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Photoacoustic Microscopy of Cerebral Hemodynamic and Metabolic Responses to General Anesthetics
10 Photoacoustic Microscopy of Cerebral Hemodynamic and Metabolic Responses to General Anesthetics Rui Cao1, Jun Li2, Zhiyi Zuo2, Song Hu1 1
DEPARTMENT OF BI OMEDICAL ENGINE ERING, UNIVERSITY OF VIRGINIA, CHARLOTTESVILLE, V A 22 90 8, UNIT ED S TATE S; 2 DE PARTMENT OF ANESTHESIOLOGY, UNIVERSITY OF VIRGINIA, C HA R L O T TE S VIL L E, V A 2 29 08, U N I TED S TA TE S
1. Introduction Recent innovations in light microscopy have revolutionized our understanding of the braindthe most complicated and least understood organ. Among them, photoacoustic microscopy (PAM) is emerging as a new enabling technology due to its unique features [1]. Based on the photoacoustic effect, which refers to ultrasound generation from light absorption, PAM combines the advantages of intrinsic optical absorption contrast and relatively weak acoustic scattering in biological tissues. Furthermore, capitalizing on the optical absorption of blood hemoglobin, PAM is uniquely capable of label-free highresolution multiparametric imaging of hemoglobin concentration (CHb), oxygen saturation (sO2), and cerebral blood flow (CBF) in the mouse brain [2,3]. This chapter introduces a latest development of PAM, termed head-restrained PAM, which enables functional and oxygen-metabolic imaging of the awake mouse brain [4]. The principle of the multiparametric measurements, instrument design and configuration, and experimental procedures for awake brain imaging are discussed in details. Besides, the application of head-restrained PAM to study cerebral hemodynamic and oxygen-metabolic responses to isoflurane, a widely used general anesthetic, is demonstrated. At the conclusion of the chapter, future developments of this enabling technology are discussed.
Neurophotonics and Biomedical Spectroscopy. https://doi.org/10.1016/B978-0-323-48067-3.00010-X Copyright © 2019 Elsevier Ltd. All rights reserved.
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2. Background and Motivation The need for high-resolution imaging of the awake brain is evident [5] because general anesthesia can significantly reduce neuronal activity and alter multiple forms of cerebral dynamics [6e8]. The profound effects may confound the readouts of conventional light microscopes, which require the preparation of anesthetized animals, and thus impose considerable limitation on the interpretation and translation of the findings. Moreover, incapable of imaging the awake brain for comparison with the anesthetized counterpart, conventional microscopies are of limited utility in examining the important yet elusive roles of clinically used general anesthetics in the progression of multiple life-threatening brain diseases, including stroke [9] and Alzheimer’s disease [10]. Recently, two complementary approaches have been developed to extend fluorescence microscopy of neuronal activity to the awake brain. First is a head-mounted approach. With innovative designs of laser excitation, scan, and detection, conventional fluorescence microscopes can be miniaturized and mounted on the head of adult mouse, thereby eliminating motion artifacts. However, its present implementations still suffer from limited field of view (FOV) and suboptimal optical performance [11]. An alternative approach is head restraint, which provides extended FOV, improved imaging performance, and the accessibility of electrode recording at the expense of freely moving condition [6]. Moreover, this setting can be integrated with the computer-generated virtual environment for controlled stimulation [12,13]. A major concern about head restraint is the possible induction of stress. Fortunately, the self-paced voluntary treadmill has been shown to help acclimate animals to the restraint [6,12,13] and thus opened the way for broad applications of the head-restrained fluorescence microscopy [12e14], including studies of the effects of general anesthetics on neural activity [15,16]. While this innovation has advanced our understanding of neural activity without the influence of anesthesia, the coevolving hemodynamics and oxygen metabolism in the awake brain remains poorly understood. With the aid of angiographic agents, fluorescence microscopy is able to image the cerebrovascular structure and CBF [7]. However, multiple hemoglobin-related hemodynamic parameters (e.g., CHb and sO2) remain inaccessible. The ever-growing gap between light microscopyebased neuronal and hemodynamic imaging in the awake animal brain presents a critical barrier to our understanding of neurovascular coupling that underlies many important cerebral diseases. Uniquely capable of comprehensive measurement of hemodynamic parameters in a label-free manner, PAM is well suited to fill this gap. However, this research is still in a nascent stage, with no report using PAM and only a couple of studies using macroscopicresolution photoacoustic tomography. Recently, wearable photoacoustic tomography devices have been developed for brain imaging in freely moving rats [17,18]. However, they are too heavy (>20 g) and bulky (40e50 mm) to be applied to mice, in which abundant disease models and genetic manipulations are available. Moreover, their spatial resolutions (>200 mm) are insufficient to resolve cerebral microvessels, which play key roles in various brain disorders [19,20]. Thus, a novel PAM implementation for
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high-resolution imaging of the hemodynamics and oxygen metabolism in the awake mouse brain is highly desired.
