Etiology of the disruption in blood-arterial wall barrier following experimental subarachnoid hemorrhage

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Etiology of the Disruption in Blood-Arterial Wall Barrier Following Experimental Subarachnoid Hemorrhage Tadayoshi Nakagomi, M .D ., D .M .Sc ., Neal F . Kassell, M .D ., Tomio Sasaki, M .D ., D.M .Sc ., R . Michael Lehman, B .A ., and Shigeru Fujiwara, M .D ., D .M .Sc . Department of Neurological Surgery, University of Tokyo Hospital, Tokyo, Japan, and Department of Neurological Surgery, University of Virginia School of Medicine, Charlottesville, Virginia

Nakagomi T, Kassell NF, Sasaki T, Lehman M, Fujiwara S . Etiology of the disruption in blood-arterial wall barrier following experimental subarachnoid hemorrhage. Sing Neurol 1990 ;34 :16-26 .

Aneurysmal subarachnoid hemorrhage is associated with a sudden rise in intracranial pressure, acute arterial hypertension, and subarachnoid blood . The role that each of these factors may play in the development of the acute barrier disruption of the major cerebral arteries following subarachnoid hemorrhage was investigated in 42 rabbits . Horseradish peroxidase was given intravenously to assess the integrity of the barrier by transmission electron microscopy . Permeation of the tracer into the vessel was noted only in animals with increased intracranial pressure . A sudden rise in intracranial pressure is suggested to trigger acute barrier disruption following subarachnoid hemorrhage . Subarachnoid hemorrhage ; Cerebral vasospasm ; Vascular permeability KEY WORDS :

Introduction In previous studies we have demonstrated disruption of the blood-arterial wall barrier of the major cerebral arteries following experimental subarachnoid hemorrhage (SAH) and have postulated that the increased vascular permeability is causally related to the pathogenesis of vasospasm [14] . This correlates with the observation in humans that development of vasospasm following aneurysmal SAH is associated with abnormal contrast enhancement of the basal subarachnoid cistern on computed tomography (CT) scan [2,3,6,161, which is likely an indication of increased permeability of the major cerebral arteries to radiographic contrast media .

Address reprint requests to . Neal . F Kassell, M .D ., Department of Neurological Surgery, Box 212, Medical Center, University of Virginia, Charlottesville, Virginia 22908 . Received December 19, 1988 ; accepted February 13, 1990 .

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The blood-arterial wall barrier disruption following experimental SAH occurs acutely as well as chronically ; indeed, it appears to be biphasic, correlating with the biphasic pattern of angiographic vasospasm in animals . Aneurysm rupture causes a sudden steep rise in intracranial pressure (ICP) and a resultant acute arterial hypertension as well as producing a subarachnoid clot . Each of these may be involved in the disruption of the blood-arterial wall barrier . The present study was conducted in order to investigate the role that each of these factors may play in the development of the barrier breakdown and to clarify the mechanism of disruption of the blood-arterial wall barrier in the major cerebral arteries in the acute stage following SAH . Materials and Methods Forty-two male New Zealand white rabbits, weighing 2 .5-3 .5 kg, were used to investigate the individual or combined influence of the three factors-subarachnoid clot, acute arterial hypertension, and sudden rise in ICP-on the barrier breakdown in the basilar artery . The animals were divided into seven groups as follows . Each group consisted of six animals . Group 1 : Control (normal animals) . Group 2 : SAH . These animals were injected with 2 .5 ml, of fresh autologous nonheparinized arterial blood into the cisterna magna . Group 3 : Mock SAH . These animals were injected with 2 .5 mL of 0 .9% physiological saline into the cisterna magna. Group 4 : Isobaric SAH [SAH without increased ICP and systemic arterial pressure (SAP)] . These animals were injected with 1.3 mL of arterial blood over 1 minute just after withdrawal of 1 .1 mL of cerebrospinal fluid in order to prevent an increase in ICP and the resultant increase in SAP . Group 5 : Acute arterial hypertension . To simulate 0090-3019/90/$3 .50



