Inhibition of p75 Neurotrophin Receptor Attenuates Isoflurane-mediated Neuronal Apoptosis in the Neonatal Central Nervous System

A CONVENTIONAL premise of general anesthesia is that anesthetics produce a nontoxic and reversible state of unconsciousness. However, recent evidence has indicated that exposure of neonatal animals to anesthetics triggers widespread neurodegeneration, leading to persistent memory and learning abnormalities during adulthood.1–3Specifically, exposure of postnatal day 5–10 (PND5-10) rat pups to a combination of isoflurane, nitrous oxide, and midazolam resulted in substantial neurodegeneration in the hippocampus and neocortex1as well as impaired electrophysiologic and behavioral function in the hippocampus weeks to months after exposure.1,3Such neurotoxicity has now been demonstrated for ketamine, midazolam,4diazepam, pentobarbital,5thiopental,3nitrous oxide,1and propofol.3,6–8The pattern of neonatal anesthetic neurotoxicity is similar to that produced by ethanol, exposure to which in utero is associated with the development of fetal alcohol syndrome. These observations have raised concerns about the potentially adverse impact of general anesthesia in the human fetus, neonate, and infant.3,9A key element of anesthetic neurotoxicity is that the brain is most susceptible to injury during the period of synaptogenesis.10An important signaling mechanism that contributes to synaptogenesis and to consolidation and maturation of synapses is the neurotrophin brain-derived neurotrophic factor (BDNF).11BDNF has dual function in that it can enhance neuronal survival or cause neuronal apoptosis through activity at either the tropomyosin receptor kinase B (TrkB) or neurotrophic receptor (p75NTR), respectively.12–14BDNF is secreted from synaptic vesicles as a proneurotrophin (proBDNF) that is proteolytically cleaved in the synaptic cleft by plasmin to a mature form (mBDNF).15Plasminogen, the precursor to plasmin, is cleaved by tissue plasminogen activator (tPA), a protease released from presynaptic vesicles.16Mature BDNF (mBDNF) preferentially binds to TrkB receptors to promote neuronal survival and synaptogenesis.15In the absence of tPA, proBDNF is uncleaved and binds with high affinity to p75NTR, resulting in reduced synaptogenesis, withdrawal of dendritic spines, and neuronal apoptosis.15Thus tPA can serve to control which effect of BDNF is predominant.17The mechanisms by which anesthetics mediate neurotoxicity are not clear. What is common among the anesthetics that have proven neurotoxicity in experimental studies is a profound suppression of neuronal activity; this has the potential to adversely affect the tPA-plasminogen-plasmin system. The release of tPA is activity-dependent,18and it is therefore conceivable that anesthetics might suppress neuronal activity, reduce tPA release, and enhance proBDNF signaling via the p75NTR, thereby leading to loss of dendritic filopodial spines and synapses and subsequent neuronal apoptosis. The current study, using in vitro neuronal cultures and in vivo mouse pups, was conducted to test this hypothesis.All studies performed on animals were approved by the Veteran Affairs San Diego Institutional Animal Care and Use Committee and conform to relevant National Institutes of Health guidelines.Neonatal mouse neurons (The Jackson Laboratory, Bar Harbor, ME) were isolated using a papain dissociation kit (Worthington Biochemical, Lakewood, NJ) as previously described.19Mix cortical and hippocampal neurons were isolated from 1-day-old pups (PND1) and grown in culture for 5 to 21 days in vitro (DIV). Neurons were cultured in Neuobasal A media supplemented with B27 (2%), 250 mm GLUTMax1, and penicillin/streptomycin (1%). Cells were cultured on poly-d-lysine/laminin (2 μg/cm2)–coated plates or coverslips at 37°C in 5% CO2for 5, 14, or 21 days before experiments. Cleaved-caspase 3 (Cell Signaling, Danvers, MA) and/or caspase-activated DNase (CAD)–positive cells (Santa Cruz Biotech, Santa Cruz, CA) were used to determine apoptosis via immunoblot and/or immunofluorescence and deconvolution microscopy. Antibodies to phospho-JNK and GAPDH were obtained from Cell Signaling and Imgenex (San Diego, CA), respectively. Cell death was normalized to total caspase-3 or the neuronal marker doublecortin (Abcam, Cambridge, MA) or NeuN (Chemicon/Millipore, Billerica, MA) of