Nonclinical and pharmacokinetic assessments to evaluate the potential of tedizolid and linezolid to affect mitochondrial function.

Prolonged treatment with the oxazolidinone linezolid is associated with myelosuppression, lactic acidosis, and neuropathies, toxicities likely caused by impairment of mitochondrial protein synthesis (MPS). To evaluate the potential of the novel oxazolidinone tedizolid to cause similar side effects, nonclinical and pharmacokinetic assessments were conducted. In isolated rat heart mitochondria, tedizolid inhibited MPS more potently than did linezolid (average [± standard error of the mean] 50% inhibitory concentration [IC50] for MPS of 0.31 ± 0.02 µM versus 6.4 ± 1.2 µM). However, a rigorous 9-month rat study comparing placebo and high-dose tedizolid (resulting in steady-state area under the plasma concentration-time curve values about 8-fold greater than those with the standard therapeutic dose in humans) showed no evidence of neuropathy. Additional studies explored why prolonged, high-dose tedizolid did not cause these mitochondriopathic side effects despite potent MPS inhibition by tedizolid. Murine macrophage (J774) cell fractionation studies found no evidence of a stable association of tedizolid with eukaryotic mitochondria. Monte Carlo simulations based on population pharmacokinetic models showed that over the course of a dosing interval using standard therapeutic doses, free plasma concentrations fell below the respective MPS IC50 in 84% of tedizolid-treated patients (for a median duration of 7.94 h) and 38% of linezolid-treated patients (for a median duration of 0 h). Therapeutic doses of tedizolid, but not linezolid, may therefore allow for mitochondrial recovery during antibacterial therapy. The overall results suggest that tedizolid has less potential to cause myelosuppression and neuropathy than that of linezolid during prolonged treatment courses. This, however, remains a hypothesis that must be confirmed in clinical studies.

Lack of neuropathological changes in rats administered tedizolid phosphate for nine months.

Tedizolid, a novel oxazolidinone antibacterial, was administered to Long Evans rats by oral gavage once daily for up to 9 months at doses near the maximum tolerated dose (MTD) to evaluate for potential neurotoxicity. Mean plasma exposures of tedizolid at the low-, medium-, and high-dose levels (7.5, 15, and 30 mg/kg of body weight/day for males; 2.5, 5, and 10 mg/kg/day for females) were similar between males and females and were 1.8-, 3.9-, and 8.0-fold greater than exposures in patients at the therapeutic dose (200 mg once daily). Evaluated endpoints included survival, clinical observations, body weight, and food consumption. At 1, 3, 6, and 9 months, ophthalmic examinations, functional observational batteries, and locomotor activity measures were conducted, brain weights/sizes were recorded, and perfusion-fixed tissues were collected from 12 rats/sex/group/time point. A detailed morphological assessment was conducted on brain, eyes, optic nerve/tract, spinal cord, peripheral nerves (includes sciatic, sural, tibial, peroneal, trigeminal), and skeletal muscle. At the end of 9 months, less body weight gain was seen in high-dose males (-6.7%) and females (-5.8%) compared with that seen in controls. There were no tedizolid-related adverse neurobehavioral effects or tedizolid-related histopathologic changes in the central/peripheral nervous systems, including the optic nerve. Results of this study indicate that tedizolid was not neurotoxic when administered long term to pigmented rats at doses near the MTD, which were up to 8-fold higher than the human therapeutic exposure.

STP position paper: Recommended practices for sampling and processing the nervous system (brain, spinal cord, nerve, and eye) during nonclinical general toxicity studies.

