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>
	<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=A+murine+model+of+inflammation-induced+cerebral+microbleeds.">A murine model of inflammation-induced cerebral microbleeds.</a> J Neuroinflammation. 13 (1), 218 (2016).</li><li>Vernooij, M. W., 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=Prevalence+and+risk+factors+of+cerebral+microbleeds+the+Rotterdam+Scan+Study.">Prevalence and risk factors of cerebral microbleeds the Rotterdam Scan Study.</a> Neurology. 70 (14), 1208-1214 (2009).</li><li>Pettersen, J. 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. L., Downing, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+single+neonatal+exposure+to+BMAA+in+a+rat+model+produces+neuropathology+consistent+with+neurodegenerative+diseases.">A single neonatal exposure to BMAA in a rat model produces neuropathology consistent with neurodegenerative diseases.</a> Toxins. 10 (1), E22 (2018).</li><li>Toth, 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=Aging+exacerbates+hypertension-induced+cerebral+microhemorrhages+in+mice:+role+of+resveratrol+treatment+in+vasoprotection.">Aging exacerbates hypertension-induced cerebral microhemorrhages in mice: role of resveratrol treatment in vasoprotection.</a> Aging Cell. 14 (3), 400-408 (2015).</li><li>Liu, 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=Comparative+analysis+of+H&amp;E+and+Prussian+blue+staining+in+a+mouse+model+of+cerebral+microbleeds.">Comparative analysis of H&amp;E and Prussian blue staining in a mouse model of cerebral microbleeds.</a> Journal of Histochemistry &amp; Cytochemistry. 62 (11), 767-773 (2014).</li><li>Zeng, J., Zh&#224;o, H., Liu, Z., Zhang, W., Huang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipopolysaccharide+induces+subacute+cerebral+microhemorrhages+with+involvement+of+Nitric+Oxide+Synthase+in+rats.">Lipopolysaccharide induces subacute cerebral microhemorrhages with involvement of Nitric Oxide Synthase in rats.</a> Journal of Stroke and Cerebrovascular Diseases. 27 (7), 1905-1913 (2018).</li><li>Greenberg, S. 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. F., 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=Sex+influences+in+behavior+and+brain+inflammatory+and+oxidative+alterations+in+mice+submitted+to+lipopolysaccharide-induced+inflammatory+model+of+depression.">Sex influences in behavior and brain inflammatory and oxidative alterations in mice submitted to lipopolysaccharide-induced inflammatory model of depression.</a> Journal of Neuroimmunol. 320, 133-142 (2018).</li><li>Souza, D. F. D., 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=Changes+in+astroglial+markers+in+a+maternal+immune+activation+model+of+schizophrenia+in+Wistar+rats+are+dependent+on+sex.">Changes in astroglial markers in a maternal immune activation model of schizophrenia in Wistar rats are dependent on sex.</a> Frontiers in Cellular Neuroscience. 9 (489), (2015).</li><li>Dutta, G., Zhang, P., Liu, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Lipopolysaccharide+Parkinson's+disease+animal+model:+mechanistic+studies+and+drug+discovery.">The Lipopolysaccharide Parkinson's disease animal model: mechanistic studies and drug discovery.</a> Fundamental &amp; Clinical Pharmacology. 22 (5), 453-464 (2008).</li><li>El-Sayed, N. S., Bayan, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Possible+role+of+resveratrol+targeting+estradiol+and+neprilysin+pathways+in+lipopolysaccharide+model+of+Alzheimer+disease.">Possible role of resveratrol targeting estradiol and neprilysin pathways in lipopolysaccharide model of Alzheimer disease.</a> Advances in Experimental Medicine and Biology. 822 (822), 107-118 (2015).</li><li>Combrinck, M. I., Perry, V. 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. Z., Garz, C., Baun, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+brain+barrier+breakdown+as+the+starting+point+of+cerebral+small+vessel+disease?+-+New+insights+from+a+rat+model.">Blood brain barrier breakdown as the starting point of cerebral small vessel disease? - New insights from a rat model.</a> Experimental &amp; Translational Stroke Medicine. 5 (1), 4 (2013).</li></ol>

