Hindlimb Ischemia-reperfusion Research Articles

Targeting CD36 With EP 80317 Reduces Remote Inflammatory Response to Hind Limb Ischemia-Reperfusion in Mice.

Reperfusion of ischemic skeletal muscle triggers oxidative stress and an immediate inflammatory reaction, leading to damage of distant organs such as the lungs. The inflammatory process implicates numerous mediators, including cytokines, chemokines, and arachidonic acid metabolites. In the orchestration of the inflammatory cascade, a critical role is played by the cluster of differentiation-36 receptor (CD36), a scavenger receptor class B protein (SR-B2) which is expressed on macrophages and functions as a Toll-like receptor coreceptor. A mouse model of hind limb ischemia-reperfusion has been used to investigate the interplay between CD36 signaling and remote inflammation: leukocyte recruitment, regulation of the nucleotide-binding domain leucin-rich repeat and pyrin-containing receptor 3 (NLRP3) inflammasome, and release of nuclear factor-kappa B (NF-ĸB) and arachidonic acid metabolites. Levels of reactive oxygen species, inflammatory mediators, and gene expression were measured in blood and lung tissue samples collected from anesthetized mice on which unilateral hind limb ischemia was induced by rubber band constriction for 30 min followed by reperfusion for 3 h. The CD36 modulator EP 80317, a member of the growth hormone releasing peptide 6 family, was employed as a pharmacological agent to mitigate distant lung injury following skeletal limb ischemia-reperfusion. Targeting CD36 on monocytes/macrophages, EP 80317 abated pro-inflammatory signaling and transcriptional activity encompassing lipid and cytokine mediators. Targeting CD36 was shown to offer promise for curtailing tissue injury following hind limb ischemia-reperfusion.

Detecting viscosity changes in the limb ischemia-reperfusion in mice with a near-infrared fluorescence probe

BackgroundLimb ischemia-reperfusion is a common phenomenon in clinical surgery, which disrupts the balanced physiological response process and ultimately leads to changes in intracellular viscosity. Intracellular viscosity is an important microenvironmental parameter that affects the normal function of organisms, and its level is closely related to many diseases. In addition, oxidative stress in the lower limbs can impair body function, and changes in pressure can lead to changes in the viscosity of limb tissues. Therefore, it is necessary to develop effective tools to detect changes in intracellular viscosity and visualize the progression of hind limb ischemia-reperfusion injury. ResultsIn order to solve this problem, a near infrared viscometry sensitive fluorescence probe (PH-XQ) with long emission wavelength and stable luminescence performance was designed and synthesized by using oxanthracene derivatives and malononitrile. The fluorescence probe (PH-XQ) has excellent selectivity, high sensitivity, low toxicity, high biocompatibility and excellent detection performance. The fluorescence intensity of the PH-XQ probe at 667 nm is highly sensitive to the change of viscosity. With the increase of viscosity, the fluorescence intensity of probe PH-XQ was significantly enhanced, and the fluorescence enhancement ratio was about 14-fold. In addition, PH-XQ can detect not only changes in viscosity between normal cells and drug-induced inflammatory cells, but also changes in the viscosity of the hind limbs of normal mice and mice after ischemia reperfusion. SignificanceIn particular, we are the first to successfully detect changes in handlimb viscosity after ischemia-reperfusion in mice using a probe. This study clearly elucidates changes in viscosity during ischemia-reperfusion of mouse limbs, providing favorable support for the relationship between viscosity and related diseases, and further providing a potential tool for the diagnosis of viscosity-related diseases.

Beneficial Effect of Heat Treatment on the Exercise Pressor Reflex in Hindlimb Ischemia-Reperfusion: Potential Role of P2X3

