An e u ry s m a l subarachnoid hemorrhage affects 8

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1 J Neurosurg 112: , 2010 Interaction of neurovascular protection of erythropoietin with age, sepsis, and statin therapy following aneurysmal subarachnoid hemorrhage Clinical article Min g-yu a n Ts e n g, M.D., Ph.D., Pe t e r J. Hu t c h i n s o n, Ph.D., F.R.C.S.(SN), a n d Pe t e r J. Ki r k pa t r i c k, F.Me d.sc i Neurosurgical Unit, Department of Clinical Neurosciences, Addenbrooke s Hospital, University of Cambridge, United Kingdom Object. In a previous randomized controlled trial, the authors demonstrated that acute erythropoietin (EPO) therapy reduced severe vasospasm and delayed ischemic deficits (DIDs) following aneurysmal subarachnoid hemorrhage. In this study, the authors aimed to investigate the potential interaction of neurovascular protection by EPO with age, sepsis, and concurrent statin therapy. Methods. The clinical events of 80 adults older than 18 years and with < 72 hours of aneurysmal subarachnoid hemorrhage, who were randomized to receive 30,000 U of intravenous EPO-β or placebo every 48 hours for a total of 3 doses, were analyzed by stratification according to age (< or 60 years), sepsis, or concomitant statin therapy. End points in the trial included cerebral vasospasm and impaired autoregulation on transcranial Doppler ultrasonography, DIDs, and unfavorable outcome at discharge and at 6 months measured with the modified Rankin Scale and Glasgow Outcome Scale. Analyses were performed using the t-test and/or ANOVA for repeated measurements. Results. Younger patients (< 60 years old) or those without sepsis obtained benefits from EPO by a reduction in vasospasm, impaired autoregulation, and unfavorable outcome at discharge. Compared with nonseptic patients taking EPO, those with sepsis taking EPO had a lower absolute reticulocyte count (nonsepsis vs sepsis, vs x 10 9 /L on Day 6; p = 0.01), suggesting sepsis impaired both hematopoiesis and neurovascular protection by EPO. In the EPO group, none of the statin users suffered DIDs (p = 0.078), implying statins may potentiate neuroprotection by EPO. Conclusions. Erythropoietin-related neurovascular protection appears to be attenuated by old age and sepsis and enhanced by statins, an important finding for designing Phase III trials. (DOI: / JNS09954) Ke y Wo r d s erythropoietin neuroprotection sepsis statin therapy subarachnoid hemorrhage An e u ry s m a l subarachnoid hemorrhage affects 8 of every 100,000 people in Great Britain every year. Unfavorable outcome is often associated with vasospasm-related DIDs. 16 Results of clinical studies have suggested that in addition to securing the culprit aneurysm, a global approach applying pluripotential neuroprotection is more likely to reduce the unfavorable outcome instead of therapies specifically targeting reversal of cerebral vasospasm. 26 In a randomized, double-blind, placebo-controlled trial, we previously demonstrated that systemic therapy using high doses of EPO within 72 hours of asah reduces severity of vasospasm, duration of impaired autoregulation, incidence of DIDs, and unfavorable outcome at discharge. 29 The neurovascular protection of EPO may involve ischemic preconditioning in both neurons and vascular endothelia and an early activation of endothelial Abbreviations used in this paper: asah = aneurysmal subarachnoid hemorrhage; DID = delayed ischemic deficit; EPO = erythropoietin; GOS = Glasgow Outcome Scale; mrs = modified Rankin Scale; TCD = transcranial Doppler. EPO receptor, resulting in stimulation of endothelial NO synthase. 20 In this study, randomization characteristics were balanced except for age, with the EPO group significantly older (mean age 59.6 vs 53.3 years; p = 0.034). It is known that systemic inflammatory response and sepsis have adverse effects on hematopoietic effects of EPO, 6 and we have discovered that statin drugs reduce vasospasm and DIDs via multiple mechanisms, including antiinflammation. 30 Whether statins can synergistically enhance neurovascular protection of EPO is unclear. The objective of this study was to investigate potential interaction of neurovascular protective effects of EPO with age, sepsis, and concurrent statin therapy. Information obtained in this study may be helpful in designing Phase III studies in the future. Methods Study Design and Treatment Details of the trial have been published previously. 29 The trial was approved by Cambridge Local Research Ethics Committee (04/Q0108/87) and Medicines 1235

