Stroke In Sickle Cell Disease Research Articles

A contemporary evaluation of the frequency & factors associated with overt stroke across the lifespan: A Ghanaian sickle cell disease registry analysis

BackgroundStroke is a devastating complication of Sickle Cell Disease (SCD) with significant mortality and substantial morbidity. The burden of prevalent stroke in SCD is highest in sub-Saharan Africa and estimated at 4.2 % to 6.4 % in the era where evidence-based prevention strategies such as use of hydroxyurea therapy and transcranial doppler ultrasound were not routine care. PurposeTo assess the contemporary frequency and factors associated with prevalent stroke across the lifespan in an SCD registry at the tertiary medical center in Ghana. MethodsThis is a cross-sectional study conducted at the Komfo Anokye Teaching Hospital, a tertiary medical center in the middle belt of Ghana. The center has comprehensive Sickle Cell Clinics for children, adolescents, and adults with a patient registry established as part of the Sickle Pan-African Research Consortium (SPARCo)-Ghana study from 2017 to date. Data captured in the registry and analyzed for the present study include demographics, stroke status using the WHO criteria supplemented by the Questionnaire for Verifying Stroke Free Status (QVSFS), use of hydroxyurea, and complete blood count. Logistic regression modeling was utilized to assess factors associated with stroke. ResultsAmong a registry cohort of 4115 individuals with confirmed SCD, 35 (0.85 %, 95 % CI: 0.59–1.18 %) had overt or clinically confirmed stroke. The frequency of stroke differed significantly across the lifespan being 0.38 % (95 % CI: 0.12–0.64 %) among children <10 years, 1.23 % (95 % CI: 0.73–1.94 %) among adolescents aged 10 to 17 years, and 1.44 % (95 % CI: 0.66–2.71 %) among adults 18 years or more, p = 0.007. In adjusted analysis, each 10-year increase in age was associated with odds ratio, OR (95 % CI) of 1.90 (1.42–2.54) and hydroxyurea use, OR of 6.09 (2.65–13.99). The association between hydroxyurea and stroke observed in this cross-sectional study is not causal. ConclusionApproximately 1 in 120 SCD patients in this large Ghanaian cohort had clinically overt stroke. The gradual uptake of hydroxyurea therapy into routine care for SCD in this resource-limited setting, may partly explain the lower frequency of stroke.

Genetic Variants Associated with the Risk of Stroke in Sickle Cell Anemia: Systematic Review and Meta-Analysis

Sickle cell anemia (SCA) is the most common cause of stroke in children. As it is a rare disease, studies investigating the association with complications like stroke in SCD have small sample sizes. Here, we performed a systematic review and meta-analysis of the studies exploring an association of genetic variants with stroke to get a better indication of their association with stroke. PubMed and Google Scholar were searched to identify studies that had performed an association analysis of genetic variants for the risk of stroke in SCA patients. After screening of eligible studies, summary statistics of association analysis with stroke and other general information were extracted. Meta-analysis was performed using the fixed effect method on the tool METAL and forest plots were plotted using the R program. The random effect model was performed as a sensitivity analysis for loci where significant heterogeneity was observed. 407 studies were identified using the search term and after screening 37 studies that cumulatively analyzed 11,373 SCA patients were included. These 37 studies included a total of 2,222 SCA patients with stroke, predominantly included individuals of African ancestry (N = 16). Three of these studies performed whole exome sequencing while 35 performed single nucleotide-based genotyping. Though the studies reported association with 132 loci, meta-analyses could be performed only for 12 loci that had data from two or more studies. After meta-analysis we observed that four loci were significantly associated with risk for stroke: -α3.7 kb Alpha-thalassemia deletion (P = 0.00000027), rs489347-TEK (P = 0.00081), rs2238432-ADCY9 (P = 0.00085), rs11853426-ANXA2 (P = 0.0034), and rs1800629-TNF (P = 0.0003396). Ethnic representation of regions with a high prevalence of SCD like the Mediterranean basin and India needs to be improved for genetic studies on associated complications like stroke. Larger genome-wide collaborative studies on SCD and associated complications including stroke need to be performed.

