Glyoxylic Acid-induced Fluorescence Research Articles

Melatonin Treatment Does Not Modify Ectopic Activity During Ischemia and Reperfusion in Rats

Introduction Ventricular tachycardia and/or ventricular fibrillation (VT/VF), a life-threatening complication of acute myocardial ischemia are based on the interaction between a vulnerable arrhythmogenic substrate and an arrhythmic trigger. Melatonin was proposed as a potential antiarrhythmic medication and was shown to ameliorate the arrhythmogenic substrate. Also, melatonin provides a sympatholytic effect and thereby can attenuate ectopic activity, which provides the triggers for VT/VF. However, little is known about a melatonin effect on the arrhythmic triggering. In the present study, we aimed at evaluation of the melatonin effects on catecholamine synthesis in normal and ischemic myocardium and heart rhythm disturbances during an episode of ischemia and reperfusion. Methods Experiments were done in 30 control and 32 melatonin-treated (10 mg/kg, daily, for 7 days) male rats. The hearts from 10 control and 10 treated animals were taken for histochemical analysis of sympathetic fibers (glyoxylic acid-induced fluorescence). Continuous ECG recording was performed in 20 control and 22 treated anesthetized animals before and during an ischemia-reperfusion episode induced by reversible 5-min occlusion of the left anterior descending coronary artery. Tyrosine hydroxylase expression in normal and ischemic myocardial regions was assessed by Western blotting analysis in 12 control and 12 melatonin-treated animals. Results No differences in the state of sympathetic innervation (density and fluorescence intensity) were observed in histochemical analysis. However, Western blotting demonstrated that melatonin treatment suppressed tyrosine hydroxylase expression in normal (p<0.05 vs control) but not in ischemic regions of myocardium. Melatonin-treated animals had longer RR-intervals in the baseline state, but this difference progressively disappeared during the period of ischemia due to tachycardic response to ischemia in the treated group (Figure). No differences in the number of extrasystoles were found between the control and treated groups during ischemia and reperfusion periods (Figure). Conclusion Chronic melatonin treatment led to the peripheral sympatholytic effect in myocardium, which was associated with decreased heart rate in preischemic conditions. However, this effect attenuated during ischemia and no differences in heart rate or extrasystolic burden were found between the control and treated animals during ischemia and reperfusion.

Melatonin pretreatment does not modify extrasystolic burden in the rat ischemia-reperfusion model.

The mechanism of reentrant ventricular tachyarrhythmias complicating acute myocardial ischemia is largely based on the interaction between an arrhythmogenic substrate and triggers. Melatonin was proposed as an antiarrhythmic medication and was shown to ameliorate the arrhythmogenic substrate. Also, melatonin provides a sympatholytic effect in different settings and might attenuate ectopic activity, which provides reentry triggers. In the present study, we aimed at evaluating the melatonin effects on cardiac sympathetic activity and the incidence of premature ventricular beats during the episode of ischemia-reperfusion. Experiments were done in a total of 26 control and 28 melatonin-treated (10 mg/kg, daily, for 7 days) male rats. Sympathetic fibers density was assessed by glyoxylic acid-induced fluorescence. Continuous electrocardiograms recording was performed during ischemia-reperfusion episodes (5 min/5 min, respectively) induced by reversible coronary occlusion. Myocardial expression of tyrosine hydroxylase, a rate-limiting enzyme of catecholamine biosynthesis was assessed by Western blotting. No differences in the state of sympathetic innervation were observed in histochemical analysis. However, Western blotting analysis demonstrated that melatonin treatment suppressed tyrosine hydroxylase expression in the non-ischemic (p < 0.05 versus control) but not ischemic regions of myocardium. The melatonin-treated animals had longer RR-intervals in the baseline state than the control animals (264 ± 48 ms versus 237 ± 33 ms, p = 0.044, respectively), but this difference decayed during the period of ischemia due to the increase of heart rate in the treated group. The number of premature ventricular beats did not differ between the control and treated groups during the ischemic and reperfusion periods. One-week melatonin pretreatment caused a slight peripheral sympatholytic effect that attenuated during ischemia and completely disappeared by the onset of reperfusion. The slight expression of sympathetic downregulation was associated with the lack of any effect of melatonin on extrasystolic burden. Collectively, the data suggest that melatonin cannot target the triggers of reentrant arrhythmias.

