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Neovascularization together with aberrant vasculature is a hallmark of glioblastomas. Understanding tumor vasculature can have a pivotal role in planning therapeutics approaches. The objective of this study was to investigate morphological, vasculature and blood flow changes induced by aggressive tumor growth, using in vivo imaging - magnetic resonance imaging (MRI) and functional ultrasound (fUS) imaging in a mouse model of orthotopic glioma. Female NMRI nude mice were xenografted orthotopically with U-87 MG, a human glioblastoma, cells (5 × 104) at the age of 8 weeks. Four weeks after implantation the mice we anesthetized with isoflurane and scanned with 11.7 T small animal MRI (Bruker) for tumor volumetry. Thereafter, the mice were imaged with Iconeus One imaging system (Iconeus, Paris, France) for structural information of brain vasculature and relative cerebral blood volume (rCBV) changes. High resolution vascular imaging was performed after intravenous injection of microbubble contrast agent (SonoVue, sulphur hexafluoride microbubbles). The average tumor size was 58.8 ± 27.6 mm3 (mean ± SD, n=7) four weeks after implantation. Aberrant vasculature could be visualized with structural fUS imaging as and the necrotic core of the tumor could be observed. A reduced rCBF was observed in ipsilateral hemisphere on the relative power doppler signal timeseries as a lower amplitude and faster decay compared to contralateral side. As a summary, in vivo fUS imaging is a noninvasive tool to visualize vascular changes induced by aggressive brain tumor growth which are not detectable with conventional imaging methods (CT, MRI, PET, SPECT) and can provide a novel readout for efficacy studies. Citation Format: Jussi Rytkönen, Kimmo Lehtimäki, Taina-Kaisa Stenius, Riikka Immonen, Ari Suhonen, Artem Shatillo. In vivo imaging of vascular pathology in mouse orthotopic glioma model using functional ultrasound [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB155.

We present MedicDeepLabv3+, a convolutional neural network that is the first completely automatic method to segment cerebral hemispheres in magnetic resonance (MR) volumes of rats with lesions. MedicDeepLabv3+ improves the state-of-the-art DeepLabv3+ with an advanced decoder, incorporating spatial attention layers and additional skip connections that, as we show in our experiments, lead to more precise segmentations. MedicDeepLabv3+ requires no MR image preprocessing, such as bias-field correction or registration to a template, produces segmentations in less than a second, and its GPU memory requirements can be adjusted based on the available resources. We optimized MedicDeepLabv3+ and six other state-of-the-art convolutional neural networks (DeepLabv3+, UNet, HighRes3DNet, V-Net, VoxResNet, Demon) on a heterogeneous training set comprised by MR volumes from 11 cohorts acquired at different lesion stages. Then, we evaluated the trained models and two approaches specifically designed for rodent MRI skull stripping (RATS and RBET) on a large dataset of 655 MR rat brain volumes. In our experiments, MedicDeepLabv3+ outperformed the other methods, yielding an average Dice coefficient of 0.952 and 0.944 in the brain and contralateral hemisphere regions. Additionally, we show that despite limiting the GPU memory and the training data, our MedicDeepLabv3+ also provided satisfactory segmentations. In conclusion, our method, publicly available at https://github.com/jmlipman/MedicDeepLabv3Plus, yielded excellent results in multiple scenarios, demonstrating its capability to reduce human workload in rat neuroimaging studies., Comment: Published in NeuroInformatics

Cognitive problems frequently accompany neurological manifestations of multiple sclerosis (MS). However, during screening of preclinical candidates, assessments of behaviour in mouse models of MS typically focus on locomotor activity. In the present study, we analysed cognitive behaviour of 9 to 10‐week‐old female C57Bl/6J mice orally administered with the toxin cuprizone that induces demyelination, a characteristic feature of MS. Animals received 400 mg/kg cuprizone daily for 2 or 4 weeks, and their performance was compared with that of vehicle‐treated mice. Cuprizone‐treated animals showed multiple deficits in short touchscreen‐based operant tasks: they responded more slowly to visual stimuli, rewards and made more errors in a simple rule‐learning task. In contextual/cued fear conditioning experiments, cuprizone‐treated mice showed significantly lower levels of contextual freezing than vehicle‐treated mice. Diffusion tensor imaging showed treatment‐dependent changes in fractional anisotropy as well as in axial and mean diffusivities in different white matter areas. Lower values of fractional anisotropy and axial diffusivity in cuprizone‐treated mice indicated developing demyelination and/or axonal damage. Several diffusion tensor imaging measurements correlated with learning parameters. Our results show that translational touchscreen operant tests and fear conditioning paradigms can reliably detect cognitive consequences of cuprizone treatment. The suggested experimental approach enables screening novel MS drug candidates in longitudinal experiments for their ability to improve pathological changes in brain structure and reverse cognitive deficits.

