Name: modeling encephalopathy of prematurity using prenatal hypoxia-ischemia with intra-amniotic lipopolysaccharide in rats
Uploaded: 2015-11-20T14:00:00.000+00:00
Duration: 7 min 36 s
Description: Watch this Scientific Journal Video about Modeling Encephalopathy of Prematurity Using Prenatal Hypoxia-ischemia with Intra-amniotic Lipopolysaccharide in Rats at JoVE.com

The authors are grateful to Dan Firl, Chris Corbett and Jesse Denson, PhD. Funding was provided by NIH NINDS R01 NS060765 to SR, the P30 CoBRE Pilot Program to LJ and the Child Health Signature Program to LJ at the University of New Mexico.

Institutional Care and Use Committees at both Boston Children&#8217;s Hospital and the University of New Mexico Health Sciences Center approved all experimental procedures.
NOTE: Prior to commencing the procedure, seal, sterilize and autoclave all surgical instruments and surgical drapes. Additionally, prepare post-operative medications in sterile vials including 0.125% bipivucaine and 0.1 mg/kg buprenorphine. Also prepare the lipopolysaccharide (LPS) solution sterilely: 0.04 mg/ml LPS (0111:B4) in sterile saline containing dilute Evan&#8217;s blue dye.
1. Anesthesia
<ol>
	<li>Induce anesthesia in an embryonic day 18 (E18) pregnant Sprague Dawley rat with a mixture of 3% isoflurane balanced 70% nitrogen and 30% oxygen.</li>
	<li>Remove rat from the induction chamber and place the rat supine on draped surgical circulating water blanket set at 37 &#176;C. Transfer anesthesia to nose cone and reduce isoflurane level to 2%.</li>
	<li>Gently apply ophthalmic ointment to each eye to prevent corneal drying. During the procedure continually monitor temperature, respiration rate and heart rate of the animal. Maternal physiology should remain stable throughout the procedure.</li>
</ol>
2. Surgical Prep and Scrub
<ol>
	<li>Using small animal clippers remove all hair in the lower abdominal region. Shave in a rectangular pattern with care to avoid nicking the nipples or generating razor rash that can be irritating for future nursing of live born pups.</li>
	<li>Prepare abdominal skin by alternating application of povidone-iodine and 70% ethanol scrub with sterile cotton swabs. Repeat the scrub such that povidone-iodine and 70% ethanol are each applied 3x in alternating fashion. Let dry.</li>
	<li>Confirm depth of anesthesia via absence of toe-pinch reflex. In the absence of reflex and stimuli to pain, reduce isoflurane level to 1%.</li>
	<li>Using sterile surgical towels, drape the animal. Take care to place the drapes at an appropriate angle such that they maximize the amount of irrigation fluid they absorb whilst not obstructing blood flow to the uterine horns.</li>
</ol>
3. Abdominal Laparotomy
<ol>
	<li>Using a scalpel make a 3 cm midline incision in the prepared abdominal skin. Bluntly dissect the skin layer from the abdominal fascia with scissors. Using forceps and surgical scissors, elevate the abdominal fascial layer and make an incision of the avascular linea alba to gain access to the peritoneal cavity.</li>
	<li>Place surgical gauze on the exterior of the incision and moisten with sterile saline. Using blunt forceps and external pressure on the abdomen, gently remove the uterine horns from the peritoneal cavity and arrange on the moistened gauze.</li>
	<li>Carefully avoid entanglement and contact with intestines. Arrange fetuses using forceps by contacting only the muscular tissue in between individual amniotic sacs. Expose and isolate the 4 uterine arteries using blunt dissection. 
	NOTE: Care must be taken to dissect the uterine arteries. Surrounding tissue and vessels themselves are extremely delicate. Damage to maternal vessels can cause bleeding, and in severe cases, fetal and maternal death.</li>
</ol>
4. Placement of Aneurysm Clips
<ol>
	<li>Place a rat 30 G aneurysm clip on each uterine artery. Ensure cessation of blood flow, including proximal and distal pulses, and darkening of the uterine vessels including individual placentas. Cover the exposed horns and entire surgical field with gauze and irrigate with sterile saline. Take care to keep the field moist with irrigation approximately every 10 min.</li>
	<li>After 60 min, remove the gauze and irrigate the field. Ensure that the uterine horns and vessels are adequately moistened for successful clip removal. Gently remove each aneurysm clip using forceps. Take care not to cause trauma to the vessel, and maintain tissue integrity during removal.</li>
	<li>Thoroughly irrigate the uterine horns and field, taking care to remove any stray threads of gauze from the amniotic sacs.</li>
</ol>
5. Injection of Lipopolysaccharide in to Amniotic Sacs
<ol>
	<li>At the base of each individual amniotic sac, just anterior to the placental plate, inject 100 &#181;l of LPS (4 &#181;g/sac) with diluted Evan&#8217;s blue dye in to the amniotic fluid. Use blunt forceps to stabilize and rotate each amniotic sac in to an optimal position for injection. Dilute Evan&#8217;s blue dye is a contrast agent that is helpful in confirming proper syringe placement and injection. 
