Towards higher sensitivity and stability of axon diameter estimation with diffusion‐weighted MRI

Diffusion‐weighted MRI is an important tool for in vivo and non‐invasive axon morphometry. The ActiveAx technique utilises an optimised acquisition protocol to infer orientationally invariant indices of axon diameter and density by fitting a model of white matter to the acquired data. In this study, we investigated the factors that influence the sensitivity to small‐diameter axons, namely the gradient strength of the acquisition protocol and the model fitting routine. Diffusion‐weighted ex. vivo images of the mouse brain were acquired using 16.4‐T MRI with high (G_max of 300 mT/m) and ultra‐high (G_max of 1350 mT/m) gradient strength acquisitions. The estimated axon diameter indices of the mid‐sagittal corpus callosum were validated using electron microscopy. In addition, a dictionary‐based fitting routine was employed and evaluated. Axon diameter indices were closer to electron microscopy measures when higher gradient strengths were employed. Despite the improvement, estimated axon diameter indices (a lower bound of ~ 1.8 μm) remained higher than the measurements obtained using electron microscopy (~1.2 μm). We further observed that limitations of pulsed gradient spin echo (PGSE) acquisition sequences and axonal dispersion could also influence the sensitivity with which axon diameter indices could be estimated. Our results highlight the influence of acquisition protocol, tissue model and model fitting, in addition to gradient strength, on advanced microstructural diffusion‐weighted imaging techniques. © 2016 The Authors. NMR in Biomedicine published by John Wiley & Sons Ltd.     

Developmental axon pruning mediated by BDNF-p75NTR-dependent axon degeneration

The mechanisms that regulate the pruning of mammalian axons are just now being elucidated. Here, we describe a mechanism by which, during developmental sympathetic axon competition, winning axons secrete brain-derived neurotrophic factor (BDNF) in an activity-dependent fashion, which binds to the p75 neurotrophin receptor (p75NTR) on losing axons to cause their degeneration and, ultimately, axon pruning. Specifically, we found that pruning of rat and mouse sympathetic axons that project to the eye requires both activity-dependent BDNF and p75NTR. p75NTR and BDNF are also essential for activity-dependent axon pruning in culture, where they mediate pruning by directly causing axon degeneration. p75NTR, which is enriched in losing axons, causes axonal degeneration by suppressing TrkA-mediated signaling that is essential for axonal maintenance. These data provide a mechanism that explains how active axons can eliminate less-active, competing axons during developmental pruning by directly promoting p75NTR-mediated axonal degeneration.     

关于轴突生长的导向分子机理的探讨

在神经系统的发育过程中,轴突的生长具有严格的方向性。轴突沿特定的路线生长、延长,并伸向与它建立突触联系的靶细胞或神经元的胞体、树突或轴突。轴突末端称为生长锥,其扇形膨大部分称作板足,在板足的表面伸出许多细小的突起,称为丝足。板足的细胞骨架主要是由微管构成,丝足的细胞骨架主要是不成束的肌动蛋白(图1)。生长锥对周围环境极其敏感,其表面受体可识别细胞外基质中或周围细胞上的导向分子。这些导向分子中,有的能够吸引生长锥向其生长,有的却是排斥生长锥向其生长,生长锥就在这样一个复杂的环境中,选择一条正确的路径到达目的地。     

Differences in sensitivity to nerve growth factor of axon formation and tyrosine hydroxylase induction in cultured sympathetic neurons

Superior cervical ganglia from 2-day-old and 3-week-old rats were maintained in vitro for up to 2 weeks in the presence of a range of concentrations of nerve growth factor up to 100 micrograms/ml. Nerve fibre length and density were measured and tyrosine hydroxylase activity of these cultures assayed after various times. Ganglia were also examined for catecholamines and neuronal numbers using fluorescence histochemistry and histology respectively. In cultures maintained without nerve growth factor, or in those containing low concentrations of nerve growth factor (3 ng/ml), tyrosine hydroxylase decreased to 5-10% of the initial levels by 14 days in vitro. The presence of the high concentration of 1 microgram/ml nerve growth factor in the culture medium or the addition of such a concentration during the culture period did not prevent an initial decrease in tyrosine hydroxylase but subsequently increased the enzyme activity. The maximal effect of nerve growth factor on nerve fibre density was at low concentrations whereas its maximal effect on neuronal survival, tyrosine hydroxylase activity or nerve fibre elongation was at high concentrations. After 2 days in culture, maximum neurite production occurred in cultures containing 10 ng/ml, while maximum nerve fibre elongation and tyrosine hydroxylase activity occurred in cultures containing 100 micrograms/ml nerve growth factor. We conclude that low concentrations of nerve growth factor, as occur in plasma, cause maximum axon formation while high concentrations of nerve growth factor, as occur in effector organs, induce maximum tyrosine hydroxylase activity and cell survival. The former process may be mediated via cell surface receptors and the latter via retrograde axonal transport of nerve growth factor to the cell body, following uptake by the terminal regions of the axons.     

