HygY Is a Twitch Radical SAM Epimerase with Latent Dehydrogenase Activity Revealed upon Mutation of a Single Cysteine Residue.

HygY is a SPASM/twitch radical SAM enzyme hypothesized to catalyze the C2'-epimerization of galacamine during the biosynthesis of hygromycin B. This activity is confirmed via biochemical and structural analysis of the derivatized reaction products using chemically synthesized deuterated substrate, high-resolution mass spectrometry and 1H NMR. Electron paramagnetic resonance spectroscopy of the reduced enzyme is consistent with ligation of two [Fe4S4] clusters characteristic of the twitch radical SAM subgroup. HygY catalyzed epimerization proceeds with incorporation of a single solvent Hydron into the talamine product facilitated by the catalytic cysteine-183 residue. Mutation of this cysteine to alanine converts HygY from a C2'-epimerase to an C2'-dehydrogenase with comparable activity. The SPASM/twitch radical SAM enzymes often serve as anaerobic oxidases making the redox-neutral epimerases in this class rather interesting. The discovery of latent dehydrogenase activity in a twitch epimerase may therefore offer new insights into the mechanistic features that distinguish oxidative versus redox-neutral SPASM/twitch enzymes and lead to the evolution of new enzyme activities.

Resolution of biliary stricture after living donor liver transplantation in a child by percutaneous trans-hepatic cholangiography and drainage: a case report.

INTRODUCTION: Intra-hepatic cholestasis arising from biliary strictures is a frequent complication in pediatric patients after liver transplantation. Minimally invasive procedures such as percutaneous drainage placement and balloon dilation are the preferred diagnostic and therapeutic modalities. CASE PRESENTATION: We report the case of a 12-month-old Caucasian boy with biliary atresia who was initially treated with hepatoportoenterostomy. In the following months, he developed biliary cirrhosis, accompanied by cystic bile retention, recurrent bile duct infections and malabsorption. Six months after the initial surgical intervention, he underwent living donor liver transplantation. Within two months, the hepatico-jejunostomy became occluded leading to progressive intra-hepatic cholestasis. Under sonographic guidance, external drainage of bile was accomplished by percutaneous trans-hepatic cholangiography and drainage. In total, our patient underwent 12 interventions under general anesthesia until balloon dilatation of the hepatico-jejunostomy was successfully performed. Finally, our patient's general condition improved and he gained weight. CONCLUSIONS: Minimally invasive techniques are preferred to surgical revisions and justify even multiple attempts. Interventions under general anesthesia, though not without risks, are still reasonable. Co-operation with parents and multidisciplinary approach to complication management by the involved surgeon, radiologist, pediatrician and anesthesiologist are important.

Evolution of pancreas transplantation: long-term results and perspectives from a high-volume center.

OBJECTIVE: To describe the evolution of pancreas transplantation from 1979 to 2011. The aim was to examine factors influencing long-term patient and graft survival, surgical methods, and risk factors influencing organ performance after transplantation. BACKGROUND: Pancreas transplantation has become the therapy of choice for patients suffering insulin-dependent diabetes and end stage renal failure. METHODS: Retrospective analysis of 509 consecutive pancreas transplants (442 simultaneous pancreas and kidney [SPK], 20 pancreas transplanted alone [PTA], and 47 pancreas transplanted after kidney [PAK]), performed at the University Hospital Innsbruck. The data were statistically analyzed using the Kaplan-Meier method and log-rank test. RESULTS: After overcoming initial immunological and technical problems between 1979 and 1988 (5-year pancreas graft survival rate, 29.7%), pancreas transplantation evolved during the second decade (1989-1996; 5-year pancreas graft survival rate, 42.2%). Technical changes, optimized immunosuppression, careful pretransplant evaluation, and improved graft monitoring have become standard in the last decade and result in excellent 5-year patient (94.3%), kidney (89.4%), and pancreas (81.5%) graft survival. Five-year graft survival was superior in SPK (68.8%) compared with PAK (62.5%) and PTA (16.4%). SPK retransplantation can be carried out safely with 5-year patient (87.5%) and pancreas graft (75.0%) survival. Overall 5-year patient survival after loss of the first pancreas graft is significantly better in patients who underwent retransplantation (89.4% vs. 67.9%, P = 0.001). Long-term pancreas graft survival is independent of donor body mass index, sex, and cause of death, anastomosis time and the number of human leukocyte antigen (HLA) mismatches, recipient age, body mass index, sex, current panel reactive antibodies, and waiting time. Significant risk factors for reduced graft survival are cold ischemia time and donor age. CONCLUSIONS: During the last 32 years, many problems in pancreas transplantation have been overcome and it may currently represent the therapeutic gold standard for some patients with diabetes and end stage renal failure.

