Metformin use is associated with longer survival in glioblastoma patients with MGMT gene silencing.

PURPOSE: New treatments are needed to improve the overall survival of patients with glioblastoma Metformin is known for anti-tumorigenic effects in cancers, including breast and pancreas cancers. In this study, we assessed the association between metformin use and overall survival in glioblastoma patients. METHODS: We retrospectively studied 241 patients who underwent surgery at diagnosis of glioblastoma between 2014 and 2018. Metformin was used for pre-existing type 2 diabetes mellitus or in the prevention or management of glucocorticoid induced hyperglycemia. Kaplan-Meier curves and log-rank p test were used for univariate analysis. Cox-proportional hazards model was used to generate adjusted hazard ratios for multivariate analysis. RESULTS: Metformin use was associated with longer survival in patients with tumors that had a methylated O6-methylguanine DNA methyltransferase gene (MGMT) promoter (484 days 95% CI: 56-911 vs. 394 days 95% CI: 249-538, Log-Rank test: 6.5, p = 0.01). Cox regression analysis shows that metformin associates with lower risk of death at 2 years in patients with glioblastoma containing a methylated MGMT promoter (aHR = 0.497, 95% CI 0.26-0.93, p = 0.028). CONCLUSION: Our findings suggest a survival benefit with metformin use in patients with glioblastomas having methylation of the MGMT promoter.

DFF40 deficiency in cancerous T cells is implicated in chemotherapy drug sensitivity and resistance through the regulation of the apoptotic pathway.

The regulation of the apoptotic pathway is one of the most studied mechanisms regarding cancer cell resistance. Many mutations have been linked to drug resistance. The DNA fragmentation factor 40 (DFF40) has been gaining interest regarding cancer cell response to chemotherapy and patient outcomes. Glioblastomas and uterine leiomyosarcomas have been shown to have a downregulation of DFF40 expression, conferring a poor patient prognosis. In concordance with these observations, in this study, we showed that DFF40 gene is also downregulated in breast, endocervical, ovarian, lung, pancreas and glioblastomas. DFF40 is the endonuclease responsible of DNA fragmentation during apoptosis. In this study, we sought to determine if a DFF40 deficiency in Jurkat T cells could impact the sensitivity to conventional chemotherapy drugs. CRISPR-cas9 generated DFF40 knockout (DFF40 KO) stable Jurkat cells and wild-type (DFF40 WT) cells were treated with different antimetabolites and topoisomerase II (TOP2) inhibitors, and cell viability was subsequently assessed. DFF40 deficient cells show chemoresistance to antimetabolites (e.g. methotrexate, 6-mercaptopurine and cytarabine) and surprisingly, they are more sensitive to TOP2 inhibitors (e.g. etoposide and teniposide). DFF40 deficient cells exposed to cytarabine present lower phosphatidylserine translocation levels to the outer cell membrane layer. Etoposide exposure in DFF40 deficient cells induces higher mortality levels and downregulation of Bcl-xL cells compared to DFF40 expressing T cells. The abolition of DFF40 expression in Jurkat cells significantly impairs histone H2AX phosphorylation following etoposide and cytarabine treatments. Our findings suggest that DFF40 is a novel key target in cancer cell resistance that potentially regulates genomic stability.

The remodelling of actin composition as a hallmark of cancer.

Actin is a key structural protein that makes up the cytoskeleton of cells, and plays a role in functions such as division, migration, and vesicle trafficking. It comprises six different cell-type specific isoforms: ACTA1, ACTA2, ACTB, ACTC1, ACTG1, and ACTG2. Abnormal actin isoform expression has been reported in many cancers, which led us to hypothesize that it may serve as an early biomarker of cancer. We show an overview of the different actin isoforms and highlight mechanisms by which they may contribute to tumorigenicity. Furthermore, we suggest how the aberrant expression of actin subunits can confer cells with greater proliferation ability, increased migratory capability, and chemoresistance through incorporation into the normal cellular F-actin network and altered actin binding protein interaction. Studying this fundamental change that takes place within cancer cells can further our understanding of neoplastic transformation in multiple tissue types, which can ultimately aid in the early-detection, diagnosis and treatment of cancer.

