Auxiliadora Espinar

Oxidative stress induced by phenylketonuria in the rat: Prevention by melatonin, vitamin E, and vitamin C.

Phenylketonuria (PKU) is an autosomal recessive disorder caused by a deficiency of the phenylalanine hydroxylation system and is characterized by a block in the conversion of phenylalanine (PHE) to tyrosine. We examined the effects of maternal hyperphenylalaninemia on the morphological and biochemical development of pup rat brain and cerebellum. In our model of PKU we evaluated a number of markers of oxidative stress such as Ehrlich adducts formation, lipid peroxidation, as well as the levels of reduced and oxidized glutathione, and the activities of the enzymes glutathione peroxidase and glutathione reductase. We also studied the expression of heme‐oxigenase‐1 and mitogen‐activated protein kinase 1/2 (MAPK 1/2) as additional markers of oxidative stress. We demonstrate that PKU strongly increased most of the oxidative stress markers studied and induced significant morphological damage. We also showed that daily administration of melatonin (20 mg/kg BW), vitamin E (30 mg/kg BW), and vitamin C (30 mg/kg BW) until delivery prevented the oxidative biomolecular damage in the rat brain and cerebellum. Although no significant differences were observed among the antioxidants studied, it should be noted that the doses of melatonin were less than those for vitamins E and C. We conclude that PKU induces a clear state of oxidative stress that is somehow involved in the brain and body damage occurring in this inborn error. Moreover, melatonin and other antioxidants are capable of preventing completely the damage induced by PKU.

HISTOLOGICAL CHANGES DURING DEVELOPMENT OF THE CEREBELLUM IN THE CHICK EMBRYO EXPOSED TO A STATIC MAGNETIC FIELD

Few studies have been performed to evaluate the ultrastructural changes that exposure to static magnetic fields (SMF) can cause to the processes of cell migration and differentiation in the cerebellum during development. Thus, we have studied the development of the cerebellum in the chick embryo (n = 144) under a uniform SMF (20 mT). All of our observations were done on folium VIc of Larsells classification. The cerebella of chick embryos, which were exposed solely on day 6 of incubation and sacrificed at day 13 of incubation [short exposure (S)1; n = 24], showed an external granular layer (EGL) that was less dense than the EGL in the control group (n = 24). The molecular layer (ML) exhibited a low number of migratory neuroblastic elements. Moreover, the internal granular layer (IGL) was immature, with the cellular elements less abundant and more dispersed than in controls. In chick embryos exposed on day 6 of incubation and sacrificed at day 17 (S2; n = 24), the outstanding feature was the regeneration of the different layers of the cerebellar cortex. The cerebellar cortex of chick embryos exposed continuously to an identical field from the beginning of the incubation up to day 13 [long exposure (L)1; n = 24] or day 17 (L2; n = 24) of incubation showed a higher number of alterations than that of group S1. Electron microscopy confirmed the findings from light microscopy and, at the same time, showed clear signs of cell degeneration and delay in the process of neuronal differentiation. This was more apparent in groups L1 (100%) and L2 (100%) than in groups S1 (95.4%) and S2 (65.2%). In conclusion, the present study showed that SMF can induce irreversible developmental effects on the processes of cell migration and differentiation of the chick cerebellar cortex.

Neuroprotection by melatonin from glutamate‐induced excitotoxicity during development of the cerebellum in the chick embryo