	NOTE: Use only an ultra-fine 0.3 ml insulin syringe with attached 8 mm 31 G needle for the intra-amniotic injections. Using larger gauge needles will result in chronic amniotic fluid loss, fetal death and reabsorption of the pregnancy. Small amounts of amniotic fluid leakage upon removal of the syringe can be mitigated by direct pressure to the amniotic sac. Some rat fetuses may tolerate a degree of oligohydramnios. However, acute amniotic fluid loss from puncture with large gauge needles, or accidental puncture resulting in chronic fluid leakage, results in fetal loss and in severe cases, loss of neighboring pregnancies.</li>
	<li>Irrigate the uterine horns 3x with sterile saline.</li>
</ol>
6. Closing the Laparotomy
<ol>
	<li>Using forceps, carefully return the uterine horns to the peritoneal cavity. Ensuring adequate space between the amniotic sacs and the midline incision, re-approximate the musculofascial layer edges using a running 3-0 silk suture. Be aware of the placement of the amniotic sacs while closing the muscle incision. Be careful to not to suture into or through a sac.</li>
	<li>Re-approximate the skin layer, using a running 3-0 silk suture, closing the skin layer. 
	NOTE: The laparotomy should be closed in two layers of continuous sutures to allow for skin and muscular expansion with increasing gestation. Continuous sutures allow for evenly distributed wound tension. Interrupted sutures are less desired as multiple knots are irritating and can be easily chewed by the rat upon recovering from anesthesia. Surgical staples are not desired. Tails of the surgical knots should be cut very short (&#60;3 mm).</li>
	<li>Inject 1 ml of 0.125% bupivacaine&#160;subcutaneously around wound edges using a 26 G needle. Administer one dose of 0.1 mg/kg buprenorphine subcutaneously at the nape of the neck.</li>
	<li>Turn off isoflurane and towel dry rat as necessary. Place in clean home cage and monitor recovery from anesthesia. Ensure rat does not become hypothermic.</li>
</ol>
7. Postoperative Recovery and Care
<ol>
	<li>Monitor the rat every 8-12 hr for 72 hr and then daily until pups are born (approximately E22 or E23). Administer additional doses of buprenorphine q8-12 hr/72 hr or prn as dictated by the IACUC.</li>
	<li>Monitor rats for signs of pain, discomfort, vaginal bleeding or bleeding from the surgical site. Inspect the sutures and incision and take care to ensure rat is not chewing or removing their sutures prematurely. Although exceptionally rare, rats that compromise their sutures are at risk for wound dehiscence. 
	NOTE: Occasionally rats may ingest excessive amounts of bedding or non-food items, termed pica, as a side effect of buprenorphine administration. Although very uncommon, rats must be monitored for pica and potential subsequent bowel obstruction.</li>
</ol>
8. Tissue Processing and Cryosectioning
<ol>
	<li>To prepare for Hematoxylin &#38; Eosin (H&#38;E) staining of the postnatal brain, remove pups from their home cage on postnatal day 2 (P2). Using surgical scissors decapitate rat pups and gently remove the brain from the skull.</li>
	<li>Drop fix brain in a 15 ml conical tube containing 7 ml of 4% paraformaldehyde in phosphate buffered saline (PBS). Place brain at 4 &#176;C and fix for 72 hr.</li>
	<li>After 72 hr, transfer brains to a sterile PBS solution containing 30% sucrose (w/v) and return to 4 &#176;C. 
	NOTE: Once brains drop in the sucrose solution they are ready to be sectioned on a cryostat.</li>
	<li>Rapidly freeze brains and mount on cryostat pestle for acquisition of frozen coronal sections. Cut 20 &#181;m frozen coronal sections and mount on slides. Ensure sections are collected serially.</li>
	<li>Allow slides to dry at room temperature overnight. Store slides at -20 &#176;C.</li>
</ol>
9. Hematoxylin &#38; Eosin Staining
<ol>
	<li>Take slide mounted frozen sections and warm to room temperature. 
	NOTE: Prepare all solutions fresh.</li>
	<li>Place slides on a slide warmer set to 50 &#176;C for 2 hr.</li>
	<li>Transfer slides to a staining rack. Dip slides 10x in double-distilled deionized water (ddH2O).</li>
	<li>Incubate slides in 100% hematoxylin for 5 min. Time in hematoxylin can be optimized depending on degree of purple staining.</li>
	<li>Dip slides 4x in tap H2O, and let stand in clean tap H2O for 1 min.</li>
	<li>Dip slides 15x in acid alcohol (250 ml 70% ethanol + 1 ml concentrated hydrochloric acid).</li>
	<li>Dip slides 4x in tap H2O, and let stand in clean tap H2O for 1 min.</li>
	<li>Incubate in 1% lithium carbonate for 2 min.</li>
	<li>Dip slides 4x in tap H2O, and let stand in clean tap H2O for 1 min.</li>
	<li>Incubate in 95% ethanol for 1 min.</li>
	<li>Dip slides 7x in 100% Eosin. Number of dips in Eosin can be modified depending on degree of&#160;pink staining.</li>
	<li>Dip slides 5x in 95% ethanol.</li>
	<li>Dip slide 5x in a fresh change of 95% ethanol.</li>
	<li>Incubate slides in 100% ethanol for 1 min.</li>
	<li>Incubate slides in a fresh change of 100% ethanol for 1 min.</li>
	<li>Incubate slides in 100% xylene for 15 min. 
	NOTE: Xylene steps and coverslipping should occur in a fume hood.</li>
	<li>Incubate slides in fresh changes of xylene for 15 min.</li>
	<li>Coverslip with Permount and let dry in fume hood.</li>
	<li>Image slides with a light microscope. &#160;</li>
</ol>

