Sealed Patch Research Articles

Fetoscopic patch coverage of experimental myelomenigocele using a two-port access in fetal sheep.

This study aims to assess the feasibility and the effectiveness of a fetoscopic myelomeningocele (MMC) coverage using a sealed inert patch through a two-port access, in the sheep model. Forty-four fetuses underwent surgical creation of a MMC defect at day 75 and were divided into four groups according to the MMC repair technique, performed at day 90. Group 1 remained untreated. Group 2 had an open surgery using suture of the defect. Groups 3 and 4 underwent defect coverage using a Gore®-polytetrafluoroethylene patch secured with surgical adhesive (Bioglue®), with an open approach (group 3) and a fetoscopic one (group 4). Lambs were killed at term, and histological examinations were performed. Fetoscopic patch coverage was achieved in all the lambs of group 4. All the fetuses of group 2 had a complete closure of the defect whereas only 38% in group 3 and 14% in group 4. Fetal loss rate seems to be lower in group 4 than in groups 2 and 3. Fetoscopic coverage of MMC defect can be performed using a sealed patch through a two-port access, but the patch and glue correction may not be the ideal technique to repair fetal MMC.

In praise of single channel kinetics.

It is more than 30 years since the advent of the gigaohm seal patch clamp technique, which enabled routine recordings of currents generated by the activity of a single ion channel molecule in its native environment. The muscle nicotinic acetylcholine (ACh) receptor (nAChR) is one of the best ligand-

Electrophysiological Characterization of TMEM16A

Sweating is an essential physiological process to regulate body temperature in humans and various disorders are associated with dysregulated sweat formation.Primary sweat secretion in human eccrine sweat glands is initiated by Ca2+-activated Cl- channels (CaCC). Recently, members of the TMEM16 family were identified as CaCCs in various secretory epithelia, however, their molecular identity in sweat glands remained elusive. Here we investigated the function of TMEM16A in sweat glands. Gene expression analysis revealed that TMEM16A is expressed in human NCL-SG3 sweat gland cells as well as in isolated human eccrine sweat gland biopsy samples. Sweat gland cells express several previously described TMEM16A splice variants, as well as one novel splice variant, TMEM16A(ace3) lacking the TMEM16A-dimerization domain. Chloride-flux assays using halide-sensitive YFP revealed that TMEM16A is functionally involved in Ca2+-dependent Cl- transport in NCL-SG3 cells. Recombinant expression in NCL-SG3 cells showed that TMEM16A(ace3) is forming a functional CaCC, with basal and Ca2+-activated Cl-permeability distinct from canonical TMEM16A(ac). Our results suggest that various TMEM16A isoforms contribute to sweat gland-specific Cl- transport providing opportunities to develop sweat gland-specific therapeutics for the treatment of sweating disorders.HEK cells expressing TMEM16a were investigated by high throughput giga seal patch clamping with the SyncroPatch 384PE. Currents were activated by internal Ca and could be blocked by Niflumic Acid.

Fetoscopic coverage of experimental myelomeningocele in sheep using a patch with surgical sealant

Objective Assess the feasibility of a fetoscopic patch coverage method for myelomeningocele repair in a sheep model. Study design Experimental study. A myelomeningocele-like defect was created in 15 fetal sheep on day 75 of gestation. Six remained untreated, whereas 9 underwent fetoscopic coverage of the defect on day 95 of gestation using an inert patch secured with surgical sealant. Clinical and histological examinations were performed after delivery. Results Four valid newborn lambs were obtained in each group. Mean fetoscopic surgical time was 26.9 (SD = 7.4) min. All untreated animals had an open lumbar defect with cerebrospinal fluid leakage, paraplegia, urinary incontinence, and Chiari malformation. All treated animals had a closed defect and were able to walk; one had weak bladder control, and another mild Chiari malformation. Conclusion In a chronic myelomeningocele model in fetal sheep, fetoscopic repair using a sealed patch results in simple, fast, satisfactory neural tube closure and averts neurological damage and Chiari malformation.

