A new model of subarachnoid hemorrhage in experimental animals with the purpose to examine cerebral vasospasm

EXPERIMENTAL

NEUROLOGY

81,257-278

(1983)

A New Model of Subarachnoid Hemorrhage in Experimental Animals with the Purpose to Examine Cerebral Vasospasm JOHN LOGOTHETIS, DIMITRIS KABACOSTAS, GEORGE KAROUTAS, NIKOS ARTEMIS, ALI MANSOURI, AND IOANNIS MILONAS’ B’ Department-of Neurology and Psychiatry, Thessaloniki and Agios Dimitrios Received

Aristotelian Hospital,

July 26, 1982: revision

received

University Thessaloniki. November

School of Medicine, Greece 24, 1982

Using 20 rabbits, we tried to establish a new model of experimental subarachnoid hemorrhage (SAH) for examining both acute and chronic cerebral vasospasm.A cranial opening was drilled, and a puncture made on the posterior branch of the middle cerebral artery. A second puncture was made in the superior sag&al sinus for additional withdrawal of subarachnoid blood. The bleeding thus induced resulted in arterial spasm which was studied by using serial electrocorticograms, cerebral blood flow measurement with “‘Xe, and videomicroscopy of the small pial vesselsat various intervals. After death of the animals, the brains were observed to identify the extention of the bleeding. It was indeed obvious that large amounts of subarachnoid blood clots had accumulated. This investigation showed that the rabbit can be used as a new experimental model of SAH. With a two-puncture method, it is possible to simulate the clinical phenomenon of a ruptured aneurysm, that seems to produce acute and chronic cerebral vasospasm. For the latter, the accumulation of blood clots in the basal surfaces plays an important role. The three methods of observation, videomicroscopy, cerebral blood flow measurements, and electrocorticography appeared to provide useful information in the study of biphasic vasospasm in the rabbit.

INTRODUCTION

The occurrence of vasospasm following subarachnoid hemorrhage (SAH) in patients with ruptured cerebral aneurysms is often associated with a poor prognosis irrespective of surgical or conservative treatment. This is a problem Abbreviations: MCA-middle cerebral artery, E&G--electrocotticogram, HVSA-high-voltage, slow activity, SAH-subarachnoid hemorrhage. CBF-cerebral blood flow, CSF-cerebrospinal fluid. ’ Many thanks are expressed to Professor Dim. Tolikas for his assistance in the computer analysis of the data and to Mrs. Aspa Kokkoura for her patience in typing this manuscript. 257 0014-4886/83

$3.00

Copyright 0 1983 by Academx Press. Inc. All rights of reproduction in any form reserved.

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of major importance in patient management and has received a great deal of investigative attention both in clinical (2, 6, 7, 19) and experimental in vivo ( 1,4, 5) and in vitro (9,24), studies. An important book has been indeed totally devoted to cerebrovasospasm by R. Wilkins (38). The vasospasm occurs as early as 0.5 to 4 h after rupture of the aneurysm and may last 1 to 2 weeks. Why vasospasm does not occur in all patients with SAH is a real enigma. It was postulated that the vasculature of some individuals is probably insensitive to the pathophysiologic mechanisms associated with SAH (37). There is experimental evidence that this may be true, because the same procedures which induce vasospasm in one animal, may fail in another, or the spasm may be mild and segmental (22) in some and of great magnitude in others ( 16). To find answers to the problem of vasospasm after SAH, we first developed an experimenta! model of SAH in rabbits. In doing so, we considered the need for an inexpensive and reliable laboratory model for studying the nature of cerebral vasospasm using two successive vascular punctures. The first was in the posterior branch of the middle cerebral artery (MCA), and the second shortly after, in the superior sagittal sinus. Thus, we were able first to simulate the mechanical stimulation at the arterial wall, and then to produce the expected subarachnoid accumulation of blood to obtain the clot at the base of the brain, as happens with rupture of an aneurysm. After the development of such an experimental SAH model we examined the animals for the development of both acute and prolonged cerebral vasospasm as follows: (a) by using videomicroscopy through a cranial opening, for direct observation of the punctured MCA branch and the pial circulation, (b) by measuring the cerebral blood flow (CBF) with ‘33Xe for the estimation of flow values in the superficial and deeper structures of the brain, and (c) by doing serial electrocorticograms (ECoGs) for the evaluation of cortical electrical activity in relation to the vascular changes seen after SAH. MATERIAL

