Neuronal Apoptosis and its Anesthetic Implications Essay
Neuronal Apoptosis and its Anesthetic Implications Essay
Although post operative psychological problems have been hypothesized in the literature for over half a century, it was not until the last few decades that the first research into the effects of anesthetics on neuronal apoptosis came into being.1 In this last decade the pace of research of the effects of anesthetics on neuronal apoptosis has skyrocketed, but as of yet no definitive answers on guidelines or strategies have been formulated1. Despite the lack of concrete answers on what, if any changes will come to the art of anesthesia from this field of research it is sure to provide an interesting subject matter for years to come.
Anatomy and Physiology
In 1999 research was performed on the effect of blockading NMDA receptors in the brains of post natal day seven rats.2 In rats post natal day seven is when peak synaptogenesis is underway, this age is used as a model for human synaptogenesis which occurs between gestational age 20-22 and continues into the infancy. 1,2 The results of NMDA antagonists on the rat brain were a marked increase in neurons undergoing apoptosis compared to the control group. 2 In addition to NMDA receptors, disrupting GABA receptors have been found capable of producing neuronal apoptosis.1Neuronal Apoptosis and its Anesthetic Implications Essay.
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Jevtovic-Todorovic et al., tested the effects of delivering GABA inhibitory on post natal day 7 rats. In their study they used the anesthetics isoflurane, nitrous oxide, and midazolam. The group receiving isoflurane had three tiers, receiving 0.75%, 1.0%, and 1.5%. This allowed a dose dependant relationship between the amount of isoflurane used and the level of neuronal apoptosis achieved to be observed. 3 The researchers also tested the effects of combining GABA inhibitory agents with NMDA antagonists such as nitrous oxide, forming an anesthetic “cocktail.” 3 The combination of isoflurane with nitrous oxide and midazolam produced a much greater level neuronal apoptosis than isoflurane alone.3 The rats who received this combination were tested later in life to see if the effects continued on to adulthood. Their spatial memory was tested via the Morris water maze and the Radial arm maze. The anesthetic combination rats performed significantly worse than the controls, showing that the neurological deficits acquired earlier in life were still present.3
Despite the replication of these rodent studies there were concerns about the generalization of rodent studies to human physiology.1 Brambrink et al., tested the effects of isoflurane on post natal day sic rhesus macaques exposed to isoflurane for five hours. 4 Compared to the control group the experimental group had over 13 times the amount of neuronal apoptosis 3 hours post surgery.4 Another study was performed testing the effects of Ketamine in non human primates as well.4 In this study there was a significant level of neuronal apoptosis in the 24 hour infusion group of postal natal day five monkeys, however those that were only exposed to 3 hour infusions did not show any changes.5 A second primate study using ketamine was published 2 years later, again showing up to 3 hour infusions of ketamine to be safe. 6 Neuronal Apoptosis was again present in the primates exposed to the longer infusion durations of 9 hours and 24 hours.6
It is difficult to get an ideal study of human neuronal apoptosis compared to the animal models but some retrospective studies have been beneficial in looking at the possible effects related to neuronal apoptosis. DiMaggio et al., examined if an early in life hernia repair under general anesthetic would increase the rates of behavioral issues later in life. 7 In this study the cohort who underwent general anesthetic prior to the age of three were more than twice as likely to be diagnosed with a developmental diagnosis than those who did not undergo surgery.7 In a second study by DiMaggio et al., used a twin cohort to view the effects of early anesthesia on developmental disorders.8 In this study the rate of diagnoses for the exposed group was more than double the unexposed group (128.2 diagnoses versus 56.3 diagnoses per 1000 person years). 8 Wilder et al. examined the effects of anesthesia prior to age 4 on the likelihood of developing a learning disorder. 9 In this study a single exposure to anesthesia did not significantly increase the likelihood of developing a learning disorder; however multiple exposures did greatly increase occurrences of learning disabilities.9 Both the length and number of exposures had effects on the probability of developing learning disabilities later in life. 1,9
