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• W2027014741 abstract "Many central nervous system (CNS) diseases display sexual dimorphism. Exposure to circulating sex steroids is felt to be a chief contributor to this phenomenon; however, CNS diseases of childhood and the elderly also demonstrate gender predominance and/or a sexually dimorphic response to therapies. Here we show that XY and XX neurons cultured separately are differentially susceptible to various cytotoxic agents and treatments. XY neurons were more sensitive to nitrosative stress and excitotoxicity versus XX neurons. In contrast, XX neurons were more sensitive to etoposide- and staurosporine-induced apoptosis versus XY neurons. The responses to specific therapies were also sexually dimorphic. Moreover, gender proclivity in programmed cell death pathway was observed. After cytotoxic challenge, programmed cell death proceeded predominately via an apoptosis-inducing factor-dependent pathway in XY neurons versus a cytochrome c-dependent pathway in XX neurons. This gender-dependent susceptibility is related to the incapacity of XY neurons to maintain intracellular levels of reduced glutathione. In vivo studies further demonstrated an incapacity for male, but not female, 17-day-old rats to maintain reduced glutathione levels within cerebral cortex acutely after an 8-min asphyxial cardiac arrest. This gender difference in sensitivity to cytotoxic agents may be generalized to nonneuronal cells, as splenocytes from male and female 16–18-day-old rats show similar gender-dependent responses to nitrosative stress and staurosporine-induced apoptosis. These data support gender stratification in the evaluation of mechanisms and treatment of CNS disease, particularly those where glutathione may play a role in detoxification, such as Parkinson's disease, traumatic brain injury, and conditions producing cerebral ischemia, and may apply to non-CNS diseases as well. Many central nervous system (CNS) diseases display sexual dimorphism. Exposure to circulating sex steroids is felt to be a chief contributor to this phenomenon; however, CNS diseases of childhood and the elderly also demonstrate gender predominance and/or a sexually dimorphic response to therapies. Here we show that XY and XX neurons cultured separately are differentially susceptible to various cytotoxic agents and treatments. XY neurons were more sensitive to nitrosative stress and excitotoxicity versus XX neurons. In contrast, XX neurons were more sensitive to etoposide- and staurosporine-induced apoptosis versus XY neurons. The responses to specific therapies were also sexually dimorphic. Moreover, gender proclivity in programmed cell death pathway was observed. After cytotoxic challenge, programmed cell death proceeded predominately via an apoptosis-inducing factor-dependent pathway in XY neurons versus a cytochrome c-dependent pathway in XX neurons. This gender-dependent susceptibility is related to the incapacity of XY neurons to maintain intracellular levels of reduced glutathione. In vivo studies further demonstrated an incapacity for male, but not female, 17-day-old rats to maintain reduced glutathione levels within cerebral cortex acutely after an 8-min asphyxial cardiac arrest. This gender difference in sensitivity to cytotoxic agents may be generalized to nonneuronal cells, as splenocytes from male and female 16–18-day-old rats show similar gender-dependent responses to nitrosative stress and staurosporine-induced apoptosis. These data support gender stratification in the evaluation of mechanisms and treatment of CNS disease, particularly those where glutathione may play a role in detoxification, such as Parkinson's disease, traumatic brain injury, and conditions producing cerebral ischemia, and may apply to non-CNS diseases as well. Sexual dimorphism exists in the normal developing mammalian brain, particularly in sex steroid-concentrating regions such as the hypothalamus (1MacLusky N.J. Naftolin F. Science. 