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(Stroke. 1997;28:382-386.)
© 1997 American Heart Association, Inc.

Articles

Phagocytic Response in Photochemically Induced Infarction of Rat Cerebral Cortex

The Role of Resident Microglia

Michael Schroeter, MD; Sebastian Jander, MD; Inge Huitinga, PhD; Otto W. Witte, MD Guido Stoll, MD

the Department of Neurology, Heinrich-Heine-Universitat, Dusseldorf, Germany, and the Department of Cell Biology and Immunology, Faculty of Medicine, Vrjie Universiteit, Amsterdam, Netherlands (I.H.).

Correspondence to Dr Guido Stoll, Department of Neurology, Heinrich-Heine-Universitat, Moorenstr 5, 40225 Dusseldorf, Germany. E-mail stoll@neurologie.uni-duesseldorf.de.

	Abstract

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Background and Purpose In this study we assessed the relative extent to which resident microglia and blood-borne macrophages contribute to the population of phagocytes after focal infarction of the rat cortex.

Methods Focal cerebral infarction was induced in rats by photothrombosis after hematogenous macrophages were depleted by means of liposomes containing dichloromethylene diphosphonate. The phagocytic activation of microglia and macrophages was monitored by immunocytochemistry with the antibody ED1.

Results In both macrophage-depleted rats and controls, ED1+ phagocytes bordered the infarct to the same extent at day 3 after photothrombosis. By contrast, at day 6 after photothrombosis ED1+ phagocytes in control rats greatly outnumbered those in macrophage-depleted rats. With the use of the antibody Ox42 directed against the CR3 receptor on the surface of microglia, it was possible to selectively document the transition of resident microglia into stellate and ameboid phagocytic microglia during the first 6 days after photothrombosis in the absence of blood-borne macrophages.

Conclusions The initial phagocytic response after focal brain ischemia is an intrinsic property of the nervous system mainly performed by resident microglia. The majority of hematogenous macrophages are recruited secondarily to participate in the removal of necrotic tissue.

Key Words: cerebral infarction, focal • leukocytes • photothrombosis • rats

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References Introduction

 
Cerebral ischemia leads to profound cellular responses in both the surround of infarcts and remote regions. This includes activation and proliferation of local glia^{1 2 3} and attraction of blood-derived leukocytes to the site of ischemic brain damage.^{4 5 6 7} Whether phagocytes that remove necrotic tissue after ischemia are of microglial origin or blood-derived macrophages is an open question.⁸ This is mainly due to the fact that activated microglia and hematogenous macrophages are indistinguishable by immunocytochemistry in which antibodies against the CR3 receptor (Ox42) or ED1, a marker for their phagocytic activity, are used.^{9 10} We used dichloromethylene diphosphonate (Cl₂MDP) containing liposomes to address this issue. Intravenous application of these liposomes leads to a reliable and almost complete depletion of macrophages from the blood stream and lymphoid organs^{11 12} but leaves resident microglia of the brain unaffected.^{13 14} In experimental autoimmune encephalomyelitis, Cl₂MDP-liposome treatment led to a dramatic reduction of the infiltration of hematogenous macrophages in the central nervous system and ameliorated clinical disease.^{14 15 16}

In this study we compared the number of ED1+ microglia/macrophages and the size of infarcts at various stages after photothrombosis between sham- and Cl₂MDP-liposome–treated rats. The photothrombosis model was used because with this technique cortical infarcts are highly reproducible in location and size,^{7 17} which is essential for quantification of cellular responses.

	Materials and Methods

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Preparation of Liposomes
Multilamellar mannosylated liposomes constructed of phosphatidylcholine, cholesterol, and mannose were prepared as described before.¹¹ Briefly, 70.9 mg phosphatidylcholine and 10.8 mg cholesterol (Sigma Chemical Co) were dissolved in 10 mL chloroform and added to 3.6 mg p-aminophenyl--D-mannopyranoside (Sigma) dissolved in 2 mL methanol. This was put into a 500-mL round-bottom flask and dried in vacuo on a rotary evaporator to form a film. Subsequently, the dried film was dissolved in 10 mL of chloroform and dried again. The total amount of lipids in the film was 140 µmol. The molar ratio of phosphatidylcholine/cholesterol/mannoside of 7:2:1 was chosen according to Umezawa and Eto.¹⁸ To prepare PBS liposomes, 10 mL of PBS (0.15 mol/L NaCl in 10 mmol/L phosphate buffer, pH 7.4) was added to the dried film, and the bottom flask was rotated until the lipid film was dispersed into liposomes; to incorporate Cl₂MDP into the liposomes, 2.5 g Cl₂MDP (maximum soluble amount, a gift of Boehringer Mannheim, Germany) was added to the 10 mL PBS. The preparations were held at room temperature for 2 hours and sonicated for 3 minutes at 20°C in a Sonicor (50 Hz) and kept at room temperature for another 2 hours or overnight at 4°C. The liposomes were centrifuged at 100 000g for 30 minutes and finally resuspended carefully in 4 mL PBS.

