Preclinical Small Animal Studies Research Articles

Whole Blood Assay with Dual Co-Stimulation for Antigen-Specific Analysis of Host Immunity to Fungal and Viral Pathogens.

Rapid and resource-efficient sample processing, high throughput, and high robustness are critical for effective scientific and clinical application of advanced antigen-specific immunoassays. Traditionally, such immunoassays, especially antigen-specific T-cell analysis by flow cytometry or enzyme-linked immunosorbent spot assays, often rely on the isolation of peripheral blood mononuclear cells. This process is time-consuming, subject to many pre-analytic confounders, and requires large blood volumes. Whole blood-based assays provide a facile alternative with increased pre-analytic robustness and lower blood volume requirements. Furthermore, whole blood-based assays allow for the preservation of inter-cellular interactions that are not captured by assays using isolated cell subsets. Recently, a refined whole blood immunoassay with dual anti-CD28 and anti-CD49d co-stimulation for comprehensive analysis of both antigen-specific T-cell functions and complex intercellular interactions in response to various fungal and viral antigens has been proposed. This protocol provides guidance for the preparation of stimulation tubes, blood stimulation, and downstream sample processing for flow cytometry, cytokine secretion assays, and transcriptional analyses. This includes a validated and functionally equivalent, previously unpublished, low-volume protocol (250 µL) to make flow cytometric and cytokine-based T-cell monitoring more accessible for studies in pediatric patients or preclinical studies in small animals (e.g., mice). Altogether, these protocols provide a versatile toolbox for complex antigen-specific immune analysis in both clinical and translational research settings.

From lab bench to hope: a review of gene therapies in clinical trials for Parkinson's disease and challenges.

Parkinson's disease (PD) is a chronic neurological disorder that is identified by a characteristic combination of symptoms such as bradykinesia, resting tremor, rigidity, and postural instability. It is the second most common neurodegenerative disease after Alzheimer's disease and is characterized by the progressive loss of dopamine-producing neurons in the brain. Currently, available treatments for PD are symptomatic and do not prevent the disease pathology. There is growing interest in developing disease-modifying therapy that can reduce disease progression and improve patients' quality of life. One of the promising therapeutic approaches under evaluation is gene therapy utilizing a viral vector, adeno-associated virus (AAV), to deliver transgene of interest into the central nervous system (CNS). Preclinical studies in small animals and nonhuman primates model of PD have shown promising results utilizing the gene therapy that express glial cell line-derived neurotrophic factor (GDNF), cerebral dopamine neurotrophic factor (CDNF), aromatic L-amino acid decarboxylase (AADC), and glutamic acid decarboxylase (GAD). This study provides a comprehensive review of the current state of the above-mentioned gene therapies in various phases of clinical trials for PD treatment. We have highlighted the rationale for the gene-therapy approach and the findings from the preclinical and nonhuman primates studies, evaluating the therapeutic effect, dose safety, and tolerability. The challenges associated with gene therapy for heterogeneous neurodegenerative diseases, such as PD, have also been described. In conclusion, the review identifies the ongoing promising gene therapy approaches in clinical trials and provides hope for patients with PD.

Visualization of Pulse-Wave Velocity on Arterial Wall of Mice Through High-Frequency Ultrafast Doppler Imaging.

