誘導および慢性-再発実験的自己免疫性脳脊髄炎の臨床スコアリング

I am Christine Beaton. I'm an assistant researcher in George Chen's lab in the Department of Physiology and Biophysics of the University of California at Irvine. And what I'm going to show you in the next few days is how to induce and monitor chronic relapsing EAE, which stands for Experimental Autoimmune Encephalomyelitis in DA rats.

This is an animal model for multiple sclerosis, or should I say, one of the animal models for multiple sclerosis. And it is characterized by an ascending flacid paralysis in those animals and histological characteristics that resemble the human disease. So this is going to take several days.

So the first day I'm going to show you how to prepare the genic emulsion with naive red spinal cord homogenate in complete trans adjuvant. The second day, I'm going to show you how to immunize the rats to UCAE, and then we're going to wait between seven and 15 days to see the first clinical signs in those animals, at which point I'm going to show you the different stages of progression of EAE in different animal. So I'm first going to show you all the reagents and material that are needed to prepare the emulsion.

So what I'm using is complete front adjuvant H 37 ra, but this, although it already has microbium tuberculosis, I'm going to supplement it to four milligrams per M with more desiccated mycobacterium tuberculosis IH 37 ra. And to prepare this desiccated bacteria, I need to put it in more tar and make a thin powder out of it with the Pele. Once that's done, I'm going to add those bacteria into the adjuvant.

Then the adjuvant is ready. The next step is to prepare my antigen. So I'm going to take out D rat spinal cords that are frozen at minus 80, and to keep them really frozen, I'm going to put them on the weighing dish in dry ice, and I'm going to take them out one by one and using simple razor blades, I'm going to mince the spinal cords into weighing dishes.

I'm going then to put my supplemented adjuvant on the vortex. And while vortexing, I'm going to add my spinal cord little by little to my adjuvant after we've mixed all of the spinal cord to the adjuvant on the vortex. We're going to vortex for further five minutes, and after that we're going to transfer the prepared immersion into five M glass syringes.

We're going to connect two five M glass syringes using a 16 G bridge, and we're going to propel the emulsion from one syringe to the other as fast as possible for that bridge. And after a few minutes, when the emulsion starts getting harder, we're going to switch to a smaller bridge. This is 18 G, and that's going to make the emulsion even harder, and we're going to keep it in those syringes with this bridge on wrapped in aluminum foil overnight in the fridge.

So I've weighed 30 milligrams of microbe tuberculosis on the weighing paper. I transferred them to a morar. And using a Pele, I've reduced the aggregates of bacteria into a thin powder.

But you don't want to press too hard with the pestle not to break the bacteria. When the bacteria in aggregate at the beginning of a procedure, they are yellow beige. If you've done thin, thin powder, you'll have in the end a white powder, and that's what you want, but you don't want to press too hard to break the bacteria.

Once I've done that, I took a 10 mil amp of complete trans adjuvant, and I've made sure that all of the bacteria is in suspension. I've broken this sample open and I've poured the whole 10 mil of complete trans adjuvant into the morta on top of the disaggregated mycobacterium tuberculosis. And now I'm going to transfer the supplemented complete trans adjuvant into a 50 mil tube.

So now I'm taking my supplemented complete trans adjuvant and putting it in a tube. So of course, the bacteria are at the bottom of the liquid, and I'm going to try to get most of them out into the tube. But if I leave some in the mortar, it's not going to be so much of a problem because I'm going to reuse this mortar without cleaning it to prepare my spinal cord emulsion.

So here are the spinal cords. They were kept frozen at minus 80. I weighed 10 grams of them to add to 10 mls of my supplemented adjuvant.

So what I'm going to do with those spinal cords while keeping them frozen as long as possible, I'm going to mince them very finely in those weighing dishes using razor blades. So to mince those spinal cords very finely, you need two razor blades and a weighing dish. So what I do is I take one spinal cord, put it in my weighing dish, and I start cutting it, and I put my hand over so that since it's frozen, its heart, it's going to start jumping around and I catch first big chunks and it's going to start thawing really fast and sticking to the side of my razor blade.

