Laser Doppler Imaging Research Articles

Peripheral Microvascular Abnormalities Associated with Open-Angle Glaucoma

To investigate peripheral microvascular abnormalities associated with patients with open-angle glaucoma (OAG). This was a cross-sectional study. Patients with OAG and controls. All subjects underwent detailed ophthalmic evaluation, including Humphrey visual field (HVF) tests and swept source OCT. To evaluate peripheral microvascular abnormalities, nailfold capillaroscopy (NFC) and laser Doppler imaging (LDI) were performed. The presence of microhemorrhages, tortuous capillaries, dilated capillaries, avascular areas, and the capillary density, among other characteristics, were recorded using NFC; fingertip blood flow (FBF) was measured using LDI at different time points, before and 1, 10, and 20 minutes after exposure to a cold stimulus. In addition, venous blood samples were collected to measure serum endothelin-1 (ET-1) concentrations as well as serum autoantibodies. Presence of microhemorrhages, tortuous capillaries, and dilated capillaries; FBF; ET-1; and autoantibodies. Sixty-eight subjects (43 patients with OAG and 25 controls) were enrolled in the study. Microhemorrhages were found in the nail bed of 65.1% of the patients with OAG compared with 25.0% of the controls (P= 0.003). There was a significant difference in the mean FBF at the baseline in patients with OAG versus controls (293.6 ± 100.2 vs 388.8 ± 52.0 perfusion units, respectively, P < 0.001), together with a significant decrease in the mean FBF 10 and 20 minutes after cold stimulus in patients with OAG in comparison to controls (P < 0.001 for all comparisons). There was a positive correlation between mean baseline FBF and HVF mean deviation (r= 0.27, P= 0.03) and between mean baseline FBF and average retinal nerve fiber layer thickness (r=0.44, P= 0.001). Neither the analysis of ET-1 concentrations (P= 0.71) nor the autoantibodies measurements (P > 0.05, for all) showed any difference between the 2 groups. Significant peripheral microvascular abnormalities were found in patients with OAG compared to controls, suggesting that microvascular changes might play a role in the pathogenesis of the disease. In addition, part of these peripheral microvascular abnormalities seems to be correlated with both functional and structural glaucomatous damage. The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Platelet-derived concentrates influence human keratinocyte proliferation in vitro and induce wound healing in a prospective case series of chronic wounds of different entities in vivo.

ObjectivesSoft tissues defects can extend into the fat layer or even deeper and can cause significant clinical disadvantages like pain, infections, and loss of function. In particular, chronic wounds are difficult to treat, as split-thickness skin grafts (STSGs) have varying success rates. To improve wound healing in chronic wounds, the authors have studied the application of platelet-mediator concentrate (PMC) in a human keratinocyte culture model in vitro and of autologous platelet concentrates (PRP) in a combination with surgical procedures in vivo as second line therapy in patients with initially failed wound closure.MethodsFor in vitro testing on keratinocytes, a PMC was processed with a commercially available bedside system (ATR®, Curasan, Germany). In a clinical, nonrandomized study, five in-house patients with chronic wounds were treated using a combination of surgical debridement and autologous PRP. Time of healing as determined by epithelization as well as laser Doppler imaging to visualize blood flow was analyzed. Additionally, changes in ease of surgical wound closure were determined. Finally, the quality of life of patients was assessed using a validated questionnaire (clinicaltrials.gov # NCT03667638).ResultsIn vitro testing shows a significant effect of PMC on keratinocyte proliferation in cell culture. Clinical studies showed that patients treated with PRP had initiation of wound closure, higher blood flow after PRP injection, and easier wound closure as well as improved quality of life.ConclusionsThe injection of platelet concentrates to treat chronic wound defects presents a favorable addition to treatment where single surgical procedures have failed and may improve current therapy options.

Sodium thiosulfate, a source of hydrogen sulfide, stimulates endothelial cell proliferation and neovascularization.

