Paw Preference Research Articles

Paw preferences in cats and dogs: Meta-analysis

ABSTRACTPredator–prey relationships have been suggested to be one of the primary evolutionary factors driving the development of functional hemispheric asymmetries. However, lateralization in many predator species is not well understood and existing studies often are statistically underpowered due to small sample sizes and they moreover show conflicting results. Here, we statistically integrated findings on paw preferences in cats and dogs, two predator species within the Carnivora order that are commonly kept as pets in many societies around the globe. For both species, there were significantly more lateralized than non-lateralized animals. We found that 78% of cats and 68% of dogs showed either left- or right-sided paw preference. Unlike humans, neither dogs nor cats showed a rightward paw preference on the population level. For cats, but not dogs, we found a significant sex difference, with female animals having greater odds of being right-lateralized compared to male animals.

Laterality as a Predictor of Coping Strategies in Dogs Entering a Rescue Shelter

It has been reported that during the first few days following entry to a kennel environment, shelter dogs may suffer poor welfare. Previous work suggests that motor bias (the preferred use of one limb over the other) can potentially be used as an indicator of emotional reactivity and welfare risk. In this study, we investigate whether paw preference could be used as a predictive indicator of stress coping (measured using cortisol levels and behavioural observation) in a sample of 41 dogs entering a rescue shelter. Cortisol levels and behavioural observations were collected for one week after admission. We scored the dogs’ paw preference during a food-retrieval task. Our results showed that increasing left-pawedness was associated with a higher expression of stress-related behaviours such as frequent change of state, vocalisations and lower body posture. These results are in keeping with previous findings showing that left-limb biased animals are more vulnerable to stress. Paw preference testing may be a useful tool for detecting different coping strategies in dogs entering a kennel environment and identifying target individuals at risk of reduced welfare.

Lack of association between paw preference and behaviour problems in the domestic dog, Canis familiaris

Evidence suggests that paw preferences are related to emotional functioning in the domestic dog. Whether motor bias in this species is related to the display of behaviour problems, which often have their basis in emotional reactivity, is still unknown. This study therefore explored the relationship between lateralised motor behaviour in the form of paw preferences and the occurrence of canine behaviour problems. Fifty-two dogs presenting with one or more behaviour problems, and 61 control animals, had their paw preferences assessed using the commonly employed Kong™ ball test. The dogs’ behavioural profile was determined using the C-BARQ, a psychometric tool designed to provide standardized evaluations of canine behaviour. Analysis revealed a roughly equal split in the proportion of dogs classified as ambilateral and lateralised, although dogs were more like to be ambilateral than left- or right-pawed. Dogs differed significantly in their behavioural profile, with animals in the behaviour problem group displaying more severe patterns of stranger- and dog-directed aggression and fear, non-social fear and touch sensitivity than those in the control group. There was no significant difference in the distribution, direction or strength of the dogs’ paw use between the two groups of animals. The dogs’ paw preference classification was significantly associated with one of the C-BARQ subscale scores, namely stranger-directed aggression, with left-pawed dogs having lower scores on this subscale than right-pawed or ambilateral animals. The direction of the dogs’ paw use in the behaviour problem group was significantly correlated with C-BARQ scores for the subscales of stranger-directed aggression, stranger-directed fear and attention-seeking behaviour, with higher subscale scores associated with increasing right-pawedness. There was no significant correlation between the direction of the dogs’ paw use and any of the other C-BARQ subscale scores. The strength of the dogs’ paw preferences was not significantly correlated with any of the C-BARQ subscale scores for either the control or behaviour problem group of animals. Canine sex was not significantly related to either the dogs’ paw preferences or the presentation of behaviour problems. Overall, this study provides little evidence in support of a relationship between paw preference and the occurrence of behaviour problems in the domestic dog. Further, longitudinal, work is recommended in an effort to unravel the ontogeny of lateralisation and its association with emotional reactivity in this species.

