Systemic proteome phenotypes reveal defective metabolic flexibility in Mecp2 mutants.

Genes mutated in monogenic neurodevelopmental disorders are broadly expressed. This observation supports the concept that monogenic neurodevelopmental disorders are systemic diseases that profoundly impact neurodevelopment. We tested the systemic disease model focusing on Rett syndrome, which is caused by mutations in MECP2. Transcriptomes and proteomes of organs and brain regions from Mecp2-null mice as well as diverse MECP2-null male and female human cells were assessed. Widespread changes in the steady-state transcriptome and proteome were identified in brain regions and organs of presymptomatic Mecp2-null male mice as well as mutant human cell lines. The extent of these transcriptome and proteome modifications was similar in cortex, liver, kidney, and skeletal muscle and more pronounced than in the hippocampus and striatum. In particular, Mecp2- and MECP2-sensitive proteomes were enriched in synaptic and metabolic annotated gene products, the latter encompassing lipid metabolism and mitochondrial pathways. MECP2 mutations altered pyruvate-dependent mitochondrial respiration while maintaining the capacity to use glutamine as a mitochondrial carbon source. We conclude that mutations in Mecp2/MECP2 perturb lipid and mitochondrial metabolism systemically limiting cellular flexibility to utilize mitochondrial fuels.

Systemic and local regulation of hematopoietic homeostasis in health and disease.

Hematopoietic stem cells (HSCs) generate all blood cell lineages responsible for tissue oxygenation, life-long hematopoietic homeostasis and immune protection. In adulthood, HSCs primarily reside in the bone marrow (BM) microenvironment, consisting of diverse cell types that constitute the stem cell 'niche'. The adaptability of the hematopoietic system is required to respond to the needs of the host, whether to maintain normal physiology or during periods of physical, psychosocial or environmental stress. Hematopoietic homeostasis is achieved by intricate coordination of systemic and local factors that orchestrate the function of HSCs throughout life. However, homeostasis is not a static process; it modulates HSC and progenitor activity in response to circadian rhythms coordinated by the central and peripheral nervous systems, inflammatory cues, metabolites and pathologic conditions. Here, we review local and systemic factors that impact hematopoiesis, focusing on the implications of aging, stress and cardiovascular disease.

Regulation of the hematopoietic stem cell pool by C-Kit-associated trogocytosis.

Hematopoietic stem cells (HSCs) are routinely mobilized from the bone marrow (BM) to the blood circulation for clinical transplantation. However, the precise mechanisms by which individual stem cells exit the marrow are not understood. This study identified cell-extrinsic and molecular determinants of a mobilizable pool of blood-forming stem cells. We found that a subset of HSCs displays macrophage-associated markers on their cell surface. Although fully functional, these HSCs are selectively niche-retained as opposed to stem cells lacking macrophage markers, which exit the BM upon forced mobilization. Macrophage markers on HSCs could be acquired through direct transfer by trogocytosis, regulated by receptor tyrosine-protein kinase C-Kit (CD117), from BM-resident macrophages in mouse and human settings. Our study provides proof of concept that adult stem cells utilize trogocytosis to rapidly establish and activate function-modulating molecular mechanisms.

Morphometric Analysis of the Thymic Epithelial Cell (TEC) Network Using Integrated and Orthogonal Digital Pathology Approaches.