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The authors have nothing to disclose.

Encephalopathy of prematurity is difficult to model in animals because of the complex interaction of etiologies, neurodevelopmental time course, intricacy of human cerebral network formation, overlapping mechanisms of injury, and the diverse phenotypes of CNS insults manifest in human preterm infants. EoP is associated with specific cell-type vulnerabilities (i.e. immature oligodendrocytes)21, as well as diverse developmentally-regulated pathways (i.e. subplate, membrane transporters and receptor subunits)12,13,22. However, significant progress can be made when animal models replicate the human condition as closely as possible. Here, we developed a model a prenatal insult that incorporates the heterogeneity of mechanisms of CNS injury observed in the preterm infant, allowing for subsequent evaluation of both gray and white matter damage and recovery. In humans, ascending bacterial infections weaken the amnion and precipitate premature rupture of membranes. Additionally, placental perfusion defects stress the placental interface and disrupt placental homeostasis. Thus, placental underperfusion compounds CNS injury from an intrauterine infection. Undeniably, it is challenging to model the common clinical scenario of ascending bacterial infections that precede chorioamnionitis in rodents as they have a duplex uterus. Each uterine horn has its own cervix, and multiple pregnancies are carried at once. Despite these challenges, preclinical models have been adapted to involve multiple components of the maternal-placental-fetal unit and incorporate in utero inflammation to various degrees. While no individual preclinical model is ideal to test every specific hypothesis, the model described herein incorporates the cellular and molecular abnormalities, behavioral and functional impairment, maternal-placental-fetal system, and the intrauterine infection and placental inflammation component common to so many preterm births20,23.
The choice of species used to model EoP impacts the interpretation of experimental data in the context of the inherent limitations posed by the species. In the simplest terms, birth does not equate to similar points of CNS development across all animals24. The model described here can be performed in both pregnant mice and rats, although pup survival in mice is significantly decreased in inexperienced or stressed dams. Consistent with our prior reports, fetal loss in rats at birth (P0) is increased in TSHI+LPS animals (approximately 40%) compared to sham, LPS and TSHI alone, but surviving pups do not exhibit significant weight differences through P2820. Similar to differences among species, the timing of injury during gestation has a crucial role in the neurodevelopmental trajectory of the offspring. The spatiotemporal regulation of neural cell developmental stages of proliferation, migration and differentiation differs amongst various mammals24-26. These cell-specific developmental programs influence the vulnerability to injury. For example, the overlap of the timing of oligodendrocyte lineage and GABAergic neuronal development with the timing of preterm birth makes these cells particularly susceptible to perinatal insults27-29. Thus, this model was developed in E18 rats (and successfully translated to E17 mice on a C57BL/6 background) as this timing corresponds to the intrauterine global prenatal insult that occurs in human infants prior to extremely preterm birth at 23-25 weeks gestation20. We previously showed O4-immunoreactive immature oligodendrocytes are most affected at this stage of development16. Their loss correlates with decreased survival and maturation14, with the most notable reductions in O4+ and O1+ stages of the lineage16, consistent with previous reports by other investigators30. Moreover, we have demonstrated premature loss of the subplate, reduced KCC2 expression, lower seizure threshold and impaired gait12,13,15 consistent with dysregulated GABAergic signaling in preterm infants31.
The model described here offers numerous benefits over previous models in rodents used to study perinatal brain injury from preterm birth23. It incorporates the entire maternal-placental-fetal system and causes both brain and placental injury. We have previously published comparisons between sham, TSHI alone, LPS alone and TSHI+LPS and differences in functional outcomes and biochemistry20, and differences with graded TSHI16. While prior investigations of unilateral carotid ligations and systemic hypoxia in neonatal rats have shed mechanistic insight into numerous pathophysiological processes (i.e. the susceptibility of immature oligodendrocytes to ischemia), direct translational and clinical relevance for such models is less robust. In addition to the applications described, the model described here may be an informative tool for investigation of other organ systems impacted by prematurity, including necrotizing enterocolitis (NEC), heart, lung, renal and hypothalamic-pituitary axis dysfunction. Owing to the complexities of LPS pharmacology and differences in maternal and fetal pharmacodynamics, intraperitoneal LPS injections in dams are less likely to produce the same fetal inflammatory response shown here. Further, LPS does not cross the placenta reliably20,32. Previously, we attempted direct cervical application of LPS and intrauterine injection similar to what has been described in other mouse models33. However, we found that mortality and inconsistency of CNS injury was significantly increased among pups within the same litter. Here, the dose of 4 &#181;g/sac was optimized using dose-response experiments. Increasing doses of LPS administered to the amniotic compartment results in increased fetal mortality. LPS has the advantage over direct infection with typical intrauterine gram-negative bacteria in that it activates inflammatory signaling through toll-like receptor 4 without causing active bacterial infection and the associated risk of pathogen spread. However, this model could be modified to include common pathogens and organisms isolated from human placentas, including group B streptococcus, which causes placental and neuropathological abnormalities, and autistic-like behavior in rats34. Similarly, Ureaplasma lipoprotein multiple-banded antigen can simulate Ureaplasma species infection. Since Ureaplasma is the most common cause of human chorioamnionitis35, this could also be an avenue for future investigation. As more inactivated-infectious agents become available, it will be informative to determine how they differentially impact neurodevelopment and the efficacy of neuro-reparative interventions.
Limitations of this model include amniotic fluid loss from the intra-amniotic injections. Although no effect is noted with intra-amniotic injections of sterile saline, the gauge of the needle used to perform the injections is a crucial technical element. Care must be taken not to use needles larger than 31 G. Surgical complications in the mother rat related to the laparotomy are extremely rare, including wound dehiscence, bowel obstruction, peritonitis and complete loss of the pregnancy, with maternal mortality less than 5%.

