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Neuroinflammation is a key factor in Parkinson's disease (PD) pathogenesis. However, the regional heterogeneity of biomarkers related to inflammation in PD is less well defined. We developed [18F]GSK PET imaging to quantify neuroinflammation via the P2X7 receptor in A53T PD male mice and wild-type (WT) male mice. Montelukast (MK) was administered to mice, and weekly behavior tests confirmed MK's efficacy. [18F]L-DOPA/[18F]GSK PET, motor testing, autoradiography, and immunofluorescence were performed after MK treatments. MK improved motor function and reduced the brain uptake of [18F]GSK, indicating resynchronization of regional microglial activity. The whole brain uptake of [18F]GSK was correlated with motor functional restoration, while [18F]L-DOPA PET was not. Overall our study indicated that brain mapping of [18F]GSK PET is beneficial for exploring P2X7R-related neuroinflammation which is correspondent to motor function in PD.
Point-to-point responses to the reviewer’s comments: Reviewer #1: 1. Line 26: The authors state that [18F]L-DOPA PET was performed before and after MK treatments, but in the manuscript it is described that [18F]L-DOPA PET was performed only after 5 weeks of treatment and not at baseline. Please clarify. Response: We appreciate your professional common. We have carefully checked and revised the expression in the revised manuscript (Page 2, line 25-26). 2. Line 54: Mention of the first P2X7R PET ligand, but there has been a larger effort toward developing a PET ligand for this target. Additional background on more promising ligands and development would be beneficial to contextualize the field. Response: Thank you for your advice! We deleted “first P2X7R PET ligand”, and We have added additional background on P2X7R in the introduction section (Page 4-5, line 69-86) 3. Line 71: Introduce M2 microglia and BV2 microglia. Response: Thank you for your advice! We have added additional background on M2 microglia and BV2 microglia in the introduction section. (Page 3, line 45-53) 4. Line 73-74: “But the evaluation of the efficacy of MK depends on whether PD symptoms have improved and is subjectively influenced by the patient and physician.” Assessment of all clinical phenotypes are subjectively influenced by the patient and physician, I’m not sure what this statement adds to the justification. Response: We appreciate the thoughtful question and suggestion! Although neuroinflammation is known to alter blood-brain barrier (BBB) permeability, its potential effects on MK's cerebral distribution and effect remain to be fully elucidated. Furthermore, conventional evaluation methods are limited, and the quantitative methods for anti-inflammatory treatments in the brain are lacking in clinical practice. We've removed the original statements and revised the corresponding expressions in the updated manuscript (Page 5-6, line 97-106) 5. Line 75-79: “We performed PET imaging and quantification to assess the neurodegenerative brain regions involved in PD and the anti-inflammatory response of MK, we performed motor testing and [18F]GSK PET scans (for P2X7R-related neuroinflammation) at baseline. We performed [18F]L-DOPA PET scans (for DA neurons), motor testing, [18F]GSK PET scans, autoradiography and immunofluorescence staining after 5 weeks of MK treatment in transgenic A53T PD mice.” Mention controls for the autoradiography and IF in PD mice. Response: We appreciate the thoughtful question and suggestion! We've added controls information in this sentence in the introduction section (Page 6, line 117-120) 6. Line 93: “Gamma spectrometry was performed 12 hours after radiochemical labeling, which confirmed that the fluorine-18 nuclide was qualified.” Unclear what this means or why it was included. Response: We appreciate the thoughtful question and suggestion! We've reworded this expression in the introduction section (Page 7, line 127-135). “Gamma spectrometry was performed 12 hours after radiochemical labeling, which confirmed the energy spectrum (511 kev) of the fluorine-18 nuclide.” (Page 7, line 129-130). 7. Line 100: Define WT at first use here. Response: We appreciate the thoughtful question and suggestion! We've added “wild-type” in the introduction section (Page 4, line 73). 