Nafamostat mesylate decreases skin flap necrosis in a mouse model of type 2 diabetes by protecting the endothelial glycocalyx.

The success rate of flap tissue reconstruction has increased in recent years owing to advancements in microsurgical techniques. However, complications, such as necrosis, are still more prevalent in diabetic patients compared to non-diabetic individuals, presenting an ongoing challenge. To address this issue, many previous studies have examined vascular anastomoses dilation and stability, primarily concerning surgical techniques or drugs. In contrast, in the present study, we focused on microvascular damage of the peripheral microvessels in patients with diabetes mellitus and the preventative impact of nafamostat mesylate. Herein, we aimed to investigate the effects of hyperglycemia on glycocalyx (GCX) levels in mice with type 2 diabetes. We examined the endothelial GCX (eGCX) in skin flap tissue of 9-12-week-old type 2 diabetic mice (db/db mice) using a perforator skin flap and explored treatment with nafamostat mesylate. The growth rates were compared after 1 week. Heterotype (db/+) mice were used as the control group. Morphological examination of postoperative tissues was performed at 1, 3, 5, and 7 days post-surgery. In addition, db/db mice were treated with 30 mg/kg/day of nafamostat mesylate daily and were evaluated on postoperative day 7. Seven days after surgery, all db/db mice showed significant partial flap necrosis. Temporal observation of the skin flaps revealed a stasis-like discoloration and necrosis starting from the contralateral side of the remaining perforating branch. The control group did not exhibit flap necrosis, and the flap remained intact. In the quantitative assessment of endothelial glycans using lectins, intensity scoring showed that the eGCX in the db/db group was significantly thinner than that in the db/+ group. These results were consistent with the scanning electron microscopy findings. In contrast, treatment with nafamostat mesylate significantly improved the flap engraftment rate and suppressed eGCX injury. In conclusion, treatment with nafamostat mesylate improves the disrupted eGCX structure of skin flap tissue in db/db mice, potentially ameliorating the impaired capillary-to-venous return in the skin flap tissue.

Recombinant antithrombin attenuates acute kidney injury associated with rhabdomyolysis: an in vivo animal study.

BACKGROUND: Rhabdomyolysis is characterized by the destruction and necrosis of skeletal muscle tissue, resulting in acute kidney injury (AKI). Recombinant antithrombin (rAT) has DNA repair and vascular endothelial-protection properties. Herein, we investigated whether rAT therapy has beneficial effects against rhabdomyolysis-induced AKI. Ten-week-old male B6 mice were injected with 5 mL/kg of 50% glycerol intramuscularly in the left thigh after 24 h of fasting to create a rhabdomyolysis mouse model. Further, 750 IU/kg rAT was injected intraperitoneally at 24 and 72 h after the rhabdomyolysis model was established. The mice were euthanized after 96 h for histological analysis. Saline was administered to mice in the control group. RESULTS: Blood tests show elevated serum creatinine, urea nitrogen, and neutrophil gelatinase-associated lipocalin levels in rhabdomyolysis. Loss of tubular epithelial cell nuclei and destruction of the tubular luminal surface structure was observed in the untreated group, which improved with rAT treatment. Immunostaining for Ki-67 showed increased Ki-67-positive nuclei in the tubular epithelial cells in the rAT group, suggesting that rAT may promote tubular epithelial cell regeneration. The microvilli of the brush border of the renal tubules were shed during rhabdomyolysis, and rAT treatment reduced this injury. The vascular endothelial glycocalyx, which is usually impaired by rhabdomyolysis, became functional following rAT treatment. CONCLUSIONS: Treatment with rAT suppressed rhabdomyolysis-induced AKI, suggesting that rAT therapy may be a novel therapeutic approach.

Decreased neutrophil counts prolong inflammation in acute pancreatitis and cause inflammation spillover to distant organs.

BACKGROUND/OBJECTIVE: Acute pancreatitis is an aseptic inflammation caused by pathologically activated pancreatic enzymes and inflammatory mediators produced secondarily by neutrophils and other inflammatory cells and is one of the most difficult diseases to treat. This study aimed to investigate the role of neutrophils in pancreatitis by examining tissue dynamics. METHODS: We created a model of caerulein-induced pancreatitis in 12-week-old male granulocyte colony-stimulating factor knockout mice (G-CSF-KO) and wild-type littermate control mice (six intraperitoneal injections of caerulein [80 µg/kg body weight] at hourly intervals for 2 days). Mice were sacrificed 0, 3, 6, 12, 24, 36, 48, 72, and 168 h after caerulein administration and examined histologically. RESULTS: The survival rate after one week of caerulein administration was 100 % in the control mice, whereas it was significantly lower (10 %) in the G-CSF-KO mice. Histological examination revealed significant hemorrhage and inflammatory cell migration in the G-CSF-KO mice, indicating prolonged inflammation. CONCLUSION: Prolonged inflammation was observed in the G-CSF-KO mice. Tissue cleanup by neutrophils during the acute phase of inflammation may influence healing through the chronic phase.

Endothelial Glycocalyx Disorders May Be Associated With Extended Inflammation During Endotoxemia in a Diabetic Mouse Model.

In diabetes mellitus (DM) patients, the morbidity of infectious disease is increased, and these infections can easily progress from local to systemic infection. Sepsis is a characteristic of organ failure related to microcirculation disorders resulting from endothelial cell injury, whose most frequent comorbidity in patients is DM. The aim of the present study was to evaluate the influence of infection on DM-induced microvascular damage on inflammation and pulmonary endothelial structure using an experimental endotoxemia model. Lipopolysaccharide (LPS; 15 mg/kg) was injected intraperitoneally into 10-week-old male C57BLKS/J Iar- + lepr db /lepr db (db/db) mice and into C57BLKS/J Iar-m + / + lepr db (db/ +) mice, which served as the littermate non-diabetic control. At 48 h after LPS administration, the survival rate of db/db mice (0%, 0/10) was markedly lower (P < 0.05) than that of the db/ + mice (75%, 18/24), whereas the survival rate was 100% in both groups 24 h after LPS administration. In control mice, CD11b-positive cells increased at 6 h after LPS administration; by comparison, the number of CD11b-positive cells increased gradually in db/db mice until 12 h after LPS injection. In the control group, the number of Iba-1-positive cells did not significantly increase before and at 6, 12, and 24 h after LPS injection. Conversely, Iba-1-positive cells continued to increase until 24 h after LPS administration, and this increase was significantly greater than that in the control mice. Expression of Ext1, Csgalnact1, and Vcan related to endothelial glycocalyx synthesis was significantly lower in db/db mice than in the control mice before LPS administration, indicating that endothelial glycocalyx synthesis is attenuated in db/db/mice. In addition, ultrastructural analysis revealed that endothelial glycocalyx was thinner in db/db mice before LPS injection. In conclusion, in db/db mice, the endothelial glycocalyx is already injured before LPS administration, and migration of inflammatory cells is both delayed and expanded. This extended inflammation may be involved in endothelial glycocalyx damage due to the attenuation of endothelial glycocalyx synthesis.