Substantia Nigra Echogenicity Signal Correlated with Clinical Features in Patients with Parkinson's Disease in Xinjiang

Background Transcranial sonography (TCS) is a noninvasive test that can reveal structural changes in the substantia nigra (SN) in Parkinson's disease (PD). The purpose of this study was to investigate the relationship between SN signatures and clinical features in PD patients in a multiethnic region of China. Methods A total of 147 patients with PD were included in the study, and all of whom had underwent a TCS examination. Clinical information was collected from PD patients, and motor and nonmotor symptoms were assessed using assessment scales. Results There were differences in the substantia nigra hyperechogenicity (SNH) area between age of onset, visual hallucinations (VH), and UPDRS3.0 II scores (P < 0.05), patients with late onset PD had a greater SNH area than early onset (0.326 ± 0.352 vs. 0.171 ± 0.194), and PD patients presenting with VH had a greater SNH area than those without hallucinations (0.508 ± 0.670 vs. 0.278 ± 0.659), and further multifactorial analysis showed that a high SNH area was an independent risk factor for development of VH. The area under the ROC curve for predicting VH from the SNH area in PD patients was 0.609 (95% CI: 0.444–0.774). There was a positive correlation between the SNH area and UPDRS3.0-II scores, but further multifactorial analysis showed that SNH was not an independent predictor of the UPDRS3.0-II score. Conclusion A high SNH area is an independent risk factor for development of VH, there is a positive correlation between the SNH area and UPDRS3.0 II score, and TCS has guiding significance in predicting clinical VH symptoms and activities of daily living in PD patients.


Introduction
Parkinson's disease (PD) is a common degenerative movement disorder of the nervous system. Te pathological changes are the progressive degeneration of nigrostriatal dopaminergic neurons in the substantia nigra and formation of Lewy vesicles [1]. It is characterized by motor symptoms and nonmotor symptoms as the main clinical manifestation. Bradykinesia, resting tremor, muscle rigidity, abnormal posture, and gait are the main manifestations of motor symptoms, and motor complications are often accompanied by middle and late stages of the disease. Its diagnosis is based on the presence of parkinsonian motor features, namely, bradykinesia, stifening, and resting tremor, and the early misdiagnosis rate is high.
Transcranial sonography (TCS) is a noninvasive, convenient, and rapid examination that utilizes weak areas of the skull as transillumination windows and allows ultrasound exploration of the intracranial vessels and structures in the substantia nigra of the midbrain using the Doppler efect of ultrasound. Since Becker [2] frst described the substantia nigra hyperechogenicity (SNH) in PD patients in 1995, a large number of studies have reported that TCS can be used as one of the imaging markers of PD, providing auxiliary evidence for the early diagnosis of PD and assisting in the identifcation of other movement disorder diseases.
Several previous studies [3][4][5][6][7] have shown the diferences in sensitivity, specifcity, and clinical characteristics of TCS in Asian and European patients with PD, and Xinjiang is located in the hinterland of Asia and Europe, which is a multiethnic region with good ethnic diversity. Tis study intends to further investigate the correlation between SNH and the clinical characteristics of PD patients through clinical studies and provide reference information for the diagnosis and treatment of PD patients.

Research Subjects.
Tis is a cross-sectional study with subjects from the Second Afliated Hospital of Xinjiang Medical University, which is a clinical medical research center for neurological disorders in Xinjiang, with patients with neurological disorders from various states in Xinjiang and high ethnic diversity. Patients with PD who attended the Neurology Treatment Centre of the Second Afliated Hospital of Xinjiang Medical University from January 2021 to October 2022 were selected. All patients were diagnosed by two experienced neurologists according to the diagnostic criteria for primary Parkinson's disease [8], and possible dementia with Lewy bodies (DLB) was excluded according to the 2017 DLB diagnostic criteria (DLBC-4) [9]. Possible secondary Parkinson's disease due to cone signs, gaze palsy, cerebellar ataxia, history of stroke, neuroleptic intake, or other possible causes was also ruled out. Two doctors were double blinded during the consultation and excluded the patient if a consistent diagnosis could not be made. All patients had MR/CT brain scans to aid in the diagnosis, and no patients underwent SPECT or PET imaging to refne the detection of dopamine transporters or F-dopa. A total of 161 patients with PD were included in the study. After completion of TCS in all patients, 14 patients were excluded due to the inability to assess the nigrostriatal echogenic signal in both temporal windows conditions, and fnally, 147 patients were included in this study. Te study was approved by the Ethics Committee of the Second Afliated Hospital of Xinjiang Medical University, and all patients signed informed consent.