Induction of Photothrombosis and Treatment
Photothrombotic cerebral infarction was induced in the rat parietal cortex according to the method of Watson et al¹⁷ as described in detail elsewhere.⁷ Male Wistar rats were anesthetized with enflurane and placed in a stereotaxic frame. A catheter was inserted into the left femoral vein, and the scalp was incised for exposure of the skull surface. For illumination, a fiber-optic bundle with a 1.5-mm aperture was placed stereotaxically onto the skull 4 mm posterior to the bregma and 4 mm lateral from the midline. The skull was illuminated with a cold, white light beam (150 W) for 20 minutes. During the first 2 minutes of illumination, the dye rose bengal (0.133 mL/kg body wt, 10 mg/mL saline) was injected intravenously. Rectal body temperature was maintained at 36.8°C to 37.2°C; temperature in the area of illumination remained below 36.5°C. Control experiments included illumination alone and infusion of rose bengal without illumination. The experimental design was in accordance with legal guidelines for animal care.

In a first series of experiments, one group of enflurane-anesthetized animals received three intravenous injections of mannosylated liposomes containing Cl₂MDP 2 days before, immediately after, and 2 days after photothrombosis. This schedule ensured depletion of macrophages at the time of the ischemic event and for at least 8 days thereafter. This was checked by examination of spleens of liposome-treated rats on the day of photothrombosis and daily thereafter until day 8 for the presence of ED1+ macrophages, which were virtually absent. Control rats received intravenous injections of either saline-filled liposomes or saline alone at the corresponding time points before and after photothrombosis. No differences were seen between these two regimens. For evaluation of the contribution of microglia and blood-derived macrophages to infarct demarcation, groups (macrophage-depleted and controls) of six animals each were killed on day 3 and groups of three animals each were killed on days 1 and 6 after photothrombosis.

In a second set of experiments, liposome treatment was started after induction of photothrombosis (injections on days 0, 2, and 4), and groups of three animals each were killed on days 6 and 14 after photothrombosis.

Tissue Processing and Immunocytochemistry
Animals were decapitated while under deep enflurane anesthesia at the time points described above. Brains and spleens were rapidly removed and frozen in isopentane at -40°C and stored at -75°C; 20-µm-thick coronal sections through the infarct region were cut sequentially at -20°C with a cryostat (Reichert-Jung cryostat CM 3000), and the first of 10 subsequent sections was collected for infarct volumetry. For evaluation of the microglia/macrophage responses by immunocytochemistry, 5-µm serial sections were cut through the center of the lesion. Similarly, representative 5-µm-thick longitudinal sections of the spleens were obtained.

For immunocytochemistry, sections were fixed with 4% phosphate-buffered paraformaldehyde followed by acetone and stained with the avidin-biotin-peroxidase technique with the use of the following primary antibodies: monoclonal antibody ED1 (1:2000), recognizing a lysosomal antigen in both activated microglia and blood-derived macrophages,¹⁰ and monoclonal antibody Ox42 (Serotec; 1:1000) directed against the CR3 receptor expressed on the surface of microglia/macrophages. Endogenous peroxidase activity was blocked by incubating spleen sections in methanol containing H₂O₂. As secondary antibody, affinity-purified biotinylated rat–absorbed anti-mouse IgG (Vector) was used.

