Rescue CNS of Multiple Sclerosis Rodent Models Experiment

Combination of Angiopoietin-1 and Integrin Alphav Beta3 Binding Peptide to Get Better Effects on Rescue CNS of Multiple Sclerosis Rodent Models

Abbreviations: BBB: blood brain barrier; EAE: experimental autoimmune encephalomyelitis; MS: multiple sclerosis; Ang-1: Angiopoietin-1; CNS: central nervous system; c-SEPs: cortical somatosensory-evoked potentials; c-MEP: cortical motor evoked potentials; LFB: luxol fast blue; PBS: phosphate-buffered saline; EB: Evan’s blue; ECs: the endothelial cells; ELISA: enzyme-linked immunosorbent assay; COX-2: Cyclooxygenase 2;NF-κB: nuclear factor kappa-light-chain-enhancer of activated B cells; Tie2:Tyrosine Kinase Receptors 2 TNF-α: tumor necrosis f actor-alpha; IFN-γ: interferon gamma; MBP: myelin basic protein; ANOVA: analysis of variance; VCAM-1: analysis of variance vascular cell adhesion molecule-1; VEGF: vascular endothelial growth factor; IL-1: cytokines interleukin-1.

Abstract

 

The breakdown of normal blood-brain barrier (BBB) function and accompanying vascular leakage are fundamental stages in the onset of multiple sclerosis (MS) and its animal counterpart, experimental allergic encephalomyelitis (EAE). In the present studies, Angiopoietin-1 (Ang-1), a member of endothelial growth factors which is well-known for its role in establishing and maintaining vascular integrity, as well as C16, a peptide which could competitively bind to integrin v3 expressed in endothelial cells, were adopted as agents to treated acute EAE model induced in Lewis rats. The results showed Ang-1 application gained better effects in ameliorating inflammation-induced vascular leakage than C16. Moreover, treatment with the combination of Ang-1 and C16 appears to obtain better effects, not only in alleviating the degree of inflammation and reducing axonal loss/demyelination, but also in down-regulating pro-inflammatory cytokines expression and achieving faster functional recovery, than either treated with Ang-1 or C16 alone. These additive protective effects inferred that Ang-1 and C16 treatments may target different specific receptors and act through different pathways.

Keywords: multiple sclerosis; experimental autoimmune encephalitis; angiopoietin-1; C16; combinated treatment.

Introduction

Disruption of the blood-brain barrier (BBB) appears to be a critical event in the disease process of experimental allergic encephalomyelitis (EAE), which is an inflammatory demyelinating disease of the central nervous system (CNS) and frequently was used as a model for multiple sclerosis (MS) [1-3]. Enhanced permeability of the BBB facilitated edema formation and recruitment of inflammatory-type cells into target tissues to mediate eventual myelin loss and neuronal dysfunction [1, 4]. Angiopoietins 1 (Ang1) is one of a novel growth factors family that can bind to Tyrosine Kinase Receptors 2 (Tie2) and regulate several aspects of the angiogenic process, which are importance for the normal development of the cardiovascular system [5]. Previous studies have showed the anti-leak effects of Ang1 have long been applied in suppressing regional edema in adjuvant-induced arthritis [6], lessening LPS-induced microvascular dysfunction in a murine model of sepsis [7-8] and reducing endotoxin-induced vascular leakage in the lung, heart and kidney [8-15]. Furthermore, currently studies suggested Ang-1 treatment also could ameliorate inflammation-induced vessels leakage in CNS and inhibit inflammatory cell infiltration into the brain and spinal cord in acute EAE model [16-17].

A peptide named C16 (KAFDITYVRLKF) could selectively bind to integrin v3 which over-expressed in the surface of endothelial cells in EAE rodent model at 2 weeks post- -immunization [18]. It was thought to competitively interfere in the binding process of leukocyte with endothelial cells that required for transmigration, therefore ameliorates the severe of inflammation [19-21]. Recent studies have revealed that this novel vascular-v3 integrin-binding peptide could cooperate with Ang-1 in producing even better outcomes in rodent models of spinal injury and uveitis, an autoimmune disease induced by injecting IRBP protein [16]. Therefore, our present studies were designed to evaluate the combined effects of C16 and Ang1 co-treatment, in order to develop even more effective therapeutic strategies of MS/EAE. Multiple histological and immunohistochemical staining studies, molecular biology and electrophysiological tests were employed to assess the blood vessel permeability, axonal loss and demyelination, pro-inflammatory cytokines expression and functional recovery of differently treated acute EAE model.

Materials and methods

Animals and EAE induction

A total of 180 adult male Lewis rats were obtained from Zhejiang University Laboratory Animal Services Center. Of these, 20 were used as normal controls and the remaining 160 were randomly assigned into four groups (one vehicle-treated group and three drug-treated groups: Ang1, C16 and Ang1+C16, n=40/group). Experiments were carried out in accordance with NIH Guidelines for the Care and Use of Laboratory Animals, with approval from the animal ethics committee at the Zhejiang University.

EAE was induced in Ang1, C16 , Ang1+C16 and vehicle-treated rats, as previously described [18,22]. Beginning on day 7, animals were weighed and assessed for clinical signs of disease, the severity of which was assessed using a scale ranging from 0 to 5: grade 0 = no signs, grade 1 = partial loss of tail tonicity, grade 2 =total loss of tail tonicity, grade 3 = unsteady gait and mild paralysis, grade 4 = hind limb paralysis and incontinence, and grade 5 =moribund or death [23]. The EAE model was generally considered a success if rats were assigned a score that exceeded 2. Disease severity was assessed until the time of sacrifice and no animal scored above grade 4.

Intravenous injection of Ang-1 and/or C16

The Ang-1 peptide (synthesized by Shanghai Science Peptide Biological Technology Co., Ltd, China) was dissolved in distilled water to a final concentration of 2mg/ml. Since previous studies have shown Ang-1 to be more effective at 200 and 400 µg/day compared to vehicle [17], we chose a dose of 400 µg/day in present experiment. The C16 peptide was prepared and administered as previously described [18]. In brief, C16 (synthesized by Shanghai Science Peptide Biological Technology Co., Ltd, China) was dissolved in distilled water with 0.3% acetic acid. The solution was sterilized through a 0.22-μm disc filter and then neutralized with NaOH to pH7.4. This solution was buffered by adding an equal volume of sterile PBS, and the final stock solution was adjusted to a concentration of 4mg/ml. The animals were treated with C16 at a dose 2mg/day, according to the results of previous dose-dependent tests. Rats in treated groups received 1mL solution which containing 400 µg of Ang1 or/and 2mg of C16 injection each day for 2 weeks. The control (vehicle) group (n=40) was treated with 1mL of the distilled water without any peptide via intravenous injection of the tail vein [16]. The first dose was given immediately after EAE induction, with access to the tail veins being achieved by beginning injections at the caudal end of the base of the tail and into one vein. Injection sites were moved to the alternate left or right side and became increasingly rostral on subsequent days.

