Superoxide dismutase activity in cisternal cerebrospinal fluid after aneurysmal subarachnoid haemorrhage

Acta Neurochir (Wien) (1997) 139:1033-1037

Acta Neurochirurgica 9 Springer-Verlag 1997 Printed in Austria

Superoxide Dismutase Activity in Cisternal Cerebrospinal Fluid After Aneurysmal Subarachnoid Haemorrhage P. Gaetani, C. Cafe l, R. Rodriguez y Baena, F. Tancioni 2, C. Torri l, E Tartara 2, and F. Marzatico t Neurosurgery, Istituto Clinico "Humanitas", Rozzano, Milan, and LDepartment of Neurosurgery, IRCCS Policlinico S. Matteo and 2institute of Pharmacology, University of Pavia, Italy

Summary It has been recognised that the level of superoxide dismutase (SOD) significantly increases in CSF as the result of cerebral ischaemic damage. The aim of this study was to correlate the CSF levels of SOD enzymatic activity to the patterns of subarachnoid haemorrhage with regards to ischaemic complications due to vasospasm. A series of 78 patients operated on for intracranial aneurysms was studied; all patients were monitored with serial TCD measurements every second day after SAH. CSF samples were obtained at surgery by cistemal puncture of the subarachnoid cistern nearest to the aneurysm. SOD activity was assayed spectrophotometrically. Mean cisternal CSF level of SOD in 12 control cases (12.99 +_ 2.33 U/ml) is significantly higher (p < 0.0l) than in 26 patients operated on between day 1 and 3 from last SAH episode (4.44 _+0.7 U/rot) and in 40 patients treated by delayed surgery (7.64 + 0.92 U/ml). In 13 patients presenting neurological deterioration related to arterial vasospasm mean cisternal SOD level was 12.23 + 1.86 U/ml; in 27 cases without vasospasm mean level was 5.43 _+0.7 U/ml (p < 001). The present results suggest that (a) cisternal CSF levels of SOD significantly decreases after SAH, probably in relation to an impaired synthesis in the brain compartment and that (b) a substantial elevation of SOD levels is evident in patients suffering ischaemic complications vasospasm-related. Biochemical events in the brain compartment could influence the expression and release of anti-oxidant enzymes in CSF after SAH.

Keywords: Superoxide dismutase; subarachnoid haemorrhage; vasospasm.

Introduction Several experimental studies have demonstrated that an enhanced production and activity of the reactive o x y g e n species plays an important role in the pathogenesis of neuronal d a m a g e and arterial vasospasm following subarachnoid h a e m o r r h a g e (SAH) [10, 13, 2 1 - 2 3 ] , as well as after brain injury and cerebral ischaemia [1-3, 7 - 9 , 24].

In the brain compartment, as well as in all cells, there are protective systems against free radical production: superoxide radicals are inactivated by superoxide dismutase (SOD), and h y d r o g e n peroxide produced in this reaction is scavenged by glutathione peroxidase and catalase. Strand and Marklund [25] in 36 patients admitted for acute cerebral ischaemia found that m e a n C u Z n S O D (the cytosolic iso-enzyme) activity in C S F rose significantly as a result o f neuronal d a m a g e and suggested that superoxide dismutase (SOD) might be considered a quantitative marker o f ischaemic damage with some prognostic significance. Furthermore, an increased S O D activity was found after acute ischaemic injury with a time lag of the 24 hours [6, 17]. Studies concerning S A H have shown that C S F levels of several c o m p o u n d s significantly increase as a result o f ischaemic complications due to v a s o s p a s m [18, 20, 22], but modifications of S O D in CSF after S A H have scarcely been investigated. Sakaki et al. [23] f o u n d a relationship between increased levels o f lipid peroxides in the first 4 days after the h a e m o r r h a g e and a m a r k e d decrease of S O D activity in patients with s y m p t o m a t i c vasospasm. However, the enzymatic levels in C S F m i g h t have been significantly influenced by the continuous draining o f cisternal C S F after surgery. In the present study, we addressed the question whether aneurysmal S A H significantly influences C S F levels of S O D and if modifications m a y be related to ischaemic complications vasospasm-related. Cisternal C S F sampling was p e r f o r m e d directly at surgery from subarachnoid cisterns before a n e u r y s m exposure and clipping in order to validate the analysis o f S O D enzymatic

1034 activity w i t h i n the same s a m p l i n g procedure and a v o i d i n g p r o b l e m s related to c o n t i n u o u s cisternal drainage.

