Masao Nakatsuka, Atsuo Masago, Hideaki Taki, and Kazuo Yamada

Department of Neurosurgery, Nagoya City University Medical School,
1 Kawasumi, Mizuho-ku, Nagoya, 467, Japan

Address correspondence to:
Masao Nakatsuka, M.D.
Department of Neurosurgery
Nagoya City University Medical School
1 Kawasumi, Mizuho-ku, Nagoya, 467, Japan

We studied mRNA expression of apoptosis-related genes in the rat model of subarachnoid hemorrhage (SAH), which was induced by endovascular perforation. The TUNEL method indicated apoptotic changes in CA1 neurons of the perforated side 48hr after SAH. The mRNA of bax, bcl-2, bcl-x and ice was induced only in the pyramidal layer and dentate gyrus of the perforated-side hippocampus. The apoptosis-regulated bax, bcl-2 and bcl-x genes were upregulated in the CA1 with peak level at 12hr. Expression of those genes, however, varies in the CA3. The bcl-2 gene was upregulated in the CA3 with the peak level at 12hr to 24hr, whereas expression of bax gene was decreased rapidly after reaching peak level at 6hr. The bcl-x mRNA was not expressed in the CA3. The mRNA of ice, apoptosis executed gene, was induced in the CA1 with peak level at 12hr, but little expression was identified in the CA3. Sustained expression of bax at CA1 might relate to apoptotic cell death, whereas rapid reduction of bax in the CA3 may avoid neuronal death at CA3. The bcl-x may be an alternative variant of bax and may influence apoptotic process. Apoptosis-like changes in the hippocampus might correlate with neurologic dysfunction after subarachnoid hemorrhage.

Key words: subarachnoid hemorrhage, apoptosis, bcl-2 family, ice, cerebral ischemia

Apoptosis-related genes and proteins were upregulated in the neurons of mild ischemia models and those neurons showed morphological features of apoptosis 1-8). Recent report indicated upregulation of bax in the focal ischemia models9), in which levels of bcl-2 mRNA in the ipsilateral hemisphere fell bellow detection level 6h following permanent MCA occlusion, whereas bax transcripts increased significantly. In the transient focal ischemia, bcl-2, bax and bcl-xL mRNA was increased in the CA1 and CA3 of the rat hippocampus 7,8). In the gerbil 10 min forebrain ischemia, bcl-2 and bcl-x mRNA was markedly induced and localizd mainly in CA1, and to a much lesser degree in CA3 at 24h. The bax mRNA remained in high level at 24h, and decreased in the CA1 at 72 h following ischemia6).
We have shown mild ischemia occurring in the rat model of subarachnoid hemorrhage (SAH), which can be developed by penetrating intracranial carotid artery through endovascular route. In this model, hsp70 mRNA was upregulated in the hippocampus and cortex of the punctured side, but no focal necrosis was detected histologically10) suggesting occurrence of mild ischemia. With this model, we analyzed expression of bax, bcl-2, bcl-x and ice mRNA with in situ hybridization and detected apoptotic neurons with TUNEL method.

Material and Methods
Animal preparation: Male Wistar rats weighing 300-350 g were subjected to the experiment. Animals were anesthetized with 2% halothane and nitrous oxide/oxygen mixture (70:30), intubated and ventilated mechanically. Temperature of the temporal muscle was monitored and maintained at 37 with a heating pad.

SAH model: The experimental SAH model was originally described by Bederson et al.11) and Veelken et al.12) and modified by us10). In brief, bifurcation of the right common carotid artery was exposed. The external carotid artery was divided and ligated, leaving a 3 to 4 mm stump. A 4-0 nylon thread was inserted into the right internal carotid artery for 1.9 to 2.5 cm. Some slight resistance was usually felt, which had to be overcome. The thread was then withdrawn and internal carotid artery was ligated immediately. Sixty-three rats were used for experiment but 28 of them were excluded, because 11 rats died within 6h and 22 rats showed pure basal subdural hematoma without SAH. Thirty-five animals with pure SAH were used for this study.
Twenty-five of them were used for detection of gene expression. After puncture, anesthesia was discontinued and the rats were allowed to move freely. The animals were re-anesthetized deeply with diethylether and sacrificed at 6 hr, 12 hr, 18 hr, 24 hr and 48 hr after SAH (n=5 for each time). For sham-operated controls, nylon thread was inserted in 5 rats in the same manner until slight resistance was felt, but no more insertion was done, and the thread was removed and the internal carotid artery was ligated. The sham operated rats were sacrificed 12 hr after the procedure. The brains were removed, frozen, embedded in Tissue-TekTM O.C.T. compound (Miles, IN, USA) and stored at -80 until use. Twenty-micron-thick coronal sections were cut on cryostat at -20, collected on silane-coated slides and processed for TUNEL method and in situ hybridization.

