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.

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.

Pain research and children and adolescents with severe intellectual disability: ethical challenges and imperatives.

Children with severe intellectual disabilities encounter inequities in pain-related care, yet little pain research involves this population. A considerable issue with pain research in this population is its ethical complexity. This Viewpoint delineates the ethical challenges of pain research involving children (aged 2-12 years) and adolescents (aged 13-21 years) with severe intellectual disabilities. There are two main issues. First, some of the standard methods for assessing pain and pain sensitivity are not suitable for individuals with severe intellectual disability, who are often non-verbal and unable to understand or follow instructions. Second, children and adolescents with severe intellectual disability cannot provide informed consent or assent to participate in pain research, and their dissent is not always recognised. The existing ethical guidelines for pain research by the International Association for the Study of Pain provide helpful, but general, guidance. This Viewpoint supplements these guidelines and uses a well established framework for assessing the ethics of clinical research to highlight points relevant to designing, doing, reviewing, and evaluating research involving children and adolescents with severe intellectual disability, focusing on issues that are unaddressed in existing guidance.

Subanesthetic ketamine: the way forward for pain management in sickle cell disease patients?

INTRODUCTION: Patients with sickle cell disease (SCD) present recurrent episodes of acute pain, the hallmark of the disease, and some will also develop chronic pain. Currently, the treatment of SCD acute pain only targets its symptoms, rather than underlying mechanisms, and is directed by expert and consensus guidelines. AREAS COVERED: While opioids remain the mainstay of therapy for acute pain and are also used to treat SCD-related chronic pain, in some patients, opioids are ineffective or are associated with severe undesirable side effects. In those instances, clinicians caring for patients with SCD face an unmet need for effective non-opioid analgesics. Recently, the use of subanesthetic ketamine has been explored as a strategy to meet this need. While definitive evidence of its efficacy is lacking, some information exists suggesting that subanesthetic ketamine improves pain control and may have opioid-sparing effects in SCD-related acute pain. However, ketamine can also yield undesirable psychotomimetic and cardiovascular effects. EXPERT OPINION: After weighing potential risks and benefits, in the absence of better alternatives and in settings where it can be administered safely, ketamine may be a reasonable option for patients with SCD-related acute refractory pain.

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.

Anesthetic Management of Children and Adolescents With Giant Axonal Neuropathy: A Large Case Series.

Giant axonal neuropathy (GAN) is a rare autosomal recessive neurodegenerative disorder caused by mutations in the GAN gene, which encodes for gigaxonin, a protein involved in intermediate filament processing in neural cells and fibroblasts. We report on 14 GAN patients who underwent 77 anesthetics during the conduct of an intrathecal gene transfer clinical trial from April 2015 to August 2020. We observed only a few nonsignificant perianesthetic complications. Our data expand the knowledge regarding safety of anesthesia for patients with this rare and potentially fatal disease and highlights the tolerability of shorter procedural sedation and anesthesia.

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.

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.

Nitric oxide and sickle cell disease-Is there a painful connection?

Sickle cell disease is the most common hemoglobinopathy and affects millions worldwide. The disease is associated with severe organ dysfunction, acute and chronic pain, and significantly decreased life expectancy. The large body of work demonstrating that hemolysis results in rapid consumption of the endogenous vasodilator nitric oxide, decreased nitric oxide production, and promotion of vaso-occlusion provides the basis for the hypothesis that nitric oxide bioavailability is reduced in sickle cell disease and that this deficit plays a role in sickle cell disease pain. Despite initial promising results, large clinical trials using strategies to increase nitric oxide bioavailability in sickle cell disease patients yielded no significant change in duration or frequency of acute pain crises. Further, recent investigations showed that sickle cell disease patients and mouse models have elevated baseline levels of blood nitrite, a reservoir for nitric oxide formation and a product of nitric oxide metabolism, regardless of pain phenotype. These conflicting results challenge the hypotheses that nitric oxide bioavailability is decreased and that it plays a significant role in the pathogenesis in sickle cell disease acute pain crises. Conversely, a large body of work demonstrates that nitric oxide, as a neurotransmitter, has a complex role in pain neurobiology, contributes to the development of central sensitization, and can mediate hyperalgesia in inflammatory and neuropathic pain. These results support an alternative hypothesis: one proposing that altered nitric oxide signaling may contribute to the development of neuropathic and/or inflammatory pain in sickle cell disease through its role as a neurotransmitter.

