Name: a ferret model of inflammation-sensitized late preterm hypoxic-ischemic brain injury
Uploaded: 2019-11-19T12:30:00
Description: Watch this Scientific Journal Video about A Ferret Model of Inflammation-sensitized Late Preterm Hypoxic-ischemic Brain Injury at JoVE.com

Development of the model was funded Bill and Melinda Gates Foundation, as well as by NIH grant 5R21NS093154-02 (NICHD).

Procedures were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals and as part of an approved protocol by the University of Washington Institutional Animal Care and Use Committee.
1. Preparation and LPS Administration
NOTE: Refer to Figure 1 for a timeline of the procedures.
<ol>
	<li>Prior to commencing the procedure, seal, sterilize and autoclave all surgical instruments and surgic...

<ol>
	<li>Martin, J. A., Hamilton, B. E., Osterman, M. J. K., Driscoll, A. K., Drake, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Births:+Final+Data+for+2017.">Births: Final Data for 2017.</a> National Vital Statistics Report. 67 (8), 1-49 (2018).</li><li>Vanhaesebrouck, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+EPIBEL+study:+outcomes+to+discharge+from+hospital+for+extremely+preterm+infants+in+Belgium.">The EPIBEL study: outcomes to discharge from hospital for extremely preterm infants in Belgium.</a> Pediatrics. 114 (3), 663-675 (2004).</li><li>Raju, T. N., 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=Long-Term+Healthcare+Outcomes+of+Preterm+Birth:+An+Executive+Summary+of+a+Conference+Sponsored+by+the+National+Institutes+of+Health.">Long-Term Healthcare Outcomes of Preterm Birth: An Executive Summary of a Conference Sponsored by the National Institutes of Health.</a> Journal of Pediatrics. , (2016).</li><li>Raju, T. N. K., Buist, A. S., Blaisdell, C. J., Moxey-Mims, M., Saigal, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adults+born+preterm:+a+review+of+general+health+and+system-specific+outcomes.">Adults born preterm: a review of general health and system-specific outcomes.</a> Acta Paediatrica. 106 (9), 1409-1437 (2017).</li><li>Bennet, 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=Chronic+inflammation+and+impaired+development+of+the+preterm+brain.">Chronic inflammation and impaired development of the preterm brain.</a> Journal of Reproductive Immunology. 125, 45-55 (2018).</li><li>Reich, B., Hoeber, D., Bendix, I., Felderhoff-Mueser, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperoxia+and+the+Immature+Brain.">Hyperoxia and the Immature Brain.</a> Developmental Neuroscience. 38 (5), 311-330 (2016).</li><li>Galinsky, R., 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=Complex+interactions+between+hypoxia-ischemia+and+inflammation+in+preterm+brain+injury.">Complex interactions between hypoxia-ischemia and inflammation in preterm brain injury.</a> Developmental Medicine &amp; Child Neurology. 60 (2), 126-133 (2018).</li><li>Empie, K., Rangarajan, V., Juul, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+the+ferret+a+suitable+species+for+studying+perinatal+brain+injury.">Is the ferret a suitable species for studying perinatal brain injury.</a> International Journal of Developlemental Neuroscience. 45, 2-10 (2015).</li><li>Snyder, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ontogeny+of+white+matter,+toll-like+receptor+expression,+and+motor+skills+in+the+neonatal+ferret.">Ontogeny of white matter, toll-like receptor expression, and motor skills in the neonatal ferret.</a> International Journal of Developlemental Neuroscience. , (2018).</li><li>Schwerin, S. 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=Progression+of+histopathological+and+behavioral+abnormalities+following+mild+traumatic+brain+injury+in+the+male+ferret.">Progression of histopathological and behavioral abnormalities following mild traumatic brain injury in the male ferret.</a> Journal of Neuroscience Research. 96 (4), 556-572 (2018).</li><li>Rafaels, K. 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+injury+risk+from+primary+blast.">Brain injury risk from primary blast.</a> Journal of Trauma and Acute Care Surgery. 73 (4), 895-901 (2012).</li><li>Wood, 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=A+Ferret+Model+of+Encephalopathy+of+Prematurity.">A Ferret Model of Encephalopathy of Prematurity.</a> Developlemental Neuroscience. , (2019).</li><li>Barnette, A. R., 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=Characterization+of+Brain+Development+in+the+Ferret+via+Magnetic+Resonance+Imaging.">Characterization of Brain Development in the Ferret via Magnetic Resonance Imaging.</a> Pediatric Research. 66 (1), 80-84 (2009).</li><li>Kroenke, C. D., Mills, B. D., Olavarria, J. F., Neil, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biology and Diseases of the Ferret. , (2014).</li><li>Eklind, 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=Bacterial+endotoxin+sensitizes+the+immature+brain+to+hypoxic--ischaemic+injury.">Bacterial endotoxin sensitizes the immature brain to hypoxic--ischaemic injury.</a> European Journal of Neuroscience. 13 (6), 1101-1106 (2001).</li><li>Falck, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neonatal+Systemic+Inflammation+Induces+Inflammatory+Reactions+and+Brain+Apoptosis+in+a+Pathogen-Specific+Manner.">Neonatal Systemic Inflammation Induces Inflammatory Reactions and Brain Apoptosis in a Pathogen-Specific Manner.</a> Neonatology. 113 (3), 212-220 (2018).</li><li>Osredkar, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypothermia+Does+Not+Reverse+Cellular+Responses+Caused+by+Lipopolysaccharide+in+Neonatal+Hypoxic-Ischaemic+Brain+Injury.">Hypothermia Does Not Reverse Cellular Responses Caused by Lipopolysaccharide in Neonatal Hypoxic-Ischaemic Brain Injury.</a> Developmental Neuroscience. 37 (4-5), 390-397 (2015).</li><li>Nakata, M., Itou, T., Sakai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+inflammatory+cytokines+expression+in+peripheral+blood+mononuclear+cells+of+the+ferret+(Mustela+putorius+furo)+using+real-time+PCR.">Quantitative analysis of inflammatory cytokines expression in peripheral blood mononuclear cells of the ferret (Mustela putorius furo) using real-time PCR.</a> Veterinary Immunology and Immunopathology. 130 (1-2), 88-91 (2009).</li><li>Christensson, M., Garwicz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time+course+of+postnatal+motor+development+in+ferrets:+ontogenetic+and+comparative+perspectives.">Time course of postnatal motor development in ferrets: ontogenetic and comparative perspectives.</a> Behavioral Brain Research. 158 (2), 231-242 (2005).</li><li>Li, Y., Dugyala, S. R., Ptacek, T. S., Gilmore, J. H., Frohlich, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+Immune+Activation+Alters+Adult+Behavior,+Gut+Microbiome+and+Juvenile+Brain+Oscillations+in+Ferrets.">Maternal Immune Activation Alters Adult Behavior, Gut Microbiome and Juvenile Brain Oscillations in Ferrets.</a> eNeuro. 5 (5), (2018).</li><li>Rice, J. E., Vannucci, R. C., Brierley, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+immaturity+on+hypoxic-ischemic+brain+damage+in+the+rat.">The influence of immaturity on hypoxic-ischemic brain damage in the rat.</a> Annals of Neurolology. 9 (2), 131-141 (1981).</li></ol>

