Application of second harmonic imaging microscopy to assess structural changes in optic nerve head structure ex vivo

Abstract

Glaucoma represents the second leading cause of blindness worldwide. While both age and intraocular pressure (IOP) are well-recognized risk factors for this disease, the underlying pathologic process involves the accelerated death of retinal ganglion cells (RGCs) that is associated with progressive loss of vision. The loss of RGCs has been postulated to occur primarily by injury to axons in the optic nerve head (ONH) due to its anatomic features and the mechanical vulnerability of the lamina cribrosa, the specialized ONH zone comprised of collagen beams that define the channels or pores through which axon bundles exit the eye. Recent advances in multiphoton microscopy using femtosecond lasers that generate second harmonic (SH) signals from collagen allows for direct optical imaging of the lamina cribrosa. We assess the application of SH generated microscopy (SHG) to the study of the ONH, and test the general hypothesis that increasing intraocular pressure in the same eye results in the movement of ONH collagen beams leading to distortion of the lamina cribrosa channels and compression of the axon bundles.

Polarization microscopy for characterizing fiber orientation of ocular tissues

“…Whereas other techniques of fiber analysis are challenged by either low [7] or high [14] collagen densities, PLM can be used to do fair comparisons independent of density.…”

“…To identify any potential effects of magnification, we compared the automated fiber orientation measurements between images acquired using the Olympus 4x and 10x objectives. Some current techniques for quantifying local connective tissue orientation are affected by tissue density [7,14]. Hence, we also split the data into sparse (lamina cribrosa beams) and dense (sclera) collagen fibers by manually segmenting the regions.…”

“…Hence, to preserve vision, it is important to characterize two elements central to connective tissues, namely the orientation and organization of the collagen fibers that form them [5,6]. Several techniques have been developed for measuring collagen fiber orientation and organization, including small angle light scattering [7,8], x-ray scattering [9][10][11][12], non-linear microscopy [7,13,14] and magnetic resonance imaging [15,16], or for estimating them using inverse numerical methods [17,18]. The complexity of the eye calls for a technique that provides data at multiple scales, including high resolution (micron-scale) and broad field of view (several mm to cm) [19].…”

The Optic Nerve Head as a Robust Biomechanical System

“…[10][11][12]34,39,[53][54][55][56][57][58] The models in this study did not account for LC microarchitecture, which in humans is not fully characterized, 44 but that animal models suggest may alter the local levels of IOPinduced stress and strain(Kodiyalam S, et al IOVS 2009;50:AR-VO E-Abstract 4893). 17,18 The authors are developing FE models that incorporate more realistic anatomies (like the variations in scleral shell thickness 59 ), material properties (anisotropic and nonlinear scleral properties, 29,33,49 lamina cribrosa anisotropy and inhomogeneity 17,43,45 ), and loading (larger IOP insult and cerebrospinal fluid pressure 57,[60][61][62][63].…”

Factors Influencing Lamina Cribrosa Microcapillary Hemodynamics and Oxygen Concentrations

“…The LC is a sieve-like network of collagenous connective tissue beams located in the optic nerve head (ONH) containing a comprehensive network of capillaries that provides nutritional and oxygen support to the RGCs. 6 The LC undergoes morphologic changes (e.g., 'cupping') and significant remodeling during OAG development [7][8][9][10] and these changes have become the focus of many studies that have attempted to link changes in IOP to RGC death. A prominent hypothesis suggests that IOP-induced ONH strains damage the RGCs either directly 11 or indirectly through a mechanical disruption of axonal transport in RGCs, thus depriving RGCs from essential trophic factors.…”