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Neuroscience

Sub-acute Cerebral Microhemorrhages Induced by Lipopolysaccharide Injection in Rats

Published: October 17th, 2018

DOI:

10.3791/58423

1Department of Neurology, Second Hospital of Shanxi Medical University, 2Department of Neurology, PLA Army General Hospital, 3Department of Neurology, NO 261 Hospital of PLA
* These authors contributed equally

We present a protocol to induce and detect CMHs caused by LPS injection in Sprague-Dawley rats, which may be utilized in future research investigations on the pathogenesis of CMHs.

Cerebral microhemorrhages (CMHs) are common in aged patients and are correlated to various neuropsychiatric disorders. The etiology of CMHs is complex, and neuroinflammation is often observed as a co-occurrence. Here, we describe a sub-acute CMHs rat model induced by lipopolysaccharide (LPS) injection, as well as a method for detecting CMHs. Systemic LPS injection is relatively simple, economical, and cost-effective. One major advantage of LPS injection is its stability to induce inflammation. CMHs caused by LPS injection could be detected by gross observation, hematoxylin and eosin (HE) staining, Perl's Prussian staining, Evans blue (EB) double-labeling, and magnetic resonance imaging-susceptibility weighted imaging (MRI-SWI) technology. Finally, other methods of developing CMHs animal models, including their advantages and/or disadvantages, are also discussed in this report.

Classical cerebral microhemorrhages (CMHs) refer to tiny perivascular deposits of blood degradation products such as hemosiderin from red blood cells in the brain1. According to the Rotterdam Scan Study, CMHs could be found in nearly 17.8% of persons aged 60–69 years and 38.3% in those over 80 years2. The prevalence of CMHs in the elderly is relatively high, and a correlation between the accumulation of CMHs and cognitive and neuropsychiatric dysfunction has been established3,4. Several animal models of CMHs have recently been reported, including rodent mod....

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All methods described here have been approved by the Animal Care and Use Committee (ACUC) of the PLA Army General Hospital.

1. Materials

  1. Preparation of LPS injection
    1. Add 25 mL of distilled water to 25 mg of LPS powder derived from S. typhimurium to a final concentration of 1 mg/mL. Store the injection in a sterile tube at 4 °C.
      CAUTION: LPS is toxic.
    2. Prepare a 2% EB solution in normal 0.9% saline solution to keep EB injection at working .......

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CMHs can be detected using various approaches. The most well-accepted methods include the following: (1) gross observation and assessment of surface CMHs (shown in Figure 1, upper panel); (2) HE staining for the detection of red blood cells (shown in Figure 2A, upper panel) or Prussian staining detecting ferric iron derived from lysis of red blood cells (Figure 2A, lower panel); (3) EB doubled staini.......

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Research studies on CMHs have increased in the past few years. However, the mechanism of CMHs remains unclear, prompting scientists to establish animal models that simulate this particular condition. For example, Hoffmann et al. developed a hypoxia-induced CMHs mouse model that shows that CMHs are caused by hypoxia and disruption of cerebrovascular autoregulation12. Reuter et al.5 established a CMHs model in APP23-transgenic mice, which showed that cerebra.......

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We thank Teacher Jian Feng Lei and colleagues from Capital Medical University for guidance during MRI. We also thank Jing Zeng from the Department of Neurology, the Second People's Hospital of Yichang for providing technical support.

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Name Company Catalog Number Comments
LPS Sigma-Aldrich L-2630 for inflammation induction
EB Sigma-aldrich E2129 for EB leakage detection
DAPI dying solution Servicbio G1012 count medium for IF
Perl’s Prussian staining Solarbio G1424 Kit for Prussian staining
HE staining Solarbio G1120 Kit for HE staining
chloral hydrate Sigma-Aldrich 47335U For anesthesia
phosphate buffer saline (PBS) Solarbio P1022 a kind of buffer solution commonly used in experiment
0.9% saline solution Hainan DonglianChangfu Pharmaceutical Co., Ltd., China solution for perfusion
paraformaldehyde Sigma-Aldrich 158127 a kind of solution commonly used for fixation
20% sucrose solution Solarbio G2461 a kind of solution commonly used for fixation
30% sucrose solution Solarbio G2460 a kind of solution commonly used for fixation
vet ointment Solcoseryl eye gel, Bacel, Switzerland for rat's eyes protection

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