- Email: [email protected]
Liposomal antioxidants in combating ischemia-reperfusion injury in rat brain
Biomed Pharmacother 2001 ; 55 : 264-71 © 2001 Éditions scientifiques et médicales Elsevier SAS. All rights reserved S0753332201000609/FLA
Dossier: Stroke
Liposomal antioxidants in combating ischemia-reperfusion injury in rat brain J. Sinha, N. Das*, M.K. Basu Biomembrane Division, Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Calcutta - 700032, India (Received and accepted 18 August 2000)
Summary – Liposome-encapsulated antioxidants have been tested in vivo to prevent oxidative attack during cerebral ischemia and reperfusion. Oxidative stress is a causal factor in the neuropathogenesis of ischemic-reperfusion injury. From the therapeutic point of view free chemical antioxidants were almost ineffective to protect cerebral tissues from those oxidative attacks. Thus an attempt has been made to prevent the oxidative damage due to the cerebral ischemic insult by the introduction of chemical antioxidants, ascorbic acid and α-tocopherol either encapsulated or intercalated in small unilamellar liposomes. The effectiveness of antioxidant-loaded liposomes was tested against an experimental in vivo rat model of global cerebral ischemia. Oxidative free radical attack on cerebral tissues by the ischemic insult and brief reperfusion was accounted for by the amount of diene production per unit of tissue protein. Diene production in ischemic reperfused rat brain increases almost twofold over that of the normal rats. Prevention of excess diene production has been attributed to rats when they were treated either with L-ascorbic acid-encapsulated liposomes or α-tocopherol intercalated liposomes 2 hours prior to the cerebral ischemic insult. Complete restriction of excess diene generation has also been achieved when a mixture of α-tocopherol and L-ascorbic acid-encapsulated liposomes were injected 3 hours before the ischemic infraction. © 2001 Éditions scientifiques et médicales Elsevier SAS antioxidants / ischemia / liposomes
Generally, neuronal cells are exposed continuously to toxic oxygen species and as a consequence trigger an effective protection mechanism against oxidative damage. Thus survival of neurons depends on the equilibrium between free radical generation and their antioxidant defense mechanism [1]. In pathogenesis as in ischemic-reperfusion, neuronal cells are not able to counter the oxidative stress [32] and succumb to irreversible damage. Experimental evidence indicates that elevation in oxygen free radical concentration during cerebral ischemia-reperfusion is a causal factor for neuronal cell damage [4, 12, 20] and thereby by introducing biological antioxidants attempts were made to protect those cells from oxy-
*Correspondence and reprints. E-mail address: [email protected] (N. Das).
gen free radical attack [28, 29]. Endogenous antioxidant therapy is not an effective approach to counter the cerebral ischemic reperfusion challenge [14, 18]. In spite of therapeutic inconvenience conjugated antioxidants have been tested against the cerebral reperfusion injury [9, 15]. Water-soluble chemical antioxidant has been reported to act synergistically with α-tocopherol in preventing lipid peroxidation [3]. Liposomes have gained considerable interest not only as site-specific drug delivery devices [2, 19] but also as encapsulators and cotransporters of water- and fatsoluble components by virtue of their hydrophobic and hydrophilic compartments. Moreover, these phospholipid vesicles are known as effective drug carriers to the central nervous system, especially during cerebral ischemia and reperfusion [6]. The aim of this report is to evaluate the therapeutic supremacy
265
Liposomal antioxidants in ischemia-reperfusion injury
of antioxidant-loaded liposomes in an experimental in vivo rat model of partial cerebral ischemia. MATERIALS AND METHODS Materials Phosphatidylcholine (PC), cholesterol, BSA, L-ascorbic acid, (±) α-tocopherol were purchased from Sigma chemicals (St. Louis, MO, USA). All other reagents are of analytic grade. Na125I (radioactive iodine) was purchased from BARC, Bombay, India. Iodination of protein Iodination of BSA was made by following the chloramine T method [10]. In brief, to the 0.20 mL of BSA solution (10 mg/mL in PBS), 20 µCi Na125I (Sp. Activity 99 mCi/mL) was added. The mixture was incubated for 30 seconds with 0.01 mL chloramine T (4 mg/mL). The reaction was terminated by the addition of 0.01 mL of sodium metabisulphite solution (4 mg/mL). The whole reaction mixture was passed through a Sephadex G-50 column (1 cm × 17 cm) and fractions were collected. An aliquot of 10 µL of each fraction was taken for radioactive counting in a gamma-radiation counter. The radioactivity was measured in terms of dpm and was converted to µCi. Preparation of liposomes Liposomes were prepared using the method described by Gregoriadis and Ryman [8] with minor modifications. In a round bottom flask egg phosphatidylcholine (PC) cholesterol (molar ratio 7:2) was dissolved in chloroform-methanol mixture (2:1 v/v). A thin dry film of these lipids was made on the inner surface of a round bottom flask by evaporating the organic solvent in a rotating flask evaporator. The dry film was dispersed with 125I-BSA(2 × 106 dpm) or L-ascorbic acid 16 mg in 2 mL PBS (pH 7.2). For the preparation of liposomal tocopherol a film with α-tocopherol (16 mg) PC and cholesterol (molar ratio PC: cholesterol: α-tocopherol 7:1:1) was made and the dry film was dispersed with 2 mL PBS. The suspension was sonicated for 30 min at 4° C. Unencapsulated 125I-BSA or antioxidants were separated from the vesicular trapped components by centrifugating
the suspension at 105,000 g for 1 hour. The pellet was resuspended in 2 mL PBS. Extrusion of liposomes (diameter < 220 nm) was carried out at room temperature by passing the suspension through a polycarbonate filter (220 nm pore size) employing nitrogen gas pressure up to 700 lb/in2 in a barreltype extruder (Lipex Biomembrane Inc., Canada). Measurement of blood-brain barrier (BBB) permeability To investigate the BBB permeability 125I-BSA encapsulated unilamellar liposomes were injected via the tail vein of normal and cerebral ischemia-reperfused rats. The BBB permeability among those two groups of rats were compared. Arterial blood was sampled until animals were decapitated at various time points (viz, 60 min, 120 min, 180 min). The brain was isolated and its surface was rinsed with cold saline. Tissue samples were punched out, weighed and processed for radioactivity counting. Radiotracer concentration in plasma sample (Cp, dpm/mL) and in tissue (CT, dpm/mg) were determined. At a time t sec, the transfer coefficient Ki for blood-to-brain diffusion of the radioactivity was calculated from the relationship [22]. Ki =
兰
CT t
Cpdt
180
The integral part represents the time integral of circulating tracers level (dpm. s/mL) obtained by multiplying Cp by circulating time t (seconds). Lipid peroxidation assay For the determination of conjugated diene, brain tissue was homogenized in 50 mm phosphate buffer pH 7.4 containing 1 mm EDTA. Lipids in tissue homogenate were extracted in a chloroform:methanol mixture (2:1, v/v). The extract was evaporated to dryness under nitrogen atmosphere at 25° C and redissolved in n-cyclohexane. Lipids in cyclohexane solvent was assayed at 234 nm and the results were expressed as mmol of lipohydroperoxide/mg protein by using an εm of 2.52 × 104 M–1·cm–1 [26]. Proteins were measured as per Lowry et al. [13].
