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J Cereb Blood Flow Metab. 2010 Apr; 30(4): 871–882.
Published online 2009 Dec 9. doi:  10.1038/jcbfm.2009.257
PMCID: PMC2949171

Inhibition of reactive astrocytes with fluorocitrate retards neurovascular remodeling and recovery after focal cerebral ischemia in mice


Glial scarring is traditionally thought to be detrimental after stroke. But emerging studies now suggest that reactive astrocytes may also contribute to neurovascular remodeling. Here, we assessed the effects and mechanisms of metabolic inhibition of reactive astrocytes in a mouse model of stroke recovery. Five days after stroke onset, astrocytes were metabolically inhibited with fluorocitrate (FC, 1 nmol). Markers of reactive astrocytes (glial fibrillary acidic protein (GFAP), HMGB1), markers of neurovascular remodeling (CD31, synaptophysin, PSD95), and behavioral outcomes (neuroscore, rotarod latency) were quantified from 1 to 14 days. As expected, focal cerebral ischemia induced significant neurological deficits in mice. But over the course of 14 days after stroke onset, a steady improvement in neuroscore and rotarod latencies were observed as the mice spontaneously recovered. Reactive astrocytes coexpressing GFAP and HMGB1 increased in peri-infarct cortex from 1 to 14 days after cerebral ischemia in parallel with an increase in the neurovascular remodeling markers CD31, synaptophysin, and PSD95. Compared with stroke-only controls, FC-treated mice demonstrated a significant decrease in HMGB1-positive reactive astrocytes and neurovascular remodeling, as well as a corresponding worsening of behavioral recovery. Our results suggest that reactive astrocytes in peri-infarct cortex may promote neurovascular remodeling, and these glial responses may aid functional recovery after stroke.

Keywords: angiogenesis, astrocytes, cerebral ischemia, high-mobility group box 1, neuroplasticity, neuroprotection


Glia are known to have key roles in how the brain responds to injury. After stroke and central nervous system injury, traditional thinking presumes that reactive astrocytes contribute to glial scarring that is detrimental to neuronal recovery (Horner and Gage, 2000; Fawcett and Asher, 1999; Silver and Miller, 2004; Giulian, 1993). Reactive glial scars produce many extracellular matrix substrates that are known to inhibit dendritic and axonal growth, such as chondroitin sulfate proteoglycans, NOGO, myelin-associated proteins, and semaphorins (Shen et al, 2008; Jones et al, 2003; GrandPre et al, 2000; Moreau-Fauvarque et al, 2003). Indeed, many strategies have been designed to enhance central nervous system recovery by targeting reactive glia and their inhibitory substrates (Horner and Gage, 2000; Fawcett and Asher, 1999; Silver and Miller, 2004; Giulian, 1993; Li et al, 2005).

Damaged brain can be surprisingly plastic, and crosstalk between various types of remodeling brain cells take place after brain injury (Chen and Swanson, 2003; Chopp et al, 2007; Arai et al, 2009). The generation of new blood vessels facilitates highly coupled neurorestorative processes including neurogenesis and synaptogenesis (Chopp et al, 2007; Zhang et al, 2000; Sun et al, 2003). And these multicellular processes of neurovascular remodeling, in turn, may lead to improved functional recovery. In this context, it has been proposed that reactive glia may also possess beneficial properties that promote recovery. For example, reactive microglia may release neurotoxic substances (Giulian, 1993), but under some conditions, microglia may also secrete neurotrophic molecules (Nakajima et al, 2002; Bessis et al, 2007). A recent study demonstrated that glial fibrillary acidic protein (GFAP) knockout mice experienced worse outcomes after brain traumatic excitotoxicity (Otani et al, 2006). Hence, the assumption that reactive glia are only damaging may not be entirely accurate, and the balance between injury and repair may depend on the timing and local environments involved after stroke (Lo, 2008).

In this study, we tested the hypothesis that reactive astrocytes may contribute to recovery after stroke. Using a model of focal cerebral ischemia in mice, we metabolically inhibited astrocytes using the well-established compound fluorocitrate (FC), and then measured various markers of neurovascular remodeling and behavioral recovery over time. Our findings suggest that reactive astrocytes in the peri-infarct cortex, including populations that coexpress HMGB1, may be required for endogenous recovery after stroke.

