Ischemia-related change of ceruloplasmin immunoreactivity in neurons and astrocytes in the gerbil hippocampus and dentate gyrus

Neurochemistry International 44 (2004) 601–607

Ischemia-related change of ceruloplasmin immunoreactivity in neurons and astrocytes in the gerbil hippocampus and dentate gyrus In Koo Hwang a , Dae-Keun Yoon a,e , Ki-Yeon Yoo a , Won Sik Eum b , Jae Hoon Bahn b , Dae Won Kim b , Jung Hoon Kang c , Hyeok Yil Kwon d , Tae-Cheon Kang a , Soo Young Choi b,1 , Moo Ho Won a,∗ a

b

Department of Anatomy, College of Medicine, Hallym University, Chunchon 200-702, South Korea Department of Genetic Engineering, Division of Life Sciences, Hallym University, Chunchon 200-702, South Korea c Department of Genetic Engineering, Chongju University, Chongju 360-764, South Korea d Department of Physiology, College of Medicine, Hallym University, Chunchon 200-702, South Korea e Department of General Surgery, Collage of Medicine, Hallym University, Chunchon 200-702, South Korea Received 30 June 2003; accepted 7 October 2003

Abstract In the present study, we investigated the temporal and spatial alterations of ceruloplasmin immunoreactivity in the gerbil hippocampus and dentate gyrus after 5 min transient forebrain ischemia. In sham-operated animals, ceruloplasmin immunoreactivity in the hippocampal CA2/3 areas was higher than that of other areas. Ceruloplasmin immunoreactivity and its protein content significantly increased and were highest in the CA1 area 1 day after ischemia-reperfusion. At this time point, the immunoreactivity was shown in pyramidal cells of the CA1 area. Four days after ischemia-reperfusion, ceruloplasmin immunoreactivity was shown in astrocytes in the hippocamapal CA1 area. These results suggest that reactive oxygen species (ROS) do not immediately damage neuronal cytosol, unlike DNA. An interval of time is required for the full expression of the cytoplasmic protein injury by ROS. This delayed neuronal injury 1 day after ischemic insult might provide a window of opportunity for therapeutic interventions using antioxidants. © 2003 Elsevier Ltd. All rights reserved. Keywords: Ceruloplasmin; Antioxidants; Transient forebrain ischemia; Hippocampus; Pyramidal cells; Immunohistochemistry; Gerbil

1. Introduction Cerebral ischemia causes delayed neuronal death in the CA1 area of the hippocampus following transient forebrain ischemia (Kirino, 1982; Pulsinelli et al., 1982; Petito et al., 1987). Ischemic injury to neurons is primarily due to the interruption of blood flow, lack of oxygenation, drop of ATP and subsequent re-oxygenation of the brain ischemia-reperfusion (Iijima et al., 2003; Milusheva and Baranyi, 2003). Cerebral ischemia may be the result of cardiac arrest and/or secondary focal ischemia following stroke, or brain hemorrhage (Petito et al., 1997; Won et al., 1999, 2001; Sims and Anderson, 2002; Schurr, 2002; Phillis and O’Regan, 2003). However, the exact mechanisms of ∗

Corresponding author. Tel.: +82-33-248-2522; fax: +82-33-256-1614. E-mail addresses: [email protected] (S.Y. Choi), [email protected] (M.H. Won). 1 Co-corresponding author. Tel.: +82-33-248-2112; fax: +82-33-241-1463. 0197-0186/$ – see front matter © 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2003.10.002

neuronal damage in ischemia remain to be elucidated. One of the acceptable hypotheses is that cellular events involving reactive oxygen species (ROS) mediated oxidative damage may evoke neurodegeneration (Numagami et al., 1996). Previous studies (Kirino, 1982; Sakamoto et al., 1991; Chan et al., 1998; Won et al., 1999; Aouffen et al., 2001; Chan, 2001; Ercal et al., 2001) have also provided evidence to support the occurrence of oxidative stress in cerebral ischemia. One of the major ROS produced in the ischemic state is the superoxide ion (Chan et al., 1998; Imai et al., 2003), which is converted into H2 O2 by superoxide dismutase in the normal state (Sakamoto et al., 1991; Chan et al., 1998; Takagi et al., 1998; Liu et al., 1999). However, it is well known that transition metals such as iron and copper may react with hydrogen peroxide to produce hydroxyl radicals through Fenton-like reactions, and with other ROS to increase oxygen radicals (Bromont et al., 1989; Yamamoto et al., 1997; Ercal et al., 2001). Trace elements tend to play a role in cardiovascular functions exerting either a harmful or beneficial influence on

