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 concentration.</li>
	</ol>
	</li>
</ol>
2. Animals
<ol>
	<li>Administer LPS to male Sprague-Dawley (SD) rats, aged 10 weeks (average weight: 280 &#177; 20 g) by intraperitoneal injection. 
	CAUTION: If rats are purchased from another organization, then the adaptive phase should not be less than 7 days.</li>
</ol>
3. LPS Injections
<ol>
	<li>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.</li>
	<li>Six hours later, inject the same dose of LPS injection into the rats.</li>
	<li>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.</li>
	<li>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.&#160;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 (&#62;20%), porphyrin staining that they be humanely euthanized prior to the 7 day EB study end point.</li>
</ol>
4. Sample Collection
<ol>
	<li>EB injection
	<ol>
		<li>Seven days after the first injection, anesthetize the rat by intraperitoneally according to your approved IACUC protocol.&#160;</li>
		<li>Wait 1&#8211;2 min until the rats do not show corneal reflex responses. Perform a 5&#8211;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.</li>
		<li>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.
		<ol>
			<li>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).</li>
		</ol>
		</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Gross observation
	<ol>
		<li>Perform cardiac perfusions using ice-cold 1 M phosphate buffer saline (PBS) to clear the cerebral vasculature and brains.
		<ol>
			<li>Using a scalpel, make an abdominal incision across the entire length of the diaphragm.</li>
			<li>Cut through ribs just left of the rib cage midline.</li>
			<li>Open up the thoracic cavity. Use clamps to expose the heart and to facilitate drainage of blood and other fluids.</li>
			<li>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.</li>
			<li>Release the valve to allow the flow ice-cold PBS solution (200&#8211;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.</li>
			<li>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&#8217;s nostrils or mouth, reposition the needle.</li>
		</ol>
		</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Fixation
	<ol>
		<li>Perform cardiac perfusions using ice-cold 0.9% saline solution (200&#8211;300 mL) to clear the cerebral vasculature and brains.</li>
		<li>Perform cardiac perfusions using ice-cold 4% paraformaldehyde for fixation.</li>
		<li>Decapitate the rat, isolate the brain, and immerse the brain in 20% sucrose solution for at least 6 h.</li>
		<li>Change the solution into 30% sucrose and fix for another 6 h.</li>
	</ol>
	</li>
	<li>Prepare 10 mm-thick brain tissues sections using a cryostat.</li>
</ol>
5. HE Staining
NOTE: This procedure is performed using a HE Staining Kit.
<ol>
	<li>Wash the slides in distilled water.</li>
	<li>Stain in hematoxylin solution for 8 min. Wash in running tap water for 5 min.</li>
	<li>Differentiate in 1% acid alcohol for 30 s. Wash in running tap water for 1 min.</li>
	<li>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.</li>
	<li>Rinse in 95% alcohol (10 dips).</li>
	<li>Counterstain with eosin solution for 30 s to 1 min.</li>
	<li>Dehydrate through 95% alcohol, two changes of absolute alcohol, for 5 min each.</li>
	<li>Clear in xylene for 30 s.</li>
	<li>Mount using Liu&#8217;s method9.</li>
	<li>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.</li>
</ol>
6. Perl Prussian Blue Staining
NOTE: This procedure is performed using a Perls Staining Kit.
<ol>
	<li>Wash slides with distilled water.</li>
	<li>Stain in reaction solution with equal parts mixture of ferrocyanide and hydrochloric acid for 10 min.</li>
	<li>Dehydrate, clear, mount, and analyze the slides as described in steps 5.7&#8211;5.10.</li>
</ol>
7. EB double-staining
NOTE: This procedure follows step 4.1.3.
<ol>
	<li>Wash slides with PBS three times for 5 min each.</li>
	<li>Incubate slides with 4&#39;,6-diamidino-2-phenylindole (DAPI) solution for 15 min at room temperature.</li>
	<li>Wash the slides with PBS solution three times for 5 min each, and mount slides with PBS-glycerol solution.</li>
	<li>Analyze images on a fluorescence microscope. EB deposition is indicated by red fluorescence; nuclei are indicated by blue fluorescence.</li>
</ol>
8. MRI-SWI
<ol>
	<li>Perform MRI on a 7-T magnet equipped with an actively shielded gradient system (16 cm inner diameter).</li>
	<li>Seven days after treatment, anesthetize the rats by facemask inhalation of 1.5&#8211;2.0% isoflurane using an isoflurane anesthesia system.</li>
	<li>Apply vet ointment onto the eyes of the rat to prevent dryness while under anesthesia.</li>
	<li>Keep the rats in prone position and perform an MRI-SWI scan.</li>
	<li>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&#176;, field of view (FOV) 35 mm &#215; 35 mm, acquisition matrix 384 &#215; 384, to acquire 1 mm-thick slices.</li>
	<li>Identify CMHs in MRI-SWI according to Greenberg et al.11, which included the following criteria: (1) a diameter of &#8804;10 mm, and (2) circular, isolated, and low-signal intensity spots.</li>
	<li>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.</li>
</ol>