Paw preferences in the Asian small-clawed otter – using an inexpensive, video-based protocol to study laterality of rare species in the zoo

ABSTRACTIt is still debated whether limb-use preferences represent a common trait in vertebrates, which is based on a shared phylogenetic history. Unravelling the evolutionary origin and pattern of paw preferences in vertebrates requires the analysis of a larger number of species within an ecologically relevant setting. We therefore investigated whether observations in a zoo enable the collection of reliable data sets by quantifying paw use in two independent groups of Asian small-clawed otters (Amblonyx cinerea). Employing a continuous focal animal sampling method, each day one of the ten individuals was video recorded from outside of the enclosure during usual activity. We selected four types of unimanual behaviour (reaching for food, reaching for non-food, reaching into a hole, carrying an object) and quantified paw use for each animal. Our study provides first evidences for individual paw preferences in otters, which were in line with previously reported forelimb use pattern in carnivoran species. Preferences differed between motor acts but for “reaching into a hole” a population-level right paw bias was detected. These data support that observations in a zoological setting are useful to explore task-dependent paw preferences and may facilitate future studies investigating paw preferences under experimentally controlled conditions.

Stability of motor bias in the domestic dog, Canis familiaris

This study explored the relationship between four measures of canine paw preference to establish whether the distribution, direction or strength of motor bias was consistent between tasks. Thirty-two dogs had their paw preferences tested using the Kong ball, tape, lift paw and First-stepping tests. A smaller sample were re-tested 6 months later. The distribution of the dogs’ paw preferences was not significantly different from that expected by chance for the Kong ball and lift paw tests; dogs were significantly more inclined towards ambilaterality on the tape and First-stepping tests. More female dogs employed their right paw on the lift paw test; males were more likely to be ambilateral or left-pawed. There was no significant correlation in the direction of dogs’ paw use for any tests. The First-stepping and lift paw tests were positively correlated for strength of paw use. Analysis revealed a significant correlation in direction and strength of dogs’ paw use between the first and second attempts of all measures, except the tape test. Findings suggest that paw preference in the dog is not consistent between tasks, although stable over time. The study raises questions as to which test of paw preference is the most appropriate to employ.

Lateralization of spontaneous behaviours in the domestic cat, Felis silvestris

Recent research has drawn attention to the link between lateral bias and cerebral functional asymmetry in animals. Most studies of animal laterality have focused on limb use arising from forced experimental challenges as opposed to spontaneous behaviours. This study explored, for the first time, the expression of lateralized spontaneous behaviour in the domestic cat, a species that exhibits motor bias in the form of paw preferences. The side used by 44 pet cats to perform three spontaneous behaviours (lying side, stepping down a flight of stairs, stepping over a raised object) was recorded. Paw preferences were also assessed using a more traditional forced food reaching challenge. Cats showed a significant lateral bias for food reaching, stepping down and stepping over. Those with a paw preference, however, did not differ significantly in their tendency towards left or right sidedness. The direction of the cats' side preferences was significantly correlated for most measures, whether forced or spontaneous. The strength of the cats' motor bias was significantly related to the task; animals displayed a weaker lateral bias for lying side than any other measure. The study revealed a sex split in the direction, although not the strength, of the cats' lateral bias for food reaching, stepping down and stepping over. Males showed a significant preference for using their left paw on these measures, while females were more inclined to use the right one. The study provides the first evidence that the domestic cat displays motor laterality on specific spontaneous behaviours, and that the direction, although not the strength, of these lateral biases is largely consistent with that of an experimental task. The results suggest that the more forced food-reaching test traditionally used to assess motor bias in the cat offers a biologically valid measure of limb use in this species.

Cognitive bias and paw preference in the domestic dog (Canis familiaris).

Limb use, an indicator of hemispheric functioning, may be a useful predictor of cognitive bias and hence vulnerability to welfare risk. The relationship between cognitive bias and motor asymmetry, however, has been subject to little investigation. This study explored the association between motor asymmetry and cognitive bias in the domestic dog, a species that displays lateral bias in the form of paw preferences and exhibits positive and negative affective states. Thirty pet dogs had their paw preferences assessed using the Kong ball test. The subjects' affective state was assessed using a cognitive bias test in which the animals' latency to approach a bowl placed in 1 of 3 ambiguous positions was recorded. Animals veering more toward a left-paw preference were found to be significantly slower to approach the bowl placed in 1 of the ambiguous positions than ambilateral or right-pawed dogs. Left-pawed subjects approached the bowl located at the 3 ambiguous positions at roughly the same speed, while ambilateral and right-pawed animals became increasingly slower to approach the bowl the further it was located from the baited food bowl. The study points to a possible relationship between cognitive bias and paw preference in the dog, with left-pawed animals being more negative or "pessimistic" in their cognitive outlook than right-pawed or ambilateral individuals. It is proposed that limb preference testing might offer a more practical and straightforward way of identifying individuals at risk from poor welfare by virtue of how they perceive the world than more time-consuming cognitive bias tests. (PsycINFO Database Record

Pawedness Trait Test (PaTRaT)-A New Paradigm to Evaluate Paw Preference and Dexterity in Rats.

