CDDO, an Anti-Inflammatory and Antioxidant Compound, Attenuates Vasospasm and Neuronal Cell Apoptosis in Rats Subjected to Experimental Subarachnoid Hemorrhage

Round 1

Reviewer 1 Report

Than you for the opportunity to review this manuscript.

Oleanolic acid derivatives with saturation in the C-ring are well known antioxidant inflammation modulators with anti-allergic, neuroprotective, antioxidative, and anti-inflammatory properties.

Synthetic oleanane triterpenoids may induce apoptosis and exert beneficial effects on blood (Konopleva M, Tsao T, Estrov Z, et al. The synthetic triterpenoid 2‐cyano‐3,12‐dioxooleana‐1,9‐dien‐28‐oic acid induces caspase‐dependent and ‐independent apoptosis in acute myelogenous leukemia. Cancer Res. 2004;64:7927‐7935) and solid (Lapillonne H, Konopleva M, Tsao T, et al. Activation of peroxisome proliferator‐activated receptor gamma by a novel synthetic triterpenoid 2‐cyano‐3,12‐dioxooleana‐1,9‐dien‐28‐oic acid induces growth arrest and apoptosis in breast cancer cells. Cancer Res. 2003;63:5926‐5939) cancers.

As Nrf2 activators, oleanane triterpenoids have role in vascular diseases (Cheng L, Zhang H, Wu F, Liu Z, Cheng Y, Wang C. Role of Nrf2 and Its Activators in Cardiocerebral Vascular Disease. Oxid Med Cell Longev. 2020 Aug 5;2020:4683943. doi: 10.1155/2020/4683943.), and have neuroprotective effects by inhibiting TNF production and modulation of glial activities (Tran TA, McCoy MK, Sporn MB, Tansey MG. The synthetic triterpenoid CDDO-methyl ester modulates microglial activities, inhibits TNF production, and provides dopaminergic neuroprotection. J Neuroinflammation. 2008 May 12;5:14. doi: 10.1186/1742-2094-5-14.) or by induction of heme oxygenase-1 (Zhang F, Wang S, Zhang M, Weng Z, Li P, Gan Y, Zhang L, Cao G, Gao Y, Leak RK, Sporn MB, Chen J. Pharmacological induction of heme oxygenase-1 by a triterpenoid protects neurons against ischemic injury. Stroke. 2012 May;43(5):1390-7. doi: 10.1161/STROKEAHA.111.647420; Li QQ, Li LJ, Wang XY, Sun YY, Wu J. Research Progress in Understanding the Relationship Between Heme Oxygenase-1 and Intracerebral Hemorrhage. Frontiers in Neurology. 2018 ;9:682. DOI: 10.3389/fneur.2018.00682. ). A recently published manuscript by Lu CC et al. is worth mentioning (Lu CC, Lin CY, Lu YY, Tsai HP, Lin CL, Wu CH. CDDO regulates central and peripheral sensitization to attenuate post-herpetic neuralgia by targeting TRPV1/PKC-δ/p-Akt signals. J Cell Mol Med. 2024 Mar;28(6):e18131. doi: 10.1111/jcmm.18131.) 

 In this manuscript, the authors stated that the effect of CCDO on SAH-related pathology has not been reported. Tsai TH et al. published a very similar article addressing the effect of second-generation semisynthetic oleanane triterpenoid, RTA 408 on  SAH-induced delayed vasospasm (Tsai TH, Lin SH, Wu CH, Tsai YC, Yang SF, Lin CL. Mechanisms and therapeutic implications of RTA 408, an activator of Nrf2, in subarachnoid hemorrhage-induced delayed cerebral vasospasm and secondary brain injury. PLoS One. 2020 Oct 5;15(10):e0240122. doi: 10.1371/journal.pone.0240122.). Furthermore, the same author has several other worth-mentioning publications (Tsai TH, Su YF, Tsai CY, Wu CH, Lee KT, Hsu YC. RTA dh404 Induces Cell Cycle Arrest, Apoptosis, and Autophagy in Glioblastoma Cells. Int J Mol Sci. 2023 Feb 16;24(4):4006. doi: 10.3390/ijms24044006. ; Tsai TH, Tsai CY, Moi SH, Wu CH, Lee KT, Hsu YC, Su YF. A Novel Synthetic Oleanolic Acid Derivative Inhibits Glioma Cell Proliferation by Regulating Cell Cycle G2/M Arrest. Pharmaceuticals (Basel). 2023 Apr 24;16(5):642. doi: 10.3390/ph16050642. )

 So, it is hard to say that CDDO and second-generation semisynthetic oleanane triterpenoid have not been investigated in this setting. The authors should emphasize the novelty of their findings in the discussion. A better literature search should be done. 

