Dear Reviewer:

Thank you for your valuable comments which are very helpful for revising and improving our paper. We have carefully studied your comments and made corresponding revision. Revised portion are marked in red in the submitted paper. The main corrections in the paper and the responds to the reviewer’s comments are as follows:

Reviewer’s comments:

1. Animal Experiment Design

Group Completeness: The study only sets up the TBI group and the TBI + propranolol group, lacking a normal control group (healthy rats that did not undergo TBI surgery). The absence of the normal control group makes it impossible to clearly determine the effect of propranolol on the cognitive function, lymphoid system, and related protein expression of normal rats, and also makes it difficult to completely distinguish the pathological damage of TBI itself from the specific effect of propranolol intervention. It is recommended to supplement the normal control group to more comprehensively evaluate the effect of propranolol.

Reply: Thanks to you for your excellent comment. We added normal control group (healthy rats that did not undergo TBI surgery) in our study. It turned out that even though propranolol could improve cognitive function after TBI, propranolol could not completely reverse the short-term memory, long-term memory and spatial learning deficits after TBI as compared to normal rats. On the other hand, propranolol could totally reverse TBI-induced p-tau/Aβ deposition. Myelin densities were significantly recovered in the brains of propranolol-treated rats, but propranolol could not completely reverse the myelin densities deficits after TBI as compared to normal rats. Also, propranolol could completely reverse the glymphatic dysfunction after TBI. Moreover, propranolol totally recovered the AQP4 accumulation around astrocyte and vessels after TBI as compared to normal group. The results showed that the level of p-STAT3/MMP2 were significantly downregulated in the propranolol-treated rats, but propranolol could not completely reverse the increased level of p-STAT3/MMP2 after TBI as compared to normal rats. In vitro, it turned out that propranolol reversed the noradrenaline-mediated increased expression of p-STAT3/MMP2 in astrocytes, but propranolol could not totally restore the noradrenaline-mediated expression of p-STAT3/MMP2 as compared to control group. We found that noradrenaline could decrease the lever of AQP4 expression of astrocytes. On the other hand, it showed that propranolol totally reversed the noradrenaline-mediated decline expression of AQP4 of astrocytes.

2. Detection Method Details

Lymphoid System Function Detection: The method of injecting 70kD dextran into the cerebellopontine cistern and detecting fluorescence density is used to evaluate the lymphoid system function. However, the accuracy control measures for the injection site (such as whether it is precisely located by a stereotactic instrument and whether there is a record of the injection position after the injection for anatomical verification) are not explained. Deviation in the injection site may lead to uneven distribution of dextran and affect the reliability of the quantitative results of fluorescence density. It is recommended to supplement the experimental details of precise control of the injection site.

Reply: Thanks to you for your excellent comment. The detail of Lymphoid System Function Detection was added as follow: Anesthetized rats were fixed in a stereotaxic frame and a 30GA needle was inserted into the central cisterna magna to avoid uneven distribution of dextran. 5µl of Dextran (Sigma, MW: 70 kD, USA) was injected into the cisterna magna of TBI rats over 5 minutes using a syringe pump at a flow rate of 2μl/min. Rats were sacrificed at 30 minutes from the start infusion. The rats were transcardially perfused with 0.9% saline followed by 4% paraformaldehyde. 80 mm thick sections were cut and imaged using a 10x objective of a laser-scanning confocal microscope (Zeiss LSM 510 NLO, Carl Zeiss, Germany). MCID image analysis system (Imaging Research, St. Catharines, Ontario, Canada) was used to quantify the fluorescence density. The data were presented as percentage of positive fluorescence area in the brain cross section.

3. Discussion and Conclusion

The discussion only compares the differences between propranolol and other β receptor blockers (such as lipophilicity), but does not fully combine the recent research progress on the regulation of the lymphoid system after TBI (such as the effects of other drugs and physical intervention methods on the lymphoid system), nor discusses the similarities and differences between the results of this study and other studies on the role of propranolol in neurological diseases (such as the research conclusions of propranolol in Alzheimer's disease and Parkinson's disease). It is recommended to expand the discussion scope, place this study in a broader research context, and highlight the innovation and limitations of the study.,This manuscript innovatively proposes a hypothesis that propranolol may regulate the function of the lymphoid system through the STAT3/MMP2 pathway to improve cognition, and this hypothesis is verified through in vitro and in vivo experiments. The research design is logically coherent, and the technical route is clear. It provides a new mechanism explanation and potential therapeutic direction for the repair of cognitive function after TBI, and has certain scientific value and clinical reference significance. However, the research still has areas that can be improved in terms of experimental design details, mechanism depth, and result interpretation. Further supplementation and optimization are required.

