Intercellular delivery of therapeutic oligonucleotides.

One promising approach to cancer therapeutics is to induce changes in gene expression that either reduce cancer cell proliferation or induce cancer cell death. Therefore, delivering oligonucleotides (siRNA/miRNA) that target specific genes or gene programs might have a potential therapeutic benefit. The aim of this study was to examine the potential of cell-based delivery of oligonucleotides to cancer cells via two naturally occurring intercellular pathways: gap junctions and vesicular/exosomal traffic. We utilized human mesenchymal stem cells (hMSCs) as delivery cells and chose to deliver in vitro two synthetic oligonucleotides, AllStars HS Cell Death siRNA and miR-16 mimic, as toxic (therapeutic) oligonucleotides targeting three cancer cell lines: prostate (PC3), pancreatic (PANC1) and cervical (HeLa). Both oligonucleotides dramatically reduced cell proliferation and/or induced cell death when transfected directly into target cells and delivery hMSCs. The delivery and target cells we chose express gap junction connexin 43 (Cx43) endogenously (PC3, PANC1, hMSC) or via stable transfection (HeLaCx43). Co-culture of hMSCs (transfected with either toxic oligonucleotide) with any of Cx43 expressing cancer cells induced target cell death (~20% surviving) or senescence (~85% proliferation reduction) over 96 hours. We eliminated gap junction-mediated delivery by using connexin deficient HeLaWT cells or knocking out endogenous Cx43 in PANC1 and PC3 cells via CRISPR/Cas9. Subsequently, all Cx43 deficient target cells co-cultured with the same toxic oligonucleotide loaded hMSCs proliferated, albeit at significantly slower rates, with cell number increasing on average ~2.2-fold (30% of control cells) over 96 hours. Our results show that both gap junction and vesicular/exosomal intercellular delivery pathways from hMSCs to target cancer cells deliver oligonucleotides and function to either induce cell death or significantly reduce their proliferation. Thus, hMSC-based cellular delivery is an effective method of delivering synthetic oligonucleotides that can significantly reduce tumor cell growth and should be further investigated as a possible approach to cancer therapy.

A study of the outward background current conductance gK1, the pacemaker current conductance gf, and the gap junction conductance gj as determinants of biological pacing in single cells and in a two-cell syncytium using the dynamic clamp.

We previously demonstrated that a two-cell syncytium, composed of a ventricular myocyte and an mHCN2 expressing cell, recapitulated most properties of in vivo biological pacing induced by mHCN2-transfected hMSCs in the canine ventricle. Here, we use the two-cell syncytium, employing dynamic clamp, to study the roles of gf (pacemaker conductance), gK1 (background K+ conductance), and gj (intercellular coupling conductance) in biological pacing. We studied gf and gK1 in single HEK293 cells expressing cardiac sodium current channel Nav1.5 (SCN5A). At fixed gf, increasing gK1 hyperpolarized the cell and initiated pacing. As gK1 increased, rate increased, then decreased, finally ceasing at membrane potentials near EK. At fixed gK1, increasing gf depolarized the cell and initiated pacing. With increasing gf, rate increased reaching a plateau, then decreased, ceasing at a depolarized membrane potential. We studied gj via virtual coupling with two non-adjacent cells, a driver (HEK293 cell) in which gK1 and gf were injected without SCN5A and a follower (HEK293 cell), expressing SCN5A. At the chosen values of gK1 and gf oscillations initiated in the driver, when gj was increased synchronized pacing began, which then decreased by about 35% as gj approached 20 nS. Virtual uncoupling yielded similar insights into gj. We also studied subthreshold oscillations in physically and virtually coupled cells. When coupling was insufficient to induce pacing, passive spread of the oscillations occurred in the follower. These results show a non-monotonic relationship between gK1, gf, gj, and pacing. Further, oscillations can be generated by gK1 and gf in the absence of SCN5A.

Lens Connexin Channels Show Differential Permeability to Signaling Molecules.

Gap junction channels mediate the direct intercellular passage of small ions as well as larger solutes such as second messengers. A family of proteins called connexins make up the subunits of gap junction channels in chordate animals. Each individual connexin forms channels that exhibit distinct permeability to molecules that influence cellular signaling, such as calcium ions, cyclic nucleotides, or inositol phosphates. In this review, we examine the permeability of connexin channels containing Cx43, Cx46, and Cx50 to signaling molecules and attempt to relate the observed differences in permeability to possible in vivo consequences that were revealed by studies of transgenic animals where these connexin genes have been manipulated. Taken together, these data suggest that differences in the permeability of individual connexin channels to larger solutes like 3',5'-cyclic adenosine monophosphate (cAMP) and inositol 1,4,5-trisphosphate (IP3) could play a role in regulating epithelial cell division, differentiation, and homeostasis in organs like the ocular lens.

