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Amelioration of hyperglycemic and hyperosmotic induced vascular dysfunction by in vivo inhibition of protein kinase C and p38 MAP kinase pathway in the rat mesenteric microcirculation.

BACKGROUND: Recently, we demonstrated in vivo effects of acutely induced hyperglycemia, diabetes and mannitol infusions on rat mesenteric microcirculation concerning leukocyte-endothelial-cell interaction (Schäffler et al. EJCI 28: 886-893, 1998). DESIGN: In this study we have investigated the possible involvement of the protein kinase C (PKC) and p38 MAP kinase cascade as signal transducing elements during hyperglycemic and osmotic stress in an in vivo rat model. RESULTS: Acutely induced hyperglycemia resulted in a significant increase in leukocyte adhesion. This effect could be mimicked by mannitol. Both PKC and p38 MAP kinase were involved in the effects mediated by glucose and mannitol. Acutely induced hyperglycemia resulted in a significant increase in leukocyte emigration. This effect could be imitated by mannitol. However, PKC and p38 MAP kinase were only involved under osmotic stimulation. The hyperglycemia-induced reduction in leukocyte rolling velocity seemed to be a glucose-specific effect, since mannitol did not influence leukocyte rolling velocity. This glucose effect on leukocyte rolling velocity was mediated by an activation of the PKC/p38 MAP kinase cascade. Both hyperglycemia and osmotic stimuli alone were able to reduce venular shear rate without recruitment of the p38 MAP kinase cascade. The observed reduction of shear rate seems to be mediated only by the osmotic effects of glucose via activation of the PKC system. CONCLUSION: The observed effects of glucose on adhesion, emigration and shear rate are due to osmotic effects. The PKC/MAP kinase cascade is involved as a signal transducing component. The reduction of leukocyte rolling velocity is a glucose-specific effect, mediated by the activation of both the PKC and the p38 MAP kinase cascade. Venular shear rate and leukocyte emigration can be influenced by glucose and mannitol due to different regulation mechanisms. It is concluded, that isoform-specific inhibitors of PKC and specific MAP kinase inhibitors represent a potential drug target for preventing microvascular dysfunction in diabetes.

Animals↗

Microcirculation: its significance in clinical and molecular medicine.

Microcirculation represents the smallest functional unit of the cardiovascular system, where the interaction between blood and tissue creates the environment necessary for cell function. Analysis of physiology and pathophysiology of this system gives a unique perspective to the disease process, and provides the link between clinical and molecular medicine. The present status and future directions of this medical and scientific frontier were assessed and projected by experts in the field at a meeting in Italy in 1995, and the conclusions are presented in this article.

Blood Gas Monitoring, Transcutaneous↗

Multiple mechanisms of early hyperglycaemic injury of the rat intestinal microcirculation.

1. Hyperglycaemia in the vast majority of humans with diabetes mellitus is the end result of profound insulin resistance secondary to obesity. For patients in treatment, hyperglycaemia is usually not sustained but, rather, occurs intermittently. In in vivo studies of the rat intestinal microcirculation, endothelial impairment occurs within 30 min at D-glucose concentrations > or = 300 mg/dL. Endothelial-dependent dilation to acetylcholine and constriction to noradrenaline is impaired. Vasodilation to exogenous nitric oxide (NO) remains normal. 2. When initiated before hyperglycaemia, suppression of oxygen radicals by both scavenging and pretreatment with cyclo-oxygenase blockade to prevent oxygen radical formation minimized endothelial impairments during hyperglycaemia. Neither treatment was effective in restoring endothelial function once it was damaged by hyperglycaemia. 3. A mechanism that may initiate the arachidonic acid- oxygen radical process is activation of specific isoforms of protein kinase C (PKC). De novo formation of diacylglycerol during hyperglycaemia activates PKC. Blockade of the beta II PKC isoform with LY-333531 prior to hyperglycaemia protected NO formation within the arteriolar wall, as judged with NO-sensitive microelectrodes. Furthermore, once suppression of endothelial dilation was present in untreated animals, PKC blockade could substantially restore endothelial-dependent dilation. 4. These results indicate that acute hyperglycaemia is far from benign and, in the rat, causes rapid endothelial impairment. Both oxygen radical scavenging and cyclo-oxygenase blockade prior to bouts of hyperglycaemia minimize endothelial impairment with limited side effects. Blockade of specific PKC isozymes protects endothelial function both as a pre- or post-treatment during moderately severe hyperglycaemia.