A-296G variant of THBS1 gene (rs1478605) is associated with a lower frequency of stroke in a Brazilian population with sickle cell anemia

ObjectivesStroke is a devastating clinical outcome that significantly contributes to the morbidity and mortality of sickle cell anemia (SCA) patients. Despite its advantages in predicting stroke risk, transcranial Doppler screening has limitations that restrict its applicability, highlighting the need for emerging prognostic tools. Thrombospondin-1 plays a crucial role in endothelial injury, platelet adhesion, and nitric oxide metabolism and may be implicated in stroke pathophysiology. Here, we aimed to evaluate the association of THBS1 genetic variations with the occurrence of stroke in SCA patients Materials and methodsBy real-time PCR, 512 SCA patients were fully genotyped for THBS1 A-296G (rs1478605) polymorphism ResultsTHBS1 GG genotype was associated with a lower risk for stroke occurrence [odds ratio (OR): 0.30; 95% confidence interval (CI): 0.11–0.78; P = 0.011], although these findings were not consistent with multivariate logistic regression analysis (OR: 0.73, 95% CI: 0.12 – 4.37; P = 0.736). In agreement, the cumulative incidence of stroke for patients with AG/AA genotypes was higher when compared to the GG genotype (P = 0.018). However, the association was not maintained in the multivariate proportional hazards model (hazard ratio: 0.67, 95% CI: 0.12–3.61; P = 0.643) ConclusionsIn summary, the present study shows that the THBS1 A-296G (rs1478605) polymorphism may be a potential modifier for stroke in SCA.

Incidence and Prevalence of Acute Stroke and its Clinical Implications and Outcomes in Patients with Sickle Cell Disease: A Systematic Review

This systematic review aims to determine the incidence and prevalence of acute stroke in patients with sickle cell disease (SCD) and explore its clinical implications and outcomes. A comprehensive search was conducted using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses 2020 guidelines to identify relevant studies published between 2013 and 2023. Databases including PubMed, Google Scholar, ResearchGate, ScienceDirect, and Wiley Online Library are searched. Inclusion criteria are applied to select studies involving human subjects, written in English, and reporting the association between acute stroke and confirmed cases of SCD. Study quality assessment and data extraction are performed by two independent reviewers using study-specific tools. A total of 9 studies met the inclusion criteria including 7 cohort studies, and two meta-analyses are included in the systematic review with a total of 24,480 participants. The prevalence of stroke in SCD patients varied across studies, ranging from 1.24% to 27.3%. The incidence rates of stroke showed significant variability, ranging from 0.31% to 31%. Long-term disabilities, recurrence, and reduced quality of life characterized the clinical outcomes of stroke in SCD patients. Mortality rates varied among studies, emphasizing the need for early detection and comprehensive care. The review provides valuable insights for health-care professionals, researchers, and policymakers involved in SCD care, emphasizing the importance of early detection, intervention, and comprehensive management to improve patient outcomes. Further research is needed to address knowledge gaps and understand the underlying mechanisms of stroke in SCD.

Insulin-like Growth Factor-1 Prevents Hypoxia/Reoxygenation-Induced White Matter Injury in Sickle Cell Mice

Occlusion of cerebral blood vessels causes acute cerebral hypoxia-an important trigger of ischemic white matter injury and stroke in sickle cell disease (SCD). While chronic hypoxia triggers compensatory neuroprotection via insulin-like growth factor-1 (IGF-1) and hypoxia inducible factor-1α (HIF-1α), severe bouts of acute hypoxia and subsequent restoration of blood flow (hypoxia/reoxygenation, H/R) overwhelm compensatory mechanisms and cause neuroaxonal damage-identified as white matter lesions-in the brain. The neuroprotective role of IGF-1 in the pathogenesis of white matter injury in SCD has not been investigated; however, it is known that systemic IGF-1 is reduced in individuals with SCD. We hypothesized that IGF-1 supplementation may prevent H/R-induced white matter injury in SCD. Transgenic sickle mice homozygous for human hemoglobin S and exposed to H/R developed white matter injury identified by elevated expression of non-phosphorylated neurofilament H (SMI32) with a concomitant decrease in myelin basic protein (MBP) resulting in an increased SMI32/MBP ratio. H/R-challenge also lowered plasma and brain IGF-1 expression. Human recombinant IGF-1 prophylaxis significantly induced HIF-1α and averted H/R-induced white matter injury in the sickle mice compared to vehicle-treated mice. The expression of the IGF-1 binding proteins IGFBP-1 and IGFBP-3 was elevated in the IGF-1-treated brain tissue indicating their potential role in mediating neuroprotective HIF-1α signaling. This study provides proof-of-concept for IGF-1-mediated neuroprotection in SCD.

The Pathogenetic Mechanism for Moyamoya Vasculopathy Including a Possible Trigger Effect of Increased Flow Velocity.