Enteroscopic biopsies in the management of pancreas transplants: a proof of concept study for a novel monitoring tool.

BACKGROUND: Although percutaneous biopsies are considered to be the gold standard in diagnosing pancreas graft rejection, they are not performed routinely because of their association with severe complications. On the other hand, correct diagnosis of rejection is essential but may be difficult in cases of enteric drainage, particularly in patients with a pancreas transplant alone or a pancreas after kidney transplant. METHODS: Pancreas recipients who underwent enteroscopy between May 2005 and September 2009 were included in this retrospective analysis. Biopsies were graded 0 to 4 for interstitial and vascular changes. RESULTS: During the study period a total of 65 simultaneous pancreas-kidney transplants, 13 pancreas after kidney transplants and 4 pancreas transplants alone were performed. Sixty-three patients underwent a single enteroscopy, 10 had two, and 6 had three or more. Indications were protocol graft monitoring (n=73), graft dysfunction (n=17), enteric hemorrhage (n=9), or other (n=3). The duodenal segment was accessed in 76 instances (75%) with abnormal findings in 23. A total of 69 biopsies were obtained and revealed normal mucosa in 49 cases (71%). Histology showed signs of acute rejection in 11 cases. The upper gastrointestinal tract was also assessed, and, in 13 cases, additional pathologies were identified including gastroduodenitis (n=10), gastric/duodenal ulcer (n=2), and hemorrhagic esophagitis (n=1). No procedure-related complication occurred. CONCLUSIONS: This series of enteroscopies demonstrates that the duodenal segment of a pancreatic graft is accessible using our implant technique, and thus permitting biopsies to be obtained and endoscopic interventions to be performed.

Single-center experience with third and fourth kidney transplants.

Kidney retransplantation is often associated with a higher immunological risk than is primary renal transplantation. Faced with increasing organ shortage and growing waiting lists, results of kidney retransplantation are of particular interest. Fifty-six third and fourth kidney transplants were analyzed retrospectively. Parameters included patient and donor demographics, operative details, incidence of surgical, immunological and infectious complications and patient and graft survival. Patients receiving third kidney grafts had 1- and 5-year patient/graft survival rates of 97.4%/72.9% and 88.9%/53.6%, respectively. Episodes of acute rejection and delayed graft function were observed in 44% and 49% of these patients. Fourth kidney transplantation was associated with 1- and 2-year patient/graft survival rates of 84.8%/68.5% and 63.6%/47%, respectively. Acute rejection and delayed graft function occurred in 33% and in 60% of cases. Acceptable patient and graft survival may be achieved after third and fourth kidney transplantation. Graft losses in this sensitized population are mainly because of rejection. Profound immunosuppression may lead to major infectious problems.

Cytomegalovirus mismatch as major risk factor for delayed graft function after pancreas transplantation.

BACKGROUND: Risk factors for delayed graft function (DGF) in pancreas transplantation (PTx) and its implications on graft survival are poorly defined. METHODS: Eighty-seven consecutive first-time PTx for type I diabetes performed between January 2003 and December 2007 were retrospectively reviewed. DGF was defined as a reversible need for exogenous insulin beyond postoperative day 10 (DGF group [DGFG]). For statistical analysis, DGFG patients were compared with patients with immediate graft function (control group [CG]). RESULTS: DGF occurred in 16 patients (18.6%). C-peptide levels and DGF were inversely correlated (r=0.24, P=0.03). In univariate analysis, donor cytomegalovirus (CMV)+ antibody status, and D+/R- CMV mismatch were significantly associated with DGF (81.3% vs. CG 52.1%, P=0.029; and 62.5% vs. CG 21.1%, P=0.002, respectively). Compared with University of Wisconsin solution, histidine tryptophan ketoglutarate-preserved grafts displayed higher DGF rates (37.5% vs. CG 12.7%, P=0.030), similar to female recipients (DGFG 68.8% vs. CG 35.2%, P=0.015). On multivariate analysis, a significantly higher DGF incidence was noted in female recipients (DGFG 68.8% vs. CG 35.2%; P=0.03) and in recipients with D+/R- CMV mismatch (DGFG 62.5% vs. CG 21.1%; P=0.03). With a median follow-up of 40.4 months (range 0.7-74.2), graft survival at 5 years did not differ between both groups (94.4% CG vs. 93.8% DGFG; P=0.791). CONCLUSION: This is the first study that identifies CMV mismatch (D+/R-) as an additional risk factor for DGF occurrence in PTx. In this particular cohort, DGF does not seem to affect graft survival.

