Department of Experimental Pathology,John Curtin School of Medical Research, tDepartment of Zoology, ...... R.J., FAHEY T.J., ZENTELLA A., ALBERT J.D.,.
Br. J. Exp. Path. (I989) 70, 293-303.
Antioxidants can prevent cerebral malaria in Plasmodium berghei-infected mice C.M. Thumwood, N.H. Hunt,* W.B. Cowden and I.A. Clarkt
Department of Experimental Pathology, John Curtin School of Medical Research, tDepartment of Zoology, Faculty of Science, Australian National University, Canberra and the *lDepartment of Pathology, Medical School, Sydney University, Australia Received for publication i 6 November I 989 Accepted for publication i February I989
Summary. A/J and CBA/H mice infected with Plasmodium berghei ANKA, a murine model of cerebral malaria, were used to see whether antioxidants influenced the outcome of this disease. Untreated, infected mice died 7 to 9 days after infection, often with cerebral symptoms. Haemorrhages, mononuclear infiltration and oedema were present in the central nervous system (CNS). Feeding a diet containing 0. 75% (w/w) butylated hydroxyanisole (BHA) greatly altered the course of this disease. Death was delayed by up to 2 weeks and mice appeared healthy at parasitaemias that would have caused cerebral symptoms and death had they been on a conventional diet. BHA-fed mice showed few or no cerebral symptoms at a time at which control mice were clearly affected, and greatly reduced haemorrhages, mononuclear infiltration and oedema when the CNS was examined. Similar, but more consistent, protective effects were seen after administration of BHA by repeated injections or in osmotic pumps. The combination of superoxide dismutase and catalase, coupled to polyethylene glycol, when administered intravenously also protected mice against death from cerebral complications. Permeability of the blood-brain barrier was monitored by the use of 1 25I-labelled bovine serum albumin, S Cr-labelled erythrocytes and the dye Evans blue, all of which are normally excluded from the CNS. Infected mice on control diet showed an increase in Evans blue staining and 1251 and 5'Cr retention in the CNS tissue itself. Feeding the diet containing BHA reduced these indices of increased blood-brain barrier permeability. In view of the potent radical scavenging activity of BHA in many other systems it is likely, but unproven, that this is its main role here. The protective effect of superoxide dismutase and catalase lends support to the idea that reactive oxygen species are involved in the pathology of experimental cerebral malaria. Keywords: cerebral malaria, reactive oxygen species; antioxidant
The mechanisms underlying the pathology of cerebral malaria, a serious complication of human infection with Plasmodium falciparum, have not been determined. Proposals have included involvement of the immune
system (Adam et al. I 98 I; Cohen & Lambert I982), release from the parasite or host gut of substances akin to endotoxin (Clark 1978; Usawattanakul et al. I985) and the sequestration of parasitized red cells (PRBC) in the
Correspondence: N.H. Hunt, Department of Pathology, Medical School, Sydney University, NSW 2006, Australia. 293
C.M. Thumwood et al. 294 cerebral microvasculature (Miller 1972; vitro (Emanuel & Lyaskovskaya I968) and in MacPherson et al. I985), leading to cerebral vivo (Dillard et al. I984; Cowden et al. I985), ischaemia and anoxia (White et al. I985; and the enzymes superoxide dismutase and Janota & Doshi 1979; Daroff et al. I967; catalase would modify the course of cerebral malaria in the mouse model. Maegraith & Fletcher I9 7I). Since opportunities to study the pathogenesis of human cerebral malaria are limited, Materials and methods various laboratories have turned to the mouse model of P. Berghei ANKA in A/J or Animals and parasites CBA mice (Rest & Wright 1979; Rest I982, and CBA/H mice of both sexes and 6 to 8 I983; Mackey et al., I980; Thumwood et al., A/J weeks of age were used in these studies. I988), which appears to be the best model Infections with the parasite Plasmodium bergfor the human condition, albeit imperfect. hei ANKA (from Australian Army Malaria Studies with these animal models (Wright Research Unit, Ingleburn, NSW and a second I968; Wright et al. 197I; Finley et al. I982, from Dr G. Grau, WHO Immunology isolate I983) have implied the need for an intact Centre, University of Research and Training immune system for development of cerebral were initiated by intraperitoneal