From early on in development, through birth and on into the first years of life the brain is undergoing massive amounts of synaptogenesis and the construction of neural circuits. 1, 10 At birth the human brain is only one third the size of an adult brain, however it will double in the first year of life and reach 90% of adult size by the age of 6.11 Apoptosis is a normal part of this development, synapse density peaks between 3 and 15 months of age and after that is pruned down via capsase cascades.11 NMDA antagonists and GABA agonist classes of anesthetics have all been implicated in abnormally effecting this natural process and causing excess neuronal apoptosis, Neuronal Apoptosis and its Anesthetic Implications Essay.particularly during peak synaptogenesis in an organism.11 The exact cause of our anesthetics effect on neuronal apoptosis is unknown, but it is known that the NMDA and GABA receptors they target mediate brain development.1,11
There are several cascades that can cause neuronal apoptosis; the two primary ones implicated in anesthetics are the mitochondrial pathway and the death receptor pathway. 1,12 The mitochondrial pathway, also known as intrinsic, is usually activated prior to the death receptor pathway, also known as the extrinsic pathway. 1 In the intrinsic pathway the downregulation of proteins results in the mitochondrial membrane becoming permeable and releases cytochrome c into the cytoplasm1 . The presence of cytochrome c in the cytoplasm triggers the caspase cascade and apoptosis1 With the extrinsic specific death receptors are triggered resulting in caspase-3 release and neuronal apoptosis1.
Anesthesia Implications
Isoflurane, Desflurane, and Sevoflurane are all GABA agonists and have been shown to produce apoptosis in rodents during their peak synaptogenesis periods.11 The NMDA antagonist nitrous oxide given alone has not been shown to cause apoptosis, however in combination with other drugs it has shown a synergistic effect causing more damage than the use of a single volatile anesthetic alone. 11 Studies have been conflicting on the results of benzodiazepines and neuronal apoptosis, some seem to show an effect while others do not in rodent models.11Propofol as with the previous drugs effecting GABA has also been shown to cause significant apoptosis.11 Interestingly Dexmedetomidine by working on alpha 2 receptors rather than GABA or NMDA has not been shown to cause neuronal apoptosis, and can even protect from the damage caused by isoflurane.11,13
Even with the knowledge of what our anesthetic agents are capable of doing in animal models it may not translate the same into humans, as a result there are no hard and fast guidelines available yet.1 Miller states that the most vulnerable group is children under the age of 4 and that it is reasonable to avoid anesthesia until synaptogenesis is completed or to at least limit general anesthesia to under 2 hours if it is required.1 Since there are multiple studies showing the use of combinations of anesthetics producing a more detrimental effect there it may pay dividends to adopt a conservative “less is more” approach.14 There is current research into neuroprotective medications that can be given concomitantly with our conventional anesthetics however it is still too early to incorporate any of these techniques into practice.1,14. In their article Chiao and Zuo also agree that it is too early to change practices based on the research available, but that it may be worthwhile to delay elective surgery until after the age of four when possible.12 The recommendations found within Practice of Anesthesia for Infants and Children state that “it would be very unwise to change practice based on concerns of anesthetic neurotoxicity, while potentially increasing the risks of cardiovascular or respiratory complications.”11
Current Literature
Duan et al. published research on the effects of ketamine injections in combination with dexmedetomidine in rodents.15 The experiment groups were divided as follows; saline and saline, ketamine and saline, dexmedetomidine and saline, and ketamine and dexmedetomidine. Neuronal Apoptosis and its Anesthetic Implications Essay. They found that the administration of dexmedetomidine prevented the neuronal apoptosis that has been consistently seen with the administration of ketamine in rodents.15 Additionally they tested the subjects at 60 days of age in a Morris water maze and found that the neuroprotective effect of dexmedetomidine was long lasting.15
