JoVE   
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Biology

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Neuroscience

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Immunology and Infection

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Clinical and Translational Medicine

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Bioengineering

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Applied Physics

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Chemistry

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Behavior

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Environment

|   

JoVE Science Education

General Laboratory Techniques

You do not have subscription access to videos in this collection. Learn more about access.

Basic Methods in Cellular and Molecular Biology

You do not have subscription access to videos in this collection. Learn more about access.

Model Organisms I

You do not have subscription access to videos in this collection. Learn more about access.

Model Organisms II

You do not have subscription access to videos in this collection. Learn more about access.

 JoVE Biology

The Hypoxic Ischemic Encephalopathy Model of Perinatal Ischemia

1, 1

1Department of Neurology and Neurological Sciences, Stanford University School of Medicine

Article
    Downloads Comments Metrics

    You must be subscribed to JoVE to access this content.

    This article is a part of   JoVE Biology. If you think this article would be useful for your research, please recommend JoVE to your institution's librarian.

    Recommend JoVE to Your Librarian

    Current Access Through Your IP Address

    You do not have access to any JoVE content through your current IP address.

    IP: 184.73.85.45, User IP: 184.73.85.45, User IP Hex: 3091813677

    Current Access Through Your Registered Email Address

    You aren't signed into JoVE. If your institution subscribes to JoVE, please or create an account with your institutional email address to access this content.

     

    Summary

    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.

    Date Published: 11/19/2008, Issue 21; doi: 10.3791/955

    Cite this Article

    Taniguchi, H., Andreasson, K. The Hypoxic Ischemic Encephalopathy Model of Perinatal Ischemia. J. Vis. Exp. (21), e955, doi:10.3791/955 (2008).

    Abstract

    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.

    Protocol

    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

    1. 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).
    2. 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).
    3. The CCA is ligated with a 5-0 silk suture (5-0 silk, Ethicon, Somer, NJ).
    4. 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.
    5. 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.
    6. At the end of 100 minutes of hypoxia, the pups are returned again to their dam for recovery.
    7. 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.
    8. 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.
    9. 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.
    10. 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.

    Representative Results

    Figure 1
    Figure 1. Representative HIE coronal levels 1-4 stained with TTC.

    TTC stained coronal sections demonstrate area of infarct (in white).

    Subscription Required. Please recommend JoVE to your librarian.

    Discussion

    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 validated 3. This model was initially perfected in rats and more recently adapted to mice 8.

    There are specific technical details that warrant mention:

    1. Before ligation of the CCA, be sure to remove connective tissue and nervus vagus which may entangle CCA. With successful ligation of the CCA, the vessel color above the ligation site should turn white. Alternatively, it is also recommended to double ligate and cut in the middle of two ligation site. Cover the incision over the ligated CCA with label tape before placing the pup back with its dam.
    2. A maximum duration of not more than 10 minutes of anesthesia for each pup is optimal for CCA ligation and successful outcome.
    3. The recuperation period after CCA ligation should be 90-120 minutes. If shorter, pups are less likely to survive, and if longer there will be more variability of infarction, tending towards smaller infarcts.
    4. Temperature is critical. Always place a thermometer in the chamber and maintain the temperature at 37°C. The length of hypoxia may vary from 90 to 120 minutes depending on the situation and severity of injury you want to achieve.
    5. TTC staining is applicable for analysis of infarct volume up to 72 hours after HIE. Longer survival time points should be assessed by sectioning and cresyl violet staining to determine the infarct size.

    Subscription Required. Please recommend JoVE to your librarian.

    Disclosures

    Acknowledgements

    Funded by American Heart Association and March of Dimes.

    Materials

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

    References

    1. J. E. Rice, 3rd, R. C. Vannucci, and J. B. Brierley, Annals of neurology 9 (2), 131 (1981).

    2. R. C. Vannucci and S. J. Vannucci, Dev Neurosci 27 (2-4), 81 (2005).

    3. E. Bona, H. Hagberg, E. M. Loberg et al., Pediatric research 43 (6), 738 (1998).

    4. K. Mishima, T. Ikeda, T. Yoshikawa et al., Behavioural brain research 151 (1-2), 209 (2004).

    5. W. H. Trescher, S. Ishiwa, and M. V. Johnston, Brain & development 19 (5), 326 (1997);

    6. R. S. Young, T. P. Olenginski, S. K. Yagel et al., Stroke; a journal of cerebral circulation 14 (6), 929 (1983).

    7. J. B. Bederson, L. H. Pitts, S. M. Germano et al., Stroke; a journal of cerebral circulation 17 (6), 1304 (1986).

    8. J. Cai, Z. Kang, W. W. Liu et al., Neuroscience letters 441 (2), 167 (2008).

    9. J. Grow and J. D. Barks, Clinics in perinatology 29 (4), 585 (2002).

    10. U. Aden, L. Halldner, H. Lagercrantz et al., Stroke; a journal of cerebral circulation 34 (3), 739 (2003).

    Comments

    6 Comments

    Thanks a lot! Different lab do HIE model have many difference.We put pups in container and container was put in baby incubator to keep the temperature inside the container with 37 degree.
    Reply

    Posted by: AnonymousDecember 14, 2008, 2:18 AM

    THANKS
    Reply

    Posted by: chenhui c.November 25, 2009, 7:53 PM

    thank you a lot it's realy useful
    Reply

    Posted by: AnonymousDecember 26, 2011, 12:13 PM

    I thank you for this useful protocol. Might you describe how you avoid the mice cannibalism when you return the mice with their dam after the surgery and after the hypoxia exposure to the cage?. I appreciate your time and answer.
    Reply

    Posted by: Carolina U.October 25, 2012, 9:42 AM

    thank you a lot it's realy useful
    Reply

    Posted by: Arthur Alexander C.April 14, 2013, 10:43 PM

    Hi, it's really a useful protocol. Unfortunately, I cann't the fulltext. Could you please send the protocol to me?
    Thank you in advance.
    Reply

    Posted by: Song Z.January 24, 2014, 8:41 PM

    Post a Question / Comment / Request

    You must be signed in to post a comment. Please or create an account.

    Metrics

    Waiting
    simple hit counter