Autoradiographic analysis of [3H]dopamine and [3H]dopa uptake in the turtle olfactory bulb

Uptake and retention of exogenous tritiated dopamine and L-dopa was observed within turtle olfactory bulb slices. In the more superficial layers, periglomerular and superficial tufted cells, as well as their processes, and intraglomerular dendrites were recognized as labeled. Within the deeper part of the bulb, some labeled cells between the tanycytes, as well as nerve fibers and terminals within the granule cell layer, are reported. The results confirm the presence of specific intrinsic dopaminergic cells within the reptilian olfactory bulb.     

Prognostic factors in the prediction of chronic wound healing by electrical stimulation

The aim of the study is to determine the effects of wound, patient and treatment attributes on the wound healing rate and to propose a system for wound healing rate prediction. Predicting the wound healing rate from the initial wound, patient and treatment data collected in a database of 300 chronic wounds is not possible. After considering weekly follow-ups, it was determined that the best prognostic factors are weekly follow-ups of the wound healing process, which alone were found to predict accurately the wound healing rate after a minimum follow-up period of four weeks (at least five measurements of wound area). After combining the follow-ups with wound, patient and treatment attributes, the minimum follow-up period was reduced to two weeks (at least three measurements of wound area). After a follow-up period of two weeks, it was possible to predict the wound healing rate of an independent test set of chronic wounds with a relative squared error of 0.347, and after three weeks, with a relative squared error of 0.181 (using regression trees with linear equations in its leaves). Regression trees with a relative squared error close to 0 produce better prediction than with an error closer to 1. Results show that the type of treatment is just one of many prognostic factors. Arranged in order of decreasing prediction capability, prognostic factors are: wound size, patient's age, elapsed time from wound appearance to the beginning of the treatment, width-to-length ratio, location and type of treatment. The data collected support former findings that the biphasic- and direct-current stimulation contributes to faster healing of chronic wounds. The model of wound healing dynamics aids the prediction of chronic wound healing rate, and hence helps with the formulation of appropriate treatment decisions.     

The pro-fibrogenic effect of nerve growth factor on conjunctival fibroblasts is mediated by transforming growth factor-β

BACKGROUND: Nerve growth factor (NGF) and nerve growth factor receptor (NGFR) expressions have been found to be increased in sub-conjunctival scarring. OBJECTIVE: The aim of this study was to investigate the in vitro effects of NGF on some pro-fibrogenic properties of human conjunctival fibroblasts. METHODS: Expression of NGF, trkA(NGFR) and p75NTR on human fibroblasts grown from conjunctival biopsies and incubated for 2 or 6 days with NGF were evaluated by immunofluorescence, RT-PCR, flow cytometry and ELISA. The fibrogenic effect of NGF on conjunctival fibroblasts was investigated by evaluating their migration (wound model), proliferation ([3H]-thymidine incorporation), collagen production (3H]-proline incorporation), expression of alpha-smooth muscle actin (alpha-SMA) (cell surface ELISA) and contraction of 3D collagen gels. RESULTS: NGF induced the expression of p75NTR in the fibroblasts that constitutively expressed only trkA(NGF) and increased the migration of wounded fibroblasts, but not their proliferation and collagen production. NGF induced the conversion of fibroblasts into myofibroblasts expressing alpha-SMA, and enhanced their contraction of a collagen matrix. Interestingly, chronic NGF treatment induced transforming growth factor-beta1 (TGF-beta1) production by fibroblasts, and following specific TGF-beta neutralization, all the NGF-induced effects were completely abrogated. CONCLUSION: Our findings indicate that NGF, via TGF-beta induction, is likely to be involved in the healing or fibrotic processes occurring in conjunctiva during some pathological conditions.     

