November 2024
Volume 65, Issue 13
Open Access
Visual Neuroscience  |   November 2024
VEP Latency Delay Reflects Demyelination Beyond the Optic Nerve in the Cuprizone Model
Author Affiliations & Notes
  • Roshana Vander Wall
    Faculty of Medicine Health and Human Sciences, Macquarie University, North Ryde, NSW, Australia
  • Devaraj Basavarajappa
    Faculty of Medicine Health and Human Sciences, Macquarie University, North Ryde, NSW, Australia
  • Viswanthram Palanivel
    Faculty of Medicine Health and Human Sciences, Macquarie University, North Ryde, NSW, Australia
  • Samridhi Sharma
    Garvan Institute of Medical Research, Sydney, NSW, Australia
  • Vivek Gupta
    Faculty of Medicine Health and Human Sciences, Macquarie University, North Ryde, NSW, Australia
  • Alexander Klistoner
    Faculty of Medicine Health and Human Sciences, Macquarie University, North Ryde, NSW, Australia
  • Stuart Graham
    Faculty of Medicine Health and Human Sciences, Macquarie University, North Ryde, NSW, Australia
    Save Sight Institute, Sydney University, Sydney, NSW, Australia
  • Yuyi You
    Faculty of Medicine Health and Human Sciences, Macquarie University, North Ryde, NSW, Australia
    Save Sight Institute, Sydney University, Sydney, NSW, Australia
  • Correspondence: Roshana Vander Wall, Department of Medicine, Health and Human Sciences, Macquarie University, L1 75 Talavera Rd., Macquarie Park, NSW 2113, Australia; [email protected]
Investigative Ophthalmology & Visual Science November 2024, Vol.65, 50. doi:https://doi.org/10.1167/iovs.65.13.50
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      Roshana Vander Wall, Devaraj Basavarajappa, Viswanthram Palanivel, Samridhi Sharma, Vivek Gupta, Alexander Klistoner, Stuart Graham, Yuyi You; VEP Latency Delay Reflects Demyelination Beyond the Optic Nerve in the Cuprizone Model. Invest. Ophthalmol. Vis. Sci. 2024;65(13):50. https://doi.org/10.1167/iovs.65.13.50.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: Remyelination therapies are advancing for multiple sclerosis, focusing on visual pathways and using visual evoked potentials (VEPs) for de/remyelination processes. While the cuprizone (CZ) model and VEPs are core tools in preclinical trials, many overlook the posterior visual pathway. This study aimed to assess functional and structural changes across the murine visual pathway during de/remyelination.

Methods: One group of C57BL/6 mice underwent a CZ diet for 6 weeks to simulate demyelination, with a subset returning to a regular diet to induce remyelination. An additional group was fed a protracted CZ diet for 12 weeks to maintain chronic demyelination. Visual function was evaluated using electrophysiological recordings, including scotopic threshold responses (STRs) and electroretinograms (ERGs), with VEPs serving as a key biomarker for overall pathway health. Tissues from eyes, brains, and optic nerves (ONs) were collected at different time points for structural analysis.

Results: Our results demonstrated significant effects on VEPs, including increased N1 latencies and reduced amplitudes in the CZ mouse model. However, retinal function remained unaffected, as evidenced by unchanged STRs, ERGs, and retinal ganglion cell counts. Analysis of ONs revealed morphological changes, characterized by a significantly decreased axon diameter in the core region compared to the subpial region. Additionally, there was a significant increase in the g-ratio of the core region at 12 weeks CZ compared to controls. Immunofluorescence further demonstrated a decrease in myelin basic protein levels at 6 and 12 weeks in CZ animals. Interestingly, the dorsal lateral geniculate nucleus and primary visual cortex (V1) exhibited similar myelin changes, correlating with VEP latency alterations.

Conclusions: These data reveal that interpreting VEP latency solely as a marker for ON demyelination is incomplete. Previous preclinical studies have overlooked the posterior visual pathways, necessitating a broader interpretation of VEP latency to cover the entire visual pathway.

