Tanshinone IIA attenuates demyelination and promotes remyelination in A. cantonensis-infected BALB/c mice

Neurobehavioral scores

At 21 d post-infection, neurological assessments for motor and sensory function were performed by staff blinded to group assignments. Motor function encompassed freedom of movement, limb symmetry, climbing, and balance. Sensory function included proprioception; tentacles reaction; and visual, olfactory, and pain responses. Every test was graded on a scale from 0 to 3, and the total for all items was 27 (for details on behavioral scoring, please refer to ¹³. The neurological function score of each mouse was the sum of the scores on all of the above tests.

A. cantonensis larvae IV (L4) collection and quantification

The mice in A. cantonensis-infected and TSII-A treatment groups were sacrificed by chloral hydrate asphyxiation and cervical dislocation at 21 d post-infection. Whole brains were removed and placed in PBS to promote the escape of A. cantonensis from the brain. The number of A. cantonensis in the solution for each mouse was counted to calculate the insect detection rate (number of A. cantonensis/30).

Histopathological observation

Mice were sacrificed by chloral hydrate asphyxiation and cervical dislocation at 21 d post-infection. The brains were quickly dissected and fixed in 4% paraformaldehyde overnight. Paraffin-embedded sections were then prepared. Hematoxylin and eosin (HE) staining ¹⁴ was performed, and the meninges and cerebral cortex were observed and imaged under microscopy (Olympus, Tokyo, Japan).

Luxol fast blue (LFB)

The brain samples were dehydrated in a gradient alcohol series, cleared in xylene, and embedded in paraffin. Sections (5-μm thick) were cut on an RM 2035 microtome (Reichert S, Leica, Wetzlar, Germany). Myelin was detected using Luxol fast blue (LFB) staining ¹⁵ (Solvent Blue 38; Sigma-Aldrich, St. Louis, MO). The sections were observed under an inverted microscope equipped with a camera (DM 2500B, Leica).

Cell count

Blood samples were collected in tubes with 100 IU/mL heparin sodium, then detected with a hematology analyzer for animals (Sysmex, Kobe, Japan). Numbers of eosinophils in the blood were recorded and compared among the different groups.

Transmission electron microscopy (TEM)

After anesthesia, the animals were sacrificed by transcardial perfusion with 4% paraformaldehyde. The eyes were quickly removed and post-fixed overnight in 2.5% glutaraldehyde. Next, the optic nerves were post-fixed in a solution containing 1% osmium tetroxide (Sigma-Aldrich). Then, they were dehydrated in an acetone series and embedded in SPIN-PON resin. Resin polymerization was performed at 60 °C for 3 days. Semi-thin sections (0.5-μm thickness) were placed on glass slides and stained with toluidine blue. Finally, demyelination detection ¹⁶ was done by using a 300KV transmission electronic microscope (FEI, Hillsboro, OR), and three image fields were taken from each specimen of every group. These images were used to measure the numbers and diameters of axons in Image-Pro Plus 6.0 software (Media Cybernetics, Rockville, MD).

Immunofluorescence

After fixing with 4% paraformaldehyde, the mouse brain and optic nerve sections were cut at 15-μm thickness at -20 °C and mounted on glass slides. Then, sections were blocked with 3% bovine serum albumin (BSA) at room temperature for 1 h before incubation with rabbit anti-mouse CC1 (OP80, Merck, Kenilworth, NJ), mouse anti-O4 (O7139, Sigma), rabbit anti-ki67 (ab833, Abcam, Cambridge, UK), rabbit-anti-arg-1 (ab91279, Abcam), mouse anti-Iba-1 (SAB2702364, Sigma), and rabbit anti-inducible nitric oxide synthase (iNOS; ab15323, Abcam) monoclonal antibodies in 1% BSA at 4 °C overnight. Sections were washed three times in PBS, incubated with tetramethylrhodamine (TRITC)-labeled and fluorescein isothiocyanate (FITC)-labeled secondary antibody (Abcam) diluted 1:500 in 1% BSA at 37 °C for 1 h, and washed again in PBS. Then, DAPI (1:1000 dilution, 4083S Beyotime Biotechnology, Haimen, China) was used to stain the nuclei for 5 min. Specimens incubated without a primary antibody was used as negative controls. Slides were observed under a confocal microscope.

