清醒小鼠全脑超快功能性光声显微成像

  神经血管偶联机制保证大脑正常运转，神经血管偶联受损会诱发脑疾病。因此，研究神经血管偶联机制对大脑功能解析和脑疾病治疗至关重要。血流动力学是神经血管偶联机制的组成部分之一，在目前可用于研究血流动力学的非侵入性成像技术中，光声显微成像相比于磁共振成像和超声成像分辨率更高，相比于光学成像对比度更强，还无需外源性造影剂，是研究血流动力学不可替代的工具。受限于光声成像设备，目前极大多数基于光声成像的脑功能研究局限于麻醉动物全脑功能研究或者基于头戴式的清醒动物局部脑区功能研究。但是麻醉剂会抑制神经血管偶联，而且大脑正常运转依靠各脑区相互协作。

  为了实现清醒动物全脑血流动力学研究，本研究结合已研发的便携式光声显微成像系统，设计了头部固定系统和辅助运动系统，实现了清醒小鼠全脑超快功能性光声显微成像。同时，利用小鼠足底电刺激模型验证了麻醉剂对血流动力学的影响，得到了清醒小鼠受足底电刺激时两侧大脑的激活规律。通过施加不同强度足底电刺激对清醒/麻醉小鼠全脑皮层的光声显微成像监测，评估了清醒/麻醉小鼠的血流动力学变化和脑激活面积覆盖率。实验结果表明，超快功能性光声显微成像可以实现清醒小鼠全脑神经血管偶联机制研究，可以监测到双边激活现象、扩散现象、延迟现象和麻醉剂对血流动力学的抑制作用。同时，超快功能性光声显微成像还可以利用其高分辨率的特点，提供更细节的血流动力学信息。清醒/麻醉小鼠受到单侧足底电刺激时，对侧脑区血流动力学变化强于同侧脑区血流动力学变化；随着刺激电流强度的增大，血流动力学变化和脑激活面积覆盖率趋于饱和。最后，超快功能性光声显微成像还可以提供其他成像技术没有的独特信息。激活的血管在同一根血管分支上，即使空间相邻但不在同一分支上的血管不会被激活。

  总而言之，本研究基于足底电刺激模型开展了清醒/麻醉小鼠全脑超快功能性光声显微成像，研究了足底电刺激诱发的清醒/麻醉小鼠全脑血流动力学变化，验证了麻醉剂对血流动力学的抑制作用，证明了光声显微成像技术用于研究清醒小鼠全脑神经血管偶联机制的可行性和独特优势，推动了研究神经血管偶联机制、揭示大脑正常功能的进程。

[1] Falkowska A, Gutowska I, Goschorska M, et al. Energy Metabolism of the Brain, Including the Cooperation between Astrocytes and Neurons, Especially in the Context of Glycogen Metabolism [J]. International Journal of Molecular Sciences, 2015, 16(11):25959-25981.
[2] Konan L M, Reddy V, Mesfin F B. Neuroanatomy, Cerebral Blood Supply [M]. Treasure Island: StatPearls Publishing, 2022.
[3] Ka, Sing, Wong, et al. Mechanisms of acute cerebral infarctions in patients with middle cerebral artery stenosis: a diffusion-weighted imaging and microemboli monitoring study [J]. Annals of Neurology, 2002, 52(1):74-81.
[4] Ghanekar S, Corey S, Lippert T, et al. Pathological links between stroke and cardiac arrest [J]. Chinese Journal of Neurosurgery, 2017, 3(1): 4.
[5] Mattson M P, Duan W, Pedersen W A, et al. Culmsee CNeurodegenerative disorders and ischemic brain diseases [J]. Apoptosis, 2001, 6:69-81.
[6] Kahl A, Blanco I, Jackman K, et al. Cerebral ischemia induces the aggregation of proteins linked to neurodegenerative diseases [J]. Rep, 2018, 8(1):2701.
[7] Iadecola, Costantino. The Neurovascular Unit Coming of Age: A Journey through Neurovascular Coupling in Health and Disease [J]. Neuron, 2017, 96(1): 17-42.
