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.

Abstract

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.

Traumatic brain injury (TBI) is a serious public health problem worldwide.31 In the US, according to estimates from the Centers for Disease Control and Prevention (CDC), there are approximately 2.5 million TBI-related emergency department visits, hospitalizations, and deaths combined each year. Among these persons, approximately 87% were treated and released from emergency departments, 11% were hospitalized and discharged, and 2% died.15 An estimated 5.3 million Americans live with TBI-related disability.38 Although significant efforts have been devoted to the development of neuroprotective agents, all of these efforts have failed to demonstrate efficacy in clinical trials of TBI.53,68,83 Clinical trials in TBI have mainly focused on targeting a single pathophysiological pathway.48 However, successful therapy may require targeting multiple injury pathways.77

N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP), a small tetrapeptide, inhibits inflammation,14 fibrosis,14 and primitive hematopoietic cell proliferation,39 and promotes endothelial cell proliferation and angiogenesis.14,44,78 It was originally purified from fetal calf bone marrow,39 and its protein precursor is thymosin β4 (Tβ4).28 AcSDKP was first reported as an endogenous hematopoiesis regulator that reversibly inhibited, in vitro and in vivo, the entry of hematopoietic pluripotent stem cells and normal early progenitors into the S phase, maintaining them in the G0 phase and thereby obstructing the DNA synthesis.26,39,50 Angiotensin I–converting enzyme (ACE) inhibitors selectively inhibit AcSDKP degradation and result in an increased plasma level of AcSDKP.6,35,62 Increased AcSDKP level dramatically restrains cardiac and renal fibrosis in hypertensive rats without affecting blood pressure or organ hypertrophy, which suggests that the antifibrotic effect of ACE inhibitors may be related to their ability to increase the level of AcSDKP in plasma.57 ACE inhibitors administered 2 hours prior to induction of stroke in the rat have been shown to reduce the infarct volume.58 Although several studies have indicated that ACE inhibitors improve recovery after cerebral ischemia,42,70,79 a previous study on TBI rats treated with ACE inhibitors, prior to injury and 7 days thereafter, showed that ACE inhibitor treatment exacerbated motor deficits following TBI, which might be related to its potential effect of increasing neuropeptide substance P activity.30 Administration of AcSDKP prevents the development of diabetic cardiomyopathy and renal damage, which underscores the end-organ protective effects of AcSDKP.12,13,67 However, the biological function of AcSDKP in the brain is unclear. We have recently demonstrated that treatment with AcSDKP initiated 1 hour after stroke onset effectively reduced infarct volume and neurological deficits in adult rats, which provides the first evidence that AcSDKP has a neuroprotective effect.88 However, the effect of AcSDKP on functional recovery in TBI rats has not been investigated. In this study, we subcutaneously infused AcSDKP to rats subjected to TBI induced by controlled cortical impact, and we investigated cognitive and sensorimotor functional recovery as well as the potential mechanisms underlying therapeutic effects of AcSDKP.

Methods

All study procedures were approved by the Institutional Animal Care and Use Committee of Henry Ford Health System. To prevent potential biases of performance and detection, the persons who performed the experiments, collected data, and assessed outcome were blinded throughout the course of the experiments and were unaware of the treatment allocation.

Animal Model and Experimental Groups

The controlled cortical impact rat model of TBI was used for this study.21 Adult male Wistar rats (2–3 months old) weighing 300 to 335 g (Charles River Laboratories) were anesthetized with chloral hydrate (350 mg/kg body weight) intraperitoneally. Rectal temperature was maintained at 37°C ± 5°C using a feedback-regulated water heating pad. The rats were placed in a stereotactic frame. Two 10-mm-diameter craniotomies were performed adjacent to the central suture, midway between the lambda and bregma. The second craniotomy allowed for lateral movement of cortical tissue. The dura mater was kept intact over the cortex. Cortical injury was delivered by impacting the left (ipsilateral) cortex with a pneumatic piston containing a 6-mm-diameter tip at a rate of 4 m/sec and 2.5 mm of compression. Velocity was measured with a linear velocity displacement transducer. For the present study, the rats were randomly divided into 3 groups (n = 15/group): Group 1, sham (craniotomies only, no injury); Group 2, TBI + vehicle (0.01 N acetic acid); and Group 3, TBI + AcSDKP (0.8 mg/kg/day, Abbiotec). AcSDKP or an equal volume of 0.01 N acetic acid was administered 1 hour after injury subcutaneously with an osmotic pump (Alzet model 1003D) for 3 days. The dose of AcSDKP used in the present study was chosen based on our previous study in rats after stroke, which was shown to provide potent neuroprotection.88 To label proliferating cells after injury, 100 mg/kg 5-bromo-2′-deoxyuridine (BrdU)86 was injected intraperitoneally into the rats daily for 10 days, starting 1 day after injury. Seven animals from each group were euthanized 1 day after injury for Western blot analysis (n = 3) and immunostaining for neuroinflammation (n = 4). The remaining rats were allowed to survive for 35 days after injury (n = 8/group).

