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Permanent focal cerebral ischemia activates erythropoietin receptor in the neonatal rat brain
Neuroscience Letters 355 (2004) 165–168 www.elsevier.com/locate/neulet
Permanent focal cerebral ischemia activates erythropoietin receptor in the neonatal rat brain Tong-Chun Wen, Marta Rogido, Tom Genetta, Augusto Sola* Division of Neonatal-Perinatal Medicine, Department of Pediatrics, Emory University School of Medicine, 1462 Clifton Road, Atlanta, GA 30322, USA Received 17 September 2003; received in revised form 14 October 2003; accepted 23 October 2003
Abstract Erythropoietin (Epo) has been shown to act as a neurotrophic and neuroprotective factor via binding to its receptor (EpoR) which is activated in adult brains following hypoxia and ischemia. However, no evidence suggests that cerebral ischemia can activate EpoR in the neonatal brain. In the present study, the changes in EpoR expression were investigated using a modified model of permanent focal cerebral ischemia (FCI) in 7-day-old rat pups. Western blot analysis with an anti-rabbit EpoR antibody revealed a significant increase in the EpoR protein in the ischemic areas, starting from 6 to 12 h after FCI. Moreover, many EpoR-positive cells were detected in the ischemic areas from 12 h after FCI, and the positive cells were identified as neurons and microglia/macrophage but not astrocytes 24 h after FCI. Additionally, double staining with a red in situ apoptosis detection kit and the EpoR antibody indicated that EpoR-positive cells were in apoptotic cell death in the ischemic area. Therefore, these results suggest that EpoR is activated in the ischemic areas of neonatal rats and plays an important role in brain injury during development. q 2003 Published by Elsevier Ireland Ltd. Keywords: Erythropoietin receptor; Permanent focal cerebral ischemia; DNA fragmentation; Neonate; Rat
Erythropoietin (Epo) is a cytokine that is commonly associated with a central role in erythropoiesis [8]. However, Epo has recently been shown to exhibit neuroprotective effects in vitro and in vivo [4,6]. For example, Epo protects primary cultured neurons against glutamate, kainic acid, hypoxia, glucose deprivation or serum deprivation-induced injuries [11 –14]. In vivo, Epo attenuates cerebral ischemic damage in gerbils, rats and mice [1 – 3,5,11 – 14,16]. Although the mechanisms by which Epo acts as a neuroprotective agent are not fully understood, Epo may act via binding to its functional receptor [6,8]. Epo receptor (EpoR) is a transmembrane receptor and is present on erythroid progenitor cell surfaces [8]. On the other hand, EpoR has recently been found in many other cell types including endothelial cells, myocardiocytes, macrophages, retinal cells, cells of the adrenal cortex and medulla, as well as in the small bowel, spleen, liver, kidney and lung [10]. Moreover, several lines of evidence suggest that EpoR is also expressed in brains of rats, mice, monkeys and * Corresponding author. Tel.: þ 1-404-727-5765; fax: þ1-404-727-3236. E-mail address: [email protected] (A. Sola). 0304-3940/03/$ - see front matter q 2003 Published by Elsevier Ireland Ltd. doi:10.1016/j.neulet.2003.10.078
humans [6]. In the brain, EpoR has been detected in neurons and astrocytes of the cortex and on capillaries [3,6]. Furthermore, recent data show that EpoR is induced by hypoxia in the central nervous system, like it occurs in the peripheral tissues [6]. For example, cerebral ischemia has been reported to upregulate EpoR expression in the ischemic brains of adult mice [2]. However, little is known about its expression in the neonatal brain with focal cerebral ischemia (FCI). In the present study, we demonstrated the EpoR expression in the ipsilateral hemisphere following permanent FCI in rat pups at postnatal day 7 (P7). Time pregnant Sprague– Dawley rats (E18) were purchased from Charles River Labs Inc. (Wilmington, MA), and P7 pups were used. All animal research was approved by the Emory University Institutional Animal Care Committee and performed in accordance with NIH guidelines. To produce permanent FCI, we used a modified intraluminal catheter technique [7] to cause the middle cerebral artery (MCA) occlusion in 40 rat pups. Briefly, each pup was weighed, anesthetized with 2% isofluorane and fixed in a supine position. Under an operative
