Ex-vivo Imaging Research Articles

A road map to manual segmentation of cerebral structures.

Manual segmentation is an essential tool in the researcher's technical arsenal. It is a frequent practice necessary for image analysis in many protocols, especially in neuroimaging and comparative brain anatomy. In the framework of emergence of studies focusing on alternative animal models, manual segmentation procedures play a critical role. Nevertheless, this critical task is often assigned to students, a process that, unfortunately, tends to be time-consuming and repetitive. Well-conducted and well-described segmentation procedures can potentially guide novice and even expert operators and enhance research works' internal and external validity, making it possible to harmonize studies and facilitate data sharing. Furthermore, recent advances in neuroimaging, such as exvivo imaging or ultra-high-field MRI, enable new acquisition modalities and the identification of minute structures that are barely visible with typical approaches. In this context of increasingly detailed and multimodal brain studies, reflecting on methodology is relevant and necessary. Because it is crucial to implement good practices in manual segmentation per se but also in the description of the segmentation procedures in research papers, we propose a general roadmap for optimizing the technique, its process and the reporting of manual segmentation. For each of them, the relevant elements of the literature have been collected and cited. The article is accompanied by a checklist that the reader can use to verify that the critical steps are being followed.

Combination chemotherapy via poloxamer 188 surface-modified PLGA nanoparticles that traverse the blood–brain–barrier in a glioblastoma model

The effect of chemotherapy for anti-glioblastoma is limited due to insufficient drug delivery across the blood–brain–barrier. Poloxamer 188-coated nanoparticles can enhance the delivery of nanoparticles across the blood–brain–barrier. This study presents the design, preparation, and evaluation of a combination of PLGA nanoparticles (PLGA NPs) loaded with methotrexate (P-MTX NPs) and PLGA nanoparticles loaded with paclitaxel (P-PTX NPs), both of which were surface-modified with poloxamer188. Cranial tumors were induced by implanting C6 cells in a rat model and MRI demonstrated that the tumors were indistinguishable in the two rats with P-MTX NPs + P-PTX NPs treated groups. Brain PET scans exhibited a decreased brain-to-background ratio which could be attributed to the diminished metabolic tumor volume. The expression of Ki-67 as a poor prognosis factor, was significantly lower in P-MTX NPs + P-PTX NPs compared to the control. Furthermore, the biodistribution of PLGA NPs was determined by carbon quantum dots loaded into PLGA NPs (P-CQD NPs), and quantitative analysis of ex-vivo imaging of the dissected organs demonstrated that 17.2 ± 0.6% of the NPs were concentrated in the brain after 48 h. The findings highlight the efficacy of combination nanochemotherapy in glioblastoma treatment, indicating the need for further preclinical studies.

Dissecting contributions of pulmonary arterial remodeling to right ventricular afterload in pulmonary hypertension.

Pulmonary hypertension (PH) is defined as an elevation in the right ventricle (RV) afterload, characterized by increased hemodynamic pressure in the main pulmonary artery (PA). Elevations in RV afterload increase RV wall stress, resulting in RV remodeling and potentially RV failure. From a biomechanical standpoint, the primary drivers for RV afterload elevations include increases in pulmonary vascular resistance (PVR) in the distal vasculature and decreases in vessel compliance in the proximal PA. However, the individual contributions of the various vascular remodeling events toward the progression of PA pressure elevations and altered vascular hemodynamics remain elusive. In this study, we used a subject-specific one-dimensional (1D) fluid-structure interaction (FSI) model to investigate the alteration of pulmonary hemodynamics in PH and to quantify the contributions of vascular stiffening and increased resistance towards increased main pulmonary artery (MPA) pressure. We used a combination of subject-specific hemodynamic measurements, ex-vivo mechanical testing of arterial tissue specimens, and ex-vivo X-ray micro-tomography imaging to develop the 1D-FSI model and dissect the contribution of PA remodeling events towards alterations in the MPA pressure waveform. Both the amplitude and pulsatility of the MPA pressure waveform were analyzed. Our results indicated that increased distal resistance has the greatest effect on the increase in maximum MPA pressure, while increased stiffness caused significant elevations in the characteristic impedance. The method presented in this study will serve as an essential step toward understanding the complex interplay between PA remodeling events that leads to the most severe adverse effect on RV dysfunction.