Evaluation of Neurological Outcome

Modified Neurological Severity Score Test

To evaluate neurological functional recovery, the modified Neurological Severity Score (mNSS) test16 was performed prior to injury and at 1, 7, 14, 21, 28, and 35 days after injury. The scale ranges from 0 to 18 (normal score 0; maximal deficit score 18). Our previous studies used the mNSS to test the motor (muscle status and abnormal movement), sensory (visual, tactile, and proprioceptive), and reflex reactions.46 For each abnormal behavior or for lack of an expected reaction, 1 point is awarded, which means that the higher the score, the more severe the injury.

Foot-Fault Test

To evaluate sensorimotor function, the foot-fault test was employed prior to injury and at 1, 7, 14, 21, 28, and 35 days after injury. With each weight-bearing step walking on a grid, a paw might fall or slip from the wires. Each fall or slip was recorded as a foot fault.8 A total of 50 steps were recorded for the right forelimb.

Morris Water Maze Test

To detect spatial learning functional recovery, a modified version of the Morris water maze (MWM) test was used.17 From Day 31 to Day 35 after injury, animals were tested with 4 trials per day consecutively. A swimming pool for rats (1.8 m in diameter) was located in a room with many clues (such as pictures on the walls, lamps, and a camera on the ceiling), which were visible to rats from the pool for spatial orientation. The position of the cues remained fixed throughout the experiment. Data were collected using the Human Visual Image (HVS) Image 2020 Plus Tracking System (US HVS Image).47 The swimming pool was divided into 4 equal quadrants formed by imaging lines. Throughout the test period, the platform was located in the northeast quadrant 2 cm below water in a randomly changing position during 4 trials every day, including locations against the wall, toward the middle of the pool, or off center but always within the target quadrant. At each trial, the rat was placed at 1 of 4 fixed starting points (designated North, South, East, and West), facing toward the wall of pool. The animal was allowed 90 seconds to find the submerged and nonvisible transparent platform. Once found, the rats were allowed to remain on the platform for 10 seconds. If the animal failed to find the platform within 90 seconds, it was placed on the platform for 10 seconds, and the trial was terminated, with 90 seconds assigned as the score. The percentage of time the animals spent swimming within the target quadrant relative to the total amount of time swimming in the maze before reaching the hidden platform or within 90 seconds for those rats that failed to find the platform was recorded for statistical analysis.

Tissue Preparation

Western Blot Analysis and Immunostaining for Neuroinflammation

On Day 1 after injury, rats were anesthetized with 4% chloral hydrate intraperitoneally and then perfused with saline solution transcardially. The ipsilateral cortical tissue from the lesion boundary zone was dissected, frozen in liquid nitrogen, and stored at −80°C until use.

Immunohistochemistry Analysis

On Day 35, rats were anesthetized with chloral hydrate intraperitoneally and then perfused with saline solution transcardially, followed by 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS) (pH 7.4). After perfusion, the brains were removed and fixed in 4% paraformaldehyde for 2 days and then each forebrain was cut into 2-mm-thick coronal blocks for a total of 7 blocks.56 All blocks were embedded in paraffin and were cut into a series of 6-μm-thick sections.

Lesion Volume Analysis

To measure the lesion volume after injury, H & E staining was used to demarcate the lesion area of each coronal section, and one 6-μm-thick section from each of 7 coronal blocks was traced by a microcomputer imaging device (MCID, Imaging Research), as described previously.87 The formula to calculate the percentage of cortical lesion volume was as follows: (contralateral cortical volume − ipsilateral cortical volume)/(contralateral cortical volume) × 100%.71

Immunohistochemical Studies

Antigen retrieval was performed by boiling sections in 10 mM citrate buffer (pH 6) for 10 minutes. After washing with PBS, sections were incubated with 0.3% H2O2 in PBS for 10 minutes, blocked with 1% bovine serum albumin containing 0.3% Triton X-100 at room temperature for 1 hour, and incubated with anti–endothelial barrier antigen (EBA, 1:1000; Covance), anti-CD68 (1:200; AbD, Serotec), anti–glial fibrillary acidic protein (GFAP, 1:1000; Dako), anti-NeuN monoclonal antibody (1:300; MAB 377, Chemicon), anti-synaptophysin antibody (1:800; MAB5258, Millipore), or anti-fibrin antibody (1:1000, Acc Chem) at 4°C overnight. For negative controls, primary antibodies were omitted. After washing, sections were incubated with biotinylated anti-mouse or anti-rabbit antibodies (1:200; Vector Laboratories, Inc.) at room temperature for 30 minutes. After an additional washing, sections were incubated with an avidin-biotin peroxidase system (ABC kit; Vector Laboratories, Inc.), visualized with diaminobenzidine (Sigma), and counterstained with hematoxylin.

