Vasomotion in the rat cerebral microcirculation recorded by laser-Doppler flowmetry.

In the present study, changes in frequency and amplitude of the rhythmic variations (vasomotion) in blood flow in the intact cerebral circulation of the rat were investigated using laser-Doppler flowmetry (LDF) during stepwise decrease in mean arterial blood pressure (MABP) and hyper- and hypocapnia. Experiments were performed on 12 adult Sprague-Dawley rats of either sex, anesthetized with alpha-chloralose. The rat's head was fixed on a stereotaxic frame and a small hole was made in the parietal bone but the dura and a thin inner bone layer were kept intact. The microvascular blood flow of the parietal cortex on the right or on both sides was continuously recorded by the laser-Doppler flowmeter (Periflux PF2B, Perimed, Stockholm, Sweden). The cerebral circulation of the rat exhibited vasomotion in control conditions with a frequency of 8-10 cycles per minute (cpm) and an amplitude of 5-10% of the cerebral blood flow (CBF). No significant changes in CBF could be detected when the MABP was above 60 mmHg, but it decreased significantly when MABP was reduced below 50 mmHg. However, during stepwise pressure reduction the vasomotion frequency decreased progressively while its amplitude showed a reversed U-shaped curve with a peak at 60-80 mmHg. During hypercapnia, the rhythmical oscillations showed a decrease in both frequency and amplitude, whereas during hypocapnia their frequency did not change but their amplitude increased. These results support the hypothesis that the vasomotion frequency might be dependent of the wall tension and cellular pH while its amplitude could be related to decreased tissue oxygenation.     

Laser-Doppler measurements of concentration and velocity of moving blood cells in rat cerebral circulation

In brain cortex all capillaries are perfused with plasma at anyone time while the flow of blood cells is heterogenous. Increased blood flow is associated with increased number of moving erythrocytes in the microcirculation, while capillary recruitment in its classical anatomical sense appears not to exist in the brain. Modulation of the concentration of flowing erythrocytes may influence the oxygen supply to the tissue. Therefore, we examined the possibility that laser-Doppler flowmetry (LDF) could be used to quantify changes in the microvascular concentration of moving blood cells (CMBC) and blood cell velocity (< v >) by comparing LDF measurements with electromagnetic flow measurements in vitro, and confocal laser-scanning microscopy in vivo in the brain of anaesthetized male Wistar rats. In vitro measurements showed that CMBC was affected by changes in haematocrit, while < v > correlated almost linearly with blood cell velocity measured electromagnetically within a relevant physiological range. In vivo studies during hypercapnia (Paco ₂ from 39 ± 4 to 66 ± 5 mmHg) with confocal laser scanning microscopy disclosed a 39 ± 10% increase of cortical capillary erythrocytes, while CMBC measured with LDF increased by 37 ± 5%. Erythrocyte flow velocity in brain cortex capillaries increased by 65 ± 17% with confocal microscopy as compared to 72 ± 8% with LDF. Local electrical stimulation of cerebellar cortex, and application of adenosine or sodium-nitroprusside, increased CMBC and < v > simultaneously, while during hypercapnia the < v > increase preceded the CMBC increase by 30 s. The CMBC rise rapidly reached a steady state in response to all types of stimulation, while < v > continued to increase during the major part, or the entire stimulation period. In conclusion, our data support the hypothesis that LDF may be useful for haemodynamic studies of brain microcirculation.     

Vasomotion in rat diaphragm microcirculation at rest and during stepwise arterial pressure reduction

The effect of haemorrhagic hypotension on the incidence, frequency and relative amplitude of vasomotion in rat diaphragm microcirculation was assessed by laser Doppler flowmetry (LDF). Graded bleeding to four hypotension levels (80, 60, 40 and 30% of the control state) were performed in 24 Sprague–Dawley rats. The incidence of vasomotion was 83% in the control state, 96% at the 80% level, 100% at the 60% level, 96% at the 40% level, and 46% at the 30% level. The median fundamental frequency of vasomotion determined manually during the control state and at the hypotension levels (in descending order) was 4.11 (range, 3.29–5.58) cycles min^?1 (cpm), 4.48 (3.21–5.92) cpm, 4.20 (3.5–5.56) cpm, 4.01 (3.33–5.36) cpm, 3.71 (3.25–4.49) cpm (P < 0.01 from the fundamental frequency at 80 and 60% hypotension levels). The median relative amplitudes determined manually during the control state and descending hypotension levels were 44.5% (range, 24.9–135.9%), 69.4% (26.6–147.2%), 84.0% (40.3–177.1%) (P < 0.01 from resting and last stage of bleeding), 90.40% (26.2–189.6%) (P < 0.01 from resting and last stage of bleeding), 69.2% (35.6–93.2%). We concluded first that during the resting condition, vasomotion was frequently present in diaphragm microcirculation, which is distinct from other vascular beds of skeletal muscles. Second, the relative amplitude of vasomotion during haemorrhagic hypotension plotted against decreasing blood pressure exhibited a reverse U-shaped curve with a maximum at 40–60% of the control blood pressure, while the frequency of vasomotion remained relatively constant until the last stage of haemorrhage and centred around 3–5 cpm.     

