Neuroimaging Correlates of Substantia Nigra Hyperechogenicity in Parkinson’s Disease

INTRODUCTION

Degeneration of the substantia nigra (SN) is one of the core pathological signs of Parkinson’s disease (PD), a disorder characterized by progressive loss of motor control and eventual cognitive impairment [1, 2]. Loss of neuromelanin (NM)-containing dopaminergic neurons and deposition of iron in the SN are frequently observed in PD and associated with disease progression [3]. Prominent loss of pigmented SN neurons is thus considered a hallmark of PD [4]. Furthermore, NM content in the pre-synaptic terminals of dopaminergic neurons is an emerging candidate neuroimaging-based biomarker for PD [5], although whether increased iron deposition is a cause or effect from the loss of NM-containing neurons is still under debate [6, 7]. Nonetheless, a relationship between increased iron deposition and decreased NM in the SN of PD patients is widely accepted, suggesting that magnetic resonance imaging (MRI) sequences sensitive to NM and iron may be of great clinical value for PD diagnosis, monitoring of disease progression, and treatment evaluation [8].

There is mounting evidence that neurodegeneration associated with PD begins years before the emergence of clinical symptoms [9]. Indeed, by the time of motor symptom onset, about 60% of dopaminergic SN neurons have already degenerated [10–12]. Reliable markers for prodromal changes in PD are thus required for early diagnosis and possible neuroprotective intervention. Transcranial sonography (TCS) is a widely applied bedside test as it allows for the rapid, inexpensive, and reliable depiction of brain structures, including the SN for differential diagnosis of movement disorders [12]. In the majority of patients with PD (∼90%), alterations of the SN manifest as a hyperechogenic midbrain signal (HE+). However, the underlying basis of the HE+ is still debated [13]. Moreover, relationships with other imaging manifestations, such as MRI signals, are also unclear.

Pilot studies have confirmed that a larger HE+ is associated with increased SN iron content [10, 14]. However, ultrasound has demonstrated limitations for identifying PD biomarkers. For instance, measurements often differ among ultrasound devices, and the measurements depend on the skill and experience of the operator to a certain extent [15]. Additionally, other neurodegenerative disorders may be associated with midbrain HE+ [15]. Previous studies have recommended HE+ as a biomarker for disease diagnosis but not for disease progression and monitoring [16, 17]. Furthermore, the causes of this change in ultrasound reflection in PD are not entirely understood due to a lack of in-vivo studies on living patients [10]. Thus, there is an urgent need for further investigation on the histopathological basis of the HE+, including the relationships of HE+ characteristics with nigral NM and iron accumulation. In contrast to TCS, various magnetic resonance- (MR-) based imaging modalities can reveal both structural and functional changes in PD with an ever-improving spatial and temporal resolution for diagnosis and disease monitoring. In this study, two particularly well suited MRI sequences, NM-weighted imaging (NMI), which exploits the iron-binding properties of NM, and susceptibility-weighted imaging (SWI), which takes advantage of the different magnetic susceptibilities of tissues to generate iron-related images with visual contrast, were used to better characterize nigral pathology associated with TCS-derived HE+ [8]. The primary goal of this study is to elucidate the MR-morphological representation of the HE+. In this context, SWI provides an indirect measure of brain iron deposition and NMI a surrogate marker for neuromelanin levels in vivo. Although both measures recapitulate two important histopathological hallmarks of PD-related neurodegeneration only indirectly, they can serve for the longitudinal and non-invasive assessment of patients with neurodegenerative diseases. One of the critical factors for NM dependence is paramagnetic behavior, which confers high signal contrast between the target and surrounding structures, thereby facilitating precise measurements of NM distribution and concentration [18–20]. Alternatively, the resolution of SWI depends on magnetic field inhomogeneities generated by iron, causing changes in the phase maps subsequently processed with the magnitude images and yielding iron-sensitive images from which iron deposition can be quantified [21–23]. Therefore, the combination of NMI and SWI could serve as a useful strategy to assess nigral neurodegeneration and serve as a surrogate marker for the pathophysiological basis of HE+, thereby improving the utility of HE+ as a biomarker for PD [8, 24]. Also, this analysis will help distinguish the primary advantages of TCS and MRI for revealing different aspects of PD pathology, including dopaminergic neuron loss in the SN [25].

