Keratanase disrupts the lateral organization of MFs in hippocampal slices
| Treatment | (ross section (μm) | Significance |
|---|---|---|
| HBSS control (n = 24) | 99.25 ± 8.49 | |
| ABC (n = 5) | 104 ± 22.47 | p < 0.977 |
| ABC + K1 + K2 (n = 20) | 319.35 ± 24.19 | *p < 0.001 |
| K1 + K2 (n = 3) | 341.67 ± 40.99 | *p < 0.006 |
| K1 (n = 5) | 303 ± 24.54 | *p < 0.001 |
| K2 (n = 4) | 221 ± 46.19 | *p < 0.008 |
| Total keratanase (n = 32) | 306.59 ± 17.57 | *p < 0.001 |
The regeneration of MFs was analyzed in cultured rat hippocampal slices that were treated with chondroitin ABC lyase (ABC), keratanase 1 (K1), or keratanase 2 (K2) as described in Materials and Methods. Regenerating MFs normally form a narrow band with a cross section that is ∼100 μm wide. MFs were stained using Micro Ruby injections (see Materials and Methods), and fluorescent images were obtained using a Spot Digital Camera. The line tool in the Spot Camera software was calibrated using an image of a micrometer, and a line was drawn across the width of the regenerated MF pathway. Cultures treated with either K1 or K2 alone or in combination with ABC or each other produced a statistically significant increase in the cross section of regenerated MFs compared with MFs in control cultures treated with HBSS. However, ABC treatment alone produced no significant increase in cross section versus HBSS control. These results demonstrate that MFs in keratanase-treated slices regenerated in a highly disorganized manner. Mann—Whitney rank sum statistical analysis was performed using SigmaStat 3.0 software. Asterisks mark enzyme treatments that are significantly different from HBSS control.