RNA isolation and real-time quantitative polymerase chain reaction (PCR)

Total RNA was extracted from cells with TRIzol reagent based on previous studies ¹⁷. For cDNA synthesis, RNA was reverse transcribed with a PrimeScript RT reagent kit (TaKaRa, Kusatsu, Japan). The expression levels of the genes encoding interleukin (IL)-1β, iNOS, tumor necrosis factor (TNF)-α, IL-6, IL-5, IL-13, CD86, CD206, Ym-1, CD11b, and Arg-1 were measured by real-time PCR with SYBR Premix Ex Taq kit (TaKaRa). cDNA amplification was performed on an ABI Prism 7900 HT cycler (Applied Biosystems, Foster City, CA). mRNA levels were measured by using the specific primer sets listed in Table ​Table11.

Western blotting

The brain tissues were washed twice with cold PBS and lysed in extraction buffer (20 mM HEPES [pH 7.4], 2 mM EDTA, 50 mM glycerophosphate, 1 mM dithiothreitol, 1 mM Na₃VO₄, 1% Triton X-100, and 10% glycerol) on ice. The lysates were centrifuged at 12,000 rpm for 15 min. Supernatants were collected, and protein concentrations were determined by bicinchoninic (BCA) Protein Assay (Bio-Rad, Hercules, CA). Proteins were boiled in sample buffer, separated in 12% sodium dodecyl sulfate-polyacrylamide gels by electrophoresis (50 μg/lane), and electroblotted onto nitrocellulose membranes (Pall Corporation, Ann Arbor, MI). Transferred blots were incubated sequentially with blocking agent (5% non-fat milk in Tris-buffered saline), anti-iNOS antibody (1:125 dilution, ab15323, Abcam), anti-Arg-1 antibody (1:500 dilution, ab91279, Abcam), and secondary antibody blots prior to development with enhanced chemifluorescence detection kits and exposure Hyperfilm (Fuji, Tokyo, Japan) according to the manufacturer's directions. The same blots were subsequently stripped and reblotted with antibodies that recognized β-actin (Sigma) as a reference to verify equal amounts of the protein among samples. Graphs of blots were obtained in the linear range of detection and were quantified for the level of specific induction with the Scion Image System.

TSIIA combined with AB protected OLs from damage and attenuated demyelination significantly more than other treatments

The above results showed that TSIIA treatment was an effective method to improve neurological behaviors and protect nerves from demyelination in the setting of angiostrongyliasis. To study the effect of different treatment methods on A. cantonensis-induced demyelination, we examined LFB staining of the brain and CC1 (mature OL marker) expression in the optic nerve. TEM was performed to visualize the optic nerve and quantify the numbers of myelin sheaths. Our results demonstrated that in the 21-d infection group, LFB staining was narrow, and vacuoles were obvious in high-power fields. This was also the case in the AB alone and AB combined with dexamethasone treatment groups. In the AB combined with CPT treatment group, LFB staining was deeper than in the 21-d infection group, but vacuoles were still obvious. In the TSIIA combined with AB treatment group, there were no differences from the normal control in the low- and high-power fields (Figure ​(Figure3A).3A). Immunofluorescence results showed that a decrease in CC1-positive cells in the 21-d infection and AB alone treatment group compared to the normal control. AB alone or in combination with dexamethasone did not increase the number of CC1-positive cells. However, the percentages of CC1-positive cells in the AB-CPT and AB-TSIIA combination groups were significantly higher than in the 21-d infection group. The effect in the AB-TSIIA combined treatment group was more obvious than in the other groups and was not significantly different to the normal control group with regard to the percentage of CC1-positive cells (Figure ​(Figure3B,3B, 3C). This indicated that A. cantonensis can induce OL damage and that TSIIA may have a protective effect. AB alone was not able to rescue these changes. The TEM analysis of nerve fibers also showed A. cantonensis infection led to demyelination and axon loss. AB with or without dexamethasone also had no obvious effect on demyelination. In contrast, AB combined with CPT or TSIIA appeared to have a good curative effect on the demyelination and axon loss caused by A. cantonensis, but TSIIA had a stronger impact (Figure ​(Figure3D,3D, 3E). We also observed a definite reduction in MBP protein expression at 21 d after A. cantonensis infection; AB with or without dexamethasone was unable to ameliorate the reduction, but AB combined with CPT or TSIIA obviously increased MBP expression (Figure ​(Figure3F,3F, 3G). These results demonstrate that AB combined with TSIIA attenuates demyelination induced by A. cantonensis infection.

