TECHNICAL NOTE |
Correspondence to: Didier Guinard, Equipe de Neurobiologie du Développement, LAPSEN-U Inserm 318, Université Joseph Fourier, BP 217, 38043 Grenoble Cedex 9, France.
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Summary |
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For detailed study of complex structures such as corpuscular mechanoreceptors, confocal microscopy can be used with multiple immunolabeling that identifies specifically different subcomponents. In addition, anatomic interpretation is enhanced by three-dimensional reconstruction. Confocal laser micrographs, reconstructed from serial images 1 µm thick of human skin Meissner corpuscles simultaneously immunostained for neurofilaments (NF 70-200) and protein S-100 (PS-100), clearly reveal the complex 3D relationship between Schwann-related lamellar cells immunoreactive for PS-100 and the nerve fibers marked by NF 70-200. The nerve fiber, after branching into the corpuscle, divides into several ramifications, presenting discoidal expansions and flattened fringed sections. The mean nerve diameter was 4 µm ± 1 (25 µm) and the mean size of the discoidal expansions was 15 µm ± 1 (730 µm). Corpuscle size varied from 30140 ± 1 µm in length and from 2060 ± 1 µm in diameter. This study confirms the presence of neural discoidal areas in Meissner's corpuscles, which are probably involved to some extent with the transduction process. Despite the accuracy of immunolabeling and imaging, an extracorpuscular neural network was never observed in the vicinity of corpuscles, thus giving doubt as to their existence. (J Histochem Cytochem 48:295302, 2000)
Key Words: Meissner corpuscles, immunolabeling, confocal microscopy, mechanoreceptors, 3D reconstruction, cutaneous innervation, finger
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Introduction |
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Since its first description, our understanding of the architecture of the Meissner's corpuscle has been enhanced by studies at the optical level (LM) and with transmission electron microscopy (TEM) (
At present, immunolabeling techniques are the most reliable means for identifying neural structures. Of the many neuropeptides and proteins that have been reported in sensory endings, protein S-100 (PS-100) has proved to be a reliable marker of Schwann cells, and nerve fibers are labeled specifically by antibodies against neurofilaments (NF 70, NF 70-200) (
For complex structures such as corpuscular mechanoreceptors, simultaneous specific labeling by different markers allows better identification of the different substructures, thus improving anatomic interpretation (
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Materials and Methods |
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Tissue Preparation
Human digital pulps of two second digits were obtained from non-replantable traumatic amputations on the dominant hand of two manual workers (43 and 51 years old) without medical antecedents who gave their informed consent in accordance with the Helsinki Declaration of 1975. Samples were immediately excised and fixed with 4% formaldehyde in pH 7.4 PBS 0.01 M for 24 hr at 4C. After rinsing in distilled water, pulp tissue was cut into small fragments perpendicular to the skin surface (1 x 6 x 3 mm) and immediately embedded in agar or paraffin. Thick serial sections (70 µm) were made from agar-embedded samples with a vibratome (Vibraslice 752 M; Campden Instruments, Oxford, UK) and treated for immunolabeling. Thin (7-µm) sections for conventional light microscopy were cut from paraffin-embedded samples with a microtome (ReichertJung 2050 Microm; Vienna, Austria).
Immunofluorescence and Immunochemistry
Agar-embedded samples were labeled using a floating technique. To enhance marker penetration, samples were pretreated by microwaving (Micromat 120 AEG; frequency 2.45 Hz, 850 W) in a 0.01 M pH 6 sodium citrate buffer (three sessions of 5 min each). After incubation in 0.01 M PBS with 2% skimmed milk powder at 4C for 1 hr and rinsing, primary antibodies were applied for 48 hr at 4C in a humid atmosphere with gentle agitation. Immunolabeling was performed with murine monoclonal antibodies (clone 2F11) to NF 70-200 (Chemical Credential; Costa Mesa, CA) at a dilution of 1:100 and with rabbit polyclonal antibodies to PS-100 (Immunotech 1071; Marseille, France) at a dilution of 1:48. After rinsing in 0.01 M PBS, pH 8.6 (three times for 20 min) and incubation in 1% bovine serum albumen (BSA) for 30 min, a second rinse in 0.01 M PBS, pH 8.6 (three times for 20 min) was done. Secondary fluorescence-conjugated antibodies were sequentially applied at 4C in a humid and dark atmosphere for 3 hr with 0.01 M PBS, pH 8.6 (1 hr) and 1% BSA (30 min) rinses between the two sequences as follows: first 1:100 rhodamine-conjugated (TRITC) goat anti-rabbit antibodies (Jackson; West Grove, PA) were used to recognize NF 70-200 and then 1:75 fluorescein-conjugated (FITC) rabbit anti-murine antibodies (Dako; Glostrup, Denmark) were applied to identify PS-100. After rinsing in 0.01 M PBS, pH 8.6 (1 hr), samples were mounted in an antifading medium (0.05 M Tris-HCl, 8%, pH 7.6, glycerol (90%), 1.4 diaziabyclo-octan (2%) (Sigma, St Louis, MO; D 2522) between a slide and a coverslip sealed with nailpolish. Control sections were prepared by substituting normal serum for primary antibodies or by eliminating the first incubation.
