Name: massive pontine hemorrhage by dual injection of autologous blood
Uploaded: 2021-05-29T12:30:00
Description: Watch this Scientific Journal Video about Massive Pontine Hemorrhage by Dual Injection of Autologous Blood at JoVE.com

This study was financially supported by the National Science Foundation of China (81471181 and 81870933) and the Opening Lab Program of Guangzhou Medical University (0506308) to Y Jiang, and by the National Science Foundation of China (81701471) and the Scientific Program of Guangzhou Municipal Health Commission (20191A011083) to Z Qiu, and by the National Science Foundation of China (81501009) to L Wu.

The protocol was reviewed and approved by the Institutional Animal Care and Use Committee of the Second Affiliated Hospital of Guangzhou Medical University. Rats were provided by the Animal Center of Southern Medical University. The experimental design is shown in Figure 1.
1. Animal and instruments
<ol>
	<li>Use 8-week-old male Sprague-Dawley rats weighing 250 &#177; 10 g.</li>
	<li>House the rats for at least 7 days before surgery under controlled environmenta...

<ol>
	<li>Charidimou, 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=Brain+hemorrhage+recurrence,+small+vessel+disease+type,+and+cerebral+microbleeds:+A+meta-analysis.">Brain hemorrhage recurrence, small vessel disease type, and cerebral microbleeds: A meta-analysis.</a> Neurology. 89 (8), 820-829 (2017).</li><li>Tao, C., 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+novel+brainstem+hemorrhage+model+by+autologous+blood+infusion+in+Rat:+White+Matter+Injury,+Magnetic+Resonance+Imaging,+and+Neurobehavioral+features.">A novel brainstem hemorrhage model by autologous blood infusion in Rat: White Matter Injury, Magnetic Resonance Imaging, and Neurobehavioral features.</a> Journal of Stroke and Cerebrovascular Diseases. 25 (5), 1102-1109 (2016).</li><li>Ichimura, 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=Surgical+treatment+for+primary+brainstem+hemorrhage+to+improve+postoperative+functional+outcomes.">Surgical treatment for primary brainstem hemorrhage to improve postoperative functional outcomes.</a> World Neurosurgery. 120, 1289-1294 (2018).</li><li>Behrouz, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prognostic+factors+in+pontine+haemorrhage:+A+systematic+review.">Prognostic factors in pontine haemorrhage: A systematic review.</a> European Stroke Journal. 3 (2), 101-109 (2018).</li><li>Guo, 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=Brainstem+iron+overload+and+injury+in+a+rat+model+of+brainstem+hemorrhage.">Brainstem iron overload and injury in a rat model of brainstem hemorrhage.</a> Journal of Stroke and Cerebrovascular Diseases. 29 (8), 104956 (2020).</li><li>Chung, Y., Haines, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+brain+stem+surgery.">Experimental brain stem surgery.</a> Neurosurgery Clinics of North America. 4 (3), 405-414 (1993).</li><li>Lekic, T., Tang, J., Zhang, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rat+model+of+pontine+hemorrhage.">A rat model of pontine hemorrhage.</a> Acta Neurochirurgica Supplement. 105, 135-137 (2008).</li><li>Lekic, T., 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=Evaluation+of+the+hematoma+consequences,+neurobehavioral+profiles,+and+histopathology+in+a+rat+model+of+pontine+hemorrhage.">Evaluation of the hematoma consequences, neurobehavioral profiles, and histopathology in a rat model of pontine hemorrhage.</a> Journal of Neurosurgery. 118 (2), 465-477 (2013).</li><li>Shrestha, B. 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=Rat+brainstem+hemorrhage+model:+Key+points+to+success+in+modeling.">Rat brainstem hemorrhage model: Key points to success in modeling.</a> World Neurosurgery. 117, 106-116 (2018).</li><li>Luo, M., Tang, X., Zhu, J., Qiu, Z., Jiang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+acute+pontine+infarction+in+rats+by+electrical+stimulation.">Establishment of acute pontine infarction in rats by electrical stimulation.</a> Journal of Visualized Experiments. (162), (2020).</li><li>Wu, L., 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=Keep+warm+and+get+success:+The+role+of+postischemic+temperature+in+the+mouse+middle+cerebral+artery+occlusion+model.">Keep warm and get success: The role of postischemic temperature in the mouse middle cerebral artery occlusion model.</a> Brain Research Bulletin. 101, 12-17 (2014).</li></ol>

