Intrauterine administration of endotoxin leads to motor deficits in a rabbit model: a link between prenatal infection and cerebral palsy

Abstract

Objective: This study was undertaken to determine whether maternal intrauterine endotoxin administration leads to neurobehavioral deficits in newborn rabbits.

Study design: Pregnant New Zealand white rabbits were injected with 1 mL saline solution (n = 8) or 20 microg/kg of lipopolysaccharide in saline solution (n = 8) into the uterine wall on day 28/31 of gestation. On postnatal day 1, kits (saline solution [n = 30] and lipolysaccharide in saline solution [n = 18] from 4 consecutive litters) underwent neurobehavioral testing. Neonatal brains were stained for microglial cells and myelin.

Results: Kits in the lipopolysaccharide in saline solution group were hypertonic and demonstrated significant impairment in posture, righting reflex, locomotion, and feeding, along with neuroinflammation indicated by activated microglia and hypomyelination in the periventricular regions. A greater mortality was noted in the lipopolysaccharide in saline solution group (16 stillbirths from 3 litters vs 3 from 1 litter).

Conclusion: Maternal intrauterine endotoxin administration leads to white matter injury and motor deficits in the newborn rabbit, resulting in a phenotype that resembles those found in periventricular leukomalacia and cerebral palsy.

FIGURE 1. C-reactive protein in maternal serum

Maternal CRP was measured at baseline (0 hours which is just before start of the laparotomy), and at 2, 6, 24 and 48 hours after administration of endotoxin or saline in utero. An increase in CRP over time was noted at 24 and 48 hours in both groups from baseline values. This increase was slightly higher in the endotoxin dams when compared to saline indicating presence of a systemic inflammation after intrauterine endotoxin administration.

FIGURE 2. Neurobehavioral scoring of control and endotoxin groups

Activity of the control and endotoxin rabbit kits on postnatal day 1 was videotaped and scored by two independent observers. The graphs depict the mean and 95% confidence interval for each score. Compared to the controls, the endotoxin kits (A) were not able to maintain prone position and right themselves rapidly and consistently (B) were hypertonic; (C) showed decreased activity; (D) demonstrated decreased circular and forward motion; and (E) showed a decrease in co-ordination of suck and swallow leading to dribbling of formula from the mouth along with a decrease in normal jerky, reflexive head movement while feeding.

FIGURE 3. Spastic posturing of the extremities in endotoxin when compared to control kits

Extremities are normally abducted in the control kit (A), however they are rigid and have fixed abnormal positions in the endotoxin kits (B and C) due to the hypertonia resulting in impairment in locomotion..

FIGURE 4. Microglial cell staining

Brain sections from post-natal day 1 control and endotoxin rabbits were stained for microglial cells using tomato lectin. An increase in activated microglial cells is noted in the endotoxin treated kits when compared to the control kits in the regions of the corpus callosum, along the borders of the lateral ventricle, region of the internal capsule and the fimbria hippocampus. Scale bar is 200 μm.

FIGURE 5. Myelin basic protein staining

Myelin basic protein staining in a representative postnatal day 8 control and endotoxin kits. Decreased myelination is seen in the kits exposed to maternal inflammation in utero when compared to the saline exposed control kits in the corpus callosum, around the lateral ventricle, internal capsule and the fimbria hippocampus. Scale bar is 200 μm.

FIGURE 6. Presence of microglial cells in rabbit white matter tracts during development and its relationship to oligodendrocyte maturation

This schematic depicts the time course of microglia and oligodendrocyte density during normal rabbit brain development. Embryonic age 28 was chosen as the time of insult in this study as this corresponds with a normal peak in the density of microglial cells in the periventricular white matter tracts as well as a period of rapid increase in the density of immature oligodendrocytes. Corpus callosum (CC), internal capsule (IC), corona radiata (CR) and anterior commissure (AC).