Phosphorylation of extracellular signal-regulated kinases in bladder afferent pathways with cyclophosphamide-induced cystitis

Abstract

Extracellular signal-regulated kinases (ERK1 and ERK2) are phosphorylated in the nervous system after somatic or visceral stimulation or inflammation and play roles in central sensitization and pain hypersensitivity. ERK1/2 activation with cyclophosphamide (CYP)-induced cystitis has been demonstrated in urinary bladder and inhibitors of ERK1/2 phosphorylation reduce CYP-induced bladder hyperreflexia. In this study, we determined pERK1/2 expression and regulation in lumbosacral dorsal root ganglia (DRG) and spinal cord with CYP-induced cystitis (4 h, 48 h, chronic) using Western blotting and immunohistochemical techniques. Phosphorylated extracellular signal-regulated kinases (pERK1/2) expression was significantly (P< or =0.01) upregulated in L6 and S1 DRG with CYP-induced cystitis with the greatest upregulation occurring at 4 h. No changes in pERK1/2 expression were observed in L1, L2 or L5 DRG or in any spinal cord segment examined (L1, L2, L5-S1) with CYP-induced cystitis. Cytoplasmic pERK1/2-immunoreactivity (IR) and pericellular pERK1/2-IR was observed in all DRG examined from control rats and cytoplasmic pERK1/2-IR was significantly (P< or =0.01) increased in L6 and S1 DRG with 4 and 48 h CYP-induced cystitis. In contrast, pericellular pERK1/2-IR in DRG was not regulated by CYP-induced cystitis. A small percentage of bladder afferent cells in lumbosacral DRG expressed pERK1/2-IR in control rats; however, CYP-induced cystitis (48 h) significantly (P< or =0.01) increased the percentage of bladder afferent cells in the L6 and S1 DRG exhibiting pERK1/2-IR. These studies suggest that activation of the ERK pathway in lumbosacral DRG may play a role in neuroplasticity in micturition reflexes with CYP-induced cystitis.

Figure 1

Upregulation of phosphorylated (p) ERK expression in lumbosacral (L6, AB; S1, C–D) dorsal root ganglia (DRG) with CYP-induced cystitis (4 hours (hr), 48 hr and chronic) using western blotting techniques. A. Representative example of a western blot of L6 DRG (20 μg) for pERK1/2 expression in control rats and those treated with cyclophosphamide (CYP) for varying duration. Total ERK staining was also determined and used as a loading control. B. Histogram of relative pERK1/2 band density in all groups examined normalized to total ERK in the same samples presented as a percentage of control ERK activation. pERK1/2 expression in L6 DRG is significantly increased at 4 hr, 48 hr and chronically with CYP treatment. C. Representative example of a western blot of S1 DRG (20 μg) for pERK1/2 expression in control rats and those treated with cyclophosphamide (CYP) for varying duration. Total ERK staining was also determined and used as a loading control. D. Histogram of relative pERK1/2 band density in all groups examined normalized to total ERK in the same samples presented as a percentage of control ERK activation. pERK1/2 expression in S1 DRG is significantly increased at 4 hr, 48 hr and chronically with CYP treatment. *, p ≤ 0.01. Data are a summary of n = 6–8 animals for each group.

Figure 2

CYP-induced cystitis increases cytoplasmic pERK1/2 immunoreactivity (IR) in lumbosacral dorsal root ganglia (DRG). Immunofluorescence images of pERK1/2-IR in L6 DRG from control (A–C) rats and those treated with 48 hr cyclophosphamide (CYP)-induced cystitis (D–I). A–C: Low power immunofluorescence images of the L6 DRG from control rats exhibiting extensive pericellular pERK1/2-IR; some examples are indicated with white arrows. In L6 DRG from control (no inflammation), few DRG cells exhibited cytoplasmic pERK1/2-IR. D–F: With CYP-induced cystitis (48 hr), an increase in the number of DRG cells exhibiting cytoplasmic pERK1/2-IR was observed (yellow arrows). Pericellular pERK1/2-IR was still apparent in L6 DRG with CYP-induced cystitis (white arrows). G–I: Higher power images of insets in images D–F showing cytoplasmic pERK1/2-IR (yellow arrows) in L6 DRG with CYP-induced cystitis (48 hr). Pericellular pERK1/2-IR in L6 DRG after 48 hr CYP-induced cystitis is also shown (white arrows). Calibration bar represents 40 μm in A–C, D–F; 20 μm in G–I.

