Stuart Gilchrist

Cytoplasmic Irradiation Induces Mitochondrial-Dependent 53BP1 Protein Relocalization in Irradiated and Bystander Cells

The accepted paradigm for radiation effects is that direct DNA damage via energy deposition is required to trigger the downstream biological consequences. The radiation-induced bystander effect is the ability of directly irradiated cells to interact with their nonirradiated neighbors, which can then show responses similar to those of the targeted cells. p53 binding protein 1 (53BP1) forms foci at DNA double-strand break sites and is an important sensor of DNA damage. This study used an ionizing radiation microbeam approach that allowed us to irradiate specifically the nucleus or cytoplasm of a cell and quantify response in irradiated and bystander cells by studying ionizing radiation-induced foci (IRIF) formation of 53BP1 protein. Our results show that targeting only the cytoplasm of a cell is capable of eliciting 53BP1 foci in both hit and bystander cells, independently of the dose or the number of cells targeted. Therefore, direct DNA damage is not required to trigger 53BP1 IRIF. The use of common reactive oxygen species and reactive nitrogen species (RNS) inhibitors prevent the formation of 53BP1 foci in hit and bystander cells. Treatment with filipin to disrupt membrane-dependent signaling does not prevent the cytoplasmic irradiation-induced 53BP1 foci in the irradiated cells, but it does prevent signaling to bystander cells. Active mitochondrial function is required for these responses because pseudo-rho(0) cells, which lack mitochondrial DNA, could not produce a bystander signal, although they could respond to a signal from normal rho+ cells.

A Focused Ultrasoft X-Ray Microbeam for Targeting Cells Individually with Submicrometer Accuracy

Abstract Folkard, M., Schettino, G., Vojnovic, B., Gilchrist, S., Michette, A. G., Pfauntsch, S. J., Prise, K. M. and Michael, B. D. A Focused Ultrasoft X-Ray Microbeam for Targeting Cells Individually with Submicrometer Accuracy. Radiat. Res. 156, 796–804 (2001). The application of microbeams is providing new insights into the actions of radiation at the cell and tissue levels. So far, this has been achieved exclusively through the use of collimated charged particles. One alternative is to use ultrasoft X rays, focused by X-ray diffractive optics. We have developed a unique facility that uses 0.2–0.8-mm-diameter zone plates to focus ultrasoft X rays to a beam of less than 1 μm diameter. The zone plate images characteristic K-shell X rays of carbon or aluminum, generated by focusing a beam of 5–10 keV electrons onto the appropriate target. By reflecting the X rays off a grazing-incidence mirror, the contaminating bremsstrahlung radiation is reduced to 2%. The focused X rays are then aimed at selected subcellular targets using rapid automated cell-finding and alignment procedures; up to 3000 cells per hour can be irradiated individually using this arrangement.

The design and application of ion microbeams for irradiating living cells and tissues

The range of radiobiological experiments requiring microirradiation techniques continues to expand and diversify, creating ever-greater challenges for the designers of such systems. A versatile microbeam for radiation biology must excel in a number of areas. For studies of intracellular cell signalling where it may be of interest to target just the cytoplasm or nuclear membrane, targeting accuracies of micron or less are desirable. Other studies may use endpoints that are rare enough to require the irradiation of hundreds of thousands of cells in order to observe and quantify the effects. Inevitably, this means automating the cell finding, aligning and irradiation steps in order to achieve high cell throughputs. For investigations related to radiation risk, the effect of single particle traversals are paramount, therefore particle counting and single particle delivery are essential. A number of improvements have been implemented to the Gray Cancer Institute charged-particle microbeam, to extend its versatility and to meet these challenges. Specifically, improvements to the speed, alignment accuracy and environmental control have enabled investigations related to cell signalling, low-dose hypersensitivity, genomic instability and the visualisation of DNA repair to be successfully addressed. 2003 Elsevier B.V. All rights reserved.

Measurements of the targeting accuracy of the Gray Laboratory charged-particle microbeam

Abstract The charged-particle microbeam provides a unique opportunity to precisely control both the dose to individual cells and the localization of dose within the cell. In the Gray Laboratory, both the performance of the detection system and the targeting system has been assessed for 3.5 MeV 3 He 2+ ions. Using an 18 μm transmission scintillator and a photomultiplier tube as the particle detection system, detection efficiency is higher than 99%. When a 5 μm collimator is used, the overall targeting accuracy (which includes the error associated with target finding and alignment) is μm for 98.7% of hits.

