Neuroscience Vol. 48, No. 3, Printed in Great Britain

0306-4522/92 SW0 + 0.00 F’ergamon Press Ltd 0 1992 IBRO

pp. 137-744,1992




*Institute of Anatomy, University of Bern, 3iihlstrasse 26, 3012 Bern, Switzerland fInstitute of Biochemistry and Molecular Biology, University of Bern, Biihlstrasse 28, 3012 Bern, Switzerland Abstract-Neural tissue of central (rat spinal cord) and peripheral origin (rat sciatic nerve, nerve fascicles of rat skin and iris and of human conjunctiva) was processed by osmium tetroxide/microwave fixation and embedded in epoxy resin. Hyaluronan-binding proteins and link proteins coupled to 15-20-nm gold particles were used as markers in a one-step post-embedding procedure for identifying hyaluronan (hyaluronic acid) at the ultrastmctural level. All my&in sheaths in both rat and human material were found to be intensely labelled. The specificity of the hy~~onan-bin~ng probes was demonstrated by the total loss of labelling following treatment of sections with hyaluronidase or by preincubating either the probes with hyaluronan oligosaccharides or the sections with unlabelled hyaluronan-binding protein. The identified hyaluronan appears to be located extracellularly, but its precise role here remains to be elucidated.


Hyaluronan (hyaluronic acid) is a glycosaminoglycan composed of simple disaccharide units (D-glucuronic acid and ~-a~tyl-D-glu~sa~ne). Its molecular structure is well documented,“*37 but many aspects of its biological role still remain to be elucidated. Hyaluronan is almost universal in its distribution amongst tissues, in which it is located predominantly in the extraceilular space; ?.lL15,22,24,2B,32,33.38,W its intra_ cellular


has, however,

Wistar rat and hnman tissue was lixed (for 2 min at ambient temperature) in 2% (v/v) osmium tetroxide solution (in 0.2 M sodium cacodylate buffer, pH 7.2, 450 mOsm). Glass vials (24 x 40 mm) containing the tissue and 10ml of fixative solution were placed in the centre of a conventional microwave oven and heated for a few seconds at maximal power output (65OW) and at a frequency of 2450 MHz; temperatures between 43 and 46°C

also been demon-

were attained.*J3Samples were maintained in the fixative at ambient temperature for 10min prior to washing in 0.1 M


In mammalian brain extracts, hyaluronan represents a major fraction of the total glycosaminoglycan content,‘.** and the biotinylated hyaluronanbinding region, in conjunction with avidin-peroxidase, has been used as a specific probe3’ to study the dist~bution of hyaluronan in the central nervous system. In newborn rats, the extracellular matrix was intensely stained, this distribution shifting to a predominantly intracellular location during brain development.j3 However, precise localization of molecules was sometimes hampered by the relatively large size of the diaminobenzidine reaction product.5 In order to improve the precision of hyaluronan localization in central and peripheral neural tissue, we directly coupled the hyaluronan-binding proteini and the link proteinI (both of which were isolated from bovine nasal cartilage by extraction with 4 M guanidinium chloride@) to gold particles, 15-20nm in diameter. ~To whom correspondence should he addressed at: Institute of Anatomy, University of Bern, P.O. Box, CH-3000 Bern 9, Switzerland. ~~~r#~a~~ons: EDTA, ethylen~iaminetetra-a~tate; HABP, hyaluronan-binding protein; LP, link protein.


Tissue processing

sodium cacodylate buffer. They were then dehydrated in a graded series of increasing ethanol concentration, and embedded in epoxy resin (Epon 812). Preparation of h~aluronan-bjnding protein-gold and link ~rotein~oid complexes Hyaluronan-binding protein (I-IABP) and link protein (LP) were prepared from bovine nasal cartilage as described by Tengblad:” following extraction of proteoglycans with 4 M guanidinium chloride, the hyaIuronan-bin~ng proteins were isolated by affinity chromatogmphy and further purified by gel chromatography. Colloidal gold particles, IS-20 nm in diameter, were prepared according to the method of Frens? The minimal concentrations of the respective proteins required to stabilize the gold soli6 at pH 7.0 were 7 and 12 fig/ml for HABP and LP, respectively. The gold complexes were prepared by adding aqueous solutions of either HABP (2 ml, 70 tin/ml) or LP (1 .j ml, 160 rg/ml) to the colloidal gold ‘sol i20 rni, pH7.0). A 2% (w/v) aqueous solution of bovine serum albumin (2Om1, fraction V, Sigma) was added after 5min, and the complexes centrifuged at 30,000 g for 45 min at 4°C (Kontron H 401, fixed angle rotor, Kontron A 8.24, Kontron, Switzerland). The loose sediments were separated from the compacted pellets (which were discarded), suspended in distilled water and re-centrifuged as described above. Thev were then resuspended in phosphate-buffered saline (I 2;



1, Myelinated

nerve fibres of rat spinal cord labelled with either HABP-gold (a,~) or LP--gold complexes. a, x 8000; b, x 8000: c, x 49,500: d, x 7 I SW.


