results of this study indicate that TUS can modulate the cerebral blood oxygenation and neurovascular coupling in peripheral sensory stimulation state mice under specific parameters. This study opens up a new area of investigation for potential applicability of TUS in brain diseases related to cerebral blood oxygenation and neurovascular coupling.

Controlling the familywise error rate in widefield optical neuroimaging of functional connectivity in mice.

Statistical inference in functional neuroimaging is complicated by the multiple testing problem and spatial autocorrelation. Common methods in functional magnetic resonance imaging to control the familywise error rate (FWER) include random field theory (RFT) and permutation testing. The ability of these methods to control the FWER in optical neuroimaging has not been evaluated. We attempt to control the FWER in optical intrinsic signal imaging resting-state functional connectivity using both RFT and permutation inference at a nominal value of 0.05. The FWER was derived using a mass empirical analysis of real data in which the null is known to be true. Data from normal mice were repeatedly divided into two groups, and differences between functional connectivity maps were calculated with pixel-wise -tests. As the null hypothesis was always true, all positives were false positives. Gaussian RFT resulted in a higher than expected FWER with either cluster-based (0.15) or pixel-based (0.62) methods. -distribution RFT could achieve FWERs of 0.05 (cluster-based or pixel-based). Permutation inference always controlled the FWER. RFT can lead to highly inflated FWERs. Although -distribution RFT can be accurate, it is sensitive to statistical assumptions. Permutation inference is robust to statistical errors and accurately controls the FWER.