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>
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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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. 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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. 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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 the signs and symptoms observed in preterm children. These neuroanatomical alterations include ventricular enlargement, thinning of the cortex and corpus callosum, and decreased brain weight but no obvious cell loss or cystic lesions 1. The behavioral alterations observed include a delayed neonatal reflex, decreased muscle strength and altered spontaneous activity 1.
To study intrauterine ischemia, animal models based on severe ischemia over a short duration with reperfusion are frequently used 10,11. When both ends of the artery arcade formed from the ovarian and uterine arteries are blocked by clipping, the blood supply to the uterine horn (including the placentas and fetuses) is completely halted. Hence, the duration of clipping must be short, or the fetuses will die. In a clinical setting, however, we speculate that prolonged mild hypoperfusion may be more frequently encountered rather than abrupt blockade of blood flow to the uterus and its complete recovery within an hour.
Rodent models involving suture ligation of one or both side(s) of the arcade of the uterine artery and those involving clipping that leaves some inner space between the bilateral ovarian arteries and the aorta have been the only available models of mild prolonged intrauterine hypoperfusion 13,14,16,19,20. These ligation and clipping models have low reproducibility, i.e., only a few fetuses survive and suffer from hypoperfusion. When the blood flow at the ovarian artery is blocked by suture ligation, the fetuses located closest to the ovary, i.e., at the top of the uterine horn, is subjected to profound ischemia, whereas the fetuses located closer to the cervix and vagina are subjected to minimal or no decreases in blood supply 12.
There are three critical steps in the protocol. Identifying the proximal portion of the main arcade of the ovarian and uterine arteries is essential. The main arterial arcade has numerous branches to the placentas. Some of these branch arteries are large in diameter, and occasionally are almost as large as the main artery. Therefore, distinguishing the main artery from among several other branching arteries can be confusing. Note that the main arterial arcade is not large in diameter. The vein running beside the arcade is several times larger in diameter than the artery. Arteries may become even narrower, owing to constriction in response to surgical stimulation. Separating the artery from the vein running alongside it is a delicate task. The adventitia of the artery is in close contact with the vein throughout most of the arcade. Be aware that the vein bleeds easily when touched by forceps. Keeping the uterus warm and wet is critical; otherwise, the dam and fetuses may suffer from stress and dehydration, and many fetuses may be lost before delivery. The limitation of this coil stenosis model technique is the long surgical duration required. A period of approximately 60 min is required to complete the surgery by applying four coils. This long surgical duration may cause additional stress to the fetuses and the dam.
We believe that the model presented in this paper is the most clinically relevant and reproducible model of MIUH. This model allows for assessment of the temporal and longitudinal impact of MIUH on IUGR.

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 details). Pups born from the dam demonstrate IUGR (Table 1).
The dams seem to be hypoactive during a few days after the surgery. Other than that, the dams do not present any noticeable complications, and fully recover in one week after the surgery. The dams are able to live for a long time after the delivery (mortality is 0 % for observation period), and to nurse pups. The coils remain in the same place.
Table 1. Body weights at birth (Postnatal day 0, P0) and on the day equivalent to embryonic day 29 (E29), which was P6-8. Pups in the no-surgery control group were born on E23, whereas those in the sham-surgery group were born 1 day earlier (E22); those in the mild intrauterine hypoperfusion (MIUH) group were born 1-2 days earlier (E21-22) via spontaneous labor. Therefore, E29 equivalents were P6 to P8 depending on the pup&#39;s gestational period. (n = 8-14 in each group, for each sex and each time point) *P &#60; 0.01 vs. a no-surgery control group as well as a sham-surgery group. One-way ANOVA, followed by a Tukey-Kramer test.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56723/56723fig1.jpg" /> 
Figure 1. Schema of the anatomic arterial system in the uterus. Four microcoils were installed around the arteries upstream of the bilateral ovarian and uterine arteries. The dotted arrows indicate the direction of arterial blood flow. This figure has been modified from the one in our previous publication 1. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56723/56723fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56723/56723fig2.jpg" /> 
Figure 2. Illustration of the application of a coil to an artery. 1. Lift up the artery with a string. Place a microcoil beside the artery. 2. By rotating the artery around the microcoil, the microcoil is wrapped around the artery. 3. After rotating the artery 3 or 4 turns (The coil has 5 turns), change the site at which the coil is held to the other edge. Hold the edge of the coil together with the artery using forceps. Change the site at which the string lifts the artery to the other side of the coil. 4. Rotate the artery around the coil so that the coil completely (5 turns) wraps the artery. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56723/56723fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56723/56723fig3.jpg" /> 
Figure 3. A representative image of a uterus produced with laser speckle flowmetry1. This image demonstrates decreased blood flow in the left uterine horn but not the right, after coil stenosis was performed in the left ovarian and uterine arteries. *: locations of coil stenosis in the arcade of the ovarian and uterine arteries; upper asterisk corresponds to the proximal part of ovarian artery, and lower one corresponds to the proximal part of uterine artery. Dotted line indicates the arcade of the ovarian and uterine arteries in the left uterine horn. Reddish color indicates more blood flow, whereas blueish color indicates less blood flow. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56723/56723fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

