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Brain trauma induces expression of diacylglycerol kinase ζ in microglia
Neuroscience Letters 461 (2009) 110–115
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Brain trauma induces expression of diacylglycerol kinase in microglia Tomoyuki Nakano a,d,∗ , Ken Iseki b , Yasukazu Hozumi a , Kaneyuki Kawamae c , Ichiro Wakabayashi d , Kaoru Goto a a
Department of Anatomy and Cell Biology, Yamagata University School of Medicine, Yamagata 990-9585, Japan Department of Emergency and Critical Care Medicine, Yamagata University School of Medicine, Yamagata 990-9585, Japan c Department of Anesthesiology, Yamagata University School of Medicine, Yamagata 990-9585, Japan d Department of Environmental and Preventive Medicine, Hyogo College of Medicine, Nishinomiya 663-8501, Japan b
a r t i c l e
i n f o
Article history: Received 18 January 2009 Received in revised form 31 March 2009 Accepted 1 June 2009 Keywords: Diacylglycerol kinase Brain cryoinjury Microglia Iba1 GLUT5
a b s t r a c t Diacylglycerol kinase (DGK) is an enzyme which phosphorylates a second messenger diacylglycerol and consists of a family of isozymes that differ in terms of structural motifs, enzymological property, and cell and tissue distribution. One of the isozymes, DGK was originally shown to be expressed in various kinds of neurons under physiological conditions. However, we unexpectedly found that under pathological conditions, such as cerebral infarction, DGK-immunoreactivity is detected in non-neuronal cells, although it remained to be elucidated in detail which cell types are responsible for the induced expression of DGK in this setting. To further elucidate functional implications of DGK in non-neuronal cells we performed detailed immunohistochemical analysis of DGK using rat brain cryoinjury model. As early as 1 h after cryoinjury, DGK-immunoreactivity was greatly decreased in the afflicted cerebral cortex and almost disappeared in the necrotic core. On day 7 after cryoinjury, however, DGK-immunoreactivity reappeared in this area. DGK-immunoreactivity was clearly detected in Iba1-immunoreactive cells of an oval or ameboid shape in the scar region, which represent activated microglia and/or macrophages. On the other hand, DGK-immunoreactivity was not detected in Iba1-immunoreactive, resting microglia of ramified and dendritic configuration in the intact cortex. Furthermore, DGK-immunoreactive cells were also positive for a microglia marker GLUT5 in the scar region, but never for an astrocyte marker GFAP. Taken together, the present study reveals that DGK is induced in activated microglia in brain trauma, suggesting the functional significance of DGK in this process. © 2009 Elsevier Ireland Ltd. All rights reserved.
An injury to the brain tissue results in the formation of a necrotic area, which is followed by the formation of a glial scar that provides supportive or inhibitory substratum for axonal repair [2,18,19,24,34,48]. The response is generally the same whatever the source of the injury, although the details are somewhat variable with distinct types of pathology. The major cell types involved in the glial reaction include microglia/macrophages, astrocytes, and oligodendrocyte precursors, with some involvement of meningeal cells and stem cells [6]. It is known that microglia/macrophages play a predominant role in the acute phase of traumatic brain injury [41]. In the later phase, glial scar is formed, which consists mainly of phagocytic cells including activated microglia and migrated macrophages in a meshwork of astrocyte processes tightly interwoven and bound together by tight and gap junctions [6,7].
∗ Corresponding author at: Department of Environmental and Preventive Medicine, Hyogo College of Medicine, Mukogawa-cho 1-1, Nishinomiya, Hyogo 6638501, Japan. Tel.: +81 798 45 6562; fax: +81 798 45 6563. E-mail address: [email protected] (T. Nakano). 0304-3940/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2009.06.001
Microglia, which contribute to about 10% of the total glial cell population in the central nervous system (CNS) of adults [44], stand sentry over pathological events in the brain. They form a network of immune alert system with a capacity for immune surveillance and control and rapidly respond to various kinds of CNS injuries. Microglial activation is a key factor in the defense of the neural parenchyma against infectious diseases, inflammation, trauma, ischemia, brain tumors and neurodegeneration [28]. From a morphological point of view, resting microglia assume a dendritic shape with a number of highly ramified processes, after which they are named ramified microglia. After activation, microglia retract their processes and are converted into an ameboid-like configuration. With regard to functional aspects, activated microglia serve mainly as scavenger cells but also perform various other functions in neural degeneration and tissue repair. It is intriguing to note that microglial activation promotes neurotoxicity and also neuroprotective effects [29]. They migrate to the site of injury and release several factors, including proinflammatory cytokines, nitric oxide, and neurotrophic factors [30], which should be under strict control. In addition to microglial activation, brain injury, such as focal cerebral ischemia and infarction, is not uncommonly accompanied
