Innervation pattern of the unclosed detrusor muscle in classic bladder exstrophy: a study of patients with urothelial overexpression of nerve growth factor.

PURPOSE: An overexpression of nerve growth factor (NGF) in the urothelium is discussed to lead to neuronal hyperinnervation of the bladder detrusor. The aim was to assess the sensory and sympathetic innervation of the detrusor in unclosed exstrophic bladders patients with known overexpression of NGF in the urothelium. METHODS: Full-thickness bladder biopsies were prospectively obtained from 34 infants at delayed primary bladder closure between 01/2015 and 04/2020. The bladder biopsies were immunohistochemically stained with antibodies against S100, calcitonin gene-related peptide (anti-CGRP), Neurofilament 200 (anti-NF200), and tyrosine-hydroxylase (anti-TH). Specimens from 6 children with congenital vesicoureterorenal reflux (VUR) served as controls. RESULTS: There was no statistically significant difference in nerve fiber density in any of the immunohistochemical assessments (anti-S100 [p = 0.210], anti-CGRP [p = 0.897], anti-NF200 [p = 0.897]), and anti-TH [p = 0.956]) between patients with BE and patients with VUR. However, we observed a trend toward lower nerve fiber densities in exstrophic detrusor. CONCLUSION: Overall our results showed an unharmed innervation pattern in this cohort but a lower density of nerve fibers in the detrusor compared to controls. Further studies in patients after successful primary closure are needed to clarify the potential impact of the urothelial overexpression of NGF modulating the innervation pattern in exstrophic bladders.

Sensory spinal interoceptive pathways and energy balance regulation.

Interoception plays an important role in homeostatic regulation of energy intake and metabolism. Major interoceptive pathways include gut-to-brain and adipose tissue-to brain signaling via vagal sensory nerves and hormones, such as leptin. However, signaling via spinal sensory neurons is rapidly emerging as an additional important signaling pathway. Here we provide an in-depth review of the known anatomy and functions of spinal sensory pathways and discuss potential mechanisms relevant for energy balance homeostasis in health and disease. Because sensory innervation by dorsal root ganglia (DRG) neurons goes far beyond vagally innervated viscera and includes adipose tissue, skeletal muscle, and skin, it is in a position to provide much more complete metabolic information to the brain. Molecular and anatomical identification of function specific DRG neurons will be important steps in designing pharmacological and neuromodulation approaches to affect energy balance regulation in disease states such as obesity, diabetes, and cancer.

Neurochemical classification of serotonin-immunoreactive neurons co-innervating motor endplates in the mouse esophagus.

Serotonin immunoreactivity was previously found in myenteric neurons co-innervating motor endplates in the mouse esophagus striated muscle and an involvement in motility control was suggested. However, it is not known if other neuroactive substances are present in these neurons and to what extent they co-localize. First, vasoactive intestinal peptide (VIP) was established as a bona fide marker for putative inhibitory myenteric neurons by evaluating co-localization with neuronal nitric oxide synthase (nNOS) and neuropeptide Y (NPY). Then, co-localization of serotonin and VIP was tested in co-innervating axons on motor endplates, which were visualized with α-bungarotoxin (α-BT) by multilabel immunofluorescence. Myenteric ganglia were also surveyed for co-localization in neuronal perikarya and varicosities. nNOS, NPY, and VIP were completely co-localized in enteric co-innervating nerve terminals on motor endplates. After co-staining with VIP, we found (a) serotonin (5-HT)-positive nerve endings without VIP (44% of 5-HT-positively innervated endplates), (b) 5-HT- and VIP-positive endings without co-localization (35%), and (c) 5-HT- and VIP-positive endings with co-localization (21%). About one-fifth of nerve terminals on motor endplates containing 5-HT originate from putative inhibitory peptidegic nitrergic neurons. However, the majority represents a different population presumably subserving different functions.

Vascularization of the dorsal base of the second metacarpal bone: an anatomical study using C-arm cone beam computed tomography.

