- Email: [email protected]
Ultrasonic Assessment of Cerebral Blood Flow Changes During Ischemia-Reperfusion in 7-Day-Old Rats
Ultrasound in Med. & Biol., Vol. 34, No. 6, pp. 913–922, 2008 Copyright © 2008 World Federation for Ultrasound in Medicine & Biology Printed in the USA. All rights reserved 0301-5629/08/$–see front matter
doi:10.1016/j.ultrasmedbio.2007.11.018
● Original Contribution ULTRASONIC ASSESSMENT OF CEREBRAL BLOOD FLOW CHANGES DURING ISCHEMIA-REPERFUSION IN 7-DAY-OLD RATS PHILIPPE BONNIN,*† HAYTHEM DEBBABI,* JEAN MARIANI,‡§ CHRISTIANE CHARRIAUT-MARLANGUE,‡ and SYLVAIN RENOLLEAU‡¶ *AP-HP, Hôpital Lariboisière, Physiologie–Explorations Fonctionnelles, Université Denis Diderot Paris 7, Paris; Cardiovascular Research Center INSERM Lariboisière, INSERM U689, Paris; ‡UMR-CNRS 7102, Equipe HICD, Université Pierre et Marie Curie Paris 6, Paris; §AP-HP, Hôpital Charles Foix, Unité d’Explorations Fonctionnelles, Ivry-sur-Seine; and ¶AP-HP, Service de Réanimation, Hôpital Armand Trousseau, Université Pierre et Marie Curie Paris 6, Paris, France †
(Received 30 July 2007; revised 15 November 2007; in final form 27 November 2007)
Abstract—A model of ischemic brain injury in 7-day-old rat pups has been developed to study perinatal ischemia. It combines permanent occlusion of the distal left middle cerebral artery (LMCA) and transient occlusion of homolateral common carotid artery (LCCA). At removal of the clip on LCCA, reflow allowed brain reperfusion through cortical anastomoses. In 10 rat pups, we measured blood flow velocities (BFV) in main cerebral arteries with 12-MHz ultrasound imaging. At basal states, peak systolic BFV in proximal LMCA was 16.0 ⴞ 3.0 cm.s–1. Occlusion of LMCA did not yield significant modifications. Occlusion of LCCA involved only a decrease in BFV to 9.5 ⴞ 2.6 cm.s–1 (p < 0.001). Indeed, LMCA was then supply by the right internal carotid and the vertebral arteries through the circle of Willis. In three rat pups, release of occlusion of LCCA was followed by restoration of BFV in the left internal carotid artery and in LMCA, in seven pups, by a reversed flow in the LICA and lower BFV in LMCA (11.9 ⴞ 2.3, p < 0.05). BFV returned to basal values from h5 to h48 in all animals. In addition, ultrasound imaging is a useful, reproducible, non invasive, easy-to-repeat, method to assess and monitor arterial cerebral blood flow supply in small animals. It helps to characterize changes occurring during cerebral ischemia and reperfusion, particularly the depth of the hypoperfusion, as well as the variability of reflow. In preclinical studies, this method could help to identify what can be assigned to a neuroprotective treatment and what depends on changes in cerebral blood flow supply. (E-mail: [email protected]) © 2008 World Federation for Ultrasound in Medicine & Biology. Key Words: Neonatal brain ischemia, Ultrasound imaging, Pulsed Doppler, Blood flow velocity, Internal carotid artery, Basilar trunk, Middle cerebral artery, Rat pup.
INTRODUCTION
was recognized in the newborn period and in 82% of those with retrospectively diagnosed HI (Lee et al. 2005). Diffuse HI is caused by transient decrease cerebral blood flow when a perinatal anoxia occurs (uterine membranes rupture, retroplacental hematoma. Focal HI is caused by the same factors that bring about the majority of cases of cerebral infarction of adults: dissection, embolism and thrombosis of cerebral vessels (Freud et al. 1968). Normal pregnancy is a procoagulant and proinflammatory condition. Around time of birth, coagulation mechanisms are already activated in anticipation of hemorrhage, which threatens the life of mother and fetus. Unfortunately, the adaptation that decreases the risk of bleeding increases the risk of clotting (Salonen et al. 2001). HI may thus result from thrombosis of intracranial vessels or from embolism from another site such as
Perinatal brain hypoxia and ischemia (HI) is a cerebrovascular event occurring during fetal or neonatal life with pathologic or radiologic evidence of focal arterial infarction of brain. (Nelson 2007). Incidence is approximately one case for 4,000 children (Ferriero 2004) and essentially involves the vascular territory supplied by the middle cerebral artery when an anatomic vascular territory is involved. Hemiplegic cerebral palsy, seizure and cognitive disorders are the common outcomes of PAS, present later in 37% of children whose cerebral injury
Address correspondence to: Dr. Philippe Bonnin, PhysiologieExplorations Fonctionnelles, Hôpital Lariboisière, 2, rue Ambroise Paré, 75 475 Paris CEDEX 10 France. E-mail: philippe.bonnin@ lrb.aphp.fr 913
914
Ultrasound in Medicine and Biology
extracranial vessels, heart, umbilical vein or placenta. Although the site of origin is usually not clearly established, it is suspected that the fetal side of the placenta may often be the source. Indeed, the presence of a patent foramen ovale enables clots from the fetal side of the placenta to embolize into the fetus, through the umbilical vein, ductus venosus, inferior vena cava, right then left auricles and aorta to reach the fetal brain. Despite recent improvement in neonatal intensive care management, the incidence of long-term disability after neonatal brain injury is not decreasing (Edwards and Azzopardi 2000; Ferriero 2004). To study cellular and molecular events during cerebral hypoxia and ischemia, several animal models have been developed (for review see Northington 2006). In all of the models with a reperfusion phase, the reperfusion time has been depicted as an important event for its potential benefits but also its deleterious effects (Benjelloun et al. 1999). We recently developed a model of ischemic brain injury in 7-day-old rat pups. This model combines a permanent occlusion of the left middle cerebral artery (LMCA), with a transient occlusion of the homolateral common carotid artery (HCCA). After the removal of the clip on the common carotid artery, reflow in the ischemic area, downstream from the occlusion of the middle cerebral artery, is allowed through cortical anastomoses providing from the middle cerebral artery upstream the occlusion, and the anterior and posterior ipsilateral cerebral arteries (Renolleau et al. 1998). In this model, the size of the ischemic lesion is not constant and varies from 45 to 60 mm3, e.g., 18 to 24% of the ipsilateral hemisphere without possibilities of prediction for size and without possibilities to control the level of reflow (Ducros et al. 2000; Joly et al. 2004; Renolleau et al. 2007). Our hypothesis was that the size variability of the lesion could depend on the efficiency of the restoration of blood flow in the carotid artery. Thus, our aim was to quantify the cerebral blood flow changes occurring during the whole procedure of cerebral ischemia–reperfusion to evidence heterogeneous cerebral blood flow supply. We thus investigated extra and intra cranial cerebral circulation with ultrasound imaging to analyze the blood flow redistribution in the main cerebral arteries, through the circle of Willis, thus, upstream from the occlusion of the LMCA and downstream from the common carotid, during and after removal of the clip on this artery. As performed in this study, the use of a 12-MHz transducer allowed visualization of the skull and brain structures in 2-D ultrasound. Colour-coded Doppler revealed the main cerebral branches, which presented sufficiently high blood velocities i.e., the internal carotid arteries, the basilar trunk, the anterior, middle and posterior cerebral arteries. We sought to measure and monitor arterial blood flow velocities changes in these arteries during the
Volume 34, Number 6, 2008
whole procedure of cerebral ischemia-reperfusion (coagulation of the LMCA – occlusion of the LCCA – release of the occlusion). Therefore, ultrasound imaging with measurements of blood flow velocities could constitute a useful, easy-to-repeat tool to monitor and characterize the circulatory changes in these experimental conditions. In that way, depth of the cerebral hypoperfusion responsible of the cerebral damage could be evaluated precisely. MATERIALS AND METHODS Anesthesia All animal experimentation was conducted in accordance with the French and European Community guidelines for the care and use of experimental animals. Isoflurane anesthesia was administered with a vaporizer (model 100-F, Ohio Medical Instruments, Cincinnati, OH, USA). Isoflurane induction was performed over 1 min in an isolation chamber with 0.50% isoflurane in 100% O2, and anesthesia was maintained during spontaneous breathing of the same mixture via a small nose cone as previously described (Bonnin et al. 2007). This procedure was sufficient to obtain sedation without cardiorespiratory depression during ultrasound study. Because hypothermia induced by anesthesia might modify cardiac function and heart rate, the animal was placed on a heating blanket (38° C) (Hoit et al. 2002). After sedation, the animal was placed in right lateral decubitus position for ultrasound acquisition. This position avoided placing pressure on the head of the rat pup with the ultrasound device. Perinatal ischemia—Surgical procedure Ischemia reperfusion was performed in ten 7-dayold rats (17–21g) of both sexes, as previously described (Renolleau et al. 1998), under isoflurane anesthesia. The anesthetized rat was positioned on its back and a median incision was made in the neck to expose the LCCA. The rat was then placed on the right side and an oblique skin incision was made between the ear and the eye. After excision of the temporal muscle, the cranial bone was removed from the frontal suture to a level below the zygomatic arch. Then, the left MCA, exposed just after its appearance over the rhinal fissure, was electrocoagulated at the inferior level of the cerebral vein. After this procedure, a clip was placed to occlude the LCCA. Rats were then placed in an incubator to avoid hypothermia. After 50 min, the clip was removed. Both neck and cranial skin incisions were then closed. During the surgical procedure, external body temperature was maintained at 36 –36.5 C. After recovery, pups were transferred to their mothers. This procedure was responsible for a stroke in the cortical and subcortical area as previously described (Renolleau et al. 1998; Joly et al. 2003).
Doppler of cerebral arteries in rat pups ● P. BONNIN et al.
915
Fig. 1. Schematic figure illustrating the general anatomy of cerebral arterial vessels for pup rat. Extra cerebral supply is supported by right and left internal carotid and vertebral arteries, intracerebral supply by right and left anterior, middle and posterior cerebral arteries. Extra cerebral supply is connected with intracerebral supply through the circle of Willis.