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cardiovascular health (Mateescu et al., 1995; Aouffen et al., 2001). Copper is a constituent of several enzyme systems that regulate oxygen transportation and utilization. The blue oxidase ceruloplasmin, which has a molecular weight of 132,000 and contains six copper atoms per molecule and 7–8% carbohydrate, plays a role in cardiovascular function exerting a beneficial influence on cardiovascular health (Osaki et al., 1966; Mateescu et al., 1995; Aouffen et al., 2001; Kang et al., 2002). Moreover, one of the most celebrated functions of ceruloplasmin is that it converts Fe2+ released from hepatocytes to Fe3+ -transferrin, as part of the regulation of hepatic iron mobilization (Aouffen et al., 2001; Osaki et al., 1966). Since the generation of oxidation products, including superoxide radical and hydrogen peroxide, is associated with conditions that increase plasma ceruloplasmin, which can serve as a scavenger of superoxide radicals (Aouffen et al., 2001), the functional properties of ceruloplasmin in vitro have led to suggestions that it serves as a serum antioxidant in vivo (Osaki et al., 1966). Despite a considerable amount of work on the serum level of this acute phase protein, its physiological role in an in vivo ischemic model remains to be elucidated. In the present study, hence, we investigated the temporal and spatial alter-

ations of ceruloplasmin immunoreactivity in the gerbil hippocampus using transient forebrain ischemic model. 2. Materials and methods 2.1. Experimental animals Male Mongolian gerbils (Meriones unguiculatus) weighing 65–75 g were placed under general anesthesia with a mixture of 2.5% isoflurane (Baxtor, USA) in 33% oxygen and 67% nitrous oxide. A midline ventral incision was made in the neck. Both common carotid arteries were isolated, freed of nerve fibers, and occluded using non-traumatic aneurysm clips. Complete interruption of blood flow was confirmed by observing the central artery in eyeballs using an ophthalmoscope. After 5 min occlusion, the aneurysm clips were removed from both common carotid arteries. Restoration of blood flow (reperfusion) was observed directly under the ophthalmoscope. Sham-operated animals (n = 10) were subjected to the same surgical procedures except that common carotid arteries were not occluded. Body temperature was monitored and maintained at 37 ± 0.5 ◦ C during the surgery and during the immediate post-operative

Fig. 1. Low magnification of ceruloplasmin immunoreactivity in the gerbil hippocampus in the sham-operated animal (A), 1 day (B), 4 days (C) and 10 days (D) after ischemic insult. Ceruloplasmin immunoreactivity is detected mainly in the CA2 area and dentate gyrus (DG) (A). Ceruloplasmin immunoreactivity significantly increases in the CA1 area (B). Ceruloplasmin immunoreactivity significantly decreases in the CA1 and CA2 areas, and dentate gyrus (C). Ceruloplasmin immunoreactivity nearly disappears in all hippocampal area (D). Bar = 800 ␮m.

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period until the animals recovered fully from anesthesia. At the designated reperfusion time, the sham-operated and operated animals were sacrificed for immunohistochemistry (Won et al., 2001). 2.2. Tissue processing and Immunohistochemistry All animals were anesthetized with pentobarbital sodium, and perfused transcardially with PBS (pH 7.4) followed by 4% paraformaldehyde in 0.1 M PB (pH 7.4) at 30 min (n = 10), 3 h (n = 10), 12 h (n = 10), 1 day (n = 10), 2 days (n = 10), 3 days (n = 10), 4 days (n = 10), 5 days (n = 10) and 10 days (n = 10) after the surgery. Brains were removed and postfixed in the same fixative for 4 h. The brain tissues were cryoprotected by infiltration with 30% sucrose overnight. Thereafter, the tissues were frozen and sectioned with a cryostat at 30 ␮m and consecutive sections were collected in six-well plates containing PBS. These free-floating sections were first incubated with 10% normal goat serum for 30 min at room temperature. The sections were then incubated in mouse anti-ceruloplasmin antiserum (DAKO, 1:100, Carpinteria, CA) in PBS containing 0.3% Triton X-100 and 2% normal goat serum overnight at room temperature. After washing three times for 10 min with PBS, the sections

