Pharyngula

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

Comments

  1. #1 Dahan
    December 21, 2007

    Next time could you give us the intro and then let us “read on” please.

  2. #2 maxi
    December 21, 2007

    I wish I had as much to show as you. So far it’s: Currently the author is recollecting all her data (even though she only has 6 months left) due to fixation problems that she can’t seem to fix (boom boom!).

    Good luck!

  3. #3 Christianjb
    December 21, 2007

    Great! Let us know if it’s possible for the Democrats to regrow their spines.
    (Too tired to read the science right now.)

  4. #4 Phill
    December 21, 2007

    “After removing four legs, frog becomes deaf.”

  5. #5 Lago
    December 21, 2007

    See the experiments being done at Harvard.. as in, gap junction genes and ion flow…

    5 bucks says you get regeneration..

  6. #6 amph
    December 21, 2007

    “Severing of the notochord was avoided because extirpation of these neural cells adversely affects zebrafish development.”

    “These neural cells?” Which ones? Someone might get the impression that you think that the notochord consists of neural cells.

    Quick! Correct before PZ sees it.

  7. #7 June
    December 21, 2007

    Hey, I have an idea! Let’s invent something called the Internet. My idea is for scientists to write an article, post it on THEIR computer or in a JOURNAL, then LINK to it on THEIR web page, with a TITLE and an ABSTRACT to summarize the article, and some KEYWORDS to locate it in the future.

    That way slower computers won’t need to spend minutes waiting for some article to download. And so many more articles could be published. And one could tell whether one is interested or not.

  8. #8 Rev. BigDumbChimp
    December 21, 2007

    That way slower computers won’t need to spend minutes waiting for some article to download. And so many more articles could be published. And one could tell whether one is interested or not.

    Somebody needs a hug

  9. #9 Cairnarvon
    December 21, 2007

    X-acto knife, not exacto knife.

    That way slower computers won’t need to spend minutes waiting for some article to download.

    The entire post is 13.7 KB in size, or slightly larger than the banner image. Even on 56K dial-up it’d only take two or three seconds to load.
    If your connection is slower than 56K, maybe you shouldn’t be on the internet in the first place.

  10. #10 Frank Oswalt
    December 21, 2007

    Jane (#7),

    great idea, but I’m afraid Al Gore beat you to it.

  11. #11 Frank Oswalt
    December 21, 2007

    JUNE, sorry.

  12. #12 Reginald Selkirk
    December 21, 2007

    You should list your real name and home address, to make it easier for PETA to track you.

  13. #13 MAJeff
    December 21, 2007

    That way slower computers won’t need to spend minutes waiting for some article to download. And so many more articles could be published. And one could tell whether one is interested or not.

    So, one takes extra time to click on the story itself, comment, and click on the “post” button rather than just scrolling past it?

  14. #14 One Eyed Jack
    December 21, 2007

    I want a T-shirt that says:

    “Life could be worse. You could be a zebra fish.”

    OEJ

  15. #15 MAJeff
    December 21, 2007

    Blue_Expo,

    As a college instructor who also engages students in doing research, I’d like to ask you, How did putting together and conducting this experiment, as well as analyzing the results, contribute to your educational experience? Not just what did you learn in the experiment, but what did you learn by doing it?

  16. #16 Umilik
    December 21, 2007

    You’re lucky you didn’t have to have the procedures approved by an Institutional Animal Care and Use Committee. I suspect you wouldn’t have been severering spinal cords if fish weren’t exempt from such reviews…..

  17. #17 bungoton
    December 21, 2007

    Interesting experiment. Thanks for sharing.
    The reading experience would be greatly improved if your spelling was fixed. I believe the substance your refer to as ‘ager’is actually agar. I see comments from others on the spelling of ‘site’ as ‘sight’ and ‘X-acto’ as ‘exacto’.

  18. #18 Asad
    December 21, 2007

    “and the deplorable cooperation of the zebrafish often resulted in deeper incisions and greater injury than intended”

    Damn those zebrafish for not cooperating :)) an interesting and funny post.

  19. #19 Leon
    December 21, 2007

    Severing the spinal cords of zebrafish? And people complained of animal abuse when a couple of us talked about exposing them to alcohol? Sheesh!

  20. #20 Jay Kanta
    December 21, 2007

    Nice write up, but you missed a tense in one of the words and now I can’t find it again….

    I would also like to see recommendations for future study of spinal regeneration, such as using a laser or glass shard. I’m also assuming that you used the X-acto by hand, further complicating the cut. What about mechanical methods of aiming and cutting with the knife?

    I would also like to see a discussion of the distance between the edges of the cut and a discussion of the front view of the cut. Was the spinal column squished or did it retain the original shape? Could this shape have an effect on the regeneration qualities of the zebrafish?

  21. #21 Mooser
    December 21, 2007

    Poor little Zebra-fish! My heart goes out to them, or maybe they could grow an extra and send it out to me. Mine is sort of wearing out about now. Broken and repaired too many times, you know, and frankly, it was junk to start with heredity-wise

  22. #22 Sven DiMIlo
    December 21, 2007

    I heard fish can’t feel pain.

    ‘course I heard it from my grandfather, who never met a largemouth, crappy or pike he didn’t want to yank out of the water with a barbed steel hook stuck through its jaw so he could bash it over the head and filet it.
    So consider the source.