Ultrasound study We used an echocardiograph (Vivid 7, GE Medical Systems, Horten, Norway) equipped with a 12-MHz linear transducer (12L). Data were transferred online to an ultrasound image workstation for subsequent analysis (PC EchoPAC, GE Medical Systems). Ultrasound study consisted in Doppler spectral recordings in the right and left internal carotid arteries (ICAs), the basilar trunk, and the right and left MCAs for further analysis during the whole procedure of ischemia-reperfusion (Fig. 1). Ultrasound study was repeated for each rat pup at different time: (i) before surgery, (ii) after coagulation of the left MCA, (iii) after placement of the clip to occlude the LCCA, (iv) after removal of the clip, then (v) five hours
after procedure onset (h5), then (vi) 24 (h24) and (7) 48 h (h48). Two-dimensional ultrasound imaging was used to visualize a cross-sectional B-mode image of the skull and the brain structures of the animal. Color-coded Doppler was then activated and, extra and intracranial arteries were then drawn and localized on the screen by their color-coded blood flow. Just below skull base, the ultrasound image crossed the right and left ICAs coded in red color. Median and just above the skull base, the basilar trunk (BT) was coded in red color. Laterally, creeping along the skull, the right and the LMCAs were coded in red color (Fig. 2). A turn of 90° with the transducer revealed an anterior longitudinal B-mode im-
Fig. 2. Color-coded ultrasound imaging of the cerebral arteries in the rat pup. (a) Two-dimensional and color-coded Doppler ultrasound imaging obtained with a cross-sectional image of the rat pup head. Below skull base, right and left ICAs were coded in red color. Median and just above the skull base, the BT was coded in redr. Laterally, creeping along the skull, the right MCA and the left MCA were coded in red. (b) Two-dimensional and color-coded Doppler ultrasound imaging obtained with a median sagittal image of the head of the rat pup. The BT was coded in redr. Imaging depth was set at 2 cm. (c) Two-dimensional and color-coded Doppler ultrasound imaging obtained with a left parasagittal image of the rat pup head. The left ICA was coded in red crossing the skull base; the left posterior cerebral artery was coded in blue color.
916
Ultrasound in Medicine and Biology
Volume 34, Number 6, 2008
Fig. 3. Pulsed Doppler with spectral analysis of the Doppler signal recorded in basal states in the cerebral arteries in the rat pup. (a) Spectral analysis of the Doppler signal recorded in the left ICA; scale of the Doppler signal was set min – 0.2m.s–1, max 0.8 m.s–1. (b) Spectral analysis of the Doppler signal recorded in the BT, scale of the Doppler signal was set min – 0.1m.s–1, max 0.6 m.s–1. (c) Spectral analysis of the Doppler signal recorded in the left MCA in basal state, scale of the Doppler signal was set min – 0.06m.s–1, max 0.25 m.s–1. Blue line represented the time-average mean blood flow velocity.
age of the head of the rat pup. With a median sagittal image of the head of the rat pup, the BT was revealed coded in red (inversion of color-codage) (Fig. 2). With a right or left parasagittal image of the rat pup head, the right or the left ICA was revealed, coded in red crossing the skull base (inversion of color-codage). The left posterior cerebral artery was coded in blue (Fig. 2). Imaging depth was set at 2 cm when applying zoom, frame-rate was 49.5 frames/s. In the configuration selected for color Doppler ultrasound acquisition, the frequency emission was 6.7 MHz, pulsed repetition frequency set at 3.5 kHz, frame rate was 37.8 frames/s and low velocity rejection at 1.65 cm s–1. A pulsed Doppler sample was then placed on the longitudinal axis of each vessel and pulsed Doppler spectrum recorded with simple repositioning of the pulsed Doppler sample from right or ICA (Fig. 3) to BT (Fig. 3) and right or left MCA (Fig. 2) in the three different 2-D ultrasound images. For the left MCA, pulsed Doppler sample was placed 1 to 2 mm upstream the level of coagulation. In the configuration selected for
Doppler ultrasound acquisition, the frequency emission was 6.7 MHz, pulsed repetition frequency was set between 2.0 and 5.25 kHz, the sample volume at 1 mm, the low-velocity rejection at 0.6 cm s–1. The minimum detectable velocity of the rat pup imaging was then about 1 cm s–1, maximum between 25 and 70 cm s–1, consistent with blood flow in peripheral arterial vessels. Two-D color-Doppler ultrasound image was refreshed between each arterial vessel acquisition. Ultrasound beam width of pulsed Doppler was small enough (1.1 mm) to ensure that flow from neighboring vessels was not superimposed on the measurement volume centered on each studied artery. Peak systolic and end-diastolic blood flow velocities waveforms were measured from the pulsed Doppler spectrum of each artery by displacing calipers on the screen and time-average mean blood flow velocities by simple redrawing of the blue line drawn on the Doppler spectrum. Blood flow velocities were measured with correction of the angle between the long axis of each vessel and the Doppler beam. Use of the steer mode of the Doppler beam helped to avoid angle correction
Doppler of cerebral arteries in rat pups ● P. BONNIN et al.
greater than 15° for MCAs and greater than 60° for ICAs and basilar trunk. The transducer was positioned on the head of the rat pup at each step of the protocol. Ultrasound imaging and pulsed-Doppler recordings were repeated in each studied artery with correction of the angle between the Doppler beam and the long axis of the artery. Blood flow velocities were measured by the same investigator. Previously to this study, repeatability of blood flow velocities measurements had been investigated in 25 mice (Bonnin et al. 2007) by calculating the repeatability coefficient (RC) (British Standards Institution 1979), according to RC2 ⫽ ⌺Di2/N, where N is the sample size, Di the relative differences calculated in two series of paired measurements separated by two-minutes intervals. The RC values for intraobserver repeatability were 1.5 cm s–1 for the peak systolic, between 2.2 and 1.7 cm s–1 for the end-diastolic and between 2.5 and 1.7 cm s–1 for the mean BFV, respectively, in ICAs and MCAs. Interobserver RC values tested in other studies were not different from intraobserver RC values for systolic, diastolic and mean blood flow velocities in all studied arteries. Statistical analysis To evaluate the effect of coagulation of the left MCA followed by the occlusion of the LCCA, a KruskalWallis nonparametric test was used to analyze differences in peak systolic, end-diastolic and time-average mean blood flow velocities measured in the right and left ICA, in the right and left MCA, and in the BT at the different steps of the protocol. The test was applied in two different groups of rat pups, one group with blood flow restoration in the left CCA (n ⫽ 3), the second group with absence of blood flow restoration in the left CCA (n ⫽ 7). Intra and intergroup comparisons were completed by using a Mann-Whitney nonparametric test with a conservative Bonferroni test. Results are expressed as mean ⫾ standard deviation and a p value of less than 0.05 was considered statistically significant. RESULTS Ten rat pups underwent the complete surgical procedure and ultrasound analysis. Four of them were male and six were female. Steady states hemodynamic values On basal state, heart rate of the rat pups was 296 ⫾ 36 beats per minute. Trepanation and coagulation of the left MCA did not modify heart rate (305 ⫾ 35 b/min), but occlusion of the LCCA was responsible for a slight decrease in heart rate, 263 ⫾ 32 beats/min (p 0.05). Just after release of LCCA occlusion, the heart rate was 310 ⫾ 38 beats/min and remained stable at h5, then increased
917
at 368 ⫾ 8 beats/min at h24 (p 0.001) and returned to basal value at h48 (310 ⫾ 34 b/min). Left ICA blood flow velocities changes In the left ICA, peak systolic, end-diastolic and time-average mean blood flow velocities were respectively 42.6 ⫾ 7.6, 12.6 ⫾ 2.5 and 12.5 ⫾ 2.7 cm.s–1 on basal state. Trepanation and coagulation of the left MCA did not modify blood flow values in the left ICA, but occlusion of the left CCA was responsible for a “circulatory stop” in the left ICA revealed by the absence of Doppler signal in color-coded Doppler mode as well as in pulsed Doppler mode (Fig. 4, Fig. 5). In three rat pups (1 male, 2 female), release of the occlusion of the LCCA was followed by restoration of perfusion in the left ICA with reappearance of colorcoded Doppler and pulsed Doppler signal in the left ICA (Fig. 4, Fig. 5). Thus, peak systolic blood flow velocity reached 31.8 ⫾ 7.9, end-diastolic blood flow velocity 8.8 ⫾ 2.1 and time-average mean blood flow velocity 8.6 ⫾ 2.3 cm.s–1 lower when compared with basal values (p 0.05). Blood flow velocities reached basal values at h24 and h48 (Fig. 6.). In seven rat pups (3 male, 4 female), release of the occlusion of the left common artery was followed by a reversed flow in the left ICA, revealed by negative blood flow velocities recorded with spectral analysis of the pulsed Doppler signal (Fig. 5.). The reserved flow increased until h24 and peak systolic blood flow velocity reached –23.5 ⫾ 8.1, end-diastolic – 6.3 ⫾ 1.2, time-average mean –7.5 ⫾ 2.6 cm.s–1 (p 0.001 vs. basal values) and persisted at h48. Right ICA blood flow velocities changes In the right ICA, peak systolic, end-diastolic and time-average mean blood flow velocities were, respectively, 38.0 ⫾ 6.7, 10.2 ⫾ 2.5 and 11.2 ⫾ 1.8 cm.s–1 on basal state. Trepanation and coagulation of the distal left MCA responsible for an increase in blood flow velocities and peak systolic, end-diastolic and time-average mean blood flow velocities reached, respectively, 47.0 ⫾ 8.4, (p 0.01 vs. basal values), 13.9 ⫾ 3.9, (p 0.05 vs. basal values) and 13.2 ⫾ 3.2 cm.s–1 (ns). During occlusion of the LCCA, peak systolic, end-diastolic and time-average mean blood flow velocities increased, respectively, up to 57.9 ⫾ 15.4 (p 0.001 vs. basal values), 17.0 ⫾ 4.1 (p 0.0001 vs. basal values) and 17.6 ⫾ 5.1 (p 0.001 vs. basal values) cm.s–1. In the group of three rat pups with restoration of blood flow in the left ICA after release of the occlusion of the LCCA, blood flow velocities in the right ICA returned to basal values. In the group of seven rats pups with absence of blood flow restoration in the left ICA after release of the occlusion of the
918
Ultrasound in Medicine and Biology
Volume 34, Number 6, 2008
Fig. 4. Colour-coded Doppler imaging of the rat pup head during procedure of cerebral ischemia reperfusion (coagulation of the left MCA – occlusion of the LCCA – release of the occlusion). Two-dimensional and color-coded Doppler ultrasound imaging obtained with a cross-sectional image of the rat pup head. During occlusion of the LCCA, the Doppler signal in the left ICA is lost or reversed, coded in blue. The right ICA, the right and left MCAs remained injected. Just after the occlusion release of the LCCA, seven rat pups presented an absence of blood flow restoration in the left ICA or a reversed flow coded in blue color, three rat pups presented blood flow restoration in the ICA. Identical ultrasound imaging was obtained 5 h after left CCA occlusion release in the two groups.
