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Open Access Open Badges Research

Systematic assessment of microneedle injection into the mouse cornea

Mario Matthaei12, Huan Meng1, Imran Bhutto1, Qingguo Xu13, Edwin Boelke4, Justin Hanes13 and Albert S Jun1*

Author Affiliations

1 The Wilmer Eye Institute, Johns Hopkins Medical Institutions, 400 N Broadway, Baltimore, MD 21231, USA

2 Department of Ophthalmology, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, Hamburg 20246, Germany

3 The Center for Nanomedicine, Johns Hopkins University, 400 N Broadway, Baltimore, MD 21231, USA

4 Department of Radiology and Radiooncology, University Medical Center Duesseldorf, Moorenstrasse 5, Duesseldorf 40225, Germany

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European Journal of Medical Research 2012, 17:19  doi:10.1186/2047-783X-17-19

Published: 20 June 2012



Corneal 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.


Forty 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.


A 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.


Intracorneal 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.

Cornea: gene therapy; Intrastromal injection