Twelve 11-month-old male SAMP8 mice were randomly divided into two groups: the control group was fed a normal diet (Fwusow Industry Co, Ltd, Taiwan), and the spirulina group was fed a normal diet with oral supplementation SP (400 mg/kg body weight) for 6 weeks. The animals were housed (in groups of four mice per cage) in a temperature-controlled room with a constant 12-hr light–dark cycle. Food and tap water were freely available throughout the experiments. The Institutional Animal Care and Use Committee of Dalin Tzu Chi Hospital approved the protocol used in this study.
Preparation of SP
SP from Far East Bio-tec Co, Ltd (Taipei, Taiwan) was prepared as follows: suspension of S. platensis powder and pure water was disrupted for 24 hours and centrifuged. The supernatant was collected and lyophilized. The lyophilized SP contained 35–45% polysaccharides, 15–25% phycobiliproteins (C-phycocyanin and allophycocyanin), 10–20% proteins other than phycobiliproteins, 10–12% ash and 5–8% water. Among that, the active compounds in the extract were sulfated polysaccharides and phycobiliproteins.
Auditory brainstem responses (ABRs)
ABRs (Intelligent Hearing Systems, Miami, FL) were measured at the start (11 months of age) and at the end of the study under general anesthesia with an intraperitoneal injection of sodium pentobarbital (65 mg/kg). Click sounds, 8- and 16-kHz tone bursts were delivered sequentially to the left ear through earphones (Telephonics Corp, Farmingdale, NY). Subdermal needles were used for recording. The active electrode was inserted at the vertex; the reference electrode was ventrolateral to the left ear; and the ground electrode to the low back above the tail. The amplified responses were then averaged by a computer and displayed on a computer screen.
ABR thresholds were obtained by reducing the stimulus intensity in 5-dB intervals and increasing the stimulus intensity in 3-dB intervals to identify the lowest intensity at which ABR waves I-V were detected by one well-trained audiologist who was blinded to all groups. The ABR data were stored digitally on disks for offline measurements and analysis of latency of ABR components later.
Histology and immunochemistry of the cochlea
At the end of the study, the mice were sacrificed by decapitation under general anesthesia with an intraperitoneal injection of pentobarbital (65 mg/kg). Before being euthanized, animals were perfused of 2.5% glutaraldehyde and 4% paraformaldehyde in phosphate buffered saline (PBS: NaCl, 150 mM; KH2PO4, 2 mM; Na2HPO4 2H2O, 8 mM, pH 7.4) via an intracardiac injection. For each mouse, the temporal bones were removed, and the cochlea was perfused with the above fixative through the oval and round windows to an outlet within the apex. After 24-hr post-fixation in the same fixative at 4 °C, the temporal bones were decalcified in 10% EDTA solution, 4% paraformaldehyde, and 2.5% glutaraldehyde in PBS (pH 7.4) for 1 week at room temperature. The temporal bones were then rinsed in PBS, dehydrated through a graded series of alcohol and xylene, and embedded in paraffin.
For histologic staining
Embedded cochleae were sectioned at 5-µm thickness at the mid-modiolar level. The cochlear structures were observed with hematoxylin and eosin (H & E) staining using a light microscope. The cell density of neurons in SG, within a central region of 50 × 50 µm2 at the middle turn of cochlea, were measured under × 400 magnification. The numbers of SGN were counted by the presence of stained nucleus. The morphology and numbers of OHCs and inner hair cells (IHCs) in the Organ of Corti of cochlea were observed under × 400 magnification.
For immunochemical staining
The sections were heat-treated at 95 °C in 10 mM sodium citrate buffer for 20 min, incubated in 3% H2O2 for 10 min at room temperature, and blocked in 5% skim milk to prevent non-specific labeling. The specimens were then incubated overnight at 4 °C with one of the following primary antibodies: rabbit monoclonal anti-PARP-1 (1:500 dilution) (Abcam plc, Cambridge, U.K.), and rabbit anti- AIF (1:1000 dilution) (R&D systems, Inc., U.S.A.), were obtained from Cell Signaling Technology (TAIGEN, TPE, R.O.C.).
Slides were washed in PBS three times, exposed to a secondary antibody for 1 h at room temperature, and stained using the DAB staining kit (R&D systems, Inc., U.S.A.) according to the manufacturer’s protocol. Slides were dehydrated, mounted, and observed through a light microscope. The images were acquired using a CCD camera (Dage-MTI Inc, Michigan City, Ind) connected to a personal computer and analyzed using image analysis software (Image-Pro Plus, version 6.0; Media Cybernetics, Silver Springs, MD).
The above features were observed blindly in two adjacent cochlear sections at 10-slides apart (equals to 50 µm apart) at the same mid-modiolar level. All data were acquired from two cochleae and averaged for each mouse by one well-trained researcher. Stained regions were quantified as the percentage of the entire target area.
The brainstem tissue of SAMP8 mice were dissected cut into small pieces (50–60 mg), homogenized with an appropriated volume of ice-cold lysis buffer with protease/phosphatase inhibitor cocktail, and incubated in the ice for 10 min. Cytoplasmic extraction reagent was added to the tube and centrifuged for 5 min (~ 16,000 xg). The supernatant was immediately transferred to a clean tube (cytoplasmic extract), and added the nuclear extraction reagent to the insoluble fraction. The tube was placed on ice and continued vortexing for 15 seconds every 10 minutes, for a total of 40 minutes, and centrifuged for 10 minutes (~ 16,000 xg). The supernatant was immediately transferred to another clean tube (nuclear extract). Total protein in the supernatant was measured by the BCA method (Bio-Rad, Hercules, CA). Equal amount of protein (20–50 µg) were separated through sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. The membranes were washed with TBST buffer (10 mM Tris-HCl, 150 mM NaCl; pH 7.5) supplemented with 0.1% Tween 20 and then blocked with TBST containing 5% nonfat dried milk. The membranes were incubated overnight with primary antibodies. After being washed three times with TBST, the membranes were exposed to secondary antibodies coupled to horseradish peroxidase for 2 h at room temperature. The membranes were washed three times with TBST at room temperature. Immunoreactivity was detected using ECL reagents. Anti-cleavaged PARP1 and anti-PARP1 (1:500, Boster, CA), anti-AIF (1:10000, Boster, CA), and anti-GADPH (1:50000, Boster, CA) antibodies, were obtained from Cell Signaling Technology (TAIGEN, TPE, ROC). Densitometric analysis of the data was performed using FusionCapt Advance Camera and FusionCapt Advance analyzer ver. 16.07 software.
ABR thresholds; counts of OHCs and IHCs, SGN cell density, ratios of PARP-1 and AIF staining in the cochlea, ratios of cleavage PARP-1versus PARP-1 protein expressions, and AIF versus GADPH protein expressions in the brainstem, were compared between two groups using a Student’s t test with Welch’s approximation. All analyses were performed using STATA 10.0 software (Stata Corp, LP, College Station, TX). P values of < 0.05 were considered significant.