The thymus, a central primary lymphoid organ of the immune system, plays a key role in T cell development. Surprisingly, the thymus is quite neglected with regards to standardized pathology approaches and practices for assessing structure and function. Most studies use multispectral flow cytometry to define the dynamic composition of the thymus at the cell population level, but they are limited by lack of contextual insight. This knowledge gap hinders our understanding of various thymic conditions and pathologies, particularly how they affect thymic architecture, and subsequently, immune competence. Here, we introduce a digital pathology pipeline to address these challenges. Our approach can be coupled to analytical algorithms and utilizes rationalized morphometric assessments of thymic tissue, ranging from tissue-wide down to microanatomical and ultrastructural levels. This pipeline enables the quantitative assessment of putative changes and adaptations of thymic structure to stimuli, offering valuable insights into the pathophysiology of thymic disorders. This versatile pipeline can be applied to a wide range of conditions that may directly or indirectly affect thymic structure, ranging from various cytotoxic stimuli inducing acute thymic involution to autoimmune diseases, such as myasthenia gravis. Here, we demonstrate applicability of the method in a mouse model of age-dependent thymic involution, both by confirming established knowledge, and by providing novel insights on intrathymic remodeling in the aged thymus. Our orthogonal pipeline, with its high versatility and depth of analysis, promises to be a valuable and practical toolset for both basic and translational immunology laboratories investigating thymic function and disease.

Validation of the Observer-Reported Communication Ability (ORCA) measure for individuals with Rett syndrome.

PURPOSE: The study goal was to validate the Observer-Reported Communication Ability (ORCA) measure for use with females with Rett Syndrome (RTT). METHODS: Qualitative interviews, including concept elicitation and cognitive interviewing methods, were conducted with 19 caregivers of individuals with RTT ages 2 and older. A quantitative study was then conducted in 279 caregivers to evaluate construct validity and reliability. RESULTS: After minor modifications were made, the modified ORCA measure was well understood and captured key communication concepts. Quantitative data showed evidence for reliable scores (α = 0.90, test-retest intraclass correlation = 0.88), minimal floor and no ceiling effects, and strong correlation with the Communication and Symbolic Behaviors Scale (r = 0.73). CONCLUSIONS: This study provided initial support that the modified ORCA measure is an acceptable caregiver-reported measure of communication ability for females with RTT. Future work should include evaluation of longitudinal validity of the measure and its associations with clinician- and performance-based measures in diverse samples.

Systemic Proteome Phenotypes Reveal Defective Metabolic Flexibility in Mecp2 Mutants.

Genes mutated in monogenic neurodevelopmental disorders are broadly expressed. This observation supports the concept that monogenic neurodevelopmental disorders are systemic diseases that profoundly impact neurodevelopment. We tested the systemic disease model focusing on Rett syndrome, which is caused by mutations in MECP2. Transcriptomes and proteomes of organs and brain regions from Mecp2-null mice as well as diverse MECP2-null male and female human cells were assessed. Widespread changes in the steady-state transcriptome and proteome were identified in brain regions and organs of presymptomatic Mecp2-null male mice as well as mutant human cell lines. The extent of these transcriptome and proteome modifications was similar in cortex, liver, kidney, and skeletal muscle and more pronounced than in the hippocampus and striatum. In particular, Mecp2- and MECP2-sensitive proteomes were enriched in synaptic and metabolic annotated gene products, the latter encompassing lipid metabolism and mitochondrial pathways. MECP2 mutations altered pyruvate-dependent mitochondrial respiration while maintaining the capacity to use glutamine as a mitochondrial carbon source. We conclude that mutations in Mecp2/MECP2 perturb lipid and mitochondrial metabolism systemically limiting cellular flexibility to utilize mitochondrial fuels.

Convergent cerebrospinal fluid proteomes and metabolic ontologies in humans and animal models of Rett syndrome.

MECP2 loss-of-function mutations cause Rett syndrome, a neurodevelopmental disorder resulting from a disrupted brain transcriptome. How these transcriptional defects are decoded into a disease proteome remains unknown. We studied the proteome of Rett cerebrospinal fluid (CSF) to identify consensus Rett proteome and ontologies shared across three species. Rett CSF proteomes enriched proteins annotated to HDL lipoproteins, complement, mitochondria, citrate/pyruvate metabolism, synapse compartments, and the neurosecretory protein VGF. We used shared Rett ontologies to select analytes for orthogonal quantification and functional validation. VGF and ontologically selected CSF proteins had genotypic discriminatory capacity as determined by receiver operating characteristic analysis in Mecp2 -/y and Mecp2 -/+ . Differentially expressed CSF proteins distinguished Rett from a related neurodevelopmental disorder, CDKL5 deficiency disorder. We propose that Mecp2 mutant CSF proteomes and ontologies inform putative mechanisms and biomarkers of disease. We suggest that Rett syndrome results from synapse and metabolism dysfunction.

