Neuropharmacology Vol. 32, No. 3, pp. 235-241, 1993
0028-3908/93 $6.00 + 0.00 Pergamon Press Ltd
Printed in Great Britain
EFFECT OF STOBADINE ON LIPID PEROXIDATION A N D PHOSPHOLIPIDS IN RABBIT SPINAL CORD AFTER ISCHAEMIA N. LUKJdX)v/~,* M. CHAVKOand G. HAL?tT Institute of Neurobiology, Slovak Academy of Sciences, grobhrova 57, 040 00 Ko~ice, Slovakia (Accepted 20 October 1992)
Sununary--Stobadine, a drug with the pyridoindol structure, was compared with thiopental and pentobarbital for its ability to inhibit stimulated peroxidation in homogenates of spinal cord/n vitro. The antioxidative capacity of the drug exceeded that of barbiturates more than 100-fold. Stobadine was also shown to inhibit the increase in formation of TBA-RS in homogenates of rabbit spinal cord, subjected to 20 min ischaemia, to the level comparable with controls. Administration of the drug (6 mg kg -~) to animals 5 min before 20 min ischaemia had no effect on level of lipid peroxidation products in the spinal cord; however, it slowed down stimulated Fe2+-dependent peroxidation after in vitro incubation of the homogenates and increased the concentration of phosphatidylserine and ethanolamine plasmalogens, as compared with non-treated animals. Application of stobadine 2 rain before the release of an aortic occlusion increased the antiradical capacity in homogenates of spinal cord and revealed an ameliorating effect on the composition of phospholipids. Key words--lipid peroxidation, phospholipids, stobadine, spinal cord ischaemia.
There is accumulating information which suggests that there is a link between oxygen radical-mediated lipid l:~eroxidation and the pathophysiology of cerebral ischaemia. Biochemical indices of lipid peroxidation during and/or after a period of cerebral ischaemia, include accumulation of the products of lipid peroxidation (Cao, Carney, Duchon, Floyd and Chevion, 1988; Watson, Busto, Goldberg, Santiso, Yoshida and Ginsberg, 1984), decrease in the level of anti-oxidants in tissue (Flamm, Demopoulos, Seligman, Poser and Ransohoff, 1978; Abe, Yoshida, Watson, Busto, Kogure and Ginsberg, 1983; Ginsberg, 'Watson, Yoshida, Busto, Abe, Goldberg and Scheinberg, 1983) and direct evidence of the generation :for free radicals during post-ischaemic reperfusion (Braughler and Hall, 1989; Kirsch, Phelan, Lange and Traystman, 1987). The role of the formation of free radicals as a causal factor in postischaemic damage, is also supported by the beneficial effect of radical scavengers or anti-oxidants on metabolic, physiological and electrophysiological recovery in the brain after ischaemia. Strong support for the deleterious effect of free radicalis was demonstrated by the ability of methylprednisolone to inhibit lipid peroxidation (Demopoulos, Flamm, Seligman, Pietronigro, Tomasula and Decrescito, 1982; Hall and Braughler, 1982) and to improve functional recovery in the spinal cord after isehaemia (Hall and Braughler, 1981). Treat*To whom correspondence and reprint requests should be addressed. NP32/~:
235
ment with superoxide dismutase in the early period of reperfusion, reduced both motor dysfunction and the incidence of infarcts after ischaemia in the spinal cord (Cuevas, Reimers, Carceller and Jimenez, 1990; Uyama, Shiratsuki, Matsuyama, Nakanishi, Matsumoto, Yamada, Narita and Sugita, 1990). Recent studies from this laboratory have shown increased susceptibility to lipid peroxidation in the spinal cord, subjected to ischaemia of more than 10min duration (Hal/tt, Chavko, Lukh~ov~i, Kluchov~i and Margala, 1989) and reduced neuropathological damage and improved functional recovery after decreasing arterial oxygen tension in the early post-ischaemic reperfusion (Danielisov/t, MarSala, Chavko and Margala, 1990; Margala, Danielisov/L, Chavko, Hort~ikov~i and MarSala, 1989). In view of the beneficial effect of some pharmacological interventions against ischaemia in the brain, it may be of importance, if some other drugs, operating as antioxidants, are equally or more efficient against ischaemic damage to the spinal cord. Stobadine, a new drug with the pyridoindole structure (gtolc, Bauer, Beneg and Tich~, 1983), was shown to maintain the level of glutathione and thiol groups and to prevent lipid peroxidation in cerebral mitochondria, synaptosomes and microsomes after oxidative stress, induced by isoprenaline (Ondrejickova, Sedlak, Macickova and Benes, 1988). In the present study, the influence of stobadine on lipid peroxidation and the composition of lipids in the spinal cord was evaluated.