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS) characterized by focal demyelinating lesions and axonal dysfunction/degeneration that results in variable clinical disability. This demyelination is naturally combatted by remyelination from mature oligodendrocytes, although after some time, these are reduced in MS, leading to worsening of the condition.1,2 Consequently, there is a growing focus on developing therapies to promote remyelination. The visual system frequently serves as a critical pathway for understanding the dynamic interplay between demyelination and axonal degeneration in the CNS. Visual evoked potential (VEP) assessments, a noninvasive technique, are frequently utilized to evaluate the functionality of the visual pathway. Originally employed to assess optic neuropathies, VEPs have since been adapted to assess demyelination within the visual pathway and subsequent remyelination, whether triggered spontaneously or induced by treatment.36 VEPs are currently the preferred method of measuring de/remyelination in patients with MS with VEP latency frequently used as the primary endpoint in clinical trials.7 Unfortunately, these endeavors have been met with limited success, and further research is required.8,9 
The cuprizone (CZ) model is a commonly utilized mouse model to evaluate the remyelination therapies for safety and effectiveness in preclinical studies. Cuprizone, a copper chelator, induces demyelination in the CNS by triggering oligodendrocyte death.10,11 Typically, CZ is incorporated into the animals’ diet, and the key feature is that upon return to a normal diet, remyelination occurs, thereby mimicking relapse remitting multiple sclerosis (RRMS).12,13 These properties have made it attractive as a model for preclinical drug trials that focus on remyelination and have been assessed in conjunction with VEPs.1417 Despite the usefulness of this combination, there have not yet been studies that addressed demyelination in the entire visual pathway, with most focusing on the optic nerve (ON).3,18,19 Therefore, based on previous research utilizing CZ models, two key time points were chosen to best illustrate both de/remyelination and chronic demyelination, employing 6-week and 12-week models, respectively.20,21 While 6 weeks is sufficient for demyelination in the posterior pathways, with remyelination occurring upon removal of CZ, it does not result in significant demyelination in the ON. Therefore, to fully explore demyelinating changes to the ON and chronic demyelination, the 12-week group was added. 
The caveat facing VEPs in preclinical trials is the common interpretation that latency delay is primarily indicative of demyelination in the ON, all but ignoring potential issues in the posterior visual pathway. Relying solely on VEP changes as a key biomarker for demyelination and axonal damage poses limitations, as it reflects alterations in the entire visual pathway without pinpointing specific affected segments. A comprehensive evaluation of functional changes along with glial activation, synaptic, and neuronal alterations across the entire visual pathway during spontaneous demyelination/remyelination and chronic demyelination provides a deeper understanding of the axonal degenerative process.22 In this study, we have evaluated the functional and structural alterations occurring in the CZ model of demyelination/remyelination along the entire murine visual pathway to gain insights into the extent to which these changes influence VEP readings and assess their potential clinical relevance. 
Methods
Animal Welfare
All animal experiments in this study were performed in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes and the guidelines of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and all procedures were approved by Macquarie University Animal Ethics Committee. Male and female C57BL/6 mice were used in this study (age 8 weeks, Animal Research Centre, Perth, Australia, n = 62). Animals were divided into a total of 8 groups (n = 8 and n = 6 controls). All animals were maintained in an air-conditioned room with a controlled temperature (21 ± 2°C) and fixed daily 12-hour light/dark cycles. 
Cuprizone Diet
Demyelination was induced by feeding mice with a diet of 0.2% (w/w) cuprizone (bis-cyclohexanone oxaldihydrazone C9012; Sigma-Aldrich, St. Louis, MO, USA) mixed into ground standard rodent chow for 6 weeks (Specialty Feeds Pty Ltd, Glen Forest, WA, Australia), and age- and sex-matched control mice (n = 6) received regular ground chow. Food and water were made available ad libitum. To establish changes in the visual system over the course of cuprizone treatment, all animals underwent baseline electrophysiological recordings before commencing cuprizone feeding, followed by repeat electrophysiological recordings at specified time points (2, 4, and 6 weeks). After each time point, the allotted cohort (n = 8) was sacrificed, and the tissues were collected for analysis. Two more time points were included (1 and 2 weeks after cuprizone treatment was ceased and animals returned to regular chow) to assess any remyelination of the visual pathway (Fig. 1). Control animals were sacrificed at the 6+2-week time point. In accordance with previous findings concerning ON demyelination, we added the 12-week CZ feeding time point with concurrent control animals and repeated the procedures conducted on the initial cohort to assess chronic demyelination. An additional subset of a 12-week diet of animals with a 2-week recovery period was included to confirm the condition. 
Figure 1.
 
Timeline of CZ treatment. (A) Demyelination and spontaneous remyelination model. All animals underwent electrophysiological measurements at each time point, with a group of n = 8 sacrificed afterward to obtain histological data of that time point. (B) Chronic demyelination model, n = 8 animals fed a CZ diet for 12 weeks, with electrophysiological measurements taken at the above time points. All run concurrently with untreated control animals.
Figure 1.
 
Timeline of CZ treatment. (A) Demyelination and spontaneous remyelination model. All animals underwent electrophysiological measurements at each time point, with a group of n = 8 sacrificed afterward to obtain histological data of that time point. (B) Chronic demyelination model, n = 8 animals fed a CZ diet for 12 weeks, with electrophysiological measurements taken at the above time points. All run concurrently with untreated control animals.
Electrophysiological Recordings
To assess visual function, animals were dark adapted overnight before testing, and all electrophysiological recordings were taken in the dark with the aid of a red light. Scotopic threshold response (STR) and electroretinogram (ERG) recordings were performed as described previously.2325 The following day, animals were anesthetized with ketamine (50 mg/kg) (Provet, Sydney, NSW, Australia) and medetomidine (0.5 mg/kg) (Troy Laboratories, Glendenning, NSW, Australia) via intraperitoneal injection and placed on a warming pad. Once sufficiently anesthetized (paw pinch test), the pupils were dilated with topical tropicamide 1% (Alcon Laboratories, Macquarie Park, NSW, Australia) eye drops. At the conclusion of the procedure, anesthesia was reversed with a subcutaneous injection of atipamezole (0.75 mg/kg; Troy Laboratories) and animals monitored. 
Mice were then placed on a three-dimensional stage with a heating pad (Phoenix Research Labs, Pleasanton, CA, USA) for maneuverability and to maintain body temperature (36–38°C) in the ERG system (Phoenix Ganzfeld ERG; Phoenix Research Labs). Electrical contact with the cornea was achieved by a gold-plated objective lens through which light passed at the tip of the equipment’s Maxwellian lens. The reference needle electrode was placed subcutaneously between the eyes, and the ground electrode was placed subcutaneously at the base of the tail. Triggering of the light stimulus and acquisition of voltage traces were accomplished with the LabScribe2 software through the Phoenix Research Labs ERG module. STRs and ERGs were recorded using flash intensities of −4.3 and 3.1 log cd⋅s/m2 with an interstimulus interval of 2 and 5 seconds, respectively. For all positive STR responses, amplitudes were measured from baseline to the positive peak observed around 150 ms and averaged. For all ERG recordings, a-wave amplitude was measured from baseline to the a-wave trough; b-wave amplitude was measured from the a-wave trough to the peak of the b-wave as described previously.26,27 
For VEP measurement, a similar technique was employed pertaining to this apparatus. Ground and reference needle electrodes remained the same, but the lead connecting to the gold-plated objective lens was removed, and a needle electrode was attached to it so it could be inserted subcutaneously above the visual cortex, as has been done previously in a number of studies.28 VEPs were recorded using 60 flashes at the intensity of 3.1 log cd⋅s/m2 with an interstimulus interval of 2 seconds. Amplitude was measured from baseline to the negative peak (N1) trough observed around 120 ms and averaged; latency was measured from baseline to the N1 trough (Fig. 2). The N1 is comparable to p100 in humans, the parameter commonly used to measure visual pathway health.18 
Figure 2.
 
