Poly(ADP-Ribose) Polymerase Inhibitor PJ34 Reduces Brain Damage after Stroke in the Neonatal Mouse Brain

Round 1

Reviewer 1 Report

Summary: Stroke lesions are comprised of an infarct core contained by a glial scar and a peri-infarct bordering the glial scar and having significantly altered blood flow. Poly(ADP-ribose) polymerase inhibition by PJ34 has been shown to increase cerebral blood flow following neonatal stroke (Bonnin et al., 2020). Maintenance of blood brain barrier (BBB) integrity is critical for the neurovascular unit (NVU) interactions and cell health. In the article “Poly(ADP-ribose) polymerase inhibitor PJ34 reduces anterior brain damage after stroke in the neonatal mouse brain,” Bonnin et al, show alterations in blood flow velocities using Doppler ultrasonography following PJ34 administration is associated with reductions in anterior lesion area after permanent middle cerebral artery occlusion (pMCAo). Bonnin et al show PJ34 increases mean blood flow velocities (mBFV) in the right intracerebral artery (ICA) at 3 hours post-pMCAo. The blood brain barrier (BBB) can become damaged during stroke and Bonnin et al. visualized BBB integrity with immunohistochemical labelling of SST2 receptors. Although Bonnin et al. show no significant difference in Iba1+ cell numbers across cortical layers at 24hours post-pMCAo, they did find increases in GFAP positive cells with stellate morphology at 8 days post-pMCAo in mice treated with PJ34. At 8 days post-pMCAo Bonnin et al. also reveal reductions in tissue area in the anterior sections of lesion in PJ34 treated mice and no significant difference in percent of total tissue loss between groups. Altogether, data presented by Bonnin et al indicate PJ34 treatment increases blood flow velocities associated with reductions in anterior lesion area and increased numbers of stellate astrocytes in a model of neonatal stroke.

Limitations: A major claim of the article, which allows for advancement of current literature, states alterations in BBB integrity are seen with PJ34 treatment, however this is qualitative in its presentation and should be quantified and shown in figure 2. Explanation of the calculation used to determine area of lesion (Figure 4B) in the methods would advance understanding of the impact of PJ34 on lesion size in the anterior brain.  TTC staining is a standard in the field and or some serial sections showing the lesions are a must for this claim to be accurate.  Kits/protocols for TTC are widely available and easier to perform than many of the techniques in this paper.

Strengths: Bonnin et al were able to show significant differences in the number of stellate astrocytes following treatment with PJ34 in a model of neonatal stroke.

Methods: The analysis of the techniques employed were not sufficient to accomplish the goals of the paper.   

Data Interpretation: Interpretation of the data presented in figures 1 and 3 was appropriate. Quantification of data in figure 2 would strengthen claim of BBB integrity. Further detail on methods used to interpret data in figure 4B would strengthen claim about reduced lesion size.

Author Response

Limitations: A major claim of the article, which allows for advancement of current literature, states alterations in BBB integrity are seen with PJ34 treatment, however this is qualitative in its presentation and should be quantified and shown in figure 2.

As requested by the reviewer, quantification of the number of cells with SST2 internalization was done at 48 h in layers I to V. The quantification in the deep cortical layer VI was almost impossible because SST2 internalization, sign of BBB defects, was seen in cells nested (small and in great number). The quantification was shown as a new figure (New Figure 3), to not alter the Figure 2.

Explanation of the calculation used to determine area of lesion (Figure 4B) in the methods would advance understanding of the impact of PJ34 on lesion size in the anterior brain. 

We modified this paragraph, and explain how was quantified the tissue loss 8 days after ischemia. We also modified the Y-axis of the Figure 4B (new Figure 5B), because this was a different presentation of the data obtained for both panels (4A and 4B) now Figure 5A and B.

TTC staining is a standard in the field and or some serial sections showing the lesions are a must for this claim to be accurate. Kits/protocols for TTC are widely available and easier to perform than many of the techniques in this paper.

We thank the reviewer for this information. However, please keep in mind that we work on neonatal mice, with a very small brain, and using this technique with only approximately 5 TTC-stained sections (≥ 1000 µm), we might miss out what we saw on 20-µm-thick Cresyl violet-stained sections. Furthermore, we determined tissue loss 8 days after ischemia, and at this time-point no pale areas were shown, but only a reduction in the volume of the ipsilateral hemisphere as compared to the contralateral.

Strengths: Bonnin et al were able to show significant differences in the number of stellate astrocytes following treatment with PJ34 in a model of neonatal stroke.

Methods: The analysis of the techniques employed was not sufficient to accomplish the goals of the paper.  We have improved the text in the reported techniques.

Data Interpretation: Interpretation of the data presented in figures 1 and 3 was appropriate. Quantification of data in figure 2 would strengthen claim of BBB integrity. Further detail on methods used to interpret data in figure 4B would strengthen claim about reduced lesion size.

Quantification on data presented in figure 2 has been done. This is now presented in a new figure (Figure 3)

Further details on the method used for the figure 4B (now fig. 5B) has been added, and the Y-axis of the panel B has been modified according to what we measured.

Reviewer 2 Report

The manuscript by Bonnin and colleagues investigates the effect of PARP inhibitor PJ34 on cerebral blood flow and subsequent tissue damage after ischemia in neonatal mice. The authors used permanent distal middle cerebral artery occlusion as a model of stroke, and performed ultrasound Doppler imaging, immunohistochemistry and infarct lesion measurements to conclude that PJ34 can partially reduce tissue damage via collateral blood flow supply.