Inert patch with bioadhesive for gentle foetal surgery of myelomeningocele in a sheep model

Objective Current techniques used in foetal myelomeningocele repair can require considerable manipulation of fragile foetal tissues to obtain tension-free closure. The aim of this study was to assess the feasibility of a simple foetal coverage method without foetal tissue manipulation to provide closure of the neural tube defect in myelomeningocele. Study design This is an experimental study performed in 15 foetal sheep with lumbar myelomeningocele, surgically created on day 75 of gestation. Five foetuses remained untreated. Ten underwent coverage with inert sheeting (5 Silastic; 5 Silastic + Marlex) secured by surgical tissue adhesive without suturing on day 95; none of them underwent foetal muscle or skin manipulation. Clinical and subsequent histological examinations were performed at 48 h after birth. The Chi-square, Fisher exact, and Mann–Whitney U tests, when appropriate, were used for the comparisons. Results The mean operating time for foetal coverage was 7.1 (SD = 1.6) min. All untreated animals were unable to walk, had sphincter incontinence, showed an open defect, histological spinal cord damage, and a large Chiari malformation. All covered animals were able to walk, had sphincter continence, showed almost complete closure of the defect with regeneration of several soft tissue layers, and minimum Chiari malformation. Conclusion In a surgical myelomeningocele model in sheep, a simple, fast and gentle coverage method using a sealed patch avoids foetal tissue manipulation and enables adequate closure of the neural tube defect, providing regeneration of several tissue layers that protect the spinal cord, and significantly reducing Chiari II malformation.

Discretionary carotid patch angioplasty leads to good results

Background This study evaluated the type and need for angioplasty in 253 consecutive carotid endarterectomies. Methods Polyester knitted gelatin sealed patch (DP) and polytetrafluoroethylene (PTFE) patches were used in, respectively, 159 and 29 patients, with 65 vessels closed primarily (no patch [NP]). Results Surgical results, estimated blood loss, and surgical time were similar in each group. Postoperative hematomas occurred in 6 DP and 3 NP patients. There were 3 strokes in the DP group. Long-term duplex evaluation was possible in 201 patients. The number of patients who had less than 15%, 15% to 50%, 50% to 79%, 80% to 99%, 100%, or an ungraded degree of narrowing were as follows for each group: DP, 117, 2, 5, 0, 1, and 2; PTFE, 18, 1, 1, 0, 0; and NP, 53, 0, 0, 0, 1. Statistical analysis failed to show any difference between groups postoperatively or in long-term follow-up evaluation. Conclusions It appears that selective patching is safe and effective in male patients who undergo carotid endarterectomy. The type of patch material also is inconsequential. Patch type and its use should be at the surgeon’s discretion.

Prevention of anastomosis leakage: An artificial connective tissue

Anastomosis leakage still causes considerable morbidity and mortality after digestive tract surgery. We report here our experience of the use of reconstituted connective tissue created from elastin, fibronectin and collagen in preventing anastomosis leakage and closing intestinal fistulas. In animal studies (56 rats), this connective tissue was used as a patch applied with fibrin sealant to the edges of a 1 cm diameter colonic defect. In a clinical evaluation, eight patients had an intestinal fistula closed by a simple sewn suture reinforced by a sealed patch and seven patients had a high risk anastomosis reinforced by a sealed patch, in most cases as an alternative to staged surgery. In animal studies, complete reconstitution of the three colonic layers was obtained without retraction and without inflammatory reaction within 40 days, while the patch was slowly resorbed. In clinical trials all 15 defects and anastomoses healed without complication. Three patients died from other causes and, at autopsy, the patch was found to have remained in place covering the suture line. If these results are confirmed by a prospective clinical trial, such biomaterial offers the possibility of reducing the occurrence of anastomosis leakage, especially in high risk circumstances.

Electrothermally actuated inline microfluidic valve

A normally open electrothermally actuated inline microvalve that has been developed, fabricated and tested with liquids is presented. These actuators use high volumetric expansion of a sealed patch of Paraffin heated above its melting point, providing large displacements and forces while using low power. The inline valve is surface micromachined on top of preformed flexible microfluidic channels using a low temperature fabrication process; therefore it is suitable for integration with microfluidic networks requiring actuation of a large number of independent valves under electrical control. Complete closure of sealed microchannels has been observed with power as low as 40 mW. Response times of 15 ms have been measured. Breakdown of the inline valve occurs at an upstream pressure of 23 psig.

