Search PubMed⌕ Search

Biomedical subjects

D W Slaaf

Publications and source records attributed to D W Slaaf.

At least 19 recordsLinked to original sources
PubMed2002

Hypoxia induces aortic hypertrophic growth, left ventricular dysfunction, and sympathetic hyperinnervation of peripheral arteries in the chick embryo.

BACKGROUND: Low birth weight is associated with an increased incidence of cardiovascular diseases, including hypertension, later in life. This suggests that antenatal insults program for fetal adaptations of the circulatory system. In the present study, we evaluated the effects of mild hypoxia on cardiac function, blood pressure control, and arterial structure and function in near-term chick embryos. METHODS AND RESULTS: Chick embryos were incubated under normoxic (21% O2) or hypoxic (15% O2) conditions and evaluated at incubation day 19 by use of histological techniques, isolated heart preparations, and in vivo measurements of sympathetic arterial tone and systemic hemodynamics. Chronic hypoxia caused a 33% increase in mortality and an 11% reduction in body weight in surviving embryos. The lumen of the ascending aorta in hypoxic embryos was 23% smaller. Left ventricular systolic pressure was 22% lower, and heart weight/body weight ratio was 14% higher. In resistance arteries of hypoxic embryos, in vivo baseline tone was 23% higher, norepinephrine sensitivity was similar, and norepinephrine release from sympathetic nerves increased 2-fold, indicating sympathetic hyperinnervation. Mean arterial pressure and heart rate were similar under resting conditions, but chronically hypoxic embryos failed to maintain blood pressure during acute stress. CONCLUSIONS: This study indicates that mild hypoxia during embryonic development induces alterations in cardiac and vascular function and structure and affects hemodynamic regulation. These findings reveal that antenatal insults have profound effects on the control and design of the circulatory system that are already established at birth and may program for hypertension and heart failure at a later age.

Animals↗
PubMed2002

Effects of experimental lower-limb ischaemia-reperfusion injury on the mesenteric microcirculation.

BACKGROUND: Ischaemia-reperfusion (I-R) of the leg is associated with functional and structural changes in the intestine. This study assessed whether acute hind-limb I-R in rats induced a reduction in perfusion and/or signs of an inflammatory response in the intestine. METHODS: Rats were subjected to 2 h of unilateral hind-limb ischaemia followed by 2 h of reperfusion (I-R group, n = 9) or to a sham procedure (control group, n = 9). Mesenteric microvascular diameters, red blood cell velocity, blood flow and leucocyte-vessel wall interactions during reperfusion were measured using intravital microscopy. RESULTS: Blood pressure and heart rate decreased from 30 min of reperfusion onwards in the I-R group compared with controls. From 15 min after the start of reperfusion, mesenteric arteriolar and venular red blood cell velocity and blood flow decreased by 40-50 per cent. Microvascular diameters and leucocyte-vessel wall interactions did not change. CONCLUSION: Restoration of blood flow to an acutely ischaemic hind limb led to a significant decline in the splanchnic microcirculatory blood flow. There were, however, no signs of an early inflammatory response in the gut.

Animals↗
PubMed2002

Capillaries and flow redistribution play an important role in muscle blood flow reserve capacity.

Perfusion of skeletal muscle varies considerably during rest, exercise, or when arteries are occluded. The extent that a muscle can adapt to changes in flow demand is often expressed as the ratio of the highest inducible flow and control flow, the microvascular blood flow reserve capacity (MBFRC). However, perfusion of the nutritive capillaries of skeletal muscle may not only be improved by the increase in blood flow proportional to the increase in arterial flow, but also by diverting originally shunted flow towards the muscle proper. Consequently, MBFRC is not a good measure of capillary flow reserve, unless the assessed flow in both conditions is purely nutritive in nature. Therefore, in critical conditions, flow measurements in large vessels are not appropriate to assess MBFRC. In muscle, capillaries are compliant, i.e., with varying transmural pressure capillary diameter varies. During high perfusion states, when capillary transmural pressure is increased, capillary compliance results in increased capillary diameter and, hence, in reduced resistance and increased exchange surface area. This results in improved perfusion and enlarged capillary exchange surface area. In low perfusion states, capillary diameter is reduced. This augments the detrimental effects of the low perfusion status. Operative restoration of perfusion pressure not only increases the driving force for perfusion, but also leads to (passive) dilatation of the capillary bed and an extra reduction in resistance to flow, and, hence, a disproportional increase in flow.

