Quantification of functional recovery in a larval zebrafish model of spinal cord injury

Abstract

Human spinal cord injury (SCI) is characterized by permanent loss of damaged axons, resulting in chronic disability. In contrast, zebrafish can regenerate axonal projections following central nervous system injury and re-establish synaptic contacts with distant targets; elucidation of the underlying molecular events is an important goal with translational potential for improving outcomes in SCI patients. We generated transgenic zebrafish with GFP-labeled axons and transected their spinal cords at 10 days post-fertilization. Intravital confocal microscopy revealed robust axonal regeneration following the procedure, with abundant axons bridging the transection site by 48 h post-injury. In order to analyze neurological function in this model, we developed and validated new open-source software to measure zebrafish lateral trunk curvature during propulsive and turning movements at high temporal resolution. Immediately following spinal cord transection, axial movements were dramatically decreased caudal to the lesion site, but preserved rostral to the injury, suggesting the induction of motor paralysis below the transection level. Over the subsequent 96 h, the magnitude of movements caudal to the lesion recovered to baseline, but the rate of change of truncal curvature did not fully recover, suggesting incomplete restoration of caudal strength over this time course. Quantification of both morphological and functional recovery following SCI will be important for the analysis of axonal regeneration and downstream events necessary for restoration of motor function. An extensive array of genetic and pharmacological interventions can be deployed in the larval zebrafish model to investigate the underlying molecular mechanisms.

Functional Trajectories during innate spinal cord repair

“…Gait analysis offers a scalable approach to quantify swim quality. Prior applications of gait analysis, such as dorsal centerline posture and tail beat kinematics, supported the model that functional recovery emerges directly from structural recovery among ray-finned fishes (12, 17, 18). These studies revealed stepwise recovery of specific gait features in correlation with axon regeneration from V2a interneurons (12, 19).…”

“…One viable option includes novelty detection, which classifies behavior episodes as inliers or outliers relative to a control distribution. Another option is posture parameter quantification, which has been used in larval zebrafish (17). Examples of posture parameter quantification from human and mouse studies used behavior and gait features, such as quantified response to stimulus and range or speed of motion, to measure functional recovery (33, 34, 35, 36).…”

“…Traditional and recently established measurements of swim capacity include swim endurance as quantified by time to exhaustion against increasing water current velocities (2,6,8,9,10), maximum speed either against increasing water current velocities or in a static tank (11,12,13,14), swim distance (7,9), time active (9,10), time swimming against flow (9), burst frequency (9,10), and average position of the fish against a water flow axis ("mean y") (9,10). Although new quality measurements have been developed for larval fish including body angle (15), power spectral densities (16), and centerline posture kinematics (17), published quality measurements for adult fish are limited to manual scoring of perceived swim ability (3,4,5), tail beat frequency and amplitude (12). Moreover, manually assigned scores are subjective and do not scale well for higher throughput.…”

Functional trajectories during innate spinal cord repair

“…Although new quality measurements have been developed for larval fish including body angle (Zeng et al, 2021), power spectral densities (Vasudevan et al, 2021), and centerline posture kinematics (Hossainian et al, 2022), published quality measurements for adult fish are limited to manual scoring of perceived swim ability (Becker et al, 1997;Goldshmit et al, 2012;Strand et al, 2016), tail beat frequency and amplitude (Huang et al, 2022). Moreover, manually assigned scores are subjective and do not scale well for higher throughput.…”

Optimizing spinal cord injury in zebrafish larvae: effects of age on the injury response

“…Embryonic development is classically defined as ending at the time of hatching, 3-4 days post fertilization (dpf) (Kimmel et al, 1995;Parichy et al, 2009) with larval development continuing for approximately six weeks. Often laboratories use late embryonic and early larval stages due to ease and ethical considerations (Tsarouchas et al, 2018) with some studies looking at larvae as old as 10 dpf (Hossainian et al, 2022). Furthermore, neurogenesis of primary motor neurons is greatly reduced at 2 dpf (Reimer et al, 2013) and nascent myelin is observed 3-4 dpf (Bin and Lyons, 2016;Kirby et al, 2006;Park et al, 2002;Preston and Macklin, 2015).…”