Spatial and Temporal Assessment of Cerebral Blood Flow in a Novel Piglet Model of Neonatal Arterial Ischemic Stroke

Spatial and Temporal Assessment of Cerebral Blood Flow in a Novel Piglet Model of Neonatal Arterial Ischemic Stroke | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Spatial and Temporal Assessment of Cerebral Blood Flow in a Novel Piglet Model of Neonatal Arterial Ischemic Stroke Qihong Wang, Mostafa Abdulrahim, Lisa Young, Larraine Lage, Sanaz Nasoohi, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7235675/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 06 Dec, 2025 Read the published version in Translational Stroke Research → Version 1 posted 13 You are reading this latest preprint version Abstract Neonatal arterial ischemic stroke (NAIS) is associated with considerable pediatric morbidity and mortality but lacks effective treatment options compared to adult stroke, highlighting the need for clinically relevant translational models. This study aimed to develop a novel middle cerebral artery occlusion (MCAO) model in neonatal piglets with exceptional clinical relevance and the opportunity for long-term survival. Piglets were randomly assigned to undergo either MCAO (n=8) or sham surgery (n=6). MCAO was achieved by occluding MCAs using 7mm aneurysm clips via craniotomy. Laser speckle contrast imaging was used to measure changes in relative cerebral blood flow (rCBF) in three cortical regions (anterior cerebral artery territory, penumbra, and ischemic core). Open field testing was performed in a subset of piglets at baseline, 24h post-MCAO, and 48h post-MCAO. 2,3,5-triphenyl tetrazolium chloride (TTC) staining was used to identify infarcts at 48h post-MCAO. By 10 minutes post-MCAO, the rCBF had risen approximately 22.4% in the ischemic core compared to immediately post-MCAO ( p <0.05), with the area of the core as a percentage of the ipsilateral hemisphere decreasing by 38%. Furthermore, MCAO piglets showed increased ipsiversive circling at 48h post-MCAO (16±4 vs 7.3±1.4 ipsiversive rotations, p <0.05) compared to baseline and higher infarct volumes compared to sham piglets (31.6±6.4%, p <0.01). Overall, our model creates a reproducible infarct with consistent neuromotor deficits, real-time assessment of rCBF dynamics, and long-term survival, thus offering insights that may inform the development of novel therapies and improve NAIS outcomes. Neonatal arterial ischemic stroke cerebral blood flow middle cerebral artery occlusion ischemia stroke laser speckle contrast imaging Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 INTRODUCTION Neonatal arterial ischemic stroke (NAIS) refers to infarction resulting from arterial large vessel occlusion in the first 28 days of life. NAIS accounts for approximately 80% of perinatal stroke cases, encompassing subtypes such as cerebral venous sinus thrombosis and hemorrhagic stroke.[ 1 ] The incidence of NAIS is estimated to be approximately 1:3,000 live births, although this is likely an underestimation given the vagaries in clinical diagnosis and limitations in magnetic resonance imaging (MRI) in low-resource settings.[ 2 ] Infants with NAIS rarely die, but generally have poor long-term outcomes and often require lifelong specialized care.[ 3 ] Long-term deficits remain in two-thirds of patients and may include hemiparetic cerebral palsy, cognitive and behavioral dysfunction, speech delay, spasticity, and epilepsy.[ 4 ], [ 5 ], [ 6 ], [ 7 ] Despite its prevalence and health impact, management of NAIS is typically supportive.[ 8 ] Treatment options are particularly limited during the acute phase of ischemia, during which treatment is most likely to have a meaningful effect. The only Food and Drug Administration (FDA)-approved therapies used in adult ischemic stroke, thrombolytics and mechanical thrombectomy, are not typically used in neonates due to safety concerns and technical difficulties.[ 9 ], [ 10 ] Moreover, the limited therapeutic time window for these therapies (< 4.5h for thrombolytics and < 24h for thrombectomy) and the associated risk of intracranial hemorrhage exclude a large population of stroke patients. The lack of safe, effective therapies that decrease stroke severity and improve neurological outcomes in neonates is a significant knowledge gap, which, until it is addressed, will limit our ability to improve the lives of children with NAIS. Establishing a preclinical model that can replicate NAIS is key to expanding our understanding of the pathophysiology of this disease and advancing the development of novel therapies that can be rapidly translated to human trials. A workshop on perinatal and childhood stroke held by the National Institute of Neurological Disorders and Stroke in 2000 emphasized the importance of increasing the implementation of clinically relevant animal models for these conditions.[ 11 ] While many stroke studies use rodent models, the anatomical and structural differences between rodent and human brains (e.g., disparities in brain size, gray/white matter ratio, and lissencephalic versus gyrencephalic morphology) have limited their translational relevance.[ 12 ] Further, the small size of neonatal rodents has made measurements of vital parameters (such as blood pressure) and cerebral blood flow (CBF) challenging. Pigs are a more clinically relevant and less ethically controversial stroke model given their cerebrovascular and neurodevelopmental similarities to human neonates, such as gyrencephalic organization and comparable brain size and gray/white matter ratio.[ 13 ] Here, we present a model of NAIS that creates a reproducible infarct via craniotomy and middle cerebral artery occlusion (MCAO) and allows for reliable measurement of CBF and vital parameters. By enabling a better understanding of CBF dynamics during ischemia, this model may help identify interventions that can reduce brain injury and improve outcomes for children affected by NAIS. MATERIALS AND METHODS Animals All procedures were approved by the Animal Care and Use Committee at Johns Hopkins University. Care and handling of animals were performed by an experienced veterinary technician in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. Fourteen healthy Yorkshire piglets (3–7 days old, 1.5-2.5kg, Archer Farms, Darlington, MD, USA) were used in this study. Animals were housed in a temperature-controlled (26.7–32.2°C, humidity 14–30%) space with 12-hour light-dark cycles and had access to milk replacer (NurseAll Milk Replacer, Manna Pro Products, Chesterfield, MO, USA) and water ad libitum. Enrichment was provided with daily human interaction and toys. Animals were co-housed to prevent separation stress except during the acute period while recovering from surgery or during behavioral testing. Surgery Milk replacer was withheld for four hours immediately before anesthesia and surgery. On the day of surgery, animals were brought to a large animal operating room suite, and anesthesia was induced using 5% isoflurane or 8% sevoflurane (depending on anesthetic availability; Piramal Critical Care Inc, Northampton County, PA, USA) in 100% O 2 . Once an adequate depth of anesthesia was reached, the laryngeal folds were sprayed with 2% lidocaine to prevent laryngospasm during intubation. The trachea was then orally intubated with a 2.5mm cuffed endotracheal tube. Mechanical ventilation was instituted to maintain normoxia (SpO 2 90–100%, pO 2 90–120 mmHg) and normocarbia (EtCO 2 /pCO 2 35–45 mmHg). The anesthetic dose was then decreased to 1-1.25% isoflurane or 2-2.5% sevoflurane and subsequently titrated to clinical effect (targeting the minimum effective dose). Doses at or below 1 minimum alveolar concentration (MAC) have been shown to have minimal effects on cerebral autoregulation.[ 14 ], [ 15 ] A 26-gauge peripheral intravenous (IV) catheter was placed in the marginal ear vein for the administration of lactated Ringer’s solution and medications. A one-time dose of enrofloxacin (7.5mg/kg, IV) and extended-release buprenorphine (0.12 mg/kg/dose, subcutaneous) were given prior to the surgical incision. A single dose of rocuronium 10 mg was given following successful intubation to prevent movement during craniotomy and brain manipulation. A femoral arterial catheter was placed to provide continuous blood pressure monitoring. Additional monitoring included end-tidal carbon dioxide (EtCO 2 ) and end-tidal anesthetic concentration, electrocardiography, heart rate, oxygen saturation (SpO 2 ), and esophageal temperature, which were continuously recorded using LabChart 8.1.30 software (AD Instruments, Colorado Springs, CO, USA). Arterial blood gases (ABG) were measured at least hourly throughout the procedure (ABL800 FLEX blood gas analyzer, Radiometer America, Brea, CA, USA). If needed, pO 2 and pCO 2 were corrected by titrating ventilator settings to optimize oxygenation and ventilation. Body temperature was maintained with a water heating blanket and a forced-air warmer (Thermacare® TC3001 Convective Warming System, Gaymar, Orchard Park, NY, USA). Lactated Ringer’s solution was administered intravenously at a maintenance dose (4 mL/kg/hour) throughout the procedure. The skin was prepared in the usual sterile fashion. Prior to all incisions, subcutaneous infiltration of 0.25% bupivacaine (maximum total volume 1 mL/kg) was performed. To perform the MCAO surgery, the scalp was shaved, a 6.5 cm midline incision was made in the scalp, and the skin was retracted laterally. A curette was used to remove fascia from the skull, and a pediatric perforator (Stryker, Kalamazoo, MI, USA) was used to drill six small burr holes into the skull. To ensure consistency, the burr holes were drilled in standardized locations relative to bregma in each animal. Two parasagittal holes were drilled 2.2 cm anterior to bregma; one was placed 0.5 cm lateral to the sagittal sinus, and the other was positioned above the orbit, 1.5 cm lateral to the sagittal sinus. An additional two holes were drilled 1.2 cm posterior to bregma; one was 0.5 cm lateral to the sagittal sinus, and the other was 2.2 cm lateral to the sinus. Two more holes were drilled between the anterior and posterior parasagittal and lateral holes. A craniotome was then used to connect the holes and create a craniotomy. The skull flap was then removed with careful attention to avoid injury to the dura and placed in sterile saline. Under a surgical microscope, the dura was incised and retracted. Cerebrospinal fluid (CSF) was aspirated to obtain a clear field of view. The brain hemisphere was gently retracted, and the MCA(s) were exposed and identified on the ventral surface of the brain. Of note, pigs are known to have multiple MCAs.[ 16 ] Two 7 mm aneurysm clips (YASARGIL® Aneurysm Clips, B. Braun Medical Inc., Bethlehem, PA, USA) were placed on all visible MCA branches at their origin from the internal carotid artery (ICA). These steps are pictured in Fig. 1 . After the clips were placed and laser speckle contrast imaging (LSCI) was performed (described in the next section), the dura was repositioned over the brain, and the bone flap was replaced and sutured to the skull. The skin was closed with a running suture technique with a 5 − 0 Vicryl suture. For sham surgeries, the piglets underwent the same steps; however, after retracting the brain and identifying the MCA branches, no aneurysm clips were placed. A second dose of extended-release buprenorphine (0.12 mg/kg/dose, IV) was given for pain control, the neuromuscular blockade was reversed with sugammadex (4mg/kg, IV), and the animal was extubated. The same veterinary technician who cared for the animal pre-surgery observed the animal during the immediate postoperative period for any surgical or anesthetic complications. During the 48h postoperative period, vital signs were monitored daily, and standard release buprenorphine (0.01–0.02 mg/kg, IV) was given every 8 hours for analgesia. LSCI acquisition Laser speckle contrast imaging (LSCI) was performed using an RFLSI-ZW (RWD, Shenzhen, Guangdong, China) LSCI device. LSCI is a commonly used imaging modality with high spatial and temporal resolution used to observe and record cerebral perfusion of exposed brain tissue before and after MCAO.[ 17 ] With the animal in the prone position, the LSCI camera was positioned 15cm from the exposed brain hemisphere and focused to obtain a high-resolution image. Imaging parameters were kept consistent at 0-5000 perfusion unit blood monitoring perfusion range, image acquisition speed of 50 frames per second, camera pixel/resolution 2048×2048, a spatial resolution of image > 3µm/pixel, effective pixels per unit area > 5,000,000 pixels/cm², and optical multiplication ratio 12:1. Five-second recordings were acquired at baseline (immediately before MCA clipping), immediately post-MCAO, and 10 minutes post-MCAO to verify the presence of successful MCAO. The average flux signal was calculated for each 5-second recording period, producing three images (gross anatomical, pseudo-color, and grayscale) at each time point ( Fig. 2 ) . LSCI analysis LSCI images were analyzed in FIJI software (Schindelin et al., 2012; https://fiji.sc ) to quantify changes in relative CBF (rCBF) following MCAO surgery.[ 18 ] For each animal, all LSCI images taken during the procedure were calibrated to a consistent reference scale, inverted, and converted to a 32-bit format. The StackReg plugin was used to align all images to the baseline image taken before MCAOs.[ 19 ] Mean pixel intensity values were used as a proxy for blood flow. Pixel-by-pixel CBF reduction maps were generated at each post-MCAO time point using the formula: rCBF reduction (%) = 100 * (baseline perfusion – post-MCAO perfusion) / baseline perfusion. Three regions of interest (ROI) were identified by the following thresholds for perfusion reduction from baseline, which were based on previously described thresholds: ischemic core (57–100% reduction from baseline), penumbra (19–55% reduction from baseline), and ACA territory (< 17% reduction from baseline).[ 20 ], [ 21 ] The small gap (~ 2%) between the threshold ranges was deliberate to establish conservative cutoff definitions. At each post-MCAO time point, the area of each ROI was measured and expressed as a percentage of the total area of the exposed ipsilateral hemisphere ( Fig. 3 ) . To assess temporal changes in CBF in the core ischemic region, penumbra, and the ACA territory, these ROIs were defined on the CBF reduction map at the immediately post-MCAO time point for each animal. These ROIs were then copied to the CBF reduction drop map at the 10-minute post-MCAO time point. To correct for slight variations in animal brain positioning between time points, the ROIs were adjusted accordingly using blood vessel landmarks. The mean intensity values in each of the three ROIs at 10-minute post-MCAO time points were then measured and reported as a percentage decrease from baseline. Behavioral testing To assess neuromotor deficits, we performed an Open Field Test (OFT) in a subset of MCAO piglets (n = 3) and sham piglets (n = 4) as previously described.