Motor evoked potentials versus Macrostimulation in predicting the postoperative motor threshold in STN Deep brain stimulation.

INTRODUCTION: Accuracy is crucial in Deep Brain Stimulation (DBS). Electrophysiological and image-based techniques are used to avoid suboptimal positioning. Macrostimulation is the gold standard to delineate the therapeutic window intraoperatively. Despite this, electrode revision rates due to malpositioning are as high as 17%. The goal was to compare motor evoked potentials (MEPs) with the gold standard of Macrostimulation. We assessed accuracy and precision as well as the correlation in predicting motor side effects at the initial mapping 4 weeks postoperatively. METHODS: In this prospective study intraoperative MEPs from 94 contacts in 16 patients undergoing STN DBS under local anesthesia were correlated to the postoperative threshold for stimulation-induced motor side effects and compared to intraoperative Macrostimulation. Analysis of accuracy, precision and correlation (Pearson) was performed. RESULTS: MEPs of the upper extremity had a mean percentage error of 25% (SD 38.8%) and correlated significantly with the motor threshold at postoperative mapping (R=0.235). Macrostimulation was less accurate and precise with a mean percentage error of - 68% (SD 78.8%) but had a higher correlation (R=0.388). MEPs rarely (3%) overestimated the threshold by maximally 1 mA. In contrast, Macrostimulation overestimated the threshold by over 1 mA in 69% leading to a false sense of security. CONCLUSION: MEPs are feasible in an awake setting during Deep Brain Stimulation in the STN for PD patients. MEPs of the upper extremity are more accurate and precise predicting the motor threshold and avoid a false sense of security in comparison to the gold standard of Macrostimulation.

Preclinical Evaluation of the Stealth Autoguide Robotic Guidance Device for Stereotactic Cranial Surgery: A Human Cadaveric Study.

INTRODUCTION: Stereotactic procedures are routinely performed for brain biopsies, deep brain stimulation, and placement of stereoelectroencephalography (SEEG) electrodes for epilepsy. The recently developed Stealth Autoguide (Medtronic, Minneapolis, MN, USA) device does not require patients to don a stereotactic frame. In this preclinical study, we sought to quantitatively compare the Stealth Autoguide robotic system to 2 devices commonly used in clinical practice: the Navigus biopsy system (Medtronic) and the Leksell stereotactic frame (Elekta Ltd., Stockholm, Sweden). METHODS: In the first experimental setup, we compared target accuracy of the Stealth Autoguide to the Navigus system by using phantom heads filled with gelatin to simulate the brain tissue. In the second experimental setup, we inserted SEEG electrodes to targets within cadaveric heads in a simulated operating room environment. RESULTS: Using a homogeneous gelatin-filled phantom 3D reconstruction of a human head, we found that using the Stealth Autoguide system, while maintaining accuracy, was faster to use than the Navigus system. In our simulated operating room environment using nonliving human cadaveric heads, we found the accuracy of the Stealth Autoguide robotic device to be comparable to that of the Leksell frame. DISCUSSION/CONCLUSION: These results compare the use of the Stealth Autoguide robotic guidance system with commonly used stereotactic devices, and this is the first study to compare its use and accuracy with the Leksell frame. These findings provide mounting evidence that Stealth Autoguide will have potential clinical uses in various stereotactic neurosurgical procedures.

Validation of 3D fluoroscopy for image-guidance registration in depth electrode implantation for medically refractory epilepsy.

BACKGROUND: Frame registration is a critical step to ensure accurate electrode placement in stereotactic procedures such as stereoelectroencephalography (SEEG) and is routinely done by merging a computed tomography (CT) scan with the preoperative magnetic resonance (MR) examination. Three-dimensional fluoroscopy (XT) has emerged as a method for intraoperative electrode verification following electrode implantation and more recently has been proposed as a registration method with several advantages. METHODS: We compared the accuracy of SEEG electrode placement by frame registration with CT and XT imaging by analyzing the Euclidean distance between planned and post-implantation trajectories of the SEEG electrodes to calculate the error in both the entry (EP) and target (TP) points. Other variables included radiation dose, efficiency, and complications. RESULTS: Twenty-seven patients (13 CT and 14 XT) underwent placement of SEEG electrodes (319 in total). The mean EP and TP errors for the CT group were 2.3 mm and 3.3 mm, respectively, and 1.9 mm and 2.9 mm for the XT group, with no statistical difference (p = 0.75 and p = 0.246). The time to first electrode placement was similar (XT, 82 ± 10 min; CT, 84 ± 22 min; p = 0.858) and the average radiation exposure with XT (234 ± 55 mGy*cm) was significantly lower than CT (1245 ± 123 mGy*cm) (p < 0.0001). Four complications were documented with equal incidence in both groups. CONCLUSIONS: The use of XT as a method for registration resulted in similar implantation accuracy compared with CT. Advantages of XT are the substantial reduction in radiation dose and the elimination of the need to transfer the patient out of the room which may have an impact on patient safety and OR efficiency.

