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 surgical drapes. Prepare pre-operative medications in sterile vials. Calculate the flow rate required to replace the air in the hypoxia/hyperoxia chamber with the experimental gas in 8&#8211;10 min.</li>
	<li>Prepare the lipopolysaccharide (LPS from E. coli 055:B5) in sterile saline to produce a concentration of 1 mg/mL. Remove P17 ferret kits from their jills. Weigh and number them. Randomize animals by litter and sex to the control or injured (or treatment) groups.</li>
	<li>Using a 300 &#181;L insulin syringe, administer 3 mg/kg of LPS intraperitoneally to kits in the injury group, and an equivalent volume of sterile saline vehicle (3 &#181;L/g) to control animals.</li>
	<li>Place animals in a chamber within a water bath at 37&#8211;40 &#176;C in order to maintain a target rectal temperature of 36&#8211;37 &#176;C throughout the surgical procedures.</li>
</ol>
2. Anesthesia
<ol>
	<li>During the procedure, continually monitor temperature, respiration rate, and heart rate of the animal.</li>
	<li>Administer buprenorphine (0.05 mg/kg) subcutaneously 30 min before the surgical procedure. Induce anesthesia in a mixture of 3% isoflurane balanced with 100% oxygen. Remove the kit from the induction chamber and place it supine on a draped surgical water blanket set to 37 &#176;C. Transfer anesthesia to the nose cone and reduce isoflurane level to 2&#8211;3%.</li>
</ol>
3. Surgical Preparation
<ol>
	<li>Using small animal clippers remove all hair on the ventral neck region. Shave in a rectangular pattern with care to avoid nicking the skin or generating razor rash. Administer local anesthesia to the shaved area using intradermal lidocaine (4 mg/kg) and bupivacaine (2.5 mg/kg).</li>
	<li>Prepare the neck by alternating application of povidone-iodine and 70% ethanol scrub with sterile cotton swabs. Repeat the scrub such that povidone-iodine and 70% ethanol are each applied 3x in alternating fashion.</li>
	<li>Confirm depth of anesthesia via absence of toe-pinch reflex. Maintain level of isoflurane at the minimum percentage required for a surgical plane of anesthesia. Using sterile disposable cut-out drapes that expose the neck region, drape the animal.</li>
</ol>
4. Bilateral Carotid Artery Ligation
<ol>
	<li>With a single-use #11 scalpel blade, make a 1.5 cm midline incision in the center of the neck. Using fine hemostats and curved forceps, bluntly dissect down to the left carotid artery. Dissect the artery away from the associated neurovascular bundle.</li>
	<li>Using a pair of curved fine forceps, pass a looped 10 cm length of sterile 5-0 silk suture under the artery. Cut the suture in half. Ligate the artery by securely tying both lengths of suture, leaving at least 2 mm between the knots. Transect the left carotid artery between the sutures, taking care to leave the nerve intact.</li>
	<li>Repeat the dissection on the right side. Reversibly ligate the right carotid artery with a single sterile 1/8 inch umbilical tie. Close the wound with surgical skin clips.</li>
	<li>Allow the animal to recover in a temperature-controlled water bath for at least 30 min before hypoxia. 
	NOTE: If the arteries are not fully isolated from the rest of the neurovascular bundle, increased mortality may be seen before or during later hypoxia.</li>
</ol>
5. Sequential Hypoxia, Hyperoxia, and Hypoxia
<ol>
	<li>During hypoxia and hyperoxia, alter water bath temperature as necessary to maintain rectal temperature during hypoxia at 37 &#176;C in the sentinel animal(s).</li>
	<li>Place animals in the injury group in an airtight chamber within a water bath. Continuously monitor oxygen concentration within the chamber, as well as rectal temperature in at least one sentinel animal. Flush the chamber with humidified 9% oxygen (91% nitrogen) then maintain a flow rate of 3&#8211;5 L/min, depending on chamber size. Once the concentration of oxygen in the chamber has reached 9%, continue for 30 min.</li>
	<li>After 30 min, switch gas supply to 80% humidified oxygen (20% nitrogen), and allow the chamber to reach the target concentration based on flow rate and chamber size. Continue for 30 min of hyperoxia. Open the chamber to allow it to more rapidly reach normoxia by equilibrating with room air.</li>
	<li>Seal the chamber, and flush with 9% humidified oxygen. Continuously monitor all animals visually, taking note of animals that display bradypnea. Once the concentration of oxygen in the chamber has reached 9%, continue for 30 min. If intra-hypoxic mortality (respiratory arrest) in any of the animals is seen before the end of the 30 min period, terminate hypoxia immediately.</li>
</ol>