Invasive growth associated with cold-inducible RNA-binding protein expression drives recurrence of surgically resected brain metastases.

BACKGROUND: Sixty percent of surgically resected brain metastases (BrM) recur within 1 year. These recurrences have long been thought to result from the dispersion of cancer cells during surgery. We tested the alternative hypothesis that invasion of cancer cells into the adjacent brain plays a significant role in local recurrence and shortened overall survival. METHODS: We determined the invasion pattern of 164 surgically resected BrM and correlated with local recurrence and overall survival. We performed single-cell RNA sequencing (scRNAseq) of >15,000 cells from BrM and adjacent brain tissue. Validation of targets was performed with a novel cohort of BrM patient-derived xenografts (PDX) and patient tissues. RESULTS: We demonstrate that invasion of metastatic cancer cells into the adjacent brain is associated with local recurrence and shortened overall survival. scRNAseq of paired tumor and adjacent brain samples confirmed the existence of invasive cancer cells in the tumor-adjacent brain. Analysis of these cells identified cold-inducible RNA-binding protein (CIRBP) overexpression in invasive cancer cells compared to cancer cells located within the metastases. Applying PDX models that recapitulate the invasion pattern observed in patients, we show that CIRBP is overexpressed in highly invasive BrM and is required for efficient invasive growth in the brain. CONCLUSIONS: These data demonstrate peritumoral invasion as a driver of treatment failure in BrM that is functionally mediated by CIRBP. These findings improve our understanding of the biology underlying postoperative treatment failure and lay the groundwork for rational clinical trial development based upon invasion pattern in surgically resected BrM.

El Perfil Epidemiológico Y Clínico De La Esclerosis Múltiple En El Ecuador / The Clinical And Epidemiological Profile Of Multiple Sclerosis In Ecuador

Resumen En el Ecuador ha habido un importante incremento en el número de publicaciones sobre Esclerosis Múltiple (EM) en los últimos años. Este interés por conocer el comportamiento clínico y epidemiológico de la enfermedad nos ha permitido establecer semejanzas y diferencias con otras cohortes de pacientes con EM que provienen de regiones en donde la prevalencia de la enfermedad es alta. El Ecuador sigue siendo un país de baja prevalencia, los estudios han demostrado que la misma fluctúa entre 3 a 5 casos por 100.000 habitantes. El comportamiento epidemiológico es muy similar a la de cohortes europeas por ejemplo el sexo femenino es el principalmente afectado. Sin embargo, el comportamiento clínico difiere en lo que respecta a deterioro cognitivo, fatiga siendo éstos menos frecuentes. Aún se desconoce el impacto de la vitamina D en nuestros pacientes debido a que, solo un estudio ha sido llevado a cabo. Al parecer, existe una alta prevalencia de deficiencia e insuficiencia de vitamina D en los pacientes ecuatorianos pero no se traduce en un incremento de prevalencia o discapacidad como ocurre en poblaciones europeas. A pesar de que tenemos una mejor comprensión de la enfermedad en el país, más estudios son necesarios y es imperativo incluir a todos los pacientes ecuatorianos con esclerosis múltiple con el fin de mejorar nuestro conocimiento sobre el comportamiento de esta patología en nuestra región.

Abstract In recent years, the number of publications on Multiple Sclerosis (MS) from Ecuador has seen a significant increase. As a result, the research on the clinical and epidemiological behaviour of the disease has allowed us to make comparisons with other cohorts of patients with MS that come from regions where the prevalence of the disease is high. Nevertheless, Ecuador is still a country in which the prevalence of MS is low with a prevalence that fluctuates between 3 to 5 cases per 100,000 inhabitants. The epidemiological behaviour of MS is very similar to that of european cohorts, for example female patients are the most affected. However, the clinical behaviour of multiple sclerosis differs in terms of cognitive impairment and fatigue being less frequent. The impact of vitamin D on patients with MS is still unknown as only one study has been carried out. This study show that there is a high prevalence of vitamin D deficiency and insufficiency in ecuadorian patients, but this does not translate into an increase in prevalence or disability as it does in european populations. Although we have a better understanding of the disease in the country, more studies are necessary, and it is imperative that all ecuadorian patients with MS be included in future studies in order to improve our knowledge about the behaviour of this disease in our region.

