Speckle Pattern Research Articles

State-dependent neurovascular modulation induced by transcranial ultrasound stimulation.

Previous studies reported baseline state-dependent effects on neural and hemodynamic responses to transcranial ultrasound stimulation. However, due to neurovascular coupling, neither neural nor hemodynamic baseline alone can fully explain the ultrasound-induced responses. In this study, using a general linear model, we aimed to investigate the roles of both neural and hemodynamic baseline status as well as their interactions in ultrasound-induced responses. Thirty Sprague-Dawley rats were randomly assigned to Hypoxia, Hyperoxia, and Normoxia groups. The baseline states were altered by changing the oxygen concentrations. Micro-electrode and laser speckle contrast imaging were used to record local field potentials and cerebral blood flow during resting, before, and after ultrasound stimulation, respectively. We found that baseline neural activity played a positive role in neural response (Coefficient = 0.634, t = 1.748, p = 0.096, = 0.133), but a negative role in hemodynamic response (Coefficient = 0.060, t = 1.996, p = 0.060, = 0.166). Baseline hemodynamic activity also had a significantly negative correlation with the hemodynamic response (Coefficient = 0.760, t = 3.947, p 0.001, = 0.438). This study enriched our understanding of state-dependent effects underlying the neurovascular activation by ultrasound stimulation.

Single-frame statistical gating in a speckle pattern generated by the dynamic scattering in disordered medium

The propagation of coherent light in a dynamic disordered medium generates dynamic speckle patterns. The dynamics intrinsic to the disordered medium result in a shorter decorrelation time for the multiple scattering component (τcM) relative to the single scattering component (τcS). In this study, we propose a single-frame statistical gating strategy for the single and multiple scattering components in reflective wide-field imaging when the exposure time T satisfies the condition τcM<T<τcS. The spatial matrix representation is modeled as the sum of a negative exponential matrix, which represents the single scattering component, and a Gaussian matrix, which represents the multiple scattering component. The Marchenko–Pastur distribution and the Tracy–Widom distribution are employed in conjunction to calculate the moments of the single and multiple scattering components. The single-frame statistical gating method is validated using an electric field Monte Carlo simulation and demonstrated in vivo in brain imaging. Based on rotational invariance, a rotational sampling strategy is proposed to significantly improve the imaging quality of the single-frame statistical gating method. The single-frame statistical gating method greatly facilitates future applications in scenarios such as clinical and complex natural environments.

Programmable scanning diffuse speckle contrast imaging of cerebral blood flow.

Cerebral blood flow (CBF) imaging is crucial for diagnosing cerebrovascular diseases. However, existing large neuroimaging techniques with high cost, low sampling rate, and poor mobility make them unsuitable for continuous and longitudinal CBF monitoring at the bedside. We aimed to develop a low-cost, portable, programmable scanning diffuse speckle contrast imaging (PS-DSCI) technology for fast, high-density, and depth-sensitive imaging of CBF in rodents. The PS-DSCI employed a programmable digital micromirror device (DMD) for remote line-shaped laser (785nm) scanning on tissue surface and synchronized a 2D camera for capturing boundary diffuse laser speckle contrasts. New algorithms were developed to address deformations of line-shaped scanning, thus minimizing CBF reconstruction artifacts. The PS-DSCI was examined in head-simulating phantoms and adult mice. The PS-DSCI enables resolving intralipid particle flow contrasts at different tissue depths. In vivo experiments in adult mice demonstrated the capability of PS-DSCI to image global/regional CBF variations induced by 8% inhalation and transient carotid artery ligations. Compared with conventional point scanning, line scanning in PS-DSCI significantly increases spatiotemporal resolution. The high sampling rate of PS-DSCI is crucial for capturing rapid CBF changes while high spatial resolution is important for visualizing brain vasculature.

