Effects of the relationship between 65Zn and blood cells. A dynamic and morphological study.

We have studied the dynamic pathway of 65Zn and its autoradiographic location in blood cells, even at the ultra-structural level. We have found evidence that tends to confirm the old biochemical postulates about the capacity of this isotope to displace iron in the haemoglobin molecule. Recently, the bibliography has demonstrated that 57Co is also able to perform this displacement, but unlike 65Zn it does not invalidate the Redox function of the molecule. In the case of 65Zn, the mentioned displacement invalidates this function because the radionuclide can only use valence 2. We have also contributed evidence of erythrocytes destruction by the spleen after the incorporation of 65Zn, as well as the clearly marked degradation of haematic pigments inside the spleen.

Exposure to ELF-pulse modulated X band microwaves increases in vitro human astrocytoma cell proliferation.

Common concern about the biological effects of electromagnetic fields (EMF) is increasing with the expansion of X-band microwaves (MW). The purpose of our work was to determine whether exposure to MW pulses in this range can induce toxic effects on human astrocytoma cells. Cultured astrocytoma cells (Clonetics line 1321N1) were submitted to 9.6 GHz carrier, 90% amplitude modulated by extremely low frequency (ELF)-EMF pulses inside a Gigahertz Transversal Electromagnetic Mode cell (GTEM-cell). Astrocytoma cultures were maintained inside a GTEM-incubator in standard culture conditions at 37+/-0.1 degrees C, 5% CO2, in a humidified atmosphere. Two experimental conditions were applied with field parameters respectively of: PW 100-120 ns; PRF 100-800 Hz; PRI 10-1.25 ms; power 0.34-0.60 mW; electric field strength 1.25-1.64 V/m; magnetic field peak amplitude 41.4-54.6 microOe. SAR was calculated to be 4.0 x 10-4 W/Kg. Astrocytoma samples were grown in a standard incubator. Reaching 70-80% confluence, cells were transferred to a GTEM-incubator. Experimental procedure included exposed human astrocytoma cells to MW for 15, 30, 60 min and 24 h and unexposed sham-control samples. Double blind method was applied. Our results showed that cytoskeleton proteins, cell morphology and viability were not modified. Statistically significant results showed increased cell proliferation rate under 24h MW exposure. Hsp-70 and Bcl-2 antiapoptotic proteins were observed in control and treated samples, while an increased expression of connexin 43 proteins was found in exposed samples. The implication of these results on increased proliferation is the subject of our current research.

Characterization by immunocytochemistry of ionic channels in Helix aspersa suboesophageal brain ganglia neurons.

The aim of this work was to characterize several ionic channels in nervous cells of the suboesophageal visceral, left and right parietal, and left and right pleural brain ganglia complex of the snail Helix aspersa by immunocytochemistry. We have studied the immunostaining reaction for a wide panel of eleven polyclonal antibodies raised against mammal antigens as follows: voltage-gated-Na+ channel; voltage-gated-delayed-rectifier-K+ channel; SK2-small-conductance-Ca2+-dependent-K+ channel apamin sensitive; SK3 potassium channel; charybdotoxin-sensitive voltage-dependent potassium channel; BKCa-maxi-conductance-Ca2+-dependent-K+ channel; hyperpolarization-activated cyclic nucleotide-gated potassium channel 4; G-protein-activated inwardly rectifying potassium channel GIRK2 and voltage-gated-calcium of L, N and P/Q type channels. Our results show positive reaction in neurons, but neither in glia cells nor in processes in the Helix suboesophageal ganglia. Our results suggest the occurrence of molecules in Helix neurons sharing antigenic determinants with mammal ionic channels. The reaction density and distribution of immunoreactive staining within neurons is specific for each one of the antisera tested. The studies of co-localization of immunoreaction, on alternate serial sections of the anterior right parietal ganglion, have shown for several recognized mapped neurons that they can simultaneously be expressed among two and seven different ionic protein channels. These results are considered a key structural support for the interpretation of Helix aspersa neuron electrophysiological activity.

Localization of connexins in neurons and glia cells of the Helix aspersa suboesophageal brain ganglia by immunocytochemistry.

The aim of the present study was to examine the distribution of cells expressing connexin 26 (Cx26) in the suboesophageal visceral, left and right parietal and left and right pleural ganglia of the snail Helix aspersa by immunocytochemistry. Altogether we have found approximately 452 immunoreactive neurons which represent the 4.7% of the total neurons counted. The stained large neurons (measured diameter 55-140 microm) occurred mostly on the peripheral surface of the ganglia while the small immunostained cells (5-25 microm diameter) were observed in groups near the neuropil. The number of large neurons giving positive Cx26-like immunostaining was small in comparison with that for medium (30-50 microm diameter) and small sized cells. The expression of Cx26 was also observed in the processes of glia cells localized among neurons somata and in the neuropil showing that the antiserum recognized epitopes in both protoplasmic and fibrous glia cells of Helix aspersa. The neuropils of all ganglia showed fibers densely immunostained. While we have observed a good specificity for Cx26-antiserum in neurons, a lack of reaction for Cx43 antiserum was observed in neurons and glia cells. The reaction for enolase antiserum in neurons was light and non-specific and a lack of reaction in glia cells and processes for GFAP antiserum was observed. Although the percentage of positive neurons for Cx26 antiserum was low is suggested that in normal physiological conditions or under stimulation the expression of connexin could be increased. The observed results can be considered of interest in the interpretation of Helix aspersa elemental two neuron networks synchronizing activity, observed under applied extremely low frequency magnetic fields.