 Minor points

1. The first sentences in section 3.5 (lines 218-2020) explaining and referencing the proliferation of microglia and apoptosis, should be moved into introduction or discussion. Furthermore, lines 265-272 in section 3.7 should be placed in the introduction and methods, respectively. 
2. Provide a reference for Jeon H et al.2009 (line 110) 

Author Response

Oleanolic acid derivatives with saturation in the C-ring are well known antioxidant inflammation modulators with anti-allergic, neuroprotective, antioxidative, and anti-inflammatory properties.

Synthetic oleanane triterpenoids may induce apoptosis and exert beneficial effects on blood (Konopleva M, Tsao T, Estrov Z, et al. The synthetic triterpenoid 2‐cyano‐3,12‐dioxooleana‐1,9‐dien‐28‐oic acid induces caspase‐dependent and ‐independent apoptosis in acute myelogenous leukemia. Cancer Res. 2004;64:7927‐7935) and solid (Lapillonne H, Konopleva M, Tsao T, et al. Activation of peroxisome proliferator‐activated receptor gamma by a novel synthetic triterpenoid 2‐cyano‐3,12‐dioxooleana‐1,9‐dien‐28‐oic acid induces growth arrest and apoptosis in breast cancer cells. Cancer Res. 2003;63:5926‐5939) cancers.

As Nrf2 activators, oleanane triterpenoids have role in vascular diseases (Cheng L, Zhang H, Wu F, Liu Z, Cheng Y, Wang C. Role of Nrf2 and Its Activators in Cardiocerebral Vascular Disease. Oxid Med Cell Longev. 2020 Aug 5;2020:4683943. doi: 10.1155/2020/4683943.), and have neuroprotective effects by inhibiting TNF production and modulation of glial activities (Tran TA, McCoy MK, Sporn MB, Tansey MG. The synthetic triterpenoid CDDO-methyl ester modulates microglial activities, inhibits TNF production, and provides dopaminergic neuroprotection. J Neuroinflammation. 2008 May 12;5:14. doi: 10.1186/1742-2094-5-14.) or by induction of heme oxygenase-1 (Zhang F, Wang S, Zhang M, Weng Z, Li P, Gan Y, Zhang L, Cao G, Gao Y, Leak RK, Sporn MB, Chen J. Pharmacological induction of heme oxygenase-1 by a triterpenoid protects neurons against ischemic injury. Stroke. 2012 May;43(5):1390-7. doi: 10.1161/STROKEAHA.111.647420; Li QQ, Li LJ, Wang XY, Sun YY, Wu J. Research Progress in Understanding the Relationship Between Heme Oxygenase-1 and Intracerebral Hemorrhage. Frontiers in Neurology. 2018 ;9:682. DOI: 10.3389/fneur.2018.00682. ). A recently published manuscript by Lu CC et al. is worth mentioning (Lu CC, Lin CY, Lu YY, Tsai HP, Lin CL, Wu CH. CDDO regulates central and peripheral sensitization to attenuate post-herpetic neuralgia by targeting TRPV1/PKC-δ/p-Akt signals. J Cell Mol Med. 2024 Mar;28(6):e18131. doi: 10.1111/jcmm.18131.)

 In this manuscript, the authors stated that the effect of CCDO on SAH-related pathology has not been reported. Tsai TH et al. published a very similar article addressing the effect of second-generation semisynthetic oleanane triterpenoid, RTA 408 on  SAH-induced delayed vasospasm (Tsai TH, Lin SH, Wu CH, Tsai YC, Yang SF, Lin CL. Mechanisms and therapeutic implications of RTA 408, an activator of Nrf2, in subarachnoid hemorrhage-induced delayed cerebral vasospasm and secondary brain injury. PLoS One. 2020 Oct 5;15(10):e0240122. doi: 10.1371/journal.pone.0240122.). Furthermore, the same author has several other worth-mentioning publications (Tsai TH, Su YF, Tsai CY, Wu CH, Lee KT, Hsu YC. RTA dh404 Induces Cell Cycle Arrest, Apoptosis, and Autophagy in Glioblastoma Cells. Int J Mol Sci. 2023 Feb 16;24(4):4006. doi: 10.3390/ijms24044006. ; Tsai TH, Tsai CY, Moi SH, Wu CH, Lee KT, Hsu YC, Su YF. A Novel Synthetic Oleanolic Acid Derivative Inhibits Glioma Cell Proliferation by Regulating Cell Cycle G2/M Arrest. Pharmaceuticals (Basel). 2023 Apr 24;16(5):642. doi: 10.3390/ph16050642. )

 So, it is hard to say that CDDO and second-generation semisynthetic oleanane triterpenoid have not been investigated in this setting. The authors should emphasize the novelty of their findings in the discussion. A better literature search should be done.

