Table 1.
Antibodies and Characterizations.
Fig 1.
Distribution of proteoglycans relative to laminin in normal rat and rabbit peripheral nerve.
Normal rat (A and B) and rabbit (C,D,E,F) sciatic nerve was subjected to immunohistochemical analysis with antibodies against laminin-2 (A), proteoglycan core proteins (B,C,D) and glycosaminoglycan chains (E and F). (A) Laminin was found within the nerve endoneurium and perineurium while only present within the basement membranes of blood vessels in the epineurium. (B) The heparan sulfate/chondroitin sulfate proteoglycan, perlecan, colocalized with laminin. (C) The chondroitin sulfate/dermatan sulfate proteoglycan, decorin, appeared primarily within the perineurium with faint immunoreactivity found amongst a subset of endoneurial tubes. (D) Versican (12C5) had a similar distribution to decorin primarily within the perineurium and faintly dispersed amongst a subset of endoneurial tubes. (E) A commonly used marker for chondroitin sulfate glycosaminoglycans, antibody CS-56, was mostly found within the endoneurium and appeared to label some components of the epineurium. (F) 473-HD, which has been reported to be specific for chondroitin sulfate/dermatan sulfate hybrid glycosaminoglycan chains, produced a similar staining pattern as decorin and versican. Scale bar: 100 μm.
Fig 2.
Endoneurial distribution of ΔCS-A and ΔCS-C relative to laminin in normal rat peripheral nerve.
Normal rat sciatic nerve was subjected to chondroitinase ABC treatment and double immunolabeled with antibodies against laminin-1 and either ΔCS-A (C4S antibody) or ΔCS-C (C6S antibody). (A) C4S immunolabeling was present within the endoneurium but appeared to be isolated to the outside of the endoneurial tubes and seemed to be absent from the perineurium. (B) Immunolabeling for laminin revealed ring-like structures within the endoneurium that represent the boundaries of basal lamina tubes. Laminin was also present within the perineurium. (C) Combining both (A) and (B) provided a complementary pattern where the C4S (Red) immunoreactive areas were distinct and separate from the laminin (Green) immunoreactive rings of the basal lamina tubes. (D) C6S immunolabeling revealed a pattern that more resembled the ring-like laminin structures of the basal lamina (E). (F) Combining both (D) and (E) confirms that both components colocalize to the basal lamina tubes of the endoneurium. Scale bar: 20 μm.
Fig 3.
Perlecan distribution relative to laminin, ΔCS-A, and ΔCS-C in normal rat peripheral nerve.
Normal rat sciatic nerve was subjected to chondroitinase ABC treatment and immunolabeled with antibodies against perlecan and laminin or ΔCS-C or ΔCS-A. (A) Perlecan was present within the endoneurium tubes and appeared to label the ring-like structures in a similar pattern to laminin (B). (C) Combination of filters revealed that both laminin and perlecan closely associate within the basal lamina tubes. (D) Perlecan also closely associated with ΔCS-C tubes (E) which was confirmed when filters were merged (F). (G) Perlecan did not colocalize with ΔCS-A (H) within the endoneurium and the combination of both filters clearly demonstrated a complementary distribution (I). Scale bar: 20 μm.
Fig 4.
Optimum conditions for chondroitinase C treatment of nerve graft.
Three-cm segments of rabbit peroneal nerve were subjected to freeze/thaw treatment and incubated in various concentrations of chondroitinase C at different time durations (1U/ml 24hours A and B; 4U/ml 16hours C and D; 4U/ml 24hours E and F). Samples were taken from the middle of the nerve segment and subjected to immunocytochemical analysis to determine which concentration/time duration revealed the C6S neoepitope (A, C, E) and completely removed the CS-56 immunoreactive glycosaminoglycan chains (B, D, F). (A) 1U/ml for 24 hours revealed the C6S neoepitope throughout the endoneurial tissue. (B) Serial tissue sections indicated that concentration was not effective at removing the CS-56 immunoreactive glycosaminoglycan chains. (C and D) 4U/ml for 16 hours incubation did not completely remove the CS-56 immunoreactive chains either. (E and F) 4 U/ml for 24 hours appeared to effectively remove the CS-56 immunoreactivity from within the endoneurial tissue. Scale bar: 100 μm.