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microscope, the left common carotid artery (CCA), the external carotid artery (ECA) and the internal carotid artery (ICA) were separated carefully from the adjacent veins and nerves. A coated suture embolus with silicone resin was chosen according to the weight of the pup and inserted through a small incision that was made on the CCA near the CCA – ECA – ICA junction. The suture embolus was advanced 7 – 8 mm to occlude the MCA. During the operation, a lubricated temperature probe connected to a digital thermometer was inserted 0.5 cm rectally to continuously monitor the pup’s body temperature. The body temperature was maintained in normal range (35.0 – 36.5 8C) with an overhead-heating lamp. During recovery from anesthesia the pups were kept for 15 min in a chamber in room air with an environmental temperature of 37 8C to maintain normal body temperature and then returned to their dams. Twenty pups were sacrificed at 1, 3, 6, 12 and 24 h (n ¼ 4 for each time point) after FCI, and homogenates were obtained from different brain areas (Fig. 1A) for Western blot analysis. They were solubilized in a sample solution containing 2% sodium dodecylsulfate. An equal amount of protein (40 mg) in the homogenates was subjected to SDSPAGE, transferred to a nitrocellulose membrane and immunoblotted with an anti-rabbit EpoR antibody (Santa Cruz, USA). For quantitative evaluation, the immunoreactive bands of EpoR were subjected to densitometric analysis.
Fig. 1. (A) Schematic drawing illustrating the brain areas for the analysis of Western blot or immunostaining. IP, IC, the periphery (IP) and the center (IC) of the ischemic cortex; IS, the ischemic striatum; NIC, NIS, the nonischemic cortex (NIC) and striatum (NIS). (B– F) Immunostaining analysis of EpoR at 24 h after FCI. No EpoR-positive cells were detected in NIC (B); however, many were found in IP (C), IC (D) and IS (E) and also on the blood vessels (F). Bar, 50 mm.
For immunostaining, 20 different pups were perfused transcardially with 4% paraformaldehyde at 1, 3, 6, 12 and 24 h (n ¼ 4 for each time point) after FCI. Serial frozen sections (30 mm thick) were cut and processed for immunostaining with the EpoR antibody diluted 1:100 with 0.1 M phosphate buffered saline (PBS, pH 7.4) containing 1% normal goat serum and 0.3% Triton X-100 for 24 h at 4 8C. Following washing, the sections were incubated with fluorescein isothiocyanate (FITC)-conjugated anti-rabbit IgG (Sigma, MO). Immunostaining analysis was focused on brain areas as shown in Fig. 1A. For double immunostaining, anti-mouse neuronal nuclei (NeuN) antibody (Chemicon, CA), anti-mouse glial fibrillary acidic protein (GFAP) antibody (Sigma), or anti-rat F4/80 antibody (Serotec, Kidlington, UK) was used to identify neurons, astrocytes, or microglia/macrophages, respectively. The section was incubated with anti-EpoR/anti-NeuN, GFAP or F4/80 antibodies for 24 h at 4 8C and then treated with FITC- or tetramethylrhodamine (TRITC)conjugated anti-rabbit IgG/TRITC-conjugated anti-mouse or FITC-conjugated anti-rat IgG (Sigma) for 2 h at room temperature. Control immunoreactions excluded the incubation of the first or second antibody. In situ detection of DNA fragmentation was performed using a red in situ apoptosis detection kit (Roche, Germany). Briefly, the sections were incubated in the permeabilization solution containing 0.1% Triton X-100 and 0.1% sodium citrate and then incubated with TUNEL reaction mixture including