The Effect of Spinal Cord Severing in Zebrafish
Blue Expo

ABSTRACT
Previous research indicates that nervous and cardiac tissue regeneration occurs in zebrafish because they lack some inhibitory characteristics found in mammals. The purpose of this project was to observe spinal cord regeneration in zebrafish of various stages of development. Zebrafish spinal cords were severed and the surviving groups were observed for visible signs of neural regeneration at that site. Revascularization was visible in zebrafish from later trials but no neural regeneration was observed in this experiment.

INTRODUCTION
The purpose of this lab was to observe the effects of spinal cord injury inflicted upon young, developing zebrafish and determine if any of spinal cord regeneration occurs. The hypothesis was that the spinal cords of the zebrafish would regenerate to some degree after being experimentally severed. Previous research has found that zebrafish neural and cardiac tissue regeneration does occur given the correct procedures and conditions. In a study on zebrafish heart regeneration by Poss, Wilson, and Keating, roughly 20% of an adult zebrafish heart is surgically removed and heart tissue regenerates through cardiomyocyte proliferation. After a period of sixty days, the regenerated portion is histologically indistinguishable from the original cardiac tissue. (Poss, Wilson, and Keating, 2002)

Another study performed by Fetcho and colleages found that some Mauthner axons in zebrafish in the presence of the second messenger, cyclic adenosine monophosphate (cAMP), begin regenerating to a small extent within two days of the zebrafish spinal cord being severed. (Bhatt, Otto, Depoister, and Fetcho 2004) To test the hypothesis that neural tissue regeneration occurs in zebrafish, they were physically immobilized and their spinal cords were carefully severed. Survival rates were recorded for each severing trial. Zebrafish surviving longer than one day were observed microscopically for evidence of neural regeneration.

METHODS

Spinal Cord Severing Procedure
The zebrafish used were from 3-12 days old were pipetted out of the 250mL beaker that they were hatched in and transferred to test tubes in 1-2 drops of water. A microwave was used to melt agar to a warm liquid, 3-4 drops of which was then added to the zebrafish test tubes and gently swirled. The zebrafish were then drawn up in 1-2 drops of the agar/water mixture and transferred onto a slide. Once on the slide, the agar cooled slightly forming a layer around the zebrafish with gel-like consistency. The purpose of the agar in this experiment was to physically restricted the movement of the zebrafish without knocking them out or harming them. With the zebrafish immobilized on a slide, spinal cord severing could be performed under the stereoscope with a steady hand using an X-acto knife blade. The slide could be rotated under the scope to achieve the best angle of approach for the dominant hand holding the blade. The goal of this step was to sever the spinal cord that is ventral to the thin black line of pigment along the back of the zebrafish in one careful and steady movement, taking care not to harm the underlying transparent notochord and adversely affect development. Early in the experiment a thin layer of water was not added to the slide on top of the agar layer containing the zebrafish during the spinal cord severing but later in the experiment a thin layer of calcium water (14g Ca/ 100mL water) was added to limit the exposure of the young zebrafish to air. Post-procedural zebrafish were pipetted using a small amount of water into a small plastic dish containing a thin layer of water. Zebrafish early in the experiment (trials 1-4) were put in normal fish water and zebrafish late in the experiment (trials 5-7) were put in calcium water for a half hour after spinal cord severing and then transferred to normal fish water. Increased calcium ion concentration water was used because calcium ions are involved in the coupling of filopodia to the actin filaments during growth cone movement. Filopodia extend or retract in response to chemotropic molecules binding to the membrane receptor of a neurite. (Matthews, 2001) Although zebrafish cannot be left in calcium water for an extended period of time because it does interesting things to their development. Post-procedural Zebrafish were fed small amounts of food after age four days when they begin feeding.

Observation and Data Collection
Post-procedural zebrafish were observed for survival rates and level of activity. Placing a pipet in the water near the zebrafish and observing whether or not they attempt to swim away is one indication of survival of the spinal cord severing. If zebrafish did not attempt to swim away then they could be observed in the plastic dish of water under the stereoscope at high power for indications of survival. The heart pulsating or sometimes light reflecting on the moving blood cells of the vascular system and revealing blood flow could be used to indicate survival. Zebrafish not surviving the spinal cord severing procedure would not have any of these indications of survival and signs of decay were obvious within one day. The hyperosmotic body fluids of the freshwater zebrafish cause continuous water gain. When zebrafish are deceased the gills and kidneys do not maintain the necessary ion/water balance with the surrounding environment causing excess water to enter the cells and often cell lysis. (Ricklefs2007, Marieb 2004) Zebrafish surviving more than one day after spinal cord severing were monitored for normality of swimming movements and microscopically for tissue regeneration.