PURPOSE: In peripheral artery disease (PAD), a greater blood pressure (BP) response during exercise causes cardiovascular and cerebrovascular risks. The exercise pressor reflex (EPR) is a key factor for why BP is exaggerated in PAD patients. Thus, we examined the effect of heat treatment (HT) with two temperature increments in skeletal muscle: 1.5 and 3oC on the EPR response following limb ischemia-reperfusion (IR) injury, one of the most common pathological conditions seen in PAD. In addition, the P2X3 (an ATP-sensitive receptor) in the muscle afferent dorsal root ganglion (DRG) was also evaluated. We hypothesized that IR upregulates the BP response to static muscle contraction and the protein expression of P2X3 in muscle afferent nerves. HT attenuates those exaggerated responses. METHODS: An experimental IR model was induced by 6 hours of ischemia followed by 18 hours of reperfusion in rats (IR rats). For HT groups, three HT sessions (muscle temperature increased by 1.5 or 3oC) for 30 mins/each time were applied. EPR response was evoked by static muscle contraction (30s). Protein expression of the P2X3 receptor in DRGs was examined. Data are presented as mean ± standard deviation. Results: The mean arterial pressure (MAP) response to the static muscle contraction was significantly exaggerated in rats of IR 18h (vs. Sham, P=0.018). With increasing Tm by 1.5°C, there was a promising trend that thus exaggerated BP response was attenuated (IR 18h + HT 1.5°C, n=5 vs. IR 18h rats, n=5, P=0.08). Meanwhile, the exaggerated BP response was significantly attenuated with the Tm increasing by 3°C (IR 18h + HT 3°C, n=5, vs. IR 18h rats, n=5, P=0.032). The expression of the P2X3 receptor was significantly enhanced in the DRGs of IR 18h rats (n=4, p<0.01 vs. sham, n=6). The upregulated P2X3 was suppressed in the DRGs of IR 18h rats +HT of 1.5°C (n=3, p<0.01 vs. IR 18h, n=4) and there was a promising trend that upregulated P2X3 was suppressed in the DRGs of IR 18h rats +HT of 3°C (n=3, P=0.079 vs. IR 18h, n=4). CONCLUSION: IR injury leads to upregulation of EPR response and this effect was attenuated by HT. The P2X3 signaling pathways are likely involved in the beneficial regulatory effect of HT on EPR in IR. Further data collection will be performed to test this conclusion. Supported by NIH R01 HL141198 & HL164571; AHA GRANT # 940567/Lu Qin/2022; and College of Medicine DOM Innovation and Inspiration Award. The authors sincerely thank Chunying Yang for the excellent technical assistance. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Inflammatory Responses of the Remote Organs After Tourniquet-Induced Ischemia-Reperfusion in Mouse Hindlimb

Tourniquet use is the most effective tool for controlling life-threatening extremity hemorrhage before other treatments and for creating bloodless operating fields in surgical procedures. However, tourniquet-induced ischemia-reperfusion (IR) also causes skeletal muscle injury and associated secondary end-organ damage. Our previous studies have demonstrated that there are involvements of inflammatory cytokines in tourniquet-induced skeletal muscle IR injuries. The objective of this study is to investigate the inflammatory responses of end-organs after tourniquet-induced mouse hindlimb IR. We hypothesized that tourniquet use would result in an inflammatory response in the remote organs. The unilateral hindlimbs of mice were subjected to 3 hours of tourniquet application by placing a rubber band at the hip joint. Then the rubber bands were released for reperfusion over different periods of time (1, 3, 7, and 14 days). The mRNA levels of IL-1β and TNFα (two inflammatory cytokines) in the lungs, liver, kidneys, and heart were measured using real-time RT-PCR analysis. In the lungs, the levels of IL-1β and TNFα mRNA were significantly elevated at day 1 after tourniquet-induced IR, then decreased at days 3 and 7, and returned to a high level at day 14. In the liver, the levels of IL-1β and TNFα mRNA were increased at day 1 and remained at a high level until day 14. However, tourniquet-induced hindlimb IR did not change the levels of IL-1β or TNFα mRNA in the kidneys or heart during the whole period of tourniquet-induced IR. In conclusion, these results suggest that the inflammatory response to tourniquet-induced hindlimb IR is different among distant organs, which will help to develop therapeutic strategies for different end-organs after tourniquet-induced hindlimb IR. NIH/NIGMS 1R01GM145736 to Y-LL. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Protective Effects of Hydrogen Gas Inhalation for Hindlimb Ischaemia–Reperfusion Injury in a Mouse Model

Ischaemia-reperfusion (I/R) injury is a severe post-operative complication that triggers an inflammatory response and causes severe damage. Hydrogen gas has anti-oxidant and anti-apoptotic properties and has been shown to be safe in humans. The study aimed to investigate whether hydrogen gas protects against skeletal muscle I/R injury. Experimental basic research using mice. A total of 160 eight to 10 week old albino laboratory bred strain of house mice (25.8 ± 0.68 g) were used in this study. The mice were cable tied to the hindlimb under anaesthesia and then placed in an anaesthesia box filled with air and 2% isoflurane (control group); 80 mice were additionally subjected to 1.3% hydrogen gas in this mix (hydrogen group). After two hours, the cable ties were removed to initiate reperfusion, and hydrogen inhalation lasted for six hours in the hydrogen group. After six hours, the mice were taken out of the box and kept in cages under standard conditions until time for observation at 16 different time points after reperfusion: zero, two, four, six, eight, and 10 hours and one, two, three, four, five, six, seven, 14, 21, and 28 days. Five mice were sacrificed using excess anaesthesia at each time point, and the bilateral hindlimb tissues were harvested. The inflammatory effects of the I/R injury were assessed by evaluating serum interleukin-6 concentrations using enzyme linked immunosorbent assay, as well as histological and immunohistochemical analyses. Untreated mice with I/R injury were used as controls. Hydrogen gas showed protective effects associated with a reduction in inflammatory cell infiltration (neutrophils, macrophages, and lymphocytes), a reduced area of damaged muscle, maintenance of normal muscle cells, and replacement of damaged muscle cells with neoplastic myocytes. Inhalation of hydrogen gas had a protective effect against hindlimb I/R injury in mice, in part by reducing inflammatory cell infiltration and in part by preserving normal muscle cells.

