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. Author manuscript; available in PMC: 2025 Dec 17.
Published in final edited form as: Brain Res. 2017 Nov 8;1678:322–329. doi: 10.1016/j.brainres.2017.10.033

Chronic Oral Methylene Blue Treatment in a Rat Model of Focal Cerebral Ischemia/Reperfusion

Lei Huang 1,1, Jianfei Lu 1,1, Bianca Cerqueira 1, Yichu Liu 1, Zhao Jiang 1, Timothy Q Duong 1,*
PMCID: PMC12707804  NIHMSID: NIHMS919110  PMID: 29108817

Abstract

A single acute low-dose methylene blue (MB), an FDA-grandfathered drug, has been shown to ameliorate behavioral deficits and reduces MRI-defined infarct volume in experimental ischemic stroke when administered intravenously or intraperitoneally. The efficacy of chronic MB treatment in ischemic stroke remains unknown. In a randomized, double-blinded and vehicle-controlled design, we investigated the efficacy of chronic oral MB administration in ischemic stroke longitudinally up to 60 days post injury using MRI and behavioral tests, with end-point histology. The major findings were chronic oral MB treatment, compared to vehicle, i) improves functional behavioral outcomes starting on day 7 and up to 60 days, ii) reduces MRI-defined total lesion volumes from day 14 and up to 60 days where some initial abnormal MRI-defined core and perfusion-diffusion mismatch were salvaged, iii) reduces white-matter damage, iv) gray matter and white matter damages are consistent with Nissl stains and Black Gold stain histology. These findings provide further evidence that long-term oral administration of low-dose MB is safe and has positive therapeutic effects in chronic ischemic stroke.

Keywords: Methylene Blue, Chronic stroke, MRI, lesion volume, white matter injury

1. Introduction

Stroke is a leading cause of death and chronic disability worldwide (Benjamin et al., 2017). The available treatments for ischemic stroke remain very limited. Recombinant tissue plasminogen activator (rt-PA) is effective, but it benefits only 3–5% of patients due to the risk of fatal hemorrhagic transformation and limited treatment window (within 4.5 hours after stroke onset) (Adeoye et al., 2011; Hacke et al., 2008). More recently, mechanical clot extraction has recently shown to be efficacious in clinical trials (Badhiwala et al., 2015; Goyal et al., 2016; Pereira et al., 2013). Mechanical thrombolytic devices can remove a clot in minutes, whereas pharmaceutical thrombolytics, even those delivered intra-arterially, may take as long as 2 hours to dissolve a thrombus. There are currently no clinically approved neuroprotective drug for treatment of acute stroke.

Methylene blue (MB), an FDA-grandfathered drug, has been used to treat methemoglobinemia and cyanide poisoning (Schirmer et al., 2011). Growing preclinical evidence has demonstrated that MB exhibits therapeutic potential in numerous neurological disorders, including but not limited to Alzheimer’s disease (Mori et al., 2014), Parkinson’s disease (Wen et al., 2011) and traumatic brain injury model (Talley Watts et al., 2014). Moreover, MB can readily cross the blood-brain barrier and distributes in the central nervous system after been intravenously or orally administrated (Walter-Sack et al., 2009). Clinical safety study has shown long-term intake of low-dose of MB has an excellent safety profile (Naylor et al., 1986).

We have previously demonstrated that acute treatment with a single-dose MB ameliorates behavioral deficits and reduces MRI-defined infarct volume in a transient (Shen et al., 2013) and permanent (Rodriguez et al., 2014) ischemic stroke models. Although these studies have shown that MB is neuroprotective for acute ischemic stroke, the efficacy of chronic MB treatment on stroke recovery in the chronic phase remains unknown.

The goal of the current study is to test the long-term effects of chronic MB oral treatment on ischemic stroke in rats. A randomized, double-blinded and vehicle-controlled design was used to avoid bias. MRI was used to select animals by excluding incomplete occlusion based on hyperacute MRI before enrolling animals into the study. MRI was used to longitudinally evaluate the effect of chronic MB treatment up to 60 days post injury along with behavioral tests, with corroboration by end-point histology.