1981; 211: 1294-1302Crossref PubMed Scopus (1325) Google Scholar). However, there is also evidence of innate gender differences at the cellular level, dependent upon the brain region, neuronal subtype, and developmental stage. For instance, phenotypic differences in XY and XX brain cells appear to be independent of gonadal phenotype (2Carruth L.L. Reisert I. Arnold A.P. Nat. Neurosci. 2002; 5: 933-934Crossref PubMed Scopus (249) Google Scholar), and cultured dopaminergic neurons from female rat fetuses are morphologically distinct and have increased capacity for dopamine uptake versus neurons from males (3Reisert I. Engele J. Pilgrim C. Cell Tissue Res. 1989; 255: 411-417Crossref PubMed Scopus (58) Google Scholar). Cell death in certain brain regions can be different in both magnitude and duration between male and female rats, supporting the notion that divergent pathways of cell death occur between genders (4Nunez J.L. Lauschke D.M. Juraska J.M. J. Comp. Neurol. 2001; 436: 32-41Crossref PubMed Scopus (89) Google Scholar). Innate sexual dimorphism independent of circulating sex steroids would have significant clinical implications in central nervous system (CNS) 1The abbreviations used are: CNS, central nervous system; ED, embryologic day; INH2BP, 5-iodo-6-amino-1,2-benzopyrone; z-VAD, N-benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethyl ketone; RT, reverse transcription; AIF, apoptosis-inducing AIF factor; ELISA, immunoabsorbant assay; SH, sulfhydryl; MTT, 3-[4,5-dimethylthiazol]-2,5-diphenyltetrazolium bromide; NMDA, N-methyl-d-aspartate; AMPA, α-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid; NAC, N-acetylcysteine; DIV, days in vitro; PND, post-natal day; PI, propidium iodide. diseases with regard to both mechanisms and treatments. Many CNS diseases also display sexual dimorphism, specifically a predilection for one gender (5Sacco R.L. Neurology. 2001; 5: S31-S34Crossref Google Scholar, 6Van Den Eeden S.K. Tanner C.M. Bernstein A.L. Fross R.D. Leimpeter A. Bloch D.A. Nelson L.M. Am. J. Epidemiol. 2003; 157: 1015-1022Crossref PubMed Scopus (1173) Google Scholar) or a gender-dependent response to treatment (7Hariz G.M. Lindberg M. Hariz M.I. Bergenheim A.T. Acta Neurol. Scand. 2003; 108: 28-37Crossref PubMed Scopus (71) Google Scholar, 8Hurn P.D. Macrae I.M. J. Cereb. Blood Flow Metab. 2000; 20: 631-652Crossref PubMed Scopus (350) Google Scholar). Exposure to circulating sex steroids is felt to be a chief contributor to this phenomenon; however, sexual dimorphism is also exhibited in many CNS diseases afflicting children and the elderly. Studies in children show that girls have more favorable neurologic outcome after traumatic brain injury (9Donders J. Hoffman N.M. Neuropsychology. 2002; 16: 491-499Crossref PubMed Scopus (87) Google Scholar) and respond more favorably to treatment for medulloblastoma (10Weil M.D. Lamborn K. Edwards M.S. Wara W.M. J. Am. Med. Assoc. 1998; 279: 1474-1476Crossref PubMed Scopus (62) Google Scholar) compared with boys but that girls demonstrate worse outcome after stroke (11Hurvitz E.A. Beale L. Ried S. Nelson V.S. Pediatr. Rehabil. 1999; 3: 43-51Crossref PubMed Scopus (42) Google Scholar). Elderly men have a higher incidence of Parkinson's disease and do not respond as well to surgical intervention compared with elderly females (6Van Den Eeden S.K. Tanner C.M. Bernstein A.L. Fross R.D. Leimpeter A. Bloch D.A. Nelson L.M. Am. J. Epidemiol. 2003; 157: 1015-1022Crossref PubMed Scopus (1173) Google Scholar, 7Hariz G.M. Lindberg M. Hariz M.I. Bergenheim A.T. Acta Neurol. Scand. 