Identification of predictive MRI and functional biomarkers in a pediatric piglet traumatic brain injury model.

Traumatic brain injury (TBI) at a young age can lead to the development of long-term functional impairments. Severity of injury is well demonstrated to have a strong influence on the extent of functional impairments; however, identification of specific magnetic resonance imaging (MRI) biomarkers that are most reflective of injury severity and functional prognosis remain elusive. Therefore, the objective of this study was to utilize advanced statistical approaches to identify clinically relevant MRI biomarkers and predict functional outcomes using MRI metrics in a translational large animal piglet TBI model. TBI was induced via controlled cortical impact and multiparametric MRI was performed at 24 hours and 12 weeks post-TBI using T1-weighted, T2-weighted, T2-weighted fluid attenuated inversion recovery, diffusion-weighted imaging, and diffusion tensor imaging. Changes in spatiotemporal gait parameters were also assessed using an automated gait mat at 24 hours and 12 weeks post-TBI. Principal component analysis was performed to determine the MRI metrics and spatiotemporal gait parameters that explain the largest sources of variation within the datasets. We found that linear combinations of lesion size and midline shift acquired using T2-weighted imaging explained most of the variability of the data at both 24 hours and 12 weeks post-TBI. In addition, linear combinations of velocity, cadence, and stride length were found to explain most of the gait data variability at 24 hours and 12 weeks post-TBI. Linear regression analysis was performed to determine if MRI metrics are predictive of changes in gait. We found that both lesion size and midline shift are significantly correlated with decreases in stride and step length. These results from this study provide an important first step at identifying relevant MRI and functional biomarkers that are predictive of functional outcomes in a clinically relevant piglet TBI model. This study was approved by the University of Georgia Institutional Animal Care and Use Committee (AUP: A2015 11-001) on December 22, 2015.

Exploring the predictive value of lesion topology on motor function outcomes in a porcine ischemic stroke model.

Harnessing the maximum diagnostic potential of magnetic resonance imaging (MRI) by including stroke lesion location in relation to specific structures that are associated with particular functions will likely increase the potential to predict functional deficit type, severity, and recovery in stroke patients. This exploratory study aims to identify key structures lesioned by a middle cerebral artery occlusion (MCAO) that impact stroke recovery and to strengthen the predictive capacity of neuroimaging techniques that characterize stroke outcomes in a translational porcine model. Clinically relevant MRI measures showed significant lesion volumes, midline shifts, and decreased white matter integrity post-MCAO. Using a pig brain atlas, damaged brain structures included the insular cortex, somatosensory cortices, temporal gyri, claustrum, and visual cortices, among others. MCAO resulted in severely impaired spatiotemporal gait parameters, decreased voluntary movement in open field testing, and higher modified Rankin Scale scores at acute timepoints. Pearson correlation analyses at acute timepoints between standard MRI metrics (e.g., lesion volume) and functional outcomes displayed moderate R values to functional gait outcomes. Moreover, Pearson correlation analyses showed higher R values between functional gait deficits and increased lesioning of structures associated with motor function, such as the putamen, globus pallidus, and primary somatosensory cortex. This correlation analysis approach helped identify neuroanatomical structures predictive of stroke outcomes and may lead to the translation of this topological analysis approach from preclinical stroke assessment to a clinical biomarker.

Semi-Automated Cell and Tissue Analyses Reveal Regionally Specific Morphological Alterations of Immune and Neural Cells in a Porcine Middle Cerebral Artery Occlusion Model of Stroke.

Histopathological analysis of cellular changes in the stroked brain provides critical information pertaining to inflammation, cell death, glial scarring, and other dynamic injury and recovery responses. However, commonly used manual approaches are hindered by limitations in speed, accuracy, bias, and the breadth of morphological information that can be obtained. Here, a semi-automated high-content imaging (HCI) and CellProfiler histological analysis method was developed and used in a Yucatan miniature pig permanent middle cerebral artery occlusion (pMCAO) model of ischemic stroke to overcome these limitations. Evaluation of 19 morphological parameters in IBA1+ microglia/macrophages, GFAP+ astrocytes, NeuN+ neuronal, FactorVIII+ vascular endothelial, and DCX+ neuroblast cell areas was conducted on porcine brain tissue 4 weeks post pMCAO. Out of 19 morphological parameters assessed in the stroke perilesional and ipsilateral hemisphere regions (38 parameters), a significant change in 38 38 measured IBA1+ parameters, 34 38   GFAP+ parameters, 32 38 NeuN+ parameters, 31 38 FactorVIII+ parameters, and 28 38 DCX+ parameters were observed in stroked vs. non-stroked animals. Principal component analysis (PCA) and correlation analyses demonstrated that stroke-induced significant and predictable morphological changes that demonstrated strong relationships between IBA1+, GFAP+, and NeuN+ areas. Ultimately, this unbiased, semi-automated HCI and CellProfiler histopathological analysis approach revealed regional and cell specific morphological signatures of immune and neural cells after stroke in a highly translational porcine model. These identified features can provide information of disease pathogenesis and evolution with high resolution, as well as be used in therapeutic screening applications.

