Chronic sodium salicylate treatment exacerbates brain neurodegeneration in rats infected with Trypanosoma brucei

Neurodegeneration by aspirin in trypanosome-infected rats

Pergamon PII: S0306-4522(99)00492-3

Neuroscience Vol. 96, No. 1, pp. 181–194, 2000 181 IBRO Published by Elsevier Science Ltd Printed in Great Britain

www.elsevier.com/locate/neuroscience

CHRONIC SODIUM SALICYLATE TREATMENT EXACERBATES BRAIN NEURODEGENERATION IN RATS INFECTED WITH TRYPANOSOMA BRUCEI N. QUAN,*† J. D. M. MHLANGA,‡ M. B. WHITESIDE,† K. KRISTENSSON‡ and M. HERKENHAM†§ †Section on Functional Neuroanatomy, National Institute of Mental Health, Building 36, Room 2D15, Bethesda, MD 20892-4070, U.S.A. ‡Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden

Abstract—We have reported previously that axonal degeneration in specific brain regions occurs in rats infected with the parasite Trypanosoma brucei. These degenerative changes occur in spatiotemporal association with over-expression of pro-inflammatory cytokine messenger RNAs in the brain. To test how aspirin-like anti-inflammatory drugs might alter the disease process, we fed trypanosome-infected rats with 200 mg/kg of sodium salicylate (the first metabolite of aspirin) daily in their drinking water. Sodium salicylate treatment in uninfected rats did not cause any neural damage. However, sodium salicylate treatment greatly exacerbated neurodegeneration in trypanosome-infected rats, resulting in extensive terminal and neuronal cell body degeneration in the cortex, hippocampus, striatum, thalamus, and anterior olfactory nucleus. The exaggerated neurodegeneration, which occurred in late stages of infection, was temporally and somewhat spatially associated with a late-appearing enhancement of messenger RNA expression of interleukin-1b, interleukin-1b converting enzyme, tumor necrosis factor-a, and inhibitory factor kBa in the brain parenchyma. Restricted areas showed elevations in messenger RNA expression of interleukin-1 receptor antagonist, interleukin-6, inducible nitric oxide synthase, interferon-g, and inducible cyclooxygenase. The association suggests that increased production of pro-inflammatory cytokines in the brain may be an underlying mechanism for neural damage induced by the chronic sodium salicylate treatment. Furthermore, the results reveal a serious complication in using aspirin-like drugs for the treatment of trypanosome infection. Published by Elsevier Science Ltd. Key words: tumor necrosis factor, interleukin-1, neurotoxicity, trypanosome, parasite, aspirin.

Aspirin and its metabolite sodium salicylate have been shown to potently inhibit prostaglandin synthesis by covalently binding cyclooxygenase. 28 More recently, they have been shown to be blockers of the activation of nuclear factor-kB (NFkB). 23,33 NF-kB is a transcription factor that is normally present in the cytoplasm in a dormant form complexed with inhibitory factor kB (IkB). Upon extracellular stimulation by inflammatory cytokines and other molecules, IkB is phosphorylated and degraded. This leads to the release of free NF-kB, which translocates to the nucleus to initiate transcription of immune genes coding for many immunoreceptors and cytokines, including interleukin-1b (IL-1b) and tumor necrosis factor-a (TNF-a). 29 Aspirin and sodium salicylate block the phosphorylation and degradation of IkB, thereby inhibiting NF-kB-initiated transcription 23 of inflammatory cytokines. This new-found activity of aspirin and sodium salicylate may be useful for treating certain neurodegenerative conditions because over-expression of inflammatory cytokines (many of them can be activated by NF-kB) has been implicated in the pathogenesis of neurodegenerative diseases such as Alzheimer’s disease and AIDS dementia. 16,39,52 Indeed, epidemiological data have shown that chronic intake of high-doses (up to 5 g/day) of aspirin is negatively correlated with the incidence of Alzheimer’s disease. 26 In addition, a

recent in vitro study showed that sodium salicylate protects neurons from excitotoxin-induced neuronal death by blocking NF-kB activation. 17 The effects of sodium salicylate treatment in an in vivo model of inflammatory neurodegeneration, however, remain to be determined. Infection by the trypanosome parasite in humans causes African trypanosomiasis—sleeping sickness—a severe inflammatory disease with involvement of the brain. Recently, we found that infection with the rodent pathogenic subspecies of the parasite Trypanosoma brucei brucei (T.b. brucei) in rats causes chronic over-expression of IL-1b and TNF-a mRNAs in the brain without significant parasitic invasion of the brain parenchyma. 35 In parallel, selective degeneration of certain nerve fibers in the vagus, lateral olfactory tract, and several other white matter tracts was found without evidence of neuronal cell body degeneration or neuronal apoptosis. 35 Therefore, trypanosomiasis in rats may represent a unique in vivo model of pro-inflammatory cytokine-induced neurodegeneration. If so, chronic treatment with sodium salicylate in trypanosome-infected rats should significantly alter the patterns of neurodegeneration. In this study, we investigated the effects of chronic treatment of sodium salicylate on neurodegeneration and cytokine mRNA expression in trypanosome-infected rats.

§To whom correspondence should be addressed. *Present address: Department of Oral Biology, 305 West 12th Avenue, Ohio State University, Columbus, OH 43210, U.S.A. Abbreviations: COX-2, inducible cyclooxygenase; GFAP, glial fibrillary acidic protein; H and E, Hematoxylin and Eosin; ICE, interleukin-1b converting enzyme; IFNg, interferon-g; IkBa, inhibitory factor kBa; IL1b, interleukin-1b; IL-1ra, interleukin-1 receptor antagonist; iNOS, inducible nitric oxide synthase; NF-kB, nuclear factor kB; PBS, phosphate-buffered saline; p.i., post-infection; SSC, standard saline citrate; T.b. brucei, Trypanosoma brucei brucei; TNF-a, tumor necrosis factor-a.

EXPERIMENTAL PROCEDURES

Animals and trypanosome infection Seventy-two male, young adult Sprague–Dawley rats (145–155 g, obtained from B and K Universal AB, Sollentuna, Sweden) were used in this study. They were kept on a 12-h light and 12-h dark schedule throughout the course of the experiments. The animals had continuous access to food and water. They were separated into four groups: group 1 was untreated and uninfected; group 2 was given 200 mg/kg of sodium salicylate daily in their drinking water (ss); group 3 was 181

