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The effects of 17β-estradiol on ischemia-induced neuronal damage in the gerbil hippocampus
Pergamon
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Neuroscience Vol. 87, No. 4, pp. 817–822, 1998 Copyright 1998 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306–4522/98 $19.00+0.00 S0306-4522(98)00198-5
THE EFFECTS OF 17â-ESTRADIOL ON ISCHEMIA-INDUCED NEURONAL DAMAGE IN THE GERBIL HIPPOCAMPUS J. CHEN, N. ADACHI,* K. LIU and T. ARAI Department of Anesthesiology and Resuscitology, Ehime University School of Medicine, Shitsukawa, Shigenobu-cho, Onsen-gun, Ehime 791-0295, Japan Abstract––The effects of 17â-estradiol, a potent estrogen, on ischemia-induced neuronal damage, membrane depolarization and changes in intracellular Ca2+ concentration were studied in gerbil hippocampi. The histological outcome evaluated seven days after 3 min of transient forebrain ischemia in hippocampal CA1 pyramidal cells was improved by high doses of 17â-estradiol (30 µg, i.c.v. and 4 mg/kg, i.p.), whereas low doses of 17â-estradiol (3 and 10 µg, i.c.v.) showed no protective effect. Administration of 17â-estradiol did not affect the changes in the direct current potential shift in ischemia in the hippocampal CA1 area at any dosage. A hypoxia-induced intracellular Ca2+ increase was evaluated by in vitro microfluorometry in gerbil hippocampal slices. Pretreatment of 17â-estradiol (4 mg/kg, injected i.p. 1 h before decapitation) suppressed the increase in the intracellular concentration of Ca2+ due to the in vitro hypoxia, affecting both the onset of the increase and the extent. The in vitro hypoxia in the Ca2+free condition induced an elevation of the intracellular concentration of Ca2+, although the increase was gradual. Pretreatment of 17â-estradiol (4 mg/kg, i.p.) also inhibited this elevation. These findings imply that high doses of 17â-estradiol protect the neurons from ischemia by inhibiting the release of Ca2+ from the intracellular Ca2+ stores, as well as by inhibiting the influx of Ca2+ from the extracellular space. 1998 IBRO. Published by Elsevier Science Ltd. Key words: Ca2+, 17â-estradiol, gerbils, hippocampus, ischemia, microfluorometry.
The mortality rate due to acute hypoxic events has been shown to be less in female mice and rats than in males.32,33 The ischemic neuronal damage has been demonstrated to be smaller in female gerbils than in males in a unilateral carotid occlusion model.15 Since a fundamental difference between male and female animals is the presence of gonadal hormones, the differences in neuronal damage could well be due to these hormones. Estrogens have been shown to protect neurons in male rats following moderate traumatic brain injury.10 The administration of 17âestradiol has also been reported to increase the cerebral blood flow during incomplete global ischemia and to ameliorate postischemic hyperemia in female animals.18 In several in vitro studies, estrogens protected cultured hippocampal neurons by attenuating the glutamate-induced accumulation of peroxides and the increase in the intracellular Ca2+ concentration ([Ca2+]i). In addition, estrogen promoted an increase in the activities of antioxidant enzymes.6,22 Estrogens have also been shown to protect cultured cortical neurons from excitatory amino acids, chemical hypoxia and hemoglobin.27,29 However, the in vivo neuroprotective effects of estrogens against ischemia have not been confirmed.
In cerebral ischemia, energy failure triggers the depolarization on the neuronal membrane,16 and the delayed onset of the direct current (d.c.) potential shift (anoxic depolarization) has been shown to be related to the reduction of energy consumption, which serves to protect the brain.1–3,5,21 In addition, the increase in [Ca2+]i in ischemia is thought to provoke the catastrophic enzymatic process leading to irreversible neuronal injury.25 In the present study, therefore, we investigated the in vivo effects of 17âestradiol on ischemia-induced neuronal damage and alterations of the d.c. potential shift, and we studied the in vitro effects of this gonadal hormone on a hypoxia-induced increase in [Ca2+]i.
*To whom correspondence should be addressed. Abbreviations: [Ca2+]i, intracellular Ca2+ concentration; IP3, inositol-1,4,5-triphosphate. 817
EXPERIMENTAL PROCEDURES
Animals This study was approved by the Committee on Animal Experimentation at Ehime University School of Medicine, Ehime, Japan. All efforts were made to minimize animal suffering, to reduce the number of animals used and to utilize alternatives to in vivo techniques, if available. Male Mongolian gerbils weighing 60–80 g (Seiwa Experimental Animals, Fukuoka, Japan) were housed in groups in a room controlled at 231C with a 12-h light/12-h dark cycle (lights on at 6.00 a.m.). Food and water were provided ad libitum. The animals were deprived of food for at least 6 h before the ischemia because of the influence of