This work investigated the ability of melatonin to prevent cell damage in the cerebellar cortex of chick embryo caused by glutamate administration. Cell injury was evaluated estimating, at ultrastructural level, the phenomenon of cell death and the synaptogenesis of the Purkinje cells and the cerebellar glomerular synaptic complex. Administration of glutamate during cerebellar development of the chick provokes excitotoxic neuronal degeneration characterized by a phenomenon of neuronal cell death that exhibits essentially the features of a death pattern described as necrosis and the deletion of synaptogenic processes. Our results show that melatonin has a neuroprotective effect against glutamate‐induced excitotoxicity. This effect is morphologically revealed by the lack of neural cell death in the embryos treated with melatonin prior to glutamate injection and also by the degree of a synaptogenesis similar to that exhibited by the control group. Likewise, we corroborate the absence of teratological effects of melatonin on chick cerebellar development. Although the possible mechanisms involved in the neuroprotective effect of melatonin are discussed, i.e., direct antioxidant effects, up‐regulating endogenous antioxidant defenses, and inhibiting nitric oxide formation activated by glutamate, further studies are required to establish the actual mechanism involved in the neuroprotective effect of melatonin.

Melatonin prevents focal rat cerebellum injury as assessed by induction of heat shock protein (HO-1) following subarachnoid injections of lysed blood

The present paper studies a marker of oxidative stress such as heme oxygenase-1 (HO-1), the main heat shock protein. HO-1 expression was induced in the focal region of the cerebellum following experimental subarachnoid hemorrhage (SAH). Lysed blood was injected into the subarachnoid space or cisterna magna region of adult rats. The experimental groups used were: (1) animals injected with lysed blood alone; (2) animals injected with saline alone; (3) lysed blood plus melatonin (10 mg/kg body weight(BW)); (4) lysed blood plus melatonin (10 mg/kg BW injected 1 h before SAH); (5) lysed blood plus melatonin (5 mg/kg BW injected 1 h before SAH); (6) lysed blood plus vitamin E (Trolox; 30 mg/kg BW injected simultaneously); (7) lysed blood plus vitamin E (30 mg/kg BW injected 1 h before SAH); and (8) lysed blood plus vitamin E (15 mg/kg BW injected 1 h before SAH). Animals were sacrificed 24 h later. Injection of lysed blood induced an overexpression of HO-1. Both, melatonin and vitamin E were able to prevent the expression of the heat shock protein. However, in terms of efficiency, the antioxidant capability of melatonin was clearly higher than that exhibited by vitamin E. The results presented in this study show that antioxidants, especially melatonin, prevent focal regions of injury as assessed by heat shock protein expression in a rat model of SAH.

Peripapillary glial cells in the chick retina: A special glial cell type expressing astrocyte, radial glia, neuron, and oligodendrocyte markers throughout development

Peripapillary glial cells of the chick are a special type of glia, not only because of their position, forming a boundary between the retina on one side and the optic nerve head (ONH) and the pecten on the other, but also because although they have the same orientation and similar shape as the retinal Müller cell (a type of radial glia) and express common markers for these cells and astrocytes, they do not express glutamine synthetase (GS) or carbonic anhydrase C (CA‐C), enzymes intensely expressed by Müller cells and astrocytes. In this study, we present further molecular characterization of these cells, using immunohistochemistry techniques. We show that peripapillary glial cells express a novel neuron antigen, 3BA8, that in the adult retina is located only in one neuron type (the amacrine cell) and in the inner plexiform layer (IPL). They also express an antigen specific to myelin and oligodendrocytes, MOSP, and a glial antigen, 3CB2, expressed by radial glia and astrocytes throughout the CNS. The study of the developmental expression of these three antigens in the peripapillary glial cell territory shows different spatiotemporal labeling patterns: 3CB2 and 3BA8 are expressed much earlier (embryonic days E3 and E5, respectively) than MOSP (E12), and during a developmental window (E6–E10) 3BA8 labels the peripapillary glial cells intensely and does not label the ONH or the optic nerve (ON), which are labeled later. The expression of 3CB2 is much more intense in the peripapillary glial cells than in Müller cells from early stages of development up to E16, and the expression of MOSP starts earlier in the peripapillary glial cells than in the Müller cells and is maintained with much higher intensity in the peripapillary glial cells throughout development. These findings show that Müller and peripapillary glial cells follow independent courses of differentiation, which together with the fact that the peripapillary glial cells express molecules typical of neurons, oligodendrocytes, radial glia, and astrocytes are evidence that peripapillary glial cells are a unique type of glia in the CNS.

