Laser Speckle Imaging Research Articles

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.

Advances in laser speckle imaging: From qualitative to quantitative hemodynamic assessment.

Laser speckle imaging (LSI) techniques have emerged as a promising method for visualizing functional blood vessels and tissue perfusion by analyzing the speckle patterns generated by coherent light interacting with living biological tissue. These patterns carry important biophysical tissue information including blood flow dynamics. The noninvasive, label-free, and wide-field attributes along with relatively simple instrumental schematics make it an appealing imaging modality in preclinical and clinical applications. The review outlines the fundamentals of speckle physics and the three categories of LSI techniques based on their degree of quantification: qualitative, semi-quantitative and quantitative. Qualitative LSI produces microvascular maps by capturing speckle contrast variations between blood vessels containing moving red blood cells and the surrounding static tissue. Semi-quantitative techniques provide a more accurate analysis of blood flow dynamics by accounting for the effect of static scattering on spatiotemporal parameters. Quantitative LSI such as optical speckle image velocimetry provides quantitative flow velocity measurements, which is inspired by the particle image velocimetry in fluid mechanics. Additionally, discussions regarding the prospects of future innovations in LSI techniques for optimizing the vascular flow quantification with associated clinical outlook are presented.

Study the effect of isoproterenol to visualize cerebral blood flow by using a laser speckle imaging system in Neonatal rats

Study the effect of isoproterenol to visualize cerebral blood flow by using a laser speckle imaging system in Neonatal rats

Neurotropin alleviates cognitive impairment by inhibiting TLR4/MyD88/NF-κB inflammation signaling pathway in mice with vascular dementia

Vascular dementia (VD) is the second most common cause of dementia after Alzheimer's disease. Neuroinflammation contributes to pathogenesis of VD. Neurotropin (NTP) is an analgesic that has been shown to suppress inflammation and neural repair. But its effects on VD are still unclear. Therefore, this study aimed to investigate the therapeutic effects and potential mechanisms of NTP in the VD model mice established by bilateral common carotid artery stenosis method. In VD mice, we found that NTP treatment increased cerebral blood flow by Laser speckle imaging, reduced neuron loss by Nissl, HE and immunochemistry staining, attenuated white matter damage by magnetic resonance imaging and ultrastructural damage by transmission electron microscope, improved cognitive functions by new object recognition test and three-chamber test, Y maze test and Morris water maze test, inhibited significantly glial activation by immunofluorescence methods, reduced the expression of TLR4, down-regulated expression of MyD88 and phosphorylation of NF-κB P65, decreased the levels of pro-inflammatory cytokines IL-1β, IL-6 and TNFα. Further, we showed that administration of a TLR4 inhibitor TAK242 had a similar effect to NTP, while the TLR4 agonist CRX-527 attenuated the effect of NTP in the VD mice. Collectively, our study suggested that NTP alleviates cognitive impairment by inhibiting TLR4/MyD88/NF-κB inflammation signaling pathway in the VD mice. Thus, NTP may be a promising therapeutic approach and a potential TLR4 inhibitor for VD.

A conceptual review on reconstructive peri-implantitis therapy: Challenges and opportunities.

The current strategies to reconstruct lost peri-implant tissues due to the disease have been largely unpredictable. The aim of this conceptual review is to discuss relevant biological and biomechanical challenges of applying reconstructive means to treat peri-implantitis. Additionally, opportunities to improve treatment predictability are presented. A narrative review was conducted to fulfill the aim. The four interrelated negative conditions hampering effective reconstruction are: inferior tissue perfusion, unfavorable bone topography, ineffective surface treatment, and unstable wound. First, peri-implant tissues resemble scars with reduced cellularity and vascularity, coupled with the absence of the periodontal ligament plexuses and the avascular implant and biomaterials, maintaining primary closure is a challenge, which is critical for regeneration. Second, defect morphology and bone topography surrounding implants determine the reconstructive potential. Unfortunately, noncontained defects are frequently encountered, with a combination of suprabony (horizontal bone loss) and infrabony (vertical usually involving circumferential bone loss) defects. Third, current attempts for implant surface decontamination are insufficient due to inaccessible macrostructure and rough surfaces in the micro-scale. Histologic evaluation has shown bacteria aggregation and calcified deposits around implants. Lastly, wound stability is difficult to achieve due to inherent soft tissue biomechanical quality and quantity deficiencies and mobile bone particulates. Opportunities to tackle the abovementioned challenges include the use of novel imaging technologies, such as high-frequency dental ultrasound and laser speckle imaging to evaluate tissue perfusion, soft tissue quality/quantity, and bone topography pre-surgically. The use of the operating microscope could allow better visualization and removal of etiologic factors. Strategies to improve soft tissue quality may include preoperative control of soft tissue inflammation and the potential use of biologics. Methods such as fixation to stabilize the biomaterials could be beneficial. A more nuanced understanding of the current challenges and opportunities can lead to more effective preoperative and postoperative care protocols, ultimately improving the success rate of reconstructive procedures.

