Preserved vascular reactivity of rat renal arteries after cold storage

In cultured renal tubular cells hypothermia results in cell damage caused by iron-dependent formation of reactive oxygen species. It is unknown whether cold preservation affects function of renal vessels. Rat renal arcuate arteries were stored in a physiological salt solution at 4 degrees C for 24h and compared to control arteries (not stored). To some of the stored arteries the iron chelator 2,2'-dipyridyl was added. Endothelium-independent vasoconstriction was assessed by cumulative concentration-response curves for potassium and phenylephrine in a small vessel myograph. Endothelium-independent vasodilation was assessed with sodium nitroprusside and endothelium-dependent vasodilation with histamine. Cold storage for 24h did not affect vascular reactivity of renal small arteries and no influence of the iron chelator was seen. Since 24h of cold storage considerable damages renal tubular cells both in vitro and after kidney transplantation, these results suggest that renal arteries are less sensitive to cold-induced damage than tubular cells.     

New approach to improving endothelial preservation in cryopreserved arterial substitutes

The endothelial loss provoked by the methods of vascular cryopreservation used at most human vessel banks is one of the main factors leading to the failure of grafting procedures performed using cryopreserved vessel substitutes. This study evaluates the effects of the storage temperature and thawing protocol on the endothelial cell loss suffered by cryopreserved vessels, and optimises the thawing temperature and protocol for cryopreserving arterial grafts in terms of that producing least endothelial loss. Segments of the common iliac artery of the minipig (n = 20) were frozen at a temperature reduction rate of 1 degrees C/min in a biological freezer. After storing the arterial fragments for 30 days, study groups were established according to the storage temperature (-80, -145 or -196 degrees C) and subsequent thawing procedure (slow or rapid thawing). Fresh vessel segments served as the control group. Once thawed, the specimens were examined by light, transmission, and scanning electron microscopy. The covered endothelial surface was determined by image analysis. Data for the different groups were compared by one way ANOVA. When cryopreservation at each of the storage temperatures was followed by slow thawing, the endothelial cells showed improved morphological features and viability over those of specimens subjected to rapid thawing. Rapidly thawed endothelial cells showed irreversible ultrastructural damage such as mitochondrial dilation and rupture, reticular fragmentation, and peripheral nuclear condensation. In contrast, slow thawing gave rise to changes compatible with reversible damage in a large proportion of the endothelial cells: general swelling, reticular dilation, mitochondrial swelling, and nuclear chromatin condensation. Gradually thawed cryopreserved arteries showed a lower proportion of damaged cells identified by the TUNEL method compared to the corresponding rapidly thawed specimens (p < 0.05, for all temperatures). In all the groups in which vessels underwent rapid thawing (except at -145 degrees C), significant differences (p < 0.05) in endothelial cover values were recorded with respect to control groups. Storage of cryopreserved vessels at -80 degrees C followed by rapid thawing led to greatest endothelial cell loss (61.36+/-9.06% covered endothelial surface), while a temperature of -145 degrees C followed by slow thawing was best at preserving the endothelium of the vessel wall (89.38+/-16.67% surface cover). In conclusion, storage at a temperature of -145 degrees C in nitrogen vapour followed by gradual automated thawing seems to be the best way of preserving the endothelial surface of the arterial cryograft. This method gives rise to best endothelial cell viability and cover values, with obvious benefits for subsequent grafting.     

Therapeutic potential of breakers of advanced glycation end product-protein crosslinks

Long-lived structural proteins, collagen and elastin, undergo continual non-enzymatic crosslinking during aging and in diabetic individuals. This abnormal protein crosslinking is mediated by advanced glycation end products (AGEs) generated by non-enzymatic glycosylation of proteins by glucose. The AGE-derived protein crosslinking of structural proteins contributes to the complications of long-term diabetes such as nephropathy, retinopathy, and neuropathy. AGE-crosslinks have also been implicated in age-related cardiovascular diseases. Potential treatment strategies for these AGE-derived complications include prevention of AGE-formation and breaking of the existing AGE-crosslinks. The therapeutic potential of the AGE-inhibitor, pimagedine (aminoguanidine), has been extensively investigated in animal models and in Phase 3 clinical trials. This review presents the pre-clinical and clinical studies using ALT-711, a highly potent AGE-crosslink breaker that has the ability to reverse already-formed AGE-crosslinks. Oral administration of ALT-711 has resulted in a rapid improvement in the elasticity of stiffened myocardium in experimental animals. Topical administration of ALT-711 was effective in improving the skin hydration of aged rats. The therapeutic potential of crosslink breakers for cardiovascular complications and dermatological alterations associated with aging and diabetes is discussed.     

