The authors investigated whether transfusion with stored erythrocytes would increase tissue injury, inflammation, oxidative stress, and mortality (adverse effects of transfusing stored erythrocytes) in a murine model of hemorrhagic shock. They tested whether the adverse effects associated with transfusing stored erythrocytes were exacerbated by endothelial dysfunction and ameliorated by inhaling nitric oxide.
The authors studied mice fed a high-fat diet (HFD-fed; to induce endothelial dysfunction) or a standard diet for 4–6 weeks. Mice were subjected to 90 min of hemorrhagic shock, followed by resuscitation with leukoreduced syngeneic erythrocytes stored less than 24 h (fresh erythrocytes) or stored for 2 weeks (stored erythrocytes).
In standard-diet–fed mice at 2 h after resuscitation, transfusion with stored erythrocytes increased tissue injury more than transfusion with fresh erythrocytes. The adverse effects of transfusing stored erythrocytes were more marked in HFD-fed mice and associated with increased lactate levels and short-term mortality. Compared with fresh erythrocytes, resuscitation with stored erythrocytes was associated with a reduction in P50, increased plasma hemoglobin levels, and increased indices of inflammation and oxidative stress, effects that were exacerbated in HFD-fed mice. Inhaled nitric oxide reduced tissue injury, lactate levels, and indices of inflammation and oxidative stress and improved short-term survival in HFD-fed mice resuscitated with stored erythrocytes.
Resuscitation with stored erythrocytes adversely impacts outcome in mice with hemorrhagic shock, an effect that is exacerbated in mice with endothelial dysfunction. Inhaled nitric oxide reduces tissue injury and improves short-term survival in HFD-fed mice resuscitated with stored erythrocytes.
Heart failure (HF) is a leading cause of hospitalization and mortality. Plasma B-type natriuretic peptide (BNP) is an established diagnostic and prognostic ambulatory HF biomarker. We hypothesized that increased perioperative BNP independently associates with HF hospitalization or HF death up to 5 yr after coronary artery bypass graft surgery.
The authors conducted a two-institution, prospective, observational study of 1,025 subjects (mean age = 64 ± 10 yr SD) undergoing isolated primary coronary artery bypass graft surgery with cardiopulmonary bypass. Plasma BNP was measured preoperatively and on postoperative days 1–5. The study outcome was hospitalization or death from HF, with HF events confirmed by reviewing hospital and death records. Cox proportional hazards analyses were performed with multivariable adjustments for clinical risk factors. Preoperative and peak postoperative BNP were added to the multivariable clinical model in order to assess additional predictive benefit.
One hundred five subjects experienced an HF event (median time to first event = 1.1 yr). Median follow-up for subjects who did not have an HF event = 4.2 yr. When individually added to the multivariable clinical model, higher preoperative and peak postoperative BNP concentrations each, independently associated with the HF outcome (log10 preoperative BNP hazard ratio = 1.93; 95% CI, 1.30–2.88; P = 0.001; log10 peak postoperative BNP hazard ratio = 3.38; 95% CI, 1.45–7.65; P = 0.003).
Increased perioperative BNP concentrations independently associate with HF hospitalization or HF death during the 5 yr after primary coronary artery bypass graft surgery. Clinical trials may be warranted to assess whether medical management focused on reducing preoperative and longitudinal postoperative BNP concentrations associates with decreased HF after coronary artery bypass graft surgery.
Anesthetics are widely used to induce unconsciousness, pain relief and immobility during surgery. It remains unclear whether the use of anesthetics has significant and long lasting effects on synapse development and plasticity in the brain. To address this question, we examined the formation and elimination of dendritic spines, postsynaptic sites of excitatory synapses, in the developing mouse cortex during and after anesthetics exposure.