So I use the other razor blade to take it off, and I try to mince it as finely as possible and as fast as possible so that it doesn't have the time to melt because it's much more difficult to cut once it's melted. So it is extremely important that the spinal cord be completely minced, very finely, because the finely fine, the most fine it is the most more homogeneous immersion will be, and the more rats will get sick and with a more homogeneous disease. So it's extremely important to mince all of those spinal cords very, very well.

So now I'm going to go on mincing every one of those spinal cords exactly the same way until I'm done with the whole 10 grams of spinal cord. So now what I'm going to do is I'm going to mix the spinal cord in the morta a little just to make sure I gather all of the mycobacterium tuberculosis that was still left in the morta after I finished supplementing the adjuvant. And what I'm going to do next is put the adjuvant on the vortex.

And while vortexing continuously as fast as possible, I'm going to add my mean spinal cord little by little to this adjuvant. So now I have my supplemented adjuvant on the vortex, and I'm going to start adding, adding my means panel cord little by little. The reason why I am adding the A, the antigen very slowly to the adjuvant is because we're making an emulsion of water in oil.

So the adjuvant is mostly mineral oil, and the antigen is an equal solution. So what we want to do is mix the antigen very slowly to the oil so that the oil completely coats the antigen each time we add it. So if we added it in one go, it wouldn't completely coat the antigen.

So now I've added all of the mean spinal cord to the adjuvant on the vortex, and I've stuck the tube on the vortex to let it spin for five more minutes to make sure everything gets completely mixed. And I adjust the speed so that I have the highest vertex possible without losing too much in the late of the tube. So now the five more minutes are vortexing are over, and I'm going to stop the vortex and transfer my merchant into glass syringes.

So I have assembled full syringe. I attached it to the larger diameter bridge, and to the other hand of the bridge, I've attached only the barrel of one syringe, and I'm now going to add the emulsion into that syringe. So now, no.

Now I'm going to take all the liquid into the lower syringe, Disconnect the top one, finish assembling the top one, make sure there is no air bubble in my lower syringe or in the bridge. And now I'm going to reattach. So now I have both syringes attached together through this bridge.

One is full of immersion, the other one is empty. And what I'm going to do now is push as fast as possible, the counter of one syringe to the other back and forth. So now I'm going to change the large diameter bridge to a smaller one to the 18 G bridge.

The smaller bridge is going to make the emulsion even thicker. So to change bridges, I put all the emulsion in one syringe. I remove the empty syringe, I make sure I lose nothing in the bridge, remove the bridge, put the new one on, chase all the air bubbles and reattach the other syringe.

And I repeat the procedure that now the emulsion is much harder to go through, and I'm careful not to break this bridge, which is very fragile. And I repeat this procedure with another set of syringes as long as I have emulsion in my tube. So I use as many syringes as necessary to use up all of the emulsion.

So now I've mixed the emulsion several times, maybe 10, 15 times, but it varies with these batches. And now it's getting extremely hard to get through. And I'll do it a couple more times, well, or maybe not.

It's really stuck now, so I'm going to leave it there. Otherwise, there's a risk of breaking either a syringe or the bridge. So when it stops going through, if it happens very early on, that means maybe the bridge is clogged.

But at this point, since I've mixed it at least 10 or 15 times, it means that the emulsion is ready. It's very thick, so I should leave it alone. Now I'm going to wrap it up in aluminum foil and put it in the fridge for the night.

So yesterday we prepared the emulsion with red spinal cords and we left the emulsion overnight in the fridge. Today we're taking it out and we are going to inject it 200 microliters per rat at the base of the tail. But before we inject it, we have to make sure that the emulsion is good.

So it should still be solid, and it is still a nice solid. The crack that you see here even tells us that it's a nice solid, it's not separated into phase, into two phases, which would be bad. So this emulsion is good to be used.

So each hutch is going to receive 200 microliters of our emulsion. And to inject it, we use three mil syringes, but we make sure we use lu lock tip syringes because if we use slipped, its syringes. The emulsion is so thick, the needle might pop out while we're trying to inject.

