Hemodynamic effects of intraoperative anesthetics administration in photothrombotic stroke model: a study using laser speckle imaging
© The Author(s) 2017
Received: 7 June 2016
Accepted: 24 December 2016
Published: 5 January 2017
Previous neuroimaging studies have shown the hemodynamic effect of either preconditioning or postconditioning anesthesia in ischemic stroke model. However, the anesthetic effect in hemodynamics during and immediately after the stroke modeling surgery remains unknown due to the lack of appropriate anesthesia-free stroke model and intraoperative imaging technology. In the present study, we utilized our recently developed photothrombotic model of focal cerebral ischemia in conscious and freely moving rats, and investigated transient hemodynamic changes with or without isoflurane administration. Laser speckle imaging was applied to acquire real-time two-dimensional full-field cerebral blood flow (CBF) information throughout the surgical operations and early after.
Significantly larger CBF reduction area was observed in conscious rats from 8 min immediately after the onset of stroke modeling, compared with anesthetized rats. Stroke rats without isoflurane administration also showed larger lesion volume identified by magnetic resonance imaging 3 h post occlusion (58.9%), higher neurological severity score 24 h post occlusion (28.3%), and larger infarct volume from 2,3,5-triphenyltetrazolium chloride staining 24 h post occlusion (46.9%).
Our results demonstrated that the hemodynamic features were affected by anesthetics at as early as during the stroke induction. Also, our findings about the neuroprotection of intraoperative anesthetics administration bring additional insights into understanding the translational difficulty in stroke research.
KeywordsLaser speckle imaging Photothrombotic stroke model Hemodynamic effect Anesthetics
Stroke is the leading cause of disability and mortality worldwide, which occurs when a cerebral vessel is either blocked or hemorrhagic. Ischemic stroke, accounting for more than 80% of all stroke cases, initiates a sequential metabolic and biochemical disorders and subsequently leads to the neuronal apoptosis and necrosis . So far, almost all the laboratorial and pre-clinical stroke studies are based on animal models, in which anesthesia is administered for the concern of animal care and ethics [2–4].
There have been a variety of studies showing the global effect of anesthetics administration in animal stroke model including alteration of neuronal and vascular functions. Inhaled anesthetics such as isoflurane can modulate synaptic transmission and neuronal excitability , augment GABA neurotransmission , and regulate cerebral blood flow (CBF) [7, 8]. The CBF reduction serves as the most important indicator in ischemic stroke, which is closely related to the volume of brain infarction . Previous neuroimaging studies have shown that the administration of isoflurane in either preconditioning  or post-conditioning following ischemia/reperfusion  altered the regional hemodynamic variations. The constrained deleterious CBF changes lead to neuroprotective effects such as decrease of brain infarction volume and reduction of intracerebral hemorrhage . However, the hemodynamic observations provided by magnetic resonance imaging (MRI) were constrained to a few time points with insufficient temporal resolution, while the CBF information obtained by laser Doppler flowmetry (LDF) was confined at very limited cerebral locations with poor spatial resolution . Furthermore, due to the lack of appropriate anesthesia-free stroke model, the intraoperative anesthetic effect during and immediately after the stroke modeling surgery remains unknown.
In our recent work  and others’ , a photothrombotic model of focal cerebral ischemia was induced in conscious and freely moving rats without introducing noticeable pain or stress to the animals. We utilized this model to investigate transient hemodynamic changes with or without isoflurane administration during the photothrombotic ischemic stroke modeling surgery. Laser speckle imaging (LSI) was applied to acquire real-time two-dimensional full-field CBF information throughout the surgical operations and early after. In addition, we measured the brain lesion by MRI 3 h post occlusion, neurological severity score (NSS) and brain infarct volume 24 h post stroke to investigate the potential neuroprotective effects of intraoperative anesthetics administered during stroke.
The experimental protocols in this study were approved by the Animal Care and Use Committee of Med-X Research Institute, Shanghai Jiao Tong University.
