Apoptosis Related Genes And Mitochondrial Signaling Pathways In Hippocampus In The Rat Model Of Post-traumatic Stress Disorder

ObjectivePost-traumatic stress disorder (PTSD) is a psychiatric disorder, which can occur following the experience or witnessing of life-threatening events such as military combat, natural disasters, terrorist incidents, serious accidents, or violent personal assaults. Symptoms of PTSD may include reliving the experience through nightmares and flashbacks, having difficulty sleeping, and feeling detached or estranged. Nowadays PTSD characteristics with high incidence rate, the long illness course, with difficulty cures and so on, having pay much more attention as the seriously physical and moral affects on people.Hypothalamic pituitary adrenal(HPA)axis is important in the course of stress. PTSD is different from other stresses or mental sicknesses, duing to suppression of plasma cortisol levels in patients. GC is an important hormone secreted by adrenal cortex. Receptor of GC includes glucocorticoid receptor and mineralocorticoid receptor. The hippocampus is an important brain structure,which is associated with learning, memory and cognition, and it is very sensitive to many types of cerebral insults. Recently, structural neuroimaging studies showed that hippocampal volumes were relatively low in PTSD patients. Several lines of evidence have demonstrated a strong relationship between atrophy of the hippocampus and PTSD. However, the mechanisms that cause such atrophy are not well understood.Apoptosis is a genetically controlled and complex process central to the development, homeostasis and disease, turned on in response to environmental signals or triggered by intrinsic factors, and designed to kill errant cells in an orderly and clean way. Apotosis can be induced by three major pathways:the intrinsic pathway(mitochondria-dependent pathway), the extrinsic pathway (death receptor- dependent pathway) and endoplasmic reticulum pathway. There are distinct mechanisms that execute apoptosis according to various apoptotic stimuli. In addition to their function in respiration, mitochondria play an important role in the regulation of cytochrome c, an apoptotic signaling element. Mitochondria act as a crossover point between caspase dependent and independent apoptotic pathways. AIF, which is located in the mitochondrial intermembrane space and is released to the cytosol and to the nucleus in response to death stimuli, is a key trigger of caspase-independent apoptosis. Both of these two major pathways are regulated by caspases. Caspases transduce the apoptotic signal cascade and engage cellular targets leading to programmed cell death.With respect to apoptotic mechanism, people pay much more attention on mitochondria. For non-genomic action, stress increases cortisol levels, which activates the GR. Stress-activated GRdirectly binds to mitochondrial membranes to regulate membrane potential. The GR will also produce a genomic action, in which GR, in interacting with several other molecules,such as heat shock proteins, translocates into the nucleus of the cell, where it binds to DNA and exerts transcriptional action. As one of the downstream modulaters of GR activation, Bax can be up regulated and translocated to the mitochondria, where it binds to modulator of apoptosis, a mitochondrial effector of Bax to cause change membrane potential. Mitochondria was impaired, resulting in the release of cytochorme c from the mitochondria into the cytoplasm. Subsequently, cytochrome c binds to Apaf-1, which in turn self-associates and binds procaspase-9, resulting in an apoptosome transactivation of the complexes procaspase-9 to active caspase-9, and the caspase then cleaves and activates downstream caspases-3 to carry out the biochemical execution of apoptosis.In China, due to earthquakes, floods and other natural disasters have occurred, some people appear anxiety and agitation for long after the disaster, of the same time as social development, accelerate the pace of life, from society, family and work pressure increasing, PTSD are a predisposing factor. It has been suggested that SPS could be an appropriate animal model of PTSD. As the development of appropriate animal models of depression promoted our understanding of the molecular pathophysiology of the disease, the pathogenesis of PTSD was revealed when an animal model of PTSD involving single prolonged stress (SPS) was developed. In the present study, we used wistar rats exposure to SPS as an animal model of PTSD. Mitochondrial pathway during apoptotic process was specifically investigated, which may provide important information for the treatment of PTSD.MethodsSPS is one of the animal models proposed for PTSD. Apoptotic cells were assessed by TUNEL method and electron microscopy. Hippocampus volume was estimated by stereological method. Expression of caspase-9, caspase-3, cytochrome c, Bcl-2 and Bax was detected by immunohistochemistry, immunofluorescence and western blotting. Expression of Bcl-2 and Bax mRNA was detected by RT-PCR. Double immunofluorescence labeling of Bcl-2 and Bax, caspase-9 and caspase-3 was detected in a confocal microscope. Cytochrome c oxidase was detected by electron microscope.Results1. Detection of neuronal apoptotic morphology(1) Apoptotic cells were assessed by electron microscopy.It demonstrated obvious abnormalities in the neuronal ultrastructure of the experiment groups, which were consistent with apoptosis. Neurons displayed characteristic morphological changes of early apoptosis, including plasma membrane blebbing, cell shrinkage, chromatin condensation, nuclear pyknosis, irregular nucleus, notching of the nuclear membrane.