The Time Course of Intracranial Pathophysiological ...

Journal of Cerebral Blood Flow and Metabolism

10:835--849 © 1990 Raven Press, Ltd., New York

The Time Course of Intracranial Pathophysiological Changes Following Experimental Subarachnoid Haemorrhage in the Rat

A. Jackowski, A. Crockard, *G. Burnstock, R. Ross Russell, and *F. Kristek Cerebral Oedema Research Group, Institute of Neurology, Queen Square, and *Department of Anatomy and Developmental Biology and Centre for Neuroscience, University College London, London, England

Summary: The rat subarachnoid haemorrhage (SAH) model was further studied to establish the precise time course of the globally reduced CBF that follows and to ascertain whether temporally related changes in cerebral perfusion pressure (CPP) and intracranial pressure (lCP) take place. Parallel ultrastructural studies were per­ formed upon cerebral arteries and their adjacent perivas­ cular subarachnoid spaces. SAH was induced by a single intracisternal injection of autologous arterial blood. Serial measurements of regional cortical CBF by hydrogen clearance revealed that experimental SAH resulted in an immediate 50% global reduction in cortical flows that per­ sisted for up to 3 h post SAH. At 24 h, flows were still significantly reduced at 85% of control values (p < 0.05), but by 48 h had regained normal values and were main­ tained up to 5 days post SAH. ICP rose acutely after haemorrhage to nearly 50 mm Hg with C-type pressure waves being present. ICP then fell slowly, only fully re­ turning to control levels at 72 h. Acute hydrocephalus was observed on autopsy examination of SAH animals but not in controls. Reductions in CPP occurred post SAH,but only in the order of 15%, which could not alone account for the fall in CBF that took place. At 48 and, to a lesser extent, 24 h post SAH, myonecrosis confined largely to smooth muscle cells of the immediately subin-

timal media was observed. No significant changes in the intima or perivascular nerve plexus were seen. Within 24 h of haemorrhage, a limited degree of phagocytosis of erythrocytes by pial lining cells took place. However, early on the second day post SAH,a dramatic increase in the numbers of subarachnoid macrophages arose from a transformation of cells of the pia-arachnoid. This period was characterised by intense phagocytic activity, eryth­ rocytes, fibrin, and other debris being largely cleared over the next 24 h. At 5 days post SAH the subarachnoid macrophage population declined,cells losing their mobile active features to assume a more typical pia-arachnoid cell appearance once more. Our studies indicate that this increasingly utilised small animal model of SAH develops global cortical flow changes only acutely, and it is likely that early vasospasm, secondary to released blood prod­ ucts rather than pressure changes per se, is responsible for the initial cerebral ischaemia that develops. Interest­ ingly, both cerebral arterial vasculopathy and perivascu­ lar macrophage phagocytic activity are most marked at -48 h following SAH in the rat, a time at which a phase of delayed cerebral arterial narrowing has previously been documented. Key Words: Cerebral arteries­ Cerebral blood flow-Macrophages-Raised intracranial pressure-Subarachnoid haemorrhage-Vasculopathy.

Subarachnoid haemorrhage (SAH) resulting from the rupture of an intracranial aneurysm in humans

may lead to decreased CBF, raised intracranial pressure (ICP) with hydrocephalus, and the devel­ opment of prolonged cerebral arterial spasm. Con­ siderable evidence points to cerebral ischaemia re­ sulting from reduced cerebral perfusion as the ma­ jor cause of delayed neurological deterioration following SAH (Heilbrun et ai., 1972; Ishii, 1979; Kutsuzawa et ai. , 1968; Voldby et ai., 1985), the peak incidence in humans being around 7-10 days after a bleed (Pickard et ai., 1989). It is likely that the cause of the cerebral ischaemia that occurs post SAH is multifactorial, delayed hypoperfusion being associated not only with angiographically demon-

Received January 29, 1 989; revised April 18, 1 990; accepted April 20, 1 990 . Address correspondence and reprint requests to Dr. A. Jack­ owski at Department of Neurosurgery, The National Hospitals for Nervous Diseases, Queen Square, London WC 1 N 3BO, En­ gland. Parts of this investigation have been presented at the XIV International Symposium on Cerebral Blood Flow and Metabo­ lism, Bologna, Italy, May 1 989. Abbreviations used: CPP, cerebral perfusion pres sure; ICP, intracranial pressure; rCBF, regional CBF; SAH, subarachnoid haemorrhage; SEM, scanning electron microscopy; TEM, trans­ mission electron microscopy.

