Name: The Hypoxic Ischemic Encephalopathy Model of Perinatal Ischemia (Video) | JoVE Journal
Uploaded: 2008-11-19T00:00:00.000+00:00
Duration: 8 min 47 s
Description: Learn to establish a hypoxic-ischemic encephalopathy model in postnatal day seven rat pups using carotid artery ligation and controlled hypoxia. This method enables researchers to study neonatal brain injury mechanisms and potential therapeutic interventions.

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5 minutes

Funded by American Heart Association and March of Dimes.

This protocol was approved by the Institutional Animal Care and Use Committee at Stanford University and abides by the National Institutes of Health guidelines for the use of experimental animals.
Neonatal Rat HIE model
<ol>
<li>Post natal day (PND) 7 Sprague-Dawley rat pups (Charles River Laboratories, Inc., Wilmington, MA) are fully anesthetized with isoflurane (Aerrane, Baxter,  Deerfield, IL; 3-4% for induction and 1-2% for maintenance).</li>
<li>A small incision is made in the neck (Extra Thin Iris Scissors #14088-10, Fine Science Tools Inc, Foster City, CA) to expose the left common carotid artery (CCA).</li>
<li>The CCA is ligated with a 5-0 silk suture (5-0 silk, Ethicon, Somer, NJ).</li>
<li>The incision is closed with cyanoacrylate adhesive (Elmers Products Inc, Columbus, OH). The incision is covered with tape and anesthesia is stopped. The pups are returned to their dam and allowed to recuperate for 1-2 hours.</li>
<li>Pups are then placed in a hypoxic chamber that contains 8% oxygen balanced with 92% nitrogen for 100 minutes at 37°C. 8% oxygen/92% nitrogen gas(Airgas, Sacramento, CA) flow via a tube into the mouse cage outfitted with a plastic cover. The cover is made to fit to the cage and has two holes of 2 cm in diameter, one of which to receive the tube connected to the 8% oxygen gas tank and another allows the gas to flowout. The chamber is placed in a water bath and is warmed up prior to use and a thermometer inside of it must register 37°C before the start of hypoxia.</li>
<li>At the end of 100 minutes of hypoxia, the pups are returned again to their dam for recovery.</li>
<li>24 hours after the end of hypoxia, pups are euthanized by deep anesthesia with isoflurane. Brain tissue is perfused with cold normal saline, followed by a solution of cold 1% TTC in phosphate buffered saline, pH 7.4 (Sigma Chemical Co, St Louis, MO). TTC must be kept protected from light.</li>
<li>Brains are removed and cooled for 1-2 minutes on ice, and four coronal sections 3 mm apart (levels 1-4) are cut, beginning rostrally at the level of the opticchiasm and infundibulum, corresponding to the bregma 1.8 ~ 2.0 mm level of the adult mouse brain.</li>
<li>Brain slices are immersed in TTC solution and incubated at 37°C for 8 minutes, followed by fixation in 4% paraformaldehyde/PBS overnight. Because TTC is light sensitive, keep slices away from light during this time.</li>
<li>The TTC stained sections are scanned and digitized. Using Image J (NIH, Bethesda, MD), the following are measured for each level: area of infarction (unstained area), the area of the ipsilateral hemisphere, and the area of the contralateral hemisphere. The percent infarcted area per slice is calculated as: (area of infarct/area of ipsilateral hemisphere) or A1, A2, A3, and A4. The percent volume of infarct is calculated as the sum (A1 +A2 + A3+ A4) * 3mm. Areas of cortical or striatal injury can also be separately quantified in a similar manner.</li>
</ol>
Representative Results
<img src="/files/ftp_upload/old/955_2008_9_2_2/955_Fig-01.png" alt="Mouse brain injury progression, experiment diagram; study of damage levels from 1 to 4 in brain tissue." width="660" height="190" data-altupdatedat="1787630408000" data-alt="Figure 1"> Figure 1. Representative HIE coronal levels 1-4 stained with TTC. 
TTC stained coronal sections demonstrate area of infarct (in white).