[8] Iadecola C. NEUROVASCULAR REGULATION IN THE NORMAL BRAIN AND IN ALZHEIMER'S DISEASE [J]. Nature reviews neuroscience, 2004, (5):5.
[9] Iadecola C. Regulation of the cerebral microcirculation during neural activity: Is nitric oxide the missing link? [J]. Trends in Neurosciences, 1993, 16(6):206-214.
[10] Rehberg B, Xiao Y H, Duch D S. Central nervous system sodium channels are significantly suppressed at clinical concentrations of volatile anesthetics [J]. Anesthesiology, 1996, 84(5):1223-1233.
[11] Ouyang, W. Depression by isoflurane of the action potential and underlying voltage-gated ion currents in isolated rat neurohypophysial nerve terminals [J]. Journal of Pharmacology & Experimental Therapeutics, 2005, 312(2):801-808.
[12] Haydon D A, Urban B W. The effects of some inhalation anaesthetics on the sodium current of the squid giant axon [J]. J Physiol, 1983, 341(1):429-439.
[13] Study R E. Isoflurane inhibits multiple voltage-gated calcium currents in hippocampal pyramidal neurons [J]. Anesthesiology, 1994, 81(1):104-16.
[14] Kamatchi G L, Chan C K, Snutch T, et al. Volatile anesthetic inhibition of neuronal Ca channel currents expressed in Xenopus oocytes [J]. Brain Research, 1999, 831(1-2):85-96.
[15] Wang H Y, Eguchi K, Yamashita T, et al. Frequency-Dependent Block of Excitatory Neurotransmission by Isoflurane via Dual Presynaptic Mechanisms [J]. The Journal of Neuroscience, 2020, 40(21):JN-RM-2946-19.
[16] Ries C R, Puil E. Ionic mechanism of isoflurane's actions on thalamocortical neurons [J]. Journal of Neurophysiology, 1999, 81(4):1802-1809.
[17] Franks N P, Eric Honoré. The TREK K2P channels and their role in general anaesthesia and neuroprotection [J]. Trends in Pharmacological Sciences, 2004, 25(11):601-608.
[18] Xie Z, McMillan K, Pike C M, et al. Interaction of anesthetics with neurotransmitter release machinery proteins [J]. J Neurophysiol, 2013, 109(3):758-767.
[19] Nagele P, Mendel J B, Placzek W J, et al. Volatile anesthetics bind rat synaptic snare proteins [J]. Anesthesiology, 2005, 103(4):768-778.
[20] Herring B E, Xie Z, Marks J, et al. Isoflurane inhibits the neurotransmitter release machinery [J]. J Neurophysiol, 2009, 102(2):1265-1273.
[21] Kaisti K K, Långsjö J W, Aalto S, et al. Effects of sevoflurane, propofol, and adjunct nitrous oxide on regional cerebral blood flow, oxygen consumption, and blood volume in humans [J]. Anesthesiology, 2003, 99(3):603-613.
[22] Martin C, Martindale J, Berwick J, et al. Investigating neural-hemodynamic coupling and the hemodynamic response function in the awake rat [J]. Neuroimage, 2006, 32(1):33-48.
[23] Qiu M, Ramani R, Swetye M, et al. Anesthetic effects on regional CBF, BOLD, and the coupling between task-induced changes in CBF and BOLD: an fMRI study in normal human subjects [J]. Magnetic Resonance in Medicine, 2010, 60(4):987-996.
[24] Tang J, Xi L, Zhou J, et al. Noninvasive high-speed photoacoustic tomography of cerebral hemodynamics in awake-moving rats [J]. Journal of cerebral blood flow and metabolism: official journal of the International Society of Cerebral Blood Flow and Metabolism, 2015, 35(8):1224-1232.
[25] Ahmmed R, Rahman M A, Hossain M F. An Advanced Algorithm Combining SVM and ANN Classifiers to Categorize Tumors with Position from Brain MRI Images [J]. Advances in Science Technology and Engineering Systems Journal, 2018, 3(2):40-48.
[26] Nicolakakis N, Hamel E. Neurovascular function in Alzheimer's disease patients and experimental models [J]. Journal of Cerebral Blood Flow, Metabolism, 2011, 31(6):1354-1370.