Neurophysiological testing

Cortical somatosensory evoked potentials (c-SEPs) were recorded 2 (the peak stage of vehicle) and 8 weeks post immunization (the recovery stage of vehicle) for five rats in each group just before animals were sacrificed. Mice were fixed into a stereotaxic frame and the surgical processes were done according to previous experiments [24]. For registration of c-SEPs, screw electrodes were implanted over primary somatosensory cortical areas, and cerebellar reference electrodes were placed over the appropriate cortical area. The contralateral forepaw was stimulated using a pair of fine sub-cutaneous electrodes with a sufficiently large amplitude (15 V) and duration (40 ms) to produce a maximum SEP (averaged over 30 stimuli). SEPs were amplified, filtered, digitally converted, and stored for post-hoc analysis. Values obtained by the three series of stimulations were processed by statistical analyses. Peak positive and negative values were measured, and results were expressed as the mean ± SEM of voltage amplitude (μV) and latency (ms) [25-27].

Cortical motor evoked potentials (c-MEP) were perform at the same time points as c-SEPs. Following anesthesia, a midline incision was made on the rat’s head skin, the tissues underneath were cleaned and the cranium exposed. Screw electrodes were implanted to a depth of 0.75 mm over primary somatomotor cortical areas, lightly contact with, but did not put pressure on or puncture the dura mater. A reference electrode was inserted in 2 mm away from the screw electrode. Sensorimotor cortex was stimulated at 10 Hz with trains of 10–25 pulses (1 ms, 1 mA) evoked a visible contralateral hindlimb movement, and signals were averaged for obtaining a c-MEP [28-29].

Perfusion and tissue processing

Animals in vehicle- and Ang-1and /or C16-treated groups were sacrificed 2 and 8 weeks (5/time point in each group) post immunization. Brain cortical and spinal tissues were collected, and sections were prepared as previously described [30]. A portion of each tissue was processed for histological assessment, immunohistological staining, and immunofluorescent staining. The remaining tissues were fixed in 2.5% glutaraldehyde solution and then examined by transmission electron microscopy.

Vascular labeling with the India ink technique

For vascular labeling experiments, India ink was filtered at 5.0 µm (Millipore, USA) before use. The femoral vein was exposed and received an intravenous injection of India ink (1 ml/kg). One hour after injection, animals of rats (n=2/each group) were then sacrificed via intracardial perfusion with saline followed by 0.1M PBS containing 4% paraformaldehyde for 5 min. For vascular labeling experiments, the spinal cord and brain tissues were carefully harvested and dissected. 1cm of the lumbar spinal cord and half of each animal’s brain were fixed in the same fixative for 4h, and then transferred into 30% sucrose in PBS until the tissue sank to the bottom of the container. Twenty m-thick sections were cut on freezing microtome through the coronal plane of the brain and the transverse plane of spinal cord using a Leica cryostat, and then mounted onto 0.02% poly-L-lysine-coated slides. photographed with a Nikon TE-300 research microscope.

Histology assessment

Cresyl violet (Nissl) staining was employed to assess inflammation and neuron survival counts. An assessment of the severity of inflammatory cell infiltration was scaled as follows [31]: 0, no inflammation; 1, cellular infiltrates only around blood vessels and meninges; 2, mild cellular infiltrates in parenchyma (1–10/section); 3, moderate cellular infiltrates in parenchyma (11–100/section); and 4, serious cellular infiltrates in parenchyma (100/section). Neuron counts were restricted to cells with a well-defined nucleolus and a cell body those displayed adequate amounts of endoplasmic reticulum. Digital images were collected using a Nikon TE-300 microscope in five sections/animal and three visual fields/section under 200× magnification bright-field viewing.

Luxol fast blue (LFB) staining was used to evaluate the degree of demyelination, as previously described [18]. Digital photomicrographs were obtained at 40×magnification, and the amount of demyelination was scored as follows [32]: 0, normal white matter; 1, rare foci; 2, a few areas of demyelination; 3, confluent perivascular or subpial demyelination; 4, massive perivascular and subpial demyelination involving one half of the spinal cord with presence of cellular infiltrates in the CNS parenchyma; and 5, extensive perivascular and subpial demyelination involving the whole cord section with presence of cellular infiltrates in the CNS parenchyma.

Bielschowsky silver staining was performed to estimate axonal loss [18,22], which was assessed using the following scale [32]: 0, no loss; 1, a few foci of superficial loss involving less than 25% of tissue; 2, foci of deep axonal loss, encompassing over 25% of tissue; and 3, diffuse and widespread axonal loss.

Luciferase Assay

To quantify the permeability of the blood–spinal barrier to proteins, rats (n=3/group) at 2 and 8 weeks post immunizationwere injected with 80 ml of a 0.5 mg/ml solution of luciferase (L9506; Sigma) in 0.05M phosphate-buffered saline (PBS)/0.001% bovine serum albumin into the jugular vein 30 min before processing [33]. After flushing out blood by PBS perfusion, a 5-mm block in brain (2.5mm in spinal cord) was collected and protein was immediately extracted from the tissue in lysis buffer (E1500; Promega). The sample was then centrifuged and the supernatant was used to measure enzyme activity with a luciferase assay kit (E1500; Promega) and a luminometer. All samples were analyzed in triplicate.

Assessment of BBB disruption with Evan’s blue extravasation

At 2 and 8 weeks postimmunization, rats (n=3/group) were randomly selected for assessment of BBB vascular permeability with a modified Evans blue extravasation method. Briefly, at 2 and 8 weeks postimmunization, rats were anesthetized with sodium pentobarbital (60 mg/kg, intraperitoneal injection) and infused with 37°C Evans blue dye (2% in 0.9% normal saline, 4 ml/kg) via the right femoral vein over 5 min. Two hours later, half of rats were perfused with 300 ml normal saline to wash out any remaining dye in the blood vessels. BBB permeability was then evaluated in the brain motor cortex and spinal cord tissue. Absorbance of Evans blue in the supernatant was then measured with a spectrophotometer (Molecular Devices OptiMax, USA) at 610 nm. Dye concentrations were expressed as μg/g of tissue weight and calculated from a standard curve obtained from known amounts of the dye [34]. Another half of rats were sacrificed by decapitation, and brains and spinal cord tissue were removed and sectioned to 20um in thickness for collected digital images.