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by breakdown of the blood brain barrier (BBB), which causes influx of extrinsic, blood-borne cells with full macrophage properties from the circulation [39]. In this regard, both activated microglia and migrated macrophages contain phagolysosomes that can be stained with the ED1 antibody [4,8], which makes it difficult to determine the relative contribution between resident microglia and invading macrophages from the blood in CNS disorders. Therefore, it is necessary to distinguish those cells and to understand the activation mechanisms at the molecular level. Diacylglycerol kinase (DGK) is an enzyme responsible for phosphorylation of diacylglycerol (DG) to phosphatidic acid, both of which are shown to serve as second messengers [31,35,42]. To date, several DGK isozymes have been cloned from various animal species, including rat [9–12,13,16,25,26,43]. Thus, DGK isozymes are supposed to play roles in a variety of intracellular signal transduction cascades in distinct cell types through the control of these lipid-derived messengers [5,14,15]. One of the isozymes, DGK is originally shown to be expressed in various kinds of neurons in the normal brain and localize to the nucleus [22]. On the other hand, under pathological conditions, such as cerebral infarction produced by 90 min of middle cerebral artery occlusion (MCAO), we unexpectedly found that DGK-immunoreactivity is detected in non-neuronal cells including ED-1-positive phagocytic cells, while another isozyme of the same class, DGK, exhibits no expression in those cells [33]. However, it remained to be determined in detail which cell types are responsible for the induced expression of DGK in brain injury. The present study aimed at this point and performed detailed investigation of DGK expression using specific markers for microglia in brain injury. We took advantage of brain cryoinjury model, which is useful to easily simulate brain injury under the same conditions. Brain cryoinjury is a well-established model that mimics traumatic brain injury in experimental animals, especially BBB disruption and vasogenic brain edema [32,38]. In this model, as observed in ischemic stroke produced by arterial occlusion, irreversible loss of tissue (infarction) occurs in the core region, while the surrounding tissue, known as the penumbral region, may either recover or progress to infarction over time. Here we show that DGK is induced in activated microglia in brain cryoinjury. This suggests that DGK might be involved in the activation process of microglia. The findings would help under-
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stand the molecular mechanism of the microglial activation under pathological conditions. All animal experiments were conducted in accordance with Yamagata University Guide for the Care and Use of Laboratory Animals. Male 8-week-old Wistar rats were used to produce cryoinjury model. Cryoinjury was produced by placing a lead probe (5 mm in diameter), pre-chilled with liquid nitrogen on the cranium for 3 min [17,21,24,32,38]. The animals were sacrificed at 1 h and 7 days after cryoinjury (n = 3). Sham operation consisted of the identical procedure except that the cryoinjury was performed. Cryoinjured and sham operated rats were anesthetized with either and fixed with a transcardiac infusion of 4% paraformaldehyde buffered by 0.1 M phosphate buffer (pH 7.4). The brain was removed, immersed in the same solution for a further 2 h at 4 ◦ C, and kept in 30% sucrose in 0.1 M phosphate buffer (pH 7.4) until use. Coronal sections (30 m) were cut on a cryostat. Sections were soaked with 0.3% Triton-X 100 in phosphate buffered saline (PBS) for 30 min at room temperature (RT) to facilitate antibody penetration. Endogenous peroxidase activity was inactivated with 0.3% H2 O2 for 10 min at RT, and non-specific binding sites were blocked with 5% normal goat serum (NGS) in PBS for 1 h at RT. The primary antibodies used were rabbit anti-DGK [22], rabbit anti-Iba-1 (Wako, Japan, 1:500), rabbit anti-GLUT5 (IBL, Japan, 1.0 g/mL) and mouse anti-GFAP (Chemicon, 1:1000) antibodies in PBS containing 5% NGS and 0.1% Tween-20. Incubation was performed for overnight at 4 ◦ C with the antibodies in a moist chamber. After washing in PBS several times, sections were incubated with a biotinylated anti-rabbit IgG antibody for 30 min at RT followed by avidin–biotin–peroxidase complex method using a kit (Vector Laboratories, Burlingame, CA, USA) for 30 min at RT. After rinsing, immunolabeling was visualized with diaminobenzidine. For immunofluorescent examination, the sections were incubated with anti-rabbit IgG-Alexa 546 (red) for anti-DGK antibody, or with anti-rabbit and anti-mouse IgG-Alexa 488 (green) for anti-Iba-1 and anti-GFAP antibodies in PBS for 30 min at RT, respectively. In some cases Zenon system (Molecular Probes Inc., Eugene, OR, USA) was used for double immunolabeling according to the protocol provided. The images were taken under the confocal laser scanning microscope (PASCAL, Carl Zeiss, Germany) and processed using Adobe Photoshop. Immunoblot analysis was performed as described previously [33]. Briefly, cryoinjured and sham
Fig. 1. Immunohistochemistry of the cerebral cortex 1 h after cryoinjury. DGK-immunoreactivity is clearly detected in neurons of the intact region (A ), while it is greatly reduced in the necrotic region (A). On the serial section, immunoreactivity for a neuronal marker NeuN, is also reduced in the necrotic region (B). Insets of the necrotic (A and B) and intact (A and B ) regions are enlarged below. Bar = 25 m.
