http://informahealthcare.com/bij ISSN: 0269-9052 (print), 1362-301X (electronic) Brain Inj, 2015; 29(1): 86–92 ! 2015 Informa UK Ltd. DOI: 10.3109/02699052.2014.968621
ORIGINAL ARTICLE
Procyanidins protects against oxidative damage and cognitive deficits after traumatic brain injury Xiang Mao1,3,4,5,6, Shuyu Hao2,5,6, Zhendan Zhu3,5,6,7, Hao Zhang1,3,5,6, Weichuan Wu8, Feifan Xu2,5,6 and Baiyun Liu2,3,4,5,6
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1
Department of Neurosurgery, The First Affiliated Hospital of Anhui Medical University, No. 218 Jixi Road, Shushan District, Hefei, Anhui, People’s Republic of China, 2Department of Neurosurgery, Beijing Tian Tan Hospital, Capital Medical University, Beijing, People’s Republic of China, 3 Neurotrauma Laboratory, Beijing Neurosurgical Institute, Capital Medical University, Beijing, People’s Republic of China, 4Department of Neurotrauma, General Hospital of Armed Police Forces, Beijing, People’s Republic of China, 5China National Clinical Research Center for Neurological Diseases, Beijing, People’s Republic of China, 6Beijing Key Laboratory of Central Nervous System Injury, Beijing, People’s Republic of China, 7General Hospital of Armed Police Forces Clinical College, Anhui Medical University, Hefei, Anhui, People’s Republic of China, and 8 Department of Neurosurgery, The Affiliated Hospital of Guangdong Medical College, Zhanjiang, People’s Republic of China Abstract
Keywords
Primary objective: Oxidative stress is the principal factor in traumatic brain injury (TBI) that initiates the events that result in protracted neuronal dysfunction and remodeling. Importantly, antioxidants can protect the brain against oxidative damage and modulate the capacity of the brain to cope with synaptic dysfunction and cognitive impairment. Research design: To date, however, no studies have investigated the effects of procyanidins (PC) on cognitive deficits after TBI. Methods and Procedures: In the present study, rats with controlled cortical impact (CCI) were used to investigate the protective effects of procyanidins. Main outcomes and results: The results showed that procyanidins reduced the level of malondialdehyde (MDA) and elevated the level of glutathione (GSH) and the activity of superoxide dismutase (SOD). In addition, treatment with procyanidins, which elevated the levels of brain-derived neurotropic factor (BDNF), phosphorylation-cAMP-response element binding protein (pCREB), total CREB, and cyclic AMP (cAMP), improved cognitive performance in the Morris water maze after TBI. Conclusions: These results suggest that procyanidins appear to counteract oxidative damage and behavioral dysfunction after TBI through antioxidant activity and the up-regulation of cAMP/CREB signaling.
Antioxidant, CREB, hippocampus, oxidative stress, procyanidins, TBI
Introduction Traumatic brain injury (TBI) is a leading cause of death and disability in industrialized countries. In the U.S., TBI is a significant public health problem. Each year approximately 1.7 million TBIs are incurred, 53 000 people die, and 3.2–5.3 million others are living with long-term disabilities. Survivors after TBI often suffer cognitive deficits, including impaired learning and memory [1]. The critical mechanisms of secondary injury after brain trauma involve a variety of progressive physiological and pathological changes. It is well-known that oxidative stress plays a significant role in the pathogenesis after TBI [2, 3].
Correspondence: Baiyun Liu, Department of Neurosurgery, Beijing Tian Tan Hospital, Capital Medical University, No. 6 Tiantan Xili, Dongcheng District; Neurotrauma Laboratory, Beijing Neurosurgical Institute, Capital Medical University; Department of Neurotrauma, General Hospital of Armed Police Forces; China National Clinical Research Center for Neurological Diseases; Beijing Key Laboratory of Central Nervous System Injury, Beijing, People’s Republic of China. Tel: +86018301605984, E-mail: [email protected]
History Received 3 May 2014 Revised 24 August 2014 Accepted 19 September 2014 Published online 1 October 2014
It is also well-known that oxidative stress causes the impairment of neurological behavior, motor function and cognition (including learning and memory abilities). Reactive oxygen species (ROS) are formed during normal cellular processes, but superoxide dismutase (SOD) and glutathione (GSH) can tightly control the production of ROS by direct or indirect ways. Neuronal membranes are rich in polyunsaturated fatty acids, which are prime targets for ROS attack, and source for lipid peroxidation reactions. Malondialdehyde (MDA) is a main breakdown product of lipid peroxidation in brain. Moreover, the reduction of ROS can protect the brain against oxidative damage and modulate the capacity of the brain to cope with cognitive impairment after TBI [2–4]. Procyanidins (PC), which are flavonoids with an oligomeric structure, are present in our diet both in foodstuffs and in beverages, such as red wine, tea and fruit juices [5]. Procyanidins contain more effective free radical scavenging capability than others, such as vitamin C, vitamin E, succinate, and beta-carotene [6]. In addition, Procyanidins have been used for antibacterial, antiviral, anti-inflammatory and anticarcinogenic treatments [7].
The effects of Procyanidins in TBI
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DOI: 10.3109/02699052.2014.968621
The mechanisms by which oxidative stress affects cognitive function have not been fully established, but it has been suggested that the impairment of cognitive function correlates with the reduction of brain-derived neurotropic factor (BDNF), and it has been shown that excessive oxidative stress can cause a reduction in BDNF and a subsequent decline in cognition [8]. BDNF appears to affect synaptic function through the modulation of its downstream effectors, such as cyclic AMP-response element binding protein (CREB), and it is CREB that is implicated in the synaptic events underlying learning and memory [9, 10] in a wide range of biologic processes when activated by phosphorylation at Ser133. The phosphorylation/activation of CREB (pCREB) on Ser133 by cyclic AMP (cAMP) or Ca2+dependent protein kinase II (CaMKII) is critical for longterm memory consolidation [11–13]. Studies have also shown that increasing cAMP and phosphorylation of CREB facilitated the induction of hippocampal long-term potentiation (LTP) [14–16]. Based on these findings, the inhibition of cAMP/CREB after TBI would lead to cognitive deficits. The aim of the present study is to determine whether or not PC can improve cognitive deficits after TBI.
