Changes in Plasma von Willebrand Factor and Cellular Fibronectin in MRI-Defined Traumatic Microvascular Injury

The neuropathology of traumatic brain injury (TB) is diverse, including primary injury to neurons, axons, glial cells, vascular structures, and secondary processes, such as edema and inflammation that vary between individual patients. Traumatic microvascular injury is an important endophenotype of TBI-related injury. We studied patients who sustained a TBI requiring ER evaluation and had an MRI performed within 48 h of injury. We classified patients into 3 groups based on their MRI findings: (1) those that had evidence of traumatic microvascular injury on susceptibility or diffusion weighted MRI sequences without frank hemorrhage [Traumatic Vascular Injury (TVI) group; 20 subjects]. (2) those who had evidence of intraparenchymal, subdural, epidural, or subarachnoid hemorrhage [Traumatic Hemorrhage (TH) group; 26 subjects], and (3) those who had no traumatic injuries detected by MRI [MRI-negative group; 30 subjects]. We then measured plasma protein biomarkers of vascular injury [von Willebrand Factor (vWF) or cellular fibronectin (cFn)] and axonal injury (phosphorylated neurofilament heavy chain; pNF-H). We found that the TVI group was characterized by decreased expression of plasma vWF (p < 0.05 compared to MRI-negative group; p < 0.00001 compared to TH group) ≤48 h after injury. cFN was no different between groups ≤48 h after injury, but was increased in the TVI group compared to the MRI-negative (p < 0.00001) and TH (p < 0.00001) groups when measured >48 h from injury. pNF-H was increased in both the TH and TVI groups compared to the MRI-negative group ≤48 h from injury. When we used the MRI grouping and molecular biomarkers in a model to predict Glasgow Outcome Scale-Extended (GOS-E) score at 30–90 days, we found that inclusion of the imaging data and biomarkers substantially improved the ability to predict a good outcome over clinical information alone. These data indicate that there is a distinct, vascular-predominant endophenotype in a subset of patients who sustain a TBI and that these injuries are characterized by a specific biomarker profile. Further work to will be needed to determine whether these biomarkers can be useful as predictive and pharmacodynamic biomarkers for vascular-directed therapies after TBI

Minocycline Synergizes with N-Acetylcysteine and Improves Cognition and Memory Following Traumatic Brain Injury in Rats

Background: There are no drugs presently available to treat traumatic brain injury (TBI). A variety of single drugs have failed clinical trials suggesting a role for drug combinations. Drug combinations acting synergistically often provide the greatest combination of potency and safety. The drugs examined (minocycline (MINO), N-acetylcysteine (NAC), simvastatin, cyclosporine A, and progesterone) had FDA-approval for uses other than TBI and limited brain injury in experimental TBI models. Methodology/Principal Findings: Drugs were dosed one hour after injury using the controlled cortical impact (CCI) TBI model in adult rats. One week later, drugs were tested for efficacy and drug combinations tested for synergy on a hierarchy of behavioral tests that included active place avoidance testing. As monotherapy, only MINO improved acquisition of the massed version of active place avoidance that required memory lasting less than two hours. MINO-treated animals, however, were impaired during the spaced version of the same avoidance task that required 24-hour memory retention. Coadministration of NAC with MINO synergistically improved spaced learning. Examination of brain histology 2 weeks after injury suggested that MINO plus NAC preserved white, but not grey matter, since lesion volume was unaffected, yet myelin loss was attenuated. When dosed 3 hours before injury, MINO plus NAC as single drugs had no effect on interleukin-1 formation; together they synergistically lowered interleukin-1 levels. This effect on interleukin-1 was not observed when th

MINO plus NAC prevented myelin loss.

<p><b>Panel A</b>, Schematic of the regions of interest (ROIs) from a coronal section located −3.36 mm from Bregma <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012490#pone.0012490-Paxinos1" target="_blank">[40]</a>. The ROIs were: corpus callosum (A), dorsal hippocampal commissure (B), stratum radiatum (C), fimbria (D), internal capsule (E), fornix (F), mammilothalamic tract (G). <b>Panel B</b>, Representative images of corpus callosum and dorsal hippocampal commissure stained with luxol fast blue. B1, Sham-CCI-saline; B2 CCI-saline; B3, CCI-MINO plus NAC. The scale bar corresponds to 100µm.</p

MINO provided a modest improvement of active place avoidance following moderate CCI.

<p>The total number of entrances into the shock zone was assayed in the 6 trials of active place avoidance training.</p

Measurements of rat behavior during open field, passive place avoidance and active place avoidance.