SAH and Cerebral Arterial Permeability

the blood pressure changes in the SAH and mock SAH groups (groups 2 and 3), SAP was initially increased by intraaortic balloon occlusion of the thoracic aorta followed by withdrawal of blood through the balloon catheter to gradually reduce the blood pressure . Group 6 : Mock SAH without increased SAP . To simulate the ICP change in SAH and mock SAH groups (groups 2 and 4), animals were injected with 2 .5 mL (low ICP-group 6a) or 5 .0 ml, (high ICP-group 6b) of physiological saline . During and after the injection, SAP was maintained constant by withdrawing the blood through an arterial catheter. In groups 2, 3, 4, and 6, arterial blood or physiological saline was injected into the cisterna magna at a rate of 0 .4 mL/s by infusion pump (Harvard model 950, Harvard Apparatus, South Natick, MA) . Preliminary experiments demonstrated that physiological saline had the same effects on the physiological parameters and cerebral arterial permeability as artificial cerebrospinal fluid [18) when used to produce mock SAH . Anesthesia for the surgical preparation was achieved with an intramuscular injection of ketamine (20 mg/kg), xylazine (5 mg/kg), and acepromazine (0 .25 mg/kg) in a ratio of 8 :1 :1 . The anesthesia was supplemented as required . Each animal was placed in the supine position and a tracheostomy was performed . Muscular paralysis was achieved with intravenous pancuronium bromide (Pavulon, 0 .08 mg/kg/30 min ; West Orange, NJ) and ventilation was maintained with a dual phase control respirator (Harvard model 683, Harvard Apparatus, South Natick, MA). After laparotomy, catheters were introduced into the aorta and into the inferior vena cava 3 cm proximal to the femoral bifurcation . Either a 16gauge catheter connected to a silicon rubber tube (Manosil-3/16 x 1/16 in) or a balloon catheter (7 SwanGanz catheter, 1 .5 cm3 cap., 93A-131H-7F) was used as an arterial line . To avoid acute arterial hypertension during and after the cisternal injection of physiological saline (group 6), the silicon rubber tube was filled with heparinized saline, which was set to overflow when the SAP went above the predetermined value of the blood pressure . An intraaortic balloon catheter was used to increase the SAP by occluding the aorta . The arterial line also served for blood pressure monitoring and blood gas sampling. A 20-gauge catheter inserted into the inferior vena cava was used for the administration of drugs and horseradish peroxidase (HRP) . Arterial blood gases were checked at least twice, 10 minutes before and 10 minutes after the cisternal injection, and pH, P ot , and Pco , were maintained within physiological range . In groups 2-6, the animal's head was positioned in the stereotactic frame, the atlantooccipital membrane was

