Human iNPC therapy leads to improvement in functional...

来自 : 发布时间：2024-05-11

Brain and BehaviorVolume 8, Issue 5 e00972 ORIGINAL RESEARCH Open Access Human iNPC therapy leads to improvement in functional neurologic outcomes in a pig ischemic stroke model Vivian W. Lau, orcid.org/0000-0002-5843-1837 Regenerative Bioscience Center, University of Georgia, Athens, GA, USA Department of Animal and Dairy Science, University of Georgia, Athens, GA, USA Department of Small Animal Medicine and Surgery, University of Georgia, Athens, GA, USASearch for more papers by this authorSimon R. Platt, Regenerative Bioscience Center, University of Georgia, Athens, GA, USA Department of Small Animal Medicine and Surgery, University of Georgia, Athens, GA, USASearch for more papers by this authorHarrison E. Grace, Regenerative Bioscience Center, University of Georgia, Athens, GA, USA Department of Animal and Dairy Science, University of Georgia, Athens, GA, USASearch for more papers by this authorEmily W. Baker, Regenerative Bioscience Center, University of Georgia, Athens, GA, USA Department of Animal and Dairy Science, University of Georgia, Athens, GA, USASearch for more papers by this authorFranklin D. West, Corresponding Author westf@uga.edu Regenerative Bioscience Center, University of Georgia, Athens, GA, USA Department of Animal and Dairy Science, University of Georgia, Athens, GA, USA Correspondence Franklin D. West, Regenerative Bioscience Center, Rhodes Center for Animal and Dairy Science, 425 River Road, Room 316, University of Georgia, Athens, GA, USA. Email: westf@uga.eduSearch for more papers by this author Vivian W. Lau, orcid.org/0000-0002-5843-1837 Regenerative Bioscience Center, University of Georgia, Athens, GA, USA Department of Animal and Dairy Science, University of Georgia, Athens, GA, USA Department of Small Animal Medicine and Surgery, University of Georgia, Athens, GA, USASearch for more papers by this authorSimon R. Platt, Regenerative Bioscience Center, University of Georgia, Athens, GA, USA Department of Small Animal Medicine and Surgery, University of Georgia, Athens, GA, USASearch for more papers by this authorHarrison E. Grace, Regenerative Bioscience Center, University of Georgia, Athens, GA, USA Department of Animal and Dairy Science, University of Georgia, Athens, GA, USASearch for more papers by this authorEmily W. Baker, Regenerative Bioscience Center, University of Georgia, Athens, GA, USA Department of Animal and Dairy Science, University of Georgia, Athens, GA, USASearch for more papers by this authorFranklin D. West, Corresponding Author westf@uga.edu Regenerative Bioscience Center, University of Georgia, Athens, GA, USA Department of Animal and Dairy Science, University of Georgia, Athens, GA, USA Correspondence Franklin D. West, Regenerative Bioscience Center, Rhodes Center for Animal and Dairy Science, 425 River Road, Room 316, University of Georgia, Athens, GA, USA. Email: westf@uga.eduSearch for more papers by this author Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URLShare a linkShare onEmailFacebookTwitterLinked InRedditWechat Abstract Introduction Stroke is the leading cause of disability in the United States but current therapies are limited with no regenerative potential. Previous translational failures have highlighted the need for large animal models of ischemic stroke and for improved assessments of functional outcomes. The aims of this study were first, to create a post-stroke functional outcome assessment scale in a porcine model of middle cerebral artery occlusion (MCAO) and second, to use this scale to determine the effect of human-induced-pluripotent-cell-derived neural progenitor cells (iNPCs) on functional outcome in this large animal stroke model. Materials and Methods Eight 6-month-old Landrace mix pigs underwent permanent MCAO. Five days following MCAO, pigs received intraparenchymal injections of either iNPCs or PBS. A post-stroke assessment scale was developed to measure functional outcome. Evaluations were performed at least 1–3days prior to MCAO and repeated 1day, 3days, and 5days post-stroke as well as 1day, 3days, 1week, 2weeks, 4weeks, 6weeks, 9weeks, and 12weeks post-injection. Comparisons of scores between animals receiving iNPCs or PBS only were compared using a two-way ANOVA and a Tukey\'s post-hoc t test.Results The developed scale was able to consistently determine differences between healthy and stroked pigs at all time points. iNPC-treated pigs showed a significantly faster recovery in their overall scores relative to PBS-only treated pigs with the parameters of appetite and body posture exhibiting the most improvement in the iNPC-treated group. Conclusions We developed a robust and repeatable functional assessment tool that can reliably detect stroke and recovery, while also showing for the first time that iNPC therapy leads to functional recovery in a translational pig ischemic stroke model. These promising results suggest that iNPCs may 1day serve as a first in class cell therapeutic for ischemic stroke. 1 INTRODUCTION Stroke is the leading cause of disability in the United States and the second leading cause of death in the world (Mozaffarian etal., 2015). Despite considerable efforts, the vast majority of laboratory-developed stroke therapies have failed to translate into clinically effective treatments (Albers etal., 2011; Saver, Jovin, Smith, & Albers, 2013). Current Food and Drug Administration (FDA)-approved stroke therapies are limited to tissue plasminogen activator (tPA) and a limited number of stent-retriever devices (Lu etal., 2014). These therapies are only effective in patients during the acute phase of injury and do not carry any regenerative potential for damaged tissues (Savitz etal., 2011). Induced-pluripotent-stem-cell-derived neural progenitor cells (iNPCs) have recently garnered significant interest as a personalized regenerative cell therapy for stroke (Hyun, 2010; Savitz etal., 2011). iNPCs can potentially be derived from the patient\'s own body, thus eliminating the potential of rejection upon transplantation (Araki etal., 2013; Guha, Morgan, Mostoslavsky, Rodrigues, & Boyd, 2013). Several groups have already demonstrated beneficial effects of iNPCs in rodent models of ischemic