Cortical AAV-CNTF Gene Therapy Combined with Intraspinal Mesenchymal Precursor Cell Transplantation Promotes Functional and Morphological Outcomes after Spinal Cord Injury in Adult Rats

Abstract

Ciliary neurotrophic factor (CNTF) promotes survival and enhances long-distance regeneration of injured axons in parts of the adult CNS. Here we tested whether CNTF gene therapy targeting corticospinal neurons (CSN) in motor-related regions of the cerebral cortex promotes plasticity and regrowth of axons projecting into the female adult F344 rat spinal cord after moderate thoracic (T10) contusion injury (SCI). Cortical neurons were transduced with a bicistronic adeno-associated viral vector (AAV1) expressing a secretory form of CNTF coupled to mCHERRY (AAV-CNTF^mCherry) or with control AAV only (AAV-GFP) two weeks prior to SCI. In some animals, viable or nonviable F344 rat mesenchymal precursor cells (rMPCs) were injected into the lesion site two weeks after SCI to modulate the inhibitory environment. Treatment with AAV-CNTF^mCherry, as well as with AAV-CNTF^mCherry combined with rMPCs, yielded functional improvements over AAV-GFP alone, as assessed by open-field and Ladderwalk analyses. Cyst size was significantly reduced in the AAV-CNTF^mCherry plus viable rMPC treatment group. Cortical injections of biotinylated dextran amine (BDA) revealed more BDA-stained axons rostral and alongside cysts in the AAV-CNTF^mCherry versus AAV-GFP groups. After AAV-CNTF^mCherry treatments, many sprouting mCherry-immunopositive axons were seen rostral to the SCI, and axons were also occasionally found caudal to the injury site. These data suggest that CNTF has the potential to enhance corticospinal repair by transducing parent CNS populations.

Figure 1

(a) Two AAV-CNTF^mCherry injections in the cerebral cortex. (b) BDA injections into the cortex revealed using immunoperoxidase; note the many labelled neurons in deeper layers. This animal had previously received cortical injections of AAV-CNTF^mCherry. (c–l) Longitudinal sections of the spinal cord—in all cases rostral is to the left of the picture. (c) Section immunostained for mCherry (red) and β-III tubulin (green) showing anterogradely labelled mCherry-positive axons (arrow) in the dorsal corticospinal tract far rostral to the lesion site. (d) Control AAV-GFP-injected rat (no MPCs injected); a small number of immunoperoxidase BDA-labelled axons and debris are visible just in front of a rostral cyst (arrow), with no axons extending beyond the injury. (e–g) Large numbers of mCherry-positive axons rostral (e, f) and running dorsally over and beyond the cyst (g); these rats received AAV-CNTF^mCherry cortical injections plus an intraspinal injection of viable rat mesenchymal precursor cells (rMPCs). Note in (e) the profusion of mCherry-positive profiles (large arrow) approximately 1 mm rostral to the lesion cavity, growing into regions dorsal to the corticospinal tract (small arrows). There appears to be considerable sprouting of axons in this zone (f). (h) Immunoperoxidase BDA-labelled axons and debris rostral to a cyst in a rat injected with AAV-CNTF^mCherry. Several axons can be seen running caudally, ventral to the cyst (arrows). (i) BDA-labelled cortical axons (arrows) visualized using a fluorescent secondary antibody (green), running over a cystic cavity. This animal also received AAV-CNTF^mCherry and viable MPC injections. (j–l) Cortical axons (arrows) double labelled (l) with both mCherry ((j), red) and BDA ((k), green); note, some axons are only mCherry or BDA immunoreactive. Scale bars: (a, e), 500 μm; (b, h, and i), 200 μm; (c, d, g, and j–l), 100 μm; and (f) 50 μm.