Sample trace of VEP illustrating amplitude and latency measurements of a healthy mouse.
Figure 2.
 
Sample trace of VEP illustrating amplitude and latency measurements of a healthy mouse.
Tissue Collection and Processing
Mice were euthanized at designated time intervals, and their tissues were collected after transcardial perfusion with PBS followed by freshly prepared 4% paraformaldehyde (PFA). The eyeballs were marked with a tissue marking dye (Polysciences, Warrington, PA, USA, #24108) to maintain a consistent orientation during tissue embedding. Half the animals from each group were processed for molecular experiments (n = 4 per group), while the other half will be used for histological analysis (n = 4 per group). 
Brains, both eyes, and the proximal half of both left and right ONs were placed in 4% PFA in PBS (pH 7) for paraffin embedding. Eyes and ONs were kept in this solution for 3 hours to allow adequate penetration into the tissue. All samples were washed three times in PBS before being stored in 70% ethanol before being processed via an automatic tissue processor (ASP200S; Leica, Germany) for paraffin wax embedding. 
The distal half of the ON was placed in 2% glutaraldehyde and 2% PFA in phosphate buffer (pH 7) for resin embedding. These were sectioned on an ultramicrotome (EM UC7 Ultracut S Ultra-microtome; Leica) using glass knives at 1 µm. Sections were stained with toluidine blue. Calculation of g-ratio was performed as previously described, using ImageJ (National Institutes of Health, Bethesda, MD, USA).3 Core and subpial regions of ONs were calculated by individual diameter in ImageJ. Based on changes seen in a previous study by Heidari et al.,3 core and subpial regions of ONs were calculated by individual diameter in ImageJ. This diameter was determined by forming a rectangle around the perimeter of the ON. Due to the slightly imperfect circle, the height and width were averaged and then halved to create a circle of that diameter within the center of the ON as the core region (Fig. 3). Subpial measurements were taken in the area outside of this circle, with core measurements taken from within (30 measurements per quadrant each). 
Figure 3.
 
Representative image of toluidine blue–stained optic nerve core-subpial sectioning for g-ratio calculation. Core diameter (D: 182.71 µm) is half of the average diameter of height (H) and width (W) (365.42 µm) and centered within the rectangle.
Figure 3.
 
Representative image of toluidine blue–stained optic nerve core-subpial sectioning for g-ratio calculation. Core diameter (D: 182.71 µm) is half of the average diameter of height (H) and width (W) (365.42 µm) and centered within the rectangle.
Sagittal and coronal sections were made of paraffin-embedded retinal and brain samples, respectively (7 µm). Cross-sectional slices of paraffin-embedded ON (5 µm) were also subject to immunofluorescence. Antigen retrieval using a citrate buffer was performed on paraffin-embedded slices for immunofluorescence. Sections were treated with a blocking buffer containing 0.3% Triton X-100 and 5% normal rat serum of the secondary species (Sigma-Aldrich) in PBS for 3 hours at room temperature. The sections were then incubated with the indicated primary antibodies overnight at 4°C prepared in antibody dilution buffer (3% BSA, 0.3% Triton X-100 in PBS). The following antibodies were used: vGlut2 (1:500, #ab2251-I; Sigma-Aldrich), myelin basic protein (MBP; 1:1000, #ab218011; Abcam, Cambridge, MA, USA), GFAP (1:1000, #3670; New England Biolabs, Notting Hill, VIC, Australia), Iba1 (1:1000, #019-19741; Fujifillm, Osaka, Japan), NeuN (1:1000, #ab104224; Abcam). Following this, sections were incubated with Alexa Fluor 488-, 555-, and 647-labeled secondary antibodies (1:500 in antibody dilution buffer; Jackson, #715-545-150, #711-165-152, #106-605-003) for 1 hour in the dark and coverslipped with Dako mounting medium (Agilent Technologies, Mulgrave, VIC, Australia).29 Cell counting for microglia was achieved by identifying DAPI (+) nuclei with the cell body displaying the immunoreactivity of Iba1. 
Retinal ganglion cell counts were obtained from the images stained with hematoxylin and eosin (H&E) (Sigma-Aldrich). Cell density in the ganglion cell layer (GCL) was determined for each eye by counting the number of cells in the middle portion of the retina over a distance of 300 µm (200–500 µm from the edge of the optic disc) using Zen Blue software (Carl Zeiss, Jena, Germany) as previously described.24,30 
Imaging for both immunofluorescence and light microscopy was performed on the Zeiss AxioImager (Carl Zeiss). 
Statistical Analysis
Statistical analysis of all data obtained in this study was performed using GraphPad Prism 9 software (GraphPad Software, San Diego, CA, USA). 
The animal tissues from the experimental groups were utilized to analyze the V1 and dorsal lateral geniculate nucleus (dLGN) changes by Western blotting of target proteins and immunofluorescence staining of brain, retinal, and ON tissues. The number of samples (n) indicated in each figure represents the tissues from the different animals of the same group. One-way ANOVA followed by Tukey's multiple comparisons test was performed to assess comparisons between the groups for both histological and electrophysiological data. All data are presented as mean ± standard deviation for given n sizes, and P < 0.05 was considered statistically significant for data analysis. 
Results
Visual Pathway Functions Are Affected in the CZ Model
To evaluate the effects of CZ on the higher visual pathway, mice were subjected to functional analysis by recording VEPs. To eliminate VEP variability between recordings, all measurements were normalized to control animal VEPs performed on the same day. We observed N1 latencies at all time points were significantly delayed compared to baseline (P < 0.0001) (Fig. 4B, Supplementary Table S1). This included the two recovery time points (P < 0.0005), although this delay does appear to be reducing. Similar results were seen in other de/remyelination studies, showing that even 2 weeks after discontinuation of the cuprizone diet, VEP latency did not recover to baseline levels.18,31 To include adequate demyelination in the ON, which is closely correlated with VEP latency increase, we included a cohort of animals fed on CZ for 12 weeks. This condition allowed the ON to demyelinate sufficiently, as stated previously.20 A significant increase in latency was observed at 12 weeks compared to both baseline and the 6-week time point measurement (Figs. 4D, 4E). 
Figure 4.
 