enzyme solution (TdT) and label solution (TRITC-labeled nucleotides) in a humidified chamber for 1 h at 37 8C. After washing with PBS, the sections were incubated with the EpoR antibody and FITC-conjugated anti-rabbit IgG. EpoR immunoreactivity was first detected in the ischemic area 6 h after FCI and gradually increased up to 24 h after FCI (Fig. 1C –E). On the other hand, EpoR immunoreactivity was not detected in the non-ischemic cortex up to 24 h after FCI (Fig. 1B). As shown in Fig. 1, many EpoR-positive cells were detected in the periphery (Fig. 1C) and the center (Fig. 1D) of the ischemic cortex and in the ischemic striatum (Fig. 1E) 24 h after FCI. However, the shapes of the positive cells in the periphery were different from those in the center of the ischemic cortex. In the periphery, the EpoR-positive cells appeared morphologically unaffected (Fig. 1C). In contrast, in the center, the positive cells were constricted and shrunk (Fig. 1D). In addition, EpoR was found on some small blood vessels (Fig. 1F). Western blot showed a single weak band in the nonischemic cortex and striatum (the first lane in Fig. 2A,B), and the intensity of EpoR-positive bands began to increase in the ischemic cortex and striatum 6 h after FCI (the fourth lane in Fig. 2A,B). Densitometric analysis is shown in Fig. 2C. Since the expression of EpoR in the non-ischemic cortex (NIC) and striatum (NIS) were the same at all different times after FCI, the data for these areas, used as
T.-C. Wen et al. / Neuroscience Letters 355 (2004) 165–168
Fig. 2. Western blot analysis of EpoR. The intensity of EpoR-positive bands was increased in the ischemic cortex (ISC) and striatum (IS) at 6 h (A,B, respectively) and in the periphery (IP) of ISC (A) at 12 h after FCI. Densitometric analysis (C) showed that EpoR protein was significantly increased in the ischemic areas, compared to the non-ischemic cortex (NIC) and striatum (NIS), used as control. *,#P , 0:05.
control, are shown only at 12 h after FCI for simplicity. EpoR protein was significantly increased from 6 to 12 h after FCI compared with the control (P , 0:05, tested by
167
analysis of variance followed by a post-hoc test of Fisher’s protected LSD). These data suggest that FCI induced a rapid increase in the expression of EpoR protein in the ischemic area of the P7 rat brain. Double staining analysis with the marker for neurons, astrocytes or microglia/macrophage showed that the EpoR immunoreactivity was present in neurons (Fig. 3A –C) and microglia/macrophages (Fig. 3D –F) but not in astrocytes (Fig. 3G –I) in the ischemic areas 24 h after FCI. These results show that FCI induces an increase in EpoR expression in the developing brain after FCI, but they are not fully consistent with previous reports regarding the expression of EpoR in the brains of humans with hypoxia/ischemia [15] and adult mice with FCI [2]. In adult mice, EpoR immunoreactivity was not found in neurons within the ischemic core after 12 h of FCI although neurons that were located outside the infarct remained positive for EpoR 12 h after FCI [2]. Moreover, in adult mice, EpoR was found on the endothelial cells as soon as 12 h, on microglia/microphages from 24 h and on astrocytes from 3 days after FCI [2]. In the human brain, EpoR has been reported to be present in neuronal somas after acute hypoxic brain injury (onset of symptoms less than 24 h before death) and also in astrocytes after chronic hypoxic damage (onset of symptoms 10– 28 days before death) [15]. In addition, EpoR has also been detected in microvessels in the normal or hypoxic/ischemic human brain [3,15]. Although direct comparisons of our present findings in the
Fig. 3. Double immunostaining analysis of EpoR. EpoR was present on neurons (A –C) and microglia/microphage (D –F) but not on astrocytes (G –I) in the ischemic areas. EpoR-positive cells were also TUNEL-positive (J –L). Arrowheads indicate double-stained cells. Bar, 50 mm.
