Evaluation of in vitro intrinsic radiosensitivity and characterization of five canine high-grade glioma cell lines.

Glioma is the most common primary brain tumor in dogs and predominantly affects brachycephalic breeds. Diagnosis relies on CT or MRI imaging, and the proposed treatments include surgical resection, chemotherapy, and radiotherapy depending on the tumor's location. Canine glioma from domestic dogs could be used as a more powerful model to study radiotherapy for human glioma than the murine model. Indeed, (i) contrary to mice, immunocompetent dogs develop spontaneous glioma, (ii) the canine brain structure is closer to human than mice, and (iii) domestic dogs are exposed to the same environmental factors than humans. Moreover, imaging techniques and radiation therapy used in human medicine can be applied to dogs, facilitating the direct transposition of results. The objective of this study is to fully characterize 5 canine glioma cell lines and to evaluate their intrinsic radiosensitivity. Canine cell lines present numerous analogies between the data obtained during this study on different glioma cell lines in dogs. Cell morphology is identical, such as doubling time, clonality test and karyotype. Immunohistochemical study of surface proteins, directly on cell lines and after stereotaxic injection in mice also reveals close similarity. Radiosensitivity profile of canine glial cells present high profile of radioresistance.

Volumetric assessment and longitudinal changes of subcortical structures in formalinized Beagle brains.

High field MRI is an advanced technique for diagnostic and research purposes on animal models, such as the Beagle dog. In this context, studies on neuroscience applications, e.g. aging and neuro-pathologies, are currently increasing. This led to a need for reference values, in terms of volumetric assessment, for the structures typically involved. Nowadays, several canine brain MRI atlases have been provided. However, no reports are available regarding the measurements' reproducibility and little is known about the effect of formalin on MRI segmentation. Here, we assessed the segmentation variability of selected structures among operators (two operators segmented the same data) in a sample of 11 Beagle dogs. Then, we analyzed, for one Beagle dog, the longitudinal volumetric changes of these structures. We considered four conditions: in vivo, post mortem (after euthanasia), ex vivo (brain extracted and studied after 1 month in formalin, and after 12 months). The MRI data were collected with a 3 T scanner. Our findings suggest that the segmentation procedure was overall reproducible since only slight statistical differences were detected. In the post mortem/ ex vivo comparison, most structures showed a higher contrast, thereby leading to greater reproducibility between operators. We observed a net increase in the volume of the studied structures. This could be justified by the intrinsic relaxation time changes observed because of the formalin fixation. This led to an improvement in brain structure visualization and segmentation. To conclude, MRI-based segmentation seems to be a useful and accurate tool that allows longitudinal studies on formalin-fixed brains.

Ex vivo susceptibility-weighted imaging anatomy of canine brain-comparison of imaging and histological sections.

Now that access of large domestic mammals to high-field MRI becomes more common, techniques initially implemented for human patients can be used for the structural and functional study of the brain of these animals. Among them, susceptibility-weighted imaging (SWI) is a recent technique obtained from gradient echo (GE) imaging that allow for an excellent anatomical tissue contrast and a non-invasive assessment of brain iron content. The goal of this study was to design an optimal GE SWI imaging protocol to be used in dogs undergoing an MRI examination of the brain in a 3-Tesla scanner. This imaging protocol was applied to ex vivo brains from four dogs. The imaging protocol was validated by visual inspection of the SWI images that provided a high anatomical detail, as demonstrated by their comparison with corresponding microscopic sections. As resolvable brain structures were labeled, this study is the first to provide an anatomic description of SWI images of the canine brain. Once validated in living animals, this GE SWI imaging protocol could be easily included in routine neuroimaging protocols to improve the diagnosis of various intracranial diseases of dogs, or be used in future comparative studies aiming at evaluating brain iron content in animals.

Diffusion Tensor Imaging Tractography of White Matter Tracts in the Equine Brain.