2. Results

2.1 Chronic MB treatment improved behavioral recovery

Prior to MCAO, foot fault scores were not significantly different between the vehicle and MB-treated groups (4.0±2.8 vs. 5.0±1.4%, t-test, P>0.05. Figure 2A). The foot fault percentage increased dramatically on day 2 followed by a reduction from day 7 to 60 for both groups. However, compared to vehicle group, the foot faults in the MB-treated group were significantly lower on day 7 through day 60 (23.9±6.8% vs. 40.7±4.8%, P<0.0001 on day 7; 21.1±5.3% vs. 31.5±5.5%, P=0.007 on day 14; 15.2±3.2% vs. 26.8±2.6%, P=0.002 on day 21; 18.2±4.4% vs. 26.0±1.6%, P=0.039 on day 35; 18.0±4.1% vs. 28.0±4.3%, P=0.001 on day 60; Two-way ANOVA). Similarly, the forelimb asymmetry scores were not statistically different between groups before the stroke and dramatically decreased 2 days after stroke (Figure 2B). No obvious improvement in the score was observed in the vehicle group from day 2 to day 60. However, the MB treatment group showed progressive improvement from day 2 to day 28, reaching significance on day 35 and day 60 compared to vehicle group (40.1 ± 7.0% vs. 24.3 ± 10.2% on day 35, P=0.01; on day 60, 43.6 ± 8.7% vs. 23.8 ± 11.8%, P= 0.02, two-way ANOVA with Bonferroni post hoc test).

Figure 2.

Figure 2

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Foot fault test (A) and cylinder test (B) at pre-MCAO and day 2, 7, 14, 21, 35, 60 post-MCAO for vehicle and methylene blue (MB) treated groups (mean±SEM, n=5 per group, *P<0.05, **P<0.01 compared to the vehicle-treated animals).

2.2 Chronic MB treatment reduced lesion volume

The MRI-defined lesion volumes before treatment and day 2 after stroke were not significantly different between groups (Figure 3). By contrast, lesion volumes of the MB-treated group were significantly lower than the vehicle group at day 14 to 60. (100.5±12.5 vs. 184.0±26.3, P=0.022 on day 14; 96.6±5.8 vs. 177.3±22.1, P=0.016 on day 35; 97.7±14.7 vs. 172.4±17.2 P=0.049 on day 60; Two-way ANOVA).

Figure 3.

Figure 3

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(A) Representative apparent diffusion coefficient (ADC) on day 0 during MCAO and T2 volumes on day 2, 14, 35 and 60 for vehicle and methylene blue (MB) treated groups. (B) Temporal evolution of ADC and T2 lesion volumes for the vehicle and MB treated groups. (mean±SEM, n=5 per group, *P<0.05 compared to vehicle-treated animals)

We further identified that MB treatment, compared to vehicle treatment, salvaged significantly more MRI-defined core tissue (40.9±4.3 vs. 26.1±4.2%, P=0.036, t-test, Figure 4) as well as mismatch tissue albeit not significantly (68.1±2.0% vs. 72.1±3.1%, P>0.05).

Figure 4.

Figure 4

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(A) Representative core (red) and mismatch (yellow) areas (determined using day 0 ADC and CBF) (B) Percentage of survived core and mismatch tissues for the vehicle and methylene blue (MB) treated groups. (mean±SEM, n=5 per group, *P<0.05 compared to vehicle-treated animals).

Nissl staining was performed to verify MRI findings at the end of the study (day 60 after stroke) (Figure 5). In both groups, Nissl stain indicated the apparent tissue loss or neuronal damage caused by stroke, which was consistent with MRI. Compared vehicle group, tissue loss was less in the MB-treated group.

Figure 5.

