Developing a Rodent Model of Disc Degeneration with Low Back Pain

Tyler Miller Author

04/05/2021 Added

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This presentation highlights the work I have done over the past year on our animal study. I was involved in development of the model, behavioral testing, and post processing.

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[00:00:00.922]Hi, my name is Tyler Miller.
[00:00:02.800]Today I will be talking about,
[00:00:04.630]my uCARE project,
[00:00:05.587]"Developing a Rodent Model of Disc Degeneration"
[00:00:08.017]"with Low Back Pain."
[00:00:10.530]The motivation for my project
[00:00:12.140]is 10% of Americans experience Chronic Low Back Pain.
[00:00:15.940]It cost the US about $100 billion each year,
[00:00:19.070]and 26-42% of Chronic Low Back Pain patients,
[00:00:22.610]have pain arising from the intervertebral disc,
[00:00:25.430]which is the area we focus on.
[00:00:28.698]The intervertebral disc
[00:00:30.510]is found between your vertebral spine.
[00:00:35.400]It is composed of the nucleus pulposus and annulus fibrosis.
[00:00:39.180]The nucleus pulposus or NP,
[00:00:41.240]is made up of the cells and proteoglycans,
[00:00:44.580]and have a lot of your liquid content in the disc.
[00:00:47.500]And then the annulus fibrosis is multiple collagen layers
[00:00:50.810]that provide a structural barrier
[00:00:54.540]for the nucleus pulposus in the body.
[00:00:59.180]The degenerated disc is what we're aiming for here
[00:01:03.060]in the bottom right.
[00:01:04.460]As you can see, there's nerve in-growth
[00:01:09.240]and inflammatory phenotype is with symbol it by red.
[00:01:13.330]We were trying to simulate this by scraping
[00:01:16.050]along the dorsal side of the disc backed by the L5DRG,
[00:01:20.290]we're hoping that innovation would occur
[00:01:22.100]so we could see pain.
[00:01:23.120]And this would be detected
[00:01:24.870]through behavioral assays like von Frey
[00:01:28.450]as the L5DRG innovates, one of the hind paws.
[00:01:34.140]So, to develop this model,
[00:01:37.040]I spent a lot of work doing mock surgery trials.
[00:01:41.910]We knew that we wanted to cause it physically
[00:01:44.140]without injection of a different factor
[00:01:47.050]and also doing it through ventral approach
[00:01:49.490]so we would lay the rat on its back
[00:01:52.090]and come in through the stomach
[00:01:53.510]to hit the ventral part of the disc
[00:01:55.210]which is the lower part of the disc in these pictures.
[00:01:59.700]So I needed to decide the needle gage,
[00:02:01.690]how deep we would go in
[00:02:03.830]and then what kind of needle movements we would need
[00:02:06.480]to cause this damage.
[00:02:07.780]So I did over 20 surgical trials
[00:02:11.860]on Sprague Dawley rat cadavers,
[00:02:13.830]injuring the lumbar disc levels L3/L4 through L6/S1.
[00:02:17.870]And then I would take the spine out,
[00:02:20.140]cut these disc out of the spine
[00:02:22.120]and then section them so that I could look at them
[00:02:25.020]under a dissecting microscope
[00:02:26.670]and then take images with the Nikon camera.
[00:02:30.100]The images you can see on the right
[00:02:33.970]is an uninjured disc and an injured disc
[00:02:36.150]and then a dissecting needle,
[00:02:37.700]the dissecting needles that are used for the final study.
[00:02:40.670]The uninjured disc is just a reference
[00:02:42.820]for the injured is so you can see
[00:02:45.955]that the injured disc has disruption of its AF layers.
[00:02:49.750]And also the nucleus pollsters is scrambled over the disc.
[00:02:53.570]This is very different than the uninjured disc
[00:02:55.673]so you can tell kind of the type of damage we're doing.
[00:02:59.730]To grade how severe these injuries were,
[00:03:03.590]I had to come up with a damaged scale
[00:03:06.680]but all these scales were based on an assumption
[00:03:09.300]that we were going up for the study.
[00:03:11.000]We were only gonna puncture the L5-L6 disc
[00:03:13.490]and have a ventral approach.
[00:03:15.440]So the rest of the variables were decided
[00:03:19.440]through this grading scale,
[00:03:22.710]it was based on different parameters you wanted like damage
[00:03:25.770]to the dorsal side and NP avoiding ventral damage
[00:03:28.680]and then avoiding damage to non-dis structures.
[00:03:31.870]So the scraping method was decided upon
[00:03:35.250]because we originally were doing multiple puncture
[00:03:38.190]and that was causing wide tracks in the ventral area
[00:03:40.380]which we did not want.
[00:03:42.250]We also had to dissecting needle for the same purpose.
[00:03:47.300]Surgical needles would core out the disc
[00:03:49.290]and make wide holes in the ventral portion.
[00:03:51.970]And this was bad
[00:03:52.803]because it might not isolate the damage area
[00:03:55.340]to where we get nerve ingrowth.
[00:03:58.820]We also wanted it to not puncture into the nerves
