Non-human Primate Stroke Research Articles

Sleep dysfunction and gut dysbiosis related amino acids metabolism disorders in cynomolgus monkeys after middle cerebral artery occlusion

IntroductionThis study aimed to explore the characteristics of post-stroke sleep dysfunction and verify their association with gut dysbiosis and the related amino acid metabolism disorders. This was achieved by using fecal microbiota transplantation (FMT) in a non-human primate stroke model. MethodsTwenty adult male cynomolgus monkeys were divided into the sham (n = 4), middle cerebral artery occlusion (MCAO, n = 5), MCAO + FMT (n = 3), and donor (n = 8) groups. The MCAO+FMT group received FMT at post-MCAO week 4. Sleep parameters, gut microbiota, gamma-aminobutyric acid (GABA), and glutamine (Gln) in the cerebrospinal fluid (CSF) were measured at baseline and postoperative weeks 4, 8, and 12. ResultsAt postoperative weeks 4, 8, and 12, the MCAO group showed decreased sleep efficiency, measured as the percentage of sleep during the whole night (82.3 ± 3.2 % vs 91.3 ± 2.5 %, 79.0 ± 3.75 % vs 90.8 ± 3.2 %, and 69.5 ± 4.8 % vs 90.5 ± 2.7 %; all P < 0.05), lower relative abundance of Lactobacillus (all P < 0.05), and reduced GABA concentrations in the CSF (317.3 ± 30.6 nmol/L vs 437.7 ± 25.6 nmol/L, 303.1 ± 48.9 nmol/L vs 4 40.9 ± 37.8 nmol/L, and 337.9 ± 49.4 nmol/L vs 457.4 ± 39.2 nmol/L; all P < 0.05) compared with the sham group. Sleep efficiency at post-FMT weeks 4 and 8 (84.7 ± 1.1 % vs 79.0 ± 3.75 %, and 84.1 ± 2.0 % vs 69.5 ± 4.8 %; both P < 0.05) and GABA concentration in the CSF at post-FMT week 4 (403.1 ± 25.4 nmol/L vs 303.1 ± 48.9 nmol/L, P < 0.05) was higher in the MCAO+FMT group than in the MCAO group. ConclusionsPost-stroke sleep dysfunction in monkeys is characterized by impaired sleep coherence, associated with decreased levels of probiotics such as Lactobacillus, GABA, and Gln in the CSF and can be ameliorated using FMT.

Comprehensive Assessment of Ischemic Stroke in Nonhuman Primates: Neuroimaging, Behavioral, and Serum Proteomic Analysis.

Ischemic strokes, prevalence and impactful, underscore the necessity of advanced research models closely resembling human physiology. Our study utilizes nonhuman primates (NHPs) to provide a detailed exploration of ischemic stroke, integrating neuroimaging data, behavioral outcomes, and serum proteomics to elucidate the complex interplay of factors involved in stroke pathophysiology. We observed a consistent pattern in infarct volume, peaking at 1-month postmiddle cerebral artery occlusion (MCAO) and then stabilized. This pattern was strongly correlated to notable changes in motor function and working memory performance. Using diffusion tensor imaging (DTI), we detected significant alterations in fractional anisotropy (FA) and mean diffusivity (MD) values, signaling microstructural changes in the brain. These alterations closely correlated with the neurological and cognitive deficits that we observed, highlighting the sensitivity of DTI metrics in stroke assessment. Behaviorally, the monkeys exhibited a reliance on their unaffected limb for compensatory movements, a common response to stroke impairment. This adaptation, along with consistent DTI findings, suggests a significant impact of stroke on motor function and spatial perception. Proteomic analysis through MS/MS functional enrichment identified two distinct groups of proteins with significant changes post-MCAO. Notably, MMP9, THBS1, MB, PFN1, and YWHAZ were identified as potential biomarkers and therapeutic targets for ischemic stroke. Our results underscore the complex nature of stroke and advocate for an integrated approach, combining neuroimaging, behavioral studies, and proteomics, for advancing our understanding and treatment of this condition.

