Journal of Cerebral Blood Flow and Metabolism
10:914-922 © 1990 Raven Press, Ltd., New York
Microvascular Changes During the Early Phase of Experimental Bacterial Meningitis H.-W. Pfister, U. Koedel, R. L. Haberl, U. Dirnagl, *W. Feiden, tG. Ruckdeschel, and K. M. Einhaupl Department of Neurology, *Institute of Neuropathology, and tMax von Pettenkofer Institute of Hygiene and Microbiology, University of Munich, Munich, F.R.C.
Summary: We investigated the temporal profile of the changes in regional CBF (rCBF) and intracranial pressure (ICP) during the early phase of pneumococcal meningitis in the rat. rCBF, as measured by laser-Doppler flowmet ry, and ICP were continuously monitored during 6 h post infection (pj.). Brain edema formation was assessed by brain water content determinations. Meningitis was in duced by intracisternal injection of 75 IJ-l of 107 colony forming units/ml pneumococci (n 7). In control animals (n 6), saline was injected. There was no change in the rCBF or ICP of controls throughout the experiment. However, there was a dramatic increase in rCBF and ICP associated with brain edema formation in untreated men ingitis animals. rCBF increased to 135.3 ± 33.8% (mean ± SD) in the untreated animals at I h pj. and reached 2 11.1 ± 40.5% at 6 h pj. (p < 0.05 compared with con trols). ICP increased from 2.9 ± 1.4 to 10.4 ± 4.7 mm Hg at 6 h pj. (p < 0.05 compared with controls). Brain water content was significantly elevated (79.69 ± 0.24 com pared with 78.94 ± 0. 16% in the control group, p < 0.05).
We investigated the effect of dexamethasone (3 mg/kg i.p.), which was given prior to the induction of meningitis (n 3) or at 2 h after pneumococcal injection (n 5), indomethacin ( 10 mg/kg i.v., n 5), and superoxide dis mutase (SOD; 132,000 U/kg i.v. per 6 h, n 6). The increases in rCBF and ICP were prevented by the pre treatment with dexamethasone and the administration of SOD, delayed and attenuated by pretreatment with in domethacin, and reversed by administration of dexameth asone 2 h pj. These findings suggest that oxygen-derived free radicals are involved as mediators in the increases of rCBF and rcp and brain edema formation during the early phase of experimental bacterial meningitis. Arachi donic acid metabolites of the cyc100xygenase pathway are partially involved in the observed changes and are one possible source for the generation of oxygen-derived free radicals in bacterial meningitis. Key Words: Bacterial meningitis-Brain edema-Cerebral blood flow Intracranial pressure-Laser-Doppler flowmetry Oxygen-derived free radicals-Superoxide dismutase.
Despite great advances in antimicrobial therapy, mortality due to bacterial meningitis remains high (Swartz, 1984). Pneumococcus-induced meningitis, the most frequent etiologic agent in bacterial men ingitis of the adult, is fatal in 25-30%. Clinical ob servations suggest that increased intracranial pres sure (ICP), brain edema, and cerebral vasculitis are associated with unfavorable clinical courses (Paul son et aI., 1974; Nugent et aI., 1979; Horwitz et aI.,
1980; Igarashi et aI., 1984; McMenamin and Volpe, 1984; Pfister, 1989). Although several investigators have tried to identify the molecular basis of the in flammatory changes in bacterial meningitis using animal models (Tauber et aI., 1985; Quagliarello et aI., 1986; Tuomanen et aI., 1987, 1989; Mustafa et aI., 1989a), the precise pathophysiological mecha nisms of the major intracranial complications are not completely understood. Multiple factors includ ing the complement alternative pathway, cytokines, prostaglandins, and platelet-activating factor have been suggested to be involved in the complex na ture of the pathophysiological alterations during bacterial meningitis (Tuomanen, 1987). The present study was designed to assess the temporal course of changes in regional CBF (rCBF), rcp, and brain edema formation during the
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Received January 4, 1990; revised March 29, 1990; accepted March 30, 199 0. Address correspondence and reprint requests to Dr . H.-W. P fister at Department of Neurology, Ludwig Maximilians Uni versity of Munich, Klinikum GroBhadem, Marchioninistr . 15, D-8000 Miinchen 70, F.R . G . Abbreviations used: I CP, intracranial pressure; LDF, laser Doppler flowmetry; p.i., post infection; r CBF, regional CBF; SOD, superoxide dismutase.
914
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CBF IN BACTERIAL MENINGITIS
early phase of pneumococcal meningitis of the rat. As known from other pathophysiological experi mental models, such as acute severe arterial hyper tension (Kontos et aI., 1981) and fluid percussion brain injury (Wei et aI., 1981), arachidonic acid me tabolites and oxygen-derived free radicals are me diators of vascular damage and brain edema. In ad dition, because of their high reactivity and capacity to produce cellular damage, the oxygen-derived free radicals have also been implicated as mediators in inflammation (McCord, 1974). Therefore, we in vestigated the effect of the blocking substances dexamethasone, the cyclooxygenase inhibitor in domethacin, and the free radical scavenger super oxide dismutase (SOD). MATERIAL AND METHODS
LDF-PROBE SUPERFUSION INLET
FIG. 1. Diagra m of the experimental setup. The laser-Doppler probe was fixed by a steel cap over the right parietal cortex. The dura was not re moved. The regional CBF measured by laser-Doppler flow metry (LDF) and the intracranial pressure were continuously recorded for 6 h after intracisternal (IC) injection.
Animal preparation
A total of 32 animals were used in this study. Adult male Wistar rats weighing 250-350 g were anesthetized with 100 mg/kg i.p. thiobutabarbiturate (lnactin). To maintain anesthesia, supplemental doses of thiobutabar biturate were given through a peritoneal catheter. An en dotracheal tube was inserted through a tracheostomy. The rats were ventilated mechanically (model 683 small animal ventilator; Harvard, South Natick, MA, U.S.A.) with room air and supplemental oxygen. The end expiratory CO2 was continuously monitored with an in frared CO2 analyzer (model 2200; Heyer, Bad Ems, F.R.G.) and maintained at a level of 35 mm Hg through out the experiment by adjusting the respiratory rate and volume. Polyethylene tubings were inserted into the fem oral artery and vein. Arterial blood pressure was mea sured with a Statham P23 pressure transducer connected to the cannula in the femoral artery. Arterial blood sam ples were collected periodically from the arterial line and analyzed for Po2, Pco2, and pH (AVL gas check, model 940). Saline was infused in the femoral vein during the experiment ( 10 m1l6 h) by an infusion pump (Precidor, type 5003; Infors HT, Bottmingen, Switzerland). Hemat ocrit investigations revealed no change during the exper iment. Body temperature was maintained at 38°C by a rectal thermometer-controlled heating pad. The animals were placed in a stereotaxic frame. A burr hole was made for the cisterna magna cannula at the angle where the sagittal and lambda sutures meet. ICP was recorded with a Statham P23 pressure transducer via a polyethylene tube. CBF was continuously measured by laser-Doppler flowmetry (LDF; model BPM 403; TSI Inc., St. Paul, MN, U.S.A.). For placement of the LD probe, a craniot omy (diameter 5 mm) was made in the mediocaudal por tion of the right parietal bone. The dura remained intact. A stainless-steel cap was implanted above the craniotomy site. This cap held the LD probe and was equipped with two openings for flushing the area under the cap (Fig. 1). The probe was placed over an area free of large pial ves sels. Saline was carefully flushed through the cap to re move stains of blood and air bubbles within the system. MABP, ICP, end-expiratory CO2, and rCBF, as mea sured by LDF (CBFLDP)' were continuously recorded on a multichannel paper strip recorder (six-channel recorder, �
model BD 10 1; Kipp & Zonen, Delft, Holland) for 6 h after induction of meningitis. Additionally, all parameters were transferred to a PC system and AD converted for digital signal processing. Because of the fluctuation of the LDF signal, the LDF data were averaged every 30 s dur ing the experiment. Bacterial inoculum The strain Streptococcus pneumoniae type 6b (no.
