Repair of astrocytes, blood vessels, and myelin in the injured brain: possible roles of blood monocytes
© Jeong et al.; licensee BioMed Central Ltd. 2013
Received: 21 March 2013
Accepted: 1 May 2013
Published: 10 June 2013
Inflammation in injured tissue has both repair functions and cytotoxic consequences. However, the issue of whether brain inflammation has a repair function has received little attention. Previously, we demonstrated monocyte infiltration and death of neurons and resident microglia in LPS-injected brains (Glia. 2007. 55:1577; Glia. 2008. 56:1039). Here, we found that astrocytes, oligodendrocytes, myelin, and endothelial cells disappeared in the damage core within 1–3 d and then re-appeared at 7–14 d, providing evidence of repair of the brain microenvironment. Since round Iba-1+/CD45+ monocytes infiltrated before the repair, we examined whether these cells were involved in the repair process. Analysis of mRNA expression profiles showed significant upregulation of repair/resolution-related genes, whereas proinflammatory-related genes were barely detectable at 3 d, a time when monocytes filled injury sites. Moreover, Iba-1+/CD45+ cells highly expressed phagocytic activity markers (e.g., the mannose receptors, CD68 and LAMP2), but not proinflammatory mediators (e.g., iNOS and IL1β). In addition, the distribution of round Iba-1+/CD45+ cells was spatially and temporally correlated with astrocyte recovery. We further found that monocytes in culture attracted astrocytes by releasing soluble factor(s). Together, these results suggest that brain inflammation mediated by monocytes functions to repair the microenvironment of the injured brain.
Brain inflammation accompanied by brain injury has been a focus of research efforts because of its possible roles in the onset and progression of a number of neurodegenerative diseases. However, most brain inflammation studies have focused on neurons, and little information on how brain inflammation affects other brain cells, including astrocytes and oligodendrocytes, is available. Astrocytes function to maintain the homeostasis of the brain microenvironment by taking up potassium, glutamate, and water from the extracellular space, and supplying nutrients and growth factors to neurons (for review, see). Oligodendrocytes myelinate axons, allowing for rapid propagation of action potentials to axon terminals. Therefore, it is important to know how these cells respond to brain injury.
Studies of systemic inflammation showed that inflammation has dual functions: a cytotoxic function to kill infected microbes and a repair function to regenerate damaged tissues[2, 3]. In the presence of proinflammatory stimulators such as IFN-γ, monocytes/macrophages are classically activated and protect the tissue from infection by producing cytotoxic inflammatory molecules[4, 5]. On the other hand, in the presence of IL-4 and IL-13, monocytes/macrophages are alternatively activated, and produce several molecules that are involved in anti-inflammation and repair/regeneration[6–9]. Therefore, in myocardial injury, monocytes/macrophages rapidly remove cell debris and lead to myofibroblast infiltration and collagen deposition through production of high levels of TGF-β and VEGF-A. In injured skin, macrophage depletion causes delays in healing processes[10, 11].
Previously, we reported that in lipopolysaccharide (LPS)- or ATP-injected brain and contusion-induced spinal cord, resident microglia as well as neurons die in the damage core and monocytes appear and fill the damage core[12–15]. In the ischemic brain, infiltration of blood monocytes has been reported[16, 17]. However, it is not clear how monocytes contribute to brain inflammation and what occurs after monocytes infiltrate into the injured brain.
In the present study, we examined how astrocytes, oligodendrocytes, and endothelial cells respond to damage in the LPS-injected substantia nigra (SN), a Parkinson’s disease-related brain area where LPS induces significant damage. We also examined the roles of infiltrating monocytes in the injured brain. The results of this study showed that damage to astrocytes, oligodendrocytes, and endothelial cells peaked at approximately 3 d. However, these cells recovered 7–14 d after infiltration from the blood of round Iba-1+/CD45+ monocytes. Importantly, these monocytes expressed repair/regeneration-related genes, suggesting that they may function to repair the damaged brain.