In the present study, we provided a protocol to generate a massive pontine hemorrhage rat model. This model can be used for the research on the pathophysiological mechanism and prognosis of massive pontine hemorrhage.
Throughout the experiment, 25 rats were used, of which only one died. The verification of MRI, gross anatomy, and the HE staining indicated that this method had a very low mortality rate and a high success rate. To establish massive pontine hemorrhage model, two problems must be ...

Intracerebral hemorrhage accounts for one-fifth of stroke patients. The prognosis of intracerebral hemorrhage depends on the speed, volume, and location of bleeding1,2. Compared to the forebrain hemorrhage, the brainstem hemorrhage has higher mortality and morbidity3. About 40% of brainstem hemorrhage occurs in the pons4. The etiology and pathophysiology of pontine hemorrhage are quite different and less studied than forebrain hemorrhage5.
There are two kinds of pontine hemorrhage animal models. One is spontaneous hemorrhage model induced by infusion of bacterial collagenase in the pons6,7,8. The biggest advantage of this model is that the bleeding is spontaneous. However, collagenase can only induce a small volume of pontine hemorrhage. Besides, collagenase might cause other injuries to the brain. The other model is induced by stereotactic injection of autologous blood into the pons9. The advantage of this model is that it is easy to master with a high success rate. Theoretically, researchers could inject any volume of blood into any location of pons. However, due to the back-leakage through the needle route, the injected volume is limited. Recently, the double-injection method has been promoted to reduce the back-leakage9. This method injects autologous blood twice with a 20-minute interval between the injections. The double-injection method is applied to induce mild (30 &#181;L) and moderate (60 &#181;L) pontine hemorrhage but not massive pontine hemorrhage. In the clinic, the majority of pontine hemorrhage patients with poor prognosis have massive hemorrhage (more than 10 mL).
In the previous study, we provided a protocol to establish a pontine ischemic stroke model in rat10. In this study, we modify the existing dual injection method, and provide a detailed protocol to induce massive pontine hemorrhage in a rat by dual injection of 100 &#181;L autologous blood at two different locations in the pons.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100ml Saline solution</td>
			<td>Guangdong yixiang</td>
			<td>191222201 C1</td>
			<td>Preparing heparin diluent</td>
		</tr>
		<tr>
			<td>100&#956;l Microinjector</td>
			<td>Shanghai Gaoge</td>
			<td></td>
			<td>Injection of autologous blood</td>
		</tr>
		<tr>
			<td>1ml Syringe</td>
			<td>Jiangsu Zhiyu</td>
			<td>20191014</td>
			<td>Withdraw autologous blood from the tail vein</td>
		</tr>
		<tr>
			<td>75% Alcohol</td>
			<td>Shandong Lierkang</td>
			<td></td>
			<td>Disinfection of rat tail</td>
		</tr>
		<tr>
			<td>Adhesive tape</td>
			<td>Shanghai Jinzhong</td>
			<td></td>
			<td>Surgicl instruments</td>
		</tr>
		<tr>
			<td>Animal anesthesia system</td>
			<td>RWD</td>
			<td>R510-31S-6</td>
			<td>Inducing and maintaining anesthesia</td>
		</tr>
		<tr>
			<td>Balance beam</td>