<ol><li>Rice, J. E. 3rd, Vannucci, R. C., Brierley, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+influence+of+immaturity+on+hypoxic-ischemic+brain+damage+in+the+rat%5BTitle%5D)+AND+%22Annals+of+neurology%22%5BJournal%5D)">The influence of immaturity on hypoxic-ischemic brain damage in the rat.</a> Annals of neurology. 9 (2), 131-141 (1981).</li>
<li>Vannucci, R. C., Vannucci, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Perinatal+Hypoxic-Ischemic+Brain+Damage%3A+Evolution+of+an+Animal+Model%5BTitle%5D)+AND+%22Dev+Neurosci%22%5BJournal%5D)">Perinatal Hypoxic-Ischemic Brain Damage: Evolution of an Animal Model.</a> Dev Neurosci. 27 (2-4), 81-86 (2005).</li>
<li>Bona, E., Hagberg, H., Loberg, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Protective+effects+of+moderate+hypothermia+after+neonatal+hypoxia-ischemia%3A+short-+and+long-term+outcome%5BTitle%5D)+AND+%22Pediatric+research%22%5BJournal%5D)">Protective effects of moderate hypothermia after neonatal hypoxia-ischemia: short- and long-term outcome.</a> Pediatric research. 43 (6), 738-738 (1998).</li>
<li>Trescher, W. H., Ishiwa, S., Johnston, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+hypothermia+and+hyperthermia+on+attentional+and+spatial+learning+deficits+following+neonatal+hypoxia-ischemic+insult+in+rats%5BTitle%5D)+AND+%22Behavioural+brain+research%22%5BJournal%5D)">Effects of hypothermia and hyperthermia on attentional and spatial learning deficits following neonatal hypoxia-ischemic insult in rats.</a> Behavioural brain research. 151 (1-2), 209-209 (2004).</li>
<li>Mishima, K., Ikeda, T., Yoshikawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Brief+post-hypoxic-ischemic+hypothermia+markedly+delays+neonatal+brain+injury%5BTitle%5D)+AND+%22Brain+development%22%5BJournal%5D)">Brief post-hypoxic-ischemic hypothermia markedly delays neonatal brain injury.</a> Brain development. 19 (5), 326-326 (1997).</li>
<li>Young, R. S., Olenginski, T. P., Yagel, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+effect+of+graded+hypothermia+on+hypoxic-ischemic+brain+damage%3A+a+neuropathologic+study+in+the+neonatal+rat%5BTitle%5D)+AND+%22Stroke%3B+a+journal+of+cerebral+circulation%22%5BJournal%5D)">The effect of graded hypothermia on hypoxic-ischemic brain damage: a neuropathologic study in the neonatal rat.</a> Stroke; a journal of cerebral circulation. 14 (6), 929-929 (1983).</li>
<li>Bederson, J. B., Pitts, L. H., Germano, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evaluation+of+2%2C3%2C5-triphenyltetrazolium+chloride+as+a+stain+for+detection+and+quantification+of+experimental+cerebral+infarction+in+rats%5BTitle%5D)+AND+%22Stroke%3B+a+journal+of+cerebral+circulation%22%5BJournal%5D)">Evaluation of 2,3,5-triphenyltetrazolium chloride as a stain for detection and quantification of experimental cerebral infarction in rats.</a> Stroke; a journal of cerebral circulation. 17 (6), 1304-1304 (1986).</li>
<li>Cai, J., Kang, Z., Liu, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hydrogen+therapy+reduces+apoptosis+in+neonatal+hypoxia-ischemia+rat+model%5BTitle%5D)+AND+%22Neuroscience+letters%22%5BJournal%5D)">Hydrogen therapy reduces apoptosis in neonatal hypoxia-ischemia rat model.</a> Neuroscience letters. 441 (2), 167-167 (2008).</li>
<li>Grow, J., Barks, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pathogenesis+of+hypoxic-ischemic+cerebral+injury+in+the+term+infant%3A+current+concepts%5BTitle%5D)+AND+%22Clinics+in+perinatology%22%5BJournal%5D)">Pathogenesis of hypoxic-ischemic cerebral injury in the term infant: current concepts.</a> Clinics in perinatology. 29 (4), 585-585 (2002).</li>
<li>Aden, U., Halldner, L., Lagercrantz , H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Aggravated+brain+damage+after+hypoxic+ischemia+in+immature+adenosine+A2A+knockout+mice%5BTitle%5D)+AND+%22Stroke%3B+a+journal+of+cerebral+circulation%22%5BJournal%5D)">Aggravated brain damage after hypoxic ischemia in immature adenosine A2A knockout mice.</a> Stroke; a journal of cerebral circulation. 34 (3), 739-739 (2003).</li>
</ol>