Knowledge transfer and retention of simulation-based learning for neurosurgical instruments: a randomised trial of perioperative nurses.

Introduction: Previous studies have shown that simulation is an acceptable method of training in nursing education. The objectives of this study were to determine the effectiveness of tablet-based simulation in learning neurosurgical instruments and to assess whether skills learnt in the simulation environment are transferred to a real clinical task and retained over time. Methods: A randomised controlled trial was conducted. Perioperative nurses completed three consecutive sessions of a simulation. Group A performed simulation tasks prior to identifying real instruments, whereas Group B (control group) was asked to identify real instruments prior to the simulation tasks. Both groups were reassessed for knowledge recall after 1 week. Results: Ninety-three nurses completed the study. Participants in Group A, who had received tablet-based simulation, were 23% quicker in identifying real instruments and did so with better accuracy (93.2% vs 80.6%, p<0.0001) than Group B. Furthermore, the simulation-based learning was retained at 7 days with 97.8% correct instrument recognition in Group A and 96.2% in Group B while maintaining both speed and accuracy. Conclusion: This is the first study to assess the effectiveness of tablet-based simulation training for instrument recognition by perioperative nurses. Our results demonstrate that instrument knowledge acquired through tablet-based simulation training results in improved identification and retained recognition of real instruments.

The accuracy of 3D fluoroscopy (XT) vs computed tomography (CT) registration in deep brain stimulation (DBS) surgery.

BACKGROUND: Stereotactic registration is the most critical step ensuring accuracy in deep brain stimulation (DBS) surgery. 3D fluoroscopy (XT) is emerging as an alternative to CT. XT has been shown to be safe and effective for intraoperative confirmation of lead position following implantation. However, there is a lack of studies evaluating the suitability of XT to be used for the more crucial step of registration and its capability of being merged to a preoperative MRI. This is the first study comparing accuracy, efficiency, and radiation exposure of XT- vs CT-based stereotactic registration and XT/MRI merging in deep brain stimulation. METHODS: Mean absolute differences and Euclidean distance between planned (adjusted for intraoperative testing) and actual lead trajectories were calculated for accuracy of implantation. The radiation dose from each scan was recorded as the dose length product (DLP). Efficiency was measured as the time between the patient entering the operating room and the initial skin incision. A one-way ANOVA compared these parameters between patients that had either CT- or XT-based registration. RESULTS: Forty-one patients underwent DBS surgery-25 in the CT group and 16 in the XT group. The mean absolute difference between CT and XT was not statistically significant in the x (p = 0.331), y (p = 0.951), or z (p = 0.807) directions. The Euclidean distance between patient groups did not differ significantly (p = 0.874). The average radiation exposure with XT (220.0 ± 0.1 mGy*cm) was significantly lower than CT (1269.3 ± 112.9 mGy*cm) (p < 0.001). There was no significant difference in registration time between CT (107.8 ± 23.1 min) and XT (106.0 ± 18.2 min) (p = 0.518). CONCLUSION: XT-based frame registration was shown to result in similar implantation accuracy and significantly less radiation exposure compared with CT. Our results surprisingly showed no significant difference in registration time, but this may be due to a learning curve effect.

Virtual Reality Surgical Simulation: Implications for Resection of Intracranial Gliomas.

Surgical simulation has the potential to play important roles in surgical training and preoperative planning. The advent of virtual reality (VR) with tactile haptic feedback has revolutionized surgical simulation, creating a novel environment for residents to learn manual skills without compromising patient safety. This concept is particularly relevant in neurosurgical training where the acquired skill set demands performance of technically challenging tasks under pressure and where the consequences of error are significant. The evolution of VR simulation is discussed here within the context of neurosurgical training and its implications for resection of intracranial gliomas. VR holds the promise of providing a useful educational tool for neurosurgical residents to hone their surgical skills and for neurosurgeons to rehearse specific segments of the surgery prior to the actual operation. Also discussed are several important issues related to simulation and simulation-based training that will need to be addressed before widespread adoption of VR simulation as a useful technology.

