White Matter Integrity and Motor Function Disruption Due to Traumatic Brain Injury in Piglets: Impacts on Motor-Related Brain Fibers.

Pediatric traumatic brain injury (TBI) often induces significant disability in patients, including long-term motor deficits. Early detection of injury severity is key in determining a prognosis and creating appropriate intervention and rehabilitation plans. However, conventional magnetic resonance imaging (MRI) scans, such as T2 Weighted (T2W) sequences, do not reliably assess the extent of microstructural white matter injury. Diffusion tensor imaging (DTI) tractography enables three-dimensional reconstruction of specific white matter tracts throughout the brain in order to detect white matter injury based on anisotropic diffusion. The objective of this study was to employ DTI tractography to detect acute changes to white matter integrity within the intersecting fibers of key motor-related brain regions following TBI. Piglets were assigned to either the sham craniectomy group (sham; n = 6) or the controlled cortical impact TBI group (TBI; n = 6). Gait and MRI were collected at seven days post-surgery (DPS). T2W sequences confirmed a localized injury predominately in the ipsilateral hemisphere in TBI animals. TBI animals, relative to sham animals, showed an increased apparent diffusion coefficient (ADC) and decreased fractional anisotropy (FA) in fiber bundles associated with key brain regions involved in motor function. TBI animals exhibited gait deficits, including stride and step length, compared to sham animals. Together these data demonstrate acute reductions in the white matter integrity, measured by DTI tractography, of fibers intersecting key brain regions that strongly corresponded with acute motor deficits in a pediatric piglet TBI model. These results provide the foundation for the further development of DTI-based biomarkers to evaluate motor outcomes following TBI.

Tanshinone IIA-loaded nanoparticles and neural stem cell combination therapy improves gut homeostasis and recovery in a pig ischemic stroke model.

Impaired gut homeostasis is associated with stroke often presenting with leaky gut syndrome and increased gut, brain, and systemic inflammation that further exacerbates brain damage. We previously reported that intracisternal administration of Tanshinone IIA-loaded nanoparticles (Tan IIA-NPs) and transplantation of induced pluripotent stem cell-derived neural stem cells (iNSCs) led to enhanced neuroprotective and regenerative activity and improved recovery in a pig stroke model. We hypothesized that Tan IIA-NP + iNSC combination therapy-mediated stroke recovery may also have an impact on gut inflammation and integrity in the stroke pigs. Ischemic stroke was induced, and male Yucatan pigs received PBS + PBS (Control, n = 6) or Tan IIA-NP + iNSC (Treatment, n = 6) treatment. The Tan IIA-NP + iNSC treatment reduced expression of jejunal TNF-α, TNF-α receptor1, and phosphorylated IkBα while increasing the expression of jejunal occludin, claudin1, and ZO-1 at 12 weeks post-treatment (PT). Treated pigs had higher fecal short-chain fatty acid (SCFAs) levels than their counterparts throughout the study period, and fecal SCFAs levels were negatively correlated with jejunal inflammation. Interestingly, fecal SCFAs levels were also negatively correlated with brain lesion volume and midline shift at 12 weeks PT. Collectively, the anti-inflammatory and neuroregenerative treatment resulted in increased SCFAs levels, tight junction protein expression, and decreased inflammation in the gut.

Tanshinone IIA-Loaded Nanoparticle and Neural Stem Cell Therapy Enhances Recovery in a Pig Ischemic Stroke Model.

