Norbert Nemeth

New guidelines for hemorheological laboratory techniques

This document, supported by both the International Society for Clinical Hemorheology and the European Society for Clinical Hemorheology and Microcirculation, proposes new guidelines for hemorheolog ...

Parameterization of red blood cell elongation index – shear stress curves obtained by ektacytometry

Abstract Measurement of red blood cell (RBC) deformability by ektacytometry yields a set of elongation indexes (EI) measured at various shear stresses (SS) presented as SS-EI curves, or tabulated data. These are useful for detailed analysis, but may not be appropriate when a simple comparison of a global parameter between groups is required. Based on the characteristic shape of SS-EI curves, two approaches have been proposed to calculate the maximal RBC elongation index (EImax) and the shear stress required for one-half of this maximal deformation (SS1/2): (i) linear Lineweaver-Burke (LB) model; (ii) Streekstra-Bronkhorst (SB) model. Both approaches have specific assumptions and thus may be subject to the measurement conditions. Using RBC treated with various concentrations of glutaraldehyde (GA) and data obtained by ektacytometry, the two approaches have been compared for nine different ranges of SS between 0.6–75 Pa. Our results indicate that: (i) the sensitivity of both models can be affected by the SS range and limits employed; (ii) over the entire range of SS-data, a non-linear curve fitting approach to the LB model gave more consistent results than a linear approach; (iii) the LB method is better for detecting SS1/2 differences between RBC treated with 0.001–0.005% glutaraldehyde (GA) and for a 40% mixture of rigid cells but is equally sensitive to SB for 10% rigid cells; and (iv) the LB and SB methods for EImax are equivalent for 0.001% and 0.003% GA and 40% rigid, with the SB better for 0.005% GA and the LB better for 10% rigid.

Effects of storage duration and temperature of human blood on red cell deformability and aggregation

Blood samples used in hemorheological studies may be stored for a period of time, the effects of storage have yet to be fully explored. This study evaluated the effects of storage temperature (i.e., 4 degrees C or 25 degrees C) and duration on RBC deformability and aggregation for blood from healthy controls and from septic patients. Our results indicate that for normal blood, RBC deformability over 0.3-50 Pa is stable up to six hours regardless of storage temperature; at eight hours there were no significant differences in EI but SS1/2 calculated via a Lineweaver-Burk method indicated impaired deformability. Storage temperature affected the stable period for RBC aggregation: the safe time was shorter at 25 degrees C whereas at 4 degrees C aggregation was stable up to 12 hours. Interestingly, blood samples from septic patients were less affected by storage. Blood can thus be stored at 25 degrees C for up to six hours for deformability studies, but should be limited to four hours for RBC aggregation; storage at 4 degrees C may prolong the storage period up to 12 hours for aggregation but not deformability measurements. Therefore, the time period between sampling and measurement should be as short as possible and reported together with results.

Influence of moderate and profound hyperventilation on cerebral blood flow, oxygenation and metabolism

OBJECTIVE The aim of the present study was to examine the impact of moderate and profound hyperventilation on regional cerebral blood flow (rCBF), oxygenation and metabolism. MATERIALS AND METHODS Twelve anesthetized pigs were subjected to moderate (mHV) and profound (pHV) hyperventilation (target arterial pO(2): 30 and 20 mmHg, respectively) for 30 min each, after baseline normoventilation (BL) for 1 h. Local cerebral extracellular fluid (ECF) concentrations of glucose, lactate, pyruvate and glutamate as well as brain tissue oxygenation (p(ti)O(2)) were monitored using microdialysis and a Licox oxygen sensor, respectively. In nine pigs, regional cerebral blood flow (rCBF) was also continuously measured via a thermal diffusion system. RESULTS Both moderate and profound hyperventilation resulted in a significant decrease in rCBF (BL: 37.9+/-4.3 ml/100 g/min; mHV: 29.4+/-3.6 ml/100 g/min; pHV: 23.6+/-4.7 ml/100 g/min; p<0.05) and p(ti)O(2) (BL: 22.7+/-4.1 mmHg; mHV: 18.9+/-4.9 mmHg; pHV: 13.0+/-2.2 mmHg; p<0.05). A p(ti)O(2) decrease below the critical threshold of 10 mmHg was induced in three animals by moderate hyperventilation and in five animals by profound hyperventilation. Furthermore, significant increases in lactate (BL: 1.06+/-0.18 mmol/l; mHV: 1.36+/-0.20 mmol/l; pHV: 1.67+/-0.17 mmol/l; p<0.005), pyruvate (BL: 46.4+/-7.8 micromol/l; mHV: 58.0+/-10.3 micromol/l; pHV: 66.1+/-12.7 micromol/l; p<0.05), and lactate/glucose ratio were observed during hyperventilation. (Data are presented as mean+/-S.E.M.) CONCLUSIONS Both moderate and profound hyperventilation may result in insufficient regional oxygen supply and anaerobic metabolism, even in the uninjured brain. Therefore, the use of hyperventilation cannot be considered as a safe procedure and should either be avoided or used with extreme caution.