3. Principle of Multiparametric Photoacoustic Microscopy 3.1
Concentration and Oxygen Saturation of Hemoglobin
The absolute value of CHb can be quantified by statistical analysis of PAM signals [21]. At 532 nm, a near-isosbestic point of hemoglobin, PAM is insensitive to sO2. Fluctuation in the amplitude of the PAM signal acquired at this wavelength encodes both the Brownian motion and the flow of red blood cells (RBCs). Since the Brownian motion of RBCs follows the Poisson distribution, the average RBC count within the detection volume (NRBC ) can be estimated as follows: EðNRBC Þ ¼
EðAmpÞ ; VarðAmpÞ VarðNoiseÞ
(10.1)
where EðÞ and VarðÞ are the mean and variance operation, respectively. Amp and Noise denote the amplitude of the photoacoustic signal and the noise of the PAM system, respectively. With this, the average RBC count can be quantified through the statistical analysis of 100 successive A-lines. Given the fact that each RBC contains w15 pg of hemoglobin [22], CHb can thus be estimated as follows: CHb ¼
15 EðNRBC Þ ; Vol
(10.2)
where Vol represents the detection volume of the PAM system. Exploiting the difference in the optical absorption spectra of oxy- and deoxyhemoglobin (HbO2 and HbR, respectively), PAM can measure the relative concentrations of HbO2 and HbR by adding a second excitation wavelength (558 nm) and using the spectroscopic analysis [23]. With this, the sO2 can be derived in absolute values as follows: sO2 ¼
3.2
½HbO2 ½HbR þ ½HbO2
(10.3)
Cerebral Blood Flow
Correlation analysis of the same 100 A-lines used for the CHb measurement allows simultaneous quantification of the blood flow speed. The flow-induced A-line decorrelation follows a second-order exponential (i.e., Gaussian) decay, and the decay constant is linearly proportional to the flow speed [24,25]. Thus, the blood flow speed can be measured by fitting the experimental data to the decay model. Given the 100-ms interval between adjacent A-lines in our current head-restrained PAM system [4], the correlation window is 10 ms. With a B-scan speed of 1 mm/s, the movement of the PAM scan head
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over this time window is only 10 mm, comparable to the average diameter of capillaries. Therefore, the head-restrained PAM is capable of measuring microvascular blood flow [26]. With the aid of vessel segmentation, the blood flow pattern in individual microvessels can be extracted. Therefore, the vascular blood flow speed and diameter can be combined to derive CBF in the volumetric unit as follows: CBF ¼
pDV 2 ; 8
(10.4)
in which D is the vessel diameter and V is the blood flow speed along the central axis of the vessel.
3.3
Oxygen Extraction Fraction and the Cerebral Metabolic Rate of Oxygen
With the measurements extracted from individual vessel segments, the average sO2 values of all feeding arteries and draining veins (saO2 and svO2, respectively) within the cortical region of interest (ROI) can be calculated to derive oxygen extraction fraction (OEF) in this region as follows: OEF ¼
sa O2 sv O2 sa O2
(10.5)
Furthermore, the cerebral metabolic rate of oxygen (CMRO2) can be derived using Fick’s law: CMRO2 ¼ x CHb sa O2 OEF
BFtotal ; W
(10.6)
where x is the oxygen binding capacity of hemoglobin (1.36 mL of oxygen per gram hemoglobin), BFtotal is the total volumetric blood flow through the region, and W is the tissue weight estimated by assuming an average cortical thickness of 1.2 mm [27] and a tissue density of 1.05 g/mL [28].
4. Instrument Design and Configuration 4.1
Multiparametric Photoacoustic Microscopy
As shown in Fig. 10.1, the multiparametric PAM contains 2 ns-pulsed lasers (BX40-2-G and BX40-2-GR, EdgeWave) operating at the repetition rate up to 10 kHz. The two laser outputs of orthogonal polarizations are combined using a polarizing beam splitter (48e545, Edmund Optics) with minimum energy loss. Through a microscope objective (M-10, Newport), the combined beam is coupled into a single-mode fiber (P1-460BFC-2, Thorlabs). To achieve high coupling efficiency and ensure that the laser pulse energy is below the damage threshold of the fiber, the beam is attenuated by a neutral density filter (NDC-50C-2 M, Thorlabs), reshaped by an iris (SM1D12D, Thorlabs), focused by a condenser lens (LA1608, Thorlabs), and filtered by a 50-mm-diameter
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FIGURE 10.1 Schematic of the multiparametric photoacoustic microscopy (PAM). AD, achromatic doublet; BS, beam sampler; CL, correction lens; NDF, neutral density filter; RT, ring-shaped ultrasonic transducer.
pinhole (P50C, Thorlabs). To monitor and compensate for the laser fluctuation, a beam sampler (BSF10-A, Thorlabs) is placed in the optical path to tap off a small portion of the laser energy to a high-speed photodiode (FDS100, Thorlabs). The beam coming out of the fiber is launched into the scan head of PAM and collimated by an achromatic doublet (AC127-025-A, Thorlabs). Then, the beam is reshaped by an iris (SM05D5, Thorlabs) and focused by another identical doublet into the mouse brain through a correction lens (LA1207-A, Thorlabs). A customized transducer (central frequency: 35 MHz; 6-dB bandwidth: 70%) with a central opening is placed beneath the correction lens for ultrasound detection. The achromatic doublet and correction lens are mounted onto a translational stage to allow for precise vertical adjustment of the optical focus, with which the optical and acoustic foci can be confocally aligned for maximum imaging sensitivity.
4.2
Head Restraint Apparatus
To stabilize the awake mouse brain for high-resolution PAM imaging, a customized head restraint apparatus is designed, manufactured, and assembled [4]. As shown in the inset in Fig. 10.1, this apparatus consists of three major parts: head-restraining nuts, adjustable head plate, and air-floated treadmill. Prior to attaching the small nut (90,730A005, McMaster-Carr), the mouse skull needs to be cleaned and fully dried to ensure adhesive strength. Then, the nut is attached to the skull using dental cement (S380, Parkell). After cement solidification, a customized head plate with a counter-bored hole is used to
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restrain the mouse head with a screw. Mounted onto a rotation mount (RSP-1T, Newport) and a right angle clamp (RA90, Thorlabs), the head plate can be angularly and vertically adjusted to align the ROI perpendicular to the scan head. Then, the mouse is placed on the surface of a spherical treadmill, which consists of two 8-inch-diameter hollow polystyrene hemispheres (03,170-1008, Blick Art Materials) and sits on a 3Dprinted cylindrical holder. Floated by slightly compressed air (15 psi), the hollow spherical treadmill provides minimal resistance for the mouse movement.
