A somato-cognitive action network alternates with effector regions in motor cortex.

Motor cortex (M1) has been thought to form a continuous somatotopic homunculus extending down the precentral gyrus from foot to face representations1,2, despite evidence for concentric functional zones3 and maps of complex actions4. Here, using precision functional magnetic resonance imaging (fMRI) methods, we find that the classic homunculus is interrupted by regions with distinct connectivity, structure and function, alternating with effector-specific (foot, hand and mouth) areas. These inter-effector regions exhibit decreased cortical thickness and strong functional connectivity to each other, as well as to the cingulo-opercular network (CON), critical for action5 and physiological control6, arousal7, errors8 and pain9. This interdigitation of action control-linked and motor effector regions was verified in the three largest fMRI datasets. Macaque and pediatric (newborn, infant and child) precision fMRI suggested cross-species homologues and developmental precursors of the inter-effector system. A battery of motor and action fMRI tasks documented concentric effector somatotopies, separated by the CON-linked inter-effector regions. The inter-effectors lacked movement specificity and co-activated during action planning (coordination of hands and feet) and axial body movement (such as of the abdomen or eyebrows). These results, together with previous studies demonstrating stimulation-evoked complex actions4 and connectivity to internal organs10 such as the adrenal medulla, suggest that M1 is punctuated by a system for whole-body action planning, the somato-cognitive action network (SCAN). In M1, two parallel systems intertwine, forming an integrate-isolate pattern: effector-specific regions (foot, hand and mouth) for isolating fine motor control and the SCAN for integrating goals, physiology and body movement.

Functional parcellation of the neonatal cortical surface.

The cerebral cortex is organized into distinct but interconnected cortical areas, which can be defined by abrupt differences in patterns of resting state functional connectivity (FC) across the cortical surface. Such parcellations of the cortex have been derived in adults and older infants, but there is no widely used surface parcellation available for the neonatal brain. Here, we first demonstrate that existing parcellations, including surface-based parcels derived from older samples as well as volume-based neonatal parcels, are a poor fit for neonatal surface data. We next derive a set of 283 cortical surface parcels from a sample of n = 261 neonates. These parcels have highly homogenous FC patterns and are validated using three external neonatal datasets. The Infomap algorithm is used to assign functional network identities to each parcel, and derived networks are consistent with prior work in neonates. The proposed parcellation may represent neonatal cortical areas and provides a powerful tool for neonatal neuroimaging studies.

Brexanolone Treatment in a Real-World Patient Population: A Case Series and Pilot Feasibility Study of Precision Neuroimaging.

PURPOSE/BACKGROUND: Brexanolone is approved for postpartum depression (PPD) by the United States Food and Drug Administration. Brexanolone has outperformed placebo in clinical trials, but less is known about the efficacy in real-world patients with complex social and medical histories. Furthermore, the impact of brexanolone on large-scale brain systems such as changes in functional connectivity (FC) is unknown. METHODS/PROCEDURES: We tracked changes in depressive symptoms across a diverse group of patients who received brexanolone at a large medical center. Edinburgh Postnatal Depression Scale (EPDS) scores were collected through chart review for 17 patients immediately prior to infusion through approximately 1 year postinfusion. In 2 participants, we performed precision functional neuroimaging (pfMRI), including before and after treatment in 1 patient. pfMRI collects many hours of data in individuals for precision medicine applications and was performed to assess the feasibility of investigating changes in FC with brexanolone. FINDINGS/RESULTS: The mean EPDS score immediately postinfusion was significantly lower than the mean preinfusion score (mean change [95% CI]: 10.76 [7.11-14.40], t (15) = 6.29, P < 0.0001). The mean EPDS score stayed significantly lower at 1 week (mean difference [95% CI]: 9.50 [5.23-13.76], t (11) = 4.90, P = 0.0005) and 3 months (mean difference [95% CI]: 9.99 [4.71-15.27], t (6) = 4.63, P = 0.0036) postinfusion. Widespread changes in FC followed infusion, which correlated with EPDS scores. IMPLICATIONS/CONCLUSIONS: Brexanolone is a successful treatment for PPD in the clinical setting. In conjunction with routine clinical care, brexanolone was linked to a reduction in symptoms lasting at least 3 months. pfMRI is feasible in postpartum patients receiving brexanolone and has the potential to elucidate individual-specific mechanisms of action.

