Julio César Rojas-Castañeda

Alterations in the spermatic function generated by obesity in rats

In order to assess the effect of obesity on epididymal and germinal epithelia in control rats and obese rats induced by a high fat diet, we evaluated the epididymal and testicular morphologies, lipid peroxidation in the epididymis, leptin serum levels, steroid hormones, insulin, cholesterol, triglycerides, glycemia and some spermatobioscopic parameters. No significant difference was observed in the levels of insulin, glucose, cholesterol and triglycerides between the two groups. Nonetheless, in the obese rats, circulating leptin and estradiol levels showed a significant increase and there was a decline in the testosterone levels. The same group showed an increase in the lipid peroxidation of the epididymis and reduced spermatobioscopic parameters. The heads of the epididymis showed morphological differences in obese rats. No significant difference was observed between the testes of both groups. There is a clear evidence of an effect on sperm in obese rats and this seems to occur in the epididymis.

Effect of a phytopharmaceutical medicine, Ginko biloba extract 761, in an animal model of Parkinson's disease: therapeutic perspectives.

Ginkgo Biloba extract 761 (EGb 761) is a patented and well-defined mixture of active compounds extracted from Ginkgo biloba leaves. This extract contains two main groups of active compounds, flavonoids (24%) and terpenoids (6%). EGb 761 is used clinically to treat dementia and vaso-occlusive and cochleovestibular disorders. This extract has neuroprotective effects, exerted probably by means of its antioxidant function. Parkinsons disease (PD) is a neurodegenerative disorder that affects 2% of the population older than 60 y. It produces a progressive loss of dopaminergic neurons and depletion of dopamine (DA), leading to movement impairment. The production of reactive oxygen species, which act as mediators of oxidative damage, is linked to PD. This disease is routinely treated with the DA precursor, L-3,4-dihydroxyphenylalanine. However, this produces severe side effects, and its neurotoxic properties can be due to a free radical production. Thus, administration of antioxidant drugs might be used to prevent neuronal death produced by oxidative mechanisms. The use of synthetic antioxidants has decreased because of their suspected activity as carcinogenic promoters. We describe the studies related to the antioxidant effect of EGb 761 in an animal model of PD. It has been shown that EGb761 can provide a neuroprotective/neurorecovery effect against the damage to midbrain DA neurons in an animal model of PD. EGb 761 also has been found to lessen the impairment of locomotion, correlating with an increase of DA and other morphologic and biochemical parameters related to its antioxidant effect in an animal model of PD. These studies suggest it as an alternative in the future treatment of PD.

EGb761 Blocks MPP+-Induced Lipid Peroxidation in Mouse Corpus Striatum

EGb761 has been suggested to be an antioxidant and free radical scavenger. Excess generation of free radicals, leading to lipid peroxidation (LP), has been proposed to play a role in the damage to striatal neurons induced by 1-methyl-4-phenylpyridinium (MPP+). We investigated the effects of EGb761 pretreatment on MPP+ neurotoxicity. C-57 black mice were pretreated with EGb761 for 17 days at different doses (0.63, 1.25, 2.5, 5 or 10 mg/kg) followed by administration of MPP+, (0.18, 0.36 or 0.72 mg/kg). LP was analyzed in corpus striatum at 30 min, 1 h, 2 h and 24 h after MPP+ administration. Striatal dopamine content was analyzed by HPLC at the highest EGb761 dose at 2 h and 24 h after MPP+ administration. MPP+-induced LP was blocked (100%) by EGb761 (10 mg/kg). Pretreatment with EGb761 partially prevented (32%) the dopamine-depleting effect of MPP+ at 24 h. These results suggest that supplements of EGb761 may be effective at preventing MPP+-induced oxidative stress.

Effect of EGb761 supplementation on the content of copper in mouse brain in an animal model of Parkinson's disease.

OBJECTIVE EGb761 is a well-defined mixture of active compounds extracted from Ginkgo biloba leaves with neuroprotective effects in an animal model of Parkinsons disease induced by 1-methyl-4-phenylpyridinium (MPP(+)). Because copper has been implicated in Parkinsons disease, we investigated whether the protective effect of EGb761 in MPP(+) neurotoxicity is related to the regulation of copper in the brain. METHODS C-57BL/6 mice were pretreated with EGb761 (10 mg/kg) daily for 17 d followed by administration of MPP(+) (0.72 mg/kg); the mice were sacrificed 24 h later. The copper content of the striatum, midbrain, hippocampus, frontal cortex, and cerebellum was analyzed by graphite furnace atomic absorption spectrophotometry. Copper content is expressed as mug of copper per gram of wet tissue. RESULTS Copper content was reduced in the corpus striatum (45%; P < 0.05), and increased in the midbrain (65%; P < 0.05) and hippocampus (116%; P < 0.001) after MPP(+) administration. EGb761 pretreatment of the MPP(+) group prevented changes in the copper content of the striatum, midbrain, and hippocampus. No significant changes were found in the copper content of the cerebellum and frontal cortex in all treatment groups. CONCLUSION We showed that the protective effect of EGb761 against MPP(+) neurotoxicity may be due in part to the regulation of copper homeostasis in the brain.

