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Immunology and Microbiology  |   May 2016
Differentiated Expression Patterns and Phagocytic Activities of Type 1 and 2 Microglia
Author Affiliations & Notes
  • Akira Haga
    Department of Ophthalmology Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan
  • Eri Takahashi
    Department of Ophthalmology Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan
  • Yasuya Inomata
    Department of Ophthalmology Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan
  • Koichi Kawahara
    Department of Pharmacology, Faculty of Pharmaceutical Sciences, Niigata University of Pharmacy and Applied Life Sciences, Niigata, Japan
  • Hidenobu Tanihara
    Department of Ophthalmology Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan
  • Correspondence: Eri Takahashi, Department of Ophthalmology, Faculty of Life Sciences, Kumamoto University, 1-1-1 Honjo, Chuo-ku, Kumamoto, Japan; eritakahashi@fc.kuh.kumamoto-u.ac.jp
Investigative Ophthalmology & Visual Science May 2016, Vol.57, 2814-2823. doi:10.1167/iovs.15-18509
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      Akira Haga, Eri Takahashi, Yasuya Inomata, Koichi Kawahara, Hidenobu Tanihara; Differentiated Expression Patterns and Phagocytic Activities of Type 1 and 2 Microglia. Invest. Ophthalmol. Vis. Sci. 2016;57(6):2814-2823. doi: 10.1167/iovs.15-18509.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: The purpose of this study was to elucidate the differentiated expression patterns and phagocytic activities of two subtypes of microglia.

Methods: A rat optic nerve crush model was used to identify the expression patterns of two subtypes of microglia. Primary microglia were isolated from rat mixed glial cultures. Subsequently, in vitro experiments evaluating the phagocytosis of axonal debris were performed to analyze responsiveness to immunologic modulators (lipopolysaccharide [LPS], interleukin [IL]-4 and interferon [IFN]-γ), and we assessed the effects of LPS and IL-4 in the optic nerve crush model. The expression levels of IL-4-associated signaling molecules were analyzed in immunoblotting experiments.

Results: In the optic nerve crush model, increased numbers of microglia were found, and a minor and transient population was identified as type 1 microglia. The type 2 microglia phagocytosed more axonal debris than the type 1 microglia. The activities of both subtypes of microglia were enhanced by treatment with LPS and IFN-γ. However, the phagocytic activity of the type 1 microglia, which showed activated STAT6 signal transduction, was significantly inhibited by stimulation with the anti-inflammatory cytokine IL-4. LPS reduced the fragmentation of axons in crushed nerve fibers, whereas the axonal debris remained in IL-4-treated rats subjected to optic nerve crush.

Conclusions: The present study revealed the time-dependent distribution of the two subpopulations of microglia in an optic nerve crush model and IL-4-dependent inhibition of the phagocytosis of axonal debris by type 1 but not type 2 microglia.

