Animals
Male Sprague–Dawley rats (weighting 180–200 g) were housed at the Laboratory Animal Center of North Sichuan Medical College (NSMC 201,810), under standard temperature, humidity and circadian rhythm conditions with ad libitum access to food and water. All animal experiments were performed in accordance of the Animal Research Committee of North Sichuan Medical College and were approved by the Chinese Animal Welfare Act for the use and care of laboratory animals.
Experimental groups
The commonly used doses of OSI-906 in in vivo interventional studies in rodents range 25–75 mg/kg [16]. Therefore, we divided the animals into the vehicle group, 25 mg/kg OSI-906 group, 50 mg/kg OSI-906 group, and 75 mg/kg OSI-906 group (n = 18 in each group, 5 for electrophysiological recording and 13 for behavioral recording and related testing). OSI-906 was diluted to a volume of 2 ml with tartaric acid solution and was administered by gavage once daily for 3 consecutive days, while the vehicle group was given 2 ml of tartaric acid solution only (Fig. 1).
Animal model of epilepsy
The pilocarpine model, which reproduces most of the seizure phenotypes, was used in this study. The electroencephalogram (EEG) data and the neuropathological characteristics of temporal lobe epilepsy were recorded as described previously [19, 20]. In brief, the rats were injected with lithium chloride (Sigma, USA; 127 mg/kg, i.p.) 20 h and atropine sulfate (1 mg/kg, i.p.) 30 min before the first pilocarpine (35 mg/kg, i.p.) administration. If rats did not develop epileptic seizures 30 min after the first injection of pilocarpine, the dose of pilocarpine was increased by 20% every 10 min until the development of level-4–5 seizures. The total number of pilocarpine injections was limited to five per animal. After 60 min of status epilepticus (SE), diazepam (DZP, 10 mg/kg), phenobarbital (25 mg/kg), and atropine sulfate (1 mg/kg) were injected intraperitoneally to terminate the convulsive seizures [21]. The seizure activity was scored according to the Racine’s standard criteria [22]. Only rats that achieved recurrent seizures of stage 4 to 5 were used for subsequent tests.
Surgical procedures and electrophysiological recordings
The rat was anesthetized with chloral hydrate (350 mg/kg, i.p.) and flurbiprofen axetil was injected (5 mg/kg, Beijing Tide Pharmaceutical Co., Ltd., Beijing) through a tail vein to relieve pain. Then, the rat was placed in a stereotaxic apparatus. A microwire array (4 × 4 array of platinum-iridium alloy electrodes, each 25 μm in diameter) was implanted in the right dorsal hippocampus (anterior–posterior –3.6 mm, medial–lateral 2.8 mm, dorsal–ventral –3.5 mm) one week before recording and fixed with dental cement as described by Paxinos and Watson (2007). Local field potentials (LFPs) were preamplified (× 1000), filtered (0.1–1000 Hz), and digitized at 4 kHz using an OmniPlex®D Neural Data Acquisition System (Plexon, Dallas, TX). Electrophysiological recordings were made with the direct current-coupled head stages, and all recordings were referenced to the two ground screws.
Analysis of electrophysiological data
We employed NeuroExplorer® (v4.0; Plexon, USA) and MATLAB software (v7.1, R2009a; MathWorks, Inc., Natick, MA) to calculate the electrophysiological data. A fast Fourier transform was applied to power spectrum analysis after digital filtering with 0.1–1000 Hz bandpass. The frequency spectra of continuous variables and the neuronal rate histograms were obtained by the power spectral density analysis of our previous method [23]. We calculated the high-frequency discharge energy during seizures including ripples (80–200 Hz). The MATLAB function in the Signal Processing Toolbox was used to compute the power of ripple oscillators and magnitude-squared coherence. MATLAB and GraphPad Prism softwares (GraphPad Software, Inc., La Jolla, CA) were used for statistical analysis and image processing.
Blood glucose monitoring
Blood glucose was measured by a Roche glucometer (Roche, Germany) in venous blood from the tail vein before treatment, 1 h after the first intragastric administration of vehicle or OSI-906, 1 h after gastric perfusion of OSI-906 on the third day, and 1 h after the epileptic seizures. Animals were fasted for more than 12 h before OSI-906 intervention. As we found that 75 mg/kg of OSI-906 treatment significantly increased blood glucose and aggravated epileptic seizures, and the mortality rate reached 100% during the acute epileptic seizure, subsequent relevant tests were not completed in this group.
