Received: 16 October 2009, Revised: 23 December 2009, Accepted: 4 January 2010, Published online in Wiley InterScience: 2010 Fractionated manganese injections: effects on MRI contrast enhancement and physiological measures in C57BL/6 mice Barbara Gru ¨ necker ay , Sebastian F. Kaltwasser ay , Yorick Peterse a , Philipp G. Sa ¨ mann a , Mathias V. Schmidt a , Carsten T. Wotjak a , Michael Czisch a * Manganese-enhanced MRI (MEMRI) is an increasingly used imaging method in animal research, which enables improved T 1 -weighted tissue contrast. Furthermore accumulation of manganese in activated neurons allows visualization of neuronal activity. However, at higher concentrations manganese (Mn 2R ) exhibits toxic side effects that interfere with the animals’ behaviour and well-being. Therefore, when optimizing MEMRI protocols, a compro- mise has to be found between minimizing side effects and intensifying image contrast. Recently, a low concentrated fractionated Mn 2R application scheme has been proposed as a promising alternative. In this study, we investigated effects of different fractionated Mn 2R dosing schemes on vegetative, behavioural and endocrine markers, and MEMRI signal contrast in C57BL/6N mice. Measurements of the animals’ well-being included telemetric monitoring of body temperature and locomotion, control of weight and observation of behavioural parameters during the time course of the injection protocols. Corticosterone levels after Mn 2R application served as endocrine marker of the stress response. We compared three MnCl 2 Á 4H 2 O application protocols: 3 times 60 mg/kg with an inter-injection interval of 48 h, six times 30 mg/kg with an inter-injection interval of 48 h, and 8 times 30 mg/kg with an inter-injection interval of 24 h (referred to as 3 T 60/48, 6 T 30/48 and 8 T 30/24, respectively). Both the 6 T 30/48 and the 8 T 30/24 protocols showed attenuated effects on animals’ well-being as compared to the 3 T 60/48 scheme. Best MEMRI signal contrast was observed for the 8 T 30/24 protocol. Together, these results argue for a fractionated application scheme such as 30 mg/kg every 24 h for 8 days to provide sufficient MEMRI signal contrast while minimizing toxic side effects and distress. Copyright ß 2010 John Wiley & Sons, Ltd. Keywords: manganese; neurotoxicity; MEMRI; mouse; contrast agent; stress INTRODUCTION Magnetic resonance imaging (MRI) is an important tool in animal research due to its high resolution and excellent soft-tissue contrast. Being non-invasive, MRI opens the possibility to pursue in vivo longitudinal studies. However, anatomical MRI of the rodent brain is limited by little native contrast between different cerebral compartments, hampering the delineation of cortical and subcortical regions. To enhance the regional contrast specificity, paramagnetic agents are frequently applied (1). One of the most promising contrast agents in animal studies is manganese (Mn 2þ ). Due to its chemical similarity to calcium (Ca 2þ ) (2), it may enter neuronal cells through voltage-gated Ca 2þ channels during depolarization (3). Mn 2þ is then taken up into the endoplasmatic reticulum (4,5). Accumulated in vesicles, it can be transported anterogradely in axonal tracts (6,7) to the synaptic cleft, where it is released and taken up by the next neuron (5,7). Mn 2þ ions reside in the brain for a prolonged period of time with a half life between 51 and 74 days (8). Its paramagnetic properties lead to an effective reduction of the spin-lattice relaxation time constant T 1 of the surrounding water molecules, resulting in positive contrast enhancement in T 1 -weighted MR images (1). These basic characteristics of Mn 2þ have led to the develop- ment of three distinctive applications of MEMRI (9): first, MEMRI is used to visualise neuroanatomical details after subcutaneous (10) or intravenous (9,11) administration of Mn 2þ . The regional MEMRI contrast depends on local neuronal cell density, differences of the local permeability of the blood brain barrier, and differences in neuronal activation (9,12). As a second application, neuronal tract tracing can be performed, exploiting the anterograde Mn 2þ transportation across synapses (4,5,7,13–16). Finally, MEMRI enables functional imaging in animal models. Since Mn 2þ enters cells through voltage-gated Ca 2þ channels, it accumulates in excitable cells. As compared to classical blood oxygenation level dependent (BOLD) methods used in functional MRI (fMRI), MEMRI is not primarily dependent on hemodynamic changes as a correlate of neuronal activity. Rather, it reflects neuronal (www.interscience.wiley.com) DOI:10.1002/nbm.1508 Research Article * Correspondence to: M. Czisch, Max Planck Institute of Psychiatry, Neuroima- ging Group, Kraepelinstr. 2, D-80804 Munich, Germany E-mail: czisch@mpipsykl.mpg.de a B. Gru ¨necker, S. F. Kaltwasser, Y. Peterse, P. G. Sa ¨mann, M. V. Schmidt, C. T. Wotjak, M. Czisch Max Planck Institute of Psychiatry, Munich, Germany y These authors contributed equally to the study. Abbreviations used: cþsc, cortical plus subcortical; CV, coefficient of vari- ation; DG, dentate gyrus; EDTA, ethylenediaminetetraacetic acid; HPA-axis, hypothalamic-pituitary-adrenal axis; LSD, least significant difference; MEMRI, manganese enhanced MRI; RI, relative intensity; ROI, region of interest; T1w, T1-weighted; T2w, T2-weighted; wb, whole brain. NMR Biomed. (2010) Copyright ß 2010 John Wiley & Sons, Ltd. 1