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Short communication
Expression of microglial response factor-1 in microglia and
macrophages following cerebral ischemia in the rat
a,b ,
*
c a c bHiroyuki Kato
, Shuuitsu Tanaka , Takanori Oikawa , Tatsuro Koike , Akira Takahashi ,
a
Yasuto Itoyama
a
Department of Neurology, Field of Neuroscience, Tohoku University Graduate School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan
b
Department of Neuroendovascular Therapy, Field of Neuroscience, Tohoku University Graduate School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai980-8574, Japan
c
Molecular Neurobiology Laboratory, Graduate Program in Biological Sciences, Hokkaido University, Sapporo 060-0810, Japan
Accepted 8 August 2000
Abstract
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Microglial response factor-1 is a newly isolated microglial gene, which encodes a Ca binding protein MRF-1 expressed in microglia and macrophages. We induced 1 h of focal cerebral ischemia or 10 min of global cerebral ischemia in the rat, and investigated the expression of MRF-1 immunoreactivity following ischemia. MRF-1 was present in resting microglia and was upregulated in response to microglial activation. MRF-1 was localized to all the cells of the mononuclear phagocyte system (microglia, monocytes, and perivascular cells) that appeared in the ischemic brain. 2000 Elsevier Science B.V. All rights reserved.
Theme: Disorders of the nervous system
Topic: Ischemia
Keywords: Microglial response factor-1; Cerebral ischemia; Microglia; Macrophage
Cerebral ischemia elicits activation of glial and in- around small to medium-sized blood vessels and separated flammatory cells and this response may play a critical role from the brain parenchyma [4,12]. The mechanisms by in the development of brain damage [6,7,13,16]. Astroglial which these glial and inflammatory cells are activated activation is characterized by hypertrophy with an increase remain to be elucidated.
in glial fibrillary acidic protein (GFAP) [16], although Recently, a new microglial gene, microglial response GFAP may decline when ischemia is severe enough [11]. factor-1 (mrf-1 ) has been isolated from microglial cells Microglia are rapidly activated within min [13]. They can activated in response to injury of cultured rat cerebellar transform into macrophages, and secrete toxic substances granule neurons [19]. The mrf-1 gene encodes a 17 kDa
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that may endanger neighboring neurons [3]. The activation protein consisting of 147 amino acids with a Ca -binding however is strictly controlled and the functional role of EF-hand motif. MRF-1 has the same amino acid sequence microglia before transforming into macrophages might be of allograft inflammatory factor-1 (AIF-1), which was
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neuroprotective [1]. Blood-derived neutrophils and mono- discovered as a novel Ca -binding peptide expressed by cytes additionally infiltrate the area of cerebral infarction activated monocytes in response to chronic rejection of rat where the blood–brain barrier is breached [9]. Another cardiac allografts [20]. Moreover, human AIF-1 is identical source of macrophages is the perivascular cells (also called to human ionized calcium binding adapter molecule-1 fluorescent granular perithelial cells), which are located (iba1), which was found in microglia and acting as an adapter molecule mediating calcium signals in activated cells of the monocytic lineage [5]. Thus, MRF-1 and its
*Corresponding author. Tel.: 181-22-717-7189; fax: 1
81-22-717-homologues may play a critical role in the regulation of
7192.
E-mail address: katoh@mail.cc.tohoku.ac.jp (H. Kato). microglial / macrophage activation. The purpose of this
H. Kato et al. / Brain Research 882 (2000) 206 –211 207
study was therefore to investigate the expression of MRF-1 partial lesioning with selective neuronal damage in the
following cerebral ischemia in the rat. transitional area surrounding the infarct (penumbra), as
Male Wistar rats (Nippon SLC Co. Ltd., Hamamatsu, reported previously [10].
