Apoptosis of endothelial cells of cerebral basilar arteries in symptomatic cerebral vasospasm rabbit models

NEURAL REGENERATION RESEARCH Volume 2, Issue 8, August 2007

Cite this article as: Neural Regen Res,2007,2(8),479-82

Basic Medicine

$SRSWRVLV RI HQGRWKHOLDO FHOOV RI FHUHEUDO EDVLODU DUWHULHV LQV\PSWRPDWLFFHUHEUDOYDVRVSDVPUDEELWPRGHOV  (OHFWURQPLFURVFRSLFREVHUYDWLRQ  Guojun Shi1, Guodong Gao2, Zhenwei Zhao2 1Fifth Endemic Area, the 89 Hospital of Chinese PLA, Weifang 261021, Shandong Province, China 2Minimally Invasive Neurosurgical Center, Tangdu Hospital, Fourth Military Medical University of Chinese PLA, Xi'an Province, China

Abstract BACKGROUND: Recent researchers report that vasospasm is caused by that, on one hand, damage of endothelial cells reduces synthesis and liberation of vessel dilator; on the other hand, defluxion of endothelial cells directly exposure vascular smooth muscles in active materials of vasoconstriction in blood. OBJECTIVE: To study whether apoptosis of cerebrovascular cells occurs in symptomatic cerebral vasospasm (CVS) rabbit models by using transmission electron microscope. DESIGN: Contrast observation. SETTINGS: The Fifth Endemic Area, the 89 Hospital of Chinese PLA; Minimally Invasive Neurosurgical Center, Tangdu Hospital, the Fourth Military Medical University of Chinese PLA. MATERIALS: A total of 24 New Zealand rabbits, of either sex, weighing 2.43.0 kg, of clear grade, were selected from the Experimental Animal Center of the Fourth Military Medical University of Chinese PLA. JEM-2000EX transmission electron microscope was made in Japan. METHODS: The experiment was carried out in the Laboratory of Anatomy (National Key Laboratory), the Preparation of Fourth Military Medical University of Chinese PLA from April 2001 to April 2002. symptomatic CVS models: Eighteen animals which were successfully modeled were randomly divided into experimental group (n =13) and control group (n =5). Animals in the experimental group were poured with blood into cavitas subarachnoidealis; while, animals in the control group were poured with the same volume of saline into cavitas subarachnoidealis. At the 5th day injection, three rabbits selected from the experimental group were anesthetized and perfused into left ventricle. And then, aorta pectoralis and caval vein were blocked by using ring clamp. Cranium was rapidly cut open to obtain cerebral basilar artery and a few of brain tissues. Both of them were fixed for 8 hours. Two rabbits selected from the control group were perfused After fixation by using optic with the same method to obtain basilar artery and brain tissues and fix. microscope, samples were stained with lead citrate uranyl acetate staining, observed with electron microscope and photographed. MAIN OUTCOME MEASURES: Morphological changes of cytoplasm and nucleus of vascular endothelial cells and smooth muscle cells in the two groups. RESULTS: Morphology of vascular endothelial cells in cerebral basilar artery was not changed in the control group. However, vascular endothelial cells in the experimental group showed that cytochondria were swelling; endocytoplasmic reticulum was amplified; chromatin margination was clear; nucleus was in pyknosis; endothelial cells fell down from basal membrane; cell-cell junction was broken. Changes of smooth muscle cells were similar to those of endothelial cells. Severely, it was shown that nuclear pyknosis was obvious, and this was like early apoptosis. CONCLUSION: Apoptosis of endothelial cells may occur in spasmodic vessels. Key Words: encephalomyelitis; models, animal; autoimmunity

710038, Shaanxi

Guojun Shi , Master, Associate chief physician, Fifth Endemic Area, the 89 Hospital of Chinese PLA, 261021, Weifang Shandong Province, China Shi GJ, Gao GD, Zhao ZW.Apoptosis of endothelial cells of cerebral basilar in symptomatic arteries cerebral vasospasm rabbit models: Electron microscopic observation. Neural Regen Res 2007;2(8):479-82 www.sjzsyj.com/Journal/ 0708/07-08-479.html

Received: 2007-05-12;Accepted: 2007-07-02 (06-S-12-1118/SHM) Corresponding author: Guojun Shi, Master, Associate chief physician, Fifth Endemic Area, the 89 Hospital of Chinese 261021, Shandong Province, China E-mail: [email protected] PLA, Weifang

