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Neuronal death and tumor necrosis factor-α response to glutamate-induced excitotoxicity in the cerebral cortex of neonatal rats
Neuroscience Letters 333 (2002) 95–98 www.elsevier.com/locate/neulet
Neuronal death and tumor necrosis factor-a response to glutamateinduced excitotoxicity in the cerebral cortex of neonatal rats V. Chaparro-Huerta a, M.C. Rivera-Cervantes b, B.M. Torres-Mendoza c, C. Beas-Za´rate a,b,* a
Laboratorio de Neurobioliologı´a Celular y Molecular, Divisio´n de Neurociencias, Centro de Investigacio´n Biome´dica de Occidente (C.I.B.O.), Instituto Mexicano del Seguro Social (IMSS), P.O. 4-160, Guadalajara, Jalisco, 44421, Mexico b Departamento de Biologı´a Celular and Molecular, Centro Universitario de Ciencias Biolo´gicas y Agropecuarias (C.U.C.B.A.), Universidad de Guadalajara (U. de G.), Guadalajara, Mexico c Divisio´n de Inmunologı´a, C.I.B.O., IMSS, Guadalajara, Mexico Received 21 May 2002; received in revised form 26 August 2002; accepted 27 August 2002
Abstract Neuronal death and lactate dehydrogenase (LDH) activity were evaluated in the cerebral cortices of neonatal rats after exposure to monosodium L-glutamate (MSG) to induce neuroexcitotoxicity. A time–response profile for tumor necrosis factor-alpha (TNF-a) expression was drawn, with measurements taken every 6 h after the first dose of MSG during the first 8 postnatal days, and at days 10 and 14 after birth. An increase in neuronal loss accompanied by high LDH activity and high TNF-a levels was observed at 8 and 10 days. These results indicate that neuronal loss may occur via an apoptosis-like mechanism directed selectively against neurons that express glutamate receptors, mainly the N-methyl-d-aspartate, which it may be strengthen by high TNF-a levels through a feedback mechanism to induce cell death via apoptosis. q 2002 Published by Elsevier Science Ireland Ltd. Keywords: Cell death; Tumor necrosis factor-a; Neuroexcitotoxicity; Glutamate; Neonatal rats
Overactivation of glutamate receptors can induce apoptosis by a mechanism involving calcium influx [1,9], and such excitotoxicity may occur in acute neurodegenerative conditions such as ischemic stroke, trauma, and severe epileptic seizures, as well as in Alzheimer’s disease and motor system disorders [7,24]. Although individual neurons may be dysfunctional for extended periods in these disorders, they may die rapidly once the apoptotic cascade is fully activated [18]. From this perspective, the progressive deficits that occur in chronic neurodegenerative disorders are the results of the progressive damage of individual neuron [18]. Several lines of evidence suggest that cell death may originate by one of two general pathways: necrosis or apoptosis [6]. Apoptosis and necrosis are generally considered distinct mechanisms of cell death with very different characteristic features, and can be distinguished by the morphological and biochemical properties of the affected cells [8]. Apoptosis is an active process of cell destruction, which is * Corresponding author. Fax: 133-3618-1756. E-mail address: [email protected] (C. Beas-Za´rate).