BACKGROUND: Vascularized bone grafts of the hand are a promising option for treatment of hand abnormalities. Therefore, the purpose of this study was to analyze the arterial anatomy of the dorsal aspect of the second metacarpal base to further investigate this possible donor site for bone grafts. METHODS: The authors examined 16 fresh frozen cadaveric hands by using a C-arm cone beam computed tomography scanner and depicted the three-dimensional course of the second dorsal metacarpal artery and measured the diameter, length, and arc of rotation of this nutritive vessel. In addition, the authors dissected six of the hands under a dissecting microscope and, after selective injection of gelatin dye solution, the authors analyzed the vessel entrances into the bones histologically. RESULTS: In all examined hands, the second dorsal metacarpal artery was a nutritive vessel to the dorsal base of the second metacarpal. The average diameter was 1.3±0.4 mm and the average length of the vascular pedicle was approximately 3.3±0.3 cm. In 14 of 16 cases, the arc of rotation was sufficient to reach the lunate without difficulty. Histologic analysis showed an intrinsic blood supply in the donor region with a vessel diameter of approximately 58 µm. A clinical case with application in Kienböck disease is presented. CONCLUSION: Pedicled vascularized bone grafts from this area are suitable for clinical application to treat Kienböck disease if standard donor sites are unavailable.

Innervation pattern of the preocular human central retinal artery.

The central retinal artery (CRA) is the main vessel for inner retinal oxygen and nutrition supply. While the intraocular branches lack autonomic innervation, the innervation pattern of the extra-ocular part of this vessel along its course within the optic nerve is poorly investigated. This part however is essential for maintenance of retinal blood supply, in physiological and pathological conditions. Therefore, the aim of this study was the characterization of the autonomic innervation of the preocular CRA in humans with morphological methods. Meeting the Declaration of Helsinki, eyes of body or cornea donors were processed for single or double immunohistochemistry against tyrosine hydroxilase (TH), dopamine-ß-hydroxylase (DBH), choline acetyl-transferase (ChAT), vesicular acetylcholine transporter (VAChT), neuronal nitric oxide synthase (nNOS), calcitonin gene-related peptide (CGRP), substance P (SP), vasoactive intestinal polypeptide (VIP), and cytochemistry for NADPH-diaphorase (NADPH-d). For documentation, light-, fluorescence-, and confocal laser-scanning microscopy were used. TH and DBH immunoreactive nerve fibres were detected in the CRA vessel wall, although a distinct perivascular plexus was missing. Further, nerve fibres immunoreactive for ChAT and VAChT were found, while CGRP, SP, and VIP were not detected. NADPH-d staining revealed scattered nerve fibres in the adventitia of the CRA and in close vicinity; however, nNOS-immunostaining could not confirm this finding. The CRA receives adrenergic and cholinergic innervations, indicating sympathetic and parasympathetic components, respectively. Remarkably, a peptidergic primary afferent innervation was missing. Since clinical results suggest an autoregulation of intraretinal vessels, further studies are needed to clarify the impact of CRA innervation for retinal perfusion.

TRPA1 and substance P mediate colitis in mice.