[27] Iadecola C. The pathobiology of vascular dementia [J]. Neuron, 2013, 80(4):844-866.
[28] Hu S, Maslov K, Tsytsarev V, et al. Functional transcranial brain imaging by optical-resolution photoacoustic microscopy [J]. Journal Of Biomedical Optics, 2009, 14(4):040503.
[29] Wang L, Maslov K, Wang L V. Single-cell label-free photoacoustic flowoxigraphy in vivo [J]. Proceedings of the National Academy of Sciences of the United States of America, 2013, 110(15):5759-5764.
[30] Gong X, Jin T, Wang Y, et al. Photoacoustic microscopy visualizes glioma-induced disruptions of cortical microvascular structure and function [J]. Journal of neural engineering, 2022(2):19.
[31] Thomas, Paul, Matthews, et al. Label-free photoacoustic microscopy of peripheral nerves [J]. Journal of Biomedical Optics, 2014, 19(1):16004.
[32] Wei Q, Tian J, Guo H, et al. Large-field-of-view optical resolution photoacoustic microscopy [J]. Optics Express, 2018, 26(4):4271-4278.
[33] Wang L V, Yao J. A Practical Guide to Photoacoustic Tomography in the Life Sciences [J]. Nature Methods, 2016, 13(8):627-638.
[34] Yao J, Maslov K I, Zhang Y, et al. Label-free oxygen-metabolic photoacoustic microscopy in vivo [J]. Journal Of Biomedical Optics, 2011, 16(7): 076003.
[35] Kim J, Jin Y K, Jeon S, et al. Super-resolution localization photoacoustic microscopy using intrinsic red blood cells as contrast absorbers [J]. Light: Science & Applications, 2019(001):008.
[36] Cao R, Li J, Zhang C, et al. Photoacoustic microscopy of obesity-induced cerebrovascular alterations [J]. Neuroimage, 2018, 188:369-379.
[37] Cao R, Li J, Ning B, et al. Functional and oxygen-metabolic photoacoustic microscopy of the awake mouse brain [J]. Neuroimage, 2017, 150:77-87.
[38] Yao J, Wang L, Yang J M, et al. High-speed label-free functional photoacoustic microscopy of mouse brain in action [J]. Nat Methods, 2015, 12:407-410.
[39] Liu C, Chen J, Zhang Y, et al. Five-wavelength optical-resolution photoacoustic microscopy of blood and lymphatic vessels [J]. Advanced Photonics, 2021, 3(1):9.
[40] Xu Z, Wang Y, Sun N, et al. Parallel Computing for Quantitative Blood Flow Imaging in Photoacoustic Microscopy [J]. Sensors, 2019, 19(18):4000.
[41] Wang Y, Hu S, Maslov K, et al. Imaging of hemoglobin oxygen saturation variations in single vessels in vivo using photoacoustic microscopy [J]. Applied Physics Letters, 2007, 90(5):1555.
[42] Zhang H F, Maslov K, Wang L V. Effects of wavelength-dependent fluence attenuation on the noninvasive photoacoustic imaging of hemoglobin oxygen saturation in subcutaneous vasculature in vivo [J]. Inverse Problems, 2007, 23(6):S113-S122.
[43] Hu S, Maslov K, Wang L V. Noninvasive label-free imaging of microhemodynamics by optical-resolution photoacoustic microscopy [J]. Optics Express, 2009, 17(9):7688-7693.
[44] Zhang H F, Maslov K, Stoica G, et al. Functional photoacoustic microscopy for high-resolution and noninvasive in vivo imaging [J]. Nature Biotechnology, 2006, 24(7):848-851.
[45] Yao J, Wang L V. Photoacoustic microscopy [J]. Laser & Photonics Reviews, 2013, 7(5):758-778.
[46] Li L, Zhu L, Ma C, et al. Single-impulse panoramic photoacoustic computed tomography of small-animal whole-body dynamics at high spatiotemporal resolution [J]. Nat Biomed Eng, 2017, 1(5): 0071.
[47] Lin L, Hu P, Tong X, et al. High-speed three-dimensional photoacoustic computed tomography for preclinical research and clinical translation [J]. Nature Communications, 2021, 12(1): 882.