643), an isolate from the blood of a patient with septic infection, was kept at - 20°C in trypticase soy broth (Ox oid, Wesel, F.R.G.), supplemented with 10% glycerol and 1% IsoVitale (BBL, Heidelberg, F.R.G.). Before use, the bacteria were subcultured on blood-agar plates, checked for purity, inoculated into brain-heart infusion broth (Ox oid), supplemented with 3% horse serum and 1% bovine albumin (Serva, Heidelberg, F .R.G .), and incubated overnight at 35°C. The broth was centrifuged for 20 min at 2,500 g; the sediment was washed once with 0.85% saline and then resuspended in saline. The final suspension was turbidometrically adjusted to a density of 0.5 (photome ter, 13-mm filter, 546 nm; Eppendorf, Hamburg, F.R.G.), thus achieving a concentration of 107 colony-forming units (cfu)/ml. �
Induction of meningitis
After recording a stable baseline of CBFLDP and ICP for 30 min, 75 ILl CSF was removed through the cisternal catheter. Meningitis was induced by intracisternal injec tion of 75 ILl of 107 cfu pneumococci (seven animals). In control animals (six animals), 75 ILl saline was injected into the cisterna magna. Administration of dexamethasone, indomethacin, and SOD
Dexamethasone (3 mg/kg i.p.; Merck Sharp & Dohme, Miinchen, F.R.G.) was given 1 h prior to the injection of pneumococci (three animals) or at 2 h post pneumococcal injection (five animals). Five animals were pretreated with indomethacin ( 10 mg/kg i.v.; Sigma Chemie, Deisen hofen, F.R.G.). Intravenous infusion of the free radical scavenger SOD (from bovine erythrocytes; Sigma Chemie) was started just before the pneumococ-
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H.-W. PFISTER ET AL.
cal injection was given continuously during the experi ment (22,000 U/kg-h) (six animals).
some selected cases, Giemsa, Gram, and (naphthol AS-D) chioroacetate esterase.
LDF
Statistical comparisons
LDF has been described in detail elsewhere (Stern, 1975; Bonner et aI., 1981; Haberl et aI., 1989a,b). The LD technique, used to assess microcirculatory blood flow in small tissue samples, has the advantage of continuous CBF measurement without disruption of brain tissue (un like H2 clearance) and without multiple infusions of tracer (unlike the umbelliferone clearance method). The method has been validated for the cerebral (Eyre et aI., 1988; Skarphedinsson et aI., 1988; Arbit et al. 1989; Dirnagl et aI., 1989; Haberl et aI., 1989a,b) and spinal (Lindsberg et aI., 1989) microvascular circulation. The CBFLDF was validated in both normal and pathophysiological states with standard techniques over a wide range of blood flow (10-200% of baseline). CBFLDF is measured in a tissue sample of -1 mm3. LDF does not provide absolute rCBF values but allows the measurement of rCBF changes ex pressed as percentage of baseline values.
Data are expressed as means ± SD. To detect signifi cant changes over time within the control group, both the CBFLDF and the ICP were compared using repeated mea sures analysis of variance. We compared the six groups for CBFLDF, ICP, and brain water content by using one way analysis of variance and Student-Newman-Keuls multiple comparisons. CBFLDF and ICP were compared across time [from the point of injection of pneumococci or saline half-hourly until 6 h post injection (p.i.)]. A p value of 0.05 compared with controls; Fig. 5). The administration of dexamethasone at 2 h p.i. reversed the initial increase of the CBFLDF after a lag of 1 h; the CBFLDF was significantly lower than the CBFLDF in the untreated meningitis group from 5 to 6 h p. i. At 6 h p.i. , CBFLDF in the animals treated with dexamethasone did not differ signifi cantly from the controls (130.6 ± 27.9 and 99.7 ± 12. 6%, respectively; Fig. 3). At 2 h after administration of dexamethasone (4 h p.i. ), ICP also began to decrease; however, there was no statistical difference between this group and the meningitis and control group (Fig. 4). Dexamethasone administration 2 h after pneumo coccal infection prevented brain edema formation at 6 h p.i. The brain water content in this group was 79.16 ± 0.19% and differed significantly from that of the untreated meningitis animals (p < 0.05; 79.69 ± 0.24%), but not from the controls (p > 0.05; 78.94 ± 0.16%; Fig. 5). Indomethacin
After injection of indomethacin, a decrease in CBFLDF was observed. Several authors have pre viously reported a decrease of CBF due to system ically applied indomethacin (Sakabe and Siesj6, 1979; Dahlgren et aI., 1981; Pickard, 1981; Nowicki et aI., 1987; Haberl et aI., 1989a). The CBFLDF de creased significantly by 21.0 ± 14. 6% at 1 h (p < 0.05 compared with controls). As compared with the untreated meningitis values, the CBFLDF in-
TABLE 1. Physiological variables
P aco2 (mm Hg) Oh 4h 6h pH Oh 4h 6h Pao2 (mm Hg) Oh 4h 6h MABP (mm Hg) Oh 4h 6h
Control (n = 6)
No therapy n ( = 7)
Oexa post (n = 5)
Oexa pre (n = 3)
34.7 ± 4.5 38 .4 ± 4.1 33.4 ±3.8
39.7 ±5.3 3 4.5 ±3.8 35.7 ±2.2
37.6 ±2.7 35.7 ±1.7 34.9 ± 3. 6
39 .3 ±7.5 38 .9 ±2.3 38 .0 ±7.0
38 .2 ±3.4 37.0±2.3 38 .3 ± 3.3
37.0 ± 5.8 34.4 ± 6.9 35. 4 ± 6.9
7 . 40±0.02 7.35 ±0.03 7.3 6 ±0.03
7.39 ±0.02 7.38 ±0.05 7.37 ±0.03
7.36 ±0.03 7.3 6 ±0.02 7.35 ±0.03
7.39 ±0.02 7.38 ±0.02 7.37 ±0.02
7.39 ±0.01 7.37 ±0.02 7 .3 6 ±0.03
7.40 ±0.03 7.39 ±0.03 7.37±0.03
103.1 ±21.7 12 6.5 ±18 .9 112.3 ±15.4
109.9 ±11.4 11 4.1 ±12.6 111.6±7.2
104.9 ±13.3 105.5 ±1 4.5 107.9 ±8 . 4
108 . 4 ±1 6.8 104.2 ±21.9 107.7 ±22.2
104.4 ±7.4 99.0±9 .8 111.4 ±15.4
107.0 ±13.9 109.8 ±11.1 11 4.7 ± 12.1
11 4.5 ±8 .2 109.2 ±10.9 108.5 ±11.8
112.7 ±2.9 110.1 ± 6.7 107.0 ±8 .8
115.2 ±8 .8 106.0 ±7.5 104.4 ±7.0
112.7 ±9 .1 105.7 ±8 .1 107.0 ± 6.1
106.6 ±2.5 95.2 ± 6.7 100.0 ± 6.8
114.3 ± 1 4.3 107.8 ± 13.9 106.0 ± 17.1
Indo (n
=
5)
n (
SO D = 6)
Values are means ±S O . No therapy, untreated animals ofthe meningitis group; Oexa post, administration ofdexamethasone 2 h post pneumococcal injection; Oexa pre, administration of dexamethasone prior to pneumococcal injection; Indo, indomethacin; SO D, superoxide dismutase .
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H.- W. PFISTER ET AL. 300
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Ooppler flowmetry (CBFLDF) in the various groups during 6 h after intracisternal (Lc.) injection. CBFLDF was expressed as percentage of the baseline values ( 100%). There was no change in the CBFLDF in the controls (n 6). CBFLDF in creased to 2 1 1. 1 ±40.5% at 6 h in the untreated meningitis animals (n 7). The increase in CBFLDF was inhibited by pretreatment with intraperitoneal dexamethasone (OEXA PRE; 3 mg/kg, n 3) and reversed by administration of in traperitoneal dexamethasone at 2 h after pneumococcal in jection (OEXA POST; n 5). Values are means±SO. *p < 0.05 compared with controls; +p < 0.05 compared with un treated meningitis animals. =
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FIG. 4. Temporal profile of the intracranial pressure (ICP) in the various groups during 6 h after intracisternal (Lc.) injec tion. There was no change of the ICP in the controls (n 6). ICP increased from a baseline of 2.9± 1.4 to 10.4 ± 4.7 mm Hg at 6 h after pneumococcal injection in the untreated men ingitis animals (n 7). The increase in ICP was inhibited by pretreatment with intraperitoneal dexamethasone (OEXA PRE; 3 mg/kg, n 3) and reversed by administration of in traperitoneal dexamethasone at 2 h after pneumococcal in jection (OEXA POST; n 5). Values are means ± SO. *p < 0.05 compared with controls; +p < 0.05 compared with un treated meningitis animals. =
=
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crease was delayed and attenuated by treatment with indomethacin (Fig. 6). The CBFLDF was sig nificantly different from control at 4.5 h p.i. (164.0 ± 34.4% in the indomethacin group compared with 111.7 ± 11.4% in the control group). Five to six hours post infection, there was also a significant difference between the untreated and indometha cin-treated meningitis animals. J Cereb Blood Flow Metab, Vol.