Effects of amlodipine on unitary non-L-type high voltage-activated Ca2+ channel currents in differentiated PC12 cells.

Effects of amlodipine on unitary non-L-type high voltage-activated Ca2+ channels (Cav2 channels) were investigated in cell-attached patches of nerve growth factor (NGF)-differentiated PC12 cells. Cav2 channels, mainly composed of N-type channel in our experimental condition, were defined in this study as high voltage-activated Ca2+ channels obtained after selection of patches without L-type channel "mode 2" activity in the presence of 1 microM BayK8644, or L-type channel block by 5 microM nifedipine. At a test potential of +20 mV, they had a unitary current amplitude of approximately 0.5 pA and open time constants of approximately 0.4 ms and approximately 1.1 ms when fit assuming double exponential components. As bath application of amlodipine was ineffective to modify Cav2 channels in the sealed patch, we analyzed the channel activity when giga-seal formation was obtained in the presence of amlodipine (10 microM both in the pipette and bath solution). Amlodipine did not modify the unitary current amplitude but suppressed the channel open probability (NPo) when holding potential was depolarized, shifting the voltage-dependent inactivation curve towards negative potentials by 25mV. Amlodipine-induced suppression of NPo was mainly due to the decreased ratio of sweeps with channel openings (availability) and was not associated with changes in open time constants. These results were consistent with the view that amlodipine prevented channel openings through the high-affinity binding to the inactivated state, as often observed when dihydropyridines block L-type Ca2+ channels.

Molecular basis of mechanotransduction in living cells.

The simplest cell-like structure, the lipid bilayer vesicle, can respond to mechanical deformation by elastic membrane dilation/thinning and curvature changes. When a protein is inserted in the lipid bilayer, an energetic cost may arise because of hydrophobic mismatch between the protein and bilayer. Localized changes in bilayer thickness and curvature may compensate for this mismatch. The peptides alamethicin and gramicidin and the bacterial membrane protein MscL form mechanically gated (MG) channels when inserted in lipid bilayers. Their mechanosensitivity may arise because channel opening is associated with a change in the protein's membrane-occupied area, its hydrophobic mismatch with the bilayer, excluded water volume, or a combination of these effects. As a consequence, bilayer dilation/thinning or changes in local membrane curvature may shift the equilibrium between channel conformations. Recent evidence indicates that MG channels in specific animal cell types (e.g., Xenopus oocytes) are also gated directly by bilayer tension. However, animal cells lack the rigid cell wall that protects bacteria and plants cells from excessive expansion of their bilayer. Instead, a cortical cytoskeleton (CSK) provides a structural framework that allows the animal cell to maintain a stable excess membrane area (i.e., for its volume occupied by a sphere) in the form of membrane folds, ruffles, and microvilli. This excess membrane provides an immediate membrane reserve that may protect the bilayer from sudden changes in bilayer tension. Contractile elements within the CSK may locally slacken or tighten bilayer tension to regulate mechanosensitivity, whereas membrane blebbing and tight seal patch formation, by using up membrane reserves, may increase membrane mechanosensitivity. In specific cases, extracellular and/or CSK proteins (i.e., tethers) may transmit mechanical forces to the process (e.g., hair cell MG channels, MS intracellular Ca(2+) release, and transmitter release) without increasing tension in the lipid bilayer.

On the discrepancy between whole-cell and membrane patch mechanosensitivity in Xenopus oocytes.