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A. JACKOWSKI ET AL.

strated luminal narrowing of vessels but also with episodes of hypovolaemia, raised intraventricular pressure, and ventricular dilatation (Ferguson et aI., 1972; Ishii, 1979; Wijdicks et aI., 1985). Increasingly, in place of larger animal models such as the dog, cat, and monkey (Brawley et aI., 1968; Kuwayama et aI., 1972; Petruck et aI., 1972; Umansky et aI. , 1983), the laboratory rat has been employed for experimental studies of SAH (Barry et aI., 1979; Lacy and Earle, 1982; Solomon et aI., 1985; Svendgaard et aI., 1985). Studies in the rat have demonstrated acute reductions in CBF, a bi­ phasic pattern of vasospasm, and changes in cere­ bral metabolic activity and perivascular neurotrans­ mitter content, consequent upon the release of au­ tologous arterial blood into the subarachnoid spaces. Surprisingly little information exists, how­ ever, regarding the subsequent time course of the globally reduced CBF that develops acutely post SAH in the rat. Similarly, few details are known regarding the temporal development of changes in intracranial and cerebral perfusion pressure (CPP) and their interrelationship with reduced CBF fol­ lowing experimental SAH in the rat model. Cerebral arterial narrowing, one factor that has been significantly associated with a reduced CBF, is known also to be related to both the patient's clinical grade and the eventual outcome (Loach and De Azevedo-Filho, 1976; Weir et aI. , 1978). Studies of cerebral vessels obtained at operation and post­ mortem in such patients have revealed the early occurrence in narrowed arterial segments of myo­ necrosis affecting a number of the smooth muscle cells of the media. This is followed in some later cases by a phase of intimal thickening and subinti­ mal proliferation (Crompton, 1964; Conway and McDonald, 1972; Hughes and Schianchi, 1978; Peerless et aI., 1980; Smith et aI. , 1985). Patholog­ ical studies in experimental SAH have taken place mainly in larger animals such as the monkey and dog, where evidence for similar changes arising within the vessel wall has also been described (Ta­ nabe et aI., 1978; Peerless et aI., 1980; Espinosa et aI., 1984). SAH both in humans and from experi­ mental studies has been additionally shown to result in the development post haemorrhage of a dysfunc­ tion of the cerebrovascular sympathetic nerves, with alterations in the levels of their contained neu­ rotransmitters (Lobato et aI., 1980; Edvinsson et aI., 1982; Tsukahara et aI., 1988; Jackowski et aI., 1989a). Whether these latter findings represent the development of actual structural damage to the neu­ ral plexus itself, or merely changes in neurochemi­ cal expression, has not hitherto been established. In humans, the release of blood into the subarachnoid J Cereb Blood Flow Metab, Vol. 10. No. 6, 1990

spaces is commonly associated with reduced CSF resorption and may in some cases lead to the devel­ opment of symptomatic communicating hydroceph­ alus with raised ICP. The mechanism by which re­ leased blood is cleared from the cisternal, perivas­ cular, and other CSF spaces following SAH has been uncertain. In the remainder of the body, mac­ rophages play a major role in the removal of extrav­ asated blood, but in the subarachnoid spaces of nor­ mal animals, cells resembling macrophages have only rarely been described (Wislocki, 1932). How­ ever, following the introduction of bacteria or for­ eign proteins in the CSF spaces, clearance occurs by phagocytosis, initially by leptomeningeal cells and subsequently by cells that resemble activated macrophages (Nelson et aI., 1962; Shabo and Max­ well, 1971). The origin of these subarachnoid mac­ rophages has been the subject of much controversy. Essick (1920) first proposed that such cells arose directly from the pia-arachnoid itself, a suggestion supported by experimental findings in the dog (Mer­ chant and Low, 1979). Others, however, have pre­ sented evidence for a haematogenous origin of lep­ tomeningeal macrophages (Koningsmark and Sid­ man, 1963; Morse and Low, 1972). This study was undertaken first to precisely de­ termine the exact time course of changes in cortical CBF occurring both within minutes and up to sev­ eral days following SAH, together with the correla­ tion of serial measurements of ICP and CPP ob­ tained over the same time period; and second to investigate if changes similar to those previously observed in the cerebral arteries of humans and some larger animals also develop following experi­ mental haemorrhage in a small animal model such as the rat. In addition, it was hoped to more clearly establish the mechanism by which released blood is subsequently cleared from the perivascular and cis­ ternal subarachnoid spaces surrounding the major cerebral arteries. MATERIALS AND METHODS Animal preparations Experiments were peIformed on a total of 174 male Sprague-Dawley rats weighing between 350 and 450 g. During operative procedures, animals were allowed to breathe spontaneously on an anaesthetic mixture consist­ ing of 70% nitrous oxide/30% oxygen, with 1.5% halo­ thane added. Halothane was reduced to 0.75% for 30 min before cisternal injections or measurements of CBF were peIformed. The left femoral artery was cannulated; arte­ rial blood pressure was monitored with a Statham P50 pressure transducer (Statham Instruments, Oxnard, CA, U.S.A.) and was recorded on a Lectromed MX6 chart recorder (Ormed Ltd., Welwyn Garden City, U.K.). Ar­ terial blood samples were obtained anaerobically in mi-

PATHOPHYSIOLOGICAL CHANGES IN EXPERIMENTAL SAH crohaematocrit tubes (0.1 ml),and systemic Paco2, Pao2, and pH were determined using an ABL 30 blood-gas anal­ yser (Radiometer, Copenhagen, Denmark). The haemat­ ocrit was measured. Body temperature was maintained at 37°C by external warming.