Morphometric Studies
For quantification of ED1+ cells, the area covered by these cells was computerized as gray-scale pictures (Mandi YG 9220 camera, 256x256 pixels, Hamamatsu DVS 3000 video processor, NIH image analyzing software on an Apple Macintosh computer) in sections through the center of the ischemic lesions. This allowed measurement of the area of infarction and the area of infiltration. Thereafter, the proportions of areas covered by ED1+ microglia/macrophages were calculated as percentage of total infarct area and compared by means of a two-tailed unpaired t test at the P<.01 level.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References Introduction

 
Effect of Systemic Macrophage Depletion on Infarct Demarcation
In the normal cerebral cortex, microglia stained with monoclonal antibody Ox42 but were negative for ED1. No changes were seen in control animals after focal illumination of the brain without infusion of rose bengal or after injection of the dye alone.

At day 1 after photothrombosis, only a few ED1+ cells were detectable in the peri-infarct region in both sham- and Cl₂MDP-liposome–treated rats. Three days after photothrombosis, a considerable number of ED1+ cells formed a ring around the ischemic focus in sham-treated animals (Fig 1A). After elimination of blood-derived macrophages by Cl₂MDP-liposome treatment, the number and morphology of ED1+ cells in the peri-infarct area were virtually unchanged (Fig 1D). This result could be substantiated further by calculating the area covered by ED1+ cells in relation to total infarct volume (Fig 2), which showed no difference between groups. These findings indicate that ED1+ cells at day 3 after photothrombosis were mainly of microglial origin.

View larger version (143K): [in this window] [in a new window]   Figure 1. Identification of phagocytic microglia/macrophages in the boundary zone of infarcts at days 3 (A and D) and 6 (B and E) after photothrombosis by immunocytochemical staining with monoclonal antibody ED1. A and B, Brains from control rats; D and E, infarcts from animals after depletion of peripheral macrophages by Cl2MDP-liposome treatment. The right and lower margins of the infarcts are shown with brain surface on top. Note that there is no difference after or without macrophage depletion in the number of ED1+ cells between A and D, indicating that microglia alone are able to demarcate infarcts. At day 6, more ED1+ cells are present in the boundary zone of the infarcts in sham-treated (B) than in macrophage-depleted (E) rats. This indicates that between days 3 and 6, hematogenous macrophages have entered the brain and contribute to the population of ED1+ phagocytes. C, Distribution and number of ED1+ macrophages in normal spleen. After Cl2MDP-liposome application, the spleen is devoid of macrophages (F; day 3 after photothrombosis), ensuring effectiveness of the depletion procedure. Bar=250 µm in A, B, D, and E; bar=100 µm in C and F.

View larger version (39K): [in this window] [in a new window]   Figure 2. Quantitative assessment of areas covered by ED1+ microglia/macrophages. The relative percentage of ED1+ areas of the total infarct area was compared 3, 6, and 14 days after photothrombosis between Cl2MDP-liposome–treated (filled columns) and sham-treated (open columns) rats. Bars indicate SD. *Statistical significance compared with control group (P<.00001).

At day 6 after photothrombosis, the number of ED1+ cells was further increased. Infarcts were surrounded by a thick wall of ED1+ phagocytes (Fig 1B). At this stage the number of ED1+ cells in sham-treated rats clearly outnumbered ED1+ cells in this region in macrophage-depleted animals (Fig 1B and 1E). Accordingly, the area covered by ED1+ cells was significantly lower in macrophage-depleted rats when formally calculated (Fig 2). This indicates that at this later stage of infarction, hematogenous ED1+ macrophages significantly contribute to the cellular infiltrate in the infarct zone. No differences were seen between rats receiving the first injection of Cl₂MDP-liposomes 2 days before or shortly after induction of photothrombosis. Two weeks after photothrombosis, the entire infarcts were infiltrated by ED1+ microglia/macrophages, and no difference was seen between sham-treated and Cl₂MDP-liposome–treated animals (not shown).

The extent of macrophage depletion at all stages was determined by staining spleens for ED1+ cells. In a screening experiment, pretreatment of rats with Cl₂MDP-liposomes 2 days in advance led to complete depletion of macrophages in spleen at the time of induction of photothrombosis on day 0. The additional injections on days 0 and 2 ensured persistent depletion of peripheral macrophages up to day 8. As shown in Fig 1, the population of ED1+ macrophages present in normal spleen (Fig 1C) was absent in Cl₂MDP-liposome–treated rats (Fig 1F). On day 14 after photothrombosis, spleens were partially repopulated by ED1+ macrophages (not shown). Infarct size assessed by volumetry showed no statistically significant differences between sham- and Cl₂MDP-treated rats on day 3 (18.3±6.6 versus 14.3±6.3 mm³) and day 6 (13.4±3.7 versus 15.1±5.1 mm³) after photothrombosis when hematogenous macrophages were consistently depleted.

