Detection Of Proteoglycans Research Articles

QUANTITATIVE HISTOCHEMICAL STUDY ON THE POSTNATAL DEVELOPMENT OF PERINEURONAL NETS IN THE RETROSPLENIAL CORTEX OF ALBINO RATS

Reticular or lattice-like perineuronal coatings of condensed extracellular matrix (ECM), termed perineuronal nets (PNs), enriched in proteoglycans (PGs) and/or glycoproteins (GPs) were demonstrated to ensheath cell surfaces of certain neuronal circuits in the central nervous system (CNS) of mammals, reptiles and fishes. In the present study, the histochemical and quantitative characteristics of postnatal ECM in the retrosplenial cortex (RSC) of albino rats were described with a cationic iron colloid (CIC) or with CIC-Bodian's technique for detection of sulfated PGs and with certain plant lectins from Vicia villosa agglutinin (VVA), Wisteria floribunda agglutinin (WFA) or Glycine max agglutinin (SBA) for demonstration of N-acetylgalactosamine containing GPs. The differentiating PNs were shown to make their development as early as 1-2 weeks postnatally (Pw1-2), which surround the surface of some neurons distributing throughout the II-V cortical layers. During the first postnatal month and onwards, the net-encapsulated neuronal cells underwent progressive increment in number and presented an inside-out pattern of migration and differentiation throughout the V-II cortical layers. At a later stage (Pw6-8), the overall density and intensity of labeled PNs progressively increased and reached the adult stage of development. In addition, they displayed their differential labeling characteristics for the lectin/CIC double staining, which were differentially identified into three types, namely; PGs-, GPs-, and PGs/GPs coats. The morphometric investigation revealed that the total density of net-associated retrosplenial neurons (RSNs) at Pw2 was about 1.80±0.29 /UA (UA = 60.15 µm2) after labeling with the WFA/CIC double staining technique, and this variant increased polynomially with progression of postnatal age reaching 9.12±1.82 /UA at Pw12. Quantitative analysis showed that the gain of net-associated RSNs increased rapidly during the first postnatal month (2.86 fold), and then increased 1.73 fold during the second month, but increased 1.02 fold at the third month. Statistical analysis revealed that throughout the postnatal period, the numerical density of net-associated RSNs increased significantly with advance of age (R = +0.992; P<0.01). In addition, the numerical densities of PGs- and GPs-coated neurons, as well as those exhibiting coat complexes (PGs/GPs coats), increased polynomially with progression of animal age (R = +0.996, +0.990 and +0.976, respectively with P < 0.01 in all parameters). At Pw2, the absolute numerical density of RSNs labeled with VVA, WFA, SBA, CIC and CIC-Bodian's staining measured about 2.12±0.17, 1.80±0.13, 0.00, 0.00 and 3.00±0.18 /UA, respectively. These parameters increased significantly with postnatal age and respectively reached 6.18±1.23, 5.00±1.03, 2.90±0.69, 3.85±0.76 and 4.30±1.13 /UA at Pw12 (R = +0.995, +0.995, +0.997, +0.996 and 0.984 with P<0.01 for all parameters). In addition, labeling indices of RSNs stained with the aforementioned staining at Pw2 was 3.60, 2.94, 0.00, 0.00 and 3.52, respectively. These variants increased significantly with advance of animal age reaching 15.84, 12.82, 7.43, 9.87 and 11.02, respectively (R = +0.994, +0.997, +0.998, +0.996 and +0.999 and P< 0.01 for all variants). This finding indicated that the perineuronal ECM components are significantly correlated with age and suggest a possible developmental and biological significance including promotion of migration and functional maturation of net-associated neurons in the RSC. In addition, the finding of present study would be contributed as baseline for future experimental or environmental studies that are expandable for other mammalian species including the human.

Detection of proteoglycans in the hairless rat skin after UVB irradiation

Detection of proteoglycans in the hairless rat skin after UVB irradiation

Detection and quantitation of proteoglycans extracted from cell culture medium and cultured cartilage slices

Detection and quantitation of extracted proteoglycans, by staining with the dye Alcian blue on cellulose acetate followed by dissolution of the stained cellulose acetate strips in dimethyl sulfoxide containing 0.5% ( v v ) sulfuric acid for absorbance measurement, is described. It is shown that, in the present system, the dye uptake by the proteoglycan is dependant only on the glycosaminoglycan content of the proteoglycan. The method is applied to the quantitation and characterization of proteoglycans and glycosaminoglycans, which have been extracted from radiolabeled bovine ankle cartilage and from mononuclear cell supernatant and which have been separated by DEAE-Sephacel column chromatography. The high sensitivity of the method allows detection of proteoglycans in 25-μl samples of solutions containing as little as 1 μg of glycosaminoglycan per milliliter of solution.

Densitometric assay of nanogram quantities of proteoglycans precipitated on nitrocellulose membrane with Safranin O

Proteoglycan (PG) and glycosaminoglycan (GAG) samples corresponding to a minimum of 10 ng of uronic acid were reliably quantified as precipitates with the cationic dye Safranin O, collected by vacuum-aided filtration onto a cellulose acetate/nitrate membrane in a standard 96-well dot assay apparatus. The reflectances of the precipitation dots were measured by automatic densitometric scanning of the membrane sheets. Standard GAGs produced reflectance values which were related to the number of anionic groups per unit disaccharide; hyaluronate and keratan sulfate gave lower values while heparin yielded values higher than those of chondroitin sulfates. The presence of 8 m urea, 1% Triton X-100, 30% sucrose, 0.02% NaN 3, or mixtures of proteinase inhibitors and various buffers did not markedly influence the reflectances, while 4 m guanidinium chloride and 3 m CsCl reduced the sensitivity of the assay to 30–50 ng. Samples containing sodium dodecyl sulfate (SDS) were not applicable because SDS precipitated with Safranin O. Proteins showed virtually no response, while nucleic acids gave significant although smaller reflectances than GAGs. Owing to its marked sensitivity and convenience the method is particularly suitable for the detection of PGs during their preparative purification and fractionation as well as in various analytical assays.