Laser Speckle Perfusion Imaging Research Articles

Dual-Modal Optical Imaging of Tissue Perfusion in Response to Cooling Stimulation Facilitates Early Detection of Pressure Ulcer.

Pressure ulcers present a significant human and economic challenge, lacking a reliable method for early detection. To address this, we developed a system capable of early detection by using cooling stimulation and dynamic data acquisition techniques to monitor blood perfusion and skin temperature. The system consists of laser speckle perfusion imaging and thermal imaging. And we performed simulations to demonstrate that the system is capable of detect tissue damage across multiple layers, from superficial to deep. Testing on a rabbit ear model demonstrated that this approach, which combines dynamic perfusion and temperature parameters, effectively distinguishes early pressure ulcer areas from normal skin with a significant p value of 0.0015. This distinction was more precise compared to methods relying solely on static parameters or one parameter. Our study thereby offers a promising advancement in the proactive management and prevention of pressure ulcers.

GPER deficiency impedes murine myocutaneous revascularization and wound healing

Estrogens regulate numerous physiological and pathological processes, including wide-ranging effects in wound healing. The effects of estrogens are mediated through multiple estrogen receptors (ERs), including the classical nuclear ERs (ERα and ERβ\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$${\\upbeta }$$\\end{document}), that typically regulate gene expression, and the 7-transmembrane G protein-coupled estrogen receptor (GPER), that predominantly mediates rapid “non-genomic” signaling. Estrogen modulates the expression of various genes involved in epidermal function and regeneration, inflammation, matrix production, and protease inhibition, all critical to wound healing. Our previous work demonstrated improved myocutaneous wound healing in female mice compared to male mice. In the current study, we employed male and female GPER knockout mice to investigate the role of this estrogen receptor in wound revascularization and tissue viability. Using a murine myocutaneous flap model of graded ischemia, we measured real-time flap perfusion via laser speckle perfusion imaging. We conducted histologic and immunohistochemical analyses to assess skin and muscle viability, microvascular density and vessel morphology. Our results demonstrate that GPER is crucial in wound healing, mediating effects that are both dependent and independent of sex. Lack of GPER expression is associated with increased skin necrosis, reduced flap perfusion and altered vessel morphology. These findings contribute to understanding GPER signaling in wound healing and suggest possible therapeutic opportunities by targeting GPER.

Experimental study on treatment of retinitis pigmentosa by inducing Müller cell reprogramming with Lycii Fructus and Salviae Miltiorrhizae Radix et Rhizoma

This study aims to explore the effect of Lycii Fructus and Salviae Miltiorrhizae Radix et Rhizoma(LFSMR), a drug pair possesses the function of nourishing Yin, promoting blood circulation, and brightening the eyes, in treating retinitis pigmentosa(RP)by inhibiting the gliosis of Müller cells(MCs) and inducing their reprogramming and differentiation into various types of retinal nerve cells. Twelve C57 mice were used as the normal control group, and 48 transgenic RP(rd10) mice were randomly divided into the model group, positive control group, and low and high dose LFSMR groups, with 12 mice in each group. HE staining was used to detect pathological changes in the retina, and an electroretinogram was used to detect retinal function. Retinal optical coherence tomography was used to detect retinal thickness and perform fundus photography, and laser speckle perfusion imaging was used to detect local retinal blood flow. Digital PCR was used to detect gene expression related to retinal nerve cells, and immunofluorescence was used to detect protein expression related to retinal nerve cells. LFSMR could significantly improve the pathological changes, increase the amplitude of a and b waves, increase the retinal thickness, restore retinal damage, and increase retinal blood flow in mice with RP lesions. LFSMR could also significantly inhibit the m RNA expression of the glial fibrillary acidic protein( GFAP) during the pathogenesis of RP and upregulate m RNA expression of sex determining region Y box protein 2(SOX2), paired box protein 6(Pax6),rhodopsin, protein kinase C-α(PKCα), syntaxin, and thymic cell antigen 1. 1(Thy1. 1). LFSMR could significantly inhibit GFAP protein expression and enhance protein expression of SOX2, Pax6, rhodopsin, PKCα, syntaxin, and Thy1. 1. It could also reverse the pathological changes in the retina of rd10 mice, improve retinal function and fundus performance, increase retinal thickness, enhance local retinal blood flow, and exert therapeutic effects on RP. The mechanism of action of LFSMR may be related to inhibiting the gliosis of MCs and promoting their reprogramming and differentiation into various types of retinal nerve cells.

