Effects of pregnancy on the extrinsic innervation of the guinea pig uterus. A histochemical, immunohistochemical and ultrastructural study

Summary The extrinsic innervation of the guinea pig uterus was studied by immunohistochemical, ultrastructural and enzyme histochemical methods.The extrinsic innervation was organized in two major ways. One consisted of nerve trunks and non-varicose nerve fibres running in the suspensory ligament, and the other of a plexus of varicose nerve fibres surrounding vessels, and non-vessel-related non-varicose nerve fibres in the mesouterus. The use of different neuronal and Schwann cell markers showed that the extrinsic innervation was predominantly adrenergic and contained only few peptidergic nerves. Acetylcholinesterase-positive (cholinergic) nerves were only found around the uterine artery.In late pregnancy, the extrinsic nerves of the mesouterus adjacent to foetus-containing uterine horns underwent pronounced degenerative changes comprising both Schwann cell and axonal structures. In comparison, no changes were found in extrinsic nerves of mesouteri adjacent to non-foetus-bearing uterine horns or in extrinsic nerves in the suspensory ligaments. Further, chemical sympathectomy produced axonal degeneration but no changes in the Schwann cells.In conclusion, the pregnancy-induced nerve degeneration is of a very special type different from that following chemical sympathectomy and represents a local phenomenon related to the conceptus. Hypothetically, this could be of importance for counteracting disturbances in placental blood flow.     

Ontogenetic development of the guinea pig uterine innervation. An immunohistochemical study of different neuronal markers, neuropeptides and S-100 protein

The ontogenetic development of the guinea pig uterine autonomic innervation was studied immunohistochemically using neurofibrillary protein (NF) and neuron specific enolase (NSE) as general neuronal markers, tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH) as specific markers for adrenergic innervation and S-100 protein as marker for Schwann cell structure and/or function. In addition, comparisons were made of the development of the different populations of peptide-containing nerves. The structure and time of appearance were similar for nerves with NF-, NSE-, TH- and DBH-immunoreactivities, which were first present in the organ periphery as coarse nerve trunks, then extending centrally and branching into non-varicose nerves. From these, varicose nerves developed first in relation to vessels and then in association with the myometrial smooth musculature. Development was completed earlier in the cervix than in the uterine horns suggesting differences in local environment. In comparison, S-100 nerve-immunoreactivity appeared later but attained complete development more rapidly than axonal structures. Neuropeptide Y-immunoreactive nerves showed a similar developmental pattern to presumed adrenergic nerves, further verifying the assumption of intraneuronal localization of NPY in uterine adrenergic nerves. Other peptide-containing nerves were developed later probably reflecting differences in neuronal growth properties.     

Ontogenetic development of the guinea pig uterine innervation

Summary The ontogenetic development of the guinea pig uterine autonomic innervation was studied immunohistochemically using neurofibrillary protein (NF) and neuron specific enolase (NSE) as general neuronal markers, tyrosine hydroxylase (TH) and dopamine -hydroxylase (DBH) as specific markers for adrenergic innervation and S-100 protein as marker for Schwann cell structure and/or function. In addition, comparisons were made of the development of the different populations of peptide-containing nerves.The structure and time of appearance were similar for nerves with NF-, NSE-, TH- and DBH-immunoreactivities, which were first present in the organ periphery as coarse nerve trunks, then extending centrally and branching into non-varicose nerves. From these, varicose nerves developed first in relation to vessels and then in association with the myometrial smooth musculature. Development was completed carlier in the cervix than in the uterine horns suggesting differences in local environment. In comparison, S-100 nerve-immunoreactivity appeared later but attained complete development more rapidly than axonal structures. Neuropeptide Y-immunoreactive nerves showed a similar developmental pattern to presumed adrenergic nerves, further verifying the assumption of intraneuronal localization of NPY in uterine adrenergic nerves. Other peptide-containing nerves were developed later probably reflecting differences in neuronal growth properties.     

