Contribution of T-Type Calcium Channels to Spinal Cord Injury-Induced Hyperexcitability of Nociceptors.

A hyperexcitable state and spontaneous activity of nociceptors have been suggested to play a critical role in the development of chronic neuropathic pain following spinal cord injury (SCI). In male rats, we employed the action potential-clamp technique to determine the underlying ionic mechanisms responsible for driving SCI-nociceptors to a hyperexcitable state and for triggering their spontaneous activity. We found that the increased activity of low voltage activated T-type calcium channels induced by the injury sustains the bulk (∼60-70%) of the inward current active at subthreshold voltages during the interspike interval in SCI-nociceptors, with a modest contribution (∼10-15%) from tetrodotoxin (TTX)-sensitive and TTX-resistant sodium channels and hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. In current-clamp recordings, inhibition of T-type calcium channels with 1 µm TTA-P2 reduced both the spontaneous and the evoked firing in response to current injections in SCI-nociceptors to a level similar to sham-nociceptors. Electrophysiology in vitro was then combined with the conditioned place preference (CPP) paradigm to determine the relationship between the increased activity of T-type channels in SCI-nociceptors and chronic neuropathic pain following SCI. The size of the interspike T-type calcium current recorded from nociceptors isolated from SCI rats showing TTA-P2-induced CPP (responders) was ∼6 fold greater than the interspike T-type calcium current recorded from nociceptors isolated from SCI rats without TTA-P2-induced CPP (non-responders). Taken together, our data suggest that the increased activity of T-type calcium channels induced by the injury plays a primary role in driving SCI-nociceptors to a hyperexcitable state and contributes to chronic neuropathic pain following SCI.SIGNIFICANCE STATEMENT Chronic neuropathic pain is a major comorbidity of spinal cord injury (SCI), affecting up to 70-80% of patients. Anticonvulsant and tricyclic antidepressant drugs are first line analgesics used to treat SCI-induced neuropathic pain, but their efficacy is very limited. A hyperexcitable state and spontaneous activity of SCI-nociceptors have been proposed as a possible underlying cause for the development of chronic neuropathic pain following SCI. Here, we show that the increased activity of T-type calcium channels induced by the injury plays a major role in driving SCI-nociceptors to a hyperexcitable state and for promoting their spontaneous activity, suggesting that T-type calcium channels may represent a pharmacological target to treat SCI-induced neuropathic pain.

Presynaptic Inhibition of Primary Nociceptive Signals to Dorsal Horn Lamina I Neurons by Dopamine.

The dorsal horn of the spinal cord represents the first relay station in the pain pathway where primary nociceptive inputs are modulated by local circuits and by descending signals before being relayed to supraspinal nuclei. To determine whether dopamine can modulate primary nociceptive Aδ- and C-fiber signals, the effects of dopamine were tested on the excitatory postsynaptic currents (EPSCs) recorded from large lamina I neurons and from retrograde-labeled spinoparabrachial lamina I neurons upon stimulation of the L4/L5 dorsal root in horizontal spinal cord slices in vitro Dopamine inhibited the EPSCs in a dose-dependent manner, with substantial inhibition (33%) at 1 µm and maximum inhibition (∼70%) at 10-20 µm Dopamine reduced the frequency of miniature EPSCs recorded from large lamina I neurons, increased the paired pulse depression ratio of paired EPSCs, and induced similar inhibition of EPSCs after dialysis of large lamina I neurons with GDP-ß-S, consistent with actions at presynaptic sites. Pharmacological experiments suggested that the inhibitory effects of dopamine were largely mediated by D4 receptors (53%). Similar inhibition (66%) by dopamine was observed on EPSCs recorded from ipsilateral large lamina I neurons 6 d after injection of complete Freund's adjuvant in the hindpaw, suggesting that dopamine downregulates primary nociceptive inputs to lamina I neurons during chronic inflammatory pain. We propose that presynaptic inhibition of primary nociceptive inputs to lamina I projection neurons is a mechanism whereby dopamine can inhibit incoming noxious stimuli to the dorsal horn of the spinal cord.SIGNIFICANCE STATEMENT Lamina I projection neurons represent the main output for the pain signals from the dorsal horn of the spinal cord to brainstem and thalamic nuclei. We found that dopamine inhibits the nociceptive Aδ- and C-fiber synaptic inputs to lamina I projection neurons via presynaptic actions. Similar inhibitory effects of dopamine on the EPSCs were observed in rats subjected to complete Freund's adjuvant to induce peripheral inflammation, suggesting that dopamine inhibits the synaptic inputs to lamina I neurons in the setting of injury. A better understanding of how primary nociceptive inputs to the dorsal horn of the spinal cord are modulated by descending monoaminergic signals may help in the development of new pharmacological strategies to selectively downregulate the output from lamina I projection neurons.