Simulation-based training for burr hole surgery instrument recognition.

BACKGROUND: The use of simulation training in postgraduate medical education is an area of rapidly growing popularity and research. This study was designed to assess the impact of simulation training for instrument knowledge and recognition among neurosurgery residents. METHODS: This was a randomized control trial of first year residents from neurosurgery residency training programs across Canada. Eighteen neurosurgery trainees were recruited to test two simulation-based applications: PeriopSim™ Instrument Trainer and PeriopSim™ for Burr Hole Surgery. The intervention was game-based simulation training for learning neurosurgical instruments and applying this knowledge to identify correct instruments during a simulated burr hole surgery procedure. RESULTS: Participants showed significant overall improvement in total score (p < 0.0005), number of errors (p = 0.019) and time saved (p < 0.0005), over three testing sessions when using the PeriopSim™ Instrument Trainer. Participants demonstrated further performance-trained improvements when using PeriopSim™ Burr Hole Surgery. CONCLUSIONS: Training in the recognition and utilization of simulated surgical instruments by neurosurgery residents improved significantly with repetition when using PeriopSim™ Instrument Trainer and PeriopSim™ for Burr Hole Surgery.

A review of bioreactor protocols for human neural precursor cell expansion in preparation for clinical trials.

Tissue-specific human neural precursor cells (hNPCs) can be isolated from various regions of the developing or adult central nervous system and may serve as a viable source of cells in cell replacement therapies for the treatment of neurodegenerative disorders. However, in order for cell replacement strategies to become a routine therapeutic option for the treatment of neurodegenerative disorders, hNPCs should be generated under standardized and controlled conditions. Studies over the last two decades have focused on developing cell growth media and cell handling protocols for expansion and differentiation of hNPCs in culture. Key studies have reported the development of serum-free growth media and large-scale computer-controlled suspension bioreactors that can support high cell proliferation rates (doubling times < 3 days), multipotentiality, and potential neurogenic differentiation (more than 60% neurons). Moreover, bioengineering studies have focused on controlling culture conditions in suspension bioreactors including inoculation, hydrodynamics of culture, oxygen and nutrients transfer to the cells, monitoring in situ physiological parameters using process control techniques, and expansion for extended periods of time. In addition, in vitro and in vivo characterization of hNPCs have been performed, providing information on stem/progenitor cell characteristics, cell surface analysis, and appropriate type of cells to use in transplantation studies.

New bioengineering insights into human neural precursor cell expansion in culture.

Understanding initial cell growth, interactions associated with the process of expansion of human neural precursor cells (hNPCs), and cellular events pre- and postdifferentiation are important for developing bioprocessing protocols to reproducibly generate multipotent cells that can be used in basic research or the treatment of neurodegenerative disorders. Herein, we report the in vitro responses of telencephalon hNPCs grown in a serum-free growth medium using time-lapse live imaging as well as cell-surface marker, aggregate size, and immunocytochemical analyses. Time-lapse analysis of hNPC initial expansion indicated that cell-surface attachment in stationary culture and the frequency of cell-cell interaction in suspension conditions are important for subsequent aggregate formation and hNPC growth. In the absence of cell-surface attachment in low-attachment stationary culture, large aggregates of cells were formed and expansion was adversely affected. The majority of the telencephalon hNPCs expressed CD29, CD90, and CD44 (cell surface markers involved in cell-ECM and cell-cell interactions to regulate biological functions such as proliferation), suggesting that cell-surface attachment and cell-cell interactions play a significant role in the subsequent formation of cell aggregates and the expansion of hNPCs. Before differentiation, about 90% of the cells stained positive for nestin and expressed two neural precursor cells surface markers (CD133 and CD24). Upon withdrawal of growth cytokines, hNPCs first underwent cell division and then differentiated preferentially towards a neuronal rather than a glial phenotype. This study provides key information regarding human NPC behavior under different culture conditions and favorable culture conditions that are important in establishing reproducible hNPC expansion protocols.

Bioreactor expansion of human neural precursor cells in serum-free media retains neurogenic potential.