Arterial pulse-wave velocity (PWV) is widely used in clinical applications to assess cardiovascular diseases. Ultrasound methods have been proposed for estimating regional PWV in human arteries. Furthermore, high-frequency ultrasound (HFUS) has been applied to perform preclinical small-animal PWV measurements; however, electrocardiogram (ECG)-gated retrospective imaging is required to achieve high-frame-rate imaging, which might be affected by arrhythmia-related problems. In this article, HFUS PWV mapping based on 40-MHz ultrafast HFUS imaging is proposed to visualize PWV on mouse carotid artery to measure arterial stiffness without ECG gating. In contrast to most other studies that used cross-correlation methods to detect arterial motion, ultrafast Doppler imaging was applied in this study to measure arterial wall velocity for PWV estimations. The performance of the proposed HFUS PWV mapping method was verified using a polyvinyl alcohol (PVA) phantom with various freeze-thaw cycles. Small-animal studies were then performed in wild-type (WT) mice and in apolipoprotein E knockout (ApoE KO) mice that were fed a high-fat diet (for 16 and 24 weeks). The Young's modulus of the PVA phantom measured through HFUS PWV mapping was 15.3 ± 0.81, 20.8 ± 0.32, and 32.2 ± 1.11 kPa for three, four, and five freeze-thaw cycles, respectively, and the corresponding measurement biases (relative to theoretical values) were 1.59%, 6.41%, and 5.73%, respectively. In the mouse study, the average PWVs were 2.0 ± 0.26, 3.3 ± 0.45, and 4.1 ± 0.22 m/s for 16-week WT, 16-week ApoE KO, and 24-week ApoE KO mice, respectively. The PWVs of ApoE KO mice increased during the high-fat diet feeding period. HFUS PWV mapping was used to visualize the regional stiffness of mouse artery, and a histology confirmed that the plaque formation in the bifurcation region increased the regional PWV. All the results indicate that the proposed HFUS PWV mapping method is a convenient tool for investigating arterial properties in preclinical small-animal studies.

Combined proton radiography and irradiation for high-precision preclinical studies in small animals.

Proton therapy has become a popular treatment modality in the field of radiooncology due to higher spatial dose conformity compared to conventional radiotherapy, which holds the potential to spare normal tissue. Nevertheless, unresolved research questions, such as the much debated relative biological effectiveness (RBE) of protons, call for preclinical research, especially regarding in vivo studies. To mimic clinical workflows, high-precision small animal irradiation setups with image-guidance are needed. A preclinical experimental setup for small animal brain irradiation using proton radiographies was established to perform planning, repositioning, and irradiation of mice. The image quality of proton radiographies was optimized regarding the resolution, contrast-to-noise ratio (CNR), and minimal dose deposition in the animal. Subsequently, proof-of-concept histological analysis was conducted by staining for DNA double-strand breaks that were then correlated to the delivered dose. The developed setup and workflow allow precise brain irradiation with a lateral target positioning accuracy of<0.26mm for in vivo experiments at minimal imaging dose of<23mGy per mouse. The custom-made software for image registration enables the fast and precise animal positioning at the beam with low observer-variability. DNA damage staining validated the successful positioning and irradiation of the mouse hippocampus. Proton radiography enables fast and effective high-precision lateral alignment of proton beam and target volume in mouse irradiation experiments with limited dose exposure. In the future, this will enable irradiation of larger animal cohorts as well as fractionated proton irradiation.

Whole-Body Photoacoustic Imaging Techniques for Preclinical Small Animal Studies.

Photoacoustic imaging is a hybrid imaging technique that has received considerable attention in biomedical studies. In contrast to pure optical imaging techniques, photoacoustic imaging enables the visualization of optical absorption properties at deeper imaging depths. In preclinical small animal studies, photoacoustic imaging is widely used to visualize biodistribution at the molecular level. Monitoring the whole-body distribution of chromophores in small animals is a key method used in preclinical research, including drug-delivery monitoring, treatment assessment, contrast-enhanced tumor imaging, and gastrointestinal tracking. In this review, photoacoustic systems for the whole-body imaging of small animals are explored and summarized. The configurations of the systems vary with the scanning methods and geometries of the ultrasound transducers. The future direction of research is also discussed with regard to achieving a deeper imaging depth and faster imaging speed, which are the main factors that an imaging system should realize to broaden its application in biomedical studies.

Microneedle Array Technique for the Longitudinal Extraction of Interstitial Fluid without Hair Removal.