Japan), 12–13 weeks-old and weighing 250–300 g, were In sham-operated rats, astrocytes with slender processes anesthetized with 1.5% halothane in a mixture of 30% were uniformly stained for GFAP (Fig. 1a). Four h after oxygen and 70% nitrous oxide. Focal cerebral ischemia reperfusion, astrocytes in the striatum appeared disinte-was induced for 1 h by occluding the right middle cerebral grated and fragmented (Fig. 1b). After 1 day, astrocytes artery (MCA) intraluminally with a 17-mm long 4-0 nylon disappeared in the striatum and in part of the cortex (Fig. suture as described previously [14]. Body temperature was 1c); astrocytes in the cortical penumbra appeared reactive maintained at approximately 378C. The animals were (Fig. 1d). After 3 days and more evidently after 7 days, a sacrificed 4 h, 1 day, 3 days and 7 days after reperfusion rim of reactive (hypertrophic) astrocytes (Fig. 1e) sur-(n54 each). Sham-operated animals (n54) were also rounded the infarct in which no astrocytes were seen.
prepared. Astrocytes in the surrounding areas (out of the MCA
Male Sprague–Dawley rats (Nippon SLC Co. Ltd.), territory) were also hypertrophied but to a lesser degree. 9–10 weeks-old and weighing 250–300 g, were used for The MRF-1 antibody visualized all types of cells of global cerebral ischemia, which was induced by 2-vessel microglia / macrophage lineage, i.e., all stages of microglia occlusion combined with hypotension essentially as de- as well as perivascular cells and invading monocytes / scribed by Smith et al. [17]. Both common carotid arteries macrophages (Figs. 1f–m and 2a–d). Isolectin staining were occluded and the mean arterial blood pressure was was essentially the same as MRF-1 immunostaining except maintained at 37–42 mmHg by withdrawal of blood from that resting microglia and normal perivascular cells were PE50 polyethylene tube inserted into the femoral artery. not well stained (Fig. 2e–h). ED1 was positive only for After 10 min of ischemia, the carotid arteries were limited subpopulations of them, which were considered to recirculated and the shed blood was infused. The animals be phagocytes / macrophages (Fig. 2i–l).
were sacrificed 1 day, 2 days and 7 days after reperfusion In sham-operated brains, MRF-1 visualized the resting (n54 each). Sham-operated animals were also prepared microglia with small cell bodies and ramified processes
(n54). (Figs. 1f and 2a). Microglia in the ischemic core appeared
The brains were perfusion-fixed with 4% paraformal- disintegrated after 4 h, and became conspicuous by 1 day dehyde in 0.1 M phosphate buffer, and then were embed- (Fig. 1g), although less prominent than astrocytic damage. ded in paraffin. Coronal sections were cut with a thickness In contrast, those in the penumbra, and in the surrounding of 5mm, and used for hematoxylin and eosin staining and areas to a lesser extent, were morphologically activated. A immunohistochemistry. Cells of the microglia / macrophage number of activated microglia appeared to be attached to lineage were visualized by histochemistry with isolectin- neurons exhibiting ischemic changes (red neurons) encircl-B4 from Griffonia simplicifolia conjugated with horserad- ing them (Fig. 1h). Microglia in the ipsilateral corpus ish peroxidase (Sigma) as reported previously [18]. Im- callosum (subjacent to frontoparietal cortex) decreased in munohistochemistry was performed using the following number, and a number of activated microglia gathered in a antibodies and the Vectastain elite ABC kit (Vector Lab- narrow zone between the corpus callosum and the striatal oratories) as reported previously [6,7]; polyclonal anti- infarction (Fig. 1j). After 3 days (Fig. 1i) and more MRF-1 antibody [19] (0.2mg / ml) for the visualization of strikingly after 7 days (Fig. 2d,h,l), the infarct was covered cells of the microglia / macrophage lineage, monoclonal by ED1-positive round cells (monocytes / macrophages). antibody ED1 for the demonstration of macrophages and The ED1-positive perivascular cells increased in number phagocytic microglia [2] (Serotec; 1:500), and monoclonal and appeared hypertrophic (Fig. 1m). In the peri-infarct anti-GFAP antibody for astrocytes (Chemicon; 1:400). penumbra, phagocytic microglia exhibiting amoeboid mor-The microglial activation was classified largely into phology and ED1stainability accumulated (Fig. 2c,g,k). In three stages according to the following criteria [8]: (1) the surrounding areas out of the MCA territory, ED1-resting microglia, which are highly ramified cells present negative, activated microglia were seen and they often lay in normal adult brain; (2) activated microglia, which are attached to normal-appearing neurons (Fig. 1k), which cells responding to ischemia with morphological and decreased after 7 days. In the contralateral corpus callosum immunophenotypic changes, but are not phagocytic. Mor- and part of the cortex, activated microglia also increased phological changes include enlarged cell bodies and con- transiently.
