GW0742 reduces mast cells degranulation and attenuates neurological impairments via PPAR_β/δ/CD300a/SHP1 pathway after GMH in neonatal rats

Abstract

Background

Activation of mast cells plays an important role in brain inflammation. CD300a, an inhibitory receptor located on mast cell surfaces, has been reported to reduce the production of pro-inflammatory cytokines and exert protective effects in inflammation-related diseases. Peroxisome proliferator–activated receptor β/δ (PPAR_β/δ), a ligand-activated nuclear receptor, activation upregulates the transcription of CD300a. In this study, we aim to investigate the role of PPAR_β/δ in the attenuation of germinal matrix hemorrhage (GMH)-induced mast cell activation via CD300a/SHP1 pathway.

Methods

GMH model was induced by intraparenchymal injection of bacterial collagenase into the right hemispheric ganglionic eminence in P7 Sprague Dawley rats. GW0742, a PPAR_β/δ agonist, was administered intranasally at 1 hour post-ictus. CD300a small interfering RNA (siRNA) and PPAR_β/δ siRNA were injected intracerebroventricularly 5 days and 2 days before GMH induction. Behavioral tests, Western blot, immunofluorescence, Toluidine Blue staining, and Nissl staining were applied to assess post-GMH evaluation.

Results

Results demonstrated that endogenous protein levels of PPAR_β/δ and CD300a were decreased, whereas chymase, tryptase, IL-17A and transforming growth factor β1 (TGF-β1) were elevated after GMH. GMH induced significant short- and long-term neurobehavioral deficits in rat pups. GW0742 decreased mast cell degranulation, improved neurological outcomes, and attenuated ventriculomegaly after GMH. Additionally, GW0742 increased expression of PPAR_β/δ, CD300a and phosphorylation of SHP1, decreased phosphorylation of Syk, chymase, tryptase, IL-17A and TGF-β1 levels. PPAR_β/δ siRNA and CD300a siRNA abolished the beneficial effects of GW0742.

Conclusions

GW0742 inhibited mast cell-induced inflammation and improved neurobehavior after GMH, which is mediated by PPAR_β/δ/CD300a/SHP1 pathway. GW0742 may serve as a potential treatment to reduce brain injury for GMH patients.

Background

Germinal matrix hemorrhage (GMH) is a leading cause of morbidity and mortality in premature infants. After GMH, secondary brain injury is brought on by the activation of inflammatory cascades, which leads to severe neurological and cognitive deficits in this patient population¹. To date, no effective therapeutic option is currently available to treat GMH. Therefore, suppression of neuroinflammatory response could be a potential therapeutic strategy for GMH treatment.

With a growing understanding of mast cells and their function, we have learned that they play an integral role in inflammatory disorders such as allergies, asthma, and immune diseases². In the brain, mast cells can be found in meninges, choroid plexus, olfactory bulb, hippocampus, parenchyma of thalamic and hypothalamic region, where they reside on the abluminal side of blood vessels and communicate with glial cells, endothelial cells and neurons³. Resting mast cells are characterized by the presence of biological preactivated granules, including histamine, chymase, tryptase, and tumor necrosis factor β (TNF-β) in their cytoplasm. Upon activation by physical and chemical stimuli, mast cells release granules and newly synthesized neuroinflammatory molecules such as leukotrienes, prostaglandins, thromboxanes, and platelet-activating factor (PAF). In brain, the increased mast cell activation and degranulation results in the onset and progression of neuroinflammation, blood-brain barrier (BBB) disruption, plasma extravasation, and neurodegeneration⁴.

Increasing evidence has shed light on the close relationship between mast cells and various central nervous system neuroinflammatory disorders, such as neuropathic pain, depression, stroke, multiple sclerosis, Austin, Parkinson’s disease, and Alzheimer’s disease^5–8. Since mast cell activation and neuroinflammation play a joint role in secondary brain injury, identifying specific molecules able to inhibit mast cell degranulation and stabilize mast cell activity may serve as a potential neuroprotective strategy to treat GMH⁸.

CD300a inhibitory receptor, part of the CD300 glycoprotein family, is found on cell surface of mast cells, with an IgV-like extracellular region and a consensus sequence for immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic tail⁹. In vivo and in vitro studies reveal that CD300a functions as an inhibitory receptor and exerts anti-inflammatory function¹⁰. The binding of CD300a to ligands leads to the phosphorylation of tyrosine residues in the ITIM domains. After tyrosine phosphorylation, ITIM is able to recruit phosphatases that mediate inhibitory signals in mast cell, which include SH2 domain-containing inositol 5-phosphatase (SHIP), SH2 domain-containing protein tyrosine phosphatase 1 (SHP-1), and SHP-2^{9, 11, 12}. Numerous evidences demonstrate that SHP1 can suppress spleen tyrosine kinase (Syk), an important signaling molecule in FcεRI-triggered mast cell activation, and downstream signaling pathway in the mast cell regulation^13–15. Recently, activation of SHP1 to inhibit Syk has been considered as a useful therapeutic strategy against mast cell-driven diseases¹⁶.

Thereby, activating CD300a may serve as a potential therapeutic target for the treatment of mast cell-associated inflammatory diseases^{10, 12, 17}. Until recently, the role of CD300a in mast cell activation after GMH remains unclear.

Peroxisome proliferator–activated receptor β/δ (PPAR_β/δ) is a ligand-activated nuclear receptor that belongs to the PPARs subfamily¹⁸. Recent studies revealed reported that PPAR_β/δ upregulates the transcription of many target genes, which include CD300a^{19, 20}. PPAR_β/δ has been shown to be associated with inflammatory regulation and plays a protective role in stroke, cerebral ischemia, spinal cord injury and Parkinson’s disease²¹.

Based on these observations, we hypothesized that PPAR_β/δ agonist GW0742 treatment would reduce mast cell degranulation and promote the release of pro-inflammatory cytokines, alleviate neurological deficits, and post-hemorrhagic hydrocephalus after the induction of GMH. We postulated that the beneficial effects of GW0742 might be meditated by PPAR_β/δ/CD300a/SHP1 pathway (Fig 9).

Fig. 9.

