Isoflurane significantly increased the number of TUNEL-positive nuclei in the INL (P < 0.0001), perhaps with a trend toward significance in the GCL and ONL (Fig. 1). The number of activated caspase-3-positive cells significantly increased in the INL after isoflurane exposure at the 5-hour time point versus air-exposed controls (P = 0.008) (Fig. 2). Given our sample size, we could not establish whether activated caspase-3 levels were increased in INL immediately after isoflurane exposure compared with air exposure (P = 0.138; 95% confidence interval [CI], −1.51 to 4.40). Isoflurane may have also increased the number of activated caspase-3-positive cells in the GCL at both time points after exposure compared with air-exposed controls, based on a trend toward significance (Fig. 2). Activated caspase-3 levels remained relatively unchanged in the ONL after isoflurane exposure (0 hour versus air: P = 0.765; 95% CI, −0.43 to 0.57; 5 hours versus air: P = 0.076; 95% CI, −0.43 to 2.05).
Isoflurane Activates the Intrinsic Apoptosis Pathway in the INL and Both the Intrinsic and Extrinsic Pathways in the GCL
Although the exact upstream mechanisms that initiate anesthesia-induced neurodegeneration are not completely known, the downstream mechanism of neurotoxicity appears to be caspase activation via the mitochondrial pathway of apoptosis followed by activation of the death receptor pathway and neurotrophic factor-activated pathways.6,25 Isoflurane-induced activation of the intrinsic pathway is mediated by downregulation of the antiapoptotic proteins, such as BCL-xL and BCL-2, permeabilization of the outer mitochondrial membrane after Bax translocation, and subsequent release of cytochrome c from mitochondria into the cytosol.6,25 Release of cytochrome c results in formation of the apoptosome and autoactivation of caspase-9, which, in turn, activates the effector caspase, caspase-3.26 The extrinsic apoptosis pathway is activated after binding of various apoptosis-inducing ligands to death receptors which then recruit and activate caspase-8.27 Caspase-8 then activates caspase-3.27 To determine the downstream mechanism of isoflurane-induced activation of caspase-3 in the developing retina, we performed immunohistochemistry for activated caspase-9 and caspase-8 immediately after 1-hour exposure to isoflurane or air on slide-mounted retinal sections. We isolated mitochondria and cytosol from retina and performed immunoblot analyses for the various proapoptotic and antiapoptotic mediators.
Consistent with the activation of the mitochondrial apoptosis pathway, isoflurane significantly increased the number of activated caspase-9-positive cells in the GCL (P = 0.005) (Fig. 2). There may have also been an increase in activated caspase-9 levels in the INL, based on a trend toward significance immediately after exposure (Fig. 2). However, isoflurane had no effect on the levels of activated caspase-9 in the ONL (P = 0.500; 95% CI, −0.35 to 0.35) (Fig. 2). Steady-state levels of Bax were relatively unchanged in the cytosolic and mitochondrial fractions after isoflurane exposure compared with controls (P = 0.101; 95% CI, −0.03 to 0.12 and P = 0.217; 95% CI, −0.19 to 0.41, respectively) suggesting that Bax translocation remained intact (Fig. 3). However, there may have been a decrease in steady-state levels of the antiapoptotic mediator, BCL-xL, in mitochondria after isoflurane exposure, based on a trend toward significance (Fig. 3). This was associated with a significant decrease in steady-state levels of mitochondrial cytochrome c after isoflurane exposure (P = 0.004) and, perhaps, an increase in cytosolic levels with a trend toward a significance indicating the release of cytochrome c from mitochondria (Fig. 3). Although steady-state levels of the antiapoptotic protein, BCL-2, slightly decreased after isoflurane exposure, this difference was not statistically significant (P = 0.073; 95% CI, −0.07 to 0.38) (Fig. 3).
With regard to the extrinsic pathway, activated caspase-8 levels significantly increased in the GCL after isoflurane exposure (P = 0.001) (Fig. 2). Although activated caspase-8 was detectable in both the INL and ONL, there was no change in levels after exposure to isoflurane compared with air-exposed controls (P = 0.486; 95% CI, −0.61 to 0.63 and P = 0.496; 95% CI, −0.24 to 0.24, respectively).
