The Expansion of Heterotopic Bone in Fibrodysplasia Ossificans Progressiva Is Activin A-Dependent.

Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal dominant disorder that is characterized by episodic yet cumulative heterotopic ossification (HO) in skeletal muscles, tendons, and ligaments over a patient's lifetime. FOP is caused by missense mutations in the type I bone morphogenetic protein (BMP) receptor ACVR1. We have determined that the formation of heterotopic bone in FOP requires activation of mutant ACVR1 by Activin A, in part by showing that prophylactic inhibition of Activin A blocks HO in a mouse model of FOP. Here we piece together a natural history of developing HO lesions in mouse FOP, and determine where in the continuum of HO Activin A is required, using imaging (T2-MRI, µCT, 18 F-NaF PET/CT, histology) coupled with pharmacologic inhibition of Activin A at different times during the progression of HO. First, we show that expansion of HO lesions comes about through growth and fusion of independent HO events. These events tend to arise within a neighborhood of existing lesions, indicating that already formed HO likely triggers the formation of new events. The process of heterotopic bone expansion appears to be dependent on Activin A because inhibition of this ligand suppresses the growth of nascent HO lesions and stops the emergence of new HO events. Therefore, our results reveal that Activin A is required at least up to the point when nascent HO lesions mineralize and further demonstrate the therapeutic utility of Activin A inhibition in FOP. These results provide evidence for a model where HO is triggered by inflammation but becomes "self-propagating" by a process that requires Activin A. © 2017 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals Inc.

Response-Derived Input Function Estimation for Dynamic Contrast-Enhanced MRI Demonstrated by Anti-DLL4 Treatment in a Murine U87 Xenograft Model.

PURPOSE: Dynamic contrast-enhanced magnetic resonance imaging (DCE MRI) is an accepted method to evaluate tumor perfusion and permeability and anti-vascular cancer therapies. However, there is no consensus on the vascular input function estimation method, which is critical to kinetic modeling and K trans estimation. This work proposes a response-derived input function (RDIF) estimated from the response of the tumor, modeled as a linear, time-invariant (LTI) system. PROCEDURES: In an LTI system, an unknown input can be estimated from the system response. If applied to DCE MRI, this method would eliminate need of distal image-derived inputs, model inputs, or reference regions. The RDIF method first determines each tumor pixel's best-fit input function, and then combines the individual fits into a single input function for the entire tumor. The method was tested with simulations and a xenograft study with anti-vascular drug treatment. RESULTS: Simulations showed successful estimation of input function expected values and good performance in the presence of noise. In vivo, significant reductions in K trans and AUC occurred 2 days following anti-delta-like ligand 4 treatment. The in vivo study results yielded K trans consistent with published data in xenograft models. CONCLUSION: The RDIF method for DCE analysis offers an alternative, easy-to-implement method for estimating the input function in tumors. The method assumes that during the DCE experiment, the changes observed by MRI result solely from vascular perfusion and permeability kinetics, and that information can be used to model the input function. Importantly, the method is demonstrated in a murine xenograft study to yield K trans results consistent with literature values and suitable for compound studies.

Molecular Simulation of Receptor Occupancy and Tumor Penetration of an Antibody and Smaller Scaffolds: Application to Molecular Imaging.

PURPOSE: Competitive radiolabeled antibody imaging can determine the unlabeled intact antibody dose that fully blocks target binding but may be confounded by heterogeneous tumor penetration. We evaluated the hypothesis that smaller radiolabeled constructs can be used to more accurately evaluate tumor expressed receptors. PROCEDURES: The Krogh cylinder distributed model, including bivalent binding and variable intervessel distances, simulated distribution of smaller constructs in the presence of increasing doses of labeled antibody forms. RESULTS: Smaller constructs <25 kDa accessed binding sites more uniformly at large distances from blood vessels compared with larger constructs and intact antibody. These observations were consistent for different affinity and internalization characteristics of constructs. As predicted, a higher dose of unlabeled intact antibody was required to block binding to these distant receptor sites. CONCLUSIONS: Small radiolabeled constructs provide more accurate information on total receptor expression in tumors and reveal the need for higher antibody doses for target receptor blockade.

Dynamic dual-isotope molecular imaging elucidates principles for optimizing intrathecal drug delivery.