The optic nerve has several components, including axons from retinal ganglion cells (RGCs), oligodendrocytes, astrocytes, and microglia. In many optic neuropathies, such as multiple sclerosis,1 optic neuritis,2 glaucoma3 and traumatic injury,4 and damage to the axons of RGCs causes retrograde cell death in the RGCs, resulting in severe impairment of visual function. Numerous investigators have reported that gliosis-related phenomena are associated with inhibited regenerative activities of damaged axons in the optic nerve.57 Several types of glial cells are present in the optic nerve, and detailed information about the regenerative and protective activities of these glial cells suggests that they play important roles.8,9 
Microglia are important cells, involved in development, regeneration, inflammation, and immune defense in the central nervous system.1013 Microglia play complicated roles due to their dual neuroprotective and neurotoxic functions.1417 In many neuronal disease models, activated microglia migrate to the inflammatory site and remove debris, similar to the activity of macrophages.18,19 Previous studies have suggested that immunologic stimulation modulates microglial activity. A recent investigation identified two subpopulations (types 1 and 2) of microglia.20 In some pathologic conditions, these two subtypes have been shown to exhibit different features. For example, only type 1 microglia are activated in the adult brain of rats following lipopolysaccharide (LPS) injection.21 Additionally, in an Alzheimer's disease mouse model, oligomeric β-amyloid is degraded selectively by activated type 2 microglia following IL-4 stimulation, preventing the progression of neurodegeneration.22,23 
In eye disorders, microglia are involved in retinal degeneration,24,25 axon regeneration,26 choroidal neovascularization,27 glaucoma,28 and optic neuropathy.29 Additionally, in animal models, activated microglia have been identified in lesions, which suggests that these cells play roles in the modulation of optic neuropathy and retinal disorders. However, information regarding the differences in function between the two subtypes of microglia is currently lacking. 
Here, we demonstrate the time-dependent distributions of the two subpopulations of microglia in an optic nerve crush model and interleukin-4 (IL-4)–dependent inhibition of the phagocytosis of axonal debris by type 1 but not type 2 microglia. 
Materials and Methods
Optic Nerve Crush Model
All rats were handled according to guidelines of the Association for Research in Vision and Ophthalmology Statement on the Use of Animals in Ophthalmic and Vision Research, and all animal experiments were approved by the experimental animal ethics committee at Kumamoto University. Optic nerve crush was unilaterally performed on male 5-week-old Sprague Dawley (SD, Kyudo, Saga, Japan) rats.30 The contralateral eyes served as negative controls. Rats were anesthetized via intraperitoneal administration of ketamine (60 mg/kg) and xylazine (10 mg/kg). The conjunctiva was incised, and the optic nerve was exposed. The optic nerve was crushed 2 mm distal to the eyeball for 10 seconds with 5-INOX forceps (Dumont, Jura, Switzerland). Rats underwent daily subtenon injection of 20 μL phosphate-buffered saline (PBS) containing 100 ng of IL-4 or intraperitoneal (i.p.) injection of LPS (1 mg/kg). The same volume of PBS was administered using the same procedure to the control. Before tissue extraction, rats were euthanized using CO2. Eyes with the injured optic nerves were enucleated 7, 14, or 21 days after optic nerve crush. The enucleated eyes were then fixed in 4% paraformaldehyde in 0.1 M PBS for 2 hours and embedded in paraffin. 
Primary Microglial Cultures
Rat primary microglial cells were harvested from primary mixed glial cell cultures prepared from neonatal SD rats as previously reported.31,32 Neonatal rats were euthanized via decapitation. After the meninges were removed, neonatal brains were dissociated by pipetting. The cell suspension was added to 75-cm2 culture flasks at a density of 1 brain per 2 flasks in 10 mL Eagle minimum essential medium (EMEM; Sigma-Aldrich Corp., St. Louis, MO, USA) supplemented with 10% bovine serum, 5 μg/mL bovine insulin, and 0.2% glucose. Type 1 microglia cells were isolated on days 14 to 16 via the shaking method at 150 rpm for 1 hour in an orbital shaker at 37°C.32 Type 2 microglia cells were isolated on days 21 to 23 using the mild trypsinization method with trypsin solution (0.25% trypsin, 1 mM ethylenediaminetetraacetic acid in Hank's balanced salt solution) diluted 1:4 in EMEM.33 For the coculture of cortical explants, microglia cells were treated with 100 ng/mL Escherichia coli LPS (Sigma-Aldrich Corp.), 10 ng/mL rat IL-4 (Sigma-Aldrich Corp.), and 20 ng/mL interferon--γ (IFN)-γ (Sigma-Aldrich Corp.) in serum-free EMEM for 4 hours. After the microglia were washed twice with serum-free EMEM, they were used for coculture with the cortical explants. For immunoblot analysis, microglia (2 ×106 cells) were incubated with 10 ng/mL IL-4 in serum-free EMEM at 37°C for 0, 10, 30, or 60 minutes. 
Coculture of Cortical Explants and Microglia
The cortices were removed from day 17 or 18 rat embryos according to previously reported methods, with slight modifications.34,35 The cortex was separated into 300–600-μm pieces using a 26-gauge needle. The cortical pieces were placed in a 35-mm poly-l-lysine–coated dish (Iwaki, Tokyo, Japan) containing 1 mL Dulbecco modified Eagle medium/nutrient mixture F-12 ham (DMEM/F12; Sigma-Aldrich Corp.) supplemented with 10% fetal bovine serum and then incubated for 7 to 10 days at 37°C under 5% CO2. The regions of extended neuritis were transected using a scalpel blade (No. 11) and incubated for 96 hours at 37°C under 5% CO2 with or without microglia (4 × 105 cells). 
Fluorescent Immunostaining
Paraffin-embedded tissue sections (5 μm) were deparaffinized using xylene and alcohol and incubated with a blocking solution containing 3% bovine serum albumin (BSA; Sigma-Aldrich Corp.) in PBS for 30 minutes. The primary microglial cultures and cocultures of the cortical explants and microglia were fixed in 4% paraformaldehyde in 0.1 M PBS for 15 minutes, then washed with PBS and incubated with a blocking solution containing 1% BSA and 0.1% Triton X-100 (Sigma-Aldrich Corp.) in PBS at 37°C for 1 hour. After samples were blocked, they were incubated overnight at 4°C with the following primary antibodies: anti-ionized calcium binding adaptor molecule 1 (Iba1; Wako, Osaka, Japan), anti-CD68 (Bio-Rad, Hercules, CA, USA), anti-9F5 (Japan patent number P4815610), anti-glial fibrillary acidic protein (GFAP; Sigma-Aldrich Corp.), anti-microtubule-associated protein 2 (MAP2; Sigma-Aldrich Corp.), and anti-β-tubulin III (Tuj1; Sigma-Aldrich Corp.). After samples were washed with PBS, they were incubated for 1 hour at room temperature with the following secondary antibodies: fluorescent dye Alexa Fluor 488-conjugated anti-mouse IgG and Alexa Fluor 594-conjugated anti-rabbit IgG (Thermo Fisher, Rockford, IL, USA). Nuclei were stained using 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA, USA). Fluorescent images were captured using a BX51 (Olympus, Tokyo, Japan) or BZ-X710 (Keyence, Osaka, Japan) fluorescence microscope. Images of the optic nerve region were taken 500 μm distal to the choroidal plane. 
Immunoblot Analysis
The primary microglial cells were homogenized in lysis buffer and incubated at 95°C for 5 minutes. Samples were then subjected to SDS-PAGE and transferred to polyvinylidene difluoride membranes (Bio-Rad). Membranes were incubated with antibodies directed against activator of interleukin-4 receptor α (IL-4Rα; Life Span Biosciences, Seattle, WA, USA), Janus kinase 1 (JAK1; Cell Signaling Technology, Danvers, MA, USA), JAK3 (Cell Signaling Technology), signal transducers and activator of transcription 6 (STAT6; Cell Signaling Technology), phospho-STAT6 (Abcam, Cambridge, UK), and β-actin (Sigma-Aldrich Corp.) overnight at 4°C. After the membranes were washed, they were incubated with horseradish peroxidase–conjugated secondary antibodies for 60 minutes at room temperature, and the labeled bands were visualized using enhanced chemiluminescence (GE Healthcare Life Sciences, Buckinghamshire, UK). 
Statistical Analysis
Statistical analyses were performed using JMP 10 software (SAS Institute, Cary, NC, USA). All data are mean ± standard deviation (SD). All analyses were conducted using unpaired Student's t-tests. Multiple comparisons were adjusted using the Bonferroni method. A P value <0.05 was considered statistically significant. 
Results
Localization of Two Subtypes of Microglia at the Optic Nerve Crush Site
The crushed optic nerve samples were stained with hematoxylin-eosin (Fig. 1A) and with antibodies for microglial markers (Fig. 1C): the 9F5 antibody was used for type 1 microglia, and the Iba1 antibody was used for both type 1 and 2 microglia. The number of Iba1-positive cells was significantly increased after injury compared with the number of Iba1-positive cells in the controls. The increased number of Iba1-positive cells persisted during the experimental period from 7 to 21 days after optic nerve crush (Figs. 1B, 1C). In contrast, the presence of 9F5-positive cells (type 1 microglia) was observed only at 14 days after the crush treatment and not at the other time points (P = 0.0009, Figs. 1B, 1C). 
Figure 1
 