Amplified P2X3 pathway activity in muscle afferent dorsal root ganglion neurons and exercise pressor reflex regulation in hindlimb ischaemia-reperfusion.

Hindlimb ischaemia-reperfusion (IR) is among the most prominent pathophysiological conditions observed in peripheral artery disease (PAD). An exaggerated arterial blood pressure (BP) response during exercise is associated with an elevated risk of cardiovascular events in individuals with PAD. However, the precise mechanisms leading to this exaggerated BP response are poorly elucidated. The P2X3 signalling pathway, which plays a key role in modifying the exercise pressor reflex (EPR), is the focus of the present study. We determined the regulatory role of P2X3 on the EPR in a rat model of hindlimb IR. In vivo and in vitro approaches were used to determine the expression and functions of P2X3 in muscle afferent nerves and EPR in IR rats. We found that in IR rats there was (1) upregulation of P2X3 protein expression in the L4-6 dorsal root ganglia (DRG); (2) amplified P2X currents in isolated isolectin B4 (IB4)-positive muscle DRG neurons; and (3) amplification of the P2X-mediated BP response. We further verified that both A-317491 and siRNA knockdown of P2X3 significantly decreased the activity of P2X currents in isolated muscle DRG neurons. Moreover, inhibition of muscle afferents' P2X3 receptor using A-317491 was observed to alleviate the exaggerated BP response induced by static muscle contraction and P2X-induced BP response by α,β-methylene ATP injection. P2X3 signalling pathway activity is amplified in muscle afferent DRG neurons in regulating the EPR following hindlimb IR.

Histological Study of the Possible Protective Role of Scutellarin on the Cardiac Muscle Injury Induced by Hind Limb Ischemia–Reperfusion in Adult Male Albino Rat

Background Ischemia–reperfusion injury is a severe disorder that can lead to dysfunctions in various organs. Scutellarin is a Chinese medical herb with many pharmacological activities, including antioxidant and a scavenger of reactive oxidative radicals that has a promising role in myocardial protection. Aim The aim is to investigate the potential protective effect of scutellarin on cardiac muscle injuries caused by hind limb I/R in adult male albino rats. Materials and methods Forty adult male albino rats were used in this study. Group I (control) were separated into two subgroups equally. Subgroup Ia did not undergo any therapy, whereas subgroup Ib was given 0.5 ml intraperitoneal injections of saline. Group II was administered scutellarin dissolved in 0.5 ml of saline, through intraperitoneal injections at a dosage of 20 mg/kg/day. Group III was subjected to hind limb ischemia by applying a rubber band tourniquet over the greater trochanter of the thigh for 4 h. Subsequently, the limb was reperfused for 24 h. Group IV received intraperitoneal injections of scutellarin dissolved in 0.5 ml saline at a dosage of 20 mg/kg/day, 15 min before induction of hind limb ischemia as outlined in group III. Cardiac muscle sections had been stained with hematoxylin and eosin. Results Group III showed discontinuation, separation, vacuolation of cardiac muscle fibers, area of hemorrhage, and cellular infiltration. Most of these histological alterations were less pronounced in group IV. Conclusion Scutellarin can protect the cardiac muscle against I/R injury. Recommendations High risk people to ischemia-reperfusion are advised to take scutellarin as a protective supplement.

NIR-II fluorescence imaging without intended excitation light.

Nowadays, second near-infrared window (NIR-II) dyes are almost excited by laser diodes, but none of the white light (400-700 nm) excited NIR-II imaging has been studied because of the lack of suitable optical probes. Herein, a novel blue-shifted NIR-II dye, TPA-TQT, has been selected for use in multi-wavelength white light emitting diode (LED) excited NIR-II imaging. This white LED barely caused photo-quenching of the dyes, especially indocyanine green (ICG), whereas the ICG's brightness decreased by 90% under continuous 808 nm laser irradiation. Compared to single-wavelength LED, multi-wavelength LED showed a lower background and similar signal-to-background ratios. This system provided high image resolution to identify blood vessels (103 μm), lymphatic capillaries (129.8 μm), and to monitor hindlimb ischemia-reperfusion and lymphatic inflammation. Furthermore, white LED excited NIR-II fluorescence imaging-guided surgery (FIGS) was successfully performed in 4T1 tumor-bearing mice. Impressively, the lighting LED-based NIR-II FIGS was found to clearly delineate small lesions of metastatic tumors of about ∼350 μm diameter and further was able to guide surgical removal. Overall, multi-wavelength LED-based NIR-II imaging is a promising imaging strategy for tumor delineation and other biomedical applications.