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neonatal rat with those findings in humans and adult mice may not be totally appropriate, one possible explanation for the discrepancies is the differences in age and species. Vulnerability to cerebral ischemia may be different between the neonate and the adult due to the different cerebral blood flow and energy consumption during development [7]. Recently, it has been reported that the activation of caspase3 (an executioner caspase) in the adult brain is different from that in the neonatal brain [9]. The activation of caspase-3 was not detected in the adult brains of rats and mice with permanent or transient FCI, but a clear induction of caspase-3 was confirmed in the P7 rat brain with hypoxia/ ischemia [9]. TUNEL staining showed that TUNEL-positive cells were detected in the ischemic cortex as early as 6 h after FCI (data not shown) and increased up to 24 h after FCI (Fig. 3K). Double staining with a TUNEL kit and the EpoR antibody showed that about 20% of the TUNEL-positive cells were also labeled with the EpoR antibody (Fig. 3J– L) in the ischemic cortex. Moreover, about 50% of the EpoRpositive cells were TUNEL-positive in the ischemic cortex 24 h after FCI. This indicates that some EpoR-positive cells were in the course of apoptotic cell death. However, the mechanism(s) of death of the EpoR-positive cells in the ischemic areas of neonatal brains is at present not clear. In conclusion, we found for the first time that FCI induces a significant increase in EpoR expression in the neonatal rat brain as shown by Western blot analysis, and that EpoR-positive cells were in apoptotic cell death in the ischemic areas. These results suggest that EpoR plays an important role in the mechanisms of injury in the developing brain. Further studies are needed to elucidate the mechanism(s) of EpoR-positive cell death, the role of EpoR and the potential beneficial effects of Epo on neonatal focal cerebral stroke.
[2]
[3]
[4]
[5]
[6]
[7] [8] [9]
[10]
[11]
[12]
[13]
Acknowledgements [14]
This study was supported in part by grants for scientific research from Children’s Research Center and Goddard Scholarship (A.S.), Emory University, Atlanta, GA 30322, USA. [15]
References [1] C. Alafaci, F. Salpietro, G. Grasso, A. Sfacteria, M. Passalacqua, A. Morabito, E. Tripodo, G. Calapai, M. Buemi, F. Tomasello, Effect of
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recombinant human erythropoietin on cerebral ischemia following experimental subarachnoid hemorrhage, Eur. J. Pharmacol. 406 (2000) 219 –225. M. Bernaudin, H.H. Marti, S. Roussel, D. Divoux, A. Nouvelot, E.T. MacKenzie, E. Petit, A potential role for erythropoietin in focal permanent cerebral ischemia in mice, J. Cereb. Blood Flow Metab. 19 (1999) 643 –651. M.L. Brines, P. Ghezzi, S. Keenan, D. Agnello, N.C. de Lanerolle, C. Cerami, L.M. Itri, A. Cerami, Erythropoietin crosses the blood-brain barrier to protect against experimental brain injury, Proc. Natl. Acad. Sci. USA 97 (2000) 10526–10531. M. Buemi, E. Cavallaro, F. Floccari, A. Sturiale, C. Aloisi, M. Trimarchi, F. Corica, N. Frisina, The pleiotropic effects of erythropoietin in the central nervous system, J. Neuropathol. Exp. Neurol. 62 (2003) 228–236. M.A. Catania, M.C. Marciano, A. Parisi, A. Sturiale, M. Buemi, G. Grasso, F. Squadrito, A.P. Caputi, G. Calapai, Erythropoietin prevents cognition impairment induced by transient brain ischemia in gerbils, Eur. J. Pharmacol. 437 (2002) 147–150. C. Dame, S.E. Juul, R.D. Christensen, The biology of erythropoietin in the central nervous system and its neurotrophic and neuroprotective potential, Biol. Neonate 79 (2001) 228–235. N. Derugin, D.M. Ferriero, Z.S. Vexler, Neonatal reversible focal cerebral ischemia: a new model, Neurosci. Res. 32 (1998) 349–353. J.W. Fisher, Erythropoietin: physiology and pharmacology update, Exp. Biol. Med. 228 (2003) 1–14. R. Gill, M. Soriano, K. Blomgren, H. Hagberg, R. Wybrecht, M.T. Miss, S. Hoefer, G. Adam, O. Niederhauser, J.A. Kemp, H. Loetscher, Role of caspase-3 activation in cerebral ischemia-induced neurodegeneration in adult and neonatal brain, J. Cereb. Blood Flow Metab. 