Localization of type 1 and 2 microglia is shown in the rat optic nerve crush model. (A) Eyes were enucleated at 7, 14, and 21 days after crush treatment and embedded in paraffin. A hematoxylin-eosin staining image from day 14 after crush treatment is shown. An asterisk indicates the site crushed with forceps. Localization of microglia in the area demarcated by a black box in sections taken 500 μm distal to the choroidal plane. Scale bar: 500 μm. (B) Numbers of 9F5+/Iba1+ (white bar) and 9F5/Iba1+ (black bars) cells were counted in the area demarcated by a solid black line. Values are mean ± SD (n = 4 eyes). **P < 0.01/4; ***P < 0.001/4 compared with control. Unpaired Student's t-test were adjusted using Bonferroni correction. (C) Representative high-magnification images show 9F5 (green), Iba1 (red), and DAPI (blue) immunostaining. Scale bars: 200 μm.
Figure 1
 
Localization of type 1 and 2 microglia is shown in the rat optic nerve crush model. (A) Eyes were enucleated at 7, 14, and 21 days after crush treatment and embedded in paraffin. A hematoxylin-eosin staining image from day 14 after crush treatment is shown. An asterisk indicates the site crushed with forceps. Localization of microglia in the area demarcated by a black box in sections taken 500 μm distal to the choroidal plane. Scale bar: 500 μm. (B) Numbers of 9F5+/Iba1+ (white bar) and 9F5/Iba1+ (black bars) cells were counted in the area demarcated by a solid black line. Values are mean ± SD (n = 4 eyes). **P < 0.01/4; ***P < 0.001/4 compared with control. Unpaired Student's t-test were adjusted using Bonferroni correction. (C) Representative high-magnification images show 9F5 (green), Iba1 (red), and DAPI (blue) immunostaining. Scale bars: 200 μm.
Identification of Isolated Type 1 and 2 Primary Microglia
Previous studies have demonstrated the existence of two subtypes of microglia (types 1 and 2). However, currently, no available studies have addressed the phagocytic activities of each subtype for axonal debris. Thus, to determine whether one type of microglia is more associated with the engulfment of axonal debris, we isolated primary microglia from neonatal rat brains. The type 1 microglia exhibited an amoeboid morphology, whereas the type 2 microglia exhibited a ramified appearance (Fig. 2). Our immunostaining experiments confirmed that the subpopulation of type 1 microglia was positive for antibodies against the type 1 microglia-specific marker 9F5, the microglia/macrophage marker Iba1,36 and the microglia marker CD6837 (Fig. 2, upper panels). Additionally, the identity of the type 2 microglia was confirmed based on the presence of immunoreactivity to Iba1 and CD68 and a lack of immunoreactivity to the 9F5 antibody (Fig. 2, lower panels). Over 95% of isolated cells were positive for Iba1 (type 1 microglia: 99.4 ± 0.1%; type 2 microglia: 96.3 ± 1.5%) and negative for both the astrocyte (GFAP)38 and neuronal (MAP2)39 markers, which suggests that these microglia fractions were appropriately isolated (Fig. 2). 
Figure 2
 
Isolation of primary type 1 and 2 microglia. Rat primary type 1 and 2 microglia (MG) were isolated from mixed glial cultures from neonatal rat brains. Cells were photographed 24 hours after isolation. After fixation, cells stained with antibodies directed against 9F5 (type 1 MG), both Iba1 and CD68 (pan MG), GFAP (astrocytes), and MAP2 (neuronal cells) were examined using fluorescence microscopy. Scale bars: 100 μm.
Figure 2
 
Isolation of primary type 1 and 2 microglia. Rat primary type 1 and 2 microglia (MG) were isolated from mixed glial cultures from neonatal rat brains. Cells were photographed 24 hours after isolation. After fixation, cells stained with antibodies directed against 9F5 (type 1 MG), both Iba1 and CD68 (pan MG), GFAP (astrocytes), and MAP2 (neuronal cells) were examined using fluorescence microscopy. Scale bars: 100 μm.
Phagocytosis of Axonal Debris by Type 1 and 2 Microglia
Next, we used cortical explant cultures to assess the effects of microglia on the engulfment of axonal debris. In the cortical explants, the axons grew radially from the explant culture after 7 to 10 days of incubation (Fig. 3A), and axotomy of the distal axon with a surgical knife resulted in the onset of axonal degeneration, as indicated by the presence of Tuj1-positive40 particles (Figs. 3B, 3C; axonal debris). The clearance of axonal debris was promoted by coculture of the damaged axons with microglia (Fig. 3C). As shown in Fig. 3D, phagocytosed axonal debris was detected in the microglial cytosol. Immunostaining for 9F5 showed that there was no contamination among the type 1 and 2 microglial cultures; type 2 microglia did not acquire reactivity against the 9F5 antibody during the coculture of cortical explants (Fig. 3E). The clearance area yielded by the type 2 microglia (4199 ± 476 μm2/cell) was significantly larger than that yielded by the type 1 microglia (2205 ± 456 μm2/cell; P = 0.0019) (Figs. 3F, 3G). In contrast, under the experimental conditions without microglial coculture, no clearance area was observed. 
Figure 3
 
