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.
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	<li>Prior to commencing the procedure, seal, sterilize and autoclave all surgical instruments and surgic...

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	<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. 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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. 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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. 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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.

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			<td height="21" style="height:21px;width:216px;">9% Oxygen</td>
			<td>Praxair</td>
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			<td height="21" style="height:21px;width:216px;">Absorbent benchtop protector</td>
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			<td height="21" style="height:21px;width:216px;">Automated catwalk</td>
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			<td height="20" style="height:20px;">Betadine surgical scrub</td>
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			<td height="20" style="height:20px;">Bupivacaine</td>
			<td>Patterson Veterinary</td>
			<td>07-888-9382</td>
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			<td height="20" style="height:20px;">Buprenorphine</td>
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			<td height="21" style="height:21px;width:216px;">Calipers</td>
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			<td>200mm range, .01 mm resolution</td>
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			<td height="20" style="height:20px;">Cotton Gauze Sponge</td>
			<td>Fisher Scientific</td>
			<td>22028556</td>
			<td></td>
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			<td height="20" style="height:20px;">Curved fine hemostat</td>
			<td>Roboz</td>
			<td>RS-7101</td>
			<td></td>
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			<td height="20" style="height:20px;">Curved forceps</td>
			<td>World Precision Instruments</td>
			<td>501215</td>
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			<td height="20" style="height:20px;">Curved suture-tying hemostat</td>
			<td>Roboz</td>
			<td>RS-7111</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethovision tracking software</td>
			<td style="width:196px;">Noldus</td>
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			<td height="21" style="height:21px;width:216px;">Ferrets (Mustela putorius furo)</td>
			<td style="width:196px;">Marshall Biosciences</td>
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			<td>Outbred (no specific strain)</td>
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			<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>
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			<td height="20" style="height:20px;">Hair Clippers</td>
			<td>Conair</td>
			<td>GMT175N</td>
			<td></td>
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		<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>
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		<tr height="20">
			<td height="20" style="height:20px;">Isoflurane</td>
			<td>Piramal</td>
			<td>66794-017-25</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Lidocaine</td>
			<td>Patterson Veterinary</td>
			<td>07-808-8202</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">LPS</td>
			<td>List Biological</td>
			<td>LPS Ultrapure #423</td>
			<td></td>
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		<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>
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		<tr height="21">
			<td height="21" style="height:21px;">Pentobarbital</td>
			<td style="width:196px;"></td>
			<td style="width:136px;"></td>
			<td></td>
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		<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>
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		<tr height="20">
			<td height="20" style="height:20px;">Saline Solution, 0.9%</td>
			<td>Hospira</td>
			<td>RL-4492</td>
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			<td height="20" style="height:20px;">Scalpel blade</td>
			<td>Integra Miltex</td>
			<td>297</td>
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			<td height="20" style="height:20px;">Scalpel handle</td>
			<td>World Precision Instruments</td>
			<td>500236</td>
			<td>#3, 13cm</td>
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			<td height="20" style="height:20px;">Sterile suture</td>
			<td>Fine Science Tools</td>
			<td>18020-50</td>
			<td>Braided Silk, 5/0</td>
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			<td>Fine Science Tools</td>
			<td>12020-09</td>
			<td></td>
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			<td height="20" style="height:20px;">Surgical clip remover</td>
			<td>Fine Science Tools</td>
			<td>12023-00</td>
			<td></td>
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			<td height="20" style="height:20px;">Surgical drapes</td>
			<td>Medline Unidrape</td>
			<td>VET3000</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Surgical gloves</td>
			<td>Ansell Perry Inc</td>
			<td>5785004</td>
			<td></td>
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			<td height="20" style="height:20px;">Surigical clips</td>
			<td>Fine Science Tools</td>
			<td>12022-09</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Thermometer (rectal)</td>
			<td>YSI</td>
			<td>Precision 4000A</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Thermometer (water)</td>
			<td>Fisher Scientific</td>
			<td>14-648-26</td>
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			<td height="20" style="height:20px;">Umbilical tape</td>
			<td>Grafco</td>
			<td>3031</td>
			<td>Sterile</td>
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			<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>
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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.