Transgenic mice expressing yellow fluorescence protein in layer 5 pyramidal neurons were used in this study. Mice at 1 month of age underwent ketamine-xylazine and isoflurane anesthesia over a period of hours. The elimination and formation rates of dendritic spines and filopodia, the precursors of spines, were followed over hours to days in the primary somatosensory cortex using transcranial two-photon microscopy. 4–5 animals were examined under each experimental condition. Student's t-test and Mann-Whitney U-test were used to analyze the data.
Administration of either ketamine-xylazine or isoflurane rapidly altered dendritic filopodial dynamics but had no significant effects on spine dynamics. Ketamine-xylazine increased filopodial formation while isoflurane decreased filopodial elimination during 4 hours of anesthesia. Both effects were transient and disappeared within a day after the animals woke up.
Our studies suggest that exposure to anesthetics transiently affects the dynamics of dendritic filopodia but has no significant effect on dendritic spine development and plasticity in the cortex of 1-month-old mice.
Inflammation of the dorsal root ganglia (DRG) may contribute to low back pain, postherpetic neuralgia, and neuropathic pain. The mineralocorticoid receptor (MR) plays a pro-inflammatory role in many non-renal tissues, but its role in peripheral pain at the DRG level is not well studied.
Local inflammation of the L5 DRG with the immune activator zymosan rapidly leads to mechanical hypersensitivity and increased excitability of sensory neurons. Using this pain model, we applied the MR antagonist eplerenone locally to the inflamed DRG. Excitability of small diameter sensory neurons was examined in acute primary culture, using patch clamp techniques.
Local eplerenone significantly reduced the mechanical hypersensitivity and shortened its duration. The same dose was ineffective systemically. Immunohistochemical studies showed the MR was present in most neurons, and rapidly translocated to the nucleus 1 day after local DRG inflammation. Activation of satellite glia (defined by expression of glial fibrillary acidic protein) in the inflamed DRG was also reduced by local eplerenone. Increased excitability of small diameter sensory neurons 1 day after inflammation could be observed in vitro. Eplerenone applied in vitro (8 – 12 hours) could reverse this increased excitability. Eplerenone had no effect in neurons isolated from normal, uninflamed DRG. The MR agonist aldosterone (10 nM) applied in vitro increased excitability of neurons isolated from normal DRG.
The MR may have a pro-nociceptive role in the DRG. Some of its effects may be mediated by neuronal MR. The MR may represent a novel therapeutic target in some pain syndromes.
Work suggests the amnesia of Dexmedetomidine (an α2-adrenergic agonist) is caused by a failure of information to be encoded into long-term memory and that dexmedetomidine might differentially affect memory for emotionally arousing material. We investigated these issues in humans using event-related neuroimaging to reveal alterations in brain activity and subsequent memory effects associated with drug exposure.
Forty-eight healthy volunteers received a computer-controlled infusion of either placebo or low-dose dexmedetomidine (target = 0.15ng/ml plasma) during neuroimaging while they viewed and rated 80 emotionally arousing (e.g., graphic war wound) and 80 nonarousing neutral (e.g., cup) pictures for emotional arousal content. Long-term picture memory was tested 4 days later without neuroimaging. Imaging data were analyzed for drug effects, emotional processing differences and memory related changes with statistical parametric mapping-8.
Dexmedetomidine impaired overall (mean ± SEM) picture memory (Placebo: 0.58 ± 0.03 vs. a dexmedetomidine: 0.45 ± 0.03, p = 0.001), but did not differentially modulate memory as a function of item arousal. Arousing pictures were better remembered for both groups. Dexmedetomidine had regionally heterogeneous effects on brain activity, primarily decreasing it in the cortex while increasing it in thalamic and posterior hippocampal regions. Nevertheless, a single subsequent memory effect for item memory common to both groups was identified only in the left hippocampus/amygdala. Much of this effect was found to be larger for the placebo than dexmedetomidine group.
Dexmedetomidine impaired long-term picture memory, but did not disproportionately block memory for emotionally arousing items. The memory impairment on dexmedetomidine corresponds with a weakened hippocampal subsequent memory effect.