2003; 108: 28-37Crossref PubMed Scopus (71) Google Scholar). Elderly men with Alzheimer's disease have an earlier onset and increased mortality compared with elderly females (12Ueki A. Shinjo H. Shimode H. Nakajima T. Morita Y. Int. J. Geriatr. Psychiatry. 2001; 16: 810-815Crossref PubMed Scopus (46) Google Scholar, 13Gambassi G. Lapane K.L. Landi F. Sgadari A. Mor V. Bernabie R. Neurology. 1999; 53: 508-516Crossref PubMed Google Scholar). In a study examining patients after cardiac arrest where the mean age was 67 years, male gender, but not age, was associated with poor outcome (14Vukmir R.B. J. Women's Health. 2003; 12: 667-673Crossref PubMed Scopus (28) Google Scholar). To determine whether innate gender differences independent of circulating sex steroids could contribute to gender-related differences in CNS disease prevalence and outcome, we developed a model system where male and female embryologic day (ED) 17 rats were segregated, and XY and XX neurons were cultured separately. The response to various cytotoxic agents and treatments and proclivity in programmed cell death pathway was then evaluated in this model system. Reagents and Materials—Neurobasal medium, RPMI 1640, and B27 supplement were from Invitrogen. Peroxynitrite (ONOO–) was from Cayman Chemical, Ann Arbor, MI. The poly-ADP-ribose polymerase inhibitor 5-iodo-6-amino-1,2-benzopyrone (INH2BP) was a kind gift from Dr. Csaba Szabó, Inotek Pharmaceuticals, Inc., Beverly, MA. The pancaspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethyl ketone (z-VAD) was from Enzyme Systems Products, Livermore, CA. The reverse transcription (RT)-PCR primers for rat SRY3 and actin were synthesized and purchased from Invitrogen. The antibody against apoptosis-inducing factor (AIF) was from Santa Cruz Biotechnology, Santa Cruz, CA. The anti-caspase-3 antibody that recognizes both the intact zymogen and the cleaved p17 fragment was from Cell Signaling, Beverly, MA. The anti-cytochrome c oxidase and anti-histone III antibodies were from BD Pharmingen. The cytochrome c enzyme-linked immunoabsorbant assay (ELISA) was from R&D Systems, Minneapolis, MN. ThioGlo-1 reagent for measurement of reduced glutathione (GSH) and protein sulfhydryls (SHs) were from Convalent Associates, Inc., Woburn, MA. The 3-[4,5-dimethylthiazol]-2,5-diphenyltetrazolium bromide (MTT) assay, hydrogen peroxide (H2O2), l-glutamate, N-methyl-d-aspartate (NMDA), kainate, α-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid (AMPA), staurosporine, etoposide, 17β-estradiol, MK801, catalase, superoxide dismutase, N-acetylcysteine (NAC), and remaining reagents were from Sigma, unless otherwise noted. Gender-segregated Neuronal Cultures, Cytotoxicity, and Pharmacological Studies—Male and female Sprague-Dawley rat fetuses (ED 16–17) were separated by visual inspection, and neurons were harvested and cultured separately using standard methods (15Du L. Zhang X. Han Y.Y. Burke N.A. Kochanek P.M. Watkins S.C. Graham S.H. Carcillo J.A. Szabo C. Clark R.S.B. J. Biol. Chem. 2003; 278: 18426-18433Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar, 16Zhang X. Chen J. Graham S.H. Du L. Kochanek P.M. Draviam R. Guo F. Nathaniel P.D. Szabo C. Watkins S.C. Clark R.S. J. Neurochem. 2002; 82: 181-191Crossref PubMed Scopus (231) Google Scholar). Male and female fetuses can be distinguished by visualization of the dorsal penile vessels and sex cords (Fig. 1a) (3Reisert I. Engele J. Pilgrim C. Cell Tissue Res. 1989; 255: 411-417Crossref PubMed Scopus (58) Google Scholar). Dissociated cell suspensions from cerebral cortices were placed in 96-well plates (5 × 104 cells/well) or in plastic dishes coated with poly-d-lysine (1.3 × 107 cells/well). Cortical neuron-enriched cultures were achieved by using neurobasal medium with serum- and estrogen-free B27 supplement (17Brewer