Integrin alpha2beta1 affects mechano-transduction in slowly and rapidly adapting cutaneous mechanoreceptors in rat hairy skin.

The role of a transmembrane protein, integrin alpha2beta1, to modulate the neural responses of cutaneous mechanoreceptors to mechanical indentation was examined using an isolated skin-nerve preparation in a rat model. Skin and its intact innervation were harvested from the medial thigh of the hindlimb and placed in a dish containing synthetic interstitial fluid. Using a standard teased nerve preparation, the neural responses of single slowly or rapidly adapting mechanoreceptors (SA or RA, respectively) were identified and the afferents categorized according to standard protocols (i.e. response to constant stimuli). The most sensitive spot of a mechanoreceptor's receptive field was identified and then stimulated using controlled compressive stress (constant or dynamic loads between threshold and saturation load for SAs and RAs, respectively). Loads were applied before, during, and after passive diffusion into the skin of a function-blocking anti-integrin alpha2 monoclonal antibody (FBmAb) or one of two types of control antibodies (immunoglobulin G or a FBmAb conjugated with a secondary antibody). The sensitivities of both SA and RA mechanoreceptors were profoundly reduced in the presence of the FBmAb, while not changing the waveforms of their action potentials or their adaptation properties. Both control antibodies had no significant effect on mechanoreceptors' sensitivities. Following removal of the FBmAb, the effects in some neurons were partially reversible. Taken together, the data from this study support the hypothesis that integrin alpha2beta1 plays a significant role in modulating mechanoreceptive response to compressive indentation.

Expression of integrin alpha2beta1 in axons and receptive endings of neurons in rat, hairy skin.

Integrin alpha2beta1 has been considered as a mechano-chemical transducer in endothelial and muscle cells. However, little data is available to show whether integrins play a role in the process of mechanical transduction in peripheral mechanosensory neurons. Using immunohistochemistry, we demonstrate that cutaneous neurons express the extracellular matrix (ECM) receptor integrin alpha2beta1. Specifically, we show that integrins alpha2 and beta1 are co-localized with peripherin in the receptive endings of cutaneous neurons in rat, hairy skin. Integrin immunofluorescence was minimal along the axons of large diameter neurons. These results, together with findings by other investigators, provide evidence suggesting that integrin alpha2beta1 may be a linking agent between mechanical stress in the ECM and modulation of the neuronal response of mechanically sensitive neurons.

Encoding of location and intensity of noxious indentation into rat skin by spatial populations of cutaneous mechano-nociceptors.

The ability of a spatial population of cutaneous, Adelta, and C mechano-nociceptors to encode the location and intensity of a noxious, cutaneous indentation was examined using an isolated preparation in a rat model. Skin and its intact innervation were harvested from the medial thigh of the rat hindlimb and placed in a dish, with the corium side down, containing synthetic interstitial fluid. The margins of the skin were coupled to an apparatus that could stretch and apply compression to the skin. The skin was suspended on top of a deformable platform whose bulk, nonlinear, compressive compliance emulated that found in vivo. The isolated preparation facilitated examination of the spatial population response by eliminating the nonlinear geometry and inhomogeneous compressive compliance present in-vivo. Spatial population responses (SPR) were formed from recordings of single neurons that were stimulated by compressing the skin with an indenter (flat cylinder, 3-mm diam) at discrete intervals from the center of their receptive fields. SPR were composed of the neural responses (z axis) at each indentation location (x, y plane), and were analyzed quantitatively using nonlinear regression to fit an equation of a Gaussian surface. Both Adelta and C SPR accurately encoded the location and intensity of noxious indentation. The intensity of the stimulus was encoded in the peak neural response of the SPR, which had a nonlinear relationship to the compressive force. The location of the stimulus was encoded in the x, y position of the peak of the SPR. The position of the peak remained constant with increasing magnitudes of compressive force. The overall form of the SPR also remained constant with changes of compressive load, suggesting a possible role for encoding in the SPR some aspects of shape of a noxious stimulus.

Raised object on a planar surface stroked across the fingerpad: responses of cutaneous mechanoreceptors to shape and orientation.