Immunofluorescent Staining

Double immunostaining was performed to identify newly generated endothelial cells (BrdU/EBA-positive) and newly formed mature neurons (BrdU/NeuN-positive) 35 days after injury. Briefly, after being deparaffinized and rehydrated, brain sections were boiled in 10 mM citric acid buffer (pH 6) for 10 minutes. After washing with PBS, sections were incubated in 2.4 N HCl at 37°C for 20 minutes. Sections were incubated with 1% bovine serum albumin containing 0.3% Triton X-100 in PBS. Sections were then incubated with mouse anti-NeuN antibody (1:200; Chemicon) or anti-EBA at 4°C overnight. For negative controls, primary antibodies were omitted. Fluorescein isothiocyanate–conjugated anti-mouse antibody (1:400; Jackson ImmunoResearch) was added to sections at room temperature for 2 hours. Sections were then incubated with rat anti-BrdU antibody (1:200; Dako) at 4°C overnight, and subsequently incubated with Cy3-conjugated goat anti-rat antibody (1:400; Jackson ImmunoResearch) at room temperature for 2 hours. Each of the steps was followed by three 5-minute rinses in PBS. Tissue sections were mounted with Vectashield mounting medium (Vector Laboratories).

Golgi-Cox Staining

To measure the number of neuronal dendritic spines, the FD Rapid Golgi Stain kit (FD NeuroTechnologies) was used to perform Golgi staining following the vendor's protocol. Briefly, the freshly dissected brains were immersed in impregnation solution (made by mixing equal volumes of Solutions A and B) and stored at room temperature for 2 weeks in the dark. The brains were then transferred into solution C and kept for 48 hours at 4°C in the dark. Vibratome sections (100 μm) were cut and stained using standard staining procedures.

Western Blot Analysis

Brain tissue was washed in PBS once and sonicated in lysis buffer (20 mM Tris, pH 7.6, 100 mM NaCl, 1% Non-idet P-40, 0.1% sodium dodecyl sulfate, 1% deoxycholic acid, 10% glycerol, 1 mM EDTA, 1 mM NaVO3, 50 mM NaF, Protease Inhibitor Cocktail Set I from Calbiochem). Soluble protein was then obtained by centrifugation at 13,000g at 4°C for 15 minutes. Bicinchoninic acid protein assay (Pierce) was used to measure total protein concentration. Equal amounts of lysate (20 μg protein/lane) were loaded to sodium dodecyl sulfate–polyacrylamide electrophoresis on Novex Trisglycine precast gels (Invitrogen), and separated proteins were then electrotransferred to polyvinylidene fluoride membranes (Millipore). SuperSignal West Pico Chemiluminescent Substrate system (Thermo Scientific) was applied to visualize different proteins by reacting to various antibodies. The band intensity was analyzed using Scion Image software (Scion). Antibodies used for Western blot included anti–transforming growth factor–β1 (TGF-β1) polyclonal antibody (Santa Cruz, SC-146), anti–nuclear factor–κB (NF-κB) antibody (Abcam, ab7970), and antiactin (1:5000, Santa Cruz Biotechnology).

Cell Counting and Quantitation

The CD68-positive microglia/macrophages, glial fibrillary acidic protein (GFAP)–positive astrocytes, neuronal nuclei (NeuN)–positive neuronal cells, NeuN/BrdU double-stained newborn mature neurons, and EBA/BrdU-colabeled newborn endothelial cells were counted in the lesion boundary zone and the dentate gyrus (DG). For analysis of neurogenesis, we counted BrdU-positive cells and NeuN/BrdU-colabeled cells in the DG and its subregions, including the subgranular zone, the granular cell layer, and the molecular layer. The fields of interest were digitized under the light microscope (Eclipse 80i, Nikon) at a magnification of either 200 or 400, using a CoolSNAP color camera (Photometrics) interfaced with the MetaMorph image analysis system (Molecular Devices), as described in detail previously.91 Briefly, 5 fields of view in the lesion boundary zone from the epicenter of the injury cavity (bregma −3.3 mm) and 9 fields of view in the ipsilateral DG were counted in each section. From our previous experience, our interrater reliability was greater than 95% when the cell counts were compared between 2 independent trained, blinded observers scoring the same sections of an animal. In the present study, 1 blinded observer performed the cell counting in all brain sections.