Interstitial fluid (ISF) bathes the cells and tissues and is in constant exchange with blood. As an exchange medium for waste, nutrients, exosomes, and signaling molecules, ISF is recognized as a plentiful source of biomolecules. Many basic and pre-clinical small animal studies could benefit from an inexpensive and efficient technique that allows for the in vivo extraction of ISF for the subsequent quantification of molecules in the interstitial space. We have previously reported on a minimally invasive technique for the extraction of ISF using a 3D-printed microneedle array (MA) platform for comprehensive biomedical applications. Previously, hairless animal models were utilized, and euthanasia was performed immediately following the procedure. Here, we demonstrate the technique in Sprague Dawley rats, without the need for hair removal, over multiple extractions and weeks. As an example of this technique, we report simultaneous quantification of the heavy metals Copper (Cu), Lead (Pb), Lithium (Li), and Nickel (Ni) within the ISF, compared with whole blood. These results demonstrate the MA technique applicability to a broader range of species and studies and the reuse of animals, leading to a reduction in number of animals needed to successfully complete ISF extraction experiments.

Mechanical ventilation restores blood gas homeostasis and diaphragm muscle strength in ketamine/medetomidine-anaesthetized rats.

What is the central question of the study? Does respiratory support ensure blood gas homeostasis and the relevance of experimental outcomes? What is the main finding and its importance? Spontaneous breathing during surgical intervention under anaesthesia results in impaired gas exchange and loss of diaphragm muscle strength in rats. Subsequent short-term mechanical ventilation restored blood gas homeostasis and diaphragm muscle strength. Blood gas homeostasis interferes substantially with experimental conditions and may alter study results. Monitoring and maintenance of blood gas balance is required to ensure quality and relevance of physiological animal experiments. In pre-clinical small animal studies with surgical interventions under general anaesthesia, animals are often left to breathe spontaneously. However, anaesthesia may impair respiratory functions and result in disturbed blood gas homeostasis. In turn, the disturbed blood gas homeostasis can affect physiological functions and thus unintentionally impact the experimental results. We hypothesized that short-term mechanical ventilation restores blood gas balance and physiological functions despite anaesthesia and surgical interventions. Therefore, we investigated variables of blood gas analyses and diaphragm muscle strength in rats anaesthetized with ketamine/medetomidine after tracheotomy and catheterization of the carotid artery under spontaneous breathing and after 20min of mechanical ventilation following the same surgical intervention. Spontaneous breathing during general anaesthesia and surgical intervention resulted in unphysiological blood oxygen partial pressure (<65mmHg) and carbon dioxide partial pressure (>55mmHg). After subsequent short-term mechanical ventilation, blood gas partial pressures were restored to their physiological ranges. Additionally, diaphragm muscle strength of animals breathing spontaneously was lower compared to animals that received subsequent mechanical ventilation (P=0.0063). We conclude that spontaneous breathing of rats under ketamine/medetomidine anaesthesia is not sufficient to maintain a physiological blood gas balance. Disturbed blood gas balance is related to reduced diaphragm muscle strength. Mechanical ventilation for only 20min restores blood gas homeostasis and muscle strength. Therefore, monitoring and maintenance of blood gas balance should be conducted to ensure quality and relevance of small animal experiments.