Reply: Thanks to you for your good comment. As reviewer suggested, we expanded the discussion scope. The changed discussion section was labeled with red in the main manuscript.

Discussion:

TBI could cause a series of symptoms, such as declined cognitive function, headaches, depression, fatigue, irritability, and anxiety, which influences living quality of TBI patients [1]. Multiple TBI patients benefit from administration of β-blockers (BBs). As one of BBs, propranolol may have more benefits than other BBs because of the lipophilic properties, increasing cerebral perfusion and decreasing catabolism and oxygen consumption of brain [20]. In this research, we conformed that propranolol could enhance defective cognitive function after TBI. But, the mechanism of propranolol-induced cognitive function recovery is still unknown.

These are numbers of proteinaceous waste products (for example: p-tau and Aβ etc.) deposition in chronic traumatic encephalopathy (CTE) and sleep disruption patients after TBI. The waste products are thought to contribute to neurodegeneration processes as Alzheimer’s disease (AD) and TBI [21]. The glymphatic system which helps to clear the waste products in brain is a promising treatment target after TBI. There are lots of factors that could influence the function of glymphatic system, like astrocyte dysfunction and perivascular spaces (PVS) disruption after TBI [22]. AQP4 is wildly distributed in capillary endothelial cells, astrocytes and ependymocytes in the brain. The level of AQP4 is significantly downregulated at 6h after TBI. The cerebral contusion is usually accompanied with cerebral edema, which takes responsible for a variety of syndromes after TBI. It has showed that the TBI-induced edema is deteriorated in the AQP4 knock-out rat which suggested that AQP4 plays an essential role in edema after TBI [23]. Meanwhile, the expression of AQP4 of vessels and astrocytes play an essential role in glymphatic system [8]. Thus, due to the ubiquitous heterogeneity of individuals, conditions, and pathophysiological mechanisms after TBI [24], treating glymphatic system dysfunction maybe a common pathway underlying these variations. The potential TBI treatment targeted at glymphatic system has made big strides these years. Sleep recovery may relieve TBI-induced cognitive impairment. Because, natural sleep or anesthesia could increase convective exchange of cerebrospinal fluid with interstitial fluid which may help to increase the rate of β-amyloid clearance[25]. Administering fingolimod could treat TBI effectively by promoting AQP4 repolarization and improving glymphatic system function [26]. Similar to fingolimod, cannabidiol, exendin-4, minocycline and interleukin-33 could all recover the function of TBI through regulating AQP4 polarization or increasing the AQP4 expression [27-30]. Multisensory gamma stimulation (40 Hz stimulation) can restore glymphatic system to clear amyloid by enhancing the polarization of AQP4, increasing the volume of meningeal lymphatic vessels (mLVs) and synchronizing multiple cells in glymphatic flow [31]. Previous studies suggest that adrenergic inhibitors promote fluid drainage from brain tissue, alleviate sleep disturbance, and decrease brain edema [32, 33]. Thus, it seems that adrenergic inhibitors, like propranolol, have a strong relationship with glymphatic system. In this study, we unexpectedly found that propranolol decreased p-tau and Aβ deposition in brain by glymphatic system recovery after TBI. The results also revealed the facts that propranolol could restore the expression of AQP4 on cerebral blood vessels and astrocytes, which may partly explain the mechanism of propranolol-induced glymphatic system recovery. To sum up, Our and previous studies suggest that AQP4 is a common regulated target that influencing the glymphatic system for the most of treating TBI drugs. Moreover, Other clinical studies have found other considerable beneficial effects of adrenergic inhibition, such as alleviating sleep disturbance [33], improving psychiatric disorders [32]. Thus, adrenergic inhibition, like propranolol, may reveal more diversified beneficial effects than other treating TBI drugs that targeted glymphatic system only. But, the clinical translation of adrenergic inhibition has been challenging, because maintaining cerebral blood perfusion is critical for the TBI patients according to existing guidelines [34]. Thus, this conflict requires further basic and clinical research to resolve.