The Society of Toxicologic Pathology charged a Nervous System Sampling Working Group with devising recommended practices to routinely screen the central nervous system (CNS) and peripheral nervous system (PNS) in Good Laboratory Practice-type nonclinical general toxicity studies. Brains should be weighed and trimmed similarly for all animals in a study. Certain structures should be sampled regularly: caudate/putamen, cerebellum, cerebral cortex, choroid plexus, eye (with optic nerve), hippocampus, hypothalamus, medulla oblongata, midbrain, nerve, olfactory bulb (rodents only), pons, spinal cord, and thalamus. Brain regions may be sampled bilaterally in rodents using 6 to 7 coronal sections, and unilaterally in nonrodents with 6 to 7 coronal hemisections. Spinal cord and nerves should be examined in transverse and longitudinal (or oblique) orientations. Most Working Group members considered immersion fixation in formalin (for CNS or PNS) or a solution containing acetic acid (for eye), paraffin embedding, and initial evaluation limited to hematoxylin and eosin (H&E)-stained sections to be acceptable for routine microscopic evaluation during general toxicity studies; other neurohistological methods may be undertaken if needed to better characterize H&E findings. Initial microscopic analyses should be qualitative and done with foreknowledge of treatments and doses (i.e., "unblinded"). The pathology report should clearly communicate structures that were assessed and methodological details. Since neuropathologic assessment is only one aspect of general toxicity studies, institutions should retain flexibility in customizing their sampling, processing, analytical, and reporting procedures as long as major neural targets are evaluated systematically.

An oral developmental neurotoxicity study of decabromodiphenyl ether (DecaBDE) in rats.

BACKGROUND: Decabromodiphenyl ether (DecaBDE; CASRN 1163-19-5) is a flame retardant used in a variety of manufactured products. A single oral dose of 20.1 mg/kg administered to mice on postnatal day 3 has been reported to alter motor activity at 2, 4, and 6 months of age. METHODS: To further evaluate these results, a developmental neurotoxicity study was conducted in the most commonly used species for studies of this type, the rat, according to international validated testing guidelines and Good Laboratory Practice Standards. DecaBDE was administered orally via gavage in corn oil to dams from gestation day 6 to weaning at doses of 0, 1, 10, 100, or 1,000 mg/kg/day. Standard measures of growth, development, and neurological endpoints were evaluated in the offspring. Motor activity was assessed at 2 months of age. Additional motor activity assessments were conducted at 4 and 6 months of age. Neuropathology and morphometry evaluations of the offspring were performed at weaning and adulthood. RESULTS: No treatment-related neurobehavioral changes were observed in detailed clinical observations, startle response, or learning and memory tests. No test substance-related changes were noted in motor activity assessments performed at 2, 4, or 6 months of age. Finally, no treatment-related neuropathological or morphometric alterations were found. CONCLUSIONS: Under the conditions of this study, the no-observed-adverse-effect level for developmental neurotoxicity of DecaBDE was 1,000 mg/kg/day, the highest dose tested.

Developmental neurotoxicity study of dietary bisphenol A in Sprague-Dawley rats.

This study was conducted to determine the potential of bisphenol A (BPA) to induce functional and/or morphological effects to the nervous system of F(1) offspring from dietary exposure during gestation and lactation according to the Organization for Economic Cooperation and Development and U.S. Environmental Protection Agency guidelines for the study of developmental neurotoxicity. BPA was offered to female Sprague-Dawley Crl:CD (SD) rats (24 per dose group) and their litters at dietary concentrations of 0 (control), 0.15, 1.5, 75, 750, and 2250 ppm daily from gestation day 0 through lactation day 21. F(1) offspring were evaluated using the following tests: detailed clinical observations (postnatal days [PNDs] 4, 11, 21, 35, 45, and 60), auditory startle (PNDs 20 and 60), motor activity (PNDs 13, 17, 21, and 61), learning and memory using the Biel water maze (PNDs 22 and 62), and brain and nervous system neuropathology and brain morphometry (PNDs 21 and 72). For F(1) offspring, there were no treatment-related neurobehavioral effects, nor was there evidence of neuropathology or effects on brain morphometry. Based on maternal and offspring body weight reductions, the no-observed-adverse-effect level (NOAEL) for systemic toxicity was 75 ppm (5.85 and 13.1 mg/kg/day during gestation and lactation, respectively), with no treatment-related effects at lower doses or nonmonotonic dose responses observed for any parameter. There was no evidence that BPA is a developmental neurotoxicant in rats, and the NOAEL for developmental neurotoxicity was 2250 ppm, the highest dose tested (164 and 410 mg/kg/day during gestation and lactation, respectively).