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were incubated sequentially, in goat anti-mouse IgG and Vectastain (Vector, USA), diluted 1:200 in the same solution as the primary antiserum. Between the incubations, the tissues were washed with PBS three times for 10 min each. The sections were visualized with 3,3 -diaminobenzidine tetrachloride (DAB, Fluka, USA) in 0.1 M Tris buffer and mounted on gelatin-coated slides. The immunoreactions were observed under the Axioscope microscope (Carl Zeiss, Germany). 2.3. Double immunofluorescent study To confirm the glial type containing ceruloplasmin immunoreactivity, double immunofluorescent staining for both the mouse anti-ceruloplasmin antiserum and the rabbit anti-glial fibrillary acidic protein (GFAP, 1:10, Biogenesis, UK) was performed. Sections were incubated in the mixture of antisera overnight at room temperature. After washing three times for 10 min with PBS, the sections were also incubated in a mixture of both indocarbocyanine (Cy3)-conjugated goat anti-rabbit IgG (1:600, Jackson ImmunoResearch, USA) and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (1:200, Jackson ImmunoResearch, USA) for 2 h at room temperature. The

Fig. 2. Immunohistochemical staining for ceruloplasmin in the gerbil hippocampal CA1 area in the sham-operated animal (A), 1 day (B), 4 days (C) and 10 days (D). Ceruloplasmin immunoreactivity is weakly stained in the hippocampal CA1 area in the sham-operated animal (A). One day after ischemic insult (B), ceruloplasmin immunoreactivity significantly increases in neurons of the stratum pyramidale (SP). Four days after ischemic insult (C), ceruloplasmin immunoreactivity is shown in glial components in the stratum oriens (SO) and stratum radiatum (SR). Ceruloplasmin immunoreactivity in glial components decreases 10 days after ischemic insult (D). Bar = 50 ␮m.

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immunoreactions were observed under the Axioscope microscope attached HBO100 (Carl Zeiss, Germany). 2.4. Quantitation of data and statistical analysis Images of immunoreactivies in the CA1 area, CA2/3 area and dentate gyrus of each animal were captured with an Applescanner. The brightness and contrast of each image file were uniformly calibrated by Adobe Photoshop version 2.4.1, followed by analysis using NIH Image 1.59 software. Values of background staining were obtained and subtracted from the immunoreactive intensities. All data obtained from the quantitative data were analyzed using one-way ANOVA to determine statistical significance. P-values below 0.01 were considered statistically significant. 2.5. Western blot analysis Three animals in each group, mentioned earlier, were used in this immunoblot study. After sacrifizing each group, the hippocampi with dentate gyrus were homogenized in 50 mM Tris containing 50 mM HEPES (pH 7.4), EGTA (pH 8.0), 0.2% NP–40, 10 mM EDTA (pH 8.0), 15 mM sodium pyrophosphate, 100 mM ␤-glycerophosphate, 50 mM NaF, 150 mM NaCl, 2 mM sodium orthvanadate, 1 mM PMSF and 1 mM DTT. After centrifugation, the protein concentration was determined in the supernatants by using the Micro BCA protein assay kit with bovine serum albumin as the standard (Pierce Chemical, USA). Aliquots containing 20 ␮M total proteins were boiled in loading buffer containing 150 mM Tris (pH 6.8), 300 mM DDT, 6% SDS, 0.3% bromophenol blue and 30% glycerol. Then, each aliquot was loaded onto a 10% polyacryamide gel. After electrophoresis, the gels were transferred to nitrocellulose transfer membranes (Schleicher and Schuell, USA). To reduced background staining, the filters were incubated with 5% non-fat dry milk in PBS containing 0.1% Tween 20 for 45 min, followed by incubation with mouse anti-ceruloplasmin, with peroxidase conjugated goat anti-mouse IgG (Sigma, USA), and then with ECL kit (Amersham, USA).