Beyond the surface: how ex-vivo diffusion-weighted imaging reveals large animal brain microstructure and connectivity.

Diffusion-weighted Imaging (DWI) is an effective and state-of-the-art neuroimaging method that non-invasively reveals the microstructure and connectivity of tissues. Recently, novel applications of the DWI technique in studying large brains through ex-vivo imaging enabled researchers to gain insights into the complex neural architecture in different species such as those of Perissodactyla (e.g., horses and rhinos), Artiodactyla (e.g., bovids, swines, and cetaceans), and Carnivora (e.g., felids, canids, and pinnipeds). Classical in-vivo tract-tracing methods are usually considered unsuitable for ethical and practical reasons, in large animals or protected species. Ex-vivo DWI-based tractography offers the chance to examine the microstructure and connectivity of formalin-fixed tissues with scan times and precision that is not feasible in-vivo. This paper explores DWI's application to ex-vivo brains of large animals, highlighting the unique insights it offers into the structure of sometimes phylogenetically different neural networks, the connectivity of white matter tracts, and comparative evolutionary adaptations. Here, we also summarize the challenges, concerns, and perspectives of ex-vivo DWI that will shape the future of the field in large brains.

Pathological cell assembly dynamics in a striatal MSN network model.

Under normal conditions the principal cells of the striatum, medium spiny neurons (MSNs), show structured cell assembly activity patterns which alternate sequentially over exceedingly long timescales of many minutes. It is important to understand this activity since it is characteristically disrupted in multiple pathologies, such as Parkinson's disease and dyskinesia, and thought to be caused by alterations in the MSN to MSN lateral inhibitory connections and in the strength and distribution of cortical excitation to MSNs. To understand how these long timescales arise we extended a previous network model of MSN cells to include synapses with short-term plasticity, with parameters taken from a recent detailed striatal connectome study. We first confirmed the presence of sequentially switching cell clusters using the non-linear dimensionality reduction technique, Uniform Manifold Approximation and Projection (UMAP). We found that the network could generate non-stationary activity patterns varying extremely slowly on the order of minutes under biologically realistic conditions. Next we used Simulation Based Inference (SBI) to train a deep net to map features of the MSN network generated cell assembly activity to MSN network parameters. We used the trained SBI model to estimate MSN network parameters from ex-vivo brain slice calcium imaging data. We found that best fit network parameters were very close to their physiologically observed values. On the other hand network parameters estimated from Parkinsonian, decorticated and dyskinetic ex-vivo slice preparations were different. Our work may provide a pipeline for diagnosis of basal ganglia pathology from spiking data as well as for the design pharmacological treatments.

UFObow: A single-wavelength excitable Brainbow for simultaneous multicolor ex-vivo and in-vivo imaging of mammalian cells.

Brainbow is a genetic cell-labeling technique that allows random colorization of multiple cells and real-time visualization of cell fate within a tissue, providing valuable insights into understanding complex biological processes. However, fluorescent proteins (FPs) in Brainbow have distinct excitation spectra with peak difference greater than 35 nm, which requires sequential imaging under multiple excitations and thus leads to long acquisition times. In addition, they are not easily used together with other fluorophores due to severe spectral bleed-through. Here, we report the development of a single-wavelength excitable Brainbow, UFObow, incorporating three newly developed blue-excitable FPs. We have demonstrated that UFObow enables not only tracking the growth dynamics of tumor cells in vivo but also mapping spatial distribution of immune cells within a sub-cubic centimeter tissue, revealing cell heterogeneity. This provides a powerful means to explore complex biology in a simultaneous imaging manner at a single-cell resolution in organs or in vivo.

