Irradiation influences the expression of substance P and enkephalin in the rat larynx

The effects of radiotherapy on neuropeptide expression in the rat larynx were studied. Irradiation was given for five days, 6 or 8 Gray daily. Ten days after the end of irradiation, the larynx, the laryngeal nerves and different ganglia related to the larynx were dissected out from irradiated and control animals and processed for neuropeptide immunohistochemistry. There was an increased immunolabelling for two of the neuropeptides tested, substance P and enkephalin, in the innervation of the subglottic glands and in the acetylcholinesterase-positive ganglionic cells of the local ganglia. These cells were interpreted as representing postganglionic parasympathetic ganglionic cells. The changes seen in the subglottic glands were interpreted as most likely being related to the changing pattern of staining seen in the local ganglia. No changes in substance P-and enkephalin expression were observed in other laryngeal structures, the nodose ganglia, superior cervical ganglia or laryngeal nerve paraganglia. Thus, in certain respects neuropeptide expression in the larynx is modulated by radiotherapy. Since neuropeptides have both neurotransmitter and/or neuromodulator effects in airway tissue and since they show effects as growth factors, the occurrence of this plasticity in neuropeptide expression should be taken into consideration in future studies examining the effects of irradiation on normal/diseased airway tissues.     

Long term effects of lipopolysaccharide on satellite glial cells in mouse dorsal root ganglia

Lipopolysaccharide (LPS) has been used extensively to study neuroinflammation, but usually its effects were examined acutely (24 h<). We have shown previously that a single intraperitoneal LPS injection activated satellite glial cells (SGCs) in mouse dorsal root ganglia (DRG) and altered several functional parameters in these cells for at least one week. Here we asked whether the LPS effects would persist for 1 month. We injected mice with a single LPS dose and tested pain behavior, assessed SGCs activation in DRG using glial fibrillary acidic protein (GFAP) immunostaining, and injected a fluorescent dye intracellularly to study intercellular coupling. Electron microscopy was used to quantitate changes in gap junctions. We found that at 30 days post-LPS the threshold to mechanical stimulation was lower than in controls. GFAP expression, as well as the magnitude of dye coupling among SGCs were greater than in controls. Electron microscopy analysis supported these results, showing a greater number of gap junctions and an abnormal growth of SGC processes. These changes were significant, but less prominent than at 7 days post-LPS. We conclude that a single LPS injection exerts long-term behavioral and cellular changes. The results are consistent with the idea that SGC activation contributes to hyperalgesia.     

Protein expression patterns of identified neurons and of sprouting cells from the leech central nervous system

It has previously been shown that cephalic, segmental, and caudal ganglia from the medicinal leech show differences in their protein composition. Here we studied whether the neuronal reorganization that occurs in cultured segmental ganglia from the medicinal leech is accompanied by detectable changes in the protein expression pattern. Using silver-stained two-dimensional gels we showed that after 5 and 12 days in culture changes in the protein patterns can be detected in isolated ganglia. The changes observed in the two-dimensional gels occurred concomitantly with a sprouting of serotoninergic neurites and a decreased transmitter content of dopaminergic neurites as shown by using the glyoxylic acid condensation reaction. In addition, we present evidence that Retzius cells, which can be identified by their characteristic morphology and action potential waveform, exhibit biochemically unique properties with respect to their protein expression pattern.     

Reorganization of the ventral nerve cord in the moth Manduca sexta (L.) (Lepidoptera : Sphingidae)

