PMC6354557_01
Personalized treatment for a patient suffering from severe neuropathic pain for >10 years.. An SFN patient was clinically characterized, pain ratings were assessed and her C-fiber spontaneous activity was evaluated by microneurography recordings. Patient's fibroblasts were reprogrammed into induced pluripotent stem cells (iPSCs). Applying a small molecule approach, iPSCs were differentiated into patient-derived sensory neurons, which were subjected to patch clamp and multielectrode array recordings. In multielectrode array the compound lacosamide was tested on its effect of suppressing spontaneous activity. After successful reduction of spontaneous activity in vitro, lacosamide treatment was started in the patient. Its remarkable effect in the patient was shown by lowered pain ratings and its peripheral site of action by reduced spontaneous activity of peripheral nociceptors via microneurography.
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Fig. 1). In order to identify potential treatment options for the patient, we reprogrammed the patient's fibroblasts into iPSC and differentiated these into peripheral sensory neurons using a small molecule approach (Fig. 1,). iPSC derived sensory neurons expressed markers for nociceptors, such as TRPV1, and the sodium channels Nav1.7, Nav1.8 and Nav1.9 shown in mRNA expression levels and protein immune-stainings (, Fig. 2).
IPSC-derived human nociceptors of patients and controls show expression of peripheral markers.. A: Representative immunofluorescence images of SFN- and Ctrl-iPSC-derived nociceptors stained for (upper row) neuron- and nociceptor-specific markers Peripherin (red), Nav 1.9 (green), V-glut (green) DAPI (blue), and (lower row) for TRPV1 (red), Tuj1 (green) and DAPI (blue). Scale bars represent 20 mum. B: mRNA expression levels of genes coding for the sodium channel subtypes NAV1.1 - NAV1.9 and TRPV1, P2X3 and TRK1 as nociceptor typical receptors.
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Fig. 2), suggesting that the Nav1.9 variant may not interfere with the response to therapy. In order to identify potential treatment options for the patient, we reprogrammed the patient's fibroblasts into iPSC and differentiated these into peripheral sensory neurons using a small molecule approach (Fig. 1,). iPSC derived sensory neurons expressed markers for nociceptors, such as TRPV1, and the sodium channels Nav1.7, Nav1.8 and Nav1.9 shown in mRNA expression levels and protein immune-stainings (, Fig. 2). Cell morphology and expression of nociceptor markers were unchanged compared to two age matched controls (Fig. 2): The amount of TRPV1+ cells out of TUJ1+ cells was 92.6% for Ctrls and 90,2% for SFN, amount of Nav1.9+ cells out of Peripherin+ cells in %: Ctrls: 99.6, SFN: 100.0.
SFN1-nociceptors exhibit enhanced spontaneous activity and increased AP firing in whole-cell current clamp recordings. A: Example traces of elicited action potentials (APs) at threshold and double threshold in SFN-nociceptors. B: AP threshold was not significantly different between controls and SFN derived nociceptors (-49.7 +- 0.7 mV (Ctrls); -49.2 +- 0.7 mV (SFN1); P > 0.05 one-way ANOVA); C: Quantification of the number of spontaneously firing IPSC-derived nociceptors of controls (grey) and the SFN patient (red) during whole cell patch clamping. 19.4% of the SFN derived nociceptors exhibit spontaneous activity (lower panels) whereas only 1.8% of the control derived nociceptors are spontaneously active (P = 0.0002; logistic regression model).
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Whereas the action potential threshold remained unaltered in SFN derived nociceptors (Fig. 3, P > 0.05; one-way ANOVA), we detected a larger number of spontaneously active SFN sensory neurons (19.4%) compared to control (1.8%) (Fig. 3, P = 0.0002; logistic regression model), mirroring the findings of microneurography.
