Diurnal Variation Induces Neurobehavioral and Neuropathological Differences in a Rat Model of Traumatic Brain Injury

Abstract

Traumatic brain injury (TBI) induces two types of brain damage: primary and secondary. Damage initiates a series of pathophysiological processes, such as metabolic crisis, excitotoxicity with oxidative stress-induced damage, and neuroinflammation. The long-term perpetuation of these processes has deleterious consequences for neuronal function. However, it remains to be elucidated further whether physiological variation in the brain microenvironment, depending on diurnal variations, influences the damage, and consequently, exerts a neuroprotective effect. Here, we established an experimental rat model of TBI and evaluated the effects of TBI induced at two different time points of the light-dark cycle. Behavioral responses were assessed using a 21-point neurobehavioral scale and the cylinder test. Morphological damage was assessed in different regions of the central nervous system. We found that rats that experienced a TBI during the dark hours had better behavioral performance than those injured during the light hours. Differences in behavioral performance correlated with less morphological damage in the perilesional zone. Moreover, certain brain areas (CA1 and dentate gyrus subregions of the hippocampus) were less prone to damage in rats that experienced a TBI during the dark hours. Our results suggest that diurnal variation is a crucial determinant of TBI outcome, and the hour of the day at which an injury occurs should be considered for future research.

Keywords: behavioral tests; circadian rhythms; diurnal variation; neuronal damage; traumatic brain injury.

FIGURE 1

The time of day at which trauma is induced determines the changes in general health parameters in rats. Effect of inducing traumatic brain injury (TBI) at two different time points, i.e., day (white symbols) and night (black symbols), on food intake (A) and body weight (B) (n = 5 per subgroup). Note the significant decrease in food intake and body weight in rats with TBI induced during the day (13:00), an effect that was maintained for up to 72 h after TBI. In rats with TBI induced at night (01:00), the food intake did not differ significantly between the TBI and sham subgroups 72 h after TBI. Data are expressed as the mean ± SEM. Two-way analysis of variance (ANOVA) and Tukey’s test were used as post-hoc tests (A,B). *p < 0.05 between TBI and sham groups; ^&p < 0.05 between day and night groups.

FIGURE 2

The time of the day at which trauma is induced determines the behavioral response. Differences in the neurobehavioral scale (A) and cylinder test (B) depending on the time at which traumatic brain injury (TBI) was induced (n = 10 per subgroup). Note the significant decrease in both behavioral tests in rats undergoing TBI during the day (13:00), a decrease that continued until 72 h after TBI. The group that underwent TBI at night (01:00) shows a decrease at 24 h after TBI that results in a score that is greater than that in the day group. However, at 72 h, there is a marked increase in the score, which becomes similar to that of the corresponding sham subgroup (cylinder test). Data are expressed as the mean ± SEM. (A) Kruskal–Wallis and Mann–Whitney U tests. (B) Two-way ANOVA and Tukey’s test as post-hoc test, *p < 0.05 between TBI and sham groups; ^&p < 0.05 between day and night groups.

FIGURE 3

Histopathology of the perilesional motor cortex reveals less morphological damage and fewer degenerating neurons (DN) in the group with traumatic brain injury (TBI) induced at night. Photomicrographs of the cerebral motor cortex in the day (A–C) and night groups (D–F) (n = 4 per column). Schematic representation of the rat motor cortex where TBI was induced (G; image modified from Paxinos and Watson, 1998). An increase in neuronal basophilia (arrow) was observed 24 h after TBI in the lamina 3 (external pyramidal cell layer) of the motor cortex, along with vasodilation (v) in both day (B) and night (E) groups. Note the presence of DN (arrowhead) in the day group and the better preservation of the neuropil (red asterisk) in the night group. At 72 h after TBI, neuronal changes were maintained with the persistence of DN (arrowhead) in the day group (C). In the night group (F), the neurons appeared normal (white arrow) and with changes (arrow); the perineuronal spaces and vasodilation were more prominent in the group with TBI induced during the day (C) than in the group with TBI induced at night (F). The total neuron count in the perilesional zone to trauma (H), note the similar decrease in both groups at 24 h after TBI, and the large neuronal loss at 72 h after trauma in the day group. Cell count per mm², according to the neuronal morphology classification in Table 1 (I). Data are expressed as the mean ± SEM. Two-way ANOVA and Tukey’s test as pos-hoc test. *p < 0.05 between TBI and sham groups; ^&p < 0.05 between day and night groups. Bars (A–F, 100 μm; bars in the insets, 25 μm).

FIGURE 4

Histopathology of the hippocampal subregion CA1 and DG reveals less morphological damage and a lower percentage of degenerating neurons (DN) in the group with traumatic brain. Injury (TBI) induced at night. CA1 hippocampal subregion in the day group (A–C) and the night group (E–G). At 24 h after TBI, the day group (B) did not show remarkable changes in the neuronal morphology, except large vasodilation (white arrowhead) which was not observed in the night group (F). However, in the night group, the layer of pyramidal neurons had more basophilia (arrow). At 72 h after TBI, the day group (C) had more dead pyramidal neurons, characterized by pyknotic nuclei, retraction, and cytoplasmic eosinophilia (arrowhead), compared with the well-preserved pyramidal neurons in the night group (G). In both the day and night groups, a large dispersion of the pyramidal neuron layer was observed both at 24 h (B,F) and 72 h (C,G) after TBI compared with their respective sham subgroup (A,E). Analysis of the percentage of DN (H) showed an increase in both the day and night groups at 24 h after trauma. However, in the day group at 72 h after TBI, the percentage of DN was higher than that in the night group. DG in the day group (I–K) and in the night group (M–O). At 24 h after TBI, the day group (J) showed few DN (arrow) in the granular layer, vasodilation (v), and astrogliosis (asterisk). In contrast, 24 h after TBI, the DG of the night group (N) showed few histological changes in the granular layer. At 72 h after TBI (K), morphological changes in the day group were accentuated, particularly astrogliosis (asterisk), and vasodilation (v), and DN (arrowhead). The night group (O) had few shrunken and basophilic neurons (arrow) in the granular layer. The percentage of DN (P) showed a large increase in the day group, particularly at 72 h after TBI; however, in the night group, the total percentage of DN remained the same at 24 and 72 h. The difference at 72 h between the day group and night groups was significant. Diagrams of the CA1 (D) and DG (L) areas where the photomicrographs from the tissue sections were acquired, and the percentage of DN was determined. (H,P) Data are expressed as the mean ± SEM. Two-way ANOVA and Tukey’s test as post-hoc test. ^&p < 0.05 between day and night groups. HE staining (bars, 25 μm).