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=
The ICP was significantly lower in the indo methacin group 3 (3.2 ± 2.0 mm Hg) and 3.5 (3.9 ± 3.5 mm Hg) h p.i. than in the untreated meningitis animals (6.2 ± 2.9 and 7.6 ± 3.4 mm Hg, respec tively; Fig. 6). From 4 to 6 h p.i., there were no
CBF IN BACTERIAL MENINGITIS
919
TABLE 2. CBFLDF• [CPo and brain water content at 6 h post infection Group and treatment
CBFLDF
(%)
I CP (mm Hg)
Brain water
(%)
3.5 ± 2.5 10.4 ± 4.7 a
7 S.9 4 ± 0.1 6 79.69 ±0.2 4a
103.7 ± 4.0b
2.1 ± O.Sb
79.03 ± O.I Ob
130.6 ±27.9 b
5.6 ±2.5
79.1 6 ±0.19 b
1 6 6.0±2 4.3"·b 103.2±11.lb
7.2 ±3.5 4.3 ±1.5b
79.25 ± O.lI a•b 79.0S±0.09 b
99.7 ±12.6 211.1± 40.5a
1. Controls (n = 6) 2. No therapy n ( = 7) 3. Dexamethasone pretreatment (n = 3) 4. Dexamethasone treatment (n = 5) 5. Indomethacin pretreatment (n = 5) 6. SOD treatment n ( = 6)
In controls saline was injected intracisternally. Values are means ± SD. n. no . of animals tested (exception: brain water content determination was performed onlyin five animals ofgroups I and 2); LDF, laser-Doppler flowmetry; I CP, intracranial pressure; SOD. superoxide dismutase. a p < 0.05 when compared with controls . b p < 0.05 when compared with untreated meningitis animals.
significant differences between these two groups and the controls. Indomethacin pretreatment attenuated brain edema formation. The brain water content in this group was 79. 25 ± 0.11% and differed significantly from that of the untreated meningitis animals (79.69 ± 0. 24%; p < 0.05) and the controls (78.94 ± 0.16%; p < 0.05; Fig. 5).
with SOD (brain water content 79.08 ± 0.09%; p 0.05 compared with controls; Fig. 5). DISCUSSION
In this study we monitored the rCBF and ICP during the early phase of pneumococcal meningitis 300 pneumococci
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CON
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FIG. 5. Brain water content determinations revealed brain
edema formation at 6 h post pneumococcal injection in the untreated meningitis animals. The brain edema formation was inhibited by treatment with superoxide dismutase (SOD). pretreatment with dexamethasone (DEXA PRE), and admin istration of dexamethasone 2 h after infection (DEXA POST) and attenuated by pretreatment with indomethacin (INDO). NO T HEA, no therapy. Values are means ± SD. *p < 0.05 compared with controls; +p < 0.05 compared with untreated meningitis animals.
+
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The increases in CBFLDF and ICP observed in the untreated meningitis animals were completely elim inated by treatment with SOD (Fig. 7). During the experiment, the CBFLDF and ICP were not signifi cantly different from those of the controls and the animals that were treated with SOD. In addition, brain edema formation was inhibited by treatment
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FIG. 6. Temporal profile of the CBF measured by laser Doppler flowmetry (CBFLDF) and the intracranial pressure (ICP) in five animals pretreated with indomethacin (INDO). The increases in CBFLDF and ICP observed in the untreated meningitis animals (Figs. 3 and 4) were delayed and attenu ated by pretreatment with indomethacin ( 10 mg/kg Lv.). Val ues are means ± SD. Lc.. intracisternal. *p < 0.05 compared with controls; +p < 0.05 compared with untreated meningitis animals.
J Cereb Blood Flow Metab, Vol.
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H.-W. PFISTER ET AL.
920 300 pneumococci
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source for the generation of oxygen-derived free radicals in bacterial meningitis. There was clear histological evidence of granulo cytic meningeal inflammation in rat brains after in tracisternal pneumococcal challenge. Inflammatory changes were not confined to the area surrounding the cisterna magna but were also evident over the hemispheres at the site of the CBF measurement. These findings were supported by previous experi ments using methylene blue dye and carrageenan (Pfister et aI. , 1989).
0 -1
0
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2 TIME
(
h
4
5
5
)
FIG. 7. Temporal profile of the CBF measured by laser Doppler flowmetry (CBFLDF) and the intracranial pressure (ICP) in six animals treated with superoxide dismutase (SOD). The intravenous infusion of SOD was started just be fore the pneumococcal injection and was given continuously over the whole experiment ( 2 2,000 U/kg-h). The increases in CBFLDF and ICP observed in the untreated meningitis ani mals (Figs. 3 and 4) were completely eliminated by SOD. There were no significant changes in the CBFLDF or ICP over time within the SOD group using repeated measures analysis of variance. Values are means±SO. i.e., intracisternal. +p < 0.05 compared with untreated meningitis animals.
in the rat. The important advantage of this model is the continuous recording of the rCBF and ICP, which provides detailed information about the tem poral profile of these changes. We found that there is a dramatic increase in rCBF and ICP associated with brain edema formation during the early phase of experimental bacterial meningitis. These changes were prevented by pretreatment with dexametha sone and treatment with SOD, delayed and attenu ated by pretreatment with indomethacin, and re versed by administration of dexamethasone 2 h pj. Our findings suggest that oxygen-derived free radi cals, including superoxide anion radical and hy droxyl radical, are involved in the increases of rCBF and ICP and the brain edema formation dur ing the early phase of experimental bacterial men ingitis. Arachidonic acid metabolites of the cyclo oxygenase pathway are partially involved as medi ators of the observed changes and are one possible J Cereb Blood Flow Metab, Vol.
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The reBF increased significantly at 1 h after pneumococcal injection and doubled within 6 h pj. (compared with controls). Brain edema formation was confirmed at 6 h pj. Several factors including alterations of intracerebral interstitial pH (Ander sen et aI., 1989), endothelial adhesion of leukocytes (Tuomanen et aI., 1989), cyclooxygenase-derived vasoactive agents (Kadurugamuwa et aI. , 1987; Tuomanen et aI., 1987; Tureen et aI., 1987), oxygen derived free radicals (McCord, 1974), cytokines (Wispelwey et aI., 1988; Saukkonen et aI., 1989), and bradykinin (Wahl et aI., 1983; Ellis et aI., 1988) have to be considered as possible causes for the observed increases of reBF and Iep and the edema formation. In this study we examined the effects of dexamethasone, the cyclooxygenase inhibitor in domethacin, and the free radical scavenger SOD. Dexamethasone, indomethacin, and SOD
Pretreatment with dexamethasone inhibited the rCBF and ICP increases that were observed in un treated meningitis animals. Administration of dexa methasone at 2 h pj. reversed these changes. In addition, brain edema formation was prevented by pretreatment with dexamethasone and reversed by treatment with dexamethasone. The mechanism by which dexamethasone acts is unknown. Dexameth asone exhibits a variety of effects. It has inhibitory effects on cachectin (tumor necrosis factor-a) and interleukin-113 in vitro. Both agents have been sug gested to produce increased blood-brain barrier permeability and brain edema (Wispelwey et aI., 1988; Saukkonen et aI. , 1989). Increased CSF levels of tumor necrosis factor-a and interleukin-113 have been detected in patients with bacterial meningitis and in animal models of bacterial meningitis (Leist et aI. , 1988; Mustafa et aI., 1989a,b; Nadal et aI., 1989; Waage et aI., 1989). The precise pathophysi ologic role of these cytokines during bacterial men ingitis has yet to be determined. Another explana tion for the observed effects of glucocorticoids in our study involves the arachidonic acid metabo lism. Glucocorticoids inhibit phospholipase A2,
CBF IN BACTERIAL MENINGITIS
which in turn prevents the production of vasoactive agents such as prostaglandins and leukotrienes. We therefore examined the effect of the cycloox ygenase inhibitor indomethacin. Our results with in domethacin support the hypothesis that cyclooxy genase derivates are mediators of the microvascular changes seen in our experiments. The rCBF and ICP increases were delayed and attenuated by pre treatment with indomethacin; in addition, brain edema formation was attenuated. The fact that in domethacin only attenuated these changes argues against the possibility that eicosanoids are the only mediators. Indomethacin prevents the generation of prostaglandins. Prostaglandins Db Eb Gb and 12 have been shown to cause vasodilation of cerebral arterioles (Ellis et aI., 1979; Morii et aI., 1989). Fur thermore, in the process of the conversion of pros taglandin G2 to prostaglandin H2, oxygen-derived free radicals may be generated. The oxygen-derived free radicals are known to cause vasodilation in ce rebral arterioles, abnormal CO2 reactivity, elimina tion of endothelium-dependent responses, and in creased permeability of the blood-brain barrier (Rosenblum, 1983, Chan et aI. , 1984; Wei et aI. , 1985; Kontos, 1989). We studied the effect of the free radical scavenger SOD. Our findings that SOD blocks the increases in rCBF and ICP and the brain edema formation suggest that the release of oxygen derived free radicals is a mqjor cause of the patho genesis of bacterial meningitis. Radical scavenging is the only known biological activity of SOD. It is not clear which of the oxygen-derived free radicals causes the changes in rCBF and ICP and brain edema during bacterial meningitis. Superoxide an ion and hydrogen peroxide can interact in the pres ence of catalytic iron or copper to form the highly reactive hydroxyl radical via the Haber-WeiB reac tion. SOD is an enzyme that converts superoxide anion to hydrogen peroxide and therefore prevents the generation of hydroxyl radical by removing a reactant of the Haber-WeiB reaction. It was reported that free SOD failed to cross the normal blood-brain barrier and did not penetrate into brain cells (Chan et aI., 1987). In addition, the half-life of free SOD in plasma is very short (-6 min) (Turrens et al. , 1984). Chemical modifications of the enzyme, such as entrapment with liposomes, have been proposed to increase half-life and blood brain barrier permeability (Chan et al., 1987). In our experiments, we found an effect of free SOD given by the intravenous route. We think that the contin uous infusion of SOD may eliminate the problem of a short half-life. The effect of SOD indicates that the agent seems to be able to reach the site of gen eration of the oxygen-derived free radicals. One
921
possibility is that oxygen-derived free radicals are scavenged by SOD intraluminally in the vessels. However, it is also conceivable that SOD passes through the blood-brain barrier if given in high doses, as was the case in our experiments. Thus, the site of generation of the oxygen-derived free radicals remains unclear. Acknowledgment: Permission for animal experiments was provided by the Regierung von Oberbayern. This study was supported by a grant from the Friedrich Baur Stiftung. We thank Pamla J. Decker for manuscript prep aration.