The Use of Systemically Absorbed Drugs to Explore An In Vitro Bioequivalence Approach For Comparing Non-Systemically Absorbed Active Pharmaceutical Ingredients in Drug Products For Use in Dogs.

PURPOSE: Currently, for veterinary oral formulations containing one or more active pharmaceutical ingredient (API) that are not systemically absorbed and act locally within the gastrointestinal (GI) tract, the use of terminal clinical endpoint bioequivalence (BE) studies is the only option for evaluating product BE. This investigation explored the use of a totality of evidence approach as an alternative to these terminal studies. METHODS: Three formulations of tablets containing ivermectin plus praziquantel were manufactured to exhibit distinctly different in vitro release characteristics. Because these APIs are highly permeable, plasma drug concentrations served as a biomarker of in vivo dissolution. Tablets were administered to 27 healthy Beagle dogs (3-way crossover) and the rate and extent of exposure of each API for each formulation was compared in a pairwise manner. These results were compared to product relative in vitro dissolution profiles in 3 media. In vivo and in vitro BE predictions were compared. RESULTS: In vivo/in vitro inconsistencies in product relative performance were observed with both compounds when considering product performance across the 3 dissolution media. Formulation comparisons flagged major differences that could explain this outcome. CONCLUSIONS: The finding of an inconsistent in vivo/in vitro relationship confirmed that in vitro dissolution alone cannot assure product BE for veterinary locally acting GI products. However, when combined with a comparison of product composition and manufacturing method, this totality of evidence approach can successfully alert scientists to potential therapeutic inequivalence, thereby supporting FDA's efforts to Replace, Reduce, and/or Refine terminal animal studies.

Network-specific selectivity of functional connections in the neonatal brain.

The adult human brain is organized into functional brain networks, groups of functionally connected segregated brain regions. A key feature of adult functional networks is long-range selectivity, the property that spatially distant regions from the same network have higher functional connectivity than spatially distant regions from different networks. Although it is critical to establish the status of functional networks and long-range selectivity during the neonatal period as a foundation for typical and atypical brain development, prior work in this area has been mixed. Although some studies report distributed adult-like networks, other studies suggest that neonatal networks are immature and consist primarily of spatially isolated regions. Using a large sample of neonates (n = 262), we demonstrate that neonates have long-range selective functional connections for the default mode, fronto-parietal, and dorsal attention networks. An adult-like pattern of functional brain networks is evident in neonates when network-detection algorithms are tuned to these long-range connections, when using surface-based registration (versus volume-based registration), and as per-subject data quantity increases. These results help clarify factors that have led to prior mixed results, establish that key adult-like functional network features are evident in neonates, and provide a foundation for studies of typical and atypical brain development.

Pharmacokinetic comparison of six anthelmintics in sheep, goats, and cattle.

This study was initiated to determine whether a comparative pharmacokinetic (PK) approach could be used to expand the pool of approved anthelmintics for minor ruminant species. Accordingly, the PK profiles of six anthelmintics (levamisole, albendazole, fenbendazole, moxidectin, doramectin, and ivermectin) in sheep, goats, and cattle were determined. The PK values determined for each anthelmintic included Tmax , Tlast , Cmax , AUC, AUC/dose, and Cmax /dose. The results of this study demonstrate that a comparative PK approach does not show commonality in the way these six anthelmintics are individually processed by these three ruminants. While some drugs demonstrated identical PK profiles between sheep and goats, none of these drugs demonstrated PK profiles in sheep and goats comparable to the PK profiles found in cattle. The results from this study suggest drug approval across these three ruminants is not a viable concept. However, the resulting PK profiles for each combination of drug and ruminant species represents a new dataset that can be used to support the US FDA Center for Veterinary Medicine's Minor Use/Minor Species indexing process for drug approvals in minor species such as sheep and goats.

