Juan José López-Costa

Progesterone neuroprotection in the Wobbler mouse, a genetic model of spinal cord motor neuron disease

Motor neuron degeneration characterizes the spinal cord of patients with amyotrophic lateral sclerosis and the Wobbler mouse mutant. Considering that progesterone (PROG) provides neuroprotection in experimental ischemia and injury, its potential role in neurodegeneration was studied in the murine model. Two-month-old symptomatic Wobbler mice were left untreated or received sc a 20-mg PROG implant for 15 days. Both light and electron microscopy of Wobbler mice spinal cord showed severely affected motor neurons with profuse cytoplasmic vacuolation of the endoplasmic reticulum and/or Golgi apparatus and ruptured mitochondria with damaged cristae, a profile indicative of a type II cytoplasmic form of cell death. In contrast to untreated mice, neuropathology was less severe in Wobbler mice receiving PROG; including a reduction of vacuolation and of the number of vacuolated cells and better conservation of the mitochondrial ultrastructure. In biochemical studies, we determined the mRNA for the alpha3 subunit of Na,K-ATPase, a neuronal enzyme controlling ion fluxes, neurotransmission, membrane potential, and nutrient uptake. In untreated Wobbler mice, mRNA levels in motor neurons were reduced by half compared to controls, whereas PROG treatment of Wobbler mice restored the expression of alpha3 subunit Na,K-ATPase mRNA. Therefore, PROG was able to rescue motor neurons from degeneration, based on recovery of histopathological abnormalities and of mRNA levels of the sodium pump. However, because the gene mutation in Wobbler mice is still unknown, further studies are needed to unveil the action of PROG and the mechanism of neuronal death in this genetic model of neurodegeneration.

Oxidative stress promotes the increase of matrix metalloproteinases-2 and -9 activities in the feto-placental unit of diabetic rats

Maternal diabetes increases the risk of congenital malformations, placental dysfunction and diseases in both the neonate and the offsprings later life. Oxidative stress has been involved in the etiology of these abnormalities. Matrix metalloproteases (MMPs), involved in multiple developmental pathways, are increased in the fetus and placenta from diabetic experimental models. As oxidants could be involved in the activation of latent MMPs, we investigated a putative relationship between MMPs activities and oxidative stress in the feto-placental unit of diabetic rats at midgestation. We found that H2O2 enhanced and that superoxide dismutase (SOD) reduced MMPs activities in the maternal side of the placenta and in the fetuses from control and diabetic rats. MMPs were not modified by oxidative status in the fetal side of the placenta. Lipid peroxidation was enhanced in the maternal and fetal sides of the placenta and in the fetus from diabetic rats when compared to controls, and gradually decreased from the maternal placental side to the fetus in diabetic animals. The activities of the antioxidant enzymes SOD and catalase were decreased in the maternal placental side, catalase activity was enhanced in the fetal placental side and both enzymes were increased in the fetuses from diabetic rats when compared to controls. Our data demonstrate changes in the oxidative balance and capability of oxidants to upregulate MMPs activity in the feto-placental unit from diabetic rats, a basis to elucidate links between oxidative stress and alterations in the developmental pathways in which MMPs are involved.

Distribution of CB2 cannabinoid receptor in adult rat retina.

Cannabinoid effects are mediated through two receptors, CB1 and CB2. In the retina CB1 has been reported in bipolar cells, gabaergic amacrine cells, horizontal cells, and inner plexiform layer. CB2 receptor mRNA localization was shown in photoreceptors, inner nuclear layer, and ganglion cell layer by using RT‐PCR. The aim of this work was to localize CB2 receptor in the rat retina by using immunocytochemistry. Our results showed that CB2 receptor was localized in retinal pigmentary epithelium, inner photoreceptor segments, horizontal and amacrine cells, cells localized in ganglion cell layer, and in fibers of inner plexiform layer. These results are in agreement with studies using RT‐PCR and provide some additional information about the distribution of CB2 receptor. Further studies are needed to clarify the role of this cannabinoid receptor in the retina. Synapse, 2010.