characterized morphologically by cell shrinkage, membrane blebbing, nuclear pyknosis, and early chromatin condensation [20]. Inflammation in the brain and activation of microglia have been associated with the pathogenesis of a variety of neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, multiple sclerosis, AIDS dementia complex, and amytrophic lateral sclerosis [10]. Activated microglia secrete a variety of proinflammatory and cytotoxic factors including nitric oxide (NO), tumor necrosis factor-alpha (TNF-a), interleukin-1 beta (IL-1 b), arachidonic acid, eicosanoids, and reactive oxygen species [2]. The production and accumulation of these factors have been attributed to participate in the neurodegeneration [5]. Cytokines are involved both in the immune response and in controlling various events in the central nervous system (CNS), i.e. they are equally immunoregulators and modulators of neural functions and neuronal survival [22]. During the last decade, neurotoxic and neuroprotective mechanisms have both been closely correlated with the balance between the pro-inflammatory and anti-inflammatory cytokines. Therefore, the expression of TNF-a is rapidly induced in
0304-3940/02/$ - see front matter q 2002 Published by Elsevier Science Ireland Ltd. PII: S03 04 - 394 0( 0 2) 01 00 6- 6
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Fig. 1. (A) Neuronal survival in the frontal cortex at different postnatal ages in rats. Neonates (1, 3, 5, and 7 days old) were exposed to MSG (4 mg/g body weight). Healthy cells were counted in 8-mm paraffin-embedded slices stained with hematoxylin–eosin. Data represent the mean ^ SD of 5–6 animals and serial slices with various fields per plate. (B) Dehydrogenase activity in the cerebral cortices from a group of animals exposed to MSG in parallel. Data represent the mean ^ SD of 5–6 animals per group. Measurements were made in duplicate. Differences relative to the control group were considered statistically significant at P , 0:001 (*).
the injured brain and clearly contributes, in the CNS, to the initiation of cascades associated with neuronal apoptosis and neurological impairment [22]. Previous findings using a model of neonatal exposure to monosodium glutamate (MSG) showed that both astrocytes and microglial cells are highly susceptible to the neuroexcitotoxic effects of MSG administered at the neonatal stage [17]. The increase in the astrocytic population in the cortex may be a consequence of early proliferation (hyperplasia). However, it is unknown whether the pro-inflammatory cytokines, such as TNF-a, have a significant role in neuronal death in the cerebral cortex in this model of neuroexcitotoxic damage. An ability to anticipate early neuronal damage could be important in avoiding further neurological and/or neurodegenerative processes. Newborn male Wistar rats from our colony were injected subcutaneously with 4 mg/g body weight MSG at 1, 3, 5, and 7 days after birth [17]. The animals were maintained in a 12/12 h light/dark cycle at 22 ^ 2 8C and 55% relative ambient humidity, with free access to balanced food (Ralston Rations, Ralston Purina, USA) and water. Animals were studied at; 8, 10, or 14 days after birth. Control and experimental groups of rats were used (n ¼ 5–6). Both experimental and control group were
using the same cortical regions for morphological analyses. These rats were perfused intracardially with 4% paraformaldehyde in phosphate-buffered saline (PBS), pH ¼ 7.2. Brains were paraffin-embedded, and coronal slices (8 mm) of motor frontal cortex of each control and those injected with MSG, 200 microscopic fields randomly were examined at 100 £ enlargement (1.6 mm 2 of tissue), through the complete cortical thickness with coordinate interaural 10.7 mm and bregma 1.7 mm (Fr1, plate 11 of Paxino’s). The cells were hematoxylin-eosin stained, analyzed and counted with double-blinded observer as described previously by Martı´nez-Contreras et al. [17]. The other group, used for biochemical analysis, was sacrificed by decapitation and the brains were maintained on ice (220 8C) until used. For the TNF-a assay, the tissue (frontal cortex) was homogenized in 500 ml PBS/Protease Inhibitor Cocktail (Calbiochem-Novabiochem, San Diego, CA) per 50 mg tissue weight. The samples were centrifuged at 4 8C for 20 min at 10 500 £ g and the supernatant was collected. Protein expression was evaluated using an enzyme-linked immunosorbent assay (ELISA) kit, Quantikine M Rat TNF-a (R&D Systems, Minneapolis, MN). Absorbances were measured at 450 nm with correction to 540 or 570 nm, using a Bio-Rad Microplate Reader Model 550. Tissue (frontal cortex)
Fig. 2. Representative microphotographs of serial slices from the cerebral cortices of rats at different postnatal ages. (A) Control group at postnatal day 8. (C) Control group at postnatal day 10, and (E) control group at postnatal day 14. (B,D and F) correspond to the treatment group. Paraffin-embedded slices, 8 mm thick, were stained with hematoxylin-eosin, and analyzed using optical microscopy. Neurons with shrinkage (bold arrow), membrane blebbing (clear arrow), nuclear pyknosis (arrow head), and chromatin condensation (*). Magnification ¼ 100 £ .
