Traumatic brain injury (TBI) often occurs when external mechanical force hits on the head. Serious TBI can result in long-term dysfunction of the brain (including motor deficit and cognitive deficit) or death. Neural cell apoptosis has been found in human brains after brain trauma and in different animal models of TBI.1 The B-cell lymphoma-2 (Bcl-2) family of proteins, including both pro- and antiapoptotic members, play a crucial role in apoptosis.2 One of the best studied antiapoptotic members in neuron is Bcl-2 and Bcl-2–associated X-protein (Bax) is one of proapoptotic members. It was found that transgenetic mice overexpressing the human Bcl-2 protein showed significantly less neuronal loss after TBI,3 and Bax null mice showed higher percentage of surviving neurons in hippocampus after TBI.4 These results suggested that regulation of Bcl-2 and Bax expression might be a potential target of therapeutic intervention after TBI.
Exosome is one type of extracellular vesicle with size range of 20–100 nm and have raised great interest in the drug delivery field, because of their ability to carry biomolecules for intercellular transportation and the nanoscaled size.5–7 Exosomes have been engineered to incorporate therapeutic molecules, including drugs, proteins, therapeutic RNA, and plasmid DNA,8,9 and have been explored as carriers for delivering therapeutic molecules to tumors5 and neural cells.10
In the current study, we incorporated plasmids expressing Bcl-2 and Bax shRNA into exosomes and sought to investigate whether these exosomes would be able to deliver their cargoes and play a role in a mouse model of TBI.
Materials and Methods
C57BL/6J mice were purchased Shanghai Lab Animal Center (Shanghai, China), at 23 ± 2°C and 12 hours light/dark cycles (light on at 7:00 A.M.), with free access to food and water. All animal-handling procedures were carried out in accordance with the Guide for the Care and Use of Laboratory Animals of Cangzhou Central Hospital.
Isolation and Transfection of Exosomes
Primary astrocytes were prepared from the cerebral cortex of newborn C57BL/6J mice by mechanical dissociation and maintained in Dulbecco's Modified Eagle's medium (DMEM) medium (Gibco, Thermo Fisher Scientific, Waltham, MA) with 10% heat-inactivated fetal bovine serum (Gibco) and 1% penicillin-streptomycin (Sigma-Aldrich, St. Louis, MO) in a humidified incubator. Culture medium from primary astrocyte cultures was centrifuged at 17,000g for 10 minutes. The resulting supernatant was passed through a 0.2 μm filter membrane and further centrifuged at 100,000g for 2 hours. The exosome pellet was resuspended in fresh culture medium and transfected with the following plasmids: EGFP-C1, pIRES2-EGFP-Bcl-2, and EGFP-C1-Bax shRNA, using Exo-Fect reagent (System Biosciences, Palo Alto, CA), according to the manufacturer’s instructions.
Primary Cortical Neuron Culture and Treatment
Cerebral cortex of newborn C57BL/6J mice was cut into small pieces and digested in phosphate-buffered saline (PBS) containing 0.25% trypsin (Sigma-Aldrich) and 100 μg/ml DNase (Sigma-Aldrich) at room temperature for 15 minutes. Then, the tissue pieces were passed through a Pasteur pipette in Neurobasal medium (Gibco, Thermo Fisher Scientific) and centrifuged at 100g for 15 minutes at 4°C. The cells were resuspended and planted onto 12-well culture plates at a density of 7.5 × 105 cells/well and were maintained in Neurobasal medium (Gibco) with 2% B27 (Invitrogen, Carlsbad, CA) and 1 mmol/L glutamine (Invitrogen) in a humidified incubator. The cells were treated with different exosomes at a dosage of 50 µg/ml for 48 hours. Then, cells were harvested for further analyses.
Real-Time Quantitative Polymerase Chain Reaction
Total RNA was extracted with Trizol RNA extraction reagent (Invitrogen, Carlsbad, CA). cDNA was synthesized using a QuantiTect Reverse Transcription kit (QIAGEN GmbH, Hilden, Germany). Real-time quantitative polymerase chain reaction was performed using SYBR Green PCR Master Mix (Applied Biosystems Inc, Foster City, CA) in a StepOnePlus real-time quantitative polymerase chain reaction system (Applied Biosystems Inc). The primers for Bax were 5′- AGC AAA CTG GTG CTC AAG GC -3′ and 5′- CCA CAA AGA TGG TCA CTG TC -3′. Primers for Bcl-2 were 5′- GTG GTG GAG GAA CTC TTC AG -3′ and 5′- GTT CCA CAA AGG CAT CCC AG -3′. Primers for β-actin were 5′-AGA GGG AAA TCG TGC GTG AC-3′ and 5′-CAA TAG TGA TGA CCT GGC CGT-3′. Data were analyzed using the 2-△△Ct method, and the values were normalized to the level of β-actin mRNA.