A 90-day repeated dose oral (gavage) toxicity study of perfluorohexanoic acid (PFHxA) in rats (with functional observational battery and motor activity determinations).

Possible toxic effects of perfluorohexanoic acid (PFHxA) were evaluated when administered orally by gavage to rats at levels up to 200mg/kg/day for 90 days. Lower body weight gains were noted in the 10, 50 and 200mg/kg/day group males (not dose-responsive) throughout dosing. Other changes included lower red blood cell parameters, higher reticulocyte counts and lower globulin in the 200mg/kg/day group males and females, higher liver enzymes in males at 50 and 200mg/kg/day, lower total protein and higher albumin/globulin ratio, and lower cholesterol, calcium in males at 200mg/kg/day. Minimal centrilobular hepatocellular hypertrophy was present in 200mg/kg/day group males and correlated with higher liver weights and slightly higher peroxisome beta oxidation activity at the end of the dosing period. Based on liver histopathology and liver weight changes, the no-observed-adverse-effect level (NOAEL) for oral administration was 50mg/kg/day for males and 200mg/kg/day for females.

Inhalation carcinogenicity study with nickel metal powder in Wistar rats.

Epidemiological studies of nickel refinery workers have demonstrated an association between increased respiratory cancer risk and exposure to certain nickel compounds (later confirmed in animal studies). However, the lack of an association found in epidemiological analyses for nickel metal remained unconfirmed for lack of robust animal inhalation studies. In the present study, Wistar rats were exposed by whole-body inhalation to 0, 0.1, 0.4, and 1.0 mg Ni/m(3) nickel metal powder (MMAD=1.8 microm, GSD=2.4 microm) for 6 h/day, 5 days/week for up to 24 months. A subsequent six-month period without exposures preceded the final euthanasia. High mortality among rats exposed to 1.0 mg Ni/m(3) nickel metal resulted in the earlier termination of exposures in this group. The exposure level of 0.4 mg Ni/m(3) was established as the MTD for the study. Lung alterations associated with nickel metal exposure included alveolar proteinosis, alveolar histiocytosis, chronic inflammation, and bronchiolar-alveolar hyperplasia. No increased incidence of neoplasm of the respiratory tract was observed. Adrenal gland pheochromocytomas (benign and malignant) in males and combined cortical adenomas/carcinomas in females were induced in a dose-dependent manner by the nickel metal exposure. The incidence of pheochromocytomas was statistically increased in the 0.4 mg Ni/m(3) male group. Pheochromocytomas appear to be secondary to the lung toxicity associated with the exposure rather than being related to a direct nickel effect on the adrenal glands. The incidence of cortical tumors among 0.4 mg Ni/m(3) females, although statistically higher compared to the concurrent controls, falls within the historical control range; therefore, in the present study, this tumor is of uncertain relationship to nickel metal exposure. The lack of respiratory tumors in the present animal study is consistent with the findings of the epidemiological studies.

28-Day oral (gavage) toxicity studies of green tea catechins prepared for beverages in rats.