Ultrastructural changes in nitric oxide synthase immunoreactivity in the brain of rats subjected to perinatal asphyxia: neuroprotective effects of cold treatment

Striatal and cortical neurons containing nitric oxide synthase (NOS) were studied in adult rats subjected to different periods of perinatal asphyxia (PA) using immunohistochemistry at both light microscopy (LM) and electron microscopy (EM). Another group was subjected to PA + hypothermia to study its neuroprotective effect. Quantitative image analysis was performed on the striatum and neocortex in order to count the number of immunoreactive neurons and to compare the pattern of staining between the different groups. Six-month-old rats that suffered subsevere and severe PA demonstrated, at LM, cytomegaly of the striatal and neocortical neurons containing NOS. Control and hypothermic neurons were more weakly immunostained than PA neurons. Subsevere and severe asphyctic rats showed an important neuronal loss that was reduced by hypothermic treatment. The PA group disclosed, at EM, dense electronic bodies distributed in terminals surrounding synaptic vesicles and in dendrites. Non-NOS-containing neurons showed signs of degeneration, such as dark cytoplasm and shrunken nuclei. Surrounding the blood vessels, we observed a clear edema. The immunolabeling in hypothermic rats resembled that observed in controls. These data suggest that subsevere and severe PA induces chronic changes in the neuronal content of NOS in the striatum and neocortex. Degeneration observed in neurons surrounding cytomegalic NOS-containing cells may be due to the excess of NO in their environment. Moreover, the chronic alterations produced by PA seem to be prevented by hypothermia.

Increased nitric oxide synthase activity in a model of serotonin depletion

Serotonin (5HT) containing cell bodies are localized in mesencephalic and rhombencephalic raphe nuclei. It has been proposed that 5HT could be involved in neuronal development and plasticity. In the central nervous system, nitric oxide (NO) has been postulated as a neurotransmitter and neuromodulator, and has been implicated in neurotoxicity as well as in neuroprotection. Using the nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) technique, NO synthesizing neurons were described in raphe nuclei. By immunohistochemistry, nitric oxide synthase (NOS) was found colocalized with 5HT in some dorsal raphe nucleus (DRN) neurons. In a model of inhibition of 5HT synthesis produced by daily administration of parachlorophenilalanine during 14 days, we have studied the relationship between 5HT and NO systems after 5HT depletion by histochemical and immunocytochemical methods. After the treatment, we observed an important reduction of 5HT immunostaining in the DRN and enhanced NOS activity demonstrated by NADPH-d technique, especially in the dorsomedial and ventromedial subgroups. In spite of the increased NOS activity, we could not observe significant changes in the NOS-immunoreactivity in the DRN after 5HT depletion. These results could indicate that 5HT depletion is concomitant with changes in NOS activity without affecting NOS expression in the DRN.

Neuronal and inducible nitric oxide synthase immunoreactivity following serotonin depletion.

Serotonin (5HT) modulates the development and plasticity of its innervation areas in the central nervous system (CNS). Astrocytic 5HT(1A) receptors are involved in the plastic phenomena by releasing the astroglial-derived neurotrophic factor S-100beta. Several facts have demonstrated that nitric oxide (NO) and the nitric oxide synthase enzyme (NOS) may also be involved in this neuroglial interaction: (i) NO, S-100beta and 5HT are involved in CNS plasticity; (ii) micromolar S-100beta concentration stimulates inducible-NOS (iNOS) expression; (iii) neuronal NOS (nNOS) immunoreactive neurons are functionally and morphologically related to the serotoninergic neurons; (iv) monoamines level, including 5HT, can be modulated by NO release. We have already shown that 5HT depletion increases astroglial S-100beta immunoreactivity, induces neuronal cytoskeletal alterations and produces an astroglial reaction, while once 5HT level is recovered, a sprouting phenomenon occurs [Brain Res. 883 (2000) 1-14]. To further characterize the relationship among nNOS, iNOS and 5HT we have analyzed nNOS and iNOS expression in the CNS after 5HT depletion induced by parachlorophenylalanine (PCPA) treatment. Studies were performed immediately after ending the PCPA treatment and during a recovery period of 35 days. Areas densely innervated by 5HT fibers were studied by means of nNOS and iNOS immunoreactivity as well as NADPH diaphorase (NADPHd) staining. All parameters were quantified by computer-assisted image analysis. Increased nNOS immunoreactivity in striatum and hippocampus as well as increased NADPHd reactivity in the striatum, hippocampus and parietal cortex were found after PCPA treatment. The iNOS immunoreactivity in the corpus callosum increased 14 and 35 days after the end of PCPA treatment. These findings showed that nNOS immunoreactivity and NADPHd activity increased immediately after 5HT depletion evidencing a close functional interaction between nitrergic and serotoninergic systems. However, iNOS immunoreactivity increased when 5HT levels were normalized, which could indicate one of the biological responses to S-100beta release.

Neuronal and macrophagic nitric oxide synthase isoforms distribution in normal rat retina.

