Effects of high-dose methylprednisolone on Na -K ATPase and b i d geroxidation after experimental subar&hnbid hemorrhage +
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Marzatico F, Gaetani P, Buratti E, Messina AL, Ferlenga P, Rodriguez y Baena R. Effects of high-dose methylprednisolone on N a + -K+ ATPase and lipid peroxidation after experimental subarachnoid hemorrhage. Acta Neurol Scand 1990: 82: 263-270. The production of oxygen-free radicals and their subsequent peroxidative action on membrane unsaturated fatty acids could be enhanced after subarachnoid hemorrhage. High-dose methylprednisolone (30 mg/Kg i.v.) treatment can antagonize acute S AH-induced brain hypoperfusion and protect the ultrastructural integrity of endothelial cell membranes. Experimental subarachnoid hemorrhage (SAH) was induced in anesthesized rats by slow injection of 0.3 ml of autologous arterial blood into cisterna magna. Tissue lipid peroxidation, quantified as thiobarbituric acid reactive matherial (TBAR) and Na -K ATPase activity were assayed in three different rat brain areas (cerebral cortex, hippocampus and brain stem) of controls (without any surgical manipulation), sham-operated (0.3 ml. of mock CSF into cisterna magna) and after SAH induction, at 1 h, 6 h and 48 h. Na -K ATPase activity decreased in the cerebral cortex at 1 h and 6 h and in brain stem at 1 h after SAH, whiIe the same enzymatic activity was unchanged in the hippocampus. High-dose methylprednisolone treatment (started immediately after SAH induction) enhanced the Na'-K' ATPase activity until control levels. There was no significant difference in lipid peroxide content between sham-operated and hemorrhagic animals; however, the injection itself induces a transient increase of TBAR (1 h after injection) and methylprednisolone treatment decreases the products of lipid peroxidation in all brain areas. +
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I
F. Marzatico', P. Gaetani', E. Buratti', A. L. Messina ', P. Ferlenga ', R. Rodriguez y Baena'
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Institute of Pharmacology, Department of Surgery, Neurosurgery, IRCCS Pofidinico S. Matteo, University of Pavia, ltaly
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Several factors have been claimed as possibly involved in the pathogenesis of vasospasm: adrenergic and serotoninergic pathways (1-9, prostaglandins and leukotrienes (6,7) or lipid peroxide (8) have been widely investigated, but the pathogenetical mechanisms of delayed vasospasm still remains unknown (9). Delayed cerebral vasospasm which complicates 15-25% of cases of aneurysmal subarachnoid hemorrhage (SAH) has been considered as the result of an inflammatory response of the arterial wall to the presence of blood in the subarachnoidal spaces (10, 11). The increase of intracranial pressure (ICP) and the arterial narrowing associated with structural derangement of the arterial wall (12-14) leads to a significant modification of local cerebral blood flow (CBF) (15-18) and causes a sequential cellular impairment and irreversibleneuronal damage similar to that occurring in cerebral ischemia. After experimental SAH, the global hypoxicischemic condition of the brain reduces the delivery of oxygen to the neuronal compartment and induces
Key words: subarachnoid hemorrhage; methylprednisolone;lipid peroxidation; NaC-KCATPase Riccardo Rodriguez y Baena, Department of Surgery, Neurosurgery, IRCCS Policlinico S. Matteo, 1-27 100 Pavia, Italy Accepted for publication May 16, 1990
a marked dissociation of normally tightly coupled electron-transport chain components (19). In this metabolic situation the production of oxygen-free radicals and their subsequent peroxidative action on membrane unsaturated fatty acids are enhanced (20). Recently, some authors have shown that a single high-dose of methylprednisolone (30 mg/Kg i.v.) can antagonize acute SAH-induced brain hypoperfusion in cats (21). Repeated administration of same methylprednisolone dose has also been shown to prevent chronic vasospasm and to protect the ultrastructural integrity of the basilar artery wall, improving the contractile response to vasoactive agents after experimental SAH in dog (10, 22). Extensive studies of high dose methylprednisolone in experimental spinal cord injury have demonstrated that the steroid probably acts by inhibiting post-traumatic lipid peroxidation (23, 24). Evidence for this antioxidant effect is seen in the protection of the lipid peroxidation sensitive membrane-bound enzyme Na -K ATPase (23, 25). +
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Marzatico et al. Although the exact mechanism(s) of glucocorticoids, as well of other steroids, in attenuating lipid peroxidative processes is unclear, the inhibitory effect on diene formation - an intermediate event in lipid peroxidative sequence - (26), suggests how the steroid may inhibit lipid peroxidation: through a physical interaction within cell membrane and preventing the initiation of lipid radical chain reactions (27). Furthermore, an anti-oxidant effect has been suggested to explain the ability of methylprednisolone to inhibit post-SAH brain hypoperfusion (21). Considering this possibility, the aim of the present study was to define whether high-dose methylprednisolone (30 mg/Kg, i.v.) administered immediately after experimental SAH induction decreases lipid peroxidation and protects the lipid peroxide sensitive enzyme Na -K ATPase activity in rat brain, during the acute and late phase of vasospasm. +
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Material and methods
