Induction of diabetes
One hundred twenty male SD rats (10-month-old, weight300-360 g) were used. Three quarters of them were randomized and type 1 diabetes was induced after overnight fasting by a single intraperitoneal (i.p.) streptozotocin administration (60 mg/kg in 10 mM citrate buffer, pH 4.5, Sigma-Aldrich, St Louis, MO, USA). Control rats received the same volume of sterile saline. Blood glucose levels were measured with a glucometer in non-fasted rats. At 72h after STZ injection, rats with blood glucose concentration higher than 16.7 mmol/L was defined diabetic.
The rats were randomly divided into 4 groups: (1) NC9W group, rats were gavaged with saline daily (4 ml/kg) and fed for 6 weeks;(2) DM9W+S group, diabetic rats were gavaged with normal saline for 6 weeks;(3) DM9W+G60 group, diabetic rats were administered with gastrodin (60 mg/kg daily, dissolved in 0.9% saline) for 6 weeks; (4) DM9W+G120 group, diabetic rats were administered with gastrodin (120 mg/kg daily; dissolved in 0.9% saline) for 6 weeks.
Western blotting analysis
Fresh tissue samples were collected from the cerebellum. To analyze the expression levels of proteins, equal amounts of total proteins were subjected to 10% (w/v) SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Darmstadt, Germany). Membranes were then blocked with 5% skim milk for 2 hours at room temperature and probed with anti-GluR2 (1:2000; Abcam, Cambridge, UK), anti-p-GluR2(phospho S880) (1: 500; Mybiosource), anti-PKC (1:1000; Abcam, Cambridge, UK) anti-BDNF (1:1000; Millipore, Darmstadt, Germany), anti-TrkB (1:750; Abcam, Cambridge, UK), anti-β-tubulin (1:1000; Santa Cruz Biotechnology, Dallas, TX) antibodies at 4°C overnight. Subsequently, membranes were incubated with horseradish peroxidase- (HRP-) conjugated goat anti-rabbit (Thermo Fisher Scientific, Waltham, MA) or rabbit anti-rat (Thermo Fisher Scientific, Waltham, MA) IgG for 2 hours at room temperature and then reacted with a pro-light HRP agent (Santa Cruz Biotechnology, Dallas, TX) after washing. The result of chemiluminescence was recorded with an imaging system and semiquantified using the ImageJ software (National Institutes of Health, Bethesda, MD).
Paraffin Embedding and Sectioning
For histology and double immunofluorescence labeling, four rats in each group were anesthetized with 10% chloral hydrate (4 mL/kg) and perfused with filtered saline (150 mL and 12 mL/min), followed by 4% paraformaldehyde in phosphate-buffered saline (PBS), pH 7.4 (500 mL and 12 mL/min). The cerebellum was removed, dehydrated, embedded in paraffin, and cut into 4 μm thick sections. The sections were transferred to silane-coated microscope slides and dewaxed.
After dewaxing and hydration, the paraffin sections were processed for routine hematoxylin and eosin (H&E) staining.
Double Immunofluorescence Labeling
The cerebellum was removed, immersed in 4% formaldehyde, dehydrated in ethanol, cleared in xylene and embedded in paraffin blocks. Paraffin-embedded tissue sections of 4μm thickness were deparaffinized and hydrated through a series of graded alcohol. The tissues sections were incubated in citrate buffer for antigen retrieval and the slices were incubated with 5% normal goat serum. The following primary antibodies were used: rabbit anti-Calbindin D-28k antibody (1:1000; Swant) mouse anti-GluR2 antibody (1:500; Abcam) mouse anti-p-GluR2 (phospho S880) (1: 200; Mybiosource), mouse anti-BDNF (BDNF, 1:500, Millipore, Darmstadt, Germany), and TrkB (1:500, Abcam, Cambridge, UK) for Purkinje cell labeling. After incubation and washing, anti-mouse and anti-rabbit Alexa Fluor 488 and 568 second antibodies (1:500; Invitrogen, were added. After mounting, images representing at least one cerebellum section each from four rats at different time points were captured under a confocal fluorescence microscope (Olympus, Tokyo, Japan). Immunofluorescence labeling for the various antibodies directed against the respective cell types was consistent and reproducible across different rats.
Data were expressed as the mean ±standard deviation (SD) and analyzed using SPSS 17.0 statistical software (SPSS, USA). Comparisons among groups were performed using one-way analysis of variance and pairwise comparison was performed using the LSD-t test. *p<0.05, **p<0.01 was considered statistically significant.