With over 12% of infants born in the United States before 37 weeks estimated gestational age1, perinatal brain injury (PBI) from prematurity is a significant cause of permanent disability. PBI from prematurity, also termed encephalopathy of prematurity (EoP), affects the entire central nervous system (CNS). CNS injury often commences in utero, and is exacerbated by antenatal processes including chorioamnionitis and postnatal complications such as hypoxia and sepsis. PBI from systemic insults alters neurodevelopment and leads to cerebral palsy, epilepsy, cognitive delay and numerous neuropsychiatric disorders affecting emotional regulation, memory and executive function1,2. Although much progress has been made, a limited understanding remains of how the cellular and molecular consequences of CNS injury from preterm birth translate to the multitude of neurological sequelae in children who are born preterm. This lack of knowledge hinders real-time diagnosis of CNS injury severity and informed dosing of emerging interventions. Additionally, age-appropriate therapeutic strategies for this vulnerable patient population remain elusive.
Intrauterine inflammation is very common in extreme prematurity and involves a complex fetal-maternal-placental inflammatory cascade3. Intrauterine infection is often subclinical. Specific placental findings consistent with acute inflammation, or histologic chorioamnionitis, are major determinants of the fetal inflammatory response and are coincident with brain injury associated with preterm birth3-5. Indeed, the fetal inflammatory response has distinct clinical implications for long-term outcomes from preterm birth. Infants who are small for gestational age (SGA) or who experience infection are exceptionally vulnerable to neurological deficits3,4. Chorioamnionitis is a typical pathological diagnosis following preterm birth6,7, and histological examination reveals signs of inflammation in 70% of placentas from infants born very preterm4. Further, chorioamnionitis is associated with cognitive impairment at two years8. Evidence of maternal vascular underperfusion in the placenta of infants born extremely preterm is also associated with cerebral palsy in childhood9. The synergistic impact of chorioamnionitis and placental perfusion defects is well illustrated by the remarkably high risk of abnormal neurologic outcomes in this patient population at two years of age10,11.
To mimic human systemic placental perfusion defects and chorioamnionitis associated with pathogen-induced inflammation, we developed a model of prenatal transient systemic hypoxia-ischemia (TSHI) combined with intra-amniotic lipopolysaccharide (LPS) in rats. Our goal was to adapt our model of TSHI alone in rats12-16 to include intrauterine inflammation, to facilitate preclinical modeling of CNS injury associated with preterm birth. TSHI alone has revealed persistent loss of oligodendroglial lineage cells, cortical neurons, increased cell death, and elevated pro-inflammatory cytokine levels, with progressive ischemic intervals leading to a graded pattern of injury consistent with prenatal brain injury16. Modifications to the ischemic components of this model have also demonstrated deficits in memory encoding, short and long-term memory and mild musculoskeletal alterations in rats as they age17-19. Indeed, we have previously demonstrated that the combination of TSHI+LPS recapitulates the pathophysiological hallmarks of EoP, including oligodendrocyte and neuronal loss, axonal injury, cellular inflammation and functional abnormalities20.