P. Gaetani et al.: Superoxide Dismutase Activity in CSF diagnosed when an overt neurological deterioration occurred accompanied by TCD velocities over 160 cm/sec and/or severe arterial narrowing at angiography. Analytical Methods

Methods and Patients Clinical material In the present study a series of 78 patients (29 males and 49 females) operated on for intracranial aneurysms was studied. In 12 cases an incidental unruptured intracranial aneurysm was diagnosed during angiographic studies performed for other reasons. These cases represent the control group: in fact, for ethical reasons, it was not possible to obtain "pure" control cistemal CSF samples and we have to consider the above mentioned cases as the best control samples. Thus, differences observed in the SAH group depend on the occurrence of haemorrhage. Sixty-six patients admitted with the diagnosis of aneurysmal SAH within the third day after the haemorrhage were included and classified according to the following parameters: timing of surgery, CT classification at diagnosis according to Fisher et al. [4], and World Federation of Neurosurgical Societies (WFNS) grading on admission. The procedures followed were in accordance with Institutional guidelines. The study protocol was approved by an Institutional Review Committee and every patient (or relatives) were infonned before surgery of cisternal CSF sampling and gave informed consent. CSF samples were obtained at surgery by cisternal puncture of the subarachnoid cistern the nearest to the aneurysm before aneurysm exclusion. Patients were subdivided into the following groups: (a) unruptured aneurysms (controls); (b) patients operated on within 72 hours after SAH; (c) patients undergoing delayed surgery because of clinical and technical considerations (general conditions, availability of intensive care unit, angiographical patterns). All patients undergoing early surgery were treated with osmotic agents (mannitol 20%), and clonidine, when required. Patients undergoing delayed surgery were treated with tranexamic acid (6/day i.v.) and osmotic agents until surgery. Vasospasm was assessed by angiographic study and trans-cranial doppler (TCD) serial measurements. Patients operated on day 1-3 from last SAH episode had their angiographic study on admission, prior to surgery, while patients operated on after day 10 fi'om last SAH episode never had angiography before day 8th after the haemorrhage; moreover, all patients were studied using serial TCD measurements evcry second day after SAH, in order to verify the occurrence of sonographic vasospasm during the maximal expectation time. The modifications of CSF levels of SOD caused by the haemorrhage were studied considering the group ot patients operated on within 72 hours after SAH, in order to avoid discrepancies due to different timing of CSF sampling in patients operated on in a delayed phase. In the same group we analyse the relationship between CSF SOD levels and the anaount of subarachnoid blood clots as classified by CT scan, comparing patients presenting thin versus consistent cisternal blood clots. Modifications of SOD levels related to vasospasm were studied only in patients undergoing delayed surgery, because sampling of CSF in this group was performed after the occurrence of ischaemic insult, while cisternal CSF samples in patients developing vasospasm after early surgery were obtained before the onset of neurological deterioration and thus were excluded from the analysis. Symptomatic vasospasm was