<p>Rats received either sham- or moderate-CCI. Beginning one hour after surgery the rats received either saline, cyclosporine (CYCLO) simvastatin (SIM), progesterone (PROG), minocycline (MINO), n-acetyl cysteine (NAC) or MINO plus NAC. Seven days later all groups were tested on the hierarchy of three behavioral tasks. In the open field test, there was no effect of treatment on total distance traveled (F_7,40 = 0.40, p>0.8). In passive place avoidance, there was no effect of treatment on either average distance over 4 trials or shock zone entrances (total distance, F_7,40 = 0.67, p>0.6; entrances, F_7,40 = 0.48, p>0.8). In massed active place avoidance testing on the 6^th trial of the first day, there was no effect of treatment on speed (F_7,40 = 0.44, p>0.8) or linearity (F_7,40 = 0.25, p>0.9)). The number of shocks per entrance in CCI-saline treated rats was not changed by the drugs individually or in combination (F_7,40 = 0.39, p>0.6). In contrast, there was a significant treatment effect with MINO or MINO plus NAC significantly improved time to first entrance (F_4,35 = 26.7, p<0.0001; **p<0.001, *p<0.05; post-hoc test).</p

MINO or MINO plus NAC prevented IL-1β formation when administered before moderate CCI.

<p>Rats were dosed with saline, MINO, NAC or both drugs 3 hours prior to sham- or moderate CCI. The rats were sacrificed one hour after surgery; and IL-1β and tubulin levels were assayed from protein extracts prepared from hippocampus. <b>Panel A</b>, Representative Il-1β and tubulin immunoblot analysis, <b>Panel B</b>, Summary of immunoblot analysis.</p

MINO plus NAC synergistically improved active place avoidance after massed training.

<p>Panel A, Rats received either CCI or sham-CCI. One hour, one or days later the sham-injured the CCI-injured rats were divided into 4 groups; one group received saline treatment. The remaining CCI-injured rats received either MINO or NAC alone, or the combination of MINO plus NAC. Saline or drug treatments were administered 1 hour, 1 day and 2 days after injury. Rats received active place avoidance training 8 and 9 days after injury. On the 8th and 9th day following CCI, the number of shock zone entrances was measured. <b>Panel B</b>, Representative tracks of rats in the sham-CCI-saline, CCI-saline or CCI-MINO plus NAC groups on the 6^th trial on the first day of active avoidance training. Red lines indicate the boundaries of the shock zone and the red circles indicate the location were a rat received a shock. <b>Panel C</b>, Summary of the number of shock zone entrances over the two days of active place avoidance.</p

Righting Reflex Predicts Long-Term Histological and Behavioral Outcomes in a Closed Head Model of Traumatic Brain Injury.

Blunt impact produces a heterogeneous brain injury in people and in animal models of traumatic brain injury. We report that a single closed head impact to adult C57/BL6 mice produced two injury syndromes (CHI-1 and CHI-2). CHI-1 mice spontaneously reinitiated breathing after injury while CHI-2 mice had prolonged apnea and regained breathing only after cardiopulmonary resuscitation and supplementation of 100% O2. The CHI-1 group significantly regained righting reflex more rapidly than the CHI-2 group. At 7 days post-injury, CHI-1, but not CHI-2 mice, acquired but had no long-term retention of an active place avoidance task. The behavioral deficits of CHI-1 and CHI-2 mice were retained one-month after the injury. CHI-1 mice had loss of hippocampal neurons and localized white matter injury at one month after injury. CHI-2 had a larger loss of hippocampal neurons and more widespread loss of myelin and axons. High-speed videos made during the injury were followed by assessment of breathing and righting reflex. These videos show that CHI-2 mice experienced a larger vertical g-force than CHI-1 mice. Time to regain righting reflex in CHI-2 mice significantly correlated with vertical g-force. Thus, physiological responses occurring immediately after injury can be valuable surrogate markers of subsequent behavioral and histological deficits

A single impact produces a heterogeneous vertical g-force.

<p>Panel A, A video still image taken at 16.8msec after impact; a time when the head was no longer moving. A color overlay shows the movement of the ipsilateral (green), midline (red) and contralateral (blue) spots after the impact. Scale bar, 10 mm. The units of g-force are newtons per kg. Panel B, Summary of high-speed video analysis of movements of spots ipsilateral, midline and contralateral to the impact site. An arrow indicates the time of the impact. Panel C, Summary of vertical g-forces computed ipsilateral, midline and contralateral to the impact site. CHI-2 mice had a significantly larger acceleration and deceleration at all three sites than CHI-1 mice (post hoc, *p<0.05). CHI-2 mice had greater rebound acceleration than CHI-1 mice (post hoc, *p<0.05). Panel D, Scatter plot of vertical g-force of individual CHI-1 and CHI-2 mice with time to regain righting reflex. The CHI-2 (<b>ρ</b> = 0.724, p = 0.05), but not CHI-1 (<b>ρ</b> = -0.468, p > 0.2) group had a significant correlation with the time needed to regain righting reflex.</p

CHI-1 mice acquire, but do not retain, a new shock zone location in conflict active place avoidance.

<p>Panel A, Representative tracks from sham-CHI and CHI-1 mice in the final trial of conflict active place avoidance. The 60° stationary shock zone is shown in red. Black circles show the location of the mouse when receiving a shock. Panel B, Summary of conflict active place avoidance. On shock zone entrances during conflict active place avoidance, group and trial did not significantly interact (F_1630,30 = 14.0, p = 0.50). The number of entrances did not significantly differ. (F_1,10 = 0.006, p = 0.94).</p