Sociability versus empathy in adolescent mice: Different or distinctive?

In recent years, a growing number of pre-clinical studies have made use of the social abilities of mice, asking how gene variants (e.g., null, transgenic or mutant alleles) give rise to abnormalities in neurodevelopment. Two distinct courses of research provide the foundation for these studies. One course has mostly focused on how we can assess "sociability" using metrics, often automated, to quantitate mouse approach and withdrawal responses to a variety of social stimuli. The other course has focused on psychobiological constructs that underlie the socio-emotional capacities of mice, including motivation, reward and empathy. Critically, we know little about how measures of mouse sociability align with their underlying socio-emotional capacities. In the present work, we compared the expression of sociability in adolescent mice from several strains versus a precisely defined behavioral model of empathy that makes use of a vicarious fear learning paradigm. Despite substantial strain-dependent variation within each behavioral domain, we found little evidence of a relationship between these social phenotypes (i.e., the rank order of strain differences was unique for each test). By contrast, emission of ultrasonic vocalizations was highly associated with sociability, suggesting that these two measures reflect the same underlying construct. Taken together, our results indicate that sociability and vicarious fear learning are not manifestations of a single, overarching social trait. These findings thus underscore the necessity for a robust and diverse set of measures when using laboratory mice to model the social dimensions of neuropsychiatric disorders.

Unbridle biomedical research from the laboratory cage.

Many biomedical research studies use captive animals to model human health and disease. However, a surprising number of studies show that the biological systems of animals living in standard laboratory housing are abnormal. To make animal studies more relevant to human health, research animals should live in the wild or be able to roam free in captive environments that offer a natural range of both positive and negative experiences. Recent technological advances now allow us to study freely roaming animals and we should make use of them.

Social Reward and Empathy as Proximal Contributions to Altruism: The Camaraderie Effect.

Natural selection favors individuals to act in their own interests, implying that wild animals experience a competitive psychology. Animals in the wild also express helping behaviors, presumably at their own expense and suggestive of a more compassionate psychology. This apparent paradox can be partially explained by ultimate mechanisms that include kin selection, reciprocity, and multilevel selection, yet some theorists argue such ultimate explanations may not be sufficient and that an additional "stake in others" is necessary for altruism's evolution. We suggest this stake is the "camaraderie effect," a by-product of two highly adaptive psychological experiences: social motivation and empathy. Rodents can derive pleasure from access to others and this appetite for social rewards motivates individuals to live together, a valuable psychology when group living is adaptive. Rodents can also experience empathy, the generation of an affective state more appropriate to the situation of another compared to one's own. Empathy is not a compassionate feeling but it has useful predictive value. For instance, empathy allows an individual to feel an unperceived danger from social cues. Empathy of another's stance toward one's self would predict either social acceptance or ostracism and amplify one's physiological sensitivity to social isolation, including impaired immune responses and delayed wound healing. By contrast, altruistic behaviors would promote well-being in others and feelings of camaraderie from others, thereby improving one's own physiological well-being. Together, these affective states engender a stake in others necessary for the expression of altruistic behavior.

Prenatal domoic acid exposure disrupts mouse pro-social behavior and functional connectivity MRI.

Domoic acid (DA) is a toxin produced by marine algae and known primarily for its role in isolated outbreaks of Amnestic Shellfish Poisoning and for the damage it inflicts on marine mammals, particularly California sea lions. Lethal effects of DA are often preceded by seizures and coma. Exposure to DA during development can result in subtle and highly persistent effects on brain development and include behavioral changes that resemble diagnostic features of schizophrenia and anomalies in social behavior we believe are relevant to autism spectrum disorder (ASD). To more fully examine this hypothesis, we chose to examine adolescent mice exposed in utero to DA for endpoints relevant to ASD, specifically changes in social behavior and network structure, the latter measured by resting state functional connectivity (rs-fcMRI). We found that male offspring exposed in utero to DA expressed reproducible declines in social interaction and atypical patterns of functional connectivity in the anterior cingulate, a region of the default mode network that is critical for social functioning. We also found disruptions in global topology in regions involved in the processing of reward, social, and sensory experiences. Finally, we found that DA exposed males expressed a pattern of local over-connectivity. These anomalies in brain connectivity bear resemblance to connectivity patterns in ASD and help validate DA-exposed mice as a model of this mental disability.

