There, it led to an increased level of caspases 3, 8, and 9 after 14 days, which suggested the involvement of the intrinsic and extrinsic apoptosis pathway. nerve, however, only an increased apoptosis rate was detectable. Therefore, the activation of caspases and the induction of an incipient immune response seem to be the main triggers for retinal degeneration in this intravitreal HSP27 model. = 1.0). In contrast, the HSP25+ staining area in the HSP27 group (1.8 0.5%) was increased (HSP27 vs. native: = 0.017 and HSP27 vs. PBS: = 0.014; Physique 1B). Open in a separate windows Physique 1 Localization and expression level of warmth shock proteins. (A) Heat shock protein 25 (HSP25, green), astrocytes (GFAP, reddish), neuronal cells (NeuN, reddish), and cell nuclei (DAPI, blue) were stained on retinal cross-sections. 1-NA-PP1 The detail view revealed that HSP25 signals were rather colocalized with GFAP signals than with NeuN signals (detail). (B) The HSP25 transmission was strongly increased in the heat shock protein 27 (HSP27) group as compared with the native (= 0.017) and PBS group (= 0.014). (C) Compared to controls, a significant increase in mRNA expression was measured in HSP27 treated retinae (HSP27 vs. native: = 0.040 and HSP27 vs. PBS: = 0.010). (D) Via RT-qPCR, a significant increase in mRNA expression was also observed in the HSP27 group as compared with the PBS group (= 0.002), but not compared with the native group. (E) In contrast, the relative mRNA expression of was comparable in all groups. Scale bars = 20 m. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer. * 0.05 and ** 0.01. Immunohistology = mean SEM, RT-qPCR = median quartile + minimum/ maximum. Dashed line represents the relative expression level of the control group. To identify the localization of HSP25, it was stained in combination with a marker against neuronal cells (NeuN) and astrocytes (GFAP). According to these overview stains, we noted that HSP25 was mainly located in the ganglion cell layer (GCL). However, a stronger colocalization between HSP25 and GFAP was observed, suggesting that HSP25 was released by astrocytes (Physique 1A, detail). In order to evaluate the HSP27 Rabbit polyclonal to ACPL2 (expression was detected in the HSP27 group (HSP27 vs. native: 1.8-fold expression, = 0.040 and HSP27 vs. PBS: 2.5-fold expression, = 0.010, Figure 1C). In contrast, the 1-NA-PP1 relative expression of in the HSP27 group was only increased as compared with the PBS group (1.5-fold expression, = 0.002, Figure 1D). No differences in the relative expression of could be measured between the HSP27 and native group (1.0-fold expression, = 0.990). Comparable results were obtained for the relative expression of = 0.108), as well as the PBS group (0.8-fold expression, = 0.377, Figure 1E). 2.2. Unaltered Quantity of Retinal Ganglion Cells but Increased Apoptosis Rate To analyze neuronal degeneration, we quantified the number of RGCs and the number of RGCs in apoptotic stage using RBPMS and Bax (Physique 2A). The number of RGCs was almost equivalent in the HSP27 group (96.6 5.6%), the PBS (98.2 6.0%, = 0.991), and the native group (100.0 12.4%, = 0.959, Figure 2B). Both control groups also demonstrated comparable RGC counts (native vs. PBS: = 0.987). Regarding the number of Bax+ and RBPMS+ cells, no differences could be observed in the HSP27 group (11.3 1.9%) when compared with both controls (PBS 10.2 1.8%, = 0.887 and native 8.4 1.0%, = 0.450). Comparable numbers of apoptotic RGCs were also observed in the PBS and the native group (= 0.730, Figure 2C). Open in a separate window 1-NA-PP1 Physique 2 Constant quantity of retinal ganglion cells but increased apoptosis rate. (A) Retinal ganglion cells (RGCs) in the retina were marked with RBPMS (green), apoptotic RGCs 1-NA-PP1 with Bax (reddish), and cell nuclei with DAPI (blue). (B) RBPMS cell count revealed no differences among.