Contribution of diacylglycerol lipase ß to pain after surgery.

BACKGROUND: Metabolism of the endocannabinoid 2-arachidonoylglycerol (2-AG) yields arachidonic acid (AA), the precursor to proalgesic eicosanoids including prostaglandin E2 (PGE2). Diacylglycerol lipase ß (DAGLß) is an enzyme that synthesizes 2-AG and its inhibition reduces eicosanoid levels and produces antinociceptive effects in models of inflammatory pain. Here we test whether inhibition of DAGLß produces antinociceptive effects in a model of postoperative pain. METHODS: Rats were administered the selective DAGLß inhibitor KT109 or vehicle and underwent plantar incision. Postsurgical pain/disability was examined using evoked (mechanical hyperalgesia), functional (incapacitance/weight bearing), and functional/spontaneous (locomotion) modalities. RESULTS: Activity-based protein profiling confirmed that KT109 inhibited DAGLß in the lumbar spinal cord (LSC) and brain, confirming that it is a systemically active DAGLß inhibitor. Treatment with KT109 reduced basal 2-AG, AA, and PGE2 levels in the liver but not the brain, indicating that DAGLß activity does not significantly contribute to basal PGE2 production within the central nervous system. Plantar incision elevated the levels of 2-AG and PGE2 in the LSC. Although KT109 did not alter postsurgical 2-AG levels in the LSC, it slightly reduced PGE2 levels. In contrast, the clinically efficacious cyclooxygenase inhibitor ketoprofen completely suppressed PGE2 levels in the LSC. Similarly, KT109 had no significant effect upon postsurgical 2-AG, AA, or PGE2 levels at the incision site, while ketoprofen abolished PGE2 production at this location. KT109 and ketoprofen reversed the weight bearing imbalance induced by plantar incision, yet neither KT109 nor ketoprofen had any significant effect on mechanical hyperalgesia. Treatment with ketoprofen partially but significantly rescued the locomotor deficit induced by incision while KT109 was without effect. CONCLUSION: DAGLß is not the principal enzyme that controls 2-AG derived AA and PGE2 production after surgery, and inhibitors targeting this enzyme are unlikely to be efficacious analgesics superior to those already approved to treat acute postoperative pain.

Intrathecal morphine administration reduces postoperative pain and peripheral endocannabinoid levels in total knee arthroplasty patients: a randomized clinical trial.