182

N. Quan et al.

infected with T.b. brucei without sodium salicylate treatment (tryp); and group 4 was infected with T.b. brucei and received the sodium salicylate treatment (tryp 1 ss). The trypanosome infection was done by intraperitoneal (i.p.) injection of pleomorphic T.b. brucei (AnTat 1/1, derived from stabilate EATRO 1125, Laboratory of Serology, Institute of Tropical Medicine `ıPrins Leopoldıˆ, Antwerpen). Approximately 30,000 trypanosomes were injected in each rat. Parasites in the blood, taken from the tip of the tail, were counted in all animals on day 8 post-infection (p.i.) and on brain collection days. 18 The rats were killed on days 22, and 41 p.i. for in situ hybridization study (n ˆ 4 in each group) and on days 36, 43, 51, and 56 p.i. for silver staining and immunohistochemistry (n ˆ 2–6 in each group). The animal experiments were approved by the Stockhomes Norra Djurforsoksetiska Namnd. All efforts were made to minimize the number of animals used and their suffering. Silver stain and immunocytochemical labeling of glial fibrillary acidic protein and immunoglobulin G Animals were deeply anesthetized and perfused through the heart with a saline and dextrose/sucrose buffer followed by 4% paraformaldehyde in phosphate buffer. The brains preserved in 4% formal saline were then processed for silver staining, immunohistochemistry, and hematoxylin and eosin (H and E) staining by NeuroScience Associates (Knoxville, Tennessee, U.S.A.). Brains were batch-processed: embedded brains (16/block) were cut into 40 mm-thick coronal sections, and equally spaced series from olfactory bulb to caudal medulla were processed. The silver stain for degeneration was a modified cupric silver stain method applied to one series of sections from all of the brains. 8 To monitor activation of astrocytes, another selected series was stained with an antibody against glial fibrillary acidic protein (GFAP; ICN Biomedical, Aurora, OH, U.S.A.) using conventional immunocytochemical procedures. Briefly, free-floating sections were fixed in 4% formaldehyde for 15 min and rinsed three times in phosphate-buffered saline (PBS); they were then incubated with the anti-GFAP (1:400) for 2 h. After three rinses in PBS, sections were incubated with the biotinylated secondary antibody (goat anti-mouse IgG, 1:200) for 1 h, and antibody bindings were visualized by the conventional avidin–biotin immunoperoxidase protocol. To assess breakdown of the blood–brain barrier, another series of sections was processed for immunoglobulin (IgG) immunohistochemistry according to published protocols. 40 Briefly, sections were stained free-floating. After treatment with hydrogen peroxide and blocking serum, the sections were immunostained for 1 h with a goat anti-rat biotinylated antibody (1:1000) (the 2nd component of the VectaStain ABC Kit, Vector, Burlingame, CA, U.S.A.). Final visualization of immunoreactivity was accomplished following the VectaStain protocol. Sections were then treated with diaminobenzidine tetrahydrochloride and mounted on gelatinized glass slides. In situ hybridization histochemistry For in situ hybridization, brains were removed immediately after decapitation, frozen by immersion in 2-methylbutane at 2408C, and stored at 2708C prior to sectioning. They were then cryostat-cut to 15 mm-thick coronal sections and thaw-mounted onto gelatin-coated slides, dried, and stored at 2358C until further processing. Brain sections were collected at the following levels: organum vasculosum of the lamina terminalis (20.02 mm relative to bregma); subfornical organ (20.92 mm); paraventricular hypothalamic nucleus (21.80 mm); central nucleus of amygdala containing also the arcuate nucleus and median eminence (23.3 mm); and area postrema containing the nucleus of the solitary tract (213.7 mm). The in situ hybridization protocols were performed as described previously for ribonucleotide (cRNA) probes. 37 First, tissue sections were processed by fixation with 4% formaldehyde solution, acetylation with 0.25% acetic anhydride in 0.1 M triethanolamine–HCl, pH 8.0 solution, dehydration with ethanol, and delipidation with chloroform. cDNAs comprising the rat IkBa (generously provided by Dr Rebecca Taub, University of Pennsylvania, U.S.A.), IL-1b and IL1ra (generously provided by Dr Ronald Hart, State University of New Jersey, U.S.A.), TNF-a (gift from Dr Karl Decker, AlbertLudwigs-Universita¨t, Freiburg, Germany), interleukin-1b converting enzyme (ICE) (generously provided by Dr Brenda Shivers, ParkeDavis, Ann Arbor, MI, U.S.A.), inducible nitric oxide synthase (iNOS; generously provided by Dr Elena Galea, Cornell University,

NY, U.S.A.), and inducible cyclooxygenase (COX-2; generously provided by Dr Daniel Hwang, Pennington Biomed. Res. Center, LA State University, U.S.A.) sequences were obtained in plasmid form. The rIL-6 (231 nucleotides) and r-interferon-g (rIFNg; (158 nucleotides) cDNA templates, capped with T7 RNA polymerase transcription sites, were purchased as components of the RiboQuant RPA Starter Package, PharMingen San Diego, CA, U.S.A. cDNAs were transcribed using the Riboprobe System (Promega Biotech, Madison, WI, U.S.A.) with T7, T3 or SP6 RNA polymerase and a- 35S-UTP (specific activity .1000 Ci/mmol; New England Nuclear, Boston, MA, U.S.A.). The radiolabeled probes were then diluted in the riboprobe hybridization buffer and applied to brain sections (500,000 c.p.m./section). After overnight incubation at 558C, slides containing brain sections were washed first in 20 mg/ml RNase solution and then in 2 × standard saline citrate (SSC) and 0.2 × SSC (55 and 608C) solutions. The slides were then dehydrated with ethanol and air-dried for autoradiography. Specificity of the ribonucleotide probes was determined previously on the basis of appropriate distribution patterns following acute or chronic immune challenges. 35–38 For all probes, sense probes gave no specific signal (data not shown). Slides and 14C plastic standards containing known amounts of radioactivity (American Radiochemicals, St Louis, MO, U.S.A.) were placed in X-ray cassettes, apposed to film (BioMax MR, Kodak, Rochester, NY, U.S.A.) for four days, and developed. To determine anatomical localization of hybridized probes at the cellular level, sections were dipped in nuclear track emulsion (NTB-2, Kodak), exposed for two weeks, developed (D19, Kodak), and counterstained with Cresyl Violet. Autoradiographic film images of brain sections and standards were digitized on a Macintosh computer-based image analysis system with IMAGE software (Wayne Rasband, National Institute of Mental Health). Light transmittance through the film was measured by outlining the structure on the TV monitor. Transmittance measurements were converted to d.p.m./mg plastic using the calibration curve (Rodbard equation) generated from the standards; in this manner the non-linear response characteristics of the film (i.e. saturation) are eliminated, and density is proportional to radioactivity. The relative density so obtained was used to represent the relative amount of mRNA expression of the labeled molecules. For five of the mRNAs quantified-IL-1ra, iNOS, COX-2, IL-6, and IFNg-calibrated area measurements were determined because increased expression appeared not as increased density of expression but rather as increased numbers of cells expressing message at fixed levels. The area measurement, determined with the aid of the density–slice function, reflected the increase in numbers of labeled cells. Statistical comparisons between groups were made using a three-way analysis of variance (infection × treatment × time). Subsequent ANOVAs were performed to identify the sources of the interactions, followed by use of the Tukey–Kramer post hoc test. RESULTS

We found previously that the loss of body weight in trypanosome-infected rats correlates temporally with mRNA expression of pro-inflammatory cytokines IL-1b and TNF-a and with the progression of terminal and axonal degeneration in the brain. 35 The mean body weights of the tryp vs tryp 1 ss groups are shown in Table 1. ANOVA analysis followed by a post hoc Tukey test revealed significant body weight loss in tryp 1 ss compared with tryp after day 36 p.i. Thus, sodium salicylate treatment accelerated the weight loss of trypanosome-infected rats, beginning at the later stages of infection. Plasma levels of parasite counts are shown in Table 1. Salicylate treatment significantly increased the parasite counts in the plasma on day 8 p.i. No significant differences in the parasite counts, however, were observed between tryp and tryp 1 ss at any of the other measured time-points (p.i. days 21, 36, 46, and 56). Thus, the sodium salicylate treatment did not alter the parasite load at the later stages of the infection. As we described previously, 35 in all infected animals at all survival times, the trypanosome parasites could be

Neurodegeneration by aspirin in trypanosome-infected rats Table 1. Body weights in g (mean ^ S.E.M.) tryp

tryp 1 ss

219 ^ 11 274 ^ 10 363 ^ 13 387 ^ 18 400 ^ 16 386 ^ 38

195 ^ 9 239 ^ 11 327 ^ 13 350 ^ 15* 333 ^ 16* 316 ^ 27*

Day 0 8 21 36 46 56

Log parasite counts/ml blood ^ S.E.M. Day tryp 8 5.26 ^ 0.04 21 7.64 ^ 0.03 36 8.06 ^ 0.15 46 7.54 ^ 0.13 56 7.50 ^ 0.11

tryp 1 ss 6.19 ^ 0.29* 7.38 ^ 0.04 7.94 ^ 0.09 7.90 ^ 0.07 7.62 ^ 0.17

*P , 0.05 compared to tryp.