Animals↗

Influence of platelet-derived growth factor on microcirculation during normal and impaired wound healing.

The aim of the current study was to evaluate the influence of platelet-derived growth factor (PDGF) on skin microcirculation during normal and impaired wound healing. Secondary healing wounds were created on the ears of hairless mice and treated once with 3 microg of PDGF-BB immediately after wound creation. Intravital fluorescence microscopy was used to quantify reepithelialization, revascularization, vessel diameters, vascular permeability, and leukocyte-endothelium interactions up to 24 days after wound creation. Microvascular perfusion was assessed by laser Doppler flowmetry. Wound healing was studied in normal (n = 15) and ischemic skin tissue (n = 15) as well as in mice (n = 17) rendered hyperglycemic by an intravenous injection of streptozotocin 7 days prior to wound creation. Treatment with PDGF accelerated reepithelialization and reduced the time for complete wound closure in ischemic skin from 14.9 +/- 2.5 (control) to 12.3 +/- 1.8 days (p < 0.03), and in hyperglycemic animals from 15.0 +/- 2.4 (control) to 12.0 +/- 3.0 days (p < 0.04). Revascularization of these wounds was also significantly enhanced after PDFG application. No other parameters were influenced by the treatment. Normal wound healing was not affected. This study confirms the positive influence of PDGF on wound healing under pathophysiological conditions. The effects in this model seem to be primarily due to the mitogenic potency of PDGF on keratinocytes and endothelial cells. A significant effect on leukocyte activation during the inflammatory process was not observed.

Animals↗

Changes in oxidative stress and microcirculation by low-density lipoprotein apheresis.

Low-density lipoprotein apheresis (LDLA) leads to an improvement of microcirculation during the very early stages of treatment, and continued treatment may produce antiatherogenic effects in patients with peripheral arterial disease (PAD). Suppression of oxidative stress, improvement of endothelial functions and alteration in the action of vasoactive compounds may occur with the improvement of the rheological property of blood as a result of aggressive removal of atherogenic factors including LDL, possibly resulting in the suppression of development of atherosclerosis. As these effects of LDLA may ameliorate not only PAD but also ischemia in other organs, it is suggested that repeated LDLA prevents the progression of atherosclerotic diseases and probably improves the long-term prognosis of patients with PAD.

Arterial Occlusive Diseases↗

Effect of ischemic preconditioning on hepatic microcirculation and function in a rat model of ischemia reperfusion injury.

Ischemic preconditioning (IPC) may protect the liver from ischemia reperfusion injury by nitric oxide formation. This study has investigated the effect of ischemic preconditioning on hepatic microcirculation (HM), and the relationship between nitric oxide metabolism and HM in preconditioning. Rats were allocated to 5 groups: 1. sham laparotomy; 2. 45 minutes lobar ischemia followed by 2-hour reperfusion (IR); 3. IPC with 5 minutes ischemia and 10 minutes reperfusion before IR; 4. L-arginine before IR; and 5. L-NAME + IPC before IR. HM was monitored by laser Doppler flowmeter. Liver transaminases, adenosine triphosphate, nitrites + nitrates, and guanosine 3'5'-cyclic monophosphate (cGMP) were measured. Nitric oxide synthase (NOS) distribution was studied using nicotinamide adeninine dinucleotide phosphate (NADPH) diaphorase histochemistry. At the end of reperfusion phase, in the IR group, flow in the HM recovered partially to 25.8% of baseline (P < .05 versus sham), whereas IPC improved HM to 49.5% of baseline (P < .01 versus IR). With L-arginine treatment, HM was 31.6% of baseline (NS versus IR), showing no attenuation of liver injury. In the preconditioned group treated with L-NAME, HM declined to 10.2% of baseline, suggesting not only a blockade of the preconditioning effect, but also an exacerbated liver injury. Hepatocellular injury was reduced by IPC, and L-arginine and was increased by NO inhibition with L-NAME. IPC also increased nitrate + nitrate (NOx) and cGMP concentrations. NOS detected by NADPH diaphorase staining was associated with hepatocytes and vascular endothelium, and was induced by IPC. IPC induced NOS and attenuated HM impairment and hepatocellular injury. These data strongly suggest a role for nitric oxide in IPC.