BACKGROUND: The primary goal of this study was to determine whether administration of intrathecal morphine reduces postoperative pain. The secondary goal was to determine the effect of intrathecal morphine upon circulating levels of the weakly analgesic endocannabinoids, anandamide (AEA) and 2-arachidonoylglycerol (2-AG), and the related lipids palmitoylethanolamide (PEA) and oleoylethanolamide (OEA). METHODS: Forty two total knee arthroplasty (TKA) patients were enrolled in a prospective, double-blinded, randomized study. The intervention consisted of intrathecal morphine (200 µg) or placebo administered at the time of the spinal anesthesia. Postoperative pain was measured during the first 4 h after surgery while serum levels of AEA, 2-AG, PEA, OEA, and cortisol were measured at baseline and 4 h after surgery. RESULTS: Administration of intrathecal morphine reduced postoperative pain 4 h after TKA surgery compared to placebo (p = 0.005) and reduced postoperative systemic opioid consumption (p = 0.001). At baseline, intrathecal morphine led to a significant reduction in AEA, 2-AG, and OEA levels but did not affect PEA or cortisol levels. In patients administered intrathecal placebo, 2-AG levels were elevated 4 h after surgery; whereas patients receiving intrathecal morphine showed reductions in AEA, PEA, and OEA when compared to placebo. At 4 h after TKA surgery cortisol levels were significantly elevated in the placebo group and reduced in those receiving morphine. CONCLUSIONS: These results indicate that intrathecal morphine reduces postoperative pain in TKA patients. Furthermore, activation of central opioid receptors negatively modulates the endocannabinoid tone, suggesting that potent analgesics may reduce the stimulus for production of peripheral endocannabinoids. This study is the first to document the existence of rapid communication between the central opioid and peripheral endocannabinoid systems in humans. TRIAL REGISTRATION: This trial was registered retrospectively. TRIAL REGISTRY: NCT02620631 . Study to Examine Pain Relief With Supplemental Intrathecal Morphine in TKA Patients, NCT02620631 , 12/03/2015.

Interleukin-6 and leptin levels are associated with preoperative pain severity in patients with osteoarthritis but not with acute pain after total knee arthroplasty.

BACKGROUND: Identifying drivers of pain that can serve as novel drug targets is important for improving perioperative analgesia. Total knee arthroplasty (TKA) is associated with significant postoperative pain. Cytokines contribute to the pathophysiology of osteoarthritis (OA) and associated pain. However, the influence of perioperative cytokine levels after TKA surgery upon postoperative pain remains unexplored. METHODS: We designed a prospective observational study to profile three proinflammatory cytokines, interleukin-6 (IL-6), tumor necrosis factor α (TNFα), and leptin in serum, synovial, and cerebrospinal fluid of TKA patients perioperatively to determine associations between cytokine levels and pain. We characterized time-trajectories in cytokines pre- and post-surgery and explored their relationships to pain across gender. RESULTS: Preoperative pain, measured by functional pain disability scores (PDQ), was predictive of postoperative pain. There were no gender differences in severity of preoperative pain or acute postoperative pain. Serum IL-6, serum leptin, and synovial fluid leptin were positively correlated with body mass index and preoperative pain severity. Stratification of patients by gender revealed strong correlations between serum IL-6, leptin, and PDQ only in females, suggesting that females may be more sensitive to the nociceptive actions of these cytokines. Although serum IL-6 increased dramatically (and TNFα increased modestly) four hours after surgery and remained elevated at 72h; they were not associated with the severity of acute postoperative pain. CONCLUSIONS: Our data suggest that while preoperative chronic pain is predictive of the severity of acute postoperative pain in TKA patients, the pre- and post-operative inflammatory status does not predict postoperative pain.

Transcriptomic evidence of a para-inflammatory state in the middle aged lumbar spinal cord.

BACKGROUND: We have previously reported elevated expression of multiple pro-inflammatory markers in the lumbar spinal cord (LSC) of middle-aged male rats compared to young adults suggesting a para-inflammatory state develops in the LSC by middle age, a time that in humans is associated with the greatest pain prevalence and persistence. The goal of the current study was to examine the transcriptome-wide gene expression differences between young and middle aged LSC. METHODS: Young (3 month) and middle-aged (17 month) naïve Fisher 344 rats (n = 5 per group) were euthanized, perfused with heparinized saline, and the LSC were removed. RESULTS: ~70% of 31,000 coding sequences were detected. After normalization, ~ 1100 showed statistically significant differential expression. Of these genes, 353 middle-aged annotated genes differed by > 1.5 fold compared to the young group. Nearly 10% of these genes belonged to the microglial sensome. Analysis of this subset revealed that the principal age-related differential pathways populated are complement, pattern recognition receptors, OX40, and various T cell regulatory pathways consistent with microglial priming and T cell invasion and modulation. Many of these pathways substantially overlap those previously identified in studies of LSC of young animals with chronic inflammatory or neuropathic pain. CONCLUSIONS: Up-modulation of complement pathway, microglial priming and activation, and T cell/antigen-presenting cell communication in healthy middle-aged LSC was found. Taken together with our previous work, the results support our conclusion that an incipient or para-inflammatory state develops in the LSC in healthy middle-aged adults.

