Dick W. Slaaf

The endothelial glycocalyx: composition, functions, and visualization.

This review aims at presenting state-of-the-art knowledge on the composition and functions of the endothelial glycocalyx. The endothelial glycocalyx is a network of membrane-bound proteoglycans and glycoproteins, covering the endothelium luminally. Both endothelium- and plasma-derived soluble molecules integrate into this mesh. Over the past decade, insight has been gained into the role of the glycocalyx in vascular physiology and pathology, including mechanotransduction, hemostasis, signaling, and blood cell–vessel wall interactions. The contribution of the glycocalyx to diabetes, ischemia/reperfusion, and atherosclerosis is also reviewed. Experimental data from the micro- and macrocirculation alludes at a vasculoprotective role for the glycocalyx. Assessing this possible role of the endothelial glycocalyx requires reliable visualization of this delicate layer, which is a great challenge. An overview is given of the various ways in which the endothelial glycocalyx has been visualized up to now, including first data from two-photon microscopic imaging.

Foot salvage and improvement of microvascular blood flow as a result of epidural spinal cord electrical stimulation

Epidural spinal cord electrical stimulation has been suggested as an alternative treatment in patients with limb-threatening ischemia in whom vascular reconstructive surgery is not possible anymore. We studied the effects of epidural spinal cord electrical stimulation on microcirculatory blood flow in 20 patients with ischemic rest pain and ulcers. Angiography showed occluded crural arteries technically unsuitable for reconstructive surgery. Intravital capillary microscopy was used to assess capillary density and diameter and red blood cell velocity before and after a 1-minute period of arterial occlusion. After epidural spinal cord electrical stimulation 18 patients claimed immediate pain relief, which was confirmed by intravital capillary microscopy. Capillary density increased from 10 to 19/mm2 (p less than 0.001), red blood cell velocity increased from 0.088 to 0.496 mm/sec (p less than 0.001), and peak red blood cell velocity after arterial occlusion increased from 0.092 to 0.548 mm/sec (p less than 0.001). Two patients had no immediate pain relief; they did not show improvement of microcirculatory perfusion, and amputation was necessary. During the follow-up period (3 months to 3 years, mean 27 months), six other patients had recurrent ischemic pain, and amputation was necessary. In 12 patients pain relief continued, and ischemic ulcers healed; capillary microscopy confirmed improved microcirculatory blood flow. Microcirculatory parameters were significantly higher in respondents than in nonrespondents (p less than 0.001). Life-table analysis revealed a cumulative foot salvage of 80% and 56% after 1 and 2 years, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Reflex sympathetic dystrophy: evolution of microcirculatory disturbances in time.

&NA; Reflex sympathetic dystrophy (RSD) is a pain syndrome that is characterised by autonomic, motor and sensory disturbances. The syndrome has often been associated with sympathetic dysfunction. Therefore, we investigated whether there are disturbances in the sympathetic function of skin microcirculation in the various clinical stages of RSD. Laser Doppler flowmetry (LDF) was used to obtain information about total (mainly thermoregulatory) skin blood flow (TSBF), since blood flow in arteriovenous anastomoses and subpapillary plexus, which are richly innervated by sympathetic nerve endings, contributes predominantly to the flow signal as obtained by LDF. Capillary microscopy was used to appraise whether the trophic changes, as observed in RSD, result from an impaired nutritive skin blood flow (NSBF). Transcutaneous oximetry (TCPO2) was employed as a measure of the oxygenation of superficial skin layers. Skin temperature (ST) was also determined. Patients were divided into 3 clinical stages: stage I in case of a chronic warmth sensation, stage II in case of an intermittent warmth and cold sensation, and stage III in case of a chronic cold sensation. As compared to controls: (1) TSBF was increased (P < 0.05) at stage I and decreased at stages II (P < 0.05) and III (P < 0.001), (2) NSBF was decreased at stages II (P < 0.05) and III (P < 0.001), (3) TCPO2 was not impaired at any stage, (4) ST was increased (P < 0.01) at stage I and decreased (P < 0.05) at stage III. The present study is the first to report an increase of TSBF at stage I of RSD, which may be caused by a decrease in efferent sympathetic nerve impulses. At stages II and III both TSBF and NSBF were decreased which may reflect increased sensitivity of skin microvessels to (circulating) catecholamines.

Epidural spinal cord electrical stimulation improves microvascular blood flow in severe limb ischemia.