[ 22 ] The test area was a 0.8 x 0.8-meter square arena enclosed by a fence. OFT was performed in a quiet, temperature-controlled room with no other piglets present. The piglet’s movements were recorded with a video camera and analyzed by an investigator blinded to groups (MCAO versus sham) using ANY-maze software (Stoelting Co, Wood Dale, IL, USA) to assess its locomotor activity. OFTs were performed at the beginning of the day, at the same time before the first feeding, to prevent animal drowsiness. To perform the test, piglets were placed into the arena for 10 minutes to acclimate to the environment, and their movements were video recorded for 12 minutes. OFTs were performed to qualitatively and quantitatively assess neuromotor behavior one day before surgery (baseline), at 24 hours post-MCAO, and 48 hours post-MCAO. TTC staining and infarct volume quantification At 48 hours post-MCAO, piglets were euthanized with a lethal dose of sodium pentobarbital/phenytoin (0.5 mL/kg, Euthasol). The brains of the piglets were removed and placed in ice-cold 0.1M phosphate-buffered saline (PBS). After the brains were checked for accuracy of MCA aneurysm clip placement ( Fig. 1 b ) , the clips were removed. Brains were placed in a tissue sectioning matrix custom-designed for neonatal piglets and covered with warm low-melt agarose to prevent tissue movement during sectioning. Coronal sectioning began at the front of the anterior pole, and 4-mm-thick coronal sections were produced, working from anterior to posterior. Freshly sliced brain sections were incubated in 1% 2,3,5-triphenyl tetrazolium chloride (TTC, Sigma Aldrich) at 37°C for 30 minutes to delineate infarcted (white) versus non-infarcted (red) tissue. Following staining, sections were washed three times with ice-cold 0.1M PBS. Once washed, sections were fixed overnight at 4°C in 4% paraformaldehyde. After 24 hours, sections were moved to 0.1M PBS for storage at 4°C. Anterior and posterior faces of coronal sections were then scanned and digitized to identify infarct volume. Analysis of TTC-stained images was performed using FIJI software by a blinded investigator to maintain consistency across the study cohort. First, the image was calibrated to distance using a ruler as a reference. Then, the boundaries of the contralateral hemisphere and the non-infarcted tissue of the ipsilateral hemisphere were manually outlined using the free-draw tool. The area of these regions was measured, and the process was repeated for all brain slices on the anterior and posterior faces. These infarct areas were multiplied by the slice thickness (4 mm) to calculate total infarct volumes. The Swanson method was used to correct for edema by calculating the percent difference between the contralateral hemisphere volume and that of the non-infarcted region of the ipsilateral hemisphere.[ 23 ] The infarct volume was expressed as a percentage of the ipsilateral brain hemisphere volume of each animal. Statistical analysis A total sample size of 14 animals (n = 8 in MCAO group, n = 6 in sham group) was selected based on prior pilot and exploratory studies in adult swine models.[ 24 ], [ 25 ], [ 26 ] The sample size in the sham group was lowered in accordance with the 3R principles of reduction, refinement, and replacement in animal research, and previous literature supporting a minimal infarct size in sham animals in invasive swine models.[ 27 ] MAP and HR data were extracted from a 5-second average every 5 minutes for the period from 15 minutes prior to clipping (or pseudo-clipping for sham piglets) and 30 minutes following (pseudo)-clipping. Since each animal had a different MAP at the start of the experiment, MAP was presented as a percentage difference from baseline to demonstrate the change in MAP before and after MCAO. The percent change in MAP from baseline was calculated, with the baseline being measured at 15 minutes prior to clipping. This data was analyzed using mixed-effects analysis followed by the Tukey test, with comparisons made between experimental groups at each time point and within each group at each time point. OFT data were analyzed by mixed-effects analysis followed by the Tukey test to compare the number of ipsiversive rotations, walking distance, and mean speed between experimental groups at each time point, and within each group at each time point. In the MCAO group, ROI area and perfusion data were compared at each time point using repeated measures ANOVA. Each animal served as its own pre-MCAO control in statistical calculations. Unpaired t-tests were used to compare ABG parameters (pH, pCO 2 , and pO 2 ) between the MCAO and sham groups at the shared time point (hour 1 of the surgery). In the MCAO group, a paired t-test was used to assess changes between hour 1 and hour 2. In the sham group, where only 4 animals had available blood gas measurements at hour 2 due to two animals having a < 2h total sham surgery length, a paired t-test was conducted only on animals with complete data (n = 4). Infarct volumes between the two groups were also compared with an unpaired t-test. Statistical analyses were performed using GraphPad Prism software (GraphPad Software, San Diego, CA, USA). Unless otherwise stated, data are presented as mean ± standard error (SEM). The threshold for statistical significance was set at * p < 0.05 and ** p < 0.01. RESULTS Fourteen neonatal piglets were randomly assigned to two surgery groups (MCAO, n = 8; sham, n = 6). On average, the piglets in the MCAO group weighed 2.05 ± 0.04 kg, and those in the sham group weighed 2.01 ± 0.05 kg; there was no statistical difference between these two groups. No piglet died during or immediately after surgery, but one piglet in the MCAO group was euthanized early at 24 hours due to severe neurological deficits and periods of non-responsiveness. On necropsy, the animal was found to have an extensive infarct lesion due to inadvertent clipping of the circle of Willis. This piglet was excluded from data analyses. Physiological Parameters Arterial blood gases obtained during the surgery are listed in Table 1 . pH, pCO 2 , and pO 2 were stable throughout the experimental period, and there were no significant differences between groups in any blood gas parameter at the shared time point (hour 1), nor between hour 1 and hour 2 within either group. ABG values were not recorded for one animal in the MCAO group, but the animal was maintained at ventilation parameters consistent with other animals during the procedure. There was no significant difference in heart rate or the percent change in MAP from baseline between MCAO and sham groups prior to and following clip placement, nor within each group at any time point (Online Resource 1) . Table 1 Intraoperative arterial blood gas (ABG) recordings Time Hour 1 Hour 2 p- value (within group) pH, units MCAO 7.43 ± 0.04 7.39 ± 0.03 0.10 Sham 7.47 ± 0.04 7.40 ± 0.05* 0.53 p -value (MCAO vs Sham) 0.46 - - pCO 2 , mmHg MCAO 37.72 ± 4.31 42.00 ± 3.48 0.14 Sham 38.35 ± 3.43 46.53 ± 6.92* 0.59 p -value (MCAO vs Sham) 0.91 - - pO 2 , mmHg MCAO 167.33 ± 17.64 174.83 ± 20.68 0.52 Sham 183.67 ± 28.01 164.25 ± 12.57* 0.42 p -value (MCAO vs Sham) 0.63 - - Arterial blood gases obtained during the MCAO and sham surgeries at hour 1 and hour 2 time points. n = 6 for all groups and time points except where indicated with asterisk (*), as the sham group had only 4 animals with available blood gas measurements at hour 2 due to a < 2h total sham surgery length. The values represent mean ± SEM. The entire MCAO surgery lasted 3-3.5 hours and was well-tolerated by piglets. All piglets survived the first 24 hours post-surgery, and 7 of 8 piglets in the MCAO group survived to 48 hours post-surgery. Cortical blood flow and infarct volumes in the MCAO group Successful occlusion of the MCAs was achieved in all piglets in the MCAO group, as assessed by LSCI immediately after the MCAs were occluded. As mentioned above, one piglet in the MCAO group was excluded due to technical difficulties placing the clip and associated damage to the brain. At the 10-minute post-MCAO time point, rCBF to the ischemic core region showed a statistically significant 22.4% recovery compared to the immediate post-MCAO time point ( p < 0.01) ( Fig. 4 ) . While not statistically significant, rCBF to the penumbra region showed a 16.8% increase at the 10-minute post-MCAO time point compared to the immediate post-MCAO time point. Compared to the immediate post-MCAO time point, there was an expansion of the ACA and penumbral regions as a percentage of the ipsilateral hemisphere area at the 10-minute post-MCAO timepoint (12.6% and 19.9% respectively) ( Fig. 5 ) . At the 10-minute post-MCAO timepoint, the relative area of the ischemic core region showed a 38% decrease (compared to the immediate post-MCAO time point) to 33.9 ± 11% of the ipsilateral hemisphere area. Analysis of TTC-stained coronal brain slices in MCAO piglets showed edematous changes and tissue damage consistent with infarction in cortical and subcortical regions of the ipsilateral hemisphere, which were not present in sham piglets ( Fig. 6 a ) . A piglet brain atlas was used to identify infarcted regions, which included the caudate nucleus, putamen, globus pallidus, anterior commissure, and internal capsule, among others.[ 28 ] The mean infarct volume was 31.6 ± 6.4% of the ipsilateral hemisphere volume at 48 hours post-MCAO, which was significantly higher than the sham group (0%) ( Fig. 6 b ). Behavioral testing in MCAO vs sham groups A small subset of animals (n = 7 total piglets) underwent pre-surgical (baseline) OFT. Analysis of ANY-maze movement tracings showed random patterns associated with normal ambulation and exploratory behaviors ( Fig. 7 a ) . Sham piglets (n = 4) showed minimal ambulatory changes on OFT at 24- and 48-hour time points after surgery. In contrast, piglets in the MCAO group (n = 3) showed considerable ambulatory deficits, including significantly increased ipsiversive circling (turns toward the side of the stroke lesion) at 48h post-MCAO compared to baseline (16 ± 4 vs 7.3 ± 1.4, p < 0.05) ( Fig. 7 b ) . There was no statistically significant difference between the number of ipsiversive rotations at 24- and 48-hours post-MCAO for either group. No significant differences were demonstrated in mean walking distance or speed between either group at any time point. However, the use of a small OFT arena makes this finding difficult to interpret. DISCUSSION The overall goal of the present study was to assess this novel approach to MCAO as a tool to perform translational NAIS research that may advance the understanding of neonatal stroke pathophysiology and allow the testing of potential therapeutics to improve outcomes for affected neonates. The experiments described herein yielded several key descriptive findings that contribute to this field. Firstly, our approach to producing MCAO in neonatal piglets is feasible and, while invasive, effective in producing a stroke with expected biological variability in lesion size in a higher mammal with a gyrencephalic brain. Recently, there has been growing interest in adjuvant neuroprotective therapies to improve outcomes for affected neonates, given the considerable morbidity and the ineligibility of this population for thrombolytics or thrombectomy. While rodents remain the most popular stroke model, they have major limitations due to rodent brains having a lissencephalic brain organization that differs from the gyrencephalic organization of humans, being considerably smaller than human brains, having a lower white matter proportion in the cortex (10–12% versus 40–45% in humans), and demonstrating limited ICP changes during stroke.[ 12 ], [ 29 ] The disparity in white matter composition is important to consider, given the differences in metabolic demands and susceptibility to ischemia between gray and white matter.[ 30 ] The limitations in these rodent studies have likely contributed to the lack of success of experimental stroke therapies in clinical trials, with the only two FDA-approved drugs being tissue plasminogen activator (alteplase) and tenecteplase.[ 31 ], [ 32 ], [ 33 ] To address this translational gap, the Stroke Academic Industry Roundtable (STAIR) and Stem Cell Therapies as an Emerging Paradigm in Stroke (STEPS) committees have recommended using large animals with human-like brain structures for therapeutic testing, in conjunction with rodent studies.[ 34 ], [ 35 ] These models have been established in a number of large animals, including sheep, dogs, pigs, and non-human primates (NHPs).[ 36 ], [ 37 ], [ 38 ] Dogs and NHPs are generally controversial due to societal and ethical implications. Pigs are a model with relatively low ethical controversy and high translational relevance given the similarities they share with humans regarding brain gyrencephalic organization, white matter composition in the cortex (28.4% versus 40–45% in humans), size, and development.[ 39 ], [ 40 ] These models have proven helpful in testing therapies such as induced pluripotent stem cell-derived neural stem cells [ 41 ] and neural stem cell extracellular vesicles.[ 42 ] However, these therapies have only been studied in adult pigs to date. Thus, our model of stroke in neonatal pigs not only adds to the body of scientific literature but also has the potential to bridge the gap between preclinical research and clinical trials. The MCA is the most frequently affected large artery in human NAIS, making it an important site to target in preclinical models.[ 43 ] Stroke models in rodents typically use an endovascular filament to mechanically occlude the MCA at its origin from the circle of Willis.[ 44 ], [ 45 ] This approach is not possible in the porcine brain due to the presence of a complex cerebral vascular anatomy, typically consisting of two or even three MCAs.[ 16 ] In addition, the presence of the rete mirabile (RM) that is present between the carotid artery and the circle of Willis makes a direct filament-based, endovascular approach impossible. The RM (Latin for “wonderful net”) is a complex network of small anastomosing blood vessels proximal to the internal carotid artery (ICA) in swine and does not exist in humans or rodents.