Focused kV X-rays for Preclinical Studies of Radiation-based Neuromodulation

Clinical observations suggest that precisely targeted irradiation can alter local neuronal function without neuronal destruction. The advancement of functional brain imaging has revealed regions of hypermetabolism that correlate with psychiatric illnesses. Radiation-based neuromodulation may offer an optimal tool to downregulate the hypermetabolic foci while preserving basic nerve functions. Achieving reliable radiomodulation in the absence of ablation, however, requires a delicate balance. Because there exists very little radiobiology information about radiomodulation and many neuroanatomical targets involved in psychiatric behaviors are believed to be similar in rodents and humans, preclinical small animal studies are critical for target identification, treatment dose planning, and treatment response evaluation. However, the target sizes in laboratory rodents are frequently below the minimum radiation field size of currently available devices. We performed Monte Carlo (MC) simulation to prove the concept of using focused kV x-ray technique to precisely deliver dose to ∼1 mm target in a rodent brain. Experiments were performed to focus 45 kVp x-rays with a polycapillary x-ray lens, which consists of a large number of small hollow curved and tapered glass tubes. The lens guides x-rays through these tubes resembling fiber optics by multiple total reflections. The x-ray transport in the lens was modeled by MC-based geometric ray tracing including defect modeling. The doses were calculated using a simulation toolkit and confirmed by phantom measurements using solid water sandwiched with films. A geometric rat brain model was created for MC treatment planning and dosimetric calculation. A focal spot size of <0.2 mm perpendicular to the beam direction can be achieved and maintained over ∼5 mm in the beam direction. The EBT3 film measured focal spot sizes and depth doses along the beam axis agree with the simulations within 10%. An example simulation was performed using multi-arc scanning for conformal irradiation to the shell region of one rat nucleus accumbens with a volume of 0.5 (LR) x 1.5 (SI) x 0.5 (AP) mm3 plus a 0.25 mm margin. The region is fundamental in brain activity related to addiction. The dose conforms to the target with fast dose fall off outside. The 70% isodose line covers 1.00 x 2.00 x 1.05 mm3. The 90% to 20% dose fall-offs are 0.2 (LR), 0.2 (SI), and 1.0 (AP) mm, respectively. AP is the depth direction of the partial arcs. Its penumbra can be improved further with arcs from more non-coplanar angles. This will also reduce the skull dose; whose current max is 47% of the prescription dose. The use of focused kV x-rays allows precise irradiation to target of just ∼1 mm without irradiating a large surrounding region, which could alter the treatment response and thus the clinical translation potential. It enables the study of radio-neuromodulation and dose volume effect to a small functional region in rat brain, which is impossible with current devices.

Magnetic Resonance Imaging-Guided Focused Ultrasound Positioning System for Preclinical Studies in Small Animals.

ObjectivesA positioning device compatible with magnetic resonance imaging (MRI) used for preclinical studies in small animals was developed that fits in MRI scanners up to 7 T. The positioning device was designed with two computer‐controlled linear stages.MethodsThe positioning device was evaluated in an agar‐based phantom, which mimics soft tissues, and in a rabbit. Experiments with this positioning device were performed in an MRI system using the agar‐based phantom. The transducer used had a diameter of 50 mm, operated at 0.5 MHz, and focused energy at 60 mm.ResultsMagnetic resonance thermometry was used to assess the functionality of the device, which showed adequate deposition of thermal energy and sufficient positional accuracy in all axes.ConclusionsThe proposed system fits in MRI scanners up to 7 T. Because of the size of the positioning device, at the moment, it can be used to perform preclinical studies on small animals such as mice, rats, and rabbits.

PHLuc, a Ratiometric Luminescent Reporter for in vivo Monitoring of Tumor Acidosis.

Even under normoxia, cancer cells exhibit increased glucose uptake and glycolysis, an occurrence known as the Warburg effect. This altered metabolism results in increased lactic acid production, leading to extracellular acidosis and contributing to metastasis and chemoresistance. Current pH imaging methods are invasive, costly, or require long acquisition times, and may not be suitable for high-throughput pre-clinical small animal studies. Here, we present a ratiometric pH-sensitive bioluminescence reporter called pHLuc for in vivo monitoring of tumor acidosis. pHLuc consists of a pH-sensitive GFP (superecliptic pHluorin or SEP), a pH-stable OFP (Antares), and Nanoluc luciferase. The resulting reporter produces a pH-responsive green 510nm emission (from SEP) and a pH-insensitive red-orange 580nm emission (from Antares). The ratiometric readout (R580/510) is indicative of changes in extracellular pH (pHe). In vivo proof-of-concept experiments with NSG mice model bearing human synovial sarcoma SW982 xenografts that stably express the pHLuc reporter suggest that the level of acidosis varies across the tumor. Altogether, we demonstrate the diagnostic value of pHLuc as a bioluminescent reporter for pH variations across the tumor microenvironment. The pHLuc reporter plasmids constructed in this work are available from Addgene.

