Deficiencies in brain derived neurotrophic factor (BDNF) reduce the brain plasticity necessary to cope with the effects of TBI. 23 BDNF activates cAMP‐responsive element‐binding protein (CREB), a multifaceted transcriptional regulator involved in synaptic plasticity essential for learning and memory. 24 BDNF is known to bind to TrkB receptors, leading to activation of Ca 2+ /calmodulin‐dependent protein kinase II (CaMKII), required for synaptic processes involved in behavior. 25 Several observations indicate that the flavonoids exert action through modulation of signaling pathways to promote synaptic and neuronal function. 26 , 27 Accordingly, in the current study, we investigated whether blueberry (BB) supplementation would counteract TBI pathology by involving BDNF‐related pathways involved in synaptic plasticity and oxidative stress to influence cognitive behaviors.

Evidence suggests that TBI is characterized by dysfunction in synaptic plasticity, elevated levels of free radicals, plasma membrane dysfunction, 18 which can contribute to the behavioral dysfunction. Oxidative stress is part of the pathology of TBI and compromises neuronal function. 19 , 20 In particular, excessive free radical formation leads to accumulation of lipid oxidation by‐products such as 4‐hydroxynonenal (4‐HNE) 21 with subsequent impairments in plasma membrane fluidity, receptor signaling across the membrane to deteriorate synaptic plasticity and reduce neuronal excitability. 22

Dietary polyphenols have significant positive effects on brain health via protecting neurons against injury and enhancing neuronal function. 5 , 6 Evidence supports the neuromodulatory effects of flavonoid‐rich blueberry, particularly in promotion of brain plasticity, 7 and counteracting behavioral deficits. 8 In the United States, demand for blueberries has increased, with 2017 fresh per capita consumption of 1.79 pounds per person. 9 Several reports indicate that blueberry dietary supplementation improves memory, learning, and general cognitive function, 10 - 14 and protects against neuronal injury associated with stroke. 15 Moreover, it has been shown that blueberries possess potent antioxidant capacity through their ability to reduce free radical formation 16 or upregulating endogenous antioxidant defenses. 17 These studies suggest that blueberry supplementation can have the potential to be used to overcome the broad pathology of TBI. Given the lack of information about the effects of blueberry intake immediately after TBI, we have performed studies to assess the effects of blueberry extracts during the acute phase of TBI.

Traumatic brain injury (TBI) accounts for approximately 90% of brain injuries, and is associated with cognitive dysfunction and long‐term disability. 1 As a result of domestic incidents, military combat, traffic accidents, and sports, TBI can compromise broad aspects of neuronal function. Patients often experience problems in the domains of learning, memory, and affective functions that can profoundly influence quality of life. 2 , 3 Existing therapeutic strategies for TBI have not been successful in counteracting the heterogeneous TBI pathology nor improving the quality of life of patients. 4 Hence, identifying interventions with broad applicability seems necessary for effective management of TBI.

Protein data are expressed as mean ± standard error of the mean (SEM). Body weight data expressed as mean ± standard deviation. Statistical analysis was performed by software GraphPad Prism 7.04. A level of 5% probability was considered as statistically significant. The Barnes maze learning data analysis ( n = 8) were analyzed by repeated measures analysis of variance (ANOVA). Protein results are expressed as percentage (%) of Sham‐RD group. One‐way ANOVA followed by Tukey post‐hoc test for multiple comparison used for protein data analysis ( n = 5–7 rats per group). The association among the endpoints were assessed using Pearson correlation (two‐tailed).

Upon completion of the experiment, hippocampal tissues were harvested frozen in dry ice, and stored at –80 °C until use for immunoblotting. The left side hippocampus (ipsilateral to injury) were homogenized in a lysis buffer containing 20 mm Tris–HCl (pH 8.0), 137 mm NaCl, 1% NP40, 10% glycerol, 1 mm phenylmethylsulfonylfluoride (PMSF), 10 μg mL −1 aprotinin, 0.1 mm benzethonium chloride, 0.5 mm sodium vanadate. The homogenates were then centrifuged (12 000 g at 4 °C) and the supernatants were collected. Total protein was then determined using a BCA Protein Assay kit (Pierce, IL, USA), using bovine serum albumin (BSA) as standard. Equal amounts of protein were separated by sodiumdocecylsulphate‐polyacrylamide gels and then transferred onto polyvinylidene difluoride membranes (Millipore, MA, USA). Membranes were probed with antiactin or anti‐BDNF, anti‐pCREB, anti‐CREB, (1:1000, Millipore, MA, USA), anti‐CaMKII, anti‐4‐hydroxynonenal (4‐HNE) (1:500; Santa Cruz Biotechnology, CA, USA) followed by secondary antibody (antirabbit or antigoat or antimouse IgG horseradish peroxidase‐conjugate, 1:10 000; Santa Cruz Biotechnology, CA, USA). Immunoreactive proteins were visualized using enhanced chemiluminescence reagents (Millipore, MA, USA). Band intensities were quantified using Image J32 Software. β‐actin (anti β‐actin; 1:5000) was used as an internal control for normalization western blot such that data were standardized according to β‐actin values. Blots for each experimental group were normalized to Sham‐RD values within the same gel.