For light microscopy, thin sections were treated with the same immunochemical process as described above for PS-100 antibodies (Immunotech 1071). Specific staining was revealed by incubation with biotinylated donkey anti-rabbit antibodies (Jackson) followed by a peroxidase-conjugated avidinbiotin complex (1: 400, PBS 7.4, BSA 1%, 1 hr) (StreptABComplex/HRP; Dako) and diaminobenzidine tetrachloride baths. Counterstaining of cell nuclei was performed with hematoxylin.
Confocal Microscopy
Confocal optical sections were collected with a confocal scanning laser microscope LSM 410 Invert (Carl Zeiss; Oberkochen, Germany) using a x40 objective lens (Plan-Neofluoar, 1.3 NA, oil immersion). Simultaneous imaging of FITC and TRITC fluorescence was performed with the following settings. Both labels were excited with the 488-nm line of an argon laser while the excitation beam was separated from emitted fluorescence by a 510-nm dichroic mirror. The two fluorescences were segregated with a 560-nm beam separator and respectively selected with a 520560-nm bandpass filter (FITC spectrum) and a 590-nm longpass filter (TRITC spectrum). The signal-to-noise ratio was improved by averaging eight ordered frames for each optical section. During the scanning session, correction of attenuation of emitted and reflected light due to sample penetration was accomplished by gain compensation of the photomultiplier (voltage magnification). Neither fluorescence bleached appreciably even during long exposures (up to 15 min/50 optical sections).
Tridimensional Reconstruction
The stacks of confocal images were edited, processed, and reconstructed with the Edit3D software developed in our laboratory (
Processing
After collecting the confocal series, it was necessary to process these data to correct for image degradation inherent to confocal microscopy. Two main image artifacts were taken into account: (a) poor signal-vs-noise ratio (SNR) and (b) loss of fluorescence intensity as we penetrated deeper inside the thick tissue sections.
A 3D median filter was used to reduce the noise. This filter has the property of removing "salt and pepper" noise while preserving edge information in images. It uses a 3 x 3 x 3 window centered on the voxel (volume element) to be filtered, sorts the intensities of the voxels within the window, and returns the median value. This median intensity value is then substituted to the original value of the current voxel.
Although the biological samples are usually transparent they tend to absorb and scatter a small amount of light. In tissue sections in which thickness is greater than 5 µm, the absorption phenomenon is no longer negligible. On the one hand, the excitation light is gradually attenuated as a function of depth on the way in. On the other hand, the emitted light is also attenuated on the way out. This results in a real decrease in the signal intensity as a function of Z. The curve of attenuation of fluorescence intensity as a function of depth can be modeled by a log-logistic function (
with Z the current depth, Zmax the deepest section of the series, Iz the voxel intensity at current depth, and Iz' the new voxel intensity. K and a are empirically defined constants.
Reconstruction
Different visual attributes of the 3D reconstruction can be selected to best visualize specific details. According to the needed attributes (shape or content of the object), a surface or a transparency-rendering approach can be used. Surface rendering is obtained by simulating bundles of parallel rays of light traversing the pixels (elementary points) of the viewing screen of the computer. When one of these rays hits a surface voxel of an object (e.g., nerve fibers), the intensity of the corresponding screen pixel was modulated as a function of the cosine of the angle between the incident ray and the normal to the object surface at the hit spot. The voxels of the object were first obtained by applying a simple intensity threshold rule: All voxels whose intensity is greater than the given value are considered to belong to the object. The other voxels are labeled as background.