Adenosine Triphosphate↗

Barium sulphate and soft-tissue radiology: allying the old and the new for the investigation of animal cutaneous microcirculation.

The study of microcirculation using angiography is essential to the advancement of flap and angiogenesis research in plastic surgery. Until the mid-1980s, barium sulphate was the most commonly used contrast material, although it did not provide optimal visualisation of the vascular tree. In 1986, a new technique using lead oxide was proposed, which permitted very high-quality imaging and rapidly became the technique of choice, despite its high toxicity. We reconsider the former technique of barium-sulphate injection and combine it with soft-tissue radiology using mammographic film to achieve a radiological definition similar to that obtained with lead oxide, and discuss the advantages and disadvantages of the two methods. We conclude that barium sulphate and the use of mammographic film is an accurate, simple and non-toxic method of analysing the cutaneous circulation in small animals.

Angiography↗

Effect of a nitric oxide donor on microcirculation of acutely denervated skeletal muscle during reperfusion.

The authors have shown that exogenous nitric oxide (NO) protects innervated skeletal muscle against reperfusion injury. This study further evaluated the effects of exogenous NO donor on denervated skeletal muscle. Forty-eight denervated rat cremaster muscles underwent 3 hr of ischemia, followed by 90 min of reperfusion, and received systemic infusion of 100 nmol/min s-nitroso-n-acetylcysteine (SNAC) or an equal amount of phosphate-buffered saline (PBS). Results showed that the average diameter in 10 to 20 microm arterioles was between 107 percent and 123 percent of baseline in the SNAC group, and between 55 percent and 84 percent in the PBS group during 90 min of reperfusion. These values in 21 to 40 microm and 41 to 70 microm arteries were between 100 percent and 110 percent in the SNAC group, and between 70 percent and 90 percent in the PBS group from 20 to 90 min of reperfusion. Compared to the PBS group, the SNAC group had a statistically significantly greater vessel diameter in both 10 to 20 microm (p<0.001) and 21 to 40 microm arterioles (p<0.01) during 90 min of reperfusion, and in 41 to 70 microm arteries (p<0.02) from 20 to 90 min of reperfusion. The overall blood flow of the muscle in the SNAC group increased from 37 percent of baseline at 10 min to 108 percent at 40 min of reperfusion, and remained above baseline thereafter. In contrast, this value in the PBS group was only between 27 percent and 68 percent of baseline during 90 min of reperfusion. The blood flow was statistically significantly (p<0.03) greater in the SNAC group than in the PBS group from 40 to 90 min of reperfusion. Among the conclusions were: (1) NO donor SNAC improves the microcirculation of denervated skeletal muscle during early reperfusion; and (2) this protection against reperfusion injury is independent of innervation in skeletal muscle.

Acetylcysteine↗

Examining microcirculation improves the angiosome theory in explaining the delay phenomenon in a rabbit model.