266
J. Sinha et al.
Induction of cerebral ischemia Female wister rats weighing 150–200 g were used for the study. The animals were kept in a temperatureand humidity-controlled housing with 12-hour light and dark cycles. They were acclimatized for 5–7 days in the new environment before use and allowed free access to food and water. Rats were subdivided into groups of 99 animals, two groups for L-ascorbic liposome formulation, two groups for α-tocopherol liposome formulation, two groups for free L-ascorbic acid treatment, two groups of saline-treated rats (one group for normal and the other group for untreated ischemia-reperfused control), one group of shamoperated animals, one group for empty liposome treatment (without antioxidants), and the last group for a liposome formulation with a mixture of α-tocopherol and L-ascorbic acid in a ratio of 1:1 (w/w). Liposome (250 µl, 2.5 mg phospholipid) containing either L-ascorbic acid or tocopherol with a dose of 8 mg/kg body weight (bw) was injected into the tail vein of rats 2–3 hours before cerebral ischemia. In control experiments the same dose of free L-ascorbic acid was injected 2–3 hours prior to ischemic insult. In another experiment, liposome containing a mixture of α-tocopherol and ascorbic acid in ratio 1:1 (w/w) (total 4 mg/kg bw) was injected 3 hours before the group of rats was made ischemic. Animals (excluding normal group) anesthetized by an intraperitoneal injection of pentobarbital (35 mg/kg) were made ischemic by bilateral clamping of the common carotid arteries (CCA) for 20 min; blood flow was restored for the next 20 min and the rats were killed by decapitation. Lipid peroxidation in brain was estimated by measuring the level of conjugated diene. Sham-operated animals were subjected to the same surgical procedure without CCAclamping.
liposome-entrapped antioxidants, e.g., α-tocopherol, L-ascorbic acid (8 mg/kg bw) or a mixture of the two antioxidants (4 mg/kg) by following the equation [25]
冉 冊
DII = nRT
− DW − 2 W0
where ∆II represents the resultant change in tissue osmolality, n denotes the osmols in an initial weight of water Wo (gm) per gm of brain tissue; ∆W, an additional amount of water (gm), enters into brain tissue and RT {1.83 ± 107 (mm Hg) cm3/mole} indicates the product of gas constant and absolute temperature. Statistical analysis One-way ANOVA analysis was performed among five different groups of rats (S1: ischemia-reperfused group; S2, S3 are two groups of rats injected with liposomal α- tocopherol either 2 or 3 hours before the ischemic reperfusion; and S4, S5 are another two groups of rats injected with liposomal L-ascorbic acid either 2 or 3 hours before ischemic reperfusion) to find out whether the conjugated diene (mmole) in whole brain due to ischemia reperfusion diminished significantly or not by the treatment of liposomal antioxidants. Two combinations were used to calculate the F-value in ANOVA, viz, S1, S2, S3 and S1, S4, S5 with the help of EPISTAT software. Regression analysis was used to show the correlation between diene amount and brain volume of ischemicreperfused rats treated either with physiological saline or antioxidant-encapsulated liposomes. RESULTS The effect of antioxidants in liposomes on diene generation in brain tissue due to induction of cerebral tissue
Quantitation of cerebral edema Cerebral edema, the entry of plasma water into swollen cerebral tissue, has been calculated by the resultant change in cerebral tissue osmolality by the induction of cerebral ischemia reperfusion. Comparison studies in brain tissue osmolality have been performed among normal, ischemic-reperfused and other groups of ischemic-reperfused rats pretreated either with free L-ascorbic acid (8 mg/kg bw) or
In sham-operated rats, the amount of diene generation in brain did not differ significantly when compared with normal groups (figure 1). But short-term reperfusion following a partial cerebral ischemia causes substantial increase in the diene level from 10.46 ± 0.73 m.mole/mg to 17.55 ± 0.21 m.mol/mg of brain proteins. Pretreatment of animals with a single dose of 8 mg free L-ascorbic acid per kg of their bw was found to be almost ineffective in pre-
267
Liposomal antioxidants in ischemia-reperfusion injury
and reperfusion was reduced to the basal level (7.1 ± 0.08 mmol/mg protein) when a mixture of α-tocopherol and ascorbic acid (1:1 w/w 4mg/kg) liposomal formulation was injected 3 hours before the ischemic insult (figure 1). Alteration in rat blood-brain barrier permeability after ischemia and reperfusion
Figure 1. Level of conjugated diene in ischemia-reperfused brain of rats treated either with free or liposome-encapsulated antioxidants 2 or 3 hours before ischemia. Conjugated diene in m.mole of lipohydroperoxide per mg of cerebral tissue protein is presented in the cases of normal (1); sham-operated (2); ischemia-reperfused (3); liposomal α-tocopherol (8 mg/kg) treated 2 hours (4) or 3 hours (5) before ischemia; liposomal ascorbic acid (8 mg/kg) treated 2 hours (6) or 3 hours (7) before ischemia; liposome (without antioxidant) treated 3 hours before ischemia (8); free L-ascorbic acid (8 mg/kg) treated 2 hours (9) or 3 hours (10) before ischemia; and liposomeencapsulated mixture of L-ascorbic acid and α-tocopherol (ratio l:1 [w/w], 4 mg/kg) treated 3 hours (11) before ischemia. Values shown as mean m.mole of lipohydroperoxide/mg of cerebral tissue protein ± S.D. (N = 9), Pa < 0.08, Pb < 0.003, Pc < 0.04, Pd < 0.005, compared to untreated group (3); Pe < 0.09 compared to group (6); Pd < 0.002 compared to group (5) or group (7).
venting the elevated diene level due to ischemic insult (figure 1). An identical amount of the antioxidants in liposomes was tested against these ischemiareperfused rats and was found to be effective in con trolling diene production. L-ascorbic acid in liposomes, when treated 3 hours prior to the ischemic insult, restricts the diene concentration nearer to the basal level, i.e., 9.66 ± 0.05 mmol/mg of brain protein. The relative effectiveness of free L-ascorbic acid, when checked against ischemia diene generation, was found to be only 22% of that in liposome encapsulated form (figure 1). The amount of diene recorded in rat brain during the period of ischemia
For measurement of BBB permeability in normal and ischemia-reperfusion states, 125I-BSA encapsulated small unilamellar liposomes were injected (iv) to three groups of rats (normal, sham-operated and ischemia-reperfused). Table I indicates the mean transfer constant (Ki) liposome distribution between cerebral tissue and blood (VD) at 1 hour and 24 hours after the injection into the tail veins of these three groups of rats. No significant difference in the liposome distribution (VD) and Ki was noticed among normal and sham-operated rats. After 1 hour of injection parameters in the case of the ischemia-reperfused rats elevated markedly when compared with the other two groups. However, those values dropped down nearly to the control level with time at 24 hours after ischemia reperfusion. Change in cerebral tissue osmolality by the induction of ischemia and reperfusion Figure 2 represents the effect of antioxidants either in free form or in liposome-encapsulated form on the decreased cerebral tissue osmolality, which resulted by the induction of cerebral ischemia and reperfusion. In sham-operated rats, osmolality in cerebral tissue did not differ significantly in comparison to the normal group (data not shown). But a substantial decrease in osmolality was noticed in neuronal cells by the short-term ischemia and reperfusion. Treatment with L-ascorbic acid liposomal formulation
Table I. Pharmacokinetic analysis of 125I-BSA encapsulated liposome uptake by brain tissue in normal and ischemic-reperfused rats. Experiment Control
Ischemic, reperfused
Time (hr)
CT
CP
Vd × 10–2
Ki × 107
1 2 3 1
1,373 ± 271 1,149 ± 152 1,004 ± 89 4,274 ± 429
43,750 ± 1,213 31,900 ± 986 28,650 ± 981 77,749 ± 1,815
3.16 ± 0.20 3.62 ± 0.25 3.51 ± 0.31 5.49 ± 0.38
87 ± 3 50 ± 7 32 ± 8 152 ± 12
Each value represents a mean ± SD radiotracer concentration CP (dpm/mL) in blood plasma, CT (dpm/gm) in brain tissue, Vd (mL/mg) and regional transfer constant Ki (mL/g/s) for blood to brain at different time periods after intravenous injection of 125I-BSA encapsulated unilamellar liposomes (2 × 106 dpm, 250 µL) to normal and ischemic-reperfused rats.
