Mouse models of sickle cell disease: Imperfect and yet very informative.

The root cause of sickle cell disease (SCD) has been known for nearly a century, however, few therapies to treat the disease are available. Over several decades of work, with advances in gene editing technology and after several iterations of mice with differing genotype/phenotype relationships, researchers have developed humanized SCD mouse models. However, while a large body of preclinical studies has led to huge gains in basic science knowledge about SCD in mice, this knowledge has not led to the development of effective therapies to treat SCD-related complications in humans, thus leading to frustration with the paucity of translational progress in the SCD field. The use of mouse models to study human diseases is based on the genetic and phenotypic similarities between mouse and humans (face validity). The Berkeley and Townes SCD mice express only human globin chains and no mouse hemoglobin. With this genetic composition, these models present many phenotypic similarities, but also significant discrepancies that should be considered when interpreting preclinical studies results. Reviewing genetic and phenotypic similarities and discrepancies and examining studies that have translated to humans and those that have not, offer a better perspective of construct, face, and predictive validities of humanized SCD mouse models.

Calcium flux alterations in erythrocytes from sickle cell mice: The relevance of mean corpuscular volume.

Red blood cells (RBC) from patients with sickle cell disease (SCD) have elevated calcium levels at baseline, which are further elevated upon deoxygenation. Here we examined baseline calcium levels and calcium flux in RBCs from a mouse model of SCD mice. We found that akin to humans with SCD, sickle (HbSS) Townes mice, have higher baseline levels and increased calcium flux in RBCs compared to control (HbAA) animals. As HbSS mice, unlike humans with SCD, have high mean corpuscular volume compared with HbAA, we highlight the importance of adjusting biochemical results to number of RBCs rather than hematocrit during the analysis and interpretation of the results. Our findings add to the face validity of humanized sickle cell mice and support its use for studies of RBC calcium flux in SCD.

Circulating mitochondrial DNA is a proinflammatory DAMP in sickle cell disease.

The pathophysiology of sickle cell disease (SCD) is driven by chronic inflammation fueled by damage associated molecular patterns (DAMPs). We show that elevated cell-free DNA (cfDNA) in patients with SCD is not just a prognostic biomarker, it also contributes to the pathological inflammation. Within the elevated cfDNA, patients with SCD had a significantly higher ratio of cell-free mitochondrial DNA (cf-mtDNA)/cell-free nuclear DNA compared with healthy controls. Additionally, mitochondrial DNA in patient samples showed significantly disproportionately increased hypomethylation compared with healthy controls, and it was increased further in crises compared with steady-state. Using flow cytometry, structured illumination microscopy, and electron microscopy, we showed that circulating SCD red blood cells abnormally retained their mitochondria and, thus, are likely to be the source of the elevated cf-mtDNA in patients with SCD. Patient plasma containing high levels of cf-mtDNA triggered the formation of neutrophil extracellular traps (NETs) that was substantially reduced by inhibition of TANK-binding kinase 1, implicating activation of the cGAS-STING pathway. cf-mtDNA is an erythrocytic DAMP, highlighting an underappreciated role for mitochondria in sickle pathology. These trials were registered at www.clinicaltrials.gov as #NCT00081523, #NCT03049475, and #NCT00047996.

Nitrite decreases sickle hemoglobin polymerization in vitro independently of methemoglobin formation.

The root cause of sickle cell disease (SCD) is the polymerization of sickle hemoglobin (HbS) leading to sickling of red blood cells (RBC). Earlier studies showed that in patients with SCD, high-dose nitrite inhibited sickling, an effect originally attributed to HbS oxidation to methemoglobin-S even though the anti-sickling effect did not correlate with methemoglobin-S levels. Here, we examined the effects of nitrite on HbS polymerization and on methemoglobin formation in a SCD mouse model. In vitro, at concentrations higher than physiologic (>1 µM), nitrite increased the delay time for polymerization of deoxygenated HbS independently of methemoglobin-S formation, which only occurred at much higher concentrations (>300 µM). In vitro, higher nitrite concentrations oxidized 100% of normal hemoglobin A (HbA), but only 70% of HbS. Dimethyl adipimidate, an anti-polymerization agent, increased the fraction of HbS oxidized by nitrite to 82%, suggesting that polymerized HbS partially contributed to the oxidation-resistant fraction of HbS. At low concentrations (10 µM-1 mM), nitrite did not increase the formation of reactive oxygen species but at high concentrations (10 mM) it decreased sickle RBC viability. In SCD mice, 4-week administration of nitrite yielded no significant changes in methemoglobin or nitrite levels in plasma and RBC, however, it further increased leukocytosis. Overall, these data suggest that nitrite at supra-physiologic concentrations has anti-polymerization properties in vitro and that leukocytosis is a potential nitrite toxicity in vivo. Therefore, to determine whether the anti-polymerization effect of nitrite observed in vitro underlies the decreases in sickling observed in patients with SCD, administration of higher nitrite doses is required.