1. Mechanical stimulation of voltage-clamped Xenopus oocytes by inflation, aspiration, or local indentation failed to activate an increase in membrane conductance up to the point of causing visible oocyte damage. 2. The absence of mechanosensitivity is not due to the vitelline membrane, rapid MG channel adaptation or tension-sensitive recruitment of new membrane. 3. Membrane capacitance measurements indicate that the oocyte surface area is at least 5 times larger than that predicted assuming a smooth sphere. We propose that this excess membrane area provides an immediate reserve that can 'buffer' membrane tension changes and thus prevent MG channel activation. 4. High-resolution images of tightly sealed patches and patch capacitance measurements indicate a smooth membrane that is pulled flat and perpendicular across the inside of the pipette. Brief steps of pressure or suction cause rapid and reversible membrane flexing and MG channel activation. 5. We propose that changes in membrane geometry induced during cell growth and differentiation or as a consequence of specific physiological and pathological conditions may alter mechanosensitivity of a cell independently of the intrinsic properties of channel proteins.

Identification of Anion-selective Channels in the Basolateral Membrane of Mitochondria-rich Epithelial Cells

Epithelial cells of toad (Bufo bufo) skin were isolated by treatments of the epidermis with collagenase and trypsin. Cl- channels in the basolateral membrane from soma or neck of mitochondria-rich cells were studied in cell-attached and excised inside-out configurations. Of a total of 87 sealed patches only 28 (32%) were electrically active, and in these we identified four different types of Cl- channels. The two major populations constituted Ohmic Cl- channels with limiting conductance (gamma125/125) of 10 pS and 30 pS, respectively. A much rarer 150 pS Ohmic Cl- channel was also characterized. From i/V relationships of individual channels the following Goldman-Hodgkin-Katz permeabilities were calculated, 2.2 (+/-0.1) x 10(-14), 5.7 (+/-0.7) x 10(-14), and 32 (+/-2) x 10(-14) cm3/sec, for the 10, 30 and 150 pS Cl- channels, respectively. The 30 pS channel was activated by hyperpolarization. The gating kinetics of the 150 pS channel was complex with burstlike closures within openings of long duration. The fourth type of Cl- channel was studied in patches generating 'noisy currents' with no discrete single-channel events, but with vanishing fluctuations at pipette potentials near ECl. Noise analysis revealed a power spectrum with cutoff frequencies of 1.2 and 13 Hz, indicating that resolution of kinetic steps was limited by small channel currents rather than fast channel gating. From the background noise level we estimated the channel conductance to be less than 1.7 pS. Despite the fact that the majority of patches did not contain electrically active Cl- channels, patches being active, generally, contained more than a single active channel. Thus, for the above three types of resolvable channels, the mean number of active channels per patch amounted to 2.1, 1.4, and 2.0, respectively. This observation, like the finding of few patches with several unresolvable channels, indicates that electrically active Cl- channels are organized in clusters.

Heterogeneity of chloride channels in the apical membrane of isolated mitochondria-rich cells from toad skin.

The isolated epithelium of toad skin was disintegrated into single cells by treatment with collagenase and trypsine. Chloride channels of cell-attached and excised inside-out apical membrane-patches of mitochondria-rich cells were studied by the patch-clamp technique. The major population of Cl- channels constituted small 7-pS linear channels in symmetrical solutions (125 mM Cl-). In cell-attached and inside-out patches the single channel i/V-relationship could be described by electrodiffusion of Cl- with a Goldmann-Hodgkin-Katz permeability of, PCl = 1.2 x 10(-14) - 2.6 x 10(-14) cm3. s-1. The channel exhibited voltage-independent activity and could be activated by cAMP. This channel is a likely candidate for mediating the well known cAMP-induced transepithelial Cl- conductance of the amphibian skin epithelium. Another population of Cl- channels exhibited large, highly variable conductances (upper limit conductances, 150-550 pS) and could be activated by membrane depolarization. A group of intermediate-sized Cl(-)-channels included: (a) channels (mean conductance, 30 pS) with linear or slightly outwardly rectifying i/V-relationships and activity occurring in distinct "bursts," (b) channels (conductance-range, 10-27 pS) with marked depolarization-induced activity, and (c) channels with unresolvable kinetics. The variance of current fluctuations of such "noisy" patches exhibited a minimum close to the equilibrium-potential for Cl-. With channels occurring in only 38% of sealed patches and an even lower frequency of voltage-activated channels, the chloride conductance of the apical membrane of mitochondria-rich cells did not match quantitatively that previously estimated from macroscopic Ussing-chamber experiments. From a qualitative point of view, however, we have succeeded in demonstrating the existence of Cl-channels in the apical membrane with features comparable to macroscopic predictions, i.e., activation of channel gating by cAMP and, in a few patches, also by membrane depolarization.

