Name: sub-acute cerebral microhemorrhages induced by lipopolysaccharide injection in rats
Uploaded: 2018-10-17T14:30:00
Description: Watch this Scientific Journal Video about Sub-acute Cerebral Microhemorrhages Induced by Lipopolysaccharide Injection in Rats at JoVE.com

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&#39;s Hospital of Yichang for providing technical support.

All methods described here have been approved by the Animal Care and Use Committee (ACUC) of the PLA Army General Hospital.
1. Materials
<ol>
	<li>Preparation of LPS injection
	<ol>
		<li>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 &#176;C. 
		CAUTION: LPS is toxic.</li>
		<li>Prepare a 2% EB solution in normal 0.9% saline solution to keep EB injection at working ...

<ol>
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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbleed+topography,+leukoaraiosis,+and+cognition+in+probable+Alzheimer+disease+from+the+Sunnybrook+dementia+study.">Microbleed topography, leukoaraiosis, and cognition in probable Alzheimer disease from the Sunnybrook dementia study.</a> Archives of Neurology. 65 (6), 790-795 (2008).</li><li>Xu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebral+microbleeds+and+neuropsychiatric+symptoms+in+an+elderly+Asian+cohort.">Cerebral microbleeds and neuropsychiatric symptoms in an elderly Asian cohort.</a> Journal of Neurology, Neurosurgery, and Psychiatry. 88 (1), 7-11 (2017).</li><li>Mcauley, G., Schrag, M., Barnes, S., Obenaus, A., Dickson, A., Kirsch, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+iron+quantification+in+collagenase-induced+microbleeds+in+rat+brain.">In vivo iron quantification in collagenase-induced microbleeds in rat brain.</a> Magnetic Resonance in Medicine. 67 (3), 711-717 (2012).</li><li>Reuter, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+cerebral+microbleeds+in+the+APP23-transgenic+mouse+model+of+cerebral+amyloid+angiopathy-a+9.4+tesla+MRI+study.">Development of cerebral microbleeds in the APP23-transgenic mouse model of cerebral amyloid angiopathy-a 9.4 tesla MRI study.</a> Frontiers in Aging Neuroscience. 8 (8), 170 (2016).</li><li>Scott, L. 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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebral+microbleeds:+A+guide+to+detection+and+interpretation.">Cerebral microbleeds: A guide to detection and interpretation.</a> Lancet Neurology. 8 (2), 165-174 (2009).</li><li>Hoffmann, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Field+MRI+reveals+a+drastic+increase+of+hypoxia-induced+microhemorrhages+upon+tissue+reoxygenation+in+the+mouse+brain+with+strong+predominance+in+the+olfactory+bulb.">High-Field MRI reveals a drastic increase of hypoxia-induced microhemorrhages upon tissue reoxygenation in the mouse brain with strong predominance in the olfactory bulb.</a> Plos One. 11 (2), e0148441 (2016).</li><li>Sumbria, R. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+phosphodiesterase+3A+modulation+on+murine+cerebral+microhemorrhages.">Effects of phosphodiesterase 3A modulation on murine cerebral microhemorrhages.</a> Journal of Neuroinflammation. 14 (1), 114 (2017).</li><li>Sumbria, R. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aging+exacerbates+development+of+cerebral+microbleeds+in+a+mouse+model.">Aging exacerbates development of cerebral microbleeds in a mouse model.</a> Journal of Neuroinflammation. 15 (1), 69 (2018).</li><li>Mello, B. S. 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H., Cunningham, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+infection+evokes+exaggerated+sickness+behaviour+in+pre-clinical+murine+prion+disease.">Peripheral infection evokes exaggerated sickness behaviour in pre-clinical murine prion disease.</a> Neuroscience. 112 (1), 7-11 (2002).</li><li>Pantoni, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebral+small+vessel+disease:+from+pathogenesis+and+clinical+characteristics+to+therapeutic+challenges.">Cerebral small vessel disease: from pathogenesis and clinical characteristics to therapeutic challenges.</a> Lancet Neurology. 9 (7), 689-701 (2010).</li><li>Rosand, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+clustering+of+hemorrhages+in+probable+cerebral+amyloid+angiopathy.">Spatial clustering of hemorrhages in probable cerebral amyloid angiopathy.</a> Annals of Neurology. 58 (3), 459-462 (2005).</li><li>Robinson, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microstructural+and+microglial+changes+after+repetitive+mild+traumatic+brain+injury+in+mice.">Microstructural and microglial changes after repetitive mild traumatic brain injury in mice.</a> Journal of Neuroscience Research. 95 (4), 1025-1035 (2017).</li><li>Kraft, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypercholesterolemia+induced+cerebral+small+vessel+disease.">Hypercholesterolemia induced cerebral small vessel disease.</a> Plos One. 12 (8), e0182822 (2017).</li><li>Schreiber, S., Bueche, C. 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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...