268
J. Sinha et al.
Figure 2. Osmolality differences between normal and ischemic brain of rats treated either with free antioxidants or antioxidantencapsulated liposomes: osmolality differences in osmole per cm3 of brain tissue are presented in the cases of ischemic reperfused rats treated with physiological saline (1); treated with liposomal α-tocopherol (8 mg/kg) 2 hours (3) and 3 hours (4) before ischemia; free ascorbic acid (8 mg/kg) treated 3 hours (4) before ischemia; liposomal L-ascorbic acid treated 2 hours (5) and 3 hours (6) before ischemia; and liposomal L-ascorbic acid and α-tocopherol mixture (4 mg/kg in the ratio 1:1 [w/w] treated 3 hours (7) before ischemia. Values shown as mean differences of osmoles per cm3 of brain tissue ± SD, N = 9.
resulted in a 95% increase in cerebral tissue osmolality from the value obtained after ischemia and reperfusion whereas free ascorbic acid was found to be almost ineffective in increasing the value. Mixture of α-tocopherol and L-ascorbic acid liposomal formulation was not only potent for increasing the value up to the basal level but was also effective with half the amount applied in the cases of individual liposomal antioxidants (figure 2). Statistical analysis ANOVA analysis was performed to study the effect of liposomal antioxidants’ pretreatment on the Table II. ANOVA analysis in relation to diene level in whole brain due to ischemia and reperfusion, and where rats were either on physiological saline or liposomal antioxidants. Compared groups
df N
df D
Calculated F value
Tabulated F value (5%)
S1,S2 and S3 S1,S4 and S5
2 2
9 9
23.28 49.63
4.26 4.26
One-way ANOVA analysis was performed among five different groups of rats to compare the diene generation in whole brain (S1: ischemia-reperfused group; S2 and S3 are two groups of rats injected with α-tocopherol either 2 or 3 hours prior to the ischemia reperfusion; S4 and S5 are another two groups of rats injected with L-ascorbic acid either 2 or 3 hours before the ischemia). ANOVA analysis in relation to find.
Figure 3. Relationship between the brain volume and amount of conjugated diene recorded after cerebral ischemia and reperfusion of rats treated either with physiological saline or liposomal antioxidants. Each point is representative of the brain volume in cm3 and the amount of lipohydroperoxide (m.mole in wet brain tissue measured in ischemic-reperfused rat treated either with saline (Ο), or liposomal antioxidants (8 mg/kg bw α-tocopherol (a), L-ascorbic acid (b) two hours ( ) and three hours (▲) before ischemia. Regression analysis revealed that both parameters are positively correlated in the cases of liposomal antioxidant treatment 2 hours before ischemia (r = 0.96 and 0.85 for liposomal α-tocopherol and liposomal L-ascorbic acid, respectively). Each point represents the mean amount of diene in m.mole lipohydroperoxide and mean brain volume in cm3 of nine experimental rats brains.
•
amount of conjugated diene generation in cerebral tissue of rats due to ischemia and reperfusion (table II). In both the compared groups (S1, S2 and S3) and (S1, S4 and S5) the calculated F-value was found to be much higher than the tabulated F0.05 value for df [2, 9], indicating a substantial change in diene concentration in the antioxidant-pretreated groups. In ischemic rats the conjugated diene level in whole brain rose significantly from basal level
Liposomal antioxidants in ischemia-reperfusion injury
211 ± 17 m.mole to 509 ± 45 mmol with n increase in brain volume from 0.78 ± 0.04 cm3 to 1.0 ± 0.06 cm3 (figure 3a, b). The amount of diene that rose from the from basal level during ischemia was reduced by 55% and 45% when rats were treated with either liposomal α-tocopherol or liposomal L-ascorbic acid 2 hours before ischemia, but at the same time the brain volume that rose above the basal level by the induction of ischemia was reduced by 77% and 90% (figure 3a, b). In regression analysis at those conditions the brain volume correlated positively with diene where r values were 0.96 and 0.85 for liposomal tocopherol and liposomal ascorbic acid, respectively. Further reduction in diene concentration in brain without any alteration of its volume was observed when rats were treated with either of the two vesicular antioxidants 3 hours prior to their ischemic condition. DISCUSSION Cerebral ischemia causes a reduction in the oxygen supply to the brain and it leads to energy failure by dropping the cellular ATP biosynthesis. The quick fall in cellular ATP causes inactivation of the biological membrane ionic pumps, which results in an increase in intracellular Ca++ concentration. This leads to the release of excitatory amino acids and causes permanent depolarization of other neurons by the activation of glutamate receptor [16]. Furthermore, the elevation of Ca++ ion during ischemia causes activation of many Ca++-dependent enzymes, e.g., phospholipases, proteases, nucleases, Na+/K+ ATPases, and adenylate cyclase, and leads to disorganization of neurons and finally exerts irreversible damage to neuronal cells [7]. Oxygen free radicals that are generated during post-ischemic reperfusion are another causative agent for damaging neuronal constituents [31]. Elevation of conjugated diene levels, the index of lipid peroxidation and cell damage were reported in the rat cerebral cortex region after ischemia and reperfusion [30], indicating the involvement of reduced oxygen species in neuronal damage. In this report we have demonstrated the efficacy of antioxidants (α-tocopherol, L-ascorbic acid and their mixture) in liposomes to prevent the generation of conjugated diene in cerebral tissues by the induction of global cerebral ischemia and reperfusion. The antioxidant-loaded liposomes was found to be greatly
269
potent compared to free ascorbic acid in controlling the conjugated diene increment in the brain of rats after ischemia and reperfused rats (figure 1). But treatment of L-ascorbic acid encapsulated in liposomes was 4.5 times more effective than an identical amount of free antioxidants in reducing the diene concentration of rat brain. The increased protection that showed in liposome-encapsulated antioxidant treatment could be explained by the observation of Michelson and Puget [17], who explained that liposomeencapsulated antioxidants penetrate cells at a much faster rate than the free antioxidants. Complete protection of the brain from lipohydroperoxide generated due to ischemia and reperfusion was noticed when rats were treated with mixed antioxidant (αtocopherol and L-ascorbic acid) liposomal formulation 3 hours prior to the ischemic insult (figure 1). The effect of liposome-encapsulated mixed antioxidants against free radicals could be synergistic as those vesicles acted in the lipid environment of the neuronal membrane where lipohydroperoxide generation might be expected by the induction of cerebral ischemia and reperfusion [21]. Our understanding of the mechanism of action of mixed antioxidants was clarified by Barclay et al. [3], who demonstrated the synergistic effect of ascorbic acid and α-tocopherol in preventing lipid peroxidation in a micellar preparation of fatty acids. The mechanism of how liposomal antioxidants prevent lipid peroxidation in neuronal cell membrane during ischemia reperfusion is not known but it may be presumed that there is a possibly greater interaction of vesicular antioxidants with neuronal cells compared to free antioxidants, as those lipid vesicles are known to fuse actively with cells [9]. But liposome with the lipid mixtures without any antioxidant was ineffective to resist the diene elevation in brain caused by ischemia and reperfusion (figure 1). Hyperpermeability of the blood-brain barrier (BBB) during ischemia had been demonstrated by Ito et al. [11]. In this report we have studied the functional aspects of BBB in the ischemia and reperfused states. Regional transfer constant (Ki) for blood-tobrain diffusion of 121I-BSA-encapsulated liposome was measured in normal, sham-operated and ischemia-reperfused rats (table I). BBB leakiness and elevation of Ki above the baseline were prominent at 1 hour after ischemia and reperfusion. But that no significant difference was noticed in Ki among the