Proteomics profiling of swine serum following lipopolysaccharide stimulation.

RATIONALE: There are no approved animal drugs for management of inflammation in swine due to lack of validated animal models. To assess efficacy, it was essential to develop proteomics approaches to identify suitable biomarkers of inflammation as presented in this study. METHODS: Serum samples were collected from a group of four pigs prior to (baseline) and 24 and 48 h following lipopolysaccharide (LPS) stimulation to reveal proteomic changes during inflammation. Two other pigs served as untreated controls. Proteins were separated by either one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) or two-dimensional (2D) gel electrophoresis (2DE) prior to analysis by nano-flow liquid chromatography (nLC) coupled to tandem mass spectrometry (MS/MS). RESULTS: We identified 165 proteins using SDS-PAGE, of which 47 proteins were also detected by 2DE prior to nLC/MS/MS. More than half (72%) of all characterized proteins were modulated as a result of LPS stimulation, many of which are known to be involved with innate and adaptive immunity. Pig serum samples obtained 24 h after LPS initiation of inflammation showed protein modulations of serum albumin, serotransferrin, light and heavy immunoglobulin chains (IGs), and major acute phase proteins including haptoglobin (HPT), serum amyloid A2 (SAA2), C-reactive protein (CRP), ß-2-glycoprotein 1 (B-2GP1), alpha-2-HS-glycoprotein (A2HS), α-1-antitrypsin (A1AT), and α-1-acid glycoprotein (A1AG). SAA2 was distinguished from the other SAA isoforms by the unique peptide sequence of SAA2. CONCLUSIONS: The results provided proteomics analysis of swine serum due to LPS stimulation and indicated the importance of SAA2, which appears to be unique and may be regarded as a potential clinical diagnostic biomarker of inflammation.

Impact of ABCB1 genotype in Collies on the pharmacokinetics of R- and S-fexofenadine.

Thirty-two Collies were used to determine the impact of ABCB1 genotype and phenotype on the plasma pharmacokinetics of fexofenadine's (Fex) R- and S-enantiomers after bolus Fex administration, as human P-gp exhibits stereoselectivity. Each Collie's ABCB1 genotype and ivermectin (IVM) sensitivity (phenotype) was determined prior to study enrolment. Wild-type (WT) Collies had lower plasma concentrations of the individual enantiomers as compared to heterozygous IVM nonsensitive (HNS), heterozygous IVM-sensitive (HS) and homozygous mutant (MUT) Collies. Based on pairwise statistical comparison, WT Collies had statistically significantly lower (AUC0-last ) and peak (Cmax ) values compared to HS, HNS and MUT Collies. Tmax was not influenced by genotype/phenotype. Inter-individual variability in PK metrics tended to be largest for WT Collies. Although the influence of genotype/phenotype on Fex PK occurred with the individual isomers, impairment of S-Fex absorption, particularly in the MUT dogs, exceeded that associated with R-Fex. Since Fex elimination occurs primarily via biliary excretion via a transporter other than P-glycoprotein, and based upon our understanding of Fex absorption kinetics, we attributed these differences primarily to the absorption portion of the profile. These differences are expressed in a stereo-specific manner. These results demonstrate the potential negative impact on estimates of drug effectiveness and toxicity, especially for P-gp substrates that do not exhibit Central Nervous System toxicities.

Influence of ABCB1 Genotype in Collies on the Pharmacokinetics and Pharmacodynamics of Loperamide in a Dose-Escalation Study.