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exposed, and a 27-gauge butterfly needle was inserted into the cisterna magna . The needle was connected to a pressure transducer with a three-way stopcock and one of the outlets served for the injection of arterial blood or saline . Intracranial pressure was monitored after the injection . Systolic arterial pressure and ICP were monitored with pressure transducers (Hewlett Packard 78342A, Andover, MA) and recorded on a chart recorder (Gould 2400, Cleveland, OH) . Five minutes before the cisternal injection, all animals received an intravenous injection of 2 % HRP solution in saline (HRP : 200 mg/kg body weight; Sigma, type II) . Twenty minutes after the cisternal injection, the animals were killed by perfusion-fixation via the left ventricle of the heart . After a brief initial perfusion with saline, perfusion-fixation with a mixture of 2% paraformaldehyde and 2 .5% glutaraldehyde in 0 .1 M phosphate buffer (pH 7 .4) was conducted, under a pressure of 130 cm H 2O . The brain was immediately removed and immersed in cacodylate-Karnovsky solution (pH 7 .3) for 5 hours at 4°C, and then kept overnight at 4°C in 0 .1 M sodium cacodylate buffer (pH 7.3). The next day the basilar artery was removed from the brain under magnification . In group 5 animals with acute arterial hypertension, 75-µm-thick brain tissue slices coronally sectioned at the level of the infundibulum were prepared on a vibratome (Lancer, Series 1000, T .P .I ., Inc ., St . Louis, MO) in order to determine whether or not the blood-brain barrier was disrupted in the cerebral cortex . Horseradish peroxidase was localized by Graham and Karnovsky's [41 procedure using a medium consisting of 5 mg of 3,3'-diaminobenzidine-tetra-HCI, 10 ml of 0 .05 M Tris-HCI buffer (pH 7.6), and 0.1 mL of 1 % hydrogen peroxide . In order to obtain optimal reactions in the basilar artery, the diaminobenzidine solution was irrigated through the vessel lumen using a 2 7-gauge needle . Using light microscopy, areas of HRP extravasation in the basilar artery and cerebral cortex were easily identified by the characteristic brown staining of the reaction product. The lower third of the basilar artery and, in the group 5 animals with acute arterial hypertension, the sections from the frontal cortex were selected for electron microscopic study . For transmission electron microscopy, samples were post-fixed in 1 % osmium tetroxide in 0 .1 M sodium cacodylate buffer at pH 7 .4, dehydrated in graded acetone, and embedded in Epon 812 . Ultrathin sections unstained or stained with uranyl acetate were examined with a Hitachi HU-12A electron microscope (Hitachi, Tokyo, Japan) .

Statistical Analysis The values of the blood pressure, ICP, and blood gases in the six groups were analyzed using analysis of variance



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Table 1 . Summary of Experimental Groups and Results Animal group I 2 3 4 5 6a 6b

Control Nonisobaric SAH Mock SAH Isobaric SAH Acute arterial hypertension Mock SAH without increased SAP, low pressure Mock SAH without increased SAP, high pressure

Time to maximum SAP (s)

MSAP before insult 69 .3 75 .0 69 .2 74 .2 67 .1 68 .2

- 2 .96 ± 4 .28 ± 2 .71 3 4 .36 t 1 .00 ± 2 .39

30 .2 ± 6 .43 37 .6 ± 8 .73 42 .8 +- 7 .98

72 .6 '- 3 .10

AMSAP (mmHg) 2 .3 ± 0 .42 58 .0 ! 5 .86° 65. x 1 .94' 3 .5- 0.76 68 .8 - 2 .44' 5 .0 = 0 .82 4 .3 ± 0.84

SICP (mmHg)

Barrier breakdown in the basilar artery

151 .7 7 .82" 71 .7 ± 5 .42' 2 .5 + 1 .57 8'

(-) (++) (++) (-1 (-)

69.7 ± 5 .02' 135 .7- 21 .34 8

(+)

Each group consists of six animals . Abbreviations : ICP, intracranial pressure; MSAP, mean systemic arterial pressure ; SAH, subarachnoid hemorrhage ; AMCP, increase in ICP after subarachnoid injection of blood or saline ; AMSAP, increase in MSAP after subarachnoid injection of blood or saline ; (-), no permeation of horseradish peroxidase into the subendothelial space ; (+), slight and localized permeation of horseradish peroxidase into the subendothelial space ; (++), marked permeation of horseradish peroxidase into the subendothelial space . ° P < 0 .01 is control . ^p < 0.01 vs group 3, p < 0 .01 vs group 2 .

(ANOVA) with the Scheffe test for critical differences . A p value of less then 0 .05 was considered significant . Data are presented as mean ± standard error of the mean (SEM) . Results Physiological Parameters As shown in Table 1, the mean arterial pressure (MAP) prior to the insult was approximately 70-75 mmHg.