2 M. Y. Tseng, P. J. Hutchinson, and P. J. Kirkpatrick and Healthcare Products Regulatory Agency (EudraCT ), and registered with ClinicalTrials.org (NCT ). Briefly, 80 adult patients older than 18 years of age with confirmation of asah within 72 hours of symptoms were randomized to receive placebo (0.9% saline) or 30,000 U of EPO (epoetin-β, NeoRecormon, Roche Products) on the day of recruitment and then every 48 hours for a total of 3 doses (90,000 U). Both patients and investigators were blind to the treatment allocation until the clinical outcome for the last recruited patient was assessed. Daily TCD ultrasonography (DWL Multi-Dop 4, Sipplingen) was performed for up to 14 days. Cerebral vasospasm on TCD was defined according to 3 criteria: 1) mean flow velocity of the middle cerebral artery > 1.2 m/ second (> 2.0 m/second for severe vasospasm); 1 2) Lindegaard ratio > 3; 15 and 3) an increase in mean flow velocity > 0.5 m/second within 24 hours. 10 Cerebral autoregulation on TCD was assessed with the transient hyperemic response test and defined as preserved or impaired. 9,25 Sepsis was defined according to the criteria of the American College of Chest Physicians/Society of Critical Care Medicine. 2 Delayed ischemic deficits was defined as new focal neurological deficits or reductions in Glasgow Coma Scale score 2 points and other causes of neurological deterioration were excluded. Outcomes at discharge and at 6 months were assessed according to the mrs 3 and GOS 11 as favorable (mrs score 0 3, or GOS classification as good recovery/moderate disability) or unfavorable (mrs score 4 6, or GOS classification as severe disability/vegetative/death). Primary end points were incidence of vasospasm on TCD and duration of impaired autoregulation on the transient hyperemic response test. Secondary end points were incidence of DIDs and outcomes at discharge and at 6 months. Statistical Analysis Analyses were performed as intention-to-treat using STATA Intercooled 8.0 for Windows. Data were presented as means ± SD with 95% CIs. All probability values were 2-sided and a probability value < 0.05 was considered statistically significant. Randomization characteristics, incidence of vasospasm, and secondary end points were compared using the chi-square or Fisher exact test (when any number of the cell was < 5). Durations of vasospasm and impaired autoregulation were compared using the Mann-Whitney U-test and separately according to the side of ruptured aneurysm. Laboratory data were examined using the ANOVA for repeated measurements. When the analysis showed significance within patients, tables of Dunnett correction were applied to identify significant changes over time. 7 End point measures were further stratified according to patient age as < or 60 years (giving roughly similar sample sizes; ratio of patients < 60 to 60 years was 50:30), concurrent statin therapy, and sepsis. Results Patient Population Results of end points have been published previously. 29 In summary, pre- and postrandomization characteristics were balanced, except for age, in which the EPO group was older (mean age 59.6 vs 53.3 years; p = 0.034). Compared with the placebo group, the incidence of severe vasospasm was significantly reduced in the EPO group from 27.5 to 7.5% (p = 0.037) and the duration of impaired autoregulation on the ipsilateral side was shortened by 3.2 days (p < 0.001) in the EPO arm. Nineteen patients had DIDs, which were reduced by EPO (16 placebo, 3 EPO; p = 0.001). Thirty patients had sepsis (12 placebo, 18 EPO; p = 0.17). Fourteen patients were taking statins (6 placebo, 8 EPO; p = 0.56). The 9 deaths during hospitalization included 5 patients with rebleeds (3 placebo, 2 EPO), 2 with immediate postoperative infarcts (1 placebo, 1 EPO), and 2 with vasospasm-related infarcts (1 placebo, 1 EPO). There were more patients achieving favorable outcome at discharge by EPO (favorable mrs score, p = 0.10; favorable GOS score, p = 0.039), although there was no difference in the outcome at 6 months. Effect of Age Only younger patients (< 60 years of age) obtained benefits of cerebrovascular protection from EPO