The intrathecal (IT) dosing route offers a seemingly obvious solution for delivering drugs directly to the central nervous system. However, gaps in understanding drug molecule behavior within the anatomically and kinetically unique environment of the mammalian IT space have impeded the establishment of pharmacokinetic principles for optimizing regional drug exposure along the neuraxis. Here, we have utilized high-resolution single-photon emission tomography with X-ray computed tomography to study the behavior of multiple molecular imaging tracers following an IT bolus injection, with supporting histology, autoradiography, block-face tomography, and MRI. Using simultaneous dual-isotope imaging, we demonstrate that the regional CNS tissue exposure of molecules with varying chemical properties is affected by IT space anatomy, cerebrospinal fluid (CSF) dynamics, CSF clearance routes, and the location and volume of the injected bolus. These imaging approaches can be used across species to optimize the safety and efficacy of IT drug therapy for neurological disorders.

Evaluation of the antitumor effects of rilotumumab by PET imaging in a U-87 MG mouse xenograft model.

INTRODUCTION: Dysregulation of the hepatocyte growth factor (HGF)/MET pathway has been implicated in various cancers. Rilotumumab is an investigational, fully human monoclonal antibody that binds and neutralizes HGF. The purpose of this study was to evaluate the efficacy of rilotumumab in a U-87 MG mouse xenograft tumor model using (18)F-FDG and (18)F-FLT PET. METHODS: U-87 MG tumor-bearing nude mice received rilotumumab or control IgG2. In the dose response study, increasing doses of rilotumumab (10, 30, 100, 300, or 500 µg) were administered, and mice were evaluated with (18)F-FDG PET at baseline and 7 days post-treatment. In the time course study, 300 µg of rilotumumab twice per week was used for the treatment, and mice were evaluated over 7 days using (18)F-FDG and (18)F-FLT PET. RESULTS: In the dose response study, rilotumumab at doses of 300 and 500 µg was similarly effective against tumor growth. Treatment with 300 and 500 µg rilotumumab inhibited (18)F-FDG accumulation with significant decreases of -37% and -40% in the percent injected dose per gram of tissue (%ID/g), respectively. In the time course study, treatment with 300 µg rilotumumab inhibited (18)F-FDG and (18)F-FLT accumulation with a maximum %ID/g of -41% and -64%, respectively. No apparent differences between the use of either tracer to evaluate rilotumumab efficacy were observed. CONCLUSIONS: Rilotumumab inhibited (18)F-FDG and (18)F-FLT accumulation as early as 2 and 4 days after treatment, respectively, in a mouse tumor model. Further studies to evaluate (18)F-FDG PET imaging as an early tumor response marker for rilotumumab are warranted. Rilotumumab is currently being tested in patients with MET-positive, advanced gastric and gastroesophageal cancer.

Regional, kinetic [(18)F]FDG PET imaging of a unilateral Parkinsonian animal model.

Positron emission tomography (PET) imaging with the glucose analog 2-deoxy-2-[(18)F]fluoro-D-glucose ([(18)F] FDG) has demonstrated clinical utility for the monitoring of brain glucose metabolism alteration in progressive neurodegenerative diseases. We examined dynamic [(18)F]FDG PET imaging and kinetic modeling of atlas-based regions to evaluate regional changes in the cerebral metabolic rate of glucose in the widely-used 6-hydroxydopamine (6-OHDA) rat model of Parkinson's disease. Following a bolus injection of 18.5 ± 1 MBq [(18)F]FDG and a 60-minute PET scan, image-derived input functions from the vena cava and left ventricle were used with three models, including Patlak graphical analysis, to estimate the influx constant and the metabolic rate in ten brain regions. We observed statistically significant changes in [(18)F]FDG uptake ipsilateral to the 6-OHDA injection in the basal ganglia, olfactory bulb, and amygdala regions; and these changes are of biological relevance to the disease. These experiments provide further validation for the use of [(18)F]FDG PET imaging in this model for drug discovery and development.

Characterization of Alisertib (MLN8237), an investigational small-molecule inhibitor of aurora A kinase using novel in vivo pharmacodynamic assays.