Mitapivat increases ATP and decreases oxidative stress and erythrocyte mitochondria retention in a SCD mouse model.

Polymerization of deoxygenated sickle hemoglobin (HbS) leads to erythrocyte sickling. Enhancing activity of the erythrocyte glycolytic pathway has anti-sickling potential as this reduces 2,3-diphosphoglycerate (2,3-DPG) and increases ATP, factors that decrease HbS polymerization and improve erythrocyte membrane integrity. These factors can be modulated by mitapivat, which activates erythrocyte pyruvate kinase (PKR) and improves sickling kinetics in SCD patients. We investigated mechanisms by which mitapivat may impact SCD by examining its effects in the Townes SCD mouse model. Control (HbAA) and sickle (HbSS) mice were treated with mitapivat or vehicle. Surprisingly, HbSS had higher PKR protein, higher ATP, and lower 2,3-DPG levels, compared to HbAA mice, in contrast with humans with SCD, in whom 2,3-DPG is elevated compared to healthy subjects. Despite our inability to investigate 2,3-DPG-mediated sickling and hemoglobin effects, mitapivat yielded potential benefits in HbSS mice. Mitapivat further increased ATP without significantly changing 2,3-DPG or hemoglobin levels, and decreased levels of leukocytosis, erythrocyte oxidative stress, and the percentage of erythrocytes that retained mitochondria in HbSS mice. These data suggest that, even though Townes HbSS mice have increased PKR activity, further activation of PKR with mitapivat yields potentially beneficial effects that are independent of changes in sickling or hemoglobin levels.

NLRP3 inflammasome and bruton tyrosine kinase inhibition interferes with upregulated platelet aggregation and in vitro thrombus formation in sickle cell mice.

The nucleotide-binding domain leucine-rich repeat containing protein 3 (NLRP3) inflammasome is a critical inflammatory mechanism identified in platelets, which controls platelet activation and aggregation. We have recently shown that the platelet NLRP3 inflammasome is upregulated in sickle cell disease (SCD), which is mediated by Bruton tyrosine kinase (BTK). Here, we investigated the effect of pharmacological inhibition of NLRP3 and BTK on platelet aggregation and the formation of in vitro thrombi in Townes SCD mice. Mice were injected for 4 weeks with the NLRP3 inhibitor MCC950, the BTK inhibitor ibrutinib or vehicle control. NLRP3 activity, as monitored by caspase-1 activation, was upregulated in platelets from SCD mice, which was dependent on BTK. Large areas of platelet aggregates detected in the liver of SCD mice were decreased when mice were treated with MCC950 or ibrutinib. Moreover, platelet aggregation and in vitro thrombus formation were upregulated in SCD mice and were inhibited when mice were subjected to pharmacological inhibition of NLRP3 and BTK. Targeting the NLRP3 inflammasome might be a novel approach for antiplatelet therapy in SCD.

Sickle cell disease mice have cerebral oxidative stress and vascular and white matter abnormalities.

Strokes are feared complications of sickle cell disease (SCD) and yield significant neurologic and neurocognitive deficits. However, even without detectable strokes, SCD patients have significant neurocognitive deficits in domains of learning and memory, processing speed and executive function. In these cases, mechanisms unrelated to major cerebrovascular abnormalities likely underlie these deficits. While oxidative stress and stress-related signaling pathways play a role in SCD pathophysiology, their role in cerebral injury remains unknown. We have shown that Townes and BERK SCD mice, while not having strokes, recapitulate neurocognitive deficits reported in humans. We hypothesized that cognitive deficits in SCD mice are associated with cerebral oxidative stress. We showed that SCD mice have increased levels of reactive oxygen species, protein carbonylation, and lipid peroxidation in hippocampus and cortex, thus suggesting increased cerebral oxidative stress. Further, cerebral oxidative stress was associated with caspase-3 activity alterations and vascular endothelial abnormalities, white matter changes, and disruption of the blood brain barrier, similar to those reported after ischemic/oxidative injury. Additionally, after repeated hypoxia/reoxygenation exposure, homozygous Townes had enhanced microglia activation. Our findings indicate that oxidative stress and stress-induced tissue damage is increased in susceptible brain regions, which may, in turn, contribute to neurocognitive deficits in SCD mice.

