Subconjunctival Bevacizumab Injection Impairs Corneal Innervations and Epithelial Wound Healing in Mice

Abstract

Citation: Dong M, Di G, Zhang X, Zhou Q, Shi W. Subconjunctival bevacizumab injection impairs corneal innervations and epithelial wound healing in mice. Invest Ophthalmol Vis Sci. 2017;58:146958: -147758: . DOI: 10.1167 PURPOSE. To investigate the effects of subconjunctival bevacizumab injection on the corneal nerve, sensitivity, and epithelial wound healing in mice.METHODS. Adult C57BL/6 mice were treated with subconjunctival injection of 1, 2, 5, or 25 mg/mL bevacizumab. The corneal nerve was observed with whole-mount anti-b3-tubulin fluorescence staining. Corneal sensitivity was measured with a Cochet-Bonnet esthesiometer. The protein levels of pigment epithelium-derived factor (PEDF), nerve growth factor (NGF), glial-derived neurotrophic factor (GDNF), and ciliary neurotrophic factor (CNTF) were measured by ELISA. The corneal epithelial wound-healing rate was evaluated by fluorescein staining. The recovery of impaired mouse corneal innervations and epithelial wound-healing rate following bevacizumab injection was evaluated with the co-injection of PEDF, NGF, or CNTF.RESULTS. Subconjunctival bevacizumab injection caused apparent corneal nerve degeneration, attenuated corneal sensitivity, and delayed corneal epithelial wound healing and nerve regeneration in normal mice, which was more significant with increased concentration and times of the bevacizumab injection. However, the corneal nerve and sensitivity gradually improved and recovered in mice with a single injection of 1 to 5 mg/mL bevacizumab. Moreover, the bevacizumab injection significantly decreased the corneal PEDF, NGF, and CNTF content, whereas exogenous PEDF, NGF, or CNTF supplement attenuated impairment of the corneal nerve, sensitivity, and epithelial wound healing after subconjunctival bevacizumab injection.CONCLUSIONS. Subconjunctival bevacizumab injection impairs corneal innervations, epithelial wound healing, and nerve regeneration in normal mice, which may be caused by the reduction of neurotrophic factor content in the cornea.

DNase I improves corneal epithelial and nerve regeneration in diabetic mice

“…Mice were anaesthetized by an intraperitoneal injection of 0.5 mL 0.9% sodium chloride solution containing 40 μL ketamine and 10 μL chlorpromazine hydrochloride, followed by topical application of proparacaine hydrochloride eye drops (Alcon) in the eye; then, an Algerbrush II remover (Alger Co) was used to scrape the central corneal epithelium (2.75 mm diameter) in mice. Fluorescein staining method was used to examine the residual epithelial defects, and the percentage of the original defect area was calculated with ImageJ software (NIH) as described in our previous studies 29‐31 . For DNase I treatment, diabetic mice were topically applied with 1 mg/mL DNase I (5 μL/eye; Roche Diagnostics GmbH) six times a day after the removal of corneal epithelium, whereas age‐matched control mice were topically applied with equal phosphate buffer saline (PBS) as the vehicle control.…”

“…Corneal mechanical sensitivity was measured by using a Cochet‐Bonnet esthesiometer (Luneau Ophtalmologie) without anaesthesia according to our previous studies 28,31 . The test began with the maximal length (6 cm) of nylon filament in the esthesiometer and shortened by 0.5 cm each time until we found the corneal touch threshold (which triggered the eye blink response).…”

PEDF Reduces the Severity of Herpetic Simplex Keratitis in Mice

“…23,24 For the treatment, mice were anesthetized and subconjunctivally injected with PEDF (R&D, San Diego, CA, USA; 16 ng/lL, 5 lL/eye), Mer 44 (Val78-Thr121), Mer34 (Asp44-Asn77; GL Biochem, Shanghai, China; 16 ng/lL, 5 lL/eye), or PBS (5 lL/eye) as vehicle control at 0 and 3 days postinfection (dpi) after HSV-1 infection, according to our preliminary experiments and previous reports. 25,26 All the dosages used in this study were evaluated and optimized in our preliminary experiments.…”

Bevacizumab and corneal pathology

“…Šest od 10 očiju razvilo je epiteliopatiju u drugom mjesecu liječenja, što upućuje na poremećaj adhezije između epitela i bazalne membrane rožnice ili poremećaja u procesu cijeljenja epitela rožnice 29 . Dong i suradnici aplicirali su subkonjunktivalne injekcije bevacizumaba u koncentracijama 1,2,5 i 25 mg/ml, te zapazili da degeneraciju živaca rožnice, smanjenu rožničnu osjetljivost i produljeno zacjeljivanje rožnice kod viših koncentracija bevacizumaba i nakon ponavljanih injekcija, a promjene su bile reverzibilne kod jedne subkonjunktivalne injekcije 1 -5 mg/ml bevacizumaba 30 . Topička primjena bevacizumaba problematična je kod liječenja NV-a rožnica s ozljedama ili ulkusima.…”