cells imaged. TAT-Pep5 was purchased from CalBiochem (Gibbstown, NJ).Neurons were placed in a plexiglass chamber within an incubator and exposed to 1.4% isoflurane delivered from a calibrated vaporizer in a gas mixture of 5% CO2, 21% O2, balance nitrogen gas at a flow rate of 2 l/min. The concentration of isoflurane was continuously monitored by a Datex Capnomac (DRE Medical, Inc., Louisville, KY).Animals were exposed to isoflurane (1.4%; air flow served as the carrier) at a flow rate of 2 l/min for 4 h. Body temperature was monitored with an 8 mm, 22-gauge temperature probe (Mon-a-therm 6510; Mallinckrodt Medical, Inc., St. Louis, MO) that was affixed to the dorsal neck. The temperature in the incubator was maintained at 37°C; this results in core body temperature in mouse pups in the range of 36.5–37.5°C (data not shown).Proteins in cell lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 10% acrylamide gels (Invitrogen, Carlsbad, CA) and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA) by electroelution. Membranes were blocked in 20 mm phosphate-buffered saline (PBS) Tween (1%) containing 4% bovine serum albumin (BSA) and incubated with primary antibody overnight at 4°C as previously described.20Primary antibodies were visualized using secondary antibodies conjugated to horseradish peroxidase (Santa Cruz Biotech) and ECL reagent (Amersham Pharmacia Biotech, Piscataway, NJ). All displayed bands are expected to migrate to the appropriate size and were determined by comparison to molecular weight standards (Santa Cruz Biotech). The amount of protein per fraction was determined using a dye-binding protein assay (Bio-Rad, Hercules, CA). Image J was used for densitometric analysis of immunoblots.∥Neurons were exposed to isoflurane for 4 h, and media from both control and isofluorane-treated neurons were frozen at –80°C before enzyme-linked immunosorbent assay (ELISA) (Innovative Research, Novi, MI). A 96-well plate was precoated with biotinylated plasminogen activator inhibitor-1 (PAI-1) for 30 min before three washes with wash buffer (provided by the supplier). tPA standard (0.05–10 ng/ml) and unknown samples (i.e. , media from control and isofluorane-exposed neurons) were added to the plate for 30 min, washed three times, and incubated with anti-tPA primary antibody for 30 min, followed by anti-rabbit horseradish peroxidase–conjugated secondary antibody for an additional 30 min. After three additional washes, tetramethylbenzidine substrate was added to the wells for 10 min, and the reaction was quenched with 1 M H2SO4and read at 450 nm on a spectrophotometer (TECAN Infinite M200, San Jose, CA).Neurons were prepared for immunofluorescence microscopy as previously described.21The following antibodies were used for immunofluorescence: cleaved caspase 3, drebrin, doublecortin (Abcam), and NeuN (Chemicon/Millipore). Tissue and cells were fixed with 4% paraformaldehyde in PBS for 10 min at room temperature, incubated with 100 mm glycine (pH. 7.4) for 10 min to quench aldehyde groups, permeabilized in buffered Triton X-100 (0.1%) for 10 min, blocked with 1% BSA/PBS/Tween (0.05%) for 20 min, and then incubated with primary antibodies (1:100) in 1% BSA/PBS/Tween (0.05%) for 24–48 h at 4°C. Excess antibody was removed by incubation with PBS/Tween (0.1%) for 15 min, and the samples were incubated with fluorescein isothiocyanate (FITC) or Alexa-conjugated secondary antibody (1:250) for 1 h. To remove excess secondary antibody, cells were washed six times at 5-min intervals with PBS/Tween (0.1%) and incubated for 20 min with the nuclear stain Dapi (1:5000) diluted in PBS. Cells were then washed for 10 min with PBS and mounted in gelvatol for microscopic imaging. Deconvolution images were obtained as described21and captured with a DeltaVision deconvolution microscope system (Applied Precision, Inc., Issaquah, WA). The system includes a Photometrics CCD (Tuscon, AZ) mounted on a Nikon TE-200 (Melville, NY) inverted epi-fluorescence microscope. Between 30 and 80 optical sections spaced by approximately 0.1–0.3 μm were taken. Exposure times were set such that the camera response was in the linear range for each fluorophore. Lenses included 100× (NA 1.4), 60× (NA 1.4), and 40× (NA 1.3). The data sets were deconvolved and analyzed using SoftWorx software (Applied Precision, Inc.) on a Silicon Graphics