Phagocytosis of axonal debris is shown by type 1 and 2 microglia. (A) A model of rat cortical explant culture is shown. After the chopped cortex (300–600 μm) was placed in a poly-l-lysine–coated dish, axons grew radially within 7 to 10 days. Explant was subjected to immunofluorescence analysis using an anti-Tuj1 antibody (green) and an anti-Iba1 antibody (red). Scale bar: 500 μm. (B) Coculture of microglia and axonal debris is shown. After primary microglia (4 × 105 cells/dish) were added to the explant culture dish, axons were transected with a scalpel blade (white dotted line). Scale bar: 200 μm. (C) Ninety-six hours after transection of the axons, immunostaining analysis was performed using anti-Tuj1 (green) and anti-Iba1 (red) antibodies. Nuclei were stained with DAPI (blue). Scale bar: 200 μm. (D) High-magnification view of the boxed region in C. Phagocytosed axonal debris was found within the microglia (arrows). Scale bar: 100 μm. (E, F) Type 1 or type 2 microglia (MG) were incubated with axonal debris for 96 hours. After fixation, immunofluorescence analyses were performed using antibodies directed against 9F5 (green) and Iba1 (red) in E and against Tuj1 (green) and Iba1 (red) in F. Nuclei were stained with DAPI (blue). All images were photographed 200 μm distal to the transection line. Scale bars: 200 μm. (G) Clearance area of axonal debris by types 1 and 2 MG. Axonal debris clearance area was measured using BZ-X analysis software. Values are mean ± SD (n = 4 samples). **P < 0.01 (unpaired Student's t-test).
Figure 3
 
Phagocytosis of axonal debris is shown by type 1 and 2 microglia. (A) A model of rat cortical explant culture is shown. After the chopped cortex (300–600 μm) was placed in a poly-l-lysine–coated dish, axons grew radially within 7 to 10 days. Explant was subjected to immunofluorescence analysis using an anti-Tuj1 antibody (green) and an anti-Iba1 antibody (red). Scale bar: 500 μm. (B) Coculture of microglia and axonal debris is shown. After primary microglia (4 × 105 cells/dish) were added to the explant culture dish, axons were transected with a scalpel blade (white dotted line). Scale bar: 200 μm. (C) Ninety-six hours after transection of the axons, immunostaining analysis was performed using anti-Tuj1 (green) and anti-Iba1 (red) antibodies. Nuclei were stained with DAPI (blue). Scale bar: 200 μm. (D) High-magnification view of the boxed region in C. Phagocytosed axonal debris was found within the microglia (arrows). Scale bar: 100 μm. (E, F) Type 1 or type 2 microglia (MG) were incubated with axonal debris for 96 hours. After fixation, immunofluorescence analyses were performed using antibodies directed against 9F5 (green) and Iba1 (red) in E and against Tuj1 (green) and Iba1 (red) in F. Nuclei were stained with DAPI (blue). All images were photographed 200 μm distal to the transection line. Scale bars: 200 μm. (G) Clearance area of axonal debris by types 1 and 2 MG. Axonal debris clearance area was measured using BZ-X analysis software. Values are mean ± SD (n = 4 samples). **P < 0.01 (unpaired Student's t-test).
Effects of LPS, IFN-γ, and IL-4 on Phagocytosis of Axonal Debris by Microglia
The above-described results revealed that both subtypes of microglia play roles in the clearance of axonal debris. Thus, we further examined the effects of inflammatory stimulation (LPS, 100 ng/mL), an anti-inflammatory cytokine (10 ng/mL IL-4) and a proinflammatory cytokine (20 ng/mL IFN-γ) on the microglial engulfment of axonal debris. Figures 4A and 5A show that the type 2 microglia did not exhibit reactivity against the 9F5 antibody in the presence of LPS, IL-4 or IFN-γ. In the experiments involving LPS-treated type 1 microglia, the clearance area was 10051 ± 3271 μm2/cell, which was significantly greater than that observed in the controls (P = 0.0063) (Figs. 4B, 4C). Additionally, in the experiment with LPS-treated type 2 microglia, the observed clearance area was 10491 ± 3185 μm2/cell, which was also significantly increased compared with the controls (P = 0.0052, Fig. 4B, 4D). 
Figure 4
 
Phagocytosis of axonal debris by microglia treated with LPS or IL-4. (A, B) Type 1 and 2 microglia (MG) were treated with LPS (100 ng/mL) or IL-4 (10 ng/mL) in serum-free medium for 4 hours before coculture with axonal debris. After 96 hours of incubation, immunostaining analyses were performed using the indicated antibodies. Nuclei were stained with DAPI. Scale bar: 200 μm. (C, D) Areas of axonal debris clearance by type 1 (C) and type 2 MG (D) 200 μm distal to the transection line were evaluated with BZ-X analysis software. Values are means ± SD (n = 4 samples). *P < 0.05/2; **P < 0.01/2 compared with control. Unpaired Student's t-test were adjusted with Bonferroni correction.
Figure 4
 
Phagocytosis of axonal debris by microglia treated with LPS or IL-4. (A, B) Type 1 and 2 microglia (MG) were treated with LPS (100 ng/mL) or IL-4 (10 ng/mL) in serum-free medium for 4 hours before coculture with axonal debris. After 96 hours of incubation, immunostaining analyses were performed using the indicated antibodies. Nuclei were stained with DAPI. Scale bar: 200 μm. (C, D) Areas of axonal debris clearance by type 1 (C) and type 2 MG (D) 200 μm distal to the transection line were evaluated with BZ-X analysis software. Values are means ± SD (n = 4 samples). *P < 0.05/2; **P < 0.01/2 compared with control. Unpaired Student's t-test were adjusted with Bonferroni correction.
Figure 5
 
Engulfment of axonal debris by type 1 and 2 microglia treated with IFN-γ. (A, B) Type 1 and 2 microglia (MG) were treated with IFN-γ (20 ng/mL) and cocultured with axonal debris, as in Figure 4. Representative images of immunostaining for 9F5 (green), Iba1 (red), and DAPI (blue) (A) and Tuj1 (green), Iba1 (red), and DAPI (blue) (B). Scale bar: 200 μm. (C, D) Clearance of axonal debris by type 1 (C) and type 2 MG (D) was quantified as in Figure 4. Values are means ± SD (n = 4 samples). **P < 0.01 (unpaired Student's t-test).
Figure 5
 