236
N. LUK~,~OV,~et al. METHODS
Operative techniques Experiments were done on adult rabbits, weighing 3.0-3.5 kg. The animals were anaesthetized with pentobarbital (30 mg kg-~) and ischaemia in the spinal cord was induced by ligation of the abdominal aorta, just below the left renal artery for 20 or 40 min. At the end of the ischaemia, the occlusion was removed and the animals were recirculated for 60 min. Care was taken to avoid changes/fluctuations in body temperature by keeping the animals on a warming blanket. The experimental model used for this study has been described in detail previously (Hal~it et aL, 1989). The following groups were investigated: (1) sham-operated controls (n = 6 - 8 ) , (2) untreated groups which underwent ischaemia for 20 or 40 min, with or without 60 min reperfusion (n = 6-8 of each), (3) a treated group in which stobadine, at a dose indicated in the legends, was given intravenously 5 min prior 20 min ischaemia (n = 8), (4) a treated group in which stobadine was given intravenously 2 min before release of the aortic occlusion (n = 8). In all experimental groups the backbone was excised, segments L4-S ~ of the spinal cord were extruded in ice-cold saline, cleaned from envelopes and dipped into liquid nitrogen.
Preparation of homogenates Frozen tissue was weighed and homogenized in 5vols of ice-cold Tris-HCI buffer (50mol 1-t, pH 7.4). For direct determination of thiobarbituric acid reactive substances (TBA-RS) and for extraction of lipids, 1 nmol 1- ~ethylene diamine tetra acetic acid (EDTA) was included in the buffer preequilibrated with nitrogen gas.
Incubation conditions For postischaemic in vitro peroxidation, homogenates were incubated in a water bath at 37°C, after addition of ferrous sulphate (0.01 mM 1-~) and ascorbic acid (0.25mM 1-~). Samples in triplicate were continuously equilibrated with a gas mixture, consisting of 95% of oxygen and 5% of carbon dioxide for 0, 15, 30, 45 and 60 min. For anti-oxidative activity, the following drugs were tested: DH 1011 (the hydrophylic) and DP 1031 (the lipophylic) form of stobadine, pentobarbital and thiopental. Both forms of stobadine were dissolved in ice-cold Tris-HCl buffer, pH 7.4. The DP 1031 was sonicated for 10 min at 20 kHz before use (Dynatech ultrasonic dismembrator). Thiopental and pentobarbital were dissolved in redistilled water. All solutions of drugs were made freshly and added just prior to the addition of the free radical initiators (ferrous sulphate and ascorbic acid). Samples in triplicate were incubated as indicated.
Analytical techniques Homogenates used for determination of the products of lipid peroxidation, were extracted with 3 ml of
1% phosphoric acid and TBA-RS were determined by the method of Uchiyama and Mihara (1978). One ml of 0.67% aqueous solution of thiobarbituric acid, prepared freshly before use, was added to all samples and vortexed. The reaction mixture was heated in boiling water for 45 rain; EDTA was added to each sample before boiling to a final concentration 1 mmol l ~. After cooling in ice 4 ml of n-butanol was added, followed by mixing and centrifugation at 1500 g for 10rain. The absorbance of the extracted chromophore in the organic layer was measured at 532 nm against extraction blanks in Specord u.v.-vis (Carl Zeiss Jena, Germany). The TBA-RS were quantified using a calibration curve, prepared with various concentrations of malondialdehyde (MDA). The concentration of protein was determined by the method of Lowry, Rosebrough, Farr and Randall 0951).