			<td>Jiangsu Saiangsi</td>
			<td></td>
			<td>For neurological deficit scores</td>
		</tr>
		<tr>
			<td>Blades</td>
			<td>Shanghai Feiying</td>
			<td>74-C</td>
			<td>For gross anatomy</td>
		</tr>
		<tr>
			<td>Bone cement</td>
			<td>Shanghai Xinshiji</td>
			<td>20180306</td>
			<td>Surgicl instruments</td>
		</tr>
		<tr>
			<td>Brain tank</td>
			<td>Shenzhen LEIYEA</td>
			<td></td>
			<td>For gross anatomy</td>
		</tr>
		<tr>
			<td>Butorphanol tartrate</td>
			<td>Jiangsu Hengrui</td>
			<td></td>
			<td>For pain management</td>
		</tr>
		<tr>
			<td>Electric cranial drill</td>
			<td>Nanjing&#160; Darwin biotechnology</td>
			<td>20180090018</td>
			<td>Making a burr hole on the skull</td>
		</tr>
		<tr>
			<td>EP tube</td>
			<td>Nantong Surui</td>
			<td></td>
			<td>Transfer autologous blood</td>
		</tr>
		<tr>
			<td>Erythromycin eye cream</td>
			<td>Yunnan pharmacy</td>
			<td></td>
			<td>Eyes protection</td>
		</tr>
		<tr>
			<td>HE dye liquor</td>
			<td>Solarbio</td>
			<td>G1120</td>
			<td>For HE staining</td>
		</tr>
		<tr>
			<td>Heating&#160;pad</td>
			<td>Dangerous Jungle</td>
			<td>JR01</td>
			<td>Keeping warm</td>
		</tr>
		<tr>
			<td>Heparin sodium injection</td>
			<td>Chengdu Haitong Pharmacal Company</td>
			<td>190701</td>
			<td>Preparing heparin diluent</td>
		</tr>
		<tr>
			<td>IndoPhors</td>
			<td>Guoyao of China</td>
			<td></td>
			<td>Sterilization</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>RWD</td>
			<td>20080701</td>
			<td>Inducing and maintaining anesthesia</td>
		</tr>
		<tr>
			<td>Light dark box</td>
			<td>Jiangsu Saiangsi</td>
			<td></td>
			<td>For neurological deficit scores</td>
		</tr>
		<tr>
			<td>Micro-injection pump</td>
			<td>Baoding Leifu</td>
			<td>TFD03-01-C</td>
			<td>Injection of autologous blood</td>
		</tr>
		<tr>
			<td>MRI system</td>
			<td>Philips</td>
			<td></td>
			<td>Confirmation of infarction in vivo</td>
		</tr>
		<tr>
			<td>Needle holder</td>
			<td>Shanghai Jinzhong</td>
			<td>J32020</td>
			<td>Surgicl instruments</td>
		</tr>
		<tr>
			<td>Penicilin</td>
			<td>Guoyao of China</td>
			<td></td>
			<td>Infection Prevention</td>
		</tr>
		<tr>
			<td>Q-tips</td>
			<td>Jiangxi Songhe</td>
			<td></td>
			<td>Surgicl instruments</td>
		</tr>
		<tr>
			<td>Scalp heedle</td>
			<td>Jiangxi Hongda</td>
			<td>20200313</td>
			<td>Withdraw autologous blood from the tail vein</td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>Shanghai Kaiyuan</td>
			<td>170902</td>
			<td>Surgicl instruments</td>
		</tr>
		<tr>
			<td>Shearing scissors</td>
			<td>Shanghai Jinzhong</td>
			<td>Y00040</td>
			<td>Surgicl instruments</td>
		</tr>
		<tr>
			<td>Stereotaxic apparatus</td>
			<td>RWD</td>
			<td>900-00001-00</td>
			<td>for surgical positioning</td>
		</tr>
		<tr>
			<td>Surgical towel</td>
			<td>Xinxiang Huakangweicai</td>
			<td>20070601</td>
			<td>Surgicl instruments</td>
		</tr>
		<tr>
			<td>Suture needle</td>
			<td>Shanghai Jinzhong</td>
			<td></td>
			<td>Surgicl instruments</td>
		</tr>
		<tr>
			<td>Suture scissors</td>
			<td>Shanghai Jinzhong</td>
			<td>J25041</td>
			<td>Surgicl instruments</td>
		</tr>
		<tr>
			<td>Tissue holding forcepts</td>
			<td>Shanghai Jinzhong</td>
			<td>J31080</td>
			<td>Surgicl instruments</td>
		</tr>
	</tbody>
</table>

