Microvascular Clip Research Articles

Prototype of Low-Cost Microvascular Clips for Laboratory Use

Vascular neurosurgical procedures require temporary or permanent surgical clips to treat cerebral aneurysms, arteriovenous malformations, or bypass surgery. In this scenario, surgical clips should have specific characteristics such as high-quality material, proper design, closing force, and biocompatibility. Due to these characteristics, the price of these clips does not allow their availability at the experimental surgery laboratory worldwide. We describe here the technique for manufacturing handcrafted clips of low cost, using dental stainless steel or titanium wire of 0.18mm, 0.20mm, or 0.22mm in diameter. We must complete six steps to obtain the clip using our hands and small electrician needle nose pliers for wire molding. These clips have a closing force of 30-60 gr/cm2 (depending on the wire diameter). They can be used in the experimental surgery laboratory to clip arteries or veins during vascular microsurgery procedures. Also, they can be used as temporary clips with confidence in low-flow bypass (v.gr. superficial temporal artery to middle cerebral artery or occipital artery to posterior inferior cerebellar artery anastomoses). Making practical low-cost clips for use in laboratory procedures or during low-flow anastomosis as temporary clips is possible. The main advantages are the low cost and the worldwide availability of the basic materials. The main disadvantage is the learning curve to get the ability to master the manufacturing of these clips.

Prophylactic Treatment of Intestinal Ischemia-Reperfusion Injury Reduces Mucosal Damage and Improves Intestinal Absorption.

Intestinal ischemia-reperfusion injury (i-IRI) involves a blood flow interruption in an intestinal segment followed by blood flow restoration. When blood flow is restored, oxidative and inflammatory molecules are distributed throughout the bloodstream, triggering both local and systemic damage. Our goal was to evaluate the potential of three antioxidant and/or anti-inflammatory compounds (curcumin, dexmedetomidine and α-tocopherol) to prevent or reverse local and systemic damage induced by i-IRI. i-IRI was induced by placing a microvascular clip in the superior mesenteric artery of female WAG/RijHsd rats; the clip was removed after 1h and reperfusion was allowed for 4h. Curcumin (200 mg/kg, orally), α-tocopherol (20 mg/kg, i.p.), and dexmedetomidine (5 or 20 µg/kg, s.c.; DEX5 and DEX20, respectively) were administered. Blood and terminal ileum specimens were collected for biochemical and histological determination. Furthermore, D-xylose absorption test was performed to evaluate intestinal absorption; after completing the 1-hour ischemia and 4-hour reperfusion period, 1 mL of aqueous D-xylose solution (0.615 mg/mL) was administered orally, and one hour later, plasma D-xylose levels were quantified. The histological injury degree (HID) measured by the Chiu scale was significantly reduced when the treatments were applied (non-treated rats, 2.6 ± 0.75; curcumin, 1.54 ± 0.8; DEX5, 1.47 ± 0.7; DEX20 1.14 ± 0.5; and α-tocopherol, 1.01 ± 0.6); intestinal absorptive capacity also improved in all cases healthy rats (2.06 ± 0.07 µg/mL; non-treated, 1.18 ± 0.07 µg/mL; curcumin 1.76 ± 0.3 µg/mL; DEX5, 2.29 ± 0.2 µg/mL; DEX20, 2.25 ± 0.26 µg/mL; and α-tocopherol 1.66 ± 0.21 µg/mL). However, it failed to reduce liver enzyme levels. Finally, only dexmedetomidine significantly reduced urea and creatinine levels compared to non-treated animals. All drugs were effective in reducing HID, although α-tocopherol was effective to a greater extent. Only dexmedetomidine reverted intestinal absorption to normal values of healthy animals.

Optogenetic Spreading Depolarizations Do Not Worsen Acute Ischemic Stroke Outcome.

Spreading depolarizations (SDs) are believed to contribute to injury progression and worsen outcomes in focal cerebral ischemia because exogenously induced SDs have been associated with enlarged infarct volumes. However, previous studies used highly invasive methods to trigger SDs that can directly cause tissue injury (eg, topical KCl) and confound the interpretation. Here, we tested whether SDs indeed enlarge infarcts when induced via a novel, noninjurious method using optogenetics. Using transgenic mice expressing channelrhodopsin-2 in neurons (Thy1-ChR2-YFP), we induced 8 optogenetic SDs to trigger SDs noninvasively at a remote cortical location in a noninjurious manner during 1-hour distal microvascular clip or proximal an endovascular filament occlusion of the middle cerebral artery. Laser speckle imaging was used to monitor cerebral blood flow. Infarct volumes were then quantified at 24 or 48 hours. Infarct volumes in the optogenetic SD arm did not differ from the control arm in either distal or proximal middle cerebral artery occlusion, despite a 6-fold and 4-fold higher number of SDs, respectively. Identical optogenetic illumination in wild-type mice did not affect the infarct volume. Full-field laser speckle imaging showed that optogenetic stimulation did not affect the perfusion in the peri-infarct cortex. Altogether, these data show that SDs induced noninvasively using optogenetics do not worsen tissue outcomes. Our findings compel a careful reexamination of the notion that SDs are causally linked to infarct expansion.