Cerebrospinalfluid handling. CSF samples obtained at surgery were immediately centrifuged at 6500 rpm (Microcentrifuge, Microcentaur, MSE). The supematants were used as specimen for the measurement of SOD activity. An aliquot of supernatants was used in order to measure eventual red and white cells residual. The specimen were frozen in liduid nitrogen and stored at -80 ~ until analysis. Measurement of SOD activity. Determination of SOD activity was assayed by spectrophotometric assay using a SOD-525 Bioxytech (Bioxytech S.A., Bonneuil, Maine, France). The SOD525 method takes some advantages as accelerated alkaline autooxidation by any catalyst having SOD activity. The auto-oxidation yields a chromophor which adsorbs visible light. Furthermore, the SOD-525 method removes the major interferences due to mercaptans in the sample, such as glutathione. The pH of 8.8 results in optimal sensitivity of the assay without significant inactivation of SOD iso-enzymes. The results are expressed as SOD-525 unit]ml of CSF. Statistical Analysis Statistical evaluation for inter group comparison was performed using one-way analysis of variance (ANOVA) and Student's t Test for unpaired data with Bonferroni correction, according to Estatix statistical package for Macintosh; statistical significance was accepted for p < .02.

Results In 53 cases patients were at a d m i s s i o n in good c l i n ical c o n d i t i o n (classified in W F N S grade 1-3); 13 patients were in poor n e u r o l o g i c a l c o n d i t i o n (grade 4 - 5 ) ; in 12 cases no radiological and clinical signs of S A H were detected. I n 12 cases patients had i n c i d e n t a l a n e u r y s m s (control cases); 26 patients were operated on b e t w e e n day 1 and 3 from last S A H episode and 40 patients treated by d e l a y e d surgery protocol. Statistical analysis ( A N O V A ) s h o w e d a s i g n i f i c a n t difference w i t h i n the groups (F = 6.25, p = 0.0035). In particular m e a n cisternal C S F S O D level in u n r u p t u r e d a n e u r y s m s was s i g n i f i c a n t l y higher (p < 0.001) than in patients operated o n day 1-3 after S A H ( F i g . l ) . C o n s i d e r i n g the group of patients operated on w i t h i n 72 hours after S A H (early surgery), we f o u n d no s i g n i f i c a n t difference b e t w e e n m e a n cisternal C S F level of S O D in 9 patients with thin s u b a r a c h n o i d b l o o d clots (2.90 + 0.62 U / m l ) and that of 17 patients with c o n s i s t e n t a m o u n t of s u b a r a c h n o i d b l o o d (5.25 + 0.9 U / m l ) (Fig. 2). A m o n g the 40 patients operated on b y d e l a y e d surgery protocol, 13 patients p r e s e n t e d ischaemic

P. Gaetani et al.: Superoxide Dismutase Activity in CSF

1035 SOD (U/ml)

SOD (U/ml)

20

15

t0

l0

r

Unruptured An.

SAH (< 72 hrs.)

Fig. 1. Bar graph representation of cisternal CSF level of SOD (U/ml) in patients with unruptured aneurysm, and patients operated on day 1-3 after SAH. Statistical analysis: Student's t test for unpaired data and Bonferroni correction: ** p < 0.01

SOD (U/ml)

No Vasospasm

Vasospasm

Fig. 3. Bar graph representation of cisternal CSF SOD activity (U/ml) in patients presenting neurological deterioration due to ischaemic complications, vasospasm-related, and patients presenting an uncomplicated course. Only patients operated on in the delayed phase were considered in the analysis (see text for details). Statistical analysis: Student's t test for unpaired data and Bonferroni correction: ** p < 00.1

15

Table 1. Cisternal CSF SOD Levels (Mean + S.E.M.) in 66 Cases of SAH Classified According to Glasgow Outcome Score Criteria

12

9 o .