Figure 3

A: Summary histogram of numbers of dorsal root ganglion (DRG) cells that exhibit pERK1/2-IR under control conditions and after induction of CYP-induced cystitis (4 hr, 48 hr) in the presence or absence of Fastblue (FB). Numbers of pERK1/2-immunoreactive cells were determined in rats with or without FB injection into the urinary bladder as a control for the potential influence of FB on pERK1/2 expression in DRG. The presence of FB did not affect the numbers of pERK1/2-immunoreactive cells observed under control (no inflammation) or CYP treatment in any DRG examined. With 4 hr and 48 hr CYP-induced cystitis, numbers of cytoplasmic pERK1/2-immunoreactive cells significantly (p ≤ 0.01) increased in L6 and S1 DRG. No differences in pERK1/2 expression in L6 or S1 were observed between 4 hr and 48 hr CYP-induced cystitis. Data are a summary of n = 6–8 animals for each group. B: Summary histogram of the percentage of bladder afferent cells expressing pERK1/2-IR in control and CYP-treated (48 hr) rats (n = 6 for each group, *, p ≤ 0.01). No difference in the percentage of bladder afferent cells exhibiting pERK1/2 expression with 48 hr CYP-induced cystitis was observed between L6 and S1 DRG.

Figure 4

Pericellular pERK1/2-IR in lumbosacral DRG is present in control and CYP-treated rats and does exhibit regulation with cystitis. Pericellular pERK1/2-IR appeared to be present in satellite cells surrounding L6-S1 DRG cells. A–C: L6 DRG section exhibiting pericellular pERK1/2-IR (red; B) was also stained for glial fibrillary acidic protein (GFAP; green; A). C: Merged image of A and B displaying some areas of overlap (yellow arrows) between pericellular pERK1/2-IR and GFAP consistent with pERK1/2 expression in satellite cells. Calibration bar represents 20 μm in A–C. D: Summary histogram of numbers of pERK1/2 cytoplasmic negative DRG neurons-pERK1/2 pericellular positive satellite (ST) cell staining in DRG. Pericellular pERK1/2-IR in L1, L2, L5-S1 DRG was not regulated by CYP-induced cystitis (4 hr or 48 hr). Data are a summary of n = 6–8 animals for each group.

Figure 5

CYP-induced cystitis increases pERK1/2-IR in bladder afferent (Fastblue, FB-labeled) cells in the lumbosacral DRG. A–C: L6 DRG section from rat treated with CYP (48 hr) showing bladder afferent cells (FB-labeled; A; white arrows) demonstrating pERK1/2-IR (B) in same section and merged image (C) demonstrating bladder afferent cells with pERK1/2-IR (pinkish-purple/magenta; white arrows). D–F: Another example of an L6 DRG section from rat with 48 hour induced cystitis showing bladder afferent cells (FB-labeled; A; white arrows) demonstrating pERK1/2-IR (B) in same section and merged image (C) demonstrating bladder afferent cells with pERK1/2-IR (pinkish-purple/magenta; white arrows). G–I: Higher power image of S1 DRG section from rat treated with CYP (48 hr) showing bladder afferent cells (FB-labeled; A; white arrows) demonstrating pERK1/2-IR (B) in same section and merged image (C) demonstrating bladder afferent cells with pERK1/2-IR (white arrows). Not all cells expressing pERK1/2-IR are bladder afferent cells (B, E, H; green arrows) and 25–29% of bladder afferent cells expressed pERK1/2-IR after 4 or 48 hr CYP-induced cystitis. Pericellular pERK1/2-IR cells are also indicated (yellow arrows); very few of these cells were bladder afferent cells. Calibration bar represents 40 μm in A–C, D–F; 20 μm in G–I.