Marie E. Rose

Oxidative stress following traumatic brain injury in rats: Quantitation of biomarkers and detection of free radical intermediates

Abstract: Oxidative stress may contribute to many pathophysiologic changes that occur after traumatic brain injury. In the current study, contemporary methods of detecting oxidative stress were used in a rodent model of traumatic brain injury. The level of the stable product derived from peroxidation of arachidonyl residues in phospholipids, 8‐epi‐prostaglandin F2α, was increased at 6 and 24 h after traumatic brain injury. Furthermore, relative amounts of fluorescent end products of lipid peroxidation in brain extracts were increased at 6 and 24 h after trauma compared with sham‐operated controls. The total antioxidant reserves of brain homogenates and water‐soluble antioxidant reserves as well as tissue concentrations of ascorbate, GSH, and protein sulfhydryls were reduced after traumatic brain injury. A selective inhibitor of cyclooxygenase‐2, SC 58125, prevented depletion of ascorbate and thiols, the two major water‐soluble antioxidants in traumatized brain. Electron paramagnetic resonance (EPR) spectroscopy of rat cortex homogenates failed to detect any radical adducts with a spin trap, 5,5‐dimethyl‐1‐pyrroline N‐oxide, but did detect ascorbate radical signals. The ascorbate radical EPR signals increased in brain homogenates derived from traumatized brain samples compared with sham‐operated controls. These results along with detailed model experiments in vitro indicate that ascorbate is a major antioxidant in brain and that the EPR assay of ascorbate radicals may be used to monitor production of free radicals in brain tissue after traumatic brain injury.

Cyclooxygenase-2 expression is induced in rat brain after kainate-induced seizures and promotes neuronal death in CA3 hippocampus

Cyclooxygenase-2 (COX-2) is the predominant isoform of cyclooxygenase in brain. COX-2 activity produces oxidative stress and results in the production of prostaglandins that have many injurious effects. COX-2 transcription is induced by synaptic activity; therefore, COX-2 activity could contribute to epileptic neuronal injury. To address this hypothesis, COX-2 protein expression and PGE2 production were determined after kainate-induced limbic seizures in rats. The effects of a specific COX-2 inhibitor, SC58125, on neuronal survival and PGE2 concentration in the hippocampus were also determined. COX-2 protein expression was increased in CA3, dentate gyrus, and cortex at 18-24 h after seizures. Hippocampal PGE2 levels were increased at 24 h following seizures, and treatment with the selective COX-2 inhibitor SC58125, 3 mg/kg p.o., attenuated the increase in PGE2 concentration. The survival of CA3 neurons at 7 days after seizures was increased in rats treated with SC58125 compared to vehicle controls. There was no effect of drug treatment on body or brain temperature, nor on the duration or rate of Type IV EEG activity. These results suggest that COX-2 activity can contribute to epileptic neuronal injury and that selective COX-2 inhibitors are neuroprotective.

Opioid neurotoxicity: fentanyl dose-response effects in rats.

Opioids, when administered in large doses, produce brain damage, primarily in the limbic system and association areas in rats.This investigation examined the relationship between opioid dose and severity and frequency of brain damage in rats. Forty male Sprague-Dawley rats were anesthetized with halothane/N2 O and underwent tracheal intubation, mechanical ventilation, arterial/venous cannulation, and insertion of a rectal temperature probe and biparietal electroencephalogram electrodes. After surgery, halothane was discontinued and O2/N2 O 30%/70% was administered for 1 h. Rats were then randomly assigned to one of eight groups. The control group received a loading dose (LD) of 4 mL/kg of 0.9% normal saline solution (NSS) and a maintenance dose (MD) of 4 mL [centered dot] kg-1 [centered dot] h-1 NSS. The other groups were given fentanyl lypophilized and reconstituted in NSS with the LD ranging from 50 to 3200 micro g/kg and the MD from 2 to 128 micro g [centered dot] kg-1 [centered dot] min-1. After 2 h of fentanyl or NSS infusion, all rats received 100% O2 and, when alert, their tracheas were extubated; after 7 days the rats underwent cerebral perfusion fixation, followed by light microscopic evaluation. Histopathologic lesions (primarily eosinophilic neuron degeneration) were subjectively graded by a pathologist unaware of the experimental treatment; the grades were based on the percentage of dead neurons. There were no lesions observed in the brain areas in any of the control or 200-8 (LD, micro g/kg; MD, micro g [centered dot] kg-1 [centered dot] min-1) groups. Eleven of 20 rats in the 400-16, 800-32, 1600-64, and 3200-18 groups showed evidence of brain damage primarily in limbic system structures and association areas (P < 0.05). Our data confirm that fentanyl produces limbic system brain damage in rats, and that the damage occurs over a broad range of doses. (Anesth Analg 1996;83:1298-306)