Sensory systems of Lineus ruber (Nemertea, Pilidiophora)

The aim of this study was to examine the organization of sensory systems in the heteronemertean Lineus ruber (Muller, 1774). Sensory systems of the head region (cerebral and frontal organs), body wall, proboscis and digestive tract of L. ruber were studied using Golgi–Colonnier silver impregnation, glyoxylic acid-induced fluorescence method for catecholamines (CAs) and immunochemical staining for serotonin (5-HT) and FMRFamide-related peptides (FaRPs). The study revealed a large number of intra- and subepithelial sensory cells in all of these structures and body regions. Only some of the sensory cells identified by silver impregnation were positive for CAs, 5-HT or FaRPs; the remaining cells appear to have different neurotransmitter modality. CA-containing (CA-C) intra- and subepithelial sensory cells were the most common cell types and were present in the epidermis and all the organs studied. All CA-C cells had a stiff cilium, which showed glyoxylic acid-induced fluorescence in the blue–green range indicative of CAs. In addition to CA-C cells, the cerebral organs had FaRP-immunoreactive sensory cells and argyrophilic cells containing an unidentified neurotransmitter and the frontal organs contained the neurites of 5-HT-immunoreactive, most likely efferent neurons. The presence of presumably mechanoreceptive CA-C cells in the frontal and cerebral organs indicates that these organs may perform not only a chemosensory, but also mechanosensory function.

Dopamine System Components in Neuroendocrine Complexes in Snail Atrium

Catecholamine dopamine (DA) is an important neurotransmitter and hormone involved in many physiological processes and stress reactions in vertebrates and invertebrates. The aim of the work was to clarify the presence and localization of dopamine system elements (tyrosine hydrokinase (TH), dopamine beta-hydrokinase (DAbetaHK), and dopamine type 1 receptors (DA-R1)) in the Achatina achatina gastropod atrial neuroendocrine complex (NEC) cells. The snail atrial NEC cells are generated by large granular cells (GCs) and nerve fibers that are in close contact with them. The methods of histochemistry, immunofluorescence staining, and immunoelectron microscopy were used. Glyoxylic acid-induced fluorescence demonstrated the presence of catecholamines in the nerve fibers and GC. TH-like and DAbetaH-like immunoreactive material was found both in nerve fibers and in GC granules. DA-R1-positive material was detected only in nerve fibers. In addition, it was demonstrated that exogenous DA induces an enhanced degranulation of GC in vivo. Data obtained indicate the involvement of dopamine system in the functioning of snail NEC.

Alpha-1B adrenoceptors mediate neurogenic constriction in mesenteric arteries of normotensive and DOCA-salt hypertensive mice

The contribution of alpha-1 adrenergic receptor (α 1-AR) subtypes to neurogenic constrictions of mesenteric resistance arteries from SHAM and deoxycorticosterone acetate-salt (DOCA-salt) hypertensive mice was assessed. Frequency-response curves (0.5–30 Hz) for transmural stimulation-evoked contractions were examined in SHAM and DOCA-salt arteries in vitro in the absence (control) and presence of prazosin (0.1 μM), PPADS (10 μM), yohimbine (1 μM), 5-methylurapidil (5-MU; 0.1 μM), L-765,314 (1 μM) and BMY-7378 (0.3 μM); selective antagonists at α 1-, P2X, α 2-, α 1A-, α 1B-, and α 1D-AR, respectively. In SHAM arteries, prazosin but not PPADS inhibited neurogenic responses. L-765,314 substantially inhibited neurogenic responses while 5-MU had a small inhibitory effect. BMY-7378 did not alter contractile responses at all. In DOCA-salt arteries, prazosin reduced neurogenic responses with no further significant inhibition seen with PPADS. L-765,314 antagonized neurogenic constrictions to a level similar to that seen in SHAM arteries. Furthermore, 5-MU and BMY-7378 did not affect these responses. The density of noradrenergic nerves (assessed using glyoxylic acid-induced fluorescence) or norepinephrine (NE) content was not altered by DOCA-salt hypertension. These results indicate that NE is the primary mediator of neurogenic constriction of murine mesenteric arteries. Nerve-released NE acts primarily at α 1B-and to a lesser extent at α 1A-ARs in SHAM arteries whereas NE mediates neurogenic constrictions in DOCA-salt arteries by acting at α 1B-ARs.