Epidural spinal cord electrical stimulation (ESES) was performed on 10 patients with severe limb ischemia due to atherosclerotic disease. Microcirculatory parameters were assessed before and after ESES. Bright field microscopy was used to assess capillary diameters and red blood cell (RBC) velocity in the dorsum of the foot. Fluorescein microscopy was used with intravenously injected sodium fluorescein to study capillary density and sodium fluorescein appearance time in the dorsum of the toe. The systolic ankle/arm pressure ratio and toe pressure measurements were used as macrocirculatory parameters. After ESES, clinical improvement was confirmed by intravital microscopy. Capillary density increased (p < 0.001), RBC velocity in capillaries already perfused before ESES increased from 0.054 mm/sec to 0.762 mm/sec (p < 0.001), and sodium fluorescein appearance time decreased from 72 to 45 seconds (p < 0.001). Capillary diameter did not change significantly so that the increase in RBC velocity may be interpreted as enhanced volume flow. Systolic ankle/arm pressure ratios and digital arterial pressure did not change significantly. The current results show that in patients with severe occlusive arterial disease of the lower limbs, ESES recruits capillaries not perfused in the control situation and enhances skin blood flow, improvements that may explain the beneficial clinical effects of ESES.

Two-Photon Microscopy for Imaging of the (Atherosclerotic) Vascular Wall: A Proof of Concept Study

Background: Understanding atherogenesis will benefit significantly from simultaneous imaging, both ex vivo and in vivo, of structural and functional information at the (sub)cellular level within intact arteries. Due to limited penetration depth and loss of resolution with depth, intravital and confocal fluorescence microscopy are not suitable to study (sub)cellular details in arteries with wall thicknesses above 50 µm. Methods: Using two-photon laser scanning microscopy (TPLSM), which combines 3D resolution and large penetration depth, we imaged mouse carotid arteries. Results: In thin slices, (sub)cellular structures identified using histochemical techniques could also be identified using TPLSM. Ex vivo, structural experiments on intact atherosclerotic arteries of Apo-E–/– mice demonstrated that in contrast to confocal or wide-field microscopy, TPLSM can be used to visualize (sub) cellular structural details of atherosclerotic plaques. In vivo, pilot experiments were carried out on healthy arteries of wild-type C57BL6 and atherosclerotic arteries of Apo-E–/– mice. As an example of functional measurements, we visualized fluorescently labeled leukocytes in vivo in the lumen. Additionally, detailed morphological information of vessel wall and atherosclerotic plaque was obtained after topical staining. Conclusions: Thus, TPLSM potentially allows combined functional and structural studies and can therefore be eminently suitable for investigating structure-function relationships at the cellular level in atherogenesis in the mouse.

NEUROGENIC INFLAMMATION IN AN ANIMAL MODEL OF NEUROPATHIC PAIN

Loose ligation of a rat sciatic nerve (chronic constriction injury (CCI) model) provokes signs and symptoms like those observed in reflex sympathetic dystrophy (RSD) patients. Primary afferent nociceptive C-fibers seem to be involved in an afferent orthodromic as well as in an efferent antidromic manner. In this study we hypothesize that consequent to development of antidromic impulses in C-nociceptive afferents, neuropeptides released from peripheral endings of these fibers, increase skin blood flow (SBF), vascular permeability, and tissue accumulation of polymorphonuclear leukocytes (PMNs). Collectively, these phenomena have been referred to as neurogenic inflammation. To investigate the presence of neurogenic inflammation in the CCI-model, we assessed skin blood flow (SBF) as well as the level of edema and accumulation of PMNs in muscle tissue obtained from the affected hindpaw. SBF was measured, by means of laser Doppler flowmetry, before ligation as well as at day 4 after ligation. At day 4, SBF measurements were performed before and after abolition of the capability of C-fibers to mediate a vasodilator response. To this end, capsaicin was applied perineurally. Increased vascular permeability was inferred from the level of edema of muscle tissue as determined by assessment of wet/dry weight ratios of muscle biopsies. PMN accumulation was investigated by enzymatic detection of myeloperoxidase (MPO) activity in muscle biopsies. Compared with preligation values, at day 4 SBF was increased more than twofold (p < 0.05). The latter response was annihilated by capsaicin application. Compared with sham operated controls, wet/dry ratios were higher in the ligated animals (1.104 vs. 1.068; p < 0.05). Likewise, when compared with sham operated controls, MPO activity was found to be increased in the ligated hindpaw (Optic Density 0.15 vs. 0.89; p < 0.001). In conclusion, the findings of this study indicate that loose ligation of a sciatic nerve induces an inflammatory response in the ipsilateral hindpaw, which most likely is mediated by release of neuropeptides from the peripheral endings of antidromically acting nociceptive C-fibres.

