- Email: [email protected]
Microglial activation and tyrosine hydroxylase immunoreactivity in the substantia nigral region following transient focal ischemia in rats
Neuroscience Letters 349 (2003) 63–67 www.elsevier.com/locate/neulet
Microglial activation and tyrosine hydroxylase immunoreactivity in the substantia nigral region following transient focal ischemia in rats Youngbuhm Huha, Ji Wook Jungb, Chan Parka, Jae Ryun Ryuc, Chan Young Shinc, Won-Ki Kimd, Jong Hoon Ryub,* a
Department of Anatomy, College of Medicine, College of Pharmacy, Kyung Hee University, 1 Hoeki-dong, Dongdeamoon-ku, Seoul 130-701, South Korea Department of Oriental Pharmaceutical Science, College of Pharmacy, Kyung Hee University, 1 Hoeki-dong, Dongdeamoon-ku, Seoul 130-701, South Korea c Department of Pharmacology, College of Pharmacy, Seoul National University, Seoul, South Korea d Department of Pharmacology, College of Medicine, Ewha Women’s University, Seoul, South Korea
b
Received 4 April 2003; received in revised form 13 June 2003; accepted 18 June 2003
Abstract The temporal profiles of the changes of dopaminergic cells and microglial activation induced by transient cerebral ischemia were investigated in the substantia nigra pars compacta (SNc) located outside ischemic areas of rat brain. Transient cerebral ischemia was induced by intraluminal occlusion of the right middle cerebral artery for 2 h and reperfusion was continued for 1, 2, 3, 4, 7, 10, 14, 28, 60, and 120 days. Dopaminergic cells immunostained with tyrosine hydroxylase (TH)-antibody in the ipsilateral SNc were significantly decreased at 7 days postischemia compared with those in the contralateral side (P , 0:05). However, at 60 and 120 days, there were no significant differences between ipsilateral and contralateral side of the SNc. Unlike the TH immunoreactivity, activated microglial cells immunostained with OX-42 antibody were significantly increased at 2 and 3 days and then decreased gradually until 10 days post-ischemia. Activated microglial cells were increased at 2 weeks post-ischemia, and this pattern remained until 60 days. These results suggest that the transient changes of TH-immunoreactive cells in the SNc caused by transient focal ischemia are correlated with a biphasic microglial cell activation response. q 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Ischemia; Substantia nigra; Tyrosine hydroxylase; Microglia; Remote area
Transient cerebral ischemia leads to neuronal damage in fronto-parietal cortex and striatum that are supplied by the middle cerebral artery. Furthermore, delayed neuronal damage has been reported in the ipsilateral thalamus and in the substantia nigra which lay outside ischemic areas of the rat brain after middle cerebral artery occlusion (MCAO) [5,12]. Neuronal damage in the ipsilateral substantia nigra is observed after striatal injury both in humans and in animals. Several lines of evidence show that lesions in the striatum cause a loss of dopaminergic neurons in the substantia nigra pars compacta (SNc) [10,13]. However, the mechanisms of these phenomena in remote areas are not well understood. Microglia, the resident immune cells in the brain, play a role in immune surveillance under normal conditions. However, they are rapidly activated in response to pathological stimuli, such as ischemic insult. During the *
Corresponding author. Tel.: þ 82-2-961-9230; fax: þ82-2-966-3885. E-mail address: [email protected] (J.H. Ryu).
early phase of activation resting microglial cells undergo morphological changes. Recently, Gao et al. reported that microglial activation induced by chronic exposure to lipopolysaccharide results in a delayed and progressive degeneration of nigral dopaminergic neurons [6]. These results suggest that microglial activation induced by a transient ischemic insult may cause a decrease in tyrosine hydroxylase (TH) immunoreactivity. On the other hand, neurotrophic factors, such as brain-derived neurotrophic factors (BDNF), glial cell line-derived neurotrophic factor (GDNF), are produced and secreted by the activated microglia [2]. Those factors increase neuronal survival in response to neuronal injury. Consequently, we believed that the correlation between microglial activation and TH immunoreactivity may be useful in the elucidation of the mechanism of remote cell death caused by transient ischemic insult. In the present study, we examined the effects of transient ischemia on TH immunoreactivity and microglial activation
0304-3940/03/$ - see front matter q 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/S0304-3940(03)00743-2
64
Y. Huh et al. / Neuroscience Letters 349 (2003) 63–67
Fig. 1. The temporal profile of the TH-immunoreactive cells in the SNc is shown. Each point represents the percentage changes in the number of THimmunoreactive cells on the ipsilateral SNc from those of the contralateral side. TH-immunoreactive cells were counted using a brightfield microscope as described in the text. Values are means ^ SEM for four rats of each time point. *P , 0:05 compared with values at Day 1 after ischemia (analysis of variance, ANOVA followed by Duncan’s test).