Recovery of the damaged microenvironment in the injured brain
Since most studies of brain injury have focused on neuronal damage, knowledge is limited on how other brain cells behave in the injured brain. In the present study, we first examined the time-dependent responses of brain cells, including astrocytes, endothelial cells, oligodendrocytes (by examining myelin) and inflammatory cells, in the injured brain. To achieve this, LPS (5 μg in 2 μl PBS) was injected into the substantia nigra pars compacta (SNpc); this region was chosen based on our previous observation that LPS induces significant damage to the SNpc but not to the cortex.
The disappearance and reappearance of GFAP+ astrocytes were not merely due to reduced GFAP immunoreactivity since other markers of astrocytes in addition to GFAP, including S100β (Ca2+-binding protein), EAAT1 (GLAST, excitatory amino acid transporter 1) and Kir4.1 (potassium channel), also disappeared at 3 d (Figure 1B-D) and reappeared at 14 d (arrows in Figure 1B-D). The areas in which each marker was absent were measured and plotted (Figure 1E-H). Using Nissl staining, we verified the death of astrocytes at 1 d (Additional file1: Figure S1).
Astrocytes and oligodendrocytes proliferate and migrate toward the damage
Possible involvement of brain inflammation in repair of damaged microenvironment of the brain
Primary antibodies used to detect microglia/monocytes
Next, we used double-immunolabeling to examine whether monocytes expressed proinflammatory genes and repair/resolution-related genes in the injured brain. Depending on the sources of antibodies used to examine expression of proteins, microglia and/or monocytes were identified by staining for Iba-1, CD11b, or CD45 in these cells (Table 1). Expression of iNOS, a major inflammatory mediator, was detected in myeloperoxidase (MPO)+ neutrophils at 12 h to 1 d after LPS injection, but not in CD11b+ or CD45+ cells (Figure 7C), as we previously reported. IL-1β expression was detected in ramified Iba-1+ cells at 3 h (Figure 7D). However, round CD45+ and Iba-1+ cells expressed neither iNOS nor IL-1β at 3–7 d (Figure 7D). Interestingly, most round CD45+ cells expressed mannose receptor (MR), which is known to play important roles in endocytosis/phagocytosis[25, 26] (Figure 7E). In addition, round but not ramified Iba-1+ cells highly expressed CD68, an indicator of lysosomal enzyme activity that is considered a marker of phagocytic activity (Figure 7F). Round CD11b+ cells also highly expressed LAMP2 (Figure 7G), a lysosomal protein that participates in the fusion of lysosomes and phagosomes. Since these monocytes have strong phagocytic activity, they may scavenge damaged cells and debris in the injured brain. These results suggest that monocytes may contribute to the recovery of impaired astrocytes in LPS-injected SNpc.
Possible spatial and temporal correlation between astrocytes and monocytes cells in the injured brain
The results in this study showed that 1) astrocytes, oligodendrocytes, myelin, and endothelial cells recovered in the injured brain; 2) the recovery of these cells and myelin occurred after infiltration of Iba-1+/CD45+ monocytes into injury sites; 3) Iba-1+/CD45+ monocytes expressed repair-related inflammatory mediators, but not cytotoxic proinflammatory mediators; and 4) in vitro, monocytes secreted soluble factor(s) that recruited astrocytes toward them. On the basis of these findings, we suggest that monocytes may function to repair the injured brain.
Many studies on cultured microglia have reported that brain inflammation is neurotoxic. However, brain inflammation in vivo is quite different from the inflammation associated with microglia in culture[12–15, 31]. Simply, in the brain, several types of cells including microglia, astrocytes, neurons, and blood inflammatory cells, both positively and negatively contribute to inflammation. Microglia continuously survey brain damage, isolate damaged areas[14, 32], and recruit monocytes. Astrocytes and/or neurons inhibit microglial activation[31, 34–37] and recruit monocytes. Neutrophils produce cytotoxic inflammatory mediators in the brain[12, 17]. However, the roles of monocytes in the brain have not been clearly defined. Since monocytes/macrophages can be differentially activated (cytotoxic/classical vs. repair/alternative activation) depending on the stimuli, their roles also differ according to the activation conditions[7, 9]. The results presented here showed that monocytes in the LPS-injected brain exhibited repair-promoting rather than neurotoxic phenotypes. In microarray, RT-PCR, and immunohistochemistry analyses, repair/resolution-related genes/proteins such as phagocytic activation markers were highly expressed in the LPS-injected brain during the period when monocytes appeared, whereas cytotoxic proinflammatory mediators were barely expressed (Figures 6,7). Phagocytosis is an important process for the repair/regeneration of damaged tissue because damaged cells and debris may act as detrimental factors that lead to further injury or hinder regeneration. In a multiple sclerosis animal model, stimulation of phagocytosis was shown to increase clearance of tissue debris, limit further destruction, and facilitate repair.