Propranolol also has significant relationship with other neurological diseases. These are studies show that propranolol could reduce Parkinson’s disease (PD) tremor effectively [35, 36]. The effect of propranolol is may influenced by beta-1 adrenergic receptor in the motor cortex.[36] Also, propranolol represents promising therapeutic effects in Alzheimer's disease (AD) and other cognitive disorders through cholinergic enhancement via butyrylcholinesterase inhibition.[37] In the contrary, there is study shown that the activation of beta-2 adrenergic receptor ameliorated amyloid-β-induced memory deficits. Inhibition of beta-2 adrenergic receptor by propranolol blocks the activation of beta-2 adrenergic receptor-induced improvements in pathological changes [38]. Thus, what role of propranolol is played in the neurological diseases is still controversial. More basic and clinical researches are also needed to explore the mechanism of propranolol for the future.

Hence, we further explored what mechanism induced propranolol-mediated AQP4 recovery of TBI. Dystroglycan (DG) widely expresses at the interface between the basement membrane and the cell membrane in various different kinds of tissues. DG plays an essential role in for maintaining the polarization of AQP4 in astrocytes. There are evidences indicate that nonspecific matrix metalloproteinase (MMP) inhibitors reverse the level of DG in astrocyte under oxygen–glucose deprivation condition, which implies that MMP takes responsible for the astrocyte-induced DG reduction. There are evidences show that MMP-2 is involved in cleavage of DG in cerebral ischemia, but the mechanism remains unclear [10, 39, 40]. Norepinephrine (NE), as a neurotransmitter and neuromodulator, plays a major role in multiple activities of neuronal or non-neuronal cells. The evidences show that NE promotes the expression of phosphorylated signal transducer and activator of transcription 3 (p-STAT3) dependent on a concentration-dependent manner in pancreatic cancer cells. NE-induced phosphorylation of STAT3 is inhibited by propranolol (aβ-adrenergic receptor inhibitor, β-ARI). Also, the level of MMP2 is downregulated through blocking STAT3 [11]. Interestingly, it is reported that clearance rate of glymphatic system is faster during sleep than during waking hours [41]. In the brain, the NE-the locus coeruleus (LC) system is shut down during sleep which may help to potentate more rapid clearance of waste products [21]. Thus, we speculated that NE/β-ARI/STAT3 signaling pathway may take responsible for deficiency of AQP4 after TBI. In the present study, we found that propranolol decreased p-STAT3/MMP2 expression after TBI in vivo which may partly explain the mechanism of propranolol-mediated AQP4 recovery of TBI. The vitro research showed that propranolol reversed the noradrenaline-mediated increased expression of p-STAT3/MMP2 and decline expression of AQP4 of astrocytes, which further confirmed our hypothesis.

Our study provides a new mechanism explanation and potential therapeutic direction of propranolol for the repair of cognitive function after TBI. But there are still lots of limitations in our study. Even though, we confirmed that propranolol improved cognitive function after TBI. But we could not explain why propranolol could not completely reverse the memory deficits, myelin densities deficits and expression of p-STAT3/MMP2 after TBI as compared to normal rats. Moreover, in our study (data not seen), high dose of propranolol (10mg/kg) may harm the cognitive function after TBI. Thus, we hypothesize if there are other mechanisms of propranolol-mediated benefits or disadvantages. As shown in other study, propranolol played an important role in regulating inflammation which was not seen in our study. There are different adrenergic receptors (α and β) in multiple types of cells in the brain. What roles of differential adrenergic receptors are played in TBI is still need further explored. Whether propranolol could influence AQP4 polarization directly still need to be further explored [7].

Thank you for your constructive comments. We tried our best to improve the manuscript and made some changes in the manuscript. These changes will not influence the content and framework of the paper. We appreciate for Reviewer’ warm work earnestly, and hope that the correction will meet with approval.

Once again, thank you very much for your comments and suggestions.

References�

1.McInnes K, Friesen CL, MacKenzie DE, Westwood DA, Boe SG. Correction: Mild Traumatic Brain Injury (mTBI) and chronic cognitive impairment: A scoping review. PloS one. 2019;14(6):e0218423. doi: 10.1371/journal.pone.0218423. PubMed PMID: 31185044; PubMed Central PMCID: PMC6559670.

2.Ramos-Cejudo J, Wisniewski T, Marmar C, Zetterberg H, Blennow K, de Leon MJ, et al. Traumatic Brain Injury and Alzheimer's Disease: The Cerebrovascular Link. EBioMedicine. 2018;28:21-30. doi: 10.1016/j.ebiom.2018.01.021. PubMed PMID: 29396300; PubMed Central PMCID: PMC5835563.

