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Age-related susceptibility to oxygen and glucose deprivation damage in rat hippocampal slices
Brain Research 1025 (2004) 226 – 230 www.elsevier.com/locate/brainres
Short communication
Age-related susceptibility to oxygen and glucose deprivation damage in rat hippocampal slices Ionara Rodrigues Siqueiraa,b, Helena Cimarostic, Cı´ntia Fochesattoc, Christianne Salbegoc, Carlos Alexandre Nettoa,c,* a
Programa de Po´s Graduac¸a˜o em Cieˆncias Biolo´gicas-Fisiologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil b Centro Universita´rio Univates, Lajeado, RS, Brazil c Departamento de Bioquı´mica, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil Accepted 10 August 2004 Available online 11 September 2004
Abstract Aging is an important risk factor for stroke. We evaluated the effects of aging on cell susceptibility to oxygen and glucose deprivation (OGD) in slices of the hippocampus from Wistar rats aged 2, 11 and 24 months. Lactate dehydrogenase (LDH) released to the incubation media and free radical content were markedly increased in the 24-month group submitted to OGD. These results confirm that hippocampal tissue from old animals is more susceptible to ischemia–reoxygenation injury. D 2004 Elsevier B.V. All rights reserved. Theme: Development and regeneration Topic: Aging process Keywords: Aging; Susceptibility to ischemia; Oxygen and glucose deprivation; Hippocampal slice; LDH release; MTT; DCF
Neurodegeneration is a prominent feature of stroke, a major cause of morbidity and mortality in the middle aged and the elderly. Global brain ischemia in rodents, as well as in humans, causes delayed cell death in hippocampal Cornus Ammon’s field 1 (CA1) neurons. CA1 pyramidal cell damage becomes irreversible after a few minutes of complete ischemia, although no histological changes are apparent until 48 h after reperfusion. The pathogenesis of cerebral ischemia/reperfusion has been associated with depletion of cellular energy sources, release of excitatory amino acids, mitochondrial dysfunction and excessive generation of free radicals [21]. As regards senescence, several lines of evidence support the free radical theory of aging, and many reports indicate * Corresponding author. Departamento de Bioquı´mica, Rua Eurı´pedes M. Duarte 10, 305, 90830-250, Porto Alegre, RS, Brazil. Tel.: +55 51 3316 5577; fax: +55 51 3316 5540. E-mail address: [email protected] (C.A. Netto). 0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2004.08.005
that free radicals and lipoperoxidation (LPO) are crucial both to the aging process [11,19] and to ischemia/ reperfusion pathophysiology [4]. Despite the fact that aging is an important risk factor for ischemic events in humans [2], most animal models of ischemia utilize young adult rodents. In addition, the induction of global brain ischemia in older animals leads to an elevated lethality [12], what complicates the design of experiments to address this question. Given that, we selected the oxygen and glucose deprivation (OGD) in slices as an in vitro model to evaluate the hypothesis that aging contributes to the susceptibility of brain tissue to ischemia. Rat forebrain slices exposed to OGD have been already used to model ischemic events and to investigate mechanisms of cell death and neuroprotection [6]. It offers important advantages because cell composition, such as functional neurons, inflammatory competent cells, locally released effectors and intercellular connections are preserved [20].