Single-channel basis of slow inactivation of Na+ channels in rat skeletal muscle

This study examined the single-channel basis of slow inactivation of Na+ currents (INa) in rat fast-twitch skeletal muscle fibers. A loose patch voltage clamp monitored changes in the maximum inward INa as the holding potential of the membrane patch changed. On a neighboring region of extrajunctional membrane of the same fiber, a gigaohm seal patch voltage clamp recorded single-channel INa. The maximum number of simultaneously open Na+ channels among a group of current traces indicated the maximum number of excitable channels. The holding potentials of the two voltage clamps were the same. Slow inactivation did not affect the open time or conductance of single Na+ channels. The number of excitable Na+ channels reversibly decreased during development of slow inactivation of INa and increased during recovery from slow inactivation of INa. Different stimulation protocols examined whether Na+ channels had to be in the closed, open, or fast-inactivated states to enter the slow-inactivated state. Na+ channels appear to be able to enter the slow-inactivated state from the closed, open, or fast-inactivated state.

Facilitated giga-seal formation with a just originated glass surface.

A simple technique of tip preparation in patch pipettes is described, which facilitates giga-seal formation. The pipettes were fabricated from thick-walled borosilicate glass tubing (external diameter 2.0 mm, internal diameter 0.5 mm) and the tips could be repeatedly broken in the bath. The pipette resistance correspondingly fell in steps of 3-20 M omega from about 80 M omega to about 2 M omega (double concentrated Tyrode). Scanning electron microscopy showed that the tip obtained after breaking was fairly plain. These broken tips were especially appropriate for patch-clamping. In cardiac myocytes in 11 out of 26 patches with Na+ channel activity, giga-seals developed spontaneously, i.e. without suction. In these patches the amplitude of the mean current with depolarizing pulses to -40 mV was significantly higher in comparison with patches formed under negative pressure. It is concluded that spontaneously sealed patches are most likely of planar configuration and the Na+ channel activity exceeds that in suction-induced patches.

Infiltration into Sealed Soils as Influenced by Gravel Cover

AbstractThe influence of stone gravel on water infiltration is not thoroughly understood, in that previous studies led to contradictory results. This field work was conducted to determine the effect of gravel on infiltration into sealed soils and to develop a regression model for infiltration based on the areas of the exposed and covered surfaces. Data were collected on 11 soils from three great groups from the fringe of the Sahara to the wet savanna zone of West Africa. A portable rainfall simulator was used to simulate rainfall on 28 undisturbed 1 by 1 m plots. Surface slope (P), portions of surface areas covered by grass vegetation (V), coarse fragments partly embedded in a surface seal (E), free coarse fragments (F), and sealed patches (S) were assessed as well as mean diameters (MD) of coarse fragments. The surface coverage by free surface gravel alone accounted for 68% of the variance of the cumulative infiltration/rainfall ratio, denoted as the infiltration ratio (IO). A simple nonlinear model taking into account S, F, and MD resulted in a high coefficient of determination of IO (R2 = 0.95). This model tested with three other sets of data was regarded as satisfactory to predict IO (r2 = 0.87). The results suggest that infiltration rate (IR) could be better related to the effective infiltrating annulus (AN) along the perimeter of free gravel than to the whole area under the free gravel (F). Moreover, simulated results indicated that IR increased with increasing F for MD < 0.029 m. For higher values of MD, IR decreased with increasing F. Thus, in the regions where large gravel‐size coarse fragments prevail, infiltration decreases with increasing stone cover, especially if the stones are embedded in a surface seal, as in desert pavements. Conversely, infiltration is enhanced by stone cover where small free coarse fragments are dominant, like those originating from a dismantled Fe pan.