Fatty-acid-binding protein inhibition produces analgesic effects through peripheral and central mechanisms.

Background Fatty-acid-binding proteins (FABPs) are intracellular carriers for endocannabinoids, N-acylethanolamines, and related lipids. Previous work indicates that systemically administered FABP5 inhibitors produce analgesia in models of inflammatory pain. It is currently not known whether FABP inhibitors exert their effects through peripheral or central mechanisms. Here, we examined FABP5 distribution in dorsal root ganglia and spinal cord and examined the analgesic effects of peripherally and centrally administered FABP5 inhibitors. Results Immunofluorescence revealed robust expression of FABP5 in lumbar dorsal root ganglia. FABP5 was distributed in peptidergic calcitonin gene-related peptide-expressing dorsal root ganglia and non-peptidergic isolectin B4-expressing dorsal root ganglia. In addition, the majority of dorsal root ganglia expressing FABP5 also expressed transient receptor potential vanilloid 1 (TRPV1) and peripherin, a marker of nociceptive fibers. Intraplantar administration of FABP5 inhibitors reduced thermal and mechanical hyperalgesia in the complete Freund's adjuvant model of chronic inflammatory pain. In contrast to its robust expression in dorsal root ganglia, FABP5 was sparsely distributed in the lumbar spinal cord and intrathecal administration of FABP inhibitor did not confer analgesic effects. Administration of FABP inhibitor via the intracerebroventricular (i.c.v.) route reduced thermal hyperalgesia. Antagonists of peroxisome proliferator-activated receptor alpha blocked the analgesic effects of peripherally and i.c.v. administered FABP inhibitor while antagonism of cannabinoid receptor 1 blocked the effects of peripheral FABP inhibition and a TRPV1 antagonist blocked the effects of i.c.v. administered inhibitor. Although FABP5 and TRPV1 were co-expressed in the periaqueductal gray region of the brain, which is known to modulate pain, knockdown of FABP5 in the periaqueductal gray using adeno-associated viruses and pharmacological FABP5 inhibition did not produce analgesic effects. Conclusions This study demonstrates that FABP5 is highly expressed in nociceptive dorsal root ganglia neurons and FABP inhibitors exert peripheral and supraspinal analgesic effects. This indicates that peripherally restricted FABP inhibitors may serve as a new class of analgesic and anti-inflammatory agents.

Restoring Pharmacologic Preconditioning in the Aging Heart: Role of Mitophagy/Autophagy.

We previously reported that pretreatment with the potent antioxidant TEMPOL improves mitochondrial function and restores preconditioning in the aging heart. Because mitophagy is implicated in cardiac preconditioning and declines with age, this study was designed to investigate how age influences mitophagy in response to preconditioning and whether TEMPOL pretreatment improves it. Old (22-24 months) rats were pretreated with or without 4-week TEMPOL and compared with young (4-6 months) untreated rats. Cardioprotection induced by isoflurane (ISO) in vivo and in isolated cardiomyocytes in vitro was assessed following ischemia/reperfusion and simulated hypoxia/reoxygenation, respectively. Mitophagy was determined by comparing the levels/subcellular locations of key mitophagic markers using Western blotting and immunofluorescence techniques. ISO preconditioned the young but not old heart in vivo and in vitro. Aging impaired ISO-induced mitochondrial accumulation of PINK1 and Parkin, as well as mitochondrial ubiquitination, and baseline and ISO-induced autophagic flux assessed by LC3 puncta, membrane associated LC3-II and p62. Pretreatment with TEMPOL improved these processes and restored ISO preconditioning. Inhibition of autophagy abolished ISO-induced protection in cardiomyocytes from young and TEMPOL pretreated old rats. Thus, antioxidant pretreatment significantly improves mitophagic response to ISO in old myocardium, which may contribute to restoration of cardioprotection in senescent animals.