traction of their processes to show a more stout morpholo- Following global cerebral ischemia, selective neuronal gy; and (3) phagocytic microglia, which are full-blown damage occurred in the CA1 subfield of the hippocampus
brain macrophages with amoeboid morphology. and the dentate hilus. The dentate hilar neurons were
Following focal cerebral ischemia, the right MCA-sup- damaged by 1 day, and the CA1 neurons died a delayed plied areas of the dorsolateral striatum and variable extent neuronal death, as reported previously [6].
Fig. 1. Astroglial and microglial responses following 1 h of middle cerebral artery occlusion in the rat. (a)–(e) Glial fibrillary acidic protein immunostaining, counterstained with hematoxylin and eosin. (a) Normal astrocyte in striatum. (b) Striatum 4 h after ischemia. Astrocytes appear disintegrated (arrowhead), and neurons appear shrunken with perineuronal vacuolization (arrows). (c) Striatum 1 day after ischemia. No astrocyes are seen and neurons are eosinophilic and shrunken (arrows). (d) Parietal cortex (penumbra) 1 day after ischemia. Astrocytes are hypertrophic. (e) Peri-infarct area 7 days after ischemia. Note strongly hypertrophic, reactive astrocytes. (f)–(m) Microglial response factor-1 immunostaining, counterstained with hematoxylin and eosin. (f) Resting microglia in control striatum. (g) One day after ischemia, microglia appear disintegrated (arrowhead) in the ischemic core and neurons appear damaged (arrows). (h) Insular cortex after 1day. An activated microglial cell appears attached to a slightly eosinophilic neuron (*). (i) After 3 days, round cells (monocytes) are infiltrating the infarction (arrows) and they are ED1-positive. (j) After 1 day, microglia are focally depleted in the corpus callosum (CC), and accumulated in subjacent striatal penumbra (arrowheads). In the ischemic core (caudate putamen; CPu), microglia are depleted. (k) Activated microglia (ED1-negative) are located very close to normal-appearing neurons in frontal cortex (area of no neuronal damage) after 3 days (arrows). (l) Perivascular cells in normal striatum (arrow). (m) In the infarction, perivascular cells (arrows) appear hypertrophic and are strongly positive for ED1. Bar in (a), 20mm and applies to all but (j). Bar in (j)5200mm.
(Fig. 3d). The reactive changes became striking in CA1 (monocytes) were seen during the entire observation after 7 days when neurons were damaged (Fig. 3b,e). Such period. Activation of perivascular cells was exceptional. reactive astrocytes were seen uniformly in the CA1 region The present study demonstrated that MRF-1 immuno-(Fig. 3b), and astrocytes in CA3 and dentate gyrus reactivity is present in all the cells of microglia / macro-remained moderately reactive at 7 days (Fig. 3b). phage lineage that appeared in response to a wide range of After 1 day, microglia appeared activated in the entire ischemic neuronal damage ranging from sublethal injury to hippocampus (Fig. 3g), especially in dentate hilus. After 2 selective neuronal damage and to infarction. The activation days, microglial staining further increased in the entire of these cells occurred in parallel with an increase in hippocampus, and was most pronounced in CA1 and MRF-1 immunoreactivity. MRF-1 is thought to function as
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dentate hilus. The damaged neurons in medial CA1 a signal-transducing molecule regulated by Ca , and is pyramidal cell layer were encircled by activated microglia not associated with microglial proliferation and
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H. Kato et al. / Brain Research 882 (2000) 206 –211 209
Fig. 2. Comparison between microglial response factor-1 (MRF-1) immunostaining (a)–(d), isolectin histochemistry (e)–(h), and ED1 immunostaining (i)–(l) following 1 h of middle cerebral artery occlusion. Counterstained with hematoxylin and eosin. (a), (e) and (i) Resting microglia in normal cortex visualized by MRF-1 (a) and isolectin (e). None visualized by ED1 (i). (b), (f) and (j) Activated microglia observed in frontal cortex (sublethal neuronal injury) after 1 day are visualized with MRF-1 (b) and isolectin (f), but not by ED1 (j). (c), (g) and (k) Phagocytic, amoeboid microglia observed in peri-infarct areas after 7 days are strongly stained for MRF-1 (c) and isolectin (g), and also positive for ED1 (k). (d), (h) and (l) Monocytes / macrophages accumulated in the infarct core after 7 days are all positive for MRF-1 (d), isolectin (h), and ED1 (l). Bar520mm and applies to all.
