A major obstacle in understanding the mechanism of ischemic stroke is the lack of a tool to noninvasively or minimally invasively monitor cerebral hemodynamics longitudinally. Here, we applied optical-resolution photoacoustic microscopy (OR-PAM) to longitudinally study ischemic stroke induced brain injury in a mouse model with transient middle cerebral artery occlusion (MCAO). OR-PAM showed that, during MCAO, the average hemoglobin oxygen saturation (sO2) values of feeder arteries and draining veins within the stroke core region dropped ~10% and ~34%, respectively. After reperfusion, arterial sO_2 recovered back to the baseline; however, the venous sO_2 increased above the baseline value by ~7%. Thereafter, venous sO_2 values were close to the arterial sO_2 values, suggesting eventual brain tissue infarction

Gong, Enhao

Gonzales, Ernie

Hu, Song

Lee, Jin-Moo

Maslov, Konstantin

Soetikno, Brian

Wang, Lihong V.

Yan, Ping

Caltech Authors - Main

PROCEEDINGS OF SPIE
SPIEDigitalLibrary.org/conference-proceedings-of-spie
Optical-resolution photoacoustic
microscopy of ischemic stroke
Song  Hu, Ernie  Gonzales, Brian  Soetikno, Enhao  Gong,
Ping  Yan, et al.
Song  Hu, Ernie  Gonzales, Brian  Soetikno, Enhao  Gong, Ping  Yan,
Konstantin  Maslov, Jin-Moo  Lee, Lihong V. Wang, "Optical-resolution
photoacoustic microscopy of ischemic stroke," Proc. SPIE 7899, Photons Plus
Ultrasound: Imaging and Sensing 2011, 789906 (10 February 2011); doi:
10.1117/12.874366
Event: SPIE BiOS, 2011, San Francisco, California, United States
Downloaded From: https://www.spiedigitallibrary.org/conference-proceedings-of-spie on 9/18/2018  Terms of Use: https://www.spiedigitallibrary.org/terms-of-use
  
Optical-resolution photoacoustic microscopy of ischemic stroke 
 
Song Hu1, Ernie Gonzales2, Brian Soetikno1, Enhao Gong1, Ping Yan2, Konstantin Maslov1, 
Jin-Moo Lee2,*, and Lihong V. Wang1,* 
 
1Department of Biomedical Engineering, Washington University in St. Louis, St. Louis, MO 63130, USA 
2Department of Neurology and the Hope Center for Neurological Disorders, Washington University School   
 of Medicine, St. Louis, MO 63110, USA 
*Correspondence should be addressed to JML (animal model) and LVW (photoacoustics). 
 
ABSTRACT 
 
A major obstacle in understanding the mechanism of ischemic stroke is the lack of a tool to noninvasively or minimally 
invasively monitor cerebral hemodynamics longitudinally. Here, we applied optical-resolution photoacoustic microscopy 
(OR-PAM) to longitudinally study ischemic stroke induced brain injury in a mouse model with transient middle cerebral 
artery occlusion (MCAO). OR-PAM showed that, during MCAO, the average hemoglobin oxygen saturation (sO2) 
values of feeder arteries and draining veins within the stroke core region dropped ~10% and ~34%, respectively. After 
reperfusion, arterial sO2 recovered back to the baseline; however, the venous sO2 increased above the baseline value by 
~7%. Thereafter, venous sO2 values were close to the arterial sO2 values, suggesting eventual brain tissue infarction. 
 
Keywords: Optical-resolution photoacoustic microscopy, ischemic stroke, vascular anatomy, hemoglobin oxygen 
saturation, oxygen extraction fraction. 
 
  
INTRODUCTION 
 
Ischemic stroke refers to the death of brain tissue resulting from an inadequate supply of blood and oxygen, usually due 
to the thrombus of major cerebral arteries [1]. Stroke-induced brain injury may lead to body paralysis, communication 
difficulty, or vision impairment. Studying cerebrovascular responses to brain ischemia has been limited by a variety of 
imaging modalities that have either good tissue penetration but low resolution, or high resolution but requiring invasive 
preparations (open-skull windows). Here, we report a study of cerebrovascular hemodynamics in a mouse model of focal 
ischemia using a novel minimally invasive, high-resolution technique known as optical-resolution photoacoustic 
microscopy (OR-PAM).   
 
Using OR-PAM [2, 3], we serially imaged cerebral blood vessels through the intact skull before, during, and after one-
hour transient middle cerebral artery occlusion (MCAO) in Swiss Webster mice up to 24 days following ischemia. 
Cerebral vessels were imaged using two different wavelengths to quantify the relative concentrations of oxy- and deoxy-
hemoglobin, from which the hemoglobin oxygen saturation (sO2) and the oxygen extraction fraction (OEF) were 
calculated.  
 
