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The long term goal of this is to compare the normal protein composition of cell bodies from spinal motoneurons the grass frog with that of motoneurons following damage to the motor axon, and to find out where the build up of protein from an inijured motoneuron comes from.
Two procedures are used in this research to get information. The first procedure is the collection of individual motoneuron cell bodies. The second procedure involves the use of an "apparatus" and the methods, with a few modifications, of Patrick H. O'Farrell(1) to use two-dimensional polyacrylamide gel electrophoresis, or 2-D PAGE, to make gels. The gels are used to resolve the major proteins of the normal and axotomized motoneurons which are compared to find out which are compared to find out which proteins change during the height of an axonal injry.
Through past research, it has been shown that changes occur in protein metaboism in motoneurons after an axonal injury. But some questions remain, such as: "How long do the changes last?" and "What are the consequences (if any) of the changes?" In conclusion, this research could help answer those questions.
Spinal motoneurons are nerve cells that are used to carry nerve impulses from the central nervous system to the skeletal muscles. There are several parts to montoneurons: dendrites, soma, and the axon. When motor axons are injured, it causes paralysis. Paralysis is the complete or patial loss of function or sensation in a part of the body (4). Even though motor axons can grow back and reinnervate skeletal muscle, they don't always reinnervate the right muscle.
Little is known about the chemistry of axotomized motoneurons. The long-term goal of the research is to understand protein changes of axotomized motoneurons. To do this, the technique of two-dimensional gel electrophoresis is used to resolve motoneuron proteins. First, motoneuron cell bodies are so isolated, Second, the proteins of the cell bodies are seperated on 2-D gels. Later, their protein patterns on the gels of injured and normal motoneurons will be compared.
My work over the summer not only included learning about motoneurons, but working with them. I learned how to isolate motoneuron cell bodies using a microscope with a built in pipet. I also learned how to modify 2-D gels for small amounts of motoneuron protein, and for better detection in staining.
During the course of this summer, I have learned more about motoneurons and the feild of research than I've ever imagined.
Lumbar regions from frog spinal cords are cleaned of meninges and spinal roots prior to cell isolation (3). Cords are minced with a razor into 1mm3 pieces. Tissue is suspended in approximately 5ml of ice cold 70% ethylene glycol and 30% 0.9M of sucrose-CG (CG=1.7mM citrate-NaOH, pH5; and 15mM glucose). Tissue remains on ice for 90 minutes before storage at-70°C or immediate use. After warming to ice temperature (4°C), the ethylene glycol solution is filtered out through Nitex 202. Tissue pieces are squished through the Nitex 202 with a spatula, then it is washed through the Nitex 202 with some 0.9M sucrose-CG.
A sucrose gradient is formed by the addition of 2ml of 2.1M sucrose-CG and 1.5M sucrose-CG. The squished homogenate in the 0.9M sucrose-CG is layered on top of the 1.5M sucrose-CG. The gradient is then centrifuged at 7000 rpm (9400 x g) for 40 minutes at 4°C. Motoneurons are found in the section of the gradient between 1.5M sucrose-CG and 2.1M sucrose-CG. A lot of large debris floats on the surface of the 0.9M sucrose-CG. Most of the nuclei will pellet through the 2.1M sucrose-CG. Next, follow these steps: Separate the fractions of sucrose and collect the 1.5M/2.1M layer of sucrose. Mix a small amount of methylene blue in the fraction. Stir the sucrose by, first, covering the tube with parafilm, and then inverting the tube. Aliquot 100 micro L volumes of the fraction. The aliquots are viewed on a cover slip with a microscope using a 10X objective. Suction, controlled by your mouth, on a micropipet is used to collect the cell bodies. The average size of a cell is approximatley 38 micro M x 74 micro M. Cells less than 30 micro M are not collected. Cells are taken up in minimal volume of about 1 to 2 micro L are then deliverd to a wash solution made of 100microL of sucrose on a cover slip. Cells are washed two times before delivering into a 400 micro L tube for analysis.
Isoelectric focusing gels, or frist dimension gels, are used to separate proteins by their molecular charge. Isoelectric focusing gels are approximatley 12cm long and are tubular shaped. At one end of the gel has an acidic section, a neutral section, and a basic section.
