Single-Prolonged Stress Induces Apoptosis by Activating Cytochrome C/Caspase-9 Pathway in a Rat Model of Post-traumatic Stress Disorder

Abstract

The purpose of this study was to provide a novel insight into the mechanism of how amygdala might participate in PTSD by investigating the changes of cytochrome c oxidase (COX), caspase-9, and caspase-3 in the amygdala of single-prolonged stress (SPS) rats. A total of 80 healthy, male Wistar rats were selected for this study. The models of post-traumatic stress disorder (PTSD) were created by SPS, which is an established animal model for PTSD. The change of COX was detected by light microscope and transmission electron microscopy (TEM). The expression of caspase-9 and caspase-3 in the basolateral amygdala was examined by immunofluorescence and reverse transcription-polymerase chain reaction (RT-PCR). SPS exposure resulted in a significant change of COX in the SPS model groups compared with the normal control group. Evaluation by enzymohistochemistry indicated translocation of COX from mitochondria to cytoplasm. The expression of both caspase-9 and caspase-3 significantly increased 1 day after SPS stimulation, then gradually increased and peaked at SPS 7d. This findings suggest changes of COX, caspase-9, and caspase-3 in the amygdala of SPS rats, which may play important roles in the pathogenesis of PTSD.

Introduction

Post-traumatic stress disorder (PTSD) is an anxiety disorder that develops after exposure to a life-threatening traumatic experience. It is characterized by symptoms that often endure for years including continuous re-experience of the traumatic event, avoidance of stimuli associated with the trauma, numbing of general responsiveness, and increased arousal (DSV-IV-R; American Psychiatric Association 1994).

Apoptosis is a genetically programed, morphologically distinct form of cell death that can be triggered by a variety of physiological and pathological stimuli (Roulston et al. 1999). Several signals can trigger apoptosis via different pathways, involving death receptor activation, mitochondria, or endoplasmic reticulum (Kidd et al. 2000).

It is well known that caspases, a family of cysteine-dependent aspartate-directed proteases, play critical roles in initiation and execution of apoptosis by cleaving a large number of proteins, which in turn lead to the typical morphology of apoptosis (Cryns and Yuan 1998; Eamshaw et al. 1999; Green and Reed 1998; Nunez et al. 1998). Caspases are aspartate-specific cysteine proteases synthesized as inactive precursors, which require proteolytic conversion to become active caspases (Zhivotovsky et al. 1999; Hengartner 2000). Caspase signaling cascade represents hallmarks in apoptosis (Degterev et al. 2003; Los et al. 2001). Results of in vitro studies concluded that the apoptotic caspase cascade is activated by cytochrome c (Mishra and Delivoria-Papadopoulos 2004). As a key step in the execution of apoptosis, the pathway of the activation of caspases involves the release of cytochrome c from mitochondria, which then binds to Apaf-1 (apoptotic protease-activating factor-1) in the apoptosome, leading to the activation of caspase-9 (Shi 2001). This, in turn, activates the downstream execution starting with caspase-3 (Amir et al. 2008). Caspase-3 is one of the known effector caspases that, once activated, irreversibly executes cell death through degradation of vital cell proteins and activation of endonucleases (Resendes et al. 2004). Therefore, activation of caspase-3 can be considered a hallmark of apoptosis (Huppertz et al. 1999).

Amygdala has been documented to play an important role in fear, rage, and emotional memory (McGaugh and Cahill 1997; LeDoux 1995). As one of the key regions in the limbic system of the brain, amygdala is usually divided into three distinct nuclear subgroups: central nucleus, corticomedial nucleus, and basolateral nucleus (Harding et al. 2002). Among these different nuclear subgroups, the basolateral nucleus is the largest nucleus of the amygdaloid complex (Sims and Williams 1990), which is a putative site of emotional memory and regulation of anxiety (Davis 1994; McGaugh et al. 2002). Thus, the basolateral nucleus (BLA) has been paid a lot of attention.

Meta-analyses showed volume of left amygdala is significantly smaller in adults with PTSD than both healthy and trauma-exposed controls (Karl et al. 2006). A study showed that police officers with PTSD had smaller amygdala volumes (Lindauer et al. 2004). Previous studies focused on the change of volume of amygdala caused by PTSD rather than the reason why the volume has changed.

Ding et al. considered that the cells apoptosis in the amygdala of a rat model of PTSD may be one of the reasons lead to amygdala atrophy, which may make the volume of amygdala become smaller (Ding et al. 2010). To clarify the mechanisms of apoptosis of the amygdala, we have detected the expression of apoptosis-related proteins in the amygdala neurons of rats with PTSD in this article.

