Review 1: "Neuroinflammation in Post-Acute Sequelae of COVID-19 (PASC) as Assessed by [11C]PBR28 PET Correlates with Vascular Disease Measures"

RR:C19 Evidence Scale rating by reviewer:

• Reliable. The main study claims are generally justified by its methods and data. The results and conclusions are likely to be similar to the hypothetical ideal study. There are some minor caveats or limitations, but they would/do not change the major claims of the study. The study provides sufficient strength of evidence on its own that its main claims should be considered actionable, with some room for future revision.

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Review: 

Brain vascular damage is suspected in patients with Post-Acute Sequelae of COVID-19 (PASC, also known as "long COVID"), linked to neuroinflammation. This neuroimaging PET study investigated mitochondrial TSPO expression using [11C]PBR28 as a biomarker of microglia activation in 12 individuals with PASC, comparing them to 43 controls. Neuroinflammation was observed in various brain regions, including the midcingulate and anterior cingulate cortex, corpus callosum, thalamus, basal ganglia, and at the boundaries of the ventricles. These findings were significantly associated with several circulating analytes related to vascular dysfunction. 

Despite some limitations, particularly the low number of included patients, the study is highly original and interesting, contributing to the understanding of the brain substrate of long COVID.
Several points require clarification:

1. As mentioned, while the study enhances our understanding of the pathophysiology of PASC, the authors should acknowledge that TSPO PET biomarkers are not readily available in medical routine, making it challenging to claim a future clinical impact in managing patients with long COVID. Among TSPO radiopharmaceuticals, those labeled with 11C are even less accessible outside research cyclotron-equipped centers. This point warrants discussion, including justification for choosing 11C- over 18F-radiolabeled TSPO tracers. What are the advantages of this methodological choice over other 18F PET TSPO tracers? The authors should also deeper discussed previous findings reported in this field with TSPO PET imaging (this article is already briefly discussed: 10.1001/jamapsychiatry.2023.1321; this preprint could also be discussed: 10.1101/2022.06.02.22275916v1; and this preclinical study (10.1186/s12974-023-02857-z).

2. Although the control group is similar in age, there is a significant difference in gender compared to patients, with more women in the latter group. The authors addressed this by comparing TSPO expression in men vs. women controls, reporting no significant effect (although data are not shown). Is this lack of difference in TSPO expression between men and women consistent with previous publications in healthy subjects?

3. TSPO expression strongly depends on genotype. The authors need to clarify the "GG vs GA" genotype count in Table 1. While the distribution is not significantly different among patients vs. controls (p=0.19), it could impact the results (5/7 in patients vs. 27/16 in controls).

4. The authors should specify the exact mask for brain analysis, indicating whether olfactory projections, white matter, brainstem, and cerebellum are included in the voxel-based analysis.

5. SUVs are normalized to cerebellum uptake. Yet the cerebellum is believed to be involved and impaired in long COVID based on FDG PET and structural MRI studies (10.1038/s41586-022-04569-5; 10.1007/s00259-021-05215-4; 10.3389/fnagi.2021.646908). This methodological choice should be better justified. They should also justify the methodological choice of performing analysis based on SUV(R), compared to a potentially more precise approach using compartmental analysis.

6. The authors should compare the locations of brain regions with neuroinflammation found in this study to other studies reporting brain impairment in various regions using different imaging methods, such as FDG/TSPO PET and structural MRI (see previous DOI references).

7. Previous FDG PET studies deserve discussion since the astrocyte dysfunction reported here through microglia activation is presumed to impact glutamate neurotransmission and glucose metabolism.

8. The authors should clarify whether TSPO findings were correlated with symptoms (severity, duration).