AND

METHODS

We used 20 rabbits weighing 2 to 3.5 kg, divided in three groups. Six rabbits made up the control group A, and seven rabbits representing group B and seven rabbits representing group C, were used in the actual experiment. Although the experimental study began with 38 rabbits (10 in the control group and 14 in each of the B and C groups), 4 animals in the control group and 6 in the other two groups were excluded for various reasons, including edema or dryness of the exposed cortex after craniotomy, or development of adhesions with the surrounding bone. These complications occurred in the early phases of the study in spite of preventative measures, such as the

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use of a glass cover over the exposed cortex and continuous drip with Ringer’s solution (33). After adequate experience was accumulated, such complications no longer occurred. In addition, eight animals of groups B and C died before completion of the experiment. All animals were anesthetized with a 10% sodium pentobarbital solution, 2 ml/kg, given half i.p. and half i.v. into the peripheral aural vein. This method, providing a deep and safe anesthesia without any significant respiratory difficulties, was used only during the initial experimental procedure. During the subsequent serial observations, we used i.v. infusion of 10 mg/ kg Pethidin to achieve adequate analgesia with a light anesthesia and thus avoid the recently described reduction of brain metabolism by prolonged barbiturate anesthesia, that was also claimed to reduce infarction in stroke models (14). The animals were placed in the supine position in a specially designed headholder, that permitted, with appropriate mechanical manipulation, all head surgical procedures in this model to be carried out without turning the animal over. Tracheostomy was carried out in all animals and the right femoral artery was exposed and cannulated with an Abocath No. 22G catheter. This catheter was used for blood sampling, for arterial PC02 measurements (Radiometer BMS-3MK2), and for systemic arterial pressure recordings by connecting it to a Statham P-23A pressure transducer. With this arrangement we were able to intervene immediately should the animals develop respiratory difficulty, by connecting the tracheal tube to a Harvard (model 6 14, dual phase control) respirator, thus keeping arterial PC02 to near baseline values (28 to 32 mmHg). After anesthesia, a parasagittal craniotomy 1 X 2 cm in size was made by an electric dental drill, always on the left side of the cranial vault, 1 mm next to the superior sagittal sinus and 1 mm behind the coronal suture. The dura was then opened with a special hook and the surface of the cortex was constantly moistened by a slow Ringer’s drip at room temperature. At the beginning of this experimental work we warmed this solution to body temperature. Eventually, we continued with solutions at room temperature without any problems, following the experience of Ross Russell’s (33) group. Observation of the control animals (group A) through the cranial opening, included study of the posterior branch of the MCA (150 to 200 pm in diameter) as well as of arterioles and capillaries at the exposed normal cortex with the videomicroscope. In addition, measurements of CBF with 133Xe and serial electrocorticograms were taken. These procedures were carried out 1, 8, 24, 48, and 72 h after the craniotomy. This certainly required considerable manpower; however, constant observation was covered in shifts by the research group. Concerning the videomicroscope method, we were able to measure the diameter of the punctured vessel (posterior branch of the MCA) at the video

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screen, by using a photographic magnification of X600, provided by our optical system, and a plastic ruler divided in millimeters. The CBF was estimated by using two Xe probes, one for each hemisphere, after the i.v. injection of 2.5 mCi ‘33Xe. The clearance of 133Xe from the brain was measured simultaneously for a lo-min period (compartmental method) by each of two collimated scintillation detectors placed symmetrically over the right (closed), and left (craniotomy side) hemispheres (2 1, 30). This method, used both in control (craniotomy only) and in SAH animals (craniotomy plus SAH), provided the CBF values for each hemisphere in the gray as well as in the white matter, thus detecting not only the superficial cortical, but also the subcortical and even deeper flow conditions as well. Estimation of these values was made by the microcomputer (type TRS 80) connected to the Meditronic Cerebrograph. There was no need to make a second craniotomy on the right side, because the skull remained intact over that side both in the control and in SAH animals, thus giving the same error factor in all animals studied. The ECoGs were obtained by using five copper wire electrodes, two on each hemisphere, implanted in the frontal and parietal cortex, respectively, and one in the middle occipital cortex; with the exception of the two electrodes introduced through the cranial opening, the others were implanted through 0.5 mm burr holes in the respective sites (Fig. 1). We used an Alvar electroencephalograph (type E.D 3) and bipolar recordings (paper speed 30 mm/ s, filter 30 Hz, calibration 50 PV, and T.C. 0.3). In group B animals, after completion of the craniotomy with a 0.020~in.diameter needle, we punctured the posterior branch of the MCA to its proximal end of the optical field. The bleeding thus induced nearly always lasted only 2 to 3 min. Additional washing of the cortex with Ringer’s solution was

