Necroptosis in a Rat Ischemia/Reperfusion Injury Flap Model
Cell death is a fundamental biological process that occurs in both physiological and pathological contexts. Traditionally, cell death has been categorized into two primary forms: necrosis and apoptosis. Necrosis is characterized by uncontrolled cell death resulting from external factors such as trauma, infection, or toxins, leading to cell membrane rupture and inflammation. In contrast, apoptosis is a highly regulated, programmed cell death process that involves controlled cellular dismantling without inducing inflammation. Recently, a novel form of cell death, termed necroptosis, has been identified. Necroptosis combines features of both necrosis and apoptosis, exhibiting a programmed yet inflammatory cell death mechanism. This study investigates the presence of necroptosis in a rat abdominal ischemia/reperfusion (I/R) injury flap model using necrostatin-1 (Nec-1), a specific inhibitor of necroptosis.
Ischemia/reperfusion injury is a critical phenomenon in which tissue damage occurs when blood flow is restored after a period of ischemia. This process is particularly relevant in plastic surgery, where I/R injury is a leading cause of flap failure. During I/R injury, cell death plays a significant role, with both apoptosis and necrosis being well-documented. However, the involvement of necroptosis in I/R injury, especially in skin flap models, remains underexplored. This study aims to fill this gap by examining the role of necroptosis in a rat abdominal I/R injury flap model.
The study utilized 20 male Sprague-Dawley rats, divided into two groups: a control group (CTL) and a Nec-1 group. An extended epigastric adipocutaneous flap (6 cm × 9 cm) was created on each rat’s abdomen. The left superficial epigastric artery and vein were ligated, while the right side was retained as the pedicle. Ischemia was induced for 3 hours by clamping the right superficial epigastric artery, followed by reperfusion. Fifteen minutes before and after reperfusion, the CTL group received intraperitoneal phosphate buffer saline (PBS), while the Nec-1 group received Nec-1 (1.65 mg/kg in 200 mL total volume). Twenty-four hours post-reperfusion, the flap was divided into 54 sections (6 lines × 9 rows) for analysis.
Flap survival and blood perfusion were evaluated using a laser Doppler flowmeter (LDF) and laser speckle contrast analysis (LSCA). The results showed that the Nec-1 group had a significantly higher flap survival rate (80.56 ± 5.40%) compared to the CTL group (70.88 ± 10.28%). Blood perfusion was also better in the Nec-1 group (64.06 ± 6.10 PU mL·100g⁻¹·min⁻¹) than in the CTL group (52.45 ± 8.63 PU mL·100g⁻¹·min⁻¹). These findings suggest that Nec-1 treatment improves flap survival and perfusion, indicating a potential role of necroptosis in I/R injury.
Histological analysis using hematoxylin and eosin (H&E) staining revealed reduced inflammatory infiltration in the Nec-1 group, particularly in rows 6 cm to 9 cm. This observation aligns with the known relationship between necroptosis and inflammation, as necroptotic cells release damage-associated molecular patterns (DAMPs) that trigger inflammatory responses. The Nec-1 group exhibited fewer inflammatory cells in the dermal and subcutaneous layers, further supporting the inhibition of necroptosis by Nec-1.
Apoptosis was assessed using TUNEL staining, which detects DNA fragmentation in apoptotic cells. The apoptosis index (AI) was significantly lower in the Nec-1 group in rows 5 cm to 9 cm, with the most notable differences in rows 5 cm, 6 cm, 7 cm, and 9 cm. This reduction in apoptosis suggests that some TUNEL-positive cells may have been necroptotic and were inhibited by Nec-1. Electron microscopy confirmed the presence of apoptotic cells, showing typical morphological changes such as cell shrinkage, increased cytoplasmic density, and chromatin condensation. Interestingly, apoptotic cells appeared earlier in the CTL group (row 3 cm) compared to the Nec-1 group (row 7 cm), indicating delayed apoptosis in Nec-1-treated flaps.
Receptor-interacting protein-1 (RIP-1), a key mediator of necroptosis, was evaluated using immunohistochemical (IHC) staining. RIP-1 expression was significantly higher in the CTL group, particularly in rows 5 cm to 9 cm, while the Nec-1 group showed elevated RIP-1 expression only in rows 8 cm to 9 cm. This differential expression pattern suggests that necroptosis is more prevalent in the distal regions of the flap, where ischemia is more severe. Western blotting further confirmed lower RIP-1 protein levels in the Nec-1 group, supporting the inhibition of necroptosis by Nec-1.
Caspase-3 activity, a hallmark of apoptosis, was measured using a fluorometric assay. While caspase-3 activity increased from row 1 cm to 9 cm in both groups, there were no significant differences between the CTL and Nec-1 groups in the same rows. This finding underscores the caspase-independent nature of necroptosis, as Nec-1 specifically targets necroptosis without affecting apoptosis.
The study’s findings collectively demonstrate the presence of necroptosis in a rat abdominal I/R injury flap model. Necroptotic cells were predominantly located in the distal regions of the flap, where ischemia is more pronounced. The inhibition of necroptosis by Nec-1 resulted in improved flap survival, better blood perfusion, and reduced inflammation, highlighting the potential therapeutic value of targeting necroptosis in I/R injury.
In conclusion, this study provides compelling evidence that necroptosis plays a significant role in I/R injury in skin flaps. The use of Nec-1 as a specific inhibitor of necroptosis offers a promising avenue for reducing flap failure and improving surgical outcomes. Future research should explore the molecular mechanisms underlying necroptosis in I/R injury and investigate the potential application of Nec-1 in clinical settings.
doi.org/10.1097/CM9.0000000000000005
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