The role of the Fracture Liaison Service (FLS) in subsequent fracture prevention in the extreme elderly.

BACKGROUND: Several guidelines recommend a bone and fall-related osteoporosis risk assessment in all patients with fracture and age > 50 years. In practice, however, there is no consensus whether screening > 85 years is useful. AIM: To evaluate the subsequent fracture risk in all patient > 85 years, comparing the two populations of Fracture Liaison Service (FLS) attenders and non-attenders. METHODS: All patients > 85 years that presented at the FLS with a non-vertebral fracture were included in the study during a 5-year period (September 2004 and December 2009). Excluded were pathologic fractures, death < 30 days, or patients on osteoporosis treatment. in patients that attended the FLS, assessment of bone mineral density and fall-risk factors were screened. In both the attenders and non-attenders groups, mortality and subsequent fracture rates were scored during a follow-up of 2 years. RESULTS: 282 patients fulfilled inclusion criteria for screening, of which 160 (57%) patients did not attend the FLS. 122 patients were screened for osteoporosis and fall-related risk of whom 72 were diagnosed with osteoporosis. Subsequent fracture risk in both groups was 19%. Medical treatment was started in 51 patients, of which 15 patients developed a subsequent fracture. Cox-regression analysis indicated a significantly lower mortality rate, but not a diminished subsequent fracture rate in the FLS screened population compared to the non-attenders. CONCLUSION: The advantage of a FLS in reducing subsequent fracture risk in patients > 85 years seems to be limited. In practice a large proportion of these patients are not screened.

Patient-reported quality of life and pain after permissive weight bearing in surgically treated trauma patients with tibial plateau fractures: a retrospective cohort study.

INTRODUCTION: A Dutch survey among orthopedic surgeons and trauma surgeons showed that almost 90% of the surgeons do not follow protocols regarding the weight bearing aftercare for tibial plateau fractures. Clinical studies comparing permissive weight bearing (PWB) versus restricted weight bearing (RWB) after surgically treated tibial plateau fractures are not available. The aim of this study was to inventory potential differences in quality of life and pain, and number of complications in patients with surgically treated tibial plateau fractures who followed a PWB regime, relative to those that followed a RWB regime. MATERIALS AND METHODS: This retrospective cohort study included surgically treated trauma patients with tibial plateau fractures, who underwent rehabilitation according to PWB or RWB between 2005 and 2015. Data such as demographics, patient-reported quality of life and pain, and patient outcome were collected. RESULTS: This cohort study included 91 patients with a tibial plateau fracture (31 and 60 patients in the PWB and RWB groups respectively). No significant between-group differences in either age or gender were found. However, a significant difference in fracture type was found between groups, (p = 0.04). No significant differences were found in either patient-reported SF-12 or VAS scores between the PWB group and RWB group. Time to full weight bearing was significantly shorter in the PWB than in the RWB group, i.e., 14.7 versus 20.7 weeks, (p = 0.02). No significant differences were found regarding postoperative complications between the PWB and the RWB groups, i.e., 6.5% versus 10.0%, respectively. CONCLUSION: PWB after surgically treated tibial plateau fractures is safe and is related to a significantly reduced time to full weight bearing with no significant differences in patient-reported quality of life and pain or complication rates.

Defining the factors that affect solute permeation of gap junction channels.

This review focuses on the biophysical properties and structure of the pore and vestibule of homotypic gap junction channels as they relate to channel permeability and selectivity. Gap junction channels are unique in their sole role to connect the cytoplasm of two adjacent cells. In general, these channels are considered to be poorly selective, possess open probabilities approximating unity, and exhibit mean open times ranging from milliseconds to seconds. These properties suggest that such channels can function as delivery pathways from cell to cell for solutes that are significantly larger than monovalent ions. We have taken quantitative data from published works concerning unitary conductance, ion flux, and permeability for homotypic connexin 43 (Cx43), Cx40, Cx26, Cx50, and Cx37, and performed a comparative analysis of conductance and/or ion/solute flux versus diffusion coefficient. The analysis of monovalent cation flux portrays the pore as equivalent to an aqueous space where hydrogen bonding and weak interactions with binding sites dominate. For larger solutes, size, shape and charge are also significant components in determining the permeation rate. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve.

