Effects of albendazole combined with TSII-A (a Chinese herb compound) on optic neuritis caused by Angiostrongylus cantonensis in BALB/c mice

Abstract

Background: Angiostrongylus cantonensis (A. cantonensis) infection can lead to optic neuritis, retinal inflammation, damage to ganglion cells, demyelination of optic nerve and visual impairment. Combined therapy of albendazole and dexamethasone is a common treatment for the disease in the clinic, but it plays no role in vision recovery. Therefore, it has been necessary to explore alternative therapies to treat this disease. Previous studies reported the neuro-productive effects of two constituents of Danshen (a Chinese herb)-tanshinone II-A (TSII-A) and cryptotanshinone (CPT), and this study aims to evaluate the impacts of TSII-A or CPT combined with albendazole on optic neuritis caused by A. cantonensis infection in a murine model.

Methods: To assess the effects of TSII-A or CPT combined with albendazole on optic neuritis due to the infection, mice were divided into six groups, including the normal control group, infection group and four treatment groups (albendazole group, albendazole combined with dexamethasone group, albendazole combined with CPT group and albendazole combined with TSII-A group). The infection group and treatment groups were infected with A. cantonensis and the treatment groups received interventions from 14 dpi (days post infection), respectively. At 21 dpi, the visual acuity of mice in each group was examined by visual evoked potential (VEP). The pathologic alteration of the retina and optic nerve were observed by hematoxylin and eosin (H&E) staining and transmission electronic microscopy (TEM).

Results: Infection of A. cantonensis caused prolonged VEP latency, obvious inflammatory cell infiltration in the retina, damaged retinal ganglions and retinal swelling, followed by optic nerve fibre demyelination and a decreasing number of axons at 21 dpi. In treatment groups, albendazole could not alleviate the above symptoms; albendazole combined with dexamethasone lessened the inflammation of the retina, but was futile for the other changes; however, albendazole combined with CPT and albendazole combined with TSII-A showed obvious effects on the recovery of prolonged VEP latency, destruction and reduction of ganglion cells, optic nerve demyelination and axon loss. Compared with albendazole-CPT compound, albendazole combined with TSII-A was more effective.

Conclusions: The current study demonstrates that albendazole combined with TSII-A plays a more effective role in treating optic neuritis caused by A. cantonensis in mice than with dexamethasone, as applied in conventional treatment, indicating that albendazole combined with TSII-A might be an alternate therapy for this parasitic disease in the clinic.

Fig. 1

Data are mean ± SEM for P2 latency alterations of VEP in in different groups. a–f: P2 latency of VEP. Black arrow indicates P2 latency (ms). a: Normal group. b: Infected group by A. cantonensis (21 dpi). P2 latency is prolonged (>100 ms, ↓points). c: Albendazole (AB) treatment for 7 days after 14 dpi with A. cantonensis. P2 latency still is prolonged (>100 ms, ↓points). d: Albendazole combined with Dexamethasone (De) treatment for 7 days after 14dpi with A. cantonensis. P2 latency is still prolonged (>100 ms, ↓points). e: Albendazole combined with CPT treatment for 7 days after 14 dpi with A. cantonensis. P2 latency recovered to normal. f: Albendazole combined with TSII-A treatment for 7 days after 14 dpi with A. cantonensis. P2 latency recovered to normal. g: Data are mean ± SEM of P2 latency of mice in different treatment groups for 7 days after 14 dpi with A. cantonensis. *Statistically significant when compared with normal control (P < 0.05); #Statistically significant when compared with 21 dpi with A. cantonensis, n = 5 per group, P < 0.05

