PREreview of In vitrosurvival and neurogenic potential of central canal-derived neural stem cells depend on spinal cord injury type

In this paper, the authors evaluate the in vitro properties of survival, proliferation, and self-organization of control and activated neural stem cells from the spinal cord central canal following two types of injuries: compression and burst fracture spinal cord injury.

The main result is that there is differential early activation of central canal neural stem cells depending on the nature of the injury. They find that dural lacerations in the spinal cord followed by compression result in reduced proliferation of neural stem cells in culture, as well as impaired maturation and self-organization properties during neural differentiation. 

The novelty of this work lies in demonstrating that, at least in vitro, these properties of central canal neural stem cells are persistently altered when the dura mater is compromised, as occurs during surgical decompression of compression spinal cord injuries.

Major issues

• It is unclear what is the rationale behind the sample sizes: ‘Group 1 – Uninjured Controls: n=16, Group 2 – Compression spinal cord injury (cSCI): n=12, Group 3 – Burst fracture spinal cord injury (bfSCI): n=11.’ The effect-sizes seem large enough to justify a smaller N, please clarify.

• In the Materials and Methods section, the authors state that 'For immunohistochemistry (IHC), heat-induced antigen retrieval (HIAR) was carried out on two series of tissue (stained with Nestin/Ki67 and Iba1/GFAP) at 60 °C for 2 hours in PB.' The rationale for using this approach should be clarified, as the tissue sections were not FFPE-embedded but instead 'sectioned coronally at 40 μm in 6 equally spaced series on a freezing microtome for immunolabeling and analysis.' Additionally, this procedure requires citation, as antigen retrieval in PB is not typically employed.

• How were positive cells for different markers counted throughout the study? A full disclosure of the procedure is needed for the sake of reproducibility.

• We recommend including a quantification of GFAP, Iba-1 and Ki67 between control and injury groups to provide stronger support for the reported data, as well as a justification for examining the inflammatory response as early as 3 days post-injury using the Iba-1 marker.

• Regarding the histological verification of the lesion and neurogenic niche activation, it would be very useful to specify whether the data labeled as SCI corresponds to a specific type of injury or if it serves as a representative example to demonstrate the injuries are adequately performed. We recommend including a final statement clarifying this point.

• Regarding the Nissl staining, it would be appropriate for the resulting observations to be further clarified in the body of the text.

• Regarding the verification of the activation of the neurogenic niche in the central canal, it would be helpful to clarify how the quantifications of EdU and Nestin, for which statistical data are provided, were performed. Were these measurements taken from the entire transverse area of the spinal cord, or were they restricted to the central region indicated by the rectangle? Additionally, does it make sense to express this increase as a fold change, given that, according to Table S2, the control value for EdU is <1?

• In Figure 2, was Iba-1 staining measured at 48-72 hpl? This microglial response appears to occur earlier than typically expected (1-2 weeks), so it would be advisable to discuss this. 

• Figure 2, B5 shows a low magnification image of Nestin-labeled cells in a spinal cord tissue section. It seems that many cell types over the CC cells became Nestin+. A higher magnification image of the area would facilitate the interpretation. The manuscript states a significant 34,9 fold increase in Nestin immunoreactivity throughout the lesioned spinal cord. It is not obvious from the control and SCI images since both show CC stained cells.  Does it refer to the CC neurogenic niche, the area included in the rectangle or the whole spinal cord? The quantification method used should also be explained as it doesn't arise from table S2. 

• Additionally, regarding Figure 2, consider moving it to a supplemental figure, as it primarily validates the SCI protocol without presenting new data.

• There are results contained in Figure 4B that deserve to be uncovered. “a small portion of the bfSCI-derived NSC cultures underwent spontaneous differentiation with 0.4% GFAP, 0.6% Beta-III-Tubulin and 0.6% Map2 immunoreactive cells at 23 DIV (Fig. 4B, 5C3, Table S4)”. Either expressing all as percentages from the total, or separating the GFAP/B3T/MAP2 data from the current plot into a new one with a different y-axis scale -as all the relevant numbers are well below 500- would greatly improve the figure. Regarding the same claim, where they refer to Figure 5C3 we were unable to see any signal whatsoever. We understand that showing a representative image of 0.6% is difficult, please consider removing panel 3 from Figure 5 into a supplementary figure as in their current form they do not provide any valuable information.

• In Figure 4c, the y-axis label does not align with the description in the text. It should read 'the average number of nodes connected to another node' instead of 'the number of filaments per node,' as these may not reflect the same information.

• In Figure 5, the scales for panels 2 and 3 are stated to be 50 microns; however, the nuclei sizes appear to differ. A revision of the stated scale or a justification for this discrepancy is necessary.

• Check the order of the figures; for example, we noticed that the flow of the manuscript would benefit from placing Figure 5 before panels a and b of Figure 4, and with panel c of Figure 4 accompanying the content shown in Figure 7.

• The Discussion section contains several references to figures that might suit better to the Results section. Removing them from the discussion might also enhance its read-flow. Also, the discussion would be enriched by adding more references that allow for the development of possible explanations for the differences observed in vitro between the types of injury.

Minor issues

• In the legend for Figure 1, it would be helpful to include a clarification regarding the meaning of the black and white boxes.

• Figure 4A readability can be improved if the y-axis is expressed in scientific notation.

• Figure S7 panel A2 seems to have an undisclosed reddish signal. Please check for jpg artifacts.

• Figures showing cell cultures would benefit from including the days-in-vitro (DIV) stated within the panels.

• Figures showing quantitative data would benefit from a layer indicating the statistical significance (p=value, for each comparison).

Competing interests

The authors declare that they have no competing interests.