Integration of orthogonal Fourier profilometry with blood flow mapping for 3D fingertip biometric authentication

We present herein the combination of Fourier transform profilometry based orthogonal projection with a laser speckle imaging method to provide three-dimensional (3D) fingertip blood flow imaging and shape reconstruction for biometric authentication purposes. In the proposed approach, orthogonal sinusoidal grating patterns with a fixed spatial frequency emanating from a digital projector first illuminate the surface of a person's finger. Then, the projector is blocked and a laser source illuminates the finger. The projector and laser work alternately such that only one source illuminates the finger at a given time allowing no illumination crosstalk between the two devices. The deformed grating patterns and the diffuse backscattered laser light, respectively, are recorded on the same CCD camera. In the computer, the 3D shape of the finger is reconstructed simultaneously with a blood flow map of the finger. Finally, the images are merged yielding a dynamic flow topography visualization of the person's finger. This dual-modality strategy utilizes the strengths of each imaging method in a complementary way, thus enabling simple 3D shape recovery and characterization of a specific user, and increasing the specificity of authentication security. The method is experimentally validated on both human and fake fingers through VanderLugt optical correlation framework. Overall, the results demonstrate the utility of our proposed approach for biometric authentication. We envision that our optoelectronic setup could be potentially integrated into a wide variety security platform.

Local transplantation of mesenchymal stem cells improves encephalo-myo-synangiosis-mediated collateral neovascularization in chronic brain ischemia

BackgroundTo explore whether local transplantation of mesenchymal stem cells (MSCs) in temporal muscle can promote collateral angiogenesis and to analyze its main mechanisms of promoting angiogenesis.MethodsBilateral carotid artery stenosis (BCAS) treated mice were administrated with encephalo-myo-synangiosis (EMS), and bone marrow mesenchymal stem cells (BMSCs) were transplanted into the temporal muscle near the cerebral cortex. On the 30th day after EMS, the Morris water maze, immunofluorescence, laser speckle imaging, and light sheet microscopy were performed to evaluate angiogenesis; In addition, rats with bilateral common carotid artery occlusion were also followed by EMS surgery, and BMSCs from GFP reporter rats were transplanted into the temporal muscle to observe the survival time of BMSCs. Then, the concentrated BMSC-derived conditioned medium (BMSC-CM) was used to stimulate HUVECs and BMECs for ki-67 immunocytochemistry, CCK-8, transwell and chick chorioallantoic membrane assays. Finally, the cortical tissue near the temporal muscle was extracted after EMS, and proteome profiler (angiogenesis array) as well as RT-qPCR of mRNA or miRNA was performed.ResultsThe results of the Morris water maze 30 days after BMSC transplantation in BCAS mice during the EMS operation, showed that the cognitive impairment in the BCAS + EMS + BMSC group was alleviated (P < 0.05). The results of immunofluorescence, laser speckle imaging, and light sheet microscopy showed that the number of blood vessels, blood flow and astrocytes increased in the BCAS + EMS + BMSC group (P < 0.05). The BMSCs of GFP reporter rats were applied to EMS and showed that the transplanted BMSCs could survive for up to 14 days. Then, the results of ki-67 immunocytochemistry, CCK-8 and transwell assays showed that the concentrated BMSC-CM could promote the proliferation and migration of HUVECs and BMECs (P < 0.05). Finally, the results of proteome profiler (angiogenesis array) in the cerebral cortex showed that the several pro-angiogenesis factors (such as MMP-3, MMP-9, IGFBP-2 or IGFBP-3) were notably highly expressed in MSC transplantation group compared to others.ConclusionsLocal MSCs transplantation together with EMS surgery can promote angiogenesis and cognitive behavior in chronic brain ischemia mice. Our study illustrated that MSC local transplantation can be the potential therapeutical option for improving EMS treatment efficiency which might be translated into clinical application.