in reducing vasospasm, severe vasospasm, and impaired autoregulation (Table 1). Both older and younger patients receiving EPO had reduced incidence of DIDs, but only the younger ones were more likely to achieve favorable outcomes at discharge (favorable mrs score, p = 0.019; favorable GOS score, p = 0.028). Both younger and older patients had similar erythropoiesis enhanced by EPO, that is, significant increments in absolute reticulocyte count from Days 3 to 12. Effect of Sepsis Although patients receiving EPO with sepsis obtained benefits in reduced durations of impaired autoregulation (ipsilateral side) and incidence of DIDs as those without sepsis (Table 2), their outcome was not improved by EPO and the absolute reticulocyte count was lower on Day 6 (nonsepsis vs sepsis, vs x 10 9 /L; p = 0.01; Fig. 1), suggesting sepsis may have attenuated both the hematopoietic and neurovascular protective effects of EPO. Effect of Statin Therapy Compared with patients not taking statins (66 patients), those receiving concurrent statin therapy (14 patients) had a lower incidence of vasospasm on TCD (28.6 vs 57.6%; p = 0.048; t-test). It was notable that none of the 8 statin users randomized to EPO experienced DIDs (0/8 vs 2/6, p = 0.078; t-test). Discussion Results of this study suggest that the strength of neurovascular protective effects of EPO may vary under different pathophysiological conditions and appears to be proportional to the hematopoietic effect. Although the trial size was not powered to detect variations among subgroups, results from the post hoc analyses imply certain populations may benefit more from EPO therapy, such as younger patients or patients taking statin drugs. 1236

3 Neurovascular protection of erythropoietin following asah TABLE 1: Results of end points according to age End Point Placebo EPO p Value Vasospasm yes no 5 12 yes no 9 12 Severe Vasospasm yes no yes no DIDs yes no yes no 8 16 Favorable mrs Score at Discharge yes no 11 3 yes no 7 8 Favorable GOS Score at Discharge yes no 11 3 yes no 9 8 Days of Impaired Autoregulation* ipsilateral side 7.2 ± ± 2.7 <0.001 ( ) ( ) contralateral side 4.8 ± ± ( ) ( ) ipsilateral side 5.6 ± 3.7 ( ) 5.0 ± 3.1 ( ) 0.76 contralateral side 3.5 ± 3.1 ( ) * Data presented as mean ± SD (95% CI). 4.7 ± 3.1 ( ) 0.24 TABLE 2: Results of end points according to sepsis End Point Placebo EPO p Value Vasospasm yes no yes no 4 7 Severe Vasospasm yes no yes no DIDs yes no yes no 6 16 Favorable mrs Score at Discharge yes no 10 2 yes no 8 9 Favorable GOS Score at Discharge yes no 12 2 yes no 8 9 Days of Impaired Autoregulation* ipsilateral side 6.3 ± ± ( ) ( ) contralateral side 4.2 ± ± ( ) ( ) ipsilateral side 7.3 ± 4.2 ( ) 4.0 ± 2.8 ( ) contralateral side 4.7 ± 4.7 ( ) * Data presented as mean ± SD (95% CI). 3.6 ± 2.5 ( )

4 M. Y. Tseng, P. J. Hutchinson, and P. J. Kirkpatrick Fig. 1. Bar graph showing a comparison of absolute reticulocyte counts between the trial groups in nonseptic and septic patients. Each box represents the median value. Each whisker represents the 75th (upper hinge) and the 25th (lower hinge) percentiles. Dots (above whiskers) represent outliers, that is, 1.5 times the interquartile range. Asterisks represent p < 0.05 between the trial groups after repeated measurement ANOVA and Dunnett adjustment. The solid horizontal line represents the upper limit of normal absolute reticulocyte counts ( /L). Erythropoietin has been shown to have antiapoptotic effects on both endothelial cells and neurons via hypoxia-inducible factor-1α mediated intracellular signaling. 4 The ability of EPO to enhance cell survival and reduce inflammation derives from blockage on the upstream pathway of apoptosis involving Janus-tyrosine kinase 2 protein and protein kinase B. 14 The neurovascular protection has been shown to play an essential role in reducing early brain injury following subarachnoid hemorrhage in animal studies. 18 However, experimental studies have also shown that EPO itself can directly dilate middle cerebral arteries and pretreatment with EPO 24 hours before subarachnoid hemorrhage enhances endothelium-mediated vasodilatation, suggesting an additional antivasospastic effect. 