Lactate and pyruvate increase the incorporation of [3H]proline into collagen [3H]hydroxyproline in liver slices of CCl4 cirrhotic rats

Lactate and pyruvate enhanced the incorporation of [3H]proline into collagen [3H]hydroxyproline when added to liver slices of CCl4-treated rats. In addition, pyruvate stimulated the accumulation of cAMP, reaching maximum values after 10 minutes of incubation. Similar results were obtained with newborn rat calvariae, a tissue that normally produces large amounts of type I collagen. In normal liver, which produces relatively small amounts of collagen, lactate had no effect on cAMP levels or collagen synthesis. Pyruvate stimulated the accumulation of cAMP, but had no effect on collagen synthesis. These results indicate that different control mechanisms are involved in regulation of collagen biosynthesis in normal as compared with cirrhotic liver; the latter resembling mesenchymal tissues specialized in collagen production such as newborn rat calvariae.     

Autoradiographic localization of [3H]RU 486 and [3H]progesterone in the uterus, pituitary and hypothalamus of the rat

Twenty-one castrated oestrogen-primed Wistar rats, which were2-months-old, were injected via the jugular vein with 100 µCi/100g body weight of [³H]RU 486 or [³H] progesterone. Some of thesereceived unlabelled compounds for competition studies. Samplesof reproductive tract, pituitary and hypothalamus were excisedafter 15 min. The 4-µm frozen sections were processedfor thaw-mounted autoradiography. The exposure time of the autoradiogramwas {small tilde}6 months. After the injection of [³H]RU 486and [³H]progesterone, the nuclear concentration of radioactivitywas most distinct in muscular and stromal cells of the uterus,and the epithelial nuclei of lumina and glands showed weak labelling.Nuclear localization was also observed in muscle cells of thevagina, cervix and oviduct. After injection of [³H]progesterone,the radioactivity was found in the nuclei and cytoplasm of anteriorpituitary cells and some cells showed a preferential nuclearconcentration of radioactivity. The distribution of [³H]RU 486in the anterior pituitary was more extensive than that of [³H]progesterone.In the hypothalamus, specific localization of [³H]RU 486 and[³H]progesterone existed in neurones accumulated in the preopticnucleus, preoptic suprachiasmatic nucleus and the periventricularnucleus. No localization was found in the diaphragm. Pretreatmentwith RU 486, but not with dexamethasone, reduced the nuclearconcentration of radioactivity of [³H]progesterone in the vagina,uterus, oviduct, pituitary and hypothalamus. The nuclear concentrationof radioactivity after injection of [³H]RU 486 was also decreasedby preinjection with progesterone. The autoradiographic resultssuggest that RU 486 and progesterone competed for the specificbinding site (possibly a progesterone receptor) in the targetcells at the levels of the uterus, pituitary and hypothalamusin vivo.     

Autoradiographic localization of [3H]RU 486 and [3H]progesterone in the uterus, pituitary and hypothalamus of the rat

Twenty-one castrated oestrogen-primed Wistar rats, which were 2-months-old, were injected via the jugular vein with 100 microCi/100 g body weight of [3H]RU 486 or [3H]progesterone. Some of these received unlabelled compounds for competition studies. Samples of reproductive tract, pituitary and hypothalamus were excised after 15 min. The 4-micron frozen sections were processed for thaw-mounted autoradiography. The exposure time of the autoradiogram was approximately 6 months. After the injection of [3H]RU 486 and [3H]progesterone, the nuclear concentration of radioactivity was most distinct in muscular and stromal cells of the uterus, and the epithelial nuclei of lumina and glands showed weak labelling. Nuclear localization was also observed in muscle cells of the vagina, cervix and oviduct. After injection of [3H]progesterone, the radioactivity was found in the nuclei and cytoplasm of anterior pituitary cells and some cells showed a preferential nuclear concentration of radioactivity. The distribution of [3H]RU 486 in the anterior pituitary was more extensive than that of [3H]progesterone. In the hypothalamus, specific localization of [3H]RU 486 and [3H]progesterone existed in neurones accumulated in the preoptic nucleus, preoptic suprachiasmatic nucleus and the periventricular nucleus. No localization was found in the diaphragm. Pretreatment with RU 486, but not with dexamethasone, reduced the nuclear concentration of radioactivity of [3H]progesterone in the vagina, uterus, oviduct, pituitary and hypothalamus. The nuclear concentration of radioactivity after injection of [3H]RU 486 was also decreased by preinjection with progesterone. The autoradiographic results suggest that RU 486 and progesterone competed for the specific binding site (possibly a progesterone receptor) in the target cells at the levels of the uterus, pituitary and hypothalamus in vivo.     

