Dynamics of central remyelination and treatment evolution in a model of Multiple Sclerosis with Optic Coherence Tomography

International Journal of Molecular Sciences Article Dynamics of Central Remyelination and Treatment Evolution in a Model of Multiple Sclerosis with Optic Coherence Tomography Rocío Benítez-Fernández 1,2,† , Carolina Melero-Jerez 1,2,†, Carmen Gil 1,3 , Enrique J. de la Rosa 1, Ana Martínez 1,3,* and Fernando de Castro 2,*   Citation: Benítez-Fernández, R.; Melero-Jerez, C.; Gil, C.; de la Rosa, E.J.; Martínez, A.; de Castro, F. Dynamics of Central Remyelination and Treatment Evolution in a Model of Multiple Sclerosis with Optic Coherence Tomography. Int. J. Mol. Sci. 2021,22, 2440. https://doi.org/10.3390/ ijms22052440 Academic Editor: Florencia Cavodeassi Received: 21 December 2020 Accepted: 24 February 2021 Published: 28 February 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1Centro de Investigaciones Biológicas Margarita Salas-CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain; [email protected] (R.B.-F.); cmeler[email protected] (C.M.-J.); [email protected] (C.G.); [email protected] (E.J.d.l.R.) 2Instituto Cajal-CSIC, Avda. Doctor Arce 37, 28002 Madrid, Spain 3Centro de Investigaciones Biomédicas en Red en Enfermedades Neurodegenerativas (CIBERNED), Instituto de Salud Carlos III, Valderrebollo 5, 28031 Madrid, Spain *Correspondence: [email protected] (A.M.); [email protected] (F.d.C.) † These authors have contributed equally to this work. Abstract: The need for remyelinating drugs is essential for healing disabling diseases such as multiple sclerosis (MS). One of the reasons for the lack of this class of therapies is the impossibility to monitor remyelination in vivo , which is of utmost importance to perform effective clinical trials. Here, we show how optical coherence tomography (OCT), a cheap and non-invasive technique commonly used in ophthalmology, may be used to assess remyelination in vivo in MS patients. Our pioneer approach validates OCT as a technique to study remyelination of the optic nerve and reflects what is occurring in non-accessible central nervous system (CNS) structures, like the spinal cord. In this study we used the orally bioavailable small molecule VP3.15, confirming its therapeutical potential as a neuroprotective, anti-inflammatory, and probably remyelinating drug for MS. Altogether, our results confirm the usefulness of OCT to monitor the efficacy of remyelinating therapies in vivo and underscore the relevance of VP3.15 as a potential disease modifying drug for MS therapy. Keywords: optic coherence tomography; multiple sclerosis; EAE; optic nerve; demyelinating diseases; remyelinating drugs 1. Introduction Multiple sclerosis (MS) is the most common primary demyelinating disease and neurological condition affecting young adults, with about 2.5 million people currently diagnosed around the world [ 1 ]. It is characterized by glial cell pathology (especially oligodendrocytes and their precursors), demyelination, inflammatory processes, and axonal damage in the central nervous system (CNS) [ 2 ]. In addition to glial cell pathology, the destruction of CNS myelin is accompanied by the activation of macrophages and microglia, cells present in MS lesions that behave either as proinflammatory or anti-inflammatory agents depending on the stage of the lesion [3–6]. An unmet challenge in demyelinating diseases like MS is reestablishing the lost myelin, thus reducing the neurological dysfunction. A proper approach should include three steps: (i) the modulation of the inflammatory response; (ii) the protection of oligodendrocytes; and (iii) the promotion of effective remyelination. Current available treatments are exclusively immunomodulatory, focused on the reduction of inflammation, which ameliorates the evolution of the non-progressive forms of the disease but does not cure them [ 7 ]. During the last decade, many studies have investigated myelin regenerating mechanisms. To date, only clemastine fumarate, an old-fashioned antihistaminic drug, has shown relative efficiency on randomized controlled clinical trials as a remyelinating drug [ 8 , 9 ]. Many other targets have been explored with this aim, such as the pathways of Notch and Wnt signaling [ 10 ], Int. J. Mol. Sci. 2021,22, 2440. https://doi.org/10.3390/ijms22052440 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021,22, 2440 2 of 20 glutamatergic receptors [ 11 ], nuclear receptors such as RXRy, PPARy, and VDR [ 12 – 14 ], and components of the extracellular matrix [ 15 , 16 ]. Furthermore, different growth factors and chemotropic molecules, like FGF-2 and Anosmin-1, secreted semaphorins and others, have been suggested to cause spontaneous remyelination in human MS [ 17 , 18 ]. However, the efficacy of remyelinating therapies is difficult to assess in vivo due to the technical limitations in exploring the CNS, not only in clinical trials but also in preclinical studies where MS animal models are used. Consequently, there is a need for novel tools