Evaluating the role of subacromial impingement in rotator cuff tendinopathy: development and analysis of a novel rat model.

Subacromial impingement of the rotator cuff caused by variations in acromial anatomy or altered glenohumeral kinematics leads to inflammation and degeneration of the rotator cuff, ultimately contributing to the development of tendinopathy. However, the underlying cellular and molecular changes in the impinged tendon remain poorly understood. Because the rat is an accepted model for rotator cuff studies, we have developed a rat model to study rotator cuff tendinopathy. Forty-four adult male Sprague-Dawley rats were allocated to one of 4 study groups: intact control group (group 1, n=11); bilateral subacromial surgical clip placement to induce supraspinatus impingement for 2 weeks (group 2, n=11), 4 weeks (group 3, n=11), and 8 weeks (group 4, n=11). Bilateral shoulder specimens were harvested for biomechanical testing, histology, and quantitative real-time polymerase chain reaction (qRT-PCR) analysis. Radiography confirmed that all microvascular clips remained in stable position in the subacromial space. Gross inspection of supraspinatus tendon specimens in the impingement groups revealed changes in tendon morphology at the enthesis and midsubstance. Biomechanical evaluation demonstrated decreased supraspinatus tendon failure force and tissue stiffness at all time points compared with control tendons. Semiquantitative scoring of histologic specimens demonstrated significant, persistent tendinopathic changes over 8 weeks. qRT-PCR analysis of impinged tendon specimens demonstrated upregulation of gene expression for Col3 and Mmp14 in the impingement groups compared with control groups. In muscle samples, significant upregulation was seen in the expression of genes that are commonly associated with muscle atrophy (MuRF1 and Ube2b) and fatty infiltration (Fabp4, Pparg2, and Klf15). This newrat subacromial impingement model creates cellular and molecular changes consistent with the development of rotator cuff tendinopathy. The results of this study may serve as a baseline for future investigation.

Poster 163: Evaluating the Role of Subacromial Impingement in Rotator Cuff Tendinopathy: Development and Analysis of a Novel Rat Model

Objectives:Subacromial impingement of the rotator cuff caused by variations in acromial anatomy is an extrinsic factor that leads to inflammation and degeneration of the rotator cuff, ultimately contributing to the development of tendinopathy. However, microscopic and molecular changes in the impinged tendon remain poorly understood, and a robust rat model for studying rotator cuff tendinopathy is still lacking.Methods:Our study was approved by the Institutional Animal Care and Use Committee. A total of 44 adult male Sprague-Dawley rats were included in the study. Animals were randomly allocated to one of four study groups: intact control group (Group 1, n=11); bilateral subacromial surgical clip placement to induce supraspinatus impingement for 2 weeks (Group 2, n=11), 4 weeks (Group 3, n=11) and 8 weeks (Group 4, n=11). At time of sacrifice, bilateral shoulder specimens were harvested for biomechanical testing, histological and quantitative real-time polymerase chain reaction (qRT-PCR) analysis. One-way analysis of variance (ANOVA) with the post-hoc Tukey test was used to analyze differences among groups.Results:Radiography confirmed that all microvascular clips remained in stable position in the subacromial space. Gross inspection of supraspinatus tendon specimens in the impingement groups revealed frayed and hazy tendon discoloration at the enthesis and mid-substance. Biomechanical evaluation demonstrated decreased supraspinatus tendon failure force and tissue stiffness at all time points compared to control group. Histologic specimens demonstrated significant, persistent tendinopathic changes over 8 weeks, with the mean modified Bonar scores significantly increased in the clip impingement groups compared to the control group (control: 1.56±0.86; 2 weeks group: 9.17±0.76; 4 weeks group: 9.11±0.65; and 8 weeks group: 6.56±0.50. P <0.001 for all comparisons). qRT-PCR analysis of impinged tendon specimens demonstrated upregulation of gene expression for Col3 and MMP14 in the impingement groups compared with control groups (P =0.001 and 0.015 respectively). In muscle samples, significant upregulation was seen in the expression of genes that are commonly associated with muscle atrophy (MURF1: P <0.001 and UBE2B: P <0.001) and fatty infiltration (FABP4: P =0.002, PPARγ2: P =0.006, and KLF15: P =0.001).Conclusions:In conclusion, we developed a novel rat model of subacromial impingement and found consistent and sustained histologic, biomechanical, and gene expression changes that are consistent with findings in human tendinopathy. This model will allow future studies to further evaluate the underlying cellular and molecular mechanisms of tendinopathy, as well as evaluation of novel therapeutic approaches for treatment of this common problem.Figure 1.Gross insepction nof the specismen at all time points.Figure 2.RadiographyFigure 3.

