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 models induced by type IV collagenase stereotaxic injection⁵, APP transgenic⁶, β-N-methylamino-L-alanine exposure⁷, and hypertension⁸, with CMHs induced by systemic inflammation as one of the most well-accepted choices. Fisher et al.⁹ first used LPS derived from Salmonella typhimurium to develop an acute CMHs mouse model. Subsequently, the same group reported the development of a sub-acute CMHs mouse model using the same approach².

LPS is considered as a standardized inflammatory stimulus via intraperitoneal injection. Previous studies have confirmed that LPS injection could cause neuroinflammation as reflected by large amounts of microglia and astrocyte activation in animal models^2,10. Furthermore, a positive correlation between activation of neuroinflammation activation and the number of CMHs has been established^2,10. Based on these previous studies, we were prompted to develop a CMHs rat model by intraperitoneal injection of LPS.

Advances in detection technologies have resulted in an increase in the number of research investigations on CMHs. The most widely acknowledged methods of detecting CMHs include the detection of red blood cells by hematoxylin and eosin (HE) staining, ferric iron detection by Prussian Blue staining⁹, detection of Evans blue (EB) deposition by immunofluorescence imaging, and 7.0 Tesla magnetic resonance imaging-susceptibility weighted imaging (MRI-SWI)¹⁰. The present study aims to develop a method of screening for CMHs.

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 (average weight: 280 ± 20 g) by intraperitoneal injection.
   CAUTION: If rats are purchased from another organization, then the adaptive phase should not be less than 7 days.

3. LPS Injections

1. Inject LPS intraperitoneally into each rat at a dose of 1 mg/kg, and return the rats to their home cage.
   NOTE: Anesthesia is not necessary.
2. Six hours later, inject the same dose of LPS injection into the rats.
3. Sixteen hours later inject the same dose of LPS into the rats.
   CAUTION: Injecting SD rats with LPS at a dose of 1 mg/kg may result in a 5% mortality rate. The mortality rate could further increase in younger or older rats or pregnant female rats.
4. Return the rats to their cages after LPS injection and provide ad libitum access to food and drink.
   NOTE: Rats will exhibit a distinct systemic inflammatory response, and thus it is essential that the cages remain clean throughout the experiment. Rats should be frequently (2x day) monitoring for weight, general appearance and attitude after the first LPS injection. If the rats start to exhibit a hunched posture, unwillingness to move, significant weight loss (>20%), porphyrin staining that they be humanely euthanized prior to the 7 day EB study end point.

4. Sample Collection

1. EB injection
   1. Seven days after the first injection, anesthetize the rat by intraperitoneally according to your approved IACUC protocol. 
   2. Wait 1–2 min until the rats do not show corneal reflex responses. Perform a 5–8 mm-deep cardiac puncture on each rat; the puncturing point should be 5 mm to the left margin of sternum at the 3rd and 4th intercostals space.
   3. Wait until blood recovery is observed, and then inject EB at a dose of 0.2 mL/100 g body weight into the left heart ventricle.
      CAUTION: Cardiac puncture and EB injection should be carefully performed. EB might be injected into thoracic cavity or pericardium instead of the left heart ventricle.
      1. Suck back with an empty tube and replace this volume with an EB tube before injection to help reduce the failure rate (e.g., injection to rib cage by error).
   4. Keep the rat in a supine position for 10 min.
      NOTE: If sample is not used to detect EB leakage in immunofluorescence imaging, then this step may be omitted.
2. Gross observation
   1. Perform cardiac perfusions using ice-cold 1 M phosphate buffer saline (PBS) to clear the cerebral vasculature and brains.
      1. Using a scalpel, make an abdominal incision across the entire length of the diaphragm.
      2. Cut through ribs just left of the rib cage midline.
      3. Open up the thoracic cavity. Use clamps to expose the heart and to facilitate drainage of blood and other fluids.
      4. While steadily holding the heart with forceps (the heart should still be beating), directly insert a needle into the protrusion of the left ventricle to extend this to about 5 mm. Secure the needle at that position by clamping near the point of entry.
         CAUTION: Do not excessively extend the needle, as it can pierce the interior wall and compromise the circulation of solutions.
      5. Release the valve to allow the flow ice-cold PBS solution (200–300 mL) at a slow and steady rate at around 20 mL/min using a perfusion pump. If the animals require fixation instead of gross observation, then use the same dose of ice-cold 0.9% saline solution.
      6. Using sharp scissors, make an incision in the atrium, ensuring that the solution continuously flows. If the fluid does not flow freely or is coming from the animal’s nostrils or mouth, reposition the needle.
   2. Screen for CMHs by gross observation.
      NOTE: If the sample is not used in calculating the number of CMHs by gross observation, then this step may be omitted.
3. Fixation
   1. Perform cardiac perfusions using ice-cold 0.9% saline solution (200–300 mL) to clear the cerebral vasculature and brains.
   2. Perform cardiac perfusions using ice-cold 4% paraformaldehyde for fixation.
   3. Decapitate the rat, isolate the brain, and immerse the brain in 20% sucrose solution for at least 6 h.
   4. Change the solution into 30% sucrose and fix for another 6 h.
4. Prepare 10 mm-thick brain tissues sections using a cryostat.

