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Young Scientist Journal

Young Scientist Journal

Gait-Based Evaluation of Zebrafish Spinal Cord Regeneration

ABSTRACT

Paralysis is a condition that affects over five million Americans, either temporarily or permanently, and we have yet to find a cure for injuries of the spine and the central nervous system. Specific organisms, such as Zebrafish, can regenerate their spinal cord and are therefore used in research on paralysis recovery. The most effective method of studying spinal regeneration is through cellular analysis: slicing a section of the spinal cord and analyzing it under a microscope. But this method requires sacrificing the zebrafish, making it difficult to track the same animal longitudinally to assess recovery over time. My project aimed to find a new way to effectively measure spinal cord regeneration by quantifying how the zebrafish recover movement as they regenerate a severed spine. Uninjured and spinal cord-injured zebrafish were evaluated and analyzed for the variation of swim height and the quality of swim after a disturbance via a tail-tap. Motor function assessment was found to be a powerful way to track recovery. Spinal cord injury decreased the zebrafish’s motivation and capability through lowered variation in swim height and poor swim quality, since paralysis impairs swimming technique. Continuing to develop methods for analyzing functional recovery can help labs by decreasing the need to sacrifice as many zebrafish as possible for regeneration research and further studying nervous system recovery.

INTRODUCTION.

Adult mammals, including humans, possess limited regenerative abilities outside of tissue repair, skeletal muscle, bone, peripheral nerves, and the liver [1]. The human body is prone to scarring over injuries, and in most cases, functional capacity is not recovered. The central nervous system, including the brain and the spinal cord, lacks the cells required for repair, so injuries scar rather than regenerate. In the case of spinal cord injury, this leads to permanent paralysis if the tissue is severely damaged [2]. Zebrafish (Danio rerio) are freshwater ray-finned fish commonly used in regenerative medicine research due to their incredible ability to regenerate multiple tissues after injury, including the spinal cord, and restore normal bodily function [3].

Commonly, spinal cord regeneration is tracked in Zebrafish using histology, analyzing the cellular behavior of resident progenitor cells, neurons, vasculature, immune cells, and other tissue-specific cell types under a fluorescent microscope [1]. However, this process is invasive for the Zebrafish and requires the animal to be euthanized to cut sections of the spinal cord for histology. While the method is incredibly accurate and preferred due to the ability to view and measure the actual regrowth of the spinal cord on a cellular level, it has various disadvantages: it does not allow to track the long-term regeneration of individual animals, it requires a large number of Zebrafish for consistent testing, and it utilizes expensive supplies such as a cryostat and a confocal microscope for sectioning and imaging, respectively. Although there are these drawbacks, histology has been used for over a century for data on cellular arrangement because of the detailed examination it provides, compared to other methods [4]. The goal of my project was to create a set of procedures that could analyze regeneration of the spinal cord as effectively as histological procedures without these limitations.  Our goal is not to replace histology, but to complement it with methodologies that reduce experimental burden where appropriate while maintaining rigorous evaluation of regenerative outcomes. To this end, common functionality tests were utilized to expand the experimental repertoire to include muscle, sensory, reactivity, and swimming quality reconstruction.

Common functional methods include the usage of a swim tunnel, allowing for the measurement of how long zebrafish swim against an increasing flow of current [5], how close they swim to the location of the flow [3], and the critical swim speed – the speed of the flow when the zebrafish can no longer maintain its ability to swim before and after injury. New methods were developed to include vertical swim height assessment and burst swim quality.

Overall, while zebrafish are well-suited for experimental studies due to their efficient reproductive capacity [6], lowering the use of lethal endpoints, further reducing cost and time to maintain a steady population, would increase efficiency. Furthermore, other model organisms used for comparable studies undergo similar experiments regarding generation, such as mice, which have longer reproduction periods, are less cost-effective and more labor-intensive to maintain [7, 8]. Therefore, developing a more efficient methodology to measure motor function recovery during spinal cord regeneration would not only streamline zebrafish research but also facilitate downstream, higher-cost studies in mammalian models.

