Proteoglycan Breakdown of Meniscal Explants Following Dynamic Compression Using a Novel Bioreactor

Motivated by our interest in examining meniscal mechanotransduction processes, we report on the validation of a new tissue engineering bioreactor. This paper describes the design and performance capabilities of a tissue engineering bioreactor for cyclic compression of meniscal explants. We showed that the system maintains a tissue culture environment equivalent to that provided by conventional incubators and that its strain output was uniform and reproducible. The system incorporates a linear actuator and load cell aligned together in a frame that is contained within an incubator and allows for large loads and small displacements. A plunger with six Teflon-filled Delrin compression rods is attached to the actuator compressing up to six tissue explants simultaneously and with even pressure. The bioreactor system was used to study proteoglycan (PG) breakdown in porcine meniscal explants following various input loading tests (0–20% strain, 0–0.1 MPa). The greatest PG breakdown was measured following 20% compressive strain. These strain and stress levels have been shown to correspond to partial meniscectomy. Thus, these data suggest that removing 30–60% of meniscal tissue will result in the breakdown of meniscal tissue proteoglycans.
INTRODUCTION
Mechanical loading of the meniscus plays a crucial role in the metabolic activity of fibrochondrocytes.7,11,12,15 Previous studies have demonstrated that increased load on meniscal tissue leads to an increase in proteoglycan and collagen levels,16 whereas unloading of meniscal tissue causes a decrease in aggrecan expression, collagen formation and cell growth.2,3 It is not fully understood how biomechanical and biochemical events interact to produce changes in the extracellular matrix. This lack in knowledge is, in part, due to the difficulties associated with performing real time meniscal loading experiments in vivo.
Recreating the physiological forces in vitro using tissue explants while measuring the biological response provides one method for observing the effect of mechanical stress on the meniscus;9,15 however, the majority of commercially available bioreactors may not be suitable for application to meniscal loading studies. The Biopress system (The Biopress system, Flexcell International, Hillsborough, NC, USA) uses air pressure applied to a flexible bottom under each well. The Biopress has been used to apply pressures of 0.1 MPa in previous studies on meniscal explants,7,15 noting strain levels of approximately 10%. Because pressures up to 10 MPa and strains ranging from 2% to 20% are seen in the meniscus13,18 this system may not generate high enough pressures to mimic the full range of meniscal strains thought to occur during active loading of healthy and damaged tissue in vivo. Stresses and strains are approximately 5–10%, and 3 MPa, respectively, in the normal healthy meniscus, but these strain levels increase with a partial meniscectomy to approximately 20% and 8 MPa.18 Another biaxial tissue-loading device, previously used to compress articular cartilage explants, is able to create a maximum 400 N axial force on as many as 12 explants at once, however, is limited to 100 μm of motion.8
Previous studies have shown that following knee trauma, such as ACL transection, prior to any articular cartilage damage, there are signs of meniscal tissue degeneration.10 We have previously shown that following partial meniscectomy, the remaining meniscal tissue is subjected to an altered loading state.18 This altered loading state may cause a direct breakdown of matrix components such as proteoglycans (PG), or indirectly affect matrix production by induction of catabolic biomolecules.
The objectives of this study were (1) to design and build a practical, cost-effective device for applying homogeneous strains to tissue explants, and (2) to utilize the system to overload and underload meniscal tissue and measure the biochemical output. In this communication, we show that the ensuing device is a simple biocompatible design that applies accurate and reproducible strains and is made of components that can be sterilized. We utilized the bioreactor to apply both load and displacement controlled dynamic compression tests. Dynamic strain compression tests showed PG breakdown following overloading of meniscal tissue. No significant changes in concentration of PG released to the conditioned media was measured following various levels of dynamic compressive stress. A comparison between stress controlled dynamic compression versus strain controlled dynamic compression was also made.
MATERIALS AND METHODS
Design of Bioreactor
The frame consists of two 2.54 cm thick parallel aluminum plates separated by 2.54 cm diameter aluminum support rods (Fig. 1). Centered on the bottom plate is a load cell that is attached to a six well dish. The system is driven by a belt-driven linear actuator utilizing a control package by Animatics (Smartmotor 1720, Ultramotion, Mattituck, NY, USA). The actuator has a maximum stroke length of 5 cm and can thrust to 2225 N. It also has a maximum speed of 50 cm/s with bi-directional repeatability of ±0.00762 mm and a unidirectional repeatability of ±0.00254 mm. Motor control was achieved by using the SmartMotor Interface (SMI). This language allows the motion of the actuator to be controlled by the signal generated by the load cell or by the displacement of the actuator. Displacement resolution for the actuator is 0.4 μm.
FIGURE 1.A 2-dimensional view of the assembly of the plunger, dish, and cap. The test frame is composed of two aluminum plates supported by aluminum rods. The actuator is positioned in a centered hole in the top plate and tighten into alignment with an adjustable collar.
A strain gage load cell (Model 1210AF, Interface, Scottsdale, AZ) with a load capacity of 1334 N (sensitivity of ∼1.3 N) was utilized. For tests that require loads near or above 1334 N, a dimensionally identical load cell with 8896 N (sensitivity ∼2.17 N) (Interface, Scottsdale, AZ, USA) capacity is interchangeable with the current load cell. This design feature of interchangeable load cells allows for a larger range of loads to be accurately measured. A 2100 series signal conditioner (Vishay Instruments, Raleigh, NC, USA) amplifies the load cell signal to produce a 5 V signal at the maximum load.
The load cell is centered on a 2.54 cm thick aluminum plate that is the base of the system frame. A threaded stud leaving the load cell connects to the aluminum dish via a quick disconnect pin. The dish has six 10 mm deep wells equally spaced in a circular orientation. Teflon-filled Delrin compression rods (diameter = 8 mm) for each well are press fit into a plunger which attaches to the actuator via a quick disconnect pin. To ensure only one plunger/dish orientation and to keep the compression rods centered in each well, the plunger also features two press-fit aluminum pins that slide into matching holes in the dish. An aluminum cap rests on the shoulder of the dish and houses a linear bearing that is press fit into the cap. Along the resting edge of the cap, four shallow grooves were machined to allow carbon dioxide supply to the explants during testing. The linear bearing allows the plunger to move up and down within the cap and restricts the plunger to vertical motion (Fig. 2). The entire frame is small enough that it can be placed in a commercially available CO2 incubator to maintain physiological conditions (Model 5015, VWR, West Chester, PA, USA).
FIGURE 2.A 2-dimensional drawing of the test frame shows the side and top view of the test system. The linear actuator is attached to the plunger using a quick-disconnect pin. The dish is attached to the load cell in the same manner. The cap improves alignment of the plunger by utilizing a linear bearing.
Accuracy Evaluation of the System
Ultra-low pressure film (Sensor Products Inc., East Hanover, NJ, USA) was used to measure well pressure during compression. First, the repeatability of the pressure film was tested by loading the film (seven times) between flat platens in a tensile testing machine (Model 8872, Instron Corp., Canton, MA, USA) to a 70 N target load, corresponding to a pressure of 0.477 MPa for the given indentor size. Calibration of the pressure film was also done using the tensile testing machine and included loading pieces of pressure film ranging from 0.2 to 1.64 MPa. Pressure film analysis was completed using commercial software (Scion Image, National Institute of Standards and Technology, Gaithersburg, MD, USA) to measure the density of the pressure film samples. Film was compressed between the platens and a piece of rubber similar to the rubber used for testing well pressure.
To determine well pressure in the bioreactor, a machined plate was set on top of the dish with a 3 mm thick piece of uniform rubber. Pressure film was placed on top of the rubber and the plunger was lowered near the surface of the film. Four tests were conducted, each to the same pressure (0.477 MPa) to determine the repeatability of the bioreactor in load control. The film from the bioreactor was analyzed and density measured to determine the difference between each compression rod. The difference in film density and the maximum percentage error was determined to demonstrate the accuracy of the system.
Determination of Displacement Repeatability
To further investigate the accuracy of the system, the gap between the bottom of the compression rods and the bottom of the wells was measured while the system was assembled into the bioreactor. This was done by using the actuator to compress commercially available Fibre-Strand body filler (6371, The Matin Senour Comp., Cleveland, OH, USA) until the gap between the bottom of the compression rod and well bottom was filled. The actuator remained at this position until the body filler hardened completely. After plunger removal, a micrometer (2.54 μm resolution) was then used to measure the thickness of the body filler. This process was repeated three times with the same plunger and dish orientation.