BACKGROUND & AIMS: The neuropeptides calcitonin gene-related peptide (CGRP) and substance P, and calcium channels, which control their release from extrinsic sensory neurons, have important roles in experimental colitis. We investigated the mechanisms of colitis in 2 different models, the involvement of the irritant receptor transient receptor potential of the ankyrin type-1 (TRPA1), and the effects of CGRP and substance P. METHODS: We used calcium-imaging, patch-clamp, and neuropeptide-release assays to evaluate the effects of 2,4,6-trinitrobenzene-sulfonic-acid (TNBS) and dextran-sulfate-sodium-salt on neurons. Colitis was induced in wild-type, knockout, and desensitized mice. RESULTS: TNBS induced TRPA1-dependent release of colonic substance P and CGRP, influx of Ca2+, and sustained ionic inward currents in colonic sensory neurons and transfected HEK293t cells. Analysis of mutant forms of TRPA1 revealed that TNBS bound covalently to cysteine (and lysine) residues in the cytoplasmic N-terminus. A stable sulfinic acid transformation of the cysteine-SH group, shown by mass spectrometry, might contribute to sustained sensitization of TRPA1. Mice with colitis had increased colonic neuropeptide release, mediated by TRPA1. Endogenous products of inflammatory lipid peroxidation also induced TRPA1-dependent release of colonic neuropeptides; levels of 4-hydroxy-trans-2-nonenal increased in each model of colitis. Colitis induction by TNBS or dextran-sulfate-sodium-salt was inhibited or reduced in TRPA1-/- mice and by 2-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydro-7H-purin-7-yl)-N-(4-isopro-pylphenyl)-acetamide, a pharmacologic inhibitor of TRPA1. Substance P had a proinflammatory effect that was dominant over CGRP, based on studies of knockout mice. Ablation of extrinsic sensory neurons prevented or attenuated TNBS-induced release of neuropeptides and both forms of colitis. CONCLUSIONS: Neuroimmune interactions control intestinal inflammation. Activation and sensitization of TRPA1 and release of substance P induce and maintain colitis in mice.

Intrinsic choroidal neurons in the chicken eye: chemical coding and synaptic input.

Intrinsic choroidal neurons (ICNs) exist in some primates and bird species. They may act on both vascular and non-vascular smooth muscle cells, potentially influencing choroidal blood flow. Here, we report on the chemical coding of ICNs and eye-related cranial ganglia in the chicken, an important model in myopia research, and further to determine synaptic input onto ICN. Chicken choroid, ciliary, superior cervical, pterygopalatine, and trigeminal ganglia were prepared for double or triple immunohistochemistry of calcitonin gene-related peptide (CGRP), choline acetyltransferase (ChAT), dopamine-beta-hydroxylase, galanin (GAL), neuronal nitric oxide synthase (nNOS), somatostatin (SOM), tyrosine hydroxylase (TH), vasoactive intestinal polypeptide (VIP), vesicular monoamine-transporter 2 (VMAT2), and alpha-smooth muscle actin. For documentation, light, fluorescence, and confocal laser scanning microscopy were used. Chicken ICNs express nNOS/VIP/GAL and do not express ChAT and SOM. ICNs are approached by TH/VMAT2-, CGRP-, and ChAT-positive nerve fibers. About 50% of the pterygopalatine ganglion neurons and about 9% of the superior cervical ganglion neurons share the same chemical code as ICN. SOM-positive neurons in the ciliary ganglion are GAL/NOS negative. CGRP-positive neurons in the trigeminal ganglion lack GAL/SOM. The neurochemical phenotype and synaptic input of ICNs in chicken resemble that of other bird and primate species. Because ICNs lack cholinergic markers, they cannot be readily incorporated into current concepts of the autonomic nervous system. The data obtained provide the basis for the interpretation of future functional experiments to clarify the role of these cells in achieving ocular homeostasis.

Enteric co-innervation of esophageal striated muscle fibers: a phylogenetic study.

Enteric co-innervation of striated muscle fibers in the esophagus occurs in several mammalian species including humans. However, the functional significance is still unknown. Phylogenetic data may be instrumental in gaining further insight. We examined the bat Glossophaga soricina and the shrew Suncus murinus as representatives for phylogenetically old mammals. As ruminants the antelope Tragelaphus imberbis, the he-goat Capra falconeri and the sheep Ovis aries were selected. As non-mammals the clawed frog Xenopus laevis as representative for the taxon amphibian and the rainbow trout Oncorhynchus mykiss as representative for the taxon fish were included. Histochemistry for nicotinamide adenine dinucleotide phosphate-diaphorase and acetylcholinesterase as well as immunofluorescence for vasoactive intestinal peptide and alpha-bungarotoxin were used to demonstrate enteric nerve fibers and motor endplates, respectively. Motor endplates were associated with enteric nerve fibers in all species investigated, although the rates of co-innervation varied from approximately 10 to 20% in shrew, antelope, he-goat, frog and fish, approximately 40% in bat to nearly 90% in sheep. These results demonstrate that enteric co-innervation, in spite of varying co-innervation rates, is conserved through vertebrate evolution, and underline the significance of this newly discovered innervation component.