Materials and Methods Materials Procyanidins (purity495%) powder was obtained from JF-NATURAL (Tianjin, China). Its chemical character (such as the composition and molecular weight), as well as other characteristics, has been reported in detail [17]. Animals Male adult Sprague-Dawley rats (weighing, 326–348 g) were purchased from Beijing Vital River Experimental Animals Technology, Ltd. (Beijing, China). They were housed in cages and kept at 24 C with a normal 12-h/12-h light-dark schedule (lights on at 7 AM). The rats had free access to food and water until 24 h before TBI was induced in the experiments. All procedures that involved animals were approved by the local ethics committee for the use of experimental animals and were conducted in accordance with institutional guidelines. The rats were randomly assigned to the following three groups: the Sham group (i.e., sham-operated treatment, n ¼ 20), the Controlled Cortical Impact (CCI) group (i.e., CCI plus physiological saline vehicle treatment, n ¼ 24), and the PC group (i.e., CCI plus procyanidins treatment, n ¼ 24). Controlled Cortical Impact (CCI) With the rats under 10% chloral hydrate anesthesia (400 mg/kg, i.p.), experimental CCI was induced using a well-established CCI model as previously described [18, 19] and modified in our laboratory. Rectal temperature was continuously monitored and maintained at 37 ± 0.5 C by a negative-feedback-controlled heating pad for the duration of the experiment. Animals were placed on a stereotaxic frame and secured using two ear bars and an incisor bar. A midline incision was made, and a 6-mm-diameter craniotomy was performed on the right side midway between the bregma and the lambda, with the medial edge of the craniotomy 1.0 mm
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lateral to the midline. A single impact device (PCI 3000, Hatteras Instruments, Inc., USA) was used to deliver an impact at a velocity of 2.0 m/sec, with a 2.5 mm deformation and a dwell time of 150 ms using an impactor tip that was 4.0 mm in diameter. After the injury, the removed skull section was immediately replaced and sealed with bone wax, and the incision was closed with interrupted 4-0 silk sutures. Following the injury, animals were treated either with PC (100 mg/kgd) or vehicle (0.9% physiological saline) within 30 min. All animals were monitored carefully for at least 4 hours after the surgery and then again daily. Sham animals underwent the same procedure as the injured rats except for the impact. The dose and timing were decided based on preliminary experiments. The body weights were measured before the surgery and at 14 days after the surgery in all animals, and the change of body weight was expressed as the body weight at 14 days after the surgery minus that before the surgery (body weight). Cognitive testing To evaluate the effects of CCI and PC on cognitive function, all of the rats were tested in the Morris Water Maze (MWM) [20]. The diameter of the MWM was 150 cm, and it was divided into four equal-sized quadrants (Q1, Q2, Q3, and Q4). The water level was 75 cm, and there was an invisible platform (diameter of 15 cm) in the pool, which was 1.5 cm below the surface. The pool was filled with water maintained at 25 ± 1 C. MWM training was recorded using the ZS Dichuang image tracking system (Beijing, China)[21]. All rats underwent a three-phase evaluation process: orientation, training and a probe trial. On post-operative day (POD) 10 (orientation), the rats were acclimated to the testing room for 30 min. Following this period, the rat was gently placed on the submerged platform located in Q1 (the escape platform was placed in the pool in the middle of Q1) and allowed to remain or swim for 60 sec. At the end of the experiment, the rats were dried and kept warm. On post-operative days (PODs) 11–14 (training), the rats were allowed to orient to the room and then underwent four 60-sec trials separated by 5 min per trial. The rats were placed in the pool facing the wall from one of the equally spaced starting locations, which were randomly changed before every trial. The rats were allowed to swim (maximum swim time ¼ 60 sec) until they found a hidden, fixed escape platform using cues provided by red stars placed around the walls. If rat failed to find the platform within 60 sec, it was guided there (latency recorded ¼ 60 sec). In each case, the rat was allowed to remain on the platform for 10 sec. At the end of the experiment, the rats were dried and kept warm. Each rat was tested with four consecutive trials per day for four consecutive days from POD 11, and the training trial parameters that were evaluated included latency to reach the platform, speed of travel, and total distance traveled. For each rat, the values for the four training trials were averaged to give a single value per parameter for each rat on each training day. A single probe trial took place 1 h following the final training session on POD 14. A 60-sec probe trial with the platform removed was conducted to evaluate the long-term memory for the platform position. The outcome measures that were evaluated included time spent in
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Q1 and the number of times the animal crossed the exact former location of the platform.
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Measurement of the levels of MDA and GSH and the activity of SOD The rat was deeply anesthetized by 10% chloride hydrate at 24 h or 14 d after CCI, and the brain was removed. The right hemisphere was collected, frozen with liquid nitrogen, and kept at 80 C until analysis. The samples frozen at 80 C were irrigated well with a solution of NaCl (0.9%), and homogenization was then achieved at a ratio of 1:10. The homogenate was centrifuged (12 000 g, 20 min, 4 C), and the supernatant was used to measure the levels of MDA and GSH and the activity of SOD by kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The level of MDA was assayed POD 14 after CCI. The level of GSH and the activity of SOD were assayed 24 h after CCI. The protein concentration of the supernatant was determined by the method described by Bradford [22]. Enzyme-linked immunosorbent assay (ELISA) After POD 14, the rats were sacrificed by decapitation, and their brains were rapidly dissected. The ipsilateral hippocampus was carefully excised and frozen on dry ice. The ipsilateral hippocampal tissues were homogenized in icecold lysis buffer (Beyotime, Jiangsu, China) with 1% phenylmethylsulfonyl fluoride (PMSF, R&D Systems Inc., Minneapolis, MN, USA). The homogenates were then centrifuged, the supernatants were collected, and the total protein concentration was determined according to the Micro BCA procedure (Pierce, Rockford, IL, USA) using bovine serum albumin as a standard. BDNF protein was quantified using an ELISA kit (ChemiKine TM BDNF Sandwich ELISA Kit, Chemicon International Inc., Temecula, CA, USA) following the manufacturer’s protocol. The levels of cAMP were determined using a cAMP Complete ELISA kit (Enzo Life Sciences, USA) according to the manufacturer’s instructions. The levels of cAMP in the sample were determined based on a standard curve and expressed as pmol/mg per each sample.