Impact of single versus dual arterial supply on perfusion and function in finger replantation after complex hand injuries

Finger amputations following complex hand injuries (CHI) pose a significant challenge in hand surgery due to severe tissue trauma and neurovascular damage, necessitating precise arterial repair. While restoring arterial perfusion is critical, it remains unclear whether reconstructing both proper palmar digital arteries is required for optimal outcomes. This study evaluates whether restoring one or both arteries in finger replantation after complex injuries impacts perfusion and overall outcomes. In this retrospective, cross-sectional, follow-up study, we investigated patients with finger amputations following CHI admitted to the high-volume regional hand trauma center between January 2013 and December 2020. Perfusion has been assessed using FLIR thermal imaging and laser speckle contrast analysis. Functional outcomes and quality of life scores were measured using standardized questionnaires. Sensory assessments, along with pain and grip strength measurements were also conducted. A total of 31 patients were included in the study. Thermal imaging showed a significantly higher finger surface temperature in patients with two-artery reconstruction. Laser speckle contrast analysis confirmed better perfusion, though not statistically significant. Functional and quality-of-life scores were similar across groups, except for significantly improved temperature sensation in the two-artery group. In conclusion, reconstructing both arteries in finger replantation following CHI isn’t essential for good outcomes if one artery provides adequate perfusion, but dual reconstruction may improve perfusion and temperature sensation.

Speckle-illumination spatial frequency domain imaging with a stereo laparoscope for profile-corrected optical property mapping.

Laparoscopic surgery presents challenges in localizing oncological margins due to poor contrast between healthy and malignant tissues. Optical properties can uniquely identify various tissue types and disease states with high sensitivity and specificity, making it a promising tool for surgical guidance. Although spatial frequency domain imaging (SFDI) effectively measures quantitative optical properties, its deployment in laparoscopy is challenging due to the constrained imaging environment. Thus, there is a need for compact structured illumination techniques to enable accurate, quantitative endogenous contrast in minimally invasive surgery. We introduce a compact, two-camera laparoscope that incorporates both active stereo depth estimation and speckle-illumination SFDI (si-SFDI) to map profile-corrected, pixel-level absorption ( ), and reduced scattering ( ) optical properties in images of tissues with complex geometries. We used a multimode fiber-coupled 639-nm laser illumination to generate high-contrast speckle patterns on the object. These patterns were imaged through a modified commercial stereo laparoscope for optical property estimation via si-SFDI. Compared with the original si-SFDI work, which required images of randomized speckle patterns for accurate optical property estimations, our approach approximates the DC response using a laser speckle reducer (LSR) and consequently requires only two images. In addition, we demonstrate 3D profilometry using active stereo from low-coherence RGB laser flood illumination. Sample topography was then used to correct for measured intensity variations caused by object height and surface angle differences with respect to a calibration phantom. The low-contrast RGB speckle pattern was blurred using an LSR to approximate incoherent white light illumination. We validated profile-corrected si-SFDI against conventional SFDI in phantoms with simple and complex geometries, as well as in a human finger in vivo time-series constriction study. Laparoscopic si-SFDI optical property measurements agreed with conventional SFDI measurements when measuring flat tissue phantoms, exhibiting an error of 6.4% for absorption and 5.8% for reduced scattering. Profile-correction improved the accuracy for measurements of phantoms with complex geometries, particularly for absorption, where it reduced the error by 23.7%. An in vivo finger constriction study further validated laparoscopic si-SFDI, demonstrating an error of 8.2% for absorption and 5.8% for reduced scattering compared with conventional SFDI. Moreover, the observed trends in optical properties due to physiological changes were consistent with previous studies. Our stereo-laparoscopic implementation of si-SFDI provides a simple method to obtain accurate optical property maps through a laparoscope for flat and complex geometries. This has the potential to provide quantitative endogenous contrast for minimally invasive surgical guidance.

Evaluation of Positive Cases for Dense Fine Speckled (DFS) Immunofluorescence Pattern and Anti-DFS70 Antibodies

Objective: The detection of anti-nuclear antibodies (ANA) is crucial in diagnosing systemic autoimmune rheumatic diseases (SARDs). The Dense Fine Speckled (DFS) nuclear pattern is one of the most common indirect immunofluorescence (IIF) patterns detected during routine ANA screening in patients with various clinical conditions. The aim of this study was to analyze the data of patients who were positive for DFS/antiDFS in our patient population and to show the possible clinical relationship. Methods: In this retrospective study, 7406 patient serum samples sent to our laboratory for routine ANA screening between May 2022-2023 were evaluated for the presence of anti-DFS. Results: In a group of patients referred for routine ANA screening using the indirect immunofluorescence method, the frequency of DFS pattern was found to be 4.55% (337/7406), with ANA positivity detected at a rate of 25.68% (1902/7406). Out of 221 patients with DFS pattern, 181 tested positive for anti-DFS antibodies in both the IIF-ANA and immunoblot (IB) tests. Additionally, 11 of these patients tested positive for other antibodies against different extractable nuclear antigens (anti-ENAs). Conversely, only seven out of the 40 patients who tested negative for anti-DFS antibodies showed positive results for other anti-ENAs. Conclusion: The DFS pattern is often positive in individuals. In patients exhibiting this pattern, anti-DFS70 antibody may be detected alone or in combination with SARD-associated autoantibodies. Therefore, we propose that in this patient cohort, it would be more prudent to screen for additional concomitant autoantibodies with anti-ENA rather than confirming isolated anti-DFS. doi: https://doi.org/10.12669/pjms.41.2.10336 How to cite this: Gurbuz M, Yıldırım BF, Cetinkol Y. Evaluation of Positive Cases for Dense Fine Speckled (DFS) Immunofluorescence Pattern and Anti-DFS70 Antibodies. Pak J Med Sci. 2025;41(2):580-584. doi: https://doi.org/10.12669/pjms.41.2.10336 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Portable six-channel laser speckle system for simultaneous measurement of cerebral blood flow and volume with potential applications in characterization of brain injury.