A fast field-cycling MRI relaxometer for physical contrasts design and pre-clinical studies in small animals

We present a fast field-cycling NMR relaxometer with added magnetic resonance imaging capabilities. The instrument operates at a maximum proton Larmor frequency of 5 MHz for a sample volume of 35 mL. The magnetic field homogeneity across the sample is 1400 ppm. The main field is generated with a notch-coil electromagnet of own design, fed with a current whose stability is 220 ppm. We show that images of reasonable quality can still be produced under such conditions. The machine is being designed for concept testing of the involved instrumentation and specific contrast agents aimed for field-cycling magnetic resonance imaging applications. The general performance of the prototype was tested through localized relaxometry experiments, T1-dispersion weighted images, temperature maps and T1-weighted images at different magnetic field intensities. We introduce the concept of positive and negative contrast depending on the use of pre-polarized or non-polarized sequences. The system is being improved for pre-clinical studies in small animals.

Preclinical ultrasound for development translatable model of metabolic syndrome in small animals

Objectives Diagnostic ultrasound (US) using general US imaging devices can be effectively used for preclinical studies in small animals providing dynamic life-time control [1], furthermore, can enhance drug delivery and therapeutic effects with visual treatment monitoring (theranostic). The Aim was to develop model of metabolic syndrome in small animals using general US machines for longitudinal in vivo observation and screening large numbers of cases for facilitating further translation. Methods The modeling of metabolic syndrome performed compliance with the ethical standards and includes conducting an experiment on laboratory animals (mice, rats, murine) with the introduction of high calorie diet or industrial fat-enriched diet; and further US monitoring using 5–20 MHz probes of diagnostic US machines in grey scale, Doppler, sonoelastography, M-mode detecting tissue movement, US-guided interventions, injection US contrast agents: 1) for precise diagnosis transabdominal US detecting signs of metabolic syndrome via detailed imaging of internal organs: liver size, echogeneicity, stiffness, kidneys structure, Doppler measuring resistance index (RI) on segmental renal arteries, spleen length, muscle thickness at the midfemoral level, assessment of visceral vessels, systemic hemodynamics, etc.; 2) for screening all involved animals we measured the visceral fat thickness (threshold considered as 1.5 mm in mice) on sagittal probe position and collected records of panoramic abdominal scans (in sagittal and transverse probe positions) and measured the largest longitudinal liver size (via subcostal approach). Weight, body size, laboratory indices (cholesterol, uric acid, glucose, etc.), microbiome, genetic markers were also determined. After sacrificing we evaluated studied organs. Results The model was successfully applied to study effects of new drugs: probiotic strains on high calorie-induced obesity model in BALB/c during 21 days [2] and prebiotic on high-calorie diet-induced obesity in rats [3]. US detected development metabolic syndrome, endogenous intoxication syndrome, visceral obesity and liver and kidney dysfunction in mouse and rats. Ultrasound data showed visceral obesity, injury of the liver and organs in all experimental animals. We revealed nephropathy signs (thinning, increasing echogenicity of kidney parenchyma, detecting increasing RI in renal arteries (over 0.7) was feasible in rats. Studies using the models demonstrated efficacy of studied strains, substances improving parameters during experiment. All observed changes were confirmed post mortem. Conclusions The method of modeling is reliable, allows to monitor metabolic syndrome signs with high translation potential reflecting development disease in humans.

Characterization of novel preclinical dose distributions for micro irradiator

This work explores and demonstrates the feasibility of utilizing new 3D printing techniques to implement advanced micro radiation therapy for pre-clinical small animal studies. 3D printed blocks and compensators were designed and printed from a strong x-ray attenuating material at sub-millimeter resolution. These techniques enable a powerful range of new preclinical treatment capabilities including grid therapy, lattice therapy, and IMRT treatment. At small scales, verification of these treatments is exceptionally challenging, and high resolution 3D dosimetry (0.5mm3) is an essential capability to characterize and verify these capabilities, Here, investigate the 2D and 3D dosimetry of several novel pre-clinical treatments using a combination of EBT film and Presage/optical-CT 3D dosimetry in rodent-morphic dosimeters.