Twenty-two male Sprague–Dawley rats (320 ± 20 g, 12 weeks of age, Slac Laboratory Animal, Shanghai, China) were used in this study. The rats were housed within a research animal facility under a 12-h reverse light/dark cycle in a comfortable environment (temperature: 21–25 °C; humidity: 20–50%) with free access to food and water. A cranial window was prepared 24 h before stroke modeling. During the window preparation, the rat was anesthetized with isoflurane (5% initial and 1.0–1.5% for maintenance) and the rectal temperature was maintained at 37.0 ± 0.2 °C using a heating pad with a control module (FHC Inc., Bowdoin, ME). After a midline incision was made over the scalp, the tissues were cleaned with a scalpel to expose the skull. A 5.0 mm × 7.0 mm window over the left hemisphere, centered at 3.5 mm posterior to the bregma and 2.5 mm lateral to the middle line, was thinned by a high-speed dental drill (Fine Science Tools, Inc., Foster City, CA) until the cortical vessels were clearly visible. A cylinder base (lab-designed, height: 4.2 mm, radius: 5.5 mm, thickness: 0.5 mm) enclosing the thinned area was fixed onto the skull with reinforced glass ionomer cements (Dental Materials Factory of Shanghai Medical Instruments Co., Shanghai, China) to form an imaging chamber. All procedures were performed under standard sterile precautions. After the cement hardened, the animals were caged and provided with sufficient food and water for 24 h to eliminate the effects of isoflurane.
Photothrombotic stroke modeling
Real-time cerebral blood flow monitoring
Brain lesion evaluation
In brain lesion evaluation, we performed MRI scanning at 3 h post stroke, which is corresponding to the hyperacute stage in stroke study . The animals were placed in an MRI scanner (Siemens MAGNETOM Trio 3T, Munich, Germany) to evaluate the brain lesion volume in vivo. The scanner was equipped with a dedicated solenoid rat coil (diameter: 60 mm), which was manually tuned and matched. The lesion site was mapped using high-resolution T2-weighted spin-echo imaging. Twenty continuous coronal and twenty continuous transversal slices (thickness: 1 mm) were acquired with the following parameters: field of view, 50 × 50 mm; matrix size, 512 × 512; repetition time, 3000 ms; echo time, 68 ms; number of averages, 2. The total imaging time was about 4 min. Computer-aided planimetric assessment of the lesion volume was performed using ImageJ software  blindly. To evaluate the lesion volume, a threshold was applied to MRI images after 3 × 3 pixels Gaussian filtering with the threshold set to 75% maximum intensity of each image. Total lesion volume was subsequently calculated by multiplying the summation of lesion area on each slice with the slice thickness.
Neurological severity scores (Modified from Chen et al. )
Beam balance tests
Reflexes absent and abnormal movements
After NSS evaluation, the rats were euthanized and the brains were removed and sectioned coronally (thickness: 3 mm) with brain matrices (Model No. 68710, RWD Life Science Co., Ltd, Shenzhen, China). All brain slices were stained with TTC (2,3,5-triphenyltetrazolium chloride, Sigma-Aldrich Co. LLC, St. Louis, MO) at 37 °C for 10 min in a dark chamber. The infarct volume was quantitated by ImageJ software as the summation of all the slice infarct area multiplied by the slice thickness.
The differences between groups in CBF changes, lesion volumes from MRI analysis, NSS, and infarct volumes from TTC staining were determined by t test using MATLAB®. Significance level was set at P < 0.05. All data were presented as the mean ± SEM.
Cerebral blood flow information
The changes of CBF+ area along with time in both groups were calculated and shown in Fig. 2c. CBF+ at each time point was also compared between two groups by independent samples t-tests. Significantly larger CBF+ was observed 1 min after illumination initiation in the anesthetized group compared with the conscious group (P < 0.05). After 15-min illumination, CBF+ showed a decreasing trend though with no significant between-group difference (P > 0.05).
Brain lesion evaluation
To address the impact of the anesthetics administered during MRI scanning on the measurement conducted 24 h following stroke, we performed an extra experiment with conscious (n = 3) and anesthetic (n = 3) rats going through all the protocol except for MRI scanning. Both NSS and infarct volume showed no significant difference in comparison with the groups from the original protocol (Fig. 4).
In this study, we compared the CBF changes throughout the photothrombotic stroke modeling of focal cerebral ischemia in conscious and isoflurane anesthetized rats, respectively. NSS, LSI, MRI analysis and TTC staining were applied to evaluate the neurologic deficits and brain lesion after stroke. We found a much smaller CBF reduction area during the surgery in the anesthetic group. Moreover, it was shown that the intraoperative anesthetics provided neuroprotective effects to the ischemic brain injury.