Organization of the guinea-pig uterine innervation. Distribution of immunoreactivities for different neuronal markers. Effects of chemical- and pregnancy-induced sympathectomy

Summary The structural organization of the guinea-pig uterine innervation was investigated by an immunofluorescence method using neurofibrillary protein (NF) and neuron-specific enolase (NSE) as general neuronal markers. NF- and NSE-immunoreactive nerve trunks and non-varicose nerves formed continuous networks similar to nerves with analogue morphology and with immunoreactivities for tyrosine hydroxylase (TH; adrenergic nerves) and neuropeptide Y (NPY). NF- and NSE-immunoreactive non-varicose nerves occurred in the myometrium and along vessels, where TH- and NPY-immunoreactive varicose nerves were also comparatively frequent. After chemical sympathectomy all TH- and NPY-immunoreactive varicose nerves and most NF- and NSE-immunoreactive non-varicose nerves disappeared, suggesting colocalization of TH, NPY, NF and NSE immunoreactivities. During pregnancy all NF-, NSE-, TH- and NPY-immunoreactive nerve structures disappeared in the foetus-bearing uterine horns whereas in the cervix and non-foetus-bearing uterine horns only the myometrial TH- and NPY-immunoreactive varicose nerves disappeared. After parturition there was a complete structural restoration of all types of immunoreactive nerves in previously non-foetus-related tissue. The reinnervation of this tissue followed a similar time-course to that after chemical sympathectomy. In contrast, the reinnervation of previously foetus-related tissue was much slower and incomplete.In conclusion, the whole autonomic uterine innervation undergoes overt structural changes during pregnancy and these changes are related to the foetus-bearing regions.     

Protein gene product 9.5 (PGP 9.5)

Summary The guinea pig uterus is supplied by different populations of nerves which can be demonstrated by specific immunocytochemical and histochemical techniques. So far, there has been no single marker displaying entire peripheral innervation patterns. Recently, protein gene product (PGP) 9.5, a cytoplasmic protein in neurons and neuroendocrine cells, was found to visualize both different populations and subtypes of nerves. This prompted the present study of using PGP 9.5 for visualization of the whole uterine innervation. This was performed by the indirect immunofluorescence method using antiserum to PGP 9.5 raised in rabbits.PGP-immunoreactivity was present in all neuronal parts of the extrinsic and intrinsic uterine innervation, including different subpopulations of nerves. This was verified by chemical sympathectomy and sensory denervation with 6-hydroxydopamine and capsaicin-treatment respectively, and double immunostaining.By term a disappearance of uterine PGP-nerve-immunoreactivity was observed which was almost complete in fetus-bearing uterine tissue and further strengthens previous assumptions of a general, pregnancy-induced uterine neuronal degeneration.The developmental time-course and morphology of PGP-immunoreactive nerve structures was similar to that for other neuronal markers and support the suggestion of PGP-immunoreactivity as a general marker for the entire uterine innervation, and suggests that the presence of PGP 9.5-immunoreactivity may coincide with functional maturation of uterine innervation.     

Co-existence and origin of peptidergic and adrenergic nerves in the guinea pig uterus

Summary The occurrence and distribution of peptidergic nerves in the guinea pig uterus was studied by means of immunocytochemistry using numerous neuropeptide antisera. Neuropeptide Y (NPY)-immunoreactive (IR) nerves were the most abundant, whereas substance P (SP)-, calcitonine gene-related peptide (CGRP)-, and neurokinin A (NKA)-IR nerves were less frequent, and peptide histidine isoleucine (PHI)-IR nerves were the most sparse. Chemical sympathectomy by means of 6-hydroxydopamine, and capsaicin treatment revealed the division of the peptidergic nerves into three separate populations: (1) NPY-IR nerves, which co-existed with adrenergic nerves, (2) SP-, CGRP-and NKA-IR nerves, which mutually co-existed, and (3) PHI-IR nerves. Parallel-running adrenergic/NPY-IR and SP-IR nerves could be found with very similar although not completely identical morphological appearance. Paracervical ganglia contained neurotensin-and dynorphin A-IR cell bodies in addition to cell bodies with immunoreactivities similar to those in prevertebral ganglia. Combined retrograde tracing with True blue and immunocytochemistry showed that the adrenergic and NPY-IR uterine nerves originate in paracervical and prevertebral ganglia. In the prevertebral ganglia the cellular origin was the same for adrenergic and NPY-IR nerves. In contrast, SP-, CGRP-,and NKA-IR nerves originated in dorsal root ganglia. At full-term pregnancy all the neuropeptide immunoreactivities had vanished, probably reflecting a fetus-induced general nerve degeneration.     