Alfentanil-induced hypermetabolism, seizure, and histopathology in rat brain.

We evaluated the effect of alfentanil on hippocampal glucose utilization and histopathology associated with alfentanil-induced seizures. Three separate experiments were performed. First, anesthetized, paralyzed Long-Evans rats (n = 15; 5 rats per group) were mechanically ventilated and randomly assigned to three groups: (a) control, 70% N2O and 30% O2continued for 1 h; (b) low-dose alfentanil (150 μg/kg IV bolus), followed by infusion at 15 μg.kg−1.min−1 for 1 h without N2O; or (c) high-dose alfentanil (1000 μg/kg IV bolus), followed by infusion at 100 μg.kg−1.min−1 for 1 h without N2O. After 1 h, [6-14C]glucose was injected intravenously for autoradiography. With high-dose alfentanil, there was increased glucose utilization in the ventral hippocampus and the lateral septal nucleus. In the second experiment, anesthetized, paralyzed Sprague-Dawley rats (n = 12; 4 rats per group) were mechanically ventilated, underwent insertion of hippocampal depth electrodes, and were randomly assigned to three groups: (a) control, 70% N2O and 30% O2;(b) low-dose alfentanil (150 μ/kg IV bolus), with 70% N2O and 30% O2; or (c) high-dose alfentanil (1000 μg/kg IV bolus), with 70% N2O and 30% O2. An epileptiform pattern was observed on hippocampal and subdermal electroencephalographic recordings in both alfentanil groups. In the third experiment, anesthetized, paralyzed Sprague-Dawley rats (n = 20) were mechanically ventilated and assigned to two groups: (a) control, 70% N2O and 30% O2 (n = 5) or 100% O2 (n = 5) continued for 1 h; or (b) alfentanil (2000 μg/kg IV bolus), followed by infusion at 33.3 μg.kg−1.min−1 for 1 h with 100% O2. After tracheal extubation, the rats recovered overnight. Light-microscopic evaluation revealed hippocampal or amygdaloid damage in 6 of the 10 alfentanil-treated rats. High doses of alfentanil administered to rats can produce limbic system seizure activity with hypermetabolism associated with neuropathologic lesions.

Disruption of Bax Protein Prevents Neuronal Cell Death but Produces Cognitive Impairment in Mice following Traumatic Brain Injury

Apoptosis contributes to delayed neuronal cell death in traumatic brain injury (TBI). To investigate if Bax plays a role in neuronal cell death and functional outcome after TBI, Bax gene disrupted (null) mice and wild-type (WT) controls were subjected to the controlled cortical impact (CCI) model of TBI. Motor function in WT and Bax null mice was evaluated using the round beam balance and the wire grip test on days 0-5. Spatial memory was assessed using a Morris Water Maze adopted for mice on days 14-18 post-injury. For histopathological analysis, animals were sacrificed 24 h and 21 days post-injury. In all three behavioral tests, the sham and TBI-injured Bax null mice performed significantly worse than their WT sham and TBI-injured counterparts. However, Bax null mice exhibited a higher percentage of surviving neurons in the CA1 and CA3 regions of hippocampus measured at 21 days post-injury. At 24 h after trauma, Bax null mice had fewer TUNEL positive cells in the CA1 and dentate regions of hippocampus as compared to WT mice, suggesting that deletion of the Bax gene ameliorates hippocampal cell death after TBI. Sham-operated Bax null mice had significantly greater brain volume as compared to WT mice. Thus, it is possible that Bax deficiency in the transgenic mice produces developmental behavioral effects, perhaps due to Baxs role in regulating cell death during development.

