Depolarization and electrical stimulation enhance in vitro and in vivo sensory axon growth after spinal cord injury

Abstract

Activity dependent plasticity is a key mechanism for the central nervous system (CNS) to adapt to its environment. Whether neuronal activity also influences axonal regeneration in the injured CNS, and whether electrical stimulation (ES) can activate regenerative programs in the injured CNS remains incompletely understood. Using KCl-induced depolarization, in vivo ES followed by ex-vivo neurite growth assays and ES after spinal cord lesions and cell grafting, we aimed to identify parameters important for ES-enhanced neurite growth and axonal regeneration. Using cultures of sensory neurons, neurite growth was analyzed after KCl-induced depolarization for 1-72h. Increased neurite growth was detected after short-term stimulation and after longer stimulation if a sufficient delay between stimulation and growth measurements was provided. After in vivo ES (20Hz, 2× motor threshold, 0.2ms, 1h) of the intact sciatic nerve in adult Fischer344 rats, sensory neurons showed a 2-fold increase in in vitro neurite length one week later compared to sham animals, an effect not observed one day after ES. Longer ES (7h) and repeated ES (7days, 1h each) also increased growth by 56-67% one week later, but provided no additional benefit. In vivo growth of dorsal column sensory axons into a graft of bone marrow stromal cells 4weeks after a cervical spinal cord lesion was also enhanced with a single post-injury 1h ES of the intact sciatic nerve and was also observed after repeated ES without inducing pain-like behavior. While ES did not result in sensory functional recovery, our data indicate that ES has time-dependent influences on the regenerative capacity of sensory neurons and might further enhance axonal regeneration in combinatorial approaches after SCI.

Keywords: CNS plasticity; Electrical stimulation; axonal regeneration; dorsal root ganglion; spinal cord injury.

Figure 1. KCl depolarization can inhibit or enhance DRG neurite growth

Naïve DRGs were isolated and cultured on PDL/Laminin (0.5μg/μl) for either (A-H) 48h, or (I-R) were replated at 72h and cultivated for an additional 24h. KCl was added to the culture medium, at the beginning of incubation, for (C) 24h or (G) 1h, and (K) 24h and then washed out and replaced with regular medium. (O) DRGs were kept in media with KCl for 72h and then immediately replated. Controls wells were incubated in (A, E, I, M) media only or (B, F, J, N) received NaCl (40mM) for osmolarity control for the same time as the KCl-treated DRGs. The longest ßIII-tubulin-labeled neurite was quantified. Average neurite length is expressed as % of control (176±80 neurons/well) (A-D) Growth is inhibited when DRGs are incubated in KCl for 24h (n=5–6 wells, 2 independent experiments). However, if (E-H) incubation is shorter (1h), total growth is enhanced (n=9–10 wells, from 3 independent experiments). (I-L) If DRGs are exposed to KCl for 24h then changed to normal media and replated at 72h in normal media, growth is enhanced (4 independent experiments). (M-P) If DRGs are exposed to KCl for 72h then replated in normal media, growth is inhibited (3 independent experiments). (Q) Neurons exposed to KCL for 24h and replated at 72h were classified by their longest neurite length (<50μm, 50–250μm, 250–500μm and >500μm) and expressed as % of the total number of neurons per condition. (R) The same classification for neurons exposed to KCl for 72h and immediately replated shows an increase in the percentage of neurons with very short neurites. (S) Growth is inhibited when 24h KCl exposed neurons were replated directly (n=3, 108±24neurons/well, 1-way ANOVA) (compare to L), (T) 72h KCl exposure is no longer inhibitory when cells are replated at 7 days (n=3, 159±35neurons/well) (compare to P). Bars represent means+SEM, One-way ANOVA, Sidak´s posthoc test, ∗p<0.05, ∗∗p<0.01, ∗∗∗p<0.001.

Figure 2. In vitro neurite growth 7 days after in vivo ES of the sciatic nerve

(A) Experimental design. DRGs (L4-L6) were isolated from (B) animals 7 days after CL, (C) naïve animals, (D) 7 days after sham or (E) after electrical stimulation. Cells were grown on PDL-laminin (5μg/ml) coated plates for 24 hours and labeled for βIII-tubulin. (F) Quantification of neurite growth demonstrates a strong effect of conditioning lesions, but 1h ES 7d prior to isolation is also growth-promoting (n=8/group, ANOVA p<0.0001, Tukey´s posthoc test). (G) Neurons classified by their longest neurites reveal that conditioning lesions enhance neurite initiation and extension, whereas electrical stimulation primarily enhances neurite extension (n=8/group, ANOVA p<0.0001, Tukey´s posthoc test). Mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 3. A delay longer than 1 day is necessary, while increased or repeated stimulation do not have additional growth-promoting effects

(A) Neurite growth of DRGs (L4-L6) isolated 1 day after ES is not enhanced (n=6, t-test p=0.68, mean ± SEM). DRGs (L4-L6) isolated (B) 7 days after a single 7h stimulation or (C) after daily 1h stimulations for 7 days show significantly increased neurite growth (n=5–8, t-test *p<0.05; mean ± SEM), but the effect size is similar to single 1h stimulation (compare to Fig.2). (D) For repeated stimulations (n=7, ANOVA p<0.01, Dunnett’s multi comparison test, *p<0.05), motor thresholds were determined on a daily basis, noticing a significant increase in the first 2 days post-surgery, with a return to baseline at later time points.

Figure 4. Axonal growth 4 weeks after dorsal column lesions (DCL) and single or repeated 1h ES

Following transection of ascending sensory axons at C4 and injection of GFP+ BMSCs into the lesion, animals received (A-C) bilaterally conditioning lesions, (D-F) no other manipulation, (G-I) electrodes with no stimulation or (J-L) electrical stimulation. (C, F, I, L) CTB-labeled axons were quantified at the caudal GFAP border (red) and within the lesion site filled with GFP⁺ BMSCs (green). Arrows mark the caudal lesion border. (B, E, H, K) Higher magnifications of boxed areas shown in (A, D, G, J). (M) The number of axonal profiles at different distances from the lesion site was quantified in 1 out of 8 serial sagittal sections. Significant differences between CL and lesion only animals (^$$p<0.01, ^$$$$ p<0.0001), and between ES and sham animals (**p<0.01, ***p<0.001, ****p<0.0001) are indicated. (N) Overall axon growth (area under the curve; AUC) into the graft shows the highest growth in animals with CLs and a smaller but significant effect after ES. (O, P) The distance of CTB-labeled growth cones/axon tips within the graft was quantified from the caudal GFAP/GFP border. (O)The percentage of axons extending for a longer distance into the graft is significantly increased in ES compared to lesion only and sham animals. Sham 1h also shows an effect compared to lesion only. Significance shown for ES 1h versus sham 1h. (P) The percentage of axons that achieve growth over a longer distance into the graft is significantly increased in ES 1hx2 compared to lesion only and sham chronic. n=6–7/group, 2-way ANOVA, Tukey´s posthoc test. Mean ± SEM, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. For comparison, naïve and CL animals shown in (O) are also shown in (P).