REFERENCES Andersen NEO, Gyring J, Hansen AJ, Laursen H, Siesj6 BK (1989) Brain acidosis in experimental pneumococcal menin gitis. J Cereb Blood Flow Metab 9:381-387 Ar bit E, DiResta GR, Bedford RF, Shah NK, Galicich JH (1989) Intraoperative measurement of cere bral and tumor blood flow with laser-Doppler flowmetry. Neurosurgery 24:166170 Bonner RF, Clem TR, Bowen PD, Bowman R L (1981) Laser Doppler continuous real-time monitor ofpulsatile and mean blood flow in tissue microcirculation. In: Scattering Tech niques Applied to Supramolecular and Nonequilibrium Sys tems, Vol 73 ( Chen SH, Chu B, Mossal R, eds), New York, Plenum, pp 685-702 Chan PH, Schmidley JW, Fishman RA, Longar SM (1984)Brain injury, edema, and vascular permea bility changes induced by oxygen-derived free radicals. Neurology 34:315-320 Chan PH, Longar S, Fishman RA (1987) Protective effects of liposome-entrapped superoxide dismutase on posttraumatic brain edema. Ann Neurol 21:540--547 Dahlgren N, Nilsson B, Saka be T, Siesj6BK (1981)The effect of indomethacin on cere bral blood flow and oxygen consump tion in the rat at normal and increased car bon dioxide ten sions. Acta Physiol Scand 111 :475-485 Dirnagl U, Kaplan B, Jacewicz M, Pulsinelli W (1989) Laser Doppler flowmetry for the estimation of CBF changes: a validation using autoradiography in a rat stroke model. J Cereb Blood Flow Metab 9:589--596 Ellis EF, Wei EP,Kontos HA (1979) Vasodilation ofcat cere bral arterioles by prostaglandins D2, E2, G2,and 12, Am J Physioi 237:H381-H385 Ellis EF, Holt SA, Wei EP, Kontos HA (1988) Kinins induce a bnormal vascular reactivity. Am J Physioi255:H397-H400 Eyre JA, Essex n, Flecknell PA, Bartholomew PH, Sinclair II (1988)A comparison ofmeasurements ofcere bral blood flow in the ra b bit using laser Doppler spectroscopy and radionu c1ide la belled microspheres. Clin Phys PhysioiMeas 9:65-74 Ha berl R L, Heizer M L, Marmarou A, Ellis EF (1989a) Laser Doppler assessment of brain microcirculation: effect ofsys temic alterations. Am J Physioi256:HI247-H1254 Ha berl R L, Heizer M L, Ellis EF (l989b) Laser-Doppler assess ment of brain microcirculation: effect of local alterations. Am J Physioi256:HI255-H1260 Horwitz SJ,Boxer baum B,O'Bell J (1980) Cere bral herniation in bacterial meningitis in childhood. Ann Neurol 7:524--528 Igarashi M, Gilmartin R C, Gerald B, Wil burn F, Ja b bour JT (1984) Cere bral arteriitis and bacterial meningitis. Arch Neu roI41:531-535 Kadurugamuwa J L, Hengstler B, Zak 0 (1987) Effects ofanti in flammatory drugs on arachidonic acid meta bolites and ce re brospinal fluid proteins during infectious pneumococcal meningitis in ra b bits. Pediatr Infect Dis J 6:1153-1154 Kontos HA (1989) Oxygen radicals in experimental brain injury.
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In: Intracranial Pressure, Vol 7 (Hoff JT, Betz A L, eds), Berlin, Springer- Verlag, pp 787- 798 Kontos HA, Wei EP, Dietrich WD, Navari WD, Povlishock JT, Ghatak NR, Ellis EF, Patterson J L ( 1981) Mechanism of cere bral arteriolar a bnormalities after acute hypertension. Am J PhysioI240:H511-H527 Leist TP, Frei K, Kam-Hansen S, Zinkemage I RM, Fontana A ( 1988) Tumor necrosis factor alpha in cere brospinal fluid during bacterial but not viral meningitis . J Exp Med 1 67: 17 43- 17 48 Linds berg PJ,O' Neill JT,Paakkari I,Hallen beck JM,Feuerstein G ( 1989) Validation oflaser-Doppler flowmetry in measure ment of spinal cord blood flow . Am J Physiol 257:H 67 4H 68 0 Mc Cord J M (1974)Free radicals and in flammation: protection of synovial fluid bysuperoxide dismutase . Science 18 5: 529- 531 McMenamin JB, Volpe 11 (1984)Bacterial meningitis in infancy: effects on intracranial pressure and cere bral blood flow ve locity. Neurology 34:500-504 Morii S, Wang R, Ohtaka H, Yada K ( 1989)The in vitro effectof iloprost, a sta ble prostacyclin analogue, on the cali ber ofthe cere bral microvessels [A bstract] . J Cereb Blood Flow Metab 9 (suppl 1): S 6 6 6 Mustafa MM, Le bel MH,Ramilo 0 ,Olsen KD,Reisch J S,Beut ler B, Mc Cracken GH (1989a) Correlation ofinterleukin- lf3 and cachectin concentrations in cere brospinal fluid and out come from bacterial meningitis . J Pediatr 115:208-213 Mustafa M, Ramilo 0, Olsen KD, Franklin P S, Hansen EJ, Beutler B, Mc Cracken GH Jr ( 1989b) Tumor necrosis factor in mediating experimental Haemophilus influenzae type b meningitis . J Clin Invest 84:1253- 1259 Nadal D, Leppert D, Frei K, Gallo P, Lamche H, Fontana A ( 1989) Tumor necrosis factor-alpha in infectious meningitis . Arch Dis Child 64:127 4- 1279 Nowicki JP, Jourdain D, MacKenzie ET ( 1987) NADH fluores cence in vivo: changes in cere bral oxidative meta bolism and perf usion induced by pento bar bital, indomethacin, and sa licylate . J Cereb Blood Flow Metab 7: 280- 288 Nugent SK, Bausher JA, Moxon ER, Rogers M C ( 1979) Raised intracranial pressure. Its management in Neisseria meningi tidismeningoencephalitis . Am J Dis Child 133: 2 60- 2 62 Paulson OB, Brodersen P, Hansen E L, Kristensen H S (1974) Regional cere bral blood flow, cere bral meta bolic rate ofox ygen, and cere brospinal fluid acid- base varia bles in patients with acute meningitis and with encephalitis. Acta Med Scand 196:191-198 P fister HW ( 1989) Complicated purulent meningitis ofthe adult: persisting high mortality caused by vasculitis and increased intracranial pressure . Nervenarzt 60:249-254 P fister HW, Dimagl U, Ha berl R, Anneser F, Kiidel U, Meh raein P, Feiden W, Einhaupl KM ( 1989) Changes of intra cranial pressure in a ratmeningitis model . Intracranial Pres sure, Vol 7 (Hoff JT,Betz A L,eds),Berlin, Springer- Verlag, pp 781-783 Pickard JD ( 1981) Role of prostaglandins and arachidonic acid derivates in the coupling ofcere bral blood flow to cere bral meta bolism. J Cereb Blood Flow Metab 1: 3 61- 38 4 Quagliarello VJ, Long WJ, Scheid WM ( 198 6) Morphological alterations ofthe blood brain barrier with experimental men ingitis in the rat. J Clin Invest 77: 1084-1095 Rosen blum W I ( 198 3)Effects offree radical generation on mouse pial arterioles: pro ba ble role of hydroxyl radicals . Am J Physiol 245:H139-HI42 Saka be T, Siesjii BK ( 1979) The effects ofindomethacin in the