  23. #23 Christianjb
    December 21, 2007

    I thought this was a good write-up. Unfortunately it’s a negative result, which is much less interesting than if you had found cases in which the spines regenerated. If this were a Mythbusters episode then the hypothesis that Zebra-fish can regenerate spines would look pretty much busted at the moment.

    It would be nice if you could suggest refinements of the experiment which would produce more useful data. For instance, if you sever the spine at a lower point would you get a greater survival rate?

    (I am not a biologist- so my opinions may be next to worthless.)

  24. #24 Ichthyic
    December 21, 2007

    You’re lucky you didn’t have to have the procedures approved by an Institutional Animal Care and Use Committee. I suspect you wouldn’t have been severering spinal cords if fish weren’t exempt from such reviews…..

    that entirely depends on the committee running the specific animal use protocols at whatever university you happen to be at.

    fish were included as “animals” one year after I started my grad program at Berkeley, for example.

    I think they were discussing making insects “animals” when I left.

    even so, I think this student project would have passed muster even with that committee. There is precedent, there is value in the project, and there really is no other way to do it.

    based on my experiences with two different animal use committees at two different unis, I don’t think he would have had to do more than present a lit review and maybe appear to answer questions once.

    of course, this being a student project done under the auspices of a university instructor (who we all know and love), it wouldn’t even be the studen’t responsibility to justify it to the committee to begin with.

    I’m sure PZ would have had no problems justifying the value of such an experiment from at the very least, a teaching perspective.

  25. #25 cyan
    December 21, 2007

    Ichthyic,

    “fish were included as “animals” one year after I started my grad program at Berkeley, for example.

    I think they were discussing making insects “animals” when I left.”

    OMG! (if I believed in one) but more appropriately, WTF!

    Fish and insects not being considered as animals?!

    What biologists were these?

    How about criteria being “on the continuum of sentience” …

  26. #26 Bill Dauphin
    December 21, 2007

    As an editor myself, I’d be the last one to argue that typos and misspellings are unimportant, but in this case I think we should lay off: Presuming this is a cut-and-paste of the paper Blue_Expo submitted to PZ, s/he has probably already heard about (and perhaps lost points over) these little hacks; no sense us piling on.

    As a practical matter…

    I believe, if you read the package, you will find the word is spelled a-g-a-r, (not “auger,” as posted previously, or “ager” as it is now)

    …this may well be a case where what the author typed was correct, and then the spellchecker automagically introduce fresh, new error. A spellchecker that wasn’t specifically science-savvy might want to replace agar with either auger or ager. Grammar checkers are even worse: In the text I edit at work, I can instantly recognize cases where the grammar checker has mindlessly flagged an “error” (e.g., passive voice) that is actually syntactically competent and offered a completely nonsensical “correction.” Writers who lack confidence may be tempted to assume the machine knows more than they do, and simply accept whatever change it offers.

    The answer, as Sam Waterston would quickly tell you, is don’t trust the robots!

    ;^)

    And speaking of robots, the censor-bot at my job has evidently figured out that Pharyngula isn’t directly related to the jet-engine business, and I can no longer access this site (not “sight”!) from my work computer… with the effect that you’ll probably be hearing less from me from now on. Just think of it as my company’s Squidmas present to all of you!

  27. #27 Ichthyic
    December 22, 2007

    Fish and insects not being considered as animals?!
    What biologists were these?

    the crucial point being…

    they weren’t.

    Most of the animal rights groups at the time (1990) hadn’t considered that fish/insects were ‘animals’, in the sense they considered warm fuzzy things to be so. I know for a fact that it was the biologists themselves that inadvertently (for better or worse) educated the morons on these “panels” as to the fact that reptiles, amphibians, fish, and yes, even insects are animals too.

    all the animal use protocol committees I’ve seen (Berkeley was one of the first to capitulate to the animal rights crowd and form one), are mostly populated with selected (don’t ask me how) members from other departments (than biology) and the general public.

    the idea was to allow voices from outside of biology to express concerns about animal research, make sure good husbandry guidelines were followed, and have a researcher provide justification for utilizing animals in an experiment. Usually, just showing that the experiments would contribute to the literature in the area of research, or have educational value was sufficient. It did take about ten pages of detailed documentation and a formal meeting for each new project, though.

    sometimes the questions were so inane one had to suppress laughing out loud, which was often the hardest thing.

    I like to look at the positive side of it, and think that the focus on good animal husbandry practices would end up making some more efficient and capable scientists in the long run. However, I rather think mostly it just added to the amount of paperwork and tedium that seems to be ever growing in a scientist’s life these days. Moreover, no matter if you even have tenure in your dept., you were still required to file a new animal use protocol for each new experiment.

    the history of the animal rights movement in Berkeley is a tumultuous one, at best, and i think the University basically thought that starting this program (the animal use committees) would help alleviate some of the irrational fears and criminal reprisals coming from some of the more strident animal rightists.

    I have a couple of good stories to tell regarding animal rightists at Berkeley, but it might be best to save those over beers.

  28. #28 Blue_Expo
    December 23, 2007

    The Grammar Nazis stormed my house this morning and threatened to take away my Christmas presents if I didn’t turn from my wicked, grammatically incorrect ways.