The neuropeptide calcitonin gene-related peptide (CGRP) prevents inflammatory liver injury in mice.

BACKGROUND/AIMS: Calcitonin gene-related peptide (CGRP) is a potent vasodilator and supposed to be responsible for neurogenic inflammation involved in migraine. Its role in inflammatory diseases of other organs is controversial and poorly investigated regarding liver inflammation, although the organ is innervated by CGRP containing primary sensory nerve fibers. METHODS: Male Balb/c and IL-10(-/-) mice were pretreated with either alphaCGRP or the CGRP receptor antagonists CGRP(8-37) or BIBN4096BS. Immune-mediated liver injury was induced by administration of lipopolysaccharide (LPS) or tumor necrosis factor-alpha (TNFalpha) to galactosamine (GalN)-sensitized mice and evaluated by serum transaminase activities and cytokine levels. Furthermore, intrahepatic CGRP receptor expression and hepatic CGRP concentrations were examined. RESULTS: CGRP receptor 1 was expressed by immune cells and hepatocytes in human and murine liver. During liver injury CGRP receptor expression was increased whereas hepatic CGRP concentrations concomitantly decreased. While CGRP receptor antagonists failed to affect liver damage, pretreatment with alphaCGRP protected mice from GalN/LPS-induced liver injury by suppression of the pro-inflammatory cytokine response independently from IL-10 but related to the induction of the transcriptional repressor inducible cAMP early repressor (ICER). In contrast, alphaCGRP failed to protect against GalN/TNFalpha-induced liver failure. CONCLUSION: In the liver, CGRP exerts anti-inflammatory properties, which are characterized by a reduced production of pro-inflammatory cytokines.

Spiny versus stubby: 3D reconstruction of human myenteric (type I) neurons.

We have compared the three-dimensional (3D) morphology of stubby and spiny neurons derived from the human small intestine. After immunohistochemical triple staining for leu-enkephalin (ENK), vasoactive intestinal peptide (VIP) and neurofilament (NF), neurons were selected and scanned based on their immunoreactivity, whether ENK (stubby) or VIP (spiny). For the 3D reconstruction, we focused on confocal data pre-processing with intensity drop correction, non-blind deconvolution, an additional compression procedure in z-direction, and optimizing segmentation reliability. 3D Slicer software enabled a semi-automated segmentation based on an objective threshold (interrater and intrarater reliability, both 0.99). We found that most dendrites of stubby neurons emerged only from the somal circumference, whereas in spiny neurons, they also emerged from the luminal somal surface. In most neurons, the nucleus was positioned abluminally in its soma. The volumes of spiny neurons were significantly larger than those of stubby neurons (total mean of stubbies 806 +/- 128 mum(3), of spinies 2,316 +/- 545 mum(3)), and spiny neurons had more dendrites (26.3 vs. 11.3). The ratios of somal versus dendritic volumes were 1:1.2 in spiny and 1:0.3 in stubby neurons. In conclusion, 3D reconstruction revealed new differences between stubby and spiny neurons and allowed estimations of volumetric data of these neuron populations.

Distribution of P2X(3) receptor immunoreactivity in myenteric ganglia of the mouse esophagus.