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Fig. 2. Immunohistochemistry of the cerebral cortex 7 days after cryoinjury. In the scar region (A), DGK-immunoreactive cells of an elongated or oval shape (arrowheads) are significantly increased in number, while it is detected solely in neurons in the intact region (A ). On the serial section, immunoreactivity for a phagocytic marker Iba1 is also detected in an increased number of cells in the scar region (B). Most of the immunoreactive cells are of an oval shape in this region, while they show ramified, dendritic configuration in the intact region (B ). Insets of the necrotic (A and B) and intact (A and B ) regions are enlarged below. Bar = 25 m.
operated cerebral cortices were freshly removed, homogenized and subjected to sodium dodecyl sulphate/10% polyacrylamide gel electrophoresis. Immunoreactive bands for anti-rat DGK and Iba1 antibodies were visualized using the chemiluminescent ECL +plus Western blotting detection kit (GE healthcare, UK). As reported previously [38], cryoinjury to the brain disrupted the BBB immediately after insult, which induced brain edema and neuronal death in our experimental model. As early as 1 h after cryoinjury, DGK-immunoreactivity was greatly decreased in the afflicted cerebral cortex and almost disappeared in the necrotic core (Fig. 1). At this time point, the immunoreactivity for a neuronal marker NeuN was also significantly decreased in the necrotic core. In our previous study on the MCAO model, DGK-immunoreactivity was also decreased in neurons of the ischemic region immediately after 90-min occlusion although NeuN-immunoreactivity remained unchanged [33]. These comparative data show that the present insult of cryoinjury was severer than that of the 90-min MCAO, suggesting that the reduction in DGK-immunoreactivity in neurons of the core was due to instantaneous neuronal death immediately after cryoinjury. On day 7 after cryoinjury, the edema subsided and many cellular components were observed in the necrotic region, suggesting that glial scar is being formed at this stage. Immunohistochemical staining revealed that much more DGK-immunoreactive cells were found in the scar region compared with the intact region where only neurons were immunoreactive (Fig. 2). The immunoreactive cells in the scar region varied in size and morphology, ranging from elongated to oval configuration. On the serial section stained with anti-Iba1 antibody, a pan-phagocyte marker, the immunoreactive cells were detected more abundantly in the scar region than in the intact region, the pattern of which resembled that of DGK-immunoreactive cells (Fig. 2). To examine the relationship between DGK-immunoreactive and Iba1-immunoreactive cells, we performed double immunostaining. Since Iba1 is a pan-phagocyte marker and identifies phagocytic cells of both resting and activated forms including microglia and macrophages, we compared the staining properties between the intact and the scar regions. In the intact region, Iba1 stained ramified cells of dendritic configuration representing resting microglia, in which DGK-immunoreactivity was
not detected (Fig. 3A upper panel). In the scar region, on the other hand, Iba1 stained oval or ameboid cells, 55% of which showed DGK-immunoreactivity (Fig. 3A lower panel, arrowheads). These data suggest that DGK is induced in activated phagocytes in the scar region. We also performed immunoblot analysis of DGK and Iba1 on samples obtained from the intact and the injured tissues. As shown in Fig. 3E, protein expression of both DGK and Iba1 was increased at 7 days after injury. These findings coincide with the results of immunohistochemical data (Figs. 2 and 3). On the other hand, it was hard to obtain consistent immunoblot data on samples at the early time (1 h) after injury, because the border between the intact and the injured tissues was macroscopically very unclear. Although primary response to neuronal death is activation of resident microglia [40], destruction of the BBB leads to infiltration of bone marrow-derived macrophages into the brain parenchyma in traumatized brain [39]. Thus, phagocytic cells of distinct origins, microglia and macrophage, may coexist in the injured brain tissue. To identify the origin of DGK-immunoreactive phagocytic cells in the scar region, we performed double staining using antibodies against DGK and glucose transporter 5 (GLUT5), a marker for microglia. Numerous GLUT5-immunoreactive cells of oval configuration were accumulated in the scar region (Fig. 3B). Double staining revealed that most of the GLUT5-immunoreactive cells also exhibited DGK-immunoreactivity (Fig. 3C). Quantitative analysis of DGK-immunoreactive cells in the scarred cortex at 7 days after injury (Fig. 3C) revealed that almost all GLUT-5-immunoreactive cells were also immunoreactive for DGK. Based on the morphological criteria and the specific marker staining, these results suggest that the oval-shaped phagocytes immunoreactive for both DGK and Iba1 (Fig. 3A) represent activated microglia. Glial scar is made up of a meshwork of astrocyte processes tightly interwoven and bound together by tight and gap junctions [6,7]. Therefore, we next asked whether or not DGK is expressed in astrocytes in the scar region. Double immunostaining with antiGFAP antibody clearly showed that DGK-immunoreactivity was never detected in GFAP-immunoreactive astrocytes (Fig. 3D), suggesting that DGK is not induced in astrocytes. Under physiological conditions DGK is shown to be expressed in neurons, but not in glial cells, in various parts of the brain [22]. However, in our previous studies we found under pathological
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Fig. 3. Characterization of DGK-immunoreactive cells in the scar region of the cerebral cortex 7 days after cryoinjury. (A) Confocal images of double-immunostaining for DGK and Iba1. In the intact region (upper panel), DGK-immunoreactivity is detected in neurons (asterisks), but not in Iba1-immunoreactive cells of a ramified shape (arrows). On the other hand, the immunoreactivity is clearly detected in Iba1-immunoreactive cells of an oval shape in the scar region (arrowheads in lower panel). Bar = 10 m. (B) GLUT5-immunoreactive cells are increased in number in the scar region. Most of the immunoreactive cells are oval-shaped (arrowheads). Inset is enlarged on the right. Bar = 25 m. (C) Confocal images of double-immunostaining for DGK and GLUT5. DGK-immunoreactivity is detected in GLUT5-immunoreactive cells of an oval configuration (asterisks). Bar = 10 m. (D) Confocal images of double-immunostaining for DGK and an astrocyte marker GFAP. DGK-immunoreactive cells (arrows) never overlap with GFAP-immunoreactive cells (arrowheads) in the scar region. Bar = 10 m. (E) Immunoblot analysis of DGK and Iba1 in the intact and the injured (7 days after cryoinjury) cortices. Forty micrograms of samples were loaded in each lane.