Ischemia In Vivo Induces Cardiolipin Oxidation in Rat Kidney Mitochondria.

Simple SummaryMitochondrial cardiolipin is a unique phospholipid that plays a vital role in ATP synthesis. It has been observed that ischemia/reperfusion causes damage to cardiolipins in the heart or brain tissues; however, very little data has been found regarding kidney cardiolipins and related damage after ischemia/reperfusion. Oxidative stress plays a key role during reperfusion. However, even during ischemia, cardiolipins may be oxidized. Therefore, our aim was to evaluate cardiolipin oxidation during renal ischemia in vivo. Renal ischemia in vivo was induced in male Wistar rats for a 30–60-min period; we then isolated kidney mitochondria, extracted the lipids and analyzed cardiolipin by applying chromatographic and mass spectrometric methods. The results showed that after even 30 min of in vivo ischemia, the amounts of the dominant species of cardiolipin decreased almost in half, and it further decreased when extending the ischemia time. Cardiolipin was oxidized with up to eight additional oxygen atoms, yielding eight different species with multiple isomeric forms. This shows that even after ischemia, cardiolipin levels are altered, and many cardiolipin oxidation products are produced, which may also potentially be modified into more harmful lipid signaling molecules that may induce more damage to mitochondria.Cardiolipin is a mitochondrial phospholipid that plays a significant role in mitochondrial bioenergetics. Cardiolipin is oxidized under conditions like oxidative stress that occurs during ischemia/reperfusion; however, it is known that even during ischemia, many reactive oxygen species are generated. Our aim was to analyze the effect of in vivo ischemia on cardiolipin oxidation. Adult male Wistar rats were anesthetized; then, their abdomens were opened, and microvascular clips were placed on renal arteries for 30, 40 or 60 min, causing ischemia. After ischemia, kidneys were harvested, mitochondria were isolated, and lipids were extracted for chromatographic and mass spectrometric analysis of tetralinoleoyl cardiolipin and its oxidation products. Chromatographic and mass spectrometric analysis revealed a 47%, 68% and 74% decrease in tetralinoleoyl cardiolipin after 30 min, 40 min and 60 min of renal ischemia, respectively (p < 0.05). Eight different cardiolipin oxidation products with up to eight additional oxygens were identified in rat kidney mitochondria. A total of 40 min of ischemia caused an average of a 6.9-fold increase in all oxidized cardiolipin forms. We present evidence that renal ischemia in vivo alone induces tetralinoleoyl cardiolipin oxidation and depletion in rat kidney mitochondria.

Human adrenomedullin and its binding protein attenuate tissue injury and inflammation following hepatic ischemia reperfusion in rabbits

BackgroundLiver injury caused by ischemia reperfusion (I/R) during surgical procedures, such as liver resection or liver transplantation, is a major cause of liver damage and graft failure. The current method of treatment is mostly preventative (i.e., ischemic preconditioning). While a number of pharmacological modalities have been studied to reduce hepatic I/R injury, none have been entirely successful. It has been demonstrated that the administration of adrenomedullin (AM) in combination with AM-binding protein (AM/AMBP-1) exerts significant protective effects in various pathological conditions. In an effort to develop AM/AMBP-1 as a novel therapeutic for hepatic I/R injury, the present study examined the effect of a low dose of human AM, which does not induce hypotension, in combination with human AMBP-1 in a rabbit model of hepatic I/R (i.e., non-rodent species). MethodsIschemia of 70% of the liver was induced by placing a microvascular clip across the hilum of the left and median lobes for 60 min. The clip was then removed to commence reperfusion. At 15 min following clip removal (i.e., reperfusion), human AM/AMBP-1 was administered intravenously via the ear marginal vein continuously for 30 min. At 20 h, blood and tissue samples were collected for various measurements. ResultsThe serum levels of liver enzymes (alanine aminotransferase and aspartate aminotransferase) and lactate dehydrogenase, were elevated following hepatic I/R. The administration of AM/AMBP-1 significantly decreased these levels by 58, 44, 41%, respectively. Hepatic I/R increased the direct and total bilirubin levels, whereas treatment with human AM/AMBP-1 decreased these levels by 60% and 69%, respectively. Treatment with AM/AMBP-1 also inhibited interleukin-6 gene expression by 95%. There were no changes in tumor necrosis factor-α (TNF-α) gene expression and myeloperoxidase activity (MPO), lactate and Suzuki scores after treatment. The treatment, however, reduced apoptosis post-hepatic I/R in the ischemic portion of the liver. ConclusionAdditional experiments with AM and AMBP-1 alone are needed to completely interpret the experimental results in this non-rodent species of hepatic I/R injury. The present study suggests that human AM/AMBP-1 may be developed as a novel therapeutic to attenuate hepatic I/R associated inflammation and liver injury.

