Neuroprotective properties of the benzodiazepine receptor, partial agonist PNU-101017 in the gerbil forebrain ischemia model.

PNU-101017 is a novel, imidazoquinoline amide and benzodiazepine receptor partial agonist that has high affinity for the GABAA receptor subtypes containing the alpha 1 and alpha 3 or alpha 5 subunits. At each of these receptors, the compound is a partial agonist with approximately 50% of the intrinsic activity of the full agonist diazepam. In view of the previously demonstrated anti-ischemic effects of some GABA agonists, the purpose of this study was to determine the ability of PNU-101017 to salvage selectively vulnerable neuronal populations in the gerbil forebrain ischemia model. In an initial set of experiments, male gerbils were pretreated 30 minutes before ischemia induction (5 minutes) with PNU-101017 (3, 10, or 30 mg/kg intraperitoneally) and again 2 hours after reperfusion. In vehicle (0.05 N HC1)-treated gerbils, the loss of hippocampal CA1 neurons at 5 days was 80%. PNU-101017 was shown to produce a dose-related increase in CA1 neuronal survival; at either 10 or 30 mg/kg, the loss of CA1 neurons was only 21% (P < 0.005 versus vehicle). A second experiment, examined the therapeutic window for PNU-101017 using the dose level of 30 mg/kg intraperitoneally. Administration of the first of two doses (2 hours apart) at the time of reperfusion resulted in an identical decrease in CA1 damage at 5 days to that seen with preischemic treatment (P < 0.003 versus vehicle). Even with a delay of the initial dosing until 4 hours after reperfusion, PNU-101017 reduced CA1 neuronal loss to only 32% (P < 0.01 versus vehicle). In a third experiment in which the duration of the ischemic insult was increased to 10 minutes and the brains were not analyzed until 28 days after ischemia, daily PNU-101017 dosing for the full 28 days still significantly preserved CA1 neurons, although less effectively than in the milder 5 minute-ischemia model. The loss of dopaminergic nigrostriatal neurons was also reduced. The neuroprotective effect of PNU-101017 was not associated with any overt CNS depression and it did not correlate with hypothermia. This benzodiazepine-receptor partial agonist may have potential for the treatment of global cerebral ischemia.

Direct measurement of hydroxyl radicals, lipid peroxidation, and blood-brain barrier disruption following unilateral cortical impact head injury in the rat.

We present data correlating the time courses of hydroxyl radical (.OH) production, lipid peroxidation, and blood-brain barrier (BBB) damage following unilateral head injury in the rat. Using a controlled cortical impact device to inflict head injury, we have directly measured brain .OH levels via the salicylate trapping method, and phosphatidylcholine hydroperoxide (PCOOH) levels via the HPLC-chemiluminescence technique, at 5, 30, and 60 min postinjury. These results were then correlated with the time course of BBB disruption, as measured by the extravasation of Evans blue dye (EB) into the injured cortex, over the same time period. In the present study, .OH levels were 62% higher than sham at 5 min postinjury, 25% higher at 15 min (both p < or = 0.05), and no different from sham at 60 min. PCOOH, on the other hand, increased linearly between 5 and 60 min postinjury. Whereas PCOOH levels were 25% greater than sham at 5 min, they were 35% and 52% higher than sham at 30 and 60 min, respectively (both p < or = 0.05 vs sham). Blood-brain barrier disruption followed a similar time course to PCOOH generation, except that the magnitude of the effect was much greater. Whereas EB extravasation was only slightly elevated in the injured cortex at 5 min postinjury, there was nearly an 8-fold increase at 30 min and an 11-fold increase at 60 min (all p < or = 0.05 vs sham). An additional experiment demonstrated that BBB damage can be attenuated by treatment with the 21-aminosteroid lipid peroxidation inhibitor, tirilazad mesylate (U-74006F). Rats were given a single i.v. injection of 3 or 10 mg/kg of U-74006F 5 min postinjury and killed 30 min postinjury. The 10 mg/kg dose of U-74006F reduced EB extravasation 52% (p < 0.025) in comparison to vehicle-treated controls. This is the first study to correlate the time courses of .OH formation, lipid peroxidation, and BBB disruption in injured brain. The results suggest that there is an immediate, posttraumatic burst in .OH formation, followed by a progressive increase in lipid peroxidation and a similar, although slightly delayed, time-related opening of the BBB. The attenuation of BBB damage by U-74006F suggests that this chain of events can be interrupted by administration of an antioxidant/lipid peroxidation inhibitor.