In vivo evaluation of liver function by multimodal imaging in an alcohol-induced liver injury model.

Visually evaluating liver function is a hot topic in hepatology research. There are few reliable and practical visualization methods for evaluating the liver function in vivo in experimental studies. In this study, we established a multimodal imaging approach for in vivo liver function evaluation and compared healthy mice with chronic alcoholic liver injury (cALI) model mice to explore its potential applicability in experimental research. In vivo fluorescence imaging (IVFI) technology was utilized to visually represent the clearance of indocyanine green from the liver of both healthy mice and mice with cALI. The reserve liver function was evaluated via IVFI using the Cy5.5-galactosylated polylysine probe, which targets the asialoglycoprotein receptor of hepatocytes. Hepatic microcirculation was assessed through laser speckle perfusion imaging of hepatic blood perfusion. The liver microstructure was then investigated by in vivo confocal laser endomicroscopy imaging. Finally, hepatic asialoglycoprotein receptor expression, histology, and the levels of serum alanine aminotransferase and aspartate aminotransferase were measured. In vivo multimodal imaging results intuitively and dynamically showed that indocyanine green clearance [mean ± standard deviation (SD): 30.83±14.71, 95% confidence interval (CI): 20.3 to 41.35], the fluorescence signal intensity (mean ± SD: 1,217.92±117.63; 95% CI: 1,148.38 to 1,290.84) and fluorescence aggregation area (mean ± SD: 5,855.80±1,271.81; 95% CI: 5,051.57 to 6,653.88) of Cy5.5-galactosylated polylysine targeting the asialoglycoprotein receptor, and hepatic blood perfusion (mean ± SD: 1,494.86±299.33; 95% CI: 1,316.98 to 1,690.16) in model mice were significantly lower than those in healthy mice (all P<0.001). Compared to healthy mice, the model mice exhibited a significant decline in liver asialoglycoprotein receptor expression (mean ± SD: 219.03±16.34; 95% CI: 208.97 to 230.69; P<0.001), increased serum alanine aminotransferase (mean ± SD: 149.70±47.89 U/L; 95% CI: 81.75 to 128.89; P=0.01) and aspartate aminotransferase levels (mean ± SD: 106.30±36.13 U/L; 95% CI: 122.01 to 180.17; P=0.021), hepatocyte swelling and deformation, disappearance of the hepatic cord structure, partial necrosis, and disintegration of hepatocytes. The imaging features of fluorescence signals in liver regions, hepatic blood perfusion and microstructure were biologically related to hepatic asialoglycoprotein receptor expression, serum indices of liver function, and histopathology in model mice. Utilizing in vivo multimodal imaging technology to assess liver function is a viable approach for experimental research, providing dynamic and intuitive visual evaluations in a rapid manner.

516-P: Lineage Tracing of Vasculogenic Fibroblasts In Vivo and Their Significance in the Rescue of Diabetic Ischemic Tissue

Our recent work reported on the identification of the vasculogenic fibroblast (VF) that is capable of generating new blood vessels during tissue repair (Pal et al. Nat Com, 2023). While these cells are physiologic and injury-inducible, the generation of VF is blunted under conditions of diabetes. Such barrier may be overcome by the inhibition of fibroblast miR-200b using tissue nanotransfection technology (PMIDs: 28785092, 34837085, 35819852, 36380587). To understand the mechanisms underlying VF function and significance, in this work we developed miR-200b-429fl/fl COL1A2creER GT(ROSA)mT/mG mice. In these mice, miR-200b regions in fibroblasts (COL1A2+ cells) are knocked out in response to tamoxifen. To gain insight into the significance of VF in vivo, ischemic hind limb studies were performed. Tamoxifen treatment not only led to Cre excision of miR-200b-429 in these mice, but also induced the constitutive expression of ROSA driven tdTomato in fibroblasts with onset of permanent GFP expression. Significant lowering of miR-200b was noted in laser capture dissected dermal fibroblast tissue elements from the wound-edge of tamoxifen treated mice (n=4). Tamoxifen treatment led to significantly enhanced perfusion in animals subjected to ischemic injury as determined by laser speckle perfusion imaging (Perimed Inc.) (n=8). This rescue was associated with increased abundance of GFP+CD31+ VF (n=5) which accounted for 26% of all CD31+ vascular cells in the laser captured blood vessels (n=9). Thus, targeted lowering of miR-200b abundance within fibroblasts resulted in substantial augmentation of VF in vivo which was associated with improved blood flow. Disclosure S.Kacar: None. K.Singh: None. S.S.Verma: None. D.Reese: None. M.Kumar: None. S.Roy: None. C.K.Sen: n/a.