Translational stiffness of the replaced shoulder joint

Results after a total shoulder arthroplasty in rheumatoid patients are poor, indicated by loosening of especially the glenoid component, bad joint functionality and the possibility of a joint dislocation. The failure mechanisms behind this are multiple, including patient, surgical and design factors. These results must be improved. At present, the optimal geometrical prosthesis component design, focused on joint conformity and constraint, still has to be investigated.

Proper understanding of the effect of geometrical design parameters on the theoretical relationship between joint translations and joint forces may contribute to improved designs. The main objective of this study is to theoretically describe this relationship and to investigate the joint translational stiffness, which can be used to investigate the effect of design parameters on joint motion. Joint translational stiffness is the gradient of the subluxation force with respect to the humeral head displacement.

For this static analysis a potential field is introduced, as the result of a joint compressive force (muscle forces) and a subluxation force (external forces). The positive and negative stiffness during articulation inside and subluxation outside the glenoid cavity, lead to stable and unstable equilibrium joint positions, respectively. A most lateral position of the humeral head centre coincides with a zero subluxation force; at this position the humerus is dislocated and a restoring force is needed to relocate the humeral head.

Joint conformity and compression force influence the joint translational stiffness during articulation inside the glenoid cavity, whereas during articulating outside the glenoid cavity this is influenced by the joint compression force and humeral radius of curvature. The glenoid radius of curvature influences the contact point and, in combination with the glenoid superior–inferior chord length, it also influences the constraintness angle, which influences the maximum allowable subluxation load to prevent a joint dislocation. This constraintness angle together with the joint conformity also influences maximum joint translations before articulation outside the glenoid cavity. Furthermore, the sign of the joint translational stiffness determines the stability of shoulder motion, which is stable and unstable if this stiffness is positive and negative, respectively.     

Fibroblast contractile force is independent of the stiffness which resists the contraction.

Using a device named the cell force monitor, the contractile force developed by fibroblasts has been studied by measuring the macroscopic contraction of porous collagen-glycosaminoglycan (GAG) matrices over the first 24 h following cell attachment. In this paper, the effect of a variation in the stiffness that resists matrix contraction by cells on the contractile force generated by the cells was determined. Data from these experiments revealed that the contractile force generated by the fibroblasts was independent of the stiffness of the resistance within the range tested (0.7-10.7 N/m). These results suggest that during the time when fibroblasts are attaching to and spreading on collagen-GAG matrices the contractile forces they generate are force limited, not displacement limited. Therefore, the cytoskeletal mechanism of force generation, corresponding with cell elongation, is capable of increasing the displacement of adhesion sites in order to develop the same level of force. Although a detailed understanding of how the passive mechanical signals provided by substrate materials affect cell processes is still unavailable, in vitro modeling of cell-mediated contraction continues to provide useful information.     

Dissociation between myocardial relaxation and diastolic stiffness in the stunned heart: its prevention by ischemic preconditioning

The effects of myocardial stunning and ischemic preconditioning on left-ventricular developed pressure and end-diastolic pressure (diastolic stiffness) as well as on coronary-perfusion pressure were examined in isolated isovolumic rabbit hearts. The isovolumic relaxation was evaluated, and the time constant of pressure decay during the isovolumic period was calculated. Our experimental protocol comprised: 1) myocardial stunning-global ischemia (15 min) followed by reperfusion (30 min); 2) myocardial stunning-global ischemia (20 min) followed by reperfusion (30 min); and 3) ischemic preconditioning — a single cycle of brief global ischemia and reperfusion (5 min each), before a second ischemic period, of 20-min duration. There was no effect upon systolic and diastolic parameters when 15 and 20 minutes of ischemia were evaluated. In both stunned groups the left ventricular developed pressure first recovered to near control values, but then stabilized at only 60% of the control values. Whereas the isovolumic relaxation time constant was increased after 5 min of reperfusion, and return to control values at late reperfusion, the end diastolic pressure remained elevated during the entire period. Values of dP/dV calculated at common pressure levels, were used as a second index of diastolic stiffness. They were increased after stunning, as also was the coronary perfusion pressure. When the heart was preconditioned with a single episode of ischemia, the systolic and diastolic alterations were completely abolished. We thus concluded that diastolic abnormalities incurred by myocardial stunning consist in both an increase in diastolic stiffness and an early impairment of isovolumic relaxation. The increase in stiffness cannot result from incomplete relaxation since these two parameters become temporally dissociated during the reperfusion period.     