Therapies to accelerate vascular repair are currently lacking. Pre-clinical studies suggest that hydrogen sulfide (H2S), an endogenous gasotransmitter, promotes angiogenesis. Here, we hypothesized that sodium thiosulfate (STS), a clinically relevant source of H2S, would stimulate angiogenesis and vascular repair. STS stimulated neovascularization in WT and LDLR receptor knockout mice following hindlimb ischemia as evidenced by increased leg perfusion assessed by laser Doppler imaging, and capillary density in the gastrocnemius muscle. STS also promoted VEGF-dependent angiogenesis in matrigel plugs in vivo and in the chorioallantoic membrane of chick embryos. In vitro, STS and NaHS stimulated human umbilical vein endothelial cell (HUVEC) migration and proliferation. Seahorse experiments further revealed that STS inhibited mitochondrial respiration and promoted glycolysis in HUVEC. The effect of STS on migration and proliferation was glycolysis-dependent. STS probably acts through metabolic reprogramming of endothelial cells toward a more proliferative glycolytic state. These findings may hold broad clinical implications for patients suffering from vascular occlusive diseases.

A chromatin signature by the methyltransferase SETD7 orchestrates angiogenic response in diabetic limb ischemia

Abstract Background Peripheral artery disease (PAD) is highly prevalent in patients with diabetes (DM) and associates with a high rate of limb amputation and poor prognosis. Surgical and catheter-based revascularization have failed to improve outcome in DM patients with PAD. Hence, a need exists to develop new treatment strategies able to promote blood vessel growth in this setting. Mono-methylation of histone 3 at lysine 4 (H3K4me1) – a specific epigenetic signature induced by the histone methyltransferase SETD7 – favours an open chromatin thus enabling gene transcription. Purpose To investigate whether SETD7-dependent epigenetic changes modulate angiogenic response in diabetes. Methodology Primary human aortic endothelial cells (HAECs) were exposed to normal glucose (NG, 5 mM) or high glucose (HG, 25 mM) concentrations for 48 hours. Unbiased gene expression profiling was performed by RNA sequencing (RNA-seq) followed by Ingenuity Pathway Analysis (IPA). In vitro assays, namely cell migration and tube formation were employed to study angiogenic properties in HAECs. SETD7 and H3K4me1 levels were investigated by Western blot and Chromatin immunoprecipitation (ChIP). Pharmacological blockade of SETD7 was achieved by using the highly selective inhibitor (R)-PFI-2. Mice with streptozotocin-induced diabetes were orally treated with (R)-PFI-2 or vehicle and underwent hindlimb ischemia by femoral artery ligation for 14 days. Blood flow recovery was analysed at 30 minutes, 7 and 14 days by laser Doppler imaging. Our experimental findings were also translated in gastrocnemius muscle samples from patients with and without diabetes. Results RNA-seq in HG-treated HAECs revealed a profound upregulation of the methyltransferase SETD7, an enzyme involved in mono-methylation of lysine 4 at histone 3 (H3K4me1). SETD7 upregulation in HG-treated HAECs was associated with increased H3K4me1 levels as well as with impaired endothelial cell migration and tube formation. Both SETD7 gene silencing and pharmacological inhibition by (R)PFI-2 rescued hyperglycemia-induced impairment of HAECs migration and tube formation, while SETD7 overexpression blunted the angiogenic response. RNA-seq and ChIP assays showed that SETD7-dependent H3K4me1 regulates the transcription of the angiogenesis inhibitor semaphorin-3G (SEMA-3G). Moreover, SEMA-3G overexpression blunted migration and tube formation in SETD7-depleted HAECs. In diabetic mice with hindlimb ischemia, treatment with (R)-PFI-2 improved limb vascularization and perfusion as compared to vehicle. Finally, SETD7/SEMA3G axis was upregulated in muscle specimens from T2D patients as compared to controls. Conclusion Targeting SETD7 represents a novel epigenetic-based therapy to boost neovascularization in diabetic patients with PAD. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): University of Zurich - Forshungskredit candoc grant

The Use of Infrared Thermography (IRT) in Burns Depth Assessment: A Diagnostic Accuracy Meta-Analysis