PURPOSE: Small-molecule inhibitors of Aurora A (AAK) and B (ABK) kinases, which play important roles in mitosis, are currently being pursued in oncology clinical trials. We developed three novel assays to quantitatively measure biomarkers of AAK inhibition in vivo. Here, we describe preclinical characterization of alisertib (MLN8237), a selective AAK inhibitor, incorporating these novel pharmacodynamic assays. EXPERIMENTAL DESIGN: We investigated the selectivity of alisertib for AAK and ABK and studied the antitumor and antiproliferative activity of alisertib in vitro and in vivo. Novel assays were used to assess chromosome alignment and mitotic spindle bipolarity in human tumor xenografts using immunofluorescent detection of DNA and alpha-tubulin, respectively. In addition, 18F-3'-fluoro-3'-deoxy-l-thymidine positron emission tomography (FLT-PET) was used to noninvasively measure effects of alisertib on in vivo tumor cell proliferation. RESULTS: Alisertib inhibited AAK over ABK with a selectivity of more than 200-fold in cells and produced a dose-dependent decrease in bipolar and aligned chromosomes in the HCT-116 xenograft model, a phenotype consistent with AAK inhibition. Alisertib inhibited proliferation of human tumor cell lines in vitro and produced tumor growth inhibition in solid tumor xenograft models and regressions in in vivo lymphoma models. In addition, a dose of alisertib that caused tumor stasis, as measured by volume, resulted in a decrease in FLT uptake, suggesting that noninvasive imaging could provide value over traditional measurements of response. CONCLUSIONS: Alisertib is a selective and potent inhibitor of AAK. The novel methods of measuring Aurora A pathway inhibition and application of tumor imaging described here may be valuable for clinical evaluation of small-molecule inhibitors.

Multimodal imaging with (18)F-FDG PET and Cerenkov luminescence imaging after MLN4924 treatment in a human lymphoma xenograft model.

UNLABELLED: Cerenkov luminescence imaging (CLI) is an emerging imaging technique that combines aspects of both optical and nuclear imaging fields. The ability to fully evaluate the correlation and sensitivity of CLI to PET is critical to progress this technique further for use in high-throughput screening of pharmaceutical compounds. To achieve this milestone, it must first be established that CLI data correlate to PET data in an in vivo preclinical antitumor study. We used MLN4924, a phase 2 oncology therapeutic, which targets and inhibits the NEDD8-activating enzyme pathway involved in the ubiquitin-proteasome system. We compared the efficacious effects of MLN4924 using PET and Cerenkov luminescence image values in the same animals. METHODS: Imaging of (18)F-FDG uptake was performed at 5 time points after drug treatment in the subcutaneously implanted diffuse large B-cell lymphoma tumor line OCI-Ly10. Data were acquired with both modalities on the same day, with a 15-min delay between CLI and PET. PET data analysis was performed using percentage injected dose per cubic centimeter of tissue (%ID/cm(3)), average standardized uptake values, and total glycolytic volume. CLI measurements were radiance, radiance per injected dose (radiance/ID), and total radiant volume. RESULTS: A strong correlation was found between PET total glycolytic volume and CLI total radiant volume (r(2) = 0.99) and various PET and CLI analysis methods, with strong correlations found between PET %ID/cm(3) and CLI radiance (r(2) = 0.83) and CLI radiance/ID (r(2) = 0.82). MLN4924 demonstrated a significant reduction in tumor volume after treatment (volume ratio of treated vs. control, 0.114 at day 29). CONCLUSION: The PET and CLI data presented confirm the correlation and dynamic sensitivity of this new imaging modality. CLI provides a preclinical alternative to expensive PET instrumentation. Future high-throughput studies should provide for quicker turnaround and higher cost-to-return benefits in the drug discovery process.

Antitumor activity of the investigational proteasome inhibitor MLN9708 in mouse models of B-cell and plasma cell malignancies.

PURPOSE: The clinical success of the first-in-class proteasome inhibitor bortezomib (VELCADE) has validated the proteasome as a therapeutic target for treating human cancers. MLN9708 is an investigational proteasome inhibitor that, compared with bortezomib, has improved pharmacokinetics, pharmacodynamics, and antitumor activity in preclinical studies. Here, we focused on evaluating the in vivo activity of MLN2238 (the biologically active form of MLN9708) in a variety of mouse models of hematologic malignancies, including tumor xenograft models derived from a human lymphoma cell line and primary human lymphoma tissue, and genetically engineered mouse (GEM) models of plasma cell malignancies (PCM). EXPERIMENTAL DESIGN: Both cell line-derived OCI-Ly10 and primary human lymphoma-derived PHTX22L xenograft models of diffuse large B-cell lymphoma were used to evaluate the pharmacodynamics and antitumor effects of MLN2238 and bortezomib. The iMyc(Cα)/Bcl-X(L) GEM model was used to assess their effects on de novo PCM and overall survival. The newly developed DP54-Luc-disseminated model of iMyc(Cα)/Bcl-X(L) was used to determine antitumor activity and effects on osteolytic bone disease. RESULTS: MLN2238 has an improved pharmacodynamic profile and antitumor activity compared with bortezomib in both OCI-Ly10 and PHTX22L models. Although both MLN2238 and bortezomib prolonged overall survival, reduced splenomegaly, and attenuated IgG2a levels in the iMyc(Cα)/Bcl-X(L) GEM model, only MLN2238 alleviated osteolytic bone disease in the DP54-Luc model. CONCLUSIONS: Our results clearly showed the antitumor activity of MLN2238 in a variety of mouse models of B-cell lymphoma and PCM, supporting its clinical development. MLN9708 is being evaluated in multiple phase I and I/II trials.

