Esperanza García

Lipid peroxidation, mitochondrial dysfunction and neurochemical and behavioural deficits in different neurotoxic models: Protective role of S-allylcysteine

Experimental evidence on the protective properties of S-allylcysteine (SAC) was collected from three models exerting striatal toxicity. In the first model, SAC (120mg kg−1×5) prevented lipoperoxidation (LP) and mitochondrial dysfunction (MD) in synaptosomal fractions from 1-methyl-4-phenyl-1,2,3,6-tetrahydropiridinium-treated mice (30mg kg−1), but without complete restoration of dopamine levels. In the second model, SAC (300mg kg−1×3), prevented LP and MD in synaptosomes from rats infused with 6-hydroxydopamine (8µg µl−1) into the substantia nigra pars compacta, but again, without total reversion of depleted dopamine levels. In the third model, SAC (100 mg kg−1×1) prevented MD in synaptosomes from rats injected with 3-nitropropionic acid (10 mg kg−1), but in contrast to the other models, it failed to prevent LP. SAC also prevented the aberrant motor activity patterns evoked by the three toxins. Altogether, the results suggest that the antioxidant properties of SAC are responsible for partial or total preservation of neurochemical, biochemical and behavioural markers, indicating that pro-oxidant reactions underlie the neurotoxicity in these models.

Ventricular injection of nerve growth factor increases dopamine content in the striata of MPTP-treated mice

Experimental depletion of dopaminergic striatal neurons was induced in mice with the neurotoxin MPTP. To investigate a possible effect of nerve growth factor on the damaged neurons, we injected 4 μg into the right cerebral ventricle of mice three days after the last administration of MPTP. We found a significant increase of dopamine and homovanillic acid in the striatum of MPTP treated mice after NGF administration when compared with dopamine and HVA levels in MPTP-treated control mice (p<0.001). The increase of dopamine and homovanillic acid seems to be related to a partial restorative effect of NGF on the damaged dopaminergic cells, since ventricular administration of NGF to normal mice did not increase dopamine or homovanillic acid contents above the levels measured in untreated controls. It appears that administration of nerve growth factor prcduces a beneficial effect on damaged dopaminergic neurons; this effect could be due to stimulation of neuron sprouting from neurons that survived the toxic effect of MPTP. The increase of dopamine levels was seen 8 days after injection of nerve growth factor and was maintained at least until day 25, showing a lasting persistence of the restorative effect.

Protective effect of L-kynurenine and probenecid on 6-hydroxydopamine-induced striatal toxicity in rats: Implications of modulating kynurenate as a protective strategy

The neuroactive metabolite at the kynunerine pathway, kynurenic acid (KYNA), is a well-known competitive antagonist at the co-agonist glycine site of the N-methyl-D-aspartate receptor (NMDAr), and also decreases the extracellular levels of glutamate by blocking α7-nicotinic acetylcholine receptor (α7-nAchr) located on glutamatergic terminals. KYNA has been often reported to be neuroprotective in different neurotoxic models. The systemic administration of L-kynurenine (L-KYN)--the precursor of KYNA--together with probenecid (PROB)--an inhibitor of organic acids transport--to rodents increases KYNA levels in the brain in a dose-dependent manner. The striatal infusion of the toxin 6-hydroxydopamine (6-OHDA) to rodents is one of the common models used to simulate Parkinsons disease (PD). Different studies have linked PD alterations with excessive glutamatergic transmission in the striatum since NMDAr antagonists exert beneficial effects in PD models. In this work we investigated the effect that a systemic administration of L-KYN+PROB exerted on the toxic model induced by 6-OHDA in rats. PROB (50 mg/kg, i.p.) + L-KYN (75 mg/kg, i.p.) were given to rats for seven consecutive days. On day two of treatment, the animals were infused with a single injection of 6-OHDA (20 μg/2 μl) into the right striatum. Fourteen days post-lesion, rotation behavior was assessed as a marker of motor impairment. The total levels of dopamine (DA) were also estimated in striatal tissue samples of 6-OHDA-treated animals as a neurochemical marker of damage. In addition, twenty eight days post-lesion, the striatal damage was assessed by hematoxylin/eosin staining and immunohistochemistry against glial fibrillary acidic protein (GFAP) in the same animals. Neurodegeneration was also assessed by Fluoro Jade staining. 6-OHDA infusion increased rotation behavior, striatal reactive gliosis and neurodegeneration, while DA levels were decreased. For all markers evaluated, we observed protective effects of L-KYN+PROB on the dopaminergic damage induced by 6-OHDA. Our results suggest that this strategy was useful to mitigate dopaminergic toxicity in the hemiparkinsonian model. The combined use of L-KYN and PROB is a valuable tool to modulate glutamatergic and cholinergic activities, presumably by means of increased levels of endogenous KYNA.