Skull-stripping and region segmentation are fundamental steps in preclinical magnetic resonance imaging (MRI) studies, and these common procedures are usually performed manually. We present Multi-task U-Net (MU-Net), a convolutional neural network designed to accomplish both tasks simultaneously. MU-Net achieved higher segmentation accuracy than state-of-the-art multi-atlas segmentation methods with an inference time of 0.35 seconds and no pre-processing requirements. We evaluated the performance of our network in the presence of skip connections and recently proposed framing connections, finding the simplest network to be the most effective. We tested MU-Net with an unusually large dataset combining several independent studies consisting of 1,782 mouse brain MRI volumes of both healthy and Huntington animals, and measured average Dice scores of 0.906 (striati), 0.937 (cortex), and 0.978 (brain mask). These high evaluation scores demonstrate that MU-Net is a powerful tool for segmentation and skull-stripping, decreasing inter and intra-rater variability of manual segmentation. The MU-Net code and the trained model are publicly available at https://github.com/Hierakonpolis/MU-Net.

Pharmacologic MRI (phMRI) is a non-invasive in vivo imaging method, which can evaluate the drug effects on the brain and provide complementary information to ex vivo techniques. The preclinical phMRI studies usually require anesthesia to reduce the motion and stress of the animals. The anesthesia, however, is a crucial part of the experimental design, as it may modulate the neural drug-induced (de)activation and hemodynamic coupling. Therefore, the aim of the present study was to address this methodologic question by performing phMRI experiments with five anesthetics (α-chloralose, isoflurane, medetomidine, thiobutabarbital, and urethane) and seven anesthesia protocols. Nicotine, a widely studied psychostimulant, was administered to rats while measuring blood oxygenation level-dependent (BOLD) signals. Notably different responses were observed depending on the anesthetic used. The highest responses were measured in urethane-anesthetized rats whereas the responses were hardly noticeable in α-chloralose group. As urethane is not commonly used in phMRI, hemodynamic coupling under urethane anesthesia was investigated with functional cerebral blood flow (CBF) and volume-weighted (CBVw) imaging, and simultaneous electrophysiologic and BOLD measurements. The BOLD, CBF, and CBVw measurements in response to nicotine were highly correlated (R(2) ≥ 0.70, p

Gliomas are the most common primary brain tumors and currently the prognosis is still poor. Due to this, it is one of the main areas in oncological research and drug development programs. Innovative therapies are vital to improve treatment outcomes but must be developed to enable trafficking across the blood brain barrier (BBB). For this advent, animal models provide important information prior to clinical studies. Among the different in vivo models, orthotopic PDX models represent best the tumor microenvironment and natural variability of tumors, hence providing the most reliable results over species. In the brain tumor field, imaging has a central role in clinical diagnosis and as a prognostic factor to monitor therapy response in patients. Magnetic resonance imaging (MRI) is widely used for clinical diagnosis and disease follow up as it offers unprecedented soft tissue contrast and high spatial resolution non-invasive way. In addition to imaging, molecular profiling, e.g. circulating immune biomarkers and local oncometabolites in the tumor environment may facilitate as important translational biomarkers in development of immunotherapy for gliomas in addition to traditional histopathological readouts. The purpose of this work was to analyze possible heterogeneity of tumors in vivo, growth rate and volume in stereotactically implanted orthotopic PDX brain tumor models using MRI/MRS imaging. In addition to imaging, neurological scoring was performed to monitor general animal welfare, cytokine profiles from plasma to observe immunological responses over time and determination of oncometabolites in plasma and brain tissues combined with traditional histopathological changes were performed. The data from orthotopic models was also compared to readouts in subcutaneous models. As a conclusion, translational in vivo imaging techniques were applied to study orthotopic tumor progression combined with circulating immune biomarkers, and general welfare scoring. These readouts provide a powerful and translational research tool together with oncological disease animal models allowing comprehensive evaluation of disease progression and treatment interventions for in vivo studies. Citation Format: Julia Schueler, Mariette Heins, Artem Shatillo, Kimmo Lehtimäki, Anne-Lise Peille, Taina-Kaisa Stenius, Timo Bragge, Jussi Rytkönen, Diana Miszczuk, Tuulia Huhtala. Longitudinal characterization of patient-derived orthotopic xenograft brain tumor models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2774.