Cerebral blood flow (CBF) and cerebral blood volume (CBV) are key metrics for regional cerebrovascular monitoring. Simultaneous, non-invasive measurement of CBF and CBV at different brain locations would advance cerebrovascular monitoring and pave the way for brain injury detection as current brain injury diagnostic methods are often constrained by high costs, limited sensitivity, and reliance on subjective symptom reporting. We aim to develop a multi-channel non-invasive optical system for measuring CBF and CBV at different regions of the brain simultaneously with a cost-effective, reliable, and scalable system capable of detecting potential differences in CBF and CBV across different regions of the brain. The system is based on speckle contrast optical spectroscopy and consists of laser diodes and board cameras, which have been both tested and investigated for safe use on the human head. Apart from the universal serial bus connection for the camera, the entire system, including its battery power source, is integrated into a wearable headband and is powered by 9-V batteries. The temporal dynamics of both CBF and CBV in a cohort of five healthy subjects were synchronized and exhibited similar cardiac period waveforms across all six channels. The potential use of our six-channel system for detecting the physiological sequelae of brain injury was explored in two subjects, one with moderate and one with significant structural brain damage, where the six-point CBF and CBV measurements were referenced to structural magnetic resonance imaging (MRI) scans. We pave the way for a viable multi-point optical instrument for measuring CBF and CBV. Its cost-effectiveness allows for baseline metrics to be established prior to injury in populations at risk for brain injury.

Separating edges from microstructure in X-ray dark-field imaging: evolving and devolving perspectives via the X-ray Fokker-Planck equation.

A key contribution to X-ray dark-field (XDF) contrast is the diffusion of X-rays by sample structures smaller than the imaging system's spatial resolution; this is related to position-dependent small-angle X-ray scattering. However, some experimental XDF techniques have reported that XDF contrast is also generated by resolvable sample edges. Speckle-based X-ray imaging (SBXI) extracts the XDF by analyzing sample-imposed changes to a reference speckle pattern's visibility. We present an algorithm for SBXI (a variant of our previously developed multimodal intrinsic speckle-tracking (MIST) algorithm) capable of separating these two physically different XDF contrast mechanisms. The algorithm uses what we call the devolving Fokker-Planck equation for paraxial X-ray imaging as its forward model and then solves the associated multimodal inverse problem to retrieve the attenuation, phase, and XDF properties of the sample. Previous MIST variants were based on the evolving Fokker-Planck equation, which considers how a reference-speckle image is modified by the introduction of a sample. The devolving perspective instead considers how the image collected in the presence of the sample and the speckle membrane optically flows in reverse to generate the reference-speckle image when the sample is removed from the system. We compare single- and multiple-exposure multimodal retrieval algorithms from the two Fokker-Planck perspectives. We demonstrate that the devolving perspective can distinguish between two physically different XDF contrast mechanisms, namely, unresolved microstructure- and sharp-edge-induced XDF. This was verified by applying the different retrieval algorithms to two experimental data sets - one phantom sample and one organic sample. We anticipate that this work will be useful in (1) yielding a pair of complementary XDF images that separate sharp-edge diffuse scatter from diffuse scatter due to spatially random unresolved microstructure, (2) XDF computed tomography, where the strong edge XDF signal can lead to strong contaminating streaking artefacts, and (3) sample preparation, as samples will not need to be embedded since the strong XDF edge signal seen between the sample and air can be separated out.