Villus tip microcirculation in the rat duodenum

Duodenal microvascular perfusion was measured in anaesthetized rats both as erythrocyte velocity (rev) in capillaries in the tip of duodenal villi and by laser-Doppler flowmetry (LDF). Rev increased transiently by about 40% during the first 5 min of luminal exposure to 10 mM (NaCl to isotonicity) hydrochloric acid, while LDF measurements only showed a transient increase of about 7%, followed by a prolonged reduction by about 11%. Since the LDF signal is a measure not only of villus microcirculation but also of blood flow in the deeper layers, our results may suggest that blood flow is transiently redistributed towards the villi from deeper layers. Hypovolaemia (bleeding by ～ 10% of the blood volume) reduced rev in the capillaries by 63% during the first 5 min of hypotension, but reduced LDF only by about 12%, a discrepancy which suggests a shift in blood flow from the tip to deeper layers. The experiments were performed under atmospheric oxygen tension, but rev in the villus capillaries exposed to abdominal Po₂ (～ 45 mmHg) did not differ significantly from the values obtained under the atmospheric oxygen condition, either in the resting situation or during hypotension. In conclusion, we have developed an animal model in which red cell velocity in the tip of the duodenal villi can be studied for several hours and in which alkaline secretion from the duodenum is similar to previously reported levels. Our results show that the villus tip microcirculation in the duodenum may respond differently from that of deeper layers of the duodenal wall.     

Responses of the cerebral blood flow to hypo-and hypercapnia after inhibition of prostaglandin biosynthesis by indomethacin

Quantitative investigation of the local cerebral blood flow by the hydrogen clearance method and of the blood flow into the brain by means of an electromagnetic flowmeter showed that inhibition of prostaglandin biosynthesis by indomethacin inhibits the response of the cerebral vessels to hypercapnia, whereas the effects of hypocapnia are not only preserved but are actually enhanced. This difference in the response of the brain vessels to hypo- and hypercapnia during inhibition of prostaglandin biosynthesis suggests that effects of hyper- and hypocapnia are produced by different mechanisms. It is postulated that a decrease in the prostaglandin concentration reduces the sensitivity of the brain vessels to hypercapnia and increases their sensitivity to hypocapnia.Problem Laboratory for Pharmacology of the Cardiovascular System, Department of Pharmacology, Erevan Medical Institute. (Presented by Academician of the Academy of Medical Sciences of the USSR V. V. Zakusov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 87, No. 3, pp. 240–243, March, 1979.     

Local cerebral blood flow velocity in newborn rats in normocapnia and hypercapnia

The local cerebral blood flow (LCBF) in the caudate nucleus was investigated in experiments on unanesthetized newborn rats by determing the rate of hydrogen saturation of the brain tissue and the cerebral blood volume was studied by plethysmography. LCBF in newborn animals was found to be considerably lower than in adults. Inhalation of CO₂ by newborn, unlike by adult rats, did not cause an increase in LCBF and the cerebral blood volume also remained unchanged.Laboratory of Brain Development, Scientific-Research Institute of Pediatrics, Academy of Medical Sciences of the USSR. (Presented by Academician M. Ya. Studenikin.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 84, No. 8, pp. 139–141, August, 1977.     