Infant rat model of the shaken baby syndrome: preliminary characterization and evidence for the role of free radicals in cortical hemorrhaging and progressive neuronal degeneration.

Infants subjected to repeated episodes of violent shaking develop brain damage characterized by intracranial hemorrhage and progressive cortical atrophy. We have developed an animal model that mimics this pathological state and investigated its etiology and treatment. Anesthetized male rats, 6 days of age, were subjected to one episode of shaking per day for 3 consecutive days. Separate groups of rats were sacrificed 1 h postinjury on the third day of shaking for HPLC quantification of cortical .OH and vitamin E levels, and histological assessment of cortical hemorrhaging. Additional groups were sacrificed 7 or 14 days postinjury to demonstrate progressive neuronal degeneration via cortical wet weight comparisons. In comparison to noninjured shams, the results indicated that cortical vitamin E and .OH levels rose 53.7% (p < 0.005) and 457.1% (p < 0.001), respectively, in shaken infant rats. Brain histologies revealed a moderate-to-severe degree of cortical hemorrhaging in these animals 1 h postinjury. By 7 and 14 days postinjury, there was a 13.3% and 28.7% (p < 0.0001 vs. sham) loss of cortical tissue in shaken infants, respectively, indicating progressive neuronal degeneration. Treatment with 10 mg/kg (ip) of the 21-aminosteroid antioxidant, tirilazad mesylate, 10 min before and 2 h after each episode of shaking, resulted in a 53.1% attenuation of cortical .OH levels and a 34.9% decrease in brain hemorrhaging (p < 0.05 vs. vehicle). Tirilazad treatment did not, however, significantly effect cortical vitamin E concentrations at 1 h postinjury or the extent of progressive neuronal degeneration at either 7 or 14 days postinjury. The present animal model mimics the brain pathology seen in abused children. Our observation that tirilazad mesylate, an antioxidant-lipid peroxidation inhibitor, significantly reduces cortical .OH levels and brain hemorrhaging in shaken infant rats supports a role for oxygen radicals in the pathophysiology of this type of CNS injury. The failure of tirilazad to block progressive cortical degeneration suggests that mechanisms other than free radicals may be of prime importance in the mediation of this aspect of the pathology.

Biochemistry and pharmacology of lipid antioxidants in acute brain and spinal cord injury.

Oxygen radical-mediated lipid peroxidation (LP) has been suggested increasingly to be an important factor in posttraumatic neuronal degeneration. Thus, numerous studies have evaluated the neuroprotective efficacy of pharmacological agents with lipid antioxidant activity in models of spinal cord and brain injury. Intensive pretreatment of animals with the endogenous lipid peroxyl radical scavenger vitamin E (i.e., alpha-tocopherol) has been shown to decrease posttraumatic spinal cord ischemia and to enhance chronic neurological recovery. However, the slow CNS tissue uptake of vitamin E requires chronic dosing, making it an impractical agent for treatment of acute neural injury. The glucocorticoid steroid, methyl-prednisolone (MP), has been shown to possess significant antioxidant efficacy and, when administered to animals or humans in antioxidant doses, improves chronic neurological recovery after spinal cord injury. This activity of MP is independent of the steroid's glucocorticoid receptor-mediated actions, as evidenced by the efficacy of the novel antioxidant 21-aminosteroids, which are devoid of glucocorticoid activity but have greater antioxidant efficacy than MP. One of these, tirilazad mesylate (U-74006F), has been shown to be effective in animal models of brain and spinal cord injury and is currently the subject of phase II clinical trials. Recently, compounds that combine the amino functionality of the 21-amino-steroids with the peroxyl radical scavenging chromanol portion of vitamin E (i.e., 2-methylaminochromans) also have shown promise as neuroprotective agents. The consistent benefit afforded by antioxidant compounds further supports the concept that LP is an important therapeutic target for acute pharmacological neuroprotection.