270
J. Sinha et al.
sham-operated, normal and 24-hour post-ischemia groups suggested a near recovery of BBB integrity by 24 hourrs after ischemia insult. Preston et al. [24] had also demonstrated the BBB opening during ischemia in rat brain and they concluded that the recovery of BBB integrity was attributed normally at 24 hours after the ischemic insult. The mechanism of BBB opening upon reperfuion after ischemia is not clear but Sujuki et al [27] described BBB opening after ischemia and reperfusion as the consequence of the loss of autoregulation in acidotic and dilated blood vessels, which on exposure to an excessive blood flow and intraluminal pressure, undergo widening of the tight junction with an induction of pinocytosis. Edema development after cerebral ischemia resulted in the decrease in neuronal osmolality and a loss of BBB integrity [27]. Treatment of antioxidants in liposomes prior to ischemia insult controls the osmolality in cerebral tissues and as a consequence protection against edema formation due to the ischemia reperfusion may be anticipated. In this regard, a liposome-encapsulated mixture of L-ascorbic acid and α-tocopherol was found to be more potent for controlling cerebral edema development during cerebral ischemia and reperfusion (figure 2). Anova analysis, which included two groups (S1, S2 and S3) and (S1, S4 and S5), resulted in statistical evidence at which both the liposomal antioxidants were effective in reducing the conjugated diene concentration and which was found to be elevated during the ischemic and reperfusion states. Furthermore, critical differences thus calculated (data not shown) indicated that liposomal ascorbic acid treatment 3 hours prior to cerebral ischemia resulted in a maximum protection for neuronal cells from the elevation of diene concentration during the progression of the neuronal disorder (table II). Cerebral ischemia and reperfusion resulted in an elevation of brain volume with the increase in diene concentration in cerebral tissue. Reduction in brain volume and a simultaneous decrease in diene content could be expected in cerebral tissue by a treatment either with α-tocopherol or L-ascorbic acid-encapsulated liposomes 2 hours before ischemia (figure 3a, b). Liposomal antioxidant treatment 3 hours before ischemia
resulted in an appreciable reduction in diene concentration in brain without altering its brain volume (figure 3a, b). Several studies have shown that antioxidants, when applied exogenously, are not capable of attenuating cerebral ischemic injury [5, 23, 28, 29]. However, some controversy over this type of treatment remains [5]. The present data suggest that lipid vesiculartrapped antioxidant therapy may be effective in combating the cerebral ischemic reperfusion oxidative damage. In summary, it has been demonstrated that liposome-encapsulated antioxidants prevented the lipid peroxidation in rat brain and in parallel with the prevention of cerebral edema formation during ischemia and reperfusion. ACKNOWLEDGEMENTS Financial assistances from University Grants Commission (India) and Department of Biotehnology (India) are gratefully acknowledged. REFERENCES 1 Ames BN, Shigenaga MK, Hagen TM. Oxidants, antioxidants and the degenerative diseases of aging. Proc Natl Acad Sci USA 1993 ; 90 : 7915. 2 Amselem S, Gabison A, Borenholz Y. Optimization and upscaling ofdoxoribicin containing liposomes for clinical use. J Pharm Sci 1990 ; 79 : 1045. 3 Barclay LRC, Locks SJ, MacNeil JM. The autooxidation of unsaturated lipids in micelles. Synergism of inhibitors vitamin C and E. Can J Chem 1983 ; 61 : 1288-90. 4 Dugan LL, Lin TS, He YY, Hsu CY, Choi DW. Detection of free radicals by nitrodialysis/spin trapping EPR following focal cerebral ischemia reperfusion and a cautionary note on the stability of 5,5 dimethyl 1-pyrroline N-oxide (DMPO). Free Radical Res 1995 ; 23 : 27. 5 Forsman M, Fleischer JE, Milde JH, Steen PA, Michenfelder JD. Superoxide dismutase and catalase failed to improve neurologic outcome after complete cerebral ischemia in the dog. Acta Anaesthesiol Scand 1988 ; 32 : 152. 6 Fresta M, Puglisi G, Giacomo CD, Russo A. Liposomes as in vivo carriers for citicoline: effects on rat cerebral post-ischemic reperfusion. J Pharm Pharmacol 1994 ; 46 : 974. 7 Ginsberg MD, Lin B, Morikawa E, Dietricu WD, Busto R, Globus MYT. Calcium antagonists in the treatment of experimental cerebral ischemia. Arzneim Forsch 1991 ; 41 : 334-7. 8 Gregoriadis G, Ryman BE. Lysosomal localization of ß-fructofuranosidase-containing liposomes injected into rats. Biochem J 1972 ; 129 : 123. 9 He YY, Hsu CY, Ezrin AM, Miller MS. Conjugated superoxide dismutase in focal cerebral ischemia reperfusion. Am J Physiol 1993 ; 265 : H-256-H-7. 10 Hunter WM. Radioimmunoassay. In: Weir DM, Ed. Handbook of experimental immunology. Oxford: Blackwell Scientific Publication; 1978. p. 141.
Liposomal antioxidants in ischemia-reperfusion injury
11 Ito U, Yamaguichi T, Tomita H, Tone O, Shishido T, Hayashi H, et al. Maturation phenomenon in cerebral-ischemia. Berlin: Springer-Verlag; 1992. p. 1. 12 Lactelot E, Callebert J, Revaud ML, Boulu RG, Plotkine M. Detection of hydroxyl radicals in rat striatum during transient focal cerebral ischemia: possible implication in tissue damage. Neurosci Lett 1995 ; 19 : 785. 13 Lowry OH, Rosebrough NJ, Farr AL, Randell RG. Protein measurement with Folin phenol reagent. J Biol Chem 1951 ; 193 : 265. 14 Mahadik SP, Makar TK, Murthy JN, Ortiz A, Wakade CG, Karp X. Temporal changes in superoxide dismutase, glutathione peroxidase and catalase levels in primary and peri-ischemic tissue. Monosialoganglioside (GM1) treatment effects. Mol Chem Neuropathol 1993 ; 18 (1). 15 Matsumija N, Koehler RC, Kirsch JR, Traystman RJ. Conjugated superoxide dismutase reduces extend to candate injury after transient focal ischemia in cats. Stroke 1991 ; 22 : 1193. 16 McCulloch J. Ischemic brain damage – prevention with competitive and non-competitive antagonists of N-methyl-D-aspartate receptors. Arzneim Forsch 1991 ; 41 : 319. 17 Michelson AM, Puget K. Cell penetration by exogenous superoxide dismutase. Acta Physiol Scand 1980 ; 492 : 67. 18 Michomiwiz SD, Melamed E, Pikarskj E, Rappaport ZH. Effect of ischemia induced by middle cerebral artery occlusion on superoxide dismutase activity in rat brain. Stroke 1990 ; 21 : 1613. 19 Mishima EV, Jusko WJ. Selected tissue distribution of liposomal methylprednisolone in rats. Res Commun Chem Part Pharma 1994 ; 84 : 47. 20 Morimoti T, Globus MYT, Busto R, Mortinez E, Ginsberg MD. Simultaneous measurement of salicylate hydroxylation and glutamate release in the penumbral cortex following transient middle cerebral artery occlusion in rats. J Cerebral Blood Flow Metab 1996 ; 16 (92). 21 Niki E. Antioxidants in relation to lipid peroxidation. Chem Phys Lip 1987 ; 44 : 227. 22 Ohno K, Pettigrew KD, Rapoport SI. Lower limits of cerebrovascular permeability to non-electrolytes in the cancious rats. Am J Physiol 1978 ; 235 : H299.
271
23 Pereira BM, Chan PH, Weinstein PR, Fishman RA. Cerebral protection during reperfusion with superoxide dismutase in focal cerebral ischemia. Adv Neurol 1990 ; 52 : 97. 24 Preston E, Sutherland G, Finsten A. Three openings of the bloodbrain barrier produced by forebrain ischemia in the rat. Neurosci Letts 1993 ; 149 : 75. 25 Rapoport SI. A mathematical model for vasogenic brain edema. J Theor Biol 1978 ; 74 : 439. 26 Recknogel RO, Glende EA. Spectrophotometric detection of lipid conjugated dienes. In: Packe X, Ed. Methods in Enzymology Vol. 105. New York: Academic Press; 1984. p. 331. 27 Suzuki R, Yamaguchi TK, Orzi F, Klatzo I. The effects of 5-minute ischemia in Mongolian gerbils 1. Blood-brain barrier, cerebral blood flow, and local cerebral glucose utilization changes. Acta Neoropathol 1983 ; 60 : 207. 28 Tasdemiroglu E, Chistenberry PD, Ardell JL, Chronister RB, Taylor AE. Effect of superoxide dismutase on acute reperfusion injury of the rabbit brain. Acta Neurochir Wien 1993 ; 120 : 180. 29 Truelove D, Shuaib A, Ijaz S, Richardson S, Kalra J. Superoxide dismutase, catalase and U78517F attenuate neuronal damage in gerbils with repeated brief ischemic insults. Neurochem Res 1994 ; 19 : 665. 30 Vanella A, Di Giacomo C, Sorrenti V, Campisi A, Castorina C, Pinturo Rhiarenza G, et al. Lipid peroxidation and xanthine dehydrogenase oxides ratio in rat cerebral cortex during post-ischemic reperfusion: effect of Ca2+ antagonist drugs. In: Krieglstein J, Oberpichles H, Eds. Pharmacology of cerebral-ischemia. Stuttgart: Wissenschaftliche Verlagsgesellschaft GmbH; 1990. p. 311. 31 Vanella A, Sorrenti V, Castorina C, Campisi A, Di Giacomo C, Runo A, et al. Lipid peroxidation in rat cerebral cortex during post-ischemic reperfusion: effect of exogenous antioxidants and Ca++ antagonist drugs. Int J Dev Neurosci 1972 ; 10 : 75. 32 Yamamoto M, Shima T, Uozumi T, Sogabe T, Yamada K, Kawasaki T. A possible role of lipid peroxidation in cellular damage caused by cerebral ischemia and protective effects of alphatocopherol administrations. Stroke 1983 ; 14 : 977.