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hyperglycemia on ischemic brain damage.12 All in vivo experiments were performed under spontaneous ventilation. In vivo experiment on histological outcome In this experiment, 25 gerbils were prepared and then randomly assigned to five groups of five animals each. The animals were anesthetized with 2% halothane and 98% oxygen. Through a ventral middle cervical incision, both common carotid arteries were exposed and silk threads (4·0) were looped around them. After the animal was placed in a stereotaxic apparatus (David Kopf Instruments, Tujunga, CA, U.S.A.) in a prone position, the skull was exposed and a small burr hole was drilled in the right hemisphere at 2 mm anterior and 2 mm lateral to bregma for the insertion of a thermocouple needle probe (TN-800; Unique Medical Corp., Tokyo, Japan). The tip of the thermocouple needle probe was positioned about 2 mm below the brain surface. An identical probe was inserted into the rectum. The temperatures were then maintained at 37.50.2C during the experimental period with a heating lamp. Another burr hole was drilled in the left hemisphere (0.5 mm posterior and 2.5 mm lateral to bregma) for drug administration. After a stabilization period of 30 min, 17â-estradiol was administered intracerebroventricularly (i.c.v.) or intraperitoneally (i.p.) as follows: Group 1, 17â-estradiol (3 µg, i.c.v.); Group 2, 17â-estradiol (10 µg, i.c.v.); Group 3, 17â-estradiol (30 µg, i.c.v.); Group 4, 17â-estradiol (4 mg/ kg, i.p.); Group 5, saline (i.c.v.). The drug was given in a constant volume of 10 µl via a 27-gauge needle to the animals in the i.c.v. groups, and in a volume of 0.2 ml to the animals in the i.p. groups. Transient forebrain ischemia (for a 3-min period) was achieved by pulling the threads with 8-g weights 60 min after drug administration, while maintaining the brain and rectal temperatures at 37.50.2C. After the 3-min ischemia, the threads were cut to restore the blood flow. The brain and rectal temperatures were maintained at 37.50.2C under halothane anesthesia for 30 min after the reflow. The thermocouple probes were then gently pulled out. The animal was removed from the stereotaxic apparatus and the surgical incisions were sutured carefully. The animal was then brought to its cage in a room maintained at constant temperature and allowed access to food and water ad libitum. Seven days after transient forebrain ischemia, the animals were anesthetized with an i.p. injection of sodium pentobarbital. The brains were perfused with heparinized saline and fixed with 10% buffered formalin. After dehydration with graded concentrations of alcohol solutions, each brain was embedded in paraffin. Brain slices (5 µm thick) were stained with hematoxylin and eosin. The numbers of preserved pyramidal cells in the hippocampal CA1 field per 1 mm length of the stratum pyramidale in each hemisphere were counted at the same level of each coronal section (1.5 mm posterior to bregma). The average of the values on both sides was then obtained for each animal. Measurement of the direct current potential Fifteen gerbils were subjected to measurement of the d.c. potential in the hippocampal CA1 area. After anesthesia, the animals were prepared for forebrain ischemia using the same procedure as described above. After the animal was fixed in the stereotaxic apparatus, a small burr hole was drilled in the right hemisphere at 2 mm posterior and 2 mm lateral to bregma. The electrode consisted of a glass micropipette with a tip diameter of about 4 µm, which was filled with 2 M NaCl with an Ag/AgCl electrode in the barrel. This local electrode was inserted through the drilled hole and was placed 2 mm below the brain surface. The remote electrode (Ag/AgCl) was inserted subcutaneously at the back of the neck. After a stabilization period of 30 min, 17â-estradiol (30 µg, i.c.v.), 17â-estradiol (4 mg/kg, i.p.) or saline was given (n=5 for each). Transient forebrain
ischemia for 3 min was performed 60 min after the injection, while the brain and rectal temperatures were maintained at 37.50.2C. The d.c. potential was recorded with a model AB-621G d.c. amplifier (Nihon Kohden, Tokyo, Japan). The difference in the d.c. potential shift was compared regarding its onset latency, amplitude, recovery time of the depolarization to half-maximal amplitude and the duration of half-maximal amplitude. Measurement of the intracellular Ca2+ concentration Gerbils were anesthetized with ether and decapitated 60 min after i.p. administration of 17â-estradiol (4 mg/kg) or saline. The brain was rapidly removed and placed in an ice-cold physiological medium (mM: NaCl 124; KCl 5; CaCl2 2; MgCl2 2; NaH2PO4 1.25; NaHCO3 26; glucose 10). Hippocampal transverse slices, approximately 300 µm thick, were cut with a vibrating slicer (DTK-1000; Dosaka Co., Kyoto, Japan); three to five slices were obtained from each hippocampus. The slices were incubated in physiological medium equilibrated with a 95% O2/5% CO2 gas mixture for 1 h at 26C. The slices were preloaded with a fluorescent indicator, rhod-2 acetoxymethyl ester (Dojin, Kumamoto, Japan), which was diluted to 20 µM in physiological medium and equilibrated with a 95% O2/5% CO2 gas mixture for 45 min at 26C. Following loading, the slices were further incubated in physiological medium for at least 30 min at 26C. The [Ca2+]i levels were measured using an inverted fluorescence microscope, a video camera and an image processor system. An objective lens (4) and a side illumination system were used to visualize the fluorescence image of the slice. The slice was transferred to a flowthrough chamber (volume 0.2 ml) mounted on a fluorescence microscope equipped with a heat plate stage (IMT2; Olympus, Tokyo, Japan) and superfused at 3 ml/min with the appropriate medium at 36.5C. The temperature of the medium in the chamber was monitored using a thermocouple needle probe. The slice was excited with 550 nm light produced by an ultraviolet (UV) lamp (100 W; Osram, Munich, Germany), filtered by an interference filter (550 nm, band width <16 nm) and conducted to the slice through an optic fiber (5 mm diameter). The fluorescence signals (>580 nm) were captured on a silicon-intensified target camera (C2400-8; Hamamatsu Photonics, Hamamatsu, Japan) and processed using an image processor (Argus-100; Hamamatsu Photonics). Prior to the measurement of [Ca2+]i, the slice loaded with rhod-2 acetoxymethyl ester was excited with 550 nm light, and the picture (on a TV monitor) was examined to confirm that the dye was distributed uniformly throughout the slice. After