Muscle blood flow disturbances produced by simultaneously elevated venous and total muscle tissue pressure

Abstract The effect of simultaneously increased mean venous ( P v ) and total intramuscular pressure ( P im ) on arteriolar diameter ( D a ) and capillary blood flow ( q c ) was investigated in rabbit tenuissimus muscle, using intravital microscopy. P v and P im were increased simultaneously by placing the hind paw of the rabbit in a sealed box and elevating the pressure in this box ( P b ) with a mixture of 5% CO 2 and 95% N 2 . The mean arterial pressure outside the box ( P a ob ), was also measured. Initial D a varied between 7.6 and 45 μm ( x = 18 μ m ). In all but one measurement D a increased when P a ob - P b was lowered. The value of P a ob - P b at which q c in the tenuissimus muscle stopped was 24.3 ± 1.26 mm Hg ( x ± SEM ). When subsequently P b was lowered, muscle q c started at a value of P a ob - P b of 30.9 ± 1.56 mm Hg ( x ± SEM ), which was significantly higher than the value at which q c stopped. These data indicate that the arterioles are not the limiting factor in the muscle blood flow disturbances due to a simultaneous increase in P im and P v as in compartmental syndromes.

Threshold selection using a minimal histogram entropy difference

A new gray-level threshold selection method for image segmentation is presented. It is based on minimizing the difference between entropies of the object and the background distributions of the gray-level histogram. The proposed method is similar to the maximum entropy method proposed by Kapur et al. (1985), however, the new method provided a good threshold value in many instances where the previous method did not. The effectiveness of our method is demonstrated by its performance on videomicroscopic images of the rat lung. Extension of the method to higher order probability density functions is described.

Imaging Collagen in Intact Viable Healthy and Atherosclerotic Arteries Using Fluorescently Labeled CNA35 and Two-Photon Laser Scanning Microscopy

We evaluated CNA35 as a collagen marker in healthy and atherosclerotic arteries of mice after both ex vivo and in vivo administration and as a molecular imaging agent for the detection of atherosclerosis. CNA35 conjugated with fluorescent Oregon Green 488 (CNA35/OG488) was administered ex vivo to mounted viable muscular (uterine), elastic (carotid), and atherosclerotic (carotid) arteries and fresh arterial rings. Two-photon microscopy was used for imaging. CNA35/OG488 labeling in healthy elastic arteries was compared with collagen type I, III, and IV antibody labeling in histologic sections. For in vivo labeling experiments, CNA35/OG488 was injected intravenously in C57BL6/J and apolipoprotein E−/− mice. Ex vivo CNA35/OG488 strongly labeled collagen in the tunica adventitia, media, and intima of muscular arteries. In healthy elastic arteries, tunica adventitia was strongly labeled, but labeling in tunica media and intima was prevented by endothelium and elastic laminae. Histology confirmed the affinity of CNA35 for type I, III, and IV collagen in arteries. Strong CNA35/OG488 labeling was found in atherosclerotic plaques. In vivo applied CNA35/OG488 minimally labeled the tunica intima of healthy carotid arteries. Atherosclerotic plaques in apolipoprotein E−/− mice exhibited large uptake. CNA35/OG488 imaging in organs revealed endothelium as a limiting barrier for in vivo uptake. CNA35/OG488 is a good molecular imaging agent for atherosclerosis.

Arteriolar vasomotion and arterial pressure reduction in rabbit tenuissimus muscle

Spontaneous arteriolar vasomotion and its relation to arterial pressure reduction was studied in the rabbit tenuissimus muscle using intravital microscopy. Vasomotion was observed in all transverse arterioles and their first-order side branches. Vasomotion frequency ranged from 5 to 32 cycles per minute (median: 25 cpm). The relative vasomotion amplitude in transverse arterioles varied from 0.06 to 0.44 (median: 0.22). Vasomotion was generally of the on-off type in first-order side branches. A gradual reduction in arterial pressure as achieved by aortic occlusion resulted in an abrupt disappearance of vasomotion. Vasomotion disappeared between 19 and 59 mm Hg (median: 30.4 mm Hg). No differences were found between the pressures at which vasomotion ceased in transverse arterioles and first-order side branches. With a further reduction of arterial pressure transverse arterioles attained a maximal diameter of 98-265% (median: 119%) of the maximal diameter during vasomotion. After release of occlusion vasomotion reappeared much earlier in the first-order side branches than in their feeding transverse arterioles. It is concluded that although vasomotion influences capillary perfusion, it does not play a major role in the adaptation of vascular resistance following a reduction in arterial pressure.