In the Netherlands Hansen et al. performed a retrospective analysis of educational outcomes on children who underwent pyloric stenosis repair before the age of 3 months. This study in particular is impressive because of its large enrollment, with 779 who underwent surgery and 14,665 children in the control.16 The study examined average grades scores in ninth grade and found no significant difference between the controls and the children who underwent surgery.16 This is particularly interesting because the age at which the children underwent surgery is coincides with peak synaptogensis and the age where you would expect to find the most effect of general anesthetics on neurodevlopment.1,11
One major study underway with a great chance to provide definitive information on the effects of anesthesia on pediatrics is the GAS study. This study is being completed presently with an anticipated end date of 1/07/2015. In this study 660 infants in two cohorts are compared, those undergoing general anesthesia for an inguinal hernia repair and those undergoing spinal anesthetic for the same surgery.17 By using the Wechsler Preschool and Primary Scale of Intelligence test to measure their I.Q. at 5 years of age this study should provide great insight into the affects of general anesthesia compared to regional in infants near their peak synaptogenesis.17
Conclusion
The occurrence of neuronal apoptosis in association with our commonly used anesthetics is an unsettling phenomenon. Unfortunately the research available is spare and sometimes provides conflicting evidence the details of process and the associations found in animal models might not extrapolate well to humans. Until more research is completed it is important for the anesthesia provider to be aware of the possibly negative side effects of our anesthetics when used during peak synaptogenesis. Despite the possibility that certain anesthetics may result in neuronal apoptosis it would be premature to change our practice. In the next few years prospective human studies will have been completed and may give us a definitive answer that we can incorporate into evidence based practices to improve patient outcomes. Once more concrete data is available we will be able to incorporate whatever risks are associated with certain anesthetics with the already known cardiovascular and respiratory factors and provide the most optimal anesthesia possible. Neuronal Apoptosis and its Anesthetic Implications Essay.
References
1. Miller RD. Miller’s Anesthesia. 8th ed. Philadelphia (PA): Elsevier; 2014: 329-345
2. Ikonomidou C, Bosch F, Miksa M, et al. Blockade of NMDA Receptors and Apoptotic Neurodegeneration in the Developing Brain. Science. 1999;283:70-74.
3. Jevtovic-Todorovic V, Hartman RE, Izumi Y, et al. Early Exposure to Common Anesthetic Agents Causes Widespread Neurodegeneration in the Developing Rat Brain and Persistent Learning Deficits. Journal of Neuroscience. 2003;23:876-882.
4. Brambrink A. Isoflurane-induced neuroapoptosis in the neonatal rhesus macaque brain. Anesthesiology. 2010;112:834-841.
5. Slikker J, William, Zou X, Hotchkiss CE, et al. Ketamine-induced neuronal cell death in the perinatal rhesus monkey. Toxicological sciences : an official journal of the Society of Toxicology. 2007;98:145-158.
6. Zou X, Patterson TA, Divine RL, et al. Prolonged exposure to ketamine increases neurodegeneration in the developing monkey brain. International Journal of Developmental Neuroscience. 2009;27:727-731.
7. DiMaggio C, Sun LS, Kakavouli A, Byrne MW, Li G. A retrospective cohort study of the association of anesthesia and hernia repair surgery with behavioral and developmental disorders in young children. J Neurosurg Anesthesiol. 2009;21:286-291.
8. DiMaggio C, Sun LS, Li G. Early childhood exposure to anesthesia and risk of developmental and behavioral disorders in a sibling birth cohort. Anesth Analg. 2011;113:1143-1151.
9. Wilder RT, Flick RP, Sprung J, et al. Early exposure to anesthesia and learning disabilities in a population-based birth cohort. Anesthesiology. 2009;110:796-804.
10. Blaylock M, Engelhardt T, Bissonnette B. Fundamentals of neuronal apoptosis relevant to pediatric anesthesia. Paediatr Anaesth. 2010;20(5):383-95.
11. Cote CJ, Lerman J, Anderson BJ. A Practice of Anesthesia for Infants and Children, Expert Consult – Online and Print. Elsevier Health Sciences; 2013. Neuronal Apoptosis and its Anesthetic Implications Essay.