Hypothermic neuroprotection by targeted cold autologous blood transfusion in a non-human primate stroke model

Over decades, nearly all attempts to translate the benefits of therapeutic hypothermia in stroke models of lower-order species to stroke patients have failed. Potentially overlooked reasons may be biological gaps between different species and the mismatched initiation of therapeutic hypothermia in translational studies. Here, we introduce a novel strategy of selective therapeutic hypothermia in a non-human primate ischemia-reperfusion model, in which autologous blood was cooled ex vivo and the cool blood transfusion was administered at the middle cerebral artery just after the onset of reperfusion. Cold autologous blood cooled the targeted brain rapidly to below 34 °C while the rectal temperature remained around 36 °C with the assistance of a heat blanket during a 2-h hypothermic process. Therapeutic hypothermia or extracorporeal-circulation related complications were not observed. Cold autologous blood treatment reduced infarct sizes, preserved white matter integrity, and improved functional outcomes. Together, our results suggest that therapeutic hypothermia, induced by cold autologous blood transfusion, was achieved in a feasible, swift, and safe way in a non-human primate model of stroke. More importantly, this novel hypothermic approach conferred neuroprotection in a clinically relevant model of ischemic stroke due to reduced brain damage and improved neurofunction. This study reveals an underappreciated potential for this novel hypothermic modality for acute ischemic stroke in the era of effective reperfusion.

CDC42 Might Be a Molecular Signature of DWI-FLAIR Mismatch in a Nonhuman Primate Stroke Model.

No definitive blood markers of DWI-FLAIR mismatch, a pivotal indicator of salvageable ischemic penumbra brain tissue, are known. We previously reported that CDC42 and RHOA are associated with the ischemic penumbra. Here, we investigated whether plasma CDC42 and RHOA are surrogate markers of DWI-FLAIR mismatch. Sixteen cynomolgus macaques (3 as controls and 13 for the stroke model) were included. Guided by digital subtraction angiography (DSA), a middle cerebral artery occlusion (MCAO) model was established by occluding the middle cerebral artery (MCA) with a balloon. MRI and neurological deficit scoring were performed to evaluate postinfarction changes. Plasma CDC42 and RHOA levels were measured by enzyme-linked immunosorbent assay (ELISA). The stroke model was successfully established in eight monkeys. Based on postinfarction MRI images, experimental animals were divided into a FLAIR (-) group (N = 4) and a FLAIR (+) group (N = 4). Plasma CDC42 in the FLAIR (-) group showed a significant decrease compared with that in the FLAIR (+) group (p < 0.05). No statistically significant difference was observed for plasma RHOA. The FLAIR (-) group showed a milder neurological function deficit and a smaller infarct volume than the FLAIR (+) group (p < 0.05). Therefore, plasma CDC42 might be a new surrogate marker for DWI-FLAIR mismatch.

Investigate the neuroprotective effects of electrical stimulation following acute ischemic stroke in non-human primates

Investigate the neuroprotective effects of electrical stimulation following acute ischemic stroke in non-human primates

A Reliable Nonhuman Primate Model of Ischemic Stroke with Reproducible Infarct Size and Long-term Sensorimotor Deficits.

A nonhuman primate model of ischemic stroke is considered as an ideal preclinical model to replicate various aspects of human stroke because of their similarity to humans in genetics, neuroanatomy, physiology, and immunology. However, it remains challenging to produce a reliable and reproducible stroke model in nonhuman primates due to high mortality and variable outcomes. Here, we developed a focal cerebral ischemic model induced by topical application of 50% ferric chloride (FeCl3) onto the MCA-M1 segment through a cranial window in the cynomolgus monkeys. We found that FeCl3 rapidly produced a stable intraarterial thrombus that caused complete occlusion of the MCA, leading to the quick decrease of the regional cerebral blood flow in 10 min. A typical cortical infarct was detected 24 hours by magnetic resonance imaging (MRI) and was stable at least for 1 month after surgery. The sensorimotor deficit assessed by nonhuman primate stroke scale was observed at 1 day and up to 3 months after ischemic stroke. No spontaneous revascularization or autolysis of thrombus was observed, and vital signs were not affected. All operated cynomolgus monkeys survived. Our data suggested that FeCl3-induced stroke in nonhuman primates was a replicable and reliable model that is necessary for the correct prediction of the relevance of experimental therapeutic approaches in human beings.