510-P: Tissue Nanotransfection–Based Endothelial-Targeted Epigenetic Gene Editing In Vivo to Rescue Diabetic Ischemic Wounds

Our previous work (PMID: 35192691) has identified that genetically silenced phospholipase Cγ2 (PLCγ2) hinders VEGF therapy of the diabetic ischemic limb. Hyperglycemia caused hypermethylation of endothelial specific gene promoter. Given that epigenetic changes are reversible, this work tests the significance of gene-targeted therapeutic DNA demethylation in improving blood flow to diabetic ischemic wounds. Bipedicle ischemic wounds were placed in streptozotocin (STZ) induced acute diabetic C57BL6 mice. Diabetic VWF+ dermal endothelial cells were isolated using flow sorting. In wound tissue, PLCγ2 promoter CpG methylation levels were analyzed using bisulfite sequencing. Next, to specifically demethylate PLCγ2 promoter in endothelial cells, a demethylation cocktail was designed: (i) (scFv)-TET1 catalytic domain (TET1CD) system capable of inducing targeted DNA methylation, and (ii) endothelial promoter driven gene specific guide RNAs. This demethylation cocktail was delivered at the diabetic ischemic wound-edge employing non-viral topical tissue nano-transfection (TNT) technology. PLCγ2 protein expression outcomes were assessed using flow cytometry. Functional outcome of such demethylation was assessed using Laser Speckle Perfusion imaging (Perimed Inc.) and ultrasonography (Vevo 2100) at days 3, 7&amp;10. Overall DNA hyper methylation was prominent in murine ischemic flaps as demonstrated by the increased ratio of 5-methylcytosine (methylation mark) to 5-hydroxymethylcytosine (demethylation mark) (n=5). Specifically, the PLCγ2 promoter was hypermethylated (n=5). TNT mediated endothelial-targeted demethylation of the PLCγ2 promoter increased its expression (n=4). Such demethylation-based upregulation of PLCγ2 improved wound tissue blood flow with increased abundance of VWF+/PLCG2+ vascular elements (n=4). TNT-based endothelial demethylation of the PLCγ2 gene promoter improved perfusion of cutaneous diabetic wounds. Disclosure S.S.Verma: None. S.Kacar: None. S.C.Gnyawali: None. L.S.Fehme: None. C.K.Sen: n/a. K.Singh: None. Funding U.S. Department of Defense (W81XWH-22-1-0146)

TIBIAL CORTEX TRANSVERSE TRANSPORT ACCELERATES WOUND HEALING VIA ENHANCED ANGIOGENESIS AND IMMUNOMODULATION

Treatment for delayed wound healing resulting from peripheral vascular diseases and diabetic foot ulcers remain a challenge. A novel surgical technique named Tibial Cortex Transverse Transport has been developed for treating peripheral ischaemia, with encouraging clinical effects. However, its underlying mechanisms remain unclear. In present study, we aimed to explore the wound healing effects after undergoing this novel technique via multiple ways.A novel rat model of Tibial Cortex Transverse Transport was established with a designed external fixator and effects on wound healing were investigated. All rats were randomized into 3 groups, with 12 rats per group: sham group (negative control), fixator group (positive control) and Tibial Cortex Transverse Transport group. Laser speckle perfusion imaging, vessel perfusion, histology and immunohistochemistry were used to evaluate the wound healing processes.Gross and histological examinations showed that Tibial Cortex Transverse Transport technique accelerated wound closure and enhanced the quality of the newly formed skin tissues. In Tibial Cortex Transverse Transport group, HE staining demonstrated a better epidermis and dermis recovery, while immune-histochemical staining showed that Tibial Cortex Transverse Transport technique promoted local collagen deposition. Tibial Cortex Transverse Transport technique also benefited to angiogenesis and immunomodulation. In Tibial Cortex Transverse Transport group, blood flow in the wound area was higher than that ofother groups according to laser speckle imaging with more blood vessels observed. Enhanced neovascularization was seen in the Tibial Cortex Transverse Transport group with double immune-labelling of CD31 and α-SMA. The M2 macrophages at the wound site in the Tibial Cortex Transverse Transport group was also increased.Tibial cortex transverse transport technique accelerated wound healing through enhanced angiogenesis and immunomodulation.

