To our knowledge, this is the first study to characterize in vivo effects in the heart of systemic 6-OHDA to rhesus monkeys, and the first to track experimental cardiac denervation with MHED PET. The main findings include a nonuniform pattern of MHED uptake in the left myocardium, as well as decrease in circulating catecholamines, suggesting that this model mimics cardiac dysautonomia in PD.
The most common nonhuman primate model of PD involves the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) 
. Systemic dosing of MPTP produces a severe parkinsonian motor syndrome; however, effects in sympathetic denervation are temporary, as observed with 6-[18
F]fluorodopamine PET imaging 
. The other frequently used PD monkey model depends on direct intracerebral delivery of 6-OHDA, in order to bypass the blood–brain barrier and affect the nigrostriatal pathway without inducing peripheral effects 
. Our method of intravenous delivery takes advantage of 6-OHDA catecholaminergic toxicity to develop a cardiac dysautonomia model.
The only previous report of systemic administration of 6-OHDA to monkeys was done in a single rhesus to compare the systemic effect of 6-OHDA to MPTP 
. Yet, the authors did not describe the 6-OHDA method of administration and only provided a 6-[18
F]fluorodopamine PET scan image and catecholamine levels 1 week after intoxication. Instead, we based our dosing regime on the reports of 6-OHDA administration to dogs, which described neurotoxin delivery over several hours to avoid clinical complications associated to the dopaminomimetic effects of the neurotoxin 
. Intravenous administration of 6-OHDA proved to require intensive monitoring and veterinary care to avoid hypertensive crisis and pulmonary edema. The adjustment of the dosing regimen to the individual animal response decreased the intensity of the symptoms and the time period needed for their normalization and ensured a safe recovery after the procedure.
Reduction in FS after 6-OHDA administration suggests an abnormality in cardiac contractility. These reductions were mild; even the lowest values were within published normal reference ranges 
, and were not associated at the time with changes in wall thickness. It should be noted that normal ranges of echocardiogram parameters are not well established for macaque monkeys and the limited data are based on single recordings with different types of anesthesia. Because FS measurements are not well established for monkeys, we evaluated the percent change of FS over time for each individual animal. Three animals in this study had recurring declines in the change of FS following 6-OHDA, suggesting that the loss of noradrenergic cardiac innervation may produce reductions in left ventricular systolic function. The animal with the greatest decrease in FS (R04094) uniquely had increased troponin I levels 1 week after toxin suggesting injury to the myocardium. This was coupled with lack of recovery in the LV MHED uptake from 1 week to 4 weeks, perhaps demonstrating a more severe hypertensive response to 6-OHDA. The other animals did not exhibit elevated cardiac troponin I levels and had no or less of an initial decrease in FS accompanied by greater increases in total MHED uptake.
Noninvasive PET imaging of cardiac sympathetic innervation allowed us to monitor over time the cardiac tissue response to 6-OHDA challenge. We used equilibrium DV relative to whole blood to analyze uptake of MHED, because the uptake index conventionally used 
declined rapidly as progressively later time windows were used for estimation. This behavior, and the success of an analysis method that assumes reversibility of the radioligand uptake, are at least partly due to progressive metabolism of MHED into compounds with no specific affinity to noradrenergic terminals. If blood metabolite analysis is not in the protocol, the DV estimates represent the most reproducible and least arbitrary way to use all the measured data to estimate a single number representing the uptake process. The use of DV – 1 to represent the innervation-specific part of the uptake is based on the observation that lesioning reduced the DV values to ~1 in all regions in all animals.
Systemic administration of 6-OHDA induced loss of catecholaminergic innervation of the heart (greater loss in inferior LV myocardium), which persisted 3 months after toxin challenge. Although nerve terminals in all regions of the LV reacted similarly to 6-OHDA at 1 week, regional rates of recovery varied over time, with an average 51% remaining deficit after 3 months. The anterior region consistently recovered most rapidly and the inferior wall most slowly. Our findings resemble those in PD: MHED PET established loss in sympathetic myocardial innervation 
F]fluorodopamine imaging suggested preservation in septal 
and anterior 
walls of the LV. The nonuniform pattern of MHED uptake may be caused by regional differences in blood perfusion that affected the distribution or metabolism of the neurotoxin. Another possibility is that the subpopulation of ganglionic cells innervating the area has a greater sensitivity to 6-OHDA. It could be argued that the regional recovery was due to re-growth of cardiac muscle tissue, instead of reinnervation. Yet, results from the echocardiogram showed no significant differences in posterior or anterior wall thickness in either diastole or systole. Bai and colleagues 
have reported recovery of cardiac sympathetic nerves following subcutaneous delivery of 6-OHDA insult in rats. In addition, sympathetic reinnervation has been described after heart transplantation, suggesting that the system has a certain plasticity that could be exploited for regenerative or neuroprotective treatments 
. Increasing animal numbers would help further define regional differences in LV MHED uptake. Further investigation of the mechanisms of regional loss is needed, as it may facilitate the identification of possible therapeutic targets.
