Abstract
Purpose :
Alpha smooth muscle actin (α-SMA), a contractile protein, mediates retinal vascular tone and neurovascular coupling. Studies report dense α-SMA surrounding retinal arterioles, but microvessel α-SMA is controversial due to complications of immunohistochemistry (IHC) including antibody penetration and rapid histological degradation. Here we characterize the extent of α-SMA expression in the retinal vasculature using ex vivo and in vivo imaging of transgenic mice.
Methods :
We used two transgenic mouse strains (FVB JAX #025406; C57BL/6J #032887) to study the structure of α-SMA fluorescence expression in vessels (mCherry or mVermilion, and GCaMP). Ex vivo, we performed epifluorescence microscopy of flat-mount retinas from mice >10 weeks old (n=7 FVB, n=5 C57). In vivo, we used a custom-build adaptive optics scanning light ophthalmoscope (AOSLO) to capture phase-contrast (796/17 nm) and fluorescence images with safe light levels (GCaMP: Ex 488 nm Em 520/35 nm; mVermilion: Ex 561 nm Em 630/92 nm) (n=3 FVB). We quantified the diameter and branch order of all vessels that could be traced to the optic disc.
Results :
Arterioles had strong α-SMA expression that diminished at progressive branch orders. Yet ex vivo retinas showed fluorescence in some of the smallest vessels (3 μm), suggesting heterogeneous organization. 41% of α-SMA+ vessels were <7 μm. Both strains had similar expression patterns and we detected α-SMA up to 7th order (5.13 ± 0.83 μm; mean ± SD) (Fig1). Similarly, in vivo imaging showed bright α-SMA in arterioles (visible at 5 μW) while microvessels required more power (134 μW). This was likely due to lower volumetric α-SMA. We observed fluorescence up to 5th order (10.54 ± 2.86 μm). Simultaneous phase-contrast imaging of the same vessels showed luminal diameter of 3.89 ± 1.19 μm necessitating single-file blood flow (Fig2).
Conclusions :
Many microvessels that accommodate single-file blood flow were α-SMA+. AOSLO imaging corroborates ex vivo findings and further overcomes histological confounds. This finding suggests microvessels have the potential to regulate hemodynamics at the scale of tens of micrometers. This is noteworthy given the retina’s high metabolic demand that may be differential in lateral and laminar organization of neural populations. Ongoing work is examining the contractile calcium response (GCaMP) to determine whether differential control exists at this vascular level.
This is a 2021 ARVO Annual Meeting abstract.