Effects Of IGF-1 On Neuronal Apoptosis And Expression Of Caspase-3 Following HIBD In Neonatal Rats

Hypoxic-ischemic encephalopathy (HIE) is an important cause of neonatal mortality and subsequent neurological handicaps among survivors. But there are no specific therapies to improve long-term outcome for HIE till now. The death of neurons following HIE is composed of two phases: primary neuronal death and delayed neuronal death. Apoptosis plays an important role in the delayed neuronal death. So anti-apoptosis treatment may become one of the important remedies for HIE.Insulin-like growth facor-1 (IGF-1) is one of the neurotrophic factors; it has non-selective neuronal nutrition support function. Experiments showed that IGF-1 could attenuate the amounts of neuronal apoptosis following several pathologic states. But there are few reports about the effects of IGF-1 on neuronal apoptosis and the mechanism of anti-apoptosis of IGF-1 following hypoxic-ischemic brain damage (HIBD) in neonatal animals.In our study, different IGF-1 dosages were injected into the left lateral ventricle following HIBD in neonatal rats. Then we studied the effects of IGF-1 on neuronal apoptosis and on the expression of apoptotic executed protease caspase-3 P20.Materials and Methods1. Unilateral HIBD model: 7-day old SD rats were subject to permanent left carotid arterial ligation, after 2h recovery they were put into a hypoxic (8% oxygen) cabin for 2h at 37 ℃.2. Groups and intracerebroventricular (ICV) injection: 2 hours after HIBD the rats were randomly divided into three groups: treatment group 1 ( 1 μ g IGF-1 ),treatment group 2 ( 5μ g IGF-1) and Control ( PBS as vehicle). The solution was infused into left lateral cerebral ventricle.The location of each injection in relation to lambda was 2.0mm rostral, 1.5mm lateral and 2.0mm deep to the skull surface. The injection speed is 1-2 μ l/min.3. Detection of apoptosis and expression of caspase-3(1) Electron microscopy: Rats were killed 24h after HIBD. Samples of cerebral cortex and hippocampus from damaged hemispheres of rats were removed and processed for electron microscope. The sections were examined using a Philips TECNAI 10 electron microscope.(2) Terminal deoxynucleotidy transferase mediated x-dUTP nick end labeling (TUNEL) staining: Rats were killed 24h after HIBD. The damaged hemispheres were removed and fixed in 10% formaldehyde, and then embedded into paraffin. Paraffin-embedded brains were sectioned at a thickness of 4 μm and processed for TUNEL staining using a TUNEL kit (staining by NBT, cells with blue-black nucleoli were TUNEL-positive cells). Negative controls had the TdT omitted. The slides of positive controls were come from lymphoma.(3)Western blots: Rats were killed 24h after HIBD. The damaged hemisphere were removed quickly and stored at -70℃ until use. After thawing, the tissue was homogenized and centrifuged at 12,000 × g at 4℃ for 10 min. Protein was denatured in SDS gel-loading buffer at 100℃ for 5 min and separated in a 12% SDS-PAGE gel using a mini-protean system with molecular weight markers. Protein measurements were performed using a protein measurement kit, equal amounts of total protein (80 μg) were loaded in each lane. After electrophoresis, the protein was transferred to a nitrocellulose membrane. The blots were incubated in blocking solution( 5% non-fat milk in TBST), followed by incubation with polyclonal goat anti-caspase-3antibody (1:100 dilution in 5% nonfat milk-TBST) at 4℃ overnight. The membranes were then washed with TBST and incubated with horseradish peroxidase-conjugated anti-goat IgG for 60 min at room temperature. Antibody dection was performed with an enhanced chemiluminescence reagent kit of Western blot. Using computer image processing to get the integrated density (ID) of caspase-3 P20 of protein.4. Statistical analysis: All the data was evaluated by the statistical program (SPSS Professional Statistical version 10.0). The difference was analyzed by rank sum test. Difference among groups was analyzed by Kruskal-Wallis test, difference between each other was analyzed by Mann-Whitney...