Moyamoya disease (MMD), which commonly exhibits moyamoya vasculopathy characterized by chronic progressive steno-occlusive lesions in the circle of Willis with "moyamoya" collateral vessels, has been well known for its unique demographic and clinical features. Although the discovery of the susceptibility gene RNF213 for MMD revealed the factor for its predominance in East Asians, the mechanisms underlying other predominant conditions (females, children, young to middle-aged adults, and anterior circulation) and lesion formation are yet to be determined. As MMD and moyamoya syndrome (MMS), which secondarily produces moyamoya vasculopathy due to pre-existing diseases, have the same vascular lesions despite differences in their original pathogenesis, they may share a common trigger for the development of vascular lesions. Thus, we herein consider a common trigger from a novel perspective on blood flow dynamics. Increased flow velocity in the middle cerebral arteries is an established predictor of stroke in sickle cell disease, which is often complicated by MMS. Flow velocity is also increased in other diseases complicated by MMS (Down syndrome, Graves' disease, irradiation, and meningitis). In addition, increased flow velocity occurs under the predominant conditions of MMD (females, children, young to middle-aged adults, and anterior circulation), suggesting a relationship between flow velocity and susceptibility to moyamoya vasculopathy. Increased flow velocity has also been detected in the non-stenotic intracranial arteries of MMD patients. In a pathogenetic overview of chronic progressive steno-occlusive lesions, a novel perspective including the trigger effect of increased flow velocity may provide insights into the mechanisms underlying their predominant conditions and lesion formation.

Comparison of Strokes in Adult Hospitalized Patients with Sickle Cell Disease and Patients without Sickle Cell Disease: A Nationwide Analysis

Background: Strokes are a common and serious complication of Sickle Cell Disease (SCD). It is the most common cause of stroke in the pediatric population. The risk of recurrent strokes continues into adulthood in SCD population. In this abstract, we quantify the burden of strokes in the SCD population and compare it against the background of strokes in the non-SCD population. Methods: Adult cases with SCD were identified by the International Classification of Disease 10th Revision Diagnosis codes from the National Inpatient Sample Database for 2016-2019. The cases were distributed into age groups based on decades (18-30, 31-40, 41-50, 51-60, 61-70, 71-80; and 81 and above). Cases were identified for the presence of a history of a prior stroke [PMHS] with residual deficits, PMHS without deficits, a new episode of a hemorrhagic stroke, and a new episode of ischemic stroke. Binary logistic regression was performed for multivariate analysis to assess the effect of risk factors that are associated with stroke (age, gender, tobacco use, alcohol, hyperlipidemia [HLD], atrial fibrillation/atrial flutter, hypertension [HTN], peripheral vascular disease [PVD], obesity, diabetes mellitus [for both type 1 and 2], drug use, previous history of venous thromboembolism, and SCD). Please note that "non-SCD” represents only the cases excluding SCD patients. For regression analysis of the "general population", both SCD and non-SCD patients are included. The results are statistically weighted. Results: 431,245 (0.36%) cases with SCD were identified from a total of 120,396,555 cases. The percentages are reported in ascending order of age by decade (18-30, 31-40, 41-50, 51-60, 61-70, 71-80; and 81 and above). The percentages of SCD cases with PMHS without deficits were 8.9, 9.1, 14.5, 8, 13.9, 14.3, and 21.2, respectively, while those for non-SCD cases were 0.3, 0.9, 3.1, 5.2, 7.1, 9.6, and 12.6 respectively. The percentages of SCD cases with PMHS with residual deficit were 1.6, 2.2, 2.8, 3.5, 5.1, 6.4, and 7.2, respectively, while those for non-SCD cases were 0.1, 0.3, 1.3, 2.4, 3.3, 3.7 and 3.7 respectively. The percentages of SCD cases with a new episode of an ischemic stroke were 2, 1.5, 1.9, 2.2, 3.8, 6.1, and 9.6, respectively, while those for non-SCD cases were 0.2, 0.5, 1.5, 2.3, 3.2, 4.3, and 4.7 respectively. The percentages of SCD cases with a new episode of hemorrhagic stroke were 0.1, 0.2, 0.3, 0.6, 0.8, 0.9, and 1.7, respectively, while those for non-SCD cases were 0.1, 0.2, 0.7, 0.8, 0.9, 1, and 1.1 respectively. The comparisons can be seen side by side in Table 1. Upon multivariate analysis of the general population to assess the effect of risk factors on PMHS, the remarkable odds ratios (OR) were: SCD with OR 5.24 (Confidence Interval 5.19-5.29, P<0.001) and HTN 2.19 (2.183-2.192, P<0.001). Multivariate analysis for a new episode of stroke in the general population showed that PMHS had an OR of 2.62 (2.61 - 2.63, P<0.001), HTN 1.745 (1.740-1.751, P<0.001), SCD 1.705 (1.67-1.74, P<0.001) and PVD 1.67 (1.65-1.70, P<0.001). On multivariate analysis for a new episode of stroke in SCD cases, notable results were PMHS with an OR of 7.47 (7.16-7.80, P<0.001), HTN 1.39 (1.32-1.46, P<0.001), HLD 1.58 (1.45-1.71, P<0.001), and tobacco use 1.25 (1.19-1.32, P<0.001). The OR for other risk factors can be reviewed in Table 2. Conclusion: The number of strokes in SCD increases with age. The difference between SCD and non-SCD groups is most pronounced in the younger population. This gap narrows in the fifth decade when the incidence of strokes rises in the general population. PMHS is strongly correlated with new episodes of stroke. Aggressive management of SCD in the younger population, along with optimal risk factor modification (control of diabetes, HTN, HLD, smoking avoidance/cessation) is desirable to decrease the risk of stroke. Further research in this direction is warranted. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Stroke in sickle cell disease in association with bilateral absence of the internal carotid arteries. Case report