There are many animal models for PTSD. Although these animal models presented behavioral alterations resembling PTSD, they failed to show the most consistent neuroendocrinologic characteristic observed in PTSD patients. Several clinical neuroendocrinological studies showed dysfunction of the hypothalamo–pituitary–adrenal (HPA) axis is one of the core neuroendocrine abnormalities related to the disorder (Yehuda et al. 2004; de Kloet et al. 2006). This neuroendocrine finding specific to PTSD has served as the basis for animal models that are useful for elucidating the pathophysiology of PTSD (Yehuda 2005). Single-prolonged stress (SPS) (Liberzon et al. 1997), an animal model of PTSD, was shown to induce enhanced inhibition of the HPA axis, which has been reliably reproduced in patients with PTSD, and also exhibit behavioral abnormalities (enhanced anxiety) that mimic the symptoms of PTSD (Imanaka et al. 2006; Khan and Liberzon 2004; Takahashi et al. 2006; Iwamoto et al. 2007). Then, SPS paradigms have been extensively developed and employed in the investigation of PTSD (Khan and Liberzon 2004; Iwamoto et al. 2007).

In the present study, we aimed to determine the changes of COX, caspase-9, and caspase-3 in the amygdala of SPS rats, and our findings may provide a novel insight into the mechanism of how amygdala might participate in PTSD.

Materials and Methods

Experimental Animals

Eighty male Wistar rats aged 7 or 8 weeks at the beginning of the study, weighing approximately 180 g, were supplied by the Animal Experimental Center, China Medical University. Rats were individually housed in an air-conditioned room (22 ± 1°C and 55 ± 5% humidity) on a 12-h light/dark schedule with free access to food and water.

The rats were raised in the laboratory for at least 7 days before conducting the experiment. Experiments were carried out in accordance with the National Institute of Health Guide for the care and use of laboratory animals. All efforts were made to reduce the number of animals used and to minimize animal suffering during the experiment.

Model establishment and grouping

Animals were divided randomly into 4 groups (n = 20 per group): 1) control group; 2) SPS 1d group; 3) SPS 4d group; 4) SPS 7d group. Control animals remained in their home cages with no handling for 7 days and were killed at the same time as SPS groups. SPS rats had SPS procedure on the first day. The SPS protocol was based on a combined plural stress paradigm (Takahashi et al. 2006; Kohda et al. 2007): immobilization (compression with plastic bags) for 2 h, forced swimming for 20 min (24 ± 1°C), rest for 15 min, and followed by ether anesthesia (until consciousness was lost).

Perfusion Based Sections

Rats of each group (n = 5) were perfused via the left ventricle with 200 ml of pre-cold heparinized 0.9% saline, followed by 300 ml of 4% paraformaldehyde in 0.01 M phosphate-buffered saline (PBS) (pH 7.2–7.4). Brains were rapidly removed and post-fixed in the same fixative for 4–6 h at 4°C, and were immersed in a 20% sucrose solution in 0.01 M phosphate buffer (PB, pH 7.4) at 4°C. Then, samples were fast frozen in liquid nitrogen and sectioned, 12 μm coronal sections were prepared for morphological studies.

Enzymohistochemistry Analysis of COX by Light Microscope

After being washed with PBS, the sections were incubated with buffer including cytochrome c 10 mg, 0.2 M PBS 5 ml, DAB 10 mg, and distilled water (ddH₂O) 5 ml. The sections were washed three times with PBS after incubation. To assess nonspecific staining, a few sections in every experiment were incubated in PBS without incubating buffer. Fifteen slides were randomly selected from each group. Each slide was randomly selected five visual fields in basolateral amygdala (×400). The optical density (OD) of cytochrome c oxidase-immunopositive cells were analyzed using a morphology image analysis system.

Assessing the Morphological Change Using Transmission Electron Microscopy

Rats of each group (n = 5) were perfused with pre-cold heparinized 0.9% saline, followed by 0.01 M PBS (pH 7.2–7.4) containing 2% paraformaldehyde and 2.5% glutaraldehyde. The brain was removed and dissected on ice, followed by 4–6 h of post-fixation in the same fixative at 4°C. The basolateral amygdala was dissected using the stereomicroscope and cut into blocks about 1 mm³. Then, the blocks were postfixed in 1% osmium tetroxide at 4°C for 2 h. Then, they were rinsed in ddH₂O several times, dehydrated in gradient series (20–100%) of ethanol and then in acetone, infiltrated with Epon 812, and finally polymerized in pure Epon 812 at 65°C for 48 h. The basolateral amygdala was localized on semi-thin sections. Ultra-thin sections were cut on an ultramicrotome, collected on copper grids, and stained with 4% uranyl acetate and lead citrate. A minimum of five sections about 250 cells from each basolateral amygdala were studied with transmission electron microscopy (TEM 1200EX, 80 kV).