8. Line 121-122: “However, the impairment of forelimb strength in A53T mice was improved by MK.” After how long? Response: We appreciate the thoughtful question and suggestion! We've added the information of the time in the revised results section (Page 7, line 146-147). “However, the impairment of forelimb strength in A53T mice was improved by MK after two weeks.” 9. Line 157: Define MRI at first use. Response: We appreciate the thoughtful question and suggestion! We've added “Magnetic Resonance Imaging” in the updated manuscript (Page 9, line 181). 10. Line 159: “Compared with those in WT mice?” what are “those”, is it meant compared with WT mice? Response: We appreciate the thoughtful question and suggestion! We have clarified “Compared with those in WT mice” as “Compared with WT mice” (Page 9, line 183-187). “Compared with WT mice, there was decreased DA synthesis observed in the cortex, basal forebrain septum, striatum, superior colliculi, and whole brain in the brains of A53T mice. Moreover, DA neuron function was improved in A53T/MK mice. Compared with A53T mice, the increased [18F]L-DOPA uptake was observed in the basal forebrain septum, striatum, and superior colliculi in the brains of A53T/MK mice.” 11. Line 168-169: Increased/decreased suggests movement from a baseline measure, reword to indicate relatively (compared to what?). Response: We appreciate the thoughtful question and suggestion! We've reworded the sentence in the updated manuscript (Page 9-10, line 193-194). “A decrease in activated microglia was observed in the striatum of A53T/MK, WT, and WT/MK mice compared to A53T mice.” 12. Line 177: Differed between the three movement disorders? Or differed between the three behavioral tests? Response: We appreciate the thoughtful question and suggestion! We've added the relevant description in the updated manuscript(Page 7, line 140-143). 13. Line 184: Again, behavioral tests are not movement disorders. Please clarify. Response: We appreciate the thoughtful question and suggestion! We've replaced “movement disorder” with “behavioral tests”or “motor behaviors”in entire revised manuscript. 14. Line 199: Please be careful with wording suggesting the mouse model is a PD brain, reword to be clear you are referring to the work performed here in the rodent models. Response: We appreciate the thoughtful question and suggestion! We've replaced “PD brain” with “the brains of A53T mice” in entire revised manuscript. 15. Line 207: “There are more brain regions with microglial activity changes in [18F]GSK that target P2X7R”. [18F]GSK PET is not a measure of microglial activity changes, reword for clarity. Response: We appreciate the thoughtful question and suggestion! We've reworded the expression in the revised manuscript. (Page 11, line 231-232). “There are more brain regions with P2X7R changes in [18F]GSK PET than brain regions with changes in [18F]L-DOPA uptake.” 16. Line 227: Again, please rephrase “motor disorders” here. Response: We appreciate the thoughtful question and suggestion! We've revised the expression in the revised manuscript. 17. Line 233: Define/explain MPTP. Response: We appreciate the thoughtful question and suggestion! We've deleted related sentences and reworded the expression in the revised manuscript. 18. Line 234-235: Again, rephrase “motor disorders” here. Response: We appreciate the thoughtful question and suggestion! We've replaced “movement disorder” with “behavioral tests”or “motor behaviors”in entire revised manuscript. 19. Line 238: “The correlation between inflammation and motor deficits was also related to the allocation of brain regions.” This sentence is confusing, please reword. Response: We appreciate the thoughtful question and suggestion! We've reworded the sentence in the revised manuscript (Page 16, line 261-262). “The observed neuroinflammation-motor behaviors association exhibited regional specificity within distinct cerebral regions.” 20. Line 244-245: “However, when there are changes in brain volume, the disease state is difficult to mitigate.” This sentence is confusing, please clarify what is meant by this. Response: We appreciate the thoughtful question and suggestion! We've reworded “brain volume” as “gray matter volume”, along with the discussion for the importance of neuroinflammation in the revised manuscript (Page 12-13, line 262-272). 