Concept of Normativity in Multi-Omics Analysis of Axon Regeneration.

Transcriptomes and proteomes can be normalized with a handful of RNAs or proteins (or their peptides), such as GAPDH, ß-actin, RPBMS, and/or GAP43. Even with hundreds of standards, normalization cannot be achieved across different molecular mass ranges for small molecules, such as lipids and metabolites, due to the non-linearity of mass by charge ratio for even the smallest part of the spectrum. We define the amount (or range of amounts) of metabolites and/or lipids per a defined amount of a protein, consistently identified in all samples of a multiple-model organism comparison, as the normative level of that metabolite or lipid. The defined protein amount (or range) is a normalized value for one cohort of complete samples for which intrasample relative protein quantification is available. For example, the amount of citrate (a metabolite) per µg of aconitate hydratase (normalized protein amount) identified in the proteome is the normative level of citrate with aconitase. We define normativity as the amount of metabolites (or amount range) detected when compared to normalized protein levels. We use axon regeneration as an example to illustrate the need for advanced approaches to the normalization of proteins. Comparison across different pharmacologically induced axon regeneration mouse models entails the comparison of axon regeneration, studied at different time points in several models designed using different agents. For the normalization of the proteins across different pharmacologically induced models, we perform peptide doping (fixed amounts of known peptides) in each sample to normalize the proteome across the samples. We develop Regen V peptides, divided into Regen III (SEB, LLO, CFP) and II (HH4B, A1315), for pre- and post-extraction comparisons, performed with the addition of defined, digested peptides (bovine serum albumin tryptic digest) for protein abundance normalization beyond commercial labeled relative quantification (for example, 18-plex tandem mass tags). We also illustrate the concept of normativity by using this normalization technique on regenerative metabolome/lipidome profiles. As normalized protein amounts are different in different biological states (control versus axon regeneration), normative metabolite or lipid amounts are expected to be different for specific biological states. These concepts and standardization approaches are important for the integration of different datasets across different models of axon regeneration.

A neural tract-inspired conduit for facile, on-demand biopsy of glioblastoma.

Background: A major hurdle to effectively treating glioblastoma (GBM) patients is the lack of longitudinal information about tumor progression, evolution, and treatment response. Methods: In this study, we report the use of a neural tract-inspired conduit containing aligned polymeric nanofibers (i.e., an aligned nanofiber device) to enable on-demand access to GBM tumors in 2 rodent models. Depending on the experiment, a humanized U87MG xenograft and/or F98-GFP+ syngeneic rat tumor model was chosen to test the safety and functionality of the device in providing continuous sampling access to the tumor and its microenvironment. Results: The aligned nanofiber device was safe and provided a high quantity of quality genomic materials suitable for omics analyses and yielded a sufficient number of live cells for in vitro expansion and screening. Transcriptomic and genomic analyses demonstrated continuity between material extracted from the device and that of the primary, intracortical tumor (in the in vivo model). Conclusions: The results establish the potential of this neural tract-inspired, aligned nanofiber device as an on-demand, safe, and minimally invasive access point, thus enabling rapid, high-throughput, longitudinal assessment of tumor and its microenvironment, ultimately leading to more informed clinical treatment strategies.

The effects of verbal and spatial working memory on short- and long-latency sensorimotor circuits in the motor cortex.

Multiple sensorimotor loops converge in the motor cortex to create an adaptable system capable of context-specific sensorimotor control. Afferent inhibition provides a non-invasive tool to investigate the substrates by which procedural and cognitive control processes interact to shape motor corticospinal projections. Varying the transcranial magnetic stimulation properties during afferent inhibition can probe specific sensorimotor circuits that contribute to short- and long-latency periods of inhibition in response to the peripheral stimulation. The current study used short- (SAI) and long-latency (LAI) afferent inhibition to probe the influence of verbal and spatial working memory load on the specific sensorimotor circuits recruited by posterior-anterior (PA) and anterior-posterior (AP) TMS-induced current. Participants completed two sessions where SAI and LAI were assessed during the short-term maintenance of two- or six-item sets of letters (verbal) or stimulus locations (spatial). The only difference between the sessions was the direction of the induced current. PA SAI decreased as the verbal working memory load increased. In contrast, AP SAI was not modulated by verbal working memory load. Visuospatial working memory load did not affect PA or AP SAI. Neither PA LAI nor AP LAI were sensitive to verbal or spatial working memory load. The dissociation of short-latency PA and AP sensorimotor circuits and short- and long-latency PA sensorimotor circuits with increasing verbal working memory load support multiple convergent sensorimotor loops that provide distinct functional information to facilitate context-specific supraspinal control.