I.R. Siqueira et al. / Brain Research 1025 (2004) 226–230
The aim of the present study was to investigate whether aging increases the susceptibility of brain tissue, as assessed by mitochondrial activity and LDH release, to experimental in vitro ischemia in the model of oxygen and glucose deprivation to rat hippocampal slices. Considering that oxidative stress has been related to brain aging process, we also investigated the free radicals content in the same condition. Male Wistar rats aged 2 (n=10), 11 (n=9) and 23–24 (n=10) months, maintained under standard conditions (12-h light/dark, 22F2 8C) with food and water ad libitum, were used. The Animal Care Committee approved all handling and experimental conditions. Rats were decapitated, the hippocampi were quickly dissected out and transverse sections (400 Am) were prepared using a McIlwain tissue chopper. Hippocampal slices were divided in two equal sets (control and OGD), placed into separate 24-well culture plates and preincubated for 15 min in a modified Krebs– Henseleit solution (KHS preincubation solution) (mM): 120 NaCl, 2 KCl, 0.5 CaCl2, 26 NaHCO3, 10 MgSO4, 1.18 KH2PO4, 11 glucose, in a tissue culture incubator at 37 8C with 95% O2/5% CO2. After preincubation, the medium in the control plate was replaced with another modified Krebs– Henseleit solution (KHS incubation solution) (mM): 120 NaCl, 2 KCl, 2 CaCl2, 2.6 NaHCO3, 1.19 MgSO4, 1.18 KH2PO4, 11 glucose (pH 7.4) and incubated for 45 min in a tissue culture incubator at 378 with 95% O2/5% CO2. OGD slices were washed twice with a KHS medium without glucose (pH 7.4), saturated with N2 and incubated for 45 min (OGD period) at 37 8C in an anaerobic chamber saturated with nitrogen. After the OGD period, the medium from both control and OGD slices was removed and the two groups received KHS with glucose. Then, the slices were incubated for 180 min (recovery period) in the culture incubator. Control and OGD experiments were run concomitantly, using four slices of the same animal in each plate [8,17,18]. At the end of the recovery period, cellular damage, cellular viability (mitochondrial activity) and free radical level assays were performed. Mitochondrial activity was evaluated by the colorimetric 3(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT) (Sigma) method. Hippocampal slices were incubated for 45 min at 37 8C in the presence of MTT (45 Ag/ml). Active mitochondrial dehydrogenases of living cells cause cleavage and reduction of the soluble yellow MTT dye to the insoluble purple formazan, which was extracted in dimethyl sulfoxide (DMSO); the optical density was measured at 560 nm [16]. Cellular damage was quantified by measuring lactate dehydrogenase (LDH) released into the medium [14]. After the recovery period, LDH activity was determined using a kit (Doles Reagents, Goiaˆnia, Brazil). Each experiment was normalized by subtracting the background levels of LDH produced from the bno-treatmentQ wells [1]. The sample values were quantified using a standard curve. Data were analyzed by ANOVA followed by Duncan multiple range test.
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The free radical content was assessed by the use of 2V-7Vdichlorofluorescein diacetate (DCFH-DA, Sigma) as probe. After the recovery period, DCFH-DA (100 AM) was added to the wells and was incubated for 45 min at 37 8C. The medium was replaced by a fresh one and the plates placed on ice. The formation of the oxidized fluorescent derivative (DCF) was monitored after homogenization, using excitation and emission wavelengths of 488 and 525 nm, respectively (fluorescence spectrophotometer Hitachi F2000) [10]. All procedures were performed in the dark, and blanks containing DCFH-DA (no sample) and sample (no DCFH-DA) were processed for measurement of autofluorescence. The results of hippocampus control slices (OGD non-exposed) from young adults were taken as 100%, data were analyzed by ANOVA followed by Duncan multiple range test. Fig. 1 shows that OGD significantly diminished cell viability (about 35%) in all experimental groups, i.e., in slices from rats of all studied ages [ F(5,27)=4.563, pb0.01], as evaluated by the decrease of mitochondrial dehydrogenase activity (MTT method). The lack of any aging effect was confirmed when data were analysed as the ratio of ischemic/ control condition for each animal, so as to account for intrinsic variability [ F(2,13)=0.02273, p=0.9776]. The OGD exposure and reoxygenation caused an increase of LDH release into the incubation media, a marker of tissue necrosis, in all studied ages, as compared to control slices [ F(5,23)=16.195, pb0.0001]; the highest level of LDH released was found in the ischemic old (24–25 months) group (Fig. 2). The analysis of data as the ratio between ischemic and control LDH release confirms the age-related effect [ F(2,11)=11.988, p=0.0029]; the ratio was higher in hippocampal slices from old rats (1.725F 0.076) than those observed for the young (1.425F0.029) and middle-aged (1.376F0.032) groups.
