PhMRI Monitoring Of GDNF Treatment And Development Of A Early Stage Model Of PD

Parkinson's disease(PD)is a neurodegenerative disorder with classical symptoms of tremor,rigidity and akinesia,Its pathological symptoms are the loss of dopaminergic neuron in substantia nigra of midbrain,the decrease of dopaminergic neuron in corpus striatum and the appearance of Lewy body in substantia nigra and locus ceruleus.According to the data from the world,the morbidity of PD is about 10-405/100000 steadily,it is 44/100000 in our country which is low.But the morbidity of PD in our country is similar to developed countries according to recent surveys. The loss and degeneration of dopaminergic neuron in substantia nigra lead to the functional disorder of corpus striatum and the dysfunction of cortex-basal ganglia-thalamus circle which is the direct reason of PD.PD was described firstly by Jame Parkinson who was a doctor in England in 1817.The cause of disease isn't clear until now.These are two kinds of treatments:internal medicine mainly aim to retrieve the disorder of transmitter metabolism,and the surgery mainly on the changing of neuro-circle.Current pharmacological and surgical treatments for PD are palliative, ameliorating the classical symptoms of tremor,rigidity and akinesia,but not altering underlying disease processes.The treatments greatly changed as the fundament research and the development of PD model.A great number of neurotrophic factors,especially GDNF are always the hot topic of research.We mainly did two aspect works according to the PD and the treatment of GDNF:(1)the animal study about the treatment of GDNF used phMRI;(2)the research of early PD model induced by micro-dose MPTP.(1)Glial cell line-derived neurotrophic factor(GDNF)is the most known neurotrophic factor in the PD treatment,and GDNF shows promise for the treatment of PD in animal experiments.GDNF not only shows good neurotrophic effect on dopaminergic neurons,but also protects and restores the nigrostriatal dopaminergic system.This trophic factor has also shown promise in PhaseⅠclinical trials for the treatment of PD.Ample evidence has proven that GDNF can protect and promote survival of pre-synaptic dopaminergic neurons in the SNc and axons in the striatum.GDNF can protect the dopaminergic neurons in midbrain greatly.Thus it may be the mainly drugs to cure PD.Clinical trials and pre-clinical trials suggested that GDNF could be effective by targeting administration.There were still many problems to be solved in the study of GDNF' treatment of PD.Such as cure time,the way of administration,dose and methods of detection.Here,we report the monitoring of GDNF-induced functional changes of the basal ganglia in hemiparkinsonian monkeys via pharmacological MRI measuring the blood oxygenation level-dependent (BOLD)response to a direct dopamine agonist(apomorphine,APO).After testing BOLD reponsiveness to APO in their normal state,two additional scans were taken with the same dose of APO stimulation after induced parkinsonism.Then,all animals were chronically treated with GDNF for 18 weeks by a programmable pump and catheter system.The catheter was surgically implanted into the right putamen and connected to the pump via flexible polyurethane tubing,phMRI scans were taken at both 6 and 18weeks while they received 22.5Ig of GDNF per day.In addition, behavioral changes were monitored throughout the entire study.The primary finding of this study was that APO-evoked activations in the DA denervated putamen were attenuated by the chronic intraputamenal infusion of GDNF accompanied by improvements of parkinsonian features,movement speed and APO-induced rotation compared to data collected before the chronic GDNF treatment.The results suggest that phMRI methods in combination with administration of a selective DA agonist may be useful for monitoring neurorestorative therapies in PD patients in the future.Methods The animals had a catheter implanted in the right putamen using magnetic resonance imaging-guided stereotactic procedures.The catheter was connected via flexible polyurethane tubing to a programmable pump,which could adjust the dose,velocity and time.At the same time,we test the treatment effect of GDNF on the unilateral PD model induced by MPTP by pharmacological MRI measuring the blood oxygenation level-dependent(BOLD)response to a direct dopamine agonist (apomorphine,APO).This is a noninvasive,highly sensitive and widely available imaging methods which can potentially be used to longitudinally monitor treatment of Parkinson's disease(PD).RESULTS All animals expressed stable,unilateral parkinsonian features from the eighth week post-MPTP administration,including bradykinesia,rigidity of the left upper and lower limbs,stooped posture,and balance instabilities.These parkinsonian features were significantly improved except for posture by 0.1 mg/kg APO treatment (Fig.3,filled columns).Additionally,APO-induced counterclockwise rotational behavior(turning toward MPTP lesioned side)was observed in all animals.No complications were encountered during catheter implantation into the putamen in any of the animals.Also,no adverse effects were observed following the surgery or during chronic GDNF treatment.While a steady improvement was evident post-GDNF delivery,there was an average of 22%improvement in the motor functions as per the nonhuman primate parkinsonian rating scale by the sixth week of the treatment(Fig. 4A),which was maintained up to 18 weeks.The improvements in motor function were also evident from increased movement speed(Fig.4B).The improvements seemed to parallel the parkinsonian rating scale.Furthermore,GDNF significantly reduced APO-induced rotations(Fig.4C)by 87%at the sixth week.A similar reduction was also seen at the 18th week posttreatment.While little changes were found in the putamen and caudate nucleus prior to MPTP treatment(Fig.5A),strong APO-induced activations were seen in the same regions on the ipsilateral side of MPTP administration(Fig.5B,Fig.7A).As predicted,chronic intraputamenal infusion of GDNF not only significantly improved motor function but also reduced the APO-induced activation in the putamen on the ipsilateral side to GDNF infusion at the sixth and 18^thweek compared to the data collected prior to GDNF treatment.The BOLD activations were significantly