Noradrenergic and cholinergic innervation of the bone marrow

Bone marrow is supplied by sensory and autonomic innervation. Although it is well established that hematopoiesis is regulated by cytokines and cell-to-cell contacts, the role played by neuromediators on the proliferation, differentiation and release of hematopoietic cells is still controversial. We studied the innervation of rat femur bone marrow by means of fluorescence histochemistry and immunohistochemistry. Glyoxylic acid-induced fluorescence was used to demonstrate catecholaminergic nerve fibers. The immunoperoxidase method with nickel amplification was applied to detect the distribution of nerve fibers using antibodies against the general neuronal marker PGP 9.5 (neuron-specific cytoplasmic protein), while the cholinacetyltransferase immunoreactivity was studied by immunohistochemistry. Our results show the presence of an extensive network of innervation in the rat bone marrow, providing a morphological basis for the neural modulation of hemopoiesis.

Localization of putative neurotransmitters in the mantle and siphuncle of the mollusc Nautilus pompilius L. (Cephalopoda).

The neurotransmitter supply in the nerve endings of the mantle and the siphuncle, i.e. in organs that are responsible for the shell formation in the ectocholeate Nautilus pompilius, were investigated with electron microscopical, fluorescence-, immuno- and enzyme histochemical methods as well as with high pressure liquid chromatography (HPLC). Using antibodies against serotonin and the tetrapeptide FMRF-amide, positive reactions were demonstrated immunohistochemically within the terminal nerve fibres of the mantle and the vessels of the siphuncle. Enzyme histochemical proof of the presence of specific acetylcholinesterase yielded positive results in the muscle fibres of the mantle and siphuncle. Additionally, in the mantle, glyoxylic acid-induced fluorescence was shown within the nerve endings indicating catecholamines as neurotransmitters, whereas in the siphuncle such fluorescence did not appear. However, the HPLC-analyses showed that in the mantle and also in the siphuncle the content of dopamine is higher than that of noradrenaline whereas only traces of adrenaline occur in both organs suggesting dopamine as a putative neurotransmitter. Transmission electron microscopical examination of the nerve endings of both organs revealed that different types of vesicles were distinguished that could be considered as cholinergic, aminergic and peptidergic structures.

Disappearance of catecholamine fluorescence from the adrenergic nerves in arterial grafts in rats: an experimental fluorescence histochemical study.

The disappearance of catecholamine fluorescence from the noradrenaline-containing sympathetic nerve fibres after arterial transplantation was studied using a femoral artery graft sutured to rat carotid artery. Glyoxylic acid-induced fluorescence was used to demonstrate adrenergic nerves histochemically. At six hours the network of fibres had started to degenerate, and catecholamine fluorescence from the adrenergic nerves had almost completely disappeared within 24 hours of grafting. Control specimens from normal femoral arteries showed a dense network of fluorescent adrenergic nerves. Based on observations of the relatively rapid liberation of catecholamines from the degenerating adrenergic nerves, we suggest that catecholamines liberated from degenerating adrenergic nerves may have an important role in early vasospasm in microvascular and coronary bypass surgery.

Early and late functional and histopathological perturbations in the rabbit ear-artery following local cold injury.

