Hypoxic-Ischemic Encephalopathy (HIE) in the brain is the leading cause of morbidity and mortality in neonates and can lead to irreparable tissue damage and cognition. Thus, investigating key mediators of the HI response to identify points of therapeutic intervention has significant clinical potential. Brain repair after HI requires highly coordinated injury responses mediated by cell-derived extracellular vesicles (EVs). Studies show that stem cell-derived EVs attenuate the injury response in ischemic models by releasing neuroprotective, neurogenic, and anti-inflammatory factors. In contrast to 2D cell cultures, we successfully isolated and characterized EVs from whole brain rat tissue (BEV) to study the therapeutic potential of endogenous EVs. We showed that BEVs decrease cytotoxicity in an ex vivo oxygen glucose deprivation (OGD) brain slice model of HI in a dose- and time-dependent manner. The minimum therapeutic dosage was determined to be 25 μg BEVs with a therapeutic application time window of 4–24 h post-injury. At this therapeutic dosage, BEV treatment increased anti-inflammatory cytokine expression. The morphology of microglia was also observed to shift from an amoeboid, inflammatory phenotype to a restorative, anti-inflammatory phenotype between 24–48 h of BEV exposure after OGD injury, indicating a shift in phenotype following BEV treatment. These results demonstrate the use of OWH brain slices to facilitate understanding of BEV activity and therapeutic potential in complex brain pathologies for treating neurological injury in neonates.
more »
« less
Organotypic whole hemisphere brain slice models to study the effects of donor age and oxygen-glucose-deprivation on the extracellular properties of cortical and striatal tissue
Abstract Background The brain extracellular environment is involved in many critical processes associated with neurodevelopment, neural function, and repair following injury. Organization of the extracellular matrix and properties of the extracellular space vary throughout development and across different brain regions, motivating the need for platforms that provide access to multiple brain regions at different stages of development. We demonstrate the utility of organotypic whole hemisphere brain slices as a platform to probe regional and developmental changes in the brain extracellular environment. We also leverage whole hemisphere brain slices to characterize the impact of cerebral ischemia on different regions of brain tissue. Results Whole hemisphere brain slices taken from postnatal (P) day 10 and P17 rats retained viable, metabolically active cells through 14 days in vitro (DIV). Oxygen-glucose-deprivation (OGD), used to model a cerebral ischemic event in vivo, resulted in reduced slice metabolic activity and elevated cell death, regardless of slice age. Slices from P10 and P17 brains showed an oligodendrocyte and microglia-driven proliferative response after OGD exposure, higher than the proliferative response seen in DIV-matched normal control slices. Multiple particle tracking in oxygen-glucose-deprived brain slices revealed that oxygen-glucose-deprivation impacts the extracellular environment of brain tissue differently depending on brain age and brain region. In most instances, the extracellular space was most difficult to navigate immediately following insult, then gradually provided less hindrance to extracellular nanoparticle diffusion as time progressed. However, changes in diffusion were not universal across all brain regions and ages. Conclusions We demonstrate whole hemisphere brain slices from P10 and P17 rats can be cultured up to two weeks in vitro. These brain slices provide a viable platform for studying both normal physiological processes and injury associated mechanisms with control over brain age and region. Ex vivo OGD impacted cortical and striatal brain tissue differently, aligning with preexisting data generated in in vivo models. These data motivate the need to account for both brain region and age when investigating mechanisms of injury and designing potential therapies for cerebral ischemia.