to real-time measure and quantify the myelin loss and the effective remyelination after a potential regenerative treatment. Over the past few decades, advanced visualization techniques such as 7-Tesla magnetic resonance imaging (MRI), positron emission tomography (PET), or visual evoked potentials have become pivotal in the diagnosis and monitoring of MS. They have allowed the study of its different aspects, such as the perivascular inflammation, the bursting of new CNS lesions, the activation of the immune response, and the inflammation of leptomeninges [ 19 ]. However, because clinical MS reflects a combination of inflammation, degeneration, and regeneration, these techniques fail to detect the remyelination process [ 20 ]. For this reason, the spectral domain optic coherence tomography (OCT), a new imaging technology, has emerged in the last few years. OCT is a reproducible and non-invasive method for retinal structure visualization in physiopathological conditions, including neuroinflammatory disorders such as neuromyelitis optica (NMO) and optic neuritis (ON), both in animal models [ 21 ] and humans [ 22 , 23 ]. In patients, OCT measurements include the macula volume and the area and thickness of the retinal nerve fiber layer (RNFL), which comprises the axons of the retinal ganglion neurons that converge to form the optic nerve [ 24 ]. Because retinal alterations have been described as a component of several MS outcomes, OCT use has been extended to MS in clinical practice for diagnostic purposes, although whether the optic nerve reflects the situation in the parenchimatous CNS remains to be addressed [25–31] . Although a correlation has been described between the decrease in RNFL thickness and brain atrophy in MS [ 32 , 33 ], it has yet to be ascertained whether the results in the RNFL clearly compile the histopathology of the CNS, in terms of inflammation, demyelination status, and oligodendrocyte-lineage cell availability and maturity. The finding of an accurate correlation between the myelin alterations in deep CNS structures and peripheral and technically approachable areas such as the retina would improve the research of remyelinating drugs, elucidating their real-time efficiency in a quantitative manner. It should be noted that, in patients, it is easier to visualize the structure of the retina and the optic nerve head taking advantage of imaging techniques, but it is more difficult to establish correlations within deeper parts of the CNS, hence the need for OCT validation in animal models. The main goal of the present work is to validate OCT as a methodology to accurately monitor neurological symptoms and histopathological hallmarks in a murine model of MS, i.e., experimental autoimmune encephalomyelitis (EAE). The second aim is to confirm the potential remyelination efficacy of a dual phosphodiesterase-7 (PDE7) and glycogen synthase kinase-3 β (GSK3 β ) inhibitor named VP3.15 [ 34 ]. This compound is a small heterocyclic drug-like molecule with safety profile and good pharmacodynamic and pharmacokinetic properties following intraperitoneal administration previously determined in two different in vivo models [ 34 ]. This property is assessed here in the EAE model. Furthermore, VP3.15 has been described as an inducer of remyelination in vitro and in vivo in oligodendrocyte precursor cells (OPCs) isolated from non-tumoral biopsies of human adult brain cortex [ 35 , 36 ]. This multitarget compound has also shown anti-inflammatory properties in vivo , both in retinal dystrophies [ 37 ] and in the EAE model of MS [ 38 ]. The data presented supports OCT as a promising and efficacious tool to evaluate the effects of potential remyelinating drugs in unobservable areas of the CNS. Moreover, a clear correlation between OCT data (retina, the papilla of the optic nerve) and immunohistochemical observations (optic nerve and spinal cord) is demonstrated. Finally, our results confirm the compound VP3.15 as a promising drug candidate for MS therapy. Int. J. Mol. Sci. 2021,22, 2440 3 of 20 2. Results 2.1. The PDE7/GSK3 Dual Inhibitor VP3.15 Ameliorates Clinical Course of Experimental Autoimmune Encephalomyelitis (EAE) The dual inhibitor VP3.15, as effective as the oral FDA-approved drug fingolimod in EAE model [ 38 ] and with remyelating effect in vitro , ex vivo, and in vivo [ 35 ], was the pharmacological tool chosen to test our hypothesis on the usefulness of OCT to monitor remyelination. Benefits of the repeated treatment with VP3.15 (see Methods) in EAE mice were evident since the beginning, with the clinical score (CS) decaying faster and to a significantly lower value in the VP3.15-treated EAE mice by day 19 after onset ( Figure 1b ). For a more quantitative estimation of the healing effect of VP3.15, we fitted the results to an exponential expression [ 39 ], and the score fraction (Sf) obtained was significantly better (0.2) than the EAE-vehicle group (0.3), thus the treatment with VP3.15 ameliorated EAE effects by 33% compared to the vehicle-treated mice (Figure 1c) by the end of the experiment. In addition to that, the estimated recovery rate ( υ ) was higher in the treated animals (EAE-Veh = 0.187 days −1 ; EAE-VP3.15 = 0.226 days −1 ) and was reflected in the fact that EAE-VP3.15 reached the endpoint Sf of EAE-VEH nearly 10 days before. Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 3 of 20 clear correlation between OCT data (retina, the papilla of the optic nerve) and immunohistochemical observations (optic nerve and spinal cord) is demonstrated. Finally, our results confirm the compound VP3.15 as a promising drug candidate for MS therapy. 2. Results 2.1. The PDE7/GSK3 Dual Inhibitor VP3.15 Ameliorates Clinical Course of Experimental Autoimmune Encephalomyelitis (EAE) The dual inhibitor VP3.15, as effective as the oral FDA-approved drug fingolimod in EAE model [38] and with remyelating effect in vitro, ex vivo, and in vivo [35], was the pharmacological tool chosen to test our hypothesis on the usefulness of OCT to monitor remyelination. Benefits of the repeated treatment with VP3.15 (see Methods) in EAE mice were evident since the beginning, with the clinical score (CS) decaying faster and to a significantly lower value in the VP3.15-treated EAE mice by day 19 after onset (Figure 1b). For a more quantitative estimation of the healing effect of VP3.15, we fitted the results to an exponential expression [39], and the score fraction (Sf) obtained was significantly better (0.2) than the EAE-vehicle group (0.3), thus the treatment with VP3.15 ameliorated EAE effects by 33% compared to the vehicle-treated mice (Figure 1c) by the end of the experiment. In addition to that, the estimated recovery rate (υ) was higher in the treated animals (EAE-Veh = 0.187 days −1 ; EAE-VP3.15 = 0.226 days −1 ) and was reflected in the fact that EAE-VP3.15 reached the endpoint Sf of EAE-VEH nearly 10 days before. Figure 1. The treatment of experimental autoimmune encephalomyelitis (EAE) mice with the dual inhibitor VP3.15 ameliorates the clinical course from the beginning of the administration; (a): Procedure outline; (b): Time course representation of the clinical performance of EAE mice from the onset of symptoms, showing a lower clinical score in the EAE-VP3.15 compared to EAE-VEH (two-way ANOVA to compare the two treatments: p < 0.05; results of Student’s t-test are represented as: * p < 0.05 from day 19 to day 22); (c): Exponential expression of the score decay from the beginning of the VP3.15 treatment, where the score (S) at every time point is normalized by the maximal score (Smax). The expression shows that VP3.15 maintains the score in a lower level than vehicle at longer times. Abbreviations: dpo = days post onset, Sf = score fraction; S∞ = score at infinite times. EAE: n = 17; EAE-VEH: n = 7; EAE-VP3.15: n = 10; SHAM: n = 13. 2.2. Retinal and Optic Nerve Changes can be Monitored using Optic Coherence Tomography (OCT) in the EAE Model Nowadays, retinal layer shrinkage observed by OCT is used in clinics to predict terms of cognitive decline and brain atrophy in MS patients [33,39,40]. Our main aim in this work is to check if remyelination, considered as the equilibrium between decreased myelin loss and enhanced new oligodendrocyte precursors cells with subsequent myelin Figure 1. The treatment of experimental autoimmune encephalomyelitis (EAE) mice with the dual inhibitor VP3.15 ameliorates the clinical course from the beginning of the administration; ( a ): Procedure outline; ( b ): Time course representation of the clinical performance of EAE mice from the onset of symptoms, showing a lower clinical score in the EAE-VP3.15 compared to EAE-VEH (two-way ANOVA to compare the two treatments: p< 0.05; results of Student’s t-test are represented as: * p< 0.05 from day 19 to day 22); ( c ): Exponential expression of the score decay from the beginning of the VP3.15 treatment, where the score (S) at every time point is normalized by the maximal score (Smax). The expression shows that VP3.15 maintains the score in a lower level than vehicle at longer times. Abbreviations: dpo = days post onset, Sf = score fraction; S ∞ = score at infinite times. EAE: n= 17; EAE-VEH: n= 7; EAE-VP3.15: n= 10; SHAM: n= 13. 2.2. Retinal and Optic Nerve Changes Can Be Monitored Using Optic Coherence Tomography (OCT) in the EAE Model Nowadays, retinal layer shrinkage observed by OCT is used in clinics to predict terms of cognitive decline and brain atrophy in MS patients [ 33 , 39 , 40 ]. Our main aim in this work is to check if remyelination, considered as the equilibrium between decreased myelin loss and enhanced new oligodendrocyte precursors cells with subsequent myelin production, can be effectively monitored in real-time using a non-invasive technique such as OCT (see Methods and Figure 2a–d). Besides the predicable stability of retinal thickness in the SHAM group along the entire experiment, we observed that the retina was significantly thinner Int. J. Mol. Sci. 2021,22, 2440 4 of 20 when EAE was induced. As expected, the treatment with VP3.15 showed better dynamics than EAE-VEH, including significant recovery from the sixth OCT onwards (Figure 2e). Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 4 of 20 production, can be effectively monitored in real-time using a non-invasive technique such as OCT (see Methods and Figure 2a–d). Besides the predicable stability of retinal thickness in the SHAM group along the entire experiment, we observed that the retina was significantly thinner when EAE was induced. As expected, the treatment with VP3.15 showed better dynamics than EAE-VEH, including significant recovery from the sixth OCT onwards (Figure 2e). Figure 2. Retina and optic nerve analysis by optical coherence tomography (OCT) of the EAE mice reveals changes associated to the concurrent disease; (a): Schematic representation of the different measurements at the fovea, optic disc, and retina levels; (b–d): Representative images obtained with the OCT; (e): Retinal layer thickness analysis along the experiment; (f): Dynamics of optic nerve width along the experiment. Scale bar represents 200 µm in a–d. Abbreviations: GCL = ganglion cell layer; IPL = inner plexiform layer; NFL = nerve fiber layer. Results of Student’s t-test are represented as: * p < 0.05 (EAE-VP3.15 vs. EAE-VEH) or # p < 0.05 (EAE-VEH vs. SHAM). EAE: n = 17; EAE-VEH: n = 7; EAE-VP3.15: n = 10; SHAM: n = 13. Figure 2. Retina and optic nerve analysis by optical coherence tomography (OCT) of the EAE mice reveals changes associated to the concurrent disease; ( a ): Schematic representation of the different measurements at the fovea, optic disc, and retina levels; ( b – d ): Representative images obtained with the OCT; ( e ): Retinal layer thickness analysis along the experiment; ( f ): Dynamics of optic nerve width along the experiment. Scale bar represents 200 µ m in a–d. Abbreviations: GCL = ganglion cell layer; IPL = inner plexiform layer; NFL = nerve fiber layer. Results of Student’s t-test are represented as: * p< 0.05 (EAE-VP3.15 vs. EAE-VEH) or # p< 0.05 (EAE-VEH vs. SHAM). EAE: n= 17; EAE-VEH: n= 7; EAE-VP3.15: n= 10; SHAM: n= 13. These results were more consistent when we measured the optic nerve width: from the fourth OCT to endpoint, the group treated with VP3.15 showed significantly larger OCT measures than non-treated EAE mice (Figure 2f; results of two-way ANOVA Bonferroni Int. J. Mol. Sci. 2021,22, 2440 5 of 20 post-hoc test were p< 0.05). Concerning the retina, there was a significant recovery of VP3.15-treated animals after the fifth OCT that was maintained and recovered to the SHAMlike levels at the endpoint. It is also observed that changes in retinal thickness are transient in EAE mice model being almost recovered at the end point. However, optic nerve width, a measure that likely does relate to myelin loss, is greater in the EAE-VEH group than in the VP3.15-treated one, showing the potential remyelinating activity of this new drug. These changes are persistent during the treatment. To further assess the potential of the damaged tissue in the EAE mice, we performed a detailed tissue analysis of the optic nerve (rostral localization, thus closer to the eye) and spinal cord (caudal localization) obtained at the endpoint. 2.3. The Remyelinating Role and the Effect in the Oligodendrocyte Lineage of VP3.15 We checked at endpoint the histopathology of two of the most myelinated structures in the CNS: the optic nerve and the spinal cord. In the optic nerve, the treatment with VP3.15 showed an increase higher than 50% in myelinated (MBP + ) axons when compared with EAE-VEH (Figure 3a,c,d,f,g). In addition, the integrity of the optic nerve axons (NFH + ) was better preserved in the VP3.15-treated animals compared to the EAE-VEH group ( Figure 3b,e,h ). This biological effect remarks the neuroprotective profile of VP3.15 and/or the recovery of myelin produced after the treatment. Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 5 of 20 These results were more consistent when we measured the optic nerve width: from the fourth OCT to endpoint, the group treated with VP3.15 showed significantly larger OCT measures than non-treated EAE mice (Figure 2f; results of two-way ANOVA Bonferroni post-hoc test were p < 0.05). Concerning the retina, there was a significant recovery of VP3.15-treated animals after the fifth OCT that was maintained and recovered to the SHAM-like levels at the endpoint. It is also observed that changes in retinal thickness are transient in EAE mice model being almost recovered at the end point. However, optic nerve width, a measure that likely does relate to myelin loss, is greater in the EAE-VEH group than in the VP3.15-treated one, showing the potential remyelinating activity of this new drug. These changes are persistent during the treatment. To further assess the potential of the damaged tissue in the EAE mice, we performed a detailed tissue analysis of the optic nerve (rostral localization, thus closer to the eye) and spinal cord (caudal localization) obtained at the endpoint. 