Mechanoporation induced by diffuse traumatic brain injury: an irreversible or reversible response to injury?

Abstract

Diffuse traumatic brain injury (DTBI) is associated with neuronal plasmalemmal disruption, leading to either necrosis or reactive change without cell death. This study examined whether enduring membrane perturbation consistently occurs, leading to cell death, or if there is the potential for transient perturbation followed by resealing/recovery. We also examined the relationship of these events to calpain-mediated spectrin proteolysis (CMSP). To assess plasmalemmal disruption, rats (n = 21) received intracerebroventricular infusion 2 h before DTBI of a normally excluded 10 kDa fluorophore-labeled dextran. To reveal plasmalemmal resealing or enduring disruption, rats were infused with another labeled dextran 2 h (n = 10) or 6 h (n = 11) after injury. Immunohistochemistry for the 150 kDa spectrin breakdown product evaluated the concomitant role of CMSP. Neocortical neurons were followed with confocal and electron microscopy. After DTBI at 4 and 8 h, 55% of all tracer-flooded neurons contained both dextrans, demonstrating enduring plasmalemmal leakage, with many demonstrating necrosis. At 4 h, 12.0% and at 8 h, 15.7% of the dual tracer-flooded neurons showed CMSP, yet, these demonstrated less advanced cellular change. At 4 h, 39.0% and at 8 h, 24.4% of all tracer-flooded neurons revealed only preinjury dextran uptake, consistent with membrane resealing, whereas 7.6 and 11.1%, respectively, showed CMSP. At 4 h, 35% and at 8 h, 33% of neurons demonstrated CMSP without dextran flooding. At 4 h, 5.5% and at 8 h, 20.9% of tracer-flooded neurons revealed only postinjury dextran uptake, consistent with delayed membrane perturbation, with 55.0 and 35.4%, respectively, showing CMSP. These studies illustrate that DTBI evokes evolving plasmalemmal changes that highlight mechanical and potential secondary events in membrane poration.

Figure 1.

Bar charts representing the mean levels of intracranial pressure measured 1 and 2 h before injury as well as 2, 3, 6, and 7 h after injury (black column, noninfused injured, n = 10; gray column, infused injured, 4 h survival, n = 10; white column, infused injured, 8 h survival, n = 11). No elevation in ICP was found in injected-non-injured sham animals, indicating that controlled infusion into the lateral ventricle does not increase ICP (data not shown). No significant difference in ICP was observed between injected-injured and non-injected-injured animals at 4 or 8 h after injury, indicating that infusion into the lateral ventricle does not complicate any ICP change associated with closed head injury. Note, however, that in this injury model, the traumatic episode was associated with a significant elevation of ICP. The ICP was elevated at each time point measured postinjury, particularly at 2, 3, 6, and 7 h after injury. Data are shown as mean ± SEM. The asterisk indicates statistical significance (p < 0.001).

Figure 2.

This double-labeled confocal image from a sham-injured animal receiving fluorophore-labeled dextran infusions demonstrates the diffusion of the tracers throughout the interstices of the neocortex without any evidence of neuronal uptake of any of the administrated dextrans. Note that in addition to their passage through the interstices of the brain parenchyma, the labeled dextrans can be easily visualized in the perivascular regions, consistent with a control pattern of dextran distribution (arrows). Scale bar, 100 μm.

Figure 3.

Confocal images of tracer-infused-injured animals. A, This double-labeled image demonstrates numerous cortical neurons (arrows) revealing intracellular tracer flooding with both the preinjury and postinjury infused dextrans. Scale bar, 100 μm. B–D, These confocal images, revealing the initially administered dextran flooding (B), the postinjury infused dextran flooding (C), and their overlay (D), demonstrate that the majority of the neurons sustaining membrane disruption and tracer uptake are double labeled both with preinjury- and postinjury-infused dextrans (arrows). Such double labeling was observed at both 4 and 8 h after injury. Note that some neurons flooding with the preinjury dextran alone can also be observed (arrowheads). Scale bar, 100 μm.

Figure 4.

A–D, Double-labeled confocal images. In A–C, neurons flooding with both dextrans reveal evidence of concomitant cellular injury, reflected in their irregular, distorted profiles and vacuolization (arrows). In those cells showing the most severe damage, dextrans are also typically found within the nucleus (arrowhead). Note that other double-labeled neurons (D) demonstrate little or no pathological damage and that despite homogenous tracer uptake, no nuclear accumulation or vacuolization occurs (arrows). Scale bars, 100 μm.

Figure 5.