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blood flow-meta bolism couple in the brain under hypercap nic and hypoxic conditions . Acta Physiol Scand 107: 283-2 8 4 Saukkonen K, Sande S, Cioffe C, Wolpe S, SherryB, Cerami A, Tuomanen E ( 1989) The role ofcytokines in the generation ofin flammation and tissue damage in experimental pneumo coccal meningitis. Presented at the 29th Interscience Con ference on Antimicro bial Agents and Chemotherapy, 17-20 Septem ber 1989, Houston, TX, a bstract no . 709 Skarphedinsson JO, Harding H, Thoren P ( 1988) Repeated mea surements ofcere bral blood flow in rats . Comparisons be tween the hydrogen clearance method and laser Doppler flowmetry. Acta Physiol Scand 134:133-142 Stem MD (1975) In vivo evaluation of microcirculation by co herent light scattering. Nature 254:56-58 Swartz M N (1984) Bacterial meningitis: more involved than just the meninges . N Engl J Med 311:912-914 Tau ber M G, Khayam-Bashi H, Sande MA (1985) Effects of ampicillin and corticosteroids on brain water content, cere brospinal fluid pressure, and cere brospinal fluid lactate lev els in experimental pneumococcal meningitis . J Infect Dis 151:528-534 Tuomanen E (1987) Molecular mechanisms of in flammation in experimental pneumococcal meningitis . Pediatr Infect Dis 6: 11 46- 11 49 Tuomanen E, Hengstler B, Rich R, BrayMA, Zak 0, Tomasz A ( 1987) Nonsteroidal antiin flammatory agents in the therapy for experimental pneumococcal meningitis . J Infect Dis 155:985-990 Tuomanen E I, Saukkonen K, Sande S, Ciof fe C, Wright SD ( 1989) Reduction ofin flammation, tissue damage, and mor talityin bacterial meningitis in ra b bits treated with monoclo nal anti bodies against adhesion-promoting receptors ofleu cocytes. J Exp Med 170:959-969 Tureen JH, Stella FB, Clyman R I, Mauray F, Sande MA ( 1987) Effect ofindomethacin on brain water content, cere brospi nal fluid white blood cell response and prostaglandin E2 lev els in cere brospinal fluid in experimental pneumococcal meningitis in ra b bits . Pediatr Infect Dis 6:1151-1153 Turrens JF, Crapo JD, Freeman BA (1984) Protection against oxygen toxicity by intravenous injection of liposome entrapped catalase and superoxide dismutase . J Clin Invest 73: 8 7- 9 5 Waage A , Halstensen A , Shala by R , Brandtzaeg P , Kierulf P, Espevik T ( 1989) Local production oftumor necrosis factor, interleukin 1, and interleukin 6 in meningococcal meningitis . Relation to the in flammatoryresponse . J Exp Med 170:18591867 Wahl M, Young AR, Edvinsson L, Wagner F (1983) Effects of bradykinin on pial arteries and arterioles in vitro and in situ . J Cereb Blood Flow Metab 3:231-237 Wei EP, Kontos HA, Dietrich WD, Povlishock JT, El1is EF (1981) Inhi bition by free radical scavengers and bycycloox ygenase inhi bitors ofpial arteriolar a bnormalities from con cussive brain injury in cats . Circ Res 48:95-103 Wei EP, Christman CW, Kontos HA, Povlishock JT (1985) Ef fects ofoxygen radicals on cere bral arterioles. Am J Physiol 2 48:H I57-H I 62 WispelweyB, Long WJ, Castracane JM, Scheid WM (1988) Ce re brospinal fluid interleukin- 1 activity fol1owing intracister nal inoculation of Haemophilus influenzae type b lipooli gosaccharide into rats . Presented at the 28th Interscience Conference on Antimicro bial Agents and Chemotherapy, Los Angeles, CA, a bstract no . 873.
10:914-922 © 1990 Raven Press, Ltd., New York
Microvascular Changes During the Early Phase of Experimental Bacterial Meningitis H.-W. Pfister, U. Koedel, R. L. Haberl, U. Dirnagl, *W. Feiden, tG. Ruckdeschel, and K. M. Einhaupl Department of Neurology, *Institute of Neuropathology, and tMax von Pettenkofer Institute of Hygiene and Microbiology, University of Munich, Munich, F.R.C.
Summary: We investigated the temporal profile of the changes in regional CBF (rCBF) and intracranial pressure (ICP) during the early phase of pneumococcal meningitis in the rat. rCBF, as measured by laser-Doppler flowmet ry, and ICP were continuously monitored during 6 h post infection (pj.). Brain edema formation was assessed by brain water content determinations. Meningitis was in duced by intracisternal injection of 75 IJ-l of 107 colony forming units/ml pneumococci (n 7). In control animals (n 6), saline was injected. There was no change in the rCBF or ICP of controls throughout the experiment. However, there was a dramatic increase in rCBF and ICP associated with brain edema formation in untreated men ingitis animals. rCBF increased to 135.3 ± 33.8% (mean ± SD) in the untreated animals at I h pj. and reached 2 11.1 ± 40.5% at 6 h pj. (p < 0.05 compared with con trols). ICP increased from 2.9 ± 1.4 to 10.4 ± 4.7 mm Hg at 6 h pj. (p < 0.05 compared with controls). Brain water content was significantly elevated (79.69 ± 0.24 com pared with 78.94 ± 0. 16% in the control group, p < 0.05).
We investigated the effect of dexamethasone (3 mg/kg i.p.), which was given prior to the induction of meningitis (n 3) or at 2 h after pneumococcal injection (n 5), indomethacin ( 10 mg/kg i.v., n 5), and superoxide dis mutase (SOD; 132,000 U/kg i.v. per 6 h, n 6). The increases in rCBF and ICP were prevented by the pre treatment with dexamethasone and the administration of SOD, delayed and attenuated by pretreatment with in domethacin, and reversed by administration of dexameth asone 2 h pj. These findings suggest that oxygen-derived free radicals are involved as mediators in the increases of rCBF and rcp and brain edema formation during the early phase of experimental bacterial meningitis. Arachi donic acid metabolites of the cyc100xygenase pathway are partially involved in the observed changes and are one possible source for the generation of oxygen-derived free radicals in bacterial meningitis. Key Words: Bacterial meningitis-Brain edema-Cerebral blood flow Intracranial pressure-Laser-Doppler flowmetry Oxygen-derived free radicals-Superoxide dismutase.
Despite great advances in antimicrobial therapy, mortality due to bacterial meningitis remains high (Swartz, 1984). Pneumococcus-induced meningitis, the most frequent etiologic agent in bacterial men ingitis of the adult, is fatal in 25-30%. Clinical ob servations suggest that increased intracranial pres sure (ICP), brain edema, and cerebral vasculitis are associated with unfavorable clinical courses (Paul son et aI., 1974; Nugent et aI., 1979; Horwitz et aI.,
1980; Igarashi et aI., 1984; McMenamin and Volpe, 1984; Pfister, 1989). Although several investigators have tried to identify the molecular basis of the in flammatory changes in bacterial meningitis using animal models (Tauber et aI., 1985; Quagliarello et aI., 1986; Tuomanen et aI., 1987, 1989; Mustafa et aI., 1989a), the precise pathophysiological mecha nisms of the major intracranial complications are not completely understood. Multiple factors includ ing the complement alternative pathway, cytokines, prostaglandins, and platelet-activating factor have been suggested to be involved in the complex na ture of the pathophysiological alterations during bacterial meningitis (Tuomanen, 1987). The present study was designed to assess the temporal course of changes in regional CBF (rCBF), rcp, and brain edema formation during the
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Received January 4, 1990; revised March 29, 1990; accepted March 30, 199 0. Address correspondence and reprint requests to Dr . H.-W. P fister at Department of Neurology, Ludwig Maximilians Uni versity of Munich, Klinikum GroBhadem, Marchioninistr . 15, D-8000 Miinchen 70, F.R . G . Abbreviations used: I CP, intracranial pressure; LDF, laser Doppler flowmetry; p.i., post infection; r CBF, regional CBF; SOD, superoxide dismutase.