In contrast to the surface model in which only the surface voxels are considered, the volume model is intended to show all the information contained in the 3D cube. This display mode might be called "see-through" or transparent viewing. The method for representing transparent volumes is called "ray casting." It consists of dividing the viewing screen into small pixels. Then we consider that each pixel of the viewing screen receives a ray of light that originated from infinity and that traversed the 3D data volume. The intensity of a given ray is the result of the information encountered along its path. In other words, the light intensities of all the traversed voxels are accumulated and contribute to the brightness and color of the pixel displayed on the screen.
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Results |
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As deduced previously with conventional histological techniques, 3D reconstruction and double immunolabeling show the characteristic organization of Meissner corpuscles with a complicated interweaving of the helical nerve fiber winding within the stacks of lamellar cells, forming a spring-like framework (Figure 1A1C). Corpuscles are usually found alone or grouped in a dermal papilla, with their long axis vertically oriented (Figure 1A). Lamellar cells are highly immunoreactive for PS-100 and are well differentiated from the nerve component labeled by antibodies against NF 70-200 (Figure 1C1D and Figure 2A2B). The afferent nerve fiber can be followed along its extra- and intracorpuscular course. A single extracorpuscular nerve fiber can branch into several sensory endings (Figure 2A and Figure 2B). Each corpuscle is penetrated through its base by dividing nerve ramifications which run in an helical fashion (Figure 2C2D). The nerve fiber presents flat, discoidal, more or less fringed regions randomly situated along the nerve course, usually one per corpuscle (Figure 2E). These formations are not easily noticeable on single optical sections and are better appreciated after 3D reconstruction, particularly when the "nerve fluorescence channel" (NF 70-200) is observed alone (Figure 2C2D).
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On the other hand, labeled lamellar cells, which are arranged in stacks intercalated with the tortuous annulospiral nerve course, are more evident on a single optical section (Figure 1C) than in a 3D reconstruction, in which they have a more diffuse appearance (Figure 2A and Figure 2B). During tomography sessions, the use of only one laser beam (argon) that activates both FITC and TRITC labels provides no blurring of the image. In addition, appropriate object rotation provides vision from another angle which modifies and corrects image interpretation (Figure 2E). Apparent discontinuities are probably artifacts caused by incomplete labeling of the sample or by photonic reflection defaults rather than a real structural defect (Figure 2A and Figure 2B). Despite the quality of labeling that is sufficient to distinguish fibers with diameters of less than 0.8 µm, no extracorpuscular network has been identified in the vicinity of observed corpuscles (Figure 2A2D). Measurements whose mean and SEM refer only to the optical 3D resolution give a nerve diameter that varies from 2 to 5 µm (mean diameter 4 ± 1 µm), with size differences which are more easily appraised on a stereoscopic view (Figure 2C2D) whereas discoidal expansions have a mean diameter of 15 ± 1 µm (from 7 to 30 µm) (Figure 2E). Corpuscle size, measured from five samples (not shown), varies from 30 to 140 ± 1 µm in length and from 20 to 60 ± 1 µm in diameter.
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Discussion |
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This study shows that double immunolabeling of thick samples observed by confocal microscopy allows reliable imaging of Meissner corpuscles. Many LM and TEM studies have demonstrated their fine structure, but analysis of this sophisticated framework could remain difficult (
Our 3D reconstructions are consistent with the results of
According to the labeling technique used, consistent visual differences can exist and image interpretation may become a matter of debate (
In 1896, Timofeew described thin accessory fibers accompanying human Meissner corpuscles in dermal papillae (
In addition to the neural component, lamellar cells are another important part whose functional role is not yet completely understood. They are considered to be Schwann cell derivatives because they are labeled by specific Schwann cell markers (
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Acknowledgments |
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We wish to thank Dr F. LabatMoleur MD for technical assistance, Prof F. Moutet (Hand Surgery Unit at Grenoble University Hospital, France) who provided us with human samples, and Prof J. Carlsson for his translation contribution.
Received for publication June 4, 1999; accepted September 29, 1999.