Immunofluorescence Analysis of Caspase-9

The sections were treated with 5% bovine serum albumin (BSA), 0.3% Triton X-100 in PBS for 30 min to block nonspecific staining at room temperature (RT). Endogenous peroxidase was inactivated with 3% H₂O₂ in ddH₂O for 5 min at room temperature. The sections were then incubated with mouse anti-caspase-9 monoclonal antibody (Santa Cruz, USA; 1:300) in 2% BSA–PBS overnight at 4°C. After being washed with PBS for three times, the sections were incubated with FITC goat anti-mouse IgG (Boster, China; 1:50) for 2 h at room temperature. To assess nonspecific staining, a few sections in every experiment were incubated in buffer without primary antibody. Slices were then mounted with glycerin and observed by fluorescence microscope.

Fifteen slides were randomly selected from each group. Each slide was randomly selected five visual fields in basolateral amygdala (×400). We recorded the OD of caspase-9-immunopositive cells in each field to evaluate the average of OD.

Reverse Transcription-Polymerase Chain Reaction (RT-PCR) to Detect Caspase-3 and Caspase-9

Total mRNA of each group (n = 5) was extracted from the basolateral amygdala according to the instructions of Trizol kit (Invitrogen, USA), and 1 μg of total RNA was reverse transcribed into cDNA. cDNA was amplified using a RNA PCR kit (AM Ver. 3.0, Takara Bio, Otsu, Japan). The primers were designed and synthesized by Shenggong Biotech Company (Shanghai, China) according to the serial number from Genbank are shown in Table 1. The reaction was started at 94°C for 2 min and amplified for 40 cycles of 30 s at 94°C, 60 s at 52°C (for caspase-3), or 45 s at 67°C (for caspase-9), or 45 s at 72°C and ended with 7 min extension at 72°C. β-actin mRNA used as an internal control was co-amplified with caspase-3- or caspase-9 mRNA. The products were observed after electrophoresis on 1.2% agarose gel, and the density of each band was analyzed on the Gel Image Analysis System (Tanon 2500R, Shanghai China). The levels of caspase-3- and caspase-9 mRNA were determined by calculating the density ratio of caspase-3 mRNA/β-actin mRNA or caspase-9 mRNA/β-actin mRNA.

Table 1.

The sequence of caspase-3, caspase-9, and β-actin

Name	Upstream primer	Downstream primer	Product size (bp)
Caspase-3	5′-ctcggtctggtac actatgtcgatg-3′	5′-ggttaacccgggta agaatgtgca-3′	284
Caspase-9	5′-atggacgaagcgg atcggcggctcc-3′	5′-ctatcctgttctct tggagagtcc-3′	330
β-actin	5′-atcacccacact gtgcccatc-3′	5′-acagagtacttg cgctcagga-3′	542

Statistical Analysis

All the data were expressed as means ± standard error. Data among groups were analyzed by one-way analysis of variance (ANOVA) using SPSS 13.0 software. A value P < 0.05 was considered statistically significant.

Results

Morphological Change of the Amygdala by Light Microscope

COX widely distributed throughout the basolateral amygdala region, mainly in the cytoplasm, appeared as buffy particle (Fig. 1a). Evaluation of COX by enzymohistochemistry indicated a significant decrease in the SPS model groups compared with the normal control group (Fig. 1b–d). The analysis results were shown in Fig. 1e (F = 34.340 df = 3.16; Tukey’s test P < 0.05).

Fig. 1.

Cytochrome c expression in basolateral amygdala in each group (a–d, magnification ×400). a Control group. b SPS 1d group. c SPS 4d group. d SPS 7d group. e Quantitative analysis of the optical density, * P < 0.05 vs. control group

Morphological Change of the Amygdala by TEM

Positive expression of COX, high electron-dense black granule, was located on mitochondrial cristae in normal control group. As shown in the figure, intracellular mitochondria had a normal structure in normal control rats (Fig. 2a). Tumescent mitochondria and mitochondrial vacuolization were found at SPS 4d (Fig. 2b). As shown in Fig. 2c, the translocation of COX, tumescent mitochondria and mitochondrial vacuolization was found at SPS 7d. Furthermore, this change was found mostly at SPS 7d (P < 0.05).