Statistical Analysis

All data are presented as the mean ± SD. ANOVA was used for repeated measurements of spatial performance, sensorimotor function, and Western blot analysis. For cell counting, a 1-way ANOVA followed by post hoc Tukey's tests was used to compare the differences between the AcSDKP-treated, acetic acid–treated, and sham groups. Differences were considered significant when the p value was < 0.05.

Results

AcSDKP Significantly Reduced Cortical Lesion Volume in Rats After TBI

Lesion volume was measured 35 days postinjury. AcSDKP treatment significantly decreased lesion volume compared with the vehicle group (Fig. 1, F2, 21 = 318.50, q = 9.990, p < 0.05), indicating a protective effect of AcSDKP on brain tissue.

FIG. 1.
FIG. 1.

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.

AcSDKP Significantly Enhanced Spatial Learning in Rats After TBI

MWM tests were performed during the last 5 days (31–35 days postinjury) before planned death to estimate the learning function of the hippocampus. The higher the percentage of time animals spend in the correct quadrant where the hidden platform is located in the water maze, the better their spatial learning function. The percentage of time spent by AcSDKP-treated rats in the correct quadrant increased significantly between 33 and 35 days compared with the vehicle group (Fig. 2A, at Day 33, F2, 21 = 34.27, q = 8.729, p < 0.05; at Day 34, F2, 21 = 29.58, q = 7.092, p < 0.05; and at Day 35, F2, 21 = 36.94, q = 8.871, p < 0.05). These data indicate that AcSDKP improves memory/learning functional recovery after TBI.

FIG. 2.
FIG. 2.

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.

AcSDKP Significantly Reduced Sensorimotor Deficits in Rats After TBI

The lower the score on the mNSS or foot-fault tests, the better the functional recovery. Spontaneous functional recovery occurred in the vehicle-treated group over the 35 days postinjury. AcSDKP treatment significantly decreased mNSS score and foot-fault steps from Day 7 postinjury compared with the vehicle group (Fig. 2B and C, for mNSS score, at Day 7, F2, 21 = 254.29, q = 19.886, p < 0.05; at Day 14, F2, 21 = 520.62, q = 16.174, p < 0.05; at Day 21, F2, 21 = 290.54, q = 13.298, p < 0.05; at Day 28, F2, 21 = 331.35, q = 15.109, p < 0.05; and at Day 35, F2, 21 = 245.87, q = 16.731, p < 0.05. For foot-fault test, at Day 7, F2, 21 = 114.28, q = 9.421, p < 0.05; at Day 14, F2, 21 = 46.68, q = 7.220, p < 0.05; at Day 21, F2, 21 = 91.57, q = 12.482, p < 0.05; at Day 28, F2, 21 = 39.87, q = 8.444, p < 0.05; and at Day 35, F2, 21 = 28.000, q = 6.792, p < 0.05). Together, these data indicate that AcSDKP treatment improves sensorimotor functional recovery after TBI.

AcSDKP Significantly Reduced Neuronal Cell Loss and Increased Neurogenesis in the Hippocampus in Rats After TBI

To investigate the effect of AcSDKP treatment on neuronal protection after TBI, anti-NeuN antibody was employed to identify neurons 35 days after injury. In the DG and cornu ammonis 3 (CA3) regions of the hippocampus, the number of NeuN-positive cells in the AcSDKP treatment group was significantly higher than that in the vehicle group (Fig. 3A–G, for DG, F2, 21 = 43.12, q = 9.708, p < 0.05; for CA3, F2, 21 = 56.12, q = 10.808, p < 0.05). In the DG, AcSDKP therapy significantly increased the number of BrdU-positive/NeuN-positive cells (newly generated mature neurons) compared with the vehicle (Fig. 3H–K, F2, 21 = 120.50, q = 12.658, p < 0.05). These data in concert suggest that AcSDKP treatment promotes neuroprotection and neurogenesis in the DG in rats after TBI.

FIG. 3.
FIG. 3.

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.

AcSDKP Significantly Increased Angiogenesis in Rats After TBI

To identify newly generated vasculature in the brain, BrdU/EBA double staining was performed 35 days after injury. BrdU-positive/EBA-positive staining showed that AcSDKP treatment significantly increased newborn endothelial cells compared with the vehicle group (Fig. 4, for cortex, F2, 21 = 244.00, q = 13.066, p < 0.05; for CC, F2, 21 = 333.23, q = 18.255, p < 0.05; for DG, F2, 21 = 244.95, q = 19.243, p < 0.05; for CA3, F2, 21 = 365.26, q = 11.593, p < 0.05; and for CA1, F2, 21 = 242.67, q = 13.064, p < 0.05), which suggests that AcSDKP promotes angiogenesis after TBI.

FIG. 4.
FIG. 4.

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.

