Sub-acute Cerebral Microhemorrhages Induced by Lipopolysaccharide Injection in Rats

Summary

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.

Abstract

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.

Introduction

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 established^3,4. Several animal models of CMHs have recently been reported, including rodent mod....

Protocol

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 concentration.

2. Animals

1. Administer LPS to male Sprague-Dawley (SD) rats, aged 10 weeks ....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

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