A. J. Haven

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

SummaryThe 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.

MYENTERIC PLEXUS IN STREPTOZOTOCIN TREATED RATS. NEUROCHEMICAL AND HISTOCHEMICAL EVIDENCE FOR DIABETIC NEUROPATHY IN THE GUT

Adrenergic, cholinergic, and serotoninergic nerves were studied in the myenteric plexus of ileum and colon from streptozotocin-treated rats, an animal model of juvenile-onset diabetes. In view of clinical reports implicating diabetic autonomic neuropathy as the cause of gastrointestinal dysfunction in diabetes mellitus, neurochemical and histochemical techniques were used to study changes in the innervation of the gut. In the myenteric plexus of the ileum from diabetic animals, adrenergic nerves displayed signs of degeneration and the brightness of fluorescence in serotoninlike immunoreactive nerves was lower. Cholinergic nerves, however, did not display any signs of reduction in the ileum, and both choline acetyltransferase and acetylcholinesterase activities per centimeter were increased. In contrast, in the proximal colon 8 wk after induction of diabetes, neurochemical assays revealed significant increases in noradrenaline and serotonin levels as well as choline acetyltransferase activity, although no obvious changes in the pattern of innervation could be detected histochemically. The results indicate that changes do occur in the innervation of the gut of the streptozotocin-diabetic model shortly after the induction of diabetes, although they differ significantly in the ileum and colon; these may be of relevance to the types of gastrointestinal dysfunction displayed in human diabetes.

Perivascular noradrenergic and peptide-containing nerves show different patterns of changes during development and ageing in the guinea-pig

The development of noradrenergic and peptide-containing perivascular nerves in common carotid, mesenteric, renal and femoral arteries of the guinea-pig was studied using the glyoxylic acid fluorescence and indirect immunofluorescence techniques on whole-mount stretch preparations at 6 stages between 6 weeks in utero and two years after birth. The noradrenergic plexus was more dense than the peptide-containing nerve plexuses in all the blood vessels, and, in general, calcitonin gene-related peptide-containing nerves were more numerous than substance P-containing nerves which in turn were more numerous than vasoactive intestinal polypeptide-containing nerves. In mesenteric and carotid arteries, noradrenergic nerve density reached a peak at about 4 weeks after birth that was maintained to old age, whereas the peptide-containing vasoactive intestinal polypeptide (VIP) and calcitonin gene-related peptide (CGRP) nerve plexuses reached a peak at birth and declined thereafter to about half maximum density in old age. In contrast, in the renal and femoral arteries, peptide-containing nerves reached a maximum density at 4 weeks after birth, while noradrenergic nerve density reached a peak around birth; both noradrenergic and peptide-containing nerve plexuses declined in density in old age. Of the 4 vessels studied, the mesenteric artery showed the greatest density of innervation for both noradrenergic and peptide-containing nerves at all stages of development, while the femoral artery was the least innervated. The possibility that some perivascular peptide-containing nerves play a trophic role during development is discussed.

Development and ageing of perivascular adrenergic nerves in the rabbit. A quantitative fluorescence histochemical study using image analysis

Age-related changes in vascular adrenergic nerves of 5 contrasting arteries in the rabbit were studied from before birth through to old age. Adrenergic nerves were demonstrated on stretch preparations using glyoxylic acid fluorescence histochemistry. Quantitative estimates of the densities of nerve fibres and varicosities were obtained by automated light microscopic image analysis. The early stages of development of vascular innervation were similar in all the vessels studied. They consisted of a period of outgrowth of axons; a period of rapid increase in density and formation of varicosities; and a later period of more gradual nerve growth. The timing of these stages varied greatly between the different vessels. The larger vessels, i.e. the carotid, renal and femoral arteries, had a well-developed innervation at birth, whilst the innervation of the smaller mesenteric and basilar arteries was sparse. Nerve loss occurred between 6 weeks and 6 months in the femoral artery and in old age (3 years or over) in the renal and carotid arteries. The large elastic arteries were, in general, more densely innervated than the smaller muscular arteries throughout life. The innervation of the different vessels became increasingly diverse between birth and adulthood, indicating a relationship between the pattern of vascular innervation and local physiological requirements. Factors which could influence age-related changes in nerve pattern and density are discussed.

Rat bladder in the early stages of streptozotocin-induced diabetes: adrenergic and cholinergic innervation

SummaryThe adrenergic and cholinergic innervation of the bladder was studied in streptozotocin-diabetic rats. The presence of hypertrophy and distension in the ‘diabetic’ bladders necessitates care in assessing changes occurring in the nerves, factors which are also relevant to clinical histochemical studies. Biochemical assays of cholinergic enzymes revealed decreased activities per g wet weight tissue. However, the total activities of choline acetyltransferase and acetylcholinesterase per whole bladder were significantly increased after 2 weeks of diabetes with greater changes by 8 weeks. Total dopamine levels per bladder were significantly higher than in control rats in the 2-week but not the 8-week group of animals; this may indicate an initial increase in adrenergic nerve activity. There was no impairment in the ability of the detrusor muscle to respond to noradrenaline, acetylcholine or to cholinergic nerve stimulation. Shortly after induction of diabetes streptozotocin-treated rats display polyuria. It is proposed that the activity of the bladder is therefore stimulated to allow greater volumes of urine to be passed. The results are discussed in relation to human diabetes mellitus where clinical studies have implicated a neuropathic origin to bladder dysfunction.