5. HE Staining

NOTE: This procedure is performed using a HE Staining Kit.

1. Wash the slides in distilled water.
2. Stain in hematoxylin solution for 8 min. Wash in running tap water for 5 min.
3. Differentiate in 1% acid alcohol for 30 s. Wash in running tap water for 1 min.
4. Stain in 0.2% ammonia water (Bluing) or saturated lithium carbonate solution for 30 s to 1 min. Wash in running tap water for 5 min.
5. Rinse in 95% alcohol (10 dips).
6. Counterstain with eosin solution for 30 s to 1 min.
7. Dehydrate through 95% alcohol, two changes of absolute alcohol, for 5 min each.
8. Clear in xylene for 30 s.
9. Mount using Liu’s method⁹.
10. Analyze using a brightfield fluorescence microscope.
    NOTE: Red blood cells released from blood vessels, which are the components of CMHs, appear in red-orange under HE staining.

6. Perl Prussian Blue Staining

NOTE: This procedure is performed using a Perls Staining Kit.

1. Wash slides with distilled water.
2. Stain in reaction solution with equal parts mixture of ferrocyanide and hydrochloric acid for 10 min.
3. Dehydrate, clear, mount, and analyze the slides as described in steps 5.7–5.10.

7. EB double-staining

NOTE: This procedure follows step 4.1.3.

1. Wash slides with PBS three times for 5 min each.
2. Incubate slides with 4',6-diamidino-2-phenylindole (DAPI) solution for 15 min at room temperature.
3. Wash the slides with PBS solution three times for 5 min each, and mount slides with PBS-glycerol solution.
4. Analyze images on a fluorescence microscope. EB deposition is indicated by red fluorescence; nuclei are indicated by blue fluorescence.

8. MRI-SWI

1. Perform MRI on a 7-T magnet equipped with an actively shielded gradient system (16 cm inner diameter).
2. Seven days after treatment, anesthetize the rats by facemask inhalation of 1.5–2.0% isoflurane using an isoflurane anesthesia system.
3. Apply vet ointment onto the eyes of the rat to prevent dryness while under anesthesia.
4. Keep the rats in prone position and perform an MRI-SWI scan.
5. Obtain SWI weighted scans using a fast-spin echo sequence using the following parameters: echo time (TE) 8 ms, repetition time (TR) 40 ms, flip angle=12°, field of view (FOV) 35 mm × 35 mm, acquisition matrix 384 × 384, to acquire 1 mm-thick slices.
6. Identify CMHs in MRI-SWI according to Greenberg et al.¹¹, which included the following criteria: (1) a diameter of ≤10 mm, and (2) circular, isolated, and low-signal intensity spots.
7. At the end of the experiment, euthanize the rats using the overdose carbon dioxide (at a flow of 6 L/min or 30% of the container volume) method.

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

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