tool to detect activation status of the cells of microglia / also do so with their detoxifying capabilities for supporting macrophage lineage. Since many membrane markers for the survival and recovery of neurons [15] remains to be microglia / macrophages work only on cryostat sections, elucidated.
MRF-1 immunoreactivity is of paramount importance Selective neuronal damage led to a strong, protracted because it works also on paraffin sections. activation of astrocytes and microglia. Activation of mi-In this study, MRF-1 immunoreactivity, in combination croglia occurred morphologically and phenotypically, fol-with other markers, delineated the temporal-spatial rela- lowed by astroglial hypertrophy (reactive astrocytosis). The tionship between glial and inflammatory responses re- activation occurred before neuronal damage became evi-sulting from cerebral ischemia. This study showed that dent, and accelerated when neuronal damage took place. these cells reacted differentially to different degrees of We have reported previously that these microglial cells ischemic brain injury. The activation of glial cells and the express the full panels of immunomolecules, suggesting recruitment of macrophages were very different between that they are fully activated, phagocytic microglia [6].
brain lesions of different severity. Macrophages may originate from resident microglia in this
Fig. 3. Astroglial and microglial responses following 10 min of global cerebral ischemia. (a)–(e) Glial fibrillary acidic protein immunostaining in the CA1 subfield and dentate gyrus of the hippocampus. (a) Control. (b) Seven days after ischemia, strongly reactive astrocytes in CA1 and moderate activation in dentate gyrus. (c)–(e) Astrocytes in the stratum radiatum of CA1 are activated after 1 day (d) and strongly hypertrophic after 7 days (e) compared to control (c). (f)–(j) Microglial response factor-1 (MRF-1) immunostaining. Only (h) is counterstained with hematoxylin and eosin. (k) isolectin histochemistry. (l) ED1 immunostaining. (g) In the stratum pyramidale of CA1, microglia are activated after 1 day compared to control (f). * in (g) CA1 neurons. (h) After 2 days, eosinophilic, damaged CA1 neurons (*, gray in this figure) are surrounded by activated microglia (black in this figure). (i)–(l) After 7 days, amoeboid microglia are accumulated prominently in the stratum pyramidale of CA1 (i), where almost all CA1 neurons are damaged. The microglia are strongly stained for MRF-1 (j), isolectin (k), and ED1 (l). o; stratum oriens, p; stratum pyramidale, r; stratum radiatum, lm; stratum lacunosum-moleculare, DG; dentate gyrus, DH; dentate hilus. Bar in (a), 100mm and applies also to (b). Bar in (c), 20mm and applies to (d)–(h), (j)–(l). Bar in (i), 500mm.
to pan-necrosis (infarction). In this sense, glial damage hypocellular. Then, round cells (monocytes) appeared and may be a prerequisite for the development of infarction. invaded the area between 3 and 7 days. The activation of Since astrocytes maintain normal neuronal environment perivascular cells also occurred after 3–7 days. Therefore, and provide supportive effects on neurons [15], it is easy to blood-derived monocytes (and perivascular cells), but not understand that a tissue with sick astrocytes cannot sur- microglia, may be the major source of macrophages in the vive. The infiltration of blood-borne leukocytes, initially infarct core. However, involvement of the penumbra into neutrophils and then monocytes, into the area of infarction infarction is likely to occur in the cortex as tissue damage has been well documented [9]. The macrophages covering progresses, and the recruitment of microglia-derived an infarct appeared to be originated predominantly from macrophages from the transitional zone into the infarct blood monocytes, and the perivascular cells. Although may occur.
H. Kato et al. / Brain Research 882 (2000) 206 –211 211
[10] Y. Li, M. Chopp, N. Jiang, F. Yao, C. Zaloga, Temporal profile of in Acknowledgements
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