Fig. 1. Effects of aging on cell viability, using MTT assay, of hippocampal slices from young adult (2–3 months), middle aged (10–11 months) and old age rats (23–24 months) exposed to oxygen and glucose deprivation (OGD) and reoxygenation—the ischemic groups. Results are expressed as percentage of the young non-OGD group. Columns represent mean FS.E.M. of quadruplicates of four to five experiments. * Values significantly different from those of non-OGD groups, + Values significantly different from those of non-OGD young group, as determined by ANOVA followed by Duncan’s test ( Pb0.05).
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Fig. 2. Effects of aging on cell injury, as assessed by LDH release to the medium, of hippocampal slices from young adult (2–3 months), middle aged (10–11 months) and old age rats (23–24 months) exposed to oxygen and glucose deprivation (OGD) and reoxygenation—the ischemic groups. Results are expressed as LDH activity (UI/l); the average LDH activity value from the young group (non-OGD) was 1.12 UI/l. Columns represent meanFS.E.M. of quadruplicates for four experiments. * Values significantly different from those of non-OGD groups, # values significantly different from those of young and middle aged OGD groups, as determined by ANOVA followed by Duncan’s test ( Pb0.05).
Interestingly, an age-dependent effect on free radical level was found [ F(5,23)=7.525, pb0.001], with a significant increase in DCF formed in control, non-OGD hippocampal slices of old rats. In addition, the ischemic event (OGD) significantly augmented the free radicals levels in slices from all ages tested; the old group exhibited a marked and consistent increase in DCF formation, as compared to OGD-young group (Fig. 3). However, given the increase in the old-group control values, ANOVA of the ratio ischemic/ control condition was not significant [ F(2,13)=0.06646, p=0.9362]. These results confirm that exposure of hippocampal slices to in vitro ischemic condition (OGD) followed by reoxygenation significantly affected the cellular viability (mitochondrial activity) and cellular damage in comparison to control (non-OGD) slices. We found that OGD and reoxygenation significantly affected cellular viability. Slices of different ages exposed to OGD showed a similar reduction on cell viability (about 30%), indicating that slices from aged rats remain viable after ischemia–reoxygenation as much as those of young and middle-aged ones. OGD induced marked cell death in hippocampal cells; hence, slices from old animals seem to be more vulnerable to this kind of injury than those from young animals. Conceivably, aged hippocampal neurons were particularly affected by OGD, since neurons are more susceptible to the metabolic challenge than glial cells [5]. It has been shown that the amount of LDH released correlates with the levels of neuronal plasma membrane damage [15]; this suggests that aged hippocampal cell membranes may be particularly susceptible to ischemic damage. Undoubtedly, LDH released from the old hippocampal slices was more severe than that of young ones. Our understanding of molecular mechanisms involved with age susceptibility to ischemic event is limited; we suggest that
the cell membranes from old animals are already altered in basal conditions, generating a condition prone to cell death. This idea is consistent with the increased ratio of polyunsaturated to monounsaturated fatty acids with aging in the plasma membrane of brain cells, which renders them highly susceptible to oxidation with advancing age [7,23]. The old membranes might be more vulnerable when exposed to acute ischemia/reoxygenation injury, possibly through oxidative stress, given that free radical content from old slices was increased in basal condition, in addition to augment OGD-induced increase. Interestingly, this result lend support to our previous work, since basal free radical content in old hippocampus homogenates rats was increased when compared to young ones [19]. The excessive formation of free radicals causes cell damage mainly through chain reactions of membrane lipid peroxidation and/or alterations in membrane fluidity. It is important to note that we observe an age-related increase in lipoperoxidation and a marked reduce of glutathione peroxidase activity and of total antioxidant capacity in homogenates of