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&#8211;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 models induced by type IV collagenase stereotaxic injection5, APP transgenic6, &#946;-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 models2,10. Furthermore, a positive correlation between activation of neuroinflammation activation and the number of CMHs has been established2,10. 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.

<table>
	<tbody>
		<tr>
			<td>LPS</td>
			<td>Sigma-Aldrich</td>
			<td>L-2630</td>
			<td>for inflammation induction</td>
		</tr>
		<tr>
			<td>EB</td>
			<td>Sigma-aldrich</td>
			<td>E2129</td>
			<td>for EB leakage detection</td>
		</tr>
		<tr>
			<td>DAPI dying solution</td>
			<td>Servicbio</td>
			<td>G1012</td>
			<td>count medium for IF</td>
		</tr>
		<tr>
			<td>Perl&#8217;s Prussian staining</td>
			<td>Solarbio</td>
			<td>G1424</td>
			<td>Kit for Prussian staining</td>
		</tr>
		<tr>
			<td>HE staining</td>
			<td>Solarbio</td>
			<td>G1120</td>
			<td>Kit for HE staining</td>
		</tr>
		<tr>
			<td>chloral hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>47335U</td>
			<td>For anesthesia</td>
		</tr>
		<tr>
			<td>phosphate buffer saline (PBS)</td>
			<td>Solarbio</td>
			<td>P1022</td>
			<td>a kind of buffer solution commonly used in experiment</td>
		</tr>
		<tr>
			<td>0.9% saline solution</td>
			<td>Hainan DonglianChangfu Pharmaceutical Co., Ltd., China</td>
			<td></td>
			<td>solution for perfusion</td>
		</tr>
		<tr>
			<td>paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>158127</td>
			<td>a kind of solution commonly used for fixation</td>
		</tr>
		<tr>
			<td>20% sucrose solution</td>
			<td>Solarbio</td>
			<td>G2461</td>
			<td>a kind of solution commonly used for fixation</td>
		</tr>
		<tr>
			<td>30% sucrose solution</td>
			<td>Solarbio</td>
			<td>G2460</td>
			<td>a kind of solution commonly used for fixation</td>
		</tr>
		<tr>
			<td>vet ointment</td>
			<td>Solcoseryl eye gel, Bacel, Switzerland</td>
			<td></td>
			<td>for rat&#39;s eyes protection</td>
		</tr>
	</tbody>
</table>

sub-acute cerebral microhemorrhages induced by lipopolysaccharide injection in rats

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&#39;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.

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...

Watch this Scientific Journal Video about Sub-acute Cerebral Microhemorrhages Induced by Lipopolysaccharide Injection in Rats at JoVE.com

Sub-acute Cerebral Microhemorrhages Induced by Lipopolysaccharide Injection in Rats

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, ...

