In Vivo Capillary Structure and Blood Cell Flux in the Normal and Diabetic Mouse Eye

Abstract

Purpose To characterize the early structural and functional changes in the retinal microvasculature in response to hyperglycemia in the Ins2 Akita mouse. Methods A custom phase-contrast adaptive optics scanning light ophthalmoscope was used to image retinal capillaries of 9 Ins2 Akita positive (hyperglycemic) and 9 Ins2 Akita negative (euglycemic) mice from postnatal weeks 5 to 18. A 15 kHz point scan was used to image capillaries and measure red blood cell flux at biweekly intervals; measurements were performed manually. Retinal thickness and fundus photos were captured monthly using a commercial scanning laser ophthalmoscope/optical coherence tomography. Retinal thickness was calculated using a custom algorithm. Blood glucose and weight were tracked throughout the duration of the study. Results Elevated blood glucose (>250 mg/dL) was observed at 4 to 5 weeks of age in Ins2 Akita mice and remained elevated throughout the study, whereas euglycemic littermates maintained normal glucose levels. There was no significant difference in red blood cell flux, capillary anatomy, lumen diameter, or occurrence of stalled capillaries between hyperglycemic and euglycemic mice between postnatal weeks 5 and 18. Hyperglycemic mice had a thinner retina than euglycemic littermates ( p < 0.001), but retinal thickness did not change with duration of hyperglycemia despite glucose levels that were more than twice times normal. Conclusions In early stages of hyperglycemia, retinal microvasculature structure (lumen diameter, capillary anatomy) and function (red blood cell flux, capillary perfusion) were not impaired despite 3 months of chronically elevated blood glucose. These findings suggest that hyperglycemia alone for 3 months does not alter capillary structure or function in profoundly hyperglycemic mice.

Differential reductions in the capillary red-blood-cell flux between retina and brain under chronic global hypoperfusion

“…However, the phase-contrast or line-scan imaging is usually conducted in a small lateral field of view (e.g., covering only the targeted capillary), which limits the data acquisition to one capillary segment at a time, making large sampling impractical. 40,43,[45][46][47] Indeed, as shown in those previous works, less than 10 capillaries per animal were measured. RBC flux can also be measured by imaging the fluorescently labeled RBCs (fRBC) with confocal microscopy.…”

“…Other imaging techniques for measuring the retinal capillary blood flow parameters have also been demonstrated. For example, optical coherence tomography (OCT) angiography-based method features a fast and label-free detection of microvascular blood flow, but it only provides a qualitative estimation instead of the absolute measurement of capillary flow parameters, hindering its application when comparing across subjects 40 , 43 . By taking advantage of the RBC-passage induced OCT signal changes, retinal capillary RBC flux can be quantitatively measured 44 .…”

“…By taking advantage of the RBC-passage induced OCT signal changes, retinal capillary RBC flux can be quantitatively measured 44 . However, the intrinsic light contrast is typically associated with a suboptimal signal-to-noise ratio (SNR), affecting the accuracy of RBC flux calculation 40 , 43 – 45 Other label-free techniques for measuring retinal capillary blood flow, such as phase-contrast scanning light ophthalmoscopy and near-confocal microscopy, have also been developed 40 , 43 , 45 …”

“… – 47 With this technique, the angle and the number of the RBC-passage-induced bright stripes in the spatiotemporal image are extracted to calculate RBC velocity and flux, respectively. However, the phase-contrast or line-scan imaging is usually conducted in a small lateral field of view (e.g., covering only the targeted capillary), which limits the data acquisition to one capillary segment at a time, making large sampling impractical 40 , 43 , 45 – 47 Indeed, as shown in those previous works, less than 10 capillaries per animal were measured.…”

Mechanical regulation of the early stages of angiogenesis

“…Blood flow is pulsatile with baseline heart rates typically in the range of 40-100 bpm and exceeding 200 bpm during exercise, which translates to frequencies in the range of 0.7-3.3 Hz. Although pulsatility is partially dampened throughout the arterial vascular tree, several studies suggest the persistence of significant unsteadiness in the microvasculature even down to capillaries in various vascular beds [200,[224][225][226][227][228]. Conceptually, pulsatile flow waveforms can be non-reversing, with positive values throughout the entire cardiac cycle; reversing, with a positive mean value but with negative (or reverse) flow during a portion of the cycle; or purely oscillatory, with periodic fluctuations around a zero mean value (figure 2e).…”