Combined [(18)F]DPA-714 micro-positron emission tomography and autoradiography imaging of microglia activation after closed head injury in mice

Abstract

Background: Traumatic brain injury (TBI) is a major cause of death and disability. Neuroinflammation contributes to acute damage after TBI and modulates long-term evolution of degenerative and regenerative responses to injury. The aim of the present study was to evaluate the relationship of microglia activation to trauma severity, brain energy metabolism, and cellular reactions to injury in a mouse closed head injury model using combined in vivo PET imaging, ex vivo autoradiography, and immunohistochemistry.

Methods: A weight-drop closed head injury model was used to produce a mixed diffuse and focal TBI or a purely diffuse mild TBI (mTBI) in C57BL6 mice. Lesion severity was determined by evaluating histological damage and functional outcome using a standardized neuroscore (NSS), gliosis, and axonal injury by immunohistochemistry. Repeated intra-individual in vivo μPET imaging with the specific 18-kDa translocator protein (TSPO) radioligand [(18)F]DPA-714 was performed on day 1, 7, and 16 and [(18)F]FDG-μPET imaging for energy metabolism on days 2-5 after trauma using freshly synthesized radiotracers. Immediately after [(18)F]DPA-714-μPET imaging on days 7 and 16, cellular identity of the [(18)F]DPA-714 uptake was confirmed by exposing freshly cut cryosections to film autoradiography and successive immunostaining with antibodies against the microglia/macrophage marker IBA-1.

Results: Functional outcome correlated with focal brain lesions, gliosis, and axonal injury. [(18)F]DPA-714-μPET showed increased radiotracer uptake in focal brain lesions on days 7 and 16 after TBI and correlated with reduced cerebral [(18)F]FDG uptake on days 2-5, with functional outcome and number of IBA-1 positive cells on day 7. In autoradiography, [(18)F]DPA-714 uptake co-localized with areas of IBA1-positive staining and correlated strongly with both NSS and the number of IBA1-positive cells, gliosis, and axonal injury. After mTBI, numbers of IBA-1 positive cells with microglial morphology increased in both brain hemispheres; however, uptake of [(18)F]DPA-714 was not increased in autoradiography or in μPET imaging.

Conclusions: [(18)F]DPA-714 uptake in μPET/autoradiography correlates with trauma severity, brain metabolic deficits, and microglia activation after closed head TBI.

Keywords: Autoradiography; Diffuse; Focal; IBA-1; Immunohistochemistry; Neuroinflammation; PET; TBI; TSPO; Weight drop.

Fig. 1

Functional outcome of mice after closed head weight-drop TBI. Outcome was assessed by using the neurological severity score (NSS), a composite score including tasks on motor function, alertness, and physiological behavior, with higher scores indicating worse deficit. Black squares indicate sham-operated mice (n = 7 on 1 h and on day 1, n = 5 on day 7), gray triangles mTBI (n = 15 1 h and on day 1, n = 9 on day 7), and open circles TBI mice (n = 10 1 h and on day 1, n = 8 on day 7), ***p < 0.001, **p < 0.01)