Adrenergic innervation of pancreatic islets and modulation of insulin secretion by the sympatho-adrenal system

Summary Morphological changes in the adrenergic innervation of pancreatic islets after chemical sympathectomy by use of 6-hydroxydopamine and the influence of the sympatho-adrenal system on insulin secretion were investigated in the mouse and rat.Fluorescence histochemistry revealed a clear-cut reduction in the number of adrenergic nerve fibers in the pancreatic islets 2 days after administration of 6-hydroxydopamine; the reduction was more pronounced in the rat than in the mouse. In the rat, a partial regeneration was seen after 6 weeks. In the pancreas of the mouse, after administration of 6-hydroxydopamine, a severe damage of unmyelinated nerve fibers was revealed electron microscopically. However, no ultrastructural or immunohistochemical alterations could be demonstrated in the endocrine cells of the islets.6-Hydroxydopamine induced a depression of basal plasma insulin concentrations in mice and an elevation in rats. Adrenalectomy depressed basal plasma insulin levels in mice.The -adrenoceptor antagonist phentolamine enhanced insulin secretion in normal mice. The secretory response of insulin to phentolamine was diminished by chemical sympathectomy and almost abolished by adrenalectomy or the combination of chemical sympathectomy and adrenalectomy. Thus, the effect of phentolamine is probably mediated by liberated catecholamines.It is concluded that basal insulin secretion is partially regulated by the sympatho-adrenal system and that species differences exist in this respect. In addition, the results suggest that endogenous catecholamines have the ability to promote insulin secretion.     

Chemical sympathectomy induced by 5.6-dihydroxytryptamine

Summary The effect of different doses of 5.6-dihydroxytryptamine—a serotonin analogue which produces a degeneration of serotonin containing nerve terminals in the rat brain—on the noradrenaline (NA) content and—storage sites of peripheral sympathetic nerves in the mouse and rat heart, spleen, rectum and vas deferens has been investigated by fluorescence—, electron microscopical and chemical methods. Moderate doses of 5.6-dihydroxytryptamine (5.6-DHT) (10–45 mg/kg ip.) cause a temporary, reversible displacement of noradrenaline from the adrenergic nerves concomitant with a significant increase in the number and opacity of small and especially large granular vesicles. The recovery of the neuronal NA concentration is, however, retarded after doses higher than 45 mg/kg (60 or 100 mg/kg ip.); a partial degeneration of varicose NA terminals is verified fluorescence- and electron microscopically. A combined treatment of animals with tyrosine hydroxylase inhibitors (-methyl-paratyrosine or -propyl-dopacetamide) and 5.6-DHT, in some instances also followed by reserpine, potentiates the destructive properties of 5.6-DHT; a similar potentiation is accomplished by reserpine posttreatment or by an additional pretreatment of animals with reserpine and nialamide.The results suggest that 5.6-DHT when given in moderate doses (up to 45 mg/kg) may be handled by sympathetic adrenergic nerves like a false neurotransmitter which displaces noradrenaline from the stores, but that it causes a chemical degeneration of noradrenaline containing nerve terminals when applied either in single high doses (60 or 100 mg/kg ip.), or when administered in moderate non-degenerative doses together with drugs that impair the neuronal inactivation mechanisms for 5.6-DHT (granular uptake and storage mechanism and/or monoamine oxidase activity) and thus provoke a temporary increase in the amount of free 5.6-DHT in the neuron's cytoplasm.The molar efficiency of 5.6-DHT in causing a chemical sympathectomy is clearly inferior to that caused by 6-hydroxydopamine. The differences are probably mainly due to differences in the affinity of both drugs to the amine uptake system located at the cell membrane and the membrane of the intraneuronal storage vesicles of the adrenergic nerve terminals.Supported by grants from the Deutsche Forschungsgemeinschaft.     

6-Hydroxydopamine produces a weight increase in toads

A large increase in body weight occurred after injection of 6-hydroxydopamine (6-OHDA, 100 mg kg-1) into the iliac lymph sac of toads. The body weight increase was apparently due to retention of fluid and only occurred when toads could not avoid contact of their ventral skin with water. Lower doses of 6-OHDA (25 or 50 mg kg-1) did not cause a weight increase although they were sufficient to eliminate fluorescence from peripheral adrenergic nerves. A peripheral injection of 6-OHDA did not affect the appearance of central aminergic nerves. Removal of the pituitary and median eminence did not affect the weight increase. Although the primary action of 6-OHDA in upsetting water balance in toads has not been revealed by this study, it is clearly not related to the chemical sympathectomy of adrenergic nerves.     