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The authors have nothing to disclose.

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 cerebral amyloid angiopathy (CAA) plays a major role in the etiology of CMHs. Fisher et al. reported a CMHs mouse model that was induced by LPS injection9, which revealed that CMHs are caused by inflammation2,13,14. Similarly, we have successfully developed a sub-acute CMHs model in SD rats using LPS injection and have found that nitric oxide synthase (NOS), especially neural NOS and endothelial NOS, are involved in the etiology of CMHs, which result in inflammation10.
The critical step of our protocol is the LPS injection, which has been extensively used in developing murine models of neuropsychiatric disorders such as depression15, schizophrenia16, Parkinson&#39;s disease17, Alzheimer&#39;s disease18, and Prion disease19. To our knowledge, our use of a single dose (1 mg/kg) is much higher than that was applied in other studies15,16,17,18,19. This might be a plausible reason for the mortality in the current study. LPS dose modification for CMHs induction may be an interesting research topic, as different doses or injection schedule have been shown to influence the number, size, distribution, and progression of CMHs2. A previous study has shown that the administration of LPS to mice at a dose of 3 mg/kg induces acute CMHs2.
Although the development of different animal models has facilitated research investigations on CMHs, we have to admit that those animal models do not simulate the process of clinical CMHs. For example, in our rat model, as well as Fisher&#39;s mice model, CMHs were observed in the cerebellum, although most were observed in lobar regions (mainly CAA-related) and deep- or infra-tentorial locations (mainly hypertension-related)20,21. We have no explanation for this discrepancy in distribution, although both Fisher and our team attribute this observation to the vulnerability of cerebellar blood vessels to inflammation.
In most clinical cases, CMHs result from more than one etiological factor, although inflammation plays an important role in its etiology. Studies focusing on multiple factors that induce CMHs, instead of pure inflammation-induced CMHs, can thus be more helpful in simulating this condition. The LPS injection model could be used with other underlying factors to investigate the mechanism of CMHs. For example, Fisher et al. have conducted a preliminary, yet interesting step with aging mice injected with LPS, and demonstrated that aging is a key factor that could make the brain more susceptible to inflammation-induced CMHs14. In our opinion, the significance of the present model is its compatibility with other factors in animal models for aging14, trauma22, as well as chronic diseases such as hypercholesterolemia23, or transgenic models such as hypertension24 because of the simplicity, time-effectiveness, and stability of this protocol.
Patients with CMHs show cognitive declines, neuropsychiatric manifestations, and vertigo, which are associated with the distribution of CMHs. One limitation of the present protocol is that after LPS injection, we could not rule out the effects of peripheral inflammation on the behavior of the rats, although a decrease in social behavior and burrowing (intrinsic natural activity of rodent reflecting the impairment of daily activities) were observed. Further studies on ways to improve our method of generating a CMHs animal model, e.g., method of injecting LPS or schedule of observation, are warranted.
Nevertheless, this simple, cost-effective, and stable CMHs murine model induced by LPS injection may be utilized by researchers in elucidating the etiology of CMHs.