Induction of experimental SAH SAH was induced in 66 rats by a posterior craniocer­ vical approach using a modification of a technique de­ scribed previously (Solomon et al., 1985). Briefly, under the operating microscope, the posterior cervical muscu­ lature was separated in the midline, exposing the arch of the atlas, occipital bone, and atlantooccipital membrane. For production of haemorrhage, 0.3 ml of arterial blood was withdrawn by syringe from the femoral arterial line and replaced with an equal volume of normal saline. The freshly withdrawn blood was then injected into the sub­ arachnoid space via a 30-gauge needle passed into the cisterna magna. Injections were given as six equal ali­ quots over a period of 3-4 min. Control groups comprised one group of 54 rats that received an intracisternal injec­ tion of 0.3 ml buffered saline (normal saline with 25 mEq/ L NaHC03 equilibrated at 37°C with 5% CO2/95% air to pH 7.4) and a second sham-operated group of 54 rats that underwent identical operative procedures including cis­ ternal needle puncture as in the other two groups but in which no injections were given.

CBF measurements Regional cortical gray matter CBF (rCBF) was mea­ sured by the hydrogen clearance technique (Auckland et al.,1964; Young,1980) using a chronically implanted plat­ inum wire microelectrode array (Jackowski et al.,1989b). In six animals from each treatment group, three pairs of microelectrodes were inserted into the parietal,occipital, and cerebellar cortical regions of both left and right hemi­ spheres. In each animal the rCBF was determined prein­ jection,after 3,15,30,60,120,and 180 min,and 1-5 days following SAH, saline, or sham operation. The values of rCBF were calculated from the hydrogen clearance curves obtained,using the initial slope method (Oleson et al.,1971; Doyle et al.,1975) between 30 and 120 s. The regional flows derived were then expressed as a percent­ age of each animal's control rCBF value obtained prior to injection on day O.

ICP, CPP, and other measurements ICP was measured via a saline-filled 30-gauge cannula placed into the cisterna magna and connected to a Statham P50 pressure transducer. Data values for ICP, CPP, MABP, haematocrit, and blood gases for the times immediately post SAH up to 3 h post event were acquired in the same animals as were employed also for the serial assessment of rCBF. Studies of these same parameters at the time points 1-5 days post SAH were performed in separate groups of animals, each group consisting of six animals, studied at those specified time points alone.

Electron microscopic studies Arteries were examined from animals that were killed at times 15 min, 1 h, 3 h, and 1,2, 3, 5, and 7 days post SAH. Under inhalational anaesthesia, the heart was ex­ posed and a cannula inserted through the left ventricle into the ascending aorta. After incision of the right atrium, the whole animal was perfused at physiological pressure with 200 ml of fixative composed of 4%

837

paraformaldehyde/0.1% glutaraldehyde in 0.1 M phos­ phate buffer, pH 7.3, followed by 500 ml of the same fixative minus glutaraldehyde. The brains were removed shortly after perfusion-fixation,immersed in the same fix­ ative for 2 h, then placed in 0.1 M phosphate buffer over­ night. Under the operating microscope, the circle of Wil­ lis was dissected from the brain and the proximal segment of the basilar artery and a distal segment of the internal carotid artery were taken and prepared for transmission (TEM) and scanning (SEM) electron microscopy. After washing in 0.1 M phosphate buffer, the specimens were postfixed in 1% osmium tetroxide in 0.1 M phosphate buffer for 30 min and then rinsed in distilled water. Spec­ imens for TEM were next stained in 1% aqueous uranyl acetate for 30 min, dehydrated through graded alcohols into propylene oxide, and flat embedded in araldite. Ul­ trathin sections were cut and mounted on 200-mesh grids to be stained with aqueous uranyl acetate followed by lead citrate and examined with a Philips-300 electron mi­ croscope. Specimens for SEM, after postfixation in os­ mium tetroxide, were further prepared both convention­ ally and following an acid-etch treatment using 8 N HCI (Fujiwara and Uehara, 1984), the latter process allowing an improved visualisation of the perivascular neural plexus and vessel wall three-dimensional structure. Spec­ imens, after dehydration in graded alcohols, were taken through amyl acetate, critical point dried in a Balzers Union CO2 bomb,mounted on SEM stubs,sputter coated with gold using a Polaron E 5000 unit, and viewed with a Hitachi S-530 microscope.

Statistical analysis All values are given as means ± SD unless otherwise stated. Statistical comparison between experimental groups at different time points was performed using a two-tailed t test. p < 0.05 was taken as the level of sig­ nificance.

RESULTS rCBF changes Regional cortical flow changes were analysed separately for parietal, occipital, and cerebellar ar­ eas. The alterations in flow observed, however, were essentially global, affecting all cortical areas to the same degree (Fig. 1). The regional flow data from each animal were therefore combined to give a mean cortical flow value that was used for further comparisons and in the statistical analysis between treatment groups (Table 1). Following experimental SAH there was an immediate and marked reduction in flow, the overall CBF falling at 15 min to 50 ± 5% of preinjection values and remaining greatly re­ duced at 57 ± 11% by 3 h post haemorrhage. At 24 h, the mean cortical flow was only just reduced as compared with control groups at 85 ± 15% of initial values. Prehaemorrhage values were regained on the second day and remained normal for up to 5 days subsequently. The mean cortical flow change in animals receiving cisternal saline injection was similar to that obtained in sham-operated animals. Neither underwent rapid or marked changes in

J Cereb Blood Flow Metab, Vol. 10, No. 6, 1990

838

A. JACKOWSKI ET AL.

120

SHAM

110 100 90 80 70 130

SALINE

120 110 LL

� 100 "2

90

C

0 u

80

'0 C

70

Qj

130

CI) u

0...