massive pontine hemorrhage by dual injection of autologous blood

We provide a protocol to establish a massive pontine hemorrhage model in a rat. Rats weighing about 250 grams were used in this study. One hundred microliters of autologous blood was taken from the tail vein and stereotaxically injected into the pons. The injection process was divided into 2 steps: First, 10 &#181;L of blood was injected into a specific location, anteroposterior position (AP) -9.0 mm; lateral (Lat) 0 mm; vertical (Vert) -9.2 mm, followed by a second injection of the residual blood located at AP -9.0 mm; Lat 0 mm; Vert -9.0 mm with a 20-minute interval. The balance beam test, limb placement test, and the modified Voestch neuroscore were used to evaluate neurological function. Magnetic Resonance Imaging (MRI) was used to assess the volume of hemorrhage in vivo. The symptoms of this model were in line with patients with massive pontine hemorrhage.

A total of 25 animals were used, 3 for control, 6 for 30 &#181;L, 6 for 60 &#181;L, and 10 for 100 &#181;L blood injections. One rat that received a 100 &#181;L injection of autologous blood (1/10) died within 24 hours after surgery.
Behavioral tests were conducted on Day 1, Day 3, Day 7 and Day 14 after surgery. The scores for the control group and blood-injection groups on different timepoints after surgery are presented in <s...

Watch this Scientific Journal Video about Massive Pontine Hemorrhage by Dual Injection of Autologous Blood at JoVE.com

Massive Pontine Hemorrhage by Dual Injection of Autologous Blood

We present a protocol to establish a massive pontine hemorrhage model in a rat via dual injection of autologous blood.

We provide a protocol to establish a massive pontine hemorrhage model in a rat. Rats weighing about 250 grams were used in this study. One hundred ...

We provide a protocol to establish massive pontine hemorrhage in a rat. This model is easy to establish, repeatable, and highly successful. Distilled injection of autologous rat method could be used to make hemorrhage in any of the brain regions.People who want to try these techniques should learn how to use a stereotaxic frame, and be familiar with pontine anatomy. Begin by placing a surgical drape over the mouse. Make an incision with a scalpel along the marked midline.Use a cotton swab to remove any potential blood. Then place a piece of forceps on each side of the scalp flap to expose the skull. Dip a cotton swab in 0.9%saline and gently remove the connective tissues from the skull bone.Use a marker pen to mark the central point of the bregma as the origin point. Perform a craniotomy using a micro drill, proceeding carefully. When finished remove the micro drill from the stereotaxic frame.Put a 100 microliter Hamilton syringe into the stereotaxic holder. Turn on the injection pump switch. Click the rapid inhalation button.Then aspirate heparin solution to 100 microliters and to drain it completely. Apply chlorhexidine and 75%alcohol surgical scrub to the whole tale from root to tip at least three times to disinfect the skin, soften the horniness and dilate the tail vein to increase the success rate of injection. Attach a scalp acupuncture to a one milliliter syringe, then insert the scalp acupuncture into a lateral tail vein three centimeters from the tail tip and to take 150 microliters of blood.Remove the scalp acupuncture from the one milliliter syringe and transfer the blood into a tube. Working quickly to prevent blood clotting. Change surgical gloves.Select the withdraw mode. Set the volume to 100 microliters and set the speed to 200 microliters per minute. Click the run button, and aspirate 100 microliters of blood into the Hamilton syringe.Switch to the infuse mode and set the speed to one microliter per minute. Advance the syringe until the tip reaches 9.2 millimeters below the surface of the brain and inject the first 10 microliters of blood. Click on the run button.Stop the injection and leave the syringe in position for 20 minutes to prevent blood from flowing into the subarachnoid space. Retract the syringe until the tip arrives at nine millimeters below the surface of the brain, then restart injection at the same speed of one microliter per minute until the residual blood has been injected completely. Leave the syringe in position for 10 minutes to avoid blood backflow.Remove the syringe from the brain slowly. Then use bone cement to cover the craniotomy hole. When the cement dries suture the wound with a four zero polyamide suture filament.After sewing three or four stitches tie 2 one-one standard surgical knots. Observe the rat every 15 minutes until it fully recovers from anesthesia, then return it to the original cage with a heating pad underneath. Provide the rat free access to food and water.A total of 25 animals were used for control 30 microliter, 60 microliter, and 100 microliter blood injections. Behavioral tests were conducted on days one, three, seven and 14 after surgery. Pontine hemorrhage caused neurological deficits like diminished corneal reflex and circling.Injection of 100 microliters of blood also induced the myotonia. MRI Scanning was performed 24 hours after surgery. In the blood injection groups, Hemorrhage was detected as a hypointense rim with an ISO to slightly hyperintense core in the Basiler part of the ponds.The volume of hemorrhage increased with the injection volume. The rats were sacrificed at 24 hours and 14 days after surgery. And two millimeter thick sections were made.Hemorrhage was detected surrounding the injection site and distributing in the base of the ponds. There was a slight edema around the hemorrhage in the 100 microliter blood injection group. Some of the rats were sacrificed three days after surgery and paraffin sections were made for HE staining.Results showed that in the blood injection groups inflammatory cells enriched in the para hemorrhage zone the hemoglobin remained contained with intact red blood cells. When attempting this protocol make sure to remember the injection position. This method can be used to induce a hemorrhage in any specific cerebral region.