5. Experimental Procedures for Awake Brain Imaging 5.1
Animal Preparation
To mimic the diversity of human population, outbred CD-1 mice (Male, 9e13 weeks old, Charles River Laboratories) are used for this study. To prepare the mouse for awake brain imaging, the first step is to remove the hair in its head and make a surgical incision to expose the skull. Povidone iodine is applied to avoid potential infection. Then, most of the exposed skull is covered by dental cement to attach the nut onto the skull on the contralateral side of the ROI. Once the cement is solidified, the nut is firmly adhered to the skull, and the mouse is ready to be transferred to the head restraint apparatus. Ketoprofen, a common analgesic, is applied to alleviate possible pain caused by the surgical preparation. Prior to the PAM imaging experiment, five training sessions are provided on five consecutive days, during which the behavior and stress level of the mouse are closely monitored. Mice that fail two or more training sessions are excluded from the awake brain imaging experiment. One day before the experiment, the skull over the ROI is thinned to create a window for high-resolution PAM imaging. Following established protocols [29,30], the skull is thinned carefully using a dental drill. Careful operation is critical to alleviate potential inflammation and avoid possible damages to the brain. Since the thickness of the thinned-skull window (w100 mm) is much larger than 20 mm, no obvious inflammation or detectable microglia activation is expected [30]. Right before the PAM experiment, the mouse is mounted onto the head restraint apparatus. Then, the mouse head is placed beneath a water tank, the bottom of which is sealed by an optically transparent polyethylene membrane. Ultrasound gel is applied between the skull window and the membrane for acoustic coupling. To maintain the brain temperature, the water tank is set at 37 C throughout the experiment. Moreover, a heating lamp is placed close to the awake mouse to maintain the body temperature. All experimental procedures are carried out in conformity with the animal protocol approved by the Animal Care and Use Committee at the University of Virginia.
5.2
Anesthesia Setting
During the PAM imaging, medical-quality air (AI M-T, Praxair) is used as the inhalation gas. To study the dose-dependent hemodynamic and oxygen-metabolic responses to
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general anesthetics, different anesthetic states are used. The minimum alveolar concentration (MAC) of isoflurane for the CD-1 mouse used herein is predetermined to be 1.6%, which represents moderate anesthesia. 0.8% (i.e., 0.5 MAC) and 2.4% (i.e., 1.5 MAC) of isoflurane are used to induce light and deep anesthesia, respectively. For the awake state, the isoflurane is simply set at 0% (i.e., 0 MAC). To ensure equilibrium after each switch of the anesthetic state, at least 15 min is waited before PAM imaging.
6. Cerebral Hemodynamic and Oxygen-Metabolic Responses to Isoflurane 6.1
Isoflurane-Induced Changes in Cerebral Hemodynamics and Oxygen Metabolism
Using the head-restrained PAM, side-by-side comparison of cerebral hemodynamics and oxygen metabolism in the absence and presence of isoflurane is performed. As shown in Fig. 10.3A, the mouse is switched between anesthesia and wakefulness by turning on (1.0 MAC) and off the isoflurane vaporizer. Two sets of multiparametric images are acquired under each state. The differences in sO2 and blood flow speed between the awake and anesthetized state are apparent. For quantitative comparison, individual feeding arteries and draining veins are segmented (Fig. 10.3B). The segmentation-based quantitative analysis shows no obvious change of CHb in response to the induction of isoflurane. Interestingly, svO2 is markedly increased in the presence of isoflurane while saO2 remains unchanged, together indicating reduced OEF under general anesthesia. Along with the elevated svO2 and reduced OEF, significant vasodilation and increase in the blood flow speed are observed in the anesthetized mouse brain, which together lead to increased CBF. Although the reduced OEF and increased CBF have opposite influences on CMRO2, the combined effect still leads to a significant suppression of CMRO2 in the anesthetized mouse brain (Fig. 10.2C). The isoflurane-induced vasodilation is observed in both arteries and veins. Furthermore, segmentation-based single-vessel analysis shows vessel typee and diameterespecific vasodilation. Small arteries (diameter: <40 mm) show much stronger dilation than arteries with larger diameters (>40 mm) and veins. Moreover, the dilation of small arteries is highly diameter-dependent, showing a negative correlation (r ¼ 0.66) between the relative dilation and the baseline diameter (Fig. 10.3).
6.2
Dose-Dependent Cerebral Responses to Isoflurane
To examine whether the multifaceted cerebral responses to isoflurane are dosedependent or not, three different dosages are used to induce light (0.5 MAC), medium (1.0 MAC), and deep (1.5 MAC) anesthesia. The awake state is induced by turning off the isoflurane vaporizer (0 MAC). Between the switch of each states, a minimum of 15 min is waited to allow for equilibrium. For statistical analysis, six head-restrained mice with
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FIGURE 10.2 (A) Head-restrained photoacoustic microscopy (PAM) of cerebral CHb, sO2 and blood flow speed in the absence (OFF) and presence (ON) of isoflurane. The white arrows in the second and third rows highlight the isoflurane-induced changes in svO2 and blood flow speed. Scale bar, 500 mm. (B) Nine feeding arteries and six draining veins in the 2.5 2.5 mm2 region of interest (ROI) identified and isolated by vessel segmentation. (C) Isoflurane-induced changes in the average CHb, sO2, diameter, and flow speed of the feeding and draining vessels and derived oxygen extraction fraction (OEF), cerebral blood flow (CBF), and cerebral metabolic rate of oxygen (CMRO2). Figure adopted from Ning B, et al. Simultaneous photoacoustic microscopy of microvascular anatomy, oxygen saturation, and blood flow. Optics Lett 2015;40(6):910 with permission. See the colored version online.
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FIGURE 10.3 Linear regression analysis of isoflurane-induced dilation of cortical microvessels (diameter: <40 mm). The relative dilation of small arteries, but not small veins, shows a negative dependence (r ¼ 0:66) on the baseline diameter measured under wakefulness.
