Effect of transient reduction of cerebral blood flow on membrane anisotropy and lipid peroxidation in different rat brain areas

Pergamon

0197-0186(94)E0038-U

Neurochem. Int. Vol. 25, No. 2, pp. 161-168,1994 Copyright © 1994ElsevierScienceLtd Printedin Great Britain.All rightsreserved 0197-0186/94$7.00+0.00

EFFECT OF TRANSIENT REDUCTION OF CEREBRAL BLOOD FLOW ON MEMBRANE ANISOTROPY A N D LIPID PEROXIDATION IN DIFFERENT RAT BRAIN AREAS MIRA MELZACKAl , NINA WEINER2, CHRISTINE HELM3*, RAINALD SCHMIDT-KASTNER4, MARIA SIEKLUCKA5, KARL-HEINZ SONTAG6 and WOLFGANG WESEMANN2 1Institute of Pharmacology, Polish Academy of Sciences, Krakow, Poland 2Department of Neurochemistry, University of Marburg, D-35043 Marburg, Germany 3Department of Psychiatry, University of GOttingen, von-Siebold-Str. 5, D-37075 G6ttingen, Germany 4Department of Neurophysiology, Ruhr-Universitiit Bochum, D-44801 Bochum, Germany 5Department of Pharmacology, Medical School, Lublin, Poland 6Department of Pharmacology, Max-Planck Institute for Experimental Medicine, D-37075 G6ttingen, Germany (Received 18 November 1993 ; accepted 3 February 1994)

AImraet--Light-microscopical studies revealed that oligemic hypoxia for 24 and 60 min as produced by bilateral clamping of the carotid arteries (BCCA) in normotension does not produce neuronal cell necrosis in the vast majority of rat brain. Less than 5% of cases showed a pattern of mild selectiveneuronal necrosis as would be expected in isehemia. However, significant changes in both lipid peroxidation (as measured by MDA formation) and membrane anisotropy (measured by DPH or TMA-DPH, respectively, as a fluorescenceprobe) in cortical and striatal, but not in hippocampal, membrane fractions could be measured in ex vivo studies. Twenty-four and 60 rain of BCCA without reperfusion decreased lipid pcroxidation in the cerebral cortex but not in the striatum. BCCA, either for 24 or 60 rain, and 60 min of reperfusion produced no changes in lipid peroxidation in either structure. However, 24 and 60 min of BCCA followed by 14 days of reperfusion led to a significant increase in MDA formation in the striatum, while lipid peroxidation in the cortex was only increased after 60 min of BCCA. Cortical as well as striatal membrane anisotropy increased significantly 14 days later in rats submitted to BCCA for 24 or 60 rain. The study shows an increased lipid peroxidation 2 weeks after a transient reduction in cerebral blood flow although no neuronal necrosis could be observed in general.

al., 1991), for example, prevented ischemic neuronal damage in the gerbil hippocampus and cerebral cortex of rats. These and other data (Chan et al., 1984 ; Abe et al., 1988) confirmed the hypothesis that ischemic brain cell necrosis can (at least partially) be induced by an elevated production of free radicals and lipid peroxidation in the brain. Recent studies by Carney et al. (1992) provided direct evidence of the production of oxygen free radicals in the post-ischemic brain. The biochemical changes were associated with both neuropathological changes in the hippocampus and cortex and significant behavioural deficits. It was therefore suggested that *Author to whom all correspondence should be addressed. subsequent cell damage occurred only in brain regions 161

Total or transient ischemia affects susceptibility to lipid peroxidation in vitro and changes membrane anisotropy in the cerebral cortices and hippocampus of gerbils (Villacara et al., 1989 ; Haba et al., 1991 ; Hara et al., 1991 ; Viani et al., 1991 ; Carney et al., 1992). The direction and intensity of these effects depend on the duration of both ischemia and reperfusion (Enseleit et al., 1984 ; Haba et al., 1991 ; Carney et al., 1992). It has also been shown that pretreatment of experimental animals with antioxidants such as x-tocopherol (Hara et al., 1990), KB-5666 or idebenone (Hara et al., 1991) and tirilazad mesylate (Hall et

