A microchip for quantitative analysis of CNS axon growth under localized biomolecular treatments

Abstract

Growth capability of neurons is an essential factor in axon regeneration. To better understand how microenvironments influence axon growth, methods that allow spatial control of cellular microenvironments and easy quantification of axon growth are critically needed. Here, we present a microchip capable of physically guiding the growth directions of axons while providing physical and fluidic isolation from neuronal somata/dendrites that enables localized biomolecular treatments and linear axon growth. The microchip allows axons to grow in straight lines inside the axon compartments even after the isolation; therefore, significantly facilitating the axon length quantification process. We further developed an image processing algorithm that automatically quantifies axon growth. The effect of localized extracellular matrix components and brain-derived neurotropic factor treatments on axon growth was investigated. Results show that biomolecules may have substantially different effects on axon growth depending on where they act. For example, while chondroitin sulfate proteoglycan causes axon retraction when added to the axons, it promotes axon growth when applied to the somata. The newly developed microchip overcomes limitations of conventional axon growth research methods that lack localized control of biomolecular environments and are often performed at a significantly lower cell density for only a short period of time due to difficulty in monitoring of axonal growth. This microchip may serve as a powerful tool for investigating factors that promote axon growth and regeneration.

Keywords: Automated measurement; Axon growth rate; Compartmentalized culture; Localized biomolecular treatment; Microfluidic culture platform; Quantitative axon length analysis.

Figure 1

(A) Schematic illustrations of the top and bottom PDMS layers composing the developed quantitative axon growth analysis microchip. (B) Illustration showing axon isolation and guidance via the array of shallow microgrooves patterned on the bottom substrate.

Figure 2

A schematic illustration showing image processing and axon tracing algorithm steps for automated axon growth quantification process.

Figure 3

(A) Sealed microgrooves (3 × 20 × 800 μm³) successfully confined neuronal somata in the soma compartment and prevented dendrites from crossing into the axon compartment. No dendrites could be observed inside the axon compartment at DIV 11 (axon: NF – green, dendrites: MAP2 – red). White dotted line indicates inlets and outlets of the sealed microgrooves. (B) Microgrooves formed on the bottom substrate physically guided axons (stained with Calcein-AM) to grow in straight lines once axons crossed into the axon compartment. Inset shows axons inside a compartmentalized microdevice having similar configuration but without the axon-guiding microgrooves, showing tangling of axons that make quantitative and automatic growth analysis challenging. Scale bar: 50 μm. (C) Retrograde staining of isolated axons by Calcein-AM loaded into the axon compartment shows that most of the somata with axons extending into the axon compartment are located in the vicinity of the inlet area of the sealed microgrooves. (D) Illustration and DAPI stained neurons showing the distribution of neurons inside the soma compartment. Well-type open compartment configuration and the ridge structure enabled most of the sealed microgrooves to have multiple neurons at inlets. (E) Isolated and guided axons inside the axon compartment without (left) and with (right) the cylindrical ridge structure. White dotted lines indicate the boundary between the sealed microgroove and the compartment. (F) Axon isolation efficiency at different neuron plating densities measured at DIV 11 (mean ± SD). More than 96.8 ± 1.9% of the sealed microgrooves were filled with axons when neurons were plated at initial density of 1000 cells/mm² (* p < 0.05 compared to 500 cells/mm²).

Figure 4

(A) Degenerated axons inside the axon compartment after 4 days of localized CSPG treatment (5 μg/ml). (B) The fluidic isolation feature and the multi-compartment configuration enabled six different concentrations of localized CSPG treatments to be performed on a single microchip for screening effective CSPG dosage (2.5 μg/ml) that caused axon degeneration. The images are representative results from more than three independent experiments.

Figure 5

(A-D) Results from key intermediate steps of the automated axon growth quantification process. (A) Original image. (B) The original image is rotated so that the microgrooves and the axons are vertically oriented. The boundary between the sealed microgrooves and the axon compartment is located and shown as the red curve (dubbed the “baseline”). (C) End points of the axons are found by sweeping the image from the bottom toward the top. Identified end points are marked with yellow circles. (D) Final result of the axon detection algorithm is shown (yellow lines indicate successfully traced axons). (E) Comparison of the measured axon length by automated (314.3 ± 85.41 pixels) and manual measurements (315.1 ± 84.01 pixels) (mean ± SD, n: number of analyzed axon compartments).

Figure 6

The effect of localized biomolecular treatments on axon growth. The results shown are obtained from 3-6 independent experiments (mean ± SD, n: number of analyzed axons). (A) The effect of ECMs and CSPG on axon growth when added only to the axon compartments. (B) The effect of ECMs and CSPG on axon growth when added only to the soma compartment. (C) The effect of Matrigel™ with BDNF on axon growth when added only to the axon compartments. (D) The effect of Matrigel™ with BDNF on axon growth when added only to the soma compartments. * p < 0.05, *** p < 0.001 to control.