Different imaging techniques have been developed in monitoring the influence of isoflurane on regional CBF during stroke. For instance, a longitudinal MRI study carried out between 6 h and 21 days after ischemia showed that isoflurane altered regional CBF and constrained the deleterious hemodynamic variation in ischemia reperfusion injury . LDF is an in vivo real-time imaging technique, which has been commonly utilized to monitor transient focal CBF throughout surgery or during the induction of ischemia [26, 27]. For example, Bleilevens et al.  observed the focal CBF in the ischemic area of isoflurane anesthetized rats by LDF at various time points before and after the onset of ischemia, finding significantly higher values at 50 min after ischemia in comparison with ketamine/xylazine anesthetized rats. Compared with LDF, LSI provides full-field CBF information with high spatial and temporal resolution . Owning to our conscious photothrombotic stroke model, for the first time, we were able to exclude the anesthetic effect during the whole modeling procedure and investigate the intraoperative anesthetic influence on the 2D CBF characteristics. The CBF information acquired during and early after stroke demonstrated that the animal hemodynamics were affected by anesthetics at as early as during the stroke induction and immediately after stroke. Also, our findings about the neuroprotective effect due to intraoperative anesthetics administration during stroke modeling bring additional insights in understanding the translational difficulty in stroke research.
In our present work, the photothrombotic stroke model was adopted. The vascular thrombosis was formed through the photoactivation of the pre-injected Rose Bengal followed by platelet aggregation. However, it was reported that inflammation after experimental stroke could lead to brain edema, blood–brain barrier injury, which would impair the recovery in stroke rats . A number of studies have reported dose-dependent increase in CBF as well as heterogeneous change in CBF distribution with isoflurane administration [30–32]. Also, it was proposed that the neuroprotective effect of isoflurane anesthesia might be caused by constrained deleterious CBF change . Moreover, the CBF reduction in early stage after stroke, both in the ischemia core and in ischemic penumbra, has been shown closely associated with infarction volume . The isoflurane-induced hemodynamic changes might be relevant to cerebral vasodilation and/or collateral circulation enhancement during ischemia, considering the fact that the isoflurane might serve as a potent cerebral vasodilator . For example, the isoflurane-mediated increase of nitric oxide could induce the depolarization of mitochondria in endothelial cells . Moreover, the dilation of arterioles in the ischemic penumbra  could be neuroprotective. It is in line with the present study that the CBF reduction area in the isoflurane anesthetized group was more constrained than that in the conscious group, which might contribute to the less brain injury after stroke. Nevertheless, the neuroprotective effect that isoflurane exerts is not solely caused by the hemodynamic changes. Alterations at molecular level, e.g. neurotransmitter concentration and neuronal excitability, could also contribute to the changes in anesthetic property [36–38]. Our work provided a useful tool for the study of early neuroprotective effect in relation to CBF changes during the procedure of stroke modeling. The detailed mechanisms underlying it merit further experimental investigations such as the alteration of neuronal excitability and its association with hemodynamic variations. Moreover, the study on dose-dependent effect of isoflurane in CBF changes could be performed in future work.
cerebral blood flow
laser Doppler flowmetry
laser speckle imaging
middle cerebral artery
magnetic resonance imaging
neurological severity score
All authors helped to draft the manuscript. In addition, ST conceived and designed the study. HLu worked on LSI and MRI protocols, data collection and analysis, and result interpretation. YL worked on protocols and result interpretation. BB performed MRI data collection and analysis. LY performed LSI data analysis. XL and HLi worked on laser speckle imaging algorithms. All authors read and approved the final manuscript.
The authors are grateful to Dr. Guo-Yuan Yang in the School of Biomedical Engineering, Shanghai Jiao Tong University for helpful discussions on animal experiments.
The authors declare that they have no competing interests.
Availability of data and material
The corresponding data and software are available on request from Prof. Tong at firstname.lastname@example.org.
Ethics approval and consent to participate
The experimental protocols in this study were approved by the Animal Care and Use Committee of Med-X Research Institute, Shanghai Jiao Tong University.
This work is partly supported by National Science Foundation of China Grant (No. 61371018).