Neuroimmune and Neuropathic Responses of Spinal Cord and Dorsal Root Ganglia in Middle Age.

Prior studies of aging and neuropathic injury have focused on senescent animals compared to young adults, while changes in middle age, particularly in the dorsal root ganglia (DRG), have remained largely unexplored. 14 neuroimmune mRNA markers, previously associated with peripheral nerve injury, were measured in multiplex assays of lumbar spinal cord (LSC), and DRG from young and middle-aged (3, 17 month) naïve rats, or from rats subjected to chronic constriction injury (CCI) of the sciatic nerve (after 7 days), or from aged-matched sham controls. Results showed that CD2, CD3e, CD68, CD45, TNF-α, IL6, CCL2, ATF3 and TGFß1 mRNA levels were substantially elevated in LSC from naïve middle-aged animals compared to young adults. Similarly, LSC samples from older sham animals showed increased levels of T-cell and microglial/macrophage markers. CCI induced further increases in CCL2, and IL6, and elevated ATF3 mRNA levels in LSC of young and middle-aged adults. Immunofluorescence images of dorsal horn microglia from middle-aged naïve or sham rats were typically hypertrophic with mostly thickened, de-ramified processes, similar to microglia following CCI. Unlike the spinal cord, marker expression profiles in naïve DRG were unchanged across age (except increased ATF3); whereas, levels of GFAP protein, localized to satellite glia, were highly elevated in middle age, but independent of nerve injury. Most neuroimmune markers were elevated in DRG following CCI in young adults, yet middle-aged animals showed little response to injury. No age-related changes in nociception (heat, cold, mechanical) were observed in naïve adults, or at days 3 or 7 post-CCI. The patterns of marker expression and microglial morphologies in healthy middle age are consistent with development of a para-inflammatory state involving microglial activation and T-cell marker elevation in the dorsal horn, and neuronal stress and satellite cell activation in the DRG. These changes, however, did not affect the establishment of neuropathic pain.

Leptin levels are negatively correlated with 2-arachidonoylglycerol in the cerebrospinal fluid of patients with osteoarthritis.

BACKGROUND: There is compelling evidence in humans that peripheral endocannabinoid signaling is disrupted in obesity. However, little is known about the corresponding central signaling. Here, we have investigated the relationship between gender, leptin, body mass index (BMI) and levels of the endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG) in the serum and cerebrospinal fluid (CSF) of primarily overweight to obese patients with osteoarthritis. METHODOLOGY/PRINCIPAL FINDINGS: Patients (20 females, 15 males, age range 44-78 years, BMI range 24-42) undergoing total knee arthroplasty for end-stage osteoarthritis were recruited for the study. Endocannabinoids were quantified by liquid chromatography - mass spectrometry. AEA and 2-AG levels in the serum and CSF did not correlate with either age or BMI. However, 2-AG levels in the CSF, but not serum, correlated negatively with CSF leptin levels (Spearman's ρ -0.48, P=0.0076, n=30). No such correlations were observed for AEA and leptin. CONCLUSIONS/SIGNIFICANCE: In the patient sample investigated, there is a negative association between 2-AG and leptin levels in the CSF. This is consistent with pre-clinical studies in animals, demonstrating that leptin controls the levels of hypothalamic endocannabinoids that regulate feeding behavior.

Endocannabinoids and acute pain after total knee arthroplasty.