hippocampus, and thus the free radicals generation could overcame antioxidant defenses levels [19], resulting in cellular injury. On the other hand, the level of basal LDH release was identical in young and old rat hippocampus; these data suggest age-induced membrane alterations itself were unable to produce cell death. However, the augmented LDH leakage from old hippocampal slices submitted to OGD clearly showed its increased vulnerability to injury, a phenomenon possibly related to the susceptibility to excitotoxic events and disturbed energy metabolism, both situations commonly found in age-related neurological disorders. This reasoning is supported by findings of Vatassery et al. [22], who postulated that older animals are
Fig. 3. Effects of aging on free radical content, using DCF assay, in hippocampal slices from young adult (2–3 months), middle aged (10–11 months) and old rats (23–24 months) exposed to oxygen and glucose deprivation (OGD) and reoxygenation—the ischemic groups. Results are expressed as percentage of the young adult non-OGD group. Columns represent meanFS.E.M. of quadruplicates for four to five experiments. * Values significantly different from those of young non-OGD group, + values significantly different from corresponding non-OGD groups, # values significantly different from OGD-young group, as determined by ANOVA followed by Duncan’s test ( Pb0.05).
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more vulnerable to excitotoxicity, since they present reduced glutamate reuptake and elevated oxidative stress induced by many factors. It is important to note that agerelated membrane alterations do not abolish the role of any event involved in the multiple and interdependent molecular pathways triggered by ischemic injury. Presented findings suggest that ischemic cell loss in aged animals OGD might be mainly a consequence of necrosis, since this process is accompanied by cell lysis and release of intracellular contents. To our knowledge, this is the first evidence to the major contribution of necrosis mechanisms in death of old cells submitted to in vitro ischemia. Consistently, data from literature show that ischemia causes brain pH to fall from 7.4 to 6.3 and that reoxygenation reverses it [13]. In the young gerbil, recovery of brain pH to normal levels occurs within 20 min, while in the old brain pH does not reach normal levels even after 1 h. Interestingly, depending upon the duration of acidosis, it induces either necrosis or apoptosis, and necrotic cell loss increase with the duration of acidosis [9]. Although studies evaluating susceptibility to ischemic damage during aging are clearly insufficient, some reports have confirmed an increased vulnerability of aged animals. Ten minutes of brain ischemia leads to 100% of lethality in older gerbils after 7 days, while just half of the younger gerbils die, even after 15 min of brain ischemia [12]. In addition, hippocampal neurons from old animals are more susceptible to glutamate and h-amyloid toxicity than young neurons, what points, as well as here presented results, to the existence of intrinsic factors rendering older neurons more susceptible to stressors [3]. In conclusion, we suggest that senescent hippocampal tissue may undergo biochemical changes favoring a greater susceptibility to ischemia–reperfusion injury. An increase in membrane damage and their interaction with other ischemia-related events could be a potential mechanism for the age-related cerebral injury caused, at least in part, by freeradical-mediated mechanisms. The membrane oxidative damage might be particularly related to increased cell death in older hippocampal slices exposed to oxygen and glucose deprivation condition; accordingly, we propose that the ischemic cellular death in older animals is probably consequent to necrosis process. The detailed mechanism by which aging modify OGD-induced cell damage remains a subject for further investigations. Future studies are necessary to determine the exact role of age-related membrane damage following ischemia. The knowledge of brain injury mechanisms seem to be particularly meaningful for the proposal of new treatments capable to decrease or restrain damage to the central nervous system. Acknowledgement We gratefully acknowledge financial support by PRONEX, FAPERGS, CAPES, CNPq and PROPESQ-UFRGS.
