Abstract
Purpose:
The complement system, as a part of the innate immune system, plays a crucial role in many neuroinflammatory diseases, such as multiple sclerosis or Alzheimer’s disease. In this study, we wanted to investigate the participation of the complement system (CS) in the loss of retinal ganglion cells (RGC) in an immune mediated glaucoma model.
Methods:
Rats were immunized with optic nerve homogenate (ONA) or S100. The control group (CO) received sodium chloride. After 14 and 28 days RGC density was quantified with Brn3a-antibody (Santa Cruz) stained flatmounts (n=6). To evaluate the activation of the CS, cross-sections of the retina were stained with C3 (Cedarlane) and membrane attack complex (MAC; Biozol) after 14 or 28 days (n=5 per group). Cell counts of RGC and the complement components were performed using Image J Software. Statistical analysis was performed using [Student] t-test.
Results:
No change in the density of the RGCs could be observed in the immunized animals compared to CO after 14 days (ONA: p=0.9; S100: p=0.8), but after 28 days there was a significant RGC loss (ONA: p=0.0005; S100: p=0.005). In the ONA group, MAC was significantly increased in the ganglion cell layer (GCL) after 14 and 28 days (14 days: CO: 0.5±0.9; ONA: 0.8±0.9; p=0.02; 28 days: CO: 0.4±0.7; ONA: 0.1±0.7;p=0.004). After 28 days the total number of MAC+ cells was also significant higher (Co: 1.1±1.2; ONA: 2.5±1.8; p=0.00001). Regarding C3, no changes could be detected in the GCL at both points in time, whereas after 28 days the total number of C3+ cells was increased (CO: 3.1±1.7; ONA: 4.0±2.0; p=0.0014). In the S100 group, no difference in MAC staining could be seen either 14 or 28 days after immunization. In contrast, the total number of C3+ cells was increased after 14 days (CO: 0.9±1.2; S100:1.5±1.6; p=0.0002), but not in the GCL (CO: 0.8±1.1; S100:0.8±0.9; p=0.696).
Conclusions:
No RGC loss could be observed at day 14, but a significant loss can be noted after 28 days. Indeed, the CS seems to be involved in apoptosis of RGC in ONA immunized animals after two weeks. Because after four weeks MAC+ cells increased in total, we assume that secondary effects lead to an additional activation of complement components, like C3, as a consequence of RGC death. The fact that, at both points in time, neither C3 nor MAC could be shown in the GCL of S100 immunized animals could indicate earlier autoimmune mechanisms that lead to the RGC loss.
Keywords: 531 ganglion cells •
426 apoptosis/cell death •
554 immunohistochemistry