Spontaneous Behavioural Recovery Following Stroke Relates to the Integrity of Parietal and Temporal Regions.

Stroke is a devastating disease that results in neurological deficits and represents a leading cause of death and disability worldwide. Following a stroke, there is a degree of spontaneous recovery of function, the neural basis of which is of great interest among clinicians in their efforts to reduce disability following stroke and enhance rehabilitation. Conventionally, work on spontaneous recovery has tended to focus on the neural reorganization of motor cortical regions, with comparably little attention being paid to changes in non-motor regions and how these relate to recovery. Here we show, using structural neuroimaging in a macaque stroke model (N = 31) and by exploiting individual differences in spontaneous behavioural recovery, that the preservation of regions in the parietal and temporal cortices predict animal recovery. To characterize recovery, we performed a clustering analysis using Non-Human Primate Stroke Scale (NHPSS) scores and identified a good versus poor recovery group. By comparing the preservation of brain volumes in the two groups, we found that brain areas in integrity of brain areas in parietal, temporal and somatosensory cortex were associated with better recovery. In addition, a decoding approach performed across all subjects revealed that the preservation of specific brain regions in the parietal, somatosensory and medial frontal cortex predicted recovery. Together, these findings highlight the importance of parietal and temporal regions in spontaneous behavioural recovery.

Quantitative behavioral evaluation of a non-human primate stroke model using a new monitoring system.

BackgroundRecently, the common marmoset (Callithrix jacchus) has attracted significant interest as a non-human primate stroke model. Functional impairment in non-human primate stroke models should be evaluated quantitatively and successively after stroke, but conventional observational assessments of behavior cannot fully fit this purpose. In this paper, we report a behavioral analysis using MarmoDetector, a three-dimensional motion analysis, in an ischemic stroke model using photosensitive dye, along with an observational behavioral assessment and imaging examination.MethodsIschemic stroke was induced in the left hemisphere of three marmosets. Cerebral infarction was induced by intravenous injection of rose bengal and irradiation with green light. The following day, the success of the procedure was confirmed by magnetic resonance imaging (MRI). The distance traveled, speed, activity time, and jumps/climbs were observed for 28 days after stroke using MarmoDetector. We also assessed the marmosets’ specific movements and postural abnormalities using conventional neurological scores.ResultsMagnetic resonance imaging diffusion-weighted and T2-weighted images showed hyperintense signals, indicating cerebral infarction in all three marmosets. MarmoDetector data showed that the both indices immediately after stroke onset and gradually improved over weeks. Neurological scores were the worst immediately after stroke and did not recover to pre-infarction levels during the observation period (28 days). A significant correlation was observed between MarmoDetector data and conventional neurological scores.ConclusionIn this study, we showed that MarmoDetector can quantitatively evaluate behavioral changes in the acute to subacute phases stroke models. This technique can be practical for research on the pathophysiology of ischemic stroke and for the development of new therapeutic methods.

Neuroprotective Effects of Electrical Stimulation Following Ischemic Stroke in Non-Human Primates.