Human neural precursor cells (hNPCs), harvested from somatic tissue and grown in vitro, may serve as a source of cells for cell replacement strategies aimed at treating neurodegenerative disorders such as Parkinson's disease (PD), Huntington's disease (HD), and intractable spinal cord pain. A crucial element in a robust clinical production method for hNPCs is a serum-free growth medium that can support the rapid expansion of cells while retaining their multipotency. Here, we report the development of a cell growth medium (PPRF-h2) for the expansion of hNPCs, achieving an overall cell-fold expansion of 10(13) over a period of 140 days in stationary culture which is significantly greater than other literature results. More importantly, hNPC expansion could be scaled-up from stationary culture to suspension bioreactors using this medium. Serial subculturing of the cells in suspension bioreactors resulted in an overall cell-fold expansion of 7.8 x 10(13) after 140 days. These expanded cells maintained their multipotency including the capacity to generate large numbers of neurons (about 60%). In view of our previous studies regarding successful transplantation of the bioreactor-expanded hNPCs in animal models of neurological disorders, these results have demonstrated that PPRF-h2 (containing dehydroepiandrosterone, basic fibroblast growth factor and human leukemia inhibitory factor) can successfully facilitate the production of large quantities of hNPCs with potential to be used in the treatment of neurodegenerative disorders.

GDNF therapy for Parkinson's disease.

With an increase in the aging population, the incidence of Parkinson's disease (PD), a disabling neurodegenerative disorder mainly affecting motor function, will inevitably present a challenge to an already overburdened healthcare system. Current medical and surgical therapies offer symptomatic relief but do not provide a cure. Experimental studies suggest that GDNF has the ability to protect degenerating dopamine neurons in PD as well as promote regeneration of the nigrostriatal dopamine system. However, clinical trials of GDNF infusion to date remain inconclusive. This review will examine the experimental and clinical evidence of GDNF use in PD with particular focus on its potential as an effective therapy in the treatment of PD.

Expansion of human neural precursor cells in large-scale bioreactors for the treatment of neurodegenerative disorders.

The transplantation of in vitro expanded human neural precursor cells (hNPCs) represents a potential new treatment alternative for individuals suffering from incurable neurodegenerative disorders such as Parkinson's disease (PD) and Huntington's disease (HD). However, in order for cell restorative therapy to have widespread therapeutic significance, it will be necessary to generate unlimited quantities of clinical grade hNPCs in a standardized method. We report here that we have developed a serum-free medium and scale-up protocols that allow for the generation of clinical quantities of human telencephalon-derived hNPCs in 500-mL computer-controlled suspension bioreactors. The average hNPC aggregate diameter in the bioreactors was maintained below a target value of 500 microm by controlling the liquid shear field. The human cells, which were inoculated at 10(5) cells/mL, exhibited a doubling time of 84 h, underwent a 36-fold expansion over the course of 18 days, and maintained an average viability of over 90%. The bioreactor-derived hNPCs retained their nestin expression following expansion and were able to differentiate into glial and neuronal phenotypes under defined conditions. It has also been demonstrated that these hNPCs differentiated to a GABAergic phenotype that has recently been shown to be able to restore functional behavior in rat models of HD and neuropathic pain (Mukhida, K. et al. Stem Cells 2007; DOI 10.1634/stemcells.2007-0326). This study demonstrates that clinical quantities of hNPCs can be successfully and reproducibly generated under standardized conditions in computer-controlled suspension bioreactors.

Survival, differentiation, and migration of bioreactor-expanded human neural precursor cells in a model of Parkinson disease in rats.

OBJECT: Fetal tissue transplantation for Parkinson disease (PD) has demonstrated promising results in experimental and clinical studies. However, the widespread clinical application of this therapeutic approach is limited by a lack of fetal tissue. Human neural precursor cells (HNPCs) are attractive candidates for transplantation because of their long-term proliferation activity. Furthermore, these cells can be reproducibly expanded in a standardized fashion in suspension bioreactors. In this study the authors sought to determine whether the survival, differentiation, and migration of HNPCs after transplantation depended on the region of precursor cell origin, intracerebral site of transplantation, and duration of their expansion. METHODS: Human neural precursor cells were isolated from the telencephalon, brainstem, ventral mesencephalon, and spinal cord of human fetuses 8-10 weeks of gestational age, and their differentiation potential characterized in vitro. After expansion in suspension bioreactors, the HNPCs were transplanted into the striatum and substantia nigra of parkinsonian rats. Histological analyses were performed 7 weeks posttransplantation. RESULTS: The HNPCs isolated from various regions of the neuraxis demonstrated diverse propensities to differentiate into astrocytes and neurons and could all successfully expand under standardized conditions in suspension bioreactors. At 7 weeks posttransplantation, survival and migration were significantly greater for HNPCs obtained from the more rostral brain regions. The HNPCs differentiated predominantly into astrocytes after transplantation into the striatum or substantia nigra regions, and thus no behavioral improvement was observed. CONCLUSIONS: Understanding the regional differences in HNPC properties is prerequisite to their application for PD cell restoration strategies.