LCCA, blood flow velocities in the right ICA remained higher than basal values and than values presented by the other group, i.e., peak systolic, enddiastolic and time-average mean blood flow velocities were, respectively, 56.7 ⫾ 15.2 (p 0.001 vs. basal values, p 0.01 vs. rat pups with left ICA blood flow restoration), 15.9 ⫾ 5.6 (p 0.001 vs. basal values, p 0.01 vs. rat pups with left ICA blood flow restoration) and 15.6 ⫾ 3.5 (p 0.001 vs. basal values, p 0.01 vs. rat pups with left ICA blood flow restoration) cm.s–1. At h5, blood flow velocities in the right ICA returned to basal values in the two groups and remained stable at h24 and h48 (Fig. 6.).
Basilar trunk blood flow velocities changes In the BT, at basal states, peak systolic, end-diastolic and time-average mean blood flow velocities were 30.5 ⫾ 1.1, 8.5 ⫾ 1.8 and 9.5 ⫾ 1.9 cm.s–1, respectively. Trepanation and coagulation of the distal left MCA did not significantly modify blood flow velocities in the BT. Nevertheless, blood flow velocities increased during occlusion of the left CCA and reached 42.2 ⫾ 8.8 (p 0.001 vs. basal values), 13.5 ⫾ 2.9 (p 0.0001 vs. basal values), 12.7 ⫾ 2.6 (p 0.01 vs. basal values) cm.s–1, respectively, for peak systolic, end-diastolic and time-average mean blood flow velocities. Blood flow velocities in the BT then returned to basal values after the release of the
Fig. 5. Pulsed Doppler with spectral analysis of the Doppler signal recorded in the left ICA during procedure of cerebral ischemia reperfusion (coagulation of the left MCA – occlusion of the LCCA – release of the occlusion). Pulsed Doppler with spectral analysis of the Doppler signal provides access to measurement of systolic, end-diastolic blood flow velocities and time-averaged mean blood flow velocities (blue line). Just after release of the occlusion of the LCCA, seven rat pups presented an absence of Doppler signal in the left ICA, and an actual reversed blood flow velocities five hours after release, three rat pups presented a blood flow restoration in the left ICA persistent 5 h after release.
Doppler of cerebral arteries in rat pups ● P. BONNIN et al.
919
Fig. 6. Time-course of changes of the time-average mean blood flow velocities recorded in the right and the left ICAs during procedure of cerebral ischemia-reperfusion (coagulation of the left MCA – occlusion of the LCCA – release of the occlusion). Coagulation of the left MCA did not modify blood flow velocities (BFV) in the left ICA. Occlusion of the LCCA annulled BFV in the left ICA and increased blood flow velocities in the right ICA in both groups. Just after occlusion release of the LCCA, blood flow injection of the left ICA was recovered in three rat pups, blood flow injection of the left ICA was not recovered in seven rats pups (**p 0.01, LCCA BFV restoration group vs. absence of LCCA BFV restoration group). BFV in the right ICA remained increased in rat pups with absence of BFV restoration in the LCCA (*p 0.05, left CCA BFV restoration group vs. absence of LCCA BFV restoration group). Five, 24 and 48 h after occlusion release, rat pups with absence of BFV restoration in the LCCA presented reversed BFVs in the left ICA. Five, 24 and 48 h after occlusion release, rats pups with BFV restoration in the LCCA presented BFV in the left ICA, reaching basal values.
occlusion of the LCCA without any significant difference between the two groups (presence or absence of blood flow restoration in the left ICA after release of the occlusion of the LCCA). Blood flow velocities then remained stable from h5 to h48 (Fig. 7.). Left MCA blood flow velocities changes In the left MCA at basal states, peak systolic, enddiastolic and time-average mean blood flow velocities
Fig. 7. Time-course of changes of the time-average mean BFVs recorded in the BT during the procedure of cerebral ischemiareperfusion (coagulation of the left MCA – occlusion of the LCCA – release of the occlusion). During the occlusion of the LCCA, BFV recorded in the BT were increased in both groups, and remained higher in group with absence of blood flow restoration in the LCCA compared with basal values, whereas BFV returned to basal values in the group with blood flow restoration in the LCCA.
were 16.0 ⫾ 3.0, 5.1 ⫾ 1.1 and 5.2 ⫾ 1.0 cm.s–1, respectively. Trepanation and electrocoagulation of the left MCA did not modify peak systolic or time-average mean blood flow velocities. End-diastolic blood flow velocity was slightly increased to 6.5 ⫾ 1.7 (p 0.05 vs. basal values) cm.s–1. The occlusion of the LCCA did not involve a “circulatory stop” in the left MCA (Fig. 8), but a decrease in peak systolic, end-diastolic and time-average mean blood flow velocities, respectively, to 9.5 ⫾ 2.6 (p 0.0001 vs. basal values), 4.0 ⫾ 0.7 (p 0.05 vs. basal values) and 3.4 ⫾ 0.7 p 0.0001 vs. basal values) cm.s–1. After release of the occlusion of the LCCA, blood flow velocities were restored at basal values in the group of three rat pups with blood flow restoration in the left ICA. In contrary, blood flow velocities remained slightly lower in the group with absence of blood flow restoration in the left ICA, i.e., peak systolic, end-diastolic and time-average mean blood flow velocities were 11.9 ⫾ 2.3 (p 0.05 vs. basal values) 4.7 ⫾ 0.5 and 4.0 ⫾ 0.5 cm.s–1, respectively. Blood flow velocities then returned to basal values from h5 to h48 (Fig. 9.). Right MCA blood flow velocities changes In the right MCA, there were no significant modifications in peak systolic, end-diastolic as well as time-average mean blood flow velocities during the whole procedure of cerebral ischemia reperfusion. Blood flow velocities remained stable (Fig. 9.). No significant difference were observed in all measured blood flow velocities in all studied arteries between the two groups (presence or absence of
920
Ultrasound in Medicine and Biology
Volume 34, Number 6, 2008
Fig. 8. Pulsed Doppler with spectral analysis of the Doppler signal recorded in the left MCA during the procedure of cerebral ischemia reperfusion (coagulation of the left MCA – occlusion of the LCCA – release of the occlusion). BFV decreased during occlusion of the LCCA and remained stable in the group of rat pups with absence of blood flow restoration in the LCCA. In the group with blood flow restoration in the LCCA, BFV in the left MCA reached basal values.
blood flow restoration in the left ICA after release of the occlusion of the LCCA), during the whole procedure. DISCUSSION To our knowledge, cerebral circulation in rat pups was investigated for the first time in this study with ultrasound imaging and we evidenced that measurements of blood flow velocities in cerebral arteries were feasible and reproducible in 7-day-old rat pups. At this stage, we characterized variations of peak systolic, end-diastolic and time-average mean blood flow velocities in the cerebral arteries, which occurred during experimental procedure of cerebral ischemia-reperfusion (electrocoagulation of the left MCA – occlusion of the LCCA – release of the occlusion). Our new approach to study cerebral circulation in the rat pup was possible thanks to technical improvement of ultrasound apparatus. This conventional method is essentially a tool for imaging the macrocirculation and is
a simple, acute and currently available technique, which could be a valuable tool to assess circulatory changes in the cerebral vasculature. The small size of the rat pup, associated with heart rates in excess of 300 beats/min, present unique methodological challenges for peripheral vessel ultrasound imaging. Recently developed transducers are capable of both high frame-rate images and improved near-field imaging, thereby generating high-quality images (Collins et al. 2003). Ultrasound biomicroscopy (UBM) use ultrasound frequency of more than 15 MHz, generally 20 to 60 MHz. Although UBM is very useful when available for looking at superficial organs and vascularization using contrast-enhanced power Doppler mode, “clinical” Doppler ultrasound systems seem to be more widely available for the study of peripheral vessels especially cerebral circulation in very small animals. Indeed, penetration depth of the ultrasound beam is inversely related to ultrasound frequency,
Fig. 9. Time-course of changes of the time-average mean blood flow velocities recorded in the right MCA and left MCA during the procedure of cerebral ischemia reperfusion (coagulation of the left MCA – occlusion of the LCCA – release of the occlusion). Coagulation of the distal left MCA did not modify BFV recorded in the proximal left MCA. During occlusion of the LCCA, BFV in the left MCA decreased in both groups. BFV in the left MCA reached basal value after release of the LCCA occlusion. No significant changes were observed in the RMCA during procedure, no difference was observed between the two groups.
Doppler of cerebral arteries in rat pups ● P. BONNIN et al.