Investigating Cerebellar Modulation of Premovement Beta-Band Activity during Motor Adaptation.

Enhancing cerebellar activity influences motor cortical activity and contributes to motor adaptation, though it is unclear which neurophysiological mechanisms contributing to adaptation are influenced by the cerebellum. Pre-movement beta event-related desynchronization (ß-ERD), which reflects a release of inhibitory control in the premotor cortex during movement planning, is one mechanism that may be modulated by the cerebellum through cerebellar-premotor connections. We hypothesized that enhancing cerebellar activity with intermittent theta burst stimulation (iTBS) would improve adaptation rates and increase ß-ERD during motor adaptation. Thirty-four participants were randomly assigned to an active (A-iTBS) or sham cerebellar iTBS (S-iTBS) group. Participants performed a visuomotor task, using a joystick to move a cursor to targets, prior to receiving A-iTBS or S-iTBS, following which they completed training with a 45° rotation to the cursor movement. Behavioural adaptation was assessed using the angular error of the cursor path relative to the ideal trajectory. The results showed a greater adaptation rate following A-iTBS and an increase in ß-ERD, specific to the high ß range (20-30 Hz) during motor planning, compared to S-iTBS, indicative of cerebellar modulation of the motor cortical inhibitory control network. The enhanced release of inhibitory activity persisted throughout training, which suggests that the cerebellar influence over the premotor cortex extends beyond adaptation to other stages of motor learning. The results from this study further understanding of cerebellum-motor connections as they relate to acquiring motor skills and may inform future skill training and rehabilitation protocols.

Pedestrian- and bicyclist-involved crashes: Associations with spatial factors, pedestrian infrastructure, and equity impacts.

INTRODUCTION: We analyze and compare the factors that influence the fatality of pedestrian and bicyclist involved crashes in New Jersey using available police-reported crash data between 2016 and 2020. Under three percent of crashes involve non-motorists statewide, but these account for about one third of all traffic fatalities in the state. METHODS: Our analysis is broken down into five parts: we (1) analyze the relationship between minority and low-income communities and non-motorist involved crashes; (2) identify spatial differences between non-motorist involved crashes and non-motorist involved fatal crashes; (3) compare the factors affecting fatal pedestrian crashes in New Jersey and in four counties in southern New Jersey for which we have data on pedestrian infrastructure; (4) compare the factors affecting fatal pedestrian crashes and fatal cyclist crashes in New Jersey; and, (5) discuss priority areas for improving safety. RESULTS: Crashes occur disproportionately more often in low-income communities. Moreover, we find that crashes are less likely to be geocoded if they take place in low-income and minority areas, a concerning finding considering that geocoded crashes are of paramount importance in identifying specific corridors for improvement. Light conditions, non-motorist age, posted speed, and vehicle type are significant factors influencing the fatality of non-motorist involved crashes. The proximity to a crosswalk or sidewalk is associated with decreased risk of a fatal crash for pedestrians. Cyclist crashes in low-income neighborhoods were more likely to be fatal - a finding that we attribute to lower access to bicycle facilities in low-income areas. CONCLUSIONS: We conclude with countermeasures, including a call for better data collection.

Metabolomics dataset of zebrafish optic nerve regeneration after injury.