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15 min. The horseradish peroxidase (HRP)-linked secondary antibody (1:5000 dilution) was incubated with the membrane for 1 h at room temperature. Excess antibody was washed off with TBST three times for 15 min before incubation in ECL (Bio-Rad) for 1 min. The images were quantified by Bio-Rad Quantity One software. The rats that underwent sham surgery were regarded as experimental controls for comparison with the experimental groups. Statistical analysis All the data were described as the mean ± standard error of the mean (SEM). The statistical analysis was performed with SPSS for Windows, version 18.0 (SPSS Inc., Chicago, IL, USA). Comparisons between groups were statistically evaluated by one-way analysis of variance (ANOVA) with a post hoc Bonferroni test (body weight loss, the levels of MDA, GSH, BDNF and cAMP and the activity of SOD). For the MWM results, the latencies in reaching the hidden platform were analyzed by repeated-measures analysis of variance (ANOVA) followed by Scheffe’s post hoc test. For the Western blot, the results are expressed as proportions of the actin value. Statistical significance was set at p50.05.
Results Effects of PC on the body weight loss after CCI Before surgery, there was no significant difference in the body weights among the groups (Figure 1). After POD 14, CCI group rats had higher amounts of weight loss compared to the sham group rats. Treatment with PC markedly reduced weight loss after CCI. Effects of PC on cognitive function in rats after CCI During the acquisition phase of the MWM task, the CCI group rats required a significantly longer amount of time (Figure 2A) to reach the platform compared to the PC group rats. During training (Figure 2B), the CCI group rats swam a greater distance, exploring the maze before reaching the platform. During the probe trial (Figure 2C), when the platform was removed from the pool, the CCI group rats spent significantly less time in Q1 where the platform was formerly
Western blot The ipsilateral hippocampal tissues were homogenized in RIPA buffer (50 mM Tris, pH 7.0, 150 mM NaCl, 1% Triton X-100) containing PMSF (R&D Systems Inc., Minneapolis, MN, USA) and phosphatase inhibitor cocktail 1 and 2 (Sigma, St. Louis, MO, USA). The homogenates were nutated at 4 C for 30 min and centrifuged at 12 000 g for 20 min. Equal amounts of protein (50 mg of total protein) were loaded into each lane and run on SDS-PAGE under reducing conditions. The samples were then electroblotted onto nitrocellulose filter membranes (Millipore Inc., MA, USA), blocked with 5% nonfat dried milk in Tris-buffered saline containing 0.1% Triton X-100 (TBST) at room temperature for 2 h, and incubated overnight with a primary antibody against antipCREB antibody (1:500, Abcam, Cambridge, UK) and antiCREB antibody (1:500, Abcam, Cambridge, UK) at 4 C. The membranes were washed with TBST three times for a total of
Figure 1. Effects of PC on the body weight loss after CCI (mean ± SEM) Data were presented as mean ± SEM. CCI: Controlled cortical impact; PC: Procyanidins. (#p50.01 vs. sham group rats; *p50.05 vs. CCI group rats; ?p50.05 vs. sham group rats).
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Figure 2. Effects of PC on cognitive function in CCI rats (mean ± SEM). Morris water maze performance after CCI and PC treatments. During the training phase of the Morris water maze, the CCI group rats required a significantly greater amount of time to find the platform (A) and swam a longer distance (B) compared to the Sham or PC groups rats. During the probe trial (C), rats after CCI spent significantly less time in the quadrant that was the former location of the platform, compared to the Sham or PC groups rats, and these rats had significantly fewer instances where they swam directly over the site where the platform had previously been located (D). Data were presented as mean ± SEM. CCI: Controlled cortical impact; PC: procyanidins. (##p50.001 vs. sham group rats; *p50.05 and **p50.01 vs. CCI group rats; ?p50.05 vs. sham group rats).
placed, and they exhibited significantly fewer crossings (Figure 2D) over the specific site where the platform had been positioned. Effects of PC on the levels of MDA and GSH and the activity of SOD Figure 3 showed the levels of MDA and GSH and the activity of SOD in all groups. CCI generated an increase in the levels of MDA and a significant reduction in the levels of GSH and the activity of SOD in the hippocampi. Effects of PC on BDNF-related plasticity: BDNF, pCREB and CREB after CCI Figure 4 showed the levels of BDNF in all groups. The level in the PC group rats is higher than that in the CCI group rats. The Western blot results showed that the immunoreactive bands of pCREB as well as CREB appeared at 43 kDa. We compared the levels of pCREB and total CREB in the different groups by Western blot analyses. The results showed
that the pCREB and total CREB levels in the CCI group rats were significant lower than those in the Sham group and PC group rats (Figure 5A and B). Although supplementation of PC led to significant increases in hippocampal pCREB and total CREB, the PC group still exhibited significantly lower hippocampal levels of pCREB and total CREB compared with Sham group rats (Figure 5A and B). Effects of PC on cAMP Levels after CCI Figure 6 showed that the cAMP levels decreased in the hippocampus in CCI group rats compared to the Sham and PC group rats (Figure 6). Thus, the results suggested that PC increased the cAMP levels and activated cAMP/CREB signaling.