Differential influence of social versus isolate housing on vicarious fear learning in adolescent mice.

Laboratory rodents can adopt the pain or fear of nearby conspecifics. This phenotype conceptually lies within the domain of empathy, a bio-psycho-social process through which individuals come to share each other's emotion. Using a model of cue-conditioned fear, we show here that the expression of vicarious fear varies with respect to whether mice are raised socially or in solitude during adolescence. The impact of the adolescent housing environment was selective: (a) vicarious fear was more influenced than directly acquired fear, (b) "long-term" (24-h postconditioning) vicarious fear memories were stronger than "short-term" (15-min postconditioning) memories in socially reared mice whereas the opposite was true for isolate mice, and (c) females were more fearful than males. Housing differences during adolescence did not alter the general mobility of mice or their vocal response to receiving the unconditioned stimulus. Previous work with this mouse model underscored a genetic influence on vicarious fear learning, and the present study complements these findings by elucidating an interaction between the adolescent social environment and vicarious experience. Collectively, these findings are relevant to developing models of empathy amenable to mechanistic exploitation in the laboratory. (PsycINFO Database Record

Social conditioned place preference in the captive ground squirrel (Ictidomys tridecemlineatus): Social reward as a natural phenotype.

Social behaviors of wild animals are often considered within an ultimate framework of adaptive benefits versus survival risks. By contrast, studies of laboratory animals more typically focus on affective aspects of behavioral decisions, whether a rodent derives a rewarding experience from social encounter, and how this experience might be initiated and maintained by neural circuits. Artificial selection and inbreeding have rendered laboratory animals more affiliative and less aggressive than their wild conspecifics, leaving open the possibility that social reward is an artifact of domestication. We compared social behaviors of wild and captive population of juvenile 13-lined ground squirrels (Ictidomys tridecemlineatus), the latter being 2nd- and 3rd-generation descendants of wild individuals. At an age corresponding to emergence from the burrow, postnatal day (PD) 38, captive squirrels engaged in vigorous social approach and play and these juvenile behaviors declined significantly by PD 56. Similarly, young wild squirrels expressed social proximity and play; affiliative interactions declined with summer's progression and were replaced by agonistic chasing behaviors. Social conditioned place preference testing (conditioned PDs 40-50) indicated that adolescent squirrels derived a rewarding experience from social reunion. Our results support the contention that undomesticated rodents have the capacity for social reward and more generally suggest the possibility that positive affective experiences may support group cohesion, social cooperation, and altruism in the wild.

Diagnosing domoic acid toxicosis in the California sea lion (Zalophus californianus) using behavioral criteria: A novel approach.

Domoic acid toxicosis in the California sea lion (Zalophus californianus) is difficult to diagnose using presence of toxin alone because the duration of domoic acid presence in blood and urine is generally less than 48 hr following exposure. Because domoic acid toxicosis is often suggested by presentation of behavioral abnormalities, we asked whether assessment of behavior might be useful for diagnostic purposes. We developed an ethogram to categorize behavioral data collected via continuous focal animal sampling. In total, 169 subjects were observed at a rehabilitation center. Sea lions with domoic acid toxicosis displayed head weaving (P < 0.0001) and muscle fasciculations (P < 0.01) significantly more often than animals in a comparison group. Dragging hind flippers and swift scanning were observed exclusively in animals from the domoic acid toxicosis group. The data show that behavioral diagnostic criteria can be effective in the diagnosis of domoic acid toxicosis in the California sea lion.

Large-scale topology and the default mode network in the mouse connectome.

Noninvasive functional imaging holds great promise for serving as a translational bridge between human and animal models of various neurological and psychiatric disorders. However, despite a depth of knowledge of the cellular and molecular underpinnings of atypical processes in mouse models, little is known about the large-scale functional architecture measured by functional brain imaging, limiting translation to human conditions. Here, we provide a robust processing pipeline to generate high-resolution, whole-brain resting-state functional connectivity MRI (rs-fcMRI) images in the mouse. Using a mesoscale structural connectome (i.e., an anterograde tracer mapping of axonal projections across the mouse CNS), we show that rs-fcMRI in the mouse has strong structural underpinnings, validating our procedures. We next directly show that large-scale network properties previously identified in primates are present in rodents, although they differ in several ways. Last, we examine the existence of the so-called default mode network (DMN)--a distributed functional brain system identified in primates as being highly important for social cognition and overall brain function and atypically functionally connected across a multitude of disorders. We show the presence of a potential DMN in the mouse brain both structurally and functionally. Together, these studies confirm the presence of basic network properties and functional networks of high translational importance in structural and functional systems in the mouse brain. This work clears the way for an important bridge measurement between human and rodent models, enabling us to make stronger conclusions about how regionally specific cellular and molecular manipulations in mice relate back to humans.