Nanyang Technological University

Research

JoVE Journal

Neuroscience

10.2K 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

Investigations on Alterations of Hippocampal Circuit Function Following Mild Traumatic Brain Injury

Traumatic Brain Injury (TBI) afflicts more than 1.7 million people in the United States each year and even mild TBI can lead to persistent neurological impairments 1. Two pervasive and disabling symptoms experienced by TBI survivors, memory deficits and a reduction in seizure threshold, are thought to be mediated by TBI-induced hippocampal dysfunction 2,3. In order to demonstrate how altered hippocampal circuit function adversely affects behavior after TBI in mice, we employ lateral fluid percussion injury, a commonly used animal model of TBI that recreates many features of human TBI including neuronal cell loss, gliosis, and ionic perturbation 4-6.
	Here we demonstrate a combinatorial method for investigating TBI-induced hippocampal dysfunction. Our approach incorporates multiple ex vivo physiological techniques together with animal behavior and biochemical analysis, in order to analyze post-TBI changes in the hippocampus. We begin with the experimental injury paradigm along with behavioral analysis to assess cognitive disability following TBI. Next, we feature three distinct ex vivo recording techniques: extracellular field potential recording, visualized whole-cell patch-clamping, and voltage sensitive dye recording. Finally, we demonstrate a method for regionally dissecting subregions of the hippocampus that can be useful for detailed analysis of neurochemical and metabolic alterations post-TBI.
	These methods have been used to examine the alterations in hippocampal circuitry following TBI and to probe the opposing changes in network circuit function that occur in the dentate gyrus and CA1 subregions of the hippocampus (see Figure 1). The ability to analyze the post-TBI changes in each subregion is essential to understanding the underlying mechanisms contributing to TBI-induced behavioral and cognitive deficits. 
	The multi-faceted system outlined here allows investigators to push past characterization of phenomenology induced by a disease state (in this case TBI) and determine the mechanisms responsible for the observed pathology associated with TBI.

A multi-faceted approach to investigating functional changes to hippocampal circuitry is explained. Electrophysiological techniques are described along with the injury protocol, behavioral testing and regional dissection method. The combination of these techniques can be applied in similar fashion for other brain regions and scientific questions.

Traumatic Brain Injury (TBI) afflicts more than 1.7 million people in the United States each year and even mild TBI can lead to persistent neurological ...

A Protocol for Measuring Cue Reactivity in a Rat Model of Cocaine Use Disorder

Cocaine use disorder (CUD) follows a trajectory of repetitive self-administration during which previously neutral stimuli gain incentive value. Cue reactivity, the sensitivity to cues previously linked with the drug-taking experience, plays a prominent role in human craving during abstinence. Cue reactivity can be assessed as the attentional orientation toward drug-associated cues that is measurable as appetitive approach behavior in both preclinical and human studies. Herein describes an assessment of cue reactivity in rats trained to self-administer cocaine. Cocaine self-administration is paired with the presentation of discrete cues that act as conditioned reinforcers (i.e., house light, stimulus light, infusion pump sounds). Following a period of abstinence, lever presses in the cocaine self-administration context accompanied by the discrete cues previously paired with cocaine infusion are measured as cue reactivity. This model is useful to explore neurobiological mechanisms underlying cue reactivity processes as well as to assess pharmacotherapies to suppress cue reactivity and therefore, modify relapse vulnerability. Advantages of the model include its translational relevance, and its face and predictive validities.&#160;The primary limitation of the model is that the cue reactivity task can only be performed infrequently and must only be used in short duration (e.g., 1 hour), otherwise rats will begin to extinguish the pairing of the discrete cues with the cocaine stimulus. The model is extendable to any positively reinforcing stimulus paired with discrete cues; though particularly applicable to drugs of abuse, this model may hold future applications in fields such as obesity, where palatable food rewards can act as positively reinforcing stimuli.