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&amp;rsquo;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&#039;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 staining for the detection of EB deposition derived from BBB leakage (Figure 3, left panel); (4) MRI-SWI detection of CMHs hypointense signals (Figure 4, left panel).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58423/58423fig1.jpg" /> 
Figure 1: Gross observation of surface CMHs. Upper panel (A): Rat model; Lower panel (B): Control animal. Red arrows signify surface CMHs. Modified and reused with permission10. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58423/58423fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58423/58423fig2.jpg" /> 
Figure 2: HE staining and Prussian staining images. (A) Reflected rat model. Red blood cells were found outside the capillaries in upper panel, and ferric iron points derived from lysis of red blood cells were detected in lower panel; (B) Reflected control animal. Neither red blood cells nor ferric iron points were found. Scale bars =&#160;100 &#181;m (left panel of A&#160;and B). Scale bars =&#160;50 &#181;m&#160;(right panel of&#160;A&#160;and B). Modified and reused with permission10. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58423/58423fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58423/58423fig3.jpg" /> 
Figure 3. Double labeled EB deposition fluorescence imaging. Left panel: Rat model. Amount of EB molecules leaked from injured BBB were detected (in red); Right panel: Control animal. No EB molecules were detected. DAPI in blue was used as mount medium. Scale bars =&#160;100 &#181;m. Modified and reused with permission10. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58423/58423fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58423/58423fig4.jpg" /> 
Figure 4. MRI-SWI images. Left panel (A): Rat model. Black arrows signify hypointense signals of CMHs; Right panel (B): Control animal. No hypointense signals were found. Modified and reused with permission10. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58423/58423fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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, ...

sub-acute-cerebral-microhemorrhages-induced-lipopolysaccharide

Nanyang Technological University

Research

JoVE Journal

Neuroscience

6.3K Views.  Second Hospital of Shanxi Medical University. Deze methode kan helpen bij het beantwoorden van belangrijke vragen in cerebrale micro-bloeding gebied, zoals de etiologie, distributie, en detectie van cerebrale micro-bloeding en ga zo maar door. Het belangrijkste voordeel van deze techniek is dat LPS-injecties stabiel ontstekingen veroorzaken. Bovendien is lps-injectie vrij eenvoudig, zuinig en kosteneffectief.Hoewel deze methode inzicht kan geven in de inductie van de hersenbloeding, kan het ook worden toegepast op andere hersenaan...

Video: Sub-acute Cerebral Microhemorrhages Induced by Lipopolysaccharide Injection in Rats

A Low Mortality Rat Model to Assess Delayed Cerebral Vasospasm After Experimental Subarachnoid Hemorrhage

Objective: To characterize and establish a reproducible model that demonstrates delayed cerebral vasospasm after aneurysmal subarachnoid hemorrhage (SAH) in rats, in order to identify the initiating events, pathophysiological changes and potential targets for treatment.
Methods: Twenty-eight male Sprague-Dawley rats (250 - 300 g) were arbitrarily assigned to one of two groups - SAH or saline control. Rat subarachnoid hemorrhage in the SAH group (n=15) was induced by double injection of autologous blood, 48 hr apart, into the cisterna magna. Similarly, normal saline (n=13) was injected into the cisterna magna of the saline control group. Rats were sacrificed on day five after the second blood injection and the brains were preserved for histological analysis. The degree of vasospasm was measured using sections of the basilar artery, by measuring the internal luminal cross sectional area using NIH Image-J software. The significance was tested using Tukey/Kramer's statistical analysis.
Results: After analysis of histological sections, basilar artery luminal cross sectional area were smaller in the SAH than in the saline group, consistent with cerebral vasospasm in the former group. In the SAH group, basilar artery internal area (.056 &mu;m &plusmn; 3) were significantly smaller from vasospasm five days after the second blood injection (seven days after the initial blood injection), compared to the saline control group with internal area (.069 &plusmn; 3; p=0.004). There were no mortalities from cerebral vasospasm.
Conclusion: The rat double SAH model induces a mild, survivable, basilar artery vasospasm that can be used to study the pathophysiological mechanisms of cerebral vasospasm in a small animal model. A low and acceptable mortality rate is a significant criterion to be satisfied for an ideal SAH animal model so that the mechanisms of vasospasm can be elucidated 7, 8. Further modifications of the model can be made to adjust for increased severity of vasospasm and neurological exams.

Aneurysmal subarachnoid hemorrhage (SAH) is bleeding that occurs into the subarachnoid space when an aneurysm ruptures. While the morbidity and mortality from this event has been on a decline due to improved treatment approaches, the risk of vasospasm after subarachnoid hemorrhage continues to be the same as it was several years ago. The importance of establishing a comprehensive and reproducible animal model to identify initiating events of cerebral vasospasm has been the focus of research since the first use of rats in an experimental vasospasm model in 1979 by Barry et al. Early work in rats demonstrated that a single injection of autologous blood into the cisterna magna led to acute (within minutes) but not delayed cerebral vasospasm 3, 6, 14. Here we characterize a low mortality SAH rat model that results in reproducible delayed vasospasm.

