Ethyl pyruvate improves white matter remodeling in rats after traumatic brain injury

Abstract

Background: Severe traumatic brain injury (TBI) results in long-term neurological deficits associated with white matter injury (WMI). Ethyl pyruvate (EP) is a simple derivative of the endogenous energy substrate pyruvate with neuroprotective properties, but its role in recovery from WMI has not been explored.

Aims: This study examines the effect of EP treatment on rats following TBI using behavioral tests and white matter histological analysis up to 28 days post-injury.

Materials and methods: Anaesthetised adult rats were subjected to TBI by controlled cortical impact. After surgery, EP or Ringers solution (RS) was administrated intraperitoneally at 15 min after TBI and again at 12, 24, 36, 48, and 60 h after TBI. Sensorimotor deficits were evaluated up to day 21 after TBI by four independent tests. Immunofluorescence and transmission electron microscopy (TEM) were performed to assess white matter injury. Microglia activation and related inflammatory molecules were examined up to day 14 after TBI by immunohistochemistry or real-time PCR.

Results: Here, we demonstrate that EP improves sensorimotor function following TBI as well as improves white matter outcomes up to 28 d after TBI, as shown by reduced myelin loss. Furthermore, EP administration during the acute phase of TBI recovery shifted microglia polarization toward the anti-inflammatoryM2 phenotype, modulating the release of inflammatory-related factors.

Conclusion: EP treatment may protect TBI-induced WMI via modulating microglia polarization toward M2.

Keywords: ethyl pyruvate; microglia; traumatic brain injury; white matter injury.

FIGURE 1

Effects of EP treatment on sensorimotor function following CCI. (A) Flow chart for the experimental design. (B) Asymmetry of forelimb usage for postural weight support was assessed in rats 3–21 days post‐CCI or sham surgery. (C) The number of foot faults expressed as a percentage of total steps was assessed to reflect the fidelity of descending sensorimotor pathways 1–21 days following CCI. (D) Rats were scored on their response to placement on the hang wire test 3–21 days following CCI. (E) Rats were subjected to rotarod testing for balance/proprioception 3–21 days post‐CCI. “Pre” represents presurgery baseline function. Data are mean ± SE, n = 10 animals/group. *p < 0.05, **p < 0.01 vs. vehicle‐treated TBI animals

FIGURE 2

Effect of EP treatment on myelin loss after TBI. (A–K) Representative images of MBP staining in white matter at different time points after TBI (MBP: green). (L, M) show the fluorescence intensity of MBP (+) in the corpus callosum (CC) and striatum (Str), respectively. Scale bar = 100 µm, n = 5 animals/group. *p < 0.05 vs. vehicle‐treated TBI animals

FIGURE 3

Changes in number of OPCs following CCI. (A–K) Representative images of OPCs in striatum up to day 28 post‐CCI (Olig2: green).(M) Negative staining image for Olig2. (L) Quantification of Olig2⁺ cells as counted number per area (mm²). Scale bar = 100 µm, n = 5 animals/group. *p < 0.05, **p < 0.01 vs. vehicle‐treated TBI animals

FIGURE 4

Ethyl pyruvate treatment improved white matter remodeling after TBI. Representative EM images from corpus callosum of a vehicle‐treated TBI brain (A), EP‐treated TBI brain (B), and sham‐operated brain (C) at day 28 post‐CCI. The arrows indicate axons covered by myelin. (D) EP treatment reduced the g‐ratio compared to the vehicle treatment. 62 myelinated axons per group were analyzed (n = 62). Scale bars = 1 μm. *p < 0.05 vs. vehicle‐treated TBI animals. The images are from three brains per group with similar results

FIGURE 5

Ethyl pyruvate modulated the polarization of microglia after TBI. (A–B) Representative images of Iba‐1/CD206 (A) or Iba‐1/CD16 (B) immunohistochemistry in impaired cortex border (CTX: cortex) at day 3 post‐CCI. Iba‐1: green, CD206: red, DAPI: blue, yellow arrow: Iba‐1 (+) CD206 (+) microglia, white arrow: Iba‐1 (+) CD16 (+) microglia. Scale bar = 10 µm. (C) The empty square box depicts the region of interest relevant to the peri‐contusion border, from where tissues were sampled for immunohistochemical images. (D–E) Quantification of CD206+/Iba‐1 + or CD16+/Iba‐1 + cells in the cortex. Data are expressed as percentages of cell numbers vs. vehicle‐treated TBI brains. *p < 0.05, n = 4 animals/group

FIGURE 6

Ethyl pyruvate modulated the level of inflammatory‐related factors after TBI. (A–D) Real‐time PCR evaluation of the pro‐inflammatory molecules TNF‐α, iNOS, CD32, and CD86 in TBI rats 3–14 days post‐injury. (E–F) Real‐time PCR measurements of the antiinflammatory molecules TGF β and CCL‐22 in TBI rats 3–14 days post‐injury. Data are mean ± SE, n = 5 animals/group. *p < 0.05, **p < 0.01 vs. vehicle‐treated TBI animals