Ovine model of neuropathic pain for assessing mechanisms of spinal cord stimulation therapy via dorsal horn recordings, von Frey filaments, and gait analysis

Figures & data

Figure 1 Flowchart showing overview of the protocols for the in vivo ovine studies.

Abbreviations: CCI, chronic constriction injury; CPN, common peroneal nerve; EVF, electronic von Frey filaments; NPP, neuropathic pain; SCS, spinal cord stimulation.

Figure 2 (A) Dissection and isolation of the peroneal nerve at the level of the stifle joint. The nerve is palpable just behind the stifle joint, coursing around the fibular head. The stifle and hock joints are marked for reference, in addition to the peroneal nerve. (B) Four suture ligatures have been placed around the nerve to create a 25% constriction (i.e., a reduction in the diameter of the constricted nerve from the normal diameter by 25%).

Figure 3 (A) Relative to a mock spinal column, from left to right are shown the micrometer-driven translation stage used to control the microelectrode insertion depth and position, the mounting tower, and the Teflon^® threaded plug used to seal the tower interior when not in use. (B) Top–down view of the apparatus from (A) anchored in place on the sheep spinal column for a dorsal horn recording. (C) H&E stain of an axial cross section of the dorsal portion of the spinal cord at the level of the lumbar enlargement (L6). The white matter tracts of the spinal cord are slightly more eosinophilic, and the neuron-containing gray matter shows the characteristic butterfly wing structure. The dashed rectangle outlines the targeted recording region within the dorsal horn. The DF, DH, LF, and the central canal are marked. (D) Example recording of a single neuron’s activity recorded at a depth of 1414 µm.

Abbreviations: DF, dorsal funiculi; DH, dorsal horn; LF, lateral funiculus.

Figure 4 Withdrawal thresholds of the hind limbs in three sheep as measured with von Frey filaments and anesthesiometer from (A, B) one animal with a sectioned nerve and (C, D) two animals with constriction nerve injuries.

Notes: (B, D) Data collected >200 days post-nerve injury. The dashed black lines indicate portions of the data that were collected while SCS was applied continuously. The SCS appears to increase the withdrawal thresholds in the injured sheep most noticeably in (A, D).
Abbreviation: SCS, spinal cord stimulation.

Figure 5 The results for the von Frey measurements for the five sheep included in the final protocol (#40014, #40015, #40016, #40021, and #40022) are presented in Table 1 and expressed in terms of means and standard errors.

Notes: The baseline threshold of the left (affected) leg is significantly lower than the right (control) leg (252.9±37.6 vs. 361.5±32.4 g, p=3×10⁻⁸). Application of SCS significantly increases the withdrawal threshold of the left leg (297.5±21.5 g, p=0.048), but is still significantly lower than the unaffected right side, either with SCS (p=0.0035) or without it (p=0.0016). There is no significant difference in the withdrawal threshold of the unaffected right side with or without SCS (357.6±20.0 vs. 361.5±32.4 g, respectively).
Abbreviation: SCS, spinal cord stimulation.

Table 1 von Frey thresholds of five sheep in standardized protocol

Sheep	Left leg		Left leg		Right leg		Right leg
	SCS off		SCS on		SCS off		SCS on
	Mean (g)	±SE	Mean	±SE	Mean	±SE	Mean	±SE
40014	169.5	19.5	266.5	23.1	295.9	53.5	323.3	36.2
40015	646.4	26.6	458.1	43.6	673.3	20.7	497.9	39.6
40016	146.5	11.7	332.6	21.0	259.7	10.1	403.6	8.1
40021	194.8	16.7	232.2	28.2	353.1	24.9	324.6	13.1
40022	107.2	5.2	198.2	36.9	225.6	17.0	238.5	28.9
Mean	252.9	37.6	297.5	21.5	361.5	32.4	357.6	20.0

Abbreviation: SCS, spinal cord stimulation; SE, standard error.

Figure 6 (A) Range of flexion–extension angle of the hock joint in the sagittal plane (leg swing angle). (B) Angular speed during the first 100 ms of the gait cycle appears to increase in the contralateral (right) limb for the sheep with the cut peroneal nerve (#40006), but this does not occur for the sheep with the constricted nerves (#40002 and #40007).

Note: Error bars represent one SD.

Figure 7 Mean height of both back hooves plotted against percent of gait cycle for the sheep with a cut peroneal nerve (#40006).

Notes: Each trace is the average of 120–130 steps time-normalized to the gait cycle percentage. No difference in height is seen in the nerve-injured (ipsilateral) limb for SCS at 0 V (off) and 0.1 V, but significant increases in hoof elevation are seen during SCS at 0.3 and 0.5 V. The mean contralateral hoof elevation remains mostly constant from SCS off to 0.3 V and increases with 0.5 V stimulation. Shaded error bars represent standard error.
Abbreviation: SCS, spinal cord stimulation.

Figure 8 Example recordings of dorsal horn neurons (A) without spontaneous activity and (B) with spontaneous activity.

Notes: Both neurons were recorded from the same sheep (#40015) with a CCI. Neuronal responses to various stimuli, including brushing, touching, pinching, and applying deep pressure to the gastrocnemius muscle and flexion and extension of the hock joint, are shown. The upper and lower panels show the digitized raw action potentials and the neuron’s firing rate (cumulative histogram, bin width=1 s), respectively.
Abbreviation: CCI, chronic constriction injury.

Figure 9 Neuronal activity of two neurons from different blocks recorded during SCS from the same sheep (#40022).

Notes: (A) One neuron was excited by SCS and (B) the other neuron inhibited. The horizontal red bars indicate the regions where 40 Hz SCS was applied at varying amplitudes. Below the raw waveform in each panel is the neuron’s firing rate (cumulative histogram, bin width=1 s). The raster plots show the neurons’ action potentials aligned to each individual pulse of the SCS. The neuron in (A) is driven by the SCS above 3 V with a latency of ~2 ms. In (B), the neuron is completely suppressed with SCS at 3 V.
Abbreviation: SCS, spinal cord stimulation.