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Figure 3

TSIIA combined with albendazole reduced demyelination more than other treatments in A. cantonensis-infected mice. (A) Luxol fast blue (LFB) staining revealed corpus callosum demyelination of mice in the control group, 21-d A. cantonensis infection group, AB treatment group, AB+De treatment group, AB+CPT treatment group, and AB+TSIIA treatment group. Black arrow indicates decreased myelin sheath after 21-d A. cantonensis infection, yellow arrows indicate deeper-staining myelin sheathes in 21-d A. cantonensis infected-mice treated with AB+TSⅡA. (B) CC1-positive cells present in the optic nerve were immunolabeled with CC1 antibodies (Green). CC1-positive cells decreased after 21-d A. cantonensis infection. AB, AB+De, and AB+CPT treatment did not obviously increase CC1-positive cells compared to AB+TSIIA treatment. (C) The percentage of CC1-positive cells (number of positive cells/DAPI-positive nuclei in one image) in the optic nerve in different groups. There were significantly fewer CC1-positive cells in the 21-d A. cantonensis infection and AB treatment groups compared to control. (D) TEM images of the optic nerve in different treatment groups. In the group infected with A. cantonensis for 21 d, optic nerve fibers exhibited obvious demyelination. (E) Quantification of optic nerve axons in the different treatment groups. (F), (G) Western blotting of MBP expression in the brain tissue of mice in the different treatment groups. Numerical results are presented as mean ± SEM. Student's t tests were used; n = 3 animals per group, experiments performed in triplicate, and at least three fields were analyzed per section in at least two non-adjacent sections per animal. Scale bars in A and C are 50 μm and 1 μm, respectively. *P < 0.05.

TSIIA combined with AB effectively promoted remyelination

We further evaluate the effects of different treatments on remyelination. At 21 d after A. cantonensis infection, there was an evident increase in large-size axons (>1.0 μm) and a decrease in small-size axons (<0.5 μm) compared with the normal control group, which indicated that A. cantonensis infection can cause optic nerve fiber swelling and damage. After treatment, the small-size axons were more obviously increased in the AB+TSIIA group compared to the other treatment groups (Figure ​(Figure4B),4B), suggesting there were more remyelinated axons in this group ^{18, 19}. In contrast, the other treatment groups had no marked change in axon diameter after A. cantonensis infection. At the same time, we quantified the g-ratio (axon circumference to myelin circumference), which provides a measure of the myelin thickness that complements axon morphology (diameter and density) in the assessment of demyelination in diseases ^{18, 20}. The results confirmed that the fully compacted myelin sheaths formed in the TSIIA combined with AB treatment group were thicker than those in other treatment groups. Moreover, the g-ratio of the AB combined with TSIIA treatment group was not significantly different from that of the normal group, whereas the other groups were significantly different from the control (Figure ​(Figure4C).4C). In addition, myelin sheath thickness in the AB combined with TSIIA and AB combined with CPT groups recovered to normal levels, but the other groups were still obviously different from the control group (Figure ​(Figure4D).4D). These observations suggest that both AB combined with TSIIA and AB combined with CPT promoted optic nerve remyelination, but TSIIA was more effective. When this ratio was plotted against the axon diameter, lower values were seen for all axon diameters in the optic nerve, and the equations for the best-fit lines obtained by linear regression for the AB combined with TSIIA treatment group datasets differed significantly from that for the 21-d infection group but there was no difference with the control group. The other treatment groups were different from the 21-d infection and control groups (Figure ​(Figure4E),4E), which indicated axons that are myelinated in the TSIIA combined with AB treatment group have thicker sheaths than axons of similar diameters in other treatment groups and 21d A. cantonensis-infected mice. Collectively, these results suggest that AB combined with TSIIA had a better effect on remyelination than other treatments.

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Figure 4

TSIIA combined with albendazole promoted remyelination in A. cantonensis-infected mice. (A) TEM images of the optic nerve in different treatment groups. The data of B, C, and D were derived from the images in A. Two-way ANOVA with Bonferroni's multiple comparisons test was used. (B) Percentages of myelinated optic nerve axons with various diameters in the different treatment groups. (C) The g-ratios of myelinated axons in the optic nerve for different groups. (D) Myelin sheath thicknesses of the optic nerve in the different treatment groups. (E) The g-ratio was plotted against the axon diameter. The best-fit lines obtained by linear regression differed significantly between the control and other groups, except for the AB+TSIIA treatment group. Moreover, the best-fit lines of the 21-d A. cantonensis infection and other treatment groups, except the AB group, were also significantly different. n = 3 animals per genotype, experiments performed in triplicate. At least 200 axons were analyzed per animal. * P < 0.05, **P < 0.01.