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with increased free radical production, thus leading to significant behavioural deficiencies (C'arney et al.. 1992). Oligemic b l o o d flow reduction as produced by bilateral clamping o f the carotid arteries (BCCA) in rats (Block et al., 1993b) does not result in necrotic cell d a m a g e in the most vulnerable brain structures. A n intensive light-microscopical analysis did not show n e u r o p a t h o l o g i c a l changes as presented in a brief report by S c h m i d t - K a s t n e r et al. (1988) (see also Heim et al., 1990; Jaspers et al., 1990; Sontag et al., 1992). However, animals after B C C A showed clearcut spatial m e m o r y deficiencies in the acquisition of a water maze (Jaspers et al., 1990) or a hole b o a r d task (Heim et al., 1990), as well as disturbances in REM-sleep a n d ultradian rhythmicity (Sontag et al., 1992: Ulrich et al., 1992). This suggests that subtle neurochemical alterations reflect the behavioural changes (Helm et al., 1990; Sieklucka et al., 1991, 1992). The question arises, therefore, whether the spatial m e m o r y deficiencies following blood flow reduction to oligemic levels might also depend on increased lipid peroxidation a n d / o r decreased m e m b r a n e fluidity, even if no necrotic cell d a m a g e occurs. Structural changes of m e m b r a n e s as a result of altered lipid b i o t r a n s f o r m a t i o n can modify enzyme activity ( B a b a et al., 1981) as well as lead to disruption of Ca ~-` homeostasis which is believed to play a crucial role in age-associated b r a i n changes ( K h a c h a t u r i a n , 1989). To this end we investigated the time course o f ex vivo lipid peroxidation in rats following 24 or 60 rain of B C C A , with a n d without reperfusion, in cortex, h i p p o c a m p u s and striatum, three brain areas vulnerable to forebrain ischemia (Pulsinelli et al., 1982 : Smith et al., 1984 ; S c h m i d t - K a s t n e r et al., 1989). This study was completed with the simultaneous measurem e n t of m e m b r a n e anisotropy in the same structures. A systematic light microscopical e x a m i n a t i o n of corresponding brain sections from 3 h up to 18 days after B C C A was performed in a parallel series of rats.

EXPERIMENTAL PROCEDURES

Oligemia experiments Experiments were carried out on 3-month-old (320 350 g) male Wistar rats. The animals were anaesthetized with pentobarbital (Nembutal, Sanofi, 60 mg/kg i.p.) and subjected to 24 or 60 rain of BCCA. The common carotid arteries were carefully prepared and clamped by using thread. During anaesthesia and surgery the rectal temperature was kept at approximately 3TC and the mean arterial blood pressure did not fall below normal levels (for further details see Block et al., 1993a). For BCCA experiments followed by reperfusion the strings were removed and the free reflow visually

inspected. Sham operated animals had their vessels prepared but not clamped. Rats were decapitated either immediatel.~ alter BCCA or after a reperfusion period of 60 rain or 14 days. The brains were removed and cortex, hlppocampus and striatum were dissected on an ice plate. Preparation of the crude membrane traction The tissues (cortex, hippocampus or striatum) were homogenized in 10 vol (10 m[ per gram of the wet tissue) of icecold sucrose (0.32 M) and centrifuged at 770 g for 10 rain. The supernatant was centrifuged at 40,000 g for 20 rain and the resulting pellet was resuspended in 20 vol of 50 mM Tris HC1 buffer (pH 7.4). Then the suspension was incubated at 37 C for 10 rain and centrifuged at 40,000 g for 20 rain. The resulting pellet was stored at - 2 0 C until it was used for assay of lipid peroxidation and membrane anisotropy. Assay of lipid peroxidation The assay of lipid peroxidation was performed according to Ohkawa et aL (1979). 250 ,ul of ascorbic acid (0.5 mM end concentration) was added to 250 ~tl of protein suspension (approx. 700-1000 mg protein per ml assayed according to Lowry et al., 1951). The samples were incubated at 37'C for 30 min and the reaction was stopped with 20% trichloroacetic acid (500 ,ul). Then the samples were centrifuged at 10,000 g for 5 rain, 500 ,ul of supernatant was transferred to another tube, mixed with 500/d of 0.67% thiobarbituric acid (TBA) and incubated at 9YC for 20 min. After centrifugation at 10,000 g for 5 rain the amount of TBA reactive material i.e. malondialdehyde (MDA) concentration as an index of lipid peroxidation was assayed with Dynatech MR5000 spectrophotometer against 1, 1,3,3-tetramethoxypropane as standard using the filter 530 rim. To exclude further lipid peroxidation during in vitro incubation we measured the degree of lipid peroxide with and without desferrioxamine and did not find any differences. The results were evaluated statistically using analysis of variance followed by the Student t-test. Membrane anisolropy measurement

