Our study discusses the value of various parameters in 123I-MIBG scintigraphy for the diagnosis and differential diagnosis of PD. Overall, the 15 min and 4 h H/M ratios, as well as the cardiac WR, demonstrate high diagnostic efficacy in both diagnosis and differential diagnosis. When evaluating organs with high sympathetic nervous activity other than the heart, traditional ratio methods may be less effective. The WR of the parotid and thyroid glands, as a new diagnostic parameter, may become a potential method for assessing peripheral sympathetic nerve reduction, especially in patients with concomitant organic heart disease.

The H/M ratio is one of the earliest widely used parameters, initially applied for evaluating heart failure and other cardiovascular diseases [19]. The H/M ratios obtained from 15 min to 4 h of 123I-MIBG scintigraphy were used to assess early and ongoing sympathetic nerve damage in the heart. Our study confirms that PD patients show significantly reduced MIBG uptake in both early and delayed scintigraphy compared to non-PD patients, demonstrating high diagnostic efficacy for PD. This finding is consistent with previous studies [15]. Additionally, the 4 h H/M ratio shows better diagnostic performance than that of 15 min, with higher sensitivity and specificity. One possible reason is that the ratio of 15 min reflects the distribution density of sympathetic nerves, while the ratio of 4 h provides comprehensive information about the uptake, storage, and release functions of neurons. Moreover, because the ratio of 4 h is less dependent on passive MIBG transfer pathways, it can more accurately reflect the condition of cardiac sympathetic nerve damage [29].

In the differential diagnosis of PD from MSA or PSP, we obtained results similar to previous studies. Our research shows that PD patients exhibit significantly reduced MIBG uptake compared to MSA or PSP patients. Furthermore, the ratio of 4 h scintigraphy also demonstrated better diagnostic performance in differential diagnosis [30,31,32,33]. It is noteworthy that there is some controversy regarding this aspect of the research. Since the pathology of MSA occurs in the pre-ganglionic sympathetic nerve fibers, despite the severe clinical manifestations of autonomic dysfunction in MSA patients, significant reductions in cardiac MIBG uptake are usually not observed. Some studies have noted that certain MSA and PSP patients may exhibit reduced MIBG uptake, and early H/M ratios may not reliably distinguish PD from PPS [32]. An autopsy-based pathological study confirmed that the cardiac sympathetic nerves of some MSA patients with slight reductions in MIBG uptake might undergo mild degeneration [34]. Additionally, some research suggests that the mild reduction in MIBG uptake in PSP patients may be related to brainstem atrophy [35]. In one autopsy-confirmed PSP patient, MIBG uptake was reduced, but pathological results showed that cardiac sympathetic nerves were intact [36]. Thus, although some patients may exhibit decreased H/M ratios, our study indicates that MIBG imaging still holds significant diagnostic value in differentiating PD. However, when clinical presentations are inconsistent, other causes of false positives should also be considered. CBD, a rare disease among Parkinson-plus syndromes, usually does not manifest autonomic dysfunction. Among the three CBD patients included in our study, no significant reduction in sympathetic nerve uptake was observed. However, due to the small number of cases, we did not perform a statistical analysis. In future studies, it will be necessary to increase the sample size of these patients and conduct statistical analyses and further investigations.

In our study, PD patients exhibited a significant increase in cardiac WR. Currently, there is insufficient basic research on elevated cardiac WR in PD patients. It is generally believed that this reflects a reduced capacity of sympathetic nerve terminals to retain norepinephrine [37], possibly related to vesicular storage defects, indicating early functional abnormalities of cardiac sympathetic nerves [38]. The increase in cardiac WR might serve as a marker indicating enhanced exocytosis mediated by sympathetic nerves [39]. Additionally, some Lewy body disease patients with normal early H/M ratios also showed increased cardiac WR [40]. This underscores the importance and clinical value of studying cardiac WR. Our results show that, whether for diagnosing PD or differential diagnosis, cardiac WR demonstrates high diagnostic efficacy comparable to H/M ratios. Since cardiac sympathetic nerve dysfunction precedes denervation, theoretically, cardiac WR has higher sensitivity, which our study further confirms. Therefore, despite the floor effect of the radioisotope in some patients with severely reduced MIBG uptake [41], cardiac WR should be included in the assessment.

A pathology-verified study also emphasized the importance of WR, suggesting that incorporating the cardiac WR parameter into the evaluation process aids diagnosis, especially when there are differences between early and delayed H/M ratios [42]. In the differential diagnosis of PD versus MSA, our results indicate that cardiac WR, along with early or delayed H/M ratios, possesses high diagnostic efficacy. Previous studies have found that cardiac WR might be more helpful than early and delayed H/M ratios in differentiating PD from MSA, though without clear pathophysiological explanations [32]; we speculate this may be due to small sample sizes.

Due to a lack of standardization, the accuracy of cardiac WR is often affected. Two representative methods for calculating the washout rate have been applied in previous studies. Method 1 calculates the ratio between the values at 15 min and 4 h, which more closely aligns with the concept of "washout." Method 2, which accounts for time decay correction and background correction, is theoretically expected to yield more accurate results. To explore the advantages and disadvantages of different washout rate calculation methods in assessment, we performed calculations using both approaches. Studies have pointed out that in heart failure patients, cardiac WR calculations without background correction and time decay correction yield the most reliable results [18]. However, our results show that in PD diagnosis and differential diagnosis, methods considering time decay correction and background correction for assessing cardiac WR are superior. Based on our findings, we recommend using this method for evaluation.