microscopically visualized in the choroid plexus where they were lodged between the blood vessel walls and the choroidal epithelium. Qualitative analysis of their abundance did not indicate any obvious difference between tryp and tryp 1 ss animals at any p.i. survival time. Neurodegeneration Confirming our earlier observations, 35 the silver-stained material showed degeneration in the olfactory tract, vagus nerve, and scattered in various other tracts in tryp animals beginning on day 46 p.i. The appearance of the impregnated axons, i.e. as beaded, swollen, and varicose, conformed to the characteristics of genuine degenerating fibers. In some infected animals, beginning on day 46 p.i., apparent degeneration of astrocytes was observed in various locations (basal forebrain, preoptic area, or hypothalamus). Features of this kind of impregnation of star-shaped cells, conforming in appearance to astrocytes, were described previously in detail. 35 These general patterns of degeneration were observed in both tryp and tryp 1 ss animals (e.g., axonal degeneration in the fimbria and stria terminalis; Fig. 1f), although some degeneration appeared to be exacerbated by the salicylate treatment. For example, the lateral olfactory tract in several tryp 1 ss animals showed a marked increase in the density of degeneration (Fig. 1b). The apparent astrocyte degeneration was not noticeably different between infected groups (data not shown). In tryp 1 ss animals, additional patterns and types of neurodegeneration were observed that were never observed in tryp animals at any survival time (Figs 1–3). At the 36- and 42-day time points, no degeneration was observed in any of the tryp 1 ss animals. On day 46 p.i., the olfactory system was preferentially affected. At this and later time-points, degenerating (i.e. fragmented and beaded) axons were observed in the lateral olfactory tract (Fig. 1b) and anterior commissure (Fig. 2b). These had been observed also in tryp cases. Unlike tryp cases, however, impregnated nerve cell bodies and their dendrites were observed throughout the anterior olfactory nuclei (Fig. 1a) and piriform cortex (Fig. 1c), typically bilaterally. Completely filled cells may represent an early stage of reactivity that renders them argyrophilic. Presumably, these cells would later take on a more broken and fragmented appearance, though more exhaustive work would have to be done to confirm this presumption. Degenerating

183

debris was seen in the olfactory nerve and mitral layer of the olfactory bulb (not shown). An additional early degeneration pattern was the appearance of regions of cell and fiber degeneration in the caudate–putamen (Fig. 1d). Later appearing degeneration (at p.i. days 51 and thereafter) was seen in the cerebral cortex, usually in patches and sometimes in laminar patterns (Fig. 1e), and the hippocampus (Fig. 1g, h). Usually these were stronger on one side than the other. In the examples shown, the cortical degeneration appeared selective for cortical pyramidal neurons in layer III (Fig. 1e), and the hippocampal degeneration was heavy in the dentate gyrus granule and molecular layer (Fig. 1g, h); it was lightest in the field CA1 where H and E stains revealed that the pyramidal neurons were completely gone. At these times (days 51 and 56 p.i.) some degeneration patterns displayed remarkable correspondence to functional anatomical groupings. Examples are shown in Figs 2 and 3. In one case, degeneration was confined to the ventral striatum at that level (Fig. 2a), and within the accumbens nucleus, the regions previously identified as the cell islands 21 were largely free of degeneration. Areas that showed the dense terminal degeneration conformed to the zones of innervation of the midline thalamic nuclei. 21 In a second case of functional anatomical grouping of degeneration patterns, the zones of the thalamus previously categorized as non-specific thalamus with on the basis of widespread ascending projections primarily to layer I of neocortex 19,20 were selectively filled with degenerating neurons and terminals. Thus, the paracentral nucleus (Fig. 2c, f), which is part of the intralaminar complex, the ventrolateral (Fig. 2c, g) and ventromedial nuclei (Fig. 2c, h), which project primarily to layer I of widespread areas of neocortex, and the lateral posterior, suprageniculate, and magnocellular medial geniculate nuclei (Fig. 2e, i), which form the caudal end of the cortical layer-I projection system, were all filled with degenerating neurons. A final example of the localization of degeneration to functional compartments is the selective degeneration of components of the striatal matrix 14 in another case (Fig. 3). At low magnification, striatal degeneration was organized in a mosaic pattern (Fig. 3a). Cortical degeneration was a mixture of cells and fibers and organized in layers (Fig. 3a). Highmagnification microscopy confirmed that the degenerating terminals and cells were primarily in the striatal matrix compartment, leaving patches free of degenerating processes (Fig. 3a, b). Highly organized degeneration in the neocortex was apparent. Whereas dust-like degeneration was seen in cortical layers I–III and V, individual pyramidal neurons in layers III and upper V were fully silver-impregnated (Fig. 3c). No neurodegeneration was found in the ss group (Fig. 3a, inset) at this or any other level or at any time-point. Additional degeneration, not marked by silver stain but rather by appearance of necrosis and rarefaction, occurred only in the tryp 1 ss animals in very late stages of infection. In two animals, early organizing infarcts were seen in the cerebral cortex (a bilateral pseudo laminar necrosis; Fig. 4d) and thalamus. In the necrotic area, vascular thickening was prominent (Fig. 4e). In another animal, complete bilateral loss of CA1 hippocampal pyramidal cells was seen (Fig. 1g). Immunohistochemical staining for GFAP was quite variable from case to case and region to region, so no clear correlation could be made between infected and uninfected animals or between tryp and tryp 1 ss groups. However, in cases of late-stage infection in tryp 1 ss animals, an elevation

184

N. Quan et al.

Neurodegeneration by aspirin in trypanosome-infected rats

185

Fig. 2. Bright-field photomicrographs show patterns of selective neuronal degeneration according to patterns of “functional” neuroanatomical circuits that share neurochemical and/or connectional properties. In the first instance (a and b), degeneration is compartmentalized in the nucleus accumbens (Acb) and extends into the olfactory tubercle (Tu). The degeneration pattern resembles the pattern of terminal afferentation of axonal projections from midline thalamic nuclei (paratenial and paraventricular). In the thalamus (c–i), neuronal degeneration selectively marks components of the non-specific thalamus that share anatomical properties such as widespread projections to layer I of cortex and projections to the striatal matrix. High-magnification views in (f–i) are located by boxes in (c–e). They are the paracentral intralaminar nucleus (PC) in (f), the ventromedial nucleus (VM) in (g), the parafascicular nucleus (Pf) in (h), and the magnocellular medial geniculate nucleus (MGm) in (i). ac, anterior commissure; LTP, lateral posterior nucleus; MD, mediodorsal nucleus; Po, posterior nucleus; SG, suprageniculate nucleus; VB, ventrobasal complex, VL, ventrolateral nucleus.

in staining intensity was seen. Interestingly, in the area of the cortical necrosis, the immunostaining abruptly ended (Fig. 4f). In situ hybridization We reported previously that induction of cytokines in the brain after trypanosome infection is characterized by two distinct patterns: an initial induction of cytokine mRNA at meninges and blood vessels between day 20 and day 36 p.i. and a later induction of cytokine mRNA in cells of the

brain parenchyma from day 41 p.i. 35 On the basis of that observation, survival times of 22 and 41 days p.i. were selected for examination in the present study. Induction of IL-b mRNA expression in the brain of tryp vs tryp 1 ss on days 22 and 41 is shown in Fig. 5. The patterns of mRNA induction for IkBa (Fig. 6) and TNF-a expression were similar to that of IL-1b. For these transcripts, induction by trypanosomes occurred early and strongly in the choroid plexus of the lateral ventricle (Figs 5, 6a, b). Elevated expression was typically found also in the regions surrounding circumventricular organs, notably the organum vasculosum of the lamina

Fig. 1. Representative bright-field microphotographs show extensive neurodegeneration in various brain regions/structures in sodium salicylate-treated trypanosome-infected (tryp 1 ss) rats. Components of the olfactory system show degeneration in (a–c), where neurons and dendrites are impregnated in the anterior olfactory nucleus (AON; a) and piriform cortex (pir; c), and broken axons are impregnated in the lateral olfactory tract (lo; b) on day 46 p.i. Degeneration in one case (51 day p.i.) in the caudate–putamen (CPu; d) is patchy and consists of filled striatal neuronal cell bodies and terminals (dust-like appearance in the inset; note also the rather sharp border, presumably at a patch–matrix interface). Box shows location of inset. Fiber fascicles passing through the CPu are unlabeled and appear as small holes. Degeneration in the cerebral cortex (e) appears as filled pyramidal cell bodies and beaded processes (46 day p.i.). Degeneration in two fiber tracts adjacent to the choroid plexus (Ch Plx) is seen in the fimbria (fi) and stria terminalis (st) in (f) (46 day p.i.). Late-appearing (56 day p.i.) degeneration in the hippocampus in one case is shown in (g) and (h). CA1–3, fields CA1–3 of Ammon’s horn; DG, dentate gyrus; ec, external capsule; gr, granule cell layer of the DG; hf, hippocampal fissure; mol, molecular layer of DG; SI Cx, somatosensory cortex. Scale bars: (a–b, e–f, h) ˆ 100 mm; (c) ˆ 200 mm; (d, g) ˆ 400 mm; (insets) ˆ 20 mm.