Transcranial Sonography.
Te patient's TCS was performed by an experienced ultrasonographer who had no knowledge of the patient's clinical profle. Te examination equipment was a Resona 7T color Doppler ultrasound diagnostic instrument (Shenzhen, China), set at a scanning depth of 16 cm and an acquisition range of 45 dB. Te examining physician performed the procedure at the typical temporal bone window above the zygomatic arch in the preauricular region and adjusted the probe angle to fnd the best imaging efect in the butterfy-shaped midbrain and fnally measured the nigrostriatal high signal area automatically with a cursor. Te study used both semiquantitative grading criteria [2] and quantitative criteria (area) [10] of the substantia nigra signal to assess the substantia nigra properties. Te semiquantitative grading criteria were grade I: the substantia nigra showed uniformly distributed hypoechogenicity; grade II: scattered dotted and fne-linear slightly strong echoes were seen in the substantia nigra; grade III: the substantia nigra echoes were patchily enhanced and lower than the foot interrogation pool echoes; grade IV: the substantia nigra echoes were patchily enhanced and equal to the foot interrogation pool echoes; grade V: the substantia nigra echoes were patchily enhanced and higher than the foot interrogation pool echoes. SNH above grade III and/or an area ≥0.2 cm 2 was considered positive nigrostriatal (SN+).

Clinical Assessment.
Relevant clinical characteristics of the study population were collected: race, age, sex, education level, and duration of disease. Te Uniform Parkinson's Disease Rating Scale (UPDRS) published in 1987 [11] and the Hoehn and Yahr Staging (H-Y staging) were used to assess the severity of Parkinson's disease in patients after 24 hours (or 72 hours for controlled-release anti-PD drugs) of discontinuation of treatment with anti-Parkinson's disease drugs. Te Non-Motor Symptom Scale (NMSS) was used to assess nonmotor features such as bladder status, constipation, and pain in PD patients, the Montreal Cognitive Assessment Scale (MoCA) was used to assess cognitive function in PD patients, and the Hamilton Depression Scale (HAMD) and the Hamilton Anxiety Scale (HAMA) were used to assess depression and anxiety status of PD patients, respectively. Te Montreal Cognitive Assessment Scale (MoCA) ≥26 is normal, ≥18 is mild cognitive impairment, ≥17 is moderate, and <10 is severe. Te Hamilton Depression Scale (HAMD) <7 is normal, ≥7 is possible depression, ≥17 is defnitely depression, and >24 is severe depression. Te Hamilton Anxiety Scale (HAMA) ≥29 may be severe anxiety, ≥21 is defnitely obvious anxiety, ≥14 points is defnitely anxiety, ≥7 points is possible anxiety, and <7 is normal. Te study further classifed PD patients into early-onset PD (≤50 years old) and late-onset PD (>50 years old) according to Chinese guidelines [12].

Statistical
Analysis. EXCEL software was used for data collection and organization, and SPSS 21.0 statistical software was used for statistical data analysis. Continuous variables were described by means and standard deviations ( ̅ x ± s), and categorical variables were described by frequencies and percentages n(n%). Comparisons of the SNH area between dichotomous variables (gender, voiding disorder, etc.) were performed using the t-test or Mann-Whitney U test. Correlation analysis between quantitative variables (UPDRS 3.0 I score, improvement rate, etc.) and the nigrostriatal high-signal area was performed using the Pearson correlation test. Logistic regression analysis of univariate and multivariate factors was used for the analysis of factors infuencing whether visual hallucinations (VH) occurred in PD patients, and linear regression analysis was used for the analysis of factors infuencing the UPDRS3.0 II score. Te signifcant level was set at α � 0.05.