Ultrastructural analysis of the dual tracer-containing neurons reveals various forms of pathologic change. A, The fluorescent image obtained via routine fluorescent microscopy. B, The anti-Alexa Fluor 488-immunostained image of the same region. C, D, An electron micrograph of the same neuron. Note that this neuron demonstrates moderately increased electron density, cytoplasmic and nuclear tracer flooding (arrows), and perinuclear organelle vacuolization (arrowheads) without overt mitochondrial damage (double arrowheads), best shown in the enlarged panel D. Scale bar, 2 μm. E, Three tracer-flooded neurons, again confirmed by routine fluorescent microscopy and followed via EM. The most severely damaged neuron (asterisk) demonstrates increased electron density, organelle vacuolization, and perisomatic glial ensheathment best illustrated in enlarged panel F. The two other cells (double and triple asterisks) demonstrate little or no pathological change. Note that the surrounding neuropil demonstrates little overt pathologic change consistent with the confocal observations. Scale bar, 5 μm

Figure 6.

A, Triple-labeled confocal image demonstrating dextran flooded neurons as well as CMSP-immunopositive neurons. Note that at both 4 and 8 h after injury, CMSP-immunoreactive neurons were observed throughout the neocortex (arrows). Scale bar, 100 μm. B–E, Confocal images of preinjury dextran flooding (B), postinjury dextran flooding (C), CMSP immunopositivity (D), and their overlay (E) demonstrate neurons showing enduring membrane disruption reflected in their content of both tracers. Note that some neurons colocalize with CMSP immunopositivity (arrow), whereas other neurons demonstrate tracer flooding without CMSP (arrowheads). Also note that at these same time points, several CMSP-immunoreactive neurons also could be identified without concomitant tracer flooding (double arrowhead). Finally, note that in the same region, a neuron demonstrating only the initial tracer flooding (big arrow), as well as a neuron revealing only secondary tracer flooding (triple arrowhead), can also be seen. Scale bar, 50 μm.

Figure 7.

A–C, A toluidine blue-stained 1-μm-thick section reacted for the visualization of CMSP is shown in A, whereas B and C reveal an EM of the same neurons. Note that in B, one CMSP-immunopositive neuron demonstrates moderate subcellular damage. Note that C, taken from that area blocked off in B, reveals the CMSP reaction product around swollen mitochondria (arrows) and dispersed throughout the cytoplasm (double arrowheads). A neuron demonstrating overt necrosis with no evidence of CMSP is also shown (arrowhead). Scale bar, 5 μm.

Figure 8.

A–C, Confocal images of preinjury-infused dextrans (A), postinjury-infused dextrans (B), and their overlay (C) demonstrate some cortical neurons flooding with the preinjury-infused dextran alone without concomitant flooding with the postinjury-administrated tracer (arrows). Note that one double-flooded neuron demonstrating severe damage (arrowhead) and one double-labeled neuron demonstrating less severe pathology (double arrowhead) are also shown. Scale bar, 50 μm. D, A routine fluorescent image that reveals a single labeled cell. E, The same neuron as in D is visualized through the use of antibodies to the fluorophore and then carried to the EM level (F, G). Note that neurons flooding with the preinjury dextran alone do not show overt pathological damage. Immunoreactive products (anti-Alexa Fluor IR) are labeled with arrows in G, which is an enlargement of the area, blocked out in F. Scale bar, 2 μm.

Figure 9.

A–C, Confocal images of preinjury-infused dextran (A), postinjury-infused dextran (B), and their overlay (C) demonstrate scattered neurons flooding with the postinjury-infused dextran alone (arrows), among with double-flooded neurons (arrowheads). Scale bar, 100 μm.

Figure 10.

Distribution of neurons with resealed, enduring, or delayed membrane perturbation within the population of neurons with DTBI-induced membrane perturbation. As membranes remain closed, open, or reseal in response to injury, tracer availability will determine the type of membrane perturbation categorically assigned to each neuron (table within figure legend). The preinjury administration of Alexa Fluor 488 and postinjury administration of Texas Red-conjugated dextrans permit the evaluation of neuronal membrane perturbation distribution at 4 and 8 h after injury. Across time points, the proportion of neurons with delayed membrane perturbation is significantly different from the proportion of resealed neurons and the proportion of enduring membrane perturbation (χ², p < 0.016). The change in the proportion of neurons with resealed and enduring membrane perturbation over time was not significant (χ², p = 0.19). The redistribution of the types of membrane perturbation between 4 and 8 h after injury may result from resealed neurons reopening to become neurons with enduring damage and/or additional neurons suffering delayed membrane perturbation. See Results for more details.