Effect of CD300a siRNA on long-term neurological function at 21–28 days after GMH with GW0742 treatment. CD300a siRNA decreased GW0742-treated GMH pup’s memory functions as shown by more swim distance (a), escape latency (b) and less time in the target quadrant (c, d) in the Morris water maze. CD300a siRNA induced deficits in rotarod tests (e, f) and foot fault tests (g). Values are expressed as mean±SD. * P < 0.05 vs. Scr siRNA, n = 8 each group.

Materials and methods

Animals

All experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of 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 complied with the Animal Research Reporting in Vivo Experiments (ARRIVE) guidelines. One hundred and forty-two Sprague-Dawley neonatal pups (weight 12–14g, Harlan, Livermore, CA) of both genders were randomly subjected to either sham (n = 34) or GMH (n = 108) group. All animals were housed in rooms with controlled temperature and 12-hour light/dark cycle and given ample access to food and water.

Experimental design

Total of 6 separate experiments were performed as follows:

Experiment I

To determine the time course of CD300a, PPAR_β/δ, chymase, tryptase, interleukin 17 (IL-17A) and TGF-β1 levels in the brain, western blot was performed to measure protein expression levels at 6 hours, 24 hours, 3 days, and at 7 days post-ictus. Thirty-six rats were numbered and randomly divided into 6 groups: sham-6h (n = 6), GMH-6h (n = 6), GMH-12h (n = 6), GMH-24h (n = 6), GMH-3d (n = 6) and GMH-5d (n = 6).

Experiment II

A dose response study was conducted to elucidate the optimal dosage of GW0742 after GMH. Righting reflex and negative geotaxis tests were performed for 3 consecutive days after surgery. Thirty rats were randomly divided into five groups: sham (n = 6), GMH + Vehicle (n = 6), GMH + GW0742 (10 μg/kg) (n = 6), GMH + GW0742 (30 μg/kg) (n = 6) and GMH + GW0742 (90 μg/kg) (n = 6). GW0742 was administered intranasally (i.n.) at 1 hour post-GMH.

Experiment III

Assess the effects of intranasal GW0742 administration on the expression of CD300a and PPAR_β/δ and the mast cell degranulation at 3 days after GMH. Eighteen rats were randomly divided into sham (n = 6), GMH + Vehicle (n = 6) and GMH + GW0742 (30 μg/kg) (n = 6). The CD300a and PPAR_β/δ cellular localization were detected via immunofluorescence. The mast cell degranulation was detected by Toluidine Blue staining assay at the 3rd after GMH.

Experiment IV

Assess the long-term effects of intranasal GW0742 administration on neurobehavioral function and post-hemorrhagic hydrocephalus after GMH. Long-term neurobehavioral tests, include Water Maze test, rotarod test and foot fault test, were performed at 21–28 days after GMH. Nissl staining was applied at the 28 days after GMH to assess the ventricular volume. Twenty-four rats were randomly divided into three groups: sham (n = 8), GMH + Vehicle (n = 8), and GMH + GW0742 (30 μg/kg) (n = 8).

Experiment V

Assess the effects of CD300a blocking on neurobehavioral function and post-hemorrhagic hydrocephalus after GW0742 treatment. CD300a small interfering RNA (siRNA) was used to specifically knock down CD300a. Short-term neurobehavioral test, including righting reflex and negative geotaxis tests were performed at 3 consecutive days after surgery. Long-term neurobehavioral tests, including Water Maze test, rotarod test and foot fault test, were performed at 21–28 days after GMH. Nissl staining was applied at the 28 days after GMH to assess the ventricular volume. Twenty-eight rats were randomly divided into two groups: GMH + GW0742(30 μg/kg) + scrambled siRNA (n = 8), and GMH + GW0742(30 μg/kg) + CD300 siRNA (30 μg/kg) (n = 8).

Experiment Ⅵ

Investigate the underlying mechanism of PPAR_β/δ/CD300a/SHP1 pathway in anti-neuroimflamation after GW0742 treatment. CD300a siRNA and PPAR_β/δ siRNA were used to specifically knock down CD300a and PPAR_β/δ, respectively. siRNA was delivered by intracerebroventricular (i.c.v.) injection 5 days and 2 days prior to GMH induction. The expression levels of CD300a, PPAR_β/δ, chymase, tryptase, IL-17A and TGF-β1 were evaluated by western blot. Tirty-six rats were randomly divided into 6 groups: sham (n = 6), GMH + Vehicle (n = 6), GMH + GW0742 (30 μg/kg) (n = 6), GMH + GW0742 (30 μg/kg) + scrambled siRNA (n = 6), GMH + GW0742 (30 μg/kg) + PPAR_β/δ siRNA (n = 6) and GMH + GW0742 (30 μg/kg) + CD300a siRNA (n = 6).

GMH model

GMH-induction in P7 neonatal SD rats using collagenase infusion was performed as previously described in Li’s study²². Briefly, rat pups were anesthetized with 3.0–5.0% isoflurane and once pups were unresponsive to stimuli they are maintained with 1.0–1.5% isoflurane for the rest of the surgery. The skin was incised on the longitudinal plane to expose the bregma. A burr hole (1 mm) was made on the skull (1.6 mm right lateral, 1.6 mm anterior to the bregma). A 27-gauge needle was inserted (2.7 mm deep from the dura) where 0.3 units (2 μl) of collagenase VII-S (Sigma-Aldrich, MO, USA) was 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 core temperature was maintained at 37 °C by a heated blanket. After infusion, the needle was left in place for additional 5 minutes to prevent possible collagenase backflow and then was withdrawn at rate of 0.5 mm/min. After surgery, the burr hole was sealed with bone wax, the incision was closed, and the pups were then placed back to their mothers for recovery. In the sham-operated animals, the same procedures were performed without collagenase infusion.

Drug and siRNA administration

Intranasal administration of GW0742

Under anesthesia, GW0742 (10, 30 and 90 μg/kg) (Tocris, USA) or vehicle (1% DMSO diluted in corn oil) were administered intranasally (i.n.) at 1 hour post-GMH. A total volume of 2 μL of GW0742 or vehicle was given in alternating nares. The dosage and treatment regimen were based on the previous study²¹.