Figure 2

Two examples (A–E and F–H) of animals that received cortical AAV-CNTF^mCherry injections and with mCherry-positive corticospinal axons distal to spinal cysts (D, E, I and J), and therefore distal to the initial injury. In all images rostral is to the left. (A, F) Low power views of cysts (β-III tubulin-immunostained sections) in each rat; the arrows in (A) and (B) point to the approximate location of the axons shown in (D, E, and H), respectively. (B, C) Large numbers of mCherry-positive axons (arrow) dorsal to the large rostral cyst (see (A)), with small numbers of irregularly organized axons distal (D, E). (G) mCherry-labelled axons rostral and dorsal to the cyst, with several axons (arrows) located distal to the injury (H). In one animal injected with AAV-CNTF^mCherry and that received a spinal rMPC injection, numerous mCherry-positive axons (arrowed) were seen distal to the most caudal lesion cavity (I, J). Scale bars: (A, F), 1 mm; (B), 500 μm; (C, G), 200 μm; (D, E, I, and J), 100 μm; and (H) = 50 μm.

Figure 3

Functional hindlimb recovery promoted after AAV-CNTF gene therapy and cellular transplantation as assessed by Ladderwalk: reduced misstepping over the Ladderwalk apparatus indicates that AAV-CNTF^mCherry therapy (red, crosses) promoted significant functional improvement after SCI compared with control AAV-GFP treatment (green). Note that at day 14, the Ladderwalk testing was carried out prior to injection of either viable (blue) or nonviable (black) cells. Post hoc tests revealed statistically significant differences between the AAV-GFP-treated control group and AAV-CNTF^mCherry + nonviable rMPCs at all time points as well as between the AAV-GFP-treated control group and AAV-CNTF^mCherry + viable rMPCs at all time points except day 56 (^∗p = 0.01–0.05, ^∗∗p = 0.005–0.01, and ^∗∗∗p = 0.001–0.005). Two-way repeated measures ANOVA was conducted using PRISM (time: p = 0.0001, treatment: p = 0.0016, and interaction: p = 0.0001). Standard deviation is shown.

Figure 4

Functional hindlimb recovery promoted after AAV-CNTF gene therapy and cellular transplantation as assessed by open-field locomotion (BBB). Significant functional improvements were observed following AAV-CNTF^mCherry (red), AAV-CNTF^mCherry + nonviable rMPC (black), and AAV-CNTF^mCherry + viable rMPC (blue) treatment compared to AAV-GFP-treated control animals (green) generally from day 21 after SCI onwards, although statistically significant differences were not maintained at all time points (^∗p = 0.01–0.05 and ^∗∗∗p = 0.001–0.005). Standard deviation is shown.

Figure 5

Ratwalk gait analysis at day 56 after SCI. Averages of arbitrary unit values of each treatment group compared to preinjury levels (dotted black lines) are shown for stance width (a), step length (b), stride length (c), and step sequence (d). Despite no statistically significant differences between any treatment group for any variable, compensatory changes such as a marked reduction in the stance width (a) of the forelimbs (Rf/Lf) between opposing fore- and hindlimbs (Rf/Lh and Lf/Rh) in all groups compared to preinjury levels were supported by data for step length (b) and stride length (c). A general decrease in the amount of patterns of coordinated fore- and hindlimb placement (“cruciate,” “alternate,” and “rotary” as indicated in (d)) and an increase in the amount of noncoordinated fore- and hindlimb placement (“none”) was observed. Standard deviation is shown.

Figure 6

Tissue sparing promoted by combined cortical AAV-CNTF^mCherry therapy and local transplantation of viable rMPCs. AAV-GFP control treatment and AAV-CNTF^mCherry treatment groups revealed similar average areas of cyst formation in spinal cord sections stained by toluidine blue, and although AAV-CNTF^mCherry + nonviable rMPC treatment resulted in slightly lower average cyst sizes, only AAV-CNTF^mCherry + viable rMPC treatment into the lesion resulted in a statistically significant reduction in average cyst size. ^∗p = 0.01–0.05.