The experiments were performed on male Sprague Dawley rats (weighmg 400-425g) in which subarachnoid hemorrhage was induced according to Solomon et al. (2), with few modifications (19). General anesthesia was induced with 3 % halothane (70% : 30% N20 : 0,) and maintained with 0.75% halothane (in the gas mixture). A burr hole by refrigerated twist-drill was performed at the interparietal/occipital suture connection. A small catheter (Clay - Adams, PB - 10) was inserted into the cisterna magna; suitable placement of the catheter was assessed by: (a) testing cerebrospinal fluid (CSF) flowing through the catheter and (b) observing (with magnification) the lower distal part of the catheter through the atlanto-occipital membrane. The tail artery was cannulated for sampling of blood to measure pH, pCO,, PO,, and monitoring of the arterial blood pressure. The body temperature, monitored by a rectal thermometer, was adjusted and maintained near 37°C by external heating. When the rats were in a steady respiratory state (arterial PO, and pCO,, 100 mm Hg and 35-40 mm Hg, respectively) autologous arterial blood was collected (0.35 d) from the femoral artery, and an aliquot of 0.30ml was injected into the cisterna magna via the catheter within about 2 min. Before SAH induction, a CSF sample of about 0.01-0.03 ml. was gently drawn in order to limit modifications of intracranial pressure. The animals were held in a 20" head down position. Experimental groups
Sixty rats were divided into the following groups: Group 1. Six rats (controls) were killed and sub264
mitted to biochemical analysis at different times after general anesthesia without surgical manipulation. Group 2. Eighteen animals (sham-operated) subjected to injection of 0.3 ml. of mock CSF (saline solution with 25 meq/L of NaHCO,) into cisterna magna and were treated with saline solution i.v. Treatment with saline solution was performed immediately after mock CSF injection for animals killed at 1h and 6 h, while four injections (every 8 h) were given to rats killed at 48 h. Group 3. Eighteen rats (SAH) were injected with 0.3 ml. of autologous arterial blood into cisterna magna and treated with saline solution, as described for rats of Group 1. Group 4. Eighteen (SAH + MP) rats were injected with 0.3 ml. if autologous arterial blood into cisterna magna and treated with high-dose (30 mg/Kg, i.v.) of methylprednisolone (MP), immediately after SAH induction for animals killed at 1 and 6 h, while animals killed at 48 h were treated with 30 mg/Kg of MP immediately after SAH induction and then with 3 other injections (10 mg/Kg i.v. every 8 hours). Analytical methods
The rats were decapitated and brains were immediately frozen in dry ice. Brain samples for Na+-K'ATPase activity and TBRA assay were prepared in a pre-refrigerated glove box maintained at - 22°C. The brain areas were dissected (28), weighed and homogenized (1/10 g/ml) in ice-cold 50 mM phosphate buffer (pH7) containing 15 mM Na+ and 145 mM K + in a pre-cooled Potter Braun S homogenizer (8 strokes up and down, 800 rpm). An aliquot of the homogenate was used to measure Na+-K'ATPase by continous monitoring of the rate of ATP hydrolysis, using the coupled enzyme system pyruvate kinase - lactate dehydrogenase as proposed by Scharschmidt et al. (29). Tissue lipid peroxidation was evaluated by measurement of TBRA (23) using the undiluted tissue homogenate prepared for ATPase assay. Aliquots of 0.5 ml of homogenate were deproteinized with 20 % thrichloroacetic acid and centrifuged at 3000 g for 15 min. The supernatant fraction was then diluted 1 : 1 with 10% trichloroacetic acid containing 0.67% thiobarbituric acid, heated in a boiling water bath for lOmin, then cooled and the absorbance read at 532 nm. Malonyldialdehyde (MDA) was evaluated on the basis of a molar extinction coefficient of 1.56 x lo5 - M - cm - expressed as nmol MDA [mg protein] - '. The protein content of homogenate was assayed according to Lowry et al. (30) with serum albumin as a standard. Materials. Enzymes were obtained from Boehringer Mannheim. Substrates, coenzymes and reagent for enzymatic assay were obtained from Sigma Che-
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Methylprednisolone and lipid peroxidation after SAH micals (St. Louis, MO). All other chemicals used were of reagent analytical grade. Statistics. Analytical runs were performed on lots of up to 6 animals, which consisted of SAH, SAHtreated and sham-operated. Statistical analysis of the results was carried out using the Analysis of Variance (ANOVA). Tukey's test for multiple comparisons was applied where ANOVA gave significant differences. Results
Physiological parameters
mental period of 1 h; the average variation of MABP was less than 8%. Four rats (one from the shamoperated group, one of the SAH-treated and two from the SAH groups) showed a change in pC0, greater than 20 % during the experimental period and these rats were excluded from the analysis of results. Three SAH animals (9%) died within 10 h after a normal recovery from anesthesia. An extensive subarachnoid blood deposition in the basal cisterns of the brain is evident at 30 min, 1 h, and 6 h after SAH induction. No hemorrhage was noted in the saline treated rats.
During SAH induction, MABP and arterial pC0, remained constant for each rat during an experi-
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Fig. 1 . Bar graph representation of Na'-K'ATPase activity (Fig. 1A) and TBA-R content (Fig. 1B) in cerebral cortex of rats subjected to experimental subarachnoid hemorrhage (SAH) induction and high-dose methylprednisolone treatment. Analysis on lot of up to 6 animals for each group. Statistics: analysis of the variance (Anova Test) and Tukey's Test for multiple comparisons. Statistical significance: Controls vs Sham-operated: A p < .05; A A p < .02; A A A p < .01. Sham-operated vs SAH: * p < .05; ** p < .02; *** p < .01. SAH vs SAH-treated: 0 p < .05; 0 0 p c .02; 0 0 0 p < .01.