Compliance of the System
The compliance of the system was determined by placing a flat stainless steel plate over the wells and running a load-deformation test in the absence of menisci. The test was repeated three times and the load-deformation data recorded.
Application of the System
Pigs used in the experiment were 18 weeks old, sacrificed 24 h after death (received from Mayo Clinic, Rochester, MN, USA). The explants were collected from both the lateral and medial meniscus using sterile technique. The explants were 6 mm in diameter and cut using a biopsy punch, perpendicular to the superior surface to maximize the amount of superior tissue preserved. To ensure two parallel flat faces on the cylindrical explants, a specialized cutting device was utilized. Explants were held such that a fixture containing two razorblades, 5 mm apart, cut the tissue perpendicular to the long axis of the cylindrical explant. This ensured that each explant was the same height with parallel faces (Fig. 3a).
FIGURE 3.a – Meniscus following biopsy, b – Maximum axial strain following different partial meniscectomy simulated by FE analysis. Various percentages (5%, 10%, 30% and 60%) removed from different portion of the medial meniscus (A – anterior, C – central, P – posterior). Knee loaded with 1200 N axial force.
Explants were incubated at 37°C (5% CO2) for 48 h in growth media (89% DMEM/F-12, 10% FBS, 1% Penn/Strep) which was changed after 24 h. For mechanical testing, the explants were placed in the six well bioreactor filled with 400 μl of the test media (97% DMEM/F-12, 2% FBS, 1% Penn/Strep). The explants were loaded for 2 h at 1 Hz, at one of the following levels: 5%, 10%, 15% or 20% strain, 0.05 or 0.1 MPa unconfined dynamic compression. Each loading group consisted of six explants taken from six different animals (except 15% test – 4 animals). Control explants were placed in wells, but not exposed to compression. Previous experiments (FE analyses) showed that under two times body weight the intact knee meniscus experiences about 10% maximum compressive strain. Removal of 5–10% of the meniscus minimally affects the maximum strain level, however, removal of 30–60% of the meniscal body increases the maximum strain to 15% or greater. Generally speaking, 0% strain is likely underloading the tissue, 5–10% is approximately physiological and 15–20% is considered overloading18 (Fig. 3b). Following compression the explants were bisected into superior and deep zone, by cutting them in half (Fig. 4), weighted and placed in 24-wells plates, in 1 ml of fresh test media. Samples were post-incubated at 37°C (5% CO2) for 24 h. Post-incubation media was collected and stored at −80°C for future analysis.FIGURE 4.Cross-section of the meniscus with the direction of the cut and explants showing superior and deep zones.
The content of sulfated glycosaminoglycan (GAG) released to the conditioned media was assayed using dimethylmethylene blue (DMMB) dye solution.5,6 The standard curve was generated with a known concentration of shark cartilage chondroitin sulfate C. All samples were run in duplicates. The concentration was normalized to the wet weight of the explant. GAG release was then normalized to the no load control samples for each animal.
Data Analysis
The concentration of the GAG released to the media was calculated using a standard curve (R2 > 0.85). The final value was an average from duplicates. All data is presented as mean ± standard error. A one way ANOVA followed by Fisher’s PLSD post-hoc testing was used to measure statistical differences (p < 0.05 was considered significant). Paired t-tests were used at each stress or strain level to determine differences between superficial and deep zone PG release to the media.
RESULTS
Accuracy Evaluation of the System
The repeatability test showed an average of 0.4773 ± 0.0003 MPa. The pressure film demonstrated equal pressure in each well for each load (Fig. 5). There was less than a 1% error (Table 1).
FIGURE 5.Pressure film impressions at 0.477 MPa under the six bioreactor compression rods.TABLE 1.Results of pressure film verification.0.477 MPaPressure (MPa)Test #Rod 1Rod 2Rod 3Rod 4Rod 5Rod 6AverageStd. Dev.10.47730.47680.47710.47710.47680.47680.47700.000220.47680.47690.47680.47680.47690.47720.47690.000130.47730.47680.47690.47700.47690.47710.47700.000240.47730.47680.47690.47680.47690.47680.47690.0002
Determination of Displacement Repeatability
The micrometer measurements from the body filler showed that the bioreactor was extremely repeatable. Well 3 had the largest standard deviation in height, 3.4 ± 0.01 mm, whereas well 1 had the lowest standard deviation 3.4 ± 0.0015.
Compliance of the System
The results of the compliance test indicate a linear load-deformation response (R2 > 0.99). The slope of the load-deformation curve was 3465 ± 200 N/mm. Thus, for the range of strains and stresses seen in this study, the compliance is negligible.
Application of the System
To determine an appropriate post incubation time, preliminary tests were run. PG concentration in the media was monitored following 2, 4, and 6 h and 1, 2, and 3 days following 10% dynamic compression and for controls samples (Fig. 6). The shortest time showing a strong signal was chosen to minimize the duration of the experiment (1 day).
FIGURE 6.PG concentration following different incubation time (n = 2 for controls (C), n = 3 for 10% compression test (10%)). Data presented is mean ± standard error.
The highest break down of PG was measured following 20% compressive strain for both the superior and deep zones of the meniscal explants (Fig. 7). There were significant differences between release of GAGs into the media for the overloaded condition (20%) versus physiological loading (10%) for both superior and deep zones. No significant differences were found between superficial and deep zones for any compression level. Explants exposed to pressures of either 0.05 or 0.1 MPa did not show any significance differences in either the superior or deep zone for PG breakdown (Fig. 8).FIGURE 7.PG concentration following different compression tests (n = 6 except 15% test (n = 4)). Data presented is mean ± standard error. *Statistically different than 20% (p < 0.05).FIGURE 8.PG concentration following different load level compression tests (n = 6). Data presented is mean ± standard error.
Displacement controlled tests showed a rapid drop in load within the first 1000 cycles with little change in the following 6200 cycles (Fig. 9). For displacement tests at or below 15% strain, loads settled just below 0.05 MPa while the 20% strain test remained above 0.1 MPa throughout the duration of the test. The change in pressure measured from the beginning of the test to the end is shown in Table 2. Load controlled tests (Fig. 10) showed a rapid increase in compressive displacement within the first 2000 cycles. The 0.05 MPa load level reached maximum displacement near 3000 cycles and remained at that level to the end of the test. The 0.1 MPa tests reached 18% strain near 3000 cycles but steadily increased to 20.7% strain by the final cycle. The differences in strain from the start to the end of the tests can be found in Table 3.FIGURE 9.Stress vs. time for four representative displacement control tests. Only peak values during each cycle are plotted. Stress was calculated by dividing the peak load by the initial cross-sectional area.Table 2.Change in pressures over the duration of the stress-relaxation tests.StrainPressure (MPa)StartEnd5%0.166 ± 0.108 *#0.038 ± 0.010 #10%1.141 ± 0.103 #0.046 ± 0.010 #15%2.185 ± 0.8270.035 ± 0.026 #20%3.548 ± 0.4290.128 ± 0.020Data represents mean ± standard deviation. n = 6 for all groups. * Significantly different than 15% (p < 0.05), # significantly different than 20% (p < 0.05).FIGURE 10.Strain vs. time for two representative load control tests. Only peak strain values during each loading cycle are plotted. Strain was calculated by dividing the peak displacement by the original height of the explant.Table 3.Change in strains over the duration of the creep tests.Pressure (MPa)Strain (%)StartEnd0.052.6 ± 0.5311.6 ± 1.360.13.0 ± 0.1220.7 ± 1.45*Data represents mean ± standard deviation. n = 6 for all groups. * Significantly different than 0.05 MPa (p < 0.05).
DISCUSSION
The explant compression system meets the criteria necessary to obtain a realistic representation of physiological forces present in the knee joint. This system is able to apply known pressures to six explants at once, which is important when trying to gather data for hypothesis testing. It is capable of applying physiological and supraphysiological levels of load and displacement, and has the ability to test in load or displacement control. SMI programming allows for flexibility in frequency, duration, amplitude, and waveform. The system is small enough to fit in a standard incubator and is made of materials that can endure autoclaving and alcohol. An important feature to this system is the ability to keep the explants and media sterile from the culture hood to the incubator. The plunger, dish, and cap form an enclosure that allows easy transport between the culture hood and incubator without allowing open air and bacteria to infect the sample. Since the cap incorporates a linear bearing it does not need to be removed for testing. Utilizing the system features and designing the correct protocol will help maintain a physiological loading sterile environment.