Functional magnetic resonance imaging (fMRI) is a powerful noninvasive tool for studying spontaneous resting state functional connectivity (RSFC) in laboratory animals. Brain function can be significantly affected by generally used anesthetics, however, rendering the need for awake imaging. Only a few different awake animal habituation protocols have been presented, and there is a critical need for practical and improved low-stress techniques. Here we demonstrate a novel restraint approach for awake rat RSFC studies. Our custom-made 3D printed restraint kit is compatible with a standard Bruker Biospin MRI rat bed, rat brain receiver coil, and volume transmitter coil. We also implemented a progressive habituation protocol aiming to minimize the stress experienced by the rats, and compared RSFC between awake, lightly sedated, and isoflurane-anesthetized rats. Our results demonstrated that the 3D printed restraint kit was suitable for RSFC studies of awake rats. During the short 4-day habituation period, the plasma corticosterone concentration, movement, and heart rate, which were measured as stress indicators, decreased significantly, indicating adaptation to the restraint protocol. Additionally, 10 days after the awake MRI session, rats exhibited no signs of depression or anxiety based on open-field and sucrose preference behavioral tests. The RSFC data revealed significant changes in the thalamo-cortical and cortico-cortical networks between the awake, lightly sedated, and anesthetized groups, emphasizing the need for awake imaging. The present work demonstrates the feasibility of our custom-made 3D printed restraint kit. Using this kit, we found that isoflurane markedly affected brain connectivity compared with that in awake rats, and that the effect was less pronounced, but still significant, when light isoflurane sedation was used instead.

Diagnosis of ongoing epileptogenesis and associated hyperexcitability after brain injury is a major challenge. Given that increased neuronal activity in the brain triggers a blood oxygenation level-dependent (BOLD) response in functional magnetic resonance imaging (fMRI), we hypothesized that fMRI could be used to identify the brain area(s) with hyperexcitability during post-injury epileptogenesis. We applied fMRI to detect onset and spread of BOLD activation after pentylenetetrazol (PTZ)-induced seizures (PTZ, 30 mg/kg, intraperitoneally) in 16 adult male rats at 2 months after lateral fluid percussion (FPI)-induced traumatic brain injury (TBI). In sham-operated controls, onset of the PTZ-induced BOLD response was bilateral and first appeared in the cortex. After TBI, 5 of 9 (56%) rats exhibited ipsilateral perilesional cortical BOLD activation, followed by activation of the contralateral cortex. In 4 of 9 (44%) rats, onset of BOLD response was bilateral. Interestingly, latency from the PTZ injection to onset of the BOLD response increased in the following order: sham-operated controls (ipsilateral 132 ± 57 sec, contralateral 132 ± 57 sec; p 0.05)TBI with bilateral BOLD onset (ipsilateral 176 ± 54 sec, contralateral 178 ± 52 sec; p 0.05)TBI with ipsilateral BOLD onset (ipsilateral 406 ± 178 sec, contralateral 509 ± 140 sec; p 0.05). Cortical lesion area did not differ between rats with ipsilateral versus bilateral BOLD onset (p 0.05). In the group of rats with ipsilateral onset of PTZ-induced BOLD activation, none of the rats showed a robust bilateral thalamic BOLD response, only 1 of 5 rats had robust ipsilateral thalamic calcifications, and 4 of 5 rats had perilesional astrocytosis. These findings suggest the evolution of the epileptogenic zone in the perilesional cortex after TBI, which is sensitive to PTZ-induced hyperexcitability. Further studies are warranted to explore the evolution of thalamo-cortical pathology as a driver of epileptogenesis after lateral FPI.

