This work was supported by the FY 2013 Research Exchange Program between JSPS and CNRS, JSPS KAKENHI Grant Number 26860858, and the Narishige Neuroscience Research Foundation. We thank Drs. Mariko Harada-Shiba and Kyoko Shioya for helpful discussions. We thank Mari Furuta, Mutsumi Sakamoto, Ritsuko Maki, and Dr. Emi Tanaka for excellent technical assistance.

All experiments were performed in accordance with protocols approved by the Experimental Animal Care and Use Committee of the National Cerebral and Cardiovascular Center, Suita, Japan.
1. Prepare the following animals and materials for MIUH surgery
<ol>
	<li>Prepare timed pregnant Sprague-Dawley rats at gestational day 17, i.e., embryonic day 17 (E17). The dams&#39; average body weights are 307.0 &#177; 40.7 g (mean &#177; SD, n = 9).</li>
	<li>Prepare metal microcoils; inner diameter 0.24 mm, made from gold-coated steel.</li>
	<li>Prepare all the materials as per the Table of Materials.</li>
</ol>
2. Prepare for MIUH surgery
<ol>
	<li>Warm a heating pad at 37 &#176;C for rat placement</li>
	<li>Place a sterile diaper on the heating pad</li>
	<li>Warm sterile saline at 37 &#176;C in a water bath</li>
</ol>
3. Perform MIUH surgery
<ol>
	<li>Perform surgical procedures under sterile condition. Sterilize coils, forceps, needle with 70% ethanol before use. Use sterile gloves, and occasionally disinfect by applying ethanol spray.</li>
	<li>Place the rat in the anesthesia induction box. Introduce 4% isoflurane into the box (approximately 5 min).</li>
	<li>Place the rat onto a sterile diaper on the heating pad in a supine position after the animal is thoroughly anesthetized in the induction box. Check for depth of anesthesia by the lack of a response to a toe pinch. Maintain 1.5-2.0% isoflurane with a nose cone.</li>
	<li>Apply depilatory foam onto the abdomen from the navel down to the region of the pelvic arch. After several minutes, wipe off the depilatory foam with a paper towel. A shaver may be used as a substitute for the depilatory foam.</li>
	<li>Apply gauze soaked in iodine disinfectant solution to the depilated region of the skin.</li>
	<li>Place a surgical drape with a round opening to cover the abdomen other than the area of the surgical incision. Several pieces of gauze may be used as a substitute for the drape.</li>
	<li>Using a surgical scalpel, make an incision approximately 2.5 cm long on the lower abdomen from the upper edge of the pubic bone straight up toward the navel. Then, make an incision through the muscle layer underneath.</li>
	<li>Place several pieces of sterile gauze around the opening of the drape, and wet the gauze with warmed saline.</li>
	<li>Push both lateral sides of the abdomen gently, so that a portion of uterine horn emerges from the incision.</li>
	<li>Disinfect the surgeon&#39;s hands with 70% ethanol.</li>
	<li>Gently pull the entire uterine horn from the abdominal cavity manually without using forceps. Using surgical forceps is not advisable, as they may easily damage the fragile uterus, fetuses, and blood vessels by compressing them strongly and unevenly.</li>
	<li>Place the uterine horn on the wet gauze.</li>
	<li>Perform the same procedure for the other uterine horn.</li>
	<li>Count the number of fetuses.</li>
	<li>Apply warmed saline frequently to keep the uterus warm and wet throughout the surgery.
	<ol>
		<li>Change the diaper when it has absorbed too much saline. To maintain stability, repeat disinfection gloves with 70% ethanol and tips of the instrument with iodine. Keeping the non-sterile fingers away from the sterile tips of the instrument is crucial.</li>
	</ol>
	</li>
	<li>Identify the proximal part of the main arcade of the ovarian and uterine arteries under a stereomicroscope. 
	NOTE: It is crucial to distinguish the main arcade from the branches to the placentas. Note that the surgical operation may be easier when the uterine horn is flipped. Occasionally, applying a coil at the very proximal part of the arcade is difficult, because it is located deep within the abdominal cavity and arteries to the placenta are very close to the proximal part of the arcade. In this case, a coil can be applied to a part of the arcade after the first branch forks off.</li>
	<li>Separate the artery from the vein running parallel to it with forceps and make space between them.</li>
	<li>Place a piece of string (silk suture 4.0, 5 cm long) underneath the artery.</li>
	<li>Lift up both ends of the string with forceps so that the artery is lifted and separated from the fragile vein.</li>
	<li>Holding the edge of the microcoil with fine forceps in the other hand, place a microcoil beside the lifted artery (Figure 2.1).</li>
	<li>Rotate the lifted artery by using the string around the microcoil to wrap the microcoil around the artery. It is easier to rotate the artery around the coil than to rotate the coil around the artery (Figure 2.2).</li>
	<li>After rotating the artery 3 or 4 turns (coils have 5 turns), change the site at which the coil is held to the other edge. Hold the edge of the coil that has been wrapped around the artery with forceps. Change the site at which the string lifts the artery to the other side of coil. Rotate the artery around the coil so that the coil completely (5 turns) wraps the artery (Figure 2.3, 2.4).</li>
	<li>Remove the string.</li>
	<li>Perform the same procedure for the other three arteries.</li>
	<li>Note that frequently applying warmed saline to the uterus is crucial; otherwise, the mortality rate of the fetuses increases, and hypothermia might be neuroprotective.</li>
	<li>Gently return the uterine horn into the abdominal cavity. Lifting an edge of the abdominal wall incision with forceps can make this procedure easier.</li>
	<li>Stich the abdominal muscle, and then stich the abdominal skin with suture (silk suture 4.0). Tie the suture after each stich, i.e. interrupted single sutures.</li>
	<li>Apply gauze soaked in iodine disinfectant solution on and around the surgical incision.</li>
	<li>Administer the analgesic meloxicam at 0.5 mg/kg body weight subcutaneously.</li>
	<li>Allow for 30 min of recovery in a warmed cage. Check animals if fully awake and moving around the cage, return to home cage.</li>
</ol>