b’4

p405

F205

FIG. 1. Electrode position sites for recording of electrocorticograms from the rabbit. F,P,frontal-parietal at right side, F2P4-frontal-parietal at lefi side, 05-middle occipital. Leads used FJ’3, P~OJ, W’4r P&,, I=,%, &OS, FFz, P3P,.

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always necessary to clear the area. Ten minutes later, we made a second puncture in the posterior end of the superior sagittal sinus in order to have a larger accumulation of subarachnoid blood (Fig. 2). This bleeding was actually more intense and more prolonged and lasted 5 to 6 min. We observed again the punctured MCA branch and the other arterioles and capillaries of the exposed cortex with the videomicroscope and measured the CBF with ‘33Xe in the same intervals as for the controls. Finally, in the group C animals, after both punctures were made as described above, we did serial ECoGs spaced in the same intervals in the control animals. In all three groups, feeding was accomplished by intraperitoneal infusion of 50 ml 5% dextrose in water by slow drip every 8 h. The amount of infusion providing adequate hydration, and blood perfusion pressure was calculated by taking into account the rabbits’ intravascular volume (150 to 200 ml), the minimal volume of surgical bleeding, and the state of the systemic arterial pressure recorded during the entire experiment. After the animal was killed, the brain was removed and the basal surface observed and photographed to identify the blood clots accumulated after the bleeding (36). The brain was placed in 10% Formalin, fixed in a paraffin block, and cut in coronal sections. These sections, stained with hematoxylineosin, were examined under light microscopy. Student’s t test was used to evaluate the numerical data.

FIG. 2. Basal surfaces from an animal with experimental subarachnoid hemorrhage (right) from a control animal (let?). Note the extensive blood clot accumulated in the former (arrow) and the clear basal surface in the latter.

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RESULTS All values are expressed as the mean f standard deviation. In the control group A, using the videomicroscope method, we observed that the diameter of the MCA branch as well as the arterioles and capillaries of the exposed cortex were significantly stable (P > 0.5 and 0.5 > P > 0.1) during the entire period of observation. Values were in the order of 7.55 f 0.41 to 7.37 f 0.34 mm in the 1st and 72nd h, respectively, as seen in Table 1 and Figs. 3 and 4 in more detail for the entire experimental period. The measurement of CBF with ‘33Xe yielded almost equal values for the two hemispheres. Mean values were in the order of 60.17 f 7.28 to 57.17 + 6.52 ml/100 g/min in the 1st and 72nd h, respectively, with P > 0.5 as seen in detail in Table 1 and in Figs. 3 and 5. The ECoGs were free of any abnormalities (Fig. 6). Systemic arterial pressure and PCOz values were significantly stable (P > 0.5 and 0.5 > P > 0.1) during the serial time periods of observation in all control animals. Values were in the order of 99.33 + 5.75 to 102.50 + 6.02 mm Hg for systemic blood pressure and 29 f 2.53 to 29.17 t- 2.64 mm Hg for arterial PCOz as seen in Table 2 and Fig. 7. TABLE

1

Measurements of the Middle Cerebral Artery and Cerebral Blood Flow in Control Rabbits and after Subarachnoid Hemorrhage Group A (N = 6): control Vessel diameter in mm (mean f SE”) Oh lh 8h 24 h 48 h 72 h