BackgroundCongenital absence of the internal carotid artery (ICA) is a highly infrequent congenital incidence and occurs in less than 0.01% of the population; bilateral absence is exceedingly rare, diagnosed below 10% of the unilateral absence of the ICA. Sickle cell disease (SCD) is a serious disorder and carries a high risk of stroke.Case presentationWe present a five-year-old child with SCD who experienced an ischemic stroke episode with epileptic seizures. Neuroimaging revealed the agenesis of both ICAs. The frequency, embryology, and collateral pathway of the vascular anomaly as the clinical presentation, of this rare hematologic disease, are discussed.ConclusionsSickle cell disease (SCD) carries a high risk of stroke. Congenital absence of ICA occurs in less than 0.01% of the population; bilateral absence is diagnosed below 10% of the unilateral absence of the ICA.

Abstract WP260: The Response Of Annexin A1 In Astrocyte Activation And White Matter Injury- A New Target For Stroke In Sickle Cell Disease

Introduction: Ischemic stroke is common in sickle cell disease (SCD). White matter injuries, incorporated into silent cerebral infarcts, are major risk factors of stroke in SCD. The pathogenesis of cerebral infarction in SCD is not known. Induced thrombogenesis may cause impaired astrocyte that stimulates demyelination leading to white matter injury. Annexin A1 (Anxa1) can regulate astrocyte function by inhibiting intracellular phospholipase A2 (PLA2). We hypothesized that astrocyte activation during thrombogenic events promotes white matter injury in SCD. We tested astrocyte dysfunction and white matter injury in a clinically relevant mouse model of SCD. Methods: Cerebral Infarctions induced by de novo thrombogenesis were identified by TTC staining in transgenic sickle (SS) and control (AA) mice with human sickle and normal hemoglobin respectively. Cerebral microvessels and astrocytes were isolated from SS and AA mice. Elisa assays were used to measure thrombin, PLA2 and Anxa1. Immunofluorescence staining was performed to determine astrocyte activation and demyelination. Results: Thrombogenesis within cerebral microvessels was elevated by ~2-fold (n=5, p&lt;0.05), and cerebral infarction became prevalent in SS mice compared to AA mice (n=7, p&lt;0.001). The infarcted brain tissue from SS mice showed neuronal cell death, whereas the brain tissue from the AA mice appeared normal. The SS mice had increased glial fibrillary acid protein positive (GFAP + ) activated astrocytes with significant increase in PLA2 accumulation. Relative abundance of non-phosphorylated neurofilament H (SMI32) and myelin basic protein (MBP) (SMI32/MBP), indicating white matter injury, was substantially induced in the SS compared to the AA. Expression of Anxa1 reduced significantly in plasma as well as within astrocytes’ end feet attached to the isolated cerebral microvessels of the SS mice. Recombinant Anxa1 prophylaxis to SS mice reduced the GFAP + astrocytes, PLA2 accumulation, demyelination and infarct score (n=6, p&lt;0.01) compared to SS mice treated with vehicle. Conclusion: White matter injury in SS mice is associated with astrocyte dysfunction and lack of Anxa1, which may be developed as potential therapeutic targets to prevent and/or treat stroke in SCD.

Preventing Stroke in Sickle Cell Disease: 2021. The Role of Transcranial Doppler Ultrasound (TCD) When the Use of Transfusion is Problematic

In 2009, as an invited Commentary to the article: “STOP” ing the Harm: When is State Intervention Justified? [1], I wrote that while TCD is an indicator of risk, not a biopsy diagnosis (such as proof of cancer), at some point in the velocity spectrum the high velocity detected by TCD reaches what many—myself included--- believe is an unacceptable risk of stroke

Epidemiology of Stroke in Sickle Cell Disease.