Effects of prenatal cocaine exposure on social development in mice.

Prenatal cocaine exposure (PCE) in humans and animals has been shown to impair social development. Molecules that mediate synaptic plasticity and learning in the medial prefrontal cortex (mPFC), specifically brain-derived neurotrophic factor (BDNF) and its downstream signaling molecule, early growth response protein 1 (egr1), have been shown to affect the regulation of social interactions (SI). In this study we determined the effects of PCE on SI and the corresponding ultrasonic vocalizations (USVs) in developing mice. Furthermore, we studied the PCE-induced changes in the constitutive expression of BDNF, egr1 and their transcriptional regulators in the mPFC as a possible molecular mechanism mediating the altered SI. In prenatal cocaine-exposed (PCOC) mice we identified increased SI and USV production at postnatal day (PD) 25, and increased SI but not USVs at PD35. By PD45 the expression of both social behaviors normalized in PCOC mice. At the molecular level, we found increased BDNF exon IV and egr1 mRNA in the mPFC of PCOC mice at PD30 that normalized by PD45. This was concurrent with increased EGR1 protein in the mPFC of PCOC mice at PD30, suggesting a role of egr1 in the enhanced SI observed in juvenile PCOC mice. Additionally, by measuring the association of acetylation of histone 3 at lysine residues 9 and 14 (acH3K9,14) and MeCP2 at the promoters of BDNF exons I and IV and egr1, our results provide evidence of promoter-specific alterations in the mPFC of PCOC juvenile mice, with increased association of acH3K9,14 only at the BDNF exon IV promoter. These results identify a potential PCE-induced molecular alteration as the underlying neurobiological mechanism mediating the altered social development in juvenile mice.

Social influences on morphine-conditioned place preference in adolescent BALB/cJ and C57BL/6J mice.

RATIONALE: Among human adolescents, drug use is substantially influenced by the attitudes and behaviors of peers. Social factors also affect the drug-seeking behaviors of laboratory animals. Conditioned place preference (CPP) experiments indicate that social context can influence the degree to which rodents derive a rewarding experience from drugs of abuse. However, the precise manner by which social factors alter drug reward in adolescent rodents remains unknown. OBJECTIVES: We employed the relatively asocial BALB/cJ (BALB) mouse strain and the more prosocial C57BL/6J (B6) strain to explore whether "low" or "high" motivation to be with peers influences the effects of social context on morphine CPP (MCPP). METHODS: Adolescent mice were conditioned by subcutaneous injections of morphine sulfate (0.25, 1.0, or 5.0 mg/kg). During the MCPP procedure, mice were housed in either isolation (Ih) or within a social group (Sh). Similarly, following injection, mice were conditioned either alone (Ic) or within a social group (Sc). RESULTS: Adolescent B6 mice expressed a robust MCPP response except when subjected to Ih-Sc, which indicates that, following isolation, mice with high levels of social motivation are less susceptible to the rewarding properties of morphine when they are conditioned in a social group. By contrast, MCPP responses of BALB mice were most sensitive to morphine conditioning when subjects experienced a change in their social environment between housing and conditioning (Ih-Sc or Sh-Ic). CONCLUSIONS: Our findings demonstrate that susceptibility to morphine-induced reward in adolescent mice is moderated by a complex interaction between social context and heritable differences in social motivation.

The autism diagnosis in translation: shared affect in children and mouse models of ASD.

