Microfluidic culture platform for studying neuronal response to mild to very mild axonal stretch injury

Abstract

A new model for studying localised axonal stretch injury is presented, using a microfluidic device to selectively culture axons on a thin, flexible poly (dimethylsiloxane) membrane which can be deflected upward to stretch the axons. A very mild (0.5% strain) or mild stretch injury (5% strain) was applied to primary cortical neurons after 7 days growth in vitro. The extent of distal degeneration was quantified using the degenerative index (DI, the ratio of fragmented axon area to total axon area) of axons fixed at 24 h and 72 h post injury (PI), and immunolabelled for the axon specific, microtubule associated protein-tau. At 24 h PI following very mild injuries (0.5%), the majority of the axons remained intact and healthy with no significant difference in DI when compared to the control, but at 72 h PI, the DI increased significantly (DI = 0.11 ± 0.03). Remarkably, dendritic beading in the somal compartment was observed at 24 h PI, indicative of dying back degeneration. When the injury level was increased (5% stretch, mild injury), microtubule fragmentation along the injured axons was observed, with a significant increase in DI at 24 h PI (DI = 0.17 ± 0.02) and 72 h PI (DI = 0.18 ± 0.01), relative to uninjured axons. The responses observed for both mild and very mild injuries are similar to those observed in the in vivo models of traumatic brain injury, suggesting that this model can be used to study neuronal trauma and will provide new insights into the cellular and molecular alterations characterizing the neuronal response to discrete axonal injury.

FIG. 1.

(a) Schematic showing experimental setup for PDMS membrane deflection characterization. Gas pressure was applied to the air channel by using an in-house built valve system which utilized a dynamic pressure regulator, USB-based controller box for the valve manifolds and Labview software. The computer sent the signal to the valve manifold through the controller box. The pneumatic valve opened and applied gas pressure to the embedded microchannel. The thin PDMS membrane deflected upward and deflection was measured with optical profiler system. (b) Photograph of assembled device comprising the Quake valve and culturing device. (c) Screen capture of optical profiler data used for quantifying deflection.

FIG. 2.

Schematic drawing of microfluidic device used for simulating axonal stretch injury. (a) A thin PDMS membrane separates the Quake valves air-channel (bottom) from the overlying culturing chamber (Top). (b) Application of gas pressure to the air channel (positioned at 200–300 μm from microgrooves), causes upward deflection of the thin PDMS, which stretches the overlying axon. (c) Rat cortical neurons at 7 days in poly-l-lysine coated culturing device showing adequate axonal extension prior to axonal stretch injury. Scale bar = 100 μm.

FIG. 3.

Relationship between applied gas pressure and membrane deflection at steady state for ∼60 μm PDMS membranes (1500 rpm) and ∼15 μm PDMS membranes (2500 rpm). A constant valve opening of 10 s was applied to ensure deflection measurements at steady state.

FIG. 4.

Immunocytochemistry images of uninjured and injured neurons 24 h following 0.5% injury. Cell bodies and dendrites ((a) MAP2 labelling), and axons ((b) tau and NFM labelling), in the control chambers were smooth and uniformly labelled for cytoskeletal markers. At 24 h post injury cortical cultures exposed to axonal stretch injury showed dendritic blebbing, and irregular MAP2 expression ((c) MAP2 labelling). The injured axons ((d) tau and NFM labelling) underwent characteristic beading and degeneration, showing punctate accumulation of tau and NFM within the swollen portions of the axon. Scale bar = 50 μm.

FIG. 5.

Axonal stretch injury to cultured cortical neurons resulted in axonal degeneration. (a) Tau-labeled (microtubule marker) axons following 0.5% injury (very mild) and 5% injury (mild) at 24 h and 72 h time point. Binarized images show fragmented axons defined by Analyze Particle function in ImageJ software. Stretch injury induced progressive distal degeneration leading to axonal beading and microtubule fragmentation. Scale bar = 50 μm. (b) The degeneration index increased significantly at 72 h following 5% injury, compared to the degeneration index following 0.5% injury. However, there was no significant difference between control and 24 h following 0.5% injury. The degeneration index increased significantly at 72 h following 0.5% injury if compared to the control. Axonal degeneration index is significantly higher than controls at both 24 h and 72 h following 5% injury. The * symbol represents a statistical difference (p < 0.05) versus control at each time point. The † symbol represents a statistical difference (p < 0.05) versus very mild stretch (0.5% injury) at post 72 h.