Buffers and solutions to make isoelectric focusing gels are:
Isoelectric focusing gels are cast in glass tubing (130 x 2.5mm inside diameter). Clean glass tubes in soapy water and rinse. Allow to soak in acid-alcohol, then rinse and dry. Start preparing solutions "G" (ammonium persulfate), "D" (30% acrylamide stock), and "E" (stock NP-40 solution).
To make the gel mixture combine 4.13g of Urea, 1.0ml of "D," 1.5 of "E," 1.5ml of water, and 375 micro L of 3.5 to 7 and 5 to 7 ampholines. After preparing the gel mixture, degas in vacuum for 30 minutes. During the 30 minute degassing period, the following things need to be prepared and done:
After 30 minute period, add catalyst to mixture. The catalyst is a mixture of 15 micro L of "G" and 8.4 ul of TEMED (tetramethylenediamine). Fill syringe with total gel mixture, then fill the tubes from the bottom up to about 5mm from the top (in most cases there will be a notch or some sort of mark already on the tubes to indicate stopping.
When pouring, try to avoid air bubbles. Air in the gels will affect the movement of current in the later steps.
Adjust the level of mixture in the tubes with twisted Kimwipes as needed. Overlay mixtures with 10-20 micro L of solution "H". Cap tubes with parafilm and let them stand for two hours. Rinse syringe and glassware throughly to aviod polymerization problems or contamination. After two hours are up, remove "H" overlay, and add 20 micro L of "A" and 10 micro L of water. Then recap the tubes with parafilm. Allow the tubes to set for another two hours. About 20 minutes before the two hours end, make 0.02M of NaOH (0.8ml of 10% NaOH and 400ml of water) and degas it.
Prepare 4L of 0.01M H3PO4 (2.72ml of concentrated phosphoric acid and 4L of water). Fill lower chamber of 2-D apparatus with phosphoric acid solution. Take parafilm off tubes, and put the tube set-up into the 2-D apparatus. Remove air bubbles from the bottom of each tube using a shepards's crook.
Replace old "A" overlay with 20 micro L of new "A" and 0.02M NaOH. Then, fill the upper chamber of the 2-D apparatus with 0.02M NaOH,and prerun for an hour at 200 volts. About 15 minutes before the prerun ends, thaw out "K" and prepare more NaOH. Replace the old upper reservior solution with new NaOH. Samples (about 20 micro L or less) are usually prepared by dissolving lyophilized sample in "A," using vortex mixing, and spinning in a microcentrifuge. Run at 400 volts for 18 hours, then at 550 volts for2 hours.
Force the gels out by applying gentle pressure using a syringe with a piece of flexible tubing connected to the tube gels. Gels are then stored at-70°C in equilibration buffer ("O" with sulfydry1 reducing agent).
In the second-dimension, the proteins are coated with a negatively charged detergent, SDS, and separated on the bases of molecular size. The gels of the second-dimension are composed of two section sections. The upper section is the stacking gel, and the lower section is the running gel. 2-D gels allow us to compare the difference in the protein composition of the axotomized and normal cells. Buffers and solutions used in the second-dimension include:
Buffers and solutions need to be kept at room temperature untill needed. The glass plates used in the second-dimension are made of window blass. The front plate 18x16cm. The beveled plate has the same measurement. The beveled plate also has a section, approximatley 2 x 14cm, cut out from the top. In that section, the top of the glass is at a 45° slant. Grease is used to glue the beveled plate to the apparatus rack so that the top of the slant is even with top part of the rack. Teflon spacers are laid around the edges of the plate, except for top. The spacers are sealed to the front plate with grease. Then clamps are used to secure everything tightly to the rack.
The lower gel buffer, running gels, is made of:12.96ml of water, 8.25ml of "L," 11.0ml of "N," and 0.66ml of 10% SDS (All measurements and calculations are done for two gels). Degas the mixture for 2 minutes. After it bubbles vigorously, cheeck the pH for 15 minutes. The pH must be btween 8.5 and 8.8. Then add 109 micro L of 10% "G,: mix it; then add 16.5 micro L of TEMED. After TEMED is added, quickly draw up the solution into a 60cc syringe. Place the bevel of the needle in the open area against the front plate and expell solution until it is up to about 1,5cm from the lower edge of the beveled plate. Then, clean syringe immediately. Using another syringe, place a few drops of water on top of the gel, then let the gel polymerize for 45 minutes.