temporal resolution is generally better on clinical systems with frame rate of 45 to 110 Hz (frame per second) in 2-D imaging mode than on actually disposable UBM systems even when improved from 8 to 10 Hz at 60 to 100 Hz using mechanical sector scanning (Phoon 2006). Furthermore, adaptation of the depth of focusing is not available on UBM (McVeigh 2006). Usefulness of the UBM technology within mouse vascular study remains then to be explored and few studies report data on arterial wall ultrasound imaging, atherosclerosis in ascending aorta (Gan et al. 2007). Interestingly, studies reporting data on blood flow velocity in the aorta of mouse fetuses or umbilical and uterine arteries are more numerous (Mu and Adamson 2006; Phoon et al. 2000, 2002). Recently, ultrahigh frame rate ultrasound micro-imaging (10 kHz) has been used for blood flow visualization in carotid arteries in mice, but after retrospective reconstruction with a prototype echograph (Cherin et al. 2006). We wondered whether nonenhanced 2-D color-coded pulsed Doppler ultrasound imaging with a 12-MHz Doppler ultrasound could be currently used for imaging cerebral vessels in rat pup. Indeed, high frame rate color Doppler mode provides a real-time color Doppler imaging of vessels allowing a good localization for pulsed Doppler acquisition. Twelve-MHz transducer provides, indeed, adapted and sufficient near-field focusing (⬍0.5 cm) and spatial resolution (axial resolution less than 0.1 mm) to recognize details as skull, brain and cerebral vascular structures of the rat pup. We wondered whether 12-MHz ultrasound imaging could be feasible in cerebral vessels and could represent an accurate and reproducible alternative tool to monitor cerebral circulatory changes in rat pups occurring during cerebral ischemia as applied in humans. Indeed, the accuracy of this technique has been validated for comparison transcranial flow velocity and cerebral blood flow during focal ischemia in adult animals (Els et al. 1999) and has been used in term human newborns with hypoxic–ischemic encephalopathy (Kirimi et al. 2002). This noninvasive method could be repeated allowing longitudinal survey during the whole duration and at each step of the protocol. This method allows analysis of rapid changes in blood flow as early as arterial occlusion is performed. Moreover, the pups could be used for long-term analysis with light limitations. Indeed, in the MCA, absolute values of systolic, diastolic and time-average mean blood flow velocities at basal state were about 16.0, 5.1 and 5.2 cm.s–1. Maximum differences between two measurements at different times of the protocol were about 8.0, 1.5 and 2.0 cm.s–1 for systolic, diastolic and mean velocities respectively. The intra or interobserver repeatability coefficient (RC) for measurement of blood flow velocity was about 1.5 cm.s–1, low enough to retain as significant variations of systolic blood flow velocities, but much too high to consider as
921
significant the diastolic and mean blood flow velocities variations, i.e., RC shared in more than 50% of the effect. Thus, small velocities in the middle cerebral could constitute a limitation to evidence relative changes with the use of 12-MHz ultrasound imaging. Nevertheless, in all the other studied arteries, changes in blood flow velocities reached three to tenfold the RC value for diastolic and mean blood flow velocities and three to 30-fold the RC value for systolic blood flow velocities, high enough to consider as significant the observed changes in blood flow velocities. Other methods, which attempted to measure cerebral blood flow, are invasive. Using iodo [14C] antipyrine method, only informations about regional cerebral blood flow are given and the sacrifice of the animal is required (Nehlig et al. 1989; Mujsce et al. 1990), then avoiding survey in the same animal. In a same manner, the hydrogen clearance technique allowed the measurement only in the region where the electrodes are inserted (Mitsufuji et al. 1996). We demonstrated the efficacy of collateral arterial supply through the Willis circle between upstream the carotid arteries and vertebral-basilar system, and downstream the intracerebral arteries. In fact, electrocoagulation of the left MCA at its median portion, which is responsible for a local distal arterial occlusion and the additional occlusion of the LCCA is responsible for a blood flow stop in the ICA. Nevertheless, we demonstrated that blood flow velocities did not fall dramatically in the left MCA, but decreased by only 35%, whereas blood flow velocities increased in the right carotid artery by 57% and in the BT by 34%. The left MCA in its proximal part own many collateral branches upstream the arterial electrocoagulation of the left MCA, which can participate to blood flow supply downstream the occlusion. This supply could occur through “very fast opening” collateral vessels at surface of the brain, even when left common carotid artery is occluded. Furthermore, we evidenced with ultrasound imaging a strong increase in blood flow velocities in the anterior communicant artery from right to left side and the increase in blood flow velocities in the left posterior communicant from BT to distal left ICA (data not shown). Additional supply could occur through the numerous anastomoses that exist in rat pups between middle cerebral artery and anterior and posterior cerebral arteries (Menzies et al. 1992). The role of the anastomoses during reperfusion has been previously reported using black ink coloration technique (Murakami et al. 1998). In addition, during the procedure of cerebral ischemia-reperfusion, blood flow supply of the left MCA through the Willis circle did not alter blood flow supply of the right MCA. So, using a simple, noninvasive method we have confirmed that in this model, the association of transient homolateral carotid artery and permanent MCA occlusion is at least necessary to create downstream a situation of low cerebral blood flow in the ipsilateral hemisphere.
922
Ultrasound in Medicine and Biology
After the release of the occlusion of the LCCA, only three rat pups obtained blood flow restoration in the left carotid artery and seven rat pups presented a persistent occlusion probably because of nonreversible parietal lesion of the artery by the clamp and thrombosis of the artery. This percentage was probably a result of the drastic conditions we used for LCCA occlusion in this study. Even when occlusion of the LCCA was persistent, rat pups presented blood flow adaptation through the Willis circle to maintain and restore the blood flow supply in the proximal part of the left MCA at least during the 48 h of the study. Moreover, blood flow velocities were reversed in the left ICA, to supply the left external carotid artery. Taken together, all these data confirm the great variability of the reflow after clip removal, which could in part explain the variability in size of the ischemic lesion. Indeed, the score lesion, as previously reported (Joly et al. 2004), presented by animal in the group with reperfusion of the LCCA was decreased compared with the others without reperfusion of the artery (data not shown). In conclusion, we propose the use of ultrasound imaging for examination of the cerebral circulation in the rat pup with relatively high-frequency ultrasound device. This clinical, useful, reproducible, noninvasive, easy-torepeat method allows the assessment and the monitoring of the cerebral blood flow in the great arteries in very small animals and could help to characterize the dramatic cerebral hemodynamic changes occurring in our model of ischemia reperfusion, particularly the depth of the hypoperfusion phase responsible of cerebral ischemia, as well as the variability of the reflow. This method could be very useful in further studies using potential neuroprotective treatments to identify in each group of animals what can be assigned to the effect of the treatment and what depends on the blood flow supply. Acknowledgments—This work was supported by a grant from “RégionIle-de-France.”
REFERENCES Benjelloun N, Renolleau S, Represa A, Ben-Ari Y, Charriaut-Marlangue C. Inflammatory responses in the cerebral cortex after ischemia in the P7 neonatal Rat. Stroke 1999;30:1916–1923, discussion 1923–1914. Bonnin P, Villemain A, Vincent F, Debbabi H, Silvestre JS, Contreres JO, Levy BI, Tobelem G, Dupuy E. Ultrasonic assessment of hepatic blood flow as a marker of mouse hepatocarcinoma. Ultrasound Med Biol 2007;33:561–570. Cherin E, Williams R, Needles A, Liu G, White C, Brown AS, Zhou YQ, Foster FS. Ultrahigh frame rate retrospective ultrasound microimaging and blood flow visualization in mice in vivo. Ultrasound Med Biol 2006;32:683– 691. Collins KA, Korcarz CE, Lang RM. Use of echocardiography for the phenotypic assessment of genetically altered mice. Physiol Genomics 2003;13:227–239. Edwards AD, Azzopardi DV. Perinatal hypoxia-ischemia and brain injury. Pediatric Res 2000;47:431– 432.
Volume 34, Number 6, 2008 Els T, Daffertshofer M, Schroeck H, Kuschinsky W, Hennerici M. Comparison of transcranial Doppler flow velocity and cerebral blood flow during focal ischemia in rabbits. Ultrasound Med Biol 1999;25:933–938. Ferriero DM. Neonatal brain injury. N Engl J Med 2004;351:1985–1995. Freud S. Infantile Cerebral Paralysis. Russin LA, trans. Coral Gables, FL: University of Miami Press; 1968. Gan LM, Gronros J, Hagg U, Wikstrom J, Theodoropoulos C, Friberg P, Fritsche-Danielson R. Non-invasive real-time imaging of atherosclerosis in mice using ultrasound biomicroscopy. Atherosclerosis 2007;190:313–320. Hoit BD, Kiatchoosakun S, Restivo J, Kirkpatrick D, Olszens K, Shao H, Pao YH, Nadeau JH. Naturally occurring variation in cardiovascular traits among inbred mouse strains. Genomics 2002;79:679–685. Joly LM, Benjelloun N, Plotkine M, Charriaut-Marlangue C. Distribution of Poly(ADP-ribosyl)ation and cell death after cerebral ischemia in the neonatal rat. Pediatr Res 2003;53:776 –782. Kirimi E, Tuncer O, Atas B, Sakarya ME, Ceylan A. Clinical value of color Doppler ultrasonography measurements of full-term newborns with perinatal asphyxia and hypoxic ischemic encephalopathy in the first 12 hours of life and long-term prognosis. Tohoku J Exp Med 2002;197:27–33. Lee J, Croen LA, Lindan C, Nash KB, Yoshida CK, Ferriero DM, Barkovich AJ, Wu YW. Predictors of outcome in perinatal arterial stroke: A population-based study. Ann Neurol 2005;58:303–308. McVeigh ER. Emerging imaging techniques. Circ Res 2006;98:879 – 886. Menzies SA, Hoff JT, Betz AL. Middle cerebral artery occlusion in rats: A neurological and pathological evaluation of a reproducible model. Neurosurgery 1992;31:100 –106, discussion 106 –107. Mitsufuji N, Yoshioka H, Okano S, Nishiki T, Sawada T. A new model of transient cerebral ischemia in neonatal rats. J Cereb Blood Flow Metab 1996;16:237–243. Mu J, Adamson SL. Developmental changes in hemodynamics of uterine artery, utero- and umbilicoplacental, and vitelline circulations in mouse throughout gestation. Am J Physiol Heart Circ Physiol 2006;291:H1421–1428. Mujsce DJ, Christensen MA, Vannucci RC. Cerebral blood flow and edema in perinatal hypoxic-ischemic brain damage. Pediatr Res 1990;27:450 – 453. Murakami K, Kondo T, Kawase M, Chan PH. The development of a new mouse model of global ischemia: Focus on the relationships between ischemia duration, anesthesia, cerebral vasculature, and neuronal injury following global ischemia in mice. Brain Res 1998;780:304 –310. Nehlig A, Pereira de Vasconcelos A, Boyet S. Postnatal changes in local cerebral blood flow measured by the quantitative autoradiographic [14C]iodoantipyrine technique in freely moving rats. J Cereb Blood Flow Metab 1989;9:579 –588. Nelson KB. Perinatal ischemic stroke. Stroke 2007;38:742–745. Northington FJ. Brief update on animal models of hypoxic-ischemic encephalopathy and neonatal stroke. Ilar J 2006;47:32–38. Phoon CK, Aristizabal O, Turnbull DH. 40 MHz Doppler characterization of umbilical and dorsal aortic blood flow in the early mouse embryo. Ultrasound Med Biol 2000;26:1275–1283. Phoon CK, Aristizabal O, Turnbull DH. Spatial velocity profile in mouse embryonic aorta and Doppler-derived volumetric flow: A preliminary model. Am J Physiol Heart Circ Physiol 2002;283:H908–916. Phoon CK. Imaging tools for the developmental biologist: Ultrasound biomicroscopy of mouse embryonic development. Pediatr Res 2006;60:14 –21. Renolleau S, Aggoun-Zouaoui D, Ben-Ari Y, Charriaut-Marlangue C. A model of transient unilateral focal ischemia with reperfusion in the P7 neonatal rat: morphological changes indicative of apoptosis. Stroke 1998;29:1454 –1460, discussion 1461. Renolleau S, Fau S, Goyenvalle C, Joly LM, Chauvier D, Jacotot E, Mariani J, Charriaut-Marlangue C. Specific caspase inhibitor QVD-OPh prevents neonatal stroke in P7 rat: A role for gender. J Neurochem 2007;100:1062–1071. Salonen R, Lichtenstein P, Bellocco R, Petersson G, Cnattingius S. Increased risks of circulatory diseases in late pregnancy and puerperium. Epidemiology 2001;12:456 – 460.