Cue reactivity is conceptualized as sensitivity to cues linked with drug-taking experiences that contribute to craving and relapse in abstinent humans. Cue reactivity is modeled in rats by measuring attentional orientation toward drug-associated cues that results in appetitive approach behavior in a cue reactivity test following self-administration and forced abstinence.

Cocaine use disorder (CUD) follows a trajectory of repetitive self-administration during which previously neutral stimuli gain incentive value. Cue ...

A Rat Model of Central Fatigue Using a Modified Multiple Platform Method

In this article, we introduced a rat model of central fatigue using the modified multiple platform method (MMPM). The Multiple Platform box was designed as a water tank with narrow platforms on the bottom. The model rats were put into the tank and stood on the platforms for 14 h (18:00 - 8:00) per day for a consecutive 21 days, with a blank control group set for contrast. At the end of modeling, rats in the model group showed an obvious fatigued appearance. To assess the model, we performed several behavioral tests, including the open field test (OFT), the elevated plus maze (EPM) test, and the exhaustive swimming (ES) test. The results showed that anxiety, spatial cognition impairment, poor muscle performance, and declined voluntary activity presented in model rats confirm the diagnosis of&#160;central fatigue. Changes of the central neurotransmitters also verified the result. In conclusion, the model successfully simulated central fatigue, and future study with the model may help reveal the pathological mechanism of the disease.

Here, we present a protocol to introduce a rat model of central fatigue using the modified multiple platform method (MMPM).

In this article, we introduced a rat model of central fatigue using the modified multiple platform method (MMPM). The Multiple Platform box was designed ...

A Novel and Translational Rat Model of Concussion Combining Force and Rotation with In Vivo Cerebral Microdialysis

Persistent cognitive and motor symptoms are known consequences of concussions/mild traumatic brain injury (mTBIs) that can be partly attributable to altered neurotransmission. Indeed, cerebral microdialysis studies in rodents have demonstrated an excessive extracellular glutamate release in the hippocampus within the first 10 min following trauma. Microdialysis offers the clear advantage of in vivo neurotransmitter continuous sampling while not having to sacrifice the animal. In addition to the aforementioned technique, a closed head injury model that exerts rapid acceleration and deceleration of the head and torso is needed, as such a factor is not available in many other animal models. The Wayne State weight-drop model mimics this essential component of human craniocerebral trauma, allowing the induction of an impact on the head of an unrestrained rodent with a falling weight. Our novel and translational rat model combines cerebral microdialysis with the Wayne State weight-drop model to study, in lightly anesthetized and unrestrained adult rats, the acute changes in extracellular neurotransmitter levels following concussion. In this protocol, the microdialysis probe was inserted inside the hippocampus as region of interest, and was left inserted in the brain at impact. There is a high density of terminals and receptors in the hippocampus, making it a relevant region to document altered neurotransmission following concussion. When applied to adult Sprague-Dawley rats, our combined model induced increases in hippocampal extracellular glutamate concentrations within the first 10 min, consistent with the previously reported post-concussion symptomology. This combined weight-drop model provides a reliable tool for researchers to study early therapeutic responses to concussions in addition to repetitive brain injury, since this protocol induces a closed-head mild trauma.

Neurotransmitter alteration is a mechanism of neural dysfunction that occurs after concussion and contributes to the sometimes-catastrophic long-term consequences. This rat model combines microdialysis, allowing in vivo neurotransmitter quantification, with a weight-drop technique exerting rapid acceleration and deceleration of the head and torso, an important factor of human craniocerebral trauma.