The rodent postnatal HIE model is an established model that recapitulates cerebral hypoxia occurring in the peri-natal period in newborns. Extensive histological and immunocytochemical characterization studies have demonstrated that the 7 day old rodent has the analogous brain maturity to model a third trimester human fetus 7. The rodent HIE model has been very informative for understanding mechanisms of brain injury from peri-natal hypoxia 2, where interventions such as hypothermia have been valid...

<table><tbody><tr><td>Sprague-Dawley Rat Pups</td><td>Animal</td><td>Charles River Laboratories</td><td></td><td></td></tr><tr><td>Isoflurane</td><td>Surgery</td><td>Baxter Internationl Inc.</td><td></td><td></td></tr><tr><td>8% Oxygen/ 92% Nitrogen Gas</td><td>Surgery</td><td>Airgas</td><td></td><td></td></tr><tr><td>2,3,5-triphenyl tetrazolium chloride </td><td>Reagent</td><td>Sigma-Aldrich</td><td>T8877</td><td></td></tr><tr><td>Phosphate buffered saline (PBS) pH 7.4 </td><td>Reagent</td><td>GIBCO, by Life Technologies</td><td></td><td></td></tr><tr><td>Paraformaldehyde</td><td>Reagent</td><td>Sigma-Aldrich</td><td>P6148</td><td></td></tr></tbody></table>

the hypoxic ischemic encephalopathy model of perinatal ischemia

The Hypoxic Ischemic Encephalopathy Model of Perinatal Ischemia

Hypoxic-Ischemic Encephalopathy (HIE) is the consequence of systemic asphyxia occurring at birth. Twenty five percent of neonates with HIE develop severe and permanent neuropsychological sequelae, including mental retardation, cerebral palsy, and epilepsy. The outcomes of HIE are devastating and permanent, making it critical to identify and develop therapeutic strategies to reduce brain injury in newborns with HIE. To that end, the neonatal rat model for hypoxic-ischemic brain injury has been developed to model this human condition. The HIE model was first validated by Vannucci et al 1 and has since been extensively used to identify mechanisms of brain injury resulting from perinatal hypoxia-ischemia 2 and to test potential therapeutic interventions 3,4. The HIE model is a two step process and involves the ligation of the left common carotid artery followed by exposure to a hypoxic environment. Cerebral blood flow (CBF) in the hemisphere ipsilateral to the ligated carotid artery does not decrease because of the collateral blood flow via the circle of Willis; however with lower oxygen tension, the CBF in the ipsilateral hemisphere decreases significantly and results in unilateral ischemic injury. The use of 2,3,5-triphenyltetrazolium chloride (TTC) to stain and identify ischemic brain tissue was originally developed for adult models of rodent cerebral ischemia 5, and is used to evaluate the extent of cerebral infarctin at early time points up to 72 hours after the ischemic event 6. In this video, we demonstrate the hypoxic-ischemic injury model in postnatal rat brain and the evaluation of the infarct size using TTC staining.

The postnatal rat model for hypoxic-ischemic brain injury is a well-established model of human neonatal hypoxic ischemic encephalopathy (HIE). In this article, we describe the model of HIE in post-natal rat pups.

Hypoxic-Ischemic Encephalopathy (HIE) is the consequence of systemic asphyxia occurring at birth. Twenty five percent of neonates with HIE develop severe ...