22 Because the latency of neosynthesis of EPO in the brain is relatively lengthy, the local inflammation induced by cerebral ischemia inhibits both EPO synthesis and expression of the EPO receptor, and severe cerebral ischemia may soon exhaust the paracrine mechanism of EPO. 23 Immediately systemic supplements of EPO following asah may secure further neuroprotection against delayed cerebral ischemia as shown by this trial. The stratification of patient age at 60 years was arbitrary, but this division was in keeping with previous studies concerning the outcome of patients following asah 13 and cerebrovascular accidents. 19 Why was EPO less effective in reducing vasospasm and impaired autoregulation among older patients? Aging is known to be associated with both progressive endothelial dysfunction 8 and defects in the signaling cascade of ischemic preconditioning, that is, reduced phospholipid cardiolipin and cytochrome c oxidase activity and impaired efficiency of oxidative phosphorylation and antioxidant systems in mitochondria. 21 Therefore, the antiapoptosis of vascular endothelia or antivasospastic effects by EPO may be less pronounced in older than younger patients, further suggesting vasospasm is not the only contributor to DIDs. 26 The more unfavorable outcome in older patients may be related to other systemic factors, that is, sepsis or cardiovascular dysfunction. 13 Sepsis and systemic inflammatory response are known to suppress the hematopoietic effect of EPO on bone marrow, a condition similar to EPO resistance. 6 Furthermore, the inflammatory cytokines incurred during sepsis can cause systemic and cerebral endothelial dysfunction and, therefore, impair cerebral vasomotor reactivity and autoregulation. 27 Although intracerebral EPO can protect neurons from phagocytosis by inhibiting local production of inflammatory cytokines and activation of microglia, 5 systemic inflammation may accentuate a similar EPO resistance in the brain. The requirement of using a higher dose of EPO for overcoming resistance needs further investigation. Why were there similar reductions in DIDs between patients < and 60 years old, and between those with and without sepsis, but not other end points observed? As mentioned above, both aging and sepsis are associated with vascular endothelial dysfunction, and therefore, the results suggest that competent cerebrovascular endothelia may be a prerequisite for EPO to execute vascular protection, but the direct neuroprotection (antiapoptosis of neurons) may be independent of the cerebrovascular effect. In this study, patients taking statins had reduced vasospasm, a finding consistent with previous trials. 17,28 It is notable that none of those on statin therapy allocated to the EPO arm experienced DIDs. Of additional interest is the finding that the antiinflammatory effect of statin drugs improves EPO responsiveness in patients receiving regular hemodialysis. 24 Observation studies also suggest that the statins may reduce occurrence or severity of sepsis. 12 These associations raise the intriguing prospect of a potential synergism between statins and EPO by targeting different components of the pathophysiological cascade leading to delayed cerebral ischemia. The main limitation of this study is a relatively small sample size and a single-center trial, and it is not powered to detect interaction of EPO with age, sepsis, and statin therapy, where data from preclinical studies are scarce. However, findings from the post hoc analyses raise the hypotheses that a further larger dose of EPO, a longer duration of EPO therapy, or a combined therapy with statins may be required in older patients or patients with sepsis following asah. Conclusions This study demonstrated that EPO-related hematopoietic and neuroprotective effects may be attenuated by age and sepsis but enhanced by statin drugs, an important finding in designing Phase III studies. Disclosure Dr. Tseng was supported by a research grant from the Roche Foundation for Anemia Research (Meggen, Switzerland, I.D. No ) from December 2005 to November

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J Neurosurg 107: , 2007 Manuscript submitted June 18, Accepted October 19, Please include this information when citing this paper: published online November 20, 2009; DOI: / JNS Address correspondence to: Ming-Yuan Tseng, M.D., Ph.D., De partment of Neurosurgery, Box 167, Level 4, Block A, Ad denbrooke s Hospital, Cambridge, United Kingdom CB2 2QQ. myt22@cam.ac.uk. 1239