Zebrafish (Danio rerio) have the capacity for successful adult optic nerve regeneration. In contrast, mammals lack this intrinsic ability and undergo irreversible neurodegeneration seen in glaucoma and other optic neuropathies. Optic nerve regeneration is often studied using optic nerve crush, a mechanical neurodegenerative model. Untargeted metabolomic studies within successful regenerative models are deficient. Evaluation of tissue metabolomic changes in active zebrafish optic nerve regeneration can elucidate prioritized metabolite pathways that can be targeted in mammalian systems for therapeutic development. Female and male (6 month to 1 year old wild type) right zebrafish optic nerves were crushed and collected three days after. Contralateral, uninjured optic nerves were collected as controls. The tissue was dissected from euthanized fish and frozen on dry ice. Samples were pooled for each category (female crush, female control, male crush, male control) and pooled at n = 31 to obtain sufficient metabolite concentrations for analysis. Optic nerve regeneration at 3 days post crush was demonstrated by microscope visualization of GFP fluorescence in Tg(gap43:GFP) transgenic fish. Metabolites were extracted using a Precellys Homogenizer and a serial extraction method: (1) 1:1 Methanol/Water and (2) 8:1:1 Acetonitrile/Methanol/Acetone. Metabolites were analyzed by untargeted liquid chromatography-mass spectrometry (LC MS-MS) profiling using a Q-Exactive Orbitrap instrument coupled with Vanquish Horizon Binary UHPLC LC-MS system. Metabolites were identified and quantified using Compound Discoverer 3.3 and isotopic internal metabolites standards.

Combined Peripheral Nerve Stimulation and Controllable Pulse Parameter Transcranial Magnetic Stimulation to Probe Sensorimotor Control and Learning.

Skilled motor ability depends on efficiently integrating sensory afference into the appropriate motor commands. Afferent inhibition provides a valuable tool to probe the procedural and declarative influence over sensorimotor integration during skilled motor actions. This manuscript describes the methodology and contributions of short-latency afferent inhibition (SAI) for understanding sensorimotor integration. SAI quantifies the effect of a convergent afferent volley on the corticospinal motor output evoked by transcranial magnetic stimulation (TMS). The afferent volley is triggered by the electrical stimulation of a peripheral nerve. The TMS stimulus is delivered to a location over the primary motor cortex that elicits a reliable motor-evoked response in a muscle served by that afferent nerve. The extent of inhibition in the motor-evoked response reflects the magnitude of the afferent volley converging on the motor cortex and involves central GABAergic and cholinergic contributions. The cholinergic involvement in SAI makes SAI a possible marker of declarative-procedural interactions in sensorimotor performance and learning. More recently, studies have begun manipulating the TMS current direction in SAI to tease apart the functional significance of distinct sensorimotor circuits in the primary motor cortex for skilled motor actions. The ability to control additional pulse parameters (e.g., the pulse width) with state-of-the-art controllable pulse parameter TMS (cTMS) has enhanced the selectivity of the sensorimotor circuits probed by the TMS stimulus and provided an opportunity to create more refined models of sensorimotor control and learning. Therefore, the current manuscript focuses on SAI assessment using cTMS. However, the principles outlined here also apply to SAI assessed using conventional fixed pulse width TMS stimulators and other forms of afferent inhibition, such as long-latency afferent inhibition (LAI).

Glycerophospholipid Analysis of Optic Nerve Regeneration Models Indicate Potential Membrane Order Changes Associated with the Lipidomic Shifts.

Purpose: Optic nerve (ON) injury causes irreversible degeneration, leading to vision loss that cannot be restored with available therapeutics. Current therapies slow further degeneration but do not promote regeneration. New regenerative factors have been discovered that are successful in vivo. However, the mechanisms of efficient long-distance regeneration are still unknown. Membrane expansion by lipid insertion is an essential regenerative process, so lipid profiles for regenerating axons can provide insight into growth mechanisms. This article's analysis aims to add to the increasingly available ON regeneration lipid profiles and relate it to membrane order/properties. Methods: In this study, we present an analysis of glycerophospholipids, one of the largest axonal lipid groups, from three mammalian ON regeneration lipid profiles: Wnt3a, Zymosan + CPT-cAMP, and Phosphatase/Tensin homolog knockout (PTENKO) at 7 and 14 days post crush (dpc). Significant lipid classes, species, and ontological properties were crossreferenced between treatments and analyzed using Metaboanalyst 5.0 and Lipid Ontology (LION). Membrane order changes associated with significant lipid classes were evaluated by C-Laurdan dye and exogenous lipids provided to a neuroblastoma cell line. Results and Conclusions: At 7 dpc, ONs show increased lysoglycerophospholipids and decreased phosphatidylethanolamines (PEs)/negative intrinsic curvature lipids. At 14 dpc, regenerative treatments show divergence: Wnt3a displays higher lysoglycerophospholipid content, while Zymosan and PTENKO decrease lysoglycerophospholipids and increase phosphatidylcholine (PC)-related species. Membrane order imaging indicates lysoglycerophospholipids decreases membrane order while PE and PC had no significant membrane order effects. Understanding these changes will allow therapeutic development targeting lipid metabolic pathways that can be used for vision loss treatments.