Persistent cognitive and motor symptoms are known consequences of concussions/mild traumatic brain injury (mTBIs) that can be partly attributable to ...

Advanced Diffusion Imaging in The Hippocampus of Rats with Mild Traumatic Brain Injury

Mild traumatic brain injury (mTBI) is the most common type of acquired brain injury. Since patients with traumatic brain injury show a tremendous variability and heterogeneity (age, gender, type of trauma, other possible pathologies, etc.), animal models play a key role in unraveling factors that are limitations in clinical research. They provide a standardized and controlled setting to investigate the biological mechanisms of injury and repair following TBI. However, not all animal models mimic the diffuse and subtle nature of mTBI effectively. For example, the commonly used controlled cortical impact (CCI) and lateral fluid percussion injury (LFPI) models make use of a craniotomy to expose the brain and induce widespread focal trauma, which are not commonly seen in mTBI. Therefore, these experimental models are not valid to mimic mTBI. Thus, an appropriate model should be used to investigate mTBI. The Marmarou weight drop model for rats induces similar microstructural alterations and cognitive impairments as seen in patients who sustain mild trauma; therefore, this model was selected for this protocol. Conventional computed tomography and magnetic resonance imaging (MRI) scans commonly show no damage following a mild injury, because mTBI induces often only subtle and diffuse injuries. With diffusion weighted MRI, it is possible to investigate microstructural properties of brain tissue, which can provide more insight into the microscopic alterations following mild trauma. Therefore, the goal of this study is to obtain quantitative information of a selected region-of-interest (i.e., hippocampus) to follow up disease progression after obtaining a mild and diffuse brain injury.

The overall goal of this procedure is to obtain quantitative microstructural information of the hippocampus in a rat with mild traumatic brain injury. This is done using an advanced diffusion-weighted magnetic resonance imaging protocol and region-of-interest based analysis of parametric diffusion maps.

Mild traumatic brain injury (mTBI) is the most common type of acquired brain injury. Since patients with traumatic brain injury show a tremendous ...

Applying the RatWalker System for Gait Analysis in a Genetic Rat Model of Parkinson's Disease

Parkinson's disease (PD) is a progressive neurodegenerative disorder caused by the loss of dopaminergic (DA) neurons in the substantia nigra pars compacta. Gait abnormalities, including decreased arm swing, slower walking speed, and shorter steps are common in PD patients and appear early in the course of disease. Thus, the quantification of motor patterns in animal models of PD will be important for phenotypic characterization during disease course and upon therapeutic treatment. Most cases of PD are idiopathic; however, the identification of hereditary forms of PD uncovered gene mutations and variants, such as loss-of-function mutations in Pink1 and Parkin, two proteins involved in mitochondrial quality control that could be harnessed to create animal models. While mice are resistant to neurodegeneration upon loss of Pink1 and Parkin (single and combined deletion), in rats, Pink1 but not Parkin deficiency leads to nigral DA neuron loss and motor impairment. Here, we report the utility of FTIR imaging to uncover gait changes in freely walking young (2 months of age) male rats with combined loss of Pink1 and Parkin prior to the development of gross visually apparent motor abnormality as these rats age (observed at 4-6 months), characterized by hindlimb dragging as previously reported in Pink1 knockout (KO) rats.

Here we describe the RatWalker system, built by redesigning the MouseWalker apparatus to accommodate for the increased size and weight of rats. This system uses frustrated total internal reflection (FTIR), high-speed video capture, and open-access analysis software to track and quantify gait parameters.

Parkinson's disease (PD) is a progressive neurodegenerative disorder caused by the loss of dopaminergic (DA) neurons in the substantia nigra pars ...

