Endothelial necrosis at 1 hour postburn predicts progression of tissue injury

Abstract

Burn injury progression has not been well characterized at the cellular level. To define burn injury progression in terms of cell death, histopathologic spatiotemporal relationships of cellular necrosis and apoptosis were investigated in a validated porcine model of vertical burn injury progression. Cell necrosis was identified by high mobility group box 1 protein and apoptosis by Caspase 3a staining of tissue samples taken 1 hour, 24 hours, and 7 days postburn. Level of endothelial cell necrosis at 1 hour was predictive of level of apoptosis at 24 hours (Pearson's r = 0.87) and of level of tissue necrosis at 7 days (Pearson's r = 0.87). Furthermore, endothelial cell necrosis was deeper than interstitial cell necrosis at 1 hour (p < 0.001). Endothelial cell necrosis at 1 hour divided the zone of injury progression (Jackson's zone of stasis) into an upper subzone with necrotic endothelial cells and initially viable adnexal and interstitial cells at 1 hour that progressed to necrosis by 24 hours and a lower zone with initially viable endothelial cells at 1 hour but necrosis and apoptosis of all cell types by 24 hours. Importantly, this spatiotemporal series of events and rapid progression resembles myocardial infarction and stroke and implicates mechanisms of these injuries, ischemia, ischemia reperfusion, and programmed cell death in burn progression.

Figure 1. Level of endothelial cell necrosis at 1h predicts further interstitial cell necrosis, apoptosis, and conversion at 24h and tissue necrosis at day 7

HMGB1 at 1h (A, B) and 24h (C, D), Caspase 3a at 24h (E, F), and HMGB1 at day 7 (G, H) are shown for representative burns made at 70°/30s, 80°/20s, and 80°/30s at low power and high power. Boxes on 80°/20s burns are shown at high power. Red dots indicate level of endothelial necrosis, with viable endothelial cells below and necrotic cells above the dots, and black dots similarly define the interface of viable and necrotic epithelial adnexae. Green arrow heads bracket zone of apoptotic cells. In 80°/20s burn in panel A, the zone between black and red dots has necrotic endothelial cells and viable epithelial adnexal cells.

Figure 2. Conversion from partial to full-thickness of a representative 80°/20s burn in 24h, confirmed at day 7

A. Low power H&E at 1h reveals viable (Green Arrow) at 1h flanked by necrotic blood vessels(red arrows, high power in Figure 3). The region of dermis with these initially viable adnexae that progress to necrosis by 24h, compromises the vertical component of the zone of stasis as defined by cellular necrosis. B. By24hnecrosis of all adnexae is apparent. C. Full-thickness injury is confirmed by complete necrosis of adnexae, infiltration of granulation tissue, and necrotic, sloughing dermis. D. High power nuclear staining HMGB1 stain of apocrine coil at 1h indicated by black arrow in A proves that these adnexal structures are not necrotic. E. Necrosis of apocrine coil indicated by black arrow in B is evident by absence of viable nuclei on coil wall and detachment of pyknotic epithelial calls. F. Completely necrotic hair follicle labeled with black arrow in C is surrounded by neutrophils and granulation tissue and has no viable epithelial cells.

Figure 3. High magnification reveals necrotic endothelial cells in deep dermal vessels labeled in Figure 2

A and B. Loss of nuclear HMGB1 staining of endothelial cell cyptoplams in vessels labeled in zone stasis in Figure 2A indicate necrosis of endothelial cell of deep dermal vessels at 1h. C. By 24h necrosis on H&E pyknotic nuclei and hypereosinophilic vessel walls. Extravasated polymorphonuclear cells and monocytes are also evident (Yellow Arrow).

Figure 4. Zone of apoptosis at 24h approximates final level of injury

A Caspase 3a staining of a non-progressing 80°/20s burn demonstrates a zone of apoptotic cells in the mid-recticular dermis. Location of the zone aproximates level of inflamatory cell infiltration and granulation tissue at day 7, but extends further. Complete necrosis at day 7 above this level is demonstrated by homogeneous pink staining due to complete absence of nuclei which contrasts sharply with intense blue hematoxlyin staining caused by inflammatory cells and granulation tissue directly below. B. At 24h, the region labeled by the black arrow in figure A is shown at high power, revealing apoptosis of many cells due to nuclear CC3a staining.

Figure 5. Progression of cell necrosis by HMGB1 in peri-burn tissue from 1 to 24h

A. At 1h (grey histograms) endothelial cell necrosis is near full-thickness in 80°/20s, 80°/30s, and 90°/20s burns, and relatively more superficial in 70°/20s and 70°/30s burns. B. Level of endothelial cell necrosis predicts full-thickness injury at 24h (black histograms) with nearly all 80°/20s burns and all 80°/30s and 90°/20s ultimately full-thickness. 80°/20s burns were notable for progression from partial thickness to full-thickness between 1h and 24h post-burn judging by necrosis of hair follicles and apocrine glands.

Figure 6. Strong correlation between endothelial cell (EC) necrosis 1h, apoptotic zone (AZ) 24h, and dermal necrosis day 7

Level of endothelial cell necrosis at 1h post injury is strongly predictive of burn depth (A), and level of apoptosis at 1h, although level apoptosis extends significantly deeper (B). Level of apoptosis is predictive of level of dermal necrosis at day 7, but overestimates it, with several burns with apoptosis to the deep dermis show significant recovery at day 7 (dashed ellipse, C).