Real-Time Imaging of Bonding in 3D-Printed Layers.

In recent times, 3D printing technology has revolutionized our ability to design and produce products, but optimizing the print quality can be challenging. The process of extrusion 3D printing involves pressuring molten material through a thin nozzle and depositing it onto previously extruded material. This method relies on bonding between the consecutive layers to create a strong and visually appealing final product. This is no easy task, as many parameters, such as the nozzle temperature, layer thickness, and printing speed, must be fine-tuned to achieve optimal results. In this study, a method for visualizing the polymer dynamics during extrusion is presented, giving insight into the layer bonding process. Using laser speckle imaging, the plastic flow and fusion can be resolved non-invasively, internally, and with high spatiotemporal resolution. This measurement, which is easy to perform, provides an in-depth understanding of the underlying mechanics influencing the final print quality. This methodology was tested with a range of cooling fan speeds, and the results showed increased polymer motion with lower fan speeds and, thus, explained the poor printing quality when the cooling fan was turned off. These findings show that this methodology allows for optimizing the printing settings and understanding the material behavior. This information can be used for the development and testing of novel printing materials or advanced slicing procedures. With this approach, a deeper understanding of extrusion can be built to take 3D printing to the next level.

Combining laser speckle imaging and spatially modulated visible illumination to study the effect of anesthetic drugs on the optical and physiological parameters of brain

This work employs an integrated optical imaging system to evaluate the effect of anesthetic drugs on several brain parameters including absorption, scattering, oxygen saturation, and cerebral blood flow (CBF). Two anesthetic drugs, namely ketamine-xylazine (KX) and thiopental, frequently used by researchers in mouse experiments, were investigated herein. The combined system is based on spatially modulated visible light illumination and a laser speckle based-technique. With this apparatus, a series of structured light (sinusoidal patterns) with phase shifts at each of two spatial frequencies (low and high) are projected onto the surface of a mouse brain and diffusely reflected light is acquired by a camera. A six-position filter wheel, equipped with five visible bandpass filters, is placed at the output of the projector. Then, structured illumination is blocked and a laser source illuminates the tissue while the diffusely reflected light is captured by the same camera through the remaining open hole in the wheel. In this manner, visible and laser speckle images are captured and processed off-line to reconstruct the cerebral tissue features. This work's major findings are six-fold: first, over a specific range of the spectrum a difference of more than 20% was found in the absorption coefficient presumably as a sensitivity to hemoglobin. Second, a difference of more than 20% was observed between drug types in the scattering coefficient along the entire spectral range. Third, CBF varied considerably in thiopental anesthesia throughout the experiment. Fourth, oxygen saturation level remains stable in both anesthetics. Fifth, thiopental keeps blood glucose in normal range while with KX glucose level increases. Sixth, the co-registered system is capable of revealing quantitative functional contrast and may be an attractive method in a variety of biomedical applications. Overall, these findings suggest that the choice of KX or thiopental should be carefully considered as a component of study design in research utilizing animal models.

Validation of Skin Perfusion Monitoring by Imaging PPG versus Laser Speckle Imaging

Abstract Assessment of skin perfusion can reveal signs of deterioration and help to prevent critical states. Commonly applied clinical tests to capture skin perfusion are subjective and often hard to quantify. Laser speckle contrast analysis (LASCA) is a technique that can capture skin perfusion at high spatial and temporal resolution. LASCA requires, however, a complex setting and is not suited for monitoring under clinical conditions. Imaging photoplethysmography (iPPG) might be an easy to use alternative. However, direct comparisons of LASCA and iPPG, and thus proves of iPPG’s capabilities to capture skin microperfusion, are rare. In this work, we compare the longitudinal development of the amplitude of green channel and near-infrared iPPG and LASCA after application of a hyperemic test. Our results show statistically significant increases in amplitude over time in all modalities. The maximum increase in median normalized amplitude is 1.281 for the green channel, 0.594 for near-infrared and 1.111 for LASCA. Median Spearman’s rank correlation coefficient of the amplitude for green channel and LASCA is r= 0.89 and r= 0.71 for near-infrared and LASCA.

