Short-term effects of an endotoxin on substantia nigra dopamine neurons

Abstract

Inflammation has been implicated in the pathology of several neurodegenerative diseases, including Parkinson׳s disease (PD). Studies using the endotoxin lipopolysaccharide (LPS), a potent inflammogen, show that systemic insults can trigger prolonged microglial activation and pro-inflammatory cytokine production leading to degeneration of substantia nigra (SN) dopamine (DA) neurons, mimicking idiopathic PD. Because rapid effects of LPS on SN neurons had not been investigated previously, the focus of this study is to assess time-dependent alterations in SN neuroinflammation, DAergic neurons, and neuronal signaling cascades following LPS administration. LPS (5mg/kg, i.p.) or saline (0.9% NaCl) was administered to 8-month-old male mice. At 3h, 5h, and 12h post-injection, the morphology of the SN was assessed using antibodies directed against tyrosine hydroxylase (TH, DAergic marker), Iba-1 (pan-microglial marker), phospho-ERK, and phospho-CREB (signaling). LPS administration significantly reduced TH-immunoreactivity (ir) at all time-points with the greatest reduction observed at 12h post-injection. Reduced TH-ir was accompanied by a significant increase in activated microglia at all time-points following LPS. By 12h post-injection, LPS-treated mice exhibited activated as well as reactive microglia, which can result in neuronal damage. These data demonstrate that the initial reduction in TH-ir observed after an LPS injection was not concomitant with morphological alterations in microglial cells, even though a significant increase in phospho-ERK was observed in glial cells as soon as 3h post-injection. It is possible that the initial alteration in DA phenotype (TH reduction) may perpetuate an inflammatory response that persists and leads to further DAergic damage.

Keywords: Dopamine; Inflammation; Lipopolysaccharide; Substantia nigra.

Figure 1

Nigral TH-ir is reduced following LPS administration. Photomicrographs of TH-ir in the SN of (A) saline-treated mice, (B) LPS-treated mice at 3 hrs, (C) LPS-treated mice at 5 hrs, and (D) LPS-treated mice at 12 hrs (magnification=10×). (E–G) Treatment groups at magnification=60×. (H) Quantification of the average TH-ir density in the SNpc. LPS-treatment, regardless of time-point, resulted in reduced TH-ir compared to saline-treatment (p<0.05). No difference existed in the density of TH-ir between 3- and 5-hr LPS treated mice, however, by 12 hrs, TH-ir was significantly reduced compared to 3- and 5-hrs post-injection (p<0.01). At 5-hrs post-LPS injection, nuclear swelling was evident as indicated by the large arrow. By 12 hrs post-LPS injection, existing TH-ir cells are showing changes in morphology, including cellular shrinking as indicated by the large arrows and punctate processes as indicated by the thin arrow in 1F. SNpc outlined by straight lines, SNpr outlined by oval shape. *p<0.05, **p<0.01, ***p<0.001.

Figure 2

Increased microglia in the SN after LPS treatment. Microglia are shown in the SN by Iba-1 labeling in (A) saline-treated mice, (B) LPS-treated mice at 3 hrs, (C) LPS-treated mice at 5 hrs, and (D) LPS-treated mice at 12 hrs (magnification 20×). (E-F) LPS-treated mice at 3- and 5-hrs at magnification =60×. (G) Quantification of the average Iba-1-ir density in the SN of the treatment groups. Iba-1-ir in the SN was significantly increased following LPS treatment compared to saline-treated mice (p<0.05). This increase in Iba-1 labeling is indicated by microglial cell body enlargement as well as a thickening of the processes (E, F). **p<0.01, ***p<0.001.

Figure 3

LPS administration resulted in increased glial phospho-ERK expression. Phospho-ERK-ir measured in the SN of (A) saline-treated mice, (B) LPS-treated mice at 3 hrs, (C) LPS-treated mice at 5 hrs, and (D) LPS-treated mice at 12 hrs (magnification 40×). (E) Quantification of phospho-ERK-ir in the SN following LPS injections. LPS treatment resulted in a significant increase of phospho-ERK-ir in the SN, regardless of time point (*p<0.001). This increase of labeling appeared to be within glial cells as indicated by morphological appearance (inset, B, magnification=60×).

Figure 4

Increased nigral phospho-CREB-ir following LPS administration. Phospho-CREB-ir as measured in (A) saline-treated mice, (B) (B) LPS-treated mice at 3 hrs, (C) LPS-treated mice at 5 hrs, and (D) LPS-treated mice at 12 hrs (magnification 40×). (E) Quantification of phospho-CREB-ir in the SN following LPS injections. (F) Nissl co-labeling (blue) with phosphor-CREB-ir (brown) in the SN as indicated by arrows (magnification=60×). LPS injections resulted in a significant increase in SN phospho-CREB labeling at 3 hrs (p<0.01) and 5 hrs (p<0.001). Phospho-CREB-ir peaked at 5 hrs and then returned to comparable levels of saline-treated mice by 12 hrs post-LPS injection. Nissl co-labeling confirmed the localization of phospho-CREB to neuronal populations (F). *p<0.05, **p<0.01, ***p<0.001.