Novel predictive biomarkers for acute injury superimposed on chronic kidney disease

Introduction and objectivesChronic kidney disease (CKD) is a risk factor for the development of acute kidney injury (AKI). Recent studies have revealed numerous biomarkers eligible for AKI prediction. However, the expression and performance of AKI biomarkers in acute injury superimposed on preexisting CKD (AonC) remain elusive. The aim of this study was to evaluate whether biomarkers which robustly expressed in acute kidney injury could predict acute injury based on CKD. Materials and methodsMice were classified into cohorts: AKI, CKD, AonC and sham. The AonC model mice were subjected to renal bilateral ischemia/reperfusion (I/R) injury fourteen days after intraperitoneally administrated with 20mg/kg aristolochic acid. Severity of acute ischemic injury was stratified by clamping the dissected bilateral renal arteries with non-traumatic microvascular clips for 20 or 35min. The AKI mice were induced with renal bilateral I/R injury and CKD mice were crafted with 20mg/kg aristolochic acid administrated intraperitoneally. Histology, genetic and protein expression of biomarkers were measured in three cohorts. ResultsWe found that serum creatinine dramatically increased in severe (sAonC) but not in moderate (mAonC) injury mice. Upregulation of Kidney injury molecule-1 (KIM-1) mRNA, tissue inhibitor of metalloproteinase-2 (TIMP-2), Syndecan-1 (SDC-1) mRNA and insulin-like growth factor binding protein-7 (IGFBP7) protein indicated the onset of mAonC. An increase in neutrophil gelatinase-associated lipocalin (NGAL), rhomboid-like protein 2 (RHBDL2), Syndecan-1 (SDC-1) mRNA and protein, and a decrease in IGFBP7 protein were associated with sAonC. ConclusionsOur study revealed the variational expression of AKI biomarkers in AonC kidneys, and uncovered IGFBP7 protein can be used as a sensitive biomarker to predict and differentiate AonC severity. The performance of RHBDL2 and SDC-1 in predicting severe AonC was promising, providing new biomarkers for predicting AonC.

Novel predictive biomarkers for acute injury superimposed on chronic kidney disease

Introduction and objectivesChronic kidney disease (CKD) is a risk factor for the development of acute kidney injury (AKI). Recent studies have revealed numerous biomarkers eligible for AKI prediction. However, the expression and performance of AKI biomarkers in acute injury superimposed on preexisting CKD (AonC) remain elusive. The aim of this study was to evaluate whether biomarkers which robustly expressed in acute kidney injury could predict acute injury based on CKD. Materials and methodsMice were classified into cohorts: AKI, CKD, AonC and sham. The AonC model mice were subjected to renal bilateral ischemia/reperfusion (I/R) injury fourteen days after intraperitoneally administrated with 20mg/kg aristolochic acid. Severity of acute ischemic injury was stratified by clamping the dissected bilateral renal arteries with non-traumatic microvascular clips for 20 or 35min. The AKI mice were induced with renal bilateral I/R injury and CKD mice were crafted with 20mg/kg aristolochic acid administrated intraperitoneally. Histology, genetic and protein expression of biomarkers were measured in three cohorts. ResultsWe found that serum creatinine dramatically increased in severe (sAonC) but not in moderate (mAonC) injury mice. Upregulation of Kidney injury molecule-1 (KIM-1) mRNA, tissue inhibitor of metalloproteinase-2 (TIMP-2), Syndecan-1 (SDC-1) mRNA and insulin-like growth factor binding protein-7 (IGFBP7) protein indicated the onset of mAonC. An increase in neutrophil gelatinase-associated lipocalin (NGAL), rhomboid-like protein 2 (RHBDL2), Syndecan-1 (SDC-1) mRNA and protein, and a decrease in IGFBP7 protein were associated with sAonC. ConclusionsOur study revealed the variational expression of AKI biomarkers in AonC kidneys, and uncovered IGFBP7 protein can be used as a sensitive biomarker to predict and differentiate AonC severity. The performance of RHBDL2 and SDC-1 in predicting severe AonC was promising, providing new biomarkers for predicting AonC.