TUNEL methods: TUNEL method was performed using in situ apoptosis detection kit (Oncor, USA). Each slide (n=3 for sham operation and SAH model at each time period) was dried at 37 for 16 hr and fixed for 10 min in Mildform (Wako, Japan). After rinsing with 0.01mol/L phosphate-buffered saline (PBS), each slide was re-fixed with ethanol acetate (ethanol: acetate = 2 : 1) and was placed in 0.01 mol/L PBS with 7mg/ml of proteinase K. After rinsed with distilled water, each slide was placed in 0.01 mol/L PBS with 3% H2O2 for 5 min. After rinsing with 0.01M PBS, each slide was placed in equilibration buffer for 15 min. Labeling reaction was performed at 37 for 1 hr with biotinylated-deoxyUTP and 50ml of terminal deoxynucleotid transferase, and each slide was placed in a stopping buffer at 37 for 30 min. After rinsing with 0.01 mol/L PBS, Anti-Digoxigenin-Peroxidase was applied to each slide for 30 min, and slides were rinsed again with 0.01 mol/L PBS. Each slide was then stained with diaminobenzidine and hydroxyperoxide. Nuclei were counterstained with hematoxylin, and slide was washed, dehydrated and coverslipped.

In situ hybridization: Techniques for in situ hybridization were the same as reported by Honkaniemi et al6). The oligonucleotide used for this study are listed in Table 1 6,13,14). They were chosen from regions of each gene present in all splicing variants suggesting the highest degree of homology among different species. These oligonucleotide probes were synthesized and purified by Nihon Gene Res. Lab. Inc. (Miyagi, Japan). Each oligonucleotide probe was labeled with [____S] dATP (Amersham, UK) at the 3' end using terminal deoxynucleotidyl transferase (Takara, Japan). Specific activity of the labeled probe was 0.5-1.0x109 dpm/_g. After warmed to room temperature, slide-mounted sections were fixed with 4% formaldehyde in 0.1 mol/L phosphate buffer (pH 7.2) for 30 min, rinsed three times in 4xSSC (pH 7.2; NaCl 35g/l, sodium citrate 17.6 g/l), and dehydrated through a graded ethanol series (70-100%). The sections were subsequently defatted with chloroform and immersed twice in 100% ethanol. For hybridization, sections were incubated with a buffer containing 4xSSC, 50% deionized formamide, 0.12 mol/L phosphate buffer (pH 7.2) 1xDenhardt's solution, 2.5 % tRNA, 10 % sarkosyl, and [_-35S]dATP-labeled probes (0.5-1.0x106 dpm/slide, 200_l/slide) for16 hr at 42. After hybridization, the sections were rinsed in 1xSSC (pH 7.2) for 10 min at room temperature, followed by rinsing 4 times in 1xSSC at 56 for 15 min. The sections were dehydrated through a graded ethanol series (70-100%), dried and exposed to a x-ray film for 5 days. The film was developed as indicated and used for evaluation.

Quantitative analysis: Macroautoradiograms were used for qualitative and quantitative analysis. For quantitative assessment, optical densities of the hippocampal CA1 and CA3 appeared at 2.8 to 3.3 mm posterior from the bregma according to the Atlas of Paxinos and Watson15) were measured with a densitometer (Konica PDA-15, Konishiroku, Tokyo, Japan). The same area of interest was measured five times and the mean value of them was used for statistical analysis, which was done with non-parametric analysis of the Scheffe's multiple comparison test. Significant level was set at p<0.05.

Histological analysis with TUNEL method: No DNA nick end-labeling was observed in the brains of sham operated rats and SAH-induced rats at 6 hr, 12 hr, 18 hr and 24 hr after SAH. At 48 hr after SAH however, rats showed TUNEL-positive neurons in the hippocampal CA1 of the perforated side. TUNEL-positive neurons were detected in the medial and lateral sides of the CA1. The TUNEL-positive neurons were accounted about one-third of the CA1 neurons (Figure 1). No TUNEL-positive neurons were detected either on the contralateral CA1 subfield nor bilateral CA3 subfield at 48 hr after SAH.