The samples ( 100 pl) containing 250 mg protein/ml (Lowry et al., 1951) were incubated with 1,6-diphenyl-t;3,5-hexatriene (DPH), (final concentration of DPH in the sample 0.5 liM) at 37'C for 30 min, or with I-[4-(trimethylammonium)phenyl]6-phenyl-1,3,5-hexatriene (TMADPH), (final concentration of TMA-DPH in the sample 0.2 ~tM) at 3TC for 4 min. The fluorescence intensity was measured with a Perkin-Elmer LS 500 luminescence spectrophotometer equipped with two glass prism polarizers, excitation 338 nm, emission 446 nm. Analysis of variance followed by Student t-test was used for statistical evaluation. Histology For neuropathological analysis, animals with 24 min of BCCA were sacrificed at 3 and 12 h, 1, 2, 4, 14 and 18 days (n = 4, each) and compared with n = 3 sham controls, Rats subjected to 60 rain of BCCA followed the same scheme with n = 4 animals per time point, except for 18 days (n = 1). This analysis was completed by a repeat series with 60 min BCCA and 3 days survival (n = 4 BCCA, n = 2 sham operated animals). All rats were infused through the ascending aorta after a rinse with Ringer's solution with neutral buff-

Membrane anisotropy and lipid peroxidation in rat brain ered 4% formaldehyde for fixation in deep pentobarbital anaesthesia. Brains were embedded in paraffin and 5 pm frontal sections were prepared through the forebraln at 12 evenly spaced levels 0aaxinos and Watson, 1982) and stained with cresyl violet or K]Over-Barrcra method. The Massonmethod was also used for colour-enhancement of necrotic neurons.

RESULTS Lipid peroxidation (as measured by M D A formation) in the cerebral cortex of rats killed directly after 24 or 60 rain o f B C C A was significantly decreased compared with control [Fig. I(A, a)], whereas 24 or 60 re_in o f B C C A followed by 60 rain of reperfusion did not produce any changes in the

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Fig. l. Effects of BCCA on MDA formation (A) and membrane anisotropy (r) measured with DPH (13) and TMADPH (C) in the cerebral cortex of rats. (a) killed directly after BCCA; (b) killed 60 rain after BCCA; (c) killed 14 days after BCCA. Results are presented as percentage of the control (sham operated animals) and as mean _+SD of 6-8 animals. The level of MDK in controls (basal lipid peroxidation in the cerebral cortex stimulated with 0.5 mM ascorbic acid): (a) 38~53+7.60; (b) 36.06+5.10; (c) 35.51 _+7.58 nmol MDA/mg protein. Basal anisotropy (r) of cerebral cortex membrane: (a) 0.2140-+0:0028 (DPH) and 0.2651 _+0.0062 (TMA-DPH); (b) 0.2120_+0.0042 (DPH) and 0.2700_+0.0038 (TMA-DPH); (c) 0.2064_+0.0026 (DPH) and 0.2642_+0.0013 (TMA-DPH), *P < 0.05; **P<0.01 vs sham; analysis of variance followed by Student t-test.

95

n b c BCCA" [] 24rain, [] 60rain Fig. 2. Effects of BCCA on MDA formation (A) and membrane anisotropy (r) measured with DPH (B) and TMADPH (C) in the striatum of rats. (a) killed directly after BCCA; (b) killed 60 min after BCCA; (c) killed 14 days after BCCA. Results are presented as a percentage of control (sham operated animals) and as mean + SD of 6-8 animals. The level of MDA in controls (basal lipid peroxidation in the striatum stimulated with 0.SmM ascorbic acid): (a) 113.21+32.17; (b) 93.6+13.77; (c) 85.49_+13.65 nmol MDA/mg protein. Basal anisotropy (r) of striatal membranes: (a) 0.2237_+0.0040 (DPH) and 0.2729_+0.0052 (TMA-DPH) ; (b) 0.2210-+0.0029 (DPH) and 0.2724-+0.0033 (TMA-DPH); (c) 0.2229_+0.0043 (DPH) and 0.2723 _+0.0022 (TMA-DPH). *P < 0.05 ; **P < 0.01 vs sham ; analysis of variance followed by Student t-test. a