Piezo1 mediates abnormal type 2 immune response to aggravate ischemia–reperfusion acute lung injury which by regulating alveolar macrophages derived IL-33: A hypothesis

The pathogenesis of ischemia–reperfusion acute lung injury (IR-ALI) is not yet fully understood. We previously reported that unwonted increased type 2 immunity plays a key role in the pathogenesis and the pathophysiological process in acute haemorrhagic inducedlung injury. Progressive lung edema after ischemia reperfusion is accompanied by the change of lung cyclical hydrostatic force. The changed lung mechanical force could activate the mechanosensitive Piezo1 channel in alveolar macrophages. Furthermore, our group had elucidated that microenvironmental stiffness exacerbated hindlimb ischemia reperfusion injury by activating Piezo1 in bone marrow-derived macrophages. Considering early findings that IL-33 is a potent driver of pulmonary type 2 immunity, that Piezo1 plays an important role in regulating macrophages biological activity and that alveolar macrophages are involved in orchestrating the early abnormal detrimental type 2 inflammatory response in lung ischemia–reperfusion injury, we herein hypothesize that the change of mechanical force in lung after ischemia–reperfusion might activate Piezo1/IL-33/type 2 immunity axis and aggravate lung ischemic injury. Mechanistically, Piezo1 mediates extracellular Ca2+influx and activates Ca2+-dependent Calcineurin-NFAT signaling in alveolar macrophages, leading to transcriptional activation of IL-33, thereby promoting abnormal type 2 immune response and participating in the occurrence and development of IR-ALI. Future validation of these mechanisms could provide a deeper explanation of the new pathogenesis of IR-ALI and ultimately serve for clinical treatment.

Abstract 12634: Amplified P2x 3 Pathway Activity in IB4 Positive Muscle DRG Neurons and Exercise Pressor Reflex Regulation in Hindlimb Ischemia-Reperfusion

Background: Hindlimb ischemia-reperfusion (IR) represents one of the most prominent pathological conditions observed in peripheral artery disease (PAD). Arterial blood pressure response (BP) during exercise, partly driven by the exaggerated exercise pressor reflex (EPR), is associated with an elevated risk of cardiovascular events in PAD patients. However, the precise underlying mechanisms leading to this exaggerated BP response are poorly elucidated. The purinergic P2X 3 signaling pathway, which plays a key role in modifying the EPR, was the focus of the present study. Objectives: To examine the regulatory role of P2X 3 on the EPR in a rat model of hindlimb IR, which was induced by 6 hours’ ischemia followed by 18 hours’ reperfusion (referred to as IR rats). Results: We found that, in IR rats, there were: 1) upregulation of P2X 3 protein expression in the L4-6 dorsal root ganglion (DRG) (1.48±0.16 in IR rats/n=8 vs. 1.00±0.41 in sham rats/n=9; P<0.05), which was localized in IB4 positive DRG neurons by the method of immunohistochemistry, 2) amplified P2X currents in isolated muscle DRG neurons (P2X transient currents: 1281.64±776.74 pA in IR rats/n=25, vs. 717.91±556.2 pA in sham rats/n=9, P<0.05; and sustain currents: 466.94±168.68 pA in IR rats/n=11, vs. 271.49±200.90 pA in sham rats/n=8, P<0.05); 3) amplification of P2X-mediated BP response (29±8 mmHg in IR rats/n=12 vs. 21±4 mmHg in sham rats/n=7; P<0.05); and 4) alleviation of the exaggerated BP response induced by static muscle contraction (18±8 mmHg in IR +A317491/n=8, vs. 29±11 mmHg in IR/n=7, P<0.05) and P2X-mediated BP response by α, β-me-ATP injection (15±4 mmHg in IR+A317491 /n=6 vs. 23±7 mmHg in IR/n=4, P<0.05) following the inhibition of muscle afferents’ P2X 3 receptor by A-317491. We further verified that both A-317491 and siRNA knockdown of P2X 3 significantly decreased the activity of P2X currents in the isolated muscle DRG neurons (all P<0.05 vs. their respective controls). Conclusion: With the above evidence from multiple approaches, we concluded that the P2X 3 signaling pathway activity is amplified following hindlimb IR via the enhancing expression and activity of P2X 3 receptors in IB4 positive muscle DRG afferents. This contributes to the exacerbated EPR responses in PAD.

