Comparative pilot study of implantation techniques for pudendal neuromodulation: technical and clinical outcome in first 20 patients with chronic pelvic pain.

PURPOSE: Neurostimulation of the pudendal nerve (PN) is considered for patients who have failed sacral neuromodulation. Previous techniques for PN localization are described to be uncomplicated and promise to achieve accuracy in electrode placement. However, in clinical use, they appear challenging. We developed a puncture technique using fixed anatomical landmarks for a fast and reproducible localization of the PN. METHODS: Full-body cadavers and dissected anatomical preparations were studied for the course of the PN. Fluoroscopically controlled fixed anatomical landmarks locating the pudendal trunk were defined. Lead placement following established techniques was performed, and the topographic relationship to the PN was documented by dissection. In a pilot series of 20 patients with chronic pelvic pain, pudendal neuromodulation (PNM) was performed uni- and bilateral using the different approaches. Technical and clinical outcomes of the various techniques were compared. RESULTS: Fixed anatomical landmarks such as ischial spine, ischial tuberosity, acetabulum and anal rim resulted in a right-angled triangle with a new start and target point for puncture. Initials of the landmarks add up to the teaching acronym STAR. STAR technique including a puncture angle of 60° and a gluteal lead exit places 3-4 electrode poles at the nerve. In clinical trial, mean operation time for bilateral PNM in STAR was 85 min with mean puncture attempts of 3.5 to reach the nerve. Pain decreased statistically significant only in bilateral PNM. CONCLUSIONS: The STAR approach appears to achieve technical standardisation and optimized reproducibility in pudendal lead placement resulting into an increased feasibility of PNM.

Organization of the actin filament cytoskeleton in the intestinal brush border: a quantitative and qualitative immunoelectron microscope study.

In the present study we have used immunogold labeling of ultrathin sections of the intact chicken and human intestinal epithelium to obtain further insight into the molecular structure of the brush-border cytoskeleton. Actin, villin, and fimbrin were found within the entire microvillus filament bundle, from the tip to the basal end of the rootlets, but were virtually absent from the space between the rootlets. This suggests that the bulk of actin in the brush border is kept in a polymerized and cross-linked state and that horizontally deployed actin filaments are virtually absent. About 70% of the label specific for the 110-kD protein that links the microvillus core bundle to the lipid bilayer was found overlying the microvilli. The remaining label was associated with rootlets and the interrootlet space, where some label was regularly observed in association with vesicles. Since the terminal web did not contain any significant amounts of tubulin and microtubules, the present findings would support a recently proposed hypothesis that the 110-kD protein (which displays properties of an actin-activated, myosin-like ATPase) might also be involved in the transport of vesicles through the terminal web. Label specific for myosin and alpha-actinin was confined to the interrootlet space and was absent from the rootlets. About 10-15% of the myosin label and 70-80% of the alpha-actinin label was observed within the circumferential band of actin filaments at the zonula adherens, where myosin and alpha-actinin displayed a clustered, interrupted pattern that resembles the spacing of these proteins observed in other contractile systems. This circular filament ring did not contain villin, fimbrin, or the 110-kD protein. Finally, actin-specific label was observed in close association with the cytoplasmic aspect of the zonula occludens, suggesting that tight junctions are structurally connected to the microfilament system.

Major loss of the 28-kD protein of gap junction in proliferating hepatocytes.

There is a reduction in the 28-kD gap junction protein detectable by immunofluorescence in livers of partially hepatectomized rats and in cultured hepatocytes stimulated to proliferate. By the coordinate use of antibodies directed to the hepatic junction protein (HJP28) and the use of a monoclonal antibody that recognizes bromodeoxyuridine (BrdU) incorporated into DNA, we have been able to study the relationship between detectable gap junction protein and cell division. Hepatocytes that label with BrdU in the regenerating liver and in cell culture show a significant reduction of HJP28. Cells that do not synthesize DNA, on the other hand, show normal levels and distribution of immunoreactive gap junction protein. We postulate that the quantitative changes in gap junction expression might play an important role in the control of proliferation in the liver.

Comparative characterization of the 21-kD and 26-kD gap junction proteins in murine liver and cultured hepatocytes.