Regional rates of brain protein synthesis are unaltered in dexmedetomidine sedated young men with fragile X syndrome: A L-[1-11C]leucine PET study.

Fragile X syndrome (FXS) is the most common inherited cause of intellectual disability. Fragile X mental retardation protein (FMRP), a putative translation suppressor, is absent or significantly reduced in FXS. One prevailing hypothesis is that rates of protein synthesis are increased by the absence of this regulatory protein. In accord with this hypothesis, we have previously reported increased rates of cerebral protein synthesis (rCPS) in the Fmr1 knockout mouse model of FXS and others have reported similar effects in hippocampal slices. To address the hypothesis in human subjects, we applied the L[1-11C]leucine PET method to measure rCPS in adults with FXS and healthy controls. All subjects were males between the ages of 18 and 24 years and free of psychotropic medication. As most fragile X participants were not able to undergo the PET study awake, we used dexmedetomidine for sedation during the imaging studies. We found no differences between rCPS measured during dexmedetomidine-sedation and the awake state in ten healthy controls. In the comparison of rCPS in dexmedetomidine-sedated fragile X participants (n = 9) and healthy controls (n = 14) we found no statistically significant differences. Our results from in vivo measurements in human brain do not support the hypothesis that rCPS are elevated due to the absence of FMRP. This hypothesis is based on findings in animal models and in vitro measurements in human peripheral cells. The absence of a translation suppressor may produce a more complex response in pathways regulating translation than previously thought. We may need to revise our working hypotheses regarding FXS and our thinking about potential therapeutics.

Sickle cell disease subjects and mouse models have elevated nitrite and cGMP levels in blood compartments.

The hypothesis of decreased nitric oxide (NO) bioavailability in sickle cell disease (SCD) proposes that multiple factors leading to decreased NO production and increased consumption contributes to vaso-occlusion, pulmonary hypertension, and pain. The anion nitrite is central to NO physiology as it is an end product of NO metabolism and serves as a reservoir for NO formation. However, there is little data on nitrite levels in SCD patients and its relationship to pain phenotype. We measured nitrite in SCD subjects and examined its relationship to SCD pain. In SCD subjects, median whole blood, red blood cell and plasma nitrite levels were higher than in controls, and were not associated with pain burden. Similarly, Townes and BERK homozygous SCD mice had elevated blood nitrite. Additionally, in red blood cells and plasma from SCD subjects and in blood and kidney from Townes homozygous mice, levels of cyclic guanosine monophosphate (cGMP) were higher compared to controls. In vitro, hemoglobin concentration, rather than sickle hemoglobin, was responsible for nitrite metabolism rate. In vivo, inhibition of NO synthases and xanthine oxidoreductase decreased nitrite levels in homozygotes but not in control mice. Long-term nitrite treatment in SCD mice further elevated blood nitrite and cGMP, worsened anemia, decreased platelets, and did not change pain response. These data suggest that SCD in humans and animals is associated with increased nitrite/NO availability, which is unrelated to pain phenotype. These findings might explain why multiple clinical trials aimed at increasing NO availability in SCD patients failed to improve pain outcomes.

RON kinase inhibition reduces renal endothelial injury in sickle cell disease mice.