Speckled Laser Pump–Thermoreflectance Microscopy Probe to Measure and Study Micro/Nanoscale Thermal Transport: Numerical Simulation

Abstract Nondiffusive thermal transport in solids and their micro/nanostructures is a key subject in the research of micro/nanoscale heat conduction. A number of laser and optical techniques to measure or capture the nondiffusive behaviors of heat carriers have been developed, such as transient thermoreflectance, time-domain thermoreflectance (TDTR), transient thermal grating (TTG), and so on. Here, we propose a novel method to study micro/nanoscale heat transport, namely, speckled laser pump–thermoreflectance microscopy probe. In this technique, micrometer to few hundred nanometer size random heat spots are generated by a speckled laser pump pulse, and the time–space evolution of heat spots are recorded by thermoreflectance microscopy images of the probe pulses arriving at different delay times. Fourier transform is applied to analyze the thermoreflectance images and extract the thermal decaying time for different spatial frequencies and along different in-plane directions. Thermal conductivity at different spatial frequencies, which includes the nondiffusive transport information, is obtained in this way. By numerically performing simulation of anisotropic Brownian motion and solving phonon Boltzmann transport equations under the initial condition of random heat spots, we retrieve the preset anisotropic thermal conductivity and the nondiffusive behavior of reduced thermal conductivity with increasing spatial frequencies, proving the validity of this technique. The innovative method can also be applied to study electron and spin transports, and holds the potential to facilitate the experimental research and understanding of nanoscale energy transport.

Early functional and structural hippocampal impairment in a bilateral common carotid artery stenosis mouse model.

Subcortical ischemic vascular dementia (SIVD) is a common subtype of vascular dementia. Currently, the bilateral common carotid artery stenosis (BCAS) mouse model is the most suitable SIVD rodent model. In this study, we investigated the functional and structural impairments in the hippocampus 1 month after BCAS. We used behavioral tests, laser speckle flowmetry, long-term potentiation, histochemical staining, molecular experiments, and voxel-based morphometry to evaluate the hippocampal impairments. Behavioral studies revealed that BCAS mice exhibited worse performance. Laser speckle flowmetry detected an obvious decrease in cerebral blood flow. The synaptic plasticity of the perforant path-dentate gyrus pathway was inhibited. Decreased fractional anisotropy and increased mean diffusivity were detected in the hippocampus via diffusion tensor imaging data. A reduction in gray matter volume, which was most prominent in the hippocampus and its surrounding areas, was detected via voxel-based morphometry analysis. Impairments in cell morphology and myelin integrity were validated using histochemical staining and molecular biology techniques. In addition, the numbers of GFAP+ astrocytes and Iba1+ microglia increased in the hippocampus. Overall, our study demonstrates early functional and structural impairments in the hippocampus contributing to learning and memory deficits after 1 month of BCAS, indicating that the hippocampus is vulnerable to chronic cerebral ischemia.

Blockade of A2AR improved brain perfusion and cognitive function in a mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is a neurodegenerative disorder that affects more than 6.2 million Americans aged 65 and older, particularly women. Along with AD's main hallmarks (formation of β-amyloid plaques and tau neurofibrillary tangles), there are vascular alterations that occurs in AD pathology. Adenosine A2 receptor (A2AR) is one of the key factors of brain vascular autoregulation and is overexpressed in AD patients. Our previous findings suggest that protein arginine methyltransferase 4 (PRMT4) is overexpressed in AD, which leads to decrease in cerebral blood flow in aged female 3xTg mice. We aimed to investigate the mechanism behind A2AR signaling in the regulation of brain perfusion and blood-brain barrier integrity in age and sex-dependent 3xTg mice, and if it is related to PRMT4. Istradefylline, a highly selective A2AR antagonist, was used to modulate A2AR signaling. Aged female 3xTg and C57BL/6J mice were evaluated for brain perfusion (via laser speckle) and cognitive function (via open field, T-maze and novel object recognition). Our results suggest that modulation of A2AR signaling in aged female 3xTg increased cerebral perfusion by decreasing PRMT4 expression, restored the levels of APP and tau, maintained blood-brain barrier integrity by maintaining the expression of tight junction proteins, and preserved functional learning/memory.