Saliva MicroRNA Differentiates Children With Autism From Peers With Typical and Atypical Development.

OBJECTIVE: Clinical diagnosis of autism spectrum disorder (ASD) relies on time-consuming subjective assessments. The primary purpose of this study was to investigate the utility of salivary microRNAs for differentiating children with ASD from peers with typical development (TD) and non-autism developmental delay (DD). The secondary purpose was to explore microRNA patterns among ASD phenotypes. METHOD: This multicenter, prospective, case-control study enrolled 443 children (2-6 years old). ASD diagnoses were based on DSM-5 criteria. Children with ASD or DD were assessed with the Autism Diagnostic Observation Schedule II and Vineland Adaptive Behavior Scales II. MicroRNAs were measured with high-throughput sequencing. Differential expression of microRNAs was compared among the ASD (n = 187), TD (n = 125), and DD (n = 69) groups in the training set (n = 381). Multivariate logistic regression defined a panel of microRNAs that differentiated children with ASD and those without ASD. The algorithm was tested in a prospectively collected naïve set of 62 samples (ASD, n = 37; TD, n = 8; DD, n = 17). Relations between microRNA levels and ASD phenotypes were explored. RESULT: Fourteen microRNAs displayed differential expression (false discovery rate < 0.05) among ASD, TD, and DD groups. A panel of 4 microRNAs (controlling for medical/demographic covariates) best differentiated children with ASD from children without ASD in training (area under the curve = 0.725) and validation (area under the curve = 0.694) sets. Eight microRNAs were associated (R > 0.25, false discovery rate < 0.05) with social affect, and 10 microRNAs were associated with restricted/repetitive behavior. CONCLUSION: Salivary microRNAs are "altered" in children with ASD and associated with levels of ASD behaviors. Salivary microRNA collection is noninvasive, identifying ASD-status with moderate accuracy. A multi-"omic" approach using additional RNA families could improve accuracy, leading to clinical application. CLINICAL TRIAL REGISTRATION INFORMATION: A Salivary miRNA Diagnostic Test for Autism; https://clinicaltrials.gov/; NCT02832557.

Spinal cord injury causes chronic bone marrow failure.

Spinal cord injury (SCI) causes immune dysfunction, increasing the risk of infectious morbidity and mortality. Since bone marrow hematopoiesis is essential for proper immune function, we hypothesize that SCI disrupts bone marrow hematopoiesis. Indeed, SCI causes excessive proliferation of bone marrow hematopoietic stem and progenitor cells (HSPC), but these cells cannot leave the bone marrow, even after challenging the host with a potent inflammatory stimulus. Sequestration of HSPCs in bone marrow after SCI is linked to aberrant chemotactic signaling that can be reversed by post-injury injections of Plerixafor (AMD3100), a small molecule inhibitor of CXCR4. Even though Plerixafor liberates HSPCs and mature immune cells from bone marrow, competitive repopulation assays show that the intrinsic long-term functional capacity of HSPCs is still impaired in SCI mice. Together, our data suggest that SCI causes an acquired bone marrow failure syndrome that may contribute to chronic immune dysfunction.

The levo enantiomer of amphetamine increases memory consolidation and gene expression in the hippocampus without producing locomotor stimulation.

