The 6-OHDA rodent model of PD is a preclinical model currently used to identify promising new therapies for PD and related side effects, such as LIDs. In order to establish the progression of lesion development, mice were unilaterally injected with 6-OHDA or ascorbic acid (vehicle control) into the MFB and nigrostriatal degeneration was assessed over 4 weeks.
6-OHDA injection induces cell loss in the SNpc over time
In this experiment there was a 20% mortality rate in 6-OHDA lesioned animals, with 5 of the 25 animals entered into the study dying prematurely. Animals that underwent sham surgery had no lasting post-operative complications. Tissue was collected 1, 2, 3, and 4 weeks post 6-OHDA injection (Fig. 1a) and dopaminergic and total neuron numbers were quantified using stereological analysis of TH and NeuN positive cells, respectively, to assess SNpc dopaminergic neurodegeneration over time. Two-way ANOVA demonstrated a significant interaction between toxin and time on TH positive cell numbers in the SNpc (F(3,32) = 10.31, p < 0.001, n = 5 per group) indicating a significant effect of 6-OHDA on dopaminergic cell survival over time. The simple main effect of toxin revealed significant loss of TH positive cell numbers at 1 (F(1,32) = 56.93, p < 0.001), 2 (F(1,32) = 171.40, p < 0.001), 3 (F(1,32) = 203.49, p < 0.001) and 4 weeks (F(1,32) = 203.35, p < 0.001) following unilateral 6-OHDA lesion compared to ascorbic acid controls (Fig. 1b, c). The simple main effect of time after 6-OHDA treatment indicated significant loss of TH positive cells with time (F(3,32) = 20.55, p < 0.001). A Bonferroni post hoc analysis revealed a significant difference in TH positive cells between 1 and 2 weeks (p < 0.001), 1 and 3 weeks (p < 0.001) and 1 and 4 weeks post lesioning (p < 0.001).
Two-way ANOVA demonstrated a significant interaction between toxin and time on NeuN positive cell numbers in the SNpc (F(3,32) = 7.02, p < 0.001, n = 5 per group) indicating a significant effect of 6-OHDA on neuronal cell survival over time. The simple main effect of toxin showed significant loss of NeuN positive cell numbers at 1 (F(1,32) = 61.36, p < 0.001), 2 (F(1,32) = 128.82, p < 0.001), 3 (F(1,32) = 184.03, p < 0.001) and 4 weeks (F(1,32) = 177.59, p < 0.001) following unilateral 6-OHDA lesion compared to ascorbic acid controls (Fig. 1d, e). The simple main effect of time after 6-OHDA treatment indicated significant loss of NeuN positive cells with time (F(3,32) = 10.16, p < 0.001). A Bonferroni post hoc analysis revealed a significant difference in TH positive cells between 1 and 3 weeks (p < 0.001) and 1 and 4 weeks post lesioning (p < 0.001).
6-OHDA injection induces early terminal loss in striatum
A loss of dopaminergic neurons in the SNpc has been shown to result in degeneration in the striatum of the projecting fibres from the nigral cell bodies [23], subsequently leading to a dopamine deficit and motor symptoms. DAT plays an important role for maintaining sufficient dopamine levels for release into the synaptic cleft in the striatum, and its expression has been shown to reduce following dopaminergic neuron loss in the SNpc [24, 25]. Thus, it is a useful indicator for lesion in the striatum following 6-OHDA. In order to confirm the extent of the lesion to the striatum we quantified TH and DAT expression in the striatum via densitometry at 1, 2, 3, and 4 weeks post 6-OHDA administration.
Two-way ANOVA of toxin and time revealed no significant interaction (F(3,32) = 0.60, p = 0.618, n = 5 per group) of TH expression. There was a significant main effect of toxin (F(1,32) = 421.73, p < 0.001) on the optical density of TH positive fibres in the striatum between 6-OHDA and ascorbic acid injected animals (Fig. 2a and b). However, there was no significant main effect of time after 6-OHDA injection (F(3,32) = 0.39, p = 0.755), suggesting that the loss of TH positive fibres in the striatum was fully established after just 1 week and didn’t increase any further over time. Similarly, two-way ANOVA of toxin and time resulted in no significant interaction (F(3,32) = 0.40, p = 0.753, n = 5 per group) on DAT expression. There was a significant main effect of toxin (F(1,32) = 1554.52, p < 0.001) on the optical density of DAT positive fibres in the striatum between 6-OHDA and ascorbic acid injected animals (Fig. 2c, d). However, there was no significant main effect of time after 6-OHDA injection (F(3,32) = 1.27, p = 0.299), suggesting the loss of DAT positive fibres in the striatum was fully established after just 1 week and didn’t increase any further over time. The results from this study therefore demonstrate that the modified 6-OHDA model described by Thiele et al. [11] results in significant and progressive dopaminergic and total neuron loss in the SNpc, as well as a loss of TH and DAT positive fibres in the striatum.
