Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The article provides a comprehensive review on the topic Ischemia-Reperfusion Injury in Stroke. It written quite well and is rather easy to follow. My main concern is the reference list (which I commented above) and I strongly reocmmend the authors to review the reference section critically and provide more appropriate references, as an important purpose of a good review article is to enable readers to further explore the topics discussed by reading the references, therefore the best and the most relevant references should always be cited.

Other minor comments include:

• please provide complete descriptions of the terms when abbreviations are first used.
• please provide legends for the abbreviations in the figures (especially figure 1), as figures should be self-contained in their own for readers to quickly glimpse the topics discussed.
• some typos: line 97: asa - as a, line 153: remove "Third bullet.", line 273: remove "– with Tracked Changes & Reference Flags".

Author Response

Dear Reviewer,

Thank you for taking the time to review our manuscript (Manuscript ID: 4219645) and for the opportunity to submit a revision. We greatly appreciate your editorial guidance, as well as the insightful comments from the reviewers. We have carefully addressed all of the feedback in the revised manuscript (new text is highlighted in red). Below, please find our point-by-point responses to the reviewers’ comments and suggestions.

Comments 1: The article provides a comprehensive review on the topic Ischemia-Reperfusion Injury in Stroke. It written quite well and is rather easy to follow. My main concern is the reference list (which I commented above) and I strongly reocmmend the authors to review the reference section critically and provide more appropriate references, as an important purpose of a good review article is to enable readers to further explore the topics discussed by reading the references, therefore the best and the most relevant references should always be cited. 

Response 1: We appreciate this suggestion and have conducted a comprehensive review of our reference section. Consequently, outdated and unrepresentative literature has been replaced with high-quality, relevant sources, and redundant citations have been removed. Notable examples include the updates to references [2] (p. 2, Line 65), [3–4] (p. 3, line 67), and [8] (p. 3, line 94). All remaining reference updates are marked in red in the revised manuscript.
References
2. Majumder, D. Ischemic Stroke: Pathophysiology and Evolving Treatment Approaches. Neurosci Insights 2024, 19, 26331055241292600, doi:10.1177/26331055241292600.
3.  Weiss, A.; Ding, Y. Beyond Reperfusion: Adjunctive Therapies Targeting Inflammation, Edema, and Blood-Brain Barrier Dysfunction in Ischemic Stroke. Cerebrovasc Dis 2025, 55, 292–301, doi:10.1159/000547092.
4.  Zhang, M.; Liu, Q.; Meng, H.; Duan, H.; Liu, X.; Wu, J.; Gao, F.; Wang, S.; Tan, R.; Yuan, J. Ischemia-Reperfusion Injury: Molecular Mechanisms and Therapeutic Targets. Sig Transduct Target Ther 2024, 9, 12, doi:10.1038/s41392-023-01688-x.
8.  Li, S.; Fisher, M. Improving Large Animal Ischemic Stroke Models for Translational Studies in the Era of Recanalization. Stroke 2023, 54, e16–e19, doi:10.1161/STROKEAHA.122.041354.

Other minor comments 2 : Please provide complete descriptions of the terms when abbreviations are first used.

Response 2 :  Thanks for your suggestion, we have ensured that all abbreviations are spelled out at their first use and then used consistently throughout the text. Examples of these corrections include:
•    Page 1, line 34: “ischemia–reperfusi injury (I/R)”
•    Page 1, line 36: “blood–brain barrier (BBB)”
•    Page 1, line 39: “cerebral ischemia/reperfusi (CIRI)”
•    Page 2, line 44 to 45: “reactive oxygen species (ROS)”
•    Page 2, line 45 to 46: “tight juncti (TJ)”
•    Page 2, line 49: “transient receptor potential melastatin 7”
•    Page 3, lines 83 to 84: “brain microvascular endothelial cells (BMECs) ”
•    Page 3, line 101: “central nervous system (CNS)”
•    Page 4, lines 128 to 129: “tumor necrosis factor-α (TNF-α)”
•    Page 4, line 129: “inducible nitric oxide synthase (iNOS)”
•    Page 4, lines 129 to 130: “matrix metalloproteinases (MMPs)”
•    Page 5, line 155: “astrocyte neur lactate shuttle (ANLS)”
•    Page 6, line 164: “Mitochdrial permeability transiti pore (mPTP)”
•    Page 6, line 166: “nuclear factor kappaB (NF-κB)”
•    Page 6, lines 190 to 190: “electr transfer chain (ETC)”
•    Page 7, line 209: “vascular endothelial growth factor (VEGF)”
•    Page 7, line 217: “extracellular matrix (ECM)”
•    Page 8, lines 257 to 258: “membrane permeabilizati (MOMP)”
•    Page 8, line 261: “death-inducing signaling complex (DISC)”
•    Page 8, line 265: “Endoplasmic reticulum (ER)”
•    Page 8, line 272: “gasdermin D (GSDMD)”
•    Page 8, line 273: “damage-associated molecular patterns (DAMPs)”
•    Page 8, line 274: “pattern recogniti receptors (PRRs)”
•    Page 10, line 336: “Transient receptor potential melastatin 7 (TRPM7)”
•    Page 14, line 482: “exosome-like nanoparticles (ELN)”
•    Page 14, line 488: “dichloroacetate (DCA)”
•    Page 14, line 489: “pyruvate dehydrogenase kinase (PDK)”
•    Page 14, line 491: “pyruvate dehydrogenase complex (PDC)”
•    Page 14, line 496: “β-hydroxybutyrate(β-HB)”
•    Page 15, line 534: “remote ischemic precditiing (RIPC)”

Other minor comments 3: Please provide legends for the abbreviations in the figures (especially figure 1), as figures should be self-contained in their own for readers to quickly glimpse the topics discussed.

Response3: We appreciate this suggestion and have carefully revised all figure captions to include a comprehensive list of abbreviations at the end. For instance, in the chart description on page 3 (lines 79 to 81), abbreviation keys for ROS, NOS, ONOO⁻, mPTP, Glu and LPO were added. In the chart description on page 5 (lines 141 to 148), abbreviation keys for IL-1β, GSDMD, TNF-α, GPX4, ACSL4, PTGS2, MDA, GSH-Px, SOD, TRPM7, NMDAR, Glu, BAX, Bcl-2, BBB and MMP9 were added. These newly added contents are marked in red in the revised manuscript. In the chart description on page 10 (lines 319 to 322), abbreviation keys for TRPM7, MMP, TJ, VEGF, EPC, BBB and ECM were added. These newly added contents are marked in red in the revised manuscript.