Analysis of Factors Infuencing the Area of SNH in PD
Patients. Tere were no diferences in the SNH area between gender, ethnicity, urinary disturbance, constipation, pain, sleep disturbance, hyposmia, hyperhidrosis, HAMD, HAMA, and cognitive impairment (P > 0.05), diferences in the SNH area between the age of onset, VH, and UPDRS3.0 II scores (P < 0.05), the SNH area was greater in patients with late-onset PD than early onset PD (0.326 ± 0.352 vs. 0.171 ± 0.194), and the SNH area was greater in PD patients presenting with VH than without hallucinations (0.508 ± 0.670 vs. 0.278 ± 0.659), see Table 2. Analysis of the correlation between the SNH area and clinical characteristics in PD patients showed that there was a positive correlation between the SNH area and the UPDRS3.0-II score (r � 0.261; P value � 0.001), as shown in Table 3 and Figure 1.

Analysis of Factors Infuencing VH in PD Patients.
To clarify whether the SNH area was an independent risk factor for VH, the study analyzed its infuencing factors with VH as the outcome variable, and the results of the single-factor analysis showed that SNH area, UPDRS3.0 I, and H-Y staging were correlated with VH (P < 0.05); further multifactor analysis showed that, controlling for constant UPDRS3.0-I and H-Y staging, the SNH area still correlated with VH (OR � 59.661; 95% CI � 2.424∼68.675; P � 0.012), and a high SNH area was an independent risk factor for the development of VH, see Table 4. Te ROC curve for predicting VH from the SNH area in PD patients yielded an area under the curve (AUC) of 0.609 (95 CI%: 0.444∼0.774), with a cutof value of 0.345 cm 2 , see Figure 2.

Analysis of Factors Infuencing UPDRS 3.0 II Scores in PD
Patients. Te study analyzed the factors infuencing the UPDRS3.0 II score as an outcome variable, and the results of the univariate analysis showed that SNH area, disease duration, voiding disorder, constipation, cognitive impairment, UPDRS3.0 I, UPDRS3.0 III, UPDRS3.0 IV, H-Y staging, and MoCA were correlated with the UPDRS3.0 II score (P < 0.05), and further multifactorial analysis showed that UPDRS3.0 III and H-Y staging still had positive correlations with UPDRS 3.0 II scores (β > 0; P < 0.05), as shown in Table 5.