Background: The timely diagnosis of burns depth is crucial to avoid unnecessary surgery and delays in adequate management of patients with burn injuries. Whilst it is mostly a clinical diagnosis, indocyanine green, laser Doppler imaging and infrared thermography have been used alongside clinical findings to support the diagnosis. Infrared thermography is a noninvasive technique which uses temperature differences to diagnose tissue burn depth. Our study aims to assess its use in differentiating between superficial and deep burns. Methods: We conducted a systematic literature review and meta-analysis using electronic databases. We used a mixed-effects logistic regression bivariate model to estimate summary sensitivity and specificity and developed hierarchical summary receiver operating characteristic (HSROC) curves. Results: We identified 6 studies reporting a total of 197 burns, of which 92 were proven to be deep burns. The reference standard was clinical assessment at the time of injury and burn healing time. The pooled estimates for sensitivity and specificity were 0.84 (95% CI 0.71–0.92) and 0.76 (95% CI 0.56–0.89), respectively. Conclusions: IRT is a promising burns assessment modality which may allow surgeons to correctly classify burn injuries at the time of presentation. This will allow a more efficient management of burns and timely surgical intervention.

Efficacy assessment of laser Doppler imager in diagnosing the pulp vitality after dental trauma

Background/purposeThis is the first paper evaluating the efficacy of laser Doppler imager in diagnosis of pulpal vitality. The purpose of this study was to evaluate and compare the diagnostic benefits of laser Doppler imaging and electric pulp test (EPT) in dental trauma. Materials and methodsSeven patients were selected for pulp vitality evaluation in Kaohsiung Medical University Hospital between 2018 and 2019. EPT and laser Doppler imager evaluation were performed for patients with traumatic injury to teeth. Statistical methods included the Kappa consistency test and the chi-square test. In addition, the receiver operating characteristic (ROC) curve, and the area under the curve (AUC) were used. ResultsThere was a significant difference in Doppler flow values between the severe trauma group and the mild trauma group, regardless of patient self-reported symptoms (P = 0.043) or physicians’ diagnostic classification (P = 0.018). For an EPT instrument, the Kappa coefficient was 0.67 and 1-year pulpal status findings were highly consistent (P < 0.001). Using a Doppler instrument, the Kappa coefficient was 0.85. According to the ROC curve, the AUC for EPT was 0.94, the AUC for Doppler was 1, and the optimal cut-off value was 31.55, indicating that both were superior diagnostic tools. ConclusionBoth laser Doppler imager and EPT can be used as tools for diagnosing traumatic pulp necrosis. Doppler imaging instruments allow for a more timely and accurate assessment of pulp vitality in dental trauma. In the future, ongoing research and related training are necessary for interpretation of Doppler data.

A hydrogen-sulfide derivative of mesalamine reduces the severity of intestinal and lung injury in necrotizing enterocolitis through endothelial nitric oxide synthase.

Necrotizing enterocolitis (NEC) remains a devastating disease that affects preterm infants. Hydrogen sulfide (H2S) donors have been shown to reduce the severity of NEC, but the optimal compound has yet to be identified. We hypothesized that oral H2S-Mesalamine (ATB-429) would improve outcomes in experimental NEC, and its benefits would be dependent on endothelial nitric oxide synthase (eNOS) pathways. NEC was induced in 5-day-old wild-type (WT) and eNOS knockout (eNOSKO) pups by formula feeding and stress. Four groups were studied in both WT and eNOSKO mice: 1) breastfed controls, 2) NEC, 3) NEC + 50 mg/kg mesalamine, and 4) NEC + 130 mg/kg ATB-429. Mesalamine and ATB-429 doses were equimolar. Pups were monitored for sickness scores and perfusion to the gut was measured by Laser Doppler Imaging (LDI). After euthanasia of the pups, intestine and lung were hematoxylin and eosin-stained and scored for injury in a blind fashion. TLR4 expression was quantified by Western blot and IL-6 expression by ELISA. P < 0.05 was significant. Both WT and eNOSKO breastfed controls underwent normal development and demonstrated milder intestinal and pulmonary injury compared with NEC groups. For the WT groups, ATB-429 significantly improved weight gain, reduced clinical sickness score, and improved perfusion compared with the NEC group. In addition, WT ATB-429 pups had a significantly milder intestinal and pulmonary histologic injury when compared with NEC. ATB-429 attenuated the increase in TLR4 and IL-6 expression in the intestine. When the experiment was repeated in eNOSKO pups, ATB-429 offered no benefit in weight gain, sickness scores, perfusion, intestinal injury, pulmonary injury, or decreasing intestinal inflammatory markers. An H2S derivative of mesalamine improves outcomes in experimental NEC. Protective effects appear to be mediated through eNOS. Further research is warranted to explore whether ATB-429 may be an effective oral therapy to combat NEC.