in the SNc at 1, 2, 3, 4, 7, 10, 14, 28, 60, and 120 days (referred to throughout as Day 1, 2, 3, …, etc.) following transient focal cerebral ischemia in the rat by immunohistochemistry and cell counting. Male Sprague –Dawley rats, weighing 260 – 270 g, were purchased from the Biogenomics Co., Ltd. of Charles River Branch (Seoul, South Korea). The rats were housed 4 or 5 per cage, allowed free access to water and food, and maintained under constant temperature (23 ^ 18C) and humidity (60 ^ 10%) under a 12-h light/dark cycle (light on 07:30 – 19:30 h). Animals were anesthetized in a chamber with a mixture of N2O and O2 (70:30) containing 2.5% isoflurane. MCA was occluded according to the method of Nagasawa and
Fig. 2. The temporal profile of the OX-42-positive cells in the ipsilateral substantia nigra pars compacta is shown. OX-42-positive cells were counted using a brightfield microscope as described in text. Values are means ^ SEM for four rats of each time points. *P , 0:05 compared with values on Day 1 after ischemia (ANOVA followed by Duncan’s test).
Kogure [8], with minor modifications. Briefly, after making a median incision in the neck skin, the right common carotid artery was exposed and a 17 mm 4-0 nylon thread with a rounded tip (coated with silicon) was inserted from the bifurcation to the right MCA. After 2 h of MCA occlusion, the thread was removed to allow complete reperfusion of the ischemic area under re-anesthesia. Neurological deficits characterized by severe left-sided hemiparesis and right Horner’s syndrome were used as criteria for evaluating the severity of the ischemic insult. Body temperature was maintained at 37 ^ 0.58C throughout surgery by a heating pad (Biomed S.L., Spain). At pre-designated times points selected for observation after transient focal ischemia (i.e. Days 1, 2, 3, 4, 7, 10, 14, 28, 60, and 120), rats were anesthetized with pentobarbital (50 mg/kg), and perfused with heparinized saline from the heart. Animals were subjected to perfusion fixation with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) and decapitated. Excised brains were immersed in the same fixative solution for overnight at 48C and then transferred to 20% sucrose in phosphate-buffered saline (pH 7.4) where they were kept for 48 h at 48C. Frozen sections (40 mm) were prepared in the coronal plane using a cryostat. The immunohistochemical staining for TH and OX-42 was carried out using a mouse monoclonal anti-TH antibody (1:1000 dilution, Roche Diagnostic GmbH, Mannheim, Germany), or mouse monoclonal anti-CD11b antibody (OX-42, 1:5000 dilution, Serotec Ltd., Oxford, UK) by free floating avidin-biotin-peroxidase complex (ABC) method (Vectastain ABC kit, Vector, Burlingame, CA, USA) as described previously [7]. To quantify TH- or OX-42-positive cells, four ischemiainduced rats and two sham-operated rats were analyzed at the designated times after ischemia. Each cell was counted under a brightfield microscope (Olympus, Japan). Three tissue sections were then used for each animal and averaged to yield a final result. In the case of OX-42 immunostaining, double staining with cresyl violet was performed to locate the SNc region, because it was difficult to identify the border of SNc from the SN. Also, the number of cresyl violet stained neurons was counted in these nigral sections. The number of TH immunoreactive neurons was counted in contralateral and ipsilateral side of the SN. As shown in Fig. 1, TH-positive cells in the ipsilateral SNc were unchanged at Days 1, 2, 3, and 4 compared with those in the contralateral SNc. However, at Day 7, a significant decrease in the number of TH-positive cells was observed (P , 0:05), and this was sustained until Day 28. This reduction recovered to the contralateral level at Day 60. However, the number of TH-positive cells in the contralateral side of substantia nigral region were unchanged throughout. Unlike TH immunoreactivity, activated microglial cells immunostained with the OX-42 antibody were not observed at Day 1 in the ipsilateral SNc (Fig. 2). At Day 2, OX-42immunoreactive microglia in the ipsilateral SNc was
Y. Huh et al. / Neuroscience Letters 349 (2003) 63–67
65
Fig. 3. OX-42-immunoreactivity in the contralateral substantia nigra pars compacta (A); and in the ipsilateral side (B) after 22 h of reperfusion after 120 min of ischemia, 3 days of reperfusion (C and D); 10 days of reperfusion (E and F); and 14 days of reperfusion (G and H). The photographs in the left column are of the contralateral side and those in the right column of the ipsilateral side. The bar represents 50 mm.