In the absence of molecular biomarkers that can be used to diagnose ASD, current diagnostic tools depend upon clinical assessments of behavior. Research efforts with human subjects have successfully utilized standardized diagnostic instruments, which include clinician interviews with parents and direct observation of the children themselves [Risi et al., 2006]. However, because clinical instruments are semi-structured and rely heavily on dynamic social processes and clinical skill, scores from these measures do not necessarily lend themselves directly to experimental investigations into the causes of ASD. Studies of the neurobiology of autism require experimental animal models. Mice are particularly useful for elucidating genetic and toxicological contributions to impairments in social function [Halladay et al., 2009]. Behavioral tests have been developed that are relevant to autism [Crawley, 2004, 2007], including measures of repetitive behaviors [Lewis, Tanimura, Lee, & Bodfish, 2007; Moy et al., 2008], social behavior [Brodkin, 2007; Lijam et al., 1997; Moretti, Bouwknecht, Teague, Paylor, & Zoghbi, 2005], and vocal communication [D'Amato et al., 2005; Panksepp et al., 2007; Scattoni et al., 2008]. Advances also include development of high-throughput measures of mouse sociability that can be used to reliably compare inbred mouse strains [Moy et al., 2008; Nadler et al., 2004], as well as measures of social reward [Panksepp & Lahvis, 2007] and empathy [Chen, Panksepp, & Lahvis, 2009; Langford et al., 2006]. With continued generation of mouse gene-targeted mice that are directly relevant to genetic linkages in ASD, there remains an urgent need to utilize a full suite of mouse behavioral tests that allows for a comprehensive assessment of the spectrum of social difficulties relevant to ASD. Using impairments in shared affect as an example, this paper explores potential avenues for collaboration between clinical and basic scientists, within an amply considered translational framework.

Rodent empathy and affective neuroscience.

In the past few years, several experimental studies have suggested that empathy occurs in the social lives of rodents. Thus, rodent behavioral models can now be developed to elucidate the mechanistic substrates of empathy at levels that have heretofore been unavailable. For example, the finding that mice from certain inbred strains express behavioral and physiological responses to conspecific distress, while others do not, underscores that the genetic underpinnings of empathy are specifiable and that they could be harnessed to develop new therapies for human psychosocial impairments. However, the advent of rodent models of empathy is met at the outset with a number of theoretical and semantic problems that are similar to those previously confronted by studies of empathy in humans. The distinct underlying components of empathy must be differentiated from one another and from lay usage of the term. The primary goal of this paper is to review a set of seminal studies that are directly relevant to developing a concept of empathy in rodents. We first consider some of the psychological phenomena that have been associated with empathy, and within this context, we consider the component processes, or endophenotypes of rodent empathy. We then review a series of recent experimental studies that demonstrate the capability of rodents to detect and respond to the affective state of their social partners. We focus primarily on experiments that examine how rodents share affective experiences of fear, but we also highlight how similar types of experimental paradigms can be utilized to evaluate the possibility that rodents share positive affective experiences. Taken together, these studies were inspired by Jaak Panksepp's theory that all mammals are capable of felt affective experiences.

Age-dependent and strain-dependent influences of morphine on mouse social investigation behavior.

Opioid-coded neural circuits play a substantial role in how individuals respond to drugs of abuse. Most individuals begin using such drugs during adolescence and within a social context. Several studies indicate that adolescent mice exhibit a heightened sensitivity to the effects of morphine, a prototypical opiate drug, but it is unclear whether these developmental differences are related to aspects of motivated behavior. Moreover, exposure to opioids within the rodent brain can alter the expression of social behavior, yet little is known about whether this relationship changes as a function of development or genetic variation. In this study, we conducted a series of experiments to characterize the relationship between genetic background, adolescent development and morphine-induced changes in mouse social investigation (SI). At two time points during adolescent development [postnatal days (PD) 25 and 45], social interactions of test mice of the gregarious C57BL/6J (B6) strain were more tolerant to the suppressive effects of morphine [effective dose 50 (ED50)=0.97 mg/kg and 2.17 mg/kg morphine, respectively] than test mice from the less social BALB/cJ (BALB) strain (ED50=0.61 mg/kg and 0.91 mg/kg morphine, respectively). By contrast, this strain-dependent difference was not evident among adult mice on PD 90 (ED50=1.07 mg/kg and 1.41 mg/kg morphine for BALB and B6 mice, respectively). An additional experiment showed that the ability of morphine to alter social responsiveness was not directly related to drug-induced changes in locomotor behavior. Finally, administration of morphine to stimulus mice on PD 25 reduced social investigation of test mice only when individuals were from the B6 genetic background. Overall, these results indicate that alterations in endogenous opioid systems are related to changes in SI that occur during adolescence, and that morphine administration may mimic rewarding aspects of social encounter.

Empathy is moderated by genetic background in mice.