Induced pluripotent stem cell-derived neural stem cells (iNSCs) are a multimodal stroke therapeutic that possess neuroprotective, regenerative, and cell replacement capabilities post-ischemia. However, long-term engraftment and efficacy of iNSCs is limited by the cytotoxic microenvironment post-stroke. Tanshinone IIA (Tan IIA) is a therapeutic that demonstrates anti-inflammatory and antioxidative effects in rodent ischemic stroke models and stroke patients. Therefore, pretreatment with Tan IIA may create a microenvironment that is more conducive to the long-term survival of iNSCs. In this study, we evaluated the potential of Tan IIA drug-loaded nanoparticles (Tan IIA-NPs) to improve iNSC engraftment and efficacy, thus potentially leading to enhanced cellular, tissue, and functional recovery in a translational pig ischemic stroke model. Twenty-two pigs underwent middle cerebral artery occlusion (MCAO) and were randomly assigned to a PBS + PBS, PBS + iNSC, or Tan IIA-NP + iNSC treatment group. Magnetic resonance imaging (MRI), modified Rankin Scale neurological evaluation, and immunohistochemistry were performed over a 12-week study period. Immunohistochemistry indicated pretreatment with Tan IIA-NPs increased iNSC survivability. Furthermore, Tan IIA-NPs increased iNSC neuronal differentiation and decreased iNSC reactive astrocyte differentiation. Tan IIA-NP + iNSC treatment enhanced endogenous neuroprotective and regenerative activities by decreasing the intracerebral cellular immune response, preserving endogenous neurons, and increasing neuroblast formation. MRI assessments revealed Tan IIA-NP + iNSC treatment reduced lesion volumes and midline shift. Tissue preservation and recovery corresponded with significant improvements in neurological recovery. This study demonstrated pretreatment with Tan IIA-NPs increased iNSC engraftment, enhanced cellular and tissue recovery, and improved neurological function in a translational pig stroke model.

Intracisternal administration of tanshinone IIA-loaded nanoparticles leads to reduced tissue injury and functional deficits in a porcine model of ischemic stroke.

BACKGROUND: The absolute number of new stroke patients is annually increasing and there still remains only a few Food and Drug Administration (FDA) approved treatments with significant limitations available to patients. Tanshinone IIA (Tan IIA) is a promising potential therapeutic for ischemic stroke that has shown success in pre-clinical rodent studies but lead to inconsistent efficacy results in human patients. The physical properties of Tan-IIA, including short half-life and low solubility, suggests that Poly (lactic-co-glycolic acid) (PLGA) nanoparticle-assisted delivery may lead to improve bioavailability and therapeutic efficacy. The objective of this study was to develop Tan IIA-loaded nanoparticles (Tan IIA-NPs) and to evaluate their therapeutic effects on cerebral pathological changes and consequent motor function deficits in a pig ischemic stroke model. RESULTS: Tan IIA-NP treated neural stem cells showed a reduction in SOD activity in in vitro assays demonstrating antioxidative effects. Ischemic stroke pigs treated with Tan IIA-NPs showed reduced hemispheric swelling when compared to vehicle only treated pigs (7.85 ± 1.41 vs. 16.83 ± 0.62%), consequent midline shift (MLS) (1.72 ± 0.07 vs. 2.91 ± 0.36 mm), and ischemic lesion volumes (9.54 ± 5.06 vs. 12.01 ± 0.17 cm3) when compared to vehicle-only treated pigs. Treatment also lead to lower reductions in diffusivity (-37.30 ± 3.67 vs. -46.33 ± 0.73%) and white matter integrity (-19.66 ± 5.58 vs. -30.11 ± 1.19%) as well as reduced hemorrhage (0.85 ± 0.15 vs 2.91 ± 0.84 cm3) 24 h post-ischemic stroke. In addition, Tan IIA-NPs led to a reduced percentage of circulating band neutrophils at 12 (7.75 ± 1.93 vs. 14.00 ± 1.73%) and 24 (4.25 ± 0.48 vs 5.75 ± 0.85%) hours post-stroke suggesting a mitigated inflammatory response. Moreover, spatiotemporal gait deficits including cadence, cycle time, step time, swing percent of cycle, stride length, and changes in relative mean pressure were less severe post-stroke in Tan IIA-NP treated pigs relative to control pigs. CONCLUSION: The findings of this proof of concept study strongly suggest that administration of Tan IIA-NPs in the acute phase post-stroke mitigates neural injury likely through limiting free radical formation, thus leading to less severe gait deficits in a translational pig ischemic stroke model. With stroke as one of the leading causes of functional disability in the United States, and gait deficits being a major component, these promising results suggest that acute Tan IIA-NP administration may improve functional outcomes and the quality of life of many future stroke patients.