22 (2002) 420–430. S.E. Juul, A.T. Yachnis, R.D. Christensen, Tissue distribution of erythropoietin and erythropoietin receptor in the developing human fetus, Early Hum. Dev. 52 (1998) 235–249. E. Morishita, S. Masuda, M. Nagao, Y. Yasuda, R. Sasaki, Erythropoietin receptor is expressed in rat hippocampal and cerebral cortical neurons, and erythropoietin prevents in vitro glutamateinduced neuronal death, Neuroscience 76 (1997) 105 –116. M. Sakanaka, T.C. Wen, S. Matsuda, S. Masuda, E. Morishita, M. Nagao, R. Sasaki, In vivo evidence that erythropoietin protects neurons from ischemic damage, Proc. Natl. Acad. Sci. USA 95 (1998) 94635–94640. A.D. Sinor, D.A. Greenberg, Erythropoietin protects cultured cortical neurons, but not astroglia, from hypoxia and AMPA toxicity, Neurosci. Lett. 290 (2000) 213–215. A.L. Siren, M. Fratelli, M. Brines, C. Goemans, S. Casagrande, P. Lewczuk, S. Keenan, C. Gleiter, C. Pasquali, A. Capobianco, T. Mennini, R. Heumann, A. Cerami, H. Ehrenreich, P. Ghezzi, Erythropoietin prevents neuronal apoptosis after cerebral ischemia and metabolic stress, Proc. Natl. Acad. Sci. USA 98 (2001) 4044–4049. A.L. Siren, F. Knerlich, W. Poser, C.H. Gleiter, W. Bruck, H. Ehrenreich, Erythropoietin and erythropoietin receptor in human ischemic/hypoxic brain, Acta Neuropathol. 101 (2001) 271–276. T.C. Wen, Y. Sadamoto, J. Tanaka, P.X. Zhu, K. Nakata, Y.J. Ma, R. Hata, M. Sakanaka, Erythropoietin protects neurons against chemical hypoxia and cerebral ischemic injury by up-regulating Bcl-xL expression, J. Neurosci. Res. 67 (2002) 795–803.
Permanent focal cerebral ischemia activates erythropoietin receptor in the neonatal rat brain Tong-Chun Wen, Marta Rogido, Tom Genetta, Augusto Sola* Division of Neonatal-Perinatal Medicine, Department of Pediatrics, Emory University School of Medicine, 1462 Clifton Road, Atlanta, GA 30322, USA Received 17 September 2003; received in revised form 14 October 2003; accepted 23 October 2003
Abstract Erythropoietin (Epo) has been shown to act as a neurotrophic and neuroprotective factor via binding to its receptor (EpoR) which is activated in adult brains following hypoxia and ischemia. However, no evidence suggests that cerebral ischemia can activate EpoR in the neonatal brain. In the present study, the changes in EpoR expression were investigated using a modified model of permanent focal cerebral ischemia (FCI) in 7-day-old rat pups. Western blot analysis with an anti-rabbit EpoR antibody revealed a significant increase in the EpoR protein in the ischemic areas, starting from 6 to 12 h after FCI. Moreover, many EpoR-positive cells were detected in the ischemic areas from 12 h after FCI, and the positive cells were identified as neurons and microglia/macrophage but not astrocytes 24 h after FCI. Additionally, double staining with a red in situ apoptosis detection kit and the EpoR antibody indicated that EpoR-positive cells were in apoptotic cell death in the ischemic area. Therefore, these results suggest that EpoR is activated in the ischemic areas of neonatal rats and plays an important role in brain injury during development. q 2003 Published by Elsevier Ireland Ltd. Keywords: Erythropoietin receptor; Permanent focal cerebral ischemia; DNA fragmentation; Neonate; Rat