S-100 protein immunoreactivity in the upper eyelid of the sheep Ovis aries.

The aim of this work was to analyse the distribution pattern of S-100-immunoreactive elements in the upper eyelid of the sheep. This pattern may be of importance regarding the diagnosis and prognosis of eyelid tumours that are linked to deregulation of S-100 gene expression. Thirty upper eyelids taken from 15 adult male Ovis aries were studied by means of the peroxidase-antiperoxidase method for light microscopy. S-100-immunopositive cells were found in the eyelid edge. S-100-immunopositive steams and thinner fibres were found throughout the eyelid. These nerve processes typically were denser around glands, hair follicles and blood vessels. S-100-immunopositive elements may play a role as neuromodulator and also in the development of the vegetative innervation of the epithelium and its derivatives.

65Zn uptake in the rat cerebellum and brainstem.

We have studied the autoradiographic uptake of 65Zn in the cerebellum and brainstem of the rat, contrasting these results with Timm's positivity in these structures. Both, autoradiographic uptake and histochemical positivity, have demonstrated Zinc in a location that could be accepted as in climbing fibres and glomeruli of the cerebellum cortex, and also in brainstem neurons that project their axons to the cerebellum cortex, suggesting a circuit where zinc may act as a neuromodulator.

Location of zinc and 65Zn in spinal ganglia of the rat.

Following the works of Velazquez et al. (1999), Jo-Seung et al. (2000), Wang et al. (2001), Danscher et al. (2001) and the criteria of Zinc-containing neurons established by Frederickson et al.(2000), we have found the presence and localisation of Zinc in the neurons of the dorsal root ganglia of Wistar rat, by using Timm's thecnique and by studying the autoradiographic uptake of 65Zn. The agreement between the results of both techniques allows us to classify these spinal ganglion neurons as Zinc-containing neurons and also, to confirm some of the results of Velazquez et al. (1999).

Acetylcholinesterase-positive and paraformaldehyde-induced-fluorescence-positive innervation in the upper eyelid of the sheep (Ovis aries).

This is the first study which describes the innervation of some eyelid structures, such as the glands of Moll and the glands of Zeiss. It is also the first to investigate the innervation pattern of the eyelid as a whole. We have studied the acetylcholinesterase-positive and paraformaldehyde-induced-fluorescence-positive (FIF+) innervation pattern of the different structures that constitute the upper eyelid of the sheep. There is widespread acetylcholinesterase-positive innervation in the epithelium, but not such an abundant FIF+ innervation. Both types of innervation are represented in the connective tissue by trunks or fibers that are distributed towards the different structures immersed within them. In the glands of Zeiss, cholinesterase-positive innervation is much more widespread than FIF innervation. On the contrary, the glands of Moll present denser FIF+ innervation than acetylcholinesterase-positive innervation. The Meibomian glands and the lachrymal glands show a rich acetylcholinesterase-positive and FIF+ innervation. Eyelid muscle innervation is mainly acetylcholinesterase-positive. In the conjunctive membrane there is no acetylcholinesterase-positive innervation, and only scarce FIF+ fibers can be demonstrated.

Innervation of the chicken esophagus wall: localization and distribution of the acetylcholinesterase-positive nervous elements and ultrastructure / Inervación de la pared esofágica del pollo: localización y distribución de los elementos nerviosos positivos a la acetilcolinesterasa y su ultraestructura

We have studied the distribution pattern of the acetylcholinesterase-positive innervation of the four-week-old chicken esophagus. We localized the different innervation plexuses and their inter-connections on sections of tissue samples. Later, we observed the three-dimensional structure of the plexuses by means of histochemical methods on whole-mount preparations, after delaminating the esophagus wall. We also studied the components of the nervous plexuses by optic microscopy on semi-thin cuts stained with toluidin blue, and studied their ultrastructure by transmission electron microscopy. In the myenteric plexus we observed compact ganglions between both muscle layers, as well as intra and inter-fascicular neuronal cell bodies in the circular muscle layer. Nervous trunks and fibers distribute along the connective tissue among the muscular bundles. The submucous plexus was found to be less abundant in nervous elements titan the myenteric plexus, and its ganglions were smaller. In the lamina propria abundant AChE+ fibers, which innervate the glands at their base, and in the surroundings of the tubules up to the epithelium were seen. On whole-mount preparations we describe that the ganglions of the myenteric plexus are larger and form a denser network than in the submucous plexus. TEM revealed varicosities with cholinergic, adrenergic, peptidergic and mixed vesicles (AU)