stroke (Chang etal., 2013; Gomi etal., 2012; Jensen, Yan, Krishnaney-Davison, Al Sawaf, & Zhang, 2013; Oki etal., 2012; Polentes etal., 2012; Tatarishvili, 2014; Tornero etal., 2013). iNPCs grafts implanted into rodents after middle cerebral artery occlusion (MCAO) were able to survive for up to 5months with no evidence of tumorigenesis (Oki etal., 2012; Tornero etal., 2013). In addition, rodents receiving iNPCs following MCAO demonstrated improved functional recovery on various tests including the sticky tape/adhesive removal test, staircase test, rotarod test, and the modified neurologic severity score (mNSS) (Chang etal., 2013; Eckert etal., 2015; Gomi etal., 2012; Oki etal., 2012; Tornero etal., 2013). While iPSC-derived therapies hold great promise in rodent stroke models, previous translational failures between rodents and humans indicate the need for further evaluation of this therapy before proceeding to clinical trials. The repeated failures in translation between the laboratory and the clinical setting prompted the development of the stroke therapy academic and industry roundtable (STAIR) recommendations (Albers etal., 2011; Fisher etal., 2009; Roundtable, 1999; Saver etal., 2013). One of the recommendations included pre-clinical testing of developed therapies in multiple animal models, preferably with inclusion of a gyrencephalic species (Roundtable, 1999). To address this need, our research group recently developed a gyrencephalic pig model of permanent right middle cerebral artery (MCA) ischemic stroke (Platt etal., 2014). Repeatable and reliable structural lesions in the pig model were demonstrated with both magnetic resonance imaging (MRI) and histology; however, functional outcome assessment was only limited to gait analysis (Duberstein etal., 2014). Another STAIR roundtable recommendation was to report the effect of proposed therapies on the acute and long-term functional outcomes of tested animal models (Fisher etal., 2009). The failure of many stroke therapy clinical trials can partially be blamed on a lack of long-term functional outcome evaluation in the preclinical animal model (Corbett & Nurse, 1998; Hunter, Mackay, & Rogers, 1998; Hunter etal., 2000; Roundtable, 1999). In humans, functional recovery can be thought of in terms of various neurologic domains: motor, sensory, vision, affection, cognition, and language (Kelly-Hayes etal., 1998). Following a stroke, these domains are assessed using a variety of scales and indices including the modified Rankin Score (mRS), American Heart Association Stroke Outcome Classification Score (AHA.SOC), Barthel index (BI), NIH Stroke Scale (NIHSS), the Glasgow Outcome Scale (GOS), and the Canadian Neurologic Scale (CNS) (Heiss & Kidwell, 2014). The goal of these evaluations is to determine the degree of neurologic recovery and function through quantification of patient neurologic deficits, quality of life, and functional independence in activities of daily living (Heiss & Kidwell, 2014). The importance of these scales is most evident in the context of randomized clinical trials, most of which use the mRS or BI as the standard to dichotomize good from poor outcomes (Weisscher, Vermeulen, Roos, & de Haan, 2008). The vast majority of ischemic strokes in humans involve the MCA, and several well-established rodent models of MCAO exist (Bederson etal., 1986; Markgraf etal., 1992; Zausinger, Hungerhuber, Baethmann, Reulen, & Schmid-Elsaesser, 2000). In MCAO, affected brain regions usually include large areas of the sensorimotor cortex and basal nuclei (Bederson etal., 1986; Modo etal., 2000; Ringelstein etal., 1992). Humans affected by MCAO can develop symptoms including, but not limited to, aphasia, hemiparesis, hemineglect, dysphagia, impaired cognition, and urinary/fecal incontinence (Banks & Marotta, 2007). Many of the criteria and scoring parameters assess language, emotion, and cognition; however, they do not translate well into non-human species. As such, several animal-specific scales and tests have been designed to assess similar representative outcomes in laboratory species (Corbett & Nurse, 1998; Hunter etal., 2000; Janssen etal., 2013). These tests have been designed to assess learning, memory, motor skills, and asymmetrical neurologic deficits (Modo etal., 2000). Functional outcome assessments in rodent models of stroke are extremely variable and a gold standard does not exist (Corbett & Nurse, 1998; Hunter etal., 1998, 2000). Many studies employ a composite scoring system (Bederson Scale or mNSS) and/or perform a multitude of specific tests (e.g., Rotarod, Morris Water Maze) and grade each individually (Corbett & Nurse, 1998; Encarnacion etal., 2011; Hunter etal., 1998; Schaar, Brenneman, & Savitz, 2010). One of the earliest composite neurologic grading systems is the Bederson Scale (Bederson etal., 1986; Hunter etal., 1998). A large number of grading systems are based on this scale, which uses limb placement and circling as measurements of sensorimotor deficits (Bederson etal., 1986; Longa, Weinstein, Carlson, & Cummins, 1989; Schaar etal., 2010; Zausinger etal., 2000). Another popular composite score scale is the mNSS, which takes into account sensorimotor function, reflex, and balance tests (Schaar etal., 2010). However, there is currently no commonly performed composite scoring system for the pig stroke model. In our recent study, MRI results demonstrated that iNPC-treated stroked pigs had improved white matter integrity, brain metabolism, and cerebral blood volume in the ipsilateral stroked hemisphere and histological results showed improved neuron survival, increased neurogenesis, and decreased immune response relative non-treated control stroked pigs (Baker etal., 2017). However, the question remained if these tissue and cellular level changes lead to functional recovery. Utilizing behavioral data collected from these same animals, the purpose of this study was to create a robust and repeatable post-stroke assessment scale for stroke pigs. In this study, we demonstrated that this scale is able to consistently determine differences between non-stroked and stroked animals with high repeatability within and between multiple assessors. In addition, we showed that iNPC treatment lead to improved