Pontamine Sky Blue Research Articles

Method for identifying adrenergic nervous elements by the glyoxylic method using pontamine sky blue

Method for identifying adrenergic nervous elements by the glyoxylic method using pontamine sky blue

Infiltration of neutrophil polymorphonuclear leucocytes into the endometrial stroma at the time of implantation of ova and the initiation of the oil decidual cell reaction in mice

Polymorphonuclear leucocytes (polymorphs) were found in the endometrial stroma adjacent to implanting blastocysts approximately 100 h after mating. Areas of uterus not containing a blastocyst had very few polymorphs. Ovariectomized mice were treated with hormones to render them sensitive to a decidual stimulus, the intraluminal injection of arachis oil. Areas of uterus responding to the oil (as indicated by the Pontamine Sky Blue reaction) showed an influx of leucocytes into the endometrial stroma between 16 and 36 h after the application of the decidual stimulus. The polymorphs were largely neutrophils. The injection of oil into non-sensitized uteri did not result in polymorph infiltration nor did the injection of physiological saline (which is not deciduogenic) into sensitized uteri. It is concluded that during the early stages of decidualization there is attraction of polymorphs from the blood into the stroma.

Somatotopical arrangement of rat spinal dorsal horn cells processing input from deep tissues

Extracellular recordings were made from 29 dorsal horn neurones which could be driven by mechanical stimulation of deep tissues, in spinal segment L 4 of rats. The recording sites were marked by iontophoresis of pontamine sky blue. According to the location of the receptive fields the cells were classified as deep neurones (12 cells), which had input from deep tissues only, and as cutaneo-deep neurones (17 cells), which had receptive fields in both skin and deep tissues. The deep cells showed a correlation between the recording sites in the dorsal horn and the location of the receptive fields on the hind-limb: the more distal a deep receptive field was situated on the hind-limb the more medial was the location of the recording site in the dorsal horn. In cutaneo-deep neurones the correlation was statistically significant for the deep receptive fields but not for the cutaneous ones.

Aminergic histofluorescence and contractile responses to transmural electrical field stimulation and norepinephrine of human middle cerebral arteries obtained promptly after death.

The responses of cerebral arteries to catecholamines and sympathetic nerve stimulation show wide variation between animal species. We examined the catecholaminergic histofluorescence and the contractile responses elicited by transmural electrical field stimulation and norepinephrine (NE) in proximal segments of human middle cerebral artery (MCA) obtained during autopsy. Twenty-four percent of the specimens were obtained within 2 hours and 76% within 4 hours of death. A moderately dense catecholaminergic histofluorescence was seen in all segments of human MCA using the glyoxylic acid technique, counterstained with pontamine sky blue. However, only seven of 35 (20%) MCA segments tested showed tetrodotoxin-blocked transmural electrical field stimulation contractions, and all of these were harvested within 4 hours of death. The responses were mostly seen in the most proximal MCA segments and, at 32 Hz, only achieved 6 +/- 1% of the maximal tissue contraction. NE caused two distinct responses in human MCA segments. At low concentrations, it acts via an alpha-like adrenoreceptor to cause contractions 20 +/- 3% of the maximal tissue response. The NE ED50s for the three successive segments were not different from each other; the value for the most-proximal segment was 7.9 +/- 0.2 x 10(-7) M. At concentrations above 10(-5) M, this catecholamine acts on low-affinity sites resistant to alpha-adrenergic antagonists causing contractions that at 10(-3) M reach 52 +/- 5% of the maximal tissue response.(ABSTRACT TRUNCATED AT 250 WORDS)

Antagonists of embryo-derived platelet-activating factor prevent implantation of mouse embryos