Other minor comments 4: Some typos: line 97: asa - as a, line 153: remove "Third bullet.", line 273: remove "– with Tracked Changes & Reference Flags".

Response 4: We have deleted “third bullet” on line 164 of page 6 and "with Tracked Changes & Reference Flags" on line 281 of page 8. Revise "asa" on line 101 of page 3 to "as a".

 

Reviewer 2 Report

Comments and Suggestions for Authors

In this review article, the authors summarize the various molecular mechanisms associated with reperfusion injury in patients treated for ischemic stroke and the potential therapeutic interventions for this pathology from a blood-brain-barrier (BBB) dysfunction-centric view. The authors see the disruption of the BBB during reperfusion injury in this patient population as a critical element that sets in motion a cascade of molecular events that are potentially actionable from the therapeutic standpoint. In short, the reperfusion event that follows the reestablishing of cerebral blood vessels post thrombolysis/thrombectomy interventions leads to biochemical imbalances in multiple cell types (starting with the endothelial cells of the affected BBB area) which translate into intracellular calcium overload and excessive production of reactive oxygen species (ROS) in the first few minutes. This sets off a cascade of biochemical changes that result in mitochondrial damage (triggered by the opening of MPT pores), the release of neuroinflammatory mediators and cell death in the affected brain tissue.

The authors nicely captured the above cascades of molecular events into a well-organized, easy to ready narrative. In my opinion, the review they wrote could certainly benefit the potential readers interested in this topic. The summary and brief discussion of each potential therapeutic intervention for reperfusion injury is also useful, although I would have preferred to read a more critical appraisal of these interventions by the authors since most of these treatment modalities failed to translate into the clinic. For instance, cyclosporine which was studied as a potential therapy intended to counteract the excessive intracellular calcium flow and the mitochondrial damage during reperfusion injury in target tissues failed to demonstrate any benefit in two clinical trials (CIRCUS and CYCLE) that were designed to test this drug’s ability to prevent reperfusion injury post-myocardial infarction. Similarly, many other therapeutic interventions mentioned by the authors performed rather poorly in clinical trials. In fact, to my knowledge, the only neuroprotective medication(s) currently approved to alleviate reperfusion injury post-ischemic stroke is the ROS scavenger Edaravone in Japan, which is also approved to be administered together with dexborneol (an anti-inflammatory agent) in China. Perhaps the authors could summarize the various medications/interventions that were tested in the clinic (and the corresponding clinical trials) for their ability to prevent or alleviate reperfusion injury events post-ischemic stroke in a separate table.

Author Response

Dear Editor and reviewer,

We would like to express our sincere gratitude to you for sparing your valuable time to review our manuscript (Manuscript ID: 4219645), and for giving us the opportunity to revise. We greatly appreciate your helpful comments and the many insightful comments of all the reviewers. We have modified the manuscript accordingly (revised text in red). Below please find our point-by-point response to the questions and suggestions raised by the reviewers.

Comments 1: The authors nicely captured the above cascades of molecular events into a well-organized, easy to ready narrative. In my opinion, the review they wrote could certainly benefit the potential readers interested in this topic. The summary and brief discussion of each potential therapeutic intervention for reperfusion injury is also useful, although I would have preferred to read a more critical appraisal of these interventions by the authors since most of these treatment modalities failed to translate into the clinic. For instance, cyclosporine which was studied as a potential therapy intended to counteract the excessive intracellular calcium flow and the mitochondrial damage during reperfusion injury in target tissues failed to demonstrate any benefit in two clinical trials (CIRCUS and CYCLE) that were designed to test this drug’s ability to prevent reperfusion injury post-myocardial infarction. Similarly, many other therapeutic interventions mentioned by the authors performed rather poorly in clinical trials. In fact, to my knowledge, the only neuroprotective medication(s) currently approved to alleviate reperfusion injury post-ischemic stroke is the ROS scavenger Edaravone in Japan, which is also approved to be administered together with dexborneol (an anti-inflammatory agent) in China. Perhaps the authors could summarize the various medications/interventions that were tested in the clinic (and the corresponding clinical trials) for their ability to prevent or alleviate reperfusion injury events post-ischemic stroke in a separate table.

Response 1:  We completely agree that a high-quality review must not only summarize potential therapeutic interventions but also critically evaluate why most have failed in clinical translation. We appreciate you pointing out the overly optimistic tone of our original manuscript, and we have substantially revised the text to address this concern.

To provide this critical analysis, we divided our revisions into two categories:

1. Interventions with clear evidence of translational failure:

We have added critical discussions explaining exactly why these specific interventions stalled, as seen in the following examples:

Page 10-11 (lines 341–345): Highlighted the lack of TRPM7-targeted agents in clinical trials due to cardiovascular side effects and limited isoform selectivity.

Page 11 (lines 367–369): Noted that MitoQ and SS-31 have failed clinical translation in stroke due to limited efficacy, short half-lives, and low bioavailability.

Page 12 (lines 419–423): Explained that ATN-161’s strict requirement for immediate post-reperfusion administration makes it clinically unfeasible given the variable delays in human stroke patients.

Page 12 (lines 438–443): Pointed out that z-VAD-fmk requires invasive intraventricular injection, fundamentally precluding its clinical translation.

Page 13 (lines 462-465): Used the failure of Deferiprone in Parkinson’s disease as a cautionary tale regarding the fundamental differences between preclinical models and human pathology.

Page 14 (lines 492-494): Noted that despite its efficacy, DCA’s unacceptable toxicity in Phase 2 trials prevents its approval.

Page 14 (lines 497–500): Highlighted that β-HB’s lack of efficacy in intracerebral hemorrhage suggests that blindly expanding its use to all stroke types will likely lead to failure.

Page 14 (lines 503–509): Discussed the biphasic effects of HIF-1α, emphasizing that the greatest clinical challenge is precisely controlling the degree and timing of its activation.