Assessing antibody pharmacokinetics in mice with in vivo imaging.

Recent advances in small-animal molecular imaging instrumentation combined with well characterized antibody-labeling chemistry have enabled detailed in vivo measurements of antibody distribution in mouse models. This article reviews the strengths and limitations of in vivo antibody imaging methods with a focus on positron emission tomography and single-photon emission computed tomography and a brief discussion of the role of optical imaging in this application. A description of the basic principles behind the imaging techniques is provided along with a discussion of radiolabeling methods relevant to antibodies. Practical considerations of study design and execution are presented through a discussion of sensitivity and resolution tradeoffs for these techniques as defined by modality, signaling probe (isotope or fluorophore) selection, labeling method, and radiation dosimetry. Images and analysis results from a case study are presented with a discussion of output data content and relevant informatics gained with this approach to studying antibody pharmacokinetics.

The relationship among tumor architecture, pharmacokinetics, pharmacodynamics, and efficacy of bortezomib in mouse xenograft models.

Understanding a compound's preclinical pharmacokinetic, pharmacodynamic, and efficacy relationship can greatly facilitate its clinical development. Bortezomib is a first-in-class proteasome inhibitor whose pharmacokinetic/pharmacodynamic parameters are poorly understood in terms of their relationship with efficacy. Here we characterized the bortezomib pharmacokinetic/pharmacodynamic/efficacy relationship in the CWR22 and H460 xenograft models. These studies allowed us to specifically address the question of whether the lack of broad bortezomib activity in solid tumor xenografts was due to insufficient tumor penetration. In vivo studies showed that bortezomib treatment resulted in tumor growth inhibition in CWR22 xenografts, but not in H460 xenografts. Using 20S proteasome inhibition as a pharmacodynamic marker and analyzing bortezomib tumor exposures, we show that efficacy was achieved only when suitable drug exposures drove proteasome inhibition that was sustained over time. This suggested that both the magnitude and duration of proteasome inhibition were important drivers of efficacy. Using dynamic contrast-enhanced magnetic resonance imaging and high-resolution computed tomographic imaging of vascular casts, we characterized the vasculature of CWR22 and H460 xenograft tumors and identified prominent differences in vessel perfusion, permeability, and architecture that ultimately resulted in variations in bortezomib tumor exposure. Comparing and contrasting the differences between a bortezomib-responsive and a bortezomib-resistant model with these techniques allowed us to establish a relationship among tumor perfusion, drug exposure, pharmacodynamic response and efficacy, and provided an explanation for why some solid tumor models do not respond to bortezomib treatment.

Application of surface roughness analysis on micro-computed tomographic images of bone erosion: examples using a rodent model of rheumatoid arthritis.

Quantifying the bone erosion in preclinical models of rheumatoid arthritis is valuable for the evaluation of drug treatments. This study introduces a three-dimensional method for bone surface roughness measurement from micro-computed tomographic data obtained from rats subjected to collagen-induced arthritis (CIA), in which the degree of bone erosion is related to the severity and the duration of the disease. In two studies of rat CIA, the surface roughness of the talus bone following 21 days of disease increased 559% and 486% from the control group. At 41 days following disease induction, the roughness of the bone surface increased 857% above baseline. The roughness of the control samples was similar from each study (less than 4% different), demonstrating the robustness of the algorithm. Treatment with methotrexate at 0.1 mg/kg daily demonstrated significant protection from bone erosion, whereas the 0.05 mg/kg daily dose was not efficacious (98% versus 22% inhibition of roughness-measured bone erosion). The main advantage of such an algorithm is demonstrated in the preclinical drug study of rat CIA with methotrexate treatment, indicating the immediate utility of this approach in drug development studies.

