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The discovery and application of induced pluripotent stem cells (iPSCs) provide a novel treatment modality for diseases, which remain incurable. Particularly, in the treatment of neurodegenerative diseases such as Parkinson's disease (PD), iPSC‑technology holds an interesting prospect for replacement therapy. Currently, the prognostic improvement of PD is limited and relies on symptomatic treatment. However, the symptomatic dopamine‑replacement therapies lose their long‑duration responses, and novel regenerative treatment modalities are needed. Animal models have provided valuable information and identified pathogenic mechanisms underlying PD but the lack of models that recapitulate the complex pathophysiology of the disease postpones further development of novel therapeutics. This review summarizes the possible uses of iPSCs in PD and discusses the future investigations needed for iPSCs as a possible treatment of PD patients.

Parkinson's disease is characterized by a progressive loss of substantia nigra (SN) dopaminergic neurons and the formation of Lewy bodies containing accumulated alpha-synuclein (alpha-syn). The pathology of Parkinson's disease is associated with neuroinflammatory microglial activation, which may contribute to the ongoing neurodegeneration. This study investigates the in vivo microglial and dopaminergic response to overexpression of alpha-syn. We used positron emission tomography (PET) and the 18 kDa translocator protein radioligand, [C-11](R)PK11195, to image brain microglial activation and (+)-alpha-[C-11]dihydrotetrabenazine ([C-11]DTBZ), to measure vesicular monoamine transporter 2 (VMAT2) availability in Gottingen minipigs following injection with recombinant adeno-associated virus (rAAV) vectors expressing either mutant A53T alpha-syn or green fluorescent protein (GFP) into the SN (4 rAAV-alpha-syn, 4 rAAV-GFP, 5 non-injected control minipigs). We performed motor symptom assessment and immunohistochemical examination of tyrosine hydroxylase (TH) and transgene expression. Expression of GFP and alpha-syn was observed at the SN injection site and in the striatum. We observed no motor symptoms or changes in striatal [C-11]DTBZ binding potential in vivo or striatal or SN TH staining in vitro between the groups. The mean [C-11](R)PK11195 total volume of distribution was significantly higher in the basal ganglia and cortical areas of the alpha-syn group than the control animals. We conclude that mutant alpha-syn expression in the SN resulted in microglial activation in multiple sub- and cortical regions, while it did not affect TH stains or VMAT2 availability. Our data suggest that microglial activation constitutes an early response to accumulation of alpha-syn in the absence of dopamine neuron degeneration.

Our two laboratories have performed PET scannings and neurosurgery using Göttingen minipigs for more than 20 years. While PET scans can be performed with only minimal effects on animal welfare, neurosurgery is mostly a welfare challenge. We would like to present data and manuals, using neurosurgical modalities on Göttingen minipigs: How to improve animal welfare to minimize the risk of euthanasia. Our two laboratories have performed PET scannings and neurosurgery using Göttingen minipigs for more than 20 years. While PET scans can be performed with only minimal effects on animal welfare, neurosurgery is mostly a welfare challenge. We would like to present data and manuals, using neurosurgical modalities on Göttingen minipigs: How to improve animal welfare to minimize the risk of euthanasia.

BACKGROUND: Stereotaxic neurosurgery in large animals is used widely in different sophisticated models, where precision is becoming more crucial as desired anatomical target regions are becoming smaller. Individually calculated coordinates are necessary in large animal models with cortical and subcortical anatomical differences.NEW METHOD: We present a convenient method to make an MRI-visible skull fiducial for 3D MRI-based stereotaxic procedures in larger experimental animals. Plastic screws were filled with either copper-sulphate solution or MRI-visible paste from a commercially available cranial head marker. The screw fiducials were inserted in the animal skulls and T1 weighted MRI was performed allowing identification of the inserted skull marker.RESULTS: Both types of fiducial markers were clearly visible on the MRÍs. This allows high precision in the stereotaxic space.COMPARISON WITH EXISTING METHOD: The use of skull bone based fiducial markers gives high precision for both targeting and evaluation of stereotaxic systems. There are no metal artifacts and the fiducial is easily removed after surgery.CONCLUSION: The fiducial marker can be used as a very precise reference point, either for direct targeting or in evaluation of other stereotaxic systems.

Background:Development of translational models for understanding and treating Parkinson’s disease (PD) are important steps towards clinical applications.The Göttingen minipig (GM) fits progressional neurological models due to a relatively low adult-weight between 20-40 kg, and a large gyrencephalic brain (6x5x4 cm) that can be examined at sufficient resolution using conventional clinical scanning modalities and neuromodulation including preclinical testing of deep brain stimulation and induced pluripotent stem cell (iPSC) grafting.Aim:Using inoculation of human or pig alpha-synuclein (aSYN) fibrils, overexpressing aSYN using Lentivirus (LV) and Adeno Assosiated Virus (AAV) vectors or proteasome inhibition in the nigrostriatal system, we hope to create a new porcine models for PD.Methods:Using conventional human-intended stereotaxic neurosurgery methods, we apply aSYN or preformed fibrils in the catecholamine nigrostriatal system of the GM.The changes are quantified by neurological tests (behavior, scoring and gait), preclinical PET, post mortem analysis of histology and autoradiography.Results:Results from LV methods show "proof of concept" on gait, surgery and histology. AAV models show decrease in activity and velocity over time. PET-imaging of the dopamine system displayed decrease in both AAV and proteasome inhibition models. Discussion:We predict that these models will be beneficial in understanding the pathological mechanisms of human PD, novel therapeutic strategies such as antiaggregant treatment, iPSC, immunotherapy and development of novel radioligands for early diagnosis and assessment of disease progression