Neurosurgical strategies for Gilles de la Tourette's syndrome.

Tourette's syndrome (TS) is a neurological disorder characterized by motor and vocal tics that typically begin in childhood and often are accompanied by psychiatric comorbidities. Symptoms of TS may be socially disabling and cause secondary medical complications. Pharmacological therapies remain the mainstay of symptom management. For the subset of patients in whom TS symptoms are medically recalcitrant and do not dissipate by adulthood, neurosurgery may offer an alternative treatment strategy. Greater understanding of the neuroanatomic and pathophysiologic basis of TS has facilitated the development of surgical procedures that aim to ameliorate TS symptoms by lesions or deep brain stimulation of cerebral structures. Herein, the rationale for the surgical management of TS is discussed and neurosurgical experiences since the 1960s are reviewed. The necessity for neurosurgical strategies to be performed with appropriate ethical considerations is highlighted.

Spinal GABAergic transplants attenuate mechanical allodynia in a rat model of neuropathic pain.

Injury to the spinal cord or peripheral nerves can lead to the development of allodynia due to the loss of inhibitory tone involved in spinal sensory function. The potential of intraspinal transplants of GABAergic cells to restore inhibitory tone and thus decrease pain behaviors in a rat model of neuropathic pain was investigated. Allodynia of the left hind paw was induced in rats by unilateral L5- 6 spinal nerve root ligation. Mechanical sensitivity was assessed using von Frey filaments. Postinjury, transgenic fetal green fluorescent protein mouse GABAergic cells or human neural precursor cells (HNPCs) expanded in suspension bioreactors and differentiated into a GABAergic phenotype were transplanted into the spinal cord. Control rats received undifferentiated HNPCs or cell suspension medium only. Animals that received either fetal mouse GABAergic cell or differentiated GABAergic HNPC intraspinal transplants demonstrated a significant increase in paw withdrawal thresholds at 1 week post-transplantation that was sustained for 6 weeks. Transplanted fetal mouse GABAergic cells demonstrated immunoreactivity for glutamic acid decarboxylase and GABA that colocalized with green fluorescent protein. Intraspinally transplanted differentiated GABAergic HNPCs demonstrated immunoreactivity for GABA and beta-III tubulin. In contrast, intraspinal transplantation of undifferentiated HNPCs, which predominantly differentiated into astrocytes, or cell suspension medium did not affect any behavioral recovery. Intraspinally transplanted GABAergic cells can reduce allodynia in a rat model of neuropathic pain. In addition, HNPCs expanded in a standardized fashion in suspension bioreactors and differentiated into a GABAergic phenotype may be an alternative to fetal cells for cell-based therapies to treat chronic pain syndromes.

Left ventricular dysfunction after acute intracranial hypertension is associated with increased hydroxyl free radical production, cardiac ryanodine hyperphosphorylation, and troponin I degradation.

BACKGROUND: In addition to generating free radicals, stress-induced activation of the sympathetic nervous system results in hyperphosphorylation of the cardiac ryanodine receptor (RyR2)/calcium (Ca2+) release channel on the sarcoplasmic reticulum, causing leaky channels. These events may contribute to cytosolic Ca2+ overload and activation of Ca2+-dependent cytotoxic processes. Because myocardial dysfunction associated with intracranial hypertension is catecholamine mediated, we sought to determine in a rat model if hemodynamic changes are associated with an increase in oxidative stress, hyperphosphorylation of RyR2, and degradation of myofilament protein cardiac troponin I (TnI). METHODS: In halothane-anesthetized rats treated with saline, dimethyl sulfoxide (DMSO), or the synthetic calpain inhibitor calpeptin (3,500 microg), a subdural balloon catheter was inflated to induce intracranial hypertension. Hearts were excised, and RyR2 phosphorylation status and TnI degradation was determined with Western blot analysis. In separate experiments, treated rats were challenged with increasing doses of dobutamine 30 minutes after subdural balloon inflation. RESULTS: Elevating the intracranial pressure resulted in an increase in plasma catecholamines, as well as in 3,4-dihydroxybenzoic acid (DHBA), an indirect marker of HO. radical production, and left ventricular dysfunction in rats treated with saline or DMSO. There was evidence of hyperphosphorylation of RyR2 and TnI degradation (27 kD immunoreactive band). Calpeptin treatment improved left ventricular function; however, this had no effect on the phosphorylation status of RyR2 or TnI degradation levels. In addition, the myocardial responsiveness to dobutamine was augmented in rats with depressed myocardial function. CONCLUSION: The present findings demonstrate that hemodynamic instability after intracranial hypertension is associated with oxidative stress and post-translational changes to RyR2 and TnI degradation. Despite this, the myocardial responsiveness to beta1 adrenergic stimulation is preserved in rats with depressed myocardial function.