Edema Measurement

In order to assess brain water content, the wet weight dry weight method was employed. At 2 weeks and 8 weeks post-immunization, animals (n=3 each group/each time point) were anaesthetised with pentobarbitone and decapitated. Brains were rapidly removed from the skull, the olfactory bulbs and cerebellum discarded and the remaining brain separated into left and right hemispheres. Each hemisphere was immediately weighed for wet brain weight. Following weighing, tissue was immediately wrapped in pre-weighed, labelled aluminium foil and placed in an oven for 72 hours at 100°C. Specimens were then removed from the oven and re-weighed to obtain dry brain water weight. Brain water content in each sample was then calculated using the wet-dry method formula [35]:

Immunohistochemical staining

Immunohistochemical stainingof anti-COX-2 (1:1000, BioVision, Milpitas, CA), anti- Anti-NF-kB p65 (1:500, AbCam, Cambridge, MA), anti-v3 integrin (1:500, eBioscience, San Diego, CA), anti-CD45 (1:500; AbCam, Cambridge, MA), and mouse anti-myelin basic protein (MBP, 1:500,AbCam, Cambridge, MA) was performed as previously described [18,22]. Five sections from the motor cortex and anterior horns of the spinal cord of each animal were randomly selected and imaged under 200× magnification for three visual fields/section.

Processing for electron microscopy

Processing for electron microscopywas performed as described previously (Fang et al., 2013; Han et al., 2013). Images were captured first at low resolution and then at higher magnification in different regions of the white matter (2550−30000×).

Cytokine quantification by enzyme-linked immunosorbent assay (ELISA)

Peripheral blood samples were collected from rats sacrificed by decapitation 2 and 8 weeks post immunization (n = 3 per time point/group).ELISA was performed as previously described (Han et al., 2013). Optical density was measured at 450 nm on a Model 680 Microplate Reader (Bio-Rad,UK), and was quantified by GraphPad Prism 4 (GraphPadSoftware,Inc, San Diego, CA).

In situ hybridization

Briefly, brain and spinal cord sections were fixed in 4% paraformaldehyde overnight at 4°C and allowed to air-dry for 30 min. Slides were then incubated in 0.2 mol/L HCl for 10 minutes followed by digestion with Proteinase K (10 μg/ml) (Sigma Chemical Co., Deisenhofen, Germany) for 10 minutes at room temperature. Following prehybridized for 5 hours at room temperature, the sections were hybridizated with a digoxigenin-labeled (Boehringer Mannheim, Mannheim, Germany) 560-bp tie-2 mRNA fragment encoding part of the rodent tie-2 extracellular domain. Hybridization with sense probe served as control. Tissue sections were incubated in a humidified chamber under glass coverslips at 70°C for 16 hours. After post-hybridization stringency twice washes, hybridized probes were detected by an anti-digoxigenin antibody conjugated to avidin–biotin peroxidase complex using DAB as substrate (Boster, China). Color reaction time ranged from 20 mins, after which slides were rinsed in PBS, counterstained with hematoxylin, dehydrated and mounted [36].

 Western blotting

Rats were sacrificed by decapitation 2 and 8 weeks post immunization (n = 3 per time point/group). The whole cortex and a 10-mm lumbar spinal cord segment were prepared from each animal for western blotting (anti-COX-2,v3 integrin, CD45, MBP and NF-kB p65), which was performed as previously described (Fang et al., 2013; Han et al., 2013). Rabbit anti--actin (1:5000, AbCam, MA) was used to normalize protein bands to a gel loading control, and the primary antibody was omitted for the negative control.

Statistical analysis

Data is presented as mean±standard deviation (SD). A Kruskal-Wallis nonparametric one-way analysis of Variance (ANOVA) was used for data presented as percentages. Differences between clinical scores and histological scores were analyzed with Mann-Whitney tests. Data was analyzed by SPSS 13.0 software and P values less than 0.05 were considered statistically significant. All statistical graphs were performed with GraphPad Prism Version 4.0 (GraphPad Prism Software, Inc. CA).

Results

Ang-1 and/or C16 treatments reduce blood vessel leakiness and BBB permeability

 The india ink and Evans blue (EB) dye leaking into tissue are indicative of compromised blood vessel integrity and tissue edema. In the current study, we saw that vasculature leakage phenomenon were severe in vehicle treated EAE rats (Fig.1 B,F, I). The Ang1 (Fig.1 C,J) and A+C (Fig.1E,G,L) treated groups could all notably reduced the leakage sign surrounding blood vessels, while in C16 (Fig.1 D,K) treated group, visible blood vessel leakiness could still be observed.

At the early stage of EAE, EB content in vehicle-treated rats reached 3 fold increases compared with normal healthy rats. As compared to those of vehicle-treated controls, Ang-1-and A+C treated EAE rats exhibited significantly reduced quantities of EB extravasation, but the C16 treated group still gain 1.5 fold increases when compared with normal rats (Fig.1 M).The samilar trend was preserved at 8 weeks post immunization (Fig. 1 N).

Blood-CNS-barrier permeability was measured by the amount of luciferase that extravasated into the tissue after intravenous injection. In vehicle-treated EAE rats, luciferase values reached 8 fold increases compared normal rats (Fig.2 A) 2 weeks post immunization. However, with daily Ang-1 and A+C injections all could clearly decreased luciferase values. Although not as evident as the effects of Ang1 and A+C, C16 treatment also decreased luciferase values when compared with vehicle control, which suggestive of a preservation in blood vessel integrity by all three agents (Fig. 2A). Eight weeks post immunization, CNS vascular permeability in vehicle-treated EAE rats decreased to 2 fold increases compared normal rats (Fig.2 B). Obvious decreases in vascular permeability were still seen in all three agents when compared with vehicle. Similar as at 2 weeks post immunization, the A+C-treated rats revealed more visible effects thanC16- and Ang1 treated groups. (Fig. 2 B).

Following EAE induction, significant increase brain water content occurred compared to normal rats (Fig.2 C, D). Treatment with both the Ang1 and A+C significantly resulted in a marked reduction (p<0.05) in brain water content (Fig.2 C, D). Evaluation of brain water content showed C16 treated rats also showed an evident reduction in brain water content occurred at 2 weeks post-immunation, but to a level that was no longer significantly different compared to vehicle-treated controls at 8 weeks post-immunation. In contrast, the Ang1 and A+C treated groups remained significantly decreased brain water content compared to control animals (Fig.2 C, D).