Isoflurane Induces Apoptosis in Amacrine Neurons in the Developing Retina
The retina is made up of several different types of neurons and glia. Thus, we attempted to determine which specific retinal cells undergo apoptosis after isoflurane exposure with double-labeling immunofluorescence on slide-mounted retinal sections 5 hours postexposure. We assessed for colocalization of activated caspase-3 with various retina cell–specific protein markers in isoflurane-exposed and air-exposed cohorts. Activated caspase-3 fluorescence in the INL colocalized with that of the bipolar neuron-specific cytosolic protein, PKC, with the amacrine neuron-specific cytosolic protein, HPC-1, and with the horizontal cell-specific protein, calbindin, in both air-exposed and isoflurane-exposed cohorts (Fig. 4). There may have been an increase in the number and percentage of cells positive for both activated caspase-3 and HPC-1 after isoflurane exposure compared with air-exposed controls, based on a trend toward significance (Fig. 4). It should be noted that we only focused on the INL and did not assess for displaced amacrine cell staining in the GCL. There was no significant effect of isoflurane versus air exposure on the number or percentage of cells positive for both activated caspase-3 and PKC (P = 0.123; 95% CI, −1.78 to 11.02 and P = 0.721; 95% CI, −16.11 to 21.67, respectively) or calbindin (P = 0.749; 95% CI, −14.00 to 18.44 and P = 0.670; 95% CI, −22.36 to 32.36, respectively) (Fig. 4). Our findings suggest that caspase-3 was activated in the cytosol of amacrine cells within the INL after exposure to isoflurane and that caspase activation in bipolar neurons likely represents natural PCD.
FITC-Annexin V Binds to Cells Undergoing Apoptosis in the Developing Retina
Translocation of phosphatidylserine (PS) from the inner leaflet of the plasma membrane to the outer leaflet is one of the earliest features of apoptosis.28 Annexin V, a 35-kD calcium-dependent phospholipid-binding protein, binds to PS with high affinity and has been used to detect apoptosis in different cell and tissue types.28 As a first step in developing a potential fluorescent probe, we aimed to determine whether the fluorophore, FITC-annexin V, could cross the blood-retinal barrier and bind to cells undergoing apoptosis in the retina. We injected FITC-annexin V via the intraperitoneal route 4 hours postexposure to isoflurane or air. One hour later, we assessed for FITC fluorescence and colocalization with activated caspase-3 on slide-mounted retinal sections. FITC-dextran served as a control fluorophore. After intraperitoneal injection, FITC-annexin V was readily detected in the INL of both air-exposed and isoflurane-exposed mice and colocalized with activated caspase-3-positive cells (Fig. 5). However, FITC-dextran was not visible (Fig. 5). The number of FITC-annexin V–positive cells significantly increased in the retina after isoflurane exposure compared with air-exposed controls (P = 0.002) (Fig. 5C). Importantly, all FITC-annexin V–positive cells were positive for activated caspase-3. However, not all caspase-3-positive cells were FITC-annexin V positive. Only about 20% of cells that were positive for activated caspase-3 also demonstrated FITC-annexin V fluorescence in the air-exposed cohort (95% CI, 17.70–25.29, calculated for the single proportion using the Newcombe-Wilson test) (Fig. 5D). However, isoflurane exposure significantly increased the percentage of cells in the retina with colocalized fluorescence (activated caspase-3 and FITC-annexin V) approximately 2-fold versus air-exposed controls (P = 0.002) (Fig. 5D). This suggests that PS translocation occurred later than activation of caspase-3 in the apoptosis pathway but that FITC-annexin V was able to gain access to the retina after systemic injection and bind specifically to cells undergoing PCD.
In this study, we demonstrate that isoflurane induces apoptosis in the developing murine retina on P7. Our findings indicate that, as with the developing brain, one of the downstream mechanisms of anesthesia-induced neurotoxicity in the retina is the activation of the intrinsic apoptosis pathway. This is evidenced by the isoflurane-induced release of cytochrome c from retinal mitochondria, caspase-9 activation in the GCL immediately after exposure, and a trend toward caspase-9 activation in the INL with subsequent activation of caspase-3.
