Peer review of: Meningeal lymphatics clear erythrocytes that arise from subarachnoid hemorrhage (2020)

Reviewers' Comments: 
 
Reviewer #1: 
Remarks to the Author: 
The manuscript by Cehn et al., entitled “Meningeal lymphatics clear erythrocytes caused 
subarachnoid hemorrhage”, presents a new evidence for the role of meningeal lymphatic vessels in 
subarachnoid hemorrhage. The role of meningeal lymphatic vessels has been recently 
demonstrated in mouse models of AD, MS mouse model of EAE, as well as in models of PD, etc. 
Here the authors provide a compelling evidence that meningeal lymphatic vessels are impacted by 
the subarachnoid hemorrhage and that impairment of their function exacerbates the outcome. The 
experiments are well-performed. It would have been interesting to see whether VEGF-c could 
improve the outcome of subarachnoid hemorrhage but these results may indeed be outside of the 
scope of the current work. I do not have any requests for additional experiments, except for 
repeat of the existing ones to solidify their data. The authors do not mention how many times each 
experiment was reproduced (or at least I missed this information) and the number of mice used in 
each group is low. The authors should repeat their studies to have at least 12 animals per groups 
combined. The authors also do not mention whether the experiments were blinded or not. It is 
extremely important for the authors to repeat their experiments with higher number of mice and 
to make sure the groups are blinded. 
 
 
 
Reviewer #2: 
Remarks to the Author: 
Chen et al present the results of experiments aimed to determine the role of the meningeal 
lymphatic system on clearance of erythrocytes after experimental subarachnoid hemorrhage in 
mice. Resolution of subarachnoid hemorrhage is an important mechanism preventing secondary 
brain injury. The role of the lymphatics in this process would have therapeutic implications and add 
to the growing body of literature on the importance of the meningeal lymphatic system in 
neurological disease. However, multiple concerns limit enthusiasm for the paper. Overall, more 
experimental rigor is needed throughout in order to convince readers of their conclusions. 
 
1. Figure 1A- Why are deep cervical lymph nodes smaller after SAH and NaCl injection than in 
control? The figure legend states the scale bar is 500 mm but this is impossible. These 
representative images also do not match the immunofluorescence in 1B or quantification in 1D. 
 
2. Figure 1 E is the only figure to attempt to show erythrocytes in the meningeal lymphatics. Since 
their primary claim is that the meningeal lymphatics are being used to clear the erythrocytes, 
showing erythrocytes in these structures is critical. However there is no control (non-SAH) image, 
only a single image is shown, there is no quantification, and the Lyve1 cells are often co-localized 
with erythrocytes suggesting engulfment (which raises the question whether they are actually 
staining some macrophages, which are also positive for Lyve1). 
 
3. They use visudyne injected into the cisterna magna followed by photoablation to determine the 
effect of lymphatic ablation on erythrocyte clearance, inflammation, and behavior. These 
experiments depend on the selective ablation of lymphatics without changes to venous sinus flow 
and damage to arachnoid granulations and therefore the direct connection from CSF to the venous 
system. What is the distribution of visudyne at 15 minutes after injection? Confirmation that this 
approach has no effect on the other major structures in the region is critical. 
 
4. What does visudyne + photoactivation do to inflammation and mouse behavior in the absence 
of SAH? All experiments need this control in order to interpret the results. 
 
5. Since all the main results and conclusions of the paper rely on their interpretation of the 
photoablation experiments, an additional method of manipulating lymphatics should be performed 

to confirm the results are not due to off target effects. Several methods for manipulation of the 
VEGFC system have been published to complement their current approach. 
 
6. Why were different time points for anti-Lyve1 antibody and fluorescent microbeads used in 
Figure 5? 
 
7. Are erythrocytes found only in the deep cervical lymph nodes that drain CSF? Did they examine 
other lymph nodes to confirm the specificity of their localization? 
 
8. Were behavioral outcomes analyzed blinded to treatment group? 
 
 
 
Reviewer #3: 
Remarks to the Author: 
This is a very interesting article that attempts to address how red blood cells may be cleared by 
cerebral lymphatics. The authors do a significant amount of immunohistochemistry to try to prove 
their point, but in the end there are many critical flaws with this manuscript. 
 
Major 
1. Using Ter119, while it is specific to murine red blood cells, does not mean that the rbcs are still 
intact when they are seen in the dCLNs. Ter119 may be recognizing a degraded rbc membrane. 
 
2. In fig 2E, to attempt to prove that the meningeal lymphatics are draining the rbcs, a laser 
visudyne system is used, but the control dCLNs look much different compared to an SAH model 
alone. The lyve and ter119 look to be partitioned across the dCLNs. The authors make no mention 
of this. 
 
3. Using the area of Ter119 is not quantitative, it is qualitative at best. What if the reason one is 
seeing more ter119 is because the rbcs are being broken down more effectively and the plasma 
membranes are spreading out across the dCLNs? The rbcs should be quantified stereologically. 
 
4. In figure 3, the increased neuroinflammation should be quantified by flow cytometry, not 
immunohistochem and western blot. Many myeloid cells can express IBa1 and it does not mean 
that they are reactive. 
 
5. In figure 4, the authors give the impression that ablation of lymphatics leads to worsened 
cognitive performance; however, no post hoc tests show a difference between L+V+SAH and 
V+SAH. 
 
6. I don't understand how dynamic lymphatic flow can be quantified based a static picture. This 
needs more explanation, ir it must be done by 2 photon microscopy. 
 
Minor 
 
There are numerous grammatical erros throughout the manuscript in tense and plurality. 
 
All data should be shown as mean and standard deviation, not standard error of the mean, which 
is much smaller than the standard deviation.