Materials and methods

Focal Cerebral Ischemia Model

Male ddY mice (25–35 g, Kiwa Experimental Animal Laboratory, Wakayama, Japan) were kept under a 12-h light/dark cycle (lights on from 0700 to 1900 h) in an air-conditioned room (23°C±2°C) with ad libitum access to food and water. All procedures regarding animal care and use were performed in compliance with the regulations established by the Experimental Animal Care and Use Committee of Fukuoka University. Focal cerebral ischemia was induced according to the standard methods (Mishima et al, 2005). Briefly, mice were anesthetized with 2% halothane and maintained with 1% halothane (Flosen, Takeda Chemical Industries, Osaka, Japan). After a midline neck incision, the left common and external carotid arteries were isolated and ligated. A nylon monofilament (8-0; Ethilon, Johnson&Johnson, Tokyo, Japan) coated with silicon resin (Xantopren, Heleus Dental Material, Osaka, Japan) was introduced through a small incision into the common carotid artery and advanced to a position 9 mm distal from the carotid bifurcation, to occlude the middle cerebral artery. Adequate cerebral ischemia was confirmed by examining the forelimb flexion after the mice recovered from anesthesia. Four hours after occlusion, the mice were reanesthetized, and reperfusion was established by withdrawal of the filament. All experiments were conducted in a masked and randomized manner with end points assessed by investigators with no knowledge of treatment group allocations. Exclusion criteria included checking for successful arterial occlusions with neurological deficits.

Behavioral Assessments

Behavioral deficits after cerebral ischemia were assessed using a standard neuroscore (Hayakawa et al, 2008a) and a rotarod test (Egashira et al, 2005) on days 1, 3, 7, and 14. Neurological scoring was performed on a 1–5 scale: 0, normal motor function; 1, flexion of the torso and of the contralateral forelimb on lifting of the animal by the tail; 2, circling to the ipsilateral side but normal posture at rest; 3, circling to the ipsilateral side; 4, rolling to the ipsilateral side; and 5, leaning to the ipsilateral side at rest (no spontaneous motor activity). Motor coordination was measured using the rotarod test, where mice were placed on a rotating rod (3 cm diameter, 5 r.p.m., Neuroscience, Tokyo, Japan) with a nonskid surface, and the latency to fall was measured for up to 2 mins.


Primary antibodies for Western blotting and immunohistochemistry were used as follows: 1:200 dilution of rabbit polyclonal anti-GFAP antibody (Ventana, Kanagawa, Japan), 1:200 dilution of goat polyclonal anti-HMGB1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), 1:200 dilution of rabbit polyclonal anti-HMGB1 antibody (Abcam, Tokyo, Japan), 1:200 dilution of rabbit polyclonal anti-synaptophysin (before synaptic marker) antibody (Santa Cruz Biotechnology), 1:200 dilution of rabbit polyclonal anti-PSD95 (after synaptic marker) antibody (Abcam), 1:100 dilution of rat monoclonal anti-mouse CD31 antibody (BD Pharmingen, Franklin Lakes, NJ, USA). Secondary antibodies for Western blotting and immunohistochemimstry were used as follows: 1:200 dilution of donkey anti-goat immunoglobulin G (IgG)-fluorescein isothiocyanate secondary antibody (Santa Cruz Biotechnology), 1:200 dilution of goat anti-rabbit IgG-Texas Red (Santa Cruz Biotechnology), 1:200 dilution of goat anti-rabbit IgG-AP (Bio-Rad, Tokyo, Japan).


Mice were killed by decapitation after perfusion with saline and 4% paraformaldehyde at 1, 3, 7, and 14 days after cerebral ischemia–reperfusion. The brains were cleared of fat and water using an auto degreasing unit (RH-12, Sakura Seiko, Tokyo, Japan) and then embedded in paraffin. Subsequently, 5-μm-thick sections were mounted on slides and dried at 37°C for 1 day. After deparaffinization and rehydration, the sections were incubated with the GFAP- and HMGB1-antibody, GFAP- and CD31-antibody, HMGB1- and CD31-antibody overnight at 4°C. The sections were then incubated with a 1:200 dilution of donkey anti-goat IgG-fluorescein isothiocyanate secondary antibody for 1 h. The sections were also incubated with a 1:200 dilution of goat anti-rabbit IgG-Texas red secondary antibody for 1 h. The density of staining was analyzed by using NIH ImageJ software (Bethesda, MD, USA) from representative staining sections, and the measurement was repeated three times in each brain as described before (Jin et al, 2009).