MRI-guided radiotherapy of the SK-N-SH neuroblastoma xenograft model using a small animal radiation research platform

Objective: Neuroblastoma has one of the lowest survival rates of all childhood cancers, despite the use of intensive treatment regimens. Preclinical models of neuroblastoma are essential for testing new multimodality protocols, including those that involve radiotherapy (RT). The aim of this study was to develop a robust method for RT planning and tumour response monitoring based on combined MRI and cone-beam CT (CBCT) imaging and to apply it to a widely studied mouse xenograft model of neuroblastoma, SK-N-SH. Methods: As part of a tumour growth inhibition study, SK-N-SH xenografts were generated in BALB/c nu/nu mice. Mice (n = 8) were placed in a printed MR- and CT-compatible plastic cradle, imaged using a 4.7-T MRI scanner and then transferred to a small animal radiation research platform (SARRP) irradiator with on-board CBCT. MRI/CBCT co-registration was performed to enable RT planning using the soft-tissue contrast afforded by MRI prior to delivery of RT (5 Gy). Tumour response was assessed by serial MRI and calliper measurements. Results: SK-N-SH xenografts formed soft, deformable tumours that could not be differentiated from surrounding normal tissues using CBCT. MR images, which allowed clear delineation of tumours, were successfully co-registered with CBCT images, allowing conformal RT to be delivered. MRI measurements of tumour volume 4 days after RT correlated strongly with length of survival time. Conclusion: MRI allowed precision RT of SK-N-SH tumours and provided an accurate means of measuring tumour response. Advances in knowledge: MRI-based RT planning of murine tumours is feasible using an SARRP irradiator.

An efficient and robust MRI-guided radiotherapy planning approach for targeting abdominal organs and tumours in the mouse

Introduction Preclinical CT-guided radiotherapy platforms are increasingly used but the CT images are characterized by poor soft tissue contrast. The aim of this study was to develop a robust and accurate method of MRI-guided radiotherapy (MR-IGRT) delivery to abdominal targets in the mouse. Methods A multimodality cradle was developed for providing subject immobilisation and its performance was evaluated. Whilst CT was still used for dose calculations, target identification was based on MRI. Each step of the radiotherapy planning procedure was validated initially in vitro using BANG gel dosimeters. Subsequently, MR-IGRT of normal adrenal glands with a size-matched collimated beam was performed. Additionally, the SK-N-SH neuroblastoma xenograft model and the transgenic KPC model of pancreatic ductal adenocarcinoma were used to demonstrate the applicability of our methods for the accurate delivery of radiation to CT-invisible abdominal tumours. Results The BANG gel phantoms demonstrated a targeting efficiency error of 0.56 ± 0.18 mm. The in vivo stability tests of body motion during MR-IGRT and the associated cradle transfer showed that the residual body movements are within this MR-IGRT targeting error. Accurate MR-IGRT of the normal adrenal glands with a size-matched collimated beam was confirmed by γH2AX staining. Regression in tumour volume was observed almost immediately post MR-IGRT in the neuroblastoma model, further demonstrating accuracy of x-ray delivery. Finally, MR-IGRT in the KPC model facilitated precise contouring and comparison of different treatment plans and radiotherapy dose distributions not only to the intra-abdominal tumour but also to the organs at risk. Conclusion This is, to our knowledge, the first study to demonstrate preclinical MR-IGRT in intra-abdominal organs. The proposed MR-IGRT method presents a state-of-the-art solution to enabling robust, accurate and efficient targeting of extracranial organs in the mouse and can operate with a sufficiently high throughput to allow fractionated treatments to be given.

A resistive heating system for homeothermic maintenance in small animals

Purpose To develop an MR-compatible resistive heater for temperature maintenance of anaesthetized animals. Materials and Methods An MR-compatible resistive electrical heater was formed from a tightly-wound twisted pair wire, interfaced to a homeothermic maintenance controller. Fat-suppressed images and localized spectra were acquired with the twisted pair heater and a near-identical single strand heater during operation at maximum power. Data were also acquired in the absence of heating to demonstrate the insensitivity of MR to distortions arising from the passage of current through the heater elements. The efficacy of temperature maintenance was examined by measuring rectal temperature immediately following induction of general anesthesia and throughout and after the acquisition of a heater artifact-prone image series. Results Images and spectra acquired in the presence and absence of DC current through the twisted pair heater were identical whereas the passage of current through the single strand wire created field shifts and lineshape distortions. Temperature that is lost during anesthesia induction was recovered within approximately 10–20 minutes of induction, and a stable temperature is reached as the animals temperature approaches the set target. Conclusion The twisted pair wire heater does not interfere with MR image quality and maintains adequate thermal input to the animal to maintain body temperature.

The Development of Technology for Effective Respiratory-Gated Irradiation Using an Image-Guided Small Animal Irradiator