Importantly, the number of TUNEL-positive nuclei exceeded the number of activated caspase-3-positive cells in all 3 layers of the retina in both air-exposed and isoflurane-exposed cohorts. This likely relates to the temporal profile of the activation of the various components of the intrinsic apoptosis pathway.29 Caspase-3 is activated at about 3 hours postrelease of cytochrome c and peaks at around 8 hours. TUNEL-positive staining begins between 3 to 8 hours poststimulus and peaks at 24 hours. Because apoptosis can occur from 24 to 48 hours in some cells, TUNEL assays will identify cells that begin DNA fragmentation due to isoflurane exposure as well as cells that were already in the process of undergoing natural PCD 24 or even 48 hours before exposure. Thus, the number of TUNEL-positive cells will be greater than activated caspase-3. Furthermore, because developmental PCD is also mediated by the intrinsic apoptosis pathway, it is unknown whether cell death results from an isoflurane-induced acceleration of the natural physiologic process within the retina or if cells destined to survive are targeted by the anesthetic. Such a question will need to be further explored.
In addition to activating the intrinsic pathway, isoflurane also activated caspase-8 in the GCL after exposure, suggesting activation of the extrinsic pathway. Although activated caspase-8 was detectable within the INL, the GCL was the only layer to demonstrate a significant increase in levels after anesthetic exposure. It is unknown why caspase-8 became activated specifically in the GCL; however, the finding suggests that isoflurane may activate the death receptor pathway in addition to the intrinsic pathway in retinal ganglion cells. Region-specific activation of the extrinsic apoptosis pathway has been demonstrated in the developing brain after anesthetic exposure.25 For example, caspase-8 became activated in neurons within the parietal and occipital cortex of 7-day-old rats after isoflurane exposure while nuclei of the anterior thalamus showed minimal activation.25 In addition, the phenomenon of GCL-specific caspase-8 activation has been demonstrated in the retina after optic nerve transection.30 Thus, retinal ganglion cells may be susceptible to activation of both pathways after exposure to proapoptotic stimuli.
Tumor necrosis factor (TNF)-α is a proinflammatory cytokine that initiates the extrinsic apoptosis pathway.27 Retinal ganglion cells express the R1 receptor of TNF-α.31 Thus, these neurons may be uniquely susceptible to death receptor ligands. Isoflurane has previously been shown to increase TNF-α levels in the brain after exposure.32 Furthermore, in experimental glaucoma, Müller glia–derived TNF-α has been shown to be a potent mediator of bystander retinal ganglion cell death.31 Thus, although we did not measure TNF-α production in this study, it is possible that isoflurane directly induced TNF-α production in the GCL, leading to activation of the death receptor pathway in ganglion cells. Alternatively, since Müller cells can become secondarily activated after primary ganglion cell death, it is possible that isoflurane directly activated the intrinsic pathway within retinal ganglion cells, leading to Müller glia–derived TNF-α production and secondary ganglion cell death via activation of the extrinsic apoptosis pathway.31
As with the developing brain, the cells in the retina undergoing natural PCD, and those that demonstrate susceptibility to the proapoptotic effect of isoflurane on P7 appear to be neurons.33 This is evidenced by colocalization of fluorescence of activated caspase-3 with that of neuron-specific proteins in both air-exposed and isoflurane-exposed cohorts. Activated caspase-3 labeling in the cytosol of retinal cells undergoing apoptosis colocalized with a bipolar neuron-specific cytosolic protein (PKC), an amacrine neuron-specific cytosolic protein (HPC-1), and rarely with a horizontal neuron-specific cytosolic protein (calbindin) within the INL of both experimental cohorts. Only the number and percentage of cells positive for both HPC-1 and activated caspase-3 increased after isoflurane exposure. These findings indicate that bipolar and amacrine neurons undergo natural PCD on P7 and that amacrine cells are the potential targets of isoflurane-induced apoptosis in the INL of the developing retina. This is consistent with the work demonstrating that most neurons undergoing physiologic apoptosis in the developing rodent retina on P7 are amacrine and bipolar cells within the INL and ganglion cells within the GCL.19,34–36 It is unclear why neurons within the ONL were relatively spared from isoflurane-induced neurodegeneration, but this may relate to the fact that photoreceptor cell death does not peak until later in development.19,35,36 Thus, developmental PCD may play a role in the susceptibility of neurons to anesthesia-induced neurotoxicity. Furthermore, the lack of anesthesia-induced photoreceptor cell death may indicate some level of protection and may provide mechanistic insights for future therapies with further investigation.