V. Chaparro-Huerta et al. / Neuroscience Letters 333 (2002) 95–98
Fig. 3. (A) Time–response curve for TNF-a levels during MSG treatment. TNF-a levels were measured by ELISA every 6 h after each MSG dose until day 9. Each point represents the mean ^ SD of 2–3 animals per group and measurements were made in duplicate. (B) TNF-a levels at postnatal days 8, 10, and 14. Each point represents the mean ^ SD of 5–6 animals per group and measurements were made in duplicate. Statistically different relative to the control group at P , 0:001 (*).
obtained from another animals group was homogenized in 1.0 ml of PBS to measure lactate dehydrogenase (LDH) activity using a commercial lactate dehydrogenase kit (Sigma, St. Louis, MO). Data are represented as means ^ SEM. Statistical differences between control and experimental groups were assessed using one-way ANOVA. Differences were considered statistically significant at P , 0:05. Our results show that neonatal exposure to glutamate induced significant neuronal loss in the cerebral cortex at 8 and 10 days of age, relative to the control group (Fig. 1A). This neuronal death was confirmed by a significant increase in LDH activity at the same ages (Fig. 1B). Morphological changes were characterized by cell shrinkage, membrane blebbing, nuclear pyknosis, and early chromatin condensation (Fig. 2B,D, and F) compared with the control group (Fig. 2A,C, and E). A time–response profile for TNF-a expression was determined 6 h after each dose of glutamate, during the first 9 days after birth (Fig. 3A). Importantly, levels of TNF-a were higher than those of the control group 24 h after the last glutamate dose (8 days old) (Fig. 3A,B). However, TNF-a levels continued to increase until day 14 of the study (Fig. 3B).
97
Programmed cell death (apoptosis) is now recognized as a fundamental biological process under normal physiological conditions, but under pathological conditions, there can be a failure to control the extent of cell death. Apoptosis acquires importance in some neurodegenerative diseases. In this study, significant neuronal loss was observed under normal conditions in the cerebral cortex during brain development (Fig. 1A). This process was accelerated by early exposure to high glutamate (Glu) concentrations, and was accompanied by the morphological characteristics of cellular injury (Fig. 2). However, at 14 days, no difference in that neuronal loss was observed. It may be due to a neuronal migration delayed by that excessive Glu concentration since it had been documented that at this age all cortical layers have reached their adult features [23]. This cell loss may occur via an apoptosis-like mechanism directed selectively against neurons expressing Glu receptors, mainly the N-methyl-D-aspartate (NMDA)-type receptors, because it is well known that Glu excitotoxicity is mediated through the activation of NMDAgated ion channels in several neurodegenerative diseases and in ischemic stroke [15,16]. These NMDA receptors are chiefly expressed from the embryonic to the neonatal developmental stages in rodents [19]. The neurotoxicity initiated by overstimulation of Glu receptors and the subsequent influx of free Ca 11 leads to an intracellular cascade of cytotoxic events, which are necessary to trigger delayed neuronal cell death [11,12]. Furthermore, NMDA-receptor activation results in an extracellular regulation of signal kinases (extracellular-regulated kinases (ERK)1/ERK2), which appear to be involved in the excitotoxicity and neuronal apoptosis-like death induced by Glu [14]. However, aamino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors may be another mechanism, by which these neurotoxic events are induced, because AMPA receptors mediate excitotoxicity in neocortical neurons at a later age (12 days) in vitro [13]. Therefore, in this in vivo model, the ages of the neonates in