changed their shape to rod-like from ramified morphology that was observed at resting microglia (Fig. 3D). As shown in Fig. 2, significant increases in the activated microglial cells were detected from Days 2 to 60. A marked increase in the number of activated microglial cells was observed from
Days 2 to 4 and this gradually decreased from Day 7 in the ipsilateral side of the SNc region. At 2 weeks, however, a secondary increase in the number of activated microglia cells was observed, though this was not as dramatic as the first increase (Figs. 2 and 3H). However, at Day 60 but not at
66
Y. Huh et al. / Neuroscience Letters 349 (2003) 63–67
Day 120, OX-42-immunoreactive microglial cells appeared in this region. No fully activated amoeboid-like microglia were observed at any time. In the contralateral side of the SN region, only scattered OX-42-immunoreactivity was found. Lesions in the striatum caused by transient forebrain ischemia induce progressive TH-positive neuron loss in the ipsilateral SNc [13]. In our studies, TH-positive neurons were unaltered up to Day 4, but were significantly decreased in the ipsilateral SNc at Day 7. Soriano et al. reported that transient focal cerebral ischemia induced a significant reduction of TH-immunoreactive cells in the ipsilateral SN at 7 and 14 days after ischemia [11]. They also reported that the reduction of TH-immunoreactivity recovered at 30 days and recovered completely at 60 days after ischemia. We also observed the recovery of THimmunoreactive neurons at Day 60 and thereafter. In this study, we did not observe the significant decrease in the number of cresyl violet stained cells in the ipsilateral SNc compared with those in the contralateral side (data not shown). In agreement with our result, Soriano et al. also demonstrated that the total cell number within the ipsilateral SNc was not significantly different from contralateral SNc [11]. These results and our findings show that TH-immunoreactive cells decrease transiently and recover completely about 2 months after ischemia. The reasons why TH-immunoreactivity in the SNc transiently decreases are unknown. Microglial cells are rapidly activated in response to pathological stimuli, including ischemic insult. Moreover, microglial activation has been proposed to be both detrimental and beneficial in certain neurodegenerative disorders [2,9]. As shown in Figs. 2 and 3, the number of activated microglial cells increased until Day 4, and thereafter decreased gradually until Day 10. At Day 14, however, a second increase in the numbers of activated microglia cells was observed. Therefore, microglial cells were found to respond to transient focal ischemia in a biphasic manner in the SNc. If microglia activated by transient focal ischemia in the substantia nigra secrete neurotoxic materials, then the number of dopaminergic neurons will be reduced in this region. However, as mentioned above, TH-immunoreactive cells in the ipsilateral SNc were transiently reduced in number and recovered to the level in the contralateral side at Day 60. Recent biochemical and neurobiological studies have revealed that activated microglia produces and secretes not only cytotoxic molecules but also neurotrophic molecules including neurotrophins and neurotrophic cytokines [9]. Aliaga et al. suggested that BDNF released by activated microglial cells might play a role in the survival of dopaminergic neuronal death [1]. Reduction of microglial recruitment following ischemia was reported to increase the infarct size in the colony stimulating factor-1 knock-out mouse [3]. These reports suggest that microglial activation is beneficial effects to the survival of dopaminergic neuronal
cells in the case of some chemical or physical injuries, such as ischemic insults. Interestingly, we found that biphasic phenomena existed in terms of the activation of microglial cells with initial peak at Days 2 –4 and a second peak at Days 14 – 60. Finkelstein et al. reported similar results in their preliminary studies [4]. They suggested that glial response to injury was biphasic with an initial peak correlating to inflammation and a second later peak associated with supporting regenerating fibers. These findings suggest that any materials secreted by the activated microglia did not resulted in the dopaminergic cell death in the transient focal ischemia induced by MCAO. However, further studies are required to clarify this issue. The mechanism by which recovery of TH-immunoreactivity in the SNc is induced is unclear, but as outlined above, the available evidences suggest that neurotrophic factors, in particular BDNF and GDNF, play an important role in the survival of nigral dopaminergic neurons. Based on these results, we suggest that microglia activated by transient focal ischemia in the SNc may be neurotoxic to dopaminergic neurons, and the other side neuroprotective to the dopaminergic neurons.