12. Chiao S, Zuo Z. A double-edged sword: volatile anesthetic effects on the neonatal brain. Brain sciences. 2014;4:273-294.
13. Sanders R. Dexmedetomidine attenuates isoflurane-induced neurocognitive impairment in neonatal rats. Anesthesiology. 2009;110:1077-1085.
14. Loftis, Grace Kline,C.R.N.A., M.S.N., Collins, Shawn, CRNA,PhD., D.N.P., McDowell, Mason,C.R.N.A., M.S.N.A. Anesthesia-induced neuronal apoptosis during synaptogenesis: A review of the literature.AANA J. 2012;80(4):291-8.
15. Duan X, Li Y, Zhou C, Huang L, Dong Z. Dexmedetomidine provides neuroprotection: impact on ketamine-induced neuroapoptosis in the developing rat brain. Acta Anaesthesiol Scand. 2014;58:1121-1126.
16. Hansen TG, Pedersen JK, Henneberg SW, Morton NS, Christensen K. Educational outcome in adolescence following pyloric stenosis repair before 3 months of age: a nationwide cohort study. Paediatr Anaesth. 2013;23:883-890.
17. Davidson, A. The effects of anaesthesia on neurodevelopmental outcome and apnoea in infants: the GAS study Available at: http://www.controlled-trials.com/isrctn12437565/Gas. Accessed November 15, 2014.
Anesthetics cause widespread apoptosis in the developing brain, resulting in neurocognitive abnormalities. However, it is unknown whether anesthesia-induced neurotoxicity occurs in humans because there is currently no modality to assess for neuronal apoptosis in vivo. The retina is unique in that it is the only portion of the central nervous system that can be directly visualized noninvasively. Thus, we aimed to determine whether isoflurane induces apoptosis in the developing retina.
METHODS
CD-1 male mouse pups underwent 1-hour exposure to isoflurane (2%) or air. After exposure, activated caspase-3, caspase-9, and caspase-8 were quantified in the retina, cytochrome c release from retinal mitochondria was assessed, and the number and types of cells undergoing apoptosis were identified.Neuronal Apoptosis and its Anesthetic Implications Essay. Retinal uptake and the ability of fluorescent-labeled annexin V to bind to cells undergoing natural cell death and anesthesia-induced apoptosis in the retina were determined after systemic injection.
RESULTS
Isoflurane activated the intrinsic apoptosis pathway in the inner nuclear layer (INL) and activated both the intrinsic and extrinsic pathways in the ganglion cell layer. Immunofluorescence demonstrated that bipolar and amacrine neurons within the INL underwent physiologic cell death in both cohorts and that amacrine cells were the likely targets of isoflurane-induced apoptosis. After injection, fluorescent-labeled annexin V was readily detected in the INL of both air-exposed and isoflurane-exposed mice and colocalized with activated caspase-3-positive cells. Neuronal Apoptosis and its Anesthetic Implications Essay.
CONCLUSIONS
These findings indicate that isoflurane-induced neuronal apoptosis occurs in the developing retina and lays the groundwork for development of a noninvasive imaging technique to detect anesthesia-induced neurotoxicity in infants and children.
Commonly used anesthetics induce widespread neuronal apoptosis in the developing mammalian brain.1–5 Vulnerability coincides with synaptogenesis, and anesthesia-induced neurodegeneration results in significant loss of neurons, cognitive impairment, and behavioral abnormalities in a variety of newborn animal models.6,7 A number of retrospective studies indicate an association between anesthesia exposure and cognitive and behavioral disorders in young children; however, anesthetics have never been shown to cause neurotoxicity in humans.8–13 Furthermore, it is unknown whether anesthesia-induced neurodegeneration occurs in humans because there is currently no modality to noninvasively assess for apoptosis in the brain of infants and children.