the placement of the slice into the chamber, the slice was perfused with the normoxic medium (a physiological medium equilibrated with a 95% O2/5% CO2 gas mixture) for 15 min, and the [Ca2+]i in the preischemic state was measured. In vitro hypoxia was induced by switching the normoxic medium to a glucose-free hypoxic medium equilibrated with a 95% N2/5% CO2 gas mixture. The fluorescence intensity was measured in the image after the induction of in vitro hypoxia. The numerical values (pixels) were then divided by the value of the corresponding element that had been taken prior to the measurement. Thus, the ratio of [Ca2+]i was obtained every 10 s. The effect of 17â-estradiol was evaluated by comparing the increase in [Ca2+]i with the control group. To investigate the effect of 17â-estradiol in a condition free from extracellular Ca2+, the slice was perfused with Ca2+-free media that were prepared by replacing the CaCl2 with MgCl2 in both the normoxic and ischemia-like media. First, the slice was perfused with the Ca2+-containing normoxic medium for 15 min, and then the medium was changed to the Ca2+-free normoxic medium. After 5 min, the medium was switched to the Ca2+-free ischemia-like medium. The effect of 17â-estradiol was evaluated by
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comparing the increase in [Ca2+]i between animals pretreated with 17â-estradiol and those injected with saline. Materials 17â-Estradiol was purchased from Sigma Chemical Co. (St Louis, MO, U.S.A.). Halothane was obtained from Takeda Chemical Industries (Osaka, Japan). Other chemicals were all of reagent grade. 17â-Estradiol was dissolved in saline, and the doses of these drugs are expressed as the weight of the respective free base. Statistical analysis The histological data were evaluated using the Kruskal– Wallis test followed by the Mann–Whitney test. The data obtained by measuring the d.c. potential were analysed by ANOVA followed by Dunnett’s test. The fluorometric data were analysed using a repeated two-way ANOVA to detect differences among groups. When differences were found, Scheffe´’s test was used post hoc to compare each value with that in the control group. RESULTS
Histology In the control animals, almost all of the pyramidal cells were degenerated seven days after ischemia. The number of preserved neurons was 2517 per mm (meanS.D., n=5). The administration of 17âestradiol (both 30 µg, i.c.v. and 4 mg/kg, i.p.) 60 min before ischemia significantly prevented damage in hippocampal CA1 pyramidal cells (17170 and 14133 per mm, respectively). However, neither 3 nor 10 µg of 17â-estradiol showed a protective effect. The numbers of preserved pyramidal neurons were 4423 and 3321 per mm, respectively (Fig. 1). Direct current potential shift
Fig. 1. Effects of 17â-estradiol (3, 10 or 30 µg, i.c.v.; 4 mg/kg, i.p.) administered 60 min before ischemia on the delayed neuronal death of gerbil hippocampal CA1 pyramidal cells. The number of preserved CA1 pyramidal cells (ordinate) was determined. Values obtained from individual animals are shown. The number of pyramidal cells in the CA1 area in intact animals was 25612 per mm (meanS.D., n=5). Saline, saline-injected control group. *P<0.01 compared with the control group.
Fig. 2. The typical change in the d.c. potential shift produced by 3 min of transient forebrain ischemia in a control animal.
In the control animals, the forebrain ischemia provoked a gradual decrease in the extracellular membrane potential in the hippocampal CA1 area. A sudden shift in the d.c. potential was then observed, at an onset latency of 5411 s (meanS.D., n=5). After reperfusion, the membrane gradually repolarized (Fig. 2). Pretreatment with 17â-estradiol produced no changes in the onset latency, amplitude, duration or recovery time (Table 1).
Table 1. Effects of 17â-estradiol on the anoxic depolarization produced in male gerbils by 3 min forebrain ischemia
Microfluorometry
The differences in the d.c. potential shift were compared regarding its onset latency, amplitude, recovery time of the depolarization to half-maximal amplitude and duration of the half-maximal amplitude. Each value represents the meanS.D. of five animals.
When hippocampal slices were perfused with the in vitro ischemia-like medium, almost no increase in the ratio of [Ca2+]i was observed in the CA1 field within 300 s after the beginning of the in vitro hypoxia. Subsequently, an acute and large increase in [Ca2+]i spread through the CA1 field 30728 s (meanS.D., n=10) after the start of hypoxia, and the ratio reached a plateau. When animals were pretreated with 17â-estradiol 60 min before decapitation, the onset of [Ca2+]i increase was significantly prolonged, the value being 38418 s, and the extent of the increase was inhibited by 10% after 600 s, respectively (Fig. 3).
Onset (s)
Amplitude (mV)
Recovery (s)
Duration (s)
Saline
5411
223
9456
20278
17â-Estradiol 30 µg, i.c.v. 4 mg, i.p.
5320 5832
236 203
10727 11325
19956 19463
When slices were perfused with the Ca2+-free in vitro ischemia-like medium, an increase in [Ca2+]i was observed in the CA1 field, although the increase was gradual and moderate. The mean value of the latency of the increase in [Ca2+]i was 29035 s. In slices from animals pretreated with 17â-estradiol, the latency at the beginning of the increase in [Ca2+]i was markedly prolonged, the value being 35746 s (Fig. 4).
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Fig. 3. Changes in the ratio of [Ca2+]i in slices of gerbil hippocampal CA1 field under in vitro ischemia-like conditions. The control group ( ) and 17â-estradiol-pretreated (4 mg/kg) group () effects are shown. Each value represents the meanS.D. of 10 slices. *P<0.05, **P<0.01, ***P<0.001 compared with the respective values in the control group.