Affinity-purified antibodies to mouse liver 26- and 21-kD gap junction proteins have been used to characterize gap junctions in liver and cultured hepatocytes. Both proteins are colocalized in the same gap junction plaques as shown by double immunofluorescence and immunoelectron microscopy. In the lobules of rat liver, the 21-kD immunoreactivity is detected as a gradient of fluorescent spots on apposing plasma membranes, the maximum being in the periportal zone and a faint reaction in the perivenous zone. In contrast, the 26-kD immunoreactivity is evenly distributed in fluorescent spots on apposing plasma membranes throughout the rat liver lobule. Immunoreactive sites with anti-21 kD shown by immunofluorescence are also present in exocrine pancreas, proximal tubules of the kidney, and the epithelium of small intestine. The 21-kD immunoreactivity was not found in thin sections of myocardium and adult brain cortex. Subsequent to partial rat hepatectomy, both the 26- and 21-kD proteins first decrease and after approximately 2 d increase again. By comparison of the 26- and 21-kD immunoreactivity in cultured embryonic mouse hepatocytes, we found (a) the same pattern of immunoreactivity on apposing plasma membranes and colocalization within the same plaque, (b) a similar decrease after 1 d and subsequent increase after 3 d of both proteins, (c) cAMP-dependent in vitro phosphorylation of the 26-kD but not of the 21-kD protein, and (d) complete inhibition of intercellular transfer of Lucifer Yellow in all hepatocytes microinjected with anti-26 kD and, in most cases, partial inhibition of dye transfer after injection of anti-21 kD. Our results indicate that both the 26-kD and the 21-kD proteins are functional gap junction proteins.

Axo-axonal coupling. a novel mechanism for ultrafast neuronal communication.

We provide physiological, pharmacological, and structural evidence that axons of hippocampal principal cells are electrically coupled, with prepotentials or spikelets forming the physiological substrate of electrical coupling as observed in cell somata. Antidromic activation of neighboring axons induced somatic spikelet potentials in neurons of CA3, CA1, and dentate gyrus areas of rat hippocampal slices. Somatic invasion by these spikelets was dependent on the activation of fast Na(+) channels in the postjunctional neuron. Antidromically elicited spikelets were suppressed by gap junction blockers and low intracellular pH. Paired axo-somatic and somato-dendritic recordings revealed that the coupling potentials appeared in the axon before invading the soma and the dendrite. Using confocal laser scanning microscopy we found that putative axons of principal cells were dye coupled. Our data thus suggest that hippocampal neurons are coupled by axo-axonal junctions, providing a novel mechanism for very fast electrical communication.

The rat hepatocyte plasma membrane organic anion binding protein is immunologically related to the mitochondrial F1 adenosine triphosphatase beta-subunit.

A 55-kD organic anion binding protein (OABP) was identified previously in liver cell plasma membrane sinusoidal subfractions. Although this protein was localized to the surface of hepatocytes by immunofluorescence, immunoblot analysis revealed reactivity toward both plasma membrane and mitochondrial fractions. To clarify these findings, an immunoreactive clone from a rat liver cDNA expression library was isolated, the 1,500-base pair cDNA insert was sequenced, and the corresponding beta-galactosidase fusion protein was expressed and purified. The resulting sequence corresponded to that of the rat mitochondrial F1-adenosine triphosphatase (F1-ATPase) beta-subunit. This protein and OABP are of similar size and are mutually immunologically cross-reactive. That the antigen was present on the cell surface as well as in mitochondria was suggested from studies of immunoprecipitation after cell-surface iodination, and light- and electron-microscopic immunocytochemistry. Photoaffinity labeling of bovine F1-ATPase with high-specific-activity [35S]sulfobromophthalein revealed binding only to the beta-subunit. Hepatocyte uptake of bilirubin and sulfobromophthalein requires cellular ATP and mitochondria also transport these organic anions, which at high doses inhibit respiration. The presence of an organic anion binding site on the F1-ATPase beta-subunit suggests that it may play a role in these processes.

Localization of the pannexin1 protein at postsynaptic sites in the cerebral cortex and hippocampus.