I have included the following statement in the discussion section of our manuscript: " In our study, CDDO attenuates vasospasm and neuronal cell apoptosis following SAH. However, related research by Tsai TH et al. on a second-generation semisynthetic oleanane triterpenoid, RTA 408, addresses its effects on SAH-induced delayed cerebral vasospasm and secondary brain injury, suggesting a broader applicative scope for these compounds in vascular and neuro-inflammatory conditions [29]. The primary distinction between CDDO and RTA 408 lies in the variation of their functional groups, indicating that the core structure itself possesses the capacity to mitigate SAH-induced physiological changes. This insight suggests that the fundamental backbone of these molecules could serve as a template for the development of new therapeutic agents targeting SAH in future research endeavors. Related research by Cheng L et al. on the Nrf2 activator properties of oleanane triterpenoids, such as CDDO, has demonstrated their role in vascular diseases [30]. Furthermore, another study by Tran TA et al. explored the CDDO-methyl ester's capacity to modulate microglial activities and inhibit TNF production, providing significant neuroprotection. This demonstrates the com-pound's potential in neuroinflammatory and neurodegenerative diseases by directly influencing key mechanisms of neurodegeneration [31]. Finally, a recent manuscript by Lu CC et al. illustrates how CDDO regulates central and peripheral sensitization to effectively reduce post-herpetic neuralgia by targeting specific neural signaling path-ways. This finding not only underlines its role in pain control but also reinforces its broader implications in neural protection [32]. These studies collectively affirm the ca-pability of the CDDO core structure to both suppress inflammation and safeguard neural cells, highlighting its potential for further development in therapies aimed at conditions involving both inflammation and neural damage." This addition highlights the novelty of our findings and aligns with the existing literature on the topic.

Minor points

The first sentences in section 3.5 (lines 218-2020) explaining and referencing the proliferation of microglia and apoptosis, should be moved into introduction or discussion. Furthermore, lines 265-272 in section 3.7 should be placed in the introduction and methods, respectively.

The first sentences in section 3.5 (lines 218-202), which explain and reference the proliferation of microglia and apoptosis, are currently positioned in the results section. This placement is intentional to provide readers with immediate context and rationale behind the observations reported in the results. It is aimed to enhance understanding by directly linking experimental findings with their potential implications, facilitating a seamless flow of information and aiding in the comprehension of the study's outcomes. Similarly, for lines 265-272 in section 3.7, their current placement is carefully considered to support the narrative and logical progression of the manuscript. By situating these details within the respective sections, we aim to maintain a clear and structured presentation of both methodological approaches and the unfolding discussion of results. Therefore, I recommend keeping these segments in their current locations to preserve the intended clarity and impact of the manuscript. This approach ensures that readers are equipped with the necessary background at the most relevant points in the text, enhancing the overall readability and effectiveness of the communication.

Provide a reference for Jeon H et al.2009 (line 110)

The reference has been added.

Author Response File: Author Response.pdf

Reviewer 2 Report

The study by Winardi et al. investigating the anti-inflammatory and neuroprotective effects of CDDO in a subarachnoid hemorrhage (SAH) model is interesting and offers a potential therapeutic strategy to reduce brain injury following SAH. The experiments themselves are well-designed. However, some points need to be addressed before publication.

Specific Comments:

1. Justification for CDDO doses: Please elaborate on the rationale behind the selection of CDDO doses used in this study.
2. H&E Staining and Brain Region Identification: To ensure consistent brain regions are compared across all experiments, consider including H&E stained whole brain slices in all figures and indicating the specific brain regions analyzed in Figures 1 (BA morphology), 2 (Iba-1 and GFAP staining), and 4 (NeuN and Caspase 3 staining).
3. Glial Cell Activation vs. Proliferation: Increased Iba-1 or GFAP staining intensity reflects glial cell activation, not necessarily proliferation. To claim glial cell proliferation, quantify the number of Iba-1 or GFAP positive cells in the regions of interest and determine if the number increases within a defined area. Additionally, BrdU staining combined with cell markers could be used as a more specific indicator of glial cell proliferation.
4. Quantification of Iba-1 and GFAP Intensity: Please provide details on how Iba-1 and GFAP intensity was quantified in the Materials and Methods section. This should include information on the software used, quantification criteria, and background and signal parameter settings.
5. High-Resolution Glial Cell Images: To better visualize morphological changes in glial cells following SAH and CDDO treatment, consider including enlarged insets of glial cells in Figure 2. The current magnification may not be sufficient to assess these changes.
6. NeuN and DNA Damage: NeuN is a neuronal nuclear protein that binds to DNA but doesn't directly indicate DNA damage. While changes in NeuN levels can suggest alterations in neuronal numbers, TUNEL assays are more appropriate for assessing DNA damage during apoptosis.
7. Abbreviations and Typos: Please spell out CDDO in full in the abstract and double-check for any typos throughout the manuscript.