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early phase of pneumococcal meningitis of the rat. As known from other pathophysiological experi mental models, such as acute severe arterial hyper tension (Kontos et aI., 1981) and fluid percussion brain injury (Wei et aI., 1981), arachidonic acid me tabolites and oxygen-derived free radicals are me diators of vascular damage and brain edema. In ad dition, because of their high reactivity and capacity to produce cellular damage, the oxygen-derived free radicals have also been implicated as mediators in inflammation (McCord, 1974). Therefore, we in vestigated the effect of the blocking substances dexamethasone, the cyclooxygenase inhibitor in domethacin, and the free radical scavenger super oxide dismutase (SOD). MATERIAL AND METHODS
LDF-PROBE SUPERFUSION INLET
FIG. 1. Diagra m of the experimental setup. The laser-Doppler probe was fixed by a steel cap over the right parietal cortex. The dura was not re moved. The regional CBF measured by laser-Doppler flow metry (LDF) and the intracranial pressure were continuously recorded for 6 h after intracisternal (IC) injection.
Animal preparation
A total of 32 animals were used in this study. Adult male Wistar rats weighing 250-350 g were anesthetized with 100 mg/kg i.p. thiobutabarbiturate (lnactin). To maintain anesthesia, supplemental doses of thiobutabar biturate were given through a peritoneal catheter. An en dotracheal tube was inserted through a tracheostomy. The rats were ventilated mechanically (model 683 small animal ventilator; Harvard, South Natick, MA, U.S.A.) with room air and supplemental oxygen. The end expiratory CO2 was continuously monitored with an in frared CO2 analyzer (model 2200; Heyer, Bad Ems, F.R.G.) and maintained at a level of 35 mm Hg through out the experiment by adjusting the respiratory rate and volume. Polyethylene tubings were inserted into the fem oral artery and vein. Arterial blood pressure was mea sured with a Statham P23 pressure transducer connected to the cannula in the femoral artery. Arterial blood sam ples were collected periodically from the arterial line and analyzed for Po2, Pco2, and pH (AVL gas check, model 940). Saline was infused in the femoral vein during the experiment ( 10 m1l6 h) by an infusion pump (Precidor, type 5003; Infors HT, Bottmingen, Switzerland). Hemat ocrit investigations revealed no change during the exper iment. Body temperature was maintained at 38°C by a rectal thermometer-controlled heating pad. The animals were placed in a stereotaxic frame. A burr hole was made for the cisterna magna cannula at the angle where the sagittal and lambda sutures meet. ICP was recorded with a Statham P23 pressure transducer via a polyethylene tube. CBF was continuously measured by laser-Doppler flowmetry (LDF; model BPM 403; TSI Inc., St. Paul, MN, U.S.A.). For placement of the LD probe, a craniot omy (diameter 5 mm) was made in the mediocaudal por tion of the right parietal bone. The dura remained intact. A stainless-steel cap was implanted above the craniotomy site. This cap held the LD probe and was equipped with two openings for flushing the area under the cap (Fig. 1). The probe was placed over an area free of large pial ves sels. Saline was carefully flushed through the cap to re move stains of blood and air bubbles within the system. MABP, ICP, end-expiratory CO2, and rCBF, as mea sured by LDF (CBFLDP)' were continuously recorded on a multichannel paper strip recorder (six-channel recorder, �
model BD 10 1; Kipp & Zonen, Delft, Holland) for 6 h after induction of meningitis. Additionally, all parameters were transferred to a PC system and AD converted for digital signal processing. Because of the fluctuation of the LDF signal, the LDF data were averaged every 30 s dur ing the experiment. Bacterial inoculum The strain Streptococcus pneumoniae type 6b (no.
643), an isolate from the blood of a patient with septic infection, was kept at - 20°C in trypticase soy broth (Ox oid, Wesel, F.R.G.), supplemented with 10% glycerol and 1% IsoVitale (BBL, Heidelberg, F.R.G.). Before use, the bacteria were subcultured on blood-agar plates, checked for purity, inoculated into brain-heart infusion broth (Ox oid), supplemented with 3% horse serum and 1% bovine albumin (Serva, Heidelberg, F .R.G .), and incubated overnight at 35°C. The broth was centrifuged for 20 min at 2,500 g; the sediment was washed once with 0.85% saline and then resuspended in saline. The final suspension was turbidometrically adjusted to a density of 0.5 (photome ter, 13-mm filter, 546 nm; Eppendorf, Hamburg, F.R.G.), thus achieving a concentration of 107 colony-forming units (cfu)/ml. �
Induction of meningitis
After recording a stable baseline of CBFLDP and ICP for 30 min, 75 ILl CSF was removed through the cisternal catheter. Meningitis was induced by intracisternal injec tion of 75 ILl of 107 cfu pneumococci (seven animals). In control animals (six animals), 75 ILl saline was injected into the cisterna magna. Administration of dexamethasone, indomethacin, and SOD
Dexamethasone (3 mg/kg i.p.; Merck Sharp & Dohme, Miinchen, F.R.G.) was given 1 h prior to the injection of pneumococci (three animals) or at 2 h post pneumococcal injection (five animals). Five animals were pretreated with indomethacin ( 10 mg/kg i.v.; Sigma Chemie, Deisen hofen, F.R.G.). Intravenous infusion of the free radical scavenger SOD (from bovine erythrocytes; Sigma Chemie) was started just before the pneumococ-
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cal injection was given continuously during the experi ment (22,000 U/kg-h) (six animals).
some selected cases, Giemsa, Gram, and (naphthol AS-D) chioroacetate esterase.
LDF
Statistical comparisons
LDF has been described in detail elsewhere (Stern, 1975; Bonner et aI., 1981; Haberl et aI., 1989a,b). The LD technique, used to assess microcirculatory blood flow in small tissue samples, has the advantage of continuous CBF measurement without disruption of brain tissue (un like H2 clearance) and without multiple infusions of tracer (unlike the umbelliferone clearance method). The method has been validated for the cerebral (Eyre et aI., 1988; Skarphedinsson et aI., 1988; Arbit et al. 1989; Dirnagl et aI., 1989; Haberl et aI., 1989a,b) and spinal (Lindsberg et aI., 1989) microvascular circulation. The CBFLDF was validated in both normal and pathophysiological states with standard techniques over a wide range of blood flow (10-200% of baseline). CBFLDF is measured in a tissue sample of -1 mm3. LDF does not provide absolute rCBF values but allows the measurement of rCBF changes ex pressed as percentage of baseline values.