G.J. J. Neurosci. Res. 1995; 42: 674-683Crossref PubMed Scopus (481) Google Scholar). Neuron survival was optimized by replacing glutamine with GlutaMaxl. Cells were incubated at 37 °C in a humidified chamber containing 5% CO2.Onthe second and sixth days in vitro (DIV) the culture media was replaced with fresh media. At 10 DIV, cultures consisted primarily of neurons with minimal glial cells detectable (>95% microtubule associated protein-2-immunoreactive cells, <5% glial fibrillary acidic protein immunoreactive cells analyzed in 8–10 fields/well, collected from 6 independent experiments). The majority of experiments were performed on the 10th DIV, with the exception of the excitotoxicity studies, which were performed on days 8–14. Primary hippocampal neuron cultures were prepared in similar fashion, with the exceptions that dissociated cell suspensions were prepared from hippocampi and glia were removed by treating cultures on DIV 2 with 5 μm cytosine arabinoside for 72 h. Nitrosative/Oxidative Stress—Neuron-enriched cultures were exposed to varying concentrations of ONOO– or H2O2 in phosphate-buffered saline for 10 min, whereupon fresh media was replaced. For excitotoxicity, neuron-enriched cultures were exposed to varying concentrations of l-glutamate with 5 μm glycine for 30 min, NMDA for 20 h or Kainate or AMPA for 30 min in culture media. Glycine was not added in the NMDA, AMPA, or kainate experiments. For apoptosis, neuron-enriched cultures were exposed to varying concentrations of staurosporine or etoposide in culture media for 4 h. For pharmacological studies cultures were pretreated with either 17β-estradiol (10–60 nm for 24 h on DIV 7, 10, and 14), the poly-ADP-ribose polymerase inhibitor INH2BP (100 μm), the NMDA antagonist MK801 (1–5 μm), the antioxidant catalase (5000 units), or the pancaspase inhibitor z-VAD (100 μm) dissolved in 20% dimethyl sulfoxide (Me2SO) or the antioxidants superoxide dismutase (300 units) or NAC (5 mm) dissolved in distilled H2O. High concentration stock solutions were diluted in culture media. Although poly-ADP-ribose polymerase inhibitors are notoriously nonspecific, INH2BP at the concentration used here significantly inhibited poly-ADP-ribosylation in neurons (15Du L. Zhang X. Han Y.Y. Burke N.A. Kochanek P.M. Watkins S.C. Graham S.H. Carcillo J.A. Szabo C. Clark R.S.B. J. Biol. Chem. 2003; 278: 18426-18433Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar) and was considerably more potent and selective than 3-aminobenzamide (18Szabo C. Virag L. Cuzzocrea S. Scott G.S. Hake P. O'Connor M.P. Zingarelli B. Salzman A. Kun E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3867-3872Crossref PubMed Scopus (281) Google Scholar). Gender-segregated, Isolated Splenocytes—Spleens were isolated from male and female post-natal day (PND) 16–18 Sprague-Dawley rats. Splenocytes were obtained by squeezing the organ in culture medium (RPMI 1640 containing 10% fetal bovine serum, 1% glutamine, 100 units of penicillin, and 100 mg/ml streptomycin). After centrifugation at 500 × g for 10 min at 4 °C, erythrocytes were removed using red blood cell lysing buffer (Sigma). Splenocytes were again centrifuged, and the pellet was washed twice with culture medium. Isolated gender-segregated splenocytes were resuspended in culture medium and cultured in 5% CO2 at 37 °C for 24-48 h at a concentration of 1 × 106 cells/ml in 96-well plates. For cytotoxicity assays varying concentrations of ONOO– or staurosporine were added to culture media, and the MTT assay was performed at 24 and 48 h. RT-PCR for SRY3—RT-PCR was performed using genomic DNA harvested from neurons in culture or brain tissue from male and female rats using standard methods (19Zhang X. Graham S.H. Kochanek P.M. Marion D.W. Nathaniel P.D. Watkins S.C. Clark