This bioreactor is capable of creating higher loads and greater displacements than previous systems used for compressing explants.7,8,15 The Biopress system (Flexcell International, Hillsborough, NC, USA) is capable of loads as high as 69 N, whereas the current systems actuator can thrust to 2225 N. An advantage of our system over the biaxial tissue-loading device presented in Frank et al. (2000), is that it can create displacements over 10 mm with a resolution of 0.4 μm.8 Our actuator also has a bi-directional repeatability of ±7.62 μm compared to the ±25 μm used in Sah et al. (2003).11 In addition, the present system is capable of 1 Hz cyclic compression in a sinusoidal wave using displacement or load control. The bioreactor can be used to compress any tissue that fits under an 8 mm compression rod and in a 10 mm deep well. All surfaces are machined to a smooth, frictionless finish, to ensure the sample is exposed to pure unconfined compression. Although we believe this bioreactor to be an improvement over others, we realize that there are some limitations that need to be compensated for. For example, this system can only perform unconfined compression currently. However, the system could be modified to run confined compression tests as well by outfitting a new plunger/well assembly.
Since the stress tests were conducted at 0.05 and 0.1 MPa, and resulted in strains between 2.6% and 20%, the compliance of the system was negligible. Similarly, for the 5% strain control tests, the loads were small enough that the compliance of the system was negligible. However, for the 10%, 15% and 20% strain control tests, stresses were between 3.548 and 0.035 MPa. Thus, at the larger stress levels, the compliance of the system was greater. For instance, during the 10% strain control test, initially stress levels of 1.1 MPa would lead to approximately 57 μm of compliance, and thus, for the first 150 of the 7200 s test, strains were closer to 8.8% instead of 10%. For the 15% strain test, strains were closer to 13% for the first 200 s of the test, and as the material relaxed, the last 1 h and 57 min of the test were at 15% strain.
We hypothesized that PG breakdown (as determined by GAG in the media) would be high for both the underloading and overloading condition. PG breakdown was significantly increased at 20% strain. Meniscal tissue is a mechanically sensitive, and mechanical loading has been shown to regulate gene expression.14 Hence, we expected that loading the tissue below normal physiological levels of magnitude (underloading) would result in PG breakdown. Lack of increased PG breakdown for control samples (underloading) is surprising and needs to be further explored. Perhaps the degraded PG is not being released into the media for the control samples, whereas the overloaded samples have mechanical compression to help move the broken down PG into the media. Future studies will measure PG breakdown in the tissue explants following compression to determine the integrity of the PG within the tissue. These data suggest that removing 30–60% of meniscal tissue, and thereby increasing tissue strains over 15% results in an increase in PG breakdown and tissue destruction. Thus, not only does meniscectomy affect the underlying articular cartilage but the remaining meniscal tissue appears to begin to breakdown, possibly leading to a change in meniscal material properties. This data is supported by previous work that showed following an ACL transection, degenerative changes were seen in the meniscus prior to any articular cartilage changes.10
DiMicco et al. showed an increase in GAG release from bovine cartilage that had been exposed to an injurious level single uniaxial, unconfined compression.1 Inhibitors of biosynthesis or degradative enzymes did not affect PG breakdown, suggesting that the breakdown was a mechanical consequence of compression. GAG release 1–7 days post injury was slowed by metalloprotease inhibitors. Shin et al.12 showed that dynamic compression (0.1 MPa for 24 h at 0.5 Hz) increased both GAG synthesis as well as release to the media compared to unloaded controls. Our loading scheme was designed to simulate approximate physiological walking conditions (2 h, 1 Hz). It is difficult to compare our GAG data to a study of a single injurious insult, or dynamic compression for 24 h. We created an approximate daily physiological loading environment that might occur during walking, and simulated loads from unloaded to overloaded due to meniscectomy. In the future, we will investigate the mechanisms of GAG release, whether it is mechanical damage or activation of enzymatic activity.
It was surprising that explants tested at 0.05 MPa showed greater PG breakdown than explants tested at 0.1 MPa for the superficial zone. One possible reason for this result might be related to the cell viability. A compression of 0.1 MPa may induce more cell death than 0.05 MPa of compression and hence fewer cells may be available for production of metalloproteases that may contribute to the breakdown of PG. Current studies are underway to document the degree of cell death in the explants.
It is interesting to compare the load control results to the displacement control results. Based on Fig. 9, the 20% strain test applied an initially very high load (∼3.5 MPa), but equilibrated at 0.1 MPa. Similarly, the 0.1 MPa test reached a steady state of 20% strain (Fig. 10). Hence, we would expect to see similar levels of PG breakdown in the 20% strain test as well as the 0.1 MPa test. This is also true for the 10% strain test and the 0.05 MPa stress tests. For example, in the deep zone the 20% strain control test resulted in 3.7 ± 1.4 ug/ml per gram of tissue, whereas the 0.1 MPa test resulted in 1.9 ± 0.5 ug/ml per gram of tissue. This large difference could be due to either “lift-off” (separation between loading platen and sample during a 1 Hz test)4 or the initially high stress that was reached initially in the 20% displacement control tests. In contrast to the differences seen above, the 10% strain test and 0.05 MPa stress test resulted in 1.3 ± 0.2 and 1.8 ± 0.5 ug/ml per gram of tissue, respectively. Previous 3-D computational studies of an entire human knee joint have shown that mean contact pressures changed from 1.57 MPa for an intact healthy meniscus to 3.09 MPa following 60% meniscectomy.18 Maximum contact pressures on the superior surface of the meniscus changed from 4.7 to 7 MPa when 60% of the meniscus was removed.18 The differences between stress and strain control studies noted above make the data presented difficult to interpret due to the non-physiological nature of in vitro unconfined compression studies. Clearly, this indicates that meniscal tissue may respond differentially to stress versus strain, the duration of strain levels, or most likely the loading history. Before definitive clinical implications can be made regarding the effects of meniscectomy on meniscal tissue, we must first determine if the levels of PG breakdown noted in this study correspond to changes in the load-bearing capacity of the tissue and its function in the knee joint.
One of the limitations of this study is lack of investigation of differences between the medial and lateral menisci as well as specific location within the meniscus from which explants were harvested. This likely resulted in higher standard deviations. Future studies with a larger set of animals are proposed in which lateral versus medial, anterior versus posterior and inner radial versus outer radial differences could be studied. Previous researchers have shown a difference between inner and outer radial explants.14 Bisection of explants has previously been shown to release many growth factors.17 Since all samples in this study were bisected, the relative differences found are still significant. It should be noted that the measured response may be due not only to mechanical stimuli but also growth factors released due to cutting the samples. In addition to bisecting the samples, other factors, such as FBS concentration and post-incubation time may effect the absolute concentrations of GAG analyzed in this study. Therefore, only relative comparisons between treatment groups should be considered.
In summary, this simple and practical experimental system allows for reproducible application and quantification of homogeneous stresses or strains to explants tissues, thereby providing a systemic and quantitative method for correlating external mechanical stimuli to cellular and molecular mechanisms of mechanotransduction.


Do dissociated or associated phoria predict the comfortable prism?

Background Dissociated and associated phoria are measures of latent strabismus under artificial viewing conditions. We examined to what extent dissociated and associated phoria predict the “comfortable prism”, i.e. the prism that appears most comfortable under natural viewing conditions.
Introduction
Since Ogle’s work [14] dissociated phoria has been distinguished from associated phoria. Dissociated phoria is defined as a deviation from the orthovergence position that occurs when no fusionable contours are provided. Associated phoria is a deviation of the eyes that appears under prism correction of fixation disparity: associated phoria equals the “aligning prism” [1] that nullifies fixation disparity. To avoid the technical requirements for measuring the eye position objectively, fixation disparity is commonly determined according to the observer’s directional perception of monocular Nonius lines, embedded in binocular contours. Both dissociated [11, 18] and associated phoria [9, 19] have been recommended as indicators for prismatic correction in the case of asthenopia, but recently associated phoria has been preferred, because the stimulus for associated phoria appears to be more natural, in that both eyes are presented with a nearly identical configuration [1, 4, 5, 16, 20]. Moreover, Yekta et al. [20] found the correlation of asthenopic complaints to be significant only with associated, not with dissociated phoria.
Kromeier et al. [8], however, suggested that dissociated and associated phoria should be similar, provided the accommodative demand in the two procedures is equal. Their argument was based on the consideration that the feedback loop for fusion is opened under both conditions. Concerning dissociated phoria, it is obvious that the feedback loop cannot operate; fusionable contours are absent, hence there is no error signal. Concerning associated phoria, the argument is more complicated because of the fundamental difference between fixation disparity and associated phoria. When the observer looks at a test for fixation disparity, any tendency to deviate from orthoposition is largely kept in check, because the disparity of the binocular contours provides an error signal: the fusional feedback loop is functioning. In the test for associated phoria, however, prisms are added. Prompt and repeated adjustment of the prismatic power nullifies any disparity of fusionable contours. This continuous and artificial resetting of the error signal to zero means that the fusional feedback loop cannot fulfill its purpose, namely stabilisation of a certain vergence angle.