Assaying metabolic activity in ageing Caenorhabditis elegans.

Accurate measures of physiological and metabolic condition could provide more insight into how longevity genes and signalling pathways affect global metabolic activity and life span. The present study is essentially a methodological treatise in which we describe and evaluate a number of methods to assess changes of metabolic activity in ageing Caenorhabditis elegans. Oxygen consumption and CO(2) production rate assays, and measurement of the heat output by microcalorimetry are performed using live worms. For other assays, frozen (-75 degrees C) samples can be used. A lucigenin-mediated light production assay provides information on the metabolic capacity (scope for metabolic activity) of the worms just before freezing. Assaying ATP and ADP levels provides a measure of the instantly available energy. The XTT assay measures the activity of enzymes that can reduce XTT. Blue fluorescence emitted at 420-470 nm is a potentially useful biomarker of the rate of ageing. A protein quantification protocol for normalising all data for quantitative comparisons is presented. We illustrate how these methods can validate or disprove models of gene action inferred from molecular identification.     

Mechanisms of axon regeneration

<正>ailure of regeneration in injured axons accounts for permanent functional deficits after CNS injury.Thus,there has been a tremendous need to understand the mechanisms underlying regeneration failure.Previously,a predominant hypothesis held that extrinsic inhibitory factors in the adult CNS environment prevent injured axons from re-growth.However,blocking these inhibitory activities genetically led to little or no axon regeneration in vivo,suggesting that removing inhibitory activities alone is insufficient     

Tissue-engineered platforms of axon guidance

Tissue engineering provides a valuable tool for in vitro investigation of complex in vivo environments. A particular application of tissue-engineered in vitro platforms in neuroscience and regenerative medicine is the fabrication of controlled microenvironments for the study of axon guidance, with the goal of informing strategies to overcome nerve injury. The innovative design of tissue-engineered scaffolds that incorporate multiple guidance cues and cell types into various environments is advancing the understanding of how neurons integrate guidance information to make growth decisions. This review focuses on recent strategies that present neurons with multiple cues with micro- and nanoscale resolution in order to study the interactions between neurons and their local environment during axon guidance.     

Intrinsic control of axon regeneration

Spinal cord injury disrupts the connections between the brain and spinal cord, often resulting in the loss of sensory and motor function below the lesion site. The most important reason for such permanent functional deficits is the failure of injured axons to regenerate after injury. In principle, the functional recovery could be achieved by two forms of axonal regrowth: the regeneration of lesioned axons which will reconnect with their original targets and the sprouting of spared axons that form new circuits and compensate for the lost function. Our recent studies reveal the activity of the mammalian target of rapamycin (mTOR) pathway, a major regulator of new protein synthesis, as a critical determinant of axon regrowth in the adult retinal ganglion neurons[1]. In this review, I summarize current understanding of the cellular and molecular mechanisms that control the intrinsic regenerative ability of mature neurons.     

Nodal axon diameter correlates linearly with internodal axon diameter in spinal roots of the cat

Nodal and internodal axon diameters of individual myelinated nerve fibres were measured electron microscopically in fibre samples from serially sectioned L₇ ventral and dorsal spinal root of young adult cats. Axon cross-sectional area at the node of Ranvier in axons more than 4 μm in diameter was reduced to less than 20% of its internodal value. Internodal and nodal axon diameters showed a rectilinear distribution and linear regression analysis gave coefficients of correlation between 0.93 and 0.99. An individual nodal diameter value could be fitted to an internodal axon diameter value in a 95% prediction interval of ± 1.06–2.41 μm.     

Axonal transport in the central axon of sensory neurons during regeneration of their peripheral axon

Following sciatic nerve injury (which provokes prompt regeneration of peripheral sensory axons) there was no change in the ratio between amounts of labeled protein conveyed by fast axonal transport into the central and peripheral axons. A change in composition of the transported protein, characteristic of regenerating peripheral axons, also occurred in the central axons. When the peripheral axon is injured, changes in axonal transport thus affect both processes, and there is no routing of fast-transported protein into or away from the regenerating process.     