Mapping Sentinel Vessels in Uveal Melanomas Using Laser Speckle Contrast Imaging

Introduction: Sentinel vessels provide an important early indication of underlying ocular neoplasm. To date, there is no non-invasive technique available for imaging and mapping of their vascular supply, which remains largely unstudied. We aimed to map sentinel vessels in uveal melanomas non-invasively by laser speckle contrast imaging (LSCI). Case presentations: This report describes a case series of four patients undergoing enucleation due to uveal melanoma. Perfusion was imaged using LSCI during the successive detachment of the four rectus muscles, with their ciliary arteries, and the optic nerve with its ophthalmic artery. Tumor location and possible extrascleral growth were analyzed histopathologically. Results: Sentinel vessels in uveal melanoma could be visualized non-invasively using LSCI, appearing broader and tortuous compared to surrounding vessels. The perfusion in the sentinel vessels was 24-94% higher than in the episcleral vessels. The origin of the sentinel vessel could be determined by perfusion monitoring during the successive detachment of the rectus muscles and the optic nerve. In three patients the sentinel vessel was supplied by the anterior ciliary arteries, and in one patient by the ophthalmic artery. In one of the cases, the sentinel vessel was not visible upon visual inspection. Conclusion: This is the first study of its kind demonstrating detailed mapping of sentinel vessels in uveal melanoma using LSCI. LSCI shows potential for the detection of sentinel vessels before they are visible in slit lamp examination. Vascular changes are a hallmark of tumor growth, and non-invasive imaging with LSCI may be a useful diagnostic tool for the detection of uveal melanoma.

Preprocessing Methods for Reducing the Impact of Image Decorrelation in Laser Speckle Digital Image Correlation

Preprocessing Methods for Reducing the Impact of Image Decorrelation in Laser Speckle Digital Image Correlation

Invariance of the speckle pattern of the transmitted wave in periodic waveguides

We report on conditions of invariance of the transmitted pattern in the propagation through a periodic waveguide, the incident wave having no effect on the intensity pattern of the transmitted field. This phenomenon is reminiscent of that observed when illuminating a disordered medium in the regime of Anderson localization, as a consequence of the contribution of a single transmission eigenchannel to the transmitted wave. It is shown that the freezing of the transmitted wave is not intrinsically related to the disorder and that, whatever the frequency, it can also be observed in a regular, periodic system, provided that at most one Bloch mode is propagating. Moreover, while all localized modes in a disordered medium are exponentially decaying, our periodic waveguide system offers the possibility of observing the freezing with propagating waves, hence without the counterpart of having a very low energy transmission.

Neuroprotective effects of hypidone hydrochloride (YL-0919) after traumatic brain injury in mice.

Neurological dysfunction is a common complication of traumatic brain injury (TBI), and early treatments are critical for the long-term prognosis. This study aimed to investigate whether hypidone hydrochloride (YL-0919) improves neurological function impairment in mice with TBI. TBI was induced in adult male C57BL/6J mice using the controlled cortical impact (CCI) method. First, the modified neurological severity score (mNSS), rotarod test, and Morris water maze (MWM) test were conducted to assess the impact of YL-0919 on neurological function in mice with TBI. Next, immunofluorescence and laser speckle contrast imaging were utilized to measure the number and activation of microglia and cerebral blood flow (CBF) after TBI. Enzyme-linked immunosorbent assays (ELISAs) were employed to assess the inflammatory factors. Finally, Western blotting was performed to measure the expression of proteins. Golgi-Cox staining was utilized to investigate the structure of pyramidal neurons. YL-0919significantly alleviated neurological dysfunction in TBI+YL-0919mice compared with TBI+Vehicle mice, increased the time spent on the rotarod (F=1.297, P <0.05), and partially relieved cognitive dysfunction in TBI mice (for mNSS, F=5.540, P <0.01; for MWM test, F=30.78, P <0.05). Additionally, YL-0919 effectively inhibited the proliferation and activation of microglia (both P<0.01), promoted the recovery of CBF around the brain injury site and inhibited the expression of tumor necrosis factor-α (F=9.142, P <0.05) and IL-1β (F=4.662, P <0.05), and increased the concentration of IL-4 (F=5.172, P <0.05). Furthermore, continuous gavage of YL-0919 (2.5mg/kg) for seven days effectively increased the protein expression of brain-derived neurotrophic factor (BDNF), promoted the phosphorylation of mammalian target of rapamycin (mTOR), increased postsynaptic density protein 95 (PSD95) and synapsin1 levels, and increased the neuronal dendritic complexity and the dendritic spine density around the brain injury site (all P <0.05). Our findings indicated that YL-0919 can ameliorate neurological dysfunction in mice after TBI through the suppression of inflammation and the stimulation of the BDNF-mTOR signaling pathway. These findings provide an insightful perspective on the potential pharmacological mechanism involved in the neuroprotective effect of YL-0919.

