Estudio de la respuesta inflamatoria en el sistema nervioso central tras el aplastamiento completo del nervio óptico en ratón

Resumen

Objectives- Unilateral Optic Nerve Crush (ONC) triggers a bilateral inflammatory reaction which extends to both injured and contralateral fellow retinas. However, few studies have investigated the spread of inflammation along visual pathway or in remote regions of Central Nervous System (RRCNS). Our aim was to study the glial response and the underlying molecular changes in retinas, superior colliculi (SCi), the main projection areas of retinal ganglion cells (RGC) in mice, or RRCNS at different distances from the lesion. Material and Methods- The left optic nerves of adult pigmented C57Bl/6 male mice were crushed (ONC) at 0.5 mm from the optic disk or underwent surgery without crushing (Sham). Intact mice were used as controls. Two studies were carried out: Molecular study: At 1,3,9 or 30 days after ONC or Sham-surgery (dpl) both retinas and SCi or RRCNS (hippocampus -H-, olfactory bulb -OB-, cerebellum -CE- and spinal cord -SC-) were freshly dissected and their RNA extracted. Using qPCR we measured the expression levels of Tgf-β1, Il-1β, Tnf-α, Iba1, Gfap, Mhc II and Tspo in retinas and in the rest of CNS regions under study. In SCi, H, OB, CE and SC extracts we also analysed Caspase-3, Cxcr1, Lcn2, Il-6, Cd206, Il-4 and Aq4. We classified genes in apoptosis (Caspase-3), pro-inflammatory (Cxcr1, Il-1, Il-1β, Lcn2, Tnf-α, Il-6), anti-inflammatory (Cd206, Il-4, Tgf-β1) and gliosis markers (Aq4, Gfap, Iba1, Mhc II, Tspo). Hptr and Gapdh were used as housekeeping genes. Anatomopathological study: Immunodetection of microglia (CMi) and macroglial (CMa) cells was performed in sagittal brain sections at 3,9 or 30 dpl. The area occupied by GFAP and MHC II signals, as well as the density of Iba1+ and CD68+ cells were quantified in both SCi and selected RRCNS (H and OB). We analysed the response in animals with ONC compared with intact or Sham animal, ipsi- vs. contralateral regions to lesion and at different survival intervals. Results- Sham surgery was sufficient to modify the expression of inflammatory and glial markers in retina and other regions of CNS that were studied. The retina of the exposed optic nerve without crushing showed an earlier and more pronounced pro-inflammatory reaction compared to the contralateral retina. In the rest of the CNS, there were a generalized increase of Iba1+ cell density or Iba1 upregulation, but no histological glial changes; Gfap overexpression with an increase of area occupied by GFAP in both SCi; and overexpression of pro-inflammatory markers and Caspase-3. Additionally, signs of glial reactivation (Tspo or Mhc II) were only present in the ipsilateral RRCNS, and the response in SC was delayed and less pronounced. ONC did not significantly modify the apoptosis, glial or inflammatory reaction that was already observed due to Sham surgery in RRCNS, which refers to areas outside the visual pathway (retina and SCi). The inflammatory and glial response was evident from the first day after ONC in both retinas, although it lasted longer in the injured retina. The macroglial reaction was exclusive to the injured retina, while the microglial response was found in both. ONC induced a stronger response in the contralateral SCi, including microglial reactivation and a pro-inflammatory response that led to apoptosis. Conclusions- After ONC, glial and inflammatory response in visual pathway occurs in relation to the connection between axotomized RGC and both retinas or retino-recipient SCis. Outside the visual pathway, the reaction is more widespread and may follow a gradient depending on the anatomical distance to the lesion. In the experimental analysis of any CNS tissue, where isolating the response to ONC or any other lesion from the effect of the surgery itself is of interest, a Sham control should be used.