P2X7R blockade prevents NLRP3 inflammasome activation and brain injury in a rat model of intracerebral hemorrhage: involvement of peroxynitrite

Animals

Sprague–Dawley (SD) male rats weighing 280–320 g were purchased from the Animal Experiment
Center of Southern Medical University (Guangzhou, China). All experimental procedures
and animal care were approved by the Southern Medical University Ethics Committee.

ICH model

After anesthetization (0.3 ml/100 g, 10 % chloral hydrate, Sigma-Aldrich, St. Louis,
MO, USA), an incision was made on the skin along the sagittal midline to expose the
skull. A burr hole (1 mm) was drilled 3 mm lateral and 1 mm anterior to the bregma,
then a 30-gauge needle was inserted through the burr hole into the striatum (6 mm
ventral from the skull surface), and ICH was induced by stereotaxic infusion of bacterial
collagenase VII-S (0.25U in 1.0 ?l sterile saline, Sigma-Aldrich) over a 10-min period.
In the Sham group, rats were subjected to only a needle insertion as described above.
The needle was kept in situ for another 10 min to prevent backflow and then slowly
removed. The craniotomies were sealed with bone wax. Rats were allowed to recover
in separate cages with free access to food and water.

Experimental protocol

Four separate experiments were conducted as shown in Additional file 1.

Experiment 1

Thirty-six rats were divided into six groups (Sham, and 6, 12, 24, 48, and 72 h after
ICH). The expression levels of P2X7R, NLRP3, ASC, and caspase-1 were detected by western
blot. The tissue for immunofluorescence (IF) was collected 24 h after ICH induction.

Experiment 2

Eighty-eight rats were randomized into four groups: Sham, Vehicle (ICH + saline, intracerebroventricular
injection), Scramble small interfering RNA (siRNA) (1000 pmol, 2 ?l, ICH + scramble
siRNA), and P2X7R siRNA (1000 pmol, 2 ?l, ICH + P2X7R siRNA). siRNA silencing efficacy
was assessed by western blot. Brain water content and modified Neurological Severity
Score (mNSS) were also measured.

Experiment 3

One hundred and thirty-two rats were randomized into four groups: Sham, Vehicle (ICH
+ saline, intraperitoneal injection), BBG (50 mg/kg), and BBG (100 mg/kg). For the
72-h study, BBG was administered daily by intraperitoneal injection. Western blot,
hematoxylin and eosin (HE) staining, immunofluorescence (IF), and terminal deoxynucleotidyl
transferase dUTP nick end labeling (TUNEL) were measured 24 h after ICH induction;
mNSS and brain water content were detected at both 24 and 72 h.

Experiment 4

Thirty-three rats were randomized into three groups: Sham, Vehicle (ICH + saline,
intraperitoneal injection), and FeTPPS (30 mg/kg). Western blotting was performed
24 h after ICH induction.

siRNA and drug delivery

BBG (Sigma-Aldrich) was diluted at 50 and 100 mg/kg in Vehicle (saline) solution.
Rats were treated intraperitoneally with either BBG or Vehicle immediately after ICH
induction and at 12, 36, and 60 h.

For in vivo siRNA administration, P2X7R siRNA or non-silencing RNA (Sigma-Aldrich)
was applied 24 h before ICH by intracerebroventricular injection as previously described
2]. A cranial burr hole (1 mm) was drilled, and a 30-gauge needle was inserted stereotaxically
into the right lateral ventricle. To improve the gene silence efficiency, two different
sequences targeting P2X7R siRNA were combined (a)sense:5?-CAGUGAAUGAGUACUACUA-3?;
antisense:5?-UAGUAGUACUCAUUCACUG-3?; (b)sense:5?-CUCUUGAGGAGCGCCGAAA-3?;antisense:5?-UUUCGGCGCUCCUCAAGAG-3?.
siRNA was dissolved in RNA free water. Scrambled control siRNA (1000 pmol, 2 ?l),
P2X7R siRNA (1000 pmol, 2 ?l), or RNA free water (2 ?l) was delivered intracerebroventricularly
for 2 min. The needle was left in place for an additional 10 min after injection and
then slowly withdrawn.

FeTPPS (Millipore, Billerica, MA, USA) was diluted to 30 mg/kg in Vehicle (saline)
solution. Rats were treated intraperitoneally with either FeTPPS or Vehicle immediately
and at 12 h after ICH induction. The FeTPPS dose was selected based on our previous
reports that showed that it is effectively protected against injury 30].

RT-PCR

Rats were anesthetized and decapitated. Lesioned tissues (about 40 mg) were obtained,
and total RNA was extracted from the tissue with GeneJETâ„¢ RNA Purification Kit (Thermo
Fisher Scientific Inc., Waltham, MA, USA). RNA (1 ?g) was reverse-transcribed to cDNA
with high capacity (Life Technologies, Carlsbad, CA, USA). RT-PCR was performed in
an ABI Prism 7500 sequence detection system (Applied Biosystems, Foster City, CA,
USA) using specific primers designed from known sequences. GAPDH was used as an endogenous
control gene. Sequence-specific primers for P2X7R, NLRP3, and GADPH were as follows:

P2X7R, 5?-CTACTCTTCGGTGGGGGCTT-3?

(forward primer),

P2X7R, 5?-CTCTGGATCCGGGTGACTTT-3?