2.3. The Remyelinating Role and the Effect in the Oligodendrocyte Lineage of VP3.15 We checked at endpoint the histopathology of two of the most myelinated structures in the CNS: the optic nerve and the spinal cord. In the optic nerve, the treatment with VP3.15 showed an increase higher than 50% in myelinated (MBP+) axons when compared with EAE-VEH (Figure 3a,c,d,f,g). In addition, the integrity of the optic nerve axons (NFH+) was better preserved in the VP3.15-treated animals compared to the EAE-VEH group (Figure 3b,e,h). This biological effect remarks the neuroprotective profile of VP3.15 and/or the recovery of myelin produced after the treatment. Figure 3. The VP3.15-treated mice preserve the optic nerve from myelin loss and axonal damage; (a–f): Detailed views of the optic nerve of vehicle (a–c) and VP3.15-treated mice (d–f), labelled for MBP (red), NFH (grey), and nuclei (Hoechst staining; blue); (g–h): Histograms showing a significant increase in the percentage of both MBP+ (j) and NFH+ (k) area in the VP3.15-treated compared to the vehicle-treated mice. Scale bar represents 50 µm in Figure 3. The VP3.15-treated mice preserve the optic nerve from myelin loss and axonal damage; ( a – f ): Detailed views of the optic nerve of vehicle ( a – c ) and VP3.15-treated mice ( d – f ), labelled for MBP (red), NFH (grey), and nuclei (Hoechst staining; blue); ( g , h ): Histograms showing a significant increase in the percentage of both MBP + ( g ) and NFH + ( h ) area in the VP3.15-treated compared to the vehicle-treated mice. Scale bar represents 50 µm in a–f. Abbreviations: MBP = myelin basic protein; NFH = neurofilament heavy. Results of Student’s t-test are represented as: ** p< 0.01; *** p< 0.001. EAE-VEH: n= 7; EAE-VP3.15: n= 10. Int. J. Mol. Sci. 2021,22, 2440 6 of 20 Similar results were obtained in the spinal cord: The demyelinated area in the EAEVP3.15 group was significantly lower and showed more MBP + staining than in the EAEVEH (Figure 4a–d,g–j), while the axons were also better preserved in the VP3.15-treated group (Figure 4e–f,k). Although it is difficult to compare both structures, our results in the optic nerve seemed slightly more robust than in the spinal cord (percentages in Figure 3g,h vs. in Figure 4i–k), and the remarkable differences in myelination and axonal density could explain them. Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 6 of 20 a–f. Abbreviations: MBP = myelin basic protein; NFH = neurofilament heavy. Results of Student’s t-test are represented as: ** p < 0.01; *** p < 0.001. EAE-VEH: n = 7; EAE-VP3.15: n = 10. Similar results were obtained in the spinal cord: The demyelinated area in the EAEVP3.15 group was significantly lower and showed more MBP+ staining than in the EAEVEH (Figure 4a–d,g–j), while the axons were also better preserved in the VP3.15-treated group (Figure 4e–f and k). Although it is difficult to compare both structures, our results in the optic nerve seemed slightly more robust than in the spinal cord (percentages in Figure 3g,h vs. in Figure 4i–k), and the remarkable differences in myelination and axonal density could explain them. Figure 4. The spinal cord of VP3.15-treated mice presented a lower level of demyelination and axonal damage; (a,b): Panoramic views of the spinal cord stained with eriochrome cyanine of vehicle (a) and VP3.15-treated mice (b). The demyelinated area is delimited by the dashed line and arrows. Scale bar represents 200 µm in a–b; (c– h): Detailed views of the spinal cords of vehicle (left) and VP3.15-treated mice (right), labelled for MBP (red), NFH (grey), and nuclei (Hoechst staining; blue). Lesions are identified as an accumulation of nuclei; (i): Graph showing a decreased percentage of demyelinated area respect to the white area in the VP3.15-treated mice; (j– k): Histograms showing a significant increase in the percentage of both MBP+ (j) and NFH+ (k) area in the VP3.15treated compared to the vehicle-treated mice. Scale bar represents 200 in a–b and 50 µm in c–h. Abbreviations: MBP = myelin basic protein; NFH = neurofilament heavy. Results of Student’s t-test are represented as: * p < 0.05. EAE-VEH: n = 7; EAE-VP3.15: n = 10. These structural differences were observed in EAE-VEH and also when the treatment with VP3.15 was applied, that resulted in a growth in both parameters (Figure 5a,b). These facts may be attributed to a combination of the neuroprotective effect of VP3.15 probably Figure 4. The spinal cord of VP3.15-treated mice presented a lower level of demyelination and axonal damage; ( a , b ): Panoramic views of the spinal cord stained with eriochrome cyanine of vehicle ( a ) and VP3.15-treated mice ( b ). The demyelinated area is delimited by the dashed line and arrows. Scale bar represents 200 µ m in a , b ; ( c – h ): Detailed views of the spinal cords of vehicle (left) and VP3.15-treated mice (right), labelled for MBP (red), NFH (grey), and nuclei (Hoechst staining; blue). Lesions are identified as an accumulation of nuclei; ( i ): Graph showing a decreased percentage of demyelinated area respect to the white