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.

Locomotor mal-performance and gait adaptability deficits in sickle cell mice are associated with vascular and white matter abnormalities and oxidative stress in cerebellum.

Patients with sickle cell disease (SCD) can develop strokes and as a result, present neurologic and neurocognitive deficits. However, recent studies show that even without detectable cerebral parenchymal abnormalities on imaging studies, SCD patients can have significant cognitive and motor dysfunction, which can present as early as during infancy. As the cerebellum plays a pivotal role in motor and non-motor functions including sensorimotor processing and learning, we examined cerebellar behavior in humanized SCD mice using the Erasmus ladder. Homozygous (sickling) mice had significant locomotor malperformance characterized by miscoordination and impaired locomotor gait/stepping pattern adaptability. Conversely, Townes homozygous mice had no overall deficits in motor learning, as they were able to associate a conditioning stimulus (high-pitch warning tone) with the presentation of an obstacle and learned to decrease steptimes thereby increasing speed to avoid it. While these animals had no cerebellar strokes, these locomotor and adaptive gait/stepping patterns deficits were associated with oxidative stress, as well as cerebellar vascular endothelial and white matter abnormalities and blood brain barrier disruption, suggestive of ischemic injury. Taken together, these observations suggest that motor and adaptive locomotor deficits in SCD mice mirror some of those described in SCD patients and that ischemic changes in white matter and vascular endothelium and oxidative stress are biologic correlates of those deficits. These findings point to the cerebellum as an area of the central nervous system that is vulnerable to vascular and white matter injury and support the use of SCD mice for studies of the underlying mechanisms of cerebellar dysfunction in SCD.

Kcnn2 blockade reverses learning deficits in a mouse model of fetal alcohol spectrum disorders.

Learning disabilities are hallmarks of congenital conditions caused by prenatal exposure to harmful agents. These include fetal alcohol spectrum disorders (FASDs) with a wide range of cognitive deficiencies, including impaired motor skill development. Although these effects have been well characterized, the molecular effects that bring about these behavioral consequences remain to be determined. We previously found that the acute molecular responses to alcohol in the embryonic brain are stochastic, varying among neural progenitor cells. However, the pathophysiological consequences stemming from these heterogeneous responses remain unknown. Here we show that acute responses to alcohol in progenitor cells altered gene expression in their descendant neurons. Among the altered genes, an increase of the calcium-activated potassium channel Kcnn2 in the motor cortex correlated with motor learning deficits in a mouse model of FASD. Pharmacologic blockade of Kcnn2 improves these learning deficits, suggesting Kcnn2 blockers as a new intervention for learning disabilities in FASD.

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.

Analgesic Requirements in Adolescents Undergoing Bariatric Surgery-an Observational Study.