An integrative multivariate approach for predicting functional recovery using magnetic resonance imaging parameters in a translational pig ischemic stroke model.

Magnetic resonance imaging (MRI) is a clinically relevant, real-time imaging modality that is frequently utilized to assess stroke type and severity. However, specific MRI biomarkers that can be used to predict long-term functional recovery are still a critical need. Consequently, the present study sought to examine the prognostic value of commonly utilized MRI parameters to predict functional outcomes in a porcine model of ischemic stroke. Stroke was induced via permanent middle cerebral artery occlusion. At 24 hours post-stroke, MRI analysis revealed focal ischemic lesions, decreased diffusivity, hemispheric swelling, and white matter degradation. Functional deficits including behavioral abnormalities in open field and novel object exploration as well as spatiotemporal gait impairments were observed at 4 weeks post-stroke. Gaussian graphical models identified specific MRI outputs and functional recovery variables, including white matter integrity and gait performance, that exhibited strong conditional dependencies. Canonical correlation analysis revealed a prognostic relationship between lesion volume and white matter integrity and novel object exploration and gait performance. Consequently, these analyses may also have the potential of predicting patient recovery at chronic time points as pigs and humans share many anatomical similarities (e.g., white matter composition) that have proven to be critical in ischemic stroke pathophysiology. The study was approved by the University of Georgia (UGA) Institutional Animal Care and Use Committee (IACUC; Protocol Number: A2014-07-021-Y3-A11 and 2018-01-029-Y1-A5) on November 22, 2017.

Dynamic Changes in the Gut Microbiome at the Acute Stage of Ischemic Stroke in a Pig Model.

Stroke is a major cause of death and long-term disability affecting seven million adults in the United States each year. Recently, it has been demonstrated that neurological diseases, associated pathology, and susceptibility changes correlated with changes in the gut microbiota. However, changes in the microbial community in stroke has not been well characterized. The acute stage of stroke is a critical period for assessing injury severity, therapeutic intervention, and clinical prognosis. We investigated the changes in the gut microbiota composition and diversity using a middle cerebral artery (MCA) occlusion ischemic stroke pig model. Ischemic stroke was induced by cauterization of the MCA in pigs. Blood samples were collected prestroke and 4 h, 12 h, 1 day, and 5 days poststroke to evaluate circulating proinflammatory cytokines. Fecal samples were collected prestroke and 1, 3, and 5 days poststroke to assess gut microbiome changes. Results showed elevated systemic inflammation with increased plasma levels of tumor necrosis factor alpha at 4 h and interleukin-6 at 12 h poststroke, relative to prestroke. Microbial diversity and evenness were reduced at 1 day poststroke compared to prestroke. Microbial diversity at 3 days poststroke was negatively correlated with lesion volume. Moreover, beta-diversity analysis revealed trending overall differences over time, with the most significant changes in microbial patterns observed between prestroke and 3 days poststroke. Abundance of the Proteobacteria was significantly increased, while Firmicutes decreased at 3 days poststroke, compared to prestroke populations. Abundance of the lactic acid bacteria Lactobacillus was reduced at 3 days poststroke. By day 5, the microbial pattern returned to similar values as prestroke, suggesting the plasticity of gut microbiome in an acute period of stroke in a pig model. These findings provide a basis for characterizing gut microbial changes during the acute stage of stroke, which can be used to assess stroke pathology and the potential development of therapeutic targets.

Characterization of tissue and functional deficits in a clinically translational pig model of acute ischemic stroke.

