用近红外光谱术评价缺血性脑梗塞的血流动力学变化
作者：陈卫国 李鹏程 骆清铭 曾绍群 胡波
单位：陈卫国.华中理工大学生物医学光子学研究所，武汉 430074
关键词：近红外光谱；脑血流; 脑梗塞；实时；预测
航天医学与医学工程000202
摘要：目的 在标准的动物脑梗塞模型上，对近红外光大脑地形图技术反映梗塞灶血流动力学变化进行评价。 方法 建立大鼠线栓脑中动脉梗塞模型和光敏剂诱导皮层梗塞模型，用近红外光大脑地形图(NCT),功能核磁共振成像(fMRI)和TTC染色成像技术分别对皮层进行定位测量和成像检测，并进行结果比较。 结果 光敏剂诱导模型，24 h组，NCT对梗塞灶成像面积和TTC染色样本的相关系数为0.897(P<0.05)；对2月组，相关系数为0.906(P<0.01)。大脑中动脉梗塞模型，常温组，NCT梗塞面积和染色样本相关系数为0.820(P<0.05)，NCT和fMRI对梗塞灶成像面积的相关系数为0.874(P<0.05)；亚低温组，NCT梗塞灶成像面积和样本梗塞灶染色面积的相关系数为0.851(P<0.05)，NCT、fMRI对梗塞灶成像面积的相关系数为0.782(P<0.05)。 结论 NCT技术能较好地反映局部脑血流的变化，该技术可望成为脑卒中趋向病人的实时预测及康复监测。
, 百拇医药
中图分类号：R319；R743 文献标识码：A 文章编号：1002-0832(2000)02-0084-06
Hemodynamic Assessment of Ischemic Stroke with Near-Infrared Spectroscopy
CHEN Wei-guo1，LI Peng-cheng1, LUO Qing-ming1, ZENG Shao-qun1,HU Bo2
(1.Institute of Biomedical Photonics, Huazhong University of Science & Technology, Wuhan, Hubei 430030, China；2.Xiehe Hospital, Tongji Medical University, Wuhan, Hubei 430074, China)
, 百拇医药
Abstract: Objective To validate near-infrared cerebral topography (NCT) as a practical toolin tracing the regional hemodynamic changes during normal ischemic stroke model of rat. Method Middle cerebral artery occlusion (MCAO) and photosensitizer induced intracranial infarction model of rat were established. The geometric shape and infarction area were measured by NCT, functional magnetic resonance imaging (fMRI), and TTC stained anatomical imaging techniques. Result In photosensitizer induced infarction model, the correlation between anatomical infarct area and NCT image area for infarct focus were r= 0.897 for 24 h group (P<0.05) and r=0.906 for 2 months group (P<0.01),respectively. The correlation between anatomical infarction area and NCT image area for infarct focus were r=0.820 for normothermia group (P<0.05) and r= 0.851 for hypothermia group (P<0.05), respectively. The correlation between fMRI and NCT image area for infarction focus were r= 0.874 for normothermia group (P<0.05) and r= 0.782 for hypothermia group (P<0.05),respectively. Conclusion Measurement with NCT for infarction focus matched well with fMRI and anatomic sample in rats. NCT technique might be a practical tool for short-term prediction of stroke and the rehabilitation after stroke in real time.
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Key words: near-infrared spectroscopy; cerebral blood flow; cerebral infarction; real time; prediction
Ischemic stroke is a dangerous cerebrovascular disease, which has high mortality and disability rate. The prediction and prevention are more important than treatment because the recovery of nervous system is very difficult[1].The prediction of stroke includes long-term and short-term prediction. The long-term prediction could be acquired from analysis of hemodynamics, cerebrovascular structure changes etc. Infact, several cerebral functional mapping techniques such as fMRI, CT, transcranial Doppler ultrasound (TCD)[2] have been used in prognosis of ischemic stroke. However, CT and brain MRI information concerning the extent of cerebral infarction usually come after the clinical onset, it may be helpful for the treatment,but is of little value in predicting the attack.
, 百拇医药
The short-term prediction or real-time prediction (e.g. in 5 min) could warn the patient on time to take action to protect the brain from stroke attack. But the major requirement for short-term prediction instruments is that it should be small enough to wear. It is obvious that current instruments could not meet these requirements. The short-term prediction and tracing of stroke hawe not got come true.