Brain stimulation has emerged as a novel therapy for ischemic stroke, a major cause of brain injury that often results in lifelong disability. Although past works in rodents have demonstrated protective effects of stimulation following stroke, few of these results have been replicated in humans due to the anatomical differences between rodent and human brains and a limited understanding of stimulation-induced network changes. Therefore, we combined electrophysiology and histology to study the neuroprotective mechanisms of electrical stimulation following cortical ischemic stroke in non-human primates. To produce controlled focal lesions, we used the photothrombotic method to induce targeted vasculature damage in the sensorimotor cortices of two macaques while collecting electrocorticography (ECoG) signals bilaterally. In another two monkeys, we followed the same lesioning procedures and applied repeated electrical stimulation via an ECoG electrode adjacent to the lesion. We studied the protective effects of stimulation on neural dynamics using ECoG signal power and coherence. In addition, we performed histological analysis to evaluate the differences in lesion volume. In comparison to controls, the ECoG signals showed decreased gamma power across the sensorimotor cortices in stimulated animals. Meanwhile, Nissl staining revealed smaller lesion volumes for the stimulated group, suggesting that electrical stimulation may exert neuroprotection by suppressing post-ischemic neural activity. With the similarity between NHP and human brains, this study paves the path for developing effective stimulation-based therapy for acute stroke in clinical studies.

Abstract WP259: Acute Stroke Neuroprotection With Intravenous Rns60 In The Cynomolgus Macaque

Introduction: RNS60 is an experimental therapy comprised of stable oxygenated nanobubbles in a sodium chloride solution with an oxygen content of 55+/-5 ppm. Phase I/II clinical trials established a safety profile for RNS60 in patients with ALS and MS. In-vitro assays in neurons have demonstrated mitochondrial biogenesis with RNS60. Therefore, we hypothesized that RNS60 would protect the ischemic penumbra and delay stroke evolution. In this study, we assessed RNS60 in a cynomolgus macaque (CM) model of transient middle cerebral artery occlusion (MCAO). Methods: Experimenters were blinded and CMs randomly allocated to treatment. In the first experiment, 22 male CMs (2.6+/-0.55 kg) were anesthetized with physiologic monitoring and MCAO followed by immediate MRI imaging to define perfusion deficit. RNS60 (5cc/kg/h IV over 1h followed by 2.5cc/kg/h for 48h) or drug vehicle were administered starting 1h following MCAO. MCA was reperfused 90min after MCAO. CMs underwent MRI diffusion-weighted (DWI) and T2 imaging at 48h and 30d after MCAO. Serial Non-Human Primate Stroke Scale (NHPSS) assessments were collected over 30 days following MCAO. In the second experiment, 10 male CMs (4.95+/-0.18 kg) underwent permanent MCAO. RNS60 or drug vehicle were infused starting 5 minutes after MCAO (5cc/kg/h IV for 1 hour followed by 2.5cc/kg/h until sacrifice). Perfusion and DWI MRI images were obtained serially over 6 hours to measure the evolution of the penumbra. CMs were sacrificed at the completion of the experiment. Results: DWI stroke volume was significantly reduced 4.3+/-1.1mL vs. 9.1+/-0.92mL (P=0.0036) at 48 hours post MCAO in RNS60 vs. placebo treated CMs. This result was conserved on T2 imaging at 30 days where stroke volume was 3.5+/-0.86mL vs. 7.3+/-0.79mL (P=0.004). RNS60 significantly improved NHPSS scores over 30 days compared to controls after removing three outlier CMs (2 mortalities and one non-reperfused MCA). The results of the second experiment demonstrated a 6h DWI volume of 5.9cc in RNS60 treated CMs compared to 10.6cc in controls. Conclusion: RNS60 administered during acute ischemic stroke significantly reduces stroke volume and improves functional outcomes in male CMs. The evolution of penumbra to stroke is slowed following RNS60 administration.

In vivo Neuroregeneration to Treat Ischemic Stroke Through NeuroD1 AAV-Based Gene Therapy in Adult Non-human Primates.