The representations of orientation and shape were studied in the responses of cutaneous mechanoreceptors to an isolated, raised object on a planar surface stroked across the fingerpad. The objects were the top portions of a sphere with a 5-mm radius, and two toroids each with a radius of 5 mm along one axis and differing radii of 1 or 3 mm along the orthogonal axis. The velocity and direction of stroking were fixed while the orientation of the object in the horizontal plane was varied. Each object was stroked along a series of laterally shifted, parallel, linear trajectories over the receptive fields of slowly adapting, type I (SA), and rapidly adapting, type I (RA) mechanoreceptive afferents innervating the fingerpad of the monkey. "Spatial event plots" (SEPs) of the occurrence of action potentials, as a function of the location of each object on the receptive field, were interpreted as the responses of a spatially distributed population of fibers. That portion of the plot evoked by the curved object (the SEPc) provided a representation of the shape and orientation of the two-dimensional outline of the object in the horizontal plane in contact with the skin. For both SAs and RAs, the major vector of the SEPc, obtained by a principal components analysis, was linearly related to the physical orientation of the major axis of each toroid. The spatial distribution of discharge rates [spatial rate surface profiles (SRSs), after plotting mean instantaneous frequency versus spatial locus within the SEPc] represented object shape in a third dimension, normal to the skin surface. The shape of the SA SRSs, well fitted by Gaussian equations, better represented object shape than that of the RA SRSs. A cross-sectional profile along the minor axis [spatial rate profile (SRP)] was approximately triangular for SAs. After normalization for differences in peak height, the falling slopes of the SA SRPs increased, and the base widths decreased with curvature of the object's minor axis. These curvature-related differences in slopes and widths were invariant with changes in object orientation. It is hypothesized that circularity in object shape is coded by the constancy of slopes of SA SRPs between peak and base and that the constancy of differences in the widths and falling slopes evoked by different raised objects encodes, respectively, the differences in their sizes and shapes regardless of differences in their orientation on the skin.

Encoding of shape and orientation of objects indented into the monkey fingerpad by populations of slowly and rapidly adapting mechanoreceptors.

The peripheral neural representation of object shape and orientation was studied by recording the responses of a spatially distributed population of rapidly and slowly adapting type I mechanoreceptors (RAs and SAs, respectively) to objects of different shapes and orientations indented at a fixed location on the fingerpad of the anesthetized monkey. The toroidal objects had a radius of 5 mm on the major axis, and 1, 3, or 5 mm on the minor axis. Each object was indented into the fingerpad for 4 s at orientations of 0, 45, 90, and 135 degrees using a contact force of 15 gwt. Estimations of the population responses (PRs) were constructed by combining the responses of 91 SA and 97 RA single afferents at discrete times during the indentation. The PR was composed of the neural discharge rates (z coordinate) plotted at x and y coordinates of the most sensitive spot of the receptive field. The shapes of the PRs were related to the shapes of the objects by fitting the PRs with Gaussian surfaces. The orientations of the PRs were determined from weighted principal component analyses. The SA PR encoded both the orientation and shape of the objects, whereas the RA PR did neither. The SA PR orientation was biased toward the long axis of the finger. The RA PR encoded orientation only for the object with the highest curvature but did so ambiguously. Only the SA PR was well fit by a Gaussian surface. The shape of the object was discriminated by the SA PR within the first 500 ms of contact, and the form of the SA PR remained constant during the subsequent 3.5 s. This was manifested by constant widths of the PR along the major and minor axes despite a peak response that decreased from its maximum at 200 ms to an asymptotic value starting at 1 s. Thus the shape and orientation of each object were coded by the shape and orientation of the SA PR.

Tensile and compressive responses of nociceptors in rat hairy skin.

Mechanically sensitive nociceptor afferents were studied in a preparation of isolated skin from rat leg. Each neuron was studied while the skin was subjected to tensile and compressive loading. The experiment was designed to create highly uniform states of stress in both tension and compression. Tensile loads were applied by pulling on the edges of the sample. Applied loads were used to determine the tensile stresses. Surface displacements were used to determine tensile strains. Compressive loads were applied by indenting the surface of the skin with flat indenter tips applied under force control. The skin was supported by a flat, hard substrate. Compressive stresses were determined from the applied loads and tip geometry. Compressive strains were determined from skin thickness and tip excursions. All nociceptors were activated by both tensile and compressive loading. There was no interaction between the responses to compressive and tensile stimuli (i.e., the responses were simply additive). Responses of nociceptors were better related to tensile and compressive stresses than to strains. Nociceptors responded better to tensile loading than to compressive loading. Response thresholds were lower and sensitivities were higher for tensile stress than for compressive stress. The response to compression was better related to compressive stress than to other stimulus parameters (i.e., load/circumference or simply load). Indentations of intact skin over a soft substrate such as muscle would be expected to cause widespread activation of nociceptors because of tensile stresses.

