Defining the role of NG2-expressing cells in experimental models of multiple sclerosis. A biofunctional analysis of the neurovascular unit in wild type and NG2 null mice
Authors: Maria Trojano
Giovanni Ferrara
Giovanna Longo
Carlotta Alias
Sara Morando
Tiziana Annese
Nicole Kerlero de Rosbo
Daniela Virgintino
Francesco Girolamo
Antonio Uccelli
Mariella Errede
Journal: PLoS ONE (2019)
Discipline: Multidisciplinary
Keywords: experimental autoimmune encephalomyelitis; oligodendrocyte precursor cells; neurovascular unit; NG2; blood-brain barrier
Affiliations: Department of Basic Medical Sciences, Neurosciences and Sense Organs, University of Bari School of Medicine, Bari, Italy., B+LabNet-Environmental Sustainability Lab, University of Brescia, Brescia, Italy., Department of Neurosciences, Ophthalmology, Genetics, Rehabilitation and Child Health, University of Genoa, Genoa, Italy., Center of Excellence for Biomedical Research (CEBR), University of Genoa, Genoa, Italy., Ospedale Policlinico San Martino-IRCCS, Genoa, Italy.

Abstract:  During experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis associated with blood-brain barrier (BBB) disruption, oligodendrocyte precursor cells (OPCs) overexpress proteoglycan nerve/glial antigen 2 (NG2), proliferate, and make contacts with the microvessel wall. To explore whether OPCs may actually be recruited within the neurovascular unit (NVU), de facto intervening in its cellular and molecular composition, we quantified by immunoconfocal morphometry the presence of OPCs in contact with brain microvessels, during postnatal cerebral cortex vascularization at postnatal day 6, in wild-type (WT) and NG2 knock-out (NG2KO) mice, and in the cortex of adult naïve and EAE-affected WT and NG2KO mice. As observed in WT mice during postnatal development, a higher number of juxtavascular and perivascular OPCs was revealed in adult WT mice during EAE compared to adult naïve WT mice. In EAE-affected mice, OPCs were mostly associated with microvessels that showed altered claudin-5 and occludin tight junction (TJ) staining patterns and barrier leakage. In contrast, EAE-affected NG2KO mice, which did not show any significant increase in vessel-associated OPCs, seemed to retain better preserved TJs and BBB integrity. As expected, absence of NG2, in both OPCs and pericytes, led to a reduced content of vessel basal lamina molecules, laminin, collagen VI, and collagen IV. In addition, analysis of the major ligand/receptor systems known to promote OPC proliferation and migration indicated that vascular endothelial growth factor A (VEGF-A), platelet-derived growth factor-AA (PDGF-AA), and the transforming growth factor-β (TGF-β) were the molecules most likely involved in proliferation and recruitment of vascular OPCs during EAE. These results were confirmed by real time-PCR that showed Fgf2, Pdgfa and Tgfb expression on isolated cerebral cortex microvessels and by dual RNAscope-immunohistochemistry/in situ hybridization (IHC/ISH), which detected Vegfa and Vegfr2 transcripts on cerebral cortex sections. Overall, this study suggests that vascular OPCs, in virtue of their developmental arrangement and response to neuroinflammation and growth factors, could be integrated among the classical NVU cell components. Moreover, the synchronized activation of vascular OPCs and pericytes during both BBB development and dysfunction, points to NG2 as a key regulator of vascular interactions. 