Acknowledgements This study was supported by a Korea Research Foundation Grant (2000-015-DP0353).
References [1] E. Aliaga, C. Carcamo, J. Abarca, L. Tapia-Arancibia, G. Bustos, Transient increase of brain derived neurotrophic factor mRNA expression in substantia nigra reticulata after partial lesion of the nigrostriatal dopaminergic pathway, Mol. Brain Res. 79 (2002) 150 –155. [2] P.E. Batchelor, G.T. Liberatore, J.Y. Wong, M.J. Porritt, F. Frerichs, G.A. Donnan, D.W. Howells, Activated macrophages and microglia induce dopaminergic sprouting in the injured striatum and express brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor, J. Neurosci. 19 (1999) 1708–1716. [3] S. Fedoroff, O. Berezovskaya, D. Maysinger, Role of colony stimulating factor-1 in brain damage caused by ischemia, Neurosci. Biobehav. Rev. 21 (1997) 187–191. [4] D.I. Finkelstein, C.L. Parish, D. Stanic, E. Borrelli, J. Drago, M.K. Horne, The role of dopamine receptors in regulating the size of terminal arbours, in: L.F.B. Nicholson (Ed.), International Basal Ganglia Society VII, Plenum, New York, 2001. [5] W. Fujie, T. Kirino, N. Tomukai, T. Iwasawa, A. Tamura, Progressive shrinkage of the thalamus following middle cerebral artery occlusion in rats, Stroke 21 (1990) 1485–1488. [6] H.M. Gao, J. Jiang, B. Wilson, W. Zhang, J.S. Hong, B. Liu, Microglial activation-mediated delayed and progressive degeneration of rat nigral dopaminergic neurons: relevance to Parkinson’s disease, J. Neurochem. 81 (2002) 1285–1297. [7] J. Lee, K. Park, S. Lee, K. Whang, M. Kang, C. Park, Y. Huh, Differential changes of calcium binding proteins in the rat striatum after kainic acid-induced seizure, Neurosci. Lett. 333 (2002) 87– 90. [8] H. Nagasawa, K. Kogure, Correlation between cerebral blood flow
Y. Huh et al. / Neuroscience Letters 349 (2003) 63–67 and histologic changes in a new rat model of middle cerebral artery occlusion, Stroke 20 (1989) 1037–1043. [9] Y. Nakamura, Regulating factors for microglial activation, Biol. Pharm. Bull. 25 (2002) 945 –953. [10] J.H. Ryu, K. Yanai, R. Iwata, T. Ido, T. Watanabe, Heterogeneous distributions of histamine H3, dopamine D1 and D2 receptors in rat brain, NeuroReport 5 (1994) 621–624. [11] M.A. Soriano, C. Justicia, I. Ferrer, E. Rodriguez-Farre, A.M. Planas, Striatal infarction in the rat causes a transient reduction of tyrosine
67
hydroxylase immunoreactivity in the ipsilateral substantia nigra, Neurobiol. Dis. 4 (1997) 376 –385. [12] A. Tamura, T. Kirino, K. Sano, K. Takagi, H. Oka, Atrophy of the ipsilateral substantia nigra following middle cerebral artery occlusion in the rat, Brain Res. 510 (1990) 154–157. [13] B.T. Volpe, A.D. Blau, T.C. Wessel, M. Saji, Delayed histopathological neuronal damage in the substantia nigra compacta (nucleus A9) after transient forebrain ischaemia, Neurobiol. Dis. 2 (1995) 119–127.