Geneva) malaria. We have suggested that this PRBC obtained from the of injection 1o6-Io7 immune response might involve activated tail veins infected mice. of other phagocytes and that, by releasing toxic subconthe erythrocytes The percentage of stances (Grau et al. I986; Clark et al. I98I) cent) parasitaemia per taining parasites (the including reactive oxygen species (Clark & Hunt I983), these cells might be central to was monitored routinely throughout the both killing of malarial parasites by the host infections by making thin blood smears oftail and the pathology of the disease. Reactive vein blood and staining with Diff Quick oxygen species are known to be secreted (Australian Hospital Supply, Sydney, Ausfrom activated phagocytes (Badwey & Kar- tralia). novsky I980; Nathan et al. I985), have been shown to damage endothelial cells in Diets supplemented with BHA or vitamin E vitro (Sacks et al. I978; Freeman et al. I983) The animal feed pellets (Doust and Ravvidge, and in vivo (DelMaestro I982; Wei et al. Sydney, Australia) were ground to a coarse I985) and are capable of parasite killing in powder in a food grinder. BHA (75 g) was vitro (Wozencraft et al. I985). Thus, acti- dissolved in 750 ml of acetone and added to vation of phagocytes in the cerebral vascula- 925 g of the ground food. The acetone was ture might, in the rodent model, cause driven off under reduced pressure in a rotary oxidative damage to the endothelium and evaporator (Buchi) at 45sC and the BHAbreakdown of the blood-brain barrier. In food mixture was then added to a further animal models of brain trauma, the produc- 9 kg of ground food, mixed thoroughly, and tion of oxygen-derived free radicals as a pelleted. The control feed was treated in a consequence of arachidonic acid metabolism similar manner, omitting the BHA. Vitamin has been shown to be a direct cause of the E was incorporated into the diet in a similar observed abnormalities in the cerebral way at 650 iu/kg. The BHA, vitamin E and microvasculature (Demopoulos et al. 1980; control food were stored at 40C. Mice were Kontos I 98 5). changed to the test diets I-2 days (A/J) or The possibility that damage to endothelial 7 days (CBA) before infection. cells in malaria could arise from reactive forms of oxygen secreted by leucocytes led us Other routes of administration of BHA to investigate whether administration of BHA, a well recognized radical scavenger in For injection, BHA was dissolved in commer-
Antioxidants and murine cerebral malaria 295 cial olive oil and injected i.p. (22 mg/kg in 25 Histology Ml) four times daily at regular 6-h intervals Brain sections were processed as described from day 2 after infection through until day previously (Thumwood et al. I988). 14. Control animals received vehicle only at the appropriate times and amounts. Osmotic pumps (model 2002, Alzet, Palo Alto, CA, USA) were implanted intra-abdominally under Avertin (2% w/v 2,2,2-tribromethanol plus 2% w/w 2-methyl-2-butanol) anaesthesia (o.s ml/25 g body weight). These pumps release 0.5 ul/h for 14 days. BHA was dissolved in polyethylene glycol 200 (BDH, Poole, England; PEG). Trolox (Aldrich) was similarly dissolved and administered in other experiments.
Treatment with polyethylene glycol conjugated enzymes
Polyethylene glycol conjugated bovine catalase (PEG-CAT) and bovine superoxide dismutase (PEG-SOD) were obtained from two sources. In two experiments the already coupled drugs were purchased from Enzon (S. Plainfield, NJ, USA) and in the third experiment bovine superoxide dismutase and catalase (both Sigma MO, USA) were coupled to methoxypolyethylene glycol (MW 5000, activated with cyanuric chloride, Sigma) using the method described by Abuchowski et al. (I977). Each CBA mouse received 6oo IU/day i.v., from the day of infection, of each of the coupled enzymes in a mixture diluted with saline. Control mice were given bovine serum albumin coupled to polyethylene glycol. Monitoring blood-brain barrier permeability Permeability of the blood-brain barrier was determined by three different methods, which are described in detail elsewhere (Thumwood et al. I988): injection of Evans blue, with subsequent monitoring of brain colour, or 1251-bovine albumin plus 51Crlabelled mouse erythrocytes with subsequent sampling of brain tissue or cerebrospinal fluid (CSF).
Statistics Data were analysed with the Blaker (I987) program, allowing for assessment by parametric and non-parametric tests as appropriate.
