Murine malaria is associated with significant hearing impairment

Schmutzhard et al. Malaria Journal 2010, 9:159 http://www.malariajournal.com/content/9/1/159 Open Access RESEARCH Murine malaria is associated with significant hearing impairment Research Joachim Schmutzhard*1, Christian H Kositz2, Peter Lackner2, Anelia Dietmann2, Marlene Fischer2, Rudolf Glueckert1, Markus Reindl2, Kurt Stephan3, Herbert Riechelmann1, Annelies Schrott-Fischer1 and Erich Schmutzhard2 Abstract Background: Plasmodium falciparum malaria has been suspected to cause hearing loss. Developmental, cognitive and language disorders have been observed in children, surviving cerebral malaria. This prospective study aims to evaluate whether malaria influences hearing in mice. Methods: Twenty mice were included in a standardized murine cerebral malaria model. Auditory evoked brainstem responses were assessed before infection and at the peak of the illness. Results: A significant hearing impairment could be demonstrated in mice with malaria, especially the cerebral form. The control group did not show any alterations. No therapy was used. Conclusion: This suggests that malaria itself leads to a hearing impairment in mice. Background With more than 247 million cases in 2006 malaria is one of the most frequent infectious diseases world wide[1]. The most severe course of the human disease is caused by Plasmodium falciparum, leading to multi-organ disease. In particular cerebral malaria (CM) is potentially leading to a wide range of neurocognitive sequelae[2]. Furthermore, it has been shown that severe falciparum malaria may lead to an acquired language disorder[2]. Language development in childhood needs an intact and perfectly functioning hearing system - from the outer ear canal to the sensory cerebral cortex. So far, in malaria research no dedicated and specific attention has been paid to the involvement of the inner ear. Only very few authors have reported an acquired hearing impairment possibly caused by falciparum malaria[3,4]. To shed light into this aspect, a cerebral malaria experiment in Plasmodium berghei ANKA infected C57BL/6J mice - a well defined model for severe/cerebral malaria - was performed evaluating the hearing threashold with auditory evoked brain stem responses (ABRs) before infection and at the peak of the disease[5,6]. * Correspondence: 1 Department of Otorhinolaryngology, Innsbruck Medical University, Anichstraße 35, 6020 Innsbruck Austria The primary aim of this study was to evaluate a possible change of the hearing threshold in mice with CM and malaria without cerebral involvement. Methods Study design The animal study conformed to the Austrian guidelines for the care and use of laboratory animals and were approved by the Austrian Ministry for Education, Science and Culture with the reference number do. Zl. A08/4102. Three different animal groups were studied. One group contains the mice suffering from cerebral malaria (CM). The second group contains mice, which are infected with malaria, show a parasitaemia comparable to the cerebral malaria mice, but did not develop a cerebral involvement (non-CM). The third group were healthy non-infected animals, which were kept at equal housing, environmental and experimental conditions. Prior to infection a baseline hearing test with ABRs was performed for the frequencies 8 kHz, 13 kHz, 36 kHz and a click sound. Only animals with clearly evokable and readable initial ABRs were included into the study and infected on the same day. To monitor the course of the disease the mice were subjected to a daily evaluation of the SHIRPA score. The CM group is expected to develop signs and symptoms Full list of author information is available at the end of the article © 2010 Schmutzhard et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Schmutzhard et al. Malaria Journal 2010, 9:159 http://www.malariajournal.com/content/9/1/159 between day 5 and 11 after infection. At the peak of the cerebral disease a second ABR measurement was performed and thereafter the animals were sacrificed. Animals surviving day 10 do not develop CM anymore [6]. These animals were used as the non-CM control group and, the second ABR measurement was done on the 11th day immediately before sacrification. The results of the ABR were compared between groups and to the baseline data. After the ABR measurements the CM mice were sacrificed and their brains examined for vascular affection. Auditory brainstem responses The auditory brain stem responses were measured with ABR machine provided by ZLE - Systemtechnik©, Munich Germany. Three frequencies 8 kHz, 13 kHz, 36 kHz and a click sound were measured to evaluate the hearing threshold. The right ear of each mouse was examined. Subsequently the ABRs were measured in an electrically shielded sound attenuating chamber. The potentials are gained by means of three subcutaneous needle-electrodes. The positive electrode is placed at the vertex, the negative electrode at the bulla and the ground electrode at the ipsilateral leg. Tones of 8, 13, 36 kHz and a click sound are used as stimuli. The clicks are produced by two different transducers. Starting with 80 dB the stimuli are decreased by 10 dB. The ABR thresholds are determined as the minimum stimulation level, that produces a clearly recognisable potential. The signals are amplified by a physiologic amplifier and filtered. The responses of 500 stimuli are averaged by means of computerized data acquisition synchronized to stimulus onset. The resulting wave responses were interpreted in a blinded manner by two independent ENT-specialists (JS, ASF). The anesthesia is performed with an Ohmeda Isotec 5 Vaporisator (Duisburg, Germany). With a continuous flow of 3 l/minute oxygen 1% isoflourane is vaporized. The nose and mouth of the mouse are loosly fitted into the dispenser mask. The excess anaestetic and air is disposed of by wide loomed noiseless suction. Animal model Six to eight week old C57BL/6J mice (Charles River Laboratories, Sulzfeld, Germany) are used as cerebral malaria susceptible strains. The mice are housed under standard conditions in the animal housing facility of the Medical University Innsbruck, Austria. After an accommodation period for approximately three to five days, the animals are taken for the study. The C57BL/6J mouse is a strain susceptible to blood stages of P. berghei ANKA. After the determination of the ABR thresholds as described above, the mice are infected with an intraperitoneal application of 1 × 106 parasitized erythrocytes of a homologous Page 2 of 5 donor, which had been infected with frozen polyclonal stocks of P. berghei [7]. The parasitaemia was monitored by daily blood smears of tail blood. The smears are stained by Wright stain. The progress o (...truncated)