FIGURE 10.4 Head-restrained photoacoustic microscopy (PAM) of sO2 and blood flow speed in the mouse brain, in the absence (0 MAC [minimum alveolar concentration]) and presence of different concentrations (0.5, 1.0, and 1.5 MAC) of isoflurane. Figure adopted from Cao R, et al. Photoacoustic microscopy of cerebral hemodynamic and oxygen-metabolic responses to anesthetics. SPIE BiOS 2017:100510V with permission. See the colored version online.
four different states are imaged. Consistent with the observation in Section 6.1, the CHb and saO2 remain unchanged under all three levels of anesthesia and wakefulness. In contrast, svO2 and blood flow speed are apparently increased as shown in representative images (Fig. 10.4). For quantitative analysis, the CHb, sO2, flow speed, and diameter of individual feeding arteries and draining veins are extracted using vessel segmentation. Then, the average hemodynamic readouts under the four states are statistically compared. Marked svO2 increase, flow upregulation, and vasodilation are observed under all three levels of anesthesia. When switching from medium to deep anesthesia, svO2 and blood flow speed partially regress, likely due to the anesthetic depression of myocardial contractility [31].
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FIGURE 10.5 Cerebral vasodilation in response to different concentrations of isoflurane (0.5, 1.0, and 1.5 MAC [minimum alveolar concentration]). Statistical comparison of the vessel diameters measured under general anesthesia with their corresponding baseline values measured under wakefulness shows significant vasodilation for all four groups across all three different anesthetic depths (significance levels are marked on the top of the columns). *, P < .05; **, P < .01; ****, P < .0001. Data are presented as mean SD. Figure adopted from Ning B, et al. Simultaneous photoacoustic microscopy of microvascular anatomy, oxygen saturation, and blood flow. Optics Lett 2015;40(6):910 with permission.
In contrast, the isoflurane-induced vasodilation is almost dose-independent. Considering the type and diameter-specific vasodilation observed in Section 6.1, the vessel segments are divided into four groups, small arteries (<40 mm in diameter), small veins (<40 mm in diameter), large arteries (>40 mm in diameter), and large veins (>40 mm in diameter). As shown in Fig. 10.5, all four groups show statistically significant vasodilation in comparison with their baselines and the diameter-dependent arterial dilation is observed under all three levels of anesthesia. However, no dose-dependent effect of isoflurane on vasodilation is observed in any of them. It is worth noting that the dose-dependent responses of svO2 and flow speed imply an inverse coupling between OEF and CBF. When the state is switched from medium to deep anesthesia, relative OEF dramatically increased from 21 8% to 37 9% along with a significant drop in relative CBFtotal from 215 39% to 180 42%. Combining the unchanged CHb and saO2, the CMRO2 maintains at a statistically constant level across different depths of anesthesia, which is only about half of that under wakefulness.
7. Discussion and Perspectives The head-restrained PAM enables, for the first time, high-resolution comprehensive imaging of cerebral hemodynamics and oxygen metabolism in the awake mouse brain. As shown in our results and other previous studies [32], CBF and CMRO2 are tightly and dynamically coupled in the awake brain. Interestingly, volatile anesthetics increase CBF
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but reduce CMRO2. The “uncoupling” of CBF and CMRO2 is known as “luxury” perfusion [33]. Capable of simultaneously quantifying the evolutions of CBF, OEF, and CMRO2 from wakefulness to anesthesia, our head-restrained PAM reveals the critical role of OEF in the flow-metabolism uncoupling. Although the head-restrained PAM shows great potential in studying cerebral hemodynamics and oxygen metabolism in the awake behaving brain, there is still plenty of room for improvement. One urgent improvement is to increase the imaging speed for visualizing rapid hemodynamic and metabolic changes. It takes, for the current headrestrained PAM system, 15 min to scan an ROI of 2.5 2.5 mm2, which prevents it from studying brain responses to neurostimulation or rapid disease onset (e.g., epilepsy). One potential solution is to integrate the head restraint apparatus and our recently developed hybrid-scan multiparametric PAM [34]. Another improvement lies in the CMRO2 quantification. Our current approach can only quantify the total CMRO2 of the entire ROI, which is millimeter in diameter. Development of novel models to extrapolate the multiparametric PAM-measured hemodynamic parameters (CHb, sO2 and CBF) from the microvascular level to the tissue level will enable high-resolution quantification of CMRO2.
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[12] Dombeck DA, Harvey CD, Tian L, Looger LL, Tank DW. Functional imaging of hippocampal place cells at cellular resolution during virtual navigation. Nat Neurosci 2010;13(11):1433e40. [13] Harvey CD, Collman F, Dombeck DA, Tank DW. Intracellular dynamics of hippocampal place cells during virtual navigation. Nature 2009;461(7266):941e6. [14] Ohmae S, Medina JF. Climbing fibers encode a temporal-difference prediction error during cerebellar learning in mice. Nat Neurosci 2015;18(12):1798e803. [15] Hentschke H, Schwarz C, Antkowiak B. Neocortex is the major target of sedative concentrations of volatile anaesthetics: strong depression of firing rates and increase of GABAA receptor-mediated inhibition. Eur J Neurosci 2005;21(1):93e102. [16] van Looij MAJ, et al. Impact of conventional anesthesia on auditory brainstem responses in mice. Hear Res 2004;193(1e2):75e82. [17] Tang J, et al. Wearable 3-D photoacoustic tomography for functional brain imaging in behaving rats. Sci Rep 2016;6:25470. [18] Tang J, Dai X, Jiang H. Wearable scanning photoacoustic brain imaging in behaving rats. J Biophotonics 2016. https://doi.org/10.1002/jbio.201500311. [19] Gould IG, Tsai P, Kleinfeld D, Linninger A. The capillary bed offers the largest hemodynamic resistance to the cortical blood supply. J Cereb Blood Flow Metab 2017;37(1):52e68. [20] Shih AY, et al. The smallest stroke: occlusion of one penetrating vessel leads to infarction and a cognitive deficit. Nat Neurosci 2013;16(1):55e63. [21] Ning B, et al. Ultrasound-aided multi-parametric photoacoustic microscopy of the mouse brain. Sci Rep 2015;5:18775. [22] Everds N. The laboratory mouse. Elsevier; 2004. https://doi.org/10.1016/B978-012336425-8/50070-4. [23] Zhang HF, Maslov K, Sivaramakrishnan M, Stoica G, Wang LV. Imaging of hemoglobin oxygen saturation variations in single vessels in vivo using photoacoustic microscopy. Appl Phys Lett 2007; 90(5):53901. [24] Chen S-L, Ling T, Huang S-W, Won Baac H, Guo LJ. Photoacoustic correlation spectroscopy and its application to low-speed flow measurement. Optics Lett 2010;35(8):1200e2. [25] Chen S-L, Xie Z, Carson PL, Wang X, Guo LJ. In vivo flow speed measurement of capillaries by