Application of microvascular clip in treatment of vascular injuries——a report in research progress

Objective Microvascular clip plays important role in microsurgery. Therefore, the development of microvascular clips and research in clamp-related vascular injuries becomes a major topic. Based on clamp-related microvascular injuries, the study investigated in 4 aspects: injury assessment, vascular characteristics and blood vessel injury, vessel repair, and development of microvascular clamp. A prospects of the current researches is also given. Key words: Microvascular clip; Blood vessel injury; Clamp-related injuries; Stanching; Microsurgical instruments

Comparison of invasive dynamic blood pressure between superior mesenteric artery and common carotid artery in rats.

The purpose of this study was to identify the consistency of invasive dynamic blood pressure (BP) monitoring between the superior mesenteric artery (SMA) and the common carotid artery (CCA). Eight male Sprague-Dawley rats were cannulated in SMA and CCA simultaneously for BP monitoring, respectively. The abdominal aorta was prepared for the induction of BP change through clamping/de-clamping by a microvascular clip. The dynamic BP monitoring was performed by a polygraph system. Systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean arterial pressure (MAP) values would be recorded during different time periods: the baseline (T1), the increasing period after clamping (T2), the platform period during clamping (T3), the decreasing period after de-clamping (T4), and the final platform period (T5). Three trials were performed on each rat with 15-minute intervals between consecutive monitoring. Systolic BP showed no significant differences between SMA and CCA. However, significant difference was found in diastolic blood pressure except at T5 (P=0.534). Mean arterial pressure of two arteries were significantly different only at T1 (P=0.015). The strength of association was significantly high between BP measurements through SMA and CCA (P<0.001). The Bland-Altman analyses showed that mean bias of MAP changed no more than 5 mmHg and standard deviation less than 8 mmHg during T2 and T4, respectively. The study indicates SMA might be an alternative site for invasive BP monitoring during abdominal aorta occlusion and release, especially in cerebrovascular-related research.

Evaluation of select biocompatible markers for labelling peripheral nerves on 11.7 T MRI

BackgroundPeripheral nerve injury is often followed by a highly variable recovery process with respect to both rapidity and efficacy. Identifying post-nerve injury phenomena is key to assessing the merit and timing of surgery as well as to tracking nerve recovery postoperatively. Diffusion Tensor Imaging (DTI) has been investigated in the clinical and research settings as a noninvasive technique to both assess and monitor each patient's unique case of peripheral nerve damage. New methodWe identify a MRI-suitable marker for tracking the exact site of either nerve injury or coaptation following surgical repair to aid with DTI analysis. ResultsDue to artefact and disruption of tractography, silver wire and microvascular clips were not suitable markers. AxoGuard®, 4-0 vicryl suture, and 10-0 polyamide suture, although detectable, did not produce a signal easily distinguished from post-surgical changes. Silicone was easily identifiable and stable in both the acute and delayed time points, exhibited negligible impact on DTI parameters, and possessed geometry to prevent nerve strangulation. Comparison with existing methodPrior studies have not assessed the efficacy of other markers nor have they assessed silicone for potential artefact with DTI parameter analysis. Furthermore, this work demonstrates the reliability and compatibility of silicone in the delayed postoperative time period and includes its unique imaging appearance on high-resolution 11.7 MRI. ConclusionSemi-cylindrical silicone tubing can be used as a safe, reliable, and readily available radiological marker to visualize and monitor a region of interest on a rodent’s peripheral nerve for aiding assessments with diffusion tensor imaging.

Analgesic effects of the novel α₂δ ligand mirogabalin in a rat model of spinal cord injury.