Blood Flow Velocity↗
PubMed2001

Microcirculatory effects of experimental acute limb ischaemia-reperfusion.

BACKGROUND: The object of this study was to develop an animal model in which changes in microvascular haemodynamics and leucocyte-vessel wall interactions due to acute limb ischaemia-reperfusion (I/R) can be measured in the skin. Furthermore, it was investigated whether these changes are related to local muscle injury. METHODS: Male Lewis rats were subjected to unilateral limb ischaemia for 1 h (n = 8) or 2 h (n = 8) by cuff inflation, or to a sham protocol (n = 6). Intravital video microscopic measurements of leucocyte-vessel wall interactions, venular diameter, red blood cell velocity and reduced velocity (which is proportional to wall shear rate) were performed in skin venules before ischaemia and at 0.5, 1, 2, 3 and 4 h after the start of reperfusion. Oedema and leucocyte infiltration of ischaemic/reperfused skeletal muscle were quantified histologically. RESULTS: In skin venules, both 1 and 2 h of ischaemia induced a significant increase in leucocyte rolling (six and five times baseline, respectively; P < 0.05) and adherence during reperfusion (eight and four times baseline; P < 0.05). No significant increase in muscular leucocyte infiltration was detected. After an initial hyperaemic response of 180 per cent of baseline values (P < 0.05), blood flow decreased to about 60 per cent after 4 h of reperfusion in skin venules of both experimental groups. I/R induced tibial muscle oedema, the severity of which depended on the ischaemic interval (wet to dry ratio: control, 4.0; 1 h, 4.5 (P not significant); 2 h, 5.8 (P < 0.05)). CONCLUSION: A non-invasive animal model was developed that enables investigation of the consequences of acute limb I/R.

Animals↗
PubMed2001

Endogenous nitric oxide and prostaglandins synergistically counteract thromboembolism in arterioles but not in venules.

It has been shown that NO and prostacyclin (prostaglandin I(2)) from cultured endothelium synergistically inhibit blood platelet aggregation in vitro. However, it is unknown whether this synergism is also effective in the inhibition of thromboembolism in vivo and, if it is, whether it differs between vessel types. Therefore, the effect of endogenous NO and prostacyclin, in combination or alone, on thromboembolism was studied in an in vivo model. Thromboembolism was induced by micropipette puncture of rabbit mesenteric arterioles and venules (diameter 18 to 40 micrometer). In addition, the influence of wall shear rate was analyzed. In arterioles, the combined inhibition of NO synthase (N(G)-nitro-L-arginine [L-NA] 0.1 mmol/L; local superfusion) and of cyclooxygenase (aspirin [ASA] 100 mg/kg IV) resulted in a pronounced, significant prolongation of embolization duration (median >600 seconds) compared with control (median 153 seconds) or treatment with either L-NA (234 seconds) or ASA (314 seconds). This combined effect of L-NA+ASA was greater than the sum of the individual effects of L-NA and ASA. In contrast, in venules L-NA+ASA had no additional effect on embolization duration (209 seconds) compared with the effect of L-NA alone (230 seconds); ASA alone had no effect (122 seconds; control 72 seconds). Interestingly, only in the L-NA+ASA arterioles did embolization correlate positively with wall shear rate (r(s)=0.687; P=0.028). In conclusion, this study indicates that in arterioles, but not in venules, endogenous NO and prostaglandins synergistically counteract ongoing thromboembolism after vessel wall injury and that the combination of endogenous NO and prostaglandins appears to protect against enhancement of arteriolar thromboembolism by wall shear rate.

Animals↗
PubMed2000

A novel model for the in vivo monitoring of uterine microcirculation and intracellular free calcium changes in rat.