Sickle cell disease is the most common cause of stroke in childhood, both ischaemic and haemorrhagic, and it also affects adults with the condition. Without any screening or preventative treatment, the incidence appears to fall within the range 0.5 to 0.9 per 100 patient years of observation. Newborn screening with Penicillin prophylaxis and vaccination leading to reduced bacterial infection may have reduced the incidence, alongside increasing hydroxyurea prescription. Transcranial Doppler screening and prophylactic chronic transfusion for at least an initial year has reduced the incidence of stroke by up to 10-fold in children with time averaged mean of the maximum velocity >200 cm/s. Hydroxyurea also appears to reduce the incidence of first stroke to a similar extent in the same group but the optimal dose remains controversial. The prevention of haemorrhagic stroke at all ages and ischaemic stroke in adults has not yet received the same degree of attention. Although there are fewer studies, silent cerebral infarction on magnetic resonance imaging (MRI), and other neurological conditions, including headache, epilepsy and cognitive dysfunction, are also more prevalent in sickle cell disease compared with age matched controls. Clinical, neuropsychological and quantitative MRI screening may prove useful for understanding epidemiology and aetiology.

Quality of Care in United States Children with Sickle Cell Anemia

Quality of Care in United States Children with Sickle Cell Anemia

Abstract P726: Cytochrome B5 Reductase 3 Modifies The Brain-Blood Axis Response To Ischemic Stroke In Mice With Sickle Cell Disease

Introduction: Stroke and silent infarcts are serious complications of sickle cell disease (SCD), occurring frequently in children. Decreased nitric oxide bioavailability and responsiveness contribute to neurovascular disease. Cytochrome b5 reductase 3 (Cyb5R3) is a heme iron reductase that reduces oxidized soluble guanylate cyclase heme iron (Fe 3+ --&gt; Fe 2+ ) to preserve nitric oxide signaling. A loss-of-function Cyb5R3 missense variant (T117S) occurs with high frequency (0.23 minor allele) in persons of African ancestry. Hypothesis: We hypothesized that impaired reductase function of T117S Cyb5R3 exacerbates brain damage after ischemic stroke in SCD. Methods: Bone marrow transplant was used to create male SCD mice with wild type (SS/WT) or T117S (SS/T117S) Cyb5R3. Blood was sampled before and after middle cerebral artery occlusion (55 minutes occlusion, 48 hours reperfusion). Infarct volume (IV) was determined by 2,3,5-triphenyltetrazolium chloride. Intravascular hemolysis and correlation (Pearson’s R) of hematology changes with IV were determined. Baseline Walk-PHaSST (NCT00492531) data were analyzed for stroke occurrence. Results: Brain IV (63 vs 27 cm 3 , P=0.003) and mortality (3/6 vs 0/8) were greater in SS/T117S vs SS/WT. Red blood cells, hemoglobin and hematocrit declined as IV increased. Plasma oxyhemoglobin increased in parallel with IV (r = 0.74, P=0.09). There were different signatures to hematologic changes that occurred with IV in SCD. Relative to wild type, T117S contracted the erythroid compartment (red blood cell: -13% vs 13%, P=0.003; hematocrit: -20% vs 1%, P=0.008; hemoglobin: -18% vs 2%, P=0.007). Mean platelet volume correlated with IV in SS/T117S (r = 0.87, P=0.06), while the inverse occurred in SS/WT (r = -0.63, P=0.09) Monocytes increased in parallel with IV in SS/T117S (r = 0.73, P=0.16), but followed the opposite trajectory in SS/WT (r = -0.77, P=0.04). WalkPHaSST participants with T117S Cyb5R3 self-reported more ischemic stroke (7.4% vs 5.1%) relative to wild type. Conclusion: Cyb5R3 is an important modifier of the evolution and outcome of ischemic brain injury in SCD and its hematologic consequences. Our findings indicate a bidirectional relationship between stroke and anemia in SCD that may axially turn on Cyb5R3 activity.

A Case Report of Dural Venous Thrombus Causing Hemorrhagic Stroke in Sickle Cell Disease

A Case Report of Dural Venous Thrombus Causing Hemorrhagic Stroke in Sickle Cell Disease

Bulk volume susceptibility difference between deoxyhemoglobin and oxyhemoglobin for HbA and HbS: A comparative study.