Real-time quantification of bowel perfusion using Laparoscopic Laser Speckle Contrast Imaging (LSCI) in a porcine model

Background/purposeReal-time quantification of tissue perfusion can improve intraoperative surgical decision making. Here we demonstrate the utility of Laser Speckle Contrast Imaging as an intra-operative tool that quantifies real-time regional differences in intestinal perfusion and distinguishes ischemic changes resulting from arterial/venous obstruction.MethodsPorcine models (n = 3) consisted of selectively devascularized small bowel loops that were used to measure the perfusion responses under conditions of control/no vascular occlusion, arterial inflow occlusion, and venous outflow occlusion using laser speckle imaging and indocyanine green fluoroscopy. Laser Speckle was also used to assess perfusion differences between small bowel antimesenteric-antimesenteric and mesenteric-mesenteric anastomoses. Perfusion quantification was measured in relative perfusion units calculated from the laser speckle perfusion heatmap.ResultsLaser Speckle distinguished between visually identified perfused, watershed, and ischemic intestinal segments with both color heatmap and quantification (p < .00001). It detected a continuous gradient of relative intestinal perfusion as a function of distance from the stapled ischemic bowel edge. Strong positive linear correlation between relative perfusion units and changes in mean arterial pressure resulting from both arterial (R2 = .96/.79) and venous pressure changes (R2 = .86/.96) was observed. Furthermore, Laser Speckle showed that the antimesenteric anastomosis had a higher perfusion than mesenteric anastomosis (p < 0.01).ConclusionsLaser Speckle Contrast Imaging provides objective, quantifiable tissue perfusion information in both color heatmap and relative numerical units. Laser Speckle can detect spatial/temporal differences in perfusion between antimesenteric and mesenteric borders of a bowel segment and precisely detect perfusion changes induced by progressive arterial/venous occlusions in real-time.

Reduced tubuloglomerular feedback activity and absence of its synchronization in a connexin40 knockout rat.

Introduction: Tubuloglomerular feedback (TGF) is the negative feedback component of renal blood flow (RBF) autoregulation. Neighbouring nephrons often exhibit spontaneous TGF oscillation and synchronization mediated by endothelial communication, largely via connexin40 (Cx40). Methods: We had a knockout (KO) rat made that lacks Cx40. One base pair was altered to create a stop codon in exon 1 of Gja5, the gene that encodes Cx40 (the strain is WKY-Gja55em1Mcwi). Blood pressure (BP)-RBF transfer functions probed RBF dynamics and laser speckle imaging interrogated the dynamics of multiple efferent arterioles that reach the surface (star vessels). Results: The distribution of wild type (WT), heterozygote, and KO pups at weaning approximated the Mendelian ratio of 1:2:1; growth did not differ among the three strains. The KO rats were hypertensive. BP-RBF transfer functions showed low gain of the myogenic mechanism and a smaller TGF resonance peak in KO than in WT rats. Laser speckle imaging showed that myogenic mechanism had higher frequency in KO than in WT rats, but similar maximum spectral power. In contrast, the TGF frequency was similar while peak power of its oscillation was much smaller in KO than in WT rats. In WT rats, plots of instantaneous TGF phase revealed BP-independent TGF synchronization among star vessels. The synchronization could be both prolonged and widespread. In KO rats TGF synchronization was not seen, although BP transients could elicit short-lived TGF entrainment. Discussion: Despite the reduced TGF spectral power in KO rats, there was sufficient TGF gain to induce oscillations and therefore enough gain to be effective locally. We conclude that failure to synchronize is dependent, at least in part, on impaired conducted vasomotor responses.

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.

Abstract P2095: Capillary Stalling by Neutrophils is a Novel Mechanism Underlying Myocardial Hypoperfusion in Heart Failure with Preserved Ejection Fraction