To induce this hypoxic encephalopathy ischemia model postnatal. Day seven, Sprague dolly rat pups undergo microsurgery and left common carotid artery ligation. After one to two hours of recuperation period, pups are put into a hypoxic chamber at 37 degrees Celsius for 100 minutes.Pups are then returned to their dam until they are euthanized. Following two systemic perfusion steps, brains are removed and sectioned to three millimeter thick slices. Slices are then stained using 1%2, 3, 5, tri fennel, tetra oleum chloride, or TTC solution in order to reveal the area of infarct.Hi, I'm Hi tango from Andre Lab Lab in Department of Neurology and Neurological Science at Stanford University School of Medicine. Today we will show you a procedure for neonatal hypoxic ischemic brain injury model. We use this procedure in our laboratory to study the mechanism of hypoxic ischemic encephalopathy in the neonatal brain with the ultimate goal of developing new treatment.So let's get started. To begin the procedure, fully anesthetize a postnatal day seven spray dolly rat pup. With ISO fluorine, we use 4%for induction and one to 2%for maintenance.After a few minutes, perform a toe pinch test to ensure the pup is anesthetized. Then sterilize the pup's neck using cotton tipped applicators to apply 10%povidone iodine, followed by 70%isopropyl alcohol. Next, use sterile, extra thin iris scissors from fine science tools to make a small incision about five millimeters left of the midline, expose the left common carotid artery or CCA via blunt dissection.Make sure to remove the connective tissue and the vagus nerve, which may entangle the CCA. Then use a five zero silk suture from Ethicon to ligate. The CCA.The vessel color above the ligation site should turn white right after the ligation. After ligation, close the incision with cyanoacrylate adhesive and cover with tape. Then stop anesthesia and ensure that the pup regains consciousness.Place the pup in a recovery box on a warm pad. Repeat the procedure on additional pups as needed. Observe the pups for at least five minutes before returning the pups to their dam.Allow pups to recuperate for one to two hours prior to the experiment. Assemble a hypoxic chamber that contains 8%oxygen, balanced with 92%nitrogen. To do this, obtain a mouse cage outfitted with a plastic cover that has two holes of two centimeters in diameter.One hole receives the tube connected to the mixed gas tank, which contains 8%oxygen and 92%nitrogen. And the other hole allows the gas to flow out, place the chamber in a water bath and then place a thermometer in the chamber. The thermometer must register 37 degrees Celsius before the start of hypoxia.Once the pups have recuperated, place them in the hypoxic chamber for 100 minutes at 37 degrees Celsius. Then return the pups to their dam 24 hours after the end of hypoxia, euthanize the pups by deep anesthesia with iso fluorine. Next perfuse intracardiac via the left ventricle using cold normal saline, followed by a solution of cold 1%TTC in P-B-S-T-T-C is light sensitive and must be kept protected from light.After the perfusion step. Place the head on ice for one to two minutes and then expose the brain using ice cooled razor blade to slice the brain tissue at four levels. Uses spatula to scoop out the brain slices and immerse the brain slices in the TTC solution.Place the slices in an incubator at 37 degrees Celsius for eight minutes to allow TTC staining to occur. Next, transfer the sections to 4%Paraform aldehyde in PBS cover the section. Since TTC is light sensitive, then place the sections at four degrees Celsius and allow them to fix overnight after the overnight fixation.Scan and digitize the TTC stain sections and proceed with the results analysis. Here are representative results of hypoxic ischemic encephalopathy, coronal levels one through four stained with TTC. The white unstained areas indicate the areas of infarct using image J.The following are measured for each level, the area of infarction A and the area of the ipsilateral hemisphere B.Based on these measurements, the percent infarcted area per slice is calculated as the area of infarct over the area of the ipsilateral hemisphere or A one over B one, A two over B two, A three over B three and A four over B four.Then the percent volume of infarct is calculated as the sum of a 1, 2, 3, and four, multiplied by three millimeters divided by the sum of B 1, 2, 3, and four multiplied by three millimeters. Areas of cortical or SAL injury can also be separately quantified in a similar manner. We've just shown you how to generate a hypoxic ischemic encephalopathy model of perinatal ischemia.When doing this procedure, it's important to remember to limit the anesthesia to no more than 10 minutes for each CCA ligation to always place a thermometer in the hypoxic chamber and to maintain the temperature at 37 cent degree. So that's it. Thanks for watching and good luck with your experiment.