recovery in postural reactions, body posture, mental status, and appetite in a pig ischemic stroke model. All procedures were conducted under guidelines approved by the University of Georgia Institutional Animal Care and Use Committee. Eight male castrated 6-month-old Landrace pigs were used in the study. All pigs were obtained from the University of Georgia Swine unit and weighed between 65 and 80kg at the start of the study and between 110 and 130kg at the end of 12weeks. Pigs were sedated with an intramuscular injection containing xylazine (5mg/kg), ketamine (5mg/kg), midazolam (0.2mg/kg), and butorphanol (0.2mg/kg). All pigs were intubated with a cuffed endotracheal tube and maintained under gas anesthesia with administration of 1–2% isoflurane and oxygen. Mechanical ventilation was performed at a rate of 8–12breaths/min using a tidal volume of 5–10ml/kg throughout anesthesia. Intravenous fluids (Lactated Ringers Solution) were administered at a rate of 5–10ml/kg throughout anesthesia. Further analgesia was provided with intramuscular injections of flunixin meglumine (Banamine®-S; Merck Animal Health Cat# 01142) 2.2mg/kg administered 30min prior to surgery and every 24hr, thereafter for 3days postoperatively. Ceftiofur sodium (Naxcel®; Zoetis Cat# 009071) 4.4mg/kg was administered intramuscularly at least 30min prior to surgery. Doses were repeated every 24hr for 3days following surgery. The surgical technique used to create a permanent right MCAO has been described in detail previously (Platt etal., 2014). Briefly, a right frontotemporal craniectomy and orbital rim ostectomy with partial zygomatic arch resection was performed on each animal. Bipolar cautery was used to permanently occlude the right MCA and some of its collateral branches near its origin. A 2cm×2cm piece of porcine urinary bladder mucosa (ACell Vet™; Cat# 07-834-9561) was placed over the craniectomy defect prior to closure. Twenty-four hours following induction of right MCAO, each pig underwent MRI evaluation of the brain using a Siemens 16-channel fixed-site 1.5T MRI system. Pigs were sedated with xylazine (5mg/kg), midazolam (0.2mg/kg), and butorphanol (0.2mg/kg) administered intramuscularly. All pigs were intubated with cuffed endotracheal tubes and maintained on 1–2% isoflurane throughout the MRI procedure. Peripheral intravenous catheters (18–22g) were placed in the left or right auricular vein and in the left or right accessory cephalic veins for administration of Lactated Ringers Solution at a rate of 5–10ml/hr during anesthesia. T1-weighted, T2-weighted, T2 fluid attenuated inversion recovery (FLAIR), and diffusion-weighted imaging (DWI) were performed in sagittal, transverse, and dorsal planes. Apparent diffusion coefficient (ADC) maps were generated from the DWI sequence. HIP™ human neural stem cells (ThermorFisher Scientific Cat# GSC-4311, RRID:CVCL_RX99; hereafter \"iNPC”) were expanded on growth factor reduced Matrigel™ (Corning™ Cat# 356231) diluted 1:100 with Neurobasal Medium (ThermoFisher Scientific Cat# 21103049) and kept under a passage number of 20. Daily media changes were performed using Neurobasal media (ThermoFisher Scientific) supplemented with 2% B-27 Supplement (ThermoFisher Scientific Cat# 17504044), 1% non-essential amino acids (ThermoFisher Scientific, catalog number 11140050), 2mmol/L l-glutamine (ThermoFisher Scientific Cat# 25030081), 1% penicillin/streptomycin (ThermoFisher Scientific Cat# 15140122), and 20ng/ml basic fibroblast growth factor (bFGF) (R and D systems Cat# 233-FG-025). Upon confluence, cells were enzymatically suspended for passage using Accutase (Innovative Cell Technologies Cat# AT104) and replated at a density of 1:4. iNPC were plated onto Matrigel-coated four-chambered glass slides for immunocytochemistry. Cells were fixed with 4% paraformaldehyde (Electron Microscopy Sciences) for 15min and permeabilized with 0.1% Triton X-100, 1% polyvinylpyrrolidone (PVP; Sigma-Aldrich) in a 3% serum blocking solution. Cells were incubated with primary antibodies diluted in blocking solution for 1hr at room temperature. Primary antibodies used were Nestin (Neuromics Cat# MO15012-100, RRID:AB_2148919; dilution 1:200) and Sox1 (R and D Systems Cat# AF3369, RRID:AB_2239879; dilution 1:20). Secondary antibodies used were Donkey anti-Goat IgG (H+L) Alexa Fluor 488 (ThermoFisher Scientific Cat# A-11055, RRID:AB_2534102; dilution 1:1000), and Goat anti-Mouse IgG (H+L) Alexa Fluor 594 (ThermoFisher Scientific Cat# A-11032, RRID:AB_2534091; dilution 1:1000). Secondary antibodies were applied for 1hr at room temperature to detect primary antibodies. Cells were washed and mounted with Prolong Gold with DAPI (ThermoFisher Scientific Cat# P36935) and imaged using SlideBook software version 4.1.0 (Intelligent Imaging innovations) on an Olympus IX-81 microscope with Disc-Spinning Unit (Olympus, Inc.). For all eight animals that underwent MCAO, the surgical site was reopened 5days post-stroke for either a PBS-only injection or an iNPC injection. Four pigs were assigned to each treatment group to receive either a PBS-only (non-treated) or an iNPC injection (iNPC-treated). Pigs were anesthetized using the same protocol as described previously and the surgical site reopened via the same incision. Tissues were gently bluntly dissected to the level of the prior craniectomy and the previously placed porcine urinary bladder mucosa (ACell Vet™) was removed to expose the brain. For cell injections, the iNPCs were suspended in PBS at a concentration of 150,000cells/μl. Two injections of 33.3μl of the cell solution were administered through a glass syringe (Hamilton Co.) and 24-g needle using a micro-injector set to deliver the volume at a rate of 2μl/min. Injections were administered at least 5mm apart in the penumbra region of the stroke (as determined through 24-hr post-stroke MRI evaluation for each individual pig) at a depth of 6mm from the surface of the brain at the junction of cortical gray and white matter. The needle was retracted at a rate of 1mm/min following injection to prevent backflow of cells. For PBS-only treatments, two injections of 33.3μl of sterile PBS were injected in lieu of the cell solution in an identical manner. Following injections, a 2cm×2cm piece of porcine