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Retinal Characterization of the Thy1-GCaMP3 Transgenic Mouse Line After Optic Nerve Transection
摘要： PURPOSE. GCaMP3 is a genetically encoded calcium indicator for monitoring intracellular calcium dynamics. We characterized the expression pattern and functional properties of GCaMP3 in the Thy1-GCaMP3 transgenic mouse retina. METHODS. To determine the specificity of GCaMP3 expression, Thy1-GCaMP3 (B6; CBA-Tg(Thy1-GCaMP3)6Gfng/J) retinas were processed for immunohistochemistry with anti-green fluorescent protein (anti-GFP, to enhance GCaMP3 fluorescence), anti-RBPMS (retinal ganglion cell [RGC]–specific marker), and antibodies against amacrine cell markers (ChAT, GABA, GAD67, syntaxin). Calcium imaging was used to characterize functional responses of GCaMP3-expressing (GCaMP+) cells by recording calcium transients evoked by superfusion of kainic acid (KA; 10, 50, or 100 μM). In a subset of animals, optic nerve transection (ONT) was performed 3, 5, or 7 days prior to calcium imaging. RESULTS. GFP immunoreactivity colocalized with RBPMS but not amacrine cell markers in both ONT and non-ONT (control) groups. Calcium transients evoked by KA were reduced after ONT (50 μM KA; ΔF/F0 [SD]; control: 1.00 [0.67], day 3: 0.50 [0.35], day 5: 0.31 [0.28], day 7: 0.35 [0.36]; P < 0.05 versus control). There was also a decrease in the number of GCaMP3+ cells after ONT (cells/mm2 [SD]; control: 2198 [453], day 3: 2224 [643], day 5: 1383 [375], day 7: 913 [178]; P < 0.05). Furthermore, the proportion of GCaMP3+ cells that responded to KA decreased after ONT (50 μM KA, 97%, 54%, 47%, and 58%; control, 3, 5, and 7 days, respectively). CONCLUSIONS. Following ONT, functional RGC responses are lost prior to the loss of RGC somata, suggesting that anatomical markers of RGCs may underestimate the extent of RGC dysfunction.
关键词： GCaMP3,retinal ganglion cells,Thy1-GCaMP3,calcium imaging,optic nerve transection
更新于2025-09-23 15:22:29
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Total Retinal Blood Flow in a Nonhuman Primate Optic Nerve Transection Model Using Dual-Beam Bidirectional Doppler FD-OCT and Microsphere Method
摘要： PURPOSE. We validated noninvasive Doppler-optical coherence tomography (OCT) blood ?ow measurements against the terminal microsphere method in a surgical induced optic nerve transection nonhuman primate model. METHODS. In 6 nonhuman primates, total retinal blood ?ow (TRBF) was measured with a custom-built dual-beam bidirectional Doppler Fourier Domain (FD)-OCT. Peripapillary retinal nerve ?ber layer thickness (RNFLT) was measured by Spectralis spectral-domain (SD)-OCT. Measurements were performed every 10 to 15 days before and after unilateral optic nerve transection (ONT) until RNFLT was reduced by more than 40% from baseline. Before the animals were killed, TRBF was measured using the microsphere technique. RESULTS. A signi?cant correlation between all arterial and venous Doppler OCT TRBF measurements was found in ONT and contralateral control eyes (both P < 0.01, n ? 6). The Bland-Altman analysis showed a bias of 0.57 in the ONT group and 0.02 in the contralateral control group. Also, excellent agreement was observed between Doppler OCT and microsphere measurements (P < 0.01, r ? 0.976, bias ? 0.54). After ONT, TRBF and RNFLT decreased by (cid:2)51% 6 42% and (cid:2)44% 6 2% (n ? 5), respectively. In the contralateral control eyes, TRBF and RNFLT were unchanged. CONCLUSIONS. Very good accordance was found between TRBF measurements, obtained with dual-beam bidirectional Doppler FD-OCT and the microsphere method. It also was possible to monitor changes over time in TRBF after ONT with Doppler OCT. These ?ndings highlight the accuracy and potential of noninvasive Doppler OCT to provide valuable information for detecting early changes in ocular disease in future.
关键词： microspheres,optic nerve transection,total retinal blood ?ow,Doppler OCT,nonhuman primate
更新于2025-09-09 09:28:46