In vivo small interfering RNA

For in vivo knockdown, rat-derived CD300a siRNA and PPAR_β/δ siRNA lentivirus (2, 000 IU/2μl, Applied Biological Materials, Canada) were administered via intracerebroventricular (i.c.v.) injection at 5 days and 2 days prior to GMH induction (at 1.0 μl/min through the Hamilton syringe). The same volume of scrambled (Scr) siRNA was used as a negative control. Intracerebroventricular drug administration was performed as previously described^{23, 24}. Briefly, rats were placed in a stereotaxic apparatus under isoflurane anesthesia (3% induction, 1.0–1.5% maintenance). The scalp area was sterilized, and bregma was exposed. Using bregma as a reference point, the following stereotactic coordinates were used: 1.0 mm left lateral and 1.0 mm rostral. A burr hole was drilled. A 27-gauge needle was inserted at a rate of 1 mm/min at the depth of 1.8 mm below the horizontal plane of the dura, where the lateral ventricle is located02.31²³.

Histological analysis

Rats pups were deeply anesthetized with isoflurane and transcardially perfused with ice-cold 0.1M PBS followed by 4% formaldehyde solution (PFA). Brains were removed and post-fixed in 4% PFA overnight at 4 °C, dehydrated in 30% sucrose at 4 °C for 72 hours (until full saturation). Brains were then sectioned at 8 μm thickness for immunofluorescence and 16 μm thickness for Toluidine Blue staining and Nissl staining using a cryostat (Leica CM3050S, Bannockburn, Germany).

Immunofluorescence staining

Immunofluorescence staining was performed as previously reported^{25, 26}. Brain sections were permeabilized with 0.3% Triton X-100 for 20 min, then blocked in 5% donkey serum at room temperature for 1 hour. After washing with 0.1M PBS for three times (10 min intervals), sections were incubated at 4 °C overnight with primary antibodies: anti-CD300a (Santa Cruz, Dallas, TX, USA), anti-PPAR_β/δ (Abcam, Cambridge, MA, USA), anti-chymase (Abcam, Cambridge, MA, USA), and anti-tryptase (Abcam, Cambridge, MA, USA). Sections were then washed again in PBS, which was followed by 1 hour incubation with the appropriate fluorescence secondary antibodies (Jackson Immunoresearch Research, West Grove, PA, USA) at 37°C and then washed again with PBS for three times (10 min intervals). Finally, slides were mounted with DAPI (Vector Laboratories Inc, USA). The sections were imaged under a fluorescent microscope (Leica DMi8, Leica Microsystems, Germany) equipped with LASX software. Six sections per pup (each section with 3 images) over a microscopic field of 20 × were averaged and expressed as cells/field of view (FOV), as described previously²³. Blinded individuals who were unaware of the animal group numbers performed quantification. For negative controls, the primary antibodies were omitted, and the same staining procedures were performed.

Toluidine Blue staining

Toluidine Blue staining was performed as previously described²⁷. Brain slice were hydrated at room temperature and stained with fresh Toluidine Blue working solution (0.1%, pH 2.0~2.5) for 2~3 minutes. The sections were washed with distilled water three times then dehydrated quickly through 2 changes of 75%, 95% and 100% Flex, cleared in xylene substitute (2 changes, 3 minutes each) and coverslip with mounting medium. The sections were then imaged and captured using an inverted microscope. The number of mast cells were counted manually under a randomized 1000× field of view in the brain parenchyma surrounding the hematoma. Six sections with hematoma were picked in each brain to be averaged.

Nissl staining and ventricular volume measurement

Nissl staining was conducted as reported previously²⁸. 10 μm thick coronal brain sections were cut every 600 μm using a cryostat (Leica Microsystems LM3050S) and were placed onto poly-L-lysine-coated slides. Brain sections were dehydrated respectively in 95% and 70% ethanol for 2 minutes, then rinsed in distilled water for 10 seconds. Sections were stained with 0.5% cresyl violet (Sigma-Aldrich, USA) for 2 minutes then washed in distilled water for 10 seconds followed by dehydration with 100% ethanol and xylene for two times (2 minutes each) before a coverslip with permount was placed. The sections were imaged by microscope (Olympus-BX51, Waltham, USA) and morphometrically analyzed using computer-assisted (ImageJ, Media Cybernetics, Silver Spring, MD) hand delineation of the ventricle system. The volumes were calculated by the following formula: volumes = average (area of coronal section) × section interval × number of sections^{23, 28, 29}. Calculations were performed by individuals who were blinded to the group of the animals.

Western blot

Western blot was performed as previously described^{30, 31}. Animals were euthanized and transcardially perfused with 4°C PBS. Brains were removed immediately, snap-frozen in liquid nitrogen and then stored at −80°C until analysis. Whole-cell lysates were obtained by homogenizing the tissue in RIPA lysis buffer (sc-24948, Santa Cruz, USA) and centrifuged at 14,000g at 4°C for 20 minutes. The supernatant was collected and aliquoted, where protein assay (Bio-Rad, USA) was used to measure protein content. Equal amounts of protein per sample were loaded onto a 10–12% sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel and electrophoresed for 90 minutes at 100 V. Then they were transferred to 0.45 mm nitrocellulose membranes at 100 V for 60 minutes. The membranes were blocked with 5% non-fat blocking milk (Bio-Rad, USA), followed by overnight incubation at 4 °C with the following primary antibodies: anti-CD300a (Santa Cruz, Dallas, TX, USA), anti-PPAR_β/δ (Abcam, Cambridge, MA, USA), anti-chymase (Abcam, Cambridge, MA, USA), anti-tryptase (Abcam, Cambridge, MA, USA), anti-TGF-β1 (Abcam, Cambridge, MA, USA), anti-IL-17A (Santa Cruz, Dallas, TX, USA), anti-phospho-SHP1 (Thermo Fisher), anti-phospho-SHP1 (Thermo Fisher), anti-phospho-Syk (spleen tyrosine kinase, Syk) (Cell Signaling Technology, signaling, Danvers, MA, USA), anti-Syk (Cell Signaling Technology, signaling, Danvers, MA, USA), anti-β-Actin (Santa Cruz, Dallas, TX, USA). The respective anti-mouse, anti-rabbit or anti-goat secondary antibodies (Santa Cruz, Dallas, TX, USA) were incubated with the membranes for 1 hour at room temperature. Bands were visualized using ECL Plus Chemiluminescence kit (Amersham Biosciences, USA) and analyzed using Image J 4.0 software (Image J 1.51, NIH, USA).