Central dexmedetomidine attenuates cardiac dysfunction in a rodent model of intracranial hypertension.

PURPOSE: To determine if central sympathetic blockade by dexmedetomidine, a selective alpha(2) adrenergic receptor agonist, prevents cardiac dysfunction associated with intracranial hypertension (ICH) in a rat model. METHODS: Following intracisternal administration of dexmedetomidine (1 microg.microL(-1), 10 microL volume) or the stereoisomer levomedetomidine (1 microg.microL(-l), 10 microL volume) in halothane-anesthetized rats, a subdural balloon catheter was inflated for 60 sec to produce ICH. Intracranial pressure, hemodynamic, left ventricular (LV) pressures and electrocardiographic (ECG) changes were recorded. Plasma and myocardial catecholamines and malondialdehyde (MDA) levels were measured. RESULTS: After levomedetomidine administration, subdural balloon inflation precipitated an increase in mean arterial pressure (149 +/- 33% of baseline), heart rate (122 +/- 19% of baseline), LV systolic pressure (LVP), LV end-diastolic pressure (LVEDP), LV developed pressure (LVDP), LV dP/dtmax and rate pressure product (RPP) (132 +/- 19%, 260 +/- 142%, 119 +/- 15%, 126 +/- 24% and 146 +/- 33% of baseline value, respectively). ICH decelerated LVP fall (tau), as tau increased from 7.75 +/- 1.1 to 14.37 +/- 4.5 msec. Moreover, plasma norepinephrine levels were elevated (169 +/- 50% of baseline) and there was the appearance of cardiac dysrhythmias and other ECG abnormalities. This response was transient and cardiac function deteriorated in a temporal manner. Intracisternal dexmedetomidine prevented the rise in plasma norepinephrine, blocked the ECG abnormalities, and preserved cardiac function. Moreover, dexmedetomidine attenuated the rise in MDA levels. CONCLUSIONS: The results demonstrate that dexmedetomidine attenuates cardiac dysfunction associated with ICH. Our results provide evidence for the role of central sympathetic hyperactivity in the development of cardiac dysfunction associated with ICH.

Rapid uptake of oxidized ascorbate induces loss of cellular glutathione and oxidative stress in liver slices.

The observation that ascorbate known to retain pro-oxidant properties induces cell death in a number of immortal cell lines, led us to examine its mechanism and whether it is involved in oxidative stress injury in such asocorbate-enriched tissue cells as hepatocytes. In rat liver homogenates, higher concentrations (1 and 3 mM) of ascorbate suppressed lipid peroxide productions but lower concentrations (0.1 and 0.3 mM) did not. In contrast to the homogenate, ascorbate increased lipid peroxide production in liver slices in a concentration dependant manner. Iso-ascorbate, the epimer of ascorbate did not cause an increase the oxidative stress in liver slices. This differential effect between homogenates and liver slices implies that cellular integrity is required for ascorbate to induce oxidative stress. Wortmannin, an inhibitor of the GLUT (glucose transporter) thought to transport dehydroascorbate into cells, inhibited [(14)C]-ascorbate uptake and suppressed oxidative stress in liver slices. Wortmannin suppressed that [(14)C]-ascorbate uptake by GLUT following oxidation to [(14)C]dehydroascorbate. Taken together, these observations support our hypothesis that ascorbate is oxidized to dehydroascorbate by molecular oxygen in solution (i.e., plasma and culture medium) which is then carried into hepatocytes (via a GLUT) where it is reduced back to ascorbate causing oxidative stress.