Abstract 17363: Loss of Skeletal Muscle Bmal1 Impairs Limb Perfusion and Muscle Regeneration in a Mouse Model of Peripheral Arterial Disease

Introduction: Peripheral arterial disease (PAD) is a growing pathologic condition worldwide and leads to severe myopathy. Traditional risk factors for PAD, including diabetes, smoking, and advanced age, are associated with disrupted circadian clock function. However, the contribution of circadian disruption to PAD remains poorly understood. Hypothesis: This study aims to investigate the role of skeletal muscle clock in the severity of PAD. Methods: We performed femoral artery ligation (FAL) surgery in male adult-life inducible skeletal muscle-specific Bmal1 knockout ( Bmal1 musc ) (n = 6) and control mice (n = 5). We measured limb perfusion with laser doppler imaging weekly and assessed post-surgical angiogenesis with CD31/αSMA co-immunostaining and muscle recovery with hematoxylin and eosin (H&E) staining and Laminin immunohistochemistry at 30 days post-surgery. We also performed RNA-sequencing, qPCR, and Western blotting assays to characterize the differentially expressed genes in both ligated and non-ligated hindlimb muscles. Results: Adult-life deletion of skeletal muscle Bmal1 reduced reperfusion recovery, with an average rate of only 50% by 22 days post-surgery in Bmal1 musc mice compared to approximately 80% in control mice. HIF1α target genes including the pro-angiogenic factor Vegfα were significantly downregulated in Bmal1 musc mice. Conversely, genes enriched in the anti-angiogenic TGF-β signaling pathways were up-regulated in Bmal1 musc mice. Myostatin, a myokine that inhibits muscle growth, was downregulated in Bmal1 musc muscles. H&E staining and Laminin immunohistochemistry revealed significantly less but enlarged newly formed myofibers in the tibialis anterior muscle in Bmal1 musc mice. Conclusions: Skeletal muscle BMAL1 plays a critical role in determining the severity of ischemic muscle injury in a mouse model of PAD. Circadian dysfunction impairs hindlimb ischemia reperfusion, decreases the number of newly formed myofibers, and promotes the formation of enlarged regenerating myofibers, which is however previously reported to be associated with compromised muscle force production. Restore muscle circadian function could be a promising strategy to ameliorate myopathic severity in PAD patients.

Remote ischemic preconditioning and hypoxia-induced biomarkers in acute myocardial infarction: study on a porcine model

Objectives. Remote ischemic preconditioning (RIPC) mitigates acute myocardial infarction (AMI). We hypothesized that RIPC reduces the size and severity of AMI and explored molecular mechanisms behind this phenomenon. Design. In two series of experiments, piglets underwent 60 min of the circumflex coronary artery occlusion, resulting in AMI. Piglets were randomly assigned into the RIPC groups (n = 7 + 7) and the control groups (n = 7 + 7). The RIPC groups underwent four 5-min hind limb ischemia-reperfusion cycles before AMI. In series I, the protective efficacy of RIPC was investigated by using biomarkers and echocardiography with a follow-up of 24 h. In series II, the heart of each piglet was harvested for TTC-staining to measure infarct size. Muscle biopsies were collected from the hind limb to explore molecular mechanisms of RIPC using qPCR and Western blot analysis. Results. The levels of CK-MBm (p = 0.032) and TnI (p = 0.007) were lower in the RIPC group. Left ventricular ejection fraction in the RIPC group was greater at the end of the follow-up. The myocardial infarct size in the RIPC group was smaller (p = 0.033). Western blot indicated HIF1α stabilization in the skeletal muscle of the RIPC group. PCR analyses showed upregulation of the HIF target mRNAs for glucose transporter (GLUT1), glucose transporter 4 (GLUT4), phosphofructokinase 1 (PFK1), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), enolase 1 (ENO1), lactate dehydrogenase (LDHA) and endothelial nitric oxidate synthase (eNOS). Conclusions. Biochemical, physiologic, and histologic evidence confirms that RIPC decreases the size of AMI. The HIF pathway is likely involved in the mechanism of the RIPC.

Mexican tea (Dysphania ambrosioides (L.) Mosyakin & Clemants) seeds attenuate tourniquet-induced hind limb ischemia–reperfusion injury by modulating ROS and NLRP3 inflammasome pathways

The main pathophysiological mechanisms of hind limb ischemia–reperfusion injury (HLIRI) are increased oxidative stress and inflammation. The present study was designed to characterize the phytocontents of Dysphania ambrosioides (DA) seeds extract via LC-MS/MS and investigate its effect on left hindlimb IR injury and the underlying mechanisms. Thirty adult rats (n = 6 per group) were divided into five groups: Control group; HLIRI group, HLIRI + DA (100 mg/kg) group, HLIRI + DA (200 mg/kg) group, and HLIRI + cilostazol (30 mg/kg) group. HE staining’s of the left gastrocnemius muscle and left kidney was done. Renal function, and both total and muscle creatine kinases were measured in serum. Oxidative stress markers (MDA, SOD, Nrf2 and HO-1), inflammatory markers (NLRP3, IL-1β and TNF-α) and caspase-3 as an apoptotic marker were measured in left gastrocnemius muscle. We found that DA extract contains 60 metabolites and its pretreatment prevented left kidney and left gastrocnemius muscle damage and decreased oxidative stress markers, inflammatory markers, and caspase-3 levels, compared to the control group. Moreover, the effect of the extract was dose-dependent and better than that of the reference drug, cilostazol. This study suggested that D. ambrosioides seeds have a protective effect on HLIRI, which may be related to the antioxidant, anti-inflammatory and antiapoptotic mechanisms.

