The optic nerve head (ONH) is the convergence point for nearly two million retinal ganglion cell axons that, having arrived, must turn 90° to enter the neural canal, passing through an opening in Bruch's membrane, choroid, and sclera.
1 Within the neural canal, bundles of axons pass through holes in a perforated series of connective tissue beams known as the lamina cribrosa.
2,3 These circular plate-like beams are attached to a circumferential ring of collagen and elastin fibers on the wall of the neural canal, and function as the load-bearing structures of the optic disc.
4,5 Also present within the lamina cribrosa are astrocytes and laminar capillaries that together sustain the environment of the extracellular matrix (ECM), and provide nutritional support to cellular elements and traversing axonal segments.
6–8
Ageing is associated with increased deposition of collagen and other ECM proteins within the lamina cribrosa.
9–11 Collagens I, III, IV, V, and elastin are increased within the cores of the cribriform plates, leading to an increase in laminar beam thickness.
9,12 This is accompanied by an increase in the thickness of the laminar astrocyte basement membrane.
9,13
Decreased collagen solubility is a good indicator of the amount of abnormal collagen present. In donors under the age of 1 year, collagen solubility in the lamina cribrosa was observed to be 100%; that is, the presence of very little damaged collagen. By the seventh decade of life, this solubility had diminished to 40%, indicative of the accumulative presence of oxidatively damaged and cross-linked collagen molecules.
11 Some of these cross-links arise from the nonenzymic glycosylation of the collagen molecule leading to the formation of advanced glycation end-products (AGEs).
14 The pentosidine-based AGE cross-link has been shown to increase with age in the lamina cribrosa.
11,14,15 The AGEs bind to their receptors (RAGE) to induce release of profibrotic cytokines, such as TGF-β, and proinflammatory cytokines, such as TNF-α and IL-6, leading to increased expression of ECM proteins.
14,15
These compositional and molecular alterations of ageing lamina cribrosa lead to increased thickness and rigidity of the laminar beams, restricted nutritional diffusion across the thickened basement membrane of laminar astrocytes, and a compromised nutritional delivery pathway to axonal segments.
9,10,13,16–19 Similar changes also occur in the peripapillary sclera and the greater rigidity of the scleral canal also contributes to the pathophysiologic changes at the ONH.
20–22 The aged ONH, therefore, is more susceptible to damage from increased IOP or non-IOP-mediated insults.
18,23–27
More advanced changes in the composition of the lamina cribrosa
7,28,29 and its elasticity
18,30 have been noted in glaucomatous samples, leading to the hypothesis that damage to ganglion axons within this region may underlie the primary pathophysiology leading to visual loss in this disease.
4,31–37
Most ECMs are maintained by coupled processes of synthesis and degradation to renew damaged components of the matrix. The degradation pathway is mediated by a family of Zn
2+-containing, Ca
2+-dependent enzymes referred to as the matrix metalloproteinases (MMPs),
38,39 together with their tissue inhibitors (TIMPs).
40 The presence of MMPs 1, 2, and 3 has been demonstrated in ONH with levels considerably increased in primary open-angle and normal-pressure glaucoma patients.
7,8,28,41
Therefore, determining the level of activity of MMPs is important in understanding the role of matrix turnover at the ONH in normal ageing and disease processes. Most MMPs are secreted into the ECM as inactive zymogens (proenzymes). These proenzymes do not possess catalytic activity, but on exposure to SDS during the process of zymography, they are partially activated and, hence, their presence can be identified on zymographic gels. Physiologic activation involves the catalytic removal of a small inhibitory peptide from the pro-MMP molecule.
42–44
Immunohistochemical studies undertaken so far cannot distinguish between active and inactive species, and the overall increase in degree of staining of glaucomatous samples has been assumed to reflect increased MMP activity. This could be an erroneous interpretation, since in human Bruch's membrane from AMD donors, active levels of MMP9 represented only 0.35% of the activity of pro-MMP9.
45
Recent work has shown the MMP system to be far more complicated than previously envisioned. In addition to the monomeric pro and active forms, high molecular weight polymeric forms, termed HMW1 and 2, also have been characterized.
46 Furthermore, large macromolecular MMP complexes, termed LMMC, and comprising pro-MMP9, and HMW1 and 2, and traces of proMMP2, also have been identified.
47 These species are interlinked to form the MMP Pathway (
Fig. 1). The functional importance of this pathway lies in its ability to regulate ageing and regeneration of the extracellular matrix. In Bruch's for example, a shift of the pathway to the left leads to accumulation of high molecular weight species reducing the regeneration potential of the membrane.
46,47 A greater shift to the left is apparent in age-related macular degeneration (AMD), compromising the level of active MMPs, thereby contributing to the structural and functional demise of Bruch's membrane.
45
Does the MMP pathway also operate at the ONH to maintain the structural and functional integrity of the extracellular matrix? In this preliminary work, we have undertaken to screen human optic nerve and rim regions for the presence and relative distribution of the various gelatinase species comprising the MMP pathway. Furthermore, the relative level of active to proenzymes has been used to assess the degree of degradative activity in comparison with activities in the ECM of Bruch's membrane from macular and peripheral regions.