The question arising from the above considerations is how monocytes have a repair function instead of a neurotoxic function in the LPS-injected brain. There is a remote possibility that monocytes are activated by LPS since cerebrospinal fluid is exchanged about 11 times daily to maintain homeostasis of the brain in the adult rat brain. Monocytes may be alternatively activated by phagocytosis of apoptotic cells. It has been reported that phagocytosis alters the phenotypes of monocytes from a proinflammatory to an anti-inflammatory phenotype. Another possibility is that neutrophils that enter the brain prior to monocytes may induce alternative activation because neutrophils express IL-4, a strong inducer of alternative activation[6, 12, 42].
Next, we examined the issue of how astrocytes, oligodendrocytes, myelin, endothelial cells, and neurites reappeared in damaged areas. We detected Ki-67+ proliferating cells in the injured area, and these cells were merged with GFAP, Vimentin, and Olig2 (Figure 4), suggesting that astrocytes and oligodendrocytes proliferate and fill the damaged area. Interestingly, Olig2 immunoreactivity was located in the cytosol of GFAP+/Vimentin+ astrocytes and in the nuclei of CC-1+ oligodendrocytes (Figure 4C). It has been reported that Olig2 is expressed in progenitor cells of oligodendrocytes and astrocytes, as well as in reactive astrocytes in the injured brain[19, 20]. As we also showed in Figure 4C, it has been reported that Olig2 is located in the cytosol and nuclei of cells that are destined to become astrocytes and oligodendrocytes, respectively[43, 44]. Therefore, newly generated Ki67+/Olig2+ progenitors may replenish astrocytes and oligodendrocytes in the injured brain. It has been reported that monocytes express several chemokines, including IL-8, IP-10, CCL1, CCL2 (MCP-1), and CCL4[45, 46]. Monocytes also produce platelet-derived growth factor (PDGF), transforming growth factor beta (TGF-β), and hepatocyte growth factor (HGF)[47–49]. Therefore, monocytes may recruit and/or promote the proliferation of astrocytes and oligodendrocytes, and induce neurite outgrowth. These findings suggest that brain inflammation plays a role in repairing damaged brain tissue. Thus, we speculate that impairment of the repair function of inflammation due to aging or genetic mutations may result in delayed recovery from damage and neurodegeneration. In our microarray analysis, mRNA expression of several genes related to cell proliferation/migration and neurogenesis was upregulated at around the times when monocytes appeared in the brain (data not shown). Furthermore, we observed that, in culture, monocytes promote astrocyte migration toward them (Figure 9), suggesting that monocytes in the LPS-injected brain may attract astrocytes toward damaged areas for recovery. We expected that the recovery of oligodendrocytes might be similar to that of GFAP+ astrocytes, since they come to occupy the same microenvironment. In addition, astrocytes may contribute to the recovery of endothelial cells and neurite outgrowths since astrocytes also express neurotrophic factors and growth factors[50, 51]. Taken together, these observations suggest that monocytes may assist in regeneration of the brain microenvironment in the injured brain.
In the present study, impaired astrocytes, endothelial cells, and neurites were recovered, and myelination occurred, after monocytes had filled the lesion sites. We thus speculate that brain inflammation mediated by monocytes serves to repair damage in the injured brain. Our results highlight the physiological importance of brain inflammation in enhancing beneficial effects while minimizing harm.
Materials and methods
All experiments were performed in accordance with the approved animal protocols and guidelines established by the Ajou University School of Medicine Ethics Review Committee, and all animal work was approved by the Ethical Committee for Animal Research of Ajou University (Amc-28).