Dossier: Stroke
Liposomal antioxidants in combating ischemia-reperfusion injury in rat brain J. Sinha, N. Das*, M.K. Basu Biomembrane Division, Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Calcutta - 700032, India (Received and accepted 18 August 2000)
Summary – Liposome-encapsulated antioxidants have been tested in vivo to prevent oxidative attack during cerebral ischemia and reperfusion. Oxidative stress is a causal factor in the neuropathogenesis of ischemic-reperfusion injury. From the therapeutic point of view free chemical antioxidants were almost ineffective to protect cerebral tissues from those oxidative attacks. Thus an attempt has been made to prevent the oxidative damage due to the cerebral ischemic insult by the introduction of chemical antioxidants, ascorbic acid and α-tocopherol either encapsulated or intercalated in small unilamellar liposomes. The effectiveness of antioxidant-loaded liposomes was tested against an experimental in vivo rat model of global cerebral ischemia. Oxidative free radical attack on cerebral tissues by the ischemic insult and brief reperfusion was accounted for by the amount of diene production per unit of tissue protein. Diene production in ischemic reperfused rat brain increases almost twofold over that of the normal rats. Prevention of excess diene production has been attributed to rats when they were treated either with L-ascorbic acid-encapsulated liposomes or α-tocopherol intercalated liposomes 2 hours prior to the cerebral ischemic insult. Complete restriction of excess diene generation has also been achieved when a mixture of α-tocopherol and L-ascorbic acid-encapsulated liposomes were injected 3 hours before the ischemic infraction. © 2001 Éditions scientifiques et médicales Elsevier SAS antioxidants / ischemia / liposomes
Generally, neuronal cells are exposed continuously to toxic oxygen species and as a consequence trigger an effective protection mechanism against oxidative damage. Thus survival of neurons depends on the equilibrium between free radical generation and their antioxidant defense mechanism [1]. In pathogenesis as in ischemic-reperfusion, neuronal cells are not able to counter the oxidative stress [32] and succumb to irreversible damage. Experimental evidence indicates that elevation in oxygen free radical concentration during cerebral ischemia-reperfusion is a causal factor for neuronal cell damage [4, 12, 20] and thereby by introducing biological antioxidants attempts were made to protect those cells from oxy-
*Correspondence and reprints. E-mail address: [email protected] (N. Das).
gen free radical attack [28, 29]. Endogenous antioxidant therapy is not an effective approach to counter the cerebral ischemic reperfusion challenge [14, 18]. In spite of therapeutic inconvenience conjugated antioxidants have been tested against the cerebral reperfusion injury [9, 15]. Water-soluble chemical antioxidant has been reported to act synergistically with α-tocopherol in preventing lipid peroxidation [3]. Liposomes have gained considerable interest not only as site-specific drug delivery devices [2, 19] but also as encapsulators and cotransporters of water- and fatsoluble components by virtue of their hydrophobic and hydrophilic compartments. Moreover, these phospholipid vesicles are known as effective drug carriers to the central nervous system, especially during cerebral ischemia and reperfusion [6]. The aim of this report is to evaluate the therapeutic supremacy
265
Liposomal antioxidants in ischemia-reperfusion injury
of antioxidant-loaded liposomes in an experimental in vivo rat model of partial cerebral ischemia. MATERIALS AND METHODS Materials Phosphatidylcholine (PC), cholesterol, BSA, L-ascorbic acid, (±) α-tocopherol were purchased from Sigma chemicals (St. Louis, MO, USA). All other reagents are of analytic grade. Na125I (radioactive iodine) was purchased from BARC, Bombay, India. Iodination of protein Iodination of BSA was made by following the chloramine T method [10]. In brief, to the 0.20 mL of BSA solution (10 mg/mL in PBS), 20 µCi Na125I (Sp. Activity 99 mCi/mL) was added. The mixture was incubated for 30 seconds with 0.01 mL chloramine T (4 mg/mL). The reaction was terminated by the addition of 0.01 mL of sodium metabisulphite solution (4 mg/mL). The whole reaction mixture was passed through a Sephadex G-50 column (1 cm × 17 cm) and fractions were collected. An aliquot of 10 µL of each fraction was taken for radioactive counting in a gamma-radiation counter. The radioactivity was measured in terms of dpm and was converted to µCi. Preparation of liposomes Liposomes were prepared using the method described by Gregoriadis and Ryman [8] with minor modifications. In a round bottom flask egg phosphatidylcholine (PC) cholesterol (molar ratio 7:2) was dissolved in chloroform-methanol mixture (2:1 v/v). A thin dry film of these lipids was made on the inner surface of a round bottom flask by evaporating the organic solvent in a rotating flask evaporator. The dry film was dispersed with 125I-BSA(2 × 106 dpm) or L-ascorbic acid 16 mg in 2 mL PBS (pH 7.2). For the preparation of liposomal tocopherol a film with α-tocopherol (16 mg) PC and cholesterol (molar ratio PC: cholesterol: α-tocopherol 7:1:1) was made and the dry film was dispersed with 2 mL PBS. The suspension was sonicated for 30 min at 4° C. Unencapsulated 125I-BSA or antioxidants were separated from the vesicular trapped components by centrifugating
the suspension at 105,000 g for 1 hour. The pellet was resuspended in 2 mL PBS. Extrusion of liposomes (diameter < 220 nm) was carried out at room temperature by passing the suspension through a polycarbonate filter (220 nm pore size) employing nitrogen gas pressure up to 700 lb/in2 in a barreltype extruder (Lipex Biomembrane Inc., Canada). Measurement of blood-brain barrier (BBB) permeability To investigate the BBB permeability 125I-BSA encapsulated unilamellar liposomes were injected via the tail vein of normal and cerebral ischemia-reperfused rats. The BBB permeability among those two groups of rats were compared. Arterial blood was sampled until animals were decapitated at various time points (viz, 60 min, 120 min, 180 min). The brain was isolated and its surface was rinsed with cold saline. Tissue samples were punched out, weighed and processed for radioactivity counting. Radiotracer concentration in plasma sample (Cp, dpm/mL) and in tissue (CT, dpm/mg) were determined. At a time t sec, the transfer coefficient Ki for blood-to-brain diffusion of the radioactivity was calculated from the relationship [22]. Ki =
兰
CT t
Cpdt
180
The integral part represents the time integral of circulating tracers level (dpm. s/mL) obtained by multiplying Cp by circulating time t (seconds). Lipid peroxidation assay For the determination of conjugated diene, brain tissue was homogenized in 50 mm phosphate buffer pH 7.4 containing 1 mm EDTA. Lipids in tissue homogenate were extracted in a chloroform:methanol mixture (2:1, v/v). The extract was evaporated to dryness under nitrogen atmosphere at 25° C and redissolved in n-cyclohexane. Lipids in cyclohexane solvent was assayed at 234 nm and the results were expressed as mmol of lipohydroperoxide/mg protein by using an εm of 2.52 × 104 M–1·cm–1 [26]. Proteins were measured as per Lowry et al. [13].