Extraction and determination of phospholipids in spinal cord
Aliquots (0.5 ml) of homogenates were extracted, according to Folch, Lees and Sloane-Stanley (1957). The final organic phase, washed with 0.9% KC1, was evaporated in vacuum and lipids were reconstituted in 5 ml of chloroform-methanol (2:1, by vol). The extracts were kept under N 2 gas at - 3 0 ° C until analysis. Butylated hydroxytoluene was used as an antioxidant during extraction and storage. Phospholipids were separated by 2D-TLC (Horrocks and Sun, 1972) on glass plates coated with Kieselgel G 60 (Merck, Germany). Plates in the first dimension were eluted with chloroformmethanol-ammonia (65:35:4, by vol), dried in a stream of air and the silica gel layer was exposed to HC1 fumes for 5min. Following hydrolysis, the fumes were blown off by a stream of air and the plates were eluted in the second dimension with chloroform-methanol-acetone-ammonium acetate (0.1 tool 1-t-acetic acid (140:50:55:10:2.5, by vol). Phospholipids were visualized by brief exposure to iodine vapour. Phospholipid fractions were scraped into test tubes and lipid phosphorus was assessed spectrophotometrically, according to Rouser, Fleischer and Yamamoto (1970). Total phospholipids were determined directly in lipid extracts. Results from the analyses were expressed as nmol phospholipid per mg protein. All chemicals were of analytical purity and organic solvents were redistilled prior to use. Both DH 1031 and DP 1031 were a gift from Dr Stolc, Institute of Experimental Pharmacology, Slovak Academy of Sciences, Bratislava. The results were evaluated statistically by Student's t-test. RESULTS
The effects of hydrophylic (DH 1011) and lipophylic (DP 1031) forms of stobadine on the rate of formation of products of lipid peroxidation in homogenates of spinal cord are shown in Fig. 1. The
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Fig. 1. Effect of ( 0 ) pentobarbital, (O) thiopental, (A) DH 1011 and (A) DP 1031 on formation of TBA-RS in control spinal cord tissue. Anti-oxidants in various concentrations were added to homogenates and incubated at 37C, in the presence of 0.01 mmol 1 ~FeSO4, 0.25 mmol 1-~ ascorbic acid, 95% 02 + 5% CO, for 60 min. Data are means of 6 experiments + SEM. MDA = malondialdehyde. addition o f both forms of stobadine to the incubation mixture significantly reduced the rate of formation of TBA-RS. At z~ concentration of 10 3mol 1 ~ the degree of inhibition was 84 and 96% for the hydrophylk and lipophylic forms, respectively, As shown, the same concentration of pentobarbital and thiopental inhibited the formation of T B A - R S by 18 and 48%, respectively (Fig. Ij. lnc abation of homogenates of ischaemic tissues, in the presence of the peroxidation couple and oxygen was u~ed to simulate the sudden restoration of blood suppl.'/ to previously ischaemic tissue. Larger values for TBA-RS, as compared to controls, were observed after incubation for 60 min in homogenates of spinal cord, subjected to 40 min ischaemia (Fig. 2). The addition of stobadine to the incubation mixture
reduced the increase in lipid peroxidation to a degree comparable with control homogenates. The concentration of total phospholipids in control spinal cord was 1.10#mol of lipid phosphorus per mg protein. This value was not significantly affected during the period of ischaemia. In vitro incubation reduced lipid phosphorus in homogenates from control and ischaemic spinal cords. The relative decrease was 92% in control and 87% in ischaemic spinal cord, respectively. The addition of stobadine to ischaemic homogenates prevented the decrease in lipid phosphorus and in a concentration of 10 -3 m o l l - l , the concentration of phospholipid almost completely recovered (94% of control, n.s.) (Fig. 3). The effect of administration of stobadine to animals, prior to and after 20 min of ischaemia, on
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Fig. 2. Effect of DH 1011 on the formation of TBA-RS in homogenates of spinal cord from rabbits after 40 min ischaemia. DH 1011 (10 -6, 10 -4, 10 -3 mol 1-~) was added to the homogenates and incubated at 37°C, in the presence of 0.01 mmol 1-1 FeSO 4 and 0.25 mmol 1-l ascorbic acid, 95% 02 + 5% CO 2 for 60 min. Data are means of 6 experiments ___SEM; ***P < 0.001, with respect to control.