Microglial activation and tyrosine hydroxylase immunoreactivity in the substantia nigral region following transient focal ischemia in rats Youngbuhm Huha, Ji Wook Jungb, Chan Parka, Jae Ryun Ryuc, Chan Young Shinc, Won-Ki Kimd, Jong Hoon Ryub,* a
Department of Anatomy, College of Medicine, College of Pharmacy, Kyung Hee University, 1 Hoeki-dong, Dongdeamoon-ku, Seoul 130-701, South Korea Department of Oriental Pharmaceutical Science, College of Pharmacy, Kyung Hee University, 1 Hoeki-dong, Dongdeamoon-ku, Seoul 130-701, South Korea c Department of Pharmacology, College of Pharmacy, Seoul National University, Seoul, South Korea d Department of Pharmacology, College of Medicine, Ewha Women’s University, Seoul, South Korea
b
Received 4 April 2003; received in revised form 13 June 2003; accepted 18 June 2003
Abstract The temporal profiles of the changes of dopaminergic cells and microglial activation induced by transient cerebral ischemia were investigated in the substantia nigra pars compacta (SNc) located outside ischemic areas of rat brain. Transient cerebral ischemia was induced by intraluminal occlusion of the right middle cerebral artery for 2 h and reperfusion was continued for 1, 2, 3, 4, 7, 10, 14, 28, 60, and 120 days. Dopaminergic cells immunostained with tyrosine hydroxylase (TH)-antibody in the ipsilateral SNc were significantly decreased at 7 days postischemia compared with those in the contralateral side (P , 0:05). However, at 60 and 120 days, there were no significant differences between ipsilateral and contralateral side of the SNc. Unlike the TH immunoreactivity, activated microglial cells immunostained with OX-42 antibody were significantly increased at 2 and 3 days and then decreased gradually until 10 days post-ischemia. Activated microglial cells were increased at 2 weeks post-ischemia, and this pattern remained until 60 days. These results suggest that the transient changes of TH-immunoreactive cells in the SNc caused by transient focal ischemia are correlated with a biphasic microglial cell activation response. q 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Ischemia; Substantia nigra; Tyrosine hydroxylase; Microglia; Remote area
Transient cerebral ischemia leads to neuronal damage in fronto-parietal cortex and striatum that are supplied by the middle cerebral artery. Furthermore, delayed neuronal damage has been reported in the ipsilateral thalamus and in the substantia nigra which lay outside ischemic areas of the rat brain after middle cerebral artery occlusion (MCAO) [5,12]. Neuronal damage in the ipsilateral substantia nigra is observed after striatal injury both in humans and in animals. Several lines of evidence show that lesions in the striatum cause a loss of dopaminergic neurons in the substantia nigra pars compacta (SNc) [10,13]. However, the mechanisms of these phenomena in remote areas are not well understood. Microglia, the resident immune cells in the brain, play a role in immune surveillance under normal conditions. However, they are rapidly activated in response to pathological stimuli, such as ischemic insult. During the *
Corresponding author. Tel.: þ 82-2-961-9230; fax: þ82-2-966-3885. E-mail address: [email protected] (J.H. Ryu).
early phase of activation resting microglial cells undergo morphological changes. Recently, Gao et al. reported that microglial activation induced by chronic exposure to lipopolysaccharide results in a delayed and progressive degeneration of nigral dopaminergic neurons [6]. These results suggest that microglial activation induced by a transient ischemic insult may cause a decrease in tyrosine hydroxylase (TH) immunoreactivity. On the other hand, neurotrophic factors, such as brain-derived neurotrophic factors (BDNF), glial cell line-derived neurotrophic factor (GDNF), are produced and secreted by the activated microglia [2]. Those factors increase neuronal survival in response to neuronal injury. Consequently, we believed that the correlation between microglial activation and TH immunoreactivity may be useful in the elucidation of the mechanism of remote cell death caused by transient ischemic insult. In the present study, we examined the effects of transient ischemia on TH immunoreactivity and microglial activation
0304-3940/03/$ - see front matter q 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/S0304-3940(03)00743-2
64
Y. Huh et al. / Neuroscience Letters 349 (2003) 63–67
Fig. 1. The temporal profile of the TH-immunoreactive cells in the SNc is shown. Each point represents the percentage changes in the number of THimmunoreactive cells on the ipsilateral SNc from those of the contralateral side. TH-immunoreactive cells were counted using a brightfield microscope as described in the text. Values are means ^ SEM for four rats of each time point. *P , 0:05 compared with values at Day 1 after ischemia (analysis of variance, ANOVA followed by Duncan’s test).