Activin A levels are increased after administration via osmotic micropump
Prior to assessing the efficacy of activin A on LID we confirmed that recombinant activin A was diffusing to the target region when administered via an intrastriatal osmotic micropump. Toxin and l-Dopa naive mice received an osmotic micropump containing either activin A or vehicle. Four regions, the ipsilateral striatum, contralateral striatum, midbrain and ipsilateral cortex, were dissected 2 days later. An ELISA was used to quantify activin A levels in these regions. Independent t-tests confirmed significantly increased activin A levels in the ipsilateral striatum (t = 3.907, p < 0.05, n = 5 per group) and elevated in the neighbouring cortex (t = 2.144, p = 0.099, n = 5 per group). However there was no increase in activin A in either the midbrain (t = 0.418, p = 0.687, n = 5 per group) or contralateral striatum (t = 0.695, p = 0.507, n = 5 per group), ensuring a region specific diffusion of activin A via striatal micropump (Fig. 3).
Activin A treatment has no effect on severity or onset of LID
To test the efficacy of activin A as a treatment to reduce the severity of LID, 6-OHDA lesioned mice were rendered dyskinetic with repeated l-Dopa injections over 3 weeks. Thereafter, mice with LIDs received treatment with vehicle or activin A together with l-Dopa, and abnormal involuntary movements (AIMs) were rated for 13 days (Fig. 4a) to investigate the ability of activin A to reduce the severity of LIDs.
We observed a 16% mortality rate, with 3 out of 19 animals deceasing following 6-OHDA injection. A two-way repeated measures ANOVA revealed no statistically significant effect of activin A on the severity of AIMs (interaction: F(4,52) = 1.978, p = 0.112; time F(4,52) = 5.088, p < 0.01; treatment: F(1,13) = 1.071, p = 0.32, control n = 7, activin A n = 8; Fig. 4b). To investigate a possible effect of activin A on AIMs expression during a single monitoring session, AIM scoring was analysed at each 20 min interval on the final test day (Day 13). Mann–Whitney tests showed no differences between activin A treatment and vehicle at any time point after l-Dopa injections (20 min: U = 27.5, p = 0.955; 40 min: U = 25.5, p = 0.779; 60 min: U = 12, p = 0.072; 80 min: U = 25, p = 0.779; 100 min: U = 23.5, p = 0.613; 120 min: U = 24, p = 0.694; Fig. 4c). Furthermore, Mann–Whitney tests showed no changes in the expression of different subtypes (axial: U = 24, p = 0.694; limb: U = 12.5, p = 0.072; orolingual: U = 16, p = 0.189) between activin A and vehicle (Fig. 4d). Together these results suggest activin A has no effect on reducing severity of established AIMs.
We next aimed to investigate if activin A can delay the onset of LID. Therefore, in a second study, animals were randomized 3 weeks following 6-OHDA injection to receive either daily treatment with l-Dopa + vehicle or l-Dopa + activin A for 13 days (Fig. 5a). In this animal cohort we observed a 19% mortality rate, with 8 out of 42 animals deceased following the 6-OHDA injection. A two-way repeated measures ANOVA revealed no statistically significant effect of activin A on delaying the onset of AIMs (interaction: F(4,120) = 1.387, p = 0.2425; time F(4,120) = 24.89, p < 0.001; treatment: F(1,30) = 0.3608, p = 0.5526, control n = 17, activin A n = 15; Fig. 5b). The significant main effect of time on global AIMs confirmed the previous established LID development profile of approximately 2 weeks [6]. Similar to the above experiments, Mann–Whitney tests on data from the last monitoring session (Day 13) showed no differences between activin A treatment and vehicle at any time point after l-Dopa injections (20 min: U = 99.5, p = 0.295; 40 min: U = 117, p = 0.710; 60 min: U = 122, p = 0.852; 80 min: U = 109, p = 0.502; 100 min: U = 127, p = 1.0; 120 min: U = 116, p = 0.682; Fig. 5c). Furthermore, Mann–Whitney tests on day 13 showed no changes in the expression of different subtypes (axial: U = 102.5, p = 0.350; limb: U = 121, p = 0.823; orolingual: U = 123.5, p = 0.88) between activin A and vehicle (Fig. 5d). Together these results suggest activin A has no effect on delaying the onset of AIMs.