Extensive surveillance promotes early diagnosis and improved survival of de novo malignancies in liver transplant recipients.

The aim of our study was to examine whether an extensive surveillance protocol will promote early diagnosis and improved survival in patients with de novo cancer following liver transplantation (LT). Of 779 consecutive LT recipients, 96 (12.3%) developed 105 malignancies. The cumulative risk for the development of de novo cancer was 10%, 24%, 32% and 42% at 5, 10, 15 and 20 years after LT, respectively. The most frequent tumor types were skin (17%), lung (16%), oropharyngeal (11%) and prostate cancer (11%). The overall standard incidence ratio as compared to that of the general population was 1.9 (95% CI: 1.5-2.3). The median survival of patients with de novo non-skin cancers was 3.1 years after diagnosis. Only patients with skin cancers and solid tumors, diagnosed at early stages, showed an excellent outcome. After introducing an intensified surveillance protocol, the detection rate of de novo cancers increased from 4.9% to 13% and more de novo malignancies were diagnosed in earlier stages. For non-skin cancers, the median tumor-related survival significantly improved from 1.2 to 3.3 years as well as the median overall survival post-LT. This study indicates that an extensive tumor surveillance program is highly recommendable in LT recipients.

A prospective randomized multicenter trial comparing histidine-tryptophane-ketoglutarate versus University of Wisconsin perfusion solution in clinical pancreas transplantation.

We aimed to evaluate early pancreas transplant graft function after histidine-tryptophan-ketoglutarate (HTK) versus University of Wisconsin (UW) perfusion. Prospective randomized multicenter study including 68 pancreas transplantations stratified according to preservation fluid used (27 HTK vs. 41 UW). Primary endpoint was pancreas graft survival at 6 months. Serum alpha-amylase, lipase, C-peptide, HbA1C and exogenous insulin requirement were compared at several time points. Mean pancreas cold ischemia time was 10.8 +/- 3.7 (HTK) vs. 11.8 +/- 3.4 h (UW) (P = 0.247). Simultaneous pancreas-kidney transplantation was performed in 95.6% of the patients, pancreas transplantation alone in 2.9%, and pancreas after kidney transplantation in 1.5%. Six months graft survival was 85.2% (HTK) vs. 90.2% (UW) (P = 0.703). Serum amylase and lipase values did not differ between both the groups during the observation period. C-peptide levels were elevated in both the groups without significant differences at each time point. Higher exogenous insulin requirement early after transplantation in the UW group had resolved at 3 months. Six month patient survival was 96.3% (HTK) vs. 100% (UW) (P = 0.397). With a mean cold ischemia time of 10 h in this study, HTK and UW solutions appear to be equally suitable for perfusion and organ preservation in clinical pancreas transplantation.

Markers of cellular senescence in zero hour biopsies predict outcome in renal transplantation.

Although chronological donor age is the most potent predictor of long-term outcome after renal transplantation, it does not incorporate individual differences of the aging-process itself. We therefore hypothesized that an estimate of biological organ age as derived from markers of cellular senescence in zero hour biopsies would be of higher predictive value. Telomere length and mRNA expression levels of the cell cycle inhibitors CDKN2A (p16INK4a) and CDKN1A (p21WAF1) were assessed in pre-implantation biopsies of 54 patients and the association of these and various other clinical parameters with serum creatinine after 1 year was determined. In a linear regression analysis, CDKN2A turned out to be the best single predictor followed by donor age and telomere length. A multiple linear regression analysis revealed that the combination of CDKN2A values and donor age yielded even higher predictive values for serum creatinine 1 year after transplantation. We conclude that the molecular aging marker CDKN2A in combination with chronological donor age predict renal allograft function after 1 year significantly better than chronological donor age alone.

Mitochondrial ischemia-reperfusion injury of the transplanted rat heart: improved protection by preservation versus cardioplegic solutions.

