RECORDING ZEBRAFISH TAIL REGENERATION FOLLOWING THE INTRODUCTION OF A CANNABINOID RECEPTOR AGONIST SOLUTION

Zebrafish possess natural regenerative capabilities, being shown to regrow tails, fins, and in some cases restore part of or an entire organ such as the heart. The introduction of a cannabinoid receptor agonist URB-597 or ‘CB-agonist’ after surgical tail removal suggests that CB-Agonist may interact with ‘Wnt’ pathways and wound response pathways to reduce apoptosis and spur healthier fin recovery. The CB-Agonist’s efforts suggest a reduction in cell death and smoother fin reconstruction along the distal tail region when examined through microscopy. Decay in zebrafish is marked by feathery, brown, and opaque flaking of the damaged body segment marking decomposition. The suggestion of less cell death is raised from the microscopy images in the CB-Agonist groups versus the untreated control wells.

HELLO, AND WELCOME TO A PRELIMINARY STUDY CONDUCTED BY ME, MATTHEW CHARTTERS, A STUDENT IN THE NEUROSCIENCE DEPARTMENT. I AM ABLE TO DISPLAY THIS WORK TO YOU THANKS TO GEORGE MASON UNIVERSITY AND THEIR OSCAR PROGRAM FOR UNDERGRADUATE RESEARCH STUDENTS LOOKING TO EXPLORE IDEAS AND CONCEPTS THEY’VE BEEN ITCHING TO EXPRESS.

MY PRESENTATION TO YOU ON ZEBRAFISH TAIL REGENERATION WHEN PLACED IN A CANNABINOID RECEPTOR AGONIST, LET’S CALL THAT A CB-AGONIST FOR SIMPLICTY, FOLLOWING A FINE AMPUTATION OF A SEGMENT OF THEIR TAIL.

LET’S LOOK AT A DAY OF RESEARCH FOR ME IN THE KRASNOW BUILDING’S NEUROSCIENCE LAB AND ITS ANIMAL FACILITY WHERE A MAJORITY OF MY RESEARCH TOOK PLACE.

TWO DAYS PRIOR TO WHEN I ANTICIPATED MY PROCEDURE’S FIRST STEPS OF CUTTING AND IMAGING, I HAD TO SET UP CROSSING CAGES, WHICH YOU WILL SEE IN THE NEXT SLIDE, WHERE MALE AND FEMALE ZEBRAFISH WOULD BE PLACED IN ONE OF THESE CROSSING CAGES OVERNIGHT. UPON DAWN’S EARLY LIGHT, OR IN THIS CASE THE ROOM’S OVERHEAD FLUORESCENT BULBS, THE FEMALE ZEBRAFISH RELEASE CLUTCHES OF EGGS AND THE MALES THEN RELEASE SPERM INTO THE CAGE WHERE THE EGGS WILL BECOME FERTILIZED AND FALL INTO A LOWER COMPARTMENT SET UP INSIDE THE CAGE. THIS HELPED MAKE COLLECTION EASIER AND PREVENTED ANY FISH BEING STUCK FLOATING AROUND AND APPEARING LIKE A SNACK.

THE NEXT DAY THE FISH ARE NOW WHAT WE CALL 24 HPF OR HOURS POST-FERTILIZATION, THAT’S HOW WE GAUGE THEIR AGE IN ADDITION TO STAGING PROCEDURES. THE METHODS OF TAIL AMPUTATION FIRST REQUIRED ME TO REMOVE THE OUTER ‘BUBBLE’ OR CHORION AS SEEN ON THE TITLE SLIDE SO THAT I COULD LET THE FISH UNCOIL AND SWIM ABOUT. IN ACCORDANCE WITH IACUC, A COUNCIL ON ANIMAL ETHICS IN LABORATORIES, THE FISH I AIMED TO CUT WERE ANESTHETIZED USING A 0.4% TRICAINE SOLUTION. WHILE ANESTHETIZED, I COULD WORK CAREFULLY WITH MY NEEDLE AND SCALPEL TO POSITION AND THEN AMPUTATE THE CAUDAL END OF THE FISH, JUST A FEW SEGMENTS FROM THE TIP OF THEIR DEVELOPING TAILS.

AFTER THAT, I SIMPLY WAITED 48 HOURS AND AT 72 HPF OR 48 HPA (POST-AMPUTATION) TO TAKE A FINAL SERIES OF IMAGES IN THE KRASNOW LAB WITH MY MILLIMETER GAUGE BEFORE TAKING THE FISH OVER TO EXPLORATORY HALL FOR FLUORESCENCE IMAGING. THIS COULD ONLY BE DONE WITH TRANSGENIC FISH THAT DISPLAYED MY TARGET FLUOROPHORE, OLIG-RED OR RED FLUORESCENT PROTEIN (RFP). MORE ON THAT LATER.

HERE’S A QUICK GLANCE AND EXPLANATION OF WHAT A TYPICAL SETUP OF CROSSING CAGES AS WELL AS THE CIRCULATING AQUATICS SYSTEM LOOK LIKE.

ON THE LEFT WE SEE 6 CROSSING CAGES LABELLED WITH THE GENOTYPE OF THE FISH INSIDE AS WELL AS THE DATE THEY WERE PLACED IN THESE CAGES. THEY WOULD REMAIN ON THE TABLE UNTIL THE NEXT MORNING UPON COLLECTION WHEN A MIDDLE CAGE IS PULLED UP AND OUT TO RETURN THE ADULT FISH AND THEN COLLECT THE EMBRYOS AT THE BOTTOM OF THE MAIN CAGE WITH A SIEVE AND SOME PURIFIED WATER TO WASH OFF ANY DEBRIS.

