Experimental Research On Apoptosis Of Neurons And Therapeutical Effect Of PACAP After Acute Traumatic Brain Injury In Rats

Background and objective Traumatic brain injury(TBI) is a serious and complex trauma including primary injury resulting from outside force, affecting central nervous system directly and secondary injury caused by tissue reaction, physiopathological changes and so on. Although the mechanism of secondary injury is still unknown, the effect of post-traumatic neuronal apoptosis in secondary injury has been attached great importance to gradually. Apoptosis , also called programmed cell death, refers to the procedure that cells end their lives following ascertain programs in a physiological or pathological condition. More and more evidences show TBI may induce apoptosis and neuronal apoptosis plays an important role in neurodegeneration and neuron loss. Caspases are a family of cysteine requiring aspartate proteases with sequence similarity to Ced-3 protein of Caenorhabditis elegans. These proteases have been found to play an important role in the morphological and biochemical changes of apoptotic cell death. Among 14 identified caspases, caspase-3 appears to be the major effector of cell apoptosis and plays critical and specific roles under the control of programmed cell death. PACAP is a member of the VIP/secretin/glucagon peptide family. PACAP is widely distributed in the central and peripheral nervous systems and exerts numerous effects. Currently available data indicate that PACAP is a promising neuroprotective peptide. PACAP plays an important role during the development of the nervous system and in regeneration following nervous injuries. It also has strong anti-apoptotic effects in several neuronal cultures and in vivo. The aim of the present study is to investigate neurons apoptosis, the changes of caspase-3 and the effects of i.c.v. administration with low does PACAP on them after acute TBI in brain tissue of rats. Methods A total of 136 Spraque-Dawley rats were randomly divided into three groups, including a normal contral group, a moderate brain injury group and a post-treatment with PACAP group. Moderate TBI models of rats were constructed with gas percussion method. The low dose PACAP(5μl) were used to inject intracerebroventricularly. The rats were sacrificed at 2 h, 12h, 24h, 48h and 72h post injury(n=8, each time-point), respectively. The specimens of the injured cerebral cortex and hippocampus were collected for detecting apoptosis of neurons and the effect of low dose PACAP on apoptosis by terminal deosxynucleotide transferase-mediated doxyuridinde-bitin nick end labeling(TUNEL),microscopy and transmission electron microscopy. The expression of caspase-3 protein was detected by immunohistochemistry and the activity of caspase-3 was detected with caspase-3 fluorescent assay kit. The expression of PACAPmRNA from the injured cerebral cortex and hippocampus was detected due to RT-PCR. Results 1. It's found neurons apoptosis at 2h after moderate TBI and reached to the peak at 24 to 48h and retained high levels, which showed significant difference with the control groups(P<0.05) 2. Caspase-3 began to increase significantly at 2h and retained at high level and reached to peak at 24h following TBI in brain tissues of rats. 3. The level of PACAPmRNA in the injured cerebral cortex and hippocampus began to decrease at 2h post injury, far lower than that of control group, and reached to the lowest value at 12h, then increased slowly. 4. The levels of caspase-3 positive neurons and TUNEL-positive neurons and the activity of caspase-3 in the injured cortex and hippocampus after treatment with low-dose PACAP decreased significantly with contrast to that of the control group in every time point (2h, 12h, 24h, 48h 72h) (P<0.05). Conclusions 1. TUNEL-positive neurons brain tissue increased reactively in early period and continued to increase until to 72h post injury. It suggested that apoptosis played an important role in the whole course of secondary brain injury after TBI. 2. The expression of caspase-3 coincided with apoptosis of neurons after acute TBI. These data indicated that caspase-3 gene participated in regulating apoptosis after traumatic brain injury. 3. The treatment of low-dose PACAP can reduce the activity of caspase-3 and protect neurons from apoptotic death caused by acute TBI.