Preliminary Experience on Laser Interstitial Thermal Ablation Therapy in the Treatment of Extra-axial Masses: Indications, Imaging Characterization and Outcomes.

Laser thermal ablation is a novel minimally invasive neurosurgical technique that has proven to be beneficial in the treatment of a select group of neurosurgical conditions such as primary brain neoplasms, brain metastases, radiation necrosis, and epileptogenic lesions such as cortical dysplasia and mesial temporal sclerosis. The applicability of laser thermal ablation and its utility in the treatment of extra-axial (EA) brain neoplasms, mainly meningioma, is another novel use of this technique. Our article discusses the use and benefits of this technique in this particular clinical scenario. We describe our experience in a group of symptomatic patients from our institution with EA masses, mainly recurrent meningiomas, that failed previous more conventional treatment therapies such as surgery and radiotherapy. Our paper emphasizes patient selection, indications for the procedure, and post-treatment imaging characteristics of the ablated lesions.

Characterization of a Clival Chordoma Xenograft Model Reveals Tumor Genomic Instability.

Patient-derived xenografts retain the genotype of the parent tumors more readily than tumor cells maintained in culture. The two previously reported clival chordoma xenografts were derived from recurrent tumors after radiation. To study the genetics of clival chordoma in the absence of prior radiation exposure we established a patient-derived xenograft at primary resection of a clival chordoma. Epicranial grafting of clival chordoma collected during surgery was performed. Tumor growth was established in a nonobese diabetic/severe combined immunodeficiency mouse and tumors have been passaged serially for seven generations. Physaliferous cell architecture was shown in the regenerated tumors, which stained positive for Brachyury, cytokeratin, and S100 protein. The tumors showed bone invasion. Single-nucleotide polymorphism analysis of the tumor xenograft was compared with the parental tumor. Copy number gain of the T gene (brachyury) and heterozygous loss of cyclin dependent kinase inhibitor 2A (CDKN2A) was observed. Heterozygous loss of the tumor-suppressor fragile histidine triad (FHIT) gene also was observed, although protein expression was preserved. Accumulation of copy number losses and gains as well as increased growth rate was observed over three generations. The patient-derived xenograft reproduces the phenotype of clival chordoma. This model can be used in the future to study chordoma biology and to assess novel treatments.

The Safety of Bevacizumab Administered Shortly after Laser Interstitial Thermal Therapy in Glioblastoma: A Case Series.

BACKGROUND: Laser interstitial thermal therapy (LITT) enables ablation of lesions using thermal energy with minimal damage to surrounding regions. Bevacizumab has been used as an adjuvant therapy in recurrent glioblastoma (GBM). At present, bevacizumab is administered at least 4-6 weeks after surgical intervention; however, given the minimally invasive nature of LITT, we suggest that bevacizumab can be safely administered at a shorter interval after LITT. METHODS: Three patients who showed evidence of recurrent GBM on magnetic resonance imaging (MRI), underwent LITT, and were subsequently administered bevacizumab within 4 weeks were identified retrospectively. Postoperative MRI was performed 24 hours after treatment and before follow-up. All 3 patients were placed on dexamethasone taper postoperatively. The ablated tumors were analyzed radiographically, and bevacizumab symptoms were monitored carefully. RESULTS: The patients ranged in age from 39 to 69 years. The median duration of follow-up was 26 weeks (range, 13-51 weeks). All 3 patients expired due to disease progression. The median progression-free survival (PFS) was 17 weeks (range, 12-22 weeks), and the median overall survival (OS) was 32 weeks (range, 12-51 weeks). There were no postoperative complications or complications due to hemorrhage, infection, or thrombosis (complication rate, 0%; 95% confidence interval 0-56%). CONCLUSIONS: This case series suggests that bevacizumab administration is safe within 1 month after LITT, thus showing promise in treating recurrent GBM. Larger studies are warranted to assess the efficacy of combined bevacizumab and LITT.

Augmented reality for the surgeon: Systematic review.