A detailed study about the distribution of nitric oxide synthase (NOS) isoforms, neuronal NOS (nNOS) and macrophagic NOS (mNOS), in normal rat retina was performed using immunocytochemistry by employing specific antibodies. The nNOS immunocytochemistry showed immunoreactive amacrine cells, fibres in inner and outer plexiform layers (IPL and OPL) and an immunostained band corresponding to inner photoreceptor segments (IPS). This was in agreement with NADPH-d histochemical results. mNOS immunoreactivity was found in cell somas localized in both, inner nuclear layer (INL) and ganglion cell layer (GCL), in slender Müller cell processes along IPL and GCL and also in the band corresponding to IPS. A different distribution of nNOS and mNOS was found in rat retina although both isoforms of NOS are co-localized in IPS.

Progesterone treatment reduces NADPH-diaphorase/Nitric oxide synthase in Wobbler mouse motoneuron disease

Previous work demonstrated that progesterone (PROG) treatment attenuates morphological, molecular and functional abnormalities in the spinal cord of the Wobbler (Wr) mouse, a genetic model of motoneuron degeneration. Wr mice show a marked up-regulation of the nitric oxide synthesizing enzyme (NOS). Since nitric oxide is a highly reactive species, it may play a role in neuropathology of Wr mice. We now studied if PROG neuroprotection involved changes of NOS activity in motoneurons and astrocytes, determined by the nicotinamide adenine dinucleotide phosphate-diaphorase (NADPHD) histochemical reaction. Two and four-month-old Wr mice at the progressive and stabilization stages of the disease, respectively, and their age-matched controls were left untreated or received a single 20-mg PROG pellet for 18 days. PROG reduced the high number of NADPHD-active motoneurons and white matter astrocytes in 2-month-old Wr mice but was unable to change the low number of NADPHD-active motoneurons in 4-month-old Wr mice or astrocytes in this age group. A large number of motoneurons in 2-month-old Wr mice showed a vacuolated phenotype, which was significantly reverted by PROG treatment. In summary, PROG treatment during the early symptomatic stage of the disease caused a significant reduction of NADPHD-active motoneurons and astrocytes and also reduced vacuolated degenerating cells, suggesting that blockade of NO synthesis and oxidative damage may contribute to steroid neuroprotection.

Long Term Changes in Nadph-Diaphorase Reactivity in Striatal and Cortical Neurons Following Experimental Perinatal Asphyxia: Neuroprotective Effects of Hypothermia

Nitric oxide (NO) is known to be involved in the neuropathological mechanisms triggered by excitatory aminoacids. NO(+) neurons in the brain may be detected histochemically by nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemical technique, as the latter readily labels NO synthase in the central nervous system (CNS). NADPH-d stained striatal and cortical sections were studied in 6-month-old male Sprague-Dawley rats exposed to perinatal asphyxia (PA) at 37 degrees C, as well as in animals subjected to PA plus hypothermia treatment at 15 degrees C. Quantitative image analysis was performed to compare the staining pattern in the various groups. NADPH-d(+) neurons in striatum and cortex from subsevere and severe asphyctic animals showed a significant increase in soma size and in dendritic processes versus controls and hypothermia-treated rats. These findings indicate that chronic NO changes are involved in postischemic striatal and cortical alterations induced by PA that may be prevented by hypothermia.

Intracerebroventricular administration of Shiga toxin type 2 altered the expression levels of neuronal nitric oxide synthase and glial fibrillary acidic protein in rat brains

Shiga toxin (Stx) from enterohemorrhagic Escherichia coli (STEC) is the main cause of hemorrhagic colitis which may derive into Hemolytic Uremic Syndrome (HUS) and acute encephalopathy, one of the major risk factors for infant death caused by the toxin. We have previously demonstrated that intracerebroventricular administration of Stx2 causes neuronal death and glial cell damage in rat brains. In the present work, we observed that the intracerebroventricular administration of Stx2 increased the expression of glial fibrillary acidic protein (GFAP) leading to astrogliosis. Confocal microscopy showed reactive astrocytes in contact with Stx2-containing neurons. Immunocolocalization of increased GFAP and Stx2 in astrocytes was also observed. This insult in the brain was correlated with changes in the expression and activity of neuronal nitric oxide synthase (nNOS) by using the NADPH-diaphorase histochemical technique (NADPH-d HT). A significant decrease in NOS/NADPH-d-positive neurons and NOS/NADPH-d activity was observed in cerebral cortex and striatum, whereas an opposite effect was found in the hypothalamic paraventricular nucleus. We concluded that the i.c.v. administration of Stx2 promotes a typical pattern of brain injury showing reactive astrocytes and an alteration in the number and activity of nNOS/NADPH-d. According to the functional state of nNOS/NADPH-d and to brain cell morphology data, it could be inferred that the i.c.v. administration of Stx2 leads to either a neurodegenerative or a neuroprotective mechanism in the affected brain areas. The present animal model resembles the encephalopathy developed in Hemolytic Uremic Syndrome (HUS) patients by STEC intoxication.