Figure 1.
Difference in oral bacterial load in African-American patients with and without glaucoma.
a) Normalized oral bacterial load (NOBL) of patients with and without glaucoma (p<0.017, t-test). Although cases were significantly different from controls in age (p<0.008, t-test), gender (p<0.02 Chi-square) and diabetes status (p<0.021, Chi square), GLM ANOVA of NOBLs (using group, gender, diabetes status and age [above or below median value] as independent variables) revealed a significant effect of group status (whether a subject belonged to cases or controls) only (p<0.024) while all other parameters did not show a statistically significant effect (p>0.26 for all). Linear regression of NOBL with age revealed a significant but low correlation (p<0.011, R2 = 0.063).b) Linear discriminant analysis of DNA amounts from various bacterial families normalized by the amount of total DNA in each sample. Cases are different from controls (MANOVA, p<0.001).
Figure 2.
Peripheral LPS administration significantly accelerates glaucomatous pathology in DBA/2J mice (but not in DBA/2J-Pe mice) as well as in the microbead-induced IOP elevation model of glaucoma in C57BL/6 mice.
LPS (60 µg) was injected into one hind footpad of 6-month old male DBA/2J and DBA/2J-Pe mice and retinal and optic nerve damage was assessed at 8 months of age. (a) Computer-assisted counts of total RGCs per retina (n = 18 and n = 24 retinas for DBA/2J and DBA/2J+LPS, respectively; n = 4 and n = 10 retinas for DBA/2J-Pe and DBA/2J-Pe+LPS, respectively). (b) Semi-automated total optic nerve axon counts, n = 9 and n = 19 optic nerves for DBA/2J and DBA/2J+LPS, respectively; n = 4 and n = 11 optic nerves for DBA/2J-Pe and DBA/2J-Pe+LPS respectively. The same amount of LPS (60 µg) or vehicle was also administered to male C57BL/6 mice that were (n = 16) or were not (n = 13) subjected to unilateral microbead-induced IOP elevation. optic nerve damage was assessed 2 months later. (c) Semi-quantitative optic nerve damage score in eyes of microbead-treated C57BL/6 animals. (d) Average IOP of eyes of microbead-treated C57BL/6 animals. Data are presented as mean ± SEM. Statistical differences were assessed by one-way ANOVA, followed by Tukey-Kramer post-hoc testing (*p<0.05, **p<0.01, ns>0.05).
Figure 3.
LPS induces up-regulation of TLR4 pathway and complement system genes in the retina but not in the brain of DBA/2J mice.
Treatment with naloxone ameliorates both optic nerve axon and RGC loss. (a) qPCR analysis of relative mRNA levels of genes in the TLR pathways in retinas (n = 9) of LPS-treated mice compared to those of control mice (n = 9) (mRNA levels in control retinas were set at one). (b) Analysis of relative gene expression of the same genes in the brains of the same control and experimental mice. (c) Analysis of TLR and complement gene expression in retinas of LPS-treated mice segregated into two groups according to the extent of damage in the optic nerve: samples from “DBA/2J+LPS” group (n = 5) had significant axon loss, whereas samples from “DBA/2J+LPS (little axonal damage)” (n = 4) had only a small degree of axon loss (comparable to that of non-LPS treated male DBA2/J animals). (d) Analysis of RGC survival in eyes from LPS and naloxone (n = 16) and LPS only treated (n = 21) animals, using semi-quantitative scoring. (e) optic nerve scores of eyes from LPS and naloxone (n = 14) and LPS only (n = 15) treated animals using semi-quantitative assessment. Data were tested for statistical differences between groups using either one-way ANOVA followed by Tukey-Kramer post-hoc testing (multiple groups) or t-test (two groups) (*p<0.05, **p<0.01, ***p<0.001). All data are presented as mean ± SEM.
Figure 4.
Peripheral LPS administration leads to microglial cell changes in the ONH and retina.
(a–d) Representative images of Iba1 (green)/DAPI (blue) (a,b), as well as CD11b (red)/DAPI(blue) (c,d)-stained optic nerve head tissue from DBA2/J animals treated without (a,c) or with LPS (b,d). “V” denotes location of the vitreous body. Arrows point to the junction between the optic nerve tissue and the sclera. Dashed lines outline the region where cell counts were performed. Scale bars are 100 µm. (e,f) Analysis of Iba1+ (e) and CD11b+ (f) microglial cell numbers in the proximal unmyelinated part of the ONH. Counts are average numbers from 6 and 7 LPS treated eyes and 3 and 4 controls respectively. (g) Correlation between RGC score and numbers of CD11b+ cells in the prelaminar region of the ONH of all eyes analyzed. (h) Sholl analysis of retinal microglial cells: representative images of microglial cell traces subjected to Sholl analysis with the overlaid concentric circles. (i) The total number of intersections was decreased in microglia from the retinas of LPS-treated animals (n = 8) as compared to controls (n = 6). (j) Skeleton analysis of retinal microglial cell: the retinal microglia of LPS-treated mice (n = 8 eyes) had fewer branches than that of control mice (n = 6 eyes). Assessments of statistically significant differences between groups were done using t-test (*p<0.05).