Dextro-amphetamine enhances memory and other cognitive functions in animals and humans. The use of d-amphetamine as a memory enhancer, however, is limited by a robust stimulatory side-effect profile caused by release of dopamine. The levo enantiomer of amphetamine has been shown to be considerably less effective as a dopamine releaser and less potent in producing the stimulatory effects characteristic of d-amphetamine. In order to determine whether l-amphetamine and the structurally related compound, l-methamphetamine, retain cognitive-enhancing effects despite their lack of stimulatory activity, we administered the compounds to rats prior to activity monitoring experiments, and in different animals, immediately after training on inhibitory avoidance and object recognition tasks. Results demonstrated that l-amphetamine and l-methamphetamine did not increase locomotion and stereotypies beyond control levels, but did produce significant memory enhancement. In addition, l-amphetamine and l-methamphetamine alleviated scopolamine-induced amnesia in the inhibitory avoidance task. In all cases, these compounds produced an effect comparable to that of d-amphetamine, but required only one quarter of the d-amphetamine dose to produce the same effect size. We also found that l-amphetamine modulates learning-induced changes in hippocampal Arc/Arg3.1 protein synthesis that correlate with memory consolidation. These results suggest that l-amphetamine and l-methamphetamine are potent memory enhancers in rats and may ultimately be useful for treating memory disorders in humans.

Human immune cells infiltrate the spinal cord and impair recovery after spinal cord injury in humanized mice.

Humanized mice can be used to better understand how the human immune system responds to central nervous system (CNS) injury and inflammation. The optimal parameters for using humanized mice in preclinical CNS injury models need to be established for appropriate use and interpretation. Here, we show that the developmental age of the human immune system significantly affects anatomical and functional outcome measures in a preclinical model of traumatic spinal cord injury (SCI). Specifically, it takes approximately 3-4 months for a stable and functionally competent human immune system to develop in neonatal immune compromised mice after they are engrafted with human umbilical cord blood stem cells. Humanized mice receiving a SCI before or after stable engraftment exhibit significantly different neuroinflammatory profiles. Importantly, the development of a mature human immune system was associated with worse lesion pathology and neurological recovery after SCI. In these mice, human T cells infiltrate the spinal cord lesion and directly contact human macrophages. Together, data in this report establish an optimal experimental framework for using humanized mice to help translate promising preclinical therapies for CNS injury.

Validation of a Salivary RNA Test for Childhood Autism Spectrum Disorder.

Background: The diagnosis of autism spectrum disorder (ASD) relies on behavioral assessment. Efforts to define biomarkers of ASD have not resulted in an objective, reliable test. Studies of RNA levels in ASD have demonstrated potential utility, but have been limited by a focus on single RNA types, small sample sizes, and lack of developmental delay controls. We hypothesized that a saliva-based poly-"omic" RNA panel could objectively distinguish children with ASD from their neurotypical peers and children with non-ASD developmental delay. Methods: This multi-center cross-sectional study included 456 children, ages 19-83 months. Children were either neurotypical (n = 134) or had a diagnosis of ASD (n = 238), or non-ASD developmental delay (n = 84). Comprehensive human and microbial RNA abundance was measured in the saliva of all participants using unbiased next generation sequencing. Prior to analysis, the sample was randomly divided into a training set (82% of subjects) and an independent validation test set (18% of subjects). The training set was used to develop an RNA-based algorithm that distinguished ASD and non-ASD children. The validation set was not used in model development (feature selection or training) but served only to validate empirical accuracy. Results: In the training set (n = 372; mean age 51 months; 75% male; 51% ASD), a set of 32 RNA features (controlled for demographic and medical characteristics), identified ASD status with a cross-validated area under the curve (AUC) of 0.87 (95% CI: 0.86-0.88). In the completely separate validation test set (n = 84; mean age 50 months; 85% male; 60% ASD), the algorithm maintained an AUC of 0.88 (82% sensitivity and 88% specificity). Notably, the RNA features were implicated in physiologic processes related to ASD (axon guidance, neurotrophic signaling). Conclusion: Salivary poly-omic RNA measurement represents a novel, non-invasive approach that can accurately identify children with ASD. This technology could improve the specificity of referrals for ASD evaluation or provide objective support for ASD diagnoses.