Fig. 2.

COX expression in basolateral amygdala by TEM. a Control group. b SPS 4d group. c SPS 7d group (bars = 200 nm)

Immunofluorescence Staining Analysis of Caspase-9

The immunofluorescence staining results were shown in Fig. 3. The caspase-9 protein was located in cytoplasm (Fig. 3a–d). In normal control group, the fluorescent intensity of caspase-9 positive cells was poor, and that of SPS rats were significantly strong and peaked at 7 days after exposure to SPS (P < 0.01) (Fig. 3e; F = 36.456 df = 3.16; Tukey’s test P < 0.05).

Fig. 3.

Caspase 9 expression in basolateral amygdala by immunofluorescence staining (a–d, magnification ×400). a Control group. b SPS 1d group. c SPS 4d group. d SPS 7d group. e Quantitative analysis of fluorescent intensity, * P < 0.05 vs. control group

RT-PCR Results of Caspase-3 and Caspase-9 mRNA

The levels of caspase-3 and caspase-9 mRNA were normalized with β-actin mRNA level. Both gradually increased after SPS stimulation than the control group, and peaked at SPS 7d (P < 0.01) (Fig. 4).

Fig. 4.

Caspase 3 and caspase 9 expression in basolateral amygdala by RT-PCR. a Representative gel pattern of caspase 3 and caspase 9. b Quantitative analysis of the optical density, * P < 0.01 vs. control group for caspase 3, ^▲  P < 0.01 vs. control group for caspase 9

Discussion

PTSD is thought to involve a disfunction in response to fear-related stimuli. Four major types of characteristic symptoms of PTSD are: re-experiencing, avoidance, numbing, and hyperarousal (APA, DSM-IV, 1994). The special role of amygdala from both animal and human investigations in the processing of threat-related stimuli, in particular anger and fear are well documented (Derntl et al. 2009; McGauqh 2004; Cahill and McGaugh 1998). Many lines of evidence have implicated the BLA as a substrate for stress-related modulation of memory (Chavez et al. 2009). So, these studies focus on observing the changes of basolateral nucleus.

Many lines of evidence now exist to suggest that mitochondrial cytochrome c release is an important control point in caspase activation and apoptosis (Solange and Martinou 2000). It is well established that released cytochrome c activates caspase-9 in concert with the cytoplasmic factors dATP and Apaf-1, and as a result, it consequently activates caspase-3 (Li et al. 1997). Thus, release of cytochrome c appears to be central step in the initiation of caspase-dependent programed cell death (Mishra et al. 2006). It is also established that caspase-9 and caspase-3 play a critical role in apoptosis.

Very little is currently known regarding apoptosis of the amygdala neurons. Ding et al. (2010) found more apoptotic cells and increased apoptosis rate in the amygdala of rats after treatment with single-prolonged stress compared with normal control group. Thus, further study devoted to clarify its mechanism(s) of apoptosis in the amygdala of rat after treatment with single-prolonged stress appears to be of remarkable importance.

In this study, we aim to explore the mechanism of apoptosis in the amygdala of rats after SPS stimuli. We found that COX translocated from mitochondria to cytoplasm by TEM. We also found tumescent mitochondria and mitochondrial vacuolization at SPS groups, which resulted in disfunction of energy metabolism necessarily. We show for the first time that both caspase-3 and caspase-9 upregulated 1d after SPS stimulation, and then gradually increased and peaked at SPS 7d by detection. Taken together, we found translocation of COX and changes of caspase-9 and caspase-3 in the amygdala of rats after treatment with single-prolonged stress. The possible reason was that changes of COX, caspase-9, and caspase-3 led to dysfunction of mitochondria, which facilitated apoptotic process. The all effects lead to apoptosis of the amygdala neurons, which might play an important role in pathobiological basis for abnormality of affect and behavior induced by PTSD. Further investigation into the cellular mechanisms that regulate apoptosis and the activation of caspase may lead to novel treatments for PTSD.

One limitation of the current study is that we didn’t examine the behavioral changes following single-prolonged stress, and this may have influenced the results, particularly the lack of emotion-related behavioral response in response to SPS stimuli, although the previous study of our discussion group has demonstrated that single-prolonged stress induces behavioral changes in rats (Wang et al. 2009; 2010). Further work will include behavioral detection to study mechanisms of PTSD.

At present, the pathogenesis of PTSD is not yet entirely clear. PTSD may cause a series of biochemical abnormalities and dysfunction of the amygdala, which leads to dysfunction of brain. Thus, the pathogenesis of PTSD needs to be further studied.