Immunocytochemical method for investigating in vivo neuronal oxygen radical-induced lipid peroxidation.

The investigation of oxygen radical-induced lipid peroxidative neuronal damage in the context of acute and chronic neurodegenerative disorders has been largely limited to the use of ex vivo analytical methodologies. These are often fraught with sensitivity or specificity problems, or they are indirect. Furthermore, none of the analytical methods allow precise anatomical identification of the cells that are undergoing peroxidative injury. This paper describes an immunocytochemical method for localization of central nervous system (CNS) lipid peroxidation (LP) that employs a rabbit-derived antibody raised against malondialdehyde (MDA)-modified rabbit serum albumin (RSA). MDA is a breakdown product of peroxidized membrane polyunsaturated fatty acids that avidly binds to cellular proteins. Using the anti-MDA-RSA, we herein illustrate increased MDA-derived immunostaining: (1) in the spinal cord of transgenic familial amyotrophic lateral sclerosis (ALS) mice; and (2) in the selectively vulnerable gerbil hippocampal CA1 region after a 5 min episode of forebrain ischemia and its relationship to the time course of neuronal degeneration.

Age-related phospholipid hydroperoxide levels in gerbil brain measured by HPLC-chemiluminescence and their relation to hydroxyl radical stress.

Phosphatidylcholine hydroperoxide (PCOOH) was directly quantified in the hippocampus, cortex and striatum from young (3 months), middle-aged (15 months) and old (20 to 24 months) gerbils by an HPLC-chemiluminescence assay. PCOOH levels in hippocampus and cortex were found between 8.05 to 8.58 pmol/mg tissue and no statistically significant difference was found across the age groups. In striatum, however, PCOOH levels were significantly higher in middle-aged and old gerbils compared to those in young animals. The regional comparison showed that PCOOH levels were significantly higher in striatum than in cortex or hippocampus for all the age groups. Moreover, this regional difference increased with aging, from approximately 20% in young animals to 30% and 40% in middle-aged and old gerbil striatum. PCOOH to phospholipid ratio is approximately the same for all age groups at the level of 1.5/10,000, although it is slightly lower in the cortex. The hydroxyl radical levels in the brain were also measured by the formation of its salicylate trapped product 2,3-DHBA and used as a measure of oxidative stress. The PCOOH levels was used as a measure of oxygen radical-induced lipid peroxidative damage. PCOOH as a function of hydroxyl radical stress was calculated and expressed as PCOOH/2,3-DHBA, representing the oxidative damage as a function of the level of oxidative stress. It also implies the tissue susceptibility to oxidative stress and the efficiency of the antioxidant systems. In hippocampus and cortex, the ratios are high in young gerbils, decrease at middle-age and significantly increase in the old.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of the lipid peroxidation inhibitor tirilazad mesylate (U-74006F) on gerbil brain eicosanoid levels following ischemia and reperfusion.