Thirty-three Collies (14 male and 19 female) were used in a dose-escalation study to determine the impact of ABCB1 genotype on loperamide pharmacokinetics (PK) and pharmacodynamics (PD). Loperamide was orally administered in four ascending doses (0.01, 0.05, 0.1, or 0.2 mg/kg) over a 4-wk period to fasted Collies. Comparisons were made within each dose to genotype, phenotype, and whether Collies received three (3D) or four (4D) loperamide doses. The 3D and 4D groupings had statistically significant differences in systemic drug exposure (defined by the area under the concentration-versus-time profile estimated from time zero to the last quantifiable drug concentration, AUC0-last). In contrast, statistical differences in AUC0-last only occurred in the comparison between wild-type (WT) Collies versus homozygous mutant (Mut) Collies administered 0.1 mg/kg. Statistical differences in the proportionality relationship were observed when comparing 3D to 4D Collies, and the WT to Mut Collies. Intersubject variability in drug exposure tended to be twice as high between Mut and WT Collies. Associations were observed between systemic drug exposure and ataxia or depression but not between systemic drug exposure and mydriasis or salivation. Thus, Collies expressing the greatest sensitivity to CNS-associated effects of loperamide (Mut) tended to have higher drug exposure compared with those less sensitive to the adverse effects of loperamide. Genotype and phenotype only partially explained differences in loperamide PK and PD, suggesting this relationship may not be straightforward and that other factors need to be considered. Accordingly, the PD and PK of one P-glycoprotein substrate only partially predicted the likelihood of adverse responses to unrelated substrates.

Challenges in exploring the cytochrome P450 system as a source of variation in canine drug pharmacokinetics.

The cytochrome P450 (CYP) superfamily constitutes a collection of enzymes responsible for the metabolism of a wide array of endo- and xenobiotic compounds. Much of the knowledge on substrate specificity and genetic identification of the various CYP isoforms is derived from research in rodents and humans and only limited information has been captured in the dog. Currently, there exist many gaps in our knowledge of canine CYP diversity as a result of the paucity of studies focusing on canine CYPs, canine CYP polymorphisms, and the therapeutic consequences of these genetic variants. Challenges engendered by this lack of information is further amplified by inter- and intraspecies differences in the specificity and affinity of substrates and inhibitors, prohibiting a simple extrapolation of probe substances used in human CYP research. This creates a need to develop and validate canine-specific CYP probes. Failure to understand this potential metabolic and pharmacogenomic diversity can also influence the interpretation of data generated in dogs to support human drug development. It is with these objectives in mind that we provide an overview of what is currently known about canine CYPs with the hope that it will encourage further exploration into this important area of research.

A Novel Cognitive Training Program Targets Stimulus-Driven Attention to Alter Symptoms, Behavior, and Neural Circuitry in Pediatric Anxiety Disorders: Pilot Clinical Trial.

