Nobuyuki Katori

The effects of platelet count on clot retraction and tissue plasminogen activator-induced fibrinolysis on thrombelastography

Clot retraction and fibrinolysis may present as a decrease in amplitude on thrombelastography (TEG®). The former represents normal or hyperactive platelet function, and the latter represents a fibrinolytic state. It is important to distinguish clot retraction from fibrinolysis because the treatment of each condition is different. To distinguish between these phenomena, we performed TEG® with platelet-poor plasma (PPP) and platelet-rich plasma (PRP) with an increasing platelet count (range, 50–1200 × 109/L) with or without abciximab. Maximum amplitude (MA) and the percentage decrease of amplitude at 30 and 60 min after MA were examined for each sample. Blood samples to which tissue plasminogen activator (tPA) was added served as positive controls for fibrinolysis. Morphological changes of clots and d-dimer levels were also examined. With higher platelet counts, the percentage decrease of amplitude after MA increased significantly at 30 and 60 min, but not in the abciximab samples. Morphological changes of clots have shown clot retraction in PRP, but not in PPP or PRP pretreated with abciximab. d-Dimer levels increased only in samples to which tPA was added, but not in native PPP or PRP samples. In conclusion, we have shown that the decrease in amplitude at 30 and 60 min can be due to platelet-mediated clot retraction and can be attenuated by sample pretreatment with abciximab, which interrupts platelet-fibrin(ogen) binding.

Electron microscopic evaluations of clot morphology during thrombelastography

In this study, we characterized clot morphology with a scanning electron microscope (SEM) at time points corresponding to the commonly used thrombelastography (TEG®) variables, illustrating the correlation of the physical clot formation with TEG® tracings. The first channel of the TEG® analyzer was used to obtain the tracings of clot formation, while the sub-samples for the SEM were obtained from the second TEG® channel. Different types of samples were examined, including whole blood, abciximab-treated whole blood, platelet-rich plasma (PRP), and abciximab-treated PRP. The SEM images were obtained at reaction time, different amplitudes (5–30 mm), maximum amplitude (MA), and at amplitude 60 min after MA. In the whole blood, coarse fibrin and activated platelets were observed at reaction time and fibrin strands progressively became more solid and intertwined at amplitude 10 mm and thereafter. Red blood cells were surrounded with fibrin strands at amplitude 30 mm and were tightly packed by fibrin strands at MA. In abciximab-treated whole blood, red blood cell shape was maintained at MA. The process of fibrin formation and platelet activation was also examined in PRP. Abciximab did not block platelet shape change, although the blockage of fibrin binding to platelets was shown on the TEG® analyzer. In summary, we have shown structural changes of the forming clot in relation to TEG® variables.

The effects of argatroban on thrombin generation and hemostatic activation in vitro

We evaluated argatroban, a direct thrombin inhibitor, as a heparin adjunct for anticoagulation. Platelet-poor plasma (PPP) was isolated from blood collected from 12 volunteers. Thrombin generation measurements were performed in donor PPP that was mixed with antithrombin (AT)-poor plasma to yield AT levels of 0%, 20%, 60%, and 100%. Effects of argatroban (0–1.0 &mgr;g/mL), heparin (0.25 U/mL), or the combination of argatroban (0.5 &mgr;g/mL) and heparin were also studied. The addition of increasing concentrations of argatroban, heparin, or both to donor PPP (AT level ∼100%) caused progressive decreases in the lag time and peak formation of thrombin generation. Heparin (0.25 U/mL) at small AT concentrations had a minimal effect on lag time or peak thrombin formation; its effectiveness of inhibiting thrombin was directly correlated with the concentration of AT. Argatroban at 0.5 &mgr;g/mL was effective in decreasing thrombin formation at both low and normal AT levels, but it was most effective when combined with heparin. Additionally, blood samples were obtained from 47 cardiac surgical patients, and the interaction of heparin (>1.5 U/mL) and AT or argatroban on clot formation was evaluated with kaolin activated clotting times (ACTs). Significant increases of ACTs at all heparin levels were observed with the addition of argatroban (0.125 and 0.25 &mgr;g/mL). The addition of AT (0.2 U/mL) to heparinized blood samples further prolonged ACTs. In summary, we showed that argatroban, unlike heparin, could effectively reduce thrombin generation regardless of AT levels and could prolong ACTs in vitro at clinically used concentrations.

