Semi-automated Sholl analysis for quantifying changes in growth and differentiation of neurons and glia

Abstract

There is a need to develop therapies that promote growth or remyelination of mammalian CNS axons. Although the feasibility of pre-clinical treatment strategies should be tested in animal models, in vitro assays are usually faster and less expensive. As a result, in vitro models are ideal for screening large numbers of potential therapeutics prior to use in more complex in vivo systems. In 1953, Sholl introduced a technique that is a reliable and sensitive method for quantifying indices of neurite outgrowth. However, application of the technique is limited because it is labor-intensive. Several methods have been developed to reduce the analysis time for the Sholl technique; but these methods require extensive pre-processing of digital images, they introduce user bias or they have not been compared to manual analysis to ensure accuracy. Here we describe a new, semi-automated Sholl technique for quantifying neuronal and glial process morphology. Using MetaMorph®, we developed an unbiased analysis protocol that can be performed ~3x faster than manual quantification with a comparable level of accuracy regardless of cell morphology. The laborious image processing typical of most computer-aided analysis is avoided by embedding image correction functions into the automated portion of the analysis. The sensitivity and validity of the technique was confirmed by quantifying neuron growth treated with growth factors or oligodendroglial maturation in the presence or absence of thyroid hormone. Thus, this technique provides a rapid and sensitive method for quantifying changes in cell morphology and screening for treatment effects in multiple cell types in vitro.

A novel cysteine-rich neurotrophic factor in Aplysia facilitates growth, MAPK activation, and long-term synaptic facilitation

“…2C). To quantify the aggregate data, we determined the areas under curves in Figure 2C (Gensel et al 2010), and found that the area for apCRNF treatment was significantly larger than the area for the control treatment (two-tailed t-test, t (17) ¼ 2.214, P , 0.05) (Fig. 2D).…”

“…Recombinant apCRNF (250 ng/mL) or vehicle (0.1% BSA in PBS) was added on cultured SNs at Day 3 in vitro and incubated for 3 d. Bright field images were captured with a Zeiss Axiovert 200 microscope. Twenty evenly spaced concentric circles with increasing radii from 30 to 315 mm were overlaid onto the center of a cell soma; neurite elongation and branchings were measured by Sholl analysis (Sholl 1953;Gensel et al 2010). …”

miR-155 Deletion in Mice Overcomes Neuron-Intrinsic and Neuron-Extrinsic Barriers to Spinal Cord Repair

“…After injury, CNS neurons fail to upregulate regeneration-associated genes (RAGs), those genes encoding proteins that promote cytoskeletal dynamics and growth-associated changes (Skene and Willard, 1981;Jacobson et al, 1986;Bonilla et al, 2002;Mason et al, 2002;Khazaei et al, 2014). Numerous neuron-extrinsic barriers also conspire to prevent efficient CNS growth and repair (McKeon et al, 1991;Brambilla et al, 2005;Donnelly and Popovich, 2008;Pineau et al, 2010;Alilain et al, 2011;Gaudet and Popovich, 2014). For example, inflammatory macrophages can cause axonal injury or inhibit axon regeneration after spinal cord injury (SCI) (Popovich et al, 1997;Kigerl et al, 2006;Kigerl et al, 2009;Prüss et al, 2011;Pool et al, 2012;Kroner et al, 2014).…”

“…Whereas little is known about miR-155-3p, miR-155-5p has been studied extensively in inflammatory diseases and cancer. In the CNS, miR-155-5p can be found in microglia/macrophages (Tili et al, 2007;Cardoso et al, 2012), astrocytes (Tarassishin et al, 2011), and neurons (Herai et al, 2014). The therapeutic potential of targeting miR-155 is exemplified by the fact that miR-155 removal improves repair and recovery in other animal models of CNS pathology, including amyotrophic lateral sclerosis (Koval et al, 2013;Butovsky et al, 2015), multiple sclerosis , and central neuroinflammation (Lopez-Ramirez et al, 2014).…”

“…miR-155-5p modulates the stability and translation of mRNAs implicated in inflammation (Tili et al, 2007;Worm et al, 2009;Liu et al, 2011b;Fonken et al, 2016) and axon growth (Wang et al, 2013;Varendi et al, 2014). In macrophages and microglia, damage-or pathogen-associated molecular patterns bind toll-like receptors and elicit inflammatory signaling pathways (Graff et al, 2012;Freilich et al, 2013;.…”