The experimental results of Kromeier et al. supported their theoretical consideration. These authors suggested that the discrepancies between dissociated and associated phoria encountered in previous studies [15, 20] might be due to different accommodative stimuli.
To clarify this issue, we compared dissociated and associated phoria using the same object for fixation, thus keeping the accommodative demand similar. We further investigated to what extent dissociated and/or associated phoria predict the “comfortable prism”. This term defines the prismatic power reached by the observers when they looked at a fully fusionable display, and tuned a pair of counterrotating prisms, called Herschel’s or Risley’s rotary prisms [17], such that the viewing of a fully fusionable object appeared most comfortable. Hence, the comfortable prism anticipated the real-life situation for which prismatic spectacles might be considered.
Methods
Apparatus
Stimuli were generated by a PowerMacintosh G4 and presented at a distance of 4.0 m on a 19” Philips GD403 CRT monitor with a resolution of 800 × 600 pixels. By means of liquid crystal shutter goggles (ELSA 3D Revelator), separate images were presented to the two eyes. The goggles were synchronized to the CRT refresh such that the frames were alternately presented to the right and left eyes. The refresh rate was 120 Hz, i.e. 60 Hz for each eye, just above the flicker fusion frequency. The phosphor persistence of the monitor, measured with a photoelectric cell, was down to 10% after 4.0 ms, i.e. within a shorter time than the frame time of 8 ms. An alternate cover test assured us that this technique provided a complete separation between the images of the two eyes. Herschel’s counterrotating prisms, manufactured by Zeiss, were mounted in front of their right eye (Fig. 1).
Fig. 1Herschel’s prisms mounted in front of the right eye. The observer, looking through shutter goggles, adjusts the prisms by means of a lever arm. A cogwheel belt transmits the prismatic power to a potentiometer
These prisms, equipped with a lever arm, allowed a variation of the prismatic power between 30 cm/m base in and 30 cm/m base out. A calibration performed with a laser beam showed that the readings on the scale of the prisms varied only about 2% around the true inflection. A cogwheel belt connected the prisms with a potentiometer that provided a signal linearly proportional to the prism power. The electric signal was directly transmitted to a computer.
During the trials the room was dark; in the intervals the room was moderately illuminated so that the test person was able to see a coloured print (Henri Matisse “Fleurs et céramique”, 35 × 40 cm, mean luminance 13.8 cd/m2) mounted directly above the monitor.
Test figures
Dissociated phoria
As depicted in Fig. 2a, the left eye was presented with a fixation cross, flanked by two O letters (“O X O”). Each of the three letters had a diameter of 10 arcmin and a line thickness of 1.5 arcmin. Above and below the cross were two Nonius lines, 20 arcmin high and 5 arcmin wide. The interval between the lines and the centre of the “O X O” was 10 arcmin. A bright circular background of 40 arcmin radius, embedded in a random dot pattern, surrounded these dark features. The right eye was presented with a bright dot of 4.3 arcmin diameter on a homogenous dark field. The luminance of the dark features was 0.2 cd/m2, and that of the bright features 22.6 cd/m2 (measured through the liquid crystal shutter goggles). The luminance outside the monitor was 0.1 cd/m2.
Fig. 2Test figures. a Test for dissociated phoria. All the elements contained in the Mallett figure, including the two Nonius lines, are displayed to the left eye. The right eye is presented with a white dot on a homogenous dark field. b Test for associated phoria, according to Mallett [9]. c Test for the comfortable prism. An identical figure is displayed to both eyes
Associated phoria
As depicted in Fig. 2b, the configuration was similar to Mallett’s test for fixation disparity [9]. The “O X O” served as a fusional stimulus. Vertical Nonius lines were used as monocular markers, in that the line above the fixation cross was shown to the right eye, and the line below the fixation cross to the left eye.
Comfortable prism
Both eyes were presented with all the Mallett elements including both Nonius lines (Fig. 2c).
Procedure and instructions
The observers were seated in a comfortable chair. During the trials, the observers had to rest their chin on a support, to lean their forehead against a bar, and to adjust Herschel’s prisms with the lever arm. Before each trial, the experimenter set the prisms to ±0. To limit prism adaptation [12], the time for the adjustment was restricted to 30 seconds. In the interval between the single trials (about 60 to 120 s), the observers were encouraged to move their head freely and to look around in the room or to behold the Matisse painting.
Each of the three conditions (dissociated phoria, associated phoria, comfortable prism) was tested in a block of eight trials. The conditions were presented in the following order:
Associated phoria. If the observers saw an offset between the Nonius lines, they had to align them with the least possible excursion of the lever arm so that they appeared as stable to each other as possible. This instruction resembles the suggestion of Karania and Evens [6] to observe whether one or both of the Nonius lines ever move. If an observer did not see an offset at the start of the trial, or the Nonius lines oscillated around zero, he or she was encouraged to “play” a little with the lever arm and set Herschel’s prisms such that the Nonius lines became aligned as stably as possible. The observers were repeatedly reminded to look at the centre of the “O X O”, not at the Nonius lines. Further, the observers were instructed to ignore if one Nonius line, or even if both of them seemed to disappear every now and then (fading). Most observers had such an experience. They had to adjust Herschel’s prisms according to the percept when they saw both Nonius lines.Dissociated phoria. The observers were asked to align the white dot with the midline of the “O X O”, neglecting any vertical offset of the white dot. While doing so, they were urged to strictly look at the centre of the “O X O”, not at the white dot.Comfortable prism. The observers were asked to look at the centre of the “O X O” and to set the prisms such that viewing appeared most relaxing. They were encouraged to start with rather bold searching excursions and to refine the tuning gradually during the available 30 seconds.
To get used to the manoeuvring of Herschel’s prisms, the experiment was preceded by a few trials in which the observers practised bringing the white dot to the midline of the “O X O” (test for dissociated phoria) and aligning the Nonius lines to each other (test for associated phoria).
To examine the reproducibility, we repeated the whole experiment for all observers in a second session after an interval of 24 to 130 days.
Observers
Twenty observers participated in the study (members of our department or recruits via a public advertisement, aged between 20 and 71 years, median 26.5 years). The observers were selected according to the following 4 criteria: (1) Visual acuity of each eye (with full spherical and cylindrical correction) at least 1.0decimal (= 6/6Snellen), (2) absence of strabismus, ascertained with the unilateral cover test, (3) absence of a prism in the spectacles, and (4) presence of random dot stereopsis (Lang Test 1). The observers were refracted without dilating their pupils, using streak retinoscopy and crossed cylinders, taking particular care to avoid any uncorrected hyperopia. During the experiment, the observers wore full spherical and cylindrical spectacle corrections. Care was taken to ensure that the glasses remained centred throughout the study. We did not inquire whether the observers had asthenopic symptoms or not.
We explained to the observers that the study intended to optimise the comfort of seeing. Otherwise, the observers were kept naive as to the purpose of the study. Each observer provided informed written consent to participate in the experiments. The study followed the tenets of the Declaration of Helsinki and was approved by the institutional human review board.
Data acquisition and analysis
The prismatic power derived from the potentiometer attached to Herschel’s prisms was recorded by PowerLab with a sampling rate of 100 Hz. To enable analysis of the dynamic behaviour of the adjustments we recorded the full 30 seconds of each trial. Offline examination was accomplished with Igor Pro® (Wavemetrics, Inc., Lake Oswego, OR, USA) and Statview® (Abacus Concepts, Inc., Berkeley, California, USA). Statistical analysis was performed with Statview® and SPSS® (SPSS Inc., Chicago, Illinois, USA). We used multifactorial ANOVA (including condition, session and trial) and paired comparisons. For post-hoc tests, Bonferroni adjustment was performed.
Results
Inspection of curves
The curves obtained from the 20 observers varied considerably between the 8 trials. For example see Fig. 3. There was no trend, e.g. with the early trials (thin lines) yielding smaller values than the late ones (progressively thicker lines), or vice versa (p = 0.34). Most observers adjusted Herschel’s prisms stepwise, reaching the final value after about 20 seconds. Therefore, we limited the numerical evaluation to the remaining 10 seconds.