area in the VP3.15-treated mice; ( j , k ): Histograms showing a significant increase in the percentage of both MBP + (j) and NFH + ( k ) area in the VP3.15-treated compared to the vehicle-treated mice. Scale bar represents 200 in a , b and 50 µ m in c – h . Abbreviations: MBP = myelin basic protein; NFH = neurofilament heavy. Results of Student’s t-test are represented as: * p< 0.05. EAE-VEH: n= 7; EAE-VP3.15: n= 10. These structural differences were observed in EAE-VEH and also when the treatment with VP3.15 was applied, that resulted in a growth in both parameters (Figure 5a,b). These facts may be attributed to a combination of the neuroprotective effect of VP3.15 probably due to its anti-inflammatory profile and its remyelination potential previously in different demyelinating animal models of MS, including EAE, and in vitro [ 35 , 36 ]. In addition to that, we found a direct and significant correlation between the normalized myelin basic Int. J. Mol. Sci. 2021,22, 2440 7 of 20 protein (MBP) area and the normalized axonal area in both treatments, both in the optic nerve (Figure 5c; EAE-Veh: r= 0.559, p< 0.01; EAE-VP3.15: r= 0.546, p< 0.01) and the spinal cord (Figure 5d; EAE-Veh: r= 0.933, p< 0.001; EAE-VP3.15: r= 0.803, p< 0.001). Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 7 of 20 due to its anti-inflammatory profile and its remyelination potential previously in different demyelinating animal models of MS, including EAE, and in vitro [35,36]. In addition to that, we found a direct and significant correlation between the normalized myelin basic protein (MBP) area and the normalized axonal area in both treatments, both in the optic nerve (Figure 5c; EAE-Veh: r = 0.559, p < 0.01; EAE-VP3.15: r = 0.546, p < 0.01) and the spinal cord (Figure 5d; EAE-Veh: r = 0.933, p < 0.001; EAE-VP3.15: r = 0.803, p < 0.001). Figure 5. Axonal integrity is higher when the myelin is better preserved; (a,b): Graphs showing the percentage of MBP (a) and NFH (b) area respect to the total white matter area between structures; (c,d): Graphs showing the results of Pearson´s correlation tests between the normalized MBP area and the normalized axonal area among treatments and in the optic nerve (c) and spinal cord (d). EAE-VEH: n = 7; EAE-VP3.15: n = 10. We wondered if there was a relationship between the histological findings at the endpoint and the OCT recordings at different time points, so a study of partial correlations was performed. We assessed the correlation found between histological parameters (percentage of neurofilament heavy-chain (NFH) area, percentage of MBP area) and OCT findings (optic nerve width). Regarding the spinal cord, a significant partial correlation was found between the histological parameters when the OCT recording was maintained constant, both at the fifth and the final OCT recordings (Table 1). However, there was no significant correlation between percentage of NFH and MBP in the nerve tissue when the OCT recordings were added to the partial correlation as the permanent variable (Table 1), suggesting that the optic nerve width is related to axon integrity. Figure 5. Axonal integrity is higher when the myelin is better preserved; ( a , b ): Graphs showing the percentage of MBP ( a ) and NFH ( b ) area respect to the total white matter area between structures; ( c , d ): Graphs showing the results of Pearson ´ s correlation tests between the normalized MBP area and the normalized axonal area among treatments and in the optic nerve ( c ) and spinal cord ( d ). EAE-VEH: n= 7; EAE-VP3.15: n= 10. We wondered if there was a relationship between the histological findings at the endpoint and the OCT recordings at different time points, so a study of partial correlations was performed. We assessed the correlation found between histological parameters (percentage of neurofilament heavy-chain (NFH) area, percentage of MBP area) and OCT findings (optic nerve width). Regarding the spinal cord, a significant partial correlation was found between the histological parameters when the OCT recording was maintained constant, both at the fifth and the final OCT recordings (Table 1). However, there was no significant correlation between percentage of NFH and MBP in the nerve tissue when the OCT recordings were added to the partial correlation as the permanent variable (Table 1), suggesting that the optic nerve width is related to axon integrity. Also, it is known that the dual PDE7-GSK3 inhibition by VP3.15 enhances murine and adult human OPC differentiation without affecting their survival or proliferation [ 35 ]. We inquired whether the remyelinating role and the neuroprotection on axons could be related to changes in the oligodendrocytes’ lineage (Olig2 positive cells, Figure 6b,f). In the optic nerve, a significant increase in the number of precursor cells, labelled as PDGFR α+ cells (Figure 6a,e,i) and mature cells, identified as CC1 + cells (Figure 6c,g,j) was observed in the VP3.15-treated animals compared to the vehicle group. Int. J. Mol. Sci. 2021,22, 2440 8 of 20 