R.S. FASEB J. 2003; 17: 1367-1369Crossref PubMed Scopus (59) Google Scholar). Total RNA was extracted using TRIzol reagent (Invitrogen), and the concentration was determined using a spectrophotometer. One μg of total RNA from each sample was reverse transcribed with 200 units of Superscript II reverse transcriptase (Invitrogen) for 50 min at 42 °C primed with 0.5 μg of random hexamer. RT products were then digested with RNase H at 37 °C for 30 min. cDNA samples (1 μl) were subjected to PCR amplification using primers specific for rat SRY gene (forward, 5′-GCTCAACAGAATCCCAGCAT-3′; reverse, 5′-TTTGTTGAGGCAACTTCACG-3′) or primers for rat actin with a Robocycler Gradient 96 PCR system (Stratagene, La Jolla, CA) using a Plantinum Taq PCRx kit (Invitrogen). Reagents were assembled in a final volume of 50 μl as follows: 1 μl of first-strand cDNA, 1 μm each primer, 1× PCR amplification buffer, 2.5 mm MgSO4, 1× PCR digoxigenin-labeling mix (Roche Applied Science), and RNase free water to volume. Samples were initially denatured at 94 °C for 3 min, and 2 units of Taq DNA polymerase were then added. Thermocycling parameters were 1 min at 94 °C, 1 min of annealing at 55 °C and 1 min of extension at 72 °C repeated for 30 cycles with a final extension step at 72 °C for 10 min. PCR products were then separated on a 1.2% agarose gel and transferred onto nylon membranes overnight in 20× SSC (1× SSC = 0.15 m NaCl and 0.015 m sodium citrate). After air drying and UV cross-linking, the membrane was blocked at room temperature for 1 h in 3% blocking reagent in 1× maleic acid buffer (Roche Applied Science) and incubated for 30 min at room temperature with alkaline phosphate-conjugated anti-digoxigenin at a concentration of 1:10,000 in 1% blocking reagent. A CDP-Star chemiluminescence reagent (PerkinElmer Life Sciences) was applied before exposing the membrane to x-ray film. Assessment of Cell Viability—Cell viability was assessed using the MTT assay, lactate dehydrogenase release, and/or flow cytometry. Duplicate 100-μl supernatant samples from 96-well plates were used for the MTT assay (15Du L. Zhang X. Han Y.Y. Burke N.A. Kochanek P.M. Watkins S.C. Graham S.H. Carcillo J.A. Szabo C. Clark R.S.B. J. Biol. Chem. 2003; 278: 18426-18433Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar, 16Zhang X. Chen J. Graham S.H. Du L. Kochanek P.M. Draviam R. Guo F. Nathaniel P.D. Szabo C. Watkins S.C. Clark R.S. J. Neurochem. 2002; 82: 181-191Crossref PubMed Scopus (231) Google Scholar). Values are expressed as the percentage of control (untreated) neurons and represent 5–8 separate experiments. In some paradigms, lactate dehydrogenase release was measured in duplicate 20-μl supernatant samples. Flow cytometry labeling of propidium iodide (PI) uptake and annexin V binding was also used to evaluate cell viability after ONOO-, glutamate/glycine, and staurosporine cytotoxicity (15Du L. Zhang X. Han Y.Y. Burke N.A. Kochanek P.M. Watkins S.C. Graham S.H. Carcillo J.A. Szabo C. Clark R.S.B. J. Biol. Chem. 2003; 278: 18426-18433Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar). Neurons were harvested using trypsin-EDTA, washed once in ice-cold phosphate-buffered saline, and resuspended in 1 ml of annexin V binding buffer (10 mm Hepes, pH 7.4, 140 mm NaCl, 2.5 mm CaCl2). 1 × 105 cells were stained with 5 μl of annexin V-fluorescein isothiocyanate and 5 μg/ml PI in binding buffer at 4 °C. After 20 min, 400 μl of binding buffer was added to each tube, and samples were analyzed using a FACSCalibur flow cytometer. Unstained cells were used to determine background fluorescence, and thresholds for normal (unlabeled) cells were determined. The percentage of cells in each quadrant was calculated. For statistical comparison, the percentage of normal cells defined as PI and annexin V for each cytotoxicity paradigm were analyzed. Subcellular Fractionation and Western Blot Analysis—Cellular proteins were separated into mitochondrial, nuclear, and cytosolic fractions (15Du L. Zhang X. Han Y.Y. Burke N.A. Kochanek P.M. Watkins S.C. Graham S.H. Carcillo J.A. Szabo C. Clark R.S.B. J. Biol. Chem. 2003; 278: 18426-18433Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar, 16Zhang X. Chen J. Graham S.H. Du L. Kochanek P.M. Draviam R. Guo F. Nathaniel P.D. Szabo C. Watkins S.C. Clark R.S. J. Neurochem. 