Objective: Pediatric anxiety disorders are associated with increased stimulus-driven attention (SDA), the involuntary capture of attention by salient stimuli. Increased SDA is linked to increased activity in the right ventrolateral prefrontal cortex (rVLPFC), especially in the portion corresponding to the ventral attention network (VAN). In this study, we present a small clinical trial using a novel attention training program designed to treat pediatric anxiety by decreasing SDA and activity in the rVLPFC. Methods: Children ages 8-12 with anxiety disorders (n = 18) participated in eight sessions of attention training over a 4-week period. At baseline and after completing training, participants completed clinical anxiety measures and a battery of cognitive tasks designed to measure three different aspects of attention: SDA, goal-oriented attention, and threat bias. A subset of participants (n = 12) underwent baseline and post-training neuroimaging while engaged in an SDA task. Brain analyses focused on activity within the rVLPFC. Results: Parent (p < 0.001)-, child (p < 0.002)-, and clinician-rated (p < 0.02) anxiety improved significantly over the course of training. Training significantly altered SDA [F(1,92) = 8.88, corrected p-value (pcor) < 0.012, uncorrected p-value (puncor) < 0.004]. Anxiety improvement correlated with improvements in goal-directed attention [r(10) = 0.60, pcor < 0.12 puncor < 0.04]. Within an area of the rVLPFC corresponding to the cingulo-opercular network (CON), there was a main effect of training [F(1,20) = 6.75, pcor < 0.16, puncor < 0.02], with decreasing signal across training. There was a significant interaction between training and anxiety on this region's activity [F(1,20) = 9.48, pcor < 0.048, puncor < 0.006]. Post hoc testing revealed that post-training activity within this CON area correlated with residual anxiety [r(10) = 0.68, p < 0.02]. Conclusions: SDA and rVLPFC neural activity may be novel therapeutic targets in pediatric anxiety. After undergoing a training paradigm aimed at modifying this aspect of attention and its underlying neural circuitry, patients showed lower anxiety, changes in SDA and goal-oriented attention, and decreased activity in the CON portion of the rVLPFC.

Multi-echo Acquisition and Thermal Denoising Advances Infant Precision Functional Imaging.

The characterization of individual functional brain organization with Precision Functional Mapping has provided important insights in recent years in adults. However, little is known about the ontogeny of inter-individual differences in brain functional organization during human development, but precise characterization of systems organization during periods of high plasticity might be most influential towards discoveries promoting lifelong health. Collecting and analyzing precision fMRI data during early development has unique challenges and emphasizes the importance of novel methods to improve data acquisition, processing, and analysis strategies in infant samples. Here, we investigate the applicability of two such methods from adult MRI research, multi-echo (ME) data acquisition and thermal noise removal with Noise reduction with distribution corrected principal component analysis (NORDIC), in precision fMRI data from three newborn infants. Compared to an adult example subject, T2* relaxation times calculated from ME data in infants were longer and more variable across the brain, pointing towards ME acquisition being a promising tool for optimizing developmental fMRI. The application of thermal denoising via NORDIC increased tSNR and the overall strength of functional connections as well as the split-half reliability of functional connectivity matrices in infant ME data. While our findings related to NORDIC denoising are coherent with the adult literature and ME data acquisition showed high promise, its application in developmental samples needs further investigation. The present work reveals gaps in our understanding of the best techniques for developmental brain imaging and highlights the need for further developmentally-specific methodological advances and optimizations, towards precision functional imaging in infants.

Neonatal neural responses to novelty related to behavioral inhibition at 1 year.

Behavioral inhibition (BI), an early-life temperament characterized by vigilant responses to novelty, is a risk factor for anxiety disorders. In this study, we investigated whether differences in neonatal brain responses to infrequent auditory stimuli relate to children's BI at 1 year of age. Using functional magnetic resonance imaging (fMRI), we collected blood-oxygen-level-dependent (BOLD) data from N = 45 full-term, sleeping neonates during an adapted auditory oddball paradigm and measured BI from n = 27 of these children 1 year later using an observational assessment. Whole-brain analyses corrected for multiple comparisons identified 46 neonatal brain regions producing novelty-evoked BOLD responses associated with children's BI scores at 1 year of age. More than half of these regions (n = 24, 52%) were in prefrontal cortex, falling primarily within regions of the default mode or frontoparietal networks or in ventromedial/orbitofrontal regions without network assignments. Hierarchical clustering of the regions based on their patterns of association with BI resulted in two groups with distinct anatomical, network, and response-timing profiles. The first group, located primarily in subcortical and temporal regions, tended to produce larger early oddball responses among infants with lower subsequent BI. The second group, located primarily in prefrontal cortex, produced larger early oddball responses among infants with higher subsequent BI. These results provide preliminary insights into brain regions engaged by novelty in infants that may relate to later BI. The findings may inform understanding of anxiety disorders and guide future research. (PsycInfo Database Record (c) 2023 APA, all rights reserved).