TUNEL Staining

After deparaffinization (100% xylene for 5 mins × 3 times) and rehydration (100% ethanol 5 mins, 100% ethanol 3 mins, 95% ethanol 3 mins, 85% ethanol 3 mins, 75% ethanol 3 mins, 50% ethanol 3 mins, phosphate-buffered saline 1 min), sections were assayed for TUNEL (terminal deoxynucleotidyl transferase-mediated 2′-deoxyuridine 5′-triphosphate-biotin nick end labeling) using direct binding of fluorescein-conjugated dUTP (green fluorochrome) with anti-mouse NeuN (Chemicon Internatinal, Temecula, CA, USA), providing the red counterstain, and using the fluorescein isothiocyanate-Apoptosis detection system (Promega, Tokyo, Japan).


The expression of HMGB1, synaptophysin, and PSD95 protein was evaluated by Western blotting after sample extraction and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Tissue samples (striatum of the ischemic core, cerebral cortex of the peri-infarct areas) were homogenized at 4°C for 1 min in lysis buffer (20 mmol/L Tris (pH 7.4), 1 mmol/L ethylenediaminetetraacetic acid, 1 mmol/L ethylene glycol bis(â-aminoethylether)-N,N,N',N',-tetraacetic acid, and 0.1% Triton X-100) with protease inhibitor cocktail for use with mammalian cell and tissue extracts (Sigma-Aldrich, Tokyo, Japan). The tissue extract was centrifuged at 15,000 g at 4°C for 30 mins. The supernatant was treated in the same manner as the tissue extract.

Sodium dodecyl sulfate sample buffer (125 mmol/L Tris (pH 6.8), 2% sodium dodecyl sulfate, 20% glycerol, 0.0001% Bromo Phenol Blue, and 10% β-mercaptoethanol) was added to aliquots of tissue extracts containing 15 μg total protein. Samples were heated at 95°C for 5 mins. Protein (15 μg) was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12% gels). Semidry blotting was performed at 2 mA/cm2 (Bio-Rad). The blots were blocked with 5% nonfat dry milk in Tris-buffered saline with 0.1% Tween 20 at 4°C, and incubated the primary antibody followed by goat anti-rabbit IgG (H+L) AP conjugate (1:1000) in TBS-T and bovine anti-goat IgG (H+L) AP conjugate (1:1000) in TBS-T. The blots were visualized by AP color reagents. The signal intensity of the blots was measured by an image analysis system (NIH Image, version 1.63).

Metabolic Inhibition of Astrocytes

To test the hypothesis that reactive astrocytes were required for stroke recovery, we metabolically inhibited astrocytes using FC. The FC solution for intracortical injection was prepared as follows: 8 mg of D,L-fluorocitric acid, Ba, salt (Sigma-Aldrich) was dissolved in 1 ml of 0.1 mmol/L HCL. Two to three drops of 0.1 mmol/L Na2SO4 were added to precipitate the Ba2+. Two milliliters of 0.1 mmol/L Na2HPO4 was added, and the suspension was centrifuged at 1,000 g for 5 mins. The supernatant was diluted with 0.9% NaCl to the final concentration, and the pH was adjusted to 7.4. The FC solution was microinjected stereotaxically into the sensory motor cortex region (anterior: −0.22 mm; lateral: 2.5 mm from bregma; depth: 2 mm from the skull surface) once every 2 days starting 5 days after ischemia. One microliter FC solution was injected continuously at a rate of 0.25 μL/min through a stainless steel cannula (28 gauge) connected to a 25-μL syringe driven by a slow-injection pump. FC optimization studies were also performed to select FC administration protocols (see Results section).

Statistical Analysis

For continuous-scale end points such as infarct size, immunostaining or Western blot densitometry, and rotarod latencies, parametric Student's t-tests, analysis of variance, and Tukey–Kramer tests were used. For nonparametric neurological scoring, Kruskal–Wallis and Mann–Whitney's U-tests were used. P<0.05 was considered to be statistically significant.


Spontaneous Functional Recovery in Mice After Focal Cerebral Ischemia

As expected, 4 h of transient focal cerebral ischemia induced significant neurological deficits in mice. But over the course of the next 14 days after stroke onset, all mice gradually and spontaneously recovered (Figures 1A and 1B). Histological examination of brains at 14 days revealed well-defined infarcts, with a necrotic core in the striatum and peri-infarct areas located in the overlying cortex (Figure 1C).