urinary bladder mucosa (ACell Vet™) was placed over the craniectomy site prior to closure. All pigs were treated with flunixin meglumine (Banamine®-S) 2.2mg/kg administered 30min prior to injection surgery and every 24hr, thereafter for 3days postoperatively. Ceftiofur sodium (Naxcel®) 4.4mg/kg was administered intramuscularly at least 30min prior to surgery with doses repeated every 24hr for 3days following surgery. A porcine post-stroke neurologic assessment scale was created to include evaluation of individual parameters such as mentation, posture, gait, postural reactions, cranial nerves, appetite, and circling (Table1). This scale was based on previously published post-stroke clinical assessment scales in both pigs and dogs (Tanaka etal., 2008; Yamaguchi, Zhou, Heistad, Watanabe, & Zhang, 2004). Postural reactions were assessed by shifting the animal\'s weight over the center of balance for each individual limb through steady pressure applied by the assessor on the contralateral side of the animal. This was meant to mimic hopping tests performed in veterinary neurologic exams in dogs to assess conscious and unconscious proprioception. The maximum score associated with the highest degree of neurologic deficits was set at 30 and a normal neurologic exam score was set as zero. Table 1. The post-stroke assessment scale. Individual parameters were scored out of a range of 2–6. The highest possible score for animals most severely affected is 30 with normal or pre-stroke animals scoring 0. More points were allotted to parameters that would have a more serious consequence for the pig such as appetite, gait, and mental status, whereas parameters such as head posture were allotted fewer points. Cranial nerves were given a higher allotment of points to incorporate the significance of brainstem deficits as assessed in coma-scores in the acute stroke patient Non ambulatory with paralysis of any of the limbs and loss of nociception (note limbs) Any of the above deficits with slow pupillary light reflexes and normal to reduced oculocephalic reflexes Pinpoint pupils with reduced to absent oculocephalic reflexes±pathologic/spontaneous nystagmus Unilateral or bilateral unresponsive mydriasis with reduced to absent oculocephalic reflexes Evaluations were performed at least 1–3days prior to induction of stroke and repeated 1day, 3days, and 5days post-stroke as well as 1day, 3days, 1week, 2weeks, 4weeks, 6weeks, 9weeks, and 12weeks post-injection. All examinations were physically performed by one individual (VL) and filmed with a digital camera (Canon Powershot D10). Filmed examinations were later viewed and scored by a non-blinded observer who was aware of the time points at which pigs were evaluated. All filmed examinations were viewed and scored again by the same non-blinded observer at a later time. Filmed exams were then relabeled and placed in random order for evaluation by an observer blinded to the treatment group and time points of each examination and to the first observer\'s scores. Only results from the scoring of the blinded observer were used in the final analysis to determine significance between iNPC-treated and non-treated pigs. All statistical analysis was performed using sas version 9.3 (SAS Institute, Inc). A two-way ANOVA and a Tukey\'s post-hoc t test were used to compare results between treatment groups and between the first and second assessments of the non-blinded observer and the blinded observer. A p value .05 was used to determine significance between groups. iNPCs were observed to have a normal monolayer growth pattern with individual cells showing large cell bodies and extended processes consistent with neural progenitor cell morphology (Figure1a). Immunocytochemistry results showed iNPCs co-expressed the neural progenitor markers SOX1 and Nestin (Figure1b–d). Greater than 95% of iNPCs were positive for SOX1 and Nestin. Figure 1Open in figure viewerPowerPoint iNPCs show typical neural progenitor cell morphology on phase contrast at 20× magnification (a). Immunocytochemistry demonstrates positive expression of neural stem cell markers Nestin (b) and SOX1 (c). Merged image with DAPI (d) 3.2 MRI 24-hr post-surgery reveals ischemic stroke in the middle cerebral artery territory Twenty-four hours following bipolar cauterization of the right MCA, all eight animals underwent MRI evaluation of the brain. An area of increased signal intensity was noted in the distribution of the right MCA in each pig on T2-weighted (Figure2a), T2-FLAIR (Figure2b), and DWI sequences (Figure2c). A corresponding region of decreased signal intensity on ADC maps consistent with cytotoxic edema confirming ischemic infarction was identified in each animal (Figure2d). Figure 2Open in figure viewerPowerPoint MRI performed 24hr following MCAO demonstrates ischemic stroke in the territory of the middle cerebral artery. The affected area is hyperintense on T2-weighted imaging (a) and T2 FLAIR (b) relative to normal grey matter. The region is hyperintense on DWI (c) with corresponding hypointensity on the ADC map (d) confirming cytotoxic edema 3.3 The post-stroke assessment scale reliably detects functional changes in pigs after MCAO stroke and iNPC treatment Overall scores obtained through the post-stroke assessment scale were able to demonstrate significant (p .05) differences between pre-stroke and post-stroke animals at every time point for both observer A (non-blinded) and observer B (blinded) at all time points (Figure3a). No detectable differences were noted between initial and repeat assessments performed by the non-blinded observer (p .05). Likewise, no significant differences were detected between the overall scores assigned by the blinded observer and the non-blinded observer at any time point (p .05). This indicates that the assessment scale is reliable both between different observers and between assessments performed by one observer. Furthermore, iNPC-treated pigs showed significant improvement in their overall total post-stroke score relative to 1day post-stroke by 2weeks post-injection (Figure3b). Non-treated pigs did not demonstrate significant improvement until 9weeks post-injection. Figure 3Open in figure viewerPowerPoint All post-stroke time points for both treatment groups were