Jo A. Oostveen

Relationship of oxygen radical‐induced lipid peroxidative damage to disease onset and progression in a transgenic model of familial ALS

Transgenic mice that overexpress a mutated human CuZn superoxide dismutase (SOD1) gene (gly93→ala) found in some patients with familial ALS (FALS) have been shown to develop motor neuron disease, as evidenced by motor neuron loss in the lumbar and cervical spinal regions and a progressive loss of voluntary motor activity. The mutant Cu,Zn SOD exhibits essentially normal dismutase activity, but in addition, generates toxic oxygen radicals as a result of an enhancement of a normally minor peroxidase reaction. In view of the likelihood that the manifestation of motor neuron disease in the FALS transgenic mice involves an oxidative injury mechanism, the present study sought to examine the extent of lipid peroxidative damage in the spinal cords of the TgN(SOD1‐G93A)G1H mice over their life span compared to nontransgenic littermates or transgenic mice that overexpress the wild‐type human Cu,Zn SOD (TgN(SOD1)N29). Lipid peroxidation was investigated in terms of changes in vitamin E and malondialdehyde (MDA) levels measured by HPLC methods and by MDA‐protein adduct immunoreactivity. Four ages were investigated: 30 days (pre‐motor neuron pathology and clinical disease); 60 days (after initiation of pathology, but predisease); 100 days (approximately 50% loss of motor neurons and function); and 120 days (near complete hindlimb paralysis). Compared to nontransgenic mice, the TgN(SOD1‐G93A)G1H mice showed blunted accumulation of spinal cord vitamin E and higher levels of MDA (P < 0.05 at 30 and 60 days) over the 30–120 day time span. In the TgN(SOD1)N29 mice, levels of MDA at age 120 days were significantly lower than in either the TgN(SOD1‐G93A)G1H or nontransgenic mice. MDA‐protein adduct immunoreactivity was also significantly increased in the lumbar spinal cord at age 30, 100, and 120 days, and in the cervical cord at 100 and 120 days. The results clearly demonstrate an increase in spinal cord lipid peroxidation in the FALS transgenic model, which precedes the onset of ultrastructural or clinical motor neuron disease. However, the greatest intensity of actual motor neuronal lipid peroxidative injury is associated with the active phase of disease progression. These findings further support a role of oxygen radical‐mediated motor neuronal injury in the pathogenesis of FALS and the potential benefits of antioxidant therapy. J. Neurosci. Res. 53:66–77, 1998.

Treatment of mice with methamphetamine produces cell loss in the substantia nigra.

Studies were conducted to determine if treatment of mice with methamphetamine (METH) would produce a loss of dopaminergic cells in the substantia nigra. The number of TH+/Nissl-stained was significantly decreased in both Swiss-Webster (S-W) and C57bl mice (approx. cell loss of 40% and 45%, respectively) 5-8 days after treatment with METH. In these same mice there was a corresponding decrease in neostriatal dopamine (DA) content (90% and 92%, respectively). In parallel studies, treatment with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) produced similar neuropathological effects. The finding that nigral cell loss occurs after METH treatment indicates that the METH-treated mouse may be a very relevant model of Parkinsons disease (PD).

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, pramipexoles 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 pramipexoles dopaminergic neuroprotection. Based on a preliminary examination of pramipexoles oxidation potential, it appears that the compound may possess significant intrinsic antioxidant properties that might contribute to its neuroprotective effects.

Increased Amyloid Protein Precursor and Apolipoprotein E Immunoreactivity in the Selectively Vulnerable Hippocampus Following Transient Forebrain Ischemia in Gerbils

The postischemic time course of amyloid protein precursor (APP), beta-amyloid protein (beta-AP), and apolipoprotein E (APO-E) immunoreactivity were examined in comparison to neuronal necrosis in the selectively vulnerable hippocampal CA1 region of gerbils subjected to 10 min of bilateral carotid occlusion-induced forebrain ischemia. Loss of 90% of the CA neurons occurred between 24 and 72 h after ischemia, after which no further neuronal necrosis was observed. At 24 h postischemia, there was a decrease in APP and beta-AP immunostaining in the CA1 region. However, beginning at 2 days, there was a dramatic increase in the staining for both proteins. This coincided with a progressive increase in the expression of APO-E and glial fibrillary acidic protein (GFAP) staining between Days 2 and 6, indicative of an activation of astrocytic protein synthesis. Each of the immunocytochemical markers also increased in the less vulnerable CA3 region. However, the peak increase in that region was much less than that in CA1 and, by 7 days, only the GFAP staining remained significantly above the sham level. It has been shown that the E4 isoform of APO-E, when oxidized, avidly binds to beta-AP and thus increases the likelihood of co-beta-AP/APO-E deposition. Therefore, it is postulated that the increased levels of amyloid proteins coincident with an increased production of APO-E in response to ischemic neuronal necrosis may provide conditions that are favorable for the postischemic formation of amyloid deposits.