Magnetic Resonance Imaging and Gait Analysis Indicate Similar Outcomes Between Yucatan and Landrace Porcine Ischemic Stroke Models.

The Stroke Therapy Academic Industry Roundtable (STAIR) has recommended that novel therapeutics be tested in a large animal model with similar anatomy and physiology to humans. The pig is an attractive model due to similarities in brain size, organization, and composition relative to humans. However, multiple pig breeds have been used to study ischemic stroke with potentially differing cerebral anatomy, architecture and, consequently, ischemic stroke pathologies. The objective of this study was to characterize brain anatomy and assess spatiotemporal gait parameters in Yucatan (YC) and Landrace (LR) pigs pre- and post-stroke using magnetic resonance imaging (MRI) and gait analysis, respectively. Ischemic stroke was induced via permanent middle cerebral artery occlusion (MCAO). MRI was performed pre-stroke and 1-day post-stroke. Structural and diffusion-tensor sequences were performed at both timepoints and analyzed for cerebral characteristics, lesion diffusivity, and white matter changes. Spatiotemporal and relative pressure gait measurements were collected pre- and 2-days post-stroke to characterize and compare acute functional deficits. The results from this study demonstrated that YC and LR pigs exhibit differences in gross brain anatomy and gait patterns pre-stroke with MRI and gait analysis showing statistical differences in the majority of parameters. However, stroke pathologies in YC and LR pigs were highly comparable post-stroke for most evaluated MRI parameters, including lesion volume and diffusivity, hemisphere swelling, ventricle compression, caudal transtentorial and foramen magnum herniation, showing no statistical difference between the breeds. In addition, post-stroke changes in velocity, cycle time, swing percent, cadence, and mean hoof pressure showed no statistical difference between the breeds. These results indicate significant differences between pig breeds in brain size, anatomy, and motor function pre-stroke, yet both demonstrate comparable brain pathophysiology and motor outcomes post-stroke. The conclusions of this study suggest pigs of these different breeds generally show a similar ischemic stroke response and findings can be compared across porcine stroke studies that use different breeds.

Neural stem cell therapy for stroke: A multimechanistic approach to restoring neurological function.

INTRODUCTION: Neural stem cells (NSCs) have demonstrated multimodal therapeutic function for stroke, which is the leading cause of long-term disability and the second leading cause of death worldwide. In preclinical stroke models, NSCs have been shown to modulate inflammation, foster neuroplasticity and neural reorganization, promote angiogenesis, and act as a cellular replacement by differentiating into mature neural cell types. However, there are several key technical questions to address before NSC therapy can be applied to the clinical setting on a large scale. PURPOSE OF REVIEW: In this review, we will discuss the various sources of NSCs, their therapeutic modes of action to enhance stroke recovery, and considerations for the clinical translation of NSC therapies. Understanding the key factors involved in NSC-mediated tissue recovery and addressing the current translational barriers may lead to clinical success of NSC therapy and a first-in-class restorative therapy for stroke patients.

The pig as a preclinical traumatic brain injury model: current models, functional outcome measures, and translational detection strategies.

Traumatic brain injury (TBI) is a major contributor of long-term disability and a leading cause of death worldwide. A series of secondary injury cascades can contribute to cell death, tissue loss, and ultimately to the development of functional impairments. However, there are currently no effective therapeutic interventions that improve brain outcomes following TBI. As a result, a number of experimental TBI models have been developed to recapitulate TBI injury mechanisms and to test the efficacy of potential therapeutics. The pig model has recently come to the forefront as the pig brain is closer in size, structure, and composition to the human brain compared to traditional rodent models, making it an ideal large animal model to study TBI pathophysiology and functional outcomes. This review will focus on the shared characteristics between humans and pigs that make them ideal for modeling TBI and will review the three most common pig TBI models-the diffuse axonal injury, the controlled cortical impact, and the fluid percussion models. It will also review current advances in functional outcome assessment measures and other non-invasive, translational TBI detection and measurement tools like biomarker analysis and magnetic resonance imaging. The use of pigs as TBI models and the continued development and improvement of translational assessment modalities have made significant contributions to unraveling the complex cascade of TBI sequela and provide an important means to study potential clinically relevant therapeutic interventions.

Controlled Cortical Impact Leads to Cognitive and Motor Function Deficits that Correspond to Cellular Pathology in a Piglet Traumatic Brain Injury Model.

