Effects of Melatonin on Neurobehavior and Cognition in a Cerebral Palsy Model of plppr5-/- Mice

Abstract

Cerebral palsy (CP), a group of clinical syndromes caused by non-progressive brain damage in the developing fetus or infant, is one of the most common causes of lifelong physical disability in children in most countries. At present, many researchers believe that perinatal cerebral hypoxic ischemic injury or inflammatory injury are the main causes of cerebral palsy. Previous studies including our works confirmed that melatonin has a protective effect against convulsive brain damage during development and that it affects the expression of various molecules involved in processes such as metabolism, plasticity and signaling in the brain. Integral membrane protein plppr5 is a new member of the plasticity-related protein family, which is specifically expressed in brain and spinal cord, and induces filopodia formation as well as neurite growth. It is highly expressed in the brain, especially in areas of high plasticity, such as the hippocampus. The signals are slightly lower in the cortex, the cerebellum, and in striatum. Noteworthy, during development plppr5 mRNA is expressed in the spinal cord, i.e., in neuron rich regions such as in medial motor nuclei, suggesting that plppr5 plays an important role in the regulation of neurons. However, the existing literature only states that plppr5 is involved in the occurrence and stability of dendritic spines, and research on its possible involvement in neonatal ischemic hypoxic encephalopathy has not been previously reported. We used plppr5 knockout (plppr5-/-) mice and their wild-type littermates to establish a model of hypoxicischemic brain injury (HI) to further explore the effects of melatonin on brain injury and the role of plppr5 in this treatment in an HI model, which mainly focuses on cognition, exercise, learning, and memory. All the tests were performed at 3-4 weeks after HI. As for melatonin treatment, which was performed 5 min after HI injury and followed by every 24h. In these experiments, we found that there was a significant interaction between genotype and treatment in novel object recognition tests, surface righting reflex tests and forelimb suspension reflex tests, which represent learning and memory, motor function and coordination, and the forelimb grip of the mice, respectively. However, a significant main effect of genotype and treatment on performance in all behavioral tests were observed. Specifically, wild-type mice with HI injury performed better than plppr5-/- mice, regardless of treatment with melatonin or vehicle. Moreover, treatment with melatonin could improve behavior in the tests for wild-type mice with HI injury, but not for plppr5-/- mice. This study showed that plppr5 knockout aggravated HI damage and partially weakened the neuroprotection of melatonin in some aspects (such as novel object recognition test and partial nerve reflexes), which deserves further study.

Keywords: cerebral palsy; hypoxic-ischemic; melatonin; neurobehavior; plppr5.

Figure 1

Generation of plppr5−/− mice and confirmation of plppr5 deficiency at the DNA level. (A) Gene sequence insertion; (B) PCR analysis; (C) Samples 8#2 and 8#1 correspond to the plppr5−/− and plppr5+/- mice, respectively. M represents the DNA ladder.

Figure 2

Timeline of the experimental schedule.

Figure 3

Photomicrographs of TTC-stained coronal slices showing brain infarction 24 h after HI: (A) plppr5−/− mice; (B) Wild-type mice. WT, wild-type mice with HI injury; KO, knockout mice with HI injury; Mel, melatonin; Veh, vehicle.

Figure 4

Behavioral test in wild-type mice with HI injury treated with vehicle or melatonin as well as knockout mice with HI injury receiving vehicle and melatonin treatments as indicated. (A) TTC staining; (B) Negative geotaxis reflex test; (C) Cliff avoidance reflex test; (D) Open field test-center duration time; (E) Open field test-locomotor score; (F) Open field test-number of grooming; (G) Step through test-number of errors; (H) Step through test-latency avoidance period; (I) Cylinder test; (J) Foot fault test-forelimb foot fault test; (K) Foot fault test-hindlimb foot fault test; (L) Seizure threshold. [Two-way ANOVA, Bonferroni post hoc test (details are shown in text)].

Figure 5

Changes in weight of the mice: **P < 0.001 for WT+Mel vs. WT; ^△△P < 0.001 for KO+Mel vs. WT+Mel; ^#P < 0.05 for KO+Mel vs. KO; ^††P < 0.001 for KO vs. WT. [Three-way ANOVA, Bonferroni post hoc test (details are shown in text)].

Figure 6

Behavioral test in wild-type mice with HI injury treated with vehicle or melatonin as well as knockout mice with HI injury receiving vehicle and melatonin treatments as indicated. (A) Novel object recognition test; (B) Surface righting reflex test; (C) Forelimb suspension reflex test. [Two-way ANOVA, Bonferroni post hoc test (details are shown in text)].

Figure 7

Timm staining results of left brain. Blue arrow means moss fiber staining. Red arrow means moss fire sprouting. Magnification is 40 times for first column (four pictures) and the others are 100 times.