Effect of ethanol on the morphohistogenesis and differentiation of cerebellar granule cells in the chick embryo

In this present study we analyse, with the help of the Golgi method, the effect of ethanol on the morphological differentiation of the cerebellar granule cells in the chick embryo. Ethanol seems to affect the process of cell migration from the early stages of differentiation. Some granule cells appear to differentiate in an inverted position. These observations also confirm, on the basis of their axon morphology, the existence of three types of granule cells in the chick cerebellum.

High activity of retinal N-acetyltransferase in the early development of the chick embryo: independence of lighting conditions

In this paper, we studied the activities of N-acetyltransferase (NAT), hydroxyindole-O-methyltranferase (HIOMT) and melatonin content in the retina of chick embryo from the 7th day of embryonic age to hatching. Our results confirm that the dependence on lighting conditions of NAT activity and melatonin content appears to develop prior to hatching, as evidenced by differences observed when chick embryos were incubated under constant darkness or constant light. Moreover, we found high NAT activity during the first stages of the embryonic development up to 13th day of incubation. This activity, unlike late retinal NAT, is not coupled to melatonin production and is not dependent on lighting conditions.

Effects of Static Electromagnetic Fields on Chick Embryo Pineal Gland Development

The effects of static electromagnetic fields on the development of the chick embryo pineal gland were studied. A total of 144 fertilized White Leghorn eggs were sacrificed after 5, 10 and 15 days of incubation. The stage of development was determined in all embryos using the Hamburger and Hamilton method [J Morphol 49: 88–92, 1951]. The various morphometric parameters (diameter and distance of the pineal gland and its lumen) were measured on serial 7-μm-thick sections. The data were obtained in a morphometer and processed statistically. The intensities of the static electromagnetic fields were 18 and 36 mT. Control and exposed embryos were equally distributed and randomly assigned. After 5 days of incubation, 25% of embryos exposed to a static electromagnetic field of 18 mT had a more advanced stage of development than controls and embryos exposed to 36 mT. On the 10th and 15th day, embryos exposed to either 18 or 36 mT tended to be more developed than controls. In the morphometric study, results were similar for the controls and exposed embryos after 5 and 10 days of incubation. However, the values of the 15-day-old embryos exposed to static magnetic fields were lower than the values of the controls (p > 0.01). These differences were more pronounced in the embryos exposed to 36 mT. These results seem to indicate that static electromagnetic fields affect the development and growth of embryos unequally, and that their action can depend not only on the intensity of the static electromagnetic field, but also on the length of exposure and the organ which is developing. It may be interesting to use these data in ultrastructural and physiological studies.

Transplantation of the chick early eye primordium into a lensectomized optic cup of a host embryo

Eye primordia of young chick embryos (stage XII) were transplanted into lensectomized optic cups of older embryos (stage XVII) to analyze the influence of the host retina on the degree of morphological differentiation attained by the donor lens. Embryos were sacrificed 24-96 h later. The donor lens primordium showed a differentiation more in correlation with the host eye cup (stage XXIII) after 24-96 h of incubation.

Pale and Dark Bipolar Cells in the Chicken Retina

Ultrastructurally, two different bipolar cell types can be distinguished in the retina of the chick embryo: one pale or electron-lucent and the other dark or electron-dense. The different electron density was seen over the whole cell, from its enclave in the outer limiting membrane to its termination in the inner plexiform layer. These observations prompted us to study the content and cytoplasmic variations of both cell types. The pale bipolar cell has a higher vacuole, vesicle and endoplasmic reticulum content and a lower number of microtubules and glycogen than the dark bipolar cell. The presence of these two cell types is probably due to a characteristic physiologic state, and the difference between the pale and dark bipolar cells can be attributed to the diverse number of gap unions which they establish with A II amacrine cells.