(A) Experimental model showing cAMP injection into reticular nucleus, graft of MSCs expressing BDNF into a T3 complete transection site, and injection of Lenti-BDNF and AAV2-BDNF viral vectors 1.5mm and 2.5mm caudal to the lesion. (B) Nissl-staining of a horizontal spinal cord section showing completeness of T3 transection lesion (Les). Inset indicates level of horizontal section. (C) The completeness of T3 transection lesion (Les) is indicated by GFAP fluorescent immunolabeling (see also Fig. 8). (D) A Nissl-stained section shows excellent survival of MSC graft and filling of lesion cavity. (E) Higher magnification view from boxed area of panel D showing excellent integration of MSC graft. Dashed lines indicate host (h)/graft (g) interface. (F) Lentiviral vectors (Lenti-BDNF-GFP) transduced mostly glia (GFAP-labeled astrocytes, red) (inset shows co-localization) and (G) AAV2 vectors expressing BDNF transduced cells with morphological features of neurons (inset). Scale bars = 400μm B-D; 120μm, E; 600μm F-G.

Transected reticulospinal motor axons regenerate into and beyond C5 hemisection lesion site after combinatorial treatment with cAMP, a cell graft in the lesion site, and BDNF gradients beyond the lesion. (A) Triple label for BDA-labeled reticulospinal axons (red), host astrocytes (GFAP, blue) and host cells expressing BDNF (green, also expressing GFP) in a horizontal section at low magnification. Main reticulospinal tract approaches lesion (Les) from left side, extends into BDNF-expressing MSC graft, and some axons regenerate beyond the graft as highlighted in next panels. Arrows indicate viral vector injection sites (IS). (B-D) Higher magnification from boxed area in (A) showing (B) axon regeneration (red) into MSC graft, and (C-D) axon crossing beyond lesion/graft site into caudal host tissue. Axons are present caudal to graft only in regions of BDNF expression (green). Dashed lines in B indicate rostral host (h)/lesion (Les) interface and in C-E indicate caudal lesion/host interface. (E) Growth of presumably bridging axons (red) into caudal host tissue is exclusively in regions of BDNF expression (green). (F) The association of regenerating axons with BDNF expression is also revealed by double light level immunolabeling for BDNF (dark blue) and BDA (brown): BDA-labeled reticulospinal axons regenerate into the lesion/graft (g) site and extend into caudal host (h) spinal cord gray matter expressing BDNF. Graft is outlined by dashed lines. (G) High magnification of boxed area from panel (F) shows BDA-labeled axons crossing caudal graft/host interface (dashed lines). (H, I) Higher magnification from boxed areas of panel G show clusters of BDA-labeled reticulospinal axons (brown, arrowheads) closely associating with BDNF-expressing host neurons (dark blue). Scale bar = 530μm A; 95μm B-E; 280μm F; 50μm G; 20μm H-I.

BDA light level immunolabeling in four experimental groups. (A-B) Lesioned control: Axons do not penetrate the lesion cavity in the absence of a cell graft, and (B) few axons are present at the caudal lesion/host interface (higher magnification of panel A). The few axons visualized arise from the contralateral, unlesioned side. (C-D) Combination treatment: Reticulospinal axons robustly regenerate into the marrow stromal cell graft in the lesion site, and (D) appear to cross the distal graft/host interface. Dashed lines indicate host (h) /graft (g) interface. (E-F) BDNF treatment: Axons also penetrate marrow stromal cell graft in lesion site, and (F) appear to emerge distally. cAMP was not injected into pons; number of axons regenerating beyond lesion is reduced compared to combination treatment, quantified in panel I. (G-H) cAMP treatment: Axons penetrate graft in lesion site, but (H) do not appear to regenerate beyond lesion. BDNF was not provided. Scale bar 300μm, A, C, E, G; 60μm, B, D, F, H. (I) Quantification of number of BDA-labeled axons crossing a vertical line placed 250μm beyond distal graft/host interface reveals significant overall group differences (ANOVA, p<0.01). Significantly greater numbers of axons regenerate beyond graft in combination group (COMB) than all other groups (post-hoc Fischer’s compared to BDNF group p<0.05; compared to each other group, **p<0.01). BDNF treatment alone also differs from other groups (p<0.05 in each case).

Functional outcomes were measured on three tasks after C5 hemisection: grid-walking, grooming and gait kinematics. (A) Functional outcomes on the grid-walking task are worse than lesioned controls in each group that received BDNF (COMB and BDNF). Lesioned groups lacking BDNF below the lesion exhibited significant improvement of right forelimb placement over time (Manova repeated measures, between groups p<0.0001; time p<0.0001, Time × Group p<0.001; *p<0.05, ***p<0.001. N=12 each group) (* in COMB dataset indicate significant difference between COMB and LES group). (B) Similar recovery patterns, and deficits in recovery in BDNF recipients, were observed in forelimb use on the grooming task (Manova with repeated measures, between groups p<0.01; time p<0.23, Time × Group p<0.05; *p<0.05, **p<0.01, ***p<0.001). (C) Kinematic analysis of forepaw placement during locomotion shows significant reduction in distance from right forepaw to hip in the combination treatment group (COMB) compared to the lesion-only control (LES, ***p<0.001). There are no significant differences in the intact left forepaw, 12 weeks post-injury. (D) Examples of a typical right forepaw step trajectory relative to ipsilateral hip position (blue square) in an intact (baseline), lesion only (LES) and combinatorial treatment (COMB) subjects. There is less joint excursion and reduced overall recovery in step trajectory in the treatment group, likely reflecting spasticity. (E) Static images and tracings (below) of joints from video Illustrating the right forepaw (arrow) in a retroflexed, grasp posture in a COMB treated subject, compared to a non-grasp posture in a LES control.