Transmission electron microscopy further revealed that there was no edema surrounding normal blood vessels (Fig. 3 A), myelin and nuclei all exhibited normal signs (Fig. 3 B,C). However, in vehicle-treated EAE rats, severe edema (Fig.3 D) and inflammatory cells leaking from blood vasculature were detected in the extracellular space surrounding the vessels (Fig.3 E), myelin displayed splitting and vacuole changes (Fig.3 F), and neurons showed signs of apoptosis (Fig. 3 G). In contrast, in Ang-1-treated EAE rats, we observed reduced localized edema (Fig. 3 H) and more lightly vacuolated myelin sheaths (Fig. 3 I). In C16 treated group, a certain degree of vesicular disintegration and edema were still visible (Fig. 3 J), but the myelin displayed relatively normal shape (Fig.3 K). The A+C-treated EAE rats exhibited little to no vesicular leakage (Fig. 3 L), and a greater number of intact myelinated fibers appeared in A+C-treated group (Fig 3 M). Meanwhile, the loose of tight junctions between endothelial cells (arrow) could be found in vehicle treated EAE rats (Fig.3 N), while normal tight junctions (arrow) appeared in A+C treated EAE rats (Fig.3 O).

Ang-1 and/or C16 treatments attenuated perivascular/parenchymal infiltration and reduced CNS inflammation

At the peak stage of acute EAE (2 weeks post immunization), there revealed a significant increase in cellular density in EAE rats treated with vehicle. Diffuse infiltration of inflammatory cells appeared not only around blood vessels, but also throughout brain and spinal cord parenchyma and under the meninges (Fig. 4C −F). However, less severe perivascular and parenchymal inflammatory cell infiltration was observed in Ang-1 (Fig. 4 G-H) and C16 (Fig. 4 I-J) -treated EAE rats, and much less infiltration was found in groups treated with A+C agent (Fig. 4 K-L). Moreover, the inflammatory score of Ang-1 and /or C16-treated groups was also significantly lower than that of the vehicle-treated EAE group both 2 and 8 weeks post immunization. This decreasing was especially prominent in A+C-treated EAE rats (P < 0.05, Fig. 4M−N).

In order to determine the phenotype of the infiltrating inflammatory cells, we performed immunostaining for CD45 (Fig. 5, a pan-leukocyte marker for leukocytes). Our results showed that cells positive for CD45 increased remarkably in both grey and white matter of vehicle-treated EAE rats, mainly expressed in infiltrated cells surrounding blood vessels and within the parenchyma (Fig.5 B,F). An evident decline of leukocyte extravagation was detected in Ang-1 and /or C16-treated groups both at 2 (Fig. 5 C-E) and 8 weeks (Fig. 5 G-I) post immunization. Westerns blot of these three markers also confirmed immunostaining results (Fig. 5 J-Q).

Both Tyrosine Kinase Receptors 2 (Tie2) and integrin av3 were expressed in endothelial cells of different agents treated EAE rats

In normal rats and each different treated groups, the results of in situ hybridization of Tie2 andimmunostaining of av3 integrin all showed evident expressions appeared in the endothelial cells of blood vessels (supplementary Fig. 1).

Ang-1 and/or C16 treatments suppressed the expression of pro-inflammatory cytokines COX-2, NF-kB, TNF-α and interferon gamma (IFN-γ)

Expression levels of COX-2 (Fig. 6), NF-kB (Fig. 7) were detected by western blot, while both TNF-α and IFN- in blood serum were measured by ELISA (Fig. 8) at early (week 2) and late (week 8) stages of EAE. We found the expression of COX-2, NF-kB, TNF-α andIFN- were all significantly increased in vehicle-treated EAE mice, and the Ang1 and C16 treatments all show remarkable suppressive effects (Fig.6,7,8). In generally, the A+C treated group gained a significantly greater effect on the attenuation of pro-inflammatory cytokines expression than other two groups (Fig. 6,7,8). Meanwhile, immunohistochemical labeling revealed that vehicle-treated EAE rats displayed greater expression of COX-2 (Fig. 6, supplementary Fig.2), NF-kB (Fig. 7, supplementary Fig.3) and TNF-α (Fig.8 E-H) in neurons and other CNS cells when compared with normal controls (Fig. 6,7), and that Ang-1 and /or C16-treated groups could marked decrease COX-2, NF-kB and TNF-α immunoreactive neurons number when compared with the vehicle-treated group (Fig. 6,7,8).

Ang-1 and/or C16 treatments inhibited demyelination and prevented axon loss

We used Luxol fast blue (LFB) staining and MBP (one of the major central myelin proteins) immunohistochemical labeling to assess the total amount of demyelination that occurred in each group. Massive confluent demyelinated areas presented in the CNS parenchyma of vehicle-treated EAE rats when compared with normal rats at 2 weeks post immunization (Fig.9 C,D, Supplementary Fig. 4 D-F). At the same time, treatment with Ang-1 exhibited a gradual decline in the visible areas of demyelination (Fig.9 E-F, Supplementary Fig. 4 G-I). Meanwhile, both MBP immunohistochemical staining and LFB revealed both the C16 and A+C treated groups revealed much more myelinated areas when compared with the Ang1 treated rats (Fig.9 G-J, Supplementary Fig. 4 J-O). At 8 weeks post immunization, myelination loss in vehicle-treated EAE rats had partially improved (Supplementary Fig.5 A-B). The demyelination was still dramatically reduced by Ang-1 and/or C16 treatments when compared with vehicle control (Supplementary Fig.5 C-H). Moreover, Ang-1 and/or C16 treatments remarkably reduced demyelization score (Fig.10 A-B) and increased MBP-expression level (as showed by western blot, supplementary Fig. 6) both at 2 and 8 weeks post immunization, the A+C-treated group still got more obviously effect than other two groups (Fig. 10 A-B).

Two weeks post immunization, impregnation of axons with Bielschowsky’s silver revealed a reduction of axonal density in the brain cortex and spinal cord of vehicle-treated EAE rats when compared to normal healthy animals (Fig.11 A−D), and the injured axons displayed swelling, deformation, and ovoid formation (Fig.11 D). Compared with vehicle-treated rats, more axons with relatively normal formation were observed at Ang-1 and/or C16 treated groups (Fig. 11E−J). Eight weeks post immunization, vehicle-treated EAE rats still displayed less axonal density in both the white and gray matter of the CNS (Supplementary Fig.7 A-B). In contrast, more axons with relatively normal formation were maintained in Ang-1, C16-treated EAE rats (Supplementary Fig.7 C-F), especially in A+C application groups (supplementary Fig.7 G-H).The measurement of axonal loss score (Fig.10 C-D) also confirmed the more evident effects of C16 and double treatments on axonal protection.