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conditions, such as cerebral ischemia/infarction, that DGK quickly disappears from ischemic neurons but appears in non-neuronal cells at a later stage [1,33], suggesting that DGK might be involved in glial functions under pathological conditions, although the details remained uncertain. In the present study on the brain cryoinjury model we show that DGK is induced in activated, but not resting, microglia in the course of glial scar formation. Brain injury, one of the major pathological conditions, is accompanied by breakdown of the BBB and elicits a strong inflammatory and glial response [39]. Microglia are activated early in the border zone of injury, partly transform into phagocytes, and rapidly remove ischemic debris in conjunction with a dramatic influx of hematogenous macrophages during the first 2 weeks after ischemia. This makes it difficult to determine the relative contribution between resident microglia and invading macrophages from the blood in the CNS disorders difficult, because upon transition into phagocytes microglia contain phagolysosomes that can be stained with the ED1 [8], which is also present in activated macrophages [4]. In this regard, recent study reveals that phagocytic microglia show positive immunoreactivity for GLUT5 in the lesions of brain infarcts while foamy macrophages predominantly derived from monocytes in the circulation do not [20,36,37]. Using this tool, the present immunohistochemical study reveals that DGK is detected in GLUT5-immunoreactive cells of an oval shape, plausibly activated microglia, in the scar region. A variety of functions upregulated in activated microglia include proliferation, migration, phagocytosis, innate immune cell surface receptor expression, and secretion of pro- and anti-inflammatory mediators, which suggests a number of roles assigned to activated microglia ranging from neuroprotective and proregenerative over immune surveillance to proinflammatory and neurotoxic [45]. Recent studies have shown that microglia express P2Y receptors [23]. Among their subtypes, P2Y12 and P2Y6 are activated by ATP and UDP, both of which are released from damaged neurons, and function as a sensor for chemotaxis and phagocytosis, respectively [27]. P2Ys are metabotropic purine or pyrimidine receptors coupled with intracellular second messenger systems through heteromeric G-proteins, suggesting a plausible involvement of DGpathway mediated by phosphatidylinositol-specific phospholipase C (PI-PLC) and DGK. In microglia, activated P2Y receptors lead to the production of DG through PI-PLC. DG is then converted by DG lipase to endocannabinoid, such as 2-arachidonoylglycerol, which is known to regulate immune response [46,47]. It is also shown that DGK is constitutively expressed in microglia and shunt PI-PLCderived DG toward PA in this process. Furthermore, DG is shown to be accumulated in the vicinity of phagosomes in the late process of phagocytosis [3]. These facts suggest that DGK might function as a competitor to DG lipase to control the regulatory mechanism in the inflammation and/or phagocytosis. Future investigation of whether the upregulation of DGK in activated microglia is involved in cascades of P2Ys and/or in phagocytic process is warranted. The removal of debris by phagocytes and re-establishment of a normal blood supply are important processes of inflammation and repair, which coincide with glial scar formation that is predominantly operated by astrocytes. Astrocytes, another glial cell type playing a major role in various pathological conditions, divide, slowly migrate into injury site, and eventually fill in the vacant space to provide supportive or inhibitory substratum for axonal repair [2,18,19,24,35,45]. However, we detected no DGK expression in GFAP-immunoreactive astrocytes in the present brain injury model, suggesting that DGK is not involved in the molecular machinery for astrocytic activation. In summary, the present study reveals that DGK is induced in activated form of microglia in brain cryoinjury. Microglial activation has been reported to promote neurotoxicity but also exert neuroprotective effects, although precise mechanism of the activa-
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Brain trauma induces expression of diacylglycerol kinase in microglia Tomoyuki Nakano a,d,∗ , Ken Iseki b , Yasukazu Hozumi a , Kaneyuki Kawamae c , Ichiro Wakabayashi d , Kaoru Goto a a
Department of Anatomy and Cell Biology, Yamagata University School of Medicine, Yamagata 990-9585, Japan Department of Emergency and Critical Care Medicine, Yamagata University School of Medicine, Yamagata 990-9585, Japan c Department of Anesthesiology, Yamagata University School of Medicine, Yamagata 990-9585, Japan d Department of Environmental and Preventive Medicine, Hyogo College of Medicine, Nishinomiya 663-8501, Japan b
a r t i c l e
i n f o
Article history: Received 18 January 2009 Received in revised form 31 March 2009 Accepted 1 June 2009 Keywords: Diacylglycerol kinase Brain cryoinjury Microglia Iba1 GLUT5