Inhibitors of platelet activation, alprazolam, iloprost and SRI 63-441, were used to demonstrate the necessity of embryo-derived platelet-activating factor (PAF) activity for the establishment of pregnancy in mice. In a splenectomized mouse bioassay 6 micrograms alprazolam inhibited, for 3 h, the thrombocytopenia induced by 0.1 micrograms PAF; 4 micrograms iloprost and 0.5 microgram SRI 63-441 were effective for 6 and 12h respectively. The administration of 2 micrograms iloprost/30 g body weight on Days 1 and 4 of pregnancy and twice daily on Days 2 and 3 caused a 50% reduction (P less than 0.0005) in the number of implantation sites in the uterus at Day 8 of pregnancy, without affecting (P greater than 0.05) the number of corpora lutea. A similar reduction in the number of implantation sites was achieved with 20 micrograms SRI 63-441/30 g body weight/day. The reduction in implantation rate was evident on Day 5 of pregnancy by visualizing the implantation sites with pontamine sky blue. SRI 63-441 had no effect on peripheral blood progesterone concentrations from Day 1 to Day 9 of pregnancy, and did not appear to inhibit implantation by blocking the preimplantation surge of oestradiol. The number and morphology of blastocysts flushed from the uterus of Day 4 inhibitor-treated mice was not different (P greater than 0.05) from the controls. The cleavage rate and morphology of embryos cultured from the 2-cell to blastocyst stage in media containing SRI 63-441 or iloprost (10 micrograms/ml) were normal, precluding a gross toxic effect. Simultaneous administration of 1 microgram PAF-acether to treated animals re-established pregnancy rates to levels not significantly different (P greater than 0.05) from the controls.

Inflammatory effects of acetylglycerylether phosphorylcholine: Vascular permeability increase and induction of pleurisy in rats

AGEPC-induced vascular permeability increase in the skins of rats, guinea pigs and rabbits was assessed by the leaked dye method, in which the dye was extracted from the skin sites injected with AGEPC after intravenous injection of pontamine sky blue. The activity was the most potent in the dorsal skin of rats and followed by guinea pigs and then rabbits. The vascular effect in rat skin was not affected by pretreatment with mepyramine and methysergide, but partially reduced by indomethacin and significantly suppressed by dexamethasone. Synergism was observed between AGEPC and bradykinin, histamine, 5-hydroxytryptamine, PGE 1, PGI 2 or PGD 2, when each agent was simultaneously injected with AGEPC into the skin. Pleurisy was induced by intrapleural injection of 1 ug of AGEPC into rats. Pleural fluid accumulation and leukocytes migration were observed. The exudate volume was not suppressed by pretreatment with indomethacin but significantly reduced by intavenous injection of CV-3988, a novel antagonist of AGEPC.

Pontamine sky blue: a counterstain for background autofluorescence in fluorescence and immunofluorescence histochemistry.

The stain pontamine sky blue (PSB) has been shown to reduce background autofluorescence in catecholamine fluorescence and immunofluorescence histochemical preparations. Using PSB as a counterstain on whole-mount stretch preparations of human mesenteric blood vessels, a medium dense noradrenergic nerve plexus is clearly revealed, which previously had been only partially visible because of background autofluorescence. Image analysis of nerve densities in whole-mount stretch preparations of guinea-pig arteries containing noradrenergic, substance P-, and vasoactive intestinal polypeptide (VIP)-positive nerve plexuses shows that PSB staining does not alter the specific neuronal fluorescence and that it improves image definition.

Distribution of Catecholamine-containing cell bodies and blood vessels in the rat nuclues tractus solitarius