Cold ischemia time and preservation of organs are limited by I/R injury leading to primary nonfunction of the graft. In a rat heart transplant model, we compared cardioplegic St Thomas (ST) to histidine-tryptophan-ketoglutarate (HTK) and University of Wisconsin preservation solutions in terms of contractile function, and mitochondrial respiratory and enzymatic defects after prolonged cold ischemia and reperfusion. Contractile function was scored after transplantation and 24 h of reperfusion. Mitochondrial function was investigated by high-resolution respirometry of permeabilized myocardial fibers. Graft performance in terms of contractile function declined with the duration of cold storage. Recovery was significantly improved after 10 h of cold storage in HTK compared with ST (cardiac scores, 3.3+/-0.5 and 1.8+/-0.8, respectively). Tissue lactate dehydrogenase was better preserved in HTK than ST. Increase of tissue water content (edema) was less pronounced in HTK than ST (3.33+/-0.14 and 3.73+/-0.21 mg/mg dry weight, respectively). Similar cardiac scores (2.6+/-0.9 and 2.9+/-1.2, respectively) and mitochondrial respiratory parameters were obtained after preservation in HTK and University of Wisconsin. Decline in contractile function of individual grafts correlated well with loss of mitochondrial respiratory capacity, whereas citrate synthase activity remained largely preserved, indicating specific damage of respiratory complexes. Our data provide evidence for the superiority of preservation solutions versus a cardioplegic solution for prolonged cold storage of the heart. The correlation of graft performance and mitochondrial function indicates the potential of high-resolution respirometry for quantitative assessment of myocardial injury upon cold I/R, providing a basis for diagnostic approaches and evaluation of improved preservation solutions for heart transplantation.

Protein levels of heme oxygenase-1 during reperfusion in human kidney transplants with delayed graft function.

INTRODUCTION: Delayed graft function (DGF) as a consequence of ischemia reperfusion injury (IRI) is associated with a decrease in long-term allograft survival. Heme oxygenase-1 (HO-1) is a stress responsive gene that is highly expressed in multiple pathological processes. The aim of our study was to analyze whether HO-1 protein levels in human kidney transplants during IRI correlate with the incidence of DGF. METHODS: Kidney biopsies were obtained from 27 kidney allografts at two time points: at the end of cold storage and shortly after reperfusion. Samples were analyzed for HO-1 protein levels by Western blot. RESULTS: Heme oxygenase-1 protein levels were significantly higher in post-reperfusion biopsies (39.4 vs. 13.7 arbitrary units, p = 0.001). In pre-reperfusion biopsies no association was observed between HO-1 protein levels and DGF. In post-reperfusion biopsies, higher levels of HO-1 protein were measured in kidneys with DGF (53.7 vs. 36.2 arbitrary units, p = 0.064). DGF kidneys showed a significantly higher increase from pre- to post-reperfusion in HO-1 protein (42.0 vs. 18.7 arbitrary units, p = 0.025). CONCLUSION: Heme oxygenase-1 protein levels shortly after allograft reperfusion are closely related with initial graft function. Assessment thereof may be considered a valuable tool to predict DGF.

Kidney transplantation in patients suffering from hereditary complete complement C4 deficiency.

Hereditary complete C4 deficiency (C4def) is a very rare condition that predisposes to immune complex disease and end-stage renal failure. Whether such patients should undergo renal transplantation is debated. The clinical outcome of five transplantations in three C4def patients is described. The first patient lost one allograft after 6 years because of chronic allograft rejection. Back on dialysis, he suffered from meningitis caused by Neisseria menigitidis and Aspergillus. One year after a second transplantation under alemtuzumab induction, he developed fulminant Kaposi's sarcoma and died. His sister is now 6 years post-transplantation without complications. The third patient lost his first graft after 3 years because of chronic allograft nephropathy and recurrence of glomerulonephritis. He has now been living with a second graft for over 9 years. He suffered from pneumonia, a generalized varicella infection and Hemophilis parainfluenzae bronchitis. Patients with complete C4def are at increased risk for infection after kidney transplantation. Under certain precautions and with judicious use of immunosuppression, good long-term results are achievable.

Cigarette smoking and chronic allograft nephropathy.