We report spontaneous hemodynamic activity termed “Spontaneous BOLD Waves” (SBWs) detected by BOLD fMRI in Sprague-Dawley rats under medetomidine anesthesia. These SBWs, which lasted several minutes, were observed in cortex, thalamus and hippocampus. The SBWs’ correlates were undetectable in electrophysiological recordings, suggesting an exclusive gliovascular phenomenon dissociated from neuronal activity. SBWs were insensitive to the NMDA receptors antagonist MK-801 but were inhibited by the α1-adrenoceptor blocker prazosin. Since medetomidine is a potent agonist of α2 adrenoceptors, we suggested that imbalance in α1/α2 receptor-mediated signalling pathways alter the vascular reactivity leading to SBWs. The frequency of SBWs increased with intensity of mechanical lung ventilation despite the stable pH levels. In summary, we present a novel type of propagating vascular brain activity without easily detectable underlying neuronal activity, which can be utilized to study the mechanisms of vascular reactivity in functional and pharmacological MRI and has practical implications for designing fMRI experiments in anesthetized animals.

Cortical spreading depression (CSD) is a phenomenon implicated in migraine with aura and associated with other neurological disorders (e.g. stroke, brain trauma). Current evidence points to the essential role of NMDA receptors in CSD mechanisms. However, the roles of multiple subunits of NMDA receptors expressed in neurons, glia and blood vessels in vivo, are little explored. Using BOLD fMRI of urethane anesthetized rats as an integrative CSD readout, we tested the involvement of different NMDA receptor subtypes in CSD induction and propagation. Rats were treated with a non-selective NMDA blocker (MK-801), NR2B antagonist (ifenprodil) or a NR2A selective antagonist (TCN-201). CSD was induced during fMRI scanning by application of KCl onto the cerebral cortex and fMRI data were collected by 9.4 T MRI. The non-specific NMDA antagonist MK-801 completely blocked CSD, which was not observed in the NR2A group where TCN-201 did not alter the CSD features. Unexpectedly, the NR2B specific antagonist ifenprodil largely promoted the initial negative phase of the BOLD CSD response, likely due to altered neurovascular coupling. Our data suggest key roles and differential involvement of NMDA receptor subtypes in CSD generation and propagation, highlighting an important role for the NR2B subtype.

We found novel types of parenchymal functional magnetic resonance imaging (fMRI) signals in the rat brain during large increases in metabolism. Cortical spreading depression (CSD), a self-propagating wave of cellular activation, is associated with several pathologic conditions such as migraine and stroke. It was used as a paradigm to evoke transient neuronal depolarization leading to enhanced energy consumption. Activation of CSD was investigated using spin-lock (SL), diffusion, blood oxygenation level-dependent and cerebral blood volume fMRI techniques. Our results show that the SL-fMRI signal is generated by endogenous parenchymal mechanisms during CSD propagation, and these mechanisms are not associated with hemodynamic changes or cellular swelling. Protein phantoms suggest that pH change alone does not explain the observed SL-fMRI signal changes. However, increased amounts of inorganic phosphates released from high-energy phosphates combined with pH changes may produce SL- power-dependent longitudinal relaxation in the rotating frame ( R1ρ) changes in protein phantoms that are similar to those observed during CSD, as seen before in acute ischemia under our experimental conditions. This links SL-fMRI changes intimately to energy metabolism and supports the use of the SL technique as a new, promising functional approach for noninvasive imaging of metabolic transitions in the active or pathologic brain.

Malignant brain tumors are the most common cause of solid cancer death in children. Innovative therapies are vital to improve treatment outcomes, but must be developed to enable trafficking across the blood brain barrier (BBB). For this advent, animal models provide important information prior to clinical studies. Among the different in vivo models orthotopic patient-derived xenograft (PDX) models represent the diversity seen in patient tumors and hence replicate response rates in the clinical trials better as compared to other more simplistic models. Especially in the brain tumor field, imaging has a central role in clinical diagnosis and as a prognostic factor to monitor therapy response. It enables longitudinal patient monitoring in a fully translational manner. Magnetic resonance imaging (MRI) and positron emission tomography (PET) are widely used for clinical diagnosis and disease follow up. Choosing the most suitable imaging application depends of the target of interest or mechanism of action. MRI offers unprecedented soft tissue contrast, high spatial resolution and non-invasive nature renders MRI in rodents a perfect tool for preclinical work in oncological applications. In case of orthotopic brain tumor models, MRI offers the state-of-the-art quantitative volumetric tumor size analysis over disease progression. PET is an excellent tool to study tumor proliferation, metabolism, metastasis as well biodistribution of novel antibodies. The purpose of this work was to analyze volumetric, metabolic and functional changes in orthotopic PDX brain tumor model using MRI, MRS and PET imaging. During the course of the experiment, volume, perfusion within the tumor as well proliferation and metastasis were monitored. Perfusion measurement indicates angiogenesis in tumor, one hallmark of most malignant gliomas. Also, alterations in glucose and amino acid metabolism between tumor and healthy tissue has been previously identified. By direct comparison of the imaging data derived from the preclinical mouse model with similar data-sets from the donor patient the translational value of the model as well as the read-out system will be achieved. As a conclusion, translational in vivo imaging techniques were applied to study orthotopic tumor progression. These readouts provide a powerful and translational research tool together with oncological disease animal models allowing comprehensive evaluation of disease progression and treatment interventions for in vivo studies. Citation Format: Tuulia Huhtala, Julia Schueler, Jussi Rytkönen, Daniela Lötsch, Dorothee Lenhard, Artem Shatillo, Kimmo Lehtimäki, Pekka Poutiainen, Diana Miszczuk, Johannes Gojol, Walter Berger. Translational imaging findings in a pediatric patient-derived orthotopic xenograft brain tumor model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1959.

