Th e rupture of intracranial aneurysms is the most. Prognostic value of histopathological findings in aneurysmal subarachnoid hemorrhage

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1 J Neurosurg 110: , 2009 Prognostic value of histopathological findings in aneurysmal subarachnoid hemorrhage Clinical article *Ma r k u s Ho l l i n g, M.D., 1 3 As t r i d Je i b m a n n, M.D., 1 Jo a c h i m Ge r s s, Ph.D., 4 Be r n h a r d R. Fi s c h e r, M.D., 2 Ha n s d e t l e f Wa s s m a n n, M.D., 2 We r n e r Pa u l u s, M.D., 1 Mar t i n Ha s s e l b l a t t, M.D., 1 a n d Fr i e d r i c h K. Al b e r t, M.D. 3 Institutes of 1 Neuropathology and 4 Medical Informatics and Biomathematics; 2 Department of Neurosurgery, University Hospital Münster; and 3 Department of Neurosurgery, Paracelsus-Klinik Osnabrück, Germany Object. Aneurysmal subarachnoid hemorrhage (SAH) carries a severe prognosis, which is often related to the development of cerebral vasospasm. Even though several clinical and radiological predictors of vasospasm and functional outcome have been established, the prognostic value of histopathological findings remains unclear. Methods. Histopathological findings in resected distal aneurysm walls were examined, as were the clinical and radiological factors in a series of 91 patients who had been neurosurgically treated for aneurysmal SAH. The impact of the histological, clinical, and radiological factors on the occurrence of vasospasm and functional outcome at discharge was analyzed. Results. Histopathological findings frequently included lymphocytic infiltrates (60%), fibrosis (60%), and necrosis (50%) of the resected aneurysm wall. On univariate analysis, clinical (Hunt and Hess grade) and radiological (aneurysm size) factors as well as histopathological features namely, lymphocytic infiltrates and necrosis of the aneurysm wall were significantly associated with the occurrence of vasospasm. On multivariate analysis, lymphocytic infiltrates (OR 6.35, 95% CI , p = ) and aneurysm size (OR 1.22, 95% CI , p = 0.009) remained the only factors predicting the development of vasospasm. A poor functional outcome at discharge was significantly associated with vasospasm, other clinical factors (Hunt and Hess grade, alcohol consumption, hyperglycemia, and elevated white blood cell count [WBC] at admission), and radiological factors (Fisher grade and aneurysm size), as well as with histopathological features (lymphocytic infiltrates [p = ] and necrosis of the aneurysm wall [p = ]). On multivariate analysis taking into account all clinical, radiological, and histological factors; vasospasm (OR 9.82, 95% CI , p = 0.008), Hunt and Hess grade (OR 5.61, 95% CI , p = ), patient age (OR 1.09, 95% CI , p = ), elevated WBC (OR 1.29, 95% CI , p = 0.04), and Fisher grade (OR 4.35, 95% CI , p = 0.015) best predicted functional outcome at discharge. Conclusions. The demonstration of lymphocytic infiltrates in the resected aneurysm wall is of independent prognostic value for the development of vasospasm in patients with neurosurgically treated aneurysmal SAH. Thus, histopathology might complement other clinical and radiological factors in the identification of patients at risk. (DOI: / JNS08789) Key Words aneurysm inflammation prognosis vasospasm Abbreviations used in this paper: BMI = body mass index; GOS = Glasgow Outcome Scale; SAH = subarachnoid hemorrhage; SMC = smooth-muscle cell; WBC = white blood cell count. *Drs. Holling and Jeibmann contributed equally to this study. Th e rupture of intracranial aneurysms is the most frequent cause of nontraumatic SAH. Because aneurysmal SAH still carries a severe prognosis, often related to the development of vasospasm, the assessment of robust prognostic markers for vasospasm and functional outcome has been a major focus of neurosurgical research. 19 Although a variety of clinical and radiological predictors have been well established, 5,9,12 16 the prognostic value of the histopathological findings frequently observed in the aneurysm wall, such as inflammatory infiltrates, 6,11 remains uncertain. We therefore analyzed the prognostic value of histopathological findings in resected distal aneurysm walls in relation to other clinical and radiological factors in a series of 91 neurosurgically treated patients with aneurysmal SAH. This article contains some figures that are displayed in color on line but in black and white in the print edition. 487

