A rat head holder for simultaneous scanning of two rats in small animal PET scanners: Design, construction, feasibility testing and kinetic validation
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.).
References (16)
- et al.
Consistent and reproducible slice selection in rodent brain using a novel stereotaxic device for MRI
Journal of Neuroscience Methods
(2004) - et al.
Small animal PET
European Neuropsychopharmacology
(2002) - et al.
Evaluation of a stereotactic frame for repositioning of the rat brain in serial positron emission tomography imaging studies
Journal of Neuroscience Methods
(2001) - et al.
A head holder for magnetic resonance imaging that allows the stereotaxic alignment of spontaneously occurring intracranial mouse tumors
Journal of Neuroscience Methods
(2002) - et al.
MicroPET: a high resolution animal PET scanner for imaging small animals
IEEE Transaction on Nuclear Science
(1997) - et al.
Estimating input functions from images for FDG-PET small animal imaging without blood sampling
- et al.
Spatial registration and normalization of images
Human Brain Mapping
(1995) - et al.
Dedicated small animal scanners: a new tool for drug development?
Current Pharmaceutical Design
(2002)
Cited by (24)
3D-printed multisampling holder for microcomputed tomography applied to life and materials science research
2021, MicronCitation Excerpt :Considering the porosity, the difference between the two scan modes was more related to the differences of the open pore volume, as the closed pore volume was almost zero. These results are corroborated with some articles in the medical field, when a holder applied to in vivo positron emission tomography/computed tomography (PET/CT) scanning simultaneously small animals did not produce a significant loss of quantitative accuracy data, as well as considered the reduction of imaging cost (Cheng et al., 2009; Habte et al., 2013; Yagi et al., 2014; Greenwood et al., 2020). The comparison of the scan duration time, imaging cost, and data storage, regarding the two scan modes, is shown in Table 3.
Open-source hardware designs for MRI of mice, rats, and marmosets: Integrated animal holders and radiofrequency coils
2019, Journal of Neuroscience MethodsCitation Excerpt :This is most notably observed in the ubiquitous solution for rodent imaging, where local surface coils are placed superior to the ear bars, thereby avoiding the ear bars, but reducing the sensitive region of the receive coil. Although commercial vendors and researchers have developed animal holders for anesthetized and awake mice (Akselrod-Ballin et al., 2016; Boretius et al., 2009; Herrmann et al., 2014; Kokuryo et al., 2010; Madularu et al., 2017), rats (Cheng et al., 2009; Lahti et al., 1998; Xu et al., 2013), and marmosets (Belcher et al., 2013; Papoti et al., 2013), they are not typically made freely available (thereby not allowing for easy reproduction) and many are not developed in conjunction with the RF coil, as required to maximize performance. This manuscript describes fully integrated solutions for mice and rat imaging, as well as an MR-compatible stereotactic frame for marmoset imaging: all computer-aided-design (CAD) files are provided for components of the animal holders, including anesthesia delivery, immobilization, and the RF coil.
A 3D-printed modular device for imaging the brain of small birds
2018, Journal of Neuroscience MethodsCitation Excerpt :Furthermore, use of this device resulted in only modest PET count losses due to addition attenuating material, as shown by our PET losses measure. Although numerous devices have been designed to hold small animals during different in vivo imaging procedures, these are typically made for laboratory rodents (Cheng et al., 2009; Yagi et al., 2014), and employ design features not compatible with the anatomy of wild species used in ecological research (e.g., rodent “bite bars” are not compatible with avian anatomy). To our knowledge, our use of CT imaging to design a customized head cone based on the actual shape, size and features of the house sparrow skull is unique, although this particular approach could be used in any species.
Characterisation of partial volume effect and region-based correction in small animal positron emission tomography (PET) of the rat brain
2012, NeuroImageCitation Excerpt :Rat brain studies with those scanners will be more likely to require a spatially variant PSF for accurate PVC if the brain is mis-positioned. Also, spatially variant PSFs will be necessary for studies where the rat brain is at a larger offset from the CFOV, for example studies with multiple rats in the FOV (Cheng et al., 2009) or a rat moving within the FOV (Zhou et al., 2010). The simulation with activity in the brain only but with attenuation from the full head showed similar RMSE and BPND bias without PVC compared to the brain only simulation.
Assessment of IP injection of [<sup>18</sup>F]fallypride for behavioral neuroimaging in rats
2011, Journal of Neuroscience MethodsCitation Excerpt :A limitation of this study was that no attenuation correction was applied to the data. We have shown that a headholder (similar to the one used in this work) induces significant attenuation (Cheng et al., 2009). In that study, when attenuation correction was not applied, whole-brain values of normalized [18F]FDG activity were ∼40% lower than values from attenuation-corrected data (see Fig. 8, Cheng et al., 2009).
Small-animal positron emission tomography as a tool for neuropharmacology
2010, Trends in Pharmacological Sciences
- 1
Address: Radiology – R2 E124, 950 W. Walnut Street, Indianapolis 46202, United States.
- 2
Address: Radiology – R2 E161, 950 W. Walnut Street, Indianapolis 46202, United States. Tel.: +1 317 278 9841.
- 3
Tel.: +1 608 265 6604.