Fig. 4. Changes in the ratio of [Ca2+]i in slices of the hippocampal CA1 field under Ca2+-free in vitro ischemialike conditions. The control group ( ) and 17â-estradiolpretreated (4 mg/kg) group () effects are shown. Each value represents the meanS.D. of 10 slices. *P<0.05, **P<0.01 compared with the respective values in the control group. DISCUSSION
In the present study, we observed protection against ischemia-induced damage in hippocampal CA1 pyramidal cells in the in vivo experiment and inhibition of the hypoxia-induced increase in [Ca2+]i by 17â-estradiol in the in vitro experiment. 17â-Estradiol protects neurons against ischemia Brain temperature was carefully maintained at 37.5C in all in vitro experiments, because brain temperature in ischemia and reperfusion plays an important role in the histological outcome. Under this normothermic condition, transient forebrain ischemia for 3 min has been demonstrated to produce marked damage in the hippocampal CA1 region.24 The administration of 17â-estradiol (30 µg, i.c.v. or 4 mg/kg, i.p.) prevented neuronal damage to a significant degree. Estrogens have been shown to attenuate neurotoxicity by glutamate in a hippocampal cell line,6 and to protect primary cultured hippocampal
neurons against excitotoxicity.13 Since the hippocampal CA1 region is innervated by glutamatergic fibers,31 the attenuation of the excitatory toxicity by 17â-estradiol may contribute to the improvement of the histological outcome. Free radicals produced in ischemia are regarded as elements that harms cells in various manners: damaging DNA and proteins, facilitating lipid peroxidation, and disturbing cellular iron metabolism.28 Estrogens possess antioxidant activity and suppress the lipid peroxidation induced by amyloid â-peptide and FeSO4, and thereby protect cultured hippocampal neurons.13,15 In addition, estrogens attenuated oxidative stress-induced cell death in a hippocampal cell line.6 Two mechanisms have been speculated to underlie the antioxidative action of estrogens: estrogens may reduce the [Ca2+]i elevation, which contributes to induction of reactive oxygen species, and they may increase the antioxidant activities of enzymes.22 The mechanism by which estrogens protect neurons against oxidative and excitotoxic injuries has been thought to involve intracellular estrogen receptors, which are related to the expression of neurotrophins,34 the induction of calcium binding proteins8 and the stabilization of microtubules.11 The improvement of long-term survival of cultured hypothalamic neurons by estrogens is speculated to be caused by these elements.7,30 Although the genomic effects of estrogens should occur during the period from 2 to 4 h after an i.p. injection of 17âestradiol,14,19 a short-term pretreatment with estrogens has also been reported to protect cultured hippocampal neurons.13 A similar phenomenon was observed in the present study. The administration of 17â-estradiol 1 h before ischemia protected against damage. These findings suggest the existence of a neuroprotective action of estrogens independent of an intracellular receptor-mediated mechanism. A high concentration of 17â-estradiol has been shown to be necessary for cellular protection in in vivo studies.6,27 Likewise, in our in vivo experiment, the very high doses of 17â-estradiol (30 µg, i.c.v. and 4 mg/kg, i.p.) showed significant neuroprotective actions, whereas low doses (3 and 10 µg, i.c.v.) did not. These findings also suggest a mechanism other than one of intracellular receptors. Although we did not examine directly the receptor-mediated neuroprotection of 17â-estradiol, both receptor-mediated and receptor-independent actions of estrogens are conceivably responsible for the neuroprotection. 17â-Estradiol does not affect the direct current potential shift The extracellular d.c. potential shift closely reflects the movement of Na+, K+, Cl and Ca2+ across the membrane.16 Because the depletion of ATP in ischemia causes sudden depolarization of the neuronal membrane due to an insufficient transportation of ions, the latency from the start of ischemia
Neuroprotection by 17â-estradiol
to the sudden depolarization represents the duration of ATP consumption in the CNS. A delayed onset of the d.c. potential shift may therefore indicate the prolongation of energy consumption.1–3,5,21 However, in the present study, 17â-estradiol protected neurons without an accompanying prolonged onset of the d.c. potential shift. Two mechanisms are conceivable to explain this discrepancy. First, 17âestradiol depolarizes hippocampal CA1 neurons rapidly.36 Second, although the d.c. potential shift reflects the energy state and ion movement before and during ischemia, it does not reflect these phenomena after reperfusion.4 The neuroprotective action of 17â-estradiol may therefore be caused by the antioxidative action after reperfusion. Mechanisms of the inhibition of the hypoxic increase in the intracellular Ca2+ concentration by 17â-estradiol It has frequently been shown that an excessive increase in [Ca2+]i leads to irreversible neuronal injury.9,20 In our present in vitro experiment, we observed a sudden and large increase in [Ca2+]i in the hippocampal CA1 area in ischemia-like conditions. The elevation of [Ca2+]i induced by ischemia is caused by both Ca2+ influx from the extracellular space and Ca2+ release from the intracellular Ca2+ stores, such as the endoplasmic reticulum and mitochondria.25 In addition, Ca2+ influx is generally presumed to be caused through voltage-gated Ca2+ channels and through agonist-gated Ca2+ channels, mainly N-methyl--aspartate-gated Ca2+ channels. It has also been shown that 17â-estradiol rapidly reduces Ca2+ influx through L-type voltage-gated channels in neostriatal neurons; this reduction is probably mediated by G-protein-coupled receptors on the membrane surface.23 Furthermore, estrogens suppress elevation of [Ca2+]i which is induced by glutamate.13 Therefore, 17â-estradiol may inhibit Ca2+ influx through both voltage-gated and agonistgated Ca2+ channels.
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In this study, an increase in [Ca2+]i in Ca2+-free hypoxic conditions was also observed, and this was inhibited by 17â-estradiol. This finding indicates that 17â-estradiol can inhibit Ca2+ release from intracellular stores. There are two mechanisms by which Ca2+ is released from the endoplasmic reticulum to the cytosol.17,35 One is the release through ryanodine receptors, which exist on the membrane of the endoplasmic reticulum. The increase in the cytosolic concentration of Ca2+, which is caused by influx of Ca2+ from the extracellular space, stimulates the release of Ca2+ from the endoplasmic reticulum by activating ryanodine receptors (Ca2+-induced Ca2+ release). The other mechanism involves inositol-1,4,5triphosphate (IP3) receptors on the endoplasmic reticulum. IP3 receptor-linked Ca2+ channels open in response to the increase in intracellular concentration of IP3, resulting in the release of Ca2+ (IP3-induced Ca2+ release). The mechanism by which 17â-estradiol reduces the release of Ca2+ from the intracellular store was not elucidated in the present study. However, since estrogens increase IP3 synthesis,26 17â-estradiol seems to inhibit Ca2+-induced Ca2+ release. Furthermore, the stabilizing effect in the cellular calcium homeostasis by estrogens may contribute to the inhibition of [Ca2+]i by maintaining plasma membrane integrity with its antioxidant action.13
CONCLUSIONS
We observed that high-dose 17â-estradiol improved the outcome in hippocampal CA1 pyramidal cells following ischemia, probably by inhibiting the release of Ca2+ from the intracellular Ca2+ stores, as well as by inhibiting the influx of Ca2+ from the extracellular space. These findings contribute to our understanding of the neuroprotective action of 17â-estradiol against ischemia-induced neuronal damage.