significantly different from pre-stroke scores (a). No significant differences were noted between observers or between different assessments by the same observer at any time point in pigs following MCAO (a). iNPC-treated animals showed significant improvement (*) from 1day post-stroke scores by 2weeks following iNPC-injection (p .05), whereas non-treated animals did not reach this improvement level until 9weeks post-injection (#) (b) 3.4 iNPC treatment hastened recovery of postural reactions, posture, mental status, and appetite following MCAO Overall, iNPC-treated MCAO stroke pigs demonstrated a faster functional recovery relative to non-treated control pigs; however, unique parameters showed varying outcomes (Figure4). An improvement in postural reactions was noted in iNPC-treated pigs in the score between 1day post-stroke and scores at 2 and 6weeks post-injection, whereas non-treated pigs did not exhibit improvement in their postural reaction scores over the 12-week testing period (Figure4a). iNPC-treated pigs also demonstrated a significant improvement in their body posture scores by 1week post-injection compared to 5days post-stroke, whereas non-treated pigs did not show any significant improvements in their body posture scores (Figure4b). A similar trend was observed in head posture scores; iNPC-treated pigs exhibited improvement in head posture 6 and 9weeks post-injection compared to 1day post-stroke, whereas non-treated pigs did not show an improvement until 12weeks post-injection (Figure4c). iNPC-treated pigs also showed more rapid recovery of their mental status scores with significant improvements by 4weeks post-injection compared to 1day post-stroke while this improvement was not observed in the non-treated group until 9weeks post-injection (Figure4d). There was also a significantly improved appetite score in the iNPC-treated pigs by 4weeks post-injection relative to 1day post-stroke while no significant improvement in appetite was ever noted in control pigs over 12weeks (Figure4e). Figure 4Open in figure viewerPowerPoint Significant improvement in postural reaction scores were noted in the iNPC treated group by 2 and 6weeks post-injection (a), whereas non-treated pigs did not exhibit any improvement over 12weeks. The body posture scores appeared to improve by 1week post-injection in iNPC-treated pigs (b) with no improvement ever noted in the non-treated pigs. Significant improvements in head posture scores were noted by 6weeks post-injection in iNPC-treated pigs but not until 12weeks post-injection in non-treated pigs (c). Improvements in mental status scores were noted by 4weeks post-injection in iNPC-treated pigs, whereas non-treated pigs did not show improvement until 9weeks post-injection (d). Appetite scores of iNPC-treated pigs improved by 4weeks post-injection while non-treated pigs did not show significantly improved appetites throughout the 12-week period (e). * represent time points in the iNPC-treated group where scores were significantly different from 1-day post-stroke scores (p .05). *In the body posture graph (b) are an exception in that these were time points significantly different from 5days post-stroke (p .05). # represents time points where the non-treated scores were significantly improved from 1day post-stroke scores 3.5 iNPC treatment does not improve rate of recovery of circling tendency, cranial nerve function, or gait For circling and gait, both treatment groups did not exhibit any significant improvement by 12weeks post-injection compared to their 1day post-stroke scores (Figure5a,b). However, the post-stroke scores for both parameters were only mildly elevated, and spontaneous recovery to pre-stroke scores for gait and circling was noted in both treatment groups by 1–5days post-stroke. No significant difference was noted between treatment groups at any time point for these parameters. Cranial nerve function scores were significantly different from pre-stroke scores at all time points with no evidence of recovery from 1day post-stroke scores over 12weeks in either treatment group (Figure5c). Figure 5Open in figure viewerPowerPoint Spontaneous recovery was noted in circling and gait scores within a few days post-MCAO (a and b). * represents time points where scores recovered to pre-stroke levels in iNPC-treated pigs. # represents time points where scores recovered to pre-stroke levels in non-treated pigs In this study, we have developed a porcine post-stroke functional outcome assessment scale that was sensitive enough to detect changes between normal and stroked animals, and between iNPC-treated and non-treated pigs with high intra- and inter-observer repeatability. Furthermore, we utilize this assessment scale to show that human iNPC therapy leads to significant improvement in functional neurologic outcome across multiple parameters in a pig ischemic stroke model. Scores for the individual parameters showed more variability between observers; however, this did not affect repeatability of the overall scale score. This supports the use of the overall score of this scale in future studies to assess the effect of iNPC therapy or other novel therapies on neurologic function in pigs following stroke. We recently demonstrated that iNPC treatment leads to tissue and cellular level recovery in stroked pigs; however, a robust and repeatable post-stroke assessment scale was needed to demonstrate functional recovery (Baker etal., 2017). The developed pig stroke assessment scale showed that animals that received iNPC-injections demonstrated significantly faster recovery of postural reactions, body posture, head posture, mental status, and appetite relative to non-treated animals. Body posture, head posture, and postural reactions are a reflection of sensorimotor status. The faster rates of recovery noted here are similar to findings in rodent studies of neural progenitor cell therapy following stroke (Chang etal., 2013; Eckert etal., 2015; Gomi etal., 2012). Head and body posture scores were designed to broadly assess the sense of balance as well as unconscious and conscious proprioception similar to the beam walk and rotarod tests used in rodents. The improvements in these parameters are similar to improvements of the aforementioned tests