21. Line 245. “Inflammation may predict disorders in muscle strength earlier in PD.” This sentence is confusing, please clarify what is meant by this. Response: We appreciate the thoughtful question and suggestion! We've reworded the sentence in the revised manuscript (Page 12-13, line 262-272). “A previous study revealed a significant association between grip strength and gray matter volume in these brain regions, including the temporal pontine cortex, hippocampus, striatum, and brain stem[47]. Gray matter atrophy in PD is driven by neuronal loss and structural damage[48]. Although compensatory mechanisms or interventions may slow disease progression[49], there was no evidence identify that PD brain atrophy is a reversible pathological outcome. In the present study, we observed that inflammation was present in the brains of A53T mice and the behavior was improved by MK. Thus, inflammation in brain regions such as cortex, hippocampus, and striatum may serve as an early warning sign of muscle strength disorders in PD. ” 22. Line 246: “between motor coordination functional restores” reword to “restored motor coordination.” Response: We appreciate the thoughtful question and suggestion! We've reworded the “between motor coordination functional restores” reword to “restored motor coordination” in the revised manuscript (Page 13, line 273). 23. Line 248-250: “The hippocampus may be involved in the encoding or retrieval of spatial information[41]. And the higher-order motor integration and planning rely on the inferior parietal cortex[41].”- Section is confusing, suggested edit to “The hippocampus may be involved in the encoding or retrieval of spatial information, whereas higher-order motor integration and planning rely on the inferior parietal cortex [41]”. Response: We appreciate the thoughtful question and suggestion! We've reworded the sentence in the revised manuscript (Page 13, line 275-276). 24. Line 272: Edit “detail” to “detailed” Response: We appreciate the thoughtful question and suggestion! We've reworded “detail” as “detailed” in the revised manuscript (Page 14, line 300). 25. Line 289: Define WT at first use and remove definition here. Response: We appreciate the thoughtful question and suggestion! We've added the full chemical name of WT (wild-type) in the introduction section (Page 4, line 73) remove definition in method section (Page 15, line 317) . 26. Line 295: What is NS? Define at first use. Response: We appreciate the thoughtful question and suggestion! We've added the full chemical name of NS “normal saline” in the revised manuscript (Page 15, line 322). 27. Line 361: “The mice were then allowed to awake for 30 minutes.” Reword. Response: We appreciate the thoughtful question and suggestion! We've reworded the sentence in the revised manuscript (Page 18, line 386-387). “Following the injection, the mice were allowed to recover from anesthesia and were maintained in a wakeful state under observation for a period of 30 minutes.” 28. Line 366: “There is a literature also indicated” Reword. Response: We appreciate the thoughtful question and suggestion! We've reworded the sentence in the revised manuscript (Page 18, line 393-394). “A PET study of an A53T mouse model indicated that static scanning may be more appropriate than dynamic scanning for A53T mice[61].” 29. Line 360-363: “The animals were anesthetized via 1.5% isoflurane before intravenous injection of [18F]GSK (238.9-336.4 MBq/kg) or [18F]L-DOPA (231.2-343.3 MBq/kg). The mice were then allowed to awake for 30 minutes. Thirty minutes after intravenous injection, the mice were anesthetized with 1.5% isoflurane, and PET scans were performed using a nanoPET scanner (Mediso Inc., Hungary).” - Is it correct that mice were anesthetized, injected, recovered and anesthetized again? Why was this done and not awake injection of the tracer or maintained anesthesia to minimize discomfort of the animals? Response: We appreciate the thoughtful question and suggestion! During the experimental process, we encountered the following technical challenges and implemented corresponding adjustments. A subset of A53T transgenic mice exhibited heightened stress sensitivity, rendering precise tail vein injection of radioligands impractical in awake states. To address this, a 5-minute 1.5% isoflurane anesthesia was administered prior to tail vein injection for stress-prone individuals. Initially, a dynamic PET/CT imaging protocol was employed, requiring sustained