a b s t r a c t Diacylglycerol kinase (DGK) is an enzyme which phosphorylates a second messenger diacylglycerol and consists of a family of isozymes that differ in terms of structural motifs, enzymological property, and cell and tissue distribution. One of the isozymes, DGK was originally shown to be expressed in various kinds of neurons under physiological conditions. However, we unexpectedly found that under pathological conditions, such as cerebral infarction, DGK-immunoreactivity is detected in non-neuronal cells, although it remained to be elucidated in detail which cell types are responsible for the induced expression of DGK in this setting. To further elucidate functional implications of DGK in non-neuronal cells we performed detailed immunohistochemical analysis of DGK using rat brain cryoinjury model. As early as 1 h after cryoinjury, DGK-immunoreactivity was greatly decreased in the afflicted cerebral cortex and almost disappeared in the necrotic core. On day 7 after cryoinjury, however, DGK-immunoreactivity reappeared in this area. DGK-immunoreactivity was clearly detected in Iba1-immunoreactive cells of an oval or ameboid shape in the scar region, which represent activated microglia and/or macrophages. On the other hand, DGK-immunoreactivity was not detected in Iba1-immunoreactive, resting microglia of ramified and dendritic configuration in the intact cortex. Furthermore, DGK-immunoreactive cells were also positive for a microglia marker GLUT5 in the scar region, but never for an astrocyte marker GFAP. Taken together, the present study reveals that DGK is induced in activated microglia in brain trauma, suggesting the functional significance of DGK in this process. © 2009 Elsevier Ireland Ltd. All rights reserved.
An injury to the brain tissue results in the formation of a necrotic area, which is followed by the formation of a glial scar that provides supportive or inhibitory substratum for axonal repair [2,18,19,24,34,48]. The response is generally the same whatever the source of the injury, although the details are somewhat variable with distinct types of pathology. The major cell types involved in the glial reaction include microglia/macrophages, astrocytes, and oligodendrocyte precursors, with some involvement of meningeal cells and stem cells [6]. It is known that microglia/macrophages play a predominant role in the acute phase of traumatic brain injury [41]. In the later phase, glial scar is formed, which consists mainly of phagocytic cells including activated microglia and migrated macrophages in a meshwork of astrocyte processes tightly interwoven and bound together by tight and gap junctions [6,7].
∗ Corresponding author at: Department of Environmental and Preventive Medicine, Hyogo College of Medicine, Mukogawa-cho 1-1, Nishinomiya, Hyogo 6638501, Japan. Tel.: +81 798 45 6562; fax: +81 798 45 6563. E-mail address: [email protected] (T. Nakano). 0304-3940/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2009.06.001
Microglia, which contribute to about 10% of the total glial cell population in the central nervous system (CNS) of adults [44], stand sentry over pathological events in the brain. They form a network of immune alert system with a capacity for immune surveillance and control and rapidly respond to various kinds of CNS injuries. Microglial activation is a key factor in the defense of the neural parenchyma against infectious diseases, inflammation, trauma, ischemia, brain tumors and neurodegeneration [28]. From a morphological point of view, resting microglia assume a dendritic shape with a number of highly ramified processes, after which they are named ramified microglia. After activation, microglia retract their processes and are converted into an ameboid-like configuration. With regard to functional aspects, activated microglia serve mainly as scavenger cells but also perform various other functions in neural degeneration and tissue repair. It is intriguing to note that microglial activation promotes neurotoxicity and also neuroprotective effects [29]. They migrate to the site of injury and release several factors, including proinflammatory cytokines, nitric oxide, and neurotrophic factors [30], which should be under strict control. In addition to microglial activation, brain injury, such as focal cerebral ischemia and infarction, is not uncommonly accompanied
