Deep brain stimulation improves central nervous system inflammation in Parkinson's disease: Evidence and perspectives

Abstract Background In Parkinson's disease (PD), inflammation may lead to the degeneration of dopaminergic (DAergic) neurons. Previous studies showed that inflammatory mediators mainly contributed to this phenomenon. On the other hand, invasive neuromodulation methods such as deep brain stimulation (DBS) have better therapeutic effects for PD. One possibility is that DBS improves PD by influencing inflammation. Therefore, we further explored the mechanisms underlying inflammatory mediators and DBS in the pathogenesis of PD. Methods We measured serum levels of two inflammatory markers, namely RANTES (regulated on activation, normal T cell expressed and secreted) and tumor necrosis factor‐alpha (TNF‐α), using Luminex assays in 109 preoperative DBS PD patients, 49 postoperative DBS PD patients, and 113 age‐ and sex‐matched controls. The plasma protein data of the different groups were then statistically analyzed. Results RANTES (p < 0.001) and TNF‐α (p = 0.005) levels differed significantly between the three groups. A strong and significant correlation between RANTES levels and Hoehn‐Yahr (H‐Y) stage was observed in preoperative PD patients (r s = 0.567, p < 0.001). Significant correlations between RANTES levels and Unified Parkinson's Disease Rating Scale III (UPDRS III) score (r s1 = 0.644, p = 0.033 and r s2 = 0.620, p = 0.042) were observed in matched patients. No correlation was observed for TNF‐α levels. Conclusion The results of this study indicate that PD patients have a persistent inflammatory profile, possibly via recruitment of activated monocytes, macrophages, and T lymphocytes to the central nervous system (CNS). DBS was shown to have a significant therapeutic effect on PD, which may arise by improving the inflammatory environment of the central nervous system.


| INTRODUC TI ON
Parkinson's disease is a common neurodegenerative disorder in middle-aged and elderly people caused by the death of DAergic neurons in the substantia nigra pars compacta (SNpc), resulting in dopamine deficiency and imbalance of dopamine and acetylcholine neurotransmitter levels. These changes cause motor symptoms such as tremors, myotonia, slow movement, postural instability, and gait difficulty. 1,2 At present, the specific pathogenesis of PD is unclear.
Some scholars believe that autophagy occurs in DAergic neurons in the basal ganglia of PD patients and dysfunction occurs in the functional structure of mitochondria and lysosomes, 3,4 while others believe that the protein transmitting signals between neurons are also abnormal in PD patients. 5,6 Recently, neuroinflammation and immunological disturbance theory has become a hotspot in PD research. [7][8][9][10][11] Early PD is mainly diagnosed by asking patients about their medical history and based on the physician's clinical experience. Most PD patients present after more than 60% of dopaminergic neurons have lost measurable function, leading to a low accuracy of early diagnosis. 12 Therefore, there is an urgent need to identify reliable markers to diagnose PD and track its progression. Of the current clinical symptomatology, imaging, genomics, and biochemistry markers of PD being explored, biologic markers are potentially the most valuable which are considered to be a breakthrough in studying the pathogenesis of PD. [13][14][15] With the development of biologic markers of PD, researchers have begun to explore protein levels in blood, urine, saliva, and other body fluids. Blood has become the ideal source for testing PD biomarkers due to its easy collection method, low contamination risk, and low invasiveness. Neuroinflammation in the CNS is characterized by the activation of microglia and astrocytes, which produce various inflammatory factors and chemokines that destroy the blood brain barrier (BBB) and promote the degeneration of dopaminergic neurons. [16][17][18] The independent or combined action of T cells also participate in this process. 19 Therefore, proinflammatory factors, such as RANTES and TNFα, may be the key to unlocking the pathogenesis of PD. RANTES is a C-C beta-chemokine with strong chemoattractant activity for T lymphocytes and monocytes. 20 Chemokines are released by activated macrophages and microglia in the process of human inflammatory immunity, and inflammatory cells secrete inflammatory factors such as RANTES to recruit additional inflammatory cells to participate in the inflammatory response. [21][22][23] TNFα has potential cytotoxic effects on DAergic neurons and is an important immune signaling molecule. It is mainly produced by astrocytes and microglia in the CNS and can induce the release of neurotoxic substances and the synthesis of inflammatory factors, whose persistent release can aggravate damage to DAergic neurons. 24 Several studies have reported a link between inflammation and PD. In autopsy studies of PD patients, pro-inflammatory cytokines, and chemokines, especially RANTES and TNFα, showed upregulated expression in brain tissue and cerebrospinal fluid. 25 27 showed that inflammatory mediators may reflect the role of systemic inflammation in the neurodegenerative process of PD. The serum RANTES level of PD patients were higher than that of controls, and the RANTES level was strong correlated with H-Y stage and disease duration in patients. Previous studies have reported significantly higher serum levels of TNFα in PD patients compared with controls, but no significant correlation with factors such as severity of disease. 26,28 Until recently, PD was mainly treated by drugs. However, with the advancement of technology and change in treatment philosophy, novel treatment modalities such as surgery and rehabilitation exercises have emerged. The advantages of DBS over nucleus disruption surgery are that it is highly effective, minimally invasive, reversible, and modifiable. Although DBS can control motor symptoms in PD patients, the complete underlying mechanism of action remains unclear due to the complexity of the brain region environment.
The current study aims to examine changes both in plasma RANTES and TNFα levels in PD patients and controls. The study goals are to validate and explore the effects of DBS on plasma biomarkers and examine whether DBS efficacy is related to neuroinflammatory responses and immune imbalance.