Discussion It has been shown that cognitive deficits after TBI are accompanied by alterations in the levels of oxidative stress and the molecules associated with learning. In this study, we
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Figure 3. Effects of PC on the levels of MDA, GSH, and the activities of SOD in the hippocampi after CCI. Vehicle or PC was injected intravenously in 30 min after CCI. (A) SOD activity; (B) GSH level; (C) MDA level; pro: protein. Data were presented as mean ± SEM. (##p50.001 vs. sham group rats; *p50.05 and **p50.01 vs. CCI group rats; ?p50.05 vs. sham group rats).
Figure 4. The effects of PC on the level of BDNF after CCI. CCI resulted in lower BDNF compared with the sham group rats (##p50.001), whereas PC substantially elevated BDNF level in the PC group rats compared with the CCI group rats (*p50.05) and the sham group rats (??p50.01).
found that PC appears to reduce oxidative stress levels and improve cognitive deficits in rats after TBI. The recent study showed that CCI resulted in an impairment of MWM performance. CCI group rats took longer to find the platform and swam farther distances before finding
the platform during the training phase and exhibited significant impairments in localizing their swim activity during the probe trial. PC significantly improved performance on measures of spatial memory and memory retrieval during the training phase or probe trial. It is well-known that oxidative stress and lipid peroxidation play important roles in the pathogenesis of TBI [2, 3, 23, 24]. ROS are highly reactive molecules, which are formed during normal cellular processes, but the production is tightly controlled by scavenging systems, including SOD and GSH. The main breakdown product of lipid peroxidation in the brain is MDA. It is reported that the reduction in the activities of SOD and glutathione peroxidase and the levels of GSH and the increases in the levels of MDA happen after TBI [3, 25, 26]. Moreover, ROS scavengers can protect the brain against experimental TBI [2, 3]. PC, which is a safe and effective antioxidant with minimal side effects, contains more effective free radical scavenging capability than many other antioxidants [6]. The present results demonstrated that the administration of PC in a rat model of TBI decreased the levels of MDA and increased the levels of GSH and the activities of SOD. The present results are consistent with the antioxidant effects of PC in other animal models [7, 17] and imply that PC can still play a significant role in antioxidant effects in a CCI model, which might be the principal mechanism for its cognitive protective effect.
The effects of Procyanidins in TBI
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Figure 5. (A, B) The effects of PC on the pCREB and CREB in the CCI rats. CCI resulted in less pCREB and CREB levels compared with the sham rats (##p50.001), whereas PC dramatically increased pCREB and CREB levels in the PC group rats compared with the CCI group rats (*p50.05 and **p50.01) and the sham group rats (?p50.05; ??p50.01). The level of pCREB and CREB was measured by Western blot analysis using actin as a standard control.
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synaptic function [30]. CREB has been identified as the key mediator for BDNF-mediated cell survival as previous studies showed that silencing the transcriptional activity of CREB impaired BDNF protection [31]. CREB is a key transcription factor that plays a role in several critical functions of the brain, such as learning, neuronal plasticity and cell survival [32]. pCREB promotes the transcription of immediate-early gene mRNA, which is then translated into proteins. These proteins are necessary for the maintenance of long-term memory [33, 34]. To identity the upstream regulators of CREBsignaling, previous studies have demonstrated that different signaling can trigger intracellular signaling cascades that phosphorylate CREB at Ser133, which is a rate-limiting step in CREB signaling [35]. Among the various signaling pathways, the most important pathway includes the stimulation of adenylyl cyclase and the accumulation of the second messenger cAMP to activate PKA and leads to the release of the catalytical subunit of PKA, which then shuttles to the nucleus and phosphorylates CREB [36]. In the current study, we measured the BDNF, pCREB, CREB and cAMP levels in the hippocampus 14 days after CCI. Our results suggested that PC increased BDNF, pCREB, CREB and cAMP levels, and the increases lasted until at least the probe trial finished. The above evidence indicated that the elevated CREB activity subsequently markedly increased BDNF protein levels in the PC supplementation group, which was also associated with the recovery of spatial learning and memory.
Conclusions In summary, we demonstrated that PC can counteract oxidative damage and behavioral dysfunction after TBI through antioxidant activity and the up-regulation of cAMP/ CREB signaling. TBI impaired hippocampus-dependent learning and memory in rats. To explore the mechanism, we observed the down-regulation of cAMP/CREB signaling, which is implicated in learning and memory, long-term potentiation, and neuroprotection. PC supplementation could activate the hippocampal cAMP/CREB signaling pathway and facilitate long-term memory formation and, consequently, ameliorated learning and memory dysfunction in rats after TBI. The study provided some theoretical basis that PC could help to counteract some of the deleterious effects of TBI on neuroplasticity and cognition and could be an effective therapeutic strategy for patients with TBI-induced cognitive deficits.
Figure 6. Effects of PC on cAMP Levels in the hippocampi after CCI. CCI reduced cAMP level compared with the sham group rats (##p50.001), whereas PC dramatically elevated cAMP levels in the PC group rats compared with the CCI group rats (**p50.01) and the sham group rats (??p50.01).
As mentioned previously, TBI-induced cognitive impairments have been demonstrated in humans [27, 28] and in animal models [29]. Oxidative stress is one of many mechanisms that can affect cognitive functioning. Although it has not been fully established, evidence has indicated that BDNF plays an important role in this process [8]. BDNF has a number of important functions in the long-term forms of synaptic plasticity in different brain areas and is also required for shortterm memory formation because it can modulate short-term
Acknowledgements We would like to express our gratitude to Mr Ming Sun for his help.
Declaration of interest The authors report no conflict of interests related to this study or the findings specified in this paper. This study was supported by National Natural Science Foundation of China (No.81171144), Beijing Nova program (No.XX2012033) and National Key Technology Research and Development Program of the Ministry of Science and Technology of China(2013BAI09B03).