Octane workstation. Image analysis was performed with Data Inspector program in SoftWorx. Maximal projection volume views or single optical sections were visualized. Colocalization of pixels was assessed quantitatively by CoLocalizer Pro 1.0 software (Tokyo, Japan). Overlap coefficient according to Manders was used to determine the degree of colocalization on whole cells or membrane regions of interest after subtracting background through normalized threshold values.22The values were defined as 0 to 1, with 1 implying that 100% of both components overlap with the other part of the image. Statistical analysis was performed using Prism 4 (GraphPad Software, Inc., La Jolla, CA).The drebrin pixels (green) were normalized to doublecortin pixels (red). Because dendritic filopodial spines are nearly completely devoid of intermediate filaments and microtubules (as indicated by doublecortin staining)23and only contain components of the actin cytoskeleton (as indicated by drebrin staining), a neuronal microtubule marker (doublecortin) was used to normalize any changes in spinal actin cytoskeletal expression. Drebrin is a filamentous (F-)actin binding protein that stabilizes the actin cytoskeleton within dendritic filopodial spines.23,24A reduction in dendritic filopodial spines is indicated by decreased drebrin protein expression. Approximately 15–20 neurons were counted per experiment (4–6 experiments total).Neurons were incubated with small interfering ribonucleic acid (siRNA) (Ambion, Inc., Foster City, CA; 200 pmol; sense: CAGCUGCAAGCAGAACAAGtt; antisense: CUUGUUCUGCUUGCAGCUGtt) using Lipofectamine 2000 transfection reagent (Invitrogen) for 72 h and then exposed to isoflurane. Scrambled siRNA served as control.Under deep pentobarbital anesthesia, a midline thoracotomy was performed and the descending thoracic aorta was occluded. A 20-gauge needle was inserted into the left ventricle, and the animal was perfused transcardially with 20 ml of heparinized saline followed by 20 ml of 4% buffered formalin. The right atrium was incised to permit free flow of perfusion fluid. The brain was removed and postfixed for 24–48 h in fixative. The brain was sectioned into 4-mm blocks, placed in tissue holders, and dehydrated in graded concentrations of ethanol in a tissue processor (Autotechnicon; Technicon Instruments, Tarrytown, NY). The tissue holders were then transferred into a paraffin bath in an oven at 37°C under vacuum suction. The paraffin blocks were separated from tissue holders and mounted in a Histostat 820S microtome (Reichert Scientific Instruments, Depew, NY); 5- to 10-μm sections were then cut, placed on glass slides, and incubated overnight at 37°C. The tissue sections were then prepared for immunohistochemistry.Brains were transcardially perfusion fixed with standard Karnovsky's fix, 4% paraformaldehyde, 1% gluteraldehyde, 0.1 M cacodylate buffer with 5 mm CaCl2. PND5-7 animals were fixed with 2% paraformaldehyde, 2.5% glutaraldehyde, 0.1 M cacodylate buffer, and 5 mm CaCl2to prevent tissue artifacts. Hippocampi were dissected from whole brains after 24 h, and 400-μm vibratome slices were prepared and refixed for an additional 24 h. Brains were blocked (i.e. , dissected) to include hippocampal areas: one hemisphere for sagittal orientation, and one hemisphere for coronal. Blocks were refixed for an additional 24 h before postfixation with 1% OsO4in 0.1 M cacodylate buffer, en bloc stained with uranyl acetate, and embedded with flat orientation to locate appropriate hippocampal regions of interest. Each block was thick sectioned, stained with toludine blue, and retrimmed to isolate hippocampal areas before preparation of grids. Grids (70-nm sections) were stained with uranyl acetate and lead nitrate for contrast and observed on the JEOL 1200 EX-II electron microscope (JEOL, Tokyo, Japan) equipped with a digital camera system. Twenty-five random low-magnification micrographs of the stratum radiatum were obtained from each specimen. Micrographs were analyzed for the quantity of synapses and for synapse abnormalities (reduction or changes in synapse and dendritic filopidal spine morphology, i.e. , degradation of cytoskeletal architecture). The dendritic profiles were by such as and and from synapses were by an associated with vesicles and that or a spine (as indicated by cytoskeletal as filopodial spines