2.1 Animals
Adult male C57BL/6 mice (8–10 weeks old, BioLasco Taiwan Co., Ltd, Taiwan) were used in this study. All animals were housed in the Animal Center of Chung Shan Medical University, Taichung, Taiwan, at an ambient temperature of 24 ± 1℃ and humidity of 55 ± 5% under a 12 h light-dark cycle. They received normal chow and water ad libitum. All surgical procedures for permanent MCAO and experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee of Chung Shan Medical University (IACUC 1637). All animal handling conformed to Directive 2010/63/EU, and all efforts were made to minimize animal suffering and to reduce the number of animals to be sacrificed. This investigation also conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication No. 85 − 23, revised 1996).
2.2 Permanent MCAO model
The method was modified from previous studies [20]. All mice were anesthetized with intraperitoneal pentobarbital (50 mg/kg in normal saline). Body temperature was maintained during surgery at 37 ± 0.5°C with a heating pad. Focal ischemic infarcts were made in the right lateral cerebral cortex in the territory of the middle cerebral artery. The bilateral common carotid arteries were exposed by midline anterior cervical incision. The mice were placed in a lateral position, and a skin incision was made at the midpoint between the right lateral canthus and the anterior pinna. The temporal muscle was retracted, and a small (3 mm in diameter) craniectomy was made at the junction of the zygoma and squamosal bone using a drill (Dremel Multipro + 5395, Dremel Company, USA) cooled with saline solution. The dura was opened with fine forceps using a dissecting microscope (OPMI-1, ZISS®, Germany), and permanently cauterized the distal MCA along with simultaneous occlusion of the bilateral common carotid arteries with microaneurysm clips for 20 min to paralyze the dominant forelimb. After 20 min of ischemia, the CCAs were unclamped, and the restoration of blood flow was visualized.
2.3 Drug administration
The selection of experimental dosage regimen was referred Zhang et al and Ji et al [18, 19]. In our preliminary experiment, we investigated that the effectiveness of various doses of lumbrokinase (from 0.1 mg/kg to 10 mg/kg) in mice subjected to ischemic stroke. We found that 1 mg/kg lumbrokinase significantly reduced infarcted volume and improved functional recovery after ischemic stroke. In order to reduce the number of animals used in the present study, we examine the neuroprotective effect of lumbrokinase at the dose of 0.1 mg/kg and 1 mg/kg for follow-up experiments. All mice underwent MCAO surgery and were randomly assigned to three groups: (1) vehicle: normal saline, (2) LK-0.1: lumbrokinase 0.1 mg/kg, and (3) LK-1: lumbrokinase 1 mg/kg. Intraperitoneal injection with lumbrokinase or vehicle was performed once a day for six days. The first treatment was 20 min after MCAO. Lumbrokinase (Canada RNA Biochemical Inc., Richmond, BC, Canada) solution was freshly prepared before administration.
2.4 Infarct volume analysis
Brain infarct size was measured seven days after the onset of MCAO-induced ischemia after decapitating each animal after the induction of anesthesia by intraperitoneal urethane injection (1.25 g/kg, Sigma-Aldrich, St. Louis, MO, USA). The brains were removed, inspected visually for MCA anatomy and signs of hemorrhage or infection, immersed in cold saline solution for 10 min, and then sectioned into standard coronal 1 mm slices using a Jacobowitz brain matrix slicer (Zivic-Miller Laboratories, Inc., Allison Park, PA, USA). The slices were immersed in vital dye 2,3,5-triphenyltetrazolium chloride (TTC, 2%, Sigma-Aldrich) for 30 min at 37°C in the dark and fixed overnight in a 10% formalin solution at room temperature. The TTC staining clearly distinguished infarcted from non-infarcted tissue. The cerebral infarction volume was measured using digital imaging and image analysis software (National Institutes of Health ImageJ software 1.42), which subtracted the ipsilateral regional volume from the contralateral regional volume. The person who analyzed the images was blinded to the treatment of each animal. The methods were modified from the method of Huang et al [21].