In rodents, dexterity is commonly analyzed in preference paradigms in which animals are given the chance to use either the left or the right front paws to manipulate food. However, paw preference and dexterity at population and individual levels are controversial as results are incongruent across paradigms. We have therefore developed a semi-quantitative method—the pawdeness trait test (PaTRaT)—to evaluate paw preference degree in rats. The PaTRaT consists in a classification system, ranging from +4 to −4 where increasingly positive and negative values reflect the bias for left or right paw use, respectively. Sprague-Dawley male rats were confined into a metal rectangular mesh cylinder, from which they can see, smell and reach sugared rewards with their paws. Due to its size, the reward could only cross the mesh if aligned with its diagonal, imposing additional coordination. Animals were allowed to retrieve 10 rewards per session in a total of four sessions while their behavior was recorded. PaTRaT was repeated 4 and 8 weeks after the first evaluation. To exclude potential bias, rats were also tested for paw fine movement and general locomotion in other behavioral paradigms as well as impulsivity (variable delay-to-signal, VDS), memory and cognitive flexibility (water maze). At the population level 54% of the animals presented a rightward bias. Individually, all animals presented marked side-preferences, >2 and <−2 for left- and right-sided bias, respectively, and this preference was stable across the three evaluations. Inter-rater consistency was very high between two experienced raters and substantial when two additional inexperienced raters were included. Left- and right-biased animals presented no differences in the ability to perform fine movements with any of the forelimbs (staircase) and general locomotor performance. Additionally, these groups performed similarly in executive function and memory tasks. In conclusion, PaTRaT is able to reliably classify rats’ pawedness direction and degree.

2080

OBJECTIVES/SPECIFIC AIMS: To assess the efficacy of exogenous administration of MSCs in animal models of HIE. METHODS/STUDY POPULATION: Adhering to the Systematic Review Protocol for Animal Intervention Studies, a systematic search of English articles was performed using MEDLINE, Web of Science, CINAHL, and Google Scholar. Search term items included mesenchymal stem/stromal cell, hypoxic ischemic encephalopathy, asphyxia, cerebral ischemia, and neonatology. We selected randomized and nonrandomized studies that examined in vivo neonatal models of induced HIE. We excluded studies that combined MSCs with an adjunct therapy. Data were collected on study specifics, MSC characteristics, and outcome measurements. The primary outcome was efficacy of MSC treatment, assessed by functional neurologic measures (cognitive, motor, sensory). Risk of bias was assessed using the SYRCLE’s risk of bias tool and we used the ARRIVE guidelines to describe the quality of study reporting. RESULTS/ANTICIPATED RESULTS: A total of 17 preclinical publications focusing on MSC therapy for HIE met our inclusion criteria. Fifteen of the studies (88%) induced HIE in rodents by ligating the common carotid artery followed by a period of hypoxic exposure. Nine (53%) studies derived their MSCs from rodent bone marrow, whereas the other investigators provided xenografts from human bone marrow or umbilical cord-derived MSCs. Range of MSC dose was between 0.25 and 3.5× 106 cells with 71% of the experiments transplanting the MSCs intranasally or intracerebral. Three studies (18%) administered multiple doses. The cylinder rearing test was the most common (73%) sensorimotor functional outcome performed in the first month following the establishment of HIE. All studies demonstrated a reduction in asymmetrical paw preference after receiving MSC therapy. Lesional size was assessed, using neuroimaging or histologic evaluations, and the majority of studies showed a decreased insult following MSC therapy. DISCUSSION/SIGNIFICANCE OF IMPACT: MSC treatment demonstrates improved functional and structural outcomes that are encouraging for future translational studies.

Supplemental Material for Cognitive Bias and Paw Preference in the Domestic Dog (Canis familiaris)

Supplemental Material for Cognitive Bias and Paw Preference in the Domestic Dog (Canis familiaris)

Lateral bias and temperament in the domestic cat (Felis silvestris).