(reverse primer);

NLRP3, 5?-CTGCATGCCGTATCTGGTTG-3?

(forward primer),

NLRP3, 5?-GCTGAGCAAGCTAAAGGCTTC-3?

(reverse primer);

GAPDH, 5?-AGACAGCCGCATCTTCTTGT-3?

(forward primer),

GAPDH, 5?- TGATGGCAACAATGTCCACT-3’

(reverse primer);

Western blot

Western blotting was performed as described previously 30]. The following primary antibodies were used: rabbit polyclonal anti-P2X7R (1:1000,
Alomone Labs, Jerusalem, Israel), rabbit polyclonal anti-NLRP3 antibody (1:1000, Santa
Cruz, Biotechnology, Santa Cruz, CA, USA), rabbit polyclonal anti-ASC antibody (1:500,
Abclonal, Cambridge, MA, USA), mouse monoclonal anti-caspase-1 p20 antibody (1:1000,
Santa Cruz Biotechnology), rabbit polyclonal anti-IL-1? antibody (1:1000, Millipore),
rabbit monoclonal anti-IL-18 antibody (1:1000, Abcam, Cambridge, UK), mouse monoclonal
anti-nitrotyrosine antibody (1:1000, Abcam), mouse monoclonal anti-iNOS antibody (1:200,
Santa Cruz Biotechnology), mouse monoclonal anti-gp91
^phox
antibody (1:2000, BD Transduction Laboratories, San Jose, CA, USA), and rabbit polyclonal
anti-myeloperoxidase antibody (MPO, 1:500, Abcam). GAPDH (1:1000, Cell Signaling Technology,
Danvers, MA, USA) was employed as the loading control. Blot bands were quantified
by densitometry with ImageJ software (National Institutes of Health, Baltimore, MD,
USA).

Paraffin section preparations

The sections were processed as previously described 30] with minor modifications. After anesthetization, rats were transcardially perfused
with 200 ml saline followed by 400 ml 4 % paraformaldehyde solution. Brain tissues
were then removed and fixed by immersion in the same solution at 4 °C for 24 h. After
dehydration and vitrification, they were embedded in paraffin, and 3-?m sections were
prepared. Sections were dewaxed, rehydrated, and then processed for IF and TUNEL.

Histological examination

Coronal sections (1 mm apart) 31] were prepared accordingly and then stained with HE. Hemorrhagic volumes were calculated
using Image Pro Plus 6.0 software (Media Cybernetics, USA) to span the entire hematoma
32].

IF

Antigen retrieval was performed by heat treatment in a microwave oven for 21 min in
Tris–EDTA buffer solution (0.05 mol/L Tris, 0.001 mol/L EDTA; pH 8.5). Sections were
incubated for 30 min in 5 % bovine serum albumin (BSA) and then incubated at 4 °C
overnight with primary antibodies (rabbit polyclonal anti-P2X7R, 1:500, Alomone labs;
mouse monoclonal anti-3-Nitrotyrosine, 1:400, Abcam; rabbit polyclonal anti-nitrotyrosine
antibody, 1:200, Millipore; rabbit polyclonal anti-MPO antibody, 1:50, Abcam; rabbit
polyclonal anti-iNOS antibody, 1:40, Santa Cruz Biotechnology; rabbit polyclonal anti-Iba-1
antibody, 1:600, WAKO, Osaka, Japan; goat polyclonal anti-Iba-1 antibody, 1:300, Abcam;
mouse monoclonal gp91
^phox
antibody, 1:400, BD Transduction Laboratories). For double-staining experiments, primary
antibodies were separately incubated overnight at 4 °C. After they were washed with
phosphate-buffered saline (PBS), sections were then incubated with secondary antibodies.
Images were obtained using confocal microscopes (FV10i-W, Olympus, Tokyo, Japan; LSM780,
Zeiss, Oberkochen, Germany).

TUNEL

At 24 h after ICH, TUNEL staining was performed with an in situ apoptosis detection
kit (Roche, Basel, Switzerland) according to the manufacturer’s instruction. For NeuN
and TUNEL co-staining, the sections were first labeled with a NeuN antibody (1:400,
Abcam), followed by TUNEL. The slides were analyzed using a fluorescence microscope
(Bx51, Olympus).

Brain water content measurement

Brain edema was evaluated by a common wet/dry method as previously described 33]. Briefly, at 24 or 72 h post-ICH, rats were anesthetized and decapitated. The brains
were removed and immediately separated into contralateral and ipsilateral hemispheres
and the cerebellum and wet weighed. The cerebellum was used as an internal control.
Brain specimens were dried in an oven at 100 °C for 24 h to obtain the dry weight.
The water content was expressed as a percentage of the wet weight: ([wet weight] –
[dry weight]) / (wet weight) × 100 %.

Behavioral testing

Behavioral tests were assessed with mNSS at 24 and 72 h after ICH by an investigator
who was blinded to the experimental groups 34].

Statistical analysis

Data are shown as mean ± SD. Statistical analysis was performed using SPSS 13.0 (SPSS
Inc., Chicago, IL, USA). Comparison between groups was determined by Student’s t tests or one-way analysis of variance (ANOVA) followed by least significant difference
(LSD) tests with multiple comparisons. The statistically significant level was P 0.05.