Beyond the initial axon segment of the spinal motor axon: fasciculated microtubules and polyribosomal clusters

Dense undercoating, microtubular fascicles and scattered polyribosomal clusters have until now been considered to be the three structural features of the initial segment, and were thought not to extend beyond the initial segment into the myelinated parts of the axon. The aim of the present study was to make clear whether there is a sudden change in morphology between the unmyelinated and myelinated part. We followed spinal motor axons from the initial segment to the first internode by conventional electron microscopy and serial sectioning, and found that the microtubular fascicles and polyribosomal clusters do exist in the internodal axoplasm. The fasciculated microtubules were observed mainly in the first paranode. The polyribosomal clusters were found along the course of the first internode at a random distance, however, they occurred mainly in the proximal part of the first internode. The proportion of sections in which ribosomes were found, i.e. the incidence of ribosomes, in the first 30-microm-long portion was 71 +/- 24% (mean +/- SD, n = 4), and significantly different from that in the second 30-microm-long portion (3.2 +/- 1.3%) (mean +/- SD, n = 4) (P < 0.005). The more distal part of the first internode was not investigated.     

Assaying 192Ir line sources using a standard length well chamber

The strength of intravascular 192Ir sources is typically measured by the manufacturer before shipment, and treatment planning is based on that assay. However, in-house verification of source strength is required at some institutions by state law or internal policy, is recommended by the AAPM TG 60 report on intravascular brachytherapy, and is considered a necessity by many medical physicists. To accommodate the long sources used in intravascular therapy, special well chambers with extended regions of constant response have been designed. To allow assays using a widely available standard well chamber, we have measured its position dependent sensitivity and derived from it a table of correction factors that account for the extended length of intravascular sources. An experimental verification shows that application of these correction factors yields assays with sufficient accuracy for routine quality assurance tests.     

Tuberous sclerosis complex proteins control axon formation

Axon formation is fundamental for brain development and function. TSC1 and TSC2 are two genes, mutations in which cause tuberous sclerosis complex (TSC), a disease characterized by tumor predisposition and neurological abnormalities including epilepsy, mental retardation, and autism. Here we show that Tsc1 and Tsc2 have critical functions in mammalian axon formation and growth. Overexpression of Tsc1/Tsc2 suppresses axon formation, whereas a lack of Tsc1 or Tsc2 function induces ectopic axons in vitro and in the mouse brain. Tsc2 is phosphorylated and inhibited in the axon but not dendrites. Inactivation of Tsc1/Tsc2 promotes axonal growth, at least in part, via up-regulation of neuronal polarity SAD kinase, which is also elevated in cortical tubers of a TSC patient. Our results reveal key roles of TSC1/TSC2 in neuronal polarity, suggest a common pathway regulating polarization/growth in neurons and cell size in other tissues, and have implications for the understanding of the pathogenesis of TSC and associated neurological disorders and for axonal regeneration.     

Midline axon guidance and human genetic disorders

In bilaterally symmetric animals, many axons cross the midline to interconnect the left and right sides of the central nervous system (CNS). This process is critical for the establishment of neural circuits that control the proper integration of information perceived by the organism and the resulting response. While neurons at different levels of the CNS project axons across the midline, the molecules that regulate this process are common to many if not all midline-crossing regions. This article reviews the molecules that function as guidance cues at the midline in the developing vertebrate spinal cord, cortico-spinal tract and corpus callosum. As well, we describe the mutations that have been identified in humans that are linked to axon guidance and midline-crossing defects.     

Temperature-sensitive conduction failure at axon branch points

1. The propagation of action potentials through the branching regions of squid axons was examined experimentally and with computer simulations over a temperature range of 5-25 degrees C. 2. Above a critical ratio of postbranch to prebranch diameters, propagation of an action potential failed. The value of this critical ratio is very sensitive to temperature and is smaller at high temperatures. The experimentally measured Q10 of the critical ratio is 0.37 +/- 0.04. 3. Evaluation of a number of parameters of action-potential propagation showed that this effect is closely related to the change in the width of the action potential with temperature (Q10 = 0.29 +/- 0.01).     

Neural regenerative strategies incorporating biomolecular axon guidance signals

There are currently no acceptable cures for central nervous system injuries, and damage induced large gaps in the peripheral nervous system have been challenging to bridge to restore neural functionality. Innervation by neurons is made possible by the growth cone. This dynamic structure is unique to neurons, and can directly sense physical and chemical activity in its environment, utilizing these cues to propel axons to precisely reach their targets. Guidance can occur through chemoattractive factors such as neurotrophins and netrins, chemorepulsive agents like semaphorins and slits, or contact-mediated molecules such as ephrins and those located in the extracellular matrix. The understanding of biomolecular activity during nervous system development and injury has generated new techniques and tactics for improving and restoring function to the nervous system after injury. This review will focus on the major neuronal guidance molecules and their utility in current tissue engineering and neural regenerative strategies.