Comparison of the Transduction Capacity of AAV5 and AAV PHP.eB Serotypes in Hippocampus Astroglia

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

1. The experimental design has certain flaws. Using AAV to transduce and study astroglia is a challenging task. Injecting viruses into the brain tissue during the process inevitably causes damage to the animal's brain tissue, leading to gliosis if injury occurs. Gliosis indicates that the observed expression of glial cells is a result of artificial damage rather than natural expression. Figures 1d and 2a of the article show a significant amount of gliosis, indicating that the tissue was damaged and not fully recovered. The author also acknowledges that they did not replicate the experiments of others effectively. The severity of the damage caused by the injection is primarily influenced by the following factors: ①. Injection speed of the virus: The faster the injection speed, the greater the tissue damage. Typically, injection speeds are kept below 50 nl/min. However, the author used a speed of 100 nl/min, which is significantly high and would lead to substantial tissue damage; ②. Injection volume: Generally, larger injection volumes lead to more tissue damage. In this case, the author used a volume of 1 ul, which is substantial and would result in significant tissue damage; ③. Quality of the AAV virus: AAV viruses with lower purity often contain impurities, mainly derived from the host cells used during virus production. These impurities can trigger an immune response in the tissue, causing inflammation and, in severe cases, leading to glial scarring; ④. Duration of virus expression: The shorter the duration of virus expression, the more severe the gliosis. Therefore, experiments of this nature are usually conducted over a longer period, typically exceeding 4 weeks. However, the author only utilized a 3-week timeframe.

2. The number of experimental animals is too limited. In some experiments, the authors used only two animals, which is insufficient. The number of animals should be at least three or more.

3. The quality of the images is subpar. The images in the manuscript lack quality and there is no indication of the equipment or objective lenses used for capturing them.

4. The author should present their data using statistical graphs and charts instead of relying solely on verbal descriptions.

Comments on the Quality of English Language

The manuscript should be carefully edited to fix issues with grammar, spelling, and usage.

Line 17 “as concentration”, should be “as the concentration”.

Line 20 “Obtained results”, should be “The obtained results”.

Line 22 “designed for transduction”, should be “designed for the transduction”.

Line 24 “In certain conditions”, should be “Under certain conditions,”.

Line 29 “in the clinical trials”, should be “in clinical trials”.

Line 31 “penetrating the blood-brain barrier,”, better to use “capable of penetrating the blood-brain barrier,”.

Line 32 “showed”, should be “shown”.

Line 37 “Significant body” should be “A significant body”.

Line 38 “was”, should be “were”.

Line 38 “cell’s population”, better to use “cell population”.

Line 39 “the glial cells”, should be “glial cells”.

Line 41 “of glial population”, should be “of the glial population”.

Line 44 “was attracted” should be “has been attracted”.

Line 45 “acting by”, better to use “the act by”.

Line 49 “regulation of synaptic density”, should be “and regulation of synaptic density”.

Line 50 “Probably, most important (and first studied) is the function of astrocytes to”, should be “Probably the most important (and first studied) function of astrocytes is to”.

Line 54 “regulating the glutamate”, should be “regulating glutamate”.

Line 55 “the synthesis of glutamate”, better to use “the glutamate synthesis”.

Line 55 “glutamine in the extracellular”, should be “glutamine into the extracellular”.

Line 56 “Most if not all astrocytes”, should be “Most, if not all, astrocytes”.

Line 57 “The other important function”, should be “Another important function”.

Line 58 “development as well as maintaining”, should be “development, as well as maintaining”.

Line 60 “Astrocyte-neuron communications”, should be “Astrocyte-neuron communication”.

Line 61 “Recent evidence suggest that”, should be “Recent evidence suggests that”.

Line 63 “aid”, should be “aiding”.

Line 67 “markers applied shows”, better to use “markers shows”.

Line 68 “and, consequently”, should be “and consequently”.

Line 69 “the list of methods of astrocytes detection rapidly increased”, better to use “the list of methods for detecting astrocytes, has rapidly increased”.

Line 70 “List of astrocyte”, should be “The list of astrocyte”.

Line 75 “under control”, should be “under the control”.

