Establishing a Mouse Model of Chlorpromazine-Induced Corneal Trigeminal Denervation

Abstract

Purpose: This study aimed to establish a mouse model of chlorpromazine-induced corneal trigeminal denervation (CCTD).

Methods: Retrobulbar chlorpromazine injections were administered to 6- to 8-week-old C57BL/6j mice to induce corneal denervation. Additionally, apoptosis was assessed in isolated primary trigeminal ganglion cells after culturing in a conditioned medium containing chlorpromazine. Finally, the success rate of model generation, mortality and complication rates, and model-preparation learning curves were compared between the CCTD model and the electrocoagulation and axotomy models.

Results: Chlorpromazine retrobulbar injections resulted in trigeminal denervation, leading to a reduced blink reflex, corneal nerve density, and corneal epithelium thickness. Furthermore, 90% (9/10) of the mice developed epithelial defects, accompanied by increased apoptosis and inhibited proliferation of corneal epithelial cells. In vitro, trigeminal ganglion cell apoptosis increased after culturing in a conditioned medium containing chlorpromazine. Moreover, the CCTD model exhibited a higher success rate, longer survival rate, and lower complication rate compared to the electrocoagulation and axotomy models. Crucially, the learning curve demonstrated that the method used to generate the CCTD model was easy to learn.

Conclusions: The CCTD model is a user-friendly mouse model for studying corneal trigeminal denervation that offers a less invasive alternative to existing models.

Translational relevance: The CCTD model serves as a valuable tool for investigating the functional mechanisms of corneal trigeminal nerves and their interactions with corneal cells.

Figure 1.

Retrobulbar chlorpromazine injections induce decreased blink reflexes and corneal epithelial defects. (A, B) Illustration of the chlorpromazine-induced corneal trigeminal denervation (CCTD) model. The needle was inserted vertically into the retrobulbar space at the outer one-third of the upper eyelid. (C) Fluorescein sodium staining revealed corneal defects following retrobulbar chlorpromazine injections. A dot-like corneal epithelial defect first appeared on day 1, progressed to a patch-like defect on days 2 and 3, and evolved into a corneal ulcer on days 5, 7, and 10.

Figure 2.

Corneal epithelial apoptosis, proliferative inhibition, and decreased corneal epithelial thickness after retrobulbar chlorpromazine injections. (A) Immunohistochemistry and (B) quantification of TUNEL-positive apoptotic cells in the corneas of both control and CCTD mice. Scale bar: 30 µm. (C) Ki-67 staining of the cornea and (D) quantitative analysis of the Ki-67–positive cells comparing the control and CCTD model groups. Scale bar: 100 µm. (E) H&E staining of the corneas and (F) comparison of corneal epithelial thickness between the control and CCTD model groups. Scale bar: 100 µm. Data are presented as mean ± SD. *P < 0.05.

Figure 3.

Reduction in corneal nerve density in the CCTD model. Whole-mount staining depicts corneal nerve density in the subbasal and the stromal plexus. Scale bar: 50 µm.

Figure 4.

Chlorpromazine increases apoptosis in primary TG cells in vitro. (A) Morphology of primary TG cells. Scale bar: 500 µm. (B) Immunofluorescence staining with DAPI (nuclear marker; blue) and III β-tubulin (neuronal marker; green) for primary TG cells. Scale bar: 300 µm. (C) TUNEL staining of primary TG cells treated with varying chlorpromazine concentrations. Scale bar: 300 µm. (D) Quantification of TUNEL-positive cells. Data are presented as mean ± SD. *P < 0.05.

Figure 5.

Comparative analysis of the CCTD, electrocoagulation, and axotomy models. (A–D) Flash visual evoked potential (f-VEP) tests of the control (A), CCTD (B), axotomy (C), and electrocoagulation (D) models for assessing the integrity of the visual pathway. P2–N2 waves reflect the functionality of the visual pathway. (E) Quantification of the P2–N2 wave amplitudes. (F–I) Flash electroretinography (f-ERG) tests of the control (F), CCTD (G), axotomy (H), and electrocoagulation (I) model groups for evaluating retinal function. The a-wave reflects photoreceptor cell function, and the b-wave reflects bipolar cell function. (J) Quantification of the a-wave and b-wave times. (K) Quantification of the a-wave and b-wave amplitudes. Data are presented as mean ± SD. *P < 0.05.

Figure 6.

The learning curves of the CCTD, electrocoagulation, and axotomy models. (A) The learning curves of the three different approaches. (B) Average time required and (C) the number of animals needed to complete the models. Data are presented as mean ± SD. *P < 0.05.