2 M. Holling et al. Patient Population Methods Ninety-one patients who had been neurosurgically treated for aneurysmal SAH by 1 neurosurgeon (F.K.A.) at the Department of Neurosurgery, Paracelsus-Klinik Osnabrück, Germany, throughout a period of 7 years ( ) were included in this retrospective observational study. We compiled clinical data on patient sex, age, BMI, nicotine consumption, known alcohol abuse, the interval from SAH to surgery, Hunt and Hess grade, serum glucose concentration, and WBC. Cerebral blood flow was monitored routinely in all patients by using transcranial Doppler ultrasonography. Vasospasm was defined as a mean flow velocity > 120 cm/second, detected at least twice by 2 independent examiners. Radiological data included aneurysm location, Fisher grade, and maximum aneurysm diameter as determined in 3 orthogonal planes on digital subtraction angiograms. Outcome at discharge was assessed using the GOS. For our purposes in the present study, a GOS score of 5 (good recovery) or 4 (moderate disability) was defined as a favorable outcome. Histopathological Examination We histopathologically examined samples that had been routinely obtained during surgery by resecting the aneurysm sac distal to the clip. Without knowing the clinical course and outcome associated with each sample, we analyzed H & E and elastic van Gieson stained sections from paraffin-embedded formalin-fixed specimens for the presence or absence of intramural lymphocytic infiltrates, vessel wall necrosis, vessel wall fibrosis, SMC hyperplasia, and the presence of hemosiderin as well as periadventitial vessels. Statistical Analysis A comparison of patient characteristics was made using the chi-square test or Mann-Whitney U-test. The impact of clinical (age, sex, BMI, known alcohol or nicotine consumption, Hunt and Hess grade, serum glucose concentration and WBC at admission, and the time to surgical intervention), radiological (Fisher grade and aneurysm size), and histopathological features (presence of lymphocytic infiltrates, necrosis, fibrosis, SMC hyperplasia, hemosiderin, or periadventitial vessels) on vasospasm and patient outcome at discharge were evaluated using logistic regression analysis. Forward stepwise model selection was performed with the Wald approach. Statistical analyses were performed using SPSS, version 16.0 (SPSS, Inc.). Results Clinical and Radiological Findings As shown in Table 1, the median age of the 30 men and 61 women at the time of surgery was 51 years (range 18 80). The median BMI was 24.2 ( ). Thirtyfour patients (37%) were smokers, and alcohol abuse was a factor in 7 patients (8%). The clinical severity of SAH was graded using the Hunt and Hess classification: Grade I, 18 patients (20%); Grade II, 25 patients (27%); Grade III, 16 patients (18%); Grade IV, 28 patients (31%); and Grade V, 4 patients (4%). At admission, hyperglycemia (> 100 mg/ dl) and an elevated WBC (> /L) were noted in 83 patients (91%) and 71 patients (78%), respectively. Most often, ruptured aneurysms were located in the territory of the middle cerebral (36 patients) and anterior communicating artery (27 patients) as well as other vessels of the anterior circulation (22 patients), whereas the posterior circulation was less frequently affected (6 patients). The amount and distribution of subarachnoid blood, as detected using CT, was classified as Fisher Grade 1 in 3 patients (3%), Grade 2 in 14 patients (15%), Grade 3 in 45 patients (50%), and Grade 4 in 29 patients (32%). The median aneurysm size was 7 mm (range 3 23 mm). In 28 patients, multiple aneurysms were encountered. The median time from hemorrhage to surgery was 2 days (range 0 15 days). Histopathological Findings Histopathological examination of the resected aneurysm wall revealed lymphocytic infiltrates in 55 cases (60%; Fig. 1A) and necrosis of the vessel wall in 45 cases (49%; Fig. 1B). Fibrosis of the aneurysm wall was present in 55 cases (60%; Fig. 1C), and 36 specimens (40%) showed