Treatment of traumatic brain injury with thymosin β₄ in rats

Abstract

Object: This study was designed to investigate the efficacy of delayed thymosin β(4) (Tβ(4)) treatment of traumatic brain injury (TBI) in rats.

Methods: Young adult male Wistar rats were divided into the following groups: 1) sham group (6 rats); 2) TBI + saline group (9 rats); 3) and TBI + Tβ(4) group (10 rats). Traumatic brain injury was induced by controlled cortical impact over the left parietal cortex. Thymosin β(4) (6 mg/kg) or saline was administered intraperitoneally starting at Day 1 and then every 3 days for an additional 4 doses. Neurological function was assessed using a modified neurological severity score (mNSS), foot fault, and Morris water maze tests. Animals were killed 35 days after injury, and brain sections were stained for immunohistochemistry to assess angiogenesis, neurogenesis, and oligodendrogenesis after Tβ(4) treatment.

Results: Compared with the saline treatment, delayed Tβ(4) treatment did not affect lesion volume but significantly reduced hippocampal cell loss, enhanced angiogenesis and neurogenesis in the injured cortex and hippocampus, increased oligodendrogenesis in the CA3 region, and significantly improved sensorimotor functional recovery and spatial learning.

Conclusions: These data for the first time demonstrate that delayed administration of Tβ(4) significantly improves histological and functional outcomes in rats with TBI, indicating that Tβ(4) has considerable therapeutic potential for patients with TBI.

Fig. 1

The effect of Tβ₄ on functional recovery after TBI. A: Effect of Tβ₄ on spatial learning function 31–35 days after TBI. TBI significantly impairs spatial learning at Days 32–35 compared to sham controls. Delayed treatment with Tβ₄ improves spatial learning performance measured by a recent version of the water maze test at Days 33–35 compared with the saline group. B: Effect of Tβ₄ on sensorimotor function (forelimb footfault) after TBI. TBI significantly impairs sensorimotor function at Days 1–35 compared with sham controls. Delayed Tβ₄ treatment significantly reduces forelimb foot faults at Days 7–35 compared with the saline-treated group. C: Effect of Tβ₄ on sensorimotor function (hindlimb footfault) after TBI. TBI significantly impairs sensorimotor function at Days 1–35 compared with sham controls. Delayed Tβ₄ treatment significantly reduces hindlimb foot faults at Days 7–35 compared with the saline-treated group. D: Line graph showing the functional improvement detected on the mNSS. Treatment with Tβ₄ significantly lowers mNSS scores at Days 7–35 compared with the saline group. Data represent mean ± SD. There were 6, 9, and 10 rats in the sham, saline, and Tβ₄ groups, respectively. Pre = preinjury level.

Fig. 2

The effect of TB4 on cell number in the ipsilateral dentate gyrus and CA3 region at 35 days after TBI. H&E staining: A-F. Delayed treatment with TB4 (C, F) significantly reduces cell loss as compared with the saline-treated group (B, E) (p < 0.05). The cell number in the dentate gyrus and CA3 region is shown in (G). Scale bar = 25μm (F, applicable to A–F). Data represent mean ± SD. *p < 0.05 vs Sham group. ^#p < 0.05 vs Saline group. N (rats/group) = 6 (Sham); 9 (Saline); and 10 (TB4).

Fig. 3

The effect of TB4 on vWF-staining vascular structure in the injured cortex, ipsilateral dentate gyrus, and CA3 region 35 days after TBI. TBI alone (B, E, and H) significantly increases the vascular density in these regions compared to sham controls (p < 0.05). TB4 treatment (C, F, and I; arrow as example showing vWF-staining vascular structure) further enhances angiogenesis after TBI compared to the saline-treated groups (p < 0.05). The density of vWF-stained vasculature is shown in (J). Scale bar = 50 μm (I, applicable to A-I). Data represent mean ± SD. *p < 0.05 vs Sham group. ^#p < 0.05 vs Saline group. N (rats/group) = 6 (Sham); 9 (Saline); and 10 (TB4).

Fig. 4

The effect of TB4 on BrdU⁺ cells in the injured cortex, ipsilateral CA3, and dentate gyrus 35 days after TBI. TBI alone (B, E, and H) significantly increases the number of BrdU⁺ cells in the ipsilateral cortex, CA3 and dentate gyrus compared to sham controls (A, D, and G) (p < 0.05). TB4 treatment significantly increases the number of BrdU⁺ cells in these regions (C, F, and I; arrow as example showing BrdU⁺ cell) compared to the saline-treated groups (p < 0.05). The number of BrdU⁺ cells is shown in (J). Scale bar = 25μm (I, applicable to A–I). Data represent mean ± SD. *p < 0.05 vs Sham group. ^#p < 0.05 vs Saline group. N (rats/group) = 6 (Sham); 9 (Saline); and 10 (TB4).

Fig. 5

The effect of TB4 on oligodendrocyte progenitor cells (NG2⁺) in the injured cortex, ipsilateral CA3 and dentate gyrus 35 days after TBI. TBI alone (B, E, and H) significantly increases the number of NG2⁺ cells in the ipsilateral cortex, CA3 and dentate gyrus compared to sham controls (A, D, and G) (p < 0.05). TB4 treatment does not affect the number of NG2⁺ cells in these regions (C, F, and I; arrow as example showing NG2⁺ cell) compared to the saline-treated groups. The number of BrdU⁺ cells is shown in (J). Scale bar = 25μm (I, applicable to A–I). Data represent mean ± SD. *p < 0.05 vs Sham group. N (rats/group) = 6 (Sham); 9 (Saline); and 10 (TB4).

Fig. 6

The effect of TB4 on mature oligodendrocytes (CNPase⁺) in the injured cortex, ipsilateral CA3, and dentate gyrus 35 days after TBI. TBI alone (B, E, and H) significantly increases the number of CNPase⁺ cells in the ipsilateral cortex, CA3, and dentate gyrus compared to sham controls (A, D, and G) (p < 0.05). TB4 treatment does not affect the number of CNPase⁺ cells in the cortex (C, arrow as example showing CNPase⁺ cell) and DG (I) but significantly increases the number of CNPase⁺ cells in the CA3 region (F) compared to saline groups. The number of BrdU⁺ cells is shown in (J). Scale bar = 25μm (I, applicable to A–I). Data represent mean ± SD. *p < 0.05 vs Sham group. ^#p < 0.05 vs Saline group. N (rats/group) = 6 (Sham); 9 (Saline); and 10 (TB4).

Fig. 7

Double immunofluorescent staining for BrdU (red) and NeuN (green) to identify newborn neurons (yellow after merge) in the brain (A–C), for BrdU (red) and NG2 (green) to identify newborn OPCs in the brain (D–F), and for BrdU (red) and CNPase (green) to identify newborn mature oligodendrocytes in the brain (G–I). The arrows indicate newborn neurons (A–C), OPCs (D–F) and mature oligodendrocytes (G–I). Bar = 25 um (for all panels). J: Bar graph showing the numbers of NeuN⁺, NG2⁺, and CNPase⁺ cells colabeled with BrdU. Scale bar = 25μm (I, applicable to A–I). Data represent mean ± SD. ^#p < 0.05 vs Saline group. N (rats/group) = 6 (Sham); 9 (Saline); and 10 (TB4).