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the rate of stimulated peroxidation in homogenates in vitro is shown at Fig. 4. There was no statistical difference in the level of TBA-RS in control and ischaemic tissue (zero time). However, the level of TBA-RS was significantly higher in samples after 20min ischaemia and 60 rain of incubation, than in those prepared from control tissue. Administration o f D H 1011 to animals 5 min before ischaemia had no effect on the level of peroxidation products in tissue; however, it slowed down stimulated lipid peroxidation in the homogenates from ischaemic spinal cord in a dose-dependent manner.
Although ischaemia itself does not increase the level of TBA-RS in the spinal cord, subsequent 60 min recirculation markedly enhanced the level of peroxidation products in tissue (Fig. 5). Stobadine, given post-ischaemia, before the release of the occlusion, reduced the increase in peroxidation as, compared with the untreated group by 87 %; however, the TBA-RS still persisted at significantly higher level, compared with controls. Decreases in phosphatidylserine and ethanolamine plasmalogens were the only changes in phospholipids, observed after ischaemia or recirculation
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Fig. 4. Effect of DH 1011, administered prior to ischaemia, on production of TBA-RS during postischaemic in vitro peroxidation. (©) Control, ( 0 ) ischaemia for 20 min, (A) ischaemia for 20 min and treatment with DH I011 (4mgkg-~), (&) ischaemia for 20min and treatment with DH 101l (6 mg kg-~ ). DH 1011 was administered intravenously 5 min before 20 min ischaemia. Homogenates were incubated at 37°C, in the presence of 0.01 mmol 1-t FeSO4, 0.25 mmol 1-t ascorbic acid, 95% 02 + 5% CO2 for 60 min. Data are means of 8 experiments + SEM, *P < 0.05, ~P < 0.01 with respect to incubated control.
Effect of stobadine on spinal cord ischaemia
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0.0 Fig. 5. Effect of DH 1011, administered after ischaemia, on production of TBA-RS in homogenates of spinal cord in vivo. (E]) Control, (m) ischaemia for 20min, (1~) ischaemia for 20 min + recirculation 60min, (IE]) ischaemia+recirculation and treatment with DH 1011. DH 101! (6mg kg ~) was administered intravenously 2 min before recirculation. Data are means of 8 experiments + SEM. *P < 0.01 with respect to control, xp < 0.01 with respect to 20 min ischaemia and 60 min rccirculation. (Fig. 6). Ischaemia of 20 min duration reduced phosphatidylserine to 96% and ethanolamine plasmalogens to 88% of control. Administration of stobadine, prior to ischaemia, did not effect the degree of the decrease. During subsequent recirculation, the concentration of phosphatidylserine fell to 90% and elhanolamine plasmalogens to 80% of controls. Stobactine injected prior to ischaemia, produced recover), of phosphatidylserine to 96% and ethanolamine plasmalogens to 89%, whereas administration of the drug prior recirculation produced complete recovery of ethanolamine plasmalogens. DISCUSSION
Stobadine, a drug with the pyridoindol structure has been tested for its protective myocardial effects
(Styk, Gabauer, Okoli6~ny, Slez~ik, Holec and BeneL 1985). As detected by spectroscopy, some of the beneficial effects can be mediated by donating hydrogen from the indole nitrogen to lipid radicals and thus terminating lipid peroxidation (OndriaL Migik, Gergel' and Sta~ko, 1989). In a previous study (Hal~.t et al., 1989) it was demonstrated that marked differences occurred in susceptibility to peroxidative degradation of lipids in the spinal cord after 20 and 40min ischaemia, as compared to 10min ischaemia or control. The pattern of the production of markers of lipid peroxidation after incubation in vitro indicates a diverse degree of exhaustion of hydrogen donors in the tissue, after the respective ischaemic intervals and, therefore, a distinct decrease in antioxidant capacity in the spinal cord after ischaemia lasting longer than 10 rain. The differences
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Fig. 6. Concentrations ofserine phospholipids (PS) and ethanolamine plasmalogens (Epls) in homogenates of spinal cord after administration of DH 1011. (U]) Control, (m) ischaemia for 20 min, (l~l) ischaemia for 20min and pretreatment with DH 1011, (Ira) ischaemia for 20min + recirculation 60rain, ([]) ischaemia + recirculation and pretreatment with DH 1011, ([]) ischaemia + recirculation and posttreatment with DH 1011. DH 1011 (6mg kg -1) was administered intravenously, 5 min before 20 min ischaemia (pretreatment) or 2 min before recirculation (post-treatment). Data are means of 8 experiments + S E M . *P < 0.05; ***P < 0.001 with respect to 20 rain ischaemia and 60 rain recirculation.