Propofol in the early postnatal period has been shown to cause brain cell death. One proposed mechanism for cognitive dysfunction after anesthesia is alteration of neural stem cell function and neurogenesis. We examined the effect of propofol on neural precursor or stem cells (NPCs) grown in vitro.
Hippocampal derived NPCs from postnatal day 2 rats were exposed to propofol or to Diprivan. NPCs were then analyzed for bromodeoxyuridine incorporation to measure proliferation. Cell death was measured by lactate dehydrogenase release. Immunocytochemistry was used to evaluate the expression of neuronal and glial markers in differentiating NPCs exposed to propofol.
Propofol dose dependently increases the release of lactate dehydrogenase from NPCs under both proliferating and differentiating conditions at supraclinical concentrations (> 7.1μM). Both Diprivan and propofol had the same effect on NPCs. Propofol mediated release of lactate dehydrogenase is not inhibited by blocking the γ-aminobutyric acid type A receptor or extracellular calcium influx and is not mediated by caspase-3/7. Direct γ-aminobutyric acid type A receptor activation did not have the same effect. In differentiating NPCs 6 h of propofol at 2.1 μM increased the number neurons but not glial cells 4 days later. Increased neuronal differentiation was not blocked by Bicuculline.
Only supraclinical concentrations of propofol or Diprivan kill NPCs in culture by a non-γ-aminobutyric acid type A, noncaspase 3 mechanism. Clinically relevant doses of propofol increase neuronal fate choice by a non-γ-aminobutyric acid type A mechanism.
Methoxycarbonyl etomidate is the prototypical soft etomidate analog. Because it has relatively low potency and is extremely rapidly metabolized, large quantities must be infused to maintain hypnosis. Consequently with prolonged infusion, metabolite reaches sufficient concentrations to delay recovery. Dimethyl-methoxycarbonyl metomidate (DMMM) and cyclopropyl-methoxycarbonyl metomidate (CPMM) are methoxycarbonyl etomidate analogs with higher potencies and slower clearance. Because of these properties, we hypothesized that dosing would be lower and electroencephalographic and hypnotic recoveries would be faster - and less context-sensitive - with DMMM or CPMM versus methoxycarbonyl etomidate or etomidate.
Etomidate, DMMM, and CPMM where infused into rats (n=6 per group) for either 5 min or 120 min. After infusion termination, electroencephalographic and hypnotic recovery times were measured. The immobilizing ED50 infusion rates were determined using a tail clamp assay.
Upon terminating 5-minute infusions, electroencephalographic and hypnotic recovery times were not different among hypnotics. However upon terminating 120-minute infusions, recovery times varied significantly with respective values (mean ± SD) 48 ± 13 min and 31 ± 6.5 min (etomidate), 17 ± 7.0 min and 14 ± 3.4 min (DMMM), and 4.5 ± 1.1 min and 4.2 ± 1.6 min (CPMM). The immobilizing ED50 infusion rates were (mean ± SD) 0.19 ± 0.03 mg/kg·min (etomidate), 0.60 ± 0.12 mg/kg·min (DMMM), and 0.89 ± 0.18 mg/kg·min (CPMM).
Electroencephalographic and hypnotic recoveries following prolonged infusions of DMMM and CPMM are faster than those following methoxycarbonyl etomidate or etomidate. In the case of CPMM infusion, recovery times are 4 min and context-insensitive.
Methoxycarbonyl etomidate is the prototypical ultra-rapidly metabolized etomidate analog. Initial studies suggest that it may be too short acting for many clinical uses. We hypothesized that its duration of action could be lengthened and clinical utility broadened by incorporating specific aliphatic groups into the molecule to sterically protect its ester moiety from esterase-catalyzed hydrolysis. To test this hypothesis, we developed a series of methoxycarbonyl etomidate analogs (spacer-linked etomidate esters) containing various aliphatic protecting groups and spacer lengths.