*Note: We also added a critical reality check on page 13 (lines 453–458) regarding disulfide death-like pathways, noting that while promising, clinical success still requires rigorous validation.*

2. Interventions with no/weak evidence of failure:

For broader interventions lacking definitive failure data, we analyzed the overarching systemic hurdles:

Page 15-16 (lines 552–564) & Page 16: We added a comprehensive summary of why most neuroprotective strategies fail to translate. This includes discrepancies between animal models and human pathology, poor blood–brain barrier (BBB) penetration, the complexity of the ischemia-reperfusion cascade, overly narrow therapeutic windows, lack of drug brain-targeting capabilities, systemic side effects, and imprecise clinical trial designs.

Page 13-14 (lines 471–475) & Page 14: We reinforced this by stating that core issues—such as appropriate therapeutic windows, feasible administration strategies, and BBB penetration efficiency—still lack systematic clinical evidence.

3. Addition of Clinical Trial Summary (Cyclosporine Example & Table 2):

Your point regarding Cyclosporine was highly incisive. Inspired by your suggestion to systematically summarize drugs tested in clinical trials, we have added a new Section 4.5 (Page 10) and created Table 2 (after line 570).

As introduced in the text: *“Table 2 systematically summarizes the drugs/interventions tested in clinical trials for the prevention or alleviation of reperfusion injury after ischemic stroke, clearly presenting the clinical research progress, mechanisms of action, and existing limitations to provide a reference for future research and clinical applications.”*

We believe these substantial additions have successfully shifted the tone of the manuscript from merely optimistic to rigorously critical. All modified text has been highlighted in red.

 

Table 2. Drugs/interventions that have been verified in clinical trials for the prevention or alleviation of reperfusion injury after ischemic stroke.”

Category	Drug	Conclusion	Clinic trail	Ref.
Restoration of Ion and Metabolic Homeostasis	Cyclosporine A	It failed to effectively reduce the size of infarction and studies have shown no benefit.	Phase II clinical trial	[140, 141]
	Nerinetide	It could slightly reduce the mortality rate of AIS, but the functional outcome was not significantly improved.	Phase III clinical trial	[142]
	Magnesium sulfate	Ultra-early administration was safe, but it had no significant neuroprotective effect.	Phase III clinical trial	[143]
	Dextromethorphan	Safe, but with no significant neuroprotective benefits, it was not advanced to Phase III and has limited clinical application.	Phase II clinical trial	[144]
	Aptiganel/ Cerestat	The high-dose group had a higher mortality rate, obvious central adverse reactions and no neuroprotective benefits.	Phase III clinical trial	[145]
	Selfotel	The therapeutic effect is not definite and it may also have neurotoxic effects in cerebral ischemia.	Phase III clinical trial	[146]
Modulation of Cell Death and Inflammatory Pathways	Edaravone dexborneol	Its therapeutic effect was superior to that of edaravone, but its safety issues were a cause for concern.	Phase III/IV clinical trials	[147, 148]
	Cerovive	The failure was declared as no positive results could be replicated.	Phase III clinical trial	[149]
	Alpha-lipoic acid	It was in the exploration stage and no clear evidence of clinical benefits had been formed yet.	Phase II exploratory clinical trial	[150]
	Minocycline	The effectiveness in improving patients' functional prognosis and good safety.	Phase III clinical trial	[151]
	glibenclamide	The primary endpoint was not reached and there was no significant improvement in functional outcomes.	Phase III clinical trial	[152]
	Fingolimod	It had good safety and can improve early neurological deficits.	Phase II exploratory clinical trial	[153, 154]
	Natalizumab	The primary endpoint was not reached and there was no significant improvement in functional outcomes.	Phase II clinical trial	[155]
	Anakinra	Preliminary exploration of anti-inflammatory effects has been conducted, but no clear evidence of reperfusion injury protection has been formed yet.	Phase II clinical trial	[156]
	DL-3-n-butylphthalide	It significantly improved neurological deficits and has good safety, making it a first-line drug for acute ischemic stroke in China.	Phase III/IV clinical trials	[157, 158]
	deferoxamine	Dose exploration and safety trials were lacking in confirmatory efficacy studies.	Phase II clinical trial	[159]
	Otaplimastat	Intravenous administration of oplimaristat as an adjuvant treatment for patients receiving rtPA was feasible and generally safe.	Phase II clinical trial	[160]
	Human Urinary kallidinogenase	It effectively improved perfusion in ischemic areas and neurological function and was used in China for reperfusion injury in AIS.	Phase IV clinical trial	[161]
Regulation of microcirculation, anticoagulation and thrombolysis	Cilostazol	For secondary prevention of non-cardiogenic ischemic stroke	Phase IV clinical trial	[162]
	rt-PA	Significantly improved prognosis	Phase III clinical trial	[163, 164]
	Tenecteplas	The recanalization rate was higher than that of alteplase and the safety was comparable.	Phase III clinical trial	[164, 165]
	Tirofiban	Early use was associated with good functional outcomes, but the risk of bleeding increased.	Phase III clinical trial	[166]
	Eptifibatide and Argatroban	Negative outcome, failed to demonstrate functional benefit	Phase III clinical trial	[167]
Physical intervention and stem cell regeneration	Remote ischemic preconditioning	Exploratory application, no standardized protocol has been formed and clinical evidence is limited.	Clinical exploratory research	[168, 169]
	Therapeutic hypothermia	Only a portion of the patients reached the target temperature and there was no difference in the efficacy analysis. Additionally, the trial was terminated due to slow enrollment and financial issues.	Phase III clinical trial	[170]
	Hyperbaric oxygen therapy	It was used for the adjuvant treatment of reperfusion injury and the therapeutic effect shows individual differences, which requires further experimental research.	Phase II clinical trial	[171]
	Mesenchymal stromal cell	It may improve functional recovery, but its therapeutic effect has not been fully confirmed.	Phase II clinical trial	[172]
	Endothelial progenitor cell	Intracarotid infusion of autologous CD34+ cells was safe and may improve the long-term prognosis of patients with acute ischemic stroke.	Phase II clinical trial	[173]

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Reviewer 3 Report

Comments and Suggestions for Authors

This is an interesting and well-structured review addressing a highly relevant topic. The manuscript covers a broad range of mechanisms involved in BBB disruption after CIRI and provides a comprehensive overview of potential therapeutic strategies. Overall, the work is valuable, but a few aspects could be further refined to enhance clarity and scientific strength.