Topographic and temporal changes in circulation were studied with laser Doppler flowmetry in rabbit dorsal skin flaps comprised of three vascular territories. The reduced caliber vessels along the dorsal median line, rather than the vessels interconnecting the thoracodorsal and deep circumflex iliac vascular trees, behaved as true choke vessels. The authors found an increased proximal circulation lasting for 48 to 72 hr. This coincided with the reported rapid phase of choke vessel dilatation. The increase in distal circulation began after postoperative day 3, coinciding with the resolution of proximal hyperemia but not with the earlier prominent vasodilatation. Secondary elevation of the flaps at 3 weeks did not disturb the homogeneous distribution of circulation and did not cause a hyperemic response at postoperative 24 hr. Based on these findings, the authors postulate that the same mechanisms may underlie the rapid choke vessel dilatation and microvascular dilatation observed at 48 to 72 hr postoperatively. Surgical delay may work not only by diminishing the resistance to blood flow along the flap, but also by diminishing the steal effect of increased proximal microcirculation, which characterizes this period, over the distal circulation.

Animals↗

Influence of transendothelial mechanisms on microcirculation: consequences for reperfusion injury after free flap transfer. Previous, current, and future aspects.

The success of a free microvascular tissue transfer is based on a sufficient microanastomosis which meets the following requirements: a pedicle placed without kinking or twisting, good drainage, a well-defined recipient vessel, integrity of the endothelium, and duration of ischemia. The extent of skin and muscle necrosis increases significantly with increases in ischemia time. Reperfusion of ischemic tissue results in local and systemic damage associated with the release of oxygen free radicals, polymorphonuclear leucocytes, and such endothelial hormones as endothelin-1, EDRF (endothelial-derived relaxing factor), thromboxane, complement, and cytokines. Ischemia-reperfusion disrupts the delicate balance that maintains homeostasis in the microcirculation. This review discusses the clinical and therapeutic aspects of such injury, concentrating on perioperative management in free flap transfer. It points out the possible influence of endothelin-1 on vasospasm at the site of anastomosis, and emphasizes the importance of the endothelium as a highly dynamic network. Finally, future diagnostic and therapeutical aspects are discussed.

Anastomosis, Surgical↗

Dose-dependent response to IFN-gamma in muscle flap microcirculation.

In this study, the authors attempted to determine the effects of intraarterial administration of various doses of Interferon-gamma (IFN-gamma) on microcirculation in a rat muscle flap model. In Group 1 (control), 0.6 ml vehicle solution-PBS-BSA, in Group 2 0.6 ml IFN-gamma (25 ng/ml), in Group 3 0.6 ml IFN-gamma (50 g/ml), in Group 4 0.6 ml IFN-gamma (100 g/ml), were injected. The diameter of the cremaster arterioles and venules, red blood cell velocities, the number of rolling leukocytes and lymphocytes, sticking leukocytes and lymphocytes, capillary perfusion, and endothelial edema index were evaluated. Deterioration of flow hemodynamics was confirmed by a significant decrease in flow velocity in the main artery (A1) (47 percent in Group 3 and 65 percent in Group 4). All dosages of IFN-gamma caused a statistically significant decrease in rolling leukocytes, but this effect was more obvious in the 25 ng/ml group. Injury to the vascular endothelium was confirmed by a two-fold increase in transmigrating leukocytes in the 100 ng/ml group. This was accompanied by 60 percent and 75 percent drops in capillary perfusion, and by 12 percent and 24 percent drops in the endothelial edema index in Groups 3 and 4, respectively. The results indicate that direct intraarterial administration of IFN-gamma in doses higher than 25 ng/ml may be toxic to muscle flaps.

Animals↗

Circulating mechanism of the "pure" venous flap: direct observation of microcirculation.

The circulating mechanism of the "pure" venous flap was studied via venography and direct observation of the microcirculation through transparent ear chambers and ear edges in a rabbit model. The flap, without arterial inflow, survived with normal hair growth and wound healing. Renewed blood circulation occurred 3 to 5 hr after transfer There were two typical flow patterns in the venules: one was a shaking movement with high frequency and short range; the other was a pendulum-like movement with low frequency and long range. The former was related to heart rate, while the later was not. The pendulum-like movement was the main flow responsible for perfusion of capillaries. Blood flow in the capillaries occurred 1 to 2 hr later than that in the venules. The pendulum-like flow perfused the capillaries in two ways: one was in the direction of venules to capillaries and then back to venules along the same route; the other was from venules to arterioles through arteriovenous shunts and then to capillaries. The first type of perfusion was gradually replaced by the second 2 days after transfer. There were extensive venous plexus flows in the flap, which lasted more than 4 weeks after transfer. Revascularized vessels ultimately supplied blood to the flap. The dynamic energy for the movement of blood at an early post-transfer stage was probably due to spontaneous venomotion.