INTRODUCTION: Since the introduction of wearable head-up displays, there has been much interest in the surgical community adapting this technology into routine surgical practice. METHODS: We used the keywords augmented reality OR wearable device OR head-up display AND surgery using PubMed, EBSCO, IEEE and SCOPUS databases. After exclusions, 74 published articles that evaluated the utility of wearable head-up displays in surgical settings were included in our review. RESULTS: Across all studies, the most common use of head-up displays was in cases of live streaming from surgical microscopes, navigation, monitoring of vital signs, and display of preoperative images. The most commonly used head-up display was Google Glass. Head-up displays enhanced surgeons' operating experience; common disadvantages include limited battery life, display size and discomfort. CONCLUSIONS: Due to ergonomic issues with dual-screen devices, augmented reality devices with the capacity to overlay images onto the surgical field will be key features of next-generation surgical head-up displays.

Technical feasibility and safety of image-guided parieto-occipital ventricular catheter placement with the assistance of a wearable head-up display.

BACKGROUND: Wearable technology is growing in popularity as a result of its ability to interface with normal human movement and function. METHODS: Using proprietary hardware and software, neuronavigation images were captured and transferred wirelessly via a password-encrypted network to the head-up display. The operating surgeon wore a loupe-mounted wearable head-up display during image-guided parieto-occipital ventriculoperitoneal shunt placement in two patients. RESULTS: The shunt placement was completed successfully without complications. The tip of the catheter ended well within the ventricles away from the ventricular wall. The wearable device allowed for continuous monitoring of neuronavigation images in the right upper corner of the surgeon's visual field without the need for the surgeon to turn his head to view the monitors. CONCLUSIONS: The adaptable nature of this proposed system permits the display of video data to the operating surgeon without diverting attention away from the operative task. This technology has the potential to enhance image-guided procedures.

Use of a Mobile Intraoperative Computed Tomography Scanner for Navigation Registration During Laser Interstitial Thermal Therapy of Brain Tumors.

OBJECTIVE: In recent years laser interstitial thermal therapy (LITT) has become the ablative neurosurgical procedure of choice. Multiple methods for registration and laser fiber verification have been described, with each method requiring multiple steps and significant time expenditure. We evaluated the use of a commercially available mobile computed tomography (CT) scanner for stereotactic registration during LITT for brain tumors in an attempt to simplify the procedure and improve intraoperative awareness of laser position. METHODS: This is a retrospective chart review comparing LITT of brain tumors in 23 patients undergoing a standard protocol requiring skull pins and transport of the patient to a CT suite to obtain a reference scan compared with 14 patients in whom the Medtronic O-arm was used intraoperatively for navigation registration and confirmation of laser position. RESULTS: Total ablation of the target was achieved in all patients with no surgical complications. Total surgery time was shorter for the O-arm group than for the standard protocol group, once experience was gained with bringing the O-arm in and out of the surgical field. Return from the magnetic resonance imaging suite to the operating room for repositioning of the laser was required for 1 patient in the standard protocol group, but for no patients in the O-arm group. Once experience was gained with using the O-arm, estimated surgical costs were lower for this group. CONCLUSIONS: Use of a mobile intraoperative CT scanner for navigation registration and confirmation of laser position during LITT may play a role in streamlining the procedure and improving patient safety and comfort.

Identification of alsterpaullone as a novel small molecule inhibitor to target group 3 medulloblastoma.

Advances in the molecular biology of medulloblastoma revealed four genetically and clinically distinct subgroups. Group 3 medulloblastomas are characterized by frequent amplifications of the oncogene MYC, a high incidence of metastasis, and poor prognosis despite aggressive therapy. We investigated several potential small molecule inhibitors to target Group 3 medulloblastomas based on gene expression data using an in silico drug screen. The Connectivity Map (C-MAP) analysis identified piperlongumine as the top candidate drug for non-WNT medulloblastomas and the cyclin-dependent kinase (CDK) inhibitor alsterpaullone as the compound predicted to have specific antitumor activity against Group 3 medulloblastomas. To validate our findings we used these inhibitors against established Group 3 medulloblastoma cell lines. The C-MAP predicted drugs reduced cell proliferation in vitro and increased survival in Group 3 medulloblastoma xenografts. Alsterpaullone had the highest efficacy in Group 3 medulloblastoma cells. Genomic profiling of Group 3 medulloblastoma cells treated with alsterpaullone confirmed inhibition of cell cycle-related genes, and down-regulation of MYC. Our results demonstrate the preclinical efficacy of using a targeted therapy approach for Group 3 medulloblastomas. Specifically, we provide rationale for advancing alsterpaullone as a targeted therapy in Group 3 medulloblastoma.