Currently, there is limited research on MIBG uptake in other organs with high sympathetic nerve activity besides the heart, and the results are somewhat controversial. Considering the stability of ROI delineation and the fact that lung MIBG uptake is easily affected by medication, we selected major salivary glands, including the parotid gland, submandibular gland, and thyroid gland, as the main targets for evaluation [43]. Studies have shown that the accumulation of α-synuclein in the cervical-thoracic sympathetic ganglia, such as the stellate ganglion or upper thoracic sympathetic ganglia, might be the cause of extracardiac organ involvement and related pathological studies have confirmed the deposition of α-synuclein in the submandibular gland [16, 17, 44].

Our study shows that using salivary gland or thyroid gland ratio parameters for evaluation did not yield statistically significant differences between PD patients and the control group, regardless of early or late imaging. The results were similar in the differential diagnosis of PD versus MSA or PSP. This finding contrasts with some recent studies where salivary gland ratio parameters were used as biomarkers for PD diagnosis, showing significantly reduced ratios in PD groups, regardless of whether the mediastinum or forehead was chosen as the background [22, 23, 45]. Additionally, our study did not observe the reduction in thyroid gland ratio parameters in PD patients reported by other studies [46, 47]. Regarding the potential reasons for these observed differences, we hypothesize the following: first, many MIBG-related studies have been conducted in Japan, where population characteristics may differ from ours. Second, due to the limitations of planar acquisition in the head and neck region with SPECT/CT, the method of ROI delineation might contribute to some discrepancies. For example, Shuangfang Li et al. utilized tomographic imaging for the delineation and acquisition of parotid and submandibular glands [23].

It is important to note that our control group differed from those in the aforementioned studies; we included a substantial number of PPS patients and conducted differential diagnoses of PD versus MSA and PSP. This approach is closer to clinical application scenarios compared to using normal subjects as controls.

Excitingly, the situation changed when we used the WR parameter for evaluation. This is the first study to focus on the salivary gland and thyroid WR in PD patients, showing a significant increase in WR in the parotid gland and thyroid gland in PD patients. Our study confirms that the WR of the parotid and thyroid glands has certain diagnostic efficacy in PD, although it is lower than that of cardiac parameters. For the evaluation of WR, previous studies have primarily focused on cardiac WR. Although relevant basic research is limited, we hypothesize that the abnormal WR observed in the parotid and thyroid glands of PD patients may share a similar mechanism with the increased cardiac WR. This could be closely related to a common pathological basis (α-synuclein deposition in peripheral sympathetic organs). Our study further emphasizes the necessity of conducting in-depth analyses of these organs. Given the actual clinical scenario where many PD patients are of advanced age and often have concomitant organic heart disease, this can affect the accuracy of assessments based solely on cardiac parameters. Therefore, adding the evaluation of parotid and thyroid WR as an auxiliary measure is of great clinical significance, especially since this parameter can be obtained simultaneously with cardiac parameters without increasing the radiation dose to the examinee. The submandibular gland did not yield satisfactory results in this study, which we speculate might be due to overlapping artifacts from oral mucosa MIBG uptake in planar imaging, leading to ROI delineation errors, despite pathological evidence often being based on submandibular gland biopsy. The distribution of salivary glands and thyroid glands varies more among individuals compared to the heart, making ROI delineation more challenging and manual delineation prone to measurement errors.

In the heart, studies have confirmed that semi-automatic delineation methods have better consistency compared to manual methods [48], while similar research is lacking for other peripheral sympathetic nerve distribution organs. Compared to ratio parameters, the WR of the parotid or thyroid gland, by maintaining consistency in pre- and post-ROI regions, can reduce result discrepancies caused by single measurement errors. This might explain the difference between our findings and other studies using ratio parameters as evaluation indicators. Notably, to avoid the impact of primary thyroid diseases on thyroid MIBG uptake in PD patients, this study excluded patients with abnormal thyroid function tests. However, a small proportion of PD patients may have hypothyroidism in clinical practice, which could introduce bias.

Finally, we studied the correlation between cardiac and salivary glands as well as thyroid MIBG scanning parameters. When using ratio parameters for evaluation, no significant correlation was observed, which is consistent with previous studies [22]. Although there was a correlation between cardiac WR and parotid or thyroid WR, the linear correlation was low. Our study suggests that the increased WR in the heart, salivary glands, and thyroid supports the possibility of a shared pathological basis. However, due to the lack of relevant basic research, the specific pathological mechanisms remain unclear. The relatively weak linear correlation observed may be attributed to the use of consistent time points for WR calculation across the heart, salivary glands, and thyroid, or it may indicate the presence of a more complex nonlinear relationship requiring further investigation.

There are several limitations to this study. First, the included patients were confirmed based on clinical standards, and a considerable number of patients already exhibited early peripheral sympathetic nerve MIBG uptake reduction before being definitively diagnosed. It is necessary to conduct examinations and follow-up studies on these patients. Therefore, larger-scale prospective studies are needed. Additionally, due to differences in disease prevalence, the number of PPS patients included was relatively small, which may lead to biased results. This issue is not uncommon in similar studies, necessitating the inclusion of a larger cohort of PPS patients in future research. Finally, in the analysis of peripheral sympathetic organ evaluations, we adopted the same time points as those used for the heart. Although no significant statistical differences were observed, the results at 4 h showed smaller differences compared to 15 min when evaluating the salivary glands or thyroid, which was inconsistent with the findings for the heart. This suggests that the early and late time points determined based on the heart may differ when assessing the function of other sympathetic nervous system organs outside of the heart. Therefore, further exploration is needed, incorporating pathophysiological research to better understand these differences.