186

N. Quan et al.

Fig. 3. Bright-field color photographs show extensive but selective neurodegeneration in a tryp 1 ss rat at day 51 p.i. (a) Low-magnification photograph shows neurodegeneration at the level of caudate–putamen (CPu), nucleus accumbens (Acb), and anterior commissure (ac) in a tryp 1 ss rat. The patchy pattern of striatal degeneration matches the patch–matrix compartmentalization revealed by histochemical stains; the pattern indicates that the degeneration is confined to the matrix compartment. Inset in (a) is taken from a sodium salicylate-treated animal without trypanosome infection on the same day. High-magnification photomicrographs (b and c; located by boxes and a) show degeneration in CPu (b) and neocortex (Cx) layer III (c). Scale bars: (a) ˆ 1 mm; (b, c) ˆ 20 mm.

terminalis, median eminence, and area postrema (not shown). Transcript expression appeared to spread from these and meningeal locations into the brain. Thus, increased parenchymal expression, notable at 41 days p.i., occurred in regions surrounding the lateral ventricle (fimbria/ventral hippocampal commissure and stria terminalis/dorsal thalamus) and in the hypothalamus (Figs 5, 6d, e). Microscopically viewed emulsion-coated sections (data not shown) revealed that labeled cells were small and typically dark staining, representing nonneuronal cell type classes (see companion study 35). Labeled cells tended to occur in clusters, giving the blotchy appearance apparent in the film autoradiographs. The types of cells labeled did not appear to differ between the tryp and tryp 1 ss animals. Expression patterns varied from brain to brain, so that great variability of quantitative densitometry was encountered. Nevertheless ANOVA showed significant main effects for infection and time. Salicylate treatment produced a significant

interaction effect at the late time-point, reflecting that in a number of regions the treatment further increased expression levels in infected animals (there was no effect of salicylate on uninfected animals). This phenomenon was seen in the autoradiographs (Fig. 5c vs d and Fig. 6c vs d). Quantitative analysis of striatal expression of IL-1b, ICE, TNF-a, and IkBa mRNAs is shown in Table 2. A number of additional transcripts for cytokines, cytokinerelated molecules, and inflammatory factors were measured in the tissue by in situ hybridization. As reported previously for trypanosome-infected animals, 35 the transcripts for iNOS, IFNg, IL-6, and IL-1ra were selectively elevated in the choroid plexus of the lateral ventricle and closely adjacent areas, notably the fimbria (Fig. 7a, b). In uninfected animals, COX-2 mRNA was constitutively expressed in widespread areas, but in infected animals it was selectively induced in blood vessels throughout the brain (Fig. 7c). These transcript levels quantified in the diencephalon responded in an irregular but

Neurodegeneration by aspirin in trypanosome-infected rats

187

Fig. 4. Photomicrographs of Nissl- and H and E-stained neocortex and GFAP immunoreactivity in an uninfected animal (top, a–c) and a tryp 1 ss animal (bottom, d–f) with a cortical infarct (day 56 p.i.). Neuronal labeling seen at high magnification (b) is replaced in the infarct with non-neuronal cellular staining and proliferation of vascular endothelia (e; arrows point to blood vessels). At the edge of the infarct (f), GFAP-positive astrocytes are prominent, but the staining ends abruptly at the edge (infarct proper is at the top). Scale bars: (left) ˆ 100 mm; (center and right) ˆ 20 mm.

generally exaggerated manner to sodium salicylate—IL-1ra, iNOS, and COX-2 mRNAs showed significant elevations at 41 days p.i. in the tryp 1 ss condition relative to the tryp condition (Table 3). Salicylate-induced elevations were recorded in the choroid plexus, but the variability of effect across animals precluded achieving statistical significance. Induction of GFAP mRNA expression is shown in Fig. 8. Expression levels in the infected animals were typically about 200–500% of control at both time-points, but variability in expression intensity across the animals resulted in statistical significance only for tryp animals in the hypothalamus at both days 22 and 41 (data not shown). In general, constitutive GFAP mRNA expression in white matter-rich areas was observed in both uninfected and infected animals, but infected animals, regardless of salicylate treatment or not, showed additional large patches of elevated signals in thalamus, hypothalamus, or amygdala, either unilaterally or bilaterally. The locations and sizes of these patches of increased signal corresponded roughly to similarly localized patterns of neuronal or astrocyte degeneration seen in other sets of animals, and to cytokine expression patterns seen in adjacent sections from the same animals. For example, in one case (tryp 1 ss, 41 days p.i.), unilateral elevation in GFAP mRNA signal was seen in the hypothalamus, medial amygdala, and internal capsule (Fig. 8f). Adjacent sections hybridized for ICE (Fig. 8g), IL-1b (Fig. 8h), and TNF-a (Fig. 8i) mRNAs showed selective elevation of transcript levels in the same region. The cases also show areas of elevated GFAP mRNA expression

that do not have elevated cytokine mRNA expression, so the correspondence is not obligatory. Blood–brain barrier breakdown To test whether the trypanosome infection, the chronic sodium salicylate treatment, or the tryp 1 ss combination had any actions on opening of the blood–brain barrier, an immunohistochemical stain for immunoglobulin (IgG) was performed on selected sections of the perfusion-fixed material. In uninfected animals, only very faint staining of the choroid plexus (Fig. 9, inset) and circumventricular organs could be seen. In all infected animals, equally for both tryp and tryp 1 ss groups, elevated staining could be seen in the choroid plexus. In some animals, additional staining was observed in the ventricular ependyma (Fig. 9). In the animals with infarcts, the region surrounding the infarct also showed staining. DISCUSSION

Unexpectedly, chronic treatment with sodium salicylate (at a dose that is compatible with high-dose aspirin treatment in humans 9) failed to ameliorate neurodegeneration in T.b. brucei-infected rats as predicted, and instead it generated additional extensive neural damage in infected rats. Thus, whereas trypanosome infection alone induced limited axonal degeneration of certain fiber tracts such as the vagus, the

188

N. Quan et al.

Fig. 5. Autoradiographs show induction of IL-1b, mRNA in tryp and tryp 1 ss rats on p.i. days 22 (a and b) and 41 (c and d) at the level of the subfornical organ. Scale bars ˆ 2 mm. cc, corpus callosum; Ch Plx, choroid plexus; fi, fimbria; hypo, hypothalamus; st, stria terminalis; thal, thalamus.

