Blood Contrast Agents Enhance Intrinsic Signals in the Retina: Evidence for an Underlying Blood Volume Component

Optical signals intrinsic to the vertebrate retina that are evoked by visual stimulation have been observed using a variety of techniques including reflectance measurements under flood illumination1–10 and, more recently, advanced imaging techniques such as adaptive optics scanning laser ophthalmoscopy (AOSLO)11 and functional optical coherence tomography (fOCT)12–14 (Suzuki W, et al. IOVS. 2010;51:ARVO E-Abstract 1034). It is likely that many of these optical signals are distinct; for some, information is available regarding the underlying cellular or anatomic origins and the biophysical mechanisms responsible. In our own studies, we have described optical imaging of changes in retinal reflectance due to patterned stimuli in the vertebrate retina in vivo (Ts'o DY, et al. IOVS. 2003;44:ARVO E-Abstract 2709). The signals reported in these studies are optical reflectance changes in the near-infrared range (NIR). They are thought to be different from those in the visible range, which show reflectance changes from photopigment bleaching in the photoreceptors.15,16 The in vivo NIR signals show the strongest activations colocalized with the stimulated region of retina, suggesting a local mechanism of action that leads to a decrease in optical reflection. The biophysical origins of these signals have yet to be fully elucidated. One plausible origin of these signals in the retina may arise from hemodynamic coupling associated with neural modulation. This neurovascular coupling has been observed in many areas of the central nervous system and forms the fundamental basis of imaging techniques such as functional magnetic resonance imaging (fMRI)17 and intrinsic signal optical imaging in the neocortex.18 Indeed, specific to studies of the cortex, evidence supports the concept that blood volume changes represent a component of the measured signal.19–22 In relation to the retina, studies primarily using laser Doppler flowmetry (LDF) have shown modulations of blood flow, velocity, and volume in response to visual stimulation.23,24 These demonstrations have confirmed the neurovascular coupling hypothesis in the retina. What remains to be elucidated, however, is the extent to which changes in blood volume contribute to the spatially evoked NIR reflectance changes in the retina. Several reports have provided suggestive evidence that hemodynamics may be a biophysical origin of the retinal signals.4,5,9,25 Our recent efforts have sought to expand this line of research to better understand intricate stimulus-response mechanisms of the retina considering signals may arise directly from events as early as phototransduction through the systemic retinal response. In this study, we test the hypothesis that a component of the retinal intrinsic signals arises from blood volume changes in response to visual stimulation. Using a modified fundus camera described in previous reports,3–5 we examined signal characteristics before and after the injection of blood contrast agents. To enhance functional blood contrast, we made systemic injections of nigrosin (India ink), which has been used to confirm effects of a blood volume component in the neocortex.20 Alternatively, we injected indocyanine green (ICG), a dye used clinically to observe the choroidal circulation,26,27 to act as a separate plasma-bound optical contrast agent. Here we present data demonstrating that injections of these agents effectively increased functional signal strength. The enhanced signal suggests that changes in blood volume in response to visual stimulation represent a component of the intrinsic optical signals reported in vivo.