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amount of TBA reactive material [Fig. I(A, b)]. Sixty min of BCCA followed by 2 weeks of rcperfusion led to a significant increase in MDA formation compared with control [Fig. 1(A, c)]. Cortical membrane anisotropy measured with DPH as a fluorescence probe increased significantly compared to control in rats exposed to 24 or 60 min of BCCA and 14 days of reperfusion [Fig. 1(B, c)]. When the assay was performed with TMA-DPH as a fluorescence probe, only 24 min of BCCA and 2 weeks of reperfusion led to a significant elevation of membrane anisotropy [Fig. I(C, c)]. Twenty-four or 60 rain of BCCA and 2 weeks of reperfusion elevated lipid peroxidation and membrane anisotropy (TMA-DPH as a fluorescence probe) in a statistically significant manner in striatal membranes of rats as compared with controls [Fig. 2(A, c and C, c)]. Neither 24 rain or 60 min of BCCA, nor reperfusion for 60 min or 14 days affected the lipid peroxidation or membrane anisotropy in the hippocampus of the rats. The very large majority of animals showed normal brain histology as studied by Nissl-, Klttver Barreraand Masson-staining at the light-microscopic level, e.g. neuronal necrosis was absent from the CAI sector and hilus of hippocampus, neocortex, dorso-lateral striatum and lateral thalamus. Fig. 3 illustrates rare examples of ischemic neuronal damage found in this series. A tiny little focus of neuronal damage was found in one case in the neocortex after 24 min of BCCA and one day of survival [Fig. 3(A, B)]. Two cases after 24 min of BCCA had loci in striatum, one at 12 h [Fig. 3(C, D)] and the other at 2 days. One case with 60 min of BCCA had damage in the tuberculum olfactorium. Selective ischemic neuronal necrosis was a rare exception: one animal with 24 rain of BCCA had damage in the hippocampus [Fig. 3(E)] and other forebrain regions ; and another with 60 min of BCCA revealed mild selective neuronal necrosis in vulnerable areas. We illustrate these changes to outline the small size of such lesions and the normal histology of the structures can be clearly seen around the lesions out-

lined in the figure. The white matter structures e.g. bundles transversing the striatum [Fig. 3(C, D)] and corpus callosum [Fig. 3(E)] appeared normal in Kltiver- Barrera stains. The repeat series with 60 rain of BCCA and 3 days survival was devoid of any neuronal damage. Taken together, 57 animals with BCCA were analysed, at different time points of survival, and 2 (3.5%) had a pattern of selective neuronal necrosis similar to global ischemia and 4 (7%) had little loci of tissue damage. DISCUSSION

Bilateral clamping of the carotid arteries (BCCA) in normotension led to changes of lipid peroxidation and membrane anisotropy in cortical and striatal membranes of rats in e x vivo experiments. The results concerning the cortex of BCCA rats are consistent with those of Haba et al. (1991) who found a decrease in lipid peroxidation in the hippocampus of gerbils submitted to transient ischemia, normal values in comparison with controls after 60 min of reperfusion and an increase in TBA reactive material formation 7 14 days later. The decrease in lipid peroxidation in the cortical membranes of rats killed directly after 24 and 60 min of BCCA might be explained by an increased activity in superoxide dismutase (SOD) (Chan et al., 1987). It is known that SOD activity in the cerebral cortex of rats submitted to partial ischemia without reperfusion is significantly elevated (Kramer et al., 1987; Horakova et al., 1991). In the brain of rats sacrificed directly after 30 min of transient ischemia the level of protective ubiquinons (Takeshige et al., 1980) was significantly increased (Yoshida et al., 1982). Also, an increased level of free fatty acids, leading to a relative decrease in esterified fatty acids as the main source of free radicals, might be involved in the phenomenon (Yoshida et al., 1982). For the BCCA model comparable results do not exist. The elevation of TBA reactive material formation

Fig. 3 (opposite). This figure illustrates a rare instance of ischemic neuronal damage after 24 min of BCCA. The lesions are outlined by arrows in the overviews(A, C, E). Normal tissue surrounds the lesions and this would be the normal anatomy referred to in the very large part of the series. Note that these were lesions found only after scrutinizing a large number of sections in a series of 57 brains and that they are exceptional findings. (A) Neocortex at 1 day, a tiny focus in the upper layers outlined by arrowheads; (B) higher magnification showing ischemiccell death, with the surrounding normal neurons marked by small arrows ; (C) Striatum at 12 h, a small focus of apparently damaged cells is outlined by arrowheads; (D) close up with dark-staining, damaged neurons, with two normal neurons marked by arrows; (E) hippocampus at 2 days in a case with selectiveneuronal necrosis in a part of the CA1 sector (between arrowheads). Klfiver Barrera stain on paraffin sections. Magnification bar (shown in E) is equivalent to 250 #m in A and C, to 125/~m in E and to 100 #m in B and D.

Membrane anisotropy and lipid pcroxidation in rat brain

Fig. 3--legend opposite.