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.

sub-acute-cerebral-microhemorrhages-induced-lipopolysaccharide

Research

JoVE Journal

Neuroscience

Focal Cerebral Ischemia Model by Endovascular Suture Occlusion of the Middle Cerebral Artery in the Rat

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable clinical state; in the development of animal models of focal ischemia, however, achieving reproducibility of experimentally induced infarct volume is essential. The rat is a widely used animal model for stroke due to its relatively low animal husbandry costs and to the similarity of its cranial circulation to that of humans2,3. In humans, the middle cerebral artery (MCA) is most commonly affected in stroke syndromes and multiple methods of MCA occlusion (MCAO) have been described to mimic this clinical syndrome in animal models. Because recanalization commonly occurs following an acute stroke in the human, reperfusion after a period of occlusion has been included in many of these models. In this video, we demonstrate the transient endovascular suture MCAO model in the spontaneously hypertensive rat (SHR). A filament with a silicon tip coating is placed intraluminally at the MCA origin for 60 minutes, followed by reperfusion. Note that the optimal occlusion period may vary in other rat strains, such as Wistar or Sprague-Dawley. Several behavioral indicators of stroke in the rat are shown. Focal ischemia is confirmed using T2-weighted magnetic resonance images and by staining brain sections with 2,3,5-triphenyltetrazolium chloride (TTC) 24 hours after MCAO.

Surgical induction of ischemic brain damage in the rat is a widely used model for stroke research. Here we demonstrate the induction of focal cerebral ischemia by occlusion of the middle cerebral artery. Visualization of the resulting infarct by histological staining and magnetic resonance imaging is also shown.

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Rat Model of Widespread Cerebral Cortical Demyelination Induced by an Intracerebral Injection of Pro-Inflammatory Cytokines

Multiple sclerosis (MS) is the most common immune-mediated disease of the central nervous system (CNS) and progressively leads to physical disability and death, caused by white matter lesions in the spinal cord and cerebellum, as well as by demyelination in grey matter. Whilst conventional models of experimental allergic encephalomyelitis are suitable for the investigation of the cell-mediated inflammation in the spinal and cerebellar white matter, they fail to address grey matter pathologies. Here, we present the experimental protocol for a novel rat model of cortical demyelination allowing the investigation of the pathological and molecular mechanisms leading to cortical lesions. The demyelination is induced by an immunization with low-dose myelin oligodendrocyte glycoprotein (MOG) in an incomplete Freund&#39;s adjuvant followed by a catheter-mediated intracerebral delivery of pro-inflammatory cytokines. The catheter, moreover, enables multiple rounds of demyelination without causing injection-induced trauma, as well as the intracerebral delivery of potential therapeutic drugs undergoing a preclinical investigation. The method is also ethically favorable as animal pain and distress or disability are controlled and relatively minimal. The expected timeframe for the implementation of the entire protocol is around 8 - 10 weeks.

The protocol presented here allows the reproduction of a widespread grey matter demyelination of both cortical hemispheres in adult male Dark Agouti rats. The method comprises of intracerebral implantation of a catheter, subclinical immunization against myelin oligodendrocyte glycoprotein, and intracerebral injection of a pro-inflammatory cytokine mixture through the implanted catheter.

Multiple sclerosis (MS) is the most common immune-mediated disease of the central nervous system (CNS) and progressively leads to physical disability and ...