Figure 5

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Nissl staining on day 60 post-MCAO. Mosaic images of the vehicle and methylene blue (MB) treated groups. The small black box indicated the corresponding T2 images. Scale bar=1mm.

2.3 Chronic MB treatment attenuated post-stroke hyperperfusion

CBF after stroke was measured by arterial spin labeling MRI. Representative CBF maps are shown in Figure 6A. Two ROIs (initial core and mismatch) were used to analyze CBF changes separately. CBF was markedly reduced following ischemic stroke in both groups on day 0. From day 2 to day 14, hyperperfusion was observed in the two ROIs of both groups. However, CBF was significantly lower in both ROIs of MB-treated group on day 14, compared to the vehicle group (1.07±0.3X vs. 1.77±0.3X of normal CBF, P=0.021 on core ROI; 0.64±0.2X vs. 0.92±0.1X of normal CBF, P=0.016 on mismatch ROI, ANOVA). From day 35 to day 60, CBF of mismatch region was close to the CBF of contralateral side, while CBF of core area persisted low. Moreover, CBF was significantly higher in initial mismatch area of MB treated group on day 35 and day 60, compared to the vehicle group (0.84±0.03X vs. 0.62±0.03X of normal CBF, P=0.024 on day 35; 0.82±0.05X vs. 0.59±0.03X of normal CBF, P=0.04 on day 60; ANOVA).

Figure 6.

Figure 6

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(A) Representative cerebral blood flow (CBF) on day 0 during MCAO, day 2, 14, 35 and 60 for the vehicle and methylene blue (MB) treated groups. (B) Normalized CBF values of initial core region for vehicle and MB treated groups. (mean±SEM, n=5 per group, *P<0.05 compared to vehicle-treated animals) (C) Normalized CBF values of initial mismatch region for vehicle and MB treated groups. (mean±SEM, n=5 per group, *P<0.05, **P<0.01 compared to the vehicle-treated animals).

2.4 Chronic MB treatment decrease white matter injury

FA was analyzed to evaluate white-matter changes after stroke (Figure 7). The corpus callosum (CC) volume was higher after MB-treatment compared to vehicle in the ipsilesional hemisphere (50.14±3.6 vs. 36.2±3.6, P=0.028, t-test), but not in the contralesional hemisphere (57.5±1.8 vs. 57.6±3.8, P=0.983, t-test) at day 60 after stroke.

Figure 7.

Figure 7

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(A) Representative fractional anisotropy (FA) maps at Day60 post MCAO for the vehicle and methylene blue (MB) treated groups. (B) The volume of the contralateral and ipsilateral corpus callosum (CC) at 60 days post-MCAO for the vehicle and MB treated groups (mean±SEM, n=5 per group, *P<0.05 compared to the vehicle-treated animals).

Black-Gold II staining was performed at the final time point of the study to corroborate the MRI findings (Figure 8). FA map was consistent with Black-Gold II staining. CC of the contralateral hemisphere was intact in both groups. However, CC of ipsilateral hemisphere showed less white-matter damages in MB treated group, compared to vehicle group.

Figure 8.

Figure 8

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Black-Gold ll staining on day 60 post-MCAO. Mosaic images of the vehicle and methylene blue (MB) treated groups. The small black box demonstrated the corresponding FA images. Scale bar=1mm.

3. Discussion

The major findings of this study are chronic MB treatment, compared to vehicle: i) improves functional behavioral outcomes starting on day 7 and up to 60 days, ii) reduces MRI-defined total lesion volume from day 14 and up to 60 days in which some initial abnormal MRI-defined core and mismatch were salvaged, iii) reduces white matter lesion volume, iv) gray matter and white matter damages detected by MRI are consistent with Nissl stains and Black-Gold II stain histology in the same animal at the final time point.

