One of the major challenges in the research for new treatments lies in the impossibility of diagnosing PD before the occurrence of widespread degeneration and establishment of long-term changes in the basal ganglia circuitry. At the beginning of the 20th century, scientists were seeking ways to regenerate the tissue or replace it with transplanted
to repair brain tissue is a very old idea, which presents its fair share of significant challenges. First, reaching a discrete area of the brain without affecting the surrounding nuclei is not an easy task and could not be achieved without the development of stereotaxic apparatus. Secondly, mature neuronal tissue does not survive transplantation well, possibly due in part to the severing of the axonal projections (Dunn, 1917). Nevertheless two schools of thought emerged, using an autograft of the patient’s own catecholamine producing adrenal gland cells, or using an allograft of embryonic dopaminergic neurons precursors collected from surgical terminations of pregnancy. The refinement of transplantation techniques for the use of embryonic neurons allowed an excellent rate of survival of the grafted cells in animals models (Le Gros Clark, 1940, Stenevi et al., 1976). Based on this data, and the poor results emanating from clinical trials of adrenal graft in PD patients, scientists started to consider foetal ventral mesencephalon (VM) tissue as a promising source of dopaminergic cells (Backlund et al., 1985, Brundin et al., 1986). The use of human foetal cells, obtained from elective abortions, as the “donor” raised important ethical questions and was, and still is, subject to active debate (Boer, 1994). Furthermore, 5-8 embryos of 6-10 weeks old are required in order transplant a patient unilaterally, a timeline that can cause practical issues, as the tissue cannot be kept for more than few days, added to the changing protocols for terminations of pregnancy from surgical to medical in some countries. Notwithstanding the ethical and practical hurdles, the first open clinical trial for foetal VM transplant for PD went on in Sweden in 1989 with 2 of the drug abusers left “frozen” by MPTP consumption. The patients transplanted exhibited clinical benefit, which correlated with increased of 18fluorodopa binding in PET scans. For the following 2 decades several open-label trials were performed and provided the proof of concept that foetal VM cells survive and innervate the striatum of PD patients, providing, an alleviation of PD motor symptoms and allowing a decrease in L-DOPA medication (Lindvall et al., 1990, Kordower et al., 1995, Hauser et al., 1999, Hagell and Brundin, 2001). Between 1980 and 2002, over 100 patients received intrastriatal VM transplants but the outcomes have been extremely variable. Although the European trials demonstrated a clear clinical benefit in most of the grafted patients, the two double-blind studies funded by the American National Institute of Health (NIH) in the mid-1990s failed to show a significant improvement in the transplanted group when compared to the placebo group (Freed et al., 2001, Olanow et al., 2003). However, further detailed analysis revealed a significant clinical benefit in younger patients (less than 60) reaching a maximum 4
years post transplantation. This highlights the importance of the choice of study end- point, as the graft needs time to mature (Ma et al., 2010). If one can argue that the design of the trials were inappropriate, the development of severe abnormal and uncontrollable movements, which persisted after the withdrawal of L-DOPA medication, raised significant concerns. Indeed, 9 out of the 70 patients transplanted in the NIH trials had to undergo subsequent DBS to alleviate these so called graft-induced dyskinesias (GID) (Greene et al., 1999).
1.3.3.1 Graft-induced dyskinesia
The first report of these severe motor side effects following intrastriatal transplantation of foetal dopaminergic neurons emanated from the first US NIH sponsored double-blind control trial performed by Freed and co-worker in the early 2000s. These abnormal movements were described as stereotypic and dystonic, mainly affecting the lower parts of the body, similar to diphasic LID (Olanow et al., 2003). In total, 30 PD patients received human embryonic VM tissue grafts and 15% of them developed severe dyskinesias during the second and third year post grafting. These dyskinesias were directly linked to transplantation and persisted after reduction or complete withdrawal of dopaminergic drug therapy (Greene et al., 1999). Similar adverse effects of the transplantation were described after the second US NIH sponsored double blind study involving 23 patients. More than half (13, 56%) of these patients developed mild off- medication dyskinesias between the 6-12 months following transplantation (Olanow et al., 2003). Lastly, a retrospective analysis of the video-based evaluation of GIDs performed on 14 grafted patients during the Swedish trials 11 years ago, underlined 8 cases of mild GIDs, which reached their peak severity after 3 years (Hagell et al., 2002). Interestingly, patients in all of these studies continued to derive benefit from the grafts, which gradually improved for up to 13-16 years post transplantation, based on the reduction of their Unified Parkinson’s Disease Rating Scale (UPDRS) scores (Hagell et al., 2002). This was a feature, which echoed through all of the studies.