The present study measured the production of eicosanoids in the gerbil brain during early reperfusion after either a 3-h unilateral carotid occlusion (UCO, model of focal ischemia) or a 10-min bilateral carotid occlusion (BCO, model of global ischemia). Arachidonic acid (AA) metabolites were examined to determine if pretreatment with the 21-aminosteroid lipid peroxidation inhibitor U-74006F (tirilazad mesylate) could influence postreperfusion synthesis of brain eicosanoids. In the 3-h UCO focal ischemia model, there was an early (5-min) postreperfusion elevation in brain levels of PGF2 alpha, TXB2 and LTC4 (P < 0.05 vs. sham for all three eicosanoids). LTB4 also rose but not significantly. On the other hand, PGE2 and 6-keto-PGF1 alpha tended to decrease during ischemia and at 5-min postreperfusion (P < 0.05 vs. sham for PGE2). Pretreatment with known neuroprotective doses of U-74006F in this model (10 mg/kg i.p. 10 min before and again immediately upon reperfusion) did not affect the increase in PGF2 alpha or TXB2 but significantly blunted the elevations in LTC4 and LTB4. The postreperfusion decrease in PGE2 was also attenuated. In the 10-min BCO global ischemia model, there was also an increase in each of the measured eicosanoids, except LTB4, at 5 min after reperfusion. Pretreatment with U-74006F (10 mg/kg i.p. 10 min before ischemia) selectively decreased the rise in LTC4 but did not significantly affect the other eicosanoids. In contrast, the antioxidant actually caused a significant enhancement of the postreperfusion increase in PGE2 vs. vehicle-treated animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Neuroprotective effects of the dopamine D2/D3 agonist pramipexole against postischemic or methamphetamine-induced degeneration of nigrostriatal neurons.

We have examined the neuroprotective efficacy of the selective dopamine (DA) D2/D3 receptor agonist pramipexole in two models of nigrostriatal (NS) degeneration. The first involves the delayed (28-day) postischemic retrograde NS degeneration that takes place in gerbils following a 10-min episode of bilateral carotid arterial occlusion-induced forebrain ischemia. In vehicle (40% hydroxypropyl cyclodextrin)-treated male gerbils, there was a 40-45% loss of NS cell bodies in the pars compacta and pars reticulata (TH immunohistochemistry and Cresyl violet histochemistry) by 28 days after ischemia/reperfusion. Daily postischemic oral dosing (1 mg/kg p.o., b.i.d., beginning at 1 h after insult) decreased the 28-day postischemic loss of NS DA neurons by 36% (P < 0.01 vs. vehicle-treated). The effect was specific for dopamine neurons since no significant salvage of hippocampal CA1 neurons was observed. In a second model, pramipexole's effects were examined on methamphetamine-induced (10 mg/kg, i.p. X 4, each 2 h apart) NS degeneration in male Swiss-Webster mice. In vehicle-treated mice, there was a 40% loss of NS neurons by day 5. In contrast, pramipexole dosing (1 mg/kg, p.o., 1 h after the last methamphetamine dose, plus daily) attenuated the NS degeneration from 40% to only 8% (P < 0.00001 vs. vehicle). We postulated that pramipexole acts in both of these models to reduce the elevated DA turnover and the associated elevation in hydroxyl radical production secondary to increased MAO activity that could be responsible for oxidative damage to the NS neurons. Indeed, in the gerbil ischemia model, we documented by HPLC-ECD a 135% postreperfusion increase in DA turnover (DOPAC + HVA/DA) at 5 min after reperfusion. Pramipexole at the 1 mg/kg, p.o., dose level was able to significantly reduce the increased DA turnover, but by only 16%. Thus, it is conceivable that other mechanisms may also contribute to pramipexole's dopaminergic neuroprotection. Based on a preliminary examination of pramipexole's oxidation potential, it appears that the compound may possess significant intrinsic antioxidant properties that might contribute to its neuroprotective effects.

Hydroxyl radical generation and nitric oxide synthase activity in Fisher 344 x brown-Norway F1 rat brain.

Hydroxyl radical production in the cerebral cortex, striatum and hippocampus of young (6 months), middle-aged (15 months) and old (28 months) Fisher 344 x Brown-Norway F1 rats was quantitated by measuring the salicylate hydroxyl radical-trapping product, 2,5-dihydroxybenzoic acid. The levels of hydroxyl radical between different age groups in each region examined were not statistically different. In all regions, with the exception of hippocampus from old rats which had a lower content of hydroxyl radical, the levels of hydroxyl radical were higher for middle-aged and old rats than young ones. There was no regional difference in the production of hydroxyl radicals, except that the level was significantly higher in striatum than in cortex for 15-month-old rats. The cerebral cortical nitric oxide synthase activities were similar in the three age groups studied.

