Article Figures & Data
Figures
- Figure 1.
Experiment overview and timeline for the main proof-of-concept experiment. Boxes indicate manipulations during the different time periods on the timeline.
- Figure 2.
Staircase-reaching task with color pellets. The images illustrate a fully baited staircase viewed from above (A) and a view of a staircase after testing showing remaining food pellets (B). Lower stairs with two or more colors indicate pellets from higher stairs that were dropped. Scale bar, 2 mm.
- Figure 3.
Suppression of PTEN expression and increased ribosomal protein S6 phosphorylation after intracortical injection of AAVshPTEN in adult Sprague Dawley rats. Shown are representative images of cortical sections from rats 3 weeks after a single injection of 109 GC of AAVshPTEN (A–C) or AAVshLuc (D–F). The sections in each set are from the same animal and are ∼20–60 μm apart. A, D, White boxes, ZsGreen reporter expression; A, Inset, high magnification of ZsGreen expression with ZsGreen-positive neurons. B, E, G, PTEN immunostaining. C, F, H, Immunostaining for the phosphorylated form of pS6. G, H, High-magnification views of the boxed areas in B and C, respectively. In G, red triangle indicates a PTEN-positive motor neuron; black arrows, motor neurons with visible H&E-stained nuclei but undetectable PTEN expression. In H, black arrows indicate pS6-immunostained neurons. Scale bars: A, D, 1 mm; A, inset, 25 μm; B, C, E, F, 0.5 mm; G, H, 50 μm.
- Figure 4.
Suppression of PTEN in the sensorimotor cortex of adult rats 3 weeks after 5 injections of AAVshPTEN. A–C, Panels illustrate coronal sections at different rostrocaudal levels through the somatosensory cortex of rats that received AAVshPTEN. The area of PTEN suppression is outlined in black and the approximate coordinates relative to bregma are indicated. D, Area of diminished PTEN suppression, the posterior boundary between the area in which PTEN is suppressed and nearby areas with normal PTEN expression. Scale bars, 0.5 mm.
- Figure 5.
PTEN suppression is maintained for 15 weeks after multiple AAVshPTEN cortical injections. A, B, DAPI-stained coronal cortical section. A, C, E, Uninjected hemisphere. B, D, F, Injected hemisphere from the same rat. C, D, PTEN immunofluorescence in the same sections as in A, B. E, F, Merged image of A, C, B, and D, respectively; G, High-magnification view of ZsGreen fluorescence from the same animal in A. H, High-magnification view of NeuN immunofluorescence in the area of PTEN deletion (same section as in G). I, Merged image of G and H; white arrows indicate cells that are positive for both ZsGreen and NeuN. Scale bars: A–F, 1 mm; G–I, 100 μm.
- Figure 6.
Preinjury performances of groups in the staircase-reaching task. Presurgery success rates at different steps for the “preferred” (A) and “nonpreferred” (B) paw. Steps are numbered 1–6, with 1 being the highest step nearest the platform. Values are group means ± SE. Green circles indicate AAVshLuc (n = 9); blue circles, AAVshLuc/fibrin (n = 9); yellow circles, AAVshPTEN (n = 9); red circles, AAVshPTEN/fibrin (n = 11).
- Figure 7.
Rats that received AAVshPTEN and salmon fibrin exhibited greater recovery of forepaw function after cervical SCI. Format of graphs and group numbers are as in Figure 6. Values are group means ± SE. A, B, Graphs illustrating the mean percentage success over time for the CL paw versus the IL paw to the vector injections for different experimental groups. Asterisk in A indicates significant differences between the AAVshPTEN/fibrin group versus other groups (one-way ANOVA with Bonferroni's correction, p < 0.0001). Asterisks in B indicate significant differences between the AAVshPTEN/fibrin group versus other groups as follows: 38 and 55–57 dpi, p = 0.001; 50 dpi, p = 0.002; 62–64 dpi, p = 0.002; 69 dpi, p = 0.004; and 71 dpi, p < 0.0001. C, D, Mean percentage success for different steps (1–6) from all dpi for CL (C) and IL (D) paws. Asterisks in C and D indicate significant differences between the AAVshPTEN/fibrin group versus other groups (p < 0.0001). D, Daggers indicate significant differences between AAVshLuc/fibrin and AAVshPTEN groups on steps 1–4 (p < 0.0001) and step 5, (p = 0.003); carets indicate significant differences between the AAVshLuc/fibrin and AAVshLuc groups on step 1 (p = 0.004), steps 2–3 (p < 0.0001), step 4 (p = 0.001), and step 5 (p = 0.007).
- Figure 8.
Larger numbers of BDA-labeled axons at the edge of the SCI lesion in rats treated with AAVshPTEN and salmon fibrin. A–C, Sagittal sections illustrating BDA-labeled axons near the lesion sites in the different groups. Lesion cavity is outlined in white and dotted vertical lines denote the rostral edge of the lesion, which is distance 0 for quantitative analysis in F. For all panels, the dorsal edge (D) is at the top and the rostral edge (R) is to the left. D, Enlargement of the boxed area in C. C, D, Asterisk indicates axons crossing into the lesion; double asterisks, BDA-labeled axons ventral to the lesion. E, Average numbers of BDA-labeled axons in cross-sections rostral to the injury. F, Average number of BDA-labeled axons in 0.5 mm increments from the injury beginning at the rostral lesion edge expressed as an index of the number of BDA axons/total number of BDA axons in rostral cross-sections. Group sizes were as follows: AAVshLuc, n = 4 (green bars); AAVshLuc/Fibrin, n = 4 (blue bars); AAVshPTEN, n = 5 (yellow bars); and AAVshPTEN/fibrin, n = 5 (red bars). Asterisks indicate significant differences between groups. Scale bars: A–C, 1 mm; D, 0.5 mm.
