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 diamet...

<ol>
	<li>Ohshima, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mild+intrauterine+hypoperfusion+reproduces+neurodevelopmental+disorders+observed+in+prematurity.">Mild intrauterine hypoperfusion reproduces neurodevelopmental disorders observed in prematurity.</a> Sci Rep. 6, 39377 (2016).</li><li>Anderson, P., Doyle, L. W., Victorian Infant Collaborative Study, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurobehavioral+outcomes+of+school-age+children+born+extremely+low+birth+weight+or+very+preterm+in+the+1990s.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+childhood+neurodevelopment+after+intrauterine+growth+restriction:+a+systematic+review.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preterm+Birth+and+Poor+Fetal+Growth+as+Risk+Factors+of+Attention-Deficit/+Hyperactivity+Disorder.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+injury+in+premature+infants:+a+complex+amalgam+of+destructive+and+developmental+disturbances.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fetal+growth+restriction:+current+knowledge.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preterm+birth+and+the+role+of+neuroprotection.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prenatal+ischemia+deteriorates+white+matter,+brain+organization,+and+function:+implications+for+prematurity+and+cerebral+palsy.">Prenatal ischemia deteriorates white matter, brain organization, and function: implications for prematurity and cerebral palsy.</a> Dev Med Child Neurol. 58, 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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postnatal+Erythropoietin+Mitigates+Impaired+Cerebral+Cortical+Development+Following+Subplate+Loss+from+Prenatal+Hypoxia-Ischemia.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+of+impaired+neuronal+migration+with+cognitive+deficits+in+extremely+preterm+infants.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mild+musculoskeletal+and+locomotor+alterations+in+adult+rats+with+white+matter+injury+following+prenatal+ischemia.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypertension+produced+by+reduced+uterine+perfusion+in+pregnant+rats+is+associated+with+increased+soluble+fms-like+tyrosine+kinase-1+expression.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduced+uterine+perfusion+pressure+(RUPP)+model+for+studying+cardiovascular-renal+dysfunction+in+response+to+placental+ischemia.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postnatal+erythropoietin+treatment+mitigates+neural+cell+loss+after+systemic+prenatal+hypoxic-ischemic+injury.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prenatal+ischemia+and+white+matter+damage+in+rats.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+changes+induced+by+graded+prenatal+systemic+hypoxic-ischemic+insults+in+rats.">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. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+periods+of+vulnerability+for+the+developing+nervous+system:+evidence+from+humans+and+animal+models.">Critical periods of vulnerability for the developing nervous system: evidence from humans and animal models.</a> Environ Health Perspect. 108, 511-533 (2000).</li><li>Basilious, A., Yager, J., Fehlings, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurological+outcomes+of+animal+models+of+uterine+artery+ligation+and+relevance+to+human+intrauterine+growth+restriction:+a+systematic+review.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroanatomical,+sensorimotor+and+cognitive+deficits+in+adult+rats+with+white+matter+injury+following+prenatal+ischemia.">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 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&#160;</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 ...

The overall goal of this surgical intervention is to use metal microcoils, wrapped around both uterine and ovarian arteries, to induce artery stenosis for the generation of mild intrauterine hypoperfusion. This method can help answer key questions in the fetal brain injury field, such as, how does mild intrauterine hypoperfusion influence neural development later in life? The main advantage of this technique is that an entire litter of fetuses can be subjected to an equal degree of mild hypoperfusion at the same time.On embryonic day 17, place an anesthetized, pregnant rat dam in the supine position on a sterile diaper placed on top of a heating pad. Confirm a lack of response to toe pinch and apply ointment to the animal's eyes. Remove the hair from the navel area to the pelvic arch, and use an iodine soaked gauze, soaked to disinfect the exposed skin.Place a sterile drape over the animal with the round opening over the surgical area, and use a scalpel to make an approximately 2.5 centimeter skin incision in the lower abdomen, from the upper edge of the public bone toward the navel. Make a secondary incision through the corresponding muscle layer and place several pieces of sterile gauze around the opening of the drape. Wet the gauze with 37 degree Celsius saline and gently push both lateral sides of the abdomen until a portion of uterine horn emerges from the incision.Using 70%ethanol-sterilized fingers, gently remove one entire uterine horn from the abdominal cavity and place the horn on the wet gauze. After removing the second uterine horn, apply 37 degree Celsius saline to keep the horns warm and place the animal under a stereo microscope. Keeping the uterus warm and wet throughout the procedure is critical.Otherwise, many fetuses may be lost before delivery. Locate the proximal region of the main arcade of the ovarian and uterine arteries, and use forceps to separate the artery from the vein running parallel to the artery. It is essential for a successful procedure to identify the proximal portion of the main arcade of the ovarian and uterine arteries, which is small in diameter but has numerous branches to the placentas.Place a 5 centimeter long piece of 4-0 silk suture under the artery and lift both ends of the suture with the forceps until the artery is separated from the vein. Using fine forceps, place a gold-coated steel microcoil next to the lifted artery and use the suture to rotate the artery three or four turns around the microcoil. Then, grasp the coil with the fine forceps at the wrapped end and wrap the artery around the naked end of the coil as just demonstrated.After wrapping the three other arteries with microcoils in the same manner, gently return the uterine horn to the abdominal cavity and use interrupted single 4-0 silk sutures to individually close the muscle and skin layers. Then, allow the rat to recover in a warmed cage with monitoring until full recovery. Applying microcoils to the bilateral uterine and ovarian arteries causes a mild stenosis of the arteries and a mild decrease in blood flow to all of the placentas and fetuses.Each fetus is then subjected to similar levels of hypoperfusion, approximately 30%from pre-stenosis level in the placenta, resulting in the consequent development of intrauterine growth-restricted pups. After its development, this technique paved the way for researchers in the field of brain injury to explore the inference of mild intrauterine hypoperfusion in a prenatal rodent.

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10.1K vistas.  National Cerebral and Cardiovascular Center. The overall goal of this surgical intervention is to use metal microcoils, wrapped around both uterine and ovarian arteries, to induce artery stenosis for the generation of mild intrauterine hypoperfusion. This method can help answer key questions in the fetal brain injury field, such as, how does mild intrauterine hypoperfusion influence neural development later in life? The main advantage of this technique is that an entire litter of fetuses can be subjected to an equal degree of mild hypop...