Pannexins (Panx) constitute a new family of gap junction type proteins. Functional expression in paired Xenopus oocytes indicated that pannexins are capable of forming communicating junctions but also proved to be active in forming of unopposed hemichannels. In the vertebrate brain pannexins have been found in neurons. However, the subcellular cerebral localization of pannexin proteins which could gain first clues on their putative function is essentially unknown. Here we demonstrate by light and electron microscopical immunohistochemistry that Panx1 reveals postsynaptic localization in rodent hippocampal and cortical principal neurons accumulating at postsynaptic densities. The postsynaptic localization was corroborated by co-localization of Panx1 with postsynaptic density protein 95 (PSD-95), a prominent postsynaptic scaffolding protein, in hippocampal neurons expressing tagged versions of these proteins. The asymmetric synaptic distribution of Panx1 suggests that it may function in neurons as non-junctional channels (pannexons) at postsynaptic sites and comprises a novel component of the postsynaptic protein complex.

Bone grafting in oblique versus prepared rectangular uncontained glenoid defects in reversed shoulder arthroplasty. A biomechanical comparison.

BACKGROUND: How the shape of the glenoid defect being reconstructed influences stability in reversed shoulder arthroplasty has never been evaluated. The purpose of this study was to compare the reconstruction of two different shaped defects in reversed shoulder arthroplasty. METHODS: Two groups (ten Sawbone scapulae each) of oblique- and rectangular-shaped glenoid defects were tested biomechanically. On the anterior half of the glenoid, bony defects (rectangular and oblique shaped) were prepared and reconstructed subsequently with a graft and reversed shoulder arthroplasty. As a control group, Sawbones without glenoid deficiency were used. In addition, these tests were reproduced in cadavers. FINDINGS: In Sawbones, no significant difference in initial stability was found between the two groups (p>0.05). Additionally, in the cadaver tests no significant difference was found between the groups with different defects (p>0.05). During the preparation, macroscopic loosening of the oblique bone grafts was found in three cases after the performance of the reversed shoulder arthroplasty due to the lack of medial support. The localization of the highest micromotion were measured primarily between the scapula bone and the graft compared to the measured micromotions between glenoid implant and the graft. INTERPRETATION: If the oblique-shaped bone graft was secured under the baseplate, the rectangular defect preparation did not show a significantly higher primary stability. However, the advantage of medial support in rectangular defects leads to more stability while placing the bone graft and baseplate during the surgical technique and should therefore be considered a preferable option.

Gap junctions in the brain: where, what type, how many and why?

Gap junctions represent well-documented means of intercellular communication in various tissues, including the brain, where they function as portals allowing the exchange of electrolytes, second messengers and metabolites between cells. In view of the enormous recent surge of information dealing with the cellular and molecular biology of gap junctions in non-nervous tissue, as well as current interest in the cell biology of glia, this review is intended to provide an overview of the molecular and functional implications of gap-junction-mediated intercellular communication in the nervous system.

X-linked dominant Charcot-Marie-Tooth disease and other potential gap-junction diseases of the nervous system.

Gap junctions play important roles in the exchange of information and metabolites in the nervous system. These roles are highlighted by peripheral neuropathy (X-linked dominant Charcot-Marie-Tooth disease) that is associated with mutations in a gap-junction protein (connexin32), resulting in loss of function, and by somatic dysfunctions where changes in expression, organization or function of gap junctions are associated with neuronal hyper- or hypoexcitability. In this review, the causes and consequences of this gap-junction-related peripheral neuropathy and other pathological conditions of the nervous system, where dysfunctions of junctional communication are considered to play a casual role, are considered.

Functional properties of channels formed by the neuronal gap junction protein connexin36.

The expression and functional properties of connexin36 (Cx36) were examined in two communication-deficient cell lines (N2A-neuroblastoma and PC-12 cells) transfected with Cx36 and in hippocampal neurons that express the connexin endogenously. Transfected cells expressed the expected 2.9 kb Cx36 transcript and Cx36 immunoreactivity, whereas nontransfected cells were devoid of Cx36. The relationship between steady-state junctional conductance (g(j)) and transjunctional voltage was well described by a two-state Boltzmann equation. The half-inactivation voltage (V(0)), the ratio of minimal to maximal g(j) (g(min)/g(max)), and the equivalent gating charge were +/- 75 mV, 0.55, and 1.75, respectively, indicating that Cx36 exhibits very low voltage sensitivity. Conductance of single Cx36 channels measured with patch pipettes containing 130 mM CsCl was 10-15 pS (n = 15 cell pairs); despite this low unitary conductance, Cx36 channels were permeable to the dye Lucifer yellow. Hippocampal neurons expressed Cx36 both in vivo and in culture. The electrophysiological properties of channels in cultured hippocampal neurons were similar to those of the channels expressed by the transfected cell lines, and the neuronal channels were similarly permeable to Lucifer yellow. The unique combination of weak voltage sensitivity, small unitary conductance, and permeation by anions as large as second messenger molecules endows Cx36 gap junction channels with properties well suited for mediating flexible electrical and biochemical interactions between neurons.

