Maternal supplementation of egg yolk modulates brain functional organization and functional outcomes of offspring.

Maternal nutrition during the perinatal stage is critical to offspring brain development. Egg yolks are a balanced and nutrient-dense food that is rich in bioactive components crucial to optimal neurodevelopment early in life. Egg consumption is often recommended to pregnant women to enhance both maternal and fetal health. We hypothesized that maternal intake of egg yolk from late gestation and throughout lactation would enhance functional organization and cognitive developmental outcomes in offspring using a pig model. Sows were fed a control diet (n = 6) or a diet containing egg yolks (n = 5, 350 mg egg yolk powder/kg BW/day, equivalent to ∼3 eggs/day for humans) from late gestation through lactation. At weaning, piglet offspring (n = 2/sow, total n = 22) underwent structural magnetic resonance imaging (MRI) and resting-state-functional MRI. Piglets underwent novel object recognition testing to assess hippocampal-dependent learning and memory. Functional MRI results demonstrated that egg yolk significantly increased functional activation in the executive network (p = 0.0343) and cerebellar network (p = 0.0253) in piglets when compared to control. Diffusion tensor imaging analysis showed that perinatal intake of egg yolks significantly increased white matter fiber length in the hippocampus (p = 0.0363) and cerebellum (p = 0.0287) in piglet offspring compared to control piglets. Furthermore, piglets from egg yolk-fed sows spent significantly more proportional frequency exploring the novel object than the familiar object in novel object recognition testing (p = 0.0370). The findings from this study support egg yolk-altered activation of specific brain networks may be associated with functional cognitive outcomes in weaning piglets.

Evaluation of Brain Function Recovery After Traumatic Brain Injury Treatment in a Porcine Model by Cross-Group Temporal-Spatial Correlation Analysis.

Traumatic brain injury (TBI), a significant global health issue, is affecting ∼69 million annually. To better understand TBI's impact on brain function and assess the efficacy of treatments, this study uses a novel temporal-spatial cross-group approach with a porcine model, integrating resting-state functional magnetic resonance imaging (rs-fMRI) for temporal and arterial spin labeling for spatial information. Our research used 18 four-week-old pigs divided into three groups: TBI treated with saline (SLN, n = 6), TBI treated with fecal microbial transplant (FMT, n = 6), and a sham group (sham, n = 6) with only craniectomy surgery as the baseline. By applying machine learning techniques-specifically, independent component analysis and sparse dictionary learning-across seven identified resting-state networks, we assessed the temporal and spatial correlations indicative of treatment efficacy. Both temporal and spatial analyses revealed a consistent increase of correlation between the FMT and sham groups in the executive control and salience networks. Our results are further evidenced by a simulation study designed to mimic the progression of TBI severity through the introduction of variable Gaussian noise to an independent rs-fMRI dataset. The results demonstrate a decreasing temporal correlation between the sham and TBI groups with increasing injury severity, consistent with the experimental results. This study underscores the effectiveness of the methodology in evaluating post-TBI treatments such as the FMT. By presenting comprehensive experimental and simulated data, our research contributes significantly to the field and opens new paths for future investigations into TBI treatment evaluations.

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.

Induced pluripotent stem cells derived from patients carrying mitochondrial mutations exhibit altered bioenergetics and aberrant differentiation potential.

BACKGROUND: Human mitochondrial DNA mutations are associated with common to rare mitochondrial disorders, which are multisystemic with complex clinical pathologies. The pathologies of these diseases are poorly understood and have no FDA-approved treatments leading to symptom management. Leigh syndrome (LS) is a pediatric mitochondrial disorder that affects the central nervous system during early development and causes death in infancy. Since there are no adequate models for understanding the rapid fatality associated with LS, human-induced pluripotent stem cell (hiPSC) technology has been recognized as a useful approach to generate patient-specific stem cells for disease modeling and understanding the origins of the phenotype. METHODS: hiPSCs were generated from control BJ and four disease fibroblast lines using a cocktail of non-modified reprogramming and immune evasion mRNAs and microRNAs. Expression of hiPSC-associated intracellular and cell surface markers was identified by immunofluorescence and flow cytometry. Karyotyping of hiPSCs was performed with cytogenetic analysis. Sanger and next-generation sequencing were used to detect and quantify the mutation in all hiPSCs. The mitochondrial respiration ability and glycolytic function were measured by the Seahorse Bioscience XFe96 extracellular flux analyzer. RESULTS: Reprogrammed hiPSCs expressed pluripotent stem cell markers including transcription factors POU5F1, NANOG and SOX2 and cell surface markers SSEA4, TRA-1-60 and TRA-1-81 at the protein level. Sanger sequencing analysis confirmed the presence of mutations in all reprogrammed hiPSCs. Next-generation sequencing demonstrated the variable presence of mutant mtDNA in reprogrammed hiPSCs. Cytogenetic analyses confirmed the presence of normal karyotype in all reprogrammed hiPSCs. Patient-derived hiPSCs demonstrated decreased maximal mitochondrial respiration, while mitochondrial ATP production was not significantly different between the control and disease hiPSCs. In line with low maximal respiration, the spare respiratory capacity was lower in all the disease hiPSCs. The hiPSCs also demonstrated neural and cardiac differentiation potential. CONCLUSION: Overall, the hiPSCs exhibited variable mitochondrial dysfunction that may alter their differentiation potential and provide key insights into clinically relevant developmental perturbations.