Knee joint passive stiffness and moment in sagittal and frontal planes markedly increase with compression

Knee joints are subject to large compression forces in daily activities. Due to artefact moments and instability under large compression loads, biomechanical studies impose additional constraints to circumvent the compression position–dependency in response. To quantify the effect of compression on passive knee moment resistance and stiffness, two validated finite element models of the tibiofemoral (TF) joint, one refined with depth-dependent fibril-reinforced cartilage and the other less refined with homogeneous isotropic cartilage, are used. The unconstrained TF joint response in sagittal and frontal planes is investigated at different flexion angles (0°, 15°, 30° and 45°) up to 1800 N compression preloads. The compression is applied at a novel joint mechanical balance point (MBP) identified as a point at which the compression does not cause any coupled rotations in sagittal and frontal planes. The MBP of the unconstrained joint is located at the lateral plateau in small compressions and shifts medially towards the inter-compartmental area at larger compression forces. The compression force substantially increases the joint moment-bearing capacities and instantaneous angular rigidities in both frontal and sagittal planes. The varus–valgus laxities diminish with compression preloads despite concomitant substantial reductions in collateral ligament forces. While the angular rigidity would enhance the joint stability, the augmented passive moment resistance under compression preloads plays a role in supporting external moments and should as such be considered in the knee joint musculoskeletal models.     

Velocity-Based Planning of Rapid Elbow Movements Expands the Control Scheme of the Equilibrium Point Hypothesis

According to the equilibrium point hypothesis of voluntary motor control, control action of muscles is not explicitly computed, but rather arises as a consequence of interaction between moving equilibrium position, current kinematics and stiffness of the joint. This approach is attractive as it obviates the need to explicitly specify the forces controlling limb movements. However, many debatable aspects of this hypothesis remain in the manner of specification of the equilibrium point trajectory and muscle activation (or its stiffness), which elicits a restoring force toward the planned equilibrium trajectory. In this study, we expanded the framework of this hypothesis by assuming that the control system uses the velocity measure as the origin of subordinate variables scaling descending commands. The velocity command is translated into muscle control inputs by second order pattern generators, which yield reciprocal command and coactivation commands, and create alternating activation of the antagonistic muscles during movement and coactivation in the post-movement phase, respectively. The velocity command is also integrated to give a position command specifying a moving equilibrium point. This model is purely kinematics-dependent, since the descending commands needed to modulate the visco-elasticity of muscles are implicitly given by simple parametric specifications of the velocity command alone. The simulated movements of fast elbow single-joint movements corresponded well with measured data performed over a wide range of movement distances, in terms of both muscle excitations and kinematics. Our proposal on a synthesis for the equilibrium point approach and velocity command, may offer some insights into the control scheme of the single-joint arm movements.     

Verapamil Block of Large-Conductance Ca-Activated K Channels in Rat Aortic Myocytes

The effects of verapamil on the large conductance Ca-activated K (BK) channel from rat aortic smooth muscle cells were examined at the single channel level. Micromolar concentrations of verapamil produced a reversible flickering block of the BK channel activity. Kinetic analysis showed that verapamil decreased markedly the time constants of the open states, without any significant change in the time constants of the closed states. The appearance of an additional closed state — specifically, a nonconducting, open-blocked state — was also observed, whose time constant would reflect the mean residence time of verapamil on the channel. These observations are indicative of a state-dependent, open-channel block mechanism. Dedicated kinetic (group) analysis confirmed the state-dependent block exerted by verapamil. D600 (gallopamil), the methoxy derivative of verapamil, was also tested and found to exert a similar type of block, but with a higher affinity than verapamil. The permanently charged and membrane impermeant verapamil analogue D890 was used to address other important features of verapamil block, such as the sidedness of action and the location of the binding site on the channel protein. D890 induced a flickering block of BK channels similar to that observed with verapamil only when applied to the internal side of the membrane, indicating that D890 binds to a site accessible from the cytoplasmic side. Finally, the voltage dependence of D890 block was assessed. The experimental data fitted with a Langmuir equation incorporating the Woodhull model for charged blockers confirms that the D890-binding site is accessed from the internal mouth of the BK channel, and locates it approximately 40% of the membrane voltage drop along the permeation pathway. Received: 11 April 2000/Revised: 17 October 2000