Longitudinally quantitative 2-deoxy-2-[18F]fluoro-D-glucose micro positron emission tomography imaging for efficacy of new anticancer drugs: a case study with bortezomib in prostate cancer murine model.

PURPOSE: The aim of this study was to validate quantitative metabolic response of tumors to a treatment measured by longitudinal 2-deoxy-2-[(18)F]fluoro-D-glucose (FDG) micro positron emission tomography (microPET) as a robust tool for preclinical evaluation of new anticancer agents. PROCEDURES: Severe combined immunodeficiency mice with CWR22 xenografts were intravenously treated with bortezomib (Velcade) at 0.8 mg/kg on days 0, 3, 7, 10, and 14 and imaged with FDG microPET before, during and after treatment. Quantitative indices of tumor FDG uptake were developed. RESULTS: FDG microPET images successfully revealed the gradual reduction of tumor FDG uptake on day 4 onward despite no absolute tumor shrinkage. The standardized uptake values of FDG in tumors was reduced to 43% of the baseline values. Using the total tumor FDG uptake as the viable tumor burden, we found 86% tumor inhibition, compared to a 55% tumor growth inhibition in tumor volume measurement. CONCLUSION: FDG microPET imaging can provide an additional dimension of the efficacy of anticancer therapies that may otherwise be underestimated by tumor volume measurement.

MRI in preclinical drug development.

This chapter outlines the challenges that the pharmaceutical industry faces during the course of drug development and discusses the role of magnetic resonance imaging in preclinical drug discovery.

Spiculated periosteal response induced by intraosseous injection of 22Rv1 prostate cancer cells resembles subset of bone metastases in prostate cancer patients.

BACKGROUND: Prostate cancer bone metastasis is distinguished by the predominance of osteoblastic lesions. This phenotype has been difficult to reproduce in animal models. Here, we describe a model utilizing the 22Rv1 human prostate cancer cell line that generates osteolytic lesions and a prominent spiculated periosteal osteoblastic response following intraosseous injection in scid mice. METHODS: We injected 22Rv1-luciferase prostate cancer cells directly into the tibiae of C.B-17 scid mice. We analyzed tumor growth and pathology every 2 weeks using radiographic and histologic techniques. RESULTS: X-ray analysis revealed that 22Rv1 tumors elicit a mixed-type lesion including some osteolysis and a robust induction of periosteal bone formation, in contrast to PC3M-luciferase intraosseous tumors which induce only extensive osteolysis. Micro-computerized tomographic imaging shows that 22Rv1 tumors exhibit both osteolytic and osteoblastic features which become apparent between 4 and 6 weeks post injection. There is initial disruption of the cortex and corresponding invasion of the periosteum which is associated with a vigorous osteoblastic response. Histological analysis of late stage tumors shows that the tumor has grown outside of the medullary cavity and surrounds the tibia underneath the periosteum and intermixed with spicules of woven bone which is detected in the radiographic analysis. CONCLUSIONS: The overall pattern of this model is suggestive of clinical cases of prostate cancer metastasis in which periosteal responses are noted, often in association with rapidly progressive disease. We expect that intraosseous injection of 22Rv1 cells will provide a new experimental model for the study of osteoblastic prostate cancer metastasis.

A prostate-specific membrane antigen-targeted monoclonal antibody-chemotherapeutic conjugate designed for the treatment of prostate cancer.

MLN2704 is an antibody-chemotherapeutic conjugate designed to target prostate-specific membrane antigen (PSMA). PSMA is a transmembrane receptor whose expression is largely restricted to prostatic epithelium and prostate cancer cells with its expression level increasing during the progression of malignancy. MLN2704 consists of a de-immunized, monoclonal antibody that is specific for PSMA conjugated to drug maytansinoid 1 (DM1), a microtubule-depolymerizing compound. After antibody binding to PSMA and the subsequent cellular internalization of this complex, DM1 is released leading to cell death. MLN2704 has an approximate half-life of 39 hours in scid mice bearing CWR22 tumor tissue, and the antibody effectively penetrates xenograft tumor tissue. Optimization of dosage and schedule of MLN2704 administration defined interdependency between these conditions that maximized efficacy with no apparent toxicity. Tumor growth delays of approximately 100 days could be achieved on the optimized schedule of one dose of 60 mg/kg MLN2704 every 14 days for five doses (q14dx5). The unconjugated antibody (MLN591) demonstrated essentially no antitumor activity and DM1 alone or a non-PSMA targeted antibody-DM1 conjugate was only weakly active. Furthermore, we show that MLN2704 is active in a novel model of osteoblastic prostate cancer metastasis.