The acute stroke phase is a critical time frame used to evaluate stroke severity, therapeutic options, and prognosis while also serving as a major tool for the development of diagnostics. To further understand stroke pathophysiology and to enhance the development of treatments, our group developed a translational pig ischemic stroke model. In this study, the evolution of acute ischemic tissue damage, immune responses, and functional deficits were further characterized. Stroke was induced by middle cerebral artery occlusion in Landrace pigs. At 24 h post-stroke, magnetic resonance imaging revealed a decrease in ipsilateral diffusivity, an increase in hemispheric swelling resulting in notable midline shift, and intracerebral hemorrhage. Stroke negatively impacted white matter integrity with decreased fractional anisotropy values in the internal capsule. Like patients, pigs showed a reduction in circulating lymphocytes and a surge in neutrophils and band cells. Functional responses corresponded with structural changes through reductions in open field exploration and impairments in spatiotemporal gait parameters. Characterization of acute ischemic stroke in pigs provided important insights into tissue and functional-level assessments that could be used to identify potential biomarkers and improve preclinical testing of novel therapeutics.

Large animal ischemic stroke models: replicating human stroke pathophysiology.

The high morbidity and mortality rate of ischemic stroke in humans has led to the development of numerous animal models that replicate human stroke to further understand the underlying pathophysiology and to explore potential therapeutic interventions. Although promising therapeutics have been identified using these animal models, with most undergoing significant testing in rodent models, the vast majority of these interventions have failed in human clinical trials. This failure of preclinical translation highlights the critical need for better therapeutic assessment in more clinically relevant ischemic stroke animal models. Large animal models such as non-human primates, sheep, pigs, and dogs are likely more predictive of human responses and outcomes due to brain anatomy and physiology that are more similar to humans-potentially making large animal testing a key step in the stroke therapy translational pipeline. The objective of this review is to highlight key characteristics that potentially make these gyrencephalic, large animal ischemic stroke models more predictive by comparing pathophysiological responses, tissue-level changes, and model limitations.

Alcohol Induced Brain and Liver Damage: Advantages of a Porcine Alcohol Use Disorder Model.

Alcohol is one of the most commonly abused intoxicants with 1 in 6 adults at risk for alcohol use disorder (AUD) in the United States. As such, animal models have been extensively investigated with rodent AUD models being the most widely studied. However, inherent anatomical and physiological differences between rodents and humans pose a number of limitations in studying the complex nature of human AUD. For example, rodents differ from humans in that rodents metabolize alcohol rapidly and do not innately demonstrate voluntary alcohol consumption. Comparatively, pigs exhibit similar patterns observed in human AUD including voluntary alcohol consumption and intoxication behaviors, which are instrumental in establishing a more representative AUD model that could in turn delineate the risk factors involved in the development of this disorder. Pigs and humans also share anatomical similarities in the two major target organs of alcohol- the brain and liver. Pigs possess gyrencephalic brains with comparable cerebral white matter volumes to humans, thus enabling more representative evaluations of susceptibility and neural tissue damage in response to AUD. Furthermore, similarities in the liver result in a comparable rate of alcohol elimination as humans, thus enabling a more accurate extrapolation of dosage and intoxication level to humans. A porcine model of AUD possesses great translational potential that can significantly advance our current understanding of the complex development and continuance of AUD in humans.

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 Extracellular Vesicles Disrupt Midline Shift Predictive Outcomes in Porcine Ischemic Stroke Model.

Magnetic resonance imaging (MRI) is a clinically relevant non-invasive imaging tool commonly utilized to assess stroke progression in real time. This study investigated the utility of MRI as a predictive measure of clinical and functional outcomes when a stroke intervention is withheld or provided, in order to identify biomarkers for stroke functional outcome under these conditions. Fifteen MRI and ninety functional parameters were measured in a middle cerebral artery occlusion (MCAO) porcine ischemic stroke model. Multiparametric analysis of correlations between MRI measurements and functional outcome was conducted. Acute axial and coronal midline shift (MLS) at 24 h post-stroke were associated with decreased survival and recovery measured by modified Rankin scale (mRS) and were significantly correlated with 52 measured acute (day 1 post) and chronic (day 84 post) gait and behavior impairments in non-treated stroked animals. These results suggest that MLS may be an important non-invasive biomarker that can be used to predict patient outcomes and prognosis as well as guide therapeutic intervention and rehabilitation in non-treated animals and potentially human patients that do not receive interventional treatments. Neural stem cell-derived extracellular vesicle (NSC EV) was a disruptive therapy because NSC EV administration post-stroke disrupted MLS correlations observed in non-treated stroked animals. MLS was not associated with survival and functional outcomes in NSC EV-treated animals. In contrast to untreated animals, NSC EVs improved stroked animal outcomes regardless of MLS severity.