Cobra Venom Factor Boosts Arteriogenesis in Mice.

Arteriogenesis, the growth of natural bypass blood vessels, can compensate for the loss of arteries caused by vascular occlusive diseases. Accordingly, it is a major goal to identify the drugs promoting this innate immune system-driven process in patients aiming to save their tissues and life. Here, we studied the impact of the Cobra venom factor (CVF), which is a C3-like complement-activating protein that induces depletion of the complement in the circulation in a murine hind limb model of arteriogenesis. Arteriogenesis was induced in C57BL/6J mice by femoral artery ligation (FAL). The administration of a single dose of CVF (12.5 µg) 24 h prior to FAL significantly enhanced the perfusion recovery 7 days after FAL, as shown by Laser Doppler imaging. Immunofluorescence analyses demonstrated an elevated number of proliferating (BrdU+) vascular cells, along with an increased luminal diameter of the grown collateral vessels. Flow cytometric analyses of the blood samples isolated 3 h after FAL revealed an elevated number of neutrophils and platelet-neutrophil aggregates. Giemsa stains displayed augmented mast cell recruitment and activation in the perivascular space of the growing collaterals 8 h after FAL. Seven days after FAL, we found more CD68+/MRC-1+ M2-like polarized pro-arteriogenic macrophages around growing collaterals. These data indicate that a single dose of CVF boosts arteriogenesis by catalyzing the innate immune reactions, relevant for collateral vessel growth.

Evaluation of Bromelain-Based Enzymatic Debridement Combined with Laser Doppler Imaging and Healing of Burn Wounds.

BackgroundAccurate estimation of burn depth is crucial for correct treatment decision making. Bromelain-based enzymatic debridement (ED) may improve clinical assessment of burn depth. Laser Doppler imaging (LDI) provides a valuable indicator of burn depth by analyzing microcirculation within tissue beds. This study aimed to evaluate bromelain-based enzymatic debridement combined with laser Doppler imaging and healing of 42 wounds in 19 patients with mixed second- and third-degree thermal burns.Material/MethodsWe included 42 wounds in 19 patients with mixed deep dermal and full-thickness thermal burns. All patients were treated with eschar-specific removal agent for ED. The perfusion of each wound after ED was assessed using LDI. Healing time was estimated by 2 experienced burn surgeons and marked by the observation of epithelization. The usefulness of the LDI performed after ED in predicting healing time was estimated. The findings were analyzed to determine a cut-off value for LDI that indicates if a burn will heal spontaneously.ResultsWe observed that burn wounds with higher mean perfusion healed faster. The analysis showed a strong relationship between perfusion after ED and healing time (Spearman rank correlation coefficient=−0.803). A mean perfusion greater than 296.89 indicated that the wound could heal spontaneously and does not require skin grafting.ConclusionsLDI examination of an already debrided wound allows for a reliable assessment of perfusion at an early stage of treatment. The use of a safe and effective debridement method in conjunction with a non-invasive diagnostic tool could improve burn management.

U-Net based Mapping from Digital Images to Laser Doppler Imaging for Burn Assessment.

The incidence of burn injuries is higher in low-and middle-income countries, and particularly in remote areas where the access to specialized burn assessment, care and recovery is limited. Given the high costs associated with one of the most used techniques to evaluate the severity of a burn, namely laser Doppler imaging (LDI), an alternative approach could be beneficial for remote locations. This study proposes a novel approach to estimate the LDI from digital images of a burn. The approach is a pixel-wise regression model based on convolutional neural networks. To minimize the dependency on the conditions in which the images are taken, the effect of two image normalization techniques is also studied. Results indicate that the model performs satisfactorily on average, presenting low mean absolute and squared errors and high structural similarity index. While no significant differences are found when changing the normalization of the images, the performance is affected by their quality. This suggests that changes in the intensity of the images do not alter the relevant information about the wound, whereas changes in brightness, contrast and sharpness do.