激光多普勒流量计观察单侧输尿管梗阻大鼠双侧肾皮质微循环血流变化

目的:用激光多普勒流量计观察单侧输尿管梗阻大鼠双侧肾皮质微循环血流的变化,探讨梗阻侧肾间质纤维化的发病机制及健侧肾的代偿机制。方法:成年雌性SD大鼠,随机分为假手术组和模型组。假手术组仅将输尿管游离但不结扎离断,模型组行单侧输尿管结扎术。术后第7、14和21天随机选取并处死各组中的6只大鼠,观察双肾病理学改变,并检测双侧肾皮质微循环血流。结果:H-E染色显示假手术组双侧肾各时间点肾单位结构正常,间质无增宽;模型组大鼠梗阻侧肾术后7 d出现早期间质纤维化的病理改变,并随梗阻时间延长逐渐加重;健侧肾小球系膜细胞增生并随代偿时间延长逐渐增多。肾皮质微循环血流,与假手术组比较,模型组大鼠健侧肾皮质微循环血流显著增加,梗阻侧肾皮质微循环灌注量先增加后进行性降低。结论:血液流变学改变、梗阻侧肾皮质微循环血流灌注量进行性减少是肾间质纤维化的发病机制之一;而健侧肾皮质微循环血流灌注量增高,在一定程度上有利于健侧肾的功能代偿。     

Effect of short-term cerebral ischemia on the mesenteric microcirculation in rats

The effect of cerebral ischemia produced by compression of both common carotid arteries on the mesenteric microcirculation was studied in experiments on rats. The extent and intensity of the microcirculatory disturbances were shown to depend on the duration of ischemia and of the postischemic period. The state of the systemic hemodynamics was compared with that of the mesenteric microcirculation. The possible mechanisms of the microcirculatory disturbances are discussed.Laboratory of General Pathology and Experimental Therapy, Institute of General Pathology and Pathological Physiology, Academy of Medical Sciences of the USSR, Moscow. Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 88, No. 7, pp. 9–12, July, 1979.     

Repeated measurements of cerebral blood flow in rats. Comparisons between the hydrogen clearance method and laser Doppler flowmetry

Changes in cerebral blood flow (CBF), in chloralose-anaesthetized spontaneously hypertensive rats, were measured simultaneously with the hydrogen clearance (HC) method and Laser Doppler flowmetry (LDFM) in order to examine the correlation between results obtained with the two techniques. To induce changes in CBF the rats were bled and CBF measured at different levels of mean arterial pressure. As would be expected, with the platinum electrode at the depth of about I mm in the somatosensory cortex, HC gave biexponential curves reflecting clearance in two distinct compartmenrs. During control situation the HC method gave the following values (mean ± SE, n= 20, mlmin^-' 100g^-1): fast (fCBF) 158.6 ± 11.5, slow (sCBF) 29.1 ± 1.6 with a weighted mean flow (mCBF) of 83.3 ± 7.4. These fCBF and sCBF values correspond well to those of others obtained with [¹⁴C]iodoantipyrine autoradiography in the cerebral cortex and corpus callosum, and better than the results of other studies using HC in rats. Our results, comparing data from HC and LDFM, show a linear relationship between relative values of blood flow changes, the coefficients being 0.658, 0.876 and 0.878 for the correlation between the LDFM data and relative changes in fCBF, sCBF and mCBF, respectively. All three regression lines were significantly different from the line of identity. Much of the discrepancy between the two methods may be related to limitations inherent in each of them, despite efforts to minimize their effects. Thus the depth sensitivity of LDFM in the brain may be greater than expected. In conclusion, the laser Doppler method seems, nevertheless, to be most useful for continuous estimations of changes in cerebral blood flow.     

Cerebral circulatory responses to hypercapnia and hypoxia in the recovery period following complete and incomplete cerebral ischemia in the rat

In this study we examined the reactions of cerebral vessels to hypercapnia and hypoxia during the recovery period following cerebral ischemia. We used ventilated, lightly anesthetized rats and induced complete ischemia by CSF compression, incomplete ischemia by bilateral carotid occlusion combined with hypotension. After 15 min of ischemia and 60 min of recirculation the animals were rendered hypercapnic or hypoxic for 2–3 min and local CBF was then measured autoradiographically with ¹⁴C-iodoantipyrine. Following complete ischemia vascular CO₂ responsiveness was abolished or attenuated in most structures analysed. However, there was a considerable interstructural heterogeneity. For example, in the cerebellum and the red nucleus flow rates were observed which approached values obtained in hypercapnic control animals, whereas CO₂ responsiveness was abolished in several cortical areas and hippocampus. The response to CO₂ following incomplete (“forebrain”) ischemia varied considerably. In the cerebral cortices areas with low flow rates were often mixed with hyperemic zones, and in most structures that had very low flow rates during ischemia, CO₂ responsiveness was lost or grossly attenuated. Structures that had suffered moderate or only mild ischemia had better retained or completely preserved CO₂ response. The cerebrovascular reaction to hypoxia was found to be attenuated in most, but not abolished in any of the structures examined. In general, the vascular response to hypoxia was better preserved than that to hypercapnia. Reactivity was similar following complete and incomplete ischemia. As observed during hypercapnia, there were pronounced interstructural variations with considerable increases in flow rates e.g. in the substantia nigra and the cerebellum.     