3.Iliff JJ, Chen MJ, Plog BA, Zeppenfeld DM, Soltero M, Yang L, et al. Impairment of glymphatic pathway function promotes tau pathology after traumatic brain injury. The Journal of neuroscience : the official journal of the Society for Neuroscience. 2014;34(49):16180-93. doi: 10.1523/JNEUROSCI.3020-14.2014. PubMed PMID: 25471560; PubMed Central PMCID: PMC4252540.

4.Khalili H, Ahl R, Paydar S, Sjolin G, Cao Y, Abdolrahimzadeh Fard H, et al. Beta-Blocker Therapy in Severe Traumatic Brain Injury: A Prospective Randomized Controlled Trial. World journal of surgery. 2020;44(6):1844-53. doi: 10.1007/s00268-020-05391-8. PubMed PMID: 32002583.

5.Solito E, Sastre M. Microglia function in Alzheimer's disease. Frontiers in pharmacology. 2012;3:14. doi: 10.3389/fphar.2012.00014. PubMed PMID: 22363284; PubMed Central PMCID: PMC3277080.

6.Acosta C, Anderson HD, Anderson CM. Astrocyte dysfunction in Alzheimer disease. Journal of neuroscience research. 2017;95(12):2430-47. doi: 10.1002/jnr.24075. PubMed PMID: 28467650.

7.O'Donnell J, Zepp

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Submitted filename: Responses to reviewer.docx

Dear Reviewer:

Thank you for your valuable comments which are very helpful for revising and improving our paper. We have carefully studied your comments and made corresponding revision. Revised portion are marked in red in the submitted paper. The main corrections in the paper and the responds to the reviewer’s comments are as follows:

Reviewer’s comments:

Reviewer #2: This reviewers commends the authors for their extensive revision. The authors have significantly improved the scientific rigor of their study by adding the normal control group as suggested by the previous reviewer, and by expanding the discussion. The only part that requires additional minor revision is a thorough review of the manuscript by preferably a native English speaker to correct the numerous minor grammatical errors.

Reply: Thanks to you for your excellent comment. We tried our best to fix all the grammatical mistakes throughout the manuscript and fully complied with all the reviewer’s requirements.

Reviewer #3: First, the statistical comparisons are unclear. The manuscript should provide clear statistical comparisons between the sham vs. TBI groups and between the sham vs. propranolol-treated TBI groups where applicable. These comparisons are necessary to determine whether TBI significantly alters each measured outcome relative to baseline, and whether propranolol restores the outcome toward sham/control levels. In the figures, please clearly mention sham/control groups and as well as add statistical comparison with Sham/control groups

Second, Figures 2, 3, 4, and 6 do not include scale bars.

the study does not directly demonstrate that the STAT3/MMP2 pathway mediates propranolol’s effects. To support this mechanism, additional functional experiments would be needed. For example, the authors should test whether a STAT3 inhibitor mimics the effect of propranolol after TBI, whether MMP2 inhibition rescues AQP4 localization and glymphatic function, and whether MMP2 overexpression blocks or reduces the beneficial effects of propranolol.

Adding comparison with blocker will make it more strong.

Reply: Thanks to you for your excellent comment. First, all statistically significant comparisons were added to the Results and Figures. Second, scale bars were also added in Figures 2, 3, 4, and 6. Third, we introduced STAT3 and MMP2 inhibitors (S31-201 and SB-3CT) in vivo and vitro experiments. The results as:

Result�

Propranolol improved cognitive function after TBI:

Compared with untreated TBI rats, propranolol, S31-201 and SB-3CT all significantly relieved short- and long-term memory deficits in the novel object recognition and odor tests, though none of the three agents fully recovered memory function compared with the normal rats. In the Morris water maze test, propranolol, S31-201, and SB-3CT all alleviated the deficits in spatial learning and memory in TBI rats. However, none of these three agents fully restored spatial cognitive function compared with the normal rats. (Fig 1)

Figure1. A) The novel object recognition test revealed markedly improved short-term memory in the propranolol, S31-201 and SB-3CT treatment groups compared with the TBI group. Nevertheless, none of the three treatments fully rescued TBI-induced short-term memory deficits compared with normal rats. B) The odor test demonstrated obvious long-term memory improvements in the propranolol, S31-201 and SB-3CT treatment groups compared with the TBI group, whereas complete reversal of long-term memory impairment was not achieved relative to normal rats. C, D) In the Morris water maze test, TBI rats receiving propranolol, S31-201 or SB-3CT displayed significantly ameliorated spatial learning and memory capacities. Still, none of the interventions fully restored spatial cognitive function compared with normal controls. (*p<0.05, n=6 per group)

Propranolol decreased p-tau/Aβ deposition and white matter (WM) damage:

To explore the underlying mechanism by which propranolol improves cognitive function, we examined cerebral p-tau and Aβ deposition levels. The results demonstrated that propranolol, S31-201, and SB-3CT all totally reduced TBI-triggered p-tau and Aβ accumulation in the brain. Moreover, no statistically significant differences were detected among the three treatment groups and the normal group (Figure 2).