SMC hyperplasia (Fig. 1D). Hemosiderin deposits were present in 33 samples (36%; Fig. 1E); periadventitial vessels were noted in 27 cases (30%; Fig. 1F). As shown in Fig. 2, the extent of lymphocytic infiltrates (absent, weak, or strong) and the timing of surgery were not correlated (p = 0.402, Spearman rank correlation coefficient). Except for necrosis of the aneurysm wall (p = 0.02), other histopathological findings (fibrosis, periadventitial vessels, SMC hyperplasia, and hemosiderin) were also unrelated to the timing of surgery. Vasospasm and Functional Outcome Postoperatively, vasospasm developed in 49 patients (54%). The outcome at discharge a median of 28 days (range days) after surgery was favorable (GOS Score 4 5) in 52 patients (57%), whereas outcome was poor (GOS Score 1 3) in 39 patients (43%). Among this latter group, 9 patients died (GOS Score 1). Prognostic Value of Clinical, Radiological, and Neuropathological Factors for the Occurrence of Vasospasm As shown in Table 1, on univariate analysis, not only clinical (Hunt and Hess grade) and radiological factors (aneurysm size), but also histopathological features (namely, lymphocytic infiltrates and necrosis of the aneurysm wall) were significantly associated with vasospasm. However, on multivariate analysis taking into account all clinical, radiological, and histological factors, aneurysm size (OR 1.22, 95% CI , p = 0.009) and lymphocytic infiltrates of the aneurysm wall (OR 6.35, 95% CI , p = ) remained the only factors independently predicting the development of vasospasm. Prognostic Value of Clinical, Radiological, and Neuropathological Factors for Functional Outcome A poor functional outcome at discharge was signifi- 488

3 Histopathological findings in subarachnoid hemorrhage TABLE 1: Clinical, radiological, and histopathological factors in 91 patients with aneurysmal SAH and their association with vasospasm and functional outcome* Factor Total No. Vasospasm Present Vasospasm Absent p Value Good Functional Outcome Poor Functional Outcome no. of patients clinical median age in yrs (range) 51 (18 80) 50 (18 80) 51 (28 79) (18 77) 51 (33 80) 0.03 male/female 30:61 33:16 28: :18 27: median BMI (range) 24.2 ( ) 24.2 ( ) 24.2 ( ) (18 42) 24 (18 35) 0.67 known nicotine use (%) 34 (37) 18 (37) 16 (38) (35) 16 (41) 0.53 known alcohol abuse (%) 7 (8) 4 (8) 3 (7) 1 1 (2) 6 (15) 0.04 median H & H grade (range) 3 (1 5) 2 (1 5) 4 (1 5) (1 5) 4 (1 4) median glucose concentration in mg/dl 132 (77 289) 131 (92 289) 132 (77 222) (77 211) 132 (92 289) (range) median WBC in 10 9 /L (range) 12 (6 29) 12 (7 27) 12 (6 29) (6 21) 12 (7 29) 0.03 median no. days to surgical intervention 2 (0 15) 2 (0 15) 2 (0 15) (0 15) 2 (0 15) 0.38 (range) radiological median Fisher grade (range) 3 (1 4) 3 (1 4) 3 (1 4) (1 4) 3 (2 4) median aneurysm size in mm (range) 7 (3 23) 7 (3 23) 7 (3 18) (3 18) 7 (3 23) 0.01 histological (%) lymphocytic infiltrates 55 (60) 38 (78) 17 (40) (38) 35 (90) necrosis 45 (50) 30 (61) 15 (36) (38) 25 (64) fibrosis 55 (60) 33 (67) 22 (52) (54) 27 (69) SMC hyperplasia 36 (40) 20 (41) 16 (38) (40) 15 (38) 0.86 hemosiderin 33 (36) 17 (35) 16 (38) (33) 16 (41) 0.41 periadventitial vessels 27 (30) 13 (27) 14 (33) (27) 13 (33) 0.51 * H & H = Hunt and Hess. Glasgow Outcome Scale Score 4 5. Glasgow Outcome Scale Score 1 3. p Value cantly associated with vasospasm as well as other clinical (age, Hunt and Hess grade, alcohol consumption, hyperglycemia, and elevated WBC at admission), radiological (Fisher grade and aneurysm size), and histopathological features (lymphocytic infiltrates and necrosis of the aneurysm wall). On multivariate analysis taking into account all clinical, radiological, and histological factors; vasospasm (OR 9.82, 95% CI , p = 0.008), Hunt and Hess grade (OR 5.61, 95% CI , p = ), age (OR 1.09, 95% CI , p = ), elevated WBC (OR 1.29, 95% CI , p = 0.04), and Fisher grade (OR 4.35, 95% CI , p = 0.015) best contributed to a prediction of functional outcome at discharge. Discussion In this series of 91 consecutively treated patients with aneurysmal SAH, histopathological findings namely, the presence of lymphocytic infiltrates in the aneurysm wall predicted the occurrence of vasospasm, which in turn most contributed to the prediction of a bad functional outcome at discharge. Inflammatory changes have been related to aneurysm formation and rupture and even have been encountered in unruptured intracranial aneurysms. 