Due to the physical and developmental similarities shared between the ferret brain and human brain, the ferret is increasingly being used to model both adult and developmental brain injury.8,9,10,11,12. However, research to date suggests that the ferret brain is both resistant to initial injury as well as highly-plastic, with behavioral deficits diminishing ov...

There is an ongoing need for large animal models that reflect the pathophysiology of prematurity and perinatal hypoxia-ischemia in which therapeutic interventions for infants can be tested. In 2017, 9.93% of the 382,726 infants born in the United States were born preterm, and 84% of these infants were born between 32 and 36 weeks of gestation1. In premature infants, perinatal exposure to infection or inflammation is common, where maternal immune activation due to viral or bacterial pathogens can initiate preterm labor. Postnatally, preterm infants are at high risk of early or late onset sepsis2. Preterm infants also frequently experience periods of hypoxia, hypotension, and hyperoxia due to their immature cardiorespiratory system, elevated oxygen tension in the atmosphere relative to those experienced in utero, and iatrogenic exposures. Additionally, in preterm infants, antioxidant defenses are immature3 and pro-apoptotic factors are naturally upregulated4. Oxidative stress and cell death lead to activation of the immune system and neuroinflammation. These combined factors are thought to contribute to developmental and physiologic vulnerability of the brain, and result in or exacerbate the encephalopathy associated with poor developmental outcomes in preterm infants5,6,7.
Due to the physical and developmental similarities that the ferret brain shares with the human brain, the ferret is an attractive species in which to model brain injury8,9,10,11,12. Ferrets are also ideal candidates to model the preterm human brain, as they are born lissencephalic and develop gyrencephalic brains postnatally, which provides a window in which to expose the developing brain to insults that mimic those experienced by infants born preterm. At birth, ferret brain development is similar to a 13 week human fetus, with postnatal-day (P) 17 kits considered to be equivalent to an infant at 32&#8211;36 weeks of gestation13.
Our group has recently published a model of extremely preterm (&#60;28 weeks&#39; gestation) brain injury in the P10 ferret by combining inflammatory sensitization with Escherichia coli lipopolysaccharide (LPS) with subsequent exposure to hypoxia and hyperoxia12. In the following protocol, we now describe a late preterm model in the P17 ferret, where LPS sensitization is followed by bilateral cerebral ischemia, hypoxia, and hyperoxia. This results in more severe injury in a subset of animals, and more closely models the complex interaction of prolonged inflammation, ischemia, hypoxia, and oxidative stress experienced in a number of preterm infants who develop brain injury.