6. Reversal of Right Carotid Artery Ligation
<ol>
	<li>Return animals to the surgical area, and induce anesthesia in a mixture of 3% isoflurane balanced with 100% oxygen. Transfer anesthesia to nose cone and reduce isoflurane level to 2&#8211;3%. Remove the surgical wound clips and re-prepare the wound area with povidone-iodine. Confirm depth of anesthesia via absence of toe-pinch reflex. Maintain the level of isoflurane at the minimum percentage required for a surgical plane of anesthesia. Using sterile disposable cut-out drapes that expose the neck region, drape the animal.</li>
	<li>Using curved forceps, identify and untie the umbilical tape from the right carotid artery. Close the wound with surgical skin clips.</li>
</ol>
7. Recovery and Temperature Management
<ol>
	<li>Return all kits to their jills for 60 min, for nursing and recovery. After 60 min, return injured animals to the water baths at 37&#8211;40 &#176;C for 6 h, adjusting water temperature as needed in order to maintain rectal temperature at 36&#8211;37 &#176;C. Return kits to their jills.</li>
	<li>Remove surgical clips 10&#8211;14 days after surgery (P27&#8211;P31).</li>
</ol>
8. Reflex Testing
<ol>
	<li>Perform all the reflex tests daily from P21&#8211;P28, and at least 3x per week from P28&#8211;P42, whilst remaining blinded to exposure (or treatment) group. Prior to reflex testing, place kits in a chamber with heat assistance (37 &#176;C water bath, heat pad, etc.) for 1 h. For each test, complete all trials per kit before testing the next kit.</li>
	<li>Negative geotaxis (25&#176;)
	<ol>
		<li>Place a flat board (16 &#189; in. x 12 in.) wrapped in an absorbent benchtop protector against an object so that the board forms a 25&#176; angle with the table. Place a kit on the board prone and facing downhill, approximately 75% of the way up the board.</li>
		<li>Ensure the kit&#8217;s body is straight and that it has all four paws gripped against the board before releasing it. As soon as the kit is placed, start the time assessment.</li>
		<li>Record the time when the kit manages to rotate its body 90&#176; relative to its starting position. Record the time at which the kit rotates its body 180&#176; and takes a full step towards the top of the board. Perform 3 trials at the 25&#176; incline before moving onto the next test.</li>
	</ol>
	</li>
	<li>Negative geotaxis (45&#176;)
	<ol>
		<li>Perform 3 trials of the previously described negative geotaxis test again, this time with the board set to a 45&#176; angle.</li>
	</ol>
	</li>
	<li>Cliff aversion
	<ol>
		<li>Place a padded platform around 1 foot below the ledge to minimize injury to kits if they fall.</li>
		<li>Place a kit facing, and perpendicular to, the edge of the lab bench. Ensure the kit&#39;s body is straight, with its front paws flush with the edge. Begin time assessment from the moment the kit is placed. Take care to differentiate between conscious movement away from the cliff and other spontaneous movements that do not involve coordinated walking.</li>
		<li>Record the time when the kit moves its body away from the edge (defined as the kit backing up, turning its body, or moving its front limbs away from the edge). Record the time the kit completes its first step in the opposite direction of the edge (defined as any direction or angle past a 90&#176; rotation from its starting position facing the edge).</li>
		<li>Perform 3 cliff aversion trials per kit before moving onto the next test.</li>
	</ol>
	</li>
	<li>Righting reflex
	<ol>
		<li>Place a kit supine on the bench, holding it gently in that position before releasing it and simultaneously beginning the stopwatch. Record the time that the kit brings itself to rest with all four paws simultaneously flat against the bench in weight-bearing positions. Record the time when the kit takes a full step in any direction (defined as the placement of all four paws to achieve progress in a given direction with no spinning or dragging of the body).</li>
		<li>Perform 5 trials of the righting reflex test per animal.</li>
		<li>After every kit has completed the 5 righting reflex trials, return the litter to the jill.</li>
	</ol>
	</li>
</ol>
9. Catwalk Testing
<ol>
	<li>On P42, remove kits from the jill. Place kits in plastic cages approximately 10 min before testing, so they can acclimate to the environment. Turn the lights off in the testing room to ensure ambient light does not affect catwalk function.</li>
	<li>Create a new experiment in the relevant software. Adjust experimental settings so that the maximum run duration does not exceed 10.00 s and the minimum run duration is not less than 1.50 s. Set the maximum speed variation so it does not exceed 60%. Set a minimum requirement of three compliant runs for each animal.</li>