Empathy, as originally defined, refers to an emotional experience that is shared among individuals. When discomfort or alarm is detected in another, a variety of behavioral responses can follow, including greater levels of nurturing, consolation or increased vigilance towards a threat. Moreover, changes in systemic physiology often accompany the recognition of distressed states in others. Employing a mouse model of cue-conditioned fear, we asked whether exposure to conspecific distress influences how a mouse subsequently responds to environmental cues that predict this distress. We found that mice are responsive to environmental cues that predict social distress, that their heart rate changes when distress vocalizations are emitted from conspecifics, and that genetic background substantially influences the magnitude of these responses. Specifically, during a series of pre-exposure sessions, repeated experiences of object mice that were exposed to a tone-shock (CS-UCS) contingency resulted in heart rate deceleration in subjects from the gregarious C57BL/6J (B6) strain, but not in subjects from the less social BALB/cJ (BALB) strain. Following the pre-exposure sessions, subjects were individually presented with the CS-only for 5 consecutive trials followed by 5 consecutive pairings of the CS with the UCS. Pre-exposure to object distress increased the freezing responses of B6 mice, but not BALB mice, on both the CS-only and the CS-UCS trials. These physiological and behavioral responses of B6 mice to social distress parallel features of human empathy. Our paradigm thus has construct and face validity with contemporary views of empathy, and provides unequivocal evidence for a genetic contribution to the expression of empathic behavior.

Differential entrainment of a social rhythm in adolescent mice.

Daily routines in animal activities range from sleep-wake cycles, to foraging bouts, to social interactions. Among animals living within groups, it is unclear whether the motivations that underlie social interactions respond to daily light-dark (LD) cycles or endogenous circadian rhythms. Employing two mouse strains (BALB/cJ [BALB] and C57BL/6J [B6]) with genetically based differences in social affect and circadian rhythms, we examined how social investigation (SI) is modulated by social deprivation and circadian factors. We found a genetic influence on SI that was moderated by the preceding duration of social deprivation, requiring 3-6 h of social isolation prior to testing. Following 6h of social deprivation, the SI responses of adolescent B6 mice were greater than those of BALB mice only when the isolation period was imposed during the dark phase of the LD cycle. When B6 mice were weaned into conditions of constant darkness, a novel, endogenous social rhythm emerged, which was characterized by two pronounced peaks of social responsiveness (relative to one peak under LD entrainment) that were separated by 12-h intervals. Irrespective of the lighting conditions during social isolation, the SI responses of adolescent BALB mice did not oscillate across the day. Similar strain-dependent patterns of sociability were evident within groups of mice that were left undisturbed in their home cage under LD entrainment or constant darkness. Overall, genetic influences on the social phenotypes of adolescent mice are thus moderated by an interaction between social deprivation and oscillations of an endogenous social rhythm that entrains to the LD cycle.

The cyclic AMP phenotype of fragile X and autism.

Cyclic AMP (cAMP) is a second messenger involved in many processes including mnemonic processing and anxiety. Memory deficits and anxiety are noted in the phenotype of fragile X (FX), the most common heritable cause of mental retardation and autism. Here we review reported observations of altered cAMP cascade function in FX and autism. Cyclic AMP is a potentially useful biochemical marker to distinguish autism comorbid with FX from autism per se and the cAMP cascade may be a viable therapeutic target for both FX and autism.

Abnormal liver development and resistance to 2,3,7,8-tetrachlorodibenzo-p-dioxin toxicity in mice carrying a mutation in the DNA-binding domain of the aryl hydrocarbon receptor.

The aryl hydrocarbon receptor (AHR) is known for its role in the adaptive and toxic responses to a large number of environmental contaminants, as well as its role in hepatovascular development. The classical AHR pathway involves ligand binding, nuclear translocation, heterodimerization with the AHR nuclear translocator (ARNT), and binding of the heterodimer to dioxin response elements (DREs), thereby modulating the transcription of an array of genes. The AHR has also been implicated in signaling events independent of nuclear localization and DNA binding, and it has been suggested that such pathways may play important roles in the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Here, we report the generation of a mouse model that expresses an AHR protein capable of ligand binding, interactions with chaperone proteins, functional heterodimerization with ARNT, and nuclear translocation, but is unable to bind DREs. Using this model, we provide evidence that DNA binding is required AHR-mediated liver development, as Ahr(dbd/dbd) mice exhibit a patent ductus venosus, similar to what is seen in Ahr(-/-) mice. Furthermore, Ahr(dbd/dbd) mice are resistant to TCDD-induced toxicity for all endpoints tested. These data suggest that DNA binding is necessary for AHR-mediated developmental and toxic signaling.