<ol><li>Ohshima, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mild+intrauterine+hypoperfusion+reproduces+neurodevelopmental+disorders+observed+in+prematurity%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Mild intrauterine hypoperfusion reproduces neurodevelopmental disorders observed in prematurity.</a> Sci Rep. 6, 39377(2016).</li>
<li>Specified Report on Vital Statistics in FY2010, Ministry of Health, Labour and Welfare, Japan. , Available from:
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<li>Anderson, P., Doyle, L. W., Victorian Infant Collaborative Study, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neurobehavioral+outcomes+of+school-age+children+born+extremely+low+birth+weight+or+very+preterm+in+the+1990s%5BTitle%5D)+AND+%22JAMA%22%5BJournal%5D)">Neurobehavioral outcomes of school-age children born extremely low birth weight or very preterm in the 1990s.</a> JAMA. 289 (24), 3264-3272 (2003).</li>
<li>Levine, T. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Early+childhood+neurodevelopment+after+intrauterine+growth+restriction%3A+a+systematic+review%5BTitle%5D)+AND+%22Pediatrics%22%5BJournal%5D)">Early childhood neurodevelopment after intrauterine growth restriction: a systematic review.</a> Pediatrics. 135 (1), 126-141 (2015).</li>
<li>Sucksdorff, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Preterm+Birth+and+Poor+Fetal+Growth+as+Risk+Factors+of+Attention-Deficit%2F+Hyperactivity+Disorder%5BTitle%5D)+AND+%22Pediatrics%22%5BJournal%5D)">Preterm Birth and Poor Fetal Growth as Risk Factors of Attention-Deficit/ Hyperactivity Disorder.</a> Pediatrics. 136 (3), e599-e608 (2015).</li>
<li>Volpe, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Brain+injury+in+premature+infants%3A+a+complex+amalgam+of+destructive+and+developmental+disturbances%5BTitle%5D)+AND+%22Lancet+Neurol%22%5BJournal%5D)">Brain injury in premature infants: a complex amalgam of destructive and developmental disturbances.</a> Lancet Neurol. 8 (1), 110-124 (2009).</li>
<li>Nardozza, L. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fetal+growth+restriction%3A+current+knowledge%5BTitle%5D)+AND+%22Arch+Gynecol+Obstet%22%5BJournal%5D)">Fetal growth restriction: current knowledge.</a> Arch Gynecol Obstet. 295 (5), 1061-1077 (2017).</li>
<li>Chang, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Preterm+birth+and+the+role+of+neuroprotection%5BTitle%5D)+AND+%22Bmj%22%5BJournal%5D)">Preterm birth and the role of neuroprotection.</a> Bmj. 350, g6661(2015).</li>
<li>Coq, J. O., Delcour, M., Massicotte, V. S., Baud, O., Barbe, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Prenatal+ischemia+deteriorates+white+matter%2C+brain+organization%2C+and+function%3A+implications+for+prematurity+and+cerebral+palsy%5BTitle%5D)+AND+%22Dev+Med+Child+Neurol%22%5BJournal%5D)">Prenatal ischemia deteriorates white matter, brain organization, and function: implications for prematurity and cerebral palsy.</a> Dev Med Child Neurol. 58, Suppl 4. 7-11 (2016).</li>
<li>Jantzie, L. L., Corbett, C. J., Firl, D. J., Robinson, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Postnatal+Erythropoietin+Mitigates+Impaired+Cerebral+Cortical+Development+Following+Subplate+Loss+from+Prenatal+Hypoxia-Ischemia%5BTitle%5D)+AND+%22Cereb+Cortex%22%5BJournal%5D)">Postnatal Erythropoietin Mitigates Impaired Cerebral Cortical Development Following Subplate Loss from Prenatal Hypoxia-Ischemia.</a> Cereb Cortex. 25 (9), 2683-2695 (2015).</li>
<li>Kubo, K. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Association+of+impaired+neuronal+migration+with+cognitive+deficits+in+extremely+preterm+infants%5BTitle%5D)+AND+%22JCI+Insight%22%5BJournal%5D)">Association of impaired neuronal migration with cognitive deficits in extremely preterm infants.</a> JCI Insight. 2 (10), (2017).</li>