Foretinib is effective therapy for metastatic sonic hedgehog medulloblastoma.

Medulloblastoma is the most common malignant pediatric brain tumor, with metastases present at diagnosis conferring a poor prognosis. Mechanisms of dissemination are poorly understood and metastatic lesions are genetically divergent from the matched primary tumor. Effective and less toxic therapies that target both compartments have yet to be identified. Here, we report that the analysis of several large nonoverlapping cohorts of patients with medulloblastoma reveals MET kinase as a marker of sonic hedgehog (SHH)-driven medulloblastoma. Immunohistochemical analysis of phosphorylated, active MET kinase in an independent patient cohort confirmed its correlation with increased tumor relapse and poor survival, suggesting that patients with SHH medulloblastoma may benefit from MET-targeted therapy. In support of this hypothesis, we found that the approved MET inhibitor foretinib could suppress MET activation, decrease tumor cell proliferation, and induce apoptosis in SHH medulloblastomas in vitro and in vivo. Foretinib penetrated the blood-brain barrier and was effective in both the primary and metastatic tumor compartments. In established mouse xenograft or transgenic models of metastatic SHH medulloblastoma, foretinib administration reduced the growth of the primary tumor, decreased the incidence of metastases, and increased host survival. Taken together, our results provide a strong rationale to clinically evaluate foretinib as an effective therapy for patients with SHH-driven medulloblastoma.

Nanosurgical resection of malignant brain tumors: beyond the cutting edge.

Advances in surgical procedures and improvements in patient outcomes have resulted from applications of new technologies in the operating room over the past three decades. All surgeons would be excited about the possibilities of improving their resections of tumors for patients with cancer if a new technology were introduced to facilitate this. In this issue of ACS Nano, Karabeber et al. use a hand-held Raman scanner to probe the completeness of resection of glioblastoma multiforme (GBM), the most malignant brain cancer, in a genetically engineered mouse model. They show that the hand-held scanner could accurately detect gold-silica surface-enhanced Raman scattering nanoparticles embedded within the GBM, resulting in a complete tumor resection. In this Perspective, we review potential applications of nanotechnologies to neurosurgery and describe how new systems, such as the one described in this issue, may be brought closer to the operating room through modifications in nanoparticle size, overcoming the obstacles presented by the blood-brain barrier, and functionalizing nanoparticle conjugates so that they reach their target at highest concentrations possible. Finally, with adaptations of the actual hand-held Raman scanner device itself, one can envision the day when "nanosurgical" procedures will be a part of the surgeon's armamentarium.

Enhanced cell volume regulation: a key mechanism in local and remote ischemic preconditioning.

We have previously shown that ischemic preconditioning (IPC) protection against necrosis in whole hearts and in both fresh and cultured cardiomyocytes, as well as the improved regulatory volume decrease to hypoosmotic swelling in cardiomyocytes, is abrogated through Cl(-) channel blockade, pointing to a role for enhanced cell volume regulation in IPC. To further define this cardioprotective mechanism, cultured rabbit ventricular cardiomyocytes were preconditioned either by 10-min simulated ischemia (SI) followed by 10-min simulated reperfusion (SR), by 10-min exposure/10-min washout of remote IPC (rIPC) plasma dialysate (from rabbits subjected to repetitive limb ischemia), or by adenoviral transfection with the constitutively active PKC-ε gene. These interventions were done before cardiomyocytes were subjected to either 60- or 75-min SI/60-min SR to assess cell necrosis (by trypan blue staining), 30-min SI to assess ischemic cell swelling, or 30-min hypoosmotic (200 mosM) stress to assess cell volume regulation. Necrosis after SI/SR and both SI- and hypoosmotic stress-induced swelling was reduced in preconditioned cardiomyocytes compared with control cardiomyocytes (neither preconditioned nor transfected). These effects on necrosis and cell swelling were blocked by either Cl(-) channel blockade or dominant negative knockdown of inwardly rectifying K(+) channels with adenoviruses, suggesting that Cl(-) and K(+) movements across the sarcolemma are critical for cell volume regulation and, thereby, cell survival under hypoxic/ischemic conditions. Our results define enhanced cell volume regulation as a key common mechanism of cardioprotection by preconditioning in cardiomyocytes.

