Limits...
Repetitive Model of Mild Traumatic Brain Injury Produces Cortical Abnormalities Detectable by Magnetic Resonance Diffusion Imaging, Histopathology, and Behavior

View Article: PubMed Central - PubMed

ABSTRACT

Noninvasive detection of mild traumatic brain injury (mTBI) is important for evaluating acute through chronic effects of head injuries, particularly after repetitive impacts. To better detect abnormalities from mTBI, we performed longitudinal studies (baseline, 3, 6, and 42 days) using magnetic resonance diffusion tensor imaging (DTI) and diffusion kurtosis imaging (DKI) in adult mice after repetitive mTBI (r-mTBI; daily × 5) or sham procedure. This r-mTBI produced righting reflex delay and was first characterized in the corpus callosum to demonstrate low levels of axon damage, astrogliosis, and microglial activation, without microhemorrhages. High-resolution DTI-DKI was then combined with post-imaging pathological validation along with behavioral assessments targeted for the impact regions. In the corpus callosum, only DTI fractional anisotropy at 42 days showed significant change post-injury. Conversely, cortical regions under the impact site (M1–M2, anterior cingulate) had reduced axial diffusivity (AD) at all time points with a corresponding increase in axial kurtosis (Ka) at 6 days. Post-imaging neuropathology showed microglial activation in both the corpus callosum and cortex at 42 days after r-mTBI. Increased cortical microglial activation correlated with decreased cortical AD after r-mTBI (r = −0.853; n = 5). Using Thy1-YFP-16 mice to fluorescently label neuronal cell bodies and processes revealed low levels of axon damage in the cortex after r-mTBI. Finally, r-mTBI produced social deficits consistent with the function of this anterior cingulate region of cortex. Overall, vulnerability of cortical regions is demonstrated after mild repetitive injury, with underlying differences of DTI and DKI, microglial activation, and behavioral deficits.

No MeSH data available.


Related in: MedlinePlus

Thy1-YFP-16 mice show axonal pathology in the cortex and corpus callosum after r-mTBI. (A and B) Thy1-YFP (green) shows labeling of neurons throughout the cortex along with axons projecting through the corpus callosum in mice perfused 3 days after r-sham (A) or r-mTBI (B) procedure. Nissl labeling (red) of the cytoplasmic rough endoplasmic reticulum along with DAPI nuclei counterstaining (blue) shows the overall cortical cytoarchitecture, which is not markedly disrupted after r-mTBI. Arrows (B) point to examples of swollen segments of damaged axons. (C and D) At higher magnification, Thy1-YFP (green) labeled axons in the corpus callosum exhibit a generally uniform longitudinal profile in r-sham mice (C) in contrast to the numerous swollen axonal segments observed in r-mTBI mice (D). (E and F) High-magnification Thy1-YFP (green) cortical regions analyzed in r-sham mice (E) and r-mTBI mice (F). Large swellings and thickened Thy1-YFP axonal profiles are evident in the cortex of r-mTBI mice (F, arrows) compared to the thin processes of Thy1-YFP axons in r-sham mice (E, arrow). Lack of DAPI nuclear labeling (blue) shows that enlarged Thy1-YFP regions (F) are not cell bodies. (G) Quantification of damaged axonal profiles (swollen, axonal bulbs, and varicosities) labeled with Thy1-YFP in the cingulate and M2 motor cortices that comprise the region of interest in the medial cortex for DTI/DKI analyses. A significant increase of damaged axons was observed in r-mTBI mice compared to r-sham. Values are mean ± standard error of the mean; n = 3; three to nine sections per animal. **p < 0.01; ****p < 0.0001. Scale bars = 200 μm (A and B) and 20 μm (C–F). Avg, average; d, days; DAPI, 4′,6-diamidino-2-phenylindole; DKI, diffusion kurtosis imaging; DTI, diffusion tensor imaging; r-mTBI, repetitive mild traumatic brain injury; r-sham, repetitive sham.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5385606&req=5