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Neuroscience Vol. 87, No. 4, pp. 817–822, 1998 Copyright 1998 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306–4522/98 $19.00+0.00 S0306-4522(98)00198-5
THE EFFECTS OF 17â-ESTRADIOL ON ISCHEMIA-INDUCED NEURONAL DAMAGE IN THE GERBIL HIPPOCAMPUS J. CHEN, N. ADACHI,* K. LIU and T. ARAI Department of Anesthesiology and Resuscitology, Ehime University School of Medicine, Shitsukawa, Shigenobu-cho, Onsen-gun, Ehime 791-0295, Japan Abstract––The effects of 17â-estradiol, a potent estrogen, on ischemia-induced neuronal damage, membrane depolarization and changes in intracellular Ca2+ concentration were studied in gerbil hippocampi. The histological outcome evaluated seven days after 3 min of transient forebrain ischemia in hippocampal CA1 pyramidal cells was improved by high doses of 17â-estradiol (30 µg, i.c.v. and 4 mg/kg, i.p.), whereas low doses of 17â-estradiol (3 and 10 µg, i.c.v.) showed no protective effect. Administration of 17â-estradiol did not affect the changes in the direct current potential shift in ischemia in the hippocampal CA1 area at any dosage. A hypoxia-induced intracellular Ca2+ increase was evaluated by in vitro microfluorometry in gerbil hippocampal slices. Pretreatment of 17â-estradiol (4 mg/kg, injected i.p. 1 h before decapitation) suppressed the increase in the intracellular concentration of Ca2+ due to the in vitro hypoxia, affecting both the onset of the increase and the extent. The in vitro hypoxia in the Ca2+free condition induced an elevation of the intracellular concentration of Ca2+, although the increase was gradual. Pretreatment of 17â-estradiol (4 mg/kg, i.p.) also inhibited this elevation. These findings imply that high doses of 17â-estradiol protect the neurons from ischemia by inhibiting the release of Ca2+ from the intracellular Ca2+ stores, as well as by inhibiting the influx of Ca2+ from the extracellular space. 1998 IBRO. Published by Elsevier Science Ltd. Key words: Ca2+, 17â-estradiol, gerbils, hippocampus, ischemia, microfluorometry.
The mortality rate due to acute hypoxic events has been shown to be less in female mice and rats than in males.32,33 The ischemic neuronal damage has been demonstrated to be smaller in female gerbils than in males in a unilateral carotid occlusion model.15 Since a fundamental difference between male and female animals is the presence of gonadal hormones, the differences in neuronal damage could well be due to these hormones. Estrogens have been shown to protect neurons in male rats following moderate traumatic brain injury.10 The administration of 17âestradiol has also been reported to increase the cerebral blood flow during incomplete global ischemia and to ameliorate postischemic hyperemia in female animals.18 In several in vitro studies, estrogens protected cultured hippocampal neurons by attenuating the glutamate-induced accumulation of peroxides and the increase in the intracellular Ca2+ concentration ([Ca2+]i). In addition, estrogen promoted an increase in the activities of antioxidant enzymes.6,22 Estrogens have also been shown to protect cultured cortical neurons from excitatory amino acids, chemical hypoxia and hemoglobin.27,29 However, the in vivo neuroprotective effects of estrogens against ischemia have not been confirmed.
In cerebral ischemia, energy failure triggers the depolarization on the neuronal membrane,16 and the delayed onset of the direct current (d.c.) potential shift (anoxic depolarization) has been shown to be related to the reduction of energy consumption, which serves to protect the brain.1–3,5,21 In addition, the increase in [Ca2+]i in ischemia is thought to provoke the catastrophic enzymatic process leading to irreversible neuronal injury.25 In the present study, therefore, we investigated the in vivo effects of 17âestradiol on ischemia-induced neuronal damage and alterations of the d.c. potential shift, and we studied the in vitro effects of this gonadal hormone on a hypoxia-induced increase in [Ca2+]i.
*To whom correspondence should be addressed. Abbreviations: [Ca2+]i, intracellular Ca2+ concentration; IP3, inositol-1,4,5-triphosphate. 817
EXPERIMENTAL PROCEDURES
Animals This study was approved by the Committee on Animal Experimentation at Ehime University School of Medicine, Ehime, Japan. All efforts were made to minimize animal suffering, to reduce the number of animals used and to utilize alternatives to in vivo techniques, if available. Male Mongolian gerbils weighing 60–80 g (Seiwa Experimental Animals, Fukuoka, Japan) were housed in groups in a room controlled at 231C with a 12-h light/12-h dark cycle (lights on at 6.00 a.m.). Food and water were provided ad libitum. The animals were deprived of food for at least 6 h before the ischemia because of the influence of
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hyperglycemia on ischemic brain damage.12 All in vivo experiments were performed under spontaneous ventilation. In vivo experiment on histological outcome In this experiment, 25 gerbils were prepared and then randomly assigned to five groups of five animals each. The animals were anesthetized with 2% halothane and 98% oxygen. Through a ventral middle cervical incision, both common carotid arteries were exposed and silk threads (4·0) were looped around them. After the animal was placed in a stereotaxic apparatus (David Kopf Instruments, Tujunga, CA, U.S.A.) in a prone position, the skull was exposed and a small burr hole was drilled in the right hemisphere at 2 mm anterior and 2 mm lateral to bregma for the insertion of a thermocouple needle probe (TN-800; Unique Medical Corp., Tokyo, Japan). The tip of the thermocouple needle probe was positioned about 2 mm below the brain