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Marzatico et al. Effects of MP on lipid peroxidation and Nat-KtATPase during subarachnoid hemorrhage
Cerebral cortex. There is a marked decrease of Na -K ATPase activity in cerebral cortex at 1 and 6 h after SAH induction, while at 2 days there is no significant difference between sham-operated and SAH rats (Fig. la). TBRA content was higher in the sham-operated animals compared to the control animals. MP treatment significantly improves Na K +ATPase activity at 1 and 6 h after SAH and generally decreases the MDA content but only at 1 h after SAH, the TBRA content was significantly lower respect to sham-operated and SAH animals (Fig. lb). +
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Hippocampus. Na+-K ATPase activity did not changed in the hippocampus (Fig. 2a). The lipid peroxide products evaluated with TBAR show a similar difference between controls and shamoperated as in cerebral cortex (Fig. 2b). In MP treated animals the Na -K ATPase activity significantly increased at 6 hours after SAH induction when compared to sham-operated rats (Fig. 2a), and the TBRA content significantly decreased at 6 h and 2 days compared with the sham-operated and SAH groups. Bruin stem. In SAH group, Na+-K'ATPase activity significantlydecreased in the brain stem only at 1 h, without any significant changes at 6 and 2 days (Fig. 3a). The TBRA content significantly in+
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Fig. 2. Bar graph representation of Na+-K ATPase activity (Fig. 1A) and TBA-R content (Fig. 1B) in hippocampus of rats subjected to experimental subarachnoid hemorrhage (SAH) induction and high-dose methylprednisolone treatment. Analysis on lot of up to 6 animals for each group. Statistics: analysis of the variance (Anova Test) and Tukey's Test for multiple comparisons. Statistical significance: Controls vs Sham-operated: A p < .05; A A p c .02; A A A p < .01. Sham-operated vs SAH: * p < .05; ** p < .02; *** p < .01. SAH vs SAH-treated: 0 p < .05; 0 0 p < .02; 0 0 0 p c .01. +
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Methylprednisolone and lipid peroxidation after SAH 75 c)
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Fig.3. Bar graph representation of Na'-K ATPase activity (Fig. 1A) and TBA-R content (Fig. 1B) in brain stem ofrats subjected to experimental subarachnoid hemorrhage (SAH) induction and high-dose methylprednisolone treatment. Analysis on lot of up to 6 animals for each group. Statistics: analysis of the variance (Anova Test) and Tukey's Test for multiple comparisons. Statistical significance: Controls vs Sham-operated: A p < .05; A A p < .02; A A A p < .01. Sham-operated vs SAH: * p < .05; ** p < .02; *** p < .01. SAH vs SAH-treated: 0 p < .05; 0 0 p < .02; 0.0 p < .01.
creased in the sham-operated animals respect to controls 1 h after the injection, while at 6 h and 2 days the TBAR content in sham-operated animals was higher, but not significantly so than observed in control animals (Fig. 3b). MP treatment increased the Na+-K' ATPase activity until values found in sham-operatedrats at 1h, and over these value at 6 h (Fig. 3a). MDA content after MP treatment was significantlyreduced at 1 h, 6 h and 2 days compared both with sham-operated and SAH animals. Discussion
Although several clinical and experimental studies have shown global changes in CBF and in brain metabolic patterns after aneurysmal SAH. there is
no evidence of a closed relationship between CBF and metabolic changes when vasospasm occur (16, 18,25,3 1). Many attempts have been made in order to reduce and/or prevent vasospasm after SAH, antagonizing spasmogenic factors such as monoamines (1, 32), arachidonic acid metabolites (33), or breakdown products of erythrocytes (34). Experimental evidences of characteristic changes in the arterial wall after vasospasm have suggested that an inflammatory response induced by blood deposition in the subarachnoidal spaces could be one of the most important pathogenetical mechanisms (10,22). The decrease of CBF due to the increase of ICP and to the onset of arterial spasm leads to anoxicischemic damage. Free radicals, enhancing lipid peroxidation of cellular and subcellular membrane 267
Marzatico et al. HIGH-DOSE METHPLPREDMSOLONE
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FUNCTIONALITY
Fig. 4. Speculative hypothesis of the methylprednisolone effects after experimental SAH.