Effect of allopurinol on damage caused by free radicals to cryptorchid testes.

Cryptorchidism causes apoptosis of germ cells. It has been suggested that the redox regulatory system is involved in this process. The free radicals produced are thought to be generated during the production of uric acid, a reaction catalyzed by xanthine oxidase. This enzyme is inhibited by allopurinol; however, the role of allopurinol in neonate rats with inguinal cryptorchidism has not been assessed yet. Sixty male Wistar rats were used and five groups were formed: a control, a sham, a sham group with allopurinol administration and two groups with surgical unilateral cryptorchidism, which either did not receive, or received, allopurinol. The rats were assessed at 40 days post-partum. Reactive oxygen species concentration and epithelial area were measured and the histopathological, apoptotic and cellular proliferation indexes were determined. We found a decrease in reactive oxygen species, histopathological and apoptotic indexes and an increase in proliferation index and epithelial area in rats with cryptorchidism treated with allopurinol in comparison with rats with untreated cryptorchidism. We suggest that the over-production of reactive oxygen species plays an important role in the damage of the cryptorchid testes. Allopurinol administration decreases reactive oxygen species concentrations as well as the damage to the germ epithelium.

Protective effect of α-tocopherol on damage to rat testes by experimental cryptorchidism.

It is thought that the degeneration of germ cells associated with an increase in the temperature due to cryptorchidism involves oxidative stress. α‐Tocopherol is a powerful antioxidant that prevents oxidation of polyunsaturated fats found in membranes and stabilizes peroxyl radicals. For this reason we were interested in determining the role of α‐Tocopherol using experimental cryptorchidism, followed by orchidopexia in neonatal rats. Eighty‐four, 10‐day‐postpartum (dpp) male rats (Wistar strain) were used and divided into 7 groups: healthy control, sham with α‐Tocopherol treated with 30 or 100 mg/kg doses, sham vehicle, cryptorchidism treated with α‐Tocopherol at 30 or 100 mg/kg doses and cryptorchidism vehicle. Cryptorchidism was surgically induced at 10 dpp. At 25 dpp the animals were treated with α‐Tocopherol and the vitamin vehicle. Lipoperoxidation and testicular morphology was determined in half of the animals at 40 dpp (short term). The remaining animals underwent orchidopexia and fertility was determined at 90 dpp. Testicular morphology was determined at 120 dpp (long term) in these animals. A significant reduction of lipoperoxidation was observed in the cryptorchid group treated with α‐Tocopherol compared to the untreated cryptorchid group, in addition to short‐term histological alterations. At long term, we observed an increase in the area and maturation of the seminiferous epithelium, a decrease in apoptosis and histological alterations and an increase in fertility from α‐Tocopherol treatment. α‐Tocopherol treatment decreased lipoperoxidation, possibly stabilizing free radicals produced during cryptorchidism, reducing morphological testicular alterations and favoring fertility.

MPTP decreases MT-I mRNA in mouse striatum.

Abstract1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a drug that induces parkinsonism in humans and non-human primates. Free radicals are thought to be involved in its mechanism of action. Recently, metallothionein has been proposed to play a role as a scavenger of free radicals. In the present work, we studied the effect of MPTP neurotoxicity on brain metallothionein-I (MT-I) mRNA expression. Male C-57 black mice were treated with MPTP (30 mg/kg, i.p., daily) for 3 or 5 days. All animals were killed by cervical dislocation 7 days after the last MPTP dose. The brains were removed quickly and immediately frozen, and quantitative in situ hybridization was performed using MT-I cDNA probe. MT-I mRNA content in striatum, a region which is known to be highly predisposed and sensitive to MPTP-induced oxidative stress, decreased by 30% (3 days) and 39% (5 days) respectively, after the last MPTP administration. These results suggest that MT-I gene expression is decreased in MPTP neurotoxicity. It is suggested that the reduction of MT, an anti-oxidant and a free radical scavenger, in the striatum by MPTP enables the neurotoxin to exert maximal oxidative damage to the striatum.

Post-ischemic administration of progesterone reduces caspase-3 activation and DNA fragmentation in the hippocampus following global cerebral ischemia.