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[19] I.R. Siqueira, C. Fochesatto, A. Andrade, M. Santos, M. Hagen, A. Bello-Klein, C.A. Netto, Total antioxidant capacity is impaired in different structures from aged rat brain, Int. J. Dev. Neurosci. (2004) (submitted for publication). [20] C.P. Taylor, S.P. Burke, M.L. Weber, Hippocampal slices: glutamate overflow and cellular damage from ischemia are reduced by sodiumchannel blockade, J. Neurosci. Methods 59 (1995) 121 – 128. [21] B.C. White, J.M. Sullivan, D.J. DeGracia, B.J. O’Neil, R.W. Neumar, L.I. Grossman, J.A. Rafols, G.S. Krause, Brain ischemia and
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Short communication
Age-related susceptibility to oxygen and glucose deprivation damage in rat hippocampal slices Ionara Rodrigues Siqueiraa,b, Helena Cimarostic, Cı´ntia Fochesattoc, Christianne Salbegoc, Carlos Alexandre Nettoa,c,* a
Programa de Po´s Graduac¸a˜o em Cieˆncias Biolo´gicas-Fisiologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil b Centro Universita´rio Univates, Lajeado, RS, Brazil c Departamento de Bioquı´mica, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil Accepted 10 August 2004 Available online 11 September 2004
Abstract Aging is an important risk factor for stroke. We evaluated the effects of aging on cell susceptibility to oxygen and glucose deprivation (OGD) in slices of the hippocampus from Wistar rats aged 2, 11 and 24 months. Lactate dehydrogenase (LDH) released to the incubation media and free radical content were markedly increased in the 24-month group submitted to OGD. These results confirm that hippocampal tissue from old animals is more susceptible to ischemia–reoxygenation injury. D 2004 Elsevier B.V. All rights reserved. Theme: Development and regeneration Topic: Aging process Keywords: Aging; Susceptibility to ischemia; Oxygen and glucose deprivation; Hippocampal slice; LDH release; MTT; DCF
Neurodegeneration is a prominent feature of stroke, a major cause of morbidity and mortality in the middle aged and the elderly. Global brain ischemia in rodents, as well as in humans, causes delayed cell death in hippocampal Cornus Ammon’s field 1 (CA1) neurons. CA1 pyramidal cell damage becomes irreversible after a few minutes of complete ischemia, although no histological changes are apparent until 48 h after reperfusion. The pathogenesis of cerebral ischemia/reperfusion has been associated with depletion of cellular energy sources, release of excitatory amino acids, mitochondrial dysfunction and excessive generation of free radicals [21]. As regards senescence, several lines of evidence support the free radical theory of aging, and many reports indicate * Corresponding author. Departamento de Bioquı´mica, Rua Eurı´pedes M. Duarte 10, 305, 90830-250, Porto Alegre, RS, Brazil. Tel.: +55 51 3316 5577; fax: +55 51 3316 5540. E-mail address: [email protected] (C.A. Netto). 0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2004.08.005
that free radicals and lipoperoxidation (LPO) are crucial both to the aging process [11,19] and to ischemia/ reperfusion pathophysiology [4]. Despite the fact that aging is an important risk factor for ischemic events in humans [2], most animal models of ischemia utilize young adult rodents. In addition, the induction of global brain ischemia in older animals leads to an elevated lethality [12], what complicates the design of experiments to address this question. Given that, we selected the oxygen and glucose deprivation (OGD) in slices as an in vitro model to evaluate the hypothesis that aging contributes to the susceptibility of brain tissue to ischemia. Rat forebrain slices exposed to OGD have been already used to model ischemic events and to investigate mechanisms of cell death and neuroprotection [6]. It offers important advantages because cell composition, such as functional neurons, inflammatory competent cells, locally released effectors and intercellular connections are preserved [20].