which neurotoxicity was induced by early Glu exposure suggest predominantly NMDAreceptor activation, rather than the activation of AMPA receptors, which react to Glu in a later phase of postnatal development. Glial cells provide mechanical and metabolic support for neurons by producing and responding to a variety of stimuli [3]. Therefore, activated glial cells produce diverse inflammatory mediators such as TNF-a [21]. There is growing evidence that toxic mediators produced by glia, astrocytes, and microglia, are involved in the pathogenesis of various neurodegenerative diseases [4]. In a previous study, significant astrocytic and microglia reactivity in the cerebral cortices of 60 days old rats was induced by the neonatal administration of Glu [17]. This reactivity was manifested by an increase in the number, hypertrophy and its complexity of the cytoplasm extension in both cells with high expression of mRNA to vimentin. This suggests that glial cell reactivity persists into adulthood in response to an early neuroexcitotoxic stimulus with Glu. For this reason, the
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TNF-a time–response profile after Glu exposure was evaluated in this study. The increase in TNF-a levels may indicate the presence of a feedback mechanism to induce cell death via apoptosis. Exposure of cells to TNF-a results in the activation of caspases, which leads to apoptosis, as well as to other signaling pathways that include transcription factor activation protein (AP-1), nuclear factor (NF)kappaB and mitogen-activated protein kinases [3]. Therefore, further studies are required to ascertain whether the cell death observed under our experimental conditions occurs via apoptosis only, or together with necrosis, or as a hybrid form somewhere along the apoptosis–necrosis continuous, as suggested by Martin et al. [16]. This work was partially supported by CONACyT grants no. 30901-M and 158761 to C.B.Z. and V.CH.H respectively. It constitutes part of the doctoral thesis of V.CH.H. prepared at the Universidad de Guadalajara (CUCS).
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[16] [1] Ankarcrona, M., Dypbukt, J.M., Bonfoco, E., Zhivotovsky, B., Orrenius, S., Lipton, S.A. and Nicotera, P., Glutamateinduced neuronal death: a succession of necrosis or apoptosis depending on mitochondrial function, Neuron, 15 (1995) 961–973. [2] Banati, R.B., Gehrmann, J., Scubert, P. and Kreutzberg, G.W., Cytotoxicity of microglia, Glia, 7 (1993) 111–118. [3] Baud, V. and Karin, M., Signal transduction by tumor necrosis factor and its relatives, Trends Cell Biol., 11 (9) (2001) 372–377. [4] Becher, B., Prat, A. and Antel, J.P., Brain–immune connection: immunoregulatory properties of CNS-resident cells, Glia, 29 (2000) 293–304. [5] Bronstein, D.M., Perez-Otano, I., Sun, V., Mullis-Sawin, S.B., Chan, J., Wu, G.C., Hudson, P.M., Kong, L.Y., Hong, J.S. and McMillian, M.K., Glia-dependent neurotoxicity and neuroprotection in mesencephalic cultures, Brain Res., 704 (1995) 112–116. [6] Cheung, N.S., Pascoe, C.J., Giardina, S.F., John, C.A. and Beart, P.M., Micromolar l-glutamate induces extensive apoptosis in an apoptotic–necrotic continuum of insultdependent, excitotoxic injury in cultured cortical neurons, Neuropharmacology, 37 (1998) 1419–1429. [7] Choi, D.W., Excitotoxic cell death, J. Neurobiol., 23 (1992) 1261–1276. [8] Clarke, P.G., Developmental cell death: morphological diversity and multiple mechanisms, Anat. Embryol., 181 (1990) 195–213. [9] Glazner, G.W., Chan, S.L., Lu, C. and Mattson, M.P., Caspase-mediated degradation of AMPA receptor subunits: a mechanism for preventing excitotoxic necrosis and ensuring apoptosis, J. Neurosci., 20 (2000) 3641–3649. [10] Gonzalez-Serrano, F. and Baltuch, G., Microglia as media-