Osteoarthritis (OA) of the knee is a progressive disease that is associated with inflammation of the joints and lower extremity pain. Total knee arthroplasty (TKA) is a surgical procedure that aims to reduce pain and restore motor function in patients suffering from OA. The immediate postoperative period can be intensely painful leading to extended recovery times including persistent pain. The endocannabinoid system regulates nociception, and the activation of cannabinoid receptors produces antinociceptive effects in preclinical models of OA. To date, the influence of the endocannabinoid tone on pain and disability in OA patients and on acute postoperative pain in humans has not been explored. In this study, we provide the first comprehensive profile of endocannabinoids in serum, cerebrospinal fluid, and synovial fluid of patients with painful end-stage OA undergoing TKA and examine correlations between endocannabinoid levels, interleukin 6, functional disability, acute postoperative pain, and postoperative opioid use. Our results reveal that central (cerebrospinal fluid) and peripheral (synovial fluid) levels of the endocannabinoid 2-arachidonoyl glycerol were significantly elevated in patients who developed higher postoperative pain after TKA. In addition, synovial fluid 2-arachidonoyl glycerol levels were positively correlated with postoperative opioid use. Similarly, synovial fluid levels of the anti-inflammatory lipid palmitoylethanolamide correlated with functional disability in OA. Taken together, our results are the first to reveal associations between central and peripheral endocannabinoid levels and postoperative pain. This suggests that endocannabinoid metabolism may serve as a target for the development of novel analgesics both for systemic or local delivery into the joint.

Inhibition of fatty acid binding proteins elevates brain anandamide levels and produces analgesia.

The endocannabinoid anandamide (AEA) is an antinociceptive lipid that is inactivated through cellular uptake and subsequent catabolism by fatty acid amide hydrolase (FAAH). Fatty acid binding proteins (FABPs) are intracellular carriers that deliver AEA and related N-acylethanolamines (NAEs) to FAAH for hydrolysis. The mammalian brain expresses three FABP subtypes: FABP3, FABP5, and FABP7. Recent work from our group has revealed that pharmacological inhibition of FABPs reduces inflammatory pain in mice. The goal of the current work was to explore the effects of FABP inhibition upon nociception in diverse models of pain. We developed inhibitors with differential affinities for FABPs to elucidate the subtype(s) that contributes to the antinociceptive effects of FABP inhibitors. Inhibition of FABPs reduced nociception associated with inflammatory, visceral, and neuropathic pain. The antinociceptive effects of FABP inhibitors mirrored their affinities for FABP5, while binding to FABP3 and FABP7 was not a predictor of in vivo efficacy. The antinociceptive effects of FABP inhibitors were mediated by cannabinoid receptor 1 (CB1) and peroxisome proliferator-activated receptor alpha (PPARα) and FABP inhibition elevated brain levels of AEA, providing the first direct evidence that FABPs regulate brain endocannabinoid tone. These results highlight FABPs as novel targets for the development of analgesic and anti-inflammatory therapeutics.

Inhibition of tetrodotoxin-resistant sodium current in dorsal root ganglia neurons mediated by D1/D5 dopamine receptors.

BACKGROUND: Dopaminergic fibers originating from area A11 of the hypothalamus project to different levels of the spinal cord and represent the major source of dopamine. In addition, tyrosine hydroxylase, the rate-limiting enzyme for the synthesis of catecholamines, is expressed in 8-10% of dorsal root ganglia (DRG) neurons, suggesting that dopamine may be released in the dorsal root ganglia. Dopamine has been shown to modulate calcium current in DRG neurons, but the effects of dopamine on sodium current and on the firing properties of small DRG neurons are poorly understood. RESULTS: The effects of dopamine and dopamine receptor agonists were tested on the tetrodotoxin-resistant (TTX-R) sodium current recorded from acutely dissociated small (diameter ≤ 25 µm) DRG neurons. Dopamine (20 µM) and SKF 81297 (10 µM) caused inhibition of TTX-R sodium current in small DRG neurons by 23% and 37%, respectively. In contrast, quinpirole (20 µM) had no effects on the TTX-R sodium current. Inhibition by SKF 81297 of the TTX-R sodium current was not affected when the protein kinase A (PKA) activity was blocked with the PKA inhibitory peptide (6-22), but was greatly reduced when the protein kinase C (PKC) activity was blocked with the PKC inhibitory peptide (19-36), suggesting that activation of D1/D5 dopamine receptors is linked to PKC activity. Expression of D1and D5 dopamine receptors in small DRG neurons, but not D2 dopamine receptors, was confirmed by Western blotting and immunofluorescence analysis. In current clamp experiments, the number of action potentials elicited in small DRG neurons by current injection was reduced by ~ 30% by SKF 81297. CONCLUSIONS: We conclude that activation of D1/D5 dopamine receptors inhibits TTX-R sodium current in unmyelinated nociceptive neurons and dampens their intrinsic excitability by reducing the number of action potentials in response to stimulus. Increasing or decreasing levels of dopamine in the dorsal root ganglia may serve to adjust the sensitivity of nociceptors to noxious stimuli.

