Technical note: fiducial markers for correlation of whole-specimen histopathology with MR imaging at 7 tesla.

PURPOSE: There is increasing interest in the registration of 3-D histopathology with 3-D in vivo imaging, for example, to validate tumor boundary delineation for targeted radiation cancer therapy. However, accurate correlation is compromised by tissue distortion induced by histopathological processing. Reference landmarks that are visible in both data sets are required. In this study, two iridescent acrylic paints, "Bronze" (containing iron oxide coated mica particles) and "Stainless Steel" (containing iron, chromium, and nickel), were evaluated for creating MRI-visible and histology-visible fiducial markers at 7 T, where resolution is more similar to histology, but artifacts are accentuated. Furthermore, a straight-line paint-track fiducial method was developed to assist in registration and 3-D histopathology reconstruction. METHODS: First, the paints were injected into ex vivo porcine tissue samples, which were MR imaged prefixation and postfixation, and subsequently prepared for hematoxylin and eosin staining to verify stability through histopathological processing. Second, the severity of marker susceptibility artifacts produced was compared while using spin-echo and gradient-echo MRI pulse sequences. Finally, multiple paint tracks were injected prefixation through an ex vivo canine prostate sample to validate the potential for line-based registration between MR images of prefixation and postfixation tissue and whole mount histology slides. RESULTS: The Stainless Steel paint produced excessive susceptibility artifacts and image distortion, while the Bronze paint created stable and appropriate markers in MRI and histology. The Bronze paint produced artifacts approximately three times larger in gradient-echo than in spin-echo MR images. Finally, the paint-track fiducials were visible in the prefixation and postfixation MRI and on whole mount histology. CONCLUSIONS: The Bronze iridescent acrylic paint is appropriate for fiducial marker creation in MRI at 7 T. The straight-line paint-track fiducials may assist 3-D histopathology reconstruction and can provide important information on the deformation effects of fixation, and hence may improve registration accuracy.

Evaluating the extent of cell death in 3D high frequency ultrasound by registration with whole-mount tumor histopathology.

PURPOSE: High frequency ultrasound imaging, 10-30 MHz, has the capability to assess tumor response to radiotherapy in mouse tumors as early as 24 h after treatment administration. The advantage of this technique is that the image contrast is generated by changes in the physical properties of dying cells. Therefore, a subject can be imaged before and multiple times during the treatment without the requirement of injecting specialized contrast agents. This study is motivated by a need to provide metrics of comparison between the volume and localization of cell death, assessed from histology, with the volume and localization of cell death surrogate, assessed as regions with increased echogeneity from ultrasound images. METHODS: The mice were exposed to radiation doses of 2, 4, and 8 Gy. Ultrasound images ivere collected from each tumor before and 24 h after exposure to radiation using a broadband 25 MHz center frequency transducer. After radiotherapy, tumors exhibited hyperechoic regions in ultrasound images that corresponded to areas of cell death in histology. The ultrasound and histological images were rigidly registered. The tumors and regions of cell death were manually outlined on histological images. Similarly, the tumors and hyperechoic regions were outlined on the ultrasound images. Each set of contours was converted to a volumetric mesh in order to compare the volumes and the localization of cell death in histological and ultrasound images. RESULTS: A shrinkage factor of 17 +/- 2% was calculated from the difference in the tumor volumes evaluated from histological and ultrasound images. This was used to correct the tumor and cell death volumes assessed from histology. After this correction, the average absolute difference between the volume of cell death assessed from ultrasound and histological images was 11 +/- 14% and the volume overlap was 70 +/- 12%. CONCLUSIONS: The method provided metrics of comparison between the volume of cell death assessed from histology and that assessed from ultrasound images. It was applied here to evaluate the capability of ultrasound imaging to assess early tumor response to radiotherapy in mouse tumors. Similarly, it can be applied in the future to evaluate the capability of ultrasound imaging to assess early tumor response to other modalities of cancer treatment. The study contributes to an understanding of the capabilities and limitation of ultrasound imaging at noninvasively detecting cell death. This provides a foundation for future developments regarding the use of ultrasound in preclinical and clinical applications to adapt treatments based on tumor response to cancer therapy.

Quantitative ultrasound characterization of responses to radiotherapy in cancer mouse models.

PURPOSE: Currently, no imaging modality is used routinely to assess tumor responses to radiotherapy within hours to days after the delivery of treatment. In this study, we show the application of quantitative ultrasound methods to characterize tumor responses to cancer radiotherapy in vivo, as early as 24 hours after treatment administration. EXPERIMENTAL DESIGN: Three mouse models of head and neck cancer were exposed to radiation doses of 0, 2, 4, and 8 Gray. Data were collected with an ultrasound scanner using frequencies of 10 to 30 MHz. Ultrasound estimates calculated from normalized power spectra and parametric images (spatial maps of local estimates of ultrasound parameters) were used as indicators of response. RESULTS: Two of the mouse models (FaDu and C666-1) exhibited large hyperechoic regions at 24 hours after radiotherapy. The ultrasound integrated backscatter increased by 6.5 to 8.2 dB (P < 0.001) and the spectral slopes increased from 0.77 to 0.90 dB/MHz for the C666-1 tumors and from 0.54 to 0.78 dB/MHz for the FaDu tumors (P < 0.05), in these regions compared with preirradiated tumors. The hyperechoic regions in the ultrasound images corresponded in histology to areas of cell death. Parametric images could discern the tumor regions that responded to treatment. The other cancer mouse model (Hep-2) was resistant to radiotherapy. CONCLUSIONS: The results indicate that cell structural changes after radiotherapy have a significant influence on ultrasound spectral parameters. This provides a foundation for future investigations regarding the use of ultrasound in cancer patients to individualize treatments noninvasively based on their responses to specific interventions.