Single channel analysis of ketamine interaction with a quisqualate receptor

The interactions of the general anaesthetic ketamine with the quisqualate-sensitive L-glutamate receptor (QUIS-R) of locust muscle have been investigated at the single channel level using a MΩ seal patch clamp technique. Low concentrations (10 −10 to 10 −9M) of ketamine did not significantly alter the kinetics of the QUIS-R channel. Higher concentrations of ketamine decreased the probability of the channel being open, the frequency of channel opening and the channel mean open time, and increased the channel mean closed time. Probability density functions of channel dwell times indicate that during application of > 10 −8 M ketamine the distribution of channel openings becomes restricted mainly to brief events. These results are consistent with the view that ketamine blocks the open, and possibly also the closed, channel of locust muscle QUIS-R and that this anaesthetic dissociates only slowly from its blocking site(s).

New techniques for the study of lens electrophysiology

The development of the gigohm seal patch clamp has extended the use of the voltage clamp technique to cells too small to study with more conventional approaches. We report the application of this technique, in its many configurations, to the study of lens epithelial cells. The technique allows measurement of membrane current at the microscopic (single channel) level or at the macroscopic (whole cell) level. At the single channel level, three configurations (on-cell, inside-out, and outside-out) are standard. The advantages and disadvantages of each are discussed. A more novel configuration, the on-cell whole-cell configuration, has several advantages over these more traditional patch configurations. Measurement of current from the entire cell membrane under voltage clamp can be achieved with the whole cell variant of the patch clamp technique. Two less invasive versions of this procedure (the perforated patch technique and droop analysis) are presented. Finally, the extension of the whole cell technique to a pair of dissociated cells is shown to allow the measurement of currents flowing through gap junctions. For each case, data is presented from lens epithelial cells (from frog, chick, rabbit and human).

Single acetylcholine-activated channel currents in developing muscle cells

The properties of single acetylcholine-activated ion channels in developing rat myoblasts and myotubes in tissue culture have been investigated using the gigaohm seal patch clamp technique. Two classes of ACh-activated channels were identified. The major class of channels (accounting for >95% of all channel openings) has a conductance of 35 pS and a mean open time of 15 msec (at room temperature and −80 mV). The minor class of channels has a larger conductance (55 pS) and a briefer mean open time (2–3 msec). Functional ACh-activated channels are present in undifferentiated mononucleated myoblasts 1–2 days in culture, although the channel density on such cells is low. Over the next week in culture, as the myoblasts fuse to form multinucleate myotubes, there is a marked increase in channel density and an increase in the proportion of large conductance channels. No significant change, however, occurs in channel conductance or open time (within a given class of channels) during this period. At high concentrations of ACh, channels desensitize and channel openings occur in groups, similar to what has been previously described in adult muscle. The rate of channel opening within a group of openings increases with increasing agonist concentration while mean open time is independent of agonist concentration, as expected from simple models of drug action. During a group of openings, the channel is open for half the time (i.e., channel opening rate is equal to channel closing rate) at a concentration of approximately 6 μm ACh.

Voltage-dependent calcium block of normal and tetramethrin-modified single sodium channels

The mechanisms by which external Ca ions block sodium channels were studied by a gigaohm seal patch clamp method using membranes excised from N1E-115 neuroblastoma cells. Tetramethrin was used to prolong the open time of single channels so that the current-voltage relationship could be readily determined over a wide range of membrane potentials. Comparable experiments were performed in the absence of tetramethrin. Increasing external Ca ions from 0.18 to 9.0 mM reduced the single channel conductance without causing flickering. From the dose-response relation the dissociation constant for Ca block at 0 mV was estimated to be 32.4 +/- 1.05 mM. The block was intensified by hyperpolarization. The voltage dependence indicates that Ca ions bind to sodium channels at a site located 37 +/- 2% of the electrical distance from the outside. The current increased with increasing external Na concentrations but showed a saturation; the concentration for half-maximal saturation was estimated to be 185 mM at -50 mV and 204 mM at 0 mV. A model consisting of a one-ion pore with four barriers and three wells can account for the observations that deviate from the independence principle, namely, the saturation of current, block by Ca ions, and rectification in current-voltage relationship. The results suggest that the Ca-induced decrease of the macroscopic sodium current results from a reduced single sodium channel conductance.