Effects of Blast-induced Neurotrauma on Pressurized Rodent Middle Cerebral Arteries

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral vascular effects. Impact (i.e., non-blast) traumatic brain injury (TBI) is known to decrease pressure autoregulation in the cerebral vasculature in both humans and experimental animals. The hypothesis that blast-induced traumatic brain injury (bTBI), like impact TBI, results in impaired cerebral vascular reactivity was tested by measuring myogenic dilatory responses to reduced intravascular pressure in rodent middle cerebral arterial (MCA) segments from rats subjected to mild bTBI using an Advanced Blast Simulator (ABS) shock tube. Adult, male Sprague-Dawley rats were anesthetized, intubated, ventilated and prepared for Sham bTBI (identical manipulation and anesthesia except for blast injury) or mild bTBI. Rats were randomly assigned to receive Sham bTBI or mild bTBI followed by sacrifice 30 or 60 min post-injury. Immediately after bTBI, righting reflex (RR) suppression times were assessed, euthanasia at the time points post-injury was completed, the brain was harvested and the individual MCA segments were collected, mounted and pressurized. As the intraluminal pressure perfused through the arterial segments was reduced in 20 mmHg increments from 100 to 20 mmHg, MCA diameters were measured and recorded. With decreasing intraluminal pressure, MCA diameters steadily increased significantly above baseline in the Sham bTBI groups while MCA dilator responses were significantly reduced (p &#60; 0.05) in both bTBI groups as evidenced by the impaired, smaller MCA diameters recorded for the bTBI groups. In addition, RR suppression in the bTBI groups was significantly (p &#60; 0.05) higher than in the Sham bTBI groups. MCA&#39;s collected from the Sham bTBI groups exhibited typical vasodilatory properties to decreases in intraluminal pressure while MCA&#39;s collected following bTBI exhibited significantly impaired myogenic vasodilatory responses to reduced pressure that persisted for at least 60 min after bTBI.

Here, we present a protocol to describe methods for ex vivo vascular reactivity determination following a primary blast traumatic brain injury (bTBI) using isolated, pressurized, rodent middle cerebral arterial (MCA) segments. bTBI induction is accomplished using a shock tube, also known as an Advanced Blast Simulator (ABS) device.

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral ...

Establishment of Acute Pontine Infarction in Rats by Electrical Stimulation

Pontine infarction is the most common stroke subtype in the posterior circulation, while there lacks a rodent model mimicking pontine infarction. Provided here is a protocol for successfully establishing a rat model of acute pontine infarction. Rats weighing about 250 g are used, and a probe with an insulated sheath is injected into the pons using a stereotaxic apparatus. A lesion is produced by the electrical stimulation with a single pulse. The Longa score, Berderson score, and beam balance test are used to assess neurological deficits. Additionally, the adhesive-removal somatosensory test is used to determine sensorimotor function, and the limb placement test is used to evaluate proprioception. MRI scans are then used to assess the infarction in vivo, and TTC staining is used to confirm the infarction in vitro. Here, a successful infarction is identified that is located in the anterolateral basis of the rostral pons. In conclusion, a new method is described to establish an acute pontine infarction rat model.

Presented here is a protocol for establishing acute pontine infarction in a rat model via electrical stimulation with a single pulse.

Pontine infarction is the most common stroke subtype in the posterior circulation, while there lacks a rodent model mimicking pontine infarction. Provided ...

Laser-Induced Brain Injury in the Motor Cortex of Rats

A common technique for inducing stroke in experimental rodent models involves the transient (often denoted as MCAO-t) or permanent (designated as MCAO-p) occlusion of the middle cerebral artery (MCA) using a catheter. This generally accepted technique, however, has some limitations, thereby limiting its extensive use. Stroke induction by this method is often characterized by high variability in the localization and size of the ischemic area, periodical occurrences of hemorrhage, and high death rates. Also, the successful completion of any of the transient or permanent procedures requires expertise and often lasts for about 30 minutes. In this protocol, a laser irradiation technique is presented that can serve as an alternative method for inducing and studying brain injury in rodent models.
When compared to rats in the control and MCAO groups, the brain injury by laser induction showed reduced variability in body temperature, infarct volume, brain edema, intracranial hemorrhage, and mortality. Furthermore, the use of a laser-induced injury caused damage to the brain tissues only in the motor cortex unlike in the MCAO experiments where destruction of both the motor cortex and striatal tissues is observed.
Findings from this investigation suggest that laser irradiation could serve as an alternative and effective technique for inducing brain injury in the motor cortex. The method also shortens the time for completing the procedure and does not require expert handlers.