U74389G prevents vasospasm after subarachnoid hemorrhage in dogs.

OBJECTIVE: Oxygen-derived free radicals may contribute to vasospasm after the rupture of an intracranial aneurysm through direct vasoconstricting effects occurring within the arterial wall or, secondarily, by causing lipid peroxidation in the subarachnoid erythrocytes with secondary induction of vasoconstriction. U74389G is a potent inhibitor of lipid peroxidation and a scavenger of oxygen-derived free radicals. This study determined the relative contributions of oxygen-derived free radicals and lipid peroxidation to vasospasm in the double-hemorrhage dog model. METHODS: Sixteen dogs underwent baseline (Day 0) cerebral angiography and induction of subarachnoid hemorrhage by two injections of blood into the cisterna magna 2 days apart. They were randomized to receive drug vehicle (n=8) or U74389G (n=8, 3 mg/kg of body weight/d) intravenously. Drug administration and end point analysis were blinded. The end points were angiographic vasospasm, as assessed by comparison of angiograms obtained before and 7 days after subarachnoid hemorrhage, and the levels of malondialdehyde and salicylate hydroxylation products (dihydroxybenzoic acids) in cerebrospinal fluid and of malondialdehyde in subarachnoid blood clots and basilar arteries 7 days after hemorrhage. RESULTS: Comparisons within groups of Day 0 and Day 7 angiograms and between groups of angiograms obtained at Day 7, showed significant vasospasm in animals in the vehicle group (mean+/-standard error, 51%+/-4) but not in the U74389G group (25%+/-11, P < 0.05, unpaired t test). High-pressure liquid chromatographic assays of malondialdehyde and dihydroxybenzoic acids in cerebrospinal fluid, subarachnoid blood clots, and basilar arteries showed no significant differences between groups. CONCLUSION: The significant prevention of vasospasm by U74389G without change in levels of indicators of free radical reactions suggests that the effect of the drug is related to other processes occurring in the arterial wall and that cerebrospinal fluid levels of oxygen radicals and lipid peroxides are not useful markers of vasospasm.

Neuroprotective efficacy of microvascularly-localized versus brain-penetrating antioxidants.

The 21-aminosteroid (lazaroid) tirilazad mesylate has been demonstrated to be a potent inhibitor of lipid peroxidation and to reduce traumatic and ischemic damage in a number of experimental models. Currently, tirilazad is being actively investigated in phase III clinical trials in head and spinal cord injury, ischemic stroke and subarachnoid hemorrhage. This compound acts in large part to protect the microvascular endothelium and consequently to maintain normal blood-brain barrier (BBB) permeability and cerebral blood flow autoregulatory mechanisms. However, due to its limited penetration into brain parenchyma, tirilazad has generally failed to affect delayed neuronal damage to the selectively vulnerable hippocampal CA1 and striatal regions. Recently, we have discovered a new group of antioxidant compounds, the pyrrolopyrimidines, which possess significantly improved ability to penetrate the BBB and gain direct access to neural tissue. Several compounds in the series, such as U-101033E, have demonstrated greater ability to protect the CA1 region in the gerbil transient forebrain ischemia model with a post-ischemic therapeutic window of at least four hours. In addition, U-101033E has been found to reduce infarct size in the mouse permanent middle cerebral artery occlusion model in contrast to tirilazad which is minimally effective. These results suggest that antioxidant compounds with improved brain parenchymal penetration are better able to limit certain types of ischemic brain damage compared to those which are localized in the cerebral microvasculature. On the other hand, microvascularly-localized agents like tirilazad appear to have better ability to limit BBB damage.