Figure 1
Transient focal cerebral ischemia induced clear infarction and neurological deficits in mice. Behavioral deficits were measured with (A) the neuroscore and (B) motor coordination on the rotarod test. Over the course of 14 days after stroke, spontaneous ...

Increased Expression of HMGB1-Positive Reactive Astrocytes in Peri-Infarct Cortex

Well-developed gliosis was apparent in all brains after stroke. By day 14, GFAP-positive astrocytes were primarily localized in peri-infarct cortex (Figure 2A). Astrogliosis seemed to be mainly restricted to these infarct boundaries. No changes were observed within the infarcted core of the striatum (Figure 2A). Peri-infarct reactive astrocytes coexpressed HMGB-1 (Figure 2B). IBA1-positive microglia were present as well, but they were not HMGB-1-positive (Figure 2C).

Figure 2
Increased expression of HMGB1-positive reactive astrocytes in peri-infarct cortex. (A) By day 14, GFAP-positive astrocytes were primarily localized in peri-infarct cortex (n=5). Astrogliosis seemed to be mainly restricted to these infarct boundaries. ...

Increased Expression of Neurovascular Markers in Peri-Infarct Cortex

Various markers of neurovascular remodeling were detected in peri-infarct cortex of mice at 14 days after focal cerebral ischemia. CD31-positive microvessels were elevated on immunostained sections (Figure 3A). The synaptic markers synaptophysin and PSD95 were increased on Western blots (Figure 3B). Neurovascular remodeling appeared to be restricted to peri-infarct cortex, and no changes were observed in the central core areas of the infarcted striatum (Figures 3A and 3B).

Figure 3
Increased expression of neurovascular markers, CD31, synaptophysin, and PSD95 in peri-infarct cortex. (A) CD31-positive microvessels were elevated on immunostained sections (n=5). (B) The synaptic markers synaptophysin and PSD95 were increased on Western ...

The Metabolic Inhibitor FC did not Affect Astrocytes in Normal Brains

Because peri-infarct areas of neurovascular remodeling seemed to coexist with increased HMGB-1-positive reactive astrocytes, we asked whether reactive astrocytes were required for remodeling to take place. FC was chosen as a metabolic inhibitor of reactive astrocytes. First, we had to assess FC in normal brains. Direct intracortical injections of FC were performed in normal mice, and brain homogenates were probed for GFAP expression. No effects were detected for 0.1 and 1 nmol concentrations (Figure 4A). But the highest FC dose of 2 nmol appeared to be gliotoxic as GFAP levels were significantly reduced (Figure 4A). Next, we assessed the feasibility of using multiple injections of FC. Selecting the 1 nmol concentration, FC was injected into the brains of normal mice, 5 times over the course of 10 days. Western blots demonstrated no effects on GFAP (Figure 4B), and established that this approach of metabolically inhibiting reactive astrocytes should have no gliotoxic artifacts in normal tissue.

Figure 4
The metabolic inhibitor fluorocitrate (FC) did not affect astrocytes in normal brains. (A) No effects were detected for 0.1 and 1 nmol concentrations. But the highest FC dose of 2 nmol appeared to be gliotoxic as GFAP levels were significantly ...

Fluorocitrate Suppressed HMGB1-Positive Reactive Astrocytes After Focal Cerebral Ischemia

Optimization studies were performed to select the FC treatment protocols. Daily FC treatments with 1 nmol per site resulted in severe mortality after cerebral ischemia (Figure 5A). So, we selected the schedule of treating every other day. FC (1 nmol) or saline was directly injected into the peri-infarct cortex of mice starting on day 5 after focal cerebral ischemia. A total of 5 and 10 injections were applied. Then brains were removed at day 14. FC did not appear to affect cell death per se as there were no clear differences in TUNEL stains (Figure 5B). There were also no clear differences in the levels of GFAP astrocytes (Figure 5C). But FC significantly decreased the expression HMGB1 (Figure 5D).

Figure 5
Fluorocitrate (FC) suppressed HMGB1-positive reactive astrocytes after focal cerebral ischemia. (A) Initial studies demonstrated that daily FC was not feasible (▴), resulting in severe mortality after cerebral ischemia (day14; 0/10 animals). But ...