EPM test was performed to assess anxiety‐like behavior 2 weeks after experimental TBI with two trials as described in Supporting Information. Specifically, rats were individually placed in the closed arm of the EPM apparatus and permitted free exploration for 5 min during which their movements were camera recorded. 31 The behaviors scored were time spent and number of entries into the open arm using automated video tracking system (AnyMaze, San Diego Instruments, CA, USA). 30 EPM testing was conducted after Barnes maze memory test.

Barnes maze testing was performed 2 weeks after experimental TBI with two trials per day with a 5‐min test period (detailed in Supporting Information). For learning assessment, rats were given two trials per day for 5 consecutive days at approximately the same time every day. Subsequently, memory retention was assessed at post‐injury day 21. Latency to finding the escape hole and search strategies were analyzed for each trial. Three search strategies were identified using following categorization: spatial, peripheral, and random using data recorded with AnyMaze software.

We employed our standard lateral FPI protocol as described earlier. 30 Briefly, 3% isoflurane (1.0 mL min −1 in 100% oxygen) was provided in a chamber (VetEquip Inc., CA, USA), and then maintained with 2–2.5% isoflurane via nose cone while rats were in a stereotaxic frame. Body temperature was controlled (37–38 °C) by a heating pad. Under aseptic surgical conditions, a midline skin incision was made to expose the skull. Using a high‐speed drill (Dremel, WI, USA), craniotomy (3.0 mm diameter) was made 3.0 mm posterior to bregma and 6.0 mm lateral (left) of midline to expose the intact dura. A hollow plastic injury cap was placed over the craniotomy, secured with dental acrylic cement and was later filled with 0.9% saline. When the dental cement hardened, the anesthesia was discontinued and the rat was attached to the FPI device via the head cap. At the first response of hind‐limb withdrawal to a paw pinch, rats received a moderate fluid percussion pulse (2.7 atm). Upon resumption of spontaneous breathing the head cap was removed and the skin was sutured. Neomycin was applied on the suture and the rats were placed in a heated recovery chamber to be fully ambulatory before being returned to their cages. The sham animals were prepared using the identical surgically procedure but without the fluid pulse.

After acclimatization, rats underwent fluid percussion injury (FPI) or sham surgery and were pair housed to a specific diet group with either regular diet (RD: regular rodent diet) or blueberry (BB) supplemented diet (5% w/w BB) for 2 weeks immediately. The BB dose was chosen based on previous in vivo studies which demonstrated that administration of blueberry offsets oxidative stress and reverses cognitive impairment. 28 , 29 The groups were: 1) Sham‐RD as control group, 2) TBI‐RD, and 3) TBI‐BB. The rats ( n = 8 per group) were subjected to FPI or sham surgery. Rats were subjected to learning on Barnes maze at post‐injury day (PID) 14 for 5 days, and after a 2‐day interval, memory was assessed at PID 21. Rats were tested for anxiety‐like behavior on elevated plus maze (EPM) on PID 22 ( Figure S1 , Supporting Information). All behavioral assessments were conducted between 9:00 and 13:00 h. Rats were provided with diets prepared daily and fed ad libitum in powder form. To determine the voluntary food intake (g), food was weighed daily to measure consumption in each cage. Since the rats were pair‐housed, food intake was divided by two to yield an approximate intake per rat.

