Development of a new surgical technique to infuse kynurenic acid to optic nerves in chickens for studying loss of myelination

Abstract

Prolonged infusion of a high dose of kynurenic acid (KYNA) reduces the myelin content in the rat spinal cord with preservation of the axonal integrity and without inducing an inflammatory response. We hypothesized that subdural infusion of a high concentration of KYNA can induce myelin loss in the optic nerves (ONs) of chickens. However, existing methods to deliver agents to the ON are inefficient, unlocalized and provide only acute exposure. Thus, we developed a surgical approach for sustained delivery of KYNA to the chicken ON. In brief, the novel surgical technique, which does not include excision of the extraocular muscles, involves incision of the skin and underlying fascial sheath to access the optic nerve within the muscle cone, implantation of a catheter in the dura of the optic nerve, the other end of which exits the orbit under the skin. The catheter runs under the skin near the lateral canthus, over the ears to the back of the neck, where a second incision is made to both implant the osmotic pump and to attach the catheter to the osmotic pump. India ink was used to confirm prolonged sustained administration to the optic nerves and across the chiasm. This surgical model was used to investigate KYNA's effect(s) on myelin loss in the ON. ONs of 7-day old chickens were infused with 50 mM KYNA or phosphate buffered saline (PBS) for seven days. Analysis of KYNA-infused contralateral ON g-ratios and protein levels indicated a reduction in myelin. These findings demonstrate the utility of our surgical approach for sustained delivery of KYNA into the ON and suggest a role for KYNA in modulating CNS myelination.

Keywords: CNS, central nervous system; Chicken; GFAP, glial fibrillary acidic protein; Infusion; KYNA, kynurenic acid; Kynurenic acid; MAG, myelin associated glycoprotein; MBP, myelin basic protein; Myelin; NC, negative control; ON, optic nerve; Optic nerve; PBS, phosphate buffered saline; PLP, proteolipid protein; Subdural.

Graphical abstract

Fig. 1

Surgical Implantation and Validation. A) Schematic and images of the surgical implantation procedure. Optic nerves were exposed by carefully spreading apart the extra ocular muscles (top left image). The catheter was then inserted under the dural sheath and fixed to the tissue with a drop of tissue glue (bottom right image). B) A schematic of the surgical protocol. Chick optic nerves were implanted at 7-days old with a pump that infused nerves for 7 days before harvesting tissue at 14 days old. Dissected negative control (C), PBS-infused (D), and India Ink-infused (E) optic pathways and brain matter. India Ink-infused nerves exhibit a significant colour change (arrows) due to the infusion of black dye under the dural sheath across the chiasm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

Subdural infusion of KYNA results in myelin loss on contralateral optic nerves. Toluidine blue (A, C, E; high magnification: A′, C′, E′) and EM (B, D, F) images for KYNA-infused (A–B), PBS-infused (C–D) and negative control (E–F) contralateral nerves. Examples of unmyelinated (*) and myelinated (arrows) regions (A′, C′ and E′) and axons (B, D and F). Large areas of unmyelinated axons of varying sizes (*) were observed in KYNA-infused contralateral nerves (A′, B). Scale bars: A, C, E, 500 μm, A′, C′, E′, 50 μm, B, D, F, 2 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

G-ratio and axon diameter distributions with Lowess spline fits. G-ratio (A, C, E) and axon diameter (B, D, F) distributions for KYNA-infused (A, B), PBS-infused (C, D) and negative control (E, F) contralateral ONs, respectively. Note the rightward shift in the distribution of g-ratios for KYNA-infused contralateral ONs.

Fig. 4

Subdural infusion of KYNA results in loss of myelin protein. A) Representative western blots of the contralateral ONs for KYNA-infused, PBS-infused and negative control (NC) birds (n = 4 each group). B–F) Band intensities as a percent relative to β-actin levels, for each of the treatment groups. Data are presented as mean ± SE. * denotes p < 0.05, ** denotes p < 0.01. One-Way ANOVA with Tukey's post-hoc test. Non-contrast adjusted and uncropped versions of western blots are available in Supplemental Fig. 3.