- Figure 9.
No significant differences in lesion volumes were seen between groups. A, B, Representative DAPI-stained sagittal sections through the lesion area from a rat in the AAVshPTEN/fibrin versus the AAVshPTEN groups, respectively. Fibrous scar dorsal to the cavity is outlined in white. Asterisks in A indicate thin cellular bridges. C, Quantification of lesions volumes in the four groups (means ± SE).
Tables
Rat ID Paw preferencea Group SCI surgery order 120206_49 Right AAV-shLuc 1 120206_39 Right AAV-shPTEN 3 120206_40 Right AAV-shPTEN 4 120206_03 Right AAV-shLuc 5 120206_45 Left AAV-shPTEN 6 120206_47 Left AAV-shLuc 7 120206_46 Left AAV-shPTEN 8 120206_48 Right AAV-shLuc 9 120206_09 Right AAV-shLuc 10 120206_57 Right AAV-shPTEN 11 120206_16 Right AAV-shPTEN 13 120206_17 Left AAV-shLuc 15 120206_30 Right AAV-shPTEN 16 120206_33 Right AAV-shLuc 17 120206_54 Left AAV-shPTEN 18 120206_56 Right AAV-shLuc 19 120206_23 Left AAV-shPTEN 20 120206_36 Left AAV-shLuc 23 120206_50 Left AAV-shPTEN/Fibrin 24 120206_51 Right AAV-shPTEN/Fibrin 25 120206_37 Left AAV-shLuc/Fibrin 26 120206_42 Left AAV-shPTEN/Fibrin 27 120206_43 Right AAV-shLuc/Fibrin 28 120206_04 Right AAV-shLuc/Fibrin 29 120206_08 Right AAV-shPTEN/Fibrin 30 120206_06 Right AAV-shPTEN/Fibrin 31 120206_20 Right AAV-shPTEN/Fibrin 33 120206_07 Left AAV-shLuc/Fibrin 34 120206_58 Left AAV-shLuc/Fibrin 35 120206_60 Right AAV-shLuc/Fibrin 36 120206_52 Left AAV-shPTEN/Fibrin 37 120206_44 Right AAV-shPTEN/Fibrin 38 120206_25 Left AAV-shLuc/Fibrin 39 120206_05 Left AAV-shPTEN/Fibrin 40 120206_27 Left AAV-shPTEN/Fibrin 41 120206_29 Right AAV-shLuc/Fibrin 42 120206_12 Right AAV-shPTEN/Fibrin 44 120206_24 Left AAV-shLuc/Fibrin 47 ↵aFor each rat, the paw with the highest success rate.
Experimental step Attrition Remaining Starting no. of rats — 60 Rats that did not meet performance criterion 7 53 Rats that died during vector administration 0 53 Rats that died during SCI surgery 4 49 Rats that removed for autophagia 5 44 Rats with incomplete SCI 6 38 Total no. of rats for behavioral analysis 38 Rat Group Day of onset Site Relative to vectora Severity 27 shPTEN-Fibrin 4 Forepaw Contralateral Mild 31 Untreated 4 Forepaw Contralateral Severe 33 shPTEN-Fibrin 7 Forepaws Both Moderate 18 shPTEN-Fibrin 11 Forepaw Contralateral Severe 8 shPTEN/shSOCS3-Fibrin 13 Forepaw Contralateral Mild 37 shPTEN/shSOCS3-Fibrin 13 Forepaw Contralateral Moderate 8 shPTEN/shSOCS3-Fibrin 14 Forepaw Ipsilateral Mild 14 shPTEN-Fibrin 14 Forepaw Ipsilateral Mild 19 shPTEN-Fibrin 14 Forepaw Contralateral Mild 5 Untreated 15 Forepaw Contralateral Mild 2 shPTEN/shSOCS3-Fibrin 19 Hindpaw Contralateral Mild 25 Untreated 22 Forepaw Ipsilateral Mild 9 shPTEN/shSOCS3-Fibrin 24 Forepaw Contralateral Mild 24 shPTEN-Fibrin 24 Hindpaw Contralateral Moderate 26 shPTEN/shSOCS3-Fibrin 24 Forepaw Contralateral Moderate 40 Untreated 28 Hindpaw Contralateral Severe 10 Untreated 29 Hindpaw Ipsilateral Mild 41 shPTEN/shSOCS3-Fibrin 30 Forepaw Contralateral Mild 46 shPTEN/shSOCS3-Fibrin 30 Hindpaw Ipsilateral Mild 18 shPTEN-Fibrin 31 Hindpaw Ipsilateral Moderate 21 Untreated 34 Forepaw Ipsilateral Mild 12 shPTEN-Fibrin 35 Hindpaw Contralateral Mild 30 Untreated 39 Forepaw Contralateral Moderate 4 shPTEN-Fibrin 46 Hindpaw Contralateral Mild 34 shPTEN-Fibrin 48 Forepaw Contralateral Mild 44 shPTEN/shSOCS3-Fibrin 54 Forepaw Ipsilateral Mild 31 Untreated 62 Hindpaw Contralateral Moderate 30 Untreated 65 Forepaw Ipsilateral Moderate 48 Untreated 66 Hindpaw Ipsilateral Moderate 41 shPTEN/shSOCS3-Fibrin 72 Hindpaw Ipsilateral Mild 42 shPTEN-Fibrin 72 Hindpaw Ipsilateral Mild 45 shPTEN/shSOCS3-Fibrin 72 Hindpaw Ipsilateral Mild 46 shPTEN/shSOCS3-Fibrin 83 Forepaw Ipsilateral Mild ↵aLaterality in terms of AAV injection.