Glyoxylic acid-induced monoamine fluorescence (GIF) was used to map the distribution of catecholamine-containing cell bodies and terminals in the rat nucleus tractus solitarius (NTS) and dorsal medulla. In addition, the relative vascularity of the NTS was quantified by examination of tissue perfused with Pontamine sky blue. The GIF-reactive cell bodies within the NTS complex were caudal to the rostral pole of the area postrema and are therefore considered to be the A2 cell body group as defined by Dahlstro¨m and Fuxe 4. The A2 cell body group was composed of 900 (980 ± 43, n= 3) small to medium sized neurons (15–25 μm diameter) located on the dorsal and lateral edges of the dorsal motor nucleus of the vagus. GIF terminal density was found to be most dense in the medial portion of the NTS as compared to the lateral or midline portions of the NTS, dorsal motor nucleus of the vagus, or hypoglossal nucleus. Quantitative blood vessel analysis revealed that the NTS is significantly less vasn the surrounding nucleus gracilius, central gray, or dorsal motor nucleus of the vagus. A full serial section analysis of NTS, stained either for GIF cell bodies, terminals or blood vessels, has been obtained; this detailed presentation of catecholamine-containing profiles and blood vessels in the dorsal medulla may facilitate futur future studies aimed at examining central control of autonomic function.

実験的歯の移動時の歯根膜における血管透過性高進反応の経時的変化について

The first purpose of this study was to study the method of separating the pressure site from the tension site in the periodontal tissues of the rat upper incisors during the experimental tooth movement, the second was to clarify the time-course of the increased periodontal vascular permeability in the pressure site and the tension site during the experimental tooth movement and the third was to study the role of bradykinin in the periodontal vascular permeability by the administration of bromelain. Pontamine sky blue was used to examine the vascular permeability quantitatively, following the method of Nitta et al. plus the addition of the decalcification of the hard tissue. The results were as follows: 1. The tension site tissue and the pressure site tissue after the experimental tooth movement could be dissected individually from the rat periodontal tissue. 2. The time-course of the increased vascular permeability shows two peaks at the tension site at 15 min-1.5 hours and at 14 hours after the beginning of the tooth movement and two peaks at the pressure site at 1. 5 and 14 hours. 3. It can be suggested that the permeability at the tension site is stronger than at the pressure site during 3 hours after the beginning of the tooth movement and weaker from 14 hours to 24 hours. 4. It can be inferred that bradykinin increases the vascular permeability at least at the tension site at 1.5 hours.

A combined electrophysiological and [ 14C]2-deoxyglucose study of the frequency organization of the inferior colliculus of the cat

Single units were recorded in the inferior colliculus of 3 young cats. A tonotopic sequence of characteristic frequencies (CF) was obtained in response to contralateral stimulation. In each cat Pontamine sky blue was deposited from the recording pipette at the site of a unit with a particular CF. [ 14C]2-Deoxyglucose was then injected into the cats, each of whom was presented with tone bursts at a frequency identical with the CF recorded at this site. Selective labelling was found in the contralateral inferior colliculus of each cat and the position of the band of marking coincided with the recording position of the chosen unit. These data demonstrated for the first time that cells in the region of [ 14C]2-deoxyglucose marking have the differential physiological properties capable of producing the selective functional labelling. The bands of selective labelling found in response to different frequencies are produced by cells tuned to the particular stimulating frequency.

Absence of cerebellar influences on hippocampal neurons in the cat

A variety of electrophysiological techniques was used to detect the presence of cerebellar influence on hippocampal neurons in the cat. Neither intracellular nor extracellular responses of 463 hippocampal neurons, nor hippocampal field potentiaLs, exhibited any changes after electrical stimulation of the cerebellar surface or of depth nuclei. Hippocampal pyramidal cells were identified electrophysiologically by evidence of fornix-evoked recurrent inhibitory postsynaptic potentials, and in some cases electrode recording sites were verified by dye marking in the hippocampus with pontamine sky blue. Electrode tracks of macroelectrode recording sites in hippocampus were verified histologically, as were tip positions of depth stimulating electrodes using a Prussian blue reaction. Stimulating currents applied to the cerebellar regions were well above the threshold determined in other experiments for evoking a motor cortex gross potential. Eleven hippocampal neurons exhibited a long-latency inhibition during high-current pulse-train stimulation of the fastigial nucleus, usually only when that stimulation evoked jaw movements. These hippocampal responses were similar to those evoked by lower currents applied to electrodes in the brain stem, and were probably due to current spread to brain stem, where the jaw movements could be evoked. These results provide evidence that there are few, if any, axons originating in the cerebellum that activate a neural pathway that directly or indirectly terminates on hippocampal neurons in the cat.

