Relative Cerebral Blood Flow Is Similar between Developing and Adult Brains after TBI
We first determined if blood flow after TBI showed age-dependent differences. The effects of age at time of injury and time after injury on relative blood flow were evaluated by two-way ANOVA (fig. ). There was no significant interaction between these factors (p = 0.13), thus the interaction term was dropped and a main effects analysis performed. Main effects analysis revealed a statistically significant effect (p < 0.001) for time after injury, but not a significant difference for age (p = 0.76). Multiple comparisons among the days after injury using Sidak's method showed that blood flow immediately after injury was significantly lower relative to all other time points in the P21 group (p < 0.001). Blood flow in the adult brain remained depressed for the first 3 days after injury. Thereafter, blood flow was significantly elevated relative to that measured immediately after injury (p < 0.002).
Fig. 1 Relative cerebral blood flow in the injured P21 (a) and adult brain (b). There is a statistically significant time-after-injury effect, but not a significant difference for age. * p < 0.002; ** p < 0.001. a Multiple comparisons among the (more ...)
The Temporal Pattern of Leukocyte Recruitment Is Age Dependent
Next we compared leukocyte recruitment in the injured P21 and adult brains at 1–35 days after injury. CD45+ cells were noted throughout the injured ipsilateral cortex as well as in the hippocampus in both age groups (fig. ). A majority of these cells were localized adjacent to the site of cortical injury, and the phenotype varied from round cells bearing no processes (fig. ) to cells with short processes (fig. ).
Fig. 2 CD45+ cells in the acutely injured brain. Localization of the panleukocytic marker CD45 in the P21 (a–c) and adult (d–f) brains at 3 days after injury. Boxes: locations of enlarged images of the cortex (b, e) and hippocampus (c, f). Note (more ...)
The distribution of GR-1+ cells was similar to that of CD45+ cells in both the developing (fig. ) and adult brains (fig. ), appearing most prominently in the cortex but also within the underlying hippocampus. To confirm the identity of these cells, adjacent sections stained with hematoxylin and eosin revealed polylobulated nuclei, a feature consistent with that of neutrophils (fig. , ).
Fig. 3 GR-1+ neutrophils in the injured P21 brain. A granulocytic marker, GR-1, is localized in the P21 brain at 1 day (a–e) and 14 days (f–j) after injury in all animals. Note the prominence of these cells at both time points in the cortex and (more ...)
Fig. 4 GR-1+ neutrophils in the injured adult brain. GR-1+ cells are identified in the adult brain at 1 day (a–e), and in some animals, at 14 days (f–j) after injury in the cortex (b, g) and hippocampus (c, h). Boxes: location of enlarged areas (more ...)
CD45+, GR-1+, CD4+ and CD8+ cells were quantified within the injured cortex (fig. ). To make the statistical assumptions of normality and equal variances, analyses were performed on square-root-transformed data. We first determined those time points in which the number of inflammatory cells in the ipsilateral cortex differed from that of the sham values, based upon one-way ANOVA and, where appropriate, Newman-Keuls multiple comparison post hoc tests. In the injured P21 brains, CD45+ and GR-1+ cell numbers were significantly higher within the first 2 weeks after injury (p < 0.05), peaking at 1–3 days, and were not statistically distinguishable from the sham values by 35 days (fig. ). CD4+ cells peaked at 1 day after injury (p < 0.001) and remained elevated over the first week (p < 0.01) (fig. ), whereas CD8+ cells were elevated early (at 3 days; p < 0.001) after injury and again during wound healing (at 14 days; p < 0.01) (fig. ). In the injured adult brains, the numbers of CD45+ and GR-1+ cells in the cortex were elevated at 1 day (GR-1; p < 0.05) and 3 days after injury (CD45, p < 0.001; GR-1, p < 0.05), but returned to the sham values thereafter (fig. ). CD4+ cells peaked 1 day after injury (p < 0.001) and remained elevated above control values for the first week (p < 0.05) (fig. ), whereas CD8+ cells were elevated at 3 and 14 days after injury (p < 0.001 and p < 0.05, respectively) (fig. ).
Fig. 5 Quantification of inflammatory cells in cortex after TBI. * p < 0.05, ** p < 0.01, *** p < 0.001, one-way ANOVA followed by Newman-Keuls multiple comparison test (sham vs. P21); # p < 0.05, ## p < 0.01, ### p < (more ...)
Next we examined the relationship between age at time of injury and time after injury. Based upon two-way ANOVA, the trajectories were not significantly different between the 2 age groups for CD45+ cells. Main effects analysis showed that, on average, the values of CD45 in the P21 group were significantly higher (p = 0.001) than in the adult group. A similar trend was seen for GR-1 (p = 0.056). In contrast, no differences were noted for CD4 and CD8. These findings demonstrate age-related differences in infiltration of the panleukocyte marker CD45 and suggest that these differences are primarily attributed to a subset of CD45+ leukocytes, namely GR-1+ granulocytes.
Temporal Pattern of Irreversibly Damaged Cells in the Cortex Is Similar between Age Groups
The time course of irreversibly injured, TUNEL+ cells was evaluated in the cortex within each of the age groups at 1, 3 and 7 days after injury (fig. ). In both age groups, the number of TUNEL+ cells in the cortical mantle was significantly elevated at 1 day after injury (one-way ANOVA; p < 0.01), returning to baseline thereafter. Based upon two-way ANOVA, the trajectories of TUNEL+ cells were indistinguishable between the age groups. Main effects analysis revealed no differences. These data suggest that age is not a determinant of early patterns of cortical cell injury.
The Kinetics of Cortical Volume Loss Are Age Dependent
Cortical volume was analyzed at both 2 and 5 weeks after injury in each age group (fig. ). Cortical volumes from sham-operated animals were similar at 2 and 5 weeks after surgery within each of the age groups.
Fig. 6 Age-dependent reduction in cortical volume after TBI. a, b Typical appearance of a coronal section stained with cresyl violet in each age group at 5 weeks after injury. A cavitation is apparent in the ipsilateral cortex in both the P21 (a) and adult ( (more ...)
The P21 brain-injured group showed a significant reduction in cortical volume to 79.69% of the sham-operated controls at 14 days after injury, and to 60.19% by 5 weeks. Importantly, there was a significant difference in cortical volumes between 2 and 5 weeks after injury (unpaired t test; p < 0.05). The adult brain-injured group also showed a reduction in cortical mantle volume to 50.68% of the sham-operated controls at 14 days after injury, and to 46.90% by 5 weeks. However, unlike in the injured P21 brains, there was no difference in cortical volumes between 2 and 5 weeks after injury (unpaired t test; p = 0.948).
We next compared the injured ipsilateral cortical volumes, as ratios to their respective shams, between the P21 and adult groups for each time point. At 2 weeks after injury, the cortical volume of the adult group was significantly reduced relative to the P21 group (unpaired t test; p < 0.01). However, by 5 weeks after injury, there was no difference between the 2 groups (unpaired t test; p = 0.316). Together, these findings suggest age-related, temporal differences in long-term loss of cortical volume. Whereas the injured adult brain undergoes a more accelerated loss within the first 2 weeks after injury, the developing brain is characterized by a more gradual long-term reduction in cortical volume.