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 ,1752'8&7,21 Cerebral vasospasm is a major cause of morbidity and mortality after aneurysmal subarachnoid hemorrhage (SAH), which shows such morphological changes as necrosis and dystrophy of smooth muscle cells, and the dystrophy and desquamation of endothelial cells[1,2]. Recently, however, some researchers reported that apoptosis of endothelial cells appeared in spasmatic cerebral arteries of a clinical autopsy, and apoptosis-like changes happened in small arterial endothelial cells in a canine two-hemorrhage cerebral vasospasm (CVS) model. And they thought apoptosis may be involved in cerebral vasospasm[3-7]. We observed apoptosis of CVS cells by transmission electron microscope on the basis of symptomatic CVS model in rabbits[8].

0$7(5,$/6$1'0(7+2'6 0DWHULDOV The experiment was carried out in the Laboratory of Anatomy (National Key Laboratory), the Fourth Military Medical University of Chinese PLA from April 2001 to April 2002. A total of 24 New Zealand rabbits, of either sex, weighing 2.43.0 kg, with average weight of 2.7 kg, of clear grade, were selected from the Experimental Animal Center of the Fourth Military Medical University of Chinese PLA (certification: 01). 0DLQUHDJHQWVDQGHTXLSPHQWV Main reagents and equipments were detailed as follows: JEM-2000EX transmission electron microscope (Japan), 40 g/L paraformaldehyde (4.00 g paraformaldehyde was dissolved in 100 mL 0.1 mol/L PB solution at 60 , filtered and maintained at 4 ), and 0.1 mol/L PB buffer solution (pH 7.4; 13.6 g KH2PO4 and 3.2 g NaOH were added with double distilled water to 1 L, and pH value was controlled at 7.4 with hydrochloric acid or sodium hydroxide). 0HWKRGV 0RGHOHVWDEOLVKLQJ Symptomatic CVS models were established based on reference[8]. All 24 rabbits underwent the ligation of bilateral common carotid arteries under general anesthesia with 0.3 mL/kg sumian xin 2 weeks later, the remaining rabbits were divided into experiment group (n =13) and control group (n =5). Animals were fed again for 14 days. Those which had death, sever nerve dysfunction or obvious thinness were excluded. The animals were placed in a prone position and a midline incision was made from the line between bases of ears to 1.5 cm cranially. 1.5 cm posteriolaterally from bregma, a pariatotomy measuring 2 mm 2 mm was performed by using dental drill on the left. The dura mater was pricked and clear CSF flowed out; then a 6F polyethlene catheter was inserted 480

along the inner surface of cranium for 22.5 cm. 0.9 mL/kg autologous arterial blood was obtained from central ear artery and injected into subarachnoid space through the catheter. At 48 hours later the same procedure was repeated, but the injection blood was 0.5 mL/kg. Animals were awaken within 1 to 2.5 hours. Rabbits in the control group were injected with the same volume of saline in subarachonid space based on above-mentioned ways. After symptomatic CVS model was performed in rabbits, 3 rabbits from SAH group and 3 from control group received general anesthesia with 0.3 mL/kg sumian xin. The animals were placed in a prone position, and hearts were opened. A small cut was made in the right auricular appendage, and perfusion needle was put into the left ventricle. After 1 L of normal saline was perfused, 1 L of 40 g/L polyoxymethylene was infused in about 2 hours at the pressure of 10 kPa. After that the skull was opened, cerebral basilar arteries were removed and embalmed in fixation fluid (2 g/L glutaraldehyde solution). Two rabbits in the control group were perfused to obtain basilar artery and brain tissues and fixed based on the same procedure. 3UHSDUDWLRQDQGREVHUYDWLRQRIVSHFLPHQ The embalmed cerebral basilar arteries in fixation fluid were washed by 0.1 mol/L phosphate buffer solution in order to clean glutaraldehyde solution. After dehydrated in a graded series of alcohol, the cerebral basilar arteries were embalmed in acetone, embedded in epon-araldite epoxy resin, cut into sections at 0.6 nm, examined with JEM-2000EX electron microscope, and were taken photographs.

