Increased intracranial pressure after diffuse traumatic brain injury exacerbates neuronal somatic membrane poration but not axonal injury: evidence for primary intracranial pressure-induced neuronal perturbation

Abstract

Increased intracranial pressure (ICP) associated with traumatic brain injury (TBI) is linked to increased morbidity. Although our understanding of the pathobiology of TBI has expanded, questions remain regarding the specific neuronal somatic and axonal damaging consequences of elevated ICP, independent of its impact on cerebral perfusion pressure (CPP). To investigate this, Fischer rats were subjected to moderate TBI. Measurements of ICP revealed two distinct responses to injury. One population exhibited transient increases in ICP that returned to baseline levels acutely, while the other displayed persistent ICP elevation (>20 mm Hg). Utilizing these populations, the effect of elevated ICP on neuronal pathology associated with diffuse TBI was analyzed at 6 hours after TBI. No difference in axonal injury was observed, however, rats exhibiting persistently elevated ICP postinjury revealed a doubling of neurons with chronic membrane poration compared with rats exhibiting only transient increases in ICP. Elevated postinjury ICP was not associated with a concurrent increase in DNA damage; however, traditional histological assessments did reveal increased neuronal damage, potentially associated with redistribution of cathepsin-B from the lysosomal compartment into the cytosol. These findings indicate that persistently increased ICP, without deleterious alteration of CPP, exacerbates neuronal plasmalemmal perturbation that could precipitate persistent neuronal impairment and ultimate neuronal death.

Figure 1

There are two distinct patterns of intracranial pressure (ICP) elevation after injury without resulting in a shift of cerebral perfusion pressure (CPP) below deleterious limits. Upon injury, half of the animals revealed a transient increase in ICP that returned to baseline levels acutely (low ICP group; ICP <20 mm Hg ⩾65% of the 6 hours after injury analysis) while the other half of the injured animals maintained a consistently elevated ICP (high ICP group; ICP >20 mm Hg ⩾65% of the 6 hours after injury analysis). A dashed line indicates the 20 mm Hg distinction. Bar graphs representing the mean (A) ICP, (B) mean arterial blood pressure (MABP), and (C) CPP over the time periods indicated of animals in the sham (black), low ICP (white), and high ICP group (dark gray), before and for 6 hours after injury. At no time over the length of the experiment did any group of animals decrease below the accepted autoregulatory limits (MABP <60 mm Hg and CPP <50 mm Hg; dashed lines in B and C). ^*P<0.05.

Figure 2

Diffuse axonal transport disruption, indicative of axonal injury, in lateral neocortical layers V and VI was unaltered by the pattern of postinjury intracranial pressure (ICP). (A) Bar graph depicting the average number of amyloid precursor protein (APP) immunoreactive axonal swellings in layers V and VI of the lateral neocortex of animals showing high or low ICP after injury. Representative photomicrographs of APP immunoreactivity from (B) sham-injured animals and animals with (C) low ICP and (D) high ICP after injury. Note the absence of APP immunoreactive axonal swellings in the sham-injured controls while the number of swellings is comparable between animals with high versus low ICP after injury. Scale bar: 1 mm.

Figure 3

The population of mechanoporated neurons shifts away from a resealing phenotype and toward the delayed opening phenotype with persistently elevated intracranial pressure (ICP) after injury. (A) Bar graph depicting the shift from resealing to delayed mechanoporated neurons in animals in the high ICP group (dark gray) as compared with those in the low ICP group (light gray). Representative photomicrographs from dextran-infused animals with (B) low ICP and (C) high ICP after injury. Note the population shift away from resealing (arrows) and toward delayed (double arrow heads) mechanoporated neurons in the high ICP group, while the enduring (arrow heads) membrane porated neurons remain comparable to the low ICP condition. ^*P<0.05. Scale bar: 100 μm.