Conventional magnetic resonance imaging approaches currently used for the detection of myelin and demyelination, are suboptimal in the quantitative imaging of myelin. Novel MRI approach exploiting RAFF4 was better associated with myelin content during demyelination. For this study 22 rats were used. Of these, demyelinating lesions were induced in 13 rats of LPC. The remaining 9 animals were control. All animals were scanned in MRI system in demyelination stage and remyelination stage. MRI data was collected for quantification of RAFF4, DTI, T1sat and MTR. Histology was performed on all animals and brain sections were Nissl and myelin stained. All MRI metrics showed statistically significant differences between LPC group in the ipsilateral cc and the corresponding area in control animals, in demyelination stage at day 3. At 37 days, RAFF4 and MTR showed clear 70–80 % return towards normal values, likely corresponding to ongoing remyelination seen in histological sections. While similar recovery was detected in DTI metrics FA showed no recovery or even further increase during remyelination phase. was only diffusion parameter which did not show significant different between groups at 37 day. Our data indicates sensitivity of RAFF4 and MT to myelin content both in demyelinated lesions and during subsequent remyelination. DTI metrices seem to provide complementary information related not only myelin content but also architecture of the myelin sheets. Combination of RAFF4 and DTI has potential to differentiate between normal, demyelinated and remyelinated axonal bundles thus providing unique non-invasive characterization of white matter pathologies in various neurological diseases.

Indirect evidence suggests the increased production of reactive oxygen species (ROS) in migraine pathophysiology. In the current study we measured lipid peroxidation product in the rat cortex, trigeminal ganglia and meninges after the induction of cortical spreading depression (CSD), a phenomenon known to be associated with migraine aura, and tested nociceptive firing triggered by ROS in trigeminal nerves ex vivo. Application of KCl to dura mater in anesthetized rats induced several waves of CSD recorded by an extracellular electrode in the cortex. Following CSD, samples of cortex (affected regions were identified with blood oxygen level-dependent (BOLD) functional magnetic resonance imaging (fMRI)), meninges from left and right hemispheres and trigeminal ganglia were taken for biochemical analysis. We found that CSD increased the level of the lipid peroxidation product malondialdehyde (MDA) in the ipsilateral cerebral cortex and meninges, but also in both ipsi- and contralateral trigeminal ganglia. In order to test the pro-nociceptive action of ROS, we applied the mild oxidant hydrogen peroxide to isolated rat hemiskull preparations including preserved trigeminal innervations. Application of hydrogen peroxide to meninges transiently enhanced electrical spiking activity of trigeminal nerves showing a pro-nociceptive action of ROS. In the presence of hydrogen peroxide trigeminal nerves still responded to capsaicin by burst of spiking activity indicating integrity of neuronal structures. The action of hydrogen peroxide was mediated by TRPA1 receptors as it was abolished by the specific TRPA1 antagonist TCS-5861528. Using dorsal root ganglion sensory neurons as test system we found that hydrogen peroxide promoted the release of the migraine mediator calcitonin gene-related peptide (CGRP), which we previously identified as a trigger of delayed sensitization of trigeminal neurons. Our data suggest that, after CSD, oxidative stress spreads downstream within the trigeminal nociceptive system and could be involved in the coupling of CSD with the activation of trigeminovascular system in migraine pathology. © 2013 IBRO.