Morphology of the ventral nerve cord of the hawkmoth, Manduca sexta (Lepidoptera : Sphingidae), changes at the larval-pupal transition as several separate larval ganglia fuse to form single ganglia characteristic of the adult. We examined in detail the time course of ganglionic fusion. Changes in the relative positions of the ganglia were studied by staining the tissue with methylene or toluidine blue. Alterations in the positions and structure of individual neurons were studied by filling neurons with a cobalt-lysine complex. The first gross morphological change, anterior movement of the first abdominal ganglion, is visible within the first 24 hr after pupal ecdysis. Adult ventral nerve cord morphology is recognizable 6 days later, approximately 12 days before the adult will emerge. The sequence in which the individual ganglia fuse is invariant. During ganglionic fusion, the neuronal cell bodies and associated neuropil move out of their former ganglionic sheath and through the sheath covering the connectives. Axons between the fusing ganglia form loops in the shortening connectives. The presence of looping axons is a morphological feature that identifies the boundaries between ganglia during intermediate stages of fusion. Some individual adult neurons also show looped axons at the boundaries of fused ganglia. These axonal loops may be a valuable morphological marker by which neurons can be characterized as conserved neurons.     

Alterations of the serotonin level in the central nervous system of Locusta migratoria during larval-adult transformation

Serotonin level was measured in the cerebral, suboesophageal, thoracic and abdominal ganglia of Locusta migratoria during larval-adult transformation. It has been established that the serotonin content shows a progressive increase in the period between the last larval stage and the first 7 days of adult life, being of the highest value in the abdominal ganglia (146%), followed by the thoracic (110%), and then by the cerebral and suboesophageal (75%) ganglia. No significant changes are manifested in the serotonin level in the course of moulting. Considerable increase is also detectable in the protein content of the ganglia during the studied development phase. No correlation is present between the protein and serotonin contents of the ganglia, the changes of the protein content was found to be faster as compared to that of serotonin.     

Structure and biometric indices of the lumbar ganglia of the sympathetic trunk of normal cats and cats subjected to hormonal stimulation]

The structure of different components from lumbar ganglia of the cat sympathetic trunk were studied in norm and 6h after intramuscular administration of prednisolone (5 mg/kg). Paraffin sections were stained with azocramine after Heidenhain, Wallart and Houette, azure II -- eosin. Biometry of different ganglial structures was performed by G.G. Avtandilov's dotted method. It was demonstrated that as a response to prednisolone administration, certain morphological changes appeared in all ganglial components. The walls of capillaries grew thicker, endothelial nuclei protruded into their lumen. Collagenous fibres of the connective tissue capsule and ganglial stroma grew thicker and acquired twisted appearance, the amount of the basilar substance increased. Simultaneously, there was a sharp increase of the nuclear section area in perineuronal ganglia. The total area of neuronal sections in the ganglia also increased at the expense of both increasing area of neuronal cytoplasm and hypertrophy of their nuclei.     

Connectivity changes in an isolated molluscan ganglion during in vivo culture

The stability of neuronal connections in the isolated buccal ganglia of Helisoma trivolvis was examined during in vivo culture for periods up to one month. After 4--8 days the characteristic IPSP input to protractor motoneurons (PMNs) was either abolished or reduced in efficacy. This is apparently due to reduced efficacy of chemical synapses, since the input resistance and resting potential of the motoneurons is unchanged and a fraction of spike-evoked IPSPs from premotor neurons (cyberchrons) onto PMNs was absent. PMNs lacking IPSP input nevertheless exhibit vigorous cyclical bursts of action potentials driven by electrical EPSPs. The IPSP of PMNs showed partial or full restoration after 14--32 days of culture despite the lack of reinnervation of normal targets. Existing electrical synapses were apparently more stable during culture, but electrical connections between cyberchrons and PMNs were strengthened. Probably because of the reinforcement of these electrical synapses, regenerative cycles of activity in both cyberchrons and PMNs may often be initiated by brief stimulating of a single PMN in cultured ganglia. This is in marked contrast to normal ganglia in which PMNs possess a limited ability to generate such activity. It is concluded that isolation of the buccal ganglia results in a predictable, functional alteration of its neuronal circuitry. Such a perturbation of connectivity indicates that a significant degree of plasticity can be exhibited by adult molluscan neurons.     

