Acute cerebral white matter damage in lethal salicylate intoxication

NeuroToxicology 28 (2007) 33–37

Acute cerebral white matter damage in lethal salicylate intoxication Helmut Rauschka a,1,*, Fahmy Aboul-Enein b,1, Jan Bauer b, Hans Nobis c, Hans Lassmann b, Manfred Schmidbauer a b

a Department of Neurology, Hospital Hietzing, Vienna, Austria Department of Neuroimmunology, Brain Research Institute, Medical University Vienna, Austria c Department of Cardiology, Hospital Hietzing, Vienna, Austria

Received 3 December 2004; accepted 30 June 2006 Available online 13 July 2006

Abstract A 34-year-old oligophrenic woman was admitted in comatose state with marked tachypnea. History revealed the oral ingestion of a large amount of acetylsalicylate to attenuate ear pain within the preceding 3 days. Laboratory investigations showed a toxic concentration of serum salicylate (668 mg/l, toxic range above 200 mg/l) and metabolic acidosis. Oxygenation, blood pressure, electrocardiography, echocardiography and CT of thorax and brain were normal. The patient was intubated, fluid and bicarbonate was given intravenously. Six hours after admission asystolia refractory to resuscitation led to death. Autopsy showed venous congestion of the brain, cardiac dilatation and pulmonary edema. Brain histopathology showed myelin disintegration and caspase-3 activation in glial cells, whereas, grey matter changes were sparse. Acute white matter damage is suggested to be the substrate of cerebral dysfunction in salicylate intoxication and possible mechanisms are discussed. # 2006 Elsevier Inc. All rights reserved. Keywords: Salicylate; Intoxication; Immunocytochemistry; Brain; Caspase-3

1. Introduction

2. Case report

Cerebral dysfunction, ranging from confusion to coma, is prominent in severe salicylate intoxication, but the responsible pathophysiological process is unknown (Hill, 1973). Neuropathological data of salicylate intoxication are sparse and previous investigations were restricted to routine autopsy, whereby cerebral edema was the sole finding (Starko and Mullick, 1983). We present here the clinical course and the brain pathology of lethal salicylate intoxication of an adult. In addition to routine histological staining methods, immunocytochemistry with antibodies for glial cells, myelin, neurons and cell death related proteins was used. A distinct pattern of white matter damage is suggested to be the morphological substrate of cerebral dysfunction in salicylate intoxication.

An unconscious 34-year-old woman was admitted to the intensive care unit. A review of patient history revealed the oral ingestion of at least 30–35 pills of aspirin within the preceding 3 days, which is a cumulative dose of more than 15 g acetylsalicylate. The patient was oligophrenic, and it was suggested, that she intended to treat ear pain, which was a common complaint after having experienced a middle ear infection years ago. Confusion and somnolence were first noticed about 3 h prior to admission. Family history and characteristic facial features suggested fetal alcohol syndrome to be the cause of her mental impairment, whereas, there was no hint regarding the presence of an inherited chromosomal or metabolic disorder (Johnson et al., 1996). Because of chronic anxiety she was prescribed alprazolam (1 mg/day) for many years. The patient was a heavy smoker, but not addicted to alcohol or illegal drugs. On examination she was deeply comatose without lateralizing neurological signs or neck stiffness. Tachypnea was prominent, but otherwise clinical signs of pulmonary or cardiac dysfunction were absent. Blood gas analysis revealed metabolic acidosis (pH 7.30; pCO2 = 19.1 mmHg; PO2 = 111 mmHg,

* Corresponding author at: Abt. fu¨r Neurologie, Krankenhaus Hietzing, Wolkersbergenstrasse 1, A-1130 Vienna, Austria. Tel.: +43 1 80110 3559; fax: +43 1 80110 3685. E-mail address: [email protected] (H. Rauschka). 1 These authors contributed equally to this study. 0161-813X/$ – see front matter # 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.neuro.2006.06.010

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HCO3 = 9.3 mmol/l; anion gap 8.8 mmol/l). Serum salicylate was 668 mg/l, whereby the therapeutic range is up to 200 mg/l and a serum concentration above 400 mg/l indicates severe intoxication. She had a leucocytosis (17,000 ml 1, 95% polymorphonuclear granulocytes) and a reduced plasma prothrombine time. Serum electrolytes, lactate, creatinine, urea, amylase, lipase, CK and blood sugar were all normal. Blood pressure was 120/90 mmHg, pulse 125 beats/min and electrocardiography showed sinus tachycardia. Echocardiography as well as cranial and thoracic computed tomography were unremarkable. Intravenous fluid and bicarbonate were given, but acidosis worsened and respiratory failure occurred. She was intubated and respirated, but 6 h after admission asystolia refractory to resuscitation led to death. 3. Pathology Due to legal reasons, autopsy was delayed until the twelfth day post mortem. During this time, the corpse was stored at 4 8C. General findings were lung edema and cardiac dilatation. The brain showed venous congestion with some perivenous blood effusion into the meninges of the posterior convexity and moderate edema with narrowed sulci and ventricles. Features of autolysis were absent (Lindenberg, 1982). Formalin-fixed and paraffin-embedded sections were examined by routine staining methods (haematoxylin–eosin, luxol fast blue (LFB) and Bielschowsky’s axonal silver impregnation) and by immunocytochemistry for glial, leukocyte, myelin and neuronal antigens as well as for markers of programmed cell death, using an avidin–biotin or an alkaline phosphatase/ anti-alkaline phosphatase technique, as described previously (Vass et al., 1986). A summary of the antibodies is given in Table 1. DNA-fragmentation within cell nuclei was determined with the method of in situ end labelling (Gold et al., 1994). In the cerebral cortex, the deep grey matter and the cerebellum, there were moderate eosinophilic neuronal changes indicative of some global-ischemic brain damage, but staining for activated caspase-3 and DNA-fragmentation were sparse or absent in the grey matter. In contrast, white matter pathology was prominent. In large areas of the centrum semiovale, but also in the deep cerebellar white matter, myelin sheaths, pre-dominantly of