Programmed cell death (PCD) is a widespread and natural phenomenon that occurs in 2 waves within the developing central nervous system (CNS).14 The early wave of PCD peaks during midembryogenesis and is important for progenitor cell size, morphogenesis, proliferation, and differentiation, whereas the postnatal wave is critical for synaptogenesis and elimination of aberrant neuronal connections.14 Thus, the prenatal wave regulates progenitor pool size and the later wave ensures proper wiring.15In the rodent brain, the postnatal peak in developmental PCD occurs within the period of peak synaptogenesis.16–18 Thus, physiologic apoptosis may influence the susceptibility of the developing brain to anesthesia-induced neurotoxicity. Neuronal Apoptosis and its Anesthetic Implications Essay. The retina is a highly specialized layer of neural tissue within the CNS that enables vision. As in the brain, neurons in the developing retina undergo developmental apoptosis.19 The early embryonic phase of PCD in the retina also coincides with neurogenesis, migration, and differentiation, whereas the postnatal phase is associated with synaptogenesis for the selective elimination of aberrant synapses.19
The retina is unique in that it is the only portion of the CNS that can be directly visualized noninvasively. Recently, high-resolution methods have been developed to image single-cell apoptosis within the retina in vivo.20 Thus, if anesthetics are shown to cause neuronal apoptosis in the developing retina, it may be possible to exploit such neurodegeneration as a surrogate for anesthesia-induced brain neurotoxicity to determine whether the phenomenon occurs in infants and children. However, the effects of anesthetics on the developing retina have not been evaluated.
In this study, we test the hypothesis that anesthetics induce neuronal apoptosis in the developing murine retina. We demonstrate that isoflurane activates caspase-3 in neurons within the inner nuclear layer (INL) of the retina via the intrinsic apoptosis pathway and within the ganglion cell layer (GCL) via both the intrinsic and extrinsic pathways. Furthermore, a systemically injected fluorescent probe readily crossed the blood-retinal barrier to bind to cells undergoing apoptosis in the INL. These findings lay the groundwork for the development of a noninvasive imaging technique to detect anesthesia-induced neuronal degeneration in vivo and indicate that isoflurane-induced neuronal apoptosis occurs in the developing retina.
METHODS
Animal Exposures
The care of the animals in this study was in accordance with the National Institutes of Health (NIH) and Institutional Animal Care and Use Committee guidelines. Study approval was granted by the Children’s National Medical Center. Six- to 8-week-old CD-1 pregnant female mice (20–30 g) were acquired (Charles River, Wilmington, MA) to yield newborn pups. On postnatal day 7 (P7), we exposed male CD-1 mouse pups to either air or isoflurane (2%) in air for 1 hour in a 7-L Plexiglas chamber (25 cm × 20 cm × 14 cm). The chamber had a port for fresh gas inlet and a port for gas outlet which was directed to a fume hood exhaust using standard suction tubing. Air (Air Products, Camden, NJ) was delivered through a variable bypass isoflurane vaporizer and exposure chamber at a flow rate of 8 to 12 L/min. Mice were kept warm with an infrared heating lamp (Cole-Parmer, Court Vernon Hills, IL). P7 was chosen because it is the time point of maximal vulnerability to anesthesia-induced neurodegeneration in the developing brain and because synaptogenesis peaks at day 7 in rodents.16,18 One hour exposure to 2% isoflurane activates brain capsase-3 in 7-day-old mice and represents a brief anesthetic exposure.21,22 Different cohorts of pups were randomly assessed either immediately postexposure or 5 hours post-exposure to isoflurane or air. Pups evaluated at the 5 hours were placed with their respective dams after exposure. Neuronal Apoptosis and its Anesthetic Implications Essay.
Fluorophore Injection
In a separate cohort of animals, pups were injected with 25 μL of either fluorescein isothiocyanate (FITC)-conjugated annexin V (BD Biosciences, San Jose, CA) or FITC-conjugated dextran (Sigma-Aldrich, St. Louis, MO) via the intraperitoneal route 4 hours after exposure to isoflurane or air. One hour postinjection, pups were euthanized via intra-peritoneal pentobarbital injection (150 mg/kg). Dose and timing of fluorophore injection were chosen based on previous work and pilot data.23
Cresyl Violet Staining
At the time of euthanasia, after pentobarbital injection (150 mg/kg, intraperitoneal), the animals were systemically perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) via left ventricle injection for 30 minutes. Both eyes were enucleated and then postfixed in additional fixative solution for 24 hours at 4°C. Paraffin-embedded whole eye serial sections were cut at a thickness of 6 μm, and individual sections were slide mounted and stained with cresyl violet for 30 minutes.
