Apoptosis and necrosis in the circumventricular organs after experimental subarachnoid hemorrhage as detected with annexin...

Methods: Three experimental groups were used: Days 1 and 2 after SAH, and a control group, seven Wistar albino rats each. Subarachnoid hemorrhage was accomplished by transclival basilar artery puncture. Rats were perfused with 0.9% NaCl and 0.1M phosphate buffer pH 7.4 until heart stoppage. Apoptosis and necrosis in CVOs were measured by flow cytometry with annexin V staining, and by caspase 3 immunostaining.

Results: Apoptosis in the organum vasculosum lamina terminalis (OVLT) , median eminence (ME), and area postrema (AP) was significantly higher in the Day 1 group than in the control group. Apoptosis in the subfornicial organ (SFO), OVLT, ME, and AP was significantly higher in the Day 2 group than in the control group. There were significant differences between the Day 1 and Day 2 groups, except for AP. Necrosis in SFO and OVLTwas significantly higher in the Day 2 group than in the Day 1 or control groups, whereas necrosis in the ME and AP did not differ between the three groups . Caspase 3-positive cell density was more intense in the Day 2 group than in the Day 1 and control groups.

Discussion: Prevention of apoptosis may potentially improve impaired functions of CVOs after SAH.

Introduction

Apoptosis is programmed cell death, which is altered in pathological conditions such as subarachnoid hemorrhage (SAH) .1 Apoptosis is believed to be a contributing factor to the pathogenesis of early brain injury,2 vasospasm,3 and the brain infarctions4 seen after SAH. In the literature, there are reports of apoptosis1,2,5,6 and necrosis7,8 after SAH, and improvements in these after administration of apoptosis inhibitors.5,6 Studies have demonstrated d that inhibition of apoptotic pathways following SAH not only reduced cellular death

but also resulted in a significant improvement in functional outcome,2,4 andthusmight havesuppressed many of the secondary injuries associated with SAH.1

Besides many valuable centers of the brain, a number of sensory circumventricular organs (CVOs), such as the subfornicial organ (SFO), organum vasculosum lamina terminalis (OVLT), area postrema (AP), and the median eminence (ME, a secretory CVO), are influenced by SAH. strong>9,10 Circumventricular organs are rich in neurotransmitters and are devoid of a blood–brain barrier.11,12

Annexin V has been shown to interact strongly and specifically with phosphatidylserine , and thus can be used to detect early apoptosis.15 Caspase 3 immunostaining is another method currently used to detect apoptosis in tissue samples.16

In this study, we investigated the presence of early and late apoptosis/necrosis by annexin V affinity and caspase 3 immunostaining in CVOs after experimental SAH in rats. In our review of the literature, we failed to find any earlier report dealing with this subject.

Materials and Methods

The study protocol was approved by Bu ¨lent Ecevit University Animal Ethics committee.

The experiments were performed at the Experimental Surgery, Research and Animal Laboratory of Bu¨lent Ecevit University, Faculty of Medicine, Zonguldak, Turkey. In total, 21 male adult Wistar albino rats, weighing 200–300 g, were included in the study. All rats were kept at 22–25uC with appropriate humidity, on a 12/12 hours light/dark cycle, and were given fluids and food ad libitum. The rats were divided into three groups as follows: Day 1 after SAH (n 5 7), Day 2 after SAH (n 5 7) and controls (n 5 7).

Rats were anesthetized by intraperitoneal injection of ketamine (60 mg/kg) and xylazine (10 mg/kg). Experimental SAH was accomplished with a technique similar to that described by Barry et al.,17 which has been described previously.9,10

Briefly, a midline cervical incision was made in the supine position, under an operating microscope (Takagi OM-5, Japan), and the clivus was exposed using the anterior parapharyngeal approach. A bony windo w in front of the basilar artery was created using large-bore needles, taking extreme care not to open the prepontine cistern. A suture needle with an outer diameter of 75 mm (Ethicon, Livingston, UK) was inserted into the basilar artery. Withdrawal of the needle caused extensive hemorrhage into the subarachnoid space, with an even distribution up to the olfactory area. The rats were kept alive for 1 and 2 days after SAH under appropriate conditions.

Rats were euthanized by perfusion. Under anesthesia, thoracotomy was performed, then the left ventricle was cannulated, the descending aorta clamped, the right atrium incised, and the blood washed from the head and cervical regions with 0.9% NaCl and 0.1M phosphate buffer (pH 7.4) until the heart stopped. The animals were decapitated, and samples of SFO, OVLT, ME, and AP were obtained using a rat stereotaxic atlas (Paxinos & Watson).