108 A Translational Approach to Suppress Local Inflammation in the Ischemic Penumbra and Deliver Local Anti-platelet Activity After Stroke

INTRODUCTION: Mechanical thrombectomy is the standard of care for ischemic stroke, but there is a significant mismatch between successful recanalization and clinical outcomes. Inflammation in the ischemic penumbra remains a target for neuroprotective interventions even in recanalized and non-recanalized patients. P-selectin (Psel) is a marker of endothelial cell activation and could be used to deliver therapeutics locally to the site of ischemic injury. METHODS: The fusion protein Pselectin-Crry was constructed using single chain antibody sequence linked to the extracellular region of mouse Crry. In-vivo testing using surface plasmon resonance for binding affinity to Psel, and zymosan assay for complement inhibitory activity. In-vivo studies were performed in C57bl/6 mice subjected to transient middle cerebral artery occlusion for 1 hour followed by administration of Psel-Crry 2 hours after reperfusion. Psel-Crry was labelled using far-red fluorescence and live-animal imaging was used to study brain-specific targeting. The modified Neurological Severity (NS) score was used for functional outcome along with survival, infarct volume and histological outcomes. RESULTS: Psel-Crry demonstrated dose-dependent complement inhibitory activity, and localized specifically to the stroke brain on in-vivo live imaging. Ex-vivo imaging of explanted brain showed Psel-Crry binding specifically to the injured hemisphere without off-target binding (Stroke vs. Sham, p < 0.001). Psel-Crry resulted in 60% reduction in infarct volume at 3 days compare to vehicle (N = 8/group, p < 0.05), reduction in NS scores compared to vehicle (Median 3 vs. 1.5, p < 0.05), and improved 1-week survival (80% vs. 50%, p < 0.05). Histological analyses demonstrated preservation of dendritic arborization in the ischemic penumbra with Psel-Crry treatment with concurrent significant reduction in microglial activation. CONCLUSIONS: Psel-Crry is a translational agent that can achieve local complement inhibitory effect and antiplatelet activity after stroke and has promise for potential use as ajuvant therapy in stroke.

Brillouin microscopic imaging of ex-vivo porcine eye using VIPA-CMOS-based spectrometer

The research on non-invasive ophthalmic imaging has become a hot topic in diagnosing ophthalmic diseases, especially assessment of corneal biomechanics has been an unmet clinical need in ophthalmology for many years. The Brillouin microscopy technique based on spontaneous Brillouin scattering has emerged as a unique elastography method due to its advantages of non-contact, label-free, and high-resolution biomechanical imaging of biological tissues. Here, we developed a three-dimensional (3D) optical microscope system for ex-vivo porcine cornea Brillouin imaging. To do this, we designed and experimentally demonstrated a setup with double-stage VIPA etalons and CMOS camera that integrate to a fully parallel dispersive Brillouin spectrometer. The designed spectrometer exhibits a smaller dispersion rate to enhance the spectral sampling rate and downsizes the size of the double-stage-based system. Using the optical microscope system, we measured and mapped the Brillouin frequency shifts of the cornea and crystalline lens in ex-vivo porcine eyes at micron-scale spatial resolution, and a spectral accuracy of < 50 MHz can be achieved without interpolating the spectra. This work is essential for future applications of Brillouin microscopy in clinical diagnosis and treatment of ophthalmic diseases.

Abstract 2840: Development, characterization, and validation of a systemic human mantle cell lymphoma model utilizing luciferase expressing Jeko-1 tumor cells