Retracted: No

PubPeer comments:
 Three separate publications appear to show the same data where it would not be expected. https: doi.org 10.1016 j.jphotochem.2020.113023 https: doi.org 10.1016 j.mssp.2020.105651 https: doi.org 10.1016 j.jcis.2020.11.011 ![file] , 
 With respect to the (stamped with the same lengths but different colors of black and wight) marker bars, from (a) to be is only 6 times, and from (b) to (c) is only 10 times more magnified! With these marker bars and views, they are not expected to be corresponding to each other, or to be for the same sample! ![file] , 
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 https: doi.org 10.1016 j.jphotochem.2020.113023 https: doi.org 10.1016 j.mssp.2020.105651 https: doi.org 10.1016 j.jcis.2020.11.011 ![file] , 
 #2 Regarding the SEM and TEM images, the size distribution of the prepared materials is very large and the analysis is at an arbitrary position. It is also impossible technically to analyze a specific site of a sample by SEM and then analyze the exact same site by TEM. As everyone knows these 2 instruments are separated and analyzing 1 sample by them cannot result in the exact position of the sample. These instruments have also different sample preparations and holders for analysis. , 
 #4 Considering the precision of the FTIR measurements due to the overlapping features of different bonds, I think the difference in the shape of IR in the lower wavenumber areas of these three graphs are sufficiently legitimate and it shows an acceptable difference in the vibration or stretching bonds of Bi-O and Fe-O in the structures. Bond of Fe-O will appear at ~ 570 cm-1 as shown below: ![file] Adopted from RSC Advances 7(38):23583-23590, DOI: 10.1039 C6RA28502C And Bi-O sits at around 580 cm-1: ![file] Adopted from: Materials 11(8):1359, DOI:10.3390 ma11081359 The remaining bonds such as O-H and C-N exist in all three studies materials in these publications. In addition, the strong relative ratio of these common bonds (O-H and C-N) in all three materials gives similar IR spectra at the longer wavenumber regions, which is the major part of these IR spectra. Also, the contribution of the undesired chemical bonds of air constituents has to take into account for the O-H bonds. A side note about the low resolution and accuracy of FTIR is the deviation in the peak assignment that normally occurs in the reported IR bonds in the database. The use of different growth methods and the use of different types of material are also sources of discrepancies of about 90 cm-1. If you look at the text of the articles and the corresponding references, the analyses seem to be re. I found this comment coming from a lack of expertise on the IR technique. We need to have realistic expectations from the FTIR technique considering its constraints ad resolutions where a tiny difference of the peaks has to be taken into account.![](http: ![](http: ))![](http: ) , 
 ![file] For the FTIR spectra, the positions of the peaks are the most important criteria not the intensity of them. In addition, the scales on Y axis (transmittance) are not generally mentioned in many published articles, as the intensity of the samples are different by different measurements, instruments, and pressure preparation. You can simply search u201cFTIR spectrau201d on u201cgoogle imagesu201d and find out many similar FTIR report without the scales. Furthermore, the FTIR of the samples are under analysis by different instruments to compare and confirm the results. In addition, these three samples are CN-based nanomaterials. All researchers and scientists know that CN possesses sharp peaks in FTIR. Furthermore, as the ranges of spectra were broad in the acquired data, we had to stretch them in the range of 400 to 4000 for the clarification as shown below. All the specimens containing Fe-O and Bi-O bindings, have peaks between 500 to 700, which are rationally similar. To make it clear, the range of 400-600 has further been enlarged for all the noted spectra and presented below. Through the enclosed images the differences between Fe-O and Bi-O peaks are crystal clear. , 
 Thank you for the responses. I m afraid you haven t addressed the issue in the above images (in post 1 and 4), which is that the noise within the data seems to have been repeated across all data. Is there a reasonable explanation for the noise being repeated? Even in similar patterns, there is expected to be some variation. , 
 The author of post 2 and 3 ( whoever this is, with access to the journal pre-proof), has created a distraction to the issue regarding this paper, and the two related papers, which is the same, or extremely similar data appearing where it would not be expected. Issues regarding magnification and axis labelling are somewhat trivial in comparison. Please address the issue of data appearing similar. , 
 This does look peculiar. The plots at the top of this thread appear identical to me. Characterisation methods dependent upon electron excitation are by their nature to a degree stochastic or random. This generates noise in measured signals. As such I am unable to explain how it would be possible (even if intentional) to replicate noise with different materials under different experimental conditions. Does anyone else know if this is possible? , 
 As an additional thought images posted in 2 and 3 seem to be watermarked  journal pre-proofs . How can these be obtained? A strange addition to this strand. Are these available to authors only with this journal? I am keen to understand how the initial similarity in post 1 can be explained - these do not address the primary issue. , 