Autoradiographic localization of binding sites for [3H]gamma-aminobutyrate, [3H]muscimol,(+)[3H]bicuculline methiodide and [3H] flunitrazepam in cultures of rat cerebellum and spinal cord

Cultures of rat cerebellum and spinal cord were used to visualize binding sites for [³Hγaminobutyrate, [³Hmuscimol, [³Hbicuculline methiodide and [³Hflunitrazepam by autoradiography. In cerebellar cultures, many large neurons (presumably Purkinje cells) and interneurons were labelled. In spinal cord cultures, these compounds were mainly bound to small and medium-sized neurons, whereas the majority of large neurons were unlabelled. No binding sites for these radioligands were found on glial cells. Binding of [³Hγ-aminobutyrate, [³Hmuscimol and [³Hbicuculline methiodide was markedly reduced or inhibited by adding unlabelled γ-aminobutyrate, muscimol and bicuculline (10^?3m) respectively to the incubation medium. Addition of a thienobenzazepine markedly reduced binding with [³Hflunitrazepam.It is concluded that tissue cultures are an excellent tool to visualize the cellular localization of binding sites for neurotransmitters and drugs using autoradiography.     

Regional differences in protein and glycoprotein synthesis and their processing in the mouse brain as revealed by the incorporation of [3H]proline, N-6-[3H]acetyl-D-glucosamine and [3H]fucose

The incorporation rate of [3H]fucose, N-6-[3H]acetyl-D-glucosamine and [3H]proline has been compared in five regions of the mouse brain on postnatal day 6. The olfactory bulbs and the cerebellum showed a prevalence of incorporation of [3H]fucose over other brain regions. Less expressed, but still well evident regional differences were observed in [3H]proline incorporation while the incorporation of N-6-[3H]acetyl-D-glucosamine was almost equal in all brain regions. The regional differences were also apparent after considering an actual pool of free isotopes in the individual regions. Gel electrophoresis of [3H]fucose-labelled membrane fraction showed that the higher incorporation of [3H]fucose in the olfactory bulbs is partly due to higher synthesis of low molecular weight glycoproteins, especially in the molecular range of 30,000. The data showed that the protein synthesis and fucosylation, and/or a fast transport of the corresponding molecules, varies more within the brain than the incorporation of N-6-[3H]acetyl-D-glucosamine and possibly also than the "core" part of glycan molecule synthesis.     

Autoradiographic comparison of [3H](-)nicotine, [3H]cytisine and [3H]epibatidine binding in relation to vesicular acetylcholine transport sites in the temporal cortex in Alzheimer's disease

The laminar binding distribution of three nicotinic receptor agonists, [3H](-)nicotine, [3H]cytisine, and [3H]epibatidine, and their relation to the [3H]vesamicol binding, which is known to represent the vesicular acetylcholine transport sites, was performed employing in vitro autoradiography on the medial temporal cortex (Brodmann area 21). Autopsied brain tissue from nine Alzheimer patients and seven age-matched controls were used. The binding pattern of the three nicotinic ligands in the normal cortex was in general similar, showing binding maxima in the cortical layers I, III and V. The binding of [3H](-)nicotine, [3H]cytisine, and [3H]epibatidine was lower in the older controls and more uniform throughout the layers as compared with younger controls. There was a significant age-related decrease in the binding of the three nicotinic ligands within the controls (age range: 58 to 89 years; P[3H](-)nicotine = 0.002, P[3H]epibatidine = 0.010, P[3H]cytisine = 0.037). In the older controls, the [3H]epibatidine binding was much decreased as compared with that of [3H](-)nicotine and [3H]cytisine. This may indicate a higher selectivity of [3H]epibatidine for a nicotinic receptor subtype that is particularly affected by aging. The laminar binding pattern of [3H]vesamicol showed one maximum in the outer cortical layers II/III. The [3H]vesamicol binding did not change with aging. The binding of all ligands was significantly decreased in all layers of the temporal cortex in Alzheimer's disease, but the [3H]vesamicol binding decreased only half as much as the nicotinic receptors. Also, choline acetyltransferase activity was percentually more reduced than [3H]vesamicol binding in Alzheimer's disease. The cortical laminar binding pattern of all 3H-ligands was largely absent in the Alzheimer's disease cases. The less severe loss of vesicular acetylcholine transport sites as compared with the loss of the nicotinic receptors and choline acetyltransferase activity may suggest that vesamicol binding sites might be more preserved in presynaptic terminals still existing and thereby expressing compensatory capacity to maintain cholinergic activity.     