doi:10.1016/j.ultrasmedbio.2007.11.018
● Original Contribution ULTRASONIC ASSESSMENT OF CEREBRAL BLOOD FLOW CHANGES DURING ISCHEMIA-REPERFUSION IN 7-DAY-OLD RATS PHILIPPE BONNIN,*† HAYTHEM DEBBABI,* JEAN MARIANI,‡§ CHRISTIANE CHARRIAUT-MARLANGUE,‡ and SYLVAIN RENOLLEAU‡¶ *AP-HP, Hôpital Lariboisière, Physiologie–Explorations Fonctionnelles, Université Denis Diderot Paris 7, Paris; Cardiovascular Research Center INSERM Lariboisière, INSERM U689, Paris; ‡UMR-CNRS 7102, Equipe HICD, Université Pierre et Marie Curie Paris 6, Paris; §AP-HP, Hôpital Charles Foix, Unité d’Explorations Fonctionnelles, Ivry-sur-Seine; and ¶AP-HP, Service de Réanimation, Hôpital Armand Trousseau, Université Pierre et Marie Curie Paris 6, Paris, France †
(Received 30 July 2007; revised 15 November 2007; in final form 27 November 2007)
Abstract—A model of ischemic brain injury in 7-day-old rat pups has been developed to study perinatal ischemia. It combines permanent occlusion of the distal left middle cerebral artery (LMCA) and transient occlusion of homolateral common carotid artery (LCCA). At removal of the clip on LCCA, reflow allowed brain reperfusion through cortical anastomoses. In 10 rat pups, we measured blood flow velocities (BFV) in main cerebral arteries with 12-MHz ultrasound imaging. At basal states, peak systolic BFV in proximal LMCA was 16.0 ⴞ 3.0 cm.s–1. Occlusion of LMCA did not yield significant modifications. Occlusion of LCCA involved only a decrease in BFV to 9.5 ⴞ 2.6 cm.s–1 (p < 0.001). Indeed, LMCA was then supply by the right internal carotid and the vertebral arteries through the circle of Willis. In three rat pups, release of occlusion of LCCA was followed by restoration of BFV in the left internal carotid artery and in LMCA, in seven pups, by a reversed flow in the LICA and lower BFV in LMCA (11.9 ⴞ 2.3, p < 0.05). BFV returned to basal values from h5 to h48 in all animals. In addition, ultrasound imaging is a useful, reproducible, non invasive, easy-to-repeat, method to assess and monitor arterial cerebral blood flow supply in small animals. It helps to characterize changes occurring during cerebral ischemia and reperfusion, particularly the depth of the hypoperfusion, as well as the variability of reflow. In preclinical studies, this method could help to identify what can be assigned to a neuroprotective treatment and what depends on changes in cerebral blood flow supply. (E-mail: [email protected]) © 2008 World Federation for Ultrasound in Medicine & Biology. Key Words: Neonatal brain ischemia, Ultrasound imaging, Pulsed Doppler, Blood flow velocity, Internal carotid artery, Basilar trunk, Middle cerebral artery, Rat pup.
INTRODUCTION
was recognized in the newborn period and in 82% of those with retrospectively diagnosed HI (Lee et al. 2005). Diffuse HI is caused by transient decrease cerebral blood flow when a perinatal anoxia occurs (uterine membranes rupture, retroplacental hematoma. Focal HI is caused by the same factors that bring about the majority of cases of cerebral infarction of adults: dissection, embolism and thrombosis of cerebral vessels (Freud et al. 1968). Normal pregnancy is a procoagulant and proinflammatory condition. Around time of birth, coagulation mechanisms are already activated in anticipation of hemorrhage, which threatens the life of mother and fetus. Unfortunately, the adaptation that decreases the risk of bleeding increases the risk of clotting (Salonen et al. 2001). HI may thus result from thrombosis of intracranial vessels or from embolism from another site such as
Perinatal brain hypoxia and ischemia (HI) is a cerebrovascular event occurring during fetal or neonatal life with pathologic or radiologic evidence of focal arterial infarction of brain. (Nelson 2007). Incidence is approximately one case for 4,000 children (Ferriero 2004) and essentially involves the vascular territory supplied by the middle cerebral artery when an anatomic vascular territory is involved. Hemiplegic cerebral palsy, seizure and cognitive disorders are the common outcomes of PAS, present later in 37% of children whose cerebral injury
Address correspondence to: Dr. Philippe Bonnin, PhysiologieExplorations Fonctionnelles, Hôpital Lariboisière, 2, rue Ambroise Paré, 75 475 Paris CEDEX 10 France. E-mail: philippe.bonnin@ lrb.aphp.fr 913
914
Ultrasound in Medicine and Biology
extracranial vessels, heart, umbilical vein or placenta. Although the site of origin is usually not clearly established, it is suspected that the fetal side of the placenta may often be the source. Indeed, the presence of a patent foramen ovale enables clots from the fetal side of the placenta to embolize into the fetus, through the umbilical vein, ductus venosus, inferior vena cava, right then left auricles and aorta to reach the fetal brain. Despite recent improvement in neonatal intensive care management, the incidence of long-term disability after neonatal brain injury is not decreasing (Edwards and Azzopardi 2000; Ferriero 2004). To study cellular and molecular events during cerebral hypoxia and ischemia, several animal models have been developed (for review see Northington 2006). In all of the models with a reperfusion phase, the reperfusion time has been depicted as an important event for its potential benefits but also its deleterious effects (Benjelloun et al. 1999). We recently developed a model of ischemic brain injury in 7-day-old rat pups. This model combines a permanent occlusion of the left middle cerebral artery (LMCA), with a transient occlusion of the homolateral common carotid artery (HCCA). After the removal of the clip on the common carotid artery, reflow in the ischemic area, downstream from the occlusion of the middle cerebral artery, is allowed through cortical anastomoses providing from the middle cerebral artery upstream the occlusion, and the anterior and posterior ipsilateral cerebral arteries (Renolleau et al. 1998). In this model, the size of the ischemic lesion is not constant and varies from 45 to 60 mm3, e.g., 18 to 24% of the ipsilateral hemisphere without possibilities of prediction for size and without possibilities to control the level of reflow (Ducros et al. 2000; Joly et al. 2004; Renolleau et al. 2007). Our hypothesis was that the size variability of the lesion could depend on the efficiency of the restoration of blood flow in the carotid artery. Thus, our aim was to quantify the cerebral blood flow changes occurring during the whole procedure of cerebral ischemia–reperfusion to evidence heterogeneous cerebral blood flow supply. We thus investigated extra and intra cranial cerebral circulation with ultrasound imaging to analyze the blood flow redistribution in the main cerebral arteries, through the circle of Willis, thus, upstream from the occlusion of the LMCA and downstream from the common carotid, during and after removal of the clip on this artery. As performed in this study, the use of a 12-MHz transducer allowed visualization of the skull and brain structures in 2-D ultrasound. Colour-coded Doppler revealed the main cerebral branches, which presented sufficiently high blood velocities i.e., the internal carotid arteries, the basilar trunk, the anterior, middle and posterior cerebral arteries. We sought to measure and monitor arterial blood flow velocities changes in these arteries during the
Volume 34, Number 6, 2008
whole procedure of cerebral ischemia-reperfusion (coagulation of the LMCA – occlusion of the LCCA – release of the occlusion). Therefore, ultrasound imaging with measurements of blood flow velocities could constitute a useful, easy-to-repeat tool to monitor and characterize the circulatory changes in these experimental conditions. In that way, depth of the cerebral hypoperfusion responsible of the cerebral damage could be evaluated precisely. MATERIALS AND METHODS Anesthesia All animal experimentation was conducted in accordance with the French and European Community guidelines for the care and use of experimental animals. Isoflurane anesthesia was administered with a vaporizer (model 100-F, Ohio Medical Instruments, Cincinnati, OH, USA). Isoflurane induction was performed over 1 min in an isolation chamber with 0.50% isoflurane in 100% O2, and anesthesia was maintained during spontaneous breathing of the same mixture via a small nose cone as previously described (Bonnin et al. 2007). This procedure was sufficient to obtain sedation without cardiorespiratory depression during ultrasound study. Because hypothermia induced by anesthesia might modify cardiac function and heart rate, the animal was placed on a heating blanket (38° C) (Hoit et al. 2002). After sedation, the animal was placed in right lateral decubitus position for ultrasound acquisition. This position avoided placing pressure on the head of the rat pup with the ultrasound device. Perinatal ischemia—Surgical procedure Ischemia reperfusion was performed in ten 7-dayold rats (17–21g) of both sexes, as previously described (Renolleau et al. 1998), under isoflurane anesthesia. The anesthetized rat was positioned on its back and a median incision was made in the neck to expose the LCCA. The rat was then placed on the right side and an oblique skin incision was made between the ear and the eye. After excision of the temporal muscle, the cranial bone was removed from the frontal suture to a level below the zygomatic arch. Then, the left MCA, exposed just after its appearance over the rhinal fissure, was electrocoagulated at the inferior level of the cerebral vein. After this procedure, a clip was placed to occlude the LCCA. Rats were then placed in an incubator to avoid hypothermia. After 50 min, the clip was removed. Both neck and cranial skin incisions were then closed. During the surgical procedure, external body temperature was maintained at 36 –36.5 C. After recovery, pups were transferred to their mothers. This procedure was responsible for a stroke in the cortical and subcortical area as previously described (Renolleau et al. 1998; Joly et al. 2003).
Doppler of cerebral arteries in rat pups ● P. BONNIN et al.
915
Fig. 1. Schematic figure illustrating the general anatomy of cerebral arterial vessels for pup rat. Extra cerebral supply is supported by right and left internal carotid and vertebral arteries, intracerebral supply by right and left anterior, middle and posterior cerebral arteries. Extra cerebral supply is connected with intracerebral supply through the circle of Willis.