Sickle cell anemia is a blood disorder that alters the morphology and the oxygen affinity of the red blood cells. Cerebral oxygen extraction fraction measurements using quantitative BOLD contrast have been used for assessing inadequate oxygen delivery and the subsequent risk of ischemic stroke in sickle cell anemia. The BOLD signal in MRI studies relies on , the bulk volume susceptibility difference between fully oxygenated and fully deoxygenated blood. Several studies have measured for normal hemoglobin A (HbA). However, it is not known whether the value is different for sickle hemoglobin. In this study, was measured for both HbA and sickle hemoglobin. Six sickle cell anemia patients and 6 controls were recruited. Various blood oxygenation levels were achieved through in vivo manipulations to keep the blood close to its natural state. To account for the differences in oxygen affinity, Hill's equations were used to translate partial pressure of oxygen to oxygen saturation for HbA, sickle hemoglobin, and fetal hemoglobin (HbF) separately. The pH and PCO2 corrections were performed. Temperature and magnetic field drift were controlled for. A multivariate generalized linear mixed model with random participant effect was used. Assuming that is similar for HbA and HbF and that is 5/4 of for HbA, it was found that the values for HbA and sickle hemoglobin were not statistically significantly different from each other. The same value can be used for both types of hemoglobin in quantitative BOLD analysis.

Transcranial Doppler Screening In Children with Sickle Cell Disease of South Gujarat Population

Background: TCD (Transcranial Doppler) is a well-established study to predict Cerebrovascular stroke in SCD (Sickle cell disease). We aim to establish baseline TCD findings in Indian children with SCD and compare the results with the available STOP (Stroke prevention trial in Sickle Cell Anemia) protocol. We would also compare TCD findings in homozygous sickle cell disease and heterozygous sickle cell trait.&#x0D; Material and Methods: Seventy nine children with SCD were included in this study for one year period. TCD was performed and TAMMV (time-averaged maximum mean) velocity in the middle cerebral, anterior cerebral, posterior cerebral and internal carotid arteries was measured. Children were divided into two groups. Group I (56 homozygos–70.88%) and group II (23 heterozygos–29.11%).&#x0D; Results: In group I, 50 children fall in normal range with average TAMM velocity of 127.59 ± 17.48 cm/s. There was 1 (1.78%) abnormal result and 5 (8.9%) conditional results in group I. All results were normal in group II with average TAMM velocity of 116.33 ± 12.412 cm/sec. Middle cerebral artery was the only affected vessels amongst all.&#x0D; Conclusions: In our study, there was low prevalence of abnormal TCD results as compared to STOP protocol. The difference was significant in TAMM velocity between two groups, with all children being within normal range in group II. Result of this study differs from previous studies, done in western countries probably due to difference in haplotype.

The T117S Variant of Cytochrome b5 Reductase 3 Increases the Risk for Ischemic Stroke with Enhanced Anemia in Mice with Sickle Cell Disease