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modeling encephalopathy of prematurity using prenatal hypoxia-ischemia with intra-amniotic lipopolysaccharide in rats

Encephalopathy of prematurity (EoP) is a term that encompasses the central nervous system (CNS) abnormalities associated with preterm birth. To best advance translational objectives and uncover new therapeutic strategies for brain injury associated with preterm birth, preclinical models of EoP must include similar mechanisms of prenatal global injury observed in humans and involve multiple components of the maternal-placental-fetal system. Ideally, models should produce a similar spectrum of functional deficits in the mature animal and recapitulate multiple aspects of the pathophysiology. To mimic human systemic placental perfusion defects, placental underperfusion and/or chorioamnionitis associated with pathogen-induced inflammation in early preterm birth, we developed a model of prenatal transient systemic hypoxia-ischemia (TSHI) combined with intra-amniotic lipopolysaccharide (LPS). In pregnant Sprague Dawley rats, TSHI via uterine artery occlusion on embryonic day 18 (E18) induces a graded placental underperfusion defect associated with increasing CNS damage in the fetus. When combined with intra-amniotic LPS injections, placental inflammation is increased and CNS damage is compounded with associated white matter, gait and imaging abnormalities. Prenatal TSHI and TSHI+LPS prenatal insults meet several of the criteria of an EoP model including recapitulating the intrauterine insult, causing loss of neurons, oligodendrocytes and axons, loss of subplate, and functional deficits in adult animals that mimic those observed in children born extremely preterm. Moreover, this model allows for the dissection of inflammation induced by divergent injury types.

Following TSHI+LPS at E18, hematoxylin and eosin staining reveals significant histopathological abnormalities in both the placenta (Figure 1) and in the brain (Figure 2). Placentas examined on E19 and E21 are grossly edematous with micro-hemorrhage, and necrosis throughout the decidua and labyrinth. Significant inflammatory infiltrate and increased vascularity is also observed. Brains examined on P2 reveal ventriculomegaly, as well as white matter and subplate neuron loss compared to shams. Previously, we have reported that TSHI+LPS induces inflammation and yields persistent white matter and axonal abnormalities concomitant with significant motor impairment in young adults20. TSHI+LPS also significantly decreases potassium chloride co-transporter 2 (KCC2) protein expression, a chloride co-transporter central to the development of &#947;-aminobutyric acid (GABA)ergic inhibition, in the cortex at P15 (Figure 3, *P &#60;0.05). These data are consistent with impaired developmental upregulation of KCC2 during a critical postnatal period of circuit maturation and these findings are consistent with our prior reports of the effects of TSHI alone in the CA3 hippocampus13.
<img alt="Figure 1" src="/files/ftp_upload/53196/53196fig1.jpg" /> 
Figure 1: TSHI+LPS induces significant histological abnormalities in the placenta. Following transient in utero hypoxia-ischemia and intra-amniotic LPS administration on embryonic day 18 (E18), placentas from E19 TSHI+LPS fetuses (B) are grossly edematous with hemorrhage (arrows), necrosis and increased inflammatory infiltrate compared to sham (A, scale bar = 100 &#181;m). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53196/53196fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53196/53196fig2.jpg" /> 
Figure 2: TSHI+LPS induces significant histological abnormalities in the brain. Following transient in utero hypoxia-ischemia and intra-amniotic LPS administration on embryonic day 18 (E18), postnatal ventriculomegaly, subplate neuron and white matter loss is observed in pups subjected to dual TSHI+LPS (B) compared to sham (A) acutely at P2. (Scale bar = 100 &#181;m; Sp = subplate; WM = white matter; LV = lateral ventricle) <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53196/53196fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53196/53196fig3.jpg" /> 
Figure 3: TSHI+LPS reduces KCC2 expression. Western blots performed from membrane preparations of micro-dissected cortical tissue, show in utero transient systemic hypoxia-ischemia and intra-amniotic LPS administration on embryonic day 18 (E18) significantly reduces expression of KCC2, a neuron-specific potassium chloride co-transporter central to the development of integrated cerebral circuits and inhibition, on postnatal day 15. (n = 6-10, mean &#177; SEM, Student&#8217;s t-test,*P &#60;0.05).