Mirogabalin, which is a novel ligand for the α₂δ subunit of voltage-gated calcium channels, is under development for the treatment of pain associated with diabetic peripheral neuropathy and postherpetic neuralgia. Mirogabalin possesses unique binding characteristics to α₂δ subunits and potent and long-lasting analgesic effects in peripheral neuropathic pain models. In the present study, we investigated the analgesic effects of mirogabalin in a rat model of spinal cord injury as an experimental animal model for central neuropathic pain. The spinal cord injury model was established by acute compression of the spinal cord at the T6/7 level with a microvascular clip in male rats. Twenty-eight days after spinal cord injury, the animals received the test compound orally, and the paw withdrawal threshold to mechanical stimulation was determined using the von Frey test at 0 (before administration), 2, 4, 6, 8, and 24 h after administration. The area under the curve of the paw withdrawal threshold (paw withdrawal threshold AUC) was also calculated. In rats subjected to spinal cord injury, mechanical allodynia was demonstrated by a decreased paw withdrawal threshold. A single oral administration of mirogabalin (2.5, 5, or 10 mg/kg) significantly increased the paw withdrawal threshold. The effects of mirogabalin were still significant 6 or 8 h after administration. The paw withdrawal threshold AUC was significantly higher in the treated animals than in the control group. In conclusion, mirogabalin showed potent and long-lasting analgesic effects in a rat model of spinal cord injury and may therefore provide effective pain relief for patients with central neuropathic pain.

Natural killer T cell ligand alpha-galactosylceramide protects against gut ischemia reperfusion-induced organ injury in mice

Background & AimsGut ischemia reperfusion (I/R) injury is a life-threatening condition. The immune response plays an important role in I/R-induced organ injury. Alpha-galactosylceramide (α-GalCer) is a potent natural killer T (NKT) cell stimulator. Activation of NKT cells by α-GalCer has been shown to reduce I/R-induced injury in the liver and heart. However, whether α-GalCer has any protective effects on gut I/R-induced organ injury remained unknown. The aim of this study was to test the hypothesis that α-GalCer attenuates gut I/R-induced local and remote organ injury through modulating immune responses. MethodsGut ischemia was induced by placing a microvascular clip across the superior mesenteric artery for 30 min in male adult mice. After removing the clip, α-GalCer (2 µg/mouse) or normal saline containing 0.5% Tween 20 (Vehicle) was administered intraperitoneally. Blood, gut, lung and mesenteric lymph node (MLN) samples were collected 4 h after reperfusion to detect bacterial translocation, tight junction protein, tissue damage, edema, apoptosis, IL-4, IL-10, IFN-γ and TNF-α levels. Resultsα-GalCer significantly reduced bacterial translocation to the MLN, restored tight junction protein and attenuated gut and lung injury after gut I/R. α-GalCer markedly stimulated the production of IL-4, IL-10 and IFN-γ, but had no obvious effects on TNF-α production in gut I/R mice. Pretreatment with anti-CD1d, IL-4 or IL-10, but not IFN-γ blocking antibodies abolished the protective effects of α-GalCer in gut I/R. Conclusionsα-GalCer treatment improved gut barrier function and attenuated gut and lung injury after gut I/R. The beneficial effects of α-GalCer in gut I/R were NKT cell dependent and mediated through upregulation of IL-4 and IL-10. Thus, activation of NKT cells by α-GalCer may serve as a novel option in the treatment of gut I/R injury.

Evaluating the role of subacromial impingement in rotator cuff tendinopathy: Development and analysis of a novel murine model.

Subacromial impingement of the rotator cuff is understood as a contributing factor in the development of rotator cuff tendinopathy. However, changes that occur in the impinged tendon are poorly understood and warrant further study. To enable further study of rotator cuff tendinopathy, we performed a controlled laboratory study to determine feasibility and baseline characteristics of a new murine model for subacromial impingement. This model involves surgically inserting a microvascular clip into the subacromial space in adult C57Bl/6 mice. Along with a sham surgery arm, 90 study animals were distributed among time point groups for sacrifice up to 6 weeks. All animals underwent bilateral surgery (total N = 180). Biomechanical, histologic, and molecular analyses were performed to identify and quantify the progression of changes in the supraspinatus tendon. Decreases in failure force and stiffness were found in impinged tendon specimens compared to sham and no-surgery controls at all study time points. Semi-quantitative scoring of histologic specimens demonstrated significant, persistent tendinopathic changes over 6 weeks. Quantitative real-time polymerase chain reaction analysis of impinged tendon specimens demonstrated persistently increased expression of genes related to matrix remodeling, inflammation, and tendon development. Overall, this novel murine subacromial impingement model creates changes consistent with acute tendonitis, which may mimic the early stages of rotator cuff tendinopathy. A robust, simple, and reproducible animal model of rotator cuff tendinopathy is a valuable research tool to allow further studies of cellular and molecular mechanisms and evaluation of therapeutic interventions in rotator cuff tendinopathy. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2780-2788, 2018.

