Multi-Hit White Matter Injury-Induced Cerebral Palsy Model Established by Perinatal Lipopolysaccharide Injection

Abstract

Cerebral palsy (CP) is a group of permanent, but not unchanging, disorders of movement and/or posture and motor function. Since the major brain injury associated with CP is white matter injury (WMI), especially, in preterm infants, we established a "multi-hit" rat model to mimic human WMI in symptomatology and at a histological level. In our WMI model, pups suffering from limb paresis, incoordination, and direction difficulties fit the performance of CP. Histologically, they present with fewer neural cells, inordinate fibers, and more inflammatory cell infiltration, compared to the control group. From the electron microscopy results, we spotted neuronal apoptosis, glial activation, and myelination delay. Besides, the abundant appearance of IBA1-labeled microglia also implied that microglia play a role during neuronal cell injury. After activation, microglia shift between the pro-inflammatory M1 type and the anti-inflammatory M2 type. The results showed that LPS/infection stimulated IBA1 + (marked activated microglia) expression, downregulated CD11c + (marked M1 phenotype), and upregulated Arg 1 + (marked M2 phenotype) protein expression. It indicated an M1 to M2 transition after multiple infections. In summary, we established a "multi-hit" WMI-induced CP rat model and demonstrated that the microglial activation correlates tightly with CP formation, which may become a potential target for future studies.

Keywords: animal model; cerebral palsy; electron microscopy; perinatal infection; white matter injury.

FIGURE 1

Administration methods for animal models. The image shows the time and dose of intraperitoneal injection of drugs in each group. Pregnant rats were given the first injection at 20 days of gestation (G20), and the second injection of LPS was conducted on neonatal rats at postnatal day 20 (P20) to construct the WMI animal model. We divided experimental mice into three groups and again intraperitoneally injected LPS consisting of low to high concentration gradients at postnatal 40 (P40), to mimic the adverse effects of multiple infections early in development.

FIGURE 2

The images and graphs of behavior experiments (n = 12 rats for the control group, n = 28 rats for the WMI group). Frame-by-frame photos were taken at 5 frames per second. (A,B) A typical rat of the WMI group. (C) WMI group on the left and control group on the right. The range of motion in the WMI group was relatively smaller than the control one. (D) The bodyweight of the WMI group was lower than that of the control group (p < 0.0001 by unpaired two-tailed t-test). Data are presented as the mean ± SE. (E) In the balance beam experiment, the WMI group scored higher than the control group (p = 0.0121 by Mann–Whitney U test). (F) Berderson score from the rats in the control and WMI groups. The comprehensive score of the WMI group was higher than that of the control group (p = 0.0170 by Mann–Whitney U test). *P < 0.05, ****P < 0.0001.

FIGURE 3

Hematoxylin-eosin staining of rat hippocampus (n = 2–3 brains per group). (A) The hippocampus of normal rats in the control group showed many neurons with a close and orderly arrangement. Scale bar = 100 μm. (B) In the WMI group, nerve cells in the hippocampus were swollen, reduced in number, and disordered. Scale bar = 100 μm. Compared with the control group (C), hippocampal neurons in the WMI group (D) were sparse and the peripheral nerve fibers were disordered. Scale bar = 200 μm.

FIGURE 4

Hematoxylin-eosin staining around the lateral ventricle in rats (n = 2–3 brains per group). (A) The rats in the control group (P20) had normal lateral ventricles, developed choroid plexus, and abundant and orderly nerve fibers in the white matter adjacent to the lateral ventricles. (B) Periventricular white matter at 4× magnification. (C,D) In the WMI group (P20), lateral ventricle development was slower, choroid plexus atrophy and nerve fibers were sparse. Scale bar = 200 μm.

FIGURE 5

Ultrastructure of neural cells under an electron microscope in the WMI group. (A) Neuronal cell injury initiates programed cell apoptosis, and chromatin in the nucleus begins to condense. Organelles such as mitochondria (“m”)and Golgi apparatus (“g”) are normal. Scale bar = 2 μm. (B) Nucleolus pyknosis, nuclear membrane, and nucleolar space enlarges (arrow), and mitochondrial swelling. Scale bar = 1 μm. (C) The oligodendrocyte has a darker cytoplasm than the surroundings and is in attachment to a myelin sheath (arrow). Its nucleus was deformed, and chromatin was highly condensed. Mitochondria appeared vacuolated with excessive swelling, even some floccule appeared in the lumens. Scale bar = 1 μm. (D) A neuron in contact with the blood vessel was degenerative with more intracytoplasmic lysosomes (“ly”). An astrocyte came to repair it (pentacle). Scale bar = 5 μm. (E) The microglia (arrow) phagocytized a neural cell and a plasma cell. The specific cell morphology of the nerve cell above disappeared, but the plasma cell below was still visible with relatively intact cell morphology, and there was also abundant Golgi in the cytoplasm. Scale bar = 2 μm. (F) Lipofuscin (“li”) appeared within the cytoplasm of an injured neuron and there was some stunted myelin in the periphery. Myelin damage (arrow). Scale bar = 2 μm.

FIGURE 6

Microglial activation in WMI group. (A,B) The control group is on the left and the WMI group is on the right. Scale bar: 200 μm. (C,D) A magnified version of A and B. IBA1 + microglia were morphologically diverse, with most cells extending processes. Scale bar: 100 μm. (E,F) Quantitative analysis of the IBA1-positive areas in each group (2–3 sections were evaluated per pup). Five regions of each specimen were randomly collected to calculate the average number of positive cells and the average optical density. Data are presented as the mean ± SE. Compared to the control group by t-test, *P < 0.05, ****P < 0.0001.

FIGURE 7

Western Blot of Arg1, CD11c, and IBA1 (n = 2–3 brains per group). (A) WB bands of the control group, low dose, medium dose, and high dose WMI + LPS groups. (B–D) The histograms represent the normalized relative density of Arg1(B), CD11c(C), and IBA1(D) between the control group and experimental groups. Data are presented as the mean ± SE. Compared the mean of each group with the mean of every other group by the Bonferroni test. ns > 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.