A combination of cAMP and BDNF supports reticulospinal motor axon regeneration beyond T3 complete transection. (A) Overview of complete transection site (horizontal section) showing BDA-labeled reticulospinal axons approaching lesion site (left side), regenerating into graft (g), and regenerating beyond lesion into caudal host (h) spinal cord beyond. IS, injection sites of BDNF-expressing vectors. (B) Higher magnification of indicated region from panel A, demonstrating BDA-labeled reticulospinal axons crossing caudal graft/host interface (dashed lines) into distal spinal cord. (C) Axons regenerate beyond lesion site only into regions that express BDNF, indicated by GFP reporter gene expression in the same section shown in panel B. GFAP labeling, red. (D) Clusters of bridging axons and (E) axons with varicosities can be found up to 4 mm caudal to lesion site in host cord, in regions of BDNF expression. Scale bars = 600μm A, 88μm B-C, 44μm D and 15μm E. (F) Quantification of axons bridging across T3 complete transection after combinatorial treatment (n=6). The number of axons crossing a virtual line 250, 500, 1000, 2000, and 3000 μm beyond the lesion site was quantified. Axons are not detectable beyond the lesion in controls with lesions alone (N=6).

(A) BDA and CS56 double immunolabeling in a horizontal section demonstrates that BDA-labeled reticulospinal axons bridge through regions of chondroitin sulfate proteoglycan labeling (CS56 antibody), at caudal graft (g) / host (h) interface (dashed lines) 3 months after T3 complete transection. (B-D) Higher magnification view of boxed area from panel (A) shows (B) merged and (C-D) individual channels. (E) 2mm caudal to T3 complete transection, BDA-labeled reticulospinal axons (red) co-localize with the pre-synaptic marker synaptophysin (SYN, green), confirmed in xz and yz plane reconstructions. A sample putative synapse in lower right corner of panel (arrow) is shown in inset. (F) Regenerating reticulospinal axons (red) express vesicular glutamate transporter-1 (VGlut1, green) in juxtaposition to a motor neuron labeled for choline acetyltransferase (ChAT, blue), suggesting that regenerating axons resume expression of an appropriate, excitatory neurotransmitter. (G-H) Immunoelectron microscopy confirms that regenerating, BDA-containing reticulospinal axons (R, dark reaction product of BDA) form synapses (arrows) with that host dendrites located 2mm below the lesion. Scale bars = 125μm A; 32μm B-D; 2μm E; 1.8μm F; 150nm G; 50nm H.

Combinatorial treatments also promote serotonergic axonal regeneration beyond sites of T3 complete transection. (A) Low magnification of a horizontal spinal cord section showing 5-HT-labeled raphespinal axons (red) approaching the lesion from the rostral (left) side, regenerating into the graft in the lesion site, and beyond. Dashed lines indicate rostral and caudal interfaces. Inset is GFAP label showing lesion site. (B) Higher magnification of boxed area from panel A, demonstrating raphespinal axons crossing caudal graft (g)/host (h) interface (dashed lines). (C) Same section as panel B now labeled for GFAP (red as pseudo-color) to show lesion border and GFP expression: serotonergic axons regenerate only into regions expressing BDNF. (D-G) Clusters of serotonergic axons regenerating up to 4 mm caudal to lesion site. Scale bars = 800μm A; 64μm B-E; 20μm F-G.

GFAP immunolabeling from a series of every sixth horizontal section demonstrates completeness of T3 complete transection from ventral through dorsal spinal cord. Scale bar = 125μm.

Intact reticulospinal axons in (A) white matter are normally myelinated (arrows), but (B) lose their myelin sheaths upon entering gray matter. BDA (red) and MBP (green) double labeling. (C) Upon regenerating into a marrow stromal cell graft in the lesion cavity, most axons are remyelinated (MAG label, arrows) by Schwann cells migrating into the lesion site (Jones et al., 2003; Lu et al., 2005; Alto et al., 2009). (D) Reticulospinal axons that regenerate beyond the T3 complete transection site, into gray matter, are not myelinated and resemble gray matter axon terminals on intact animals, as seen in panel B. (E) Higher magnification image of a myelinated axon in a graft. (arrow points to a node of Ranvier). Scale bar = 7μm A; 6μm B, D; 12μm C; 2.5μm E.

Functional outcomes were measured on the BBB scale and in a spasticity model. (A) BBB testing shows significantly higher scores in hindlimb locomotion among subjects that received combinatorial treatment (COMB) compared to lesioned controls (Student’s t-test, ***p<0.0001), three months post-injury. However, re-transection (arrow) at the rostral interface of graft and host failed to reduce locomotor recovery when assessed one day later, indicating a likely trophic factor effect of the graft rather than a functional benefit from axonal bridging. Values are mean ± SEM. (B) A validated model of spasticity (Bennett et al., 2004) indicates greater tail spasticity among BDNF recipients. Beginning with tail rest (I, IV), stimulation leads to a marked extension (II) followed by a prolonged flexion coil (III) in BDNF treated rats, whereas only a moderate flexion is see in control rats (V). AAV2-BDNF administration significantly increases tail spasticity compared to lesioned controls (**p<0.001; VI).