[ 46 ] The location of the RM between the extracranial and intracranial vasculature and its winding tortuosity are major impediments to catheter or filament navigation. Recent studies have attempted to overcome this obstacle by using a liquid embolic to occlude the RM wing up to the origin of the MCAs or by injecting thrombin proximally to the RM.[ 24 ], [ 25 ] However, these endovascular stroke models have significant morbidity and mortality, with the former having a 33% mortality rate within the first 24 hours after stroke induction [ 25 ] and the latter having a 28% mortality rate within the same time period.[ 24 ] This high mortality rate is likely due to cerebral edema and swelling, which leads to rapid neurological deterioration. In contrast, despite the invasive nature of our approach, there was no mortality in any animal in which the MCAs were properly clipped. This more closely replicates the mortality rate of neonatal stroke, which was estimated to be approximately 3% in a summary review.[ 47 ] This is likely due to the presence of open fontanelles in neonates with ischemic stroke, which has a similar effect to a craniotomy in allowing them to better tolerate elevated ICPs. In addition, endovascular embolization approaches introduce a foreign substance that likely has direct effects on vascular physiology. Finally, these approaches are irreversible, which makes studying reperfusion impossible. The ability to study reperfusion is critical in NAIS because most neonates who have a stroke spontaneously recanalize.[ 48 ] The use of removable aneurysm clips in our model will allow the study of the effects of reperfusion in NAIS after varying periods of ischemia, potentially offering insights into therapies that can improve NAIS outcomes when administered in the acute ischemic period or after reperfusion. In our model, the actual procedure was relatively short, lasting 3-3.5 hours in total. In all cases, the time from the start of the craniotomy to clipping was less than an hour. There was no significant difference in MAP or heart rate after MCAO compared to baseline, suggesting that this surgical approach does not significantly impact hemodynamics. Of note, the mean heart rate in the MCAO group did increase slightly from around 180 beats per minute (bpm) before MCAO to around 200 bpm after MCAO; however, this change is unlikely to be clinically significant and is consistent with vital parameter changes that have been reported previously in piglets undergoing surgeries.[ 49 ] Although our study used a craniotomy approach to expose the MCAs, another approach that has been studied in adult swine involves enucleation of the eyeball (i.e., transorbital approach), followed by either permanent (i.e., ligation or electrocoagulation) or transient (i.e., clipping) occlusion.[ 50 ] Advantages of using the craniotomy approach over the transorbital approach include the ability to perform behavioral testing (given the intact eye and preserved binocular vision and depth perception), to perform direct measurements of tissue oxygenation, and to perform direct visualization and assessment of microvascular flow (through LSCI, for example).[ 51 ] Our model allows investigation of all of these factors, thus potentially lending key insights into long-term functional outcomes and time-dependent changes in ischemic injury and rCBF following neonatal stroke. The stroke lesions in our model varied in size, with a mean infarct volume of 31.6 ± 6.4% of hemisphere volume. Two animals had infarct volumes of less than 16%. Infarct volume is frequently presented as a percentage of brain volume in pediatric patients, given the range of brain sizes during development.[ 52 ] This variability in infarct size was expected and consistent with the biological variability reported in clinical studies.[ 53 ] Specifically, a study of 31 neonates (64.5% of whom had isolated MCA strokes) demonstrated a wide range of infarct volumes on diffusion-weighted imaging (DWI) scans, with a median infarct volume as a percent of supratentorial brain volume of 6.9% (IQR 2.4–17.2%).[ 54 ] The regions of the brain that were affected by infarction in our model included cortical and subcortical regions of the ipsilateral hemisphere, involving the putamen, globus pallidus, caudate nucleus, and internal capsule.[ 28 ] These regions are associated with motor function, which is consistent with the neuromotor deficits seen in our model. These affected regions were consistent with a previous study using a similar pig MCAO model that demonstrated infarcts in the putamen, globus pallidus, insular cortex, somatosensory cortices, temporal gyri, claustrum, and visual cortices.[ 55 ] While the inherent limitations of TTC staining prevented the exact identification of the structures affected by infarction, more exact localization can be performed in future studies with histological staining or advanced MRI. Additionally, our study showed that dynamic, real-time changes in CBF are demonstrable in our model and are consistent with what is expected in the setting of acute focal cerebral ischemia. LSCI is a non-invasive imaging technique capable of measuring blood flow with high spatial (~ 10 µm) and temporal (10 ms–10 s) resolution.[ 17 ] The mechanism involves analysis of the speckle pattern produced by scattering of coherent light back from moving particles (e.g., erythrocytes); the faster an object moves, the greater the degree of scattering it produces. While this technique has been widely used in adult animal stroke models to assess blood flow activity, its application in neonatal models is novel.[ 56 ], [ 57 ], [ 58 ] In our model, LSCI provided a reliable tool for evaluating the spatial and temporal aspects of CBF in different brain regions, including the ACA, penumbral, and ischemic core regions. The device used in this study was capable of high-resolution, high-speed imaging and micron-sized spatial resolution.[ 59 ] Using this approach, we observed in real-time the effect of MCAO on blood flow to the ipsilateral brain hemisphere. Perfusion to the penumbra and ischemic core regions was significantly reduced immediately after clipping, confirming accurate clip placement. As early as 10 minutes post-MCAO, blood flow to the ischemic core region recovered significantly, and the ischemic core area shrank considerably in this interval. A similar pattern was seen in the penumbra, though to a lesser extent. One possible explanation is the recruitment of pial collateral vessels, or leptomeningeal anastomoses (LMAs), which are small arterial connections between major vascular territories like the ACA and MCA.[ 60 ] Under normal physiological conditions, there is little to no flow through LMAs due to similar flow pressures in the MCA and ACA vasculature. However, during large vessel occlusion, the differential pressure gradient can trigger retrograde flow through LMAs, allowing for preserved perfusion and even gradual reperfusion to ischemic areas.[ 61 ] In humans, optimal LMA function and more LMAs have been associated with smaller infarct volume at 24h post-MCAO, greater benefit from thrombolytic treatment, and improved stroke outcomes in adults.[ 62 ], [ 63 ], [ 64 ] The potential engagement of collateral circulation even 10 minutes post-MCAO, combined with the familiar adage “time is brain,” suggests a possible time window after intra-arterial occlusion where neuroprotective therapies may be effective at limiting infarct progression. However, the structure and function of LMAs in human neonates remain poorly understood. The role of LMAs in stroke pathophysiology and their potential as a therapeutic target merits further research and can be explored with our novel neonatal piglet MCAO model. Third, we demonstrated that our model of NAIS in piglets produces a reproducible and characteristic neuromotor phenotype consistent with what prior studies have shown in animals after MCAO.[ 55 ] Previous studies have shown similar somatotopic organization of the motor cortices between pigs and humans [ 40 ], along with a similar affected MCA territory in permanent craniotomy-based MCAO models.[ 55 ] Therefore, it is expected and appropriate that the impairments in motor performance seen in this model mirror those seen in human neonates.[ 65 ] OFT is commonly used to characterize and quantify behavioral changes after stroke in preclinical research models.[ 22 ], [ 66 ] In our model, piglets that underwent MCAO demonstrated increased ipsiversive circling, which is classically associated with brain injury post-unilateral stroke. This behavior was not observed in sham-operated piglets, supporting the notion that the ischemic brain injury from the MCAO surgery itself and not the craniotomy resulted in these neurological deficits. The similar number of ipsiversive rotations in an open field seen in MCAO piglets between 24h and 48h post-MCAO suggests the presence of a stable neuromotor deficit. These findings are consistent with reports of reduced ambulatory activity and depressive symptoms, such as anhedonia and apathy, in rodents and pigs post-stroke.[ 67 ], [ 68 ] One study in adult pigs demonstrated a significant decrease in exploratory perimeter sniffing at 48h post-stroke.[ 69 ] These findings validate the study approach by suggesting that produced infarcts in this model localize to specific brain structures contributing to characteristic neuromotor outcomes. The lack of significant difference in mean walking speed or distance traveled in an open field was consistent with a previous study in adult pigs that found no significant difference in mean walking speed or distance traveled on OFT before or after MCAO.[ 69 ] However, this same study also demonstrated reduced pressure placement, stride length, and swing phases in the hemiplegic limb, and decreased overall velocity and cadence when piglets were motivated to move at a jog pace versus a walking pace. The self-selected walking pace during OFT in our study likely limited our ability to detect more subtle gait deficits following stroke. A larger open field arena may have enhanced our ability to assess these deficits. A recent systematic review demonstrated that open field arenas used in adult pig studies were typically larger (a median open field area of 9 m 2 vs 0.64m 2 in our study), and that there was not a positive correlation between age, body weight, and open field dimensions.[ 22 ] Additional functional assessments that assess spatiotemporal gait parameters (e.g., velocity, cadence, swing time, stride length, and weight distribution) will be important to integrate into future studies, as gait analysis is frequently used to assess these parameters in patients following stroke.[ 70 ], [ 71 ] Specifically, changes in stride length, walking speed, and weight-bearing distribution are frequently exhibited by patients following stroke and are useful indicators of stroke severity and therapeutic recovery.[ 72 ], [ 73 ], [ 74 ] Changes in appetite and vocalizations are other behavioral outcomes that have been assessed in previous studies.[ 75 ] A number of cognitive tests have also been developed and validated in pigs, although they have thus far been underutilized in the field of stroke research.[ 76 ] Future studies are needed to further categorize neuromotor and neurocognitive outcomes, allowing for a more in-depth interpretation comparing this model to stroke in human neonates. Limitations The present study had several limitations. Firstly, our sample size was limited, although it was selected based on prior pilot and exploratory studies in adult swine models.[ 24 ], [ 26 ], [ 77 ] While this study demonstrated the reproducibility and utility of this model in a small number of animals, studies with a larger sample size are needed to confirm these findings. Additionally, the use of volatile anesthesia must always be considered as a confounder when measuring CBF. To minimize the impact of anesthesia on cerebrovascular reactivity, the dose of inhaled anesthesia was maintained at or below 1 MAC throughout the experimental procedure, which has been shown to preserve cerebral autoregulation.[ 14 ], [ 15 ] Additionally, the change in ICP and brain blood flow dynamics due to the open craniotomy and durotomy may have influenced our results, though replacement of the skull flap immediately following MCAO mitigates this concern. For example, a previous study using a similar craniotomy approach in sheep demonstrated that ICP spontaneously returned to pre-craniotomy levels after dural and bone reconstruction and with CSF reaccumulation, as was performed in our study.[ 78 ] This will be particularly important when performing future studies during which acute cerebral blood flow during MCAO will be studied with an exposed brain. Moreover, the brain retraction and manipulation involved in placing the clips may contribute to tissue damage and swelling unrelated to the actual MCAO. However, we did not note significant brain trauma during necropsy, and no animal in the sham group demonstrated an infarct at 48h. Current and future studies will continue to refine our approach to make it as minimally invasive as possible. Finally, a high degree of technical and surgical expertise is required to perform the procedure, which may limit its broad implementation. CONCLUSION In sum, we have developed a novel model of MCAO using a craniotomy approach in neonatal piglets. This model produced consistent infarctions in the MCA territory and neuromotor deficits that mimic those of human stroke. These characterizations suggest that our model provides a reliable and reproducible platform for studying NAIS in a large animal with brain anatomy and development similar to that of humans. The understanding we obtain of neonatal stroke pathophysiology and CBF dynamics using this model may inform the development and testing of novel therapeutics for neonates, thus helping us improve outcomes for our most vulnerable patients. Declarations Acknowledgments: BioRender was used to create some figures for this manuscript. Funding This study was supported by internal funding from the Department of Anesthesiology and Critical Care Medicine and the Department of Neurosurgery at Johns Hopkins University. Conflicts of interest The authors have no relevant financial or non-financial interests to disclose. Ethics approval All procedures performed in this animal study were in accordance with the ethical standards of the Johns Hopkins University institutional research board. Care was taken to promote the welfare of animals and minimize unnecessary harm and discomfort during this study. Data availability The experimental data generated in this study are available upon reasonable request. Author contributions Conceptualization: Emmett Whitaker, Risheng Xu; Methodology: Qihong Wang, MostafaAbdulrahim, Lisa Young, Larraine Lage, Sanaz Nasoohi, Navid Modiri; Formal analysis and investigation: Qihong Wang, Lisa Young, Saif Ansari; Writing - original draft preparation: Qihong Wang, Mostafa Abdulrahim, Lisa Young; Writing - review and editing: Emmett Whitaker, Risheng Xu, Qihong Wang, Mostafa Abdulrahim, Lisa Young, Larraine Lage, Sanaz Nasoohi, Navid Modiri, George Hong, Saif Ansari; Funding acquisition: Emmett Whitaker, Risheng Xu; Resources: Emmett Whitaker, Risheng Xu; Supervision: Emmett Whitaker, Risheng Xu References L. L. Lehman et al. , “Workup for Perinatal Stroke Does Not Predict Recurrence,” Stroke , vol. 48, no. 8, pp. 2078–2083, Aug. 2017, https://doi.org/ 10.1161/STROKEAHA.117.017356 M. Dunbar, A. Mineyko, M. Hill, J. Hodge, A. Floer, and A. 