Fig. 4. Antisense oligos to MAPs disrupt the orderly spacing of the ChAT-positive cells and scatter these cells at different retinal depths. (A) Delaunay segments link array cells (dots) with their surrounding neighbours. (B) Examples of the altered distributions of Delaunay segments (DS) associated to the GCL ChAT-positive array 24 hours after treatment with MAP2 phosphorothiated antisense oligos (black circles, 25 µM PPRM21; black squares, 25 µM PPRM38), or with MAP2 antisense with phosphorothioate links only at the 3' and 5' ends (continuous line indicates 50 µM ppRM21). The DS associated with normal and vehicle injected cases are shown as grey lines. Each curve is the average DS distribution of a single retina: as shown, the extent of the oligo effect varies significantly between treated retinas, but it always includes DS lengths outside the normal range. Each treated DS plot is significantly different from the normal DS histogram (KS test; P<0.0001). The difference between the treated and the normal cases was confirmed by the bootstrap method, which allows the comparison of datasets, taking into consideration their internal variability (see Materials and Methods). (C) Examples showing that the DS distribution of the GCL ChAT-positive array did not vary significantly with: (1) the phosphorothioate sense sequences complementary to the antisense oligos used (black circles, 25 µM RM21 sense; black squares, 25 µM RM11 sense); (2) blocking antibodies to ßFGF (white circles, 10 µg/ml), used to simulate potential nonspecific effects of PP oligos. Normal DS distributions are grey lines. Each treated DS plot was not significantly different from the normal DS histogram (KS test P<0.01). This result was confirmed by bootstrap analysis (see Materials and Methods). (D) Examples of the alteration of the DS distribution after treatment with a phosphorothioate antisense to tau (PPRT11: upward facing triangles, 12 µM; downward facing triangles, 25 µM). Normal DS are the grey curves. Each treated DS plot is significantly different from the normal DS histogram (KS test; P<0.0001). The difference between the treated and the normal cases was confirmed by the bootstrap method, which allows the comparison of datasets, taking into consideration their internal variability (see Materials and Methods). (E) Schematic representation of array cells at different retinal depths. Dots represent cells, the line an arbitrary reference depth. (F-H) Examples of the scatter of the GCL ChAT-positive cells at different retinal depths after treatment with MAP2 antisense (F), control treatments (G) and tau antisense (H). Each curve represents data from a single retina (see above). Symbols are as in B-D. The treated cases are illustrated upside-down to facilitate comparison with the normal controls (grey curve). The statistical significance of the effects was assessed by means of the bootstrap method and the K-S test (P<0.001). Between 5 and 20% of each retina was sampled for this analysis.

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