Tibial cortex transverse transport accelerates wound healing via enhanced angiogenesis and immunomodulation

AimsTreatment for delayed wound healing resulting from peripheral vascular diseases and diabetic foot ulcers remains a challenge. A novel surgical technique named ‘tibial cortex transverse transport’ (TTT) has been developed for treating peripheral ischaemia, with encouraging clinical effects. However, its underlying mechanisms remain unclear. In the present study, we explored the potential biological mechanisms of TTT surgery using various techniques in a rat TTT animal model.MethodsA novel rat model of TTT was established with a designed external fixator, and effects on wound healing were investigated. Laser speckle perfusion imaging, vessel perfusion, histology, and immunohistochemistry were used to evaluate the wound healing processes.ResultsGross and histological examinations showed that TTT technique accelerated wound closure and enhanced the quality of the newly formed skin tissues. In the TTT group, haematoxylin and eosin (H&E) staining demonstrated a better epidermis and dermis recovery, while immunohistochemical staining showed that TTT technique promoted local collagen deposition. The TTT technique also benefited to angiogenesis and immunomodulation. In the TTT group, blood flow in the wound area was higher than that of other groups according to laser speckle imaging with more blood vessels observed. Enhanced neovascularization was seen in the TTT group with double immune-labelling of CD31 and α-Smooth Muscle Actin (α-SMA). The number of M2 macrophages at the wound site in the TTT group was also increased.ConclusionThe TTT technique accelerated wound healing through enhanced angiogenesis and immunomodulation.Cite this article: Bone Joint Res 2022;11(4):189–199.

Application of laser speckle blood perfusion imaging in the evaluation of erectile function in rats.

Intracavernosal pressure measurement is the gold standard for evaluating erectile function in experimental animals, but it has the shortcoming of being invasive. This study aimed to explore the application of laser speckle perfusion imaging in evaluating erectile function in rats. Sixty Sprague Dawley rats were randomly divided into the sham operation and model groups (n=30 each). A rat model of neuroinjury erectile dysfunction was established by surgically damaging the bilateral cavernous nerves in the model group. Simulated surgery was performed in the sham operation group; the nerves were not damaged. Erectile function was evaluated by comparing the changes in intracavernosal pressure and blood flow fluctuations when the cavernous nerve was stimulated using the same voltage parameters. Intracavernosal pressure in the model group was significantly lower than that in the other group when using 2.5V. No significant difference was found in cavernous blood flow fluctuation between the two groups when using 0.5V. Cavernous blood flow fluctuation in the model group after 2.5V, 5V and 7.5V stimulations was significantly lower than that in the sham operation group. Evaluating erectile function in rats is feasible by measuring the cavernous blood flow using laser speckle perfusion imaging.

Effect of electroacupuncture on the hemorheology and hepatic microcirculation in mice with chronic alcoholic liver injury