lateral olfactory tract, and subcortical white matter, 35 chronic sodium salicylate treatment did not alter this pattern and additionally elicited extensive neuronal cell body degeneration in cerebral cortex, corpus striatum, hippocampus, diencephalon, and certain olfactory structures. The salicylate treatment also produced a significant drop in body weight at the later stages of infection relative to the tryp animals. This kind of deleterious effect of salicylate has been reported previously in other animal models of infection. 51,53,54 As previously reported, 42 we did not find evidence of trypanosome parasites in the brain parenchyma at any post-infection time-point (data not shown). We also did not observe evidence of parenchymal reactions such as perivascular cuffing or widespread meningitis, though we did observe proliferative reactions in the choroid plexus where the parasites are lodged, in the ventricular ependyma, and variably in blood vessels. We propose therefore that the observed neural damage may have been caused by diffusable toxic substances produced within the brain at the later stages of infection. The observed neurodegeneration in tryp 1 ss animals showed remarkable selectivity. Often the degeneration was bilaterally symmetrical. Occasionally, functional neuroanatomical circuits appeared to be targeted for degeneration. For example, the olfactory system consisting of components of the olfactory bulb, the anterior olfactory nuclei, the lateral olfactory tract, and piriform cortex were targets in many tryp 1 ss animals. In several animals the patch–matrix system

of the striatum was selectively targeted: terminal degeneration formed matrix-like patterns, leaving patches unaffected. In one of these cases, neuronal degeneration in the overlying neocortex was restricted to layer III and superficial layer V neurons, apparently constituting the cortical component of the degenerating cortex-to-matrix projection system (Fig. 3). It has been shown that the striatal patches marked by m opiate receptors receive cortical projections from deep layer V and layer VI neurons, whereas the surrounding acetylcholinesterase-rich matrix receives projections from more superficial layers of the cortex. 14 In the thalamus, projection nuclei can be designated as specific or non-specific, depending on the patterns of cortical connectivity. 20 In one tryp 1 ss case (Fig. 2), components of the non-specific thalamus showed selective neurodegeneration. Neurons from these regions form widespread projections to striatum (from the paratenial, paraventricular, and parafascicular thalamic nuclei) and cortical layer I (from the paracentral, ventrolateral, ventromedial, magnocellular medial geniculate, and suprageniculate nuclei). It is possible that these neurons are selectively vulnerable because their widespread projections renders them more accessible by toxins present in the interstitial fluid of the brain. Another possible mechanism for the manifestation of selective neural damage is the flow pattern of neurotoxins. Molecules flowing in the cerebrospinal fluid of the ventricles and subarachnoid spaces might have access to the cortical surface,

Neurodegeneration by aspirin in trypanosome-infected rats

189

Fig. 6. Autoradiographs show induction of IkBa mRNA in tryp and tryp 1 ss rats on p.i. days 22 (b and c) and 41 (d and e) at the level of the hypothalamic paraventricular nucleus. Constitutive expression in an uninfected animal is shown in (a). Scale bars ˆ 2 mm.

just like they may enter structures adjacent to the lateral ventricles where the parasites are predominantly lodged. Cerebrospinal fluid flow patterns along the ventral surface of the brain 34 may allow access to the hypothalamus, preoptic area, basal forebrain, and ventral striatum—all sites of degeneration observed in this study. In a rather general fashion, sites of neuronal degeneration in tryp 1 ss animals were associated with locations of elevated mRNA expression for the cytokines and inflammatory molecules evaluated in this study. Thus, degeneration appearing in the white matter tracts close to the lateral

ventricles, the subcortical white matter, striatal systems, and olfactory and preoptic/hypothalamic areas showed proximity to elevated pro-inflammatory cytokine mRNA expression in those white matter tracts and more diffusely throughout the diencephalon. Conversely, the lower brainstem showed little evidence of degeneration or cytokine mRNA induction. Importantly, both the exacerbated degeneration and the elevated IL-1b, ICE, TNF-a, IL-1ra, iNOS, and COX-2 mRNA expression levels in cells of the brain parenchyma were seen in the tryp 1 ss animals at similar points of the infection, i.e. at later stages of the trypanosome infection,

Fig. 7. Autoradiographs show induction of iNOS (a), IL-1ra (b), and COX-2 (c) mRNAs in tryp 1 ss rats on p.i. day 41 at the level of the subfornical organ. Arrow in (c) points to a labeled blood vessel. Scale bars ˆ 2 mm. Ch Plx, choroid plexus; PVN, paraventricular hypothalamic nucleus.

190

N. Quan et al.

Table 2. Increases in hybridization density (means ^ S.E.M., calculated as percent of control) measured in designated brain areas at two post-infection survival times IL-1 Level Str/BF Choroid plexus Fimbria Thalamus Medial hypothalamus Lateral hypothalamus Paraventricular nucleus Arcuate Meninges TNF-a Str/BF Choroid plexus Fimbria Thalamus Medial hypothalamus Lateral hypothalamus Paraventricular nucleus Arcuate Meninges Ik-B Str/BF Choroid plexus Fimbria Thalamus Medial hypothalamus Lateral hypothalamus Paraventricular nucleus Arcuate Meninges ICE Choroid plexus Fimbria Thalamus Medial hypothalamus Lateral hypothalamus Paraventricular nucleus Arcuate Meninges

Day 22

tryp 1 ss

tryp 154 ^ 18 1338 ^ 270 142 ^ 15 116 ^ 2 188 ^ 8** 125 ^ 17 146 ^ 20 318 ^ 19** 1024 ^ 134

125 ^ 7 872 ^ 383 126 ^ 3 106 ^ 3 176 ^ 13** 132 ^ 15 176 ^ 26* 370 ^ 17** 743 ^ 62 tryp 1 ss

tryp 204 ^ 16**,*** 7878 ^ 2206 107 ^ 7 115 ^ 8 150 ^ 12** 130 ^ 7* 114 ^ 10 193 ^ 31** 3151 ^ 808

133 ^ 14 8994 ^ 3984 118 ^ 13 113 ^ 6 122 ^ 3 128 ^ 8* 113 ^ 13 158 ^ 11 978 ^ 172

tryp 139 ^ 13 626 ^ 59* 112 ^ 9 103 ^ 11 181 ^ 15** 127 ^ 19 79 ^ 16 399 ^ 31**,**** 324 ^ 57** tryp 4471 ^ 3985 208 ^ 35 177 ^ 28 267 ^ 42** 240 ^ 59 244 ^ 44** 475 ^ 65** 3069 ^ 964*

Day 41 tryp 126 ^ 8 1573 ^ 506 277 ^ 56* 109 ^ 10 136 ^ 11 109 ^ 9 114 ^ 10 281 ^ 30** 970 ^ 88 tryp 130 ^ 10 4431 ^ 1336 158 ^ 21 126 ^ 10 132 ^ 11 117 ^ 8 140 ^ 13 125 ^ 14 1369 ^ 352*

tryp 1 ss

tryp

124 ^ 28 366 ^ 87** 127 ^ 15 107 ^ 10 133 ^ 9 133 ^ 11 122 ^ 14 228 ^ 14* 266 ^ 14

214 ^ 6* 782 ^ 98** 241 ^ 30** 144 ^ 15 131 ^ 14 140 ^ 14 133 ^ 21 198 ^ 37 196 ^ 36

tryp 1 ss

tryp

4447 ^ 1036 176 ^ 9 143 ^ 3 235 ^ 8* 178 ^ 17 220 ^ 19* 362 ^ 62** 1518 ^ 165

4310 ^ 1831 186 ^ 33 115 ^ 9 181 ^ 10* 150 ^ 5 148 ^ 9 242 ^ 18 1451 ^ 63

tryp 1 ss 215 ^ 45**,*** 1875 ^ 119** 458 ^ 38**,**** 163 ^ 25**,*** 161 ^ 13** 147 ^ 10* 140 ^ 8 275 ^ 65** 1269 ^ 229 tryp 1 ss 204 ^ 20**,**** 7508 ^ 1277* 197 ^ 33** 134 ^ 16 140 ^ 13 119 ^ 7 130 ^ 9 109 ^ 15 2358 ^ 699 tryp 1 ss 267 ^ 53** 1055 ^ 255* 325 ^ 31** 186 ^ 40* 166 ^ 10** 163 ^ 29 134 ^ 9 214 ^ 30* 279 ^ 77 tryp 1 ss 7678 ^ 1297 319 ^ 35**,**** 177 ^ 27**,*** 199 ^ 18** 183 ^ 10* 183 ^ 23* 242 ^ 43 1362 ^ 558

Values vs control *P , 0.05 and **P , 0.01. Values vs treatment ***P , 0.05 and ****P , 0.01. Str/BF, striatum and basal forebrain at rostral striatal level. n ˆ 4 animals/group.