Our findings are in general agreement with three previous acute MB treatment studies on ischemic stroke using different methods of delivery. One study found that s single low-dose MB (1mg/kg given at 30–180 mins after MCAO) prolonged the ischemic penumbra during occlusion in the hyperacute phase of permanent stroke(Rodriguez et al., 2014). A single low-dose of MB (1 mg/kg) given during reperfusion after 60-min MCAO reduced infarct volume by 30% at 48 hours compared to vehicle (Shen et al., 2013). Another study also found an acute MB dose following by intraperitoneal 1 mg/kg MB injection on day 2, 7, and 14 reduced lesion volume in a 60-min MCAO model at 28-day readout (Rodriguez et al., 2016).

The current studies extended previous studies as followed: (1) Using daily oral chronic MB administration (day 1 to 21) and a double-blinded experimental design, we further demonstrated that the improvement in infarct volumes and functional recovery persisted at least up to 60 days. Based on the Stroke Therapy Academic Industry Roundtable (STAIR) guideline for pre-clinical stroke study, long-term outcome of the drug must be monitored to avoid the misleading short-term beneficial effect, which may be caused by barely slowing the pathophysiologic process of stroke (Fisher et al., 2009). We tracked the effects of MB treatment on the behavioral functions and infarction up to 2 months, which was adequately covered the 30 days period, a STAIR suggested time windows for measuring the long-term effect. More importantly, there are no side effects with long-term administration of an equivalent dose of MB in human (Naylor et al., 1986), opening the possibility of doing similar studies in clinical trials.

(2) Chronic low-dose MB reduced behavioral deficits starting from 7 days after stroke and sustained up to 60 days. No improvement was detected on day 2 after stroke. While, our previous studies have been demonstrated the amelioration of behavioral deficits on 48h and 7 days post stroke, after intravenous administration of 1mg/Kg MB within 3-hour treatment time windows (Shen et al., 2013). The difference in the behavioral improvement at 48h probably due to the administration protocol of MB. Under current oral treatment, MB might take some time for functional recovery to differentiate. The data on day 7 post-stroke suggested that MB improved behavioral functions, administered either in the hyperacute phase or chronic phase of ischemic stroke. Future studies are needed to explore the mechanism of functional recovery induced by MB under different administration protocol.

(3) Chronic low-dose MB by daily oral administration up to 21 days reduced lesion volume starting from 14 days and persisted up to 60 days. The delayed neuroprotection effects of MB are consistent with the pattern of improvement on behavioral functions, which indicated the therapeutic effects of MB were delayed by this oral administration protocol. However, the mechanism of such delayed protective impact with chronic MB administration might be different from acute MB treatment. It is possible that acute MB treatment decreases infarct volume through reducing neural apoptosis and increasing autophagy (Jiang et al., 2015) by its unique antioxidant effect. Additional mechanisms (i.e., post-stroke neurogenesis) may be involved in chronic MB treatment. Recent evidence has shown that 5 days continuously intraperitoneal injection of 0.5mg/Kg MB increases cortical neurogenesis on 12 days after photothrombotic stroke (Ahmed et al., 2016).

(4) We further identified that MB treatment, compared to vehicle treatment, significantly salvaged more abnormal ADC tissue as well as mismatch tissue albeit not significantly. The lack of significant improvement between groups in the mismatch could be due to the nature of high variability of the mismatch tissue and the precise threshold used.

(5) We found that the extents of hyperperfusion in the initially defined core and mismatch tissue were reduced in the MB-treated group on day 2 and 14 after stroke. The possible mechanism for this is that MB may minimize the leakage of blood-brain-barrier (BBB) and dysfunction of vessels. Indeed, a study indicated that MB reduces BBB disruption in a pig cardiac arrest model (Miclescu et al., 2010). MB has also been found to have effects on vascular dynamics(Huang et al., 2013).