Ultrasound study We used an echocardiograph (Vivid 7, GE Medical Systems, Horten, Norway) equipped with a 12-MHz linear transducer (12L). Data were transferred online to an ultrasound image workstation for subsequent analysis (PC EchoPAC, GE Medical Systems). Ultrasound study consisted in Doppler spectral recordings in the right and left internal carotid arteries (ICAs), the basilar trunk, and the right and left MCAs for further analysis during the whole procedure of ischemia-reperfusion (Fig. 1). Ultrasound study was repeated for each rat pup at different time: (i) before surgery, (ii) after coagulation of the left MCA, (iii) after placement of the clip to occlude the LCCA, (iv) after removal of the clip, then (v) five hours
after procedure onset (h5), then (vi) 24 (h24) and (7) 48 h (h48). Two-dimensional ultrasound imaging was used to visualize a cross-sectional B-mode image of the skull and the brain structures of the animal. Color-coded Doppler was then activated and, extra and intracranial arteries were then drawn and localized on the screen by their color-coded blood flow. Just below skull base, the ultrasound image crossed the right and left ICAs coded in red color. Median and just above the skull base, the basilar trunk (BT) was coded in red color. Laterally, creeping along the skull, the right and the LMCAs were coded in red color (Fig. 2). A turn of 90° with the transducer revealed an anterior longitudinal B-mode im-
Fig. 2. Color-coded ultrasound imaging of the cerebral arteries in the rat pup. (a) Two-dimensional and color-coded Doppler ultrasound imaging obtained with a cross-sectional image of the rat pup head. Below skull base, right and left ICAs were coded in red color. Median and just above the skull base, the BT was coded in redr. Laterally, creeping along the skull, the right MCA and the left MCA were coded in red. (b) Two-dimensional and color-coded Doppler ultrasound imaging obtained with a median sagittal image of the head of the rat pup. The BT was coded in redr. Imaging depth was set at 2 cm. (c) Two-dimensional and color-coded Doppler ultrasound imaging obtained with a left parasagittal image of the rat pup head. The left ICA was coded in red crossing the skull base; the left posterior cerebral artery was coded in blue color.
916
Ultrasound in Medicine and Biology
Volume 34, Number 6, 2008
Fig. 3. Pulsed Doppler with spectral analysis of the Doppler signal recorded in basal states in the cerebral arteries in the rat pup. (a) Spectral analysis of the Doppler signal recorded in the left ICA; scale of the Doppler signal was set min – 0.2m.s–1, max 0.8 m.s–1. (b) Spectral analysis of the Doppler signal recorded in the BT, scale of the Doppler signal was set min – 0.1m.s–1, max 0.6 m.s–1. (c) Spectral analysis of the Doppler signal recorded in the left MCA in basal state, scale of the Doppler signal was set min – 0.06m.s–1, max 0.25 m.s–1. Blue line represented the time-average mean blood flow velocity.
age of the head of the rat pup. With a median sagittal image of the head of the rat pup, the BT was revealed coded in red (inversion of color-codage) (Fig. 2). With a right or left parasagittal image of the rat pup head, the right or the left ICA was revealed, coded in red crossing the skull base (inversion of color-codage). The left posterior cerebral artery was coded in blue (Fig. 2). Imaging depth was set at 2 cm when applying zoom, frame-rate was 49.5 frames/s. In the configuration selected for color Doppler ultrasound acquisition, the frequency emission was 6.7 MHz, pulsed repetition frequency set at 3.5 kHz, frame rate was 37.8 frames/s and low velocity rejection at 1.65 cm s–1. A pulsed Doppler sample was then placed on the longitudinal axis of each vessel and pulsed Doppler spectrum recorded with simple repositioning of the pulsed Doppler sample from right or ICA (Fig. 3) to BT (Fig. 3) and right or left MCA (Fig. 2) in the three different 2-D ultrasound images. For the left MCA, pulsed Doppler sample was placed 1 to 2 mm upstream the level of coagulation. In the configuration selected for
Doppler ultrasound acquisition, the frequency emission was 6.7 MHz, pulsed repetition frequency was set between 2.0 and 5.25 kHz, the sample volume at 1 mm, the low-velocity rejection at 0.6 cm s–1. The minimum detectable velocity of the rat pup imaging was then about 1 cm s–1, maximum between 25 and 70 cm s–1, consistent with blood flow in peripheral arterial vessels. Two-D color-Doppler ultrasound image was refreshed between each arterial vessel acquisition. Ultrasound beam width of pulsed Doppler was small enough (1.1 mm) to ensure that flow from neighboring vessels was not superimposed on the measurement volume centered on each studied artery. Peak systolic and end-diastolic blood flow velocities waveforms were measured from the pulsed Doppler spectrum of each artery by displacing calipers on the screen and time-average mean blood flow velocities by simple redrawing of the blue line drawn on the Doppler spectrum. Blood flow velocities were measured with correction of the angle between the long axis of each vessel and the Doppler beam. Use of the steer mode of the Doppler beam helped to avoid angle correction
Doppler of cerebral arteries in rat pups ● P. BONNIN et al.
greater than 15° for MCAs and greater than 60° for ICAs and basilar trunk. The transducer was positioned on the head of the rat pup at each step of the protocol. Ultrasound imaging and pulsed-Doppler recordings were repeated in each studied artery with correction of the angle between the Doppler beam and the long axis of the artery. Blood flow velocities were measured by the same investigator. Previously to this study, repeatability of blood flow velocities measurements had been investigated in 25 mice (Bonnin et al. 2007) by calculating the repeatability coefficient (RC) (British Standards Institution 1979), according to RC2 ⫽ ⌺Di2/N, where N is the sample size, Di the relative differences calculated in two series of paired measurements separated by two-minutes intervals. The RC values for intraobserver repeatability were 1.5 cm s–1 for the peak systolic, between 2.2 and 1.7 cm s–1 for the end-diastolic and between 2.5 and 1.7 cm s–1 for the mean BFV, respectively, in ICAs and MCAs. Interobserver RC values tested in other studies were not different from intraobserver RC values for systolic, diastolic and mean blood flow velocities in all studied arteries. Statistical analysis To evaluate the effect of coagulation of the left MCA followed by the occlusion of the LCCA, a KruskalWallis nonparametric test was used to analyze differences in peak systolic, end-diastolic and time-average mean blood flow velocities measured in the right and left ICA, in the right and left MCA, and in the BT at the different steps of the protocol. The test was applied in two different groups of rat pups, one group with blood flow restoration in the left CCA (n ⫽ 3), the second group with absence of blood flow restoration in the left CCA (n ⫽ 7). Intra and intergroup comparisons were completed by using a Mann-Whitney nonparametric test with a conservative Bonferroni test. Results are expressed as mean ⫾ standard deviation and a p value of less than 0.05 was considered statistically significant. RESULTS Ten rat pups underwent the complete surgical procedure and ultrasound analysis. Four of them were male and six were female. Steady states hemodynamic values On basal state, heart rate of the rat pups was 296 ⫾ 36 beats per minute. Trepanation and coagulation of the left MCA did not modify heart rate (305 ⫾ 35 b/min), but occlusion of the LCCA was responsible for a slight decrease in heart rate, 263 ⫾ 32 beats/min (p 0.05). Just after release of LCCA occlusion, the heart rate was 310 ⫾ 38 beats/min and remained stable at h5, then increased
917
at 368 ⫾ 8 beats/min at h24 (p 0.001) and returned to basal value at h48 (310 ⫾ 34 b/min). Left ICA blood flow velocities changes In the left ICA, peak systolic, end-diastolic and time-average mean blood flow velocities were respectively 42.6 ⫾ 7.6, 12.6 ⫾ 2.5 and 12.5 ⫾ 2.7 cm.s–1 on basal state. Trepanation and coagulation of the left MCA did not modify blood flow values in the left ICA, but occlusion of the left CCA was responsible for a “circulatory stop” in the left ICA revealed by the absence of Doppler signal in color-coded Doppler mode as well as in pulsed Doppler mode (Fig. 4, Fig. 5). In three rat pups (1 male, 2 female), release of the occlusion of the LCCA was followed by restoration of perfusion in the left ICA with reappearance of colorcoded Doppler and pulsed Doppler signal in the left ICA (Fig. 4, Fig. 5). Thus, peak systolic blood flow velocity reached 31.8 ⫾ 7.9, end-diastolic blood flow velocity 8.8 ⫾ 2.1 and time-average mean blood flow velocity 8.6 ⫾ 2.3 cm.s–1 lower when compared with basal values (p 0.05). Blood flow velocities reached basal values at h24 and h48 (Fig. 6.). In seven rat pups (3 male, 4 female), release of the occlusion of the left common artery was followed by a reversed flow in the left ICA, revealed by negative blood flow velocities recorded with spectral analysis of the pulsed Doppler signal (Fig. 5.). The reserved flow increased until h24 and peak systolic blood flow velocity reached –23.5 ⫾ 8.1, end-diastolic – 6.3 ⫾ 1.2, time-average mean –7.5 ⫾ 2.6 cm.s–1 (p 0.001 vs. basal values) and persisted at h48. Right ICA blood flow velocities changes In the right ICA, peak systolic, end-diastolic and time-average mean blood flow velocities were, respectively, 38.0 ⫾ 6.7, 10.2 ⫾ 2.5 and 11.2 ⫾ 1.8 cm.s–1 on basal state. Trepanation and coagulation of the distal left MCA responsible for an increase in blood flow velocities and peak systolic, end-diastolic and time-average mean blood flow velocities reached, respectively, 47.0 ⫾ 8.4, (p 0.01 vs. basal values), 13.9 ⫾ 3.9, (p 0.05 vs. basal values) and 13.2 ⫾ 3.2 cm.s–1 (ns). During occlusion of the LCCA, peak systolic, end-diastolic and time-average mean blood flow velocities increased, respectively, up to 57.9 ⫾ 15.4 (p 0.001 vs. basal values), 17.0 ⫾ 4.1 (p 0.0001 vs. basal values) and 17.6 ⫾ 5.1 (p 0.001 vs. basal values) cm.s–1. In the group of three rat pups with restoration of blood flow in the left ICA after release of the occlusion of the LCCA, blood flow velocities in the right ICA returned to basal values. In the group of seven rats pups with absence of blood flow restoration in the left ICA after release of the occlusion of the
918
Ultrasound in Medicine and Biology
Volume 34, Number 6, 2008
Fig. 4. Colour-coded Doppler imaging of the rat pup head during procedure of cerebral ischemia reperfusion (coagulation of the left MCA – occlusion of the LCCA – release of the occlusion). Two-dimensional and color-coded Doppler ultrasound imaging obtained with a cross-sectional image of the rat pup head. During occlusion of the LCCA, the Doppler signal in the left ICA is lost or reversed, coded in blue. The right ICA, the right and left MCAs remained injected. Just after the occlusion release of the LCCA, seven rat pups presented an absence of blood flow restoration in the left ICA or a reversed flow coded in blue color, three rat pups presented blood flow restoration in the ICA. Identical ultrasound imaging was obtained 5 h after left CCA occlusion release in the two groups.