photoacoustic correlation spectroscopy. Optics Lett 2011;36(20):4017e9. [26] Ning B, et al. Simultaneous photoacoustic microscopy of microvascular anatomy, oxygen saturation, and blood flow. Optics Lett 2015;40(6):910. [27] DeFelipe J. The evolution of the brain, the human nature of cortical circuits, and intellectual creativity. Front Neuroanat 2011;5(May):29. [28] Chong SP, Merkle CW, Leahy C, Srinivasan VJ. Cerebral metabolic rate of oxygen (CMRO_2) assessed by combined Doppler and spectroscopic OCT. Biomed Optics Express 2015;6(10):3941. [29] Drew PJ, et al. Chronic optical access through a polished and reinforced thinned skull. Nat Methods 2010;7:981e4. [30] Yang G, Pan F, Parkhurst CN, Grutzendler J, Gan W-B. Thinned-skull cranial window technique for long-term imaging of the cortex in live mice. Nat Protoc 2010;5(2):201e8. [31] Rusy BF, Komai H. Anesthetic depression of myocardial contractility: a review of possible mechanisms. Anesthesiology 1987;67(5):745e66. [32] Fox PT, Raichle ME. Focal physiological uncoupling of cerebral blood flow and oxidative metabolism during somatosensory stimulation in human subjects. Proc Natl Acad Sci USA 1986;83(4): 1140e4.
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[33] Kaisti KK, et al. Effects of surgical levels of propofol and sevoflurane anesthesia on cerebral blood flow in healthy subjects studied with positron emission tomography. J Am Soc Anesthesiol 2002; 96(6):1358e70. [34] Wang T, et al. Multiparametric photoacoustic microscopy of the mouse brain with 300-kHz A-line rate. Neurophotonics 2016;3(4). https://doi.org/10.1117/1.NPh.3.4.045006. [35] Cao R, et al. Photoacoustic microscopy of cerebral hemodynamic and oxygen-metabolic responses to anesthetics. SPIE BiOS 2017:100510V.
DEPARTMENT OF BI OMEDICAL ENGINE ERING, UNIVERSITY OF VIRGINIA, CHARLOTTESVILLE, V A 22 90 8, UNIT ED S TATE S; 2 DE PARTMENT OF ANESTHESIOLOGY, UNIVERSITY OF VIRGINIA, C HA R L O T TE S VIL L E, V A 2 29 08, U N I TED S TA TE S
1. Introduction Recent innovations in light microscopy have revolutionized our understanding of the braindthe most complicated and least understood organ. Among them, photoacoustic microscopy (PAM) is emerging as a new enabling technology due to its unique features [1]. Based on the photoacoustic effect, which refers to ultrasound generation from light absorption, PAM combines the advantages of intrinsic optical absorption contrast and relatively weak acoustic scattering in biological tissues. Furthermore, capitalizing on the optical absorption of blood hemoglobin, PAM is uniquely capable of label-free highresolution multiparametric imaging of hemoglobin concentration (CHb), oxygen saturation (sO2), and cerebral blood flow (CBF) in the mouse brain [2,3]. This chapter introduces a latest development of PAM, termed head-restrained PAM, which enables functional and oxygen-metabolic imaging of the awake mouse brain [4]. The principle of the multiparametric measurements, instrument design and configuration, and experimental procedures for awake brain imaging are discussed in details. Besides, the application of head-restrained PAM to study cerebral hemodynamic and oxygen-metabolic responses to isoflurane, a widely used general anesthetic, is demonstrated. At the conclusion of the chapter, future developments of this enabling technology are discussed.
Neurophotonics and Biomedical Spectroscopy. https://doi.org/10.1016/B978-0-323-48067-3.00010-X Copyright © 2019 Elsevier Ltd. All rights reserved.
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2. Background and Motivation The need for high-resolution imaging of the awake brain is evident [5] because general anesthesia can significantly reduce neuronal activity and alter multiple forms of cerebral dynamics [6e8]. The profound effects may confound the readouts of conventional light microscopes, which require the preparation of anesthetized animals, and thus impose considerable limitation on the interpretation and translation of the findings. Moreover, incapable of imaging the awake brain for comparison with the anesthetized counterpart, conventional microscopies are of limited utility in examining the important yet elusive roles of clinically used general anesthetics in the progression of multiple life-threatening brain diseases, including stroke [9] and Alzheimer’s disease [10]. Recently, two complementary approaches have been developed to extend fluorescence microscopy of neuronal activity to the awake brain. First is a head-mounted approach. With innovative designs of laser excitation, scan, and detection, conventional fluorescence microscopes can be miniaturized and mounted on the head of adult mouse, thereby eliminating motion artifacts. However, its present implementations still suffer from limited field of view (FOV) and suboptimal optical performance [11]. An alternative approach is head restraint, which provides extended FOV, improved imaging performance, and the accessibility of electrode recording at the expense of freely moving condition [6]. Moreover, this setting can be integrated with the computer-generated virtual environment for controlled stimulation [12,13]. A major concern about head restraint is the possible induction of stress. Fortunately, the self-paced voluntary treadmill has been shown to help acclimate animals to the restraint [6,12,13] and thus opened the way for broad applications of the head-restrained fluorescence microscopy [12e14], including studies of the effects of general anesthetics on neural activity [15,16]. While this innovation has advanced our understanding of neural activity without the influence of anesthesia, the coevolving hemodynamics and oxygen metabolism in the awake brain remains poorly understood. With the aid of angiographic agents, fluorescence microscopy is able to image the cerebrovascular structure and CBF [7]. However, multiple hemoglobin-related hemodynamic parameters (e.g., CHb and sO2) remain inaccessible. The ever-growing gap between light microscopyebased neuronal and hemodynamic imaging in the awake animal brain presents a critical barrier to our understanding of neurovascular coupling that underlies many important cerebral diseases. Uniquely capable of comprehensive measurement of hemodynamic parameters in a label-free manner, PAM is well suited to fill this gap. However, this research is still in a nascent stage, with no report using PAM and only a couple of studies using macroscopicresolution photoacoustic tomography. Recently, wearable photoacoustic tomography devices have been developed for brain imaging in freely moving rats [17,18]. However, they are too heavy (>20 g) and bulky (40e50 mm) to be applied to mice, in which abundant disease models and genetic manipulations are available. Moreover, their spatial resolutions (>200 mm) are insufficient to resolve cerebral microvessels, which play key roles in various brain disorders [19,20]. Thus, a novel PAM implementation for
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high-resolution imaging of the hemodynamics and oxygen metabolism in the awake mouse brain is highly desired.