Reconstruction Method for In Vivo Bioluminescence Tomography Based on the Split Bregman Iterative and Surrogate Functions.

Bioluminescence tomography (BLT) can provide in vivo three-dimensional (3D) images for quantitative analysis of biological processes in preclinical small animal studies, which is superior than the conventional planar bioluminescence imaging. However, to reconstruct light sources under the skin in 3D with desirable accuracy and efficiency, BLT has to face the ill-posed and ill-conditioned inverse problem. In this paper, we developed a new method for BLT reconstruction, which utilized the mathematical strategies of the split Bregman iterative and surrogate functions (SBISF) method. The proposed method considered the sparsity characteristic of the reconstructed sources. Thus, the sparsity itself was regarded as a kind of a priori information, and the sparse regularization is incorporated, which can accurately locate the position of the sources. Numerical simulation experiments of multisource cases with comparative analyses were performed to evaluate the performance of the proposed method. Then, a bead-implanted mouse and a breast cancer xenograft mouse model were employed to validate the feasibility of this method in in vivo experiments. The results of both simulation and in vivo experiments indicated that comparing with the L1-norm iteration shrinkage method and non-monotone spectral projected gradient pursuit method, the proposed SBISF method provided the smallest position error with the least amount of time consumption. The SBISF method is able to achieve high accuracy and high efficiency in BLT reconstruction and hold great potential for making BLT more practical in small animal studies.

Bioluminescence-Based Tumor Quantification Method for Monitoring Tumor Progression and Treatment Effects in Mouse Lymphoma Models

Although bioluminescence imaging (BLI) shows promise for monitoring tumor burden in animal models of cancer, these analyses remain mostly qualitative. Here we describe a method for bioluminescence imaging to obtain a semi-quantitative analysis of tumor burden and treatment response. This method is based on the calculation of a luminoscore, a value that allows comparisons of two animals from the same or different experiments. Current BLI instruments enable the calculation of this luminoscore, which relies mainly on the acquisition conditions (back and front acquisitions) and the drawing of the region of interest (manual markup around the mouse). Using two previously described mouse lymphoma models based on cell engraftment, we show that the luminoscore method can serve as a noninvasive way to verify successful tumor cell inoculation, monitor tumor burden, and evaluate the effects of in situ cancer treatment (CpG-DNA). Finally, we show that this method suits different experimental designs. We suggest that this method be used for early estimates of treatment response in preclinical small-animal studies.

Bioluminescence-Based Tumor Quantification Method for Monitoring Tumor Progression and Treatment Effects in Mouse Lymphoma Models

Although bioluminescence imaging (BLI) shows promise for monitoring tumor burden in animal models of cancer, these analyses remain mostly qualitative. Here we describe a method for bioluminescence imaging to obtain a semi-quantitative analysis of tumor burden and treatment response. This method is based on the calculation of a luminoscore, a value that allows comparisons of two animals from the same or different experiments. Current BLI instruments enable the calculation of this luminoscore, which relies mainly on the acquisition conditions (back and front acquisitions) and the drawing of the region of interest (manual markup around the mouse). Using two previously described mouse lymphoma models based on cell engraftment, we show that the luminoscore method can serve as a noninvasive way to verify successful tumor cell inoculation, monitor tumor burden, and evaluate the effects of in situ cancer treatment (CpG-DNA). Finally, we show that this method suits different experimental designs. We suggest that this method be used for early estimates of treatment response in preclinical small-animal studies.

