All animal protocols were carried out according 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). Male C57BL/6 mice (age 8–12 weeks, weight 22–28 g) were housed under conditions of controlled temperature (22–25 °C) and humidity (40–60%) with a 12-h/12-h light-dark cycle, and were allowed free access to water and food.
A total of 224 mice were used. Mice that had neurological deficit scores greater than 15 or less than 3 at 3 h post-ICH were excluded from the study. Forty-six mice were excluded due to neurological deficit standards or death after ICH (vehicle: 22/90; DHF20; 5/23, DHF40; 19/87). Twenty-four additional sham-operated control mice were used for metabolic characteristics, histology examination and biochemical assays.
The animals were randomized to different treatment groups by using computer-generated random numbers. All outcome measurements and analyses were performed in a blinded manner. Three studies were conducted. The first study was to determine the optimal dose of 7,8-DHF. Following ICH, animals were randomized into 3 groups: 1) ICH + vehicle, 2) ICH + 20 mg/kg 7,8-DHF (DHF20), and 3) ICH + 40 mg/kg 7,8-DHF (DHF40). Next, 7,8-DHF (TCI America, Portland, OR, USA) dissolved in 60% DMSO (0.1 ml) or a corresponding volume of vehicle (60% DMSO) was administered intraperitoneally (ip) 10 min after ICH and subsequently daily for 3 days (10 min, 24 h, 48 h, and 72 h), and behavior tests (n = 12/group) were evaluated as the main outcomes. The dose and route of 7,8-DHF were selected based on our previous work on experimental traumatic brain injury . The 4-dose regimen was chosen because the previous study showed that apoptosis-related signals last for over 3 days after ICH . The results of the first study showed that 40 mg/kg DHF produced more protection against behavior deficits than 20 mg/kg. Therefore, a dose of 40 mg/kg was chosen for all subsequent histology and biochemical experiments.
The second study was to evaluate the effect of DHF40 on brain edema, histological damage, apoptosis, and TrkB-related signals. Testing was as follows: 1) blood biochemistry and histology at day 3 or day 28 (n = 6/group); 2) brain water content and hemoglobin assay at day 3 (n = 6–7/group), and 3) western blot analysis and enzyme-linked immunosorbent assay (ELISA) at day 1 and day 3 (n = 5–7/group). The third study was to investigate the therapeutic potential of delayed administration of 7,8-DHF after ICH. Either DHF40 or vehicle was administered ip at 3 h following ICH and subsequently daily for 3 days (3, 27, 51 and 75 h), and protective effects were assessed using cresyl violet staining and Fluoro-Jade B (FJB) histology (n = 7/group).
Intracerebral hemorrhage model
ICH model was according to a previous study . Briefly, mice were anesthetized with sodium pentobarbital (65 mg/kg, ip; Rhone Merieux, Harlow, UK) and placed into a stereotaxic frame. After retracting the scalp, a Hamilton syringe with a 30-gauge needle was implanted through a 1-mm-diameter burr hole into the right striatum (stereotaxic coordinates: 0.8 mm anterior and 2.5 mm lateral to bregma, 2.5 mm in depth). Bacterial collagenase (0.0375 U in 1 μL of saline; type VII-S; Sigma-Aldrich, St. Louis, MO, USA) was infused into the brain at a rate of 0.1 μL/min over 10 min with an infusion pump to induce ICH, and the needle was left in place for an additional 20 min to prevent reflux. After surgery, the craniotomy was sealed with dental cement to close the scalp. Mice were placed on a heated pad throughout the surgery and recovery period to maintain body temperature at 37.0 ± 0.5 °C. Sham-operated mice received an equal volume of normal saline in the same manner.
Metabolic characteristics assessment
Following terminal anesthesia, venous blood was collected via direct right atrial puncture. The obtained blood was centrifuged (3500 rpm for 5 min), and the serum was stored at − 20 °C until analysis. Serum blood urea nitrogen (BUN), creatinine (CRE), alanine aminotransferase (ALT), were measured by a chemistry autoanalyzer (Synchron Clinical System LX20; Beckman Coulter, Fullerton, CA) to assess renal and liver functions. Hematological determinations were performed using an automatic hematology analyzer ADVIA 2120i (Siemens, Germany). Total white blood cell (WBC), red blood cell (RBC), hemoglobin (HGB), and platelet (PLT) were determined.
