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Retinitis pigmentosa (RP) is a group of diseases affecting 1.5 million people worldwide. It involves apoptosis of retinal photoreceptor cells and results in
eventual blindness. The bile acid tauroursodeoxycholic acid (TUDCA) is anti-apoptotic in rodent neurodegeneration models. We hypothesize that TUDCA
will similarly be anti-apoptotic in photoreceptor cells in the rd10 mouse, a model of RP. To test this hypothesis, rd10 mice were subcutaneously injected with
varying concentrations of TUDCA or vehicle. Afterwards, retinal function was assessed by electroretinograms (ERG). Apoptosis in retina sections was assessed
via TUNEL immunohistochemistry. Vehicle-injected mice had severely suppressed ERG amplitudes and massive loss of photoreceptor cells via caspase-mediated
apoptosis. These deficits were prevented in TUDCA-treated mice. Given that hydrophilic bile acids are FDA-approved for treating cirrhosis and are well-tolerated
by humans, these data suggest that TUDCA may be useful in the ophthalmic clinic. Optimally this would entail local delivery, but toxicity associated with this
approach is unstudied. Accordingly, wild-type C57/Bl6 mice were intravitreally injected with varying concentrations of TUDCA in one eye and vehicle in the other.
ERGs were taken at various times post-injection. No diminution in ERGs was detected between vehicle and TUDCA eyes, suggesting lack of toxicity within the
concentration range tested. Clinical trials testing the effects of TUDCA on RP patients may be warranted.
RP leads to blindness via apoptosis of photoreceptor cells in the outer nuclear layer (ONL) of the retina. There is currently no effective treatment for RP.
Tauroursodeoxycholic acid (TUDCA), a bile acid, is known to prevent apoptosis of liver cells (Rodrigues et al., 2000), and pilot studies by the Boatright lab
suggest that TUDCA treatment preserves retinal function and rod cell morphology in the rd10 mouse model of RP (Boatright et al., 2003). The rd10/rd10 mice
have a mutation in the gene that codes for the β subunit of cGMP phosphodiesterase, as essential component in the phototransduction cascade (Chang et al.,
2002). Although subcutaneous injections at a specific concentration of TUDCA imply neuroprotection in these pilot studies, a dose-response curve has not yet
been studied to find optimal concentration. In this study, our first aim (AIM 1) will be to deduce the minimal effective concentration of TUDCA with the maximal
neuroprotective effect. We hypothesized that TUDCA will be increasingly neuroprotective to photoreceptor cells with increasing doses. Injecting a drug
systemically may not be the best means of delivery as there may be negative side effects to the surrounding vital organs. Recent studies in the Boatright
laboratory suggest that intraocular injections of oligonucleotides and nanoparticles have been non-toxic to wild-type mice, and may be better suited for clinical
use as they deliver the drug locally, increasing the likelihood for the drug to reach its target. Preliminary work has been done to see the effect of intraocular
injections in wild-type mice at a low concentration of TUDCA (Figure 1), and the results show a promising future for toxicity studies (AIM 2). We hypothesize
that higher concentrations of TUDCA administered intravitreally (intraocularly) will be toxic to mouse eyes as reflected in declining amplitudes. This model was
chosen because preliminary studies have established results on TUDCAs apoptosis inhibition properties, allowing further testing to refine the optimal
concentration and testing alternative and more clinically practical means of drug introduction. If found neuroprotective, it can be a cost effective over the counter
pharmaceutical to treat people with RP, allowing them a longer period of functional vision.
AIM 1: rd10 mice were subcutaneously treated with various concentrations of TUDCA (50 mg/kg, 100 mg/kg, 500 mg/kg, and 1000 mg/kg in 0.15M NaHCO3
1mL/kg) or Vehicle (0.15M NaHCO3 1mL/kg) every 3 days starting on postnatal day 6 (P6), before any retinal degeneration began, and continued until P24.
Electroretinogram (ERG) recordings were taken on P18 and P24, when the mouse eyes are large enough to measure ERGs (Figure 2). Dark adapted and light
adapted a-waves and b-waves were analyzed (Figure 3). Eyes were enucleated on P24, processed, embedded, and superior-inferior cross sections were taken.
Sections near the optic nerve were assayed using TUNEL Fluorormetric Labeling to mark apoptotic cells, and photographed (Figure 4).
AIM 2: Wild-type adult mice (C57Bl6) were treated with various concentrations of an intravitreal injection of TUDCA in one eye (0.5 mg/mL, 5 mg/mL, 50 mg/mL,
dissolved in sterile 1xPBS), with Vehicle (1x PBS) in the other eye to test for a toxicity response. ERG recordings were taken one week before the injection
(baseline), and one and two weeks after the injection (Figure 2). Dark-adapted and light-adapted a-waves and b-waves were analyzed using repeated
measures ANOVA (Figure 5).