Introduction. Stroke and silent infarcts are serious complications of sickle cell disease (SCD), occurring frequently in children between 3 and 14 years old. A vast amount of clinical and experimental evidence has concluded that decreased nitric oxide (NO) bioavailability and/or NO responsiveness, as is seen in SCD, is a major contributing factor in the pathogenesis of neurovascular disease. NO responsiveness, which occurs via NO-induced activation of soluble guanylate cyclase (sGC), requires reduced heme iron (Fe2+) in the sGC active site. We recently identified cytochrome b5 reductase 3 (Cyb5R3) as an sGC heme iron reductase in vascular smooth muscle (VSM), where it reverses the oxidized heme iron of sGC (Fe3+ --&amp;gt; Fe2+) to preserve NO sensing/signaling under conditions of oxidative stress. In a mouse model of SCD we have shown that knockdown of Cyb5R3 in VSM accelerates the development of pulmonary hypertension and cardiac remodeling. A missense variant of Cyb5R3 (T117S) that results in loss-of-function methemoglobin reductase activity occurs at a high frequency in persons of African ancestry (0.23 minor allele frequency). Unpublished baseline data from the Walk-PHaSSt trial (NCT00492531) reveals that persons with SCD who carry the T117S variant are at increased risk of ischemic stroke; these individuals self-reported almost 50% more (74 vs 51 cases per 1000 individuals) ischemic stroke than those with wild-type (WT) Cyb5R3. Hypothesis. We hypothesized that impaired reductase function of Cyb5R3 T117S leads to sustained sGC heme oxidation, which drives cerebral vascular dysfunction and exacerbates brain damage after ischemic stroke in SCD. Methods. Bone marrow transplant was used to create SCD mice with global expression of WT or T117S Cyb5R3, hereafter referred to as SS/WT or SS/T117S, respectively. All mice were male, C57Bl/6 background, and &amp;gt;85% engrafted with SS Hb for 12 weeks. Ischemic stroke was induced using transient middle cerebral artery occlusion (MCAO: 55 min occlusion, 48 hr reperfusion), after which brains were stained with 2,3,5-triphenyltetrazolium chloride (TTC,1%) to determine infarct volume. Blood was sampled before and after MCAO to assess effects of brain infarct on hematological parameters. Student's t-test was used for analysis of 2 groups and Pearson's R used for correlation analyses of brain infarct volume with hematology changes [(post-pre/pre) * 100]. Results. Global expression of T117S Cyb5R3 in SCD caused increased cerebral infarct volume (62.9 vs 26.7 cm3, P=0.003) and mortality (3/6 vs 0/6) relative to WT Cyb5R3. WT and T117S Cyb5R3 mice with SCD were similar in that both showed declining red blood cells (RBC), hemoglobin (Hgb) and hematocrit (Hct) as infarct volumes increased. In the SS/T117S group, the anemia was more severe in keeping with larger infarct volumes. There were different signatures to the hematologic changes that occurred with cerebral infarct in SCD. When compared to WT Cyb5R3, T117S caused the erythroid compartment to contract (RBC: -12.97% vs 13.41%, P=0.01; Hct: -19.75% vs 0.31%, P=0.025; Hgb: -17.93% vs 2.78%, P=0.017). In SS/WT mice platelet numbers increased more relative to SS/T117S (17.5 vs 9.7 * 103 cells/uL); and MPV, a measure of platelet activation, inversely correlated with brain infarct volume (r = -0.94, P=0.006), the opposite of what was seen in SS/T117S (r = 0.87, P=0.056). Monocytes seem to play an important role in the volume of brain infarct in SS/T117S as their numbers increased in parallel with infarct volume (r = 0.73, P=0.16), but followed the opposite trajectory in SS/WT mice (r = -0.75, P=0.14). Conclusion. These results indicate that Cyb5R3 is an important modifying factor in the evolution and outcome of ischemic brain injury in SCD. Our findings also raise questions on just how cerebral infarct modifies the anemia of SCD, as well as the role played by Cyb5R3 in the dynamics of that relationship. To what extent is the sGC-cGMP-PKG pathway involved at the cerebrovascular and erythropoietic levels? Does Cyb5R3 contribute resilience to ischemic stroke in SCD? The development and application of targeted therapies for effectively preventing and treating cerebrovascular disease in SCD rely on finding the answers to these questions. Disclosures No relevant conflicts of interest to declare.

Hydroxyurea to Reduce Stroke Risk in Tanzanian Children with Sickle Cell Anemia

Introduction: Sickle cell anemia (SCA) is highly prevalent in sub-Saharan Africa with &amp;gt;300,000 annual births, and substantial morbidity and mortality due to limited resources. The burden of stroke in this population is of particular concern, given the devastating clinical and neurocognitive sequelae of these events. Hydroxyurea, a potent disease modifying therapy for SCA, is safe and feasible for low-resource and malarial endemic countries within sub-Saharan Africa and when used at maximum tolerated dose (MTD), decreases the incidence of acute painful vaso-occlusive events, infections, malaria, transfusions, hospitalizations, and death. Whether hydroxyurea can prevent primary stroke in SCA within Africa has not yet been determined, due in part to lack of stroke screening programs using transcranial Doppler (TCD) ultrasonography. If effective, hydroxyurea would have even more therapeutic benefits for children with SCA, particularly in settings where blood is not available, affordable, or safe. We designed the Stroke Prevention with Hydroxyurea Enabled through Research and Education (SPHERE) trial to determine the stroke risk among Tanzanian children using TCD screening and to investigate the effects of hydroxyurea to reduce that risk. Methods: The SPHERE trial (NCT03948867) is a single center prospective phase 2 open-label screening and treatment pilot study at Bugando Medical Centre, a teaching and referral hospital in Mwanza, Tanzania. Children 2-16 years old with SCA consented to TCD screening by locally trained and certified examiners; recent febrile illness, red cell transfusion, or hospitalization were temporary exclusions. Study participants with maximum Time-Averaged Mean Velocity (TAMV) on TCD exam categorized as conditional (170-199 cm/sec) or abnormal (≥200 cm/sec) are offered hydroxyurea with escalation to MTD, while those with normal TCD screening exams will be rescreened annually. Hydroxyurea is initiated at ~20 mg/kg/day using 500 mg capsules and a weekly dosing calculator, then escalated every 8 weeks by 5 mg/kg/day up to 35 mg/kg/day. Children on hydroxyurea are seen monthly during dose escalation and every 3 months after reaching MTD. The primary endpoint is change in TCD velocity after 12 months of hydroxyurea therapy. Secondary endpoints include changes in splenic volume and filtrative function; change in renal function; incidence of infection, especially malaria; hydroxyurea pharmacokinetics; and genetic modifiers of disease including pharmacogenomics. Results: From April 2019 to April 2020, a total of 202 children underwent TCD screening, exceeding the projected enrollment pace and goal (Figure). The average age (mean ± SD) at enrollment was 6.8 ± 3.5 years, and 53% were female. A majority had previous dactylitis (75%), painful vaso-occlusive episode (93%), blood transfusion (68%), and malaria (89%). Recurrent hospitalization was common with 30% having &amp;gt;5 previous hospitalizations. Only 4% had previously used hydroxyurea. Baseline labs included hemoglobin = 7.8 ± 1.3 g/dL, HbF = 9.3 ± 5.4 %, and ANC = 5.5 ± 2.4 x 109/L. Baseline assessment revealed a palpable spleen in 46 children (23%), and most of these (29) were ≥5 cm below the costal margin. Abdominal ultrasonography documented splenic tissue in 91% of children with an average volume of 101 ± 123 mL (range 8-1045). TCD examinations were performed in all children at enrollment with average TAMV of 148 ± 27 cm/sec [median 144, IQR 130-169 cm/sec] with 76% normal, 21% conditional, 2% abnormal, and 1% inadequate exams. Of 47 children eligible for hydroxyurea for elevated TCD velocities, 45 successfully initiated treatment, while 1 lived too far away for regular visits, and 1 had low blood counts from acute splenic sequestration and died before initiating study treatment. Conclusion: Children with SCA in Tanzania have a high risk for primary stroke. Identification of elevated TCD velocities through screening by local trained certified examiners, coupled with initiation of hydroxyurea treatment with dose escalation to MTD, offers a feasible and affordable means by which to lower TCD velocities and reduce primary stroke risk. Now fully enrolled, SPHERE has built local clinical capacity, research infrastructure and high-quality TCD screening, and will prospectively determine the benefits of hydroxyurea for stroke prevention, as a prelude for expanding hydroxyurea access for children with SCA in Tanzania. Figure Disclosures No relevant conflicts of interest to declare.

