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Annals of Clinical & Laboratory Science 35:79-85 (2005)
© 2005 Association of Clinical Scientists

The Influence of Subarachnoid Hemorrhage on Neurons: An Animal Model

Lin Wang, Ji Xin Shi, Hong Xia Yin, Chi Yuan Ma and Qing Rong Zhang
Neurosurgery Department, Jinling Hospital, Nanjing University Medical School, Nanjing, China

Address correspondence to Lin Wang or Ji-Xin Shi, Department of Neurosurgery, Jinling Hospital, 305 East Zhongshan Road, Nanjing 210002, P.R. China; tel 86 25 8596 2075; fax 86 25 8480 6839; e-mail wanglin_77{at}hotmail.com.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgement
 Reference
 
Subarachnoid hemorrhage (SAH) has considerable mortality and morbidity, but the pathophysiologic mechanism is not entirely clear. Following SAH, blood or its lysate enters the subarachnoid space. This study examined how blood lysate influences the vulnerable brain following SAH. Heparinized hemolysate was slowly injected into the cisterna magna of 10 female rabbits, while a control group of 10 rabbits received a similar injection of heparinized isotonic sodium chloride solution without hemolysate. The basilar artery and brain tissue were excised after perfusion fixation. The degree of cerebral vasospasm was evaluated by measuring the cross-sectional area of the basilar artery, and brain damage was investigated by TUNEL staining. In the SAH group, the apoptosis index of neuronal cells located at the base of the temporal lobe averaged 26% (range = 3 to 56%), which was significantly higher than the corresponding apoptosis index in the control group (mean 0.5%, range = 0 to 4%, p <0.001). The mean cross-sectional area of the basilar artery in the SAH group did not differ significantly from that in the control group. These results suggest that SAH induces apoptosis of neuronal cells by a mechanism that is independent of cerebral vasospasm.

(received 24 September 2004; accepted 21 December 2004)

Keywords: subarachnoid hemorrhage, brain damage, apoptosis, basilar artery, cerebral vasospasm


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Acknowledgement
 Reference
 
Subarachnoid hemorrhage (SAH) is a common condition with relatively poor clinical outcome, as 20–35% of SAH patients die within a few days [1]. Unfortunately, there is no effective therapy that specifically targets the pathophysiologic mechanisms of SAH. A serious barrier to developing SAH therapies is the current lack of understanding about the pathogenesis of SAH.

The cerebral vasospasm that follows SAH has been a focus of research in this field. Ischemia that is induced by cerebral vasospasm is believed to play a major role in SAH, but cerebral vasospasm is only one aspect of SAH pathophysiology, and fails to account for all of the related clinical manifestations. Some SAH patients with little or no symptoms have obvious cerebral vasospasm, while others with severe symptoms do not show evidence of cerebral vasospasm [2]. To elucidate the pathogenesis of SAH, research on mechanisms other than cerebral vasospasm is warranted. Following SAH (especially after hemolysis), the properties of cerebrospinal fluid (CSF) change acutely (eg, pH, osmotic pressure, ion concentrations) and potentially harmful substances enter the CSF (eg, complement components, iron, hemoglobin). It is unclear whether or not these changes damage the vulnerable neural tissue. The aim of this research was to study this question in an experimental model of SAH in rabbits.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Acknowledgement
 Reference
 
Animals.  Female New Zealand rabbits (n = 20, age 5 mo, body wt 2.4 to 2.6 kg) were purchased from the Experimental Animal Centre of Jiangsu Province. The rabbits were randomly assigned to 2 groups: control group (n = 10) and SAH group (n = 10). The rabbits had free access to food and water during the experiment. The feeding room and lab room were kept at 25°C. The experimental protocols complied with the Laboratory Animal Care and Use Guidelines of Nanjing University Medical School.

Hemolysate preparation.  Rabbit blood cells were lysed by freezing and thawing [3] to prepare hemolysate. Arterial blood was withdrawn from the central artery of the ear with a sterile syringe. The blood was placed in freezer (–20°C) for 20 min. The hemolysate was then kept at 39°C until use. Aseptic precautions were strictly observed.