Human Neural Stem Cell Extracellular Vesicles Improve Recovery in a Porcine Model of Ischemic Stroke.

BACKGROUND AND PURPOSE: Recent work from our group suggests that human neural stem cell-derived extracellular vesicle (NSC EV) treatment improves both tissue and sensorimotor function in a preclinical thromboembolic mouse model of stroke. In this study, NSC EVs were evaluated in a pig ischemic stroke model, where clinically relevant end points were used to assess recovery in a more translational large animal model. METHODS: Ischemic stroke was induced by permanent middle cerebral artery occlusion (MCAO), and either NSC EV or PBS treatment was administered intravenously at 2, 14, and 24 hours post-MCAO. NSC EV effects on tissue level recovery were evaluated via magnetic resonance imaging at 1 and 84 days post-MCAO. Effects on functional recovery were also assessed through longitudinal behavior and gait analysis testing. RESULTS: NSC EV treatment was neuroprotective and led to significant improvements at the tissue and functional levels in stroked pigs. NSC EV treatment eliminated intracranial hemorrhage in ischemic lesions in NSC EV pigs (0 of 7) versus control pigs (7 of 8). NSC EV-treated pigs exhibited a significant decrease in cerebral lesion volume and decreased brain swelling relative to control pigs 1-day post-MCAO. NSC EVs significantly reduced edema in treated pigs relative to control pigs, as assessed by improved diffusivity through apparent diffusion coefficient maps. NSC EVs preserved white matter integrity with increased corpus callosum fractional anisotropy values 84 days post-MCAO. Behavior and mobility improvements paralleled structural changes as NSC EV-treated pigs exhibited improved outcomes, including increased exploratory behavior and faster restoration of spatiotemporal gait parameters. CONCLUSIONS: This study demonstrated for the first time that in a large animal model novel NSC EVs significantly improved neural tissue preservation and functional levels post-MCAO, suggesting NSC EVs may be a paradigm changing stroke therapeutic.

Human Neural Stem Cell Extracellular Vesicles Improve Tissue and Functional Recovery in the Murine Thromboembolic Stroke Model.

Over 700 drugs have failed in stroke clinical trials, an unprecedented rate thought to be attributed in part to limited and isolated testing often solely in "young" rodent models and focusing on a single secondary injury mechanism. Here, extracellular vesicles (EVs), nanometer-sized cell signaling particles, were tested in a mouse thromboembolic (TE) stroke model. Neural stem cell (NSC) and mesenchymal stem cell (MSC) EVs derived from the same pluripotent stem cell (PSC) line were evaluated for changes in infarct volume as well as sensorimotor function. NSC EVs improved cellular, tissue, and functional outcomes in middle-aged rodents, whereas MSC EVs were less effective. Acute differences in lesion volume following NSC EV treatment were corroborated by MRI in 18-month-old aged rodents. NSC EV treatment has a positive effect on motor function in the aged rodent as indicated by beam walk, instances of foot faults, and strength evaluated by hanging wire test. Increased time with a novel object also indicated that NSC EVs improved episodic memory formation in the rodent. The therapeutic effect of NSC EVs appears to be mediated by altering the systemic immune response. These data strongly support further preclinical development of a NSC EV-based stroke therapy and warrant their testing in combination with FDA-approved stroke therapies.