Intracranial and Extracranial Vascular Stenosis as Risk Factors for Stroke in Sickle Cell Disease

BackgroundPrevalence and contribution of intracranial and extracranial arterial stenosis to stroke risk were assessed prospectively in children and young adults with sickle cell disease. MethodsIn this cross-sectional study, children and young adults (mean = 19.4 years) with sickle cell disease underwent neurological examination, brain MRI, and magnetic resonance angiography of the head and neck. Two neuroradiologists independently recorded infarcts and arterial stenosis. Clinical features and stroke outcomes were compared between participants with and without stenosis and between children and young adults. Logistic regression analysis assessed the association of variables of interest with overt stroke and silent cerebral infarct. ResultsOf 167 participants (79 children and 88 young adults), 20 (12.0%) had intracranial stenosis, all in the anterior circulation, and nine had concurrent extracranial stenosis. No participants had isolated extracranial stenosis. Participants with intracranial stenosis were more likely than those without stenosis to have an overt stroke (70% vs 5%, P < 0.001) or silent cerebral infarct (95% vs 35%, P < 0.001). Logistic regression analysis indicated that intracranial stenosis was strongly associated with overt stroke when compared with participants with silent cerebral infarct alone and strongly associated with silent cerebral infarct when compared with participants with normal brain MRI; male sex and age were also significant predictors of silent cerebral infarct. ConclusionsIntracranial stenosis was strongly associated with both overt stroke and silent cerebral infarct; prevalence of intracranial stenosis was similar to prior estimates in sickle cell disease. Extracranial stenosis without concurrent intracranial stenosis did not occur and thus could not be evaluated as an independent risk factor for stroke.

Blood Cell Disorders and the Nervous System.

This article discusses the epidemiology, diagnosis, treatment, and prevention of neurologic complications of common and rare blood cell disorders. A growing number of preventive treatment options are available for stroke in sickle cell disease. Paroxysmal nocturnal hemoglobinuria and immune thrombocytopenia can lead to stroke. Thrombotic thrombocytopenic purpura frequently causes neurologic symptoms and should be considered in the differential diagnosis of a patient with neurologic symptoms, thrombocytopenia, and hemolytic anemia. Polycythemia vera and essential thrombocythemia are rare causes of stroke. This article discusses sickle cell disease and the most recent advances in stroke preventive therapy as well as neurologic complications of paroxysmal nocturnal hemoglobinuria, immune thrombocytopenia, thrombotic thrombocytopenic purpura, polycythemia vera, and essential thrombocythemia.