RESULTS
Early in the experiment survival rates after 48 hours of post-procedural zebrafish were zero so the methods of capturing the zebrafish, transferring them to and from the slide, and spinal cord severing were examined for flaw. The discovery was made that not adding a thin layer of water on the slide over the agar that immobilized the zebrafish allows increased air exposure and can damage the yolk of the developing zebrafish. Survival rates after 48 hrs. increased from zero to 60-67% after the methods were changed to add a thin layer of calcium water over the agar during the spinal cord severing.

Trial 1
Date of Trial: 11/6/07
Number of Zebrafish in Trial: 15
Age of Zebrafish During Trial: 4 days
Zebrafish surviving initially: 2
Zebrafish surviving after 24 hrs: 2
Zebrafish surviving after 48 hrs: 0
Survival Rate after 48 hrs: 0%
Longest post-procedural survival (days): 1 day

Trial 2
Date of Trial: 11/11/07
Number of Zebrafish in Trial: 15
Age of Zebrafish During Trial: 6 days
Zebrafish surviving initially: 2
Zebrafish surviving after 24 hrs: 0
Zebrafish surviving after 48 hrs: 0
Survival Rate after 48 hrs: 0%
Longest post-procedural survival (days): 0 days

Trial 3
Date of Trial: 11/14/07
Number of Zebrafish in Trial: 20
Age of Zebrafish During Trial: 9 days
Zebrafish surviving initially: 11
Zebrafish surviving after 24 hrs: 0
Zebrafish surviving after 48 hrs: 0
Survival Rate after 48 hrs: 0%
Longest post-procedural survival (days): 0 days

Trial 4
Date of Trial: 11/17/07
Number of Zebrafish in Trial: 15
Age of Zebrafish During Trial: 12 days
Zebrafish surviving initially: 2
Zebrafish surviving after 24 hrs: 2
Zebrafish surviving after 48 hrs: 0
Survival Rate after 48 hrs: 0%
Longest post-procedural survival (days): 1 day

Trial 5
Date of Trial: 12/2/07
Number of Zebrafish in Trial: 15
Age of Zebrafish During Trial: 4 days
Zebrafish surviving initially: 11
Zebrafish surviving after 24 hrs: 9
Zebrafish surviving after 48 hrs: 9
Survival Rate after 48 hrs: 60%
Longest post-procedural survival (days): 6 days

Trial 6
Date of Trial: 12/4/07
Number of Zebrafish in Trial: 15
Age of Zebrafish During Trial: 6 days
Zebrafish surviving initially: 13
Zebrafish surviving after 24 hrs: 12
Zebrafish surviving after 48 hrs: 10
Survival Rate after 48 hrs: 67%
Longest post-procedural survival (days): 10 days

Trial 7
Date of Trial: 12/6/07
Number of Zebrafish in Trial: 15
Age of Zebrafish During Trial: 8 days
Zebrafish surviving initially: 11
Zebrafish surviving after 24 hrs: 11
Zebrafish surviving after 48 hrs: 9
Survival Rate after 48 hrs: 60 %
Longest post-procedural survival (days): 8 days