Immunocytochemical localization of S-100b protein in degenerating and regenerating rat sciatic nerves

We studied the cellular and subcellular distribution of S-100b protein in normal, crushed, and transected rat sciatic nerves by an immunocytochemical procedure. In uninjured nerves, S-100b protein was restricted to the cytoplasm and membranes of Schwann cells, with no reaction product present in the nucleus or in axons. Similar images were seen from the first to the thirtieth day after the crush in activated Schwann cells during the degeneration period, i.e., up to the seventh post-lesion day, and in normal Schwann cells reappearing during the regeneration period, i.e., after the seventh post-lesion day, in the zone of the crush and proximal and distal to it. By the technique employed, there seemed to be no differences in the intensity of the immune reaction product in normal and activated Schwann cells. Also, similar images were seen in the proximal stump of transected nerves. Only a slight S-100b protein immune reaction product could be observed in the rare activated Schwann cells present in the distal stump around the seventh post-lesion day, the majority of cell types being represented by fibroblasts and elongated cells at this stage and thereafter. By immunochemical assays, similar results as those presented here have been reported and interpreted as indicative of the presence of S-100 protein in axons or, alternatively, of axonal control over expression of S-100 protein in Schwann cells. Our immunocytochemical data clearly show that the strong reduction in the S-100 protein content of the distal stump of transected nerves is owing to the paucity of Schwann cells and to the decrease in the S-100 protein content of these cells, rather than to degeneration of axons.     

Effects of infantile/prepubertal chronic estrogen treatment and chemical sympathectomy with guanethidine on developing cholinergic nerves of the rat uterus.

The innervation of the uterus is remarkable in that it exhibits physiological changes in response to altered levels in the circulating levels of sex hormones. Previous studies by our group showed that chronic administration of estrogen to rats during the infantile/prepubertal period provoked, at 28 days of age, an almost complete loss of norepinephrine-labeled sympathetic nerves, similar to that observed in late pregnancy. It is not known, however, whether early exposure to estrogen affects uterine cholinergic nerves. Similarly, it is not known to what extent development and estrogen-induced responses in the uterine cholinergic innervation are affected by the absence of sympathetic nerves. To address this question, in this study we analyzed the effects of infantile/prepubertal chronic estrogen treatment, chronic chemical sympathectomy with guanethidine, and combined sympathectomy and chronic estrogen treatment on developing cholinergic nerves of the rat uterus. Cholinergic nerves were visualized using a combination of acetylcholinesterase histochemistry and the immunohistochemical demonstration of the vesicular acetylcholine transporter (VAChT). After chronic estrogen treatment, a well-developed plexus of cholinergic nerves was observed in the uterus. Quantitative studies showed that chronic exposure to estrogen induced contrasting responses in uterine cholinergic nerves, increasing the density of large and medium-sized nerve bundles and reducing the intercept density of fine fibers providing myometrial and perivascular innervation. Estrogen-induced changes in the uterine cholinergic innervation did not appear to result from the absence/impairment of sympathetic nerves, because sympathectomy did not mimic the effects produced by estrogen. Estrogen-induced responses in parasympathetic nerves are discussed, considering the direct effects of estrogen on neurons and on changes in neuron-target interactions.     

Decentralisation of neurones in the pelvic ganglion of the guinea-pig: Reinnervation by adrenergic nerves

An electron-microscopic study has been made of adrenergic and cholinergic nerve fibres and synapses in the pelvic ganglion of the guinea-pig at intervals of up to 60 days following section of the hypogastric and pelvic nerves. Transection of the hypogastric nerves led to degeneration of 80-90% of the cholinergic nerve profiles and synapses in the ganglion. The small number of adrenergic nerves and synapses did not change, but 30-60 days after section, this number increased 8-10 times. Transection of the pelvic nerves led to degeneration of about 15% of the cholinergic nerve terminals, but no change in adrenergic terminals. After transection of both hypogastric and pelvic nerves, only about 1% of cholinergic nerves survived, but after 30-60 days, the number of adrenergic nerves increased 8-10 times. It is concluded that following cholinergic nerve degeneration in the ganglion, adrenergic nerves, probably originating as collateral sprouts from postganglionic neurones and granule-containing cells, can replace them to some extent.     