<li>Delcour, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mild+musculoskeletal+and+locomotor+alterations+in+adult+rats+with+white+matter+injury+following+prenatal+ischemia%5BTitle%5D)+AND+%22Int+J+Dev+Neurosci%22%5BJournal%5D)">Mild musculoskeletal and locomotor alterations in adult rats with white matter injury following prenatal ischemia.</a> Int J Dev Neurosci. 29 (6), 593-607 (2011).</li>
<li>Gilbert, J. S., Babcock, S. A., Granger, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hypertension+produced+by+reduced+uterine+perfusion+in+pregnant+rats+is+associated+with+increased+soluble+fms-like+tyrosine+kinase-1+expression%5BTitle%5D)+AND+%22Hypertension%22%5BJournal%5D)">Hypertension produced by reduced uterine perfusion in pregnant rats is associated with increased soluble fms-like tyrosine kinase-1 expression.</a> Hypertension. 50 (6), 1142-1147 (2007).</li>
<li>Granger, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reduced+uterine+perfusion+pressure+%28RUPP%29+model+for+studying+cardiovascular-renal+dysfunction+in+response+to+placental+ischemia%5BTitle%5D)+AND+%22Methods+Mol+Med%22%5BJournal%5D)">Reduced uterine perfusion pressure (RUPP) model for studying cardiovascular-renal dysfunction in response to placental ischemia.</a> Methods Mol Med. 122, 383-392 (2006).</li>
<li>Mazur, M., Miller, R. H., Robinson, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Postnatal+erythropoietin+treatment+mitigates+neural+cell+loss+after+systemic+prenatal+hypoxic-ischemic+injury%5BTitle%5D)+AND+%22J+Neurosurg+Pediatr%22%5BJournal%5D)">Postnatal erythropoietin treatment mitigates neural cell loss after systemic prenatal hypoxic-ischemic injury.</a> J Neurosurg Pediatr. 6 (3), 206-221 (2010).</li>
<li>Olivier, P., Baud, O., Evrard, P., Gressens, P., Verney, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Prenatal+ischemia+and+white+matter+damage+in+rats%5BTitle%5D)+AND+%22J+Neuropathol+Exp+Neurol%22%5BJournal%5D)">Prenatal ischemia and white matter damage in rats.</a> J Neuropathol Exp Neurol. 64 (11), 998-1006 (2005).</li>
<li>Robinson, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Developmental+changes+induced+by+graded+prenatal+systemic+hypoxic-ischemic+insults+in+rats%5BTitle%5D)+AND+%22Neurobiol+Dis%22%5BJournal%5D)">Developmental changes induced by graded prenatal systemic hypoxic-ischemic insults in rats.</a> Neurobiol Dis. 18 (3), 568-581 (2005).</li>
<li>Rice, D., Barone, S. Jr <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Critical+periods+of+vulnerability+for+the+developing+nervous+system%3A+evidence+from+humans+and+animal+models%5BTitle%5D)+AND+%22Environ+Health+Perspect%22%5BJournal%5D)">Critical periods of vulnerability for the developing nervous system: evidence from humans and animal models.</a> Environ Health Perspect. 108, Suppl 3. 511-533 (2000).</li>
<li>Basilious, A., Yager, J., Fehlings, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neurological+outcomes+of+animal+models+of+uterine+artery+ligation+and+relevance+to+human+intrauterine+growth+restriction%3A+a+systematic+review%5BTitle%5D)+AND+%22Dev+Med+Child+Neurol%22%5BJournal%5D)">Neurological outcomes of animal models of uterine artery ligation and relevance to human intrauterine growth restriction: a systematic review.</a> Dev Med Child Neurol. 57 (5), 420-430 (2015).</li>
<li>Delcour, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neuroanatomical%2C+sensorimotor+and+cognitive+deficits+in+adult+rats+with+white+matter+injury+following+prenatal+ischemia%5BTitle%5D)+AND+%22Brain+Pathol%22%5BJournal%5D)">Neuroanatomical, sensorimotor and cognitive deficits in adult rats with white matter injury following prenatal ischemia.</a> Brain Pathol. 22 (1), 1-16 (2012).</li>
</ol>