1.3.3.2 Animal models of graft-induced dyskinesia
After the development of transplantation side effects in patients and the variable lack of efficacy, scientists went back to the bench. Transplantation studies in non-human primates and rodents were performed and carefully monitored for dyskinesia in the absence of anti-parkinsonian drugs. Only infrequent, spontaneous GIDs were reported
in the MPTP-treated primates (Redmond et al., 2008). Similarly, in the 6-OHDA- lesioned rat model, very few spontaneous dyskinesias were observed in the absence of medication and were considered too inconsistent and unreliable to constitute a good model of GID (Lane et al., 2006, Vinuela et al., 2008). Although, it has been established that VM dopaminergic grafts are capable of reducing AIMs induced by L-DOPA in the 6-OHDA-lesioned rat, a few studies have described the appearance of a novel stereotypic behaviour limited to the forelimb and facial region described as a specific GID-type behaviour. It is however important to note that this forelimb-facial stereotypy appears under L-DOPA treatment and are associated with single site ‘hot-spot’ VM transplants (Steece-Collier et al., 2003, Maries et al., 2006, Steece-Collier et al., 2009). These facial-forelimb and forelimb stereotypies were not observed in rats receiving multiple sites deposition of the same number of foetal cells producing a more evenly innervating graft. This observation was consistent with the proposal from the Denver- Columbia clinical trial, which identified ‘hot spots’ of DA in their GID patients (Ma et al., 2002, Maries et al., 2006). However, this movement is hard to evaluate as it can be easily masked by mild LIDs.
An alternative approach, which still relies on drug stimulation, is that of amphetamine- induced abnormal movements. The amphetamine-induced rotation test is commonly used to assess the severity of DA depletion following 6-OHDA lesions and as a crude estimate of foetal VM transplantation success. Closer observation, following description of GIDs in the American trials, demonstrated that dyskinetic movements, generally resembling mild to moderate LIDs, were evoked by amphetamine administration in 6- OHDA lesioned rats, transplanted with VM tissue (Carlsson et al., 2006, Lane et al., 2006). Typically, the amphetamine-induced dyskinesias are hyperkinetic limb and/or orofacial dyskinetic movements, reaching their peak severity at 12-16 weeks post transplantation and disappearing if the graft is completely rejected by the host brain (Carlsson et al., 2006, Lane et al., 2006, Lane et al., 2008). Although this model is now commonly used to study the parameters influencing the development of GIDs (Carlsson et al., 2006) it is not fully representative of the clinical situation. Indeed, transplanted patients receiving amphetamine for 11C raclopride PET imaging scans did not show the appearance or worsening of GIDs (Piccini et al., 1999, Carlsson et al., 2006, Smith et al., 2012). Despite the efforts made to reproduce some important clinical conditions (i.e. extremely severe DA depletion mimicking late stage patients, and long-term, high dose
of L-DOPA to induce severe LID prior to grafting), in neither the rodent, nor the primate, are we able to establish true spontaneous GIDs. One hypothesis that will be addressed in this thesis is that these models have still failed to recreate a situation close enough to the clinical experience, omitting specific critical factors. For instance, the impact of L-DOPA treatment post-graft and non- fully immunologically compatible transplants have never been rigorously assessed in the context of GIDs (Steece-Collier et al., 1990, Steece-Collier et al., 1995, Maries et al., 2006, Lane et al., 2008, Soderstrom et al., 2008, Steece-Collier et al., 2009, Garcia et al., 2011).