Hepatocyte innervation in primates

The efferent innervation and some characteristics of nerve fibers of the liver lobule in the tree shrew, a primate, are described. Nerve endings on hepatocytes were encountered regularly and were determined to be efferent adrenergic nerves. Transmission electron microscopy revealed nerve endings and varicosities in close apposition to the hepatocytes adjacent to the connective tissue of the triads as well as within the liver lobule in the space of Disse. Fluorescence microscopy indicated the existence of adrenergic nerves with a similar distribution. Autoradiography of the avid uptake of exogenous [3H]norepinephrine indicated that all intralobular nerves are potentially norepinephrinergic (adrenergic). Chemical sympathectomy with 6-OH-dopamine resulted in the degeneration of all intralobular liver nerve fibers as revealed by fluorescence microscopy and electron microscopy. Substantial regeneration occurred after 60-90 days but was not completed by that time. Some nerves were also observed in close association with von Kupffer cells and endothelial cells. The functional significance of the efferent liver innervation is discussed.     

Plasticity in developing rat uterine sensory nerves: the role of NGF and TrkA

In the present study we investigated the effects of infantile/prepubertal chronic oestrogen treatment, chemical sympathectomy with guanethidine and combined sympathectomy and chronic oestrogen treatment on developing sensory nerves of the rat uterus. Changes in sensory innervation were assessed quantitatively on uterine cryostat tissue sections stained for calcitonin gene-related peptide (CGRP). Uterine levels of NGF protein, using immunohistochemistry and ELISA, and mRNA, using Northern blots and in situ hybridization, were also measured. Finally, levels of TrkA NGF receptor in sensory neurons of T13 and L1 dorsal root ganglia (DRG), which supply the uterus, were assessed using densitometric immunohistochemistry. These studies showed that: (1) chronic oestrogen treatment led to an 83% reduction in the intercept density of CGRP-immunoreactive nerves; (2) sympathectomy had no effect on the density of uterine sensory nerves or on the pattern of oestrogen-induced changes; (3) NGF mRNA and protein increased following sympathectomy or chronic oestrogen treatment; and (4) oestrogen produced increased intensity of labelling (28%) for TrkA receptors in small-diameter sensory neurons, but decreased labelling (13%) in medium-sized neurons, which represent the large majority of the DRG neurons supplying the upper part of the uterine horn. Contrary to expectations, increased levels of NGF after sympathectomy and oestrogen treatment did not lead to increased sensory innervation of the uterus. The possibility that alterations in neuronal levels of TrkA contribute to the lack of response of uterine sensory nerves to the oestrogen-induced increase in NGF levels is discussed.This work was supported by The Wellcome Trust, UK (CRIG Grant 058122/Z/99/Z/JC/KO), and PEDECIBA, Universidad de la República, Montevideo, Uruguay     

OCCURRENCE AND PROPERTIES OF MONOAMINE OXIDASE IN ADRENERGIC NEURONS

—Monoamine oxidase activity of peripheral organs of various species has been examined after surgical, chemical and immunological sympathectomy to assess the proportion of enzyme activity in adrenergic neurons and in extraneuronal cells. Significant falls in monoamine oxidase activity of vas deferens, submaxillary gland, iris and spleen were seen after sympathetic denervation although not in heart, small intestine and kidney. It was suggested that a correlation exists between the extent of the fall in monoamine oxidase activity after sympathectomy and the density of sympathetic innervation of the control organ. Studies of monoamine oxidase activity in vas deferens after inhibition with clorgyline suggested multiple forms of monoamine oxidase. Differences in inhibitor sensitivity, substrate specificity and thermal inactivation of monoamine oxidase in normal and denervated vas deferens were found and it was suggested that differences exist in the properties of the neuronal and extraneuronal monoamine oxidase.     

Immunoelectron microscopic demonstration of S-100b protein-like in centriole, cilia, and basal body

We report here the ultrastructural localization of S-100b protein-like immunoreactivity in the centriole, cilia, and basal body. Duodenum and trachea of guinea pigs and rats were fixed and immunostained by the protein A-gold method. All centrioles, cilia, and basal bodies observed showed clear S-100b protein-like immunoreactivity. Specific colloidal gold particles were located over the microtubules in these cell organelles. However, other microtubules scattered throughout the cytoplasm were devoid of immunoreactivity. Although the functional significance of S-100b protein-like immunoreactivity in the centriole, cilia, and basal bodies remains to be elucidated, the present results introduce new perspectives into the investigation of localization and function of S-100 proteins.     