Tumor necrosis factor-α mediates hyperglycemia-augmented gut barrier dysfunction in endotoxemia

Objective:To examine whether hyperglycemia would augment gut barrier dysfunction and inflammatory responses in endotoxemic rats, and simultaneously to clarify the roles of tumor necrosis factor (TNF)-α in alterations of gut mucosal permeability associated with hyperglycemia. Design:Prospective randomized animal study. Setting:University research laboratory. Subjects:Male Wistar rats treated with lipopolysaccharide (LPS) injection. Interventions:After LPS injection (4 mg/kg), rats were randomly allocated into group S (n = 6), group G (n = 7), or group GI (n = 8) with continuous infusion of different fluid solutions: normal saline, 40% glucose or 10% glucose mixed with insulin, respectively. Blood glucose, insulin, and proinflammatory cytokines, accompanied by gut mucosal permeability using an in situ loop preparation of gut with fluorescence isothiocyanate-conjugated dextran, were measured. Bacterial growth or alterations in mesenteric lymph nodes and cecal contents were also assessed. We further determined the roles of TNF-α using an inhibitor of TNF-α converting enzyme in gut barrier dysfunction under the same experimental settings. Measurements and Main Results:Hyperglycemia over 400 mg/dL was achieved and kept in group G during the study period whereas normoglycemia was preserved in group S and GI, the latter of which showed the similar extent of hyperinsulinemia to group G. Plasma concentrations of fluorescence-labeled dextran and TNF-α in group G were significantly higher vs. group S and GI, and the number of bacteria found in mesenteric lymph nodes in group G was greater compared with group S. Intestinal environments including microflora and organic acids were not altered by blood glucose or insulin level. Inhibiting conversion of membrane-bound to soluble type of TNF-α restored gut mucosal permeability augmented by hyperglycemia. Conclusions:These findings indicate that hyperglycemia deteriorates LPS-elicited gut barrier dysfunction and bacterial translocation independently of plasma insulin level, and that TNF-α mediates such mucosal dysfunction of gut in endotoxemia.

The effects of vasoactive agents, platelet agonists and anticoagulation on thrombelastography

Background:u2002 Platelet activation is a critical step in primary hemostasis and clot formation. We tested a hypothesis that platelet stimulating effects of vasoactive agents or platelet agonists could be shown using thrombelastography (TEG®) as faster onset or increased clot strength. We further examined if TEG® could be modified to evaluate activated platelets as a reversal of anticoagulation in the presence of partial thrombin inhibition.

A Novel Method to Assess Platelet Inhibition by Eptifibatide with Thrombelastograph

We examined a novel method to detect platelet inhibition with thrombelastography (TEG®). We hypothesized that this method would be suitable for monitoring the antiplatelet effects of eptifibatide (Integrilin®). Whole blood from healthy volunteers was anticoagulated with 3.2% citrate or unfractionated heparin (7 IU/mL). For the platelet aggregation test, both citrate and heparinized samples were spiked with increasing concentrations of eptifibatide (0, 0.2, 0.4, 0.8, 1.6, and 4 &mgr;g/mL). Conventional kaolin TEG® was performed with citrated samples, and batroxobin-modified TEG® was performed with heparinized samples, which were spiked with eptifibatide at concentrations of 0, 0.4, 0.8, 1.6, 4, 8, and 24 &mgr;g/mL. Adenosine 5′-diphosphate-induced platelet aggregation was reduced to 6.4% ± 2.9% (citrate) and 10.3% ± 4.8% (heparin) with eptifibatide at the concentration of 4 &mgr;g/mL. The kaolin TEG® showed a decrease in maximum amplitude (MA) only at the eptifibatide concentration of 24 &mgr;g/mL and no change in &agr; angle, whereas with the batroxobin-based TEG®, the difference in MA and &agr; angle was observed at concentrations ≥0.8 &mgr;g/mL. Additionally, the time to achieve maximum MA was much shorter for batroxobin TEG® than for kaolin TEG®. We conclude that the batroxobin-modified TEG® is a sensitive method that detects platelet inhibition induced by eptifibatide.

Epidural Cooling Minimizes Spinal Cord Injury after Aortic Cross-clamping through Induction of Nitric Oxide Synthase

Background:By using a U-shaped lumen catheter, the authors examined the effects of epidural cooling on spinal cord injury after aortic cross-clamping (ACC), with a focus on changes of spinal cord blood flow and expression of inducible nitric oxide synthase. Methods:Sixteen pigs were randomized into two groups: Control group (n = 8) or Cooling group (n = 8). In the latter, epidural cooling started at 30 min (baseline) before 45 min of ACC and persisted for the next 30 min of reperfusion period. Spinal cord blood flow and somatosensory-evoked potentials were assessed during peri-ACC period. At 48 h, we evaluated hind limb function by using Tarlov score and expression of inducible nitric oxide synthase on spinal cord using immunohistochemistry. Results:After ACC, spinal cord blood flow dropped to a similar extent in both groups. During the reperfusion period, spinal cord blood flow increased up to 113% (103–124%), median (interquartile range), level transiently and decreased to 32% (22–47%) level versus baseline in the Control group, whereas it increased and remained at 92% (86–97%) level in the Cooling group. Simultaneously, somatosensory-evoked potentials showed that onset of loss time was delayed and recovery time was shortened in the Cooling group. Tarlov scores in the Cooling group were significantly higher and accompanied by normal-appearing motor neurons and significantly greater expression of inducible nitric oxide synthase on spinal cord versus the Control group. Conclusions:This study shows that epidural cooling during ACC minimized the risk of spinal cord injury, possibly by preventing delayed hypoperfusion and upregulating inducible nitric oxide synthase expression.