Neurobehavioral tests

Neurobehavioral tests were performed by two blinded researchers in a random and unbiased system as previously reported^32–34. To evaluate short-term neurological function, negative geotaxis tests and righting reflex were performed at 1 day to 3 days post-GMH. To evaluate long-term neurological function, water maze, rotarod and foot-fault tests were performed at 21 d to 28 d post-GMH.

Righting reflex

The time needed for the rat pups to completely rollover onto four limbs after being placed in the supine position was recorded. The trial was performed 3 times per pup per day. The average values of all three trials were calculated.

Negative geotaxis

Pups were placed head downward onto an inclined board (40°), the duration time for the pups to rotate their heads in a 90° and 180° turn was recorded, respectively. The maximum recording time was 60 seconds (3 trials/pup/day). The average values of all three trials were calculated.

Water maze test

Rats’ spatial learning and memory function was tested by the Morris water-maze test, which was conducted and modified as previously reported^{35, 36}. The apparatus consists of a plastic pool (110 cm in diameter) and an escape platform (11 cm in diameter) for the SD rats to climb on. On day 1, animals were trained using a visible platform. The rats were allowed to find the platform in 60 seconds durations and manually guided to the platform if they had not found it. On day 2 to day 6, the platform was submerged by 1 cm of water. Escape distance, latency and swimming speed were recorded and analyzed by a computerized tracking system (Noldus Ethovision; Noldus, Tacoma, WA, USA). The probe trial, in which the platform was removed, was conducted on the seventh day, where the swim path and time spent in the target quadrant was measured.

Rotarod test

Rotarod test is used to evaluate the test subject’s motor-coordination, where each rat remains on an accelerating rod until it falls off the rod. Rats were placed on an accelerating rotarod (SD Instruments, USA) and had to keep walking forward after being placed on the cylinder (7cm diameter). Animals were tested at 5 revolutions per minute (RPM) or 10 RPM with an increasing acceleration of 2 RPM per 5 seconds. The average latency and speed to fall off was recorded and analyzed (3 trials/rat/day), the maximum recording time was 60 seconds.

Foot fault test

Foot fault was used to further assess motor coordination. Rats were placed on a horizontal wire grid (20 × 40 cm) 1 foot above the ground. The numbers of limb missteps where steps fall through the grid openings were recorded over 60 seconds’ intervals (3 trials/pup/day). The average values of all three trials were calculated.

Statistical analysis

All data was presented as mean ± SD. Statistical analysis was performed using GraphPad Prism 6 (GraphPad Software). One-way analysis of variance (ANOVA) was used followed by multiple comparisons between groups using Tukey’s HSD post hoc test. Two-way ANOVA was used to analyze the long-term neurobehavioral function. Statistically significance was defined as P <0.05.

Results

Time course expression of CD300a, PPAR_β/δ, chymase, tryptase, IL-17A and TGF-β1 after GMH

CD300a protein level was significantly decreased at 6 hours and gradually tend to recover, reached the highest level at 3 days and decreased at 5 days (Fig.1 a, b) after GMH. PPAR_β/δ protein level was significantly decreased at 6 hours when compared to the sham group, then gradually increased at 24 hours, and then decreased at 3 and 5 days after GMH (Fig.1 a, c). Chymase and tryptase, mast cell degranulation markers, were significantly increased at 6 hours and continuously increased for 5 days after GMH when compared to the sham group (Fig.1 a, d, e). IL-17A expression was increased from 12 hours to 3 days after GMH. TGF-β1 showed a continuous elevation at 12 hours to 5 days after GMH (Fig.1 a, f, g).

Fig. 1.

Expression time course of CD300a, PPAR_β/δ, chymase, tryptase, IL-17A and TGF-β1 after GMH. Representative Western blots bands of time course (a) and densitometric quantification of endogenous CD300a (b), PPAR_β/δ (c), chymase (f), tryptase (e), IL-17A (f) and TGF-β1 (g) after GMH. Values are expressed as mean±SD. * P< 0.05 vs. sham, one-way ANOVA, Tukey’s test, n = 6 each group.

Double immunofluorescence staining showed the cellular localization of CD300a and PPAR_β/δ in rats’ brain 3 days post GMH

Staining demonstrated, in both sham and GMH group, CD300a was co-expressed with mast cell chymase (Fig. 2a). CD300a positive cells was more in GMH group than in the sham group. Additionally, PPAR_β/δ was shown to be co-expressed with mast cell marker tryptase (Fig. 2b), where PPAR_β/δ positive cells was more in GMH group than in the sham group. Lastly, immunofluorescence staining showed CD300a was co-expressed with PPAR_β/δ in the pups’ brains with or without GMH (Fig. 2c).

Fig. 2.

Immunohistochemistry staining of CD300a and PPAR_β/δ in rats’ brain. (a) CD300a (red) was co-localized with mast cell chymase (green) in sham and GMH vehicle-treated animal groups (3 days after GMH). (b) PPAR_β/δ (green) was co-localized with mast cell tryptase (red) in sham and GMH vehicle-treated animals (3 days after GMH). (c) CD300a (red) was co-expressed with PPAR_β/δ (green) in sham and GMH vehicle-treated groups (3 days after GMH). (d) The black four-sided frame indicates the location of obtained images in (a), (b) and (c). Arrowhead part was amplified. n = 3 each group, scale bar = 50 μm.

Intranasal administration of GW0742 ameliorated short-term neurological function and decreased mast cell degranulation 3 days post-GMH

At 1 to 3 days post-GMH, the vehicle group spent longer durations rotating the head in an upward position during the negative geotaxis test and flipping to the prone position during the righting reflex (Fig. 3a, b, c), when compared to the sham group. A dose-response study was conducted to evaluate the efficacy and safety of GW0742, three doses (10, 30 and 90 μg/kg) were intranasally administered 1-hour post-ictus. No significant improvement was observed in low dose GW0742 animal group, compared to GMH group. Medium and high-dose GW0742 treated groups showed a significant improvement in motor-coordination when examined in the negative geotaxis test compared to the vehicle and low-dose of GW0742 (Fig. 3a, b, c). Yet, only the medium-dose of GW0742 treated groups showed a significant improvement in motor coordination during the righting reflex (Fig. 3c). Among these three GW0742-treated groups, medium-dose regimen of GW0742 demonstrated the best efficacy. Therefore, 30 μg/kg of GW0742 was selected as the optimal dose in the following studies.