CZ diet affects visual evoked potentials. Top: Original cohort. (A) Average VEPs of all time points. At each time point, n ≥ 8. (B) Average VEP latency at each time point normalized to control readings at respective time points. n ≥ 8 at each time point. (C) Average amplitude of a-wave of VEPs normalized to control readings at each time point. Bottom: 12-week cohort. (D) Average VEPs of 12-week animals at key time points. n = 8. (E) Average VEP latency at key time points normalized to control readings at respective time points. n = 8 at each time point. (F) Average amplitude of a-wave of VEPs normalized to control readings at key time points. Amplitude (µV), latency (ms), *P < 0.05, ****P < 0.0001 compared to baseline. #P < 0.05 compared to 6 weeks.
Figure 4.
 
CZ diet affects visual evoked potentials. Top: Original cohort. (A) Average VEPs of all time points. At each time point, n ≥ 8. (B) Average VEP latency at each time point normalized to control readings at respective time points. n ≥ 8 at each time point. (C) Average amplitude of a-wave of VEPs normalized to control readings at each time point. Bottom: 12-week cohort. (D) Average VEPs of 12-week animals at key time points. n = 8. (E) Average VEP latency at key time points normalized to control readings at respective time points. n = 8 at each time point. (F) Average amplitude of a-wave of VEPs normalized to control readings at key time points. Amplitude (µV), latency (ms), *P < 0.05, ****P < 0.0001 compared to baseline. #P < 0.05 compared to 6 weeks.
VEP N1 amplitude was significantly reduced at 6 weeks of treatment compared to baseline (P < 0.0001) (Fig. 4C). We observed a significant, near-baseline recovery after cessation of the CZ diet at the 6+1-week and 6+2-week time points compared to 6 weeks (P < 0.05). These results demonstrated that animals on the CZ diet displayed functional abnormalities in their visual pathway, with recovery observed upon returning to a normal diet for 1 to 2 weeks. We sought to investigate if a comparable recovery could occur after 12 weeks of chronic demyelination. However, animals subjected to a 12-week CZ diet followed by a 2-week recovery period did not exhibit significant functional improvements (Supplementary Fig. S1). 
No Functional or Pathological Changes in the Retina in CZ-Treated Mice
We next assessed the retinal function to determine the effect of the CZ diet. The STR and ERG recordings were performed on all cohorts to assess inner retinal and gross retinal function, respectively.32,33 Multiple comparisons revealed no changes across all groups, indicating that there was no impact of CZ on the inner GCL function (Figs. 5A, 5B, Supplementary Table S2). Further GCL cell density analysis using H&E sections of the retinas revealed no significant changes in GCL cell counts between controls and any other time point or across time points (Figs. 5C, 5D). These data suggest no significant impact of CZ diet on the retina, which aligns with the existing evidence that CZ does not affect the retina.20,22 
Figure 5.
 
Functional and structural analysis of the mice retinas treated with CZ. Electrophysiological recordings and retinal ganglion cell count (RGC) in all groups. (A) Average ERGs at each time point, n = 8. (B) Average STRs at each time point, n = 8. (C) Average RGC count per group; control, 2 weeks, 4 weeks, 6 weeks, 6+1 weeks, 6+2 weeks, 12 weeks. Counts taken over a distance of 300 µm (200–500 µm from the edge of the optic disc). (D) Representative images of H&E-stained retinas from all groups (12 weeks in supplementary data), n = 3 animals per group. Scale bar: 50 µm and 100 µm, respectively.
Figure 5.
 
Functional and structural analysis of the mice retinas treated with CZ. Electrophysiological recordings and retinal ganglion cell count (RGC) in all groups. (A) Average ERGs at each time point, n = 8. (B) Average STRs at each time point, n = 8. (C) Average RGC count per group; control, 2 weeks, 4 weeks, 6 weeks, 6+1 weeks, 6+2 weeks, 12 weeks. Counts taken over a distance of 300 µm (200–500 µm from the edge of the optic disc). (D) Representative images of H&E-stained retinas from all groups (12 weeks in supplementary data), n = 3 animals per group. Scale bar: 50 µm and 100 µm, respectively.
CZ Affects Myelin Morphology in the Optic Nerve
Structural changes to the ON in CZ models are the most frequently documented portion of the visual pathway, using the g-ratio (axon diameter ÷ axon + myelin sheath diameter) to determine de/remyelination status. Therefore, 1-µm-thick sections of ON from controls and 6-week, 6+2-week, and 12-week groups were stained with the toluidine blue for myelin and the g-ratio of each region measured.34 Using Šídák's multiple comparisons ANOVA, we sought to determine if changes were occurring between the core and subpial regions of the ON within time points, as shown previously.3 Changes were most apparent in the core of the ON compared to the subpial zone, which appeared unchanged across treatment groups. We noted no changes between regions in controls or 6 weeks or 6+2 weeks, but the 12-week chronic demyelination time point showed a significant difference between subpial and core regions (Fig. 6C, P < 0.001). A change in morphology was observed in the core region in 6-week CZ-treated animals, and after performing a t-test between regions of each respective time point, the axon diameter of the core was significantly smaller than the subpial zone in the 6-week time point and was similarly altered at 12 weeks, although not significantly (Supplementary Fig. S2). This trend reversed after the 6+2-week recovery time point. Despite this change in axon diameter, there was no difference in g-ratio observed between regions within time points. Therefore, Tukey multiple comparisons ANOVA was performed between all groups, comparing core and subpial treatment groups to respective controls, resulting in a significant difference in g-ratio between control and 12-week core regions of the ON (Fig. 6C, P < 0.001). This result aligns with previous studies in this model.20 
Figure 6.
 