Effects of treatment with Ang-1and /or C16 treatments on neuron loss in the CNS of EAE rats

There was marked neuron loss in the CNS of the vehicle-treated EAE rats, especially 8 weeks post immunization (Fig. 12 C, D) when compared with normal animals (Fig. 12 A, B). However, in Ang-1and /or C16-treated EAE rats, more neurons were present in the anterior horn of the spinal cord and in the motor cortices (Fig. 12 E–J).Similarly, the A+C-treated group gained more normal shaped neurons other two groups (Fig.12 K).

Ang-1and/or C16 treatments reversed electrophysiological dysfunction of EAE rats, postponed the onset of motor symptoms andalleviated the disease severity

EAE induction resulted in an increase in the latency-to-waveform initiation and a decrease in peak amplitude (Fig 13, 14) both in c-SEP (Table 1, 3) and MEP (Table 2, 4 ) record. A decrease in the waveform slope in vehicle-treated mice was also detected in c-SEP record (Fig. 13). However, the application of Ang1 and /or C16 could significantly prevent electrophysiological disturbances, change the disease-related latency delays which are related to the speed of conduction (Fig. 13,14), and reversed the decrease of amplitude with are related to the fiber numbers survived (Fig.13,14). Besides, the A+C treated group was revealed to achieve more remarkable effects than other two groups. In line with this, the functional scoring showed that in vehicle-treated rats, disease symptoms appeared on days 7–9 postimmunization, and that the acute phase of the disease began with a sharp increase of motor symptoms (average clinical score 3.0–4.0) which peaked at 2 weeks postimmunization (Fig. 15 A-C). Thereafter, the clinical score gradually declined and the acute EAE disease process underwent a spontaneous recovery. At 8 weeks postimmunization, the clinical score of survival in vehicle-treated animals returned to a level that ranged from 1.5 to 2. Animals treated with Ang-1 and/or C16 showed a similar disease course to the vehicle group, but with an obvious delay in the onset of clinical signs; motor symptoms began on days 10-14 and peaked 3–4 weeks postimmunization with an average clinical score of 2–2.5. Additionally, the later stages in the Ang-1, C16 and A+C-treated groups showed similar trends as the vehicle group, the clinic score in each time point was remarkably declined compared with the vehicle control (Fig. 15 A-C). Furthermore, the A+C treated rats exhibited a more obvious suppressed clinical score at the clinical peak stages, but not at the onset and recovery stage (Fig.15 C).

Table 1: C16 and Ang1 treatment reduced the c-SEP latencies and increased c-SEP amplitudes. *P< 0.05 versus vehicle-treated EAE mice. ** P< 0.01 versus vehicle-treated EAE mice.

2 W	Latency (ms)
Groups	N	P	Wave amplitude (µV mean±SD)
Normal	13.57 ± 1.36	16.3±0.79	14.33 ± 3.06
Vehicle	37.63 ± 0.81	44.37 ± 1.29	1.93 ± 0.11
Ang1	20.37 ± 0.51	26.77 ±2.63	6.2 ± 0.72**
C16	21.83 ± 0.7	28.9±0.36	7.73 ± 0.64**
C16+Ang1	14.87 ± 0.8	21.97 ± 0.76	9.07 ± 0.59**

Table 2: C16 and Ang1 treatment reduced the MEP latencies and increased c-MEP amplitudes. *P< 0.05 versus vehicle-treated EAE mice. ** P< 0.01 versus vehicle-treated EAE mice.

2 W	Latency (ms)
Groups			Wave amplitude (µV mean±SD)
Normal	1.85 ± 0.74		5.33 ± 2.35
Vehicle	5.17 ± 0.9		0.07 ± 0.006
Ang1	3.23 ± 0.47		1.27 ± 0.65*
C16	2.23 ± 0.15		1.63 ± 0.21*
C16+Ang1	2.37 ± 0.12		3.7 ± 1.45**

Table 3: C16 and Ang1 treatment reduced the c-SEP latencies and increased c-SEP amplitudes. *P< 0.05 versus vehicle-treated EAE mice. ** P< 0.01 versus vehicle-treated EAE mice.

8W	Latency (ms)
Groups	N	P	Wave amplitude (µV mean±SD)
Normal	13.97 ± 2.05	18.2±2.86	11.41± 2.95
Vehicle	28.57 ± 5	36.07 ± 3.47	2± 0.23
Ang1	15.9 ± 0.92	20.87 ±6.03	4.67 ± 1.66*
C16	14.73 ± 0.47	21.57±2.29	4.98 ± 1.13*
C16+Ang1	14.23 ± 0.15	19.57 ± 1.24	7.69 ± 0.56**

Table 4: C16 and Ang1 treatment reduced the MEP latencies and increased c-MEP amplitudes. *P< 0.05 versus vehicle-treated EAE mice. ** P< 0.01 versus vehicle-treated EAE mice.

8 W	Latency (ms)
Groups			Wave amplitude (µV mean±SD)
Normal	0.88 ± 0.12		11.3 ± 3.54
Vehicle	5.57 ± 0.45		0.6 ± 0.36
Ang1	3.25 ± 0.64		1.15 ± 0.07*
C16	2.13 ± 0.24		2.65 ± 0.07**
C16+Ang1	1.17 ± 0.3		3.55 ± 0.49**

Discussion

During MS or EAE, loss of BBB function occurs [1]. Clinical signs of paralysis have been shown to depend on the sensitization of T lymphocytes on myelin basic protein (MBP), and correlate with the extent of perivascular cellular inflammation and edema in the CNS [1-2]. One particular morphological characteristic of the BBB is the presence of tight junctions which form the physical link between endothelial cells and prevent the non-specific passage of molecules into CNS tissues [4]. Previous studies have shown an increase in endothelial transcytotic activity associated with decreased mitochondrial content, which was evident for BBB dysfunction in EAE [1]. During MS and EAE, changes occurred in the capillary bed and lead to CNS edema and clinical signs [1]. Several potent mechanisms of passive leakage, including increased tight junctional permeability, increased interendothelial space and leakage alongside migrating inflammatory cells have been demonstrated [3]. At present, the major events mediating neuroimmune-directed BBB breakdown were believed to be: (1) Release of mediators, including interleukin-1 (IL-1) and tumour necrosis factor-a (TNF-a), from neuroantigen-activated inflammatory cells to instigate BBB disruption. (2) Migration of inflammatory cells into CNS parenchyma with passage of plasma constituents across leaky neurovasculature. (3) Perpetuation of BBB damage caused by permeability inducing factors released by infiltrated inflammatory cells, thus turn on a vicious circle [1].