were as dendritic in and at as previously electron micrographs per animal were analyzed in a for total synapse per data were analyzed by or comparison as were by was set at Statistical analysis was performed using Prism 4 (GraphPad Software, and hippocampal neurons were isolated on and were grown in culture to 5, 14, and 21 days in vitro and 4 exposure cleaved caspase-3 to total caspase-3 in neurons not in 3, or 3, These data are with the neurotoxicity of isoflurane that has been demonstrated is a of release is activity-dependent,18and anesthetics suppress neuronal activity; therefore that isoflurane tPA primary neurons were exposed to isoflurane 4 and tPA in the culture were by Neurons exposed to isoflurane tPA in the media to control of tPA to the in at 2 h after of which can potentially proBDNF to and which to not reduce neuronal apoptosis 3 tPA reduced 3, 3, apoptosis. tPA of 3, a signaling of TrkB of plasmin neuronal apoptosis 3, the of both plasmin and tPA were blocked by the protease primary neurons were exposed to isoflurane 4 and stained for dendritic (i.e. , filopodial using a neuronal drebrin, and the dendritic microtubule drebrin dendritic common to Exposure of neurons to isoflurane reduced the of dendritic filopodial with the plasmin or tPA 5, the loss of neuronal dendritic is as drebrin pixels (green) normalized to doublecortin immunofluorescence the only immunofluorescence was not images that of tPA 3, the 3, reduction in drebrin protein isoflurane proBDNF signaling via the p75NTR, then or of this receptor reduce to this neurons were with the a only the of the p75NTR, or an of or siRNA for isoflurane exposure 4 with 3, or 3, neuronal apoptosis. resulted in 3, of the neurotrophin receptor after 72 h and apoptosis 3, control to neurons TAT-Pep5 was in neurotoxicity and loss of synapses in in vivo were TAT-Pep5 10 15 min before isoflurane exposure 4 cleaved caspase 3 in the and of the hippocampus in 2 h after TAT-Pep5 in in both the and isoflurane exposure not enhance in that the brains of at this are to in the hippocampus to and from to of neonatal mouse pups 2 h after isoflurane exposure 4 was by electron microscopy. with and presynaptic vesicles are in control images was a reduction in total synapses in PND5-7 pups exposed to isoflurane reduction in synapses was by with TAT-Pep5 5, electron microscopy images for control isoflurane and TAT-Pep5 are in of data is by the in These results from in vitro studies that PND5-7 animals are to neurotoxicity and from isoflurane can by TAT-Pep5 in vivo results of the current are with the neurotoxicity of isoflurane that has been demonstrated neurons and mouse pups were to injury during the period of synaptogenesis, the was not at the of and 21 days and in vivo vitro , isoflurane exposure reduced tPA release, and the of either tPA or plasmin reduced of either with of with with isoflurane vivo , injury to the and of the hippocampus was of TAT-Pep5 before isoflurane exposure the results that isoflurane neurotoxicity is at in part by a reduction of tPA release and signaling of proBDNF via the is a body of evidence to that the brain is to anesthetic neurotoxicity during the synaptic with appropriate neurons are for neuronal survival neurons are on from of synaptic to apoptosis. important element to synaptogenesis and to consolidation and maturation of synapses is the neurotrophin are as which to the mature preferentially to the TrkB receptor to promote neuronal survival and as a for the neuronal to of the secreted are in the plasmin and proteolytically the most protease is as and the key that plasminogen is tPA, which is secreted from presynaptic and is data that isoflurane the amount of tPA in culture media of primary tPA is released from neurons in an this reduction in tPA to neuronal suppression by isoflurane. proBDNF both and by neuronal activity during the key period of synaptogenesis, anesthetics might reduce tPA release and subsequent plasmin thereby proBDNF signaling via in the of a of isoflurane exposure this The of this is neuronal either to a of synaptic and consolidation the subsequent of neurons to factor or apoptosis. of tPA to the of a kinase of TrkB signaling to neuronal neurotoxicity as well as the loss of synaptic filopodial spines in The studies used both and animal to the of in anesthetic to tPA has a of tPA binds to the