There were no significant differences among the six groups . In groups 2, 3, and 5, the MAP increased abruptly just after the insult and reached its maximum value at 30 ± 6 s (mean ± SEM), 38 ± 9 s, and 43 ± 8 s, respectively (Figure 1 A, B, and D) . The magnitudes of the MAP changes were 58 ± 6 mmHg, 65 ± 5 mmHg, and 69 ± 2 mm Hg. There was no significant difference between the three groups for these two parameters . The length of time during which the SAP remained elevated was variable . In groups 1, 4, and 6, no peak of SAP was

Figure 1. (A-E) Representative traces afjyjtemic arterial pressure (SAP) and (F andG) intracranial pressure (ICP) . Systemic arterial pressures increased just after the injection (Ir!) of either (A) arterial blood or (B) physiological saline, However, no peak ofSAP was noticed in the animals that (C) received isobaric blood injection or in the (E) animals in which SAP was kept constant by withdrawal (W) of the blood. Acute arterial hypertension was introduced by inflating the balloon (INF) of the catheter placed in the aorta . The ICPrfie lasted longer in the (F) animals that received blood injection than in (G) those that received saline injection . HRP . horseradish peroxidase; S, sacrif ce. @10.~ NNI ,.

S

1 reo tm~ 0 ~-



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Table 2 . Blood Gases of Experimental Groups Before and After Insults Before Animal group

pH

PC .,

1 2 3 4 5 6a 6b

7 .41 ± 0.03 7 .45 ± 0.03 7 .47'-0.02 7 .48 ± 0.03 7 .44 ! 0.03 7 .38 ! 0 .03 7 .39 ! 0 .02

36 .9-' 2 .16 33 .7 - 1 .16 34 .3±4.27 30 .6 ± 1_95 39 .1 ± 1 .87 33 .0 ± 2 .23 37 .8 ± 3 .24

After pH 128_1±12 .12 98 .9 ± 12 .66 117 .6±14 .58 128 .6 ± 20 .03 93 .5 ± 12 .09 104 .1 ± 7 .72 114 .5 ± 7 .57

7 .41 7 .35 7 .38 7 .43 7 .43 7 .48 7 .37

± 0 .04 '- 0 .02 ± 0 .05 ± 0 .02 *- 0 .02 *- 0 .04 1 0 .03

38.8 35 .5 42 .2 35 .9 33 .6 33 .5 42 .3

± '± -' ± ! ±

1 .53 2 .79 3 .37 4 .00 4 .43 3 .57 5 .27

126.1 101 .1 119.8 105 .6 132 .2 100.9 97 .8

! 14 .47 ± 9.74 '_ 11 .35 2 11 .36 ± 14 .06 ± 5 .21 ± 6.44

Each group consists of six animals .

noticed (Figure 1 C and E) . The ICP prior to the insult ranged from I to 2 mmHg . In groups 2, 4, 6a, and 6b, there was an abrupt transient increase in ICP of 152 ± 8 mmHg, 72 ± 5 mmHg, 70 ± 5 mmHg, and 136 ± 21 mmHg, respectively . Intracranial pressure changes between groups 2 and 6b, and between groups 3 and 6a, were not significantly different . In group 2 animals, it took a longer time to return to the base ICP level than in the other groups (Figure 1 F and G) . The ICP change in group 4 animals was minimal . In group 5, ICPs of three animals were monitored and they ranged between 0 and 4 mmHg. Arterial blood gases were all within physiological ranges and were not significantly different among the experimental groups both before and after the insult (Table 2) . Gross Observation

In group 2 SAH animals, a thick subarachnoid clot was observed on the basal surface of the brain surrounding the major cerebral arteries . The subarachnoid clot in group 4 isobaric SAH animals was confined to the subarachnoid space over the brain stem . However, the clot was wide and thick enough to cover the entire basilar artery . Basilar arteries in group 2 SAH animals showed marked constriction, especially in the lower region . In the other groups, such marked constriction was not noticed . Basilar arteries of control animals were not stained macroscopically with HRP-reaction product . In the animals that received a cisternal injection of arterial blood (group 2), the basilar arteries were diffusely and markedly stained with HRP-reaction products, whereas the basilar arteries of groups 3 (mock SAH), 5 (acute arterial hypertension), and 6 (mock SAH without increased SAP) were diffusely but lightly stained . In two specimens taken from the group 3 animals (mock SAH), marked staining of portions of the basilar arteries was noticed . Basilar arteries in the group 4 animals (isobaric SAH) were not visibly stained . Brain slices in group 5 animals