Experimental subarachnoid hemorrhage.  Each rabbit was anesthetized by im injection of a mixture of ketamine (25 mg/kg) and droperidol (1.0 mg/ kg). The local hair on the neck was shaved and the skin was sterilized with 75% ethanol. The SAH group was subjected to percutaneous puncture into the cisterna magna. After withdrawal of 0.9 ml of CSF, 0.9 ml of autologous heparinized hemolysate (39°C) was slowly injected into the cisterna magna during 20 sec. The control group received heparinized isotonic sodium chloride solution instead of hemolysate. Then each rabbit was placed in a head-down position (30° angle, 30 min). After recovery from anesthesia, the rabbits were returned to the vivarium.

Perfusion-fixation.  At 48 hr post-injection, the rabbits were anesthetized by im injection of a mixture of ketamine (40 mg/kg) and droperidol (2.5 mg/kg). Each rabbit was intubated with an endo-tracheal tube (3.5 mm diameter) and mechanically ventilated using a rodent ventilator (SGC, China). A 22-gauge butterfly needle (BD, China) was placed in the central artery of the ear and the mean arterial pressure (MAP) and the heart rate (HR) were recorded with a monitor (Spacelabs Medical, USA). Blood samples were obtained at 0, 15, 30, and 45 min to measure arterial pH, pCO2, pO2, and O2 saturation. Perfusion-fixation was then performed as previously described [4–6]. After the thorax was opened, a cannula was placed in the left ventricle, the descending thoracic aorta was clamped, and the right atrium was opened. Perfusion was begun with 300 ml of Hank’s balanced salt solution (HBSS, pH 7.4, 39°C), followed by 400 ml of 10% buffered formaldehyde under a perfusion pressure of 120 cm H2O. The brain was then removed and immersed in the same fixative.

TUNEL staining.  The formalin-fixed brain tissues were embedded in paraffin and sectioned at 4 µm with a microtome. The sections were examined for apoptotic cells by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) method, using an in situ cell death detection kit (ISCDD, Boehringer Mannheim, Germany), according to the manufacturer’s protocol. Briefly, sections were deparaffinized, rehydrated, and washed with distilled water. The tissues were digested with 20 µg/ml proteinase K (Boehringer Mannheim, Mannheim, Germany) at room temperature for 15 min. Endogenous peroxidase activity was blocked by incubation in 0.3% hydrogen peroxide/methanol in PBS at 37°C for 30 min. The sections then were incubated with terminal deoxynucleotidyl transferase at 37°C for 60 min to add dioxigenin-conjugated dUTP to the 3'-OH ends of fragmented DNA. Anti-dioxigenin antibody peroxidase was applied to the sections in order to detect the labeled nucleotides. The sections were stained with DAB and counterstained lightly with hematoxylin. The apoptotic cells were identified and counted by light microscopy, performed by an investigator who was blinded to the grouping. The extent of neurological damage was evaluated by the apoptotic index (ie, the mean number of positive neuronal cells per 100 neuronal cells, based on examination of 1000 neuronal cells).

Blood vessel cross-sectional areas.  As previously described [7–11], the degree of cerebral vasospasm was evaluated by measuring the cross-sectional luminal area of the basilar artery. The formalin-fixed basilar artery was embedded in paraffin, sectioned at 4 µm with a microtome, and stained with hematoxylin and eosin. Micrographs of the basilar arteries were scanned into a computer and the cross-sectional areas of the vessels were measured by an investigator (blinded to the grouping) using a High Definition Medical Image Analysis Program (HMIAP-2000, developed by Tongji Medical University, China). The areas were calculated by measuring the perimeter of the vessel lumen and then calculating the area of an equivalent circle (area = r2, where r = radius), based on the calculated equivalent r value from the perimeter measurement (r = perimeter/2 ), thus correcting for vessel deformation and off-transverse sections. For each vessel, at each midpoint of the proximal, middle, and distal third, 3 sequential sections were measured and averaged. The results were expressed as mean ± SD.