The authors have nothing to disclose.

The microcoil stenosis of both ovarian and uterine arteries in both uterine horns produces consistent and reproducible intrauterine hypoperfusion in all placentas and fetuses. The level of hypoperfusion can be modified by using microcoils with different inner diameters. Rat pups born from a dam on which artery stenosis has been performed with microcoils 0.24 mm in inner diameter demonstrate IUGR and premature birth (see reference 1 for details). The pups exhibit neuroanatomical and behavioral alterations that resemble th...

Infants with intrauterine growth restriction (IUGR) (also known as fetal growth restriction) (birth weight &#60;10th percentile for gestational age), preterm birth (born at &#60; 37 weeks of gestation), and/or low birth weight (&#60; 2500 g) account for nearly 10% of all newborns 2,3. Many of these infants present with neurological problems such as cerebral palsy and developmental disorders (e.g., attention-deficit/hyperactivity disorder (ADHD) and learning disorders) 3,4,5. Those conditions have similarities and differences in their etiology and outcomes. The etiology of IUGR is multifactorial, and placental insufficiency associated with intrauterine hypoperfusion is considered to be the most common cause in non-anomalous fetuses 7. The etiology of preterm is multifactorial as well, and chorioamnionitis is the most frequent cause 8.
The influence of mild intrauterine hypoperfusion (MIUH) on the developing brain is unclear. Currently available animal models of intrauterine hypoperfusion/ischemia primarily involve either severe hypoperfusion or gradient degree of hypoperfusion with or without reperfusion 9,10,11. In clinical settings, however, cases of MIUH are considered to be far more frequent than those involving such severe conditions. The currently available models of MIUH are a rodent model involving suture ligation of either the uterine or the ovarian artery and a rodent model involving clipping the bilateral ovarian arteries and aorta having patency 12,13,14,15,16,17. One of the disadvantages of these models is the extensive inter-fetus variability, ranging from fetuses with profound hypoperfusion to fetuses with nearly intact perfusion, depending on the position of the fetus within the arterial arcade of the uterine and ovarian arteries. Another disadvantage of these models is their inability to distinguish the position of each fetus after birth; hence, researchers cannot distinguish the severity of intrauterine hypoperfusion experienced by an individual pup after its birth.
We have developed a rat model of MIUH involving multiple artery stenosis 1. Wrapping metal micro-coils with an inner diameter of 0.24 mm around the ovarian and uterine arteries causes stenosis, but not obstruction, of the blood vessels (Figure 1). Applying these microcoils at the proximal parts of all of the arteries supplying the uterus, i.e., the bilateral uterine and ovarian arteries, on embryonic day 17 (equivalent to embryonic weeks 20-25 in humans 18) induces a significant but mild decrease in blood flow to the placentas and fetuses. The decreases in blood flow after coils are applied to all four arteries feeding the uterus are largely the same across each placenta and fetus. The fetus mortality rate is less than 20%. The pups are born via spontaneous labor 1-2 days earlier (embryonic day 21-22) than normal. Almost all pups are born exhibiting significantly low birth weights 1. Gray and white matter volumes are decreased without obvious tissue damage 1. Pups present with delayed acquisition of newborn reflexes, muscle weakness, and altered spontaneous activity 1. This model mimics the clinical signs and symptoms of children born prematurely or with IUGR; children born preterm exhibit reduced gray and white matter volume with or without white matter injury 6, present delayed milestones of neurological development, and may present behavioral problems such as ADHD 3,5; children with IUGR exhibit minimal neuroanatomical alterations, and have an increased risk of impaired neurological development such as motor and cognitive delay 4,7. Preterm birth and IUGR are different conditions, but the two conditions share the basic mechanism, i.e. insults to immature brains before full-term gestational age.

<table><tbody><tr><td>Stereomicroscope</td><td></td><td></td><td></td></tr><tr><td>Isoflurane anesthesia machine</td><td></td><td></td><td></td></tr><tr><td>Anesthesia induction box</td><td></td><td></td><td></td></tr><tr><td>Heating pad</td><td></td><td></td><td></td></tr><tr><td>Diaper 30x40 cm</td><td></td><td></td><td></td></tr><tr><td>Depilatory foam or shaver</td><td></td><td></td><td></td></tr><tr><td>Iodine disinfectant solution</td><td></td><td></td><td></td></tr><tr><td>Gauze 10x20 cm&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Surgical drape 45x45 cm with a round opening 5 cm in diameter</td><td></td><td></td><td></td></tr><tr><td>Spray bottle with ethanol for disinfection</td><td></td><td></td><td></td></tr><tr><td>Cotton swab</td><td></td><td></td><td></td></tr><tr><td>Forceps with large blunt tips</td><td></td><td></td><td></td></tr><tr><td>Forceps with angled fine tips</td><td></td><td></td><td></td></tr><tr><td>Scissors</td><td></td><td></td><td></td></tr><tr><td>Surgical scalpel, blade size is 27mm long (no.10, Axel, AS ONE Corporation, Osaka, Japan)</td><td></td><td></td><td></td></tr><tr><td>Surgical suture needle</td><td></td><td></td><td></td></tr><tr><td>Metal microcoils; inner diameter 0.24 mm, made from gold-coated steel (SAMINI Co. Ltd., Shizuoka, Japan)</td><td></td><td></td><td></td></tr><tr><td>Silk suture 4-0</td><td></td><td></td><td></td></tr><tr><td>Sterile saline (0.9% sodium chloride)</td><td></td><td></td><td></td></tr><tr><td>Heating water bath</td><td></td><td></td><td></td></tr><tr><td>Plastic syringes (50ml) and needles (18G)</td><td></td><td></td><td></td></tr></tbody></table>