anesthesia for approximately 50 minutes (including 5 minutes for injection, 5 minutes for positioning, 10 minutes for CT scanning, and 30 minutes for dynamic PET acquisition). However, preliminary experiments revealed a high mortality rate (66.6%) in cohorts subjected to anesthesia exceeding 30 minutes. To optimize animal survival, the imaging protocol was modified as follows. First, for cooperative A53T mice, tail vein injection was performed without anesthesia, with anesthesia induced solely during PET scanning. For stress-prone A53T mice, brief 1.5% isoflurane anesthesia (<10 minutes) was administered for injection, followed by immediate recovery to an awake state post-procedure. Second, dynamic imaging was replaced with static PET/CT scanning, reducing total anesthesia duration to 25 minutes (5 minutes for positioning, 10 minutes for CT, and 10 minutes for PET). Third, respiratory rate was strictly maintained at 40–60 breaths per minute. Scanning was immediately halted and animals were revived if respiratory depression occurred. Following these optimizations, no intra-procedural mortality was observed in experimental cohorts. Preliminary validation confirmed that static imaging retained sufficient sensitivity to acquire reliable data. 30. Line 385: Define VOIs. Response: We appreciate the thoughtful question and suggestion! We've added the full chemical name of VOIs (Volumes of Interest) in the revised manuscript (Page 19, line 412-413). 31. Line 400-401: “At least 10 regions of interest (ROIs) were outlined in each brain region.” Why were 10 ROIs outlined in each region? Response: We appreciate the thoughtful question and suggestion! We've explained the reasons in the revised manuscript (Page 20, line 428-430). “To ensure comprehensive data acquisition, at least ten serial sections were systematically collected from specific brain regions of each mouse. A representative region of interest (ROI) was delineated within each section, resulting in a minimum of 10 independent ROI data points per group. ” 32. Line 408-409: Indicate secondary antibody dilution. Response: We appreciate the thoughtful question and suggestion! We've added secondary antibody dilution in the revised manuscript (Page 20, line 437-441). “After incubation with primary antibodies, the sections were labeled with the corresponding species-specific secondary antibodies for 1 hour at room temperature under light-protected conditions: Alexa Fluor 488-conjugated goat anti-mouse IgG (1:500, Bs-0296G-AF488, Bioss, China) and Alexa Fluor 647-conjugated goat anti-rat IgG (1:500, 112-605-143, Jackson, USA). Then, the slides were incubated with DAPI (1:1000, D1306, Thermo Fisher, USA). ” 33. Line 420: Organize abbreviation table alphabetically. Response: We appreciate the thoughtful question and suggestion! We've organized abbreviation table alphabetically in the revised manuscript (Page 1-22). 34. Overall suggestion to remove all “baseline” data, there is no comparison or discussion of data between baseline and week 5, therefore it does not add to the manuscript to show repeated experimental data with different n numbers multiple times throughout the manuscript. Comparison only seems to be made between the four groups after 5 weeks of treatment. Response: We appreciate the thoughtful question and suggestion! We've remove all baseline data in the revised manuscript. 35. Data is repeated between Figure 2 and Table 2, although nice to have the absolute values in the table this may make the paper too cumbersome and I suggest the table be moved to SI. Figure 2h is labelled as 2i in figure caption, please make this edit. Response: We appreciate the thoughtful question and suggestion! We've removed Table 2 and the “Figure 2i” was reworded as “Figure 2h” in the revised manuscript (Page 30, line 685). 36. Table 1 is redundant to Figure 1e, I suggest Table 1 be moved to SI or removed from the manuscript. Response: We appreciate the thoughtful question and suggestion! We've moved Table 1 to supplementary materials (Table S1 in Supplementary materials). 37. Figure 3 and Table 3 are redundant. Include “*p < 0.05, **p < 0.01 and ***p < 0.001” at the end of Figure 3 caption. Response: We appreciate the thoughtful question and suggestion! We've deleteded Table 3 and added “*p < 0.05, **p < 0.01 and ***p < 0.001”at the end of Figure 3 caption (Page 33, line 701-702). 