5(68/76 4XDQWLWDWLYHDQDO\VLVRIWKHH[SHULPHQWDODQLPDOV  After the ligation of bilateral common carotid arteries, all 24 rabbits stayed under observation for the following 2 weeks. Six rabbits were discarded, including 2 did not wake up, 1 woke up but suddenly died, 2 woke up but ate less and died, and 1 became too thin. 0RUSKRORJLFDO FKDQJHV RI EDVLODU DUWHULHV LQ WKH WZR JURXSV  The endothelial cells in the control group showed normal morphology, and had tight junctions with each other and with the basal membrane. The peripheral heterochromatin was present in all cells without any condensation. No blebbing or vacuolization was noticed. No corrugation was observed in the internal elastic lamina. There were no abnormal changes in the smooth muscle cells and the adventitial layer (Figure 1). Some chromatin condensation, blebbing, condensation of cytoplasm and karyopyknosis, desquamation of the endothelial cells from the membrane were observed in the experimental group. Corrugation was present in the internal elastic lamina. Some smooth muscle cells had the similar changes to the endothelial cells, which looked like early

Shi GJ, et al. / Neural Regeneration Research,2007,2(8):479-82

apoptosis (Figure 2).

Figure 1 Normal endothelial cells in the control group (Lead citrate uranyl acetate staining, 5 000)

Figure 2 Apoptosis-like endothelial cells in the experimental group (Lead citrate uranyl acetate staining, 6 000)

',6&866,21 Lots of clinical and experimental studies demonstrated that

after SAH the spasmatic cerebral arteries showed microscopic or electron microscopic morphological changes, which was regarded as the main reason for cerebral ischemia[9], though some author thought otherwise [10]. Up to now, endothelial cellular dystrophy and desquamation, vacuolization, and the necrosis of smooth muscle cells were thought to be the most characteristic changes [1,2]. The endothelial cells form a continuous monolayer overlying the internal elastic lamina and have physical functions such as anti-coagulation, anti-thrombosis or fibrinolysis, and regulation of vessel tension [11]. So it is the key point of cerebral vasospasm. Many researchers concluded that dystrophy or desquamation of endothelial cells lead to the decrease of such vasodilators as nitric oxide and prostacyclin, and expose smooth muscle cells to vasoconstrictors in the blood [12-14], resulting in artery constriction. Many experimental and clinical studies showed the spasmatic arteries had less nitric oxide synthase, and nitric oxide-releasing substances or endothelin receptor antagonists could reverse or decrease vasospasm [12-17]. Recently, Zubkov et al [3] reported apoptosis was confirmed by electron microscope and DNA fragmentation in a patient who died from a ruptured aneurysm and severe vasospasm. Both Ogihara [4] and Meguro[5] induced apoptosis by oxyhemoglobin in cultured bovine endothelial cells. In 2000, Zubkov et al [18,19] observed that vasospasm and apoptosis also occurred in cerebral penetrating arteries, consistent with the time course of angiographic vasospasm in a canine double hemorrhage model, and he thought the vasospasm and apoptosis of penetrating arteries might be the reason for cerebral ischemia and delayed neurological deficits, which could explain the paradoxical phenomenon that occurred in some cases: angiographic vasospasm without clinical symptoms, or clinical deterioration without angiographic vasospasm. Other people proved apoptosis really appeared in spasmatic cerebral basilar arteries and drug could reverse arterial constriction[6,20]. Apoptosis is a type of cell death controlled by gene and has become the key point in biological field. Different from necrosis, the features of apoptosis are the compaction and segregation of the chromatin at the periphery of the nucleus, and condensation of the cytoplasm, often followed by apoptotic bodies[21]. More and more evidence demonstrated apoptosis is essential in physiological and pathological process. On the basis of symptomatic CVS model in rabbits, we found that the basilar arteries became marked spasmatic, with vacuolization in cytoplasm, condensation of chromatin, and some smooth muscle cells showed the similar changes, which was never reported before. The above-mentioned changes were very early apoptosis-like. But unfortunately apoptotic bodies were not found[22]. Some researcher thought that the 481

Shi GJ, et al. / Neural Regeneration Research,2007,2(8):479-82

apoptotic bodies were phagocytized by nearby histomonocytes or epithelial cells. Other people[3] proved that the apoptotic bodies were washed away by the blood, and was hard to observe. In our opinion, besides these reasons, apoptosis has its own time course and we may miss the best observation time. So we concluded that apoptosis may be involved in the pathology of cerebral vasospasm, and it may open a new avenue for CVS treatment, but it also needs further study.

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