Development of a New Scoring System in Higher Animals for Testing Cognitive Function in the Newborn Period: Effect of Prenatal Hypoxia-Ischemia

Abstract

Introduction: Enhanced models for assessing cognitive function in the neonatal period are imperative in higher animals. Postnatal motor deficits, characteristic of cerebral palsy, emerge in newborn kits within our prenatal rabbit model of hypoxia-ischemia (HI). In humans, prenatal HI leads to intellectual disability and cerebral palsy. In a study examining cognitive function in newborn rabbits, we explored several questions. Is there a distinction between conditioned and unconditioned kits? Can the kits discern the human face or the laboratory coat? Do motorically normal kits, born after prenatal HI, exhibit cognitive deficits?

Methods: The conditioning protocol was randomly assigned to kits from each litter. For conditioning, the same human, wearing a laboratory coat, fed the rabbit kits for 9 days before the cognitive test. The 6-arm radial maze was chosen for its simplicity and ease of use. Normally appearing kits, born after uterine ischemia at 79% or 92% term in New Zealand White rabbits, were compared to naïve kits. On postpartum day 22/23 or 29/30, the 6-arm maze helped determine if the kits recognized the original feeder from bystander (test 1) or the laboratory coat on bystander (test 2). The use of masks of feeder/bystander (test 3) assessed confounding cues. A weighted score was devised to address variability in entry to maze arms, time, and repeated-trial learning.

Results: In conditioned kits, both naïve and HI kits exhibited a significant preference for the face of the feeder but not the laboratory coat. Cognitive deficits were minimal in normal-appearing HI kits.

Conclusion: The weighted score was amenable to statistical manipulation.

Introduction: Enhanced models for assessing cognitive function in the neonatal period are imperative in higher animals. Postnatal motor deficits, characteristic of cerebral palsy, emerge in newborn kits within our prenatal rabbit model of hypoxia-ischemia (HI). In humans, prenatal HI leads to intellectual disability and cerebral palsy. In a study examining cognitive function in newborn rabbits, we explored several questions. Is there a distinction between conditioned and unconditioned kits? Can the kits discern the human face or the laboratory coat? Do motorically normal kits, born after prenatal HI, exhibit cognitive deficits?

Methods: The conditioning protocol was randomly assigned to kits from each litter. For conditioning, the same human, wearing a laboratory coat, fed the rabbit kits for 9 days before the cognitive test. The 6-arm radial maze was chosen for its simplicity and ease of use. Normally appearing kits, born after uterine ischemia at 79% or 92% term in New Zealand White rabbits, were compared to naïve kits. On postpartum day 22/23 or 29/30, the 6-arm maze helped determine if the kits recognized the original feeder from bystander (test 1) or the laboratory coat on bystander (test 2). The use of masks of feeder/bystander (test 3) assessed confounding cues. A weighted score was devised to address variability in entry to maze arms, time, and repeated-trial learning.

Results: In conditioned kits, both naïve and HI kits exhibited a significant preference for the face of the feeder but not the laboratory coat. Cognitive deficits were minimal in normal-appearing HI kits.

Conclusion: The weighted score was amenable to statistical manipulation.

Keywords: Anoxia; Conditioning; Fetus; Infant; Intellectual disability; Learning maze; Newborn; Operant.

Fig. 1.

6-arm maze. Newborn kit placed in the center with head toward west. South is 5 and north is 2. The head of the kit is positioned with head toward west. Weighted scores for every action of the kit in either direction with scores increasing toward arm 5 and decreasing toward arm 2. Maximum score is 50, and minimum score is 0.

Fig. 2.

Weighted scoring system. Directionality of every action of the kit is scored, and time and number of attempts are factored in.

Fig. 3.

Arm 5 entry over 5 trials depicted on the ordinate. The three tests depicted in the left column. Rows show the results of each test. The two right columns show the data from P22 (a–c) and P29 (d–f) as box and whisker plots of all the means of the 5 trials shown as black circles. The abscissa shows 4 groups of animals, naïve and post-hypoxia-ischemia (HI), unconditioned (-U) and conditioned (-C). By chance, naïve kits would enter 1–3.8 times in arm 5 (range of means). Conditioning significantly affects the response to entry of arm 5 (*p < 0.05, **Bonferroni correction for multiple comparisons, p < 0.004). Simple main effects analysis showed that both conditioning and HI had a statistically significant effect at P22 in test 1, conditioning alone at P22 and P29 in test 2, and HI alone at P22 and P29 in test 3. There is a statistically significant interaction between the conditioning and HI at P29 in test 1, and at P22 for test 3 (^#p < 0.05 by two-way ANOVA, SAS PROC GLM). The directionality of this interaction shown by dashed light green lines for naïve and solid lines for HI. c, d Both suggest that cognition is worsened following HI. Statistically significant interaction between age, conditioning, and HI was found in tests 1 and 2 (^§p < 0.05, three-way ANOVA, SAS PROC GLM), suggesting P29 to be better age to detect effects of HI on cognition. Power of statistical tests in ANOVA in f 7–38%.