Statistical analyses.  Software SPSS 11.0 was used for statistical computations, including the non-paired Student’s t-test, the Mann-Whitney U test, and Pearson’s correlation coefficient, as appropriate. The level of significance was p <0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Acknowledgement
 Reference
 
General observations.  After the intracisternal injection, the rabbits in the SAH group appeared dispirited, hypoactive, and anorexic, compared to the control group. At post-mortem examination, the SAH rabbits showed no clots over the basal surface of the brain stem, but there were obvious blood stains at the base of temporal lobe (Fig. 1Go).



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  		Fig. 1. Gross appearance of rabbit brain. Panel A: brain of a control rabbit shows neither blood stains nor clots. Panel B: brain of a SAH rabbit shows blood stains at the base of temporal lobe (large arrow), but no clots over the brain stem (small arrow).

 
Physiological parameters.  Table 1Go lists the results (mean ± SD) of MAP and blood gas measurements in the 2 groups at 48 hr after injection of hemolysate or heparinized NaCl solution into the cisterna magna. There were no significant differences in respect to MAP, or blood pH, pCO2, pO2, or O2 saturation in the 2 groups (p >0.05 by t-test).


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  		Table 1. Summary of physiological parameters measured in the 2 experimental groups (mean ± SD, n = 10/group)
 
Evaluation of brain damage.  In the SAH group, TUNEL staining showed numerous apoptotic cells mainly in the cortex at the base of temporal lobe, where blood staining was obvious (Fig. 2AGo). The hippocampus showed no apoptotic cells (Fig. 2BGo). In the control group, there were either no apoptotic cells or a few apoptotic cells irregularly distributed in the brain tissue (Fig. 2CGo). As listed in Table 1Go, the apoptosis index of the SAH group averaged 25.8% (range = 3 to 56%) and that of the control group averaged 0.5% (range = 0 to 4%; p <0.001 by Mann-Whitney U test). From the TUNEL results, it was evident that brain was severely damaged after SAH.



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  		Fig. 2. Photomicrographs of TUNEL staining of brain tissue, magnification x400. Apoptotic cells are stained brown and normal cells are light blue. Panel A: cortex of temporal lobe of a SAH rabbit, showing dystrophic, brown-stained apoptotic cells (arrow). Panel B: hippocampus of a SAH rabbit, showing light blue-stained cells with normal structure. Panel C: cortex of temporal lobe of a control rabbit, showing light blue-stained cells with normal structure (arrow).

 
Cross-sectional areas of the basilar arteries.  The mean cross-sectional luminal area of the basilar artery in the SAH group was 3.36 x105 ± 1.54 x105 µm2, which did not differ significantly from the corresponding mean value in the control group (3.65 x1.05 ± 1.70 x105 µm2) (p = 0.69 by Student’s t-test). This suggests that there was no significant difference of cortical blood-supply between the control and SAH groups. Moreover, the cross-sectional areas of basilar arteries showed no significant correlation with the apoptosis index in either group (Fig. 3Go). Hence, it is unlikely that the difference of pathological findings in the 2 groups is attributable to cerebral vasospasm.



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  		Fig. 3. The cross-sectional areas of the basilar arteries (µm2) show no significant correlation with the apoptosis index in the control or the SAH groups (p >0.1, Pearson’s correlation analysis).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Acknowledgement
 Reference
 