Fig. 2

The alteration of mouse retina and ganglion cells before and after treatment regimen following infection with A. cantonensis. a1-f1: retina in different treatment groups with HE staning. bar = 100 μm.↑ points ganglion cell layer of retina. a2-f2: Electron microscopy images of ganglion cells in different treatment groups. ↓ points cytoplasm of ganglion cells; ▲ points the nucleusof ganglion cells. bar = 1 μm. a1-a2: retina and ganglion cell in normal group. There was a layer of ganglion cells (↑ in A1). The cytoplasm and nucleus of ganglion cells were intact (↓ and ▲ in A2). b1-b2: retina and ganglion cell in groups infected with A. cantonensis at 21 dpi. The ganglion cell layer showed inflammatory cell infiltration (↑ in B1). Swelling was pronounced in cytoplasm of ganglion cells (↓in B2) and nucleus diminished. c1-c2: Albendazole (AB) treatment for 7 days after 14 dpi with A. cantonensis. Inflammation was alleviated in ganglion cell layer of retina (↑ in C1). Swelling can still be observed in cytoplasm of ganglion cell (↓ in C2) with an intact nuclear as usual (▲ in C2). d1-d2: Albendazole combined with Dexamethasone treatment (AB + De) for 7 days after 14 dpi with A. cantonensis. The inflammation of the retina almost resolved (↑ in D2). Swelling of ganglion cytoplasm relieved (↓ in C2). The nucleus was still obviously observed (▲ in D2). e1-e2: Albendazole combined with CPT (AB + CPT)treatment for 7 days after 14 dpi with A. cantonensis. Inflammation was alleviated in ganglion cell layer of retina (↑ in E1). Swelling decreased greatly in cytoplasm of ganglion cell (↓ in E2). f1-f2: Albendazole combined with TSII-A (AB + TSII-A) treatment for 7 days after 14 dpi with A. cantonensis. Inflammation was alleviated slightly in ganglion cell layer of retina (↑ in F1). Swelling diminished in cytoplasm of ganglion cell (↓ in F2) and the cells regained normal structure as in A2. g: Retinal thickness measured under a light microscope in normal group, 21 dpi infection group, albendazole treatment group (AB), albendazole combined with dexamethasone (AB + De) treatment group, albendazole combined with CPT (AB + CPT) treatment and albendazole combined with TSII-A (AB + TSII-A) treatment group. Data are mean ± SEM. *Statistically significant when compared with control (0 dpi) (P < 0.05); # Statistically significant when compared with 21 dpi (P < 0.05)

Fig. 3

TSII-A promotes RGC survival of mice infected with A. cantonensis. a-f: Immunofluorescence reinforced the image of RGC cells labeled with Brn-3A (Green). Scale bar = 100 μm. a Normal control. b: Untreated mice infected with A. cantonensis (21 dpi). c: Albendazole (AB) for 7 days after 14 dpi with A. cantonensis. d: Albendazole combined with Dexamethasone (AB + De) for 7 days after 14 dpi with A. cantonensis. e: Albendazole combined with CPT (AB + CPT) for 7 days after 14 dpi with A. cantonensis. f: Albendazole combined with TSII-A (AB + TSII-A) for 7 days after 14 dpi with A. cantonensis. g: Data are mean ± SEM of ganglion cell numbers in normal group, untreated group for 21 dpi with A. cantonensis, and treated group for 7 days after 14 dpi with A. cantonensis infection, respectively. *Statistically significant when compared with control (0 dpi); # statistically significant when compared with 21 dpi; △ statistically significant when compared with albendazole treatment group, ▲ statistically significant when compared with Albendazole combined with Dexamethasone treatment group; N = 3 coverslips per group, with 5fields analyzed per coverslip, P < 0.05

Fig. 4

Histological alteration of optic nerve in A. cantonensis-infected mice after different treatments. a-f: Electron microscopy images of optic nerve, scale bar = 1 μm. ↑ points to myelin sheath; ★ marks axon. a: normal group. The myelin sheathes and axons are intact. b: infected group by A. cantonensis (21 dpi). Demyelination (red ↑) and impaired axons (red ★) can be observed. c: Albendazole (AB) treatment for 7 days after 14 dpi with A. cantonensis. Demyelination become lessened, but still can be observed. d: Albendazole combined with Dexamethasone (AB + De) treatment for 7 days after 14 dpi with A. cantonensis. Demyelination become lessened, but still can be observed (red ↑ and ★). e: Albendazole combined with CPT treatment for 7 days after 14 dpi with A. cantonensis. The myelin sheathes regain intact structure (red ★), but swelling can be observed in axons (red ). f: Albendazole combined with TSII-A treatment for 7 days after 14 dpi with A. cantonensis. Demyelinated axons almost recover to intact structure (red ↑ and ★). Scale bar = 1 μm. g: Data are mean ± SEM of axon numbers in different treatment groups for 7 days after 14 dpi with A. cantonensis, respectively. *Statistically significant when compared with control (0 dpi); # statistically significant when compared with 21 dpi; △ statistically significant when compared with albendazole treatment group, ▲ statistically significant when compared with albendazole combined with Dexamethasone treatment group; ★ statistically significant when compared with albendazole combined with CPT treatment group. n = 3 per group, with 5 fields analysed per section. P < 0.05