Subarachnoidal and intraventricular human neurocysticercosis: application of an antigen detection assay for the diagnosis and follow-up.

Background  Neurocysticercosis (NC) is a parasitic disease of the central nervous system caused by the larval stage of Taenia solium. Although imaging studies are recommended for diagnosis and follow‐up of patients, their high cost and restricted availability limit their use. Among various immunological tests, the detection of HP10 antigen in cerebral spinal fluid (CSF) has proved to be a useful tool for the diagnosis of NC in the case of viable but not dead parasites.

Immunodominant synthetic peptides of Taenia crassiceps in murine and human cysticercosis

The screening of a cDNA library of Taenia crassiceps revealed a clone designated KETc7 that induced high levels of protection against murine cysticercosis in previous experiments. The molecular structure of the deduced 100-amino acid sequence of the corresponding proline-rich polypeptide was studied to detect potentially immunologically active epitopes. Several candidate epitopes were identified, three of which were synthesized by solid-phase peptide synthesis and used as antigens in enzyme-linked immunosorbent assay (ELISA) for detection of specific antibodies in a selected panel of sera from mice infected with Taenia crassiceps and pigs infected with Taenia solium, as well as in the serum and cerebrospinal fluid of human patients with neurocysticercosis. The three peptides detected antibodies in serum from all infected mice. Seven of nine sera from patients with neurocysticercosis reacted strongly with peptide GK-3, and four of them with peptides GK-1 and GK-2. A lower reactivity was observed in sera from experimentally infected pigs. Peptide GK-3 reacted also with 45 out of 77 cerebrospinal fluids (CSF) from patients with confirmed neurocysticercosis and with 14 out of 68 CSF from control patients with other neurological disorders. This is the first report on synthetic peptides that are prominent in the humoral response of murine, porcine and human cysticercosis. Their identification implies finer molecular tools in the exploration of this form of host-parasite relationship, as well as hints to their application in immunodiagnosis and in vaccine design.

Application of synthetic peptides to the diagnosis of neurocysticercosis

We tested the possible diagnostic utility of five Taenia saginata oncosphere‐derived synthetic peptides in T. solium neurocysticercosis (NC). The five peptides correspond to protein sequences with high antigenic indexes that were cloned from a T. saginata oncosphere cDNA library. The test samples consisted of cerebrospinal fluid (CSF) samples randomly collected from patients referred from Mexican and Brazilian neurological institutes. Indirect enzyme‐linked immunosorbent assays (ELISA) were carried out with the peptides either unconjugated or coupled to carrier proteins, and were compared with results obtained using T. solium cyst fluid as a positive control. For active inflammatory NC, the higher sensibility (93%) and specificity (85%) was obtained with peptides HP6‐2 and Ts45W‐1, respectively, coupled to ovalbumin, in both Mexican and Brazilian patients. Examining the results of the individual peptide assays in combination, in some instances, improved the sensitivity to 100%.

Neuroprotective effect of silymarin in a MPTP mouse model of Parkinson's disease

Parkinsons disease (PD) is a neurodegenerative disease secondary to the loss of dopaminergic neurons in the substantia nigra. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) produces in mice and primates histopathological changes similar to PD in humans. A common feature of PD and MPTP models is neuronal death and dopamine depletion. Silymarin is a complex of flavonolignans derived from the seeds of the plant Silybum marianum and has mainly antioxidant, anti-inflammatory, cytoprotective and neuroprotective effects. In order to explore whether silymarin has a neuroprotective effects in a mouse model of PD we determined the concentration of striatal dopamine by HPLC, the number of apoptotic cells by in situ Tunel assay and the number of tyrosine hydroxylase positive neurons by immunohistochemistry in substantia nigra of vehicle-treated, silymarin-treated, MPTP-intoxicated and MPTP-silymarin treated C57BL/6J male mice. MPTP (30 mg/kg) and silymarin doses (25, 50, 100, 200, 250, 300 or 400mg/kg) were administered intraperitoneally once daily for five consecutive days. Silymarin treatment showed a non-monotonic dose-response curve and only 50 and 100mg/kg doses preserved dopamine levels (62% and 69%, respectively) after MPTP intoxication. Additionally, 100mg/kg silymarin treatment significantly diminished the number of apoptotic cells and preserved dopaminergic neurons in the substantia nigra of MPTP-intoxicated mice. These results show the neuroprotective properties of 100mg/kg silymarin and may be of interest in the treatment of PD.