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ORIGINAL ARTICLE
Procyanidins protects against oxidative damage and cognitive deficits after traumatic brain injury Xiang Mao1,3,4,5,6, Shuyu Hao2,5,6, Zhendan Zhu3,5,6,7, Hao Zhang1,3,5,6, Weichuan Wu8, Feifan Xu2,5,6 and Baiyun Liu2,3,4,5,6
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1
Department of Neurosurgery, The First Affiliated Hospital of Anhui Medical University, No. 218 Jixi Road, Shushan District, Hefei, Anhui, People’s Republic of China, 2Department of Neurosurgery, Beijing Tian Tan Hospital, Capital Medical University, Beijing, People’s Republic of China, 3 Neurotrauma Laboratory, Beijing Neurosurgical Institute, Capital Medical University, Beijing, People’s Republic of China, 4Department of Neurotrauma, General Hospital of Armed Police Forces, Beijing, People’s Republic of China, 5China National Clinical Research Center for Neurological Diseases, Beijing, People’s Republic of China, 6Beijing Key Laboratory of Central Nervous System Injury, Beijing, People’s Republic of China, 7General Hospital of Armed Police Forces Clinical College, Anhui Medical University, Hefei, Anhui, People’s Republic of China, and 8 Department of Neurosurgery, The Affiliated Hospital of Guangdong Medical College, Zhanjiang, People’s Republic of China Abstract
Keywords
Primary objective: Oxidative stress is the principal factor in traumatic brain injury (TBI) that initiates the events that result in protracted neuronal dysfunction and remodeling. Importantly, antioxidants can protect the brain against oxidative damage and modulate the capacity of the brain to cope with synaptic dysfunction and cognitive impairment. Research design: To date, however, no studies have investigated the effects of procyanidins (PC) on cognitive deficits after TBI. Methods and Procedures: In the present study, rats with controlled cortical impact (CCI) were used to investigate the protective effects of procyanidins. Main outcomes and results: The results showed that procyanidins reduced the level of malondialdehyde (MDA) and elevated the level of glutathione (GSH) and the activity of superoxide dismutase (SOD). In addition, treatment with procyanidins, which elevated the levels of brain-derived neurotropic factor (BDNF), phosphorylation-cAMP-response element binding protein (pCREB), total CREB, and cyclic AMP (cAMP), improved cognitive performance in the Morris water maze after TBI. Conclusions: These results suggest that procyanidins appear to counteract oxidative damage and behavioral dysfunction after TBI through antioxidant activity and the up-regulation of cAMP/CREB signaling.
Antioxidant, CREB, hippocampus, oxidative stress, procyanidins, TBI
Introduction Traumatic brain injury (TBI) is a leading cause of death and disability in industrialized countries. In the U.S., TBI is a significant public health problem. Each year approximately 1.7 million TBIs are incurred, 53 000 people die, and 3.2–5.3 million others are living with long-term disabilities. Survivors after TBI often suffer cognitive deficits, including impaired learning and memory [1]. The critical mechanisms of secondary injury after brain trauma involve a variety of progressive physiological and pathological changes. It is well-known that oxidative stress plays a significant role in the pathogenesis after TBI [2, 3].
Correspondence: Baiyun Liu, Department of Neurosurgery, Beijing Tian Tan Hospital, Capital Medical University, No. 6 Tiantan Xili, Dongcheng District; Neurotrauma Laboratory, Beijing Neurosurgical Institute, Capital Medical University; Department of Neurotrauma, General Hospital of Armed Police Forces; China National Clinical Research Center for Neurological Diseases; Beijing Key Laboratory of Central Nervous System Injury, Beijing, People’s Republic of China. Tel: +86018301605984, E-mail: [email protected]
History Received 3 May 2014 Revised 24 August 2014 Accepted 19 September 2014 Published online 1 October 2014
It is also well-known that oxidative stress causes the impairment of neurological behavior, motor function and cognition (including learning and memory abilities). Reactive oxygen species (ROS) are formed during normal cellular processes, but superoxide dismutase (SOD) and glutathione (GSH) can tightly control the production of ROS by direct or indirect ways. Neuronal membranes are rich in polyunsaturated fatty acids, which are prime targets for ROS attack, and source for lipid peroxidation reactions. Malondialdehyde (MDA) is a main breakdown product of lipid peroxidation in brain. Moreover, the reduction of ROS can protect the brain against oxidative damage and modulate the capacity of the brain to cope with cognitive impairment after TBI [2–4]. Procyanidins (PC), which are flavonoids with an oligomeric structure, are present in our diet both in foodstuffs and in beverages, such as red wine, tea and fruit juices [5]. Procyanidins contain more effective free radical scavenging capability than others, such as vitamin C, vitamin E, succinate, and beta-carotene [6]. In addition, Procyanidins have been used for antibacterial, antiviral, anti-inflammatory and anticarcinogenic treatments [7].
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DOI: 10.3109/02699052.2014.968621
The mechanisms by which oxidative stress affects cognitive function have not been fully established, but it has been suggested that the impairment of cognitive function correlates with the reduction of brain-derived neurotropic factor (BDNF), and it has been shown that excessive oxidative stress can cause a reduction in BDNF and a subsequent decline in cognition [8]. BDNF appears to affect synaptic function through the modulation of its downstream effectors, such as cyclic AMP-response element binding protein (CREB), and it is CREB that is implicated in the synaptic events underlying learning and memory [9, 10] in a wide range of biologic processes when activated by phosphorylation at Ser133. The phosphorylation/activation of CREB (pCREB) on Ser133 by cyclic AMP (cAMP) or Ca2+dependent protein kinase II (CaMKII) is critical for longterm memory consolidation [11–13]. Studies have also shown that increasing cAMP and phosphorylation of CREB facilitated the induction of hippocampal long-term potentiation (LTP) [14–16]. Based on these findings, the inhibition of cAMP/CREB after TBI would lead to cognitive deficits. The aim of the present study is to determine whether or not PC can improve cognitive deficits after TBI.