9

l

9

9

|

Outcome (GOS)

No. of cases

SOD (U/ml)

Full recovery Minor impairment Severe impairment Vegetative and dead

22 14 17 13

8.42 5.54 6.84 3.88

_+ 1.37 + 1.31 + 1.24 + 0.68

9

I 9

Analysis of variance (ANOVA) showed that there is no significant difference within the subgroups (F = 2.13, p = 0.106).

i

Thin

Consistent

Fig. 2. Plot representation of cisternal CSF SOD activity (U/ml) in patients admitted and operated on within 72 hours after SAH (early surgery) and classified according to CT scan (Fisher's criteria): The amount of cisternal blood was considered "thin" for patients classified as Fisher's grade 1 and "consistent" for patients in Fisher's grade 2 and 3. No statistical difference was shown

ference in cisternal CSF levels of SOD within subgroups of patients classified according to GOS criteria (F = 2.212; p = 0.106) (Table 1). Discussion

complications due to vasospasm: in these cases mean cisternal SOD activity was significantly higher than

in 27 patients without symptomatic vasospasm (Fig. 3). In 45 (57.6%) cases at a 3 months follow-up examination patients had a good recovery (as classified with Glasgow Outcome Score criteria); in 19 cases (24.4%) a severe disability was evident, while in 14 cases (18%) patients were in vegetative status or dead. Analysis of variance showed that there is no dif-

SAH has a complex pathophysiological spectrum and the primary damage to the brain and ischaemic complications due to vasospasm are considered the most important problems. The analysis of SOD levels in cisternal CSF reflects this situation: in fact the present results show that (a) cisternal levels of the enzyme significantly decrease in the early phases after the haemorrhage and (b) a substantial elevation of SOD levels is evident in patients operated on in a

delayed phase and suffering ischaemic complications, vasospasm-related.

1036 A discrepancy may result considering that after SAH, anti-oxidant enzymes (such as SOD) may be at first supplied by the extravasated blood; in the present study, cisternal CSF levels of SOD are not related to the degree of subarachnoid blood, suggesting that unknown factors might be involved in maintaining a dynamic equilibrium of SOD concentration in CSF with extracellular fluid of the brain. Sakaki et al. [22, 23] found that SOD activity is detectable and stable even in patients operated on for unruptured aneurysms, but CSF level of SOD cannot derive from plasma which contains approximately 10 fold lower concentrations of the enzyme [11, 25]. In a recent experimental study we showed that the activity of CuZn SOD in cerebral cortex of rats subjected to experimental SAH procedure significantly decreases after SAH induction [5]. The changes in superoxide scavenging activity by CSF might be related mainly to CuZn SOD that is the cytosolic 32 Kd iso-enzyme of SOD [15]. Thus biochemical events in the brain compartment could influence the expression and release of anti-oxidant enzymes in CSF, changing the temporal presence of this CSF marker of tissue damage. The results of the present study confirm those reported by Sakaki et al. [22] which showed in 25 patients operated on within the first 72 hours that SOD activity started to decrease rapidly until day 4 after SAH, and by Kuwabara et al. [10] which found that SOD loses its activity within 5 days after incubation at 37 ~ in a mixture of CSF and arterial blood, indirectly favouring the vasospastic activity of free radicals and lipid peroxides. The CSF SOD activity reflects a dynamic homeostasis between intra- and extra-cellular compartments: thus, in the earty phase after SAIl, the Significantly lower SOD activity reflects the impairment of intracellular anti-oxidant capacity because of the effects of the haemorrhage "per se" on cerebral metabolism, as shown in a recent experimental study [5]. However, the impairment of enzymatic activity does not depend on the amount of subarachnoid blood as measured by CT scan (Fig 2), confirming previous observations [5]. The mechanism of impaired intracellular SOD synthesis is unknown: in previous studies we showed that the altered synthesis of anti-oxidant enzymes in the brain compartment after experimental SAH may be related to the impaired mitochondrial function [12, 14, 16]. On the other hand, clinical studies about SOD activity in cerebral ischaemia have shown that the highest CSF levels were found in the early phase after