Abstract Mantle cell lymphoma (MCL) is an aggressive subtype of B-cell non-Hodgkin lymphoma that is clinically and biologically heterogenous. MCL murine models are scarce and reliable in vivo models are necessary for pre-clinical testing and identification of improved novel therapeutics. To address this need, we established and characterized a human MCL in vivo model by systemic implantation of the luciferase-expressing Jeko-1 cell line (Jeko-1-luc) into immunodeficient NCG mice. Tumor growth kinetics was evaluated by testing multiple cell inoculums, and disease progression was monitored by in vivo serial bioimaging, three-dimensional optical imaging (3D), and ex-vivo endpoint analyses. Jeko-1-luc tumor growth profile revealed moderate latency with a median survival of 40 days post implant. Subsequently, we utilized 3D optical imaging and ex-vivo imaging that confirmed the localization of tumor growth predominantly in lungs, spleen, and skeletal regions. Additionally, we determined the surface expression of selected B-cell lineage markers including CD19, CD20, and CD22. Next, we evaluated the efficacy and effect on survival in response to various standard of care agents including Vincristine, Rituximab, Palbociclib, Temsirolimus, and Cyclophosphamide. The quantification of tumor burden by in vivo and ex-vivo bioluminescent imaging demonstrated that the highest therapeutic activity and overall survival was achieved by Palbociclib and Vincristine. Flow cytometric analyses further demonstrated that tumor cells obtained from blood and selected tissues including spleen and bone marrow showed reduced tumor burden in treatment groups relative to the control group. Taken together, these results illustrate the translational value of the Jeko-1-luc model and provides a platform for evaluating novel treatment modalities for mantle cell lymphoma. Citation Format: David Harris, Andrew Wong, Elizabeth Rainbolt, Nicole Westrick, Justin Avery, Joseph Kolb, Aswani Vunnava, Zachary Ward, Chassidy Hall, Bincy John. Development, characterization, and validation of a systemic human mantle cell lymphoma model utilizing luciferase expressing Jeko-1 tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2840.

Abstract 4166: TSPO-targeted near-infrared optical probe for early identification and localization of high-risk premalignant pancreatic lesions

Abstract Background: Pancreatic cancer is the third most prevalent cause of cancer death in the U.S. with a ~10% survival rate. Surgery represents one of the best opportunities for extending survival and potential cures. Fluorescence-guided surgery (FGS) employing near-infrared (NIR) fluorescence-based targeting agents improves resection margins, allowing removal of small lesions not visible under white light. We previously reported a targeted NIR tracer (V-1520) of translocator protein (TSPO) expression that demonstrated proof-of-principle utility in high-grade pancreatic lesions arising in Ptf1a-Cre; LSL-KrasG12D/+; Smad4flox/flox (KCS) of pancreatic cancer. The purpose of the present study is to evaluate the utility of V-1520 to identify low-grade, premalignant yet high risk lesions in KCS mice; in this scenario we attempted to model use of V-1520 to remove lesions likely to manifest as future recurrence. We compared the uptake of the tracer in these mice from weaning to adulthood to systematically evaluate the efficacy of FGS as a function of lesion grade. Experimental: The tracer, V-1520, was synthesized and KCS mice were generated as reported.1 Mice were injected with 30μg of V-1520 intravenously 24 hours before imaging. Images were acquired using the 800nm channel. KCS and WT mice were imaged as a function of from 4 to 11 weeks age. After imaging, tumor tissues and major organs were harvested for ex-vivo imaging, histopathology and Cryo-Fluorescent Tomography (CFT). Summary: We found, V-1520 prominently localized in low-grade, premalignant PanIN cystic lesions with malignancy potential, as well as pancreatic tumors with and without local invasion. In contrast, V-1520 did not accumulate in healthy pancreata of KCS or WT mice. Mice as young as 4 weeks exhibited localized accumulation of the tracer with low grade PanIN and early dysplasia. The fluorescence intensity was further enhanced in mice with higher grade disease, whereby the pathology was characterized by high-grade cyst formation, increased nuclear/cytoplasm ratio, acinar to ductal metaplasia and invasion. Low and high-grade neoplasia was characterized by high TSPO immunoreactivity, consistent with in-vivo imaging. Confocal microscopy was used to colocalize V-1520 accumulation and TSPO immunoreactivity; V-1520 was found to prominently localized to premalignant epithelia pancreatic cancer cells. CFT illustrated that the primary localization of V-1520 included the liver, kidneys, and pancreatic diseases. Conclusion: These studies suggest the utility of V-1520 to identify high-risk pancreatic lesions that could be removed with surgery. Encouragingly, the V-1520 was capable of differentiating tumors from surrounding non-tumor tissue at an early stage, which could lead to better resections and delay and/or prevent recurrence in the future. 1. Clinical Cancer Research 26(22), 15 Nov. 2020 5914-5925 Citation Format: Shilpa Sharma, Denise Hernandez, Xiaoxia Wen, Jianbo Wang, Cong-Dat Pham, Beibei Huang, Henry Charles Manning. TSPO-targeted near-infrared optical probe for early identification and localization of high-risk premalignant pancreatic lesions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4166.