more »
« less
- Award ID(s):
- 1934292
- NSF-PAR ID:
- 10339820
- Date Published:
- Journal Name:
- Journal of Biological Engineering
- Volume:
- 16
- Issue:
- 1
- ISSN:
- 1754-1611
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Introduction: Computed tomography perfusion (CTP) imaging requires injection of an intravenous contrast agent and increased exposure to ionizing radiation. This process can be lengthy, costly, and potentially dangerous to patients, especially in emergency settings. We propose MAGIC, a multitask, generative adversarial network-based deep learning model to synthesize an entire CTP series from only a non-contrasted CT (NCCT) input. Materials and Methods: NCCT and CTP series were retrospectively retrieved from 493 patients at UF Health with IRB approval. The data were deidentified and all images were resized to 256x256 pixels. The collected perfusion data were analyzed using the RapidAI CT Perfusion analysis software (iSchemaView, Inc. CA) to generate each CTP map. For each subject, 10 CTP slices were selected. Each slice was paired with one NCCT slice at the same location and two NCCT slices at a predefined vertical offset, resulting in 4.3K CTP images and 12.9K NCCT images used for training. The incorporation of a spatial offset into the NCCT input allows MAGIC to more accurately synthesize cerebral perfusive structures, increasing the quality of the generated images. The studies included a variety of indications, including healthy tissue, mild infarction, and severe infarction. The proposed MAGIC model incorporates a novel multitask architecture, allowing for the simultaneous synthesis of four CTP modalities: mean transit time (MTT), cerebral blood flow (CBF), cerebral blood volume (CBV), and time to peak (TTP). We propose a novel Physicians-in-the-loop module in the model's architecture, acting as a tunable layer that allows physicians to manually adjust the amount of anatomic detail present in the synthesized CTP series. Additionally, we propose two novel loss terms: multi-modal connectivity loss and extrema loss. The multi-modal connectivity loss leverages the multi-task nature to assert that the mathematical relationship between MTT, CBF, and CBV is satisfied. The extrema loss aids in learning regions of elevated and decreased activity in each modality, allowing for MAGIC to accurately learn the characteristics of diagnostic regions of interest. Corresponding NCCT and CTP slices were paired along the vertical axis. The model was trained for 100 epochs on a NVIDIA TITAN X GPU. Results and Discussion: The MAGIC model’s performance was evaluated on a sample of 40 patients from the UF Health dataset. Across all CTP modalities, MAGIC was able to accurately produce images with high structural agreement between the entire synthesized and clinical perfusion images (SSIMmean=0.801 , UQImean=0.926). MAGIC was able to synthesize CTP images to accurately characterize cerebral circulatory structures and identify regions of infarct tissue, as shown in Figure 1. A blind binary evaluation was conducted to assess the presence of cerebral infarction in both the synthesized and clinical perfusion images, resulting in the synthesized images correctly predicting the presence of cerebral infarction with 87.5% accuracy. Conclusions: We proposed a MAGIC model whose novel deep learning structures and loss terms enable high-quality synthesis of CTP maps and characterization of circulatory structures solely from NCCT images, potentially eliminating the requirement for the injection of an intravenous contrast agent and elevated radiation exposure during perfusion imaging. This makes MAGIC a beneficial tool in a clinical scenario increasing the overall safety, accessibility, and efficiency of cerebral perfusion and facilitating better patient outcomes. Acknowledgements: This work was partially supported by the National Science Foundation, IIS-1908299 III: Small: Modeling Multi-Level Connectivity of Brain Dynamics + REU Supplement, to the University of Florida.more » « less
-
more » « less
Abstract Kidney ischemia reperfusion injury (IRI) poses a major global healthcare burden, but effective treatments remain elusive. IRI involves a complex interplay of tissue‐level structural and functional changes caused by interruptions in blood and filtrate flow and reduced oxygenation. Existing in vitro models poorly replicate the in vivo injury environment and lack means of monitoring tissue function during the injury process. Here, a high‐throughput human primary kidney proximal tubule (PT)‐microvascular model is described, which facilitates in‐depth structural and rapid functional characterization of IRI‐induced changes in the tissue barrier. The PREDICT96 (P96) microfluidic platform's user‐controlled fluid flow can mimic the conditions of IR to induce pronounced changes in cell structure that resemble clinical and in vivo phenotypes. High‐throughput trans‐epi/endo‐thelial electrical resistance (TEER) sensing is applied to non‐invasively track functional changes in the PT‐microvascular barrier during the two‐stage injury process and over repeated episodes of injury. Notably, ischemia causes an initial increase in tissue TEER followed by a sudden increase in permeability upon reperfusion, and this biphasic response occurs only with the loss of both fluid flow and oxygenation. This study demonstrates the potential of the P96 kidney IRI model to enhance understanding of IRI and fuel therapeutic development.