(6) Another novel finding is that less white matter damage was observed in MB treated group, which suggests the white matter protective role of chronic MB treatment. Chronic cerebral hypoperfusion has been reported to increase the level of reactive oxygen species within white matter and suppress differentiation of oligodendrocyte precursor cell (Miyamoto et al., 2013). Other antioxidant agents (e.g. ebselen, cinnamophilin) have been shown to successfully protect white matter damage after ischemic stroke (Chen et al., 2011; Imai et al., 2001). Due to the antioxidant properties of MB, it is possible that MB treatment protects the stroke-affected white matter through reducing oxidative stress. However, it is unclear if the protective effect of MB treatment related to the reduction of oligodendrocyte cell death or enhancement of white matter regeneration. Future studies need to explore the mechanism underlying MB-induced white matter protection.

Limitations and future perspectives

Because the ultimate goal of this study is to translate this approach into clinical trials, we mainly focused on using MRI and behavioral scores to longitudinally evaluate the treatment efficacy of chronic MB treatment on stroke, and we did not investigate the molecular mechanism under the beneficial impact. While the anti-oxidant mechanisms of MB are well known, its role in brain recovery, such as angiogenesis, neurogenesis, and white matter reorganization, in the chronic phase remains to be explored. We performed MB treatment for 21 days, which was sufficiently covered the period that infarction underwent a dramatic change, based on our previous MRI studies on stroke development in the rat. It may be of interest to explore MB combination therapy with mechanical clot retrieval treatment, given that mechanical clot retrieval has been shown to be beneficial in recent clinical trials (Campbell et al., 2016; Muir et al., 2017).

4. Conclusions

Long-term oral methylene blue treatment reduces brain lesion volume and white matter damage in the chronic phase of stroke, and improves behavioral function and regional CBF in transient focal ischemia in rats. Oral treatment is shown to be effective and should have broader application. These findings provided further supportive evidence that low-dose MB has positive therapeutic effects in the treatment of chronic stroke.

5. Experimental Procedure

5.1 Animal Preparation

All experimental protocol in this study was evaluated and approved by the Institutional Animal Care and Use Committees. All experiments followed guideline and regulations consistent with the Guide for the Care and Use of Laboratory Animals, Public Health Service Policy on Humane Care and Use of Laboratory Animals, and the Animal Welfare Act and Animal Welfare Regulations. Animal experiments were approved by Institutional Animal Care and Use Committee of the University of Texas Health Science Center San Antonio. Transient (60-minutes) focal cerebral ischemia was induced by intraluminal filament middle cerebral artery occlusion (MCAO), as previously described (Huang et al., 2017). Animals were anesthetized initially with 5% isoflurane mixed with room air and maintained at 1.5% isoflurane throughout all surgical and imaging procedures. Under operating microscope, the right common carotid artery, internal carotid artery (ICA), and external carotid artery (ECA) were surgically exposed. Then the ECA was isolated and ligated. A 4-0 nylon suture (diameter 0.29mm) with silicon rubber coated (coating length 3 mm, diameter 0.37 mm) was inserted into the ICA through the ECA stump and gently advanced to occlude the MCA. After 1 hour of MCAO, the nylon suture was carefully removed, and then the neck incision was sutured. The rectal temperature was carefully maintained at 37.0° C by a heating pad during the postoperative period until the animal completely recovered from the anesthesia. After the stroke, soft gel food (BioServ, Flemington, NJ) was supplied every day.

The experimental timeline is shown in Figure 1. In a randomized double-blinded, vehicle-controlled design, total eighteen male Sprague-Dawley rats (250–350g body weight) were subjected to MCAO surgery. Hyperacute MRI (30 minutes after MCAO) was performed before group assignment, and six rats were excluded due to incomplete occlusion. Then, twelve rats with similar initial infarct lesion were randomly assigned to two groups after stroke. No animal dropped in the following studies. Subject selection, which has been demonstrated to be critical in clinical trials on stroke treatment, would be impossible to achieve with terminal histological methods. The food dye and MB solution were prepared by another person who had access to the randomization list, but who was not involved in performing experiments or analyzing data. One group of the stroke animals was provided with gel food (Nutra-Gel Diet, #S5769-TRAY, Bio-Serv) mixed with blue food dye (FD&C blue) as vehicle control, and the other was supplied with same gel food mixed with MB (4 mg/kg, USP Pharmaceutical grade; American regent Inc, Shirley, NY) from 1 day to 21 days post stroke. Daily oral 4mg/kg MB has been reported safely in the clinical trial (Naylor et al., 1986). One rat from each group died during the follow-up study, and motility rate was equal in the two group.