The cyclic AMP cascade is altered in the fragile X nervous system.

Fragile X syndrome (FX), the most common heritable cause of mental retardation and autism, is a developmental disorder characterized by physical, cognitive, and behavioral deficits. FX results from a trinucleotide expansion mutation in the fmr1 gene that reduces levels of fragile X mental retardation protein (FMRP). Although research efforts have focused on FMRP's impact on mGluR signaling, how the loss of FMRP leads to the individual symptoms of FX is not known. Previous studies on human FX blood cells revealed alterations in the cyclic adenosine 3', 5'-monophosphate (cAMP) cascade. We tested the hypothesis that cAMP signaling is altered in the FX nervous system using three different model systems. Induced levels of cAMP in platelets and in brains of fmr1 knockout mice are substantially reduced. Cyclic AMP induction is also significantly reduced in human FX neural cells. Furthermore, cAMP production is decreased in the heads of FX Drosophila and this defect can be rescued by reintroduction of the dfmr gene. Our results indicate that a robust defect in cAMP production in FX is conserved across species and suggest that cAMP metabolism may serve as a useful biomarker in the human disease population. Reduced cAMP induction has implications for the underlying causes of FX and autism spectrum disorders. Pharmacological agents known to modulate the cAMP cascade may be therapeutic in FX patients and can be tested in these models, thus supplementing current efforts centered on mGluR signaling.

Affiliative behavior, ultrasonic communication and social reward are influenced by genetic variation in adolescent mice.

Social approach is crucial for establishing relationships among individuals. In rodents, social approach has been studied primarily within the context of behavioral phenomena related to sexual reproduction, such as mating, territory defense and parental care. However, many forms of social interaction occur before the onset of reproductive maturity, which suggests that some processes underlying social approach among juvenile animals are probably distinct from those in adults. We conducted a longitudinal study of social investigation (SI) in mice from two inbred strains to assess the extent to which genetic factors influence the motivation for young mice to approach one another. Early-adolescent C57BL/6J (B6) mice, tested 4-6 days after weaning, investigated former cage mates to a greater degree than BALB/cJ (BALB) mice, irrespective of the sex composition within an interacting pair. This strain difference was not due to variation in maternal care, the phenotypic characteristics of stimulus mice or sensitivity to the length of isolation prior to testing, nor was it attributable to a general difference in appetitive motivation. Ultrasonic vocalization (USV) production was positively correlated with the SI responses of mice from both strains. Interestingly, several USV characteristics segregated with the genetic background of young mice, including a higher average frequency and shorter duration for the USVs emitted by B6 mice. An assessment of conditioned place preference responses indicated that there was a strain-dependent difference in the rewarding nature of social contact. As adolescent mice aged, SI responses gradually became less sensitive to genetic background and more responsive to the particular sex of individuals within an interacting pair. We have thus identified a specific, genetic influence on the motivation of early-adolescent mice to approach one another. Consistent with classical theories of motivation, which propose a functional relationship between behavioral approach and reward, our findings indicate that reward is a proximal mechanism through which genetic factors affect social motivation during early adolescence.

An experimental model for improving fat graft viability and purity.

BACKGROUND: Autologous fat is an excellent soft-tissue filler, given its abundance, ease of harvest, and natural appearance. However, graft longevity is unpredictable and is reported in the literature to be between 3 months and 8 years. METHODS: A genetically identical, age- and sex-matched mouse experiment was used to develop a model. Inguinal fat pads were subjected to different harvest and preparatory techniques. Primary endpoints-viability and purity-were assessed with the trypan blue viability assay and component counting with a hemocytometer. RESULTS: Viability and purity were highest after excisional harvest versus blunt or needle harvest, presumably secondary to differences in cellular trauma. Saline wash or centrifugation after harvest produced modest but statistically significant improvements in viability and purity. However, if grafts harvested in any fashion were treated with an initial collagenase digestion followed by an idealized centrifugation regimen and a single wash step, viability and purity were consistently 96 percent and 93 percent, respectively. CONCLUSIONS: Using an in vitro murine model, the authors have systematically developed a clinically practical model for creating a pure single-cell suspension of viable adipocytes that is reproducible, regardless of tissue harvest method.