Erythropoietin (Epo) is a cytokine that is commonly associated with a central role in erythropoiesis [8]. However, Epo has recently been shown to exhibit neuroprotective effects in vitro and in vivo [4,6]. For example, Epo protects primary cultured neurons against glutamate, kainic acid, hypoxia, glucose deprivation or serum deprivation-induced injuries [11 –14]. In vivo, Epo attenuates cerebral ischemic damage in gerbils, rats and mice [1 – 3,5,11 – 14,16]. Although the mechanisms by which Epo acts as a neuroprotective agent are not fully understood, Epo may act via binding to its functional receptor [6,8]. Epo receptor (EpoR) is a transmembrane receptor and is present on erythroid progenitor cell surfaces [8]. On the other hand, EpoR has recently been found in many other cell types including endothelial cells, myocardiocytes, macrophages, retinal cells, cells of the adrenal cortex and medulla, as well as in the small bowel, spleen, liver, kidney and lung [10]. Moreover, several lines of evidence suggest that EpoR is also expressed in brains of rats, mice, monkeys and * Corresponding author. Tel.: þ 1-404-727-5765; fax: þ1-404-727-3236. E-mail address: [email protected] (A. Sola). 0304-3940/03/$ - see front matter q 2003 Published by Elsevier Ireland Ltd. doi:10.1016/j.neulet.2003.10.078
humans [6]. In the brain, EpoR has been detected in neurons and astrocytes of the cortex and on capillaries [3,6]. Furthermore, recent data show that EpoR is induced by hypoxia in the central nervous system, like it occurs in the peripheral tissues [6]. For example, cerebral ischemia has been reported to upregulate EpoR expression in the ischemic brains of adult mice [2]. However, little is known about its expression in the neonatal brain with focal cerebral ischemia (FCI). In the present study, we demonstrated the EpoR expression in the ipsilateral hemisphere following permanent FCI in rat pups at postnatal day 7 (P7). Time pregnant Sprague– Dawley rats (E18) were purchased from Charles River Labs Inc. (Wilmington, MA), and P7 pups were used. All animal research was approved by the Emory University Institutional Animal Care Committee and performed in accordance with NIH guidelines. To produce permanent FCI, we used a modified intraluminal catheter technique [7] to cause the middle cerebral artery (MCA) occlusion in 40 rat pups. Briefly, each pup was weighed, anesthetized with 2% isofluorane and fixed in a supine position. Under an operative
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microscope, the left common carotid artery (CCA), the external carotid artery (ECA) and the internal carotid artery (ICA) were separated carefully from the adjacent veins and nerves. A coated suture embolus with silicone resin was chosen according to the weight of the pup and inserted through a small incision that was made on the CCA near the CCA – ECA – ICA junction. The suture embolus was advanced 7 – 8 mm to occlude the MCA. During the operation, a lubricated temperature probe connected to a digital thermometer was inserted 0.5 cm rectally to continuously monitor the pup’s body temperature. The body temperature was maintained in normal range (35.0 – 36.5 8C) with an overhead-heating lamp. During recovery from anesthesia the pups were kept for 15 min in a chamber in room air with an environmental temperature of 37 8C to maintain normal body temperature and then returned to their dams. Twenty pups were sacrificed at 1, 3, 6, 12 and 24 h (n ¼ 4 for each time point) after FCI, and homogenates were obtained from different brain areas (Fig. 1A) for Western blot analysis. They were solubilized in a sample solution containing 2% sodium dodecylsulfate. An equal amount of protein (40 mg) in the homogenates was subjected to SDSPAGE, transferred to a nitrocellulose membrane and immunoblotted with an anti-rabbit EpoR antibody (Santa Cruz, USA). For quantitative evaluation, the immunoreactive bands of EpoR were subjected to densitometric analysis.
Fig. 1. (A) Schematic drawing illustrating the brain areas for the analysis of Western blot or immunostaining. IP, IC, the periphery (IP) and the center (IC) of the ischemic cortex; IS, the ischemic striatum; NIC, NIS, the nonischemic cortex (NIC) and striatum (NIS). (B– F) Immunostaining analysis of EpoR at 24 h after FCI. No EpoR-positive cells were detected in NIC (B); however, many were found in IP (C), IC (D) and IS (E) and also on the blood vessels (F). Bar, 50 mm.