Cells were lysed in cold radioimmunoprecipitation assay (RIPA) lysis buffer (Sigma-Aldrich) supplemented with protease inhibitors for 20 minutes on ice. Total proteins were isolated by centrifugation at 12,000 rpm at 4°C for 4 minutes. Protein extracts (30 µg) were subjected to sodium dodocyle sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a polyvinylidine fluoride membrane (Emd Millipore, Billerica, MA). After blocking in 5% skim milk in tris-buffered saline and 0.1% Tween 20 (TBST) for 1 hour, the membranes were probed with indicated antibodies and visualized using SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific). The bands on the film were scanned and analyzed using Image J software. The primary antibodies were anti-Bax (1:500; Santa Cruz Biotechnology, Santa Cruz, CA), anti-Bal-2 (1:500; Santa Cruz Biotechnology), anti-Bad (1:500; Santa Cruz Biotechnology), and anti-β-actin (1:1,000; Santa Cruz Biotechnology). For Cytochrome c assays, we utilized Cytochrome c Releasing Apoptosis Assay Kit (Biovision) and followed the manufacturer’s instructions. The values of protein level were normalized to the level of β-actin protein.
Traumatic Brain Injury Model and Treatment With Exosomes
Traumatic brain injury was induced via unilateral controlled cortical impact injury. Animals were anesthetized with 4% isoflurane and maintained with 2% isoflurane during surgery. The scalp was incised to expose the skull, and a 3 mm craniotomy was performed in the center of the right parietal bone. After craniotomy, a single impact was produced using a 2 mm flat impactor (Air-Power Inc, High Point, NC) discharged at 5 m/second at a depth of 1.5 mm. Animals received intraventricular injection of exosomes (10 μg exosomes in 20 μl PBS/mouse) at 1 hour after surgery. Forty-eight hours later, animals were sacrificed, and the ipsilateral brain tissues were harvested. The protein levels of Mcl-1, XIAP, Survivin, and Cytochrome c were measured following above procedures (primary antibodies purchased from Santa Cruz Biotechnology).
Electrophysiological tests were carried out at 14 days after surgery. After anesthetized with isoflurane, mouse brains were quickly removed and immersed in oxygenated ice-cold cutting solution (composition in mmol/L: 234 sucrose, 2.5 KCl, 1.25 NaH2PO4, 10.0 MgSO4, 1 CaCl2, 26 NaHCO3, and 20 glucose). Then, the brain was sliced into 400 µm thick coronal slices using a Vibratome 3000 (Technical Products International, St. Louis, MO). Slides from hippocampus were dissected and incubated in oxygenated external recording solution (composition in mmol/L: 124 NaCl, 2.5 KCl, 2.5 CaCl2, 1.3 MgSO4, 26 NaHCO3, 1 NaH2PO4, and 10 glucose) at 22°C for at least 1 hour. Then, slices were transferred to the recording chamber and held fixed by a grid of parallel nylon threads under constant perfusion with oxygenated external recording solution. Miniature excitatory postsynaptic currents (mEPSCs) of hippocampal CA1 pyramid neurons were recorded using whole-cell recording. The patch electrode (3–4 MΩ) was filled with internal solution (containing in mmol/L: 120 Cs-gluconate, 20 CsCl, 10 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 4 MgATP, 0.3 Na3GTP, 0.2 ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), and 10 phosphocreatine, pH 7.3 with osmolarity 290–300 mOsm). The membrane potential was held at −68 mV under a voltage-clamp mode to record the mEPSCs. For recording of field excitatory postsynaptic potentials (fEPSPs), a patch electrode (3–4 MΩ) filled with internal solution was placed in the stratum radiatum of area CA1 and fEPSPs were evoked by 0.1 Hz square pulses in Schaffer collateral afferents by a bipolar tungsten stimulating electrode (FHC, Bowdoin, ME). Stimulations were generated with a pulse generator (Grass S-88X; AstroNova, West Warwich, RI). After 15 minute period of baseline fEPSP recording, LTP was induced by high-frequency stimulation (4 trains of 100 Hz, 25 pulses per train at 0.1 Hz). Data were acquired using a MultiClamp 700B amplifier (Axon Instruments, Foster City, CA) with a digitizer (DigiData 1322A; Axon Instruments, Foster City, CA).