Major comments
1. A more nuanced discussion of the level of evidence would strengthen the therapeutic section. Many interventions are presented as promising, but at times the distinction between preclinical evidence and clinical applicability is not entirely clear. In some paragraphs, the tone may appear slightly stronger than what the current evidence fully supports. It would be helpful to more explicitly indicate which approaches remain experimental and to briefly acknowledge current translational limitations. This would make the review more balanced and clinically meaningful.
2. The therapeutic section could benefit from a clearer organizing framework. While the content is rich, it sometimes reads as a sequence of different strategies rather than a fully integrated discussion. A more structured approach (for example grouping interventions by mechanism, timing, or level of evidence) could help guide the reader and improve the overall flow.
3. The reference list would benefit from a careful revision. There appear to be a few duplicated entries and some inconsistencies in formatting. A thorough check of the bibliography, including alignment between statements and supporting references, would improve the overall quality of the manuscript.

Minor comments
- Some minor language and formatting adjustments are recommended. For example, “Stem cell therapeutic” could be revised to “Stem cell therapy”, and “Future Direction” to “Future Directions”. A general language polishing would further improve readability.
- A few repetitions and small inaccuracies should be corrected. The sentence describing the immunomodulatory effects of RIPC appears repeated, and the abbreviation “MACO” likely should be corrected to “MCAO”.

Author Response

Dear Editor and reviewer,

Thank you for taking the time to review our manuscript (Manuscript ID: 4219645) and for giving us the opportunity to submit a revision. We greatly appreciate your helpful comments, as well as the insightful feedback from all the reviewers. We have carefully revised the manuscript accordingly, with all new and modified text highlighted in red. Below, please find our point-by-point response to the reviewers’ questions and suggestions.

This is an interesting and well-structured review addressing a highly relevant topic. The manuscript covers a broad range of mechanisms involved in BBB disruption after CIRI and provides a comprehensive overview of potential therapeutic strategies. Overall, the work is valuable, but a few aspects could be further refined to enhance clarity and scientific strength.

Major comments 1: A more nuanced discussion of the level of evidence would strengthen the therapeutic section. Many interventions are presented as promising, but at times the distinction between preclinical evidence and clinical applicability is not entirely clear. In some paragraphs, the tone may appear slightly stronger than what the current evidence fully supports. It would be helpful to more explicitly indicate which approaches remain experimental and to briefly acknowledge current translational limitations. This would make the review more balanced and clinically meaningful.

Response 1: We completely agree that a high-quality review article must not only summarize potential therapeutic interventions but also critically evaluate why most of them fail to translate into the clinic. We appreciate you pointing out the overly optimistic tone of our original manuscript, and we have substantially revised the relevant sections to address this concern.

To provide this critical analysis, we divided our approach into two categories:

1. Critical analysis of interventions with clear evidence of translational failure:

We have added specific discussions explaining exactly why these interventions have stalled in clinical development:

TRPM7 inhibitors (Page 10-11, lines 341–345): Highlighted that no agents have entered clinical stroke trials due to cardiovascular side effects and limited isoform selectivity.

MitoQ and SS-31 (Page 11, lines 367–369): Explicitly stated that clinical translation has failed due to limited therapeutic effects, short half-lives, and low bioavailability.

ATN-161 (Page 12, lines 419–423): Explained that its requirement for immediate post-reperfusion administration makes it clinically unfeasible given the variable and often delayed onset-to-reperfusion times in human patients.

z-VAD-fmk (Page 13, lines 438–443): Pointed out that its required invasive intraventricular injection fundamentally precludes clinical translation.

Deferiprone (Page 13, lines 462-465): Used its failure in Parkinson’s disease as a cautionary tale regarding the fundamental differences between preclinical models and human pathology.

DCA (Page 14, lines 492–494): Noted that despite its efficacy, unacceptable toxicity in Phase 2 clinical trials prevents its approval.

β-HB (Page 14, lines 497-500): Highlighted that its lack of efficacy in intracerebral hemorrhage models suggests that blindly expanding its use to all stroke types will likely lead to failure.

HIF-1α modulation (Page 14, lines 503–509): Discussed the biphasic effects of HIF-1α-regulated growth factors, emphasizing that the greatest clinical challenge is precisely controlling the degree and timing of its activation.

*(Note regarding Page 13, lines 453–458: While we mentioned that the alcohol-withdrawal drug [disulfiram] shows promise for repurposing due to its established safety profile, we ensured this is framed strictly as a theoretical repurposing candidate, rather than a guaranteed clinical success, to maintain our newly revised, critical tone.)*

2. Systematic analysis of broader translational hurdles:

For interventions lacking definitive failure data, we analyzed the overarching systemic reasons why most neuroprotective strategies fail:

Page 15-16 (lines 552–564) & Page 16: We added a comprehensive summary explaining that failures are collectively driven by discrepancies between animal models and human pathology, poor blood-brain barrier (BBB) penetration, the complexity of the ischemia-reperfusion cascade, overly narrow therapeutic windows, lack of drug brain-targeting capabilities, systemic side effects, and imprecise clinical trial designs.

Page 13-14 (lines 471–475) & Page 14: We reinforced this by stating that core issues—such as appropriate therapeutic windows, feasible administration strategies, and BBB penetration efficiency—still lack systematic clinical evidence.

3. Addition of Clinical Trial Summary (Cyclosporine Example & Table 2):

Your point regarding Cyclosporine was highly incisive. Inspired by your suggestion to systematically summarize drugs tested in clinical trials, we have added a new Section 4.5 (beginning on Page 10) and created Table 2 (after line 570).

As introduced in the text: *“Table 2 systematically summarizes the drugs/interventions tested in clinical trials for the prevention or alleviation of reperfusion injury after ischemic stroke, clearly presenting the clinical research progress, mechanisms of action, and existing limitations to provide a reference for future research and clinical applications.”*

We believe these substantial additions have successfully shifted the manuscript from a purely optimistic summary to a rigorously critical evaluation. All modified text has been highlighted in red.