DISCUSSION
The hypothesis of this project was that spinal cord neurons of the Zebrafish would regenerate to some degree after being experimentally severed. Previous research has found that neural regeneration occurs in some zebrafish central nervous system (CNS) neurons provided the chemical environment in the tissue is facilitating for neural growth. Human neural regeneration in the CNS does not occur because of chemical inhibitors in these tissues and characteristics of the nervous tissue that disable it from successfully regenerating. The affects of cyclic adenosine monophosphate (cAMP) on regeneration in 5-7 day old zebrafish of the Mauthner cell, a myelinated neuron that functions in escape behavior, has also been studied. Fetcho and colleagues found that with the addition of cAMP, about a third of Mauthner axons in zebrafish display some degree of regeneration within two days after being severed along with part of the spinal cord. These partially regenerating Mauthner axons typically deviate away from the spinal cord injury rather than growing through indicating that if spinal cord regeneration occurs the severed region will not appear the same histologically as before the procedure. (Bhatt, Otto, Depoister, and Fetcho 2004) There was no observed zebrafish spinal cord regeneration throughout this project, however, there was observed revascularization at the sight of spinal cord severing in two zebrafish from trial 6 and one zebrafish from trial 7. The blood flow, visible under the microscope, travelled ventrally toward the notochord around the severed region . Although it is unlikely, especially since cAMP was not used in this experiment, undetected spinal cord regeneration may have occurred in zebrafish from the later trials in which water with increased calcium ion concentration was used.

Severing of the notochord was avoided because extirpation of these cells adversely affects zebrafish development. The research done by Greenspoon and colleagues, in which the notochord of zebrafish embryos were ablated before axon generation using lasers, resulted in development errors of growth cones. They discovered that the accuracy of growth cones at the ventral midline of the spinal cord is dependent on both the floor plate (a group of cells at the ventral midline) and the notochord. Growth cones remain accurate either without the floor plate or the notochord but not without both. (Greenspoon, Patel, Hashmi, Bernhardt, and Kuwada 1995) Given that exacto blades are far less accurate than lasers, caution was taken during spinal cord severing not to make incisions too deep. This would likely damage the nerve tissue that growth cones depend on during development. Zebrafish surviving more than one day after spinal cord severing often had decreased tail functionality in swimming.

Diminished immune systems or disease may have been a factor affecting zebrafish survival in this experiment. The X-acto knife was washed before each trial but was not sterilized so the zebrafish could possibly have been infected with a disease during the spinal cord severing procedure. Deceased fish were removed from containers upon their discovery. Aside from the beaker that the zebrafish hatched in, the number of fish per container throughout the experiment did not exceed fifteen fish so epidemic disease is not probable.

The limited precision of the X-acto blade under the stereoscope at high power during the spinal cord severing procedure and the deplorable cooperation of the zebrafish often resulted in deeper incisions and greater injury than intended. This project did not yield neural regeneration, however, zebrafish tissue regeneration remains a beneficial area of research. Zebrafish heart regeneration research found that if roughly 20% of an adult zebrafish heart is surgically removed, heart tissue regenerates through cardiomyocyte proliferation and after a period of sixty days, the regenerated portion is histologically indistinguishable from the original cardiac tissue (Poss, Wilson, Keating). A subsequent study lead by Keating has found that signaling of the PDGF gene induces DNA synthesis in the cells and is required for cardiomyocyte proliferation during heart regeneration. (Gross 2006) Further understanding the underlying processes in both neural and cardiac zebrafish tissue regeneration could lead to knowledge of how to stimulate these processes in mammals.

LITERATURE CITED

1) Kenneth D. Poss, Lindsay G. Wilson, Mark T. Keating. 2002. Heart Regeneration in Zebrafish. Science. 13 December 2002: Vol. 298. no. 5601, pp. 2188 – 2190

2) Ricklefs, R.E. 2007. The Economy of Nature 5th ed. W.H. Freeman and CO. New York,
NY. pp. 180-198. 3) Marieb, Elaine N. 2004. Human Anatomy & Physiology 6th ed. Pearson Benjamin Cummings. San Francisco, CA. pp

4) Bhatt, H., Otto, S.J., Depoister, B., Fetcho, J.R. Cyclic AMP-Induced Repair of Zebrafish Spinal Circuits. Science. 9 July 2004: Vol. 305. no. 5681, pp. 254 – 258

5) S Greenspoon, CK Patel, S Hashmi, RR Bernhardt and JY Kuwada. 1995. The notochord and floor plate guide growth cones in the zebrafish spinal cord. Journal of Neuroscience, Vol 15, 5956-5965

6) Gross L. Regenerating Zebrafish Hearts Reveal the Molecular Agents of Repair. 2006. PloS Biol 4(8): e281 doi:10.1371/journal.pbio.0040281

7) Matthews, G. Neurobiology. Molecules, Cells, and Systems. Second Edition. Blackwell Science. 2001