MATERIALS AND METHODS

Demographics

Thirty-four PD patients (mean age: 68.2±7.5 years) and 29 age- and sex-matched healthy controls (mean age: 69.9±6.3 years) were enrolled (Fig. 1). PD was diagnosed according to the well validated Movement Disorders Society clinical criteria by trained movement disorders specialists [26].

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Fig. 1

Schematic MRI and TCS data processing and analytical workflow. Panel A illustrates the analytical workflow of the susceptibility-weighted imaging (SWI). In accordance with A, B illustrates the workflow of neuromelanin-weighted imaging (NMI). In A.I and B.I, both raters have to visually identify the midbrain (red-dashed line) on respective axial slices, which contain the Substantia nigra (SN). In A.II and B.II, seeds (red) with a diameter of circa 6 mm are placed in non-SN midbrain tissues for both sides, and for all axial slices were the SN could be identified. Based on the mean signal intensities and the standard deviation (SD) of the placed seeds, thresholds with different rigor are defined ±(3SD): yellow; ±(4SD): orange; ±(5SD): red; see A.III and B.III). C demonstrates the detection of hyperechogenicity (HE+) via transcranial sonography (TCS). TCS has to be performed on both sides (C.I and C.II) to measure the ipsilateral HE+ planimetrically. The midbrain is marked with a red-dashed line and the expected location of the SN with a red-dotted line. The gray element depicts the direction of the ultrasound transducer.

RESULTS

DISCUSSION

In this study, we employed three imaging modalities, TCS, NMI, and SWI to investigate pathological alterations of the SN in patients with PD. Our primary goal was to examine whether NM loss as measured by NMI and (or) iron accumulation as measured by SWI can account for the HE+ observed by TCS. In addition to elucidating the pathophysiology underlying HE+, it was hoped that these analysis would help support the utility of SWI and NMI for patient diagnosis and monitoring given that several previous studies have implicated nigral NM loss and iron deposition in PD [11, 13]. However, our findings strongly suggest that the HE+ is unrelated to either process and thus these modalities should be viewed as providing complementary independent clinical information on patient status.

These weak correlations are unlikely due to poor measurement reliability as TCS measures of HE+ have shown excellent IRR in larger cohorts [27], while all IRR values measured in the current study were highly significant. However, the correlations between measures became weaker when more strict thresholds were employed (4SD) and (5SD), which emphasizes the need for an a priori definition of the optimal threshold. More standardized MRI processing pipelines may also enhance the reliability of these metrics to facilitate elucidation of SN pathology during PD progression.

While the majority of correlations between measures were weak, there was a moderate correlation between SN area as measured by NMI and TCS (ρ= 0.361, p < 0.039 among PD patients and ρ= 0.201, p < 0.116 for the entire cohort; Table 4). Alternatively, the weak correlations between most measures are inconsistent with postmortem studies implicating NM loss and (or) iron deposition in the HE+ [13, 14]. These weak correlations with SWI-derived measures are particularly surprising as several studies have suggested that iron dysregulation is a key factor leading to increased SN echogenicity [13, 14]. Our results are also in contrast to previous epidemiological studies implicating genetic alterations of ferritin metabolism in the nigral HE+ [30–32]. Further, NM is directly involved in PD pathogenesis [5, 34] and several studies have reported that NM content within the SN can serve as a reliable marker for the assessment of disease duration and progression, whereas HE+ does not reflect individual disease progression [20, 35]. In contrast to recent reports, we were not able to find significant group differences based on the assessment of the SN via SWI or NMI. This can mainly be explained by the different methodological approach employed in this study. Here, we used a segmentation-based analysis (with three different thresholds derived from non-SN midbrain tissue, see Fig. 1). However, our segmentation-based approach provides a more meaningful correlation target for the assessment of the HE+ (which can be interpreted as an area as well). For the demonstration of, e.g., a peak-intensity based analyses by manually placed ROIs within the SN, the interested reader may refer to Prasuhn et al. (2021) [36]. The absence of relevant group differences may also stem from the lack of normalization in the preprocessing of NMI (or SWI) images as there are no widely accepted normalization procedures for TCS images and different SN imaging modalities.