During the 45 minute period, begin work on the upper (stacking) gel solutions. The upper gel buffer is made of: 6.94ml of water, 3.0ml of "M," 1.9ml of "N," 120 micro L of 10% SDS. Degas the mixture for two minutes. After vigorous bubbling, check the pH. The pH should be between 6.5 and 6.8. When ready to pour gel, add 36 micro L of 10% "G," mix it; then add 12.0 micro L of TEMED, and mix. Fill 60cc syringe with this solution. Remove the overlay of water. Pour solution until the middle along the bevel is filled. Then overlay with some water, and let it polymerize for 45 minutes.
Next, begin making "q" and "P." "Q" is 4L total. "Q" is made of 56.7g of glycine, 4.0g of SDS, 12.12g of Tris, and water. "P" is1g of agarose/ 100ml of "O." Let "P" boil untill needed, and leave the lid on partially to prevent contamination. Now, take 500ml of "Q" and add a few drops of 0.1% bromophenol blue. Pour "Q" into apparatus and remove bubbles. Next, take off the bottom clamps and bottom spacer, and place the set-up into the apparatus.
Thaw out the IEF, if needed too. Remove the water overlay from the upper gel. Place some "P" alnog the bevel with a Pasteur pipet. At this point work must be done quickly. Place an IEF gel along the bevel. Fix the IEF gel's positioning with a shepard's crook,then add more "P" along the top of the IEF gel. Wait a couple of minutes, then fill upper reservior with 0.1% bromophenol blue/ "Q" solution. Then attach top parts of the apparatus and anode, and run it at 40mA overnight. Power settings: voltage-max, current-max, output-constant current, and display-volts or mA.
Staining is the method that allows us to detect spot patterns of proteins. The method of staining used was developed by R. C. Switzer et al. and modified by B. R. Oakley et al.(2).
The silver staining process takes two days, and requires agitation throught the whole process. On the first day, 2-D gels are put in a solution of 50% methanol and 10% acetic acid which the gels sit in overnight. The next day the gels are washed in a solution of 5% methanol and 7% acetic acid. Then the gels are soaked in 10% unbuffered glutaraldehyde for 30 minutes. Next, the gels are rinsed in water three times for five minutes each. Then they are washed four more times for 30 minutes each.
After water is drained off, the gels are put in a freshly made ammonical silver solution. The total volume of thew solution is 100ml. It is composed of: 21ml of 0.36% NaOH, 1.4 ml of concentracted NH4OH, 4ml of AgNO3, and enough water to bring the solution to volume. The gels set in this for 10 to 15 minutes. Then they are washed three times in water for two minutes each.
The developing soution is made fresh out of 0.005% citric acid, 0.019% formaldehyde, and water. The total volume of the solution is 250ml. The gels are to sit in this solution untill spot patterns are visible. If over stained, a destaining solution of 60ml of Kodak Rapid Fixer made up to 250ml with water can be used to lighten patterns and background. Next, 2.5g of Kodak Hypo Clearing Agent dissolved in 100ml of water is used to remove all traces of fixer. This prevents gels from fading if stored for long periods. The gels are soaked in the solution for 5 minutes. Finally, gels are washed in three different washes of water for 10 minutes each. If gels are to be dried, 1% glycerol can be added to prevent cracking.
The first couple of test run gels came out with some mechanical damage, and had a lot a streaking in the patterns. The second group of gels turned out to better with little damage done to gels, and the problem with streaking had improved a lot compared to the first group. The next group of gels was made thinner in the second dimension to see if detection would increase with a thinner gel. Detection increased a lot with the thinner gels, but the gels were so fragile that a lot of of the gels were damaged during the staining procedure. After several runs with the thinner gels, it became easier to handle them without a lot of damage to the gels.
During the staining procedure, it was discovered that it was easier to agitate solutions over the gels with a magnetic stirring bar. The stirring bar was set up in the corner of a dish big enough for the gel and the bar to fit with room to spare. The bar was covered with the bottom portion of a plastic cup in which holes had been punched to allow liquids to flow underneath it, and then a weight was put on top of the cup to prevent the bar from damaging the gels. This set up was more convenient than a shaker dish because it was smaller and easter to move around.
Due to my short time here, I will not see any of the long term results. At most, I will be able to see the protein pattern of control motoneurons.
Over a period of seven weeks of researching and studying, I have learned much about the results of the results of a spinal injury and the spinal cord itself. I doubt that my high school would have been able to provide me with the experience this program has given to me.
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