f9: Thy1-YFP-16 mice show axonal pathology in the cortex and corpus callosum after r-mTBI. (A and B) Thy1-YFP (green) shows labeling of neurons throughout the cortex along with axons projecting through the corpus callosum in mice perfused 3 days after r-sham (A) or r-mTBI (B) procedure. Nissl labeling (red) of the cytoplasmic rough endoplasmic reticulum along with DAPI nuclei counterstaining (blue) shows the overall cortical cytoarchitecture, which is not markedly disrupted after r-mTBI. Arrows (B) point to examples of swollen segments of damaged axons. (C and D) At higher magnification, Thy1-YFP (green) labeled axons in the corpus callosum exhibit a generally uniform longitudinal profile in r-sham mice (C) in contrast to the numerous swollen axonal segments observed in r-mTBI mice (D). (E and F) High-magnification Thy1-YFP (green) cortical regions analyzed in r-sham mice (E) and r-mTBI mice (F). Large swellings and thickened Thy1-YFP axonal profiles are evident in the cortex of r-mTBI mice (F, arrows) compared to the thin processes of Thy1-YFP axons in r-sham mice (E, arrow). Lack of DAPI nuclear labeling (blue) shows that enlarged Thy1-YFP regions (F) are not cell bodies. (G) Quantification of damaged axonal profiles (swollen, axonal bulbs, and varicosities) labeled with Thy1-YFP in the cingulate and M2 motor cortices that comprise the region of interest in the medial cortex for DTI/DKI analyses. A significant increase of damaged axons was observed in r-mTBI mice compared to r-sham. Values are mean ± standard error of the mean; n = 3; three to nine sections per animal. **p < 0.01; ****p < 0.0001. Scale bars = 200 μm (A and B) and 20 μm (C–F). Avg, average; d, days; DAPI, 4′,6-diamidino-2-phenylindole; DKI, diffusion kurtosis imaging; DTI, diffusion tensor imaging; r-mTBI, repetitive mild traumatic brain injury; r-sham, repetitive sham.

Mentions: Next, we further examined the cortical changes after r-mTBI using Thy1-YFP-16 mice (Fig. 9), which have a high density of neurons with yellow fluorescent labeling. The labeling in cortical neurons enables visualization of the dendritic arbor and axonal processes, including callosal axons.50 Nissl staining in r-mTBI mice at 3 days post-injury and in matched r-sham mice showed a low density of neuronal cell bodies in layer I and a much higher density in layers II/II, indicating generally normal neuronal cytoarchitecture in the superficial cortex under the site of impact (Fig. 9A,B). In deeper cortical regions, the Thy1-YFP labeling was very dense without overt disruption of cytoarchitecture in r-mTBI, which appeared similar to r-sham (Fig. 9A,B). In r-sham mice, normal YFP distribution in axons is evident in higher-magnification images of the corpus callosum (Fig. 9C) and the medial cortex (Fig. 9E). In cortical regions of r-mTBI mice, YFP accumulation in some labeled axons was observed as axonal swellings (Fig. 9B,F). YFP expression revealed conspicuous axonal swellings in the corpus callosum of r-mTBI mice (Fig. 9D). Quantitative analysis of Thy1-YFP mice in the medial cortical region analyzed by MRI, which was comprised of cingulate cortex and M2 motor cortex, showed a significant increase of damaged YFP-labeled axonal segments in r-mTBI mice compared to the r-sham condition (Fig. 9G). As a point of validation, a parallel cohort of Thy1-YFP mice was examined with the s-mTBI model (Supplementary Fig. 5A,B) (see online supplementary material at http://www.liebertpub.com), in which axon damage in the corpus callosum has been previously characterized by electron microscopy.31 The s-mTBI mice, in which more-extensive corpus callosum pathology was expected, indeed exhibited more marked axon damage in the corpus callosum (Supplementary Fig. 5A,B) (see online supplementary material at http://www.liebertpub.com), compared to the r-mTBI mice (Fig. 9D). However, in the medial cortex, levels of damaged axonal segments labeled with YFP were similar in r-mTBI (Fig. 9G) and s-mTBI (Supplementary Fig. 5C) (see online supplementary material at http://www.liebertpub.com) models. Therefore, axonal damage in the cortex may contribute to DTI effects, but does not fully explain the reduction in cortical AD, which was only detected in this r-mTBI model.


