Araceli Diaz-Ruiz

Copper and Copper Proteins in Parkinson’s Disease

Copper is a transition metal that has been linked to pathological and beneficial effects in neurodegenerative diseases. In Parkinsons disease, free copper is related to increased oxidative stress, alpha-synuclein oligomerization, and Lewy body formation. Decreased copper along with increased iron has been found in substantia nigra and caudate nucleus of Parkinsons disease patients. Copper influences iron content in the brain through ferroxidase ceruloplasmin activity; therefore decreased protein-bound copper in brain may enhance iron accumulation and the associated oxidative stress. The function of other copper-binding proteins such as Cu/Zn-SOD and metallothioneins is also beneficial to prevent neurodegeneration. Copper may regulate neurotransmission since it is released after neuronal stimulus and the metal is able to modulate the function of NMDA and GABA A receptors. Some of the proteins involved in copper transport are the transporters CTR1, ATP7A, and ATP7B and the chaperone ATOX1. There is limited information about the role of those biomolecules in the pathophysiology of Parkinsons disease; for instance, it is known that CTR1 is decreased in substantia nigra pars compacta in Parkinsons disease and that a mutation in ATP7B could be associated with Parkinsons disease. Regarding copper-related therapies, copper supplementation can represent a plausible alternative, while copper chelation may even aggravate the pathology.

Antioxidant, antiinflammatory and antiapoptotic effects of dapsone in a model of brain ischemia/reperfusion in rats

Although dapsone (4,4′‐diaminodiphenylsulfone) has been described as a neuroprotective agent in occlusive focal ischemia in rats, its mechanism of action is still unknown. To explore this mechanism, oxidative, inflammatory and apoptotic processes were evaluated in the striatum of adult rats using a model of ischemia‐reperfusion (I/R), either with or without dapsone treatment. Male Wistar rats were submitted to transient middle cerebral artery occlusion for 2 hr, followed by reperfusion. Rats were dosed either with dapsone (12.5 mg/kg i.p.) or vehicle 30 min before or 30 min after the ischemia onset. Lipid peroxidation (LP) and nitrotyrosine contents were measured 22 hr after reperfusion, and myeloperoxidase activity was evaluated 46 hr after I/R. Different markers for apoptosis and necrosis were also evaluated both at 24 and 72 hr after I/R experimental procedure. LP increased by 37% in ischemic animals vs controls, and this effect was reversed by dapsone treatments. A similar effect was observed regarding nitrotyrosine striatal contents. Myeloperoxidase activity, a marker of inflammatory response, increased 3.7‐fold in ischemic animals vs. control rats, and dapsone treatment antagonized that effect. Although apoptosis was increased by the effect of ischemia at both evaluation times, dapsone antagonized that effect only at 72 hr after surgery. Dapsone antagonized all of the I/R end points measured, showing a remarkable ability to decrease markers of damage through antioxidant, antiinflammatory, and anti‐apoptotic effects.

Early metabolic reactivation versus antioxidant therapy after a traumatic spinal cord injury in adult rats.

Disability after traumatic spinal cord injury (TSCI) results from physical trauma and from “secondary mechanisms of injury” such as low metabolic energy levels, oxidative damage and lipid peroxidation. In order to prove if early metabolic reactivation is a better therapeutic option than antioxidant therapy in the acute phase of TSCI, spinal cord contusions were performed in adult rats using a well‐characterized weight drop technique at thoracic 9 level. After TSCI, pyrophosphate of thiamine or non‐degradable cocarboxylase (NDC) enzyme was used to maintain energy levels, antioxidants such as superoxide dismutase and catalase (ANT) were used to decrease oxidative damage and methylprednisolone (MP), which has both therapeutic properties, was used as a control. Rats were divided into one sham group and six with TSCI; one of them received no treatment, and the rest were treated with NDC, MP, NDC + MP, NDC + ANT or ANT. The ANT group decreased lactate and creatine phosphokinase levels and increased the amount of preserved tissue (morphometric analysis) as well as functional recovery (Basso, Beattie and Bresnahan or BBB motor scale). In contrast, NDC treatment increased lipid peroxidation, measured through thiobarbituric acid reactive substances (TBARS) levels, as well as spinal cord tissue destruction and functional deficit. Early metabolic reactivation after a TSCI may be deleterious, while natural early metabolic inhibition may not be a “secondary mechanism of injury” but a “secondary neuroprotective response”. While increased antioxidant defence after a TSCI may currently be an ideal therapeutic strategy, the usefulness of metabolic reactivation should be tested in the sub‐acute or chronic phases of TSCI and new strategies must continue to be tested for the early ones.