in the SNc at 1, 2, 3, 4, 7, 10, 14, 28, 60, and 120 days (referred to throughout as Day 1, 2, 3, …, etc.) following transient focal cerebral ischemia in the rat by immunohistochemistry and cell counting. Male Sprague –Dawley rats, weighing 260 – 270 g, were purchased from the Biogenomics Co., Ltd. of Charles River Branch (Seoul, South Korea). The rats were housed 4 or 5 per cage, allowed free access to water and food, and maintained under constant temperature (23 ^ 18C) and humidity (60 ^ 10%) under a 12-h light/dark cycle (light on 07:30 – 19:30 h). Animals were anesthetized in a chamber with a mixture of N2O and O2 (70:30) containing 2.5% isoflurane. MCA was occluded according to the method of Nagasawa and
Fig. 2. The temporal profile of the OX-42-positive cells in the ipsilateral substantia nigra pars compacta is shown. OX-42-positive cells were counted using a brightfield microscope as described in text. Values are means ^ SEM for four rats of each time points. *P , 0:05 compared with values on Day 1 after ischemia (ANOVA followed by Duncan’s test).
Kogure [8], with minor modifications. Briefly, after making a median incision in the neck skin, the right common carotid artery was exposed and a 17 mm 4-0 nylon thread with a rounded tip (coated with silicon) was inserted from the bifurcation to the right MCA. After 2 h of MCA occlusion, the thread was removed to allow complete reperfusion of the ischemic area under re-anesthesia. Neurological deficits characterized by severe left-sided hemiparesis and right Horner’s syndrome were used as criteria for evaluating the severity of the ischemic insult. Body temperature was maintained at 37 ^ 0.58C throughout surgery by a heating pad (Biomed S.L., Spain). At pre-designated times points selected for observation after transient focal ischemia (i.e. Days 1, 2, 3, 4, 7, 10, 14, 28, 60, and 120), rats were anesthetized with pentobarbital (50 mg/kg), and perfused with heparinized saline from the heart. Animals were subjected to perfusion fixation with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) and decapitated. Excised brains were immersed in the same fixative solution for overnight at 48C and then transferred to 20% sucrose in phosphate-buffered saline (pH 7.4) where they were kept for 48 h at 48C. Frozen sections (40 mm) were prepared in the coronal plane using a cryostat. The immunohistochemical staining for TH and OX-42 was carried out using a mouse monoclonal anti-TH antibody (1:1000 dilution, Roche Diagnostic GmbH, Mannheim, Germany), or mouse monoclonal anti-CD11b antibody (OX-42, 1:5000 dilution, Serotec Ltd., Oxford, UK) by free floating avidin-biotin-peroxidase complex (ABC) method (Vectastain ABC kit, Vector, Burlingame, CA, USA) as described previously [7]. To quantify TH- or OX-42-positive cells, four ischemiainduced rats and two sham-operated rats were analyzed at the designated times after ischemia. Each cell was counted under a brightfield microscope (Olympus, Japan). Three tissue sections were then used for each animal and averaged to yield a final result. In the case of OX-42 immunostaining, double staining with cresyl violet was performed to locate the SNc region, because it was difficult to identify the border of SNc from the SN. Also, the number of cresyl violet stained neurons was counted in these nigral sections. The number of TH immunoreactive neurons was counted in contralateral and ipsilateral side of the SN. As shown in Fig. 1, TH-positive cells in the ipsilateral SNc were unchanged at Days 1, 2, 3, and 4 compared with those in the contralateral SNc. However, at Day 7, a significant decrease in the number of TH-positive cells was observed (P , 0:05), and this was sustained until Day 28. This reduction recovered to the contralateral level at Day 60. However, the number of TH-positive cells in the contralateral side of substantia nigral region were unchanged throughout. Unlike TH immunoreactivity, activated microglial cells immunostained with the OX-42 antibody were not observed at Day 1 in the ipsilateral SNc (Fig. 2). At Day 2, OX-42immunoreactive microglia in the ipsilateral SNc was
Y. Huh et al. / Neuroscience Letters 349 (2003) 63–67
65
Fig. 3. OX-42-immunoreactivity in the contralateral substantia nigra pars compacta (A); and in the ipsilateral side (B) after 22 h of reperfusion after 120 min of ischemia, 3 days of reperfusion (C and D); 10 days of reperfusion (E and F); and 14 days of reperfusion (G and H). The photographs in the left column are of the contralateral side and those in the right column of the ipsilateral side. The bar represents 50 mm.
