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

Abstract

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-β3-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.

Vascular endothelial growth factor (VEGF) is well-known for being involved in physiological and pathologic angiogenesis as a vascular permeability factor and endothelial cell mitogen.^1,2 However, VEGF also possesses neurotrophic and neuroprotective capacities in both the central and peripheral nervous systems, and it appears directly on neuronal cells, independent of its vascular actions.³ Previous reports have demonstrated that VEGF stimulates proliferation, protects against hypoxia-induced death, and promotes axonal outgrowth of the central and peripheral neurons,^4–7 including in the cornea.^8–10 The neurotrophic and neuroprotective effects of VEGF depend on the expression of VEGF receptor (VEGFR)-1, VEGFR-2, and neuropilins in neuronal cells.^4,5,9,11 The dual roles of VEGF in the vascular and nervous systems led to consideration of the potential neuronal risk of anti-VEGF therapies, which are being widely used to treat ocular diseases.^12–15 

The cornea is the most densely innervated tissue in the human body and is dominantly supplied by the sensory nerve fibers derived from the neurons located in the trigeminal ganglion. In the subbasal layer, the corneal nerve fibers form a distinctive pinwheel pattern plexus and project toward the apical surface of the corneal epithelium.^16,17 The intact corneal innervations play an important role in the regulation of blink reflex, epithelial homeostasis, and tear production and secretion.¹⁰ However, the attenuation of corneal innervations, caused by herpetic viral infection, trigeminal nerve damage, or diabetic mellitus, can cause impaired corneal sensation, chronic inflammation, delayed corneal epithelial wound healing, and even persistent defects.^18–23 

Corneal neovascularization (CNV) is a major sight-threatening complication of various ocular injuries. In recent years, the topical application or subconjunctival injection of bevacizumab has been confirmed for CNV treatment in both animals and humans.^14,24–27 Compared with topical application, subconjunctival injection is assumed to enable easier penetration of bevacizumab into the corneal stroma in the presence of an intact epithelium, suggesting that subconjunctival injection is a more reliable administration route for CNV treatment.^28,29 Previous reports have confirmed the safety of topical application of bevacizumab, including for corneal epithelial wound healing and subbasal nerve fiber density.^30,31 However, the potential effect of subconjunctival injection on corneal innervations, epithelial wound healing, and nerve regeneration remains unclear. In the present study, we examined the effects of subconjunctival bevacizumab injection on the corneal nerve, sensitivity, neurotrophic factor expression, and epithelial wound healing in mice. 

Methods and Materials

Subconjunctival Bevacizumab Injection in Mice

Adult C57BL/6 mice (6–8 weeks old, Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences [CAMS] and Peking Union Medical College [PUMC], Beijing, China) were maintained in the animal facility of the Shandong Eye Institute. All experiments were carried out in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The mice were anesthetized and injected subconjunctivally (at the 2–3 o'clock position) with 1, 2, 5, or 25 mg/mL bevacizumab (Avastin; Roche Pharma, Basel, Switzerland; 5 μL per eye) one, two, or four times (injection every 2 days). For the recovery experiment, mice were injected simultaneously with bevacizumab (5 mg/mL) and recombinant mouse pigment epithelium-derived factor (PEDF; R&D, San Diego, CA, USA; 15 ng/μL, 5 μL per eye), nerve growth factor (NGF; R&D; 20 ng/μL, 5 μL per eye) or ciliary neurotrophic factor (CNTF; R&D; 10 ng/μL, 5 μL per eye), in accordance with previous reports^32–34 and our preliminary experiments. Normal saline (0.9% NaCl) was injected as vehicle control. For all experiments, only one eye of each mouse was used for injection. 

Corneal Sensitivity Measurement

Corneal sensitivity was measured using a Cochet-Bonnet esthesiometer (Luneau Ophtalmologie, Chartres Cedex, France) in unanesthetized mice at 2, 7, and 14 days after the final injection. The testing began with the maximal length (6 cm) of nylon filament and was shortened by 0.5 cm each time until the corneal touch threshold was found. The longest filament length resulting in a positive response was considered as the corneal sensitivity threshold, which was verified four times (n = 10 per group). 