Optic nerve g-ratio analysis. (A) representative optic nerve cross sections stained with toluidine blue of control and 6-, 6+2-, and 12-week time points. Scale bar: 10 µm. (B) The g-ratio graphed to correlate with axon diameter (≥110 measurements per animal, n = 3 per time point). (C) Average g-ratio of core and subpial zone of optic nerves in panel B (**P < 0.001 compared to control region, #P < 0.001 compared to subpial, n = 3 per group).
Figure 6.
 
Optic nerve g-ratio analysis. (A) representative optic nerve cross sections stained with toluidine blue of control and 6-, 6+2-, and 12-week time points. Scale bar: 10 µm. (B) The g-ratio graphed to correlate with axon diameter (≥110 measurements per animal, n = 3 per time point). (C) Average g-ratio of core and subpial zone of optic nerves in panel B (**P < 0.001 compared to control region, #P < 0.001 compared to subpial, n = 3 per group).
Immunofluorescence staining of ONs revealed significantly lower MBP at the 6-week time point compared to control and 6+2-week recovery animals (Figs. 7A, 7B). The normalized VEP latencies were correlated to the average fluorescence intensity of MBP from respective animals (Fig. 7C) and had a significant inverse relationship (r = −0.65, P < 0.005). 
Figure 7.
 
Optic nerve de/remyelination and latency correlation. (A) Representative images of optic nerve immunofluorescent staining for MBP magnified 100×. Scale bar: 10 µm. (B) Quantified average fluorescent intensity of MBP staining. (C) Relationship between average above myelin quantity and respective latency of animals. Each dot represents one animal; the average MBP fluorescence intensity from three ON sections per animal compared to the VEP latency obtained at the time point before sacrifice (r = −0.65, P < 0.005). *P < 0.05, compared to controls.
Figure 7.
 
Optic nerve de/remyelination and latency correlation. (A) Representative images of optic nerve immunofluorescent staining for MBP magnified 100×. Scale bar: 10 µm. (B) Quantified average fluorescent intensity of MBP staining. (C) Relationship between average above myelin quantity and respective latency of animals. Each dot represents one animal; the average MBP fluorescence intensity from three ON sections per animal compared to the VEP latency obtained at the time point before sacrifice (r = −0.65, P < 0.005). *P < 0.05, compared to controls.
To assess glial activity, Iba1 counts were performed on ON cross sections, although they were not significantly changed between groups but exhibited an upward trend as CZ treatment progressed, with a decrease in recovery animals (Supplementary Fig. S3). 
Demyelination and Spontaneous Remyelination in the Posterior Visual Pathway Changes in CZ Mice
We next evaluated myelin changes in the visual pathway centers of the brain by immunofluorescence staining with MBP antibody for myelin changes, and vesicular glutamate transporter 2 (vGlut2) for excitatory synaptic changes in the dLGN was confirmed by significant corresponding MBP immunofluorescence staining in the dLGN across cohorts (one-way multiple comparison ANOVA, P < 0.0001) (Figs. 8A, 8B). vGlut2 was stained to confirm synaptic plasticity alterations in the dLGN in CZ-treated mice (one-way multiple comparison ANOVA, P < 0.0001). Results were consistent with previous findings.22 The Western blot data follow a similar trend as the immunofluorescence (Supplementary Fig. S4). Correlation of average fluorescence intensity of MBP in dLGN with the respective animals’ latencies showed a significant inverse relationship (r = −0.48, P < 0.05, Fig. 8C). 
Figure 8.
 
Changes in the dorsal lateral geniculate nucleus. (A) Representative immunofluorescence staining of dLGN, vGlut2, and MBP. A representative image of the region with schematic overlay in Supplementary Fig. 6. (B) Quantification of the average MBP and GFAP fluorescence intensity from ≥3 dLGN sections per animal, ≥3 animals per time point. (C) Relationship between average above myelin quantity and respective latency of animals. Each dot represents one animal; the average MBP fluorescence intensity from ≥3 dLGN sections compared to the VEP latency obtained at the time point before sacrifice (r = −0.48, P < 0.05). *P < 0.05, compared to controls.
Figure 8.
 
Changes in the dorsal lateral geniculate nucleus. (A) Representative immunofluorescence staining of dLGN, vGlut2, and MBP. A representative image of the region with schematic overlay in Supplementary Fig. 6. (B) Quantification of the average MBP and GFAP fluorescence intensity from ≥3 dLGN sections per animal, ≥3 animals per time point. (C) Relationship between average above myelin quantity and respective latency of animals. Each dot represents one animal; the average MBP fluorescence intensity from ≥3 dLGN sections compared to the VEP latency obtained at the time point before sacrifice (r = −0.48, P < 0.05). *P < 0.05, compared to controls.
The V1 region of the visual cortex exhibited a similar pattern of myelin changes (Figs. 9A, 9B), showing a significant decrease in MBP at 6 and 12 weeks, followed by a recovery (one-way multiple comparison ANOVA), confirmed with Western blot data (P < 0.05, Supplementary Fig. S5). The opposite was true for GFAP, showing a significant increase at 6 weeks compared to baseline, indicating increased glial activity, P < 0.05. Unlike the dLGN, the V1 exhibited an unaccounted-for increase in vGlut2 at 12 weeks. The relationship between demyelination and VEP latency in the V1 conforms with both ON and dLGN results (r = −0.64, P < 0.005, Fig. 9C), revealing that along the length of the visual pathway, demyelination in each of these key regions affects the VEP latency. 
Figure 9.
 