负极性驻极体与5-氟尿嘧啶对大鼠创面愈合的影响

目的:比较不同表面电位的负极性驻极体与5-氟尿嘧啶(5-FU)对大鼠创面愈合的影响。方法:利用电晕充电技术与药剂学方法制备不同表面电位的负极性驻极体贴剂、5-FU贴剂、-2 000 V驻极体5-FU贴剂,借助等温表面电位测量、光学显微镜等手段,研究负极性驻极体贴剂与-2 000 V驻极体5-FU贴剂外静电场的稳定性以及上述三类贴剂对大鼠创面愈合的影响。结果:不同表面电位负极性驻极体贴剂与-2 000 V驻极体5-FU贴剂的等效表面电位随时间增加,均按指数规律衰减,能够给创面提供较为稳定的外静电场;不同表面电位负极性驻极体贴剂对创面愈合具有促进作用,而且等效表面电位越大,创面愈合越快;5-FU对创面愈合具有抑制作用,而将-2 000 V驻极体与5-FU联用可减轻5-FU对创面愈合的抑制作用。结论:负极性驻极体对创面愈合有促进作用,5-FU能延缓创面愈合,若合理利用两者,可以调控创面愈合和抑制增生性瘢痕生长。     

The effect of 5-fluorouracil on [3H]nucleoside incorporation into the DNA of mouse embryos and maternal tissues

Pregnant albino mice (ICR) were administered, ip, 40 mg/kg 5-fluorouracil (5-FU) on Day 10 of gestation. This dosage produced 96.3% embryolethality and 100% of the surviving fetuses were malformed. This dosage also produced a pronounced inhibition of 6-[³H]dUrd incorporation into the DNA of the embryo, chorioallantoic placenta, maternal intestine, and maternal spleen. There was no recovery in the embryo or placenta over the 48 hr time period analyzed. In the maternal tissues, however, incorporation levels returned to control values by 48 hr after 5-FU administration. 5-FU had no effect on 6-[³H]dThd incorporation into the DNA of any of the mitotically active tissues with the exception of the embryo at the 48 hr time point. Concomitant histological studies of the embryo neural tube showed that 40 mg/kg 5-FU produced mitotic inhibition 2 hr after treatment. Cell death began at 4 hr with heteropycnosis in the DNA synthetic zone of the primitive ependymal layer. As with the incorporation experiment, there was no histological recovery in the neural epithelium and the pycnosis became progressively more severe with time after 5-FU treatment.     

Autoradiographic and morphological studies on the uptake of the triglyceride [3H]-glyceroltrioleate by acanthocephalans

Two eoacanthocephalans, four palaeacanthocephalans, including the infective larvae of one species, and one archiacanthocephalan were exposed in vitro to a labelled triglyceride. The nutrient was almost exclusively incorporated by the tegument of the presoma. The uptake of [³H]-glyceroltriolete started at the anterior half of the presoma, followed by a consecutive labelling of the entire presoma tegument and the lemnisci. In the archiacanthocephalan, however, the general uptake was comparatively low. In the final host of the acanthocephalans, the absorbed nutrient must derive from the host's intestinal wall, injured by the parasite. In the eoacanthocephalans and the palaeacanthocephalans, the labelled nutrient was most intensively taken up by the tegument of the hooks. Inside the hook tegument, the basal membrane and the outer membrane form a labyrinth of entangling crypts and protuberances. In the surrounding proboscis tegument as well as in the neck tegument, the lipid seemed to be transported along these two membranes. In addition to its absorption by the presoma tegument, the labelled nutrient was intensively incorporated by the apical organ and the paired lateral organ of the eoacanthocephalan presoma and by the terminal part of the uterine endothelium of all female eoacanthocephalans and palaeacanthocephalans. The use of the nutrient by the parasites is discussed.Supported by a grant from the Deutsche Forschungsgemeinschaft (DFG)     