Drug development for neurodevelopmental disorders: lessons learned from fragile X syndrome.

Neurodevelopmental disorders such as fragile X syndrome (FXS) result in lifelong cognitive and behavioural deficits and represent a major public health burden. FXS is the most frequent monogenic form of intellectual disability and autism, and the underlying pathophysiology linked to its causal gene, FMR1, has been the focus of intense research. Key alterations in synaptic function thought to underlie this neurodevelopmental disorder have been characterized and rescued in animal models of FXS using genetic and pharmacological approaches. These robust preclinical findings have led to the implementation of the most comprehensive drug development programme undertaken thus far for a genetically defined neurodevelopmental disorder, including phase IIb trials of metabotropic glutamate receptor 5 (mGluR5) antagonists and a phase III trial of a GABAB receptor agonist. However, none of the trials has been able to unambiguously demonstrate efficacy, and they have also highlighted the extent of the knowledge gaps in drug development for FXS and other neurodevelopmental disorders. In this Review, we examine potential issues in the previous studies and future directions for preclinical and clinical trials. FXS is at the forefront of efforts to develop drugs for neurodevelopmental disorders, and lessons learned in the process will also be important for such disorders.

Arbaclofen in fragile X syndrome: results of phase 3 trials.

BACKGROUND: Arbaclofen improved multiple abnormal phenotypes in animal models of fragile X syndrome (FXS) and showed promising results in a phase 2 clinical study. The objective of the study is to determine safety and efficacy of arbaclofen for social avoidance in FXS. METHODS: Two phase 3 placebo-controlled trials were conducted, a flexible dose trial in subjects age 12-50 (209FX301, adolescent/adult study) and a fixed dose trial in subjects age 5-11 (209FX302, child study). The primary endpoint for both trials was the Social Avoidance subscale of the Aberrant Behavior Checklist-Community Edition, FXS-specific (ABC-CFX). Secondary outcomes included other ABC-CFX subscale scores, Clinical Global Impression-Improvement (CGI-I), Clinical Global Impression-Severity (CGI-S), and Vineland Adaptive Behavior Scales, Second Edition (Vineland-II) Socialization domain score. RESULTS: A total 119 of 125 randomized subjects completed the adolescent/adult study (n = 57 arbaclofen, 62 placebo) and 159/172 completed the child study (arbaclofen 5 BID n = 38; 10 BID n = 39; 10 TID n = 38; placebo n = 44). There were no serious adverse events (AEs); the most common AEs included somatic (headache, vomiting, nausea), neurobehavioral (irritability/agitation, anxiety, hyperactivity), decreased appetite, and infectious conditions, many of which were also common on placebo. In the combined studies, there were 13 discontinuations (n = 12 arbaclofen, 1 placebo) due to AEs (all neurobehavioral). The adolescent/adult study did not show benefit for arbaclofen over placebo for any measure. In the child study, the highest dose group showed benefit over placebo on the ABC-CFX Irritability subscale (p = 0.03) and Parenting Stress Index (PSI, p = 0.03) and trends toward benefit on the ABC-CFX Social Avoidance and Hyperactivity subscales (both p < 0.1) and CGI-I (p = 0.119). Effect size in the highest dose group was similar to effect sizes for FDA-approved serotonin reuptake inhibitors (SSRIs). CONCLUSIONS: Arbaclofen did not meet the primary outcome of improved social avoidance in FXS in either study. Data from secondary measures in the child study suggests younger patients may derive benefit, but additional studies with a larger cohort on higher doses would be required to confirm this finding. The reported studies illustrate the challenges but represent a significant step forward in translating targeted treatments from preclinical models to clinical trials in humans with FXS.

Arbaclofen in Children and Adolescents with Autism Spectrum Disorder: A Randomized, Controlled, Phase 2 Trial.