Fecal microbial transplantation limits neural injury severity and functional deficits in a pediatric piglet traumatic brain injury model.

Pediatric traumatic brain injury (TBI) is a leading cause of death and disability in children. Due to bidirectional communication between the brain and gut microbial population, introduction of key gut bacteria may mitigate critical TBI-induced secondary injury cascades, thus lessening neural damage and improving functional outcomes. The objective of this study was to determine the efficacy of a daily fecal microbial transplant (FMT) to alleviate neural injury severity, prevent gut dysbiosis, and improve functional recovery post TBI in a translational pediatric piglet model. Male piglets at 4-weeks of age were randomly assigned to Sham + saline, TBI + saline, or TBI + FMT treatment groups. A moderate/severe TBI was induced by controlled cortical impact and Sham pigs underwent craniectomy surgery only. FMT or saline were administered by oral gavage daily for 7 days. MRI was performed 1 day (1D) and 7 days (7D) post TBI. Fecal and cecal samples were collected for 16S rRNA gene sequencing. Ipsilateral brain and ileum tissue samples were collected for histological assessment. Gait and behavior testing were conducted at multiple timepoints. MRI showed that FMT treated animals demonstrated decreased lesion volume and hemorrhage volume at 7D post TBI as compared to 1D post TBI. Histological analysis revealed improved neuron and oligodendrocyte survival and restored ileum tissue morphology at 7D post TBI in FMT treated animals. Microbiome analysis indicated decreased dysbiosis in FMT treated animals with an increase in multiple probiotic Lactobacilli species, associated with anti-inflammatory therapeutic effects, in the cecum of the FMT treated animals, while non-treated TBI animals showed an increase in pathogenic bacteria, associated with inflammation and disease such in feces. FMT mediated enhanced cellular and tissue recovery resulted in improved motor function including stride and step length and voluntary motor activity in FMT treated animals. Here we report for the first time in a highly translatable pediatric piglet TBI model, the potential of FMT treatment to significantly limit cellular and tissue damage leading to improved functional outcomes following a 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.

Detecting functional connectivity disruptions in a translational pediatric traumatic brain injury porcine model using resting-state and task-based fMRI.

Functional magnetic resonance imaging (fMRI) has significant potential to evaluate changes in brain network activity after traumatic brain injury (TBI) and enable early prognosis of potential functional (e.g., motor, cognitive, behavior) deficits. In this study, resting-state and task-based fMRI (rs- and tb-fMRI) were utilized to examine network changes in a pediatric porcine TBI model that has increased predictive potential in the development of novel therapies. rs- and tb-fMRI were performed one day post-TBI in piglets. Activation maps were generated using group independent component analysis (ICA) and sparse dictionary learning (sDL). Activation maps were compared to pig reference functional connectivity atlases and evaluated using Pearson spatial correlation coefficients and mean ratios. Nonparametric permutation analyses were used to determine significantly different activation areas between the TBI and healthy control groups. Significantly lower Pearson values and mean ratios were observed in the visual, executive control, and sensorimotor networks for TBI piglets compared to controls. Significant differences were also observed within several specific individual anatomical structures within each network. In conclusion, both rs- and tb-fMRI demonstrate the ability to detect functional connectivity disruptions in a translational TBI piglet model, and these disruptions can be traced to specific affected anatomical structures.

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.

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.

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.

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.

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.

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.