The beneficial health effects associated with drinking green tea are widely considered to be due primarily to tea catechins. Heat treatment of marketed green tea beverages for sterilization causes epimerization and/or polymerization of tea catechins. Safety studies on heat-treated tea catechins are limited. The objective of the present study was to evaluate potential adverse effects, if any, of two standardized green tea catechin (GTC) preparations: one that underwent heat sterilization (GTC-H) and one that was not heat-sterilized (GTC-UH). A decaffeinated preparation of the GTC-H (GTC-HDC) was also evaluated to ascertain if any effects were due to caffeine. The GTC preparations were administered to rats once daily at levels up to 2000 mg/kg/day for 28 days. There were no deaths attributable to the GTC preparations. The clinical condition of the animals, functional observational battery, motor activity, clinical pathology evaluations, organ weights, and gross necropsy findings were unaffected by any of the GTC preparations. GTC-HDC or GTC-UH dosing had no effects on body weights or microscopic findings, whereas lower body weights and food consumption were observed in the 1000 and 2000 mg/kg/day GTC-H group males. The no observed-adverse-effect level (NOAEL) for localized gastric effects for GTC-H was 1000 mg/kg/day. No other target organs were identified. Thus, the NOAEL for systemic toxicity following oral administration was 2000 mg/kg/day for GTC-H, GTC HDC, and GTC-UH under the conditions of this study.

Critical time window for intra-arrest cooling with cold saline flush in a dog model of cardiopulmonary resuscitation.

BACKGROUND: Mild hypothermia improves outcome when induced after cardiac arrest in humans. Recent studies in both dogs and mice suggest that induction of mild hypothermia during cardiopulmonary resuscitation (CPR) greatly enhances its efficacy. In this study, we evaluate the time window for the beneficial effect of intra-arrest cooling in the setting of prolonged CPR in a clinically relevant large-animal model. METHODS AND RESULTS: Seventeen dogs had ventricular fibrillation cardiac arrest no flow of 3 minutes, followed by 7 minutes of CPR basic life support and 50 minutes of advanced life support. In the early hypothermia group (n=9), mild hypothermia (34 degrees C) was induced with an intravenous fluid bolus flush and venovenous blood shunt cooling after 10 minutes of ventricular fibrillation. In the delayed hypothermia group (n=8), hypothermia was induced at ventricular fibrillation 20 minutes. After 60 minutes of ventricular fibrillation, restoration of spontaneous circulation was achieved with cardiopulmonary bypass for 4 hours, and intensive care was given for 96 hours. In the early hypothermia group, 7 of 9 dogs survived to 96 hours, 5 with good neurological outcome. In contrast, 7 of 8 dogs in the delayed hypothermia group died within 37 hours with multiple organ failure (P=0.012). CONCLUSIONS: Early application of mild hypothermia with cold saline during prolonged CPR enables intact survival. Delay in the induction of mild hypothermia in this setting markedly reduces its efficacy. Our data suggest that if mild hypothermia is used during CPR, it should be applied as early as possible.

Developmental neurotoxicity study of styrene by inhalation in Crl-CD rats.

