Neuroprotective and neurorestorative effects of thymosin β4 treatment initiated 6 hours after traumatic brain injury in rats

Laboratory investigation

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Object

Thymosin β4 (Tβ4) is a regenerative multifunctional peptide. The aim of this study was to test the hypothesis that Tβ4 treatment initiated 6 hours postinjury reduces brain damage and improves functional recovery in rats subjected to traumatic brain injury (TBI).

Methods

Traumatic brain injury was induced by controlled cortical impact over the left parietal cortex in young adult male Wistar rats. The rats were randomly divided into the following groups: 1) saline group (n = 7); 2) 6 mg/kg Tβ4 group (n = 8); and 3) 30 mg/kg Tβ4 group (n = 8). Thymosin β4 or saline was administered intraperitoneally starting at 6 hours postinjury and again at 24 and 48 hours. An additional group of 6 animals underwent surgery without TBI (sham-injury group). Sensorimotor function and spatial learning were assessed using the modified Neurological Severity Score and the Morris water maze test, respectively. Animals were euthanized 35 days after injury, and brain sections were processed to assess lesion volume, hippocampal cell loss, cell proliferation, and neurogenesis after Tβ4 treatment.

Results

Compared with saline administration, Tβ4 treatment initiated 6 hours postinjury significantly improved sensorimotor functional recovery and spatial learning, reduced cortical lesion volume and hippocampal cell loss, and enhanced cell proliferation and neurogenesis in the injured hippocampus. The high dose of Tβ4 showed better beneficial effects compared with the low-dose treatment.

Conclusions

Thymosin β4 treatment initiated 6 hours postinjury provides both neuroprotection and neurorestoration after TBI, indicating that Tβ4 has promising therapeutic potential in patients with TBI. These data warrant further investigation of the optimal dose and therapeutic window of Tβ4 treatment for TBI and the associated underlying mechanisms.

Abbreviations used in this paper:BSA = bovine serum albumin; CCI = controlled cortical impact; mNSS = modified Neurological Severity Score; PBS = phosphate-buffered saline; Tβ4 = thymosin β4.

Article Information

Address correspondence to: Ye Xiong, M.D., Ph.D., Henry Ford Health System, Department of Neurosurgery, E&R Building, Room 3096, 2799 West Grand Boulevard, Detroit, Michigan 48202. email: yxiong1@hfhs.org.

Please include this information when citing this paper: published online February 10, 2012; DOI: 10.3171/2012.1.JNS111729.

© AANS, except where prohibited by US copyright law.

Headings

Figures

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    The effect of Tβ4 on functional recovery after TBI. Left: Graph showing the effect of Tβ4 on spatial learning function 31–35 days after TBI. Traumatic brain injury significantly impairs spatial learning at Days 32–35 compared with results in controls that were subjected to surgery without CCI (sham group) (p < 0.05). Thymosin β4 improves spatial learning performance (increased percentage of time spent in the correct quadrant of the Morris water maze) measured by a modified version of the Morris water maze test at Days 33–35 compared with the saline group (p < 0.05). Right: Graph showing the effect of Tβ4 on sensorimotor function detected on the mNSS. Thymosin β4 treatment significantly lowers mNSS at Days 4–35 compared with results in the saline-treated group (p < 0.05). D = Day. Data points are mean values; error bars indicate SD. *p < 0.05 versus the saline group. #p < 0.05 versus the 6 mg/kg Tβ4 group. n (rats/group) = 6 (sham), 7 (saline), 8 (Tβ4–6 mg/kg), and 8 (Tβ4–30 mg/kg).

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    Graph and corresponding photomicrographs showing the effect of Tβ4 on cortical lesion volume examined 35 days after TBI. Traumatic brain injury caused significant cortical tissue loss. Thymosin β4 treatment significantly reduced lesion volume caused by TBI compared with results in the saline-treated group (p < 0.05). Bar = 3 mm. Data are presented as means ± SDs. *p < 0.05 versus the saline group. #p < 0.05 versus the 6 mg/kg Tβ4 group. n = 7 (saline), 8 (Tβ4–6 mg/kg), and 8 (Tβ4–30 mg/kg).

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    The effect of Tβ4 on cell number in the ipsilateral dentate gyrus (DG) and CA3 region at 35 days after TBI. a–h: Photomicrographs of H & E–stained sections showing that treatment with Tβ4 (c, d, g, and h) significantly reduces cell loss as compared with saline administration (b and f). Arrows indicate cell loss. i: Graph showing the numbers of cells per millimeter in the dentate gyrus and CA3 regions. Scale bar = 50 μm (applicable to a–h). Data represent means ± SDs. *p < 0.05 versus the saline group. #p < 0.05 versus the 6 mg/kg Tβ4 group. n = 6 (sham), 7 (saline), 8 (Tβ4–6 mg/kg), and 8 (Tβ4–30 mg/kg).

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    Photomicrographs and bar graph showing the effect of Tβ4 on cell proliferation in the injured cortex (CT) and dentate gyrus 35 days after TBI. Traumatic brain injury without Tβ4 treatment (b and f) significantly increased the number of BrdU+ cells in the ipsilateral cortex and dentate gyrus compared with results in animals subjected to surgery without CCI (sham) (a and e) (p < 0.05). The arrows (in a and b) indicate examples of BrdU+ cells. Thymosin β4 treatment significantly increases the number of BrdU+ cells in these regions (c, d, g, h, and i). Scale bar = 25 μm (applicable to a–h). Data represent means ± SDs. *p < 0.05 versus saline group. #p < 0.05 versus the 6 mg/kg Tβ4 group. n = 6 (sham), 7 (saline), 8 (Tβ4–6 mg/kg), and 8 (Tβ4–30 mg/kg).

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    a–d: Photomicrographs showing the results of double immunofluorescent staining for BrdU (red) and NeuN (green) to identify newborn neurons (yellow after merge) in the brain. Scale bar = 25 μm (d, applicable to a–d). e: Bar graph showing the number of NeuN+ cells colabeled with BrdU. Data represent means ± SDs. *p < 0.05 versus saline group. #p < 0.05 versus the 6 mg/kg Tβ4 group. n = 6 (sham), 7 (saline), 8 (Tβ4–6 mg/kg), and 8 (Tβ4–30 mg/kg).

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    Graphs showing correlation of functional recovery with lesion volume, cell loss, and cell proliferation and neurogenesis examined 35 days after TBI. A: The mNSS was significantly correlated with lesion volume. B: Spatial learning was significantly correlated with cell loss in the DG and CA3 region. C: Spatial learning was significantly correlated with the number of with BrdU+ cells in the DG. D: The mNSS was significantly correlated with the number of BrdU+ cells in the cortex. E: Spatial learning was significantly correlated with the number of newborn neurons in the DG. n = 6 (sham), 7 (saline), 8 (Tβ4–6 mg/kg), and 8 (Tβ4–30 mg/kg).

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