2002; 82: 181-191Crossref PubMed Scopus (231) Google Scholar). Neurons at 0, 2, and 24 h after cytotoxic treatment were lysed in buffer. Samples were centrifuged at 600 × g for 15 min at 4 °C. The supernatants were centrifuged at 17,200 × g for 20 min at 4 °C. These supernatants were used for assessment of cytosolic proteins. The pellets containing mitochondria were lysed in buffer and sonicated. The initial pellet was re-suspended in lysis buffer then centrifuged at 16,000 × g for 25 min at 4 °C with the supernatant used for assessment of nuclear proteins. Protein concentration was determined using bicinchoninic acid (Pierce). Protein samples (50 μg) were then boiled in loading buffer for 5 min followed by chilling on ice, then electrophoretically separated on SDS-PAGE gels and transferred onto polyvinylidene difluoride membranes (Bio-Rad). After blocking overnight in 5% nonfat milk in phosphate-buffered saline, the membranes were incubated in 1:100 dilutions of antibodies against AIF or caspase-3 at room temperature for 1 h, washed, then incubated in appropriate secondary antibody. Protein bands were visualized using a chemiluminescence detection system (PerkinElmer Life Sciences) and exposed to x-ray film. The relative optical density of detected peptides was semi-quantified using an Eastman Kodak Co. imaging system. To determine the purity of individual cellular compartments, immunoblotting for cytochrome c oxidase and histone III was performed pre hoc. Cytochrome c Enzyme-linked ELISA—Cellular proteins were separated into mitochondrial and cytosolic fractions (15Du L. Zhang X. Han Y.Y. Burke N.A. Kochanek P.M. Watkins S.C. Graham S.H. Carcillo J.A. Szabo C. Clark R.S.B. J. Biol. Chem. 2003; 278: 18426-18433Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar, 16Zhang X. Chen J. Graham S.H. Du L. Kochanek P.M. Draviam R. Guo F. Nathaniel P.D. Szabo C. Watkins S.C. Clark R.S. J. Neurochem. 2002; 82: 181-191Crossref PubMed Scopus (231) Google Scholar). Cytochrome c concentrations were determined by ELISA within 96-well plates using triplicate samples as per manufacturer's instruction. DNA Analysis—Chromosomal DNA samples were prepared in agarose plugs using a CHEF Mammalian Genomic DNA Plug Kit (Bio-Rad). Samples were homogenized in cell suspension buffer, mixed with preheated (50 °C) 2% low melting point agarose, and transferred into agarose plug molds. After solidification at room temperature the plugs were incubated with 1 mg/ml proteinase K overnight at 50 °C without agitation. Deproteinized DNA-containing agarose plugs were washed in wash buffer, added to wells, and sealed with low melting point agarose. Pulsed field gel electrophoresis was performed as described (16Zhang X. Chen J. Graham S.H. Du L. Kochanek P.M. Draviam R. Guo F. Nathaniel P.D. Szabo C. Watkins S.C. Clark R.S. J. Neurochem. 