Several lines of emerging data point to an imbalance between neuronal excitation and inhibition in at least a subgroup of individuals with autism spectrum disorder (ASD), including in those with fragile X syndrome (FXS), one of the most common genetic syndromes within ASD. In animal models of FXS and of ASD, GABA-B agonists have improved both brain and behavioral phenotypes, including social behavior. A phase 2 randomized, placebo-controlled, crossover trial found that the GABA-B agonist arbaclofen improved social avoidance symptoms in FXS. A pilot open-label trial of arbaclofen suggested similar benefits in ASD. We therefore evaluated arbaclofen in a randomized, placebo-controlled, phase 2 study of 150 participants, aged 5-21 years, with ASD. No difference from placebo was detected on the primary outcome measure, the parent-rated Aberrant Behavior Checklist Social Withdrawal/Lethargy subscale. However, a specified secondary analysis found improvement on the clinician-rated Clinical Global Impression of Severity. An exploratory post hoc analysis of participants with a consistent rater across the trial revealed greater improvement in the Vineland Adaptive Behavior Scales II socialization domain in participants receiving arbaclofen. Affect lability (11%) and sedation (9%) were the most common adverse events. In this exploratory study, secondary analyses suggest that arbaclofen may have the potential to improve symptoms in some children with ASD, but further study will be needed to replicate and extend these initial findings.

Transgenic expression of HuR increases vasogenic edema and impedes functional recovery in rodent ischemic stroke.

BACKGROUND AND PURPOSE: Ischemic stroke produces significant morbidity and mortality, and acute interventions are limited by short therapeutic windows. Novel approaches to neuroprotection and neurorepair are necessary. HuR is an RNA-binding protein (RBP) which modulates RNA stability and translational efficiency of genes linked to ischemic stroke injury. METHODS: Using a transgenic (Tg) mouse model, we examined the impact of ectopic HuR expression in astrocytes on acute injury evolution after transient middle cerebral artery occlusion (tMCAO). RESULTS: HuR transgene expression was detected in astrocytes in perilesional regions and contralaterally. HuR Tg mice did not improve neurologically 72h after injury, whereas littermate controls did. In Tg mice, increased cerebral vascular permeability and edema were observed. Infarct volume was not affected by the presence of the transgene. CONCLUSIONS: Ectopic expression of HuR in astrocytes worsens outcome after transient ischemic stroke in mice in part by increasing vasogenic cerebral edema. These findings suggest that HuR could be a therapeutic target in cerebral ischemia/reperfusion.

High-dose estrogen treatment at reperfusion reduces lesion volume and accelerates recovery of sensorimotor function after experimental ischemic stroke.

Estrogens have previously been shown to protect the brain against acute ischemic insults, by potentially augmenting cerebrovascular function after ischemic stroke. The current study hypothesized that treatment with sustained release of high-dose 17ß-estradiol (E2) at the time of reperfusion from middle cerebral artery occlusion (MCAO) in rats would attenuate reperfusion injury, augment post-stroke angiogenesis and cerebral blood flow, and attenuate lesion volume. Female Wistar rats underwent ovariectomy, followed two weeks later by transient, two-hour right MCAO (tMCAO) and treatment with E2 (n=13) or placebo (P; n=12) pellets starting at reperfusion. E2 treatment resulted in significantly smaller total lesion volume, smaller lesions within striatal and cortical brain regions, and less atrophy of the ipsilateral hemisphere after six weeks of recovery. E2-treated animals exhibited accelerated recovery of contralateral forelimb sensorimotor function in the cylinder test. Magnetic resonance imaging (MRI) showed that E2 treatment reduced the formation of lesion cysts, decreased lesion volume, and increased lesional cerebral blood flow (CBF). K(trans), a measure of vascular permeability, was increased in the lesions. This finding, which represents lesion neovascularization, was not altered by E2 treatment. Ischemic stroke-related angiogenesis and vessel formation was confirmed with immunolabeling of brain tissue and was not altered with E2 treatment. In summary, E2 treatment administered immediately following reperfusion significantly reduced lesion size, cyst formation, and brain atrophy while improving lesional CBF and accelerating recovery of functional deficits in a rat model of ischemic stroke.

