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. 2008 Sep;29(5):864-70.
doi: 10.1016/j.neuro.2008.02.014. Epub 2008 Mar 13.

Endotoxin induces a delayed loss of TH-IR neurons in substantia nigra and motor behavioral deficits

Yuxin Liu  1 , Liya Qin, Belinda Wilson, Xuefei Wu, Li Qian, Ann-Charlotte Granholm, Fulton T Crews, Jau-Shyong Hong
Affiliations

Endotoxin induces a delayed loss of TH-IR neurons in substantia nigra and motor behavioral deficits

Yuxin Liu et al. Neurotoxicology. 2008 Sep.

Abstract

We have previously reported that a single injection of endotoxin, lipopolysaccharide (LPS, 5mg/kg, i.p.), causes a delayed and progressive loss of TH-IR neurons in the substantia nigra (SN) in C57BL/six male mice. In this study, we determined sex differences and behavioral deficits accompanying the loss of TH-IR neurons in response to peripheral LPS injection. A single injection of LPS (5mg/kg, i.p.) failed to produce any loss of TH-IR neurons in the SN of female mice over a 12-month period. To determine if multiple-injections were required, female mice received five injections of LPS (5mg/kg, i.p.) at either weekly or monthly intervals. Behavioral motor ability and TH-IR neuronal loss were determined after the first injection of LPS. We found significant differences in both behavioral activities and neuronal loss between these two injection paradigms. Between 7 and 20 months after the first injection of LPS, progressive behavioral changes, measured by rotor-rod and open-field activities, and neuronal loss in SN were observed in monthly injected, but not in weekly injected mice. In addition, reduced rotor-rod ability in monthly injected mice were restored following treatment of l-dopa/carbidopa (30 mg/3mg/kg), i.p.). Approximately 40 and 50% loss of TH-IR neurons at 9 and 20 months, respectively, was observed after exposure to LPS, suggesting that the behavioral deficit is related to loss of dopamine function in the nigra-striatal pathway. More intense immuno-staining of alpha-synuclein and inflammatory markers were detected in brain sections exposed to LPS. In conclusion, these results show that multi-LPS monthly injections can induce a delayed and progressive loss of TH-IR neurons and motor deficits which resemble the progressive nature of Parkinson's disease. Further, the present study reveals a clear sex difference: female mice are more resistant to LPS than male mice. Repeated monthly LPS injections are required to cause both motor behavioral deficits and DA neuronal loss in female mice.

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Figures

Fig. 1
Fig. 1
The different response to LPS injection between male and female mice. Male or female C57BL/six mice were treated with saline or LPS (5 mg/kg, i.p.) and then maintained under normal conditions for the indicated time points. (A) Number of TH-IR neurons in the SN of control and LPS-treated mice at different time points. Single-injected mice (male and female) were sacrificed and brain sections (35 µm) were cut through the nigral complex. After immunostaining with TH antibody, the number of TH-IR neurons in the SN was counted as described in Section 2. Results are expressed as percentage of the corresponding saline controls. (B) Multi-injected (once a month) female mice were made rotarod test at 7 months after the first injection. *P < 0.05 and **P < 0.01 were compared to the saline controls (n = 5–10 per group).
Fig. 2
Fig. 2
The latency comparison between the monthly and weekly LPS injections in female mice on the rotarod test. The female C57BL/six mice were injected with saline or LPS (5 mg/kg, i.p.) weekly or monthly up to five times. Mice were maintained under normal conditions, and tested on rotarod at different time points: (A) monthly injection group; (B) weekly injection group. **P < 0.01 compared to the saline controls.
Fig. 3
Fig. 3
Decreased locomotor behavior in five monthly LPS-injected mice. Female C57BL/six mice were injected with saline or LPS (5 mg/kg, i.p.) monthly up to five times, maintained under normal conditions, open-field test was performed on the 9th month after the first injection. Total activity, ambulatory activity and distance traveled were recorded in the open-field test. *P < 0.05 compared to the saline controls.
Fig. 4
Fig. 4
l-dopa/carbidopa administration can reverse the reduction of rotarod motor ability. Mice injected with five monthly saline and LPS were administed l-dopa/ carbidopa (30 mg/3 mg/kg, i.p.) 12 months following the first saline or LPS treatment. Rotarod testing was done at 0 h, 2 h and 1 week after l-dopa/carbidopa injection. **P < 0.01 compared to the saline controls. #P < 0.05 compared to the same group at 0 h or 1 week after l-dopa/carbidopa administration.
Fig. 5
Fig. 5
Delayed loss of nigral DA neurons following systemic LPS injection. Female C57BL/six mice were monthly administered saline or LPS (5 mg/kg, i.p.) for five times, 9 or 20 months after the first LPS injection, mice were sacrificed, brains were harvested and sectioned, and 24 sections (rostral to caudal: 4.52–5.36 mm posterior mm to bregma) were collected as described in methods. Eight evenly spaced brain sections from saline or LPS-injected animals were immunostained with an anti-TH antibody, and the number of TH-IR neurons in the SN was counted. Data were expressed as percent loss compared to saline-injected controls. (A) Number of TH-IR neurons in the SN in control and LPS-treated mice at 9 and 20 months. **P < 0.001, compared with the corresponding control group. (B) Visualization of TH-IR neurons in the substantia nigra and ventral tegmental area in saline and LPS-treated animals at 20 months.
Fig. 6
Fig. 6
Enhanced immunostaining of α-synuclein, CD45 and IL-6 in LPS-treated mice. Female C57BL/six mice were monthly administered saline or LPS (5 mg/kg, i.p.) for five times. At 20 months after the first saline or LPS treatment, brain sections were immunostained with α-synuclein, CD45 and IL-6 antibody as described in methods. Five monthly repeated LPS exposure significantly increased immunostaining of α-synuclein (A), CD45 (B) and IL-6 (C) compared with the saline control group.
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