Abstract
Purpose :
Appropriate communication between neurons and blood vessels is essential for normal retinal function. Inter-pericyte tunnelling nanotubes (IPTNTs) are structures vital for neurovascular communication. IPTNTs connect pericytes, the cells surrounding vessels that regulate capillary diameter and blood flow. The interruption of blood supply, or ischemia, occurs in several retinal disorders, including diabetic retinopathy, retinal vascular occlusion, and glaucoma. Re-establishment of blood flow, or reperfusion, often does not prevent neurodegeneration, suggesting that transient ischemia triggers pathophysiology that persists after reperfusion. Here, we use two-photon laser scanning microscopy (TPLSM) in the retina of living mice to study the role of IPTNTs and pericytes after ischemia-reperfusion injury.
Methods :
We induced transient retinal ischemia in BALB/c mice by tying a ligature between the optic nerve and the dural sheath occluding the central retinal artery. We released the ligature after 60 minutes. For permanent ischemia, we left the ligature in place. After one week, we used in vivo TPLSM to image IPTNTs, pericytes, and capillaries. We labelled IPTNTs and pericytes with fluorescent-lectin and capillaries with fluorescein. Ex vivo unbiased stereology analysis was performed to quantify ruptured and intact IPTNTs.
Results :
Ischemia for 60 minutes led to IPTNT rupture (sham: 41±2 ruptured IPTNT/mm2, N=5 mice; ischemia: 96±5 ruptured IPTNT/mm2, N=4 mice; two-tailed Student’s t-test p<0.0001). Permanent ischemia caused almost the complete elimination of IPTNTs (sham: 211±5 total number of IPTNTs/mm2, N=5 mice; one-week ischemia: 24±3 total number of IPTNTs/mm2, N=6 mice, two-tailed ANOVA Tukey’s test p<0.001). Critically, reperfusion promoted the regrowth of IPTNTs, with similar numbers of ruptured IPTNT as sham animals (sham: 39±1 ruptured IPTNT/mm2, N=5 mice; reperfusion: 53±7 ruptured IPTNT/mm2, N=9 mice, two-tailed ANOVA Tukey’s test p=0.30). In vivo experiments showed reduced vessel diameter and dysfunctional capillaries, with reduced blood flow (video) (sham: 5.3±0.1, n=142 vessels, N=9 mice; reperfusion: 4.4±0.1 µm, n=181 vessels, N=6 mice, two-tailed Mann-Whitney test p<0.0001).
Conclusions :
IPTNTs can regrow after transient ischemic insult and potentially reconnect pericytes in distal capillaries. Preserving IPTNT health during ischemia could improve neurovascular function post injury and protect from vision loss.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.