Effect of mepyramine, a histamine H1-, and burimamide, a histamine H2-receptor antagonist, on ovum implantation in the rat.

Systemic administration to rats of a combination of mepyramine, a histamine H1-, and burimamide, a histamine H2-receptor antagonist, over a period which included the time of implantation (late Day 5 to early Night 5), increased the number of blastocysts recovered from the uterus and reduced the number and intensity of Pontamine Sky Blue (PSB) sites obtained at autopsy on Night 5. Histological examination of the PSB sites taken on Night 5 from animals receiving the histamine antagonists revealed that the stromal oedema, which is characteristic of the attachment phase of pregnancy, had been inhibited.

Responses of thoracic dorsal horn interneurons to cutaneous stimulation and to the administration of algogenic substances into the mesenteric artery in the spinal cat

The effects of the injection of algogenic substances (bradykinin, acetylcholine) into the inferior mesenteric artery were studied at the thoracic level on 47 dorsal horn interneurons responding to cutaneous stimulation. Each unit was characterized by its electrophysiological properties and carefully located within the cord by extracellular injection of pontamine sky blue. Twenty cells, driven only by non-noxious cutaneous stimulation and mainly located in lamina IV, were not affected by the administration of algogenic substances. The activity of 25/27 cells, excited by both non-noxious and noxious cutaneous stimulation and mainly located in lamina V, was strongly modified by nociceptive visceral stimulation, induced by bradykinin and acetylcholine: 8/27 cells were activated, 14/27 were inhibited, 3/27 had a mixed inhibitory-excitatory response. From our study it clearly appears that nociceptive visceral messages only project on dorsal horn cells receiving noxious cutaneous afferents. Thus viscerosomatic convergence seems only to concern nociceptive messages; the existence of this kind of convergence reinforces the hypothesis suggested by several authors to explain referred pain from a neurophysiological point of view.

An analysis of response properties of spinal cord dorsal horn neurones to nonnoxious and noxious stimuli in the spinal rat.

Electrophysiological properties of neurones in the spinal cord dorsal horn were studied in decerebrated, immobilized spinal rats. Extracellular recordings were performed at the thoraco-lumbar junction level. Each track was systematically located by extracellular injection of pontamine sky blue. According to their responses to mechanical peripheral stimuli, cells were classified in four classes: Class 1 cells: Cells activated only by nonnoxious stimuli. They were divided into - 1A: hair movement and/or touch and 1B: hair movement and/or touch and pressure or pressure only. Class 2 cells: Cells driven by both nonnoxious and noxious stimuli, divided into - 2A: hair movement and/or touch, pressure, pinch and/or pin-prick, and 2B: pressure, pinch and/or pin-prick. Class 3 cells: Cells only activated by noxious stimuli (pinch and/or pin-prick). Class 4 cells: Cells responding to joint movement or pressure on deep tissues. Peripheral transcutaneous or sural nerve stimulation clearly showed that class 1 cells were activated only by A fiber input while 68% of classes 2 and 3 cells received A and C input. Histological examination indicated that cells driven only by noxious input were located either in the deepest part or in the marginal zone (lamina I) of the dorsal horn. Nevertheless, some lamina I cells were also driven by both nonnoxious and noxious stimuli. In addition, there is a great deal of overlap between class 1 and class 2 cells. This fact was confirmed by considering the wide distribution in the dorsal horn of cells receiving A and C input. However, spinal organization of the different classes of cells consists of a preferential distribution rather than a strict lamination. This study indicates that properties of dorsal horn inter-neurones in the rat have a high degree of similarity with those previously described in other species (cat and monkey).

