Methylene blue exerts a neuroprotective effect against traumatic brain injury by promoting autophagy and inhibiting microglial activation

Abstract

Traumatic brain injury (TBI) leads to permanent neurological impairment, and methylene blue (MB) exerts central nervous system neuroprotective effects. However, only one previous study has investigated the effectiveness of MB in a controlled cortical impact injury model of TBI. In addition, the specific mechanisms underlying the effect of MB against TBI remain to be elucidated. Therefore, the present study investigated the neuroprotective effect of MB on TBI and the possible mechanisms involved. In a mouse model of TBI, the animals were randomly divided into sham, vehicle (normal saline) or MB groups. The treatment time‑points were 24 and 72 h (acute phase of TBI), and 14 days (chronic phase of TBI) post‑TBI. The brain water content (BWC), and levels of neuronal death, and autophagy were determined during the acute phase, and neurological deficit, injury volume and microglial activation were assessed at all time‑points. The injured hemisphere BWC was significantly increased 24 h post‑TBI, and this was attenuated following treatment with MB. There was a significantly higher number of surviving neurons in the MB group, compared with the Vehicle group at 24 and 72 h post‑TBI. In the acute phase, the MB‑treated animals exhibited significantly upregulated expression of Beclin 1 and increased LC3‑II to LC3‑I ratios, compared with the vehicle group, indicating an increased rate of autophagy. Neurological functional deficits, measured using the modified neurological severity score, were significantly lower in the acute phase in the MB‑treated animals and cerebral lesion volumes in the MB‑treated animals were significantly lower, compared with the other groups at all time‑points. Microglia were activated 24 h after TBI, peaked at 72 h and persisted until 14 days after TBI. Although the number of Iba‑1‑positive cells in the vehicle and MB groups 24 h post‑TBI were not significantly different, marked microglial inhibition was observed in the MB group 72 h and 14 days after ‑TBI. These results indicated that MB exerts a neuroprotective effect by increasing autophagy, decreasing brain edema and inhibiting microglial activation.

Figure 1

Effect of MB on brain edema and neurological function following TBI. (A) MB reduced BWC 24 h post-TBI. At 72 h post-TBI, the BWC decreased. Data are expressed as the mean ± standard deviation (n=6; ^#P<0.05, vs. Sham). (B-D) MB significantly reduced the neurological deficits, compared with the vehicle group at 24 and 72 h post-TBI. (n=7; ^#P<0.05, vs. Sham; ^*P<0.05, vs. vehicle). MB, methylene blue; TBI, traumatic brain injury; BWC, brain water content; NS, normal saline; mNSS, modified neurological severity score.

Figure 2

MB reduces cerebral lesion volume and microglial activation following TBI. (A) Representative images of HE-stained coronal sections 24 h, 72 h and 14 days post-TBI (Scale bar=1 mm). (B) Representative images of Iba-1-stained coronal sections 24 h, 72 h and 14 days post-TBI (Scale bar=50 µm). (C) Quantitative analysis of injury volume revealed a significant decrease in loss of tissue following treatment with MB (n=7; ^*P<0.05). (D and E) Quantitative analysis demonstrated reduced Iba-1-positive cell numbers and fluorescence intensity following treatment with MB. (n=6; ^*P<0.05). The data are presented as the mean ± standard deviation. MB, methylene blue; TBI, traumatic brain injury; NS, normal saline; HE, hematoxylin and eosin.

Figure 3

Methylene blue prevents neuronal death and promotes autophagy at the perilesional area following TBI. (A and B) Representative images of NeuN-stained coronal tissue sections 24 and 72 h post-TBI. (Scale bar=50 µm). (C) Quantitative analysis revealed a significant increase in the number of NeuN-positive cells 24 and 72 h post-TBI following treatment with MB. The data are presented as the mean ± standard deviaton (n=6; ^*P<0.05). (D and E) Representative images of Beclin 1-stained coronal tissue sections 24 and 72 h post-TBI demonstrating marked cytoplasmic staining in the MB-treated cells (Scale bar=50 µm). MB, methylene blue; TBI, traumatic brain injury; NS, normal saline.

Figure 4

Effect of MB on autophagy, determined using western blot analysis. (A-D) Representative western blot analyses of expression levels of Beclin 1 and LC3 in the ipsilateral cortex 24 and 72 h post-TBI. (E and F) Relative band densities of LC3 and Beclin 1 were determined 24 and 72 h post-TBI. MB significantly upregulated the expression of Beclin 1 and increased the LC3-II/LC3-I ratio, compared with the vehicle control group. The protein band densities were analyzed and normalized against β-actin. Lanes 1 and 2, sham group; lanes 3–5, vehicle group; lanes 6–8, MB group. The data are expressed as a percentage of the sham group and are presented as the mean ± standard deviation (n=6; ^#P<0.05, vs. sham; ^*P<0.05, vs. vehicle). MB, methylene blue; TBI, traumatic brain injury; NS, normal saline.