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Lo EH, Dalkara T, Moskowitz MA. Mechanisms, challenges and opportunities in stroke. Nat Rev Neurosci. 2003;4(5):399–415.View ArticlePubMedGoogle Scholar
- Ginsberg MD, Busto R. Rodent models of cerebral ischemia. Stroke. 1989;20(12):1627–42.View ArticlePubMedGoogle Scholar
- Dirnagl U, Iadecola C, Moskowitz MA. Pathobiology of ischaemic stroke: an integrated view. Trends Neurosci. 1999;22(9):391–7.View ArticlePubMedGoogle Scholar
- Carmichael ST. Rodent models of focal stroke: size, mechanism, and purpose. NeuroRx. 2005;2(3):396–409.View ArticlePubMedPubMed CentralGoogle Scholar
- Ying SW, et al. Isoflurane modulates excitability in the mouse thalamus via GABA-dependent and GABA-independent mechanisms. Neuropharmacology. 2009;56(2):438–47.View ArticlePubMedGoogle Scholar
- Grasshoff C, Antkowiak B. Effects of isoflurane and enflurane on GABAA and glycine receptors contribute equally to depressant actions on spinal ventral horn neurones in rats. Br J Anaesth. 2006;97(5):687–94.View ArticlePubMedGoogle Scholar
- Sicard K, et al. Regional cerebral blood flow and BOLD responses in conscious and anesthetized rats under basal and hypercapnic conditions: implications for functional MRI studies. J Cereb Blood Flow Metab. 2003;23(4):472–81.View ArticlePubMedPubMed CentralGoogle Scholar
- Berwick J, et al. Hemodynamic response in the unanesthetized rat: intrinsic optical imaging and spectroscopy of the barrel cortex. J Cereb Blood Flow Metab. 2002;22(6):670–9.View ArticlePubMedGoogle Scholar
- Li Y, et al. Predicting the ischemic infarct volume at the first minute after occlusion in rodent stroke model by laser speckle imaging of cerebral blood flow. J Biomed Opt. 2013;18(7):76024.View ArticlePubMedGoogle Scholar
- Zheng S, Zuo Z. Isoflurane preconditioning induces neuroprotection against ischemia via activation of P38 mitogen-activated protein kinases. Mol Pharmacol. 2004;65(5):1172–80.View ArticlePubMedGoogle Scholar
- Zhao H, Sapolsky RM, Steinberg GK. Interrupting reperfusion as a stroke therapy: ischemic postconditioning reduces infarct size after focal ischemia in rats. J Cereb Blood Flow Metab. 2006;26(9):1114–21.PubMedGoogle Scholar
- Taheri S, et al. Isoflurane reduces the ischemia reperfusion injury surge: a longitudinal study with MRI. Brain Res. 2014;1586:173–83.View ArticlePubMedGoogle Scholar
- Frerichs KU, Feuerstein GZ. Laser-Doppler flowmetry. A review of its application for measuring cerebral and spinal cord blood flow. Mol Chem Neuropathol. 1990;12(1):55–70.View ArticlePubMedGoogle Scholar
- Lu H, et al. Induction and imaging of photothrombotic stroke in conscious and freely moving rats. J Biomed Opt. 2014;19(9):96013.View ArticlePubMedGoogle Scholar
- Seto A, et al. Induction of ischemic stroke in awake freely moving mice reveals that isoflurane anesthesia can mask the benefits of a neuroprotection therapy. Front Neuroenerg. 2014;6:1.View ArticleGoogle Scholar
- Watson BD, et al. Cerebral blood flow restoration and reperfusion injury after ultraviolet laser-facilitated middle cerebral artery recanalization in rat thrombotic stroke. Stroke. 2002;33(2):428–34.View ArticlePubMedGoogle Scholar
- Rosenblum WI, El-Sabban F. Platelet aggregation in the cerebral microcirculation: effect of aspirin and other agents. Circ Res. 1977;40(3):320–8.View ArticlePubMedGoogle Scholar
- Watson BD, et al. Induction of reproducible brain infarction by photochemically initiated thrombosis. Ann Neurol. 1985;17(5):497–504.View ArticlePubMedGoogle Scholar
- Briers JD, Richards G, He XW. Capillary blood flow monitoring using laser speckle contrast analysis (LASCA). J Biomed Opt. 1999;4(1):164–75.View ArticlePubMedGoogle Scholar