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in the cortex and in the striatum of rats after BCCA and 14 days of reperfusion might be connected with the decreased brain levels of important antioxidants during reperfusion (Sato and Hall, 1992) and/or with the decreased activity of SOD caused by reperfusion lasting longer than 7 days (Michowiz et aL. 1990). As the present results correspond well with those obtained after ischemia (Haba et al., 1991) one might suggest that comparable pathophysiological conditions exist (for the relevant structures) following either oligemia or ischemia. Elevated lipid peroxidation and free radical processes have been claimed to be consistently involved in membrane failure and delayed neuronal death (Siesj6 et al., 1989, 1992; Carney et al., 1992). However, in the BCCA model systematic inspection of the different brain areas in a time course from 3 h up to 18 days later showed no light-microscopically detectable tissue damage, although lipid peroxidation was markedly increased. The neuropathological analysis was carried out in a large number of animals (57 in total). A time course of neuropathology was studied which allows exclusion of the possibility that subtle neuronal loss was overlooked, since damaged neurons must go through a phase of enhanced staining with Nissl- or Massonmethods. Less than 5% of cases showed a pattern of mild selective neuronal necrosis as would occur in ischemia, which might be due to an undetected fall in blood pressure (Smith et al., 1984). However, such cases are exceptional as documented by physiological measurements (Block et al., 1993a). The tiny loci of damage in a few cases (7%) probably resulted from thrombic events following carotid ligation. In comparison to animals with deep ischemia produced by four-vessel occlusion (Schmidt-Kastner and Hossmann, 1988), the exceptional changes in oligemia were very mild. These minor lesions would be totally diluted in a tissue sample and could not account for the changes in the present measurements. As a histological examination discovered no reproducible neuronal cell necrosis in either of the three brain structures investigated by neurochemical methods the results demonstrate that a long-lasting increase in lipid peroxidation can occur without deleterious neuropathological consequences. This finding contrasts with conclusions obtained from studies on consequences of ischemic conditions (Carney et al., 1992 ; Haba et al., 1991). However, increased lipid peroxidation alone seems not to be responsible for the observed cognitive deficiencies (Helm et at., 1990; Jaspers et al., 1990). If animals after 60 min of BCCA were tested 14 days

after surgery, learning and memory deficiencies could not be observed (Helm and Sontag, in preparation) in contrast to 24 min of BCCA even though lipid peroxidation is increased 14 days alter 24 min and 60 rain. respectively, of BCCA. Deficiencies in rats treated with 60 rain of BCCA could be measured (1 to 9 months after the BCCA procedure (Helm and Sontag, in preparation). The hippocampus which is reported to be highly important for learning and memory tasks (Olton et al., 1979; Whishaw, 1987; Morris ~,t al., 1982; Zola-Morgan et at., 1992) shows no aherations in lipid peroxidation after BCCA in both cases. Thus, the correlation between behavioural disturbances (Helm et al., 1990; Jaspers et al., 1990; Sontag et aL, 1992; Ulrich et al., 1992) and lipid peroxidation per se as well as the consequences of increased lipid peroxidation are still unclear. Further studies arc necessary to investigate which mechanisms' if ai all. are involved in disturbances of learning and memory processes after BCCA. Furthermore, it seems of interest to analyse which mechanisms may protect the hippocampus in contrast to cortex and striatum against further lipid peroxidation stimulated by the consequences of the BCCA treatment. The hippocampus is one of the most vulnerable brain structures suffering from a long-lasting decrease in regional pO2 (Block et al., 1993a) and changed transmitter activities after BCCA (Helm et al., 1990; Sieklucka et a/.. 1991. 1992). in summary, oligemic hypoxic events produced by bilateral clamping of the carotid arteries in normotensive rats lead to a significant increase in both lipid peroxidation and membrane anisotropy in the cortex and striatum. The time course of alterations in lipid peroxidation of cortical structures after B C C A in rats is comparable to the changes obtained in hippocampal tissue after forebrain ischemia in the gerbil (Haba et al., 1991). However, light-microscopically detectable tissue damage is not observable in animals after BCCA, suggesting that stimulation of lipid peroxidation does not necessarily result in neuronal necrosis. Furthermore the most important structure for learning and memory processes, the hippocampus. is not affected by lipid peroxidation although rats have spatial memory deficits after BCCA. skilful technical assistance of I. Kurz and R. Ropte is gratefully acknowledged. We thank Dr N. N. Osborne for helpful comments on the manuscript. This study was supported by the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 330 "Organprotektion" and BMFT Schwerpunkt "Morbus Parkinson und andere Basalganglienerkrankungen". F6rderkennzeichen 01 KL 9101/0. Acknowledgements--The

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