(SAP increase) showed cortical or subcortical minute foci of HRP-reaction products . Electron Microscopic Observation

In control animals, no acute morphological abnormalities were observed in the basilar arteries . Horseradish peroxidase-reaction products were observed mainly in the luminal pits of endothelial cells . A few plasmalemmal vesicles in the cytoplasm were also stained with HRP . However, neither abluminal vesicles nor interendothelial spaces were stained with HRP . No HRP-reaction product was observed in the subendothelial space or smooth muscle layers (Figure 2) . In group 2 SAH animals, marked corrugation of the elastic lamina was frequently observed and endothelial cells were compressed between tight folds of the elastic lamina. These morphological alterations were not seen in the othergroups . In spite of such findings, no degenerative change in the endothelial cells was noticed in the group 2 animals . Horseradish peroxidase-reaction products were frequently found at the luminal front, in the plasmalemmal vesicles, and at the abluminal front of endothelial cells as well as in the subendothelial space (Figure 3 A and B) . In some areas of these basilar arteries, HRP-reaction products were observed in the myointimal region and smooth muscle layers (Figure 3 C) . Occasionally, interendothelial spaces were stained, but only partially (Figure 3 B) . In group 3 mock SAH, HRP-reaction products were consistently visualized at the luminal front, in the plasmalemmal vesicles, and at the abluminal front of endothelial cells . In several animals ; HRP-reaction products were observed in the subendothelial spaces and in the myointimal regions (Figure 4) . The HRP-labeled vesicles or pits were also observed to be connected to the subendothelial spaces . In contrast, only occasional staining of the abluminal portion of the interendothelial spaces was noticed (Figure 4) . In group 4 and group 5 animals, which had isobaric SAH and acute arterial hypertension, respectively, no



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permeation of HRP into the subendothelial space was observed, although luminal pits or vesicles and a few plasmalemmal vesicles were stained with HRP-reaction products (Figures 5 and 6 A)- In group 5 acute arterial hypertension animals, interendothelial spaces were always free from HRP-reaction products (Figure 6 B) . In the frontal cortex of these animals, blood-brain barrier disruption was demonstrated both in capillaries and in precapillary arterioles . Horseradish peroxidase-reaction products were observed in the basement membrane of the endothelial cells and smooth muscle layers (Figure 6 C) . Numerous pinocytotic vesicles containing HRP were present at the abluminal front of the muscle cells and the endothelial cells . Finally, in group 6 animals, which had mock SAH without increased SAP, HRP-reaction products were evident at the luminal front, at the abluminal front of endothelial cells, and also in the subendothelial space and in the myointimal region (Figure 7) . In particular, a number of plasmalemmal vesicles of various sizes were stained with HRP-reaction products (Figure 7 B) . Interendothelial spaces were only occasionally and partially stained with HRP-reaction products (Figure 7 B) . In places, channel-like structures with HRP were observed near the interendothelial spaces . Electron microscopic findings were the same in group 6a (low ICP) and 6b (high ICP) animals .

Figure 2 . Unstained electron micrograph of the basilar artery from a control rabbit . Horseradish peroxidase-reaction products are observed at the luminal front of the endothelial cells, but not ire the subendothelial space or in the smooth muscle layer . The interendotbelial space (ti,l is devoid of HRPreaction products (bar 7 pm) . E . endothelium; VL, vascular lumen, el, elas i lamina : sm . smooth muscle .

