PEMFs were generated using a system composed of a pulsed signal generator and two-array Helmholtz coils, as shown in Figure 1 (GHY-III, FMMU, Xi`an, China; Chinese patent no. ZL02224739.4). The waveforms generated by the PEMF system consisted of a pulse burst (burst width, 5 ms; pulse width, 0.2 ms; pulse wait, 0.02 ms; burst wait, 60 ms; pulse rise, 0.3 μs; pulse fall, 2.0 μs) repeated at 15 Hz as described in our previous studies35. The distance between the two-array coils (20 cm diameter) was 10 cm, the turn number of the enamel-coated copper wire was 80, and the diameter of the copper wire was 1.0 mm. An oscilloscope was connected to the two sides of a 2-Ω resistor that was connected in series with the coils to display the voltage-current waveform. The current waveform could be obtained by dividing the voltage waveform by the 2-Ω resistor value. The current flowing through the resistor and the Helmholtz coils was the same. The waveform of the output magnetic fields was obtained on the basis of the current waveform. The determined peak intensity of the PEMF of the coils was 5 Gauss (0.5 mT). The bottom of the culture plates containing BV2 cells was aligned with the center of the coils. The PEMF treatment group received PEMF treatment immediately after red blood cell (RBC)- or Hb-induced injury for 4 hours, and the cells were collected after 24 hours. The untreated group was placed in a different chamber under the same conditions with inactivated Helmholtz coils.
For the animal study, the mice that underwent ICH surgery were placed in the center of the coils 4 hours after surgery and for 4 hours per day. The untreated ICH group mice were placed in a different chamber with inactivated Helmholtz coils.
The experimental protocol was approved by the Animal Care and Use Committee of Air Force Medical University. A total of 100 8- to 10-week-old C57BL/6J mice were purchased from the Animal Center of Air Force Medical University. The mice were group-housed in individually ventilated cages on a 12-hour light-dark cycle with ad libitum access to standard mouse feed and water.
The collagenase injection ICH model was constructed as described previously. After the mice were anesthetized, 1 ml 0.03 U type IV-S collagenase (Sigma-Aldrich) was injected into the right striatum (coordinates: -0.2 mm anterior, 2.5 mm lateral, 3.5 mm deep; relative to bregma). A heating pad was applied to maintain a rectal temperature of 37.0 °C. The animals were returned to their cages after recovery from anesthesia. PEMF treatment was applied 4 hours after ICH and for 4 hours per day for 7 days. On day 3 and day 7, all mice were subjected to the corner turn test and forelimb use asymmetry test.
Western blot analysis
The protocol was the same as that described in previous articles. Briefly, the protein samples were quantified by the BCA method. Then, equal amount of the samples (50 mg) were loaded and subjected to electrophoresis on an SDS-PAGE gel. After the proteins were transferred to a membrane, the PVDF membrane was blocked with 5% fat-free milk and then incubated with primary antibody overnight at 4°C. The images were analyzed with ImageJ software.
After anesthesia, mice were perfused with ice-cold 0.01 M phosphate-buffered saline (PBS) and then with 4% paraformaldehyde. The brains were collected and postfixed for another 4 hours and then dehydrated with 30% sucrose. Frozen coronal slices (30 mm thick) were obtained with a freezing microtome. The sections were washed with 0.01 M PBS and blocked with 5% normal goat serum for 30 minutes. Then, they were incubated with primary antibody (CD36, Iba1) at 4 °C overnight and washed three times with 0.01 M PBS. We applied appropriate secondary antibodies at room temperature for 2 hours. Antifade solution with or without DAPI was applied, and the sections were observed with an Olympus microscope after they were completely dried.
BV2 cell erythrophagocytosis was evaluated as previously described21. For the microglia with CD36 or IgG group, CD36 neutralized antibody or IgG were pretreated on each cell (2 mg/ml) 4 hours before Erythrophagocytosis assay. Briefly, RBCs (2´107) were isolated from the whole blood of donor mice. Then, they were washed with 0.01 M PBS, centrifuged at 750 rpm/min for 5 minutes, resuspended in diluent, and stained with PKH-26 for 4 minutes. Serum was applied for 1 minute to stop the staining process. Then, the PKH-26-labeled RBCs were washed three times to remove the residual dye. The cells were counted for further experiments and added to BV2 cells, and erythrophagocytosis was assessed.
