A Novel Topical Ophthalmic Formulation to Mitigate Acute Mustard Gas Keratopathy In Vivo: A Pilot Study

Abstract

Purpose: This pilot study investigated the in vivo therapeutic potential and tolerability of a multimodal ophthalmic formulation, topical eye drops (TED), for acute mustard gas keratopathy (MGK) using a rabbit model.

Methods: Twenty New Zealand White rabbits were used. Only right eyes of 18 rabbits (oculus dexter [OD]) received single sulfur mustard gas (SM) vapor injury, whereas contralateral eyes were left untreated or received TED for tolerabilty evaluation. Two rabbit eyes received no treatment and served as age-matched naive control. The four groups were: Naive (oculus sinister [OS] untreated eyes; n = 9); TED (OS treated only with TED BID for 3 days; n = 9); SM (OD exposed to SM vapor; n = 9); and SM+TED (OD exposed to SM+TED BID for 3 days; n = 9). Ocular examination in live rabbits were performed utilizing slit-lamp biomicroscopy, Fantes grading system, fluorescein staining, Schirmer's tests, pachymetry, and applanation tonometry. Cellular and molecular changes in rabbit corneas were assessed after humane euthanasia on day-3 and day-7 with histopathological and real-time polymerase chain reaction PCR techniques.

Results: TED to rabbit eyes was found tolerable in vivo. SM-exposed eyes showed significant increase in Fantes scores, central corneal thickness (CCT), Schirmer's test, epithelium-stroma separation, and corneal edema. TED mitigated clinical symptoms by reducing corneal edema, Fantes scores, CCT, and Schirmer's test. Further, TED decreased SM-induced corneal haze, inflammatory and profibrotic markers, transforming growth factor-TGF-β1 and cyclooxygenase-2COX-2, and damage to corneal structure, including epithelial-stromal integrity.

Conclusions: The developed multimodal eyedrop formulation, TED, has potential to mitigate acute MGK effectively in vivo.

Translational relevance: TED is effective against MGK.

Keywords: cornea; corneal edema; intraocular pressure; ocular inflammation; sulfur mustard.

Figure 1.

Representative fluorescein dye test images showing the extent/area of corneal epithelial erosion/defect in rabbit eyes. SM-vapor severely damaged corneal epithelium at day-3 (C, arrowheads) and day-7 (G, arrowhead), and topical TED effectively reduced SM toxicity on day-3 (D, arrows) and day-7 (H, arrow). TED dosing to SM-unexposed eye did not show any damage to the corneal epithelium at day-3 (B) or day-7 (F), and corneal health was akin to the na10ve eyes at day-3 (A) and day-7 (E). Scale bar: 2.0 mm.

Figure 2.

Representative slit-lamp narrow beam images exhibiting that SM-vapor caused inflammation in rabbit cornea at day-3 (C, arrowhead) and day-7 (G, arrowhead). TED decreased SM-induced corneal toxicity in vivo and reduced inflammation at day-3 (D, arrow) and day-7 (H, arrow). TED on SM-unexposed eye did not show any corneal inflammation at day-3 (B) or day-7 (F), and corneal health was similar to the naive corneas at day-3 (A) and day-7 (E). Scale bar: 2.0 mm.

Figure 3.

Representative stereo biomicroscopy digital images describing corneal opacity. SM-vapor caused severe corneal haze in rabbits in vivo at day-3 (C, arrowhead) and day-7 (G, arrowhead). TEDs diminished SM-induced corneal haze at day-3 (D, arrow) and day-7 (H, arrow). TED on SM-unexposed eye did not generate any corneal haze or inflammation at day-3 (B) or day-7 (F) and showed ocular health akin to the naive corneas at day-3 (A) and day-7 (E). Scale bar: 2.0 mm.

Figure 4.

Quantification of mean corneal haze using the Fantes score in naive, TED, SM, and SM+TED. The corneal haze score was significantly increased in rabbit eyes after SM exposure compared to the naive and decreased in the SM+TED as compared to the SM exposure at day-3, and day-7. The TED alone treatment to the cornea did not cause corneal haze and scored 0.00 at the Fantes scale. Results are expressed as Mean ± SEM (****p < 0.0001 **p = 0.0014, and *p = 0.0344, One-way ANOVA).

Figure 5.

Representative H&E stained images showing that SM-vapor caused severe corneal edema in rabbits in vivo at day-3 (C) and day-7 (G). TEDs attenuated SM-induced edema at day-3 (D) and day-7 (H). TED on SM-unexposed eye did not cause corneal edema or structural abnormalities at day-3 (B) or day-7 (F) and showed corneal morphology akin to the naive corneas at day-3 (A) and day-7 (E). e, epithelium, s, stroma, en, endothelium. Scale bar: 100 µM.

Figure 6.

Representative H&E stained images displaying that SM-vapor caused severe epithelium-stroma separation in rabbits in vivo at day-3 (C, arrowheads) and day-7 (G, arrowheads). TEDs diminished SM-induced epithelium-stroma separation at day-3 (D, arrows) and day-7 (H, arrows). TED on SM-unexposed eye did not generate any corneal epithelium-stroma separation at day-3 (B) or day-7 (F) and showed corneal composition analogous to the naive corneas at day-3 (A) and day-7 (E). Scale bar: 100 µM.

Figure 7.

Representative immunofluorescence images stained with anti-Integrin-β4 antibody indicating that SM-vapor caused severe corneal epithelium-stroma separation in rabbits in vivo at day-3 (C, arrowheads) and day-7 (G, arrowheads). TEDs mitigated epithelium-stroma separation at day-3 (D, arrows) and day-7 (H, arrows). TED on SM-unexposed eye did not alter corneal epithelium-stromal integrity at day-3 (B, arrows) or day-7 (F, arrows) and showed corneal composition analogous to the naive corneas at day-3 (A, arrows) and day-7 (E, arrows). Scale bar: 100 µM.

Figure 8.

Quantitative gene expression analysis. (A) qRT-PCR analysis results showing upregulation of the TGF-β1 expression in SM-exposed corneas compared with naive at day-3 and day-7. TED treatment to the SM-exposed corneas downregulates TGF-β1 expression compared with SM-exposed at day-7 postexposure but not at day-3 (***P = 0.0002, **P = 0.0027, ns = no significance, one-way ANOVA). No significant changes in the expression of profibrotic marker, TGF-β1 gene in TED vs. naive rabbits at day-3 and day-7 (P > 0.05). (B) qRT-PCR analysis showing significant upregulation of the COX-2 in SM-exposed corneas compared with naive. SM+TED corneas showed downregulation of COX-2 expression compared with SM on day-3 and day-7 postexposure. Results are expressed as mean ± SEM (****P < 0.0001, ***P = 0.0001, one-way ANOVA). No significant change in the expression of inflammatory marker COX-2 gene in TED versus naive rabbit corneas at day-3 and day-7 (P > 0.05).