The development of image-guided small animal irradiators represents a significant improvement over standard irradiators by enabling preclinical studies to mimic radiotherapy in humans. The ability to deliver tightly collimated targeted beams, in conjunction with gantry or animal couch rotation, has the potential to maximize tumor dose while sparing normal tissues. However, the current commercial platforms do not incorporate respiratory gating, which is required for accurate and precise targeting in organs subject to respiration related motions that may be up to the order of 5 mm in mice. Therefore, a new treatment head assembly for the Xstrahl Small Animal Radiation Research Platform (SARRP) has been designed. This includes a fast X-ray shutter subsystem, a motorized beam hardening filter assembly, an integrated transmission ionization chamber to monitor beam delivery, a kinematically positioned removable beam collimator and a targeting laser exiting the center of the beam collimator. The X-ray shutter not only minimizes timing errors but also allows beam gating during imaging and treatment, with irradiation only taking place during the breathing cycle when tissue movement is minimal. The breathing related movement is monitored by measuring, using a synchronous detector/lock-in amplifier that processes diffuse reflectance light from a modulated light source. After thresholding of the resulting signal, delays are added around the inhalation/exhalation phases, enabling the “no movement” period to be isolated and to open the X-ray shutter. Irradiation can either be performed for a predetermined time of X-ray exposure, or through integration of a current from the transmission monitor ionization chamber (corrected locally for air density variations). The ability to successfully deliver respiratory-gated X-ray irradiations has been demonstrated by comparing movies obtained using planar X-ray imaging with and without respiratory gating, in addition to comparing dose profiles observed from a collimated beam on EBT3 radiochromic film mounted on the animals chest. Altogether, the development of respiratory-gated irradiation facilitates improved dose delivery during animal movement and constitutes an important new tool for preclinical radiation studies. This approach is particularly well suited for irradiation of orthotopic tumors or other targets within the chest and abdomen where breathing related movement is significant.

Improving In Vivo High-Resolution CT Imaging of the Tumour Vasculature in Xenograft Mouse Models through Reduction of Motion and Bone-Streak Artefacts

Introduction Preclinical in vivo CT is commonly used to visualise vessels at a macroscopic scale. However, it is prone to many artefacts which can degrade the quality of CT images significantly. Although some artefacts can be partially corrected for during image processing, they are best avoided during acquisition. Here, a novel imaging cradle and tumour holder was designed to maximise CT resolution. This approach was used to improve preclinical in vivo imaging of the tumour vasculature. Procedures A custom built cradle containing a tumour holder was developed and fix-mounted to the CT system gantry to avoid artefacts arising from scanner vibrations and out-of-field sample positioning. The tumour holder separated the tumour from bones along the axis of rotation of the CT scanner to avoid bone-streaking. It also kept the tumour stationary and insensitive to respiratory motion. System performance was evaluated in terms of tumour immobilisation and reduction of motion and bone artefacts. Pre- and post-contrast CT followed by sequential DCE-MRI of the tumour vasculature in xenograft transplanted mice was performed to confirm vessel patency and demonstrate the multimodal capacity of the new cradle. Vessel characteristics such as diameter, and branching were quantified. Results Image artefacts originating from bones and out-of-field sample positioning were avoided whilst those resulting from motions were reduced significantly, thereby maximising the resolution that can be achieved with CT imaging in vivo. Tumour vessels ≥ 77 μm could be resolved and blood flow to the tumour remained functional. The diameter of each tumour vessel was determined and plotted as histograms and vessel branching maps were created. Multimodal imaging using this cradle assembly was preserved and demonstrated. Conclusions The presented imaging workflow minimised image artefacts arising from scanner induced vibrations, respiratory motion and radiopaque structures and enabled in vivo CT imaging and quantitative analysis of the tumour vasculature at higher resolution than was possible before. Moreover, it can be applied in a multimodal setting, therefore combining anatomical and dynamic information.

Prospective gating control for highly efficient cardio-respiratory synchronised short and constant TR MRI in the mouse

Purpose Cardiac and respiratory motion derived image artefacts are reduced when data are acquired with cardiac and respiratory synchronisation. Where steady state imaging techniques are required in small animals, synchronisation is most commonly performed using retrospective gating techniques but these invoke an inherent time penalty. This paper reports the development of prospective gating techniques for cardiac and respiratory motion desensitised MRI with significantly reduced minimum scan time compared to retrospective gating. Methods Prospective gating incorporating the automatic reacquisition of data corrupted by motion at the entry to each breath was implemented in short TR 3D spoiled gradient echo imaging. Motion sensitivity was examined over the whole mouse body for scans performed without gating, with respiratory gating, and with cardio-respiratory gating. The gating methods were performed with and without automatic reacquisition of motion corrupted data immediately after completion of the same breath. Prospective cardio-respiratory gating, with acquisition of 64 k-space lines per cardiac R-wave, was used to enable whole body DCE-MRI in the mouse. Results Prospective cardio-respiratory gating enabled high fidelity steady state imaging of physiologically mobile organs such as the heart and lung. The automatic reacquisition of data corrupted by motion at the entry to each breath minimised respiratory motion artefact and enabled a highly efficient data capture that was adaptive to changes in the inter-breath interval. Prospective cardio-respiratory gating control enabled DCE-MRI to be performed over the whole mouse body with the acquisition of successive image volumes every 12–15 s at 422 μm isotropic resolution. Conclusions Highly efficient cardio-respiratory motion desensitised steady state MRI can be performed in small animals with prospective synchronisation, centre-out phase-encode ordering, and the automatic reacquisition of data corrupted by motion at the entry to each breath. The method presented is robust against spontaneous changes in the breathing rate. Steady state imaging with prospective cardio-respiratory gating is much more efficient than with retrospective gating, and enables the examination of rapidly changing systems such as those found when using DCE-MRI.