Behavioral testing was performed before ICH and at 1, 4, 7, 14, 21, and 28 days post-ICH. Animals were pre-trained for 3 days for both the rotarod and beam walking tests.
Modified neurological severity score
The modified neurological severity score (mNSS) included sensory, reflex, motor and balance tests. The neurological function was scored on a scale of 0–18. One point was given for the inability to perform each test or for absence of a testing reflex.
The speed of an accelerating rotarod was gradually increased from 6 rpm to 42 rpm within 7 min to observe motor function and balance; the time for mice to fall off was recorded.
The test was used to assess fine motor coordination and function by measuring the ability of the animals to cross an elevated beam. The time for mice to traverse the beam (not to exceed 60 s) and the hindlimb performance as it crossed the beam (based on a 1 to 7 rating scale) were recorded. A score of 7 was given when animals traversed the beam with two or fewer footslips; 6 was given when animals traversed the beam with less than 50% footslips; 5 was given when animals traversed the beam for more than 50% but less than 100% footslips; 4 was given when animals traversed the beam for 100% footslips; 3 was given for traversal with the affected limb extended and not reaching the surface of the beam; 2 was given when the animal was able to balance on the beam but not traverse it; 1 was given when the animal could not balance on the beam.
Brain water content
Brain edema was examined by measuring brain water content using the wet-dry/wet brain weight method . After decapitation (under anesthesia), the brains were immediately removed and divided into five parts, consisting of the ipsilateral and contralateral cortex, ipsilateral and contralateral basal ganglia and the cerebellum (which served as an internal control). The samples were weighed (wet weight), then baked at 100 °C for 24 h and reweighed (dry weight). Water content was determined as [(wet weight-dry weight)/wet weight] × 100%.
The hemoglobin content of brains that had undergone ICH were quantified using a spectrophotometric assay according to previously described methods . Mice were transcardially perfused and the ipsilateral striatum regions were collected following ICH. Distilled water (300 μL) was added to the hemorrhagic hemisphere, followed by homogenization for 30 s and sonication on ice for 1 min. After centrifugation at 13,000 rpm for 30 min, 60 μL of supernatant was reacted with Drabkin’s reagent (240 μL; Sigma-Aldrich) for 15 min at room temperature. Optical density was then measured at a wavelength of 545 nm to assess the concentration of cyanmethemoglobin. To generate a standard curve, blood was collected by cardiac punctures from anesthetized control mice. Incremental volumes of this blood (0, 0.5, 1.0, 2.0, 4.0, and 8.0 μL) were then added to 300 μL of tissue lysate from a normal hemispheric sample.
Tissue processing and histology
Following terminal anesthesia, mice were transcardially perfused with PBS followed by 4% paraformaldehyde. Brains were removed, post-fixed in 4% paraformaldehyde overnight, cryoprotected with 30% sucrose, and then sectioned coronally (10 μm) from the level of the olfactory bulbs to the visual cortex.
Fluoro-jade B staining
Fluoro-Jade B (FJB; Chemicon, Temecula, CA, USA) is a polyanionic fluorescein derivative that binds with high sensitivity and specificity to degenerating neurons. Briefly, sections were rehydrated in graded ethanol solutions (100 and 70%, 5 min each) and distilled water, incubated in 0.06% KMnO4 for 30 min, rinsed in distilled water for 2 min, incubated in a 0.001% solution of FJB for 30 min, and observed under a fluorescence microscope (Olympus BX-51; Olympus, Tokyo, Japan) at 450–490 nm.
Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay is used for the detection of fragmented DNA by labeling it with fluorescein isothiocyanate (In situ Cell Death Detection Kit; Roche Molecular Biochemicals, Mannheim, Germany). Sections were incubated in TUNEL reaction mixture containing terminal deoxynucleotidyl transferase (TdT) for 60 min at 37 °C. Sections were then observed and photographed under a fluorescence microscope (Olympus BX-51) with blue (450~490 nm) excitation light. Negative controls were prepared by omission of the enzyme TdT.
