Quantitative T₂ MRI mapping of the rat brain in vivo offers unique opportunities to provide non-invasive biomarkers of neurotoxicity related changes in non-clinical studies [1, 2]. To better understand the potential performance of this type of biomarker baseline characteristics of T₂ mapping need to be determined. The aim of this study was to characterize the between- and within-subject reproducibility of T₂ mapping in control (untreated) rats over time and to assess the statistical power of such an approach using a relatively large population of research animals.An animal use protocol was approved by the National Center for Toxicological Research IACUC. Male Sprague-Dawley rats (N = 138, 360 ± 40 g) were used. All animals were untreated and scanned in the MRI one time except for a separate cohort (N = 21), in which the animals were scanned, injected with saline (once, ip, 2 ml/kg) and imaged again at later time points: 2 hr (N = 5), 24 hr (N = 7), 48 hr (N = 5), 144 hr (N = 5), or 288 hr (N = 4). MRI was performed using a 7 tesla Bruker Biospec AV III equipped with a 4-channel array rat brain RF coil. Animals were anesthetized using isoflurane (3% induction, 1-2% maintenance in oxygen) and body temperatures maintained at 37.3 ± 0.6°C. For T₂ mapping a multi-echo spin echo sequence was used (MTX 192 x 192 x 24, FOV = 3.84 x 3.84 x 2.4 cm, echo spacing = 15 ms, 16 echoes, TR = 6 s, NA = 1). T₂ maps were skull stripped and co-registered to the template image using the surface registration feature in Analyze 12.0. The resultant T₂ maps were segmented using an in-house developed rat brain atlas that was co-registered to the same template. The average T₂ relaxation values were calculated in all segmented regions. Statistical analysis (one-way ANOVA with Student-Neuman-Keuls post hoc tests, and power statistical analysis) was performed using SigmaStat.The between-rat reproducibility of the T₂ mapping was excellent as can be seen in figure 1 that shows the T₂ maps of a representative rat brain (A) as well as the T₂ maps averaged for all rats (B) with corresponding CV% maps (C). Note that the variability in T₂ values in the areas away from the borders and CSF is below 10%, while in the places close to tissue interfaces the variability increases significantly, which is most likely caused by imperfect co-registration and variations in brain sizes and shapes. Table 1 shows the results of the segmentation of all baseline T₂ maps with corresponding estimates of the statistical power (number of animals required to detect a 5% change at 80% power and a significance level of 0.05). Figure 2 shows the changes in mean T₂ values in some anatomical regions (striatum, hippocampus, thalamus, and olfactory bulb) over time. These values were significantly elevated at the 24 hr time point in all brain regions except the olfactory bulb and could be a result of the protracted effect of general anesthesia received during the previous imaging session (at the 0 time point). At all other time points (2 hr, 2, 6, and 12 days) no significant differences were observed with respect to baseline (time point 0).It has been established that T₂ relaxation values for water protons in the normal rat brain are very stable and reproducible with high statistical power. This allows for the detection of very small changes using small group sizes.and constitutes the basis for the further development of the quantitative T₂ mapping method as a biomarker of neurotoxicity with the prospect of future formal qualification.