Allopurinol Prevents Erythrocyte Deformability Impairing but Not the Hematological Alterations After Limb Ischemia–Reperfusion in Rats

The measurement of red blood cell deformability provides a possible method for detecting the effect of ischemia–reperfusion on erythrocytes. In our study the effect of 1-h ischemia–reperfusion with or without allopurinol pretreatment on hematological parameters and red blood cell deformability was investigated in a follow-up experiment of 26 male CD outbred rats that were subjected to unilateral hind-limb ischemia by microvascular clips on femoral vessels for 1 h (IR, n = 6), some rats received allopurinol pretreatment under the same conditions (50 mg/kg, AP + IR, n = 8), others were subjected to sham operation (n = 6), and the rest of animals served as control (n = 6). Measurement of erythrocyte deformability using a bulk filtrometer with special setting of cell suspension hematocrit (1%), and determination of hematological parameters were performed daily for one week. In the IR group, relative cell transit time increased significantly on postoperative days 1 and 2, which was not observed in the other groups. Settings for the measurement of erythrocyte deformability by reducing the blood sample volume gave the possibility of monitoring the resulting changes in rats. Mean corpuscular volume and hemoglobin, platelet count, and platelet volume were higher in the IR and AP + IR groups than in the other groups. In summary, short-term ischemia and reperfusion induced lower red blood cell deformability in the early postoperative period, which could be prevented by allopurinol pretreatment.

Hemorheological changes in ischemia-reperfusion: An overview on our experimental surgical data

Blood vessel occlusions of various origin, depending on the duration and extension, result in tissue damage, causing ischemic or ischemia-reperfusion injuries. Necessary surgical clamping of vessels in vascular-, gastrointestinal or parenchymal organ surgery, flap preparation-transplantation in reconstructive surgery, as well as traumatological vascular occlusions, all present special aspects. Ischemia and reperfusion have effects on hemorheological state by numerous ways: besides the local metabolic and micro-environmental changes, by hemodynamic alterations, free-radical and inflammatory pathways, acute phase reactions and coagulation changes. These processes may be harmful for red blood cells, impairing their deformability and influencing their aggregation behavior. However, there are still many unsolved or non-completely answered questions on relation of hemorheology and ischemia-reperfusion. How do various organ (liver, kidney, small intestine) or limb ischemic-reperfusionic processes of different duration and temperature affect the hemorheological factors? What is the expected magnitude and dynamics of these alterations? Where is the border of irreversibility? How can hemorheological investigations be applied to experimental models using laboratory animals in respect of inter-species differences? This paper gives a summary on some of our research data on organ/tissue ischemia-reperfusion, hemorheology and microcirculation, related to surgical research and experimental microsurgery.

Hemorheological, morphological, and oxidative changes during ischemia-reperfusion of latissimus dorsi muscle flaps in a canine model

Although ischemia‐reperfusion (I/R) strongly influences muscle flap survival in reconstructive surgery, there is limited knowledge about its relation to hemorheological parameters and oxidative stress markers in flaps. In the present study we investigated these changes during I/R of latissimus dorsi muscle (LDM) flaps in beagle dogs. In four animals LDM flaps were prepared bilaterally. The right side served as control, while the left sides vascular pedicle was clamped for 60 minutes, and a 60‐minute reperfusion was allowed afterward. Blood samples (0.5 ml each) were taken from the pedicles vein bilaterally before and after the ischemia, and at the 5th, 15th, 30th, 45th, and 60th minutes of the reperfusion, for hematological and erythrocyte aggregation tests. In muscle biopsies, taken before and after I/R, histological investigations and tests for measuring gluthation‐peroxidase (GSH‐PX) activity, glutathione (GSH) and carbonyl concentrations, and thiobarbituric acid reactive substances (TBARS) content were carried out. In I/R side leukocyte count increased during the reperfusion with a peak at the 30th minute. Hematocrit continuously increased from the 15th minute. In the first 5 minutes of the reperfusion, erythrocyte aggregation increased, than tented to be normalized. In muscle homogenates GSH‐PX activity did not change markedly, GSH content slightly decreased, carbonyl and TBARS content increased during reperfusion. A 1‐hour ischemia and reperfusion of LDM flaps caused local changes of leukocyte distribution and erythrocyte aggregation, supposedly due to the metabolic and inflammatory reactions. Oxidative damage during reperfusion was also demonstrated.