Retinal Ganglion Cell Axon Fractionation.

Retinal ganglion cell (RGC) axon regeneration in mammals can be stimulated through gene knockouts, pharmacological agents, and biophysical stimulation. Here we present a fractionation method to isolate regenerating RGC axons for downstream analysis using immunomagnetic separation of cholera toxin subunit B (CTB)-bound RGC axons. After optic nerve tissue dissection and dissociation, conjugated CTB is used to bind preferentially to regenerated RGC axons. Anti-CTB antibodies crosslinked to magnetic sepharose beads are used to isolate CTB-bound axons from a nonbound fraction of extracellular matrix and neuroglia. We provide a method of verifying fractionation by immunodetection of conjugated CTB and the RGC marker, Tuj1 (ß-tubulin III). These fractions can be further analyzed with lipidomic methods, such as LC-MS/MS to gather fraction-specific enrichments.

Isolation of Mitochondrial Lipids and Mass Spectrometric Analysis.

Mitochondria participate in many important metabolic processes in the body. The lipid profile of mitochondria is especially important in membrane regulation and pathway signaling. The isolation and study of these lipids can provide unparalleled information about the mechanisms behind these cellular processes. In this chapter, we describe a protocol to isolate mitochondrial lipids from homogenized murine optic nerves. The lipid extraction was performed using butanol-methanol (BUME) and subsequently analyzed using liquid chromatography-mass spectrometry. Further analysis of the raw data was conducted using LipidSearch™ and MetaboAnalyst 4.0.

Analyses and Localization of Phosphatidylcholine and Phosphatidylserine in Murine Ocular Tissue Using Imaging Mass Spectrometry.

Imaging mass spectrometry (IMS) allows for spatial visualization of proteins, lipids, and metabolite distributions in a tissue. Identifying these compounds through mass spectrometry, combined with mapping the compound distribution in the sample in a targeted or untargeted approach, renders IMS a powerful tool for lipidomics. IMS analysis for lipid species such as phosphatidylcholine and phosphatidylserine allows researchers to pinpoint areas of lipid deficiencies or accumulations associated with ocular disorders such as age-related macular degeneration and diabetic retinopathy. Here, we describe an end-to-end IMS approach from sample preparation to data analysis for phosphatidylcholine and phosphatidylserine analysis.

C-Laurdan: Membrane Order Visualization of HEK293t Cells by Confocal Microscopy.

Membrane order is a biophysical characteristic dependent on cellular lipid makeup. Cells regulate the membrane structure as it affects membrane-bound protein activity levels and membrane stability. Spatial organization of membrane lipids, such as lipid rafts, is a proposed theory that has been indirectly measured through polarity-sensitive fluorescent dyes. C-Laurdan is one such dye that penetrates plasma and internal membranes. C-Laurdan is excited by a single 405 nm photon and emits in two distinct ranges depending on membrane order. Herein, we present a protocol for staining HEK293t cells with C-Laurdan and acquiring ratiometric images using a revised ImageJ macro and confocal microscopy. An example figure is provided depicting the effects of methyl-ß-cyclodextrin, known to remove lipid rafts through cholesterol sequestration, on HEK293t cells. Further image analysis can be performed through region of interest (ROI) selection tools.

Analyses and Localization of Serotonin and L-DOPA in Ocular Tissues by Imaging Mass Spectrometry.

Imaging mass spectrometry (IMS) allows for visualization of the spatial distribution of proteins, lipids, and other metabolites in a targeted or untargeted approach. The identification of compounds through mass spectrometry combined with the mapping of compound distribution in the sample establishes IMS as a powerful tool for metabolomics. IMS analysis for serotonin will allow researchers to pinpoint areas of deficiencies or accumulations associated with ocular disorders such as serotonin selective reuptake inhibitor optic neuropathy. Furthermore, L-DOPA has shown great promise as a therapeutic approach for disorders such as age-related macular degeneration, and IMS allows for localization, and relative magnitudes, of L-DOPA in the eye. We describe here an end-to-end approach of IMS from sample preparation to data analysis for serotonin and L-DOPA analysis.