Autoradiographic localization of binding sites for [3H]histamine and H1- and H2-antagonists on cultured neurones and glial cells

By means of autoradiography we have studied the cellular localization of binding of [3H]histamine and H1- and H2-antagonists in explant cultures of rat cerebellum, brain stem and spinal cord. In brain stem and spinal cord cultures, a relatively great number of neurones revealed binding sites for [3H]histamine and to a lesser extent also for the H1-antagonist [3H]pyrilamine and for the H2-antagonist [3H]tiotidine. In contrast, only a small number of labelled neurones was found in cerebellar cultures. The intensity of labelling was usually much stronger for [3H]histamine than for its antagonists, suggesting that binding sites for histamine might reflect both H1- and H2-receptors. Glial cells also showed binding sites for [3H]histamine and the H1- and H2-antagonists, the number of labelled astrocytes by these radioligands was, however, smaller than that observed with [3H]noradrenaline and alpha- and beta-adrenergic antagonists. It is suggested that in addition to alpha- and beta-adrenoceptors, glial cells also possess receptors for histamine.     

Specific binding of [3H]diazepam in mouse glioblastoma: the influence of clonazepam and Ro 5-4864 on [3H]diazepam binding

The reversible specific binding of [3H]diazepam was observed by a radioligand method in homogenates of cultured cells of mouse glioblastoma. It was characterized by an equilibrium dissociation constant Kd = 91 +/- 5 nM and the number of maximal binding sites (Bmax) of 1006 +/- 100 fmol/mg protein. The half-saturation and half-degradation periods for the ligand-receptor complex were 15 and 10 s, respectively. The specific binding sites from glioblastoma are similar to the peripheral-type receptors as their inhibition constant for Ro 5-4864 Ki = 16 nM and that for clonazepam Ki = 30 microM.     

Autoradiographic analysis of cyclic adenosine monophosphate phosphodiesterase using [3H]rolipram in the postischemic rat brain.

Postischemic alteration of cyclic adenosine monophosphate phosphodiesterase was investigated in the rat brain, using [3H]rolipram in vitro autoradiography. After 90 min of middle cerebral artery (MCA) occlusion, [3H]rolipram binding sites decreased rapidly in the brain areas supplied by the occluded MCA. Moreover, 3 days after the ischemia, significant decreases of [3H]rolipram binding sites were observed in the thalamus and the substantia nigra, both areas had not been directly affected by the original ischemic insult. These results suggest that alteration of second messenger pathways may be involved in neuronal degeneration caused by transsynaptic process and that alteration of intracellular signal transduction may precede the neuronal damage in the exo-focal postischemic brain areas.     

Autoradiographic distribution of phencyclidine receptors in the rat brain using [3H]1-(1-(2-thienyl)cyclohexyl)piperidine ([3H]TCP)

The distribution of phencyclidine (PCP) receptors in the rat brain was determined by autoradiography using 1-(1-(2-thienyl)cyclohexyl)piperidine ([3H]TCP). [3H]TCP appeared to bind to PCP receptors as only PCP-like drugs and sigma-opioids inhibited the binding of [3H]TCP. The areas of the rat brain with the highest density of radiolabeled binding sites were the superficial layers of cerebral cortex, hippocampus and dentate gyrus. Moderate densities of binding sites were found in the medial geniculate nuclei, caudate nucleus, nucleus accumbens, interpeduncular nucleus, superior colliculus, periaqueductal gray and cerebellum. Low densities of binding sites were observed in spinal cord, most of the brainstem, the substantia nigra and most of the hypothalamus.