Measurement of oxygen radicals and lipid peroxidation in neural tissues.

An important role for oxygen radical-mediated neuronal damage has been implicated in a number of acute and chronic neurodegenerative disorders. Particular interest has centered upon oxygen radical-induced, iron-catalyzed lipid peroxidation (LP) as the principal mechanism of the neuronal injury associated with oxygen radicals. Thus, there has been a growing interest in methods for monitoring increased oxygen radical levels as an index of oxidative stress as well as markers of LP-associated oxidative injury in a number of in vitro and in vivo model systems. This unit provides a detailed description of the salicylate trapping method for the measurement of the most highly reactive oxygen radical, the hydroxyl radical, as well as several direct or indirect methods for assessment of cellular LP in either cell cultures or in in vivo models.

Age-related regional changes in hydroxyl radical stress and antioxidants in gerbil brain.

The levels of hydroxyl radicals and oxidized GSH have been examined as indices of oxidative stress in young (3 months), middle-aged (15 months), and old (20-24 months) gerbil brain hippocampus, cortex, and striatum. The hydroxyl radical stress was estimated by measuring the salicylate hydroxyl radical trapping products 2,5- and 2,3-dihydroxybenzoic acid. The stress was significantly higher in all three brain regions in middle-aged and old gerbils versus young animals (< or = 66.0%). Regional comparisons showed that the stress was significantly higher in cortex than in either the hippocampus or striatum of the middle-aged and old gerbils (< or = 32.0%). The ratio of oxidized to total GSH also increased progressively in middle-aged and old animals in all three brain regions (p < 0.05, < or = 41.1%), further indicating a general age-related increase in oxidative stress. Parallel to this age-related increase in oxidative stress, a significant, albeit slight (8%), decrease in neuronal number in hippocampal CA1 region was observed in both the middle-aged and old animals. Possible differences in antioxidant levels were also examined. Total GSH levels were similar across age groups (variance < 12%). However, the regional comparison showed that it was highest in striatum in all age groups. The levels of alpha-tocopherol (vitamin E) were significantly higher in the middle-aged and old animals in all three regions (< or = 70.4%). Vitamin E was highest in the hippocampus and the differences between the hippocampus and the cortex and striatum increased with age.(ABSTRACT TRUNCATED AT 250 WORDS)

Brain hydroxyl radical generation in acute experimental head injury.

The time course and intensity of brain hydroxyl radical (.OH) generation were examined in male CF-1 mice during the first hour after moderate or severe concussive head injury. Hydroxyl radical production was measured using the salicylate trapping method in which the production of 2,3- and/or 2,5-dihydroxybenzoic acid (DHBA) in brain 15 min after salicylate administration was used as an index of .OH formation. In mice injured with a concussion of moderate severity as defined by the 1-h posttraumatic neurologic recovery (grip score), a 60% increase in 2,5-DHBA formation was observed by 1 min after injury compared with that observed in uninjured mice. The peak in DHBA formation occurred at 15 min after injury (+67.5%; p < 0.02, compared with uninjured). At 30 min, the increase in DHBA lost significance, indicating that the posttraumatic increase in brain .OH formation is a transient phenomenon. In severely injured mice, the peak increase in DHBA (both 2,3- and 2,5-) was observed at 30 min after injury, but also fell off thereafter as with the moderate injury severity. Preinjury dosing of the mice with SKF-525A (50 mg/kg i.p.), an inhibitor of microsomal drug oxidations, did not blunt the posttraumatic increase in salicylate-derived 2,5-DHBA, thus showing that it is not due to increased metabolic hydroxylation. Neither injury nor SKF-525A administration affected the DHBA plasma levels. However, saline perfusion of the injured mice to remove the intravascular blood before brain removal eliminated the injury-induced increase in 2,5-DHBA, but did not affect the baseline levels seen in uninjured mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Protective efficacy of a hypothermic pharmacological agent in gerbil forebrain ischemia.