Data are expressed as means ± SD. To detect signifi cant changes over time within the control group, both the CBFLDF and the ICP were compared using repeated mea sures analysis of variance. We compared the six groups for CBFLDF, ICP, and brain water content by using one way analysis of variance and Student-Newman-Keuls multiple comparisons. CBFLDF and ICP were compared across time [from the point of injection of pneumococci or saline half-hourly until 6 h post injection (p.i.)]. A p value of 0.05 compared with controls; Fig. 5). The administration of dexamethasone at 2 h p.i. reversed the initial increase of the CBFLDF after a lag of 1 h; the CBFLDF was significantly lower than the CBFLDF in the untreated meningitis group from 5 to 6 h p. i. At 6 h p.i. , CBFLDF in the animals treated with dexamethasone did not differ signifi cantly from the controls (130.6 ± 27.9 and 99.7 ± 12. 6%, respectively; Fig. 3). At 2 h after administration of dexamethasone (4 h p.i. ), ICP also began to decrease; however, there was no statistical difference between this group and the meningitis and control group (Fig. 4). Dexamethasone administration 2 h after pneumo coccal infection prevented brain edema formation at 6 h p.i. The brain water content in this group was 79.16 ± 0.19% and differed significantly from that of the untreated meningitis animals (p < 0.05; 79.69 ± 0.24%), but not from the controls (p > 0.05; 78.94 ± 0.16%; Fig. 5). Indomethacin
After injection of indomethacin, a decrease in CBFLDF was observed. Several authors have pre viously reported a decrease of CBF due to system ically applied indomethacin (Sakabe and Siesj6, 1979; Dahlgren et aI., 1981; Pickard, 1981; Nowicki et aI., 1987; Haberl et aI., 1989a). The CBFLDF de creased significantly by 21.0 ± 14. 6% at 1 h (p < 0.05 compared with controls). As compared with the untreated meningitis values, the CBFLDF in-
TABLE 1. Physiological variables
P aco2 (mm Hg) Oh 4h 6h pH Oh 4h 6h Pao2 (mm Hg) Oh 4h 6h MABP (mm Hg) Oh 4h 6h
Control (n = 6)
No therapy n ( = 7)
Oexa post (n = 5)
Oexa pre (n = 3)
34.7 ± 4.5 38 .4 ± 4.1 33.4 ±3.8
39.7 ±5.3 3 4.5 ±3.8 35.7 ±2.2
37.6 ±2.7 35.7 ±1.7 34.9 ± 3. 6
39 .3 ±7.5 38 .9 ±2.3 38 .0 ±7.0
38 .2 ±3.4 37.0±2.3 38 .3 ± 3.3
37.0 ± 5.8 34.4 ± 6.9 35. 4 ± 6.9
7 . 40±0.02 7.35 ±0.03 7.3 6 ±0.03
7.39 ±0.02 7.38 ±0.05 7.37 ±0.03
7.36 ±0.03 7.3 6 ±0.02 7.35 ±0.03
7.39 ±0.02 7.38 ±0.02 7.37 ±0.02
7.39 ±0.01 7.37 ±0.02 7 .3 6 ±0.03
7.40 ±0.03 7.39 ±0.03 7.37±0.03
103.1 ±21.7 12 6.5 ±18 .9 112.3 ±15.4
109.9 ±11.4 11 4.1 ±12.6 111.6±7.2
104.9 ±13.3 105.5 ±1 4.5 107.9 ±8 . 4
108 . 4 ±1 6.8 104.2 ±21.9 107.7 ±22.2
104.4 ±7.4 99.0±9 .8 111.4 ±15.4
107.0 ±13.9 109.8 ±11.1 11 4.7 ± 12.1
11 4.5 ±8 .2 109.2 ±10.9 108.5 ±11.8
112.7 ±2.9 110.1 ± 6.7 107.0 ±8 .8
115.2 ±8 .8 106.0 ±7.5 104.4 ±7.0
112.7 ±9 .1 105.7 ±8 .1 107.0 ± 6.1
106.6 ±2.5 95.2 ± 6.7 100.0 ± 6.8
114.3 ± 1 4.3 107.8 ± 13.9 106.0 ± 17.1
Indo (n
=
5)
n (
SO D = 6)
Values are means ±S O . No therapy, untreated animals ofthe meningitis group; Oexa post, administration ofdexamethasone 2 h post pneumococcal injection; Oexa pre, administration of dexamethasone prior to pneumococcal injection; Indo, indomethacin; SO D, superoxide dismutase .
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8 6
TIME ( h ) FIG. 3. Temporal profile of the CBF measured by laser
Ooppler flowmetry (CBFLDF) in the various groups during 6 h after intracisternal (Lc.) injection. CBFLDF was expressed as percentage of the baseline values ( 100%). There was no change in the CBFLDF in the controls (n 6). CBFLDF in creased to 2 1 1. 1 ±40.5% at 6 h in the untreated meningitis animals (n 7). The increase in CBFLDF was inhibited by pretreatment with intraperitoneal dexamethasone (OEXA PRE; 3 mg/kg, n 3) and reversed by administration of in traperitoneal dexamethasone at 2 h after pneumococcal in jection (OEXA POST; n 5). Values are means±SO. *p < 0.05 compared with controls; +p < 0.05 compared with un treated meningitis animals. =
4 2 0 -1
0
3
=
=
=
=
TIME
4
5
6
(h)
FIG. 4. Temporal profile of the intracranial pressure (ICP) in the various groups during 6 h after intracisternal (Lc.) injec tion. There was no change of the ICP in the controls (n 6). ICP increased from a baseline of 2.9± 1.4 to 10.4 ± 4.7 mm Hg at 6 h after pneumococcal injection in the untreated men ingitis animals (n 7). The increase in ICP was inhibited by pretreatment with intraperitoneal dexamethasone (OEXA PRE; 3 mg/kg, n 3) and reversed by administration of in traperitoneal dexamethasone at 2 h after pneumococcal in jection (OEXA POST; n 5). Values are means ± SO. *p < 0.05 compared with controls; +p < 0.05 compared with un treated meningitis animals. =
=
=
crease was delayed and attenuated by treatment with indomethacin (Fig. 6). The CBFLDF was sig nificantly different from control at 4.5 h p.i. (164.0 ± 34.4% in the indomethacin group compared with 111.7 ± 11.4% in the control group). Five to six hours post infection, there was also a significant difference between the untreated and indometha cin-treated meningitis animals. J Cereb Blood Flow Metab, Vol.
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=
The ICP was significantly lower in the indo methacin group 3 (3.2 ± 2.0 mm Hg) and 3.5 (3.9 ± 3.5 mm Hg) h p.i. than in the untreated meningitis animals (6.2 ± 2.9 and 7.6 ± 3.4 mm Hg, respec tively; Fig. 6). From 4 to 6 h p.i., there were no
CBF IN BACTERIAL MENINGITIS
919
TABLE 2. CBFLDF• [CPo and brain water content at 6 h post infection Group and treatment
CBFLDF
(%)
I CP (mm Hg)
Brain water
(%)
3.5 ± 2.5 10.4 ± 4.7 a
7 S.9 4 ± 0.1 6 79.69 ±0.2 4a
103.7 ± 4.0b
2.1 ± O.Sb
79.03 ± O.I Ob
130.6 ±27.9 b
5.6 ±2.5
79.1 6 ±0.19 b
1 6 6.0±2 4.3"·b 103.2±11.lb
7.2 ±3.5 4.3 ±1.5b
79.25 ± O.lI a•b 79.0S±0.09 b
99.7 ±12.6 211.1± 40.5a
1. Controls (n = 6) 2. No therapy n ( = 7) 3. Dexamethasone pretreatment (n = 3) 4. Dexamethasone treatment (n = 5) 5. Indomethacin pretreatment (n = 5) 6. SOD treatment n ( = 6)
In controls saline was injected intracisternally. Values are means ± SD. n. no . of animals tested (exception: brain water content determination was performed onlyin five animals ofgroups I and 2); LDF, laser-Doppler flowmetry; I CP, intracranial pressure; SOD. superoxide dismutase. a p < 0.05 when compared with controls . b p < 0.05 when compared with untreated meningitis animals.
significant differences between these two groups and the controls. Indomethacin pretreatment attenuated brain edema formation. The brain water content in this group was 79. 25 ± 0.11% and differed significantly from that of the untreated meningitis animals (79.69 ± 0. 24%; p < 0.05) and the controls (78.94 ± 0.16%; p < 0.05; Fig. 5).
with SOD (brain water content 79.08 ± 0.09%; p 0.05 compared with controls; Fig. 5). DISCUSSION
In this study we monitored the rCBF and ICP during the early phase of pneumococcal meningitis 300 pneumococci
�
SOD
3: 0 -.J u.. 0::: W -.J n. n. 0 D
80.0
f-z W f-z 0 u 0::: W
79.8
!;(
3: Z «
0::: en
w 0::: => (/) (/) w
+
79.4
•
+
79.2
0:::
n. -.J �
z < 0::: u < 0::: f--
79.0 78.8 78.6
CON
NO THER
DEXA POST
DEXA PRE
INDO
SOD
FIG. 5. Brain water content determinations revealed brain
edema formation at 6 h post pneumococcal injection in the untreated meningitis animals. The brain edema formation was inhibited by treatment with superoxide dismutase (SOD). pretreatment with dexamethasone (DEXA PRE), and admin istration of dexamethasone 2 h after infection (DEXA POST) and attenuated by pretreatment with indomethacin (INDO). NO T HEA, no therapy. Values are means ± SD. *p < 0.05 compared with controls; +p < 0.05 compared with untreated meningitis animals.
+
+ •
150
50 -1
E E
+
•
:3
'" I
79.6
+
200
0::: W (/)
80.2 �
INDO
i.e.