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.

Interactions of GSK-3ß with mitochondrial permeability transition pore modulators during preconditioning: age-associated differences.

Anesthetic preconditioning (APC) and ischemic preconditioning (IPC) are lost with normal aging. Here, we investigated age-related difference between phosphoglycogen synthase kinase-3beta (pGSK-3ß) and pGSK-3ß with modulators of mitochondrial permeability transition pore, including adenine nucleotide translocase (ANT), cyclophilin-D, or voltage-dependent anion channel. APC or IPC significantly increased pGSK-3ß in the young groups in both the cytosol and the mitochondria and also significantly increased pGSK-3ß in co-immunoprecipitates with ANT. Importantly, the level of cyclophilin-D in co-immunoprecipitates with ANT was significantly decreased in the young APC and IPC groups, but not in old rats. We also found that APC or IPC significantly prolonged mitochondrial permeability transition pore opening time in the young cardiomyocytes under oxidative stress, but not in the elderly. Attenuation of APC or IPC protection in the aging heart is associated with failure to reduce ANT-cyclophilin-D interactions and to decreased pGSK-3ß responsiveness of ANT, critical modulators of mitochondrial permeability transition pore.

Cardioprotection of the aged rat heart by GSK-3beta inhibitor is attenuated: age-related changes in mitochondrial permeability transition pore modulation.

It is well established that inhibition of glycogen synthase kinase (GSK)-3ß in the young adult myocardium protects against ischemia-reperfusion (I/R) injury through inhibition of mitochondrial permeability transition pore (mPTP) opening. Here, we investigated age-associated differences in the ability of GSK-3ß inhibitor [SB-216763 (SB)] to protect the heart and to modulate mPTP opening during I/R injury. Fischer 344 male rats were assigned from their respective young or old age groups. Animals were subjected to 30 min ischemia following 120 min reperfusion to determine myocardial infarction (MI) size in vivo. Ischemic tissues were collected 10 min after reperfusion for nicotinamide adenine dinucleotide (NAD(+)) measurements and immunoblotting. In parallel experiments, ventricular myocytes isolated from young or old rats were exposed to oxidative stress through generation of reactive oxygen species (ROS), and mPTP opening times were measured by using confocal microscopy. Our results showed that SB decreased MI in young SB-treated rats compared with young untreated I/R animals, whereas SB failed to significantly affect MI in the old animals. SB also significantly increased GSK-3ß phosphorylation in young rats, but phosphorylation levels were already highly elevated in old control groups. There were no significant differences observed between SB-treated and untreated old animals. NAD(+) levels were better maintained in young SB-treated animals compared with the young untreated group during I/R, but this relative improvement was not observed in old animals. SB also significantly prolonged the time to mPTP opening induced by ROS in young cardiomyocytes, but not in aged cardiomyocytes. These results demonstrate that this GSK-3ß inhibitor fails to protect the aged myocardium in response to I/R injury or prevent mPTP opening following a rise in ROS and suggest that healthy aging alters mPTP regulation by GSK-3ß.

Age-associated differences in activation of Akt/GSK-3beta signaling pathways and inhibition of mitochondrial permeability transition pore opening in the rat heart.

Pretreatment with isoflurane decreased myocardial infarction size in young rats (3-5 months) but not in old rats (20-24 months). To understand the mechanisms underlying the failure to protect the old myocardium, differences in phosphorylation of Akt/GSK-3beta and age-associated differences in mitochondrial permeability transition pore (mPTP) opening in the aging heart in vivo were measured. Isoflurane significantly increased Akt and GSK-3beta phosphorylation in the young groups. In contrast, levels of p-Akt and p-GSK-3beta were highly elevated in the old sham control groups. Isoflurane preconditioning significantly reduced the fall in NAD(+) levels induced by ischemia/reperfusion injury in the young animals, reflecting the inhibition of mPTP opening. In the old animals, however, isoflurane failed to prevent the fall in NAD(+) levels induced by ischemia/reperfusion injury. Lack of isoflurane-induced cardioprotective effects, seen in the old animals, can be explained by age-related differences in Akt/GSK-3beta signaling pathway and the inability to reduce mPTP opening following ischemia/reperfusion injury.