	<li>Adjust the width of the walkway relative to the size of the animal so that it is able to freely locomote without touching the walls while remaining narrow enough to discourage turning. Add a new detection setting in the Detection settings profiles tab using auto detection. Use the same detection settings for all litters and animals at a given age.</li>
	<li>Thoroughly clean the walkway with a low-lint paper tissue and 70% ethanol before and after each animal. Clean the ferret&#39;s paws regularly to improve accuracy of detection and classification. Once the walkway and animal are prepared, begin trial acquisition.</li>
	<li>Pause acquisition to clean the catwalk if footprints accumulate on the glass, or if animals pass urine or feces. Stop acquisition once the catwalk software has recognized three compliant runs based on the pre-determined experiment settings.</li>
</ol>
10. Open Field Testing (P42)
<ol>
	<li>Use a non-porous acrylic box (55 cm x 55 cm x 40 cm high) painted matte-white. Position the camera so that it is centered directly above box and all four walls are captured. Clean the testing arena with 70% ethanol before first use and between animals.</li>
	<li>In the relevant software, select New From Template and Apply a Pre-defined Template. Continue the set-up procedure by sequentially selecting Subject type: Other; Arena template: Open field, square; Zone template: Center, border, corners; Features to track: Center-point.</li>
	<li>Open Arena Settings and grab the background image from the camera input, making sure that the tops of the arena walls are visible. Calibrate the dimensions of the arena using the scale tool.</li>
	<li>Adjust the pre-defined arena zones by resizing the outlines to fit the wall zones (NW, NE, SW, SE) and floor zones (left top, middle top, right top, left middle, center, right middle, left bottom, middle bottom, right bottom). Validate the set-up to confirm that no zones overlap.</li>
	<li>Open the Acquisition window and press Start Acquisition. Place the ferret at the center of the testing arena oriented in such a way that is consistent for every test subject. Allow the ferret to move freely throughout the arena for a period of 5 min. At the end of the testing period, press Stop Acquisition. Repeat the procedure with the next ferret. 
	NOTE: All experimenters in the room should position themselves to be unobservable by the ferret and remain quiet during the testing period.</li>
</ol>
11. Fixation-perfusion
<ol>
	<li>On P42, deeply anesthetize kits with 5% isoflurane. Administer a pentobarbital overdose (120&#8211;150 mg/kg i.p.). Ensure deep anesthesia by lack of response to toe pinch and loss of respiratory movements.</li>
	<li>Transfer the animal to a fume hood. Open the thorax and clamp the descending aorta using fine hemostats. Cut the right atrium. Using a perfusion pump, perfuse the left ventricle with 60 mL of sterile saline at a rate of 30 mL/min. Perfuse with 60 mL of formalin (10% formaldehyde) at a rate of 30 mL/min.</li>
	<li>Decapitate the carcass, and remove the brain from the skull using scissors, forceps, rongeurs, and a spatula. Take high-resolution photographs of the dorsal, ventral, and lateral aspects of each brain. Post-fix the brain in formalin for at least 48 h.</li>
</ol>
12. Ex Vivo Brain Measurement
<ol>
	<li>Remove the brain from formalin (step 11.3) and place onto a paper towel to absorb excess liquid.</li>
	<li>Using an electronic caliper, measure the height of the brain by placing the tips of the caliper at the dorsal and ventral aspects of the brain. Measure the length of the brain by placing the tips of the caliper at the olfactory bulb and the most posterior border of the occipital lobe. Measure the width of the brain by placing the tips of the caliper at the most lateral portions of the temporal lobes. Weigh the brain.</li>
	<li>Measure the longitudinal fissure (anterior and posterior to the cruciate sulcus), lateral sulci, suprasylvian sulci, coronal sulci, pseudosylvian sulci, ansinate sulci, cruciate sulci, presylvian sulci, lateral gyri, suprasylvian gyri, sigmoid gyri (anterior and posterior), coronal gyri, ectosylvian gyri (anterior and posterior), and orbital gyri. Measure all sulci from the beginning and end of the most distinct portion of the corresponding sulcus. Measure all gyri from the widest aspect of each corresponding gyrus.</li>
	<li>Measure the amount of cerebellum exposed by placing one tip of the caliper at the most posterior point of the longitudinal fissure and placing the other tip of the caliper at the most posterior part of the cerebellum. 