Previous study pointed out one of the crucial systems regulating vascular cell integrity is the Ang1-Tie2 system. It has been recognized that Ang-1 serves as a Tie2 receptor agonist by phosphorylating Tie2 on tyrosine residues [37-38]. Ang-1 mediated Tie2 signaling is required to maintain cellular integrity and quiescence of the endothelial barrier [37]. Moreover, Ang1 could suppress the expressions of some adhesion molecules: intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin, thus counteracts vascular endothelial growth factor (VEGF) -induced inflammatory cells adhesion and infiltration [39]. Previous studies have revealed Ang-1 exhibits anti-inflammatory properties by interfering with the process of leukocyte transendothelial migration and changing vascular permeability [6, 11]. It has been showed to ameliorate endotoxin-induced lung injury and improve functional impairment in a rodent model of acute experimental autoimmune encephalomyelitis [15, 17, 40].

Previous studies showed a synthetic peptide named C16, representing a functional laminin domain, could selectively binds to v3 and 51 integrin, and promotes angiogenesis in vitro and in the chick chorioallantoic assay in vivo [41-43]. Furthermore, since v3 integrin is involved in the transition between tight adhesion of monocytes to the vascular endothelium [19-21], and occupancy of the v3 integrin could competitively decrease monocyte binding to ICAM-1 and inhibit the process of monocytes migration across the endothelium [16-18], we have observed the effects of C16 by performing continuously intravenous injection in rodent spinal cord injury and EAE model [16, 22, 24]. The results suggested that it might act as a protective agent by attenuating inflammatory progression and improving micro-environment

In present study, we observed the targets of Ang1 and C16 application— Tie2 and integrin av3 all exhibited notably high expression in the endothelial cells (ECs) of blood vessels.Our results (india ink and Evens blue labeling, and Evens blue and luciferase assay, nissel and CD45 immunstaining) also revealed Ang-1 and/or C16 treatments all could attenuate perivascular/parenchymal infiltration and suppress CNS inflammation, but the Ang-1 application gained better effects on ameliorating inflammation-induced vascular leakage and edema. This phenomenon might be due to the different target and mechanisms through which these two agents give play to effects. Although Ang1could inhibit the expressions of adhesion molecules and block the adhesion of inflammatory cells, its main effects could still be attributed to the maintenance of the normal cellular integrity of vascular ECs, promoting EC survival and reducing blood vessel leakage through Ang1-Tie2 system [44]. However, the main action mechanism of C16 is competitively blocking the v3 integrin and reduces inflammatory cells binding to endothelium, finally inhibit leukocytes migrated across the blood vessels [16]. Its function on endothelial cell survival, angiogenesis and reducing vascular leakage are of secondary importance [42-43]. The combinated treatment of Ang1+C16 possesses relatively normal micro-structure of tight junctions and double effects of Ang1 and C16, therefore confirmed the synergistic effects of two drugs which target different pathways.

NF-B is a transcription factor that plays a pivotal role in the processes of CNS inflammation. It contributes to the regulation of CNS inflammation by positively regulating the transcription of numerous genes, including cytokines interleukin-1 (IL-1), IL-6, tumor necrosis factor (TNF-), proinflammatory enzymes cyclooxygenase 2 (COX-2) and some adhesion factors. TNF-alpha is a key pro-inflammatory cytokine and critical regulator of inflammation, while COX-2 is a highly inducible enzyme by proinflammatory cytokines that contributes to the generation of chronic inflammation, the expression of these two genes is all regulated by NFkB [45]. Moreover, induction of COX-2 could be inhibited by antibody to IFN-γ, and IFN-γ could also work as an inflammatory mediator and/or tissue damaging agent to mediate the inflammatory process of EAE [46]. In our study, the expression of COX-2, NF-kB, TNF-α and IFN-γ all remarkably decreased in CNS tissue and/or in serum levels, which were in according with the suppressed inflammatory cells infiltration in each group, especially in the Ang1+C16 combination treated group.

The myelins and axons of CNS are vulnerable to the damages effects of inflammatory milieu and toxic inflammatory factors. Extensive demyelination and loss of normal axons are observed in our vehicle -treated EAE rats, which lies behind the clinical signs such as sensory/motor impairment and paralysis [47]. The lost of neurons in spinal cord and motor cortex at late stage (8 weeks post-immunation), as well as the increase of latency-to-waveform initiation and decrease in peak amplitude of c-SEP and MEP, all confirm the sensory/motor symptoms in EAE model caused by demyelination, axons degeneration and neuronal loss in a devastating inflammatory micro-environment. However, the application of Ang1 and/or C16 could effectively inhibit BBB breakdown, reduce proinflammatory cytokines which induced by NF-B pathway and released by infiltrated inflammatory cells, thus improved the inflammatory milieu. Finally, the decrease of demyelinated axons and increase of survival neurons with intact axons significantly reversed the reduction in waveform amplitude and prevented the increase in latency, as showed byneurophysiological testing. This phenomenon inferred increases of the intact fiber numbers and speed of axonal conduction after Ang1 and C16 applications, especially the combination of two treatments.

Most importantly, our present demonstrated a prominent combinated effects in Ang1 and C16 co-application. In A+C group, the brain water content, amount of luciferase and Evans blue that extravasated into the CNS were all significantly decreased just as in Ang1-treated group, the tight junctions between endothelial cells were also kept normal structure. Moreover, the decrease of infiltrated inflammatory cells in A+C group was more significant than in Ang1 or C16 group, which suggested an obvious additive effect come from two drugs target which different specific receptors and play anti-inflammation role through different pathways. The synergistic effects in anti-inflammation lead to more evident amelioration in CNS internal micro-environment and the vicious circle was turn off. The further improvements in myelin/axon morphology and functions could all be due to the reduplicated effects in combinated treated group.

Acknowledgements

This work was funded by the Zhejiang Provincial Natural Science Foundation of China no. R2110025 and the National Natural Science Foundation of China, project no. 81271333.

