Molecular Brain Research 100 (2002) 103–117 www.elsevier.com / locate / bres Research report Matrix metalloproteases and their inhibitors are produced by overlapping populations of activated astrocytes E.M. Muir, K.H. Adcock, D.A. Morgenstern, R. Clayton, N. von Stillfried, K. Rhodes, * C. Ellis, J.W. Fawcett, J.H. Rogers Department of Physiology, University of Cambridge, Cambridge CB23EG, UK Accepted 29 January 2002 Abstract Matrix metalloproteases (MMPs) and tissue inhibitors of metalloproteases (TIMPs) are involved in many cell migration phenomena and produced by many cell types, including neurons and glia. To assess their possible roles in brain injury and regeneration, we investigate their production by glial cells, after brain injury and in tissue culture, and we investigate whether they are capable of digesting known axon-inhibitory proteoglycans. To determine the action of MMPs, we incubated astrocyte conditioned medium with activated MMPs, then did western blots for several chondroitin sulphate proteoglycans. MMP-3 digested all five proteoglycans tested, whereas MMP-2 digested only two and MMP-9 none. To determine whether MMPs or TIMPs are produced by astrocytes in vitro, we tested both primary cultures and astrocyte cell lines by western blotting, and compared them with Schwann cells. All cultures produced at least some MMPs and TIMPs, with no obvious correlation with the ability of axons to grow on those cells. Both MMP-9 and TIMP-3 were regulated by various cytokines. To determine which cells produce MMPs and TIMPs after brain injury, we made lesions of adult rat cortex, and did immunohistochemistry. MMP-2 was seen to be induced in activated astrocytes through the whole thickness of the cortex but not deeper, but MMP-3 was not seen in the injured brain. TIMP-2 and TIMP-3 immunoreactivities were induced in activated astrocytes in deep cortex and the underlying white matter. In situ hybridisation confirmed induction of TIMP-2 in glia as well as neurons, but showed no expression of TIMP-4. These results show that both MMPs and TIMPs are produced by some astrocytes, but TIMP production is particularly strong, especially in deep cortex and white matter which is more inhibitory for axon regeneration. Conversely the MMPs produced may not be adequate to promote migration of cells and axons within the glial scar.  2002 Elsevier Science B.V. All rights reserved. Keywords: Brain injury; Cortex; Cytokines; Glia; MMPs; TIMP proteases 1. Introduction cifically following a mechanical injury. MMPs degrade various extracellular matrix molecules and could thus play After brain injury, major changes occur in the surround- significant roles for both good and ill. MMPs and TIMPs ing brain tissue, due to reactions of both leukocytes and, are involved in many cell migration phenomena and especially, glial cells. The resulting environment is crucial produced by many cell types [7,10,12,30,34,41,98]. for loss or retention of neural function, and for the failure Peripheral neuron growth cones secrete MMPs and other of severed axons to regenerate. At least in the case of proteases which promote their migration through extracel- mechanical injury, this environment includes chondroitin lular matrix [19,59,63,68,71,72]. Likewise but pathologi- sulfate proteoglycans (CSPGs), produced by glial cells, cally, MMPs are important for the invasiveness of some which are an important barrier to axon regeneration. A gliomas [12,41,98]. We therefore investigate whether zone of CSPGs is induced around the injury, and axons fail MMPs and TIMPs are produced in mechanically injured to regenerate past it [4,6,55,60]. brain, and in particular whether they are produced by the In this study we investigate the possible roles of matrix major cell type of the glial scar, viz. astrocytes. We also metalloproteases (MMPs) and tissue inhibitors of metallo- ask whether MMPs would be capable of degrading the proteases (TIMPs) in this post-injury environment, spe- known axon-inhibitory CSPGs, if they were to be either expressed endogenously or delivered exogenously in the injured brain tissue. *Corresponding author. Fax: 144-1223-333-840. E-mail address: jr@mole.bio.cam.ac.uk (J.H. Rogers). The main soluble MMPs produced in the central nervous 0169-328X / 02 / $ – see front matter  2002 Elsevier Science B.V. All rights reserved. PII: S0169-328X(02)00132-8