Real-Time Noninvasive Bioluminescence, Ultrasound and Photoacoustic Imaging in NFκB-RE-Luc Transgenic Mice Reveal Glia Maturation Factor-Mediated Immediate and Sustained Spatio-Temporal Activation of NFκB Signaling Post-Traumatic Brain Injury in a Gender-Specific Manner

Abstract

Neurotrauma especially traumatic brain injury (TBI) is the leading cause of death and disability worldwide. To improve upon the early diagnosis and develop precision-targeted therapies for TBI, it is critical to understand the underlying molecular mechanisms and signaling pathways. The transcription factor, nuclear factor kappa B (NFκB), which is ubiquitously expressed, plays a crucial role in the normal cell survival, proliferation, differentiation, function, as well as in disease states like neuroinflammation and neurodegeneration. Here, we hypothesized that real-time noninvasive bioluminescence molecular imaging allows rapid and precise monitoring of TBI-induced immediate and rapid spatio-temporal activation of NFκB signaling pathway in response to Glia maturation factor (GMF) upregulation which in turn leads to neuroinflammation and neurodegeneration post-TBI. To test and validate our hypothesis and to gain novel mechanistic insights, we subjected NFκB-RE-Luc transgenic male and female mice to TBI and performed real-time noninvasive bioluminescence imaging (BLI) as well as photoacoustic and ultrasound imaging (PAI). Our BLI data revealed that TBI leads to an immediate and sustained activation of NFκB signaling. Further, our BLI data suggest that especially in male NFκB-RE-Luc transgenic mice subjected to TBI, in addition to brain, there is widespread activation of NFκB signaling in multiple organs. However, in the case of the female NFκB-RE-Luc transgenic mice, TBI induces a very specific and localized activation of NFκB signaling in the brain. Further, our microRNA data suggest that TBI induces significant upregulation of mir-9-5p, mir-21a-5p, mir-34a-5p, mir-16-3p, as well as mir-155-5p within 24 h and these microRNAs can be successfully used as TBI-specific biomarkers. To the best of our knowledge, this is one of the first and unique study of its kind to report immediate and sustained activation of NFκB signaling post-TBI in a gender-specific manner by utilizing real-time non-invasive BLI and PAI in NFκB-RE-Luc transgenic mice. Our study will prove immensely beneficial to gain novel mechanistic insights underlying TBI, unravel novel therapeutic targets, as well as enable us to monitor in real-time the response to innovative TBI-specific precision-targeted gene and stem cell-based precision medicine.

Keywords: Bioluminescence imaging; Glia maturation factor; NFκB; Photoacoustic imaging; Traumatic brain injury; Ultrasound; microRNA.

Fig. 1

Lack of NFκB activation in non-TBI mice: Real-time noninvasive BLI performed on NFκB-RE-Luc mice immediately prior to TBI indicates that there is lack of NFκB activation in non-TBI mice especially in the head region. Since these are global transgenic mice, certain regions like extremities as well as nose tip reveals minimal NFκB activation. As such, there is no observable difference between the male and female mice. These BLI data serve as proper baseline control prior to TBI induction

Fig. 2

TBI induces rapid activation of NFκB signaling: Real-time noninvasive BLI performed on NFκB-RE-Luc mice within 30 min post-TBI revealed a rapid activation of NFκB signaling cascade post-TBI. In the case of male NFκB-RE-Luc mice in addition to the brain region, BLI signals could be detected in the region of the heart, liver, as well as kidneys. However, in the case of the female NFκB-RE-Luc mice, the BLI signals remained very localized in the brain region throughout the duration of the study

Fig. 3

TBI induces sustained activation of NFκB signaling: Real-time noninvasive BLI performed on NFκB-RE-Luc mice 24 h post-TBI revealed an enhanced and sustained activation of NFκB signaling cascade. In the case of male NFκB-RE-Luc mice in addition to the brain region, BLI signals could be detected in the region of the heart, liver, as well as kidneys. In the case of the female NFκB-RE-Luc mice, the BLI signals remained very localized in the brain and heart region throughout the duration of the study

Fig. 4

TBI Induces Sustained Activation of NFκB signaling: Real-time noninvasive BLI performed on NFκB-RE-Luc mice 48 h post-TBI revealed an enhanced and sustained activation of NFκB signaling cascade in one of the male mouse. In the case of male NFκB-RE-Luc mice in addition to the brain region, BLI signals could be detected in the region of the heart, liver, as well as kidneys. In the case of the female NFκB-RE-Luc mice, the BLI signals remained very localized in the brain and the heart region throughout the duration of the study