Concerning the importance of changes in hemorheological parameters caused by acid-base and blood gas alterations in experimental surgical models

Acid-base equilibrium and pH of blood have important clinical consequences in numerous diseases and pathophysiological conditions. The micro-rheological parameters of blood, such as red blood cell deformability and red blood cell aggregation are influenced by several metabolic factors, and provide information regarding inflammatory, septic and tissue or organ ischemia-reperfusion processes. Despite the anticipated logical relation of the blood acid-base condition, blood gas parameters and pH to red blood cell deformability and aggregation, controversial data can be found in the literature. Furthermore, related to ischemia-reperfusion hemorheological studies little is known about this issue. In this paper we aimed to thought-provokingly overview some aspect of acid-base changes, blood pH and hemorheological parameters, discussing certain results from ischemia-reperfusion experimental surgical models (local versus systemic changes), laboratory technical and experimental design protocols related to in vitro and in vivo studies.

Changes of local and systemic hemorheological properties in intestinal ischemia‐reperfusion injury in the rat model

The aim of this study was to investigate intestinal ischemia‐reperfusion and its local and systemic hemorheological relations in the rat. Ten anaesthetized female CD outbred rats were equally divided into 2 experimental groups. (1) Ischemia‐reperfusion (I/R): the superior mesenterial artery was clipped for 30 minutes. After removing the clip, 60 minutes of the reperfusion was observed before extermination. Blood samples were taken from the caudal caval vein and from the portal vein before ischemia, 1 minute before and after clip removal, and at the 15th, 30th, and 60th minutes of the reperfusion. (2) Sham operation: median laparotomy and blood sampling were done according to the timing as in I/R group. Hematological parameters, red blood cell aggregation, and deformability were determined. Leukocyte count and mean volume of erythrocytes increased slightly but continuously in portal venous samples during the reperfusion period. Red blood cell aggregation values were higher in portal blood by the end of ischemia, and then became elevated further comparing to the caval venous blood. Both in caval and portal venous samples of I/R group red blood cell deformability significantly worsened during the experimental period compared to its base and Sham group. In portal blood red blood cell deformability was impaired more than in caval vein samples. Histology showed denuded villi, dilated capillaries, and the inflammatory cells were increased after a 30 minutes ischemia. In conclusion, intestinal ischemia‐reperfusion causes changes in erythrocyte deformability and aggregation, showing local versus systemic differences in venous blood during the first hour of reperfusion.

New Insights into the Neuromuscular Anatomy of the Ileocecal Valve

The neuroanatomy of the ileocecal valve (ICV) is poorly understood. A better understanding of this important functional component of the gastrointestinal tract would enable surgeons to reconstruct an effective valve following surgical resection of the ICV. ICVs were examined in young pigs (N = 5) using frontal and transverse paraffin embedded and frozen sections. Hematoxylin+Eosin (H+E) staining, acetylcholinesterase (AchE), and NADPH‐diaphorase (NADPH‐d) histochemistry and protein gene product 9.5 (PGP 9.5) and C‐kit immunohistochemistry were performed. The H+E staining revealed that the ICV consists of three muscle layers: an external circular muscle layer continuous with that of the ileal circular muscle layer, an inner circular muscle layer continuous with that of the cecal circular muscle layer, and a single longitudinal muscle layer, which appears to be secondary to a fusion of the ileal and cecal longitudinal muscle layers. The AchE, NADPH‐d, and PGP 9.5 staining revealed two distinct coaxial myenteric plexuses, together with superficial and deep submucosal plexuses. The C‐kit immunostaining showed a continuous myenteric ICC network within the ICV. The structure of the neuromuscular components within the ICV suggests that the valve is a result of a simple intussusception of the terminal ileum into the cecum. This knowledge may help surgeons in their future attempts at reconstructing more anatomically and functionally suitable ICVs following surgical resection of native ICVs. Anat Rec 2009.