This study was conducted to assess potential adverse functional and/or morphological effects of styrene on the neurological system in the F2 offspring following F0 and F1 generation whole-body inhalation exposures. Four groups of male and female Crl:CD (SD)IGS BR rats (25/sex/group) were exposed to 0, 50, 150, and 500 ppm styrene for 6 hr daily for at least 70 consecutive days prior to mating for the F0 and F1 generations. Inhalation exposure continued for the F0 and F1 females throughout mating and through gestation day 20. On lactation days 1 through 4, the F0 and F1 females received styrene in virgin olive oil via oral gavage at dose levels of 66, 117, and 300 mg/kg/day (divided into three equal doses, approximately 2 hr apart). Inhalation exposure of the F0 and F1 females was re-initiated on lactation day 5 and continued through weaning of the F1 or F2 pups on postnatal day (PND) 21. Developmental landmarks were assessed in F1 and F2 offspring. The neurological development of randomly selected pups from the F2 generation was assessed by functional observational battery, locomotor activity, acoustic startle response, learning and memory evaluations, brain weights and dimension measurements, and brain morphometric and histologic evaluation. Styrene exposure did not affect survival or the clinical condition of the animals. As expected from previous studies, slight body weight and histopathologic effects on the nasal olfactory epithelium were found in F0 and F1 rats exposed to 500 ppm and, to a lesser extent, 150 ppm. There were no indications of adverse effects on reproductive performance in either the F0 or F1 generation. There were exposure-related reductions in mean body weights of the F1 and F2 offspring from the mid and high-exposure groups and an overall pattern of slightly delayed development evident in the F2 offspring only from the 500-ppm group. This developmental delay included reduced body weight (which continued through day 70) and slightly delayed acquisition of some physical landmarks of development. Styrene exposure of the F0 and F1 animals had no effect on survival, the clinical condition or necropsy findings of the F2 animals. Functional observational battery evaluations conducted for all F1 dams during the gestation and lactation periods and for the F2 offspring were unaffected by styrene exposure. Swimming ability as determined by straight channel escape times measured on PND 24 were increased, and reduced grip strength values were evident for both sexes on PND 45 and 60 in the 500-ppm group compared to controls. There were no other parental exposure-related findings in the F2 pre-weaning and post-weaning functional observational battery assessments, the PND 20 and PND 60 auditory startle habituation parameters, in endpoints of learning and memory performance (escape times and errors) in the Biel water maze task at either testing age, or in activity levels measured on PND 61 in the 500-ppm group. Taken together, the exposure-related developmental and neuromotor changes identified in F2 pups from dams exposed to 500 ppm occurred in endpoints known to be both age- and weight-sensitive parameters, and were observed in the absence of any other remarkable indicators of neurobehavioral toxicity. Based on the results of this study, an exposure level of 50 ppm was considered to be the NOAEL for growth of F2 offspring; an exposure level of 500 ppm was considered to be the NOAEL for F2 developmental neurotoxicity.

Suspended animation can allow survival without brain damage after traumatic exsanguination cardiac arrest of 60 minutes in dogs.

BACKGROUND: We have previously shown in dogs that exsanguination cardiac arrest of up to 120 minutes without trauma under profound hypothermia induced by aortic flush (suspended animation) can be survived without neurologic deficit. In the present study, the effects of major trauma (laparotomy, thoracotomy) are explored. This study is designed to better mimic the clinical scenario of an exsanguinating trauma victim, for whom suspended animation may buy time for resuscitative surgery and delayed resuscitation. METHODS: Fourteen dogs were exsanguinated over 5 minutes to cardiac arrest. Flush of saline at 2 degrees C into the femoral artery was initiated at 2 minutes of cardiac arrest and continued until a tympanic temperature of 10 degrees C was achieved. The dogs were then randomized into a control group without trauma (n = 6) or a trauma group (n = 8) that underwent a laparotomy and isolation of the spleen before hemorrhage and then, at the start of cardiac arrest, spleen transection and left thoracotomy. During cardiac arrest, splenectomy was performed. After 60 minutes of no-flow cardiac arrest, reperfusion with cardiopulmonary bypass was followed by intensive care to 72 hours. RESULTS: All 14 dogs survived to 72 hours with histologically normal brains. All control dogs were functionally neurologically intact. Four of eight trauma dogs were also functionally normal. Four had neurologic deficits, although three required prolonged mechanical ventilation because of airway edema and evidence of multiple organ failure. Blood loss from the chest and abdomen was variable and was associated with poor functional outcomes. CONCLUSION: Rapid induction of profound hypothermic suspended animation (tympanic temperature, 10 degrees C) can enable survival without brain damage after exsanguination cardiac arrest of 60 minutes even in the presence of trauma, although prolonged intensive care may be required. This technique may allow survival of exsanguinated trauma victims, who now have almost no chance of survival.

Mild hypothermia during prolonged cardiopulmonary cerebral resuscitation increases conscious survival in dogs.