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Zory, “Variations in kinematics during clinical gait analysis in stroke patients,” PloS One , vol. 8, no. 6, p. e66421, 2013, https://doi.org/10.1371/journal.pone.0066421 L. R. Nascimento, C. Q. De Oliveira, L. Ada, S. M. Michaelsen, and L. F. Teixeira-Salmela, “Walking training with cueing of cadence improves walking speed and stride length after stroke more than walking training alone: a systematic review,” J. Physiother. , vol. 61, no. 1, pp. 10–15, Jan. 2015, https://doi.org/10.1016/j.jphys.2014.11.015 L. Hak, H. Houdijk, P. van der Wurff, M. R. Prins, P. J. Beek, and J. H. van Dieën, “Stride frequency and length adjustment in post-stroke individuals: influence on the margins of stability,” J. Rehabil. Med. , vol. 47, no. 2, pp. 126–132, Feb. 2015, https://doi.org/10.2340/16501977-1903 A. M. De Nunzio et al. , “Biofeedback rehabilitation of posture and weightbearing distribution in stroke: a center of foot pressure analysis,” Funct. Neurol. , vol. 29, no. 2, pp. 127–134, 2014 Y. Tanaka et al. , “Experimental Model of Lacunar Infarction in the Gyrencephalic Brain of the Miniature Pig: Neurological Assessment and Histological, Immunohistochemical, and Physiological Evaluation of Dynamic Corticospinal Tract Deformation,” Stroke , vol. 39, no. 1, pp. 205–212, Jan. 2008, https://doi.org/10.1161/STROKEAHA.107.489906 B. R. Kornum and G. M. Knudsen, “Cognitive testing of pigs (Sus scrofa) in translational biobehavioral research,” Neurosci. Biobehav. Rev. , vol. 35, no. 3, pp. 437–451, Jan. 2011, https://doi.org/10.1016/j.neubiorev.2010.05.004 T. G. N. D. S. Nielsen, N. Dancause, T. A. M. Janjua, F. R. Andreis, B. Kjærgaard, and W. Jensen, “Porcine Model of Cerebral Ischemic Stroke Utilizing Intracortical Recordings for the Continuous Monitoring of the Ischemic Area,” Sensors , vol. 24, no. 10, p. 2967, May 2024, https://doi.org/10.3390/s24102967 A. J. Wells et al. , “A surgical model of permanent and transient middle cerebral artery stroke in the sheep,” PloS One , vol. 7, no. 7, p. e42157, 2012, https://doi.org/10.1371/journal.pone.0042157 Additional Declarations No competing interests reported. Supplementary Files PigletPaperSupplementalFigure.docx Cite Share Download PDF Status: Published Journal Publication published 06 Dec, 2025 Read the published version in Translational Stroke Research → Version 1 posted Editorial decision: Revision requested 06 Oct, 2025 Reviews received at journal 29 Sep, 2025 Reviews received at journal 25 Sep, 2025 Reviews received at journal 23 Sep, 2025 Reviewers agreed at journal 16 Sep, 2025 Reviewers agreed at journal 13 Sep, 2025 Reviews received at journal 20 Aug, 2025 Reviewers agreed at journal 30 Jul, 2025 Reviewers agreed at journal 30 Jul, 2025 Reviewers invited by journal 30 Jul, 2025 Editor assigned by journal 29 Jul, 2025 Submission checks completed at journal 29 Jul, 2025 First submitted to journal 28 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7235675","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":506038386,"identity":"0adb352c-27b2-4ef6-aa65-dee1ee1b6b97","order_by":0,"name":"Qihong Wang","email":"","orcid":"","institution":"Johns Hopkins University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Qihong","middleName":"","lastName":"Wang","suffix":""},{"id":506038387,"identity":"86bf2def-3f85-4f3f-a299-a0a89e9a4e2b","order_by":1,"name":"Mostafa Abdulrahim","email":"","orcid":"","institution":"Johns Hopkins University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Mostafa","middleName":"","lastName":"Abdulrahim","suffix":""},{"id":506038388,"identity":"eb7cdb5f-983f-40ad-bdc3-020d026c6c24","order_by":2,"name":"Lisa Young","email":"","orcid":"","institution":"Johns Hopkins University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Lisa","middleName":"","lastName":"Young","suffix":""},{"id":506038389,"identity":"4db4cf0b-c8cb-4c5d-bba7-4bca591b85fd","order_by":3,"name":"Larraine Lage","email":"","orcid":"","institution":"Johns Hopkins University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Larraine","middleName":"","lastName":"Lage","suffix":""},{"id":506038390,"identity":"64472611-cc8a-42f2-bb30-ca12181f4d08","order_by":4,"name":"Sanaz Nasoohi","email":"","orcid":"","institution":"Johns Hopkins University School of 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Medicine","correspondingAuthor":false,"prefix":"","firstName":"Saif","middleName":"","lastName":"Ansari","suffix":""},{"id":506038394,"identity":"24c97b15-a8ff-4d32-9691-0cfb227e6bd6","order_by":8,"name":"Risheng Xu","email":"","orcid":"","institution":"Johns Hopkins University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Risheng","middleName":"","lastName":"Xu","suffix":""},{"id":506038395,"identity":"4734ea09-b5d1-4162-8e42-13eecd16ac7d","order_by":9,"name":"Emmett Whitaker","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAtUlEQVRIiWNgGAWjYPACG2YDBsYGBgY2YjUcSEgjXcthBgMwixgt/GKnEx9//HGe3VzscAPDh7LDhLVIzs7dbHAg4Taz5ezEBsYZ54jQYnA7d5sESIvB7cQGZt42IrTY387d/uNAwjmIlr/EaDGQzt0G9P4BiBZGYrRI3M7dLHEmLRms5WDPuXTCWvhn5278UGFjl2xwO/3hgx9l1oS1wEAyiDhAvHogsCNJ9SgYBaNgFIwsAABEsD/HR5O6ggAAAABJRU5ErkJggg==","orcid":"","institution":"Johns Hopkins University School of Medicine","correspondingAuthor":true,"prefix":"","firstName":"Emmett","middleName":"","lastName":"Whitaker","suffix":""}],"badges":[],"createdAt":"2025-07-28 15:53:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7235675/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7235675/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s12975-025-01392-8","type":"published","date":"2025-12-06T15:57:59+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":90284369,"identity":"9ac57af2-24f0-4fa5-ad63-19fceac50c3b","added_by":"auto","created_at":"2025-09-01 05:52:28","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":394116,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMCAO surgery in neonatal piglet\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Marking of the midline skin incision\u003c/p\u003e\n\u003cp\u003e(b) Craniotomy with intact dura exposed\u003c/p\u003e\n\u003cp\u003e(c) Durotomy with intact brain underneath\u003c/p\u003e\n\u003cp\u003e(d) Location of burr holes on the piglet skull in relation to sagittal suture (SS) and orbital rim (OR)\u003c/p\u003e\n\u003cp\u003e(e) Location of aneurysm clip placement at the origin of the middle cerebral artery (MCA) branches in the circle of Willis (CoW) in relation to the anterior cerebral artery (ACA)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAbbreviations\u003c/strong\u003e: ACA, Anterior communicating artery; MCA, Middle cerebral artery; MCAO, Middle cerebral artery occlusion; SS, sagittal suture; OR, orbital rim; CoW, Circle of Willis.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7235675/v1/94a693f0982e5241f0b0d318.png"},{"id":90284379,"identity":"9dba430e-643b-4eb7-bc5f-09d7487fa97c","added_by":"auto","created_at":"2025-09-01 05:52:28","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":289459,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLSCI output\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRepresentative anatomical, grayscale, and pseudo-color LSCI images depicting cortical cerebral blood flow in one piglet’s brain at baseline (before MCAO surgery), from left to right. The color map to the right provides a reference scale from high to low flow, with warmer colors (red-orange) indicating regions of higher perfusion, while cooler colors (blue) indicating lower perfusion.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAbbreviations\u003c/strong\u003e: LSCI, laser speckle contrast imaging; P, posterior; A, anterior; M, medial; L, lateral\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7235675/v1/8a1c8c408b034592be14c457.png"},{"id":90284382,"identity":"d74c3687-a39d-4dc5-952d-5049232133d4","added_by":"auto","created_at":"2025-09-01 05:52:28","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":233067,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRepresentative LSCI images and CBF reduction maps of one piglet’s brain at baseline, immediately post-MCAO, and 10 minutes post-MCAO\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTop panel: Representative gray-scale LSCI images that were obtained at post-MCAO time points.\u003c/p\u003e\n\u003cp\u003eBottom panel: Representative CBF reduction maps that were created by calculating the percent difference in LSCI intensity (relative to pre-MCAO baseline) for each pixel at each time point post-MCAO. Ischemic core (blue) was defined as a 57-100% CBF decrease, penumbra (green) was defined as a 19-55% decrease, and ACA (yellow) was defined as less than 17% decrease from baseline based on rCBF thresholds defined in prior literature.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAbbreviations: \u003c/strong\u003eLSCI, laser speckle contrast imaging; CBF, cerebral blood flow; MCAO, middle cerebral artery occlusion; ACA, anterior cerebral artery, rCBF, relative cerebral blood flow\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7235675/v1/7c32fd404ff76cf9e6c2038f.png"},{"id":90284380,"identity":"1a2c4f6f-d404-4647-a6ad-3011a59e5de3","added_by":"auto","created_at":"2025-09-01 05:52:28","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":97727,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical representation of changes in rCBF (measured by LSCI blood flow index) compared to baseline in three cortical ROIs in seven neonatal piglets with MCAO\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBy 10 minutes post-MCAO, there was a statistically significant increase of ~22.4% in perfusion of the ischemic core region but no statistically significant changes in perfusion of the penumbral or ACA region. \u003cem\u003ep-\u003c/em\u003evalues: \u003cem\u003e** = \u003c/em\u003e\u0026lt;0.01, * = \u0026lt;0.05\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAbbreviations\u003c/strong\u003e: rCBF, relative cerebral blood flow; LSCI, laser speckle contrast imaging; ACA, anterior cerebral artery; MCAO, middle cerebral artery occlusion; ROIs, regions of interest\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7235675/v1/4ce76add4c0e9a77f1e3420e.png"},{"id":90284370,"identity":"7d6826dc-2932-4405-b449-fdcee853d076","added_by":"auto","created_at":"2025-09-01 05:52:28","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":76891,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical representation of changes in relative area of cortical ROIs as a percentage of the ipsilateral hemisphere area in seven neonatal piglets with MCAO\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe area of the ACA, penumbral, and ischemic core regions on LSCI images (relative to the total area of the ipsilateral hemisphere) was assessed immediately post-MCAO and 10 minutes post-MCAO. By 10 minutes post-MCAO, there was an average decrease in the relative area of the core by ~38%, and an average increase in the relative area of the penumbra and ACA territory by 19.9% and 12.6%, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAbbreviations\u003c/strong\u003e: ACA, anterior cerebral artery; MCAO, middle cerebral artery occlusion; ROIs, regions of interest; LSCI, laser speckle contrast imaging\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7235675/v1/02b3a49e8f6194c38900dc04.png"},{"id":90284383,"identity":"a66ccb71-4a79-41ec-a316-bbc3bb138a6f","added_by":"auto","created_at":"2025-09-01 05:52:28","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":265829,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInfarct volume in MCAO (n=7) vs sham piglets (n=6)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Representative TTC images are shown for piglet brains at 48 hours post-surgery in sham and MCAO group. TTC staining was used to delineate infarcted (white) versus non-infarcted (red) tissue. Increased hemispheric swelling and infarct-related damage was seen in the MCAO group compared to the sham group.\u003c/p\u003e\n\u003cp\u003e(b) The average infarct volume corrected for edema was 31.6±6.4% of hemisphere volume in the MCAO group and 0% in the sham group. (**, \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAbbreviation\u003c/strong\u003e: 2,3,5-triphenyl tetrazolium chloride, TTC\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7235675/v1/49318822f528bff9bf1381f3.png"},{"id":90285358,"identity":"8f725ebc-ae4b-4ad9-a9ee-55e3fbaca2b5","added_by":"auto","created_at":"2025-09-01 06:00:29","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":155492,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNeurobehavioral outcomes in subset of MCAO (n=3) vs sham piglets (n=4)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Representative movement trace in an open field test (OFT) of a representative MCAO and sham piglet before surgery, 24h post-surgery, and 48h post-surgery. Constrained movement and circular rotations are observed more frequently in the MCAO piglet’s movement trace.\u003c/p\u003e\n\u003cp\u003e(b) Graphical representation of number of ipsiversive rotations (turns toward the side of the stroke lesion) in MCAO and sham piglets. There was a statistically significant increase in ipsiversive rotations in the MCAO group at 48h post-MCAO compared to baseline and compared to the sham group (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05). There was no significant difference in the number of ipsiversive rotations between 24h and 48h post-MCAO, suggesting the presence of a stable motor deficit. There were no significant differences in mean walking speed (meters per second) or mean walking distance (meters) between MCAO and sham groups at any time point.