2.5 Neurological evaluation
Neurological evaluations were performed in all animals before MCAO surgery, and the first and third days after ischemic stroke.
Rotarod test. The accelerating rotarod assesses motor deficits [22]. The mice were placed on the rungs of the accelerating rotarod cylinder, and the duration (in seconds) of the animals remaining on the rotarod was measured. The speed was slowly increased from 4 to 40 rev/min within 4 min. A trial would end if the animal fell off the rungs. Each animal underwent three consecutive trials.
Grip test. Motor function and coordination were evaluated by the grip test [23]. Each mouse was placed on a taut, 43 cm-long string suspended 34 cm high from the bracket between both sides of the string. The experimenters ensured that both front paws came in contact with the string, allowing the mice an equal chance to grasp the string if they could. The tail was then released, at which time the mice either fell immediately or remained on the string. Neurological status was evaluated according to a grip test, which measured the length of time the mice could remain on the string in some manner (i.e., using 1–4 paws, tail, or paws plus tail) within a 30-s time period. Each mouse was placed on a string midway between two supports and evaluated according to the following scale: 0, fell off; 1, held on in some way for 30 s; 2, held on with four paws for at least 5 s; 3, held on with four paws and placed the tail on the string for at least 5 s; 4, held on with four paws, placed the tail on the string, and traveled along the string in either direction for at least 5 s; and 5, traveled to one of the vertical sides within the 30-s test period.
Adhesive removal test. The adhesive removal test was modified from a previous experiment [24]. Patches of adhesive tape (4 mm × 4 mm) were attached to the animal’s paws in alternating sequence and with equal pressure. The animals were individually released into the testing cage, and the latency times of the contact and removal of the adhesive patch were recorded. Contact was recorded when either shaking of the paw or mouth contact occurred. The trial ended after the removal of the adhesive patch or after 2 min had elapsed. Preoperative training was conducted for three days for one trial per day prior to pretesting. The animals were tested for three trials postoperatively. The best two trials from each mouse were averaged for statistical analysis.
2.6 Western blotting
Brains were homogenized in T-PER Tissue Protein Extraction Reagent (Thermo Scientific, USA) containing the Protease Inhibitor Cocktail (Sigma, USA). The homogenates were centrifuged at 12,000 g for 10 min at 4℃. Protein concentration was determined with protein assay kits (BioRad, USA) using bovine serum albumin (BSA) as the standard. The samples were mixed with an equal volume of loading buffer (62.5 mM Tris-HCl, pH 6.8, 10% v/v glycerol, 2% SDS, 5% v/v 2-mercaptoethanol, and 0.05% w/v bromophenol blue) and heated for 10 min at 95℃. The mixture was subjected to SDS-PAGE and transferred electrophoretically to nitrocellulose membranes at a constant voltage of 30 V at 4°C overnight. The membranes were blocked with 5% w/v non-fat milk in PBS containing 0.1% v/v Tween 20 (PBST) for 90 min at room temperature. The membranes were reacted with primary antibodies (1:3,000 dilutions) at 4°C overnight and washed three times with PBST. HRP-conjugated secondary antibody (1:20,000 dilutions) was added, and the membranes were incubated at room temperature for 40 min to detect the primary bound antibody. Reactive proteins were detected by enhanced chemiluminescence (Amersham, UK), and the density of specific immunoreactive bands was quantified by densitometric scanning.