Table 1. Correlations between histological parameters and OCT data. Veh (n= 7) VP3.15 (n= 10) Spinal cord Pearson’s coefficient (r) MBP area—NFH area 0.933 0.803 MBP area—optic nerve width at fifth OCT −0.212 −0.024 MBP area—optic nerve width at endpoint OCT −0.413 −0.186 NFH area—optic nerve width at fifth OCT −0.364 −0.228 NFH area—optic nerve width at endpoint OCT −0.533 −0.362 Multiple correlation at fifth OCT (two-tailed multicomparison test) 5.52246 3.78265 p(fifth OCT) <0.01 <0.05 Multiple correlation at endpoint OCT (two-tailed multicomparison test) 4.87270 3.56740 p(endpoint OCT) <0.01 <0.05 Optic Nerve Pearson’s coefficient (r) MBP area—NFH area 0.559 0.546 MBP area—optic nerve width at fifth OCT 0.579 −0.385 MBP area—optic nerve width at endpoint OCT 0.120 0.222 NFH area—optic nerve width at fifth OCT 0.579 0.390 NFH area—optic nerve width at endpoint OCT 0.077 0.202 Multiple correlation at fifth OCT (two-tailed multicomparison test) 0.71492 1.39569 p(fifth OCT) NS NS Multiple correlation at endpoint OCT (two-tailed multicomparison test) 1.33580 1.63117 p(endpoint OCT) NS NS Abbreviations: n: mice number; NS: not significant. In the spinal cord, the same effect, an increase in the precursor cells (Figure 7a,c,g,i,m) and mature oligodendrocytes (Figure 7b,e,h,k,n) was also observed. In all the cases, differences are related with oligodendrocytes’ lineage (Olig2 positive cells, Figure 6d,f,j,l). We applied a two-way ANOVA to unravel the differences among treatments and structures, showing no significant differences for PDGFR α+ cells (Figure 8a; p= 0.072). We did find a significant difference between structures in the case of CC1 + cells, being the proportion lower in the optic nerve than in the spinal cord (Figure 8d; p< 0.001). We then looked for a relationship between the myelin (MBP + cells) and the two stages of the oligodendrocyte lineage under study (Figure 8b,c,e,f). We found a significant and direct correlation in the case of CC1 + cells in the optic nerve of vehicle-treated EAE mice, ( Figure 8b,e ; EAE-Veh: r = 0.470, p= 0.0422; EAE-VP3.15: r = 0.254, p= 0.22). The relation between a lower proportion of CC1 + cells and higher levels of myelination that we previously showed could be explained by a difference in the rate of differentiation to mature phenotypes, that might be higher in the case of a highly myelinated tissue such as the optic nerve. Int. J. Mol. Sci. 2021,22, 2440 9 of 20 Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 9 of 20 endpoint OCT (two-tailed multicomparison test) p (endpoint OCT) NS NS Abbreviations: n: mice number; NS: not significant. Also, it is known that the dual PDE7-GSK3 inhibition by VP3.15 enhances murine and adult human OPC differentiation without affecting their survival or proliferation [35]. We inquired whether the remyelinating role and the neuroprotection on axons could be related to changes in the oligodendrocytes’ lineage (Olig2 positive cells, Figure 6b,f). In the optic nerve, a significant increase in the number of precursor cells, labelled as PDGFRα+ cells (Figure 6a,e,i) and mature cells, identified as CC1+ cells (Figure 6c,g,j) was observed in the VP3.15-treated animals compared to the vehicle group. Figure 6. VP3.15 promotes the presence of both precursor and mature oligodendrocytes in optic nerve; (a–h): Detailed views of the optic nerve of vehicle (a–d) and VP3.15-treated mice (e–h), labelled for PDGFRα (green), Olig2 (red), CC1 (grey), and merged includes nuclei (Hoechst; blue). Arrows point to PDGFRα+Olig2+ cells and asterisks to CC1+Olig2+ cells. Scale bar represents 50 µm in a–h; (i–j): Graphs showing the significant increase in the percentage of PDGFRα+Olig2+ cells (i) and CC1+ Olig2+ cells (j) after the treatment with VP3.15 compared to the vehicle. Results of Student’s t-test are represented as: *** p < 0.001. EAE-VEH: n = 7; EAE-VP3.15: n = 10. In the spinal cord, the same effect, an increase in the precursor cells (Figure 7a,c,g,i,m) and mature oligodendrocytes (Figure 7b,e,h,k,n) was also observed. In all the cases, differences are related with oligodendrocytes’ lineage (Olig2 positive cells, Figure 6d,f,j,l). We applied a two-way ANOVA to unravel the differences among treatments and structures, showing no significant differences for PDGFRα+ cells (Figure 8a; p = 0.072). We did find a significant difference between structures in the case of CC1+ cells, being the proportion lower in the optic nerve than in the spinal cord (Figure 8d; p < 0.001). We then looked for a relationship between the myelin (MBP+ cells) and the two stages of the oligodendrocyte lineage under study (Figure 8b,c,e,f). We found a significant and direct correlation in the case of CC1+ cells in the optic nerve of vehicle-treated EAE mice, (Figure Figure 6. VP3.15 promotes the presence of both precursor and mature oligodendrocytes in optic nerve; ( a – h ): Detailed views of the optic nerve of vehicle ( a – d ) and VP3.15-treated mice ( e – h ), labelled for PDGFR α (green), Olig2 (red), CC1 (grey), and merged includes nuclei (Hoechst; blue). Arrows point to PDGFRα+Olig2+cells and asterisks to CC1+Olig2+cells. Scale bar represents 50 µm in a–h; ( i , j ): Graphs showing the significant increase in the percentage of PDGFR α+ Olig2 + cells ( i ) and CC1 + Olig2 + cells ( j ) after the treatment with VP3.15 compared to the vehicle. Results of Student’s t-test are represented as: *** p< 0.001. EAE-VEH: n= 7; EAE-VP3.15: n= 10. 