TH-AB-204-12: Practical/ultrasensitive Benchtop Gold L-Shell XRF Imaging System with a Kilowatt-Range X-Ray Source and Silicon Drift Detector

Purpose: To improve the sensitivity and throughput of a benchtop x-ray fluorescence (XRF) imaging system capable of locating and quantifying distributions of gold nanoparticles (GNPs) by detecting gold L-shell XRF photons from subcentimeter-sized ex vivo samples or superficial tumors for preclinical small animal studies. Methods: A proof-of-principle L-shell XRF imaging system has been developed previously. Originally, the system was configured with a low-power (∼50 W) x-ray source (62 kVp, 0.8 mA) and a silicon PIN diode detector. The detection limit of the system was approximately 0.02 mg/cm3 (20 ppm) for an acquisition time of 10 min. Currently, the system has been retrofitted with a high-power (∼3 kW) x-ray source. The detector was replaced with a silicon drift detector (SDD) to handle the increased photon flux at the new settings (62 kVp, 45 mA). Calibration was performed using phantoms containing water/GNPs at a wide range of concentrations (0 and 0.0001–10 mg/cm3). The acquisition time was set to 10 s (60 times shorter than that used with the former configuration, to maintain the same overall x-ray fluence and dose). The XRF/scatter spectrum at 90° was acquired from each phantom. The data were processed to extract XRF signal from background, which was then corrected for attenuation using a Compton-scatter-based normalization algorithm. The corrected XRF signals were plotted vs. GNP concentration and analyzed to determine the detection limit. Results: The detection limit with the current configuration was found to be 0.007 mg/cm3 (7 ppm), a three-fold improvement compared to the former configuration for the same dose. Conclusion: By adopting a high-power x-ray source and SDD, the material detection limit has been improved to a level comparable to that of a synchrotron-based system. Concurrently, much shorter acquisition time afforded by the higher photon flux makes the current setup practical for routine preclinical imaging tasks.

Erroneous cardiac ECG-gated PET list-mode trigger events can be retrospectively identified and replaced by an offline reprocessing approach: first results in rodents

The assessment of left ventricular function, wall motion and myocardial viability using electrocardiogram (ECG)-gated [18F]-FDG positron emission tomography (PET) is widely accepted in human and in preclinical small animal studies. The nonterminal and noninvasive approach permits repeated in vivo evaluations of the same animal, facilitating the assessment of temporal changes in disease or therapy response. Although well established, gated small animal PET studies can contain erroneous gating information, which may yield to blurred images and false estimation of functional parameters. In this work, we present quantitative and visual quality control (QC) methods to evaluate the accuracy of trigger events in PET list-mode and physiological data. Left ventricular functional analysis is performed to quantify the effect of gating errors on the end-systolic and end-diastolic volumes, and on the ejection fraction (EF). We aim to recover the cardiac functional parameters by the application of the commonly established heart rate filter approach using fixed ranges based on a standardized population. In addition, we propose a fully reprocessing approach which retrospectively replaces the gating information of the PET list-mode file with appropriate list-mode decoding and encoding software. The signal of a simultaneously acquired ECG is processed using standard MATLAB vector functions, which can be individually adapted to reliably detect the R-peaks. Finally, the new trigger events are inserted into the PET list-mode file. A population of 30 mice with various health statuses was analyzed and standard cardiac parameters such as mean heart rate (119 ms ± 11.8 ms) and mean heart rate variability (1.7 ms ± 3.4 ms) derived. These standard parameter ranges were taken into account in the QC methods to select a group of nine optimal gated and a group of eight sub-optimal gated [18F]-FDG PET scans of mice from our archive. From the list-mode files of the optimal gated group, we randomly deleted various fractions (5% to 60%) of contained trigger events to generate a corrupted group. The filter approach was capable to correct the corrupted group and yield functional parameters with no significant difference to the optimal gated group. We successfully demonstrated the potential of the fully reprocessing approach by applying it to the sub-optimal group, where the functional parameters were significantly improved after reprocessing (mean EF from 41% ± 16% to 60% ± 13%). When applied to the optimal gated group the fully reprocessing approach did not alter the functional parameters significantly (mean EF from 64% ± 8% to 64 ± 7%). This work presents methods to determine and quantify erroneous gating in small animal gated [18F]-FDG PET scans. We demonstrate the importance of a quality check for cardiac triggering contained in PET list-mode data and the benefit of optionally reprocessing the fully recorded physiological information to retrospectively modify or fully replace the cardiac triggering in PET list-mode data. We aim to provide a preliminary guideline of how to proceed in the presence of errors and demonstrate that offline reprocessing by filtering erroneous trigger events and retrospective gating by ECG processing is feasible. Future work will focus on the extension by additional QC methods, which may exploit the amplitude of trigger events and ECG signal by means of pattern recognition. Furthermore, we aim to transfer the proposed QC methods and the fully reprocessing approach to human myocardial PET/CT.