Engulfment of axonal debris by type 1 and 2 microglia treated with IFN-γ. (A, B) Type 1 and 2 microglia (MG) were treated with IFN-γ (20 ng/mL) and cocultured with axonal debris, as in Figure 4. Representative images of immunostaining for 9F5 (green), Iba1 (red), and DAPI (blue) (A) and Tuj1 (green), Iba1 (red), and DAPI (blue) (B). Scale bar: 200 μm. (C, D) Clearance of axonal debris by type 1 (C) and type 2 MG (D) was quantified as in Figure 4. Values are means ± SD (n = 4 samples). **P < 0.01 (unpaired Student's t-test).
The IL-4-stimulated type 1 microglia exhibited a clearance area of 403 ± 173 μm2/cell, which was significantly decreased compared with the controls (P = 0.0008) (Figs. 4B, 4C). In contrast, the type 2 microglial clearance area was 3700 ± 190 μm2/cell, which was similar to that observed in the controls (P = 0.08) (Figs. 4B, 4D). In the presence of IFN-γ, both subtypes of microglia promoted the engulfment of axonal debris (type 1 microglia: 6567 ± 1081 μm2/cell; P = 0.0012; type 2 microglia: 8709 ± 815 μm2/cell; P = 0.0011) (Figs. 5C, 5D). Considered together, our quantitative analyses revealed that the phagocytosis of axonal debris was modulated by inflammatory responses and that responsiveness to the anti-inflammatory cytokine IL-4 varied with the microglial subtype. 
IL-4 Signal Transduction in Microglia
To examine differences in responsiveness to IL-4 stimulation between type 1 and type 2 microglia, we conducted immunoblotting experiments using specific antibodies directed against the IL-4 receptor and JAK/STAT signaling molecules. Immunoblot analysis revealed that JAK1 expression was higher in type 1 than in type 2 microglia. The expression levels of IL-4Rα, JAK3, and STAT6 were similar between the two subtypes (Figs. 6A, 6B). The phosphorylation of STAT6 due to IL-4 stimulation exhibited a greater increase in the type 1 microglia than in the type 2 microglia (Figs. 6B, 6C). 
Figure 6
 
IL-4 induced phosphorylation of STAT6 in microglia. (A) Equal amounts of protein from type 1 and 2 microglia (MG) were subjected to immunoblot analysis with the indicated antibodies. (B) Type 1 and 2 microglia were treated with IL-4 (10 ng/mL) in serum-free medium at the indicated times. Immunoblot analyses of phospho-STAT6 (p-STAT6), STAT6, and β-actin are shown. (C) Quantitative analyses of the levels of phosphorylated STAT6 in the microglia subtypes (type 1 [white bars]; type 2 [black bars]). Values are means ± SD (n = 3). **P < 0.01; ***P < 0.001 compared with type 2 microglia, using unpaired Student's t-test.
Figure 6
 
IL-4 induced phosphorylation of STAT6 in microglia. (A) Equal amounts of protein from type 1 and 2 microglia (MG) were subjected to immunoblot analysis with the indicated antibodies. (B) Type 1 and 2 microglia were treated with IL-4 (10 ng/mL) in serum-free medium at the indicated times. Immunoblot analyses of phospho-STAT6 (p-STAT6), STAT6, and β-actin are shown. (C) Quantitative analyses of the levels of phosphorylated STAT6 in the microglia subtypes (type 1 [white bars]; type 2 [black bars]). Values are means ± SD (n = 3). **P < 0.01; ***P < 0.001 compared with type 2 microglia, using unpaired Student's t-test.
Decreased Axon Fragmentation due to LPS and IL-4 Injection
Finally, we administered LPS or IL-4 to optic nerve crush model rats and evaluated the appearance of axons via immunostaining for Tuj1. The crush treatment induced fragmentation of axon nerve fibers, as detected in images showing Tuj1-positive dot staining, and swelling of axonal fibers, with strong fluorescent staining, as reported by Qu and Jakobs.41 Swelling and fragmentation of axons were observed in the control (i.p. PBS), and these changes were detected more clearly on day 14 than on day 7 after crush treatment. Following LPS injection, the axon fibers became swollen, whereas dot staining for Tuj1 was reduced on day 7 after crush treatment. On day 14 in the LPS group, axon atrophy had progressed markedly (residual nerve fiber area on day 14 in the LPS injection group: 54129 ± 10525 μm2; P = 0.015; compared with day 14 in the control) (Fig. 7A). In the group subjected to optic nerve crush with subtenon PBS injection, fragmentation was observed in addition to axon swelling on days 7 and 14. There were no remarkable differences in the appearance of axons between the PBS and IL-4 injection groups on day 14 (Fig. 7B). 
Figure 7
 
Effects of IL-4 and LPS on axonal degeneration in the nerve crush model. (A, B) Rats were injected with LPS (1 mg/kg) i.p. (A) or 100 ng of IL-4 subtenonly (B) at the same time as the nerve crush treatment and daily until the end of experiments. PBS was injected as a control (CTL). Representative immunostained images are shown of Tuj1 at 500 μm distal to choroid on days 7 and 14 after crush treatment. Scale bar: 100 μm. Each graph shows quantification of the residual nerve fibers in an area 300 × 500 μm from the choroidal plane, determined using BZ-X analysis software. Values are mean ± SD (n = 3 eyes). *P < 0.05 compared with control (PBS i.p.) on 14 day (unpaired Student's t-test).
Figure 7
 
