Treatment of traumatic brain injury in rats with N-acetyl-seryl-aspartyl-lysyl-proline

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OBJECTIVE

The authors' previous studies have suggested that thymosin beta 4 (Tβ4), a major actin-sequestering protein, improves functional recovery after neural injury. N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) is an active peptide fragment of Tβ4. Its effect as a treatment of traumatic brain injury (TBI) has not been investigated. Thus, this study was designed to determine whether AcSDKP treatment improves functional recovery in rats after TBI.

METHODS

Young adult male Wistar rats were randomly divided into the following groups: 1) sham group (no injury); 2) TBI + vehicle group (0.01 N acetic acid); and 3) TBI + AcSDKP (0.8 mg/kg/day). TBI was induced by controlled cortical impact over the left parietal cortex. AcSDKP or vehicle was administered subcutaneously starting 1 hour postinjury and continuously for 3 days using an osmotic minipump. Sensorimotor function and spatial learning were assessed using a modified Neurological Severity Score and Morris water maze tests, respectively. Some of the animals were euthanized 1 day after injury, and their brains were processed for measurement of fibrin accumulation and neuroinflammation signaling pathways. The remaining animals were euthanized 35 days after injury, and brain sections were processed for measurement of lesion volume, hippocampal cell loss, angiogenesis, neurogenesis, and dendritic spine remodeling.

RESULTS

Compared with vehicle treatment, AcSDKP treatment initiated 1 hour postinjury significantly improved sensorimotor functional recovery (Days 7–35, p < 0.05) and spatial learning (Days 33–35, p < 0.05), reduced cortical lesion volume, and hippocampal neuronal cell loss, reduced fibrin accumulation and activation of microglia/macrophages, enhanced angiogenesis and neurogenesis, and increased the number of dendritic spines in the injured brain (p < 0.05). AcSDKP treatment also significantly inhibited the transforming growth factor–β1/nuclear factor–κB signaling pathway.

CONCLUSIONS

AcSDKP treatment initiated 1 hour postinjury provides neuroprotection and neurorestoration after TBI, indicating that this small tetrapeptide has promising therapeutic potential for treatment of TBI. Further investigation of the optimal dose and therapeutic window of AcSDKP treatment for TBI and the associated underlying mechanisms is therefore warranted.

ABBREVIATIONSACE = Angiotensin I–converting enzyme; AcSDKP= N-acetyl-seryl-aspartyl-lysyl-proline; BBB = blood-brain barrier; BrdU = 5-bromo-2′-deoxyuridine; CA1, CA2, CA3 = cornu ammonis region 1, 2, 3; CDC = Centers for Disease Control and Prevention; DG = dentate gyrus; EBA = endothelial barrier antigen; GFAP = glial fibrillary acidic protein; mNSS = modified Neurological Severity Score; MWM = Morris water maze; NeuN = neuronal nuclei; NF-κB = nuclear factor–κB; PBS = phosphate-buffered saline; TBI = traumatic brain injury; TGF-β1 = transforming growth factor–β1; Tβ4 = thymosin β4.

Article Information

INCLUDE WHEN CITING Published online May 20, 2016; DOI: 10.3171/2016.3.JNS152699.

Correspondence Ye Xiong, Department of Neurosurgery, Henry Ford Health System, E&R Bldg., Rm. 3096, 2799 West Grand Blvd., Detroit, MI 48202. email: yxiong1@hfhs.org.

© AANS, except where prohibited by US copyright law.

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Figures

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    The effect of AcSDKP on cortical lesion volume examined 35 days after TBI. TBI caused significant cortical tissue loss (B and C) compared with no injury (A). AcSDKP treatment significantly reduced lesion volume caused by TBI compared with the vehicle-treated groups (D). Scale bar = 3 mm. Data represent mean ± SD. There were 8 rats per group. Figure is available in color online only.

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    The effect of AcSDKP on spatial learning in the MWM test (A), sensorimotor function measured by mNSS (B), and right forelimb foot-fault test scores (C) in rats after TBI (8 rats per group). Data represent the mean ± SD. Figure is available in color online only.

