Per-ischemic changes in penumbral blood supply and its microscopic distribution

Reviewer #1 (Public Review):

Iversen et al. performed middle cerebral artery occlusion in rats to evaluate microscopic changes in the blood flow in the ischemic region. By using measures for global (laser speckle) and local capillary blood flow (two-photon imaging), their results show that the capillary transient time/directionality is affected in this model of ischemic stroke. There are several points that need to be addressed, including what vessels authors considered as capillaries and how they controlled/compensated for the capillary blood flow heterogeneity in their analysis. The authors also proposed that the pericytes are not contributing to these functional deficits by doing morphological analysis, more functional studies are needed to confirm this conclusion.

Reviewer #2 (Public Review):

Novelty: The concept that capillary stalls occur in the ischemic penumbra is not new. However, there are several interesting findings in the current study.
1- Flow reversal, 2- the effect of flow disturbances on oxygenation, and 3- capillary pericytes do not affect the hemodynamics in the penumbra.
However, more in-depth analysis is needed and the underlying mechanism of flow reversal and the link between flow reversal and pericytes is unclear.

Strengths:
1. The study employs a combination of techniques including Laser speckle imaging, two photon microscopy and biophysical modelling to specifically examine hemodynamic and metabolic changes in the penumbra following experimental stroke.
2. The importance of following microvascular flow changes during hours after stroke.
3. The authors used a rat model of stroke and confirmed previous work that has been performed in mice about capillary stalls and flow disturbance in the ischemic penumbra.

Weaknesses:
1- The reliance on laser speckle to define the ischemic core and penumbra is not convincing.
2- The mechanisms behind microvascular flow disturbance are poorly defined.
3- The inability to measure capillary flow simultaneously in the regions of interest: e.g, Bessel beam imaging or volumetric imaging.
4- Lack of baseline measurements.

Reviewer #3 (Public Review):

In the present study, Iversen et al investigate the effect of middle cerebral artery occlusion (MCAo) on penumbral capillary blood flow in rat brains. Using Laser Speckle Contrast imaging and two-photon microscopy, they found that during MCAo the red blood cell dynamics become chaotic in penumbral capillaries despite an apparent constant residual blood flow. They further conclude that these disturbances would cause decreases in steady-state cerebral metabolic rate of oxygen (CMRO2), and tissue oxygen tension (PtO2) using a post hoc biophysical model for oxygen extraction. Interestingly, the authors present data excluding a role for pericytes in altering capillary blood flow. From this observation, the study raises potentially interesting questions on the origin of the disturbance but fails to address them by not investigating the upstream arteriolar behavior. Increased vasomotion, palpability, or intermittent vasospasm may trigger capillary blood flow disturbances without necessarily impacting residual blood flow resting as measured by Laser Speckle Contrast imaging. Furthermore, the data are very poorly presented, here are some examples:
Fig 1b is incorrectly labeled and, assuming this is the "first" 1f panel, the scale bar shows 500 µm while the legend says 200.
Fig 1d is poorly convincing as pink or grey, as detailed in the legend, are not visible. It also looks like there is a second core and penumbra on the more rostral left part of the brain.
Line 219 time is misspelled.
Fig 2, what does "percent of alle capillaries" on the y axes mean? 2d is presented before 2c in the text.
What is the rationale for presenting the statistics from Fig 3 in Fig 4? Panels 4e and 4f are not discussed. The reference in the Fig 4 legend is not formatted.
Fig 6 is presented before Fig 5.
The overall lack of a central hypothesis combined with the aforementioned weaknesses prevents the study from achieving its proposed goal "to characterize microvascular flow disturbances in penumbral tissue in a rat model of acute ischemic stroke".