Changes in the visual cortex. (A) Representative immunofluorescence staining of V1, vGlut2, and MBP. (B) Quantification of the average MBP and GFAP fluorescence intensity from ≥3 V1 sections per animal, ≥3 animals per time point. (C) Relationship between average above myelin quantity and respective latency of animals. Each dot represents one animal; the average MBP quantity from immunofluorescence compared to the VEP latency obtained at the time point before sacrifice (r = −0.64, **P < 0.005). *P < 0.05, **P < 0.005, ****P < 0.0001.
Figure 9.
 
Changes in the visual cortex. (A) Representative immunofluorescence staining of V1, vGlut2, and MBP. (B) Quantification of the average MBP and GFAP fluorescence intensity from ≥3 V1 sections per animal, ≥3 animals per time point. (C) Relationship between average above myelin quantity and respective latency of animals. Each dot represents one animal; the average MBP quantity from immunofluorescence compared to the VEP latency obtained at the time point before sacrifice (r = −0.64, **P < 0.005). *P < 0.05, **P < 0.005, ****P < 0.0001.
Discussion
This study has documented the functional, molecular, and morphological changes caused by CZ in both the demyelination/spontaneous remyelination and chronic demyelination conditions. We tracked myelin density changes along the visual pathway aligning with the VEP latencies at the time points of CZ treatment and established a significant negative correlation. We observed alterations in VEP waveforms in the 6-week CZ treatment group despite limited g-ratio change, indicating that VEPs are representative of more than ON demyelination/damage. 
A number of studies have investigated the effects of CZ in the CNS, with several considering the visual pathway, with a particular focus on the visual pathway. However, until now, documentation of myelin changes throughout this pathway has been incomplete.20,22,35 Few studies have integrated these findings with functional changes represented by VEPs, and those that have tend to concentrate on the ON while overlooking changes in the posterior visual pathway.3,17,18 This bias is partly due to the fact that 25% of patients with MS experience their first clinical demyelinating event in the ON, making it a prominent area of study compared to other visual pathway structures.36 Admittedly, more clinicians are pushing for the ON to be included in the current diagnostic criteria for MS, and VEPs are being used more frequently as a diagnostic tool.37,38 However, it would be remiss to neglect the remaining aspects of the visual pathway and how changes thereto affect VEPs. To address these discrepancies, this study has compiled the comprehensive effects of CZ within the visual pathway in both spontaneous remyelination and chronic conditions, correlating these effects with functional VEP output. In doing so, we have established changes in myelin density and structural morphology at key landmarks in the visual pathway. 
In this study, we observed a significant increase in VEP latency in CZ-treated animals at the 6- and 12-week time point. This phenomenon, noted in several prior studies utilizing demyelinating models, is the metric for ON health assessment in humans.3943 Accompanying these functional changes was an interesting morphological change in the core region of the ON. In a study, Marenna et al.17 demonstrated that the ON suffers some demyelination and morphological changes by way of altered longitudinal thickness, with remyelinated mouse ONs found to be significantly thicker than healthy and CZ-treated animals. Heidari et al.3 noted gradient changes to the core of the ON in a de/remyelinating model in cats. Importantly, in the disease state, the distal end of the ON was most severely demyelinated, especially at the core, which contains a higher proportion of large-diameter axons. Another pertinent finding from their study was that small/medium-diameter axons of the subpia were preferentially remyelinated over those in the core. Combined with the ON core changes in our 6- and 12-week cohort, this fosters the possibility that demyelination occurs from the inside out in such models. 
To illustrate the efficacy of using VEPs as a preclinical tool for remyelination outcomes, Cordano et al.18 performed an in-depth remyelinating drug study in demyelinating models, including CZ, in the ON. By combining the needle electrode technique, similar to that employed here, with electron microscopy to accurately determine g-ratios and corresponding VEP latencies, they successfully demonstrated that increased latency was coupled with an increase in g-ratio, with the inverse occurring during remyelination. However, they did not include any data pertaining to VEP amplitude. 
CZ damage appears to spread in a retrograde fashion, showing demyelination in the V1 but almost none in the ON within the 6-week timeframe.20,21 This study was protracted to include a 12-week CZ treatment time point, at which both significant demyelination and VEP latency increase were observed compared to control. This poses the question of why the ON resists demyelination for longer compared with the cerebrum. There are a few postulations such as regional differences in cytokine expression from astrocytes and their effects on oligodendrocyte precursor cells, the origin of oligodendrocyte development and differentiation at various developmental stages, or even the length of the myelin sheaths.4448 
A portion of the visual pathway not commonly referred to in VEP-related studies are optic radiations. Abnormalities of optic radiations are also represented by VEP latency.49,50 Given that we did not see a sufficient decrease in the latency of the recovery groups, it is possible that they are not yet completely remyelinated. Further experiments will need to be conducted to determine more in-depth changes in optic radiations. Another factor that can reveal optic radiation malfunction is decreased VEP amplitude, due to possible demyelination-related conduction block. Numerous studies have shown that optic neuritis is correlated with latency increase and amplitude decrease, where latency reflects myelin health and amplitude indicates axonal damage.42,51,52 In the current study, a decrease in VEP amplitude, typically associated with axonal loss, was observed, despite the absence of significant axonal damage.53 
One possible explanation for this phenomenon is the occurrence of conduction block.54 Myelin-related conduction block is the result of voltage-gated sodium channel disruption combined with a lack of saltatory conduction along the demyelinated axon.55 Oligodendrocytes preferentially myelinate electrically active axons, with vGlut2 decreasing when electrical activity is diminished.56 We observed more severe demyelination in the posterior visual pathway (dLGN and V1) at both the 6- and 12-week time points, supported by other studies reporting similar results around the 5-week period.21,35 These results were coupled with a significant reduction in amplitude, which was then recovered at 6+1-week and 6+2-week time points. With the gradual loss of myelin and subsequent diminishment of electrical activity in higher-order axons, it is possible that the resultant vGlut2 reduction during demyelination in this study is a by-product of conduction block. However, the chronic CZ model confounds this, with vGlut2 increased at 12 weeks in the V1. vGLUT2, typically reflective of glutamatergic innervation, has been observed to increase with CZ feeding in the hippocampus at 6 weeks. This study posits that this is likely the result of the engulfment of this and other synaptic proteins by CZ-activated microglia.57 The quantification for this result is based on fluorescence and therefore will need to be quantified in future studies using Western blots or enzyme-linked immunosorbent assays to determine a more accurate result. 
The variance in myelin alterations between the anterior and posterior visual pathways might stem from the diverse morphologies of oligodendrocytes across these structures. Variations such as internodal length and myelin thickness can significantly influence conduction.48 Additionally, it has also been shown that the sodium channel clusters that are broken up during demyelination reform during remyelination, accounting for the amplitude recovery at the 6+1 and 6+2 time points.47,54 The recovery of amplitude is most likely due to a combination of both sodium channel reformation and new oligodendrocyte production. Pfeiffer et al.58 noted that the number of nodes of Ranvier increased following CZ treatment, suggesting that newer myelin sheaths are shorter but more numerous. 
To better illustrate changes in the visual pathway in the future, immunofluorescent staining with transferrin receptor 1 (Tfr1) or sodium channel markers and CASPR would provide greater insight into cell survival and axonal changes occurring during de/remyelination, respectively.47,59 
Conclusions
By integrating in vivo functional tracking of disease progression with histological and molecular analyses at critical anatomical checkpoints along the visual pathway, this study achieved a deeper understanding of subtle alterations. Our results revealed a significant negative correlation between latency delay and myelin, not only in the ON but also throughout the visual pathway, while VEP amplitude reduction is reflective of demyelination/demyelination-related conduction block in the posterior visual pathway in mice. Therefore, it would be prudent for preclinical and clinical studies going forward to also include demyelination of the posterior visual pathway in VEP analysis. 
Acknowledgments
The authors thank Arthur Chein and Sue Lindsay of the Macquarie University Microscopy Unit for their assistance with the resin-embedded optic nerve experiment and use of their facilities. 
Supported in part by a grant from the National Multiple Sclerosis Society. 
Data supporting the findings of this study are available upon reasonable request to the corresponding author. 
Disclosure: R. Vander Wall, None; D. Basavarajappa, None; V. Palanivel, None; S. Sharma, None; V. Gupta, None; A. Klistoner, None; S. Graham, None; Y. You, None 
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Figure 1.
 