7.55 f 7.32 f 7.53 * 7.28 + 7.37 -t

0.17 0.12 0.15 0.17 0.14

Group B (N = 7): after hemorrhage

Comparison 0.5 > P > 0.1 P>

0.5

0.5 > P > 0.1 0.5 > P > 0.1

Cerebral blood flow in ml/ 100 g/min Oh lh 8h 24 h 48 h 72 h

60.17 f 2.97 61.67 k 2.53 61 f 1.93 58.17 + 2.46 57.17 -+ 2.66

Vessel diameter in mm (mean k SE”) 8.43 6.76 7 5.23 4.86 4.61

+ 0.24 of:0.23 + 0.28 + 0.24 1?:0.20 zk 0.18

Comparison 0.01 > Pz 0.001 0.01 > P > 0.001 P < 0.001 P
Cerebral blood flow in ml/l00 g/min P> P > P > P >

0.5 0.5 0.5 0.5

65.57 45.86 46.29 35 32.29 26.29

LtStandard error; statistical comparison by Student’s t test.

f 3.14 + 3.03 + 3.34 + 3.02 f 2.85 -c 2.56

0.01 > P > 0.001 0.01 > P > 0.001 P < 0.001 P < 0.001 P < 0.001

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FIG .4 . Cerebral cortex of a control animal during the 1st (A) and 72nd (B) hours of ob sen The pl .astic ruler was placed on the TV screen to show the method of measurement. The tdia of the er;posed vessel remained stable.

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FIG. 5. Measurement of cerebral blood flow with “‘Xe in a control animal during the 1st (A) and 24th (B) hours after craniotomy. Photographs were obtained from the microcomputer (type TRS 80), connected to the Meditronic Cerebrograph. Note the almost equal values for the two hemispheres.

The seven rabbits of group B, after the experimental model of SAH was completed, showed a 20 to 30% decrease in the diameter of the punctured arterial branch. Values were 8.43 f 0.63 mm before SAH to 6.76 + 0.60 mm in the first hour after SAH, with 0.01 > P > 0.001, as seen in Table 1 and Figs. 3 and 8. This vascular constriction appeared in the 1st hour after SAH, remained almost the same in the 8th hour, and became worse (40%) in the 24th hour. Values were 8.43 + 0.63 mm before SAH to 5.23 + 0.65

1.1

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SAH

TABLE 2 Arterial PC02 and Systemic Arterial Blood Pressure in Control Rabbits (Group A, N = 6) Systemic arterial pressure in mm Hg (mean f SE”) Cb

lh 8h 24 h 48 h 12 h

99.33 98.33 93 102 98.83 102.50

+ + f f + +

2.35 2.84 2.13 2.13 2.32 2.46

Comparison -

P > 0.5 0.5 > P > 0.1 0.5 1 P > 0.1 P > 0.5 0.5 > P > 0.1

PC02in mm Hg Cb

lh 8h 24 h 48 h 72 h

29 28.5 29.33 28.50 28.33 29.17

+ f f + f +

1.03 1.18 1.26 1.43 1.17 1.08

-

P> P1 P> P> P>

0.5 0.5 0.5 0.5 0.5

a Standard error; statistical comparison by Student’s t test, * c = craniotomy.

mm in the 24th hour after SAH with P < 0.000 1. Thereafter, the punctured branch was partially thrombosed, and several areas on the exposed cortex appeared pale and many capillaries disappeared from the optical field in the next 48 and 72 h of observation. The CBF measurements in all seven animals proved that the flow decreased 30% 1 h after SAH in the ipsilateral hemisphere. On the contralateral side the flow was almost within normal limits. Mean CBF values were 65.57 f 9.03 ml/100 g/min before SAH and 45.86 k 8.01 ml/100 g/min in the first hour after SAH, with 0.01 > P > 0.001 as seen in Table 2 and Figs. 3 and 9. At 8 h after SAH, the CBF values remained relatively stable, whereas 24, 48, and 72 h later the values had decreased significantly (40 to 60%) in both hemispheres. Mean CBF values were 65.57 + 9.03 ml/100 g/min before SAH to 26.29 + 6.78 ml/100 g/min in the 72nd hour after SAH with P < 0.001.

FIG. 6. Representative electrocorticograms from a control animal in the 1st (top) and 72nd (bottom) hours after craniotomy. Note that all leads were free of changes in cortical activity. Time = 30 mm/s, filter = 30 Hz, TC = 0.3, calibration = 50 pV.