Fig. 2

[¹⁸F]FDG- and [¹⁸F]DPA-714-μPET imaging after closed head weight-drop TBI in mice. a Representative transverse images of [¹⁸F]FDG-μPET, 2 days after TBI show an area of no [¹⁸F]FDG uptake within the focal cortical lesion of a TBI mouse (white arrow head), in contrast to a uniform [¹⁸F]FDG uptake in sham-operated and mTBI mice. b–d Representative transverse images of ¹⁸F]DPA-714-μPET on the left and semi-quantitative analyses of these data on the right. Arrowheads in the μPET images in panels. c, d The visible overlap of [¹⁸F]DPA-714 uptake on days 7 and 16 with the reduction of the [¹⁸F]FDG uptake on day 2 (a). b [¹⁸F]DPA-714-μPET on day 1 after TBI shows no significant difference in [¹⁸F]DPA-714 uptake between sham (n = 5), mTBI (n = 12), and TBI (n = 8) mice. c [¹⁸F]DPA-714-μPET on day 7 after TBI. Compared to the sham animals (0.7 ± 0.2, mean ± SD, n = 6), the lesion-to-cerebellum (L/C) ratio was not significantly increased in mTBI mice (0.8 ± 0.3, n = 12), but it was significantly increased in TBI mice (1.9 ± 0.5, n = 10) as compared to both sham and mTBI mice. d [¹⁸F]DPA-714-μPET on day 16 after TBI. The L/C ratio of three mTBI and three TBI mice. e Time-course of the L/C ratio in the three groups (data identical to bar graphs in b–d. The L/C ratio of TBI mice significantly increased from 0.61 on day 1 to 1.85 on day 7 as compared to both sham-operated and mTBI mice and the decreased to 1.38 on day 16. In mTBI mice the L/C ratio was not statistically different from that in sham group even if it tended to increase from 0.67 on day 1 to 0.82 on day 16. f Linear regression analysis between [¹⁸F]FDG uptake on days 2–5 and [¹⁸F]DPA-714 accumulation on day 7 show a highly significant correlation between reduced brain metabolic activity and [¹⁸F]DPA-714 uptake.***p < 0.001 in c and e

Fig. 3

Combined ex vivo autoradiography and IBA-1 immunohistochemistry show increased [¹⁸F]DPA-714 uptake in microglia after closed head weight-drop TBI in mice. a Representative images of [¹⁸F]DPA-714 autoradiography and IBA-1 immunohistochemical staining on the same section of a sham, mTBI, and TBI mouse on day 7. The middle panel show merged images in each case. Red asterisks on the low magnification images denote areas depicted in higher magnification below each image. Red arrows mark IBA-1 positive cells, scale bar = 50 μm. b Box-plots representing calculated TBI/N ratios of [¹⁸F]DPA-714 binding in sham operated (n = 5), mTBI (n = 11), and TBI (n = 6) mice on day 7 and in c on day 16 in three mTBI and three TBI mice. d IBA-1 positive cell counts on day 7 were derived from the sections used for calculation of the TBI/N ratios in b in sham (n = 5), mTBI (n = 11), and TBI (n = 6) mice. e show IBA-1 counts on day 16 in two mTBI and three TBI mice used for TBI/N ratio in c. ***p < 0.001, **p < 0.01, *p < 0.05

Fig. 4

Correlation of [¹⁸F]DPA-714 brain uptake with outcome and IBA-positive cell counts after closed head weight-drop TBI in mice. Linear regression analysis between NSS and L/C ratios of [¹⁸F]DPA-714 accumulation in μPET (a), between NSS and TBI/N ratios of [¹⁸F]DPA-714 accumulation in ex vivo autoradiography (b), between IBA-1 counts and L/C ratios of [¹⁸F]DPA-714 (c), and between IBA-1 counts and TBI/N ratios of [¹⁸F]DPA-714 (d)

Fig. 5

Astrogliosis and diffuse axonal injury in relation to in vitro binding of [¹⁸F]DPA-714 on day 7 after closed head weight-drop TBI in mice. a Representative images of [¹⁸F]DPA-714 in vitro autoradiography and the initial trauma severity (NSS at 1 h after TBI) plotted in relation to positive/negative [¹⁸F]DPA-714 accumulation. b, c Representative images of GFAP-positive astrocytes (b) or SMI-32 staining of axons (c) on the left and on the right counts of GFAP-positive cells or SMI-32 positive damaged axons (retraction bulbs/brain section) in sham-operated (n = 4), mTBI (n = 6), and TBI (n = 6) mice. Scale bar = 50 μm. d, e Linear regression analyses between the initial trauma severity and number of GFAP-positive cells (d) or SMI-32 positive retraction bulbs (e). f, g GFAP-positive cells counts (f) or damaged axons (g) plotted in relation to [¹⁸F]DPA-714 uptake in TBI mice by in vitro autoradiography (negative n = 6, positive n = 6). ***p < 0.001, **p < 0.01, *p < 0.05