Discussion The disruption of the blood-arterial wall barrier in the major cerebral arteries following experimental SAH has been demonstrated in our previous studies, and we suggested that this barrier disruption may be involved in the pathogenesis of vasospasm [14) . Three important factors-presence of a subarachnoid clot, acute arterial hypertension, and sudden rise in ICP-are considered as possible mechanisms responsible for the disruption of the blood-arterial wall barrier in the acute stage following SAH . The present experiments revealed the following : (1) the cisternal injection of either arterial blood or physiological saline with increased ICP and SAP produced extensive disturbance in the blood-arterial wall barrier of the basilar arteries ; (2) subarachnoid clots surrounding the basilar arteries do not contribute to the acute breakdown of the barrier in isobaric experimental SAH where ICP is not elevated ; (3) acute arterial hypertension alone, of the magnitude induced in these experiments, cannot



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Figure3. Electron micrographs of the basilar artery from rabbits that received cisternal injection of arterial blood (group 2), (A) Various-sized plasmalemmal vesicles in the cytoplasm of the endothelial cells arefslled with HRP-reaction products (uranyl acetate stained, bar = I µm) . (B) Marked corrugation of the elastic lamina is demonstrated . Horseradish peroxidase-reaction products are observed in the vesicles in the abluminal front ofthe endothelial tells and in the subendothelial space . Interendothelial space (tzi is stained with HRP-reaction product in its lower region (arrowheads) (unstained, bar = I µm) . (C) Horseradish peroxidase-reaction products are observed in the subendothelial space and in the smooth muscle layer (arrowheads) . Horseradish peroxidase-reaction products are also seen at the luminal front of the smooth muscle cell (arrows) . Interendothelial spaces (*) are free from HRPreaction products (uranyl acetate stained, bar = I µm) . E, endothelium ; VL, vascular lumen ; el. elastic lamina ; sm . smooth muscle .

disrupt the blood-arterial wall barrier ; (4) sudden rise in ICP is responsible for the acute barrier disruption, even in the low ICP group, as well as in the high ICP group . In the acute stage following experimental SAH, the subarachnoid clot alone around the basilar arteries is not sufficient to induce the breakdown of the blood-arterial wall barrier . This observation is reinforced in the present experiments by the findings that cisternal injection of physiological saline as well as arterial blood injection produced an extensive disturbance of barrier function in the basilar arteries . However, the present result seems

to be different from that previously obtained in our laboratory in a chronic canine double hemorrhage model [14] . The study demonstrated morphological changes of the arterial wall including degenerative changes of endothelial cells, and complete filling of the interendothelial spaces with HRP-reaction products in both isovolemic and nonisovolemic SAH 3 days after the second SAH. In the present experiments, however, arterial blood, enough to fully cover the entire brain stem, was injected very slowly in the isobaric SAH animals and changes in both SAP and ICP were negligibly small . Microscopic studies did not demonstrate HRP perme-



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ation into the subendothelial space of the basilar artery . Absence of morphological changes-in particular, lack of degenerative changes of the endothelial cells-might explain the different results . It is well known that acute arterial hypertension increases the permeability of the brain capillaries [5,6,10,11,13,19] . Based on these results, it is reasonable to assume that acute arterial hypertension in the setting of SAH is an essential factor in producing blood-arterial wall barrier breakdown . It has been demonstrated that the effectiveness of an acute hypertensive insult in inducing blood-brain barrier breakdown is directly proportional to the magnitude of the blood pressure rise and inversely proportional to the length of time it takes to elevate it [8] . Therefore, we compared the SAP changes in group 5 animals with acute arterial hypertension with those animals with nonisobaric SAH or nonisobaric mock SAH . The increase in SAP in animals with acute arterial hypertension is enough to induce blood-brain barrier breakdown, as documented in previous reports [5,11,131 . In fact, in this group, the present study revealed barrier breakdown both in the precapillary arterioles and in capillaries in the cerebral cortex . However, we could not detect any permeability change in the basilar arteries, except the presence of many vesicles containing HRP-reaction products at the luminal