massive-pontine-hemorrhage-by-dual-injection-of-autologous-blood

Research

JoVE Journal

Neuroscience

Gene Delivery to Postnatal Rat Brain by Non-ventricular Plasmid Injection and Electroporation

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals are not viable in those cases where the modified gene is expressed or deleted in the whole organism. Moreover, a variety of compensatory mechanisms often make it difficult to interpret the results. The compensatory effects can be alleviated by either timing the gene expression or limiting the amount of transfected cells.
The method of postnatal non-ventricular microinjection and in vivo electroporation allows targeted delivery of genes, siRNA or dye molecules directly to a small region of interest in the newborn rodent brain. In contrast to conventional ventricular injection technique, this method allows transfection of non-migratory cell types. Animals transfected by means of the method described here can be used, for example, for two-photon in vivo imaging or in electrophysiological experiments on acute brain slices.

This protocol describes a non-viral method of delivery of genetic constructs to a certain area of living rodent brain. The method consists of plasmid preparation, micropipette fabrication, neonatal rat pup surgery, microinjection of the construct, and in vivo electroporation.

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals ...

Autologous Blood Injection to Model Spontaneous Intracerebral Hemorrhage in Mice

Investigation of the pathophysiology of injury after intracerebral
hemorrhage (ICH) requires a reproducible animal model. While ICH
accounts for 10-15% of all strokes, there remains no specific
effective therapy. The autologous blood injection model in mice
involves the stereotaxic injection of arterial blood into the basal
ganglia mimicking a spontaneous hypertensive hemorrhage in man. The
response to hemorrhage can then be studied in vivo and the
neurobehavioral deficits quantified, allowing for description of the
ensuing pathology and the testing of potential therapeutic agents. The
procedure described in this protocol uses the double injection
technique to minimize risk of blood reflux up the needle track, no
anticoagulants in the pumping system, and eliminates all dead space
and expandable tubing in the system.

The autologous blood injection model of intracerebral hemorrhage in mice described in this protocol uses the double injection technique to minimize risk of blood reflux up the needle track, no anticoagulants in the pumping system, and eliminates all dead space and expandable tubing in the system.

Investigation of the pathophysiology of injury after intracerebral
hemorrhage (ICH) requires a reproducible animal model.  While ICH
accounts for 10-15% ...