Results Time of death Infection of CBA/H (Finley et al. I982; Thumwood et al. I988) or A/J (Rest I982; Mackey et al. I980; Thumwood et al. I988) mice, fed a normal diet, with P. berghei ANKA causes a biphasic pattern of mortality. Approximately 85-95% of the animals die 7-9 days after a standard inoculum of parasites and exhibit cerebral involvement: neurological symptoms, increased permeability of the blood-brain barrier and mononuclear cell infiltration into the brain. Parasitaemias rarely exceed 25% at the time of death and are often lower than io% (Thumwood et al. I988). The minority of mice which survive this period die later, around days 20-24, with high parasitaemias and extensive haemolysis but no obvious cerebral pathology. CBA/H and A/J mice fed the control diet mostly died by day 9 (Fig. i), as expected from previous work (Thumwood et al. I 988). However, the great majority of the infected A/J mice fed the BHA supplemented diet did not die until 20-24 days after infection (Fig. i), and the comparable CBA mice mostly died after day 25. Neurological signs were not observed, and death may have been a consequence of chronic anaemia. BHA intake slightly delayed the progressive increase in parasitaemia in A/J (Fig. 2a) and CBA (not shown) mice. Both strains of mice were subject to a slight reduction of body weight in response to the diet (Fig. 2b for A/J,
C.M. Thumwood et al.
296
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Fig. ii. Both A/J mice (a, n 20) and CBA mice (b, n -, the diet containing BHA sur2) fed, vived the early mortality seen in infected mice fed 5 0). 3 0, CBA: n -, the control diet (A/J: n =
=
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the BHA diet protection
6 2 4 8 Time post-infection (days)
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Fig. 2. (a) The rise in the percentage parasitaemia was slower in, * the BHA-fed A/J mice (n = 20) when compared to 0, mice on control diet (n=45). (b) BHA-fed A/J mice also showed an increase in weight-loss in the first 7 days of infection compared with mice on control diet. Symbols and number of animals as in (a). Points and vertical bars are mean ± s.e.m.
=
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death with cerebral involvement was delayed by a few days, or in some cases a substantial number of mice were not protected. Therefore two other routes of administration of BHA were tested. Repeated i.p. injections of BHA in A/J mice were able to delay, but not prevent, the onset of symptoms and death in infected A/J mice (Fig. 3a). BHA in osmotic pumps decreased both the number of mice showing cerebral symptoms and the number dying at the first phase ofthe infection (Fig. 3b). The rise in parasitaemia in the early stages of infection was delayed by about 24 h (Fig. 4a) in all mice bearing pumps. The presence of pumps had little influence on the rate of weight loss (Fig. 4b). In the CBA mice injected with the combination of PEG-CAT and PEG-SOD, nine out of I 5 (pooled data from three experiments) survived past day 9 of infection and showed
no cerebral symptoms, whereas all 1 5 of the BSA-PEG group were dead by this time. In A/J mice fed a vitamin E-enriched diet eight out of i 8 survived past day 9 after infection and three out of i 8 survived altogether the early phase of mortality, whereas I0 out of io animals fed control diet died before day 9. Administration of another antioxidant, trolox, at 200 sg/h via osmotic pumps to IO CBA mice increased to 40% their survival past day io post-infection. Gross anatomy and light microscopy Infected mice fed the control diet and showing cerebral symptoms had haemorrhages and oedema, as do animals fed the normal diet (Thumwood et al. I988), BHA administration greatly reduced the severity of these manifestations of the disease. Infected mice with cerebral symptoms showed mononuclear leucocyte infiltration (into brain tissue), extravasated erythrocytes
Antioxidants and murine cerebral malaria
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Fig. 3. BHA was administered by two routes other than in the diet. (a) --, BHA dissolved in olive oil and injected i.p. into A/J mice (n = io) delayed the vehicle alone mortality compared with (n = io). (b) In abdominally implanted osmotic pumps in CBA mice, BHA delayed mortality up to day I9 (at which time the mice were killed to remove the pump). No pump; -, pump + PEG + BHA; pump + PEG. For all three
297
diet showed varying degrees of staining by Evans blue of CNS tissue on days 7-9 after infection, when cerebral symptoms would be expected. The brains of infected mice fed the BHA diet were, at the same time-point after infection, much less strongly stained, indicating a considerably less permeable bloodbrain barrier. This was true even until 17 days after infection in some animals. The brains of these mice were never as intensely blue as were those, at 7-9 days after infection, from infected mice fed the control diet. The same was true when BHA was administered by injection or osmotic pumps. In an attempt to quantify blood-brain barrier permeability and erythrocyte retention in the CNS, 5 1Cr-RBC and 125I-BSA were injected intravenously together. The retention of 125I-BSA and 51Cr-RBC, and the increase in 125I-BSA in the CSF of infected A/J mice, were substantially less on the BHA diet (Table ia), though these effects were less marked in CBA mice (Table ib).