with elevated mRNA expression preceding the appearance of degeneration by one to two weeks. The surprising finding that chronic sodium salicylate treatment resulted in exacerbated degeneration and elevated mRNA expression may be explained by several possible mechanisms: (i) sodium salicylate treatment might have worsened the course of infection by increasing the rate of trypanosome proliferation, perhaps by suppressing the immune response to the parasites; (ii) it may have compromised the integrity of the blood–brain barrier to allow higher levels of parasites or toxic molecules into the brain; 32 (iii) it might have altered NF-kB-mediated events; and (iv) it might have inhibited the synthesis of prostaglandins, which themselves can inhibit certain immune responses. 28 These mechanisms will be discussed below. Although we did find a significant increase in parasite

counts in the plasma on day 8 p.i. in rats treated with sodium salicylate, the parasite counts were not different thereafter between tryp and tryp 1 ss. Because the enhanced neurodegeneration induced by sodium salicylate occurred only at the later stage of the disease (after 43 days p.i.), the early increase of trypanosome proliferation is not likely to be the cause of this phenomenon. In addition, the fact that the weight loss of the trypanosome-infected rats did not increase significantly until day 46 p.i. after sodium salicylate treatment also indicates that more profound changes might have been induced in these rats at the later stages of the infection. The site of action of sodium salicylate is not known. It does not appear to be affecting the parasite directly, as indicated by the normal levels in blood. There was no evidence that it affected the course of blood–brain barrier breakdown because

Neurodegeneration by aspirin in trypanosome-infected rats

191

Fig. 8. Autoradiographs show examples of the varied patterns of induction of GFAP mRNA at the level of the median eminence and arcuate nucleus. In nearadjacent sections from the case shown in f (tryp 1 ss, 41 days p.i.), hybridization for ICE (g), IL-1b (h) and TNF-a (i) mRNAs is shown for comparison of pattern of hybridization in the hypothalamus shown by the arrows. Scale bars ˆ 2 mm.

Table 3. Increases in hybridization density (means ^ S.E.M., calculated as % of control) measured in designated brain areas at two post-infection survival times Choroid plexus Day 22 tryp IL-1ra IL-6 iNOS IFNg COX-2

2258 ^ 1 063 1575 ^ 523 1965 ^ 999 2969 ^ 1553 1387 ^ 223**

tryp 1 ss

tryp

2700 ^ 601 2661 ^ 421* 2500 ^ 642* 736 ^ 182 3726 ^ 876 2451 ^ 632 2688 ^ 916 2970 ^ 326 1300 ^ 329** 928 ^ 181

Day 41 tryp 1 ss 3968 ^ 736** 1240 ^ 379* 4354 ^ 1705** 5760 ^ 2 477* 1289 ^ 359*

Diencephalon Day 22 tryp IL-1ra IL-6 iNOS IFNg COX-2

184 ^ 19 319 ^ 49 175 ^ 21 533 ^ 289 232 ^ 45

tryp 1 ss

187 ^ 43 233 ^ 36 255 ^ 28 333 ^ 98 214 ^ 50

tryp 293 ^ 139 117 ^ 27 128 ^ 25 80 ^ 25 302 ^ 42

Values vs control *P , 0.05 and **P , 0.01. Values vs treatment ***P , 0.05 and ****P , 0.01. n ˆ 4 animals/group.

Day 41 tryp 1 ss 1099 ^ 416**,*** 257 ^ 70* 303 ^ 67**,**** 193 ^ 50 601 ^ 120**,***

elevated IgG immunostaining in the choroid plexus and elsewhere was similar in tryp and tryp 1 ss animals. Interestingly, immunostaining of immunoglobulins along the ventricular ependyma illustrates how peripheral proteins can gain access to the cerebrospinal flow pathways that disseminate the proteins from their probable entry site in the choroid plexus throughout the ventricular spaces. This delivery system is proposed to explain the locations of some of the pathology found. Under normal conditions, salicylate enters the brain and cerebrospinal fluid and remains in relatively high concentrations because of a poor clearance rate compared to other organic acids. 45 By itself (as judged from the ss animals), salicylate had no effect on the brain, and it did not have a major effect on the integrity of the blood–brain barrier (as seen by IgG immunostaining). However, it may have indirect actions that promote the necrosis seen in the late stages of infection in tryp 1 ss animals (discussed below). Further work with other pharmacological agents may address the issue of mechanism and site of action. The ability of sodium salicylate treatment to induce additional neurodegeneration and enhance induced expression of inflammatory molecule mRNAs suggests an unexpected mechanism of sodium salicylate action in vivo. Evidence from in vitro work shows that: (i) sodium salicylate can

192

N. Quan et al.

Fig. 9. Sites where the blood–brain barrier has been compromised by trypanosome infection are shown at the level of the subfornical organ. Immunohistochemical stain for immunoglobulin in the choroid plexus and ependymal epithelia of a tryp 1 ss rat at 42 day’s survival. Scale bar ˆ 500 mm. Inset shows a higher magnification photomicrograph of the choroid plexus in a non-infected rat. Scale bar ˆ 50 mm. cc, corpus callosum; Ch Plx, choroid plexus; fi, fimbria; sfo, subfornical organ; III, 3rd ventricle.

block NF-kB activation of gene expression 12,13,41 by preventing the degradation of IkB protein, 23,33 which retains NF-kB in the cytosol, away from the kB activation site in the nucleus; (ii) sodium salicylate reduces the stimulated expression of cytokines including IL-1b and TNF-a; 2,30 and (iii) the mRNA expression of IL-1b, TNF-a, and IkBa can all be initiated by NF-kB activation. 29 Therefore, one would expect that the treatment of sodium salicylate would reduce rather than enhance the mRNA expression of IL-1b, TNF-a and IkBa. The contrary effects of sodium salicylate observed in the present study, however, have precedent in in vivo studies. Thus, Endres et al. 11 showed that chronic oral intake of aspirin and ibuprofen for two weeks in humans increased lipopolysaccharide-induced synthesis of both IL-1b and TNF-a by peripheral blood mononuclear cells measured three weeks after discontinuation of drug. Whether chronic treatment of sodium salicylate leads to a switch or rebound effect remains to be determined. Similarly, anti-inflammatory drugs such as indomethacin can have pro-inflammatory actions at delayed time-points in an in vivo carrageenininduced pleurisy model. 15 A mechanism for our unexpected results may be that prostaglandin inhibition by sodium salicylate blocked the

normally inhibitory role that prostaglandins have on proinflammatory cytokine production. 4,44 It has been suggested from in vitro work that microglial prostaglandin E2, through a combination of actions including pro-inflammatory cytokine inhibition, is neuroprotective. 24 Because prostaglandins can block induced IL-1b synthesis, 3 iNOS expression, 27 and microglia-mediated neurotoxicity, 49 the observed enhancements in pro-inflammatory gene expression and neurodegeneration may be a consequence of blocking prostaglandin synthesis by sodium salicylate. The induction of iNOS and IFNg mRNAs in the choroid plexus may reflect a complex interaction with the parasites, possibly a means by which the parasite creates immunosuppression, a feature of its survival. 46,47 Augmented expression of these cytokines by salicylate could further promote survival of parasites. Expression of these mRNAs in the choroid plexus was intense and may reflect the presence of lymphocytes and macrophages as well as parasites in these locations. Microscopic analysis of emulsion-coated sections containing the choroid plexus indicated that the cells expressing iNOS and IFNg mRNAs appeared to be predominantly choroidal epithelial cells (not shown, but see Ref. 35). Expression of mRNAs in brain parenchyma may reflect an infiltration of inflammatory cells as revealed by markers to such cells. 43 However, again, microscopic analysis indicated that the labeled cell types were predominantly of the glial (IFNg) and endothelial (iNOS) categories rather than leucocytederived (not shown, but see Ref. 35). It is not clear whether the extensive neurodegeneration in tryp 1 ss was caused by the enhanced expression of proinflammatory molecules in the brain. High levels of IL-1b and TNF-a have been found to be toxic to neurons in vitro, 6,48 and over-expression of TNF-a in transgenic mice has been found to cause neurodegeneration. 1,5 The timecourse of IL-1b and TNF-a induction slightly preceded the onset of widespread neurodegeneration after day 43 p.i. The increased levels of cytokine gene expression in tryp 1 ss animals correlated with the increased amount of degeneration in these animals. The locations of cytokine expression roughly correlated with the locations of neurodegeneration when extracellular diffusion and cerebrospinal flow pathways were taken into account. Therefore, IL-1b, ICE, and TNF-a are good candidates as the cause of the observed neurodegeneration in trypanosome-infected rats treated with sodium salicylate. An alternative explanation for the data is that the inflammation associated with the neurodegeneration induced a rise in brain cytokine expression. Salicylate can be toxic to hepatocytes in culture. 50 In tryp 1 ss animals, impaired liver function may allow higher levels of salicylate to elicit similar kinds of hypoxic effects on neurons; their distress may elicit a surrounding cytokine response that may precede frank degeneration. However, the temporal sequence of events—cytokine mRNA expression preceding neurodegeneration and in fact falling at the late time p.i. points associated with degeneration 35 —argues against this explanation. Interestingly, the circumventricular organs and the regions surrounding them, which showed early elevations in cytokine mRNA expression levels, were not characterized by neurodegeneration. Circumventricular organs have a deficient blood–brain barrier, so they normally encounter potentially destructive peripheral immune signals. Evidence suggests that they have mechanisms to protect themselves from these