1) To assess the cellular source of TrkB, double immunofluorescence labeling was performed by simultaneous incubation of sections with rabbit anti-TrkB (1:100; Cell Signaling Danvers, MA, USA) overnight at 4 °C with mouse anti-NeuN (a neuronal marker; 1:100; Millipore, Billerica, MA, USA), rat anti-GFAP (an astrocyte marker; 1:200; Invitrogen, Camarillo, CA, USA), and mouse anti-F4/80 (a microglia/macrophage marker; 1:100; Serotec, Düsseldorf, Germany).
2) To assess protein expression of pTrkB, pAkt Ser473 or BDNF, double immunofluorescence labeling was performed by simultaneous incubation of sections with rabbit anti-pTrkB (1:100; Cell Signaling), rabbit anti-pAkt Ser473 (1:100; Cell Signaling) or rabbit anti-BDNF (1:100; Santa Cruz, CA, USA) overnight at 4 °C with mouse anti-NeuN (1:100; Millipore) or rat anti-GFAP (1:200; Invitrogen).
Sections were washed, followed by incubation with Alexa Fluor 488- or Alexa Fluor 594-conjugated secondary antibodies (1:500; Molecular Probes, Eugene, OR, USA) for 2 h.
Injury volume and hemispheric enlargement assessment
Injury volume, hemispheric atrophy, and hemispheric enlargement ratios were quantified using coronal sections stained with cresyl violet at 20 rostral-caudal levels that were spaced 200 μm apart. Sections were analyzed using ImageJ software version 1.50i (ImageJ, National Institutes of Health, Bethesda, MD, USA). Volume measurement was computed by a summation of the areas multiplied by the interslice distance (200 μm). Hemispheric atrophy was assessed using the following formula: ([Contralateral hemisphere or striatal volume − ipsilateral hemisphere or striatal volume]/contralateral hemisphere or striatal volume) × 100%. Hemispheric enlargement was assessed using the following formula: ([ipsilateral hemisphere volume − contralateral hemisphere volume]/contralateral hemisphere volume) × 100%. Analysis was performed by two experimenters who were blinded to all animal groups. Inter-rater reliability was within 10%.
Quantification of FJB, TUNEL and co-localization staining
FJB, TUNEL assay and double immunofluorescence were quantified on three consecutive sections from the hemorrhagic core at a level of 0.24 mm from the bregma. The number of positive cells was counted in an area of 920 × 860 μm2 in 10–12 non-overlapping fields immediately adjacent to the hematoma using a magnification of 200× as previously described . The total number of FJB-positive cells, pTrkB-positive neurons, pAkt Ser473-positive neurons, BDNF-positive neurons or BDNF-positive astrocytes were expressed as the mean number per field of view. Quantification of TUNEL staining was expressed as (TUNEL-stained nuclei/ DAPI-stained nuclei) × 100%. Analysis was performed by two experimenters who were blinded to all animal groups. Inter-rater reliability was within 10%.
Western blot analysis was performed as previously described . A 3–5-mm coronal section from the injured hemisphere was collected following ICH or sham surgery. Primary neuron cultures were collected at 3 h or 24 h after hemin-induced injury. All samples were centrifuged at 14,000 g for 30 min, and supernatants were used for further protein analysis. Protein concentration was determined by Bradford reagent at 595 nm. Protein samples were denatured in gel-loading buffer at 100 °C for 5 min, separated by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels, and transferred to Immobilon-P membranes (Millipore). Membranes were blocked with 5% milk in PBS-XT and probed overnight at 4 °C with primary antibodies including rabbit anti- cleaved caspase-3 (cCP-3, 1:1000), rabbit anti-pTrkB (1:1000), rabbit anti-pAkt Ser473 (1:1000), rabbit anti-pAkt Thr308 (1:1000), rabbit anti-total Akt (1:1000), rabbit anti-p-extracellular signal-regulated kinases (pErk 44/42; 1:1000), rabbit anti-total Erk (1:2000), rabbit anti-pAsk-1 (1:1000), rabbit anti-total Ask-1 (1:1000) and rabbit anti-pFOXO-1 (1:1000) from Cell Signaling; rabbit anti-total trkB (1:1000), rabbit anti-Bcl-2 (1:1000) and rabbit anti-Bax (1:1000) from Santa Cruz; rabbit anti-Smac/DIABLO (1:500) and rabbit anti-VDAC (1:1000) from Abcam (Cambridge, MA, USA); mouse anti-XIAP (1:1000) and rabbit anti-Cytochrome C (CytoC; 1:1000) from BD Biosciences (San Jose, CA, USA); and mouse anti-β-actin (1:10,000, Sigma-Aldrich).