Sickle cell disease patients are at increased risk of developing a chronic kidney disease. Endothelial dysfunction and inflammation associated with hemolysis lead to vasculopathy and contribute to the development of renal disease. Here we used a Townes sickle cell disease mouse model to examine renal endothelial injury. Renal disease in Townes mice was associated with glomerular hypertrophy, capillary dilation and congestion, and significant endothelial injury. We also detected substantial renal macrophage infiltration, and accumulation of macrophage stimulating protein 1 in glomerular capillary. Treatment of human cultured macrophages with hemin or red blood cell lysates significantly increased expression of macrophage membrane-associated protease that might cleave and activate circulating macrophage stimulating protein 1 precursor. Macrophage stimulating protein 1 binds to and activates RON kinase, a cell surface receptor tyrosine kinase. In cultured human renal glomerular endothelial cells, macrophage stimulating protein 1 induced RON downstream signaling, resulting in increased phosphorylation of ERK and AKT kinases, expression of Von Willebrand factor, increased cell motility, and re-organization of F-actin. Specificity of macrophage stimulating protein 1 function was confirmed by treatment with RON kinase inhibitor BMS-777607 that significantly reduced downstream signaling. Moreover, treatment of sickle cell mice with BMS-777607 significantly reduced glomerular hypertrophy, capillary dilation and congestion, and endothelial injury. Taken together, our findings demonstrated that RON kinase is involved in the induction of renal endothelial injury in sickle cell mice. Inhibition of RON kinase activation may provide a novel approach for prevention of the development of renal disease in sickle cell disease.

The role of nitrite in muscle function, susceptibility to contraction injury, and fatigability in sickle cell mice.

Sickle cell disease (SCD) patients can have limited exercise capacity and muscle dysfunction characterized by decreased force, atrophy, microvascular abnormalities, fiber distribution changes, and skeletal muscle energetics abnormalities. Growing evidence suggests that in SCD there is alteration in nitric oxide (NO) availability/signaling and that nitrate/nitrite can serve as a NO reservoir and enhance muscle performance. Here, we examined effects of nitrite on muscle strength, exercise capacity, and on contractile properties of fast-(extensor digitorum longus, EDL) and slow-twitch (soleus) muscles in SCD mice. Compared to controls, homozygotes (sickling) had decreased grip strength, impaired wheel running performance, and decreased muscle mass of fast-twitch, but not slow-twitch muscle. Nitrite treatment yielded increases in nitrite plasma levels in controls, heterozygotes, and homozygotes but decreases in muscle nitrite levels in heterozygotes and homozygotes. Regardless of genotype, nitrite yielded increases in grip strength, which were coupled with increases in specific force in EDL, but not in soleus muscle. Further, nitrite increased EDL, but not soleus, fatigability in all genotypes. Conversely, in controls, nitrite decreased, whereas in homozygotes, it increased EDL susceptibility to contraction-induced injury. Interestingly, nitrite yielded no changes in distances ran on the running wheel. These differential effects of nitrite in fast- and slow-twitch muscles suggest that its ergogenic effects would be observed in high-intensity/short exercises as found with grip force increases but no changes on wheel running distances. Further, the differential effects of nitrite in homozygotes and control animals suggests that sickling mice, which have altered NO availability/signaling, handle nitrite differently than do control animals.

Patient characteristics affect the response to ketamine and opioids during the treatment of vaso-occlusive episode-related pain in sickle cell disease.

BackgroundN-methyl-D-aspartate receptor activation has been implicated in the pathobiology of inflammatory, nociceptive and neuropathic pain, opioid tolerance, opioid-induced hyperalgesia, and central sensitization. Some of those mechanisms underlie sickle cell disease(SCD)-associated pain.MethodsWe conducted an exploratory cohort study of SCD patients who during vaso-occlusive episodes (VOEs) received subanesthetic doses of the N-methyl-D-aspartate receptor antagonist, ketamine, as an adjunct to opioids. We sought to identify predictors of changes in pain scores and of the percentage of ketamine infusions associated with meaningful changes (≥20% reduction) in pain and opioid consumption.ResultsEight-five patients received 181 ketamine infusions for VOE-associated pain. Combined with opioids, ketamine yielded significant decrease in pain scores and opioid consumption. Ketamine administered to males and to younger patients yielded greater pain score decrease compared with females (P=0.013) and older patients (P=0.018). Fifty-four percent of infusions yielded meaningful reductions in pain scores, and in multivariate analysis, sex, age group, pain location, and infusion duration independently predicted pain score changes.ConclusionThis study suggests that in SCD patients admitted with VOE-associated pain, ketamine has age- and sex-dependent effects. These data can inform sample and effect size calculations for controlled trials to determine which SCD patients would benefit most from ketamine.