An experimental model of SAH that induces severe cerebral vasospasm cannot be used to delineate the neural pathophysiologic changes caused by blood components, because vasospasm-related ischemia would confound interpretation of the results. Therefore several modifications of the rabbit SAH model were adopted in this study. First, rabbits were observed at 48 hr post-SAH, since cerebral vasospasm usually occurs after the third day post-SAH in the rabbit model [12]. Second, a single-hemorrhage model was used instead of the commonly used two-hemorrhage model [12–15]. The injection volume was reduced to 0.9 ml from the more commonly used 1.2–3.0 ml [12–15]. These measures were designed to minimize blood vessel stimulation and decrease the odds of vasospasm. Third, blood lysate was injected instead of blood, so that blood-borne toxins that may mediate SAH might be more precisely controlled. Blood cells typically begin lysis in the CSF on the third day after aneurysm rupture [16], which implies that potential intracellular toxins (eg, iron, potassium, hemoglobin, oxidative enzymes) are unlikely to be present in the subarachnoid space at 48 hr. Therefore, in the present study, blood cells were lysed in vitro by freezing and thawing [3] before injection. Lastly, the blood lysate was heparinized to prevent blood clot formation, which is known to potentiate cerebral vasospasm. These modifications were intended to facilitate studying the blood lysate-mediated toxic injury, independent of cerebral vasospasm.

The results of TUNEL staining showed obvious neural damage after SAH. Potential factors that could induce the neural damage include vasospasm-related ischemia, hypoxemia, hypotension during operation, low perfusion pressure after SAH, and the effects of toxic blood components. Measurements showed no difference in the cross-sectional area of the basilar artery in the 2 groups. Furthermore, there was no correlation between the cross-sectional areas of the basilar artery and the apoptosis index. Therefore, the brain damage in this study was unlikely to be caused by vasospasm-related ischemia.

The physiological parameters (mean arterial pressure, blood pH, pCO2, pO2, O2 saturation) did not differ significantly in the 2 groups, which indicates that the brain damage of the SAH group was unlikely to result from hypoxemia or hypotension. The volume of fluid that was injected into the subarachnoid space was the same as the volume of CSF that was withdrawn. This precaution was intended to avoid high intracranial pressure or low perfusion pressure, which could damage the brain. These considerations suggest that the brain damage in the SAH group was induced by blood lysate. Another finding that indirectly supports this conclusion is that the apoptotic cells mainly collected at the inferior basal temporal lobe where blood stains were obvious (Fig. 1Go). This suggests that the brain damage was related to blood toxins or lysate products. If the brain damage were caused by vasospasm-related ischemia, hypoxemia, hypotension, or low perfusion pressure, apoptotic cells would have been widely distributed in the brain (especially in the hippocampus, which is highly sensitive to ischemia and hypoxia).

Previous work suggested that blood or its lysate could influence SAH brain injury. First, blood or its lysate is known to damage normal tissues. Thus, subretinal hemorrhage may induce atrophy of retina [17], hemosiderin may damage the lungs of patients with thalassemia [18], heme released from red cells has cytotoxic properties [19–21], and bilirubin can cause CNS dysfunction in hemolysis patients [22–24]. It has been reported that blood or its lysate could cause pathological changes or inflammation of the basilar artery [25–28]. Second, neural cells are particularly vulnerable to toxins and their normal function and survival depends on strict homeostasis, including cerebrospinal fluid (CSF) stabilization. Besides its cushioning function, the CSF provides essential nutritive substances to the central nervous system [29]. The normal electrophysiological activity of the brain depends on various ion concentrations in the CSF. Following SAH, some important CSF characteristics change substantially, including osmotic pressure and ion concentrations, and some potentially harmful substances enter the CSF, such as complement components, iron, and hemoglobin. This study in a rabbit model of SAH indicates that the presence of blood lysate in the subarachnoid space can damage the brain tissue.

While vasospasm clearly plays a central role in the later stages of SAH, it alone does not adequately explain the clinical manifestations and outcomes of patients with SAH. The present study showed apoptosis primarily in the temporal lobe of rabbits injected with hemolysate, consistent with blood lysate-related injury, rather than ischemia. Further research is warranted to elucidate the molecular pathogenesis of brain injury and apoptosis in this experimental model of SAH.


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  		Table 2. The apoptosis index of neuronal cells in the control and SAH groups (10 rabbits/group)
 

   Acknowledgement
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgement
Reference
 
We thank Dr. Wu Bo for assistance with pathological examinations and Dr. Liu Yuxiu for statistical advice.


   Reference
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgement
 Reference
 

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