All experimental procedures were approved by the Institutional Animal Care and Use Committee at Loma Linda University. All studies were conducted in accordance with the United States Public Health Service’s Policy on Humane Care and Use of Laboratory Animals and reported according to the ARRIVE guidelines. One hundred and sixty-four P7 Sprague-Dawley neonatal pups (weight = 12–14 g, Harlan, Livermore, CA) were randomly divided into Sham (n = 34) and GMH (n = 130) groups (Supplementary table 1). All pups were housed with controlled temperature and 12-hour light/dark cycle, and given ad libitum access to food and water. Neither collagenase-induced GMH nor administration of rh-relaxin-2 caused mortality in this study. Investigators were blinded to the experimental groups when performing neurological tests, immunofluorescence, toluidine blue staining and quantitation Western blot density.
Gmh Model And Experimental Protocol
The procedure for the GMH model in unsexed P7 rats using collagenase infusion was performed as previously described (14). Briefly, pups were anesthetized with isoflurane (3.0% induction, 1-1.5% maintenance on a stereotaxic frame. After the skin was incised on the longitudinal plane and the bregma was exposed, a 27-gauge needle with 0.3U clostridial collagenase (0.3 units of clostridial collagenase VII-S, Sigma-Aldrich, MO) was inserted (2.7 mm deep from the dura) through a burr hole drilled on the skull (1.6 mm lateral, 1.5 mm anterior to the Bregma) and infused (1 µl/min) using a 10 µl Hamilton syringe (Hamilton Co, Reno, NV, USA) guided by a microinfusion pump (Harvard Apparatus, Holliston, MA). The needle was kept in place for an extra 10 min to avoid leakage and withdrawn at a speed of 0.5 mm/min. The pups were placed back to a heated blanket after infusion and euthanized at different time points according to the experimental design.
Intracerebroventricular drug administration was performed as previously described (15). Briefly, rats were placed in a stereotaxic apparatus under 2.5% isoflurane stereotactic anesthesia. The scalp area was sterilized, and bregma was exposed. Using bregma as a reference point, the following stereotactic coordinates were measured: 1.0 (rostral) and 1.0 mm (lateral). A bur hole (1 mm) was drilled. A 27-gauge needle was inserted at a rate of 1 mm/min at the depth of 1.7 mm from the dura, where the lateral ventricle is located.
Recombinant human relaxin-2 (rh-relaxin-2, Sigma-Adrich) was dissolved within phosphate-buffered saline (PBS). Pups were administered at different dosages (30 µg /kg, 60 µg/kg and 90 µg/kg) or PBS via intraperitoneal injection at 1 hour after GMH and then once again after 12 hours.
Rat-derived RXFP1 siRNA (0.5 nm/2 µl, Thermo Fisher), Scramble siRNA (0.5 nm/2 µl, Thermo Fisher) and clodronate liposome (15 µg/3 µl/rat, Encapsula Nano Sciences) were infused via intracerebroventricular injection (i.c.v.) at 24 hours prior to GMH induction (1.0 mm anterior, 1.0 mm lateral to the bregma and 1.7 mm deep on the contralateral hemisphere).
Phosphotidylinsitol-3-Kinase (PI3K) inhibitor LY294002 (50 mM, 2 µl, Sigma) was infused i.c.v. at 1 hour prior to GMH induction (1.0 mm anterior, 1.0 mm lateral to the Bregma and 1.7 mm deep on the contralateral hemisphere).
Experiment 1. The time course of endogenous relaxin-2, its receptor RXFP1, and the mast cells markers chymase and tryptase in the whole brain at 0.5, 1, 3, 5, and 7 days after GMH was analyzed by Western blot. The cellular localization of receptor RXFP1 and tumor necrosis factor-α induced protein 3 (TNFAIP3) was detected at 1 day after GMH by double immunofluorescence staining on mast cells. Thirty-six rat whole brains were collected at 0 (naive), 0.5, 1, 3, 5 and 7 days after GMH for Western blot (Supplementary Fig. 2).