38. It is not necessary to have both sets of baseline data in Figure 2 and 4, Figure 2 can be revised to demonstrate the behavioral tasks, quantitation removed and the figure moved to SI, where Figure 4 can remain as it contains the same quantitative information of the baseline animals. Addition of stats to the figure caption. What are the asterisks above each timepoint representing? Significant differences between which groups? Response: We appreciate the thoughtful question and suggestion! We've modified original Figure 2 and moved it to supplementary materials as Figure S1(see Figure S1 in Supplementary materials). We have renumbered the original Figure 4 as Figure 2 and added the description of the motor tests in the Figure 2 caption (Page 30-31, line 680-701). 39. Again, there is more redundancy in Figure 5. It is not necessary to show the same sets of baseline data in 3 separate figures/tables throughout the manuscript. I suggest the removal of Figure 3, as Figure 5 contains the same “baseline” imaging data. Add in statistics statement to figure caption when significance is indicated in the figure. Response: We appreciate the thoughtful question and suggestion! We've removed original Figure 3. We have renumbered the original Figure 5 as Figure 3 in the revised manuscript and added the statistics statement in the Figure 3 caption (Page 33, line 709-710). 40. Figure 6 caption. Add in statistics statement. Response: We appreciate the thoughtful question and suggestion! We have renumbered the original Figure 6 as Figure 4 in the revised manuscript and added the statistics statement in the Figure 4 caption (Page 33-34, line 717-719). 41. Figure 7 caption: Remove “There was no difference in the DAPI fluorescence intensity among the four groups.” Not necessary here. Response: We appreciate the thoughtful question and suggestion! We've removed the expression of DAPI in the revised manuscript (Page 35, line 725-726). 42. Table S1 shows the same data as Table 3. Consolidate. Response: We appreciate the thoughtful question and suggestion! The original Table 3 refers to [18F]GSK SUVRLV at baseline, and the original Table S1 refers to [18F]GSK SUV at baseline. We have deleted original Table 3 and original Table S1 in the revised manuscript and supplementary material. 43. Table S13 and Table 5 show the same data. Consolidate. Response: We appreciate the thoughtful question and suggestion! The original Table 5 refers to [18F]GSK SUVRLV after MK treatment, and the original Table 13 refers to [18F]GSK SUV after MK treatment. We have renumbered the original Table 5 as Table 2 in the revised manuscript and deleted original Table S13 in the supplementary material. 44. Table S15, “At least 8 ROIs were outlined in each brain region.” Unclear what is meant by this. In the methods it is indicated that 10 ROIs were drawn. Response: We appreciate the thoughtful question and suggestion! We have revised the expression and renumbered the original Table S15 as Table S13 in the revised supplementary material (see Table S13 in Supplementary materials). 45. In Figure 6 each ROI is outlined in a white dashed line, encompassing the entire region so what does it mean for there to be 8-10 ROIs indicated? Response: We appreciate the thoughtful question and suggestion! We've added the reason for outlining 10 ROIs in autoradiographic image analysis in the revised manuscript (Page 20, line 428-430). 46. I don’t think it’s necessary to have Table S17 and S18 showing SUV and SUVR, if SUVR is the parameter of interest, Table S17 can be removed. Response: We appreciate the thoughtful question and suggestion! We've removed Table S17 and other basaline data in the revised manuscript and the revised supplementary material. ? Reviewer #2: 1. The descriptions related to TableS in the manuscript do not match the actual TableS data, so the consistency of correspondence should be adjusted. For example, Tables S16-18 in the manuscript should actually be Tables S13-14, and Tables S17 and S18 should actually be Tables S15 and S16. There are other inconsistencies beyond those listed in the examples, so authors should check the correspondence of tables throughout the manuscript. Response: Thanks for the recommendation! We have carefully checked and revised the correspondence of tables in the revised manuscript.
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