Functional parcellation of the neonatal brain.

The cerebral cortex is organized into distinct but interconnected cortical areas, which can be defined by abrupt differences in patterns of resting state functional connectivity (FC) across the cortical surface. Such parcellations of the cortex have been derived in adults and older infants, but there is no widely used surface parcellation available for the neonatal brain. Here, we first demonstrate that adult- and older infant-derived parcels are a poor fit with neonatal data, emphasizing the need for neonatal-specific parcels. We next derive a set of 283 cortical surface parcels from a sample of n=261 neonates. These parcels have highly homogenous FC patterns and are validated using three external neonatal datasets. The Infomap algorithm is used to assign functional network identities to each parcel, and derived networks are consistent with prior work in neonates. The proposed parcellation may represent neonatal cortical areas and provides a powerful tool for neonatal neuroimaging studies.

Effects of swine microRNA mimics on lipopolysaccharide (LPS) induced inflammatory changes in 3D4/21 cells.

There have been limited studies focused on validation of swine microRNAs (miRNA) with mRNA targets. The objective of this study was to validate a defined set of targets using artificial miRNA mimics transfected into cell lines to confirm specific targets of endogenous miRNAs after administration of Escherichia coli lipopolysaccharide (LPS). Sixteen hours after mimic transfection of 3D4/21 cell lines, the cells were stimulated with 1 µg/ml LPS or phosphate-buffered saline (PBS). The cells were harvested and collected at 0, 1, 3, and 8 h post administration. The selected genes DAD1, IL8, and ESR, which are involved in known pathways of inflammation. and are predicted or validated human targets of either miR-146a, let-7a, or miR-22-3p. These were then evaluated by quantitative real-time-PCR (qRT-PCR) to verify microRNA-mRNA interaction in swine. Using the ROX reference dye, mRNA changes in expression were assessed using the comparative CT Method (ΔΔCT method) for normalization against the PBS control group. DAD1 and ESR1 were negatively regulated by miR-22-3p and miR-146a-5p, respectively in 3D4/21 cells after LPS stimulation. However, miR-146a-5p may play an indirect positive regulatory role of both DAD1 and IL8 mRNA expression. Furthermore, we found an inverse relationship between LPS stimulation compared with the let-7a-5p overexpression with DAD1. Our inflammation study provides new evidence on the roles and predicted targets of miR-146a, let-7a, and miR-22-3p in swine.

Lipopolysaccharide (LPS) induced inflammatory changes to differentially expressed miRNAs of the host inflammatory response.

In veterinary medicine, inflammation in swine is evaluated principally by clinical signs. This method is often unreliable when assessing large animal populations because of inconsistent interpretations of clinical observations. This study examined whether changes in miRNA expression can predict the severity of the inflammatory response in swine after administration of Escherichia coli lipopolysaccharide (LPS). Whole blood from swine challenged with LPS at 0.125 µg/kg to 2.0 µg/kg body weight was collected at 0, 1, 3, and 8 h post LPS-challenge. Mature miRNAs were extracted from plasma and quantitative real-time-PCR (qRT-PCR) was used to evaluate the 84 most abundant swine miRNAs found in plasma. The miRNA changes in expression were assessed using the comparative CT Method (ΔΔCT method) for normalization with an exogenous control. The results revealed that expression of ssc-let-7e-5p, ssc-mir-22-3p, and ssc-miR-146a-5p were the most significantly changed miRNA over the time course. At 1 h post-LPS, ssc-let-7e-5p decreased as the LPS dosage levels increased from 0.125 to 1.0 µg/kg. Similarly, as the LPS doses increased from 0.125 to 0.5 µg/kg, ssc-miR-22-3p levels significantly decreased at 1 h post-LPS. In the 2.0 µg/kg LPS, ssc-miR-146a-5p levels increased between 0 and 3 h post-LPS; however, expression was downregulated with a 145 % decrease from 3 to 8 h. The three miRNA biomarkers suggest potentially useful surrogate endpoints for the evaluation of inflammatory and endotoxemia responses in swine.