 

Table 2. Drugs/interventions that have been verified in clinical trials for the prevention or alleviation of reperfusion injury after ischemic stroke.”

Category	Drug	Conclusion	Clinic trail	Ref.
Restoration of Ion and Metabolic Homeostasis	Cyclosporine A	It failed to effectively reduce the size of infarction and studies have shown no benefit.	Phase II clinical trial	[140, 141]
	Nerinetide	It could slightly reduce the mortality rate of AIS, but the functional outcome was not significantly improved.	Phase III clinical trial	[142]
	Magnesium sulfate	Ultra-early administration was safe, but it had no significant neuroprotective effect.	Phase III clinical trial	[143]
	Dextromethorphan	Safe, but with no significant neuroprotective benefits, it was not advanced to Phase III and has limited clinical application.	Phase II clinical trial	[144]
	Aptiganel/ Cerestat	The high-dose group had a higher mortality rate, obvious central adverse reactions and no neuroprotective benefits.	Phase III clinical trial	[145]
	Selfotel	The therapeutic effect is not definite and it may also have neurotoxic effects in cerebral ischemia.	Phase III clinical trial	[146]
Modulation of Cell Death and Inflammatory Pathways	Edaravone dexborneol	Its therapeutic effect was superior to that of edaravone, but its safety issues were a cause for concern.	Phase III/IV clinical trials	[147, 148]
	Cerovive	The failure was declared as no positive results could be replicated.	Phase III clinical trial	[149]
	Alpha-lipoic acid	It was in the exploration stage and no clear evidence of clinical benefits had been formed yet.	Phase II exploratory clinical trial	[150]
	Minocycline	The effectiveness in improving patients' functional prognosis and good safety.	Phase III clinical trial	[151]
	glibenclamide	The primary endpoint was not reached and there was no significant improvement in functional outcomes.	Phase III clinical trial	[152]
	Fingolimod	It had good safety and can improve early neurological deficits.	Phase II exploratory clinical trial	[153, 154]
	Natalizumab	The primary endpoint was not reached and there was no significant improvement in functional outcomes.	Phase II clinical trial	[155]
	Anakinra	Preliminary exploration of anti-inflammatory effects has been conducted, but no clear evidence of reperfusion injury protection has been formed yet.	Phase II clinical trial	[156]
	DL-3-n-butylphthalide	It significantly improved neurological deficits and has good safety, making it a first-line drug for acute ischemic stroke in China.	Phase III/IV clinical trials	[157, 158]
	deferoxamine	Dose exploration and safety trials were lacking in confirmatory efficacy studies.	Phase II clinical trial	[159]
	Otaplimastat	Intravenous administration of oplimaristat as an adjuvant treatment for patients receiving rtPA was feasible and generally safe.	Phase II clinical trial	[160]
	Human Urinary kallidinogenase	It effectively improved perfusion in ischemic areas and neurological function and was used in China for reperfusion injury in AIS.	Phase IV clinical trial	[161]
Regulation of microcirculation, anticoagulation and thrombolysis	Cilostazol	For secondary prevention of non-cardiogenic ischemic stroke	Phase IV clinical trial	[162]
	rt-PA	Significantly improved prognosis	Phase III clinical trial	[163, 164]
	Tenecteplas	The recanalization rate was higher than that of alteplase and the safety was comparable.	Phase III clinical trial	[164, 165]
	Tirofiban	Early use was associated with good functional outcomes, but the risk of bleeding increased.	Phase III clinical trial	[166]
	Eptifibatide and Argatroban	Negative outcome, failed to demonstrate functional benefit	Phase III clinical trial	[167]
Physical intervention and stem cell regeneration	Remote ischemic preconditioning	Exploratory application, no standardized protocol has been formed and clinical evidence is limited.	Clinical exploratory research	[168, 169]
	Therapeutic hypothermia	Only a portion of the patients reached the target temperature and there was no difference in the efficacy analysis. Additionally, the trial was terminated due to slow enrollment and financial issues.	Phase III clinical trial	[170]
	Hyperbaric oxygen therapy	It was used for the adjuvant treatment of reperfusion injury and the therapeutic effect shows individual differences, which requires further experimental research.	Phase II clinical trial	[171]
	Mesenchymal stromal cell	It may improve functional recovery, but its therapeutic effect has not been fully confirmed.	Phase II clinical trial	[172]
	Endothelial progenitor cell	Intracarotid infusion of autologous CD34+ cells was safe and may improve the long-term prognosis of patients with acute ischemic stroke.	Phase II clinical trial	[173]

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119. Adeoye, O.; Broderick, J.; Derdeyn, C.P.; Grotta, J.C.; Barsan, W.; Bentho, O.; Berry, S.; Concha, M.; Davis, I.; Demel, S.; et al. Adjunctive Intravenous Argatroban or Eptifibatide for Ischemic Stroke. N Engl J Med 2024, 391, 810–820, doi:10.1056/NEJMoa2314779.
120. Zhao, T.; Li, M.; Yan, Q.; Gu, J.; Liu, L. Effect of Remote Ischemic Preconditioning Intervention on Serum Levels of microRNA-582-5p/HMGB1 in Patients with Acute Cerebral Infarction. Clin Neurol Neurosurg 2024, 241, 108291, doi:10.1016/j.clineuro.2024.108291.
121. An, J.; Wei, M.; Wang, D.; Zhao, C.; Yuan, X.; Kimberly, W.T.; Luo, G.; Li, G. Long-Term Efficacy of Remote Ischaemic Postconditioning after Rt-PA Intravenous Thrombolysis in Patients with Acute Ischaemic Stroke. BMJ Open 2025, 15, e094001, doi:10.1136/bmjopen-2024-094001.
122. van der Worp, H.B.; Macleod, M.R.; Bath, P.M.W.; Demotes, J.; Durand-Zaleski, I.; Gebhardt, B.; Gluud, C.; Kollmar, R.; Krieger, D.W.; Lees, K.R.; et al. EuroHYP-1: European Multicenter, Randomized, Phase III Clinical Trial of Therapeutic Hypothermia plus Best Medical Treatment vs. Best Medical Treatment Alone for Acute Ischemic Stroke. Int J Stroke 2014, 9, 642–645, doi:10.1111/ijs.12294.
123. Li, W.; Lan, J.; Wei, M.; Liu, L.; Hou, C.; Qi, Z.; Li, C.; Jiao, L.; Yang, Q.; Chen, W.; et al. Normobaric Hyperoxia Combined with Endovascular Treatment for Acute Ischaemic Stroke in China (OPENS-2 Trial): A Multicentre, Randomised, Single-Blind, Sham-Controlled Trial. Lancet 2025, 405, 486–497, doi:10.1016/S0140-6736(24)02809-5.
124. Bang, O.Y.; Lee, J.S.; Lee, P.H.; Lee, G. Autologous Mesenchymal Stem Cell Transplantation in Stroke Patients. Ann Neurol 2005, 57, 874–882, doi:10.1002/ana.20501.
125. Lin, H.-S.; Sung, P.-H.; Huang, S.-H.; Lin, W.-C.; Chiang, J.Y.; Ma, M.-C.; Chen, Y.-L.; Chen, K.-H.; Lee, F.-Y.; Ko, S.-F.; et al. Long Term Outcomes of Intracarotid Arterial Transfusion of Circulatory-Derived Autologous CD34 + Cells for Acute Ischemic Stroke Patients—A Randomized, Open-Label, Controlled Phase II Clinical Trial. Stem Cell Res Ther 2024, 15, 443, doi:10.1186/s13287-024-04021-7