Exploring the potential of ex-vivo 7-T magnetic resonance imaging on patients with clinically significant prostate cancer: visibility and size perspective

BackgroundDespite progress in multiparametric magnetic resonance imaging (MRI), issues of prostate cancer invisibility and underestimated tumor burden persist. This study investigates the potential of an ultra-high field MRI at 7-T in an ex-vivo setting to address these limitations. MethodsThis prospective study included 54 tumors from 20 treatment-naïve clinically significant prostate cancer patients, confirmed by biopsy, despite negative findings on preoperative 3-T MRI. Ex-vivo 7-T MRI of resected prostates was performed, with assessment on tumor visibility and size. Factors influencing visibility were analyzed using logistic regression analyses. ResultsTumor visibility was confirmed in 80% of patients, and 48% of all tumors on ex-vivo imaging. Gleason pattern 4 percentage (odds ratio 1.09) and tumor size on pathology (odds ratio 1.36) were significantly associated with visibility (P < 0.05). Mean MRI-visible and invisible tumor sizes were 10.5 mm and 5.3 mm, respectively. The size discrepancy between MRI and pathology was 2.7 mm. ConclusionTumor visibility on ex-vivo 7-T MRI was influenced by tumor grade and size. The notable tumor visibility initially overlooked on 3-T MRI, along with small size discrepancy with pathology, suggests potential improvements in resolution.

A toolkit for stroke infarct volume estimation in rodents

Stroke volume is a key determinant of infarct severity and an important metric for evaluating treatments. However, accurate estimation of stroke volume can be challenging, due to the often confined 2-dimensional nature of available data. Here, we introduce a comprehensive semi-automated toolkit to reliably estimate stroke volumes based on (1) whole brains ex-vivo magnetic resonance imaging (MRI) and (2) brain sections that underwent immunofluorescence staining. We located and quantified infarct areas from MRI three days (acute) and 28 days (chronic) after photothrombotic stroke induction in whole mouse brains. MRI results were compared with measures obtained from immunofluorescent histologic sections of the same brains. We found that infarct volume determined by post-mortem MRI was highly correlated with a deviation of only 6.6 % (acute) and 4.9 % (chronic) to the measurements as determined in the histological brain sections indicating that both methods are capable of accurately assessing brain tissue damage (Pearson r > 0.9, p < 0.001). The Dice similarity coefficient (DC) showed a high degree of coherence (DC > 0.8) between MRI-delineated regions of interest (ROIs) and ROIs obtained from histologic sections at four to six pre-defined landmarks, with histology-based delineation demonstrating higher inter-operator similarity compared to MR images. We further investigated stroke-related scarring and post-ischemic angiogenesis in cortical peri‑infarct regions and described a negative correlation between GFAP+fluorescence intensity and MRI-obtained lesion size.

Treatment affects load to failure and microdamage accumulation in healthy and osteolytic rat vertebrae

Bone turnover and microdamage are impacted by the presence of skeletal metastases which can contribute to increased fracture risk. Treatments for metastatic disease may further impact bone quality. This exploratory study aimed to establish an initial understanding of microdamage accumulation and load to failure in healthy and osteolytic rat vertebrae following focal and systemic cancer treatment (docetaxel (DTX), stereotactic body radiotherapy (SBRT), or zoledronic acid (ZA)). Osteolytic spine metastases were developed in 6-week-old athymic female rats via intracardiac injection of HeLa human cervical cancer cells (day 0). Additional rats served as healthy controls. Rats were either untreated, received SBRT to the T10-L6 vertebrae on day 14 (15 Gy, two fractions), DTX on day 7 or 14, or ZA on day 7. Rats were euthanized on day 21. Tumor burden was assessed with bioluminescence images acquired on day 14 and 21, histology of the excised T11 and L5 vertebrae, and ex-vivo μCT images of the T13-L4. Microstructural parameters (bone volume/total volume, trabecular number, spacing, thickness, and bone mineral density) were measured from L2 vertebrae. Load to failure was measured with axial compressive loading of the L1-L3 motion segments. Microdamage accumulation was labeled in T13 vertebrae with BaSO4 staining and was visualized with high resolution μCT imaging. Microdamage volume fraction was defined as the ratio of BaSO4 to bone volume. DTX administered on day 7 reduced tumor growth significantly (p < 0.05). Microdamage accumulation was found to be increased by the presence of metastases but was reduced by all treatments with ZA showing the largest improvement in HeLa cell injected rats. Load to failure was decreased in untreated and SBRT HeLa cell injected rats compared to healthy controls (p < 0.01). There was a moderate negative correlation between load to failure and microdamage volume fraction in vertebrae from rats injected with HeLa cells (R = −0.35, p = 0.031). Strong correlations were also found between microstructural parameters and load to failure and microdamage accumulation. Several factors, including the presence of osteolytic lesions and use of cancer therapies, influence microdamage accumulation and load to failure in rat vertebrae. Understanding the impact of these treatments on fracture risk of metastatic vertebrae is important to improve management of patients with spinal metastases.