Terminal Deoxynucleotidyl Transferase-Mediated UTP Nick End-Labeling Staining
Five hours postexposure, after euthanasia, the animals were systemically perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) via left ventricle injection for 30 minutes. Both eyes were enucleated and then postfixed in additional fixative solution for 24 hours at 4°C. Paraffin-embedded whole eye sections were cut into 6-μm sections, slide mounted, and stained with terminal deoxynucleotidyl transferase (TdT) UTP nick end labeling (TUNEL). Sections were incubated in 0.5% Triton at room temperature, followed by proteinase K at 37°C, then immersed in TdT buffer (30 mmol/L Tris-HCl buffer, pH 7.2, 140 mmol/L sodium cacodylate, and 1 mmol/L cobalt chloride) at room temperature. This was followed by incubation with TdT and biotin-16-dUTP for 60 minutes at 37°C. The reaction was terminated with termination buffer (300 mmol/L sodium chloride with 30 mmol/L sodium citrate) at room temperature, followed by immersion in 3% hydrogen peroxide and 2% fetal bovine serum at room temperature. The sections were then covered with an avidin-biotin complex (1:200 dilution) for 30 minutes at room temperature, incubated with FITC-Avidin D for detection and counterstained with 4′,6-dimidino-2-phenylindole (DAPI). Six to 8 regions of interest were randomly selected under fluorescence microscopy (Olympus BX41 microscope; Olympus America Inc., Melville, NY) in a blinded manner (performed by VP) in 3 to 4 nonserial sections per mouse (15–30 μm from the optic nerve bundle). The numbers of TUNEL-positive nuclei were manually quantified in the outer nuclear layer (ONL), INL, and GCL per square micrometer at 10× magnification and corrected for the calculated area in the region of interest using NIH Image J v. 1.33 in 4 to 8 animals per group (based on sample sizes used previously22).
Immunohistochemistry
In separate cohorts, after euthanasia with pentobarbital injection (150 mg/kg, intraperitoneal), the animals were systemically perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) via left ventricle injection for 30 minutes either immediately after or 5 hours postexposure to isoflurane or air. Both eyes were enucleated and then postfixed in additional fixative solution for 24 hours at 4°C. Paraffin-embedded whole eye serial sections were cut at a thickness of 6 μm, and individual sections were slide mounted. Sections were deparaffinized and rehydrated with xylene, ethanol, and water. Antigen retrieval was performed with 0.01 M sodium citrate (pH 6.0) for 10 minutes at 100°C. Immunohistochemistry was performed using polyclonal rabbit anti-human–activated caspase-3 antibody (9661; Cell Signaling Technology, Beverly, MA), polyclonal rabbit anti-mouse–activated caspase-9 antibody (9509; Cell Signaling Technology), or monoclonal rabbit anti-mouse–activated caspase-8 antibody (8592; Cell Signaling Technology) with biotinylated secondary antibody (goat anti-rabbit; Cell Signaling Technology) and developed with diaminobenzidine. Ten to 12 regions of interest were randomly selected under light microscopy (Olympus BX41 microscope; Olympus America Inc.) in a blinded manner (performed by VP) in 3 to 4 nonserial sections per mouse (15–30 μm from the optic nerve bundle). The numbers of activated caspase-positive cells were manually quantified per square micrometer in the ONL, INL, and GCL at 10× magnification and corrected for the calculated area in the region of interest using NIH Image J v. 1.33 in 3 to 8 animals per group (based on sample sizes used previously22).
