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 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.
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.
Institutional Care and Use Committees at both Boston Children’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’s blue dye.
1. Anesthesia
2. Surgical Prep and Scrub
3. Abdominal Laparotomy
4. Placement of Aneurysm Clips
5. Injection of Lipopolysaccharide in to Amniotic Sacs
6. Closing the Laparotomy
7. Postoperative Recovery and Care
8. Tissue Processing and Cryosectioning
9. Hematoxylin & Eosin Staining
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 γ-aminobutyric acid (GABA)ergic inhibition, in the cortex at P15 (Figure 3, *P <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.
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 µm). Please click here to view a larger version of this figure.
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 µm; Sp = subplate; WM = white matter; LV = lateral ventricle) Please click here to view a larger version of this figure.
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 ± SEM, Student’s t-test,*P <0.05).
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 µg/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%.
The authors have nothing to disclose.
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.
Saline Solution, 0.9% | Sigma | S8776 | |
LPS 011B4 | Sigma | L2630 | |
Evan's Blue Dye | Sigma | E2129 | |
Surgical gloves | Biogel | 40870 | |
OR Towels | Cardinal Health | 287000-008 | Sterile |
PDI Alcohol Prep Pads | Fisherbrand | 06-669-62 | |
Mini Arco Rechargeable Clippers | Kent Scientific Corp. | CL8787 | |
Betadine surgical scrub | Purdue Products L.P. | 67618-151-17 | |
Eye Lubricant | Refresh Lacri Lube | 00023-0312 | |
Blunt Forceps | Roboz | RS-8100 | |
Scissors | Roboz | RS-6808 | |
Surgical Scissors | Roboz | RS-5880 | |
Surgical Scissors | F.S.T. | 14002-16 | |
Syringe | BD | 309628 | 1 ml |
Needle | BD | 305122 | 25G 5/8 |
Needle | BD | 305128 | 30G 1 |
Cotton-tipped Applicators | Fisherbrand | 23-400-114 | Small, 6 inch sterile |
Cotton Gauze Sponge | Fisherbrand | 22-362-178 | |
Needle Holders | Kent Scientific Corp. | INS600109 | 12.5 CM STR |
Vessel Clips | Kent Scientific Corp. | INS600120 | 30G Pressure |
3-0 Perma Hand Silk Sutures | Ethicon | 1684G | Black braided, 3-0 (2 metric), 18", non-absorbable, PS-1 24mm needle, 3/8 circle |
Insulin Syringes | BD | 328438 | 0.3cc 3mm 31G |
Pentobarbital | |||
Buprenorphine | |||
Bupivacaine | |||
Isoflurane | |||
Lithium Carbonate | Acros Chemicals | 554-13-2 | |
Superfrost Plus Microscope Slides | VWR | 48311-703 | |
Hematoxylin | Leica | 3801521 | Surgipath Gill II Hematoxylin |
Eosin | Leica | 3801601 | Surgipath Eosin |
Xylenes | Fisherbrand | X3S-4 | Histological Grade |
Permount | Fisherbrand | SP15-100 | |
Coverglass | Fisherbrand | 12-548-5P | Fisher Finest Premium Coverglass |