Background Simultaneous EEG-fMRI is a valuable tool in the clinic as it provides excellent temporal and spatial information about normal and diseased brain function. In pre-clinical research with small rodents, obtaining simultaneous EEG-fMRI in longitudinal studies faces a number of challenges, including issues related to magnetic susceptibility artifacts. New method Here, we demonstrate a method for permanent MRI RF-coil and EEG electrode implantation in rats that is suitable for long-term chronic follow-up studies in both stimulus and resting-state fMRI paradigms. Results Our findings showed that the screw-free implantation method is well suited for long-term follow-up studies in both freely moving video-EEG settings and fMRI without causing MRI susceptibility artifacts. Furthermore, the results demonstrated that a multimodal approach can be used to track the progression of structural and functional changes. Comparison with existing methods The quality of both MRI and EEG data were comparable to those obtained with traditional methods with the benefit of combining them into artifact-free simultaneous recordings. The signal-to-noise ratios of the MRI images obtained with the implanted RF-coil were similar to those using a quadrature coil and were therefore suitable for resting-state fMRI experiments. Similarly, EEG data collected with the RF-coil/electrode set-up were comparable to EEG recorded with traditional epidural screw electrodes. Conclusion This new multimodal EEG-fMRI approach provides a novel tool for concomitant analysis and follow-up of anatomic and functional MRI, as well as electrographic changes in a preclinical research.

Sleep is essential for nervous system functioning and sleep disorders are associated with several neurodegenerative diseases. However, the macroscale connectivity changes in brain networking during different sleep states are poorly understood. One of the hindering factors is the difficulty to combine functional connectivity investigation methods with spontaneously sleeping animals, which prevents the use of numerous preclinical animal models. Recent studies, however, have implicated that urethane anesthesia can uniquely induce different sleep-like brain states, resembling rapid eye movement (REM) and non-REM (NREM) sleep, in rodents. Therefore, the aim of this study was to assess changes in global connectivity and topology between sleep-like states in urethane anesthetized rats, using blood oxygenation level dependent (BOLD) functional magnetic resonance imaging. We detected significant changes in corticocortical (increased in NREM-like state) and corticothalamic connectivity (increased in REM-like state). Additionally, in graph analysis the modularity, the measure of functional integration in the brain, was higher in NREM-like state than in REM-like state, indicating a decrease in arousal level, as in normal sleep. The fMRI findings were supported by the supplementary electrophysiological measurements. Taken together, our results show that macroscale functional connectivity changes between sleep states can be detected robustly with resting-state fMRI in urethane anesthetized rats. Our findings pave the way for studies in animal models of neurodegenerative diseases where sleep abnormalities are often one of the first markers for the disorder development.

Experimental tumors raised in rodents represent an important preclinical tool to develop innovative anticancer compounds before clinical testing. Amongst others such models include solid tumors raised in syngeneic fully immunocompetent hosts and tumors spontaneously growing in genetically engineered mice (GEM) and derivate thereof. These model platforms have gained additional value since the manipulation of the immune system to fight cancer has led to tangible benefits for cancer patients. Imaging has become important part of the basic and translational research. It enables e.g. metabolic activity, volumetric and biodistribution monitoring within same individual over time. Magnetic resonance imaging (MRI) and positron emission tomography (PET) are widely used for clinical diagnosis and disease follow up. Choosing the most suitable imaging application depends of the prioritization of features. MRI offers unprecedented soft tissue contrast, high spatial resolution and non-invasive nature renders MRI in rodents a perfect tool for preclinical work in oncological applications. Similarly to clinical setting, MRI in rodents is used for detection of the tumors, evaluation of therapeutic response and induced changes, with longitudinal follow-up for early detection of possible tumor recurrence. PET is an excellent tool to study tumor metabolism, location as well biodistribution of novel antibodies. The purpose of this work was to study molecular as well volumetric, metabolic and functional changes in syngeneic oncological mouse model using MRI, MRS and PET imaging. Our panel consists of 32 fully characterized mouse tumor models covering 12 major cancer types. We characterized those models by molecular profiling (whole exome sequencing and RNAseq), histopathological examination (tissue micro array as well as whole slide analysis of Ki67, CD31, SMA, TIL infiltration) and sensitivity towards checkpoint inhibitors. Amongst other findings we saw an enhanced activity of anti-CTLA-4 treatment in combination with anti-PD1 in 4T1 orthotopically implanted into the mammary fat pad in comparison to single agent therapy. In a follow-up study we implanted 14 mice with breast cancer cell line 4T1 orthotopically into the mammary fat pad. When tumors were palpable treatment with the checkpoint inhibitor combination started. During the course of the experiment we determined tumor volume of the primary lesion twice weekly. In addition to molecular markers, MRI, MRS and PET were applied to study changes in tumor over cancer progression. As a conclusion, several syngeneic oncological mouse models have been characterized using molecular profiling, histological techniques and in vivo imaging. These readouts provide a powerful and translational research tool together with oncological disease animal models allowing comprehensive evaluation of disease progression and treatment interventions for in vivo studies. Citation Format: Julia Schueler, Artem Shatillo, Jussi Rytkönen, Nina Zanella, Antti Nurmi, Tuulia Huhtala. Molecular, histopathologic, MRI and PET findings in syngeneic oncologic mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3041.