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    The effects of AcSDKP on the number of neuronal cells and BrdU/NeuN-positive newborn mature neurons in the DG 35 days after TBI. A–F: Anti-NeuN staining was performed to identify neuronal cells in the DG. Compared with the vehicle group (C and D, arrows indicate cell loss in the DG and CA3), AcSDKP treatment (E and F) significantly increased NeuN-positive neuronal cells in the DG and CA3 regions 35 days after TBI. Scale bar = 40 µm. G: Bar graph showing the number of NeuN-positive neuron cells. H–J: Compared with the vehicle group, AcSDKP treatment significantly increased newborn mature neurons identified with BrdU/NeuN double immunofluorescent staining in the DG as indicated by arrows (H–J), at 35 days postinjury. Scale bar = 20 µm. K: Bar graph showing the number of BrdU/NeuN-positive newborn mature neurons. Data in the graphs represent mean ± SD. There were 8 rats per group. Figure is available in color online only.

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    The effect of AcSDKP on the number of newly generated vessels in rats 35 days after TBI (8 rats per group). Double staining for BrdU (A, arrows) and EBA (B, green signal) was performed to identify newly formed mature vessels (C, arrows) in the brain at Day 35 after TBI in the lesion boundary zone and DG area. Scale bar = 20 µm. Data in the bar graph represent the mean ± SD (D). Figure is available in color online only.

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    The effect of AcSDKP on the number of dendritic spines in the injured brain 35 days after TBI. A–E: Golgi staining was used to identify neuronal dendritic spines in the brain. Compared with the vehicle group, AcSDKP treatment significantly increased the number of neuronal dendritic spines in the brain 35 days after TBI. F: Bar graph showing the number of neuronal dendritic spines. There were 3–4 rats per group. Figure is available in color online only.

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    The effect of AcSDKP on synaptophysin expression 35 days after TBI. AcSDKP treatment (C, F, I, and L) significantly increased synaptophysin expression in various brain regions 35 days after TBI compared with the vehicle group (B, E, H, and K). Scale bar = 20 µm. The bar graph (M) shows that synaptophysin-positive area. Data represent mean ± SD. There were 8 rats per group. Figure is available in color online only.

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    The effect of AcSDKP on microglia/macrophages in the injured brain 35 days after TBI. CD68 staining was performed to detect activation of microglia/macrophages 35 days after TBI. AcSDKP treatment (C, F, I, L, and O) significantly decreased CD68-positive cells in various brain regions 35 days after TBI compared with the vehicle group (B, E, H, K, and N). Scale bar = 20 µm. The bar graph (P) shows the CD68-positive cells. Data represent mean ± SD. There were 8 rats per group. Figure is available in color online only.

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    The effect of AcSDKP on astrocyte activation. GFAP staining was performed to detect activation of astrocytes 35 days after TBI. Some weak expression of GFAP was observed in brain regions of sham animals (A, D, G, J, and M). AcSDKP treatment (C, F, I, L, and O) significantly decreased GFAP-positive cells in various brain regions 35 days after TBI compared with the vehicle group where prominent astrogliosis exists (B, E, H, K, and N). CC = corpus callosum. Scale bar = 20 µm. The data on GFAP-positive cells are shown in the bar graph (P). Data represent mean ± SD. There were 8 rats per group. Figure is available in color online only.

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    The effect of AcSDKP on brain fibrin accumulation 1 day after TBI. Fibrin was weakly expressed in brain regions of sham animals (A, D, G, J, and M). Compared with the vehicle group (B, E, H, K, and N), AcSDKP treatment (C, F, I, L, and O) significantly reduced fibrin accumulation in various brain regions 1 day after TBI. Scale bar = 20 µm. The data on fibrin-positive areas are shown in the bar graph (P). Data represent mean ± SD. There were 4 rats per group. Figure is available in color online only.

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    The effects of AcSDKP on the expression of TGF-β1 and NF-κB 1 day after TBI. Protein levels of TGF-β1 and NF-κB were measured by Western blot analysis in the injured cortical tissue (A). Bar graphs (B and C) show that AcSDKP treatment significantly reduced the expression of TGF-β1 and NFκB 1 day after TBI. There were 3 rats per group. Figure is available in color online only.

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