surface. An identical probe was inserted into the rectum. The temperatures were then maintained at 37.50.2C during the experimental period with a heating lamp. Another burr hole was drilled in the left hemisphere (0.5 mm posterior and 2.5 mm lateral to bregma) for drug administration. After a stabilization period of 30 min, 17â-estradiol was administered intracerebroventricularly (i.c.v.) or intraperitoneally (i.p.) as follows: Group 1, 17â-estradiol (3 µg, i.c.v.); Group 2, 17â-estradiol (10 µg, i.c.v.); Group 3, 17â-estradiol (30 µg, i.c.v.); Group 4, 17â-estradiol (4 mg/ kg, i.p.); Group 5, saline (i.c.v.). The drug was given in a constant volume of 10 µl via a 27-gauge needle to the animals in the i.c.v. groups, and in a volume of 0.2 ml to the animals in the i.p. groups. Transient forebrain ischemia (for a 3-min period) was achieved by pulling the threads with 8-g weights 60 min after drug administration, while maintaining the brain and rectal temperatures at 37.50.2C. After the 3-min ischemia, the threads were cut to restore the blood flow. The brain and rectal temperatures were maintained at 37.50.2C under halothane anesthesia for 30 min after the reflow. The thermocouple probes were then gently pulled out. The animal was removed from the stereotaxic apparatus and the surgical incisions were sutured carefully. The animal was then brought to its cage in a room maintained at constant temperature and allowed access to food and water ad libitum. Seven days after transient forebrain ischemia, the animals were anesthetized with an i.p. injection of sodium pentobarbital. The brains were perfused with heparinized saline and fixed with 10% buffered formalin. After dehydration with graded concentrations of alcohol solutions, each brain was embedded in paraffin. Brain slices (5 µm thick) were stained with hematoxylin and eosin. The numbers of preserved pyramidal cells in the hippocampal CA1 field per 1 mm length of the stratum pyramidale in each hemisphere were counted at the same level of each coronal section (1.5 mm posterior to bregma). The average of the values on both sides was then obtained for each animal. Measurement of the direct current potential Fifteen gerbils were subjected to measurement of the d.c. potential in the hippocampal CA1 area. After anesthesia, the animals were prepared for forebrain ischemia using the same procedure as described above. After the animal was fixed in the stereotaxic apparatus, a small burr hole was drilled in the right hemisphere at 2 mm posterior and 2 mm lateral to bregma. The electrode consisted of a glass micropipette with a tip diameter of about 4 µm, which was filled with 2 M NaCl with an Ag/AgCl electrode in the barrel. This local electrode was inserted through the drilled hole and was placed 2 mm below the brain surface. The remote electrode (Ag/AgCl) was inserted subcutaneously at the back of the neck. After a stabilization period of 30 min, 17â-estradiol (30 µg, i.c.v.), 17â-estradiol (4 mg/kg, i.p.) or saline was given (n=5 for each). Transient forebrain
ischemia for 3 min was performed 60 min after the injection, while the brain and rectal temperatures were maintained at 37.50.2C. The d.c. potential was recorded with a model AB-621G d.c. amplifier (Nihon Kohden, Tokyo, Japan). The difference in the d.c. potential shift was compared regarding its onset latency, amplitude, recovery time of the depolarization to half-maximal amplitude and the duration of half-maximal amplitude. Measurement of the intracellular Ca2+ concentration Gerbils were anesthetized with ether and decapitated 60 min after i.p. administration of 17â-estradiol (4 mg/kg) or saline. The brain was rapidly removed and placed in an ice-cold physiological medium (mM: NaCl 124; KCl 5; CaCl2 2; MgCl2 2; NaH2PO4 1.25; NaHCO3 26; glucose 10). Hippocampal transverse slices, approximately 300 µm thick, were cut with a vibrating slicer (DTK-1000; Dosaka Co., Kyoto, Japan); three to five slices were obtained from each hippocampus. The slices were incubated in physiological medium equilibrated with a 95% O2/5% CO2 gas mixture for 1 h at 26C. The slices were preloaded with a fluorescent indicator, rhod-2 acetoxymethyl ester (Dojin, Kumamoto, Japan), which was diluted to 20 µM in physiological medium and equilibrated with a 95% O2/5% CO2 gas mixture for 45 min at 26C. Following loading, the slices were further incubated in physiological medium for at least 30 min at 26C. The [Ca2+]i levels were measured using an inverted fluorescence microscope, a video camera and an image processor system. An objective lens (4) and a side illumination system were used to visualize the fluorescence image of the slice. The slice was transferred to a flowthrough chamber (volume 0.2 ml) mounted on a fluorescence microscope equipped with a heat plate stage (IMT2; Olympus, Tokyo, Japan) and superfused at 3 ml/min with the appropriate medium at 36.5C. The temperature of the medium in the chamber was monitored using a thermocouple needle probe. The slice was excited with 550 nm light produced by an ultraviolet (UV) lamp (100 W; Osram, Munich, Germany), filtered by an interference filter (550 nm, band width <16 nm) and conducted to the slice through an optic fiber (5 mm diameter). The fluorescence signals (>580 nm) were captured on a silicon-intensified target camera (C2400-8; Hamamatsu Photonics, Hamamatsu, Japan) and processed using an image processor (Argus-100; Hamamatsu Photonics). Prior to the measurement of [Ca2+]i, the slice loaded with rhod-2 acetoxymethyl ester was excited with 550 nm light, and the picture (on a TV monitor) was examined to confirm that the dye was distributed uniformly throughout the slice. After the placement of the slice into the chamber, the slice