Repetitive Model of Mild Traumatic Brain Injury Produces Cortical Abnormalities Detectable by Magnetic Resonance Diffusion Imaging, Histopathology, and Behavior
Thy1-YFP-16 mice show axonal pathology in the cortex and corpus callosum after r-mTBI. (A and B) Thy1-YFP (green) shows labeling of neurons throughout the cortex along with axons projecting through the corpus callosum in mice perfused 3 days after r-sham (A) or r-mTBI (B) procedure. Nissl labeling (red) of the cytoplasmic rough endoplasmic reticulum along with DAPI nuclei counterstaining (blue) shows the overall cortical cytoarchitecture, which is not markedly disrupted after r-mTBI. Arrows (B) point to examples of swollen segments of damaged axons. (C and D) At higher magnification, Thy1-YFP (green) labeled axons in the corpus callosum exhibit a generally uniform longitudinal profile in r-sham mice (C) in contrast to the numerous swollen axonal segments observed in r-mTBI mice (D). (E and F) High-magnification Thy1-YFP (green) cortical regions analyzed in r-sham mice (E) and r-mTBI mice (F). Large swellings and thickened Thy1-YFP axonal profiles are evident in the cortex of r-mTBI mice (F, arrows) compared to the thin processes of Thy1-YFP axons in r-sham mice (E, arrow). Lack of DAPI nuclear labeling (blue) shows that enlarged Thy1-YFP regions (F) are not cell bodies. (G) Quantification of damaged axonal profiles (swollen, axonal bulbs, and varicosities) labeled with Thy1-YFP in the cingulate and M2 motor cortices that comprise the region of interest in the medial cortex for DTI/DKI analyses. A significant increase of damaged axons was observed in r-mTBI mice compared to r-sham. Values are mean ± standard error of the mean; n = 3; three to nine sections per animal. **p < 0.01; ****p < 0.0001. Scale bars = 200 μm (A and B) and 20 μm (C–F). Avg, average; d, days; DAPI, 4′,6-diamidino-2-phenylindole; DKI, diffusion kurtosis imaging; DTI, diffusion tensor imaging; r-mTBI, repetitive mild traumatic brain injury; r-sham, repetitive sham.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5385606&req=5

