Effect Of Anti-TNF-α Antibody On Apoptosis-related Molecules (Caspase-3. Bax. Bcl-2) In Different Brain Areas Of Rats After Subarachnoid Hemorrhage

Part I Gene expression of apoptotic molecule caspase-3 and bax and the anti-apoptotic molecule bcl-2 at different time points of SAH in hippocampusObjectiveTo investigate time course of apoptosis-related molecules (Caspase3,Bax,Bcl-2) changed in hippocampus after subarachnoid hemorrhage (SAH) and find out the appropriate time point to improve apoptosis after SAH.Material and methodAnimals Wistar rats (2-month-old males,250-300 g, n=48) were caged individually at 22℃under 12 hr light/dark cycles. Water and food were available ad libitum. SAH was produced one week later. All procedures were in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the institutional animal care and use committee of Shandong University.Method Rats were randomly divided into six groups. Each group contained eight rats. SAH was produced using male Wistar rats (250 to 300g).The rats were anesthetized with 10% chloral hydrate (4ml/kg, i.p.) and allowed to breathe spontaneously. A small suboccipital incision was made, exposing the arch of the atlas, the occipital bone, and the atlantooccipital membrane. The atlantooccipital membrane was tapped carefully into the cisterna magna using a 27-gauge needle. Fresh nonheparinized blood (0.3 ml) from autologous femoral artery was injected during a period exceeding 2 min. The rats were then placed in a head-down prone position at a 30-degree angle for 30min in order to hold the blood in the basal cisterns. For the second injection, the same method was repeated after 48h (day 2).The animal recovered from the effects of anesthesia and was returned to its cage. These rats were decapitated at 12h, day 1, day 2, day 3 and day 7, respectively, followed by RNA extraction and Real time PCR to test changes of different apoptosis-related molecules during this period. Sham-operated rats were sacrificed one day without injection of blood.Results1. Nerve function deficits were found in rats suffering from SAH.2. Caspase-3 mRNA began to rise at 12 hr and on the first, second and third day it has statistical significance compared with control group (p< 0.01), what's more, Bax mRNA reached to the top at a later peak on the second day (p< 0.01). Different from other two molecules above, bcl-2 mRNA which is an anti-apoptosis molecule did not change during all the test days (p=0.162).Conclusion1. In this study, the second blood injection of SAH through the cisterna magna make bad behavior and diet of rats compared with control group. The expression of apoptosis molecules also has statistical significance compared with control group which means that this is an effective model for SAH study.2.48h after SAH, caspase-3 and bax mRNA rise up to the top while bcl-2 which is an anti-apoptosis molecule has no change through time point study.Part II Effect of anti-TNF-a antibody on caspase-3, bax, and bcl-2 in different brain areas after subarachnoid hemorrhageObjectiveTo investigate the effect of TNF-a inhibitor, anti-TNF-a antibody, on apoptosis (Caspase3,Bax,Bcl-2) in different brain areas after SAH. To identify the mechanism of signal transduction during cell apoptosis.Material and methodAnimalsWistar rats (2-month-old males,250-300 g, n=48) were caged individually. Water and food were available ad libitum. SAH was produced one week later. All procedures were in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the institutional animal care and use committee of Shandong University.MethodRats were randomly divided into three groups:1) Naive control group (n=16) which received no treatment.2) PBS group:rats received PBS microinfusion into brain regions were performed as vehicle control.3) TNF group:anti-TNF-a antibody was microinjected into different brain regions to inhibit the role of TNF-a. Each group contains 16 rats. Anti-TNF-a antibody was injected into brain regions 30 min before the first surgery of SAH. The protocol was preformed following the process below. Rats were anesthetized with 10% chloral hydrate, restrained in a stereotaxic. After sterilize, expose the cranium and find out the bregma, inject the antibody or vehicle 10 ul at the rate of 0.5 ul/min following the coordinates below (coordinates in reference to bregma):anteroposterior (AP),-0.8mm, lateral (L),±1.5 mm, dorsoventral (V),-4.0 mm. Injection cannulas were left in the position for an additional 2 min after the infusion was completed to minimize dragging of the injected liquid along the injection tract. The method of SAH is the same as described above. These rats were decapitated two days after SAH, followed by RNA extraction, Real time PCR to test gene expression changes of different apoptosis-related molecules during this period. In addition, protein levels of three apoptosis-related molecules and caspase-3 mRNA were detected using western blot and ISH, respectively. Data were analyzed with one-way or two-way ANOVA, followed by LSD post hoc comparisons. The significance level was set to 0.05 for all statistical analyses, and all values in the text and figures represent means±S.E.M. Data analyses were performed using SPSS statistical program version 13.0. Results1. In PBS group, there was statistical significance 48 h after SAH in hippocampus compared with naive control and TNF group for both mRNA and protein expression of caspase-3 and bax, but not bcl-2.2. Significant changes of both mRNA and protein were only observed in the PBS group in hypothalamus, while no changes were found in caspase-3, bcl-2 among the three groups (naive control, PBS group, and TNF group).3. Levels of bax mRNA and protein were significantly elevated in PBS group. No noticeable changes were in bcl-2 gene expression. However, significant increase of bcl-2 protein was found in both PBS and TNF group compared with naive group. In addition, changes of caspse-3 were found neither in gene expression nor in protein among the three groups in hypothalamus.ConclusionAnti-TNF-a antibody can inhibit expression of both caspase-3 in hippocampus and bax in hippocampus and prefrontal cortex which suggest that TNF can facilitate apoptosis through activation of bax.