To observe the effect of electroacupuncture (EA) on the hemorheology and hepatic microcirculation in mice with chronic alcoholic liver injury (cALI), so as to explore the microcirculation mechanism of EA underlying regulating liver function in cALI mice. Forty Kunming mice were randomly assigned to control, model, acupoint EA and non-acupoint EA groups, with 10 mice in each group. The cALI model was established by gavage with 50% ethanol (15 mL/kg，2 times per day with 8 hours' interval) for 28 d. Mice in the acupoint EA group received EA at bilateral "Zusanli" (ST36) and "Ganshu"(GB11) for 20 min, once daily for 14 d. And mice in the non-acupoint EA group received EA the spot about 1 cm lateral to GB11 and 0.5 cm lateral to ST36 respectively for 20 min, once daily for 14 d. The hepatic blood perfusion (HBP) of mice was detected by laser speckle perfusion imaging. The index of hemorheology, serum markers of liver function and hepatic histology in mice were observed by automatic blood rheometer, automatic bio-analysis machine and H.E. staining, respectively. Compared with the control group, the HBP of the model group was significantly decreased (P<0.05); the low-cut whole blood viscosity, plasma viscosity, and erythrocyte sedimentation level were significantly increased （P<0.05）, and the red blood cell deformation index was significantly decreased (P<0.05); serum alanine aminotransferase (ALT) and gamma-glutamyl transferase (GGT) increased significantly (P<0.05) in the model group. Hepatocyte steatosis accompanied by inflammatory cell infiltration and focal necrosis was observed in the model group. After EA at ST36 and GB11, and in comparison with the model group showed that the HBP was significantly increased (P<0.05); the low-cut whole blood viscosity, plasma viscosity, erythrocyte sedimentation rate and serum GGT were significantly decreased (P<0.05), and the red blood cell deformation index was significantly increased (P<0.05）; the degree of fatty degeneration in the liver tissue was reduced, and the focal necrosis was reduced. The plasma viscosity, erythrocyte sedimentation rate were significantly decreased (P<0.05), and the red blood cell deformation index was significantly increased in the non-acupoint EA group relevant to the model group (P<0.05). The erythrocyte sedimentation rate and the red blood cell deformation index of the acupoint EA group was significantly lower than that of the non-acupoint EA group (P<0.05). EA at ST36 and GB11 could improve the hemorheological disorder and the hepatic microcirculation, and inhibit the deve-lopment of liver injury in cALI mice.

Peri-Infarct Hot-Zones Have Higher Susceptibility to Optogenetic Functional Activation-Induced Spreading Depolarizations.

Spreading depolarizations (SDs) are recurrent and ostensibly spontaneous depolarization waves that may contribute to infarct progression after stroke. Somatosensory activation of the metastable peri-infarct tissue triggers peri-infarct SDs at a high rate. We directly measured the functional activation threshold to trigger SDs in peri-infarct hot zones using optogenetic stimulation after distal middle cerebral artery occlusion in Thy1-ChR2-YFP mice. Optogenetic activation of peri-infarct tissue triggered SDs at a strikingly high rate (64%) compared with contralateral homotopic cortex (8%; P=0.004). Laser speckle perfusion imaging identified a residual blood flow of 31±2% of baseline marking the metastable tissue with a propensity to develop SDs. Our data reveal a spatially distinct increase in SD susceptibility in peri-infarct tissue where physiological levels of functional activation are capable of triggering SDs. Given the potentially deleterious effects of peri-infarct SDs, the effect of sensory overstimulation in hyperacute stroke should be examined more carefully.

600-P: Single-Cell RNA Sequencing Identifies Novel Molecular Targets of Endothelial MicroRNA-200b in Diabetic and Ischemic Wounds