Isolation of mitochondria
Dissected hemispheres (prepared as in western blot analysis) were immediately homogenized in 300 μL of ice-cold cytosol extraction buffer (Cytosol/Mitochondria Fractionation kit; Merck, Rockland, Massachusetts, USA) with a protease inhibitor cocktail and DTT. The homogenates were then centrifuged at 700 g for 10 min at 4 °C, and the supernatant was further centrifuged at 10000 g for 30 min at 4 °C. The supernatant obtained after centrifugation at 10000 g was collected as the cytosolic fraction, and the pellet contained the mitochondria. The pellet was resuspended in 50 μL mitochondrial extraction buffer mix containing protease inhibitors and DTT for 10 s and saved as mitochondrial fraction, or maintained intact in PBS at − 80 °C until use.
A 3–5-mm coronal section was taken from the injured hemisphere or sham animals post-ICH. BDNF was measured in brain homogenates using a commercially available ELISA kit (KA0331, Abnova, Walnut, CA, USA).
Primary cortical neuron cultures, cell viability and cytotoxicity assessment
All media supplies for culture work were purchased from Invitrogen. Primary neuronal cultures were prepared from embryonic C57BL/6 mice at day 15.5 as previously described. Cortical-striatal tissue from 8 to 10 embryos were isolated and digested in 0.5 mg/mL papain, dissociated in Hibernate-A medium (containing B27 supplement), and cultured on 6-well plates at a density of 1 × 106 cells/well. Cultures were maintained in Neurobasal medium supplemented with B27, 10 units/mL penicillin, 10 mg/mL streptomycin, and 0.5 mg/ml glutamine. Three days after plating, arabinofuranosyl cytidine was added to inhibit proliferation of glial cells, and half of the medium was removed and replaced with fresh medium at 4 days. The cells were incubated at 37 °C in an atmosphere containing 10% O2, 85% N2, and 5% CO2 and neurons were used at day 10 in vitro. The purity of neurons was 95% as determined by NeuN immunohistochemical staining. Primary neurons were treated with 10 μM hemin or co-treated with 7,8-DHF and hemin for 24 h and then analyzed by following assessments. Cell viability and cytotoxicity were assessed 24 h post-injury using the 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2-tetrazolium bromide (MTT) reduction assay (Sigma-Aldrich; St. Louis, MO) and lactate dehydrogenase (LDH) release assay (LDH assay kit; Roche), respectively. Cells were incubated at 37 °C for 2 h with MTT (0.5 mg/mL; Sigma-Aldrich), and then a solution of anhydrous isopropanol, HCl (0.1 N), and 0.1% Triton X-100 was added to dissolve the water-insoluble formazan. The optical density was determined at 570 nm. Cell viability was expressed as a percentage of the control culture. LDH release was used to quantify cytotoxicity in cultured neurons. Culture supernatants were collected, incubated with substrate mixtures, allowed to undergo a coupled enzymatic reaction that results in the conversion of iodonitrotetrazolium to formazan, and assessed spectrophotometrically for LDH activity at 500 nm. Percent cytotoxicity was calculated by subtracting LDH content in injured cells from total LDH in undamaged controls.
The experiments were repeated 4 times with different batches of primary cultures.
Values are expressed as mean with standard error of the mean (mean ± SEM). Student’s t-test was used to evaluate the difference between two groups. One-way or two-way analysis of variance (ANOVA) followed by post-hoc Bonferroni t-test was used for multiple groups to determine significant differences. Statistical significance was set at P < 0.05.