Timeline of CZ treatment. (A) Demyelination and spontaneous remyelination model. All animals underwent electrophysiological measurements at each time point, with a group of n = 8 sacrificed afterward to obtain histological data of that time point. (B) Chronic demyelination model, n = 8 animals fed a CZ diet for 12 weeks, with electrophysiological measurements taken at the above time points. All run concurrently with untreated control animals.
Figure 1.
 
Timeline of CZ treatment. (A) Demyelination and spontaneous remyelination model. All animals underwent electrophysiological measurements at each time point, with a group of n = 8 sacrificed afterward to obtain histological data of that time point. (B) Chronic demyelination model, n = 8 animals fed a CZ diet for 12 weeks, with electrophysiological measurements taken at the above time points. All run concurrently with untreated control animals.
Figure 2.
 
Sample trace of VEP illustrating amplitude and latency measurements of a healthy mouse.
Figure 2.
 
Sample trace of VEP illustrating amplitude and latency measurements of a healthy mouse.
Figure 3.
 
Representative image of toluidine blue–stained optic nerve core-subpial sectioning for g-ratio calculation. Core diameter (D: 182.71 µm) is half of the average diameter of height (H) and width (W) (365.42 µm) and centered within the rectangle.
Figure 3.
 
Representative image of toluidine blue–stained optic nerve core-subpial sectioning for g-ratio calculation. Core diameter (D: 182.71 µm) is half of the average diameter of height (H) and width (W) (365.42 µm) and centered within the rectangle.
Figure 4.
 
CZ diet affects visual evoked potentials. Top: Original cohort. (A) Average VEPs of all time points. At each time point, n ≥ 8. (B) Average VEP latency at each time point normalized to control readings at respective time points. n ≥ 8 at each time point. (C) Average amplitude of a-wave of VEPs normalized to control readings at each time point. Bottom: 12-week cohort. (D) Average VEPs of 12-week animals at key time points. n = 8. (E) Average VEP latency at key time points normalized to control readings at respective time points. n = 8 at each time point. (F) Average amplitude of a-wave of VEPs normalized to control readings at key time points. Amplitude (µV), latency (ms), *P < 0.05, ****P < 0.0001 compared to baseline. #P < 0.05 compared to 6 weeks.
Figure 4.
 
CZ diet affects visual evoked potentials. Top: Original cohort. (A) Average VEPs of all time points. At each time point, n ≥ 8. (B) Average VEP latency at each time point normalized to control readings at respective time points. n ≥ 8 at each time point. (C) Average amplitude of a-wave of VEPs normalized to control readings at each time point. Bottom: 12-week cohort. (D) Average VEPs of 12-week animals at key time points. n = 8. (E) Average VEP latency at key time points normalized to control readings at respective time points. n = 8 at each time point. (F) Average amplitude of a-wave of VEPs normalized to control readings at key time points. Amplitude (µV), latency (ms), *P < 0.05, ****P < 0.0001 compared to baseline. #P < 0.05 compared to 6 weeks.
Figure 5.
 