Materials and Methods Materials Procyanidins (purity495%) powder was obtained from JF-NATURAL (Tianjin, China). Its chemical character (such as the composition and molecular weight), as well as other characteristics, has been reported in detail [17]. Animals Male adult Sprague-Dawley rats (weighing, 326–348 g) were purchased from Beijing Vital River Experimental Animals Technology, Ltd. (Beijing, China). They were housed in cages and kept at 24 C with a normal 12-h/12-h light-dark schedule (lights on at 7 AM). The rats had free access to food and water until 24 h before TBI was induced in the experiments. All procedures that involved animals were approved by the local ethics committee for the use of experimental animals and were conducted in accordance with institutional guidelines. The rats were randomly assigned to the following three groups: the Sham group (i.e., sham-operated treatment, n ¼ 20), the Controlled Cortical Impact (CCI) group (i.e., CCI plus physiological saline vehicle treatment, n ¼ 24), and the PC group (i.e., CCI plus procyanidins treatment, n ¼ 24). Controlled Cortical Impact (CCI) With the rats under 10% chloral hydrate anesthesia (400 mg/kg, i.p.), experimental CCI was induced using a well-established CCI model as previously described [18, 19] and modified in our laboratory. Rectal temperature was continuously monitored and maintained at 37 ± 0.5 C by a negative-feedback-controlled heating pad for the duration of the experiment. Animals were placed on a stereotaxic frame and secured using two ear bars and an incisor bar. A midline incision was made, and a 6-mm-diameter craniotomy was performed on the right side midway between the bregma and the lambda, with the medial edge of the craniotomy 1.0 mm
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lateral to the midline. A single impact device (PCI 3000, Hatteras Instruments, Inc., USA) was used to deliver an impact at a velocity of 2.0 m/sec, with a 2.5 mm deformation and a dwell time of 150 ms using an impactor tip that was 4.0 mm in diameter. After the injury, the removed skull section was immediately replaced and sealed with bone wax, and the incision was closed with interrupted 4-0 silk sutures. Following the injury, animals were treated either with PC (100 mg/kgd) or vehicle (0.9% physiological saline) within 30 min. All animals were monitored carefully for at least 4 hours after the surgery and then again daily. Sham animals underwent the same procedure as the injured rats except for the impact. The dose and timing were decided based on preliminary experiments. The body weights were measured before the surgery and at 14 days after the surgery in all animals, and the change of body weight was expressed as the body weight at 14 days after the surgery minus that before the surgery (body weight). Cognitive testing To evaluate the effects of CCI and PC on cognitive function, all of the rats were tested in the Morris Water Maze (MWM) [20]. The diameter of the MWM was 150 cm, and it was divided into four equal-sized quadrants (Q1, Q2, Q3, and Q4). The water level was 75 cm, and there was an invisible platform (diameter of 15 cm) in the pool, which was 1.5 cm below the surface. The pool was filled with water maintained at 25 ± 1 C. MWM training was recorded using the ZS Dichuang image tracking system (Beijing, China)[21]. All rats underwent a three-phase evaluation process: orientation, training and a probe trial. On post-operative day (POD) 10 (orientation), the rats were acclimated to the testing room for 30 min. Following this period, the rat was gently placed on the submerged platform located in Q1 (the escape platform was placed in the pool in the middle of Q1) and allowed to remain or swim for 60 sec. At the end of the experiment, the rats were dried and kept warm. On post-operative days (PODs) 11–14 (training), the rats were allowed to orient to the room and then underwent four 60-sec trials separated by 5 min per trial. The rats were placed in the pool facing the wall from one of the equally spaced starting locations, which were randomly changed before every trial. The rats were allowed to swim (maximum swim time ¼ 60 sec) until they found a hidden, fixed escape platform using cues provided by red stars placed around the walls. If rat failed to find the platform within 60 sec, it was guided there (latency recorded ¼ 60 sec). In each case, the rat was allowed to remain on the platform for 10 sec. At the end of the experiment, the rats were dried and kept warm. Each rat was tested with four consecutive trials per day for four consecutive days from POD 11, and the training trial parameters that were evaluated included latency to reach the platform, speed of travel, and total distance traveled. For each rat, the values for the four training trials were averaged to give a single value per parameter for each rat on each training day. A single probe trial took place 1 h following the final training session on POD 14. A 60-sec probe trial with the platform removed was conducted to evaluate the long-term memory for the platform position. The outcome measures that were evaluated included time spent in
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Q1 and the number of times the animal crossed the exact former location of the platform.
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Measurement of the levels of MDA and GSH and the activity of SOD The rat was deeply anesthetized by 10% chloride hydrate at 24 h or 14 d after CCI, and the brain was removed. The right hemisphere was collected, frozen with liquid nitrogen, and kept at 80 C until analysis. The samples frozen at 80 C were irrigated well with a solution of NaCl (0.9%), and homogenization was then achieved at a ratio of 1:10. The homogenate was centrifuged (12 000 g, 20 min, 4 C), and the supernatant was used to measure the levels of MDA and GSH and the activity of SOD by kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The level of MDA was assayed POD 14 after CCI. The level of GSH and the activity of SOD were assayed 24 h after CCI. The protein concentration of the supernatant was determined by the method described by Bradford [22]. Enzyme-linked immunosorbent assay (ELISA) After POD 14, the rats were sacrificed by decapitation, and their brains were rapidly dissected. The ipsilateral hippocampus was carefully excised and frozen on dry ice. The ipsilateral hippocampal tissues were homogenized in icecold lysis buffer (Beyotime, Jiangsu, China) with 1% phenylmethylsulfonyl fluoride (PMSF, R&D Systems Inc., Minneapolis, MN, USA). The homogenates were then centrifuged, the supernatants were collected, and the total protein concentration was determined according to the Micro BCA procedure (Pierce, Rockford, IL, USA) using bovine serum albumin as a standard. BDNF protein was quantified using an ELISA kit (ChemiKine TM BDNF Sandwich ELISA Kit, Chemicon International Inc., Temecula, CA, USA) following the manufacturer’s protocol. The levels of cAMP were determined using a cAMP Complete ELISA kit (Enzo Life Sciences, USA) according to the manufacturer’s instructions. The levels of cAMP in the sample were determined based on a standard curve and expressed as pmol/mg per each sample.