Molecular and functional diversity of neural connexins in the retina.

Electrical synapses (gap junctions) in neuronal circuits have become a major focus in the study of network properties such as synchronization and oscillation (Galarreta and Hestrin, 1999; Gibson et al., 1999). Despite the recent progress made in unraveling the contribution of gap junctions to network behavior, little is known about the molecular composition of the junctional constituents. By cloning gap junction proteins [connexins (Cxs)] from zebrafish retina and through functional expression, we demonstrate that the retina possesses a high degree of connexin diversity, which may account for differential functional properties of electrical synapses. Three new Cxs, designated as zebrafish Cx27.5 (zfCx27.5), zfCx44.1, and zfCx55.5, and the carp ortholog of mammalian Cx43 were cloned. By in situ hybridization and in situ RT-PCR, we demonstrate that the four fish connexin mRNAs show differential localization in the retina. Transient functional expression in paired Xenopus oocytes and in the neuroblastoma N2A cell line indicate an extreme range of electrophysiological properties of these connexins in terms of voltage dependence and unitary conductance. For instance, the new zfCx44.1 exhibited high sensitivity to voltage-induced closure with currents decaying rapidly for transjunctional potentials >10 mV, whereas zfCx55.5 channels showed an opposite voltage dependence in response to voltage steps of either polarity. Moreover, although zfCx44.1 channels showed unitary conductance as high as any previously reported for junctional channels (nearly 300 pS), zfCx55. 5 and zfCx27.5 exhibited much lower unitary conductances (<60 pS).

Molecular anatomy of the blood-brain barrier as defined by immunocytochemistry.

This review outlines the recent developments and improvements of our knowledge concerning the molecular composition of the BBB as revealed by immunocytochemistry. Data have been accumulated which show that the BBB exhibits a specific collection of structural and metabolic properties which are also found in tight transporting epithelia. This conclusion is substantiated by (i) the implementation of antibodies which recognize proteins of non-BBB origin, to show that these biochemical markers and the functions that they represent are localized in the BBB endothelium; and (ii) the characterization of target molecules to which polyclonal or monoclonal antibodies which have been generated to epitopes of the BBB endothelium or brain homogenates. According to these data the protein assemblies comprising the phenotypical appearance of the BBB can therefore be defined by the particular selection as well as topological expression of common epithelial antigens, rather than the expression of BBB-unique molecular species. In this respect the immunocytochemical data corroborate the physiological assumption that the BBB possesses the character of a specific polarized epithelium. Attention is also given to the description of developmental expression of BBB-related immunomarkers. By collecting the data from different sources we introduce a classification of the BBB marker proteins according to their developmental appearance. Three groups of proteins are classified with respect to their sequential expression around the time of BBB closure: Phase E (early) markers which appear before BBB closure, phase I (intermediate) markers which are expressed at the time of BBB tightening, and phase L (late) markers which are detectable after the closure of the BBB. Such a scheme may to be useful in better defining the maturation process of BBB, which apparently is not a momentary event in brain development, but rather consists of a temporally sequenced process of hierarchically structured gene expression which finally define the molecular properties of the BBB. This process continues even after parturition, especially with regard to the achievement of immunological properties of the mature BBB. By examining the developmental spatio-temporal expression of different BBB markers we conclude that the mechanisms governing the pattern of BBB maturation are not limited to the interactions occurring between glial and endothelial cells. We therefore suggest a heuristic model in a triangular interrelationship that includes differentiation effects of neurons on glia and of glia cells on the BBB endothelium.(ABSTRACT TRUNCATED AT 400 WORDS)

Construction of an apparatus for perfusion cell cultures which enables in vitro experiments under organotypic conditions.