Fig. 3.

Intranasal administration of GW0742 improved short-term neurological function at 1–3 days after GMH. Negative Geotaxis reflex (a, b) and righting reflex (c) showed GMH induced neurological deficits, when compared with sham group. Medium dose (30 μg/kg) of GW0742 significantly improved neurological function compared to GMH vehicle treated pups after GMH (a, b, c). Values are expressed as mean±SD. * P < 0.05 vs. sham, ^# P < 0.05 vs. GMH vehicle, one-way ANOVA, ^@ P < 0.05 vs. medium dose of GW0742, Tukey’s test, n = 6 each group.

Toluidine blue, a synthetic dye that is specific for mast cells, was used to determine if GW0742 inhibits mast cell degranulation after GMH¹³. In the sham animal group, positive mast cells displayed an elongated spindle shape and contained intracytoplasmic granule contents (Fig. 4a). The vehicle group had enlarged with an irregular shaped mast cells; this is due to the mast cells having an increased number of disorganized intracytoplasmic granules which were distributed inside and outside the cell (Fig. 4a). GW0742 treated group showed a decreased number of mast cells with normal elongated shape (Fig. 4a). Statistical analysis demonstrated that the number of mast cells increased significantly in GMH + vehicle group compared with in sham group, and GW0742 reversed the increase in mast cells significantly (Fig. 4b). These results demonstrated that mast cells were undergoing degranulation in the brain after GMH. GW0742 treatment decreased mast cells degranulation post-ictus.

Fig. 4.

Effects of GW0742 treatment on mast cell degranulation 3 days after GMH. (a) Toluidine Blue staining of mast cell in the brain of sham, GMH vehicle-treated and GW0742-treated GMH animals. The lower row showes the amplified images of the long arrowhead indicating area in the upper row. (b) Quantitative analysis of the number of mast cells. (c) The black four-sided frame indicates the location of obtained images in (a). Values are expressed as mean ± SD. * P < 0.05 vs. Sham, ^# P < 0.05 vs. GMH + vehicle, n = 6 each group, scale bar = 50 μm.

Intranasal administration of GW0742 ameliorated long-term neurological deficits and reduced ventricular dilation at 4 weeks after GMH

To investigate the effects of GW0742 treatment on the long-term neurological deficits induced by GMH, neurological functions were evaluated by Morris water maze, rotarod and foot-fault tests at 21–28 days post-GMH. In the Morris water maze test, GMH vehicle rats, from the second to fourth day, had significantly longer swim durations (Fig. 5a), spent more time finding the platform (Fig. 5b), and spent less time in the target quadrant in the probe trials when compared to sham rats (Fig. 5c, 5d). In contrast, GW0742-treated animals performed significantly better than GMH vehicle-treated animals in the hidden platform test and in the probe trial (Fig. 5a, b, c, d). Meanwhile, there was no significant difference in velocity among all groups (Fig. 5e), indicating that neurological deficits were associated with spatial memory impairment, not swimming ability.

Fig. 5.

GW0742 administration restored memory and learning function at 21–28 days post GMH. GW0742 improved GMH pup’s memory functions as shown by less swim distance (a), escape latency (b) and more time in the target quadrant (c, d) in the Morris water maze. Swim velocity showed no significant difference among the three groups. GMH induced deficits in rotarod tests (f, g) and foot fault test (h) and GW0742 improved pups’ neurological functions. Values are expressed as mean±SD. * P < 0.05 vs. sham, ^# P < 0.05 vs. GMH + vehicle, n.s no significance. Two-way ANOVA, Tukey’s test, n = 8 each group.

In the rotarod test, GW0742 treatment significantly reduced the falling latency at both 5 RPM and 10 RPM acceleration when compared to the GMH vehicle group (Fig. 5f, g). The foot fault test showed that GMH vehicle group rats displayed significantly more foot slips on the left side compared to the sham group, which in contrast was improved by GW0742 treatment (Fig. 5h).

Ventricular dilation is a major long-term complication of GMH³⁷, therefore we assessed the effects of GW0742 administration in the long-term. Nissl staining demonstrated significant ventricular dilation after GMH when compared to sham rats (Fig. 6a, b). The hippocampus loss and white matter damage is obvious in the GMH group (Fig. 6a). GW0742 significantly decreased GMH-induced ventricular dilation at 28 days after GMH (Fig. 6a, b) and alleviated the hippocampus loss and white matter damage(Fig. 6a).

Fig. 6.

GW0742 treatment attenuated ventricular dilation at 28 days after GMH. (a) Representative Nissl staining images. (b) Ventricular volume. Values are expressed as mean±SD. * P < 0.05 vs. sham, ^# P < 0.05 vs. GMH + vehicle. One-way ANOVA, Tukey’s test, n = 8 each group.

Selective knockdown of CD300a reversed the protective effects of GW0742 in neurological deficits and ventricular dilation

To investigate the role of CD300a blocking on neurobehavioral function and post-hemorrhagic hydrocephalus after GW0742 treatment, the neurobehavioral tests and ventricular volume were evaluated by righting reflex, negative geotaxis tests, Morris water maze, rotarod, foot-fault tests and Nissl staining analysis.

Immunofluorescence staining was used to evaluate the effectiveness of CD300a siRNA’s distribution in the brain. Results showed that Scramble siRNA and CD300a siRNA were colocalized with one of the markers of mast cells, tryptase, in the rat pup brain, respectively (Fig. 7a, b)

Fig. 7.

Immunofluorescence staining of siRNA at 1 day after GMH. (a) Colocalization of scrambled (Scr) siRNA (green) with tryptase (red). (b) CD300a siRNA (green) with tryptase (red). (c) Colocalization of PPAR_β/δ siRNA (green) with tryptase (red). n = 3 each group, scale bar = 50 μm.

In the short-term neurobehavioral tests, the time length of righting reflex and negative geotaxis tests at 1 to 3 days post-GMH in GMH + GW0742 + CD300a SiRNA group were longer significantly than in GMH + GW0742 + Scramble SiRNA group (Fig. 8).