Animals↗

The effect of endothelin-1 on muscle microcirculation and its attenuation by verapamil.

The vascular endothelium plays an important role in the regulation of vascular tone, by producing both vasodilating and vasoconstricting mediators. Using intravital videomicroscopy, this study examined the in vivo responses of the rat cremaster muscle microcirculation to topical application of endothelin-1 (ET-1), as well as the effect of verapamil pretreatment on these responses. ET-1 produced a potent and persistent vasoconstriction in arteries and veins which lasted for 150 min. The contractile response to ET-1 was significantly more prolonged in arterioles (11 to 30 microns) than in small arteries (40 to 70 microns). Pretreatment of the muscle with verapamil did not inhibit the initial vasoconstrictive action of ET-1, but produced a significant (p < 0.0001) reduction in the recovery time required for vessel relaxation in both small arteries and arterioles. The results suggest that Ca2+ channels contribute to the prolonged vasoconstriction induced in microvessels by ET-1. Because this prolonged vasoconstriction may be responsible for the "no-reflow" phenomenon which occurs in microsurgical procedures, therapy with a calcium antagonist may be useful in attenuating its duration.

Analysis of Variance↗

Study of microcirculation of rat femoral nerve and development of a new vascularized nerve graft model.

This study was designed to investigate the microcirculation of the rat femoral nerve and to develop a vascularized nerve graft (VNG) model which is reliable, consistent, and simple. The rat was chosen for considerations of cost and simplicity of the model. One and 2 cm long femoral nerve segments were transected proximally and distally and left attached to the femoral artery and vein in all animals. Experimental groups included the following procedures: 1) the femoral artery and vein were ligated distally and an arteriovenous (AV) fistula just proximal to the ligature was constructed; 2) distal ligation only of the femoral artery and vein was done; 3) ligation of the femoral artery and vein was done both distally and proximally; 4) control: no ligation or construction of an AV fistula were done. Neurovascular segments in all groups were wrapped with a thin silicone sheath to prevent neurovascularization from the surrounding tissues. At seven days postoperatively, viability of the segments was assessed by fluorescein dye and microangiography studies, followed by histologic evaluation. Results showed that a 1- or 2-cm long femoral nerve segment, when left attached to distally ligated femoral vessels and an intact neurovascular sheath, is a simple design to reproduce and can serve reliably as a VNG model in the rat.

Animals↗

[Microcirculation of the nasal mucosa during use of balloon tamponade].

BACKGROUND: Nasal packings are commonly accepted in the treatment of severe epistaxis. Cuffed catheters are known to cause damage to the nasal mucosa most likely by interfering with tissue perfusion. In this study the effect of different pressure levels on local perfusion of septal mucosa is investigated. METHOD: In 15 healthy subjects the blood flow in septal mucosa was measured by laser doppler flowmetry by positioning a cuffed epistaxis catheter into the nasal cavity with a laser probe attached to it. Increasing pressure was administered by injecting saline solution while continuously recording intraluminal pressure, perfusion, and filling volume. The local pressure affecting the septal mucosa at the moment of stalling perfusion was determined by subtracting the extranasal cuff pressure from the current intranasal cuff pressure at same inflation volumes. RESULTS: Microcirculation of the septal mucosa stopped when the local pressure exceeded a value of Pmean = 42 mmHg. Individual variations (n = 15) were small (s = 9 mmHg). The intraluminal cuff pressure was measured to be about ten times higher due to the retraction force of the cuff. Spontaneous oscillations of the blood flow were reduced with increasing pressure to the blood vessels. Filling volumes up to 3.2 ml were sufficient to stop perfusion. CONCLUSIONS: Cuffed nasal packings stop the blood flow in nasal mucosa even at low local pressures. Depending on the material characteristics of different cuffs the pressure to dilate the cuff may, however, be several times higher than the actual local pressure. This effect may cause problems in the proper use of cuffed catheters. Laser doppler flowmetry proved to be helpful in determining reproducible perfusion values.