Rhythmical oscillations in rat testicular microcirculation as recorded by laser Doppler flowmetry

We have earlier reported that local testicular blood flow, recorded by laser Doppler flowmetry, shows large oscillations with a frequency of 5-10 min-1. In the present study it is proposed that the recorded oscillations represent mainly local microvascular blood flow variations rather than variations in total testicular blood flow or tissue movements. The reasons for this are: (a) Blood flow simultaneously measured at two separate sites showed oscillations with different frequencies. (b) A local subcapsular injection of room-tempered saline under one probe site eradicated oscillations under that probe but not under another adjacent probe. (c) When the testicular capsule was split open, recordings of blood flow continued to show oscillations. (d) The amplitude of the oscillations was rather large (peak to peak value about 50% of mean flow value). No movements of the testicular surface were seen. A 20 min continuous infusion of 0.4 microgram/min noradrenaline did induce a decrease in plasma testosterone concentration, but did not change the mean blood flow. However, the oscillations nearly completely disappeared during the infusion period. The present study also shows that laser Doppler flowmetry is a versatile method and the rat testis provides a suitable organ in the study of the origin and functional importance of these oscillations.     

Measurement by laser-Doppler flowmetry of microcirculation in lower leg muscle at different blood fluxes in relation to electromyographically determined contraction and accumulated fatigue

Single-fibre percutaneous laser-Doppler flowmetry (LDF) of the tibialis anterior muscle was performed continuously for measurement of the microcirculation during different blood fluxes, as well as in relation to different muscle activities and fatigue determined electromyographically (EMG). The laser-Doppler power spectrum density function was studied in a frequency range of 0–8.2 Hz as representing the blood flow most selectively. Reduced blood flow from tourniquet inflation caused a decrease in signal power density, compared to that of intact blood flow at rest. During postocclusion reactive hyperaemia an increased signal power was recorded. This reached its maximum within 4.4 (SD 1.88) s after deflation of the tourniquet. The different fluxes were recorded at high sensitivity and disturbances were small. Periods of 1-min static dorsi-flexion of the foot at 10, 20, 30, 40, and 50% MVC (maximal voluntary contraction) with 1-min rest between were associated with a significant increase in LDF, the recordings obtained during the rest periods showing a tendency towards an increase. A decrease in the EMG mean power frequency (MPF) indicated accumulated fatigue. The LDF. for the rest periods that followed upon continuous contractions up to the same MVC levels showed a tendency towards an increase but variability was large. With further development, these techniques may be useful in the evaluation of insufficiency of the peripheral circulation.     

Characterization of the cortical laser-Doppler flow and hippocampal degenerative patterns after global cerebral ischaemia in the goat

 Large-animal models offer several advantages in the study of cerebral ischaemia: easier control of physiological variables, easier neuropathological evaluation, etc. In the present study we have taken advantage of the unique cerebrovascular anatomy of the goat to reproduce a model of reversible, incomplete, global cerebral ischaemia in a large-sized animal species, in which the effects of successive manoeuvres to stop and re-start cerebral blood flow can be recorded continuously. Early cortical laser-Doppler flow response (up to 2 h) and delayed neuronal degeneration (7 days) in the hippocampal CA1 subfield have been analysed in goats undergoing 5, 10 or 20 min of transient, global cerebral ischaemia. Bilateral occlusion of the external carotid artery plus compression of jugular veins reduced cortical laser-Doppler flow to 11 ± 8% of preischaemic values (P<0.01), flattened the electrocorticogram, and increased mean arterial blood pressure by 17 ± 23% (P<0.01) and intracranial pressure by 161 ± 136% (P<0.01). A rather heterogeneous response was obtained during reperfusion: 14 out of 31 goats showed the ”classical” pattern consisting of hyperaemia followed by delayed hypoperfusion. The remaining goats showed neither hyperaemia (11 goats) nor delayed hypoperfusion (6 goats). The duration of the ischaemic insult did not correlate with the magnitude of hyperaemia or delayed hypoperfusion, but influenced neurodegeneration: while no loss of hippocampal CA1 neurons was observed at 7 days after 5 or 10 min ischaemia, a 68% cell loss was observed in the 20-min ischaemia group. Our goat model has thus proven to be very suitable for the induction of global cerebral ischaemia in a large-animal species without extensive surgery. It allows reproducible reductions of cerebral blood flow, long-term recovery, low mortality rate, and high incidence of neuronal damage. The results reported here support the view that delayed hypoperfusion is not an important determinant of neuronal injury. Received: 23 September 1997 / Received after revision and accepted: 3 December 1997     