Cysticercosis: towards the design of a diagnostic kit based on synthetic peptides

Cysticercosis caused by Taenia solium is a very common disease in developing countries that seriously affects human health. Diagnosis can only be confirmed with the aid of computerized tomography or nuclear magnetic resonance (NMR) creating obvious difficulties for epidemiological studies. Reliable immunoassays employing cerebrospinal fluid (CSF) have been developed, based on the use of cysticercal antigens. However, the reliance on parasite material is restrictive. Herein, we report the advances in the design of a diagnostic kit based on immunodominant synthetic peptides, targeting four candidate epitopes KETc1, KETc12, 410 and 413 which were identified from three different clones (KETc1, 12 and 4) selected from a cDNA library of Taenia crassiceps. CSF antibodies against T. solium cysticercal antigens (TCA) as well as the four peptides were determined by enzyme-linked immunoabsorbent assays (ELISA) using two panels of CSF from patients with confirmed neurocysticercosis and other neurological diseases. In the first CSF panel which included patients with high level of antibodies against TCA, KETc12 exhibited almost the same sensitivity (87.5%) as TCA (93.7%) and 100% specificity. In the second panel of 110 CSF collected at random, two peptides (KETc1 and KETc12) exhibited sensitivities of 40 and 36% respectively, and were 100% specific.

A new complement fixation test for the diagnosis of neurocysticercosis in cerebrospinal fluid.

SummaryAs a modification of the classical complement fixation test, a new test for immunodiagnosis in cerebrospinal fluid (CSF) was developed. When the assay was used for the diagnosis of neurocysticercosis in 149 CSF samples from patients and 1036 from controls, results showed 93% concordance with positives and 97% with negatives when compared with the results obtained by ELISA; in addition, it was positive in 12 of 16 cases of neurocysticercosis in which negative results had been obtained by ELISA. The new complement fixation test is particularly useful in CSF because of the immunological peculiarities of the subarachnoid space, where local synthesis of oligoclonal antibodies is induced by infectious agents. Reagents used for the assay are easy to produce and preserve, and the test is inexpensive, reliable and easy to perform. As this test detects the immunobiological consequence of the antigen-antibody reaction, it can be used in conjunction with other assays, such as ELISA, that measure the reaction directly, thus increasing the diagnostic possibilities. It may also be used where financial or technical limitations hinder access to other immunodiagnostic tests.

S-allylcysteine reduces the MPTP-induced striatal cell damage via inhibition of pro-inflammatory cytokine tumor necrosis factor-α and inducible nitric oxide synthase expressions in mice

We have recently demonstrated that S-allylcysteine (SAC) induces protection on neurochemical, biochemical and behavioral markers of striatal damage in different neurotoxic animal models - including a murine model induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropiridinium (MPTP) injection to mice - indicating that pro-oxidant reactions underlie neurotoxicity in these models (García et al. 2008). In this work we investigated whether SAC can protect the striatum of mice from the morphological alterations in the MPTP toxic model, and if this response is correlated with a reduction in pro-inflammatory cytokine tumor necrosis factor-α (TNF-α) and inducible nitric oxide synthase (iNOS) expressions, and further reduction in astrocyte activation (glial fibrillary acidic protein (GFAP) expression). The striatal tissue from MPTP injected animals (30 mg/kg, i.p., ×5 days) showed a significant degree of cell damage and enhanced immunoreactivities to GFAP, TNF-α and iNOS, as well as an enhanced number of apoptotic nuclei. Treatment of mice with SAC (120 mg/kg, i.p., ×5 days) in parallel to MPTP significantly reduced or prevented all these markers. Our results suggest that MPTP-induced morphological alterations recruit a pro-inflammatory component triggered by cytokine TNF-α release and nitric oxide formation, which is sensitive to the antioxidant properties of SAC. This antioxidant is an effective experimental tool to reduce the brain lesions associated with oxidative damage and inflammatory responses.