Legends

• Figure 1. The CNS of EAE rats exhibited evident Blood-brain barrier (BBB) damage and increased leakage, as revealed by India ink (A-G) and Evens blue ( H-L), counterstained with cresyl viollet. At 2 weeks postimmunization, the treatment of Ang1 and C16 exhibits an evident inhibitory effect on blood vessel permeability, especially in Ang1 and A+C treated groups. (A, H): normal rats (B, I): vehicle-treated EAE rats (arrow show leakage of India ink and Evens blue). (C,J): Ang-1-treated EAE rats; (D,K), C16-treated EAE rats (arrow show leakage of India ink and Evens blue); (E,L) Ang-1+C16-treated EAE rats. (F): The vehicle-treated EAE rats showed obviously BBB disruption phenomenon, the leaked india ink resembling medusa head; while in the A+C treatment (G), the increased leakage have been visibly alleviated and the BBB was similar to normal formation. (M,N): Evans blue extravasation was significantly increased in vehicle-treated EAE rats, while reduced mainly by Ang1 and C16 at 2 (M) and 8 (N) weeks postimmunization.(a), P < 0.05 versus the normal control; (b), P < 0.05 versus vehicle-treated EAE mice;(c), P<0.05 versus Ang-1-treated EAE rats; (d), P < 0.05 versus C16-treated EAE rats.

Figure 2: Using Luciferase (A,B) as a marker of BBB disruption treatment, results showed treated with both Ang1and A+C all resulted in a remarkable reduction of BBB permeability, which also showed much more decreased blood vessel leakage than in C16 treated group. Similarly, Ang1 and A+C treatment resulted in a significant reduction of brain water content (C, D) compared to vehicle treated rats and C16 treated rats at both 2 (A) and 8 (B) weeks postimmunization. (a), P < 0.05 versus the normal control; (b), P < 0.05 versus vehicle-treated EAE mice;(c), P<0.05 versus Ang-1-treated EAE rats; (d), P < 0.05 versus C16-treated EAE rats.

Figure 3: Electron micrograph demonstrating prevention of perivascular edema, demyelination/axon loss, and neuronal apoptosis in Ang-1 and C16-treated EAE rats. (A–C): Normal control group (A), blood vessel with normal shape, arrow indicates an endothelial cell; (B),normal myelinated axons exhibiting dark, ring-shaped myelin sheaths surrounding axons; (C),normal nuclei of neurons with uncondensed chromatin; (D–G),vehicle-treated EAE rats 2 weeks postimmunization.(D),Severe leakage out of the blood vessels and tissue edema was detected in the extracellular space surrounding the vessels; (E) shows an infiltrated inflammatory cell. (F),A myelin sheath displaying splitting, vacuoles, loose and fused changes, and an axon that is shrunken and dissolving.(G),Neuron showing apoptotic signs of a shrunken nucleus with condensed, fragmented, and marginated nuclear chromatin.Ang-1 (H,I), C16(J,K), and A+C (L,M)-treated EAE rats 2 weeks postimmunization, the perivascular edema and leakage were reduced, especially in Ang1 and A+C-treated group(H,L).Meanwhile, the loose of tight junctions between endothelial cells (arrow) could be found in vehicle treated EAE rats (N), while normal tight junctions (arrow) appeared in A+C treated EAE rats (O).(m), scale bar = 0.2 μm; (a,d-h,j,l), scale bar = 1 μm; (c,),scale bar = 2 μm; (b,I,k), scale bar = 0.5 μm, (n,o): scale bar=100nm.

Figure 4: Two weeks postimmunization, numerous infiltration of inflammatory cells was observed surrounding blood vessels and in the parenchyma of the brain and spinal cord of vehicle-treated EAE rats. Ang-1 and C16 application could all alleviated this phenomenon, but the combination treatment of Ang1 and C16 got more significant effects. Nissl staining, scale bar = 100μm. (A,C,D,G,I,K): coronal sections of brain motor cortex (BC); (B,D,E,F,H,J,L): traverse sections through the anterior horn of the lumbar spinal cord (SC). (A,B) normal rats (C–F): vehicle-treated EAE rats (arrow show the infiltrated inflammatory cells surrounding blood vessels and spreading in the parenchyma). (G,H), Ang-1-treated EAE rats; (I,J), C16-treated EAE rats; (K,L) A+C-treated EAE rats. (M,N): Ang-1 and C16 treatments attenuated inflammation in the CNS at weeks 2 (M) and 8 (N) postimmunization, as shown by the inflammation score. (a), P < 0.05 versus the normal control; (b), P < 0.05 versus vehicle-treated EAE mice;(c), P<0.05 versus Ang-1-treated EAE rats; (d), P < 0.05 versus C16-treated EAE rats.

Figure 5: Ang-1 and C16 treatment attenuated perivascular/parenchymal CD45⁺ leukocytes infiltration. (A-I): CD45 Immunostaining was performed 2 weeks and 8 weeks postimmunization to measure infiltration of leukocytes surrounding blood vessels in the brain motor cortex of vehicle-, Ang-1-, C16- and A+C-treated EAE rats, counterstained with hematoxylin. Two weeks postimmunization, (A), normal rats. (B), vehicle- (C), Ang-1- (D), C16-, and (E) A+C-treated EAE rats. Eight weeks postimmunization, vehicle-treated EAE rats (F), Ang-1-treated EAE rats (G), C16-treated rats (H), and A+C-treated EAE rats (I). Scale bar = 100 μm. ( J-Q): Ang-1 and/or C16 treatment inhibited the expression of leukocytes-specific marker CD45 at 2 (J,K,N,O) and 8 weeks (L, M, P, Q) postimmunization both in spinal cord and brain cortex, as shown by western blotting. (a), P < 0.05 versus the normal control; (b), P < 0.05 versus vehicle-treated EAE mice;(c), P<0.05 versus Ang-1-treated EAE rats; (d), P < 0.05 versus C16-treated EAE rats.

Figure 6: Treatment with Ang-1 and C16 decreased the expression of pro-inflammatory kinase COX-2 in both the spinal cord and brain at 2 weeks postimmunization. (A-J): COX-2immunostaining, counterstained with hematoxylin. (A,C,E,G,L): traverse sections through the anterior horn of the lumbar spinal cord (SC). (B,D,F,H,J): coronal sections of brain motor cortex (BC). Scale bar = 100 μm. (A, B) Normal rats. Two weeks postimmunization, density of COX-2 expression appeared in neuronal cells of vehicle-treated EAE rats (C, D), but the COX-2+ neural cells were obviously reduced in Ang1 (E,F), C16 (G, H) and A+C (I,J) treated groups when compared with vehicle-treated EAE rats. (K-P): Ang-1 and C16 treatment inhibited the expression of COX-2 at 2 (K,L,O,P) and 8 weeks (M,N,Q,R) postimmunization both in spinal cord and brain cortex, as shown by western blotting. (a), P < 0.05 versus the normal control; (b), P < 0.05 versus vehicle-treated EAE mice;(c), P<0.05 versus Ang-1-treated EAE rats; (d), P < 0.05 versus C16-treated EAE rats.