En este trabajo hemos estudiado el patrón de distribución de la inervación acetilcolinesterasa-positiva (AChE+) del esófago de pollos de cuatro semanas de edad. Localizamos los distintos plexos de inervación y sus interconexiones sobre secciones de muestras de tejido. Posteriormente, observamos la estructura tridimensional de los plexos mediante métodos histoquímicos en preparaciones después de deslaminar la pared esofágica. También estudiamos los componentes de los plexos nerviosos mediante microscopía óptica sobre cortes semifinos teñidos con azul de toluidina y estudiamos su ultraestructura mediante microscopía electrónica de transmisión (MET). En el plexo mientérico, observamos ganglios compactos entre ambas capas musculares así como somas neuronales intra- e inter-fasciculares en la capa circular de músculo. Troncos y fibras nerviosas se distribuyen a lo largo del tejido conectivo entre los haces musculares. Se vio que el plexo submucoso contenía menos elementos nerviosos que el plexo mientérico y sus ganglios eran más pequeños. En la lámina propia, se apreciaron abundantes fibras AChE-positivas; éstas inervaban las glándulas en sus bases y también en la vecindad de los túbulos hasta el epitelio.En las preparaciones microscópicas completas describimos que los ganglios del plexo mientérico son más grandes y que forman una red más densa que en el plexo submucoso. El estudio con MET reveló la presencia de varicosidades con vesículas colinérgicas, adrenérgicas, peptidérgicas y mixtas (AU)

Histochemical, immunohistochemical, and electron microscopy study of the caudal portion of the chicken intestinal nerve of Remak.

In order to deepen our knowledge of the different components of the chicken intestinal nerve of Remak (I.N.R.), we have studied it by means of histochemical, immunohistochemical and electron microscopy techniques to distinguish the different neurotransmitters. We have found cholinergic cell bodies, as well as acetylcholinesterase (AChE) positive neuronal fibers, forming part of the web that constitutes the I.N.R. in its caudal portion, with a higher density of neuronal bodies in the ganglia. We also observed catecholaminergic neuronal bodies and fibers, located fundamentally in the periphery of the nerve, and a low density of catecholaminergic cell bodies. With respect to the vasoactive intestinal peptide (VIP) and substance P (SP) positive peptidergic innervation, we found more abundant neuronal bodies positive for the V.I.P. than for S.P. Electron microscopy corroborated the results observed under the optic microscope, showing the various types of vesicles containing different neurotransmitters.

Intrinsic innervation of the chicken lower digestive tract.

We have studied the different components of the enteric nervous system in the rectum and cloaca of the chicken by means of histochemical and immunohistochemical techniques. We found cholinergic neuronal bodies as well as nervous fibers, which constitute part of the Meissner and Auerbach plexuses. We also observed plentiful catecholaminergic fibers in both plexuses, though there were no catecholaminergic neuronal bodies. With respect to the Vasoactive Intestinal Peptide (VIP) and substance P (SP) positive peptidergic innervation, only positive fibers were found, which were less abundant than in the other zones of the gastrointestinal tract. The optic microscopy results were confirmed by electron microscopy.

Zinc in hypothalamus and hypophysis of the rat.

Zinc has been located using both histochemical and autoradiographic procedures in the neurons of the nuclei of the hypothalamic medial area and in some adenohypophisary cells. Some suggestions about the functional significance of the presence of Zn in these places are made.

65Zn in studies of the neurobiology of zinc.

The presence of zinc in the mossy fibre system of the Hippocampus is the most thoroughly studied of zinc relation to the CNS, but many other areas of the CNS are zinc-containing. Many methods have been used in order to investigate the above mentioned relation, most of them being based on histochemistry and physical measurement. 65Zn trace has also been used, but scarcely, probably due to the difficulty of radioisotope handling. In the present review we focus on 65Zn studies in the CNS and comment on their advantages and disadvantages.

Presence and possible function of Zn in the hyaline cartilage.

Using histochemical and autoradiographical techniques the location of Zn in the hyaline cartilage of the Wistar rat was studied. A triple location was observed in the territorial matrix; in the chondrocyte; and in the perichondrio (cellular area). We believe that the molecules which were observed carrying Zn could be alkaline phosphatase, timidin-kinase and chondroitin-sulphuric acid.

Location of Zn in the testicle of the rat.

We studied the location of Zn in the testicle of the Wistar rat using the Timm histochemical technique, autoradiography with 65Zn and the dynamic pathway of 65Zn in the organ. Locations of Zn in several cellular components of the spermatogenic chain and in the Leydig cells were observed. This location was proved statistically by a quantitative analysis of the autoradiographs. The presence of Zn in the testicle, prostate and the adrenal gland could be related to the activation of the enzyme beta-hydroxy-steroid dehydrogenase although this fact does not exclude any other function of this metal, as far as we know.