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Large animal models have good translational values in the examination of physiology and pharmacology of neonatal asphyxia. Using newborn piglets, we develop an experimental protocol to simulate neonatal asphyxia which has advantages of studying the systemic and regional hemodynamics, oxygen transport with biochemical and pathologic pathways and correlations.

A Swine Model of Neonatal Asphyxia

The video demonstrates the procedure for creating a mouse model to study neonatal hypoxic-ischemic brain injury. It outlines the steps for surgically inducing ischemia and subjecting the animal to hypoxia to exacerbate brain damage. Finally, it shows how to assess the resulting brain lesions, ensuring the mouse model is prepared for further scientific investigation.

Establishing a Mouse Model of Neonatal Hypoxic-Ischemic Brain Injury

Source: Jantzie, L. L., et. al. Modeling Encephalopathy of Prematurity Using Prenatal Hypoxia-ischemia with Intra-amniotic Lipopolysaccharide in Rats. J. Vis. Exp. (2015)This video demonstrates a protocol to induce hypoxic-ischemic and inflammatory conditions in fetal rats by restricting uterine blood flow and injecting LPS, modeling neuroinflammation and disrupted brain development relevant to preterm birth studies.

Inducing Hypoxic-Ischemic and Inflammatory Conditions to Model Encephalopathy in Prenatal Rats

Hypoxic-ischemic encephalopathy following perinatal asphyxia can be studied using animal models. We demonstrate the procedures necessary for establishing a piglet model of neonatal hypoxic-ischemic encephalopathy.

A Piglet Model of Neonatal Hypoxic-Ischemic Encephalopathy

Thromboembolic stroke models are vital tools for optimizing the recanalization therapy. Here we report a murine thrombotic stroke model based on transient cerebral hypoxic-ischemic (tHI) insult, which triggers thrombosis and infarction, and responds favorably to tissue plasminogen activator (tPA)-mediated fibrinolysis in a therapeutic window similar to those in stroke patients.

A Thrombotic Stroke Model Based On Transient Cerebral Hypoxia-ischemia

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.

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

Neonatal stroke is a significant cause of early brain injury requiring a translational model with consistent focal injury patterns and high reproducibility in order to enable study. This study describes the detailed surgical procedure for creating a non-hemorrhagic, unilateral focal ischemia-reperfusion injury in full-term-equivalent rodents.

Transient Middle Cerebral Artery Occlusion Model of Neonatal Stroke in P10 Rats

We performed unilateral carotid artery occlusion on postnatal day 7-10 CD-1 mouse pups to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of HI brain injury. We studied neurobehavioral functions in these mice compared to non-operated normal mice.

Neurobehavioral Assessments in a Mouse Model of Neonatal Hypoxic-ischemic Brain Injury

The method describes inflammation-sensitized hypoxic-ischemic and hyperoxic brain injury in the P17 ferret to model the complex interaction between prolonged inflammation and oxidative brain injury experienced in a number of late preterm infants.

A Ferret Model of Inflammation-sensitized Late Preterm Hypoxic-ischemic Brain Injury

This manuscript describes a method for continuous video EEG recordings using multiple depth electrodes in neonatal mice undergoing hypoxia-ischemia.

Continuous Video Electroencephalogram during Hypoxia-Ischemia in Neonatal Mice

Learn to establish a hypoxic-ischemic encephalopathy model in postnatal day seven rat pups using carotid artery ligation and controlled hypoxia. This method enables researchers to study neonatal brain injury mechanisms and potential therapeutic interventions.

The Hypoxic Ischemic Encephalopathy Model of Perinatal Ischemia

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