The aim of this work was to develop a model to study the microcirculation and relative levels of intracellular free calcium in the myometrium of pregnant rats. On Day 21 of gestation a lobe of uterus was prepared free, flipped over, and mounted in a superfusion chamber leaving the radix and thereby the innervation and circulation intact. RBC velocity and arteriolar diameters were determined by means of intravital video microscopy before and after stimulation (norepinephrine). To study intracellular free calcium changes, the fluorescent dye Indo-1 AM was added to the superfusate in the chamber. Fluorescence images were recorded and ratios of the images collected at 400 and 506 nm were calculated and changes thereof were assumed to represent intracellular free calcium changes. RBC velocity and arteriolar diameter did not change for at least 1 h, while the response to norepinephrine was similar at the beginning of the experiment and after 120 min. In four separate interventions, the uterus was challenged with 5 x 10(-4) IU/ml oxytocin, 4.5 mM calcium, 5 x 10(-4) IU/ml oxytocin with 4.5 mM calcium, and 5 microM ionomycin, resulting in an increase of the 400/506 nm ratio of 27, 31, 76, and 103%, respectively, representing a relative increase in intracellular free calcium. This novel in vivo model is suitable for monitoring intracellular free calcium changes and to record RBC velocities and blood vessel diameters in the myometrium of pregnant rats.

Animals↗
PubMed2000

Ischemia/reperfusion-induced changes in intracellular free Ca2+ levels in rat skeletal muscle fibers--an in vivo study.

Accumulation of intracellular free calcium (Ca2+i) may play an essential role in the ischemia/reperfusion injury of skeletal muscle. Although it has been shown that Ca2+i levels significantly increase during ischemia/reperfusion, it is still a matter of debate whether Ca2+i increases during ischemia alone. It was the aim of this study to monitor the in vivo Ca2+i levels in the rat spinotrapezius muscle during ischemia of varying duration and reperfusion, using a ratiometric fluorescence technique, and to investigate the relationship between the postischemic flow patterns and Ca2+i, if any. The muscle was loaded with Indo-1/AM and imaged by a cooled digital camera. Pre- and postischemic tissue perfusion was assessed by means of an analogue camera. Our results show that short-term ischemia (5, 15 and 30 min) and subsequent reperfusion (60 min) does not alter Ca2+i homeostasis and that tissue perfusion promptly recovers after the insult. One or two hours of ischemia resulted in changes in Ca2+i levels, varying from preparation to preparation; increases in some and no changes in others. In these preparations three distinct flow patterns - normal, compromised and no-reflow - could be distinguished during the 60-min reperfusion. Our main conclusion is that in skeletal muscle Ca2+i levels may increase, the increase probably depending on the muscle fiber type exposed.

Animals↗
PubMed2000

Effect of repetitive asphyxia on leukocyte-vessel wall interactions in the developing chick intestine.

BACKGROUND/PURPOSE: Information on leukocyte-vessel wall interactions (LVWI) during development of the immature intestine is scarce. The authors designed an experimental model for studying the microcirculation in the developing intestine of chick fetuses at days 13 (n = 12), 15 (n = 17), and 17 (n = 19) of incubation (0.6, 0.7, and 0.8 of the incubation time, respectively) using intravital microscopy. METHODS: The authors investigated whether episodes of asphyxia increase LVWI and induce tissue damage in the developing intestine. Asphyxia was induced by clamping of the chorioallantoic vein for 6 periods of 5 minutes each, with 5-minute intervals, whereas in sham groups a sham procedure was performed. Video recordings were made before as well as 10, 20, and 30 minutes after the end of the asphyxia or sham protocol. RESULTS: Baseline number of rolling leukocytes per minute significantly increased (P < .001) from 0 at 0.6 incubation to 1.5 and to 4 at 0.7 and 0.8 incubation time, respectively. At 0.6 and 0.7 incubation no adherent leukocytes were observed under baseline conditions, whereas at 0.8 incubation single leukocytes adhered to the venular wall. LVWI variably increased during the course of the experiments. Asphyxia neither enhanced LVWI nor induced histological damage in the intestine. CONCLUSIONS: These findings indicate that (1) leukocyte-vessel wall interactions mature during fetal development, and (2) repetitive episodes of asphyxia induce neither an inflammatory response nor histological tissue injury in the developing intestine from 0.6 to 0.8 incubation. The authors hypothesize that immaturity of leukocyte-vessel wall interactions, as part of the nonspecific host defense to invading bacteria, might play a role in the development of necrotizing enterocolitis in premature neonates.