Neurodegeneration by aspirin in trypanosome-infected rats

challenges. 31 Thus, it is possible that peripheral or central signals of a toxic nature have no adverse effect on these regions. The present findings are reminiscent of a serious and mysterious complication of aspirin known as Reye’s syndrome. Reye’s syndrome appears to be induced by the combined effects of aspirin treatment and influenza infection, but not by aspirin alone. 9 Typical side effects of aspirin or sodium salicylate alone are upper gastrointestinal symptoms, dizziness, and drowsiness, 10 but the use of these drugs during influenza infection may result in Reye’s syndrome in children, which is associated with a high death rate. 7 Pathology includes multifocal infarcts in the cortex, basal ganglia, and brainstem. 25 Interestingly, our results show that treatment of sodium salicylate alone does not cause any neurodegeneration, but combining sodium salicylate treatment with trypanosome infection causes extensive neurodegeneration, with occasional patterns of infarcts at the latest stages. Because rapid progression of encephalopathy is a common feature of Reye’s syndrome, findings in the present study may suggest common mechanisms by which sodium salicylate causes neural damage in different diseases.

193 CONCLUSIONS

The present study reveals a unique side effect of chronic sodium salicylate treatment: it worsens the neural damage induced by trypanosome infection. The data also show that chronic sodium salicylate treatment enhances proinflammatory cytokine gene expression in the brain under certain disease conditions, which may significantly exacerbate neuronal damage. Whereas non-steroidal anti-inflammatory drugs may be useful in managing complications associated with late stages of trypanosome infection, 22 the present data caution against the use of these agents to treat trypanosomiasis.

Acknowledgements—This study was supported by the NIMH Intramural Research Program and grants from UNDP/World Bank/ WHO Special Program for Research and Training in Tropical Diseases #970468 and SMFR #4480. Eileen Briley carried out assays and data analysis. Dr Nancy Tresser analysed and interpreted H and E-stained material. Dr Robert Switzer helped interpret silver stains.

REFERENCES

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.

Akassoglou K., Probert L., Kontogeorgos G. and Kollias G. (1997) Astrocyte-specific but not neuron-specific transmembrane TNF triggers inflammation and degeneration in the central nervous system of transgenic mice. J. Immunol. 158, 438–445. Bitko V., Velazquez A., Yang L., Yang Y. C. and Barik S. (1997) Transcriptional induction of multiple cytokines by human respiratory syncytial virus requires activation of NF-kB and is inhibited by sodium salicylate and aspirin. Virology 232, 369–378. Caggiano A. O. and Kraig R. P. (1998) Prostaglandin E2 and 4-aminopyridine prevent the lipopolysaccharide-induced outwardly rectifying potassium current and interleukin-1b production in cultured rat microglia. J. Neurochem. 70, 2357–2368. Caggiano A. O. and Kraig R. P. (1999) Prostaglandin E receptor subtypes in cultured rat microglia and their role in reducing lipopolysaccharide-induced interleukin-1b production. J. Neurochem. 72, 565–575. Campbell I. L., Stalder A. K., Chiang C. S., Bellinger R., Heyser C. J., Steffensen S., Masliah E., Powell H. C., Gold L. H., Henriksen S. J. and Siggins G. R. (1997) Transgenic models to assess the pathogenic actions of cytokines in the central nervous system. Molec. Psychiat. 2, 125–129. Chao C. C., Hu S., Ehrlich L. and Peterson P. K. (1995) Interleukin-1 and tumor necrosis factor-a synergistically mediate neurotoxicity: involvement of nitric oxide and of N-methyl-d-aspartate receptors. Brain Behav. Immun. 9, 355–365. Corey L., Rubin R. J., Hattwick M. A., Noble G. R. and Cassidy E. (1976) A nationwide outbreak of Reye’s Syndrome. Its epidemiologic relationship of influenza B. Am. J. Med. 61, 615–625. DeOlmos J. S. and Ingram W. R. (1971) An improved cupric-silver method for impregnation of axonal and terminal degeneration. Brain Res. 33, 523–529. Deshmukh D. R., Maassab H. F. and Mason M. (1982) Interactions of aspirin and other potential etiologic factors in an animal model of Reye syndrome. Proc. natn. Acad. Sci. U.S.A. 79, 7557–7560. Eisenberg E., Berkey C. S., Carr D. B., Mosteller F. and Chalmers T. C. (1994) Efficacy and safety of nonsteroidal antiinflammatory drugs for cancer pain: a meta-analysis. J. clin. Oncol. 12, 2756–2765. Endres S., Whitaker R. E., Ghorbani R., Meydani S. N. and Dinarello C. A. (1996) Oral aspirin and ibuprofen increase cytokine-induced synthesis of IL1b and of tumour necrosis factor-a ex vivo. Immunology 87, 264–270. Farivar R. S. and Brecher P. (1996) Salicylate is a transcriptional inhibitor of the inducible nitric oxide synthase in cultured cardiac fibroblasts. J. biol. Chem. 271, 31585–31592. Frantz B. and O’Neill E. A. (1995) The effect of sodium salicylate and aspirin on NF-kB. Science 270, 2017–2019. Gerfen C. R. (1992) The neostriatal mosaic: multiple levels of compartmental organization. Trends Neurosci. 15, 133–139. Gilroy D. W., Colville-Nash P. R., Willis D., Chivers J., Paul-Clark M. J. and Willoughby D. A. (1999) Inducible cyclooxygenase may have antiinflammatory properties. Nature Med. 5, 698–701. Griffin W. S., Sheng J. G., Royston M. C., Gentleman S. M., McKenzie J. E., Graham D. I., Roberts G. W. and Mrak R. E. (1998) Glial–neuronal interactions in Alzheimer’s disease: the potential role of a ‘cytokine cycle’ in disease progression. Brain Path. 8, 65–72. Grilli M., Pizzi M., Memo M. and Spano P. (1996) Neuroprotection by aspirin and sodium salicylate through blockade of NF-kB activation. Science 274, 1383–1385. Herbert W. J. and Lumsden W. H. R. (1976) Trypanosoma brucei: a rapid “matching” method for estimating the host’s parasitaemia. Expl Parasitol. 40, 427–431. Herkenham M. (1980) Laminar organization of thalamic projections to the rat neocortex. Science 207, 532–535. Herkenham M. (1986) New perspectives on the organization and evolution of nonspecific thalamocortical projections. In Cerebral Cortex, (eds Jones E. G. and Peters A.), Vol. 5, pp. 403–445. Plenum, New York. Herkenham M., Edley S. M. and Stuart J. (1984) Cell clusters in the nucleus accumbens of the rat, and the mosaic relationship of opiate receptors, acetylcholinesterase and subcortical afferent terminations. Neuroscience 11, 561–593. Hunter C. A., Jennings F. W., Kennedy P. G. and Murray M. (1992) The use of azathioprine to ameliorate post-treatment encephalopathy associated with African trypanosomiasis. Neuropath. appl. Neurobiol. 18, 619–625. Kopp E. and Ghosh S. (1994) Inhibition of NF-kB by sodium salicylate and aspirin. Science 265, 956–959. Levi G., Minghetti L. and Aloisi F. (1998) Regulation of prostanoid synthesis in microglial cells and effects of prostaglandin E2 on microglial functions. Biochimie 80, 899–904. Manz H. J. and Colon A. R. (1982) Neuropathology, pathogenesis, and neuropsychiatric sequelae of Reye syndrome. J. neurol. Sci. 53, 377–395. McGeer P. L. and McGeer E. G. (1996) Anti-inflammatory drugs in the fight against Alzheimer’s disease. Ann. N.Y. Acad. Sci. 777, 213–220. Minghetti L., Nicolini A., Polazzi E., Creminon C., Maclouf J. and Levi G. (1997) Inducible nitric oxide synthase expression in activated rat microglial cultures is downregulated by exogenous prostaglandin E2 and by cyclooxygenase inhibitors. Glia 19, 152–160.