For immunostaining, 20 different pups were perfused transcardially with 4% paraformaldehyde at 1, 3, 6, 12 and 24 h (n ¼ 4 for each time point) after FCI. Serial frozen sections (30 mm thick) were cut and processed for immunostaining with the EpoR antibody diluted 1:100 with 0.1 M phosphate buffered saline (PBS, pH 7.4) containing 1% normal goat serum and 0.3% Triton X-100 for 24 h at 4 8C. Following washing, the sections were incubated with fluorescein isothiocyanate (FITC)-conjugated anti-rabbit IgG (Sigma, MO). Immunostaining analysis was focused on brain areas as shown in Fig. 1A. For double immunostaining, anti-mouse neuronal nuclei (NeuN) antibody (Chemicon, CA), anti-mouse glial fibrillary acidic protein (GFAP) antibody (Sigma), or anti-rat F4/80 antibody (Serotec, Kidlington, UK) was used to identify neurons, astrocytes, or microglia/macrophages, respectively. The section was incubated with anti-EpoR/anti-NeuN, GFAP or F4/80 antibodies for 24 h at 4 8C and then treated with FITC- or tetramethylrhodamine (TRITC)conjugated anti-rabbit IgG/TRITC-conjugated anti-mouse or FITC-conjugated anti-rat IgG (Sigma) for 2 h at room temperature. Control immunoreactions excluded the incubation of the first or second antibody. In situ detection of DNA fragmentation was performed using a red in situ apoptosis detection kit (Roche, Germany). Briefly, the sections were incubated in the permeabilization solution containing 0.1% Triton X-100 and 0.1% sodium citrate and then incubated with TUNEL reaction mixture including enzyme solution (TdT) and label solution (TRITC-labeled nucleotides) in a humidified chamber for 1 h at 37 8C. After washing with PBS, the sections were incubated with the EpoR antibody and FITC-conjugated anti-rabbit IgG. EpoR immunoreactivity was first detected in the ischemic area 6 h after FCI and gradually increased up to 24 h after FCI (Fig. 1C –E). On the other hand, EpoR immunoreactivity was not detected in the non-ischemic cortex up to 24 h after FCI (Fig. 1B). As shown in Fig. 1, many EpoR-positive cells were detected in the periphery (Fig. 1C) and the center (Fig. 1D) of the ischemic cortex and in the ischemic striatum (Fig. 1E) 24 h after FCI. However, the shapes of the positive cells in the periphery were different from those in the center of the ischemic cortex. In the periphery, the EpoR-positive cells appeared morphologically unaffected (Fig. 1C). In contrast, in the center, the positive cells were constricted and shrunk (Fig. 1D). In addition, EpoR was found on some small blood vessels (Fig. 1F). Western blot showed a single weak band in the nonischemic cortex and striatum (the first lane in Fig. 2A,B), and the intensity of EpoR-positive bands began to increase in the ischemic cortex and striatum 6 h after FCI (the fourth lane in Fig. 2A,B). Densitometric analysis is shown in Fig. 2C. Since the expression of EpoR in the non-ischemic cortex (NIC) and striatum (NIS) were the same at all different times after FCI, the data for these areas, used as
T.-C. Wen et al. / Neuroscience Letters 355 (2004) 165–168
Fig. 2. Western blot analysis of EpoR. The intensity of EpoR-positive bands was increased in the ischemic cortex (ISC) and striatum (IS) at 6 h (A,B, respectively) and in the periphery (IP) of ISC (A) at 12 h after FCI. Densitometric analysis (C) showed that EpoR protein was significantly increased in the ischemic areas, compared to the non-ischemic cortex (NIC) and striatum (NIS), used as control. *,#P , 0:05.