AcSDKP Significantly Increased the Number of Dendritic Spines in the Injured Brain Regions After TBI

Neuronal dendritic spines were detected following Golgi-Cox staining 35 days after injury. Compared with the vehicle group, the AcSDKP treatment group showed a significantly increased number of dendritic spines after TBI (Fig. 5, for cortex, F2, 9 = 19.13, q = 6.852, p < 0.05; for DG, F2, 9 = 12.80, q = 4.828, p < 0.05; for CA3, F2, 9 = 67.75, q = 7.778, p < 0.05; and for CA1, F2, 9 = 29.38, q = 7.575, p < 0.05), which suggests that AcSDKP has protective and/or neural plasticity effects on dendritic spines in TBI rats.

FIG. 5.
FIG. 5.

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.

AcSDKP Significantly Increased Synaptophysin Expression in the Injured Brain After TBI

Synaptophysin was used to measure the density of synapses 35 days after injury. In the injured rats treated with vehicle, the density of synapses was remarkably decreased. However, AcSDKP administration after injury significantly decreased the loss of synapses (Fig. 6, for cortex, F2, 21 = 58.77, q = 7.013, p < 0.05; for DG, F2, 21 = 45.57, q = 7.283, p < 0.05; for CA3, F2, 21 = 28.58, q = 10.688, p < 0.05; and for CA1, F2, 21 = 41.85, q = 7.126, p < 0.05), which suggests that AcSDKP reduces synapse loss and/or promotes synaptic plasticity after TBI.

FIG. 6.
FIG. 6.

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.

AcSDKP Significantly Reduced the Number of CD68-Positive Microglia/Macrophages in the Injured Brain Regions After TBI

To evaluate effects of AcSDKP on neuroinflammation, CD68 staining was used to detect microglia/microphages in TBI rats.87 Thirty-five days after injury, inflammation was notably increased, while AcSDKP treatment significantly decreased the CD68-positive cells compared with the vehicle group (Fig. 7, for cortex, F2, 21 = 600.39, q = 32.950, p < 0.05; for corpus callosum, F2, 21 = 259.80, q = 23.001, p < 0.05; for DG, F2, 21 = 658.24, q = 21.296, p < 0.05; for CA3, F2, 21 = 700.83, q = 29.112, p < 0.05; and for CA1, F2, 21 = 265.47, q = 28.610, p < 0.05), which indicates that AcSDKP significantly attenuates neuroinflammation after TBI.

FIG. 7.
FIG. 7.

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.

AcSDKP Decreased GFAP Expression in the Injured Brain After TBI

Astrocytes were activated post-TBI, as also shown in our previous studies.90,92 GFAP staining was performed to detect astrocytes 35 days after injury. There was a significantly decreased GFAP-positive cell number in AcSDKP treatment group compared with vehicle group (Fig. 8, for cortex, F2, 21 = 195.31, q = 17.726, p < 0.05; for corpus callosum, F2, 21 = 386.08, q = 25.169, p < 0.05; for DG, F2, 21 = 181.07, q = 15.360, p < 0.05; for CA3, F2, 21 = 192.40, q = 14.777, p < 0.05; and for CA1, F2, 21 = 572.92, q = 26.673, p < 0.05), which suggests that AcSDKP reduces reactive astrogliosis after TBI.

FIG. 8.
FIG. 8.

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.

AcSDKP Significantly Reduced Fibrin in the Injured Brain After TBI

To investigate the effects of AcSDKP on fibrin deposition, staining with fibrinogen/fibrin antibody was performed on brain sections 1 day after TBI. Compared with vehicle-treated rats, the percentage of fibrin-positive area was significantly decreased after AcSDKP treatment in TBI rats (Fig. 9, for cortex, F2, 9 = 42.88, q = 8.908, p < 0.05; for corpus callosum, F2, 9 = 50.53, q = 12.312, p < 0.05; for DG, F2, 9 = 23.32, q = 7.978, p < 0.05; for CA3, F2, 9 = 87.16, q = 16.905, p < 0.05; and for CA1, F2, 9 = 14.36, q = 5.686, p < 0.05), which indicates that AcSDKP may have protective effects of vascular and blood-brain barrier (BBB) integrity after TBI.

FIG. 9.
FIG. 9.

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.

AcSDKP Significantly Reduced Expression of TGF-β1 and NF-κB After TBI

Compared with vehicle treatment, AcSDKP significantly reduced the expression of TGF-β1 and NF-κB protein in the lesion boundary zone 1 day after TBI (Fig. 10, for TGF-β1, F2, 6 = 7.69, q = 5.075, p < 0.05; and for NF-κB, F2, 6 = 9.21, q = 5.612, p < 0.05), which suggests that AcSDKP contributes to neuroprotection by suppressing the TGF-β1 and NF-κB expression, which modulates cerebral vascular patency and integrity and neuroinflammation after brain injury.29

FIG. 10.
FIG. 10.

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.