Animals↗
PubMed2000

Tumor angiogenesis factors reduce leukocyte adhesion in vivo.

Leukocyte-endothelium interactions are diminished in tumors. It is reported here that, in a tumor-free in vivo model, angiogenic factors can down-regulate leukocyte adhesion to endothelium. Slow releasing pellets were loaded with either basic fibroblast growth factor (bFGF), vascular endothelial cell growth factor (VEGF) or vehicle alone and were placed in the scrotum of mice. After 3 days, a single intrascrotal injection of 1 microg/kg IL-1beta was given 4 h before vessels of the cremaster muscle were investigated for leukocyte rolling and adhesion by means of intravital microscopy. Exposure of normal tissue to either bFGF or VEGF resulted in markedly decreased levels of cytokine-induced leukocyte adhesion. Suppression of leukocyte rolling was not observed. Instead a moderate enhancement of rolling by VEGF was found. The observed differences could not be explained by differences in fluid dynamic parameters or systemic leukocyte counts. In conclusion, evidence is presented that, in vivo, angiogenic factors significantly reduce leukocyte adhesion, the final step preceding leukocyte infiltration. This observation may explain why tumors escape from immune surveillance.

Angiogenesis Inducing Agents↗
PubMed2000

Detection of leukocytes in contact with the vessel wall from in vivo microscope recordings using a neural network.

Leukocytes play an important role in the host defense as they may travel from the blood stream into the tissue in reacting to inflammatory stimuli. The leukocyte-vessel wall interactions are studied in post capillary vessels by intravital video microscopy during in vivo animal experiments. Sequences of video images are obtained and digitized with a frame grabber. A method for automatic detection and characterization of leukocytes in the video images is developed. Individual leukocytes are detected using a neural network that is trained with synthetic leukocyte images generated using a novel stochastic model. This model makes it feasible to generate images of leukocytes with different shapes and sizes under various lighting conditions. Experiments indicate that neural networks trained with the synthetic leukocyte images perform better than networks trained with images of manually detected leukocytes. The best performing neural network trained with synthetic leukocyte images resulted in an 18% larger area under the ROC curve than the best performing neural network trained with manually detected leukocytes.

Animals↗
PubMed2000

Development of vasomotor responses in fetal mesenteric arteries.

Changes in mesenteric arterial diameters were studied using intravital microscopy in chick fetuses at days 13 and 17 of incubation, corresponding to 0.6 and 0.8 fetal incubation time, both during 5 min of hypoxia followed by 5 min of reoxygenation and after topical administration of increasing concentrations (10(-6)-10(-2) M) of norepinephrine (NE) and acetylcholine (ACh). Baseline diameters of second-order mesenteric arteries increased from 56 microm at 0.6 incubation to 75 microm at 0.8 incubation. Acute hypoxia induced a reduction in arterial diameter to 87 +/- 4.4% of baseline at 0.6 incubation and to 44 +/- 6.7% at 0.8 incubation (P < 0.01). During reoxygenation, mesenteric arteries dilated to 118 +/- 6.5% and 121 +/- 7.5% of baseline at 0.6 and 0.8 fetal incubation time, respectively. Phentolamine did not affect the vasoconstriction during hypoxia at 0.6 incubation, whereas this alpha-adrenergic antagonist significantly attenuated the vasoconstrictor response at 0.8 incubation (to 93 +/- 2.7% of baseline, P < 0.01). Topical NE induced maximal vasoconstriction to 71 +/- 3% of baseline at 0.6 incubation and to 35 +/- 3.8% at 0.8 incubation (P < 0.01). Maximal vasodilation to topical ACh was 113 +/- 4.4% and 122 +/- 4.8% of baseline at 0.6 and 0.8 incubation, respectively. These in vivo findings show that fetal mesenteric arteries constrict in response to acute hypoxia and that the increase in magnitude of this vasoconstrictor response from 0.6 to 0.8 of fetal development results from an increase in adrenergic constrictor capacity.