HN1 Functions in Protein Synthesis Regulation via mTOR-RPS6 Axis and Maintains Nucleolar Integrity.

Haematological and Neurological Expressed 1 (HN1) is an oncogene for various cancers and previously has been linked with centrosome clustering and cell cycle pathways. Moreover, HN1 has recently been reported to activate mTOR signalling, which is the regulator of ribosome biogenesis and maintenance. We explored the role of HN1 in mTOR signalling through various gain- and loss-of-function experiments using biochemical approaches in different cell lines. We demonstrated for the first time that HN1 is required for nucleolar organiser region (NOR) integrity and function. Immunoprecipitation-based association and colocalization studies demonstrated that HN1 is an important component of the mTOR-RPS6 axis, and its depletion results with reduced mRNA translation in mammalian cancer cell lines. This study also demonstrated that the depletion of HN1 leads to the irregular distribution of nucleolar structures, potentially leading to cell cycle deregulation as reported previously. Accordingly, components of the translation machinery aggregate with a distinct speckled pattern, lose their essential interactions and ultimately impair mRNA translation efficiency when the HN1 is depleted. These results suggest that HN1 is an essential component of the nucleolus, required for ribosome biogenesis as well as global mRNA translation.

Snapshot Imaging of Stokes Vector Polarization Speckle in Turbid Optical Phantoms and In Vivo Tissues

Significance: We present a system to measure and analyze the complete polarization state distribution of speckle patterns generated from in vivo tissue. Accurate measurement of polarization speckle requires both precise spatial registration and rapid polarization state acquisition. A unique measurement system must be designed to achieve accurate images of polarization speckle patterns for detailed investigation of the scattering properties of biological tissues in vivo. Aim and approach: This system features a polarization state analyzer with no moving parts. Two pixel-polarizer cameras allow for the instantaneous acquisition of the spatial Stokes vector distribution of polarization speckle patterns. System design and calibration methods are presented, and representative images from measurements on liquid phantoms (microsphere suspensions) and in vivo healthy and tumor murine models are demonstrated and discussed. Results and Conclusions: Quantitative measurements of polarization speckle from microsphere suspensions with controlled scattering coefficients demonstrate differences in speckle contrast, speckle size, and the degree of polarization. Measurements on in vivo murine skin and xenograft tumor tissue demonstrate the ability of the system to acquire snapshot polarization speckle images in living systems. The developed system can thus rapidly and accurately acquire polarization speckle images from different media in dynamic conditions such as in vivo tissue. This capability opens the potential for future detailed investigation of polarization speckle for in vivo biomedical applications.

Rnd3 Ameliorates Diabetic Cardiac Microvascular Injury via Facilitating Trim40-mediated Rock1 Ubiquitination.

Diabetic microvascular dysfunction is evidenced by disrupted endothelial cell junctions and increased microvascular permeability. However, effective strategies against these injuries remain scarce. In this study, the type 2 diabetes mouse model was established by high-fat diet combined with streptozotocin injection in Rnd3 endothelial- specific transgenic and knockout mice. Echocardiography was employed to evaluate cardiac function. Microvascular corrosion casts, lanthanum nitrate perfusion, trans- endothelial electrical resistance, FITC-dextran permeability assay and laser speckle contrast imaging were performed to evaluate the integrity of endothelial cell junctions and microvascular function. RNA sequencing, mass spectrometry, co-immunoprecipitation, immunofluorescence and molecular docking were used to explore the downstream regulators of Rnd3. Evidence from gain/loss-of-function studies denoted a protective role for Rnd3 against microvascular dysfunction in diabetic heart. Endothelial-specific deletion of Rnd3 significantly exacerbated coronary microvascular barrier dysfunction under diabetic conditions, while Rnd3 overexpression effectively prevented these effects. Furthermore, Rnd3 overexpression also attenuated cardiac dysfunction in diabetic mice, as indicated by increased LVEF, LVFS, and E/A ratio. Rnd3 overexpression inhibited CMECs apoptosis and increased CMECs migration in response to HG-PA challenge. Rnd3 overexpression inhibited Rock1 activity and MLC phosphorylation in CMECs treated with HG-PA stimulus. Mechanically, Rnd3 recruited and interacted with the E3 ubiquitin ligase Trim40 which further facilitated the degradation of Rock1, thus inhibiting endothelial barrier hyperpermeability in HG-PA-stimulated CMECs. However, the cardioprotective effects of Rnd3 were largely abrogated by Trim40 deficiency in diabetic conditions. Collectively, Rnd3 alleviates microvascular hyperpermeability, maintains endothelial barrier integrity, and mitigates cardiac dysfunction by regulating the Rock1/MLC signaling pathway in the state of DCM.