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15 min. The horseradish peroxidase (HRP)-linked secondary antibody (1:5000 dilution) was incubated with the membrane for 1 h at room temperature. Excess antibody was washed off with TBST three times for 15 min before incubation in ECL (Bio-Rad) for 1 min. The images were quantified by Bio-Rad Quantity One software. The rats that underwent sham surgery were regarded as experimental controls for comparison with the experimental groups. Statistical analysis All the data were described as the mean ± standard error of the mean (SEM). The statistical analysis was performed with SPSS for Windows, version 18.0 (SPSS Inc., Chicago, IL, USA). Comparisons between groups were statistically evaluated by one-way analysis of variance (ANOVA) with a post hoc Bonferroni test (body weight loss, the levels of MDA, GSH, BDNF and cAMP and the activity of SOD). For the MWM results, the latencies in reaching the hidden platform were analyzed by repeated-measures analysis of variance (ANOVA) followed by Scheffe’s post hoc test. For the Western blot, the results are expressed as proportions of the actin value. Statistical significance was set at p50.05.
Results Effects of PC on the body weight loss after CCI Before surgery, there was no significant difference in the body weights among the groups (Figure 1). After POD 14, CCI group rats had higher amounts of weight loss compared to the sham group rats. Treatment with PC markedly reduced weight loss after CCI. Effects of PC on cognitive function in rats after CCI During the acquisition phase of the MWM task, the CCI group rats required a significantly longer amount of time (Figure 2A) to reach the platform compared to the PC group rats. During training (Figure 2B), the CCI group rats swam a greater distance, exploring the maze before reaching the platform. During the probe trial (Figure 2C), when the platform was removed from the pool, the CCI group rats spent significantly less time in Q1 where the platform was formerly
Western blot The ipsilateral hippocampal tissues were homogenized in RIPA buffer (50 mM Tris, pH 7.0, 150 mM NaCl, 1% Triton X-100) containing PMSF (R&D Systems Inc., Minneapolis, MN, USA) and phosphatase inhibitor cocktail 1 and 2 (Sigma, St. Louis, MO, USA). The homogenates were nutated at 4 C for 30 min and centrifuged at 12 000 g for 20 min. Equal amounts of protein (50 mg of total protein) were loaded into each lane and run on SDS-PAGE under reducing conditions. The samples were then electroblotted onto nitrocellulose filter membranes (Millipore Inc., MA, USA), blocked with 5% nonfat dried milk in Tris-buffered saline containing 0.1% Triton X-100 (TBST) at room temperature for 2 h, and incubated overnight with a primary antibody against antipCREB antibody (1:500, Abcam, Cambridge, UK) and antiCREB antibody (1:500, Abcam, Cambridge, UK) at 4 C. The membranes were washed with TBST three times for a total of
Figure 1. Effects of PC on the body weight loss after CCI (mean ± SEM) Data were presented as mean ± SEM. CCI: Controlled cortical impact; PC: Procyanidins. (#p50.01 vs. sham group rats; *p50.05 vs. CCI group rats; ?p50.05 vs. sham group rats).
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Figure 2. Effects of PC on cognitive function in CCI rats (mean ± SEM). Morris water maze performance after CCI and PC treatments. During the training phase of the Morris water maze, the CCI group rats required a significantly greater amount of time to find the platform (A) and swam a longer distance (B) compared to the Sham or PC groups rats. During the probe trial (C), rats after CCI spent significantly less time in the quadrant that was the former location of the platform, compared to the Sham or PC groups rats, and these rats had significantly fewer instances where they swam directly over the site where the platform had previously been located (D). Data were presented as mean ± SEM. CCI: Controlled cortical impact; PC: procyanidins. (##p50.001 vs. sham group rats; *p50.05 and **p50.01 vs. CCI group rats; ?p50.05 vs. sham group rats).
placed, and they exhibited significantly fewer crossings (Figure 2D) over the specific site where the platform had been positioned. Effects of PC on the levels of MDA and GSH and the activity of SOD Figure 3 showed the levels of MDA and GSH and the activity of SOD in all groups. CCI generated an increase in the levels of MDA and a significant reduction in the levels of GSH and the activity of SOD in the hippocampi. Effects of PC on BDNF-related plasticity: BDNF, pCREB and CREB after CCI Figure 4 showed the levels of BDNF in all groups. The level in the PC group rats is higher than that in the CCI group rats. The Western blot results showed that the immunoreactive bands of pCREB as well as CREB appeared at 43 kDa. We compared the levels of pCREB and total CREB in the different groups by Western blot analyses. The results showed
that the pCREB and total CREB levels in the CCI group rats were significant lower than those in the Sham group and PC group rats (Figure 5A and B). Although supplementation of PC led to significant increases in hippocampal pCREB and total CREB, the PC group still exhibited significantly lower hippocampal levels of pCREB and total CREB compared with Sham group rats (Figure 5A and B). Effects of PC on cAMP Levels after CCI Figure 6 showed that the cAMP levels decreased in the hippocampus in CCI group rats compared to the Sham and PC group rats (Figure 6). Thus, the results suggested that PC increased the cAMP levels and activated cAMP/CREB signaling.