large fibres, disintegrated, whereas, axons were relatively preserved (Fig. 1a and b). In addition, there were venous congestion and pronounced edema. Expression of activated caspase-3 was present in numerous glial cells both in the areas of the white matter, which showed myelin disintegration and in areas without visible myelin damage (Fig. 1c). In contrast, the nuclei appeared unremarkable and DNAfragmentation, as indicated by the in situ tailing reaction, was generally absent. The morphology of a proportion of caspase-3 expressing glia was characteristic of astrocytes, in part with clearly visible glia limitans contacting small blood vessels (Fig. 1e). Other caspase-3 positive cells showed morphology characteristic of oligodendrocytes, with processes in connection with disintegrating myelin sheaths, the latter also showing some caspase-3 expression (Fig. 1f). The cells positive for activated caspase-3 did not show the characteristics of NG2 positive oligodendrocyte progenitor cells (Chang et al., 2000). Some large axons with damaged myelin sheaths were reactive for amyloid precursor protein (APP), indicative of acute axonal injury. Within respective lesions of the white matter, microglial cells were found activated (Fig. 1d), but ingestion of myelin breakdown products was absent. Double staining for glial markers and activated caspase-3, using light microscopy and confocal fluorescence microscopy showed a proportion of caspase-3 positive cells expressing S100. The latter is a general glial marker present both in astrocytes and oligodendrocytes (Fig. 1g and h). In contrast, despite extensive efforts including variations of pre-treatment, antibody concentration and sequence of antibody-staining, the caspase-3 positive cells could not be double-stained with any cell-type specific glial marker, such as GFAP (astrocytes) or CNP (oligodendrocytes). 4. Controls The delay of brain autopsy raises doubt on the validity of histopathology. Therefore, we studied white matter changes of three control cases with an identical autolysis time of 11–12 days and comparable storing conditions. Cause of death was cardiac failure in all control cases. Acute white matter changes were sparse and consisted of scattered myelin swelling and

Table 1 Antibodies used for immunocytochemistry Antigen

Ab-type

Target

Source

APP CLA CNPase GFAP MBP MHC II Neurofilament 68 kDa p18 (CM1) p17 and p12 S-100

mAb mAb mAb mAb polyAb mAb polyAb polyAb mAb polyAb

Neuron Leukocytes Myelin/oligodendrocytes Astrocytes Myelin Monocytes/microglia Neuron Activated caspase-3 Activated caspase-3 Astro/oligodendrocytes

Chemicon, Temecula, CA Dako, Glostrup, DK Affinity Res. Prod., UK Neomarkers, USA Dako, Glostrup, DK Dako, Glostrup, DK Chemicon, Temecula, CA IDUN, La Lolla, USA BD Pharmingen, USA Dako, Glostrup, DK

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Fig. 1. (a) LFB, showing myelin disintegration, especially of large fibres; (b) Bielschowsky’s silver staining, showing acute axonal degeneration; (c) activated caspase-3 (p17) in numerous glial cells; (d) HLA-DR, showing activated microglia; (e) activated caspase-3 (p17), showing glial cell with process contacting a small vessel (indicative of astrocyte); (f) activated caspase-3 (p17), showing glial cell connecting to myelin sheat (indicative of oligodendrocyte); (g) and (h) confocale fluorescence image showing glial cell double stained (yellow) for S-100 (red) and caspase-3 (p17) (green); (i) LFB of control case 1, showing white matter edema but no myelin disintegration; (j) SM 31 of control case 1, showing normal axons; (k) activated caspase-3 (p17) in control case 1 without specific staining. Original magnifications: (a–d) 312.5; (e and f) 800; (g and h) 1000; (i–k) 200. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)