BACKGROUND AND PURPOSE: The novel muscarinic cholinergic partial agonist U-80816E was tested in the gerbil brief bilateral carotid occlusion ischemia model based on the rationale that the compound's hypothermic properties might afford effective protection of the selectively vulnerable hippocampal CA1 region. METHODS: Male gerbils were subjected to either 10 or 15 minutes of bilateral carotid occlusion, followed by histopathological assessment of the CA1 neuronal survival 7 days later. RESULTS: In saline-treated animals, 10 minutes of bilateral carotid occlusion resulted in a 30.5% loss of CA1 neurons, whereas a 15-minute insult resulted in a 49.6% loss. Administration of U-80816E (6 mg/kg i.p. 30 minutes before bilateral carotid occlusion and again 2 hours after reperfusion) resulted in a significant protective effect of the CA1 neuronal population with either duration of ischemia; neuronal loss was reduced to 12.6% in the milder model (p < 0.05 versus saline-treated) and 24.9% in the more severe model (p < 0.04 versus saline). However, the 6 mg/kg i.p. dose of U-80816E was found to produce a 1.0 degree C decrease in brain temperature (measured with a tympanic temperature probe) at 10 minutes of ischemia compared with that of saline-treated gerbils. At 10 minutes of reperfusion, after the 10-minute episode of ischemia, the brain temperature of the U-80816E-treated gerbils was 2.2 degrees C lower than that of saline-treated animals. When the U-80816E-treated gerbils were subjected to either 10 or 15 minutes of ischemia but placed in a heated chamber that prevented the hypothermic effects, no cerebroprotection was observed. CONCLUSIONS: These results show that the anti-ischemic efficacy of U-80816E is mediated through its hypothermic properties, thus suggesting the feasibility of pharmacologically induced hypothermia as a cerebroprotective approach.

Hydroxyl radical production and lipid peroxidation parallels selective post-ischemic vulnerability in gerbil brain.

The salicylate trapping method was used to investigate the changes in hydroxyl radical (.OH) levels in the selectively vulnerable hippocampus compared to the cerebral cortex of gerbils subjected to a 10 min period of near complete forebrain ischemia. Salicylate-derived 2,5-dihydroxybenzoic acid (2,5-DHBA) was measured in sham-operated animals and at 1, 5, and 15 min of reperfusion. A basal level of 2,5-DHBA was also seen in non-ischemic gerbil brain, both in the hippocampus and cortex. The hippocampal basal level was 160% higher than in the cortex (P < .01). Treatment with the cytochrome P450 inhibitor SKF-525A (50 mg/kg s.c. 30 min before measurement) did not affect this basal level in either hippocampus or cortex, which argues against a contribution of metabolic salicylate hydroxylation as its source. In contrast, pretreatment with the arachidonic acid cyclo-oxygenase inhibitor ibuprofen (20 mg/kg s.c.) decreased (-68.8%) the level of salicylate hydroxylation in the hippocampus, but not the cortex. In animals subjected to 10 min of forebrain ischemia, a selective increase in 2,5-DHBA was observed in the hippocampus at 1 min of reperfusion which subsided by 5 min. No increase in salicylate hydroxylation was apparent in the cortex within the same time frame. The increase in .OH in the hippocampus at 1 min of reperfusion was accompanied by a significant decrease (-15.7%; P < .03) in the hippocampal levels of vitamin E. No loss of vitamin E was observed in the cortex at the same time.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein oxidative damage in a transgenic mouse model of familial amyotrophic lateral sclerosis.

