Activation of the Large-Conductance, Voltage, and Ca2+- Activated K+ (BK) Channel in Acute Spinal Cord Injury in the Wistar Rat Is Neuroprotective

Figures

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  Figure 1. Treatment of SCI with ISO maintains the myelination of axonal tracts. Spinal cords were sectioned and stained with NF (red), MBP (green), and DAPI for nuclear stain (blue) as described in Materials and Methods. (A–C) Injured non-treated (Inj-No-Tx); (D–F) IbTx treated; (G–I) ISO treated; (B,E,H) ROIs rostral to the injury site; (C,F,I) ROIs of the injury site of the spinal cords. (J,K) A semi quantitative comparison between study groups measuring MPB and NF ratios to DAPI.

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  Figure 2. Electrophysiological set-up. Electrophysiological studies were conducted using a suction electrode. (A) Diagram demonstrating the electrophysiology set up with the stimulating electrode on the rostral side of the injury (8 weeks post-SCI) and the suction electrode (recording electrode) on the caudal side of the injury site. This allowed for local recordings of the compound action potential propagation through the injury site, as described in Materials and Methods. The initial deflection represents the stimulus artifact, while the second spike is due to the generation of action potentials along axonal tract. (B) Representative traces of CAPs on a health spinal cord stimulated at 1 mA for 0.1 ms before (top) and after (bottom) cutting the spinal cord between the two electrodes, demonstrating that the CAP recordings were successful. When the spinal cord was severed, there was no measured CAP present.

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  Figure 3. ISO-treated animals exhibit greater axonal survival and CAP velocities compared to injured non-treated animals. Eight weeks post-injury, animals were sacrificed, and spinal cords removed for electrophysiological analysis. Representative traces are shown of CAPs after stimulation of spinal cords at 1 mA for 0.1 ms. (A) LAM (laminectomy only); (B) injured non-treated (Inj-No-Tx); (C) IbTx treated; (D) ISO treated. The arrows indicate the artifact upon stimulation of the spinal cord followed by the CAP. Peak Amplitude of CAPs (E) and conduction velocity (F) were measured. Injured non-treated animals (Inj-No-Tx) exhibited significantly lower peak amplitudes and CAP velocities compared to laminectomised (LAM) animals and animals treated with ISO. Tukey box plots are shown. LAM n = 3; Inj = 4; IbTx = 4; ISO = 6.

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  Figure 4. Representative stick figures of angles between the 4 kinematic joints depicting one (LAM) or two (Inj-No-Tx, IbTx, and ISO treated) step cycles. Stick figures of angles between the hip (red), knee (green), ankle (blue), and toe (purple) are depicted of one representative from each treatment group. ISO treated animals demonstrated two clear step cycles, similar to the one step cycle demonstrated from the LAM group. The Inj-No-Tx and IbTx treated representative shows no clear swing phase in the step cycle.

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  Figure 5. Improved neuro-protection translates to improved ROM in spinal cord injured rats treated with ISO. Eight weeks post-injury, 2D gait analysis was performed as described in Materials and Methods. Maximum thigh (A), leg (B), and foot (C) segment angles were assessed. Laminectomised rats and SCI rats treated with ISO had significantly greater ROM of thigh and leg segment angles compared to injured non-treated animals. There were no significant differences in foot segment angle ROM. Tukey box plots are shown. LAM n = 3; Inj = 6; IbTx = 6; ISO = 13.

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  Figure 6. Improved neuro-protection translates to improved motor function in spinal cord injured rats treated with ISO. Eight weeks post-injury, 2D gait analysis was performed as described in Materials and Methods. Maximum velocity of thigh (A), leg (B), and foot (C) segment angles were assessed. Laminectomised rats and SCI rats treated with ISO had significantly greater ROM of thigh and leg segment angles compared to injured non-treated animals. There were no significant differences in foot segment angle velocity. Tukey box plots are shown. LAM n = 3; Inj = 6; IbTx = 6; ISO = 13.

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  Figure 7. Generalized Additive Mixed Model of joint segment angles through a step cycle. The 2D gait analysis was used to calculate angles of the thigh, leg and foot segments through the proportion of the step cycle, and modeled using a GAM. Inj-No-Tx (red) and IbTx treated (green) animals demonstrated no swing phase in gait cycle in thigh and leg segment angles. Foot segment angles in the Inj-No-Tx and IbTx treated animals demonstrated some range of movement, although the mobility was less than in LAM (purple) and ISO (blue) treated animals. ISO treated animals demonstrated a classic swing phase in thigh, leg, and foot segment angles through the proportion of the step cycle, mirroring the LAM animals.