Fig. 4.

Preliminary arm entry depicted as a pie chart. Total number of entries = 943.

Fig. 5.

Multiple entries to arms other than arms 2 and 5.40% entered arms 2 or 5 and stayed there, but others entered other arms. Some kits would enter the other arms as many as 4 times. Total number of entries = 918.

Fig. 6.

Descriptive statistics of the time before entering the stimulus arms. Graphs generated by PROC UNIVARIATE, SAS. Ordinate. The time was censored at 1,966 s, and testing was then stopped at that time. Also the skewed distribution of time in the box and whisker plot. Two-factor ANOVA shows no significant interaction between conditioning and HI in any age or test.

Fig. 7.

Weighted score shown as box and whisker plots of all the means of the 5 trials shown as black circles. The setup of three tests depicted in the left column. Columns show age, and rows show the results of each test. The abscissa shows 4 groups of animals, naïve and post-hypoxia-ischemia (HI), unconditioned (-U) and conditioned (-C). Conditioning significantly affects the response to entry of arm 5 (*p < 0.05, **Bonferoni correction for multiple comparisons, p < 0.004). Simple main effects analysis showed both conditioning and HI had a statistically significant effect at P22 in test 1 (a), conditioning alone at P22 and P29 in test 2 (b, e) (ANOVA). There is a statistically significant interaction between the conditioning and HI at both ages in test 2, and at P22 for test 3 (^#p < 0.05 by two-way ANOVA, SAS PROC GLM). Dashed light green lines for naïve and solid lines for HI show directionally of interaction. In b, it seems that cognition is improved, while in c and e, cognition is worsened following HI. There is also statistically significant interaction between age, conditioning, and HI in all the tests (^§p < 0.05, ^§§p < 0.004 Bonferroni correction, three-way ANOVA, SAS PROC GLM). Interestingly, there was significant interaction between age and conditioning only in test 3. Power of statistical tests in ANOVA in 5–73% (d) and in 12–27% (f).

Fig. 8.

Effect of Trials. Weighted score depicted on the ordinate. Rows show the results of each test. The columns show the data from P22 and P29 with means (circles) and 95% confidence intervals (a–l, shaded portion). The abscissa shows trials. A neutral score is 25. Higher than 25 favors the feeder in tests 1 and 2, and a score <25 favors the feeder in test 3. The arrows depict the implications of deviation from 25. Yellow lines joining yellow circles depict lines joining the means in unconditioned groups, while blue lines joining blue circles depict conditioned group means’ line. The conditioned groups are offset a little for better clarity. Naïve groups are in light colors and HI in darker shades. There was no significant change between trial 1 versus trial 5 using Bonferroni correction for α error. However, * for p < 0.05 is shown for trends, shown in (e, d, l). The trend in (e) if anything indicates the rabbit is learning less with more trials, while the trends in (f) and (l) indicate the rabbit could be learning with more trials. Also, most of the area in the confidence intervals in blue is over 25 in tests 1 and 2, and lower than 25 in test 3 in the conditioned groups (compared to unconditioned groups), indicating that the kits seem to recognize the face of the feeder, confirming the findings in Figure 7.

Fig. 9.

Paired comparisons of tests. Box and whisker plots with distribution of data in black circles. The difference between the weighted scores of 2 tests is depicted on the ordinate. The columns show the data from P22 and P29. Naïve groups on the top row and HI on the bottom row. The zero line represents a neutral result. The green shaded area shows the preference for the face of the feeder. The implications of the changes are shown in C with arrows. A preference for the face is observed mostly in conditioned kits by the significant deviations from zero (*p < 0.05, **p < 0.004 Bonferroni correction, paired t test). a, c The significant effects of conditioning are confirmed in P22 (^#p < 0.05, two-sample t test). There does not seem to be a preference for the laboratory coat (b, d). A significant interaction of age and conditioning was only observed for tests 1–3 (^§p < 0.05, three-way ANOVA, SAS PROC GLM) but not for other pairings.

Fig. 10.

a–d No difference with sex. Weighted score in ordinate. Pink circles are female and blue circles represent male. Yellow lines represent naïve, and purple lines represent HI. For convenience, the dashed yellow line shows the neutral score of 25. Means and standard errors of mean depicted. There was no significant interaction with sex, age, HI, and conditioning.