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by breakdown of the blood brain barrier (BBB), which causes influx of extrinsic, blood-borne cells with full macrophage properties from the circulation [39]. In this regard, both activated microglia and migrated macrophages contain phagolysosomes that can be stained with the ED1 antibody [4,8], which makes it difficult to determine the relative contribution between resident microglia and invading macrophages from the blood in CNS disorders. Therefore, it is necessary to distinguish those cells and to understand the activation mechanisms at the molecular level. Diacylglycerol kinase (DGK) is an enzyme responsible for phosphorylation of diacylglycerol (DG) to phosphatidic acid, both of which are shown to serve as second messengers [31,35,42]. To date, several DGK isozymes have been cloned from various animal species, including rat [9–12,13,16,25,26,43]. Thus, DGK isozymes are supposed to play roles in a variety of intracellular signal transduction cascades in distinct cell types through the control of these lipid-derived messengers [5,14,15]. One of the isozymes, DGK is originally shown to be expressed in various kinds of neurons in the normal brain and localize to the nucleus [22]. On the other hand, under pathological conditions, such as cerebral infarction produced by 90 min of middle cerebral artery occlusion (MCAO), we unexpectedly found that DGK-immunoreactivity is detected in non-neuronal cells including ED-1-positive phagocytic cells, while another isozyme of the same class, DGK, exhibits no expression in those cells [33]. However, it remained to be determined in detail which cell types are responsible for the induced expression of DGK in brain injury. The present study aimed at this point and performed detailed investigation of DGK expression using specific markers for microglia in brain injury. We took advantage of brain cryoinjury model, which is useful to easily simulate brain injury under the same conditions. Brain cryoinjury is a well-established model that mimics traumatic brain injury in experimental animals, especially BBB disruption and vasogenic brain edema [32,38]. In this model, as observed in ischemic stroke produced by arterial occlusion, irreversible loss of tissue (infarction) occurs in the core region, while the surrounding tissue, known as the penumbral region, may either recover or progress to infarction over time. Here we show that DGK is induced in activated microglia in brain cryoinjury. This suggests that DGK might be involved in the activation process of microglia. The findings would help under-
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stand the molecular mechanism of the microglial activation under pathological conditions. All animal experiments were conducted in accordance with Yamagata University Guide for the Care and Use of Laboratory Animals. Male 8-week-old Wistar rats were used to produce cryoinjury model. Cryoinjury was produced by placing a lead probe (5 mm in diameter), pre-chilled with liquid nitrogen on the cranium for 3 min [17,21,24,32,38]. The animals were sacrificed at 1 h and 7 days after cryoinjury (n = 3). Sham operation consisted of the identical procedure except that the cryoinjury was performed. Cryoinjured and sham operated rats were anesthetized with either and fixed with a transcardiac infusion of 4% paraformaldehyde buffered by 0.1 M phosphate buffer (pH 7.4). The brain was removed, immersed in the same solution for a further 2 h at 4 ◦ C, and kept in 30% sucrose in 0.1 M phosphate buffer (pH 7.4) until use. Coronal sections (30 m) were cut on a cryostat. Sections were soaked with 0.3% Triton-X 100 in phosphate buffered saline (PBS) for 30 min at room temperature (RT) to facilitate antibody penetration. Endogenous peroxidase activity was inactivated with 0.3% H2 O2 for 10 min at RT, and non-specific binding sites were blocked with 5% normal goat serum (NGS) in PBS for 1 h at RT. The primary antibodies used were rabbit anti-DGK [22], rabbit anti-Iba-1 (Wako, Japan, 1:500), rabbit anti-GLUT5 (IBL, Japan, 1.0 g/mL) and mouse anti-GFAP (Chemicon, 1:1000) antibodies in PBS containing 5% NGS and 0.1% Tween-20. Incubation was performed for overnight at 4 ◦ C with the antibodies in a moist chamber. After washing in PBS several times, sections were incubated with a biotinylated anti-rabbit IgG antibody for 30 min at RT followed by avidin–biotin–peroxidase complex method using a kit (Vector Laboratories, Burlingame, CA, USA) for 30 min at RT. After rinsing, immunolabeling was visualized with diaminobenzidine. For immunofluorescent examination, the sections were incubated with anti-rabbit IgG-Alexa 546 (red) for anti-DGK antibody, or with anti-rabbit and anti-mouse IgG-Alexa 488 (green) for anti-Iba-1 and anti-GFAP antibodies in PBS for 30 min at RT, respectively. In some cases Zenon system (Molecular Probes Inc., Eugene, OR, USA) was used for double immunolabeling according to the protocol provided. The images were taken under the confocal laser scanning microscope (PASCAL, Carl Zeiss, Germany) and processed using Adobe Photoshop. Immunoblot analysis was performed as described previously [33]. Briefly, cryoinjured and sham
Fig. 1. Immunohistochemistry of the cerebral cortex 1 h after cryoinjury. DGK-immunoreactivity is clearly detected in neurons of the intact region (A ), while it is greatly reduced in the necrotic region (A). On the serial section, immunoreactivity for a neuronal marker NeuN, is also reduced in the necrotic region (B). Insets of the necrotic (A and B) and intact (A and B ) regions are enlarged below. Bar = 25 m.