-
null (Ed.)Abstract Skeletal muscle is a tissue that is directly involved in the progression and persistence of type 2 diabetes (T2D), a disease that is becoming increasingly common. Gaining better insight into the mechanisms that are affecting skeletal muscle dysfunction in the context of T2D has the potential to lead to novel treatments for a large number of patients. Through its ability to emulate skeletal muscle architecture while also incorporating aspects of disease, tissue-engineered skeletal muscle (TE-SkM) has the potential to provide a means for rapid high-throughput discovery of therapies to treat skeletal muscle dysfunction, to include that which occurs with T2D. Muscle precursor cells isolated from lean or obese male Zucker diabetic fatty rats were used to generate TE-SkM constructs. Some constructs were treated with adipogenic induction media to accentuate the presence of adipocytes that is a characteristic feature of T2D skeletal muscle. The maturity (compaction and creatine kinase activity), mechanical integrity (Young's modulus), organization (myotube orientation), and metabolic capacity (insulin-stimulated glucose uptake) were all reduced by diabetes. Treating constructs with adipogenic induction media increased the quantity of lipid within the diabetic TE-SkM constructs, and caused changes in construct compaction, cell orientation, and insulin-stimulated glucose uptake in both lean and diabetic samples. Collectively, the findings herein suggest that the recapitulation of structural and metabolic aspects of T2D can be accomplished by engineering skeletal muscle in vitro. Impact Statement The tissue engineering of skeletal muscle to model disease and injury has great promise to provide a tool to develop and/or improve therapeutic approaches for improved health care. A tissue-engineered skeletal muscle model of one of the most common and debilitating diseases, type 2 diabetes, has been developed in vitro as evidenced by the structural and metabolic alterations that are consistent with the disease phenotype in vivo.more » « less
-
more » « less
Abstract Astrocytes are actively involved in a neuroprotective role in the brain, which includes scavenging reactive oxygen species to minimize tissue damage. They also modulate neuroinflammation and reactive gliosis prevalent in several brain disorders like epilepsy, Alzheimer's, and Parkinson's disease. In animal models, targeted manipulation of astrocytic function via modulation of their calcium (Ca2+) oscillations by incorporating light‐sensitive cation channels like Channelrhodopsin‐2 (ChR2) offers a promising avenue in influencing the long‐term progression of these disorders. However, using adult animals for Ca2+imaging poses major challenges, including accelerated deterioration of
in situ slice health and age‐ related changes. Additionally, optogenetic preparations necessitate usage of a red‐shifted Ca2+indicator like Rhod‐2 AM to avoid overlapping light issues between ChR2 and the Ca2+indicator during simultaneous optogenetic stimulation and imaging. In this article, we provide an experimental setting that uses live adult murine brain slices (2‐5 months) from a knock‐in model expressing Channelrhodopsin‐2 (ChR2(C128S)) in cortical astrocytes, loaded with Rhod‐2 AM to elicit robust Ca2+response to light stimulation. We have developed and standardized a protocol for brain extraction, sectioning, Rhod‐2 AM loading, maintenance of slice health, and Ca2+imaging during light stimulation. This has been successfully applied to optogenetically control adult cortical astrocytes, which exhibit synchronous patterns of Ca2+activity upon light stimulation, drastically different from resting spontaneous activity. © 2020 Wiley Periodicals LLC.Basic Protocol 1 : Experimental preparation, setup, slice preparation and Rhod‐2 AM stainingBasic Protocol 2 : Image acquisition and analysis
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1186/s13036-022-00293-w