3. Principle of Multiparametric Photoacoustic Microscopy 3.1
Concentration and Oxygen Saturation of Hemoglobin
The absolute value of CHb can be quantified by statistical analysis of PAM signals [21]. At 532 nm, a near-isosbestic point of hemoglobin, PAM is insensitive to sO2. Fluctuation in the amplitude of the PAM signal acquired at this wavelength encodes both the Brownian motion and the flow of red blood cells (RBCs). Since the Brownian motion of RBCs follows the Poisson distribution, the average RBC count within the detection volume (NRBC ) can be estimated as follows: EðNRBC Þ ¼
EðAmpÞ ; VarðAmpÞ VarðNoiseÞ
(10.1)
where EðÞ and VarðÞ are the mean and variance operation, respectively. Amp and Noise denote the amplitude of the photoacoustic signal and the noise of the PAM system, respectively. With this, the average RBC count can be quantified through the statistical analysis of 100 successive A-lines. Given the fact that each RBC contains w15 pg of hemoglobin [22], CHb can thus be estimated as follows: CHb ¼
15 EðNRBC Þ ; Vol
(10.2)
where Vol represents the detection volume of the PAM system. Exploiting the difference in the optical absorption spectra of oxy- and deoxyhemoglobin (HbO2 and HbR, respectively), PAM can measure the relative concentrations of HbO2 and HbR by adding a second excitation wavelength (558 nm) and using the spectroscopic analysis [23]. With this, the sO2 can be derived in absolute values as follows: sO2 ¼
3.2
½HbO2 ½HbR þ ½HbO2
(10.3)
Cerebral Blood Flow
Correlation analysis of the same 100 A-lines used for the CHb measurement allows simultaneous quantification of the blood flow speed. The flow-induced A-line decorrelation follows a second-order exponential (i.e., Gaussian) decay, and the decay constant is linearly proportional to the flow speed [24,25]. Thus, the blood flow speed can be measured by fitting the experimental data to the decay model. Given the 100-ms interval between adjacent A-lines in our current head-restrained PAM system [4], the correlation window is 10 ms. With a B-scan speed of 1 mm/s, the movement of the PAM scan head
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over this time window is only 10 mm, comparable to the average diameter of capillaries. Therefore, the head-restrained PAM is capable of measuring microvascular blood flow [26]. With the aid of vessel segmentation, the blood flow pattern in individual microvessels can be extracted. Therefore, the vascular blood flow speed and diameter can be combined to derive CBF in the volumetric unit as follows: CBF ¼
pDV 2 ; 8
(10.4)
in which D is the vessel diameter and V is the blood flow speed along the central axis of the vessel.
3.3
Oxygen Extraction Fraction and the Cerebral Metabolic Rate of Oxygen
With the measurements extracted from individual vessel segments, the average sO2 values of all feeding arteries and draining veins (saO2 and svO2, respectively) within the cortical region of interest (ROI) can be calculated to derive oxygen extraction fraction (OEF) in this region as follows: OEF ¼
sa O2 sv O2 sa O2
(10.5)
Furthermore, the cerebral metabolic rate of oxygen (CMRO2) can be derived using Fick’s law: CMRO2 ¼ x CHb sa O2 OEF
BFtotal ; W
(10.6)
where x is the oxygen binding capacity of hemoglobin (1.36 mL of oxygen per gram hemoglobin), BFtotal is the total volumetric blood flow through the region, and W is the tissue weight estimated by assuming an average cortical thickness of 1.2 mm [27] and a tissue density of 1.05 g/mL [28].
4. Instrument Design and Configuration 4.1
Multiparametric Photoacoustic Microscopy
As shown in Fig. 10.1, the multiparametric PAM contains 2 ns-pulsed lasers (BX40-2-G and BX40-2-GR, EdgeWave) operating at the repetition rate up to 10 kHz. The two laser outputs of orthogonal polarizations are combined using a polarizing beam splitter (48e545, Edmund Optics) with minimum energy loss. Through a microscope objective (M-10, Newport), the combined beam is coupled into a single-mode fiber (P1-460BFC-2, Thorlabs). To achieve high coupling efficiency and ensure that the laser pulse energy is below the damage threshold of the fiber, the beam is attenuated by a neutral density filter (NDC-50C-2 M, Thorlabs), reshaped by an iris (SM1D12D, Thorlabs), focused by a condenser lens (LA1608, Thorlabs), and filtered by a 50-mm-diameter
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FIGURE 10.1 Schematic of the multiparametric photoacoustic microscopy (PAM). AD, achromatic doublet; BS, beam sampler; CL, correction lens; NDF, neutral density filter; RT, ring-shaped ultrasonic transducer.
pinhole (P50C, Thorlabs). To monitor and compensate for the laser fluctuation, a beam sampler (BSF10-A, Thorlabs) is placed in the optical path to tap off a small portion of the laser energy to a high-speed photodiode (FDS100, Thorlabs). The beam coming out of the fiber is launched into the scan head of PAM and collimated by an achromatic doublet (AC127-025-A, Thorlabs). Then, the beam is reshaped by an iris (SM05D5, Thorlabs) and focused by another identical doublet into the mouse brain through a correction lens (LA1207-A, Thorlabs). A customized transducer (central frequency: 35 MHz; 6-dB bandwidth: 70%) with a central opening is placed beneath the correction lens for ultrasound detection. The achromatic doublet and correction lens are mounted onto a translational stage to allow for precise vertical adjustment of the optical focus, with which the optical and acoustic foci can be confocally aligned for maximum imaging sensitivity.