changed their shape to rod-like from ramified morphology that was observed at resting microglia (Fig. 3D). As shown in Fig. 2, significant increases in the activated microglial cells were detected from Days 2 to 60. A marked increase in the number of activated microglial cells was observed from
Days 2 to 4 and this gradually decreased from Day 7 in the ipsilateral side of the SNc region. At 2 weeks, however, a secondary increase in the number of activated microglia cells was observed, though this was not as dramatic as the first increase (Figs. 2 and 3H). However, at Day 60 but not at
66
Y. Huh et al. / Neuroscience Letters 349 (2003) 63–67
Day 120, OX-42-immunoreactive microglial cells appeared in this region. No fully activated amoeboid-like microglia were observed at any time. In the contralateral side of the SN region, only scattered OX-42-immunoreactivity was found. Lesions in the striatum caused by transient forebrain ischemia induce progressive TH-positive neuron loss in the ipsilateral SNc [13]. In our studies, TH-positive neurons were unaltered up to Day 4, but were significantly decreased in the ipsilateral SNc at Day 7. Soriano et al. reported that transient focal cerebral ischemia induced a significant reduction of TH-immunoreactive cells in the ipsilateral SN at 7 and 14 days after ischemia [11]. They also reported that the reduction of TH-immunoreactivity recovered at 30 days and recovered completely at 60 days after ischemia. We also observed the recovery of THimmunoreactive neurons at Day 60 and thereafter. In this study, we did not observe the significant decrease in the number of cresyl violet stained cells in the ipsilateral SNc compared with those in the contralateral side (data not shown). In agreement with our result, Soriano et al. also demonstrated that the total cell number within the ipsilateral SNc was not significantly different from contralateral SNc [11]. These results and our findings show that TH-immunoreactive cells decrease transiently and recover completely about 2 months after ischemia. The reasons why TH-immunoreactivity in the SNc transiently decreases are unknown. Microglial cells are rapidly activated in response to pathological stimuli, including ischemic insult. Moreover, microglial activation has been proposed to be both detrimental and beneficial in certain neurodegenerative disorders [2,9]. As shown in Figs. 2 and 3, the number of activated microglial cells increased until Day 4, and thereafter decreased gradually until Day 10. At Day 14, however, a second increase in the numbers of activated microglia cells was observed. Therefore, microglial cells were found to respond to transient focal ischemia in a biphasic manner in the SNc. If microglia activated by transient focal ischemia in the substantia nigra secrete neurotoxic materials, then the number of dopaminergic neurons will be reduced in this region. However, as mentioned above, TH-immunoreactive cells in the ipsilateral SNc were transiently reduced in number and recovered to the level in the contralateral side at Day 60. Recent biochemical and neurobiological studies have revealed that activated microglia produces and secretes not only cytotoxic molecules but also neurotrophic molecules including neurotrophins and neurotrophic cytokines [9]. Aliaga et al. suggested that BDNF released by activated microglial cells might play a role in the survival of dopaminergic neuronal death [1]. Reduction of microglial recruitment following ischemia was reported to increase the infarct size in the colony stimulating factor-1 knock-out mouse [3]. These reports suggest that microglial activation is beneficial effects to the survival of dopaminergic neuronal
cells in the case of some chemical or physical injuries, such as ischemic insults. Interestingly, we found that biphasic phenomena existed in terms of the activation of microglial cells with initial peak at Days 2 –4 and a second peak at Days 14 – 60. Finkelstein et al. reported similar results in their preliminary studies [4]. They suggested that glial response to injury was biphasic with an initial peak correlating to inflammation and a second later peak associated with supporting regenerating fibers. These findings suggest that any materials secreted by the activated microglia did not resulted in the dopaminergic cell death in the transient focal ischemia induced by MCAO. However, further studies are required to clarify this issue. The mechanism by which recovery of TH-immunoreactivity in the SNc is induced is unclear, but as outlined above, the available evidences suggest that neurotrophic factors, in particular BDNF and GDNF, play an important role in the survival of nigral dopaminergic neurons. Based on these results, we suggest that microglia activated by transient focal ischemia in the SNc may be neurotoxic to dopaminergic neurons, and the other side neuroprotective to the dopaminergic neurons.