Stereotaxic surgery and drug injection
Male Sprague–Dawley rats (230–250 g, 7 weeks old) were anesthetized by ketamine (40–80 mg/kg) and xylazine (5–10 mg/kg), and positioned in a stereotaxic apparatus (David Kopf Instruments, Tujunga, CA, USA). LPS (5 μg in 2 μl sterile PBS; Sigma, St. Louis, MO, USA) was unilaterally administered into the right SNpc (AP, -5.3 mm; ML, -2.3 mm; DV, -7.6 mm from the bregma), according to the atlas of Paxinos and Watson. All animals were injected using a Hamilton syringe equipped with a 30-gauge blunt needle and attached to a syringe pump (KD Scientific, New Hope, PA, USA). LPS was infused at a rate of 0.4 μl/min. After injection, the needle was held in place for an additional 5 min before removal.
For immunostaining, rats were anesthetized and transcardially perfused with saline solution containing 0.5% sodium nitrate and heparin (10 U/ml), followed by 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2) for tissue fixation. Brains were obtained and post-fixed overnight at 4°C in 4% paraformaldehyde. Fixed brains were stored at 4°C in a 30% sucrose solution until they sank. Six separate series of 30-μm coronal brain sections (50 μm for stereological counting) were obtained using a sliding microtome (Microm, Walldorf, Germany).
For RNA preparation, rats were anesthetized and transcardially perfused with saline solution without paraformaldehyde. Brains were obtained and sliced with a Rat Brain Slicer Matrix (1.0 mm slice intervals, RBM-4000C; ASI Instruments, Warren, MI, USA) and a razor blade. A slice that included the needle injection spot was selected, and tissue blocks (2 × 2 × 2 mm) just below the needle tip were collected and stored at −70°C until use.
Primary antibodies used in immunohistochemistry
Reverse transcriptase-polymerase chain reaction (RT-PCR)
Primer sequences for RT-PCR
Sample preparation and labeling
Microarray experiments were performed in duplicate. For each sample, total RNA was extracted from tissue blocks (2 × 2 × 2 mm) obtained from four LPS-injected and uninjected rat brains using RNeasy mini kits (Qiagen GmbH, Hilden, Germany). Quantity and quality of RNA were assayed by UV spectrometry and RNA gel electrophoresis. RNA was labeled and hybridized to a GeneChip according to Standard Affymetrix Protocols (GeneChip Whole Transcript Sense Target Labeling Assay Manual, Version 4; Affymetrix, Santa Clara, CA). Affymetrix GeneChip Rat Gene 1.0 ST Arrays were used in this study. Each reaction involving a single GeneChip hybridization was initiated with 200 ng RNA. cDNA and cRNA were generated using a GeneChip WT cDNA Synthesis and Amplification Kit (900673, Affymetrix); cRNA cleanup was performed using a GeneChip IVT cRNA Cleanup Kit (900547, Affymetrix). After the second cDNA synthesis, cRNA was hydrolyzed by RNase H treatment, and biotin-labeled sense strand DNA fragments were generated using a GeneChip WT Terminal Labeling Kit (900671, Affymetrix).
Hybridization and scanning
Biotin-labeled DNA fragments (target) or controls in a hybridization cocktail were hybridized to the GeneChip array by incubating for 16 h in a GeneChip Hybridization Oven 640. Immediately following hybridization, the array was washed and stained with a streptavidin-phycoerythrin conjugate on the GeneChip Fluidics Station 450 using an automated protocol, followed by scanning on a GeneChip Scanner 3000 (7G). The GeneChip Hybridization, Wash, and Stain Kit (900720, Affymetrix) was used in this procedure.
Data measurement and analysis
An Affymetrix GeneChip scanner operated by GeneChip Operating Software (GCOS ver1.4; Affymetrix) was used to generate original array images. The average difference for each probe set (a measure of the relative abundance of a transcript) and signals and detection calls (i.e., present or absent) were computed using GCOS. Data were analyzed using Silicon Genetics Genespring 10.1 Software.
Eriochrome cyanine RC staining
Myelin was visualized with Eriochrome Cyanine RC (ECRC; Sigma). Brain sections were mounted on slides and air-dried overnight at room temperature. After dehydration and rehydration in graded ethanol solutions, sections were stained with ECRC solution (0.2% in 0.5% H2SO4) for 10 min, rinsed in running tap water, placed in 1% NH4OH for 30 s, and rinsed in distilled water. After dehydration, sections were treated with xylene and mounted using Permount (Fisher Scientific Co., Morris Plains, NJ, USA).