MATERIALS AND METHODS.

Zebrafish Culturing.

Wild-type zebrafish of the Ekkwill (EK) strain were used for all experiments. Uninjured, injured, and scoliosis animals were female and male clutch mates aged 3 to 12 months, measuring ~2 cm in length. Animal studies were approved by the Institutional Animal Care and Use Committee and the Institutional Biosafety Committee of Vanderbilt University.

Zebrafish Spinal Cord Injuries.

Adult zebrafish were anesthetized using 0.02% tricaine. Scales were scraped off, and fine scissors were used to make a small incision through the dorsal muscle to expose the vertebral column, which was transected halfway between the dorsal fin and the operculum. Complete transection and loss of swimming capacity were visually confirmed both at the time of surgery and at 1-day post-injury (dpi). The scoliosis condition occurred naturally post-surgery in zebrafish, as is common due to spinal tissue stiffening, causing an increase in spinal curvature [9].

Experimental Groups.

Three experimental groups of zebrafish were used: seven uninjured as the control, four injured, and one scoliosis-developed zebrafish. All zebrafish were held in the Vanderbilt Light Hall Building’s aquatic facility and injured before the research began. All zebrafish were housed and cared for under the same conditions, according to the Institutional Animal Care and Use Committee and the Institutional Biosafety Committee of Vanderbilt University.

Vertical Swim Height Test.

Vertical swim distance was assessed to analyze the functional regeneration and motivation of the zebrafish. A ruler was adhered to the back of a laboratory zebrafish tank, and a 720-1080 progressive scan resolution camera was used to record 10 minutes of undisturbed zebrafish swim patterns (Fig. 1A). The videos were analyzed by hand to decide at what heights the zebrafish tended to stay and the variation of their swim height capabilities. It was predicted that the injured zebrafish would reside in the lowest region of the tank for most of the time and have a low swim height variation in comparison to the uninjured, as paralysis would be detrimental to vertical swim, like all other forms of movement. As regeneration occurs, it was predicted that the zebrafish would spend their time spread equally across the three sections and have a higher swim variation as they regain functionality akin to when they were previously uninjured.

Figure 1. A. Vertical swim height test, measuring the most common swim height of occupation and the variation of swim height over 10 minutes of undisturbed swim time. Digitally, the tank was split into three equal vertical sections (top, middle, bottom). B. Burst swim quality test, measuring the number of c-shapes (providing a burst of energy to swim away from stressors) and the distance swam after being tapped on the tail to illicit a disturbance. Each fish was tail-tapped 5 times with an interval of 30 seconds between each, beginning after they finished swimming, to lessen the possibility of desensitization. Figure made using BioRender.

Burst Swim Quality Test.

Burst swim quality and caudal fin sensitivity were tested throughout regeneration using a ‘tap test.’ Using a 100mm x 15mm petri dish filled with ~15 mL of system water, the tap test was performed individually by transferring a single zebrafish from the tank to the dish. A minute was dedicated to allowing acclimation to the dish for the zebrafish to work out most of the initial stress caused by the researcher’s interference. Then, using the blunt, thin back end of a laboratory stainless steel forceps, the middle of the caudal fin was tapped (Fig. 1B). This elicited a bodily C-shape tactic by the zebrafish, turning its head towards its tail, allowing it to quickly turn away from the disturbance and gain a burst of swimming distance. This maneuver response is common in multiple organisms as a reaction to stress or disturbance, such as freshwater planaria [10] and the zebrafish [11, 12]. Using this knowledge, it was decided to collect data on the number of c-shapes and the distance swam after each tap to determine the number of attempted bursts and the distance swam for each, respectively. Five tail taps for each zebrafish were performed, recording the functional behavior for each using a 720-1080 progressive scan resolution camera. The end of each tail tap burst swim would end once the zebrafish either stopped swimming fully or eased the speed of the swim. In some instances, the uninjured zebrafish did not always completely stop swimming, as is normal. Because of this, the end of the stress period caused by the disturbance for the uninjured was deemed to be when they finished the initial bursts and began to slow down. After they stopped burst-swimming, 30 seconds were given to alleviate desensitization and stress in the zebrafish after each tail-tap. This method was performed for each zebrafish – five tail taps per fish – in each group.