Objective: To characterize and establish a reproducible model that demonstrates delayed cerebral vasospasm after aneurysmal subarachnoid hemorrhage (SAH) ...

Medicine

Modeling Intracerebral Hemorrhage in Mice: Injection of Autologous Blood or Bacterial Collagenase 

Spontaneous intracerebral hemorrhage (ICH) defines a potentially life-threatening neurological malady that accounts for 10-15% of all stroke-related hospitalizations and for which no effective treatments are available to date1,2. Because of the heterogeneity of ICH in humans, various preclinical models are needed to thoroughly explore prospective therapeutic strategies3. Experimental ICH is commonly induced in rodents by intraparenchymal injection of either autologous blood or bacterial collagenase4. The appropriate model is selected based on the pathophysiology of hemorrhage induction and injury progression. The blood injection model mimics a rapidly progressing hemorrhage. Alternatively, bacterial collagenase enzymatically disrupts the basal lamina of brain capillaries, causing an active bleed that generally evolves over several hours5. Resultant perihematomal edema and neurofunctional deficits can be quantified from both models. In this study, we described and evaluated a modified double injection model of autologous whole blood6 as well as an ICH injection model of bacterial collagenase7, both of which target the basal ganglia (corpus striatum) of male CD-1 mice. We assessed neurofunctional deficits and brain edema at 24 and 72 hr after ICH induction. Intrastriatal injection of autologous blood (30 &mu;l) or bacterial collagenase (0.075U) caused reproducible neurofunctional deficits in mice and significantly increased brain edema at 24 and 72 hr after surgery (p&lt;0.05). In conclusion, both models yield consistent hemorrhagic infarcts and represent basic methods for preclinical ICH research.

Modeling Intracerebral Hemorrhage in Mice: Injection of Autologous Blood or Bacterial Collagenase

Clinically relevant animal models of intracerebral hemorrhage (ICH) are needed to extend our knowledge of hemorrhagic stroke and to examine novel therapeutic strategies. In this study, we describe and evaluate two ICH models that implement unilateral injections of either autologous whole blood or bacterial collagenase into the basal ganglia (corpus striatum) of mice.

Spontaneous intracerebral hemorrhage (ICH) defines a potentially life-threatening neurological malady that accounts for 10-15% of all stroke-related ...

Intrastriatal Injection of Autologous Blood or Clostridial Collagenase as Murine Models of Intracerebral Hemorrhage

Intracerebral hemorrhage (ICH) is a common form of cerebrovascular disease and is associated with significant morbidity and mortality. Lack of effective treatment and failure of large clinical trials aimed at hemostasis and clot removal demonstrate the need for further mechanism-driven investigation of ICH. This research may be performed through the framework provided by preclinical models. Two murine models in popular use include intrastriatal (basal ganglia) injection of either autologous whole blood or clostridial collagenase. Since, each model represents distinctly different pathophysiological features related to ICH, use of a particular model may be selected based on what aspect of the disease is to be studied. For example, autologous blood injection most accurately represents the brain&#39;s response to the presence of intraparenchymal blood, and may most closely replicate lobar hemorrhage. Clostridial collagenase injection most accurately represents the small vessel rupture and hematoma evolution characteristic of deep hemorrhages. Thus, each model results in different hematoma formation, neuroinflammatory response, cerebral edema development, and neurobehavioral outcomes. Robustness of a purported therapeutic intervention can be best assessed using both models. In this protocol, induction of ICH using both models, immediate post-operative demonstration of injury, and early post-operative care techniques are demonstrated. Both models result in reproducible injuries, hematoma volumes, and neurobehavioral deficits. Because of the heterogeneity of human ICH, multiple preclinical models are needed to thoroughly explore pathophysiologic mechanisms and test potential therapeutic strategies.

Preclinical models of intracerebral hemorrhage are utilized to mimic certain aspects of clinical disease. Thus, mechanisms of injury and potential therapeutic strategies may be explored. In this protocol, two models of intracerebral hemorrhage are described, intrastriatal (basal ganglia) injections of autologous blood or collagenase.

Intracerebral hemorrhage (ICH) is a common form of cerebrovascular disease and is associated with significant morbidity and mortality. Lack of effective ...