Fig. 3Adjustment of Herschel’s prisms by observer #10 for dissociated phoria, associated phoria, and the comfortable prism. First session. Positive values indicate base out prisms (eso deviation), negative values base in prisms (exo deviation). The upper panels show the original recordings of the eight single trials. The lower panels show the mean values (solid lines) with the standard error of the mean (SEM, dashed lines). Note that the scale of the ordinate for the comfortable prism differs from those in the two phoria conditions
For dissociated phoria, most observers saw an offset right at the beginning of each trial and promptly moved the lever arm accordingly.
For associated phoria, some observers initially did not see an offset between the Nonius lines. In this case, the observers followed the instruction to “play” a little with the lever arm and find a position in which the lines appeared as stable as possible.
For the comfortable prism, most observers started with rather bold searching excursions and refined the tuning gradually (Fig. 3, right panels). Only exceptionally, an observer ended up with a rather large range for the comfortable prism.
Statistical analysis
We averaged the 1000 values recorded during the last 10 seconds in each of the eight trials, as most observers reached the final value after about 20 seconds. As stated above, there was no trend between the eight trials, e.g. with the early trials yielding smaller values than the late ones, or vice versa (p = 0.34). This allowed us to treat the 8 trials together and calculate an intertrial mean±SEM (standard error of the mean) for each observer (Fig. 4). Among the 20 observers, the range of the intertrial mean was for dissociated phoria from +9.3 eso to −5.9 cm/m exo deviation, for associated phoria from +11.2 eso to −3.3 cm/m exo deviation, and for the comfortable prism from +4.8 eso to −4.1 cm/m exo deviation.
Fig. 4Mean of the last 10 seconds of eight trials ±SEM for each of the 20 observers. The values obtained in the first session are represented in the left columns, those obtained in the second session in the right columns. DP = dissociated phoria, AP = associated phoria, CP = comfortable prism. The observers are identified with #1 to #20. Ordinate: positive values = eso deviation, negative values = exo deviation. Note that the ordinates in #7 and #19 are scaled down
To assess the intertrial variability over all 20 observers, we averaged the 95% confidence interval (±1.96 SD) per session. The values were as follows. Dissociated phoria: first session ±1.3 cm/m, second session ±1.0 cm/m; associated phoria: first session ±1.6 cm/m, second session ±1.6 cm/m; comfortable prism: first session ±2.0 cm/m, second session ±2.2 cm/m. Hence, the variability was similar in the two sessions. Combining the two sessions resulted in a greater overall scatter (95% confidence interval = ±1.96 SD): ±5.8 cm/m for dissociated phoria, ±5.4 cm/m for associated phoria, and ±4.3 cm/m for the comfortable prism. The marked increase in the overall scatter indicates that the values changed between the two sessions. In some cases the mean values of the second session were even outside the 95% confidence interval of the first session: 5/20 for dissociated phoria, 9/20 for associated phoria, and 6/20 for the comfortable prism. The change was not always concordant in the three conditions. For example, in observer #13, the associated phoria changed from +0.1 ± SEM 0.4 cm/m to +4.6 ± SEM 0.5 cm/m, i.e. in the eso direction, and the comfortable prism changed from −0.1 ± SEM 0.4 cm/m to −1.8 ± SEM 0.2 cm/m, i.e. in the exo direction.
Are these changes clinically relevant? As several practitioners consider prescribing prisms from a minimum of about 1.0 cm/m onwards [13], we identified the observers in whom the change was greater than 1.0 cm/m: for dissociated phoria 6/20 (up to 3.2 cm/m), for associated phoria 11/20 (up to 4.5 cm/m), and for the comfortable prism 12/20 (up to 3.3 cm/m). To find out whether the observers who changed their values by more than 1.0 cm/m could be recognized at the first session, we compared the intertrial variability at the first session in two groups: the observers who changed their values more than 1.0 cm/m with those who changed them less than 1.0 cm/m. There was no statistically significant difference between the two groups (p = 0.67 for dissociated phoria, 0.11 for associated phoria, 0.52 for the comfortable prism).
Comparison between dissociated phoria, associated phoria, and the comfortable prism
The ANOVA over all trials of all 20 observers (n = 8 × 2 × 20 = 320) revealed that dissociated and associated phoria differed significantly (p < 0.01). In 11 observers, the dissociated phoria was lower, in four higher than the associated phoria; in five observers, dissociated and associated phoria were similar.
Figure 5 demonstrates that the majority of observers had an eso deviation for dissociated and associated phoria (13/20 and 17/20, respectively), but an exo deviation for the comfortable prism (17/20). The tendency towards eso deviation in the two phoria conditions was also obvious in the average ±SD over all trials of all 20 observers (n = 8 × 2 × 20 = 320): +1.0 ± 2.9 cm/m eso deviation for dissociated phoria, and +2.0 ± 2.7 cm/m eso deviation for associated phoria, versus −0.6 ± 2.2 cm/m exo deviation for the comfortable prism. The difference between dissociated phoria and comfortable prism, on the one hand, and the difference between associated phoria and comfortable prism on the other were both significant (p < 0.05 and p < 0.001 respectively).
Fig. 5Mean of the two sessions ±SEM for associated phoria, dissociated phoria, and comfortable prism. The first column in each graph represents observer #1, followed by #2 etc
To investigate whether the tendency towards eso deviation in the two phoria conditions, as compared with the comfortable prism, was brought about by the sequence of tests, we made a spot check. We chose observer #10, because she had a marked eso deviation in the two phoria conditions, but an exo deviation in the comfortable prism. In an extra session, this observer determined the comfortable prism before the tests for dissociated and associated phoria. The values obtained were of the same magnitude as those in the two previous sessions (ANOVA p = 0.084 for tested difference).
Discussion
We compared three vergence parameters: dissociated phoria, associated phoria, and the comfortable prism. To avoid any (possibly prejudiced) influence of the experimenter, we recorded the prismatic power set by the observer with a potentiometer. The comfortable prism anticipates the real-life situation in which prismatic spectacles are worn. Therefore, the comfortable prism is very close to the endpoint of therapeutical considerations, i.e. prescribing prismatic spectacles. In contrast, dissociated and associated phoria, determined under artificial viewing conditions, are surrogate measures of the comfortable prism. We studied whether these surrogate measures predict the comfortable prism, assuming that dissociated phoria, associated phoria, and the comfortable prism might be similar, because all three parameters are determined by opening the feedback loop for fusional vergence. When measuring dissociated phoria, fusionable contours are absent; hence, there is no disparity error signal. When associated phoria is measured, any disparity of the (defective) fusional pattern, which initially constitutes an error signal, is promptly nullified by repeated prism adjustment. Concerning the comfortable prism, one has to realize that the fusional feedback loop, which uses disparity as its error signal, can stabilize the vergence angle only if the stimuli are presented to the two eyes in a fixed angle, e.g. through a certain unchanging prism. If, however, the observer is asked to adjust a variable prism according to his or her comfort, the fusional feedback loop is open and cannot stabilize the vergence angle. Instead, the fusional feedback loop is replaced by another feedback loop whose error signal is discomfort. Accordingly, the vergence position reached with the comfortable prism may also be described as the “vergence position of rest when both eyes are exposed to identical pictures”.
To our surprise, we found marked differences between the three vergence parameters: as indicated in Fig. 5, the majority of observers had an eso deviation under the two phoria conditions (13/20 for dissociated and 17/20 for associated phoria), but preferred base-in for the comfortable prism, corresponding to an exo deviation (17/20). The maximal difference occurred in observer #10 who showed in his second session an eso deviation for associated phoria of +4.9 cm/m, and an exo deviation for the comfortable prism of −3.0 cm/m.
The tendency towards eso deviation in the two phoria conditions was also evident in the significant difference between the averages ±SD over all 20 observers: +1.0 ± 2.9 cm/m eso deviation for dissociated phoria, +2.0 ± 2.7 cm/m eso deviation for associated phoria, but −0.6 ± 2.2 cm/m exo deviation for the comfortable prism. Although the test condition for associated phoria is commonly regarded as being more natural than that for dissociated phoria, the associated phoria was not closer to the comfortable prism than the dissociated phoria. This means that the difference in the overall luminance for the two eyes, present in our test for dissociated phoria, did not push the vergence away from the value obtained with the comfortable prism. This finding supports the finding of Kromeier et al. [7, 8] that unequal luminance of the images for the two eyes does not necessarily influence the vergence position.