Ex-vivo 1.5T MR Imaging versus CT in Estimating the Size of the Pathologically Invasive Component of Lung Adenocarcinoma Spectrum Lesions.

The purpose of this study was to investigate whether ex-vivo MRI enables accurate estimation of the invasive component of lung adenocarcinoma. We retrospectively reviewed 32 patients with lung adenocarcinoma who underwent lung lobectomy. The specimens underwent MRI at 1.5T. The boundary between the lesion and the normal lung was evaluated on a 5-point scale in each three MRI sequences, and a one-way analysis of variance and post-hoc tests were performed. The invasive component size was measured histopathologically. The maximum diameter of each solid component measured on CT and MR T1-weighted (T1W) images and the maximum size obtained from histopathologic images were compared using the Wilcoxon signed-rank test. Inter-reader agreement was evaluated using intraclass correlation coefficients (ICC). T1W images were determined to be optimal for the delineation of the lesions (P < 0.001). The histopathologic invasive area corresponded to the area where the T1W ex-vivo MR image showed a high signal intensity that was almost equal to the intravascular blood signal. The maximum diameter of the solid component on CT was overestimated compared with the maximum invasive size on histopathology (mean, 153%; P < 0.05), while that on MRI was evaluated mostly accurately without overestimation (mean, 108%; P = 0.48). The interobserver reliability of the measurements using CT and MRI was good (ICC = 0.71 on CT, 0.74 on MRI). Ex-vivo MRI was more accurate than conventional CT in delineating the invasive component of lung adenocarcinoma.

Immunomodulatory effects of copper nanoparticles against mitogen-stimulated rat splenic and thymic lymphocytes

In the present study, we have evaluated the effects of copper (Cu) nanoparticles (NPs) on the primary B-and T-lymphocytes proliferation, cytokine levels, and bio-distribution through in vitro, in vivo and ex-vivo studies to allow the possible exploitations of CuNPs in biomedical applications. CuNPs were characterized by UV–Visible spectroscopy, transmission electron microscopy (TEM), and nanoparticle tracking analysis (NTA). The proliferative response of lymphocytes was studied by 3H-thymidine incorporation assay and lymphocyte viability through trypan blue assay. The bio-distribution of CuNPs into lymphoid organs was examined by using ex-vivo imaging system. Cytokine levels in plasma of control and CuNPs treated animal groups were determined by enzyme-linked immunosorbent assay (ELISA) method along with other biochemical analysis. CuNPs significantly suppressed the proliferation of primary splenic and thymic lymphocytes in a dose dependent manner. Ex-vivo imaging exhibited the distribution of CuNPs in spleen and thymus. Oral administration of CuNPs (2 mg and 10 mg/kg body weight) significantly inhibited the proliferation of splenic and thymic lymphocytes along with lowered cytokines levels (TNF-alpha and IL-2) on comparison with controls. The results indicated the significant inhibition of lymphocytes proliferative response and secretion of cytokines, thus unveiling the immunomodulatory effects of CuNPs.