Morphological Changes in Microglia
Elimination of blood-derived macrophages allowed us to study selectively the morphological transition from resting to activated phagocytic microglia after cerebral ischemia. Typical ramified microglia were seen in the normal cerebral cortex after staining with monoclonal antibody Ox42 (Fig 3A). In the boundary zone of the infarcts, subtle morphological changes in microglia were already seen after day 1. Ox42 staining was more intense and cell processes were shortened, features typical for "stellate" microglia. These changes were more pronounced on day 3 after photothrombosis (Fig 3B). In addition, some Ox42+ "ameboid" microglia with large and round cell bodies devoid of processes were present at the transition zone to necrotic tissue. On day 6, Ox42+ ameboid microglia predominated (Fig 3C and 3D). There was a sharp border between Ox42+ ameboid microglia of the peri-infarct region and Ox42+ ramified microglia of the adjacent normal cortex. In contrast, another morphological type of Ox42+ microglia ("rod" cells) appeared toward the white matter. Rod cells represent activated microglia longitudinal to nerve fibers undergoing wallerian degeneration (Fig 3C).

View larger version (127K): [in this window] [in a new window]   Figure 3. Microglia identified by immunocytochemical staining with monoclonal antibody Ox42 in the normal cortex (A) and at the boundary zone of the infarcts at days 3 (B) and 6 (C and D) after photothrombosis in macrophage-depleted rats. Note more intense Ox42 labeling of "stellate" microglia with shortened processes at day 3 after photothrombosis (C, arrows) compared with ramified microglia of the normal cerebral cortex (A, arrows). Six days after photothrombosis, Ox42+ "ameboid" microglia with a large and round cell body predominate in the peri-infarct region (open arrows in C; D). C, Ox42+ microglia longitudinal to degenerating fibers ("rod" cells) at the white matter underlying the infarct. Bar=20 µm in A through D.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References Introduction

 
This study shows that the initial phagocytic response in the peri-infarct region of photochemically induced ischemia of the rat cortex is mainly of microglial origin. Microglia showed signs of activation and phagocytic transformation during the first days after ischemia, while hematogenous macrophages were recruited with a remarkable delay between days 3 and 6 to participate in the removal of necrotic tissue.

The extent to which resident microglia and blood-derived macrophages contribute to the population of phagocytes after cerebral infarction was controversial because of lack of a distinctive marker. Under normal and pathological conditions, microglia represent an extremely sessile glial cell population with a low turnover and replacement rate from bone marrow–derived cells of macrophage lineage.¹⁹ However, microglia respond rapidly to brain injury by proliferation and upregulation of surface markers, including major histocompatibility complex class I and II antigens and complement receptor-3 recognized by monoclonal antibody Ox42,^{9 20 21} which is also expressed by hematogenous macrophages. Microglia similar to blood-derived monocytes can transform into phagocytes. This phagocytic transformation is accompanied by the de novo expression of a lysosomal antigen recognized by monoclonal antibody ED1.¹⁰ It is therefore possible to monitor the phagocytic response of microglia/macrophages in tissue sections by ED1 immunolabeling. Recently, using bone marrow chimeras, Bauer and colleagues¹⁴ established in an elegant study that Cl₂MDP-liposome treatment dramatically reduced the number of hematogenous macrophages in inflammatory foci in the spinal cord of rats with experimental autoimmune encephalomyelitis, whereas resident microglia were not eliminated by this treatment. Thus, systemic depletion of macrophages allowed us to study responses of microglia to photochemically induced ischemia in the absence of hematogenous macrophages. We were able to show that a significant number of ED1+ phagocytes in the boundary zone after cerebral infarction were of microglial origin. However, we cannot exclude the possibility that a small proportion of these phagocytes were blood-derived macrophages because of limitations of the depletion procedure. A major role for resident microglia in the early phagocytic response was further substantiated by the observed morphological transition from ramified microglia into stellate microglia with retraction of processes into finally typical ameboid microglia in macrophage-depleted rats.