What is the reason for the tendency towards eso deviation in the two phoria conditions? A different accommodative demand can be excluded, because we used a similar target in all three conditions. Rather, we suggest that the shift towards eso deviation is brought about by the dissimilarity between the images of the two eyes. This suggestion pertains even to the associated phoria condition, in which ample fusionable contours are available, and the dissimilarity is limited to the monocular Nonius lines. Nevertheless, this dissimilarity was conspicuous for the observers: most of them reported that one or both Nonius lines seemed to disappear every now and then. Correspondingly, the observers reported occasional disappearance of the white dot when they determined their dissociated phoria, although this was less pronounced than the disappearance of the Nonius lines in the associated phoria condition. These observations raise the possibility that the irritating perception of binocular rivalry drives the eso shift in the two phoria conditions. Compatible with this idea is that the eso shift was stronger in the associated than in the dissociated phoria condition, i.e. in the condition with the more pronounced fading.
Considering the open feedback loop for fusional vergence in all three conditions, it is not surprising that the values obtained with all three methods (dissociated phoria, associated phoria, and comfortable prism) were rather variable. This was true in each of the two sessions.
The values changed in many observers from the first to the second session beyond the 95% confidence interval of the eight trials in the first session. Comparing the three conditions, they did not always change in the same direction (eso or exo). We do not see a specific reason for these changes, as there was no hint that the general health or attentiveness had changed between the two sessions. Rather, the variability might be understandable as there is no need for the ocular motor system to keep the vergence angle stable under open-loop conditions. Surely, adaptation maintains the open-loop vergence in a certain range [10, 12], but this range appears to be rather wide.
What are the practical inferences of our study?
As mentioned in the introduction, both dissociated [11, 18] and associated phoria [9, 19] have been recommended as indicators for prismatic correction in the case of asthenopia. We purposely did not select our observers according to the presence or absence of asthenopic complaints. Rather, we avoided using the relief from asthenopic complaints as a criterion, because this approach would have required prolonged wearing of various test prisms, including placebo, in a controlled design, to face the following four problems. First, a causal relationship between heterophoria and asthenopia is hard to establish, because the prevalence of both conditions is high in the general population, so that an unrelated coincidence can easily occur. Second, wearing prisms for hours or days leads to adaptation, so that the patient may later value even prisms that he or she initially disliked. Third, most asthenopic symptoms are waxing and waning. It is highly likely that patients seek remedy at a time when their symptoms are relatively intense. Hence, a subsequent improvement may well be due to a regression to the mean, rather than to a prism [21]. Fourth, a relief from symptoms may be brought about by the supportive behaviour of the therapist.
Although we did not use the relief from asthenopic complaints as a criterion, we think that our findings relate to the prescription of therapeutic prisms for such patients. The discrepancy between dissociated and associated phoria on the one hand and the comfortable prism on the other hand would probably also occur in patients with asthenopia, and it is plausible to assume that these patients would benefit more from the comfortable prism, chosen by themselves under natural viewing conditions, than from a prescription based on one of the phoria parameters, which carry artefacts due to the artificial test conditions.
Another practical aspect of our study is the finding that both phoria parameters and the comfortable prism can change from one session to the next. Are these changes clinically relevant? Several practitioners consider prescribing prisms from 1.0 cm/m onwards [13]. On this background, our finding that 12 of the 20 observers changed their comfortable prism by more than 1.0 cm/m (up to 3.3 cm/m) within an interval of a few weeks is important. We examined whether patients tending to a long-term change of their open-loop vergence might be identifiable in the first session by a large scatter. Unfortunately, our results did not support this notion: the intertrial variability at the first session was not larger in the observers who changed their values more than 1.0 cm/m, as compared with observers who changed their values less than 1.0 cm/m. Hence, repeated determinations of the comfortable prism on different days appear advisable to identify patients in whom the values remain relatively stable. Only in these patients would a prescription of prisms be reasonable.
What are the limitations of our study?
Towards the end of our experiments, we considered that it might have been preferable to randomize the sequence of tests between the 20 observers. However, it is unlikely that such a randomization would have produced significantly different results, for the following two reasons: (1) an influence of training or fatigue is unlikely since there was no trend between the eight trials of each test, e.g. with the early trials yielding smaller values than the late ones, or vice versa (p = 0.34), and (2) a spot check in observer #10 with the reverse order of tests confirmed this observer’s marked discrepancy between an eso deviation in the two phoria conditions, and an exo deviation in the comfortable prism (ANOVA p = 0.084 for tested difference).
The angles encountered in our 20 observers were relatively small: the dissociated phoria ranged from +9.3 cm/m eso to −5.9 cm/m exo deviation, the associated phoria from +11.2 cm/m eso to −3.3 cm/m exo deviation, and the comfortable prism from +4.8 cm/m eso to −4.1 cm/m exo deviation. An extrapolation of our conclusions to observers with larger angles may not be justified.
Our tests corresponded to just one of many “natural” viewing conditions: with respect to luminance, visual angle, and distance (4 meters), the conditions were similar to watching television. According to previous work [2, 3], it is likely that, at closer viewing distances, the values would be shifted in the exo direction. Hence, we suggest that the practitioner should determine the comfortable prism in the viewing distance, for which therapeutic prisms are being considered. This recommendation corresponds with the concept of Jaschinski [4], who suggested that observers should choose their comfortable viewing distance for computer screens in the real-life situation.

fixation disparity
aligning prism
asthenopia
heterophoria
mallett unit


Connexons and cell adhesion: a romantic phase

Recent evidence indicates, that gap junction forming proteins do not only contribute to intercellular communication (Kanno and Saffitz in Cardiovasc Pathol 10:169–177, 2001; Saez et al. in Physiol Rev 83:1359–1400, 2003), ion homeostasis and volume control (Goldberg et al. in J Biol Chem 277:36725–36730, 2002; Saez et al. in Physiol Rev 83:1359–1400, 2003). They also serve biological functions in a mechanical sense, supporting adherent connections between neighbouring cells of epithelial and non-epithelial tissues (Clair et al. in Exp Cell Res 314:1250–1265, 2008; Shaw et al. in Cell 128:547–560, 2007), where they stabilize migratory pathways in the developing central nervous system (Elias et al. in Nature 448:901–907, 2007; Malatesta et al. in Development 127:5253–5263, 2000; Noctor et al. in Nature 409:714–720, 2001; Rakic in Brain Res 33:471–476, 1971; J Comp Neurol 145:61–83 1972; Science 241:170–176, 1988), or mediate polarized movements and directionality of neural crest cells during organogenesis (Kirby and Waldo in Circ Res 77:211–215, 1995; Xu et al. in Development 133:3629–3639, 2006). Since, most data describing adhesive properties of gap junctions delt with connexin 43 (Cx43) (Beardslee et al. in Circ Res 83:629–635, 1998), we will focus our brief review on this isoform.
Introduction
For those readers who are not familiar with the molecular composition of gap junctions, we will briefly recollect the substructure of this cell contact and refer to recent detailed reviews (Duffy et al. 2002; Gaietta et al. 2002; Goodenough and Paul 2003; Meier and Dermietzel 2006; Willecke et al. 2002).
Gap junctions are formed by hemichannels (connexons), which consist of an oligomer of six proteins (connexins). At present, at least 20 genes seem to be present in the human and rodent genome (Willecke et al. 2002), which may oligomerize in monomeric or heteromeric patterns to form a hemichannel. A complete gap junction channel is formed by two hemichannels in mirror symmetry (Fig. 1). Heterotypic configurations of different isoforms are allowed for some connexins while others occur exclusively in homotypic configuration. In the history of gap junctions, the junctional plaque has always been considered to occur in a “naked” form without cytoplasmic adjuncts like scaffolding proteins or cytoskeletal elements (Hirokawa and Heuser 1982). However, accruing evidence indicates that gap junctions are associated with a complex system of scaffolding and cytoskeletal proteins, which seem to assemble in cell specific patterns (Duffy et al. 2002 for recent review).Fig. 1General structure of a gap junction plaque. Gap junctions are formed by paired hemichannels (connexons) of two adjacent cells. A single connexon is made by a polymer of six connexins. Only apposed connexons allow intercellular transfer of ions (ionic coupling) and small metabolites (metabolic coupling). Unapposed connexons seem to perform per se functions
In the following, we will put main emphasis on heart and brain tissues for which most of the data on gap junctions and cell adhesion have been reviewed.