Discussion

The principal findings of the present study are as follows: AcSDKP treatment 1 hour after TBI significantly 1) improved cognitive and sensorimotor functional recovery compared with vehicle treatment; 2) reduced lesion volume, hippocampal neuronal cell loss, fibrin deposit, and the number of activated microglia/macrophages and reactive astrogliosis in the injured brain; 3) increased the number of newborn vessels and mature neurons as well as synaptophysin expression and the number of dendritic spines in the injured brain; and 4) reduced protein expression of TGF-β1 and NF-κB in the injured brain. These findings suggest that continuous infusion of AcSDKP initiated 1 hour postinjury for 3 days not only provides neuroprotection but also promotes neurovascular remodeling in rats with TBI. These dual effects may maximize functional recovery in TBI rats, implying that AcSDKP is a multifunctional agent and has promising therapeutic potential for treatment of TBI.

Increasing evidence suggests that neuroinflammation occurs in both acute and chronic stages of TBI,1,32,73 which may impact the pathophysiology and treatment of TBI. Neuroinflammation is present after injury and persists for a long period of time,72 which may provide a wide therapeutic window for antiinflammatory intervention after TBI. After TBI, the rapid inflammatory response is followed by neuronal injury and BBB rupture.52,69 Within minutes, activated microglial cells are detected and resemble peripheral macrophages by proinflammatory cytokines and chemokines.10 In our study, CD68, a marker for microglia/macrophages,33 was used to evaluate neuroinflammation after TBI. Compared with vehicle-treated rats, the number of CD68-positive microglia/macrophages was dramatically decreased in the AcSDKP-treated rats, suggesting that infusion of AcSDKP initiated 1 hour postinjury and continued for 3 days effectively suppresses activation of microglia/macrophages after TBI and reduces neuroinflammation.

Astrocytes secrete inflammatory mediators, which play an important role in secondary injury after TBI.23 GFAP is a sensitive marker for activated astrocytes.23 Our study showed that few astrocytes expressed GFAP in normal brain tissue, while a robust increase in GFAP was expressed in astrocytes after TBI, a finding consistent with our earlier study.9 AcSDKP administration significantly reduced numbers of GFAP-reactive astrocytes. A previous clinical study indicates that TBI patients who die within the first 24 hours have a significantly higher GFAP concentration in the blood at 6, 12, and 24 hours after TBI; moreover, a high level of GFAP in the blood was associated with poor outcome at 1–6 months.20

From our study, newly generated vessels identified with BrdU/EBA-positive staining were detected in the lesion boundary zone and DG of the rats after TBI, indicating that TBI induces angiogenesis, which is consist with previous studies.51,82,85 AcSDKP further enhances angiogenesis in these regions. AcSDKP promotes formation of capillary-like structures in vitro by activating the proliferation and migration of endothelial cells and increases capillary density in rat heart with myocardial infarction.44,78 MRI indices, including cerebral blood volume, cerebral blood flow, and blood-to-brain transfer constant, were used to monitor development of angiogenesis after TBI noninvasively, which found that new vessels are permeable at the early phase of angiogenesis and become less permeable as they mature.40,41 On the blood-to-brain transfer constant map, angiogenic areas became apparent 3–4 weeks after TBI.40 Furthermore, in the ipsilateral DG regions after TBI, elevated cerebral blood volume was observed starting from Day 1 to 2 weeks after injury, which suggests that newly generated vessels by TBI-induced angiogenesis are functional. These newly born vessels may contribute to functional recovery after TBI, since angiogenesis is coupled with and may drive neurogenesis in the DG.75,82