Background: Chronic inflammation and myocardial hypoperfusion are implicated in the pathogenesis of heart failure with preserved ejection fraction (HFpEF), but the cellular and molecular mechanisms are unknown. Intravital cardiac multiphoton microscopy (MPM) enables direct visualization of blood flow and inflammatory cells within the capillary bed of the actively beating heart. We hypothesize that transient arrest of neutrophils in myocardial capillaries leads to perfusion deficits, resulting in hypoxia and driving disease progression. Methods: Male and female 8-12-week-old C57BL/6 mice received a high fat diet and L-NAME in drinking water (HFpEF) or standard diet and water (Chow) for 15 weeks. Intravital imaging of the actively beating heart was performed in mechanically ventilated mice to visualize neutrophils and capillary perfusion. An antibody against the neutrophil surface marker Ly6G (αLy6G) was administered intraperitoneally to test the effect of neutrophil depletion. Results: In vivo MPM revealed increases in arrested neutrophils in myocardial capillaries in HFpEF mice compared to Chows. Myocardial hypoxia, assessed by pimonidazole staining, and diastolic function, assessed as mitral e/e’ using echocardiography, were rescued with both extended neutrophil depletion (2mg/kg every 3d for 4 wk) and acutely (24 hrs after a single dose of 4mg/kg). To further test the immediate effects of αLy6G we implemented multi-exposure laser speckle imaging for perfusion and spectral imaging for tissue and blood oxygenation with electrocardiograph-driven reconstruction. Conclusion: Arrested neutrophils within capillaries appear to compromise myocardial perfusion in HFpEF. Neutrophil depletion to restore blood flow improved myocardial oxygenation and diastolic function rapidly. Effects were sustained with prolonged treatment. Capillary stall perfusion deficits may be a future therapeutic target to slow disease progression and offer rapid symptomatic relief.

Effect of 10.6 μm laser moxibustion on inflammation in diabetic peripheral neuropathy rats.

Diabetic peripheral neuropathy (DPN) is the main cause of disability in diabetes patients but the efficacy of available drugs is poor. Moxibustion is an adjunctive treatment for DPN that can reduce symptoms. The peak value of the far infrared wavelength of 10.6 μm laser moxibustion is close to the infrared radiation spectrum of traditional moxibustion. Its effect is similar to that of moxibustion and does not cause pain, infection or produce irritating smoke. Twenty-four male SD rats were divided into control (Con), DPN, laser moxibustion (LM), and pyrrolidine dithiocarbamate (PDTC) groups (n=6/group). The DPN, LM and PDTC group rats were intraperitoneally injected with 1% streptozotocin (STZ) to induce a model of DPN. LM group rats were irradiated with a laser at bilateral ST36 acupoints for 15min, once every other day, for 14 days. PDTC group rats were intraperitoneally injected with PDTC once a day. Body weight, blood glucose, and paw withdrawal mechanical threshold (PWMT) were measured and laser speckle imaging (LSI) performed before and after modeling and at 1 and 2 weeks after intervention. Two weeks after intervention, changes in serum interleukin 1β (IL1β), interleukin 6 (IL6), tumor necrosis factor α (TNFα) and nerve growth factor (NGF) were analyzed, and the abundance of NF-κB and IκB-α proteins and levels of NF-κB and IκB-α mRNAs in the sciatic nerve were observed. The results showed that 10.6 μm laser moxibustion can relieve pain, improve microcirculation, and alleviate inflammation in DPN rats, possibly via the NF-κB inflammatory pathway.

Employing optoelectronic laser speckle imaging to reveal molecular dynamics followed by the thermal expansion of liquid water

Employing optoelectronic laser speckle imaging to reveal molecular dynamics followed by the thermal expansion of liquid water

Edaravone dexborneol alleviates cerebral ischemia-reperfusion injury through NF-κB/NLRP3 signal pathway.

Inflammatory injury following ischemia-reperfusion (I/R) severely limits the efficacy of stroke treatment. Edaravone dexborneol (C.EDA) has been shown to reduce inflammation following a cerebral hemorrhage. However, the precise anti-inflammatory mechanism of C.EDA is unknown. In this study, we investigated whether C.EDA provides neuroprotection after I/R in rats, as well as the potential mechanisms involved. A middle cerebral artery occlusion/reperfusion (I/R) model was created using Sprague-Dawley rats. The blood flow of the central cerebral artery was monitored by a laser speckle imaging system. The neurological score was used to assess behavioral improvement. Cerebral infarction volume was measured by TTC staining. And the integrity of the blood-brain barrier was detected by Evan's blue staining. The expression of the nuclear factor kappa-B (NF-κB)/ the NOD-like receptor protein (NLRP3) inflammasome signal pathway and microglia polarization were detected by immunofluorescence and Western blotting. The cerebral blood flow ratio indicates that the cerebral I/R model was successfully established. After reperfusion for 72 h, the improvement of neurological scores, infarct volume reduction, and integrity of the blood-brain barrier was observed in I/R rats with C.EDA treatment. Meanwhile, the immunofluorescence result showed that the expression of iNOS, NLRP3, and NF-κB protein was decreased and the level of Arg1 was increased. Western blot analysis showed that the expression of NF-κB/NLRP3 signal pathway-related protein was decreased. In conclusion, this study indicates that C.EDA alleviates I/R injury by blocking the activation of the NLRP3 inflammasome and regulating the polarization of M1/M2 microglia via the NF-κB signal pathway.