Cross-hemicord spinal fiber reorganization associates with cortical sensory and motor network expansion in the rat model of hemicontusion cervical spinal cord injury

Magnetic resonance imaging plays an important role in characterizing microstructural changes and reorganization after traumatic injuries to the nervous system. In this study, we tested the feasibility of ex-vivo spinal cord diffusion tensor imaging (DTI) in combination with in vivo brain functional MRI to characterize spinal reorganization and its supraspinal association after a hemicontusion cervical spinal cord injury (SCI). DTI parameters (fractional anisotropy [FA], mean diffusion [MD]) and fiber orientation changes related to reorganization in the contused cervical spinal cord were compared to sham specimens. Altered fiber density and fiber directions occurred across the ipsilateral and contralateral hemicords but with only ipsilateral FA and MD changes. The hemicontusion SCI resulted in ipsilateral fiber breaks, voids and vivid fiber reorientations along the injury epicenter. Fiber directional changes below the injury level were primarily inter-hemispheric, indicating prominent below-level cross-hemispheric reorganization. In vivo resting state functional connectivity of the brain from the respective rats before obtaining the spinal cord samples indicated spatial expansion and increased connectivity strength across both the sensory and motor networks after SCI. The consistency of the neuroplastic changes along the neuraxis (both brain and spinal cord) at the single-subject level, indicates that distinctive reorganizational relationships exist between the spinal cord and the brain post-SCI.

IMMU-52. LOW-INTENSITY PULSED ULTRASOUND CONDUCTS ANTIGEN-INDEPENDENT T CELL HOMING INTO THE BRAIN PARENCHYMA WITH THE INTACT BLOOD-BRAIN BARRIER

Abstract The blood-brain barrier (BBB) protects the brain parenchyma from excessive inflammation but also leads to poor immune cell homing, hampering the success of immunotherapies, such as ones for low-grade glioma, where the BBB is intact. Low-intensity pulsed ultrasound (LIPU) combined with the application of microbubbles allows the non-invasive, transient opening of the BBB and recent studies have observed immune cell infiltration into malignant gliomas following LIPU. While these data warrant mechanistic investigations on the effects of LIPU in preclinical settings, most mouse models of malignant glioma already display BBB disruption. Therefore, to study whether LIPU affects antigen-specific immune cell trafficking through the BBB, we created a novel immunocompetent mouse model of non-tumor bearing mice that display an intact BBB and express a minigene cassette encoding for four immunogenic peptides in glia cells under the glial fibrillary acidic protein (GFAP) promotor. These peptides are either presented by major histocompatibility complex (MHC) class I: human melanoma antigen glycoprotein (gp) 100 and lymphocytic choriomeningitis virus (LCMV)gp31) or class II: LCMV gp66 and tyrosinase-related protein 1. Minigene expression in astrocytes induced antigen-specific T cell proliferation and Interferon-γ release in vitro. Intravenous adoptive T cell transfer of naïve or respectively CD3/CD28-coactivated antigen-specific CD 8 T cells targeting gp100 peptide followed by immediate LIPU treatment induced T cell homing into the CNS parenchyma, and astrocyte-T cell interaction was confirmed by ex-vivo two-photon imaging. Furthermore, some animals that underwent immediate LIPU treatment after transfer of preactivated antigen-specific T cells developed neurological deficits such as ataxia, while no deficits were observed in those animals that underwent LIPU but did not receive T cells or received activated T cells but did not undergo LIFU treatment. In conclusion, LIPU might be a novel effective tool to support antigen-independent immune cell homing with the intact BBB.

Abstract 14675: The Role of Biomechanics in Diagnosis and Evaluation of Coronary Artery Disease