phospholipids, have been suggested as the triggers of neuronal damage (19, 35, 36). Treatment with high-dosemethylprednisolonehas been proposed for the prevention of delayed cerebral vasospasm (10, 22): in dogs subjected to experimental SAH the ultrastructural integrity of smooth muscle cells in the arterial wall is preserved; moreover, a preliminary clinical study has reported beneficial effects in preventing vasospasm occurrence (1 l), but the pharmacological mechanism is not well understood. In a previous study in the same experimental model, we have shown that after SAH there is a significant impairment of Na+-K'ATPase activity in cerebral cortex and in the brain stem, while in the hippocampus, the area more sensitive to ischemic insult, the enzymatic activity does not significantly change. Lipid peroxidative processes, as quantified by TBAR measurement, are not significantly enhanced after SAH compared to shamoperated rats, and changes of Na -K ATPase activity are not closely related to the magnitudo of lipid peroxidation (37). Moreover, the present results show that the injection per se into the cistema magna, induces a transient increase of lipoperoxidative products (MDA) in all brain areas tested: this event is independent from the presence of iron which in case of SAH are liberated from red cells and could be a stimulus triggering MDA formation (38, 39). Otherwise, Na -K ATPase activity is significantly affected only after SAH induction, when blood is present in the subarachnoidal spaces suggesting that other pathophysiological mechanisms act on this enzyme activity. In the acute phase of the SAH the increase of ICP, and the consequent decrease of CBF (12), the presence of the blood in the +
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subarachnoid spaces' and the decrease of cerebral energy potential (3 1, 37) are, together, possible factors triggering the sequence of various metabolic events, which mimick the brain response to an anoxic-ischemic insult. It has been demonstrated that a low concentration of ATP itself, may limit the Na+-K'pump (40),and all these factors may contribute to the inhibition of Na+-K+ATPase. Treatment with high-dose methylprednisolone decreases the MDA content in all brain areas at different times comparing hemorrhagic and shamoperated rats with control values: thus, we may hypothesize that the increased lipid peroxidation seen after SAH in our experiment until 2 days after blood injection should be related to metabolic events and surgical manipulation. Meanwhile, the results of the present study show that the beneficial effect of high-dose methylprednisolone after SAH could be related to the reduction of peroxide lipid products in all brain areas with a major stability of cellular membrane: this would influence the activity of lipid-dependent enzyme Na -K ATPase, reduce the derangement of mitochondria1 membrane preserving the electrochemical ion gradient (37) and maintain cellular energy metabolism at levels sufficient to preserve the activity of Na -K +pump (Fig. 4). Although the results of the present study have provided some interesting data regarding peroxidative events of brain cell membrane after SAH, further studies are ongoing in order to verify the relationship between CBF, oxidative and peroxidative metabolism and the effects of glucocorticoid treatment. +
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Methylprednisolone and lipid peroxidation after SAH References 1. ALKSNE JF, GREENBOOT JH. Experimental cathecolamineinduced chronic cerebral vasospasm. Myonecrosis in vessel wall. J Neurosurg 1974: 41: 440-445. RA, MCCORMACK MB, LOVITZRN, SWIFTDM, 2. SOLOMON HEGEMANN MT. Elevation of brain norepinephrine concentration after experimental subarachnoid hemorrhage. Neurosurgery 1986: 19: 363-366. NA, BRISMARJ, DELGADOTJ. Subarachnoid 3. SVENGAARD hemorrhage in the rat: effect on the development of vasospasm of selective lesions of the catecholamine systems in the lower brainstem. Stroke 1985: 16: 602-608. NA, BRISMAR J, DELGADO TJ, ROSENGREN E, 4. SVENCAARD STENEVIU. Subarachnoid hemorrhage in the rat: effect on the development of cerebral vasospasm of lesions in the central serotoninergic and dopaminergic system. Stroke
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SILVANIV, PAOLETTI P, BENZIG. Bioenergetics of different brain areas after experimental subarachnoid hemorrhage in rats. Stroke 1988: 19: 378-384. DEMOPOULOSHB, FLAMMES, PIETRONIGRODD, SELIGMAN ML. The free radical pathology and the microcirculation in the major central nervous system disorders. Acta Physiol Scand 1980: (Supp. 492): 91-119. HALLED, TRAVIS MA. Attenuation of progressive brain hypoperfusion following experimental subarachnoid hemorrhage by large intravenous doses ofmethylprednisolone. Exp Neurol 1988: 99: 594-606. CHYATTE D, RUSCHN, SUNDTTM Jr. Prevention of chronic experimental cerebral vasospasm with ibuprofen and highdose methylprednisolone. J Neurosurg 1983: 59: 925-932. BRAUGHLER MJ, HALL ED. Correlation of methylprednisolone levels in cat spinal cord with its effects on (Na+-K' )ATPase, lipid peroxidation and alpha motor neuron function. J Neurosurg 1982: 56: 838-844. HALLED, BRAUGHLER MJ. Effects of intravenous methylprednisolone on spinal cord lipid peroxidation and (Na+-K' ) ATPase activity. Dose response analysis during the 1st hour after conclusion injury in the cat. J Neurosurg
on cisternal CSF levels of arachidonic acid metabolites after aneurysmal subarachnoid hemorrhage. J Neurol Sci 1988:
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SASAKIT, ASANOT. Identification of 5-hydroxy-eicosatetraenoic acid (5-HETE) in cerebrospinal fluid after subarachnoid hemorrhage. J Neurochem 1983: 41: 1186-1189. 9. KASSELLNF, SASAKIT, COLOHANART, NAZARG. Cerebral vasospasm following aneurysmal subarachnoid hemorrhage. Stroke 1985: 16: 562-572. 10. CHYATTED, SUNDTTM Jr. Response of chronic experimental cerebral vasospasm to methylprednisolone and dexamethasone. J Neurosurg 1984: 60: 923-926. 11. CHYATTE D, FODENC, NICHOLSDA, SUNDTTM Jr. Preliminary report: effect of high dose methylprednisolone on delayed cerebral ischemia in patients at high risk for vasospasm after aneurysmal subarachnoid hemorrhage. Neurosurgery 1987: 21: 157-160. A, CROCKARDHA, Ross RUSSELLRW, 12. JACKOWSKY BURNSTOCKG. SAH in a rat model: the time course of changes in cerebral blood flow, cerebral perfusion, and intracranial pressure. J Cereb Blood Flow Metaboll989: 9 (supp. 1): 455. 13. LISZCZAKTM, VARSOSVG, BLACKPM, ZERVASNT. Cerebral arterial constriction after experimental subarachnoid hemorrhage is associated with blood components within the arterial wall. J Neurosurg 1983: 58: 18-26. 14. TANABE Y, SAKATAK, YAMADAH. et al. Cerebral vasospasm and ultrastructural changes in cerebral arterial wall: An experimental study. J Neurosurg 1978: 49: 229-238. 15. DELGADOTJ, ARBABMAR, DIEMERNH, SVENGAARD NA. Subarachnoid hemorrhage in the rat: cerebral blood flow andglucose metabolism during thelate phase ofcerebral vasospasm. J Cereb Blood Flow Metab 1986: 6: 590-599. GG, HARPERAH, FITCHW, ROWANJO, 16. FERGUSON JENNETT AL. Cerebral blood flow measurement after spontaneous subarachnoid hemorrhage. Eur Neurol 1972: 8: 15-22. RA, LOBOANTUNESJ, CHENRYZ, BLANDL, 17. SOLOMON
CHIEN S. Decrease in cerebral blood flow in rats after experimental subarachnoid hemorrhage. A new model. Stroke 1985: 16: 58-64. 18. VOLDBY B, ENEVOLDSEN EM, JENSENFT. Regional CBF,
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Protein measurement with the folin phenol reagent. J Biol Chem 1951: 193: 265-275. FEINJM. Brain energetics and circulatory control after subarachnoid hemorrhage. J Neurosurg 1976: 45: 498-506. WHITERP, ROBERTSON JT. Pharmacodynamic evaluation of human cerebral arteries in the genesis of vasospasm. Neurosurgery 1987: 21: 523-531. MORGANH, WHITE RP, PENNINKM, ROBERTSONJT. Prostaglandins and experimental cerebral vasospasm. Surg Forum 1972: 23: 447-448. OSAKA K. Prolonged vasospasm produced by the breakdown products of erythrocytes. J Neurosurg 1977: 47:
403-41 1. JL, EL-MOFTYSK. The biochemical pathology of 35. FABER liver cell necrosis. Am J Pathol 1975: 81: 237-250. 36. SIESJOBK. Cell damage in the brain: a speculative synthesis. J Cereb Blood Flow Metabol 1981: 1: 155-185. F, GAETANI P, RODRIGUEZY BAENAR. et al. 37. MARZATICO
Experimental subarachnoid hemorrhage. Lipid peroxidation and Na+-K' ATPase in different rat brain areas. Mol Chem Neuropathol 1990: 11: 99-107. 38. WILLMORELJ, HURD RW, SYPERTGW.: Epileptiform activity initiated by pial iontophoresis of ferrous and ferric
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levels in isolated rat brain synaptosomes. J Neurochem 1987: 49: 1229-1240. 41. BRAUGHLER JM, HALLED. Acute enhancement of spinal cord synaptosomal (Na+-K' )ATPase activity in cats following intravenous methyl-prednisolone. Brain Res 1981: 219: 464-469.
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Marzatico F, Gaetani P, Buratti E, Messina AL, Ferlenga P, Rodriguez y Baena R. Effects of high-dose methylprednisolone on N a + -K+ ATPase and lipid peroxidation after experimental subarachnoid hemorrhage. Acta Neurol Scand 1990: 82: 263-270. The production of oxygen-free radicals and their subsequent peroxidative action on membrane unsaturated fatty acids could be enhanced after subarachnoid hemorrhage. High-dose methylprednisolone (30 mg/Kg i.v.) treatment can antagonize acute S AH-induced brain hypoperfusion and protect the ultrastructural integrity of endothelial cell membranes. Experimental subarachnoid hemorrhage (SAH) was induced in anesthesized rats by slow injection of 0.3 ml of autologous arterial blood into cisterna magna. Tissue lipid peroxidation, quantified as thiobarbituric acid reactive matherial (TBAR) and Na -K ATPase activity were assayed in three different rat brain areas (cerebral cortex, hippocampus and brain stem) of controls (without any surgical manipulation), sham-operated (0.3 ml. of mock CSF into cisterna magna) and after SAH induction, at 1 h, 6 h and 48 h. Na -K ATPase activity decreased in the cerebral cortex at 1 h and 6 h and in brain stem at 1 h after SAH, whiIe the same enzymatic activity was unchanged in the hippocampus. High-dose methylprednisolone treatment (started immediately after SAH induction) enhanced the Na'-K' ATPase activity until control levels. There was no significant difference in lipid peroxide content between sham-operated and hemorrhagic animals; however, the injection itself induces a transient increase of TBAR (1 h after injection) and methylprednisolone treatment decreases the products of lipid peroxidation in all brain areas. +
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F. Marzatico', P. Gaetani', E. Buratti', A. L. Messina ', P. Ferlenga ', R. Rodriguez y Baena'
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Institute of Pharmacology, Department of Surgery, Neurosurgery, IRCCS Pofidinico S. Matteo, University of Pavia, ltaly