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7235675/v1/8ad42f96e13a4a7295e36ff5.png"},{"id":97723923,"identity":"087666a8-a7dc-4dd1-a685-e8d9b1b81917","added_by":"auto","created_at":"2025-12-08 16:09:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2736066,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7235675/v1/728ae3ee-396f-4cd8-87f2-8dcc1a347137.pdf"},{"id":90284368,"identity":"c7bac4da-8247-49c9-bff8-d65f21125cdd","added_by":"auto","created_at":"2025-09-01 05:52:28","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":148207,"visible":true,"origin":"","legend":"","description":"","filename":"PigletPaperSupplementalFigure.docx","url":"https://assets-eu.researchsquare.com/files/rs-7235675/v1/53471d4ea19297b7b9e1ae6a.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Spatial and Temporal Assessment of Cerebral Blood Flow in a Novel Piglet Model of Neonatal Arterial Ischemic Stroke","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eNeonatal arterial ischemic stroke (NAIS) refers to infarction resulting from arterial large vessel occlusion in the first 28 days of life. NAIS accounts for approximately 80% of perinatal stroke cases, encompassing subtypes such as cerebral venous sinus thrombosis and hemorrhagic stroke.[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] The incidence of NAIS is estimated to be approximately 1:3,000 live births, although this is likely an underestimation given the vagaries in clinical diagnosis and limitations in magnetic resonance imaging (MRI) in low-resource settings.[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] Infants with NAIS rarely die, but generally have poor long-term outcomes and often require lifelong specialized care.[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] Long-term deficits remain in two-thirds of patients and may include hemiparetic cerebral palsy, cognitive and behavioral dysfunction, speech delay, spasticity, and epilepsy.[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] Despite its prevalence and health impact, management of NAIS is typically supportive.[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] Treatment options are particularly limited during the acute phase of ischemia, during which treatment is most likely to have a meaningful effect. The only Food and Drug Administration (FDA)-approved therapies used in adult ischemic stroke, thrombolytics and mechanical thrombectomy, are not typically used in neonates due to safety concerns and technical difficulties.[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] Moreover, the limited therapeutic time window for these therapies (\u0026lt;\u0026thinsp;4.5h for thrombolytics and \u0026lt;\u0026thinsp;24h for thrombectomy) and the associated risk of intracranial hemorrhage exclude a large population of stroke patients. The lack of safe, effective therapies that decrease stroke severity and improve neurological outcomes in neonates is a significant knowledge gap, which, until it is addressed, will limit our ability to improve the lives of children with NAIS.\u003c/p\u003e\u003cp\u003eEstablishing a preclinical model that can replicate NAIS is key to expanding our understanding of the pathophysiology of this disease and advancing the development of novel therapies that can be rapidly translated to human trials. A workshop on perinatal and childhood stroke held by the National Institute of Neurological Disorders and Stroke in 2000 emphasized the importance of increasing the implementation of clinically relevant animal models for these conditions.[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] While many stroke studies use rodent models, the anatomical and structural differences between rodent and human brains (e.g., disparities in brain size, gray/white matter ratio, and lissencephalic versus gyrencephalic morphology) have limited their translational relevance.[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] Further, the small size of neonatal rodents has made measurements of vital parameters (such as blood pressure) and cerebral blood flow (CBF) challenging. Pigs are a more clinically relevant and less ethically controversial stroke model given their cerebrovascular and neurodevelopmental similarities to human neonates, such as gyrencephalic organization and comparable brain size and gray/white matter ratio.[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/p\u003e\u003cp\u003eHere, we present a model of NAIS that creates a reproducible infarct via craniotomy and middle cerebral artery occlusion (MCAO) and allows for reliable measurement of CBF and vital parameters. By enabling a better understanding of CBF dynamics during ischemia, this model may help identify interventions that can reduce brain injury and improve outcomes for children affected by NAIS.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e\u003cb\u003eAnimals\u003c/b\u003e\u003c/p\u003e\u003cp\u003e All procedures were approved by the Animal Care and Use Committee at Johns Hopkins University. Care and handling of animals were performed by an experienced veterinary technician in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. Fourteen healthy Yorkshire piglets (3\u0026ndash;7 days old, 1.5-2.5kg, Archer Farms, Darlington, MD, USA) were used in this study. Animals were housed in a temperature-controlled (26.7\u0026ndash;32.2\u0026deg;C, humidity 14\u0026ndash;30%) space with 12-hour light-dark cycles and had access to milk replacer (NurseAll Milk Replacer, Manna Pro Products, Chesterfield, MO, USA) and water ad libitum. Enrichment was provided with daily human interaction and toys. Animals were co-housed to prevent separation stress except during the acute period while recovering from surgery or during behavioral testing.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSurgery\u003c/b\u003e\u003c/p\u003e\u003cp\u003eMilk replacer was withheld for four hours immediately before anesthesia and surgery. On the day of surgery, animals were brought to a large animal operating room suite, and anesthesia was induced using 5% isoflurane or 8% sevoflurane (depending on anesthetic availability; Piramal Critical Care Inc, Northampton County, PA, USA) in 100% O\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\u003cp\u003eOnce an adequate depth of anesthesia was reached, the laryngeal folds were sprayed with 2% lidocaine to prevent laryngospasm during intubation. The trachea was then orally intubated with a 2.5mm cuffed endotracheal tube. Mechanical ventilation was instituted to maintain normoxia (SpO\u003csub\u003e2\u003c/sub\u003e 90\u0026ndash;100%, pO\u003csub\u003e2\u003c/sub\u003e 90\u0026ndash;120 mmHg) and normocarbia (EtCO\u003csub\u003e2\u003c/sub\u003e/pCO\u003csub\u003e2\u003c/sub\u003e 35\u0026ndash;45 mmHg). The anesthetic dose was then decreased to 1-1.25% isoflurane or 2-2.5% sevoflurane and subsequently titrated to clinical effect (targeting the minimum effective dose). Doses at or below 1 minimum alveolar concentration (MAC) have been shown to have minimal effects on cerebral autoregulation.[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] A 26-gauge peripheral intravenous (IV) catheter was placed in the marginal ear vein for the administration of lactated Ringer\u0026rsquo;s solution and medications. A one-time dose of enrofloxacin (7.5mg/kg, IV) and extended-release buprenorphine (0.12 mg/kg/dose, subcutaneous) were given prior to the surgical incision. A single dose of rocuronium 10 mg was given following successful intubation to prevent movement during craniotomy and brain manipulation. A femoral arterial catheter was placed to provide continuous blood pressure monitoring. Additional monitoring included end-tidal carbon dioxide (EtCO\u003csub\u003e2\u003c/sub\u003e) and end-tidal anesthetic concentration, electrocardiography, heart rate, oxygen saturation (SpO\u003csub\u003e2\u003c/sub\u003e), and esophageal temperature, which were continuously recorded using LabChart 8.1.30 software (AD Instruments, Colorado Springs, CO, USA). Arterial blood gases (ABG) were measured at least hourly throughout the procedure (ABL800 FLEX blood gas analyzer, Radiometer America, Brea, CA, USA). If needed, pO\u003csub\u003e2\u003c/sub\u003e and pCO\u003csub\u003e2\u003c/sub\u003e were corrected by titrating ventilator settings to optimize oxygenation and ventilation. Body temperature was maintained with a water heating blanket and a forced-air warmer (Thermacare\u0026reg; TC3001 Convective Warming System, Gaymar, Orchard Park, NY, USA). Lactated Ringer\u0026rsquo;s solution was administered intravenously at a maintenance dose (4 mL/kg/hour) throughout the procedure.\u003c/p\u003e\u003cp\u003eThe skin was prepared in the usual sterile fashion. Prior to all incisions, subcutaneous infiltration of 0.25% bupivacaine (maximum total volume 1 mL/kg) was performed. To perform the MCAO surgery, the scalp was shaved, a 6.5 cm midline incision was made in the scalp, and the skin was retracted laterally. A curette was used to remove fascia from the skull, and a pediatric perforator (Stryker, Kalamazoo, MI, USA) was used to drill six small burr holes into the skull. To ensure consistency, the burr holes were drilled in standardized locations relative to bregma in each animal. Two parasagittal holes were drilled 2.2 cm anterior to bregma; one was placed 0.5 cm lateral to the sagittal sinus, and the other was positioned above the orbit, 1.5 cm lateral to the sagittal sinus. An additional two holes were drilled 1.2 cm posterior to bregma; one was 0.5 cm lateral to the sagittal sinus, and the other was 2.2 cm lateral to the sinus. Two more holes were drilled between the anterior and posterior parasagittal and lateral holes. A craniotome was then used to connect the holes and create a craniotomy. The skull flap was then removed with careful attention to avoid injury to the dura and placed in sterile saline. Under a surgical microscope, the dura was incised and retracted. Cerebrospinal fluid (CSF) was aspirated to obtain a clear field of view. The brain hemisphere was gently retracted, and the MCA(s) were exposed and identified on the ventral surface of the brain. Of note, pigs are known to have multiple MCAs.[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] Two 7 mm aneurysm clips (YASARGIL\u0026reg; Aneurysm Clips, B. Braun Medical Inc., Bethlehem, PA, USA) were placed on all visible MCA branches at their origin from the internal carotid artery (ICA). These steps are pictured in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAfter the clips were placed and laser speckle contrast imaging (LSCI) was performed (described in the next section), the dura was repositioned over the brain, and the bone flap was replaced and sutured to the skull. The skin was closed with a running suture technique with a 5\u0026thinsp;\u0026minus;\u0026thinsp;0 Vicryl suture. For sham surgeries, the piglets underwent the same steps; however, after retracting the brain and identifying the MCA branches, no aneurysm clips were placed. A second dose of extended-release buprenorphine (0.12 mg/kg/dose, IV) was given for pain control, the neuromuscular blockade was reversed with sugammadex (4mg/kg, IV), and the animal was extubated. The same veterinary technician who cared for the animal pre-surgery observed the animal during the immediate postoperative period for any surgical or anesthetic complications. During the 48h postoperative period, vital signs were monitored daily, and standard release buprenorphine (0.01\u0026ndash;0.02 mg/kg, IV) was given every 8 hours for analgesia.\u003c/p\u003e\u003cp\u003e\u003cb\u003eLSCI acquisition\u003c/b\u003e\u003c/p\u003e\u003cp\u003eLaser speckle contrast imaging (LSCI) was performed using an RFLSI-ZW (RWD, Shenzhen, Guangdong, China) LSCI device. LSCI is a commonly used imaging modality with high spatial and temporal resolution used to observe and record cerebral perfusion of exposed brain tissue before and after MCAO.[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] With the animal in the prone position, the LSCI camera was positioned 15cm from the exposed brain hemisphere and focused to obtain a high-resolution image. Imaging parameters were kept consistent at 0-5000 perfusion unit blood monitoring perfusion range, image acquisition speed of 50 frames per second, camera pixel/resolution 2048\u0026times;2048, a spatial resolution of image\u0026thinsp;\u0026gt;\u0026thinsp;3\u0026micro;m/pixel, effective pixels per unit area\u0026thinsp;\u0026gt;\u0026thinsp;5,000,000 pixels/cm\u0026sup2;, and optical multiplication ratio 12:1. Five-second recordings were acquired at baseline (immediately before MCA clipping), immediately post-MCAO, and 10 minutes post-MCAO to verify the presence of successful MCAO. The average flux signal was calculated for each 5-second recording period, producing three images (gross anatomical, pseudo-color, and grayscale) at each time point \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eLSCI analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eLSCI images were analyzed in FIJI software (Schindelin et al., 2012; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://fiji.sc\u003c/span\u003e\u003cspan address=\"https://fiji.sc\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e)\u003c/span\u003e to quantify changes in relative CBF (rCBF) following MCAO surgery.[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] For each animal, all LSCI images taken during the procedure were calibrated to a consistent reference scale, inverted, and converted to a 32-bit format. The StackReg plugin was used to align all images to the baseline image taken before MCAOs.[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] Mean pixel intensity values were used as a proxy for blood flow. Pixel-by-pixel CBF reduction maps were generated at each post-MCAO time point using the formula: rCBF reduction (%)\u0026thinsp;=\u0026thinsp;100 * (baseline perfusion \u0026ndash; post-MCAO perfusion) / baseline perfusion. Three regions of interest (ROI) were identified by the following thresholds for perfusion reduction from baseline, which were based on previously described thresholds: ischemic core (57\u0026ndash;100% reduction from baseline), penumbra (19\u0026ndash;55% reduction from baseline), and ACA territory (\u0026lt;\u0026thinsp;17% reduction from baseline).[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] The small gap (~\u0026thinsp;2%) between the threshold ranges was deliberate to establish conservative cutoff definitions. At each post-MCAO time point, the area of each ROI was measured and expressed as a percentage of the total area of the exposed ipsilateral hemisphere \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo assess temporal changes in CBF in the core ischemic region, penumbra, and the ACA territory, these ROIs were defined on the CBF reduction map at the immediately post-MCAO time point for each animal. These ROIs were then copied to the CBF reduction drop map at the 10-minute post-MCAO time point. To correct for slight variations in animal brain positioning between time points, the ROIs were adjusted accordingly using blood vessel landmarks. The mean intensity values in each of the three ROIs at 10-minute post-MCAO time points were then measured and reported as a percentage decrease from baseline.