Stroke may cause severe death and disability but many clinical trials have failed in the past, partially because the lack of an effective method to regenerate new neurons after stroke. In this study, we report an in vivo neural regeneration approach through AAV NeuroD1-based gene therapy to repair damaged brains after ischemic stroke in adult non-human primates (NHPs). We demonstrate that ectopic expression of a neural transcription factor NeuroD1 in the reactive astrocytes after monkey cortical stroke can convert 90% of the infected astrocytes into neurons. Interestingly, astrocytes are not depleted in the NeuroD1-converted areas, consistent with the proliferative capability of astrocytes. Following ischemic stroke in monkey cortex, the NeuroD1-mediated astrocyte-to-neuron (AtN) conversion significantly increased local neuronal density, reduced microglia and macrophage, and surprisingly protected parvalbumin interneurons in the converted areas. Furthermore, the NeuroD1 gene therapy showed a broad time window in AtN conversion, from 10 to 30 days following ischemic stroke. The cortical astrocyte-converted neurons showed Tbr1+ cortical neuron identity, similar to our earlier findings in rodent animal models. Unexpectedly, NeuroD1 expression in converted neurons showed a significant decrease after 6 months of viral infection, indicating a downregulation of NeuroD1 after neuronal maturation in adult NHPs. These results suggest that in vivo cell conversion through NeuroD1-based gene therapy may be an effective approach to regenerate new neurons for tissue repair in adult primate brains.

Assessment of Hand Motor Function in a Non-human Primate Model of Ischemic Stroke.

Ischemic stroke results from arterial occlusion and can cause irreversible brain injury. A non-human primate (NHP) model of ischemic stroke was previously developed to investigate its pathophysiology and for efficacy testing of therapeutic candidates; however, fine motor impairment remains to be well-characterized. We evaluated hand motor function in a cynomolgus monkey model of ischemic stroke. Endovascular transient middle cerebral artery occlusion (MCAO) with an angiographic microcatheter induced cerebral infarction. In vivo magnetic resonance imaging mapped and measured the ischemia-induced infarct lesion. In vivo diffusion tensor imaging (DTI) of the stroke lesion to assess the neuroplastic changes and fiber tractography demonstrated three-dimensional patterns in the corticospinal tract 12 weeks after MCAO. The hand dexterity task (HDT) was used to evaluate fine motor movement of upper extremity digits. The HDT was modified for a home cage-based training system, instead of conventional chair restraint training. The lesion was localized in the middle cerebral artery territory, including the sensorimotor cortex. Maximum infarct volume was exhibited over the first week after MCAO, which progressively inhibited ischemic core expansion, manifested by enhanced functional recovery of the affected hand over 12 weeks after MCAO. The total performance time decreased with increasing success rate for both hands on the HDT. Compensatory strategies and retrieval failure improved in the chronic phase after stroke. Our findings demonstrate the recovery of fine motor skill after stroke, and outline the behavioral characteristics and features of functional disorder of NHP stroke model, providing a basis for assessing hand motor function after stroke.

Intranasal salvinorin A improves neurological outcome in rhesus monkey ischemic stroke model using autologous blood clot

Salvinorin A (SA) exerts neuroprotection and improves neurological outcomes in ischemic stroke models in rodents. In this study, we investigated whether intranasal SA administration could improve neurological outcomes in a monkey ischemic stroke model. The stroke model was induced in adult male rhesus monkeys by occluding the middle cerebral artery M2 segment with an autologous blood clot. Eight adult rhesus monkeys were randomly administered SA or 10% dimethyl sulfoxide as control 20 min after ischemia. Magnetic resonance imaging was used to confirm the ischemia and extent of injury. Neurological function was evaluated using the Non-Human Primate Stroke Scale (NHPSS) over a 28-day observation period. SA significantly reduced infarct volume (3.9 ± 0.7 cm3 vs. 7.2 ± 1.0 cm3; P = 0.002), occupying effect (0.3 ± 0.2% vs. 1.4 ± 0.3%; P = 0.002), and diffusion limitation in the lesion (−28.2 ± 11.0% vs. −51.5 ± 7.1%; P = 0.012) when compared to the control group. SA significantly reduced the NHPSS scores to almost normal in a 28-day observation period as compared to the control group (P = 0.005). Intranasal SA reduces infarct volume and improves neurological outcomes in a rhesus monkey ischemic stroke model using autologous blood clot.