The principle of the annexin V method. is to measure by flow cytometry the affinity of annexin V to phosphatidylserine, which is translocated from the plasma inner membrane to the outer leaflet during

apoptosis.15 Cells were harvested from the various CVOs separately. We used 100 ml cell suspension for each sample. Annexin V conjugated with fluorescein isothiocyanate (FITC)-stained apoptotic cells in the presence of propidium iodide (PI). This reaction enables the detection of phosphatidylserine on the surface of apoptotic cells. Both PI-positive and annexin V-positive cells indicated percentage necrotic status. We used a commercial annexin-FITC kit (Beckman-Coulter, Fullerton, CA, USA) in accordance with the manufacturer's instructions, and a Coulter FC500 > flow cytometer (Beckman-Coulter). A schematic presentation of the loss of membrane lipid asymmetry during early apoptosis and annexin V specific binding to phosphatidylserine is shown in Fig. 1. PI-positive and annexin V-positive cells indicate late apoptosis or necrosis (Fig. 1).

For histopathological and immunohistochemical analysis, CVOs Obtained from the rat brains were used for all groups. Circumventricular organs samples from each rat were fixed in 10% neutral formalin solution, and then embedded in paraffin wax. Sections were cut on a cryostat at 5–6 mm thickness Tissue sections were then dewaxed and stained with hematoxylin and eosin (H&E) and cresyl violet for histomorphological analysis. Circumventricular organs were evaluated under light microscopy by a single pathologist (FB)blinded to the study groups. Immunohistochemical analysis using the polyclonal antibody anticaspase 3 (CPP32) (Neomarkers, Cat #RB-1197R7, Fremont, CA, USA) was also performed to evaluate apoptosis. Immunostaining was based on the streptavidin biotin peroxidase complex technique with microwave antigen retrieval using formalin-fixed paraffin wax-embedded tissues. Wax-embedded sections were collected onto slides and were then dewaxed and rehydrated. After dewaxing, sections were treated with 10% hydrogen peroxidase in filtered water to block endogenous peroxidase activity. For antigen retrieval, slides were boiled with 10 mmol/L citrate buffer (pH 7) for 10 minutes in a microwave. The slides were then incubated with primary antisera, including caspase 3 rabbit polyclonal antibody (Neomarkers, Cat #RB1197-R7, Fremont, CA, USA). After washing in phosphate-buffered saline, the tissues on the slides were incubated with a biotin-conjugated secondary antibody, followed by incubation in the streptavidin biotin system components for 30 minutes at room temperature. The reactions became visible after immersion of the samples in 3,39-diaminobenzidine tetrahydrochloride (DAB; Lab Vision, Fremont, CA, USA). Afterwards, the sections were counterstained with hematoxylin, then rinsed and mounted. The negative control had the primary antibody omitted, and tonsil tissue was used as the positive control. Caspase 3 immunoreactivity was observed predominantly in the cytoplasm with some nuclear staining. The slides were evaluated in a blinded fashion by a single pathologist (FB). Apoptosis, as measured by caspase 3-positive cell density, was evaluated in the CVOs for each group.

Statistical Analysis

The Statistical Package for Social Sciences (version 19.0; SPSS Inc, Chicago, IL, USA) was used for all data analyses. The results were expressed as median and range. The Shapiro–Wilk test was used for normality testing for variables without normal distribution, and the Kruskal–Wallis test was used for comparisons between the three groups. The Conover test was used for post hoc comparisons. For all statistical comparisons, 0.05 was considered statistically.

Results

Flow-cytometric findings are summarized in Table 1. Graphics obtained from PI/annexin significant V flow cytometry analysis of the SFO, OVLT, ME, and AP (control and Day 2 groups) are given in Fig. 2. In the Day 1 group, the early apoptosis results, shown as median (range), were as follows: OVLT 0.50% (0.08–1.89), ME 0.55% (0.21–0.84), and AP 0.46% (0.20–1.86), apoptosis in theSFO did not differ significantly from that in the control group. For the Day 2 group, early apoptosis was as follows: SFO 1.88% (0.97–2.99), OVLT 1.85% (0.77–3.16), ME 0.79% (0.54–1.43), and AP 0.82% (0.19–1.49). ), which was significantly higher in all four areas than in the control group. In the control group, early apoptosis was0.12% (0.02–0.35) in the SFO, 0.15% (0.04–0.71)in the OVLT, 0.18% (0.09–0.34) > in the ME, and 0.06% (0.01–0.24) in the AP.

Necrosis in the SFO and OVLT in the Day 2 group was significantly higher than in both the Day 1 and the control groups, and the results for the ME and AP did not differ between the Day 1 and the control groups

Histopathological examination of the SFO, OVLT, ME, and AP sections exhibited more prominent cell loss in the Day 2 group than in the other groups.

Caspase 3-positive cell density was more intense in the Day 2 group than in the other two groups. There were few caspase 3-positive cells in the control group, and the Day 1 group had fewer caspase 3-positive cells than the Day 2 group .

Discussion

Apoptosis is one of the mechanisms in the evolution of cell death, which may be seen after clinical and experimental SAH.

In this study of CVOs after experimental SAH, in rats, apoptosis was seen in the SFO, OVLT, AP, and ME. In the Day 1 study group, we found increased annexin V affinity to phosphatidylserine (early apoptosis)in all CVOs except the SFO. In the Day 2 g