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Several factors have been claimed as possibly involved in the pathogenesis of vasospasm: adrenergic and serotoninergic pathways (1-9, prostaglandins and leukotrienes (6,7) or lipid peroxide (8) have been widely investigated, but the pathogenetical mechanisms of delayed vasospasm still remains unknown (9). Delayed cerebral vasospasm which complicates 15-25% of cases of aneurysmal subarachnoid hemorrhage (SAH) has been considered as the result of an inflammatory response of the arterial wall to the presence of blood in the subarachnoidal spaces (10, 11). The increase of intracranial pressure (ICP) and the arterial narrowing associated with structural derangement of the arterial wall (12-14) leads to a significant modification of local cerebral blood flow (CBF) (15-18) and causes a sequential cellular impairment and irreversibleneuronal damage similar to that occurring in cerebral ischemia. After experimental SAH, the global hypoxicischemic condition of the brain reduces the delivery of oxygen to the neuronal compartment and induces
Key words: subarachnoid hemorrhage; methylprednisolone;lipid peroxidation; NaC-KCATPase Riccardo Rodriguez y Baena, Department of Surgery, Neurosurgery, IRCCS Policlinico S. Matteo, 1-27 100 Pavia, Italy Accepted for publication May 16, 1990
a marked dissociation of normally tightly coupled electron-transport chain components (19). In this metabolic situation the production of oxygen-free radicals and their subsequent peroxidative action on membrane unsaturated fatty acids are enhanced (20). Recently, some authors have shown that a single high-dose of methylprednisolone (30 mg/Kg i.v.) can antagonize acute SAH-induced brain hypoperfusion in cats (21). Repeated administration of same methylprednisolone dose has also been shown to prevent chronic vasospasm and to protect the ultrastructural integrity of the basilar artery wall, improving the contractile response to vasoactive agents after experimental SAH in dog (10, 22). Extensive studies of high dose methylprednisolone in experimental spinal cord injury have demonstrated that the steroid probably acts by inhibiting post-traumatic lipid peroxidation (23, 24). Evidence for this antioxidant effect is seen in the protection of the lipid peroxidation sensitive membrane-bound enzyme Na -K ATPase (23, 25). +
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Marzatico et al. Although the exact mechanism(s) of glucocorticoids, as well of other steroids, in attenuating lipid peroxidative processes is unclear, the inhibitory effect on diene formation - an intermediate event in lipid peroxidative sequence - (26), suggests how the steroid may inhibit lipid peroxidation: through a physical interaction within cell membrane and preventing the initiation of lipid radical chain reactions (27). Furthermore, an anti-oxidant effect has been suggested to explain the ability of methylprednisolone to inhibit post-SAH brain hypoperfusion (21). Considering this possibility, the aim of the present study was to define whether high-dose methylprednisolone (30 mg/Kg, i.v.) administered immediately after experimental SAH induction decreases lipid peroxidation and protects the lipid peroxide sensitive enzyme Na -K ATPase activity in rat brain, during the acute and late phase of vasospasm. +
+
Material and methods
The experiments were performed on male Sprague Dawley rats (weighmg 400-425g) in which subarachnoid hemorrhage was induced according to Solomon et al. (2), with few modifications (19). General anesthesia was induced with 3 % halothane (70% : 30% N20 : 0,) and maintained with 0.75% halothane (in the gas mixture). A burr hole by refrigerated twist-drill was performed at the interparietal/occipital suture connection. A small catheter (Clay - Adams, PB - 10) was inserted into the cisterna magna; suitable placement of the catheter was assessed by: (a) testing cerebrospinal fluid (CSF) flowing through the catheter and (b) observing (with magnification) the lower distal part of the catheter through the atlanto-occipital membrane. The tail artery was cannulated for sampling of blood to measure pH, pCO,, PO,, and monitoring of the arterial blood pressure. The body temperature, monitored by a rectal thermometer, was adjusted and maintained near 37°C by external heating. When the rats were in a steady respiratory state (arterial PO, and pCO,, 100 mm Hg and 35-40 mm Hg, respectively) autologous arterial blood was collected (0.35 d) from the femoral artery, and an aliquot of 0.30ml was injected into the cisterna magna via the catheter within about 2 min. Before SAH induction, a CSF sample of about 0.01-0.03 ml. was gently drawn in order to limit modifications of intracranial pressure. The animals were held in a 20" head down position. Experimental groups
Sixty rats were divided into the following groups: Group 1. Six rats (controls) were killed and sub264
mitted to biochemical analysis at different times after general anesthesia without surgical manipulation. Group 2. Eighteen animals (sham-operated) subjected to injection of 0.3 ml. of mock CSF (saline solution with 25 meq/L of NaHCO,) into cisterna magna and were treated with saline solution i.v. Treatment with saline solution was performed immediately after mock CSF injection for animals killed at 1h and 6 h, while four injections (every 8 h) were given to rats killed at 48 h. Group 3. Eighteen rats (SAH) were injected with 0.3 ml. of autologous arterial blood into cisterna magna and treated with saline solution, as described for rats of Group 1. Group 4. Eighteen (SAH + MP) rats were injected with 0.3 ml. if autologous arterial blood into cisterna magna and treated with high-dose (30 mg/Kg, i.v.) of methylprednisolone (MP), immediately after SAH induction for animals killed at 1 and 6 h, while animals killed at 48 h were treated with 30 mg/Kg of MP immediately after SAH induction and then with 3 other injections (10 mg/Kg i.v. every 8 hours). Analytical methods