 
METHODS AND MATERIALS  
1.1 System description 
 
In OR-PAM (Fig. 1), the cerebral vasculature is irradiated through intact mouse skulls with a short-pulsed laser beam 
generated by a wavelength-tunable laser system, consisting of an Nd:YLF pump laser and a dye laser. Wideband 
ultrasonic waves are induced as a result of transient thermo-elastic expansion due to the laser excitation, and then 
detected by a high-frequency ultrasonic transducer. To maximize the sensitivity, the optical illumination and the 
ultrasonic detection in our system are configured confocally by an acoustic-optical beam combiner, where a right-angle 
prism and a rhomboid prism form a cube with a thin layer of silicone oil in between. This novel design offers high 
acoustic detection sensitivity. A microscopic objective lens is employed to achieve nearly diffraction-limited optical 
Photons Plus Ultrasound: Imaging and Sensing 2011, edited by Alexander A. Oraevsky, Lihong V. Wang,
Proc. of SPIE Vol. 7899, 789906 · © 2011 SPIE · CCC code: 1605-7422/11/$18 · doi: 10.1117/12.874366
Proc. of SPIE Vol. 7899  789906-1
Downloaded From: https://www.spiedigitallibrary.org/conference-proceedings-of-spie on 9/18/2018
Terms of Use: https://www.spiedigitallibrary.org/terms-of-use
  
resolution (~2.6 µm in clear media). The detailed system description and performance can be found in our paper 
included in the same proceeding [4]. 
 
 
 
Figure 1. Schematic of the optical-resolution photoacoustic microscope system.  
 
 
1.2 Animal preparation 
 
A Swiss Webster mouse (Hsd:ND4, Harlan Co., ~30 g) was longitudinally monitored for up to 24 days post MCAO 
procedures. Before the first-day experiment, the mouse was anesthetized with intraperitoneal injection of an anesthesia 
cocktail [recipe: 1 ml ketamine (100 mg/ml), 0.1 ml xylazine (100 mg/ml), and 8.9 ml saline; dosage: 0.1 ml/10 g]. Then 
the hair in the mouse head was shaved, and the residual hair was further depilated with Surgi Cream before cleaning with 
deionized water. Right before functional brain imaging at each monitoring point (i.e., before, during, after MCAO 
procedure, and 2 days, 7 days, and 24 days post MCAO), the experimental mouse was anesthetized with 3% isoflurane 
vaporized by the inhalation gas (flow rate: 1 l/min), and then transferred to a custom-made stereotaxic imaging stage. 
The anesthesia was maintained with 1% isoflurane throughout the experiments. Medical-grade air was used as the 
inhalation gas to maintain the mouse at normal physiological status. The scalp of the mouse was surgically cut open 
along the sagittal line, and the exposed skull was cleaned with 0.9% sodium chloride irrigation solution right before 
imaging. Ultrasonic gel was used for ultrasound coupling and maintaining skull hydration. Throughout the experiment, 
the body temperature of the animal was maintained at 37ºC by a temperature controlled heating pad. After each 
monitoring experiment, the scalp of the experimental mouse was sutured before applying antibacterial lotion. After 24-
day monitoring, the mouse was euthanatized for histology analysis of brain infarction. 
 
 
RESULTS  
 
Using dual-wavelength (563nm and 570 nm) OR-PAM, the vascular anatomy (Figs. 2A–2F) and sO2 (Figs. 2G–2L) were 
simultaneously monitored for 24 days following the MCAO-induced focal ischemia. The detailed method for sO2 
computation can be found in our published papers [5, 6]. It is clearly shown in the anatomical images that, during 60-min 
MCAO, a majority of the microvessels in the stroke region disappeared, which indicated the MCAO-induced hypo-
perfusion of downstream microvasculature (Fig. 2B). After releasing the suture blocking the MCA blood flow, some of 
the microvessels showed up again, indicating the reperfusion of MCA (Fig. 2C). Starting from two days following 
ischemia, we can clearly see the dilation of draining veins, which was likely due to the accumulation of CO2 in the blood 
stream (Figs.2D–2F). More interestingly, from the longitudinal sO2 monitoring, we can clearly see that arteries could be 
easily distinguished from veins based on the baseline image shown in Fig. 2G (average arterial sO2: 0.86; average 
Proc. of SPIE Vol. 7899  789906-2
Downloaded From: https://www.spiedigitallibrary.org/conference-proceedings-of-spie on 9/18/2018
Terms of Use: https://www.spiedigitallibrary.org/terms-of-use
  
venous sO2: 0.73). During MCAO, the average sO2 values of arteries and veins within the stroke core region dropped 
~10% and ~34%, respectively (Fig. 2H). After reperfusion, arterial sO2 recovered back to baseline; however, the venous 
sO2 increased above the baseline value by ~7% (Fig. 2I). Thereafter, venous sO2 values were close to the arterial sO2 
values (Figs.2J–2L).  
 