Effects of IL-4 and LPS on axonal degeneration in the nerve crush model. (A, B) Rats were injected with LPS (1 mg/kg) i.p. (A) or 100 ng of IL-4 subtenonly (B) at the same time as the nerve crush treatment and daily until the end of experiments. PBS was injected as a control (CTL). Representative immunostained images are shown of Tuj1 at 500 μm distal to choroid on days 7 and 14 after crush treatment. Scale bar: 100 μm. Each graph shows quantification of the residual nerve fibers in an area 300 × 500 μm from the choroidal plane, determined using BZ-X analysis software. Values are mean ± SD (n = 3 eyes). *P < 0.05 compared with control (PBS i.p.) on 14 day (unpaired Student's t-test).
Discussion
Under physiological and pathologic conditions, glial cells are thought to play important roles in the optic nerve. Because optic nerve disorders are a major cause of blindness, investigations of optic neuropathy should be conducted to understand the pathophysiology of ocular disorders, such as glaucoma, optic neuritis, optic atrophy, and traumatic injury. A number of animal models have been used in such investigations.1,2,2830 In some of these studies, optic nerve damage induced by crushing with forceps (i.e., an optic nerve crush model) has been used to study neuronal death, reactive gliosis, and axon regeneration.30 Previous studies have demonstrated that microglia exhibit a reactive morphology and proliferative activity in the damaged optic nerve during optic nerve remodeling following injury.41 Therefore, we used the optic nerve crush model for our investigation of microglia in the damaged optic nerve. However, although phagocytosis is regarded as an important step in the regeneration of axons, to our knowledge, this study is the first to report the different phagocytic functions of each subtype of microglia. 
We found that the numbers of Iba1-positive cells (microglia/macrophages) were increased significantly at the proximal site after optic nerve crush during the experimental period. However, the presence of 9F5-positive cells (type 1 microglia) was observed only at 14 days after the crush treatment. These observations suggest that most microglia in the damaged optic nerve are type 2 microglia not type 1 microglia. Although we are unable to exclude the possibility that macrophages might have constituted a portion of the Iba1-positive cell population, the transient characteristics of the localization of the 9F5-positive cells (type 1 microglia) differed from those of the type 2 microglia (and/or macrophages). 
In the nervous system, the phagocytic removal of damaged axons following injury is essential for axonal regeneration, and the axonal debris is engulfed by microglia through p38 MAPK activation.35 In our in vitro experiments, we investigated the phagocytic activities of each microglial subtype on axonal debris, thus providing the first report on this topic. Our results revealed that the cultured type 1 and 2 microglia were appropriately isolated. Comparison of the two microglial subtypes revealed that the type 2 microglia showed greater phagocytic activity than the type 1 microglia, although both subtypes exhibited phagocytosis. 
Microglia have been reported to phagocytose apoptotic cells,42 debris,35 and amyloid plaques19 in many disease models, and these activities are promoted by several cytokines in each pathogenic condition. Among these cytokines, IL-4 plays an inhibitory role in the engulfment of neuronal apoptotic cells.42 Our experiments revealed that responsiveness to IL-4 was very different between the two subtypes of microglia. In the minor population of microglia (type 1) in the damaged optic nerve, a high level of responsiveness to IL-4 and activation of STAT6 were observed, unlike the response observed in the type 2 microglia. IL-4 receptor and JAK complexes containing JAK1 and JAK3 result in intracellular signaling leading to STAT6 phosphorylation.43 IL-4 responsive microglia (type 1) exhibited high levels of JAK1 compared with type 2 microglia; however, IL-4 receptor expression was similar between type 1 and 2 microglia. Further experiments are required to clarify why different cellular events occur in response to IL-4 in each subtype of microglia. 
In contrast, the two subtypes were similarly responsive to LPS and IFN-γ treatments in terms of phagocytic activity. Previously, Tanaka et al.35 reported that the engulfment of axonal debris by microglia is enhanced by treatment with LPS, and this observation is consistent with our results. These findings suggest that IL-4 acts as a selective activator of type 1 microglia and that phagocytic activity under inflammatory conditions is shared by the two subtypes of microglia. Thus, these microglial subtypes might play different roles in the modulation of optic nerve damage. 
Our findings from optic nerve crush experiments indicated that LPS facilitated inflammation following nerve injury and accelerated the clearance of axonal debris by phagocytic cells. Conversely, fragmented axons persisted in optic nerves treated with IL-4, although we believe that daily subtenon injection itself triggered inflammation based on the observation that axons subjected to subtenon injection of PBS became swollen more than those subjected to the i.p. injection procedure. Although the phagocytosis of axonal debris by microglia was not observed in our in vivo experiments, previous electron microscopy studies have found that axonal debris was enclosed by microglia/macrophages and astrocytes in a rat crush model44 and that myelin debris in the extracellular spaces was phagocytosed by astrocytes in NMDA-induced Wallerian-like degeneration.45 The in vivo phagocytic behavior of microglia remained unclear in our experiments and requires further studies. 
Th1 CD4+ T cells secrete proinflammatory cytokines such as IFN-γ, tumor necrosis factor-α (TNF-α), and IL-2. In contrast, Th2 cells produce IL-4, IL-6, and IL-10.46 The in vivo crush model did not clarify the expression of IL-4/IFN-γ or activation of an IL-4-associated signal pathway; however, the infiltration of T cells at the injury site47 is thought to be caused by an increase in IFN-γ and IL-4 production. In addition, a recent study showed that CD4+ Th2 cells are the primary origin of IL-4 production in injured axonal cord and that IL-4 contributes to not only axon regrowth but also retinal ganglion cell survival.48 Consideration that the removal of axonal debris is important for nerve regeneration,35 skewing of the T cell population is associated with the phagocytic activity of each subtype of microglia and with axonal regeneration. However, whether IL-4-dependent inhibition of phagocytosis in type 1 microglia is associated with nerve degeneration remains unclear. Further studies are required to understand the role of microglial phagocytosis of axonal debris in nerve degeneration or regeneration. 
In conclusion, we revealed that type 1 microglia differ from type 2 microglia in terms of expression patterns, IL-4 responsiveness, and signal transduction. 
Acknowledgments
This work was supported in part by Japan Society for the Promotion of Science (JPS) KAKENHI grant 25462721. The sponsor or funding organization had no role in the design or conduct of this research. 
KK holds a patent related to antibody 9F5 (Japan patent number P4915610, 2011-09-09). The authors alone are responsible for the content and writing of the paper. 
Disclosure: A. Haga, None; E. Takahashi, None; Y. Inomata, None; K. Kawahara, P; H. Tanihara, None 
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Figure 1
 
