Effects and mechanism of myeloperoxidase on microglia in the early stage of intracerebral hemorrhage

Abstract

Objectives: (1) To clarify the dynamic relationship between the expression of myeloperoxidase (MPO) and microglial activation of intracerebral hemorrhage (ICH), (2) to explore the effect of inhibition of MPO on microglial activation, and (3) to observe the improvement in the neurobehavior of mice with inhibition of MPO.

Methods: C57 BL/6 mice and CX3CR1 + /GFP mice were used to establish a phosphate-buffered saline (PBS) group, an ICH group, and a 4-aminobenzoic acid hydrazide (ABAH) group. Longa score, open field locomotion, hind-limb clasping test, immunohistochemistry, immunofluorescence, blood routine detection, and flow cytometry were used.

Results: The neurobehavior of the mice was significantly impaired following ICH (P < 0.01); the expression of MPO was significantly increased following ICH, and reached a peak value at 6 h post-injury (P < 0.001). Moreover, the microglial activation increased significantly following ICH, and reached a peak level at 24 h post-injury (P < 0.01). Following inhibition of MPO, the activation of microglia in the ICH group decreased significantly (P < 0.001). Moreover, the neurobehavior of the ICH group was significantly improved with MPO inhibition (P < 0.05).

Conclusion: MPO may be an upstream molecule activated by microglia and following inhibition of MPO can improve secondary injury resulting from ICH.

Keywords: activation; inhibition; intracerebral hemorrhage; microglia; myeloperoxidase; neurobehavior.

FIGURE 1

Inhibition of myeloperoxidase (MPO) activity promotes recovery of motor function following intracerebral hemorrhaging (ICH). (A) Establishment of intracerebral hemorrhage model; (B,C) Longa scores of PBS, ICH, and ABAH groups (*P < 0.05, **P < 0.01, n = 6 in each group); open field locomotion in the PBS, ICH, and first and second ABAH treatment groups. The times of movement and rest of the mice in the open field were recorded. The total time was 900 s (*P < 0.05, **P < 0.01, n = 6 in each group); (D) open field mode diagram; (E) hind-limb clasping test in the PBS, ICH, and first and second ABAH treatment groups. The degrees of hind-limb opening and closing in mice were observed.

FIGURE 2

ICH can induce changes in the number and proportion of inflammatory cells in peripheral blood. (A) Quantity of neutrophils in peripheral blood (**P < 0.01, n = 18 in each group); (B) neutrophils/peripheral blood ratio (*P < 0.05, n = 18 in each group); (C) quantity of peripheral blood mononuclear cells (*P < 0.05, n = 18 in each group); (D) ratio of mononuclear cells to peripheral blood (**P < 0.01, n = 18 in each group).

FIGURE 3

Inhibition of MPO activity reduces microglia proliferation following ICH. (A) Flow cytometry FITC channel, to detect the content of microglia in the control, PBS, ICH, and ABAH groups (set the total number of living cells to 20,000, n = 1 in each group); (B) proportion of FITC in the control, PBS, ICH, and ABAH groups; (C) number of FITC in the control, PBS, ICH, and ABAH groups.

FIGURE 4

Microglia and MPO were activated following ICH. (A) Immunohistochemistry of the PBS and ICH groups at 6, 12, and 24 h post-injury. The labeled molecule was iba1 (10×, 20×); (B) statistics of iba1 positive expression (**P < 0.01, n = 6 in each group); (C) immunohistochemistry of iba1 in the PBS and ICH groups at 24 h post-injury (10×, 40×); (D) immunofluorescence of MPO in the PBS and ICH groups at 6, 12, and 24 h post-injury (10×, 20×); (E) immunohistochemistry of MPO in the PBS and ICH groups at 6 h post-injury (10×, 40×); (F) statistics of MPO molecular expression (**P < 0.01, ***P < 0.001, n = 6 in each group).

FIGURE 5

Inhibition of MPO activity reduces microglial proliferation following ICH. (A) Immunofluorescence double staining was performed in the PBS, ICH, and ABAH groups at 24 h post-injury. The labeled molecules were microglia and MPO; (B) microglia expression (***P < 0.001, n = 6 in each group); (C) MPO expression (***P < 0.001, n = 6 in each group).