2002; 82: 181-191Crossref PubMed Scopus (231) Google Scholar) using a CHE DR II pulsed field gel electrophoresis system (Bio-Rad). Fragments were separated on a 1.2% agarose gel at 14 °C for 20 h. Field strength was set at 6 V/cm, and initial switching time was set at 60 s for 15 h followed by final switching time 90 s for 9 h. The gel was stained with ethidium bromide and visualized under UV light. DNA laddering was performed using standard methods. Fluorescence Assay of GSH and Protein-SH—The concentrations of GSH and protein-SH were determined using ThioGlo-1, a maleimide reagent that produces a highly fluorescent product upon its reaction with sulfhydryl groups (20Bayir H. Kagan V.E. Tyurina Y.Y. Tyurin V. Ruppel R.A. Adelson P.D. Graham S.H. Janesko K. Clark R.S. Kochanek P.M. Pediatr. Res. 2002; 51: 571-578Crossref PubMed Scopus (207) Google Scholar), modified for use in culture and brain tissue. A standard curve was established using 0.04–4 μm GSH in 50 mm Na2PO4 buffer, pH 7.4, containing 10 μm Thio-Glo-1. GSH content was estimated by an immediate fluorescence response observed upon the addition of ThioGlo-1 to each sample. Levels of total protein-SH were determined as an additional fluorescence response after adding of 4 mm SDS to the same sample. A Cytofluor 2350 fluorescence plate reader (Millipore Corp., Marlborough, MA) was used to detect fluorescence at excitation and emission wavelengths of 388 and 500 nm, respectively. Asphyxial Cardiopulmonary Arrest in Post-natal Day 17 Rats—Cortical brain tissue samples were obtained from PND 17 rats after 8-min of asphyxial cardiac arrest. This model produces hypoxic-ischemic injury at a time when circulating levels of reproductive hormones are similar between genders (21Fink E.L. Alexander H. Marco C.D. Dixon C.E. Kochanek P.M. Jenkins L.W. Lai Y. Donovan H.A. Clark R.S. Pediatr. Crit. Care Med. 2004; 5: 139-144Crossref PubMed Scopus (69) Google Scholar). At 6 or 24 h after asphyxia or in sham controls, rats were perfused with ice-cold saline, and cerebral cortices were dissected and frozen. Gender was verified at necropsy. The concentrations of GSH and protein-SH were determined as above. For analysis, 6- and 24-h samples were combined for each gender. Data Analysis—Data are presented as the mean ± S.D. Comparisons between genders, treatment groups, and time points were made using two-factor analysis of variance with Tukey's post hoc tests unless otherwise specified. If data failed tests of normality and/or equal variance, data were ranked before analysis. For cell viability studies, 3–9 wells/condition and a minimum of 3 independent experiments were performed. For Western blot, ELISA and DNA gels 3–6 wells/condition and a minimum of 2 independent experiments were performed. DNA gels were qualitatively analyzed. A p < 0.05 was considered significant. Gender Proclivity in Response to Cytotoxicity—There were no obvious gross morphologic differences between XY and XX neurons. Accurate gender separation was verified using RT-PCR for the Y-chromosome gene SRY3 (Fig. 1b). The sensitivity of XY and XX neurons to multiple cytotoxic insults is shown in Fig. 1c. The conversion of MTT to formazan as the indicator of cell viability was used because the assay could be performed in a high-throughput fashion. Cytotoxicity models of nitrosative/oxidative stress using exogenous ONOO– or H2O2, excitotoxicity (supraphysiologic glutamate and glycine), cell-signal mediated apoptosis (staurosporine), and apoptosis mediated via topoisomerase II inhibition (etoposide) were tested. Remarkably, for most cytotoxicity paradigms, XY and XX neurons were differentially susceptible. XY neurons were more sensitive to nitrosative stress and excitotoxicity, whereas XX neurons were more sensitive to etoposide and staurosporine. Responses to H2O2 were similar between genders. Because ONOO– and other oxidants affect mitochondrial function thereby influencing the results of the MTT assay, other measures of cell viability were performed. Gender-dependent responses to nitrosative stress, excitotoxicity, and staurosporine were confirmed by flow-cytometric analysis using PI and Annexin V binding as markers (Fig. 1d and e), as well via measurement of lactate dehydrogenase release (data not shown). Hippocampal neurons at ED 17 are also non-sex steroid producing (1MacLusky N.J. Naftolin F. Science. 