So always go with the lu lock type syringe. And as a needle, we try to go with the smallest needle possible, but with an emulsion that thick, we have to go with a 20 gauge needle. Since we need to inject very precisely at the base of the tail, we anize the rats for a few minutes and we take the opportunity that those rats are fast asleep to mark them and to mark them.

We use ear punches, and each rat is identified by specific pattern of your punching. So now we're ready to inject the rats, and we're going to give each rat 200 microliters of emulsion at the base of the tail, and we're going to mark the ears at the same time. I only prepare emulsion in the in the injection syringe for one rat at a time because it's often really difficult to inject and I'm pressing really hard, and I don't want to inject more to a rat than it needs.

So I only fill the syringe for one hu at a time. To fill the syringe, I connect my plastic injection syringe to the bridge that's itself connected to the glass syringe that contains the emulsion. I put about 300 microliters of emulsion in my plastic syringe.

Then I connect the needle and I press the air out of the needle so that I don't lose this 50 microliters lost space in the needle. And I make sure I just have 200 microliters, and then I inject it at the base of the tail of the rat. So now that we've injected all of our hearts, we're going to monitor them daily.

So we're going to weigh them every morning and check them twice daily for clinical signs of EAE. The first signs, as I'll show you when they appear, will be a flacid tail, and we expect to see those signs appearing for the first rats around day seven, day eight post-injection. Most of the rats will get sick around day 10 to day 12, and the latest rats will get sick around day 15 to 17.

We are now on day 13 after we immunized our hearts with the spinal cord emulsion, and I'm now going to monitor those animals to check their clinical score. But before I do that, let me introduce you to how we house our animals. So autoimmune diseases are this characteristic that any pathogen present in the animal is going to reduce the autoimmune response.

So to avoid that to a maximum, we house our rats in specific conditions. So the house in this cubicle, which contains filtered air and it's under positive pressure, the animals are themselves housed into autoclaved cages. So absolutely everything in the cage is autoclaved before we put the rat net.

And they have filters on top of the cages. The food is the standard rodent chow. It's I originated.

And we also give our rats acidified water. We take normal drinking water and add hydrochloric acid following standard protocols, and we use acidified waters to maintain the intestinal flora to minimum, which will reduce the risk of impairing our autoimmune response. So now I'm ready to do the clinical scoring of my rats.

The scoring ranges from a score of zero, which is no clinical signs to six, which is a morbid state at which case state with sacrifice the animals. And now I'm going to take you step by step showing you examples of each clinical score we can find in this model. So we are now on day 13 after immunization of the rats to UCAE.

And in that cage I have two rats that have not gotten sick yet. So they have absolutely no clinical signs of yay. So both rats are still healthy.

They're running around, run around, and as you can see, when they move, they lift their tail from the bedding. So sometimes they let the tail dry, but most of the time they'll try to lift it from the bedding. And if given the chance, they'll also climb out of the cage, which means they have full control of all four limbs and they can stand up if they need to.

So the first clinical sign of EA is a limp tail. So if I pick up a rat and I press my finger against the tail, the rat will have a tendency of grabbing with the, with the tip of the tail. So as you just saw, this rat doesn't have a li tail, and I can do it with the other one.

The tail grabs the finger, which means the, those rats have a full reactive tail. They have a clinical score of zero, which means actually no a e at all. So here is a rat that has a clinical score of O 0.5.

So if you just look at it in the cage, it behaves absolutely, normally it's running around, it's climbing up on the side of the cage, but the tip of the tail never gets off the bedding. So this rat might have a partial limb tail. So what I do is I grab the rat and I do the same test, and although the tail has some tone, the end doesn't completely curl on my finger.

So I'll score this rat with a score of 1.5, which is partial li tail. So clinical score of one is characterized by complete flad tail, so no part of the tail has any tone, but the red still can use their hind lines. So as you can see, this rat is standing up against the side of the cage, which shows that his back legs are functioning normally.