Figure 1.

Figure 1

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Experimental design.

5.2 Functional assessment

Sensorimotor function by foot-fault test (Talley Watts et al., 2014) and cylinder test were evaluated at 2, 7, 14, 21, 35 and 60 days post-MCAO preceding MRI scanning in the same animals. The behavioral test was not performed on the day of MCAO surgery owing to incomplete recovery from the anesthetic condition. The foot-fault test was to assess limb misplacement during locomotion. In this test, the rat was placed on a grid floor (size 45.7 × 27.9cm with grid openings of 3.96 × 2.54 cm2) and allowed to move freely for 5 minutes, or until 50 steps were taken. Videotaping was carried out during the test. The limb fell through the grid during movement was regarded as foot faults. The number of “foot faults” for left forelimb (stroke affected) and the total number of left forepaw steps were counted separately. The percentage of foot faults for the left forepaw was calculated as the number of left forelimb foot faults divided by the total number of steps taken. The cylinder test was performed to determine the asymmetrical use of the contralateral (affected) forearm. The animal was video-recorded in a transparent cylinder (20cm diameter by 30cm height) for 5mins. Forelimb placement on the wall was counted by an observer blinded to experimental groups. The frequency of left forelimb placement to total placement was calculated and expressed, as previously described (Huang et al., 2017).

5.3 MRI

MRI was performed on a Bruker 7-T BioSpec Scanner with a 40 G/cm BGA12S gradient insert (Billerica, MA, USA). A custom-made surface coil (2.3-cm ID) and a neck coil were used for brain imaging and perfusion labeling separately. MRI was acquired at 30 mins after MCAO, and again on 2, 14, 35 and 60 days after MCAO.

Diffusion

Diffusion-weighted images were acquired using single-shot, spin-echo, echo-planar imaging sequence with matrix = 96×96 and reconstructed to 128×128, field of view (FOV) 2.56×2.56 cm, seven 1.5-mm slices, repetition time (TR) = 3 s, echo time (TE) = 37 ms. Two levels of diffusion sensitization (b = 0 and 1200 s/mm2), applied along x, y, z-direction separately, were used to calculate the ADC map (Shen et al., 2013). Diffusion tensor imaging (DTI) was also performed with following parameters: B-value (10 and 1200 s/mm2), 30 gradient directions, FOV = 2.56 × 2.56 cm, Matrix = 128X128, TR = 3000 ms, TE = 32 ms, number of average = 2. FA map was generated as previously described (Long et al., 2015).

CBF

Continuous arterial spin-labeling (cASL) technique was used to measure CBF, as previously described (Shen et al., 2005). Continuous arterial spin labeling employed a 2.7-second square radiofrequency pulse to the labeling coil. Single-shot, gradient-echo, echo-planar imaging (EPI) sequence was used with followed parameters: matrix = 96×96 and reconstructed to 128×128, FOV = 2.56×2.56 cm, seven 1.5-mm slices, TR = 3 s, flip angle = 90°, and TE=14 ms. Pair images with and without tagging were acquired. For CBF, 60 repetitions were obtained and averaged.

T2

T2-weighted images were acquired using fast spin- echo sequence, with TR = 3 s and four effective TE (25, 40, 75 and 120 ms) to generate T2 maps. Other parameters were: seven 1.5-mm coronal images, FOV = 2.56 × 2.56 cm, matrix 96 × 96 and reconstructed to 128 × 128, and 8 transients for signal averaging.