Fig. 2. Myelinated nerve fibres of rat sciatic nerve (a,b), skin (c) and iris (d) labelled with either HABP-gold (a 10”) per gold particle may be estimated to be 75 and 2, respectively.‘” Cytochemical labeiling

Ultrathin sections were preincubated for 10 min by floating on 1% (w/v) bovine serum albumin in phosphatebuffered saline to block nonspecific staining and then incubated on an undiluted drop of either HABP-gold or LP-gold for 2 h at ambient temperature. They were subsequently washed by floating initially on phosphate-buffered saline (six changes) and then on distilled water (four changes), and finally dried. Sections were stained with uranyl acetate and lead citrate and examined in a Philips EM 400 electron microscope. Cytochemical controls of .~peciJicity’ Digestion with Streptomyces hyaluronidase.6~‘2~2U Ultrathin

sections were digested for 4 h at 37°C in a humidified chamber with 100 TRU/ml Streptomyces hyaluronidase (Calbiochem) in 50mM sodium acetate buffer, pH 5.0, containing 1 mM iodoacetic acid (Calbiochem), 1 mM EDTA (Sigma), 1 mM phenylmethyl sulphonylfluoride (Calbiochem), 250 ng/ml ovomucoid (Sigma) and 1 pg/ml pepstatin A (Calbiochem) as protease inhibitors.29 Digestion with testicular hyaluronidase.‘2~4’ Ultrathin sections were digested for 4 h at 37°C in a humidified chamber with 10 mg/ml testicular hyaluronidase (Fluka) in Soerensen’s phosphate buffer (0.1 M, pH 5.66). All digestion experiments included controls (absence of the respective enzymes) incubated under similar conditions. Blocking of labellingwith hyaluronan oligosaccharides.i2 A 2% (w/v) solution of rooster comb hyaluronan (Sigma) in Soerensen’s phosphate buffer (0.1 M in 0.15 M NaCl. pH 5.0) was digested with 4 mg/ml testicular hyaluronidase (Fluka, Switzerland) for 24 h at 37°C to obtain hyaluronan oligosaccharides, and the mixture heated in a boiling water bath for 15 min to inactivate and precipitate the enzyme.34 The oligosaccharides were combined (1: 1)with HABP-gold or LP-gold for 2 h at ambient temperature prior to incubation of sections. Blocking of tabelling by preincubation of sections with unlabelled hyaluronan -binding protein. Sections were prein-

cubated with HABP (70 ng/ml) for 2 h at ambient temperature, washed, and labelled with HABP-gold as described above. RESULTS

Hyaluronan-binding gold complex


and link protein-


HABP-gold complexes suspended in physiological buffers were stable for an indefinite period of time, whereas the LP-gold complexes precipitated within a

When resuspended in distilled water, the latter remained stable for several weeks. After labelling of sections for a period of 2 h with the respective hyaluronan-binding probes, HABP-gold complexes were resolved predominantly as single-particle entities, and occasionally as small aggregates (Fig. la,c), whereas LP-gold complexes tended to form large clumps (Fig. lb,d), the size of which increased as a function of incubation time. The labelling specificity of each probe for myelin sheaths (as well as for other intra- and extracellular compartments) was, however, identical. few hours.

Localization human neural


in myelin sheaths ofrat and


The myelin sheaths in neural tissue of both central (rat spinal cord) and peripheral origin (rat sciatic nerve, skin and iris; human conjunctiva) exhibited intense labelling with HABP- (Figs la,c, 2a-c, 3a,b) and LP-gold complexes (Figs 1b,d, 2d, 4) on ultrathin sections derived from tissue processed by osmium tetroxide/microwave fixation and embedded in epoxy resin. The axons of myelinated and unmyelinated nerve fibres, the cytoplasm of central and peripheral glial cells, in general, and particularly at the node of Ranvier (Fig. 4) and at the myelin clefts of Schmidt-Lanterman (Fig. 3b), and the extracellular matrix between nerve fibres, were only occasionally labelled, and then very weakly. At high magnifications, labelling of the periaxonal space was revealed (Fig. 2b). Controls

Digestion of hyaluronan with Streptomyces hyaluronidase, in the presence of protease inhibitors (Fig. 5a) or with testicular hyaluronidase, completely inhibited labelling of myelin sheaths, as did preincubation of sections with unlabelled HABP followed by addition of the HABP-gold complex (Fig. 5c), or preincubation of the gold complexes with hyaluronan oligosaccharides (Fig. 5b).


The hyaluronan-binding regions of LP, a hydrophobic glycoprotein,27,36 and HABP, a large, highly hydrophilic proteoglycan (also termed aggrecan4’.42),

Fig. 3. Myelinated nerve fibre of human conjunctiva labelled with HABP-gold complexes. Labelling intensity over the myelin clefts of Schmidt-Lanterman (SL in b) is significantly lower than that associated with the myelin sheath. a, x 24,500; b, x 38,200. Fig. 4. Rat sciatic nerve labelled with LP-gold complexes. Labelhng is seldom observed around the axon at the node of Ranvier. P, cytoplasmic processes of Schwann’s cells. x 11,500. Fig. 5. Cytochemical controls for specificity of HABP-gold and LP-gold probes for hyaluronan. (a) Section of rat sciatic nerve digested with Streptomyces hyaluronidase prior to incubation with HABP-gold. (b) Appearance of rat sciatic nerve (unstained section) after application of an LP-gold complex preincubated with hyaluronan oligosaccharides. (c) Section of rat spinal cord (unstained) preincubated with uncomplexed HABP before treatment with HABP-gold. In all controls, labelhng with gold particles is sparse (arrowheads). a, x 11,700; b, x 12,800; c, x 31,300.

Hyaluronan in myelin sheaths

Figs 3-S.




Ultrastructural localization of hyaluronan in myelin sheaths of the rat central and rat and human peripheral nervous systems using hyaluronan-binding protein-gold and link protein-gold.

Neural tissue of central (rat spinal cord) and peripheral origin (rat sciatic nerve, nerve fascicles of rat skin and iris and of human conjunctiva) wa...
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