Double-check typos

Author Response

The study by Winardi et al. investigating the anti-inflammatory and neuroprotective effects of CDDO in a subarachnoid hemorrhage (SAH) model is interesting and offers a potential therapeutic strategy to reduce brain injury following SAH. The experiments themselves are well-designed. However, some points need to be addressed before publication.

Specific Comments:

1. Justification for CDDO doses: Please elaborate on the rationale behind the selection of CDDO doses used in this study.

I have provided the data in Figure 1 to justify and explain the rationale behind the selection of CDDO doses used in this study. The figure illustrates the dose-response relationship and highlights the optimal concentrations for achieving protective effects against oxidative stress in neuron cells.

2. H&E Staining and Brain Region Identification: To ensure consistent brain regions are compared across all experiments, consider including H&E stained whole brain slices in all figures and indicating the specific brain regions analyzed in Figures 1 (BA morphology), 2 (Iba-1 and GFAP staining), and 4 (NeuN and Caspase 3 staining).

I have included schematic diagrams of whole brain slices in all figures to ensure consistent comparison of brain regions across all experiments. These diagrams clearly indicate the specific brain regions analyzed in Fig 2 (Iba-1 and GFAP staining) and Fig 4 (NeuN and Caspase 3 staining).

3. Glial Cell Activation vs. Proliferation: Increased Iba-1 or GFAP staining intensity reflects glial cell activation, not necessarily proliferation. To claim glial cell proliferation, quantify the number of Iba-1 or GFAP positive cells in the regions of interest and determine if the number increases within a defined area. Additionally, BrdU staining combined with cell markers could be used as a more specific indicator of glial cell proliferation.

I have added graphs displaying the counts of Iba-1 positive and GFAP positive cells within the defined areas of interest. This data will help clarify the distinction between glial cell activation and proliferation in our study.

4. Quantification of Iba-1 and GFAP Intensity: Please provide details on how Iba-1 and GFAP intensity was quantified in the Materials and Methods section. This should include information on the software used, quantification criteria, and background and signal parameter settings.

I have added the requested details on how the Iba-1 and GFAP intensity was quantified to the Materials and Methods section.

5. High-Resolution Glial Cell Images: To better visualize morphological changes in glial cells following SAH and CDDO treatment, consider including enlarged insets of glial cells in Figure 2. The current magnification may not be sufficient to assess these changes.

I have updated the images with an increased magnification of 200X to better visualize the morphological changes in glial cells following SAH and CDDO treatment. These changes are now included as enlarged insets in Figure 2, allowing for a clearer assessment.

6. NeuN and DNA Damage: NeuN is a neuronal nuclear protein that binds to DNA but doesn't directly indicate DNA damage. While changes in NeuN levels can suggest alterations in neuronal numbers, TUNEL assays are more appropriate for assessing DNA damage during apoptosis.

When comparing the use of cleaved caspase-3 and the TUNEL assay to detect apoptosis, cleaved caspase-3 offers certain advantages that can make it a more effective choice under specific circumstances. Caspase-3 is a critical executioner protein in the apoptosis pathway. Its activation is a definitive step in the apoptotic process, making it a highly specific marker for identifying apoptotic cells. This specificity is particularly important because, unlike TUNEL, cleaved caspase-3 does not mistakenly identify cells undergoing other forms of cell death like necrosis or autolysis, which can also result in DNA fragmentation. Furthermore, the TUNEL assay, which tags DNA strand breaks, can sometimes produce false positives by labeling DNA damages that are not necessarily indicative of apoptosis, such as those that will be repaired or those caused by necrotic processes. This means that TUNEL might not always distinguish between apoptosis and other cellular events that lead to DNA breaks.

7. Abbreviations and Typos: Please spell out CDDO in full in the abstract and double-check for any typos throughout the manuscript.

It has been addressed.

Please finds the attached file for detail.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Thank you for addressing my previous comments. There are no more issues.