AIM: To examine the impact of preexisting psychiatric/psychological diagnoses on perioperative analgesic requirements in adolescents with morbid obesity undergoing bariatric surgery. METHODS: A retrospective cohort study of adolescents with morbid obesity undergoing bariatric surgery. Primary and secondary outcomes included perioperative analgesic intake and pain scores (Numerical Rating Scale (0-10) NRS) throughout the hospital stay. RESULTS: From our Bariatric Surgery Research Registry, we identified 17 adolescents with psychiatric/psychological diagnoses prior to undergoing bariatric surgery. Fifteen patients from the same registry and without such diagnosis undergoing bariatric surgery during the same time interval served as controls. In both groups, there was a predominance of female patients. During the perioperative period, in both groups, oral morphine equivalent and ketorolac and acetaminophen intake were similar. Notably, the perioperative median pain scores at the times examined were below 5 for all patients. The median pain scores in the PACU, day of surgery, and first postoperative day were similar. Conversely, on postoperative day 2, pain scores were higher in patients with diagnoses of psychiatric/psychological disorders (p = 0.004) compared to those without. CONCLUSION: In this cohort of morbidly obese adolescents undergoing bariatric surgery, patients with and without preexisting psychiatric/psychological diagnoses had similar analgesic requirements during the perioperative period. This finding appears contrary to those suggesting that preexisting depression and/or anxiety might be associated with increased analgesic requirements during the perioperative period.

The platelet NLRP3 inflammasome is upregulated in sickle cell disease via HMGB1/TLR4 and Bruton tyrosine kinase.

A key inflammatory mechanism recently identified in platelets involves the Nod-like receptor nucleotide-binding domain leucine-rich repeat containing protein 3 (NLRP3) and Bruton tyrosine kinase (BTK), which control activation of caspase-1 within inflammasome complexes. We investigated platelet caspase-1 activity in the context of sickle cell disease (SCD) directly in platelets isolated from SCD patients (n = 24) and indirectly by incubating platelets from healthy subjects with plasma obtained from SCD patients (n = 20), both in steady state and during an acute pain crisis (paired samples). The platelet NLRP3 inflammasome was upregulated in SCD patients under steady state conditions compared with healthy controls, and it was further upregulated when patients experienced an acute pain crisis. The results were consistent with indirect platelet assays, in which SCD plasma increased caspase-1 activity of platelets from healthy subjects in an NLRP3-dependent fashion. The damage-associated molecular pattern molecule high-mobility group box 1 (HMGB1) was elevated in plasma of SCD subjects compared with healthy controls and correlated with caspase-1 activity in platelets. Pharmacological or antibody-mediated inhibition of HMGB1, Toll-like receptor 4, and BTK interfered with sickle plasma-induced platelet caspase-1 activation. In Townes SCD mice, caspase-1 activity and aggregation of circulating platelets were elevated, which was suppressed by IV injection of an NLRP3 inhibitor and the BTK inhibitor ibrutinib. Activation of the platelet NLRP3 inflammasome in SCD may have diagnostic and therapeutic implications.

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.

The corticosteroid compounds prednisolone and vamorolone do not alter the nociception phenotype and exacerbate liver injury in sickle cell mice.

Clinicians often hesitate prescribing corticosteroids to treat corticosteroid-responsive conditions in sickle cell disease (SCD) patients because their use can be associated with complications (increased hospital readmission, rebound pain, strokes, avascular necrosis, acute chest syndrome). Consequently, SCD patients may receive suboptimal treatment for corticosteroid-responsive conditions. We conducted a preclinical trial of dissociative (vamorolone) and conventional (prednisolone) corticosteroid compounds to evaluate their effects on nociception phenotype, inflammation, and organ dysfunction in SCD mice. Prednisolone and vamorolone had no significant effects on nociception phenotype or anemia in homozygous mice. Conversely, prednisolone and vamorolone significantly decreased white blood cell counts and hepatic inflammation. Interestingly, the effects of vamorolone were milder than those of prednisolone, as vamorolone yielded less attenuation of hepatic inflammation compared to prednisolone. Compared to controls and heterozygotes, homozygotes had significant liver necrosis, which was significantly exacerbated by prednisolone and vamorolone despite decreased hepatic inflammation. These hepatic histopathologic changes were associated with increases in transaminases and alkaline phosphatase. Together, these results suggest that, even in the setting of decreasing hepatic inflammation, prednisolone and vamorolone were associated with significant hepatic toxicity in SCD mice. These findings raise the possibility that hepatic function deterioration could occur with the use of corticosteroids (conventional and dissociative) in SCD.