I only have minor comments:

1. There is a discrepancy in how results shown in Fig. 1 are described in the body text vs. figure legend:

• Left ICA/text: mBFV remained stable in PJ34-treated mice; Left ICA/legend: increased mBFV in PJ34-treated mice

• Further, for right ICA, the authors claim no modification in mBFV for PBS-treated mice, however, the individual data points are scattered over a wide range (-35 through +25). The statement should therefore be revised and the distribution/outliers in each direction be discussed.

2. To put numbers of Iba1+ cells in Fig. 3C into perspective, it would helpful to understand basal levels before ischemia. The authors may reference data from other studies (own or others) that established ischemia-induced neuroinflammation as measured by increased Iba-1 immunoreactivity.

3. The statement in l. 128/129 “[…] vessels with GFAP-positive endfeet were still observed (Fig. 4D).” cannot be made without a counterstain for endothelium. The image only stained for GFAP/astrocytes which therefore doesn’t provide any evidence for presence of intact vessels. The authors should either re-phrase their statement, or provide endothelial/astrocyte co-stainings to support their conclusion.

4. The statement in l. 143-145 refers to Supplemental Figure 2, not Supplemental Figure 1.

5. The following statements should be replaced by more scientific verbiage:

• 101/102: “[…] a huge number of Iba-1+-macrophages were observed around the lesion.”
• 128: “[…] with more or less strong GFAP immunoreactivity in the processes. […]”
• 148: “Fortunatly, early establishment of this collateral supply […]”; also, please correct typo.

Author Response

1. There is a discrepancy in how results shown in Fig. 1 are described in the body text vs. figure legend:

• Left ICA/text: mBFV remained stable in PJ34-treated mice; Left ICA/legend: increased mBFV in PJ34-treated mice
• Further, for right ICA, the authors claim no modification in mBFV for PBS-treated mice, however, the individual data points are scattered over a wide range (-35 through +25). The statement should therefore be revised and the distribution/outliers in each direction be discussed.

In the revised manuscript, we have taken into account these comments, and modified accordingly the text and figure 1 legend (in red in the revised manuscript).

2. To put numbers of Iba1+ cells in Fig. 3C into perspective, it would helpful to understand basal levels before ischemia. The authors may reference data from other studies (own or others) that established ischemia-induced neuroinflammation as measured by increased Iba-1 immunoreactivity.

Basal levels of microglia before ischemia have not been classically referenced. However, we have modified the text and given references about microglial cells in the normal brain (references 8 and 9), and reported previous studies (with the use of the same model) showing microglial activation after ischemia in the neonatal brain (references 10 and 11).

3. The statement in l. 128/129 “[…] vessels with GFAP-positive endfeet were still observed (Fig. 4D).” cannot be made without a counterstain for endothelium. The image only stained for GFAP/astrocytes, which therefore doesn’t provide any evidence for presence of intact vessels. The authors should either re-phrase their statement, or provide endothelial/astrocyte co-stainings to support their conclusion.

The text has been modified accordingly.

4. The statement in l. 143-145 refers to Supplemental Figure 2, not Supplemental Figure 1.

We thank the reviewer for its careful reading and modified the text.

5. The following statements should be replaced by more scientific verbiage:

• 101/102: “[…] a huge number of Iba-1+-macrophages were observed around the lesion.”
• 128: “[…] with more or less strong GFAP immunoreactivity in the processes. […]”
• 148: “Fortunatly, early establishment of this collateral supply […]”; also, please correct typo.

Our apologies for theses incorrect sentences. The text has been modified and carefully reading to avoid typo and verbiage.

Reviewer 3 Report

Bonnin et al. conducted this experimental study in new born mice. They occluded the MCA of the pups which were treated with either PJ34 or PBS (controls). PJ34 treatment revealed a prevention from reduction of blood flow in the ICA, and reduce disruption of BBB and astrocyte demise. However, total brain tissue loss was not reduced (whereas lesioned area was reduced in the anterior brain).

Overall, this is an excellent paper. The study is well structured and technically sound. Authors strictly adhered to good lab practise and CAMARADES reporting guidelines whith all details to animal well-fare well reported. Furthermore, figures are very illustrative, well organized and appear where necessary. I would like to congratulate the authors for their great work.

Just a few issues that should be addressed before publication:

• The last sentence of the conclusion leans a bit far out of the window. There is no data presented in the manuscript that suggest a better effect if PJ34 is applied in a higher dose or multiple times.
• I think the work would be even stronger, if you could provide a paragraph (and maybe a conclusion) with a more translation focus.
• Please explain more distinctively your ratio to use neonatal mice (thus eventually addressing neonantal ischemia) and why this treatment could not be used for adolescent/adult cerebral haemorrhage.

Author Response

Overall, this is an excellent paper. The study is well structured and technically sound. Authors strictly adhered to good lab practice and CAMARADES reporting guidelines with all details to animal well-fare well reported. Furthermore, figures are very illustrative, well organized and appear where necessary. I would like to congratulate the authors for their great work.

We are very grateful to the reviewer’s comments and thank him.

Just a few issues that should be addressed before publication:

• The last sentence of the conclusion leans a bit far out of the window. There is no data presented in the manuscript that suggest a better effect if PJ34 is applied in a higher dose or multiple times.

We have modified the last sentence of the conclusion.

• I think the work would be even stronger, if you could provide a paragraph (and maybe a conclusion) with a more translation focus.

We agree with the reviewer and provide a paragraph with a more translational focus.