FIG. 1. Percentage changes in regional CBF

(rCBF) following subarachnoid haemorrhage (SAH), saline injection, or sham operation. Values are means ± SO (e, parietal cortex; 0, occipital cortex; A, cerebellar cortex). The x-axis is the log scale of time in hours. SAH

120 110 100 90 80 70 60 50 40 C

3

15

30

2 34 5

60 1 20 1 80

Minutes

Days

TABLE 1. CBF as a percentage of control flow following cisternal injection of 0.3 ml blood or buffered saline or

sham operation Time

Sham ( 1 )

Saline (2)

Control 3 min 15 min 30 min 60 min 1 20 min 1 80 min I day 2 days 3 days 4 days 5 days

1 00 1 05 ± 8 1 03 ± 8 93 ± 7 89 ± 8 87 ± 1 0 84 ± 1 1 102 ± 8 1 04 ± 9 1 04 ± 7 1 02 ± 7 1 04 ± 8

1 00 101 ± 100 ± 95 ± 87 ± 80 ± 79 ± 1 04 ± 111 ± 1 06 ± 1 06 ± 1 12 ±

Values are means p < 0 . 00 1 . b P < 0.01 . p < 0.05.

±

SD, n

=

SAH (3)

p ( 1 -3)

p (2-3)

NS NS NS NS NS NS NS NS NS NS NS

1 00 58 ± 5 50 ± 5 51 ± 6 55 ± 8 55 ± 8 57 ± 1 1 85 ± 1 5 98 ± 1 0 1 08 ± 9 1 08 ± 6 1 07 ± 6

b

b

NS NS NS NS

NS NS NS NS

4-6 (pooled regional flow percentages). SAH, subarachnoid haemorrhage.

a

c

J

15 10 9 6 7 11 9 3 7 9 19

p ( 1 -2)

Cereb Blood Flow Metab, Vol. 10, No. 6, 1990

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PATHOPHYSIOLOGICAL CHANGES IN EXPERIMENTAL SAH

flow; in both groups after 30 min of the acute study, there was a gradual reduction in the mean cortical flow that reached 80-85% of prehaemorrhage val­ ues at 3 h. In both groups of control animals, the mean cortical flow had fully recovered to prehaem­ orrhage values by 24 h.

arachnoid space of 0.3 ml of blood as six aliquots was accompanied by a step-like rise in ICP that reached a maximum value of �50 mm Hg and only slowly declined upon completion of the injection (Fig. 2). During the period of acutely raised ICP, pressure waves were commonly observed; these occurred at a frequency of �8/min and were usually closely related to similar variations in the arterial pressure. Also during this period, an increased CSF pulse pressure was often evident. In those animals in which 0.3 ml of buffered saline was administered in place of blood, the ICP attained roughly the same immediate peak values, but fell rapidly toward control values both between individual aliquots and upon final completion of injection. The subsequent

ICP changes The changes in ICP differed markedly in animals that underwent experimental SAH from those in an­ imals that received cisternal saline injection or un­ derwent sham procedures. Differences in response occurred in the periods both during and acutely fol­ lowing injection as well as in the time course of subsequent recovery. Administration into the sub-

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-250 FIG. 2. Representative arterial (upper trace) and intracranial pressure (lower trace) re­ cordings from experimental animals: during and immediately following 0.3 ml (6 x 0.05 ml aliquots) cisternal saline injection (A)j during and immediately following a 0.3-ml subarachnoid haemorrhage (SAH) (B)j dur­ ing a 0.3-ml SAH in an animal that demon­ strates well the pressure wave phenomena frequently observed (C).

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Cereb Blood Flow Metab, Vol. 10, No. 6, 1990

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A. JACKOWSKI ET AL.

time course of ICP recovery differed also between the groups (Fig. 3). Following experimental SAH the ICP remained elevated for up to 3 h at a pres­ sure of 16 mm Hg compared with a normal value of only 4 mm Hg in the rat. At 24 h the ICP was still mildly elevated at 8 mm Hg, and it only fully re­ turned to control levels after 48 h. In the saline­ injected animals the ICP normalised far more rap­ idly, falling to 8 mm Hg by 15 min and returning fully to control values by 2 h. There were no signif­ icant changes in the ICP of sham-operated animals. No formal analysis of ventricular volumes was per­ formed in this study; however, on sectioning of the brains of animals used in delayed ICP studies, it was observed that hydrocephalus occurred in ani­ mals 24 h post SAH, but not following control pro­ cedures.

MABP and CPP changes (Fig. 3) In both the SAH and the saline-injected animals, there was a rapid rise in MABP following the injec­ tion of a 0.3-ml volume load into the subarachnoid space via the cisterna magna. The pressure reached a mean peak value of -125 mm Hg, then fell slowly after 3 min, recovering to normal values by 3 h in both groups of animals. CPP fell immediately by -20% following either SAH or saline injection as the rise in MABP pro­ duced failed to match the peak ICP that resulted. In the SAH animals, CPP then recovered to normal within 3 min only to fall again by 15% at 1-3 h post SAH as ICP remained elevated in the presence of a now normal MABP. In the saline-injected animals, as the ICP returned rapidly to normal values, the CPP after its initial fall became elevated by 20%,

MABP

140 130 120 110 100 90 130 120 110 Q")

I E E

FIG. 3. Sequential changes in MABP, cerebral perfusion pressure (CPP), and intracranial pressure (ICP) in the three experimental groups of animals. Values are means ± SO (e, subarachnoid haemorrhage animals; 0, saline-injected animals; A, sham-operated animals). The xaxis is the log scale of time in hours. C, preinjection values; P, values at time of peak ICP.