a rat model of mild intrauterine hypoperfusion with microcoil stenosis

Intrauterine hypoperfusion/ischemia is one of the major causes of intrauterine/fetal growth restriction, preterm birth, and low birth weight. Most studies of this phenomenon have been performed in either models with severe intrauterine ischemia or models with gradient degree of intrauterine hypoperfusion. No study has been performed in a model on uniform mild intrauterine hypoperfusion (MIUH). Two models have been used for studies of MIUH: a model based on suture ligation of either side of the arterial arcade formed with the uterine and ovarian arteries, and a transient model based on clipping the bilateral ovarian arteries and aorta having patency. Those two rodent models of MIUH have some limitations, e.g., not all fetuses are subjected to MIUH, depending on their position in the uterine horn. In our MIUH model, all fetuses are subjected to a comparable level of intrauterine hypoperfusion. MIUH was achieved by mild stenosis of all four arteries feeding the uterus, i.e., the bilateral uterine and ovarian arteries.
Arterial stenosis was induced by metal microcoils wrapped around the feeding arteries. Producing arterial stenosis with microcoils allowed us to control, optimize, and reproduce decreased blood flow with very little inter-animal variability and a low mortality rate, thus enabling accurate evaluation. When microcoils with an inner diameter of 0.24 mm were used, the blood flow in both the placenta and fetus was mildly decreased (approximately 30% from the pre-stenosis level in the placenta). The offspring of our MIUH model clearly demonstrates long-lasting alterations in neurological, neuroanatomical and behavioral test results.

After applying microcoils to all of the arteries feeding the uterus, i.e., the bilateral uterine and ovarian arteries, all fetuses are subjected to comparable levels of hypoperfusion. The application of microcoils with an inner diameter of 0.24 mm causes mild stenosis of those arteries, thereby causing a mild decrease in blood flow to the placentas and fetuses (Figure 3; approximately 30% from the pre-stenosis level in the placenta, see reference 1 for detail...

Watch this Scientific Journal Video about A Rat Model of Mild Intrauterine Hypoperfusion with Microcoil Stenosis at JoVE.com

A Rat Model of Mild Intrauterine Hypoperfusion with Microcoil Stenosis

Mild intrauterine hypoperfusion was produced by artery stenosis with metal microcoils wrapped around the uterine and ovarian arteries in rats at embryonic day 17. This procedure produced prenatal hypoperfusion and intrauterine growth restriction.

Intrauterine hypoperfusion/ischemia is one of the major causes of intrauterine/fetal growth restriction, preterm birth, and low birth weight. Most studies ...

a-rat-model-of-mild-intrauterine-hypoperfusion-with-microcoil-stenosis

Research

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Neuroscience

10.3K Views.  National Cerebral and Cardiovascular Center. Mild intrauterine hypoperfusion was produced by artery stenosis with metal microcoils wrapped around the uterine and ovarian arteries in rats at embryonic day 17. This procedure produced prenatal hypoperfusion and intrauterine growth restriction.

Video: A Rat Model of Mild Intrauterine Hypoperfusion with Microcoil Stenosis

The Hypoxic Ischemic Encephalopathy Model of Perinatal Ischemia

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

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

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

Biology

Fetal Echocardiography and Pulsed-wave Doppler Ultrasound in a Rabbit Model of Intrauterine Growth Restriction