ProBDNF Upregulation in Murine Hind Limb Ischemia Reperfusion Injury: A Driver of Inflammation.

Brain-derived neurotropic factor (BDNF) has been shown to be expressed in many nonneuronal tissues including skeletal muscle. Skeletal muscle BDNF has been studied regarding its function in metabolism and exercise; however, less is known about its role in skeletal muscle injury. The precursor to BDNF, proBDNF, has an unknown role in skeletal muscle. The levels of proBDNF, mature BDNF, and their receptors were compared in the skeletal muscle and brain tissues of C57BL/6J mice. Tourniquet-induced hind limb ischemia-reperfusion injury was used to assess the function of skeletal muscle-derived proBDNF in skeletal muscle injury. Skeletal muscle-specific knockout of BDNF and pharmacological inhibition of p75NTR, the proBDNF receptor, were used to determine the role of proBDNF-p75NTR signaling. We show for the first time that proBDNF is the predominantly expressed form of BDNF in skeletal muscle and that proBDNF is significantly upregulated in skeletal muscle following hind limb ischemia-reperfusion injury. Skeletal muscle-specific knockout of BDNF blunted the inflammatory response in the injured tissue and appears to be mediated by the proBDNF-p75NTR pathway, as shown by the pharmacological inhibition of p75NTR. These findings suggest that skeletal muscle proBDNF plays a critical role in driving the inflammatory response following skeletal muscle injury.

Endothelin-1 Enhances Activities of Cav1.2 in Arterial Vascular Smooth Muscle Cells of Peripheral Artery Disease with Hindlimb Ischemia-Reperfusion

Background and Objective: The ischemia-reperfusion (IR) injury is a main feature of pathophysiological process in peripheral artery disease (PAD). In PAD, an exaggerated blood pressure response increases cardiovascular and cerebrovascular risks for the disease. Considering that L-type voltage-gated calcium channel (Cav1.2) in arterial vascular smooth muscle cells (VSMCs) plays a crucial role in regulating blood pressure, the purpose of this study was to determine the activities of Cav1.2 following hindlimb IR as well as a contribution of endothelin-1 (ET-1), a potent vasoconstrictor, to the activities of Cav1.2. Hypothesis: We hypothesized that IR injury of PAD upregulates ET-1 signaling pathways in limb muscle and thereby amplifies the activities of Cav1.2 in VSMCs. Methods: An experimental PAD model with IR was induced by 6 hours of ischemia followed by 18 hours of reperfusion in rats (IR18h rats). ELISA and western blot analysis were used to examine the levels of ET-1 in the hindlimb muscle, and protein expression of Cav1.2 and ET-1 receptor ETA in VSM. Patch clamp was used to determine the Cav1.2 currents. Data are presented as mean ± standard deviation. Results: The hindlimb IR increased the levels of ET-1 in the hindlimb muscle, and expression of Cav1.2 and ET-1’s ETA in VSMCs. In particular, compared with the control group, there was a promising trend for the increasing Cav1.2 in VSM of the IR18h group (1.00±0.61 in control/n=4 vs. 8.77±8.04 in IR18h/n=5, P=0.09). Meanwhile, ETA expression was significantly higher in IR18h group than control group (1.00±1.12 in control/n=4 vs. 3.71±1.59 in IR18h/n=5, P<0.05). The hindlimb IR also increased amplitude of Cav1.2. In addition, ET-1 (1 μM) enhanced Cav1.2 current density in VSMCs of control rats from 0.47± 0.14 pA/pF to 0.68 ± 0.14 pA/pF (n=3; P<0.05) and from 0.54 ± 0.17 pA/pF to 0.74 ± 0.15 pA/pF (n=5; P<0.05) in the VSMCs of IR18h rats, respectively. Pretreatment of the VSMCs with an ETA antagonist, BQ-123 (1 μM), prevented the increasing effects of ET-1 on Cav1.2 current. In contrast, an ETB antagonist, BQ-788 (1 μM), had no distinct effects on the ET-1 increased-Cav1.2 current. Conclusions: ET-1 increases the activities of Cav1.2 in VSMCs via ETA receptor and upregulation of ET-1 signaling pathways in VSMCs of PAD is likely to lead to amplified blood pressure response via increasing the activities of Cav1.2. NIH R01 HL141198 & HL164571; and American Heart Association grant # 940567 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Systemic glucose-insulin-potassium reduces skeletal muscle injury, kidney injury, and pain in a murine ischaemia-reperfusion model.