RESULTS.

Vertical Swim Tests.

When uninjured, the zebrafish demonstrated high variation in the vertical height where they spent their time (Fig. 2). There was no set pattern in their swim, and each zebrafish could be located at different heights in the tank at the same time. The zebrafish developing scoliosis showed an exceptionally low vertical swim height variation: throughout the ten-minute test period, the zebrafish mainly remained at the bottom of the tank. The injured zebrafish demonstrated a lower swim height variation than the uninjured, but higher than the scoliosis, with some of the zebrafish occasionally gathering a burst of energy to swim up the tank, inevitably falling back down to rest at the bottom.

Figure 2. Images acquired from the 10-minute videos of the Vertical Swim Distance Tests recorded on a 720-1080 progressive scan resolu-tion camera. Images of the three groups are shown. From left to right: injured (n=7), scoliosis (n=1), regenerating zebrafish (n=4).

Burst Swim Quality Test.

To quantify the burst swim quality of the zebrafish, the distance swam in centimeters, and the number of c-shapes performed by each zebrafish were found, as well as per tap for the three groups (Fig. 3). The ratio of the average distance swam to the average quantity of c-shapes for each group was then found. The average total distance swam per fish was 63 cm for the scoliosis, 172 cm for the injured, and 574.5 cm for the uninjured fish, respectively. There was variation across each fish, but the data depicted a large and significant difference between the groups by scoliosis injury, especially between the scoliosis-to-injured and the injured-to-uninjured. This same difference between groups occurred with the average distance per tap of each of the fish. With the number of c-shapes that dictated a burst-start movement, the average quantity for the scoliosis fish was 9, 49.4 for the injured, and 14.71 for the uninjured. Unlike the distance swam, where the lowest-to-highest averages went from scoliosis, injured, to uninjured, the injured fish had the highest number of c-start bursts, a significantly higher quantity than the other two groups. The difference between the c-starts was not significantly different between the scoliosis group and the uninjured group.

Figure 3. Graphs created from data collected from the burst swim quality tests comparing distance swam (cm), c-shape quantity, and gait quality ratio. Green bars are scoliosis (n=1), brown bars are regenerating (n=4), and yellow bars are uninjured (n=7).

For the final method of data collection on the burst swim quality test, the ratio of each group between the average distance swam and the number of c-starts was calculated. These ratios were deemed to be the gait quality averages. The ratio was the smallest for the injured zebrafish (2.86) and was highest for the uninjured (39.04). The scoliosis zebrafish were between the two, with 2.86, but it was closer to the injured, being significantly lower than the uninjured control.

DISCUSSION.

The uninjured zebrafish, in contrast to both the injured and scoliosis groups, had high motivation and sufficient capabilities to fulfill it. The uninjured zebrafish had a high swim height variation while undisturbed in the tank, while their normal swim habits have high variation and aren’t restricted by a vertical pattern. They are free to swim at whatever height and speed they choose, while the injured and scoliosis fish are inhibited by their spinal cord injury. So, the difference between the groups is evident because of the functional capabilities determined by whether a SCI or abnormality has occurred. This is also depicted by the data gathered from the tap test, as the uninjured fish had a high gait quality ratio of average distance swam to the quantity of c-shapes – they engaged in a lower number of c-shapes per tap because they had the functional ability to swim a further distance per burst. The tap test is a transparent functional capability test for zebrafish because it determines whether they can efficiently use a common escape maneuver to swim away from disturbances, shown by the greater ratio of distance swam per c-shapes performed by the injured and scoliosis groups.

