Validating visual evoked potentials as a preclinical, quantitative biomarker for remyelination efficacy

Abstract

Many biomarkers in clinical neuroscience lack pathological certification. This issue is potentially a significant contributor to the limited success of neuroprotective and neurorestorative therapies for human neurological disease-and is evident even in areas with therapeutic promise such as myelin repair. Despite the identification of promising remyelinating candidates, biologically validated methods to demonstrate therapeutic efficacy or provide robust preclinical evidence of remyelination in the CNS are lacking. Therapies with potential to remyelinate the CNS constitute one of the most promising and highly anticipated therapeutic developments in the pipeline to treat multiple sclerosis and other demyelinating diseases. The optic nerve has been proposed as an informative pathway to monitor remyelination in animals and human subjects. Recent clinical trials using visual evoked potential have had promising results, but without unequivocal evidence about the cellular and molecular basis for signal changes on visual evoked potential, the interpretation of these trials is constrained. The visual evoked potential was originally developed and used in the clinic as a diagnostic tool but its use as a quantitative method for assessing therapeutic response requires certification of its biological specificity. Here, using the tools of experimental pathology we demonstrate that quantitative measurements of myelination using both histopathological measures of nodal structure and ultrastructural assessments correspond to visual evoked potential latency in both inflammatory and chemical models of demyelination. Visual evoked potential latency improves after treatment with a tool remyelinating compound (clemastine), mirroring both quantitative and qualitative myelin assessment. Furthermore, clemastine does not improve visual evoked potential latency following demyelinating injury when administered to a transgenic animal incapable of forming new myelin. Therefore, using the capacity for therapeutic enhancement and biological loss of function we demonstrate conclusively that visual evoked potential measures myelin status and is thereby a validated tool for preclinical verification of remyelination.

Keywords: clemastine; demyelination; remyelination; visual evoked potential.

Figure 1

Effect of clemastine on remyelination in the EAE model. (A) Prolongation of VEP latency precedes clinical onset (grey area) of EAE (grey dots represent EAE score). VEP latency (in orange) is delayed 5 days post-immunization when compared with sham-immunized mice (wine red). N1 latency increases through Day 18 with subsequent improvement. (B) Optic nerve CASPR quantification in EAE mice. This mirrors A showing the histopathological correlate of N1 delay. (C–E) Examples of CASPR doublets from healthy optic nerve (C) and EAE mice 15 days and 40 post-immunization. (D and E). (F) Effect of clemastine on VEP latency in EAE mice. (G and H) Examples of ON EM micrographs from EAE mice treated with vehicle (G) and clemastine (H) 28 days post-immunization. Three mice were analysed per group. Note unmyelinated axons (yellow asterisk) and remyelinating axons (light blue asterisk). (I) G-ratios of optic nerve axons 28 days post-immunization. (J) Quantification of unmyelinated (g-ratio = 1) and remyelinating (0.8 < g-ratio < 1) axons. (K) CASPR quantification shows improvement of paranodal density (count × 10³/mm²) in mice treated with clemastine compared with vehicle. Error bars describe SEM for latency change and EAE score graphs, SD for the other graphs.

Figure 2

Effect of clemastine on remyelination after toxic demyelination by cuprizone including a model with no capacity for forming new myelin. (A) Cuprizone diet provokes N1 latency delay. The graph shows VEP latency in healthy subjects (average N1 = 86 ms, SD 6.7) and mice after 5 weeks of cuprizone diet (average N1 = 101.6 ms, SD 15), P = 0.002. (B) CASPR staining of ONs (5 weeks of cuprizone diet, followed by 2 weeks of treatment with clemastine/vehicle) shows a higher density (count × 10³/mm²) of paranodes in clemastine-treated mice. (C) Clemastine enhances the degree and pace of latency recovery (P = 0.007 at 14 days). (D and E) Examples of EM micrographs of mouse ONs after 5 weeks of cuprizone followed by 2 weeks with vehicle (D) or clemastine (E). (F) G-ratios of ON axons from the same experiment (five mice analysed per group). (G) Quantification of unmyelinated (g-ratio = 1) and remyelinating (0.8 < g-ratio < 1) axons. (H) Absence of improvement in delay of N1 latency after discontinuing cuprizone diet in NG2creERT^+/−Myrf^flox/flox mice, even if treated with a remyelinating compound (clemastine). Clemastine enhances the degree and pace of latency recovery in wild-type [mean (SEM) −14.3 ms (4.1)] mice but not in NG2creERT^+/−Myrf^flox/flox mice [mean (SEM) 0.8 ms (1.3)]. Error bars represent SEM for latency change.

Figure 3

Schematic representation of experiment documenting the contingency of VEP improvement on remyelination. The provision of clemastine that induces OPC differentiation leads to enhanced VEP recovery following chemical demyelination with cuprizone. Blocking OPC’s capacity to differentiate via the inducible conditional knockout of MyRF from oligodendrocyte lineage cells leads to complete abrogation of the capacity to recover VEP latency following cuprizone-induced demyelination. (A) Following cuprizone-induced demyelination for 5 weeks, spontaneous OPC differentiation and resultant remyelination leads to moderate improvement in VEP latency. Vehicle (20% kleptose, 10 ml/kg) was dosed per os daily. (B) Conditional knockout of MyRF^flox/flox following tamoxifen dosing (300 mg tamoxifen/kg body for four consecutive days starting 7 days before the start of the cuprizone diet) in creERT⁺ animals on an NG2 promoter. Vehicle-treated MyRF icKO animals cannot form new myelin and show no VEP latency improvement after cuprizone discontinuation. Vehicle (20% kleptose, 10 ml/kg) was dosed per os daily. (C) Daily administration of clemastine (10 mg/kg, prepared in 20% kleptose) to wild-type mice leads to increased remyelination and a significant enhancement of the improvement in VEP latency. (D) Despite the administration of clemastine as in C the enhanced recovery of VEP latency is lost in animals unable to form new myelin. This group reproduces the same results seen in condition (B). Clemastine was dosed per os daily (10 mg/kg prepared in 20% kleptose).