AAV9-mediated KCC2 upregulation enhances functional recovery following cervical spinal cord injury

Figures

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  Fig. 1. Traumatic C6/7 SCI significantly elevates the forearm MEP range (n = 6 per group). A Schematic overview of the forelimb MEP recording experiment (adapted from BioRender.com with permission). B, C Mean forearm MEP recordings in sham and SCI animals following 10 constant stimuli (10 mA and 0.3 ms). Each line represents the mean of 10 MEPs induced by 10 mA and 0.3 ms pulses in an individual animal. The group average and 95% confidence interval are represented by the central red line and the faded red area, respectively. D C6/7 SCI presents with a significantly higher mean MEP range. E MEP range across varying stimuli intensity. F MEP area under the curve (AUC) across varying stimulus intensities. G MEP range across varying stimulus durations. H MEP AUC across varying stimuli durations. I MEP range following a single pulse and a paired pulse with a 2 ms interval, demonstrating synaptic facilitation. J MEP range following consecutive paired pulses with varying intervals. Statistical inferences were based on unpaired two-tailed t-tests in (D) and repeated-measure two-way ANOVA with Geisser-Greenhouse correction for (E–J). All data in (D–J) are presented as mean ± SEM and were assessed…

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  Fig. 2. Both acute and subacute localized intrathecal AAV9 injections can induce episomal expression. A Schematic overview of the localized intrathecal AAV9 injection at acute (total n = 16) and subacute (total n = 19) timepoints (adapted from BioRender.com with permission). B Slc12a5 expression at the acute time point in the injury epicenter and caudal thoracic spinal cord. C Slc12a5 expression at the subacute time point in the injury epicenter and caudal thoracic spinal cord. D Difference in Slc12a5 expression between acute and subacute injections. E 10× confocal images of the injured spinal cord demonstrate the efficacy of localized intrathecal AAV9 administration to induce episomal EGFP expression. The cell nucleus is represented by DAPI. All data are presented as mean ± SEM (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).

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  Fig. 3. KCC2 upregulation enhances functional recovery following traumatic cervical SCI (Sham n = 3; AAV9-Syn-EGFP: n = 9; AAV9-Syn-KCC2: n = 10). A Representative FLAS images showing enhanced forelimb recovery following KCC2 upregulation. B Averaged FLAS score by two independent observers, compared with one-way ANOVA with Dunnett multiple comparison. C Forelimb grip strength quantified by grip strength meter (SDI Grip strength system) reveals incremental improvement in KCC2-treated rats compared to EGFP control. D Average inclined plane measurement reveals gradual improvements in trunk stability. E Improved hindlimb function recorded by open-field BBB following AAV9-mediated KCC2 upregulation. F Representative CATWALK runs for each group. G, H CatWalkTM analysis demonstrated enhanced recovery in stride length following KCC2 gene therapy (two-way ANOVA using FDR according to the two-stage linear set-up procedure of Benjamini, Krieger, and Yekutieli to correct for multiple comparisons). Time-coursed data analyzed with repeated-measure two-way ANOVA using FDR according to the two-stage linear set-up procedure of Benjamini, Krieger, and Yekutieli to correct for multiple comparisons.…

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  Fig. 4. KCC2 gene therapy restores forelimb motor-evoked potential (MEP) threshold following traumatic spinal cord injury (Sham n = 3; AAV9-Syn-EGFP: n = 9; AAV9-Syn-KCC2: n = 10). A Mean forearm MEP recordings in sham, AAV9-Syn-EGFP, and AAV9-Syn-KCC2, using 10 MEPs involving pulses with 10 mA and 0.3 ms in duration. B MEP range quantification in the mean forearm MEP. C MEP AUC quantification in the mean forearm MEP. D Peak latency quantification in mean forearm MEP. E, F Increasing stimulus intensity at 0.1 ms pulse duration increases the MEP range and AUC. KCC2 gene therapy restores the MEP range and AUC. G, H Increasing stimulus duration at 10 mA intensity similarly increases MEP range and AUC. KCC2 gene therapy restores the forelimb MEP range back to an uninjured level. I, J Paired-pulse quantifications. Statistical analysis included the Shapiro–Wilk test to assess Gaussian distribution, followed by either one-way ANOVA or repeated-measure two-way ANOVA with Geisser-Greenhouse correction. All data are presented as mean ± SEM (*p < 0.05, **p < 0.01).

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  Fig. 5. Immunohistochemical analysis of the impact of AAV9-mediated gene therapy across the spinal cord (n = 4 per group). A The transgenic expression of EGFP demonstrates the extent of AAV9 diffusion in the injured spinal cord in a longitudinal slide. B, C Higher magnification images of regions shown in (A). D Axial section at the injury epicenter demonstrating the extent of AAV9-induced EGFP expression. E Representative confocal images of the cervical spinal cord stained for β3-tubulin (Blue) and 5HT (Red) in sham, EGFP/non-treated, and KCC2-treated rats, collected 12 weeks post-SCI. The intensity of F β3-tubulin and G 5HT staining was quantified using Fiji software. H The ratio of 5HT and β3-tubulin was calculated across the rostro-caudal region 2 mm from the SCI epicenter in EGFP/non-treated and KCC2-treated spinal cords. All data are presented as mean ± SEM.

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  Fig. 6. KCC2 upregulation enhances neuronal preservation in the lesional and perilesional cervical spinal cord (n = 5 per group). A Representative confocal images for NeuN, GAD67 (threshold), VGAT (threshold), and VGlut2 (threshold) at 960 µm. B–E The mean quantification across −1920 µm, −960 µm, 0 µm, 960 µm, and 1920 µm relative to the SCI epicenter. B Elevated NeuN cell counts at and around the lesion site. Total NeuN+ cell count. C GAD67+ area is significantly higher in the KCC2-treated group compared to the EGFP control. D Similarly, the VGAT+ area is significantly higher in the KCC2-treated group compared to the EGFP control. E No statistically significant alteration in the Vglut2+ area was observed. All data are presented as mean ± SEM, normalized to the uninjured sham group, checked for Gaussian distribution using the Shapiro–Wilk test, and analyzed using t-test following log2 transformation (ns: non-significant, *p < 0.05, **p < 0.01).

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  Fig. 7. KCC2 upregulation alters the neuroglial response in the injured spinal cord. A Heatmap with hierarchical clustering performed based on cosine similarity. Statistically significantly altered cytokines/chemokines are indicated by asterisks (*), as determined by ANOVA using FDR according to the two-stage linear set-up procedure of Benjamini, Krieger, and Yekutieli to correct for multiple comparisons. B KCC2 upregulation elevates the level of IP-10. C Cxcl10 expression coding for IP-10 expression was significantly upregulated in the injured spinal cord receiving AAV9-Syn-KCC2. D Representative images of GFAP and IBA1 immunohistochemical staining at 1920 µm rostral to the SCI epicenter. E KCC2 upregulation does not alter GFAP+ area. F KCC2 upregulation significantly reduces IBA1+ area. G, H AAV9-mediated KCC2 upregulation reduces the expression of KCC2-related genes and alters the expression of Mecp2 transcription factor. All data are presented as mean ± SEM (ns: non-significant, *p < 0.05).