Sprague Dawley male rats were purchased from Charles River Laboratories (Wilmington, MA) at 10 weeks of age and were acclimatized for vivarium 1 week prior to commencement of experimental procedures. Rats were housed in environmentally controlled conditions (temperature 22–24 °C and humidity) with 12‐h light/dark cycle in a controlled room with free access to food and water. All procedures were approved by the University of California at Los Angeles (UCLA) Chancellor's Animal Research Committee (ARC) and were conducted with adherence to the guidelines set out by the United States National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Freeze‐dried highbush whole blueberry fruit powder ( Vaccinium corymbosum L.; 50:50 blend of Tifblue and Rubel; U.S. Highbush Blueberry Council, Folsom, CA). This blend (per 100 g) contained bioactive phytocompounds (33 mg/g total phenolics; 10.2 mg/g anthocyanins; 85.9 IU β‐carotene), and other macro‐ and micronutrients (2.38 mg proteins; 32.2 g fructose; 18.8 mg vitamin C). Diet supplemented with 5% w/w BB was mixed with pulverized standard rodent chow (#5001, Lab Diet, St. Louis, MO). 1.6% fructose, 1.45% glucose, and 0.0009% vitamin C were mixed with the standard rodent chow to match the levels of sugars in the BB supplemented diet and used as the rodent control diet (RD).

Oxidative stress is known to play a role in TBI pathology. 35 We assessed the carbonyl‐containing molecule 4‐HNE which is the end product of lipid peroxidation affecting plasma membrane integrity and neuronal survival and function. The ANOVA for 4‐HNE revealed a significant group effect (F 2,14 = 3.96, p = 0.043). As depicted in Figure 5 A,B, TBI enhanced the levels of 4‐HNE in rats fed a regular diet compared with Sham animals fed regular diet. In turn, BB supplementation diminished the levels of 4‐HNE in TBI rats. Linear regression analysis showed a positive correlation between the latency in the memory test of the Barnes maze and levels of 4‐HNE, suggesting that higher oxidative stress may reduce spatial memory performance ( r = 0.467, p = 0.050; Figure 5 C).

We also studied CaMKII phosphorylation in our paradigm based on the involvement of CaMKII on hippocampal memory consolidation, 33 and its close interaction with the BDNF system. 34 The ANOVA showed a significant group effect for CaMKII phosphorylation levels (F 2,16 = 9.05, p = 0.002). Subsequent analyses revealed that TBI reduced phosphorylation of CaMKII which was ameliorated by the BB supplementation ( Figure 4 A). We found that the CaMKII phosphorylation negatively correlated with latency time in Barnes maze test ( r = −0.613, p = 0.005; Figure 4 B), suggesting that CaMKII contributed to the observed memory performance.

ANOVA revealed a significant group effect on BDNF levels (F 2,12 = 6.38, p = 0.012). As shown in Figure 2 A and confirmed by the Tukey's comparisons test, TBI rats showed significant reduction in BDNF levels compared to sham rats fed regular diet. BB supplementation was able to maintain BDNF levels near Sham in rats exposed to TBI. We found that the BDNF levels increased in proportion to a reduction in latency time in the Barnes maze memory test ( r = −0.638, p = 0.011; Figure 2 B), suggesting that BDNF was a factor for the memory performance. To assess the effects of BB supplementation on molecular systems involved with the action of BDNF, we evaluated the protein levels of cyclic‐AMP response element binding protein (CREB). The CREB family of transcription factors plays a major role in regulating synaptic plasticity and cognition. 32 The ANOVA revealed a significant group effect for CREB phosphorylation (F 2,16 = 9.69, p = 0.001). Tukey's comparison test showed that TBI reduced levels of CREB phosphorylation ( Figure 3 A) in rats fed a regular diet whereas BB supplementation counteracted these effects. In addition, we found that the levels of CREB phosphorylation increased in proportion to a reduction in memory latency ( r = −0.492, p = 0.038, Figure 3 B), suggesting that CREB may contribute to the effects of BB supplementation on memory performance. Moreover, CREB phosphorylation changed in proportion to the levels of BDNF ( r = 0.614, p = 0.016, Figure 3 C) and suggests that the regulations of BDNF and CREB may be coordinated.

We performed the search strategy analysis to evaluate the efficiency of rats to locate the escape hole. Sham‐RD animals used spatial strategies starting the first day and continued progressing over time (Figure 1 B). TBI rats appeared to have lost their capacity to navigate using spatial learning cues throughout the test time and exhibited reliance mostly on random strategies (Figure 1 C). In turn, rats exposed to BB supplementation appeared to regain the capacity to use spatial leaning cues (Figure 1 D).