a-ferret-model-inflammation-sensitized-late-preterm-hypoxic-ischemic

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 mortality. Based on the nature of primary injury following TBI, complex and heterogeneous secondary consequences result, which are followed by regenerative processes 1,2. Primary injury can be induced by a direct contusion to the brain from skull fracture or from shearing and stretching of tissue causing displacement of brain due to movement 3,4. The resulting hematomas and lacerations cause a vascular response 3,5, and the morphological and functional damage of the white matter leads to diffuse axonal injury 6-8. Additional secondary changes commonly seen in the brain are edema and increased intracranial pressure 9. Following TBI there are microscopic alterations in biochemical and physiological pathways involving the release of excitotoxic neurotransmitters, immune mediators and oxygen radicals 10-12, which ultimately result in long-term neurological disabilities 13,14. Thus choosing appropriate animal models of TBI that present similar cellular and molecular events in human and rodent TBI is critical for studying the mechanisms underlying injury and repair.
Various experimental models of TBI have been developed to reproduce aspects of TBI observed in humans, among them three specific models are widely adapted for rodents: fluid percussion, cortical impact and weight drop/impact acceleration 1. The fluid percussion device produces an injury through a craniectomy by applying a brief fluid pressure pulse on to the intact dura. The pulse is created by a pendulum striking the piston of a reservoir of fluid. The percussion produces brief displacement and deformation of neural tissue 1,15. Conversely, cortical impact injury delivers mechanical energy to the intact dura via a rigid impactor under pneumatic pressure 16,17. The weight drop/impact model is characterized by the fall of a rod with a specific mass on the closed skull 18. Among the TBI models, LFP is the most established and commonly used model to evaluate mixed focal and diffuse brain injury 19. It is reproducible and is standardized to allow for the manipulation of injury parameters. LFP recapitulates injuries observed in humans, thus rendering it clinically relevant, and allows for exploration of novel therapeutics for clinical translation 20.
We describe the detailed protocol to perform LFP procedure in mice. The injury inflicted is mild to moderate, with brain regions such as cortex, hippocampus and corpus callosum being most vulnerable. Hippocampal and motor learning tasks are explored following LFP.

Lateral fluid percussion (LFP), an established model of traumatic brain injury in mice, is demonstrated. LFP fulfills three major criteria for animal models: validity, reliability and clinical relevance. The procedure, consisting of surgical craniotomy, fixation of hub followed by induction of injury, resulting in focal and diffuse injuries, is described.

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 monitoring of brain function in preterm and term babies. Most common indications are the diagnosis of epileptic seizures, assessment of brain maturity, and recovery from hypoxic-ischemic events. EEG recording techniques and the understanding of neonatal EEG signals have dramatically improved, but these advances have been slow to penetrate through the clinical traditions. The aim of this presentation is to bring theory and practice of advanced EEG recording available for neonatal units. 
In the theoretical part, we will present animations to illustrate how a preterm brain gives rise to spontaneous and evoked EEG activities, both of which are unique to this developmental phase, as well as crucial for a proper brain maturation. Recent animal work has shown that the structural brain development is clearly reflected in early EEG activity. Most important structures in this regard are the growing long range connections and the transient cortical structure, subplate. Sensory stimuli in a preterm baby will generate responses that are seen at a single trial level, and they have underpinnings in the subplate-cortex interaction. This brings neonatal EEG readily into a multimodal study, where EEG is not only recording cortical function, but it also tests subplate function via different sensory modalities. Finally, introduction of clinically suitable dense array EEG caps, as well as amplifiers capable of recording low frequencies, have disclosed multitude of brain activities that have as yet been overlooked.
In the practical part of this video, we show how a multimodal, dense array EEG study is performed in neonatal intensive care unit from a preterm baby in the incubator. The video demonstrates preparation of the baby and incubator, application of the EEG cap, and performance of the sensory stimulations.

This video explains the background theory of the neonatal EEG activity and the sensory responses, followed by a live demonstration of their recording in neonatal intensive care unit.

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

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 degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

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

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

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

Traumatic brain injury (TBI) affects millions of people each year, causing impairment of physical, cognitive, and behavioral functions and death. Studies using Drosophila have contributed important breakthroughs in understanding neurological processes. Thus, with the goal of understanding the cellular and molecular basis of TBI pathologies in humans, we developed the High Impact Trauma (HIT) device to inflict closed head TBI in flies. Flies subjected to the HIT device display phenotypes consistent with human TBI such as temporary incapacitation and progressive neurodegeneration. The HIT device uses a spring-based mechanism to propel flies against the wall of a vial, causing mechanical damage to the fly brain. The device is inexpensive and easy to construct, its operation is simple and rapid, and it produces reproducible results. Consequently, the HIT device can be combined with existing experimental tools and techniques for flies to address fundamental questions about TBI that can lead to the development of diagnostics and treatments for TBI. In particular, the HIT device can be used to perform large-scale genetic screens to understand the genetic basis of TBI pathologies.