Discussion It has been shown that cognitive deficits after TBI are accompanied by alterations in the levels of oxidative stress and the molecules associated with learning. In this study, we
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Figure 3. Effects of PC on the levels of MDA, GSH, and the activities of SOD in the hippocampi after CCI. Vehicle or PC was injected intravenously in 30 min after CCI. (A) SOD activity; (B) GSH level; (C) MDA level; pro: protein. Data were presented as mean ± SEM. (##p50.001 vs. sham group rats; *p50.05 and **p50.01 vs. CCI group rats; ?p50.05 vs. sham group rats).
Figure 4. The effects of PC on the level of BDNF after CCI. CCI resulted in lower BDNF compared with the sham group rats (##p50.001), whereas PC substantially elevated BDNF level in the PC group rats compared with the CCI group rats (*p50.05) and the sham group rats (??p50.01).
found that PC appears to reduce oxidative stress levels and improve cognitive deficits in rats after TBI. The recent study showed that CCI resulted in an impairment of MWM performance. CCI group rats took longer to find the platform and swam farther distances before finding
the platform during the training phase and exhibited significant impairments in localizing their swim activity during the probe trial. PC significantly improved performance on measures of spatial memory and memory retrieval during the training phase or probe trial. It is well-known that oxidative stress and lipid peroxidation play important roles in the pathogenesis of TBI [2, 3, 23, 24]. ROS are highly reactive molecules, which are formed during normal cellular processes, but the production is tightly controlled by scavenging systems, including SOD and GSH. The main breakdown product of lipid peroxidation in the brain is MDA. It is reported that the reduction in the activities of SOD and glutathione peroxidase and the levels of GSH and the increases in the levels of MDA happen after TBI [3, 25, 26]. Moreover, ROS scavengers can protect the brain against experimental TBI [2, 3]. PC, which is a safe and effective antioxidant with minimal side effects, contains more effective free radical scavenging capability than many other antioxidants [6]. The present results demonstrated that the administration of PC in a rat model of TBI decreased the levels of MDA and increased the levels of GSH and the activities of SOD. The present results are consistent with the antioxidant effects of PC in other animal models [7, 17] and imply that PC can still play a significant role in antioxidant effects in a CCI model, which might be the principal mechanism for its cognitive protective effect.
The effects of Procyanidins in TBI
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DOI: 10.3109/02699052.2014.968621
Figure 5. (A, B) The effects of PC on the pCREB and CREB in the CCI rats. CCI resulted in less pCREB and CREB levels compared with the sham rats (##p50.001), whereas PC dramatically increased pCREB and CREB levels in the PC group rats compared with the CCI group rats (*p50.05 and **p50.01) and the sham group rats (?p50.05; ??p50.01). The level of pCREB and CREB was measured by Western blot analysis using actin as a standard control.
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synaptic function [30]. CREB has been identified as the key mediator for BDNF-mediated cell survival as previous studies showed that silencing the transcriptional activity of CREB impaired BDNF protection [31]. CREB is a key transcription factor that plays a role in several critical functions of the brain, such as learning, neuronal plasticity and cell survival [32]. pCREB promotes the transcription of immediate-early gene mRNA, which is then translated into proteins. These proteins are necessary for the maintenance of long-term memory [33, 34]. To identity the upstream regulators of CREBsignaling, previous studies have demonstrated that different signaling can trigger intracellular signaling cascades that phosphorylate CREB at Ser133, which is a rate-limiting step in CREB signaling [35]. Among the various signaling pathways, the most important pathway includes the stimulation of adenylyl cyclase and the accumulation of the second messenger cAMP to activate PKA and leads to the release of the catalytical subunit of PKA, which then shuttles to the nucleus and phosphorylates CREB [36]. In the current study, we measured the BDNF, pCREB, CREB and cAMP levels in the hippocampus 14 days after CCI. Our results suggested that PC increased BDNF, pCREB, CREB and cAMP levels, and the increases lasted until at least the probe trial finished. The above evidence indicated that the elevated CREB activity subsequently markedly increased BDNF protein levels in the PC supplementation group, which was also associated with the recovery of spatial learning and memory.
Conclusions In summary, we demonstrated that PC can counteract oxidative damage and behavioral dysfunction after TBI through antioxidant activity and the up-regulation of cAMP/ CREB signaling. TBI impaired hippocampus-dependent learning and memory in rats. To explore the mechanism, we observed the down-regulation of cAMP/CREB signaling, which is implicated in learning and memory, long-term potentiation, and neuroprotection. PC supplementation could activate the hippocampal cAMP/CREB signaling pathway and facilitate long-term memory formation and, consequently, ameliorated learning and memory dysfunction in rats after TBI. The study provided some theoretical basis that PC could help to counteract some of the deleterious effects of TBI on neuroplasticity and cognition and could be an effective therapeutic strategy for patients with TBI-induced cognitive deficits.
Figure 6. Effects of PC on cAMP Levels in the hippocampi after CCI. CCI reduced cAMP level compared with the sham group rats (##p50.001), whereas PC dramatically elevated cAMP levels in the PC group rats compared with the CCI group rats (**p50.01) and the sham group rats (??p50.01).
As mentioned previously, TBI-induced cognitive impairments have been demonstrated in humans [27, 28] and in animal models [29]. Oxidative stress is one of many mechanisms that can affect cognitive functioning. Although it has not been fully established, evidence has indicated that BDNF plays an important role in this process [8]. BDNF has a number of important functions in the long-term forms of synaptic plasticity in different brain areas and is also required for shortterm memory formation because it can modulate short-term
Acknowledgements We would like to express our gratitude to Mr Ming Sun for his help.
Declaration of interest The authors report no conflict of interests related to this study or the findings specified in this paper. This study was supported by National Natural Science Foundation of China (No.81171144), Beijing Nova program (No.XX2012033) and National Key Technology Research and Development Program of the Ministry of Science and Technology of China(2013BAI09B03).
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