Intramural neurones of the guinea-pig urinary bladder: histochemical localization of putative neurotransmitters in cultures and newborn animals

Histochemical methods have been used to study the distribution of putative neurotransmitters in the urinary bladder of newborn guinea-pigs and in cultures of intramural ganglia. Following the nicotinamide adenine dinucleotide (NADH)-diaphorase reaction which specifically labels nerve cell bodies, up to 66 ganglia were observed in stretch preparations of the newborn urinary bladder. Each ganglion contained 2-50 nerve cell bodies. Vasoactive intestinal polypeptide was localized in a few nerve cell bodies of intramural ganglia both in in situ and culture preparations. In the in situ preparations it was widely distributed in nerve fibres to the muscle, being most dense at the base of the bladder, and in some mucosal epithelial cells. Somatostatin was contained in numerous neuronal cell bodies in the detrusor muscle both in situ and in culture. Extensively distributed varicose fibres were found in culture and in the muscle, submucous and mucosal layers in situ. Substance P immunofluorescence was demonstrated in a few neuronal cell bodies in ganglia both in situ and in vitro, particularly in those of the mucosa at the base of the bladder. In the in situ preparations varicose nerve fibres containing substance P were seen in the muscle coats with greatest density in the bladder base. Met-enkephalin-immunoreactive nerve cell bodies were not seen either in situ or in culture. Nerve fibres in in situ preparations were found largely enveloping neuronal cell bodies within the ganglia. Neither serotonin-immunoreactive nor catecholamine-containing neuronal cell bodies were seen in the in situ bladder preparation. However, some nerve cell bodies in culture showed positive staining, possibly as a result of selective uptake of serotonin and catecholamine known to be contained in foetal calf serum in the culture medium or possibly as the result of increased synthetic activity in certain neurones in the culture situation. In whole-mount stretch preparations, no serotonin-immunoreactive nerve fibres were seen, but catecholamine-containing small intensely fluorescent cells and nerve fibres were observed. Acetylcholinesterase-positive nerve cell bodies and nerve fibres were observed both in in situ and culture preparations of the bladder. Quinacrine-positive nerve cell bodies (as an indicator of purinergic neurones) were found in numerous intramural neurones examined. in situ; however, under the culture conditions used, non-selective staining of all cell types occurred.

Regional differences in the density of perivascular nerves and varicosities, noradrenaline content and responses to nerve stimulation in the rabbit ear artery.

Quantitative image analysis of fluorescent nerve following histochemical localization of monoamines in stretch preparations of the rabbit ear artery (REA) reveals marked differences in the density of innervation between the proximal and distal regions. The innervation in the proximal region is about twice as dense as that in the distal region and there are approximately 10,500 and 6,500 varicosities per mm2 vessel area in these two regions, respectively. These varicosities have approximately the same mean diameter throughout the length of the vessel. The noradrenaline contents per gram wet weight of tissue in the proximal and distal regions are 1.93 and 0.94 microgram, respectively. It is estimated that noradrenaline contents per mm2 nerve plexus area are 0.30 and 0.08 ng and that the nerve endings consist 2.8 x 10(-14) and 1.2 x 10(-14) g per varicosity in the proximal and distal REA, respectively. Sympathetic nerve stimulation in vitro with frequencies up to 8 Hz elicits larger and faster contractions in the proximal REA and the threshold frequency is less than in the distal region. This study also indicates that care should be taken to use the same region when using the REA for pharmacological and physiological studies.

Reduction in noradrenergic perivascular nerve density in the left and right cerebral arteries of old rabbits

With the use of fluorescence and acetylcholinesterase histochemistry, marked reductions have been shown in the noradrenergic and acetylcholinesterase-positive innervation of the right (RMC) and left (LMC) middle cerebral arteries of old compared with young adult rabbits. The decrease in noradrenergic nerve density tended to be greater in LMC than in RMC: Nerve density fell by ∼45% in LMC and by ∼30% in RMC. The reductions in acetylcholinesterase-positive nerves were similar in both LMC and RMC (29 and 33%, respectively). Vessel circumference and cross-sectional wall area appeared to increase in old age in LMC and RMC.

Effect of oestrogen and progesterone on noradrenergic nerves and on nerves showing serotonin-like immunoreactivity in the basilar artery of the rabbit

The effects of oestrogen and progesterone on noradrenergic nerves and nerves with serotonin (5-HT)-like immunoreactivity in the basilar artery were investigated in the rabbit using whole-mount stretch preparations. The noradrenergic nerves were demonstrated by glyoxylic acid fluorescence histochemistry and nerves with 5-HT-like immunoreactivity by indirect immunofluorescence techniques. Quantitative image analysis of fluorescent nerve fibres revealed that nerve density and varicosity diameter of nerves with 5-HT-like immunoreactivity were significantly (P less than 0.01 and P less than 0.05 respectively) reduced after 4-week administration of oestrogen and the intensity of fluorescence was also reduced. However, there were no significant changes after progesterone. Neither oestrogen nor progesterone had any effect on noradrenergic innervation. The findings are discussed in relation to higher incidence of migraine headaches in females taking oral contraceptives.

Functional and Non-Functional Nerve-Smooth Muscle Transmission in the Renal Arteries of the Newborn and Adult Rabbit and Guinea-Pig

Isolated renal arteries from newborn and adult guinea-pigs failed to respond to electrical stimulation of the intramural nerves, using parameters identical to those which elicit large neurogenic contractions in renal arteries from newborn and adult rabbits. The threshold stimulation frequency was lower and responses were greater in adult compared with neonatal rabbits. Comparisons of noradrenaline EC50 values showed that guinea-pig renal arteries are significantly less sensitive to noradrenaline that rabbit vessels. Quantitative fluorescence histochemistry of the perivascular adrenergic nerves in renal arteries revealed that the density of innervation gradually declines in the guinea-pig but substantially increases in the rabbit during postnatal development. The lack of neurogenic response of the guinea-pig renal artery, despite the existence of a varicose, adrenergic nerve plexus, is discussed.