Primate Version of Modified Rankin Scale for Classifying Dysfunction in Rhesus Monkeys.

Background and Purpose- Nonhuman primates are increasingly used in translational studies of ischemic stroke. However, current scoring systems in monkeys (eg, Nonhuman Primate Stroke Scale) do not focus on impairments in activities of daily living, so clinically relevant data are scarce for evaluating functional deficits in this model. Methods- Here, we referenced the modified Rankin Scale to provide a primate version of Rankin Scale (pRS) for ranking neurological dysfunction in monkeys following stroke. We selected hand function and strength, level of activity, and general mobility as the main components of pRS. We also analyzed interobserver variability. Results- pRS is a simple scale with only 6 levels. Functional deficit can be easily classified into none (category 0), slight (categories 1-2), moderate (category 3-4), and severe disabilities (category 5) based on pRS. We validated this scoring system on 11 monkeys, all with varying levels of neurological dysfunction following stroke, assessed by blinded testers. After a short training period, both technicians and neurology residents were able to achieve a high level of consistency using this scoring system. Conclusions- pRS is a simple and reliable functional scale, similar to the widely used modified Rankin Scale, for evaluating long-term neurological dysfunction in nonhuman primates. We recommend further validation studies and analyses.

Abstract 122: Selective Intra-Arterial Cooling (SI-AC) Improve Recovery in a Nonhuman Primate Model of Ischemic Stroke

Background and Purpose: Recent findings from our group prove that selective intra-arterial cooling (SI-AC) treatment leads to prominent and persistent functional improvements after stroke in both rodents and non-human primate (NHPs) stroke models. In this study, neuro-protective potentials of SI-AC treatment were further explored at brain network and histopathological levels in preclinical higher-order brains. Methods: Cerebral ischemia/reperfusion was induced by thrombus and thrombolysis (t-PA) in adult rhesus monkeys (n=10). SI-AC treatment was randomly given to 5 models. Conventional MRI and diffusion tensor imaging scans were performed 4 h, 7 and 30 days post focal ischemia. Effects on functional recovery were also assessed through longitudinal neurologic scores and behavior tests. Animals were euthanized after the final scan for histological analysis. Results: SI-AC treatment led to a higher rate of infarct reversal, significantly mitigated neurologic deficits, and more preserved dexterity when compared with those only receiving t-PA during a 30-day observation period. More fiber numbers, a higher FA values based on DTI analysis, and a higher integrity of white matter based on immunochemistry in ipsilateral side were observed in models receiving t-PA plus SI-AC during the same observation period. Histological findings, consistent with MRI images in the infarct size and reversal, indicated that myelin damage, spheroids, and spongiosis were significantly improved in models receiving SI-AC treatment than those with t-PA alone treatment. Conclusions: Our findings further supported that SI-AC as an adjunctive treatment could significantly improve neural tissue and functional preservation in a large animal model, suggesting its potential application in ischemic stroke treatments.

Abstract 179: Significant Efficacy of Adenosine A3 Receptor Agonist AST-004 in Rat and Non-human Primate Models of Transient Middle Cerebral Artery Occlusion