Spacer-linked etomidate esters were synthesized and their hypnotic potencies and durations of action following bolus administration were measured in rats using a loss of righting reflexes assay. Octanol:water partition coefficients and metabolic half-lives in pooled rat blood were determined chromatographically.
All spacer-linked etomidate esters produced hypnosis rapidly and in a dose-dependent manner. ED50s for loss of righting reflexes ranged from 0.69 ± 0.04 mg/kg for cyclopropyl-methoxycarbonyl metomidate to 11.1 ± 0.8 mg/kg for methoxycarbonyl metomidate. The slope of a plot of the duration of loss of righting reflexes versus the logarithm of the dose ranged 12-fold among spacer-linked etomidate esters, implying widely varying brain clearance rates. The in vitro metabolic half-lives of these compounds in rat blood varied by more than two orders of magnitude and were diastereometrically-selective.
We created 13 new analogs of methoxycarbonyl etomidate and identified two that have significantly higher potency and potentially address methoxycarbonyl etomidate’s too brief duration of action. This work may provide a blueprint for optimizing the pharmacological properties of other soft drugs.
Multiple lines of evidence suggest that the adrenergic system can modulate sensitivity to anesthetic-induced immobility and anesthetic-induced hypnosis as well. However, several considerations prevent the conclusion that the endogenous adrenergic ligands norepinephrine and epinephrine alter anesthetic sensitivity.
Using dopamine β-hydroxylase (Dbh−/−) mice genetically engineered to lack the adrenergic ligands and their siblings with normal adrenergic levels, we test the contribution of the adrenergic ligands upon volatile anesthetic induction and emergence. Moreover, we investigate the effects of intravenous dexmedetomidine in adrenergic-deficient mice and their siblings using both righting reflex and processed electroencephalographic measures of anesthetic hypnosis.
We demonstrate that the loss of norepinephrine and epinephrine and not other neuromodulators copackaged in adrenergic neurons is sufficient to cause hypersensitivity to induction of volatile anesthesia. However, the most profound effect of adrenergic deficiency is retarding emergence from anesthesia, which takes two to three times as long in Dbh−/− mice for sevoflurane, isoflurane, and halothane. Having shown that Dbh−/− mice are hypersensitive to volatile anesthetics, we further demonstrate that their hypnotic hypersensitivity persists at multiple doses of dexmedetomidine. Dbh−/− mice exhibit up to 67% shorter latencies to loss of righting reflex and up to 545% longer durations of dexmedetomidine-induced general anesthesia. Central rescue of adrenergic signaling restores control-like dexmedetomidine sensitivity. A novel continuous electroencephalographic analysis illustrates that the longer duration of dexmedetomidine-induced hypnosis is not due to a motor confound, but occurs because of impaired anesthetic emergence.
Adrenergic signaling is essential for normal emergence from general anesthesia. Dexmedetomidine-induced general anesthesia does not depend upon inhibition of adrenergic neurotransmission.
We sought whether subjects with pathophysiological conditions that are characterized by elevated levels of aldosterone have increased susceptibility to the side effects of neonatal anesthesia with sevoflurane.
Postnatal day 4–20 (P4–P20) rats were exposed to 6% and 2.1% sevoflurane for 3 min and 60–360 min, respectively. Exogenous aldosterone was administered to imitate pathophysiological conditions with elevated levels of aldosterone.