\u003c/p\u003e\u003cp\u003e\u003cb\u003eBehavioral testing\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo assess neuromotor deficits, we performed an Open Field Test (OFT) in a subset of MCAO piglets (n\u0026thinsp;=\u0026thinsp;3) and sham piglets (n\u0026thinsp;=\u0026thinsp;4) as previously described.[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] The test area was a 0.8 x 0.8-meter square arena enclosed by a fence. OFT was performed in a quiet, temperature-controlled room with no other piglets present. The piglet\u0026rsquo;s movements were recorded with a video camera and analyzed by an investigator blinded to groups (MCAO versus sham) using ANY-maze software (Stoelting Co, Wood Dale, IL, USA) to assess its locomotor activity. OFTs were performed at the beginning of the day, at the same time before the first feeding, to prevent animal drowsiness. To perform the test, piglets were placed into the arena for 10 minutes to acclimate to the environment, and their movements were video recorded for 12 minutes. OFTs were performed to qualitatively and quantitatively assess neuromotor behavior one day before surgery (baseline), at 24 hours post-MCAO, and 48 hours post-MCAO.\u003c/p\u003e\u003cp\u003e\u003cb\u003eTTC staining and infarct volume quantification\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAt 48 hours post-MCAO, piglets were euthanized with a lethal dose of sodium pentobarbital/phenytoin (0.5 mL/kg, Euthasol). The brains of the piglets were removed and placed in ice-cold 0.1M phosphate-buffered saline (PBS). After the brains were checked for accuracy of MCA aneurysm clip placement \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e, the clips were removed. Brains were placed in a tissue sectioning matrix custom-designed for neonatal piglets and covered with warm low-melt agarose to prevent tissue movement during sectioning. Coronal sectioning began at the front of the anterior pole, and 4-mm-thick coronal sections were produced, working from anterior to posterior. Freshly sliced brain sections were incubated in 1% 2,3,5-triphenyl tetrazolium chloride (TTC, Sigma Aldrich) at 37\u0026deg;C for 30 minutes to delineate infarcted (white) versus non-infarcted (red) tissue. Following staining, sections were washed three times with ice-cold 0.1M PBS. Once washed, sections were fixed overnight at 4\u0026deg;C in 4% paraformaldehyde. After 24 hours, sections were moved to 0.1M PBS for storage at 4\u0026deg;C. Anterior and posterior faces of coronal sections were then scanned and digitized to identify infarct volume.\u003c/p\u003e\u003cp\u003eAnalysis of TTC-stained images was performed using FIJI software by a blinded investigator to maintain consistency across the study cohort. First, the image was calibrated to distance using a ruler as a reference. Then, the boundaries of the contralateral hemisphere and the non-infarcted tissue of the ipsilateral hemisphere were manually outlined using the free-draw tool. The area of these regions was measured, and the process was repeated for all brain slices on the anterior and posterior faces. These infarct areas were multiplied by the slice thickness (4 mm) to calculate total infarct volumes. The Swanson method was used to correct for edema by calculating the percent difference between the contralateral hemisphere volume and that of the non-infarcted region of the ipsilateral hemisphere.[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] The infarct volume was expressed as a percentage of the ipsilateral brain hemisphere volume of each animal.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eA total sample size of 14 animals (n\u0026thinsp;=\u0026thinsp;8 in MCAO group, n\u0026thinsp;=\u0026thinsp;6 in sham group) was selected based on prior pilot and exploratory studies in adult swine models.[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] The sample size in the sham group was lowered in accordance with the 3R principles of reduction, refinement, and replacement in animal research, and previous literature supporting a minimal infarct size in sham animals in invasive swine models.[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/p\u003e\u003cp\u003eMAP and HR data were extracted from a 5-second average every 5 minutes for the period from 15 minutes prior to clipping (or pseudo-clipping for sham piglets) and 30 minutes following (pseudo)-clipping. Since each animal had a different MAP at the start of the experiment, MAP was presented as a percentage difference from baseline to demonstrate the change in MAP before and after MCAO. The percent change in MAP from baseline was calculated, with the baseline being measured at 15 minutes prior to clipping. This data was analyzed using mixed-effects analysis followed by the Tukey test, with comparisons made between experimental groups at each time point and within each group at each time point. OFT data were analyzed by mixed-effects analysis followed by the Tukey test to compare the number of ipsiversive rotations, walking distance, and mean speed between experimental groups at each time point, and within each group at each time point.\u003c/p\u003e\u003cp\u003eIn the MCAO group, ROI area and perfusion data were compared at each time point using repeated measures ANOVA. Each animal served as its own pre-MCAO control in statistical calculations. Unpaired t-tests were used to compare ABG parameters (pH, pCO\u003csub\u003e2\u003c/sub\u003e, and pO\u003csub\u003e2\u003c/sub\u003e) between the MCAO and sham groups at the shared time point (hour 1 of the surgery). In the MCAO group, a paired t-test was used to assess changes between hour 1 and hour 2. In the sham group, where only 4 animals had available blood gas measurements at hour 2 due to two animals having a\u0026thinsp;\u0026lt;\u0026thinsp;2h total sham surgery length, a paired t-test was conducted only on animals with complete data (n\u0026thinsp;=\u0026thinsp;4). Infarct volumes between the two groups were also compared with an unpaired t-test.\u003c/p\u003e\u003cp\u003eStatistical analyses were performed using GraphPad Prism software (GraphPad Software, San Diego, CA, USA). Unless otherwise stated, data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error (SEM). The threshold for statistical significance was set at * \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and ** \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01.\u003c/p\u003e\u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003eFourteen neonatal piglets were randomly assigned to two surgery groups (MCAO, n\u0026thinsp;=\u0026thinsp;8; sham, n\u0026thinsp;=\u0026thinsp;6). On average, the piglets in the MCAO group weighed 2.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 kg, and those in the sham group weighed 2.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 kg; there was no statistical difference between these two groups. No piglet died during or immediately after surgery, but one piglet in the MCAO group was euthanized early at 24 hours due to severe neurological deficits and periods of non-responsiveness. On necropsy, the animal was found to have an extensive infarct lesion due to inadvertent clipping of the circle of Willis. This piglet was excluded from data analyses.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePhysiological Parameters\u003c/b\u003e\u003c/p\u003e\u003cp\u003eArterial blood gases obtained during the surgery are listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. pH, pCO\u003csub\u003e2\u003c/sub\u003e, and pO\u003csub\u003e2\u003c/sub\u003e were stable throughout the experimental period, and there were no significant differences between groups in any blood gas parameter at the shared time point (hour 1), nor between hour 1 and hour 2 within either group. ABG values were not recorded for one animal in the MCAO group, but the animal was maintained at ventilation parameters consistent with other animals during the procedure. There was no significant difference in heart rate or the percent change in MAP from baseline between MCAO and sham groups prior to and following clip placement, nor within each group at any time point \u003cb\u003e(Online Resource 1)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eIntraoperative arterial blood gas (ABG) recordings\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTime\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHour 1\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eHour 2\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003ep-\u003c/em\u003evalue (within group)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003epH, units\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMCAO\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.10\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSham\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.53\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003ep\u003c/em\u003e-value (MCAO vs Sham)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003epCO\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e, \u003cb\u003emmHg\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMCAO\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e37.72\u0026thinsp;\u0026plusmn;\u0026thinsp;4.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e42.00\u0026thinsp;\u0026plusmn;\u0026thinsp;3.48\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.14\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSham\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e38.35\u0026thinsp;\u0026plusmn;\u0026thinsp;3.43\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e46.53\u0026thinsp;\u0026plusmn;\u0026thinsp;6.92*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.59\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003ep\u003c/em\u003e-value (MCAO vs Sham)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.91\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003epO\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e, \u003cb\u003emmHg\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMCAO\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e167.33\u0026thinsp;\u0026plusmn;\u0026thinsp;17.64\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e174.83\u0026thinsp;\u0026plusmn;\u0026thinsp;20.68\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.52\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSham\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e183.67\u0026thinsp;\u0026plusmn;\u0026thinsp;28.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e164.25\u0026thinsp;\u0026plusmn;\u0026thinsp;12.57*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.42\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003ep\u003c/em\u003e-value (MCAO vs Sham)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.63\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"4\"\u003eArterial blood gases obtained during the MCAO and sham surgeries at hour 1 and hour 2 time points. n\u0026thinsp;=\u0026thinsp;6 for all groups and time points except where indicated with asterisk (*), as the sham group had only 4 animals with available blood gas measurements at hour 2 due to a\u0026thinsp;\u0026lt;\u0026thinsp;2h total sham surgery length. The values represent mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe entire MCAO surgery lasted 3-3.5 hours and was well-tolerated by piglets. All piglets survived the first 24 hours post-surgery, and 7 of 8 piglets in the MCAO group survived to 48 hours post-surgery.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCortical blood flow and infarct volumes in the MCAO group\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSuccessful occlusion of the MCAs was achieved in all piglets in the MCAO group, as assessed by LSCI immediately after the MCAs were occluded. As mentioned above, one piglet in the MCAO group was excluded due to technical difficulties placing the clip and associated damage to the brain. At the 10-minute post-MCAO time point, rCBF to the ischemic core region showed a statistically significant 22.4% recovery compared to the immediate post-MCAO time point (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. While not statistically significant, rCBF to the penumbra region showed a 16.8% increase at the 10-minute post-MCAO time point compared to the immediate post-MCAO time point.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eCompared to the immediate post-MCAO time point, there was an expansion of the ACA and penumbral regions as a percentage of the ipsilateral hemisphere area at the 10-minute post-MCAO timepoint (12.6% and 19.9% respectively) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. At the 10-minute post-MCAO timepoint, the relative area of the ischemic core region showed a 38% decrease (compared to the immediate post-MCAO time point) to 33.9\u0026thinsp;\u0026plusmn;\u0026thinsp;11% of the ipsilateral hemisphere area.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAnalysis of TTC-stained coronal brain slices in MCAO piglets showed edematous changes and tissue damage consistent with infarction in cortical and subcortical regions of the ipsilateral hemisphere, which were not present in sham piglets \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e. A piglet brain atlas was used to identify infarcted regions, which included the caudate nucleus, putamen, globus pallidus, anterior commissure, and internal capsule, among others.[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] The mean infarct volume was 31.6\u0026thinsp;\u0026plusmn;\u0026thinsp;6.4% of the ipsilateral hemisphere volume at 48 hours post-MCAO, which was significantly higher than the sham group (0%) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb\u003cb\u003e).\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eBehavioral testing in MCAO vs sham groups\u003c/b\u003e\u003c/p\u003e\u003cp\u003eA small subset of animals (n\u0026thinsp;=\u0026thinsp;7 total piglets) underwent pre-surgical (baseline) OFT. Analysis of ANY-maze movement tracings showed random patterns associated with normal ambulation and exploratory behaviors \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e. Sham piglets (n\u0026thinsp;=\u0026thinsp;4) showed minimal ambulatory changes on OFT at 24- and 48-hour time points after surgery. In contrast, piglets in the MCAO group (n\u0026thinsp;=\u0026thinsp;3) showed considerable ambulatory deficits, including significantly increased ipsiversive circling (turns toward the side of the stroke lesion) at 48h post-MCAO compared to baseline (16\u0026thinsp;\u0026plusmn;\u0026thinsp;4 vs 7.