This prospective, randomized animal study was approved by the Institutional Animal Care and Use Committee (approval number ESM13-0235). 4-week-old healthy male Sprague–Dawley rats (Orient Bio, Seongnam, Korea) weighing 80–90 g supplied by a single-source breeder were used in this study. 4-week-old rats were used in this study as this age corresponds to the teenagers and young adults in rats, which is a common age of mTBI occurrence in humans [12] Only male rats were used in this study as the hormonal levels could influence the cortical excitability and the neurotransmitter levels could affect the tDCS response [13,14,15] The male would receive more current at the cortex than the female due to the cortical bone structure [16] Furthermore, males make up a larger percentage of cases than females in mTBI [17] For these reasons, in this study, only male rats were included. The animals were under standard conditions with 12-h light–dark cycle, and had free access to tap water and regular rat chow. All animals received human care in compliance with the National Institutes of Health guidelines for the use of experimental animals. This study was carried out in compliance with the ARRIVE guidelines. This study was performed using protocols approved by the Institutional Animal Care and Use Committee (Approval number ESM13-0235).
Repetitive mTBI models
mTBI was induced in rats using a modified weight-drop device and a protocol previously described by Tang et al [18] Through previous studies, we have demonstrated that the weight-drop device used in this study causes only mild traumatic brain injury without causing histological changes or imaging changes such as cerebral hemorrhage [8, 19, 20] The animals were anesthetized by intramuscular injection of tiletamine/zolazepam (10 mL/kg; zoletilⓇ, Vibac, France), and placed on the wooden platform of the device in the prone position. A 175 g steel weight was briefly dropped from a height of 30 cm through a polyvinyl chloride tube with an inner diameter of 11 mm terminating on the bregma of the rat. NCAA Concussion Study (1999–2001) showed the average interval between first and repeat concussion was 5.59 days. [21] This is a time interval equivalent to 0.89 h in rats. And atheletes reporting a history of 3 or more previous concussions were 3.0 times more likely to have an incident concussion than atheletes with no concussion history [22] Based on these previous studies, the mTBI procedure was performed 3 times at 1-h intervals for a repetitive mTBI model.
Experimental design
Forty rats underwent repetitive mTBIs (Day 1) and were randomly assigned to one of four groups: amantadine group (n = 10) that received intraperitoneal injections of amantadine alone, tDCS group (n = 10) that underwent anodal tDCS alone, combination of amantadine and anodal tDCS (amantadine + tDCS) group (n = 10) that underwent both amantadine intraperitoneal injections and anodal tDCS, and control group (n = 10) that did not undergo additional treatment. In the combination group, amantadine injection was administered first, followed by tDCS. All treatments were performed 4 times for four consecutive days (Days 2–5), once a day. The treatment and test schedules are shown in Fig. 1.
Experimental procedure
The day after three inductions of mTBI (Day 2), amantadine injection and anodal tDCS were conducted (Fig. 1a). The amantadine and combination groups received daily intraperitoneal injections of amantadine hydrochloride (10 mg/kg; Sigma Chemical Co., St. Louis, MO, USA) [23]. Amantadine hydrochloride was dissolved in 0.9% sterile saline and administered. Amantadine hydrochloride was injected once a day for 4 days (Days 2–5). Ion combination group, amantadine injection was administered before tDCS application.
In tDCS and combination groups, anodal tDCS was performed under isoflurane-induced anesthesia (2% isoflurane in a 1:2 mixture of O2/N2O) [20] A tDCS was applied using a constant-current stimulator, PhoresorIIⓇ (IOMED, Salt Lake City, UT, USA). A constant direct current was delivered for 30 min at an intensity of 0.2 mA and a density of 0.255 mA/cm2 (0.2 mA/0.785 cm2). The fur around the bregma was removed to ensure tight attachment of the anodal electrode. A 1 cm-diameter cup–shaped anodal electrode was placed on the scalp over the left motor cortex, 3 mm to the left of the bregma, and 2 mm in front of the interaural line, using a high-conductivity fixation cream (Fig. 1b). A 30 mm × 30 mm rectangular rubber cathodal electrode was affixed to the abdomen [8, 20, 24]. Anodal tDCS was conducted once a day for 4 days (Day 2–5, Fig. 1a). tDCS was performed by a single experienced physiatrist.
Measurements
Behavioral tests
In this study, two behavioral tests were performed; the rota rod test and the novel object test. These tests were conducted pre-mTBI (Day 1), post-mTBI (Day 2) and one day after the last session of treatment (Day 6), to eliminate anesthetic effects (Fig. 1a). For evaluation of the balance control and motor coordination, the rotarod test was used [25] A rat was placed on the rotarod treadmill. The rotation speed was started at 4 rpm and accelerated to 40 rpm over a 4 min period [7] The duration of the rolling rotarod before falling and the maximal speed on the rotarod were recorded and analyzed. Three trials were performed, and the average value was calculated. For the evaluation of the memory function, the modified version of the novel object test developed by Ennaceur and Delacour was used [10, 26, 27] Two identical objects were placed in an acryl room with black walls and floors, and the rats were allowed to explore for 5 min. One hour later, one of the objects was swapped for a different object, the rat was placed back in the room, and the time spent exploring each object was measured. The ratio between the times spent exploring the new object and the time spent exploring the familiar object was then compared. The objects were changed in every trial. In the first trial, the familiar object was a tetrahedron, and the new object was a cuboid. In the second trial, the familiar object was a cylinder, and the new object was a conical flask model. In the third trial, the familiar object was a narrow tetrahedron, and the new object was a narrow cuboid. A higher ratio meant that more time was spent exploring the new object, which was interpreted as a better memory function.