A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

Here we describe a method to inflict closed head traumatic brain injury (TBI) in Drosophila. This method provides a gateway to investigate the cellular and molecular mechanisms that underlie TBI pathologies using the vast array of experimental tools and techniques available for flies.

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 other anatomically, it is important to identify an appropriate model to use for the study of axon regeneration. By using a suitable model, we can formulate a specific treatment based on the severity of injury, the neuronal cell type of interest, and the desired spinal tract for assessing regeneration. Within the sensory pathway, DRG neurons are responsible for relaying sensory information from the periphery to the CNS. We present here a protocol that uses a DRG injection with a viral vector and a concurrent dorsal root crush injury in the lower cervical spinal cord of an adult rat as a model to study sensory axon regeneration. As demonstrated using a control virus, AAV5-GFP, we show the effectiveness of a direct DRG injection in transducing DRG neurons and tracing sensory axons into the spinal cord. We also show the effectiveness of the dorsal root crush injury in denervating the forepaw as an injury model for evaluating axon regeneration. Despite the requirement for specialized training to perform this invasive surgical procedure, the protocol is flexible, and potential users can modify many parts to accommodate their experimental requirements. Importantly, it can serve as a foundation for those in search of a suitable animal model for their studies. We believe that this article will help new users to learn the procedure in a very efficient and effective manner.

This protocol presents the use of a dorsal root ganglion (DRG) injection with a viral vector and a concurrent dorsal root crush injury in an adult rat as a model to study sensory axon regeneration. This model is suitable for investigating the use of gene therapy to promote 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 brain injury by studying neurobehavioral functions in these mice compared to non-operated (i.e., normal) mice. During the study, Rice-Vannucci&#39;s method was used to induce neonatal HI brain damage in postnatal day 7-10 (P7-10) mice. The HI operation was performed on the pups by unilateral carotid artery ligation and exposure to hypoxia (8% O2 and 92% N2 for 90 min). One week after the operation, the damaged brains were evaluated with the naked eye through the semi-transparent skull and were categorized into subgroups based on the absence (&#34;no cortical injury&#34; group) or presence (&#34;cortical injury&#34; group) of cortical injury, such as a lesion in the right hemisphere. On week 6, the following neurobehavioral tests were performed to evaluate the cognitive and motor functions: passive avoidance task (PAT), ladder walking test, and grip strength test. These behavioral tests are helpful in determining the effects of neonatal HI brain injury and are used in other mouse models of neurodegenerative diseases. In this study, neonatal HI brain injury mice showed motor deficits that corresponded to right hemisphere damage. The behavioral test results are relevant to the deficits observed in human neonatal HI patients, such as cerebral palsy or neonatal stroke patients. In this study, a mouse model of neonatal HI brain injury was established and showed different degrees of motor deficits and cognitive impairment compared to non-operated mice. This work provides basic information on the HI mouse model. MRI images demonstrate the different phenotypes, separated according to the severity of brain damage by motor and cognitive tests.

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.

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

Traumatic brain injury is a leading cause of death and disability in military and civilian populations. Blast traumatic brain injury results from the detonation of explosive devices, however, the mechanisms that underlie the brain damage resulting from blast overpressure exposure are not entirely understood and are believed to be unique to this type of brain injury. Preclinical models are crucial tools that contribute to better understand blast-induced brain injury. A novel in vitro blast TBI model was developed using an open-ended shock tube to simulate real-life open-field blast waves modelled by the Friedlander waveform. C57BL/6N mouse organotypic hippocampal slice cultures were exposed to single shock waves and the development of injury was characterized up to 72 h using propidium iodide, a well-established fluorescent marker of cell damage that only penetrates cells with compromised cellular membranes. Propidium iodide fluorescence was significantly higher in the slices exposed to a blast wave when compared with sham slices throughout the duration of the protocol. The brain tissue injury is very reproducible and proportional to the peak overpressure of the shock wave applied.