Neurogenesis occurs in the subventricular zone of the lateral ventricle and the subgranular zone of the DG in mammals during adulthood. It also occurs in the disease process of different neurological disorders.25,74,93 Neurogenesis is stimulated by TBI in rodents and humans.60,94 Newly generated neuroblasts in the subventricular zone can migrate to the injured area and become mature neurons after TBI.11,18,27 AcSDKP not only significantly reduced neuronal cell loss in the hippocampus, but it also increased the number of newly born neurons and dendritic spines, which might contribute to memory recovery after TBI. Additionally, cortical lesion volume of the brain was significantly reduced with AcSDKP treatment initiated 1 hour after TBI, which is consistent with our previous study, showing that Tβ4 administered 6 hours after TBI significantly reduced lesion volume.86 Further studies to investigate the effects of delayed, e.g., 6-hour AcSDKP treatment post-TBI, time points that are more realistic for preclinical and clinical trials are now warranted. The protection of the cortex by AcSDKP may contribute to improving sensorimotor functional recovery (reduced mNSS and number of foot faults). Synaptophysin is a well-established marker for a presynaptic vesicle membrane protein.80 The elevated expression of synaptophysin in our study with AcSDKP treatment is consistent with that reported in previous studies,24 suggesting that increased synaptophysin level in the rat brain is related to improved spatial memory. This is also in agreement with our Golgi-Cox staining, showing that AcSDKP treatment increased the number of dendritic spines. Based on our current study, AcSDKP amplifies angiogenesis, neurogenesis, and synaptogenesis in the hippocampus, which may contribute to recovery of cognitive function after TBI. However, increased neurogenesis is not always good for brain function. In animal experiments, status epilepticus can also cause a marked increase in adult neurogenesis.61 It has been speculated that seizure-induced aberrant neurogenesis contributes to deficits in hippocampal learning and memory that are associated with status epilepticus.61 However, the present study showed an enhancement in spatial memory, suggesting that AcSDKP-induced newborn neurons are functionally integrated into or enhanced neuronal circuitry. Thus, an AcSDKP-induced increase in neurogenesis may be favorable for spatial learning after TBI. Our present data are in agreement with a recent study that demonstrated that intrahippocampal infusion of AcSDKP facilitated the generation of new neurons in the hippocampus and enhanced spatial memory in normal mice.37 In our current study, the level of AcSDKP was not determined in the plasma and the brain after TBI. AcSDKP can pass the BBB in normal rats and stroke rats,88 indicating that AcSDKP is a promising potential treatment for neural injuries.

Fibrinogen is known for its role as the protein component of blood clots and is normally excluded from the brain parenchyma via the BBB.43,63,65 BBB damage occurs after TBI.76 One of the earliest events after TBI is leakage of blood components into brain parenchyma in areas that correlate with the formation of reactive astrocytes.59,66 The soluble blood protein fibrinogen is converted to insoluble fibrin by the action of thrombin and is deposited in the nervous system promptly following vascular damage or BBB disruption.2 Fibrinogen plays a causative role in nervous system disease as a regulator of inflammation,2,55 remyelination,3 and neurodegeneration.19,64 Fibrinogen regulates TGF-β1–mediated signal transduction within CNS tissues after vascular damage and induces reactive astrogliosis and deposition of chondroitin sulfate proteoglycans (CSPGs).65 Mice genetically or pharmacologically depleted of fibrinogen show a dramatic reduction in TGF-β1 and reduced astrogliosis and neurocan deposition after injury.65 In primary astrocyte cultures, fibrinogen is a potent inducer of secretion of proteoglycans, and conditioned medium of fibrinogen-treated astrocytes inhibits neurite outgrowth.65 Our data indicate that fibrin accumulates in the injured brain regions after TBI, and AcSDKP decreases the accumulation and improves functional recovery, suggesting that fibrin plays a critical role in pathophysiology contributing to functional deficits after TBI.

NF-κB can be activated by inflammatory mediators,7 and its activation results in the induction of inflammatory responsive genes in TBI.89 Activation of NF-κB is present in astrocytes after injury and induces astrocyte swelling/brain edema after TBI.34 Correspondingly, NF-κB inhibition significantly reduced trauma-induced astrocyte swelling.34 In our study, the NF-κB level was notably decreased to a nearly normal level in the AcSDKP treatment group 1 day after TBI compared with vehicle-treated injured rats. This is in agreement with our previous study that demonstrated that inactivation of the TGF-β1/NF-κB signaling by AcSDKP may contribute to its neuroprotection in stroke rats.88

TGF-β1 has multiple functions in various organs, regulating the growth, differentiation, and survival of different cell types.22 The role of TGF-β1 is complex. TGF-β1 has neuroprotective effects,22 but it also participates in neuroinflammation and the scarring response in the rat brain.45 A recent study indicated that the blood protein fibrinogen, after leaking into the CNS after BBB disruption, served as an early signal for the induction of glial scar formation via the TGF-β1 signaling pathway.65 Our data suggest that brain TGF-β1 expression was increased after TBI. More importantly, our data show that AcSDKP treatment significantly reduced fibrin accumulation, TGF-β1 expression, neuroinflammation, and astrogliosis. These data suggest that TGF-β1 plays a critical role in brain damage after TBI and AcSDKP can attenuate these detrimental effects mediated by TGF-β1. Together, our findings strongly suggest that: 1) the fibrin-mediated TGF-β1 signaling pathway contributes to brain damage; and 2) AcSDKP provides neuroprotection and promotes neurovascular remodeling likely by inhibiting TGF-β1 signaling. These neuroprotective and neurorestorative effects of AcSDKP, in concert, may contribute to improved functional recovery after TBI.