Intraganglionic laminar endings (IGLEs) represent the major vagal afferent terminals throughout the gut. Electrophysiological experiments revealed a modulatory role of ATP in the IGLE-mechanotransduction process and the P2X(2)-receptor has been described in IGLEs of mouse, rat and guinea pig. Another purinoceptor, the P2X(3)-receptor, was found in IGLEs of the rat esophagus. These findings prompted us to investigate occurrence and distribution of the P2X(3)-receptor in the mouse esophagus. Using multichannel immunofluorescence and confocal microscopy, P2X(3)-immunoreactivity (-iry) was found colocalized with the vesicular glutamate transporter 2 (VGLUT2), a specific marker for IGLEs, on average in three-fourths of esophageal IGLEs. The distribution of P2X(3) immunoreactive (-ir) IGLEs was similar to that of P2X(2)-iry and showed increasing numbers towards the abdominal esophagus. P2X(3)/P2X(2)-colocalization within IGLEs suggested the occurrence of heteromeric P2X(2/3) receptors. In contrast to the rat, where only a few P2X(3)-ir perikarya were described, P2X(3) stained perikarya in ~80% of myenteric ganglia in the mouse. Detailed analysis revealed P2X(3)-iry in subpopulations of nitrergic (nNOS) and cholinergic (ChAT) myenteric neurons and ganglionic neuropil of the mouse esophagus. We conclude that ATP might act as a neuromodulator in IGLEs via a (P2X(2))-P2X(3) receptor-mediated pathway especially in the abdominal portion of the mouse esophagus.

The normal human choroid is endowed with a significant number of lymphatic vessel endothelial hyaluronate receptor 1 (LYVE-1)-positive macrophages.

PURPOSE: Lymphatic vessel endothelial hyaluronic acid receptor (LYVE-1) is a newly discovered lymphatic endothelium-specific marker that is also expressed by a subpopulation of macrophages. To date, there is no report on its expression in the posterior human uvea. The purpose of this study was to investigate the expression of LYVE-1 in normal human choroids. METHODS: Eyes of body/cornea donors (55-89 years of age; 4-9 hours postmortem) were obtained. Choroids were dissected and prepared for cryosections followed by immunohistochemistry with anti-human LYVE-1 antiserum and immunogold labeling. In addition, anti-human antibodies against macrophage markers (CD68, MHC class II) and lymphatic (podoplanin) and blood vascular endothelium (CD31, vWF) were used. For documentation, light-, fluorescence-, confocal laser scanning-, and electron-microscopy were used. RESULTS: The normal human choroidal stroma contained 274 +/- 86 LYVE-1 positive cells/mm(2). The cells displayed irregular shapes with a relatively uniform diameter of 32 mum. Costaining with CD68 and negativity for CD31, podoplanin, and melan-A/HMB45, as well as electron microscopic features, suggest these LYVE-1(+) cells to be macrophages. Besides that, no classic LYVE-1(+)/podoplanin(+) lymphatic vessels were detected within the normal adult human choroid. CONCLUSIONS: The normal adult human choroid does not contain typical lymph vessels, but is endowed with a significant number of LYVE-1 positive macrophages. These cells may be involved in choroidal hyaluronic acid metabolism or contribute to temporary formation of lymphatic channels under inflammatory conditions.

The autonomic facial nerve pathway in birds: a tracing study in chickens.

PURPOSE: In birds, the parasympathetic innervation of the choroid is via the ciliary (cranial nerve III) and pterygopalatine (cranial nerve VII) ganglia, the latter consisting of a chain of microganglia within the orbit. Because of the scattered nature of these microganglia, lesions of this nerve pathway in birds have not been attempted, making interpretation of the functional contribution of this parasympathetic input to the avian eye uncertain. The purpose of this study was to find an extraorbital approach to the preganglionic part of cranial nerve VII and to reveal its peripheral terminals and its site of origin in the brain stem. METHODS: The radix autonomica cranial nerve VII was accessed via the tympanic cavity and injected with dextran coupled to Texas red (DTxR). Orbital structures and the brain stem were prepared for tracer detection and immunohistochemistry for neuronal nitric oxide synthase (nNOS), choline acetyl transferase (ChAT), vasoactive intestinal polypeptide (VIP), calcitonin gene-related peptide (CGRP), galanin (GAL), and somatostatin (SOM). For documentation, fluorescence and confocal laser scanning microscopy were used. RESULTS: Anterogradely labeled DTxR-positive nerve fibers were detected within the orbital pterygopalatine microganglionic chain, forming boutons closely associated with nNOS-positive neurons. Retrogradely labeled DTxR-positive neurons with cell diameters of approximately 20 microm were found in the brain stem. These were positive for ChAT, but negative for nNOS, VIP, SOM, GAL, and CGRP. They most likely represent the preganglionic neurons of the superior salivatory nucleus. In close proximity, there were larger (40 microm) unlabeled neurons that were positive for ChAT and CGRP, but negative for GAL. These most likely represent motoneurons of the facial nerve. CONCLUSIONS: This surgical approach offers excellent opportunities for lesioning experiments for the study of the autonomic facial nerve pathway in birds in terms of both its anatomic organization and its function.