BACKGROUND: Smoking has been demonstrated to decrease patient and graft survival after kidney transplantation. Data on histological changes associated with smoking in renal allografts are lacking. METHODS: Smoking habits before and after renal transplantation were evaluated by questionnaire in 279 patients. A transplant biopsy was performed more than 1 year after transplantation in 76 of them. Histological changes were classified according to Banff 97 criteria. Linear regression analysis and proportional odds models for histological changes including the factors age, gender, diabetes, body mass index, donor age, time since transplantation, history of acute rejection and smoking status were calculated. RESULTS: Overall 22% of patients continued smoking after transplantation, with the proportion decreasing from 38% of those transplanted before 1990 to 13% of those transplanted after 2000. Serum creatinine was non-significantly higher in smokers (2.3 +/- 2.7 mg/dl vs 1.8 +/- 1.4 mg/dl, P = 0.21). A renal biopsy was performed in 24% of non-smokers and 39% of smokers (P = 0.02), and smokers were biopsied on average 1.5 years earlier. Among biopsied patients current smokers tended to suffer more often from diabetes (25.0% vs 13.5%, P = 0.33), to develop transplant failure (33.3% vs 21.2%, P = 0.25) or experience a cardiovascular event (29.2% vs 15.4%, P = 0.16). The frequency of acute rejection was comparable between smokers and non-smokers (25.0% vs 34.6%, P = 0.40). Glomerular sclerosis was associated with diabetes (P = 0.03). Severity of vascular intimal fibrous thickening was associated with current smoking (P = 0.004), whereas the degree of arteriolar hyalinosis (P < 0.001) and chronic/sclerosing nephropathy (P = 0.05) were associated with time since transplantation. CONCLUSIONS: The number of patients who continue cigarette smoking after renal transplantation has decreased in recent years. The main allograft lesion associated with smoking is fibrous intimal thickening of small arteries.

A novel technique for heterotopic vascularized pancreas transplantation in mice to assess ischemia reperfusion injury and graft pancreatitis.

BACKGROUND: Although various suture techniques for murine pancreas transplantation have been described, severe limitations have limited their widespread use. We therefore designed a surgical model for cervical heterotopic pancreas transplantation using a cuff technique. METHODS: C57BL6 mice were used as donor and recipient pairs. Recipients were rendered diabetic with streptozotocin and subsequently transplanted. The donor pancreas was isolated using a no-touch technique and then placed in the recipient's cervical region. Vascular anastomoses were completed by pulling the portal vein over the external jugular vein cuff and the donor aortic segment over the carotid cuff and fixed with an 8-0 ligature thereby facilitating a nonsuture technique. To test applicability of this model, graft microcirculation was evaluated by intravital microscopy after prolonged cold ischemia (16 h). RESULTS: The immediate success rate was >90%. Donor operation lasted 40 +/- 5 min; dissection of recipient vessels lasted 20 +/- 4 min. Revascularization time was 4 to 6 min, resulting in a total pancreas ischemia time of 33 +/- 6 min. No thromboembolic complications on the cuff side were observed. Preoperative glucose levels were 518 +/- 59 mg/dl and returned to normal by postoperative day 1 (88 +/- 13 mg/dl). Histology on postoperative days 10 and 30 showed almost normal islet cell and acinar architecture of all grafts. In groups with prolonged cold ischemia, graft microcirculation was significantly reduced and paralleled by increased inflammation, interstitial edema, hemorrhage, acinar vacuolization, and focal areas of necrosis compared with nonischemic controls. CONCLUSIONS: This new model may provide an excellent tool to further investigate the pathophysiology as well as novel therapeutic strategies of preservation, ischemia reperfusion injury, and graft pancreatitis.

Laparoscopically implanted gastric pacemaker after kidney-pancreas transplantation: treatment of morbid obesity and diabetic gastroparesis.

Combined kidney-pancreas transplantation is the treatment of choice for end-stage diabetic nephropathy. Weight gain post-transplant increases the risk for post-transplant complications and death due to cardiovascular events. Gastric pacemakers have been used for therapy of diabetic gastropathy and for the treatment of moderate morbid obesity. We report a patient who experienced significant weight gain following successful kidney-pancreas transplantation and was thereafter successfully treated for diabetic gastroparesis and morbid obesity by use of a laparoscopically implanted gastric pacemaker.

Malignancies of the colorectum and anus in solid organ recipients.