Morris Water Maze
The water maze apparatus was a circular tank (120 cm in diameter) filled with opaque water. In training sessions, mice were released into the water and allowed to navigate in the tank to find the hidden platform, which was fixed in the center of 1 quadrant. Each mouse performed four training trials from different starting quadrants per day for 5 consecutive days. The latency to find the platform was recorded, and the four trials per day were averaged. Then, the platform was removed and mice were subjected to a 30 second probe test at 24 hours and 7 days after the last training trial. The time spent in each quadrant was recorded by Ethovision XT software (Noldus, Wageningen, Netherlands).
Beam-Balance, Beam-Walk, and Rotarod Tests
The apparatus for beam-balance test was a 1.5 cm wide wooden beam elevated at 90 cm from the ground. Mice were placed on the beam for a maximum of 60 seconds. Latency to fall was recorded. In the beam-walk test, mice were trained to traverse a narrow wooden beam (2.5 cm wide, 100 cm long, 90 cm elevated from the ground) to escape bright light on one end and enter a darkened goal box at the opposite end. The time to traverse the beam was recorded. For Rotarod test, an automated Rotarod apparatus (Ugo Basile, Comerio, Italy) was used. Mice were tested on at an accelerating rotational speed (4–40 rpm) for three trials per day (inter-trial interval 15 minutes). The average latency to fall was recorded. For all these tests, mice were trained and tested to get a baseline on the day before surgery, and then tested at 1, 2, 3, 4, 5, 6, 7, 11, and 14 days after surgery.
All the values are expressed as mean ± standard error of the mean. When 2 groups were compared, data were analyzed by Student’s t-test. Other data were analyzed by one-way analysis of variance with post-hoc Bonferroni test to compare the differences among groups. Significance level was set at p < 0.05 for all these analyses.
Exosome-Mediated Intracellular Delivery of B-Cell Lymphoma-2 cDNA and Bcl-2-Associated X-Protein shRNA Into Primary Cultured Neurons
As shown in Figure 1, A–E, compared with control or cell treated with exosomes carrying EGFP-C1 plasmid, cells treated with modified exosomes (exosomes carrying both pIRES2-EGFP-Bcl-2 plasmid and EGFP-C1-Bax shRNA plasmid) resulted in higher Bcl-2 mRNA expression and protein level, as well as downregulated Bax mRNA expression and protein level.
Modified Exosomes Reduced Apoptosis Induced by Traumatic Brain Injury
As shown in Figure 2, A–D, compared with sham group, protein levels of apoptotic markers, Mcl-1, XIAP, and Survivin in the brain were significantly decreased in TBI mice treated with exosomes carrying EGFP-C1 plasmid, and treatment with modified exosomes could attenuate the decrease. In contract, the protein level of proapoptotic marker Bad significantly increased in the TBI group, but this increase was suppressed by the treatment with modified exosomes (Figure 2, E). Treatment with modified exosomes also significantly reduced the ratio of Cytochrome c levels in cytosol versus in mitochondria, indicating reduced Cytochrome c release from mitochondria to cytosol (Figure 2, F and G).
Modified Exosomes Attenuated Impairments of Miniature Excitatory Postsynaptic Current and LTP in the Hippocampus of Traumatic Brain Injury Mice
Results of electrophysiological tests showed that the amplitude and frequency of mEPSC in hippocampal CA1 pyramid neurons were impaired after TBI (Figure 3, A–C), and treatment with modified exosomes could attenuate these impairments. Modified exosomes also attenuated the TBI-induced deficits in LTP in hippocampus (Figure 3, D and E).
Modified Exosomes Improved Cognitive Function in Traumatic Brain Injury Mice
Treatment with modified exosomes significantly reduced the escape latency compared with TBI mice treated with exosomes carrying EGFP-C1 plasmid (Figure 4, A and B). In the probe tests 24 hours and 7 days after the training, there was no difference between time spent in the target quadrant (where the hidden platform was previous in) and in other quadrants in TBI mice treated with exosomes carrying EGFP-C1 plasmid. Treatment with modified exosomes could rescue this deficit (Figure 4, C and D).