| Sample collection and measurement
Peripheral venous blood samples (5 mL) were collected between 8:00 AM and 10:00 AM from participants in a resting state, following a 12 h washout period (OFF-medication/ON-DBS). Blood samples were also collected 1 month after surgery to avoid any influence of postoperative inflammation. The samples were allowed to clot for 30 min at room temperature before centrifugation for 15 min at 3000 rpm. Next, the supernatant was collected, transferred to a 1.5 mL cryopreservation tube, and stored at −80 °C until further analysis. Analysis was performed using Luminex kits obtained from Millipore (Billerica, MA). Assays were performed as per the manufacturer's instructions to determine the plasma levels of 40 proteins. Properly diluted plasma samples were incubated with antibody-coupled microspheres and then with the biotinylated detection antibody before the addition of streptavidin-phycoerythrin.
The captured bead complexes were measured with an FLEXMAP 3D system (Luminex Corporation) using the following instrument settings: events/bead, 40; sample size, 50 μL; discriminator gate, 8000-15,000. The raw data (mean fluorescence intensity) were collected and further processed to calculate the protein concentration.

| Data preprocessing
Before statistical analysis, quality checks (QC) were performed for each assay. The median fluorescent intensity (MFI) was measured using xPONENT 5.1 (Luminex Corporation) and exported into Milliplex Analyst 5.1 (VigeneTech) for estimation of the protein concentrations using a five-parameter logistic fit. Briefly, all analytes that passed QC checks based on the four criteria (standard curve linearity, intra-assay coefficient of variation, interassay coefficient of variation for reference sample, and percentage of missing data) underwent further analysis. Next, we performed a differential analysis of 40 plasma proteins using the log 2 value of the protein content by heatmap (package pheatmap version 1.0.12, Figure 1). The RANTES and TNFα levels showed upregulation. The volcano plot displays the relationship between the log 2 fold change and −log 10 pvalue (package ggplot2 version 3.3.6, Figure 2, Table 1). Differently expressed plasma proteins were screened using a threshold fold change of 1.50. Similar results were observed. Any inconsistencies in the results are explained in the discussion.