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Literature Cited |
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Björklund H, Dalsgaard CJ, Jonsson CE, Hermasson A (1986) Sensory and autonomic innervation of non-hairy and hairy human skin. An immunohistochemical study. Cell Tissue Res 243:51-57[Medline]
Bolton CF, Winkelmann RK, Dyck PJ (1966) A quantitative study of Meissner's corpuscles in man. Neurology 16:1-9[Medline]
Breatnach AS (1977) Electron microscopy of cutaneous nerves and receptors. J Invest Dermatol 69:8-26[Abstract]
Carlsson K (1991) The influence of specimen refractive index, detector signal integration, and non-uniform scan speed on the images properties in confocal microscopy. J Microsc 163:167-178
Castano P (1974) Further observations on the Wagner-Meissner's corpuscle of man. An ultrastructural study. J Submicrosc Cytol 6:327-337
Castano P, Marcucci A, Miani A, Jr, Morini M, Veraldi S, Rumio C (1993) Central and peripheral nervous structures as seen at the confocal scanning laser microscope. J Microsc 175:229-237
Castano P, Rumio C, Morini M, Miani A, Jr, Castano SM (1995) Three-dimensional reconstruction of the Meissner's corpuscle of man after silver impregnation and immunofluorescence with PGP 9.5 antibodies using confocal scanning laser microscopy. J Anat 186:261-270[Medline]
Castano P, Ventura RG (1978) The Meissner's corpuscle of the green monkey. (Cercopithecus aetiops L.). I. The organization of nervous component. J Submicrose Cytol 10:327-344
Castano P, Ventura RG (1979) The Meissner's corpuscle of the green monkey (Cercopithecus aetiops L.). II. The connective tissue components. Some considerations from a functional standpoint. J Submicrose Cytol 11:185-191
Castano P, Ventura RG, Maddalone M (1985) Notes on morpho-functional differences between the Meissner's corpuscles of man and the green monkey (Cercopithecus aetiops L.). Folia Morphol 33:294-298
Castano P, Ventura RG, Pizzini G, Marcucci A, Morini M (1991) Unmyelinated nerve fibers associated with Meissner's corpuscle in the green monkey (Cercopithecus aetiops L.). Funct Dev Morphol 1:51-54[Medline]
Cauna N (1956a) Structure and origin of the capsule of Meissner's corpuscles. Anat Rec 124:77-92[Medline]
Cauna N (1956b) Nerve supply and nerve endings in Meissner's corpuscles. Am J Anat 99:315-327
Cauna N, Ross LL (1960) The fine structure of Meissner's touch corpuscles of human fingers. J Biophys Biochem Cytol 8:467-482
Cheng PC, Summers RG (1990) Image contrast in confocal light microscopy. In Pawley JB, ed. Handbook of Biological Confocal Microscopy. New York, Plenum Press, 179-195
Chouchkov ChN (1973) Further observations of the fine structure of Meissner's corpuscles in human digital skin and rectum. Z Mikrosk Anat Forsch 87:33-45[Medline]
Dalsgaard CJ, Björklund H, Jonsson CE, Hermasson A, Dahl D (1984) Distribution of neurofilament-immunoreactive fibers in human skin. Histochemistry 81:111-114[Medline]
Dubövy P, Rosario CM, Aldsögius H (1993) Combination of non-specific cholinesterase histochemistry and immunofluorescence staining for the study of the sensory innervation of skin and muscle. Histochem J 25:112-118[Medline]
Hachisuka H, Mori O, Sakamoto F, Sasai Y, Nomura H (1984) Immunohistochemical demonstration of S-100 protein in the cutaneous nervous system. Anat Rec 210:639-646[Medline]
Halata Z (1975) The mechanoreceptors of the mammalian skin. Ultrastructure and morphological classification. In Brodal A, Hild W, van Limborgh J, Ortmann R, Schlieber TH, Töndury G, Wolff E, eds. Advances in Anatomy, Embryology and Cell Biology. Vol 50. Fasc 5. Berlin, Springer-Verlag, 20-26
Haro JJ, Vega JA, Del Valle ME, Calzada B, Zaccheo D, Malinovsky L (1991) Immunohistochemical study of sensory nerve formations in human glabrous skin. Eur J Morphol 29:271-284[Medline]
Haschimoto K (1973) Fine structure of the Meissner's corpuscle of human palmar skin. J Invest Dermatol 60:20-28[Medline]