Nakagomi et at

Figure 4 . The barilar artery of a rabbit that receired cisternal injection of phwiological saline (group 3) . Horseradish peroxidase-reaction products are ohrerved in the sabendothelial space and in the rnynrnttmal region . Horseradish peroxida .e-reaction products are seen at the ablum ;nal front of the endothelium and at the luminalfront of the smooth muscle cell (arrotes) . The interendothelial space (?U is filled with HRP-reaction products in its ahitemenal portion (arrou'beads) (uranyl acetate stained. bar - I.µn)F, endothelium ; VL, macular lumen ; el, elastic lamina ; son, smooth muscle.

surface of the endothelial cells . Thus, major cerebral arteries seem to be more resistant to the acute arterial hypertension than the arterioles or capillaries in the cerebral cortex . However, in animals with nonisobaric SAH and in animals with nonisobaric mock SAH, breakdown of the blood-arterial wall barrier was more marked than in animals with mock SAH without increased SAP . The possibility that acute arterial hypertension might reinforce the barrier disruption by facilitating the transendothelial vesicular transport cannot be ruled out . In the present study, it is demonstrated that a sudden rise in ICP triggers the disruption of the blood-arterial wall barrier following SAH . It has been reported that the ICP is increased by approximately 150 mmHg after rebleeding of aneurysms [12,17] . In animal models in which SAH was produced by arterial puncture, the ICP was reported to increase up to 150-250 mmHg [1,15] . In the present studies, the increase in the ICP in animals



SAH and Cerebral Arterial Permeability

with nonisobaric SAH and in animals with mock SAH without increased SAH (high ICP) is consistent with these reports . The more pronounced increase in ICP in animals with nonisobaric SAH than that in animals with nonisobaric mock SAH is probably due to the viscosity of the blood, which enhances the ICP elevation . In the present studies, no significant permeability changes could be'seen between the subgroups with mock SAH without increased SAP . These results suggest that barrier change occurs just after the ICP goes over the critical level and is not proportional to the increase in ICP. Such a level seems to be near the SAP of the animals before insult, since the major cerebral arteries start to shrink when the ICP goes over the SAP . Increased ICP can bring about a decrease in transmural pressure with subsequent diffuse short-term reduction in arterial wall caliber resulting in corrugation of the elastic lamina . The corrugated lamina may squeeze the endothelial cells and inhibit the metabolism of the cells [9], which probably induces the barrier breakdown . Thus, a sudden rise in ICP triggers the disruption of the blood-arterial wall barrier . A subarachnoid clot further prolongs the ICP elevation. In this sense, the subarachnoid clot is a cofactor that exacerbates the barrier disruption . Histamine may also play a role in the disruption of the barrier . Previous studies [7] demonstrated that, in regions of

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Figure5. Unstained electronmicrographofthebasilararteryfromarabbit that received isobaric blood injection (group 4) . Plasmalemmal vesicles in the cytoplasm of the endothelial cell are filled with HRP-reaction products . Interendothelial space (*) is devoid of HRP-reaction products (bar = I gm) . E, endothelium; VL . vascular lumen ; el, elastic lamina ; sm, smooth muscle.

increased hemodynamic shearing stress, there is increased histamine synthesis in the endothelial layer and an increase in vascular permeability . Electron microscopy revealed that in animals with mock SAH without increased SAP, HRP permeated into the subendothelial space . Horseradish peroxidasereaction products were frequently observed in luminal pits, in plasmalemmal vesicles in the cytoplasm, and at the abluminal front of the endothelial cells . In places, HRP-reaction products were also found in the channellike structures of the endothelial cells and in the subendothelial space . On the other hand, interendothelial spaces were only occasionally labeled with HRP-reaction products . These results suggest that HRP permeated into the subendothelial space through the transendothelial routes rather than into interendothelial spaces . The present studies, together with the previous studies [ 14] in chronic experimental SAH, suggest that there are two types of disruption of the blood-arterial wall