To further verify whether propranolol, S31-201, and SB-3CT could rescue TBI-induced white matter (WM) integrity damage, brain myelin density was quantified. As illustrated in Figure 2, myelin density was markedly restored in rats treated with propranolol, S31-201, or SB-3CT relative to untreated TBI rats. Nevertheless, none of the three interventions fully restored myelin density to the level observed in normal rats.

Figure2. (A–F) Propranolol, S31-201, and SB-3CT all totally alleviated TBI-induced cerebral p-tau accumulation. No statistically significant differences were observed among the three treatment groups and the normal control group. G–L) Similarly, propranolol, S31-201, and SB-3CT totally lowered TBI-triggered brain Aβ accumulation, with no significant differences detected among the treatment groups and normal controls. (M–R) Myelin density was significantly recovered in drug-treated rats compared with untreated TBI rats. However, none of the three interventions restored myelin density completely to the level of normal rats. (*p<0.05, n=6/group,10×40 objective lens).

Propranolol recovered glymphatic dysfunction in TBI rats:

Since propranolol was found to reduce cerebral p-tau and Aβ deposition following TBI, we further examined glymphatic system function. The results revealed that propranolol, S31-201, and SB-3CT totally rescued TBI-induced delay in metabolic waste clearance. No significant differences were detected among the three treatment groups and the normal group. (Fig3)

Figure3. (A-F) Propranolol, S31-201 and SB-3CT totally rescued TBI-induced delay in metabolic waste clearance. No significant differences were detected among the three treatment groups and the normal group. (*p<0.05, n=5/group)

Propranolol restored AQP4 expression after TBI:

To elucidate the mechanism by which propranolol ameliorates TBI-induced impairment of glymphatic function, we quantified perivascular AQP4 expression and astrocyte levels in the brain. The results demonstrated that propranolol, S31-201, and SB-3CT totally restored perivascular and astrocytic AQP4 expression relative to untreated TBI rats. No statistically significant differences were identified among the three treatment groups and the normal group. (Fig 4)

Figure4. (A–C, G–I, M–O, S–U, Y–a, e) Propranolol, S31-201, and SB-3CT totally restored perivascular AQP4 (green) expression compared with untreated TBI rats. No statistically significant differences were detected among the three treatment groups and the normal group. Blood vessels were labeled with vWF (von Willebrand factor, red), and cell nuclei were counterstained with DAPI. (D–F, J–L, P–R, V–X, b–d, f) Propranolol, S31-201, and SB-3CT totally restored astrocytic AQP4 (green) expression compared with untreated TBI rats. No statistically significant differences were detected among the three treatment groups and the normal group. Astrocytes were labeled with glial fibrillary acidic protein (GFAP, red), and cell nuclei were counterstained with DAPI. (*p<0.05, n=6/group, 10×40 objective lens)

Propranolol decreased p-STAT3/MMP2 expression after TBI:

To further explore the mechanism of propranolol-induced improvement of cognitive function and glymphatic system recovery after TBI, Western blot was used to test the expression of phosphorylated STAT3 (p-STAT3) and MMP2. The results showed that the levels of p-STAT3 and MMP2 were significantly downregulated in the propranolol-treated TBI rats, but propranolol could not completely reverse the increased levels of p-STAT3 and MMP2 after TBI compared with normal rats. (Fig5)

Propranolol reversed the noradrenaline-mediated increased expression of p-STAT3/MMP2 in astrocytes:

To further clarify the mechanism of propranolol-induced reduction in p-STAT3/MMP2 expression in TBI rats, we treated astrocytes with noradrenaline and propranolol. Western blot data showed that the levels of p-STAT3 and MMP2 were upregulated after astrocytes were incubated with noradrenaline. Subsequent addition of propranolol reversed the noradrenaline-induced elevation in p-STAT3 and MMP2 expression; however, propranolol failed to fully restore p-STAT3 and MMP2 levels to those of the control group. No significant difference was observed between the propranolol-only group and the control group. (Fig5)