Modification of ubiquitin-C-terminal hydrolase-L1 by cyclopentenone prostaglandins exacerbates hypoxic injury.

Cyclopentenone prostaglandins (CyPGs), such as 15-deoxy-Δ(12,14) -prostaglandin J(2) (15d-PGJ(2)), are active prostaglandin metabolites exerting a variety of biological effects that may be important in the pathogenesis of neurological diseases. Ubiquitin-C-terminal hydrolase L1 (UCH-L1) is a brain specific deubiquitinating enzyme whose aberrant function has been linked to neurodegenerative disorders. We report that [15d-PGJ(2)] detected by quadrapole mass spectrometry (MS) increases in rat brain after temporary focal ischemia, and that treatment with 15d-PGJ(2) induces accumulation of ubiquitinated proteins and exacerbates cell death in normoxic and hypoxic primary neurons. 15d-PGJ(2) covalently modifies UCH-L1 and inhibits its hydrolase activity. Pharmacologic inhibition of UCH-L1 exacerbates hypoxic neuronal death while transduction with a TAT-UCH-L1 fusion protein protects neurons from hypoxia. These studies indicate that UCH-L1 function is important in hypoxic neuronal death and that excessive production of CyPGs after stroke may exacerbate ischemic injury by modification and inhibition of UCH-L1.

Cyclooxygenase-2 Activity Following Traumatic Brain Injury in the Developing Rat

Cyclooxygenase (COX) is the rate-limiting enzyme in the production of prostaglandins. COX-2, the predominant COX isoform in brain, is induced by synaptic activity. COX-2–generated prostaglandins are important regulators for a range of activities under physiologic conditions. However, under pathologic conditions, COX-2 activity can produce reactive oxygen species and toxic prostaglandin metabolites that can exacerbate brain injury. In this study, we examine the developmental production of COX-2 and test the ability of a COX-2 inhibitor, SC58125, to attenuate traumatic brain injury in developing rats. We show that constitutive COX-2 concentration is low (0.5-fold adult concentration) during the first postnatal week and then increases to 3-fold of adult levels between days 14–60. Controlled cortical impact (CCI) at postnatal day (PND) 17, but not PND 7, caused an additional 3-fold increase in COX-2 content and was associated with an increase in the COX-2 product PGE2. Treatment with the COX-2 inhibitor SC58125 in PND17 rats exposed to CCI attenuated the rise in PGE2 but did not attenuate lesion volume or improve performance in the Morris water maze.

Prolonged opportunity for neuroprotection in experimental stroke with selective blockade of cyclooxygenase-2 activity

The post-treatment effects of the selective cyclooxygenase (COX)-2 inhibitor, valdecoxib, were investigated in a rat model of temporary focal ischemia. Valdecoxib reduced basal brain prostaglandin E(2) concentrations at dosages that did not affect serum thromboxane B(2), consistent with a selective COX-2 effect. Temporary focal cerebral ischemia was produced in rats by middle cerebral artery occlusion for 90 min. There was increased expression of COX-2 protein detected by Western blot and immunocytochemistry within neurons in the ischemic cortex at 4 and 24 h after ischemia. Rats were treated with vehicle or valdecoxib 15 min before or 1.5, 3 and 6 h after cerebral ischemia. Rats were sacrificed and brain infarction volume determined 24 h after ischemia. Valdecoxib treatment was associated with a decrease in infarction volume when administered 15 min before, and 1.5 or 3 h but not 6 h after cerebral ischemia. There were no differences in physiological parameters during the procedure. Valdecoxib administered at 1.5 h after ischemia significantly reduced the concentrations of prostaglandin E(2) in ischemic penumbral cortex as compared to the vehicle-treated group and contralateral non-ischemic cortex. These results suggest that COX-2 inhibition with valdecoxib is effective when initiated both before and after middle cerebral artery occlusion.