noted in rodents following iNPC therapy as shown by Eckert etal. (2015) and Chang etal. (2013). Postural reaction assessments in the pigs in this study were scored similarly to the sensory test portion of the rodent mNSS and were designed to test unconscious and conscious proprioception. Rodent mNSS scores were previously demonstrated to improve more rapidly following iNPC injections by Gomi etal. (2012) and Chang etal. (2013). In contrast to the rodent studies, however, the rapid recovery in the iNPC-treated pigs did not result in significant differences in neurologic scores between iNPC-treated and non-treated groups by the endpoint of the study (12weeks post-stroke) due to spontaneous recovery of the non-treated pigs. This difference may be due to the much shorter follow-up times in rodent studies with endpoints being 9weeks or fewer, thus allowing less time for spontaneous recovery to occur in the chronic stage post-stroke. An exception to this is the study by Polentes etal. in which rodents were followed for 4months following stroke and cell transplantation (Polentes etal., 2012). In their study, animals receiving cell grafts demonstrated sustained improvements over vehicle-injected animals for tape-removal and apomorphin-induced rotation behavioral tests. However, both grafted and non-grafted animals demonstrated spontaneous recovery simultaneously on assessments of the Montaya stair case test and the mNSS within 1month post-stroke (Polentes etal., 2012). Scores for individual parameters within the stroke scale did not retain significant differences from pre-stroke scores throughout the 12-week test period indicating spontaneous recovery in both treatment groups. This is similar to spontaneous recovery rates in humans which has been reported to occur by about 10weeks post-stroke; albeit the degree of recovery in humans is significantly less (Kwakkel & Kollen, 2013). In addition to faster sensorimotor recovery, we show that iNPC-treated pigs also demonstrated faster recovery of appetite over control animals. Human post-stroke outcome assessment scales such as the BI and Functional Independence Measure (FIM) account for activities of daily living such as feeding and urinary/fecal continence (Dromerick, Edwards, & Diringer, 2003; Kwakkel & Kollen, 2013). It has been proposed that scales incorporating activities of daily living, such as feeding, are more sensitive to the level of disability and recovery following ischemic stroke than scales like the Modified Rankin Score (MRS) (Dromerick etal., 2003). If tasks like feeding are more sensitive, the rapid improvement of appetite noted in the iNPC-treated pigs and lack of improvement in the control pigs may be the most compelling indicator that iNPC therapy improved recovery in pigs following MCAO. Cranial nerve function did not exhibit significant improvement in either treatment group over the 12-week testing period. The most common cranial nerve deficits were contralateral menace response and facial hypalgesia, which are consistent with injury to the sensorimotor cortex. The lack of significant improvement may be due to the grading scheme for cranial nerve deficits where higher scores were more reflective of midbrain and medullary dysfunction, which are areas that are not injured by MCAO. Future cranial nerve scores may be more sensitive if more weight is placed on menace response and facial hypalgesia rather than testing cranial nerves that originate in the midbrain or medulla. Both treatment groups showed spontaneous improvement in their gait and circling scores within a few days post-stroke before iNPC treatment occurred. The spontaneous recovery of gait scores by 3days post-MCAO in this study may be a reflection of the predominant role of extrapyramidal brainstem centers in gait generation in pigs rather than corticospinal tracts (King, 1987). In addition, the method of gait assessment used in this study (gross visual observation) may not have been as sensitive to dysfunction as computerized gait analysis performed with limb stride or step height measurements (Duberstein etal., 2014). More specialized gait analyses, however, require special equipment and setup, which would make the post-stroke assessment scale less user-friendly and globally transferable. Circling was placed in the scale as a crude measurement of cognitive dysfunction and hemi-inattention and was graded on a scale from 0 to 2, making it one of the smallest contributors to the overall score. The narrow grading scale for this parameter may have altered its sensitivity and accuracy. Specifically, this test may not have been sensitive if the pigs were not observed for prolonged periods of walking. An induced-rotation test, such as the apomorphine test used in rodents, may need to be developed to detect more subtle differences between treatment groups (Li etal., 2004; Schaar etal., 2010; Vorhees & Williams, 2006). The development of such tests for pigs following MCAO warrants further investigation. The faster recovery noted in the overall score of iNPC-treated pigs could be a reflection of the anti-inflammatory and trophic factors secreted by iNPCs. The rapid onset of improvement in the cell-treated group and lack of significant difference between treatment groups at 12weeks post-injection would argue against neuronal regeneration being the major mechanism of action. iNPCs have previously been reported to reduce inflammatory cytokines such as TNF-α, IL-6, and IL-1β, in addition to reducing microglial activation and mitigating neuronal loss which is correlated to improved neurologic outcome (Eckert etal., 2015; Oki etal., 2012; Polentes etal., 2012; Tatarishvili, 2014). Given the spontaneous recovery seen in gait and circling, these parameters may be excluded from future post-stroke functional outcome assessment scales. Alternatively, more sensitive testing for these parameters, possibly through development of a porcine apomorphine-induced rotation test and/or computerized and measured gait analyses, may allow for better detection of disability and differences between treatment groups in future studies. The disparity between appetite scores of the iNPC-treated and non-treated pigs may indicate that this is the most sensitive parameter in the functional