Idé C (1976) The fine structure of the digital corpuscle of the mouse toe pad, with special references to nerve fibers. Am J Anat 147:329-356[Medline]
Iggo A (1974) Cutaneous receptors. In Hubbard JI, ed. Peripheral Nervous System. New York, Plenum Press, 347-404
Iwanaga T, Fujita T, Takahashi Y, Nakajima T (1982) Meissner's and Pacinian corpuscles as studied by immunohistochemistry for S-100 protein, neuron-specific enolase and neurofilament protein. Neurosci Lett 31:117-121[Medline]
Karanth SS, Springall DR, Kuhn DM, Levene MM, Polak JM (1991) An immunohistochemical study of cutaneous innervation and the distribution of neuropeptides and protein gene product 9.5 in man and commonly employed laboratory animals. Am J Anat 191:369-383[Medline]
Kennedy WR, WendelschaferCrabb G (1993) The innervation of human epidermidis. J Neurol Sci 115:184-190[Medline]
Matsuoka S, Suzuki H, Morioka S, Ogawa Y, Kojima T (1983) Quantitative and qualitative studies of Meissner's corpuscles in human skin with special reference to alterations caused by aging. J Dermatol 10:205-216[Medline]
Mayhew TM (1992) A review of recent advances in stereology for quantifying neural structure. J Neurocytol 21:313-328[Medline]
Munger BL, Idé C (1988) The structure and function of cutaneous sensory receptors. Arch Histol Cytol 51:1-34[Medline]
Navarro X, Verdü E, WendelschaferCrabb G, Kennedy WR (1995) Innervation of cutaneous structures in the mouse hind paw: a confocal microscopy immunohistochemical study. J Neurosci Res 41:111-120[Medline]
Parazza F, Humbert C, Usson Y (1993) Method for 3D volumetric analysis of intranuclear fluorescence distribution in confocal microscopy. Comp Med Imag Graph 17:189-200[Medline]
Ramieri G, Stella M, Calcagni M, Teich-Alesia S, Cellino G, Panzica GC (1992) Morphology of corpuscular receptors in hairy and non-hairy human skin as visualized with an antiserum to protein gene product 9.5 compared to antineuron-specific enolase and anti S-100 protein. Acta Anat 144:343-347[Medline]
Rigaut JP, Vassy J (1991) High-resolution three-dimensional images from confocal scanning laser microscopy. Anal Quant Cytol Histol 13:223-232[Medline]
Ruffini A (1902) SullÄ apparato nervoso di Timofeew od apparato ultraterminale nei corpuscoli di Meissner della cute umana. Biblio Anat 11:267-281
Rumio C, Castano P, Veraldi S, Morini M, Castano M (1995a) The innervation of human skin studied with confocal scanning laser microscopy: a comparison between PGP 9.5 immunofluorescence and silver impregnation. Neuroimage 2:102-111[Medline]
Rumio C, Morini M, Miani A, Jr., Barajon I, Castano P (1995b) A simple method for overcoming some problems when observing thick reflective biological samples with a confocal scanning laser microscope. J Microsc 177:85-89[Medline]
Saxod R (1996) Ontogeny of the cutaneous sensory organs. Microsc Res Tech 34:313-333[Medline]
Schimirgk K, Rüttinger H (1980) The touch corpuscles of plantar surface of the big toe. Histological and histometrical investigations with respect to age. Eur Neurol 19:49-60[Medline]
Shotton MD (1989) Confocal scanning optical microscopy and its applications for biological specimens. J Cell Sci 94:175-206
Stefansson K, Wollman RL, Moore BW (1982) Distribution of S-100 protein outside the central nervous system. Brain Res 234:309-317[Medline]
Tachibana T, Nawa T, Mizuhira V, Yoshida Y (1992) Ultrastructural localization of calcium in mechanoreceptors of the oral mucosa. J Neurocytol 21:745-753[Medline]
Timofeew D (1896) Über eine besondere Art von eingekapselten Nervenendigungen in den männlichen Geschlechtsorganen bei Säugetieren. Anat Anz 11:44-49
Vega JA, Llamosas MM, Huerta JJ, GarcíaFernández JM (1996) Study of human cutaneous sensory corpuscles using double immunolabeling and confocal laser scanning microscopy. Anat Rec 246:557-560[Medline]
Ventura RG, Castano P (1977) Meissner's corpuscle of the green-monkey. Electron microscope three-dimensional reconstruction and quantitative analysis of the nervous supply. Acta Anat 99:312