Longitudinal MRI studies in the isoflurane-anesthetized rat: long-term effects of a short hypoxic episode on regulation of cerebral blood flow as assessed by pulsed arterial spin labelling

MRI is a powerful tool for measuring cerebral blood flow (CBF) longitudinally. However, most animal studies require anesthesia, potentially interfering with normal physiology. Isoflurane anesthesia was used here to study CBF regulation during repetitive scanning in rats. MR perfusion images were acquired using FAIR (flow-sensitive alternating inversion recovery) arterial spin labeling, and absolute CBF was calculated. CBF changes in response to a hypoxic (12% O2) and hypercapnic (5% CO2) gas stimulus were monitored. Hypercapnia led to a robust increase in CBF compared with baseline (195.5+/-21.5 vs 123.6+/-17.9 ml/100 g/min), and hypoxia caused a smaller non-significant increase in mean CBF values (145.4+/-13.4 ml/100 g/min). Strikingly, when measurements were repeated 5 days later, CBF was dramatically reduced in hypoxia (93.2+/-8.1 ml/100 g/min) compared with the first imaging session. Without application of the hypoxic and hypercapnic gases during the first MRI, baseline CBF and CBF changes in response to hypoxia at the second MRI were similar to naive rats. Blood gas analyses revealed a slight reduction in arterial oxygenation during the second period of anesthesia compared with the first. These findings indicate that, in isoflurane-anesthetized rats, even a short hypoxic episode can have long-lasting effects on cerebrovascular regulation.     

Discrimination of capillary and arterio-venular blood flow in skin by laser Doppler flowmetry

The scattering and absorption of light by tissue and blood is wavelength dependent; the tissue penetration of green light (λ=543·5nm) is about 60 per cent of that of red light (λ=632·8 nm) but the absorption of green light by blood is about 20 times greater than for red light. The effect of this difference has been studied by observing the responses of skin blood flow to heat and weal, measured by laser Doppler flowmetry at the two wavelengths. By using time autocorrelation function analysis (ACF) of the scattered light measured, low and high frequency components have been associated with capillary and larger vessel flow, respectively. The comparison of ACF from scattered green and red light has shown that measurements cannot be interpreted by only considering light penetration depth through a homogeneous tissue. Light absorption and multiple scattering by blood at the individual microvessel level, blood rheology and vessel morphology are parameters which are considered for greater attention.     

Efferent renal sympathetic nerve stimulation in vivo. Effects on regional renal haemodynamics in the Wistar rat,studied by laser-Doppler technique

Intrarenal blood flow regulation probably affects long-term blood pressure homeostasis. We have previously shown that 5 Hz renal sympathetic stimulation inhibits a humoral renal depressor mechanism, otherwise activated when increasing perfusion pressure to an isolated kidney in a cross-circulation set-up. This inhibition was suggested to occur as a result of a reduction of renomedullary blood flow. Little is known about nervous blood flow regulation within the medulla. Therefore in this study, total renal (RBF), cortical (CBF) and papillary (PBF) blood flows were separately measured by ultrasonic and laser-Doppler techniques in Wistar rats during graded renal sympathetic stimulations. Periods of 15 min stimulation at 0.5, 2 and 5 Hz were performed in random order. RBF decreased at 0.5 Hz by 1%, at 2 Hz by 16% (P < 0.001) and at 5 Hz by 49% (P < 0.001). In a similar fashion (r = 0.73, P < 0.001), CBF decreased by 1%, 10% (P < 0.001) and 37% (P < 0.001), respectively. By contrast, PBF increased by 2% at 0.5 Hz and 4% at 2 Hz, while it decreased at 5 Hz, by 4% (P < 0.05, compared with 2 Hz). It seems therefore, that superficial renocortical and total renal blood flows are closely regulated by renal sympathetic nerves with increasing vasoconstriction at higher frequencies, while medullary blood flow, on the other hand, seems to be under strong local control, tending to offset neurogenic flow restrictions.