Vascular changes underlying headache in migraine patients induced by Glyceryl trinitrate (GTN) were previously studied with various imaging techniques. Despite the long history of medical and experimental use of GTN, its effects on the brain vasculature are still poorly understood presumably due to low spatial resolution of the imaging modalities used so far. We took advantage of the micrometer-scale vertical resolution of two-photon microscopy to differentiate between the vasodynamic effects of GTN on meningeal versus cortical vessels imaged simultaneously in anesthetized rats through either thinned skull or glass-sealed cranial window. Intermediate and small calibre vessels were visualized in vivo by imaging intravascular fluorescent dextran, and detection of blood flow direction allowed identification of individual arterioles and venules. We found that i.p.-injected GTN induced a transient constriction of meningeal arterioles, while their cortical counterparts were, in contrast, dilated. These opposing effects of GTN were restricted to arterioles, whereas the effects on venules were insignificant. Interestingly, the NO synthase inhibitor L-NAME did not affect the diameter of meningeal vessels but induced a constriction of cortical vessels. The different cellular environment in cortex versus meninges as well as distinct vessel wall anatomical features probably play crucial role in the observed phenomena. These findings highlight differential region- and vessel-type-specific effects of GTN on cranial vessels, and may implicate new vascular mechanisms of NO-mediated primary headaches.

Cortical spreading depression (CSD) is a slowly propagating wave of depolarization of neurons and glia and has a less characterized vascular component. CSD is a commonly used phenomenon to test new methods of live brain imaging. Application of a blood pulsations imaging (BPI) technique to study of CSD induced with high-potassium solution in rat cortex allowed us to visualize for the first time the novel vascular component of a CSD wave. In our study, this wave component propagated in the limited part of the cortex along the bow-shaped trajectory in sharp contrast with concentric development of CSD measured by concurrently applied optical intrinsic signal (OIS) imaging technique. It was associated with a significant increase of the blood pulsations amplitude (BPA), started with a delay of 20 to 90 s comparing to signal measured with OIS, and propagated 40% faster than OIS signal. These findings suggest that the BPA and slower change of the cerebral blood volume are not directly related to each other even though both characterize the same vascular system. Our study indicates that the BPI technique could be used for characterization of the new pulsatile vascular component of CSDs in animal models of migraine, stroke, and brain trauma.

Manual segmentation of rodent brain lesions from magnetic resonance images (MRIs) is an arduous, time-consuming and subjective task that is highly important in pre-clinical research. Several automatic methods have been developed for different human brain MRI segmentation, but little research has targeted automatic rodent lesion segmentation. The existing tools for performing automatic lesion segmentation in rodents are constrained by strict assumptions about the data. Deep learning has been successfully used for medical image segmentation. However, there has not been any deep learning approach specifically designed for tackling rodent brain lesion segmentation. In this work, we propose a novel Fully Convolutional Network (FCN), RatLesNet, for the aforementioned task. Our dataset consists of 131 T2-weighted rat brain scans from 4 different studies in which ischemic stroke was induced by transient middle cerebral artery occlusion. We compare our method with two other 3D FCNs originally developed for anatomical segmentation (VoxResNet and 3D-U-Net) with 5-fold cross-validation on a single study and a generalization test, where the training was done on a single study and testing on three remaining studies. The labels generated by our method were quantitatively and qualitatively better than the predictions of the compared methods. The average Dice coefficient achieved in the 5-fold cross-validation experiment with the proposed approach was 0.88, between 3.7% and 38% higher than the compared architectures. The presented architecture also outperformed the other FCNs at generalizing on different studies, achieving the average Dice coefficient of 0.79., Comment: Accepted to Machine Learning in Medical Imaging (MLMI 2019)