Systematic assessment of microneedle injection into the mouse cornea

Mario Matthaei,Imran Bhutto,Huan Meng,Edwin Boelke,Justin Hanes,Qingguo Xu,Albert S Jun

doi:10.1186/2047-783x-17-19

Abstract

BackgroundCorneal intrastromal injection is an important mode of gene-vector application to subepithelial layers. In a mouse model, this procedure is substantially complicated by the reduced corneal dimensions. Furthermore, it may be difficult to estimate the corneal area reached by the volume of a single injection. This study aimed to investigate intrastromal injections into the mouse cornea using different microneedles and to quantify the effect of injecting varying volumes. A reproducible injection technique is described.MethodsForty eyes of 20 129 Sv/J mice were tested. India ink was intrastromally injected using 30° beveled 33 G needles, tri-surface 25° beveled 35 G needles, or hand-pulled and 25° beveled glass needles. Each eye received a single injection of a volume of 1 or 2 μL. Corneoscleral buttons were fixed and flat mounted for computer-assisted quantification of the affected corneal area. Histological assessment was performed to investigate the intrastromal location of the injected dye.ResultsA mean corneal area of 5.0 ±1.4 mm2 (mean ± SD) and 7.7 ±1.4 mm2 was covered by intrastromal injections of 1 and 2 μL, respectively. The mean percentage of total corneal area reached ranged from 39% to 53% for 1 μL injections, and from 65% to 81% for 2 μL injections. Injections using the 33 G needles tended to provide the highest distribution area. Perforation rates were 8% for 30° beveled 33 G needles and 44% for tri-surface beveled 35 G needles. No perforation was observed with glass needle; however, intrastromal breakage of needle tips was noted in 25% of these cases.ConclusionsIntracorneal injection using a 30° beveled 33 G needle was safe and effective. The use of tri-surface beveled 35 G needles substantially increased the number of corneal perforations. Glass needles may break inside the corneal stroma. Injections of 1 μL and 2 μL resulted in an overall mean of 49% and 73% respectively of total corneal area involved.

Highlights

Corneal intrastromal injection is an important mode of gene-vector application to subepithelial layers
Lacrimation, mucus, blinking of the eyelid, and tight junctions of the corneal epithelium are a significant obstacle to genetic vectors [1,2]
Some studies describing the technique of intrastromal injections in mice have been conducted [5-17]

Summary

Introduction

Corneal intrastromal injection is an important mode of gene-vector application to subepithelial layers. This study aimed to investigate intrastromal injections into the mouse cornea using different microneedles and to quantify the effect of injecting varying volumes. Intrastromal or intracameral injections are generally used for delivery of genetic therapeutics to subepithelial corneal layers [1]. Corneal thickness substantially varies among different animal species [3]. Some studies describing the technique of intrastromal injections in mice have been conducted [5-17]. According to the method first described by Epstein and Stulting [9], most of these studies used a 33 gauge (G) needle with a bevel of 30° or 45° and the injected volume generally varied up to 2 μL [5,6,7,9-13]. It is important to consider that this variation may cause differences in the corneal area reached by the injected fluid or therapeutic agent

Objectives

Methods

Results

Discussion

Conclusion