Watch this Scientific Journal Video about Modeling Encephalopathy of Prematurity Using Prenatal Hypoxia-ischemia with Intra-amniotic Lipopolysaccharide in Rats at JoVE.com

Modeling Encephalopathy of Prematurity Using Prenatal Hypoxia-ischemia with Intra-amniotic Lipopolysaccharide in Rats

Encephalopathy of prematurity encompasses the central nervous system abnormalities associated with injury from preterm birth. This report describes a clinically relevant rat model of in utero transient systemic hypoxia-ischemia and intra-amniotic lipopolysaccharide administration (LPS) that mimics chorioamnionitis, and the related impact of infectious stimuli and placental underperfusion on CNS development.

Encephalopathy of prematurity (EoP) is a term that encompasses the central nervous system (CNS) abnormalities associated with preterm birth. To best ...

modeling-encephalopathy-prematurity-using-prenatal-hypoxia-ischemia

Nanyang Technological University

Research

JoVE Journal

Medicine

11.3K Views.  University of New Mexico. Encephalopathy of prematurity encompasses the central nervous system abnormalities associated with injury from preterm birth. This report describes a clinically relevant rat model of in utero transient systemic hypoxia-ischemia and intra-amniotic lipopolysaccharide administration (LPS) that mimics chorioamnionitis, and the related impact of infectious stimuli and placental underperfusion on CNS development.

Video: Modeling Encephalopathy of Prematurity Using Prenatal Hypoxia-ischemia with Intra-amniotic Lipopolysaccharide in Rats

Mouse Models of Periventricular Leukomalacia

We describe a protocol for establishing mouse models of periventricular leukomalacia (PVL). PVL is the predominant form of brain injury in premature infants and the most common antecedent of cerebral palsy. PVL is characterized by periventricular white matter damage with prominent oligodendroglial injury. Hypoxia/ischemia with or without systemic infection/inflammation are the primary causes of PVL. We use P6 mice to create models of neonatal brain injury by the induction of hypoxia/ischemia with or without systemic infection/inflammation with unilateral carotid ligation followed by exposure to hypoxia with or without injection of the endotoxin lipopolysaccharide (LPS). Immunohistochemistry of myelin basic protein (MBP) or O1 and electron microscopic examination show prominent myelin loss in cerebral white matter with additional damage to the hippocampus and thalamus. Establishment of mouse models of PVL will greatly facilitate the study of disease pathogenesis using available transgenic mouse strains, conduction of drug trials in a relatively high throughput manner to identify candidate therapeutic agents, and testing of stem cell transplantation using immunodeficiency mouse strains.

We established mouse models of periventricular leukomalacia (PVL), the predominant brain injury in premature infants characterized by periventricular white matter lesions. Hypoxia/ischemia with/without systemic infection are the primary causes of PVL. Unilateral carotid ligation and hypoxia exposure with/without lipopolysaccharide injection creates PVL-like lesions in P6 mice.

We describe a protocol for establishing mouse models of periventricular leukomalacia (PVL). PVL is the predominant form of brain injury in premature ...