A062 Dye-less, Real-time Visualization of Tissue Perfusion in Porcine Gastric Tube Model using Laser Speckle Contrast Imaging (LSCI) and Laser Doppler Imaging (LDI)

A062 Dye-less, Real-time Visualization of Tissue Perfusion in Porcine Gastric Tube Model using Laser Speckle Contrast Imaging (LSCI) and Laser Doppler Imaging (LDI)

Perineural Capsaicin Treatment Inhibits Collateral Sprouting of Intact Cutaneous Nociceptive Afferents

Perineural treatment of peripheral nerves with capsaicin produces a long-lasting selective regional thermo- and chemo-analgesia and elimination of the neurogenic inflammatory response involving degeneration of nociceptive afferent fibers. In this study, we examined longitudinal changes in mustard oil–induced sensory neurogenic vasodilatation and plasma extravasation following perineural capsaicin treatment of the rat saphenous nerve utilizing scanning laser Doppler imaging and vascular labeling with colloidal silver. Capsaicin treatment resulted in a marked decrease in mustard oil–induced vasodilatation in the skin area served by the saphenous nerve. Repeated imaging of the vasodilatatory response showed no recovery for at least 7 weeks. However, following transection and ligation of the capsaicin-treated saphenous nerve, a substantial recovery of the vasodilatatory response was observed, suggesting a reinnervation of the chemodenervated skin area by collateral sprouting of neighboring intact sciatic nerve afferents. Elimination of the recovered vascular reaction by capsaicin treatment of the sciatic nerve supported this conclusion. Similar results have been obtained by using the vascular labeling technique. These findings indicate an inhibitory effect of persisting cutaneous nerve fibers on the collateral sprouting of intact nerve fibers into the chemodenervated skin area. These observations may bear implications for the development of sensory disturbances following peripheral nerve injuries.

Soluble guanylate cyclase stimulator praliciguat promotes ischemic leg reperfusion in db/db mice

Lower-limb peripheral artery disease (PAD) is a prevalent and challenging complication of diabetes, requiring innovating therapies. Praliciguat is an orally available stimulator of soluble guanylate cyclase (sGC) reported to have favorable effects on metabolic and hemodynamic endpoints in preclinical and clinical studies, suggesting the potential for benefit in PAD. We evaluated the effect of praliciguat on hindlimb ischemia recovery in a mouse model of diabetes and investigated the molecular mechanism of sGC stimulation. Hindlimb ischemia was induced in db/db mice by ligation and excision of the left femoral artery. Praliciguat 10 mg/kg/day ( n = 10 mice) or vehicle ( n = 10) were administered in the diet for 31 days starting 3 days before surgery. Foot perfusion was assessed with a Laser Doppler Imager and reported as a ratio in the ischemic versus non-ischemic limb. Ischemic leg function was assessed with a 4-point scale: 0, plantar/toe flexion in response to tail traction; 1, plantar but not toe flexion; 2, no flexion; 3, foot dragging. Ischemic foot perfusion and function were better 28 days after surgery in praliciguat than in vehicle treated mice (mean ± SD: perfusion 1.05 ± 0.23 vs. 0.38 ± 0.16; P < 0.0001; function 1.14 ± 0.36 vs. 1.73 ± 0.80; P = 0.03). Praliciguat did not impact angiogenesis. Arteriolar diameter was higher (9.88 ± 0.96 vs. 8.57 ± 0.64 μm; P = 0.004) and ICAM-1 expression lower (186 ± 41 vs. 403 ± 177% of healthy muscle; P = 0.005) in the ischemic limb of mice treated with praliciguat than with vehicle. Praliciguat attenuated accumulation of oxidative pro-angiogenic and pro-inflammatory muscle fibers. In cultured myoblasts praliciguat significantly downregulated Myh2 and Cxcl12 mRNA expression. Conditioned medium from praliciguat-treated myoblast decreased ICAM-1 mRNA expression in HUVECs. These results suggest that praliciguat restored perfusion and function in the ischemic muscle of db/db mice by increasing arteriolar diameter and decreasing ICAM-1 expression in endothelial cells via downregulation of Cxcl12 in myocytes.