1981; 211: 1294-1302Crossref PubMed Scopus (1325) Google Scholar). Similar gender-dependent sensitivity to ONOO–, glutamate/glycine, and staurosporine was seen in hippocampal neurons compared with cortical neurons (Fig. 1g). However, both XY and XX hippocampal neurons were more sensitive to similar concentrations of glutamate/glycine and staurosporine than their cortical neuron counterparts. To determine whether results in neurons could be generalized to non-neuronal cells, the sensitivity of splenocytes to nitrosative stress and staurosporine-induced apoptosis was also examined. Remarkably, similar gender-dependent sensitivity to ONOO– and staurosporine was seen in splenocytes as was seen in neurons (Fig. 1 h; representing n = 10 wells/condition and 2 independent experiments). However, splenocytes of both genders appeared more sensitive to equivalent concentrations of ONOO– and staurosporine versus cortical neurons. In addition, gender differences were not detected in splenocytes after staurosporine treatment until 48 h after exposure (versus 2 h in neurons). Gender Proclivity in Programmed Cell Death Pathway— Pathways of programmed cell death were examined by measuring mitochondrial release of cytochrome c and AIF. We and others have previously reported that nitrosative/oxidative stress induces programmed cell death in mixed-gender neurons via an AIF-mediated, poly-ADP-ribose polymerase-dependent and caspase-independent pathway (15Du L. Zhang X. Han Y.Y. Burke N.A. Kochanek P.M. Watkins S.C. Graham S.H. Carcillo J.A. Szabo C. Clark R.S.B. J. Biol. Chem. 2003; 278: 18426-18433Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar, 22Yu S.W. Wang H. Poitras M.F. Coombs C. Bowers W.J. Federoff H.J. Poirier G.G. Dawson T.M. Dawson V.L. Science. 2002; 297: 259-263Crossref PubMed Scopus (1577) Google Scholar). After ONOO–, nuclear translocation of AIF was more prominent in XY neurons in contrast to cytochrome c release, which was more prominent in XX neurons (Fig. 2, a–c). Similar to our previous studies, a concomitant reduction in mitochondrial AIF was not detected using this dose of ONOO– (15Du L. Zhang X. Han Y.Y. Burke N.A. Kochanek P.M. Watkins S.C. Graham S.H. Carcillo J.A. Szabo C. Clark R.S.B. J. Biol. Chem. 2003; 278: 18426-18433Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar). With higher concentrations of ONOO-, a reduction in mitochondrial AIF is seen (16Zhang X. Chen J. Graham S.H. Du L. Kochanek P.M. Draviam R. Guo F. Nathaniel P.D. Szabo C. Watkins S.C. Clark R.S. J. Neurochem. 2002; 82: 181-191Crossref PubMed Scopus (231) Google Scholar). The predilection for XY neurons to undergo an AIF-mediated programmed cell death pathway was confirmed by the detection of 50-kbp DNA fragments. Large scale DNA fragments are the biochemical hallmark of AIF-mediated cell death (23Susin S.A. Lorenzo H.K. Zamzami N. Marzo I. Snow B.E. Brothers G.M. Mangion J. Jacotot E. Costantini P. Loeffler M. Larochette N. Goodlett D.R. Aebersold R. Siderovski D.P. Penninger J.M. Kroemer G. Nature. 1999; 397: 441-446Crossref PubMed Scopus (3464) Google Scholar) and were increased in XY neurons to a greater degree than in XX neu" @default.
• W2027014741 created "2016-06-24" @default.
• W2027014741 creator A5007238584 @default.
• W2027014741 creator A5058521832 @default.
• W2027014741 creator A5060016417 @default.
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• W2027014741 date "2004-09-01" @default.
• W2027014741 modified "2023-10-17" @default.
• W2027014741 title "Innate Gender-based Proclivity in Response to Cytotoxicity and Programmed Cell Death Pathway" @default.
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