<table border="0" cellpadding="0" cellspacing="0" style="width:775px;" width="775">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">80% Oxygen</td>
			<td style="width:196px;">Praxair</td>
			<td style="width:136px;"></td>
			<td style="width:227px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">9% Oxygen</td>
			<td>Praxair</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Absorbent benchtop protector</td>
			<td style="width:196px;">Kimtech</td>
			<td align="right" style="width:136px;">7546</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Automated catwalk</td>
			<td style="width:196px;">Noldus</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Betadine surgical scrub</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bupivacaine</td>
			<td>Patterson Veterinary</td>
			<td>07-888-9382</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Buprenorphine</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Calipers</td>
			<td style="width:196px;">SRA Measurement Products</td>
			<td style="width:136px;">ME-CAL-FP-200</td>
			<td>200mm range, .01 mm resolution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cotton Gauze Sponge</td>
			<td>Fisher Scientific</td>
			<td>22028556</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Curved fine hemostat</td>
			<td>Roboz</td>
			<td>RS-7101</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Curved forceps</td>
			<td>World Precision Instruments</td>
			<td>501215</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Curved suture-tying hemostat</td>
			<td>Roboz</td>
			<td>RS-7111</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethovision tracking software</td>
			<td style="width:196px;">Noldus</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Eye Lubricant</td>
			<td>Rugby</td>
			<td>NDC 0536-1970-72</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ferrets (Mustela putorius furo)</td>
			<td style="width:196px;">Marshall Biosciences</td>
			<td style="width:136px;"></td>
			<td>Outbred (no specific strain)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Formalin</td>
			<td style="width:196px;">Fisher Scientific</td>
			<td style="width:136px;">SF100-4</td>
			<td>10% (Phosphate Buffer/Certified)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hair Clippers</td>
			<td>Conair</td>
			<td>GMT175N</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Insulin Syringes</td>
			<td>BD</td>
			<td>329461</td>
			<td>0.3 cc 3 mm 31G</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Isoflurane</td>
			<td>Piramal</td>
			<td>66794-017-25</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lidocaine</td>
			<td>Patterson Veterinary</td>
			<td>07-808-8202</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">LPS</td>
			<td>List Biological</td>
			<td>LPS Ultrapure #423</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Oxygen sensor</td>
			<td>BW Gas Alert</td>
			<td>GAXT-X-DL-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pentobarbital</td>
			<td style="width:196px;"></td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Plastic chamber</td>
			<td>Tellfresh</td>
			<td align="right">1960</td>
			<td>10L; 373x270x135mm</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Saline Solution, 0.9%</td>
			<td>Hospira</td>
			<td>RL-4492</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Scalpel blade</td>
			<td>Integra Miltex</td>
			<td>297</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Scalpel handle</td>
			<td>World Precision Instruments</td>
			<td>500236</td>
			<td>#3, 13cm</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sterile suture</td>
			<td>Fine Science Tools</td>
			<td>18020-50</td>
			<td>Braided Silk, 5/0</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Surgical clip applicator</td>
			<td>Fine Science Tools</td>
			<td>12020-09</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Surgical clip remover</td>
			<td>Fine Science Tools</td>
			<td>12023-00</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Surgical drapes</td>
			<td>Medline Unidrape</td>
			<td>VET3000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Surgical gloves</td>
			<td>Ansell Perry Inc</td>
			<td>5785004</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Surigical clips</td>
			<td>Fine Science Tools</td>
			<td>12022-09</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Thermometer (rectal)</td>
			<td>YSI</td>
			<td>Precision 4000A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Thermometer (water)</td>
			<td>Fisher Scientific</td>
			<td>14-648-26</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Umbilical tape</td>
			<td>Grafco</td>
			<td>3031</td>
			<td>Sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Water bath</td>
			<td style="width:196px;">Thermo Scientific</td>
			<td style="width:136px;">TSCOL19</td>
			<td>19L</td>
		</tr>
	</tbody>
</table>

a ferret model of inflammation-sensitized late preterm hypoxic-ischemic brain injury