Glucose-insulin-potassium (GIK) is protective following cardiac myocyte ischaemia-reperfusion (IR) injury, however the role of GIK in protecting skeletal muscle from IR injury has not been evaluated. Given the similar mechanisms by which cardiac and skeletal muscle sustain an IR injury, we hypothesized that GIK would similarly protect skeletal muscle viability. A total of 20 C57BL/6 male mice (10 control, 10 GIK) sustained a hindlimb IR injury using a 2.5-hour rubber band tourniquet. Immediately prior to tourniquet placement, a subcutaneous osmotic pump was placed which infused control mice with saline (0.9% sodium chloride) and treated mice with GIK (40% glucose, 50 U/l insulin, 80 mEq/L KCl, pH 4.5) at a rate of 16 µl/hr for 26.5 hours. At 24 hours following tourniquet removal, bilateral (tourniqueted and non-tourniqueted) gastrocnemius muscles were triphenyltetrazolium chloride (TTC)-stained to quantify percentage muscle viability. Bilateral peroneal muscles were used for gene expression analysis, serum creatinine and creatine kinase activity were measured, and a validated murine ethogram was used to quantify pain before euthanasia. GIK treatment resulted in a significant protection of skeletal muscle with increased viability (GIK 22.07% (SD 15.48%)) compared to saline control (control 3.14% (SD 3.29%)) (p = 0.005). Additionally, GIK led to a statistically significant reduction in gene expression markers of cell death (CASP3, p < 0.001) and inflammation (NOS2, p < 0.001; IGF1, p = 0.007; IL-1β, p = 0.002; TNFα, p = 0.012), and a significant reduction in serum creatine kinase (p = 0.004) and creatinine (p < 0.001). GIK led to a significant reduction in IR-related pain (p = 0.030). Systemic GIK infusion during and after limb ischaemia protects murine skeletal muscle from cell death, kidneys from reperfusion metabolites, and reduces pain by reducing post-ischaemic inflammation.

Lyophilized Extracellular Vesicles from Adipose-Derived Stem Cells Increase Muscle Reperfusion but Degrade Muscle Structural Proteins in a Mouse Model of Hindlimb Ischemia-Reperfusion Injury.

Ischemia-reperfusion (I/R) injury is a complication impacting multiple organs and tissues in clinical conditions ranging from peripheral arterial disease to musculoskeletal trauma and myocardial infarction. Stem cell-derived extracellular vesicles (EVs) may represent one therapeutic resource for preventing the tissue damage associated with I/R injury. Here we tested the hypothesis that lyophilized extracellular vesicles derived from adipose stem cells could serve as an "off-the-shelf" treatment modality for I/R injury in a mouse hindlimb ischemia model. Ischemia was induced for 90 min using a rubber band tourniquet and extracellular vesicles (0, 50, or 100 µg) administered via tail vein injection immediately prior to reperfusion. Perfusion was measured prior to, during, and after ischemia using laser Doppler imaging. Serum and tissue were collected 24 h after reperfusion. Mass spectrometry (MS)-based proteomics was used to characterize the EV cargo and proteins from the ischemic and non-ischemic hindlimb. Inflammatory cytokines were measured in muscle and serum using a multiplex array. Results indicate that EVs significantly increase reperfusion and significantly increase expression of the anti-inflammatory factor annexin a1 in skeletal muscle; however, the increased reperfusion was also associated with a marked decrease in muscle structural proteins such as dystrophin, plectin, and obscurin. Circulating inflammatory cytokines TNF-alpha and IL-6 were increased with EV treatment, and serum TNF-alpha showed a significant, positive correlation with reperfusion level. These findings suggest that, while EVs may enhance reperfusion, the increased reperfusion can negatively impact muscle tissue and possibly remote organs. Alternative approaches, such as targeting mitochondrial permeability, may be more effective at mitigating I/R injury.

DNase I and Sivelestat Ameliorate Experimental Hindlimb Ischemia-Reperfusion Injury by Eliminating Neutrophil Extracellular Traps.

Neutrophil extracellular traps (NETs) play an important role in ischemia-reperfusion injury (IRI) of the hindlimb. The aim of this study was to investigate the effect of recombinant DNase I and sivelestat in eliminating NETs and their effects on IRI limbs. An air pump was used to apply a pressure of 300 mmHg to the root of the right hindlimb of the rat for 2 h and then deflated to replicate the IRI model. The formation of NETs was determined by the detection of myeloperoxidase (MPO), neutrophil elastase (NE), and histone H3 in the skeletal muscles of the hindlimbs. Animals were administered 2.5 mg/kg bw/d DNase I, 15 or 60 mg/kg bw/d sivelestat by injection into the tail vein or intramuscularly into the ischemic area for 7d. Elimination of NETs, hindlimb perfusion, muscle fibrosis, angiogenesis and motor function were assessed. DNase I reduced NETs, attenuated muscle fibrosis, promoted angiogenesis in IRI area and improved limb motor function. Local administration of DNase I improved hindlimb perfusion more than intravenous administration. Sivelestat at a dose of 15 mg/kg bw/d increased perfusion, counteracted skeletal muscle fibrosis, promoted angiogenesis and enhanced motor function. However, sivelestat at a dosage of 60 mg/kg bw/d had an adverse effect on tissue repair, especially when injected locally. Both DNase I and moderate doses of sivelestat can eliminate IRI-derived NETs. They improve hindlimb function by improving perfusion and angiogenesis, preventing muscle fibrosis. Appropriate administration mode and dosage is the key to prevent IRI by elimination of NETs. DNase I is more valid when administered topically and sivelestat is more effective when administered intravenously. These results will provide a better strategy for the treatment of IRI in clinical.