Alvimopan: an oral, peripherally acting, mu-opioid receptor antagonist for the treatment of opioid-induced bowel dysfunction--a 21-day treatment-randomized clinical trial.

Alvimopan has been shown to reverse the inhibitory effect of opioids on gastrointestinal transit without affecting analgesia. We evaluated oral alvimopan, 0.5 or 1 mg, versus placebo, once daily for 21 days, in 168 patients with opioid-induced bowel dysfunction (OBD) who were receiving chronic opioid therapy (minimum, 1 month) for nonmalignant pain (n = 148) or opioid dependence (n = 20). The primary outcome was the proportion of patients having at least one bowel movement (BM) within 8 hours of study drug on each day during the 21-day treatment period. Averaged over the 21-day treatment period, 54%, 43%, and 29% of patients had a BM within 8 hours after alvimopan 1 mg, 0.5 mg, or placebo, respectively (P < .001). Secondary outcomes of median times to first BM were 3, 7, and 21 hours after initial doses of 1 mg, 0.5 mg, and placebo, respectively (P < .001; 1 mg vs placebo). Weekly BMs and overall patient satisfaction were increased after the 1-mg dose (P < .001 at weeks 1 and 2 vs placebo, and P = .046, respectively). Treatment-emergent adverse events were primarily bowel-related, occurred during the first week of treatment, and were of mild to moderate severity. Alvimopan was generally well tolerated and did not antagonize opioid analgesia. Patients treated with chronic opioid therapy often experience opioid-induced bowel dysfunction as a result of undesirable effects on peripheral opioid receptors located in the gastrointestinal tract. Alvimopan, a novel peripheral opioid mu-receptor antagonist, has demonstrated significant efficacy for the management of opioid-induced bowel dysfunction without compromise of centrally mediated opioid-induced analgesia.

Traumatic spinal cord injury in mice with human immune systems.

Mouse models have provided key insight into the cellular and molecular control of human immune system function. However, recent data indicate that extrapolating the functional capabilities of the murine immune system into humans can be misleading. Since immune cells significantly affect neuron survival and axon growth and also are required to defend the body against infection, it is important to determine the pathophysiological significance of spinal cord injury (SCI)-induced changes in human immune system function. Research projects using monkeys or humans would be ideal; however, logistical and ethical barriers preclude detailed mechanistic studies in either species. Humanized mice, i.e., immunocompromised mice reconstituted with human immune cells, can help overcome these barriers and can be applied in various experimental conditions that are of interest to the SCI community. Specifically, newborn NOD-SCID-IL2rg(null) (NSG) mice engrafted with human CD34(+) hematopoietic stem cells develop normally without neurological impairment. In this report, new data show that when mice with human immune systems receive a clinically-relevant spinal contusion injury, spontaneous functional recovery is indistinguishable from that achieved after SCI using conventional inbred mouse strains. Moreover, using routine immunohistochemical and flow cytometry techniques, one can easily phenotype circulating human immune cells and document the composition and distribution of these cells in the injured spinal cord. Lesion pathology in humanized mice is typical of mouse contusion injuries, producing a centralized lesion epicenter that becomes occupied by phagocytic macrophages and lymphocytes and enclosed by a dense astrocytic scar. Specific human immune cell types, including three distinct subsets of human monocytes, were readily detected in the blood, spleen and liver. Future studies that aim to understand the functional consequences of manipulating the neuro-immune axis after SCI should consider using the humanized mouse model. Humanized mice represent a powerful tool for improving the translational value of pre-clinical SCI data.