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In the seven rabbits of group C, after the experimental model of SAH was carried out, two major electrocorticographic changes appeared to be indicative of ischemic brain damage. In the first hour after SAH, high-voltage, slow activity (HVSA) was found in four animals ipsilateral to the punctured side. Later (8, 24,48, and 72 h after SAH), there was bilateral HVSA in the same animals (Fig. 10). The second observed change was a marked suppression of rhythms in the remaining three animals, with a very low amplitude on the experimental side during the first hour of examination, that continued for the entire period of observation, or was replaced by HVSA (Fig. 11). The eight animals excluded from the study because of early death (between 20 and 62 h) after SAH, showed the same changes during the period they were alive, as did the animals that survived longer. Comparisons were made of course at the same time intervals. All SAH animals showed an immediate rise in systemic arterial pressure

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just after bleeding (150 to 170 mm Hg) that returned to baseline (90 to 110 mm Hg) approximately 1 h later. Respiratory disturbances that necessitated the use of the respirator developed in the first hour after SAH in all 14 animals (arterial PC02 values were 48 +- 4 mm Hg in nine animals and 18 f 3 mm Hg in five animals). Respiration returned to a normal rhythm about 30 min later (PC02 values averaged 29 f 2.5 mm Hg in all animals). Due to the rapid return of the arterial pressure and PC02 values to baseline values, these initial changes were considered rather insignificant in affecting the videomicroscopic, CBF, and ECoG results. The animals were killed by an i.v. Pentothal (20%) injection 96 h after craniotomy. In the 14 animals of groups B and C, after removal of the brains, cerebral edema was observed in almost half of them (8 of 14) on the side of the craniotomy, as evidenced by an increase in hemispheral volume. None of the control animals showed any evidence of brain edema. The SAH was apparent in all 14 experimental animals together with marked accumulation of blood clots, mainly ipsilateral to the puncture side. In spite of unilateral bleeding, the pressure effects observed on the brain stem were possibly severe enough to account for the bilateral changes seen in the CBF measurements and ECoG recordings. There were no SAH or blood clots in the control animals. Ipsilaterally to the punctured side, 2 of 14 animals (groups B and C) presented cerebral infarcts, whereas no brain infarcts were found in any control animal (Fig. 12). DISCUSSION It is well established that prolonged, often delayed, vasoconstriction of the cerebral arteries may accompany SAH from an aneurysm rupture (2, 6, 20). The most commonly considered etiologic factors include (a) mechanical stimulation or injury of intradural arteries (3) (b) action of a vasoconstrictor agent (or agents) in the blood released during platelet aggregation as shown by Linder and Alskne (24) (c) a vasomotor mechanism (16, 29, 39) and (d) a combination of all three (26). Simeone et al. (34), using an angiographic technique, were the first to demonstrate experimentally that acute and prolonged vasospasm can be produced by puncturing with a needle a major branch of the circle of Willis that caused “a brief period of arterial bleeding.” On the other hand, Landaw and Ransohoff (20) observed in monkeys that the combination of vessel puncture and the presence of blood in the subarachnoid space consistently produced marked vasospasm. Among the most important hypotheses advanced regarding spasmogenic substances in the presence of subarachnoid blood are those implicating serotonin (1) catecholamines, hemoglobin, thrombin, prostaglandins (19) thromboxanes BZ, and recently cyclic nucleotide metabolism (25) and an

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I3G. 8. (Cerebral cortex of a group B animal before subarachnoid hemorrhage (A) and the 1st (B)I and 24th (C) hours of observation. Note the diameter of the vessel change tim le, the r bale areas, and the disappearance of capillaries.

ing vith

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3-Continued.

unidentified vasoconstrictor material in the cerebrospinal fluid (CSF) (7). In addition, the blood clots presumably impede the flow of CSF in the basal cisterns and entrap many substances, preventing their rapid outflow from the CSF into the circulation. The fact that saline wash of the subarachnoid space can temporarily relieve an experimentally induced vasospasm in monkeys (12, 36) and dogs (15), suggests that spasmogens accumulate in these clots converting this space into a chemical factory (38). In previous experimental studies, investigators seeking answers for the vasospasm in SAH have utilized mechanical stroking of a vessel (10, 31), topical application of serotonin on the vessels (1, 32), autologous blood introduced into the subarachnoid space ( 1, 18), cutting arteries in the subarachnoid space (16) directly puncturing vessels with needles (5, 34), or a combination of vessel puncturing and repeated injections of blood into the subarachnoid space (20). Those investigators evaluated the resultant vasospasm either by direct observation of pial vessels through cranial openings (IO, 32), by direct observation of the basilar artery through a transclival approach ( 11, 16), or by angiographic techniques (20, 34). Our study presents an attempt to establish a satisfactory and inexpensive new experimental model of SAH in rabbits with the purpose of examining the resultant acute and chronic vasospasm by means of videomicroscopy, measurements of CBF with ‘33Xe, or serial ECoGs. Using two punctures, we tried to simulate as closely as possible the clinical phenomenon of an aneurysm rupture in humans, with the first puncture mimicking the me-