Establishment of a Rat Model of Superior Sagittal-Sinus Occlusion via a Thread-Embolism Method

The mechanisms contributing to the natural onset of cerebral venous sinus thrombosis (CVST) are mostly unknown, and a variety of uncontrollable factors are involved in the course of the disease, resulting in great limitations in clinical research. Therefore, the establishment of stable CVST animal models that can standardize a variety of uncontrollable confounding factors have helped to circumvent shortcomings in clinical research. In recent decades, a variety of CVST animal models have been constructed, but the results based on these models have been inconsistent and incomplete. Hence, in order to further explore the pathophysiological mechanisms of CVST, it is necessary to establish a novel and highly compatible animal model, which has important practical value and scientific significance for the diagnosis and treatment of CVST. In the present study, a novel Sprague-Dawley (SD) rat model of superior sagittal sinus (SSS) thrombosis was established via a thread-embolization method, and the stability and reliability of the model were verified. Additionally, we evaluated changes in cerebral venous blood flow in rats after the formation of CVST. Collectively, the SD-rat SSS-thrombosis model represents a novel CVST animal model that is easily established, minimizes trauma, yields good stability, and allows for accurately controlling ischemic timing and location.

Here, we establish a novel Sprague-Dawley (SD) rat model of superior sagittal sinus (SSS) thrombosis via a thread-embolization method, and the stability and reliability of the model were verified.

The mechanisms contributing to the natural onset of cerebral venous sinus thrombosis (CVST) are mostly unknown, and a variety of uncontrollable factors ...

A Rat Model of EcoHIV Brain Infection

It has been well studied that the EcoHIV infected mouse model is of significant utility in investigating HIV associated neurological complications. Establishment of the EcoHIV infected rat model for studies of drug abuse and neurocognitive disorders, would be beneficial in the study of neuroHIV and HIV-1 associated neurocognitive disorders (HAND). In the present study, we demonstrate the successful creation of a rat model of active HIV infection using chimeric HIV (EcoHIV). First, the lentiviral construct of EcoHIV was packaged in cultured 293 FT cells for 48 hours. Then, the conditional medium was concentrated and titered. Next, we performed bilateral stereotaxic injections of the EcoHIV-EGFP into F344/N rat brain tissue. One week after infection, EGFP fluorescence signals were detected in the infected brain tissue, indicating that EcoHIV successfully induces an active HIV infection in rats. In addition, immunostaining for the microglial cell marker, Iba1, was performed. The results indicated that microglia were the predominant cell type harboring EcoHIV. Furthermore, EcoHIV rats exhibited alterations in temporal processing, a potential underlying neurobehavioral mechanism of HAND as well as synaptic dysfunction eight weeks after infection. Collectively, the present study extends the EcoHIV model of HIV-1 infection to the rat offering a valuable biological system to study HIV-1 viral reservoirs in the brain as well as HAND and associated comorbidities such as drug abuse.

Here, we present a protocol to establish a new rat model of active HIV infection using chimeric HIV (EcoHIV), which is critical for enhancing our understanding of HIV-1 viral reservoirs in the brain and offering a system to study HIV-associated neurocognitive disorders and associated comorbidities (i.e., drug abuse).

It has been well studied that the EcoHIV infected mouse model is of significant utility in investigating HIV associated neurological complications. ...

Induction and Assessment of Levodopa-induced Dyskinesias in a Rat Model of Parkinson's Disease

Levodopa (L-DOPA) remains the gold-standard therapy used to treat Parkinson&#39;s disease (PD) motor symptoms. However, unwanted involuntary movements known as L-DOPA-induced dyskinesias (LIDs) develop with prolonged use of this dopamine precursor.&#160;It is estimated that the incidence of LIDs escalates to approximately 90% of individuals with PD within 10&#8211;15 years of treatment. Understanding the mechanisms of this malady and developing both novel and effective anti-dyskinesia treatments requires consistent and accurate modeling for pre-clinical testing of therapeutic interventions. A detailed method for reliable induction and comprehensive rating of LIDs following 6-OHDA-induced nigral lesioning in a rat model of PD is presented here. Dependable LID assessment in rats provides a powerful tool that can be readily utilized across laboratories to test emerging therapies focused on reducing or eliminating this common treatment-induced burden for individuals with PD.

This article describes methods to induce and evaluate levodopa-induced dyskinesias in a rat model of Parkinson&#39;s disease. The protocol offers detailed information regarding the intensity and frequency of a range of dyskinetic behaviors, both dystonic and hyperkinetic, providing a reliable tool to test treatments targeting this unmet medical need.

Levodopa (L-DOPA) remains the gold-standard therapy used to treat Parkinson&#39;s disease (PD) motor symptoms. However, unwanted involuntary movements ...

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

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

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

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