450-P: Soluble Guanylate Cyclase Stimulator Praliciguat Promotes Ischemic Leg Reperfusion in db/db Mice

Background: Lower-limb peripheral artery disease (PAD) is a prevalent and challenging complication of diabetes, requiring innovating therapies. Praliciguat is an orally available stimulator of soluble guanylate cyclase reported to have favorable effects on metabolic and hemodynamic endpoints in preclinical and clinical studies, suggesting the potential for benefit in PAD. We evaluated the effect of praliciguat on hindlimb ischemia recovery in a mouse model of diabetes. Methods: Hindlimb ischemia was induced in db/db mice by ligation and excision of the left femoral artery. Praliciguat 10mg/kg/day (N=mice) or vehicle (N=10) were administered in the diet for 31 days. Foot perfusion was assessed with a Laser Doppler Imager and reported as a ratio in the ischemic versus non-ischemic limb. Ischemic leg function was assessed with a 4-point scale: 0, plantar/toe flexion in response to tail traction; 1, plantar but not toe flexion; 2, no flexion; 3, foot dragging. Results: Ischemic foot perfusion and function were better 28 days after surgery in praliciguat than in vehicle treated mice (mean ± SD: perfusion 1.± 0.23 vs. 0.38 ± 0.16; p&amp;lt;0.0001; function 1.14 ± 0.36 vs. 1.73 ± 0.80; p=0.03) . Praliciguat did not impact angiogenesis. Arteriolar diameter was higher (9.88 ± 0.96 vs. 8.57 ± 0.64 µm; p=0.004) and ICAM-1 expression lower (186 ± 41 vs. 403 ± 177 % of healthy muscle; p=0.005) in the ischemic limb of mice treated with praliciguat than with vehicle. Praliciguat attenuated accumulation of oxidative pro-angiogenic and pro-inflammatory muscle fibers. In cultured myoblasts praliciguat significantly downregulated Myh2 and Cxcl12 mRNA expression. Conditioned medium from praliciguat-treated myoblast decreased ICAM-1 mRNA expression in HUVECs. Conclusion: These results suggest that praliciguat restored perfusion and function in the ischemic muscle of db/db mice by increasing arteriolar diameter and decreasing ICAM-1 expression in endothelial cells via downregulation of Cxcl12 in myocytes. Disclosure N.Foussard: None. E.S.Buys: Employee; Cyclerion Therapeutics, Inc. K.Mohammedi: Board Member; Lilly, Novo Nordisk, Sanofi, Research Support; Cyclerion Therapeutics, Inc., Speaker's Bureau; Abbott, AstraZeneca. M.Renault: None.

Research advances on the techniques for diagnosing burn wound depth

The accurate diagnosis of burn wound depth is particularly important for evaluating the disease prognosis of burn patients. In the past, the diagnosis of burn wound depth often relied on the subjective judgment of doctors. With the continuous development of diagnostic technology, the methods for judging the depth of burn wound have also been updated. This paper mainly summarizes the research progress in the applications of indocyanine green angiography, laser Doppler imaging, laser speckle contrast imaging, and artificial intelligence in the diagnosis of burn wound depth, and compares the advantages and disadvantages of these techniques, so as to provide ideas for accurate diagnosis of burn wound depth.

Formononetin Improves the Survival of Random Skin Flaps Through PI3K/Akt-Mediated Nrf2 Antioxidant Defense System.