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FIG. 9. Measurement of cerebral blood flow in a group B animal, during the 1st (A) and 72nd (B) hours after experimental subarachnoid hemorrhage. Note the decrease in the value of the left hemisphere in the 1st hour and the further decrease of both hemispheres in the 72nd hour.

chanical stimulation of the vessel wall (3,8), and the second puncture causing a large accumulation of blood clots in the base of the brain (12, 13). Rabbits have been used occasionally to study certain aspects of cerebral vasospasm (9,24), but none of those methods was as close to the physiologic condition as in our study. Linder and Alskne (24), used an in vitro study, and Duckles et al. (9) studied the phenomenon of vasospasm also in vitro by utilizing an isolated basilar artery. In our experimental model, as close to the natural condition as possible, there are still certain limitations as would be expected with any animal model. The first limitation is the open skull technique that significantly minimizes the effects on spasm of intracranial pressure change. A second problem arises from the fact that we were obligated to puncture only a branch of the MCA

FIG. 10. Representative tracings showing the first electrocorticographic sign in a group C animal. Note the high-voltage, slow activity ipsilateral to the punctured side (leads 2-4 and 45) in the 1st hour (top) after subarachnoid hemorrhage. Bilateral high-voltage, slow activity in the same animal appeared during the later hours of observation (bottom). Time, Tc, filter, calibration as in Fig. 5. 273

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FIG. 11. Representative tracings showing the second ECoG sign in a group C animal. Note the marked suppression of rhythms in the punctured side (leads 2-4 and 4-5) during the 1st hour (top) after subarachnoid hemorrhage. This suppression was replaced (bottom) by highvoltage, slow activity in the same animal (leads 2-4 and 4-5) during the later hours of observation.

and in no case its main trunk. The reason was technical in that the main trunk of the MCA in rabbits (and other animals) is well hidden behind the temporal lobe. In order to expose this main trunk either retraction of the

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FIG. 12. Light microscopy in an animal with subarachnoid hemorrhage. Note that the brain’s parenchyma is infiltrated with foci of lymphocytes and leucocytes indicative of an early stage of a cerebral infarct (hematoxylin and eosin, X 16).

temporal lobe (35), or a transorbital approach is necessary both of which are traumatic surgical procedures. A third limitation is the need for continuous observation to maintain the cortex in the best possible condition and also to maintain anesthesia for 72 h together with a feeding every 8 h during this period. We offset costs of personnel by using our research team in 4-h shifts. In spite of these deficiencies, the relative simplicity of the surgical procedures used, the similarity of the lesion to the natural occurrence of SAH, combined with the low cost and availability of rabbits, makes this model a useful experimental tool. Unlike other investigators utilizing conventional arteriography (34,36,40) for the visualization of spasm, the videomicroscopy method (4, 27) used in this study permitted direct observation of the punctured vessel and other pial vessels exposed through a cranial opening (10, 32) and, at the same time, measurement of their diameter as well. Moreover, the observer can directly examine other pathologic changes such as thromboses and disappearance of capillaries, that occur during observation over the exposed cortical surface. In our study we found a 20 to 30% decrease in the diameter of the punctured vessel in the 1st hour after SAH that remained the same in the 8th hour and became worse (40%) in the 24th hour. This is in agreement to the well known biphasic time course in vasospasm that follows SAH (8). The CBF measurement with ‘33Xe, used also in experimental models of SAH by others such as in the study by Weir’s group in Canada (30) proved in our study to follow a biphasic course with a decrease of 30% 1 h after SAH in the ipsilateral hemisphere. Eight hours later the values remained relatively stable, and later they decreased significantly (40 to 60%) in both hemispheres. The fact that the CBF decreased to 30% 1 h after SAH, with

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