Permissive weight bearing in trauma patients with fracture of the lower extremities: prospective multicenter comparative cohort study.

BACKGROUND: The standard aftercare treatment in surgically treated trauma patients with fractures around or in a joint, known as (peri)- or intra-articular fractures of the lower extremities, is either non-weight bearing or partial weight bearing. We have developed an early permissive weight bearing post-surgery rehabilitation protocol in surgically treated patients with fractures of the lower extremities. In this proposal we want to compare our early permissive weight bearing protocol to the existing current non-weight bearing guidelines in a prospective comparative cohort study. METHODS/DESIGN: The study is a prospective multicenter comparative cohort study in which two rehabilitation aftercare treatments will be contrasted, i.e. permissive weight bearing and non-weight bearing according to the AO-guideline. The study population consists of patients with a surgically treated fracture of the pelvis/acetabulum or a surgically treated (peri)- or intra-articular fracture of the lower extremities. The inclusion period is 12 months. The duration of follow up is 6 months, with measurements taken at baseline, 2,6,12 and 26 weeks post-surgery. PRIMARY OUTCOME MEASURE: ADL with Lower Extremity Functional Scale. Outcome variables for compliance, as measured with an insole pressure measurement system, encompass peak load and step duration. DISCUSSION: This study will investigate the (cost-) effectiveness of a permissive weight bearing aftercare protocol. The results will provide evidence whether a permissive weight bearing protocol is more effective than the current non-weight bearing protocol. TRIAL REGISTRATION: The study is registered in the Dutch Trial Register ( NTR6077 ). Date of registration: 01-09-2016.

Exposure to TiO2 nanoparticles increases Staphylococcus aureus infection of HeLa cells.

BACKGROUND: Titanium dioxide (TiO2) is one of the most common nanoparticles found in industry ranging from food additives to energy generation. Approximately four million tons of TiO2 particles are produced worldwide each year with approximately 3000 tons being produced in nanoparticulate form, hence exposure to these particles is almost certain. RESULTS: Even though TiO2 is also used as an anti-bacterial agent in combination with UV, we have found that, in the absence of UV, exposure of HeLa cells to TiO2 nanoparticles significantly increased their risk of bacterial invasion. HeLa cells cultured with 0.1 mg/ml rutile and anatase TiO2 nanoparticles for 24 h prior to exposure to bacteria had 350 and 250 % respectively more bacteria per cell. The increase was attributed to bacterial polysaccharides absorption on TiO2 NPs, increased extracellular LDH, and changes in the mechanical response of the cell membrane. On the other hand, macrophages exposed to TiO2 particles ingested 40 % fewer bacteria, further increasing the risk of infection. CONCLUSIONS: In combination, these two factors raise serious concerns regarding the impact of exposure to TiO2 nanoparticles on the ability of organisms to resist bacterial infection.

Effect of spinal immobilisation devices on radiation exposure in conventional radiography and computed tomography.

Trauma patients at risk for, or suspected of, spinal injury are frequently transported to hospital using full spinal immobilisation. At the emergency department, immobilisation is often maintained until radiological work-up is completed. In this study, we examined how these devices influence radiation exposure and noise, as a proxy for objective image quality. Conventional radiographs (CR) and computer tomography (CT) scans were made using a phantom immobilised on two types of spineboard and a vacuum mattress and using two types of headblocks. Images were compared for radiation transmission and quantitative image noise. In CR, up to 23 % and, in CT, up to 11 % of radiation were blocked by the devices. Without compensation for the decreased transmission, noise increased by up to 16 % in CT, depending on the device used. Removing the headblocks led to a statistically significant improvement in transmission with automatic exposure control (AEC) enabled. Physicians should make an informed decision whether the increased radiation exposure outweighs the risk of missing a clinically significant injury by not making a CR or CT scan. Manufacturers of immobilisation devices should take radiological properties of their devices into account in the development and production process.

Effect of spineboard and headblocks on the image quality of head CT scans.