These experiments aimed to study the in vivo short and long term neurovascular regeneration after frostbite. The rabbit central ear-artery was used as the experimental model. The effects on the noradrenergic innervation of the artery were measured in isolated vascular ring segments the first day and 2, 3-4, and 8-10 or 10-20 weeks following freezing at -9 degrees C or -18 degrees C for 15 min with slow rewarming for 7 min at room temperature. Two days after freezing the sympathetic nerves were completely degenerated, as observed with glyoxylic acid-induced fluorescence. The vascular isometric tension responses to exogenous noradrenaline and endogenously released noradrenaline by electrical stimulation in vitro were abolished. A varying degree of necrosis of the vascular wall was observed. Two weeks after freezing at -18 degrees C in vitro responses to exogenous noradrenaline and electrical stimulation were still abolished, then gradually approaching control levels after 10-20 weeks of in vivo regeneration. Eight and 10 weeks after injury at -9 degrees C increased vascular tension responses to exogenous noradrenaline was found. In spite of a long regeneration period the total uptake and the spontaneous and K+ (75 mM) evoked releases of [3H]noradrenaline were persistently decreased after frostbite at -18 degrees C, but they were regenerated to control levels already 10-20 weeks after -9 degrees C. Regeneration of noradrenergic nerve function, expressed as [3H]noradrenaline uptake and release and responsiveness to electrical stimulation, expressed as vascular contraction, was slower than the regeneration of the vascular smooth muscle. Myointimal hyperplasia developed in response to -9 degrees C and -18 degrees C frostbite. The uptake and the K+ evoked release of [3H]noradrenaline were particularly sensitive parameters for autonomic nerve function. The present findings may demonstrate important neurovascular reactions to local frostbite and may explain human sequelae following frostbite.

Growth factors effects on the expression of morphological and biochemical properties of avian embryonic sympathetic cells. Emphasis on NGF

Growth factors are known to be important agents in the differentiation and modulation of neuronal phenotypes. We have analyzed the effect of several growth factors on the modulation of morphological and biochemical properties of avian embryonic sympathetic neurons. The growth factors studied include: nerve growth factor (NGF), neurotrophin-3 (NT-3), brain derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), basic fibroblast growth factor (bFGF) and transforming growth factor β-1 (TGF-β1). Morphological properties were analyzed by immunocytochemistry to neurofilament proteins and visualization of fibers after glyoxylic acid-induced fluorescence. Biochemical modulation was determined by radioimmunoanalysis for the peptides enkephalin (ENK), somatostatin (SS) and neuropeptide Y (NPY) and by HPLC-electrochemistry quantification of catecholamines. Similar to previous results using chromaffin cell cultures [R. Ramı́rez-Ordóñez, J.E. Garcı́a-Arrarás, Peptidergic, catecholaminergic and morphological properties of avian chromaffin cells are modulated distinctively by growth factors, Dev. Brain Res., 87 (1995) 160–171], we found a dissociation in the modulation of biochemical and morphological properties, however, the effect of specific factors differed between the chromaffin and sympathetic cultures. We have focused on NGF to analyze its effect on the sympathetic peptide phenotypes and its lack of an effect on the chromaffin cell peptide phenotypes. The results presented here, establish interesting differences between chromaffin cells and sympathetic neurons that are of importance to studies of cell lineage and differentiation.

Heart autonomic innervation during the acute phase of experimental American trypanosomiasis in the dog.

Heart autonomic innervation was studied in dogs during the acute phase of the experimental infection with the Berenice-78 strain of Trypanosoma cruzi. A glyoxylic acid-induced fluorescence method for catecholamines and a thiocholine method for demonstrating acetylcholinesterase activity showed the sympathetic and the parasympathetic nerve fibers, respectively. At day 34 of infection, moderate-to-intense rarefaction of both cholinergic and noradrenergic nerve fibers occurred in the atria of all animals coincident with moderate to intense myocarditis. In the ventricles, sympathetic denervation was clearly present only when the inflammatory processes were moderate to intense. Preliminary results on the chronic phase indicate that normal autonomic innervation coexists with an incipient chronic fibrosing myocarditis.