240
N. Lux<~t~ovA etal.
between lipid peroxidation in control and ischaemic spinal cord correspond with the degree of functional impairment, as demonstrated by the neurological deficit occurring in animals subjected to ischaemia of more than 10min duration (Danielisovfi et al., 1990). It would therefore be interesting to test some antioxidants for their ability to suppress the rate of peroxidation in tissue with a decreased defence against oxidative damage. As shown here, the capacity of stobadine to suppress stimulated peroxidation in homogenates from spinal cord exceeded that of thiopental by more than 100 times. The results are in accord with the powerful effect of stobadine against lipid peroxidation in liposomes (Ondriag et al., 1989). In addition, stobadine, at a concentration of 10 3 1 ~, was found to inhibit completely the peroxidation in homogenates after 40rain ischaemia and to ameliorate the decrease in lipid phosphorus. The results indicate the remarkable antioxidative effect of the drug against lipid peroxidation in vitro. In vivo, despite the decrease in some lipids (ethanolamine plasmalogens), after 20 min of ischaemia, it was not possible to detect an increase in the products of lipid peroxidation, originating from endoperoxy hydroperoxides, as reported by others in whole brain (Yoshida, Busto, Watson, Santiso and Ginsberg, 1985). It is assumed that lipolysis was mainly due to the action of phospholipase; however, the present data do not reflect possible differences in susceptibility of individual structures of the spinal cord to ischaemia. Stobadine, administered before ischaemia, inhibited the rate of formation of TBA-RS in homogenates of spinal cord, after 20 min of ischaemia; however, it did not influence the initial level of TBA-RS in the spinal cord (zero time). It appears, that stobadine can prevent an excessive peroxidation, during reoxygenation, following ischaemia. The treatment protocol was targeted to the early reperfusion period, following 20 min of ischaemia. The procedure was based on the hypothesis that this duration would increase the probability for early reversible changes to be present in the time of administration of stobadine. Longer periods of aortic occlusion may induce irreversible changes prior to treatment with stobadine. Although ischaemia itself does not affect lipid peroxidation in the spinal cord, 60min recirculation significantly increased the level of TBARS in tissue. As the increase in TBA-RS was significantly less in treated animals, administration of stobadine during early reperfusion seemed to prevent, at least partially, the deteriorating process which leads to the final damage. Further studies may help to understand the effect of stobadine on the ischaemic spinal cord. The present data suggest that stobadine could be useful for the treatment of ischaemic damage.
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Uchiyama M. and Mihara M. (1978) Determination of malondialdehyde precursor in tissues by thiobarbituric acid test. Analyt. Biochem. 86: 271-278. Uyama O., Shiratsuki N., Matsuyama T., Nakanishi T., Matsumoto Y., Yamada T., Narita M. and Sugita M. (1990) Protective effects of superoxide dismutase on acute reperfusion injury of gerbil brain. Free Radical Biol. Med. 8: 265-268. Watson B. D., Busto R., Goldberg W. J., Santiso M., Yoshida S. and Ginsberg M. D. (1984) Lipid peroxidation in vivo induced by reversible global ischemia in rat brain. J. Neurochem. 42: 268-274. Yoshida S., Busto R., Watson B. D., Santiso M. and Ginsberg M. D. (1985) Postischemic cerebral lipid peroxidation in vivo: modification by dietary vitamin E. J. Neurochem. 44: 1593-1601.