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. Author manuscript; available in PMC: 2016 Nov 1.
Published in final edited form as: Anesth Analg. 2015 Nov;121(5):1325–1335. doi: 10.1213/ANE.0000000000000714

Figure 4.

Figure 4

Bipolar neurons and amacrine cells undergo developmental programmed cell death within the inner nuclear layer (INL) on P7 while isoflurane induces apoptosis only in amacrine cells. Double-labeling immunofluorescence was performed on retinal sections 5 hours after exposure to air or isoflurane (Iso). A, Representative INL sections imaged at 40× are depicted. Bipolar neurons were labeled with PKC. Amacrine cells were labeled with HPC-1. Horizontal cells were labeled with calbindin. Müller glia were labeled with GLAST. Green activated capsase-3 staining indicates cells undergoing apoptosis. Arrowheads indicate cells with colocalization of fluorescence with activated caspase-3. Activated caspase-3 staining was rarely associated with calbindin (horizontal cells) and did not colocalize with GLAST in the cytosol of Müller glia. Scale bar, 50 μm. The outer nuclear layer (ONL), INL, and ganglion cell layer (GCL) are labeled. Sections are oriented with ONL at the top of the image and GCL at the bottom. B, Representative images magnified at 100× demonstrating colocalization (arrowheads) of activated caspase-3 (casp-3) green fluorescence with trimethylrhoadmine isothiocyanate (TRITC) (red) fluorescence of PKC in the cytosol of bipolar neurons and HPC-1 of amacrine neurons in air-exposed and isoflurane-exposed cohorts. There was rare colocalization of fluorescence of activated caspase-3 with calbindin of horizontal cells and no colocalization with GLAST of Müller glia. Scale bar, 20 μm. C, Quantification of cells positive for both activated caspase-3 and TRITC within the INL of air-exposed (white bars) and isoflurane-exposed (black bars) cohorts is demonstrated. Percentage of activated caspase-3-positive cells demonstrating colocalization is also depicted. Values are expressed as means + SD. *P = 0.028 versus air-exposed cohort; 95% CI, 5.5–57.2. †P = 0.022 versus air-exposed cohort; 95% CI, 8.8–67.1. PKC = bipolar neuron-specific cytosolic protein; HPC-1 = amacrine neuron-specific cytosolic protein; DAPI = 4′,6-dimidino-2-phenylindole.