Specific sensorimotor interneuron circuits are sensitive to cerebellar-attention interactions.

Background: Short latency afferent inhibition (SAI) provides a method to investigate mechanisms of sensorimotor integration. Cholinergic involvement in the SAI phenomena suggests that SAI may provide a marker of cognitive influence over implicit sensorimotor processes. Consistent with this hypothesis, we previously demonstrated that visual attention load suppresses SAI circuits preferentially recruited by anterior-to-posterior (AP)-, but not posterior-to-anterior (PA)-current induced by transcranial magnetic stimulation. However, cerebellar modulation can also modulate these same AP-sensitive SAI circuits. Yet, the consequences of concurrent cognitive and implicit cerebellar influences over these AP circuits are unknown. Objective: We used cerebellar intermittent theta-burst stimulation (iTBS) to determine whether the cerebellar modulation of sensory to motor projections interacts with the attentional modulation of sensory to motor circuits probed by SAI. Methods: We assessed AP-SAI and PA-SAI during a concurrent visual detection task of varying attention load before and after cerebellar iTBS. Results: Before cerebellar iTBS, a higher visual attention load suppressed AP-SAI, but not PA-SAI, compared to a lower visual attention load. Post-cerebellar iTBS, the pattern of AP-SAI in response to visual attention load, was reversed; a higher visual attention load enhanced AP-SAI compared to a lower visual attention load. Cerebellar iTBS did not affect PA-SAI regardless of visual attention load. Conclusion: These findings suggest that attention and cerebellar networks converge on overlapping AP-sensitive circuitry to influence motor output by controlling the strength of the afferent projections to the motor cortex. This interaction has important implications for understanding the mechanisms of motor performance and learning.

Proteomics and systems biology in optic nerve regeneration.

We present an overview of current state of proteomic approaches as applied to optic nerve regeneration in the historical context of nerve regeneration particularly central nervous system neuronal regeneration. We present outlook pertaining to the optic nerve regeneration proteomics that the latter can extrapolate information from multi-systems level investigations. We present an account of the current need of systems level standardization for comparison of proteome from various models and across different pharmacological or biophysical treatments that promote adult neuron regeneration. We briefly overview the need for deriving knowledge from proteomics and integrating with other omics to obtain greater biological insight into process of adult neuron regeneration in the optic nerve and its potential applicability to other central nervous system neuron regeneration.

Effect of Ezogabine on Cortical and Spinal Motor Neuron Excitability in Amyotrophic Lateral Sclerosis: A Randomized Clinical Trial.