Expression of Apoptosis-related genes: The bax gene was induced in the pyramidal layer and dentate gyrus of the hippocampus of the perforated side (Figure 2). The bax mRNA was induced substantially high level in the CA1 with a peak level at 12hr. The signal of bax mRNA in the CA3 reached peak level at 6 hr and it declined rather rapidly (Figure 3). The bcl-2 gene was induced in the CA1 and CA3 pyramidal layer and dentate gyrus of the perforated side (Figure 2). The bcl-2 gene was upregulated both in the CA1 and CA3 with the peak at 12hr, and the expression was sustained at significantly high level till 24hr (Figure 3). The bcl-x gene expression was detectable in the dentate gyrus and pyramidal layer of the perforated side hippocampus (Figure 2). Significant level of bcl-x gene expression was detectable in the CA1 with the peak at 12hr after SAH (Figure 3). In the CA3 however, signal increase did not reached to the statistical significance (Figure 3), though some signal was detectable (Figure 2). The ice gene was induced in the pyramidal neurons and dentate gyrus of the perforated side (Figure 2). The induction of ice gene in the CA1 was upregulated significantly between 6hr and 24hr with its peak at 12hr after SAH (Figure 3). In the CA3, ice gene signals were upregulated significantly only at 6h (Figure 3).

In opposition to necrosis, apoptosis was characterized by series of nuclear morphological changes16) and internucleosomal DNA fragmentation17). In the present study, we detected non-necrotic cell death in the CA1 pyramidal neurons of hippocampus evaluated by DNA nick end-labeling method. TUNEL-positive neurons were detected only in the side of arterial perforation, though SAH is distributed diffusely and intracranial pressure rose uniformly and rapidly after SAH 10,11). Accordingly, unilateral distribution of TUNEL-positive neurons did not relate to the blood in the subarachnoid space nor high intracranial pressure. Instead, mild ischemia caused by this model is of importance. Veelken et al12) reported that regional cerebral blood flow (rCBF) reduced to the 30-35ml/100g /min which is the level of mild ischemia and might cause cellular dysfunction. This blood flow reduction was reversed to approximately 60% of the preSAH level within two hours. In the contralateral side, the blood flow reduction was less evident. Therefore, mild ischemia in the side of arterial penetration might be caused by combination of arterial trauma and early vasospasm. The mild ischemia can therefore be attributable to the presense of TUNEL-positive neurons on the side of arterial perforation. There present other models of SAH, such as injection of autologous blood into the cisterna magna18,19) or puncture of the basilar artery through the transclival approach20). Yet, those models have not shown TUNEL-positive neurons.
In this study, we could detect TUNEL-positive neurons 48hr after SAH. Several previous reports have detected TUNEL-positive neurons between 24 and 48 hr after global21) and focal22-24) ischemia. Appearance of DNA fragmentation in this SAH model is later than previous reports, suggesting that degree of ishcemia in this model is milder than other ischemia model. We propose that SAH model with endovascular perforation is suitable for studing apoptosis caused by mild ischemia.
The bcl-2 family members, bcl-2, bax and bcl-x, interact with each other to regulate apoptosis. The bcl-2 which binds bax and bak25) is a well-known inhibitor of apoptosis,. The bax is the accelerator of apoptosis, binding to bax and bcl-x26). The bcl-x was originally described as having short (bcl-xS) and long (bcl-xL) isoforms. The bcl-xL inhibits apoptosis, whereas bcl-xS accelerates apoptosis, and they bind to bax and bak 27).
In the present study, expression pattern of bcl-2 gene family might be similar to those of global ischemia rather than focal ischemia6-9). Expression of bcl-2 mRNA in the perforated-side CA3 continued till 24 h, whereas bax mRNA decreased at 12 h after SAH. The bcl-2 dominancy in the CA3 might avoid apoptosis-like cell death. In vitro investigations28) suggested that bcl-2 gene suppressed expression of ice mRNA.
In our study, bcl-2 dominancy over bax might be related to suppression of ice at CA3. In the CA1, bcl-2, bax and bcl-x genes were induced in fairly high level. As the cause of CA1 neurons leading to apoptosis is concerned, bax/bcl-2 ratio might be modified by bcl-x, especially bcl-xS. Though this study did not investigate status of protein synthesis, impediment in protein synthesis should be considered, and this might be another cause of apoptosis29). Less expression of bcl-2 might lead to expression of ice mRNA, and accerated apoptosis-like cell death. Indeed, we found the same peak point of mRNA expression between ice and bcl-2.
Ice forms ice gene family with other homologous, and is recognized as executioner of apoptosis. The ice play a role in the ischemic injury. The ice mRNA was upregulated in the global ischemia model6). The ice inhibitor attenuated ischemic brain damage in the rat model of middle cerebral artery occulusion30). Transgenic mice expressing a mutant ice protein attenuated ischemic brain damage in the focal ischemia model31). In the present study, expression of ice mRNA was apparent in the perforated side CA1 with the peak level at 12 hr. The present data along with previous data indicated definite role of ice for apoptosis in this SAH model.
In conclusion, this study demonstrated apoptosis-related genes and apoptosis-like change in the CA1 neurons after experimental SAH. Genetic programs which maintain cellular viability are disrupted and apoptotic cell death are activated by SAH, and the change is similar to that of cerebral ischemia.

Nagoya City University
Medical School