Minimally invasive ultrasound-guided technique for central venous catheterization via the external jugular vein in pigs.

OBJECTIVE: To describe an ultrasound-guided technique for central venous catheter placement via the external jugular vein (EJV) in pigs. ANIMALS: 96 healthy Landrace-Poland China barrows (approx 16 weeks old with a mean weight of 70 kg). PROCEDURES: Pigs were anesthetized. With ultrasound guidance, a needle was inserted into the EJV without a large incision or cutdown procedure. A guidewire was inserted through the needle into the vein. A modified Seldinger technique was used to advance a catheter into the vessel until the tip was in the cranial vena cava near the right atrium. A trocar was used to create a tunnel through the subcutaneous tissues from the catheter insertion site to between the dorsal borders of the scapulae. The free end of the catheter was passed through that tunnel. An extension was attached to the catheter and secured to the skin. Pigs were euthanized and underwent necropsy at completion of the study for which they were catheterized. RESULTS: Central venous catheters were successfully placed in all 96 pigs and facilitated collection of serial blood samples with minimal stress. Catheters remained in place for a mean of 6 days (range, 4 to 10 days). Necropsy revealed abscesses along the subcutaneous catheter tract in 9 pigs. Twenty pigs had histologic evidence of phlebitis and fibroplasia in the cranial vena cava. CONCLUSIONS AND CLINICAL RELEVANCE: The described technique, in combination with extensive socialization, allowed serial collection of blood samples with minimal stress and restraint and is an alternative to surgical cutdown procedures for catheter placement.

Microfluidic Separation of Canine Adipose-Derived Mesenchymal Stromal Cells.

Mesenchymal stromal cells (MSCs) are potential treatments for a variety of veterinary medical conditions. However, clinical trials have often fallen short of expectations, due in part to heterogeneity and lack of characterization of the MSCs. Identification and characterization of subpopulations within MSC cultures may improve those outcomes. Therefore, the functional heterogeneity of different-sized subpopulations of MSCs was evaluated. A high-throughput, biophysical, label-free microfluidic sorting approach was used to separate subpopulations of canine adipose-derived MSCs (Ad-MSCs) based on size for subsequent characterization, as well as to evaluate the impact of culture conditions on their functional heterogeneity. We found that culture-expanded canine Ad-MSCs comprise distinct subpopulations: larger MSCs (mean diameter of 18.6 ± 0.2 µm), smaller MSCs (mean diameter of 15.3 ± 0.2 µm), and intermediate MSCs (mean diameter of 16.9 ± 0.1 µm). In addition, proliferation characteristics, senescence, and differentiation potential of canine Ad-MSCs are also dependent on cell size. We observed that larger MSCs proliferate more slowly, senesce at earlier passages, and are inclined to differentiate into adipocytes compared with smaller MSCs. Most importantly, these size-dependent functions are also affected by the presence of serum in the culture medium, as well as time in culture. Cell surface staining for MSC-specific CD44 and CD90 antigens showed that all subpopulations of MSCs are indistinguishable, suggesting that this criterion is not relevant to define subpopulations of MSCs. Finally, transcriptome analysis showed differential gene expression between larger and smaller subpopulations of MSCs. Larger MSCs expressed genes involved in cellular senescence such as cyclin-dependent kinase inhibitor 1A and smaller MSCs expressed genes that promote cell growth [mechanistic target of rapamycin 1 (mTORC1) pathway] and cell proliferation [myelocytomatosis (myc), e2f targets]. These results suggest that different subpopulations of MSCs have specific properties. Impact statement Clinical trials of mesenchymal stromal cells (MSCs) from veterinary species have often fallen short of expectations, due in part to heterogeneity and lack of characterization of the MSCs. A high-throughput, biophysical, label-free microfluidic sorting approach was used to separate subpopulations of canine adipose-derived MSCs (Ad-MSCs) based on size for subsequent characterization. Proliferation characteristics, senescence, and differentiation potential of canine Ad-MSCs are also dependent on cell size. Cell surface staining for MSC-specific cell surface markers showed that all subpopulations of MSCs are indistinguishable, suggesting that this criterion is not relevant to define subpopulations of MSCs.