was perfused with the normoxic medium (a physiological medium equilibrated with a 95% O2/5% CO2 gas mixture) for 15 min, and the [Ca2+]i in the preischemic state was measured. In vitro hypoxia was induced by switching the normoxic medium to a glucose-free hypoxic medium equilibrated with a 95% N2/5% CO2 gas mixture. The fluorescence intensity was measured in the image after the induction of in vitro hypoxia. The numerical values (pixels) were then divided by the value of the corresponding element that had been taken prior to the measurement. Thus, the ratio of [Ca2+]i was obtained every 10 s. The effect of 17â-estradiol was evaluated by comparing the increase in [Ca2+]i with the control group. To investigate the effect of 17â-estradiol in a condition free from extracellular Ca2+, the slice was perfused with Ca2+-free media that were prepared by replacing the CaCl2 with MgCl2 in both the normoxic and ischemia-like media. First, the slice was perfused with the Ca2+-containing normoxic medium for 15 min, and then the medium was changed to the Ca2+-free normoxic medium. After 5 min, the medium was switched to the Ca2+-free ischemia-like medium. The effect of 17â-estradiol was evaluated by
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comparing the increase in [Ca2+]i between animals pretreated with 17â-estradiol and those injected with saline. Materials 17â-Estradiol was purchased from Sigma Chemical Co. (St Louis, MO, U.S.A.). Halothane was obtained from Takeda Chemical Industries (Osaka, Japan). Other chemicals were all of reagent grade. 17â-Estradiol was dissolved in saline, and the doses of these drugs are expressed as the weight of the respective free base. Statistical analysis The histological data were evaluated using the Kruskal– Wallis test followed by the Mann–Whitney test. The data obtained by measuring the d.c. potential were analysed by ANOVA followed by Dunnett’s test. The fluorometric data were analysed using a repeated two-way ANOVA to detect differences among groups. When differences were found, Scheffe´’s test was used post hoc to compare each value with that in the control group. RESULTS
Histology In the control animals, almost all of the pyramidal cells were degenerated seven days after ischemia. The number of preserved neurons was 2517 per mm (meanS.D., n=5). The administration of 17âestradiol (both 30 µg, i.c.v. and 4 mg/kg, i.p.) 60 min before ischemia significantly prevented damage in hippocampal CA1 pyramidal cells (17170 and 14133 per mm, respectively). However, neither 3 nor 10 µg of 17â-estradiol showed a protective effect. The numbers of preserved pyramidal neurons were 4423 and 3321 per mm, respectively (Fig. 1). Direct current potential shift
Fig. 1. Effects of 17â-estradiol (3, 10 or 30 µg, i.c.v.; 4 mg/kg, i.p.) administered 60 min before ischemia on the delayed neuronal death of gerbil hippocampal CA1 pyramidal cells. The number of preserved CA1 pyramidal cells (ordinate) was determined. Values obtained from individual animals are shown. The number of pyramidal cells in the CA1 area in intact animals was 25612 per mm (meanS.D., n=5). Saline, saline-injected control group. *P<0.01 compared with the control group.
Fig. 2. The typical change in the d.c. potential shift produced by 3 min of transient forebrain ischemia in a control animal.
In the control animals, the forebrain ischemia provoked a gradual decrease in the extracellular membrane potential in the hippocampal CA1 area. A sudden shift in the d.c. potential was then observed, at an onset latency of 5411 s (meanS.D., n=5). After reperfusion, the membrane gradually repolarized (Fig. 2). Pretreatment with 17â-estradiol produced no changes in the onset latency, amplitude, duration or recovery time (Table 1).
Table 1. Effects of 17â-estradiol on the anoxic depolarization produced in male gerbils by 3 min forebrain ischemia
Microfluorometry
The differences in the d.c. potential shift were compared regarding its onset latency, amplitude, recovery time of the depolarization to half-maximal amplitude and duration of the half-maximal amplitude. Each value represents the meanS.D. of five animals.
When hippocampal slices were perfused with the in vitro ischemia-like medium, almost no increase in the ratio of [Ca2+]i was observed in the CA1 field within 300 s after the beginning of the in vitro hypoxia. Subsequently, an acute and large increase in [Ca2+]i spread through the CA1 field 30728 s (meanS.D., n=10) after the start of hypoxia, and the ratio reached a plateau. When animals were pretreated with 17â-estradiol 60 min before decapitation, the onset of [Ca2+]i increase was significantly prolonged, the value being 38418 s, and the extent of the increase was inhibited by 10% after 600 s, respectively (Fig. 3).
Onset (s)
Amplitude (mV)
Recovery (s)
Duration (s)
Saline
5411
223
9456
20278
17â-Estradiol 30 µg, i.c.v. 4 mg, i.p.
5320 5832
236 203
10727 11325
19956 19463
When slices were perfused with the Ca2+-free in vitro ischemia-like medium, an increase in [Ca2+]i was observed in the CA1 field, although the increase was gradual and moderate. The mean value of the latency of the increase in [Ca2+]i was 29035 s. In slices from animals pretreated with 17â-estradiol, the latency at the beginning of the increase in [Ca2+]i was markedly prolonged, the value being 35746 s (Fig. 4).
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Fig. 3. Changes in the ratio of [Ca2+]i in slices of gerbil hippocampal CA1 field under in vitro ischemia-like conditions. The control group ( ) and 17â-estradiol-pretreated (4 mg/kg) group () effects are shown. Each value represents the meanS.D. of 10 slices. *P<0.05, **P<0.01, ***P<0.001 compared with the respective values in the control group.