The cells were suspended at a density of 106 cells/ml and seeded in 24-well plates. The cells were incubated for 24 hours in the absence or in the presence of Hb (20 mM). Mouse brain was collected at the end of treatment, and suspended cells were collected and centrifuged at 1000 × g for 10 minutes. The levels of the proinflammatory cytokines TNF-a and IL-1b were determined with a specific quantitative sandwich ELISA kit according to the manufacturer's instructions. The reaction was developed with streptavidin-horseradish peroxidase, and the optical density was read at a wavelength of 450 nm.
Hematoma volume measurement
On day 3 and day 7 after ICH, the mice were perfused with 0.01 M PBS 50 ml after being anesthetized. Their brains were collected and sectioned with 1-mm coronal mouse brain matrices. The brain slices were digitalized and analyzed, and the cubic volume of the hematoma was calculated using ImageJ in a blinded manner.
For quantification of neuronal apoptosis, TUNEL (green) staining was performed using the In Situ Apoptosis Detection Kit (Roche, Indianapolis, IN, USA) according to the manufacturer’s instructions at 72 hours after ICH. The number of TUNEL-positive neurons in the perihematomal area in six sections per brain at 20× magnification was counted manually using ImageJ software (ImageJ 1.4, NIH). The data are expressed as the number of TUNEL-positive cells.
Corner turn test
Each mouse was placed in a 30° corner, and the direction that the mouse exited from the corner was recorded 10 times. The percentage of right turns was calculated. The behavioral tests were conducted by a blinded investigator.
Forelimb use asymmetry
A forelimb use asymmetry test was performed as previously described. The mouse was placed in a cylinder (15 cm ´ 9 cm), and forelimb usage during at least 15 exploratory movements (less than 20) within a period of up to 10 minutes was recorded. The forelimb use asymmetry score was calculated as [I-C]/[I+C+B] ´ 100, where I is the number of times the ipsilateral forelimb was used, C is the number of times the contralateral forelimb was used, and B is the number of times the bilateral forelimbs were used.
Transcriptome resequencing analysis
The mice were divided into four groups: the sham, PEMF, ICH, and ICH with PEMF groups (n = 3). After 3 days, the ipsilateral brain hemispheres of all mice were collected for RNA-seq. Total RNA was extracted using a TRIzol reagent kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. RNA quality was assessed with an Agilent 2100 Bioanalyzer and checked using RNase-free agarose gel electrophoresis. After total RNA was extracted, eukaryotic mRNA was enriched by oligo(dT) beads, while prokaryotic mRNA was enriched by rRNA removal with a Ribo-ZeroTM Magnetic Kit. Then, the enriched mRNA was fragmented into short fragments using fragmentation buffer and reverse transcribed into cDNA with random primers. Second-strand cDNA was synthesized by DNA polymerase I, RNase H, dNTPs, and buffer. Then, the cDNA fragments were purified with a QiaQuick PCR extraction kit (Qiagen, Venlo, The Netherlands), end-repaired, poly(A) added, and ligated to Illumina sequencing adapters. The ligation products were size selected by agarose gel electrophoresis and PCR amplified. Finally, the sample library products were sequenced using an Illumina NovaSeq6000 device (Gene Denovo Biotechnology Corporation, Guangzhou, China).
The data are expressed as the mean±SD of at least three independent experiments. When relative values were compared between the sham, ICH, and ICH with PEMF groups, statistical analysis was performed by one-way ANOVA. In all cases, P values of <0.05 and <0.01 were considered significant and are indicated as * and #, respectively. When values between two groups were compared, statistical significance was assessed using paired two-way repeated-measures ANOVA. Statistical analysis was performed using Prism 6.0.