Injury induced transient downregulation of endothelial miR-200b is required to jump-start wound angiogenesis but the underlying mechanisms remain unclear. To interrogate the transcriptional changes, single-cell RNA sequencing was performed on ∼15000 human endothelial cells following treatment with miR-200b inhibitor or its mimic. The 3’ scRNA-seq analysis was conducted using the Chromium single cell system (10x Genomics). Unsupervised clustering using CellRanger identified 13 cell clusters, few of which were miR-200b responsive. To verify the novel potential targets of miR-200b identified in endothelial cells, we used Stable Isotope Labelling by Amino acids in Cell culture (SILAC) based proteomic analysis using mass spectrometry. A total of 3818 proteins were detected which were then filtered using statistical cut-offs (p value &amp;lt; 0.05; % change &amp;gt; 10%) identifying 319 proteins targeted by miR-200b. To dissect mechanisms involved in vascular function post-miR-200b silencing in vivo, we generated miR-200b-429fl/fl-Tie2 Cre mice where endothelial miR-200b levels was depleted. The therapeutic significance of miR-200b inhibition was studied using ischemic hind limb in these mice. Perfusion imaging was performed using Laser Speckle Perfusion imaging. Color-coded perfusion maps were acquired at different time-points post-surgery (d3, d7, d10, d14) and average perfusion was calculated using PimSoft v1.4 software. Inhibition of endothelial miR-200b rescued hindlimb ischemia with improved perfusion (n=5). This rescue was associated with increased abundance of CD31+/vWF+ vasculogenic cells at the site of injury (n=5). This work identified miR-200b regulated endothelial cell clusters, the functionality of which are explained by SILAC proteomics. These findings provide insight into novel mechanisms explaining how transient inhibition of endothelial miR-200b helps switch vascular homeostasis into an angiogenic fate in response to injury. Disclosure K. Singh: None. E. Hernandez: None. M. Kumar: None. Y. Rustagi: None. S.K. Mohanty: None. A.S. Abouhashem: None. S. Khanna: None. S. Roy: None. C.K. Sen: None. Funding National Institutes of Health (GM108014); Eli Lilly and Company; Indiana Collaborative Initiative for Talent Enrichment (to C.K.S., S.R.)

Application of laser speckle perfusion imaging in predicting wound healing time of burn patients

Objective: To explore the application effect of laser speckle perfusion imaging (LSPI) in predicting wound healing time of burn patients. Methods: LSPI was performed in 84 adult burn patients hospitalized in department of burns of Tongren Hospital of Wuhan University & Wuhan Third Hospital within post injury hour (PIH) 24 to 72 to detect the blood perfusion values of the wounds. The wound healing time was recorded. The 128 wounds were divided into superficial group (wound healing time shorter than or equal to 14 d, n=57) and deep group (wound healing time longer than 14 d and shorter than or equal to 28 d, n=71) according to the healing time. The blood perfusion values of the two groups were compared. Data were processed with t test or chi-square test. The receiver operating characteristic (ROC) curve was drawn and Youden index was calculated to determine the optimal critical blood perfusion value of wound healing time of the two groups, and the validity of the critical value was assessed by Kappa consistency test. Results: (1) The blood perfusion value of woundsin superficial group was (6.8±1.8) perfusion unit (PU), which was significantly higher than (3.5±1.3) PU in deep group (t=11.404, P<0.01). (2) The total area under ROC curve of blood perfusion value to predict wound healing time was 0.931 (with 95% confidence interval 0.887-0.975, P<0.01). Combined with Youden index, 5.52 PU was chosen as the optimal critical value of wound healing time of the two groups, with sensitivity of 76.9% and specificity of 94.7%. (3) The healing time of 44 wounds predicted was shorter than or equal to 14 d, and the healing time of 84 wounds predicted was longer than 14 d and shorter than or equal to 28 d, while the actual number of wounds was 57 and 71, respectively. The Kappa coefficient of consistency test was 0.754 (P<0.01). Conclusion: LSPI is a useful method to predict the healing time of burn wounds.

Evaluation of liver function and electroacupuncture efficacy of animals with alcoholic liver injury by the novel imaging methods.

Imaging methods to evaluate hepatic microcirculation (HM) and liver function (LF) by directly monitoring overall liver tissue remain lacking. This study establish imaging methods for LF that combines Laser speckle perfusion imaging (LSPI) and in vivo optical imaging (IVOI) technologies to investigate changes of hepatic microcirculation and reserve function in the animals gavaged with 50% ethanol (15 ml/kg·bw) for a model of acute alcoholic liver injury (ALI), and for evaluation of electroacupuncture (EA) effect. The liver blood perfusion and indocyanine green (ICG) distribution were observe by LSPI and IVOI separately. After EA, the livers were collected to measure the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), thromboxane A (TXA2), prostacyclin (PGI2) and endothelin (ET). The acquisitions of newly established LSPI of liver and ICG in vivo fluorescence imaging (ICG-IVFI), combining the results of other indexes showed: hepatic microcirculation perfusion (HMP) significantly reduced, ICG metabolism reduced, and ALT/AST increased in animal model with acute ALI. EA can reverse these changes. The use of LSPI of liver and ICG-IVFI, which was novel imaging methods for LF established in this study, could display the LF characteristics of ALI and the EA efficacy.