The Rabbit Blood-shunt Model for the Study of Acute and Late Sequelae of Subarachnoid Hemorrhage: Technical Aspects

Early brain injury and delayed cerebral vasospasm both contribute to unfavorable outcomes after subarachnoid hemorrhage (SAH). Reproducible and controllable animal models that simulate both conditions are presently uncommon. Therefore, new models are needed in order to mimic human pathophysiological conditions resulting from SAH.
This report describes the technical nuances of a rabbit blood-shunt SAH model that enables control of intracerebral pressure (ICP). An extracorporeal shunt is placed between the arterial system and the subarachnoid space, which enables examiner-independent SAH in a closed cranium. Step-by-step procedural instructions and necessary equipment are described, as well as technical considerations to produce the model with minimal mortality and morbidity. Important details required for successful surgical creation of this robust, simple and consistent ICP-controlled SAH rabbit model are described.

The experimental intracranial pressure-controlled blood shunt subarachnoid hemorrhage (SAH) model in the rabbit combines the standard procedures &#8212; subclavian artery cannulation and transcutaneous cisterna magna puncture, which enables close mimicking of human pathophysiological conditions after SAH. We present step-by-step instructions and discuss key surgical points for successful experimental SAH creation.

Early brain injury and delayed cerebral vasospasm both contribute to unfavorable outcomes after subarachnoid hemorrhage (SAH). Reproducible and ...

Modeling Encephalopathy of Prematurity Using Prenatal Hypoxia-ischemia with Intra-amniotic Lipopolysaccharide in Rats

Encephalopathy of prematurity (EoP) is a term that encompasses the central nervous system (CNS) abnormalities associated with preterm birth. To best advance translational objectives and uncover new therapeutic strategies for brain injury associated with preterm birth, preclinical models of EoP must include similar mechanisms of prenatal global injury observed in humans and involve multiple components of the maternal-placental-fetal system. Ideally, models should produce a similar spectrum of functional deficits in the mature animal and recapitulate multiple aspects of the pathophysiology. To mimic human systemic placental perfusion defects, placental underperfusion and/or chorioamnionitis associated with pathogen-induced inflammation in early preterm birth, we developed a model of prenatal transient systemic hypoxia-ischemia (TSHI) combined with intra-amniotic lipopolysaccharide (LPS). In pregnant Sprague Dawley rats, TSHI via uterine artery occlusion on embryonic day 18 (E18) induces a graded placental underperfusion defect associated with increasing CNS damage in the fetus. When combined with intra-amniotic LPS injections, placental inflammation is increased and CNS damage is compounded with associated white matter, gait and imaging abnormalities. Prenatal TSHI and TSHI+LPS prenatal insults meet several of the criteria of an EoP model including recapitulating the intrauterine insult, causing loss of neurons, oligodendrocytes and axons, loss of subplate, and functional deficits in adult animals that mimic those observed in children born extremely preterm. Moreover, this model allows for the dissection of inflammation induced by divergent injury types.

Encephalopathy of prematurity encompasses the central nervous system abnormalities associated with injury from preterm birth. This report describes a clinically relevant rat model of in utero transient systemic hypoxia-ischemia and intra-amniotic lipopolysaccharide administration (LPS) that mimics chorioamnionitis, and the related impact of infectious stimuli and placental underperfusion on CNS development.

Encephalopathy of prematurity (EoP) is a term that encompasses the central nervous system (CNS) abnormalities associated with preterm birth. To best ...

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 ...

Pre-Chiasmatic, Single Injection of Autologous Blood to Induce Experimental Subarachnoid Hemorrhage in a Rat Model

Despite advances in treatment over the last decades, subarachnoid hemorrhage (SAH) continues to carry a high burden of morbidity and mortality, largely afflicting a fairly young population. Several animal models of SAH have been developed to investigate the pathophysiological mechanisms behind SAH and to test pharmacological interventions. The pre-chiasmatic, single injection model in the rat presented in this article is an experimental model of SAH with a predetermined blood volume. Briefly, the animal is anesthetized, intubated, and kept under mechanical ventilation. Temperature is regulated with a heating pad. A catheter is placed in the tail artery, enabling continuous blood pressure measurement as well as blood sampling. The atlantooccipital membrane is incised and a catheter for pressure recording is placed in the cisterna magna to enable intracerebral pressure measurement. This catheter can also be used for intrathecal therapeutic interventions. The rat is placed in a stereotaxic frame, a burr hole is drilled anteriorly to the bregma, and a catheter is inserted through the burr hole and placed just anterior to the optic chiasm. Autologous blood (0.3 mL) is withdrawn from the tail catheter and manually injected. This results in a rise of intracerebral pressure and a decrease of cerebral blood flow. The animal is kept sedated for 30 min and given subcutaneous saline and analgesics. The animal is extubated and returned to its cage. The pre-chiasmatic model has a high reproducibility rate and limited variation between animals due to the pre-determined blood volume. It mimics SAH in humans making it a relevant model for SAH research.