Corneal Epithelial Wound Healing

The corneal epithelium (2.5-mm diameter) was scraped with Algerbrush II corneal rust ring remover (Alger Co, Lago Vista, TX, USA) immediately after the subconjunctival bevacizumab injection (1, 2, 5, 25 mg/mL). Corneal epithelium defects were visualized at 24, 30, 36, and 48 hours by instilling 0.25% fluorescein sodium and photographing them under a BQ900 slit lamp (Haag-Streit, Bern, Switzerland). The staining area was analyzed using ImageJ software (http://imagej.nih.gov/ij/; provided in the public domain by the National Institutes of Health, Bethesda, MD, USA) and calculated as the percentage of residual epithelial defects (n = 10 per group). 

Whole-Mount Corneal Staining

Full-thickness corneal flat mounts were fixed in acetone for immunofluorescence staining. To block nonspecific staining, the samples were incubated with normal serum for 20 minutes at room temperature and incubated with rhodamine-conjugated anti-β3-tubulin (Millipore, Billerica, MA, USA) overnight at 4°C. Subsequently, they were washed three times with PBS for 5 minutes. Finally, the flat mounts were examined and captured under an epifluorescence microscope (E800; Nikon, Tokyo, Japan). The defective area of corneal nerve fibers was analyzed using ImageJ software and calculated as the percentage of the full photo (n = 10 per group). For the measurement of corneal nerve regeneration, the subbasal nerve fiber density was calculated as the percentage of area positive for β-tubulin III staining immediately, 2 and 7 days after the central corneal epithelial removal (2.5 mm diameter, n = 5 per group). 

Enzyme-Linked Immunosorbent Assay

Corneas were collected from the control and treated mice 2 days after the final bevacizumab injection. The samples were homogenized and centrifuged (10,000g for 30 minutes at 4°C). The supernatants were used for ELISA analysis according to the manufacturer's instructions for PEDF (USCN, Wuhan, China), CNTF (USCN), GDNF (USCN), and NGF (Millipore). The neurotrophic factor concentrations (n = 18 per group) were calculated for total corneal proteins, which were measured using a BCA kit (Beyotime, Shanghai, China). 

Statistical Analysis

Data in this study are representative of at least three independent experiments and are presented as means ± SD. Statistical analysis was performed using SPSS 17.0 software (SPSS, Chicago, IL, USA) and 1-way ANOVA. Differences were considered statistically significant at P < 0.05. 

Results

Bevacizumab Injection Causes Corneal Nerve Fiber Degeneration

A previous study reported that topical bevacizumab application had no effect on corneal nerve fiber density in mice.³⁰ To explore the effect of subconjunctival bevacizumab injection on the corneal innervations, mice received 1, 2, 5, or 25 mg/mL bevacizumab or saline solution injection one or two times, and corneal nerve fibers were stained with neurofilament β-tubulin antibody. Compared with the intact vortex pattern of corneal subbasal nerve fibers in normal mice that had the vehicle injection (Fig. 1A), significant nerve degeneration was detected at 2 days after bevacizumab injection, which was more severe with increased injection concentration and times (Figs. 1B, 1C). Moreover, the mice with a single bevacizumab injection showed apparent regeneration of corneal nerve fibers at 7 days and the vortex pattern was partially recovered at 14 days, even at 25 mg/mL concentration (Fig. 1B). However, although the mice with double injection showed the same regeneration of corneal nerve fibers at 7 days, there were still persistent defects and no typical vortex pattern reformation at 5 and 25 mg/mL concentrations, even after 14 days (Fig. 1C). 