Functional and structural analysis of the mice retinas treated with CZ. Electrophysiological recordings and retinal ganglion cell count (RGC) in all groups. (A) Average ERGs at each time point, n = 8. (B) Average STRs at each time point, n = 8. (C) Average RGC count per group; control, 2 weeks, 4 weeks, 6 weeks, 6+1 weeks, 6+2 weeks, 12 weeks. Counts taken over a distance of 300 µm (200–500 µm from the edge of the optic disc). (D) Representative images of H&E-stained retinas from all groups (12 weeks in supplementary data), n = 3 animals per group. Scale bar: 50 µm and 100 µm, respectively.
Figure 5.
 
Functional and structural analysis of the mice retinas treated with CZ. Electrophysiological recordings and retinal ganglion cell count (RGC) in all groups. (A) Average ERGs at each time point, n = 8. (B) Average STRs at each time point, n = 8. (C) Average RGC count per group; control, 2 weeks, 4 weeks, 6 weeks, 6+1 weeks, 6+2 weeks, 12 weeks. Counts taken over a distance of 300 µm (200–500 µm from the edge of the optic disc). (D) Representative images of H&E-stained retinas from all groups (12 weeks in supplementary data), n = 3 animals per group. Scale bar: 50 µm and 100 µm, respectively.
Figure 6.
 
Optic nerve g-ratio analysis. (A) representative optic nerve cross sections stained with toluidine blue of control and 6-, 6+2-, and 12-week time points. Scale bar: 10 µm. (B) The g-ratio graphed to correlate with axon diameter (≥110 measurements per animal, n = 3 per time point). (C) Average g-ratio of core and subpial zone of optic nerves in panel B (**P < 0.001 compared to control region, #P < 0.001 compared to subpial, n = 3 per group).
Figure 6.
 
Optic nerve g-ratio analysis. (A) representative optic nerve cross sections stained with toluidine blue of control and 6-, 6+2-, and 12-week time points. Scale bar: 10 µm. (B) The g-ratio graphed to correlate with axon diameter (≥110 measurements per animal, n = 3 per time point). (C) Average g-ratio of core and subpial zone of optic nerves in panel B (**P < 0.001 compared to control region, #P < 0.001 compared to subpial, n = 3 per group).
Figure 7.
 
Optic nerve de/remyelination and latency correlation. (A) Representative images of optic nerve immunofluorescent staining for MBP magnified 100×. Scale bar: 10 µm. (B) Quantified average fluorescent intensity of MBP staining. (C) Relationship between average above myelin quantity and respective latency of animals. Each dot represents one animal; the average MBP fluorescence intensity from three ON sections per animal compared to the VEP latency obtained at the time point before sacrifice (r = −0.65, P < 0.005). *P < 0.05, compared to controls.
Figure 7.
 
Optic nerve de/remyelination and latency correlation. (A) Representative images of optic nerve immunofluorescent staining for MBP magnified 100×. Scale bar: 10 µm. (B) Quantified average fluorescent intensity of MBP staining. (C) Relationship between average above myelin quantity and respective latency of animals. Each dot represents one animal; the average MBP fluorescence intensity from three ON sections per animal compared to the VEP latency obtained at the time point before sacrifice (r = −0.65, P < 0.005). *P < 0.05, compared to controls.
Figure 8.
 
Changes in the dorsal lateral geniculate nucleus. (A) Representative immunofluorescence staining of dLGN, vGlut2, and MBP. A representative image of the region with schematic overlay in Supplementary Fig. 6. (B) Quantification of the average MBP and GFAP fluorescence intensity from ≥3 dLGN sections per animal, ≥3 animals per time point. (C) Relationship between average above myelin quantity and respective latency of animals. Each dot represents one animal; the average MBP fluorescence intensity from ≥3 dLGN sections compared to the VEP latency obtained at the time point before sacrifice (r = −0.48, P < 0.05). *P < 0.05, compared to controls.
Figure 8.
 
Changes in the dorsal lateral geniculate nucleus. (A) Representative immunofluorescence staining of dLGN, vGlut2, and MBP. A representative image of the region with schematic overlay in Supplementary Fig. 6. (B) Quantification of the average MBP and GFAP fluorescence intensity from ≥3 dLGN sections per animal, ≥3 animals per time point. (C) Relationship between average above myelin quantity and respective latency of animals. Each dot represents one animal; the average MBP fluorescence intensity from ≥3 dLGN sections compared to the VEP latency obtained at the time point before sacrifice (r = −0.48, P < 0.05). *P < 0.05, compared to controls.
Figure 9.
 
Changes in the visual cortex. (A) Representative immunofluorescence staining of V1, vGlut2, and MBP. (B) Quantification of the average MBP and GFAP fluorescence intensity from ≥3 V1 sections per animal, ≥3 animals per time point. (C) Relationship between average above myelin quantity and respective latency of animals. Each dot represents one animal; the average MBP quantity from immunofluorescence compared to the VEP latency obtained at the time point before sacrifice (r = −0.64, **P < 0.005). *P < 0.05, **P < 0.005, ****P < 0.0001.
Figure 9.
 
Changes in the visual cortex. (A) Representative immunofluorescence staining of V1, vGlut2, and MBP. (B) Quantification of the average MBP and GFAP fluorescence intensity from ≥3 V1 sections per animal, ≥3 animals per time point. (C) Relationship between average above myelin quantity and respective latency of animals. Each dot represents one animal; the average MBP quantity from immunofluorescence compared to the VEP latency obtained at the time point before sacrifice (r = −0.64, **P < 0.005). *P < 0.05, **P < 0.005, ****P < 0.0001.
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