A model of cerebral aspergillosis in nonimmunosup

Authors: Stefan Zimmerli Urspeter Knecht Stephen L Leib

Publish Date: 2007/06/30

Volume: 114, Issue: 4, Pages: 411-418

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Abstract

Central nervous system aspergillosis is an often fatal complication of invasive Aspergillus infection Relevant disease models are needed to study the pathophysiology of cerebral aspergillosis and to develop novel therapeutic approaches This study presents a model of central nervous system aspergillosis that mimics important aspects of human disease Elevendayold nonimmunosuppressed male Wistar rats were infected by an intracisternal injection of 10 μl of a conidial suspension of Aspergillus fumigatus An inoculum of 718 log10 colonyforming units CFU consistently produced cerebral infection and resulted in death of all animals n = 25 within 3–10 days Median survival time was 3 days Histomorphologically all animals developed intracerebral abscesses 2–26 per brain containing abundant fungal hyphae and neutrophils Fungal culture of cortical homogenates yielded maximal growth on day 3 after infection 54 log10 CFU/g n = 15 that declined over time Galactomannan concentrations in cortical homogenates assessed as an index for hyphal burden peaked on days 3–5 Fungal infection spread to peripheral organs in 83 of animals Fungal burden in lung liver spleen and kidney was two orders of magnitude lower than in the brain The successful establishment of a model of cerebral aspergillosis in a nonimmunosuppressed host provides the opportunity to investigate mechanisms of disease and to develop novel treatment regimens for this commonly fatal infectionInvasive aspergillosis is a major cause of illness and death in immunocompromised subjects including patients with hematologic cancers and recipients of bone marrow and solidorgan transplants Risk factors are prolonged neutropenia graftversushost disease highdose corticosteroid treatment and other immunosuppressive regimens Cerebral aspergillosis occurs at a frequency of 14–42 in patients with acute leukemia or allogeneic stemcell transplantation making it the most common organ manifestation of hematogenous dissemination from primary foci usually in the lungs 16 24 Alternatively cerebral aspergillosis can arise by direct invasion from adjacent infectious foci eg sinusitis The pathophysiology of cerebral aspergillosis is incompletely understood Ischemic and hemorrhagic infarcts are thought to result from arteriolitis and arteritis with fungal thrombosis and obliteration of the vessel lumen Cerebral abscesses characterized by necrotic parenchyma a paucity of inflammatory cells and fungal hyphae are commonly found but their pathogenesis has remained largely unknown 30 Until recently cerebral aspergillosis was considered to be universally lethal 16 24 30 Recently introduced antifungal drugs with enhanced central nervous system penetration and novel mechanisms of action offer promising treatment options Indeed the combination of voriconazole and neurosurgery resulted in complete or partial responses in 28 of 81 patients with central nervous system CNS aspergillosis 27 The role of posaconazole and the echinocandins in the treatment of cerebral aspergillosis remains to be defined Relevant disease models are needed to study the pathophysiology of cerebral aspergillosis and to develop improved treatment regimensA recently established model of aspergillosis in immunosuppressed mice uses direct intracerebral injection of conidia to induce cerebral abscesses 3 This model has been used successfully for studies on drug efficacy and disease progression 4 5 6 8 14 15 29 However persistent pancytopenia induced by the high level of immunosuppression necessary to establish infection and the significant systemic disease that likely contributes to mortality may be considered as the disadvantages of this modelBuilding on our experience with experimental CNS infection in rats we have established a model of cerebral aspergillosis in nonimmunosuppressed nursing rats that mimics important aspects of human disease 1 10 22 23 28 To allow sufficient time to study disease progression and potential treatment effects CNS infection in untreated animals was intended to be universally and reproducibly lethal within several daysThe strain of Aspergillus fumigatus used for infection was isolated from a patient with invasive pulmonary aspergillosis The isolate was cultivated on Sabouraud’s agar plates at 37°C for 3 days 31 Conidia were harvested by washing the plates with phosphatebuffered saline PBS supplemented with 005 Tween 80 and filtering the resulting suspension through a BD Falcon 40 μm nylon cell strainer BD Biosciences Bedford MA USA to eliminate hyphal fragments and conidial clumps The concentration of the resulting stock suspension of predominately single spores was repeatedly verified by culturing serial dilutions on Sabouraud’s agar plates Colonyforming units CFU were counted after 20 h at 37°C Three days before infection the stock suspension stored at 4°C was diluted with 005 Tween 80 in PBS to the desired concentration which was verified by culture In addition the fungal titer of the inoculum was routinely confirmed by quantitative culture on the day of infectionNursing Wistar rat pups Charles River Laboratories Sulzfeld Germany were kept in groups of four to eight with their dam for 5 days before the experiments Water and food were provided ad libitum during a 12h day–night cycle The animal studies were approved by the Animal Care and Experimentation Committee of the Kanton of Bern Switzerland and followed National Institutes of Health guidelines for the performance of animal experimentsNo immunosuppressive drugs were used The relative immaturity of the immune system of infant rats permits establishment of invasive aspergillosis without iatrogenic immunosuppression To produce cerebral Aspergillus infection the inoculum was injected into the cisterna magna of the rats The procedure that does not produce structural brain damage was well tolerated by all animals and no deaths were observed during the first 12 h after injection On postnatal day 11 when the rats n = 97 weighed 265 ± 39 g they were infected by a direct intracisternal injection with a 32gauge needle of 10 μl of saline containing Aspergillus conidia 1 10 22 23 28 In preliminary experiments the rats n = 12 were given inocula of 50 60 and 70 log10 CFU n = 4 per group One animal of each group was killed on day 4 for quantitative cerebral fungal culture resulting in 32 34 and 46 log10 CFU/g respectively and three out of four were observed for survival to day 7 after infection when the experiment was censored The median survival of animals infected with inocula ≤70 log10 CFU was 7 days range 3–7 For an inoculum of 50 log10 CFU survival was 100

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