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tors of inflammatory and degenerative diseases, Ann. Rev. Neurosci., 22 (1999) 219–240. Hartley, D. and Choi, D.W., Delayed rescue of N-methyl-Daspartate receptor-mediated neuronal injury in cortical culture, J. Pharmacol. Exp. Ther., 250 (1989) 752–758. Hirashima, Y., Kurimoto, M., Nogami, K., Endo, S., Saitoh, M., Ohtami, O., Nagata, T., Muraguchi, A. and Takaku, A., Correlation of glutamate-induced apoptosis with caspase activities in cultured rat cerebral cortical neurons, Brain Res., 849 (1999) 109–118. Jensen, J.B., Schousboe, A. and Pickering, D.S., AMPA receptor-mediated excitotoxicity in neocortical neurons is developmentally regulated and dependent upon receptor desensitization, Neurochem. Int., 32 (1998) 505–513. Jiang, Q., Gu, Z., Zhang, G. and Jing, G., N-Methyl-D-aspartate receptor activation results in regulation of extracellular signal-regulated kinases by protein kinases and phosphatases in glutamate-induced neuronal apoptotic-like death, Brain Res., 887 (2000) 285–292. Kobayashi, T. and Mori, Y., Ca 21 channel antagonists and neuroprotection from cerebral ischemia, Eur. J. Pharmacol., 363 (1998) 1–15. Martin, L.J., Al-Abdulla, N.A., Brambrink, M.A., Kirsh, J.R., Sieber, F.E. and Portera-Cailliau, C., Neurodegeneration in excitotoxicity, global cerebral ischemia, and target deprivation: a perspective on the contributions of apoptosis and necrosis, Brain Res. Bull., 46 (1998) 281–309. Martı´nez-Contreras, A., Huerta, M., Lopez-Perez, S., Garcı´aEstrada, J., Luquı´n, S. and Beas-Za´ rate, C., Astrocytic and microglia cells reactivity induced by neonatal administration of glutamate in cerebral cortex of adult rats, J. Neurosci. Res., 67 (2002) 200–210. Mattson, M.P., Apoptosis in neurodegenerative disorders, Nat. Rev. Mol. Cell Biol., 1 (2000) 120–129. Ozawa, S., Kamiya, H. and Tsuzuki, K., Glutamate receptors in the mammalian central nervous system, Prog. Neurobiol., 54 (1998) 581–618. Saraste, A. and Pulkki, K., Morphologic and biochemical hallmarks of apoptosis, Cardiovasc. Res., 45 (2000) 528– 537. Sawada, M., Kondo, N., Suzumura, A. and Marunouchi, T., Production of tumor necrosis factor-alpha by microglia and astrocytes in culture, Brain Res., 491 (1989) 394–397. Szele´ nyi, J., Cytokines and the central nervous system, Brain Res. Bull., 54 (2001) 329–338. Van Eden, C.G., Kros, J.M. and Uylings, H.B.M., The development of the rat prefrontal cortex; Its size and development of connections with thalamus, spinal cord and other cortical areas, In H.M.B. Uylings, C.G. Van Den, J.P.C. De Bruin, M.A. Corner and M.G.P. Freenstra (Eds.), Progress in Brain Research, 85, Elsevier Science, Amsterdam, 1990, pp. 169–183. Wong, P.C., Rothstein, J.D. and Price, D.L., The genetic and molecular mechanisms of motor neuron disease, Curr. Opin. Neurobiol., 8 (1998) 791–799.
Neuronal death and tumor necrosis factor-a response to glutamateinduced excitotoxicity in the cerebral cortex of neonatal rats V. Chaparro-Huerta a, M.C. Rivera-Cervantes b, B.M. Torres-Mendoza c, C. Beas-Za´rate a,b,* a
Laboratorio de Neurobioliologı´a Celular y Molecular, Divisio´n de Neurociencias, Centro de Investigacio´n Biome´dica de Occidente (C.I.B.O.), Instituto Mexicano del Seguro Social (IMSS), P.O. 4-160, Guadalajara, Jalisco, 44421, Mexico b Departamento de Biologı´a Celular and Molecular, Centro Universitario de Ciencias Biolo´gicas y Agropecuarias (C.U.C.B.A.), Universidad de Guadalajara (U. de G.), Guadalajara, Mexico c Divisio´n de Inmunologı´a, C.I.B.O., IMSS, Guadalajara, Mexico Received 21 May 2002; received in revised form 26 August 2002; accepted 27 August 2002