Six hours of anesthesia with sevoflurane on P4–P5 resulted in more than 30-fold increase in serum levels of aldosterone (7.02 ± 1.61 ng/dl vs. 263.75 ± 22.31 ng/dl, mean ± SE, n = 5–6) and reduced prepulse inhibition of the acoustic startle response (F(2,37)= 5.66, P<0.001). Administration of exogenous aldosterone during anesthesia with sevoflurane further enhanced seizure-like electroencephalogram patterns in neonatal rats (48.25±15.91 s vs. 222.00 ± 53.87 s, mean± SE, n = 4), but did not affect electroencephalographic activity in older rats. Exogenous aldosterone increased activation of caspase-3 (F(3,28)=11.02, P<0.001) and disruption of prepulse inhibition of startle (F(3,46)=6.36; P= 0.001) caused by sevoflurane. Intracerebral administration of oxytocin receptor agonists resulted in depressed seizure-like electroencephalogram patterns (F(2,17)=6.37, P=0.009), reduced activation of caspase-3 ((t(11) = 2.83, P = 0.016) and disruption of PPI of startle (t(7) = −2.9; P = 0.023) caused by sevoflurane.
These results suggest that adverse developmental effects of neonatal anesthesia with sevoflurane may involve both central and peripheral actions of the anesthetic. Subjects with elevated levels of aldosterone may be more vulnerable, while intracerebral oxytocin receptor agonists may be neuroprotective.
Volatile anesthetics (VAs) alter the function of key central nervous system proteins but it is not clear which, if any, of these targets mediates the immobility produced by VAs in the face of noxious stimulation. A leading candidate is the glycine receptor, a ligand-gated ion channel important for spinal physiology. VAs variously enhance such function, and blockade of spinal GlyRs with strychnine affects the minimal alveolar concentration (an anesthetic EC50) in proportion to the degree of enhancement.
We produced single amino acid mutations into the glycine receptorα1 subunit that increased (M287L, third transmembrane region) or decreased (Q266I, second transmembrane region) sensitivity to isoflurane in recombinant receptors, and introduced such receptors into mice. The resulting knockin mice presented impaired glycinergic transmission, but heterozygous animals survived to adulthood, and we determined the effect of isoflurane on glycine-evoked responses of brain stem neurons from the knockin mice, and the minimal alveolar concentration for isoflurane and other VAs in the immature and mature knockin mice.
Studies of glycine-evoked currents in brain stem neurons from knock-in mice confirmed the changes seen with recombinant receptors. No increases in the minimal alveolar concentration were found in knockin mice, but the minimal alveolar concentration for isoflurane and enflurane (but not halothane) decreased in 2-week-old Q266I mice. This change is opposite to the one expected for a mutation that decreases the sensitivity to volatile anesthetics.
Taken together, these results indicate that glycine receptors containing the α1 subunit are not likely to be crucial for the action of isoflurane and other VAs.
Intraoperative awareness with explicit recall occurs in approximately 0.15% of all surgical cases. Efficacy trials based on the Bispectral Index™ (BIS) monitor and anesthetic concentrations have focused on high-risk patients, but there are no effectiveness data applicable to an unselected surgical population.
We conducted a randomized controlled trial of unselected surgical patients at three hospitals of a tertiary academic medical center. Surgical cases were randomized to alerting algorithms based on either BIS values or anesthetic concentrations. The primary outcome was the incidence of definite intraoperative awareness; prespecified secondary outcomes included postanesthetic recovery variables.
The study was terminated due to futility. At interim analysis the incidence of definite awareness was 0.12% (11/9376) (95% CI 0.07 to 0.21%) in the anesthetic concentration group and 0.08% (8/9460) (95% CI 0.04 to 0.16%) in the BIS group (p = 0.48). There was no significant difference between the two groups in terms of meeting criteria for recovery room discharge or incidence of nausea and vomiting. By post hoc secondary analysis, the BIS protocol was associated with a 4.7-fold reduction in definite or possible awareness events compared to a cohort receiving no intervention (p = 0.001; 95% CI 1.7 to 13.1).
This negative trial could not detect a difference in the incidence of definite awareness or recovery variables between monitoring protocols based on either BIS values or anesthetic concentration. By post hoc analysis, a protocol based on BIS monitoring reduced the incidence of definite or possible intraoperative awareness compared to routine care.