2.7 Primary antibodies
The primary antibodies used were as follows: mouse anti-β-actin monoclonal antibody, rabbit anti-LC3 polyclonal antibody, rabbit anti-p62 polyclonal antibody, rabbit anti-interleukin (IL)-1β polyclonal antibody, mouse anti-caspase-1 monoclonal antibody, mouse anti-cytochrome C monoclonal antibody (Novus, USA), rabbit anti-p-IRE1 polyclonal antibody, rabbit anti-IRE1 polyclonal antibody, rabbit anti-Bax monoclonal antibody, rabbit anti-Bcl-2 monoclonal antibody, rabbit anti-Beclin-1 monoclonal antibody, rabbit anti-XBP1 polyclonal antibody, rabbit anti-NLRP3 polyclonal antibody, rabbit anti-COX-2 polyclonal antibody (Abcam, UK), rabbit anti-caspase-12 polyclonal antibody, mouse anti-GRP78 polyclonal antibody (BD Biosciences, USA), rabbit anti-caspase-3 polyclonal antibody, rabbit anti-cleaved-caspase-3 polyclonal antibody, rabbit anti-p-nuclear factor kappa B (NF-κB) polyclonal antibody, and rabbit anti-NF-κB polyclonal antibody (Cell Signaling, USA).
2.8 Secondary antibodies
HRP-conjugated goat anti-mouse IgG (H + L) antibody, HRP-conjugated bovine anti-goat IgG (H + L) antibody, and HRP-conjugated goat anti-rabbit IgG (H + L) antibody were all purchased from Jackson ImmunoResearch Laboratories, Inc. (USA).
2.9 Gelatin zymography
Stroke-induced proteinase in mouse brain was analyzed by gelatin zymography, as described previously [25], with slight modifications. In brief, heart homogenates were loaded onto SDS-PAGE [7.5% (w/v) polyacrylamide gel copolymerized with 0.1% w/v gelatin (Sigma, USA)]. The stacking gels were 4% w/v polyacrylamide and did not contain a gelatin substrate. Electrophoresis was performed in a running buffer (25 mM Tris, 250 mM glycine, 1% SDS) at room temperature. The gel was washed twice in double-distilled water containing 2.5% TritonX-100 for 30 min each time and then incubated in a reaction buffer (50 mM Tris-HCl, pH 7.5, 200 mM NaCl, 10 mM CaCl2, 0.02% w/v Brij® − 35, 0.01% w/v NaN3) at 37℃ for 18 h. The gel was stained with 0.25% w/v Coomassie Brilliant Blue R-250 (Sigma, USA) for 1 h and destained in 15% v/v methanol and 7.5% v/v acetic acid. Gelatinase activity was detected as unstained bands on a blue background. Quantitative analysis was performed with a computer-assisted imaging densitometer system (UN-SCAN-ITTM gel version 5.1, Silk Scientific, UT, USA).
2.10 Immunofluorescence assay
Mouse brain was removed as fresh and fixed directly with 4% paraformaldehyde. All the samples were embedded in an optimal cutting temperature compound, and sections were cut at a 25-µm thickness. For immunostaining, frozen brain sections were fixed with methanol for 20 min at − 20°C and washed repeatedly. The tissue sections were blocked with 5% BSA in PBS for 1 h and incubated with 2 N HCl at 37°C for 30 min, followed by blocking the non-specific binding sites in 0.1 M PBS containing 0.3% Triton X-100 and 2% fetal bovine serum (FBS). The sections were incubated with a rabbit anti-p-IRE1 monoclonal antibody and doubly stained with another antibody against various cellular marker proteins, including mouse anti-neuronal nuclei (NeuN) and rabbit anti-glial fibrillary acidic protein (GFAP), all diluted in PBS containing 0.3% Triton X-100 and 2% FBS. The secondary antibodies were Alexa Fluor 488-conjugated goat anti-rabbit and TRITC-conjugated donkey anti-mouse antibody. Nuclei were stained with antifade reagent (Molecular Probes Inc., UK). The slides were examined under a confocal microscope (Zeiss LSM 510 META; Carl Zeiss, Oberkochen, Germany) and a fluorescence microscope (ZEISS Axio Imager A2).
2.11 Statistical analysis
Data analysis was performed using SigmaPlot Version 11.0. All data are expressed as the mean ± standard error of the mean. Between-group differences were assessed using one-way analysis of variance, followed by Student’s t tests with the post hoc Bonferroni's comparison tests. The threshold for statistical significance was set to p < 0.05 using a two-tailed hypothesis test.