Neuroscience

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

A Piglet Model of Neonatal Hypoxic-Ischemic Encephalopathy

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the world&#8217;s 2.9 million neonatal deaths. Animal models of HIE have contributed to the understanding of the pathophysiology in HIE, and have highlighted the dynamic process that occur in brain injury due to perinatal asphyxia. Thus, animal studies have suggested a time-window for post-insult treatment strategies. Hypothermia has been tested as a treatment for HIE in pdiglet models and subsequently proven effective in clinical trials. Variations of the model have been applied in the study of adjunctive neuroprotective methods and piglet studies of xenon and melatonin have led to clinical phase I and II trials1,2. The piglet HIE model is further used for neonatal resuscitation- and hemodynamic studies as well as in investigations of cerebral hypoxia on a cellular level. However, it is a technically challenging model and variations in the protocol may result in either too mild or too severe brain injury. In this article, we demonstrate the technical procedures necessary for establishing a stable piglet model of neonatal HIE. First, the newborn piglet (&#60; 24 hr old, median weight 1500 g) is anesthetized, intubated, and monitored in a setup comparable to that found in a neonatal intensive care unit. Global hypoxia-ischemia is induced by lowering the inspiratory oxygen fraction to achieve global hypoxia, ischemia through hypotension and a flat trace amplitude integrated EEG (aEEG) indicative of cerebral hypoxia. Survival is promoted by adjusting oxygenation according to the aEEG response and blood pressure. Brain injury is quantified by histopathology and magnetic resonance imaging after 72 hr.

Hypoxic-ischemic encephalopathy following perinatal asphyxia can be studied using animal models. We demonstrate the procedures necessary for establishing a piglet model of neonatal hypoxic-ischemic encephalopathy.

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the ...

Neurobehavioral Assessments in a Mouse Model of Neonatal Hypoxic-ischemic Brain Injury

We performed unilateral carotid artery occlusion on CD-1 mice to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of neonatal HI brain injury by studying neurobehavioral functions in these mice compared to non-operated (i.e., normal) mice. During the study, Rice-Vannucci&#39;s method was used to induce neonatal HI brain damage in postnatal day 7-10 (P7-10) mice. The HI operation was performed on the pups by unilateral carotid artery ligation and exposure to hypoxia (8% O2 and 92% N2 for 90 min). One week after the operation, the damaged brains were evaluated with the naked eye through the semi-transparent skull and were categorized into subgroups based on the absence (&#34;no cortical injury&#34; group) or presence (&#34;cortical injury&#34; group) of cortical injury, such as a lesion in the right hemisphere. On week 6, the following neurobehavioral tests were performed to evaluate the cognitive and motor functions: passive avoidance task (PAT), ladder walking test, and grip strength test. These behavioral tests are helpful in determining the effects of neonatal HI brain injury and are used in other mouse models of neurodegenerative diseases. In this study, neonatal HI brain injury mice showed motor deficits that corresponded to right hemisphere damage. The behavioral test results are relevant to the deficits observed in human neonatal HI patients, such as cerebral palsy or neonatal stroke patients. In this study, a mouse model of neonatal HI brain injury was established and showed different degrees of motor deficits and cognitive impairment compared to non-operated mice. This work provides basic information on the HI mouse model. MRI images demonstrate the different phenotypes, separated according to the severity of brain damage by motor and cognitive tests.

We performed unilateral carotid artery occlusion on postnatal day 7-10 CD-1 mouse pups to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of HI brain injury. We studied neurobehavioral functions in these mice compared to non-operated normal mice.

We performed unilateral carotid artery occlusion on CD-1 mice to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of neonatal HI ...

A 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.

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

There is an ongoing need for clinically relevant models of perinatal infection and hypoxia-ischemia (HI) in which to test therapeutic interventions for infants with the neurological sequela of prematurity. Ferrets are ideal candidates for modeling the preterm human brain, as they are born lissencephalic and develop gyrencephalic brains postnatally. At birth, ferret brain development is similar to a 13 week human fetus, with postnatal-day (P) 17 kits considered to be equivalent to an infant at 32&#8211;36 weeks&#39; gestation. We describe an injury model in the P17 ferret, where lipopolysaccharide administration is followed by bilateral cerebral ischemia, hypoxia, and hyperoxia. This simulates the complex interaction of prolonged inflammation, ischemia, hypoxia, and oxidative stress experienced in a number of neonates who develop brain injury. Injured animals display a range of gross injury severity, with morphological changes in the brain including narrowing of multiple cortical gyri and associated sulci. Injured animals also show slowed reflex development, slower and more variable speed of locomotion in an automated catwalk, and decreased exploration in an open field. This model provides a platform in which to test putative therapies for infants with neonatal encephalopathy associated with inflammation and HI, study mechanisms of injury that affect cortical development, and investigate pathways that provide resilience in unaffected animals.

The method describes inflammation-sensitized hypoxic-ischemic and hyperoxic brain injury in the P17 ferret to model the complex interaction between prolonged inflammation and oxidative brain injury experienced in a number of late preterm infants.

There is an ongoing need for clinically relevant models of perinatal infection and hypoxia-ischemia (HI) in which to test therapeutic interventions for ...