ON THE RIGHT IS THE AQUATICS SETUP I BECAME ACCUSTOMED TO USING. THE BASICS IS WELL, BASIC, WATER CIRCULATES THROUGH THE SYSTEM AND IS FILTERED BY A NUMBER OF FILTRATION METHODS DOWN BELOW AND EACH DAY NEW WATER IS ADDED INTO THE SYSTEM FROM AN ACCESSORY TANK.

SO, WHAT DID I DO? WELL, USING A FLAT PLASTIC BOARD WITH GRIDLINES, I PLACED A DROP OF WATER AND THE ANESTHETIZED FISH TO LIE ALONG A STRAIGHT LINE AND THEN USED A FINE SCALPEL TO CUT ANY GIVEN NUMBER OF SEGMENTS OFF, AND THROUGHOUT I TESTED MORE INTENSE AND LAXED CUTTING TO SEE WHAT THE LIMITS TO TAIL REGENERATION REALLY WERE.

WHAT WE SEE, IN OUR CONTROL GROUP, IS THAT ZEBRAFISH DO POSSESS NATURAL REGENERATIVE ABILITIES, THE QUESTION HERE IS, DO THE CB-AGONISTS WORK TO ACTIVATE THE KNOWN PATHWAYS IN REGENERATION.

TO TEMPER THESE FINDINGS, I WOULD TELL YOU THIS, THE INTRODUCTION OF THE CB-AGONIST SUGGESTS INCREASED REGENERATION THROUGH TWO METHODS, REDUCING CELL DEATH AT THE CUT SITE, WHERE IN THE CONTROL THE BUD TAPERS OFF WITHOUT MUCH FIN REGROWTH TO COVER THE BLASTOMA. THE SECOND METHOD WAS PROMOTING FASTER REGENERATION THROUGH HEIGHTENED RECEPTOR ACTIVITIY. THIS OBSERVATIONAL STUDY SUGGESTS SUCH POSSIBILITIES, BUT FURTHER WORK IS REQUIRED TO DRAW ANY STATISTICAL CONCLUSIONS.

NOW, Comparing the 3 trial concentration groups against the control: 1.25 mg/mL, 2.5 mg/mL, and the 5.0 mg/mL trials, the 2.5 mg/mL group was measurably more effective at regrowth length by micrometer than the other groups, as this table suggests.

What differentiated them upon imaging was the exhibition of greater fin regrowth and more pronounced tail buds in an observational setting.

The CB-Agonist’s efforts suggest a reduction in cell death and smoother fin reconstruction along the distal tail region when examined through microscopy.

Decay in zebrafish is marked by feathery, brown, and opaque flaking of the damaged body segment marking decomposition.

LASTLY, AS I SAID BEFORE, A LOOK AT SOME ZEBRAFISH EXHIBITING THE RED FLUROESCENT PROTEIN (RFP) AND SOME STANDARD PHOTOS OF THE SAME FISH, TO HELP EXPRESS THE BENEFITS OF FLUORESCENT IMAGING WITH TRANSGENIC FISH, AS WELL AS WHAT HAPPENS WHEN SOME TRANSGENES DON’T QUITE EXPRESS THEMSELVES AS PROMINENTLY AS OTHERS.

WE SEE A CONTROL TAIL ON THE BOTTOM ROW, THE FIRST TWO IMAGES FROM LEFT TO RIGHT, AND ON THE TOP ROW WE SEE FROM LEFT TO RIGHT A COMPARISON OF THE 5.0 MG/ML CONCENTRATION GROUP THAT PROVED LARGELY UNSUCCESSFUL AT REGENERATION AND ITS FAILURE TO GENERATE THE NEEDED PERIPHERAL NEURON OUTREACH AS IN THE FINE POINT CONTROL. IN THE TOP RIGHT WE SEE A TRANSGENIC FISH THAT SHOWS FAINT, VERY FAINT, FLUOROPHORES, MEANING IT DIDN’T QUITE TAKE ON THE GENES OF ITS PARENTS THAT WE ANTICIPATED.

HAVE NO FEAR, IN THE BOTTOM RIGHT A 2.5 MG/ML TAIL SHOWING NERVES PROTRUDING FROM THE SPINAL CORD INTO THE TAIL BUD DISPLAYS A TAIL THAT CAN, WELL, FEEL.

I’VE HAD AN EXCELLENT TIME WORKING WITH THE TOOLS AND TECHNIQUES THIS SEMESTER AND AIM TO CONTINUE RESEARCH IN SOME SHAPE OR FORM LOOKING AHEAD. MY HOPE, TO BRING REGENERATION TO ANIMALS UNLIKE THE ZEBRAFISH THAT CANNOT REGROW LIMBS OR EVEN ORGANS AND MAKE THAT A POSSIBILITY FOR FUTURE SCIENTISTS TO EXPLORE, MYSELF INCLUDED. NEXT STOP, LIZARDS.

I WANT TO END THIS PRESENTATION BY SAYING THANK YOU FOR TAKING THE TIME TO WATCH THIS PRESENTATION AND TAKE A PEEK AT A PRELIMINARY STUDY THAT SUGGESTS THAT EXTERNAL WORK AND SOLUTIONS MAY SPEED UP REGENERATION AND WOUND RECOVERY. I’D LOVE TO CONTINUE THIS WORK IN SOME CAPACITY WITH CRISPR AND CANNOT WAIT TO GET THE OPPORTUNITY TO DO JUST THAT.