Acknowledgements This study was supported by a Korea Research Foundation Grant (2000-015-DP0353).
References [1] E. Aliaga, C. Carcamo, J. Abarca, L. Tapia-Arancibia, G. Bustos, Transient increase of brain derived neurotrophic factor mRNA expression in substantia nigra reticulata after partial lesion of the nigrostriatal dopaminergic pathway, Mol. Brain Res. 79 (2002) 150 –155. [2] P.E. Batchelor, G.T. Liberatore, J.Y. Wong, M.J. Porritt, F. Frerichs, G.A. Donnan, D.W. Howells, Activated macrophages and microglia induce dopaminergic sprouting in the injured striatum and express brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor, J. Neurosci. 19 (1999) 1708–1716. [3] S. Fedoroff, O. Berezovskaya, D. Maysinger, Role of colony stimulating factor-1 in brain damage caused by ischemia, Neurosci. Biobehav. Rev. 21 (1997) 187–191. [4] D.I. Finkelstein, C.L. Parish, D. Stanic, E. Borrelli, J. Drago, M.K. Horne, The role of dopamine receptors in regulating the size of terminal arbours, in: L.F.B. Nicholson (Ed.), International Basal Ganglia Society VII, Plenum, New York, 2001. [5] W. Fujie, T. Kirino, N. Tomukai, T. Iwasawa, A. Tamura, Progressive shrinkage of the thalamus following middle cerebral artery occlusion in rats, Stroke 21 (1990) 1485–1488. [6] H.M. Gao, J. Jiang, B. Wilson, W. Zhang, J.S. Hong, B. Liu, Microglial activation-mediated delayed and progressive degeneration of rat nigral dopaminergic neurons: relevance to Parkinson’s disease, J. Neurochem. 81 (2002) 1285–1297. [7] J. Lee, K. Park, S. Lee, K. Whang, M. Kang, C. Park, Y. Huh, Differential changes of calcium binding proteins in the rat striatum after kainic acid-induced seizure, Neurosci. Lett. 333 (2002) 87– 90. [8] H. Nagasawa, K. Kogure, Correlation between cerebral blood flow
Y. Huh et al. / Neuroscience Letters 349 (2003) 63–67 and histologic changes in a new rat model of middle cerebral artery occlusion, Stroke 20 (1989) 1037–1043. [9] Y. Nakamura, Regulating factors for microglial activation, Biol. Pharm. Bull. 25 (2002) 945 –953. [10] J.H. Ryu, K. Yanai, R. Iwata, T. Ido, T. Watanabe, Heterogeneous distributions of histamine H3, dopamine D1 and D2 receptors in rat brain, NeuroReport 5 (1994) 621–624. [11] M.A. Soriano, C. Justicia, I. Ferrer, E. Rodriguez-Farre, A.M. Planas, Striatal infarction in the rat causes a transient reduction of tyrosine
67
hydroxylase immunoreactivity in the ipsilateral substantia nigra, Neurobiol. Dis. 4 (1997) 376 –385. [12] A. Tamura, T. Kirino, K. Sano, K. Takagi, H. Oka, Atrophy of the ipsilateral substantia nigra following middle cerebral artery occlusion in the rat, Brain Res. 510 (1990) 154–157. [13] B.T. Volpe, A.D. Blau, T.C. Wessel, M. Saji, Delayed histopathological neuronal damage in the substantia nigra compacta (nucleus A9) after transient forebrain ischaemia, Neurobiol. Dis. 2 (1995) 119–127.