The distribution and origin of nerve fibers immunoreactive for substance P and neurokinin A in the anterior buccal gland of the rat

The distribution and origin of substance P (SP) and neurokinin A (NKA) were studied in rat in the anterior buccal glands, which are minor mucous salivary glands. Indirect immunofluorescence staining showed moderate SP and NKA innervation of salivary acini and interlobular ducts, whereas blood vessels were more sparsely innervated, and there were few nerve fibers in the stroma and around the intralobular ducts. About 10%–20% of the trigeminal ganglion cells showed equally strong immunoreactivity to both SP and NKA. Unilateral denervation of the branches of the trigeminal nerve caused complete disappearance of the stromal fibers and greatly reduced the number of all other SP-immunoreactive and NKA-immunoreactive nerve fibers. In the superior cervical ganglia, SP and NKA immunoreactivity was restricted to small intensely fluorescent cells; SP and NKA immunoreactivity was absent from principal ganglionic cells, and thus sympathectomy had no any effect on the number or distribution of fibers immunoreactive for SP and NKA in the anterior buccal glands. The fibers remaining after sensory denervation could have been of parasympathetic origin, indicating a dual origin of nerves immunoreactive for SP and NKA in these glands. The present data demonstrate that the major part of the glandular SP and NKA innervation in the minor salivary glands derives from the trigeminal ganglia. The distribution of the peripheral nerve fibers indicates that they may play a role in the delivery of potent neuropeptides involved in the vascular, secretory, and motor (myoepithelial cells) functions of salivary glands.     

Nerves immunoreactive to vasoactive intestinal polypeptide in the porcine female genital tract

The immunoreactivity of vasoactive intestinal polypeptide (VIP) was localized at the light-microscopical level in cryostat sections using a peroxidase-antiperoxidase technique or at the electron-microscopical (EM) level in glutaraldehyde-fixed, resin-embedded sections, using an immunogold technique, of tissue samples from the genital tract of cycling pigs. X-ray micro-analysis of glutaraldehyde-dichromate-fixed sections was used to discriminate noradrenaline-containing nerves. VIP immunoreactivity was localized to nerves associated to some degree with epithelial cells, blood vessels and non-vascular smooth muscle. VIP nerves were most concentrated in the cervix and the uterus, localized in the submucosa, the muscle layers and the adventitia. Nerve profiles were also seen accompanying blood vessels in the endometrium, running close to the uterine glands. In the oviduct, VIP nerves had a similar localization though less dense. At the EM level, the immunogold localization confirmed the above-mentioned results, VIP being localized in synaptic vesicles. Nerve terminals without VIP reactivity had an EM appearance of cholinergic nerve terminals or were chrome positive (noradrenaline-containing) at X-ray micro-analysis, thus being adrenergic terminals. It is concluded that the porcine female genital tract is well innervated, along with adrenergic and cholinergic components, by VIP-containing nerves.     

Observations on the loss of catecholamine fluorescence from intrauterine adrenergic nerves during pregnancy in the guinea-pig

During unilateral pregnancy in the guinea-pig there is loss of formaldehyde-induced fluorescence from the adrenergic nerves supplying the uterus and its vasculature. This loss occurs initially near the site of implantation at about Day 20 of gestation and spreads progressively. Implantation of wax pellets containing progesterone into the uterine lumen or the gastrocnemius muscle of virgin guinea-pigs for 7 days produced loss of fluorescence from all local adrenergic nerves. No diminution of fluorescence was seen when pellets containing oestradiol were substituted. Chronic denervation studies showed that the adrenergic axons supplying the uterus and its arteries originated from both the ovarian artery and the pelvic region. Our results suggest that loss of adrenergic fluorescence within the uterus during pregnancy is due to an effect of placental progesterone which is localized to the uterus because the high concentration of progesterone necessary to cause fluorescence loss is not attained in the systemic circulation.     

Uptake of circulating hyaluronic acid by the rat liver

Summary The present study deals with the localization and development of S-100 protein-like immunoreactivity in the retina, ciliary body and iris of human fetuses. In the retina, numerous astrocytes, densely distributed in the nerve-fiber layer and ganglion-cell layer, were stained strongly with the S-100 antiserum. The first immunoreactive astrocytes occurred at the posterior pole of the retina and spread gradually outward and toward the ora serrata with increasing age. Müller cells were not immunoreactive for S-100 during development, except in the retina of the latest fetus examined. S-100 immunoreactivity was also found in the nonpigmented ciliary epithelium and posterior epithelium of the iris, both of which are developed from the inner wall of the optic cup. On the other hand, the pigmented epithelium extending from retina to iris, derived from the outer layer of the optic cup, was free of S-100 immunoreactivity.