Fig. 4. Changes in the ratio of [Ca2+]i in slices of the hippocampal CA1 field under Ca2+-free in vitro ischemialike conditions. The control group ( ) and 17â-estradiolpretreated (4 mg/kg) group () effects are shown. Each value represents the meanS.D. of 10 slices. *P<0.05, **P<0.01 compared with the respective values in the control group. DISCUSSION
In the present study, we observed protection against ischemia-induced damage in hippocampal CA1 pyramidal cells in the in vivo experiment and inhibition of the hypoxia-induced increase in [Ca2+]i by 17â-estradiol in the in vitro experiment. 17â-Estradiol protects neurons against ischemia Brain temperature was carefully maintained at 37.5C in all in vitro experiments, because brain temperature in ischemia and reperfusion plays an important role in the histological outcome. Under this normothermic condition, transient forebrain ischemia for 3 min has been demonstrated to produce marked damage in the hippocampal CA1 region.24 The administration of 17â-estradiol (30 µg, i.c.v. or 4 mg/kg, i.p.) prevented neuronal damage to a significant degree. Estrogens have been shown to attenuate neurotoxicity by glutamate in a hippocampal cell line,6 and to protect primary cultured hippocampal
neurons against excitotoxicity.13 Since the hippocampal CA1 region is innervated by glutamatergic fibers,31 the attenuation of the excitatory toxicity by 17â-estradiol may contribute to the improvement of the histological outcome. Free radicals produced in ischemia are regarded as elements that harms cells in various manners: damaging DNA and proteins, facilitating lipid peroxidation, and disturbing cellular iron metabolism.28 Estrogens possess antioxidant activity and suppress the lipid peroxidation induced by amyloid â-peptide and FeSO4, and thereby protect cultured hippocampal neurons.13,15 In addition, estrogens attenuated oxidative stress-induced cell death in a hippocampal cell line.6 Two mechanisms have been speculated to underlie the antioxidative action of estrogens: estrogens may reduce the [Ca2+]i elevation, which contributes to induction of reactive oxygen species, and they may increase the antioxidant activities of enzymes.22 The mechanism by which estrogens protect neurons against oxidative and excitotoxic injuries has been thought to involve intracellular estrogen receptors, which are related to the expression of neurotrophins,34 the induction of calcium binding proteins8 and the stabilization of microtubules.11 The improvement of long-term survival of cultured hypothalamic neurons by estrogens is speculated to be caused by these elements.7,30 Although the genomic effects of estrogens should occur during the period from 2 to 4 h after an i.p. injection of 17âestradiol,14,19 a short-term pretreatment with estrogens has also been reported to protect cultured hippocampal neurons.13 A similar phenomenon was observed in the present study. The administration of 17â-estradiol 1 h before ischemia protected against damage. These findings suggest the existence of a neuroprotective action of estrogens independent of an intracellular receptor-mediated mechanism. A high concentration of 17â-estradiol has been shown to be necessary for cellular protection in in vivo studies.6,27 Likewise, in our in vivo experiment, the very high doses of 17â-estradiol (30 µg, i.c.v. and 4 mg/kg, i.p.) showed significant neuroprotective actions, whereas low doses (3 and 10 µg, i.c.v.) did not. These findings also suggest a mechanism other than one of intracellular receptors. Although we did not examine directly the receptor-mediated neuroprotection of 17â-estradiol, both receptor-mediated and receptor-independent actions of estrogens are conceivably responsible for the neuroprotection. 17â-Estradiol does not affect the direct current potential shift The extracellular d.c. potential shift closely reflects the movement of Na+, K+, Cl and Ca2+ across the membrane.16 Because the depletion of ATP in ischemia causes sudden depolarization of the neuronal membrane due to an insufficient transportation of ions, the latency from the start of ischemia
Neuroprotection by 17â-estradiol
to the sudden depolarization represents the duration of ATP consumption in the CNS. A delayed onset of the d.c. potential shift may therefore indicate the prolongation of energy consumption.1–3,5,21 However, in the present study, 17â-estradiol protected neurons without an accompanying prolonged onset of the d.c. potential shift. Two mechanisms are conceivable to explain this discrepancy. First, 17âestradiol depolarizes hippocampal CA1 neurons rapidly.36 Second, although the d.c. potential shift reflects the energy state and ion movement before and during ischemia, it does not reflect these phenomena after reperfusion.4 The neuroprotective action of 17â-estradiol may therefore be caused by the antioxidative action after reperfusion. Mechanisms of the inhibition of the hypoxic increase in the intracellular Ca2+ concentration by 17â-estradiol It has frequently been shown that an excessive increase in [Ca2+]i leads to irreversible neuronal injury.9,20 In our present in vitro experiment, we observed a sudden and large increase in [Ca2+]i in the hippocampal CA1 area in ischemia-like conditions. The elevation of [Ca2+]i induced by ischemia is caused by both Ca2+ influx from the extracellular space and Ca2+ release from the intracellular Ca2+ stores, such as the endoplasmic reticulum and mitochondria.25 In addition, Ca2+ influx is generally presumed to be caused through voltage-gated Ca2+ channels and through agonist-gated Ca2+ channels, mainly N-methyl--aspartate-gated Ca2+ channels. It has also been shown that 17â-estradiol rapidly reduces Ca2+ influx through L-type voltage-gated channels in neostriatal neurons; this reduction is probably mediated by G-protein-coupled receptors on the membrane surface.23 Furthermore, estrogens suppress elevation of [Ca2+]i which is induced by glutamate.13 Therefore, 17â-estradiol may inhibit Ca2+ influx through both voltage-gated and agonistgated Ca2+ channels.
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In this study, an increase in [Ca2+]i in Ca2+-free hypoxic conditions was also observed, and this was inhibited by 17â-estradiol. This finding indicates that 17â-estradiol can inhibit Ca2+ release from intracellular stores. There are two mechanisms by which Ca2+ is released from the endoplasmic reticulum to the cytosol.17,35 One is the release through ryanodine receptors, which exist on the membrane of the endoplasmic reticulum. The increase in the cytosolic concentration of Ca2+, which is caused by influx of Ca2+ from the extracellular space, stimulates the release of Ca2+ from the endoplasmic reticulum by activating ryanodine receptors (Ca2+-induced Ca2+ release). The other mechanism involves inositol-1,4,5triphosphate (IP3) receptors on the endoplasmic reticulum. IP3 receptor-linked Ca2+ channels open in response to the increase in intracellular concentration of IP3, resulting in the release of Ca2+ (IP3-induced Ca2+ release). The mechanism by which 17â-estradiol reduces the release of Ca2+ from the intracellular store was not elucidated in the present study. However, since estrogens increase IP3 synthesis,26 17â-estradiol seems to inhibit Ca2+-induced Ca2+ release. Furthermore, the stabilizing effect in the cellular calcium homeostasis by estrogens may contribute to the inhibition of [Ca2+]i by maintaining plasma membrane integrity with its antioxidant action.13
CONCLUSIONS
We observed that high-dose 17â-estradiol improved the outcome in hippocampal CA1 pyramidal cells following ischemia, probably by inhibiting the release of Ca2+ from the intracellular Ca2+ stores, as well as by inhibiting the influx of Ca2+ from the extracellular space. These findings contribute to our understanding of the neuroprotective action of 17â-estradiol against ischemia-induced neuronal damage.
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