Age-associated changes in cardiac gene expression after preconditioning.

BACKGROUND: Cardiac protection afforded by ischemic preconditioning (IPC) and anesthetic preconditioning (APC) are significantly reduced in the senescent myocardium. The authors hypothesized that age would differentially modulate gene expression induced by IPC and APC in vivo. METHODS: Affymetrix RAT EXON ST 1.0 gene chips (Affymetrix, Santa Clara, CA) were used to explore the transcriptional response to IPC and APC in Fisher 344 male rats (young, 3-5 months, and old, 20-24 months, respectively). Both cohorts, young and old, were divided into three groups: (1) sham control, (2) IPC, and (3) APC. After a total of 90 min, the heart was removed, and the total RNA and protein were extracted. RESULTS: Thirty-one transcripts were increased in the young animals subjected to IPC, particularly transcriptional regulators (Atf3, Egr-1, Btg2, Egr2), cytokines (interleukin 6, CSF1, Myd88), chemokines (Cxcl10, Ccl2, Ccl7), regulators of growth and inflammation (Reg3g, Hamp), remodeling and cell adhesion migration (Cyr61, Tfpi2, Timp1), regulators of apoptosis/cell death (Birc3, Arntl, Hamp, Phlda1), and cell cycle control/DNA repairs (Rrad, Gadd45b, Gadd45g). In contrast, only one transcript increased (Atf3) in the old animals subjected to IPC. No changes in gene expression were found in the young or the old animals subjected to APC. CONCLUSIONS: Early-phase IPC and APC induced different genomic responses. The absence of detectable changes associated with early-phase APC suggests a posttranscriptional or posttranslational mechanism. The absence of a genomic response in the senescent myocardium (except for IPC-induced Atf3) could underlie the failure of IPC to provide any cardiac protective benefit to older animals.

Attenuation of isoflurane-induced preconditioning and reactive oxygen species production in the senescent rat heart.

BACKGROUND: Although attenuation of anesthetic preconditioning in aged ex vivo heart models has been studied extensively, there are no comparable in vivo studies. To extend previous work and to address a possible mechanism underlying age-related differences, we investigated isoflurane-induced preconditioning and reactive oxygen species (ROS) production in the aged rat heart in vivo. METHODS: Male Fisher 344 rats were assigned from their respective age groups (young, 3-5 mo; old, 20-24 mo) to either receive 30 min of 1.0 minimum alveolar concentration isoflurane or to a control group. Rats were subjected to coronary artery occlusion for 30 min followed by 2 h of reperfusion. A fluorescent probe for superoxide anion production (dihydroethidium, 1 mg) was administered in the absence of the isoflurane or just before isoflurane exposure in four additional groups. Myocardial infarct size and superoxide anion production were assessed using triphenyltetrazolium staining and epifluorescence microscopy, respectively. RESULTS: Isoflurane decreased myocardial infarct size of young rats (26.7% +/- 3.0%) compared with young controls (50.9% +/- 1.9%; P < 0.001), whereas isoflurane did not significantly affect myocardial infarct size of old rats (39.1% +/- 0.9%) compared with old controls (46.5% +/- 2.4%; P > 0.05). Isoflurane increased ROS levels in young rats (430.5 +/- 95.9 arbitrary units [AU]) compared with young controls (162.7 +/- 25.5 AU; P < 0.01). In contrast, no significant changes in ROS levels were observed in old animals (316.4 +/- 56.3 AU isoflurane versus 233.8 +/- 59.2 AU control). CONCLUSIONS: Reduction in the cardioprotective effects of isoflurane and attenuation of isoflurane-stimulated ROS production were observed in the senescent myocardium in vivo.