Fig. 8.

Effect of CD300a siRNA on short-term neurological function at 1–3 days after GMH with GW0742 treatment. CD300a siRNA significantly decreased neurological function on negative geotaxis reflex (a, b) and righting reflex (c) compared to Scr siRNA GW0742 treated pups after GMH. Values are expressed as mean±SD. * P < 0.05 vs. Scr siRNA, n = 6 each group.

In the Morris water maze test, the rats in GMH + GW0742 + CD300a SiRNA group, from the second to third day, had significantly longer swim distances (Fig. 9a), spent more time finding the platform (Fig. 9b), and spent less time in the target quadrant in the probe trials when compared to GMH + GW0742 + Scramble SiRNA group (Fig. 9c, d).

In the rotarod test, CD300a SiRNA delivery significantly decreased the falling latency at both 5 RPM and 10 RPM acceleration when compared to the GMH + GW0742 + Scramble SiRNA group (Fig. 9e, f). The foot fault test showed that the rats in GMH + GW0742 + CD300a SiRNA displayed significantly more foot slips on the left side compared to the GMH + GW0742 + Scramble SiRNA group (Fig. 9g).

Nissl staining demonstrated ventricular dilation, hippocampus loss and white matter damage are worse in GMH + GW0742 + CD300a SiRNA group than in GMH + GW0742 + Scramble SiRNA group (Fig. 10).

Fig. 10.

CD300a siRNA treatment increased ventricular dilation at 28 days after GMH with GW0742 treatment. (a) Representative Nissl staining images. (b) Ventricular volume. Values are expressed as mean±SD. * P < 0.05 vs. Scr siRNA, n = 8 each group.

Selective knockdown of CD300a and PPAR_β/δ abolished the CD300a/PPAR_β/δ/p-SHP1 axis, downregulation of p-Syk, and the anti-inflammatory effects induced by GW0742 at 24 hours after GMH

To investigate the role of CD300a and its downstream signaling molecules after GW0742’s treatment, PPAR_β/δ and CD300a siRNA were injected intracerebroventricularly into the CNS before GMH induction. Immunofluorescence staining was used to evaluate the effectiveness of siRNA’s distribution in the brain. Results showed that Scramble siRNA, CD300a siRNA and PPAR_β/δ siRNA were colocalized with one of the markers of mast cells, tryptase, in the rat pup brain, respectively (Fig. 7a, b, c)

Western Blots results showed that GW0742 significantly increased the protein levels of PPAR_β/δ and CD300a after GMH. PPAR_β/δ and CD300a expression, when compared to GW0742-treated pups and scramble siRNA group, were significantly decreased after the administration of PPAR_β/δ and CD300a siRNA, respectively, which confirmed the knockdown efficacy of the siRNA’s in this study (Fig. 11a, b, c).

Fig. 11.

Selective inhibition of PPAR_β/δ/CD300a/SHP1 signaling abolished the effect of GW0742 on inhibiting neuroinflammation at 24 hours after GMH. (a) Representative images of Western blot bands. Quantification of PPAR_β/δ (b), CD300a (c), p-SHP1 (d), p-Syk (e), chymase (f), tryptase (g), IL-17A (h) and TGF-β (i). Values are expressed as mean±SD. * P < 0.05 vs. sham, ^# P < 0.05 vs. GMH + vehicle, ^@ P < 0.05 vs. GMH + GW0742. One-way ANOVA, Tukey’s test, n = 6 each group.

GMH induced chymase and tryptase expression levels increasing, whereas the levels were significantly reduced in GW0742-treated pups (Fig. 11a, f, g). However, knockdown of CD300a and PPAR_β/δ, significantly reversed the inhibitory effect of GW0742 on the expression of chymase and tryptase when compared to GW0742-treated and Scr siRNA groups (Fig. 11a, f, g), indicating that GW0742 plays a role in the suppression of mast cell degranulation via the upregulation of CD300a.

In vivo and in vitro studies show that SHP1/Syk signaling molecules play an important role in the regulation of mast cell-related inflammation^{16, 38}. Therefore we investigated whether SHP1/Syk played a role in GW0742 regulation of GMH-induced inflammation. p-SHP1 expression level was decreased after GMH, whereas the level of p-SHP1 was significantly increased in GW0742-treated rats (Fig. 11a, d). Knockdown of CD300a and PPAR_β/δ, respectively, significantly reversed the effect of GW0742 on p-SHP1 expression (Fig. 11a, d). Meanwhile, p-Syk expression level was increased after GMH, whereas the level of p-Syk was significantly decreased in GW0742-treated rats (Fig. 11a, e). Knockdown of CD300a and PPAR_β/δ, respectively, significantly increased p-Syk expression level compared to GW0742-treated and Scr siRNA groups (Fig. 11a, e).

IL-17A and TGF-β1 play important roles in neuroinflammation in the brain^{38, 39}. Western Blots results showed that IL-17A was increased at 12 hours to 3 days after GMH, compared with sham animals. TGF-β1 showed a continuous elevation over 5 days after GMH that started as early as 12 hours (Fig. 1a, f, g). GW0742 decreased IL-17A and TGF-β1 expression levels after GMH (Fig. 11a, h, i). Knockdown of CD300a and PPAR_β/δ, respectively, significantly increased IL-17A and TGF-β1 expression level, compared to GW0742-treated and Scr siRNA groups (Fig. 11a, h, i).

Discussion

Germinal matrix haemorrhages (GMH) refers to bleeding occurs in the subependymal or periventricular germinal region of premature brains⁴⁰. Attributing to structures fragility of germinal matrix and the lacking of auto-adaptability to modulate the premature vasculatrue lumen under fluctuant hemodynamics⁴⁰, any stress experienced by a premature infant after birth may cause rapid fluctuation of blood pressure and a periventricular hemorrhage (PVH). If germinal matrix in the periventricular area bleeding persists, the haemorrhage will expand into the adjacent lateral ventricles and lead to intraventricular hemorrhage (IVH), so GMH is also known as periventricular-intraventricular haemorrhages (PVIH) or germinal matrix-intraventricular hemorrhage (GMH-IVH)⁴¹. GMH is a devastating perinatal disease, which can result in severe and permanent damages to the premature brain such as post-hemorrhagic hydrocephalus(PHH), cerebral palsy, seizures, hemiplegia, and even death⁴². In this study, the GMH model was induced by collagenase-injection in P7 neonatal rodent animals to mimic the human CNS pathophysiology, PHH and neurological impairments^{43, 44}.

