J. J. Glerum

In vitro comparison of isometric and stop-test contractility parameters for the urinary bladder

SummaryContractility parameters in the urinary bladder can be calculated from isometric contractions (no extra patient load as compared to routine cystometry) or from stop-tests (more accurate, simpler analysis). A stop-test involves a voluntarily interrupted micturition with pressure and flow measurement. In a series of measurements in vitro on pig urinary bladder strips, parameters of the first type, obtained either by analyzing isometric contractions in terms of the Hill model, or by making phase plots, were compared to parameters of the second type. A good correlation was found. The parameter correlating best with the maximal contraction velocity of the bladder, normalized for differences in initial muscle length, as obtained from stop-test, is the isometric contraction force, which can be obtained from an isometric contraction by either of the two analysis techniques. Clinically, making phase plots seems more promising than analyzing contractions in terms of the Hill model.

Mechanical Properties of Mammalian Single Smooth Muscle Cells. III. Passive Properties of Pig Detrusor and Human a Terme Uterus Cells

SummaryCells isolated from pig urinary bladders and pregnant full term human uteruses were attached longitudinally between a microforce transducer and a length displacement apparatus. Cells were stretched by applying a series of ramp-like length changes of 0.2 s duration and 10.0μm amplitude at intervals of 15 min. Passive forces upon straining were as high as 70–100μN. Following these peak forces stress relaxation occurred, levelling off approximately 50% of the maximum peak force. The maximum elastic modulus estimated for single cells was found to be at least a tenfold higher than was previously estimated from intact bladder strips. The relation between the increase in length and the increase in initial force increment was found to be approximately linear. An exponential equation was fitted to a selected number of stress relaxation curves. Relaxation curves of bladder cells show a clearly different time course as compared to bladder tissue strips, suggesting that a significant amount of relaxation in strips has to be contributed to the connective tissue components or to structural changes in these strips.

Isolation and individual electrical stimulation of single smooth-muscle cells from the urinary bladder of the pig

SummaryIn contrast to striated muscle, measurements on strips of smooth muscle cannot be uniquely interpreted in terms of an array of contractile units. Therefore scaling down to the single-cell level is necessary to gain detailed understanding of the contractile process in this type of muscle. The present study describes the development of a method for isolating contractile single smooth muscle cells from pig urinary bladders. Contractile responses evoked by individual electrical stimulation were used as a measure of cell quality during development of the method. Responses were evaluated by measuring latency, contraction and relaxation times, as indicated by visible length changes, and stored on-line in a computer. Initial length, relative shortening and shortening speed were determined by measuring cell lengths in previously timed still video frames using a computer-controlled crosshair device. Increase of stimulus pulse duration resulted in improved responses, indicating that the observed shortening represented a physiological contractile response. Ultimately this method of evaluation was applied to two sets of cell preparations obtained by two different methods, one using only collagenase digestion, the other using mechanical manipulation as well. Both sets showed two main patterns of response to electrical stimulation: a pattern of contraction upon stimulation followed by enhanced contraction when stimulation was switched off (CK), and a pattern of contraction upon stimulation followed by relaxation when the stimulus was switched off (CR). The set of preparations containing the highest percentage of CR cells was found to be superior (i.e. greater initial length, shorter latency and contraction times, increased shortening and higher shortening speed). The method of isolation used for this set gives a high yield of contractile cells available for experimental use over a long span of time.

Mechanical properties of mammalian single smooth muscle cells. I. A low cost large range microforce transducer.

SummaryA transducer has been developed for measuring the minute forces generated during isometric contractions (1.0–10.0μN) of single smooth muscle cells from the pig urinary bladder and the human uterus. In addition to its high sensitivity, resolution and stability (100 mVμN−1, <0.1μN and <2.0μN h−1), the transducer features a very wide range (100–140μN) with good linearity, enabling measurement of contractions as well as passive force-length characteristics within one uninterrupted measurement session. Since the transducer features an independent and interchangeable force to displacement conversion system, different force ranges can be realized by inserting force conversion systems with different compliances.

Mechanical properties of mammalian single smooth muscle cells II. Evaluation of a modified technique for attachment of cells to the measurement apparatus

SummaryA method is described for attaching isolated single smooth muscle cells to an apparatus designed for measuring the longitudinal forces developed passively and actively by the cell upon straining, electrical or pharmacological stimulation.Primary attachment of the cell is based on its natural negative surface charge in combination with a positive surface charge on the micro-tools used for attaching. Definite attachment is obtained by a knotting technique. Results show that this method of attachment is reliable and strong enough to withhold forces exceeding those necessary to break or tear the cell.Although this method allows relatively short cells to be attached (L >80μm). alternative methods e.g. glueing, are necessary to attach the shortest smooth muscle cells.

Variation of passive mechanical properties of the ureter along its length.

In order to test whether there is a significant variation of passive mechanical properties along the length of the ureter, pig ureters were cut into eight equal segments. These were simultaneously str

Influence of Temperature and Stimulus Interval Variations on the Propagation of Contractions in the Pig Ureter

The influences of ureter temperature and time interval between stimulations on the velocity of propagation of contractions in the pig ureter were studied in vitro. The propagation velocity was calculated from electromyogram signals measured at regular distances along the ureter. It was found that a temperature decrease of 3 degrees C causes a velocity decrease of nearly 5 mm/s. The influence of the interstimulus interval is much smaller: a reduction from 300 to 10 s causes a 10% drop in propagation velocity. Both phenomena can be understood from changes in the strength-duration curve describing the excitability of the tissue and clarify the discrepancies between propagation velocity measurements in vitro and in vivo.

Measurement of passive and active force in single isolated smooth muscle cells

In urodynamics, a subspecialism of urology, the function of the urinary tract is studied from a physical point of view, and methods are developed to objectively (and eventually automatically) diagnose the condition of the urinary tract. An important aspect of the function of the lower urinary tract is the contractility of the urinary bladder. Methods to measure this contractility are based on models developed using mechanical measurements on samples of bladder tissue. Such methods do not lead to a complete understanding of contractile properties of the (smooth muscle) tissue as the organization and structure of this tissue are irregular. Therefore measurements on single smooth muscle cells are necessary. Such measurements would also enable the use of smooth muscle biopsies for diagnostic reasons. In this chapter the development and first results of a practical method for measuring active and passive mechanical forces in single smooth muscle cells of the urinary bladder are described. Basically, such a method requires that viable and contractile cells can be isolated from the surrounding tissue and be attached to a transducer with sufficient resolution and stability to measure the very small forces involved. The three main issues isolation, attachment and force measurement will be separately discussed, and some first results will be shown.