Acute Ischemic Stroke (AIS) is the second-leading cause of global mortality with an estimated 6.7 million victims dying each year. Our research has focused on a non-neuronal cell type, the astrocyte, and has demonstrated that GPCR receptor-mediated maintenance of astrocyte function enhances critical homeostatic mechanisms in the brain including protection against AIS-associated edema, glutamate excitotoxicity and oxidative stress. AST-004 is a small-molecule agonist of the GPCR Adenosine A3 receptor (ADORA3), demonstrating excellent pharmacokinetics and blood-brain barrier permeability in preclinical species. The efficacy and dose-response of AST-004 was assessed in rat and non-human primate stroke models of transient occlusion of the middle cerebral artery (tMCAO). In rats, a 1.5-hour tMCAO was conducted with a 24-hour steady-state intravenous infusion of vehicle or AST-004 initiated at time of reperfusion (vehicle or three AST-004 dose levels, n=12/group); neurological deficit and stroke lesion volume (TTC staining/image analysis) were assessed 24 hours post-reperfusion. Compared to vehicle, AST-004 reduced brain lesion volume by 50% (p=0.04) and improved neurological function by 36% (p=0.03). In non-human primates (cynomolgus macaque), a 4-hour tMCAO was conducted with a 22-hour steady-state intravenous infusion initiated 2 hours prior to reperfusion (vehicle or three AST-004 dose levels, n=4/group). Stroke lesion, perfusion deficits and penumbral endpoints were assessed by FLAIR, T2, MRA, DWI and ASL at multiple timepoints out to 5 days post-occlusion. Neurological deficit was assessed at 5-days post-occlusion. AST-004 treatment resulted in statistically significant improvements in outcome relative to vehicle, up to a 44% decrease in lesion volume (p=0.02) and a 72% decrease in percentage of lesion growth (p=0.0006) over the 5-day study period. AST-004 treatment during occlusion also reduced the slope of lesion evolution prior to reperfusion by 72% (p=0.05) relative to vehicle. Together, these studies indicate the potential of a non-neuronal approach to reducing stroke lesion damage via the ADORA3 receptor. AST-004 is a first-in-class candidate that warrants further evaluation in human clinical stroke trials.

Poly-Arginine Peptide-18 (R18) Reduces Brain Injury and Improves Functional Outcomes in a Nonhuman Primate Stroke Model.

Poly-arginine peptide-18 (R18) is neuroprotective in different rodent middle cerebral artery occlusion (MCAO) stroke models. In this study, we examined whether R18 treatment could reduce ischemic brain injury and improve functional outcome in a nonhuman primate (NHP) stroke model. A stroke was induced in male cynomolgus macaques by MCAO distal to the orbitofrontal branch of the MCA through a right pterional craniotomy, using a 5-mm titanium aneurysm clip for 90min. R18 (1000nmol/kg) or saline vehicle was administered intravenously 60min after the onset of MCAO. Magnetic resonance imaging (MRI; perfusion-weighted imaging, diffusion-weighted imaging, or T2-weighted imaging) of the brain was performed 15min, 24h, and 28days post-MCAO, and neurological outcome was assessed using the NHP stroke scale (NHPSS). Experimental endpoint was 28days post-MCAO, treatments were randomized, and all procedures were performed blinded to treatment status. R18 treatment reduced infarct lesion volume by up to 65.2% and 69.7% at 24h and 28days poststroke, respectively. Based on NHPSS scores, R18-treated animals displayed reduced functional deficits. This study confirms the effectiveness of R18 in reducing the severity of ischemic brain injury and improving functional outcomes after stroke in a NHP model, and provides further support for its clinical development as a stroke neuroprotective therapeutic.

Increased CD68/TGFβ Co-expressing Microglia/ Macrophages after Transient Middle Cerebral Artery Occlusion in Rhesus Monkeys.

The function of microglia/macrophages after ischemic stroke is poorly understood. This study examines the role of microglia/macrophages in the focal infarct area after transient middle cerebral artery occlusion (MCAO) in rhesus monkeys. We measured infarct volume and neurological function by magnetic resonance imaging (MRI) and non-human primate stroke scale (NHPSS), respectively, to assess temporal changes following MCAO. Activated phagocytic microglia/macrophages were examined by immunohistochemistry in post-mortem brains (n=6 MCAO, n=2 controls) at 3 and 24 hours (acute stage), 2 and 4 weeks (subacute stage), and 4, and 20 months (chronic stage) following MCAO. We found that the infarct volume progressively decreased between 1 and 4 weeks following MCAO, in parallel with the neurological recovery. Greater presence of cluster of differentiation 68 (CD68)-expressing microglia/macrophages was detected in the infarct lesion in the subacute and chronic stage, compared to the acute stage. Surprisingly, 98~99% of transforming growth factor beta (TGFβ) was found colocalized with CD68-expressing cells. CD68-expressing microglia/macrophages, rather than CD206+ cells, may exert anti-inflammatory effects by secreting TGFβ after the subacute stage of ischemic stroke. CD68+ microglia/macrophages can therefore be used as a potential therapeutic target.