The protocol presented here shows a technique to create a rodent model of brain injury. The method described here uses laser irradiation and targets motor cortex.

A common technique for inducing stroke in experimental rodent models involves the transient (often denoted as MCAO-t) or permanent (designated as MCAO-p) ...

Detrusor Underactivity Model in Rats by Conus Medullaris Transection

The goal of the presented protocol was to establish a detrusor underactivity (DU) model in the rat through conus medullaris transection. Laminectomy was performed in a total of 40 female Wistar rats (control group: 10 rats; test group: 30 rats) weighing 200&#8211;220 g, and the conus medullaris was transected at the L4&#8210;L5 level in the test group. All the rats were housed and fed under the same environmental conditions for six weeks. In the test group, urine voiding was performed twice daily for six weeks, and mean residual urine volume was recorded. A cystometrogram was performed in both groups. Maximum cystometric capacity (MCC), detrusor opening pressure (DOP), and compliance of the bladder were recorded and calculated. The test group showed significant urinary retention after the surgery, both during and after the spinal shock. However, no abnormality was observed in the control group. When compared to the control group, the MCC and compliance of bladder in the test group was significantly higher than that of the test group (3.24 &#177; 2.261 mL versus 1.04 &#177; 0.571 mL; 0.43 &#177; 0.578 mL/cmH2O versus 0.032 &#177; 0.016 mL/cmH2O), whereas DOP in the test group was lower than control (20.28 &#177; 14.022 cmH2O versus 35 &#177; 13.258 cmH2O). This method of establishing an animal model of DU by the conus medullaris transection offers an excellent opportunity to understand DU&#8217;s pathophysiology in a better manner.

We present a method for establishing a detrusor underactivity model by conus medullaris transection in rats. Detrusor underactivity was successfully stimulated in these animals. The model can be used for studying urinary tract function.

The goal of the presented protocol was to establish a detrusor underactivity (DU) model in the rat through conus medullaris transection. Laminectomy was ...

Modeling Neonatal Intraventricular Hemorrhage Through Intraventricular Injection of Hemoglobin

Neonatal intraventricular hemorrhage (IVH) is a common consequence of premature birth and leads to brain injury, posthemorrhagic hydrocephalus (PHH), and lifelong neurological deficits. While PHH can be treated by temporary and permanent cerebrospinal fluid (CSF) diversion procedures (ventricular reservoir and ventriculoperitoneal shunt, respectively), there are no pharmacological strategies to prevent or treat IVH-induced brain injury and hydrocephalus. Animal models are needed to better understand the pathophysiology of IVH and test pharmacological treatments. While there are existing models of neonatal IVH, those that reliably result in hydrocephalus are often limited by the necessity for large-volume injections, which may complicate modeling of the pathology or introduce variability in the clinical phenotype observed.
Recent clinical studies have implicated hemoglobin and ferritin in causing ventricular enlargement after IVH. Here, we develop a straightforward animal model that mimics the clinical phenotype of PHH utilizing small-volume intraventricular injections of the blood breakdown product hemoglobin. In addition to reliably inducing ventricular enlargement and hydrocephalus, this model results in white matter injury, inflammation, and immune cell infiltration in periventricular and white matter regions. This paper describes this clinically relevant, simple method for modeling IVH-PHH in neonatal rats using intraventricular injection and presents methods for quantifying ventricle size post injection.

We present a model of neonatal intraventricular hemorrhage using rat pups that mimics the pathology seen in humans.

Neonatal intraventricular hemorrhage (IVH) is a common consequence of premature birth and leads to brain injury, posthemorrhagic hydrocephalus (PHH), and ...