Measurement of damaged areas
Damaged areas were measured by staining every sixth brain section of the whole midbrain (AP, -4.3 to −6.5 mm from the bregma) with antibodies for specific markers of astrocytes (GFAP, S100β, EAAT1, and Kir4.1), endothelial cells (SMI 71), and neurites (MAP2). Myelin was stained with ECRC. Specific marker-negative areas in serial sections were measured on 4×-magnified images using Axiovision image-analysis software (version 4.7.2; Zeiss) and summed as shown in Additional file3: Figure S3.
Primary astrocytes were cultured from the cerebral cortices of 1-day-old Sprague–Dawley rats, as described previously[53, 54]. In brief, cortices were triturated in MEM (Sigma) containing 10% FBS (HyClone, Logan, UT, USA), plated in 75 cm2 T-flasks (0.5 hemisphere/flask), and incubated for 2–3 weeks. Microglia were removed from flasks by mild shaking, and astrocytes were cultured in serum-free MEM for 2–3 d. Astrocytes were harvested with 0.1% trypsin, plated, and cultured in MEM containing 10% FBS before use. Purity of astrocytes (>95%) was confirmed using GFAP antibodies.
Rat blood monocytes were isolated as described previously. Briefly, blood was obtained by cardiac puncture and mixed with 2.5% dextran in PBS for 1 h at RT. The plasma layer was centrifuged at 300× g for 12 min. To remove red blood cells (RBC), the pellet was suspended in PBS containing 0.15 M NH4Cl, 10 mM NaHCO3, and 0.1 mM EDTA, and centrifuged at 350× g for 6 min. This process was repeated twice. Pellets containing monocytes and lymphocytes were suspended in PBS and placed in 15-ml polystyrene conical tubes (BD Falcon, NJ, USA). An equal volume of Ficoll-Paque PLUS (GE Healthcare, Uppsala, Sweden) was carefully added to the bottom of cell-containing tubes so as to prevent mixing with PBS. After centrifugation at 450× g for 30 min, cells between the Ficoll and PBS layers were collected, washed with PBS, suspended in HBSS containing calcium (140 mg/l), and plated in a Petri dish for 30 min. Unattached lymphocytes were removed and adherent monocytes were collected and cultured in MEM containing 10% FBS.
Astrocyte migration assay
Astrocyte migration was examined using a polydimethylsiloxane (PDMS) device that was a gift from N. Jeon (Seoul National University). Briefly, PDMS device was comprised of two compartments. Each compartment had two reservoirs in the form of 8-mm-diameter holes, one at either end of the compartment, which served as a loading gate and medium reservoir, respectively (Figure 9A). The two holes in each compartment were connected by a main channel that was 1.5 mm wide, 100 μm high, and 7 mm in length. The two compartments were connected by 100 grooves, each 10 μm wide, 5 μm high, and 450 μm in length. Primary astrocytes (3 × 104 cells/125-150 μl for each hole) were plated in one compartment, and culture media (150 μl for each hole) was added to the other compartment. The following day, the media was removed from the media-filled compartment and then monocytes (2.5 × 104 cells/125-150 μl for each hole) were added to the compartment. The size of the grooves was sufficiently small that cells could not pass over to the opposite compartment during loading. The volume difference (50 μl) between compartments leads to convective flow from the higher volume side to the lower volume side. The difference in volume slowly decreased over time, but was still 10 μl at 7 d. Phase contrast images were taken every day and calcein AM-labeled fluorescence images were taken at 7 d using an AxioVision fluorescence light microscope (Zeiss Axiovert 200 M).
All values are expressed as means ± SEMs. The statistical significance of differences between mean values was assessed by Student’s t-test using the Statistical Package for Social Sciences 8.0 (SPSS Inc., Chicago, IL, USA).
This work was supported by a KOSEF NRL Program grant (no. 2-2008025-0) funded by the Korean government (MEST), a grant (NRF-2012R1A5A2051429) from KOSEF through the Chronic Inflammatory Disease Research Center (CIDRC) at Ajou University, and a National Research Foundation of Korea Grant (NRF-2011-355-E00087) funded by the Korean Government (Ministry of Education, Science and Technology).
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