2,6 Indeed, results from animal studies have suggested that an inflammatory reaction in response to endothelial injury represents the basic step in the pathogenesis of aneurysm formation. 10 Even though inflammatory infiltrates occur more frequently in ruptured than in unruptured aneurysms, 6,11 the prognostic implications of their presence in ruptured aneurysms has not been investigated. Importantly, in the present study, the histopathological finding of lymphocytic infiltrates of the aneurysm wall remained an independent predictor for the occurrence of vasospasm on multivariate analysis, exceeding the prognostic value of aneurysm size, the only clinical variable identified in this series as being independently associated with vasospasm. Notably, the presence of lymphocytic infiltrates was also independent from the timing of surgery, arguing against the possibility that these infiltrates merely reflect reactive changes. The routine histopathological examination of a resected aneurysm wall for the presence of lymphocytic infiltrates on H & E staining thus may provide reliable and easily accessible additional information for the identification of patients at risk for vasospasm. 489

4 M. Holling et al. Fig. 1. Photomicrographs showing lymphocytic infiltrates (A), necrosis (B), fibrosis (C), SMC hyperplasia (D), and hemosiderin (E) within the aneurysm wall as well as periadvential vessels in the vicinity of the aneurysm wall (F). H & E (A, D, E, and F) and van Gieson (B and C). Original magnification 200 and 400 (insets). It is tempting to speculate that the observed inflammatory changes are causally linked to the later development of vasospasm. Early activation of inflammatory mediators by inflammatory infiltrates in the aneurysm wall (and possibly the cerebral vasculature) may predispose one to the later development of vasospasm. On the other hand, these findings could also reflect high local levels of cytokines attracting lymphocytes to the aneurysm wall. Indeed, results from animal studies have highlighted the role of proinflammatory cytokines such as interleukin-61 in the development of vasospasm.4 In humans, increased CSF concentrations of interleukin-6 herald the development of vasospasm and can be used as an early predictive marker.17 Thus far, however, clinical trials have failed to demonstrate a beneficial effect of antiinflammatory treatment.7 With respect to functional outcome, vasospasm was the strongest determinant, and additional factors were age, preoperative Hunt and Hess grade, Fisher grade, and leukocytosis. These findings are well in line with previ490 ous observations.3,8,9,14 Even though on univariate analysis, histopathological findings, such as lymphocytic infiltrates and necrosis of the aneurysm wall, were associated with outcome, our data do not support an independent predictor role for histopathological findings with regard to functional outcome. Given the observational nature of this retrospective study, however, functional outcome could only be assessed at discharge, and the effect of histopathological findings on functional outcome may have been underestimated. The prognostic role of histopathological findings on long-term functional outcome will need to be evaluated further in future prospective studies. Conclusions The results of this retrospective study suggest that the presence of lymphocytic infiltrates on histopathological examination of the resected aneurysm wall is an independent prognostic marker for the development of vasospasm