Our data indicate that continuous infusion of AcSDKP initiated 1 hour postinjury for 3 days not only provides neuroprotection but also promotes neurovascular remodeling in rats with TBI. These dual effects may maximize functional recovery in TBI rats. We are aware that in the vast majority of preclinical research, the treatment compounds are administered early and, frequently, even prior to the TBI.48 The administration of a compound early by prehospital care personnel may be problematic because of the practical time constraints associated with injury and patient management and the difficulty in obtaining informed consent.49 Our previous study suggested that delayed Tβ4 treatment initiated 24 hours postinjury significantly improves histological and functional outcomes in rats with TBI.84 Thus, investigation of whether AcSDKP delivered in the subacute (e.g., at or > 24 hours postinjury) period after TBI improves functional outcome is warranted. Treatments targeting neurovascular remodeling at a greatly extended treatment window, if successful, can be made available for all brain injuries.

Several characteristics of AcSDKP indicate that this tetrapeptide is a promising neurovascular protective and remodeling agent for TBI, and therefore merits further preclinical development and evaluation. First, while the BBB poses significant challenges to the intraparenchymal delivery of neuroprotective agents, AcSDKP with a low molecular weight of 488 D can readily cross the BBB in the intact and injured brain, as demonstrated in our previous study.88 Second, as a naturally occurring peptide, the pharmacokinetics and metabolism of AcSDKP have been well established, and there is no apparent toxicity in rodents.5,36,67,81 More importantly, with the identification of multiple molecular mechanisms and mediators on the pathogenesis of TBI, the multitargeted effects of AcSDKP on the neurovascular unit could achieve optimized therapeutic efficacy. AcSDKP is cleared almost exclusively by ACE.4 An alternative approach to increase AcSDKP levels in plasma is the use of ACE inhibitors.4 Some centrally acting ACE inhibitors may slow disease progression in patients with Alzheimer's disease.54 However, ACE inhibitors have multiple other effects and exacerbate the histological damage and motor deficits in a rat model of diffuse TBI induced by impact acceleration, likely by regulating substance P–mediated neuronal injury.30 Our data imply that administration of AcSDKP may be a novel treatment of TBI.

Conclusions

In the present study, we demonstrate that early (1 hour postinjury) administration of AcSDKP provides neuroprotection (reduced neuronal loss and cortical lesion size, fibrin accumulation), promotes neurovascular remodeling (increased angiogenesis, neurogenesis, and dendritic spines), reduces neuroinflammation and astrogliosis, as well as improves functional recovery in rats after TBI, which may be mediated, in part, by AcSDKP's inhibition of TGF-β1/NF-κB signal pathway. Thus, AcSDKP is a multifunctional neuroprotective, neurorestorative, and antiinflammatory agent for treatment of TBI. Further investigation of the optimal dose and therapeutic window of AcSDKP treatment for TBI and the associated underlying mechanisms of action is warranted.

Acknowledgments

This work was partially supported by National Institutes of Health Grant R01 NS 079612 (to Zheng Gang Zhang).

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    Zhang YChopp MMeng YZhang ZGDoppler EWinter S: Cerebrolysin improves cognitive performance in rats after mild traumatic brain injury. J Neurosurg 122:8438552015

  • 93

    Zhao CDeng WGage FH: Mechanisms and functional implications of adult neurogenesis. Cell 132:6456602008

  • 94

    Zheng WZhuGe QZhong MChen GShao BWang H: Neurogenesis in adult human brain after traumatic brain injury. J Neurotrauma 30:187218802013

Disclosures

The authors report no conflict of interest concerning the materials or methods used in this study or the findings specified in this paper.

Author Contributions

Conception and design: Xiong, ZG Zhang, Chopp, L Zhang, Mahmood. Acquisition of data: Xiong, Y Zhang, Meng, L Zhang. Analysis and interpretation of data: all authors. Drafting the article: Xiong, Y Zhang, ZG Zhang, Chopp. Critically revising the article: Xiong, Y Zhang, ZG Zhang, Chopp. Reviewed submitted version of manuscript: all authors. Approved the final version of the manuscript on behalf of all authors: Xiong. Statistical analysis: Xiong, Y Zhang. Administrative/technical/material support: Xiong. Study supervision: Xiong, Chopp.

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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.

Headings

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.

  • View in gallery

    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.

  • View in gallery

    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.

  • View in gallery

    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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Zhang YChopp MMeng YZhang ZGDoppler EMahmood A: Improvement in functional recovery with administration of Cerebrolysin after experimental closed head injury. J Neurosurg 118:134313552013

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Zhang YChopp MMeng YZhang ZGDoppler EWinter S: Cerebrolysin improves cognitive performance in rats after mild traumatic brain injury. J Neurosurg 122:8438552015

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Zhao CDeng WGage FH: Mechanisms and functional implications of adult neurogenesis. Cell 132:6456602008

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Zheng WZhuGe QZhong MChen GShao BWang H: Neurogenesis in adult human brain after traumatic brain injury. J Neurotrauma 30:187218802013

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