reduced at both 6 and 18 weeks after the treatment,falling much closer to the baseline levels in DA denervated putamen(Fig. 6A).However,GDNF-induced functional changes in the caudate nucleus seemed to be biphasic(i.e.,APO-induced activation was slightly higher at the sixth week and significantly decreased at the 18th week posttreatment)(Fig.6B)).There was little change in BOLD signal in the SN(data not shown).Typical changes of BOLD signal in response to GDNF treatment are shown in Figure 7(monkey #445).The BOLD effect was superimposed on a T1-weighted image from the same monkey collected on the same day.The size of APO-evoked activation was significantly reduced compared with data collected before chronic GDNF treatment.Postmortem analysis showed the radius of diffusion from the catheter.Positive GDNF staining was observed in most of the treated putamen(Fig.8).The spread of GDNF along the catheter track was seen in all monkeys with a diffusion radius of at least 3 mm at the tip of the catheter.CONCLUSION:The results suggest that phMRI methods in combination with administration of a selective DA agonist may be useful for monitoring neurorestorative therapies in PD patients.This study would determine whether the amplitude of activation induced by APO could be attenuated continuesly by the treatment of GDNF in the putamen damaged by MPTP in details.(2)The treatment strategy of PD is changing from palliative cure which has serious complication and side effects to new neuroprotective/restorative treatment which can delay the disease.As the best treated period of PD,how to cure and estimate the therapeutic efficacy earlyier were considered as a very important topic in the new neuroprotective/restorative treatment.Therefore,a nonhuman primate model of the earlier stages of PD would be useful for assessing the benefits and risks of early intervention.This earlier stages of PD model should meet some conditions as follows:1.This animal model should mimic pathological and biochemical changes in the early stages of idiopathic PD.2.This animal model should mimic the early stages of idiopathic PD without spontaneous recovery.3.This model can mimic the reaction to the treatment of PD patients.Iin our study,we want to develop a steable nonhuman primate model of the earlier stages of PD by controlling the dose of MPTP,the way of administration and the age of animals.Methods We used 27 midage monkeys(16-19 years old)to develop the nonhuman primate model of the earlier stages of PD by injecting low dose of MPTP(0.12mg/kg). All animals showed stable parkinsonian features throughout the 12-month behavioral evaluation.Compared with late-stage PD animals,postmortem analysis demonstrated that more dopaminergic neurons remained in the substantia nigra compacta,and more fibers were found in the striatum.RESULTS Unilateral parkinsonian features emerged by two weeks and were stable by six-eight weeks after MPTP treatment.The movement dysfunctions were continuously expressed throughout the study without evidence of either behavioral recovery or progressive functional decline(Figure 1A close bars).None of the animals required an additional dose of MPTP to maintain their level of parkinsonism. The parkinsonian features expressed included bradykinesia and rigidity of upper and lower limbs on the contralateral side of MPTP administration(Figure 1B,open bars). In addition,stooped posture and mild postural instability were evident in all animals. Severelylesioned rhesus monkeys can express an action tremor.This feature was either absent or rarely expressed in the monkeys in this study.As shown in figure 1A, the total(cumulative)parkinsonian score of the early-stage animals was 5.3±0.15 points(close bars)compared to a significantly more severe score of 8.3±0.45(the open bar)for the late-stage parkinsonian animals.The PD scores were consistent with the results collected using an automated video-tracking system showing the stability of the model over 12 months(Figure 2 A&B).Both distance traveled and movement speed were reduced by～44%by one month post MPTP treatment and remained lower over the twelve month test period.In addition,the parkinsonian features were levodopa responsive,with significant improvements measured following levodopa administration five months after the MPTP lesion(Figure 1B,close bars).In addition, rotational behavior was observed in 22 out of 27 animals.The average total number of turns over 60 min was 34.5±9.6.Furthermore,all animals showed a significant increase of locomotor activity assessed by distance traveled and movement speed in response to levodopa challenge(Figure 2A&B,right hatched bar).In accordance with the other effects on the nigrostriatal DA system,significant declines in DA and DA metabolite levels were found using both MPTP dose regimens.Normal values are shown in Table 1 for comparisons.In the early-stage model,tissue DA levels in the putamen were decreased by 98%,while DOPAC and HVA levels were reduced by～95%and 83%,respectively(Table 1).In the animals modeling late-stage PD,DA, DOPAC and HVA levels were reduced by up to 99%in the putamen(Table 1). Consistent with a milder lesion,HVA levels measured in the early-stage model were significantly greater than those found in the late-stage model(P＜0.05,Table 1).The HVA/DA ratios were significantly larger in the putamen of early-and late-stage animals versus the normal putamen.In addition,the DOPAC/DA ratios were significantly larger in the early-stage model versus normal putamen,but the DOPAC/DA ratios in the putamen of the late stage animals,while larger,did not reach statistical significance.In the animals with early-stage PD,no significant differences were seen between the caudate nucleus and putamen in DA and DOPAC levels(see Figure 7A).However,levels of HVA were statistically higher in the putamen than in the caudate nucleus(Figure 7A).In the animals with late-stage PD, no significant differences were seen between the putamen and caudate nucleus. Although the levels of DA and DOPAC were lower in the putamen than in the caudate nucleus,they did not reach significance due to a large variance in the caudate nucleus (Figure 7B).Conclusion:Our results suggest that a milder but stable PD model can be produced in middle-aged rhesus monkeys by unilateral injecting the low dose of MPTP(0.12mg/kg).