Identifying the appropriate measurement environment for laser speckle flowmetry of cerebral blood flow in rats.

Laser speckle flowmetry (LSF) is a noninvasive tool for cerebral blood flow (CBF) measurement via a cranial bone window. LSF is influenced by various factors including the extent of removal of bone and dura mater and tissue wetness in the bone window. In this study, we aimed to characterize the effect of these conditions on LSF signals and identify optimal measurement conditions for CBF LSF measurements in rats. Three bone windows were created over the Sprague-Dawley rat brains including (i) bone removal until the brain surface was visible through the thin skull, (ii) complete bone removal for dura mater exposure, and (iii) dura mater removal for cortical surface exposure. We investigated the difference in the LSF signals of these windows under dry and wet conditions. The differences between signals obtained using artificial cerebrospinal fluid (aCSF) and mineral oil for wet conditions were also examined. Furthermore, we investigated the stability of repeated CBF measurements in thinned skulls over 15days and the effects of gentamicin ointment. No significant difference was observed in the LSF values of the three bone windows under dry and wet conditions. Moreover, mineral oil may provide better LSF signal stability. CBF LSF measurements with minimum signal fluctuation were possible for 15days using the thinned skull window with gentamicin ointment. In conclusion, CBF LSF measurements are feasible in rats using thinned skulls or dura matter in dry or wet environments, preferably with mineral oils. Relatively repetitive CBF LSF measurements were possible for long duration using gentamicin ointment for daily wound closure.

Transcranial Cortex-Wide Imaging of Murine Ischemic Perfusion With Large-Field Multifocal Illumination Microscopy.

Ischemic stroke is a common cause of death worldwide and a main cause of morbidity. Presently, laser speckle contrast imaging, x-ray computed tomography, and magnetic resonance imaging are the mainstay for stroke diagnosis and therapeutic monitoring in preclinical studies. These modalities are often limited in terms of their ability to map brain perfusion with sufficient spatial and temporal resolution, thus calling for development of new brain perfusion techniques featuring rapid imaging speed, cost-effectiveness, and ease of use. We report on a new preclinical high-resolution angiography technique for murine ischemic stroke imaging based on large-field high-speed multifocal illumination fluorescence microscopy. We subsequently showcase the proposed method by monitoring therapeutic effects of thrombolysis in stroke (n=6), further performing cross-strain comparison of perfusion dynamics (n=6) and monitoring the therapeutic effects of sensory stimulation-based treatment (n=11). Quantitative readings of hemodynamic and structural changes in cerebral vascular network and pial vessels were attained with 14.4-µm spatial resolution at 80-Hz frame rate fully transcranially. The in vivo perfusion maps accurately delineated the ischemic core and penumbra, further exhibiting a strong correlation (86.1±4.5%) with ex vivo triphenyl tetrazolium chloride staining, significantly higher than for the conventional laser speckle contrast imaging method. Monitoring of therapeutic effects of thrombolysis confirmed that early recanalization could effectively save the penumbra while reducing the infarct area. Cross-strain comparison of perfusion dynamics affirmed that C57BL/6 mice feature a larger penumbra and smaller infarct core as compared with BALB/c mice, which have few or no collaterals. Sensory stimulation-based treatment could effectively enhance blood flow and abolish perfusion deficits in the ischemic core and penumbra regions. A high-speed fluorescence-based angiography method for transcranial stroke imaging in mice is introduced, which is capable of localizing brain perfusion changes and accurately assessing the ischemic penumbra. Compared with the whole-brain x-ray computed tomography and magnetic resonance imaging methods, which are conventionally used for stroke diagnosis and therapeutic monitoring, the new approach is simple and cost-effective, further offering high resolution and speed for in vivo studies. It thus opens new venues for brain perfusion research under various disease conditions such as stroke, neurodegeneration, or epileptic seizures.