AFM analysis of ECM softening during glial scar formation

Lead Research Organisation: Newcastle University
Department Name: Sch of Engineering

Abstract

Goal
To use atomic force microscopy to investigate brain tissue softening during glial scarring in vitro.

Hypothesis
An in vitro model of glial scar ECM can be used to identify drugs to suppress softening in glial scar.

Summary
After traumatic brain injury, astrocytes become "activated", proliferating and forming scar tissue; this tissue is referred to as a "glial scar" (1).

Glial scar is both beneficial and detrimental to wound recovery (2). A combination of neuroprotective, neurotoxic, growth inhibitory and growth stimulatory factors are released; division of astrocytes is upregulated and growth of neural axons and dendrites is dysregulated (3).

It was assumed until recently that glial scar is stiffer than the original brain tissue; most scar tissue resists cellular penetration, resulting in a less functional tissue after healing (4).
Recent research indicates that glial scar tissue is substantially softer than healthy brain, meaning that it cannot be acting as a physical barrier to healing (2).

Neurons, microglia and astroglia are affected by mechanical cues in their environment such as ECM stiffness (5,6). Neural stem cells proliferate in injured tissue, preferentially differentiating into glial cells on softer substrates and into neurons on stiffer substrates (7); this is one of the most promising indicators that restoring normal tissue stiffness following brain injury may result in better health outcomes.

Chondroitin Sulphate Proteoglycans (CSPGs) are upregulated during reactive gliosis and appear to be a promising target for preventing softening during glial scarring; this can be achieved via delivery of chondroitinase enzyme to the tissue (8). Additionally, the recently developed drug fluorosamine is a potential candidate for reducing CSPG production (9).

This project will develop an in vitro model of the glial scarring process, using atomic force microscopy (AFM) to evaluate mechanical properties of ECM when treated with various drugs intended to prevent the softening process.

Cell-derived ECM mechanics are not well understood (unlike ECM from decellularised tissue) and AFM techniques for investigating these challenging samples are not well established. This project will develop techniques for investigating these materials.

1. Silver J, Schwab ME, Popovich PG. Central nervous system regenerative failure: role of oligodendrocytes, astrocytes, and microglia. Cold Spring Harb Perspect Biol. 2014 Dec 4;7(3):a020602.
2. Moeendarbary E, Weber IP, Sheridan GK, Koser DE, Soleman S, Haenzi B, et al. The soft mechanical signature of glial scars in the central nervous system. Nat Commun. 2017 Mar 20;8:14787.
3. Chew DJ, Fawcett JW, Andrews MR. The challenges of long-distance axon regeneration in the injured CNS. Prog Brain Res. 2012;201:253-94.
4. Dingal PCDP, Bradshaw AM, Cho S, Raab M, Buxboim A, Swift J, et al. Fractal heterogeneity in minimal matrix models of scars modulates stiff-niche stem-cell responses via nuclear exit of a mechanorepressor. Nat Mater. 2015 Sep;14(9):951-60.
5. Jiang FX, Yurke B, Firestein BL, Langrana NA. Neurite outgrowth on a DNA crosslinked hydrogel with tunable stiffnesses. Ann Biomed Eng. 2008 Sep;36(9):1565-79.
6. Koser DE, Thompson AJ, Foster SK, Dwivedy A, Pillai EK, Sheridan GK, et al. Mechanosensing is critical for axon growth in the developing brain. Nat Neurosci. 2016;19(12):1592-8.
7. Pathak MM, Nourse JL, Tran T, Hwe J, Arulmoli J, Le DTT, et al. Stretch-activated ion channel Piezo1 directs lineage choice in human neural stem cells. Proc Natl Acad Sci U S A. 2014 Nov 11;111(45):16148-53.
8. Bradbury EJ, Moon LDF, Popat RJ, King VR, Bennett GS, Patel PN, et al. Chondroitinase ABC promotes functional recovery after spinal cord injury. Nature. 2002 Apr 11;416(6881):636-40.
9. Keough MB, Rogers JA, Zhang P, Jensen SK, Stephenson EL, Chen T, et al. An inhibitor of chondroitin sulfate proteoglycan synthesis promotes central nervous s

Publications

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Studentship Projects

Project Reference Relationship Related To Start End Student Name
EP/R51309X/1 30/09/2018 29/09/2023
2132772 Studentship EP/R51309X/1 30/09/2018 30/12/2022 John Exton