Because the retina can be directly visualized noninvasively, the opportunity exists to develop a technique to image anesthesia-induced retinal neurotoxicity in vivo. After intraperitoneal injection, FITC-annexin V was readily visible in the INL of the retina and colocalized with activated caspase-3-positive cells in both air-exposed and isoflurane-exposed mice. Our findings indicate that systemically injected FITC-annexin V crosses the blood-retina barrier and binds to cells undergoing apoptosis, providing a potential opportunity to image natural PCD and isoflurane-induced apoptosis in the developing retina. A method has recently been developed using fluorescent-labeled annexin V to detect neuronal apoptosis in the retina in vivo with noninvasive confocal laser scanning ophthalmoscopy.23 This approach has been shown to enable visualization of single-cell apoptosis within the retina in animal models of glaucoma, optic nerve transection, and after intravitreal injection of staurosporine and is currently in clinical trials in adults.23 In order for such a technique to be of value in the context of anesthesia-induced toxicity, however, a method will need to be developed to differentiate cells dying from natural PCD versus those dying from anesthesia-induced apoptosis. Furthermore, FITC-annexin V labeled only 40% of activated caspase-3-positive cells after isoflurane exposure. Differences in fluorescent-labeled annexin V staining after isoflurane exposure versus models of ocular hypertension and optic nerve transection, for example, likely relate to the route of fluorophore injection, the degree of apoptosis induced by the stimulus, and the retinal tissue layer affected. We injected FITC-annexin V systemically, whereas others have injected annexin V directly into the intravitreal fluid.23 Furthermore, isoflurane exposure on P7 affected neurons mostly within the INL, whereas experimental glaucoma and other models of retinal neurodegeneration appear to affect primarily retinal ganglion cells within the GCL.23 Intravitreal injection permits annexin V to access retinal ganglion cells directly, whereas intraperitoneal injection requires that the fluorophore cross the blood-retinal barrier and gain access to the inner retinal layers. Thus, further development and refinement of this approach will likely be necessary. However, in time, we may be able to translate this novel approach to the human condition to determine whether neuronal apoptosis occurs in the retina after anesthesia exposure.
It should be noted that attempting to extrapolate experimental findings in rodents to humans has major limitations.37 It has been suggested by some that the structural maturity of the murine brain on P7 corresponds to that of the human fetus at 36 weeks postconception, whereas others, using mathematical modeling,37–39 predict that P7 correlates to an even earlier time point in human gestation. In the developing murine retina, with regard to synapse elimination, P7 is thought to correspond to a time point just before birth in humans.40 Thus, the findings presented here may only have direct relevance for the fetal retina and may not necessarily be applicable to the postnatal developing retina. Therefore, in order for these findings to have a clinical impact, future investigation should explore later stages in development more relevant to infants and children.
Furthermore, with regard to developing an imaging tool for anesthesia-induced neurotoxicity, retinal cell apoptosis will carry importance only if there is correlation with the degree of neurodegeneration in the brain. Although we did not directly compare apoptosis in the retina with cell death in the brain of the same animals after isoflurane exposure, we can make some comparisons with a recently studied cohort.22 Compared with the neocortex, hippocampus, and hypothalamus/thalamus, the numbers of TUNEL-positive nuclei and activated caspase-3-positive cells in the retina were relatively increased after an identical 1-hour exposure to isoflurane.22 Although there are limitations with such a comparison, the relative greater degree of neurodegeration seen in the retina may relate to the smaller area of this very thin structure compared with many of the brain regions previously examined but may be because anesthetics are known to affect specific CNS regions differently.22
Despite the limitations, the importance of this work is that we demonstrate for the first time that anesthetics induce neuronal apoptosis in the developing retina. Because the retina provides a window to the CNS and can be imaged noninvasively, our findings create an opportunity to explore anesthesia-induced neuronal degeneration in the developing retina as a potential surrogate for neurotoxicity in the brain. Ultimately, we may be able to develop a noninvasive imaging modality to determine whether anesthesia-induced neuronal apoptosis occurs in infants and children.
Name: Ying Cheng, MS.
Contribution: This author helped conduct the study and analyze the data.
Attestation: Ying Cheng has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Linda He.
Contribution: This author helped conduct the study.
Attestation: Linda He has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Vidhya Prasad, MS.
Contribution: This author helped conduct the study.
Attestation: Vidhya Prasad has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Shuang Wang, PhD.
Contribution: This author helped analyze the data and write the manuscript.
Attestation: Shuang Wang has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Richard J. Levy, MD.
Contribution: This author helped design the study, conduct the study, analyze the data, and write the manuscript.
Attestation: Richard J. Levy has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.
This manuscript was handled by: Gregory J. Crosby, MD.
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© 2015 International Anesthesia Research Society
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