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e. There was no statistically significant difference between the number of ipsiversive rotations at 24- and 48-hours post-MCAO for either group. No significant differences were demonstrated in mean walking distance or speed between either group at any time point. However, the use of a small OFT arena makes this finding difficult to interpret.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe overall goal of the present study was to assess this novel approach to MCAO as a tool to perform translational NAIS research that may advance the understanding of neonatal stroke pathophysiology and allow the testing of potential therapeutics to improve outcomes for affected neonates. The experiments described herein yielded several key descriptive findings that contribute to this field.\u003c/p\u003e\u003cp\u003eFirstly, our approach to producing MCAO in neonatal piglets is feasible and, while invasive, effective in producing a stroke with expected biological variability in lesion size in a higher mammal with a gyrencephalic brain. Recently, there has been growing interest in adjuvant neuroprotective therapies to improve outcomes for affected neonates, given the considerable morbidity and the ineligibility of this population for thrombolytics or thrombectomy. While rodents remain the most popular stroke model, they have major limitations due to rodent brains having a lissencephalic brain organization that differs from the gyrencephalic organization of humans, being considerably smaller than human brains, having a lower white matter proportion in the cortex (10\u0026ndash;12% versus 40\u0026ndash;45% in humans), and demonstrating limited ICP changes during stroke.[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] The disparity in white matter composition is important to consider, given the differences in metabolic demands and susceptibility to ischemia between gray and white matter.[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] The limitations in these rodent studies have likely contributed to the lack of success of experimental stroke therapies in clinical trials, with the only two FDA-approved drugs being tissue plasminogen activator (alteplase) and tenecteplase.[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] To address this translational gap, the Stroke Academic Industry Roundtable (STAIR) and Stem Cell Therapies as an Emerging Paradigm in Stroke (STEPS) committees have recommended using large animals with human-like brain structures for therapeutic testing, in conjunction with rodent studies.[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] These models have been established in a number of large animals, including sheep, dogs, pigs, and non-human primates (NHPs).[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] Dogs and NHPs are generally controversial due to societal and ethical implications. Pigs are a model with relatively low ethical controversy and high translational relevance given the similarities they share with humans regarding brain gyrencephalic organization, white matter composition in the cortex (28.4% versus 40\u0026ndash;45% in humans), size, and development.[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] These models have proven helpful in testing therapies such as induced pluripotent stem cell-derived neural stem cells [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] and neural stem cell extracellular vesicles.[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] However, these therapies have only been studied in adult pigs to date. Thus, our model of stroke in neonatal pigs not only adds to the body of scientific literature but also has the potential to bridge the gap between preclinical research and clinical trials.\u003c/p\u003e\u003cp\u003eThe MCA is the most frequently affected large artery in human NAIS, making it an important site to target in preclinical models.[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] Stroke models in rodents typically use an endovascular filament to mechanically occlude the MCA at its origin from the circle of Willis.[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e] This approach is not possible in the porcine brain due to the presence of a complex cerebral vascular anatomy, typically consisting of two or even three MCAs.[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] In addition, the presence of the rete mirabile (RM) that is present between the carotid artery and the circle of Willis makes a direct filament-based, endovascular approach impossible. The RM (Latin for \u0026ldquo;wonderful net\u0026rdquo;) is a complex network of small anastomosing blood vessels proximal to the internal carotid artery (ICA) in swine and does not exist in humans or rodents.[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] The location of the RM between the extracranial and intracranial vasculature and its winding tortuosity are major impediments to catheter or filament navigation. Recent studies have attempted to overcome this obstacle by using a liquid embolic to occlude the RM wing up to the origin of the MCAs or by injecting thrombin proximally to the RM.[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] However, these endovascular stroke models have significant morbidity and mortality, with the former having a 33% mortality rate within the first 24 hours after stroke induction [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] and the latter having a 28% mortality rate within the same time period.[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] This high mortality rate is likely due to cerebral edema and swelling, which leads to rapid neurological deterioration. In contrast, despite the invasive nature of our approach, there was no mortality in any animal in which the MCAs were properly clipped. This more closely replicates the mortality rate of neonatal stroke, which was estimated to be approximately 3% in a summary review.[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e] This is likely due to the presence of open fontanelles in neonates with ischemic stroke, which has a similar effect to a craniotomy in allowing them to better tolerate elevated ICPs. In addition, endovascular embolization approaches introduce a foreign substance that likely has direct effects on vascular physiology. Finally, these approaches are irreversible, which makes studying reperfusion impossible. The ability to study reperfusion is critical in NAIS because most neonates who have a stroke spontaneously recanalize.[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e] The use of removable aneurysm clips in our model will allow the study of the effects of reperfusion in NAIS after varying periods of ischemia, potentially offering insights into therapies that can improve NAIS outcomes when administered in the acute ischemic period or after reperfusion.\u003c/p\u003e\u003cp\u003eIn our model, the actual procedure was relatively short, lasting 3-3.5 hours in total. In all cases, the time from the start of the craniotomy to clipping was less than an hour. There was no significant difference in MAP or heart rate after MCAO compared to baseline, suggesting that this surgical approach does not significantly impact hemodynamics. Of note, the mean heart rate in the MCAO group did increase slightly from around 180 beats per minute (bpm) before MCAO to around 200 bpm after MCAO; however, this change is unlikely to be clinically significant and is consistent with vital parameter changes that have been reported previously in piglets undergoing surgeries.[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e] Although our study used a craniotomy approach to expose the MCAs, another approach that has been studied in adult swine involves enucleation of the eyeball (i.e., transorbital approach), followed by either permanent (i.e., ligation or electrocoagulation) or transient (i.e., clipping) occlusion.[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e] Advantages of using the craniotomy approach over the transorbital approach include the ability to perform behavioral testing (given the intact eye and preserved binocular vision and depth perception), to perform direct measurements of tissue oxygenation, and to perform direct visualization and assessment of microvascular flow (through LSCI, for example).[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e] Our model allows investigation of all of these factors, thus potentially lending key insights into long-term functional outcomes and time-dependent changes in ischemic injury and rCBF following neonatal stroke.\u003c/p\u003e\u003cp\u003eThe stroke lesions in our model varied in size, with a mean infarct volume of 31.6\u0026thinsp;\u0026plusmn;\u0026thinsp;6.4% of hemisphere volume. Two animals had infarct volumes of less than 16%. Infarct volume is frequently presented as a percentage of brain volume in pediatric patients, given the range of brain sizes during development.[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e] This variability in infarct size was expected and consistent with the biological variability reported in clinical studies.[\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e] Specifically, a study of 31 neonates (64.5% of whom had isolated MCA strokes) demonstrated a wide range of infarct volumes on diffusion-weighted imaging (DWI) scans, with a median infarct volume as a percent of supratentorial brain volume of 6.9% (IQR 2.4\u0026ndash;17.2%).[\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e] The regions of the brain that were affected by infarction in our model included cortical and subcortical regions of the ipsilateral hemisphere, involving the putamen, globus pallidus, caudate nucleus, and internal capsule.[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] These regions are associated with motor function, which is consistent with the neuromotor deficits seen in our model. These affected regions were consistent with a previous study using a similar pig MCAO model that demonstrated infarcts in the putamen, globus pallidus, insular cortex, somatosensory cortices, temporal gyri, claustrum, and visual cortices.[\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e] While the inherent limitations of TTC staining prevented the exact identification of the structures affected by infarction, more exact localization can be performed in future studies with histological staining or advanced MRI.\u003c/p\u003e\u003cp\u003eAdditionally, our study showed that dynamic, real-time changes in CBF are demonstrable in our model and are consistent with what is expected in the setting of acute focal cerebral ischemia. LSCI is a non-invasive imaging technique capable of measuring blood flow with high spatial (~\u0026thinsp;10 \u0026micro;m) and temporal (10 ms\u0026ndash;10 s) resolution.[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] The mechanism involves analysis of the speckle pattern produced by scattering of coherent light back from moving particles (e.g., erythrocytes); the faster an object moves, the greater the degree of scattering it produces. While this technique has been widely used in adult animal stroke models to assess blood flow activity, its application in neonatal models is novel.[\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e], [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e], [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e] In our model, LSCI provided a reliable tool for evaluating the spatial and temporal aspects of CBF in different brain regions, including the ACA, penumbral, and ischemic core regions. The device used in this study was capable of high-resolution, high-speed imaging and micron-sized spatial resolution.[\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e] Using this approach, we observed in real-time the effect of MCAO on blood flow to the ipsilateral brain hemisphere. Perfusion to the penumbra and ischemic core regions was significantly reduced immediately after clipping, confirming accurate clip placement. As early as 10 minutes post-MCAO, blood flow to the ischemic core region recovered significantly, and the ischemic core area shrank considerably in this interval. A similar pattern was seen in the penumbra, though to a lesser extent. One possible explanation is the recruitment of pial collateral vessels, or leptomeningeal anastomoses (LMAs), which are small arterial connections between major vascular territories like the ACA and MCA.[\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e] Under normal physiological conditions, there is little to no flow through LMAs due to similar flow pressures in the MCA and ACA vasculature. However, during large vessel occlusion, the differential pressure gradient can trigger retrograde flow through LMAs, allowing for preserved perfusion and even gradual reperfusion to ischemic areas.[\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e] In humans, optimal LMA function and more LMAs have been associated with smaller infarct volume at 24h post-MCAO, greater benefit from thrombolytic treatment, and improved stroke outcomes in adults.[\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e], [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e], [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e] The potential engagement of collateral circulation even 10 minutes post-MCAO, combined with the familiar adage \u0026ldquo;time is brain,\u0026rdquo; suggests a possible time window after intra-arterial occlusion where neuroprotective therapies may be effective at limiting infarct progression. However, the structure and function of LMAs in human neonates remain poorly understood. The role of LMAs in stroke pathophysiology and their potential as a therapeutic target merits further research and can be explored with our novel neonatal piglet MCAO model.\u003c/p\u003e\u003cp\u003eThird, we demonstrated that our model of NAIS in piglets produces a reproducible and characteristic neuromotor phenotype consistent with what prior studies have shown in animals after MCAO.[\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e] Previous studies have shown similar somatotopic organization of the motor cortices between pigs and humans [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], along with a similar affected MCA territory in permanent craniotomy-based MCAO models.