Fluorocitrate Suppressed Neurovascular Markers in Peri-Infarct Cortex and Worsened Neurological Function After Focal Cerebral Ischemia

Various markers of neurovascular remodeling were elevated in peri-infarct cortex on day 14 after focal cerebral ischemia. CD31-positive microvessels were found in close proximity with GFAP- and HMGB1-positive reactive astrocytes (Figures 6A and 6B). FC significantly suppressed the fluorescent density of CD31 and HMGB1 (Figure 6C). But there were no effects on GFAP levels (Figure 6C). In parallel with these vascular responses, peri-infarct levels of the synaptic proteins synaptophysin and PSD95 were also suppressed by FC (Figures 6D and 6E). Corresponding to these effects on neurovascular markers, FC seemed to affect neurological recovery as well. There was significantly worsening of the neuroscore at day 14 (Figure 7A). And motor deficits on the rotarod test was significantly exacerbated by FC on day 14 (Figure 7B).

Figure 6
Fluorocitrate (FC) suppressed neurovascular markers in peri-infarct cortex. Various markers of neurovascular remodeling were elevated in peri-infarct cortex on day 14 after focal cerebral ischemia. CD31-positive microvessels were found in close proximity ...
Figure 7
Fluorocitrate (FC) worsened neurological function after focal cerebral ischemia. (A) There was significantly worsening of the neuroscore, *P<0.05; saline- versus FC-treated group in middle cerebral artery (MCA) occlusion (Mann–Whitney's ...


In this study, spontaneous neurological recovery occurred in mice after transient focal cerebral ischemia. Examination of peri-infarct cortex revealed an elevation in various markers of neurovascular remodeling, such as CD31-positive microvessels and the synaptic proteins synaptophysin and PSD95. Treatment with the astrocytic metabolic inhibitor FC significantly decreased HMGB1-expressing astrocytes, suppressed neurovascular markers, and correspondingly worsened neurological outcomes. These results suggest that certain subsets of reactive astrocytes in peri-infarct cortex may promote functional recovery through brain remodeling after stroke.

Astrocytes have critical supportive roles for neurons. Without astrocytic glutamate reuptake mechanisms, neurons would be especially vulnerable to excitotoxicity (Rossi et al, 2007; Voloboueva et al, 2007). Without astrocytic radical scavenging enzymes, neurons become vulnerable to ischemia–reperfusion (Ouyang et al, 2007). Conversely, sick or dysfunctional astrocytes would induce neurotoxicity. For example, astrocytes from the SOD1 mutant mice are directly neurotoxic (Nagai et al, 2007). In contrast to beneficial supportive actions under normal conditions, reactive astrocytes after brain injury may have detrimental properties. Reactive astrocytes have been traditionally thought to contribute to scarring after stroke and central nervous system injury (Horner and Gage, 2000; Fawcett and Asher, 1999; Silver and Miller, 2004; Giulian, 1993). Under these pathological conditions, reactive astrocytes can secrete many substrates that are inhibitory against neuronal plasticity and axonal growth (Shen et al, 2008; Jones et al, 2003). Indeed, many efforts have focused on blocking these inhibitory glial factors and substrates to promote central nervous system recovery after injury and disease (Horner and Gage, 2000; Fawcett and Asher, 1999; Silver and Miller, 2004; Li et al, 2005).

More recently, however, emerging data suggest that the role of the reactive astrocyte may be more complex. In GFAP knockout mice, brain injury after trauma was significantly worsened, suggesting that these cells may in fact also possess beneficial actions (Otani et al, 2006). In this study, we found that a specific subset of reactive astrocytes expressing HMGB1 may be involved in peri-infarct recovery. HMGB1 is a nonhistone DNA-binding protein that is widely expressed in various tissues, including the brain. HMGB1 is a multifunctional molecule that can also act as an extracellular trigger and/or modulator of critical cell processes such as inflammation, proliferation, migration, and survival. Recently, HMGB1 was shown to be released into the extracellular space in massive amounts immediately after an ischemic insult (Qiu et al, 2008). Others reported that HMGB1 mediated neuroinflammation and microglial activation in the ischemic core after focal cerebral ischemia (Kim et al, 2006). Hence, blockade of HMGB1 signaling during the acute phases of stroke may be beneficial (Liu et al, 2007). In an earlier study, we reported that early inhibition of microglia expressing HMGB1 by treatment with minocycline significantly reduced infarction (Hayakawa et al, 2008b).