Cardiomyocytes: directed trafficking of connexin43 involves the cytoskeleton and adhesion plaques
In the terminal intercalated discs between cardiomyocytes, Cx43 celebrates an example for integrating its hemichannel and cell coupling functions (Gros and Jongsma 1996; Shaw and Rudy 1997; van Veen et al. 2001). Asides the terminal intercalated discs, gap junctions are also localised in the lateral sarcolemma of the heart (Fig. 2), and thus described to form site-to-site and end-to-end connections (Yao et al. 2003). Cardiomyocytes also represent unopposed connexons with hemichannel function in the lateral sarcolemma (Saez et al. 2003; Schulz and Heusch 2006; Yao et al. 2003).Fig. 2a shows cultured cardiomyocytes immunolabelled with an anti-Cx43 antibody (red). Immunolabelling is prevalent in apposed cell membranes, but also in some unapposed domains. Nuclei are counterstained with Hoechst dye. b Immunolabelling of heart tissue with Cx43 antibody. Intercalated discs (red) are intensivley stained. Bar indicates 25 μm
To ensure its’ trafficking and functional integration into a gap junction plaque or in form of unpaired connexons (hemichannels) into the plasma membrane, Cx43 has to interact with other proteins. In the intercalated discs of coupled cardiomyocytes for instance, gap junction plaques are embedded into adherens junctions, which are primarily formed by cadherins (Matsuda et al. 2006; Niessen 2007; Zuppinger et al. 2000). Whilst the adherens junction supports the mechanical coupling (Gutstein et al. 2003; Niessen 2007), the gap junction ensures the propagation of action potentials along the cardiomyocytes (Gros and Jongsma 1996; Shaw and Rudy 1997).
Multiple models describe the pathway from connexon assembly to the initial gap junction formation and interaction with cadherins in the adherens junction. The most common examples are based on half-life time determined trafficking and junctional protein (cadherins) mediated activation of Cx43. The half-life time of Cx43 is restricted between 1 and 3 h, and implicates a dynamic process of assembly, insertion and replacement of connexons, and pairing of connexons to form gap junctions (Beardslee et al. 1998; Hofer and Dermietzel 1998; Laird et al. 1991). Cx43 synthesis was shown to be located on membrane bound ribosomes, where connexin proteins are rapidly oligomerized into homo or heteromeric connexons (Evans et al. 1999; Martin and Evans 2004). Final packing into hemichannel loaded vesicles occurs in the trans-Golgi network as shown by Musil and Goudenough (1993) followed by directed transport along microtubules to multiple insertion sites in the membrane (Akhmanova and Hoogenraad 2005; Jordan et al. 1999; Lauf et al. 2002; Mimori-Kiyosue et al. 2005; Shaw et al. 2007). Once arrived at the gap junction borders at the membrane, connexons are assumed to be inserted via flipping events into the membrane and to diffuse into the centre of the plaque, whilst elderly connexin proteins are shifted to the plaque periphery for subsequent disposal (Gaietta et al. 2002; Laird 2005; Segretain and Falk 2004).
The finding that mislocated Cx43 gap junction plaques in the ischemic myocardium are associated with similarly misplaced adherens junctions (Matsushita et al. 1999), underlines an interdependence between Cx43 and the adherens junction related cadherins (Angst et al. 1997; Li et al. 2005; Luo and Radice 2003; Matsushita et al. 1999). E-cadherin transfections into gap junction incompetent cells, allowed the transfectants to build out functional gap junctions (Matsushita et al. 1999). Furthermore, N-cadherin knockout mice (Luo and Radice 2003) and conditional knockdown of N-cadherin in the heart caused mislocalisation and compromized expression of gap junctions. Conditional knockdown of N-cadherin in the heart was additionally shown to lead to arrhythmogenic death (Li et al. 2005), which may involve aberrant regulation of gap junction function (for reviews see: Duffy et al. 2007)
Recently, Shaw et al. (2007) described microtubule-mediated target-delivered transport of Cx43 via microtubule plus-end-tracking proteins (+TIPs) and interaction partners such as p150(GLUED) (Berrueta et al. 1999), a component of the dynein/dynactin complex, which in turn is potent to tether microtubules at the adherens junctions (Chausovsky et al. 2000; Ligon et al. 2001). Studies implicating fluorescence recovery after photobleaching (FRAP) on Cx43-YFP transfected HeLa cells that do not endogenously express Cx43, revealed a rapid Cx43 delivery to gap junction plaques. Deconvolution clarified that microtubules extend directly to the gap junction plaques at the cell’s border and total internal reflection fluorescence (TIRF) microscopy and time lapse imaging revealed the appearance of a preferential and prolonged association of microtubule plus ends with the plaques. Most strikingly, Shaw et al. (2007) were able to show that gap junction plaque formation was disrupted by siRNA knockdown of the dimeric +TIP EB1. EB1 associates directly with the plus ends of microtubules and provides, in turn, dual binding sites for adherens junction related proteins like p150(GLUED) and β-catenin. Furthermore, in this setting gap junction plaques could also be disrupted via Nocodazol and Taxol treatment, peptides, which compromise the homophilic cadherin-cadherin interaction in adherens junctions. This setting according to the recent paper by Shaw et al. (2007) is sketched in the cartoon (Fig. 3). While Nocodazol interrupts formation of microtubules by depolimerization, Taxol lets microtubules remain stable, but interferes with their EB1 interaction partner (Nakata and Hirokawa 2003).Fig. 3Model for microtubulus mediated delivery of vesicle-bound connexons to adherens junctions (adapted from Shaw et al. 2007). Microtubules bind via their +end to EB1. EB1 in turn binds to P150 (GLUED), a component of the dynein/dynactin complex, which interacts with β-catenin through P120-catenin with the adherens junction. This interaction is understood to tether the microtubule to the junction and to serve as a gateway for connexon delivery
In this context, actin is discussed to act as an initial sensor of cell-cell interaction, driving the localisation of adherens junctions with assistance from Rho-GTPases (Noren et al. 2001, 2003).
Spread of Shigella flexneri requires connexin43 hemichannels
How close cytoskeletal (re-)organization and intracellular connexin distribution are related is demonstrated for polarized intestinal cells during Shigella flexneri invasion (Clair et al. 2008). For invading the colonic mucosa (Labrec et al. 1964), the gram negative enteric bacillus requires RhoGTPases, Src and Abl/Arg tyrosine kinases for actin polymerization and formation of cytoplasmic extensions of surrounding cells (Burton et al. 2003; Tran Van Nhieu et al. 2000). The invasion and dissemination of the bacteria causes intense inflammatory responses, and especially ATP-dependent paracrine signalling induced by Cx hemichannel opening (Tran Van Nhieu et al. 2000). E-cadherins were indispensable for the intercellular spreading of S. flexneri (Sansonetti et al. 1994). It is assumed that a cytoskeletal reorganization toward the formation zone of gap junctions is induced in this process to allow the spread of the bacteria (Clair et al. 2008; Tran Van Nhieu et al. 2000). On the epithelial level there is mounting evidence that connexin hemichannels regulate intercellular signalling (Stout et al. 2004), which might be of importance for incoming phagocytic cells during bacterial infection (Ferrari et al. 1997; Griffiths et al. 1995; John et al. 2001; Korcok et al. 2004).
The developing brain and hemichannel adhesion
The necessity of gap junction adhesion via regulation by its cytoskeleton interaction partners p120 catenin, integrin and actin has become well identified in the developing brain (Xu et al. 2001, 2006). Here, stem cells of the developing neocortex give rise to neurons (Malatesta et al. 2000; Noctor et al. 2001) and provide guidance of the developing neurons to the target zones of the cortical plate, where they are meant to become pyramidal cells of the adult cortex (Rakic 1971, 1972, 1988). Electron microscopy showed, that during the process of migration of neuronal precursors, gap junctions occur between radial fibres and migrating neurons and nestin− and nestin+ cells (Huang et al. 1998a). The most important gap junction protein isoforms are Cx43 and Cx26 in developing brain tissue (Dermietzel et al. 1989). Until now there was evidence that gap junctions between radial glia and migrating neurons served for chemical and electrical communication. Elias et al. (2007) recently found that Cx26 and Cx43 are expressed in β-III tubulin positive migrating neurons in the contacting regions close to vimentin positive radial glial fibres. Using a RNA knockdown of Cx43 and Cx26 by short hairpin RNA (shRNA) constructs in rat, the authors were able to demonstrate a reduced fractioning of neurons in the cortical plate. In addition, transplantation of Cx26 and Cx43 shRNA knocked down donor cells into E17 wildtype mice revealed an intact engrafting of the donor cells into the host brain, but no migration. Immunocytochemistry of the shRNA knocked down transplanted neurons in the recipient brains showed no compromised cell cycle exit and no alterations of differentiation. Furthermore, the expression of the adherens related proteins ZO-1, N-cadherin and β-integrin was not altered, indicating that gap junctions mediate glial-guided radial migration of developing neurons in the cortex. This migration was additionally demonstrated to rely on the adhesive and not on the channel properties of Cx26 or Cx43 (Elias et al. 2007). Dominant negative connexin mutants lacking channel properties were still able to form adhesive contacts. In reverse experiments, the authors demonstrated that channel, but no adhesion forming mutants, were unable to rescue the Cx43 shRNA induced migration defect. Finally, time lapse imaging of Cx43/Cx26 shRNA expressing neurons affirmed their inability to stabilize their processes and to continue to extend along the radial glia.