Modified Exosomes Improved Motor Function in Traumatic Brain Injury Mice
Compared with TBI mice treated with exosomes carrying EGFP-C1 plasmid, treatment with modified exosomes attenuated TBI-induced motor impairments in beam-balance test, beam-walk test, and Rotarod test, showing significantly increased latency to fall in beam-balance (Figure 5, A) and Rotarod (Figure 5, C) and decreased time to traverse the beam (Figure 5, B) on day 11 and 14 after TBI.
Apoptosis contributes to the pathogenesis of TBI.11 The Bcl-2 family of genes, including both proapoptotic members such as Bax, and antiapoptotic members such as Bcl-2, play a crucial role in apoptosis. Postmortem histological and immunochemical analyses of the brains of TBI patients found increased Bax expression.12 Increased Bax expression and decreased Bcl-2 expression were observed in rat models of ischemia and TBI.13,14 The alteration of gene expression implies a target for therapeutic intervention for apoptosis after TBI. In the current study, we injected exosomes containing plasmids expressing Bcl-2 and Bax shRNA into the brains after TBI. These modified exosomes could significantly upregulate Bcl-2 expression and silence Bax gene in cultured primary neurons and thus was expected to play a therapeutic role in TBI by inhibiting the apoptosis. As expected, we found that these modified exosomes suppressed the TBI-induced increase of proapoptotic marker Bad and attenuate the decrease of protein levels of apoptotic markers, Mcl-1, XIAP, and Survivin in the mice brain after TBI. Mcl-1 is another antiapoptotic protein that belongs to the Bcl-2 family.15 XIAP and Survivin are members of the inhibitor of apoptosis family and function to inhibit caspase activation and thereby prevent apoptosis.16 Meanwhile, treatment with modified exosomes also significantly reduced Cytochrome c release from mitochondria to cytosol release of Cytochrome c from mitochondria is a key step in apoptotic process, which could be promoted by Bax and blocked by Bcl-2.17 Thus, our results indicated that the modified exosomes might reduce TBI-induced apoptosis in vivo. On the other hand, apoptosis is a complicated process which contains downstream process such as activation of caspase cascade, thus further studies are warranted to draw a more definite conclusion. Along with these results, we found that the modified exosomes improved motor and cognitive functions in TBI mice. Taken together, our results suggested that the modified exosomes might exert therapeutic effect after TBI by suppressing apoptosis.
Gene therapy has been investigated as potential treatment for apoptosis after TBI, for the ability to control the expression of specific apoptosis-related gene and consequently inhibit neuronal cell apoptosis.18 One method of reversing apoptosis using gene therapy was to overexpress antiapoptotic proteins, such as Bcl-2. A previous study found that after delivered into the brain by a recombinant adenovirus vector, the Bcl-2 fusion protein could suppress apoptosis in the brain after TBI, and promote the behavioral recovery.19 Others showed that rats received injection of defective herpes simplex virus vectors containing Bcl-2 showed significantly greater neuron survival after focal ischemia.20 In the current study, we used exosomes, rather than viral vectors, to deliver the therapeutic nucleic acids. Exosomes are nanoscaled extracellular vesicle with ability to carry biomolecules for intercellular transportation. We found that intraventricular injection of the modified exosomes in our study resulted in inhibition of apoptosis and improved neurological and functional outcome of TBI mice. These results were similar to that observed when viral vectors were used to overexpress Bcl-2 in the brain.19,20 A major clinical barrier of viral vector-mediated gene therapy is that the immunological defense system would recognize the viral vectors as foreign and be activated against them.21 Compared with viral vectors, exosomes could be isolated from the patient’s body fluids or cell cultures and then transferred back to the same patient after modification, thus are less likely to evoke immune response.7 Thus, the modified exosomes might serve as a useful tool in gene therapy for TBI.
In conclusion, we incorporated plasmids expressing Bcl-2 and Bax shRNA into exosomes to overexpress Bcl-2 and silence Bax gene in the recipient cells. Data from our study indicated that these modified exosomes could reduce apoptosis and ameliorate neural and functional deficits in mouse models of TBI.
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