Trauma patients at risk for, or suspected of, spinal injury are frequently transported to hospital using full spinal immobilisation. At the emergency department, immobilisation is often maintained until radiological work-up is completed. In this study, we examined how these devices for spinal stabilization influence visual image quality. Image quality was judged for both patient CT scans and phantom CT scans. CT scans of 217 patients were assessed retrospectively by two radiologists for visual scoring of image quality, scoring both quantity and impact of artifacts caused by the immobilization devices. For the phantom CT scans, eight set-ups were made, using a vacuum mattress without headblocks and a rigid and a soft-layered spineboard without headblocks, with standard soft-foam headblocks, or with new design headblocks. Overall, artifacts were found in 67 % of CT scans of patients on immobilization devices, which hampered diagnosis in 10 % of the cases. In the phantom CT scans, artifacts were present in all set-ups with one or more devices present and were seen in 20 % of all scan slices. The presence of headblocks resulted in more artifacts in both the patient CT scans and the phantom CT scans. Considerable effort should therefore be made to adjust the design of the immobilization devices and to remove the headblocks before CT scans are made.

Articular chondrocyte network mediated by gap junctions: role in metabolic cartilage homeostasis.

OBJECTIVE: This study investigated whether chondrocytes within the cartilage matrix have the capacity to communicate through intercellular connections mediated by voltage-gated gap junction (GJ) channels. METHODS: Frozen cartilage samples were used for immunofluorescence and immunohistochemistry assays. Samples were embedded in cacodylate buffer before dehydration for scanning electron microscopy. Co-immunoprecipitation experiments and mass spectrometry (MS) were performed to identify proteins that interact with the C-terminal end of Cx43. GJ communication was studied through in situ electroporation, electrophysiology and dye injection experiments. A transwell layered culture system and MS were used to identify and quantify transferred amino acids. RESULTS: Microscopic images revealed the presence of multiple cellular projections connecting chondrocytes within the matrix. These projections were between 5 and 150 µm in length. MS data analysis indicated that the C-terminus of Cx43 interacts with several cytoskeletal proteins implicated in Cx trafficking and GJ assembly, including α-tubulin and ß-tubulin, actin, and vinculin. Electrophysiology experiments demonstrated that 12-mer oligonucleotides could be transferred between chondrocytes within 12 min after injection. Glucose was homogeneously distributed within 22 and 35 min. No transfer was detected when glucose was electroporated into A549 cells, which have no GJs. Transwell layered culture systems coupled with MS analysis revealed connexins can mediate the transfer of L-lysine and L-arginine between chondrocytes. CONCLUSIONS: This study reveals that intercellular connections between chondrocytes contain GJs that play a key role in cell-cell communication and a metabolic function by exchange of nutrients including glucose and essential amino acids. A three-dimensional cellular network mediated through GJs might mediate metabolic and physiological homeostasis to maintain cartilage tissue.

Improved fracture healing in patients with concomitant traumatic brain injury: proven or not?

Over the last 3 decades, scientific evidence advocates an association between traumatic brain injury (TBI) and accelerated fracture healing. Multiple clinical and preclinical studies have shown an enhanced callus formation and an increased callus volume in patients, respectively, rats with concomitant TBI. Over time, different substances (cytokines, hormones, etc.) were in focus to elucidate the relationship between TBI and fracture healing. Until now, the mechanism behind this relationship is not fully clarified and a consensus on which substance plays the key role could not be attained in the literature. In this review, we will give an overview of current concepts and opinions on this topic published in the last decade and both clinical and pathophysiological theories will be discussed.

Functional outcome and cost-effectiveness of pulsed electromagnetic fields in the treatment of acute scaphoid fractures: a cost-utility analysis.

BACKGROUND: Physical forces have been widely used to stimulate bone growth in fracture repair. Addition of bone growth stimulation to the conservative treatment regime is more costly than standard health care. However, it might lead to cost-savings due to a reduction of the total amount of working days lost. This economic evaluation was performed to assess the cost-effectiveness of Pulsed Electromagnetic Fields (PEMF) compared to standard health care in the treatment of acute scaphoid fractures. METHODS: An economic evaluation was carried out from a societal perspective, alongside a double-blind, randomized, placebo-controlled, multicenter trial involving five centres in The Netherlands. One hundred and two patients with a clinically and radiographically proven fracture of the scaphoid were included in the study and randomly allocated to either active bone growth stimulation or standard health care, using a placebo. All costs (medical costs and costs due to productivity loss) were measured during one year follow up. Functional outcome and general health related quality of life were assessed by the EuroQol-5D and PRWHE (patient rated wrist and hand evaluation) questionnaires. Utility scores were derived from the EuroQol-5D. RESULTS: The average total number of working days lost was lower in the active PEMF group (9.82 days) compared to the placebo group (12.91 days) (p = 0.651). Total medical costs of the intervention group (€1594) were significantly higher compared to the standard health care (€875). The total amount of mean QALY's (quality-adjusted life year) for the active PEMF group was 0.84 and 0.85 for the control group. The cost-effectiveness plane shows that the majority of all cost-effectiveness ratios fall into the quadrant where PEMF is not only less effective in terms of QALY's but also more costly. CONCLUSION: This study demonstrates that the desired effects in terms of cost-effectiveness are not met. When comparing the effects of PEMF to standard health care in terms of QALY's, PEMF cannot be considered a cost-effective treatment for acute fractures of the scaphoid bone. TRIAL REGISTRATION: Netherlands Trial Register (NTR): NTR2064.