Interaction of δ and κ opioid receptors with adenosine A1 receptors mediates cardioprotection by remote ischemic preconditioning.

Multiple initiatives are underway to harness the clinical benefits of remote ischemic preconditioning (rIPC) based on applying non-invasive, brief, intermittent limb ischemia/reperfusion using an external occluder. However, little is known about how rIPC induces protection in cardiomyocytes, particularly through G-protein coupled receptors. In these studies, we determined the role of opioid and adenosine receptors and their functional interactions in rIPC cardioprotection. In freshly isolated cardiomyocytes subjected to 45-min simulated ischemia followed by 60-min simulated reperfusion, we examined the ability of plasma dialysate (derived from blood obtained from rabbits remotely preconditioned by application of brief cycles of hind limb ischemia/reperfusion, rIPC dialysate) to protect cells against necrosis. rIPC dialysate and selective activation of either δ-opioid receptors or κ-opioid receptors significantly reduced the % of dead cells after simulated ischemia and simulated reperfusion. Inhibition of adenosine A1 receptors, but not adenosine A3 receptors, blocked the protection by rIPC dialysate, δ-opioid receptor and κ-opioid receptor activation. In HEK293 cells expressing either hemagglutinin A-tagged δ-opioid receptors or hemagglutinin A-tagged κ-opioid receptors, selective immunoprecipitation of adenosine A1 receptors pulled down both δ-opioid and κ-opioid receptors. This molecular association of adenosine A1 receptors with δ-opioid and κ-opioid receptors was confirmed by reverse pull-down assays. These findings strongly suggest that rIPC cardioprotection requires the activation of δ-opioid and κ-opioid receptors and relies on these receptors functionally interacting with adenosine A1 receptors.

Focused ultrasound disruption of the blood-brain barrier: a new frontier for therapeutic delivery in molecular neurooncology.

Recent advances in molecular neurooncology provide unique opportunities for targeted molecular-based therapies. However, the blood-brain barrier (BBB) remains a major limitation to the delivery of tumor-specific therapies directed against aberrant signaling pathways in brain tumors. Given the dismal prognosis of patients with malignant brain tumors, novel strategies that overcome the intrinsic limitations of the BBB are therefore highly desirable. Focused ultrasound BBB disruption is emerging as a novel strategy for enhanced delivery of therapeutic agents into the brain via focal, reversible, and safe BBB disruption. This review examines the potential role and implications of focused ultrasound in molecular neurooncology.

Challenges and solutions for total hip arthroplasty in treatment of patients with symptomatic sequelae of developmental dysplasia of the hip.

Conditions that adversely affect a child's hip alignment, joint congruity, or articular surfaces often result in joint destruction associated with pain and motion limitation later in life. The usual culprits are developmental dysplasia of the hip (DDH), slipped capital femoral epiphysis, Legg-Calvé-Perthes disease, juvenile rheumatoid arthritis, infection, trauma, and neoplasm. In this review, we address DDH, the most common cause of secondary osteoarthritis of the hip. Symptomatic sequelae of DDH present challenges for total hip arthroplasty, including excessive proximal femoral anteversion, narrowing of the medullary canal, acetabular anteversion, verticality, hypoplasia and incongruity, pseudoacetabulum, and neurovascular shortening. Presiding corrective femoral and/or acetabular osteotomies, as well as retained hardware, further complicate total hip arthroplasty. This review emphasizes evaluation of hip morphology while considering reconstructive techniques and implants.

Direct evidence of chloride ion efflux in ischaemic and pharmacological preconditioning of cultured cardiomyocytes.