Experiment 2. The outcome of rh-relaxin-2 treatment was assessed on the first three days and between 21 and 28 days after GMH. The pups were randomly divided into 5 groups: Sham, GMH + PBS, GMH + rh-relaxin-2 (30 µg/kg), GMH + rh-relaxin-2 (60 µg/kg), GMH + rh-relaxin-2 (90 µg/kg). Exogenous rh-relaxin-2 (Sigma) was dissolved in phosphate-buffered saline (PBS) and administered in a total volume of 60 µl intraperitoneally at 1 hour and 13 hours after GMH. Short-term (negative geotaxis and body righting reflex) and long-term (rotarod test, foot fault and water maze) neurological tests were examined during the first 3 days and between 21 and 28 days after GMH, respectively. The Nissl staining was also performed between 21 and 28 days after GMH (Supplementary Fig. 2).
Experiment 3. To evaluate the mast cells activation, the number of mast cells was quantified in the perihematoma area and thalamus on the first day after GMH by toluidine staining. Eighteen pups were divided into sham (n = 6), GMH + vehicle (n = 6), and GMH + rh-relaxin-2 (60 µg/kg, n = 6) (Supplementary Fig. 2).
Experiment 4. To evaluate the effect of RXFP1 in vivo on mast cell degranulation after administration of rh-relaxin-2 post-GMH. Clodronate liposome was administered intracerebroventricularly on the left side of brain to inhibit the microglial activation at 24 hours prior to GMH induction. Meanwhile, RXFP1 small interfering RNA (RXFP1 siRNA) and scramble siRNA (Scr siRNA) were also infused via intracerebroventricular injection (i.c.v.) on the right side of the brain. The whole brain samples were collected to conduct Western blot on the first day after GMH. The pups were randomly divided into six groups: Sham, GMH + Vehicle, GMH + Vehicle + clodronate liposome, GMH + clodronate liposome + rh-relaxin-2 (i.p. 60 µg/kg), GMH + clodronate liposome + rh-relaxin-2 (i.p. 60 µg/kg) + Scr siRNA and GMH + clodronate liposome + rh-relaxin-2 (i.p. 60 µg/kg) + RXFP1 siRNA (Supplementary Fig. 2).
Experiment 5. To assess the role of PI3K-AKT pathway in vivo on mast cell degranulation after administration of rh-relaxin-2 post-GMH. Clodronate liposome was administered intracerebroventricularly on the left side of the brain to inhibit the microglial activation at 24 hours prior to GMH induction. At the same time, LY294002 was administered via intracerebroventricular injection at 1 hour on the left side of the brain prior to GMH induction. The whole brains were collected for Western blot on the first day after GMH. The pups were divided randomly into Sham, GMH + Vehicle, GMH + Vehicle + clodronate liposome, GMH + clodronate liposome + rh-relaxin-2 (i.p. 60 µg/kg), GMH + clodronate liposome + rh-relaxin-2 (i.p. 60 µg/kg) + DMSO and GMH + clodronate liposome + rh-relaxin-2 (i.p. 60 µg/kg) + LY294002 (Supplementary Fig. 2) .
Double fluorescence staining was performed as described previously (16). Sections were blocked with 5% donkey serum for 1 hour and incubated at 4 °C overnight with primary antibodies: rabbit anti-RXFP1 (1:100, biorbyt, orb157275 ), mouse anti-tryptase (1:200, Abcam, ab2378), mouse anti-chymase (1:200, santa cruz, sc-59586), and rabbit anti-TNFAIP3 (1:100, lifespan, LS-C352948) followed by incubation with appropriate fluorescence-conjugated secondary antibodies for 2 hours at room temperature. Negative control staining was performed by omitting the primary antibody. Fluorescence microscopy and LASX software were used to image the sections (Leica DMi8; Leica Microsystems, Wetzlar, Germany).
Neurological tests were performed in a random and blinded setup as previously reported (17). Short-term neurological tests, namely negative geotaxis and righting reflex, were conducted from Day 1 to Day 3 after GMH. Long-term neurological tests, including rotarod, foot fault and water maze, were performed from Day 21 to Day 28 after GMH.
In detail, Negative geotaxis was tested to record the duration of the pups to turn 90° and 180° when positioned head downward on a 45° inclined plane. The maximum recording time was 60 s (three trials/pup/day). For righting reflex, the pups were placed on the back onto a horizontal plane, and the time needed for the pup to right itself in a prone position on its four paws was recorded. The maximum recording time was 20 s (three trials/pup/day).
