TSG6 null (TSG6-/-) or heterozygous (TSG6+/-) mice and animal maintenance
Transgenic Tsg-6 null mice (Tnfip6D/D), hereafter referred to as Tsg-6-/- mice, and heterozygous mice, hereafter referred to as Tsg-6+/- mice, were maintained as previously described (52). Our previously published work demonstrated that Tsg-6+/- mice present a similar distribution of astrocytes throughout the brain to wt mice (42). Moreover, Tsg-6+/- mice have previously been shown not to display a phenotype and present similar TSG-6 expression levels as wt mice, and, therefore, were used as littermate controls in our study (52). Experimental procedures for handling the mice were approved by the Institutional Animal Care and Use Committee (IACUC), University of Houston under protocol 16-036.
Mice (7 to 8 weeks old) were anesthetized with ketamine (80-100 mg/kg - Vedco INC, Catalog# 07-890-8598) and xylazine (5-10 mg/kg, Akorn INC, Catalog# 07-808-1947) by IP injection and allowed to go into full anesthetic state. A sterile surgical drill (Precision Tools, Model Craft PPV2237) was used to make a hole of approximately 1.5 mm in diameter in the skull over the right frontal cortex at the stereotaxic coordinates AP: 1.0mm, ML: 1.5mm, and DV: 1.5mm, according to Franklin and Paxinos (62). A 30-gauge needle (Exel, Catalog# 26437) was then used to make a puncture wound at a depth of 2 mm. After injury, the skin at the surgical site was closed with two sutures. The area was then cleaned with 70% ethanol, and mice were placed on a heating pad and monitored until they regained consciousness prior to being transferred to a clean cage. All surgeries were carried out at the same time of day to minimize bias. Mice were monitored daily and did not show any decrease in weight ≥ 15% when compared to their pre-surgical weight. Mice were euthanized at 1, 3 and 5 days post injury to study the acute effects of brain injury, and at 10 and 14 days to study long-term/chronic effects. Five mice per experimental group were used for the real-time PCR analysis and at least seven mice per experimental group were used for immunofluorescence analysis.
Perfusion fixation and brain tissue processing
Brain samples were collected at 1, 3, 5, 10, and 14 days post injury for immunofluorescence analyses. Briefly, mice were initially injected with a lethal dose of combined anesthetics, ketamine and xylazine. Once mice were under deep anesthesia, abdominal excisions were performed to expose the heart, which was used to perfuse 2% formalin (Fisher Scientific, Catalog# SF100-4) throughout the whole body via a gravity-driven flow system for whole body fixation. Subsequently, the brain was isolated from the skull and further immerse fixed for 2 days in 2% paraformaldehyde (Electron Microscopy Sciences, Catalog# 15710). For cryosection processing, brains were immersed in 30% sucrose for 2 days, embedded in OCT embedding medium (Fisher Healthcare, Catalog# 4585) and frozen. Sections 10 μm thick were obtained, mounted on superfrost slides (VWR, Catalog# 48311-703) and stored at -20°C until use.
Upon use, the slides were heated at 65oC for 30 minutes and, subsequently, sections were washed with PBS to remove tissue freezing medium. Sections were then treated with 0.1% glycine (Fisher Chemical, Catalog# G46-500), blocked with 5% FBS (Seradigm, Catalog# 3100-500) and permeabilized with 0.1% saponin prepared in PBS. Sections were then incubated with the primary antibodies anti-Tenascin (Abcam, Ab108930), anti-GFAP (Abcam, Ab4647), anti-CD68 (Ab31630, Abcam) and anti-b III tubulin (Ab18207, Abcam). Sections were washed and incubated with appropriate secondary donkey antibodies conjugated with Alexa Fluor® 488 (Life Technologies) or Alexa Fluor® 555 (Life Technologies) for one hour at 18oC. For HA staining, tissues were incubated with biotinylated HA binding protein (385911, Millipore) followed by NeutrAvidin®Alexa 555 (Life Technologies). The tissues were then washed and nuclei stained with 4’,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich). Sections were mounted in Prolong®Gold (Molecular Probes) and imaged using a ZEISS LSM 800 Confocal microscope with Airyscan. Secondary controls were done with a goat IgG isotype control (ab37388; Abcam) in place of the primary antibody and did not yield any significant staining (results not shown). For imaging, multiple z-stack tiles were captured of entire brain sections and frames were processed together into a single image (using the stitching mode followed by full orthogonal projection) using Zen Software (Zeiss). The number of GFAP+ and CD68+ cells in and around the injury site were counted by two independent investigators in a blinded manner and the relative Fluorescent intensity was measured using the Zen Software (Zeiss). At least 2 sections were scanned and analyzed from each animal for each set of antibodies and representative images shown in the figures.
RNA extraction from brains and real-time PCR analysis
Brains collected from injured mice at 1, 5 and 10 days post injury were used for RNA extraction. At least 5 mice were used per experimental group and each animal was analyzed separately. Briefly, mice were euthanized and brain tissue was immediately isolated from each mouse. Injury sites (A samples) were dissected from the rest of the injured right hemisphere, transferred into a labeled Eppendorf tube and immediately immersed in liquid nitrogen. The remaining right hemisphere brain tissue (B samples) from each animal was transferred into a different tube and also frozen as described. The samples were kept at -80oC until RNA extraction. Total RNA was isolated from these tissue samples using Trizol® Reagent (Invitrogen, Carlsbad, CA) and chloroform extraction (Sigma-Aldrich, Catalog# 650498). First strand cDNA was reverse transcribed using 1.5 to 2 μg of total RNA and the high capacity cDNA Reverse Transcription kit (Applied Biosystems, catalog# 4368814, lot 00593854) according to the manufacturer’s instructions. Quantitative real-time PCR amplification was performed on 1 μl or 50 ng of the cDNA (1:5) using the PowerUp SYBR Green Master Mix kit (Applied Biosystems, Catalog# A25918) in a CXF Connect Real-time System from BIO-RAD, using an activation cycle of 95°C for 10 min, 40 cycles of 95°C for 15 seconds and 60°C for 1 min. A complete list of primers used in this study is shown in Table 1. Gene expression levels were normalized against Actb and Gapdh using the 2−ΔCt and/or 2−ΔΔCt methods.
All values are presented as the mean ± standard deviation of the mean. The difference between the two groups was compared by means of the Student’s t-test. P ≤ 0.05 was considered to be statistically significant. Statistical analysis was performed using the GraphPad Prism version 7 software package (GraphPad Software, San Diego, CA, USA). * was used to indicate the statistical differences of ≤ 0.05. Unless indicated otherwise, * indicates the statistical difference of Tsg-6-/- mice compared to Tsg-6+/- mice.