Major Comments 2: The therapeutic section could benefit from a clearer organizing framework. While the content is rich, it sometimes reads as a sequence of different strategies rather than a fully integrated discussion. A more structured approach (for example grouping interventions by mechanism, timing, or level of evidence) could help guide the reader and improve the overall flow.

Response 2: We have adopted your suggested mechanism-based approach to structuring the interventions, which significantly improves the logical flow and will greatly assist the reader. Because these structural changes were extensive, we kindly direct you to the revised text from line 300 on page 9 through line 571 on page 18.

To summarize our reorganization, the section now follows this streamlined structure:

4.1 Restoration of Ion and Metabolic Homeostasis: This section integrates the original sections on ion channel/exchanger regulation and mitochondrial protection. The core focus is on correcting ionic imbalances, improving mitochondrial function, reversing energy depletion in BBB endothelial cells, and blocking the initiation of damage.

4.2 Inhibition of Excessive Proteolysis and Matrix Remodeling: This integrates the previous sections on MMP inhibition and endothelial cell/basement membrane protection. The core focus is on preventing basement membrane degradation, promoting neurovascular unit (NVU) structural repair, and maintaining the physical integrity of the BBB.

4.3 Immune Modulation and Redox Balance Regulation: This combines the previous sections on metabolic regulation, programmed cell death inhibition, and anti-inflammatory strategies. The core focus is on reshaping the immune and oxidative microenvironment to block the inflammation-oxidative stress-cell death cascade, thereby alleviating secondary BBB damage.

4.4 Dual-Action Agents and Non-pharmacological Interventions: This consolidates the previous sections on dual-effect drugs and non-drug interventions. The core focus is on multi-mechanism synergistic protection, combining pharmacological and non-pharmacological measures to enhance BBB protection.

4.5 Clinical Translation and Evidence Evaluation: This is a newly added section designed to critically evaluate why the therapies discussed in this review have largely failed to advance into clinical trials. As discussed in our previous response, we have also included a new table summarizing 28 drugs and interventions that have undergone clinical testing for ischemic stroke reperfusion injury, explicitly detailing the reasons for their translational failures.

Major Comments 3:  The reference list would benefit from a careful revision. There appear to be a few duplicated entries and some inconsistencies in formatting. A thorough check of the bibliography, including alignment between statements and supporting references, would improve the overall quality of the manuscript.

Response 3: We appreciate you pointing this out. We have thoroughly re-examined and revised the entire reference section. Outdated or unrepresentative citations have been replaced with high-quality references, and redundant ones have been removed. Specific examples of these updates include references [2] (page 2, line 65), [3–4] (page 3, line 67), and [8] (page 3, line 94). All other modifications to the reference list are highlighted in red in the revised manuscript.

References

2. Majumder, D. Ischemic Stroke: Pathophysiology and Evolving Treatment Approaches. Neurosci Insights 2024, 19, 26331055241292600, doi:10.1177/26331055241292600.
3. Weiss, A.; Ding, Y. Beyond Reperfusion: Adjunctive Therapies Targeting Inflammation, Edema, and Blood-Brain Barrier Dysfunction in Ischemic Stroke. Cerebrovasc Dis 2025, 55, 292–301, doi:10.1159/000547092.
4. Zhang, M.; Liu, Q.; Meng, H.; Duan, H.; Liu, X.; Wu, J.; Gao, F.; Wang, S.; Tan, R.; Yuan, J. Ischemia-Reperfusion Injury: Molecular Mechanisms and Therapeutic Targets. Sig Transduct Target Ther 2024, 9, 12, doi:10.1038/s41392-023-01688-x.
5. Li, S.; Fisher, M. Improving Large Animal Ischemic Stroke Models for Translational Studies in the Era of Recanalization. Stroke2023, 54, e16–e19, doi:10.1161/STROKEAHA.122.041354.

Minor Comments: Some minor language and formatting adjustments are recommended. For example, “Stem cell therapeutic” could be revised to “Stem cell therapy”, and “Future Direction” to “Future Directions”. A general language polishing would further improve readability. A few repetitions and small inaccuracies should be corrected. The sentence describing the immunomodulatory effects of RIPC appears repeated, and the abbreviation “MACO” likely should be corrected to “MCAO”.

Response: We have made the necessary language and formatting corrections, including changing “Future Direction” to “Future Directions” (page 18, line 572) and correcting the typo “MACO” to “MCAO” (page 19, line 636). Additionally, we removed a redundant sentence regarding the immunomodulatory effects of RIPC on page 15 (line 534). Finally, as a result of the structural reorganization of Chapter Four, the standalone “Stem Cell Therapy” subsection has been removed.