Fig 5.
Chondroitinase C treatment on normal peripheral nerve reveals ΔCS-C only.
Normal rabbit nerves were subjected to chondroitinase C treatment only (A and C) or by an additional treatment of chondroitinase ABC (B and D). Tissue was then labeled with ΔCS-C (A and B) or ΔCS-A (C and D) antibodies (C6S or C6S respectively). (A) C6S was present exclusively within the endoneurium of chondroitinase C treated nerve. (B) A secondary chondroitinase ABC treatment did not reveal any additional C6S immunoreactivity indicating that chondroitinase C effectively degraded the CS-C glycosaminoglycan chains. (C) Chondroitinase C treatment did not create ΔCS-A immunoreactivity, which provided evidence of substrate specificity. (D) A secondary chondroitinase ABC treatment did reveal the C4S neoepitope confirming that chondroitinase C is selective for CS-C glycosaminoglycan chains. Scale bar: 100μm.
Fig 6.
Chondroitinase C effectively removed endoneurial chondroitin sulfate glycosaminoglycans.
Normal human (A and C) and normal rabbit (B and D) were treated with chondroitinase C (C and D) and immunolabeled for CS-56 (A and C) or 473-HD (B and D). (A) CS-56 was present throughout the normal human nerve endoneurium. (B) 473-HD immunolabeled a subset of endoneurial basal laminae tubes within the rabbit nerve. Chondroitinase C treatment effectively removed immunoreactivity of both CS-56 (C) and 473-HD (D) antibodies which indicated the glycosaminoglycan chains were cleaved off the core protein. Scale bar: 100μm.
Table 2.
Cryoculture results.
Fig 7.
Cryoculture demonstrates differential functional effects from chondroitinase ABC and C treated nerve tissue.
Normal human nerve was tranversely sectioned and treated with vehicle control (A and A’), ChABC (B and B’), or chondroitinase C (C and C’) and subjected to cryoculture bioassay. Cultures were double immunolabeled for Gap43 (A, B, C) and laminin (A’, B’,C’). (A) Vehicle treated tissue sections provided a poor substrate for neurite extention either within or outside the nerve fascicles. (B) ChABC treated tissue significantly enhanced the ability for neurite extension and arborization within the nerve fascicles. Furthermore, neurites readily cross the perineurial and epineurial tissue to extend neurites from one fascicle to another (B arrow). (C) ChC treatment moderately improved neuritic growth throughout the endoneurium. Interestingly, neurites did not cross the perineurium surrounding the fascicles (C arrows) indicating that ChC selectively removed endoneurial inhibitory proteoglycan glycosaminoglycan chains while retaining the inhibitory properties of the perineurium and epineurium. (A’, B’, C’) Laminin immunolabeled tissue demarkated nerve fascicles. Scale bar: 100μm.
Fig 8.
SDS-PAGE analysis of normal rabbit nerve proteoglycan extraction with Alcian Blue and Coomassie Blue.