In the present study, we investigated the anti-inflammatory effects of GW0742 in GMH rat pups and explored the underlying mechanisms involved. (1) CD300a and PPAR_β/δ were expressed on mast cell in the neonatal rat CNS and their protein level both demonstrated an initial decrease followed by a gradually increase after GMH. (2) Protein expression levels for mast cell granule markers, chymase and tryptase, were increased after GMH. (3) GW0742 treatment decreased mast cell degranulation 3 days after GMH. (4) GW0742 attenuated short- and long-term neurological impairments and ventricular dilation at 4 weeks after GMH. (5) Furthermore, GW0742 treatment significantly upregulated the expression of PPAR_β/δ, CD300a, and phosphorylated-SHP1, whereas it decreased the expression of phosphorylated-Syk, chymase, tryptase and inflammatory cytokines, IL-17A and TGF-β1, at 24h after GMH. PPAR_β/δ siRNA and CD300a siRNA reversed the effects of GW0742 on the inhibition of mast degranulation and improvements of neurological functions after GMH. These results suggested that GW0742 could attenuate mast cell degranulation and neuroinflammation through the PPAR_β/δ/CD300a/SHP1 signaling pathway.

Previous studies demonstrated, in both human and rodents, that CD300a is expressed on the surface of mast cells¹⁷. In the present study, double immunofluorescence staining results showed that CD300a was co-localized with mast cell marker chymase and tryptase⁴⁵, which indicates the presence of CD300a in mast cell in the neonatal rat brain. CD300a plays an integral role in allergic and non-allergic inflammatory response. To date, numerous studies demonstrated that activation of CD300a leads to the decrease of the production of inflammatory cytokines and chemokines factors in mast cell, thus, to inhibit global inflammation¹⁷. In this study, Western blot results showed that the protein expression levels of CD300a was initially decreased at 6 h followed by a gradual increase for up to 3 days after GMH, indicating a possible role in the inflammatory response post-ictus. It is reported that CD300a is a direct target of PPARs^{20, 46}. There are three members of the PPAR subfamily: α, β/δ), γ⁴⁷. PPARs expression has been observed extensively in all major cell types in the CNS, including neurons, astrocytes, microglia, endothelial cells, and oligodendrocytes. So PPAR_β/δ has been demonstrated to act on multiple mechanisms and was shown to activate multiple protective pathways related to inflammation, apoptosis, BBB protection, neurogenesis, and oxidative stress in literature reports⁴⁷. In the past decade, our research group has observed the beneficial effects of PPAR agonist in cerebrovascular disease animal models and investigated the possible mechanisms involved, including attenuating neuronal apoptosis via PPAR/miR-17/TXNIP pathway²¹, reducing neuroinflammatory effect through PPAR/SIRT6/FoxO3a pathway⁴⁸, ameliorating brain injury by regulating microglia M1/M2 polarization⁴⁹, and so on. GW0742 is a specific synthetic high-affinity PPAR_β/δ agonist and has been shown to upregulate both nuclear and protein expression of PPAR_β/δ^{18, 21, 50}. To ascertain whether PPAR_β/δ plays a role after GMH, we attempted to promote the expression of PPAR_β/δ by GW0742 in this study. Immunofluorescence staining showed that PPAR_β/δ was expressed in mast cell and neuron in neonatal rodent CNS. Meanwhile, Western blots showed that the protein level of PPAR_β/δ was increased along with CD300a activation after GW0742 treatment.

Mast cell degranulation results in the rapid and efficiently release of specific set of bioactive mediators in response to external and internal environmental cues⁵¹. Chymase and tryptase markers have been readily used as the diagnostic biomarkers of mast cell degranulation and pharmacological targets in several pathological conditions^{45, 52, 53}. In the present study, protein levels of chymase and tryptase were increased after GMH, while GW0742 decreased the expression of chymase and tryptase after GMH. In accordance with protein expression, Toluidine blue staining demonstrated a significant increase in the number of mast cells and granules after GMH. Whereas GW0742 prevented mast cell morphological changes and decreased intracytoplasmic granule content after GMH. Toluidine blue staining and Western blot results demonstrate that GMH activates and triggered degranulation in mast cells, while GW0742 inhibited mast cell activation and degranulation after GMH.

The blood clot after GMH results in mechanical pressure on the surrounding brain tissue resulting in cell death, development of post hemorrhagic hydrocephalus, and short- and long-term neurocognitive disabilities^{1, 54}. In this study, neurobehavioral results showed that GW0742 treatment reduced short- and long-term neurological impairments induced by GMH. Moreover, inhibition mast cell activation and inflammation led to the reduction in post-hemorrhagic ventricular dilation after GMH via GW0742 treatment. These results revealed that neurological and morphological improvement was attributed to GW0742 treatment. Our results about the protective effects of GW0742 in GMH model is consistent with the research in a collagenase-induced intracerebral hemorrhage mouse model⁵⁵.

Mast cells play an integral role in neuroinflammation, through the close interactions between mast cells and glial and neuronal cells⁵. When mast cell is activated, secretory proteases are released and the production of inflammatory cytokines is increased. TGF-β is a powerful inflammatory simulator that mediates various biological events that involve inflammatory responses, leading to the development brain injury^{56, 57}. Mature TGF-β is secreted constitutively by most tissues and cells, including epithelial cells⁵⁸, microglia⁵⁹, astrocytes⁶⁰, et al. Previous study discovered that after activation of mast cells, chymase triggers the rapid production of the TGF-β and converts TGF-β from the latent to active form^{39, 61, 62}. Additionally, IL-17 leads to the production of numerous pro-inflammatory cytokines such as IL-8, CXCL1, CXCL6, IL-1, IL-6, TNF-α, GM-CSF, and macrophage inflammatory protein-2 (MIP-2) which affects the function of a wide range of cells in the CNS (e.g. astrocytes, neurons and microglia). Consequently, neuroinflammatory responses in the CNS can directly induce neuronal degeneration and disruption of BBB leading to secondary brain injury in neurological diseases⁶³. Mast cells not only release IL-17 directly, but also promote the release of IL-17 by other immune cells. For example, studies have shown that mast cells specifically increase the number of Th17 cells through release of IL-1β in a caspase independent manner, and this enhancement of Th17 cell number triggers the release of IL-17 by Th17 cells⁶⁴. Inhibiting the function of the th17 cells and secretion of IL-17 could have a neuroprotective effect in inflammatory diseases of the CNS⁶⁵.