2.4. VP3.15 Treatment Modifies Microglial Activation State To get a deeper insight into the inflammatory component of the CNS at the end of VP3.15 treatment, we analyzed the composition of the microglial population, being divided into three groups: ramified, stellate-shaped, and amoeboid microglia [ 41 , 42 ]. In the optic nerve, there was an increase in the proportion of the stellate-shaped cells in the EAE-VP3.15 group compared to vehicle (Figure 9A,B,E; EAE-VEH = 0.043 ±0.007% ; EAE-VP 3.15 = 0.082 ±0.006% ), while the other two subpopulations remained similar between groups (Figure 9A,B,E; ramified microglia: EAE-VEH = 0.004 ± 0.010%; EAE-VP3.15 = 0.045 ±0.006% ; amoeboid microglia: EAE-VEH = 0.044 ± 0.0147%; EAE-VP3.15 = 0.027% ± 0.014%). Regarding the spinal cord, the microglial population was enriched in stellate-shaped cells in the VP3.15-treated mice in contrast to the vehicle-treated group (Figure 9C,D,F; EAE-VEH = 0.025 ± 0.003%; EAE-VP3.15 = 0.045 ± 0.002%), being the amoeboid subpopulation significantly decreased (Figure 9C,D,F; EAE-VEH = 0.004 ± 0.003%; EAE-VP3.15 = 0.266 ± 0.003%). Finally, that of the ramified microglia remained constant in both groups, as in the optic nerve. Altogether, our present results show that the neuroprotective profile of VP3.15 is accompanied by a switch of the microglial content to anti-inflammatory phenotypes, underlying the important role of the immunomodulatory effects of this drug candidate. Int. J. Mol. Sci. 2021,22, 2440 16 of 20 oligodendrocytes, we quantified the PDGFR α+ Olig2 + and the CC1 + Olig2 + . Total Iba-1 + cells were sorted out into three morphological subtypes (ramified microglia, stellate-shaped microglia, and amoeboid microglia cells) [ 73 – 75 ]. Briefly, ramified microglia have a small and rounded cell body with processes in the form of tree branches; the stellate-shaped microglia have a large oval cell body with less extensive and thick processes; and the amoeboid microglia does not have a completely rounded cell body and its processes are not as well-defined. 4.7. Statistical Analysis The data were expressed as the mean ± SEM and analyzed with Sigma Plot version 11.0 (Systat Software, San Jose, CA, USA). Student’s t-test was used to compare pairs of the different groups of mice with a Mann–Whitney U test for non-parametric data. A two-way ANOVA with multiple comparisons using a Bonferroni post-hoc test was used for the comparison of three groups (EAE-VP3.15 vs. EAE-VEH vs. SHAM), obtaining the area under the curve. Correlation analyses were performed using the Pearson ´ s correlation test. Multiple correlations were made using the Pearson’s correlation test, and applying the following formula, as previously described [76]: t= r12.3 √n−3 q1−r212.3 Minimal statistical significance was set at p< 0.05, and illustrated as * or # (p< 0.05), ** or ## (p< 0.01), and ***or ### (p< 0.001). Author Contributions: Conceptualization, A.M. and F.d.C.; methodology, R.B.-F., C.G., E.J.d.l.R., F.d.C., and A.M.; formal analysis, R.B.-F., C.M.-J., C.G., E.J.d.l.R., A.M., and F.d.C.; investigation, R.B.-F. and C.M.-J.; resources, A.M. and F.d.C.; data curation, E.J.d.l.R., A.M., and F.d.C.; writing— original draft preparation, R.B.-F. and C.M.-J.; writing—review and editing, C.G., E.J.d.l.R., A.M., and F.d.C.; supervision, C.G., A.M., and F.d.C.; funding acquisition, A.M. and F.d.C. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by Asociación Española de Esclerosis MúltipeConfederación Española de Personas con Discapacidad Física y Orgánica (AEDEM-COCEMFE; Dña. Angela Sastre legacy) and Ministerio de Ciencia, Innovación y Universidades (grant no. SAF2015-72325-EXP, SAF2016-77575-R to F.d.C and RD16-0019 to F.d.C). Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the CSIC Ethics Committee (protocol code PROEX 143/16 21/07/2016). Data Availability Statement: The data presented in this study are available on request from the corresponding author. Acknowledgments: The authors would like to thank the invaluable help of Sergio Casas from Instituto Cajal with the IMARIS equipment, the animal facility at CIB Margarita Salas for their technical assistance and support, Eduardo Sanz from Universidad Complutense for the exponential adjustment and Carmen Hernández and Belén García from the Confocal Microscopy facility at Instituto Cajal for their help in image acquisition. We want to dedicate this work to our friend Gerardo García Perales, unforgettable President of AEDEM-COCEMFE, who always fought to improve the quality of life of mS patients and supported research. 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