OBJECTIVE: Therapeutic hypothermia during cardiac arrest and after restoration of spontaneous circulation enables intact survival after prolonged cardiopulmonary cerebral resuscitation (CPCR). The effect of cooling during CPCR is not known. We hypothesized that mild to moderate hypothermia during CPCR would increase the rate of neurologically intact survival after prolonged cardiac arrest in dogs. DESIGN: Randomized, controlled study using a clinically relevant cardiac arrest outcome model in dogs. SETTING: University research laboratory. SUBJECTS: Twenty-seven custom-bred hunting dogs (19-29 kg; three were excluded from outcome evaluation). INTERVENTIONS: Dogs were subjected to cardiac arrest no-flow of 3 mins, followed by 7 mins of basic life support and 10 mins of simulated unsuccessful advanced life support attempts. Another 20 mins of advanced life support continued with four treatments: In control group 1 (n = 7), CPCR was with normothermia; in group 2 (n = 6, 1 of 7 excluded), with moderate hypothermia via venovenous extracorporeal shunt cooling to tympanic temperature 27 degrees C; in group 3 (n = 6, 2 of 8 excluded), the same as group 2 but with mild hypothermia, that is, tympanic temperature 34 degrees C; and in group 4 (n = 5), with normothermic venovenous shunt. After 40 mins of ventricular fibrillation, reperfusion was with cardiopulmonary bypass for 4 hrs, including defibrillation to achieve spontaneous circulation. All dogs were maintained at mild hypothermia (tympanic temperature 34 degrees C) to 12 hrs. Intensive care was to 96 hrs. MEASUREMENTS AND MAIN RESULTS: Overall performance categories and neurologic deficit scores were assessed from 24 to 96 hrs. Regional and total brain histologic damage scores and extracerebral organ damage were assessed at 96 hrs. In normothermic groups 1 and 4, all 12 dogs achieved spontaneous circulation but remained comatose and (except one) died within 58 hrs with multiple organ failure. In hypothermia groups 2 and 3, all 12 dogs survived to 96 hrs without gross extracerebral organ damage (p < .0001). In group 2, all but one dog achieved overall performance category 1 (normal); four of six dogs had no neurologic deficit and normal brain histology. In group 3, all dogs achieved good functional outcome with normal or near-normal brain histology. Myocardial damage scores were worse in the normothermic groups compared with both hypothermic groups (p < .01). CONCLUSION: Mild or moderate hypothermia during prolonged CPCR in dogs preserves viability of extracerebral organs and improves outcome.

Survival without brain damage after clinical death of 60-120 mins in dogs using suspended animation by profound hypothermia.