| Statistical analysis
Shapiro-Wilk's test and Levene's test, respectively, were used to confirm the assumptions of normality and homogeneity of variance for all the variables. Owing to deviations from the normal distribution or heterogeneity of variances, nonparametric tests (Mann-Whitney test, Kruskal-Wallis test) were used to compare RANTES and TNFα levels between groups. Student's t-test was used when the data met normality, unless otherwise specified. Spearman's correlation coefficient was used to evaluate the relationships between all data. One-way analysis of variance (ANOVA) and the chi-squared test were also used when appropriate. The data are presented as the

| RE SULTS
The participants' clinical and demographical characteristics are summarized in Table 2. There were no significant differences with respect to sex, age, or H-Y stage between groups, but there was a significant difference for duration (p = 0.005). The PD group showed significantly different RANTES and TNFα levels compared with the control group (p < 0.001 and p = 0.005, respectively). Pairwise comparisons showed significant increases in RANTES and TNFα levels between the pre-operation and postoperation group and the control group (*p < 0.001, △ p < 0.001, ✦ p = 0.005, ▲ p = 0.002, Figures 3 and 4). The relationship be-

| DISCUSS ION
The onset of PD is usually unilateral, presenting with N-type develop-   Note: Data are presented as mean ± SD or median (25th-75th percentile).
Abbreviations patients was 1.7 times higher than that of healthy controls, further supporting this theory. Interestingly, after surgical intervention, plasma RANTES levels in PD patients decreased compared to before surgery, and there was no significant difference between post-surgery patients and healthy controls, suggesting that STN-DBS (subthalamic nucleus deep brain stimulation) may improve plasma RANTES levels in PD patients. In further analyses, we performed a more refined patient segmentation. We found that the RANTES content in PD patients with rigidity, H-Y stage 2.5 and 3 patients before and after surgery were not statistically significant but meaningful. The reason that statistical significance was not achieved may be because the sample size was somewhat small.
TNFα is an important inflammatory factor that is produced by abnormally activated glial cells during CNS inflammation.  42 In contrast, knockdown of the TNFα receptor gene had a protective effect in PD mice. 42 In the present study, we found a significant difference in TNFα levels between PD patients and healthy subjects, and this difference disappeared between patients and healthy subjects after surgery. Furthermore, we observed a correlation between TNFα and RANTES levels, suggesting a synergistic effect of these two plasma proteins in PD. TNFα may induce the expression of chemokines such as C-X-C Motif Chemokine the researchers detected differences in these proteins. 27 However, IL-6 and CRP concentrations that were found to be increased in PD have also been shown to be in normal physiology and increased in the aged brain. 43 Evidence has been presented to show that chronic neuroinflammation occurs in the aging brain which are hallmarked by elevated immunoreactivity and low-scale constant production of cytokines. 44 We believe that this concomitant effect must be considered. In the subsequent research, we will focus on exploring the relationship between them.

| LI M ITATI O N S
The study still has some limitations. As this study was a retrospective study and the surgical methods and procedures were optimized, more samples could not be added to support the experimental results. In addition, due to the inadequate study design, which resulted in a small number of matched patients, further studies should have more matched patients. Finally, the relationship between inflammatory proteins associated with the nervous system still needs to be supported by more experimental results. Systematic evaluation should be included in future studies.

| CON CLUS IONS
This study showed significant differences in serum RANTES and TNFα levels in PD patients compared to matched controls. Our findings indicate that PD patients have an on-going systemic inflammatory profile and that RANTES and TNFα levels are potential plasma biomarkers for PD. Moreover, elevated RANTES and TNFα levels were associated with the severity of PD and, in paired comparisons, RANTES levels correlated with UPDRS scores. These results support the effectiveness of DBS for PD and suggest that its mechanism of action may involve altering the systemic inflammatory response.
Unfortunately, we were unable to further explore cellular-molecular mechanisms. The source of plasma inflammatory factors in PD patients should be elucidated in future studies and the mechanism of DBS should be further investigated to contribute to delaying or even curing PD.

AUTH O R CO NTR I B UTI O N S
All authors contributed to the study conception and design. Data collection and analysis were performed by BL, CQ, and YL. The first draft of the manuscript was written by LC and WWD. WBZ edited and revised the manuscript. All authors contributed to and approved the final manuscript.

ACK N OWLED G M ENT
We thank all participants for their cooperation in this study.

FU N D I N G I N FO R M ATI O N
This study was supported by the grant from Subtopic of the 13th

CO N FLI C T O F I NTER E S T S TATEM ENT
The authors declare that they have no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.

CO N S ENT TO PA RTI CI PATE
All patients or their family members provided written informed consent for participation in the study.

CO N S E NT FO R PU B LI C ATI O N
Not applicable.