-
-,
-,
- -
-,
groups, n = 1 3.
and microhaemorrhages (Fig. sa and Thumwood et al. I988). In mice treated with BHA, far fewer, if any, haemorrhages were observed (Fig. 5b) and there was far less infiltration of mononuclear leucocytes. Figure 5 shows results from A/J mice, but similar observations were made in CBA mice.
Permeability of the blood-brain barrier Uninfected mice, both CBA/H and A/J, excluded intravenously administered Evans blue from CSF and central nervous system (CNS), whether fed normal, control or BHA diet. In contrast, infected mice fed the control
Discussion In this study we have shown that administration of BHA by any of three routes can protect A/J or CBA/H mice against cerebral malaria caused by P. berghei ANKA infection. BHA prevented, reduced or delayed early mortality and breakdown of the blood-brain barrier without killing the parasite itself. Since this phenolic compound is a resonance-stabilized free radical scavenger (Emanuel & Lyaskovskaya I968), this result is consistent with our proposal that oxygenderived free radicals could have an essential role at some stage in the pathogenesis of this condition. This conclusion is substantiated by the observed protective effect of PEG-SOD plus PEG-CAT. Superoxide dismutase is generally considered to be specific for one substrate, 02 , and catalase for H202. Administration of the BHA diet caused a small reduction in body weight, which may have been a consequence of reduced food intake. This conceivably could have led to a reduction in parasite growth because of a
298
C.M. Thumwood et al.
0
40
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30
.
XL 20 CU
10
-
25
._0 o
mn
22
Time post-infection (days)
Fig. 4. (a) Insertion of pumps, whether 0, containing PEG only or *, BHA + PEG, slowed the progression of the parasite compared to its growth in 0, CBA mice with no pumps (n = I 3 in each case). (b) Mice with pumps containing PEG lost weight at a similar rate to mice bearing pumps ifiled with BHA and mice with no pumps. Symbols and numbers of animals as in (a). Points and vertical bars are mean ± s.e.m.
decrease in available nutrients. However, pumps containing PEG alone induced a comparable weight loss, despite which this treatment did not prevent the cerebral involvement. In separate experiments (Thumwood I987) dietary restriction was found to inhibit the development of murine cerebral malaria, but a substantially greater degree of weight loss was required to be effective. We have previously (Thumwood et a]. I988) discussed the similarities and differences between this mouse model of cerebral malaria and the human disease. The major difference is that autopsy material from humans shows extensive sequestration of parasitized RBC in cerebral venules (Thomas
197I), which is not a feature of the murine model. The attachment of P. falciparum infected RBC to human endothelial cells, in vitro at least, depends on the presence of parasite-induced 'knobs' on the erythrocyte surface (Udeinya et aL. I98I; Leech et al. I984). However, as discussed elsewhere (Thumwood et al. I988; Clark I987), this factor alone cannot explain the aetiology of human cerebral malaria. In several other respects, the mouse model parallels reasonably closely the human condition (Rest I982; Mackey et al. I980; Thumwood et al. I988). The movement of albumin and erythrocytes into the brain during the period in which neurological symptoms and mortality
Antioxidants and murine cerebral malaria
299
Fig. 5. Light microscopy sections of cerebellum from P. berghei ANKA infected A/J mice on day 8 after inoculation. (a) Haemorrhages (H) and mononuclear cell infiltration (-.) were prominent in infected mice consuming the normal control diet. x i6o. (b) Some mononuclear cell infiltration and very few, if any, haemorrhages were observed in infected mice on the BHA-supplemented diet. x i6o.
C.M. Thumwood et al.
300
Table i. The retention of radioactivity in parts ofthe CNS ofinfected A/J (a) or CBA (b) mice on control diet (CD) and BHA diet when injected with 125 I-BSA and 51CrRBC on day 8 of infection. Values are mean i s.e.m. of the ratio of radioactivity in samples from infected animals to that in samples from uninfected animals. For tissues, values are derived from whole anatomical areas. For CSF, values were expressed per unit volume before calculation of ratios. In (c) actual radioactivity in samples from control, uninfected mice eating CD are given as mean i s.e.m.