Cooperative effect of biliverdin and carbon monoxide on survival of mice in immune-mediated liver injury.

Induction of the heme-degrading enzyme heme oxygenase-1 (HO-1) has been shown to be beneficial in terms of improvement of liver allograft survival and prevention of CD95-mediated apoptosis in the liver. In the present study, we investigated the effects of HO-1, and its products carbon monoxide (CO), biliverdin (BV), and iron/ferritin, in a mouse model of inflammatory liver damage inducible by lipopolysaccharide (LPS) in mice sensitized with the hepatocyte-specific transcription inhibitor D-galactosamine (GalN). Our results show that HO-1 induction by cobalt-protoporphyrin-IX (CoPP) reduced cytokine expression, protected mice from liver injury, and prolonged survival. While in contrast to ferritin overexpression, single administration of the CO donor methylene chloride (MC) or of BV also protected mice from liver damage, only coadministration of both HO products prolonged survival and reduced the expression of cytokines, e.g., tumor necrosis factor (TNF) and interferon gamma (IFN-gamma). In conclusion, HO-1-induced prolongation of survival, but not the protection from liver damage, seems to be dependent on down-regulation of cytokine synthesis.

Neurokinin-1 receptor antagonists protect mice from CD95- and tumor necrosis factor-alpha-mediated apoptotic liver damage.

Previously, we have shown that primary afferent neurons are necessary for disease activity in immune-mediated liver injury in mice. These nerve fibers are detectable by substance P (SP) immunocytochemistry in the portal tract of rodent liver. Antagonists of the neurokinin-1 receptor (NK-1R), which is the prime receptor of SP, prevented liver damage by suppressing the synthesis of proinflammatory cytokines. Here, we investigated the influence of primary afferent nerve fibers, SP, and NK-1 receptor antagonists on hepatocyte apoptosis in vivo induced by administration of activating anti-CD95 monoclonal antibody (mAb) to mice. Depletion of primary afferent nerve fibers by neonatal capsaicin treatment prevented CD95-mediated activation of caspase-3, measured as enzymatic activity in liver homogenates or by demonstration of hepatocellular immunoreactivity for active caspase-3 in liver slices, and liver damage. This effect was reversed by administration of SP to anti-CD95 mAb-treated mice depleted from primary afferent neurons. The presence of the NK-1R on mouse hepatocytes was demonstrated by immunocytochemistry and flow cytometry. Intraperitoneal pretreatment with the NK-1 receptor antagonists (2S,3S)-cis-2-(diphenylmethyl)-N-([2-methoxyphenyl]-methyl)-1-azabicyclo(2.2.2.)-octan-3-amine (CP-96,345) or (2S,3S)3-([3,5-bis(trifluoromethyl)phenyl]methoxy)-2-phenylpiperadine (L-733,060) dose dependently protected mice from CD95-mediated liver injury. Similar results were obtained when apoptotic liver damage was induced by administration of tumor necrosis factor-alpha to d-galactosamine-sensitized mice. In conclusion, SP, probably by binding to its receptor on hepatocytes, might aggravate apoptotic signals in these cells. Because NK-1 receptor antagonists not only suppress the proinflammatory cytokine response in the liver but also prevent liver cell apoptosis in vivo, they might be suitable drugs for treatment of immune-mediated liver disease.

Intrinsic choroidal neurons in the human eye: projections, targets, and basic electrophysiological data.