Anesthetic preconditioning protects cardiomyocytes from oxidative stress-induced injury, but it is ineffective in patients with diabetes mellitus. To address the role of hyperglycemia in the inability of diabetic individuals to be preconditioned, we used human cardiomyocytes differentiated from induced pluripotent stem cells generated from patients with or without type 2 diabetes mellitus (DM-iPSC- and N-iPSC-CMs, respectively) to investigate the efficacy of preconditioning in varying glucose conditions (5, 11, and 25 mM).
Induced pluripotent stem cells were induced to generate cardiomyocytes by directed differentiation. For subsequent studies, cardiomyocytes were identified by genetic labeling with enhanced green fluorescent protein driven by a cardiac-specific promoter. Cell viability was analyzed by lactate dehydrogenase assay. Confocal microscopy was utilized to measure opening of the mitochondrial permeability transition pore and the mitochondrial adenosine-5′-triphosphate-sensitive potassium channels.
Isoflurane (0.5 mM) preconditioning protected N-iPSC- and DM-iPSC-CMs from oxidative stress-induced lactate dehydrogenase release and mitochondrial permeability transition pore opening in 5 mM and 11 mM glucose. Isoflurane triggered mitochondrial adenosine-5′-triphosphate-sensitive potassium channel opening in N-iPSC-CMs in 5 mM and 11 mM glucose and in DM-iPSC-CMs in 5 mM glucose. 25 mM glucose disrupts anesthetic preconditioning-mediated protection in DM-iPSC- and N-iPSC-CMs.
The opening of mitochondrial adenosine-5′-triphosphate-sensitive potassium channels are disrupted in DM-iPSC-CMs in 11 mM and 25 mM glucose and in N-iPSC-CMs in 25 mM glucose. Cardiomyocytes derived from healthy donors and patients with a specific disease, such as diabetes in this study, open possibilities in studying genotype and phenotype related pathologies in a human relevant model.
Ischemia reperfusion (I/R) injury involves sterile inflammation and is commonly associated with diverse clinical situations such as hemorrhage followed by resuscitation, transient embolic events, and organ transplantation. I/R injury can induce lung dysfunction whether the I/R occurs in the lung itself or in a remote organ. Recently, evidence has emerged that receptors and pathways of the innate immune system are involved in recognizing sterile inflammation and overlap considerably with those involved in recognition and response to pathogens.
We used a mouse surgical model of transient unilateral left pulmonary artery occlusion without bronchial involvement to create ventilated lung I/R injury. Additionally, we mimicked nutritional I/R injury in vitro by transiently depriving cells of all nutrients.
Compared with sham-operated mice, mice subjected to ventilated lung I/R injury had upregulated lung expression of inflammatory mediator messenger RNA for IL-1β, IL-6, and CXCL1 and 2, paralleled by histologic evidence of lung neutrophil recruitment, and increased plasma levels of IL-1β, IL-6 and HMGB1 proteins. This inflammatory response to I/R required toll-like receptor-4. Furthermore, we demonstrated in vitro cooperativity and cross-talk between macrophages and endothelial cells, resulting in augmented inflammatory responses to I/R. Remarkably, we found that selective depletion of alveolar macrophages rendered mice resistant to ventilated lung I/R injury.
Our data reveal that alveolar macrophages and the pattern recognition receptor, toll-like receptor-4 are required for the generation of the early inflammatory response to lung I/R injury.
The development of arterial spin labeling methods, has allowed measuring regional cerebral blood flow (rCBF) quantitatively and to show the pattern of cerebral activity associated with any state such as a sustained pain state or changes due to a neurotropic drug.
We studied the differential effects of three pain conditions in ten healthy subjects on a 3T scanner during resting baseline, heat, cold and ischemic pain using continuous arterial spin labeling.