Protective effect of cyclosporin-A in spinal cord injury: an overview.

Cyclosporin-A (CsA) is a potent and selective immunosupressive agent that, due to its mechanism of action, may be used to inhibit both the inflammatory reaction and the synthesis of nitric oxide (NO), a well-known neurotoxic agent. By these means CsA may diminish overproduction of free radicals and secondarily, lipid peroxidation (LP), both observed after acute spinal cord (SC) injury. Studies performed on reliable experimental models, using a well-standardized CsA dosing scheme, showed that a low dose of this drug inhibits the expression and activity of constitutive and inducible nitric oxide synthases (NOS), two enzymes strongly involved in the production of NO after SCI. Likewise, this compound inhibits LP. This inhibition is equivalent to the one induced by methylprednisolone (MP) at a high dose, but without the deleterious effects of the latter upon the survival of the animals. Moreover, inhibition of LP by CsA significantly correlates with a decrease in the demyelination process at the epicenter of the lesion, a significant survival of neurons in the red nucleus and enhanced motor recovery in animals submitted to a severe SC contusion. CsA acts as a neuroprotector agent after SC injury; hence, this drug may be useful in the treatment of acute SCI. CsA deserves further study in experimental animal models and in humans.

Delayed administration of dapsone protects from tissue damage and improves recovery after spinal cord injury.

After spinal cord injury (SCI), a complex cascade of pathophysiological processes increases the primary damage. The inflammatory response plays a key role in this pathology. Recent evidence suggests that myeloperoxidase (MPO), an enzyme produced and released by neutrophils, is of special importance in spreading tissue damage. Dapsone (4,4′‐diaminodiphenylsulfone) is an irreversible inhibitor of MPO. Recently, we demonstrated, in a model of brain ischemia/reperfusion, that dapsone has antioxidant, antiinflammatory, and antiapoptotic effects. The effects of dapsone on MPO activity, lipid peroxidation (LP) processes, motor function recovery, and the amount of spared tissue were evaluated in a rat model of SCI. MPO activity had increased 24.5‐fold 24 hr after SCI vs. the sham group, and it had diminished by 38% and 19% in the groups treated with dapsone at 3 and 5 hr after SCI, respectively. SCI increased LP by 45%, and this increase was blocked by dapsone. In rats treated with dapsone, a significant motor function recovery (Basso‐Beattie‐Bresnahan score, BBB) was observed beginning during the first week of evaluation and continuing until the end of the study. Spontaneous recovery 8 weeks after SCI was 9.2 ± 1.12, whereas, in the dapsone‐treated groups, it reached 13.6 ± 1.04 and 12.9 ± 1.17. Spared tissue increased by 42% and 33% in the dapsone‐treated groups (3 and 5 hr after SCI, respectively) vs. SCI without treatment. Dapsone significantly prevented mortality. The results show that inhibition of MPO by dapsone significantly protected the spinal cord from tissue damage and enhanced motor recovery after SCI.

Cyclosporin-A inhibits inducible nitric oxide synthase activity and expression after spinal cord injury in rats

Nitric oxide is generated from l-arginine by a family of three distinct nitric oxide synthase (NOS) enzymes playing a crucial role in the physiopathology of spinal cord injury (SCI). Cyclosporin-A (CsA), an immunosupressive agent, may be used to inhibit the activity of iNOS and perhaps to protect against neural tissue destruction. Rats were submitted to SCI by contusion, and killed 4, 24 and 72 h after lesion. Results showed an increase in the activity of iNOS at 72 h after the SCI, inhibited by CsA (2.5 mg/kg) administered 12 h after trauma. iNOS Western blot assay showed an increase in the expression of iNOS after trauma, also antagonized by CsA administration.