PURPOSE: The chemical coding of intrinsic choroidal neurons (ICNs) has features in common with extrinsic fibers (e.g., from the pterygopalatine ganglion) making it impossible to assess whether a neuronal nitric oxide synthase (nNOS)/vasoactive intestinal polypeptide (VIP)-immunoreactive nerve fiber is of intrinsic or extrinsic origin. Neurobiotin injections into single neurons allow the visualization of projections of these cells and the determination of the origin of target innervation. Thus, this technique was used in the present study to help characterize the organization of the ICN in the human eye. METHODS: ICNs were visualized with the fluorescent vital dye 4-Di-2-ASP. Electrophysiological properties were determined by means of intracellular recordings. The impaled neurons were iontophoretically filled with neurobiotin. After fixation, immunohistochemistry for neuronal nitric oxide synthase (nNOS), alpha-smooth muscle actin, and calcitonin gene-related peptide (CGRP) was conducted. RESULTS: ICN processes were traced over distances of up to 2.612 micro m. They were found in the immediate vicinity of other nNOS-positive or -negative ICNs and were also found apposed to smooth muscle fibers (vascular and stromal nonvascular). CGRP-positive fibers forming boutons were observed closely associated with ICNs. Electrophysiological recording showed phasic firing without slow afterhyperpolarization, no spontaneous activity, an input resistance of 136 +/-73 MOmega, and a membrane time constant of 7 +/- 1 ms. CONCLUSIONS: Apart from the first functional characterization of ICNs, this study provided more precise evidence of reciprocal ICN-to-ICN contacts and innervation of both choroidal nonvascular and vascular smooth muscle. The presented technique offers promising perspectives to further investigate the function of ICNs in ocular homeostasis.

Vasoactive intestinal and calcitonin gene-related peptides, tyrosine hydroxylase and nitrergic markers in the innervation of the rat central retinal artery.

The vascular supply of the optic nerve has been studied with different methods including corrosion casts both in humans and in other mammals. In man, primates and some other mammals, such as the rat, a distinct central retinal artery accompanies the optic nerve, and runs through the lamina cribosa to reach the optic nerve head. Similarities between human and rat central retinal artery could serve to understand changes in the autonomic perivascular innervation in glaucoma using the rat as an animal model. Nitric oxide, calcitonin gene-related peptide, neuropeptide Y, substance P and vasoactive intestinal peptide have been identified around the monkey central retina artery. Innervation of the rat central artery, however, has not been described in detail. Using immuno- and histochemical methods, the present study investigates the peptidergic, adrenergic and nitrergic innervation of the rat posterior ciliary artery as well as the central retina artery. Numerous nitric oxide positive nerve fibers were visualized posterior and anterior to the lamina cribosa of the optic nerve. They colocalized with NADPH-diaphorase positive fibers, which could also be observed in two of six specimens studied at the level of the optic nerve head. Calcitonin gene-related peptide, tyrosine hydroxylase, and VIP positive fibers were also observed surrounding the vessels of the rat optic nerve. The presence of neuronal nitric oxide/NADPH-diaphorase and vasoactive intestinal peptide positive nerve fibers surrounding the posterior ciliary and central retinal arteries indicates a vasodilator effect in the rat optic nerve. Tyrosine hydroxylase positive innervation indicates the presence of sympathetic activity, and calcitonin gene-related peptide positive fibers indicate sensory innervation by trigeminal primary efferents.

Neurokinin-1 receptor antagonists CP-96,345 and L-733,060 protect mice from cytokine-mediated liver injury.