In this study, IL-17A and TGF-β1 expression levels were increased after GMH and GW0742 treatment decreased the protein levels of both respectively after GMH. Meanwhile, the inflammation was deteriorated by knockdown of CD300a and PPAR_β/δ after GMH. These results indicated that neuroinflammation was triggered after GMH and GW0742 treatment suppressed the expression of pro-inflammatory mediators via PPAR_β/δ/CD300a pathway.

We further investigated the potential molecular mechanism of PPAR_β/δ/CD300a signaling and its anti-inflammatory effects after GMH. Mast cells express a substantial number of activating and inhibitory receptors. After being activated, mast cells can rapidly release preformed granule-store mediators which contribute to the rapid activation of neuroinflammation in response to activating signal^{7, 16}. CD300a contains a single extracellular immunoglobulin domain, a transmembrane domain and a long cytoplasmic caudal tail that containing various ITIMs motifs. The binding of ligands to CD300a leads to phosphorylation of tyrosine residues at the ITIMs domains⁶⁶. After phosphorylation, ITIMs provide a docking site for the recruitment of protein tyrosine phosphatase, including SHIP, SHP-1, and SHP-2, and block activation signals by dephosphorylating intracellular molecules at the earliest step of the activation response mediated by the high affinity IgE receptor (FcεRI) pathway. Recently, the most in depth study in the molecular regulation of mast cell activation is the FcεRI receptor mediated signaling pathway^{38, 67}. Syk is the most upstream signaling molecule in FcεRI-triggered mast cell activation and plays a critical role in mast cell-induced inflammatory cytokine production^13–15. SHP1 is an important immunomodulator which suppresses Syk and downstream signaling pathway in the mast cell. Suppression of Syk directly by promoting the effects of SHP1 could serve as a useful therapeutic strategy against mast cell-driven diseases¹⁶.

To investigate the underlying protective mechanism of GW0742 after GMH, we detected the effects of GW0742 on the expression level of SHP1 and Syk. The protein level of p-SHP was decreased after GMH. The protein level of p-Syk was increased after GMH. GW0742 treatment increased the level of p-SHP and decreased the level of p-Syk and, subsequently, decreased the levels of inflammatory factors IL-17A and TGF-β1 in GMH. However, the effects of GW0742 on the expression of p-SHP, p-Syk and inflammatory factors on GMH were abolished by treatment of specific siRNAs that targeted the neuroprotective signaling pathway.

Based on the results above, our results suggest that GW0742 decreased GMH-induced neuroinflammation which induced by the upregulation of the PPAR_β/δ/CD300a/SHP1 signaling pathway in mast cell.

There are some limitations in the present study. Firstly, as shown in the present study, PPAR_β/δ is expressed not only in the mast cells in the CNS, but also in other cell types, such as neurons. Second, several neuroprotective mechanisms might be linked to GW0742 and its receptors. The observed protective effects of GW0742 in the present study may have occurred because of a combined effect that involves the inhibition of inflammation, oxidative stress and neuron apoptosis. Third, the potential roles of PPAR_β/δ on neurons and glia cells need to be further investigated. Fourth, while we focused the effects of mast cell on GMH in the present study, it is important to keep in mind that some other cells, e.g. astrocyte and microglia also have detrimental effects on neuroinflammation in GMH. Much remains to be elucidated about the roles of other cells.

Conclusions

In conclusion, the present study demonstrated that GW0742 reduces mast cell-induced degranulation and subsequent neuroinflammation, and attenuate neurological impairments via, at least in part, PPAR_β/δ/CD300a/SHP1 pathway after GMH in neonatal rats (Fig.9). The underlying mechanism of GW0742 signaling is complex and is dynamically regulated through its crosstalk with potential other several signaling pathways. Further research is needed to elucidate the precise mechanism by which GW0742 regulates PPAR_β/δ/CD300a signaling in mast cells and other cells. Our results indicate a neuroprotective role of GW0742 in reducing inflammation in an experimental model of GMH. Therefore, we propose that GW0742 may serve as a potential treatment to reduce neuroinflammation and brain injury for GMH patients.

Fig. 12.

Mechanism representation in mast cells after GMH. GMH induces FcεRI/Syk activation thus leading to an increase in degranulation of chymase and tryptase and producing a neuroinflammatory response. Administration of GW0742 activates PPAR_β/δ, CD300a and phosphorylated SHP1, thus inhibiting FcεRI/Syk signaling and reducing inflammation. Administration of PPAR_β/δ and CD300a siRNA reduce the effects of GW0742/PPAR_β/δ/CD300a/SHP1 thus reducing the beneficial effects of GW0742 on GMH-induced inflammation.

Highlights.

1. The level of endogenous CD300a and PPAR_β/δ decreased post GMH.

2. GW0742 increased expression of PPAR_β/δ and CD300a after GMH.

3. GW0742 inhibited mast cell-induced inflammation post GMH.

4. The protective effects of GW0742 are mediated by the PPAR_β/δ/CD300a/SHP1 pathway.

Abbreviations

BBB

blood-brain barrier

GMH

germinal matrix hemorrhage

interleukin 17

IL-17

ITIM

immunoreceptor tyrosine-based inhibition motif

PAF

platelet-activating factor

PPARβ/δ

Peroxisome proliferator–activated receptor β/δ

SD

Standard Deviation

RPM

revolutions per minute

SDS-PAGE

sodium dodecylsulfate polyacrylamide gel electrophoresis

SHIP

SH2 domain-containing inositol 5-phosphatase

SHP-1

SH2 domain-containing protein tyrosine phosphatase 1

siRNA

small interfering RNA

Syk

suppress spleen tyrosine kinase

TGF-β

transforming growth factor β

Availability of data and materials

The data used in this study are available from the corresponding author on reasonable request.