Using Lean Six Sigma Methodology to Improve Quality of the Anesthesia Supply Chain in a Pediatric Hospital.

Six Sigma and Lean methodologies are effective quality improvement tools in many health care settings. We applied the DMAIC methodology (define, measure, analyze, improve, control) to address deficiencies in our pediatric anesthesia supply chain. We defined supply chain problems by mapping existing processes and soliciting comments from those involved. We used daily distance walked by anesthesia technicians and number of callouts for missing supplies as measurements that we analyzed before and after implementing improvements (anesthesia cart redesign). We showed improvement in the metrics after those interventions were implemented, and those improvements were sustained and thus controlled 1 year after implementation.

Cognitive and behavior deficits in sickle cell mice are associated with profound neuropathologic changes in hippocampus and cerebellum.

Strokes are perhaps the most serious complications of sickle cell disease (SCD) and by the fifth decade occur in approximately 25% of patients. While most patients do not develop strokes, mounting evidence indicates that even without brain abnormalities on imaging studies, SCD patients can present profound neurocognitive dysfunction. We sought to evaluate the neurocognitive behavior profile of humanized SCD mice (Townes, BERK) and to identify hematologic and neuropathologic abnormalities associated with the behavioral alterations observed in these mice. Heterozygous and homozygous Townes mice displayed severe cognitive deficits shown by significant delays in spatial learning compared to controls. Homozygous Townes also had increased depression- and anxiety-like behaviors as well as reduced performance on voluntary wheel running compared to controls. Behavior deficits observed in Townes were also seen in BERKs. Interestingly, most deficits in homozygotes were observed in older mice and were associated with worsening anemia. Further, neuropathologic abnormalities including the presence of large bands of dark/pyknotic (shrunken) neurons in CA1 and CA3 fields of hippocampus and evidence of neuronal dropout in cerebellum were present in homozygotes but not control Townes. These observations suggest that cognitive and behavioral deficits in SCD mice mirror those described in SCD patients and that aging, anemia, and profound neuropathologic changes in hippocampus and cerebellum are possible biologic correlates of those deficits. These findings support using SCD mice for studies of cognitive deficits in SCD and point to vulnerable brain areas with susceptibility to neuronal injury in SCD and to mechanisms that potentially underlie those deficits.

Rapamycin increases fetal hemoglobin and ameliorates the nociception phenotype in sickle cell mice.

Fetal hemoglobin-inducing therapies are disease-modifying and ameliorate the pain phenotype in sickle cell disease (SCD). Rapamycin, a mammalian target of rapamycin (mTOR) inhibitor, increases HbF in erythroid precursor cells in vitro. We hypothesized that rapamycin would increase HbF levels and improve nociception phenotype in SCD mice. We used sine-wave electrical stimulation to examine nocifensive phenotype and evaluate myelinated [2000Hz (Aß-fiber) and 250Hz (Aδ-fiber)] and unmyelinated (5Hz C-fibers)] sensory fiber function. Rapamycin significantly increased γ-globin mRNA and HbF levels [+2.3% (0.7, 3.9), mean increase (95% confidence interval, CI), p=0.006]. In homozygous (sickling) mice, long- (16 weeks), but not short-term (6 weeks), rapamycin treatment increased 2000Hz and 250Hz current thresholds in a pattern that varied according to sex. In male, but not female mice, rapamycin (compared with vehicle) was associated with increases in 2000Hz [21Units (7, 35), mean difference (95% CI), p=0.009 for sex∗treatment interaction] and 250Hz [9Units (1, 16), p=0.01] current thresholds. In rapamycin-treated homozygotes, HbF levels directly correlated with myelinated [2000Hz(Aß-fiber, r=0.58, p=0.01) and 250Hz(Aδ-fiber, r=0.6, p=0.01)] but not unmyelinated sensory fiber current thresholds. These findings suggest that in SCD mice, rapamycin increases HbF and modulates current thresholds of myelinated fibers. Therefore, mTOR signaling might be implicated in the pathobiology of SCD.

Validation of a method to directly and specifically measure nitrite in biological matrices.