The proteoglycan content of 40 normal rabbit sciatic nerves were extracted and isolated through DAEA column chromatography. Samples were treated with vehicle buffer (V), chondroitinase C (C), or chondroitinase ABC (ABC) and subjected to 4–15% SDS-PAGE separation. Gels were then stained for Alcian Blue or Coomassie Blue to label glycosaminoglycans or protein content respectively. Vehicle treated samples resulted in a broad band when stained with Alcian Blue which indicated the presence of glycosaminoglycan chains of various molecular masses including three bands at the top of the gel interface, a broad band corresponding to Mr = 300-450kD, and band between 100 and 130kD. Coomassie Blue staining of the corresponding column also visualized several broad bands at lower molecular weight including several strong bands between Mr = 100-130kD, 65kD, and 45-55kD. Chondroitinase C treated samples removed the Alcian Blue stained bands at the gel interface but retained the bands between Mr = 300-450kD and 100-130kD. Coomassie Blue staining provided evidence of increased intensity of the Mr = 65kD and 45-55kD band. Chondroitinase ABC treated samples were devoid of Alcain Blue staining which indicated the absence of glycosaminoglycans. Coomassie Blue staining showed a reduction in staining intensity for the Mr = 100-130kD and 65kD band while the 45-55kD band shifted downward to aproximately 35-45kD.
Fig 9.
Western analysis of neoepitope immunolabeling of enzyme or vehicle treated proteoglycan extract.
Proteoglycan enriched nerve extract (PENE) was obtained as described in Methods and Materials and were pretreated with vehicle buffer (V), chondroitinase C (C), or chondroitinase ABC (ABC) prior to 4–15% SDS-PAGE separation and transferred to nitrocellulose for western analysis with neoepitope antibodies. Vehicle treated samples did not reveal any immunoreactivity as expected. Chondroitinase C and chondroitinase ABC treated PENE produced differential immunoreactive band indicating selective degradation of glycosaminoglycans and the production of ΔCS-A and ΔCS-C. Surprisingly, Chondroitinase C treated PENE was immunoreactive for C4S producing three distinct bands of high molecular mass and a broad band at Mr = 45-55kD. Chondroitinase ABC eliminated the high molecular weight C4S bands, created a doublet at Mr = 100kD, and several lower mass bands including a very intense band at approximately 45kD. C6S labeling produced several bands for chondroitinase C treated PENE. Chondroitinase ABC treated PENE produced several identical C6S immunoreactive bands as observed in chondroitinase C treated PENE with a few exceptions including the elimination of the 75kD band and the creation of a 30kD band.
Fig 10.
Identification of proteoglycan core proteins and glycosaminoglycan chains in western analysis of enzyme or vehicle treated proteoglycan extract.
Proteoglycan enriched nerve extract (PENE) was obtained as described in Methods and Materials. Western analysis was performed using core protein antibodies against decorin and perlecan as well as the glycosaminoglycan antibodies CS-56 and 473-HD. Vehicle (V) treated PENE provided various immunoreactive bands of different molecular weight against CS-56 and 473-HD. Chondroitinase C (C) treatment eliminated all the higher molecular weight bands of CS-56 but retained a CS-56 immunoreactive doublet at Mr = 60-65kD and a faint band at 45kD that was observed in the vehicle treated PENE. Meanwhile, chondroitinase ABC (ABC) treatment completely eliminated CS-56 immunoreactivity from the PENE. Decorin appeared as a very broad band between Mr = 140-350kD and an isolated 60kD band in the vehicle treated sample. Chondroitinase C treated PENE did not provide remarkable changes in the decorin immunoreactive bands but chondroitinase ABC modified the immunoreactivity slightly by creating a few bands at 90kD and 70KD while eliminating the 60kD band. 473-HD immunoreactivity was observed in several bands at 300kD, 90kD, 55kD, and at the gel interface of the vehicle treated PENE while chondroitinase C treatment reduced the intensity of the higher weight bands but retained the 90kD and 55kD bands. Chondroitinase ABC treated PENE did not contain any 473-HD immunoreactivity. Perlecan antibody bound to several bands at Mr = 250kD, 165kD, and 155kD as well as a strong band at 90kD and a weak band at 55kD in the vehicle treated PENE. Chondroitinase C treated PENE increased the intensity of perlecan immunoreactivity in the Mr = 250kD band while reducing the intensity of the 165kD and 155kD but retained the 90kD and 55kD band. Interestingly, chondroitinase ABC treatment did not modify the higher molecular weight bands of perlecan but did remove the 90kD and 55kD bands.