Abstract Neuronal death and lactate dehydrogenase (LDH) activity were evaluated in the cerebral cortices of neonatal rats after exposure to monosodium L-glutamate (MSG) to induce neuroexcitotoxicity. A time–response profile for tumor necrosis factor-alpha (TNF-a) expression was drawn, with measurements taken every 6 h after the first dose of MSG during the first 8 postnatal days, and at days 10 and 14 after birth. An increase in neuronal loss accompanied by high LDH activity and high TNF-a levels was observed at 8 and 10 days. These results indicate that neuronal loss may occur via an apoptosis-like mechanism directed selectively against neurons that express glutamate receptors, mainly the N-methyl-d-aspartate, which it may be strengthen by high TNF-a levels through a feedback mechanism to induce cell death via apoptosis. q 2002 Published by Elsevier Science Ireland Ltd. Keywords: Cell death; Tumor necrosis factor-a; Neuroexcitotoxicity; Glutamate; Neonatal rats
Overactivation of glutamate receptors can induce apoptosis by a mechanism involving calcium influx [1,9], and such excitotoxicity may occur in acute neurodegenerative conditions such as ischemic stroke, trauma, and severe epileptic seizures, as well as in Alzheimer’s disease and motor system disorders [7,24]. Although individual neurons may be dysfunctional for extended periods in these disorders, they may die rapidly once the apoptotic cascade is fully activated [18]. From this perspective, the progressive deficits that occur in chronic neurodegenerative disorders are the results of the progressive damage of individual neuron [18]. Several lines of evidence suggest that cell death may originate by one of two general pathways: necrosis or apoptosis [6]. Apoptosis and necrosis are generally considered distinct mechanisms of cell death with very different characteristic features, and can be distinguished by the morphological and biochemical properties of the affected cells [8]. Apoptosis is an active process of cell destruction, which is * Corresponding author. Fax: 133-3618-1756. E-mail address: [email protected] (C. Beas-Za´rate).
characterized morphologically by cell shrinkage, membrane blebbing, nuclear pyknosis, and early chromatin condensation [20]. Inflammation in the brain and activation of microglia have been associated with the pathogenesis of a variety of neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, multiple sclerosis, AIDS dementia complex, and amytrophic lateral sclerosis [10]. Activated microglia secrete a variety of proinflammatory and cytotoxic factors including nitric oxide (NO), tumor necrosis factor-alpha (TNF-a), interleukin-1 beta (IL-1 b), arachidonic acid, eicosanoids, and reactive oxygen species [2]. The production and accumulation of these factors have been attributed to participate in the neurodegeneration [5]. Cytokines are involved both in the immune response and in controlling various events in the central nervous system (CNS), i.e. they are equally immunoregulators and modulators of neural functions and neuronal survival [22]. During the last decade, neurotoxic and neuroprotective mechanisms have both been closely correlated with the balance between the pro-inflammatory and anti-inflammatory cytokines. Therefore, the expression of TNF-a is rapidly induced in
0304-3940/02/$ - see front matter q 2002 Published by Elsevier Science Ireland Ltd. PII: S03 04 - 394 0( 0 2) 01 00 6- 6
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V. Chaparro-Huerta et al. / Neuroscience Letters 333 (2002) 95–98
Fig. 1. (A) Neuronal survival in the frontal cortex at different postnatal ages in rats. Neonates (1, 3, 5, and 7 days old) were exposed to MSG (4 mg/g body weight). Healthy cells were counted in 8-mm paraffin-embedded slices stained with hematoxylin–eosin. Data represent the mean ^ SD of 5–6 animals and serial slices with various fields per plate. (B) Dehydrogenase activity in the cerebral cortices from a group of animals exposed to MSG in parallel. Data represent the mean ^ SD of 5–6 animals per group. Measurements were made in duplicate. Differences relative to the control group were considered statistically significant at P , 0:001 (*).