Cold pain showed the greatest absolute rCBF increases in left anterior cingulate cortex, left amygdala, left angular gyrus, and Brodmann Area 6, and a significant rCBF decrease in the cerebellum. Changes in rCBF were characteristic of the type of pain condition: cold and heat pain showed increases, while the ischemic condition showed a reduction in mean absolute gray matter flow compared to rest. An association of subjects’ pain tolerance and cerebral blood flow was noted.
The observation that quantitative rCBF changes are characteristic of the pain task employed and that there is a consistent rCBF change in Brodman area 6, an area responsible for the integration of a motor response to pain, should provide extremely useful information in the quest to develop an imaging biomarker of pain. Conceivably, response in BA6 may serve as an objective measure of analgesic efficacy.
We have recently shown that post-ischemic administration of intralipid protects the heart against ischemia/reperfusion injury. Here we compared the cardioprotective effects of intralipid with cyclosporine-A, a potent inhibitor of the mitochondrial permeability transition pore opening.
In-vivo rat hearts or isolated Langendorff-perfused mouse hearts were subjected to ischemia followed by reperfusion with Intralipid (0.5%, 1% and 2% ex-vivo and 20% in-vivo), cyclosporine-A (0.2μM, 0.8μM and 1.5μM ex-vivo and 10mg/kg in-vivo) or vehicle. The hemodynamic function, infarct size, calcium retention capacity, mitochodrial superoxide production and phosphorylation levels of Akt/GSK-3β were measured. The values are mean±SEM.
Administration of intralipid at reperfusion significantly reduced myocardial infarct size compared with cyclosporine-A in-vivo ((infarct size/area at risk)%: 22.9±2.5% vs. 35.2±3.5%; p=0.030, n=7/group). Postischemic administration of intralipid at its optimal dose (1%) was more effective than cyclosporine-A (0.8μM) in protecting the ex-vivo heart against ischemia/reperfusion injury as the rate pressure product at the end of reperfusion was significantly higher (mmHg*beats/min:12740±675(n=7) vs. 9203±10781(n=5), p=0.024), and the infarct size was markedly smaller (17.3±2.9(n=7) vs. 29.2±2.7(n=5), p=0.014). Intralipid was as efficient as cyclosporine-A in inhibiting the mPTP opening (calcium retention capacity=280±8.2 vs. 260.3±2.9nmol/mg-mitochondria-protein in cyclosporine-A, p=0.454, n=6) and in reducing cardiac mitochondrial superoxide production. Unlike intralipid, which increased phosphorlyation of Akt (6-fold) and GSK-3β (5-fold), cyclosporine-A had no effect on the activation of these pro-survival kinases.
Although intralipid inhibits the opening of the mitochondrial permeability transition pore as efficiently as cyclosporine-A, intralipid is more effective in reducing the infarct size and improving the cardiac functional recovery.
Neutrophils are one of the predominant immune cells migrating to surgical wound edges initially. They produce mediators both associated with supporting (interleukin [IL]-1β, C5a) and reducing (opioid peptides) pain. Studies demonstrate neutrophil depletion/blockade reduces nociceptive sensitization after nerve injury and carrageenan administration, but enhance sensitization in Complete Freund's Adjuvant inflammation. This research identifies the contribution of infiltrating neutrophils to incisional pain and inflammation.
Antibody-mediated Gr1+ neutrophil depletion preceded hind paw incisions. Sensitization to mechanical and thermal stimuli, effects on edema and local levels of IL-1β and C5a were measured. Local effects of C5a or IL-1 receptor antagonists, PMX53 and Anakinra on sensitization after neutrophil depletion were examined. Groups of 4–8 mice were used.
Anti-Gr1 antibody depleted >90% of circulating and infiltrating skin neutrophils after incision. Neutrophil depletion did not change magnitude or duration of mechanical hypersensitivity in incised mice. However, paw edema was significantly reduced and heat hypersensitivity was slightly increased in depleted animals. In depleted animals IL-1β levels were half of controls 24 h after incision, while C5a levels were elevated in both. Prominent IL-1β immunohistochemical staining of epidermis was seen in both groups. PMX53 and Anakinra reduced incisional mechanical and heat nociceptive sensitization to the same extent regardless of neutrophil depletion.