control, are shown only at 12 h after FCI for simplicity. EpoR protein was significantly increased from 6 to 12 h after FCI compared with the control (P , 0:05, tested by
167
analysis of variance followed by a post-hoc test of Fisher’s protected LSD). These data suggest that FCI induced a rapid increase in the expression of EpoR protein in the ischemic area of the P7 rat brain. Double staining analysis with the marker for neurons, astrocytes or microglia/macrophage showed that the EpoR immunoreactivity was present in neurons (Fig. 3A –C) and microglia/macrophages (Fig. 3D –F) but not in astrocytes (Fig. 3G –I) in the ischemic areas 24 h after FCI. These results show that FCI induces an increase in EpoR expression in the developing brain after FCI, but they are not fully consistent with previous reports regarding the expression of EpoR in the brains of humans with hypoxia/ischemia [15] and adult mice with FCI [2]. In adult mice, EpoR immunoreactivity was not found in neurons within the ischemic core after 12 h of FCI although neurons that were located outside the infarct remained positive for EpoR 12 h after FCI [2]. Moreover, in adult mice, EpoR was found on the endothelial cells as soon as 12 h, on microglia/microphages from 24 h and on astrocytes from 3 days after FCI [2]. In the human brain, EpoR has been reported to be present in neuronal somas after acute hypoxic brain injury (onset of symptoms less than 24 h before death) and also in astrocytes after chronic hypoxic damage (onset of symptoms 10– 28 days before death) [15]. In addition, EpoR has also been detected in microvessels in the normal or hypoxic/ischemic human brain [3,15]. Although direct comparisons of our present findings in the
Fig. 3. Double immunostaining analysis of EpoR. EpoR was present on neurons (A –C) and microglia/microphage (D –F) but not on astrocytes (G –I) in the ischemic areas. EpoR-positive cells were also TUNEL-positive (J –L). Arrowheads indicate double-stained cells. Bar, 50 mm.
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neonatal rat with those findings in humans and adult mice may not be totally appropriate, one possible explanation for the discrepancies is the differences in age and species. Vulnerability to cerebral ischemia may be different between the neonate and the adult due to the different cerebral blood flow and energy consumption during development [7]. Recently, it has been reported that the activation of caspase3 (an executioner caspase) in the adult brain is different from that in the neonatal brain [9]. The activation of caspase-3 was not detected in the adult brains of rats and mice with permanent or transient FCI, but a clear induction of caspase-3 was confirmed in the P7 rat brain with hypoxia/ ischemia [9]. TUNEL staining showed that TUNEL-positive cells were detected in the ischemic cortex as early as 6 h after FCI (data not shown) and increased up to 24 h after FCI (Fig. 3K). Double staining with a TUNEL kit and the EpoR antibody showed that about 20% of the TUNEL-positive cells were also labeled with the EpoR antibody (Fig. 3J– L) in the ischemic cortex. Moreover, about 50% of the EpoRpositive cells were TUNEL-positive in the ischemic cortex 24 h after FCI. This indicates that some EpoR-positive cells were in the course of apoptotic cell death. However, the mechanism(s) of death of the EpoR-positive cells in the ischemic areas of neonatal brains is at present not clear. In conclusion, we found for the first time that FCI induces a significant increase in EpoR expression in the neonatal rat brain as shown by Western blot analysis, and that EpoR-positive cells were in apoptotic cell death in the ischemic areas. These results suggest that EpoR plays an important role in the mechanisms of injury in the developing brain. Further studies are needed to elucidate the mechanism(s) of EpoR-positive cell death, the role of EpoR and the potential beneficial effects of Epo on neonatal focal cerebral stroke.
[2]
[3]
[4]
[5]
[6]
[7] [8] [9]
[10]
[11]
[12]
[13]
Acknowledgements [14]
This study was supported in part by grants for scientific research from Children’s Research Center and Goddard Scholarship (A.S.), Emory University, Atlanta, GA 30322, USA. [15]
References [1] C. Alafaci, F. Salpietro, G. Grasso, A. Sfacteria, M. Passalacqua, A. Morabito, E. Tripodo, G. Calapai, M. Buemi, F. Tomasello, Effect of
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