In the photothrombosis model originally described by Watson and colleagues,¹⁷ infarctions are induced by a local photochemical reaction between the systemically injected dye rose bengal and a light beam transmitted through the intact skull, leading to endothelial alterations. These are followed by an early disruption of the blood-brain barrier, vasogenic edema, platelet activation, formation of clots, and thrombotic occlusion of cerebral vessels.^{22 23} An ischemic component in lesion development was confirmed by direct measurements showing a dramatic reduction of the cerebral blood flow in the center of lesions that were surrounded by well-demarcated areas of mild to moderate hyperemia.²⁴ The cellular responses to photochemically induced focal infarction were virtually indistinguishable from the reactions to infarction after permanent occlusion of the middle cerebral artery.^{6 7} In both conditions, polymorphonuclear leukocytes, T cells, ED1+ phagocytes, and a population of yet undefined CD8+ cells appear in the areas of infarction and the boundary zone. These similarities make a direct effect of the photosensitized dye on leukocyte or macrophage functions unlikely. It is therefore likely that our findings in photochemically induced infarction also apply to other stroke models. Accordingly, similar changes of microglial morphology have been described after transient global²⁰ and focal^{2 25} cerebral ischemia. With a delay of 3 to 6 days after photothrombosis, hematogenous macrophages significantly contributed to the population of ED1+ phagocytes in our study. These findings correspond very well to a comprehensive histological study performed by Clark and colleagues,⁸ who described a great profusion of macrophages in the area of infarction from day 5 through resolution of the lesion 30 days later after permanent occlusion of the middle cerebral artery.

In view of the early microglial response after photothrombosis, it is not surprising that systemic depletion of macrophages had no influence on infarct volume in our study. Similarly, antibodies against the CD11b/18 complex critically involved in the migration of neutrophils and macrophages through the endothelial wall failed to reduce infarct volume after permanent occlusion of the middle cerebral artery.²⁶ Among other possibilities, the lack of a therapeutic effect could be explained by the fact that resident activated microglia can transform into phagocytes and exert immunological and neurotoxic functions identical to those of blood-derived macrophages.^{9 27 28}

The question of what signal activates resident microglia to transform after cerebral infarction is intriguing. Since polymorphonuclear leukocytes and T cells are present early after infarction,^{5 6 7} leukocyte-derived cytokines could provide the necessary stimuli. The fact that in contrast to polymorphonuclear leukocytes and T cells the majority of hematogenous macrophages are recruited with delay points to a complex interplay between local glia and infiltrating leukocytes that is not yet understood. Moreover, it is unclear at present whether the local microglial response is harmful by releasing neurotoxic factors²⁷ or beneficial by protecting remote brain areas from dysfunction through rapid demarcation and clearance of necrotic tissue.

	Acknowledgments

 
This study was supported by the Deutsche Forschungsgemeinschaft SFB 194 (B2 and B6). Dr Stoll holds a Hermann and Lilly-Schilling professorship. We thank Annette Tries for technical assistance and U. Vollmer for photographic work.

Received May 10, 1996; revision received September 9, 1996; accepted October 2, 1996.

	References

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Editorial Comment

The Role of Resident Microglia

W. Dalton Dietrich, PhD, Guest Editor

Department of NeurologyUniversity of Miami School of MedicineMiami, Fla

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References Introduction

 
The interesting study by Schroeter and colleagues examined the relative extent to which resident microglia and blood-borne macrophages contribute to the population of phagocytes after cortical infarction in rats. Using immunocytochemical techniques, the authors report that the initial phagocytic response is mainly composed of resident microglia, with hematogenous macrophages recruited secondarily.

The rationale and experimental approach used in the investigation are good, and the authors present new data regarding phagocytic response to thrombotic infarction. Various inflammatory processes have been implicated in the pathophysiology of delayed neuronal injury after cerebral ischemia and brain trauma. The present data will be valuable to future treatment studies that may target these cellular reactions for pharmacological intervention.

The blood-brain barrier consequences of photothrombotic cortical injury differ significantly from those reported in conventional focal ischemia models. For example, in contrast to permanent middle cerebral artery occlusion in which the blood-brain barrier remains intact for several hours, the blood-brain barrier is immediately disrupted after thrombotic infarction. One might therefore ask whether differences in the temporal profile of microvascular damage between models would affect the phagocytic response to injury.

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