 

Reviewer 4 Report

Comments and Suggestions for Authors I have read the paper and make the following comments:

• Scientific writing should be in third person, past tense, not first person as per the following quotation, taken from the abstract, "...We comprehensively..."
• "In addition" is superfluous in the following quotation and the use of first person is repeated, "...In addition, we assessed potential...". Scientific writing should demonstrate economy of wordage and any sentence starting with unnecessary words, followed by a comma, should have those words removed. A perfect example arises with, "...Consequently, therapeutic strategies specifically..." in which "Consequently" is superfluous as is also the case with "However" in line 118. I will not offer further examples but advocate the authors revisit the paper to correct the grammar, syntactic and language errors. I will not offer further examples that are prolific throughout the paper.
• When using examples, introduced by "such as", there should be a comma before such, namely the last word  before "such" and it be used to reflect an imaginary set of brackets, as per "...Although revascularization therapies such as thrombolysis and thrombectomy can restore blood flow, about 30% of patients experience cerebral..." which should read, "...Although revascularization therapies, such as thrombolysis and thrombectomy, can restore blood flow, about 30% of patients experience cerebral..." or in "...injuries such as hemorrhage and cerebral edema..." from line 100.
• As a rule there should not be a comma before a conjunctive, such as "and" or "but", as per the following quotation, "...that form the capillary wall, and is structurally and functionally...".
• In the following quotation, there should be a space between      , "...The BBB functions asa crucial guardian...", to read "...functions as  a crucial...".
•  Having identified these issues with language, the paper is both interesting and very informative and should be accepted once the language issues have been rectified with minor revision.

Author Response

Dear Editor and reviewer,

We would like to express our sincere gratitude to you for sparing your valuable time to review our manuscript (Manuscript ID: 4219645), and for giving us the opportunity to revise. We greatly appreciate your helpful comments and the many insightful comments of all the reviewers. We have modified the manuscript accordingly (revised text in red). Below please find our point-by-point response to the questions and suggestions raised by the reviewers.

Comments 1: Scientific writing should be in third person, past tense, not first person as per the following quotation, taken from the abstract, "...We comprehensively...". "In addition" is superfluous in the following quotation and the use of first person is repeated, "...In addition, we assessed potential...". 

Response 1: We sincerely appreciate the time and effort you invested in identifying specific language, grammar, and syntax issues. We have carefully addressed your feedback regarding verb tense consistency.
Moving forward, we have applied a strict rule: the simple present tense is used exclusively for defining generally known facts and accepted pathological mechanisms, while the past tense is now used when describing the specific research of previous scholars.
Examples of shifts to the past tense for previous research include:
Page 10 (lines 355–358): “In MCAO models, S0859 and HOE642 improved neurological outcomes and NHE inhibitors had been evaluated in clinical trials…”
Page 12 (lines 387–389): “Their study indirectly suggested that inhibiting MMP-9 during the critical window period…”
Page 13 (lines 438–443): “…siRNA-mediated knockdown of the pro-apoptotic protein Bax, had also shown robust BBB protection in animal models…”
Page 15 (lines 514–516): “Edaravone was already approved clinically in Japan…”
Page 15 (lines 547–550): “…various stem/progenitor cells… had been evaluated as potential cell therapies…”
Conversely, we have maintained the simple present tense for established facts. For example, on page 2 (lines 59–61): “Stroke… represents a major global public health burden… Epidemiological evidence indicates that stroke is the leading cause of death in China.”
Finally, in response to your suggestion regarding the abstract, we have comprehensively removed all first-person expressions and uniformly adjusted them to objective, third-person narration to ensure a rigorous tone. Examples include the opening of the abstract (page 1-2, lines 37–43): “This article comprehensively analyzes the literature… systematically elaborates on the main pathological pathways… and evaluates potential intervention measures…” A similar objective revision was made on page 6 (lines 169–172): “This process, together with oxidative stress, disrupts the balance…” All modifications have been highlighted in red.

Comments 2: Scientific writing should demonstrate economy of wordage and any sentence starting with unnecessary words, followed by a comma, should have those words removed. A perfect example arises with, "...Consequently, therapeutic strategies specifically..." in which "Consequently" is superfluous as is also the case with "However" in line 118. I will not offer further examples but advocate the authors revisit the paper to correct the grammar, syntactic and language errors. I will not offer further examples that are prolific throughout the paper.

Response 2: We strongly believe that scientific writing should be concise and to the point. Any sentence that begins with redundant words followed by a comma should have such superfluous words removed. We have already deleted unnecessary words such as "Consequently", "However", and "Therefore" from the article.

Comments 3: When using examples, introduced by "such as", there should be a comma before such, namely the last word  before "such" and it be used to reflect an imaginary set of brackets, as per "...Although revascularization therapies such as thrombolysis and thrombectomy can restore blood flow, about 30% of patients experience cerebral..." which should read, "...Although revascularization therapies, such as thrombolysis and thrombectomy, can restore blood flow, about 30% of patients experience cerebral..." or in "...injuries such as hemorrhage and cerebral edema..." from line 100.

As a rule there should not be a comma before a conjunctive, such as "and" or "but", as per the following quotation, "...that form the capillary wall, and is structurally and functionally...".

In the following quotation, there should be a space between"...The BBB functions asa crucial guardian...", to read "...functions as a crucial...".

Response: We appreciate your point this and have systematically revised the document to ensure commas are correctly used before “such as” when introducing non-restrictive examples (e.g., page 2, lines 65–67). We have also removed superfluous punctuation before conjunctions. Finally, we apologize for the typographical error (“asa”) on page 3-4 (lines 101–104); this has been corrected to “as a”.

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I thank the authors for the amendments, which have made major improvements to the article. They need to review the article critically once more and work with the journal editorial office to improve the text before going to the publisher. I give some examples below but I will leave the authors and the editors to work on the final version. 