Fig. 5

TBI induces sustained activation of NFκB: Real-time noninvasive BLI performed on NFκB-RE-Luc mice 72 h post-TBI revealed an enhanced and sustained activation of NFκB signaling cascade in one of the male mouse. In the case of male NFκB-RE-Luc mice in addition to the brain region, BLI signals could be detected in the region of the heart, liver, as well as kidneys. In the case of the female NFκB-RE-Luc mice, the BLI signals remained very localized in the brain and the heart region throughout the duration of the study

Fig. 6

TBI induces alterations in cerebrovascular function: Real-time noninvasive PAI revealed minimal alterations in the cerebrovascular function immediately post-TBI. Especially, the blood flow appeared to be consistently normal in all the areas post-TBI. In M1 mouse, the Circle of Willis was relatively more clear

Fig. 7

TBI induces alterations in cerebrovascular function: Real-time noninvasive PAI 24 h post-TBI revealed alterations in the cerebrovascular function in TBI mice as compared to the TBI mice at 0 h post-TBI. Especially, the blood flow appeared to be consistently reduced in the area representing TBI in a time-dependent manner. In the brain, there was enhanced blood flow in the TBI region in the male as well as female mice

Fig. 8

TBI induces alterations in cerebrovascular function: Real-time noninvasive PAI 48 h post-TBI revealed alterations in the cerebrovascular function in TBI mice as compared to the TBI mice at 0 and 24 h post-TBI. Especially, the blood flow appeared to be consistently reduced in the area representing TBI in a time-dependent manner especially in the M2 mouse brain. In the brain, there was enhanced blood flow in the TBI region in the two males as well as two female mice. However, there was a significant reduction in the blood flow in one of the female mice

Fig. 9

TBI induces alterations in cerebrovascular function: Real-time noninvasive PAI 72 h post-TBI revealed alterations in the cerebrovascular function in TBI mice as compared to the TBI mice at 0, 24, and 48 h post-TBI. Especially, the blood blow appeared to be consistently reduced in the area representing TBI in a time-dependent manner in all the three males and one female mice

Fig. 10

TBI-induced NFκB activation perturbs microRNA expression: Changes in the levels of microRNAs were determined by TaqMan advanced miRNAs assays. Quantitative data were analyzed using the 2^−ΔΔCt method. A comparative analysis of microRNAs in the control and TBI serum revealed a significant upregulation of mir-9-5p, mir-16-3p, mir-21a-5p, mir-34a-5p, as well as mir-155-5p within 24 h post-TBI. However, the most of the microRNA levels except mir-16-3p and mir-34a-5p declined to near baseline levels within 72 h post-TBI. Comparisons between different groups were performed by one-way analyses of variance (ANOVA) with Tukey’s multiple comparisons test was used to determine the statistical significance between different groups. (***P < 0.0001, **P < 0.005, *P < 0.01 versus the control group, values represent mean + SD, n = 3 mice/group)

Fig. 11

Immunofluorescence analysis reveals TBI-induced neuropathology: a, b The representative brain sections from the control and TBI mice subjected to GFAP immunostaining reveal TBI-induced neuropathology by confocal microscopic analysis. As compared to the control brain, TBI brain exhibits significant upregulation of GFAP as well as GMF expression 72 h post-TBI. Enhanced GFAP expression indicates glial activation post-TBI. *P < 0.01 control vs 72 h post-TBI. c, d Microglia in Iba1 stained representative brain sections in the TBI brain reveal alterations in numbers, staining intensity, as well as morphology. *P < 0.05 control vs 72 h post-TBI. e, f In comparison to the control mouse brain, representative NeuN staining exhibits neuronal damage as well as neuronal loss post-TBI. In comparison to the control, TBI brain exhibits upregulation of GMF expression post-TBI. *P < 0.05 control vs 72 h post-TBI. Furthermore, TBI induces significant overexpression of GMF which is colocalized with GFAP, Iba1 and NeuN and is responsible for TBI-associated neuroinflammation and neuropathology. Results are representative analyses of 4–5 brain sections from each group (n = 3 mice per group). Statistical analyses were conducted by performing Student’s t test. *P < 0.05 versus control group. Scale bar = 50 µm