[48] Na S, Russin J J, Lin L, et al. Massively parallel functional photoacoustic computed tomography of the human brain [J]. Nature Biomedical Engineering, 2022, 6(5):584-592.
[49] Mohesh M, Manojit P. Performance Characterization of a Switchable Acoustic Resolution and Optical Resolution Photoacoustic Microscopy System [J]. Sensors, 2017, 17(2): 357.
[50] Cai D, Wong T T W, Zhu L, et al. Dual-view photoacoustic microscopy for quantitative cell nuclear imaging [J]. Optics Letters, 2018, 43(20):4875-4878.
[51] Yang J M, Maslov K, Yang H C, et al. Photoacoustic Endoscopy [J]. Optics Letters, 2009, 34:1591-1593.
[52] Yang J M, Li C Y, Chen R M, et al. Label-free optical-resolution photoacoustic endomicroscopy in vivo[C]// Photons Plus Ultrasound: Imaging and Sensing 2015. International Society for Optics and Photonics, 2015.
[53] Salehi H S, Li H, Merkulov A, et al. Coregistered photoacoustic and ultrasound imaging and classification of ovarian cancer:ex vivoandin vivostudies [J]. Journal of Biomedical Optics, 2016, 21(4): 046006.
[54] Min W, Springeling G, Lovrak M, et al. Real-time volumetric lipid imaging in vivo by intravascular photoacoustics at 20 frames per second [J]. Biomedical Optics Express, 2017, 8(2):943-953.
[55] Peng, Lei, Xue, et al. Ultrafine intravascular photoacoustic endoscope with a 0.7  mm diameter probe [J]. Optics letters, 2019, 44(22):5406-5409.
[56] Ji X, Xiong K, Yang S, et al. Intravascular confocal photoacoustic endoscope with dual-element ultrasonic transducer [J]. Optics Express, 2015, 23(7):9130-9136.
[57] Yang J M, Favazza C, Yao J, et al. Three-dimensional photoacoustic endoscopic imaging of the rabbit esophagus [J]. PLos One, 2015, 10(4):0120269.
[58] Attia A B E, Balasundaram G, Moothanchery M, et al. A review of clinical photoacoustic imaging: Current and future trends [J]. Photoacoustics, 2019, 16: 100144.
[59] Qu Y, Li C, Shi J, et al. Transvaginal fast-scanning optical-resolution photoacoustic endoscopy [J]. Journal Of Biomedical Optics, 2018, 23(12): 121617.
[60] Hu S, Rao B, Maslov K, et al. Label-free photoacoustic ophthalmic angiography [J]. Optics Letters, 2010, 35(1): 1-3.
[61] Na S, Russin J J, Lin L, et al. Massively parallel functional photoacoustic computed tomography of the human brain [J]. Nat Biomed Eng, 2022, 6(5): 584-92.
[62] Matsumoto Y, Asao Y, Yoshikawa A, et al. Label-free photoacoustic imaging of human palmar vessels: a structural morphological analysis [J]. Scientific Reports, 2018, 8(1): 786.
[63] Toi M, Asao Y, Matsumoto Y, et al. Visualization of tumor-related blood vessels in human breast by photoacoustic imaging system with a hemispherical detector array [J]. Scientific Reports, 2017, 7(1): 41970.
[64] Favazza C P, Jassim O, Cornelius L A, et al. In vivo photoacoustic microscopy of human cutaneous microvasculature and a nevus [J]. Journal Of Biomedical Optics, 2011, 16(1):016015.
[65] Lin L, Hu P, Shi J, et al. Single-breath-hold photoacoustic computed tomography of the breast [J]. Nature Communications, 2018, 9(1): 2352.
[66] Huang N, Guo H, Qi W, et al. Whole-body multispectral photoacoustic imaging of adult zebrafish [J]. Biomedical Optics Express, 2016, 7(9):3543-3550.
[67] Yao J, Wang L, Yang J M, et al. High-speed label-free functional photoacoustic microscopy of mouse brain in action [J]. Nature Methods, 2015, 12(5):407-410.