Abstract WP348: Astrocyte Reaction After Ischemic Stroke in Nonhuman Primate

In the mammalian central nervous system, gliogenesis and neurogenesis constitutively occurs in the subventricular zone (SVZ) and the hippocampus albeit on a much lower scale in comparison to the other organs. In response to stroke and presumably as an endogenous mechanism of brain self repair, the neural precursors located in the SVZ and the hippocampus are induced to proliferate and migrate towards stroke-damaged tissue. This innate neural reaction in adult brain may represent a mechanism of neuroplasticity and structural remodeling that leads to functional recovery. The rodent animal model of ischemic stroke (IS) showed a clear and pronounced neurogenesis and gliogenesis. However, to date little is known about this innate response in human and nonhuman primates to IS. We have developed a nonhuman primate model of IS with well-defined anatomical infarct lesions and motor behavioral deficit. After a small craniotomy, the dura was opened and with microsurgical technique and the M3 segment of the middle cerebral artery was occluded for 90 minutes followed by reperfusion. Animals with IS showed significant (p&lt;0.005) motor deficit in right hand. To label endogenous neural precursors undergoing cell proliferation in vivo we injected all animals with the S-phase marker 5-bromo-2-deoxyuridine (BrdU). Postmortem analysis demonstrated significant increase of GFAP+ astrocytes (P&lt;0.0001) in the infarct area. These astrocytes exhibited unique re-organization around the infarct zone and expressed various types of morphologies and phenotypes. Less than 1% incorporated BrdU. Sixty-five % of astrocytes with elongated processes oriented toward the IS co-expressed vimentin while tissue from control animals expressed no vimentin. Approximately 35% of astrocytes co-expressed Sox-2, while control animals showed about 4% in the same area of the cortex. Both vimentin and Sox-2 are markers for neural stem cells. These data suggests that astrocytes may be more heterogeneous than previously thought and may undertake various roles in response to IS. A subpopulation of astrocytes may be reprogrammed to an early developmental stage under IS conditions to promote tissue repair and homeostasis.

Abstract WP156: Poly-arginine Peptides R18 and R18D Improve Functional Outcomes After Endothelin-1-induced Stroke in the Sprague Dawley Rat

Aims: We have previously demonstrated that the poly-arginine peptides R18 and R18D are neuroprotective at 24 hours following intraluminal middle cerebral artery occlusion (MCAO) in the rat. Therefore, using an independent laboratory we examined the ability of R18 and R18D to improve functional outcomes after endothelin-1 (ET-1) induced MCAO in rats over 56 days post-stroke. Methods: Peptides were administered intravenously at 100, 300 or 1000 nmol/kg, 60 minutes after ET-1 induced MCAO. Multiple forelimb placing tests and horizontal ladder tests were performed post-stroke, and the neuroprotective peptide NA-1 (TAT-NR2Bpc), which is being assessed in Phase 3 clinical stroke trials, was used as a benchmark. Results: R18 (300 and 1000nmol/kg, respectively) was the most effective peptide to improve functional outcomes, followed by R18D (300 and 1000 nmol/kg, respectively), and NA-1 (300 and 100 nmol/kg, respectively). Furthermore, R18 at 300 and 1000 nmol/kg was the most effective treatment to restore pre-stroke animal body weight (post-stroke day 4 versus day 7 for saline, and days 5-7 for all other treatments), while R18 and R18D at 300 and 1000 nmol/kg, but not NA-1 also significantly reduced the number of animals requiring special care (hand feeding) 48 hours after stroke. Conclusion: In conclusion, this study confirms that R18 and R18D can improve long-term functional outcomes after stroke, and given the superior effectiveness of R18 over NA-1 in the present, as well as in our previous studies, justifies further evaluation in a non-human primate stroke model and in a clinical stroke trial.