f9: Thy1-YFP-16 mice show axonal pathology in the cortex and corpus callosum after r-mTBI. (A and B) Thy1-YFP (green) shows labeling of neurons throughout the cortex along with axons projecting through the corpus callosum in mice perfused 3 days after r-sham (A) or r-mTBI (B) procedure. Nissl labeling (red) of the cytoplasmic rough endoplasmic reticulum along with DAPI nuclei counterstaining (blue) shows the overall cortical cytoarchitecture, which is not markedly disrupted after r-mTBI. Arrows (B) point to examples of swollen segments of damaged axons. (C and D) At higher magnification, Thy1-YFP (green) labeled axons in the corpus callosum exhibit a generally uniform longitudinal profile in r-sham mice (C) in contrast to the numerous swollen axonal segments observed in r-mTBI mice (D). (E and F) High-magnification Thy1-YFP (green) cortical regions analyzed in r-sham mice (E) and r-mTBI mice (F). Large swellings and thickened Thy1-YFP axonal profiles are evident in the cortex of r-mTBI mice (F, arrows) compared to the thin processes of Thy1-YFP axons in r-sham mice (E, arrow). Lack of DAPI nuclear labeling (blue) shows that enlarged Thy1-YFP regions (F) are not cell bodies. (G) Quantification of damaged axonal profiles (swollen, axonal bulbs, and varicosities) labeled with Thy1-YFP in the cingulate and M2 motor cortices that comprise the region of interest in the medial cortex for DTI/DKI analyses. A significant increase of damaged axons was observed in r-mTBI mice compared to r-sham. Values are mean ± standard error of the mean; n = 3; three to nine sections per animal. **p < 0.01; ****p < 0.0001. Scale bars = 200 μm (A and B) and 20 μm (C–F). Avg, average; d, days; DAPI, 4′,6-diamidino-2-phenylindole; DKI, diffusion kurtosis imaging; DTI, diffusion tensor imaging; r-mTBI, repetitive mild traumatic brain injury; r-sham, repetitive sham.
Mentions: Next, we further examined the cortical changes after r-mTBI using Thy1-YFP-16 mice (Fig. 9), which have a high density of neurons with yellow fluorescent labeling. The labeling in cortical neurons enables visualization of the dendritic arbor and axonal processes, including callosal axons.50 Nissl staining in r-mTBI mice at 3 days post-injury and in matched r-sham mice showed a low density of neuronal cell bodies in layer I and a much higher density in layers II/II, indicating generally normal neuronal cytoarchitecture in the superficial cortex under the site of impact (Fig. 9A,B). In deeper cortical regions, the Thy1-YFP labeling was very dense without overt disruption of cytoarchitecture in r-mTBI, which appeared similar to r-sham (Fig. 9A,B). In r-sham mice, normal YFP distribution in axons is evident in higher-magnification images of the corpus callosum (Fig. 9C) and the medial cortex (Fig. 9E). In cortical regions of r-mTBI mice, YFP accumulation in some labeled axons was observed as axonal swellings (Fig. 9B,F). YFP expression revealed conspicuous axonal swellings in the corpus callosum of r-mTBI mice (Fig. 9D). Quantitative analysis of Thy1-YFP mice in the medial cortical region analyzed by MRI, which was comprised of cingulate cortex and M2 motor cortex, showed a significant increase of damaged YFP-labeled axonal segments in r-mTBI mice compared to the r-sham condition (Fig. 9G). As a point of validation, a parallel cohort of Thy1-YFP mice was examined with the s-mTBI model (Supplementary Fig. 5A,B) (see online supplementary material at http://www.liebertpub.com), in which axon damage in the corpus callosum has been previously characterized by electron microscopy.31 The s-mTBI mice, in which more-extensive corpus callosum pathology was expected, indeed exhibited more marked axon damage in the corpus callosum (Supplementary Fig. 5A,B) (see online supplementary material at http://www.liebertpub.com), compared to the r-mTBI mice (Fig. 9D). However, in the medial cortex, levels of damaged axonal segments labeled with YFP were similar in r-mTBI (Fig. 9G) and s-mTBI (Supplementary Fig. 5C) (see online supplementary material at http://www.liebertpub.com) models. Therefore, axonal damage in the cortex may contribute to DTI effects, but does not fully explain the reduction in cortical AD, which was only detected in this r-mTBI model.

View Article: PubMed Central - PubMed

ABSTRACT

Noninvasive detection of mild traumatic brain injury (mTBI) is important for evaluating acute through chronic effects of head injuries, particularly after repetitive impacts. To better detect abnormalities from mTBI, we performed longitudinal studies (baseline, 3, 6, and 42 days) using magnetic resonance diffusion tensor imaging (DTI) and diffusion kurtosis imaging (DKI) in adult mice after repetitive mTBI (r-mTBI; daily&thinsp;&times;&thinsp;5) or sham procedure. This r-mTBI produced righting reflex delay and was first characterized in the corpus callosum to demonstrate low levels of axon damage, astrogliosis, and microglial activation, without microhemorrhages. High-resolution DTI-DKI was then combined with post-imaging pathological validation along with behavioral assessments targeted for the impact regions. In the corpus callosum, only DTI fractional anisotropy at 42 days showed significant change post-injury. Conversely, cortical regions under the impact site (M1&ndash;M2, anterior cingulate) had reduced axial diffusivity (AD) at all time points with a corresponding increase in axial kurtosis (Ka) at 6 days. Post-imaging neuropathology showed microglial activation in both the corpus callosum and cortex at 42 days after r-mTBI. Increased cortical microglial activation correlated with decreased cortical AD after r-mTBI (r&thinsp;=&thinsp;&minus;0.853; n&thinsp;=&thinsp;5). Using Thy1-YFP-16 mice to fluorescently label neuronal cell bodies and processes revealed low levels of axon damage in the cortex after r-mTBI. Finally, r-mTBI produced social deficits consistent with the function of this anterior cingulate region of cortex. Overall, vulnerability of cortical regions is demonstrated after mild repetitive injury, with underlying differences of DTI and DKI, microglial activation, and behavioral deficits.

No MeSH data available.


Related in: MedlinePlus