outcome scale (Dromerick etal., 2003). In addition, appetite may also be the most translatable to human functional outcome scales, and future modifications to the porcine scale should be weighted accordingly. The post-stroke assessment scale designed in this study offers a robust and repeatable means of evaluating functional outcomes in a large animal model of stroke. This will be of significant value to the field as future studies focus on the pig as a translational animal model for neural disease and injury. In addition, we demonstrated for the first time that iNPC therapy shortened functional recovery time in a large animal stroke model. Shorter recovery times could have significant effects on human quality of life and the cost associated with post-stroke hospitalization and long-term care. We thank Lisa Reno for surgical expertise and support, Kelly Parham and Richard Utley for animal assistance. Research reported in this publication was supported, in part, by the National Institute of Neurological Disorders and Stroke of the National Institutes of Health under award number R01NS093314 as well as the University of Georgia Office of the Vice President for Research. Albers, G. W., Goldstein, L. B., Hess, D. C., Wechsler, L. R., Furie, K. L., Gorelick, P. B., … STAIR VII Consortium. (2011). Stroke Treatment Academic Industry Roundtable (STAIR) recommendations for maximizing the use of intravenous thrombolytics and expanding treatment options with intra-arterial and neuroprotective therapies. Stroke, 42, 2645– 2650. https://doi.org/10.1161/STROKEAHA.111.618850 Araki, R., Uda, M., Hoki, Y., Sunayama, M., Nakamura, M., Ando, S., … Nifuji, A. (2013). Negligible immunogenicity of terminally differentiated cells derived from induced pluripotent or embryonic stem cells. Nature, 494, 100– 104. https://doi.org/10.1038/nature11807 Baker, E. W., Platt, S. R., Lau, V. W., Grace, H. E., Holmes, S. P., Wang, L., … Hess, D. C. (2017). Induced pluripotent stem cell-derived neural stem cell therapy enhances recovery in an ischemic stroke pig model. Scientific Reports, 7, 10075. https://doi.org/10.1038/s41598-017-10406-x Banks, J. L., & Marotta, C. A. (2007). Outcomes validity and reliability of the modified Rankin scale: Implications for stroke clinical trials: a literature review and synthesis. Stroke, 38, 1091– 1096. https://doi.org/10.1161/01.STR.0000258355.23810.c6 Bederson, J. B., Pitts, L. H., Tsuji, M., Nishimura, M. C., Davis, R. L., & Bartkowski, H. (1986). Rat middle cerebral artery occlusion: Evaluation of the model and development of a neurologic examination. Stroke, 17, 472– 476. https://doi.org/10.1161/01.STR.17.3.472 Chang, D.-J., Lee, N., Park, I.-H., Choi, C., Jeon, I., Kwon, J., … Lee, H. (2013). Therapeutic potential of human induced pluripotent stem cells in experimental stroke. Cell Transplantation, 22, 1427– 1440. https://doi.org/10.3727/096368912X657314 Corbett, D., & Nurse, S. (1998). The problem of assessing effective neuroprotection in experimental cerebral ischemia. Progress in Neurobiology, 54, 531– 548. https://doi.org/10.1016/S0301-0082(97)00078-6 Dromerick, A. W., Edwards, D. F., & Diringer, M. N. (2003). Sensitivity to changes in disability after stroke: A comparison of four scales useful in clinical trials. Journal of Rehabilitation Research and Development, 40, 1– 8. https://doi.org/10.1682/JRRD.2003.01.0001 Duberstein, K. J., Platt, S. R., Holmes, S. P., Dove, C. R., Howerth, E. W., Kent, M., … West, F. D. (2014). Gait analysis in a pre- and post-ischemic stroke biomedical pig model. Physiology & Behavior, 125, 8– 16. https://doi.org/10.1016/j.physbeh.2013.11.004 Eckert, A., Huang, L., Gonzalez, R., Kim, H.-S., Hamblin, M. H., & Lee, J.-P. (2015). Bystander effect fuels human induced pluripotent stem cell-derived neural stem cells to quickly attenuate early stage neurological deficits after stroke. Stem Cells Translational Medicine, 4, 841– 851. https://doi.org/10.5966/sctm.2014-0184 Wiley Online Library Encarnacion, A., Horie, N., Keren-Gill, H., Bliss, T. M., Steinberg, G. K., & Shamloo, M. (2011). Long-term behavioral assessment of function in an experimental model for ischemic stroke. Journal of Neuroscience Methods, 196, 247– 257. https://doi.org/10.1016/j.jneumeth.2011.01.010 Fisher, M., Feuerstein, G., Howells, D. W., Hurn, P. D., Kent, T. A., Savitz, S. I., … STAIR Group. (2009). Update of the stroke therapy academic industry roundtable preclinical recommendations. Stroke, 40, 2244– 2250. https://doi.org/10.1161/STROKEAHA.108.541128 Gomi, M., Takagi, Y., Morizane, A., Doi, D., Nishimura, M., Miyamoto, S., & Takahashi, J. (2012). Functional recovery of the murine brain ischemia model using human induced pluripotent stem cell-derived telencephalic progenitors. Brain Research, 1459, 52– 60. https://doi.org/10.1016/j.brainres.2012.03.049 Guha, P., Morgan, J. W., Mostoslavsky, G., Rodrigues, N. P., & Boyd, A. S. (2013). Lack of immune response to differentiated cells derived from syngeneic induced pluripotent stem cells. Cell Stem Cell, 12, 407– 412. https://doi.org/10.1016/j.stem.2013.01.006 Heiss, W.-D., & Kidwell, C. S. (2014). Imaging for prediction of functional outcome and assessment of recovery in ischemic stroke. Stroke, 45, 1195– 1201. https://doi.org/10.1161/STROKEAHA.113.003611 Hunter, A. J., Hatcher, J., Virley, D., Nelson, P., Irving, E., Hadingham, S. J., & Parsons, A. A. (2000). Functional assessments in mice and rats after focal stroke. Neuropharmacology, 39, 806– 816. https://doi.org/10.1016/S0028-3908(99)00262-2 Hunter, A. J., Mackay, K. B., & Rogers, D. C. (1998). To what extent have functional studies of ischaemia in animals been useful in the assessment of potential neuroprotective agents? Trends in Pharmacological Sciences, 19, 59– 66. https://doi.org/10.1016/S0165-6147(97)01157-7 Hyun, I. (2010). The bioethics of stem cell research and therapy. The Journal of Clinical Investigation, 120, 71– 75. https://doi.org/10.1172/JCI40435 Janssen, H., Speare, S., Spratt, N. J., Sena, E. S., Ada, L., Hannan, A. J., … Bernhardt, J. (2013). Exploring the efficacy of constraint in animal models of stroke: Meta-analysis and systematic review of the current evidence. Neurorehabilitation and Neural Repair, 27, 3– 12. https://doi.org/10.1177/1545968312449696 