A Novel In Vitro Model of Blast Traumatic Brain Injury

This paper describes a novel model of primary blast traumatic brain injury. A compressed-air driven shock tube is used to expose in vitro mouse hippocampal slice cultures to a single shock wave. This is a simple and rapid protocol generating a reproducible brain tissue injury with a high throughput.

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 vascular effects. Impact (i.e., non-blast) traumatic brain injury (TBI) is known to decrease pressure autoregulation in the cerebral vasculature in both humans and experimental animals. The hypothesis that blast-induced traumatic brain injury (bTBI), like impact TBI, results in impaired cerebral vascular reactivity was tested by measuring myogenic dilatory responses to reduced intravascular pressure in rodent middle cerebral arterial (MCA) segments from rats subjected to mild bTBI using an Advanced Blast Simulator (ABS) shock tube. Adult, male Sprague-Dawley rats were anesthetized, intubated, ventilated and prepared for Sham bTBI (identical manipulation and anesthesia except for blast injury) or mild bTBI. Rats were randomly assigned to receive Sham bTBI or mild bTBI followed by sacrifice 30 or 60 min post-injury. Immediately after bTBI, righting reflex (RR) suppression times were assessed, euthanasia at the time points post-injury was completed, the brain was harvested and the individual MCA segments were collected, mounted and pressurized. As the intraluminal pressure perfused through the arterial segments was reduced in 20 mmHg increments from 100 to 20 mmHg, MCA diameters were measured and recorded. With decreasing intraluminal pressure, MCA diameters steadily increased significantly above baseline in the Sham bTBI groups while MCA dilator responses were significantly reduced (p &#60; 0.05) in both bTBI groups as evidenced by the impaired, smaller MCA diameters recorded for the bTBI groups. In addition, RR suppression in the bTBI groups was significantly (p &#60; 0.05) higher than in the Sham bTBI groups. MCA&#39;s collected from the Sham bTBI groups exhibited typical vasodilatory properties to decreases in intraluminal pressure while MCA&#39;s collected following bTBI exhibited significantly impaired myogenic vasodilatory responses to reduced pressure that persisted for at least 60 min after bTBI.

Here, we present a protocol to describe methods for ex vivo vascular reactivity determination following a primary blast traumatic brain injury (bTBI) using isolated, pressurized, rodent middle cerebral arterial (MCA) segments. bTBI induction is accomplished using a shock tube, also known as an Advanced Blast Simulator (ABS) device.

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 United States. In fact, TBI is a contributing factor to over a third of all injury-related mortality. Nonetheless, the cellular and molecular mechanisms underlying the pathophysiology of TBI are poorly understood. Thus, preclinical models of TBI capable of replicating the injury mechanisms pertinent to TBI in human patients are a critical research need. The controlled cortical impact (CCI) model of TBI utilizes a mechanical device to directly impact the exposed cortex. While no model can full recapitulate the disparate injury patterns and heterogeneous nature of TBI in human patients, CCI is capable of inducing a wide range of clinically applicable TBI. Furthermore, CCI is easily standardized allowing investigators to compare results across experiments as well as across investigative groups. The following protocol is a detailed description of applying a severe CCI with a commercially available impacting device in a murine model of TBI.

Here we describe a protocol for the induction of murine traumatic brain injury via an open-head controlled cortical impact.

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 understanding the mechanisms of how a TBI alters brain function. The availability of multiple animal models of TBI is necessary to model the different aspects and severities of TBI seen in people. This manuscript describes the use of a midline closed head injury (CHI) to develop a mouse model of mild TBI. The model is considered mild because it does not produce structural brain lesions based on neuroimaging or gross neuronal loss. However, a single impact creates enough pathology that cognitive impairment is measurable at least 1 month after injury. A step-by-step protocol to induce a CHI in mice using a stereotaxically guided electromagnetic impactor is defined in the paper. The benefits of the mild midline CHI model include the reproducibility of the injury-induced changes with low mortality. The model has been temporally characterized up to 1 year after the injury for neuroimaging, neurochemical, neuropathological, and behavioral changes. The model is complementary to open skull models of controlled cortical impact using the same impactor device. Thus, labs can model both mild diffuse TBI and focal moderate-to-severe TBI with the same impactor.

The protocol describes mild traumatic brain injury in a mouse model. In particular, a step-by-step protocol to induce a mild midline closed head injury and the characterization of the animal model is fully explained.

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