Background:Parkinson's disease (PD) animal models are important translational steps toward clinical applications. The Göttingen minipig(GM) has a large gyrencephalic brain (6x5x4cm) that can be examined using conventional scanning modalities. Preclinical neuromodulatory devices can be evaluated in GM models. aSYN is a known pathological protein in PD. Aggregation or overexpression occurs in human PD and has been used to induce PD in other animal models.Aim:We aim to develop and validate novel models of PD in the GM based on abnormal protein aggregation.Methods:Using human intended MRI-guided stereotaxic neurosurgery, we are inducing a PD-syndrome in GM via overexpression of mutated human aSYN using viral vectors or intracerebroventricular administration of an ubiquitin proteasome system inhibitor.The resulting pathological changes are quantified by neurological tests, PET-imaging of monaminergic function and neuroinflammation, HPLC and post mortem histological evaluation for aSYN and neuroinflammatory markers.Results:Data from ongoing studies with the different models demonstrate gait disturbances, bradykinesia, deficits in monoaminergic neurotransmission, alterations in the aSYN levels in CNS and histological signs of PD including aSYN accumulation. Discussion:We predict that these animal models will be beneficial in the understanding of pathological mechanisms of human PD and in the testing of novel therapeutic strategies.

Objectives:Some of the debilitating non-motor aspects of Parkinson’s disease (PD) are related to the serotonin system1. To investigate the involvement of the brain serotonergic system in a PD animal model, we measured the in vivo binding of [11C]-DASB to the serotonin transporter (SERT) as a marker of serotonergic neurons. In this study, we use the in vivo capabilities of PET imaging to study serotonin neurotransmission in a minipig model of PD induced by the intracerebroventricular injection of lactacystin, an inhibitor of the ubiquitin proteasome system.Methods: Five female Göttingen minipigs were implanted in the cisterna magna with a catheter connected to a subcutaneous titanium injection port under sterile conditions. Six-eight weeks after recovery from the catheter implant, and after injections of sterile saline alone to verify patency, minipigs were scanned at baseline with [11C]-3-amino-4-(2-dimethylaminomethyl-phenylsulfanyl)-benzonitrile (11C-DASB), a label of SERT availability. Four pigs then received eight weekly injections of lactacystin dissolved in sterile saline, and one pig received saline alone, directly into the CSF through the access port. They were scanned with DASB again after a cumulative dose of 200μg lactacystin. PET data were registered to an average minipig MRI atlas and processed using PMOD software. The binding potential (BPND) of DASB was obtained with the Logan graphical analysis and cerebellum activity as a region of non-displaceable binding.Results:Lactacystin administration induced behavioural symptoms including weakness of hindlimbs and decreased motor activity. SERT binding potential was decreased by 35-40% in striatal brain regions and by 20% in thalamic regions compared to the baseline scans. Conclusions: Our imaging data suggests a loss of brain serotonergic innervation in response to protein aggregation. The decreased striatal binding of DASB observed in this minipig model of PD is to some extend consistent with previous studies done in PD patients2. The proteasome inhibition model may therefore be useful in the investigation of non-motor deficits in PD and LDOPA-induced dyskinesia.

Parkinson disease (PD) is a common neurodegenerative disorder, resulting from a progressive dopaminergic neuron loss in the substantia nigra (SN). Alpha-synuclein positive neuronal inclusion bodies and progressive loss of dopaminergic striatal terminals is also well described in PD. Attempts to discover effective compounds halting PD progression have so far failed in clinical trials, perhaps because current animal models do not imitate the neuropathological progression of PD well enough. We recently established a progressive large animal PD model in Göttingen minipigs based on chronic infusion of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and here present the neuropatholigical findings.Sixteen Göttingen minipigs were used in the study. Twelve were implanted subcutaneously with infusion pumps for continuous intramuscular chronic MPTP delivery for 11 weeks (4-18 mg MPTP/day, n=12) or acute MPTP intoxication for 11 days (24 mg MPTP/day, n=2) and 9 weeks of recovery. Four pigs served as normal controls. Animals were euthanized with intracardial pentobarbital injections, transcardially perfused with 5 L 4% paraformaldehyde and the brains removed. The striatae and brain stems including the SN were paraffin embedded and immunohistochemically stained for tyrosine hydroxylase (TH) and alpha-synuclein.Stereological examination of the SN showed progressive nigral neuron loss with increased MPTP dosages. Occasional neuronal staining confined to the cytoplasm and cell membrane was observed in the SN but no neuronal inclusions. In the striatum, alpha-synuclein positive staining occurred in the acutely intoxicated animals and increased in intensity with increasing doses of chronic MPTP infusion. The 12 and 18 mg animals showed swollen TH-positive terminals in the striatum with a reduction in the number of fibers and terminals in the 18 mg group. Some sense of direction of fibers was preserved in these groups, but this was completely lost in the 24 mg animals, which additionally presented with smaller, fewer terminals. In the locus coeruleus, the 18 mg animals showed decreased TH fiber staining and increased intensity in the neuronal staining.We conclude that chronic infusion of MPTP in this large animal PD model shows neuropathological changes resembling those seen in the clinical state of PD. Animals receiving 18 mg MPTP/day for 11 weeks thus showed a decrease in nigral TH-positive neuron number and striatal terminals, intense striatal alpha-synuclein staining and pathology in the locus coeruleus. Use of this model thus holds promise for future neuroprotective studies in PD.