TH-A-141-04: Localization and Quantification of Gold Nanoparticles Using L-Shell Fluorescence Imaging with a Benchtop X-Ray Source

Purpose: To develop a high sensitivity L-shell x-ray fluorescence imaging system that locates and quantifies sparse concentrations of gold nanoparticles (GNPs) using a polychromatic benchtop x-ray source. Methods: An L-shell x-ray fluorescence imaging system was built with a benchtop polychromatic x-ray source and a Si-PIN detector. Water-filled cylindrical tubes (12 mm in diameter) loaded with GNPs at 2%, 1%, 0.5%, 0.05%, and 0.005% GNP by weight were served as calibration phantoms. An imaging phantom was created using the same cylindrical tube but filled with water-equivalent gel containing structures mimicking GNP-loaded blood vessel and 1 cc tumor. The phantoms were irradiated by a 3-mm diameter 65 kVp x-rays filtered by 1 mm aluminum. Fluorescence/scatter photons from the phantoms were detected at 90° with respect to the beam direction using a Si-PIN detector with a pin-hole collimation. For the imaging phantom, the detector was translated horizontally and vertically in 0.3-mm steps to create a 2-D fluorescence image of the phantom. The net L-shell fluorescence signal from GNPs was extracted from background, and then corrected for detector efficiency and in-phantom attenuation using a fluorescence-to-scatter normalization algorithm. The corrected signal from the calibration phantoms was used to create a calibration curve showing a linear relationship between corrected fluorescence signal and GNP mass per image voxel. Results: The results suggest that the current setup can detect a GNP mass of 500 ng (or 0.5 ppm) contained within each image voxel (0.0173 cc). The 2-D fluorescence image properly correlated the known spatial distribution and amount of GNPs within the imaging phantom. Conclusion: L-shell fluorescence imaging can be a highly sensitive tool that has the capability of simultaneously imaging the spatial distribution and determining the local concentration of GNPs presented within ex-vivo samples and superficial tumors during pre-clinical small animal studies. Supported by NIH/NCI grant R01CA155446; NIH/NCI grant R01CA155446

Harnessing the Power of Radionuclides for Optical Imaging: Cerenkov Luminescence Imaging

Over the past several years, nuclear imaging modalities such as PET and SPECT have received much attention because they have been instrumental not only in preclinical cancer research but also in nuclear medicine. Yet nuclear imaging is limited by high instrumentation cost and subsequently low availability to basic researchers. Cerenkov radiation, a relativistic physical phenomenon that was discovered 70 years ago, has recently become an intriguing subject of study in molecular imaging because of its potential in augmenting nuclear imaging, particularly in preclinical small-animal studies. The intrinsic capability of radionuclides emitting luminescent light from decay is promising because of the possibility of bridging nuclear imaging with optical imaging-a modality that is much less expensive, is easier to use, and has higher throughput than its nuclear counterpart. Thus, with the maturation of this novel imaging technology using Cerenkov radiation, which is termed Cerenkov luminescence imaging, it is foreseeable that advances in both nuclear imaging and preclinical research involving radioisotopes will be significantly accelerated in the near future.