Neural crest cells and colonization
Gap junction regulated polarized cell movements and directional migration are not restricted to developmental processes within the central nervous system (Elias et al. 2007; Schaar and McConnell 2005). Studies focusing on Cx43 expression of cardiac neural crest cells indicated a clear relationship between their migratory properties and Cx43 expression (Huang et al. 1998a, b; Li et al. 2002; Lo et al. 1999; Reaume et al. 1995; Sullivan and Lo 1995; Xu et al. 2001, 2006). Neural crest cells are ectomesenchymal cells emerging from epithelial mesenchymal cell transformation in the dorsal neural tube from where they disperse throughout the embryo to generate a variety of tissues (Kirby and Waldo 1995; Xu et al. 2006). Neural crest cells from different axial levels of the neural tube use multiple migratory pathways to reach their terminal destinations. Cardiac neural crest cells (CNCs) have been shown to migrate along a circumpharyngeal pathway to reach the aortic arches and the heart (Kirby et al. 1983; Lumsden et al. 1991). This deployment has been shown to be modulated by Cx43 and cytoskeletal interaction partners with the extracellular matrix (Xu et al. 2006). The finding, that dynamic di- and reassembly of focal contacts is essential for polarized cell movements and directional cell migration moved the heterodimeric receptor group of integrins into the centre of related studies. Integrins cluster to form focal domains within the cell membrane, linking the extracellular matrix to the actin cytoskeleton. Since, it could be shown that neural crest cells express multiple integrins (Delannet et al. 1994; Monier-Gavelle and Duband 1997) and perturbation studies provide evidence that integrins modulate the migratory behaviour of neural crest cells, (Strachan and Condic 2003, 2004, 2008) the question arose whether integrin signalling might be affected in Cx43 expressing versus Cx43 knock out cells (Xu et al. 2006).
In neural tube explants of the post-otic hindbrain folds from E8.5 mice, underlying either an Cx43 knockout or Cx43 overexpression, neural crest cells were generated that emerge from the same axial level as CNCs, which migrate to the heart. In contrast to overexpressing CNCs, the Cx43 deficient CNC type was characterized by a severe loss of directionality and reduced adhesion whilst being cultured on a fibronectin matrix. Furthermore, an increase in the fibronectin matrix density leads to reductions in the migratory speed of Cx43 deficient CNCs, as shown via time lapse videomicroscopy (Xu et al. 2001). In fact double-immunostaining against β1-integrin and vinculin as markers for focal adhesion, was significantly reduced in Cx43 deficient CNCs, indicating a reduction in the actin-cytoskeletal linkage for matrix adhesion. A modulatory influence of Cx43 on the actin cytoskeleton became evident in rhodamine-phalloidine stainings, where Cx43 knockout CNCs represented shorter stress fibre bundles (Xu et al. 2001). Additionally, these bundles exhibit no anchoring via vinculin to focal adhesions, as being observed for Cx43 overexpressing CNCs. Furthermore, adhesion and migration of Cx43 deficient CNCs on a fibronectin matrix could be inhibited by semaphorin application, which is described to act as a potent blocker of integrin activation (Brown et al. 2001). This approach also confirmed that Cx43 modulates the retraction of cellular processes. Immunoprecipitation, Western blot and immunocytochemistry pointed out that Cx43 in CNCs does not co-localise with β1-integrin, but with vinculin and actin-filaments (Osborne et al. 2005; Pasterkamp and Kolodkin 2003; Serini et al. 2003; Xu et al. 2006). This finding is supported by the co-localisation of Cx43 with several actin binding proteins, such as ezrin, IQGAP, α-actinin and drebrin (Butkevich et al. 2004). No correlation between gap junctional coupling properties and the density of fibronectin matrix was found for Cx43 knockouts and Cx43 overexpressing CNCs in dye coupling experiments, although an upregulation of gap junction communication with altered integrin-matrix interactions has previously been described for other cell types (Czyz et al. 2005; Lampe et al. 1998; Shanker et al. 2005).
In summary, asides their function as gap junction forming elements, unpaired connexons have been shown to modulate the cells’ migratory and adhesive functions whilst being in permanent crosstalk with an elaborate complex of cytoskeletal interaction partners.
Perspectives on pathology
How important the connexin-cytoskeleton interaction is, becomes elucidated in case of pathology. For instance, cadherin-cadherin interactions might be critically affected during tumour formation. The interaction normally becomes active in cell sorting mechanisms during development (Wheelock and Johnson 2003). Since, Shaw et al. (2007) have shown that Cx43 can reach adherens junctions via microtubule directed delivery, it may be suggested that gap junctions are formed preferentially with cells, expressing the same type of cadherin (Wheelock and Johnson 2003). Additional studies provide evidence, that loss of E-cadherin or upregulation of N-cadherin can increase tumour invasiveness and Cx43 downregulation in malignant cells (Mesnil 2002). It is thought that gap junction channels and their interactions with molecules such as p120 catenin, integrin and the actin cytoskeleton are important for neural crest cell migration (Xu et al. 2001, 2006), and that glioblastoma invasion of the brain parenchyma requires functional gap junctions between tumour cells and astrocytes (Lin et al. 2002). Furthermore, the migration of lung and skin cancer cells has also been associated with gap junction expression, although no clear mechanism has been proposed so far (Ito et al. 2000; Lois et al. 2002).
Developmental defects are also related to mutations of the Cx43 gene. As already indicated, Cx43 knockout mice reveal comprised conotruncal heart development, which is associated with a reduction in the number of cardiac neural crest cells targeted to the heart (Xu et al. 2006). Mutations in Cx43 which seem to influence cytoskeletal organization in a strong manner range from disease patterns like deafness, cataracts, germ cell developmental defects, ocludentodigital dysplasia to cardial outflow abnormalities (Polontchouk et al. 2002) and left ventricular remodelling (Kanno et al. 2003).
At its final extent, gap junction channels, in particular in form of hemichannels, constitute a new player in the complex interaction of cell adhesion and cytoskeletal activation, which underlies directed migration during development and in mature tissue. It is a romantic phase where anything may go, but time has to approve what will remain forever.

cell adhesion
gap junction
hemichannel
heart
developing brain


Hemangioblastomatosis in a patient with von Hippel-Lindau disease

Case presentation
A woman developed a cerebellar hemangioblastoma in 1991 at the age of 17 and von Hippel-Lindau disease was diagnosed. She underwent five craniotomies for recurrent hemangioblastoma. In 2001 she had a laminectomy for an intramedullar hemangioblastoma at C2-C3 level. In 2004 a renal cell carcinoma was found and a partial nephrectomy was performed. A few weeks after her last craniotomy in July 2005 she developed progressive right hemisensory disturbances and sensory ataxia. Spinal MRI showed leptomeningeal contrast enhancement around the entire spinal cord (see Figure). The sensory ataxia progressed and she got tetraparesis, most severe proximally in her arms, especially myotome C4. Although hemangioblastoma are not very radiosensitive, radiotherapy was started because of the rapid clinical deterioration. High-dose total neuraxis radiotherapy was considered to toxic. It was judged that most of her clinical complaints could be attributed to the cervical spine. Also the MRI showed the most severe lesions at the cervical region. Therefore she was treated from C1 to Th1 (30 × 1.8 Gy) combined with dexamethasone (2 × 8 mg). During radiotherapy she developed also symptoms outside the radiotherapy field (thoracal radiculopathy).
As the main problem in VHL is uncontrolled angiogenesis by overexpressing VEGF and VEGF receptors, Thalidomide (400 mg, later 600 mg) was added. Thalidomide inhibits angiogenesis induced by VEGF [1]. Despite the treatment she developed a total tetraplegia and died 3 months after diagnosis as a result of respiratory failure.
This syndrome appears to result from delayed growth of subarachnoid tumor cells disseminated by surgery. Hemangioblastomatosis is also seen in hemangioblastoma patients without von Hippel-Lindau disease and it is thought that additional genes are likely to be the source of this malignant behaviour [2].

hemangioblastoma
von hippel-lindau disease