Pericyte-derived fibrotic scarring is conserved across diverse central nervous system lesions

Fibrotic scar tissue limits central nervous system regeneration in adult mammals. The extent of fibrotic tissue generation and distribution of stromal cells across different lesions in the brain and spinal cord has not been systematically investigated in mice and humans. Furthermore, it is unknown whether scar-forming stromal cells have the same origin throughout the central nervous system and in different types of lesions. In the current study, we compared fibrotic scarring in human pathological tissue and corresponding mouse models of penetrating and non-penetrating spinal cord injury, traumatic brain injury, ischemic stroke, multiple sclerosis and glioblastoma. We show that the extent and distribution of stromal cells are specific to the type of lesion and, in most cases, similar between mice and humans. Employing in vivo lineage tracing, we report that in all mouse models that develop fibrotic tissue, the primary source of scar-forming fibroblasts is a discrete subset of perivascular cells, termed type A pericytes. Perivascular cells with a type A pericyte marker profile also exist in the human brain and spinal cord. We uncover type A pericyte-derived fibrosis as a conserved mechanism that may be explored as a therapeutic target to improve recovery after central nervous system lesions.


Statistics
For all statistical analyses, confirm that the following items are present in the figure legend, table legend, main text, or Methods section.
n/a Confirmed The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement A statement on whether measurements were taken from distinct samples or whether the same sample was measured repeatedly The statistical test(s) used AND whether they are one-or two-sided Only common tests should be described solely by name; describe more complex techniques in the Methods section.
A description of all covariates tested A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons A full description of the statistical parameters including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals) For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted Give P values as exact values whenever suitable.

For Bayesian analysis, information on the choice of priors and Markov chain Monte Carlo settings
For hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes Estimates of effect sizes (e.g. Cohen's d, Pearson's r), indicating how they were calculated Our web collection on statistics for biologists contains articles on many of the points above.

Software and code
Policy information about availability of computer code Data collection Immunofluorescence images were acquired with a Leica DM5500 B bright-field microscope coupled to the LAS X 3.7.2.22383 software, a Zeiss Axioplan 2 upright epifluorescent microscope equipped with the ZEN 2 software (version 2.0.14283.302) or a Leica TCS SP8X confocal microscope equipped with the LAS X 3.5.7.23225 software. Electron microscopic images were acquired with a Tecnai 12 electron microscope (FEI) at 80 kV equipped with ITEM FEI version 5.1 software (Olympus Soft Imaging Solutions, Münster, Germany).

Data analysis
Image analysis, processing, assembly and cell counting were performed with the open source software ImageJ/Fiji (version 2.0.0rc-43/1.51j for Mac), Adobe Photoshop CC 2018 19.1.1 release and Illustrator CC 2018 22.0.1 release for Mac. GraphPad Prism version 6.0g software for Mac was used for all statistical analysis.
For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors/reviewers. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Research guidelines for submitting code & software for further information.

Data
Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability The authors declare that all data supporting the findings of this study are included in this published article (and its supplementary information files  (2011)), groups of 3 to 4 animals are sufficient for quantitative analyses, but in most cases, more animals were used.
Data exclusions As pre-established, for complete crush spinal cord injury experiments, animals undergoing surgery that lost more than 15% of their preoperative body-weight were euthanized.

Replication
Replicate measurements were obtained by analyzing 3 or more alternate tissue sections per animal or human biospecimen. Three or more biological replicates were used per condition. All attempts at replication were successful.

Blinding
The investigators were blinded to group allocation during data collection and analyses.

Reporting for specific materials, systems and methods
We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response. RL-1062, respectively) was used to label human endothelial cells. Biotinylated Lycopersicon Esculentum (Tomato) lectin (biotinylated, Vector Labs, B-1175-1) was used to label blood vessels in mouse tissue. FluoroMyelin Red fluorescent myelin stain (1:500, Invitrogen, F34652) was used to label myelin in mouse tissue.

Authentication
The cell line has been authenticated by the provider. Confirmed as mouse by species-PCR and COI barcoding; inbred strain analysis excluded DBA/s, Swiss, Balb/c, C3H and 129Sv.

Mycoplasma contamination
Cell line tested negative for Mycoplasma contamination.
Commonly misidentified lines (See ICLAC register) No commonly misidentified cell lines have been used in the study.

Animals and other organisms
Policy information about studies involving animals; ARRIVE guidelines recommended for reporting animal research Laboratory animals The following adult (8 weeks of age or older) female and male mice were used in this study: -GLAST-CreERT2 transgenic mice (Slezak, M. et al. Transgenic mice for conditional gene manipulation in astroglial cells. Glia 55, 1565Glia 55, -1576Glia 55, , doi:10.1002Glia 55, /glia.20570 (2007) in a C57Bl/6J genetic background.

Wild animals
The study did not involve wild animals.

Field-collected samples
The study did not involve samples collected from the field. -Stroke post mortem and glioblastoma tissue samples: For stroke samples, tissue collection was performed in such cases that had a history of a supratentorial territorial or lacunar stroke involving cerebral cortical and subcortical areas and who died from non-neurological cause. Healthy occipital cortex and healthy tissue from the contralateral hemisphere in corresponding topography served as control tissue. For glioblastoma samples, initial histological analysis for verification of tumor type and WHO classification was performed by local experienced neuropathologists on formalin embedded tissue pieces. Standard hematoxylin and eosin staining, and immunohistochemical analyses for GFAP, MAP2 and pan cytokeratin-1 (KL-1) confirmed tumor types (glioblastoma, WHO grade IV).
-Multiple sclerosis post mortem tissue samples: Multiple sclerosis tissue samples and associated clinical and neuropathological data were supplied by the UK Multiple Sclerosis Tissue Bank, supported by the Multiple Sclerosis Society of Great Britain and Northern Ireland, in partnership with Imperial College London. All tissue sample characterization and identification were done by independent neuropathologist following standard criteria. For healthy controls, tissue had no pathological conditions and diagnosis.
-Spinal cord injury post mortem tissue samples: Human spinal cord injury samples and related clinical and neuropathologial information were obtained from the International Spinal Cord Injury Biobank (ISCIB), which is housed in Vancouver, BC, Canada.

Recruitment
All biospecimens were collected from consented participants or their next-of-kin for use in future research.

Ethics oversight
This study complied with all relevant ethical regulations regarding experiments involving human tissue samples. Ethical permission for this study was granted by the Regional Ethics Committee of Sweden (2010/313-31/3).
Stroke and glioblastoma samples: The institutional review boards and the local ethics committee of the Medical Faculty of the University of Erlangen-Nürnberg, Erlangen, Germany, approved the study (issued ethical votes No. 4821, 104_1313 and 331_14B) and informed consent was obtained from the relatives of all analyzed patients.
-Multiple sclerosis samples: The ethical permission was granted by the Regional Ethics Committee for UK (Research Ethics Committee for Wales, 08/ MRE09/31+5). All samples have been donated with informed consent for use in future research.
-Spinal cord injury samples: Permission for post-mortem spinal cord acquisition and for sharing of biospecimens was granted by the Clinical Research Ethics Board (CREB) of the University of British Columbia, Vancouver, Canada (Ethics certificate of full board approval H19-00690). All biospecimens were collected from consented participants or their next-of-kin.