Plasma polypyrrole implants recover motor function in rats after spinal cord transection

We studied the use of three biocompatible materials obtained by plasma polymerization of pyrrole (PPy), pyrrole doped with iodine (PPy/I) and a copolymer formed with pyrrole and polyethylene glycol (PPy/PEG), implanted, separately, after a complete spinal cord transection in rats. Motor function assessed with the BBB scale and somatosensory evoked potentials (SEPs) in the implanted rats were studied. Results showed that the highest motor recovery was obtained in rats with PPy/I implants. They also showed a significant reduction in the latency of SEPs. Histological analyses showed no signs of implant rejection; on the contrary, implants based on PPy improved the SEPs conduction and motor function after lesion.

Nitric oxide synthase immunolocalization and expression in the rat hippocampus after sub-acute lead acetate exposure in rats

Interference with nitric oxide production is a possible mechanism for lead neurotoxicity. In this work, we studied the effects of sub-acute lead administration on the distribution of NOS isoforms in the hippocampus with respect to blood and hippocampal lead levels. Lead acetate (125, 250 and 500ppm) was given via drinking water to adult male Wistar rats for 14 days. We determined blood and hippocampal lead levels by atomic absorption spectrophotometry. Antibodies against three isoforms of NOS were used to analyze expression and immunolocalization using western blotting and immunohistochemistry, respectively. Blood and hippocampal lead levels were increased in a dose-dependent manner in groups treated with lead acetate. We found diminished expression and immunoreactivity of nNOS and eNOS at 500ppm as compared to the control group. No expression and immunoreactivity was observed in hippocampus for iNOS. The observed high levels of lead in the blood reflect free physiological access to this metal to the organism and were related to diminished expression and immunoreactivity for nNOS and eNOS.

Metallothionein-II Inhibits Lipid Peroxidation and Improves Functional Recovery after Transient Brain Ischemia and Reperfusion in Rats

After transient cerebral ischemia and reperfusion (I/R), damaging mechanisms, such as excitotoxicity and oxidative stress, lead to irreversible neurological deficits. The induction of metallothionein-II (MT-II) protein is an endogenous mechanism after I/R. Our aim was to evaluate the neuroprotective effect of MT-II after I/R in rats. Male Wistar rats were transiently occluded at the middle cerebral artery for 2 h, followed by reperfusion. Rats received either MT (10 μg per rat i.p.) or vehicle after ischemia. Lipid peroxidation (LP) was measured 22 h after reperfusion in frontal cortex and hippocampus; also, neurological deficit was evaluated after ischemia, using the Longa scoring scale. Infarction area was analyzed 72 hours after ischemia. Results showed increased LP in frontal cortex (30.7%) and hippocampus (26.4%), as compared to control group; this effect was fully reversed by MT treatment. Likewise, we also observed a diminished neurological deficit assessed by the Longa scale in those animals treated with MT compared to control group values. The MT-treated group showed a significant (P < 0.05) reduction of 39.9% in the infarction area, only at the level of hippocampus, as compared to control group. Results suggest that MT-II may be a novel neuroprotective treatment to prevent ischemia injury.

Additive effect of dl-penicillamine plus Prussian blue for the antidotal treatment of thallotoxicosis in rats

DL-penicillamine (DL-P) and Prussian blue (PB) given alone or in combination were tested as possible treatments against acute thallium toxicity. Rats were intoxicated by i.p. injection of thallium (I) acetate at LD(50) (32 mg/kg). A day later, pharmacological treatment was administered until day 4 as follows: (1) vehicles, (2) PB 50mg/kg, by oral route, twice a day, (3) DL-P 25mg/kg i.p. route, twice daily and (4) PB+DL-P. The Estimated Probability Survival (EPS) was recorded during the experiment for each treatment. DL-P alone did not show a significant effect on survival. However, when it was used in combination with PB, it increased the survival significantly (EPS=0.8, P<0.05) as compared to the control group (EPS=0.4). In a different experiment, using 16 mg/kg of Thallium I acetate, the metal levels were analyzed in blood, body organs and brain regions after treatments. DL-P given alone decreased slightly the thallium content in blood, organs and brain. Meanwhile, its administration in combination with PB diminished the thallium levels significantly (P<0.05) in the majority of tissues, at levels lower than those achieved in the PB group. Those results indicate that DL-P administered alone did not prevent the mortality nor accumulation of the metal in body tissues. Its combination with PB could be considered an alternative antidotal treatment in thallium toxicity, because this chelating agent given alone did not cause thallium redistribution to the brain. When given in combination with PB it has an additive effect in the treatment of acute thallotoxicosis.