(2) TUNEL resultsCompared with the control group, the total number of marked cells significantly increased in experiment groups. Particularly, the number of TUNEL-stained cells peaked at 7 days after exposure to SPS. Then decreased gradually.2. Changes of hippocampus volumeIn our study, there was a significant difference between SPS and normal groups. The hippocampal volume was obviously reduced in SPS groups.3. Expression of Bcl-2 and Bax(1) Results of immunohistochemistryVery slight immunoreactive cells of Bcl-2 and Bax were observed in control groups. Marked Bcl-2 immunoreactivity was detected at 4 days after exposure to SPS. At 7 days, much more increases of Bax immunostaining were noticed. (2) Results of double immunofluorescence labelingConfocal microscopic examination showed that the majority of Bax-positive neurons co-localized with Bcl-2. Both of them were abundant at 4 days after exposed to SPS.(3) Results of Western blottingThe intensity of Bcl-2 and Bax was significantly high, compared with control group. The intensity of Bcl-2 was relatively enhanced during early PTSD. Bcl-2 was down regulated at 7 days and then recovered gradually. Bax showed the deepest intensity at 7 days after exposure to SPS. The Bcl-2/Bax ratio was down regulated, compared with control group. At 7days after exposure to SPS, the ratio reached the lowest.(4) Results of RT-PCRThe mRNA of Bcl-2 and Bax was significantly high, compared with control group. Bcl-2 mRNA was enhanced during early PTSD and it peaked at 4days after exposure to SPS. Bax mRNA was up-regulted and peaked at 7days after exposure to SPS.4. Changes of cytochrome c(1) Results of immunohistochemistryVery slight immunoreactive cells of cytochrome c was observed in control groups. Marked immunoreactivity was detected at 4 days and 7days after exposure to SPS. Then decreased gradually.(2) sults of Western blottingCompared with the control group, cytochrome c tended to decrease in mitochondrial fractions. In cytosolic fraction samples, the band showed the deeper intensity at 4 days and 7 days after exposure to SPS. Then it recovered to the normal level gradually.5. Cytochrome c enzyme histochemistry resultsCytochrome c oxidase was obviously located in exterior and interior membrane of mitochondria in control group. Many abnormal mitochondria were observed in experiment groups. The micrographs showed that mitochondria were markedly enlarged and swollen, vacuolar degeneration and cristae fragmentation. Seriouly, some mitochondria appeared changes like-ballon, exterior membrane disruption and cytochrome c was released from mitochondria to cytosol. 6. Expression of caspase-9 and caspase-3(1) Results of caspase-9 immunofluorescenceVery slight immunoreactive cells of caspase-9 were observed in control groups. Increase of caspase-9 was occurred at 1 day after exposure to SPS and it reached the highest level at 7 days after exposure to SPS.(2) Results of caspase-3 immunohistochemistryVery slight immunoreactive cells of caspase-3 were observed in control groups. An increase in caspase-3 immunoreactivity was observed after exposure to SPS. Marked caspase-3 immunoreactivity was detected at 7 days after exposure to SPS and many neurons showed strong immunoreactivity in their cytoplasm. Then the immunoreactivity was gradually decreased.(3) Results of double immunofluorescence labelingConfocal microscopic examination showed that the majority of caspase-9-positive neurons co-localized with caspase-3. Both of them were abundant at 7days after exposed to SPS, but caspase-3 was obvious stronger than caspase-9..(4) Results of Western blottingThe activated form of caspase-9 and caspase-3, while not detected in normal group, were present in model groups. The intensity of cleaved caspase-9 was relatively enhanced during early PTSD. And it reached its peak level at 7 days after exposure to SPS. A significant greater level of cleaved caspase-3 was observed at 1 day after exposure to SPS, reaching its peak level at 7 days after exposure to SPS.Conclusion1. The hippocampal volumes were relatively low in PTSD rats. Apoptosis may be one of the reasons inducing hippocampus atrophy.2. The changes of apoptosis-related proteins and genes might be involved in early molecular regulatory mechanism of apoptosis in PTSD.3. These data suggest cytochrome c was released from mitochondria to cytosol in hippocampal neurons after exposed to SPS.4. It indicated that cytochrome c may play a critical role in regulating apoptosis induced by SPS.5. We concluded that apoptosis induced by SPS was accompanied by an increase of caspase-9 and caspase-3 activities at the protein level. And the mitochondrial pathway was involved in the process of SPS induced apoptosis.