Localized delivery of brain-derived neurotrophic factor-expressing mesenchymal stem cells enhances functional recovery following cervical spinal cord injury

Abstract

Neurotrophins, such as brain-derived neurotrophic factor (BDNF), are important in modulating neuroplasticity and promoting recovery after spinal cord injury. Intrathecal delivery of BDNF enhances functional recovery following unilateral spinal cord hemisection (SH) at C2, a well-established model of incomplete cervical spinal cord injury. We hypothesized that localized delivery of BDNF-expressing mesenchymal stem cells (BDNF-MSCs) would promote functional recovery of rhythmic diaphragm activity after SH. In adult rats, bilateral diaphragm electromyographic (EMG) activity was chronically monitored to determine evidence of complete SH at 3 days post-injury, and recovery of rhythmic ipsilateral diaphragm EMG activity over time post-SH. Wild-type, bone marrow-derived MSCs (WT-MSCs) or BDNF-MSCs (2×10(5) cells) were injected intraspinally at C2 at the time of injury. At 14 days post-SH, green fluorescent protein (GFP) immunoreactivity confirmed MSCs presence in the cervical spinal cord. Functional recovery in SH animals injected with WT-MSCs was not different from untreated SH controls (n=10; overall, 20% at 7 days and 30% at 14 days). In contrast, functional recovery was observed in 29% and 100% of SH animals injected with BDNF-MSCs at 7 days and 14 days post-SH, respectively (n=7). In BDNF-MSCs treated SH animals at 14 days, root-mean-squared EMG amplitude was 63±16% of the pre-SH value compared with 12±9% in the control/WT-MSCs group. We conclude that localized delivery of BDNF-expressing MSCs enhances functional recovery of diaphragm muscle activity following cervical spinal cord injury. MSCs can be used to facilitate localized delivery of trophic factors such as BDNF in order to promote neuroplasticity following spinal cord injury.

Keywords: diaphragm muscle; neuroplasticity; neurotrophin; respiration; spinal hemisection.

FIG. 1.

Delivery of mesenchymal stem cells engineered to produce releasable brain-derived neurotrophic factor (BDNF)-green fluorescent protein (GFP) (BDNF-expressing mesenchymal stem cells [BDNF-MSCs]) to the spinal cord was confirmed by GFP expression. Representative stitched image composed of 91 fields of view of a longitudinal spinal cord section at ∼C2–C4. The left side of the spinal cord is on the bottom and the right side is on the top of the image. Autofluorescence at 488 nm was used to create the background gray scale image. Note that white matter appears darker than the gray matter. Cholera toxin subunit B-labeled phrenic motoneurons were converted to gray scale and inverted to facilitate visualization (white). Phrenic motoneurons were present on the right side of the spinal cord starting near C3, and were not located on the left side of the spinal cord in this section. Intraspinal injection of BDNF-MSCs (into right side of spinal cord, rostral and caudal to C2; n=6) resulted in GFP immunoreactivity (red) between C2 and C3, and primarily in the white matter. Some background fluorescence was visible in this section contralateral to the injections (left side), but overall GFP expression was not evident in the contralateral side. Bar, 1 mm. Color image is available online at www.liebertpub.com/neu

FIG. 2.

Representative raw diaphragm electromyographic (EMG) recordings and root mean squared (RMS) EMG tracings after C2 hemisection (SH) and injection with wild-type mesenchymal stem cells (WT-MSCs) or brain-derived neurotrophic factor (BDNF)-green fluorescent protein (GFP) expressing MSCs (BDNF-MSCs). Intraspinal WT-MSCs treated SH (SH+WT-MSCs; n=4) and intraspinal BDNF-MSCs treated SH (SH+BDNF-MSCs; n=7) animals were monitored under anesthesia using EMG recordings obtained during eupnea via chronically implanted diaphragm electrodes. (A) EMG recordings obtained prior to SH (pre-SH), 3 days after SH (SH 3D) and 14 days after SH (SH 14D) for one animal from each group are shown. Diaphragm EMG activity occurred in rhythmic bursts. Electrocardiographic activity is visible in some recordings as narrow spikes. In this example, there is a lack of diaphragm EMG activity in the SH+WT-MSCs group. Untreated SH animals (n=6) displayed qualitatively similar results to the WT-MSCs treated SH group and are not shown. (B) Expanded EMG recordings for SH+WT-MSCs and SH+BDNF-MSCs groups, obtained pre-SH and at SH 14D. Each tracing is the first burst of the corresponding EMG recording in A. Notice multiple active motor units (with varying spike morphology) in the diaphragm EMG signal at SH 14D with intraspinal BDNF-MSCs treatment.

FIG. 3.

Proportion of animals displaying functional recovery of ipsilateral hemidiaphragm eupneic electromyographic (EMG) activity after C2 hemisection (SH). Chronic diaphragm EMG recordings were used to establish the proportion of animals displaying functional recovery after SH. The criteria for classification of functional recovery are detailed in the Methods section. A subset of animals were treated with wild-type mesenchymal stem cells (WT-MSCs) (n=4) and were not different than untreated SH animals (n=6); therefore, these groups were combined into one group (SH+Control/MSCs). Diaphragm EMG activity was absent in all animals at 3 days after SH (SH 3D), confirming complete interruption of descending ipsilateral drive to phrenic motoneurons and resulting diaphragm muscle paralysis. At 7 days after SH (SH 7D), 2 out of 7 animals with intraspinal injection of brain-derived neurotrophic factor (BDNF)-MSCs (SH+BDNF-MSCs) displayed functional recovery, compared with 2 out of 10 SH+Control/MSCs treated animals (p=0.68). By SH 14D, all of the BDNF-MSCs treated SH animals displayed functional recovery, compared with 3 out of 10 SH+Control/MSCs treated animals (*p=0.004).

FIG. 4.

Extent of functional recovery of ipsilateral rhythmic phrenic activity at 7 days (7D) and 14 days (14D) after C2 hemisection (SH). Individual animal values are depicted by different symbols within each group; filled symbols denote animals that displayed recovery at any point beyond 3 days after SH. Diaphragm root mean squared (RMS) electromyographic (EMG) amplitude was measured during eupnea at 3, 7, and 14 days after SH, and normalized to the pre-SH RMS EMG amplitude for the same animal. In all animals, RMS EMG was 0 at 3 days after SH. Data are shown as box plots (25th to 75th percentile), with the thicker line in the box representing the median value. In the Control/wild-type mesenchymal stem cells (WT-MSCs) treated SH animals (SH+Control/MSCs; n=10), RMS EMG amplitude was reduced at SH 14D compared with pre-SH. Intraspinal injection of BDNF-MSCs (SH+BDNF-MSCs; n=6) increased the extent of diaphragm activity at SH 14D compared with SH+Control/MSCs (p=0.008).