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Fig. 2. Immunohistochemistry of the cerebral cortex 7 days after cryoinjury. In the scar region (A), DGK-immunoreactive cells of an elongated or oval shape (arrowheads) are significantly increased in number, while it is detected solely in neurons in the intact region (A ). On the serial section, immunoreactivity for a phagocytic marker Iba1 is also detected in an increased number of cells in the scar region (B). Most of the immunoreactive cells are of an oval shape in this region, while they show ramified, dendritic configuration in the intact region (B ). Insets of the necrotic (A and B) and intact (A and B ) regions are enlarged below. Bar = 25 m.
operated cerebral cortices were freshly removed, homogenized and subjected to sodium dodecyl sulphate/10% polyacrylamide gel electrophoresis. Immunoreactive bands for anti-rat DGK and Iba1 antibodies were visualized using the chemiluminescent ECL +plus Western blotting detection kit (GE healthcare, UK). As reported previously [38], cryoinjury to the brain disrupted the BBB immediately after insult, which induced brain edema and neuronal death in our experimental model. As early as 1 h after cryoinjury, DGK-immunoreactivity was greatly decreased in the afflicted cerebral cortex and almost disappeared in the necrotic core (Fig. 1). At this time point, the immunoreactivity for a neuronal marker NeuN was also significantly decreased in the necrotic core. In our previous study on the MCAO model, DGK-immunoreactivity was also decreased in neurons of the ischemic region immediately after 90-min occlusion although NeuN-immunoreactivity remained unchanged [33]. These comparative data show that the present insult of cryoinjury was severer than that of the 90-min MCAO, suggesting that the reduction in DGK-immunoreactivity in neurons of the core was due to instantaneous neuronal death immediately after cryoinjury. On day 7 after cryoinjury, the edema subsided and many cellular components were observed in the necrotic region, suggesting that glial scar is being formed at this stage. Immunohistochemical staining revealed that much more DGK-immunoreactive cells were found in the scar region compared with the intact region where only neurons were immunoreactive (Fig. 2). The immunoreactive cells in the scar region varied in size and morphology, ranging from elongated to oval configuration. On the serial section stained with anti-Iba1 antibody, a pan-phagocyte marker, the immunoreactive cells were detected more abundantly in the scar region than in the intact region, the pattern of which resembled that of DGK-immunoreactive cells (Fig. 2). To examine the relationship between DGK-immunoreactive and Iba1-immunoreactive cells, we performed double immunostaining. Since Iba1 is a pan-phagocyte marker and identifies phagocytic cells of both resting and activated forms including microglia and macrophages, we compared the staining properties between the intact and the scar regions. In the intact region, Iba1 stained ramified cells of dendritic configuration representing resting microglia, in which DGK-immunoreactivity was
not detected (Fig. 3A upper panel). In the scar region, on the other hand, Iba1 stained oval or ameboid cells, 55% of which showed DGK-immunoreactivity (Fig. 3A lower panel, arrowheads). These data suggest that DGK is induced in activated phagocytes in the scar region. We also performed immunoblot analysis of DGK and Iba1 on samples obtained from the intact and the injured tissues. As shown in Fig. 3E, protein expression of both DGK and Iba1 was increased at 7 days after injury. These findings coincide with the results of immunohistochemical data (Figs. 2 and 3). On the other hand, it was hard to obtain consistent immunoblot data on samples at the early time (1 h) after injury, because the border between the intact and the injured tissues was macroscopically very unclear. Although primary response to neuronal death is activation of resident microglia [40], destruction of the BBB leads to infiltration of bone marrow-derived macrophages into the brain parenchyma in traumatized brain [39]. Thus, phagocytic cells of distinct origins, microglia and macrophage, may coexist in the injured brain tissue. To identify the origin of DGK-immunoreactive phagocytic cells in the scar region, we performed double staining using antibodies against DGK and glucose transporter 5 (GLUT5), a marker for microglia. Numerous GLUT5-immunoreactive cells of oval configuration were accumulated in the scar region (Fig. 3B). Double staining revealed that most of the GLUT5-immunoreactive cells also exhibited DGK-immunoreactivity (Fig. 3C). Quantitative analysis of DGK-immunoreactive cells in the scarred cortex at 7 days after injury (Fig. 3C) revealed that almost all GLUT-5-immunoreactive cells were also immunoreactive for DGK. Based on the morphological criteria and the specific marker staining, these results suggest that the oval-shaped phagocytes immunoreactive for both DGK and Iba1 (Fig. 3A) represent activated microglia. Glial scar is made up of a meshwork of astrocyte processes tightly interwoven and bound together by tight and gap junctions [6,7]. Therefore, we next asked whether or not DGK is expressed in astrocytes in the scar region. Double immunostaining with antiGFAP antibody clearly showed that DGK-immunoreactivity was never detected in GFAP-immunoreactive astrocytes (Fig. 3D), suggesting that DGK is not induced in astrocytes. Under physiological conditions DGK is shown to be expressed in neurons, but not in glial cells, in various parts of the brain [22]. However, in our previous studies we found under pathological
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Fig. 3. Characterization of DGK-immunoreactive cells in the scar region of the cerebral cortex 7 days after cryoinjury. (A) Confocal images of double-immunostaining for DGK and Iba1. In the intact region (upper panel), DGK-immunoreactivity is detected in neurons (asterisks), but not in Iba1-immunoreactive cells of a ramified shape (arrows). On the other hand, the immunoreactivity is clearly detected in Iba1-immunoreactive cells of an oval shape in the scar region (arrowheads in lower panel). Bar = 10 m. (B) GLUT5-immunoreactive cells are increased in number in the scar region. Most of the immunoreactive cells are oval-shaped (arrowheads). Inset is enlarged on the right. Bar = 25 m. (C) Confocal images of double-immunostaining for DGK and GLUT5. DGK-immunoreactivity is detected in GLUT5-immunoreactive cells of an oval configuration (asterisks). Bar = 10 m. (D) Confocal images of double-immunostaining for DGK and an astrocyte marker GFAP. DGK-immunoreactive cells (arrows) never overlap with GFAP-immunoreactive cells (arrowheads) in the scar region. Bar = 10 m. (E) Immunoblot analysis of DGK and Iba1 in the intact and the injured (7 days after cryoinjury) cortices. Forty micrograms of samples were loaded in each lane.