Figure 1

 

Corneal nerve fiber degeneration after bevacizumab injection. (A) The intact vortex pattern of corneal nerve fibers in mice with vehicle injection. (B) Representative morphologic changes and defect measurement of corneal nerve fibers after single bevacizumab injection. (C) Representative morphologic changes and defect measurement of corneal nerve fibers after double bevacizumab injections.

Bevacizumab Injection Impairs Corneal Sensitivity

To investigate the possible influence of unilateral bevacizumab injection on the corneal sensation of both eyes, bilateral corneal sensitivity in mice was measured at 2, 7, and 14 days after the final injection. The results showed that either single or double vehicle (normal saline) injection did not cause any reduction of corneal sensitivity (5.98 ± 0.02 cm versus 5.96 ± 0.04 cm before injection). However, a single bevacizumab injection caused the injected eye with transient impaired corneal sensitivity at 2 days to partially recover at 7 days, and finally return to normal levels after 14 days (except for the 25 mg/mL; Fig. 2A). However, the corneal sensitivity of the contralateral eye without injection showed no significant impairment, even with the 25 mg/mL single injection (Fig. 2B). Compared with the single injection, double bevacizumab injections caused more severe impairment in both the injected and contralateral eyes, which remained at lower than normal levels at 5 and 25 mg/mL (injected eye) or 25 mg/mL (contralateral eye) after 14 days (Figs. 2C, 2D). In addition, we also found that impairment of bilateral corneal sensitivity in mice after four bevacizumab injections showed no significant recovery even after 14 days (data not shown). The results suggest that a single bevacizumab injection causes only transient corneal sensitivity impairment in the injected eye, whereas multiple bevacizumab injections significantly delay the recovery of bilateral corneal sensitivity, which was consistent with the severity of corneal nerve fiber degeneration. 

Figure 2

 

Corneal sensitivity impairment after bevacizumab injection. Corneal sensitivity of the injected eye (A) and contralateral eye (B) after single bevacizumab injection. Corneal sensitivity of the injected eye (C) and contralateral eye (D) after double bevacizumab injections (*P < 0.05, compared with the corneal sensitivity of normal mice).

Bevacizumab Injection Impairs Corneal Epithelial Wound Healing

To investigate the effect of subconjunctival-injected bevacizumab on corneal epithelial wound healing, mouse central corneal epithelium was removed after being injected with different concentrations of bevacizumab or normal saline. The corneal epithelial healing rate exhibited an apparent difference ranging from 24 to 48 hours between the control and bevacizumab-injected mice. At 48 hours, the corneal epithelium remained defective in mice injected with 2 to 25 mg/mL bevacizumab, whereas mice injected with normal saline or 1 mg/mL bevacizumab showed complete re-epithelialization (Fig. 3A). According to analysis of the corneal epithelial defect area, the percentage of corneal epithelial defect increased from 7.90% ± 1.09% of vehicle control mice to 26.58% ± 8.79% of 1 mg/mL bevacizumab-injected mice, 47.56% ± 7.14% of 2 mg/mL bevacizumab-injected mice, 52.58% ± 2.59% of 5 mg/mL bevacizumab-injected mice, and 69.89% ± 7.88% of 25 mg/mL bevacizumab-injected mice at 36 hours after the epithelial removal (Fig. 3B). 

Figure 3

 

Corneal epithelial wound healing after bevacizumab injection. (A) Corneal fluorescence staining of the mice after bevacizumab injection. (B) Quantitative analysis of the corneal epithelial defect area (*P < 0.05, compared with the control group with normal saline injection).

Bevacizumab Injection Impairs Corneal Nerve Regeneration

To evaluate the effect of subconjunctival-injected bevacizumab on corneal nerve fiber regeneration, mouse central corneal epithelium was removed after the injection with 5 mg/mL of bevacizumab or normal saline. As shown in Figure 4A, the corneal epithelial removal caused the debridement of subbasal nerve fibers, whereas the corneal stromal nerve fibers remained undamaged. At 2 and 7 days after the epithelial removal, the corneal subbasal nerve fibers in control mice showed apparent regeneration, whereas mouse corneas with bevacizumab injection exhibited persistent defects in the sub-basal nerve fibers. According to the density analysis of corneal subbasal nerve fibers, there was a significant difference at 2 and 7 days between the control and bevacizumab-injected mice (Fig. 4B). 