was highest (Fig. 6). However, at this time point, ceruloplasmin immunoreactivity was not observed in glia. Thereafter, the number of ceruloplasmin immunoreactive neurons decreased, and nearly disappeared 4 days after ischemic insult (Fig. 2C and D). Four days after ischemic insult, the massive neuronal loss was detected in CA1 area, however, ceruloplasmin immunoreactivity was significantly expressed in the glia (Fig. 2C). Thereafter, the immunoreactivity in glia decreased (Fig. 2D). Judging from the double immunofluorescent study, the ceruloplasmin immunoreactive structures were confirmed to be astrocytes (Fig. 3). Ten days after ischemic insult, the ceruloplasmin immunoreactivity had nearly disappeared (Figs. 1D and 2D). At this time point after ischemic insult, the protein content of ceruloplasmin was slightly lower than that of the sham-operated animal (Fig. 6). 3.2. CA2/3 areas In the sham-operated animals, ceruloplasmin immunoreactivity in the CA2/3 areas was higher as compared with other regions (Fig. 1A). From 3 h to 4 days after ischemic insult, the density of ceruloplasmin immunoreactivity in the CA2/3 areas was similar to that of the sham-operated

3. Results 3.1. CA1 area In the sham-operated animals, ceruloplasmin immunoreactivity was weakly distributed in the hippocampal CA1 area (Figs. 1A and 2A). At 12 h after ischemic insult, ceruloplasmin immunoreactivity was expressed in pyramidal cells of the CA1 area (Fig. 5). One day after ischemic insult, the number of ceruloplasmin immunoreactive neurons in the pyramidal cell layer of the CA1 area significantly increased as compared with 12 h post-ischemic group (Figs. 1B, 2B and 5). At this time point, protein content of ceruloplasmin

Fig. 3. The double immunofluorescent staining for ceruloplasmin (A, red) and glial fibrillary acidic protein (GFAP) (B, green) within the CA1 area 4 days after ischemia-reperfusion. Ceruplasmin immunoreactivity is colocalized in astrocytes stained with GFAP. SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum. Bar = 200 ␮m.

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Fig. 4. Immunohistochemical staining for ceruloplasmin in dentate gyrus in the sham-operated animal (A), 1 day (B), 4 days (C) and 10 days (D) after ischemic insult. Ceruloplasmin immunoreactivity is observed in the subgranular zone (SGZ) of dentate gyrus and hippocampal CA3c region in the sham-operated animal (A). Ceruloplasmin immunoreactivity is mainly detected in SGZ (D). Ceruloplasmin immunoreactivity in SGZ and CA3c region is nearly disappeared (C and D). Bar = 50 ␮m.

Fig. 5. The densitometric analysis of ceruloplasmin immunoreactivity in the gerbil hippocampus and dentate gyrus after ischemic insult. Asterisk indicates P < 0.01.

animals (Figs. 1B and 5). Thereafter, the intensity of ceruloplasmin immunoreactivity decreased, and nearly disappeared 10 days after ischemic insult ((Figs. 1D and 5). 3.3. Dentate gyrus In the sham-operated animals, ceruloplasmin immunoreactive neurons were detected in the hippocampal CA3c area and subgranular zone of the dentate gyrus (Figs. 1A and 4A). One day after ischemic insult, the number of ceruloplasmin immunoreactive neurons increased as compared with the sham-operated animal (Fig. 1B). At this time point,

ceruloplasmin immunoreactivity was detected in the granular layer as well as hippocampal CA3c area and subgranular zone of the dentate gyrus (Fig. 4B). Thereafter, ceruloplasmin immunoreactivity significantly decreased, and was very low 10 days after ischemic insult (Figs. 4C, D and 5).

4. Discussion In the present study, we identified temporal and spatial changes of ceruloplasmin immunoreactivity in the hippocampus and dentate gyrus of the gerbil. Ceruloplasmin is

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Fig. 6. Western blot analysis of ceruloplasmin in hippocampus with dentate gyrus derived from sham-operated and operated animals after ischemia-reperfusion. (Lane 1) sham-operated animals; (Lane 2) 1 day after ischemic insult; (Lane 3) 4 days after ischemic insult; (Lane 4) 10 days after ischemic insult.