Interprofessional communication failures in acute care chains: How can we identify the causes?

Although communication failures between professionals in acute care delivery occur, explanations for these failures remain unclear. We aim to gain a deeper understanding of interprofessional communication failures by assessing two different explanations for them. A multiple case study containing six cases (i.e. acute care chains) was carried out in which semi-structured interviews, physical artifacts and archival records were used for data collection. Data were entered into matrices and the pattern-matching technique was used to examine the two complementary propositions. Based on the level of standardization and integration present in the acute care chains, the six acute care chains could be divided into two categories of care processes, with the care chains equally distributed among the categories. Failures in communication occurred in both groups. Communication routines were embedded within organizations and descriptions of communication routines in the entire acute care chain could not be found. Based on the results, failures in communication could not exclusively be explained by literature on process typology. Literature on organizational routines was useful to explain the occurrence of communication failures in the acute care chains. Organizational routines can be seen as repetitive action patterns and play an important role in organizations, as most processes are carried out by means of routines. The results of this study imply that it is useful to further explore the role of organizational routines on interprofessional communication in acute care chains to develop a solution for failures in handover practices.

Stem cell-based biological pacemakers from proof of principle to therapy: a review.

Electronic pacemakers are the standard therapy for bradycardia-related symptoms but have shortcomings. Over the past 15 years, experimental evidence has demonstrated that gene and cell-based therapies can create a biological pacemaker. Recently, physiologically acceptable rates have been reported with an adenovirus-based approach. However, adenovirus-based protein expression does not last more than 4 weeks, which limits its clinical applicability. Cell-based platforms are potential candidates for longer expression. Currently there are two cell-based approaches being tested: (i) mesenchymal stem cells used as a suitcase for delivering pacemaker genes and (ii) pluripotent stem cells differentiated down a cardiac lineage with endogenous pacemaker activity. This review examines the current achievements in engineering a biological pacemaker, defines the patient population for whom this device would be useful and identifies the challenges still ahead before cell therapy can replace current electronic devices.

Barriers to implementation of a redesign of information transfer and feedback in acute care: results from a multiple case study.

BACKGROUND: Accurate information transfer is an important element of continuity of care and patient safety. Despite the demonstrated urge for improvement of communication in acute care, there is a lack of data on improvements of communication. This study aims to describe the barriers to implementation of a redesign of the existing model for information transfer and feedback. METHODS: A case study with six cases (i.e. acute care chains), using mixed methods was carried out in the Netherlands. The redesign was implemented in one acute care chain while the five other acute care chains served as control groups. Focus group interviews were held with members of the acute care chains and questionnaires were sent to care providers working in the acute care chains. RESULTS: Respondents reported three sets of barriers for implementation of the model: (a) existing routines for information transfer and feedback in organizations within the acute care chain; (b) barriers related to the implementation method and time period; and (c) the absence of a high 'sense of urgency' amongst providers in the acute care chain which would aid in improving the communication process. CONCLUSIONS: This study shows that organizational factors play an important role in the success or failure of redesigning a communication process. Organizational routines can hamper implementation of a redesign if it differs too much from the routines of care providers involved. Besides focussing on provider characteristics in the implementation of a redesigned process, specific attention should be paid to unlearning existing organizational routines.

The human Cx26-D50A and Cx26-A88V mutations causing keratitis-ichthyosis-deafness syndrome display increased hemichannel activity.