Exaggerated blood pressure response to static exercise in hindlimb ischemia-reperfusion.

Peripheral artery disease (PAD) reduces the blood flow supply in the affected limbs as one of the significant cardiovascular concerns. Revascularization surgery in the femoral artery plays a central role in treating PAD. Exercise is also a rehabilitation strategy suggested for PAD patients to improve vascular functions. However, the effects of limb ischemia-reperfusion (IR), one of the most predominant complications in revascularization surgery, on exercise-induced arterial blood pressure (BP) response are poorly understood. In the present study, we determined 1) the blood flow status in the hindlimb muscles of rats (plantar muscle, red and white portions of gastrocnemius) with different time points of the hindlimb IR; and 2) the BP response to static muscle contraction in rats at different time points after the blood flow reperfusion procedure. Results of this study indicated that, compared with the Sham group, the blood flow in the hindlimb muscles evaluated by Evans blue concentration was significantly reduced at 6h of femoral artery occlusion (FAO 6h) (vs. sham control, p < 0.05). The decreased blood flow was gradually recovered after the blood flow reperfusion for 18 (IR 18h), 66 (IR 66h), and 114 (IR 114h) hours (p < 0.05 vs. FAO 6h for all IR groups). The response of mean arterial pressure was 20 ± 4mmHg in Sham rats (n = 7); 32 ± 10mmHg in IR 18h rats (n = 10); 27 ± 7mmHg in IR 66h rats (n = 13); 26 ± 4mmHg in IR 114h rats (n = 9) (p < 0.05 vs. Sham for all groups). No significant difference was observed in the peak-developed tension during muscle contraction among all the groups (p > 0.05). In conclusion, static exercise-induced BP response is exaggerated following IR. Whereas the BP response is not statistically significant but tends to decrease with a prolonged IR time, the exaggerated BP response remains through time points from post-IR 18h-114h.

Assessment of the Effects of Quercetin on Lung Injury After Hind Limb Ischemia Reperfusion in Rats

Background: Quercetin has antioxidant and anti-inflammatory effects. Although there are previous animal experi-ments investigating Quercetin’s effect on ischemia reperfusion (IR) injury in the literature, studies involving effect of lower extremity IR on remote organ are rare.Materials and Methods: 18 male Wistar Albino rats were randomly divided into 3 groups, 6 in each group as; Cont-rol (C), Ischemia-reperfusion (IR), IR-Quercetin, (IR-Q). Their weights were between 200-250 g. 30 minutes before the procedure20 mg/kgQuercetin was administered via intraperitoneal route. In the IR groups, infrarenal abdomi-nal aorta was clamped by an atraumatic microvascular clamp. After 120 minutes of ischemia and reperfusion was achieved for another 120 minutes. When reperfusion period ended, tissue samples were taken from the lungs. Malondialdehyde (MDA) level, superoxide dismutase (SOD) and catalase (CAT) enzyme activity and histopathological parameters were compared.Results: We found the MDA level in the IR group higher than the control group (p&amp;lt;0.0001). Lower MDA level was found in the IR-Q group compared tothe IR group (p=0.012). SOD and CAT enzyme activity in the IR group was notably lower in the control group (p&amp;lt;0.0001, p&amp;lt;0.001, respectively). Higher SOD and CAT enzyme activities were found in the IR-Q group compared to the IR group (p=0.012, p&amp;lt;0.001, respectively). Neutrophil infiltra-tion/aggregation, alveolar wall thickness and total lung injury score were notably higher in IR group than in C group (p=0.001, p=0.002, p&amp;lt;0.0001, respectively). In addition, a statistically significant decrease was observed in the Quercetin treated group in neutrophil infiltration/aggregation, alveolar wall thickness and total lung injury score compared to the IR group (p=0.023, p=0.022, p=0.002, respectively).Conclusions: We determined that intraperitoneally administered Quercetin at a dose of 20 mg/kg30 minutes before ischemia in rats reduces lipid peroxidation, oxidative stress and reduces the damage caused by IR in lung histo-pathology.Study findings suggest that Quercetin has a lung protective effect when administered before IR.