Subarachnoid hemorrhage continues to carry a high burden of mortality and morbidity in man. To facilitate further research into the condition and its pathophysiology, a pre-chiasmatic, single injection model is presented.

Despite advances in treatment over the last decades, subarachnoid hemorrhage (SAH) continues to carry a high burden of morbidity and mortality, largely ...

Modeling Posthemorrhagic Hydrocephalus of Prematurity in Rats

Posthemorrhagic hydrocephalus of prematurity (PHHP) is a serious sequela of severe intraventricular hemorrhage (IVH) in very preterm infants less than 32 weeks gestational age (GA). PHHP is defined by the accumulation of cerebrospinal fluid (CSF) associated with clinical symptoms of elevated intracranial pressure (ICP). Infants with PHHP suffer lifelong shunt dependence, with half requiring repeat surgery in the first year of life and many requiring multiple additional surgeries throughout the lifespan. Prenatal chorioamnionitis predisposes preterm infants to severe IVH and the need for surgical treatment of PHHP trends with neonatal sepsis. These clinical features suggest that systemic inflammation is an integral component of PHHP pathophysiology. 
Here, we define an animal model that recapitulates all clinical aspects and essential features of PHHP in rats. The goal of this protocol is to illustrate how in utero chorioamnionitis and postnatal IVH using lysed red blood cells can be combined to yield PHHP. This preclinical approach yields progressive macrocephaly and domed craniums, elevated intracranial pressure, and ventriculomegaly that can be detected via magnetic resonance imaging (MRI) or via microscopy. In addition to sustained disruption in CSF dynamics, rats also have cognitive delay and functional disability into adulthood. Accordingly, this preclinical platform facilitates unique and unparalleled translational studies of PHHP that can incorporate molecular, cellular, biochemical, histologic, imaging, and functional outcome measures. It can also be used for rigorous analysis of the choroid plexus, ependymal motile cilia, and glymphatic system in parallel. Last, it can also be an invaluable preclinical tool for the investigation of novel surgical intervention strategies and non-surgical therapeutic approaches for the treatment of hydrocephalus.

Posthemorrhagic hydrocephalus of prematurity (PHHP) can be modeled in neonatal rats by combining chorioamnionitis and intraventricular hemorrhage. The combination of these prenatal and postnatal events accurately recapitulates the clinical hallmarks of PHHP, including macrocephaly, ventriculomegaly, and elevated intracranial pressure, through the lifespan.

Posthemorrhagic hydrocephalus of prematurity (PHHP) is a serious sequela of severe intraventricular hemorrhage (IVH) in very preterm infants less than 32 ...

Induction of Cerebral Microhemorrhages in a Rat Model Using Lipopolysaccharide

Source: Li, D., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/58423">Sub-acute Cerebral Microhemorrhages Induced by Lipopolysaccharide Injection in Rats.</a> J. Vis. Exp. (2018).
This video demonstrates the process of inducing blood-brain barrier (BBB) disruption and cerebral microhemorrhages in a rat model using lipopolysaccharide (LPS). It highlights the sequence of immune responses that lead to increased BBB permeability and subsequent brain tissue damage.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

JoVE Encyclopedia of Experiments

Neuropathology

Cerebrovascular Diseases

Detection of Cerebral Microhemorrhages in a Rat Model

Source: Li, D., Zh&#224;o, H., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/58423">Sub-acute Cerebral Microhemorrhages Induced by Lipopolysaccharide Injection in Rats.</a> J. Vis. Exp. (2018).
This video demonstrates an assay to detect Cerebral microhemorrhages (CMHs) in a rat model. CMHs are brain bleeds due to blood-brain barrier disruption, identified by leaked red blood cells (RBCs). In a rat model, fixed brain tissue is cryoprotected, sectioned, and stained with hematoxylin-eosin for RBCs, allowing microscopic visualization of CMHs. Leaked RBCs appear as red-orange under the microscope.

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Materials

	Preparation of ...

Sub-acute Cerebral Microhemorrhages Induced by Lipopolysaccharide Injection in Rats

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