Figure5. A, B) MMP2 protein level was markedly decreased in propranolol-treated rats compared with TBI model rats. Nevertheless, propranolol could not fully restore MMP2 upregulation caused by TBI to the level of normal rats. GAPDH was applied as the loading reference. A, C) Western blot quantification demonstrated that p-STAT3 expression was significantly lower in the propranolol intervention group than in the TBI group, whereas complete recovery of TBI-induced abnormal p-STAT3 expression was not achieved by propranolol treatment relative to normal rats. Total STAT3 was set as the loading control. D, E) Incubation with noradrenaline led to significant upregulation of MMP2 in astrocytes. Additional propranolol treatment rescued the noradrenaline-mediated elevation in MMP2 expression, though full normalization to the control group level was not attained. No statistical difference was detected between the single propranolol group and the control group. GAPDH served as the loading control. D, F) Noradrenaline stimulation significantly increased p-STAT3 levels in astrocytes, and this upregulation was partially counteracted by propranolol supplementation. However, p-STAT3 expression could not be fully restored to the baseline level of the control group. No statistical difference was observed between the single propranolol group and the control group. Total STAT3 was used as the loading control. (*p < 0.05)

Propranolol antagonized the noradrenaline-mediated decline expression of AQP4 of astrocytes:

To further elucidate how propranolol elevates AQP4 expression following TBI, astrocytes were subjected to treatments with noradrenaline alone or combined with propranolol, S31-201 or SB-3CT. Immunohistochemical staining was utilized to quantify AQP4 expression in astrocytes. Noradrenaline treatment markedly downregulated AQP4 levels in astrocytes, whereas co-administration of propranolol, S31-201 or SB-3CT fully rescued this noradrenaline-triggered AQP4 downregulation. (Fig6)

Figure6 D–F, S) AQP4 (red) expression was significantly decreased in astrocytes following noradrenaline treatment relative to the control group. G–I, S) Treatment with propranolol fully restored the noradrenaline-evoked downregulation of AQP4 (red) in astrocytes. J–L, S) No statistically significant difference in AQP4 (red) expression was detected between the propranolol single-treatment group and the control group. M–O, S) S31-201 fully rescued the noradrenaline-induced reduction in astrocytic AQP4 (red) expression. P–R, S) SB-3CT also completely reversed the noradrenaline-triggered decline in AQP4 (red) expression in astrocytes. Nuclei were counterstained with DAPI. *p<0.05, 10×20 objective lens, 10 of fields and 6 of cells per field were counted).

Reviewer #4: 1. The authors have comprehensively addressed the three major issues raised in the previous round. They have added a normal healthy control group to differentiate TBI pathology from drug effects, provided precision and stereotaxic instrumentation details for the glymphatic system detection method, and extensively expanded their discussion to integrate broader clinical frameworks, limitations, and relevant neurological disease models.

2. By incorporating a healthy normal rat control group alongside the TBI and TBI + Propranolol groups, the in vivo experimental structure is now complete and rigorous. The added findings confirm that while propranolol significantly improves spatial learning, memory, and white matter integrity, it does not completely reverse all TBI deficits compared to healthy controls, which demonstrates a realistic and data-supported conclusion. The matching in vitro model using astrocytes treated with norepinephrine further confirms the mechanistic role of the STAT3/MMP2 pathway.

3. The statistical evaluation is robust. An investigator blinded to the experimental groupings conducted the quantification. Multiple brain slices (five slides per sample, eight fields per slide) were digitized uniformly via an objective MCID image analysis system using density thresholds to eliminate bias. Student’s t-test was appropriately utilized to evaluate variations between distinct groups, and data are standardly presented as mean ± SE.

4.The authors state that they have added the missing statistical baselines and control configurations directly into the main text and figures (e.g., Figure 1 revisions) to ensure compliance with PLOS data sharing policies. (Note: Please do a quick final check of the full manuscript PDF to confirm no separate raw data repository was omitted).

5. The revision reads clearly and follows standard scientific English formatting. The expanded text and author replies are organized logically. A minor typographical error exists on Page 2 / Page 6 where the section thickness is written as "80 mm thick sections"—this should be corrected to micrometers (µm) as seen elsewhere in the histology protocol.

Reply: Thanks to you for your good comment. The "80 mm thick sections" was corrected to micrometers (µm).

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