The effects of epidural anesthesia on growth of Escherichia coli at pseudosurgical site: The roles of the lipocalin-2 pathway

BACKGROUND:Neutrophil-derived lipocalin-2 exerts bacteriostatic effects through retardation of iron uptake by the Gram-negative organisms like Escherichia coli. We tested the hypothesis that the expression of lipocalin-2, a bacteriostatic protein, was upregulated by induction of surgical site infection (SSI) with E coli in healthy and diseased rats and that epidural anesthesia modulated its expression. METHODS:Male Wistar rats were randomized into a healthy or disease group, the latter of which was administered lipopolysaccharide. Both groups were further divided into 3 subgroups, the control, saline, and lidocaine groups: group healthy control (n = 10), healthy saline (n = 10), and healthy lidocaine (n = 10) versus group disease control (n = 15), disease saline (n = 18), and disease lidocaine (n = 19), respectively. While saline was epidurally administered to the control and saline groups, lidocaine was administered to the lidocaine groups. Except for the control groups, E coli was injected to the pseudosurgical site to mimic SSI after abdominal surgery. Plasma concentrations of inflammatory cytokine and lipocalin-2 were measured. At 72 hours, the surgical site tissues were obtained to evaluate mRNA expression of lipocalin-2 and E coli DNA expression. RESULTS:All disease subgroups showed markedly increased plasma inflammatory cytokines versus the healthy subgroups. Among the disease subgroups, plasma concentrations of lipocalin-2 and tissue mRNA expression of lipocalin-2 were significantly increased in group disease lidocaine versus the others. Concurrently, E coli DNA expression in the tissue specimens was also significantly lower in group disease lidocaine as compared with group disease saline. CONCLUSIONS:Epidural anesthesia was associated with an increase in the expression lipocalin-2 and a decrease in the expression of E coli DNA at pseudosurgical sites in sick but not healthy rats. These observations suggest a potential mechanism by which epidural anesthesia could reduce the risk of SSI.

Perioperative Factors Associated with Chronic Central Pain after the Resection of Intramedullary Spinal Cord Tumor

Objective: Some patients experience severe chronic pain after intramedullary spinal cord tumor (IMSCT) resection, but the underlying mechanisms have yet to be fully elucidated. We aimed to investigate perioperative factors associated with chronic pain after IMSCT resection. Materials and Methods: We analyzed data from a postal survey and the medical records of patients who had undergone IMSCT resection in our institution between 2000 and 2008. Chronic pain was assessed using the Neuropathic Pain Symptom Inventory score, and its associations with factors related to tumor pathology, patient demographics, neurological findings, surgery, anesthesia, and perioperative management were determined. Results: Seventy-eight consecutive patients (55 men and 23 women; age 17 to 79 y) were included in the statistical analysis of the present study. In univariate analyses, sex, body mass index, preoperative tumor-related pain, preoperative nonsteroidal anti-inflammatory drugs, intraoperative hypotension, postoperative corticosteroids, and decrease in Japanese Orthopaedic Association (JOA) scores were found to be associated with postsurgical chronic central pain. Logistic regression analysis identified 3 significant factors: a decline in JOA scores compared with preoperative values (odds ratio [OR], 3.33; 95% confidence interval [CI], 1.18-9.42; P=0.023), intraoperative hypotension (OR, 3.01; 95% CI, 1.02-8.97; P=0.047), and postoperative corticosteroids (OR, 3.21; 95% CI, 1.02-10.09; P=0.046). Discussion: Decline in JOA score, intraoperative hypotension, and postoperative corticosteroids are independently associated with postsurgical chronic central pain. Intraoperative hypotension and the use of postoperative corticosteroids can be avoided or modified during perioperative management. As results from animal studies have indicated that the administration of corticosteroids may intensify chronic pain, further studies in larger cohorts are required to definitively determine the effect of corticosteroids on postsurgical central pain.

Beneficial effects of nasal high flow oxygen therapy after weaning from non­invasive ventilation: A prospective observational study

It remains unknown whether application of nasal high flow (NHF) is effective after liberation from non-invasive ventilation (NIV). This study was aimed at investigating the effect of NHF in patients ready for weaning from NIV. With institutional ethic committee approval, patients receiving NIV due to hypoxemic respiratory failure for more than 24 hours were enrolled. After passing the weaning criteria with continuous positive airway pressure (CPAP) mode [fraction of inspiratory oxygen (FIO2) ≦0.5, SIGNA VITAE 2016; 11(1):