Robust critical limb ischemia porcine model involving skeletal muscle necrosis

This work sought to develop a robust and clinically relevant swine model of critical limb ischemia (CLI) involving the onset of ischemic muscle necrosis. CLI carries about 25–40% risk of major amputation with 20% annual mortality. Currently, there is no specific treatment that targets the ischemic myopathy characteristic of CLI. Current swine models of CLI, with tolerable side-effects, fail to achieve sustained ischemia followed by a necrotic myopathic endpoint. Such limitation in experimental model hinders development of effective interventions. CLI was induced unilaterally by ligation-excision of one inch of the common femoral artery (CFA) via infra-inguinal minimal incision in female Yorkshire pigs (n = 5). X-ray arteriography was done pre- and post-CFA transection to validate successful induction of severe ischemia. Weekly assessment of the sequalae of ischemia on limb perfusion, and degree of ischemic myopathy was conducted for 1 month using X-ray arteriography, laser speckle imaging, CTA angiography, femoral artery duplex, high resolution ultrasound and histopathological analysis. The non-invasive tissue analysis of the elastography images showed specific and characteristic pattern of increased muscle stiffness indicative of the fibrotic and necrotic outcome expected with associated total muscle ischemia. The prominent onset of skeletal muscle necrosis was evident upon direct inspection of the affected tissues. Ischemic myopathic changes associated with inflammatory infiltrates and deficient blood vessels were objectively validated. A translational model of severe hindlimb ischemia causing ischemic myopathy was successfully established adopting an approach that enables long-term survival studies in compliance with regulatory requirements pertaining to animal welfare.

Targeting mitochondria in the aged cerebral vasculature with SS-31, a proteomic study of brain microvessels

Cognitive impairment and dementias during aging such as Alzheimer’s disease are linked to functional decline and structural alterations of the brain microvasculature. Although mechanisms leading to microvascular changes during aging are not clear, loss of mitochondria, and reduced efficiency of remaining mitochondria appear to play a major role. Pharmacological agents, such as SS-31, which target mitochondria have been shown to be effective during aging and diseases; however, the benefit to mitochondrial- and non-mitochondrial proteins in the brain microvasculature has not been examined. We tested whether attenuation of aging-associated changes in the brain microvascular proteome via targeting mitochondria represents a therapeutic option for the aging brain. We used aged male (> 18 months) C57Bl6/J mice treated with a mitochondria-targeted tetrapeptide, SS-31, or vehicle saline. Cerebral blood flow (CBF) was determined using laser speckle imaging during a 2-week treatment period. Then, isolated cortical microvessels (MVs) composed of end arterioles, capillaries, and venules were used for Orbitrap Eclipse Tribrid mass spectrometry. CBF was similar among the groups, whereas bioinformatic analysis revealed substantial differences in protein abundance of cortical MVs between SS-31 and vehicle. We identified 6267 proteins, of which 12% were mitochondria-associated. Of this 12%, 107 were significantly differentially expressed and were associated with oxidative phosphorylation, metabolism, the antioxidant defense system, or mitochondrial dynamics. Administration of SS-31 affected many non-mitochondrial proteins. Our findings suggest that mitochondria in the microvasculature represent a therapeutic target in the aging brain, and widespread changes in the proteome may underlie the rejuvenating actions of SS-31 in aging.

Laser speckle imaging for visualization of hidden effects for early detection of antibacterial susceptibility in disc diffusion tests.

Rapid identification of effective antibiotic treatment is crucial for increasing patient survival and preventing the formation of new antibiotic-resistant bacteria due to preventative antibiotic use. Currently utilized "gold standard" methods require 16-24 h to determine the most appropriate antibiotic for the patient's treatment. The proposed technique of laser speckle imaging with subpixel correlation analysis allows for identifying dynamics and changes in the zone of inhibition, which are impossible to observe with classical methods. Furthermore, it obtains the resulting zone of inhibition diameter earlier than the disk diffusion method which is recommended by the European Committee on Antimicrobial Susceptibility Testing (EUCAST). These results could improve mathematical models of changes in the diameter of the zone of inhibition around the disc containing the antimicrobial agent, thereby speeding up and facilitating epidemiological analysis.