Tractography, a noninvasive technique tracing brain pathways from diffusion tensor magnetic resonance imaging (DTI) data, is increasingly being used for brain investigation of domestic mammals. In the equine species, such a technique could be useful to improve our knowledge about structural connectivity or to assess structural changes of white matter tracts potentially associated with neurodegenerative diseases. The goals of the present study were to establish the feasibility of DTI tractography in the equine brain and to provide a morphologic description of the most representative tracts in this species. Postmortem DTI and susceptibility-weighted imaging (SWI) of an equine brain were acquired with a 3-T system using a head coil. Association, commissural, and projection fibers, the three fiber groups typically investigated in tractography studies, were successfully reconstructed and overlaid on SWI or fractional anisotropy maps. The fibers derived from DTI correlate well with their description in anatomical textbooks. Our results demonstrate the feasibility of using postmortem DTI data to reconstruct the main white matter tracts of the equine brain. Further DTI acquisitions and corresponding dissections of equine brains will be necessary to validate these findings and create an equine stereotaxic white matter atlas that could be used in future neuroimaging research.

Anatomy, histology, and ultrasonography of the normal adrenal gland in brown lemur: Eulemur fulvus.

BACKGROUND: The medical care currently to brown lemurs (Eulemur fulvus) is limited by a lack of knowledge of their anatomy. The aim of this study was to describe the anatomy and histology and obtain ultrasonographic measurements of normal adrenal glands in these animals. METHODS: The adrenal glands of four lemurs cadavers were used for the anatomical and histological studies, and those of 15 anesthetized lemurs were examined by ultrasonography. RESULTS: Anatomically, the adrenal glands of brown lemurs are comparable to those of other species. The histological findings showed that the cortex is organized into three distinct layers, whereas most domestic mammals have an additional zone. The surface area of the adrenal glands increased with body weight, and the area of the right adrenal was slightly larger than the left. CONCLUSIONS: We suggest using ultrasonography to aid the etiological diagnosis of behavioral abnormalities that might be due to dysfunctions of the adrenal gland.

Presence of neural progenitors in spontaneous canine gliomas: A histopathological and immunohistochemical study of 20 cases.

Gliomas are the most common primary brain tumours in humans and are associated with a poor prognosis. An accurate animal model of human glioma tumorigenesis is needed to test new treatment strategies. Dogs represent a promising model because they develop spontaneous diffusely-infiltrating gliomas. This study investigated whether spontaneous canine gliomas contain cancer stem cells previously identified in all grades of human gliomas. Twenty spontaneous cases of canine gliomas were graded according to the human WHO classification. The expression of different markers of lineage differentiation was evaluated with immunohistochemistry as follows: nestin and CD133 for neural stem cells, doublecortin for neuronal progenitor cells, Olig2 for glial progenitor cells, glial fibrillary acidic protein, vimentin and S-100 for mature glial cells, and NeuN and ßIII-tubulin for mature neurons. Gliomas were characterised as follows: five grade II (oligodendrogliomas); nine grade III (seven anaplastic oligodendrogliomas, one anaplastic astrocytoma, one anaplastic oligoastrocytoma); six grade IV (glioblastomas). Immunohistochemical evaluation revealed that (1) nestin and CD133 were expressed in all grades of gliomas with a higher proportion of positive cells in high-grade gliomas; (2) the expression of S-100 protein and Olig2 did not differ substantially between astrocytic and oligodendroglial tumours, and (3) all gliomas were negative for mature neuron markers. The results demonstrated the presence of undifferentiated neural progenitors in all grades of spontaneous canine gliomas, confirming the relevance of this animal model for further studies on cancer stem cells.

Voxel-based evidence of perfusion normalization in glioblastoma patients included in a phase I-II trial of radiotherapy/tipifarnib combination.

We previously showed that the farnesyl transferase inihibitor, Tipifarnib induced vascularization normalization, oxygenation and radiosensitization in a pre-clinical glioblastoma (GBM) model. The aim of this study was to assess by dynamic-susceptibility-contrast MRI (DSC-MRI) the effect of radiotherapy (RT) and Tipifarnib combination on tumor perfusion in GBM patients. Eighteen patients with newly diagnosed GBM, enrolled in a phase I-II clinical trial associating RT with Tipifarnib, underwent anatomical MR imaging and DSC-MRI before (M0) and two months after treatment (M2). Anatomic volumes of interest (VOIs) were delineated according to contrast-enhanced and hyper-intense signal areas on T1-Gd and T2 images, respectively. Perfusion variations between M0 and M2 were assessed with median relative cerebral blood volume (rCBV) inside these VOIs. Another voxel by voxel analysis of CBV values classified 405,117 tumor voxels into High_, Normal_ and Low_CBVTUMOR according to the distribution of CBV in the contralateral normal tissue. These three categories of CBVTUMOR voxels were color-coded over anatomical MRI. Variations of median rCBV were significantly different for two groups of patients (P < 0.013): rCBV decreased when initial rCBV was ≥ 1.0 (Group_rCBV_M0 > 1) and rCBV increased when initial rCBV was < 1.0 (Group_rCBV_M0 < 1). Mapping of color-coded voxels provided additional spatial and quantitative information about tumor perfusion: Group_rCBV_M0 > 1 presented a significant decrease of High_CBVTUMOR volume (P = 0.015) simultaneously with a significant increase of Normal_CBVTUMOR volume (P = 0.009) after treatment. Group_rCBV_M0 < 1 presented a decrease of Low_CBVTUMOR volume with an increase of Normal_ and High_CBV TUMOR volume after treatment. Pre and post-treatment CBV measurements with DSC-MRI characterized tumor perfusion evolution in GBM patients treated with RT combined to Tipifarnib; showing variations in favour of tumor perfusion normalization in agreement with our pre-clinical results of vascular normalization.