Immunoblot Analysis
After euthanasia with pentobarbital injection (150 mg/kg, intraperitoneal), retina mitochondria and cytosol were isolated by differential centrifugation immediately after exposure to isoflurane or air. Retina was harvested and homogenized in ice-cold H medium (70 mM sucrose, 220 mM mannitol, 2.5 mM HEPES, pH 7.4, and 2 mM EDTA). The homogenate was spun at 1500 g for 10 minutes at 4°C. Supernatant was removed and centrifuged at 10,000g for 10 minutes at 4°C. Cytosolic supernatant was collected, and pellet was resuspended in H medium and centrifuged again at 10,000g for 10 minutes at 4°C. Pellet was again resuspended in H medium, and mitochondrial and cytosolic protein concentrations subsequently determined using the method of Lowry.
Ten microgram samples of homogenized retina mitochondrial or cytosolic protein were subjected to SDS-acrylamide gel electrophoresis and immunoblotting. Blots were labeled with a primary rabbit polyclonal anti-equine cytochrome c antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA) or rabbit polyclonal anti-human Bax antibody (Millipore Corporation, Billerica, MA). Mitochondrial blots were labeled with rabbit monoclonal anti-human BCL-xL antibody (Cell Signaling Technology) or rabbit polyclonal anti-human BCL-2 antibody (GeneTex Inc., Irvine, CA). Blots were secondarily exposed to goat anti-rabbit Immunoglobulin G (IgG; Cell Signaling Technology). Mitochondrial protein loading was assessed with a primary monoclonal antibody to mouse Voltage-dependent Anion Channel (Molecular Probes, Eugene, OR) and secondarily exposed to rabbit anti-mouse IgG (Santa Cruz Biotechnology Inc.). Cytosolic protein loading was assessed with a primary monoclonal antibody to mouse glyceraldehyde 3-phosphate dehydrogenase (Thermo Fisher Scientific Inc., Rockford, IL) and secondarily exposed to rabbit anti-mouse IgG (Santa Cruz Biotechnology Inc.). Signal was detected with enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ), and density was measured using scanning densitometry. Five to 6 animals per cohort were evaluated (based on sample sizes used previously24).
Immunofluorescence
After euthanasia with pentobarbital injection (150 mg/kg, intraperitoneal), the animals were systemically perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) via left ventricle injection for 30 minutes 5 hours after exposure to isoflurane or air. Both eyes were enucleated and then postfixed in additional fixative solution for 24 hours at 4°C. Paraffin-embedded whole eye serial sections were cut at a thickness of 6 μm, and individual sections were slide mounted. Sections were deparaffinized and rehydrated with xylene, ethanol, and water. Antigen retrieval was performed with 0.01 M sodium citrate (pH 6.0) for 10 minutes at 100°C. Immunofluorescence was performed using monoclonal mouse anti-human PKC α antibody, monoclonal mouse anti-rat HPC-1 antibody, polyclonal goat anti-human calbindin antibody, or polyclonal goat anti-human Glutamate Aspartate Transporter (GLAST) antibody (Santa Cruz Biotechnology Inc.). Trimethylrhoadmine isothiocyanate (TRITC)-conjugated goat anti-mouse IgG secondary antibody (Jackson Immunoresearch Laboratories, Inc., West Grove, PA) was used for PKC and HPC-1, and donkey anti-goat IgG secondary antibody (Jackson Immunoresearch Laboratories, Inc.) for calbindin and GLAST. Double immunofluorescence was performed with polyclonal rabbit anti-human–activated caspase-3 antibody (9661; Cell Signaling Technology) and either FITC-conjugated goat anti-rabbit IgG secondary antibody (Jackson Immunoresearch Laboratories, Inc.) (for PKC and HPC-1) or FITC-conjugated donkey anti-rabbit IgG secondary antibody (Jackson Immunoresearch Laboratories, Inc.) (for calbindin and GLAST). Sections were counter-stained with DAPI, and colocalization of fluorescence was assessed at 40× and 100× magnification (oil immersion) using inverted confocal microscopy (Olympus Fluoview FV1000 confocal laser scanning microscope; Olympus America Inc.) in 3 to 4 animals per group (based on sample sizes used previously for immunohistochemistry22). Excitation/emission settings were 488/515 to 700 nm for FITC, 559/515 to 700 nm for TRITC, and 405/420 to 700 nm for DAPI. Six to 8 regions of interest were randomly selected in a blinded manner (performed by VP) in 3 to 4 nonserial sections per mouse (15–30 μm from the optic nerve bundle). The number of activated caspase-3-positive cells was manually quantified per square micrometer in INL at 40× magnification and corrected for the calculated area in the region of interest using NIH Image J v. 1.33. The number and percentage of cells with colocalized fluorescence were then manually quantified.