4.2
Head Restraint Apparatus
To stabilize the awake mouse brain for high-resolution PAM imaging, a customized head restraint apparatus is designed, manufactured, and assembled [4]. As shown in the inset in Fig. 10.1, this apparatus consists of three major parts: head-restraining nuts, adjustable head plate, and air-floated treadmill. Prior to attaching the small nut (90,730A005, McMaster-Carr), the mouse skull needs to be cleaned and fully dried to ensure adhesive strength. Then, the nut is attached to the skull using dental cement (S380, Parkell). After cement solidification, a customized head plate with a counter-bored hole is used to
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restrain the mouse head with a screw. Mounted onto a rotation mount (RSP-1T, Newport) and a right angle clamp (RA90, Thorlabs), the head plate can be angularly and vertically adjusted to align the ROI perpendicular to the scan head. Then, the mouse is placed on the surface of a spherical treadmill, which consists of two 8-inch-diameter hollow polystyrene hemispheres (03,170-1008, Blick Art Materials) and sits on a 3Dprinted cylindrical holder. Floated by slightly compressed air (15 psi), the hollow spherical treadmill provides minimal resistance for the mouse movement.
5. Experimental Procedures for Awake Brain Imaging 5.1
Animal Preparation
To mimic the diversity of human population, outbred CD-1 mice (Male, 9e13 weeks old, Charles River Laboratories) are used for this study. To prepare the mouse for awake brain imaging, the first step is to remove the hair in its head and make a surgical incision to expose the skull. Povidone iodine is applied to avoid potential infection. Then, most of the exposed skull is covered by dental cement to attach the nut onto the skull on the contralateral side of the ROI. Once the cement is solidified, the nut is firmly adhered to the skull, and the mouse is ready to be transferred to the head restraint apparatus. Ketoprofen, a common analgesic, is applied to alleviate possible pain caused by the surgical preparation. Prior to the PAM imaging experiment, five training sessions are provided on five consecutive days, during which the behavior and stress level of the mouse are closely monitored. Mice that fail two or more training sessions are excluded from the awake brain imaging experiment. One day before the experiment, the skull over the ROI is thinned to create a window for high-resolution PAM imaging. Following established protocols [29,30], the skull is thinned carefully using a dental drill. Careful operation is critical to alleviate potential inflammation and avoid possible damages to the brain. Since the thickness of the thinned-skull window (w100 mm) is much larger than 20 mm, no obvious inflammation or detectable microglia activation is expected [30]. Right before the PAM experiment, the mouse is mounted onto the head restraint apparatus. Then, the mouse head is placed beneath a water tank, the bottom of which is sealed by an optically transparent polyethylene membrane. Ultrasound gel is applied between the skull window and the membrane for acoustic coupling. To maintain the brain temperature, the water tank is set at 37 C throughout the experiment. Moreover, a heating lamp is placed close to the awake mouse to maintain the body temperature. All experimental procedures are carried out in conformity with the animal protocol approved by the Animal Care and Use Committee at the University of Virginia.
5.2
Anesthesia Setting
During the PAM imaging, medical-quality air (AI M-T, Praxair) is used as the inhalation gas. To study the dose-dependent hemodynamic and oxygen-metabolic responses to
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general anesthetics, different anesthetic states are used. The minimum alveolar concentration (MAC) of isoflurane for the CD-1 mouse used herein is predetermined to be 1.6%, which represents moderate anesthesia. 0.8% (i.e., 0.5 MAC) and 2.4% (i.e., 1.5 MAC) of isoflurane are used to induce light and deep anesthesia, respectively. For the awake state, the isoflurane is simply set at 0% (i.e., 0 MAC). To ensure equilibrium after each switch of the anesthetic state, at least 15 min is waited before PAM imaging.
6. Cerebral Hemodynamic and Oxygen-Metabolic Responses to Isoflurane 6.1
Isoflurane-Induced Changes in Cerebral Hemodynamics and Oxygen Metabolism
Using the head-restrained PAM, side-by-side comparison of cerebral hemodynamics and oxygen metabolism in the absence and presence of isoflurane is performed. As shown in Fig. 10.3A, the mouse is switched between anesthesia and wakefulness by turning on (1.0 MAC) and off the isoflurane vaporizer. Two sets of multiparametric images are acquired under each state. The differences in sO2 and blood flow speed between the awake and anesthetized state are apparent. For quantitative comparison, individual feeding arteries and draining veins are segmented (Fig. 10.3B). The segmentation-based quantitative analysis shows no obvious change of CHb in response to the induction of isoflurane. Interestingly, svO2 is markedly increased in the presence of isoflurane while saO2 remains unchanged, together indicating reduced OEF under general anesthesia. Along with the elevated svO2 and reduced OEF, significant vasodilation and increase in the blood flow speed are observed in the anesthetized mouse brain, which together lead to increased CBF. Although the reduced OEF and increased CBF have opposite influences on CMRO2, the combined effect still leads to a significant suppression of CMRO2 in the anesthetized mouse brain (Fig. 10.2C). The isoflurane-induced vasodilation is observed in both arteries and veins. Furthermore, segmentation-based single-vessel analysis shows vessel typee and diameterespecific vasodilation. Small arteries (diameter: <40 mm) show much stronger dilation than arteries with larger diameters (>40 mm) and veins. Moreover, the dilation of small arteries is highly diameter-dependent, showing a negative correlation (r ¼ 0.66) between the relative dilation and the baseline diameter (Fig. 10.3).