Spontaneous activity of SFN iPSC derived nociceptors in multi electrode array is reduced by lacosamide application.. A: Upper: nociceptors on MEA chip. Lower: Snapshot of a real time spike firing rate heat map of spontaneously active SFN- (right side) and Ctrl-(left side) nociceptors either mock treated (upper part) or with 500 muM lacosamide (lower part). The colour-coded scale bar shows spikes/s. B: Raster plot examples of spontaneous activity of Control (upper row) and SFN (lower row) iPSC derived nociceptors in multi electrode array with either mock treatment (left side) or with 500 muM lacosamide (right side) over 120 s. Each plot represents one well with 16 electrodes (rows). Each small vertical line indicates activity. Blue means high frequency bursting. Spontaneous activity is more pronounced in SFN iPSC derived nociceptors (upper row) in comparison to control nociceptors. Spontaneous activity is reduced by lacosamide application (left). C: Quantification of number of spikes, active electrodes, burst frequency and burst duration per well during 2 min recordings. First panel: Total number of spikes of all active electrodes during mock and lacosamide treatment are shown. Lacosamide significantly reduced number of spikes in SFN iPSC derived nociceptors (mock: Ctrl: 380.6 +- 207.9, SFN: 1206.0 +- 177.4, Lacosamide: Ctrl: 282.8 +- 184.2, SFN: 480.4 +- 310.2, P < 0.05, students t-test). Lacosamide did not significantly alter other parameters: Second panel: number of active electrodes (mock: Ctrl: 2.25 +- 1.01, SFN: 8.5 +- 2.23; Lacosamide: Ctrl: 2.20 +- 1.06, SFN: 5.13 +- 2.15; P > 0.05, students t-test.) Third panel: Burst frequency (in Hz: Mock: Ctrl: 0.008 +- 0.000, SFN: 0.060 +- 0.021; Lacosamide: Ctrl: 0.016 +- 0.004, SFN: 0.024 +- 0.016, P > 0.05, students t-test). Fourth panel: Burst duration (in s: Mock: Ctrl: 0.271 +- 0.007, SFN: 0.327 +- 0.0571, Lacosamide: Ctrl: 0.285 +- 0.033, SFN: 0.205 +- 0.043, P > 0.05, students t-test).
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To assess the overall cellular excitability in larger cell populations, we seeded patient- and control-derived nociceptors onto multielectrode array (MEA) chips and assessed their spontaneous activity (Fig. 4A, B). Patient-derived nociceptors displayed a significantly increased excitability: more spikes (Ctrl: 380.6 +- 207.9, SFN: 1206.0 +- 177.4, P < 0.05; one-way ANOVA) and more active electrodes (Ctrl: 2.25 +- 1.01, SFN: 8.5 +- 2.23, P < 0.05, students t-test) were detected (Fig. 4C). In MEA recordings of patient-derived nociceptors, lacosamide strongly reduced the number of spikes, but did not have an effect on controls (Fig. 4C, Ctrl: 282.8 +- 184.2, SFN: 480.4 +- 310.2, P < 0.05, students t-test), indicating that pathological hyperactivity was impaired, but the general action potential generation was not inhibited.
Microneurography reveals effective, individualized treatment of the patient.. A: Pain ratings of the SFN patient with and without lacosamide treatment. Pain ratings were considerably reduced during lacosamide treatment. Lacosamide treatment was interrupted due to side effects in combination with anti-allergic medication during summer and the same pain level was reported as before lacosamide treatment. After the end of allergic season lacosamide treatment again reduced the pain rating substantially.. B: Example traces of C-fiber activity during microneurography recordings. Depicted are response latencies to electrical test pulses (20 pulses every 8 s, 20 pulses every 4 s, 30 pulses every 2 s and then pulses every 4 s): each black point and triangle represents the latency of one electrically induced action potential, symbols arranged in a vertical line (means same response latency) indicate that the respective action potentials belong to one single C-fiber; left panel: irregular latency responses of two spontaneously active C-fibers before lacosamide, black arrows point to latency shifts indicating spontaneous activity between the electrically induced action potentials, the black inset shows the original recording traces from the C-fiber responses depicted in the black square besides. Action potentials are marked in red. Right panel: regular C-fibers responses to electrical test pulses of three C-fibers during lacosamide treatment without spontaneous activity.. C: Microneurography recordings show a large proportion of spontaneously active C-fibers (26%) before lacosamide treatment, which considerably decreased during treatment to 7% which is in the upper range of age matched healthy controls.
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Within five days the patient's pain was tremendously reduced, and peak pain ratings in the evenings dropped from VAS 7.5 to 1.5 (Fig. 5A). Re-start of lacosamide treatment in fall again dramatically decreased the patient's pain to VAS 1.5 (Fig. 5A). The proportion of spontaneously active C-fibers was significantly reduced to 7% (from 26.3% reported previously,Fig. 5B, C), which represents a range comparable to healthy age-matched volunteers.