Traumatic brain injury (TBI) is a leading cause of death and disability in the United States, with children who sustain a TBI having a greater risk of developing long-lasting cognitive, behavioral, and motor function deficits. This has led to increased interest in utilizing large animal models to study pathophysiologic and functional changes after injury in hopes of identifying novel therapeutic targets. In the present study, a controlled cortical impact (CCI) piglet TBI model was utilized to evaluate cognitive, motor, and histopathologic outcomes. CCI injury (4 m/sec velocity, 9 mm depression, 400 msec dwell time) was induced at the parietal cortex. Compared with normal pigs (n = 5), TBI pigs (n = 5) exhibited appreciable cognitive deficiencies, including significantly impaired spatial memory in spatial T-maze testing and a significant decrease in exploratory behaviors followed by marked hyperactivity in open field testing. Additionally, gait analysis revealed significant increases in cycle time and stance percent, significant decreases in hind reach, and a shift in the total pressure index from the front to the hind limb on the affected side, suggesting TBI impairs gait and balance. Pigs were sacrificed 28 days post-TBI and histological analysis revealed that TBI lead to a significant decrease in neurons and a significant increase in microglia activation and astrogliosis/astrocytosis at the perilesional area, a significant loss in neurons at the dorsal hippocampus, and significantly increased neuroblast proliferation at the subventricular zone. These data demonstrate a strong relationship between TBI-induced cellular changes and functional outcomes in our piglet TBI model that lay the framework for future studies that assess the ability of therapeutic interventions to contribute to functional improvements.

Controlled Cortical Impact Severity Results in Graded Cellular, Tissue, and Functional Responses in a Piglet Traumatic Brain Injury Model.

A number of pre-clinical rodent models have been developed in an effort to recapitulate injury mechanisms and identify potential therapeutics for traumatic brain injury (TBI), which is a major cause of death and long-term disability in the United States. The lack of restorative treatments for TBI, however, has led to considerable criticism of current pre-clinical therapeutic development strategies-namely, the translatability of widely used rodent models to human patients. The use of large animal models, such as the pig, with more brain anatomy and physiology comparable to humans may enhance the translational capacity of current pre-clinical animal models. The objective of this study was to develop and characterize a graded piglet TBI model with quantitative pathological features at the cellular, tissue, and functional level that become more prominent with increasing TBI severity. A graded TBI was produced by controlled cortical impact (CCI) in "toddler-aged" Landrace piglets by increasing impact velocity and/or depth of depression to 2 m/sec; 6 mm; 4 m/sec; 6 mm; 4 m/sec; 12 mm; or 4 m/sec; 15 mm, producing a range of neural injury responses that corresponded to injury severity. Quantitative gait analysis was performed pre-TBI and one, three, and seven days post-TBI, and piglets were sacrificed seven days post-TBI. Increasing impact parameters correlated to increases in lesion size with piglets that sustained a 6 mm depth of depression exhibiting significantly smaller lesions than piglets that sustained a depth of depression of 12 mm or 15 mm. Similarly, the extent of neuronal loss, astrogliosis/astrocytosis, and white matter damage became more prominent as CCI parameters were increased. These cellular and tissue-level changes correlated with motor function deficits including swing/stance time, stride velocity, and two- versus three-limb support. The piglet TBI model described here could serve as a translational platform for studying TBI sequelae across injury severities and identifying novel therapeutics.

Traumatic Brain Injury Results in Dynamic Brain Structure Changes Leading to Acute and Chronic Motor Function Deficits in a Pediatric Piglet Model.

Traumatic brain injury (TBI) is a leading cause of death and disability in children. Pediatric TBI patients often suffer from crippling cognitive, emotional, and motor function deficits that have negative lifelong effects. The objective of this study was to longitudinally assess TBI pathophysiology using multi-parametric magnetic resonance imaging (MRI), gait analysis, and histological approaches in a pediatric piglet model. TBI was produced by controlled cortical impact in Landrace piglets. MRI data, including from proton magnetic resonance spectroscopy (MRS), were collected 24 hours and 12 weeks post-TBI, and gait analysis was performed at multiple time-points over 12 weeks post-TBI. A subset of animals was sacrificed 24 hours, 1 week, 4 weeks, and 12 weeks post-TBI for histological analysis. MRI results demonstrated that TBI led to a significant brain lesion and midline shift as well as microscopic tissue damage with altered brain diffusivity, decreased white matter integrity, and reduced cerebral blood flow. MRS showed a range of neurochemical changes after TBI. Histological analysis revealed neuronal loss, astrogliosis/astrocytosis, and microglia activation. Further, gait analysis showed transient impairments in cadence, cycle time, % stance, step length, and stride length, as well as long-term impairments in weight distribution after TBI. Taken together, this study illustrates the distinct time course of TBI pathoanatomic and functional responses up to 12 weeks post-TBI in a piglet TBI model. The study of TBI injury and recovery mechanisms, as well as the testing of therapeutics in this translational model, are likely to be more predictive of human responses and clinical outcomes compared to traditional small animal models.