Research points to a relationship between lateralization and emotional functioning in humans and many species of animal. The present study explored the association between paw preferences and emotional functioning, specifically temperament, in a species thus far overlooked in this area, the domestic cat. Thirty left-pawed, 30 right-pawed, and 30 ambilateral pet cats were recruited following an assessment of their paw preferences using a food-reaching challenge. The animals' temperament was subsequently assessed using the Feline Temperament Profile (FTP). Cats' owners also completed a purpose-designed cat temperament (CAT) scale. Analysis revealed a significant relationship between lateral bias and FTP and CAT scale scores. Ambilateral cats had lower positive (FTP+) scores, and were perceived as less affectionate, obedient, friendly, and more aggressive, than left or right-pawed animals. Left and right pawed cats differed significantly on 1 trait on the CAT scale, namely playfulness. The strength of the cats' paw preferences was related to the animals' FTP and CAT scores. Cats with a greater strength of paw preference had higher FTP+ scores than those with a weaker strength of paw preference. Animals with stronger paw preferences were perceived as more confident, affectionate, active, and friendly than those with weaker paw preferences. Results suggest that motor laterality in the cat is strongly related to temperament and that the presence or absence of lateralization has greater implications for the expression of emotion in this species than the direction of the lateralized bias. (PsycINFO Database Record

Positively reinforcing an operant task using tactile stimulation and food – a comparison in horses using clicker training

A widely used method for testing lateralization in domestic dogs is the Kong test. This study examined the Kong test and the individual characteristics of dogs associated with performance on this task. Assessments of paw preference were conducted on 96 dogs, with additional demographic and temperament data collected from the owners. To standardize the Kong test for this study, each dog was presented with a Kong filled with the same frozen food, and the criterion for completion of the test was 50 touches, within a 1-hour time limit. The results showed that 60% of dogs completed the test, a further 15% engaged with the task but failed to reach the required 50 touches, and almost 25% did not engage with it at all. Dogs with higher owner-rated levels of “neuroticism” were less likely to complete the task. Completion of the task was more likely in taller dogs and those that slept apart from the owners. Performance was unrelated to the dog's age, sex, whether the dog was from a single or multiple dog household, and other temperament measures. The results suggest that the Kong test is most appropriate for larger breeds. Future research may further investigate factors that influence performance on the Kong test of motor laterality.

Relationship between visuospatial attention and paw preference in dogs.

The relationship between visuospatial attention and paw preference was investigated in domestic dogs. Visuospatial attention was evaluated using a food detection task that closely matches the so-called “cancellation” task used in human studies. Paw preference was estimated by quantifying the dog’s use of forepaws to hold a puzzle feeder device (namely the “Kong”) while eating its content. Results clearly revealed a strong relationship between visuospatial attention bias and motor laterality, with a left-visuospatial bias in the left-pawed group, a right-visuospatial bias in the right-pawed group and with the absence of significant visuospatial attention bias in ambi-pawed subjects. The current findings are the first evidence for the presence of a relationship between motor lateralization and visuospatial attentional mechanisms in a mammal species besides humans.

Assessing lateralization in domestic dogs: Performance by Canis familiaris on the Kong test

Abstract A widely used method for testing lateralization in domestic dogs is the Kong test. This study examined the Kong test and the individual characteristics of dogs associated with performance on this task. Assessments of paw preference were conducted on 96 dogs, with additional demographic and temperament data collected from the owners. To standardize the Kong test for this study, each dog was presented with a Kong filled with the same frozen food, and the criterion for completion of the test was 50 touches, within a 1-hour time limit. The results showed that 60% of dogs completed the test, a further 15% engaged with the task but failed to reach the required 50 touches, and almost 25% did not engage with it at all. Dogs with higher owner-rated levels of neuroticism were less likely to complete the task. Completion of the task was more likely in taller dogs and those that slept apart from the owners. Performance was unrelated to the dog's age, sex, whether the dog was from a single or multiple dog household, and other temperament measures. The results suggest that the Kong test is most appropriate for larger breeds. Future research may further investigate factors that influence performance on the Kong test of motor laterality.

Ancestral Exposure to Stress Generates New Behavioral Traits and a Functional Hemispheric Dominance Shift.

In a continuously stressful environment, the effects of recurrent prenatal stress (PS) accumulate across generations and generate new behavioral traits in the absence of genetic variation. Here, we investigated if PS or multigenerational PS across 4 generations differentially affect behavioral traits, laterality, and hemispheric dominance in male and female rats. Using skilled reaching and skilled walking tasks, 3 findings support the formation of new behavioral traits and shifted laterality by multigenerational stress. First, while PS in the F1 generation did not alter paw preference, multigenerational stress in the F4 generation shifted paw preference to favor left-handedness only in males. Second, multigenerational stress impaired skilled reaching and skilled walking movement abilities in males, while improving these abilities in females beyond the levels of controls. Third, the shift toward left-handedness in multigenerationally stressed males was accompanied by increased dendritic complexity and greater spine density in the right parietal cortex. Thus, cumulative multigenerational stress generates sexually dimorphic left-handedness and dominance shift toward the right hemisphere in males. These findings explain the origins of apparently heritable behavioral traits and handedness in the absence of DNA sequence variations while proposing epigenetic mechanisms.