Neutrophil-derived IL-1β and C5a do not appear to contribute critically to peri-incisional nociceptive signaling. Other sources of mediators like epidermal cells may need to be considered. Controlling inflammatory activation of resident cells in epidermis/deeper structures may show therapeutic efficacy in reducing pain from surgical incisions.
After incision keratinocytes in the epidermis become activated to produce a range of pain-related mediators. microRNA 203 (miR-203) is known to be involved in keratinocyte growth, differentiation and skin inflammation. We hypothesized that one or more of these mediators might be under the control of miR-203.
The expression of miR-203 and its target gene, phospholipase A2 activating protein (PLAA) were examined after hindpaw incision in mice. We investigated the local effect of intraplantar PLAA peptide injection in normal mice and the effects of a selective secretory phospholipase A2 inhibitor (HK064) on PLAA or incision-induced mechanical allodynia. Last, we investigated the role of substance P signaling in regulating miR-203 and PLAA expression in vitro and in vivo.
Levels of miR-203 were strongly down-regulated in keratinocytes after incision. Informatics-based approaches identified PLAA as a likely candidate for regulation by miR-203. PLAA caused mechanical allodynia and conditioned place aversion but not thermal sensitization. HK064 reduced mechanical allodynia after incision and after intraplantar injection of PLAA. Using preprotachykinin gene knockout mice or with neurokinin-1 selective antagonist LY303870 treatment, we observed that substance P-mediated signaling was also required for miR-203 and PLAA regulation after incision. Finally, using the rat epidermal keratinocyte cell line we observed that a miR-203 mimic molecule could block the substance P induced increase in PLAA expression observed under control conditions.
miR-203 may regulate expression of the novel nociceptive mediator PLAA after incision. Furthermore, the regulation of miR-203 and PLAA levels is reliant upon intact substance P signaling.
Retrospective analysis of patients undergoing cancer surgery suggests the use of regional anesthesia may reduce cancer recurrence and improve survival. Amide-linked local anesthetics have anti-inflammatory properties, although the mechanism of action in this regard is unclear. As inflammatory processes involving Src tyrosine protein kinase and intercellular adhesion molecule-1 are important in tumor growth and metastasis, we hypothesized that amide-linked local anesthetics may inhibit inflammatory Src-signaling involved in migration of adenocarcinoma cells.
NCI-H838 lung cancer cells were incubated with Tumor Necrosis Factor-α in absence/presence of ropivacaine, lidocaine, or chloroprocaine (1nM-100μM). Cell migration and total cell lysate Src-activation and Intercellular Adhesion Molecule-1 phosphorylation were assessed. The role of voltage-gated sodium-channels in the mechanism of local anesthetic effects was also evaluated.
Ropivacaine treatment (100μM) of H838 cells for 20 minutes decreased basal Src activity by 62% (p=0.003), and both ropivacaine and lidocaine co-administered with Tumor Necrosis Factor-α statistically significantly decreased Src-activation and Intercellular Adhesion Molecule-1 phosphorylation, whereas chloroprocaine had no such effect. Migration of these cells at 4 hours was inhibited by 26% (p=0.005) in presence of 1μM ropivacaine and 21% by 1μM lidocaine (p=0.004). These effects of ropivacaine and lidocaine were independent of voltage-gated sodium-channel inhibition.
This study indicates that amide-, but not ester-linked local anesthetics may provide beneficial anti-metastatic effects. The observed inhibition of NCI-H838 cell migration by lidocaine and ropivacaine was associated with the inhibition of Tumor Necrosis Factor-α-induced Src-activation and Intercellular Adhesion Molecule-1 phosphorylation, providing the first evidence of a molecular mechanism which appears to be independent of their known role as sodium-channel blockers.