[68] Wang X, Geng K, Wegiel M A, et al. Noninvasive photoacoustic angiography of animal brains in vivo with near-infrared light and an optical contrast agent [J]. Optics Letters, 2004, 29(7):730-732.
[69] Wang Z, Yang F, Cheng Z, et al. Photoacoustic-guided photothermal therapy by mapping of tumor microvasculature and nanoparticle [J]. Nanophotonics, 2021, 10(12):3359-3368.
[70] Jin T, Guo H, Yao L, et al. Portable optical-resolution photoacoustic microscopy for volumetric imaging of multiscale organisms [J]. Journal of Biophotonics, 2018, 11(4):e201700250.
[71] Qin W, Gan Q, Yang L, et al. High-resolution in vivo imaging of rhesus cerebral cortex with ultrafast portable photoacoustic microscopy [J]. Neuroimage, 2021, 238: 118260.
[72] Cha S S, Bucklin M E, Han X. Removable cranial window for sustained wide-field optical imaging in mouse neocortex [M]. Cold Spring Harbor Laboratory, 2020.
[73] Silva A C, Lee S P, Yang G, et al. Simultaneous blood oxygenation level-dependent and cerebral blood flow functional magnetic resonance imaging during forepaw stimulation in the rat [J]. J Cereb Blood Flow Metab, 1999, 1999(8):871-879.
[74] Seuwen A, Schroeter A, Grandjean J, et al. Functional spectroscopic imaging reveals specificity of glutamate response in mouse brain to peripheral sensory stimulation [J]. Scientific Reports, 2019, 9(1):10563.
[75] Bosshard S C, Baltes C, Wyss M T, et al. Assessment of brain responses to innocuous and noxious electrical forepaw stimulation in mice using BOLD fMRI [J]. Pain, 2012, 151(3):655-663.
[76] Nair G, Duong T Q. Echo-planar BOLD fMRI of mice on a narrow-bore 9.4 T magnet [J]. Magnetic Resonance in Medicine, 2010, 52(2):430-434.
[77] Kim J, Kim G, Li L, et al. Deep learning acceleration of multiscale superresolution localization photoacoustic imaging [J]. Light: Science & Applications, 2022, 11(1):131.
[78] Qin W, Qi W, Xi L. Quantitative investigation of vascular response to mesenteric venous thrombosis using large-field-of-view photoacoustic microscopy [J]. Journal of Biophotonics, 2019, 12(12):e201900198.
[79] Schroeter A, Schlegel F, Seuwen A, et al. Specificity of stimulus-evoked fMRI responses in the mouse: the influence of systemic physiological changes associated with innocuous stimulation under four different anesthetics [J]. Neuroimage, 2014, 94(1):372-384.
[80] Xiong B, Li A, Lou Y, et al. Precise Cerebral Vascular Atlas in Stereotaxic Coordinates of Whole Mouse Brain [J]. Front Neuroanat, 2017, 11:128.
[81] Dorr A, Sled J G, Kabani N. Three-dimensional cerebral vasculature of the CBA mouse brain: a magnetic resonance imaging and micro computed tomography study [J]. Neuroimage, 2007, 35(4):1409-1423.
[82] Tang P, Li Y, Rakymzhan A, et al. Measurement and visualization of stimulus-evoked tissue dynamics in mouse barrel cortex using phase-sensitive optical coherence tomography [J]. Biomedical Optics Express, 2020, 11(2):699-710.
[83] Yi Y, Yanchao Z, Hongshuai J, et al. Cortical Hemodynamic Responses Under Focused Ultrasound Stimulation Using Real-Time Laser Speckle Contrast Imaging [J]. Frontiers in Neuroence, 2018, 12:269.
[84] Silva A C, Koretsky A P, Duyn J H. Functional MRI impulse response for BOLD and CBV contrast in rat somatosensory cortex [J]. Magnetic Resonance in Medicine, 2010, 57(6):1110-1118.
[85] Ivanov K P, Kalinina M K, Levkovich Y I. Blood flow velocity in capillaries of brain and muscles and its physiological significance [J]. Microvascular Research, 1981, 22(2):143-155.
[86] Sicard K, Shen Q, Brevard M E, 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]. Journal of Cerebral Blood Flow & Metabolism, 2003, 23(4):472-481.