Properties of the beta-nerve growth factor receptor in development

The cell surface receptor for beta-nerve growth factor was used as a probe to study the development of embryonic chick sensory ganglia. The ganglia were shown to lose their responsiveness to nerve growth factor in vitro between 14 and 16 days of embryonic age. This loss occurred by a decrease in the magnitude of the maximum biological response, not by a shifting of the response to higher concentrations. Binding assays for the beta-nerve growth factor receptor, using 125I-radiolabelled beta-nerve growth factor, were performed with cells from sensory ganglia 8, 12, 14, 16, 18, and 21 days of age. The assays revealed a twofold increase in the number of receptor sites per ganglion between 8 and 14 days and a sixfold drop between 14 and 16 days of embryonic life. Neither increase nor decrease was accompanied by a large change in the affinity of the receptor for the protein. Together with the results of the bioassay, the data show that the loss of biological responsiveness is correlated with and may be due to a loss of the cells' ability to bind beta-nerve growth factor. Correlation of the results of the binding assays with the known ontogeny of the chick embryo provides a hint at the role of nerve growth factor in normal development.     

Testosterone,gonadotropins and androgen receptor during spermatogenesis of Biomphalaria alexandrina snails (Pulmonata: Basommatophora)

Endocrine regulation of reproductive processes of the snail Biomphalaria alexandrina is poorly recognized. Thus, the aims of the study were: (1) to acquire histological images of the ovotestis; (2) to determine the hemolymph concentrations of testosterone (T) and gonadotropic hormones (luteinizing hormone: LH and follicle stimulating hormone: FSH), (3) to demonstrate androgen receptor (AR) immunolocalization in the ovotestis, and (4) to show LH and FSH protein expression in cerebral ganglia of small (diameter shell: 4–6 mm), medium (7–11 mm) and large (12–16 mm) B. alexandrina snails. These three groups represented different reproductive stages of the snail. The AR immunoexpression was found in the periphery and inside the acini of small (immature) snails as well as in spermatocytes, spermatids, Sertoli cells, the interstitial cells and the acinus lining epithelium of medium (mature) snails. Low AR immunoexpression was demonstrated in the interstitial cells of large (aged) snails. The neurons at the periphery of the cerebral ganglia and connective sheath of the ganglia showed a positive FSH and LH immunostaining. T concentration in the hemolymph was higher in medium snails than in small and large snails. In contrast, LH concentration was higher in medium snails than in small and large snails. These data suggests that gonadotropins and T play a role in the gonadal development in B. alexandrina.     

Temporal Changes of CB1 Cannabinoid Receptor in the Basal Ganglia as a Possible Structure-Specific Plasticity Process in 6-OHDA Lesioned Rats

The endocannabinoid system has been implicated in several neurobiological processes, including neurodegeneration, neuroprotection and neuronal plasticity. The CB1 cannabinoid receptors are abundantly expressed in the basal ganglia, the circuitry that is mostly affected in Parkinson’s Disease (PD). Some studies show variation of CB1 expression in basal ganglia in different animal models of PD, however the results are quite controversial, due to the differences in the procedures employed to induce the parkinsonism and the periods analyzed after the lesion. The present study evaluated the CB1 expression in four basal ganglia structures, namely striatum, external globus pallidus (EGP), internal globus pallidus (IGP) and substantia nigra pars reticulata (SNpr) of rats 1, 5, 10, 20, and 60 days after unilateral intrastriatal 6-hydroxydopamine injections, that causes retrograde dopaminergic degeneration. We also investigated tyrosine hydroxylase (TH), parvalbumin, calbindin and glutamic acid decarboxylase (GAD) expression to verify the status of dopaminergic and GABAergic systems. We observed a structure-specific modulation of CB1 expression at different periods after lesions. In general, there were no changes in the striatum, decreased CB1 in IGP and SNpr and increased CB1 in EGP, but this increase was not sustained over time. No changes in GAD and parvalbumin expression were observed in basal ganglia, whereas TH levels were decreased and the calbindin increased in striatum in short periods after lesion. We believe that the structure-specific variation of CB1 in basal ganglia in the 6-hydroxydopamine PD model could be related to a compensatory process involving the GABAergic transmission, which is impaired due to the lack of dopamine. Our data, therefore, suggest that the changes of CB1 and calbindin expression may represent a plasticity process in this PD model.     

