Animals
All animals (male C57BL/6 mice, 18–28 g, 7–8 weeks of age, and maternal SD rats, TP16) were bred and housed in the animal facility at 21°C under 12-h light/12-h dark cycles (lights on at 6 a.m.) with access to food pellets and water ad libitum. All the experiments were performed in compliance with the National Institutes of Health guidelines for animal research.
Primary culture of hippocampal neurons
Rat hippocampal neurons were prepared from TP 17 SD rats (Figure 1D). Fetuses were removed on embryonic day 17 from maternal rats anesthetized with 16.5% urethane. Hippocampi were dissected and placed in Ca2+- and Mg2+-free HEPES buffered Hank’s salt solution (pH 7.45). Cells were dissociated by trituration through a series of flame-narrowed Pasteur pipettes. Then, dissociated cells were plated at a density of 16,000 cells/well onto a 25-mm-round cover glass containing neurobasal medium with L-glutamine, 2% B27 supplement, 0.25% Glutamax I, and penicillin/streptomycin/amphotericin B (100 U/mL, 100 µg/mL, and 0.025 µg/mL, respectively). The cover glass was coated with Matrigel (0.2 mg/mL; BD Bioscience) for 1 h. Neurons were grown in a humidified atmosphere of 10% CO2 and 90% air (pH 7.4) at 37°C and fed at 4-day intervals by replacing 75% of the media with a fresh media. All in vitro experiments consisted of six independent cultures.
Following the manufacturer’s instructions, transfection with small interfering RNA (siRNA) was initiated by adding a mixture (1:1) of Lipofectamine 2000 Reagent diluted in Opti‐MEM Medium (1:50) and the siRNA (abx925999, Abbexa), also diluted in Opti‐MEM Medium (1:50) for 5 min. Then, the cells were incubated with the siRNA‐Lipid complex for 24 h at 37°C.
PI-induced chemoconvulsant models
Male C57BL/6 mice 7–8 weeks of age were administered scopolamine methyl nitrate (intraperitoneally (i.p.); 1.5 mg/kg; Sigma-Aldrich) and terbutaline hemisulfate salt (i.p.; 1.5 mg/kg; Sigma-Aldrich) to block peripheral effects of PI and dilate the respiratory tract, respectively. PI hydrochloride (i.p.; Sigma-Aldrich) at 320 mg/kg was injected 30 min later (Figure 1E). After continuous tonic clonic convulsive seizures were initiated, mice were placed at room temperature for 8 h and monitored with video recoding. Acute seizures were behaviorally scored using a modified Racine scale (Supplemental video, stages: 0, no abnormality; 1, exploring, sniffing, and grooming ceased, becoming motionless; 2, forelimb and/or tail extension, appearance of rigid posture; 3, myoclonic jerks of the head and neck, with brief twitching movement, or repetitive movements with head bobbing; 4, forelimb clonus and partial rearing, or occasional rearing and falling; 5, forelimb clonus, continuous rearing and falling; and 6, tonic-clonic movements with loss of posture tone, often resulting in death) (27-29). Only mice showing multiple stage 5 or above behavioral seizures were selected for further processing as status epilepticus.
PBM irradiation
Irradiation was performed using an 825 nm laser with total irradiance at skin surface of 40 mW/cm2 and a total energy density of 15.2 J/cm2 for a 380 s exposure (Figure 1A-B, Table 1). Mice were maintained motionless by restraint in the restrainer and the laser was manually applied at 4 cm from the skin of the shaved head. Laser transmission ratio to skin and skull was measured before the laser treatment (penetration rate of PBM; Figure 1C), which was applied once 4 h after PI injection (Figure 1E). Hippocampal primary neurons were exposed for 600 sec to a 850 nm LED device with a power density of 16 mW/cm2 and a total energy density of 9.6 J/cm2 (Figure 1A, Table 2). Cells were irradiated 30 min after the treatment with kainic acid on day in vitro (DIV) 13 in the dark, followed by a fixative 4 h after treatment with kainic acid (Figure 1D). A powermeter (PD300 and VEGA, Ophir PD, Ophir Photonics) was used to measure the light source power density.
Neuronal death based on Nissl staining analysis
After PI-induced seizures, mice were anesthetized 3 and 7 days later with ethyl ether and transcardially perfused with cold saline using a pump and fixed in 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. The brains were removed from the skulls, fixed for at least 48 h, then cryoprotected in 30% sucrose in phosphate-buffered saline. Sequential coronal sections (18-µm-thick) through the hippocampus were prepared using a cryocut microtome (CM3050S, Leica). The tissues were mounted on gelatin-coated slides overnight before use. After dehydration in a graded alcohol series, hippocampal sections were stained for 20 min with pre-warmed 0.3% CV solution at room temperature. After destaining with 95% ethanol and 0.3% glacial acetic acid solution, the sections were dehydrated using 100% ethanol followed by 100% xylene. Then, the sections were mounted with DPX. Unbiased cell counting was obtained in the CA1, CA3, and hilus. Only cells displaying Nissl-stained cytoplasm with a nucleus top completely within the section were counted. Cells were included if they were partly or entirely within the dissector frame and did not cross the exclusion lines. The counted average range for CA1, CA3, and hilus was 200 μm, 200 μm, and 1 mm2, respectively.