Delayed death of hippocampal CA1 pyramidal neurons following global cerebral ischemia/reperfusion may be mediated, in part, by caspase-3 activation resulting in DNA fragmentation. Progesterone (P4) is known to exert neuroprotective effects in several models of brain injury. This study was designed to assess the effect of P4 on caspase-3 levels and activation, and DNA fragmentation in the hippocampus following global cerebral ischemia/reperfusion. Adult male Sprague-Dawley rats were subjected to global ischemia by the four-vessel occlusion model. P4 (8 mg/kg), or its vehicle were administered i.v. at 15 min, 2, 6, 24, 48 and 70 h of reperfusion. Remaining pyramidal neurons were assesed by the Nissl staining technique, caspase-3 levels and activation by immunohistochemistry and an in situ activity assay, and DNA fragmentation by the TUNEL method. Post-ischemic progesterone treatment significantly reduced the ischemia/reperfusion-induced increase in caspase-3 levels and activation at 72 h, and DNA fragmentation and CA1 neuronal loss at 7 days. Present results suggest the reduction of caspase-3 levels/activation, and DNA fragmentation, as a part of the neuroprotective effects of progesterone against global cerebral ischemia/reperfusion injury.

Alterations induced by chronic lead exposure on the cells of circadian pacemaker of developing rats

Lead (Pb) exposure alters the temporal organization of several physiological and behavioural processes in which the suprachiasmatic nucleus (SCN) of the hypothalamus plays a fundamental role. In this study, we evaluated the effects of chronic early Pb exposure (CePbe) on the morphology, cellular density and relative optical density (OD) in the cells of the SCN of male rats. Female Wistar rats were exposed during gestation and lactation to a Pb solution containing 320 ppm of Pb acetate through drinking water. After weaning, the pups were maintained with the same drinking water until sacrificed at 90 days of age. Pb levels in the blood, hypothalamus, hippocampus and prefrontal cortex were significantly increased in the experimental group. Chronic early Pb exposure induced a significant increase in the minor and major axes and somatic area of vasoactive intestinal polypeptide (VIP)‐ and vasopressin (VP)‐immunoreactive neurons. The density of VIP‐, VP‐ and glial fibrillary acidic protein (GFAP)‐immunoreactive cells showed a significant decrease in the experimental group. OD analysis showed a significant increase in VIP neurons of the experimental group. The results showed that CePbe induced alterations in the cells of the SCN, as evidenced by modifications in soma morphology, cellular density and OD in circadian pacemaker cells. These findings provide a morphological and cellular basis for deficits in circadian rhythms documented in Pb‐exposed animals.

Analysis of POU5F1, c-Kit, PLAP, AP2γ and SALL4 in gonocytes of patients with cryptorchidism.

Cryptorchidism is a risk factor for the development of testicular germ cell tumors (TGCTs). The most common type of TGCT in cryptorchidism is seminoma. The intratubular germ cell neoplasia unclassified (ITGCNU) is a histological pattern preceding the development of seminomas and non-seminomas. It was suggested that in patients with cryptorchidism, the gonocytes remained undifferentiated with pluripotent abilities expressing proteins like POU domain class 5 transcription factor 1 (POU5F1), tyrosine kinase receptor c-Kit, placental-like alkaline phosphatase (PLAP), the transcription factor AP2γ and sal-like protein 4 (SALL4) that confer to the gonocytes this ability and therefore make them susceptible to develop ITGCNU. The aim of the present study was to determine if the gonocytes of patients with cryptorchidism express POU5F1, c-Kit, PLAP, AP2γ and SALL4 proteins after their differentiation period. Based on this, we evaluated samples of testicular tissue from newborns to 16-year old subjects with or without cryptorchidism in search of POU5F1, c-Kit, PLAP, AP2γ and SALL4 using immunocytochemical method, the results of which were validated by RT-PCR. The results showed that control subjects witnessed a down-regulation in the expression of these five proteins in the first year of life, which eventually disappeared. On the other hand, it was determined that 21.6% (8/37) of the patients with cryptorchidism continued to express, at least, one of the proteins analyzed in this study after the second year of life. And only 5.4% (2/37) of the patients were positive to the five markers. These data sustain the proposed hypothesis that in cryptorchid patients, ITGCNU arises from gonocytes that fail in their differentiation process to spermatogonia with conservation of the proteins (POU5F1, c-Kit, PLAP, AP2γ and SALL4) that maintain pluripotency and undifferentiated characteristics and which are responsible for making the gonocytes susceptible to malignancy. However, we cannot guarantee that these patients present neoplastic transformation.