Figure 4

Injury-induced persistently elevated intracranial pressure (ICP) exacerbates chronic neuronal somatic mechanoporation. (A) Bar graph depicting the percentage of dextran-flooded NeuN+ neocortical neurons per total neurons analyzed. The high ICP group had double the number of mechanoporated neurons that took up the postinjury infused dextran as compared with the low ICP group, while the number of membrane porated neurons that took up the preinjury infused dextran was analogous. Representative photomicrographs of dextran-infused (preinjury infused dextran: green and postinjury infused dextran: red) animals in the (B) sham, (C) low ICP, or (D) high ICP group labeled with NeuN (blue in B–D) to identify neurons. Notice the stark contrast between nonmechanoporated (arrow heads) and mechanoporated (arrows) neurons. ^*P<0.05. Scale bar: 100 μm.

Figure 5

Injury-induced intracranial pressure (ICP) elevation does not precipitate DNA fragmentation but is associated with cell perturbation. Representative photomicrographs from animals sustaining (B, G) sham injury and animals with either (C, H) low ICP or (D, I) high ICP after injury immunolabeled with (A–D) TUNEL to visualize the number of cells with fragmented DNA, and (F–I) hematoxylin and eosin (H&E) to visualize damaged neurons (arrows). Corresponding bar graphs depicting the (E) percent of total Dapi+ cells that were also TUNEL labeled and (J) the percent of total neurons that were damaged visualized with H&E within layers V and VI of the lateral neocortex. While there are very few TUNEL-positive cells observed after injury (irrespective of ICP pattern), H&E analysis indicated increased neuronal damage in animals exhibiting persistent ICP elevation after TBI. ^*P<0.05. Scale bar: 100 μm. TUNEL, terminal deoxynucleotidyl transferase-mediated 2′-deoxyuridine 5′-triphosphate-biotin nick end labeling.

Figure 6

Ultrastructural analysis of chronically mechanoporated neurons revealed morphological heterogeneity. Representative electron micrographs of postinjury infused dextran labeled neurons. (A) Many neurons with cytoplasmic dextran incorporation (arrow heads in B) without nuclear flooding exhibit intact nuclear membranes (arrows in B) and show no overt mitochondrial damage (^*), best shown in the enlarged panel (B). (C) Note that some neurons with both cytoplasmic and nuclear dextran flooding show ultrastructural perturbation including perinuclear organelle vacuolization (arrows in C). (D) Some neurons containing the postinjury infused dextran also show moderately increased electron density. (E) An additional subset of late flooded neurons show overt necrotic change. These neurons undergoing necrotic change, comprised predominantly of the neuronal population exhibiting enduring mechanoporation, as assessed by fluorescent microscopy (arrow in F denotes the neuron in E) displayed a redistribution of cathepsin-B from the lysosomal compartment into the cytoplasm surrounding what appear to be ruptured lysosomes (double arrow heads). Scale bars: 1 μm A–E, 10 μm F.

Figure 7

Chronically membrane porated neurons display a redistribution of cathepsin-B. (A) Bar graph depicting the percentage of neurons showing punctate (i.e., lysosomal) or diffuse (i.e., cytosolic) localization of cathepsin-B. The majority of neurons exhibiting either no mechanoporation (small white-dotted black bar) or membrane resealing (gray bar) had punctate localization of cathepsin-B, indicative of intact lysosomes. Conversely, the majority of neuron sustaining chronic membrane poration, either enduring (black bar) or delayed opening (large black-dotted white bar), showed diffuse cytoplasmic localization of cathepsin-B, suggestive of lysosomal rupture. Representative confocal photomicrographs of (B) the postinjury infused dextran, (C) cathepsin-B immunoreactivity of the same region, and (D) an overlay showing the redistribution of cathepsin-B from punctate lysosomal compartments, typical of nonmechanoporated neurons (arrow heads), to diffuse cytosolic localization in neurons sustaining chronic membrane poration (arrows). Enlarged photomicrographs of cathepsin-B labeling in (E) nonmechanoporated and (F) chronically mechanoporated neurons demarcated with white boxes in (B). ^*P<0.05. Scale bar: 100 μm.