The timeline of our experimental design was based on our previous experience with administration of neurotoxins in the central nervous system of nonhuman primates. For example, an observation period of 3 months after MPTP challenge allows for the dopaminergic nigral cell neurodegeneration to be completed and defines a stable syndrome 
. Lack of significant changes in MHED uptake between 10 and 14 weeks suggests that as predicted, by 3 months the catecholaminergic lesion was stabilized and recovery mechanisms were completed. The characterization of the timeline for recovery and stabilization of the lesion in this model will be helpful when designing a study to test disease-modifying strategies for the heart. Follow-up experiments with endpoints exceeding 14 weeks after toxin administration would further confirm that 6-OHDA produces a stable lesion in the LV.
Dopamine, epinephrine, norepinephrine, and DOPAC circulating levels were significantly decreased after 6-OHDA, which indicates that the neurotoxin affected peripheral catecholaminergic sources such as adrenal medulla, facilitating onset of cardiac dysautonomia. The positive correlation found between catecholamine levels and cardiac MHED uptake further suggests a similar toxic effect of 6-OHDA in different peripheral catecholaminergic cells. The drop in circulating catecholamines did not seem to affect animal health, probably because of adaptive sympathetic presynaptic supersensitivity 
. Studies in dogs did not find changes in catecholamine levels, suggesting a species difference in the sensitivity to neurotoxin effects 
Cardiac dysautonomia in PD patients is clinically characterized by orthostatic hypotension, an increase in corrected QT intervals (QTc), and reductions in heart rate variability 
. The presence of these symptoms was not confirmed in this study, because they are detected in an awake state and the cardiac and blood pressure evaluations were performed under anesthesia. Nonuniform cardiac innervation, affects cardiac repolarization and has been associated with arrhythmias 
. Future preclinical experiments using telemetric measurements of heart rate, blood pressure, and locomotive activity 
will allow their identification in awake animals. Injections of vasoactive pharmaceuticals such as phenylephrine or sodium nitroprusside may also help characterize the cardiovascular response.
The monkeys presented abnormal feces and weight loss that became significant 4 weeks after 6-OHDA. The frequency of loose stools and diarrhea correlated with the amount of weight loss. Supplementation of feedings with chow soaked in protein-enriched drink (standard practice in our facility for animals loosing weight) enticed feeding, increased fluid intake and helped prevent further weight loss. The abnormal feces could have been the result from stress (each animal underwent procedures at least monthly), but their presence were also described in 6-OHDA-treated dogs that did not undergo those evaluations, suggesting an effect of 6-OHDA in the gastrointestinal tract 
. The enteric nervous system consists of dopaminergic neurons (myenteric and submucosal plexus), which are potentially susceptible to the toxic effects of systemic 6-OHDA 
. In that regard, rats treated with 6-OHDA have decreased TH mRNA levels in the duodenum 
. Reduced sympathetic innervation of the intestinal tract can lead to bowel dysmotility and, therefore, abnormal feces.
Collectively, the changes in cardiac innervation, catecholamine levels and feces suggest that systemic dosing of 6-OHDA affects multiple vulnerable catecholaminergic peripheral cells, and this property can be applied to model dysautonomias in nonhuman primates. Similar to CNS neurotoxic models, a 6-OHDA-induced dysautonomia model presents limitations like acute onset and the risk for spontaneous recovery 
. Intravenous administration of 6-OHDA does not induce a PD motor syndrome, therefore a comprehensive PD model would require supplementation with systemic MPTP or direct intracerebral dosing of 6-OHDA to induce dopaminergic nigral cell loss. Postmortem analysis and quantification of catecholaminergic cell and terminal loss in susceptible tissues, as well as evaluation of pathologies typical of PD–related neurodegeneration, such as inflammatory cell response and intracytoplasmic accumulation of alpha synuclein 
, are warranted to further characterize the effect of 6-OHDA in peripheral catecholaminergic cells.
To conclude, systemic administration of 6-OHDA to rhesus monkeys mimics features of cardiac dysautonomia in PD that can be tracked and mapped in vivo using PET imaging. We hope that these results will facilitate model development to study this symptom and to identify new therapeutic alternatives.