Attention Alterations in Pediatric Anxiety: Evidence From Behavior and Neuroimaging.

BACKGROUND: Pediatric anxiety disorders involve greater capture of attention by threatening stimuli. However, it is not known if disturbances extend to nonthreatening stimuli, as part of a pervasive disturbance in attention-related brain systems. We hypothesized that pediatric anxiety involves greater capture of attention by salient, nonemotional stimuli, coupled with greater activity in the portion of the inferior frontal gyrus (IFG) specific to the ventral attention network (VAN). METHODS: A sample of children (n = 129, 75 girls, mean 10.6 years of age), approximately half of whom met criteria for a current anxiety disorder, completed a task measuring involuntary capture of attention by nonemotional (square boxes) and emotional (angry and neutral faces) stimuli. A subset (n = 61) completed a task variant during functional magnetic resonance imaging. A priori analyses examined activity in functional brain areas within the right IFG, supplemented by a whole-brain, exploratory analysis. RESULTS: Higher clinician-rated anxiety was associated with greater capture of attention by nonemotional, salient stimuli (F1,125 = 4.94, p = .028) and greater activity in the portion of the IFG specific to the VAN (F1,57 = 10.311, p = .002). Whole-brain analyses confirmed that the effect of anxiety during capture of attention was most pronounced in the VAN portion of the IFG, along with additional areas of the VAN and the default mode network. CONCLUSIONS: The pathophysiology of pediatric anxiety appears to involve greater capture of attention to salient stimuli, as well as greater activity in attention-related brain networks. These results provide novel behavioral and brain-based targets for treatment of pediatric anxiety disorders.

Whole-Brain Resting-State Functional Connectivity Patterns Associated With Pediatric Anxiety and Involuntary Attention Capture.

BACKGROUND: Pediatric anxiety disorders are linked to dysfunction in multiple functional brain networks, as well as to alterations in the allocation of spatial attention. We used network-level analyses to characterize resting-state functional connectivity (rs-fc) alterations associated with 1) symptoms of anxiety and 2) alterations in stimulus-driven attention associated with pediatric anxiety disorders. We hypothesized that anxiety was related to altered connectivity of the frontoparietal, default mode, cingulo-opercular, and ventral attention networks and that anxiety-related connectivity alterations that include the ventral attention network would simultaneously be related to deviations in stimulus-driven attention. METHODS: A sample of children (n = 61; mean = 10.6 years of age), approximately half of whom met criteria for a current anxiety disorder, completed a clinical assay, an attention task, and rs-fc magnetic resonance imaging scans. Network-level analyses examined whole-brain rs-fc patterns associated with clinician-rated anxiety and with involuntary capture of attention. Post hoc analyses controlled for comorbid symptoms. RESULTS: Elevated clinician-rated anxiety was associated with altered connectivity within the cingulo-opercular network, as well as between the cingulo-opercular network and the ventral attention, default mode, and visual networks. Connectivity between the ventral attention and cingulo-opercular networks was associated with variation in both anxiety and stimulus-driven attention. CONCLUSIONS: Pediatric anxiety is related to aberrant connectivity patterns among several networks, most of which include the cingulo-opercular network. These results help clarify the within- and between-network interactions associated with pediatric anxiety and its association with altered attention, suggesting that specific network connections could be targeted to improve specific altered processes associated with anxiety.