Previously, we have shown that primary afferent sensory neurons are necessary for disease activity in T cell-mediated immune hepatitis in mice. In the present study, we analyzed the possible role of substance P (SP), an important proinflammatory neuropeptide of these nerve fibers, in an in vivo mouse model of liver inflammation. Liver injury was induced by bacterial lipopolysaccharide (LPS) in D-galactosamine (GalN)-sensitized mice. Depletion of primary afferent nerve fibers by neonatal capsaicin treatment down-regulated circulating levels of the proinflammatory cytokines tumor necrosis factor-alpha (TNFalpha) and interferon-gamma (IFNgamma) and protected mice from GalN/LPS-induced liver injury. Likewise, pretreatment of mice with antagonists of the SP-specific neurokinin-1 receptor (NK-1R), i.e., (2S,3S)-cis-2-(diphenylmethyl)-N-((2-methoxyphenyl)-methyl)-1-azabicyclo(2.2.2.)-octan-3-amine (CP-96,345) and (2S,3S)3-([3,5-bis(trifluoromethyl)phenyl]methoxy)-2-phenylpiperidine (L-733,060), dose dependently protected mice from GalN/LPS-induced liver injury. The presence of the NK-1R in the murine liver was demonstrated by reverse transcription-polymerase chain reaction, sequence analysis, and immunocytochemistry. NK-1R blockade reduced inflammatory liver damage, i.e., edema formation, neutrophil infiltration, hepatocyte apoptosis, and necrosis. To get further insight into the mechanism by which receptor blockade attenuated GalN/LPS-induced liver damage, we analyzed plasma levels and intrahepatic expression of TNFalpha, IFNgamma, interleukin (IL)-6, and IL-10. NK-1R blockade clearly inhibited GalN/LPS-induced production of TNFalpha and IFNgamma, whereas synthesis of the hepatoprotective cytokines IL-6 and IL-10 was increased. NK-1 receptor antagonists might be potent drugs for treatment of inflammatory liver disease, most likely by inhibiting SP effects.

Impaired uptake of apoptotic cells into tingible body macrophages in germinal centers of patients with systemic lupus erythematosus.

OBJECTIVE: To investigate the fate of apoptotic cells in the germinal centers (GCs) of patients with systemic lupus erythematosus (SLE). METHODS: Lymph node biopsy specimens obtained from 7 SLE patients with benign follicular hyperplasia, 5 non-SLE patients with benign follicular hyperplasia (non-SLE), 5 patients with malignant follicular lymphoma, and 3 patients with dermatopathic lymphadenitis were stained with monoclonal antibodies against macrophages (CD68) and follicular dendritic cells (CR2/CD21). TUNEL staining and transmission electron microscopy were performed to detect apoptotic cells. Confocal microscopy was used to evaluate the in vivo capacity of tingible body macrophages to remove apoptotic cell material. RESULTS: In a subgroup of patients with SLE, apoptotic cells accumulated in the GCs of the lymph nodes. The number of tingible body macrophages, which usually contained engulfed apoptotic nuclei, was significantly reduced in these patients. In contrast to what was observed in all controls, TUNEL-positive apoptotic material from SLE patients was observed to be directly associated with the surfaces of follicular dendritic cells (FDCs). CONCLUSION: Our findings suggest that in a sub-group of SLE patients, apoptotic cells are not properly cleared by tingible body macrophages of the GCs. Consequently, nuclear autoantigens bind to FDCs and may thus provide survival signals for autoreactive B cells. This action may override an important control mechanism for B cell development, resulting in the loss of tolerance for nuclear antigens.

PGE(2) selectively blocks inhibitory glycinergic neurotransmission onto rat superficial dorsal horn neurons.

Despite the crucial role that prostaglandins (PGs) have in the sensitization of the central nervous system to pain, their cellular and molecular targets leading to increased pain perception have remained elusive. Here we investigated the effects of PGE(2) on fast synaptic transmission onto neurons in the rat spinal cord dorsal horn, the first site of synaptic integration in the pain pathway. We identified the inhibitory (strychnine-sensitive) glycine receptor as a specific target of PGE(2). PGE(2), but not PGF(2 alpha), PGD(2) or PGI(2), reduced inhibitory glycinergic synaptic transmission in low nanomolar concentrations, whereas GABAA, AMPA and NMDA receptor-mediated transmission remained unaffected. Inhibition of glycine receptors occurred via a postsynaptic mechanism involving the activation of EP2 receptors, cholera-toxin-sensitive G-proteins and cAMP-dependent protein kinase. Via this mechanism, PGE(2) may facilitate the transmission of nociceptive input through the spinal cord dorsal horn to higher brain areas where pain becomes conscious.