Discussion
Of the 161 PD patients collected in the study, 14 (8.6%) were excluded due to poor sound transmission through the temporal bone window, and no nigrostriatal-related information was obtained, which is consistent with the 6.9%-15.5% [5] rate reported in the literature for Caucasians and inconsistent with a reported rate of 20.5%-30.2% in Asians [4]. Te results of the study [4] showed that the proportion of women in the group with poor temporal bone ultrasound penetration was signifcantly higher than that in the group with good temporal bone ultrasound penetration (74.71% vs. 30.85%, P < 0.001). It has also been shown [13] that frontal lobe hypertrophy develops with age, particularly in females. In contrast, the relatively high proportion of males in this study, with a male-to-female ratio of (57.8% vs. 42.2%), may have contributed to the lower proportion of poor ultrasound penetration of the temporal bone in this study. In addition, the ethnic diversity of this study may have contributed to some degree of disparity. Te proportion of 147 PD patients with SNH above grade III and/or an area of 0.2 cm 2 or more was 60.5%, which is much less sensitive than earlier reports in the literature [6] and not inconsistent with reports of similar sensitivity in Asians and Europeans [7] but similar to the reported 61.69% in Chinese [4]. Tis may be related to a variety of factors such as the selection of the optimal section during TCS examination related to operator experience, diferent ultrasound equipment accuracy, subjectivity in the determination of nigrostriatal echo intensity, human error in the calculation of the SN + area, and the diverse pathogenesis of PD patients.
Tis study investigated the correlation between clinical characteristics and the SNH area in 147 PD patients, and it was found that the SNH area was not related to gender and ethnicity but to age of onset, VH, and UPDRS II scores. Te study divided PD patients into two groups, early-onset and late-onset, and the SNH area of late-onset PD patients was signifcantly higher than that of early-onset PD patients (P < 0.05). Te mechanism may be the accumulation of various metallic elements in the midbrain substantia nigra with age, with iron ions, manganese ions, and calcium ions [14], which are thought to be responsible for SNH. Te age of onset and the echogenicity of the substantia nigra are still highly controversial. It has been reported in the literature [15] that younger PD patients have larger hyperechoic areas than older PD patients and that SNH is positively correlated with age in healthy individuals. Te Berg [16] study, on the other hand, suggests that the odds of SNH gradually increase with age. It has also been suggested [17] that the area of SNH increases with the severity of the disease after 5 years of patient onset. Te mean duration of disease in PD patients in the study was 5.267 ± 3.913 years, and no correlation between the duration of disease and SN echo was found, probably due to the relative difculty of early diagnosis of  PD and some error in the defnition of the duration of disease, and further follow-up studies are still needed. 16 of 147 patients with PD had VH, with a prevalence of 10.9%, which is consistent with 8.8%-44% [18,19] reported in the literature. Te mechanism of VH is not fully understood, and previous studies [3,20,21] have shown that VH is associated with disease duration, cognitive dysfunction, sleep quality, anticholinergic drugs, and severity (H-Y staging). Te present study confrmed that VH was associated with SNH, UPDRS I scores, and H-Y staging. Te study showed that the area of SNH in patients with VH was signifcantly larger than that without VH, and further    Parkinson's Disease 5 multifactorial analysis revealed that a high SNH area was an independent risk factor for VH, which is consistent with the results of related studies. Te main mechanisms associated with the occurrence of SNH in PD patients include iron deposition [1] and microglia activation [22]. Tere is no direct evidence that either iron accumulation in the substantia nigra or microglia activation is associated with PD with VH, so the mechanism of SNH area associated with VH needs further study. At the same time, the study used the ROC curve to determine the value of the SNH area to predict VH, and the AUC value was 0.609, suggesting that the value of the SNH area alone to predict VH is low; the SNH area still needs to be combined with other relevant factors for VH prediction. In addition, it was found that after dividing all PD patients into VH and no VH groups, all PD patients in the VH group had late onset of VH, but it was not related to the duration of the disease, indicating that the patients' age of onset was related to the occurrence of VH, which is consistent with related studies [21,23]. Tis may be related to rapid sensory loss and age-related drug side efects [24][25][26]. Tis study found a correlation between VH and the UPDRS I score, which may be related to the fact that the UPDRS I score is mainly used to determine the degree of mental activity and behavioral and emotional disturbances in patients with PD. Several previous studies [4,27] have confrmed the correlation between SNH and the UPDRS II score, which has a clear correlation with disease duration and severity (H-Y staging), but SNH did not show a clear correlation with disease duration and severity (H-Y staging) in the present study. Te study found that the UPDRS III score and severity (H-Y stage) were independent risk factors for the UPDRS II score by further multifactorial analysis, while the area was not an independent risk factor, and the contradictory relationship between them was not well understood, probably because diferent PD subtypes lead to diferent mechanisms of SNH; further exploratory studies are needed.

Limitations
Tere are still some methodological limitations to this study. First, due to the number of factors contributing to hallucinations, although the study population was assessed 24 hours after stopping the drug, the efects of the drug could still not be completely excluded. Second, given the small sample size and ethnic diversity ratio in this study, statistical power remains limited, and a larger multiethnic sample size is needed to validate the current work.

Conclusion
TCS, as a noninvasive test, has a certain value in the diagnosis of PD patients. Te study found that a high SNH area was an independent risk factor for the development of VH by statistical analysis of the SNH area and clinical characteristics of PD, and there was a positive correlation between the SNH area and the UPDRS3.0 II score. TCS has some guiding signifcance in predicting clinical VH symptoms and activities of daily living in PD patients.

Data Availability
Te data that support the fndings of this study are available on request from the corresponding author.

Ethical Approval
Te study was approved by the Ethics Committee of the Second Afliated Hospital of Xinjiang Medical University, and all methods were carried out in accordance with relevant guidelines and regulations.

Disclosure
Rurui Wei, Yan Zhang, and Peishan Li are the co-frst authors.

Conflicts of Interest
Te authors declare no conficts of interest.