I.R. Siqueira et al. / Brain Research 1025 (2004) 226–230
The aim of the present study was to investigate whether aging increases the susceptibility of brain tissue, as assessed by mitochondrial activity and LDH release, to experimental in vitro ischemia in the model of oxygen and glucose deprivation to rat hippocampal slices. Considering that oxidative stress has been related to brain aging process, we also investigated the free radicals content in the same condition. Male Wistar rats aged 2 (n=10), 11 (n=9) and 23–24 (n=10) months, maintained under standard conditions (12-h light/dark, 22F2 8C) with food and water ad libitum, were used. The Animal Care Committee approved all handling and experimental conditions. Rats were decapitated, the hippocampi were quickly dissected out and transverse sections (400 Am) were prepared using a McIlwain tissue chopper. Hippocampal slices were divided in two equal sets (control and OGD), placed into separate 24-well culture plates and preincubated for 15 min in a modified Krebs– Henseleit solution (KHS preincubation solution) (mM): 120 NaCl, 2 KCl, 0.5 CaCl2, 26 NaHCO3, 10 MgSO4, 1.18 KH2PO4, 11 glucose, in a tissue culture incubator at 37 8C with 95% O2/5% CO2. After preincubation, the medium in the control plate was replaced with another modified Krebs– Henseleit solution (KHS incubation solution) (mM): 120 NaCl, 2 KCl, 2 CaCl2, 2.6 NaHCO3, 1.19 MgSO4, 1.18 KH2PO4, 11 glucose (pH 7.4) and incubated for 45 min in a tissue culture incubator at 378 with 95% O2/5% CO2. OGD slices were washed twice with a KHS medium without glucose (pH 7.4), saturated with N2 and incubated for 45 min (OGD period) at 37 8C in an anaerobic chamber saturated with nitrogen. After the OGD period, the medium from both control and OGD slices was removed and the two groups received KHS with glucose. Then, the slices were incubated for 180 min (recovery period) in the culture incubator. Control and OGD experiments were run concomitantly, using four slices of the same animal in each plate [8,17,18]. At the end of the recovery period, cellular damage, cellular viability (mitochondrial activity) and free radical level assays were performed. Mitochondrial activity was evaluated by the colorimetric 3(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT) (Sigma) method. Hippocampal slices were incubated for 45 min at 37 8C in the presence of MTT (45 Ag/ml). Active mitochondrial dehydrogenases of living cells cause cleavage and reduction of the soluble yellow MTT dye to the insoluble purple formazan, which was extracted in dimethyl sulfoxide (DMSO); the optical density was measured at 560 nm [16]. Cellular damage was quantified by measuring lactate dehydrogenase (LDH) released into the medium [14]. After the recovery period, LDH activity was determined using a kit (Doles Reagents, Goiaˆnia, Brazil). Each experiment was normalized by subtracting the background levels of LDH produced from the bno-treatmentQ wells [1]. The sample values were quantified using a standard curve. Data were analyzed by ANOVA followed by Duncan multiple range test.
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The free radical content was assessed by the use of 2V-7Vdichlorofluorescein diacetate (DCFH-DA, Sigma) as probe. After the recovery period, DCFH-DA (100 AM) was added to the wells and was incubated for 45 min at 37 8C. The medium was replaced by a fresh one and the plates placed on ice. The formation of the oxidized fluorescent derivative (DCF) was monitored after homogenization, using excitation and emission wavelengths of 488 and 525 nm, respectively (fluorescence spectrophotometer Hitachi F2000) [10]. All procedures were performed in the dark, and blanks containing DCFH-DA (no sample) and sample (no DCFH-DA) were processed for measurement of autofluorescence. The results of hippocampus control slices (OGD non-exposed) from young adults were taken as 100%, data were analyzed by ANOVA followed by Duncan multiple range test. Fig. 1 shows that OGD significantly diminished cell viability (about 35%) in all experimental groups, i.e., in slices from rats of all studied ages [ F(5,27)=4.563, pb0.01], as evaluated by the decrease of mitochondrial dehydrogenase activity (MTT method). The lack of any aging effect was confirmed when data were analysed as the ratio of ischemic/ control condition for each animal, so as to account for intrinsic variability [ F(2,13)=0.02273, p=0.9776]. The OGD exposure and reoxygenation caused an increase of LDH release into the incubation media, a marker of tissue necrosis, in all studied ages, as compared to control slices [ F(5,23)=16.195, pb0.0001]; the highest level of LDH released was found in the ischemic old (24–25 months) group (Fig. 2). The analysis of data as the ratio between ischemic and control LDH release confirms the age-related effect [ F(2,11)=11.988, p=0.0029]; the ratio was higher in hippocampal slices from old rats (1.725F 0.076) than those observed for the young (1.425F0.029) and middle-aged (1.376F0.032) groups.