Compressive behavior of articular cartilage is not completely explained by proteoglycan osmotic pressure.

It has been hypothesized that applied mechanical or osmotic loads which decrease cartilage volume by 5% or more are sufficient to relieve all collagen tensile forces, and that further changes in the applied load are completely supported by changes in proteoglycan osmotic pressure. In this view, cartilage should behave mechanically like a concentrated solution of proteoglycans. We tested this hypothesis by measuring the equilibrium axial and radial stresses in bovine articular cartilage during uniaxial confined compression. If the hypothesis is correct, the observed changes in the radial and axial stresses in confined compression should be equal for compression greater than 5%. However, the observed change in axial stress was always substantially greater than the change in radial stress over the range of strains (5-26%) and saline concentrations (0.05-0.15 M) tested. This indicates that the mechanical behavior of cartilage in confined compression cannot solely be explained by changes in proteoglycan osmotic pressure even for strains as large as 26%. A linear isotropic model was found to describe the observed equilibrium behavior adequately. In addition, the inferred shear modulus was found to be independent of saline concentration and similar to measurements by others of the flow-independent shear modulus. Our results have implications regarding the relative contribution of the proteoglycans and collagen to the mechanical properties of the tissue in compression, and suggest that tensile forces in the collagen network may play an important role in determining tissue behavior in confined compression even for relatively large volume changes.

Mechanical states encoded by stretch-sensitive neurons in feline joint capsule.

1. The sensitivity of group II joint afferents innervating cat knee joint capsule to in-plane stretch was studied in vitro. Single afferents were recorded from teased filaments of the posterior articular nerve. The capsule was stretched by applying forces through tabs along the edges of the capsule (3 tabs/edge) with the use of an apparatus that allowed for independent control of each load. The relationships between the neural responses of these afferents and the local continuum mechanical state of the joint capsule have been investigated. By appropriately loading the tissue margins, it was possible to establish states of uniaxial and biaxial tension, including shear. 2. Plane stress was calculated from the loads along the tissue margins. Stress at the location of the mechanoreceptor ending was estimated by interpolation. Strain was calculated from deformations of the capsule measured by tracking markers on its surface. Full characterization of tissue stress and strain made it possible to determine strain energy density and the magnitudes of other coordinate invariant mechanical quantities. 3. Individual afferents (n = 15) exhibited pronounced selectivity to the direction of applied stress and strain. There was no overall preferred orientation across neurons, and simple correlation of individual stress or strain components with the neuronal response revealed no consistent relationship between neuronal response and any single tensor component. However, linear multiple regression of the combined stress and strain components with the neuronal response revealed high correlation (mean R = 0.91), indicating that the measured mechanical states strongly determine the neuronal response. There was a much stronger relationship between neuronal response and stress variables than with strain variables. Simple correlation of the first invariant of the stress tensor with neuronal response had the highest mean correlation of the tensor quantities (R = 0.51). On average, strain energy density was only modestly correlated with the neural response (R = 0.28). 4. These findings indicate that capsule mechanoreceptors are encoding the local continuum mechanical state in the joint capsule. The neural response of these mechanoreceptors is more strongly correlated to local stress than to local strain.

Responses of mechanoreceptor neurons in the cat knee joint capsule before and after anterior cruciate ligament transection.

It is known that afferent neurons play a protective role in knees made unstable by transection of the anterior cruciate ligament. However, it is not known whether cutting the anterior cruciate ligament has an effect on the response of the sensory neurons that innervate the joint. In this study, the responsiveness (activation threshold and position sensitivity) of single, mechanically sensitive afferent neurons from the cat knee was evaluated by a series of extension, internal, and external rotations. The anterior cruciate ligament then was cut and the same procedure was repeated. Transection of the ligament increased joint laxity for all types of rotation. The responsiveness of the neurons was not changed significantly by cutting the ligament (p > 0.05). Therefore, capsule afferents continue to behave normally in joints in which the anterior cruciate ligament has been transected.