Abstract 328: Effects of Unilateral Hindlimb Venous Occlusion in a Murine Model

Introduction: Acute and chronic venous occlusion from thrombosis or other causes leads to long-term morbidity in a significant number of individuals. Understanding mechanisms of venous flow restoration may lead to future treatments and prevention of morbidity. Authors have previously hypothesized that this occurs by a combination of thrombus recanalization and neovascularization with collateral formation. A model of permanent hindlimb venous occlusion may allow the study of flow restoration independent of vein recanalization. Methods: Adult C57Bl/6 mice (n=9 total, n=6 operated, n=3 unoperated controls) were subjected to identical unilateral hindlimb venous occlusive surgeries (n=6 limbs total) involving both vein ligation and permanent ablation using an external thermal device. Contralateral limbs underwent no surgery and served as in-vivo controls (n=6 limbs total). Laser speckle perfusion imaging of hindlimbs was conducted for 10 post-operative days. Real-time qPCR was used to measure tissue mRNA expression of venous angiogenesis-related signaling molecules. Results: Mean treated limb perfusion was 165.0 perfusion units (PU), 179.9 PU, and 154.2 PU on post-operative days 3, 5 and 7, respectively. This compares to 121.5 PU, 125.0 PU, and 117.2 PU in the untreated limb (p=0.01, 0.03 and 0.04 respectively). Relative VEGFa, EphB4 and COUP-TFII expression in treated limbs immediately following surgery and at postoperative day 10 was not significantly different compared to unoperated controls. Conclusions: Surgical occlusion of unilateral hindlimb venous drainage leads to tissue hyperemia by laser speckle imaging 3, 5 and 7 days after surgery which resolves by post-operative day 10. Gene expression reveals no evidence of induced venous angiogenesis to account for restoration of limb drainage. Re-establishment of venous outflow may be due to recruitment of existing venous collateral tributaries rather than true neovascularization in this model.

Effects of Electroacupuncture Stimulation at "Zusanli" Acupoint on Hepatic NO Release and Blood Perfusion in Mice.

The study is to observe the influence of electroacupuncture (EA) stimulation at “Zusanli” (ST36) on the release of nitric oxide (NO) and blood perfusion (BP) in the liver and further explore whether the hepatic blood perfusion (HBP) changes were regulated by EA ST36 induced NO in nitric oxide synthase inhibited mice. The HBP change of the mice was detected by laser speckle perfusion imaging (LSPI) before and after being given interventions, and the NO in liver tissue was detected by nitric acid reductase in each group. The NO levels and HBP in the L-NAME group were significantly lower than those in the control group (P < 0.01). The NO level and HBP increase in EA group were significantly higher than those in control group (P < 0.05). The NO level in the L-NAME EA group was slightly higher than that in the L-NAME group. The HBP increase in the L-NAME EA group was not statistically significant. These results showed that EA could accelerate the synthesis of NO and thereby increase HBP via vasodilation in liver tissue.

Vascular structure and function in the medial collateral ligament of anterior cruciate ligament transected rabbit knees.

To determine if decreased vascular responsiveness in the medial collateral ligament (MCL) of anterior cruciate ligament transected (ACL-t) rabbit knees is due to pericyte deficiency associated with angiogenesis. Vascular responses to potassium chloride (KCl), phenylephrine, acetylcholine, and sodium nitroprusside (SNP) were evaluated in ACL-t rabbit knees (n = 6) and control knees (n = 5) using laser speckle perfusion imaging. Ligament degeneration was determined by ultrasound imaging. Vascular and pericyte volume were measured using quantitative immunohistochemical volumetric analysis using CD31 and α-smooth muscle actin antibodies with co-localization analysis. Perfusion was increased in the ACL-t rabbits 2.5-fold. Responsiveness to phenylephrine, SNP, and acetylcholine was significantly decreased in the ACL knee while no change in KCl responses was seen. MCL ultrasound imaging revealed decreased collagen organization, increased ligament thickness, and increased water content in the ACL-t MCL. Vascular Volume was increased fourfold in ACL deficient knees, while pericyte volume to endothelial volume was not changed. No difference in CD31 and α-SMA co-localization was found. Blood vessels in the MCL of ACL-t knees do not lack smooth muscle. The MCL vasculature can undergo constrictive response to KCl, but have impaired receptor mediated responses and impaired nitric oxide signaling.