Foot fault test was recorded as the total numbers of missteps. When the animal's forelimb or hind limb fell into one of the grid openings, a foot fault was recorded. The maximum recording time was 60 s.
In rotarod test, the animals were placed on a rotating wheel (Columbus Instruments) and tested at a starting speed of 5-RMP and 10 RMP with acceleration at 2RPM per 5 s. The time latency for the animals to remained on the rotating wheel and the speed at which animals fell down from the rotarod were measured and averaged from 3 repeated trials.
The water maze test used a circular pool (diameter: 110 cm) filled with water at 24 ± 1℃. A transparent escape platform (diameter: 11 cm) was submerged 1 cm beneath the water and placed at a fixed position at the center of one of the quadrants. On Day 6, a probe trial was performed to assess spatial memory retention. During this trial, animals were allowed to swim freely for 60 s, but no platform was present. Swim distance, latency, velocity and the percentage of time in target quadrant were digitally recorded and analyzed by a tracking software (Noldus Ethovision).
Frozen sections were stained in toluidine blue working solution for 2–3 minutes. Sections were dehydrated quickly through 95% and 2 changes of 100% alcohol (10 dips in each since stain fades quickly in alcohol) after being washed in distilled water for 3 times. Finally, sections were cleared in xylene and covered with resinous mounting medium.
Nissl staining was conducted and analyzed as previously (4, 18). Brain sections were dehydrated in 95% and 70% ethanol for 2 min, and then washed in distilled water for 2 min. Sections were stained with 0.5% cresyl violet (Sigma-Aldrich, USA) for 2 min and washed in distilled water for 10 s followed by dehydration with 100% ethanol and xylene for 2 min twice respectively before a coverslip with permount was placed. The volume of ventricular, gray matter loss, relative cortical thickness and relative white matter area were calculated with Image J 4.0 (Media Cybernetics). Calculations were performed in a blinded fashion.
Brain tissues were collected and stored in -80 oC freezer after being perfused with cold PBS (0.1M, pH 7.4). Western blot was performed as described previously (14) (19). After extraction of protein samples, protein quantification was performed using Lowry methodology (BioRad, USA). Each sample containing 50 µg of protein were separated by SDS-PAGE gel electrophoresis, and then transferred onto nitrocellulose membranes. Membranes were blocked with 5% milk and incubated with the following primary antibodies overnight at 4 °C: rabbit anti-RXFP1 antibody (biorbyt, USA, orb157275), rabbit anti-Relaxin 2 Antibody (invitrogen, USA, PA5-76483), mouse anti-Mast Cell Tryptase antibody (abcam, USA, ab2378), rabbit anti-Mast Cell chymase antibody (santa cruz, USA, sc-59586), rabbit anti PI3K (CST, USA, #4249), rabbit anti phospho-AKT (CST, USA, #9271s), rabbit anti-AKT(CST, USA, #9272), rabbit anti TNFAIP3 Antibody (lifespan, USA, LS-C352948), rabbit anti-NF-κB (Novusbio, USA, NBP1-87760), rabbit anti-phospho-NF-κB (CST, USA, #3033S), rabbit anti IL-6 (Abcam, USA, ab9324), rabbit anti TNF-α (Abcam, USA, ab9755), goat anti-β-actin (Santa Cruz Biotechnology, USA, sc-1616). β-actin was used as the internal loading control. Then, membranes were incubated with horseradish-peroxidase conjugated secondary antibodies for 1 h at room temperature. Membranes were probed with an ECL Plus chemiluminescence reagent kit (Amersham Biosciences, USA). The relative density of protein was analyzed by ImageJ software (ImageJ 1.5, NIH, USA).
All data were presented as a mean ± SD. All analyses were performed using GraphPad Prism 6 (GraphPad software). Normal distribution was first confirmed using the ShapiroWilk normality test. For the data that passed the normality test, the statistical differences among groups were further analyzed using one-way ANOVA followed by Tukey’s multiple comparison post-hoc analysis. For the data that failed the normality test, Kruskal-Wallis one-way ANOVA on Ranks was used, followed by Tukey’s multiple comparison post hoc analysis. P < 0.05 was considered statistically significant.