Mutations in the human gene encoding connexin 26 (Cx26 or GJB2) cause either nonsyndromic deafness or syndromic deafness associated with skin diseases. That distinct clinical disorders can be caused by different mutations within the same gene suggests that different channel activities influence the ear and skin. Here we use three different expression systems to examine the functional characteristics of two Cx26 mutations causing either mild (Cx26-D50A) or lethal (Cx26-A88V) keratitis-ichthyosis-deafness (KID) syndrome. In either cRNA-injected Xenopus oocytes, transfected HeLa cells, or transfected primary human keratinocytes, we show that both Cx26-D50A and Cx26-A88V form active hemichannels that significantly increase membrane current flow compared with wild-type Cx26. This increased membrane current accelerated cell death in low extracellular calcium solutions and was not due to increased mutant protein expression. Elevated mutant hemichannel currents could be blocked by increased extracellular calcium concentration. These results show that these two mutations exhibit a shared gain of functional activity and support the hypothesis that increased hemichannel activity is a common feature of human Cx26 mutations responsible for KID syndrome.

Can gap junctions deliver?

In vivo delivery of small interfering RNAs (siRNAs) to target cells via the extracellular space has been hampered by dilution effects and immune responses. Gap junction-mediated transfer between cells avoids the extracellular space and its associated limitations. Because of these advantages cell based delivery via gap junctions has emerged as a viable alternative for siRNA or miRNA delivery. Here we discuss the advantages and disadvantages of extracellular delivery and cell to cell delivery via gap junction channels composed of connexins. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.

MATLAB implementation of a dynamic clamp with bandwidth of >125 kHz capable of generating I Na at 37 °C.

We describe the construction of a dynamic clamp with a bandwidth of >125 kHz that utilizes a high-performance, yet low-cost, standard home/office PC interfaced with a high-speed (16 bit) data acquisition module. High bandwidth is achieved by exploiting recently available software advances (code-generation technology and optimized real-time kernel). Dynamic-clamp programs are constructed using Simulink, a visual programming language. Blocks for computation of membrane currents are written in the high-level MATLAB language; no programming in C is required. The instrument can be used in single- or dual-cell configurations, with the capability to modify programs while experiments are in progress. We describe an algorithm for computing the fast transient Na(+) current (I Na) in real time and test its accuracy and stability using rate constants appropriate for 37 °C. We then construct a program capable of supplying three currents to a cell preparation: I Na, the hyperpolarizing-activated inward pacemaker current (I f) and an inward-rectifier K(+) current (I K1). The program corrects for the IR drop due to electrode current flow and also records all voltages and currents. We tested this program on dual patch-clamped HEK293 cells where the dynamic clamp controls a current-clamp amplifier and a voltage-clamp amplifier controls membrane potential, and current-clamped HEK293 cells where the dynamic clamp produces spontaneous pacing behavior exhibiting Na(+) spikes in otherwise passive cells.

Chronic Tempol treatment restores pharmacological preconditioning in the senescent rat heart.

Cardioprotective effects of anesthetic preconditioning and cyclosporine A (CsA) are lost with aging. To extend our previous work and address a possible mechanism underlying age-related differences, we investigated the role of oxidative stress in the aging heart by treating senescent animals with the oxygen free radical scavenger Tempol. Old male Fischer 344 rats (22-24 mo) were randomly assigned to control or Tempol treatment groups for 2 or 4 wk (T×2wk and T×4wk, respectively). Rats received isoflurane 30 min before ischemia-reperfusion injury or CsA just before reperfusion. Myocardial infarction sizes were significantly reduced by isoflurane or CsA in the aged rats treated with Tempol (T×4wk) compared with old control rats. In other experiments, young (4-6 mo) and old rats underwent either chronic Tempol or vehicle treatment, and the levels of myocardial protein oxidative damage, antioxidant enzymes, mitochondrial Ca(2+) uptake, cyclophilin D protein, and mitochondrial permeability transition pore opening times were measured. T×4wk significantly increased MnSOD enzyme activity, GSH-to-GSSH ratios, MnSOD protein level, mitochondrial Ca(2+) uptake capacity, reduced protein nitrotyrosine levels, and normalized cyclophilin D protein expression in the aged rat heart. T×4wk also significantly prolonged mitochondrial permeability transition pore opening times induced by reactive oxygen species in old cardiomyocytes. Our studies demonstrate that 4 wk of Tempol pretreatment restores anesthetic preconditioning and cardioprotection by CsA in the old rat and that this is associated with decreased oxidative stress and improved mitochondrial function. Our results point to a new protective strategy for the ischemic myocardium in the high-risk older population.