Localization of type 1 and 2 microglia is shown in the rat optic nerve crush model. (A) Eyes were enucleated at 7, 14, and 21 days after crush treatment and embedded in paraffin. A hematoxylin-eosin staining image from day 14 after crush treatment is shown. An asterisk indicates the site crushed with forceps. Localization of microglia in the area demarcated by a black box in sections taken 500 μm distal to the choroidal plane. Scale bar: 500 μm. (B) Numbers of 9F5+/Iba1+ (white bar) and 9F5/Iba1+ (black bars) cells were counted in the area demarcated by a solid black line. Values are mean ± SD (n = 4 eyes). **P < 0.01/4; ***P < 0.001/4 compared with control. Unpaired Student's t-test were adjusted using Bonferroni correction. (C) Representative high-magnification images show 9F5 (green), Iba1 (red), and DAPI (blue) immunostaining. Scale bars: 200 μm.
Figure 1
 
Localization of type 1 and 2 microglia is shown in the rat optic nerve crush model. (A) Eyes were enucleated at 7, 14, and 21 days after crush treatment and embedded in paraffin. A hematoxylin-eosin staining image from day 14 after crush treatment is shown. An asterisk indicates the site crushed with forceps. Localization of microglia in the area demarcated by a black box in sections taken 500 μm distal to the choroidal plane. Scale bar: 500 μm. (B) Numbers of 9F5+/Iba1+ (white bar) and 9F5/Iba1+ (black bars) cells were counted in the area demarcated by a solid black line. Values are mean ± SD (n = 4 eyes). **P < 0.01/4; ***P < 0.001/4 compared with control. Unpaired Student's t-test were adjusted using Bonferroni correction. (C) Representative high-magnification images show 9F5 (green), Iba1 (red), and DAPI (blue) immunostaining. Scale bars: 200 μm.
Figure 2
 
Isolation of primary type 1 and 2 microglia. Rat primary type 1 and 2 microglia (MG) were isolated from mixed glial cultures from neonatal rat brains. Cells were photographed 24 hours after isolation. After fixation, cells stained with antibodies directed against 9F5 (type 1 MG), both Iba1 and CD68 (pan MG), GFAP (astrocytes), and MAP2 (neuronal cells) were examined using fluorescence microscopy. Scale bars: 100 μm.
Figure 2
 
Isolation of primary type 1 and 2 microglia. Rat primary type 1 and 2 microglia (MG) were isolated from mixed glial cultures from neonatal rat brains. Cells were photographed 24 hours after isolation. After fixation, cells stained with antibodies directed against 9F5 (type 1 MG), both Iba1 and CD68 (pan MG), GFAP (astrocytes), and MAP2 (neuronal cells) were examined using fluorescence microscopy. Scale bars: 100 μm.
Figure 3
 
Phagocytosis of axonal debris is shown by type 1 and 2 microglia. (A) A model of rat cortical explant culture is shown. After the chopped cortex (300–600 μm) was placed in a poly-l-lysine–coated dish, axons grew radially within 7 to 10 days. Explant was subjected to immunofluorescence analysis using an anti-Tuj1 antibody (green) and an anti-Iba1 antibody (red). Scale bar: 500 μm. (B) Coculture of microglia and axonal debris is shown. After primary microglia (4 × 105 cells/dish) were added to the explant culture dish, axons were transected with a scalpel blade (white dotted line). Scale bar: 200 μm. (C) Ninety-six hours after transection of the axons, immunostaining analysis was performed using anti-Tuj1 (green) and anti-Iba1 (red) antibodies. Nuclei were stained with DAPI (blue). Scale bar: 200 μm. (D) High-magnification view of the boxed region in C. Phagocytosed axonal debris was found within the microglia (arrows). Scale bar: 100 μm. (E, F) Type 1 or type 2 microglia (MG) were incubated with axonal debris for 96 hours. After fixation, immunofluorescence analyses were performed using antibodies directed against 9F5 (green) and Iba1 (red) in E and against Tuj1 (green) and Iba1 (red) in F. Nuclei were stained with DAPI (blue). All images were photographed 200 μm distal to the transection line. Scale bars: 200 μm. (G) Clearance area of axonal debris by types 1 and 2 MG. Axonal debris clearance area was measured using BZ-X analysis software. Values are mean ± SD (n = 4 samples). **P < 0.01 (unpaired Student's t-test).
Figure 3
 
Phagocytosis of axonal debris is shown by type 1 and 2 microglia. (A) A model of rat cortical explant culture is shown. After the chopped cortex (300–600 μm) was placed in a poly-l-lysine–coated dish, axons grew radially within 7 to 10 days. Explant was subjected to immunofluorescence analysis using an anti-Tuj1 antibody (green) and an anti-Iba1 antibody (red). Scale bar: 500 μm. (B) Coculture of microglia and axonal debris is shown. After primary microglia (4 × 105 cells/dish) were added to the explant culture dish, axons were transected with a scalpel blade (white dotted line). Scale bar: 200 μm. (C) Ninety-six hours after transection of the axons, immunostaining analysis was performed using anti-Tuj1 (green) and anti-Iba1 (red) antibodies. Nuclei were stained with DAPI (blue). Scale bar: 200 μm. (D) High-magnification view of the boxed region in C. Phagocytosed axonal debris was found within the microglia (arrows). Scale bar: 100 μm. (E, F) Type 1 or type 2 microglia (MG) were incubated with axonal debris for 96 hours. After fixation, immunofluorescence analyses were performed using antibodies directed against 9F5 (green) and Iba1 (red) in E and against Tuj1 (green) and Iba1 (red) in F. Nuclei were stained with DAPI (blue). All images were photographed 200 μm distal to the transection line. Scale bars: 200 μm. (G) Clearance area of axonal debris by types 1 and 2 MG. Axonal debris clearance area was measured using BZ-X analysis software. Values are mean ± SD (n = 4 samples). **P < 0.01 (unpaired Student's t-test).
Figure 4
 