Figure 2. Longitudinal transcranial OR-PAM monitoring of (A–F) cerebral vascular anatomy and (G–L) hemoglobin oxygen 
saturation (sO2) during stroke-induced ischemic brain injury. 
Proc. of SPIE Vol. 7899  789906-3
Downloaded From: https://www.spiedigitallibrary.org/conference-proceedings-of-spie on 9/18/2018
Terms of Use: https://www.spiedigitallibrary.org/terms-of-use
  
Further quantification of OEF based on the average sO2 values of the MCA and draining veins showed that, the OEF of 
the stroke region increased dramatically from 0.15 (baseline) to 0.48 during the MCAO to compensate for the decreased 
cerebral blood flow. After MCA reperfusion, the OEF progressively dropped and maintained at near-zero levels 
throughout the 24-day monitoring, which indicated the stroke-induced death of brain tissues. 
 
 
 
Figure 3. Longitudinal transcranial OR-PAM monitoring of oxygen extraction fraction (OEF) changes due to stroke-induced ischemic 
brain injury. SaO2 and SvO2 are the average sO2 in MCA and the draining veins, respectively. 
 
 
CONCLUSION 
 
We demonstrate, for the first time, longitudinal transcranial OR-PAM monitoring of morphological (i.e., vessel 
diameter) and functional (i.e., sO2 and OEF) hemodynamic alterations during transient focal ischemia in mice. 
Consistent with previous PET studies in human stroke [7], we found OEF increased during acute ischemia, but 
progressively decreased after reperfusion, suggesting eventual brain tissue infarction. OR-PAM can be a valuable tool to 
longitudinally study cerebrovascular hemodynamics in intact mouse models of ischemic stroke. 
 
 
Proc. of SPIE Vol. 7899  789906-4
Downloaded From: https://www.spiedigitallibrary.org/conference-proceedings-of-spie on 9/18/2018
Terms of Use: https://www.spiedigitallibrary.org/terms-of-use
  
ACKNOWLEDGMENTS 
 
This work was sponsored by National Institutes of Health Grants R01 EB000712, R01 EB008085, R01 CA134539, R01 
NS48283, P01 NS32636, U54 CA136398, and 5P60 DK02057933. Prof. Lihong V. Wang has a financial interest in 
Microphotoacoustics, Inc. and Endra, Inc., which, however, did not support this work. 
 
 
REFERENCES 
 
[1] J. M. Lee, G. J. Zipfel and D. W. Choi, "The changing landscape of ischaemic brain injury mechanisms," Nature 
399, A7-14 (1999). 
[2] S. Hu, K. Maslov, V. Tsytsarev and L. V. Wang, "Functional transcranial brain imaging by optical-resolution 
photoacoustic microscopy," J. Biomed. Opt. 14(4), 040503 (2009). 
[3] K. Maslov, H. F. Zhang, S. Hu and L. V. Wang, "Optical-resolution photoacoustic microscopy for in vivo imaging 
of single capillaries," Opt. Lett. 33(9), 929-931 (2008). 
[4] K. Maslov, S. Hu and L. V. Wang, "Second-generation optical-resolution photoacoustic microscopy," SPIE 
Proceeding, (in press). 
[5] S. Hu and L. V. Wang, "Photoacoustic imaging and characterization of the microvasculature," Journal of 
Biomedical Optics 15(1), 011101 (2010). 
[6] H. F. Zhang, K. Maslov, M. Sivaramakrishnan, G. Stoica, and L. V. Wang, "Imaging of hemoglobin oxygen 
saturation variations in single vessels in vivo using photoacoustic microscopy," Applied Physics Letters 90(5), 
053901 (2007). 
[7] W.-D. Heiss, "Ischemic Penumbra: Evidence From Functional Imaging in Man," Journal of Cerebral Blood Flow & 
Metabolism 20, 1276–1293 (2000). 
 
 
 
Proc. of SPIE Vol. 7899  789906-5
Downloaded From: https://www.spiedigitallibrary.org/conference-proceedings-of-spie on 9/18/2018
Terms of Use: https://www.spiedigitallibrary.org/terms-of-use


Optical-resolution photoacoustic microscopy of ischemic stroke

https://authors.library.caltech.edu/89809/1/789906.pdf

Optical-resolution photoacoustic microscopy of ischemic stroke

Abstract

Similar works

Full text

Available Versions

Caltech Authors - Main