Preclinical Multi-Modality Data Analysis

Experimental Design
To explore the effects of reinforcing exposures to a widely abused volatile solvent, male Sprague Dawley rats were scanned prior to and following a conditioned place preference (CPP) to inhaled toluene (12 pairings of 5000 ppm for 30 mins each, every other day). The new radiotracer, 11C-toluene, was used to map the distribution and accumulation of toluene in the brain of each animal. Metabolic effects of toluene exposure were identified with FDG, while changes in brain structure were examined with Magnetization Transfer Ratio MRI imaging. Following the second MTR MRI scan, brains were harvested and stained using the Weil method for myelin. Control animals received two 11C-toluene, FDG and MTR MRI scans separated by 20 days, after which their brains were harvested and stained for myelin.

Image Processing
Images from each modality were realigned to each other, co-registered and normalized to Paxinos and Watson space using modality-specific templates.

Results

Multiple imaging modalities were used to show that 11C-toluene accumulates in white matter-rich and lipophilic regions of the brain. Structural deficits were observed in these regions following a reinforcing regimen of toluene. MTR microMRI was used to observe these deficits, and substantiated by histological evidence from the same animals. FDG microPET data revealed cortical metabolic deficits with little functional change in the thalamus and other subcortical structures.

Radioactivity distribution of 11C-toluene.
Summed time frames from the last 30 min of an 11C-toluene PET study in the rat superimposed on the MTR MRI scan from the same animal. The time activity of 11C-toluene in the striatum and white matter regions are shown on the right. Regions of highest uptake were the pons, colliculi and internal capsule. Below, the radioactivity distribution of 11C-toluene in the papio anubis brain has been superimposed onto D & H atlas space using the templates provided by Black et al. (1998).


C11-Toluene in a rat brain


		5 minutes after C11 Toluene
		25 minutes after C11 Toluene
C11 Toluene in a primate brain

Magnetization Transfer Ratio MRI
Proton density (PD, no saturation) and magnetization transfer contrast (MTC, with saturation of bound protons) images were acquired on a 9.4T microMRI (Magnex Scientific). For parametric MTR images shown below, the percentage change between PD and MTC images were calculated using the Image Algebra function in PFUS. MTR histograms from the entire cranial vault of an animal scanned 20 days apart with no intervention (brain parenchyma and ventricular space). Insets are coronal slices though the caudal striatum. Figure b. presents the pixel to pixel correspondence in MTR values between Scan 1 and Scan 2 for all of the regions in the ROI template. The fit to the data is shown as a solid line (R2 = 0.78). All microMRI work was performed in collaboration with Helene Benveniste, with the help of Bernd Foerster and David Smith.

All animals showed some degree of ventricular enlargement
Below, transaxial and coronal magnetization transfer ratio (MTR) images from a toluene-exposed animal indicate significantly enlarged ventricular spaces in the right, but not the left, hemispheres.
Images are in the neurological convention, where right = right.


MT MRI in animal #2 before toluene		MT MRI in animal #2 after 12 pairings with 5000 ppm toluene

MTR micro MRI

Weil stain for myelin performed on the same toluene-exposed animals and a separate group of controls
All seven brains (four treated and three control) were sent to Neuroscience Associates (www.neuroscienceassociates.com), where they were sectioned into 40 micron slices and block-set in a group of 7.

Four animals treated with toluene were placed on the top row while the three control animals were set into the second row. Each slide was scanned and imported into PMOD using the PVIEW tool. Slides were converted to individual image files (8 bit, Analyze format) and co-registered to each other using landmarks in the block. The resulting image file was 7 brains of 63 slices each.

Histology

Volumetric

Histology - Volumetric

The PFUS tool was used to separate each brain from the blockset and co-register it to the same animal's’ MTR MRI (below). Each brain was centered in a bounding box dictated by the template and extracted using the Resize tool. Once in identical bounding boxes, each Weil stained volume was coregistered to the corresponding MTR MRI volume from the same animal.

		MTR MRI Animal #1
		Histology Animal #1

FDG MicroPET
Metabolic activity in the same four animals was scanned at baseline and again after toluene. Parametric SUV images were generated using Image Algebra tool in PFUS, co-registered to MTR MRI and superimposed for display. Arrows indicate relatively high cortical metabolism prior to toluene-induced place preference and diminished metabolism after the CPP to toluene.


Mean Images from 4 animals before toluene	Mean Images from the same 4 animals after toluene

FDG micro PET