In contrast to these negative effects, it has also been reported that HMGB1 may also possess beneficial actions. HMGB1 signaling can promote endothelial activation (Treutiger et al, 2003) and sprouting (Schlueter et al, 2005). And it has also been reported that HMGB1 may increase neurite outgrowth and cell survival in neurons (Passalacqua et al, 1998; Huttunen et al, 2000; Huttunen et al, 2002). Recently, stimulated astrocytes have been shown to induce and release HMGB1 protein into the extracellular medium (Passalacqua et al, 1998; Kim et al, 2008). In addition, HMGB1 immunoreactivity was also observed in reactive astrocytes, which are concentrated in the ischemic penumbra in an in vivo ischemic model (Kim et al, 2008). In this context, HMGB1 may provide a potential missing link between reactive astrocytes and stroke recovery. Our findings here are consistent with this idea. Metabolic inhibition of astrocytes with FC suppressed HMGB1-positive astrocytes without broadly affecting neuronal damage in the peri-infarct cortex after cerebral ischemia.

An important caveat for this study involves the question of specificity. We used FC to metabolically inhibit astrocytes. But of course, FC is not specific for HMGB1-expressing astrocytes alone. Overall, metabolic suppression of astrocytes will lead to many downstream effects. For example, astrocytic metabolic failure can lead to a massive release of glutamate and other excitatory amino acids, resulting in seizures and brain injury (Broberg et al, 2008). Although we did not observe any seizures with the FC doses used here, we acknowledge that the worsening of outcomes induced by FC in our experiments may also have an excitotoxic component as well. Furthermore, we do not know exactly how much metabolic inhibition occurs with our FC dosing protocols. Injections of FC at 1 nmol concentrations inhibits metabolism without outright destruction of astroglial cells. Others showed that this type of metabolic suppression is transient. At 24 h after injection of 1 nmol FC, astroglial cells mostly recovered, whereas 2 nmol FC resulted in irreversible degeneration of both neurons and glial cells (Paulsen et al, 1987). We selected our FC protocol on the basis of a series of optimization studies. Daily FC treatments with 1 nmol per site resulted in severe mortality after cerebral ischemia. Whereas treating every other day seemed to decrease HMGB1 expression up to 14 days without affecting the number of TUNEL-positive cells. But it is acknowledged that we do not know exactly how much and for how long astrocytes are being suppressed in our experiments.

In our study, we noted that astrocytic suppression with FC appeared to decrease HMGB1 subsets without affecting overall GFAP levels. In our mouse model, GFAP-positive astrocytes without HMGB1 expression showed up early and persisted up to 14 days after cerebral ischemia. Because FC was infused every 2 days beginning on day 5, there is at least the theoretical possibility that these astrocytes could recover and reconstruct the cytoskeleton GFAP protein in between injections of FC. Conversely, HMGB1-positive astrocytes were only observed at later times from day 7 onwards. Our data suggest that these delayed populations of astrocytes may somehow be more susceptible to FC-mediated metabolic suppression. But the mechanisms underlying this differential effects on GFAP versus HMGB1 signals are unknown at this time.

Taken together, our results suggest that certain types of reactive astrocytes, including HMGB1-expressing subsets, may contribute to neurovascular remodeling and functional recovery after stroke. But we acknowledge that some of our findings are correlational and we cannot prove mechanisms at this time. Our data only indirectly implicate HMGB1. As discussed above, we cannot unequivocally establish causality because FC is nonspecific. It remains possible that HMGB1 is not causative, but rather a ‘bystander' phenomenon that reflects the degree of neurovascular remodeling. Furthermore, although our main focus was on astrocytes, many other cells can secrete HMGB1, including monocytes, macrophages, and microglia (Passalacqua et al, 1998; Wang et al, 1999; Andersson et al, 2000). How cross-talk with HMGB1 and other mediators affects beneficial versus detrimental substrates in the remodeling peri-infarct brain remains to be determined.

In conclusion, our present study demonstrated that metabolic inhibition of certain subsets of reactive astrocytes suppressed neurovascular remodeling in peri-infarct cortex after focal cerebral ischemia in mice. Further studies are warranted to dissect how HMGB1 and other molecular signals are regulated in the dynamic balance between injury and repair during stroke recovery.


This study was supported in part by a Grant-in-Aid for Scientific Research (no. 20590552) from the Ministry of Education, Science and Culture of Japan, the Advanced Materials Institute of Fukuoka University, the Mochida Memorial Foundation for Medical and Pharmaceutical Research, a Postdoctoral Fellowship for Research Abroad from the Japan Society for the Promotion of Science, and NIH Grants R37-NS37074, P01-NS55104, and a Bugher award from the American Heart Association.


Conflict of interest

The authors declare no conflict of interest.


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