The value of cultured cells in cell biological, pharmaceutical or biotechnological research depends on the degree of terminal cell differentiation. In conventional Petri dishes or tissue culture plates it is often difficult to achieve culture conditions which resemble the in situ situation of intact tissue, as regards optimal cell adhesion, exchange of nutrients and metabolic products. These limitations prompted us to develop simple laboratory tools which optimize the environment of cultured cells. A perfusion apparatus with various culture containers and compatible cell holder sets was constructed which allows the simulation of organotypic conditions. (i) The cells can be kept on individual and interchangeable support materials for an optimal cell attachment. (ii) Culture medium can be perfused during the whole culture period. (iii) One type of the new culture container can be perfused with different media at the apical and basal side of the cells, thus mimicking the organotypic environment that applies for epithelial monolayers. Cell culture experiments with renal collecting duct epithelia exhibited an excellent morphological appearance showing typical features of principal and intercalated cells.

Cytoplasmic and cell surface structure of purified liver gap junctions revealed by freeze-drying.

After freeze-drying of purified liver gap junction plaques and vesicles the structural features of the inner and outer aspects of purified gap junctions were investigated. No structural details were seen on the cytoplasmic side of the connexions whereas on the cell surface side the connexions were organized in a paracrystalline pattern and exhibited a central depression or pore. We conclude that the central pore through each connexion varies in diameter along its length and that the closing site is located near the cytoplasmic face.

Large conductance channel in plasma membranes of astrocytic cells is functionally related to mitochondrial VDAC-channels.

Large conductance anion channels with similar electrophysiological characteristics were found in plasma membranes and in outer mitochondrial membranes of various cell types. Although their large conductance and their peculiar voltage dependence point to a close relation, it was questioned whether they belong to the same family. We therefore compared some biochemical features of a plasmalemmal channel with those known from the mitochondrial channel. Current events were recorded from excised patches of plasma membranes of a rat astrocytic cell line (RGCN). The underlying channels exhibited a conductance of 401 +/- 50 pS. Open probability was highest between +/- 10 mV and gradually approached zero beyond +/- 25 mV. Activity as induced by voltage ramps between +/- 40 mV appeared after a delay of up to several min. The delay could be reduced by bathing either side of the patch in an acidic Ringer solution (pH 6.2). 1 mM Al3+ increased the open time at potentials more positive than 20 mV. 10 mM dextran sulfate (MW 8000) caused reversible flickering, increasing the closed probability. 4,4'-diisothiocyanatostilbene-2,2' disulfonic acid (DIDS) also caused a reversible flickering into the closed state, reducing the apparent single channel amplitude by up to 70% at 0.5 mM DIDS. Application of 5 mM ATP resulted in reversible blockade; ATP was more effective from the outside than from the inside (blocking activity 65% vs. 16% of the patches). We conclude that the large conductance anion channel from astrocytic cells displays electrophysiological and pharmacological characteristics that resemble those of VDAC (Voltage Dependent Anion Channel) from the outer mitochondrial membrane.

Regulation of a blood-brain barrier-specific enzyme expressed by cerebral pericytes (pericytic aminopeptidase N/pAPN) under cell culture conditions.

In this study we show that the aminopeptidase N of cerebral pericytes (pAPN) associated with the blood-brain barrier (BBB) is downregulated in pericytic cell cultures. This observation is in accordance with previous data describing comparable in vitro effects for BBB-specific enzymes of endothelial or pericytic origin, such as gamma-glutamyl transpeptidase or alkaline phosphatase. By polymerase chain reaction and in situ hybridization we were able to determine that the down-regulation of pAPN occurs at the posttranscriptional level. The mRNA of pAPN was found to be constitutively expressed even when the protein is no longer detectable. Culturing the pericytes in an endothelial cell-conditioned medium allowed pAPN to be reexpressed. However, the reexpression effect depended largely on the culturing conditions of the pericytes. Although purified pericytes deprived of endothelial cells did not reveal a reexpression effect, pericytes that were kept in contact with endothelial cells were able to acquire a pAPN-positive phenotype, indicating that endothelial cells constitute an essential requirement for the in vitro reexpression of pAPN. Astrocytes, however, were insufficient in exerting any reexpression effect.