Evaluation of the lactate-to-N-acetyl-aspartate ratio defined with magnetic resonance spectroscopic imaging before radiation therapy as a new predictive marker of the site of relapse in patients with glioblastoma multiforme.

PURPOSE: Because lactate accumulation is considered a surrogate for hypoxia and tumor radiation resistance, we studied the spatial distribution of the lactate-to-N-acetyl-aspartate ratio (LNR) before radiation therapy (RT) with 3D proton magnetic resonance spectroscopic imaging (3D-(1)H-MRSI) and assessed its impact on local tumor control in glioblastoma (GBM). METHODS AND MATERIALS: Fourteen patients with newly diagnosed GBM included in a phase 2 chemoradiation therapy trial constituted our database. Magnetic resonance imaging (MRI) and MRSI data before RT were evaluated and correlated to MRI data at relapse. The optimal threshold for tumor-associated LNR was determined with receiver-operating-characteristic (ROC) curve analysis of the pre-RT LNR values and MRI characteristics of the tumor. This threshold was used to segment pre-RT normalized LNR maps. Two spatial analyses were performed: (1) a pre-RT volumetric comparison of abnormal LNR areas with regions of MRI-defined lesions and a choline (Cho)-to- N-acetyl-aspartate (NAA) ratio ≥ 2 (CNR2); and (2) a voxel-by-voxel spatial analysis of 4,186,185 voxels with the intention of evaluating whether pre-RT abnormal LNR areas were predictive of the site of local recurrence. RESULTS: A LNR of ≥ 0.4 (LNR-0.4) discriminated between tumor-associated and normal LNR values with 88.8% sensitivity and 97.6% specificity. LNR-0.4 voxels were spatially different from those of MRI-defined lesions, representing 44% of contrast enhancement, 64% of central necrosis, and 26% of fluid-attenuated inversion recovery (FLAIR) abnormality volumes before RT. They extended beyond the overlap with CNR2 for most patients (median: 20 cm(3); range: 6-49 cm(3)). LNR-0.4 voxels were significantly predictive of local recurrence, regarded as contrast enhancement at relapse: 71% of voxels with a LNR-0.4 before RT were contrast enhanced at relapse versus 10% of voxels with a normal LNR (P<.01). CONCLUSIONS: Pre-RT LNR-0.4 in GBM indicates tumor areas that are likely to relapse. Further investigations are needed to confirm lactate imaging as a tool to define additional biological target volumes for dose painting.

Choroidal melanocytoma in a cat.

An 11-year-old male, neutered European cat was presented for anisocoria due to pupillary dilation in the right eye. Ophthalmic findings were restricted to this eye and consisted of a raised, darkly pigmented, retrolental mass associated with retinal detachment. Ultrasonography identified the mass lesion protruding into the vitreous cavity from the posterior pole of the eyeball and confirmed the detachment of the retina. A tentative diagnosis of an intraocular tumor was made. Radiographic evaluation and retromandibular lymph node cytology did not reveal evidence of distant metastasis. Orbital exenteration of the affected eye was performed and the tumor was diagnosed as a choroidal melanocytic tumor with no criteria of malignancy (melanocytoma). The cat died 5 months later from renal lymphoma, and necropsy did not detect metastasis of the melanocytic tumor. To our knowledge this is the first reported case of feline choroidal melanocytoma.