Vascular Morphometry of the Retina in Antarctic Fishes is Dependent upon the Level of Hemoglobin in Circulation

Antarctic notothenioids express the circulating oxygen-binding protein hemoglobin (Hb) over a broad range of blood concentrations. White-blooded icefishes (Suborder: Notothenioidei, Family: Channichthyidae) are the only known adult vertebrates to lack Hb completely. In addition to its role in oxygen transport, Hb is the primary reactant in degradation of nitric oxide (NO). Thus, NO should be degraded at a slower rate in Hb-lacking icefishes than in Hb-expressing notothenioids, leading to higher steady-state levels of NO in the former group. Increased levels of NO should stimulate upregulation of angiogenesis, the growth and proliferation of new blood vessels from existing vasculature. Based upon these relationships, our laboratory proposes that an inverse correlation exists between vascular density and Hb concentration. Morphometric parameters of retinal vascular anatomy of Antarctic fishes with varying hematocrit (Hct) were quantified and the relationship between vascular density and Hct was assessed. Retinal tissue is particularly amenable to quantification because the entire vascular array is essentially a two-dimensional sheet, eliminating the need for three-dimensional reconstructions. Digital images of retinal vessels filled with a silicone rubber compound ( ~ i c r o f i lw~e~re) analyzed using a macro for MATLAB 7.1 developed for this study. Icefishes display mean blood vessel length densities (Chaenocephalus aceratus, 5.5150.32 mm/mm2; Champsocephalus gunnari, 5.15f0.50 mm/mm2) that are greater than those observed in red-blooded species (Gymnodraco acuticeps, 5.20H.46 mm/mm2; Parachaenichthyes charcoti, 4.40H.30 mm/mm2). Hemoglobinless (-Hb) fishes have average vessel diameters that are -1.5 times larger than vessel diameters of +Hb species (-Hb, 0.19350.006 mm; +Hb, 0.125H.005 mm). The combination of greater length densities and larger diameter vessels results in fractional image areas (i.e., vessel surface areas) that are greater in -Hb icefishes (C. aceratus, 49.1+2.23%; C. gunnari, 43.853.6%) than those seen in +Hb fishes (G. acuticeps, 33.0&4.1%; P. charcoti, 23.851.0%). Vascular density index (VDI), a stereological indicator of vessel number and length, is greatest in -Hb C. aceratus (3.51H.20) and lowest in +Hb Notothenia coriiceps (1.58k0.14). Among four species of +Hb fishes with a \u3e2.3-fold range of Hct, retinal VDI is inversely correlated with Hct (R2=0.934) and intervessel distance in retinal tissue is positively correlated with Hct (R2=0.898). Within this group of closely related fishes, vascular capacity to supply blood to the retina increases as Hct decreases. The direct relationship between VDI and Hct is consistent with the hypothesis of NO-mediated angiogenesis