TSIIA combined with AB contributed to OL proliferation and differentiation

The results above revealed that TSIIA combined with AB more effectively protected OLs and prevented demyelination than the other treatments. However, it is unknown whether these therapy methods also promote remyelination. This process of demyelination protection is initiated by activation of OL progenitor cells, so we investigated OL proliferation and differentiation. We examined O4 (OL progenitor marker) and Ki67 expression in optic neuritis and found that the percentage of O4 and Ki67 double-positive cells increased slightly in the AB combined with dexamethasone treatment group compared with the normal control, 21d-A. cantonensis infection, and AB alone treatment groups. Notably, the AB combined with TSIIA group had the most O4-Ki67 double-positive cells compared to the other treatment groups. These findings indicate that both dexamethasone and TSIIA can protect OLs from death and contribute to OL proliferation, but TSIIA may have a stronger effect on remyelination than dexamethasone (Figure ​(Figure5A,5A, 5B).

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Figure 5

TSIIA combined with albendazole contributed to OL proliferation and differentiation in A. cantonensis-infected mice. (A) O4 and Ki67 double-positive cells present in the optic nerve were immunolabeled with antibodies for O4 (Green) and ki67 (red). White arrows indicate double-positive cells. (B) The percentage of O4-Ki67 double-positive cells present in the optic nerves of different groups. Numerical results are presented as the mean ± SEM. Student's t tests were used; n = 3 animals per group, experiments performed in triplicate, and at least three fields were analyzed per section in at least two non-adjacent sections per animal. Scale bar: 50 μm. * P < 0.05, **P < 0.01, ***P < 0.001.

TSIIA-induced remyelination was not achieved by inhibiting eosinophil infiltration and reducing A. cantonensis but by restraining inflammatory cytokine expression in the brain

To explore how TSIIA can inhibit demyelination and promote remyelination in A. cantonensis-infected mice, we performed HE staining of the brain, cell counting with a hematology analyzer, and real-time PCR to detect inflammatory infiltration of the brain, the number of eosinophils in blood, and mRNA levels of eosinophil-related cytokines (IL-5 and IL-13) and inflammatory factors (iNOS, TNF-α, IL-6, and IL-1β). To examine the effect of TSIIA on A. cantonensis, we measured survival rate of worms in A. cantonensis-infected and only TSIIA treatment groups. The experimental groups were normal control, 21-d A. cantonensis-infected group, AB treatment group, and AB combined with TSIIA treatment. A. cantonensis infection caused obvious eosinophil infiltration of the cerebral meninges, and eosinophils increased in the blood of 21-d infection group. AB alone or in combination with TSIIA had no apparent effect on eosinophil infiltration or quantity (Figure ​(Figure6A,6A, 6B). These results indicate that the alleviating effect of TSIIA on demyelination is not achieved by inhibiting eosinophil infiltration into the brain. Moreover, real-time PCR showed increased expression of the eosinophil-related cytokines IL-5 and IL-13 upon A. cantonensis infection, but there was no apparent difference before and after treatment with AB alone or in combination with TSIIA (Figure ​(Figure6C).6C). However, the increased expression levels of inflammation-related factors iNOS, IL-6, IL-1β and TNF-α upon A. cantonensis infection were not affected by AB treatment but decreased obviously after AB combined with TSIIA treatment. Moreover, iNOS expression was down-regulated much more apparently than other cytokines and even reached normal levels (Figure ​(Figure5D).5D). Finally, the survival rate of worms in the A. cantonensis-infected and only TSIIA treatment groups was not significantly different, suggesting that TSIIA did not kill A. cantonensis. These results suggest that the demyelination and remyelination effects of TSIIA are achieved via effects on inflammation-related signaling pathways rather than by inhibiting eosinophil infiltration into the brain or reducing the number of A. cantonensis.

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Figure 6

The remyelination effect of TSIIA was not achieved by inhibiting eosinophil infiltration into the brains of A. cantonensis-infected mice. (A) HE staining of the brain. In the 21-d A. cantonensis infection group, there was obvious inflammatory cell infiltration of the meninges and cerebral cortex. In the AB and AB+TSIIA treatment groups, inflammation was still observed in the meninges (black arrows). (B) The number of eosinophils in the blood. (C) mRNA expression of IL-5 and IL-13. (D) mRNA expression of iNOS, IL-1β, TNF-α, and IL-6. Numerical results are presented as mean ± SEM. Student's t tests were used; n = 3 animals per group, experiments performed in triplicate, and at least three fields were analyzed per section in at least two non-adjacent sections per animal. (E)Survival rate of worms in 21-d A. cantonensis infection group and TSIIA treatment groups. There were no significant differences between the two groups. Scale bar: 50 μm. * P < 0.05, **P < 0.01, ***P < 0.001.

Thanks Dr. Xiang Zeng for his suggesting amendments to the manuscript. He is the researcher of Histology &Embryology department, Zhongshan Medical College, Sun Yat-sen University.