Comments on the Quality of English Language

The editorial office need to work with the authors to improve the article:

1. In general, the text need to be more precise.
2. There are some duplications: lines 71-73, line 105 (repeating lines 99-100), lines 343-347).
3. Line 381: delayed inhibition should "compromises" rather than "preserves" angiogenesis.
4. Lines 438-440 are awkward. A more precise description will be "z-VAD-fmk required administration by invasive intraventricular injection due to its physicochemical properties, problematic for translation into clinical practice[99].
5. Line 455: The term "disulfide death-like[106]." is incomplete.
6. Lines 472-475: " The key lies in the fact that core issues..." would better be changed to "Core issues such as the appropriate therapeutic window, clinically feasible drug administration strategies and the efficiency of drug penetration through the BBB are still awaiting supportive clinical data."
7. Line 488: "pharmacological agents" is redundant.
8. Line 499: "the clinical "translation" of β-HB requires "better selection" of the target population" is better.
9. Line 500 "Blindly expanding it to all types of stroke may lead to failure." will be redundant with the above change.
10. Line 505: "and vice versa" seems not appropriate and may be deleted. 
11. Line 508: the "dosage and timing of HIF-1α treatment" may be better.
12. Ref 163 and 165: The author lists for the ATTEST-2 and TEMPO-2 trials are incorrect. They are not Chinese.

Author Response

Comments 1: In general, the text need to be more precise

Response 1: Thanks for your comments and suggestion.  The manuscript has been thoroughly revised to enhance clarity, logical flow, and scientific accuracy. We have reworded ambiguous passages and vague descriptions to ensure a precise and accurate presentation of our research.

Comments 2: There are some duplications: lines 71-73, line 105 (repeating lines 99-100), lines 343-347).

Response 2: Thanks you for  pointing those out. We have carefully reviewed the manuscript and streamlined the text by removing or condensing redundant content at the specified locations, ensuring all necessary information is preserved (page 2, lines 71–72; pages 3–4, lines 97–107; page 10, lines 340–341)

Comments 3: Line 381: delayed inhibition should "compromises" rather than "preserves" angiogenesis.

Response 3: We appreciate this suggestion and have corrected “preserves” to “compromises” on page 11, line 375

Comments 4: Lines 438-440 are awkward. A more precise description will be "z-VAD-fmk required administration by invasive intraventricular injection due to its physicochemical properties, problematic for translation into clinical practice[99].

Response 4: We greatly appreciate the corrections and have revised the text as suggested on page 13, lines 433–434.

Comments 5: Line 455: The term "disulfide death-like[106]." is incomplete.

Response 5: Thanks you for point this out and we have revised  this  to "…reducing disulfidptosis-like cytoskeletal and mitochondrial damage."(line 447-449 , pg 13)

Comments 6: Lines 472-475: " The key lies in the fact that core issues..." would better be changed to "Core issues such as the appropriate therapeutic window, clinically feasible drug administration strategies and the efficiency of drug penetration through the BBB are still awaiting supportive clinical data."

Response 6: Thanks for your suggestion ,We have revised the text as suggested on page 13, lines 466–468.

Comments 7: Line 488: "pharmacological agents" is redundant.

Response 7: Thank you for point this out. We have deleted "pharmacological agents" on page 14, line 481.

Comments 8: Line 499: "the clinical "translation" of β-HB requires "better selection" of the target population" is better.

Response 8: Thanks for your suggestion. We have revised this on page 14, lines 491-492.

Comments 9: Line 500 "Blindly expanding it to all types of stroke may lead to failure." will be redundant with the above change.

Response 9: Thanks your for point this out. We have deleted "Blindly expanding it to all types of stroke may lead to failure." on page 14, line 492.

Comments 10: Line 505: "and vice versa" seems not appropriate and may be deleted.

Response 10: We have deleted "and vice versa" on page 14, line 497.

Comments 11: Line 508: the "dosage and timing of HIF-1α treatment" may be better.

Response 11: Task for your suggestion. We have revised the text on page 17, lines 499-500.

Comments 12: Ref 163 and 165: The author lists for the ATTEST-2 and TEMPO-2 trials are incorrect. They are not Chinese.

Response 12: We are very grateful for your careful review and have corrected these to: 

163. Muir, K.W.; Ford, G.A.; Ford, I.; Wardlaw, J.M.; McConnachie, A.; Greenlaw, N.; Mair, G.; Sprigg, N.; Price, C.I.; MacLeod, M.J.; et al. Tenecteplase versus Alteplase for Acute Stroke within 4·5 h of Onset (ATTEST-2): A Randomised, Parallel Group, Open-Label Trial. The Lancet Neurology 2024, 23, 1087–1096, doi:10.1016/S1474-4422(24)00377-6.

 

165. Coutts, S.B.; Ankolekar, S.; Appireddy, R.; Arenillas, J.F.; Assis, Z.; Bailey, P.; Barber, P.A.; Bazan, R.; Buck, B.H.; Butcher, K.S.; et al. Tenecteplase versus Standard of Care for Minor Ischaemic Stroke with Proven Occlusion (TEMPO-2): A Randomised, Open Label, Phase 3 Superiority Trial. Lancet 2024, 403, 2597–2605, doi:10.1016/S0140-6736(24)00921-8.

 

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have clearly improved the manuscript in response to the previous comments. The structure is now more coherent, the therapeutic section is better organized, and the discussion of translational limitations is more balanced and clinically meaningful. The addition of the clinical trial summary table is particularly valuable.

Only a few minor points could further improve the manuscript:
- In the therapeutic section (e.g., TRPM7 paragraph, pages ~10–11), there is a small degree of redundancy with closely repeated concepts that could be streamlined for better readability.
- A light language polishing would be beneficial, particularly to simplify a few long or complex sentences and ensure a more consistent academic tone throughout.

These are minor adjustments that can be addressed quickly.
Overall, the manuscript is now suitable for publication after minor revision.

Author Response

Comments 1: In the therapeutic section (e.g., TRPM7 paragraph, pages ~10–11), there is a small degree of redundancy with closely repeated concepts that could be streamlined for better readability.

Response 1:  Thanks you for pointing this out, we have deleted the repeat.

Comment 2:  A light language polishing would be beneficial, particularly to simplify a few long or complex sentences and ensure a more consistent academic tone throughout.

Response 2:  We sincerely appreciate the reviewer’s constructive suggestions regarding language polishing and manuscript revision. We have carefully revised and polished the entire manuscript, improving the grammar, sentence structure, word choice, logical expression, and adherence to academic writing norms. Specifically, several long and complex sentences have been rewritten for clarity (Line 66, 98, 107, 152, 195, 261, 410, 418, 494, 522, 561).