5 Histopathological findings in subarachnoid hemorrhage Fig. 2. Graph depicting the relationship between lymphocytic infiltrates and the timing of surgery. The extent of lymphocytic infiltrates in the aneurysm wall (0 = absent, 1 = weak, 2 = strong) and the timing of surgery are not correlated (p = 0.402, Spearman rank correlation coefficient). in patients with neurosurgically treated aneurysmal SAH. Thus, histopathological examination of the resected aneurysm wall may complement other clinical and radiological factors in identifying patients at risk for vasospasm. Disclaimer The authors report no conflict of interest concerning the materials or methods used in this study or the findings specified in this paper. Acknowledgments We thank Maria Leisse and Andrea Rothaus for expert technical assistance. References 1. Bowman G, Bonneau RH, Chinchilli VM, Tracey KJ, Cockroft KM: A novel inhibitor of inflammatory cytokine production (CNI-1493) reduces rodent post-hemorrhagic vasospasm. Neurocrit Care 5: , Chyatte D, Bruno G, Desai S, Todor DR: Inflammation and intracranial aneurysms. Neurosurgery 45: , Dhar R, Diringer MN: The burden of the systemic inflammatory response predicts vasospasm and outcome after subarachnoid hemorrhage. Neurocrit Care 8: , Dumont AS, Dumont RJ, Chow MM, Lin CL, Calisaneller T, Ley KF, et al: Cerebral vasospasm after subarachnoid hemorrhage: putative role of inflammation. Neurosurgery 53: , Fergusen S, Macdonald RL: Predictors of cerebral infarction in patients with aneurysmal subarachnoid hemorrhage. Neurosurgery 60: , Frösen J, Piippo A, Paetau A, Kangasniemi M, Niemela M, Hernesniemi J, et al: Remodeling of saccular cerebral artery aneurysm wall is associated with rupture: histological analysis of 24 unruptured and 42 ruptured cases. Stroke 35: , Hansen-Schwartz J, Vajkoczy P, Macdonald RL, Pluta RM, Zhang JH: Cerebral vasospasm: looking beyond vasoconstriction. Trends Pharmacol Sci 28: , Hijdra A, van Gijn J, Nagelkerke NJ, Vermeulen M, van Crevel H: Prediction of delayed cerebral ischemia, rebleeding, and outcome after aneurysmal subarachnoid hemorrhage. Stroke 19: , Hoh BL, Topcuoglu MA, Singhal AB, Pryor JC, Rabinov JD, Rordorf GA, et al: Effect of clipping, craniotomy, or intravascular coiling on cerebral vasospasm and patient outcome after aneurysmal subarachnoid hemorrhage. Neurosurgery 55: , Jamous MA, Nagahiro S, Kitazato KT, Tamura T, Aziz HA, Shono M, et al: Endothelial injury and inflammatory response induced by hemodynamic changes preceding intracranial aneurysm formation: experimental study in rats. J Neurosurg 107: , Kataoka K, Taneda M, Asai T, Kinoshita A, Ito M, Kuroda R: Structural fragility and inflammatory response of ruptured cerebral aneurysms. A comparative study between ruptured and unruptured cerebral aneurysms. Stroke 30: , Lagares A, Gomez PA, Lobato RD, Alen JF, Alday R, Campollo J: Prognostic factors on hospital admission after spontaneous subarachnoid haemorrhage. Acta Neurochir (Wien) 143: , Macdonald RL, Rosengart A, Huo D, Karrison T: Factors associated with the development of vasospasm after planned surgical treatment of aneurysmal subarachnoid hemorrhage. J Neurosurg 99: , Niskanen MM, Hernesniemi JA, Vapalahti MP, Kari A: Oneyear outcome in early aneurysm surgery: prediction of outcome. Acta Neurochir (Wien) 123:25 32, Rosengart AJ, Schultheiss KE, Tolentino J, Macdonald RL: Prognostic factors for outcome in patients with aneurysmal subarachnoid hemorrhage. Stroke 38: , Salary M, Quigley MR, Wilberger JE Jr: Relation among aneurysm size, amount of subarachnoid blood, and clinical outcome. J Neurosurg 107:13 17, Schoch B, Regel JP, Wichert M, Gasser T, Volbracht L, Stolke D: Analysis of intrathecal interleukin-6 as a potential predictive factor for vasospasm in subarachnoid hemorrhage. Neurosurgery 60: , Solenski NJ, Haley EC Jr, Kassell NF, Kongable G, Germanson T, Truskowski L, et al: Medical complications of aneurysmal subarachnoid hemorrhage: a report of the multicenter, cooperative aneurysm study. Participants of the Multicenter Cooperative Aneurysm Study. Crit Care Med 23: , Suarez JI: Treatment of ruptured cerebral aneurysms and vasospasm after subarachnoid hemorrhage. Neurosurg Clin N Am 17 (Suppl 1):57 69, van Gijn J, Rinkel GJ: Subarachnoid haemorrhage: diagnosis, causes and management. Brain 124: , 2001 Manuscript submitted June 24, Accepted August 25, Please include this information when citing this paper: published online December 1, 2008; DOI: / JNS Address correspondence to: Martin Hasselblatt, M.D., Institute of Neuropathology, University Hospital Münster, Domagkstrasse 19, Münster, Germany. hasselblatt@uni-muenster.de. 491