This method can help answer key questions in cerebral micro-hemorrhage field such as the etiology, distribution, and detection of cerebral micro-hemorrhage and so on. The main advantage of this technique is that LPS injections stably induce inflammation. In addition, LPS injection is quite simple, economical, and cost-effective.Though this method can provide insight into cerebral hemorrhage induction, it can also be applied to other brain disorders such as depression, schizophrenia, Parkinson's disease, Alzheimer's disease, and prion disease. Administer LPS at a dose of one milligram per kilogram of body weight to 10-week-old male Sprague Dawley rats by intraperitoneal injection, then return each rat to the home cage. Repeat the LPS injection after six hours and 16 hours.Please note that injecting SD rats with LPS at a dose of one milligram per kilogram may result in a 5%mortality. The mortality could further increase in younger or older rats or pregnant female rats. Return the rats to their cages after LPS injection, and provide ad libitum access to food and drink.Under terminal anesthesia, perform a five to eight-millimeter deep cardiac puncture on each rat. The puncture point should be five millimeters to the left margin of the sternum at the third and fourth intercostal space. Hold the needle in place and replace the empty tube with a tube containing Evans blue.Wait until blood recovery is observed, and then inject Evans blue at a dose of 0.2 milliliters per 100 grams of body weight into the left heart ventricle. Keep the rat in a supine position for 10 minutes if assessing Evans blue leakage through the blood/brain barrier by immunofluorescence imaging. Perform cardiac perfusions using ice-cold 200 to 300 milliliters of 0.9%saline solution to clear the cerebral vasculature and brains.Switch to ice-cold 4%paraformaldehyde for fixation. Then, after isolating the brain, immerse it in 20%sucrose in PBS for at least six hours or until the brain sinks to the bottom of the tube. Change the solution to 30%sucrose and fix it for another six hours.Finally, prepare 10-millimeter thick brain tissue sections using a cryostat. Wash the slides with PBS three times for five minutes each, then incubate slides with 4, 6'diamidino-2-phenylindole or DAPI solution for 15 minutes at room temperature. After washing the slides in PBS as before, mount the slides with PBS-glycerol solution.Capture images on a fluorescence microscope. EB deposition is indicated by red fluorescence, nuclei are indicated by blue fluorescence. Begin H&E staining by washing the slides in distilled water.Then immerse in hematoxylin solution for eight minutes. Alternatively, for Perl Prussian blue staining, stain in reaction solution with equal-parts mixture of ferrocyanide and hydrochloric acid for 10 minutes. Following staining, wash in running tap water for five minutes.Differentiate in 1%acid alcohol for 30 seconds. After washing in running tap water for one minute, stain in 0.2%ammonia water or saturated lithium carbonate solution for 30 seconds to one minute. Next, wash in running tap water for five minutes.Counter-stain with eosin solution for 30 seconds to one minute. Dehydrate through 90%alcohol, then 95%alcohol, and absolute alcohol for 0.5 to two minutes each. After dehydration, clear in xylene for 30 seconds.After mounting, analyze the H&E staining using a brightfield fluorescence microscope. The red blood cells released from blood vessels, which are components of the CMHs, appear in red-orange under H&E staining. Surface CMHs can be detected by gross observation as indicated by the red arrows.Evans blue staining reveals visible red fluorescence where Evans blue molecules leaked from an injured blood-brain barrier. H&E staining shows red blood cells found outside the capillaries, and Prussian staining reveals ferric ion points derived from lysis of red blood cells. While attempting this procedure, it is important to remember that the dose of LPS to induce cerebral micro-hemorrhage is larger than that used in Parkinson's disease or schizophrenia model.Therefore, the environment of animals should be kept clean to reduce mortality rate. Following this procedure, other methods like electron microscopy, magnetic resonance imaging, and immunofluorescent imaging could be performed to determine the damage to outer structure of blood-brain barrier in cerebral micro-hemorrhage, distribution of cerebral micro-hemorrhages in brain parenchyma in vivo, as well as glial cell activation.

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