Neuroprotective effects of the dopamine agonists pramipexole and bromocriptine in 3-acetylpyridine-treated rats

The neuroprotective effects of pramipexole, a dopamine agonist, were investigated in 3-acetylpyridine (3-AP)-treated Wistar rats. Bromocriptine was used as a reference compound to compare the results obtained with pramipexole. A significant reduction (P < 0.01) in cerebellar cGMP and ATP was observed 96 h after treatment with 3-AP (500 micromol/kg, i.p.). Both pramipexole and bromocriptine significantly attenuated 3-AP-induced reduction in cerebellar cGMP and ATP. Consistent with the neurochemical effect, both pramipexole and bromocriptine prevented 3-AP-induced loss of motor coordination. 3-Acetylpyridine produced a significant (P < 0.01) loss of neurons in the inferior olivary nucleus. Treatment with pramipexole and bromocriptine partially, but significantly (P < 0.01), prevented the loss of inferior olivary neurons. There was no reduction in the temperature of the animals, indicating that hypothermia was not responsible for neuroprotection.

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.

Induction of cobalt accumulation by excitatory amino acids within neurons of the hippocampal slice

Computer-assisted image analysis was used to establish the dose response of excitatory amino acid (EAA) analogs on the induction of cobalt accumulation within pyramidal and granule cell neurons in 400 microns slices of gerbil hippocampus. Slices were incubated 20 min at 22 degrees C in a solution containing 5 mM CoCl2 and 0-1,000 microM EAA analog. The cobalt was visualized by development in (NH4)2S, and the slices were digitized for quantitative densitometry. Kainic acid (KA) had the largest effect and induced cobalt accumulation in the dentate gyrus and CA1, 180% and 150% above control, respectively, with an ED50 = 30 microM. alpha-Amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) induced accumulations of cobalt in CA1 and hilar neurons 130% above control with an ED50 = 30 microM, but had little effect on dentate granule cells. The accumulations induced by KA and AMPA were blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), but not by DL-2-amino-5-phosphonovaleric acid (AP5) or voltage-dependent calcium channel blockers. N-Methyl-D-aspartate (NMDA) induced accumulation in the dentate and CA1 150% above control in a pattern similar to KA, but with an ED50 of 100 microM. The accumulation was blocked by both AP5 and CNQX. These data indicate that cobalt-permeable, receptor-activated divalent cation channels are differentially distributed within the gerbil hippocampus, and have differential sensitivities to non-NMDA agonists. The localization of KA-activated, cobalt-permeable channels appears to be coincident with the flop form of the AMPA-selective calcium-permeable glutamate receptor-activated channel.(ABSTRACT TRUNCATED AT 250 WORDS)

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‐β‐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. J. Neurosci. Res. 47:650–654, 1997.

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.

Inhibition of lipid peroxidation attenuates axotomy-induced apoptotic degeneration of facial motor neurons in neonatal rats

The purpose of this study was to investigate the role of oxygen radical‐induced lipid peroxidative mechanisms in trophic deprivation‐induced apoptotic motor neuronal degeneration by testing the ability of the 21‐aminosteroid lipid peroxidation inhibitor tirilazad mesylate (U‐74006F) to attenuate the retrograde degeneration of facial motor neurons following axotomy in 14‐day‐old rat pups. On day 0, the right facial nerve of each rat was transected at its point of exit from the stylomastoid foramen. Pups were treated orally with either 10 or 30 mg/kg U‐74006F or cyclodextrin vehicle 10 min before axotomy, and post‐treated once a day from days 1 to 6, and then once every other day from days 8 to 21. The rats were sacrificed 3 weeks post‐transection and the surviving motor neurons, identified through choline acetyl‐transferase immunocytochemistry, were counted in three regions (planes) in the facial nucleus. In vehicle‐treated rats, 56.2% (region A), 50.6% (region B), and 57.4% (region C) of the motor neurons in the ipsilateral facial nucleus survived 21 days following facial nerve axotomy in comparison to the non‐axotomized contralateral nucleus (P < 0.0001). Treatment with 10 mg/kg U‐74006F significantly enhanced motor neuron survival in regions B and C to 72.8% (P < 0.01) and 66.7% (P < 0.02%), respectively. The 30 mg/kg dose level also increased survival rates to 64.2% (P < 0.02) and 67.9% (P < 0.01), respectively. A second experiment demonstrated that oral dosing with U‐74006F (30 mg/kg), when limited to the first 5 days after axotomy, also significantly blunted retrograde degeneration measured at 21 days post‐axotomy. The efficacy of the lipid peroxidation inhibitor U‐74006F in protecting a portion of the facial motor neuron pool from post‐axotomy degeneration suggests that lipid peroxidation may play a mechanistic role in trophic deprivation‐induced apoptotic neuronal death.