The scoliosis fish also experiences a decrease in swimming quality because of a decrease in motivation and capabilities, as the scoliosis of the spine decreases the efficiency of swim propulsion and increases the energy needed to perform normal swimming. The data gathered from the scoliosis fish depicts this, as it had a very low variation in swim height and a low gait ratio. The scoliosis in the spine makes it incredibly difficult to obtain enough force efficiently to swim, which is why the fish primarily remained resting at the bottom of the tank.

The injured fish experience current paralysis that inhibits the usage of their caudal region and fin, which significantly prohibits the main component of gathering enough propulsive force to swim efficiently, hindering swim quality. The injured zebrafish had a higher swim height variation than the scoliosis fish, but it was significantly lower than the uninjured. The fish would occasionally use a burst of energy to swim up to the top of the tank but did not have the physical capability to maintain the height and would fall back to the bottom of the tank. While the scoliosis fish did this a couple of times, the injured fish did it more frequently, depicting a higher motivation to attempt to swim but still being inhibited by their SCI. We can also see this in the data with the large number of c-shapes executed during the tap test. However, with the higher quantity of c-shapes performed by the injured in comparison to the uninjured, they had a low average distance swam. This data shows that while the injured fish are attempting to undergo this burst swim with the starting c-shape, the burst is inefficient, and they are not getting a lot of distance for each burst, as depicted by the ratio of the average distance swam to the average quantity of c-shape. While the injured zebrafish are trying to swim away from disturbances, they are unable to do so effectively, so they compensate by undergoing more bursts for the small amount of distance swam for each tap.

Limitations and Challenges.

The subject groups included zebrafish previously injured in the lab, a fish that naturally developed scoliosis post-SCI, and a population of uninjured fish. The groups were only used for these tests but were not injured specifically for this research. Gathering a group of zebrafish that are specifically and only for this research would be more beneficial for results, as you could track recovery and regeneration post-SCI of the same sample rather than just analyzing locomotion at a singular time point of fish with different conditions, as was completed. This research, however, acts as preliminary data for a greater project and will be expanded on.

CONCLUSION.

Analyzing and recording data on regeneration appears to be possible using functional assessments. Laboratory teams can assess functionality by creating and using tests that focus on swimming capability and motivation. A new set of methods was created that can be used to collect such data, placing a steppingstone for discussion on expanding and collecting further information on physical and functional tests for analyzing spinal cord regeneration. This way, laboratory research with zebrafish and regeneration can be more efficient, as less of a consistent flow of model organisms would be needed to keep up with the fatalities caused by histology. While histological analysis is still vital for the field, as it gives incredible cellular data unattainable by functional tests, it is not needed for all research, and finding areas to limit it, such as tracking recovery and regeneration, is important.

Future Directions.

Further research is being conducted in the Cigliola Lab to highlight these methods and enhance the results. The aim is to find the correlation between these and an additional gait test and histology to see if tracking functional recovery can be accurately used to track cellular regeneration. More research should be done to discover a wider range of functional methodologies that can also have a high correlation and connection with cellular regeneration of the spinal cord to not only improve the efficiency of the SCI field, but also across regeneration and recovery in zebrafish and other model organisms. Developing a functional methodology of analyzing or tracking recovery can also be used for research in muscle degeneration [13], and we could further expand to using these tests for mice, as it’s incredibly inefficient to have to sacrifice so many research subjects for data that can be obtained other ways with alternative methods.

ACKNOWLEDGMENTS.

Thank you to Dr. Valentina Cigliola, Nicolas Noel, the Cigliola lab, and Dr. Menton Deweese for your mentorship and collaboration. Thank you to Vanderbilt students and lab members Barera Ajaz, Christina Avila, and Ava Aria for assisting with training. Thank you to the School for Science and Math at Vanderbilt and Vanderbilt University for research support.

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Posted by on Thursday, May 14, 2026 in May 2026.

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