Live Imaging of Drosophila Larval Neuroblasts

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given their relevance to development and disease, understanding the mechanisms that govern asymmetric stem cell division has been a robust area of study. Because they are genetically tractable and undergo successive rounds of cell division about once every hour, the stem cells of the Drosophila central nervous system, or neuroblasts, are indispensable models for the study of stem cell division. About 100 neural stem cells are located near the surface of each of the two larval brain lobes, making this model system particularly useful for live imaging microscopy studies. In this work, we review several approaches widely used to visualize stem cell divisions, and we address the relative advantages and disadvantages of those techniques that employ dissociated versus intact brain tissues. We also detail our simplified protocol used to explant whole brains from third instar larvae for live cell imaging and fixed analysis applications.

Live Imaging of Drosophila Larval Neuroblasts

This protocol details a streamlined method used to conduct live cell imaging in the context of an intact larval brain. Live cell imaging approaches are invaluable for the study of asymmetric neural stem cell divisions as well as other neurogenic and developmental processes, consistently uncovering mechanisms that were previously overlooked.

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given ...

Experimental Demyelination and Remyelination of Murine Spinal Cord by Focal Injection of Lysolecithin

Multiple sclerosis is an inflammatory demyelinating disease of the central nervous system characterized by plaque formation containing lost oligodendrocytes, myelin, axons, and neurons. Remyelination is an endogenous repair mechanism whereby new myelin is produced subsequent to proliferation, recruitment, and differentiation of oligodendrocyte precursor cells into myelin-forming oligodendrocytes, and is necessary to protect axons from further damage. Currently, all therapeutics for the treatment of multiple sclerosis target the aberrant immune component of the disease, which reduce inflammatory relapses but do not prevent progression to irreversible neurological decline. It is therefore imperative that remyelination-promoting strategies be developed which may delay disease progression and perhaps reverse neurological symptoms. Several animal models of demyelination exist, including experimental autoimmune encephalomyelitis and curprizone; however, there are limitations in their use for studying remyelination. A more robust approach is the focal injection of toxins into the central nervous system, including the detergent lysolecithin into the spinal cord white matter of rodents. In this protocol, we demonstrate that the surgical procedure involved in injecting lysolecithin into the ventral white matter of mice is fast, cost-effective, and requires no additional materials than those commercially available. This procedure is important not only for studying the normal events involved in the remyelination process, but also as a pre-clinical tool for screening candidate remyelination-promoting therapeutics.

Demyelinating diseases can be modeled in animals by focal application of lysolecithin into the CNS. A single injection of lysolecithin into mouse spinal cord produces a lesion that spontaneously repairs over time. The goal is to study factors involved in de- and remyelination, and to test agents for enhancing repair.

Multiple sclerosis is an inflammatory demyelinating disease of the central nervous system characterized by plaque formation containing lost ...

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 ...

Minimally Invasive Endoscopic Intracerebral Hemorrhage Evacuation

Intracerebral hemorrhage (ICH) is a subtype of stroke with high mortality and poor functional outcomes, largely because there are no evidence-based treatment options for this devastating disease process. In the past decade, a number of minimally invasive surgeries have emerged to address this issue, one of which is endoscopic evacuation. Stereotactic ICH Underwater Blood Aspiration (SCUBA) is a novel endoscopic evacuation technique performed in a fluid-filled cavity using an aspiration system to provide an additional degree of freedom during the procedure. The SCUBA procedure utilizes a suction device, endoscope, and sheath and is divided into two phases. The first phase involves maximal aspiration and minimal irrigation to decrease clot burden. The second phase involves increasing irrigation for visibility, decreasing aspiration strength for targeted aspiration without disturbing the cavity wall, and cauterizing any bleeding vessels. Using the endoscope and aspiration wand, this technique aims to maximize hematoma evacuation while minimizing collateral damage to the surrounding brain. Advantages of the SCUBA technique include the use of a low-profile endoscopic sheath minimizing brain disruption and improved visualization with a fluid-filled cavity rather than an air-filled one.