Acetylcholine↗
PubMed1999

An in vivo model for studying the dynamics of intracellular free calcium changes in slow- and fast-twitch muscle fibres.

The understanding of the regulation of the free cytosolic [Ca2+] ([Ca2+]i) in skeletal muscle is hampered by the lack of techniques for quantifying free [Ca2+]i in muscle fibres in situ. We describe a model for studying the dynamics of free [Ca2+]i in the fast-twitch extensor digitorum longus (EDL) and the slow-twitch soleus (SOL) muscles of the rat in vivo using caffeine superfusion to induce changes in free [Ca2+]i. We assumed that differences in sensitivity between the two muscle types for this substance reflect differences in intracellular Ca2+ handling in the fibres of which these muscles consist. The Indo-1 ratiometric method, using intravital microscopy with incident light, was adapted to measure free [Ca2+]i in vivo. Fluorescence images were collected by means of a digital camera. Caffeine superfusion at 37 degrees C for 2 min, at concentrations of 1, 2, 5, 10 or 20 mmol/l, induced a concentration-dependent increase in free [Ca2+]i and revealed differences in caffeine sensitivity between the muscle types, with the SOL being more sensitive. In a separate set of experiments the contracture threshold, as assessed by topical application of caffeine, was determined in both muscle types. EDL had a higher threshold for developing contracture than SOL. These finding are in agreement with previous in vitro studies. We may conclude that the dynamics of free [Ca2+]i can be assessed reliably in intact mammalian muscle in vivo.

Animals↗
PubMed1998

Motor denervation induces altered muscle fibre type densities and atrophy in a rat model of neuropathic pain.

Loose ligation of a sciatic nerve in rats (chronic constriction injury; CCI) provokes sensory, autonomic, and motor disturbances like those observed in humans with partial peripheral nerve injury. So far, it is unknown whether these motor disturbances result from (mechanical) allodynia or from damage to the motor neuron. These considerations prompted us to assess, in CCI rats, the density of motor axons in both the ligated sciatic nerve and the ipsilateral femoral nerve. To this end, we determined the number of cholinesterase positive fibres. It has been demonstrated previously that muscle fibre type density may be used as a measure of motor denervation and/or hypokinesia. Therefore, the myofibrillar ATPase reaction was employed to assess fibre type density in biopsies obtained from the lateral gastrocnemius muscle (innervated by sciatic nerve) and rectus femoris muscle (innervated by femoral nerve). We observed axonal degeneration of motor fibres within the loosely ligated sciatic nerve, both at an intermediate (day 21) and at a late stage (day 90) after nerve injury. The reduction in the number of motor nerve fibres was more pronounced distal to the site of the ligatures than proximal. A (less pronounced) reduction of motor fibres was observed in the ipsilateral (non-ligated) femoral nerve. In line with these findings, we observed altered fibre type densities in muscle tissue innervated by the ligated sciatic nerve as well as the non-ligated femoral nerve indicative of motor denervation rather than hypokinesia. The findings of this study suggest that the motor disorder induced by partial nerve injury involves degeneration of motor nerve fibres not only within the primarily affected nerve but also within adjacent large peripheral nerves. This spread outside the territory of the primarily affected nerve suggests degeneration of motor neurons at the level of the central nervous system.

Acetylcholinesterase↗
PubMed1998

Effects of surgical sympathectomy on skin blood flow in a rat model of chronic limb ischemia.