Near-infrared spectroscopy (NIRS), as a novel noninvasive optical technique can measure changes in concentration of oxygenated and deoxygenated hemoglobin ([oxy-Hb], [deoxy-Hb]) in cerebral blood. The changes in total hemoglobin concentration [t-Hb] reflect those in blood volume which in the optical field, are closely related to those in cerebral blood flow. Many researchers have elucidated the optical mechanism of hemoglobin state changes, which reflect the CBF and have set up non-animal models to verify the hypothesis[3]. Even more, some mapping and spectroscopy studies on human nervous diseases and activities have been made[4]. However, no proper animal model has been established to confirm the theory. Meanwhile, these were no consideration of the brain structure, thus localization of the focus was impossible. In fact, a certain cerebral function involves many functional areas. Without cerebral structure mapping, it is difficult to localize the functional areas involved in cerebral diseases or psychic activities. The main purpose of this study was to establish a localizing diagnostic technique of ischemic stroke and to analyze the spatial characteristics of the Near-infrared cerebral topography(NCT) images corresponding to the hemodynamic alternations during ischemia.
, 百拇医药
Method
The principle and system description of NCT The theory of [oxy-Hb], [deoxy-Hb] absorption in near-infrared spectrum has been elucidated by many authors[3,4]. NCT is an optical method for measuring concentration changes of oxy-Hb and deoxy-Hb in cerebral vessels by means of characteristic absorption spectra of hemoglobin in the near-infrared range. Changes in total-Hb (sum of oxy-Hb and deoxy-Hb) indicate blood volume changes and are correlated with CBF changes under condition with constant hematocrit and perfusion. Thus, NCT can measure both blood oxygenation and hemodynamics in the brain noninvasively. In our system, hemodynamic changes in cerebral cortex could be localized by the combination of the NCT signals with relative cortex points by neutrona-vigator in rats or humans.
, http://www.100md.com
The NCT developed by us is shown in figure 1. Two laser diodes were used as the continuous light sources; their wavelengths are 760 nm and 850 nm respectively and their intensities were modulated at 1 kHz. The distance between the end surface of incident and receiving (for reflected light) fiberoptic bundles on the rat's scalp is about 4 mm which is just the length of the path of the reflected light from the cerebral cortex, passing through the skin and the skull. We then will refer to the middle point from the incident and the received points as the measurement position. The reflected light was collected in the receiving fiberoptic bundle and transmitted to a photomultiplier tube(PMT). The PMT output was separated into two modulated signals for the corresponding wavelengths with two lock-in amplifiers. After analog-to-digital conversion, the signals were fed to a computer at a sampling rate of 1 s.
, http://www.100md.com
Fig.1 Experimental setup of NCT
Nylon monofilament for intraluminal middle cerebral artery occlusion 20 male sprague-dawley rats weighing 200～250 g were used. All processes were performing in according with institutional guidelines. Intraluminal ligature was made of 4-0 surgical nylon monofilament (Nitcho Kogyo Co,Inc).The tips of the ligatures were rounded by flame heating and coated with silicone.
Animals were initially anesthetized with 400 mg/kg IP chloral hydrate, then the anesthetic state was maintained with 0.5% to 2% halothane delivered in an air oxygen mixture gas through a facemask. The ostium of the left MCAO in each rat was occluded intraluminally[6].A ligature was introduced through the left CCA into the internal carotid artery, then advanced approximately 17mm intracranially from the CCA bifurcation. After the model was set up, the rats were randomly assigned to either normothermia (37℃,n=10) or hypothermia (33℃,n=10) condition created by servoregulation of pericranial temperature to the indicated values. The MCA was occluded for 1 h. After drawing out the ligature, the wound was closed, and normothermia (37℃) or hypothermia (33℃) was continued for 1 h[7].
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Photosensitizer induced intracranial infarct model 20 male spargue-dawley rats weighing 200～250g were anesthetized according to the methods described above. The rats were pronely positioned on an operation table. A YAG:Nd pumped dye laser (wavelength, 562 nm, peak power, 325 mw) was focused onto the right prefrontal lobe for 30 min.Simultaneously,the photosensitizer dye rose Bengal [2%(v/v) in 0.9%saline, 0.125 ml/100g weight] was injected intravenously into the circulation over 90 s. This irradiation procedure has been shown to produce cortical infarction. After the irradiation, wounds were sutured, and the rats were randomly assigned to either chronic (2months,n=10) or acute (24 h, n=10) group, and then were allowed to recover in their cages[8].