Adolescent↗

[Various aspects of the existing relationship between retinal neuronal function, oxidative metabolism and the constant O2 supply by the microcirculation].

Retinal excitation implies transmembrane ion movement in the retinal neurones. In order to maintain excitability, the neurones utilize energy derived from the hydrolysis of ATP. ATP is produced in the mitochondria, which consume O2 and carbohydrates for this purpose. Thus, O2 consumption is essential for maintaining ATP in the retinal neurons and, therefore, for maintaining excitability. O2 is delivered to the retinal mitochondria by the microcirculation, where variations of the blood flow cause dramatic fluctuations in the local PO2 in the tissue. The retinal blood flow can be impaired by experimental changes in the pH of the glial cells surrounding the arterioles. According to a hypothesis, intraglial pH modulates the release of a mediator (prostaglandins) which in turn acts on the smooth musculature of the arteriole wall and thus controls vascular motility.

Adenosine Triphosphate↗

[Cardiac function and the skin microcirculation in acromegaly].

The microcirculation was measured by nail-fold capillary video microscopy in 21 patients (12 men, 9 women; mean age 54.7 [29-74] years) with acromegaly. Levels of growth hormone (12.0-71.7 microU/ml) and of somatomedin C (2.4-10.5 IU/ml) were elevated in 10 patients, despite preceding treatment. Eleven patients had an increase in myocardial thickness and nine had impairment of left ventricular function, although only slight in most. Left ventricular hypertrophy was demonstrable even in the absence of hypertension. No patient had evidence of coronary heart disease. Nail-fold capillary video microscopy (capillary density, torque index, reactive hyperaemia, epidermal blood flow) failed to distinguish between successfully treated patients and those with persistently elevated growth hormone concentrations or disease duration of over 5 years. There was no evidence of inadequate capillary blood flow as a cause of abnormal function in hypertrophied organs.

Acromegaly↗

Blunted vasodilatory responses in the cutaneous microcirculation of cigarette smokers.

OBJECTIVE: The present study was undertaken to investigate whether chronic smoking alters the vasodilatory capacity in the microcirculation. METHODS: We assessed, in habitual cigarette smokers, the forearm skin blood flow response to iontophoretically applied acetylcholine (endothelium-dependent vasodilator) and sodium nitroprusside. Postocclusive forearm reactive hyperemia was also explored. The skin blood flow responses were determined with a laser-Doppler flowmeter that allowed us to scan the surface after acetylcholine and sodium nitroprusside application. RESULTS: Forty healthy male volunteers were included. Twenty subjects were aged 20 to 35 years and 20 subjects were aged 40 to 60 years. We studied the following 4 groups of 10 subjects each: group 1, younger smokers (mean of 7.2 pack-years); group 2, older smokers (mean of 30 pack-years); group 3, younger nonsmokers; and group 4, older nonsmokers. On the day of the experiment, the subjects of groups 1 and 2 were asked to smoke at least 15 cigarettes from the morning until the afternoon, when the experiments were performed. No significant difference in the studied parameters was observed between younger smokers and younger nonsmokers. In older smokers, however, both acetylcholine- and sodium nitroprusside-induced skin blood flow increases were significantly attenuated in comparison with nonsmokers. Heart rate also was significantly blunted by long-term cigarette smoking in older subjects. CONCLUSION: These data show that the vasodilatory response of the skin microvasculature is impaired in subjects who have smoked cigarettes for many years. This abnormality involves both endothelium-dependent and endothelium-independent responses.

Adult↗