For cohorts injected with FITC-conjugated annexin V or dextran, pups were euthanized with pentobarbital injection (150 mg/kg, intraperitoneal) 1 hour after intraperitoneal injection. The animals were systemically perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) via left ventricle injection for 30 minutes, and eyes were postfixed in additional fixative solution for 24 hours at 4°C. Paraffin-embedded whole eye serial sections were cut at a thickness of 6 μm, and individual sections were slide mounted. Immunofluorescence was performed with polyclonal rabbit anti-human–activated caspase-3 antibody (9661; Cell Signaling Technology) and TRITC-conjugated goat anti-rabbit IgG secondary antibody (Jackson Immunoresearch Laboratories, Inc.). Sections were counterstained with DAPI, and colocalization of fluorescence was assessed at 40× and 100× magnification (oil immersion) using inverted confocal microscopy in 5 to 6 animals per group (based on sample sizes used previously for immunohistochemistry22). Six to 8 regions of interest were randomly selected in a blinded manner (performed by V.P.) in 3 to 4 nonserial sections per mouse (15–30 μm from the optic nerve bundle). The number of activated caspase-3-positive cells and FITC-positive cells was manually quantified per square micrometer at 40× magnification and corrected for the calculated area in the region of interest using NIH Image J v. 1.33. The number and percentage of cells with colocalized fluorescence was then quantified.
Statistical Analysis
Although we did not perform a power analysis for this investigation, we used sample sizes from previous work for each end point.22,24 Data are presented in bar plots as mean ± SD.
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Statistical significance was assessed using a 2-sample t test for the 2-group comparisons for immunohistochemistry, immunofluorescence, immunoblot, and TUNEL data. One-sided t test was used for TUNEL, immunohistochemistry, and immunoblot data, given that only unidirectional change for each parameter would be considered relevant after isoflurane exposure. This is based on evidence from previous work indicating that anesthetics induce apoptosis and do not decrease it.1–5,22 Furthermore, natural apoptosis occurs in 2% (or less) of neurons at any given time during development.18 Because this value is physiologically close to 0, a reduction <0 would not be likely. In addition, anesthetics are known to induce apoptosis in developing neurons by activating caspases (not inactivating them) and causing cytochrome c release from mitochondria (not enhancing cytochrome c levels in mitochondria).6 Thus, with regard to TUNEL, immunohistochemistry, and immunoblot analyses, results from the experimental group could not be any less than that of the controls. Therefore, 1-tailed t test was used. However, 2-sided t test was used for immunofluorescence. Significance for each t test was set at P < 0.01. Statistical analyses were conducted using SAS 9.3 (SAS Institute, Inc.).
RESULTS
Isoflurane Induces Apoptosis and Activates Caspase-3 in the Developing Retina
Isoflurane has previously been shown to induce widespread neuronal apoptosis in the developing rodent brain coincident with the peak in synaptogenesis.25 Thus, we determined whether isoflurane induces PCD and activates caspase-3 in the developing retina with TUNEL staining and immunohistochemistry on slide-mounted retinal sections. Different cohorts of pups were evaluated immediately after 1-hour exposure to isoflurane in air (activated caspase-3) or 5 hours after exposure (both TUNEL and activated caspase-3) on P7. Air-exposed cohorts served as controls.
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).
Neuronal Apoptosis and its Anesthetic Implications Essay
