IS MRI-BASED VOLUME A MEDIATOR OF THE ASSOCIATION OF CUMULATIVE LEAD DOSE WITH COGNITIVE FUNCTION?

This work considers the pathway through which past occupational lead exposure impacts cognitive function using cross-sectional data. It is motivated by studies linking cumulative lead dose with brain volumes, volumes with cognitive function, and lead dose with cognitive function. It is hypothesized that the brain regions associated with lead mediate a portion of the association between lead dose and cognitive function. The data were derived from an ongoing study of 513 former organolead manufacturing workers. Using MRIs, a novel analysis was performed to investigate mediation. Volumes associated with cognitive function and lead dose were derived using registered images and used in a subsequent mediation analysis. Cumulative lead dose was associated with adverse function in the visuo-construction, executive functioning and eye-hand coordination domains. Of these, there was strong evidence of volumetric mediation of lead’s effect on cognition in the visuo-construction domain, a moderate amount for eye-hand coordination, and limited evidence for executive functioning. A second path analysis based approach was also performed. To address the possibility that chance associations explained these findings, a permuted analysis was conducted, the results of which support the mediation inferences. The approach to the evaluation of volumetric mediation may have general applicability in epidemiologic neuroimaging settings. Page 4 of 29 http://biostats.bepress. com/jhubiostat/paper140 MeSH headings: cognitive manifestations; epidemiologic factors; epidemiologic methods; lead; magnetic resonance imaging; spectrometry, X-ray emission Page 5 of 29 Hosted by The Berkeley Electronic Press We previously reported that past occupational exposure to organic and inorganic lead was associated with a decline in cognitive function (1) and with variation in the volume of brain structures as measured with magnetic resonance imaging (MRI) (2). Specifically, past cumulative absorption of lead, estimated by measurement of tibia lead (by Cd-induced X-ray fluorescence), was associated with pronounced longitudinal decline in verbal memory and learning, visual memory, and executive function, and with variation in volume of total brain, parietal white and gray matter, temporal white matter, and two relatively small paralimbic system structures (cingulate gyrus, insula), among others. These findings raise questions about the extent to which variation in cognitive function measures that are associated with tibia lead are mediated through brain volumes measured by MRI. Our previous work indicated that (peak cumulative) lead dose was associated with persistent and possibly progressive changes in the brain, both in the terms of cognition and brain structure, long after lead levels had declined in brain and blood (19). Based on these findings, we hypothesized that the brain regions associated with lead should mediate a portion of the association between lead and measures of cognitive function, recognizing that MRI only measures volume differences, not neurobiological and more subtle changes to brain structure. For this paper, we refer to these as overt (i.e., variation in MRI volumes) and covert (i.e., not detected by MRI) effects of lead (figure 1). More specifically, we hypothesize that variation in lead dose is associated with measurable variation in brain volume that, in turn, mediates variation in cognitive function. The variation in cognitive function attributable to lead that is not mediated by volume differences is assumed to be due to covert changes. Page 6 of 29 http://biostats.bepress. com/jhubiostat/paper140 Mediation is often assessed from a comparison of results from longitudinal models with and without the hypothesized mediator (10). In our case, we assume that cognitive function is mediated through brain structure and neurobiology, whether or not we are able to observe all aspects of mediation. It is hypothesized that a component of the cross-sectional variation in brain volume associated with lead dose mediates a decline in cognitive function. We argue that this decline can be assessed, indirectly, by variation in cognitive function at cross-section. That is, because our evidence indicates that the effect of lead is at least persistent, and possibly progressive, the variation in cognitive function observed at cross-section incorporates historical changes attributable to past lead exposure, as well as other factors; the same assumption holds for MRI measures of volume. Given this framework, we test the hypothesis of mediation using cross-sectional measures of historical lead dose, brain volumes, and cognitive function. We present a voxel-by-voxel analysis to understand mediation that combines the attractive qualities of region of interest analysis while not being constrained by anatomical boundaries. Finally, we propose a method using permuted analysis that addresses inherent limitations