There is an ongoing need for clinically relevant models of perinatal infection and hypoxia-ischemia (HI) in which to test therapeutic interventions for infants with the neurological sequela of prematurity. Ferrets are ideal candidates for modeling the preterm human brain, as they are born lissencephalic and develop gyrencephalic brains postnatally. At birth, ferret brain development is similar to a 13 week human fetus, with postnatal-day (P) 17 kits considered to be equivalent to an infant at 32&#8211;36 weeks&#39; gestation. We describe an injury model in the P17 ferret, where lipopolysaccharide administration is followed by bilateral cerebral ischemia, hypoxia, and hyperoxia. This simulates the complex interaction of prolonged inflammation, ischemia, hypoxia, and oxidative stress experienced in a number of neonates who develop brain injury. Injured animals display a range of gross injury severity, with morphological changes in the brain including narrowing of multiple cortical gyri and associated sulci. Injured animals also show slowed reflex development, slower and more variable speed of locomotion in an automated catwalk, and decreased exploration in an open field. This model provides a platform in which to test putative therapies for infants with neonatal encephalopathy associated with inflammation and HI, study mechanisms of injury that affect cortical development, and investigate pathways that provide resilience in unaffected animals.

Of 34 (n = 18 males, n = 16 females) animals from six litters exposed to the insult, eight animals (24%; n = 4 males, n = 4 females) in the injured group died during the second hypoxia period (n = 5), during temperature management (n = 2), or overnight after the insult (n = 1). In the injured group, nine of 26 survivors (35%) had visible gross injury. Five animals (n = 5 males) had moderate injury, and four animals (n = 2 males, n = 2 females) had severe injury, defined as gross pathology scores of 2&#8211;5 and 6&#8211;...

Watch this Scientific Journal Video about A Ferret Model of Inflammation-sensitized Late Preterm Hypoxic-ischemic Brain Injury at JoVE.com

A Ferret Model of Inflammation-sensitized Late Preterm 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.

There is an ongoing need for clinically relevant models of perinatal infection and hypoxia-ischemia (HI) in which to test therapeutic interventions for ...