Comparing lateral bias in dogs and humans using the Kong™ ball test

The Kong™ ball test has been used extensively to assess lateral bias in the domestic dog. Implicit in this challenge is the assumption that dogs use their dominant paw to stabilise the ball. This study examined whether or not this is the case. A comparative approach was adopted, exploring limb use in dogs and humans. In Experiment 1, the paw preference of 48 dogs was assessed on the Kong™ ball test. Analysis revealed an equal distribution of paw use, although significantly more dogs were paw-preferent than ambilateral. Significantly more male dogs were classified as right-pawed, while more females were ambilateral. There was no significant effect of canine sex or castration status on the dogs' paw preferences. In Experiment 2, 94 adult humans were assessed on their ability to remove a piece of paper from a Kong™ ball with their mouth, using their left, right or both hands to stabilise the ball. 76% of the right-handed people used their left hand, and 82% of the left-handed participants used their right hand, to hold the Kong™ steady. It is concluded that dogs, like humans, are most likely using their non-dominant limb to stabilise the Kong™ ball and their dominant side for postural support. This has potential applied implications from an animal welfare perspective.

Evaluation of the anatomical and functional consequences of repetitive mild cervical contusion using a model of spinal concussion

Spinal cord concussion is characterized by a transient loss of motor and sensory function that generally resolves without permanent deficits. Spinal cord concussions usually occur during vehicular accidents, falls, and sport activity, but unlike brain concussions, have received much less attention despite the potential for repeated injury leading to permanent neurological sequelae. Consequently, there is no consensus regarding decisions related to return to play following an episode of spinal concussion, nor an understanding of the short- and long-term consequences of repeated injury. Importantly, there are no models of spinal concussion to study the anatomical and functional sequelae of single or repeated injury. We have developed a new model of spinal cord concussion focusing on the anatomical and behavioral outcomes of single and repeated injury. Rats received a very mild (50kdyn, IH impactor) spinal contusion at C5 and were separated into two groups three weeks after the initial injury — C1, which received a second, sham surgery, and C2, which received a second contusion at the same site. To track motor function and recovery, animals received weekly behavioral tests — BBB, CatWalk™, cylinder, and Von Frey. Analysis of locomotor activity by BBB demonstrated that rats rapidly recovered, regaining near-normal function by one week after the first and second injury, which was confirmed using the more detailed CatWalk™ analysis. The cylinder test showed that a single contusion did not induce significant deficits of the affected limb, but that repeated injury resulted in significant alteration in paw preference, with animals favoring the unaffected limb. Intriguingly, Von Frey analysis demonstrated an increased sensitivity in the contralateral hindlimb in the C2 group vs. the C1 group. Anatomical analyses revealed that while the lesion volume of both groups was minimal, the area of spared white matter in the C2 group was significantly reduced 1 and 2mm rostral to the lesion epicenter. Reactive astrocytes were present in both groups, with the majority found at the lesion epicenter in the C1 group, whereas the C2 group demonstrated increased reactive astrocytes extending 1mm caudal to the lesion epicenter. Macrophages accumulated within the injured, dorsal and ipsilateral spinal cord, with significant increases at 2 and 3mm rostral to the epicenter in the C2 group. Our model is designed to represent the clinical presentation of spinal cord concussion, and highlight the susceptibility and functional sequelae of repeated injury. Future experiments will examine the temporal and spatial windows of vulnerability for repeated injuries.