18F-FDG-microPET imaging
MicroPET is a non-invasive imaging technique that is often used to record and analyze the correlation between neural function changes and brain energy metabolism in small animals. To determine the effect of IR/IGF-1R inhibitor OSI-906 on glucose uptake in the brain after the epileptic seizures, a 18F-FDG-PET-CT scanner (SiemensInveon Multi-Modality System, Erlangen, Germany) was used to detect cerebral glucose uptake at 1 h after the pilocarpine-induced seizures. First, the rats were fixed and injected with 18F-FDG (500 µCi) through the tail vein as a tracer to detect the relative cerebral glucose metabolism rate. Thirty minutes later, they were anesthetized with isoflurane (induction period: 5% isoflurane + 1 L/min O2, maintenance period: 1% isoflurane + 1 L/min O2) using an inhaled anesthesia system. Then the rats were scanned in the prone position in a three-dimensional model for 8 min (field of vision: 85.62 mm × 125.58 mm), and the static PET data were collected for 7 min. The CT signal was mainly used for anatomical description and attenuation correction of 18F-FDG-PET images.
PET data analysis
The imaging data were imported into the Inveon Research Workplace software for analysis, and data were collected in the form of lists. The cerebral metabolism rate was reconstructed using the maximum posterior probability algorithm, with a pixel size of 0.4 × 0.4 × 1.2 mm3. Glucose metabolism in the brain was evaluated as the whole-brain average standard uptake values (SUV) using the following equation: SUV = Ct/ID*Wt.Ct(MBq/cm3), in which Ct indicates the activity concentration after the decay correction of the measured brain region; ID (mCi) indicates the injection dose of 18F-FDG; and Wt (kg) represents the weight of the rat [24]. We recorded the maximum and the mean SUV values of normal rats, as well as SUV value 1 h post-seizure, and that post the OSI-906 intervention (OSI-906 50 mg/kg + SE).
Projection electron microscopy
The ultrastructural changes of nucleus and mitochondria in rat hippocampal neurons were observed by projection electron microscopy in the vehicle control, SE, and OSI-906 + SE groups (n = 3). All animals were deeply anesthetized with 10% chloral hydrate and then injected with fresh normal saline to the heart. The rat was cardinally perfused with a mixture of glutaraldehyde (2%) and paraformaldehyde for heart reperfusion and tissue fixation, and then the brain was collected. The hippocampal tissue was isolated immediately, and the CA1 area of the hippocampus was cut into 1 mm3 pieces. Three specimens were taken from each rat and fixed in 4% glutaraldehyde solution. The fixed tissues were sent to the electron microscope room of Chongqing Medical University for unified specimen preparation. Images under projection electron microscopy (JEOL JEM-1400, Japan) were captured.
Western blotting
Hippocampal tissues were collected 24 h after pilocarpine administration and stored at -80 °C for western blotting. The tissues were homogenized in lysate buffer containing phosphatase and protease inhibitors (Sigma, St. Louis, MO), centrifuged (Sigma-Aldrich, USA) and the supernatant collected. The total protein concentration was determined with the BCA protein assay (Beyotime Institute of Biotechnology, China). The total protein samples were loaded at 40 μg/lane for sodium dodecyl sulfate polyacrylamide gel (5% spacer gel; 10% separating gel) electrophoresis, followed by electro-transfer onto polyvinylidene fluoride membranes (Millipore).
The membranes were blocked with 5% non-fat milk at room temperature for 120 min, then incubated with anti-p-IR (Tyr1361), anti-IR, anti-pIGF-1R (Tyr1135), anti-IGF-1R, anti-p-AKT (Ser473), anti-AKT or anti-β-actin (1:500–1000, Cell Signaling Technology, Inc Boston, MA, USA) antibody for 16 h at 4 °C. After TBST wash, the membranes were incubated with goat anti-rabbit IgG (1:5000, Cell Signaling Technology, Inc Boston, MA) for 1 h. Electrochemiluminescence system (Bio-Rad, CA, USA) was used to visualize the protein bands with a chemiluminescence kit (Biosharp, Beijing, China). Blot intensities were analyzed with QuantityOne software (Bio-Rad Laboratories, version 4.6.2, Hercules, CA).
Statistical analysis
All data were analyzed using the SPSS 19.0 statistical software (IBM Corp., Armonk, NY), and the values are expressed as mean ± SEM. All data were tested for normality and if non-normal, a non-parametric test was used. One-way analysis of variance (ANOVA) was used for comparisons between more than two groups; the Student’s t-test was used for comparison between groups of multiple samples, and the Bonferroni test was used for post-test analysis. The Wilcoxon rank-sum test (Mann–Whitney U-test) was performed to compare two groups of ordinal variables. The K-W test was used to compare the classification data of multiple groups, and Fisher’s exact test was also used to compare classification variables between two groups. P < 0.05 was considered as statistically significant.