Multilevel synchronization of tubuloglomerular feedback is modulated by nitric oxide (692.10)

Previously we showed that NO modulates synchronization of both mechanisms of renal autoregulation. We did not, however, provide a mechanism to explain TGF synchronization after L‐NAME. L‐NAME changed TGF synchronization in a way we did not understand. Here we show that TGF synchronization may become stronger within cortical lobules after L‐NAME, resulting in two mechanisms of synchronization within a synchronized cluster. Male Long‐Evans rats (N=8) were anaesthetized (isoflurane) and their left kidneys were exposed. Cortical perfusion during control and NOS inhibition (L‐NAME, 10mg/kg i.v.) was monitored by laser speckle perfusion imaging. The TGF frequency range (0.01‐0.06 Hz) was isolated with a band pass filter. Clusters of synchronized nephrons were identified based on phase coherence (PC) between all pairs of imaged pixels. During control, TGF was synchronized moderately over large areas (~5x7 mm). After L‐NAME, each cluster had areas with high PC (strong synchronization) in the center and lower, but significant, PC in the surround. When the PC reference pixel was moved systematically there was strong synchronization within smaller ~800 x 800 um areas. The position of the high PC areas was consistent with an anatomical basis for synchronization. We conclude that TGF synchronization becomes stronger within cortical lobules while remaining weakly synchronized among lobules within the larger clusters.Grant Funding Source: Supported by CIHR MOP‐102694

Investigation of Hepatic Blood Perfusion by Laser Speckle Imaging and Changes of Hepatic Vasoactive Substances in Mice after Electroacupuncture

The study was conducted to observe the effect of electroacupuncture (EA) on hepatic blood perfusion (HBP) and vascular regulation. We investigated 60 male anesthetized mice under the following 3 conditions: without EA stimulation (control group); EA stimulation at Zusanli (ST36 group); EA stimulation at nonacupoint (NA group) during 30 min. The HBP was measured using the laser speckle perfusion imaging (LSPI). The level of nitric oxide (NO), endothelin-1 (ET-1), and noradrenaline (NE) in liver tissue was detected by biochemical methods. Results were as follows. At each time point, HBP increase in ST36 group was higher than that in the NA group in anesthetized mice. HBP gradually decreased during 30 min in control group. The level of NO in ST36 group was higher than that in NA group. The level of both ET-1 and NE was the highest in control group, followed by NA group and ST36 group. It is concluded that EA at ST36 could increase HBP possibly by increasing the blood flow velocity (BFV), changing vascular activity, increasing the level of NO, and inhibiting the level of ET-1 in liver tissue.

Detecting physiological systems with laser speckle perfusion imaging of the renal cortex

Laser speckle perfusion imaging (LSPI) has become an increasingly popular technique for monitoring vascular perfusion over a tissue surface. However, few studies have utilized the full range of spatial and temporal information generated by LSPI to monitor spatial properties of physiologically relevant dynamics. In this study, we extend the use of LSPI to analyze renal perfusion dynamics over a spatial surface of ~5 × 7 mm of renal cortex. We identify frequencies related to five physiological systems that induce temporal changes in renal vascular perfusion (cardiac flow pulse, respiratory-induced oscillations, baroreflex components, the myogenic response, and tubuloglomerular feedback) across the imaged surface and compare the results with those obtained from renal blood flow measurements. We find that dynamics supplied from global sources (cardiac, respiration, and baroreflex) present with the same frequency at all locations across the imaged surface, but the local renal autoregulation dynamics can be heterogeneous in their distribution across the surface. Moreover, transfer function analysis with forced blood pressure as the input yields the same information with laser speckle imaging or renal blood flow as the output during control, intrarenal infusion of N(ω)-nitro-L-arginine methyl ester to enhance renal autoregulation, and intrarenal infusion of the rho-kinase inhibitor Y-27632 to inhibit vasomotion. We conclude that LSPI measurements can be used to analyze local as well as global renal perfusion dynamics and to study the properties of physiological systems across the renal cortex.