Phagocytosis of axonal debris by microglia treated with LPS or IL-4. (A, B) Type 1 and 2 microglia (MG) were treated with LPS (100 ng/mL) or IL-4 (10 ng/mL) in serum-free medium for 4 hours before coculture with axonal debris. After 96 hours of incubation, immunostaining analyses were performed using the indicated antibodies. Nuclei were stained with DAPI. Scale bar: 200 μm. (C, D) Areas of axonal debris clearance by type 1 (C) and type 2 MG (D) 200 μm distal to the transection line were evaluated with BZ-X analysis software. Values are means ± SD (n = 4 samples). *P < 0.05/2; **P < 0.01/2 compared with control. Unpaired Student's t-test were adjusted with Bonferroni correction.
Figure 4
 
Phagocytosis of axonal debris by microglia treated with LPS or IL-4. (A, B) Type 1 and 2 microglia (MG) were treated with LPS (100 ng/mL) or IL-4 (10 ng/mL) in serum-free medium for 4 hours before coculture with axonal debris. After 96 hours of incubation, immunostaining analyses were performed using the indicated antibodies. Nuclei were stained with DAPI. Scale bar: 200 μm. (C, D) Areas of axonal debris clearance by type 1 (C) and type 2 MG (D) 200 μm distal to the transection line were evaluated with BZ-X analysis software. Values are means ± SD (n = 4 samples). *P < 0.05/2; **P < 0.01/2 compared with control. Unpaired Student's t-test were adjusted with Bonferroni correction.
Figure 5
 
Engulfment of axonal debris by type 1 and 2 microglia treated with IFN-γ. (A, B) Type 1 and 2 microglia (MG) were treated with IFN-γ (20 ng/mL) and cocultured with axonal debris, as in Figure 4. Representative images of immunostaining for 9F5 (green), Iba1 (red), and DAPI (blue) (A) and Tuj1 (green), Iba1 (red), and DAPI (blue) (B). Scale bar: 200 μm. (C, D) Clearance of axonal debris by type 1 (C) and type 2 MG (D) was quantified as in Figure 4. Values are means ± SD (n = 4 samples). **P < 0.01 (unpaired Student's t-test).
Figure 5
 
Engulfment of axonal debris by type 1 and 2 microglia treated with IFN-γ. (A, B) Type 1 and 2 microglia (MG) were treated with IFN-γ (20 ng/mL) and cocultured with axonal debris, as in Figure 4. Representative images of immunostaining for 9F5 (green), Iba1 (red), and DAPI (blue) (A) and Tuj1 (green), Iba1 (red), and DAPI (blue) (B). Scale bar: 200 μm. (C, D) Clearance of axonal debris by type 1 (C) and type 2 MG (D) was quantified as in Figure 4. Values are means ± SD (n = 4 samples). **P < 0.01 (unpaired Student's t-test).
Figure 6
 
IL-4 induced phosphorylation of STAT6 in microglia. (A) Equal amounts of protein from type 1 and 2 microglia (MG) were subjected to immunoblot analysis with the indicated antibodies. (B) Type 1 and 2 microglia were treated with IL-4 (10 ng/mL) in serum-free medium at the indicated times. Immunoblot analyses of phospho-STAT6 (p-STAT6), STAT6, and β-actin are shown. (C) Quantitative analyses of the levels of phosphorylated STAT6 in the microglia subtypes (type 1 [white bars]; type 2 [black bars]). Values are means ± SD (n = 3). **P < 0.01; ***P < 0.001 compared with type 2 microglia, using unpaired Student's t-test.
Figure 6
 
IL-4 induced phosphorylation of STAT6 in microglia. (A) Equal amounts of protein from type 1 and 2 microglia (MG) were subjected to immunoblot analysis with the indicated antibodies. (B) Type 1 and 2 microglia were treated with IL-4 (10 ng/mL) in serum-free medium at the indicated times. Immunoblot analyses of phospho-STAT6 (p-STAT6), STAT6, and β-actin are shown. (C) Quantitative analyses of the levels of phosphorylated STAT6 in the microglia subtypes (type 1 [white bars]; type 2 [black bars]). Values are means ± SD (n = 3). **P < 0.01; ***P < 0.001 compared with type 2 microglia, using unpaired Student's t-test.
Figure 7
 
Effects of IL-4 and LPS on axonal degeneration in the nerve crush model. (A, B) Rats were injected with LPS (1 mg/kg) i.p. (A) or 100 ng of IL-4 subtenonly (B) at the same time as the nerve crush treatment and daily until the end of experiments. PBS was injected as a control (CTL). Representative immunostained images are shown of Tuj1 at 500 μm distal to choroid on days 7 and 14 after crush treatment. Scale bar: 100 μm. Each graph shows quantification of the residual nerve fibers in an area 300 × 500 μm from the choroidal plane, determined using BZ-X analysis software. Values are mean ± SD (n = 3 eyes). *P < 0.05 compared with control (PBS i.p.) on 14 day (unpaired Student's t-test).
Figure 7
 
Effects of IL-4 and LPS on axonal degeneration in the nerve crush model. (A, B) Rats were injected with LPS (1 mg/kg) i.p. (A) or 100 ng of IL-4 subtenonly (B) at the same time as the nerve crush treatment and daily until the end of experiments. PBS was injected as a control (CTL). Representative immunostained images are shown of Tuj1 at 500 μm distal to choroid on days 7 and 14 after crush treatment. Scale bar: 100 μm. Each graph shows quantification of the residual nerve fibers in an area 300 × 500 μm from the choroidal plane, determined using BZ-X analysis software. Values are mean ± SD (n = 3 eyes). *P < 0.05 compared with control (PBS i.p.) on 14 day (unpaired Student's t-test).
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