Patients undergoing solid organ transplantation (SOT) are at increased risk for developing malignancies due to the long term immunosuppression. Data on malignancies of the large intestine after various types of SOT are rare. A total of 3595 SOTs were performed between 1986 and 2005 at our center and retrospectively analyzed with regard to the incidence and course of malignancies of the colon, rectum, and anus. Standard immunosuppression consisted of calcineurin inhibitors in combination with azathioprine or mycophenolate mofetil and steroids with or without antithymocyte globulin or IL-2 receptor antagonist induction. A total of 206 patients (5.7%) developed malignancies. Colorectal adenocarcinoma was diagnosed in nine patients (0.25%; mean age at diagnosis 65 years) at a mean of 5.3 years after transplantation. Five patients (55%) died 7.2 years post-transplant due to cardiovascular disease (n = 4) and tumor progression (n = 1). Four patients developed anal neoplasia (0.11%) 7 years post-transplant with 100% 1-year survival. Five patients showed post-transplant lymphoproliferative disorders (PTLD) with intestinal involvement. The incidence of anal but not of colorectal cancers in our transplant recipients differed from that of immunocompetent individuals of corresponding age (0.11% vs. 0.002% and 0.25% vs. 0.3%). PTLD may involve the colon.

Spectrum of surgical complications after simultaneous pancreas-kidney transplantation in a prospectively randomized study of two immunosuppressive protocols.

BACKGROUND: Simultaneous pancreas-kidney transplantation (SPK) has evolved as an effective treatment for patients with end-stage nephropathy due to type 1 diabetes mellitus. This report analyses the spectrum of surgical complications among patients receiving tacrolimus and cyclosporin microemulsion (ME)-based therapy for SPK transplantation. METHODS: The analysis included 205 patients randomly assigned to tacrolimus (n = 103) or cyclosporin-ME (n = 102) in the Euro-SPK 001 study. Surgical complications were defined as any intervention in the 3-month post-operative course related to the transplant procedure. RESULTS: In the tacrolimus vs cyclosporin-ME group, repeat laparotomy was required by fewer patients (26 vs 43%, respectively; P = 0.01) and at a later stage post-transplant (26+/-26 vs 14+/-17 days; P = 0.05). Also, thrombosis of graft vessels (2 vs 9%; P = 0.03) and repeat laparotomy for intra-abdominal haemorrhage within the first 3 months (8 vs 22%; P = 0.005) occurred significantly less frequently with tacrolimus vs cyclosporin-ME. A donor age of > or =45 years was a significant determinant for surgical complications requiring repeat laparotomy, regardless of the type of immunosuppression. Portal anastomosis was the safest method of endocrine venous drainage, and Roux-en-Y loop for enteric exocrine drainage was associated with a higher re-operation rate than duodenoenterostomy. Repeat laparotomy had no impact on patient survival, but significantly reduced kidney and pancreas graft survival in the cyclosporin-ME group (kidney: P<0.01; pancreas: P<0.001) and in both groups combined (P < or = 0.05 and P<0.001, respectively). CONCLUSIONS: The immunological benefits of tacrolimus compared with cyclosporin-ME treatment result in a lower incidence of repeat laparotomies post-transplant and a reduced in-hospital stay. Fewer repeat laparotomies translate into improved pancreas and kidney graft survival.

17beta-estradiol attenuates glutamate-induced apoptosis and preserves electrophysiologic function in primary cortical neurons.

Glutamate toxicity causes neuronal death in neurodegenerative diseases; hence, there is a need for therapeutic agents rendering functional neuroprotection. We tested the effects of 17beta-estradiol (estrogen) in rat primary cortical neurons after glutamate exposure. Wright staining and ApopTag assays indicated that 0.5 microM glutamate for 24 hr caused apoptosis. Glutamate-induced apoptosis correlated with upregulation of calpain, a proapoptotic shift in the Bax:Bcl-2 ratio, and increased activation of caspase-3. Pretreatment with 10 nM estrogen prevented apoptosis, attenuated calpain upregulation, shifted the Bax:Bcl-2 ratio toward survival, and decreased caspase-3 activation. Single-cell voltage-clamp techniques were used to record whole-cell currents associated with Na+ channels, N-methyl-D-aspartate receptor channels, and kainate receptor channels. No significant differences were recorded in membrane capacitance at -70 mV in neurons treated with estrogen or estrogen plus glutamate, relative to controls. Notably, no changes in capacitance indicated that neurons treated with estrogen and glutamate did not experience apoptosis-associated cell shrinkage. No membrane potential could be recorded in the neurons treated with glutamate due to apoptosis. All recorded currents were similar in amplitude and activation/inactivation kinetics in control neurons and neurons treated with estrogen plus glutamate. Estrogen thus preserved both neuronal viability and function in this in vitro glutamate toxicity model.