The Gly93-->Ala mutation in the Cu,Zn superoxide dismutase (Cu,Zn-SOD) gene (SOD1) found in some familial amyotrophic lateral sclerosis (FALS) patients has been shown to result in an aberrant increase in hydroxyl radical production by the mutant enzyme that may cause oxidative injury to spinal motor neurons. In the present study, we analyzed the extent of oxidative injury to lumbar and cervical spinal cord proteins in transgenic FALS mice that overexpress the SOD1 mutation [TgN(SOD1-G93A)G1H] in comparison with nontransgenic mice. Total protein oxidation was examined by spectrophotometric measurement of tissue protein carbonyl content by the dinitrophenylhydrazine (DNPH) assay. Four ages were investigated: 30 (pre-motor neuron pathology and clinical disease), 60 (after initiation of pathology, but pre-disease), 100 (approximately 50% loss of motor neurons and function), and 120 (near complete hindlimb paralysis) days. Protein carbonyl content in 30-day-old TgN(SOD1-G93A)G1H mice was twice as high as the level found in age-matched nontransgenic mice. However, at 60 and 100 days of age, the levels were the same. Then, between 100 and 120 days of age, the levels in the TgN(SOD1-G93A)G1H mice increased dramatically (557%) compared with either the nontransgenic mice or transgenic animals that overexpress the wild-type human Cu,Zn-SOD [TgN(SOD1)N29]. The 100-120-day increase in spinal cord protein carbonyl levels was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoretic separation and western blot immunoassay, which enabled the identification of heavily oxidized individual proteins using a monoclonal antibody against DNPH-derivatized proteins. One of the more heavily oxidized protein bands (14 kDa) was identified by immunoprecipitation as largely Cu,Zn-SOD. Western blot comparison of the extent of Cu,Zn-SOD protein carbonylation revealed that the level in spinal cord samples from 120-day-old TgN(SOD1-G93A)G1H mice was significantly higher than that found in age-matched nontransgenic or TgN(SOD1)N29 mice. These results suggest that the increased hydroxyl radical production associated with the G93A SOD1 mutation and/or lipid peroxidation-derived radical species (peroxyl or alkoxyl) causes extensive protein oxidative injury and that the Cu,Zn-SOD itself is a key target, which may compromise its antioxidant function.

Neuroprotective effects of the novel brain-penetrating pyrrolopyrimidine antioxidants U-101033E and U-104067F against post-ischemic degeneration of nigrostriatal neurons.

A 10-min period of bilateral carotid occlusion (BCO)-induced forebrain ischemia in gerbils triggers a delayed retrograde degeneration of 35-40% of dopaminergic nigrostriatal (NS) neurons. The mechanism of the NS degeneration is believed to involve oxygen radical formation secondary to a postischemic increase in dopamine turnover (monoamine oxidase, MAO). If the oxygen radical increase is sufficiently severe, lipid peroxidative injury to the striatal NS terminals is followed by retrograde degeneration of the NS cell bodies. In the present study, we examined whether the novel brain-penetrating lipid antioxidant pyrrolopyrimidine, U-101033E, and its aromatized analog, U-104067F, could attenuate dopaminergic neurodegeneration in this model. Male Mongolian gerbils were dosed with U-101033E (1.5, 5, or 15 mg/kg, by mouth, twice daily) or U-104067F (5 or 15 mg/kg, by mouth, twice daily) for 27 days beginning on the day of the 10-min ischemic insult. Preservation of NS neurons was assessed by tyrosine hydroxylase immunohistochemistry at 28 days. In vehicle (40% hydroxypropyl-beta-cyclodextrin)-treated animals, there was a 42% loss of NS neurons. In contrast, gerbils that received 5 or 15 mg/kg U-101033E twice daily had only a 23% or 28% loss of NS neurons, respectively (P < 0.002 vs. vehicle). U-104067F showed little effect at sparing neurons at the 10 mg/kg dose, but did significantly attenuate neuronal loss to only 20% at the 30 mg/kg dose (P < 0.01 vs. vehicle). The results show that both the pyrrolopyrimidines (U-101033E and U-104067F) significantly attenuate the postischemic loss of NS dopaminergic neurons and further support the involvement of a dopamine metabolism-derived, oxygen radical-induced lipid peroxidative mechanism.