Scaled traumatic brain injury results in unique metabolomic signatures between gray matter, white matter, and serum in a piglet model.

Traumatic brain injury (TBI) is a leading cause of death and long-term disability in the United States. The heterogeneity of the disease coupled with the lack of comprehensive, standardized scales to adequately characterize multiple types of TBI remain to be major challenges facing effective therapeutic development. A systems level approach to TBI diagnosis through the use of metabolomics could lead to a better understanding of cellular changes post-TBI and potential therapeutic targets. In the current study, we utilize a GC-MS untargeted metabolomics approach to demonstrate altered metabolism in response to TBI in a translational pig model, which possesses many neuroanatomical and pathophysiologic similarities to humans. TBI was produced by controlled cortical impact (CCI) in Landrace piglets with impact velocity and depth of depression set to 2m/s;6mm, 4m/s;6mm, 4m/s;12mm, or 4m/s;15mm resulting in graded neural injury. Serum samples were collected pre-TBI, 24 hours post-TBI, and 7 days post-TBI. Partial least squares discriminant analysis (PLS-DA) revealed that each impact parameter uniquely influenced the metabolomic profile after TBI, and gray and white matter responds differently to TBI on the biochemical level with evidence of white matter displaying greater metabolic change. Furthermore, pathway analysis revealed unique metabolic signatures that were dependent on injury severity and brain tissue type. Metabolomic signatures were also detected in serum samples which potentially captures both time after injury and injury severity. These findings provide a platform for the development of a more accurate TBI classification scale based unique metabolomic signatures.

Human iNPC therapy leads to improvement in functional neurologic outcomes in a pig ischemic stroke model.

Introduction: Stroke is the leading cause of disability in the United States but current therapies are limited with no regenerative potential. Previous translational failures have highlighted the need for large animal models of ischemic stroke and for improved assessments of functional outcomes. The aims of this study were first, to create a post-stroke functional outcome assessment scale in a porcine model of middle cerebral artery occlusion (MCAO) and second, to use this scale to determine the effect of human-induced-pluripotent-cell-derived neural progenitor cells (iNPCs) on functional outcome in this large animal stroke model. Materials and Methods: Eight 6-month-old Landrace mix pigs underwent permanent MCAO. Five days following MCAO, pigs received intraparenchymal injections of either iNPCs or PBS. A post-stroke assessment scale was developed to measure functional outcome. Evaluations were performed at least 1-3 days prior to MCAO and repeated 1 day, 3 days, and 5 days post-stroke as well as 1 day, 3 days, 1 week, 2 weeks, 4 weeks, 6 weeks, 9 weeks, and 12 weeks post-injection. Comparisons of scores between animals receiving iNPCs or PBS only were compared using a two-way ANOVA and a Tukey's post-hoc t test. Results: The developed scale was able to consistently determine differences between healthy and stroked pigs at all time points. iNPC-treated pigs showed a significantly faster recovery in their overall scores relative to PBS-only treated pigs with the parameters of appetite and body posture exhibiting the most improvement in the iNPC-treated group. Conclusions: We developed a robust and repeatable functional assessment tool that can reliably detect stroke and recovery, while also showing for the first time that iNPC therapy leads to functional recovery in a translational pig ischemic stroke model. These promising results suggest that iNPCs may 1 day serve as a first in class cell therapeutic for ischemic stroke.

Induced Pluripotent Stem Cell-Derived Neural Stem Cell Therapy Enhances Recovery in an Ischemic Stroke Pig Model.

Induced pluripotent stem cell-derived neural stem cells (iNSCs) have significant potential as an autologous, multifunctional cell therapy for stroke, which is the primary cause of long term disability in the United States and the second leading cause of death worldwide. Here we show that iNSC transplantation improves recovery through neuroprotective, regenerative, and cell replacement mechanisms in a novel ischemic pig stroke model. Longitudinal multiparametric magnetic resonance imaging (MRI) following iNSC therapy demonstrated reduced changes in white matter integrity, cerebral blood perfusion, and brain metabolism in the infarcted tissue. The observed tissue level recovery strongly correlated with decreased immune response, enhanced neuronal protection, and increased neurogenesis. iNSCs differentiated into neurons and oligodendrocytes with indication of long term integration. The robust recovery response to iNSC therapy in a translational pig stroke model with increased predictive potential strongly supports that iNSCs may be the critically needed therapeutic for human stroke patients.