OBJECTIVES: This study explored the limits of good outcome of brain and organism achievable after cardiac arrest (no blood flow) of 60-120 mins, with preservation (suspended animation) induced immediately after the start of exsanguination cardiac arrest. DESIGN: Prospective experimental comparison of three arrest times, without randomization. SETTING: University research laboratory. SUBJECTS: Twenty-seven custom-bred hunting dogs (17-25 kg). INTERVENTIONS: Dogs were exsanguinated over 5 mins to cardiac arrest no-flow of 60 mins, 90 mins, or 120 mins. At 2 mins of cardiac arrest, the dogs received, via a balloon-tipped catheter, an aortic flush of isotonic saline at 2 degrees C (at a rate of 1 L/min), until tympanic temperature reached 20 degrees C (for 60 mins of cardiac arrest), 15 degrees C (for 60 mins of cardiac arrest), or 10 degrees C (for 60, 90, or 120 mins of cardiac arrest). Resuscitation was by closed-chest cardiopulmonary bypass, postcardiac arrest mild hypothermia (tympanic temperature 34 degrees C) to 12 hrs, controlled ventilation to 20 hrs, and intensive care to 72 hrs. MEASUREMENTS AND MAIN RESULTS: We assessed overall performance categories (OPC 1, normal; 2, moderate disability; 3, severe disability; 4, coma; 5, death), neurologic deficit scores (NDS 0-10%, normal; 100%, brain death), regional and total brain histologic damage scores at 72 hrs (total HDS >0-40, mild; 40-100, moderate; >100, severe damage), and morphologic damage of extracerebral organs. For 60 mins of cardiac arrest (n = 14), tympanic temperature 20 degrees C (n = 6) was achieved after flush of 3 mins and resulted in two dogs with OPC 1 and four dogs with OPC 2: median NDS, 13% (range 0-27%); and median total HDS, 28 (range, 4-36). Tympanic temperature of 15 degrees C (n = 5) was achieved after flush of 7 mins and resulted in all five dogs with OPC 1, NDS 0% (0-3%), and HDS 8 (0-48). Tympanic temperature 10 degrees C (n = 3) was achieved after flush of 11 mins and resulted in all three dogs with OPC 1, NDS 0%, and HDS 16 (2-18). For 90 mins of cardiac arrest (n = 6), tympanic temperature 10 degrees C was achieved after flush of 15 mins and resulted in all six dogs with OPC 1, NDS 0%, and HDS 8 (0-37). For 120 mins of cardiac arrest (n = 7), three dogs had to be excluded. In the four dogs within protocol, tympanic temperature 10 degrees C was achieved after flush of 15 mins. This resulted in one dog with OPC 1, NDS 0%, and total HDS 14; one with OPC 1, NDS 6%, and total HDS 20; one with OPC 2, NDS 13%, and total HDS 10; and one with OPC 3, NDS 39%, and total HDS 22. CONCLUSIONS: In a systematic series of studies in dogs, the rapid induction of profound cerebral hypothermia (tympanic temperature 10 degrees C) by aortic flush of cold saline immediately after the start of exsanguination cardiac arrest-which rarely can be resuscitated effectively with current methods-can achieve survival without functional or histologic brain damage, after cardiac arrest no-flow of 60 or 90 mins and possibly 120 mins. The use of additional preservation strategies should be pursued in the 120-min arrest model.

Antioxidant Tempol enhances hypothermic cerebral preservation during prolonged cardiac arrest in dogs.

The authors are systematically exploring pharmacologic preservation for temporarily unresuscitable exsanguination cardiac arrest in dogs. They hypothesized that the antioxidant Tempol improves cerebral outcome when added to aortic saline flush at the start of cardiac arrest. In study A, no drug (n = 8), Tempol 150 mg/kg (n = 4), or Tempol 300 mg/kg (n = 4) was added to 25 mL/kg saline flush at 24 degrees C (achieving mild cerebral hypothermia) at the start of 20-minute cardiac arrest. In study B, no drug (n = 8) or Tempol 300 mg/kg (n = 7) was added to 50 mL/kg saline flush at 2 degrees C (achieving moderate cerebral hypothermia) at the start of 40-minute cardiac arrest. Cardiac arrest was reversed with cardiopulmonary bypass. Mild hypothermia lasted for 12 hours, controlled ventilation was sustained to 24 hours, and intensive care was provided for up to 72 hours. In study A, overall performance category 1 or 2 (good outcome) was achieved in all eight dogs treated with Tempol compared with three of eight dogs in the control group ( P = 0.03). In study B, good outcome was achieved in all seven dogs treated with Tempol versus only two of 8 dogs in the control group ( P = 0.007). In both studies, neurologic deficit scores were significantly better in the Tempol group, but not total histologic damage scores. At 72 hours, electron paramagnetic resonance spectroscopy of Tempol revealed direct evidence for its presence in the brain. Single- and double-strand DNA damage, nitrotyrosine immunostaining, total antioxidant reserve, and ascorbate acid levels were similar between groups, and thiol levels were decreased after Tempol in study B. The authors conclude that when added to aortic saline flush at the start of prolonged cardiac arrest, the antioxidant Tempol can enhance mild or moderate hypothermic cerebral preservation in terms of improved functional outcome. The mechanisms involved in this beneficial effect need further clarification.