The bioactivity of nitric oxide (NO) is influenced by chemical species generated through reactions with proteins, lipids, metals, and its conversion to nitrite and nitrate. A better understanding of the functions played by each of these species could be achieved by developing selective assays able of distinguishing nitrite from other NO species. Nagababu and Rifkind developed a method using acetic and ascorbic acids to measure nitrite-derived NO in plasma. Here, we adapted, optimized, and validated this method to assay nitrite in tissues. The method yielded linear measurements over 1-300 pmol of nitrite and was validated for tissue preserved in a nitrite stabilization solution composed of potassium ferricyanide, N-ethylmaleimide and NP-40. When samples were processed with chloroform, but not with methanol, ethanol, acetic acid or acetonitrile, reliable and reproducible nitrite measurements in up to 20 sample replicates were obtained. The method's accuracy in tissue was ≈ 90% and in plasma 99.9%. In mice, during basal conditions, brain, heart, lung, liver, spleen and kidney cortex had similar nitrite levels. In addition, nitrite tissue levels were similar regardless of when organs were processed: immediately upon collection, kept in stabilization solution for later analysis or frozen and later processed. After ip nitrite injections, rapidly changing nitrite concentrations in tissue and plasma could be measured and were shown to change in significantly distinct patterns. This validated method could be valuable for investigations of nitrite biology in conditions such as sickle cell disease, cardiovascular disease, and diabetes, where nitrite is thought to play a role.

Subanesthetic ketamine infusions for the treatment of children and adolescents with chronic pain: a longitudinal study.

BACKGROUND: Chronic pain is common in children and adolescents and is often associated with severe functional disability and mood disorders. The pharmacological treatment of chronic pain in children and adolescents can be challenging, ineffective, and is mostly based on expert opinions and consensus. Ketamine, an N-methyl-D-aspartate receptor antagonist, has been used as an adjuvant for treatment of adult chronic pain and has been shown, in some instances, to improve pain and decrease opioid-requirement. We examined the effects of subanesthetic ketamine infusions on pain intensity and opioid use in children and adolescents with chronic pain syndromes treated in an outpatient setting. METHODS: Longitudinal cohort study of consecutive pediatric patients treated with subanesthetic ketamine infusions in a tertiary outpatient center. Outcome measurements included self-reported pain scores (numeric rating scale) and morphine-equivalent intake. RESULTS: Over a 15-month period, 63 children and adolescents (median age 15, interquartile range 12-17 years) with chronic pain received 277 ketamine infusions. Intravenous administration of subanesthetic doses of ketamine to children and adolescents on an outpatient basis was safe and not associated with psychotropic effects or hemodynamic perturbations. Overall, ketamine significantly reduced pain intensity (p < 0.001) and yielded greater pain reduction in patients with complex regional pain syndrome (CRPS) than in patients with other chronic pain syndromes (p = 0.029). Ketamine-associated reductions in pain scores were the largest in postural orthostatic tachycardia syndrome (POTS) and trauma patients and the smallest in patients with chronic headache (p = 0.007). In 37% of infusions, patients had a greater than 20 % reduction in pain score. Conversely, ketamine infusions did not change overall morphine-equivalent intake (p = 0.3). CONCLUSIONS: These data suggest that subanesthetic ketamine infusion is feasible in an outpatient setting and may benefit children and adolescents with chronic pain. Further, patients with CRPS, POTS, and a history of trauma-related chronic pain are more likely to benefit from this therapeutic modality.

Dexmedetomidine as an Adjuvant to Analgesic Strategy During Vaso-Occlusive Episodes in Adolescents with Sickle-Cell Disease.

Patients with sickle-cell disease (SCD) can experience recurrent vaso-occlusive episodes (VOEs), which are associated with severe pain. While opioids are the mainstay of analgesic therapy, in some patients with SCD, increasing opioid use is associated with continued and increasing pain. Dexmedetomidine, an α2 -adrenoreceptor agonist with sedative and analgesic properties, has been increasingly used in the perioperative and intensive care settings and has been shown to reduce opioid requirement and to facilitate opioid weaning. Therefore, there might be a role for dexmedetomidine in pain management during VOEs in patients with SCD. Here, we present the hospital course of 3 patients who during the course of VOEs had severe pain unresponsive to opioids and ketamine and were treated with dexmedetomidine. Dexmedetomidine infusions that lasted for 3 to 6 days were associated with marked reduction in daily oral morphine-equivalent intake and decreases in pain scores (numeric rating scale). There were no hemodynamic changes that required treatment with vasoactive or anticholinergic agents. These preliminary findings of possible beneficial effects of dexmedetomidine in decreasing opioid requirements support the hypothesis that dexmedetomidine may have a role as a possible analgesic adjuvant to mitigate VOE-associated pain in patients with SCD.