Wallerian degeneration is required for both neuropathic pain and sympathetic sprouting into the DRG

Chronic loose constriction of the sciatic nerve produces mechanoallodynia and thermal hyperalgesia in rats and mice, and the behaviour develops during the time in which the nerve distal to the ligature site is undergoing Wallerian degeneration. There is a sympathetic component to the pain generated by this and other rodent models of neuropathic pain, yet the site at which this sympathetic-sensory coupling remains unknown. It has been shown that following sciatic nerve transection or spinal nerve lesion, sympathetic axons invade the dorsal root ganglion (DRG) where they sometimes form pericellular baskets around mostly large diameter DRG neurons – a possible anatomical substrate for sympathetically maintained pain (SMP). The signal for the sympathetic invasion of the DRG has not yet been shown, but associated with Wallerian degeneration is the upregulation of nerve growth factor (NGF) in the distal stump of the partially injured nerve, which may be retrograde-transported to the DRG in uninjured sensory axons to induce sprouting of sympathetic axons. To investigate the role of Wallerian degeneration in the development of neuropathic pain and sympathetic sprouting in the DRG, we have made use of a strain of mouse (C57Bl/Wld) in which Wallerian degeneration following nerve injury is delayed. We gave wild-type or Wld mice chronic constriction injuries (CCI) by loosely ligating the sciatic nerve with 3 ligatures, and allowed these mice to survive for a further 1, 2 or 3 weeks, during which time we assessed mechanoallodynia and thermal hyperalgesia. At the end of the testing period, the lumbar DRGs were removed for glyoxylic acid-induced fluorescence of catecholamines to determine the extent to which sympathetic axons had invaded the DRG. We found that both indices of neuropathic pain were significantly attenuated in Wld mice compared to wild-type mice, with the wild-type mice increasing in sensitivity to both thermal and mechanical stimulation in the first week post-operative (PO), while Wld mice showed marked hypoalgesia following CCI. Histological examination of the DRG showed that sympathetic sprouting into the DRG was also markedly delayed in Wld mice compared to wild-type mice: 1 week following injury, sympathetic fibres had invaded the ipsilateral DRG of wild-type mice, while sprouting in ipsilateral DRG of Wld mice was only slightly increased at 3 weeks PO. These results show that Wallerian degeneration is tightly linked to the development of both pain and sympathetic sprouting following CCI, and we speculate on the possible role of NGF as a mediator of both phenomena.

β‐Adrenoceptor‐mediated effects in rat cultured thymic epithelial cells

1. Sympathetic nerves were visualized in sections from rat thymus by immunostaining of tyrosine hydroxylase, the rate-limiting enzyme of catecholamine biosynthesis, and by glyoxylic acid-induced fluorescence of catecholamines. Catecholaminergic nerve fibres were detected in close connection to thymic epithelial cells which therefore might be preferred target cells. To evaluate this, rat immunocytochemically defined, cultured thymic epithelial cells were investigated for adrenoceptors and adrenergic effects. 2. In rat cultured thymic epithelial cells mRNA for beta 1- and beta 2-adrenoceptors was detected by reverse transcription-polymerase chain reaction by use of sequence-specific primers. Specific, saturable binding to the cultivated cells was observed with the beta-adrenoceptor agonist CGP 12177. 3. Adrenaline, noradrenaline or the beta-adrenoceptor agonist, isoprenaline, increased intracellular adenosine 3':5'-cyclic monophosphate (cyclic AMP) levels in cultivated thymic epithelial cells dose-dependently about 25 fold. The pharmacological properties revealed that this response was mediated by receptors of the beta 1- and the beta 2-subtypes. The selective beta 3-adrenoceptor agonist BRL 37344 had no effect on cyclic AMP levels. The increase in cyclic AMP was downregulated by preincubation with glucocorticoids like dexamethasone or cortisol which also changed the relative importance of beta 1-/beta 2-adrenoceptors to the response. 4. Incubation with isoprenaline or the adenylate cyclase activator forskolin decreased basal and serum-stimulated proliferation of thymic epithelial cells. However, adrenergic stimulation of thymic epithelial cells did not induce interleukin 1 production. Since thymic epithelial cells create a microenvironment which influences the maturation and differentiation of thymocytes to T-lymphocytes, their observed capacity to respond to catecholamines provides novel evidence for the suggestion that adrenergic stimulation may interfere with the regulation of immune functions.