The role of lumbar sympathectomy in the treatment of limb ischemia secondary to arteriosclerosis obliterans has been controversial. Increased temperature and rubor of the skin, which usually follow sympathectomy, have generally been interpreted as indicative of improved nutritive skin blood flow. However, the existence of a (nonnutritive) thermoregulatory level of skin microcirculation makes such an extrapolation questionable. We investigated the total (mainly thermoregulatory) skin blood flow (TSBF) in the hindlimb of 15 male Lewis rats by means of laser Doppler flowmetry and the nutritive skin blood flow (NSBF) by means of capillary microscopy (red blood cell velocity). Transcutaneous oximetry was used to assess skin oxygenation (SO). Measurements were performed before and 2 and 28 days after ligation of the common iliac and iliolumbar artery. Subsequently, either a surgical resection of the sympathetic chain (L2-L6) was performed or a sham operation. Measurements were repeated 2 and 28 days later. For the group of 15 rats as a whole, TSBF (p < 0.05), NSBF (p < 0.05), and SO (p < 0.05) were found to be drastically reduced at day 2 after litigation compared to preligation values. This reduction partially recovered during the following weeks. TSBF (p < 0.05) and NSBF (p < 0.05), however were still reduced at day 28 after ligation compared to preligation values, whereas the SO at this time tended to be lower (p = 0.11). In the sympathectomy group the TSBF was found to be increased at day 2 (p < 0.05) and day 28 (p < 0.05) after sympathectomy, both compared to values obtained at day 28 after ligation. Sympathectomy did not have an effect on NSFB and SO. The sham procedure had no effect on the TSBF, NSBF, or SO. These results indicate that in case of lower limb ischemia, sympathectomy improves skin blood flow at the thermoregulatory but not the nutritive level of skin microcirculation. This may be related to the fact that the thermoregulatory vessels are mainly sympathetically controlled, whereas the nutritive capillaries are mainly controlled by local (nonneural) factors.

Animals↗
PubMed1998

The role of mast cells and histamine in leukocyte-endothelium interactions in four rat strains.

The objective of the present study was to determine the role of mast cells and histamine in leukocyte-endothelium interactions in mesenteric venules of four rat strains: Brown Norway, Lewis, Sprague-Dawley and Wistar. Intravital microscopy showed that the mast cell stabilizer cromoglycate (5 mg/kg i.v. just before exteriorization of the mesentery) did not affect the baseline level and velocity of leukocyte rolling in any of the four strains. This finding is in agreement with the observation that cromoglycate pretreatment only slightly influenced mast cell degranulation in all strains except the Brown Norway. After mast cell stabilization, only in Sprague-Dawley did topical administration of histamine (10(-4) M) result in a significant increase in the level of leukocyte rolling and a decrease in the rolling velocity compared with the time control. Histamine induced leukocyte adhesion only in the Brown Norway strain. In conclusion, the hypothesis presented in other studies, that degranulation of mast cells, and more specifically the release of histamine, is of major importance for the induction of leukocyte-endothelium interactions in rat mesenteric venules is not generally applicable; the present study shows a clear strain dependency.

Animals↗
PubMed1998

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.

Animals↗
PubMed1998

Quantitative assessment of [Ca2+]i levels in rat skeletal muscle in vivo.

Intracellular free Ca2+ concentration ([Ca2+]i) plays an essential role in physiological regulatory processes and common pathological conditions. Better understanding of these phenomena is still hampered by problems encountered in the quantitative assessment of [Ca2+]i changes, especially in blood-perfused organs. This study demonstrates that the ratiometric fluorescence technique can be adapted for quantitative in vivo [Ca2+]i determinations. The rat spinotrapezius muscle was topically loaded with indo 1-AM and imaged by a cooled digital camera. Ratio images were calculated in small regions (100 micrometers x 100 micrometers) practically devoid of large vessels in the resting state, after 30 min of ischemia, 20 min of reperfusion, or ionomycin or manganate treatments. When we assumed an average [Ca2+]i of 100 nM in the resting blood-perfused muscle, ischemia increased [Ca2+]i to approximately 200 nM. During reperfusion [Ca2+]i decreased to approximately 140 nM. Ionomycin induced an increase in [Ca2+]i to well above 750 nM. Manganate reduced Ca2+-dependent fluorescence to virtually zero. Our main conclusion is that changes in [Ca2+]i can be monitored and quantitatively determined in vivo.

Animals↗