In vivo Positron Emission Tomography to Reveal Activity Patterns Induced by Deep Brain Stimulation in Rats

Deep brain stimulation (DBS) is an invasive neurosurgical technique based on the application of electrical pulses to brain structures involved in the patient&#39;s pathophysiology. Despite the long history of DBS, its mechanism of action and appropriate protocols remain unclear, highlighting the need for research aiming to solve these enigmas. In this sense, evaluating the in vivo effects of DBS using functional imaging techniques represents a powerful strategy to determine the impact of stimulation on brain dynamics. Here, an experimental protocol for preclinical models (Wistar rats), combined with a longitudinal study [18F]-fluorodeoxyclucose positron emission tomography (FDG-PET), to assess the acute consequences of DBS on brain metabolism is described. First, animals underwent stereotactic surgery for bilateral implantation of electrodes into the prefrontal cortex. A post-surgical computerized tomography (CT) scan of each animal was acquired to verify electrode placement. After one week of recovery, a first static FDG-PET of each operated animal without stimulation (D1) was acquired, and two days later (D2), a second FDG-PET was acquired while animals were stimulated. For that, the electrodes were connected to an isolated stimulator after administering FDG to the animals. Thus, animals were stimulated during the FDG uptake period (45 min), recording the acute effects of DBS on brain metabolism. Given the exploratory nature of this study, FDG-PET images were analyzed by a voxel-wise approach based on a paired T-test between D1 and D2 studies. Overall, the combination of DBS and imaging studies allows describing the neuromodulation consequences on neural networks, ultimately helping to unravel the conundrums surrounding DBS.

In vivo Positron Emission Tomography to Reveal Activity Patterns Induced by Deep Brain Stimulation in Rats

We describe a preclinical experimental method to evaluate metabolic neuromodulation induced by acute deep brain stimulation with in vivo FDG-PET. This manuscript includes all experimental steps, from stereotaxic surgery to the application of the stimulation treatment and the acquisition, processing, and analysis of PET images.

Deep brain stimulation (DBS) is an invasive neurosurgical technique based on the application of electrical pulses to brain structures involved in the ...

A Model for Encephalomyosynangiosis Treatment after Middle Cerebral Artery Occlusion-Induced Stroke in Mice

There is no effective treatment available for most patients suffering with ischemic stroke, making development of novel therapeutics imperative. The brain's ability to self-heal after ischemic stroke is limited by inadequate blood supply in the impacted area. Encephalomyosynangiosis (EMS) is a neurosurgical procedure that achieves angiogenesis in patients with moyamoya disease. It involves craniotomy with placement of a vascular temporalis muscle graft on the ischemic brain surface. EMS has never been studied in the setting of acute ischemic stroke in mice. The hypothesis driving this study is that EMS enhances cerebral angiogenesis at the cortical surface surrounding the muscle graft. The protocol shown here describes the procedure and provides initial data supporting the feasibility and efficacy of the EMS approach. In this protocol, after 60 min of transient middle cerebral artery occlusion (MCAo), mice were randomized to either MCAo or MCAo + EMS treatment. The EMS was performed 3-4 h after occlusion. The mice were sacrificed 7 or 21 days after MCAo or MCAo + EMS treatment. Temporalis graft viability was measured using nicotinamide adenine dinucleotide reduced-tetrazolium reductase assay. A mouse angiogenesis array quantified angiogenic and neuromodulating protein expression. Immunohistochemistry was used to visualize graft bonding with brain cortex and change in vessel density. The preliminary data here suggest that grafted muscle remained viable 21 days after EMS. Immunostaining showed successful graft implantation and increase in vessel density near the muscle graft, indicating increased angiogenesis. Data show that EMS increases fibroblast growth factor (FGF) and decreases osteopontin levels after stroke. Additionally, EMS after stroke did not increase mortality suggesting that protocol is safe and reliable. This novel procedure is effective and well-tolerated and has the potential to provide information of novel interventions for enhanced angiogenesis after acute ischemic stroke.

The protocol aims to provide methods for encephalomyosynangiosis-grafting of a vascular temporalis muscle flap on the pial surface of ischemic brain tissue-for the treatment of non-moyamoya acute ischemic stroke. The approach's efficacy in increasing angiogenesis is evaluated using a transient middle cerebral artery occlusion model in mice.

There is no effective treatment available for most patients suffering with ischemic stroke, making development of novel therapeutics imperative. The ...