Effects of Juvenile or Adolescent Working Memory Experience and Inter-Alpha Inhibitor Protein Treatment after Neonatal Hypoxia-Ischemia

Abstract

Hypoxic-Ischemic (HI) brain injury in the neonate contributes to life-long cognitive impairment. Early diagnosis and therapeutic interventions are critical but limited. We previously reported in a rat model of HI two interventional approaches that improve cognitive and sensory function: administration of Inter-alpha Inhibitor Proteins (IAIPs) and early experience in an eight-arm radial water maze (RWM) task. Here, we expanded these studies to examine the combined effects of IAIPs and multiple weeks of RWM assessment beginning with juvenile or adolescent rats to evaluate optimal age windows for behavioral interventions. Subjects were divided into treatment groups; HI with vehicle, sham surgery with vehicle, and HI with IAIPs, and received either juvenile (P31 initiation) or adolescent (P52 initiation) RWM testing, followed by adult retesting. Error rates on the RWM decreased across weeks for all conditions. Whereas, HI injury impaired global performance as compared to shams. IAIP-treated HI subjects tested as juveniles made fewer errors as compared to their untreated HI counterparts. The juvenile group made significantly fewer errors on moderate demand trials and showed improved retention as compared to the adolescent group during the first week of adult retesting. Together, results support and extend our previous findings that combining behavioral and anti-inflammatory interventions in the presence of HI improves subsequent learning performance. Results further indicate sensitive periods for behavioral interventions to improve cognitive outcomes. Specifically, early life cognitive experience can improve long-term learning performance even in the presence of HI injury. Results from this study provide insight into typical brain development and the impact of developmentally targeted therapeutics and task-specific experience on subsequent cognitive processing.

Keywords: Inter-alpha Inhibitor Proteins; early behavioral intervention; eight-arm water maze; encephalopathy; hypoxia-ischemia; immune modulator; inflammation; working memory.

Figure 1

Timeline for surgery, exposure to Hypoxic-Ischemic (HI), Inter-alpha Inhibitor Proteins (IAIP) treatment, Radial Water Maze (RWM) experience, and tissue collection by post-natal day (P#). Subject numbers post-surgery separated into experience groups (Group 1 = Juvenile, Group 2 = Adolescent) are indicated in boxes. All subjects were male rats.

Figure 2

(a) Body weight of animals (±SEM) over time of study. Total n = 67 after surgery, Sham (22), HI (20), HI + IAIPs (25). Repeated measures ANOVA indicates an overall effect of HI injury on body weight, * indicates that HI is significantly lighter than Sham and ** indicates both HI and HI + IAIP cohorts are significantly lighter than Sham animals. (b) Brain weights for Sham, HI, and HI + IAIPs subjects at time of collection. Horizontal bars are mean values and vertical bars indicate SEM. Total n = 66 after brain collection, Sham (22), HI (20), HI + IAIPs (24). ANOVA with Tukey’s homologous subsets indicate that brain weights were significantly lower in both injured treatment groups as compared to shams. HI = Hypoxic-Ischemic injury; IAIPs = Inter-alpha Inhibitory Proteins; PND = post-natal day.

Figure 3

Treatment effects on WMI errors (±SEM), a moderate cognitive demand task component. Total n = 67, Sham (22), HI (20), HI + IAIPs (25). Horizontal bars indicate mean and vertical error bars represent SEM. ANOVA and post-hoc tests indicate both HI and HI + IAIPs treatment groups performed worse than Shams in overall WMI errors across all trials and weeks (a) * indicate significant differences by Tukey’s homologous subsets and (b) ** indicates both HI and HI + IAIPs performed significantly worse than Sham in later trials of the maze protocol over all weeks by paired comparison with Sidak correction.

Figure 4

Effects of juvenile versus adolescent experience on performance. (a) Interaction of experience group and trial WMI error performance (±SEM). ANOVA indicates a significant difference between experience groups at specific trials. (b) Interaction of experience group and week WMI performance (±SEM). * Indicates a significant paired comparison in performance overall between the experience groups at this week, with Sidak correction for multiple comparisons.

Figure 5

Expanded representation of WMI error performance (±SEM) on individual trials collapsed across weeks. Top two rows (a–e): juvenile first RWM experience starting at P31. Total n = 34, Sham (11), HI (10), HI + IAIPs (13). Bottom two rows (f–j): adolescent first RWM experience starting at P52. Total n = 33, Sham (11), HI (10), HI + IAIPs (12). Each plot is aligned by that experience group’s number of weeks of experience. A three-week break occurs between Week 3 and Week 4 for both experience groups. Paired comparisons were made between both treatments within each graph and between experience groups at the same relative trial, for example, Figure 5a,f. * indicates a trial where one injured treatment group, either HI or HI + IAIPs, performed significantly worse than Sham. ** indicates a trial where both HI and HI+IAIP treatment groups performed worse than Sham. † indicates a trial where the HI-treated, juvenile experience cohort (d) significantly differs from the HI-treated, adolescent experience cohort (i). All corrected by Sidak correction for multiple comparisons.