 #1 Here are the graphs replotted from the raw data with a magnified inset showing the questioned part confirming the legitimacy of the acquired data of all three materials. There is no similarity between the noises of these three graphs as can be clearly observed in the inset of the graph below. We d be happy to share the raw data if the request would be sent from an academic email address. At the same time, we appreciate unbiased and true scientific discussions; however, we have to request a legitimate email address with a proper affiliation to further disclose the data. Unfortunately, we and many other non-relevant people have been receiving threatening emails with harassment towards some of the co-authors. ![file] , 
 #11 Preproofs are sometimes available from repositories such as Sci-Hub. , 
 Regarding the XPS valence band spectrum, it should be explained that after some scale expansion the area which seemed too similar shows significant differences in details. Another important point is that because all samples were semiconductors with a bandwidth smaller than 3.2, therefore, the parts beyond linear sections did not affect or interfere with the presented calculation in papers. In many scientific reports, the parts of XPS VBS recorded at high values of Eb showed a high degree of similarity because the background noise is predominant, which in some cases may not pure white noise. If high-frequency noise is superimposed on a low-frequency one the observed noise pattern may seem similar. For example, if the wavelength of the incident x-ray changed in comparison to the d the number of inelastic collisions increased or decreased and the background level as well. , 
 ![file] The three aforementioned XPS valence band spectra have been extracted from a published article in a reputable journal, showing clearly various potential energies. It should be noted that at the first glance all these patterns seem to be completely similar in terms of fluctuations. , 
 ![file] In addition, the spectra of WO3 and MoO3 WO3 look very similar in another article published in u200ean accredited journal. The only difference is the existing shifted along the x axis, while the nature u200eof these samples are utterly different. Accordingly, the similarity in XPS valence bond diagrams u200ecan happen frequently. Furthermore, the similarities in our cases are located in the regions 4 eV u200ethat are not used in calculations. Importantly, noises are different in high magnifications even in u200esuch areas.u200e , 
 ![file] It should also be noted that the XPS valence band spectra in all four aforementioned papers, were not used for identifying the nature of specimens, but for presenting a broad range of examinations. For instance, as presented below, XPS survey spectra are provided for nanocomposites of gCN-NS MgBi2O6, gCN-NS AgBi, gCN-NS FeOCl, and ZnO ZnSnO3 CD, clearly providing the existing of the expected elements in the prepared nanocomposites. The presence of the constituent elements is apparent and confirmative by looking at the spectra. Therefore, the XPS results ascertain the composition of the samples and eliminate any doubt on the prepared nanocomposites. , 
 #12 Regarding post number 12. All four sets of data appear to be the same to me, albeit some are slightly shifted. I am quite surprised this was provided as raw data and proof that the data are different. If it wasn t clear, see below: ![file] , 
 It appears that the noise present in the XPS data is present across 5 different papers. Is there a reasonable explanation for noise being repeatable in this way for 5 different material types? https: www.sciencedirect.com science article pii S1369800120315900 https: www.sciencedirect.com science article pii S0021979720315149 https: www.sciencedirect.com science article pii S1010603020308200 https: www.sciencedirect.com science article pii S1876107021000134 https: www.sciencedirect.com science article pii S0025540821000441 ![file] , 
 Considering the noise in XPS valence band spectrum, you can refer to the above-mentioned explanations. If you send your request through an academic or institutional email, I can send the source files of all papers. , 
 #19 There is in fact no similarities between the four graphs. All the raw data were obtained, re-examined, and replotted using another software (Origin). It is noted that, the similarities are the artifact of compressing the graphs in a small area. Looking closely to the replotted data using Origin, clearly the differences in these four graphs can be observed. This closeness of the noises in the latter part of the graphs can be due to the high level of white noise available with the raw data that when the graphs are compressed, they look quite similar. Some of the coauthors have presented other sets of data published in reputable journals (by other authors) that this trend and similarities can also be observed; please see the above links. , 
 I do not doubt that the journals in question are reputable and the other papers are not my concern, at the moment. The matter at hand (5 re-uses of the same data and rebranding) is. The authors are systematically avoiding the simple matter that the data is identical and there is no logical reason for this to be the case. They either don t or won t understand. Either is contemptable at this stage of the discussion. Ultimately our opinion at this point is moot as the journal editors publisher will likely make a call. I am eager to see how compelling they find the arguments presented. I wonder if any of the other authors wish to comment? The corresponding and first author Van-Huy Nguyen (International Editorial Board, Materials Science in Semiconductor Processing) is notable by his absence in this discussion. Further Quyet Van Le (Associate Editor: Solar Energy ) is also yet to contribute. Are these real people? They cannot be found and this chain would certainly benefit from their opinion given their outstanding recent form in these journals. [Moderator: this is going around in circles; readers will be perfectly able to draw their own conclusions.] ,