5.4 MRI Data Analysis

ADC, CBF, T2, FA maps were generated and analyzed using Matlab (MathWorks Inc., Natick, MA, USA) and STIMULATE (University of Minnesota)/Mango (University of Texas Health Science Center at San Antonio) as previously described (Huang et al., 2017; Shen et al., 2013). Image maps of individual subjects were co-registered across time points by QuickVol and MRIAnalysisPak software. The stroke-induced initial lesion was defined by abnormal ADC after 30mins post MCAO with an established threshold (0.53×10−3 mm2/s) (Meng et al., 2004; Shen et al., 2005). The ischemic core and perfusion-diffusion mismatch were defined based on 30-min ADC, and CBF maps using previous measurements described (Shen et al., 2013). The fate of initial ischemic core and mismatch were tracked over time. Lesion volume was calculated based on T2 map at two days post MCAO, due to the better resolution than the ADC map. The lesion area was defined by the pixels with the T2 value higher than mean value plus two times the standard deviation (Mean+2 SD) obtained from the contralateral side. Edema correction was applied for day 2 data (Huang et al., 2017; Shen et al., 2013). Two regions of interest (ROIs) were generated to separate initial core and mismatch area, based on Day 0 MRI data. CBF value was quantified on these two areas across time and normalized to CBF value of the contralateral corresponding area. Investigators performing image analysis were blinded to experimental groups. ROIs of the contralateral and ipsilateral corpus callosum(CC) were manually drawn on the FA map by an investigator blinded to experimental grouping. A FA threshold of 0.19 was used to select white matter tissue. The volume of the CC on ipsilateral and contralateral hemisphere to stroke was calculated based on FA maps at the end of the study.

5.5 Histology

5.5.1 Nissl staining

At the end of the study, the rat was anesthetized and transcardially perfused with ice-cold 0.01M phosphate buffered saline (PBS), followed by ice-cold 4% paraformaldehyde in 0.1M phosphate buffer (PB) (pH=7.4). Brains were removed and postfixed with 4% paraformaldehyde, and then cryoprotected in 30% sucrose in 0.1M PB for over 24h at 4°C. Coronal brain sections (25μm thick) were sliced on a cryostat affixed to gelatin-coated slides and dried overnight at 37°C. Sections were hydrated through a series of graded alcohols to distilled water followed by 0.1% cresyl violet acetate for 30min at 37°C, as previously described (Huang et al., 2014). Brain sections were then dehydrated through a series of graded alcohols, cleared in xylene and cover-slipped with mounting medium. Images were acquired on an Olympus BX60F microscope equipped with an Olympus DP70 camera.

5.5.2 Black-Gold ll staining

Black-Gold ll stain is an aurohalophosphate complex which stains specifically for myelin within the central nervous system (Schmued et al., 2008). Sections were hydrated in distilled water for 2 mins, and then incubated for 15 mins in 0.3% Black-Gold ll solution dissolved in 0.9% saline and heated to 60–65°C, rinsed with distilled water, and transferred to a 1% sodium thiosulfate solution for 3 mins. Sections were rinsed with tap water three times for 5 mins each. The slides were then dehydrated through a series of graded alcohols, cleared in xylene and cover-slipped with mounting medium. Images were acquired on an Olympus BX60F microscope equipped with an Olympus DP70 camera with a 5X objective.

5.6 Statistical Analysis

Repeated-measures ANOVA (with Bonferroni’s post hoc tests) were used to compare the percentage of foot fault, lesion volumes, and CBF values between vehicle- and MB-treated groups. Unpaired two-tailed t-tests were used to compare the percentage of tissue been rescued, and CC volume between vehicle- and MB-treated groups. Data were presented as Mean ± SEM. Statistical significance was set at P<0.05.

Highlights.

  • Chronic oral Methylene blue treatment improves functional recovery in the chronic phase of ischemic stroke.

  • Chronic oral Methylene blue treatment reduces lesion development and white mater injury after ischemic stroke.

Acknowledgments

This work was supported in part by NIH/NINDS (R01-NS45879).

Footnotes

Disclosure

The authors declare no conflict of interest.

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