266
J. Sinha et al.
Induction of cerebral ischemia Female wister rats weighing 150–200 g were used for the study. The animals were kept in a temperatureand humidity-controlled housing with 12-hour light and dark cycles. They were acclimatized for 5–7 days in the new environment before use and allowed free access to food and water. Rats were subdivided into groups of 99 animals, two groups for L-ascorbic liposome formulation, two groups for α-tocopherol liposome formulation, two groups for free L-ascorbic acid treatment, two groups of saline-treated rats (one group for normal and the other group for untreated ischemia-reperfused control), one group of shamoperated animals, one group for empty liposome treatment (without antioxidants), and the last group for a liposome formulation with a mixture of α-tocopherol and L-ascorbic acid in a ratio of 1:1 (w/w). Liposome (250 µl, 2.5 mg phospholipid) containing either L-ascorbic acid or tocopherol with a dose of 8 mg/kg body weight (bw) was injected into the tail vein of rats 2–3 hours before cerebral ischemia. In control experiments the same dose of free L-ascorbic acid was injected 2–3 hours prior to ischemic insult. In another experiment, liposome containing a mixture of α-tocopherol and ascorbic acid in ratio 1:1 (w/w) (total 4 mg/kg bw) was injected 3 hours before the group of rats was made ischemic. Animals (excluding normal group) anesthetized by an intraperitoneal injection of pentobarbital (35 mg/kg) were made ischemic by bilateral clamping of the common carotid arteries (CCA) for 20 min; blood flow was restored for the next 20 min and the rats were killed by decapitation. Lipid peroxidation in brain was estimated by measuring the level of conjugated diene. Sham-operated animals were subjected to the same surgical procedure without CCAclamping.
liposome-entrapped antioxidants, e.g., α-tocopherol, L-ascorbic acid (8 mg/kg bw) or a mixture of the two antioxidants (4 mg/kg) by following the equation [25]
冉 冊
DII = nRT
− DW − 2 W0
where ∆II represents the resultant change in tissue osmolality, n denotes the osmols in an initial weight of water Wo (gm) per gm of brain tissue; ∆W, an additional amount of water (gm), enters into brain tissue and RT {1.83 ± 107 (mm Hg) cm3/mole} indicates the product of gas constant and absolute temperature. Statistical analysis One-way ANOVA analysis was performed among five different groups of rats (S1: ischemia-reperfused group; S2, S3 are two groups of rats injected with liposomal α- tocopherol either 2 or 3 hours before the ischemic reperfusion; and S4, S5 are another two groups of rats injected with liposomal L-ascorbic acid either 2 or 3 hours before ischemic reperfusion) to find out whether the conjugated diene (mmole) in whole brain due to ischemia reperfusion diminished significantly or not by the treatment of liposomal antioxidants. Two combinations were used to calculate the F-value in ANOVA, viz, S1, S2, S3 and S1, S4, S5 with the help of EPISTAT software. Regression analysis was used to show the correlation between diene amount and brain volume of ischemicreperfused rats treated either with physiological saline or antioxidant-encapsulated liposomes. RESULTS The effect of antioxidants in liposomes on diene generation in brain tissue due to induction of cerebral tissue
Quantitation of cerebral edema Cerebral edema, the entry of plasma water into swollen cerebral tissue, has been calculated by the resultant change in cerebral tissue osmolality by the induction of cerebral ischemia reperfusion. Comparison studies in brain tissue osmolality have been performed among normal, ischemic-reperfused and other groups of ischemic-reperfused rats pretreated either with free L-ascorbic acid (8 mg/kg bw) or
In sham-operated rats, the amount of diene generation in brain did not differ significantly when compared with normal groups (figure 1). But short-term reperfusion following a partial cerebral ischemia causes substantial increase in the diene level from 10.46 ± 0.73 m.mole/mg to 17.55 ± 0.21 m.mol/mg of brain proteins. Pretreatment of animals with a single dose of 8 mg free L-ascorbic acid per kg of their bw was found to be almost ineffective in pre-
267
Liposomal antioxidants in ischemia-reperfusion injury
and reperfusion was reduced to the basal level (7.1 ± 0.08 mmol/mg protein) when a mixture of α-tocopherol and ascorbic acid (1:1 w/w 4mg/kg) liposomal formulation was injected 3 hours before the ischemic insult (figure 1). Alteration in rat blood-brain barrier permeability after ischemia and reperfusion
Figure 1. Level of conjugated diene in ischemia-reperfused brain of rats treated either with free or liposome-encapsulated antioxidants 2 or 3 hours before ischemia. Conjugated diene in m.mole of lipohydroperoxide per mg of cerebral tissue protein is presented in the cases of normal (1); sham-operated (2); ischemia-reperfused (3); liposomal α-tocopherol (8 mg/kg) treated 2 hours (4) or 3 hours (5) before ischemia; liposomal ascorbic acid (8 mg/kg) treated 2 hours (6) or 3 hours (7) before ischemia; liposome (without antioxidant) treated 3 hours before ischemia (8); free L-ascorbic acid (8 mg/kg) treated 2 hours (9) or 3 hours (10) before ischemia; and liposomeencapsulated mixture of L-ascorbic acid and α-tocopherol (ratio l:1 [w/w], 4 mg/kg) treated 3 hours (11) before ischemia. Values shown as mean m.mole of lipohydroperoxide/mg of cerebral tissue protein ± S.D. (N = 9), Pa < 0.08, Pb < 0.003, Pc < 0.04, Pd < 0.005, compared to untreated group (3); Pe < 0.09 compared to group (6); Pd < 0.002 compared to group (5) or group (7).
venting the elevated diene level due to ischemic insult (figure 1). An identical amount of the antioxidants in liposomes was tested against these ischemiareperfused rats and was found to be effective in con trolling diene production. L-ascorbic acid in liposomes, when treated 3 hours prior to the ischemic insult, restricts the diene concentration nearer to the basal level, i.e., 9.66 ± 0.05 mmol/mg of brain protein. The relative effectiveness of free L-ascorbic acid, when checked against ischemia diene generation, was found to be only 22% of that in liposome encapsulated form (figure 1). The amount of diene recorded in rat brain during the period of ischemia
For measurement of BBB permeability in normal and ischemia-reperfusion states, 125I-BSA encapsulated small unilamellar liposomes were injected (iv) to three groups of rats (normal, sham-operated and ischemia-reperfused). Table I indicates the mean transfer constant (Ki) liposome distribution between cerebral tissue and blood (VD) at 1 hour and 24 hours after the injection into the tail veins of these three groups of rats. No significant difference in the liposome distribution (VD) and Ki was noticed among normal and sham-operated rats. After 1 hour of injection parameters in the case of the ischemia-reperfused rats elevated markedly when compared with the other two groups. However, those values dropped down nearly to the control level with time at 24 hours after ischemia reperfusion. Change in cerebral tissue osmolality by the induction of ischemia and reperfusion Figure 2 represents the effect of antioxidants either in free form or in liposome-encapsulated form on the decreased cerebral tissue osmolality, which resulted by the induction of cerebral ischemia and reperfusion. In sham-operated rats, osmolality in cerebral tissue did not differ significantly in comparison to the normal group (data not shown). But a substantial decrease in osmolality was noticed in neuronal cells by the short-term ischemia and reperfusion. Treatment with L-ascorbic acid liposomal formulation
Table I. Pharmacokinetic analysis of 125I-BSA encapsulated liposome uptake by brain tissue in normal and ischemic-reperfused rats. Experiment Control
Ischemic, reperfused
Time (hr)
CT
CP
Vd × 10–2
Ki × 107
1 2 3 1
1,373 ± 271 1,149 ± 152 1,004 ± 89 4,274 ± 429
43,750 ± 1,213 31,900 ± 986 28,650 ± 981 77,749 ± 1,815
3.16 ± 0.20 3.62 ± 0.25 3.51 ± 0.31 5.49 ± 0.38
87 ± 3 50 ± 7 32 ± 8 152 ± 12
Each value represents a mean ± SD radiotracer concentration CP (dpm/mL) in blood plasma, CT (dpm/gm) in brain tissue, Vd (mL/mg) and regional transfer constant Ki (mL/g/s) for blood to brain at different time periods after intravenous injection of 125I-BSA encapsulated unilamellar liposomes (2 × 106 dpm, 250 µL) to normal and ischemic-reperfused rats.
268
J. Sinha et al.
Figure 2. Osmolality differences between normal and ischemic brain of rats treated either with free antioxidants or antioxidantencapsulated liposomes: osmolality differences in osmole per cm3 of brain tissue are presented in the cases of ischemic reperfused rats treated with physiological saline (1); treated with liposomal α-tocopherol (8 mg/kg) 2 hours (3) and 3 hours (4) before ischemia; free ascorbic acid (8 mg/kg) treated 3 hours (4) before ischemia; liposomal L-ascorbic acid treated 2 hours (5) and 3 hours (6) before ischemia; and liposomal L-ascorbic acid and α-tocopherol mixture (4 mg/kg in the ratio 1:1 [w/w] treated 3 hours (7) before ischemia. Values shown as mean differences of osmoles per cm3 of brain tissue ± SD, N = 9.
resulted in a 95% increase in cerebral tissue osmolality from the value obtained after ischemia and reperfusion whereas free ascorbic acid was found to be almost ineffective in increasing the value. Mixture of α-tocopherol and L-ascorbic acid liposomal formulation was not only potent for increasing the value up to the basal level but was also effective with half the amount applied in the cases of individual liposomal antioxidants (figure 2). Statistical analysis ANOVA analysis was performed to study the effect of liposomal antioxidants’ pretreatment on the Table II. ANOVA analysis in relation to diene level in whole brain due to ischemia and reperfusion, and where rats were either on physiological saline or liposomal antioxidants. Compared groups
df N
df D
Calculated F value
Tabulated F value (5%)
S1,S2 and S3 S1,S4 and S5
2 2
9 9
23.28 49.63
4.26 4.26
One-way ANOVA analysis was performed among five different groups of rats to compare the diene generation in whole brain (S1: ischemia-reperfused group; S2 and S3 are two groups of rats injected with α-tocopherol either 2 or 3 hours prior to the ischemia reperfusion; S4 and S5 are another two groups of rats injected with L-ascorbic acid either 2 or 3 hours before the ischemia). ANOVA analysis in relation to find.
