Axonal transport following acute intraocular pressure elevation in mice

Abstract

 

Purpose

 

The aim of this study is to clarify the effects of an acute intraocular pressure (IOP) injury on axonal transport along retinal ganglion cells (RGCs) in mice. It has been established that IOP elevation of 50mmHg for 30 minutes causes reduced inner retinal function (measured by positive scotopic threshold response (pSTR) on electroretinogram), with near complete recovery of pSTR at 7 days following IOP elevation in 3 month-old mice. We aimed to measure axonal transport at a point where inner retinal function has recovered.

 

Methods

 

C57BL/6J mice (n=10, 5 male) at age 3 months were used and each mouse received identical treatment. At day 0, mice were anaesthetized and acute IOP elevation was administered to the right eye via anterior chamber (AC) cannulation (50mmHg for 30 minutes). The left eye received AC cannulation and was held at physiological IOP (12mmHg) for 3 minutes. At day 5, mice were anaesthetized and received bilateral 1µL intravitreal injections of 1% choleratoxin subunit B (CTB) conjugated to Alexafluor 488. At day 7, mice were perfused with paraformaldehyde, eyes and brains were harvested and stored in sucrose overnight. Retinal flatmounts were prepared from enucleated eyes. The superior colliculi (SC) were dissected from each brain and prepared on a slide. CTB uptake was imaged in the retina and SC using fluorescent microscopy and graded (masked) using an intensity score of 0-4. A delta score for each eye was calculated by comparing the difference in fluorescent intensity between each retina and its corresponding superior colliculus.

 

Results

 

Delta scores did not differ significantly between injured and uninjured eyes (p=0.23). The majority of delta scores were 0 or 1, indicating no difference or small difference, respectively, in fluorescent intensity between retina and SC.

 

Conclusions

 

These results suggest that there was no major disruption of axonal transport in RGCs following an acute sub-ischemic pressure injury where there is full recovery of the pSTR. Future work will determine whether axonal transport is also maintained in older animals where there is more persistent loss of pSTR.

 

 

Figure 1. Graph comparing mean±SD of delta scores between uninjured (left) and injured (right) eyes.

 

 

Figure 2. Example of CTB in a right retina (flatmount, above) and corresponding left SC (below). This retina was graded at 3 for fluorescence and the SC was graded at 4, producing a delta score of 1.

 

Keywords: 568 intraocular pressure • 531 ganglion cells • 413 aging