Dexmedetomidine preconditioning protects against lung injury induced by ischemia-reperfusion through inhibition of autophagy.

The present study aimed to evaluate the role of autophagy in the protective effect of dexmedetomidine in lung injury caused by ischemia-reperfusion (IR) in rats. In total 48 adult male Sprague-Dawley rats were randomly divided into 6 groups (n=8) as follows: i) Sham group; ii) the IR group; iii) IR + 1 µg/kg dexmedetomidine preconditioning group (pre-LD); iv) IR + 10 µg/kg dexmedetomidine preconditioning group (pre-HD); v) IR + 1 µg/kg dexmedetomidine postconditioning group (post-LD); and vi) IR + 10 µg/kg dexmedetomidine postconditioning group (post-HD). After the rats were anesthetized, the hilum of the left lung was occluded with a non-invasive microvascular clip for 30 min to induce ischemia. The clip was then removed and the left lung was allowed to regain ventilation and blood for 2 h. The rats were then sacrificed, the left lung removed and the wet/dry (W/D) lung weight ratio was determined. Pathological changes to the lungs were evaluated by light and transmission electron microscopy. Furthermore, the rate of lung cell apoptosis was determined by the TUNEL assay. The expression of hypoxia-inducible factor 1α (HIF-1α), Bcl-2/adenovirus E1B 19-kDa interacting protein 3 (BNIP3), BNIP3 like (BNIP3 L) and microtubule-associated protein 1A/1B light chain 3B (LC3II) was determined by western blotting. Compared with the sham group, a significant increase in the W/D lung weight ratio, and malondialdehyde (MDA), BNIP3, BNIP3 L and LC3II levels were observed in the IR group, and HIF-1α levels and superoxide dismutase (SOD) activity were decreased. Furthermore, the W/D ratio was lower in the pre-LD and pre-HD groups than in the IR group. Additionally, SOD activity was significantly higher and MDA expression was significantly lower in the pre-LD and pre-HD groups compared with the IR group. BNIP3, BNIP3 L and LC3II protein levels were significantly lower in the pre-LD and pre-HD groups compared with the IR group, while HIF-1α was notably upregulated in the pre-LD and pre-HD groups compared with the IR group. In conclusion, the results of the present study indicate that dexmedetomidine preconditioning protects against lung injury induced by IR through inhibition of autophagy and apoptosis.

Impact of groin flap ischemia-reperfusion on red blood cell micro-rheological parameters in a follow-up study on rats.

Flap hypoperfusion or ischemia-reperfusion (I/R) may occur during preparation-transposition procedures and by postoperative thrombotic complications. Behind the microcirculatory disturbances micro-rheological alterations are also supposed. We aimed to investigate the groin flap I/R with following-up micro-rheological parameters. Anesthetized rats were subjected to Control or I/R groups. Groin flaps were prepared bilaterally, pedicled on the superficial epigastric vessels. In the Control group the flaps were re-sutured after one hour, while in I/R group microvascular clips were applied on the pedicles for 60 minutes, then the flaps were repositioned. Besides daily wound control, before the operation and on the 1st, 3rd, 5th, 7th and 14th postoperative days blood samples were collected for testing red blood cell (RBC) deformability (rotational ektacytometry) and aggregation (light-transmission aggregometry). RBC deformability significantly worsened by the 3rd-7th postoperative day in I/R group. RBC aggregation enhanced significantly by the 1st day, in I/R group it remained elevated on the 3rd day as well. In a complicated case with unilateral flap necrosis, RBC deformability and aggregation worsening was outlined from its group (base, 1st, 3rd day). Wound healing affected micro-rheological parameters in the early postoperative period. Flap I/R exacerbated the alterations. The parameters markedly worsened in case of flap necrosis.

Nonhuman primate models of focal cerebral ischemia.

Rodents have been widely used in the production of cerebral ischemia models. However, successful therapies have been proven on experimental rodent stroke model, and they have often failed to be effective when tested clinically. Therefore, nonhuman primates were recommended as the ideal alternatives, owing to their similarities with the human cerebrovascular system, brain metabolism, grey to white matter ratio and even their rich behavioral repertoire. The present review is a thorough summary of ten methods that establish nonhuman primate models of focal cerebral ischemia; electrocoagulation, endothelin-1-induced occlusion, microvascular clip occlusion, autologous blood clot embolization, balloon inflation, microcatheter embolization, coil embolization, surgical suture embolization, suture, and photochemical induction methods. This review addresses the advantages and disadvantages of each method, as well as precautions for each model, compared nonhuman primates with rodents, different species of nonhuman primates and different modeling methods. Finally it discusses various factors that need to be considered when modelling and the method of evaluation after modelling. These are critical for understanding their respective strengths and weaknesses and underlie the selection of the optimum model.