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conditions, such as cerebral ischemia/infarction, that DGK quickly disappears from ischemic neurons but appears in non-neuronal cells at a later stage [1,33], suggesting that DGK might be involved in glial functions under pathological conditions, although the details remained uncertain. In the present study on the brain cryoinjury model we show that DGK is induced in activated, but not resting, microglia in the course of glial scar formation. Brain injury, one of the major pathological conditions, is accompanied by breakdown of the BBB and elicits a strong inflammatory and glial response [39]. Microglia are activated early in the border zone of injury, partly transform into phagocytes, and rapidly remove ischemic debris in conjunction with a dramatic influx of hematogenous macrophages during the first 2 weeks after ischemia. This makes it difficult to determine the relative contribution between resident microglia and invading macrophages from the blood in the CNS disorders difficult, because upon transition into phagocytes microglia contain phagolysosomes that can be stained with the ED1 [8], which is also present in activated macrophages [4]. In this regard, recent study reveals that phagocytic microglia show positive immunoreactivity for GLUT5 in the lesions of brain infarcts while foamy macrophages predominantly derived from monocytes in the circulation do not [20,36,37]. Using this tool, the present immunohistochemical study reveals that DGK is detected in GLUT5-immunoreactive cells of an oval shape, plausibly activated microglia, in the scar region. A variety of functions upregulated in activated microglia include proliferation, migration, phagocytosis, innate immune cell surface receptor expression, and secretion of pro- and anti-inflammatory mediators, which suggests a number of roles assigned to activated microglia ranging from neuroprotective and proregenerative over immune surveillance to proinflammatory and neurotoxic [45]. Recent studies have shown that microglia express P2Y receptors [23]. Among their subtypes, P2Y12 and P2Y6 are activated by ATP and UDP, both of which are released from damaged neurons, and function as a sensor for chemotaxis and phagocytosis, respectively [27]. P2Ys are metabotropic purine or pyrimidine receptors coupled with intracellular second messenger systems through heteromeric G-proteins, suggesting a plausible involvement of DGpathway mediated by phosphatidylinositol-specific phospholipase C (PI-PLC) and DGK. In microglia, activated P2Y receptors lead to the production of DG through PI-PLC. DG is then converted by DG lipase to endocannabinoid, such as 2-arachidonoylglycerol, which is known to regulate immune response [46,47]. It is also shown that DGK is constitutively expressed in microglia and shunt PI-PLCderived DG toward PA in this process. Furthermore, DG is shown to be accumulated in the vicinity of phagosomes in the late process of phagocytosis [3]. These facts suggest that DGK might function as a competitor to DG lipase to control the regulatory mechanism in the inflammation and/or phagocytosis. Future investigation of whether the upregulation of DGK in activated microglia is involved in cascades of P2Ys and/or in phagocytic process is warranted. The removal of debris by phagocytes and re-establishment of a normal blood supply are important processes of inflammation and repair, which coincide with glial scar formation that is predominantly operated by astrocytes. Astrocytes, another glial cell type playing a major role in various pathological conditions, divide, slowly migrate into injury site, and eventually fill in the vacant space to provide supportive or inhibitory substratum for axonal repair [2,18,19,24,35,45]. However, we detected no DGK expression in GFAP-immunoreactive astrocytes in the present brain injury model, suggesting that DGK is not involved in the molecular machinery for astrocytic activation. In summary, the present study reveals that DGK is induced in activated form of microglia in brain cryoinjury. Microglial activation has been reported to promote neurotoxicity but also exert neuroprotective effects, although precise mechanism of the activa-
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