the injured brain and clearly contributes, in the CNS, to the initiation of cascades associated with neuronal apoptosis and neurological impairment [22]. Previous findings using a model of neonatal exposure to monosodium glutamate (MSG) showed that both astrocytes and microglial cells are highly susceptible to the neuroexcitotoxic effects of MSG administered at the neonatal stage [17]. The increase in the astrocytic population in the cortex may be a consequence of early proliferation (hyperplasia). However, it is unknown whether the pro-inflammatory cytokines, such as TNF-a, have a significant role in neuronal death in the cerebral cortex in this model of neuroexcitotoxic damage. An ability to anticipate early neuronal damage could be important in avoiding further neurological and/or neurodegenerative processes. Newborn male Wistar rats from our colony were injected subcutaneously with 4 mg/g body weight MSG at 1, 3, 5, and 7 days after birth [17]. The animals were maintained in a 12/12 h light/dark cycle at 22 ^ 2 8C and 55% relative ambient humidity, with free access to balanced food (Ralston Rations, Ralston Purina, USA) and water. Animals were studied at; 8, 10, or 14 days after birth. Control and experimental groups of rats were used (n ¼ 5–6). Both experimental and control group were
using the same cortical regions for morphological analyses. These rats were perfused intracardially with 4% paraformaldehyde in phosphate-buffered saline (PBS), pH ¼ 7.2. Brains were paraffin-embedded, and coronal slices (8 mm) of motor frontal cortex of each control and those injected with MSG, 200 microscopic fields randomly were examined at 100 £ enlargement (1.6 mm 2 of tissue), through the complete cortical thickness with coordinate interaural 10.7 mm and bregma 1.7 mm (Fr1, plate 11 of Paxino’s). The cells were hematoxylin-eosin stained, analyzed and counted with double-blinded observer as described previously by Martı´nez-Contreras et al. [17]. The other group, used for biochemical analysis, was sacrificed by decapitation and the brains were maintained on ice (220 8C) until used. For the TNF-a assay, the tissue (frontal cortex) was homogenized in 500 ml PBS/Protease Inhibitor Cocktail (Calbiochem-Novabiochem, San Diego, CA) per 50 mg tissue weight. The samples were centrifuged at 4 8C for 20 min at 10 500 £ g and the supernatant was collected. Protein expression was evaluated using an enzyme-linked immunosorbent assay (ELISA) kit, Quantikine M Rat TNF-a (R&D Systems, Minneapolis, MN). Absorbances were measured at 450 nm with correction to 540 or 570 nm, using a Bio-Rad Microplate Reader Model 550. Tissue (frontal cortex)
Fig. 2. Representative microphotographs of serial slices from the cerebral cortices of rats at different postnatal ages. (A) Control group at postnatal day 8. (C) Control group at postnatal day 10, and (E) control group at postnatal day 14. (B,D and F) correspond to the treatment group. Paraffin-embedded slices, 8 mm thick, were stained with hematoxylin-eosin, and analyzed using optical microscopy. Neurons with shrinkage (bold arrow), membrane blebbing (clear arrow), nuclear pyknosis (arrow head), and chromatin condensation (*). Magnification ¼ 100 £ .
V. Chaparro-Huerta et al. / Neuroscience Letters 333 (2002) 95–98
Fig. 3. (A) Time–response curve for TNF-a levels during MSG treatment. TNF-a levels were measured by ELISA every 6 h after each MSG dose until day 9. Each point represents the mean ^ SD of 2–3 animals per group and measurements were made in duplicate. (B) TNF-a levels at postnatal days 8, 10, and 14. Each point represents the mean ^ SD of 5–6 animals per group and measurements were made in duplicate. Statistically different relative to the control group at P , 0:001 (*).
obtained from another animals group was homogenized in 1.0 ml of PBS to measure lactate dehydrogenase (LDH) activity using a commercial lactate dehydrogenase kit (Sigma, St. Louis, MO). Data are represented as means ^ SEM. Statistical differences between control and experimental groups were assessed using one-way ANOVA. Differences were considered statistically significant at P , 0:05. Our results show that neonatal exposure to glutamate induced significant neuronal loss in the cerebral cortex at 8 and 10 days of age, relative to the control group (Fig. 1A). This neuronal death was confirmed by a significant increase in LDH activity at the same ages (Fig. 1B). Morphological changes were characterized by cell shrinkage, membrane blebbing, nuclear pyknosis, and early chromatin condensation (Fig. 2B,D, and F) compared with the control group (Fig. 2A,C, and E). A time–response profile for TNF-a expression was determined 6 h after each dose of glutamate, during the first 9 days after birth (Fig. 3A). Importantly, levels of TNF-a were higher than those of the control group 24 h after the last glutamate dose (8 days old) (Fig. 3A,B). However, TNF-a levels continued to increase until day 14 of the study (Fig. 3B).