[\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e] Therefore, it is expected and appropriate that the impairments in motor performance seen in this model mirror those seen in human neonates.[\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e] OFT is commonly used to characterize and quantify behavioral changes after stroke in preclinical research models.[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e] In our model, piglets that underwent MCAO demonstrated increased ipsiversive circling, which is classically associated with brain injury post-unilateral stroke. This behavior was not observed in sham-operated piglets, supporting the notion that the ischemic brain injury from the MCAO surgery itself and not the craniotomy resulted in these neurological deficits. The similar number of ipsiversive rotations in an open field seen in MCAO piglets between 24h and 48h post-MCAO suggests the presence of a stable neuromotor deficit. These findings are consistent with reports of reduced ambulatory activity and depressive symptoms, such as anhedonia and apathy, in rodents and pigs post-stroke.[\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e], [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e] One study in adult pigs demonstrated a significant decrease in exploratory perimeter sniffing at 48h post-stroke.[\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e] These findings validate the study approach by suggesting that produced infarcts in this model localize to specific brain structures contributing to characteristic neuromotor outcomes.\u003c/p\u003e\u003cp\u003eThe lack of significant difference in mean walking speed or distance traveled in an open field was consistent with a previous study in adult pigs that found no significant difference in mean walking speed or distance traveled on OFT before or after MCAO.[\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e] However, this same study also demonstrated reduced pressure placement, stride length, and swing phases in the hemiplegic limb, and decreased overall velocity and cadence when piglets were motivated to move at a jog pace versus a walking pace. The self-selected walking pace during OFT in our study likely limited our ability to detect more subtle gait deficits following stroke. A larger open field arena may have enhanced our ability to assess these deficits. A recent systematic review demonstrated that open field arenas used in adult pig studies were typically larger (a median open field area of 9 m\u003csup\u003e2\u003c/sup\u003e vs 0.64m\u003csup\u003e2\u003c/sup\u003e in our study), and that there was not a positive correlation between age, body weight, and open field dimensions.[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] Additional functional assessments that assess spatiotemporal gait parameters (e.g., velocity, cadence, swing time, stride length, and weight distribution) will be important to integrate into future studies, as gait analysis is frequently used to assess these parameters in patients following stroke.[\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e], [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e] Specifically, changes in stride length, walking speed, and weight-bearing distribution are frequently exhibited by patients following stroke and are useful indicators of stroke severity and therapeutic recovery.[\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e], [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e], [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e] Changes in appetite and vocalizations are other behavioral outcomes that have been assessed in previous studies.[\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e] A number of cognitive tests have also been developed and validated in pigs, although they have thus far been underutilized in the field of stroke research.[\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e] Future studies are needed to further categorize neuromotor and neurocognitive outcomes, allowing for a more in-depth interpretation comparing this model to stroke in human neonates.\u003c/p\u003e\u003cp\u003e\u003cb\u003eLimitations\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe present study had several limitations. Firstly, our sample size was limited, although it was selected based on prior pilot and exploratory studies in adult swine models.[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], [\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e] While this study demonstrated the reproducibility and utility of this model in a small number of animals, studies with a larger sample size are needed to confirm these findings. Additionally, the use of volatile anesthesia must always be considered as a confounder when measuring CBF. To minimize the impact of anesthesia on cerebrovascular reactivity, the dose of inhaled anesthesia was maintained at or below 1 MAC throughout the experimental procedure, which has been shown to preserve cerebral autoregulation.[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] Additionally, the change in ICP and brain blood flow dynamics due to the open craniotomy and durotomy may have influenced our results, though replacement of the skull flap immediately following MCAO mitigates this concern. For example, a previous study using a similar craniotomy approach in sheep demonstrated that ICP spontaneously returned to pre-craniotomy levels after dural and bone reconstruction and with CSF reaccumulation, as was performed in our study.[\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e] This will be particularly important when performing future studies during which acute cerebral blood flow during MCAO will be studied with an exposed brain. Moreover, the brain retraction and manipulation involved in placing the clips may contribute to tissue damage and swelling unrelated to the actual MCAO. However, we did not note significant brain trauma during necropsy, and no animal in the sham group demonstrated an infarct at 48h. Current and future studies will continue to refine our approach to make it as minimally invasive as possible. Finally, a high degree of technical and surgical expertise is required to perform the procedure, which may limit its broad implementation.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eIn sum, we have developed a novel model of MCAO using a craniotomy approach in neonatal piglets. This model produced consistent infarctions in the MCA territory and neuromotor deficits that mimic those of human stroke. These characterizations suggest that our model provides a reliable and reproducible platform for studying NAIS in a large animal with brain anatomy and development similar to that of humans. The understanding we obtain of neonatal stroke pathophysiology and CBF dynamics using this model may inform the development and testing of novel therapeutics for neonates, thus helping us improve outcomes for our most vulnerable patients.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u0026nbsp;\u003c/strong\u003eBioRender was used to create some figures for this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by internal funding from the Department of Anesthesiology and Critical Care Medicine and the Department of Neurosurgery at Johns Hopkins University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll procedures performed in this animal study were in accordance with the ethical standards of the Johns Hopkins University institutional research board. Care was taken to promote the welfare of animals and minimize unnecessary harm and discomfort during this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experimental data generated in this study are available upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: Emmett Whitaker, Risheng Xu; Methodology: Qihong Wang, MostafaAbdulrahim, Lisa Young, Larraine Lage, Sanaz Nasoohi, Navid Modiri; Formal analysis and investigation: Qihong Wang, Lisa Young, Saif Ansari; Writing - original draft preparation: Qihong Wang, Mostafa Abdulrahim, Lisa Young; Writing - review and editing: Emmett Whitaker, Risheng Xu, Qihong Wang, Mostafa Abdulrahim, Lisa Young, Larraine Lage, Sanaz Nasoohi, Navid Modiri, George Hong, Saif Ansari; Funding acquisition: Emmett Whitaker, Risheng Xu; Resources: Emmett Whitaker, Risheng Xu; Supervision: Emmett Whitaker, Risheng Xu\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eL. 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Knudsen, \u0026ldquo;Cognitive testing of pigs (Sus scrofa) in translational biobehavioral research,\u0026rdquo; \u003cem\u003eNeurosci. Biobehav. Rev.\u003c/em\u003e, vol. 35, no. 3, pp. 437\u0026ndash;451, Jan. 2011, https://doi.org/10.1016/j.neubiorev.2010.05.004\u003c/li\u003e\n \u003cli\u003eT. G. N. D. S. Nielsen, N. Dancause, T. A. M. Janjua, F. R. Andreis, B. Kj\u0026aelig;rgaard, and W. Jensen, \u0026ldquo;Porcine Model of Cerebral Ischemic Stroke Utilizing Intracortical Recordings for the Continuous Monitoring of the Ischemic Area,\u0026rdquo; \u003cem\u003eSensors\u003c/em\u003e, vol. 24, no. 10, p. 2967, May 2024, https://doi.org/10.3390/s24102967\u003c/li\u003e\n \u003cli\u003eA. J. Wells \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;A surgical model of permanent and transient middle cerebral artery stroke in the sheep,\u0026rdquo; \u003cem\u003ePloS One\u003c/em\u003e, vol. 7, no. 7, p. e42157, 2012, https://doi.org/10.1371/journal.pone.0042157\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":true,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"translational-stroke-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"trsr","sideBox":"Learn more about [Translational Stroke Research](http://jcmr-online.biomedcentral.com)","snPcode":"12975","submissionUrl":"https://submission.nature.com/new-submission/12975/3","title":"Translational Stroke Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Neonatal arterial ischemic stroke, cerebral blood flow, middle cerebral artery occlusion, ischemia, stroke, laser speckle contrast imaging","lastPublishedDoi":"10.21203/rs.3.rs-7235675/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7235675/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNeonatal arterial ischemic stroke (NAIS) is associated with considerable pediatric morbidity and mortality but lacks effective treatment options compared to adult stroke, highlighting the need for clinically relevant translational models. This study aimed to develop a novel middle cerebral artery occlusion (MCAO) model in neonatal piglets with exceptional clinical relevance and the opportunity for long-term survival. Piglets were randomly assigned to undergo either MCAO (n=8) or sham surgery (n=6). MCAO was achieved by occluding MCAs using 7mm aneurysm clips via craniotomy. Laser speckle contrast imaging was used to measure changes in relative cerebral blood flow (rCBF) in three cortical regions (anterior cerebral artery territory, penumbra, and ischemic core). Open field testing was performed in a subset of piglets at baseline, 24h post-MCAO, and 48h post-MCAO. 2,3,5-triphenyl tetrazolium chloride (TTC) staining was used to identify infarcts at 48h post-MCAO. By 10 minutes post-MCAO, the rCBF had risen approximately 22.4% in the ischemic core compared to immediately post-MCAO (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05), with the area of the core as a percentage of the ipsilateral hemisphere decreasing by 38%. Furthermore, MCAO piglets showed increased ipsiversive circling at 48h post-MCAO (16±4 vs 7.3±1.4 ipsiversive rotations, \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05) compared to baseline and higher infarct volumes compared to sham piglets (31.6±6.4%, \u003cem\u003ep\u003c/em\u003e\u0026lt;0.01). Overall, our model creates a reproducible infarct with consistent neuromotor deficits, real-time assessment of rCBF dynamics, and long-term survival, thus offering insights that may inform the development of novel therapies and improve NAIS outcomes.\u003c/p\u003e","manuscriptTitle":"Spatial and Temporal Assessment of Cerebral Blood Flow in a Novel Piglet Model of Neonatal Arterial Ischemic Stroke","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-01 05:52:22","doi":"10.21203/rs.3.rs-7235675/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-06T14:19:46+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-29T18:49:28+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-25T16:47:59+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-23T14:18:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"127720769493868388383386460236304293696","date":"2025-09-16T16:42:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"274007356632660568613079284986146319926","date":"2025-09-13T15:59:04+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-20T16:45:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"174755684773745960379498743908882711557","date":"2025-07-30T13:05:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"316124687957023809458103203881397760566","date":"2025-07-30T11:43:21+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-30T11:36:31+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-30T02:06:38+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-29T08:15:27+00:00","index":"","fulltext":""},{"type":"submitted","content":"Translational Stroke Research","date":"2025-07-28T15:44:47+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"translational-stroke-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"trsr","sideBox":"Learn more about [Translational Stroke Research](http://jcmr-online.biomedcentral.com)","snPcode":"12975","submissionUrl":"https://submission.nature.com/new-submission/12975/3","title":"Translational Stroke Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"4f2bac48-4055-44b3-8405-453195533a31","owner":[],"postedDate":"September 1st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-08T16:02:44+00:00","versionOfRecord":{"articleIdentity":"rs-7235675","link":"https://doi.org/10.1007/s12975-025-01392-8","journal":{"identity":"translational-stroke-research","isVorOnly":false,"title":"Translational Stroke Research"},"publishedOn":"2025-12-06 15:57:59","publishedOnDateReadable":"December 6th, 2025"},"versionCreatedAt":"2025-09-01 05:52:22","video":"","vorDoi":"10.1007/s12975-025-01392-8","vorDoiUrl":"https://doi.org/10.1007/s12975-025-01392-8","workflowStages":[]},"version":"v1","identity":"rs-7235675","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7235675","identity":"rs-7235675","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}