Figure 3. Relationship between the brain volume and amount of conjugated diene recorded after cerebral ischemia and reperfusion of rats treated either with physiological saline or liposomal antioxidants. Each point is representative of the brain volume in cm3 and the amount of lipohydroperoxide (m.mole in wet brain tissue measured in ischemic-reperfused rat treated either with saline (Ο), or liposomal antioxidants (8 mg/kg bw α-tocopherol (a), L-ascorbic acid (b) two hours ( ) and three hours (▲) before ischemia. Regression analysis revealed that both parameters are positively correlated in the cases of liposomal antioxidant treatment 2 hours before ischemia (r = 0.96 and 0.85 for liposomal α-tocopherol and liposomal L-ascorbic acid, respectively). Each point represents the mean amount of diene in m.mole lipohydroperoxide and mean brain volume in cm3 of nine experimental rats brains.
•
amount of conjugated diene generation in cerebral tissue of rats due to ischemia and reperfusion (table II). In both the compared groups (S1, S2 and S3) and (S1, S4 and S5) the calculated F-value was found to be much higher than the tabulated F0.05 value for df [2, 9], indicating a substantial change in diene concentration in the antioxidant-pretreated groups. In ischemic rats the conjugated diene level in whole brain rose significantly from basal level
Liposomal antioxidants in ischemia-reperfusion injury
211 ± 17 m.mole to 509 ± 45 mmol with n increase in brain volume from 0.78 ± 0.04 cm3 to 1.0 ± 0.06 cm3 (figure 3a, b). The amount of diene that rose from the from basal level during ischemia was reduced by 55% and 45% when rats were treated with either liposomal α-tocopherol or liposomal L-ascorbic acid 2 hours before ischemia, but at the same time the brain volume that rose above the basal level by the induction of ischemia was reduced by 77% and 90% (figure 3a, b). In regression analysis at those conditions the brain volume correlated positively with diene where r values were 0.96 and 0.85 for liposomal tocopherol and liposomal ascorbic acid, respectively. Further reduction in diene concentration in brain without any alteration of its volume was observed when rats were treated with either of the two vesicular antioxidants 3 hours prior to their ischemic condition. DISCUSSION Cerebral ischemia causes a reduction in the oxygen supply to the brain and it leads to energy failure by dropping the cellular ATP biosynthesis. The quick fall in cellular ATP causes inactivation of the biological membrane ionic pumps, which results in an increase in intracellular Ca++ concentration. This leads to the release of excitatory amino acids and causes permanent depolarization of other neurons by the activation of glutamate receptor [16]. Furthermore, the elevation of Ca++ ion during ischemia causes activation of many Ca++-dependent enzymes, e.g., phospholipases, proteases, nucleases, Na+/K+ ATPases, and adenylate cyclase, and leads to disorganization of neurons and finally exerts irreversible damage to neuronal cells [7]. Oxygen free radicals that are generated during post-ischemic reperfusion are another causative agent for damaging neuronal constituents [31]. Elevation of conjugated diene levels, the index of lipid peroxidation and cell damage were reported in the rat cerebral cortex region after ischemia and reperfusion [30], indicating the involvement of reduced oxygen species in neuronal damage. In this report we have demonstrated the efficacy of antioxidants (α-tocopherol, L-ascorbic acid and their mixture) in liposomes to prevent the generation of conjugated diene in cerebral tissues by the induction of global cerebral ischemia and reperfusion. The antioxidant-loaded liposomes was found to be greatly
269
potent compared to free ascorbic acid in controlling the conjugated diene increment in the brain of rats after ischemia and reperfused rats (figure 1). But treatment of L-ascorbic acid encapsulated in liposomes was 4.5 times more effective than an identical amount of free antioxidants in reducing the diene concentration of rat brain. The increased protection that showed in liposome-encapsulated antioxidant treatment could be explained by the observation of Michelson and Puget [17], who explained that liposomeencapsulated antioxidants penetrate cells at a much faster rate than the free antioxidants. Complete protection of the brain from lipohydroperoxide generated due to ischemia and reperfusion was noticed when rats were treated with mixed antioxidant (αtocopherol and L-ascorbic acid) liposomal formulation 3 hours prior to the ischemic insult (figure 1). The effect of liposome-encapsulated mixed antioxidants against free radicals could be synergistic as those vesicles acted in the lipid environment of the neuronal membrane where lipohydroperoxide generation might be expected by the induction of cerebral ischemia and reperfusion [21]. Our understanding of the mechanism of action of mixed antioxidants was clarified by Barclay et al. [3], who demonstrated the synergistic effect of ascorbic acid and α-tocopherol in preventing lipid peroxidation in a micellar preparation of fatty acids. The mechanism of how liposomal antioxidants prevent lipid peroxidation in neuronal cell membrane during ischemia reperfusion is not known but it may be presumed that there is a possibly greater interaction of vesicular antioxidants with neuronal cells compared to free antioxidants, as those lipid vesicles are known to fuse actively with cells [9]. But liposome with the lipid mixtures without any antioxidant was ineffective to resist the diene elevation in brain caused by ischemia and reperfusion (figure 1). Hyperpermeability of the blood-brain barrier (BBB) during ischemia had been demonstrated by Ito et al. [11]. In this report we have studied the functional aspects of BBB in the ischemia and reperfused states. Regional transfer constant (Ki) for blood-tobrain diffusion of 121I-BSA-encapsulated liposome was measured in normal, sham-operated and ischemia-reperfused rats (table I). BBB leakiness and elevation of Ki above the baseline were prominent at 1 hour after ischemia and reperfusion. But that no significant difference was noticed in Ki among the