100 90 80 70 60

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15 30 60 120 1 80 Minutes

Cereb Blood Flow Metab, Vol. 10, No. 6, 1990

,

'

"

2 3 45 Days

841

PATHOPHYSIOLOGICAL CHANGES IN EXPERIMENTAL SAH

only to then fall in parallel with the recovery in MABP. In both SAH and saline-injected animals, the MABP and CPP were maintained subsequently at normal levels from 24 h onward. There were no significant changes in the MABP or CPP of sham­ operated animals.

always present on the luminal surface, with the long axis of the cells being oriented in the direction of blood flow. Acutely (up to 3 h) following SAH, the endothelial cells tended to be more rounded and to possess an increased number of bleb-like and mi­ crovillus surface projections compared with those from control animals, although no other evidence of epithelial cell loss or damage was apparent at any time. By 1 day following experimental SAH, the endothelial cell appearance was once again identical to controls.

Other physiological parameters The blood gases and haematocrit for the experi­ mental groups at different times are given in Table 2. These parameters remained largely constant; ap­ proximate mean values were as follows: haemat­ ocrit 45%, Paco2 37 mm Hg, Pao2 139 mm Hg, and pH 7.4. There were no significant differences in these values between the experimental groups.

Media A striking feature was the development 1 day post SAH of a small number of smooth muscle cells that displayed an overall increased electron density, contained dense bodies and degenerate mitochon­ dria, and showed loss of their normal internal struc­ ture (Fig. 4A). By 2 days the sarcolemma of such necrotic cells had broken down, with the appear­ ance of aggregated myofilament complexes, free ri­ bosomes and membrane debris lying freely in the extracellular space between smooth muscle cells

Pathological alterations Endothelium SEM revealed the intimal endothelium of arteries taken from animals following experimental SAH as being largely similar in appearance to the endothe­ lium taken from control animals. A well-defined po­ lygonal pavement-like layer of endothelial cells was

TABLE 2. Physiological measurements in experimental groups (mean ± SD)

Arterial blood gases Haematocrit

(%)

Sham operated Control 3 min 1 5 min 30 min 60 min 1 20 min 1 80 min I day 3 days 5 days Saline injected Control 3 min 1 5 min 30 min 60 min 1 20 min 1 80 min I day 3 days 5 days SAH animals Control 3 min 1 5 min 30 min 60 min 1 20 min 1 80 min I day 3 days 5 days

45

43 45 44 45 46

±

1

±

I 2 2 3

±

0.5

± ± ±

44 45 45 46

±

1 2 3 2

46

±

I

44 46 45 46

± ± ±

± ± ± ±

I 2 2 2

Pco2 (mm Hg)

38.4 37.5 36.6 36. 1 38.4 36.3 35.9 37.6 37. 1 38.4 38.9 37.4 37.8 37.6 40.9 35.6 36.2 35.9 40 .7 37.8 37.0 35.7 36.8 38. 1 34.7 36.5 36.6 37.3 38.8 35.5

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

P02 (mm Hg)

3.6 4.0 4.9 3.5 2.4 1 .8 4.8 3.8 4.9 4.3

136 1 39 141 1 39 1 39 1 40 141 138 1 39 143

2.4 3.2 2.0 4.5 6.4 3.8 3.5 4.2 5.7 2.4

141 1 32 1 40 138 141 1 36 1 39 143 1 32 138

2.7 4.0 2.7 5.1 3.1 2.5 3.5 4.5 3.8 3.8

1 36 1 37 1 40 131 1 39 1 40 138 1 42 1 35 1 43

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

n

pH

3.5 9.3 9.8 12.5 7.0 5.9 1 5 .4 8.5 7.6 1 1 .5

7 . 38 7 .43 7 . 42 7 . 44 7 .43 7.41 7 . 39 7 . 39 7 . 37 7 . 42

6.7 7.2 3.7 9.9 1 1 .6 6. 1 5.8 10.4 7.8 7.3

7 . 43 7 . 45 7 . 40 7 . 40 7.38 7 . 43 7 . 39 7 . 44 7 . 43 7 . 40

9 10 11 II 7 II 15 9 9 10

7 . 36 7.38 7.41 7 . 42 7.41 7 . 40 7 . 44 7.41 7 . 39 7 . 40

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0 . 05 0 . 04 0 . 07 0 . 04 0 . 03 0 . 05 0 . 05 0 . 09 0 . 05 0 . 04

5 5 5 5 5 5 5 6 6 6

0 . 05 0 . 05 0 . 04 0 . 07 0 . 09 0 . 04 0 . 07 0 . 05 0.04 0 . 08

6 5 5 5 5 5 5 5 5 5

0 . 06 0 . 05 0 . 07 0.04 0 . 03 0 . 04 0.03 0 . 04 0.05 0.03

6 6 6 6 5 5 5 6 6 6

SAH, subarachnoid haemorrhage.

