A rat head holder for simultaneous scanning of two rats in small animal PET scanners: Design, construction, feasibility testing and kinetic validation

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Abstract

To reduce imaging costs, we designed a head holder for scanning two rats simultaneously in small animal PET scanners. Our goals were (i) to maintain high sensitivity and (ii) to minimize repositioning error between scans.

Methods

A semi-stereotaxic dual rat head holder was designed and constructed for dual rat scanning in our IndyPET-II scanner and the commercial microPET P4. It was also used for single rat scanning in a small-bore, high-resolution animal scanner (“ISAP”). Positional repeatability was validated via multiple [11C]Raclopride scans of a single rat on different days. Accuracy of repositioning was determined by visual comparison of images, and by metrics derived through image alignment.

Kinetic validation was assessed via analysis of [18F]Fluorodeoxyglucose ([18F]FDG) dynamic PET studies of six rats. Each rat was scanned twice: once individually, with brain positioned at the center of field of view (CFOV), and once with a partner, with brain away from CFOV. Both rats were injected with FDG during each dual rat session. Patlak uptake constants (Ki) were calculated from whole brain images. Effects of attenuation and scatter correction on single versus dual scan images were explored.

Results

Image comparison and alignment metrics indicated excellent repositioning of rats. Scaled time-activity-curves from single and dual rat scans were indistinguishable. Average single and dual scan Ki values differed by only 6.3 ± 7.5%.

Conclusion

Dual rat scanning in a semi-stereotaxic holder is practical for economical small animal scanning and does not compromise kinetic accuracy of [18F]FDG dynamic scan data.

Introduction

Positron emission tomography (PET) is prized for its unique capability in imaging physiological processes in vivo. Using molecularly specific tracers, PET images can be used to track the local kinetics of physiological processes by extracting time activity curves (TACs) from regions of interest (ROIs). Fitting models to TACs leads to estimation of physiologically relevant kinetic parameters. In preclinical studies, rodents are often scanned with small animal PET scanners. As the use of small animal PET proliferates, interest in high-throughput, quantitative imaging is growing.

A significant limitation of PET experiments is cost. PET scans of short lived tracers with moderate to low specific activity are particularly expensive because the radioactivity from a single synthesis is often insufficient or the mass too great to support a second tracer experiment. Functional studies that focus on repeated measures of activity concentration in small regions may suffer a second possible limitation. Repeatable positioning of the animal with respect to the field of view (FOV) is important to avoid subtle confounds due to the spatially varying response of the PET scanner (i.e., spatially variant point-spread-function). We addressed both concerns by designing and building a dual rat head holder to enable simultaneous scanning of two rats while maintaining repeatable repositioning of the brains from scan to scan.

Concomitant with the interest in small animal scanning is a great interest in holders and positional reproducibility (Lecomte et al., 1994, Cherry et al., 1997, Jeavons et al., 1999, Hume and Myers, 2002, Myers and Hume, 2002). Previous work by Rubins et al. (2001) examined use of sharp and blunt ear bars in a single rat holder for repeatable positioning and recommended sharper ear bars for less positional variation. Tada et al. (2002) and Fricke et al. (2004) introduced holders that could be used in MRI scanners. Much of the work of other investigators in small animal scanning has been on slice or positional reproducibility within a single rat. Our focus has been on the feasibility of simultaneous dual rat scanning and positional repeatability.

Possible drawbacks of placing two rats simultaneously in a PET scanner include increased signal attenuation and scatter due to increased object mass in the FOV, and increased dead time due to the doubling of injected activity within the scanner. To fit two rats simultaneously in a gantry, the brains have to be positioned away from the center of the field of view (CFOV), where performance of the scanner is optimal. As the subjects are moved away from CFOV, we can expect some loss of resolution and sensitivity. Loss of resolution could make it more difficult to identify small brain structures. Loss of sensitivity could lead to unwanted mass effects if additional injected activity were needed to achieve sufficient signal to noise ratio. Therefore, scanning two rats simultaneously could impact the quality of our images, and subsequently degrade the accuracy of the estimated kinetic parameters. We validated the dual rat head holder through comparisons of [18F]FDG uptake constants (Ki), derived from single and dual rat scans of the same rats in two small animal scanners, the IndyPET-II and the microPET P4. If the holder has only limited effects on Ki, we infer that it will have little effect on other kinetic parameters, such as those derived for neuroligand tracers.

Half of the rat holder can be used by itself to hold one rat (“single rat mode”). Repeatability of positioning a single rat was tested using the holder in single rat mode in a small-bore small animal scanner.

Section snippets

Holder design and fabrication

To assure repeatable positioning, the dual rat head holder was designed based on the concept of positioning a rat’s head via semi-stereotaxic methods. That is, we chose to immobilize each rat using two ear bars that are inserted, one through each ear canal, and pressed against an indentation in the skull, and a bite bar over which the incisors are hooked. The dual rat holder, based partly on the Kopf Model 900M (David Kopf Instruments, Tujunga, CA), is comprised of four ear-bars, four ear-bar

Holder design and fabrication

The dual rat head holder was used in both IndyPET-II and microPET P4. In IndyPET-II, the holders were oriented obliquely to the main axis of the bore, so that brains could be positioned 60 mm radially and 8 mm axially away from each other. Distances between the thru-holes at one end and the slots at the other end were designed and machined such that when the holder was positioned in IndyPET-II via these mechanisms, each brain lay equidistant from the CFOV. The intent was to place each brain

Effect of dual versus single mode scanning

We tested our rat head holder in two different scanners with and without attenuation and scatter corrections and compared its use in dual and single modes. In dual mode, the presence of the second animal contributes negligibly to the estimate of FDG uptake constant in either animal. This finding was insensitive to scanner or to the application of scatter and/or attenuation correction.

Experimental procedures using the dual rat head holder

For experienced users, ear-bar insertion typically takes less than a minute. For that short period of time, the

Conclusions

Kinetic data derived from scanning two rats simultaneously are comparable to those derived from scans of individual rats. The difference in Ki caused by dual scanning as opposed to single scanning is not statistically significant. Dual rat scanning in a semi-stereotaxic holder is practical for economical small animal scanning and does not compromise kinetic accuracy of [18F]FDG dynamic scan data.

Acknowledgements

The authors wish to thank Brian McCarthy for help with PET scanning, Larry Corbin and Joe Huerkamp for help with machining and Wendy Winkle and Dr. Gary Hutchins for their help in administering the Indiana Center of Excellence in Biomedical Imaging. This work was funded in part by NIH grants R21 AA015077 (to EDM), P60 AA007611-16 (to the Indiana Alcohol Research Center) and Indiana Genomics Initiative (INGEN, supported in part by the Lilly Endowment, Inc.).

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