Motor-evoked potentials (MEP)
To evaluate the functional integrity of the motor system, transcranial MEP was evaluated. MEPs are muscle action potentials elicited by transcranial magnetic brain stimulation [28] In this study, MEP measurements were evaluated pre-mTBI (day 1), post-mTBI (day 2), and post-tDCS (day 6) to evaluate the excitability of the corticospinal pathway (Fig. 1). MEP at the right tibialis anterior muscle of the hindlimb was evaluated. The MEP was recorded from the tibialis anterior muscle of the right hindlimb, which resulted from left motor cortex stimulation. The monopolar uninsulated stainless-steel active needle electrode was inserted into the belly of the tibialis anterior muscle, and the reference needle electrode was inserted into the distal part of the tibialis anterior muscle. The ground electrode was placed on an opposite footpad. The MEP test was performed using a Medtronic KeypointⓇ laboratory computer (Medtronic Inc., Jacksonville, FL, USA). The measurement settings were a sweep velocity of 5 ms with a sensitivity of 200 μV, and the bandpass filter setting was 20 Hz–10 kHz. Single-pulse transcranial magnetic stimulation was administered over the left motor cortex, which was anterior and left lateral to the bregma, with a figure-of-eight magnetic coil (diameter of one widening = 50 mm, peak magnetic field = 4.0Tesla) using a magnetic stimulator, MagstimⓇ (Magstim Company, Whiteland, Wales, United Kingdom). The center of the coil was positioned on the left motor cortex, whose center was anterior and lateral to the bregma on the right side of the hindlimb, where the active needle electrode was inserted. A total of 20 MEPs were recorded at 10 s inter-stimulus intervals [29] TMS intensity was recorded as percent machine output (MO), with 100% corresponding to the maximal amplitude electrical current conducted through the magnetic coil. We set the stimulation intensity to 100% MO [20]. The intensity of the stimulation was maintained constant throughout the procedure. The average latency and largest peak-to-peak amplitude of the MEP waves were recorded and analyzed.
Glial fibrillary acidic protein (GFAP) immunohistochemistry
GFAP is a protein found only in the central nervous system. GFAP levels are increased when astrocytes are damaged [30]. A low GFAP integral intensity indicates a decrease in reactive astrocytosis, which can play a neurotoxic role and aggravate neural death [31, 32] Previous study showed that GFAP could determine patients with mTBI with subtle injuries detected only through MRI [33] 30 days after all treatments and evaluations (Day 36), 12 rats (3 rats from each group were randomly selected) were euthanized by carbon dioxide inhalation using an approved standard protocol. The brain tissue washed PBS for removing the bound and unbound reagents/serum component. The fresh brain is fixed in 10% formalin to prevent deformation or deterioration due to autolysis. Formalin-fixed tissue undergoes tissue processing and then is embedded in paraffin wax to create the paraffin block. Embedding is important in preserving tissue morphology and giving the tissue support during sectioning. Brain slices were sectioned to a thickness of 4 μm. The tissues incubated using the free floating method. Immunohistochemical staining was performed to assess axonal damage and astrocytes. The slices were incubated with primary antibody against GFAP ( 1:500 dilution, Ab4674, Abcam, Cambridge, United Kingdom) at room temperature for 30 min, and with conjugated secondary antibodies (1: 200 dilution, Ab6877, Abcam, Cambridge, United Kingdom) for 20 min [20] After staining, the tissue sections were washed with running water and mounted using a universal mount (Dako, Carpinteria, CA, U.S.A.). Left motor cortex and hippocampus were assessed and averaged in a blinded manner. In this study, respectively one representative left motor cortex tissue slice and hippocampus slice were selected and analyzed in each brain. Total twelve left motor cortex slices and twelve hippocampal slices were included. After tissue sections were obtained. Immunohistochemical study was performed with Bond Max (Leica Biosystems, Newcastle, UK). The mean integral intensity of GFAP was calculated and analyzed. A computer-assisted image analysis program, AnalySISⓇ (Soft Imaging System, GmbH, Munster, Germany) was used to measure GFAP expression [20] Images were captured from left motor cortex and hippocampus. The software automatically changed the color of all immunolabeled elements beyond the threshold range into red pixels and changed the color of the rest of the image into gray pixels. The software then estimated the intensity of pure red pixels [20, 34]
Statistical analysis
To verify the effects of mTBI, Wilcoxon signed-rank test was performed to compare the results of pre-mTBI and post-mTBI evaluation results for all rats. After treatment, to compare the effects among the four groups, each treatment effect was measured with changes in each parameter (post-treatment values minus post-mTBI (pre-treatment) values; △). The Kruskal–Wallis test was used to compare the effects between groups. If the Kruskal–Wallis test was positive, the Mann–Whitney test was used for comparison between the two groups. In addition, to analyze the within-group effectiveness of treatments, we used the Wilcoxon signed test. Statistical analysis was performed using SPSS version 21.0 (IBM SPSS, Armonk, NY, USA), and p-values of 0.05 or less, were considered statistically significant.