Line 81 “ensures a binding”, should be “ensures binding”.

Line 86 “it was repeatedly”, better to use “it has been repeatedly”.

Line 88 “allow the development”, should be “allow for the development”.

Line 92 “Then it”, should be “Then, it”.

Line 94 “become very”, should be “become a very”.

Line 97 “for efficient targeting”, better to use “for efficiently targeting”.

Line 97 “cells including”, should be “cells, including”.

Line 98 “well examined”, should be “well-examined”.

Line 101 “Pairing”, should be “By pairing”.

Line 102 “, or”, should be “or”.

Line 103 “utilized intravenous”, should be “utilized an intravenous”.

Line 109 “work work”, should be “work”.

Line 210-211 “Morphological examination of frontal brain 210 slices revealed in hippocampal area an irregular pattern of transduced astrocyte’s distribution consisting of one to three clusters.”, better to use ” Morphological examination of 210 frontal brain slices revealed an irregular pattern of transduced astrocyte distribution in the hippocampal area, consisting of one to three clusters”.

Line 212 “main population”, should be “the main population”.

Line 212 “and in stratum”, should be “and stratum”.

Line 215 “the third”, should be “a third”.

Line 224 “the AAV5”, should be “AAV5”.

Line 236 “relative the”, should be “relative to the”.

Line 272 “the AAV”, should be “AAV”.

Line 273 “focused mainly on”, should be “mainly focused on”.

Line 281 “but, to”, better to use “but also, to”.

Line 283 “Though”, better to use “While”.

Line 290 “partly”, better to use “partially”.

Line 308 “areas including”, should be “areas, including”.

Line 313 “the typical cortex”, better to use “a typical cortex”.

Line 320 “Basing on”, should be “Based on”.

Line 338 “were focused”, better to use “have been focused”.

Line 345 “known, that”, should be “known that”.

Line 352 “restricted”, better to use “limited”.

Line 361 “restricted”, better to use “limited”.

Line 388,397 “GfaABC1D-driven”, should be “The GfaABC1D-driven”.

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This paper presents the findings from an experiment to assess the comparison of the transduction capacity of AAV5 and AAV 3 PHP.eB serotypes in hippocampus astroglia, through local injection into hip13 pocampus (AAV5, AAV PHP.eB), or by systemic injection in the retro-orbital venous sinus (AAV 14 PHP.eB). I believe there is much merit to the data presented in this paper and the findings are interesting for readers. According to the authors, the results suggest that the usage of AAV5 is more reliable for astrocyte labeling than AAV PHP.eB, and that intrahippocampal injection is more suitable than the systemic one for preferential labeling of hippocampal cells. However, I recommend authors fix some points in the manuscript to improve its presentation. I would encourage the authors to adjust the manuscript to publish it in this important journal. In this context, the following comments should be addressed:

- Line 12: “In vivo” should be in italic

- Lines 23 to 25 – “… inconsistent cellular tropism…” These sentences should be better explained, and for me, the authors could include their conclusion presented in lines 436 to 438 at the end of the abstract section.

- Line 26- Keywords, authors should avoid repeating terms present in the title, for example, "hippocampus" is replaced by "Central Nervous System (CNS)", This helps the article indexing and increases its visibility.

- Line 172 – rephrase “for further processing for immunocytochemistry” by for further immunocytochemistry processing.

- Line 174 – it is missing a comma “…cells, we…”

- Lines 202 to 208 – Figure 1 – The authors should set the number of scale bars in each photomicrograph for better comprehension (since in this case there are different scales)

 

The results section is confusing because the authors discuss the findings and compare them with other research. In my opinion, this section should present only results and the discussion should be set properly in the “discussion section” or the author could design one section with both “Results and Discussion”. On the other hand, many times the author described how to get those findings in the results section, it sounds like a methodology (not results).

Comments on the Quality of English Language

Minor editing of English language is required

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Thank you to the author for addressing all of my questions. Although the conclusions in the manuscript are negative, I still find these negative results to be important. If other laboratories were considering similar experiments, they might reconsider after reading this manuscript. This is a significant contribution to research, saving both time and research funds. I have no further comments or suggestions on the current version.