Morphologic indices of the rat heart after colchicine application to the vagus nerve

By means of the light optic and electron microscopic methods atrial ganglia, myocytes, vessels of the right cardiac chambers have been studied in rats 2 days--3 weeks after application of 100 mcg of colchicine on the right nervus vagus. Certain changes of the neural fibers have been described at the area of the application. In the myocardium the microcirculatory bed, focal edema and hypoxic alterations of the myocyte ultrastructure have been revealed. In the ventrical ganglia destruction of some terminals of the preganglionar fibers, chromatolysis and vacuolization of single neurocytes, as well as intraganglionar granule-containing cells have been found. The changes described take place for 7 days and they nearly completely disappear in 10 days. A suggestion is made that some phenomena, in particular, destruction of the preganglionar fibers and changes of the cardiac microcirculatory bed are connected with certain disturbances of the quick transport of substances in the nervus vagus fibers.     

Ontogenetic development of the brain of the platyhelminth Fasciola hepatica

During ontogenetic development in the definitive host, the cerebral ganglia of the parasitic flatworm Fasciola hepatica lose their cell rind integrity and develop specialized nerve processes. The organization and cytological features of the central nervous system were examined during three developmental stages in the parasitic life cycle of F. hepatica to determine when the changes occur. The cerebral ganglion cell bodies of migrating juvenile worms (5 days post-infection) are organized into a one-cell-thick rind that surrounds a central neuropile composed of small unmyelinated nerve processes (less than 3 microns in diameter). In young, sexually-immature adult worms (30 days post-infection), the cell bodies of the ganglia are no longer organized into a complete or tight cell rind around the ganglia. In addition, large diameter ('giant') unmyelinated nerve processes (greater than 12 microns) are found in the neuropile area. These giant nerve processes are also found in the transverse commissure and the longitudinal nerve cords. In mature adult worms (4-6 months post-infection), the rind of nerve cell bodies has completely disappeared and cell bodies are scattered around and within the neuropile. More than half of the volume of the mature adult neuropile is composed of giant nerve processes. The three developmental stages of the parasite that were used in this study differ significantly in their sizes, behaviours and microhabitat locations in the host. The results suggest that the organizational and morphological changes in the ganglia reflect selective adaptations to changes in the parasitic microenvironment.     

The differential effect of thyrotropin on the electrical responses of thyroid cells in monolayer cultures of varying duration.

The acute effects of thyrotropin on the membrane potential of thyroid cells maintained in the presence or absence of thyrotropin (0.2 U/ml) in the culture medium was determined. Monolayer cultures were prepared from porcine thyroid glands and cultured for 4--17 days after which the culture medium was exchanged for a buffered salt solution for intracellular measurements of the membrane potential. Cells were serially impaled with a microelectrode, first in the absence and then in the presence of 10 mU/ml thyrotropin. Cells cultured for 4--9 days depolarized from --29.6 +/- 1.7 (mean +/- S.E.) to --19.3 +/- 1.3 mV within 10 min after acute addition of 10 mU/ml thyrotropin. From 11 to 17 days of culture, basal membrane potentials were lower and, in most instances, cell hyperpolarization occurred within 30 min in response to thyrotropin. There was no difference in the electrical response of cells maintained in culture with or without thyrotropin. However, cells cultured with thyrotropin formed follicle-like structures in contrast to the monolayer formation of cells cultured without thyrotropin. The changes in the basal and stimulated electrical responses occur within a time frame similar to that reported for changes in the biosynthetic capacity of thyroid cells in culture. The data further emphasize the possible regulatory role of the cell membrane in stimulus-secretion coupling in the thyroid.     