receptor and triggers signaling that lead to of this effect of tPA is of The receptor is in neurons and can to and the activator receptor and that the reduction in anesthetic neurotoxicity might a function of the of tPA of survival is a protease of the activity of that the of tPA in that the effect of tPA is by activity and not by a from that of neurons with a that to enhance release from presynaptic cell death in a that neuronal activity can the from isoflurane. plasmin reduced that the activity of proBDNF to is for of anesthetic plasmin is the protease that other such as can to and can To determine a in the of isoflurane added or to neurons exposed to isoflurane. The of either of to reduce isoflurane that not proBDNF to in the premise that it is tPA and plasmin that have to signaling (i.e. , decreased isoflurane synaptic and and it neuronal dendritic which is can reduced by a effect of dendritic spine a that to and BDNF and therefore which that of or siRNA neuronal that anesthesia results in neuronal apoptosis in the neonatal by proBDNF at TAT-Pep5 of neurotoxicity that the from anesthetics in part to to the actin The that is isoflurane neurotoxicity is to the loss of or a combination of the development of to prevent anesthetic a is are the potential of the effect of anesthetic of by with by with or by of siRNA was reduced by the not indicated by the that immunoblot was similar in the control and A is that might is a in the brain that is for the of excess of apoptosis therefore might produce the effect of this of data was in isoflurane the of apoptosis was not in the control and These data that of not have and not that injury was only in the hippocampus and not in other that are by anesthesia, such as the and adversely the and is not clear. the effect of of synaptogenesis the of anesthetic neurotoxicity studies on However, the current that the injury is not to neuronal it includes the loss of dendritic filopodial spines and The quantity of dendritic filopodial spines was reduced after isoflurane exposure both in vitro and in vivo of the synapses are on dendritic filopodial loss of filopodial spines therefore reduce and lead to loss of electron microscopy of the stratum radiatum in the hippocampus and demonstrated a reduction in the of synapses as well as of synapses to control state However, a to the is the absence of of presynaptic or of synaptic vesicles in to in and dendritic analysis in studies a of changes that with isoflurane and other anesthetics during The current results are with by a general anesthetic of midazolam to a reduction in the quantity of associated with synapses , interest is the that the reduction in synapse in the to was the of apoptosis in either the or the The synapse loss and neuronal apoptosis recent that proBDNF and is released in the mature which are in to previously by the of the of proBDNF to isoflurane at within the that has been of data that with an antibody that is for the proBDNF is in the neuronal tPA or plasmin are the of proBDNF is isoflurane exposure not affect proBDNF data that neurons exposed to isoflurane are exposed to proBDNF and that tPA and plasmin, which isoflurane reduce has were to proBDNF in the culture media of control and isofluorane-exposed The most kit not have the to the small of proBDNF that might in the culture the absence of proBDNF the for a of proBDNF in is However, a key of evidence that is the effect of a that binds reduced isoflurane and effect was to tPA, plasmin, and siRNA data that an important in isoflurane A is that the by which isoflurane reduced tPA release are not clear. tPA release is and isoflurane neuronal are data to the neuronal suppression effect of isoflurane in vivo , such for in vitro neuronal cultures to the of activity data in cultured data a in tPA in the culture media after isoflurane exposure a reduction in neuronal the in most of the in vitro studies are the of an However, the of the data the in vitro neuronal cultures and the in vivo studies were this that neurotoxicity in the neonatal mouse system is a function of decreased synaptic tPA release, reduced signaling via and proBDNF signaling via The results that TAT-Pep5 or other that or enhance TrkB serve as potential for the of neurotoxicity in the are for the from the of San Diego La Jolla, in of of San Diego, La Jolla, of San of San are for the from and of San