Random-pattern skin flap is widely used in plastic and reconstructive surgery. However, its clinical effect is limited by ischemia necrosis occurs at the distal part of flap. Previous studies have proved that the protective effect of formononetin was associated with its antioxidant, anti-inflammatory ability. However, further research is still needed on the effect of formononetin on flap viability. The purpose of our study was to investigate the effect of formononetin on flap survival and the underlying mechanisms. Two doses (25 mg/kg, 50 mg/kg)of formononetin were administered for seven consecutive days on flap model. Flap tissues were collected on postoperative day 7. Our results revealed that formononetin promoted skin flap viability in a dose-dependent manner. Using immunohistochemical staining and western blot, we found that formononetin significantly reduced oxidative stress and inflammation. Hematoxylin and eosin (H and E) staining, laser Doppler images and immunofluorescence staining showed the enhancement of angiogenesis after formononetin treatment. Mechanistically, we demonstrated that the antioxidation of formononetin was mediated by activation and nuclear translocation of nuclear factor-E2-related factor 2 (Nrf2), while down-regulating cytoplasmic Kelch-like ECH-associated protein 1 (Keap1) expression. Co-treatment with formononetin and LY294002 (15 mg/kg), a potent Phosphatidylinositol-3-kinase (PI3K) inhibitor, which aborted nuclear Nrf2 expression and phosphorylated Akt, indicating that formononetin-mediated Nrf2 activation was related to PI3K/Akt pathway. Overall, our findings revealed that formononetin increased angiogenesis, reduced oxidative stress and inflammation, thus promoting flap survival. We highlighted the antioxidant effects of formononetin since the Nrf2 system was activated. Therefore, formononetin might be a promising candidate drug that can enhance survival of skin flaps.

Catalpol Enhances Random-Pattern Skin Flap Survival by Activating SIRT1-Mediated Enhancement of Autophagy.

Random-pattern skin flap necrosis limits its application in the clinic. It is still a challenge for plastic surgeons. Catalpol is an effective ingredient extracted from Rehmannia glutinosa, which is reported to promote angiogenesis and protect against ischemic cerebral disease. The aim of our experiment is to assess whether catalpol can facilitate random flap survival and the underlying mechanisms. Male “McFarlane flap” rat models were employed to explore the protective effects of catalpol. The range of necrosis in the flap was calculated 7 days after the models were established. The flap specimens were harvested for further experiments, including angiogenesis, apoptosis, oxidative stress, and autophagy evaluation. Catalpol-treated group promoted the average survival area of the flap than that in the control group. Based on immunohistochemical staining, Western blotting, and ROS detection, we found that catalpol significantly reduces oxidative stress and apoptosis and increases angiogenesis. Hematoxylin and eosin (H&E) staining and laser Doppler images further clarified the enhancement of angiogenesis after catalpol treatment. The impact of catalpol in flap was switched by using 3-methyladenine (3MA), proving the important role of autophagy in curative effect of catalpol on skin flaps. Importantly, the ability of catalpol to regulate autophagy is mediated by the activation of sirtuin 1 (SIRT1) based on its high affinity for SIRT1. Our findings revealed that catalpol improved the viability of random skin flaps by activating SIRT1-mediated autophagy pathway.

Reproducibility of high-resolution laser speckle contrast imaging to assess cutaneous microcirculation for wound healing monitoring in mice.

Laser speckle contrast imaging (LSCI) combines an excellent spatial and temporal resolution, with excellent reproducibility in humans. Recently, high-resolution LSCI (LSCI-HR), coupled or not with oximetry, have been marketed. They are promising approaches to assess wound healing, especially in rodents. However, their reproducibility and performance against a reference technique remain unknown. Healthy skin perfusion was evaluated at day 0 and repeated at day 2, using LSCI-HR, high-resolution LSCI with oximetry by reflectance spectrometry (LSCI-OX), compared with laser Doppler imaging (LDI) as a reference. In a second experiment, cutaneous perfusion was measured daily during 8days after wounding at two different sites. The reproducibility of haemoglobin oxygenation with LSCI-OX was also assessed in the two experiments. Reproducibility was expressed as within-subject coefficients of variation (CV, in %). The inter-day reproducibility of healthy skin perfusion was better when assessed with LSCI-HR and LSCI-OX, compared to LDI (CVs between 12 and 17% and between 26 and 29%, respectively). Inter-site reproducibility of perfusion during wound healing was also better with LSCI-HR compared to LDI (CV=12% and 23%, respectively). Finally, we observed a good, positive correlation between perfusion measured with LDI and LSCI-HR on the periulcer area (average r=0.77±0.24). Recently developed high-resolution LSCI devices provide good reproducibility to assess healthy and wounded skin perfusion in mice. However, the reproducibility of haemoglobin oxygenation is poor.

Gene therapy with Pellino-1 improves perfusion and decreases tissue loss in Flk-1 heterozygous mice but fails in MAPKAP Kinase-2 knockout murine hind limb ischemia model.