High-frequency ultrasound for monitoring changes in liver tissue during preservation.

Currently the only method to assess liver preservation injury is based on liver appearance and donor medical history. Previous work has shown that high-frequency ultrasound could detect ischemic cell death due to changes in cell morphology. In this study, we use high-frequency ultrasound integrated backscatter to assess liver damage in experimental models of liver ischemia. Ultimately, our goal is to predict organ suitability for transplantation using high-frequency imaging and spectral analysis techniques. To examine the effects of liver ischemia at different temperatures, livers from Wistar rats were surgically excised, immersed in phosphate buffer saline and stored at 4 and 20 degrees C for 24 h. To mimic organ preservation, livers were excised, flushed with University of Wisconsin (UW) solution and stored at 4 degrees C for 24 h. Preservation injury was simulated by either not flushing livers with UW solution or, before scanning, allowing livers to reach room temperature. Ultrasound images and corresponding radiofrequency data were collected over the ischemic period. No significant increase in integrated backscatter (approximately 2.5 dBr) was measured for the livers prepared using standard preservation conditions. For all other ischemia models, the integrated backscatter increased by 4-9 dBr demonstrating kinetics dependent on storage conditions. The results provide a possible framework for using high-frequency imaging to non-invasively assess liver preservation injury.

Ultrasound imaging of apoptosis: spectroscopic detection of DNA-damage effects at high and low frequencies.

A new noninvasive method for the detection of DNA damage using mid-to high-frequency ultrasound (10-60 MHz) has been developed. Ultrasound imaging and quantitative analysis methods are used to detect cell death occurring in response to anticancer therapies in cell samples in vitro, in rat brain tissue ex vivo, and in cancer mouse models in vivo. Experimental evidence indicates that the mechanism behind this ultrasonic detection is linked to changes in the size and acoustic properties of the cell nucleus occurring with forms of cell death, and in particular apoptosis. Nuclear changes associated with cell death can result in up to 16-fold increase in ultrasound backscatter intensity and changes in spectral slope that are consistent with theoretical predictions. Furthermore, color-coded images can be generated based on specific ultrasound parameters in order to identify the regions of cell death in tumor ultrasound images with treatments. These results provide a foundation for future investigations regarding the use of ultrasound in preclinical and clinical settings to noninvasively monitor tumor responses to specific interventions that induce cell death.

An increase in cellular size variance contributes to the increase in ultrasound backscatter during cell death.

This study aims to explain the contribution of changes in cellular size variance (CSV) to increases in ultrasound-integrated backscatter (UIB) measured from cell samples undergoing cell death. A Monte Carlo algorithm was used to compare simulations of 2D distributions of cells, uniform (CSV = 0) versus heterogeneous (CSV > 0) and the same mean cellular size (M ). UIB increased in arrangements with heterogeneous cellular sizes from 3.6dB (M = 20 mum, CSV = 0 microm/CSV = 18 microm) to 5.6 dB (M =10 microm, CSV = 0 microm/CSV = 8 microm). Experimentally, UIB (10 to 30 MHz) was measured from cell samples of four tumor cell lines viable and undergoing cell death after radiotherapy and chemotherapy treatment. An increase of 3.8-7.5 dB (p < 0.001) in UIB was measured from three cell lines. No increase in UIB was measured from one cell line. An increase in CSV was found for all cell samples after cell death. The results suggest that an increase in CSV could have a significant contribution to the increases measured in UIB after cell death in cell samples exposed to anticancer therapies.

Quantitative ultrasound characterization of cancer radiotherapy effects in vitro.

PURPOSE: Currently, no routinely used imaging modality is available to assess tumor responses to cancer treatment within hours to days after radiotherapy. In this study, we demonstrate the preclinical application of quantitative ultrasound methods to characterize the cellular responses to cancer radiotherapy in vitro. METHODS AND MATERIALS: Three different cell lines were exposed to radiation doses of 2-8 Gy. Data were collected with an ultrasound scanner using frequencies of 10-30 MHz. As indicators of response, ultrasound integrated backscatter and spectral slope were determined from the cell samples. These parameters were corrected for ultrasonic attenuation by measuring the attenuation coefficient. RESULTS: A significant increase in the ultrasound integrated backscatter of 4-7 dB (p < 0.001) was found for radiation-treated cells compared with viable cells at all radiation doses. The spectral slopes decreased in the cell samples that predominantly underwent mitotic arrest/catastrophe after radiotherapy, consistent with an increase in cell size. In contrast, the spectral slopes did not change significantly in the cell samples that underwent a mix of cell death (apoptosis and mitotic arrest), with no significant change in average cell size. CONCLUSION: The changes in ultrasound integrated backscatter and spectral slope were direct consequences of cell and nuclear morphologic changes associated with cell death. The results indicate that this combination of quantitative ultrasonic parameters has the potential to assess the cell responses to radiation, differentiate between different types of cell death, and provide a preclinical framework to monitor tumor responses in vivo.