194

N. Quan et al.

28. Mitchell J. A., Saunders M., Barnes P. J., Newton R. and Belvisi M. G. (1997) Sodium salicylate inhibits cyclo-oxygenase-2 activity independently of transcription factor (nuclear factor kappa B) activation: role of arachidonic acid. Molec. Pharmac. 51, 907–912. 29. Miyamoto S. and Verma I. M. (1995) Rel/NF-kB/IkB story. Adv. Cancer Res. 66, 255–292. 30. Osnes L. T., Foss K. B., Joo G. B., Okkenhaug C., Westvik A. B., Ovstebo R. and Kierulf P. (1996) Acetylsalicylic acid and sodium salicylate inhibit LPS-induced NF-kB/c-Rel nuclear translocation, and synthesis of tissue factor (TF) and tumor necrosis factor alpha (TNF-a) in human monocytes. Thromb. Haemost. 76, 970–976. 31. Pedersen E. B., McNulty J. A., Castro A. J., Fox L. M., Zimmer J. and Finsen B. (1997) Enriched immune-environment of blood–brain barrier deficient areas of normal adult rats. J. Neuroimmunol. 76, 117–131. 32. Philip K. A., Dascombe M. J., Fraser P. A. and Pentreath V. W. (1994) Blood–brain barrier damage in experimental African trypanosomiasis. Ann. trop. Med. Parasitol. 88, 607–616. 33. Pierce J. W., Read M. A., Ding H., Luscinskas F. W. and Collins T. (1996) Salicylates inhibit IkB-a phosphorylation, endothelial-leukocyte adhesion molecule expression, and neutrophil transmigration. J. Immunol. 156, 3961–3969. 34. Proescholdt M. G., Hutto B., Brady L. S. and Herkenham M. (1999) Studies of cerebrospinal fluid flow and penetration into brain following lateral ventricle and cisterna magna injections of the tracer [ 14C]inulin in rat. Neuroscience 95, 577–592. 35. Quan N., Mhlanga J. D. M., Whiteside M. B., McCoy A. N., Kristensson K. and Herkenham M (1999) Chronic over-expression of pro-inflammatory cytokines and histopathology in the brains of rats infected with Trypanosoma brucei. J. comp. Neurol. 44, 114–130. 36. Quan N., Stern E. L., Whiteside M. B. and Herkenham M. (1999) Induction of pro-inflammatory cytokine mRNAs in the brain after peripheral injection of subseptic doses of lipopolysaccharide in the rat. J. Neuroimmunol. 93, 72–80. 37. Quan N., Whiteside M. and Herkenham M. (1998) Time course and localization patterns of interleukin-1b messenger RNA expression in brain and pituitary after peripheral administration of lipopolysaccharide. Neuroscience 83, 281–293. 38. Quan N., Whiteside M., Kim L. and Herkenham M. (1997) Induction of inhibitory factor kBa mRNA in the central nervous system after peripheral lipopolysaccharide administration: an in situ hybridization histochemistry study in the rat. Proc. natn. Acad. Sci. U.S.A. 94, 10985–10990. 39. Quan N., Zhang Z., Emery M., Lai E., Bonsall R., Kalyanaraman V. S. and Weiss J. M. (1996) In vivo induction of interleukin-1 bioactivity in brain tissue after intracerebral infusion of native gp 120 and gp 160. Detection of interleukin-1 bioactivity in various brain regions of normal healthy rats. Neuroimmunomodulation 3, 56–61. 40. Richmon J. D., Fukuda K., Maida N., Sato M., Bergeron M., Sharp F. R., Panter S. S. and Noble L. J. (1998) Induction of heme oxygenase-1 after hyperosmotic opening of the blood–brain barrier. Brain Res. 780, 108–118. 41. Sakitani K., Kitade H., Inoue K., Kamiyama Y., Nishizawa M., Okumura T. and Ito S. (1997) The anti-inflammatory drug sodium salicylate inhibits nitric oxide formation induced by interleukin-1b at a translational step, but not at a transcriptional step, in hepatocytes. Hepatology 25, 416–420. 42. Schultzberg M., Ambatsis M., Samuelsson E.-B., Kristensson K. and van Meirvenne N. (1988) Spread of Trypanosoma brucei to the nervous system: early attack on circumventricular organs and sensory ganglia. J. Neurosci. Res 21, 56–61. 43. Schultzberg M., Olsson T., Samuelsson E. B., Maehlen J. and Kristensson K. (1989) Early major histocompatibility complex (MHC) class I antigen induction in hypothalamic supraoptic and paraventricular nuclei in trypanosome-infected rats. J. Neuroimmunol. 24, 105–112. 44. Seldon P. M., Barnes P. J., Meja K. and Giembycz M. A. (1995) Suppression of lipopolysaccharide-induced tumor necrosis factor-alpha generation from human peripheral blood monocytes by inhibitors of phosphodiesterase 4: interaction with stimulants of adenylyl cyclase. Molec. Pharmac. 48, 747–757. 45. Spector R. and Lorenzo A. V. (1973) The transport and metabolism of salicylate in the central nervous system: in vivo studies. J. Pharmac. exp. Ther. 185, 276–286. 46. Sternberg J. and McGuigan F. (1992) Nitric oxide mediates suppression of T cell responses in murine Trypanosoma brucei infection. Eur. J. Immunol. 22, 2741–2744. 47. Sternberg J. M. and Mabbott N. A. (1996) Nitric oxide-mediated suppression of T cell responses during Trypanosoma brucei infection: soluble trypanosome products and interferon-g are synergistic inducers of nitric oxide synthase. Eur. J. Immunol. 26, 539–543. 48. Strijbos P. J. and Rothwell N. J. (1995) Interleukin-1b attenuates excitatory amino acid-induced neurodegeneration in vitro: involvement of nerve growth factor. J. Neurosci. 15, 3468–3474. 49. The´ry C., Dobbertin A. and Mallat M. (1994) Downregulation of in vitro neurotoxicity of brain macrophages by prostaglandin E2 and a b-adrenergic agonist. Glia 11, 383–386. 50. Trost L. C. and Lemasters J. J. (1997) Role of the mitochondrial permeability transition in salicylate toxicity to cultured rat hepatocytes: implications for the pathogenesis of Reye’s syndrome. Toxicol. appl. Pharmac. 147, 431–441. 51. Vaughn L. K., Veale W. L. and Cooper K. E. (1980) Antipyresis: its effect on mortality rate of bacterially infected rabbits. Brain Res. Bull. 5, 69–73. 52. Vitkovic L. (1997) Neuropathogenesis of HIV-1 infection: interactions between interleukin-1 and transforming growth factor-beta 1. Molec. Psychiat. 2, 111–112. 53. Xiao L., Patterson P. S., Yang C. and Lat A. A. (1999) Role of eicosanoids in the pathogenesis of murine cerebral malaria. Am. J. Trop. Med. Hyg. 60, 668–673. 54. Yeh S. Y. (1997) Effects of salicylate on 3,4-methylenedioxymethamphetamine (MDMA)-induced neurotoxicity in rats. Pharmac. Biochem. Behav. 58, 701–708. (Accepted 28 September 1999)