Blueberry (BB) supplementation protects against spatial learning deficits after TBI, A) as indicated by the latency to locate the escape hole on Barnes maze during acquisition training (Days 1–5), B) percent time using spatial, serial, or random search strategies among Sham/RD, C) TBI/RD, and D) TBI/BB groups, and E) latency to locate escape hole during memory test on Barnes maze. F) BB supplementation appeared to counteract a reducing trend in time spent in the open arms of the elevated plus maze to assess anxiety‐like behavior. Data presented as means (± SEM). * p < 0.05, compared to Sham‐RD, # p < 0.05, compared to TBI‐RD; Student's t ‐test or ANOVA followed by post hoc Tukey test, as appropriate.

4 Discussion

In the present study, we found that BB supplementation can attenuate important aspects of the acute TBI pathology. We report that BB supplementation immediately following TBI mitigates behavioral deficits in spatial learning and memory. BB supplementation counteracted the effects of TBI on proteins associated with the action of BDNF (CREB and CaMKII) on plasticity and behavior. In addition, BB supplementation counteracted the increase of the end product of lipid peroxidation, 4‐HNE. The results showing that markers of neuronal plasticity and lipid peroxidation change in proportion to memory performance suggest a possible association between these molecular parameters and behavior. Taken together, the present findings emphasize the beneficial effects of BB supplementation in fostering brain plasticity in the TBI pathology.

4.1 Impact of BB Supplementation on Behavior In agreement with previous reports,18, 30 we found that TBI impairs spatial learning as evidenced by an increase in latency in the Barnes maze, while BB supplementation decreased latency time to find the escape hole at each training day. We assessed the use of spatial learning strategies in our paradigm to provide a complementary measure of cognitive function less dependent on motor behavior. Interestingly, we found that BB supplementation appeared to counteract a lost capacity of TBI rats to employ spatial leaning cues. This information together with results of the shorter latencies strongly suggest that BB supplementation protects TBI animals from a loss in spatial learning performance. In this regard, recent functional neuroimaging study in humans has established a connection between BB intake and cognitive function.36 Further, in the EPM test, rats exposed to TBI showed a tendency to reduce time spent in the open arms, which encompasses with clinical reports that psychiatric disorders are often observed in TBI patients.37 TBI‐induced behavioral deficits probably stems from the impairments in BDNF‐TrkB signaling that has been implicated in various cognitive and affective disorders.38 We cannot ascertain the cellular identity of the reported protein alterations. Although neuronal cells are the primary locus for learning and memory processing, non‐neuronal cell types such as astrocytes and microglia can also contribute to these alterations.39, 40 Moreover, it known that astrocytes and microglia provide support to synaptic transmission that is fundamental for neuronal function involved in cognitive processing.41-43

4.2 Effects of BB Supplementation on Plasticity Markers In the present investigation, we also found that TBI significantly reduced levels of hippocampal BDNF, and that BB dietary supplementation normalized these levels. Previous report indicated that deficiencies in BDNF signaling is associated with impairments in cognition.44 Alternatively, cognition is strongly reliant on long‐term potentiation (LTP) and hippocampal BDNF, and the interaction between BDNF and its tyrosine kinase receptor (TrkB) is required for induction of LTP.45 Previously, we have shown the protective effects of BDNF on the TBI pathology.46 Presently, our findings show that BB supplementation counteracted the BDNF reduction induced by TBI, paralleling improvements in cognitive function. It is well established that BDNF regulates synaptic plasticity and learning through interaction with the transcription factor CREB.47 Interestingly, our results also showed that BB supplementation normalized levels of CREB in TBI animals, and that these changes were proportional to changes in BDNF levels. These findings are consistent with reports showing that BB dietary supplementation enhances BDNF‐mediated plasticity with improved spatial and object recognition memory.48, 49 Moreover, the significant positive correlation between levels of BDNF and CREB indicates that BDNF and CREB are co‐regulated in our paradigm. In addition, evidence indicates an association between BDNF and CREB, and this interaction is important for regulation of learning and memory.50 The latter possibility can also be inferred from our results showing a negative correlation between CREB signaling and latency in the Barnes maze. We also found that BB supplementation preserves levels of hippocampal CaMKII phosphorylation after TBI, and changes in CaMKII correlated negatively with latency to locate the escape hole in the Barnes maze memory test. CaMKII, the main protein of the postsynaptic density and key BDNF signaling element, upon autophosphorylation increases synaptic efficacy51 and long‐term synaptic memory.52 In fact, CaMKII dysregulation has been associated with several neuropsychiatric diseases.53 It is possible that the effects of BB on the BDNF levels results in autophosphorylation of tyrosine residues that rise intracellular calcium levels leading to CaMKII activation.54