in the voxel-based method and the traditional approach to evaluation of mediation. MATERIALS AND METHODS Study Population, Design, and Data Overview. The data for this manuscript were derived from an ongoing prospective study of past occupational lead exposure and its associated health effects in central nervous system structure and function. Subjects were recruited from a chemical manufacturing plant that previously produced gasoline additives (7). Subject recruitment occurred in two phases between 1994 and 2003. Annual tests of Page 7 of 29 Hosted by The Berkeley Electronic Press neurobehavioral function were performed. Current tibia lead was measured by Cdinduced K-shell X-ray fluorescence in the third year for subjects recruited in Phase 1 (1994 to 1997) and in Phase 2 (2001 to 2003). Additional exposure information, such as the duration of exposure and the time since the last exposure, were also collected. Brain MRIs were collected during Phase 2. Detailed methods for Phase 1 and Phase 2 can be found in (2, 3) while the relevant aspects are summarized below. The study was reviewed and approved by the Johns Hopkins Bloomberg School of Public Health Committee on Human Research and written informed consent was obtained from all participants. Subject recruitment. Individuals recruited for this study were former workers in the organolead area of a chemical plant, involved in the manufacture of tetraethyl lead from 1923 to 1991 and tetramethyl lead from 1960 to 1983, but were not occupationally exposed to lead at the time of study enrollment. All study participants were previously employed in the facility on or after January 1, 1950, were male, and were between the ages of 40 and 70 years in 1995. In Phase 1, a total of 703 former lead workers were enrolled and completed one to four visits. In Phase 2, another 276 former lead workers were enrolled and completed one or two visits. During Phase 2, MRIs were completed on 589 of the 979 former lead workers. Tibia lead was measured on 532 of the 589 individuals who completed MRI acquisition. Analysis was limited to the 513 subjects who had no errors in the processing of their images and had key covariate data. One subject was eliminated because of a potential neurological disorder. Cognitive function did not differ by MRI status and the relations of tibia lead with neurobehavioral test scores did not differ in those with and without MRIs (2). Page 8 of 29 http://biostats.bepress. com/jhubiostat/paper140 Data collection. Data collection, cognitive assessment, and MRI acquisition protocols for the Phase 1 and Phase 2 studies have been previously described (1, 2). All subjects were scanned on the same General Electric 1.5 T Signa Model. Neurobehavioral test scores were aggregated into six cognitive domain scores (table 1), as previously described (11) according to neuropsychological theory and empirical evaluation of inter-test correlations and variation. Before creating domains specific measures, individual test scores were normalized. Current tibia lead was used to derive peak tibia lead levels, the estimated level at the end of occupational lead exposure, by previously published methods (3, 12). Peak tibia lead was found to be the lead dose measure most associated with cognitive test scores (3), decline in cognitive function over time (1), brain volumes, and white matter lesions (2). Image processing. Images were preprocessed using previously published methods (2, 13) and subsequently segmented (into gray matter, white matter, and cerebrospinal fluid) using a Bayesian algorithm (14). Regional analysis of volumes in normalized space (RAVENS) was used to elastically warp brain images into a standard template space while retaining volumetric information (15). The RAVENS images contained absolute volumes in standardized Talairach space (16), with separate gray and white matter images, that provide the means for inter-subject voxel-wise analysis. The principal benefit of the so called voxel-based morphometry approach, is the ability to assess associations of predictor variables in highly localized volumetric analyses without relying on anatomically constrained regions of interest (17, 18). In contrast, analysis of anatomy-based regions of interest requires assumptions on the resolution of the anatomy to be considered. For example, total brain volume can be Page 9 of 29 Hosted by The Berkeley Electronic Press decomposed into total gray and white mater volumes, which can be decomposed into smaller substructures, and so on. Voxel-based morphometry, i.e. the voxel-by-voxel statistical analysis of the RAVENS images, avoids challenges to these assumptions, as well as complications from the anatomical definitions required by traditional region of interest-based analysis, and enables an unbiased evaluation of morphometry and post hoc determination of regions of interest that are not known a priori. (In this study, these are regions that mediate the association between lead exposure and cognition). Statistical Analysis Overv