AIM 1: Untreated rd10 animals showed declining retinal function over time as opposed to wild type (C57/Bl6) animals as reported by ERGs. TUDCA
treated rd10 animals showed maintenance of retinal function with increasing doses as reported by ERGs (Figure 3). Higher doses also showed increased
outer nuclear layer (ONL) thickness and reduced apoptotic signal as reported by the TUNEL assay (Figure 4).
AIM 2: Increasing concentrations of intravitreally injected TUDCA showed comparable retinal function to Vehicle injected eyes (Figure 5). Repeated
measures ANOVA indicated no significant differences across all treatments (p > 0.05).
AIM 1: According to ERG data (Figure 3), overall, higher concentrations of TUDCA seem to be more neuroprotective to retinal cells than lower concentrations.
500 mg/kg treatment shows the highest level of retinal function, supporting our hypothesis. Since all 1000 mg/kg treated mice have lead to premature deaths,
ERG results could not be taken of these mice. According to the TUNEL labeling assay (Figure 4), 1000 mg/kg treatment seems to be most neuroprotective to
the outer nuclear layer of the retina, also supporting our hypothesis. Further studies will be performed replicating this experiment.
AIM 2: According to ERG data and repeated measures ANOVA (Figure 5), all concentrations of TUDCA and Vehicle injected intravitreally show similar r
esponses to increasing light intensities, which suggest that TUDCA is not toxic to the mouse retina when delivered locally. Further studies will include testing
TUDCA intravitreally at concentrations of 500 mg/mL and above, and testing the non-toxic concentrations in retinal degeneration mouse models to test
neuroprotective effects.
This material is based upon work supported by the Howard Hughes Medical Institute under Grant No.52003727 and by RPB, FFB, NIH
Grant No. R01EY014026, P30EY06360.
1 Boatright JH, Rengarajan K, Pardue MT, German Moring AJ, Nickerson JM, Hawes NL, Chang B. (2003). Developmental analysis of the rd10 mouse. ARVO
e-abstract, 4536.
2 Chang B, Hawes NL, Hurd RE, Davisson MT, Nusinowitz S, Heckenlively JR. (2002). Retinal degeneration mutants in the mouse. Vision Res. 42:517-25.
3 Rodrigues CM, Stieers CL, Keene CD, Ma X, Kren BT, Low WC, Steer CJ. (2000). Tauroursodeoxycholic acid partially prevents apoptosis induced by
3-nitropropionic acid: evidence for a mitochondrial pathway independent of the permeability transition. J Neurochem. 75:2368-2379.
Retinitis pigmentosa (RP) is a blinding genetic disease that leads to thinning of the retina (the part of the eye that lets you see). It acts by programmed cell death,
also known as apoptosis. There is a certain bile acid called TUDCA (tauroursodeoxycholic acid) that has been previously found to keep cells from undergoing
apoptosis in other diseases that target the nervous system. Researchers have found a certain strain of mouse with the same disease of the retina that humans
have, and others in our lab have tested this bile acid drug on these mice by injecting them under their skin with a certain dose of the acid. The results indicate
that the bile acid does indeed help reduce apoptosis in the retina, and causes the retina to be thicker overall. Since they have only tested the drug at one dose,
we decided to try and test the drug at other doses higher and lower than the previously tested dose to find out if there is a better dose to treat people who are
going blind. We found that the retina was thicker at the highest dose tested, and not very thick at the lower doses. We also conducted ERG (electroretinogram)
testing, where we flashed the mice with light. The ERG apparatus then showed us waves that indicated whether or not the retina was functional. Our results
showed us that higher doses of the drug lead to larger waves, meaning greater retinal function. Since we had been injecting these mice under the skin, or
systemically, the drug could possibly have negative side effects in other parts of the body. To avoid this complication, we thought we should try to inject the
drug directly into the eyes, or locally. Before we could do that, we decided to test a range of concentrations to see if the drug would be toxic to the eyes of normal
mice that were not going blind. After taking ERG recordings of the mice, the results indicated that the drug was not toxic at any of the concentrations tested
because all of the treatments, including those injected with just a buffer, showed similar sized waves. Our future studies will include testing even higher
concentrations for toxicity in normal mice, and testing the drug in the mice that are going blind to see if it can reduce apoptosis this way as well.
subcutaneous injections, intravitreal injections, electroretinograms (ERGs), paraffin tissue sectioning, TUNEL fluorometric assay, immunohistochemistry,
confocal microscopy
subcutaneous injections, intravitreal injections, electroretinograms (ERGs), paraffin tissue sectioning, TUNEL fluorometric assay,
immunohistochemistry, confocal microscopy
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