Alternatively, NMI and TCS may reflect distinct pathophysiological properties of the SN, in accordance with the observation that nigral HE+ does not serve as a neuroimaging marker for individual disease progression [17, 37] while NMI does [20, 35]. However, it should be noted that SWI can only be considered a surrogate marker of brain iron deposition in general. The involvement of iron dyshomeostasis in PD mechanisms is most likely multi-faceted and is not limited to the total brain iron level in a given neuroanatomical structure. For example, the oxidative state of iron (ferrous vs. ferric iron) or the complexation of iron atoms within macromolecules substantially determines its pathophysiological role [38]. Furthermore, activated microglia may be a major contributor to nigral HE+ since epidemiological, animal model, and cell culture studies support microglia as key drivers of PD progression [39–41]. Microglial activation may be of particular importance to SWI manifestation as these cells contain large amounts of ferritin, the primary iron storage protein in mammals [31]. However, most of these results are from post-mortem studies, which can only reflect the final pathways of neurodegeneration and miss ongoing pathophysiological mechanisms, such as neuroinflammation, potentially more strongly related to HE+ [34].

Although our study used well-established approaches for NMI and SWI analysis, we cannot rule out the possibility that other image analysis approaches may yield more reliable SN segmentation. In future studies, the in-vivo assessment of nigrosome-1 may provide further insights into the origins of the HE+ in patients with PD. However, reliable and standardized segmentation methods for this substructure of the SN are currently lacking. The visual inspection of the nigrosome-1 concerning the presence of the so-called swallow-tail sign (STS) is still under debate for 3 T MRI [42]. The STS, as a dichotomous radiological sign, may only provide negligible benefits to enhance our understanding of the origin of the HE+. Potential alternatives are structural and functional connectivity-based segmentation algorithms of the SN or the combination of additional MRI sequences [43]. The combination of complementary MRI sequences, such as high-resolution quantitative multiparameter mapping (through R1, PD^*, T2^*, and R2^* sequences) [44, 45], MR elastography [46], or advanced diffusion tensor imaging techniques [37, 47], could be more useful for revealing the MR-morphological origin of the HE+. Improvements in MRI acquisition schemes or studies on associations between the imaging manifestations from existing sequences and histopathology are therefore warranted. Additionally, MRI sequences directly sensitive to cytophysiological alterations, such as magnetic resonance spectroscopy imaging [48–51] or chemical exchange saturation transfer [52–54], may provide more relevant information on PD pathophysiology and should also be used to investigate fundamental neuroanatomical structures in vivo. The utility of ultra-high field MRI (7T or above) has already been demonstrated for microstructural imaging of the midbrain [55–57]. However, sparse availability and patient safety concerns have hindered widespread use for clinical research [58].

CONCLUSION

Neurodegenerative changes in the SN produce distinct manifestations on TCS and MRI images, although both modalities can be used to show differences between PD patients and HCs for diagnosis. Further, these NM- and iron-sensitive MRI modalities may advance our understanding of PD pathophysiology and identify new neuroimaging markers for improved diagnostic accuracy or to map disease progression [18, 22]. However, improved MRI sequences, defining suitable thresholds, and more advanced segmentation algorithms, among other possible improvements, should be considered. The lack of correlation between HE+ and our MRI findings is inconsistent with prior post-mortem studies implicating NM loss and iron accumulation in PD pathogenesis [13, 14]. Nonetheless, TSC and specific MRI modalities (e.g., with multiparameter mapping [45]) or higher magnetic field strengths [57]) may provide complementary information for clinical research and patient management.