- Boas DA, Dunn AK. Laser speckle contrast imaging in biomedical optics. J Biomed Opt. 2010;15(1):011109.View ArticlePubMedPubMed CentralGoogle Scholar
- Miao P, et al. High resolution cerebral blood flow imaging by registered laser speckle contrast analysis. IEEE Trans Biomed Eng. 2010;57(5):1152–7.View ArticlePubMedGoogle Scholar
- Miao P, et al. Random process estimator for laser speckle imaging of cerebral blood flow. Opt Express. 2010;18(1):218–36.View ArticlePubMedGoogle Scholar
- Lapchak PA, Zhang JH. Translational stroke research: from target selection to clinical trials. New York: Springer; 2012.View ArticleGoogle Scholar
- Schneider CA, Rasband WS, Eliceiri KW. NIH Image to ImageJ: 25 years of image analysis. Nat Methods. 2012;9(7):671–5.View ArticlePubMedGoogle Scholar
- Chen J, et al. Therapeutic benefit of intravenous administration of bone marrow stromal cells after cerebral ischemia in rats. Stroke. 2001;32(4):1005–11.View ArticlePubMedGoogle Scholar
- Zhu W, et al. Isoflurane preconditioning neuroprotection in experimental focal stroke is androgen-dependent in male mice. Neuroscience. 2010;169(2):758–69.View ArticlePubMedPubMed CentralGoogle Scholar
- Bleilevens C, et al. Effect of anesthesia and cerebral blood flow on neuronal injury in a rat middle cerebral artery occlusion (MCAO) model. Exp Brain Res. 2013;224(2):155–64.View ArticlePubMedGoogle Scholar
- Miao P, et al. Laser speckle contrast imaging of cerebral blood flow in freely moving animals. J Biomed Opt. 2011;16(9):090502.View ArticlePubMedGoogle Scholar
- Denes A, Ferenczi S, Kovacs KJ. Systemic inflammatory challenges compromise survival after experimental stroke via augmenting brain inflammation, blood–brain barrier damage and brain oedema independently of infarct size. J Neuroinflamm. 2011;8:164.View ArticleGoogle Scholar
- Todd MM, Weeks J. Comparative effects of propofol, pentobarbital, and isoflurane on cerebral blood flow and blood volume. J Neurosurg Anesthesiol. 1996;8(4):296–303.View ArticlePubMedGoogle Scholar
- Hansen TD, et al. Distribution of cerebral blood flow during halothane versus isoflurane anesthesia in rats. Anesthesiology. 1988;69(3):332–7.View ArticlePubMedGoogle Scholar
- Hendrich KS, et al. Cerebral perfusion during anesthesia with fentanyl, isoflurane, or pentobarbital in normal rats studied by arterial spin-labeled MRI. Magn Reson Med. 2001;46(1):202–6.View ArticlePubMedGoogle Scholar
- Lenzarini F, et al. Time course of isoflurane-induced vasodilation: a Doppler ultrasound study of the left coronary artery in mice. Ultrasound Med Biol. 2016;42(4):999–1009.View ArticlePubMedGoogle Scholar
- Katakam PV, et al. Depolarization of mitochondria in endothelial cells promotes cerebral artery vasodilation by activation of nitric oxide synthase. Arterioscler Thromb Vasc Biol. 2013;33(4):752–9.View ArticlePubMedPubMed CentralGoogle Scholar
- Terpolilli NA, et al. Inhalation of nitric oxide prevents ischemic brain damage in experimental stroke by selective dilatation of collateral arterioles. Circ Res. 2012;110(5):727–38.View ArticlePubMedGoogle Scholar
- Liu L, et al. Baclofen mediates neuroprotection on hippocampal CA1 pyramidal cells through the regulation of autophagy under chronic cerebral hypoperfusion. Sci Rep. 2015;5:14474.View ArticlePubMedPubMed CentralGoogle Scholar
- Tang H, et al. Neuroprotective effects of scutellarin and scutellarein on repeatedly cerebral ischemia–reperfusion in rats. Pharmacol Biochem Behav. 2014;118:51–9.View ArticlePubMedGoogle Scholar
- Elsersy H, et al. Selective γ-aminobutyric acid type A receptor antagonism reverses isoflurane ischemic neuroprotection. J Am Soc Anesthesiol. 2006;105(1):81–90.View ArticleGoogle Scholar