The rats were decapitated and brains were immediately frozen in dry ice. Brain samples for Na+-K'ATPase activity and TBRA assay were prepared in a pre-refrigerated glove box maintained at - 22°C. The brain areas were dissected (28), weighed and homogenized (1/10 g/ml) in ice-cold 50 mM phosphate buffer (pH7) containing 15 mM Na+ and 145 mM K + in a pre-cooled Potter Braun S homogenizer (8 strokes up and down, 800 rpm). An aliquot of the homogenate was used to measure Na+-K'ATPase by continous monitoring of the rate of ATP hydrolysis, using the coupled enzyme system pyruvate kinase - lactate dehydrogenase as proposed by Scharschmidt et al. (29). Tissue lipid peroxidation was evaluated by measurement of TBRA (23) using the undiluted tissue homogenate prepared for ATPase assay. Aliquots of 0.5 ml of homogenate were deproteinized with 20 % thrichloroacetic acid and centrifuged at 3000 g for 15 min. The supernatant fraction was then diluted 1 : 1 with 10% trichloroacetic acid containing 0.67% thiobarbituric acid, heated in a boiling water bath for lOmin, then cooled and the absorbance read at 532 nm. Malonyldialdehyde (MDA) was evaluated on the basis of a molar extinction coefficient of 1.56 x lo5 - M - cm - expressed as nmol MDA [mg protein] - '. The protein content of homogenate was assayed according to Lowry et al. (30) with serum albumin as a standard. Materials. Enzymes were obtained from Boehringer Mannheim. Substrates, coenzymes and reagent for enzymatic assay were obtained from Sigma Che-
'
Methylprednisolone and lipid peroxidation after SAH micals (St. Louis, MO). All other chemicals used were of reagent analytical grade. Statistics. Analytical runs were performed on lots of up to 6 animals, which consisted of SAH, SAHtreated and sham-operated. Statistical analysis of the results was carried out using the Analysis of Variance (ANOVA). Tukey's test for multiple comparisons was applied where ANOVA gave significant differences. Results
Physiological parameters
mental period of 1 h; the average variation of MABP was less than 8%. Four rats (one from the shamoperated group, one of the SAH-treated and two from the SAH groups) showed a change in pC0, greater than 20 % during the experimental period and these rats were excluded from the analysis of results. Three SAH animals (9%) died within 10 h after a normal recovery from anesthesia. An extensive subarachnoid blood deposition in the basal cisterns of the brain is evident at 30 min, 1 h, and 6 h after SAH induction. No hemorrhage was noted in the saline treated rats.
During SAH induction, MABP and arterial pC0, remained constant for each rat during an experi-
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Fig. 1 . Bar graph representation of Na'-K'ATPase activity (Fig. 1A) and TBA-R content (Fig. 1B) in cerebral cortex of rats subjected to experimental subarachnoid hemorrhage (SAH) induction and high-dose methylprednisolone treatment. Analysis on lot of up to 6 animals for each group. Statistics: analysis of the variance (Anova Test) and Tukey's Test for multiple comparisons. Statistical significance: Controls vs Sham-operated: A p < .05; A A p < .02; A A A p < .01. Sham-operated vs SAH: * p < .05; ** p < .02; *** p < .01. SAH vs SAH-treated: 0 p < .05; 0 0 p c .02; 0 0 0 p < .01.
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Marzatico et al. Effects of MP on lipid peroxidation and Nat-KtATPase during subarachnoid hemorrhage
Cerebral cortex. There is a marked decrease of Na -K ATPase activity in cerebral cortex at 1 and 6 h after SAH induction, while at 2 days there is no significant difference between sham-operated and SAH rats (Fig. la). TBRA content was higher in the sham-operated animals compared to the control animals. MP treatment significantly improves Na K +ATPase activity at 1 and 6 h after SAH and generally decreases the MDA content but only at 1 h after SAH, the TBRA content was significantly lower respect to sham-operated and SAH animals (Fig. lb). +
+
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Hippocampus. Na+-K ATPase activity did not changed in the hippocampus (Fig. 2a). The lipid peroxide products evaluated with TBAR show a similar difference between controls and shamoperated as in cerebral cortex (Fig. 2b). In MP treated animals the Na -K ATPase activity significantly increased at 6 hours after SAH induction when compared to sham-operated rats (Fig. 2a), and the TBRA content significantly decreased at 6 h and 2 days compared with the sham-operated and SAH groups. Bruin stem. In SAH group, Na+-K'ATPase activity significantlydecreased in the brain stem only at 1 h, without any significant changes at 6 and 2 days (Fig. 3a). The TBRA content significantly in+
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Fig. 2. Bar graph representation of Na+-K ATPase activity (Fig. 1A) and TBA-R content (Fig. 1B) in hippocampus of rats subjected to experimental subarachnoid hemorrhage (SAH) induction and high-dose methylprednisolone treatment. Analysis on lot of up to 6 animals for each group. Statistics: analysis of the variance (Anova Test) and Tukey's Test for multiple comparisons. Statistical significance: Controls vs Sham-operated: A p < .05; A A p c .02; A A A p < .01. Sham-operated vs SAH: * p < .05; ** p < .02; *** p < .01. SAH vs SAH-treated: 0 p < .05; 0 0 p < .02; 0 0 0 p c .01. +
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Methylprednisolone and lipid peroxidation after SAH 75 c)
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Fig.3. Bar graph representation of Na'-K ATPase activity (Fig. 1A) and TBA-R content (Fig. 1B) in brain stem ofrats subjected to experimental subarachnoid hemorrhage (SAH) induction and high-dose methylprednisolone treatment. Analysis on lot of up to 6 animals for each group. Statistics: analysis of the variance (Anova Test) and Tukey's Test for multiple comparisons. Statistical significance: Controls vs Sham-operated: A p < .05; A A p < .02; A A A p < .01. Sham-operated vs SAH: * p < .05; ** p < .02; *** p < .01. SAH vs SAH-treated: 0 p < .05; 0 0 p < .02; 0.0 p < .01.