J

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FIG. 4. Transmission electron micrographs of the rat basilar artery demonstrating myo­ necrotic changes in the media following ex­ perimental subarachnoid haemorrhage (SAH). A: Artery from an animal killed 24 h post SAH. sm, normal smooth muscle cell; dm, degenerating smooth muscle cell dis­ playing increased electron density, dense bodies, and mitochondrial necrosis. Bar, 3 fLm. B: Artery from an animal killed 48 h post SAH. el, elastic lamina; Nm, remains of a ne­ crosed smooth muscle cell in which the sar­ colemma has largely disappeared. Note the myofilament complexes (cpx) and other cel­ lular debris lying freely in the intercellular space. Bar, 1.5 fLm.

(Fig. 4B). These changes were most commonly ob­ served in, although not wholly confined to, the me­ dial layers closest to the elastic lamina. Perivascular nerves a nd adventitia The Schwann cell/neural plexus was most readily visualised in the acid-etched SEM preparations. No differences were apparent between experimental animals and controls, either in the structure of the plexus itself or in the extent of its distribution within the adventitial layer, at any of the times stud­ ied. J

Cereb Blood Flow Metab, Vol. 10, No. 6, 1990

Subarachnoid spaces

Immediately following SAH, the perivascular and cisternal subarachnoid spaces adjacent to the basi­ lar and internal carotid arteries were occupied by a dense blood clot that consisted largely of erythro­ cytes trapped within a fibrin mesh (Fig. SA). Little change was detected on the first day, although oc­ casionally erythrocytes were seen that appeared to have been directly phagocytosed by cells of the pial membrane. Early on the second day post SAH, large numbers of rounded cells became evident.

PATHOPHYSIOLOGICAL CHANGES IN EXPERIMENTAL SAH

843

FIG. 5. Scanning electron micrographs of perivascular subarachnoid spaces adjacent to the major cerebral arteries. A: At 60 min post subarachnoid haemorrhage (SAH), the subarachnoid space is entirely filled with blood clot (Bc). Pm, pia mater; Tr, arachnoid trabeculae; Ar, arachnoid mater. Bar, 35 f.Lm. B: At 48 h post SAH, large numbers of rounded cells (arrows), some possessing mi­ croappendages, are now present within the blood clot. Zones (Tz) within the pia have ap­ peared, where defects in the membrane are occupied by cells of intermediate forms of these rounded cells. Bar, 40 f.Lm.

Fully matured forms displayed a variety of ruffled membranes and microappendages and possessed the characteristics of subarachnoid macrophages. This emergent cell population appeared to originate principally from the pial and, to a lesser extent, the arachnoid membranes. Within these structures, transition zones appeared (Fig. 5B), from which in­ termediate cell types could be seen in various stages of transformation, initially rounding up, then gain­ ing microappendages upon their cell surface. De­ fects in the normally continuous sheet of overlap­ ping pial cells became evident, as maturing cells

migrated out into the surrounding subarachnoid blood clot. These cells behaved subsequently in a manner characteristic of activated macrophages and could be seen avidly phagocytosing erythro­ cytes and other debris from the subarachnoid spaces (Fig. 6). Already by 3 days post SAH, the perivascular CSF spaces were largely cleared of blood cells and fibrin. At 5 days post SAH the mac­ rophage population declined and sites were increas­ ingly observed upon the pia-arachnoid membranes and arachnoid trabeculations, where subarachnoid macrophages appeared to be losing their mobile acJ

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FIG. 6. Scanning electron micrograph of the perivascular subarachnoid space 3 days post subarachnoid haemorrhage. At higher mag­ nification, the rounded cells seen previously display the ruffled membranes and microap­ pendages characteristic of subarachnoid macrophages (Ma) and are seen actively en­ gaged in erythrophagocytosis. Er, erythro­ cyte. Bar, 10 IJ-m. Inset: Transmission elec­ tron micrograph of one such cell. Internally. these transformed pial cells d isplay in­ creased perinuclear cytoplasm and clear vacuoles. A pseudopod process is in close contact with an erythrocyte. Bar, 5 IJ-m.

tive characteristics, flattening out to resemble typ­ ical pia-arachnoid cells once more (Fig. 7). DISCUSSION

After SAH �30% of patients will develop delayed cerebral ischaemia, the peak incidence being at �7-10 days post bleed (Pickard et aI., 1989). In hu­ mans CBF tends to decline in the first week after SAH and to remain low over the next 2 weeks. The reduction in flow is usually most marked in older patients and in those that are drowsy or in poor clinical grades (Meyer et aI., 1982, 1983; Mickey et aI., 1984). A significant global reduction in CBF during the period acutely following experimental SAH has been demonstrated in animal studies per­ formed in the monkey, dog, cat (Brawley et aI., 1968; Petruck et aI., 1972; Umansky et aI., 1983), and more recently in the rat (Solomon et aI., 1985). J