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myelin degradation. Expression of activated caspase-3 was generally absent (Fig. 1i–k). Since the distinct pattern of white matter damage could be causally related to cerebral edema, we studied three routine autopsy cases with acute brain edema due to severe terminal cardiac insufficiency. In every case, the edematous tissue changes involved pre-dominantly the hemispheric white matter, most severely in perivascular areas. Myelin disintegration was generally sparse or absent and, when present, did not resemble the pattern found in our patient. Expression of activated caspase-3 was generally absent. In addition, since double labelling of caspase-3 and specific astrocyte- or oligodendrocyte markers was not possible in the present case, we investigated this technique in a case of severe acute disseminated encephalomyelitis (ADEM), using light microscopy and confocal fluorescence microscopy on formalin-fixed and paraffin-embedded tissue blocks. There, single labelling showed numerous cells expressing activated caspase3 and the morphology of a considerable proportion of these cells was characteristic of astrocytes and oligodendrocytes (data not shown). In contrast, double labelling of specific glial markers with activated caspase-3 was possible in very few scattered cells, although numerous variations of the staining technique have been tested. 5. Discussion In the presented case, the white matter changes are most prominent and consist of pronounced myelin disintegration associated with glial cells expressing activated caspase-3. Since only axons with advanced disintegration of the myelin sheath show some morphologic and immunocytochemical signs of acute degeneration, the latter may be viewed as secondary to the break down of myelin. The morphologic features of the caspase-3 positive cells are characteristic of either astrocytes or oligodendrocytes, but we were not able to prove this by double labelling, whereby the latter technique also was only marginally successful in the ADEM-control case with regular autolysis time. Although activated caspase-3 is a central executioner enzyme of programmed cell death, the absence of DNAfragmentation excludes apoptosis. The limitations of a retrospective post mortem case study hinder the interpretation of caspase-3 activation and its role in the observed myelin disintegration. Whereas, the cortical and deep grey matter changes reflect pre-terminal ischemia due to asystolia and failed resuscitation, acute white matter damage is generally not a feature of global ischemia (Auer and Benveniste, 1997). The absence of autolytic features and the absence of relevant white matter changes in a number of control cases with similar delay of brain autopsy argue strongly against an autolytic etiology of this distinct white matter pathology. However, the clinical and laboratory features of this case are proof of severe salicylate intoxication, which we regard to be causal for the acute white matter damage. This is substantiated by neuropathological investigations in Reye’s syndrome, where swelling of myelin sheaths and glial cells as well as mitochondrial disturbances

have been found (Brown and Imam, 1991). Reye’s syndrome is an entity characterised by acute hepatic and cerebral dysfunction, which in most cases is preceded by salicylate intake in the course of an otherwise harmless viral infection. Since in some cases of Reye’s syndrome salicylate levels are reported to be within the toxic range, the contributions of salicylate, viral infection and possible inborn errors of metabolism are unclear, and there seems to be some overlap with salicylate intoxication (Belay et al., 1999; Linnemann et al., 1974; Starko and Mullick, 1983). Our patients characteristic dentatofacial anomalies combined with mental retardation suggest fetal alcohol syndrome. In addition to neuronal loss and disruption of synaptogenesis, in utero alcohol exposure disturbs myelin and glial cell development, which may have pre-disposed white matter to toxic damage (Guerri et al., 2001). Regarding the pathophysiology of white matter disturbance in salicylate overdose, it is conceivable that several mechanisms may be acting in concert. Toxicity of salicylate, which is the active metabolite of aspirin, is related to the induction of mitochondrial permeability transition by opening pores in the inner membrane (Trost and Lemasters, 1996). This process not only results in the uncoupling of oxidative phosphorilation and metabolic acidosis, but may also release proteins such as ‘‘apoptosis inducing factor’’ (AIF) or cytochrome c, which are able to initiate the caspases cascade (Rosenfeld and Liebhaber, 1976; Wilson, 1998). Salicylate may also initiate cell death by activation of p38 mitogen-activated protein kinase (Schwenger et al., 1997). In addition to direct salicylate toxicity, tissue acidosis is known to induce selective glial cell death and directly destabilizes myelin sheaths (Giffard et al., 1990; Young et al., 1988). In summary, neuropathological investigations in this case of salicylate intoxication reveal acute white matter pathology, characterized by myelin disintegration and glial caspase-3 activation, which may constitute the pathological substrate of the cerebral dysfunction observed in severe salicylate intoxication. Acknowledgements The authors wish to thank Mrs. M. Leisser, Prof. H. Breitschopf, Mrs. E. Katzenschlager and Mrs. S. Bariszlovits for excellent technical assistance. References Auer RN, Benveniste H. Hypoxia and related conditions.In: Graham DI, Lantos PL, editors. 6th ed. Greenfield’s Neuropathology, vol. I. London, UK: Arnold; 1997. p. 262–314. Belay ED, Bresee JS, Holman RC, Kahn AS, Shahriari A, Schonberger LB. Reye’s syndrome in the United States from 1991 through 1997. N Engl J Med 1999;349:1377–82. Brown JK, Imam H. Interrelationship of liver and brain with special reference to Reye syndrome. J Inher Metab Dis 1991;14:436–58. Chang A, Nishiama A, Peterson J, Prineas J, Trapp B. NG2-positive oligodendrocyte progenitor cells in adult human brain and multiple sclerosis lesions. J Neurosci 2000;20:6404–12.

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