Figure 4

 

Corneal nerve regeneration after bevacizumab injection. (A) Representative corneal nerve staining of the mice after normal saline or bevacizumab injection. (B) Quantitative analysis of corneal nerve fiber density (*P < 0.05, compared with the control group with normal saline injection).

Bevacizumab Injection Reduces Neurotrophic Factor Levels in the Cornea

Although the maintenance mechanism of corneal innervations remains unclear, multiple neurotrophic factors have been found to promote the regeneration of corneal nerve fibers in mice. Therefore, we examined the corneal protein levels of several neurotrophic factors, including PEDF, NGF, GDNF, and CNTF, in the mice 2 days after the single injection of bevacizumab (1, 2, 5, 25 mg/mL) or normal saline. The ELISA results showed that subconjunctival bevacizumab injection significantly decreased corneal CNTF content (with 1–25 mg/mL bevacizumab injection), PEDF and NGF content (with 5 and 25 mg/mL bevacizumab injection), and GDNF content (with 25 mg/mL bevacizumab injection) in the cornea (Fig. 5). In addition, we found that the reduced corneal content of PEDF, NGF, and CNTF exhibited no significant recovery until 3 days after the injection of 5 mg/mL bevacizumab (data not shown). 

Figure 5

 

Corneal neurotrophic factor content after bevacizumab injection. Corneal contents of four neurotrophic factors were measured with ELISA in the mice 2 days after the injection with bevacizumab (*P < 0.05, compared with the control group with normal saline injection).

Pigment Epithelium-Derived Factor, NGF, or CNTF Attenuates Corneal Impairment After Bevacizumab Injection

To explore the importance of reduced corneal neurotrophic factors after subconjunctival bevacizumab injection, the mice received simultaneous injection of bevacizumab (5 mg/mL) and PEDF, NGF, or CNTF, with normal saline as the vehicle control. The results showed that all three neurotrophic factors significantly attenuated the degeneration of corneal nerve fibers (Figs. 6A, 6B) and the impairment of corneal sensitivity (Fig. 6C) caused by bevacizumab injection. Moreover, according to analysis of the corneal epithelial defect area, the percentage of corneal epithelial defects decreased from 37.2% ± 0.14% of bevacizumab-injected mice to 11.4% ± 7.95% of bevacizumab- and PEDF-injected mice, 9.56% ± 7.63% of bevacizumab and NGF-injected mice, and 7.27% ± 1.41% of bevacizumab- and CNTF-injected mice at 36 hours after the epithelial removal, which reached a level of epithelial healing rate (10.57% ± 3.39%) equal to the normal control mice (Fig. 6D). 

Figure 6

 

Effects of exogenous neurotrophic factors on corneal impairment after bevacizumab injection. Representative morphologic changes (A) and defect measurement (B) of corneal nerve fibers. (C) Corneal sensitivity at 2 days after 5 mg/mL bevacizumab combined with PEDF, NGF, or CNTF injection (*P < 0.05, compared with bevacizumab and normal saline injection group). (D) Corneal fluorescence staining and quantitative analysis of corneal epithelial defect area (*P < 0.05, compared with the bevacizumab and normal saline injection group).

Discussion

Bevacizumab is a recombinant humanized monoclonal IgG antibody that inhibits abnormal blood vessel formation and decreases vascular permeability through being directed against VEGF-A. In the cornea, multiple studies have reported the successful application of both topical and subconjunctival bevacizumab for the inhibition of corneal neovascularization. The corneal safety profile of topical bevacizumab application has been investigated in mice.³⁰ In the present study, we explored the effects of subconjunctival bevacizumab injection on the corneal nerve, sensitivity, epithelial wound healing, and nerve regeneration in normal mice. The results showed that bevacizumab injection significantly impaired mouse corneal nerve fiber distribution, corneal sensitivity, and epithelial and nerve regeneration. Therefore, the corneal safety of subconjunctival bevacizumab injection differs from topical application in mice. 