Cereb Blood Flow Metab. Vol. 10. No. 6. 1990

However, details of the subsequent time course to recovery of such alterations in CBF and of any sub­ sequent development of delayed ischaemic changes have been described far less commonly. In the mon­ key, persistent reductions in CBF have been dem­ onstrated up to 3 days post SAH (Jakubowski et aI., 1982; Sahlin et aI., 1987), while in the rat a uniform 20% reduction in CBF has been reported to occur at 2 days post haemorrhage (Delgado et aI., 1986). The development of a delayed narrowing of major cerebral arteries following SAH due to aneurysmal rupture has been known since the original angio­ graphic description by Ecker and Riemenschneider (1951). The time course of this constriction in hu­ mans has been detailed (Kwak et aI., 1979); its on­ set is usually delayed until the fourth or fifth day post bleed and has a maximum incidence between 10 and 14 days. Experimental studies in the dog (Brawley et aI., 1968; Kuwayama et aI., 1972; Nagai

PATHOPHYSIOLOGICAL CHANGES IN EXPERIMENTAL SAH

845

FIG. 7. Scanning electron micrograph of the pia mater 6 days post subarachnoid haemorrhage. Cells possessing macrophage features are still present (Ma). However, new zones of transformation (Tz) have appeared, where intermediate cell forms are seen in various stages of flattening out and assuming more typical pial cell morphology. Bar, 25 f.l.m.

et aI., 1974) revealed that cerebral arteries can re­ spond to SAH in a biphasic manner, with an acute reversible phase of narrowing immediately post bleed, followed in some instances by a more persis­ tent phase that develops after a variable delay. Fol­ lowing experimental SAH in the rat, Delgado and co-workers (1985) also described a biphasic pattern of vasospasm with a maximum acute spasm at 10 min and a maximum late spasm at 48 h. In contrast, an earlier study (Barry et aI., 1979) documented only an immediate onset of spasm, which steadily recovered to normal by day 3 and was followed by vasodilatation from day 5 onward. Study of angio­ graphically observed narrowed segments of cere­ bral arteries, taken either at operation or at autopsy from patients dying following SAH, have demon­ strated the occurrence of varying degrees of medial necrosis, subintimal proliferation, and other patho­ logical changes (Crompton, 1964; Conway and Mc­ Donald, 1972; Hughes and Schianchi, 1978; Peer­ less et aI., 1980; Smith et aI., 1985). While most investigators of experimental SAH in the monkey have documented similar changes (Peerless et aI., 1980; Espinosa et aI., 1984), studies in dogs have been more variable. Some authors observed patho­ logical changes in the walls of cerebral vessels (Ta­ nabe et aI., 1978; Tani et aI., 1978), whilst others found few or none at all (Pickard et aI., 1985). The measurements of CBF obtained in our study

clearly confirm the occurrence of an acute decrease in mean cortical flow of �50% for up to 3 h follow­ ing haemorrhage. A smaller but statistically signif­ icant reduction in CBF persisted up to 24 h post SAH. However, at 48 h, during the period when a second lesser phase of vasospasm has been de­ scribed, the mean cortical CBF was fully recovered to normal values. Such a lack of absolute correla­ tion between the time course of a reduced CBF and previously described angiographic changes in the rat is not altogether surprising. Studies of SAH in humans have shown the precise relationship of va­ sospasm to reductions in CBF to be uncertain. Many investigators have found the presence of ce­ rebral vasospasm in patients to be associated with a reduced CBF as both a global and a focal phenom­ enon (Heilbrun et aI., 1972; Ishii, 1979; Voldby et aI., 1985). Other authors, however, have shown a less-well-defined relationship (Bergvall et aI., 1973; Grubb et aI. , 1977; Mickey et aI., 1984). Several patient studies have shown a relationship post SAH between hydrocephalus, raised ICP, and reduced rCBF (Ferguson et aI., 1972; Hartmann et aI. , 1977; Ishii, 1979). Similarly, hydrocephalus and vasospasm following SAH have been found to be significantly associated (Black, 1986), and the ther­ apeutic benefits from ventricular drainage in cases of raised ICP upon CBF and clinical grade have been well described (Kusske et aI., 1973; Suzuki et J

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A. JACKOWSKI ET AL.

al., 1974; Hartmann, 1980). One finding from our experimental work has been an emphasis on the difference in ICP response following the adminis­ tration of a blood or saline volume load into the rat cisterna magna. Injection of 0. 3 ml saline caused only a short-lived elevation of ICP to a peak of �50 mm Hg. An equal volume of blood produced the same peak ICP, but the pressure subsequently fell considerably more slowly. Concomitant with the rise in ICP, pressure wave phenomena were ob­ served following SAH, but not after control proce­ dures. These waves, most closely resembling Traube-Hering-Mayer-related "C" waves, were seen only when the pulse pressure amplitude was increased in the presence of an elevated ICP and are almost certainly evidence of a greatly reduced in­ tracranial compliance post SAH. The changes in CBF observed following experi­ mental SAH in the rat cannot be explained simply on the basis of concurrent changes in ICP and CPP alone. In the saline-injected control animals, the CBF remained relatively constant, despite 20% fluctuations in CPP, while in the SAH animals, the maximum sustained reduction in CPP was 15%, which, even in the presence of impaired autoregu­ lation, would not alone account for the 50% decline in CBF that took place. The time course of reduced CBF in the rat does, however, correlate well with previous observations made by ourselves and oth­ ers (Barry et aI., 1979; Jackowski et aI., 1989a) that subarachnoid-released blood diminishes and is largely resorbed within 48 h in the rat. This suggests that the reduction in cerebral flow that occurs is closely related to the actual presence of blood and blood breakdown products within the perivascular CSF spaces themselves, acting either directly upon the cerebral vessel wall or perhaps more indirectly via perivascular nerves and central brainstem affer­ ent connections (Solomon et aI., 1987; Svendgaard et aI., 1985; Jackowski et aI., 1989a) to produce an early phase of vasospasm. The development of pathological changes affect­ ing the cerebral arteries of the rat after SAH has not been previously described. In the present study, however, we found evidence of changes similar to those described previously in larger animal models. Myonecrosis of limited numbers of smooth muscle cells of the media of cerebral arteries was observed, commencing the day following haemorrhage. A number of studies, both in humans and upon a va­ riety of animals including the rat, have now clearly demonstrated that a dysfunction in both endothelial cell- and perivascular nerve-related cerebrovascu­ lar reactivity commonly develops post SAH (Pick­ ard and Perry, 1983; Sahlin et aI., 1985; Nakagomi J

