Traumatic brain injury results in acute rarefication of the vascular network

Abstract

The role of the cerebrovascular network and its acute response to TBI is poorly defined and emerging evidence suggests that cerebrovascular reactivity is altered. We explored how cortical vessels are physically altered following TBI using a newly developed technique, vessel painting. We tested our hypothesis that a focal moderate TBI results in global decrements to structural aspects of the vasculature. Rats (naïve, sham-operated, TBI) underwent a moderate controlled cortical impact. Animals underwent vessel painting perfusion to label the entire cortex at 1 day post TBI followed by whole brain axial and coronal images using a wide-field fluorescence microscope. Cortical vessel network characteristics were analyzed for classical angiographic features (junctions, lengths) wherein we observed significant global (both hemispheres) reductions in vessel junctions and vessel lengths of 33% and 22%, respectively. Biological complexity can be quantified using fractal geometric features where we observed that fractal measures were also reduced significantly by 33%, 16% and 13% for kurtosis, peak value frequency and skewness, respectively. Acutely after TBI there is a reduction in vascular network and vascular complexity that are exacerbated at the lesion site and provide structural evidence for the bilateral hemodynamic alterations that have been reported in patients after TBI.

Figure 1

Traumatic brain injury results in vascular loss. The top three rows display representative axial images from wide field epifluorescent vessel painting, classical vascular analysis (AngioTool), and fractal analysis (FracLac) maps. The bottom two rows illustrate coronal images of vessel painted brain tissue and complimentary susceptibility weighted magnetic resonance imaging (SWI MRI) to monitor the extent of extravascular bleeding. Each column is representative for naïve, sham, and TBI brains. The images from the naïve animal demonstrate a diffuse and uniform pattern of vessels without vascular disruptions. The sham animal exhibits slight vascular alterations due to the craniotomy. In contrast, the TBI animal has a clear and overt a region of vessel disruption at the site of injury on both the axial and coronal vessel painted microscopic images. These disruptions to the vascular system after TBI are also visible on the classical vascular analysis maps and on the fractal analysis images.

Figure 2

Confocal images of vessel painted vasculature at the perilesional region. (A) Sham animal exhibited uniform staining of large, intermediate and small vessels at the perimeter of the craniotomy site. (B) The vessels of a TBI animal demonstrate marked sparsity in vessel numbers and dramatic loss of small vessels adjacent to the injury site. Cal bar = 200 um.

Figure 3

Classical vascular analysis reveals a decrease in the number of junctions and total vessel length following TBI. (A) An axial AngioTool image where vessels (red) and junctions (blue) are displayed. Whole cortex and specific concentric radial ROIs projecting outward from the injury site (circles 1–3), were analyzed to quantify vascular alterations. TBI animals exhibited a decrease in the number of vessel junctions and length, even moving outward from the site of injury (1–3 mm). (B) Analysis of the entire whole cortex demonstrated a significant reduction in the both number of junctions (t-test, p < 0.05) and in the total vessel length (t-test, p < 0.05) in TBI animals compared to sham animals. (C) TBI animals also exhibited a significant decline in the number vascular junctions moving radially outward from the injury site (ROIs 1 to 3) (t-test, p < 0.05). A similar finding was also found in TBI animals where a significant decline in total vessel length moved radially outward from the injury site compared to sham animals (t-test, p < 0.05).

Figure 4

Fractal analysis reveals a quantitative reduction in both vascular complexity and frequency in TBI animals. (A) A binary image of the axial vascular network of a representative sham animal with radial ROIs radiating outward from the injury or sham surgery site (ROI1–3). The right panel illustrates the complexity changes in the vasculature from the concentric circles as you move radially outward from the injury site. These fractal images are colorized based on the resultant fractal dimension with a gradient from lower local fractal dimension (LFD) in red (less complex network) to higher LFD in purple (more complex network). (B) Histogram distribution of the LFD and peak frequency from the whole cortex of TBI animals reveals a decrease in complexity of the vascular network (leftward LFD shift) along with a corresponding decrease in the peak frequency value (decreased numbers). (C) Distribution of the LFD at the injury site (ROI 1) mimics what is observed within the whole brain analysis. At the injury site there is a reduction in the complexity of the vascular networks (leftward LFD shift) along with a concomitant decrease in amplitude of the LFD histogram signifying decreased numbers of vascular components. (D) Quantitative analysis of the distribution of LFD histogram from the whole cortex revealed a significant reduction in skewness (t-test, p < 0.03), kurtosis (t-test, p < 0.03) and peak value frequency (t-test, p < 0.03) in TBI animals. The significant change in skewness and kurtosis quantitates the degree to which the complexity of vasculature has been simplified (reduced skewness) and diminished (reduced kurtosis). The peak frequency value synthesizes information on the vascular network and its relative complexity. (E) Quantitative analysis of the distribution of LFD at the injury site (ROI 1) reveals similar finding as seen from whole cortex analysis. Significant differences between TBI and sham animals were found only in skewness (t-test, p < 0.05) and not kurtosis or peak frequency values (t-test, p < 0.06).

Figure 5

Fractal analyses of the ipsilateral and contralateral brain demonstrate widespread alterations in vasculature complexity and frequency after brain injury. (A) The average skewness for radial concentric circles located in the ipsilateral injury site and contralateral corresponding brain region demonstrate a widespread decrease in vascular complexity (skewness) compared to shams. (B) Similarly, the average kurtosis from ipsilateral and contralateral brain regions displayed a similar distribution and pattern as found for skewness. (C) The average peak frequency value for each concentric circle ipsilateral and contralateral to the injury site confirms a reduction in the complexity of the vasculature in TBI animals compared to shams. Interestingly, the focal injury reductions in the complexity of the vasculature are observed uniformly not only at the ipsilateral injury site but also on identical contralateral brain tissues.

Figure 6

Magnetic Resonance Imaging (MRI) of lesion and hemorrhage volumes in vessel painted brains. (A) Representative coronal slices from naïve, sham, and TBI animals at 1d post injury. T2 Weighted Imaging (T2WI) was performed for lesion (blood and edema) volumes while Susceptibility Weighted Imaging (SWI) was used to derive hemorrhage volumes (top and bottom row respectively). Note that the SWI vividly demonstrates hemorrhage in the TBI animal (red arrow) in comparison to the T2WI. Note the lack of bleeding at the craniotomy site in shams (asterisk). (B) Coronal SWI images in an anterior to posterior progression from a sham and TBI animal illustrating the anterior-posterior extent of lesion hemorrhage through the cortex (left). Sagittal view of MRI with red dash indicates the site of injury (top-right). A three-dimensional reconstruction from SWI data set shows the expanse of hemorrhage (red, bottom-right). (C) Automated lesion and hemorrhage analysis (hierarchical region splitting, HRS) was used to determine the group average from all TBI animals. Edema constituted the largest component of the lesion at 1d after injury. Lesion and hemorrhage volumes were significantly higher in TBI animals when compared to naïve and sham groups (p < 0.05). (D) Correlations were performed to determine if individual fractal properties were more predictive of vascular loss and how these were related TBI lesions. Only the fractal peak value correlated to the extent of lesion and approached significance (r² = 0.6364, p = 0.057) but no significance was found for fractal kurtosis (p = 0.093), skewness (p = 0.095) or LFD (p = 0.082).