[00:04:03.380]in the back of the disc
[00:04:04.380]so I had to find a depth
[00:04:06.410]so that we weren't coming out the back of the disc,
[00:04:08.270]which was sort of three millimeters was decided.
[00:04:10.690]And then the number of scrapes for each damage model
[00:04:12.720]was decided by looking at the damage to the drills
[00:04:16.210]to sudden NP in relation to damaged quantification.
[00:04:21.160]And both of these parameters had low ventral damage
[00:04:24.380]and low damaged and under structures
[00:04:26.790]but the low damage model
[00:04:29.750]or one scrape had lower NP scrambling
[00:04:32.340]and total area damage then the high srape method.
[00:04:35.140]The reason that high scrape is set six
[00:04:36.960]is because once we wouldn't be on that,
[00:04:38.560]we were seeing higher ventral damage.
[00:04:41.620]So the animal study was a 36 females Sprague Dawley rats
[00:04:46.800]that were a less than 18 weeks.
[00:04:50.870]There were 12 sham, 12 a low injury model
[00:04:53.450]and 12 high injury models so one in six scrape.
[00:04:56.400]And then I assisted with a grip strength
[00:04:58.430]which is one of our pain essays
[00:05:01.092]microcomputed tomography of the discs
[00:05:04.400]for disc degeneration calculation.
[00:05:05.940]And then I'm currently working on histology of the IVD.
[00:05:10.560]For grip strength,
[00:05:12.340]I met with Dave Lumen every two weeks
[00:05:16.100]and we allow the animals to grip.
[00:05:20.670]The bar is shown below,
[00:05:22.460]pulled on their tail until they let go
[00:05:24.310]and then recorded the max force.
[00:05:26.290]Each animal was tested three times
[00:05:28.150]and we averaged all three values,
[00:05:33.790]three general and then average all the animals together
[00:05:36.280]for each group.
[00:05:37.640]And then the data can be shown on the top left
[00:05:39.710]or it can be seen in the top left.
[00:05:41.730]You can see that the high damaged model
[00:05:44.730]was lower than the sham every week
[00:05:48.200]but it did come up a little bit in the fourth week.
[00:05:52.740]And then the ones group was pretty close to sham
[00:05:56.520]throughout the study but it did differentiate quite a bit
[00:05:59.350]on week 12 and 16.
[00:06:02.180]And that just seems to show
[00:06:04.590]that we might've been experiencing pain.
[00:06:08.230]The disc height index is the most common used way
[00:06:12.470]to measure disc height in rat studies using micro CT.
[00:06:19.250]So we use a volume method with micro CT
[00:06:23.370]and we wanted to compare this method to the DHI Methodist.
[00:06:29.560]So I went in and calculate DHI for all our time points
[00:06:34.010]by taking multiple measurements
[00:06:36.140]of the either adjacent vertebrae
[00:06:39.430]and then also the disc in the middle left and right.
[00:06:43.560]And then I would use the calculation below
[00:06:45.880]to calculate the DHI.
[00:06:48.430]The data between the two shown here.
[00:06:50.790]As you can see the disc volume
[00:06:52.730]and disc index showed the same patterns
[00:06:56.930]throughout the weeks, relatively sharing the same thing.
[00:07:00.170]And then they both also show a return
[00:07:02.960]to baseline as weeks go on,
[00:07:05.920]which we are,
[00:07:09.610]I both sizing is a fibrosis in the disc
[00:07:12.470]but that has not been investigated yet.
[00:07:16.110]And it also seems that some of the error bars
[00:07:19.150]are higher in the disc index
[00:07:22.050]but I have not analyzed the full data samples
[00:07:24.650]against each other yet,
[00:07:25.800]so it cannot say that it is more variable.
[00:07:30.870]Lastly, I've been working on IVD histology
[00:07:33.860]for the last couple of weeks and we're getting ready
[00:07:36.200]to do it on the samples from the study.
[00:07:39.830]I've had to optimize the process for section thickness
[00:07:44.030]to get the assembles are essentially section retention.
[00:07:47.430]We were losing samples of slides
[00:07:49.010]and then also permounting to make sure
[00:07:51.230]that there's no air bubbles in our cover slips.
[00:07:55.120]After spending a couple trials doing this,
[00:07:58.000]I've gotten samples like the one shown below.
[00:08:00.400]And I think we're ready to move on soon.
[00:08:06.360]Lastly, I'd like to thank uCARE
[00:08:08.730]for allowing me to do this research this year,
[00:08:10.970]as well as Dr. Rebecca Wachs for being my PI and mentor
[00:08:17.930]and also David Lillyman for being my graduate mentor
[00:08:21.900]and then everyone else in the one lab
[00:08:23.470]that's helped me throughout the year.
[00:08:25.810]And I would like to thank you for listening to me today
[00:08:29.000]and hope you have a great day.

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Developing a Rodent Model of Disc Degeneration with Low Back Pain

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