	NOTE: The ferret brain atlas, as found in Biology and Diseases of the Ferret14, was used to develop the ex vivo ferret brain measurements.</li>
</ol>
13. Gross Injury Scoring
<ol>
	<li>Using the photographs taken in step 11.3., apply the scoring criteria in steps 13.2&#8211;13.4 to assess gross brain injury (0&#8211;9 scale) whilst remaining blinded to exposure (or treatment) groups.</li>
	<li>Assess the longitudinal fissure. If it appears normal, assign a score of 0. If it is mildly widened (approximately 2x normal width), but the increase in width is incomplete along length of fissure, assign a score of 1. If it is moderately widened (approximately 2&#8211;3x normal), assign a score of 2. If it is markedly widened, with a visible gap &#62;3x normal width along most of the length of fissure, apply a score of 3.</li>
	<li>Assess the lateral sulci. If they show normal definition, with separation of the lateral gyri and suprasylvian gyri, assign a score of 0. If mild unilateral or bilateral reduced definition of sulcus is seen, particularly in the caudal portion, with minimal narrowing of frontal and temporal lobes relative to the occipital lobes, assign a score of 1.
	<ol>
		<li>If moderately reduced definition of the sulci is seen, with depression of the suprasylvian gyri, narrowing of the coronal and ectosylvian gyri, and mild narrowing of frontal and temporal lobes relative to occipital lobes, assign a score of 2. If unilateral cystic degeneration is seen, with minimal change of the contralateral hemisphere, assign a score of 3. If poor definition of the lateral sulci is present, with bilateral cystic or severe degeneration of the occipital and temporal lobes, assign a score of 4.</li>
	</ol>
	</li>
	<li>Assess the visible portion of the cerebellum. If it appears normal, with (&#60;75% of the vermis and &#60;66% of the hemispheres visible, assign a score of 0. If 75&#8211;90% of the vermis and &#8805;66% of the hemispheres are visible, assign a score of 1. If most of the cerebellum is visible, showing all of the vermis and &#8805;66% of hemispheres, assign a score of 2.</li>
</ol>
14. Data Analysis
<ol>
	<li>For reflex testing data, assign failures a score of 61 s to allow them to be compared to successes at the end of time (60 s), but with a worse ranking in the statistical analysis. Calculate an area under the curve for each animal over time in each of the reflex tests.</li>
	<li>Adjust catwalk data that involves paw size of pressure by the weight of the animal.</li>
	<li>Analyze data using non-parametric statistical methods, describing data using the median and interquartile range (IQR).</li>
</ol>

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The authors have nothing to disclose.

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;">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 ...

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

Research

JoVE Journal

Neuroscience

7.4K Views.  University of Washington. 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.

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

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 ...

Stereotactic Atlas-Guided Laser Capture Microdissection of Brain Regions Affected by Traumatic Injury

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial distribution. Such techniques also potentially skew gene expression analysis because the process itself can alter expression patterns in individual cells. Here we describe a laser capture microdissection (LCM) method to selectively collect specific brain regions affected by traumatic brain injury (TBI) by using a modified Nissl (cresyl violet) staining protocol and the guidance of a rat brain atlas. LCM provides access to brain regions in their native positions and the ability to use anatomical landmarks for identification of each specific region. To this end, LCM has been used previously to examine brain region specific gene expression in TBI. This protocol allows examination of TBI-induced alterations in gene and microRNA expression in distinct brain areas within the same animal. The principles of this protocol can be amended and applied to a wide range of studies examining genomic expression in other disease and/or animal models.

We describe the use of laser capture microdissection to obtain samples of distinct cell populations from different brain regions for gene and microRNA analysis. This technique allows the study of differential effects of traumatic brain injury in specific regions of the rat brain.

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial ...

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 ...