Fig. 1. Effects of aging on cell viability, using MTT assay, of hippocampal slices from young adult (2–3 months), middle aged (10–11 months) and old age rats (23–24 months) exposed to oxygen and glucose deprivation (OGD) and reoxygenation—the ischemic groups. Results are expressed as percentage of the young non-OGD group. Columns represent mean FS.E.M. of quadruplicates of four to five experiments. * Values significantly different from those of non-OGD groups, + Values significantly different from those of non-OGD young group, as determined by ANOVA followed by Duncan’s test ( Pb0.05).
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Fig. 2. Effects of aging on cell injury, as assessed by LDH release to the medium, of hippocampal slices from young adult (2–3 months), middle aged (10–11 months) and old age rats (23–24 months) exposed to oxygen and glucose deprivation (OGD) and reoxygenation—the ischemic groups. Results are expressed as LDH activity (UI/l); the average LDH activity value from the young group (non-OGD) was 1.12 UI/l. Columns represent meanFS.E.M. of quadruplicates for four experiments. * Values significantly different from those of non-OGD groups, # values significantly different from those of young and middle aged OGD groups, as determined by ANOVA followed by Duncan’s test ( Pb0.05).
Interestingly, an age-dependent effect on free radical level was found [ F(5,23)=7.525, pb0.001], with a significant increase in DCF formed in control, non-OGD hippocampal slices of old rats. In addition, the ischemic event (OGD) significantly augmented the free radicals levels in slices from all ages tested; the old group exhibited a marked and consistent increase in DCF formation, as compared to OGD-young group (Fig. 3). However, given the increase in the old-group control values, ANOVA of the ratio ischemic/ control condition was not significant [ F(2,13)=0.06646, p=0.9362]. These results confirm that exposure of hippocampal slices to in vitro ischemic condition (OGD) followed by reoxygenation significantly affected the cellular viability (mitochondrial activity) and cellular damage in comparison to control (non-OGD) slices. We found that OGD and reoxygenation significantly affected cellular viability. Slices of different ages exposed to OGD showed a similar reduction on cell viability (about 30%), indicating that slices from aged rats remain viable after ischemia–reoxygenation as much as those of young and middle-aged ones. OGD induced marked cell death in hippocampal cells; hence, slices from old animals seem to be more vulnerable to this kind of injury than those from young animals. Conceivably, aged hippocampal neurons were particularly affected by OGD, since neurons are more susceptible to the metabolic challenge than glial cells [5]. It has been shown that the amount of LDH released correlates with the levels of neuronal plasma membrane damage [15]; this suggests that aged hippocampal cell membranes may be particularly susceptible to ischemic damage. Undoubtedly, LDH released from the old hippocampal slices was more severe than that of young ones. Our understanding of molecular mechanisms involved with age susceptibility to ischemic event is limited; we suggest that
the cell membranes from old animals are already altered in basal conditions, generating a condition prone to cell death. This idea is consistent with the increased ratio of polyunsaturated to monounsaturated fatty acids with aging in the plasma membrane of brain cells, which renders them highly susceptible to oxidation with advancing age [7,23]. The old membranes might be more vulnerable when exposed to acute ischemia/reoxygenation injury, possibly through oxidative stress, given that free radical content from old slices was increased in basal condition, in addition to augment OGD-induced increase. Interestingly, this result lend support to our previous work, since basal free radical content in old hippocampus homogenates rats was increased when compared to young ones [19]. The excessive formation of free radicals causes cell damage mainly through chain reactions of membrane lipid peroxidation and/or alterations in membrane fluidity. It is important to note that we observe an age-related increase in lipoperoxidation and a marked reduce of glutathione peroxidase activity and of total antioxidant capacity in homogenates of hippocampus, and thus the