6.2
Dose-Dependent Cerebral Responses to Isoflurane
To examine whether the multifaceted cerebral responses to isoflurane are dosedependent or not, three different dosages are used to induce light (0.5 MAC), medium (1.0 MAC), and deep (1.5 MAC) anesthesia. The awake state is induced by turning off the isoflurane vaporizer (0 MAC). Between the switch of each states, a minimum of 15 min is waited to allow for equilibrium. For statistical analysis, six head-restrained mice with
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FIGURE 10.2 (A) Head-restrained photoacoustic microscopy (PAM) of cerebral CHb, sO2 and blood flow speed in the absence (OFF) and presence (ON) of isoflurane. The white arrows in the second and third rows highlight the isoflurane-induced changes in svO2 and blood flow speed. Scale bar, 500 mm. (B) Nine feeding arteries and six draining veins in the 2.5 2.5 mm2 region of interest (ROI) identified and isolated by vessel segmentation. (C) Isoflurane-induced changes in the average CHb, sO2, diameter, and flow speed of the feeding and draining vessels and derived oxygen extraction fraction (OEF), cerebral blood flow (CBF), and cerebral metabolic rate of oxygen (CMRO2). Figure adopted from Ning B, et al. Simultaneous photoacoustic microscopy of microvascular anatomy, oxygen saturation, and blood flow. Optics Lett 2015;40(6):910 with permission. See the colored version online.
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FIGURE 10.3 Linear regression analysis of isoflurane-induced dilation of cortical microvessels (diameter: <40 mm). The relative dilation of small arteries, but not small veins, shows a negative dependence (r ¼ 0:66) on the baseline diameter measured under wakefulness.
FIGURE 10.4 Head-restrained photoacoustic microscopy (PAM) of sO2 and blood flow speed in the mouse brain, in the absence (0 MAC [minimum alveolar concentration]) and presence of different concentrations (0.5, 1.0, and 1.5 MAC) of isoflurane. Figure adopted from Cao R, et al. Photoacoustic microscopy of cerebral hemodynamic and oxygen-metabolic responses to anesthetics. SPIE BiOS 2017:100510V with permission. See the colored version online.
four different states are imaged. Consistent with the observation in Section 6.1, the CHb and saO2 remain unchanged under all three levels of anesthesia and wakefulness. In contrast, svO2 and blood flow speed are apparently increased as shown in representative images (Fig. 10.4). For quantitative analysis, the CHb, sO2, flow speed, and diameter of individual feeding arteries and draining veins are extracted using vessel segmentation. Then, the average hemodynamic readouts under the four states are statistically compared. Marked svO2 increase, flow upregulation, and vasodilation are observed under all three levels of anesthesia. When switching from medium to deep anesthesia, svO2 and blood flow speed partially regress, likely due to the anesthetic depression of myocardial contractility [31].
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FIGURE 10.5 Cerebral vasodilation in response to different concentrations of isoflurane (0.5, 1.0, and 1.5 MAC [minimum alveolar concentration]). Statistical comparison of the vessel diameters measured under general anesthesia with their corresponding baseline values measured under wakefulness shows significant vasodilation for all four groups across all three different anesthetic depths (significance levels are marked on the top of the columns). *, P < .05; **, P < .01; ****, P < .0001. Data are presented as mean SD. Figure adopted from Ning B, et al. Simultaneous photoacoustic microscopy of microvascular anatomy, oxygen saturation, and blood flow. Optics Lett 2015;40(6):910 with permission.
In contrast, the isoflurane-induced vasodilation is almost dose-independent. Considering the type and diameter-specific vasodilation observed in Section 6.1, the vessel segments are divided into four groups, small arteries (<40 mm in diameter), small veins (<40 mm in diameter), large arteries (>40 mm in diameter), and large veins (>40 mm in diameter). As shown in Fig. 10.5, all four groups show statistically significant vasodilation in comparison with their baselines and the diameter-dependent arterial dilation is observed under all three levels of anesthesia. However, no dose-dependent effect of isoflurane on vasodilation is observed in any of them. It is worth noting that the dose-dependent responses of svO2 and flow speed imply an inverse coupling between OEF and CBF. When the state is switched from medium to deep anesthesia, relative OEF dramatically increased from 21 8% to 37 9% along with a significant drop in relative CBFtotal from 215 39% to 180 42%. Combining the unchanged CHb and saO2, the CMRO2 maintains at a statistically constant level across different depths of anesthesia, which is only about half of that under wakefulness.
7. Discussion and Perspectives The head-restrained PAM enables, for the first time, high-resolution comprehensive imaging of cerebral hemodynamics and oxygen metabolism in the awake mouse brain. As shown in our results and other previous studies [32], CBF and CMRO2 are tightly and dynamically coupled in the awake brain. Interestingly, volatile anesthetics increase CBF
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but reduce CMRO2. The “uncoupling” of CBF and CMRO2 is known as “luxury” perfusion [33]. Capable of simultaneously quantifying the evolutions of CBF, OEF, and CMRO2 from wakefulness to anesthesia, our head-restrained PAM reveals the critical role of OEF in the flow-metabolism uncoupling. Although the head-restrained PAM shows great potential in studying cerebral hemodynamics and oxygen metabolism in the awake behaving brain, there is still plenty of room for improvement. One urgent improvement is to increase the imaging speed for visualizing rapid hemodynamic and metabolic changes. It takes, for the current headrestrained PAM system, 15 min to scan an ROI of 2.5 2.5 mm2, which prevents it from studying brain responses to neurostimulation or rapid disease onset (e.g., epilepsy). One potential solution is to integrate the head restraint apparatus and our recently developed hybrid-scan multiparametric PAM [34]. Another improvement lies in the CMRO2 quantification. Our current approach can only quantify the total CMRO2 of the entire ROI, which is millimeter in diameter. Development of novel models to extrapolate the multiparametric PAM-measured hemodynamic parameters (CHb, sO2 and CBF) from the microvascular level to the tissue level will enable high-resolution quantification of CMRO2.
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