AIMS: We have previously shown that reduction of ischaemic cell swelling via enhanced cell volume regulation is a key mechanism of ischaemic preconditioning (IPC) in cardiomyocytes. We have also shown that pharmacological blockade of Cl(-) channels abolishes cardioprotection achieved by IPC in Langendorff-perfused hearts and freshly isolated cardiomyocytes, thus suggesting that Cl(-) plays a key role in IPC cardioprotection. However, direct evidence of Cl(-) channel activation resulting in transsarcolemmal Cl(-) efflux by IPC had been lacking. To address this issue, 24 h cultured rabbit cardiomyocytes were loaded with 5 mM 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ), a specific fluorescence indicator that is quenched by Cl(-) so that cellular efflux of Cl(-) results in an increase in SPQ fluorescence. METHODS AND RESULTS: After stabilization for 10 min, cardiomyocytes were preconditioned either with 10 min simulated ischaemia/10 min simulated reperfusion or with 10 min treatment with 1 microM N(6)-2-(4-aminophenyl)ethyladenosine (APNEA). IPC and APNEA significantly (P < 0.001) reduced the intracellular Cl(-) concentration ([Cl(-)](i)) to 31.9 +/- 3.2 mM (mean +/- SEM) and 32.5 +/- 2.8 mM, respectively, from an initial [Cl(-)](i) (pooled stabilization 61.5 +/- 7.1 mM). [Cl(-)](i) did not change in control (non-preconditioned) cardiomyocytes (control 58.1 +/- 1.9 mM and control + vehicle 62.6 +/- 4.9 mM, P = 0.98 and 0.99 vs. pooled pre-treatment baseline, respectively). Inhibition of Cl(-) channels with 50 microM indanyloxyacetic acid 94 completely blocked preconditioning-induced Cl(-) efflux. Thus, a net Cl(-) efflux of 29.6 and 29.0 mM was triggered by IPC and APNEA. CONCLUSION: These findings provide the first direct evidence of activation of sarcolemmal Cl(-) channels by ischaemic and pharmacological preconditioning in cardiomyocytes.

Transient limb ischaemia remotely preconditions through a humoral mechanism acting directly on the myocardium: evidence suggesting cross-species protection.

rIPC (remote ischaemic preconditioning) is a phenomenon whereby short periods of ischaemia and reperfusion of a tissue or organ (e.g. mesentery, kidney) can protect a distant tissue or organ (e.g. heart) against subsequent, potentially lethal, ischaemia. We, and others, have shown that transient limb ischaemia can provide potent myocardial protection experimentally and clinically during cardiac surgery. Nonetheless, our understanding of the signal transduction from remote stimulus to local effect remains incomplete. The aim of the present study was to define the humoral nature of rIPC effector(s) from limb ischaemia and to study their local effects in isolated heart and cardiomyocyte models. Using a Langendorff preparation, we show that infarct size after coronary artery ligation and reperfusion was substantially reduced by rIPC in vivo, this stimulus up-regulating the MAPKs (mitogen-activating protein kinases) p42/p44, and inducing PKCepsilon (protein kinase Cepsilon) subcellular redistribution. Pre-treatment with the plasma and dialysate of plasma (obtained using 15 kDa cut-off dialysis membrane) from donor rabbits subjected to rIPC similarly protected against infarction. The effectiveness of the rIPC dialysate was abrogated by passage through a C18 hydrophobic column, but eluate from this column provided the same level of protection. The dialysate of rIPC plasma from rabbits and humans was also tested in an isolated fresh cardiomyocyte model of simulated ischaemia and reperfusion. Necrosis in cardiomyocytes treated with rIPC dialysate was substantially reduced compared with control, and was similar to cells pre-treated by 'classical' preconditioning. This effect, by rabbit rIPC dialysate, was blocked by pre-treatment with the opiate receptor blocker naloxone. In conclusion, in vivo transient limb ischaemia releases a low-molecular-mass (<15 kDa) hydrophobic circulating factor(s) which induce(s) a potent protection against myocardial ischaemia/reperfusion injury in Langendorff-perfused hearts and isolated cardiomyocytes in the same species. This cardioprotection is transferable across species, independent of local neurogenic activity, and requires opioid receptor activation.