Hemorrhagic shock after experimental traumatic brain injury in mice: effect on neuronal death

Abstract

Traumatic brain injury (TBI) from blast injury is often complicated by hemorrhagic shock (HS) in victims of terrorist attacks. Most studies of HS after experimental TBI have focused on intracranial pressure; few have explored the effect of HS on neuronal death after TBI, and none have been done in mice. We hypothesized that neuronal death in CA1 hippocampus would be exacerbated by HS after experimental TBI. C57BL6J male mice were anesthetized with isoflurane, mean arterial blood pressure (MAP) was monitored, and controlled cortical impact (CCI) delivered to the left parietal cortex followed by continued anesthesia (CCI-only), or either 60 or 90 min of volume-controlled HS. Parallel 60- or 90-min HS-only groups were also studied. After HS (+/-CCI), 6% hetastarch was used targeting MAP of > or =50 mm Hg during a 30-min Pre-Hospital resuscitation phase. Then, shed blood was re-infused, and hetastarch was given targeting MAP of > or =60 mm Hg during a 30-min Definitive Care phase. Neurological injury was evaluated at 24 h (fluorojade C) or 7 days (CA1 and CA3 hippocampal neuron counts). HS reduced MAP to 30-40 mm Hg in all groups, p < 0.05 versus CCI-only. Ipsilateral CA1 neuron counts in the 90-min CCI+HS group were reduced at 16.5 +/- 14.1 versus 30.8 +/- 6.8, 32.3 +/- 7.6, 30.6 +/- 2.2, 28.1 +/- 2.2 neurons/100 mum in CCI-only, 60-min HS-only, 90-min HS-only, and 60-min CCI+HS, respectively, all p < 0.05. CA3 neuron counts did not differ between groups. Fluorojade C staining confirmed neurodegeneration in CA1 in the 90-min CCI+HS group. Our data suggest a critical time window for exacerbation of neuronal death by HS after CCI and may have implications for blast injury victims in austere environments where definitive management is delayed.

FIG. 1.

Diagram depicting the overall scheme and timen course of experiment protocol used in this study (CCI, controlled cortical impact; HS, hemorrhagic shock; FJC, Fluoro-Jade C; H&E, hematoxylin and eosin; MAP, mean arterial blood pressure).

FIG. 2.

Average number of surviving CA1 hippocampal neurons per 100-μm length for each experimental group, both ipsilateral (light bars) and contralateral (dark bars). Data are mean and SEM, n = 6 for each group. *p < 0.05 compared to all other groups (HS, hemorrhagic shock; CCI, controlled cortical impact).

FIG. 3.

Average number of surviving CA3 hippocampal neurons per 100-μm length for each experimental group, both ipsilateral (light bars) and contralateral (dark bars). Data are mean and SEM, n = 6 for each group. There were no differences between groups (HS, hemorrhagic shock; CCI, controlled cortical impact).

FIG. 4.

Representative microphotographs (original magnification, ×20), stained with hematoxylin and eosin (H&E), depicting the CA1 hippocampal subfield in 90HS-only (A), CCI-only (B), 60CCI+HS (C), and 90CCI+HS (D). 60HS is not shown. Pyramidal neuron loss is evident within the medial region of CA1 in the 90CCI+ HS group. HS, hemorrhagic shock; CCI, controlled cortical impact.

FIG. 5.

(A) Representative medium-high-power micrograph (×20 objective) of a 90CCI+HS mouse hippocampus demonstrating normal pyramidal neurons (arrowhead) stained blue with DAPI and degenerating neurons stained yellow-green with Fluoro-Jade C (FJC; long arrow) within the CA1 subfield at 24 h. (B) High-power micrograph (×40 objective) of a hematoxylin and eosin (H&E)-stained section demonstrating normal pyramidal neurons (arrowheads) interspersed with degenerating eosinophilic neurons (long arrows) at 7 days in the CA1 hippocampal subfield of a 90CCI+ HS mouse. (C) Low-power microphotograph (×4 objective) of the most severe spectrum of hippocampal damage sustained by several mice in the 90CCI+ HS group. Robust microglial and PMN infiltration is noted along the superior aspect (arrows), and there is substantial loss of neurons in the CA1 subfield (asterisks). (D) Higher magnification (×20 objective) of the boxed region in C demonstrates the absence of viable pyramidal neurons and loss of architecture within much of the CA1 subfield in this example. CCI, controlled cortical impact; HS, hemorrhagic shock; PMN, polymorphonuclear leukocyte.