Olfactory lobe
Cerebrum
Cerebellum
Brain stem
CSF
(a) Retention of radioactivity (infected/uninfected ratio) for A/J mice CD (n = I9) 1251
8±I
i8+2
I3+1
i6+3
51Cr
7±1
1 ±2
I0±2
I2+±3
BHA (n = I9) 125i
4±1
6±i
541
4±1
51Cr
3 I
3± I
3± I
3± I
79±26
8+i
(b) Retention of radioactivity (infected/uninfected ratio) for CBA mice CD (n= 8) 1251
14±3
I3±3
10±3
8±2
*
5
Cr BHA (n=8) 125i
i8±4
7±2
9+2
6±2
*
14±3
9+2
9+2
II ±2
3± 1
3+ 1
7±2 3± I
i6±5
51Cr
(c) Radioactivity retained (c.p.m. or c.p.m./ul for CSF)
A/J (n = io)
0125i
51Cr CBA (n = 5)
i85t5 32±3
1251
8i±i6
51Cr
I5±6
702±85 88+20
257+57
227±25
36±io
35±9
2±I 0
567±3I I48+30
I43±25 41+Il
I57±2I
2±1
46±I5
0
*No CSF obtainable. are evident suggests that breakdown of the blood-brain barrier is an important event in P. berghei ANKA-induced cerebral malaria. Indeed, electron microscopy showed disruption of the endothelial layer in the cerebral microvasculature (Thumwood et al. I988). Mononuclear cell infiltration was marked during the progression of the pathological changes, suggesting the involvement of an inflammatory process (Thumwood et al. I988). Others have shown that a hostmediated immune response (Wright I968;
Wright etal. 197I;Finleyetal. I982, I983), involving particularly the L3T4 + subset of T lymphocytes (Grau et al. I986), is involved in the aetiology of the cerebral lesions in this mouse model of cerebral malaria. T cells produce cytokines, for example IFN-y, which can enhance the activities, including oxygen radical generation, of mononuclear phagocytes (Nathan et al. I983; Nathan I987). Antibodies to the monocyte/macrophage-derived tumour necrosis factor/cachectin (TNF) prevent
Antioxidants and murine cerebral malaria murine cerebral malaria (Grau et al. I987). TNF has a wide spectrum of activities (Le & Vilcek I987) which make it an important regulator of inflammation (Tracy et al. I986). These activities include the ability to promote adherence of phagocytes to endothelial cells (Gamble et al. I985), to damage endothelial cells directly (Stophen et al. I986; Sato et al. I986), and to potentiate the release of oxygen-derived free radicals by monocytes/macrophages (Hoffman & Weinberg I987). Recombinant TNF induces pathological changes in mice which closely parallel those seen during malaria (Clark et al. I987). Taken together these phenomena point to an inflammatory aetiology for cerebral malaria, in the murine model at least. We have argued that phagocyte-derived reactive oxygen species are involved in the immunopathology of malaria (Clark et al. I986), and the prevention of cerebral malaria by the free-radical scavenger BHA and the enzymes superoxide dismutase and catalase is consistent with this idea. Administration of two other antioxidants, vitamin E and trolox, partially protected against cerebral malaria. There are other ways in which BHA could influence the development of murine cerebral malaria. Agents which scavenge oxygen radicals, such as BHA, inhibit T lymphocyte proliferation in vitro (Chaudhri et al. I986, I988) and may also do so in vivo; this would be consistent with the reduction in mononuclear infiltration seen in the mice treated with BHA. In-vivo administration of BHA at the doses employed in the present study, however, does not appear to inhibit the subsequent in-vitro proliferation of lymphocytes isolated from the treated animals, nor to affect the phenotypic distribution of lymphocytes in lymph nodes (manuscript in preparation). Also, PEG-coupled superoxide dismutase and catalase provide some protection against murine cerebral malaria but do not inhibit lymphocyte proliferation in vitro (Hunt, unpublished observation). The precise mode of action of BHA in this system needs further investigation, but the
30I
results suggest a role for free radicals in murine cerebral malaria. This has important implications for our understanding of the pathology of this disease. Acknowledgements We thank Ms F. Ferguson for her expert secretarial skills in preparing this manuscript. This investigation received the financial support of the UNDP/World Bank/WHO Special Programme for Research Training in Tropical Diseases, the National Health and Medical Research Council of Australia and an ANU postgraduate scholarship (to CMT).
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