creased in the sham-operated animals respect to controls 1 h after the injection, while at 6 h and 2 days the TBAR content in sham-operated animals was higher, but not significantly so than observed in control animals (Fig. 3b). MP treatment increased the Na+-K' ATPase activity until values found in sham-operatedrats at 1h, and over these value at 6 h (Fig. 3a). MDA content after MP treatment was significantlyreduced at 1 h, 6 h and 2 days compared both with sham-operated and SAH animals. Discussion
Although several clinical and experimental studies have shown global changes in CBF and in brain metabolic patterns after aneurysmal SAH. there is
no evidence of a closed relationship between CBF and metabolic changes when vasospasm occur (16, 18,25,3 1). Many attempts have been made in order to reduce and/or prevent vasospasm after SAH, antagonizing spasmogenic factors such as monoamines (1, 32), arachidonic acid metabolites (33), or breakdown products of erythrocytes (34). Experimental evidences of characteristic changes in the arterial wall after vasospasm have suggested that an inflammatory response induced by blood deposition in the subarachnoidal spaces could be one of the most important pathogenetical mechanisms (10,22). The decrease of CBF due to the increase of ICP and to the onset of arterial spasm leads to anoxicischemic damage. Free radicals, enhancing lipid peroxidation of cellular and subcellular membrane 267
Marzatico et al. HIGH-DOSE METHPLPREDMSOLONE
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PRESERVATION OF
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ARTERIALWALL XNTEGRm
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APROVEMENT
PRESERVATIONOF
OF CBF
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FUNCTIONALITY
Fig. 4. Speculative hypothesis of the methylprednisolone effects after experimental SAH.
phospholipids, have been suggested as the triggers of neuronal damage (19, 35, 36). Treatment with high-dosemethylprednisolonehas been proposed for the prevention of delayed cerebral vasospasm (10, 22): in dogs subjected to experimental SAH the ultrastructural integrity of smooth muscle cells in the arterial wall is preserved; moreover, a preliminary clinical study has reported beneficial effects in preventing vasospasm occurrence (1 l), but the pharmacological mechanism is not well understood. In a previous study in the same experimental model, we have shown that after SAH there is a significant impairment of Na+-K'ATPase activity in cerebral cortex and in the brain stem, while in the hippocampus, the area more sensitive to ischemic insult, the enzymatic activity does not significantly change. Lipid peroxidative processes, as quantified by TBAR measurement, are not significantly enhanced after SAH compared to shamoperated rats, and changes of Na -K ATPase activity are not closely related to the magnitudo of lipid peroxidation (37). Moreover, the present results show that the injection per se into the cistema magna, induces a transient increase of lipoperoxidative products (MDA) in all brain areas tested: this event is independent from the presence of iron which in case of SAH are liberated from red cells and could be a stimulus triggering MDA formation (38, 39). Otherwise, Na -K ATPase activity is significantly affected only after SAH induction, when blood is present in the subarachnoidal spaces suggesting that other pathophysiological mechanisms act on this enzyme activity. In the acute phase of the SAH the increase of ICP, and the consequent decrease of CBF (12), the presence of the blood in the +
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subarachnoid spaces' and the decrease of cerebral energy potential (3 1, 37) are, together, possible factors triggering the sequence of various metabolic events, which mimick the brain response to an anoxic-ischemic insult. It has been demonstrated that a low concentration of ATP itself, may limit the Na+-K'pump (40),and all these factors may contribute to the inhibition of Na+-K+ATPase. Treatment with high-dose methylprednisolone decreases the MDA content in all brain areas at different times comparing hemorrhagic and shamoperated rats with control values: thus, we may hypothesize that the increased lipid peroxidation seen after SAH in our experiment until 2 days after blood injection should be related to metabolic events and surgical manipulation. Meanwhile, the results of the present study show that the beneficial effect of high-dose methylprednisolone after SAH could be related to the reduction of peroxide lipid products in all brain areas with a major stability of cellular membrane: this would influence the activity of lipid-dependent enzyme Na -K ATPase, reduce the derangement of mitochondria1 membrane preserving the electrochemical ion gradient (37) and maintain cellular energy metabolism at levels sufficient to preserve the activity of Na -K +pump (Fig. 4). Although the results of the present study have provided some interesting data regarding peroxidative events of brain cell membrane after SAH, further studies are ongoing in order to verify the relationship between CBF, oxidative and peroxidative metabolism and the effects of glucocorticoid treatment. +
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