Modeling Neonatal Intraventricular Hemorrhage Through Intraventricular Injection of Hemoglobin

Neonatal intraventricular hemorrhage (IVH) is a common consequence of premature birth and leads to brain injury, posthemorrhagic hydrocephalus (PHH), and lifelong neurological deficits. While PHH can be treated by temporary and permanent cerebrospinal fluid (CSF) diversion procedures (ventricular reservoir and ventriculoperitoneal shunt, respectively), there are no pharmacological strategies to prevent or treat IVH-induced brain injury and hydrocephalus. Animal models are needed to better understand the pathophysiology of IVH and test pharmacological treatments. While there are existing models of neonatal IVH, those that reliably result in hydrocephalus are often limited by the necessity for large-volume injections, which may complicate modeling of the pathology or introduce variability in the clinical phenotype observed.
Recent clinical studies have implicated hemoglobin and ferritin in causing ventricular enlargement after IVH. Here, we develop a straightforward animal model that mimics the clinical phenotype of PHH utilizing small-volume intraventricular injections of the blood breakdown product hemoglobin. In addition to reliably inducing ventricular enlargement and hydrocephalus, this model results in white matter injury, inflammation, and immune cell infiltration in periventricular and white matter regions. This paper describes this clinically relevant, simple method for modeling IVH-PHH in neonatal rats using intraventricular injection and presents methods for quantifying ventricle size post injection.

We present a model of neonatal intraventricular hemorrhage using rat pups that mimics the pathology seen in humans.

Neonatal intraventricular hemorrhage (IVH) is a common consequence of premature birth and leads to brain injury, posthemorrhagic hydrocephalus (PHH), and ...

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 ...

Modeling Posthemorrhagic Hydrocephalus of Prematurity in Rats

Posthemorrhagic hydrocephalus of prematurity (PHHP) is a serious sequela of severe intraventricular hemorrhage (IVH) in very preterm infants less than 32 weeks gestational age (GA). PHHP is defined by the accumulation of cerebrospinal fluid (CSF) associated with clinical symptoms of elevated intracranial pressure (ICP). Infants with PHHP suffer lifelong shunt dependence, with half requiring repeat surgery in the first year of life and many requiring multiple additional surgeries throughout the lifespan. Prenatal chorioamnionitis predisposes preterm infants to severe IVH and the need for surgical treatment of PHHP trends with neonatal sepsis. These clinical features suggest that systemic inflammation is an integral component of PHHP pathophysiology. 
Here, we define an animal model that recapitulates all clinical aspects and essential features of PHHP in rats. The goal of this protocol is to illustrate how in utero chorioamnionitis and postnatal IVH using lysed red blood cells can be combined to yield PHHP. This preclinical approach yields progressive macrocephaly and domed craniums, elevated intracranial pressure, and ventriculomegaly that can be detected via magnetic resonance imaging (MRI) or via microscopy. In addition to sustained disruption in CSF dynamics, rats also have cognitive delay and functional disability into adulthood. Accordingly, this preclinical platform facilitates unique and unparalleled translational studies of PHHP that can incorporate molecular, cellular, biochemical, histologic, imaging, and functional outcome measures. It can also be used for rigorous analysis of the choroid plexus, ependymal motile cilia, and glymphatic system in parallel. Last, it can also be an invaluable preclinical tool for the investigation of novel surgical intervention strategies and non-surgical therapeutic approaches for the treatment of hydrocephalus.

Posthemorrhagic hydrocephalus of prematurity (PHHP) can be modeled in neonatal rats by combining chorioamnionitis and intraventricular hemorrhage. The combination of these prenatal and postnatal events accurately recapitulates the clinical hallmarks of PHHP, including macrocephaly, ventriculomegaly, and elevated intracranial pressure, through the lifespan.

Posthemorrhagic hydrocephalus of prematurity (PHHP) is a serious sequela of severe intraventricular hemorrhage (IVH) in very preterm infants less than 32 ...

Induction of Cerebral Microhemorrhages in a Rat Model Using Lipopolysaccharide

Source: Li, D., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/58423">Sub-acute Cerebral Microhemorrhages Induced by Lipopolysaccharide Injection in Rats.</a> J. Vis. Exp. (2018).
This video demonstrates the process of inducing blood-brain barrier (BBB) disruption and cerebral microhemorrhages in a rat model using lipopolysaccharide (LPS). It highlights the sequence of immune responses that lead to increased BBB permeability and subsequent brain tissue damage.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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Modeling Encephalopathy of Prematurity Using Prenatal Hypoxia-ischemia with Intra-amniotic Lipopolysaccharide in Rats

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