Neuronal Cyclooxygenase-2 Activity and Prostaglandins PGE2, PGD2, and PGF2α Exacerbate Hypoxic Neuronal Injury in Neuron-enriched Primary Culture

Cyclooxygenase-2 (COX-2) activity has been implicated in the pathogenesis of cerebral ischemia. To determine whether COX-2 activity within the neuron itself exacerbates hypoxic neuronal injury, neuron-enriched cultures were subjected to anoxia. Treatment with COX-2 selective antagonists decreased cell death. Neurons cultured from homozygous COX-2 gene disrupted mice were resistant to hypoxia compared to those of heterozygotes. Infection of primary neurons with AAV expressing COX-2 exacerbated cell death compared to neurons infected with enhanced green fluorescent protein (EGFP) control vector. Addition of PGE2, PGD2 or PGF2α to the medium exacerbated injury, suggesting that the deleterious effects of COX-2 overexpression in hypoxia could be mediated by direct receptor mediated effects of prostaglandins. Overexpression of COX-2 did not increase expression of cyclin D1 or phosphoretinoblastoma protein (pRb), or cleavage of caspase 3 suggesting that this cell cycle mechanism does not mediate COX-2 toxicity in this model.

Soluble epoxide hydrolase inhibitor trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid is neuroprotective in rat model of ischemic stroke.

Soluble epoxide hydrolase (sEH) diminishes vasodilatory and neuroprotective effects of epoxyeicosatrienoic acids by hydrolyzing them to inactive dihydroxy metabolites. The primary goals of this study were to investigate the effects of acute sEH inhibition by trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (t-AUCB) on infarct volume, functional outcome, and changes in cerebral blood flow (CBF) in a rat model of ischemic stroke. Focal cerebral ischemia was induced in rats for 90 min followed by reperfusion. At the end of 24 h after reperfusion rats were euthanized for infarct volume assessment by triphenyltetrazolium chloride staining. Brain cortical sEH activity was assessed by ultra performance liquid chromatography-tandem mass spectrometry. Functional outcome at 24 and 48 h after reperfusion was evaluated by arm flexion and sticky-tape tests. Changes in CBF were assessed by arterial spin-labeled-MRI at baseline, during ischemia, and at 180 min after reperfusion. Neuroprotective effects of t-AUCB were evaluated in primary rat neuronal cultures by Cytotox-Flour kit and propidium iodide staining. t-AUCB significantly reduced cortical infarct volume by 35% (14.5 ± 2.7% vs. 41.5 ± 4.5%), elevated cumulative epoxyeicosatrienoic acids-to-dihydroxyeicosatrienoic acids ratio in brain cortex by twofold (4.40 ± 1.89 vs. 1.97 ± 0.85), and improved functional outcome in arm-flexion test (day 1: 3.28 ± 0.5 s vs. 7.50 ± 0.9 s; day 2: 1.71 ± 0.4 s vs. 5.28 ± 0.5 s) when compared with that of the vehicle-treated group. t-AUCB significantly reduced neuronal cell death in a dose-dependent manner (vehicle: 70.9 ± 7.1% vs. t-AUCB0.1μM: 58 ± 5.11% vs. t-AUCB0.5μM: 39.9 ± 5.8%). These findings suggest that t-AUCB may exert its neuroprotective effects by affecting multiple components of neurovascular unit including neurons, astrocytes, and microvascular flow.