Fetal intrauterine growth restriction (IUGR) results in abnormal cardiac function that is apparent antenatally due to advances in fetoplacental Doppler ultrasound and fetal echocardiography. Increasingly, these imaging modalities are being employed clinically to examine cardiac function and assess wellbeing in utero, thereby guiding timing of birth decisions. Here, we used a rabbit model of IUGR that allows analysis of cardiac function in a clinically relevant way. Using isoflurane induced anesthesia, IUGR is surgically created at gestational age day 25 by performing a laparotomy, exposing the bicornuate uterus and then ligating 40-50% of uteroplacental vessels supplying each gestational sac in a single uterine horn. The other horn in the rabbit bicornuate uterus serves as internal control fetuses. Then, after recovery at gestational age day 30 (full term), the same rabbit undergoes examination of fetal cardiac function. Anesthesia is induced with ketamine and xylazine intramuscularly, then maintained by a continuous intravenous infusion of ketamine and xylazine to minimize iatrogenic effects on fetal cardiac function. A repeat laparotomy is performed to expose each gestational sac and a microultrasound examination (VisualSonics VEVO 2100) of fetal cardiac function is performed. Placental insufficiency is evident by a raised pulsatility index or an absent or reversed end diastolic flow of the umbilical artery Doppler waveform. The ductus venosus and middle cerebral artery Doppler is then examined. Fetal echocardiography is performed by recording B mode, M mode and flow velocity waveforms in lateral and apical views. Offline calculations determine standard M-mode cardiac variables, tricuspid and mitral annular plane systolic excursion, speckle tracking and strain analysis, modified myocardial performance index and vascular flow velocity waveforms of interest. This small animal model of IUGR therefore affords examination of in utero cardiac function that is consistent with current clinical practice and is therefore useful in a translational research setting.

We describe examination of fetal cardiac function with contemporary functional fetal echocardiography and fetoplacental Doppler ultrasound using the VisualSonics VEVO 2100 microultrasound in a surgically induced model of intrauterine fetal growth restriction in a rabbit.

Fetal intrauterine growth restriction (IUGR) results in abnormal cardiac function that is apparent antenatally due to advances in fetoplacental Doppler ...

Medicine

Transuterine Fetal Tracheal Occlusion Model in Mice

Fetal tracheal occlusion (TO), an established treatment modality, promotes fetal lung growth and survival in severe congenital diaphragmatic hernia (CDH). Following TO, retention of the secreted epithelial fluid increases luminal pressure and induces lung growth. Various animal models have been defined to understand the pathophysiology of CDH and TO. All have their own advantages and disadvantages such as the difficulty of the technique, the size of the animal, cost, high mortality rates, and the availability of genetic tools. Herein, a novel transuterine model of murine fetal TO is described. Pregnant mice were anesthetized, and the uterus exposed via a midline laparotomy. The trachea of selected fetuses were ligated with a single transuterine suture placed behind the trachea, one carotid artery, and one jugular vein. The dam was closed and allowed to recover. Fetuses were collected just before parturition. Lung to body weight ratio in TO fetuses was higher than that in control fetuses. This model provides researchers with a new tool to study the impact of both TO and increased luminal pressure on lung development.

Various animal models of congenital diaphragmatic hernia and fetal tracheal occlusion present advantages and disadvantages regarding ethical issues, cost, surgical difficulty, size, survival rates, and availability of genetic tools. This model provides a new tool to study the impact of both tracheal occlusion and increased luminal pressure on lung development.

Fetal tracheal occlusion (TO), an established treatment modality, promotes fetal lung growth and survival in severe congenital diaphragmatic hernia (CDH). ...

Developmental Biology

A Thrombotic Stroke Model Based On Transient Cerebral Hypoxia-ischemia

Stroke research has endured many setbacks in translating neuroprotective therapies into clinical practice. In contrast, the real-world therapy (tPA thrombolysis) rarely produces benefits in mechanical occlusion-based experimental models, which dominate preclinical stroke research. This split between the bench and bedside suggests the need to employ tPA-responsive models in preclinical stroke research. To this end, a simple and tPA-reactive thrombotic stroke model is invented and described here. This model consists of transient occlusion of the unilateral common carotid artery and delivery of 7.5% oxygen through a face mask in adult mice for 30 min, while maintaining the animal rectal temperature at 37.5 &#177; 0.5 &#176;C. Although reversible ligation of the unilateral carotid artery or hypoxia each suppressed cerebral blood flow only transiently, the combination of both insults caused lasting reperfusion deficits, fibrin and platelet deposition, and large infarct in the middle cerebral artery-supplied territory. Importantly, tail-vein injection of recombinant tPA at 0.5, 1, or 4 hr post-tHI (10 mg/kg) provided time-dependent reduction of the mortality rate and infarct size. This new stroke model is simple and can be standardized across laboratories to compare experimental results. Further, it induces thrombosis without craniectomy or introducing pre-formed emboli. Given these unique merits, the tHI model is a useful addition to the repertoire of preclinical stroke research.

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

Stroke research has endured many setbacks in translating neuroprotective therapies into clinical practice. In contrast, the real-world therapy (tPA ...