Noradrenergic and peptidergic innervation of the mouse femur bone marrow

The innervation of bone marrow from femur bones of BALB/c mice was studied by means of immunohistochemistry and fluorescence histochemistry. The immunoperoxidase method with nickel amplification was applied to visualize the topographical distribution of nerve fibers using antibodies against the general neuronal marker PGP 9.5 (neuron-specific cytoplasmic protein), catecholamine synthesizing enzyme tyrosine hydroxylase (TH) and neuropeptide Y (NPY). Glyoxylic acid-induced fluorescence was also applied to demonstrate catecholamine-containing nerves. Both staining methods revealed dense innervation by fibers seen predominantly around blood vessels but also ramifying among marrow cells. Recent findings on adrenergic and peptidergic influences on marrow physiology combined with anatomical data indicate the existence of a neural modulation of hematopoiesis.

Monoaminergic and peptidergic contributions of the superior and the inferior spermatic nerves to the innervation of the testis in the rat.

Sections of the rat testis and whole-mounts of the testicular capsule were studied microscopically using the glyoxylic acid-induced fluorescence method, to detect monoamines, and immunohistochemical procedures for the detection of immunoreactivities to protein gene-product 9.5 (PGP 9.5), the C-terminal accompanying peptide of neuropeptide Y (CPON), and vasoactive intestinal polypeptide (VIP). Monoaminergic nerves were only observed around the intracapsular blood vessels: the initial segment of the testicular artery and the superior venous plexus, and in the anterior aspect of the upper and lower testicular poles. These capsular nerve networks were associated with the superior and inferior ligaments of the testis. Nerves displaying PGP 9.5 and CPON immunoreactivity appeared in the same sites and followed the same distribution as monoaminergic nerves. By contrast, VIP-immunoreactive fibers were only found in the nerve network of the lower pole. Observations done after different surgical denervation procedures demonstrated that the superior spermatic nerve was the source of fibers for testicular vessels and for the nerve network of the upper pole. On the other hand, fibers from the inferior spermatic nerve were restricted to the nerve network of the lower pole.

The distribution and colocalization of neuropeptides and catecholamines in nerves supplying the gall bladder of the toad, Bufo marinus.

The indirect immunofluorescence technique was used to determine the distribution of peptide-containing axons in the gall bladder of the cane toad, Bufo marinus. In addition, the adrenergic innervation of the gall bladder was examined by use of immunoreactivity to the catecholamine-synthesizing enzyme, tyrosine hydroxylase, and glyoxylic acid-induced fluorescence. On the basis of peptide coexistence, two intrinsic populations of neurones and their projecting fibres could be distinguished substance P neurones and vasoactive intestine peptide neurones. Neither of these two types of neurones contained any other colocalized neuropeptides. Four populations of nerve fibres arising from cell bodies outside the gall bladder were identified: nerves containing colocalized galanin, somatostatin and vasoactive intestinal peptide; nerves containing colocalized calcitonin gene-related peptide and substance P; adrenergic nerves containing neuropeptide Y; and nerves containing only adrenaline.