250
The increases in CBFLDF and ICP observed in the untreated meningitis animals were completely elim inated by treatment with SOD (Fig. 7). During the experiment, the CBFLDF and ICP were not signifi cantly different from those of the controls and the animals that were treated with SOD. In addition, brain edema formation was inhibited by treatment
>
�
0
2
3
4
5
6
16 pneumococci
14
i.e.
12
INDO
,
10 +
B + 6 4
2
indo
l
o -1
I
,......-V
r---' .--
,-/
V
L
TT
o
2
TIME
3
4
5
6
(h)
FIG. 6. Temporal profile of the CBF measured by laser Doppler flowmetry (CBFLDF) and the intracranial pressure (ICP) in five animals pretreated with indomethacin (INDO). The increases in CBFLDF and ICP observed in the untreated meningitis animals (Figs. 3 and 4) were delayed and attenu ated by pretreatment with indomethacin ( 10 mg/kg Lv.). Val ues are means ± SD. Lc.. intracisternal. *p < 0.05 compared with controls; +p < 0.05 compared with untreated meningitis animals.
J Cereb Blood Flow Metab, Vol.
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H.-W. PFISTER ET AL.
920 300 pneumococci
�
i.c.
250 :;: 0 "-
--'
,
200
0:: W
--'
SOD
CL CL 0 0
150
a::
100
w (/)
5 50 -1
'" I
E E
12
6
« 0:: 0 « 0::
6
5
Changes in reBF and Iep i.c.
--' z
4
:3
pneumococci
14
10
�
2
16
w 0:: ::J (/) (/)
w 0:: CL
0
, SOD
8
4 2
;-
�
source for the generation of oxygen-derived free radicals in bacterial meningitis. There was clear histological evidence of granulo cytic meningeal inflammation in rat brains after in tracisternal pneumococcal challenge. Inflammatory changes were not confined to the area surrounding the cisterna magna but were also evident over the hemispheres at the site of the CBF measurement. These findings were supported by previous experi ments using methylene blue dye and carrageenan (Pfister et aI. , 1989).
0 -1
0
:3
2 TIME
(
h
4
5
5
)
FIG. 7. Temporal profile of the CBF measured by laser Doppler flowmetry (CBFLDF) and the intracranial pressure (ICP) in six animals treated with superoxide dismutase (SOD). The intravenous infusion of SOD was started just be fore the pneumococcal injection and was given continuously over the whole experiment ( 2 2,000 U/kg-h). The increases in CBFLDF and ICP observed in the untreated meningitis ani mals (Figs. 3 and 4) were completely eliminated by SOD. There were no significant changes in the CBFLDF or ICP over time within the SOD group using repeated measures analysis of variance. Values are means±SO. i.e., intracisternal. +p < 0.05 compared with untreated meningitis animals.
in the rat. The important advantage of this model is the continuous recording of the rCBF and ICP, which provides detailed information about the tem poral profile of these changes. We found that there is a dramatic increase in rCBF and ICP associated with brain edema formation during the early phase of experimental bacterial meningitis. These changes were prevented by pretreatment with dexametha sone and treatment with SOD, delayed and attenu ated by pretreatment with indomethacin, and re versed by administration of dexamethasone 2 h pj. Our findings suggest that oxygen-derived free radi cals, including superoxide anion radical and hy droxyl radical, are involved in the increases of rCBF and ICP and the brain edema formation dur ing the early phase of experimental bacterial men ingitis. Arachidonic acid metabolites of the cyclo oxygenase pathway are partially involved as medi ators of the observed changes and are one possible J Cereb Blood Flow Metab, Vol.
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The reBF increased significantly at 1 h after pneumococcal injection and doubled within 6 h pj. (compared with controls). Brain edema formation was confirmed at 6 h pj. Several factors including alterations of intracerebral interstitial pH (Ander sen et aI., 1989), endothelial adhesion of leukocytes (Tuomanen et aI., 1989), cyclooxygenase-derived vasoactive agents (Kadurugamuwa et aI. , 1987; Tuomanen et aI., 1987; Tureen et aI., 1987), oxygen derived free radicals (McCord, 1974), cytokines (Wispelwey et aI., 1988; Saukkonen et aI., 1989), and bradykinin (Wahl et aI., 1983; Ellis et aI., 1988) have to be considered as possible causes for the observed increases of reBF and Iep and the edema formation. In this study we examined the effects of dexamethasone, the cyclooxygenase inhibitor in domethacin, and the free radical scavenger SOD. Dexamethasone, indomethacin, and SOD
Pretreatment with dexamethasone inhibited the rCBF and ICP increases that were observed in un treated meningitis animals. Administration of dexa methasone at 2 h pj. reversed these changes. In addition, brain edema formation was prevented by pretreatment with dexamethasone and reversed by treatment with dexamethasone. The mechanism by which dexamethasone acts is unknown. Dexameth asone exhibits a variety of effects. It has inhibitory effects on cachectin (tumor necrosis factor-a) and interleukin-113 in vitro. Both agents have been sug gested to produce increased blood-brain barrier permeability and brain edema (Wispelwey et aI., 1988; Saukkonen et aI. , 1989). Increased CSF levels of tumor necrosis factor-a and interleukin-113 have been detected in patients with bacterial meningitis and in animal models of bacterial meningitis (Leist et aI. , 1988; Mustafa et aI., 1989a,b; Nadal et aI., 1989; Waage et aI., 1989). The precise pathophysi ologic role of these cytokines during bacterial men ingitis has yet to be determined. Another explana tion for the observed effects of glucocorticoids in our study involves the arachidonic acid metabo lism. Glucocorticoids inhibit phospholipase A2,
CBF IN BACTERIAL MENINGITIS
which in turn prevents the production of vasoactive agents such as prostaglandins and leukotrienes. We therefore examined the effect of the cycloox ygenase inhibitor indomethacin. Our results with in domethacin support the hypothesis that cyclooxy genase derivates are mediators of the microvascular changes seen in our experiments. The rCBF and ICP increases were delayed and attenuated by pre treatment with indomethacin; in addition, brain edema formation was attenuated. The fact that in domethacin only attenuated these changes argues against the possibility that eicosanoids are the only mediators. Indomethacin prevents the generation of prostaglandins. Prostaglandins Db Eb Gb and 12 have been shown to cause vasodilation of cerebral arterioles (Ellis et aI., 1979; Morii et aI., 1989). Fur thermore, in the process of the conversion of pros taglandin G2 to prostaglandin H2, oxygen-derived free radicals may be generated. The oxygen-derived free radicals are known to cause vasodilation in ce rebral arterioles, abnormal CO2 reactivity, elimina tion of endothelium-dependent responses, and in creased permeability of the blood-brain barrier (Rosenblum, 1983, Chan et aI. , 1984; Wei et aI. , 1985; Kontos, 1989). We studied the effect of the free radical scavenger SOD. Our findings that SOD blocks the increases in rCBF and ICP and the brain edema formation suggest that the release of oxygen derived free radicals is a mqjor cause of the patho genesis of bacterial meningitis. Radical scavenging is the only known biological activity of SOD. It is not clear which of the oxygen-derived free radicals causes the changes in rCBF and ICP and brain edema during bacterial meningitis. Superoxide an ion and hydrogen peroxide can interact in the pres ence of catalytic iron or copper to form the highly reactive hydroxyl radical via the Haber-WeiB reac tion. SOD is an enzyme that converts superoxide anion to hydrogen peroxide and therefore prevents the generation of hydroxyl radical by removing a reactant of the Haber-WeiB reaction. It was reported that free SOD failed to cross the normal blood-brain barrier and did not penetrate into brain cells (Chan et aI., 1987). In addition, the half-life of free SOD in plasma is very short (-6 min) (Turrens et al. , 1984). Chemical modifications of the enzyme, such as entrapment with liposomes, have been proposed to increase half-life and blood brain barrier permeability (Chan et al., 1987). In our experiments, we found an effect of free SOD given by the intravenous route. We think that the contin uous infusion of SOD may eliminate the problem of a short half-life. The effect of SOD indicates that the agent seems to be able to reach the site of gen eration of the oxygen-derived free radicals. One
921
possibility is that oxygen-derived free radicals are scavenged by SOD intraluminally in the vessels. However, it is also conceivable that SOD passes through the blood-brain barrier if given in high doses, as was the case in our experiments. Thus, the site of generation of the oxygen-derived free radicals remains unclear. Acknowledgment: Permission for animal experiments was provided by the Regierung von Oberbayern. This study was supported by a grant from the Friedrich Baur Stiftung. We thank Pamla J. Decker for manuscript prep aration.
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