While there is a strong need to assess the safety and efficacy of novel therapies prior to evaluation in human patients, the ability to accurately model the complexities of coronary disease in-vitro and/or animal models is somewhat limited. Animal models, even those with genetic predispositions toward coronary disease do not exhibit high grade stenoses, similar to those that require treatment in human patients. At the same time, most in-vitro models and simulations of fluid-structure interactions cannot simultaneously capture the complex geometries, hemodynamics, and biomechanical response of human coronary disease. Here, we introduce a robust workflow to replicate an in-vitro platform incorporating compliant, realistic diseased coronary arteries in a flow loop under physiological conditions. Through ex-vivo imaging of cadaveric specimens, coupled with co-registered measurements of coronary biomechanics, we developed protocols that allow for fabrication and testing of highly realistic 3D printed models. The anatomical and biomechanical features of the coronary arteries, including local variations associated with the observed disease burdens were extracted and 3D printed, in high spatial resolution, using commercially available printers, making this methodology conveniently reproducible. Subsequently, the models were incorporated in a testing platform made of flow loops and valve resistance to mimic circulation and microvascular resistance respectively. Under physiological boundary conditions, we successfully collected measurements along the length of the vessels that exhibit a range of biomechanical characteristics from low to high values of modulus and assessed the impact of biomechanics on gold standard diagnostic and prognostic measures such as Fractional Flow Reserve. We demonstrated that incorporating the effects of local biomechanics significantly improves the predicted hemodynamics metrics with respect to ex-vivo coronaries.

Mapping the neuroanatomical abnormalities in a phenotype of male compulsive rats

Compulsivity is considered a transdiagnostic dimension in obsessive–compulsive and related disorders, characterized by heterogeneous cognitive and behavioral phenotypes associated with abnormalities in cortico-striatal-thalamic-cortical circuitry. The present study investigated the structural morphology of white and gray matter in rats selected for low- (LD) and high- (HD) compulsive drinking behavior on a schedule-induced polydipsia (SIP) task. Regional brain morphology was assessed using ex-vivo high-resolution magnetic resonance imaging (MRI). Voxel-based morphometry of segmented MRI images revealed larger white matter volumes in anterior commissure and corpus callosum of HD rats compared with LD rats. HD rats also showed significantly larger regional volumes of dorsolateral orbitofrontal cortex, striatum, amygdala, hippocampus, midbrain, sub-thalamic nucleus, and cerebellum. By contrast, the medial prefrontal cortex was significantly smaller in HD rats compared with LD rats with no significant group differences in whole brain, ventricular, or cerebrospinal fluid volumes. These findings show that limbic cortico-basal ganglia structures implicated in impulse control disorders are distinct in rats that are vulnerable to develop compulsive behavior. Such abnormalities may be relevant to the etiology of compulsive disorders in humans.

Longitudinal x-ray based lung function measurement for monitoring Nintedanib treatment response in a mouse model of lung fibrosis

Lung fibrosis (LF) is a chronic progressive, incurable, and debilitating condition of the lung, which is associated with different lung disease. Treatment options are still sparse. Nintedanib, an oral tyrosine kinase inhibitor, significantly slows the LF progression. However, there is a strong need of further research and the development of novel therapies. In this study, we used a correlative set-up that combines X-ray based lung function (XLF) with microCT and whole body plethysmography (WBP) for a comprehensive functional and structural evaluation of lung fibrosis (LF) as well as for monitoring response to orally administered Nintedanib in the mouse model of bleomycin induced LF. The decline in lung function as early as one week after intratracheal bleomycin instillation was reliably detected by XLF, revealing the lowest decay rate in the LF mice compared to healthy ones. Simultaneously performed microCT and WBP measurements corroborated XLF findings by exhibiting reduced lung volume V^{insp}_{mu CT} and tidal volume TV_{WBP}. In LF mice XLF also revealed profound improvement in lung function one week after Nintedanib treatment. This positive response to Nintedanib therapy was further substantiated by microCT and WBP measurements which also showed significantly improved V^{insp}_{mu CT} and TV_{WBP} in the Nintedanib treated mice. By comparing the XLF data to structural features assessing the extent of fibrosis obtained by ex-vivo high-resolution synchrotron radiation-based imaging and classical histology we demonstrate that: (1) a simple low dose x-ray measurement like XLF is sensitive enough to pick up treatment response, (2) Nintedanib treatment successfully improved lung function in a bleomycin induced LF mouse model and (3) differences between the fully restored lung function and the partially reduced fibrotic burden compared to healthy and untreated mice. The presented analysis pipeline underlines the importance of a combined functional and anatomical readout to reliably measure treatment response and could easily be adapted to other preclinical lung disease models.