P. Gaetani et al.: SuperoxideDismutaseActivityin CSF cerebrovascular accidents [6, 17, 25]. In the study by Strand and Marklund [25] the maximal peak of SOD level in CSF was found in 75% of cases in the first lumbar sample (8-36 hours after symptoms onset) and the level of SOD was significantly related with the size of ischaemic damage (as measured at CT scan), to the gravity of neurological deterioration and mortality and finally with overall mortality rate at three months follow-up. After SAH, a similar situation may be suggested, with elevation of SOD in CSF, when delayed cerebral ischaemia due to arterial vasospasm occurs. According to this hypothesis, the higher cisternal CSF levels of SOD found in the present study in patients with vasospasm might be related to the massive cytosolic leakage of the enzyme due to ischaemic damage [19, 25] which completely alters the dynamic homeostasis of the enzymatic activity in CSF. In conclusion, the results of the present study confirm that an impairment of anti-oxidant SOD activity might be involved in pathophysiological aspects of SAH in humans and that biochemical events in the brain compartment could influence the expression and release of antioxidant enzymes in CSF.

Acknowledgements The present work was granted by IRCCSPoliclinicoS. Malteo. Authors are indebted to M. Brunelli, mecenate of Neurosurgical research. References 1. Braughler JM, Hall ED (1989) Central nervous system trauma and stroke. I. Biochemical considerations for oxygen radical formation and lipid peroxidation. Free Radic Biol Med 6: 289-301 2. Caf~ C, Marzatico F (1993) Oxygen radicals and other toxic oxygen metabolites as key mediators of the central nervous system tissue injury. Funct Neurol 8:51-66 3. Cord JM (1985) Oxygen-derived three radicals in postischemic tissue injury. N Engl J Med 312:159-163 4. Fisher CM, Kistler JP, Davis JM (1980) Relation of cerebral vasospasm to subarachnoid hemorrhage visualized by computerized tomographic scanning. Neurosurgery 6:1-9 5. Gaetani P, Rodriguez y Baena R, Quaglini S, et al (1994) Experimental subarachnoid hemorrhage: events related to antioxidant enzymatic systems and eicosanoid peroxide enhancement. Neurochem Res 19- 839-844 6. Gross GB, Gozlan O, Barak M (1994) Increase in superoxide dismurase after cerebrovascular accident. Life Sci 54: 711713 7. Hall ED, Braughler JM (1989) Central nervous system trauma and stroke. II. Physiological and pharmacological evidence for the involvement of oxygen radicals and lipid peroxidatlon. Free Rad Biol Med 6:303-313 8. Kitagawa K, Matsumom M, Oda T, et al (1990) Free radical

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Comments This is an interesting study extending the authors' previous experimental studies and confirming the clinical evidence that superoxide dismutase in the cerebral spinal fluid is decreased primarily after subarachnoid haemonhage and then increases later together with developing ischaemia. H. J. Steiger

In the study presented in the above-mentioned paper the authors measured the activity of superoxide dismutase (SOD) in the cerebrospinal fluid (CSF) of patients suffering from subarachnoid haemorrhage (SAH). In contrast to previous studies, samples of CSF were not obtained by means of a ventricular drain but during neurosurgery from that cistern which appeared most affected by the bleeding (controls: by the presence of an unruptered aneurysm) The main reselts are: i) SOD activity is higher in patients operated on non-ruptured aneurysms than in those suffering fiom SAH, ii) SOD activity is lowest in patients who underwent early surgery (i.e., within the first 3 days following SAIl) with no apparent correlation with the amount of extravasated blood in CT scans, and iii) after SAH the SOD activity is higher in patients with signs of vasospasm than in those without. Although the authors did not study the measured SOD subtype in any detail they presume that it mainly represents the intracellular type of iso-enzyme. Therefore, they suspect an increase in SOD activity in patients presenting with signs of vasospasm to reflect ischaemic cell damage. However, this explanation obviously does not work in the case of a decreased SOD activity in the early phase after SAH without signs of ischaemia. Therefore, the hypothesis for explaining the low level of SOD activity in the early phase after SAH offered by the authors in the Discussion is questionable. P. Schmiedek

Correspondence: Paolo Gaetani, M.D., Neurosurgery, Istituto Clinico "Humanitas", Via A. Manzoni, 56, 1-20089 Rozzano, Milan, Italy.