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Figure 6. Stained electron micrographs of the basilar artery and the precapillary arteriole in thefrontal cortex of a rabbit in which SAP was increased using a balloon catheter (group 5) . (A) Horseradish peroxidase-reaction products are observed in the luminal front of the endothelium (bar = I gm) . (B) The interendothelial space (u) is free from HRP-reaction products (bar 0 .5 µm) . (C) Horseradish peroxidase-reaction products are observed not only in the subendothelial basement membrane but also in the basement membranes surrounding the one or two layers of smooth muscle cells . E, endothelium: VL, vascular lumen : (bar = 2 wool el. elastic lamina .

barrier in the basilar arteries following SAH . One is a breakdown of the barrier in the acute stage, which is triggered by the sudden rise in ICP . The main route of protein leakage into the subendothelial spaces and muscle layers may be transendothelial . The other is a barrier disruption due to the vasoactive substances released from the subarachnoid clot after some latent period of time . Vasoactive substances seem to produce the degenerative changes in endothelial cells, which may facilitate the transendothelial permeation of HRP and also cause the opening of the junctions between endo-

thelial cells . Therefore, SAH presumably induces biphasic disruption of the blood-arterial wall barrier in different ways . In conclusion, the present results revealed that a sudden rise in ICP triggers the disruption of the blood-arterial wall barrier of the basilar artery . Both subarachnoid clot and acute arterial hypertension seem to have a reinforcing role in the development of the barrier disruption . One of the main routes for the protein leakage was shown by electron microscopy to be transendothelial vesicular transport .



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Figure

7 . Electron micrographs of the hasilar arteries from rabbits in which intracranial pressure alone was increased (group 6) . (A) Horseradish peroxidase-reaction products are observed in the abluminal resides (arrowheads) and in the myointimal region . Horseradish peroxidase-reaction products are also seen at the lumtnal front of the smooth muscle cell (arrows) (unstained. group 6a animal, bar = I ton) . (B) Plasmalemmal vesicles of various sizes are stained with HRP-reaction products, but the interendothelial space (u) is only partially stained (arrowheads) (uran)l acetate stained, group 6b animal, bar = 0 .5 µm) . (C) Horseradish peroxidase-reaction products are noticed at the abluminal front of the endothelium (arrows) (uranyl acetate stained, group 6a animal, bar = 0 .5 µm) . (D) In the subendothelial space . HRP-reaction products are seen attached to the endothelium (arrow) (unstained, group 6a animal, bar = 0 .5 µm) . E. endothelium, VL, vascular lumen ; el, elastic lamina; sm. smooth muscle.

The authors thank John Povlishock, Ph .D., for valuable discussion, William Maggio, M .D ., for reviewing the manuscript, Sarah B . Hudson, for technical assistance, and Lucille Staiger, for manuscript preparation .

References I . Asano T, Sano K . Pathogenetic role of no-reflow phenomenon in experimental subarachnoid hemorrhage in dogs . J Neurosurg 1977 ;46 :454-66. 2 . D6czi T, Ambrose J, O'Laoire S . Significance of contrast enhancement in cranial computerized tomography after subarachnoid hemorrhage . J Neurosurg 1984 ;60 :335-42 .

3 . Fox JL, Ko JP . Cerebral vasospasm : a clinical observation . Surg Neurol 1978 ;10 :269-75 . 4 . Graham RC, Karnovsky MJ . The early stage of absorption of injected horseradish peroxidase in the proxymal tubes of mouse kidney . Ultracytochemistry by a new technique . J Histochem Cyrochem 1966 ;14 :291-302 . 5 . Hansson HE, Johansson B, Blomstrand C . Ultrastructural studies on cerebrovascular permeability in acute hypertension . Acta Neuropathol 1975 ;32 :187-98 . 6 . HirataY, MatsukadoY, FukumuraA . Subarachnoid enhancement secondary to subarachnoid hemorrhage with special reference to the clinical significance and pathogenesis . Neurosurgery 1982 ;11 :367-71 . 7 . Hollis TM, Ferrone RA . Effects of shearing stress on aortic histamine synthesis . Exp Mol Pathol 1974 ;20 :1-10 .



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