Nonuniform Cardiac Denervation Observed by 11C-meta-Hydroxyephedrine PET in 6-OHDA-Treated Monkeys

Parkinson's disease presents nonmotor complications such as autonomic dysfunction that do not respond to traditional anti-parkinsonian therapies. The lack of established preclinical monkey models of Parkinson's disease with cardiac dysfunction hampers development and testing of new treatments to alleviate or prevent this feature. This study aimed to assess the feasibility of developing a model of cardiac dysautonomia in nonhuman primates and preclinical evaluations tools. Five rhesus monkeys received intravenous injections of 6-hydroxydopamine (total dose: 50 mg/kg). The animals were evaluated before and after with a battery of tests, including positron emission tomography with the norepinephrine analog 11C-meta-hydroxyephedrine. Imaging 1 week after neurotoxin treatment revealed nearly complete loss of specific radioligand uptake. Partial progressive recovery of cardiac uptake found between 1 and 10 weeks remained stable between 10 and 14 weeks. In all five animals, examination of the pattern of uptake (using Logan plot analysis to create distribution volume maps) revealed a persistent region-specific significant loss in the inferior wall of the left ventricle at 10 (P<0.001) and 14 weeks (P<0.01) relative to the anterior wall. Blood levels of dopamine, norepinephrine (P<0.05), epinephrine, and 3,4-dihydroxyphenylacetic acid (P<0.01) were notably decreased after 6-hydroxydopamine at all time points. These results demonstrate that systemic injection of 6-hydroxydopamine in nonhuman primates creates a nonuniform but reproducible pattern of cardiac denervation as well as a persistent loss of circulating catecholamines, supporting the use of this method to further develop a monkey model of cardiac dysautonomia

Systolic and diastolic blood pressure measurements during 6-OHDA administration.

<p>Blood pressure results from a representative animal (RH2318). A, Both pressures increase immediately after each 6-OHDA dosing (arrows). B, Change in systolic and diastolic blood pressure from peak of pressure following 6-OHDA until levels normalized at which time the next injection of 6-OHDA was administered. The amount of time to normalize paralleled the change in blood pressure. At the accumulated dose of 2.0 mg/kg, this animal required 61 minutes to regulate blood pressure. bp, blood pressure.</p

Circulating plasma catecholamine levels (pg/mL) at baseline (0) and at 1, 4, 10, and 14 weeks after 6-OHDA in three animals.

<p>The values in parentheses are the natural logarithms of the concentration values and used to graph correlations in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035371#pone-0035371-g005" target="_blank">Figure 5</a>. For statistical analysis, non-detectable levels (nd) levels were considered as the lowest detection level with 1.5 pg/mL for norepinephrine and 3.0 pg/mL for epinephrine. Post hoc analysis with Bonferroni multiple comparisons detected significance at all timepoints compared to baseline for norepinephrine and DOPAC (*<i>P</i><0.05, ** <i>P</i><0.01).</p

Distribution volume maps before and after systemic 6-OHDA dosing.

<p>Maps express regional capacity of the tissue for MHED uptake relative to whole blood, thus providing a measure of the density of nerve terminals. Each individual map consists of 8 sectors and 7 rings (apex of the heart at the center, base of the LV at the edge), totaling to 56 blocks of data per timepoint. MHED uptake significantly decreased at each timepoint after 6-OHDA compared to baseline (<i>P</i><0.001). Scales (mL whole blood/g tissue) are similar between animals and identical across time points for each individual. The compass rose indicates regions: A, anterior; S, septal; I, inferior; L, lateral.</p

Echocardiogram measurements at baseline, 10 and 14 weeks after 6-OHDA for each animal.

<p>FS is calculated as [(LVD_d – LVD_s)/LVD_d]×100. Three animals experienced large decreases in FS compared to baseline, but there was no significant change in left ventricle diameter or in anterior and posterior wall thickness. This suggests that the increased luminal dimensions of the left ventricle were not due to the loss of cardiac muscle. AWd, anterior wall thickness in diastole. AWs, anterior wall thickness in systole. PWd, posterior wall thickness in diastole. PWs, posterior wall thickness in systole. LVD_d, left ventricle diameter in diastole. LVD_s, left ventricle diameter in systole. FS, fractional shortening.</p

Weekly animal weights (kg) and average group % weight loss before and after 6-OHDA dosing.

<p>A significant loss of weight was found starting at four weeks post 6-OHDA challenge and persisting throughout the study.</p>*<p><i>P</i><0.05,</p>**<p><i>P</i><0.01.</p

Electrocardiogram (ECG) measurements at baseline, 1, 4, 10 and 14 weeks after 6-OHDA for each animal.

<p>QTc was calculated using the Bazett's formula as QT interval/sqrt (RR interval), where the RR interval was calculated 60/HR. ΔQTc was calculated as QTc of each time point minus baseline. No significant differences were found in PR, QRS and QTc intervals obtained in anesthetized animals. HR, heart rate. QTc, corrected QT interval.</p

Experimental timeline.

<p>All procedures were performed in 5 adult rhesus monkeys following a within-subject experimental design. MHED PET scans, plasma catecholamines, troponin I and ECGs (*) were performed at baseline and 1, 4, 10 and 14 weeks after 6-OHDA. Echocardiograms (Echo) were obtained at baseline and 10 and 14 weeks after 6-OHDA. Clinical rating (CR) was performed at baseline and 4, 10 and 14 weeks following toxin administration. Food consumption and feces were monitored daily and body weight was measured weekly throughout the course of the study (shading). Animals were euthanized 14 weeks following 6-OHDA administration.</p

6-OHDA dosing scheme (mg/kg) for each individual animal.

<p>The rhesus monkeys received 7–9 injections of 6-OHDA solution in one session, accumulating to a total final dose of 50 mg/kg. Dosing and timeline of injections varied based on the individual reaction to 6-OHDA. The normalization (return to baseline measures) of vital signs, ECG, heart rate, and blood pressure was used to define when the next dose could be given.</p

Quantitative analysis of MHED PET.

<p>A, Group mean global MHED % retention deficit comparing 1, 4, 10, and 14 weeks after toxin to baseline. B, Group mean regional MHED uptake comparing anterior, inferior, septal, and lateral regions of the left ventricle at baseline and at 1, 4, 10, and 14 weeks after toxin. *<i>P</i><0.05, ** <i>P</i><0.01, *** <i>P</i><0.001.</p