Predictability and randomness of paw choices are critical elements in the behavioural plasticity of mouse paw preference

Lateralized paw usage of mice, Mus musculus, is a learned behaviour, based on a gradual reinforcement of randomly occurring weak asymmetries in paw choice early in training. The reinforcement relies on strain-dependent, short-term and long-term memory. We characterized the skills of information accumulation by quantifying the predictability of each reach of initially naive mice from past behaviour in two training sessions of 50 reaches, separated by a 1-week interval. We studied six mouse strains, including 9XCA and BTBR with absent corpus callosum and severely reduced hippocampal commissure, and compared them to a null model with random, unbiased paw preference. We found that each paw choice was based on a limited, strain-specific number of previous choices. Also, there was a limited, strain-specific degree of predictability of each choice. Consequently, there was a strain-specific degree of randomness that was not lost with training. After 1 week for consolidation of memory of learned biases, paw choices became more predictable and made use of fewer previous choices, except in 9XCA and BTBR; nevertheless, a degree of randomness remained. We conclude that paw choices are regulated by short-term memory of a small number of previous choices and by long-term memory that affects future behaviour patterns and decreases, but does not remove, the usage of short-term memory. Both short-term and long-term memory skills are strain dependent. Importantly, a degree of randomness is not removed by training and this may be a critical element for behavioural plasticity in paw preference in changing environments, supplying constant adaptability in paw preferences.

Preclinical evaluation of recombinant T cell receptor ligand RTL1000 as a therapeutic agent in ischemic stroke.

Recombinant T cell Receptor Ligand 1000 (RTL1000), a partial human major histocompatibility complex (MHC) molecule coupled to a human myelin peptide, reduces infarct size after experimental stroke in HLA-DRB1*1502 transgenic (DR2-Tg) mice. In this study, we characterized the therapeutic time window of opportunity for RTL1000; we explored the efficacy of a single dose of RTL1000 administration and determined if RTL1000 affords long-term neurobehavioral functional improvement after ischemic stroke. Male DR2-Tg mice underwent 60min of intraluminal reversible middle cerebral artery occlusion (MCAO). RTL1000 or vehicle was injected 4, 6, or 8h after MCAO, followed by three daily injections. In the single-dose study, one-time injection of RTL1000 was applied 4h after MCAO. Cortical, striatal, and hemispheric infarct sizes were measured 24 or 96h after stroke. Behavioral testing, including neuroscore evaluation, open field, paw preference, and novel object recognition, was performed up to 28days after stroke. Our data showed that RTL1000 significantly reduced the infarct size 96h after MCAO when the first injection was given at 4 and 6h, but not 8h, after the onset of stroke. A single dose of 400 or 100μg RTL1000 also significantly reduced the infarct size 24h after MCAO. Behavioral testing showed that RTL1000 treatment used 4h after MCAO improved long-term cognitive outcome 28days after stroke. Taken together, RTL1000 protects against acute injury if applied within a 6-h time window and improves long-term functional recovery after experimental stroke in DR2-Tg mice.

Abstract W P92: Preclinical Evaluation of Recombinant T-cell Receptor Ligand RTL1000 as a Therapeutic Agent in Stroke

Objective: RTL1000, a partial human MHC molecule coupled to a human myelin peptide, reduces infarct size after experimental stroke in HLA-DR2 transgenic mice expressing the human MHC class II allele. In this study, we characterized the therapeutic window of RTL1000. We then determined if RTL1000 efficacy is altered when combined with t-PA, a standard therapy for stroke, and if it affords long-term neurobehavioral functional improvement after stroke in mice. Methods: Male DR2 mice underwent 60 min of intraluminal MCA occlusion (MCAO). RTL1000 (100 μg) or vehicle (100 μl) was given S.C. at 4, 6 or 8 h after MCAO, followed by 3 daily S.C. injections. t-PA (10 mg/kg) or vehicle was infused I.V. over 30 min starting at 15 min into ischemia. Brains were collected at either 24 or 96 h of reperfusion. Cortical, striatal and hemispheric infarcts were measured after staining with 2, 3, 5-triphenyltetrazolium chloride. Behavioral testing neuroscore, open field, paw preference and novel object recognition was performed up to 28 days after MCAO. Results: RTL1000 significantly reduced infarct size when administered at 4 and 6, but not 8 h after MCAO. Protection was more robust when RTL1000 was given at 4 h, and was limited to cortex when first administration was delayed for 6 h. When administered alone, t-PA reduced infarct size when measured at 24 but not 96 h after MCAO. Importantly, RTL1000 alone reduced infarct size at 96 h after MCAO, and the addition of t-PA did not reduce its ability to protect against ischemic injury. Behavioral testing showed RTL1000 had no effect on acute cognitive impairment on day 7, but improved long term cognitive outcome 28 days after MCAO. Conclusions: RTL1000 is protective against ischemic stroke with a therapeutic window of up to 6 h. Protection by RTL1000 is preserved in the presence of t-PA, is long-lasting and improves neurobehavioral functional outcomes after experimental cerebral ischemia.