A rat pup model of cerebral palsy induced by prenatal inflammation and hypoxia

Abstract

Animal models of cerebral palsy established by simple infection or the hypoxia/ischemia method cannot effectively simulate the brain injury of a premature infant. Healthy 17-day-pregnant Wistar rats were intraperitoneally injected with lipopolysaccharide then subjected to hypoxia. The pups were used for this study at 4 weeks of age. Simultaneously, a hypoxia/ischemia group and a control group were used for comparison. The results of the footprint test, the balance beam test, the water maze test, neuroelectrophysiological examination and neuropathological examination demonstrated that, at 4 weeks after birth, footprint repeat space became larger between the forelimbs and hindlimbs of the rats, the latency period on the balance beam and in the Morris water maze was longer, place navigation and ability were poorer, and the stimulus intensity that induced the maximal wave amplitude of the compound muscle action potential was greater in the lipopolysaccharide/hypoxia and hypoxia/ischemia groups than in the control group. We observed irregular cells around the periventricular area, periventricular leukomalacia and breakage of the nuclear membrane in the lipopolysaccharide/hypoxia and hypoxia/ischemia groups. These results indicate that we successfully established a Wistar rat pup model of cerebral palsy by intraperitoneal injection of lipopolysaccharide and hypoxia.

Keywords: animal models; brain injury; cerebral palsy; grants-supported paper; histopathology; hypoxia; lipopolysaccharide; neural regeneration; neuroelectrophysiology; neuroregeneration; photographscontaining paper; water maze test.

Figure 1

The behavioral outcomes observation in each group at 4 weeks of age. (A) Footprint analysis results. The space between the footprints of the forelimbs and hindlimbs of the rats in the lipopolysaccharide/hypoxia (LPS/H, n = 14) and hypoxia/ischemia (H/I, n = 13) groups was longer than that in the control group (n = 12, ^aP < 0.05). (B) Balancing beam test results. The latency period was longer and the number of the hindlimbs slipped was higher in the LPS/H and H/I groups than that in the control group (^aP < 0.05). The data are expressed as mean ± SEM. Two-sample t-test was used.

Figure 2

Outcomes of the Morris water maze test in each group at about 4 weeks of age. The escape latency of the lipopolysaccharide/hypoxia (LPS/H, n = 14) and hypoxia/ischemia (H/I, n = 13) groups was significantly longer than that of the control group (n=12) at different times (A: postnatal day 29; B: postnatal day 31; C: postnatal day 33; ^aP < 0.05); however, there was no difference between the LPS/H and H/I groups (P > 0.05). The data are expressed as mean ± SEM. Two-sample t-test was used.

Figure 3

Outcomes of neuroelectrophysiological examination of hypoxia rats at 4 weeks of age. The wave amplitude of the compound muscle action potential (CMAP) in the lipopolysaccharide/hypoxia (LPS/H, n = 14) and hypoxia/ischemia (H/I, n = 13) groups was lower than that in the control group (n=12) at 4 weeks of age (^aP < 0.05). There was no statistically significant difference in the latency of the CMAP between the three groups (P > 0.05). The data are expressed as mean ± SEM. Two-sample t-test was used.

Figure 4

Severity of white matter changes in the periventricular area in each group at 4 weeks of age. (A) Control group; (B) hypoxia/ischemia group; (C) lipopolysaccharide/hypoxia group. Coronal sections (6 μm) were obtained for hematoxylin-eosin staining. Control littermates did not show any abnormal findings Scale bars: 50 μm.

Figure 5

Ultra-thin slices of left brain specimens under transmission electron microscopy at 4 weeks of age. (A) An abundant cytoplasm, integrated nuclear membrane and a well-distributed chromoplasm of the cellular nucleus can be seen in the control group (scale bar: 5 μm). (B) A breakdown in the nuclear membrane can clearly be seen in the hypoxia/ischemia group (scale bar: 2 μm). (C) A breakdown in the nuclear membrane can also be seen in the lipopolysaccharide/hypoxia group (scale bar: 2 μm).