an abundant plasma protein that contains six copper atoms per molecule and accounts for 95% of the total circulating copper in healthy adults (Rydén, 1984; Fox et al., 1995). The physiological function of ceruloplasmin is not well known; roles in copper transport, coagulation, angiogenesis, defense against oxidant stress, and iron homeostasis have been proposed (Rydén, 1984; Saenko et al., 1994). The human ceruloplasmin concentration in the plasma increases during pathological processes such as inflammation (Fleming et al., 1991) and decreases with aging (Musci et al., 1993). A marked increase in the extent of plasma membrane peroxidation was observed in the patients with aceruloplasminemia (Miyajima et al., 1996). The mechanisms of delayed neuronal death in pyramidal cells of the hippocampal CA1 area, which are vulnerable to ischemic damage after transient forebrain ischemia, have been extensively studied (Kirino, 1982; Pulsinelli et al., 1982; Petito et al., 1987). Two major factors, glutamate neurotoxicity and reactive oxygen species (ROS), are attributed to delayed neuronal death (Won et al., 1999, 2001). In the present study, we focused upon ROS because ischemia-reperfusion supplies oxygen to ischemic regions of the brain in which oxygen is required by mitochondria to generate adenosine triphosphate, and superoxide radicals and H2 O2 are produced as by-products (Liu et al., 1999). The enhanced expression of ROS produced by reperfusion may attack DNA and cytoplasmic proteins (Won et al., 1999; Liu et al., 1999). In the previous study (Won et al., 1999), we observed the changes of 8-hydroxy-2 -deoxyguanosine (8-OHdG) levels in the gerbil hippocampal CA1 area after 5 min transient forebrain ischemia. The 8-OHdG immunoreactivity significantly increased (about fourfold) at 3 h after ischemia-reperfusion and sustained for at least 12 h. Thereafter the 8-OHdG level in the CA1 area decreased. On the other hand, the significant change of 8-OHdG level was not observed within the hippocampal CA2/3 areas. In the present study, we concentrated on the cytoplasmic protein damage and the defense mechanisms associated with this change. Ceruloplasmin immunoreactive neurons were found to confine to the cytoplasm and to be consistently increased in number of immunoreactive neurons 1 day after ischemic insult. The number of ceruloplasmin immunoreactive neurons was highest 1 day after ischemia-reperfusion, suggesting that superoxide production in the hippocampal CA1 area is well-developed 1 day after ischemia-reperfusion, because the production of ROS creates hydroxyl radicals. As the hydroxyl radicals are very unstable, they attack the intracellular proteins and lipids (Yamamoto et al., 1997). And it has been reported that 4-hydroxynonnenal, one

of the most physiologically active lipid peroxides, was strongly detected in neurons and astrocytes in the brains of patients with aceruloplasminemia by immunostaining (Kaneko et al., 2002). In the present study, we found that ceruloplasmin immunoreactivity was detected in the astrocytes in the hippocampal CA1 area 4 days after ischemic insult. This result suggests that the astrocytes are very important in reducing the lipid peroxidation. Astrocytes are known to play an important role in maintaining the neuronal environment and are crucial for the survival of neurons (Ridet et al., 1997; Tacconi, 1998). Although the role of astrocytes in reactive gliosis is not clear, mice lacking the GFAP gene showed significantly larger cortical infarct volume and a more extensive and profound decrease in cortical cerebral blood flow after ischemia (Nawashiro et al., 2000). It is also thought that the Fenton reaction causes the delayed neuronal death after ischemic insult. The ceruloplasmin creates superoxide radicals that do not react with Fe2+ , because it oxidizes Fe2+ into Fe3+ , which does not produce hydroxyl radicals (Osaki et al., 1966; Sakamoto et al., 1991; Fox et al., 1995). Hence, the enhanced expression of ceruloplasmin may allow hippocampal CA1 neurons to escape protein damage. This study reinforces the findings of a previous study that transgenic mice overexpressing superoxide dismutase attenuate superoxide radical levels 1 day after ischemia-reperfusion (Chan et al., 1998). Together with our results, this suggests that unlike their action on DNA, ROS do not immediately damage pyramidal neurons in the CA1 area. In conclusion, cytoplasmic damages are occurred for 1 day interval of time after ischemia-reperfusion, this interval might provide a window of opportunity for therapeutic interventions using antioxidant.

Acknowledgements This work was supported by grant (R01-2000-000-00127-0) from the Basic Research Program of the Korea Science & Engineering Foundation. This research was supported by the Research Grant from Hallym University, Korea.

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