Investigation of techniques to quantify in vivo lesion volume based on comparison of water apparent diffusion coefficient (ADC) maps with histology in focal cerebral ischemia of rats.

Stroke lesion-volume estimates derived from calculated water apparent diffusion coefficient (ADC) maps provide a quantitative surrogate end-point for investigating the efficacy of drug treatment or studying the temporal evolution of cerebral ischemia. Methodology is described for estimating ischemic lesion volumes in a rat model of permanent middle cerebral artery occlusion (MCAO) based on absolute and percent-reduction threshold values of the water ADC at 3 h post-MCAO. Volume estimates derived from average ADC (ADC(av)) maps were compared with those derived from post-mortem histological sections. Optimum ADC thresholds were established as those that provided the best correlation and one-to-one correspondence between ADC- and histologically derived lesion-volume estimates. At 3 h post-MCAO, an absolute-ADC(av) threshold of 47 x 10(-5) mm(2)/s (corresponding to a 33% reduction in ADC(av) based on a contralateral hemisphere comparison) provided the most accurate estimate of percent hemispheric lesion volume (%HLV). Experimental and data analysis issues for improving and validating the usefulness of DWI as a surrogate endpoint for the quantification of ischemic lesion volume are discussed.

Quantitative analysis of micro-CT imaging and histopathological signatures of experimental arthritis in rats.

Micro-computed tomographic (micro-CT) imaging provides a unique opportunity to capture 3-D architectural information in bone samples. In this study of pathological joint changes in a rat model of adjuvant-induced arthritis (AA), quantitative analysis of bone volume and roughness were performed by micro-CT imaging and compared with histopathology methods and paw swelling measurement. Micro-CT imaging of excised rat hind paws (n = 10) stored in formalin consisted of approximately 600 30-mum slices acquired on a 512 x 512 image matrix with isotropic resolution. Following imaging, the joints were scored from H&E stained sections for cartilage/bone erosion, pannus development, inflammation, and synovial hyperplasia. From micro-CT images, quantitative analysis of absolute bone volumes and bone roughness was performed. Bone erosion in the rat AA model is substantial, leading to a significant decline in tarsal volume (27%). The result of the custom bone roughness measurement indicated a 55% increase in surface roughness. Histological and paw volume analyses also demonstrated severe arthritic disease as compared to controls. Statistical analyses indicate correlations among bone volume, roughness, histology, and paw volume. These data demonstrate that the destructive progression of disease in a rat AA model can be quantified using 3-D micro-CT image analysis, which allows assessment of arthritic disease status and efficacy of experimental therapeutic agents.

The macrosphere model: evaluation of a new stroke model for permanent middle cerebral artery occlusion in rats.

BACKGROUND AND PURPOSE: The suture middle cerebral artery occlusion (MCAO) model is widely used for the simulation of focal cerebral ischemia in rats. This technique causes hypothalamic injury resulting in hyperthermia, which can worsen outcome and obscure neuroprotective effects. Herein, we introduce a new MCAO model that avoids these disadvantages. METHODS: Permanent MCAO was performed by intraarterial embolization using six TiO(2) macrospheres (0.3-0.4 mm in diameter) or by the suture occlusion technique. Body temperature was monitored, functional and histologic outcome was assessed after 24 h. Additional 16 rats were subjected to macrosphere or suture MCAO. Lesion progression was evaluated using magnetic resonance imaging (MRI). RESULTS: The animals subjected to suture MCAO developed hyperthermia (>39 degrees C), while the temperature remained normal in the macrosphere MCAO group. Infarct size, functional outcome and model failure rate were not significantly different between the groups. Lesion size on MRI increased within the first 90 min and remained unchanged thereafter in both groups. CONCLUSIONS: The macrosphere MCAO model provides reproducible focal cerebral ischemia, similar to the established suture technique, but avoids hypothalamic damage and hyperthermia. This model, therefore, may be more appropriate for the preclinical evaluation of neuroprotective therapies and can also be used for stroke studies under difficult conditions, e.g., in awake animals or inside the MRI scanner.