Functional Relevance of Von Willebrand Factor in Mouse Model of Hepatic Ischemia- Reperfusion Injury

Hepatic ischemia-reperfusion (I/R) injury is a liver damage occurring during liver surgeries such as hepatic resection or transplantation, and denotes the major basis for graft dysfunction after transplantation. Although cellular and molecular mechanisms of hepatic I/R injury are complex and remain to be clarified, an excessive inflammatory response is assumed to play a role in this regard. An adhesive protein von Willebrand factor (VWF) plays a pivotal role in platelet thrombus formation and is recently understood as a key protein in a cross-talk between inflammation and thrombosis. In this context, we assumed that VWF may also be involved in the pathophysiology of hepatic I/R injury, in which VWF-dependent platelet activation or inflammatory responses could play a role at liver sinusoidal milieu. To test this hypothesis, we have used a mouse experimental model of hepatic I/R injury. Mice were anesthetized with sodium pentobarbital and a midline laparotomy was then performed on a heating pad. Blood supply for left lateral and median lobes of liver (approximately 70% of the liver mass) was interrupted by cross-clamping the hepatic artery and portal vein with a microvascular atraumatic clip for 90 min. Then a clip was taken off to provoke the reperfusion of hepatic blood flow, which was monitored on the surface of left lateral lobe by Laser Doppler flowmetry (ALF21, Advance Co, Tokyo, Japan). The hepatic blood flow was measured again 24 h after reperfusion and mice were then sacrificed for blood collection and histological analysis of liver tissue. We compared 16 wild-type (WT) and 12 VWF-gene deleted (knock-out; KO) mice (from The Jackson Laboratory, Bar Harbor, ME), all of which were 8-12 weeks of age, healthy and fertile. Excess blood loss was not observed in all mice (WT or KO) during whole surgical process. As compared to WT mice, restoration of hepatic blood flow was significantly greater in VWF-KO mice at 24 h after reperfusion (WT; 61± 17% vs. KO; 87 ± 17%, expressed as the percentage of pre-ischemic value). Consistent with the hepatic blood flow, the time-course analysis of serum alanine aminotransferase (ALT) at several time points after reperfusion revealed the lesser liver damages of KO mice (WT; 6898 ± 3270 and 1313 ± 621 IU/L vs. KO; 3043 ± 1320 and 478 ± 330 IU/L, at 3 h and 24 h after reperfusion, respectively). In addition, histological analysis confirmed that neutrophil infiltration in the liver tissue of KO mice was significantly reduced as compared to WT mice at 24 h after reperfusion. These impaired hepatic blood flow and ALT values as well as intensified neutrophil infiltration in WT mice were significantly improved to an extent comparable to those of KO mice by the bolus injection of recombinant human ADAMTS13 (3 μg/mouse equivalent to 2800 U/kg, n=12) just prior to the I/R operation. Our results altogether indicate that VWF-dependent inflammatory responses with neutrophil recruitment at ischemic sites are involved in pathophysiology of hepatic I/R injury, and functional regulation of VWF by ADAMTS 13 may serve as a promising therapeutic option for hepatic I/R injury. DisclosuresNo relevant conflicts of interest to declare.

A modified method to reduce variable outcomes in a rat model of four-vessel arterial occlusion

Objectives: Rat models of global cerebral ischemia (GCI) are essential to study potential treatment strategies against stroke in humans. However, nonvisual electrocoagulation of vertebral arteries often leads to incomplete occlusion that may prevent the creation of a successful model.Methods: Therefore, we used a modified method in which the vertebral arteries were safely visualized and electrocauterized between the first and second transverse processes through a dorsal incision. Twenty-four hours later, GCI was induced by transient bilateral common carotid arterial occlusion using microvascular clips. The clips were removed after 10, 20, or 30 min of four-vessel occlusion.Results: Digital subtraction angiography demonstrated complete occlusion of the rat vertebral arteries. In the 20-min GCI group, the rats were monitored for 1, 3, 5, or 7 days followed by histological analysis. The results showed that neuronal death in the CA1 subregion occurred almost linearly between 1 and 3 days of recovery. Tape removal test was used for up to 7 days after 20 min of GCI or sham operation. The latency time was found to be significantly increased 1 to 7 days after GCI compared with sham.Conclusions: In conclusion, this modified model provides a reliable tool for ischemic stroke research.