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

A number of animal models have been used to study hypoxic-ischemic injury, traumatic injury, global hypoxia, or permanent ischemia in both the immature and mature brain. Stroke occurs commonly in the perinatal period in humans, and transient ischemia-reperfusion is the most common form of stroke in neonates. The reperfusion phase is a critical component of injury progression, which occurs over a period of days to weeks, and of the endogenous response to injury. This postnatal day 10 (p10) rat model of transient middle cerebral artery occlusion (tMCAO) creates a unilateral, non-hemorrhagic focal ischemia-reperfusion injury that can be utilized to study the mechanisms of focal injury and repair in the full-term-equivalent brain. The injury pattern that is produced by tMCAO is consistent and highly reproducible and can be confirmed with MRI or histological analyses. The severity of injury can be manipulated through changes in occlusion time and other methods that will be discussed.

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

A number of animal models have been used to study hypoxic-ischemic injury, traumatic injury, global hypoxia, or permanent ischemia in both the immature ...

Murine Model of Central Venous Stenosis using Aortocaval Fistula with an Outflow Stenosis

Central venous stenosis is an important entity contributing to arteriovenous fistula (AVF) failure. A murine AVF model was modified to create a partial ligation of the inferior vena cava (IVC) in the outflow of the fistula, mimicking central venous stenosis. Technical aspects of this model are introduced. The aorta and IVC are exposed, following an abdominal incision. The infra-renal aorta and IVC are dissected for proximal clamping, and the distal aorta is exposed for puncture. The IVC at the midpoint between the left renal vein and the aortic bifurcation is carefully dissected to place an 8-0 suture beneath the IVC. After clamping the aorta and IVC, an AVF is created by puncturing the infra-renal aorta through both walls into the IVC with a 25 G needle, followed by ligating a 22 G intra-venous (IV) catheter and IVC together. The catheter is then removed, creating a reproducible venous stenosis without occlusion. The aorta and IVC are unclamped after confirming primary hemostasis. This novel model of central vein stenosis is easy to perform, reproducible, and will facilitate studies on AVF failure.

An aortocaval fistula was created by puncturing the murine infra-renal aorta through both walls into the inferior vena cava and was followed by creation of a stenosis in its outflow via partial ligation of the inferior vena cava. This reproducible model can be used to study central venous stenosis.

Central venous stenosis is an important entity contributing to arteriovenous fistula (AVF) failure. A murine AVF model was modified to create a partial ...

Real-Time Assessment of Spinal Cord Microperfusion in a Porcine Model of Ischemia/Reperfusion

Spinal cord injury is a devastating complication of aortic repair. Despite developments for the prevention and treatment of spinal cord injury, its incidence is still considerably high and therefore, influences patient outcome. Microcirculation plays a key role in tissue perfusion and oxygen supply and is often dissociated from macrohemodynamics. Thus, direct evaluation of spinal cord microcirculation is essential for the development of microcirculation-targeted therapies and the evaluation of existing approaches in regard to spinal cord microcirculation. However, most of the methods do not provide real-time assessment of spinal cord microcirculation. The aim of this study is to describe a standardized protocol for real-time spinal cord microcirculatory evaluation using laser-Doppler needle probes directly inserted in the spinal cord. We used a porcine model of ischemia/reperfusion to induce deterioration of the spinal cord microcirculation. In addition, a fluorescent microsphere injection technique was used. Initially, animals were anesthetized and mechanically ventilated. Thereafter, laser-Doppler needle probe insertion was performed, followed by the placement of cerebrospinal fluid drainage. A median sternotomy was performed for exposure of the descending aorta to perform aortic cross-clamping. Ischemia/reperfusion was induced by supra-celiac aortic cross-clamping for a total of 48 min, followed by reperfusion and hemodynamic stabilization. Laser-Doppler Flux was performed in parallel with macrohemodynamic evaluation. In addition, automated cerebrospinal fluid drainage was used to maintain a stable cerebrospinal pressure. After completion of the protocol, animals were sacrificed, and the spinal cord was harvested for histopathological and microsphere analysis. The protocol reveals the feasibility of spinal cord microperfusion measurements using laser-Doppler probes and shows a marked decrease during ischemia as well as recovery after reperfusion. Results showed comparable behavior to fluorescent microsphere evaluation. In conclusion, this new protocol might provide a useful large animal model for future studies using real-time spinal cord microperfusion assessment in ischemia/reperfusion conditions.

Spinal cord microcirculation plays a pivotal role in spinal cord injury. Most methods do not allow real-time assessment of spinal cord microcirculation, which is essential for the development of microcirculation-targeted therapies. Here, we propose a protocol using Laser-Doppler-Flow Needle probes in a large animal model of ischemia/reperfusion.

Spinal cord injury is a devastating complication of aortic repair. Despite developments for the prevention and treatment of spinal cord injury, its ...