Changes in the elemental content of rat heart as a result of the fixation in formalin analyzed by synchrotron radiation X-ray fluorescent analysis

The aim of the study was to investigate possible changes in the elemental content of small biological tissue samples (with low weight) during the short period of fixation in formalin. Therefore, the effect of the fixation time (4 h to 6 days) was studied on the elemental content of the tissue. Synchrotron radiation X-ray fluorescent analysis (SRXRF) technique was used for the determination of the elemental concentrations. The levels of Cl, K, Ca, Br, Rb, and Sr in the samples fixed for 4 h (and longer) show significant differences in comparison with levels in fresh samples, because these ions have a high mobility and capacity for moving through the cell membrane. The content of Zn, Se, Fe, and Cu in the samples fixed by formalin solution during 4 h do not show significant differences compared with fresh samples. The ions of these elements can form complexes with the large protein molecules in the tissue and can hardly be removed from it.     

Morphometric study of the superior cervical and stellate ganglia of spontaneously hypertensive rats during the prehypertensive stage

To compare the functional state of the superior cervical (SCG) and stellate sympathetic ganglia (SG) of spontaneously hypertensive rats (SHR) with those of age-matched normotensive Wistar Kyoto rats (WKY), ganglion cell volume and area occupied by ganglion cells relative to each whole ganglionic area were morphometrically examined using the Texture Analyse System (TAS) in rats at 0, 10 and 30 days of age. The weight of each ganglion relative to animal weight was also measured. The ganglion cell volume and the relative area of ganglionic cells in both ganglia of SHR were significantly larger (P less than 0.05) than those of age-matched WKY at ages 0 and 10 days after birth. The relative ganglionic weights of SHR were significantly larger (P less than 0.01) compared with those of WKY at all ages examined, except for SG at 0 days after birth. These results show that the relative volume of sympathetic ganglion cells is greater in both SCG and SG of SHR than that of WKY, suggesting that hyperfunction of sympathetic ganglia occurs at the prehypertensive stage as a primary factor in the development of hypertension in SHR.     

Morphometric study of the superior cervical and stellate ganglia of spontaneously hypertensive rats during the prehypertensive stage

To compare the functional state of the superior cervical (SCG) and stellate sympathetic ganglia (SG) of spontaneously hypertensive rats (SHR) with those of age-matched normotensive Wistar Kyoto rats (WKY), ganglion cell volume and area occupied by ganglion cells relative to each whole ganglionic area were morphometrically examined using the Texture Analyse System (TAS) in rats at 0, 10 and 30 days of age. The weight of each ganglion relative to animal weight was also measured. The ganglion cell volume and the relative area of ganglionic cells in both ganglia of SHR were significantly larger (P<0.05) than those of age-matched WKY at ages 0 and 10 days after birth. The relative ganglionic weights of SHR were significantly larger (P<0.01) compared with those of WKY at all ages examined, except for SG at 0 days after birth. These results show that the relative volume of sympathetic ganglion cells is greater in both SCG and SG of SHR than that of WKY, suggesting that hyperfunction of sympathetic ganglia occurs at the prehypertensive stage as a primary factor in the development of hypertension in SHR.     

Effect of spermidine and its metabolites on the activity of pea seedlings diamine oxidase and the problems of biosensing of biogenic amines with this enzyme

Spermidine is one of the several biogenic amines, produced during the microbial decarboxylation of proteins. Individual biogenic amines in the formed mixtures are frequently analyzed with oxygen sensor based biosensors, as their content serves as a good biomarker for the determination of food quality. In these biosensors, diamine oxidase from pea seedlings (PSAO), catalyzing the oxidation of various biogenic amines by dissolved oxygen is commonly used for the bio-recognition of amines. However, in the presence of spermidine and/or its metabolite 1,3-diaminopropane, the activity of PSAO and the sensitivity of PSAO-based biosensors decrease due to inhibition. The inhibition constant of soluble spermidine, acting as an inhibiting substrate toward PSAO, was found to be (40 ± 15) mM in freshly prepared solution and (0.28 ± 0.05) mM in solution, incubated 30 days at room temperature. The inhibition constant of 1,3-diaminopropane, acting as a competitive inhibitor, was (0.43 ± 0.12) mM as determined through the oxidation reaction of cadaverine. The metabolic half-life of soluble spermidine was 7 days at room temperature and 186 days at 4 °C. The kinetic measurements were carried out with an oxygen sensor; the composition of the solution of degraded spermidine was analyzed with MS.     