Importance: Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease of the motor nervous system. Clinical studies have demonstrated cortical and spinal motor neuron hyperexcitability using transcranial magnetic stimulation and threshold tracking nerve conduction studies, respectively, although metrics of excitability have not been used as pharmacodynamic biomarkers in multi-site clinical trials. Objective: To ascertain whether ezogabine decreases cortical and spinal motor neuron excitability in ALS. Design, Setting, and Participants: This double-blind, placebo-controlled phase 2 randomized clinical trial sought consent from eligible participants from November 3, 2015, to November 9, 2017, and was conducted at 12 US sites within the Northeast ALS Consortium. Participants were randomized in equal numbers to a higher or lower dose of ezogabine or to an identical matched placebo, and they completed in-person visits at screening, baseline, week 6, and week 8 for clinical assessment and neurophysiological measurements. Interventions: Participants were randomized to receive 600 mg/d or 900 mg/d of ezogabine or a matched placebo for 10 weeks. Main Outcomes and Measures: The primary outcome was change in short-interval intracortical inhibition (SICI; SICI-1 was used in analysis to reflect stronger inhibition from an increase in amplitude) from pretreatment mean at screening and baseline to the full-dose treatment mean at weeks 6 and 8. The secondary outcomes included levels of cortical motor neuron excitability (including resting motor threshold) measured by transcranial magnetic stimulation and spinal motor neuron excitability (including strength-duration time constant) measured by threshold tracking nerve conduction studies. Results: A total of 65 participants were randomized to placebo (23), 600 mg/d of ezogabine (23), and 900 mg/d of ezogabine (19 participants); 45 were men (69.2%) and the mean (SD) age was 58.3 (8.8) years. The SICI-1 increased by 53% (mean ratio, 1.53; 95% CI, 1.12-2.09; P = .009) in the 900-mg/d ezogabine group vs placebo group. The SICI-1 did not change in the 600-mg/d ezogabine group vs placebo group (mean ratio, 1.15; 95% CI, 0.87-1.52; P = .31). The resting motor threshold increased in the 600-mg/d ezogabine group vs placebo group (mean ratio, 4.61; 95% CI, 0.21-9.01; P = .04) but not in the 900-mg/d ezogabine group vs placebo group (mean ratio, 1.95; 95% CI, -2.64 to 6.54; P = .40). Ezogabine caused a dose-dependent decrease in excitability by several other metrics, including strength-duration time constant in the 900-mg/d ezogabine group vs placebo group (mean ratio, 0.73; 95% CI, 0.60 to 0.87; P < .001). Conclusions and Relevance: Ezogabine decreased cortical and spinal motor neuron excitability in participants with ALS, suggesting that such neurophysiological metrics may be used as pharmacodynamic biomarkers in multisite clinical trials. Trial Registration: ClinicalTrials.gov Identifier: NCT02450552.

Differential experiences of embodiment between body-powered and myoelectric prosthesis users.

Prosthesis embodiment, the perception of a prosthesis as part of one's body, may be an important component of functional recovery for individuals with upper limb absence. This work determined whether embodiment differs between body-powered and myoelectric prosthesis users. In a sample of nine individuals with transradial limb absence, embodiment was quantified using a survey regarding prosthesis ownership and agency. The extent to which the prosthesis affected the body schema, the representation of the body's dimensions, was assessed using limb length estimation. Because body-powered prostheses offer proprioceptive feedback that myoelectric prostheses do not, it was hypothesized that both measures would reveal stronger embodiment of body-powered prostheses. However, our results did not show differences across the two prosthesis designs. Instead, body schema was influenced by several patient-specific characteristics, including the cause of limb absence (acquired or congenital) and hours of daily prosthesis wear. These results indicate that regular prosthesis wear and embodiment are connected, regardless of the actual prosthesis design. Identifying whether embodiment is a direct consequence of regular prosthesis use would offer insight on how individuals with limb absence could modify their behavior to more fully embody their prosthesis.

The Motor skills At Playtime intervention improves children's locomotor skills: A feasibility study.

BACKGROUND: Interventions are needed to teach fundamental motor skills (FMS) to preschoolers. There is a need to design more practical and effective interventions that can be successfully implemented by non-motor experts and fit within the existing gross motor opportunities such as outdoor free play at the preschool. The purpose of this study was to evaluate the feasibility and efficacy of a non-motor expert FMS intervention that was implemented during outdoor free play, Motor skills At Playtime (MAP). METHODS: Participants were preschoolers from two Head Start centres (N = 46; Mage = 4.7 ± 0.46 years; 41% boys) and were divided into a MAP (n = 30) or control (outdoor free play; n = 16) group. Children completed either a 1,350-min MAP intervention or control condition (outdoor free play) from January to April of 2018. FMS were assessed before and after each programme using both the Test of Gross Motor Development-3rd Edition and skill outcome measures (running speed, hopping speed, jump distance, throwing speed, kicking speed and catching percentage). Intervention implementation feasibility was measured through daily fidelity checks. Fidelity was evaluated as the percentage of intervention sessions that included all explicit intervention criteria. FMS data were analysed using linear mixed modelling. Models were fit with fixed effects of time and treatment, covariates of sex and height, and a random intercept for each individual. RESULTS: The non-motor expert was feasibly able to implement MAP with high fidelity (>93%). There was a significant treatment effect for MAP on process and product locomotor FMS (P < 0.05) and a trend for a treatment effect for MAP on total process FMS (P = 0.07). CONCLUSION: Results support that MAP was successfully implemented by a non-motor expert and led to improvements in children's FMS, especially locomotor FMS.