Jensen, M. B., Yan, H., Krishnaney-Davison, R., Al Sawaf, A., & Zhang, S. C. (2013). Survival and differentiation of transplanted neural stem cells derived from human induced pluripotent stem cells in a rat stroke model. Journal of Stroke and Cerebrovascular Diseases, 22, 304– 308. https://doi.org/10.1016/j.jstrokecerebrovasdis.2011.09.008 Kelly-Hayes, P. M., Robertson, J. T., Broderick, J. P., Duncan, P. W., Hershey, L. A., Roth, E. J., … Trombly, C. A. (1998). The American Heart Association stroke outcome classification. Stroke, 29, 1274– 1280. https://doi.org/10.1161/01.STR.29.6.1274 King, A. S. (1987). Physiological and clinical anatomy of the domestic mammals. Volume 1. Central nervous system. Oxford: Oxford University Press. Kwakkel, G., & Kollen, B. J. (2013). Predicting activities after stroke: What is clinically relevant? International Journal of Stroke, 8, 25– 32. https://doi.org/10.1111/j.1747-4949.2012.00967.x Wiley Online Library Li, X., Blizzard, K. K., Zeng, Z., DeVries, A. C., Hurn, P. D., & McCullough, L. D. (2004). Chronic behavioral testing after focal ischemia in the mouse: Functional recovery and the effects of gender. Experimental Neurology, 187, 94– 104. https://doi.org/10.1016/j.expneurol.2004.01.004 Longa, E. Z., Weinstein, P. R., Carlson, S., & Cummins, R. (1989). Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke, 20, 84– 91. https://doi.org/10.1161/01.STR.20.1.84 Lu, A., Ansari, S., Nyström, K., Damisah, E., Amin, H., Matouk, C., … Bulsara, K. (2014). Intra-arterial treatment of acute ischemic stroke: The continued evolution. Current Treatment Options in Cardiovascular Medicine, 16, 1– 10. Markgraf, C. G., Green, E. J., Hurwitz, B. E., Morikawa, E., Dalton Dietrich, W., McCabe, P. M., … Schneiderman, N. (1992). Sensorimotor and cognitive consequences of middle cerebral artery occlusion in rats. Brain Research, 575, 238– 246. https://doi.org/10.1016/0006-8993(92)90085-N Modo, M., Stroemer, R. P., Tang, E., Veizovic, T., Sowniski, P., & Hodges, H. (2000). Neurological sequelae and long-term behavioural assessment of rats with transient middle cerebral artery occlusion. Journal of Neuroscience Methods, 104, 99– 109. https://doi.org/10.1016/S0165-0270(00)00329-0 Mozaffarian, D., Benjamin, E. J., Go, A. S., Arnett, D. K., Blaha, M. J., Cushman, M., … Huffman, M. D. (2015). Heart disease and stroke statistics–2015 update: A report from the American Heart Association. Circulation, 131, e29– e322. https://doi.org/10.1161/CIR.0000000000000152 Oki, K., Tatarishvili, J., Wood, J., Koch, P., Wattananit, S., Mine, Y., … Brüstle, O. (2012). Human-induced pluripotent stem cells form functional neurons and improve recovery after grafting in stroke-damaged brain. Stem Cells, 30, 1120– 1133. https://doi.org/10.1002/stem.1104 Wiley Online Library Platt, S. R., Holmes, S. P., Howerth, E. W., Duberstein, K. J. J., Dove, C. R., Kinder, H. A., … Hill, W. D. (2014). Development and characterization of a Yucatan miniature biomedical pig permanent middle cerebral artery occlusion stroke model. Experimental and Translational Stroke Medicine, 6, 5. https://doi.org/10.1186/2040-7378-6-5 Polentes, J., Jendelova, P., Cailleret, M., Braun, H., Romanyuk, N., Tropel, P., … Turnovcova, K. (2012). Human induced pluripotent stem cells improve stroke outcome and reduce secondary degeneration in the recipient brain. Cell Transplantation, 21, 2587– 2602. https://doi.org/10.3727/096368912X653228 Ringelstein, E. B., Biniek, R., Weiller, C., Ammeling, B., Nolte, P. N., & Thron, A. (1992). Type and extent of hemispheric brain infarctions and clinical outcome in early and delayed middle cerebral artery recanalization. Neurology, 42, 289– 298. https://doi.org/10.1212/WNL.42.2.289 Roundtable, S. T. A. I. (1999). Recommendations for standards regarding preclinical neuroprotective and restorative drug development. Stroke, 30, 2752– 2758. Saver, J. L., Jovin, T. G., Smith, W. S., & Albers, G. W. (2013). Stroke treatment academic industry roundtable: Research priorities in the assessment of neurothrombectomy devices. Stroke, 44, 3596– 3601. https://doi.org/10.1161/STROKEAHA.113.002769 Savitz, S. I., Chopp, M., Deans, R., Carmichael, T., Phinney, D., Wechsler, L., & Participants, S. (2011). Stem cell therapy as an emerging paradigm for stroke (STEPS) II. Stroke, 42, 825– 829. https://doi.org/10.1161/STROKEAHA.110.601914 Schaar, K. L., Brenneman, M. M., & Savitz, S. I. (2010). Functional assessments in the rodent stroke model. Experimental and Translational Stroke Medicine, 2, 13. https://doi.org/10.1186/2040-7378-2-13 Tanaka, Y., Imai, H., Konno, K., Miyagishima, T., Kubota, C., Puentes, S., … Saito, N. (2008). 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, 39, 205– 212. https://doi.org/10.1161/STROKEAHA.107.489906 Tatarishvili, J. (2014). Human induced pluripotent stem cells improve recovery in stroke-injured aged rats. Restorative Neurology and Neuroscience, 32, 547– 558. Tornero, D., Wattananit, S., Grønning Madsen, M., Koch, P., Wood, J., Tatarishvili, J., … Hevner, R. F. (2013). Human induced pluripotent stem cell-derived cortical neurons integrate in stroke-injured cortex and improve functional recovery. Brain, 136, 3561– 3577. https://doi.org/10.1093/brain/awt278 Vorhees, C. V., & Williams, M. T. (2006). Morris water maze: Procedures for assessing spatial and related forms of learning and memory. Nature Protocols, 1, 848– 858. https://doi.org/10.1038/nprot.2006.116 Weisscher, N., Vermeulen, M., Roos, Y. B., & de Haan, R. J. (2008). What should be defined as good outcome in stroke trials; a modified Rankin score of 0–1 or 0–2? Journal of Neurology, 255, 867– 874. https://doi.org/10.1007/s00415-008-0796-8 Yamaguchi, M., Zhou, C., Heistad, D. D., Watanabe, Y., & Zhang, J. H. (2004). Gene transfer of extracellular superoxide dismutase failed to prevent cerebral vasospasm after experimental subarachnoid hemorrhage. Stroke, 35, 2512– 2517. https://doi.org/10.1161/01.STR.0000145198.07723.8e Zausinger, S., Hungerhuber, E., Baethmann, A., Reulen, H.-J., & Schmid-Elsaesser, R. (2000). Neurological impairment in rats after transient middle cerebral artery occlusion: A comparative study under various treatment paradigms. 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