This protocol is significant because it offers the first model of inflammation sensitized premature brain injury in the ferrets that results in significant and sustained injury. This technique involves a prolonged insults that incorporates many of the triggers and biochemical mechanisms thought to contribute to the types of brain injuries seen in premature human infants. Demonstrating the brain measurement procedure will be Kylie Corry, a research scientist in our lab and Olivia White, an undergraduate student.Thirty minutes before starting surgery, administer three milligrams per kilogram LPS IP to kits in the injury group and an equivalent volume of sterile saline of three microliters per gram to control animals. Before beginning the procedure, use small animal clippers to remove all of the hair on the ventral neck region of the ferret in a rectangular pattern, taking care to avoid nicking the skin or generating heat rash. Administer local anesthesia to the shaved area and disinfect the skin with three alternating swabs of povidone iodine and seventy percent ethanol.After confirming an appropriate level of sedation by toe pinch, cover the animal with sterile, disposable cut out drapes that expose the neck region. For bilateral carotid artery ligation, use a single use number ten scalpel blade to make a 1.5 centimeter midline incision in the center of the neck and use fine hemostats and curved forceps to bluntly dissect down to the left carotid artery. Dissect the exposed artery away from the associated neurovascular bundle.And use a pair of curved fine forceps to pass a looped ten centimeter length of sterile 5/0 silk suture under the artery. Cut the suture in half and securely tie both lengths of suture to ligate the artery, leaving at least two millimeters between the knots. Transect the left carotid artery between the sutures, taking care to leave the nerve intact and repeat the dissection on the right.Reversibly ligate the right carotid artery with a single sterile 1/8 inch umbilical tie. And close the wound with surgical skin clips. Then, allow the animal to recover in a temperature controlled water bath for at least thirty minutes with monitoring before hypoxia.For sequential hypoxia, hyperoxia and hypoxia treatment, place the animals in the injury group in an airtight chamber within a water bath. Continuously monitor the oxygen concentration within the chamber, as well as the rectal temperature in at least one sentinel animal. Flush the chamber with humidified nine percent oxygen and 91 percent nitrogen and maintain a flow rate of three to five liters per minute, depending on chamber size.Once the concentration of oxygen in the chamber has reached nine percent, continue the delivery for thirty minutes. At the end of the hypoxia treatment, switch the gas supply to 80 percent humidified oxygen and 20 percent nitrogen. After thirty minutes of hyperoxia, open the chamber to allow it to more rapidly reach normoxia, by equilibrating with room air, before sealing the chamber and flushing with nine percent humidified oxygen again.Continuously monitor all of the animals during this second round of hypoxia, taking note of any animals that display bradypnea. Once the concentration of oxygen in the chamber has reached nine percent, continue the hypoxia treatment for thirty minutes. At the end of the treatment, used curved forceps to identify and untie the umbilical tape from the right carotid artery.Reclose the wound with surgical skin clips. Then return all of the kits to the jills for 60 minutes for nursing and recovery. At the end of the recovery period, return the injured animals to the water baths at 37 to 40 degrees Celsius for six hours, adjusting water temperature as needed, to maintain rectal temperatures of 36 to 37 degrees Celsius.Then, return kits to their jills again. After brain harvest and fixation at the appropriate experimental endpoint, place the tips of an electronic caliper on the dorsal and ventral aspects of each brain to measure the height of each injured and non-injured brain. Place the tips of the caliper at the most lateral portions of the temporal lobes to measure the width of each brain and weigh each brain.Use the caliper to measure the longitudinal fissure, all of the sulci from the beginning and end of the most distinct portion of the corresponding sulcus, and all of the gyri from the widest aspect of each corresponding gyrus. Then, place one tip of the caliper at the most posterior point of the longitudinal fissure and the other tip of the caliper at the most posterior part of the cerebellum to measure the amount of exposed cerebellum. Of the 34 animals from six litters exposed to the insult in this study, five animals had moderate injury and four animals had severe injury.With increasing injury, narrowing of the gyri in the temporal and/or occipital lobes is observed, with an associated sulcul shortening and widening of the longitudinal fissure and larger areas of cystic tissue loss in the most severely injured animals. Over the reflex testing period, the injured animals display a slower time to rotate in the negative geotaxis task. A slower time to rotate away from the edge in the Cliff Aversion task and a slower time to right.In the catwalk, injured animals demonstrate a similar average speed to controls but display a significantly greater degree of speed variation during each run. The weight adjusted distance between the paws and hind paws is significantly greater in injured animals, with less pressure exerted per unit paw area, through the four paws. In the open field, the injured animals cover less total distance, stop more frequently and spend significantly more time in the center of the field and less time in the corners.Here, representative heat maps of control and injured animals can be observed. Take care to dissect away the artery from the accompanying nerve and allow the maximum possible hypoxia time. These steps will ensure that the majority of animals survive, whilst also sustaining an injury.Following this procedure, any biochemical, pathological or behavioral outcomes can be assessed, including assessments that are normally restricted to rodent models of brain injury.

Research

JoVE Journal

Neuroscience

Lateral Fluid Percussion: Model of Traumatic Brain Injury in Mice

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and ...

Preterm EEG: A Multimodal Neurophysiological Protocol

Since its introduction in early 1950s, electroencephalography (EEG) has been widely used in the neonatal intensive care units (NICU) for assessment and ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

Traumatic brain injury (TBI) affects millions of people each year, causing impairment of physical, cognitive, and behavioral functions and death. Studies ...

Dorsal Root Ganglion Injection and Dorsal Root Crush Injury as a Model for Sensory Axon Regeneration

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each ...

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

We performed unilateral carotid artery occlusion on CD-1 mice to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of neonatal HI ...

A Novel In Vitro Model of Blast Traumatic Brain Injury

A Novel In Vitro Model of Blast Traumatic Brain Injury

Traumatic brain injury is a leading cause of death and disability in military and civilian populations. Blast traumatic brain injury results from the ...

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

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

Murine Model of Controlled Cortical Impact for the Induction of Traumatic Brain Injury

The Centers for Disease Control and Injury Prevention estimate that almost 2 million people sustain a traumatic brain injury (TBI) every year in the ...

Electromagnetic Controlled Closed-Head Model of Mild Traumatic Brain Injury in Mice

Highly reproducible animal models of traumatic brain injury (TBI), with well-defined pathologies, are needed for testing therapeutic interventions and ...