The effect of zinc and phytoestrogen supplementation on the changes in mineral content of the femur of rats with chemically induced mammary carcinogenesis

The aim of this study was to assess skeletal effects of zinc or zinc with phytoestrogen (resveratrol or genistein) supplementation in an animal model of rats with DMBA-induced mammary carcinogenesis. The changes in bone parameters such as the length and mass were examined, as well as the changes in concentrations of selected minerals: calcium, magnesium, zinc, iron and phosphorus. Moreover, the investigations focused on finding the differences between the levels of iron and zinc in other tissues: the liver, spleen and serum of the examined rats.Fifty-six female Sprague–Dawley rats, 40 days old, were divided into four groups, regardless of the diets: standard (77 mg Zn kg/food), zinc (4.6 mg/mL via gavage), zinc (4.6 mg/mL) plus resveratrol (0.2 mg/kg bw), and zinc (4.6 mg/mL) plus genistein (0.2 mg/kg bw) for a period from 40 days until 20 weeks of age. The study rats were also treated with 7,12-dimethyl-1,2-benz[a]anthracene (DMBA) to induce mammary carcinogenesis.The applied diet and the advanced mammary cancer did not affect macrometric parameters of the rats’ bones, but they strongly affected their mineral content. It was found that mammary cancer, irrespectively of the applied diet, significantly modified the iron level in the femur, liver, spleen and serum of the examined rats. In addition, zinc supplementation significantly lowered the levels of calcium, magnesium and phosphorus in the femur of rats with mammary cancer as compared with respective levels in the control group. So, it was found that additional supplementation with zinc, which is generally considered to be an antioxidant, with the co-existing mammary carcinoma, increased the unfavorable changes as concerns the stability of bone tissue. The appropriate combination of zinc and phytoestrogens (resveratrol or genistein) could help prevent or slow bone loss associated with a range of skeletal disorders in breast cancer.     

Ganglia implantation as a means of supplying neurotrophic stimulation to the newt regeneration blastema: cell-cycle effects in innervated and denervated limbs.

Regulation of blastema cell proliferation during amphibian limb regeneration is poorly understood. One unexplained phenomenon is the relatively low level of active cell cycling in the adult newt blastema compared to that of larval axolotls. In the present study, we used ganglia implantation as a means of "superinnervating" normally innervated adult newt blastemas to test whether blastema cell subpopulations are responsive to nerve augmentation. The effectiveness of implanted ganglia to provide neurotrophic stimulation was demonstrated in denervated blastemas. Blastemas implanted with 2 dorsal root ganglia and simultaneously denervated 14 days after amputation exhibited control levels of cell cycle activity 6 days later, as measured by 3H-thymidine pulse labeling. Denervated blastemas that were sham-operated or implanted with pituitary glands exhibited cell-cycle declines similar to those of denervated blastemas without implanted ganglia. Thus, 2 implanted ganglia provide neurotrophic stimulation equivalent to that of the normal nerve supply. Dorsal root ganglia implanted into normally innervated blastemas, which should effectively double neurotrophic activity to the blastema, had no effect on cell-cycle activity, innervated blastemas implanted with ganglia for 6 days exhibited pulse labeling indices similar to those of normally innervated blastemas. These data indicate that neurotrophic stimulation is not normally limiting in innervated limbs, and that some other factor, whether extrinsic or intrinsic to blastema cells, accounts for the relatively low level of active cell cycling in the adult newt blastema.