This paper details the surgical protocol for minimally invasive endoscopic intracerebral hemorrhage evacuation using the SCUBA technique.

Intracerebral hemorrhage (ICH) is a subtype of stroke with high mortality and poor functional outcomes, largely because there are no evidence-based ...

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.

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, ...

In Vivo Direct Reprogramming of Resident Glial Cells into Interneurons by Intracerebral Injection of Viral Vectors

Converting resident glia in the brain into functional and subtype-specific neurons in vivo provides a step forward towards the development of alternative cell replacement therapies while also creating tools to study cell fate in situ. To date, it has been possible to obtain neurons via in vivo reprogramming, but the precise phenotype of these neurons or how they mature has not been analyzed in detail. In this protocol, we describe a more efficient conversion and cell-specific identification of the in vivo reprogrammed neurons, using an AAV-based viral vector system. We also provide a protocol for functional assessment of the reprogrammed cells&#8217; neuronal maturation. By injecting flip-excision (FLEX) vectors, containing the reprogramming and synapsin-driven reporter genes&#160;to specific cell types in the brain that serve as the target for cell reprogramming. This technique allows for the easy identification of newly reprogrammed neurons. Results show that the obtained reprogrammed neurons functionally mature over time, receive synaptic contacts and show electrophysiological properties of different types of interneurons. Using the transcription factors Ascl1, Lmx1a and Nurr1, the majority of the reprogrammed cells have properties of fast-spiking, parvalbumin-containing interneurons.

This protocol aims at generating directly reprogrammed interneurons in vivo, using an AAV-based viral system in the brain and a FLEX synapsin-driven GFP reporter, which allows for cell identification and further analysis in vivo.

Converting resident glia in the brain into functional and subtype-specific neurons in vivo provides a step forward towards the development of alternative ...

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 ...

Double Direct Injection of Blood into the Cisterna Magna as a Model of Subarachnoid Hemorrhage

Among strokes, subarachnoid hemorrhage (SAH) consecutive to the rupture of a cerebral arterial aneurysm represents 5-9% but is responsible for about 30% of the total stroke-related mortality with an important morbidity in terms of neurological outcome. A delayed cerebral vasospasm (CVS) may occur most often in association with a delayed cerebral ischemia. Different animal models of SAH are now being used including endovascular perforation and direct injection of blood into the cisterna magna or even the prechiasmatic cistern, each exhibiting distinct advantages and disadvantages. In this article, a standardized mouse model of SAH by double direct injection of determined volumes of autologous whole blood into the cisterna magna is presented. Briefly, mice were weighed and then anesthetized by isoflurane inhalation. Then, the animal was placed in a reclining position on a heated blanket maintaining a rectal temperature of 37 &#176;C and positioned in a stereotactic frame with a cervical bend of about 30&#176;. Once in place, the tip of an elongated glass micropipette filled with the homologous arterial blood taken from carotid artery of another mouse of the same age and gender (C57Bl/6J) was positioned at a right angle in contact with the atlanto-occipital membrane by means of a micromanipulator. Then 60 &#181;L of blood was injected in the cisterna magna followed by a 30&#176; downward tilt of the animal for 2 minutes. The second infusion of 30 &#181;L of blood into the cisterna magna was performed 24 h after the first one. The individual follow-up of each animal is carried out daily (careful evaluation of weight and well-being). This procedure allows a predictable and highly reproducible distribution of blood, likely accompanied by intracranial pressure elevation that can be mimicked by an equivalent injection of an artificial cerebral spinal fluid (CSF), and represents an acute to mild-model of SAH inducing low mortality.

We described in this protocol a standardized subarachnoid hemorrhage (SAH) mouse model by a double injection of autologous whole-blood into the cisterna magna. The high degree of standardization of the double-injection procedure represents a middle-to-acute model of SAH with relative safety regarding mortality.

Among strokes, subarachnoid hemorrhage (SAH) consecutive to the rupture of a cerebral arterial aneurysm represents 5-9% but is responsible for about 30% ...