Dopaminergic acceleration and GABAergic inhibition in extrinsic neural control of the hermit crab heart

The acceleratory and inhibitory cardio-regulatory nerves of hermit crabs (Aniculus aniculus, Dardanus crassimanus) were studied using histochemical, immunocytochemical and pharmacological tests. Glyoxylic acid-induced fluorescence was observed in two of three axons of the dorsal cardiac nerve. One axon of the nerve showed gamma-aminobutyric acid-like immunoreactivity. Effects of stimulation of cardio-acceleratory axons were blocked by the dopaminergic antagonists, haloperidol and chlorpromazine, but not by cholinergic, adrenergic or serotonergic blockers, suggesting that dopamine is the primary potential candidate for the neurotransmitter of cardio-accelerator neurons. Picrotoxin antagonized inhibition of the cardiac ganglion induced by gammaam-inobutyric acid and by cardio-inhibitory axons. Both small and large ganglionic cells may receive dopaminergic and GABAergic extrinsic neural control.

Short communication: distal adrenergic innervation remains normal after perivascular sympathectomy.

Perivascular sympathectomy, which has been used for vasospastic disorders of the hands, was examined in experimental models. Perivascular sympathectomy was performed on the central ear vessels and forepaw metacarpal vessels of the rabbit and on the common digital vessels in the monkey. The effects on adrenergic nerves were studied using a glyoxylic acid-induced fluorescence method. Adrenergic nerves were removed only at the site of the perivascular sympathectomy and the distal adrenergic innervation appeared normal. Because the distal sympathetic nerves appeared normal after the operation, it should rather be called adventitectomy and we feel its clinical effects should be reevaluated.

An immunohistochemical study of the innervation of the large intestine of the toad (Bufo marinus).

The distribution of intrinsic enteric neurons and extrinsic autonomic and sensory neurons in the large intestine of the toad, Bufo marinus, was examined using immunohistochemistry and glyoxylic acid-induced fluorescence. Three populations of extrinsic nerves were found: unipolar neurons with morphology and location typical of parasympathetic postganglionic neurons containing immunoreactivity to galanin, somatostatin and 5-hydroxytryptamine were present in longitudinally running nerve trunks in the posterior large intestine and projected to the muscle layers and myenteric plexus throughout the large intestine. Sympathetic adrenergic fibres supplied a dense innervation to the circular muscle layer, myenteric plexus and blood vessels. Axons containing colocalized calcitonin gene-related peptide immunoreactivity and substance P immunoreactivity distributed to all layers of the large intestine and are thought to be axons of primary afferent neurons. Five populations of enteric neurons were found. These contained immunoreactivity to vasoactive intestinal peptide, which distributed to all layers of the large intestine; galanin/vasoactive intestinal peptide, which projected to the submucosa and mucosa; calcitonin gene-related peptide/vasoactive intestinal peptide, which supplied the circular muscle, submucosa and mucosa; galanin, which projected to the submucosa and mucosa; and enkephalin, which supplied the circular muscle layer.

Combined Trypan Blue and Glyoxylic Acid-Induced Catecholamine Fluorescence in the Female Rat Urinary Bladder After Distension

The effect of short-term urinary bladder distension on bladder perfusion and adrenergic innervation was studied in Sprague-Dawley rats. Distension was induced for three hours by forced diuresis and balloon obstruction. A 2% solution of trypan blue was injected into the tail vein 12, 24, 36 and 48 hours after distension and the animals were killed 5-10 min after the injection. Whole thick biopsies were taken from the dome, anterior body and base. A combination of trypan blue and catecholamine fluorescence (GIF method) was used to correlate the distension induced changes in blood vessel perfusion and permeability with changes in adrenergic innervation. Both ischaemic damage and adrenergic hypoinnervation were observed after distension. Later marked extravasation of trypan blue was observed in the whole bladder after 12 hours and after 24 hours distension the dome was almost necrotic in appearance and highly ischaemic. After 36 hours a few exploded small, intensely fluorescent cells (SIF cells) were found to be scattered along the blood vessels, and degranulated mast cells had invaded the anterior body and dome. The adrenergic hypoinnervation reached its maximum 48 hours after distension. The hypoinnervation and possibly also the damage to SIF cells, would seem to be related to ischaemia during distension, probably attributable in turn to overstretching of the organ and its blood vessels. This may provide an explanation for the prolonged micturition problems found after bladder overdistension.