free radicals generation could overcame antioxidant defenses levels [19], resulting in cellular injury. On the other hand, the level of basal LDH release was identical in young and old rat hippocampus; these data suggest age-induced membrane alterations itself were unable to produce cell death. However, the augmented LDH leakage from old hippocampal slices submitted to OGD clearly showed its increased vulnerability to injury, a phenomenon possibly related to the susceptibility to excitotoxic events and disturbed energy metabolism, both situations commonly found in age-related neurological disorders. This reasoning is supported by findings of Vatassery et al. [22], who postulated that older animals are
Fig. 3. Effects of aging on free radical content, using DCF assay, in hippocampal slices from young adult (2–3 months), middle aged (10–11 months) and old rats (23–24 months) exposed to oxygen and glucose deprivation (OGD) and reoxygenation—the ischemic groups. Results are expressed as percentage of the young adult non-OGD group. Columns represent meanFS.E.M. of quadruplicates for four to five experiments. * Values significantly different from those of young non-OGD group, + values significantly different from corresponding non-OGD groups, # values significantly different from OGD-young group, as determined by ANOVA followed by Duncan’s test ( Pb0.05).
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more vulnerable to excitotoxicity, since they present reduced glutamate reuptake and elevated oxidative stress induced by many factors. It is important to note that agerelated membrane alterations do not abolish the role of any event involved in the multiple and interdependent molecular pathways triggered by ischemic injury. Presented findings suggest that ischemic cell loss in aged animals OGD might be mainly a consequence of necrosis, since this process is accompanied by cell lysis and release of intracellular contents. To our knowledge, this is the first evidence to the major contribution of necrosis mechanisms in death of old cells submitted to in vitro ischemia. Consistently, data from literature show that ischemia causes brain pH to fall from 7.4 to 6.3 and that reoxygenation reverses it [13]. In the young gerbil, recovery of brain pH to normal levels occurs within 20 min, while in the old brain pH does not reach normal levels even after 1 h. Interestingly, depending upon the duration of acidosis, it induces either necrosis or apoptosis, and necrotic cell loss increase with the duration of acidosis [9]. Although studies evaluating susceptibility to ischemic damage during aging are clearly insufficient, some reports have confirmed an increased vulnerability of aged animals. Ten minutes of brain ischemia leads to 100% of lethality in older gerbils after 7 days, while just half of the younger gerbils die, even after 15 min of brain ischemia [12]. In addition, hippocampal neurons from old animals are more susceptible to glutamate and h-amyloid toxicity than young neurons, what points, as well as here presented results, to the existence of intrinsic factors rendering older neurons more susceptible to stressors [3]. In conclusion, we suggest that senescent hippocampal tissue may undergo biochemical changes favoring a greater susceptibility to ischemia–reperfusion injury. An increase in membrane damage and their interaction with other ischemia-related events could be a potential mechanism for the age-related cerebral injury caused, at least in part, by freeradical-mediated mechanisms. The membrane oxidative damage might be particularly related to increased cell death in older hippocampal slices exposed to oxygen and glucose deprivation condition; accordingly, we propose that the ischemic cellular death in older animals is probably consequent to necrosis process. The detailed mechanism by which aging modify OGD-induced cell damage remains a subject for further investigations. Future studies are necessary to determine the exact role of age-related membrane damage following ischemia. The knowledge of brain injury mechanisms seem to be particularly meaningful for the proposal of new treatments capable to decrease or restrain damage to the central nervous system. Acknowledgement We gratefully acknowledge financial support by PRONEX, FAPERGS, CAPES, CNPq and PROPESQ-UFRGS.
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