97
Programmed cell death (apoptosis) is now recognized as a fundamental biological process under normal physiological conditions, but under pathological conditions, there can be a failure to control the extent of cell death. Apoptosis acquires importance in some neurodegenerative diseases. In this study, significant neuronal loss was observed under normal conditions in the cerebral cortex during brain development (Fig. 1A). This process was accelerated by early exposure to high glutamate (Glu) concentrations, and was accompanied by the morphological characteristics of cellular injury (Fig. 2). However, at 14 days, no difference in that neuronal loss was observed. It may be due to a neuronal migration delayed by that excessive Glu concentration since it had been documented that at this age all cortical layers have reached their adult features [23]. This cell loss may occur via an apoptosis-like mechanism directed selectively against neurons expressing Glu receptors, mainly the N-methyl-D-aspartate (NMDA)-type receptors, because it is well known that Glu excitotoxicity is mediated through the activation of NMDAgated ion channels in several neurodegenerative diseases and in ischemic stroke [15,16]. These NMDA receptors are chiefly expressed from the embryonic to the neonatal developmental stages in rodents [19]. The neurotoxicity initiated by overstimulation of Glu receptors and the subsequent influx of free Ca 11 leads to an intracellular cascade of cytotoxic events, which are necessary to trigger delayed neuronal cell death [11,12]. Furthermore, NMDA-receptor activation results in an extracellular regulation of signal kinases (extracellular-regulated kinases (ERK)1/ERK2), which appear to be involved in the excitotoxicity and neuronal apoptosis-like death induced by Glu [14]. However, aamino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors may be another mechanism, by which these neurotoxic events are induced, because AMPA receptors mediate excitotoxicity in neocortical neurons at a later age (12 days) in vitro [13]. Therefore, in this in vivo model, the ages of the neonates in which neurotoxicity was induced by early Glu exposure suggest predominantly NMDAreceptor activation, rather than the activation of AMPA receptors, which react to Glu in a later phase of postnatal development. Glial cells provide mechanical and metabolic support for neurons by producing and responding to a variety of stimuli [3]. Therefore, activated glial cells produce diverse inflammatory mediators such as TNF-a [21]. There is growing evidence that toxic mediators produced by glia, astrocytes, and microglia, are involved in the pathogenesis of various neurodegenerative diseases [4]. In a previous study, significant astrocytic and microglia reactivity in the cerebral cortices of 60 days old rats was induced by the neonatal administration of Glu [17]. This reactivity was manifested by an increase in the number, hypertrophy and its complexity of the cytoplasm extension in both cells with high expression of mRNA to vimentin. This suggests that glial cell reactivity persists into adulthood in response to an early neuroexcitotoxic stimulus with Glu. For this reason, the
98
V. Chaparro-Huerta et al. / Neuroscience Letters 333 (2002) 95–98
TNF-a time–response profile after Glu exposure was evaluated in this study. The increase in TNF-a levels may indicate the presence of a feedback mechanism to induce cell death via apoptosis. Exposure of cells to TNF-a results in the activation of caspases, which leads to apoptosis, as well as to other signaling pathways that include transcription factor activation protein (AP-1), nuclear factor (NF)kappaB and mitogen-activated protein kinases [3]. Therefore, further studies are required to ascertain whether the cell death observed under our experimental conditions occurs via apoptosis only, or together with necrosis, or as a hybrid form somewhere along the apoptosis–necrosis continuous, as suggested by Martin et al. [16]. This work was partially supported by CONACyT grants no. 30901-M and 158761 to C.B.Z. and V.CH.H respectively. It constitutes part of the doctoral thesis of V.CH.H. prepared at the Universidad de Guadalajara (CUCS).
[11]
[12]
[13]
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