Myocardial Infarction by Percutaneous Embolization Coil Deployment in a Swine Model

Myocardial infarction (MI) is the leading cause of mortality worldwide. Despite the use of evidence-based treatments, including coronary revascularization and cardiovascular drugs, a significant proportion of patients develop pathological left-ventricular remodeling and progressive heart failure following MI. Therefore, new therapeutic options, such as cellular and gene therapies, among others, have been developed to repair and regenerate injured myocardium. In this context, animal models of MI are crucial in exploring the safety and efficacy of these experimental therapies before clinical translation. Large animal models such as swine are preferred over smaller ones due to the high similarity of swine and human hearts in terms of coronary artery anatomy, cardiac kinetics, and the post-MI healing process. Here, we aimed to describe an MI model in pig by permanent coil deployment. Briefly, it comprises a percutaneous selective coronary artery cannulation through retrograde femoral access. Following coronary angiography, the coil is deployed at the target branch under fluoroscopic guidance. Finally, complete occlusion is confirmed by repeated coronary angiography. This approach is feasible, highly reproducible, and emulates the pathogenesis of human non-revascularized MI, avoiding the traditional open-chest surgery and the subsequent postoperative inflammation. Depending on the time of follow-up, the technique is suitable for acute, sub-acute, or chronic MI models.

Myocardial infarction (MI) animal models that emulate the natural process of the disease in humans are crucial to understanding pathophysiological mechanisms and testing the safety and efficacy of new emergent therapies. Here, we describe an MI swine model created by deploying a percutaneous embolization coil.

Myocardial infarction (MI) is the leading cause of mortality worldwide. Despite the use of evidence-based treatments, including coronary revascularization ...

Early Pathological and Magnetic Resonance Detection of Cerebral Injury Using a Rat Model of Neonatal Hypoxic Ischemic Encephalopathy

Perinatal hypoxic-ischemic encephalopathy (HIE) is an acute disease that may afflict newborns, resulting in variable long- and short-term neurodevelopmental outcomes. Early diagnosis is critical to identifying infants who may benefit from intervention; however, early diagnosis relies heavily on clinical criteria. No molecular or radiological tests have shown promise in detecting early cerebral injury. Studies have shown that magnetic resonance imaging (MRI) can show changes in both blood flow/ischemia and metabolic disruption. However, they have all been used to evaluate the secondary phase of the disease (&gt;12 h) after the onset of the injury. Early diagnosis is critical to rapidly starting therapeutic hypothermia in eligible infants, which is currently recommended to be initiated within 6 h of birth. The rat model of hypoxic-ischemic injury was developed in 1981 and has been validated and used extensively to study changes in brain perfusion, cerebral injury markers, and morphology. However, it has primarily been used as a "late model", evaluating injury several days after the initial ischemic insult. The model has been known to have poor sensitivity in evaluating reliable and reproducible early cerebral changes. The objective of this study was to develop a reliable model to study early gross morphological and radiological markers of HIE using pathological staining and cerebral magnetic resonance imaging/magnetic resonance spectroscopy.

The present protocol describes a rodent model of newborn hypoxic-ischemic injury for identifying early changes in cerebral tissue by gross morphology and magnetic resonance imaging. This has benefits over existing models, which can be used to study late injury but do not allow the evaluation of reproducible early changes.

Perinatal hypoxic-ischemic encephalopathy (HIE) is an acute disease that may afflict newborns, resulting in variable long- and short-term ...

Ascending Aortic Banding for Studying Pressure-Overload Induced Heart Failure

A Minimally Invasive Model of Aortic Stenosis in Swine

Large animal models of heart failure play an essential role in the development of new therapeutic interventions due to their size and physiological similarities to humans. Efforts have been dedicated to creating a model of pressure-overload induced heart failure, and ascending aortic banding while still supra-coronary and not a perfect mimic of aortic stenosis in humans, closely resembling the human condition.
The purpose of this study is to demonstrate a minimally invasive approach to induce left ventricular pressure overload by placing an aortic band, precisely calibrated with percutaneously introduced high-fidelity pressure sensors. This method represents a refinement of the surgical procedure (3Rs), resulting in homogenous trans-stenotic gradients and reduced intragroup variability. Additionally, it enables swift and uneventful animal recovery, leading to minimal mortality rates. Throughout the study, animals were followed for up to 2 months after surgery, employing transthoracic echocardiography and pressure-volume loop analysis. However, longer follow-up periods can be achieved if desired. This large animal model proves valuable for testing new drugs, particularly those targeting hypertrophy and the structural and functional alterations associated with left ventricular pressure overload.

Author Spotlight: Development of a Minimally Invasive Large-Animal Model for Reliable and Reproducible Cardiovascular Research 

This protocol describes a minimally invasive surgical procedure for ascending aortic banding in swine.

Large animal models of heart failure play an essential role in the development of new therapeutic interventions due to their size and physiological ...