In the United States, over 8.5 million people suffer from peripheral arterial disease (PAD). Previously we reported that Pellino-1(Peli1) gene therapy reduces ischemic damage in the myocardium and skin flaps in Flk-1 [Fetal Liver kinase receptor-1 (Flk-1)/ Vascular endothelial growth factor receptor-2/VEGFR2] heterozygous (Flk-1+/--) mice. The present study compares the angiogenic response and perfusion efficiency following hind limb ischemia (HLI) in, Flk-1+/- and, MAPKAPKINASE2 (MK2-/-) knockout (KO) mice to their control wild type (WT). We also demonstrated the use of Peli1 gene therapy to improve loss of function following HLI. Femoral artery ligation (HLI) was performed in both Flk-1+/- and MK2-/- mice along with their corresponding WT. Another set of Flk-1+/- and MK2-/- were injected with either Adeno-LacZ (Ad.LacZ) or Adeno-Peli1 (Ad.Peli1) after HLI. Hind limb perfusion was assessed by laser doppler imaging at specific time points. A standardized scoring scale is used to quantify the extent of ischemia. Histology analysis performed includes capillary density, fibrosis, pro-angiogenic and anti-apoptotic proteins. Flk-1+/- and MK2-/- had a slower recovery of perfusion efficiency in the ischemic limbs than controls. Both Flk-1+/- and MK2-/- KO mice showed decreased capillary density and capillary myocyte ratios with increased fibrosis than their corresponding wild types. Ad.Peli1 injected ischemic Flk-1+/- limb showed improved perfusion, increased capillary density, and pro-angiogenic molecules with reduced fibrosis compared to Ad.LacZ group. No significant improvement in perfusion was observed in MK2-/- ischemic limb after Ad. Peli1 injection. Deletion of Flk-1 and MK2 impairs neovascularization and perfusion following HLI. Treatment with Ad. Peli1 results in increased angiogenesis and improved perfusion in Flk-1+/- mice but fails to rectify perfusion in MK2 KO mice. Overall, Peli1 gene therapy is a promising candidate for the treatment of PAD.

96.5 Using Computer Vision-Based Algorithms Trained on Mobile-Device Camera Images for Monitoring Burn Wound Healing

IntroductionThe appropriate characterization of burn depth and healing is paramount. Unfortunately, the accuracy of approximating thermal injury depth among all physicians is poor. While tools to improve detection accuracy, including laser doppler imaging and laser speckle imaging exist, these technologies are expensive and limited to specialized burn referral centres. They also do not provide an easy means for quantitative, interval tracking of burn healing. Considering these limitations, the application of artificial intelligence has garnered significant interest. We herein present the use of three novel machine learning and computer vision-based algorithms to track burn wound healing.MethodsConvolutional neural network (CNN) models, were trained on 1800 2D color burn images, to classify them into four burn severities. These CNNs were used to develop saliency algorithms that identify the highest “attention” pixels used to recognize burns. Image-based algorithms that count these attention pixels of the CNN, count pixels representing red granulation of burns, and measure burns, were also developed. As proof-of-concept, we tracked the healing of a localized burn on a 25-year-old female patient. The patient suffered a scald on the dorsum of the foot, resulting in a deep partial-thickness burn. Opting out of surgical intervention, the patient visited the hospital over a 6-week period for treatment with non-adhesive dressings and silver nitrate. High-resolution images of the burn, with and without a fiducial marker, were captured with a smartphone camera every 7-days. Images were taken under institutional lighting and used as algorithmic inputs.ResultsData analyses indicate that the healing of the open-wound area was accurately measured in millimetres (+/- 1.7 mm error) using a fiducial marker (18.3 mm diameter). The open-wound area shrank consistently from week 1 to week 6 seen in (Figure 1. a-b). The normalized, 2D colour images, where the “red” pixel value was counted (Figure 1. a-b), confirms the reduction of the red granulation in the wound. The saliency algorithm also measured a percentage reduction in the machine learning model’s total attention pixels over the 6-week period (Figure 1. c-d). This suggests that the model was less discerning of the healing burn wound over time, suggesting burn healing, which was also clinically validated.