Figure 7: Treatment with Ang-1 and C16 decreased the expression of transcription factor NF-kB in both the spinal cord and brain at 2 weeks postimmunization. (A-J): NF-kB immunostaining, counterstained with hematoxylin. (A,C,E,G,L): traverse sections through the anterior horn of the lumbar spinal cord (SC). (B,D,F,H,J): coronal sections of brain motor cortex (BC). Scale bar = 100 μm. Two weeks postimmunization, clear NF-kB expression appeared in neuronal cells of vehicle-treated EAE rats (C, D) when compared with normal rats (A,B). However, the Ang1 (E,F), C16 (G, H) and A+C (I, J) treatment evidently reduced NF-kB + neural cells compared with vehicle-treated EAE rats. (K-R): Ang-1 and C16 treatment inhibited the expression of COX-2 at 2 (K,L,O,P) and 8 weeks (M,N,Q,R) postimmunization both in spinal cord and brain cortex, as shown by western blotting. (a), P < 0.05 versus the normal control; (b), P < 0.05 versus vehicle-treated EAE mice;(c), P<0.05 versus Ang-1-treated EAE rats; (d), P < 0.05 versus C16-treated EAE rats.

Figure 8: The Ang1 and C16 treatments, especially A+C treatment could effectively reduced the pro-inflammatory cytokines IFN- and TNF- expression, which were up-regulated in vehicle treated EAE rats. (A,B): Quantitation of the pro-inflammatorycytokine, IFN- in serum samplesat weeks 2 (A) and 8 (B) postimmunization by ELISA. (C,D), The expression of the pro-inflammatorycytokine TNF-α was measured 2 (C) and 8 (D) weeks postimmunization by ELISA. (a), P < 0.05 versus the normal control; (b), P < 0.05 versus vehicle-treated EAE mice;(c), P<0.05 versus Ang-1-treated EAE rats; (d), P < 0.05 versus C16-treated EAE rats. (E-H): TNF-αimmunostaining in spinal cord anterior horn at 8 weeks post-immunation, counterstained with hematoxylin, scale bar = 100 μm. (E): vehicle- (F), Ang-1- (G), C16-, and (H) A+C-treated EAE rats.

Figure 9: Ang-1 and C16 treatment inhibited demyelination in the CNS. (A,C,E,G,L): traverse sections through the anterior horn of the lumbar spinal cord, (B,D,F,H,J): coronal sections of brain motor cortex were stained with MBP and counterstained with hematoxylin. Scale bar = 100 μm. Images show that MBP-labeled myelin was evidently reduced in vehicle-treated EAE rats 2 weeks postimmunization (C,D) when compared with normal rats, while Ang-1 and/or C16-treated rats still obtained much more MBP+ myelin than rats of the vehicle group (E-J).

Figure 10: (A,B): Demyelination was measured by the estimated demyelination score. The C16 and A+C treated group revealed more notable myelin protective effect at both 2 (A) and 8 (B) weeks postimmunization. (C, D): The C16 and A+C treated group showed more distinct axonal loss preventing effects at 2 (C) and 8 (D) weeks postimmunization by estimated axonal loss score . (a), P < 0.05 versus the normal control; (b), P < 0.05 versus vehicle-treated EAE mice;(c), P<0.05 versus Ang-1-treated EAE rats; (d), P < 0.05 versus C16-treated EAE rats.

Figure 11: Ang-1 and C16 treatment alleviated axonal loss in the spinal cord and cerebral cortex revealed by Bielschowsky staining at 2 weeks postimmunization. (A,C,E,G,I): transverse sections through the anterior horn of the lumbar spinal cord (SC). (B,D,F,H,J): coronal sections of the motor cortex (BC). Scale bar=100m. Two weeks postimmunization, when compared with normal control group (A, B), vehicle-treated EAE rats (C, D) showed numerous axons undergoing gradual loss and exhibited deformed and ovoid formation. Ang1 (E, F), C16 (G,H) and A+C (I,J) treated EAE rats revealed more axons were kept when compared with the vehicle-treated group.

Figure 12: Treatment with Ang-1 and C16 reduced the loss of neurons in the spinal cord and brain. Nissl staining, 8 weeks after immunization, scale bar = 100μm. (A,C,E,G,I): transverse sections through the anterior horn of the lumbar spinal cord (SC). (B,D,F,H,J): coronal sections of the motor cortex (BC). K: Surviving neural cells calculated in different groups at 8 weeks after immunization following Nissl staining (each group is presented as a % of the normal control). (a), P<0.05 versus the normal control;(b),P < 0.05 versus vehicle-treated EAE rats.

Figure 13: Ang-1 and C16 treatments reduced the clinical severity of EAE in rats at 2 (A, C) and 8 (B, D) weeks post-immunation measured by determining somatosensory-evoked potential (c-SEP) latencies and amplitudes (measured from peak to peak between negative deflection (N) and positive deflection (P). The amplitude of c-SEP was obviously lower in vehicle-treated EAE rats and the latency was also significantly prolonged, while the treatments of Ang1 and/or C16 could effectively reverse these phenomenon. (A-B), Waveform of c-SEP. (C-D), c-SEP amplitude. (a), P < 0.05 versus the normal control; (b), P < 0.05 versus vehicle-treated EAE mice;(c), P<0.05 versus Ang-1-treated EAE rats; (d), P < 0.05 versus C16-treated EAE rats.

Fig14: Ang-1 and C16 treatments reduced the clinical severity of EAE in rats at 2 (A, C) and 8 (B, D) weeks post-immunation measured by determining motor-evoked potential (MEP) latencies and amplitudes. (A-B), Waveform of c-SEP. (C-D), c-SEP amplitude. The amplitude of MEP was significantly lower in vehicle-treated EAE rats and the latency was also significantly prolonged. However, these phenomenon were reversed following Ang1 and/or C16 treatments, especially in C16 and A+C groups. (a), P < 0.05 versus the normal control; (b), P < 0.05 versus vehicle-treated EAE mice;(c), P<0.05 versus Ang-1-treated EAE rats; (d), P < 0.05 versus C16-treated EAE rats.

Fig15: Clinical progression of EAE was attenuated after Ang1- (a), C16- (b), and A+C (c) treatment as measured by disease score. a, P <0.05 versus the vehicle-treated group at the same time point. aa, P <0.01 versus the vehicle-treated group at the same time point