But if I check the tail of those rats by picking them up, there's absolutely no tone in their tail. Another test you can do is while the RU is in the bottom of the cage, if you lift the tail with a normal ru, the tail will come down slowly or maybe not even reach the ground with the RU with a score of one. If you lift the tail, it'll drop immediately to the to the ground.

This RU here can move all four limbs, but the hind limbs are too weak to allow this RU to stand up. So I'll classify this RU with a clinical score of two. So the main difference between a score of one and two is that the score of one is only a limp tail.

The back legs are fully functional, and the score of three, the rat would be falling on its side. Two is intermediate, so the rat can use it back, its back legs, but they're much weaker than that of a normal rat. So this rat has an unusual feature of EAE.

Normally EAE is characterized as an ascending flacid paralysis, but in some cases, which are pretty rare, like this one, the rats develop first front limb paralysis before the hind limb gets paralyzed. And this is the case of this rat. So if you look at it, it's, it's head is lower than the rest of its body because the right has difficulties with his front limbs and it cannot to climb on the side of the cage.

And although his back limbs are working normally, he cannot push with his front limbs. So I would still score this rat with a score of two, which would correspond to mild paras, although we normally see that with hind limbs in this ru, we see it with the four limbs. So this RU is, has a much more severe phenotype than the previous ones.

It has a clinical score of three. So as I'm going to show you, the front limbs are functioning absolutely normally, but the two hind limbs have a present moderate parais. So both hy limbs are usable by the RU to move, but the RU falls on its side and it cannot con completely control those legs.

Those spores, they're getting much weaker than they normally would be without EE.And at that stage, we don't worry about limps of the tail anymore because many of those rats after a while, start having a very wiry tail. So this one still has a very limp tail, but sometimes you can see some dystonia. So the tail starts moving in a uncoordinated manner.

So we don't look at the tail anymore at this level, we only look at how the limbs function. We score this right 3.5 because if we look at his hind limbs, one moves, but the other is completely paralyzed and doesn't at all help in the movement forward of the ru. So this is considered a com complete paralysis of one hind line and therefore score of 3.5.

This RU has a clinical score of four, which means complete hind line paralysis, and this is characterized by a total inability to move his back legs. The rat we have in this cage has a clinical score of five, so that corresponds to a complete hind line paralysis with an additional incontinence. This animal is incontinent, as we can see, its completely weight in this area, which we do not see in the other animals that are not in continent.

When our HUS have scores of three or more, we give give them water bottles with long sipper tubes to make sure they can access the water. And we also put food at the bottom of the cage, and we also give them gel packs. But even though with all that, each time we take the rats out to monitor them, which is twice a day, we always offer them water in case they didn't access enough water during the day.

Here I have a rat with a clinical score of four. And at that stage, those animals need to be a little encouraged to eat. So what I do is I wet their food, which makes it much easier for them to chew on.

So over the past two weeks, I have showed you how to induce and monitor chronic relapsing EAE in DA rats. So in the first step, I showed you how to prepare the spinal cord immersion, then how to inject it at the base of the tail of those rats. And today I've shown you how to score those rats with different levels of disease.

Of course, this is a chronic relapsing model, so we expect the rats to have fluctuating scores. Disease is going to go into remission in a few days and going to relapse over time. Some rats are going to go into a more chronic phase of disease.

It'll vary from animal to animal. Now, before we end this video, I want to emphasize one thing about testing drugs in those models. The very important point is to randomize the animals as best as possible.

One very important thing to know is the earliest the animal gets sick, the most severe it's disease. So you do not want to group all your control animals in the first day, and you are treated animals in the next few days. What we do is as soon as an animal gets sick and the day we want to start treatment, we place the animals randomly in two different groups.

If we have, for example, one control and one treated group, the first heart will go in the control group, the second one in a treated group, and so on until all the animals are placed in their own group. But we make sure this randomization happens as early as possible in the protocol. So I hope this video was helpful for you and that you'll be able to induce and monitor chronic relapsing EAE in D rats.

And good luck with your Experiments.