LCCA, blood flow velocities in the right ICA remained higher than basal values and than values presented by the other group, i.e., peak systolic, enddiastolic and time-average mean blood flow velocities were, respectively, 56.7 ⫾ 15.2 (p 0.001 vs. basal values, p 0.01 vs. rat pups with left ICA blood flow restoration), 15.9 ⫾ 5.6 (p 0.001 vs. basal values, p 0.01 vs. rat pups with left ICA blood flow restoration) and 15.6 ⫾ 3.5 (p 0.001 vs. basal values, p 0.01 vs. rat pups with left ICA blood flow restoration) cm.s–1. At h5, blood flow velocities in the right ICA returned to basal values in the two groups and remained stable at h24 and h48 (Fig. 6.).
Basilar trunk blood flow velocities changes In the BT, at basal states, peak systolic, end-diastolic and time-average mean blood flow velocities were 30.5 ⫾ 1.1, 8.5 ⫾ 1.8 and 9.5 ⫾ 1.9 cm.s–1, respectively. Trepanation and coagulation of the distal left MCA did not significantly modify blood flow velocities in the BT. Nevertheless, blood flow velocities increased during occlusion of the left CCA and reached 42.2 ⫾ 8.8 (p 0.001 vs. basal values), 13.5 ⫾ 2.9 (p 0.0001 vs. basal values), 12.7 ⫾ 2.6 (p 0.01 vs. basal values) cm.s–1, respectively, for peak systolic, end-diastolic and time-average mean blood flow velocities. Blood flow velocities in the BT then returned to basal values after the release of the
Fig. 5. Pulsed Doppler with spectral analysis of the Doppler signal recorded in the left ICA during procedure of cerebral ischemia reperfusion (coagulation of the left MCA – occlusion of the LCCA – release of the occlusion). Pulsed Doppler with spectral analysis of the Doppler signal provides access to measurement of systolic, end-diastolic blood flow velocities and time-averaged mean blood flow velocities (blue line). Just after release of the occlusion of the LCCA, seven rat pups presented an absence of Doppler signal in the left ICA, and an actual reversed blood flow velocities five hours after release, three rat pups presented a blood flow restoration in the left ICA persistent 5 h after release.
Doppler of cerebral arteries in rat pups ● P. BONNIN et al.
919
Fig. 6. Time-course of changes of the time-average mean blood flow velocities recorded in the right and the left ICAs during procedure of cerebral ischemia-reperfusion (coagulation of the left MCA – occlusion of the LCCA – release of the occlusion). Coagulation of the left MCA did not modify blood flow velocities (BFV) in the left ICA. Occlusion of the LCCA annulled BFV in the left ICA and increased blood flow velocities in the right ICA in both groups. Just after occlusion release of the LCCA, blood flow injection of the left ICA was recovered in three rat pups, blood flow injection of the left ICA was not recovered in seven rats pups (**p 0.01, LCCA BFV restoration group vs. absence of LCCA BFV restoration group). BFV in the right ICA remained increased in rat pups with absence of BFV restoration in the LCCA (*p 0.05, left CCA BFV restoration group vs. absence of LCCA BFV restoration group). Five, 24 and 48 h after occlusion release, rat pups with absence of BFV restoration in the LCCA presented reversed BFVs in the left ICA. Five, 24 and 48 h after occlusion release, rats pups with BFV restoration in the LCCA presented BFV in the left ICA, reaching basal values.
occlusion of the LCCA without any significant difference between the two groups (presence or absence of blood flow restoration in the left ICA after release of the occlusion of the LCCA). Blood flow velocities then remained stable from h5 to h48 (Fig. 7.). Left MCA blood flow velocities changes In the left MCA at basal states, peak systolic, enddiastolic and time-average mean blood flow velocities
Fig. 7. Time-course of changes of the time-average mean BFVs recorded in the BT during the procedure of cerebral ischemiareperfusion (coagulation of the left MCA – occlusion of the LCCA – release of the occlusion). During the occlusion of the LCCA, BFV recorded in the BT were increased in both groups, and remained higher in group with absence of blood flow restoration in the LCCA compared with basal values, whereas BFV returned to basal values in the group with blood flow restoration in the LCCA.
were 16.0 ⫾ 3.0, 5.1 ⫾ 1.1 and 5.2 ⫾ 1.0 cm.s–1, respectively. Trepanation and electrocoagulation of the left MCA did not modify peak systolic or time-average mean blood flow velocities. End-diastolic blood flow velocity was slightly increased to 6.5 ⫾ 1.7 (p 0.05 vs. basal values) cm.s–1. The occlusion of the LCCA did not involve a “circulatory stop” in the left MCA (Fig. 8), but a decrease in peak systolic, end-diastolic and time-average mean blood flow velocities, respectively, to 9.5 ⫾ 2.6 (p 0.0001 vs. basal values), 4.0 ⫾ 0.7 (p 0.05 vs. basal values) and 3.4 ⫾ 0.7 p 0.0001 vs. basal values) cm.s–1. After release of the occlusion of the LCCA, blood flow velocities were restored at basal values in the group of three rat pups with blood flow restoration in the left ICA. In contrary, blood flow velocities remained slightly lower in the group with absence of blood flow restoration in the left ICA, i.e., peak systolic, end-diastolic and time-average mean blood flow velocities were 11.9 ⫾ 2.3 (p 0.05 vs. basal values) 4.7 ⫾ 0.5 and 4.0 ⫾ 0.5 cm.s–1, respectively. Blood flow velocities then returned to basal values from h5 to h48 (Fig. 9.). Right MCA blood flow velocities changes In the right MCA, there were no significant modifications in peak systolic, end-diastolic as well as time-average mean blood flow velocities during the whole procedure of cerebral ischemia reperfusion. Blood flow velocities remained stable (Fig. 9.). No significant difference were observed in all measured blood flow velocities in all studied arteries between the two groups (presence or absence of
920
Ultrasound in Medicine and Biology
Volume 34, Number 6, 2008
Fig. 8. Pulsed Doppler with spectral analysis of the Doppler signal recorded in the left MCA during the procedure of cerebral ischemia reperfusion (coagulation of the left MCA – occlusion of the LCCA – release of the occlusion). BFV decreased during occlusion of the LCCA and remained stable in the group of rat pups with absence of blood flow restoration in the LCCA. In the group with blood flow restoration in the LCCA, BFV in the left MCA reached basal values.
blood flow restoration in the left ICA after release of the occlusion of the LCCA), during the whole procedure. DISCUSSION To our knowledge, cerebral circulation in rat pups was investigated for the first time in this study with ultrasound imaging and we evidenced that measurements of blood flow velocities in cerebral arteries were feasible and reproducible in 7-day-old rat pups. At this stage, we characterized variations of peak systolic, end-diastolic and time-average mean blood flow velocities in the cerebral arteries, which occurred during experimental procedure of cerebral ischemia-reperfusion (electrocoagulation of the left MCA – occlusion of the LCCA – release of the occlusion). Our new approach to study cerebral circulation in the rat pup was possible thanks to technical improvement of ultrasound apparatus. This conventional method is essentially a tool for imaging the macrocirculation and is
a simple, acute and currently available technique, which could be a valuable tool to assess circulatory changes in the cerebral vasculature. The small size of the rat pup, associated with heart rates in excess of 300 beats/min, present unique methodological challenges for peripheral vessel ultrasound imaging. Recently developed transducers are capable of both high frame-rate images and improved near-field imaging, thereby generating high-quality images (Collins et al. 2003). Ultrasound biomicroscopy (UBM) use ultrasound frequency of more than 15 MHz, generally 20 to 60 MHz. Although UBM is very useful when available for looking at superficial organs and vascularization using contrast-enhanced power Doppler mode, “clinical” Doppler ultrasound systems seem to be more widely available for the study of peripheral vessels especially cerebral circulation in very small animals. Indeed, penetration depth of the ultrasound beam is inversely related to ultrasound frequency,
Fig. 9. Time-course of changes of the time-average mean blood flow velocities recorded in the right MCA and left MCA during the procedure of cerebral ischemia reperfusion (coagulation of the left MCA – occlusion of the LCCA – release of the occlusion). Coagulation of the distal left MCA did not modify BFV recorded in the proximal left MCA. During occlusion of the LCCA, BFV in the left MCA decreased in both groups. BFV in the left MCA reached basal value after release of the LCCA occlusion. No significant changes were observed in the RMCA during procedure, no difference was observed between the two groups.
Doppler of cerebral arteries in rat pups ● P. BONNIN et al.