Topical application of drug is the preferred method to treat various corneal diseases, as it is feasible and has few systemic side effects; however, the effective drug concentration depends on its capacity to penetrate through ocular barriers. For the duration of bevacizumab use, topical application was carried out only on the superficial layer of the intact epithelium, whereas the subconjunctivally injected bevacizumab penetrated well into the cornea stroma with an intact epithelium.²⁹ A previous study confirmed that the topical neutralization of VEGF-A showed no significant side effects on normal corneal epithelial wound healing and nerve fiber density.³⁰ In the present study, we found that subconjunctival bevacizumab injection caused significant impairment of corneal nerve fiber density, corneal sensitivity, and epithelial and nerve regeneration in normal mice. Moreover, impairments following subconjunctival injection worsened with increased concentration and times of bevacizumab. The results suggest that a higher concentration of bevacizumab in subconjunctival injection than used in topical application may cause the significant impairments of corneal innervations and epithelial wound healing in mice. It is worth noting that we also found unilateral, multiple bevacizumab injections caused attenuation of bilateral corneal sensitivity, but contralateral corneal sensitivity was higher than in the injected cornea. Although the molecular basis of corneal sensitivity remains unclear, previous reports have described the bilateral responses in the patients with unilateral diseases or peripheral unilateral nerve lesions, including the infectious keratitis, which may be related to the sympathetic reaction caused by the central nervous system.^19,35–37 Considering that intravitreal-injected bevacizumab could be detected in the cornea,³⁸ multiple intravitreal injections of bevacizumab may cause impairment of corneal innervations and epithelial functions, which needs to be evaluated in the future. 

Neurotrophic factors are regulatory molecules that play important roles in the growth and survival of developing neurons and the maintenance of mature neurons in both the central and peripheral nervous systems.^3,5,9,39,40 In the cornea, previous studies have found the patterned expression of neurotrophic factors and their receptors in normal and injured conditions.^41–44 Although their exact roles in the maintenance of proper corneal innervations remain unclear, the decreased content of NGF, PEDF, and CNTF in the cornea may be one of the important factors in the degeneration of corneal nerve fibers following subconjunctival bevacizumab injection. In addition, previous research has reported that soluble VEGFR1 in the cornea sequesters the potent pro-angiogenic molecule VEGF-A, which is also contained in the avascular corneal tissue.⁴⁵ Peripheral nerve axon expresses membrane-bound VEGFR1, VEGFR2, and the neuropilin receptors.^8,46 Vascular epithelial growth factor can promote the recovery of injured corneal nerve fibers in anatomical, sensory, and trophic functions, which requires the activation of various VEGF receptors.⁹ Here, we also confirmed that bevacizumab injection impaired the regeneration of corneal nerve fibers in normal mice. Therefore, the degeneration of corneal nerve fibers following bevacizumab injection may be also caused by the impaired availability of corneal VEGF-A bounded previously by the VEGFRs of corneal nerve fibers, which needs further study. 

In summary, our study found that subconjunctival bevacizumab injection impaired corneal innervations, epithelial wound healing, and nerve regeneration in mice. This may be caused by the reduction of neurotrophic factor contents in the cornea, and it should be followed up in future research on clinical treatment for corneal diseases. 

Acknowledgments

Supported in part by the National Natural Science Foundation of China (81530027, 81470610, 81470611), Shandong Provincial Nature Science Fund for Distinguished Young Scholars (JQ201518), Taishan Scholar Program (20150215, 20161059) (WS, QZ), and the Innovation Project of Shandong Academy of Medical Sciences (WS, QZ). 

Disclosure: M. Dong, None; G. Di, None; X. Zhang, None; Q. Zhou, None; W. Shi, None