Age is a determinant of leukocyte infiltration and loss of cortical volume after traumatic brain injury

Abstract

There is increasing evidence that the inflammatory response differs in the injured developing brain as compared to the adult brain. Here we compared cerebral blood flow and profiled the inflammatory response in mice that had been subjected to traumatic brain injury (TBI) at postnatal day (P)21 or at adulthood. Relative blood flow, determined by laser Doppler, revealed a 30% decrease in flow immediately after injury followed by prominent hyperemia between 7 and 35 days after injury in both age groups. The animals were euthanized at 1-35 days after injury and the brains prepared for the immunolocalization and quantification of CD45-, GR-1-, CD4- and CD8-positive (+) cells. On average, the number of CD45+ leukocytes in the cortex was significantly higher in the P21 as compared to the adult group. A similar trend was seen for GR-1+ granulocytes, whereas no age-related differences were noted for CD4+ and CD8+ cells. While CD45+ and GR-1+ cells in the P21 group remained elevated, relative to shams, over the first 2 weeks after injury, the adult group showed a time course limited to the first 3 days after injury. The loss of ipsilateral cortical volumes at 2 weeks after injury was significantly greater in the adult relative to the P21 group. While the adult group showed no further change in cortical volumes, there was a significant loss of cortical volumes between 2 and 5 weeks after injury in the P21 group, reaching values similar to that of the adult group by 5 weeks after injury. Together, these findings demonstrate age-dependent temporal patterns of leukocyte infiltration and loss of cortical volume after TBI.

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 days after injury using Sidak's method shows that blood flow immediately after injury (day 0) is significantly lower relative to other time points for the P21 group. b Blood flow in the adult brain remains depressed for the first 3 days after injury. Thereafter, blood flow is significantly elevated relative to day 0.

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 the prominent accumulation in both the cortex and hippocampus of both age groups. Phenotypes of these cells include round cells with no processes as well as cells bearing a few short processes.

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 hippocampus of both groups. Boxes: location of enlarged areas of the cortex (b, g) and hippocampus (c, h). Adjacent sections stained with hematoxylin and eosin show cells with lobulated nuclei in the cortex (d, i) and hippocampus (e, j). Scale bars = 200 μm (a, f) and 20 μm (c, e, h, j).

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 of the cortex (b, g) and hippocampus (c, h). Based upon staining with hematoxylin and eosin, neutrophils are confirmed by their lobulated nuclei at both time points and in the cortex (d, i) and hippocampus (e, j). Scale bars = 200 μm (a, f) and 20 μm (c, e, h, j).

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 < 0.001, one-way ANOVA followed by Newman-Keuls multiple comparison test (sham vs. adult). a, b In the injured P21 brains, CD45 (a) and GR-1 (b) are significantly higher within the first 2 weeks after injury relative to shams. In the injured adult brains, CD45 is elevated at 3 days and GR-1 is elevated at 1 and 3 days after injury. c, d In the injured P21 brains, the number of CD4+ cells peaks at 1 day after injury and remains elevated at 14 days. In the injured adult brains, CD4 remains elevated over the first week. CD8 is elevated at 3 and 14 days after injury in both age groups. e TUNEL+ cells quantified within the cortex. In both age groups, the number of TUNEL+ cells in the cortical mantle is significantly elevated at 1 day after injury.

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 (b) brain. c, d The ipsilateral cortical mantle volume was determined in cresyl-violet-stained sections. Injury leads to a significant reduction in cortical mantle volume at 2 and 5 weeks after injury, as compared to sham-operated controls, in both the P21 (c) and adult groups (d). In the injured P21 group, there is a significant reduction in cortical mantle volume between 2 and 5 weeks. * p < 0.05, ** p < 0.01, *** p < 0.001, unpaired t test.