Prolonged perioperative infusion of low-dose ketamine does not alter opioid use after pediatric scoliosis surgery.

BACKGROUND: Opioid consumption after posterior spinal fusion is known to be high and often exceeds those reported in other major surgical procedures. A number of clinical trials provide evidence that the perioperative use of subanesthetic doses of ketamine reduces pain and opioid requirements in some surgical procedures, but the effect of prolonged perioperative low-dose ketamine infusion in patients undergoing posterior spinal fusion for pediatric scoliosis surgery is unknown. OBJECTIVE: To test the hypothesis that a 72-h perioperative low-dose ketamine infusion would decrease opioid use in pediatric patients undergoing posterior spinal fusion. METHODS: In a double-blind prospective controlled trial, patients undergoing posterior spinal fusion for scoliosis were randomized to receive perioperative low-dose ketamine or placebo control. Patients received general anesthesia, intraoperative remifentanil, and morphine patient-controlled analgesia postoperatively. Daily opioid consumption, self-reported pain scores, and sedation scores were measured. RESULTS: Fifty-four patients were enrolled and 50 completed the study. Contrary to our hypothesis, ketamine- and control-treated patients had similar postoperative opioid use, pain scores, and sedation scores measurements. In contrast, ketamine-treated patients required less intraoperative remifentanil compared with control (mean 2.9 mg vs. 4 mg, P = 0.0415). Number of vertebrae instrumented, time between end-of-surgery and 24 h assessment, or remifentanil doses did not impact on postoperative opioid use. Over 96-h postoperatively, morphine-equivalent consumption was lower (-0.40, P = 0.006) and sedation score was higher (0.47, P = 0.0211) in male patients, compared with female patients. CONCLUSIONS: These findings do not support the use of perioperative low-dose ketamine to decrease opioid use in children with scoliosis undergoing posterior spinal fusion.

Disruption of adaptive energy metabolism and elevated ribosomal p-S6K1 levels contribute to INCL pathogenesis: partial rescue by resveratrol.

The infantile neuronal ceroid lipofuscinosis (INCL) is a devastating neurodegenerative lysosomal storage disease. Despite our knowledge that palmitoyl-protein thioesterase-1 (PPT1)-deficiency causes INCL, the molecular mechanism(s) of neurodegeneration and the drastically reduced lifespan of these patients remain poorly understood. Consequently, an effective treatment for this disease is currently unavailable. We previously reported that oxidative stress-mediated abnormality in mitochondria activates caspases-9 pathway of apoptosis in INCL fibroblasts and in neurons of Ppt1-knockout (Ppt1-KO) mice, which mimic INCL. Since mitochondria play critical roles in maintaining cellular energy homeostasis, we hypothesized that oxidative stress-mediated disruption of energy metabolism and homeostasis may contribute to INCL pathogenesis. We report here that, in cultured INCL fibroblasts and in the brain tissues of Ppt1-KO mice, the NAD(+)/NADH ratio, the levels of phosphorylated-AMPK (p-AMPK), peroxisome proliferator-activated receptor-γ (PPARγ) coactivator-1α (PGC-1α) and Silent Information Regulator T1 (SIRT1) are markedly down-regulated. This suggested an abnormality in AMPK/SIRT1/PGC-1α signaling pathway of energy metabolism. Moreover, we found that, in INCL fibroblasts and in the Ppt1-KO mice, phosphorylated-S6K-1 (p-S6K1) levels, which inversely correlate with lifespan, are markedly elevated. Most importantly, resveratrol (RSV), an antioxidant polyphenol, elevated the NAD(+)/NADH ratio, levels of ATP, p-AMPK, PGC-1α and SIRT1 while decreasing the level of p-S6K1 in both INCL fibroblasts and in Ppt1-KO mice, which showed a modest increase in lifespan. Our results show that disruption of adaptive energy metabolism and increased levels of p-S6K1 are contributing factors in INCL pathogenesis and provide the proof of principle that small molecules such as RSV, which alleviate these abnormalities, may have therapeutic potential.