Neurodegeneration based on FJB staining
Coronal sections (18-µm-thick) were stained with FJB solution. The tissues were mounted on gelatin-coated slides overnight before use. After dehydration in a graded alcohol series, hippocampal sections were incubated in 0.06% potassium permanganate solution for 10 min. Next, brain sections were stained with 0.0004% FJB solution containing 0.1% glacial acetic acid for 20 min at room temperature. Then, the sections were washed with distilled water, dried, and mounted with DPX. Unbiased cell counting was also obtained in the CA1, CA3, and hilus and counted average range was 200 μm, 200 μm, and 1 mm2, respectively.
Immunocytochemistry and immunohistochemistry
Primary hippocampal neurons were fixed with chilled methanol for 8 min at −20°C. Hippocampal tissue sections and primary neurons were permeabilized with 0.3% Triton X-100 for 5 min. The tissues and cells were blocked with 10% bovine serum albumin (BSA) and incubated overnight at 4°C with the following primary antibodies: mouse anti-MAP2 (Sigma-Aldrich, M9942), rabbit anti-PSD95 (Abcam, ab18258), mouse anti-GFAP (Millipore, MAB360), rat anti-CD11b (Abcam, ab8878). After treatment with primary antibodies, the tissues and cells were incubated with secondary antibodies conjugated with Alexa Fluor 488-conjugated anti-rabbit IgG (ThermoFisher, A11008), Alexa Fluor 555-conjugated anti-mouse IgG (ThermoFisher, A21422), or Alexa Fluor 488-conjugated anti-rat IgG (ThermoFisher, A11006) for 1.5 h at room temperature. Immunostained tissues and cells were mounted with VECTASHIELD Antifade mounting medium with DAPI (H-1200; Vector Laboratories). Alexa Fluor 488 (excitation, 488 nm; emission, 520 nm) and Alexa Fluor 555 (excitation, 561 nm; emission, 568 nm)-labeled tissues and cells were imaged using a confocal microscope (FV3000, Olympus).
Western blot analysis
Proteins of primary cultured neurons and homogenized hippocampal tissues were extracted in RIPA buffer (ThermoFisher) supplemented with a protease and phosphatase inhibitor cocktail (ThermoFisher). After centrifugation (4°C, 13,000 rpm, 15 min), the supernatant was collected into a new 1.5 mL tube and stored at −80°C. Total proteins (15–30 μg) were resolved on 10–15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and electrotransferred with transfer buffer to polyvinylidene fluoride transfer membrane (Bio-Rad). Then, the membranes were blocked in TBST (10 mM Tris- HCl, pH 7.4, 150 mM NaCl, 0.1% Tween 20) containing 5% BSA and incubated with the appropriate primary and secondary antibodies. PSD-95, MAP2, GFAP, and Iba1 were detected in the membranes using anti-PSD95 (Abcam, ab18258), anti-MAP2 (Sigma-Aldrich, M9942), anti-GFAP (Millipore, MAB360), and anti-Iba1 (Invitrogen, PA5-27436), respectively. β-actin antibody (Sigma-Aldrich, A1978) was used as the standard. Protein bands were detected using the Clarity™ Western ECL Substrate (Bio-Rad). Bands were captured using Image Lab 6.0.1 (Bio-Rad). Densitometry analyses are presented as the ratio of protein to β-actin protein, which was compared with the controls and normalized.
Microscopic analysis and quantification
Primary hippocampal neurons were imaged using the confocal microscope (FV3000, Olympus) and observed with 20× and 60× objective lenses. All multiple optical sections spanning 10 µm in the z-dimension were collected (1-µm steps) and the optical sections were combined through the z-axis into a compressed z-stack; 3–4 images from randomly selected fields were taken. The PSD95 puncta was counted in primary dendrites within proximal dendrites (30). The puncta counting algorithm was performed using ImageJ software to quantify PSD95-positive puncta as previously described (31, 32). Briefly, the maximum z-projection PSD95 image was applied with the maximum z-projection MAP2 image using a threshold set of one standard deviation (SD) above the image mean. This 1-bit created projection was used as a mask with the PSD95 maximum z-projection. Structures between 8–80 pixels (approximately 0.4–4.0 μm in diameter) were counted as PSD95. The structures were then dilated on the MAP2 maximum z-projection for visualization. The PSD95 puncta counts were presented as the mean ± standard error of the mean (SEM), where n is the number of dendrites from six different cultures. The 20× objective MAP2 confocal images were analyzed to evaluate dendritic morphological changes in primary hippocampal neurons using Metamorph software (Molecular Devices).