The location of neurones with pontamine sky blue

Pontamine Sky Blue was used to mark the position of the recording site of multi-barrelled micro-electrodes. For light microscopy the dye mark was found in frozen sections. An image of a section was projected with an enlarger by using the section as the object; permanent photographic records and drawings were made from this image. For electron microscopy the dye mark was first found in thick sections. Thin sections were viewed in a Phillips EM 300 at low (100 ×) and high magnifications. Stained cells could be indentified and were surrounded by damaged tissue.

Localization with pontamine sky blue of neurones in the brainstem responding to microiontophoretically applied compounds

A development of the technique of marking electrode tip positions with Pontamine Sky Blue is described. The locations of a number of neurones in the medullary and pontine brainstem of the rat which were found to be sensitive to microiontophoretically applied noradrenaline, 5-hydroxytryptamine or morphine are presented, and the difficulties of interpretation of the results discussed.

Coccidiosis: Development of new techniques in coccidiostat evaluation

Four new techniques are discussed which may prove useful for assessing the effect of themotherapeutic agents on Eimeria. Changes in the permeability of the intestine during coccidial infection can be demonstrated by the intravenous injection of pontamine sky blue (PSB). In moderate infections of E. acervulina serum proteins can be demonstrated in the gut lumen; edema of the intestinal wall was observed and lesions caused by E. acervulina were clearly seen. Infection of the chorioallantoic membranes of the developing chick embryo may be extremely useful in assessing the effect of drugs against Eimeria, especially E. tenella. Embryos are highly susceptible to this infection and provide a sensitive system for the evaluation of anticoccidials. The technique is of particular value in the quantitative titration of drug-tolerant strains of E. tenella. A tissue culture method combining embryo infection and culture of E. tenella-infected chorioallantoic membrane cells may also be of value in assessing the action of anticoccidial drugs. Freeze preservation of sporozoites and sporocysts are techniques helpful for the investigation of species and strains of Eimeria in poultry. It is hoped that these techniques will be fully examined to determine whether they could play an important part in building information on the activity of existing and future coccidiostats.

Increase in length of the uterus at the time of implantation in the mouse.

Summary. When unilaterally ovariectomized mice were mated, the horn containing blastocysts increased in length during the early stages of implantation, at the same time as the onset of the Pontamine Sky Blue reaction and before implantation swellings were visible macroscopically. Similarly, the horns of pseudopregnant entire mice injected with oil to induce a decidual cell reaction were longer than the uninjected horns when the response had reached the stage at which a Pontamine Sky Blue reaction could be elicited. It is concluded that growth in length of the uterus occurs during the implantation reaction and this may assist in the accommodation of blastocysts along the length of the uterus.

Studies on aminoacetonitrile and its related compounds. II. Infiltration of pontamine sky blue into poisoned mouse liver cells, and its protection by aminoacetonitrile

Studies on aminoacetonitrile and its related compounds. II. Infiltration of pontamine sky blue into poisoned mouse liver cells, and its protection by aminoacetonitrile

In vivo staining by dis and trisazo dyes, especially of bone and elastic tissue.

The localization and retention of dis and trisazo dyes in connective tissues and bone was studied in rats and rabbits. Chlorazol fast pink, 5% in 0.9% NaCl. was injected intraperitoneally, 25 mg/kg each day for 2 days in newborn, growing, mature and 17-18 day pregnant rats, and up to 5 days in young rabbits. The dye was also injected at different time intervals during the development of strontium-induced rickets in growing rats, and in animals with abscess walls following subcutaneous injection of 0.1 ml turpentine. Animals were killed at-intervals thereafter, and comparison of in vivo staining of 5% solutions of chlorazol fast pink, chlorantine fast red, chlorazol black E, chlorazol sky blue, chlorazol sky pink, chlorazol green, chlorazol violet, pontamine green and pontamine sky blue was made by intraperitoneal injection in rats. Soft and hard tissue specimens were embedded in polyester resin or in paraffin wax and sectioned at 5-7 μ. Chlorazol fast pink stained some connective tissues and growing bones...