Repetitive Neonatal Erythropoietin and Melatonin Combinatorial Treatment Provides Sustained Repair of Functional Deficits in a Rat Model of Cerebral Palsy

Abstract

Cerebral palsy (CP) is the leading cause of motor impairment for children worldwide and results from perinatal brain injury (PBI). To test novel therapeutics to mitigate deficits from PBI, we developed a rat model of extreme preterm birth (<28 weeks of gestation) that mimics dual intrauterine injury from placental underperfusion and chorioamnionitis. We hypothesized that a sustained postnatal treatment regimen that combines the endogenous neuroreparative agents erythropoietin (EPO) and melatonin (MLT) would mitigate molecular, sensorimotor, and cognitive abnormalities in adults rats following prenatal injury. On embryonic day 18 (E18), a laparotomy was performed in pregnant Sprague-Dawley rats. Uterine artery occlusion was performed for 60 min to induce placental insufficiency via transient systemic hypoxia-ischemia, followed by intra-amniotic injections of lipopolysaccharide, and laparotomy closure. On postnatal day 1 (P1), approximately equivalent to 30 weeks of gestation, injured rats were randomized to an extended EPO + MLT treatment regimen, or vehicle (sterile saline) from P1 to P10. Behavioral assays were performed along an extended developmental time course (n = 6-29). Open field testing shows injured rats exhibit hypermobility and disinhibition and that combined neonatal EPO + MLT treatment repairs disinhibition in injured rats, while EPO alone does not. Furthermore, EPO + MLT normalizes hindlimb deficits, including reduced paw area and paw pressure at peak stance, and elevated percent shared stance after prenatal injury. Injured rats had fewer social interactions than shams, and EPO + MLT normalized social drive. Touchscreen operant chamber testing of visual discrimination and reversal shows that EPO + MLT at least partially normalizes theses complex cognitive tasks. Together, these data indicate EPO + MLT can potentially repair multiple sensorimotor, cognitive, and behavioral realms following PBI, using highly translatable and sophisticated developmental testing platforms.

Keywords: cerebral palsy; chorioamnionitis; cognition; gait; hypoxia-ischemia; inflammation; social interaction; touchscreen.

Figure 1

Open field testing of locomotion and disinhibition. (A) After prenatal injury, vehicle-treated rats are more mobile than shams, particularly in the last 5 min interval. Erythropoietin (EPO) + melatonin (MLT), but not EPO alone, normalizes the activity. (B) Rats typically avoid open areas. Vehicle-treated adult rats following prenatal injury spend more time immobile in the center zone than sham rats or EPO + MLT-treated rats. (C) Sham and EPO + MLT-treated injured rats spent more time in the peripheral zone than vehicle-treated or EPO-treated injured rats. (D) Similarly, vehicle-treated injured rats spent more time in the neutral zone than sham or EPO + MLT-treated rats. EPO treatment by itself did not improve disinhibition (two-way ANOVA with Bonferroni correction, *p < 0.05, **p < 0.01, ***p < 0.001).

Figure 2

Prenatal injury impairs gait performance. (A) After prenatal injury, vehicle-treated rats contact their hindpaws with less area than sham or erythropoietin (EPO) + melatonin (MLT)-treated injured rats. (B) Similarly, vehicle-treated injured rats contact hindpaws with less pressure than shams or EPO + MLT-treated rats. (C) The percent of shared stance is elevated in vehicle-treated rats with prenatal injury compared to shams, and EPO + MLT treatment normalizes gait and posture (two-way ANOVA with Bonferroni correction, *p < 0.05, **p < 0.01, ***p < 0.001).

Figure 3

Prenatal injury impairs social interaction. After prenatal injury, pairs of vehicle-treated rats of the same sex but different litters have fewer social interactions than pairs of shams, or pairs of erythropoietin (EPO) + melatonin (MLT)-treated injured rats (two-way ANOVA with Bonferroni correction, *p < 0.05, **p < 0.01).

Figure 4

Touchscreen operant chamber testing reveals impaired cognition. (A) Fewer vehicle-treated rats after prenatal injury successfully reached passing criterion for visual discrimination, compared to shams or erythropoietin (EPO) + melatonin (MLT)-treated injured rats (*p < 0.05). (B) Shams, vehicle-treated rats with prenatal injury, and EPO + MLT-treated rats with prenatal injury all committed approximately the same number of errors during visual discrimination testing. (C) The reaction latency between the three groups did not differ. (D) Likewise, the magazine latency was similar for all three groups. (E) Significantly fewer vehicle-treated rats after prenatal injury were able to pass criterion for VD and reversal (*p < 0.05). (F) After prenatal injury, vehicle-treated rats required more correction trials in reversal learning paradigms than shams.