Detection of cell viability based on CCK-8 assay
Protection against cell death was assayed using the CCK-8 kit (Dojindo). Primary hippocampal neurons (10,000 cells/well) were plated in 96-well plates and incubated for 24 h in 100 μL DMEM medium. Cells were seeded in triplicate for each treatment group. Kainic acid (150 μM) was added to the cells 30 min after, followed by 850 nm LED. Cells were incubated for an additional 24 h. Next, 10 μL CCK-8 solution was added to each well and the cell culture plate was incubated for 1–4 h. The absorbance at 450 nm was detected using a plate reader. Blank wells (culture media and CCK) and control wells (untreated cells, culture media, and CCK) were also detected.
RNA-sequencing data analysis
The quality of sequenced reads (paired-end) was estimated using FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and the sequenced reads were trimmed using Trim Galore (https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) with default settings. Trimmed reads were mapped to the mouse reference genome (mm10) using STAR (33) with default settings. Normalized expression levels of genes were calculated as transcripts per million (TPM) values using StringTie (34), and differentially expressed transcripts (adjusted p-value cutoff < 0.05 and |log2 fold-change| > 1) were identified using DESeq2 (35) after correcting potential batch-effects using RUVSeq (36).
Gene ontology analysis
Gene ontology analysis was performed with the Metascape application (https://metascape.org/gp/index.html#/main/step1) (37). Upregulated or downregulated differentially expressed transcripts were used as input.
Behavioral tests
Seven days after PI injection, mice were subjected to behavioral tests in the following order: OFT, novel object recognition test, and Y-maze spontaneous alternation. All behavioral tests occurred during the light phase. Mice were allowed to acclimate to each behavioral test once per day, for 3 days, 1 week before the behavioral experiment. The experiment was only performed if no motor seizure was observed for at least 1 h before the test. Prior to behavioral tests, mice were acclimated to the procedure room for at least 1 h. Cages were then moved to an adjacent room and mice were transported to the procedure room for tests one at a time. After tests were completed, mice were returned to their home cage and the maze was cleaned using a 70% EtOH solution between trials. All behavior tests were recorded using a mounted video camera and analyzed with EthovisionXT 12 software (Noldus Information Technology). After behavioral tests, mice were used in other experiments including immunohistochemistry, histology, and western blot.
Open field test
Locomotor and explorative activities and anxiety-like behavior were measured using the OFT, which was conducted in an opaque-sided box (measuring 50 cm long × 50 cm wide × 50 cm high) under indirect low illumination. Mice were individually placed in the center of the box and allowed to explore the maze for 5 min. Total distance traveled and number of line crossings were recorded for analysis of locomotor and explorative activities. Time spent in the center zone was recorded with percentage time spent in the center zone/(center + peripheral side zone) of the OFT used as a measure of anxiety-like behavior.
Novel object recognition test
The novel object recognition test was used to analyze the effects of tPBM on recognition memory. During habituation, all mice were introduced into an empty opaque-sided box (measuring 50 cm long × 50 cm wide × 50 cm high) for 10 min on the first day of testing. On the next day (training), the mice were exposed to two identical objects placed in the arena for 10 min. On test day (24 h later), the mice were allowed to explore one of the familiar objects and a novel object for 10 min. The time spent exploring the two objects was scored by the blinded observer using video-recorded sessions. Exploration was defined as pointing the head toward an object at a distance < 2 cm from the object. The exploration time was defined as the percentage of time that mice spent exploring familiar or novel objects with respect to the total exploration time.
Y maze spontaneous alternation
Working memory was assessed using the Y-maze spontaneous alternation test. The Y-maze apparatus consisted of three arms made of white plastic joined in the middle to form a “Y” shape. The diagonal walls of the identical arms were 15-cm-high from the ground, allowing the mouse to see distal spatial landmarks. This ethologically relevant test is based on the rodents’ innate curiosity to explore novel areas and presents no negative or positive stimulators and very little stress for the mice. The Y maze design was based on published protocols with modifications to adapt the system to mice. Briefly, mice were placed into the center of the maze and allowed to explore the maze for 3 days (habituation). The next day, mice were returned to the Y maze by placing them in the center of the maze. Then, the mice were allowed to freely explore all three arms of the maze for 5 min (test trial). Due to habituation, the maze was likely to be less stressful during the test trial. The recording of entry to each arm during trials was considered spontaneous alternation. Mice were subjected to a three-arm Y maze for 10 min with all three arms open. The number and the sequence of arms entered were recorded. The dependent variables were activity, defined as the number of arms entered, and percent alternation, calculated as the number of alternations (entries into three different arms consecutively) divided by the total possible alternations (i.e., the number of arms entered minus 2) and multiplied by 100.
Statistics
Statistical analyses were performed using Prism 7.0 (GraphPad). All experimental data are presented as means ± SEM. A P value less than 0.05 was considered significant. Detailed statistical information for all Figures is described in Table 3.