270
J. Sinha et al.
sham-operated, normal and 24-hour post-ischemia groups suggested a near recovery of BBB integrity by 24 hourrs after ischemia insult. Preston et al. [24] had also demonstrated the BBB opening during ischemia in rat brain and they concluded that the recovery of BBB integrity was attributed normally at 24 hours after the ischemic insult. The mechanism of BBB opening upon reperfuion after ischemia is not clear but Sujuki et al [27] described BBB opening after ischemia and reperfusion as the consequence of the loss of autoregulation in acidotic and dilated blood vessels, which on exposure to an excessive blood flow and intraluminal pressure, undergo widening of the tight junction with an induction of pinocytosis. Edema development after cerebral ischemia resulted in the decrease in neuronal osmolality and a loss of BBB integrity [27]. Treatment of antioxidants in liposomes prior to ischemia insult controls the osmolality in cerebral tissues and as a consequence protection against edema formation due to the ischemia reperfusion may be anticipated. In this regard, a liposome-encapsulated mixture of L-ascorbic acid and α-tocopherol was found to be more potent for controlling cerebral edema development during cerebral ischemia and reperfusion (figure 2). Anova analysis, which included two groups (S1, S2 and S3) and (S1, S4 and S5), resulted in statistical evidence at which both the liposomal antioxidants were effective in reducing the conjugated diene concentration and which was found to be elevated during the ischemic and reperfusion states. Furthermore, critical differences thus calculated (data not shown) indicated that liposomal ascorbic acid treatment 3 hours prior to cerebral ischemia resulted in a maximum protection for neuronal cells from the elevation of diene concentration during the progression of the neuronal disorder (table II). Cerebral ischemia and reperfusion resulted in an elevation of brain volume with the increase in diene concentration in cerebral tissue. Reduction in brain volume and a simultaneous decrease in diene content could be expected in cerebral tissue by a treatment either with α-tocopherol or L-ascorbic acid-encapsulated liposomes 2 hours before ischemia (figure 3a, b). Liposomal antioxidant treatment 3 hours before ischemia
resulted in an appreciable reduction in diene concentration in brain without altering its brain volume (figure 3a, b). Several studies have shown that antioxidants, when applied exogenously, are not capable of attenuating cerebral ischemic injury [5, 23, 28, 29]. However, some controversy over this type of treatment remains [5]. The present data suggest that lipid vesiculartrapped antioxidant therapy may be effective in combating the cerebral ischemic reperfusion oxidative damage. In summary, it has been demonstrated that liposome-encapsulated antioxidants prevented the lipid peroxidation in rat brain and in parallel with the prevention of cerebral edema formation during ischemia and reperfusion. ACKNOWLEDGEMENTS Financial assistances from University Grants Commission (India) and Department of Biotehnology (India) are gratefully acknowledged. REFERENCES 1 Ames BN, Shigenaga MK, Hagen TM. Oxidants, antioxidants and the degenerative diseases of aging. Proc Natl Acad Sci USA 1993 ; 90 : 7915. 2 Amselem S, Gabison A, Borenholz Y. Optimization and upscaling ofdoxoribicin containing liposomes for clinical use. J Pharm Sci 1990 ; 79 : 1045. 3 Barclay LRC, Locks SJ, MacNeil JM. The autooxidation of unsaturated lipids in micelles. Synergism of inhibitors vitamin C and E. Can J Chem 1983 ; 61 : 1288-90. 4 Dugan LL, Lin TS, He YY, Hsu CY, Choi DW. Detection of free radicals by nitrodialysis/spin trapping EPR following focal cerebral ischemia reperfusion and a cautionary note on the stability of 5,5 dimethyl 1-pyrroline N-oxide (DMPO). Free Radical Res 1995 ; 23 : 27. 5 Forsman M, Fleischer JE, Milde JH, Steen PA, Michenfelder JD. Superoxide dismutase and catalase failed to improve neurologic outcome after complete cerebral ischemia in the dog. Acta Anaesthesiol Scand 1988 ; 32 : 152. 6 Fresta M, Puglisi G, Giacomo CD, Russo A. Liposomes as in vivo carriers for citicoline: effects on rat cerebral post-ischemic reperfusion. J Pharm Pharmacol 1994 ; 46 : 974. 7 Ginsberg MD, Lin B, Morikawa E, Dietricu WD, Busto R, Globus MYT. Calcium antagonists in the treatment of experimental cerebral ischemia. Arzneim Forsch 1991 ; 41 : 334-7. 8 Gregoriadis G, Ryman BE. Lysosomal localization of ß-fructofuranosidase-containing liposomes injected into rats. Biochem J 1972 ; 129 : 123. 9 He YY, Hsu CY, Ezrin AM, Miller MS. Conjugated superoxide dismutase in focal cerebral ischemia reperfusion. Am J Physiol 1993 ; 265 : H-256-H-7. 10 Hunter WM. Radioimmunoassay. In: Weir DM, Ed. Handbook of experimental immunology. Oxford: Blackwell Scientific Publication; 1978. p. 141.
Liposomal antioxidants in ischemia-reperfusion injury
11 Ito U, Yamaguichi T, Tomita H, Tone O, Shishido T, Hayashi H, et al. Maturation phenomenon in cerebral-ischemia. Berlin: Springer-Verlag; 1992. p. 1. 12 Lactelot E, Callebert J, Revaud ML, Boulu RG, Plotkine M. Detection of hydroxyl radicals in rat striatum during transient focal cerebral ischemia: possible implication in tissue damage. Neurosci Lett 1995 ; 19 : 785. 13 Lowry OH, Rosebrough NJ, Farr AL, Randell RG. Protein measurement with Folin phenol reagent. J Biol Chem 1951 ; 193 : 265. 14 Mahadik SP, Makar TK, Murthy JN, Ortiz A, Wakade CG, Karp X. Temporal changes in superoxide dismutase, glutathione peroxidase and catalase levels in primary and peri-ischemic tissue. Monosialoganglioside (GM1) treatment effects. Mol Chem Neuropathol 1993 ; 18 (1). 15 Matsumija N, Koehler RC, Kirsch JR, Traystman RJ. Conjugated superoxide dismutase reduces extend to candate injury after transient focal ischemia in cats. Stroke 1991 ; 22 : 1193. 16 McCulloch J. Ischemic brain damage – prevention with competitive and non-competitive antagonists of N-methyl-D-aspartate receptors. Arzneim Forsch 1991 ; 41 : 319. 17 Michelson AM, Puget K. Cell penetration by exogenous superoxide dismutase. Acta Physiol Scand 1980 ; 492 : 67. 18 Michomiwiz SD, Melamed E, Pikarskj E, Rappaport ZH. Effect of ischemia induced by middle cerebral artery occlusion on superoxide dismutase activity in rat brain. Stroke 1990 ; 21 : 1613. 19 Mishima EV, Jusko WJ. Selected tissue distribution of liposomal methylprednisolone in rats. Res Commun Chem Part Pharma 1994 ; 84 : 47. 20 Morimoti T, Globus MYT, Busto R, Mortinez E, Ginsberg MD. Simultaneous measurement of salicylate hydroxylation and glutamate release in the penumbral cortex following transient middle cerebral artery occlusion in rats. J Cerebral Blood Flow Metab 1996 ; 16 (92). 21 Niki E. Antioxidants in relation to lipid peroxidation. Chem Phys Lip 1987 ; 44 : 227. 22 Ohno K, Pettigrew KD, Rapoport SI. Lower limits of cerebrovascular permeability to non-electrolytes in the cancious rats. Am J Physiol 1978 ; 235 : H299.
271
23 Pereira BM, Chan PH, Weinstein PR, Fishman RA. Cerebral protection during reperfusion with superoxide dismutase in focal cerebral ischemia. Adv Neurol 1990 ; 52 : 97. 24 Preston E, Sutherland G, Finsten A. Three openings of the bloodbrain barrier produced by forebrain ischemia in the rat. Neurosci Letts 1993 ; 149 : 75. 25 Rapoport SI. A mathematical model for vasogenic brain edema. J Theor Biol 1978 ; 74 : 439. 26 Recknogel RO, Glende EA. Spectrophotometric detection of lipid conjugated dienes. In: Packe X, Ed. Methods in Enzymology Vol. 105. New York: Academic Press; 1984. p. 331. 27 Suzuki R, Yamaguchi TK, Orzi F, Klatzo I. The effects of 5-minute ischemia in Mongolian gerbils 1. Blood-brain barrier, cerebral blood flow, and local cerebral glucose utilization changes. Acta Neoropathol 1983 ; 60 : 207. 28 Tasdemiroglu E, Chistenberry PD, Ardell JL, Chronister RB, Taylor AE. Effect of superoxide dismutase on acute reperfusion injury of the rabbit brain. Acta Neurochir Wien 1993 ; 120 : 180. 29 Truelove D, Shuaib A, Ijaz S, Richardson S, Kalra J. Superoxide dismutase, catalase and U78517F attenuate neuronal damage in gerbils with repeated brief ischemic insults. Neurochem Res 1994 ; 19 : 665. 30 Vanella A, Di Giacomo C, Sorrenti V, Campisi A, Castorina C, Pinturo Rhiarenza G, et al. Lipid peroxidation and xanthine dehydrogenase oxides ratio in rat cerebral cortex during post-ischemic reperfusion: effect of Ca2+ antagonist drugs. In: Krieglstein J, Oberpichles H, Eds. Pharmacology of cerebral-ischemia. Stuttgart: Wissenschaftliche Verlagsgesellschaft GmbH; 1990. p. 311. 31 Vanella A, Sorrenti V, Castorina C, Campisi A, Di Giacomo C, Runo A, et al. Lipid peroxidation in rat cerebral cortex during post-ischemic reperfusion: effect of exogenous antioxidants and Ca++ antagonist drugs. Int J Dev Neurosci 1972 ; 10 : 75. 32 Yamamoto M, Shima T, Uozumi T, Sogabe T, Yamada K, Kawasaki T. A possible role of lipid peroxidation in cellular damage caused by cerebral ischemia and protective effects of alphatocopherol administrations. Stroke 1983 ; 14 : 977.