temporal resolution is generally better on clinical systems with frame rate of 45 to 110 Hz (frame per second) in 2-D imaging mode than on actually disposable UBM systems even when improved from 8 to 10 Hz at 60 to 100 Hz using mechanical sector scanning (Phoon 2006). Furthermore, adaptation of the depth of focusing is not available on UBM (McVeigh 2006). Usefulness of the UBM technology within mouse vascular study remains then to be explored and few studies report data on arterial wall ultrasound imaging, atherosclerosis in ascending aorta (Gan et al. 2007). Interestingly, studies reporting data on blood flow velocity in the aorta of mouse fetuses or umbilical and uterine arteries are more numerous (Mu and Adamson 2006; Phoon et al. 2000, 2002). Recently, ultrahigh frame rate ultrasound micro-imaging (10 kHz) has been used for blood flow visualization in carotid arteries in mice, but after retrospective reconstruction with a prototype echograph (Cherin et al. 2006). We wondered whether nonenhanced 2-D color-coded pulsed Doppler ultrasound imaging with a 12-MHz Doppler ultrasound could be currently used for imaging cerebral vessels in rat pup. Indeed, high frame rate color Doppler mode provides a real-time color Doppler imaging of vessels allowing a good localization for pulsed Doppler acquisition. Twelve-MHz transducer provides, indeed, adapted and sufficient near-field focusing (⬍0.5 cm) and spatial resolution (axial resolution less than 0.1 mm) to recognize details as skull, brain and cerebral vascular structures of the rat pup. We wondered whether 12-MHz ultrasound imaging could be feasible in cerebral vessels and could represent an accurate and reproducible alternative tool to monitor cerebral circulatory changes in rat pups occurring during cerebral ischemia as applied in humans. Indeed, the accuracy of this technique has been validated for comparison transcranial flow velocity and cerebral blood flow during focal ischemia in adult animals (Els et al. 1999) and has been used in term human newborns with hypoxic–ischemic encephalopathy (Kirimi et al. 2002). This noninvasive method could be repeated allowing longitudinal survey during the whole duration and at each step of the protocol. This method allows analysis of rapid changes in blood flow as early as arterial occlusion is performed. Moreover, the pups could be used for long-term analysis with light limitations. Indeed, in the MCA, absolute values of systolic, diastolic and time-average mean blood flow velocities at basal state were about 16.0, 5.1 and 5.2 cm.s–1. Maximum differences between two measurements at different times of the protocol were about 8.0, 1.5 and 2.0 cm.s–1 for systolic, diastolic and mean velocities respectively. The intra or interobserver repeatability coefficient (RC) for measurement of blood flow velocity was about 1.5 cm.s–1, low enough to retain as significant variations of systolic blood flow velocities, but much too high to consider as
921
significant the diastolic and mean blood flow velocities variations, i.e., RC shared in more than 50% of the effect. Thus, small velocities in the middle cerebral could constitute a limitation to evidence relative changes with the use of 12-MHz ultrasound imaging. Nevertheless, in all the other studied arteries, changes in blood flow velocities reached three to tenfold the RC value for diastolic and mean blood flow velocities and three to 30-fold the RC value for systolic blood flow velocities, high enough to consider as significant the observed changes in blood flow velocities. Other methods, which attempted to measure cerebral blood flow, are invasive. Using iodo [14C] antipyrine method, only informations about regional cerebral blood flow are given and the sacrifice of the animal is required (Nehlig et al. 1989; Mujsce et al. 1990), then avoiding survey in the same animal. In a same manner, the hydrogen clearance technique allowed the measurement only in the region where the electrodes are inserted (Mitsufuji et al. 1996). We demonstrated the efficacy of collateral arterial supply through the Willis circle between upstream the carotid arteries and vertebral-basilar system, and downstream the intracerebral arteries. In fact, electrocoagulation of the left MCA at its median portion, which is responsible for a local distal arterial occlusion and the additional occlusion of the LCCA is responsible for a blood flow stop in the ICA. Nevertheless, we demonstrated that blood flow velocities did not fall dramatically in the left MCA, but decreased by only 35%, whereas blood flow velocities increased in the right carotid artery by 57% and in the BT by 34%. The left MCA in its proximal part own many collateral branches upstream the arterial electrocoagulation of the left MCA, which can participate to blood flow supply downstream the occlusion. This supply could occur through “very fast opening” collateral vessels at surface of the brain, even when left common carotid artery is occluded. Furthermore, we evidenced with ultrasound imaging a strong increase in blood flow velocities in the anterior communicant artery from right to left side and the increase in blood flow velocities in the left posterior communicant from BT to distal left ICA (data not shown). Additional supply could occur through the numerous anastomoses that exist in rat pups between middle cerebral artery and anterior and posterior cerebral arteries (Menzies et al. 1992). The role of the anastomoses during reperfusion has been previously reported using black ink coloration technique (Murakami et al. 1998). In addition, during the procedure of cerebral ischemia-reperfusion, blood flow supply of the left MCA through the Willis circle did not alter blood flow supply of the right MCA. So, using a simple, noninvasive method we have confirmed that in this model, the association of transient homolateral carotid artery and permanent MCA occlusion is at least necessary to create downstream a situation of low cerebral blood flow in the ipsilateral hemisphere.
922
Ultrasound in Medicine and Biology
After the release of the occlusion of the LCCA, only three rat pups obtained blood flow restoration in the left carotid artery and seven rat pups presented a persistent occlusion probably because of nonreversible parietal lesion of the artery by the clamp and thrombosis of the artery. This percentage was probably a result of the drastic conditions we used for LCCA occlusion in this study. Even when occlusion of the LCCA was persistent, rat pups presented blood flow adaptation through the Willis circle to maintain and restore the blood flow supply in the proximal part of the left MCA at least during the 48 h of the study. Moreover, blood flow velocities were reversed in the left ICA, to supply the left external carotid artery. Taken together, all these data confirm the great variability of the reflow after clip removal, which could in part explain the variability in size of the ischemic lesion. Indeed, the score lesion, as previously reported (Joly et al. 2004), presented by animal in the group with reperfusion of the LCCA was decreased compared with the others without reperfusion of the artery (data not shown). In conclusion, we propose the use of ultrasound imaging for examination of the cerebral circulation in the rat pup with relatively high-frequency ultrasound device. This clinical, useful, reproducible, noninvasive, easy-torepeat method allows the assessment and the monitoring of the cerebral blood flow in the great arteries in very small animals and could help to characterize the dramatic cerebral hemodynamic changes occurring in our model of ischemia reperfusion, particularly the depth of the hypoperfusion phase responsible of cerebral ischemia, as well as the variability of the reflow. This method could be very useful in further studies using potential neuroprotective treatments to identify in each group of animals what can be assigned to the effect of the treatment and what depends on the blood flow supply. Acknowledgments—This work was supported by a grant from “RégionIle-de-France.”
REFERENCES Benjelloun N, Renolleau S, Represa A, Ben-Ari Y, Charriaut-Marlangue C. Inflammatory responses in the cerebral cortex after ischemia in the P7 neonatal Rat. Stroke 1999;30:1916–1923, discussion 1923–1914. Bonnin P, Villemain A, Vincent F, Debbabi H, Silvestre JS, Contreres JO, Levy BI, Tobelem G, Dupuy E. Ultrasonic assessment of hepatic blood flow as a marker of mouse hepatocarcinoma. Ultrasound Med Biol 2007;33:561–570. Cherin E, Williams R, Needles A, Liu G, White C, Brown AS, Zhou YQ, Foster FS. Ultrahigh frame rate retrospective ultrasound microimaging and blood flow visualization in mice in vivo. Ultrasound Med Biol 2006;32:683– 691. Collins KA, Korcarz CE, Lang RM. Use of echocardiography for the phenotypic assessment of genetically altered mice. Physiol Genomics 2003;13:227–239. Edwards AD, Azzopardi DV. Perinatal hypoxia-ischemia and brain injury. Pediatric Res 2000;47:431– 432.
Volume 34, Number 6, 2008 Els T, Daffertshofer M, Schroeck H, Kuschinsky W, Hennerici M. Comparison of transcranial Doppler flow velocity and cerebral blood flow during focal ischemia in rabbits. Ultrasound Med Biol 1999;25:933–938. Ferriero DM. Neonatal brain injury. N Engl J Med 2004;351:1985–1995. Freud S. Infantile Cerebral Paralysis. Russin LA, trans. Coral Gables, FL: University of Miami Press; 1968. Gan LM, Gronros J, Hagg U, Wikstrom J, Theodoropoulos C, Friberg P, Fritsche-Danielson R. Non-invasive real-time imaging of atherosclerosis in mice using ultrasound biomicroscopy. Atherosclerosis 2007;190:313–320. Hoit BD, Kiatchoosakun S, Restivo J, Kirkpatrick D, Olszens K, Shao H, Pao YH, Nadeau JH. Naturally occurring variation in cardiovascular traits among inbred mouse strains. Genomics 2002;79:679–685. Joly LM, Benjelloun N, Plotkine M, Charriaut-Marlangue C. Distribution of Poly(ADP-ribosyl)ation and cell death after cerebral ischemia in the neonatal rat. Pediatr Res 2003;53:776 –782. Kirimi E, Tuncer O, Atas B, Sakarya ME, Ceylan A. Clinical value of color Doppler ultrasonography measurements of full-term newborns with perinatal asphyxia and hypoxic ischemic encephalopathy in the first 12 hours of life and long-term prognosis. Tohoku J Exp Med 2002;197:27–33. Lee J, Croen LA, Lindan C, Nash KB, Yoshida CK, Ferriero DM, Barkovich AJ, Wu YW. Predictors of outcome in perinatal arterial stroke: A population-based study. Ann Neurol 2005;58:303–308. McVeigh ER. Emerging imaging techniques. Circ Res 2006;98:879 – 886. Menzies SA, Hoff JT, Betz AL. Middle cerebral artery occlusion in rats: A neurological and pathological evaluation of a reproducible model. Neurosurgery 1992;31:100 –106, discussion 106 –107. Mitsufuji N, Yoshioka H, Okano S, Nishiki T, Sawada T. A new model of transient cerebral ischemia in neonatal rats. J Cereb Blood Flow Metab 1996;16:237–243. Mu J, Adamson SL. Developmental changes in hemodynamics of uterine artery, utero- and umbilicoplacental, and vitelline circulations in mouse throughout gestation. Am J Physiol Heart Circ Physiol 2006;291:H1421–1428. Mujsce DJ, Christensen MA, Vannucci RC. Cerebral blood flow and edema in perinatal hypoxic-ischemic brain damage. Pediatr Res 1990;27:450 – 453. Murakami K, Kondo T, Kawase M, Chan PH. The development of a new mouse model of global ischemia: Focus on the relationships between ischemia duration, anesthesia, cerebral vasculature, and neuronal injury following global ischemia in mice. Brain Res 1998;780:304 –310. Nehlig A, Pereira de Vasconcelos A, Boyet S. Postnatal changes in local cerebral blood flow measured by the quantitative autoradiographic [14C]iodoantipyrine technique in freely moving rats. J Cereb Blood Flow Metab 1989;9:579 –588. Nelson KB. Perinatal ischemic stroke. Stroke 2007;38:742–745. Northington FJ. Brief update on animal models of hypoxic-ischemic encephalopathy and neonatal stroke. Ilar J 2006;47:32–38. Phoon CK, Aristizabal O, Turnbull DH. 40 MHz Doppler characterization of umbilical and dorsal aortic blood flow in the early mouse embryo. Ultrasound Med Biol 2000;26:1275–1283. Phoon CK, Aristizabal O, Turnbull DH. Spatial velocity profile in mouse embryonic aorta and Doppler-derived volumetric flow: A preliminary model. Am J Physiol Heart Circ Physiol 2002;283:H908–916. Phoon CK. Imaging tools for the developmental biologist: Ultrasound biomicroscopy of mouse embryonic development. Pediatr Res 2006;60:14 –21. Renolleau S, Aggoun-Zouaoui D, Ben-Ari Y, Charriaut-Marlangue C. A model of transient unilateral focal ischemia with reperfusion in the P7 neonatal rat: morphological changes indicative of apoptosis. Stroke 1998;29:1454 –1460, discussion 1461. Renolleau S, Fau S, Goyenvalle C, Joly LM, Chauvier D, Jacotot E, Mariani J, Charriaut-Marlangue C. Specific caspase inhibitor QVD-OPh prevents neonatal stroke in P7 rat: A role for gender. J Neurochem 2007;100:1062–1071. Salonen R, Lichtenstein P, Bellocco R, Petersson G, Cnattingius S. Increased risks of circulatory diseases in late pregnancy and puerperium. Epidemiology 2001;12:456 – 460.