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et aI., 1987; Kim et aI., 1988). In our study, the ultrastructural morphology of both the endothelium and the neural plexus appeared to be largely unaf­ fected following SAH. This suggests that such al­ terations reflect either an impairment of smooth muscle responsiveness or some qualitative change in endothelial cell and nerve function rather than simple physical damage to the latter structures alone. Clearance of blood from the subarachnoid spaces in humans can take from 6 to 30 days (Tourtellotte et aI., 1964), the continued presence of blood clot within these spaces predisposing to impaired CSF resorption, hydrocephalus, raised ICP, and delayed cerebral ischaemia (Ellington and Margolis, 1969; Vassilouthis and Richardson, 1979; Butler et aI., 1980; Mohsen et aI., 1984). In humans most eryth­ rocytes rapidly become enmeshed within the arach­ noid trabeculae of the subarachnoid spaces, and it was naturally hypothesised that they were then re­ moved by lysis and phagocytosis (Sprang, 1934; Hammes, 1944). Early studies of erythrocyte clear­ ance in animals, however, seemed initially to sug­ gest that cells might instead be removed largely in­ tact via perineural lymphatic-type channels (Ken­ nady, 1967; McQueen et aI., 1974). Separate studies had suggested a system of tubular spaces and pores across the arachnoid villi (Welch and Friedman, 1960), and it was proposed that whole erythrocytes might pass directly into the bloodstream via this route (Simmond, 1953). Ultrastructural studies have since shown the endothelium of the arachnoid villi to be continuous, thereby preventing such a mode of clearance (Alksne and White, 1965). Stud­ ies in the dog (Alksne and Lovings, 1972) then sug­ gested that removal of erythrocytes from the sub­ arachnoid spaces occurred mainly by lysis and pos­ sibly by phagocytosis of resultant debris by cells of the arachnoid villi. An increase also in the numbers of free subarachnoid macrophages was shown to occur after SAH by Julow et al. (1979), who ob­ served limited erythrophagocytosis to take place. In our studies upon the mode of clearance of blood from the perivascular subarachnoid spaces in the rat, erythrocytes immediately following haem­ orrhage occasionally underwent phagocytosis by cells of the pial membrane. With increased survival time, there followed a period characterised by a burst of intense phagocytic activity by mobile cells possessing the typical features of activated mac­ rophages. This greatly increased population of sub­ arachnoid macrophages appeared to arise largely by the transformation of cells of the pial membrane. Upon clearance of blood clot from the perivascular subarachnoid space, these activated macrophages

PATHOPHYSIOLOGICAL CHANGES IN EXPERIMENTAL SAH

flattened out, assuming the characteristics of typi­ cal pia-arachnoid cells once more. The development of pathological changes affect­ ing the media of rat cerebral arteries and a period of intense macrophage erythrophagocytic activity within perivascular subarachnoid blood clot were both maximal at �48 h post haemorrhage. It is of considerable interest that this period coincides also with the time at which delayed basilar arterial lumi­ nal narrowing has previously been described (Del­ gado et aI., 1985). The finding of such an association may be of importance inasmuch as macrophage ac­ tivity and oxyhaemoglobin autooxidation are both well known to be associated with the generation and release of free radicals (Sutton et aI., 1976; Babior, 1978). Work by a number of authors has suggested that an excess of free radicals may be involved in the genesis of arterial wall damage and the angio­ graphically visible delayed cerebral arterial narrow­ ing that may follow SAH (Sakaki et aI., 1988; Steinke et aI., 1989; Zuccarello et aI., 1989). Although from our study it now appears that sig­ nificant delayed cerebral ischaemia fails to develop in the rat after haemorrhage, the simple single­ haemorrhage rat model remains a cheap and readily reproducible one that demonstrates many of the pathophysiological changes that occur following SAH in man. In particular, the occurrence of a glob­ al reduction in CBP, the development of an ele­ vated ICP with reduced intracranial compliance, and evidence of delayed cerebral arterial vasculop­ athy are found. Use of the rat for further investiga­ tive studies upon the mechanisms arising out of and treatment of SAH would appear to be relevant, pro­ vided the observed difference in the rat CBP re­ sponse compared with that resulting in humans re­ mains recognised. Acknowledgment: Andre Jackowski was supported by a Medical Research Council Training Fellowship Award. This work was supported in part by The Welton Founda­ tion.

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