A 16 kDa protein co-isolating with gap junctions from brain tissue belonging to the class of proteolipids of the vacuolar H+-ATPases.

A 16 kDa protein from an enriched gap junction preparation was isolated from bovine brain tissues. N-terminal amino acid microsequencing of the first 20 amino acids showed a complete homology with a recently published sequence of a proteolipid from a vacuolar H+-ATPase from chromaffin granules. Incubation of the brain gap junction preparation with 14C-N,N'-dicyclohexylcarbodiimide showed a significant binding of this compound to the 16 kDa protein, indicating that a proton binding site also occurs within that particular protein. The data suggest that this 16 kDa protein, which has also been described in gap junction preparations from various other tissues, belongs to the proton transporting ATPase.

Distribution of connexin43 immunoreactivity in the retinas of different vertebrates.

The distribution of Connexin43 (Cx43) was examined by immunoblotting and immunofluorescence microscopy in the retinas of five different vertebrates by using a C-terminal specific peptide antibody. The specificity of the antibody was proved on immunoblots, in which it showed cross reactivity with a 43-kDa protein in rat heart homogenates as well as in homogenates of rabbit, rat, chicken, turtle, and fish (carp and zebrafish) retinas. Immunofluorescence histochemistry with retinal cryosections revealed the presence of Cx43 in the retinal pigment epithelium cells of all tested species and in blood vessels of vascularized retinas (fish and rat). Cx43 immunoreactivity was further localized in the stria medullaris of rabbit retina, in the nerve fiber layer of rat retina, most likely in astrocytes, and in the area of the outer limiting membrane of the fish retina, most likely representing Cx43 in Müller glia cells. A punctate Cx43-immunoreactive pattern consistent with gap junctions was also detected in the outer plexiform layer of carp and zebrafish retinas, and a specific amacrine cell type, which ramified in two layers of the inner plexiform layer, was labeled in the zebrafish retina. The present results are in accordance with previous findings showing the abundance of Cx43 in astrocytes, endothelium, and epithelial cells. However, the presence of Cx43 immunoreactivity in a specific population of amacrine cells of the zebrafish retina might indicate that a Cx43-like protein is also expressed in neurons.

Synapses between slowly adapting lung stretch receptor afferents and inspiratory beta-neurons in the nucleus of the solitary tract of cats: a light and electron microscopic analysis.

Previous neuroanatomic and physiologic studies indicated that afferent fibres from slowly adapting pulmonary stretch receptors (SAR) project to the nuclei of the solitary tract and terminate on inspiratory beta-neurons. In the present study we combined electrophysiologic and morphologic approaches to verify the presumed monosynaptic connections between SARs and beta-neurons. Single identified beta-neurons and single identified SAR afferent fibres were labelled intrasomally and intraaxonally, respectively, with horseradish peroxidase (HRP) in the same anesthetized cats. Under the light microscope, we analyzed the morphology of beta-neurons and their dendritic fields and of the terminal projection pattern of fibres from SARs and identified potential synaptic connections between boutons of SAR afferent fibres and the soma and dendrites of beta-neurons. The identified tissue was then processed further for electron microscopic analysis. On average, beta-neurons had 6 primary dendrites that bifurcated 3-8 times. The dendritic trees extended 1.5 mm both rostrocaudally in the ventrolateral nucleus of the solitary tract and medially into the intermediate subnucleus. Axons of beta-neurons curved toward the midline and no collateral branches were evident over its stained length (2.5-3.4 mm). Axodendritic synaptic contacts between SAR fibres and beta-neurons were identified electron microscopically in four of six tissue samples chosen by light microscopy. In addition, we located 2 axodendritic and 2 axosomatic synaptic contacts that were not observed under light microscopic screening. The boutons of SAR fibres contained clear, round vesicles and formed asymmetrical synapses with beta-neurons. Multiple synaptic connections were found between collaterals of a single SAR and single beta-neurons, indicating a dense terminal projection of single SAR afferent fibres onto beta-neurons. These morphologic data prove monosynaptic connections between electrophysiologically identified SAR afferent fibres and beta-neurons.