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Magnetic resonance spectroscopy protocols All MRI images were obtained on a Bruker Biospec 47/30 spectrometer. The probe was inserted into a Bruker Biospec200 spectrometer with a vertical bore and a magnetic field strength of 4.7T. The magnetic field homogeneity was optimized using the water signal. Homogeneity of 0.5 parts per million (PPM) was obtained in all experiments, leading to a constant line width. The excitation pulse was optimized by maximizing the water signal in the 1H spectra[13]. A T2-weighted MRI (Slice thickness, 1 mm; interlice separation, 1 mm;repeat time [TR], 2.05 s; echo time [TE], 25 ms;) was used to identify the location of the stroke and for lesion volume estimation.
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The defined 25 measurement points on the rat's scalp corresponding to relevant cortex points are shown in figure 2[5]. Pre-and post-MCAO, changes in the concentration of oxy-and deoxy-and total hemoglobin in occluded and controlled hemisphere were recorded, and a color map was reconstructed with these data. According to the rat's cerebral cortex map, the NCT overlapped on the rat's cerebral cortex. An NCT, which mapped total-Hb concentration distribution, was acquired and compared with a MRI image and anatomical sample from the same rat. We were thereby able to validate the NCT images with the corresponding ischemic area and to analyze the spatial characteristics of the hemodynamic alternations caused by the ischemia.
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Fig.2 With neutronavigator, 25 white circles on the scalp representing different cortex points were selected to measure with NCT
R—rat head right side, L—rat head left side
For each rat with acute photosensitizer induced intracranial infarction model and MCAO model (including normothermia group and hypothermia group), NCT and fMRI were obtained at preoperation and at 24 h postoperation. After recovering for 2 months, NCT and MRI images for another 10 rats with chronic photosensitizer induced intracranial infarcton model were made.
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Neurological evaluation was performed 24 h after the induction of ischemia and scored on a 6-point scale, as described previously by Zea LE et al[6]. For the acute photosensitizer induced intracranial infarction model and MCAO model, animals were anesthetized with 300 mg/kg IP chloral hydrate and decapitated. The brains were quickly removed and inspected to confirm the position of the infarction focus, the absence of subarachnoid hemorrhage and/or arterial penetration with the ligature. The brains were sectioned coronarily with for 30 min in 2% solution of TTC at 37℃ for vital staining. The occlusion focus areas were calculated with the following formula:
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Infarction focus area (%) = infarction cortex lattice work number/ whole cortex latticework number
For the chronic photosensitizer induced intracranial infarct model (2 months), same procedures were performed for obtaining brain samples as described above.
Physiological values and infarction area ratio were compared between groups with Chi test analysis of variance and with one way ANOVA analysis. The association between infarction size and neurological deficit score was analyzed with correlation coefficient. Parametric values are represented as mean(
±s). Significance was assumed at the value P<0.05.
, 百拇医药
Results
To evaluate the spatial resolution of NCT, we established the photosensitizer introduced cortex infarction model that had a small infarction focus. Now, the spatial characteristics of changes in the concentration of Hb caused by infarction are described as follows. To visualize these characteristics, we mapped the static topograms of the changes in concentration of Hb during pre-and post-ischemic models. Meanwhile, a series of fMRI images were obtained on the same rats for identifying the reliability of the NCT images.
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The correlation of geometric shape with image areas of different techniques was compared with. In the photosensitizer induced infarction model (Fig.3.a), one way ANONAs analysis showed that there were no significant difference between anatomical samples, fMRI images and NCT images for infarction focus [F(2,27)=3.10, P>0.05 for 24 h group; F(2,27)= 2.68, P>0.05 for 2 months group]. Comparing with 24 h group, fMRI images for infarction area showed a significant decrease in the 2 months group (P<0.01); Similar phenomenon was observed in NCT images and in anatomical samples (P<0.01;P<0.01, respectively). The correlation in infarction area between anatomical samples and NCT image area for infarction focus were r=0.897 for 24 h group (P<0.05) and r=0.906 for 2 months group (P<0.01), respectively.
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For the hypothermia MCAO group (Fig.3.b), the fMRI mean image area of infarction focus was 16.03%±2.1%;the NCT images mean image area for infraction focus was 19.36%±2.82%;the infarction focus mean area of anatomical samples was 16.93%±1.57%. There were no significant difference among NCT images, fMRI mean image and anatomical samples [F(2,27)=2.47, P>0.05]; In the normothermia MCAO group, the fMRI mean image area of infarction focus was 40.32%±2.16%;the NCT images mean image area of infraction focus was 42.48%±5.23%; The infract focus mean area of anatomical samples was 36.65%±4.35%. There were no significant difference among NCT images, fMRI mean image and anatomical samples [F(2,27)= 2.64,P>0.05], similarly.
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Comparing with hypothermia group, infraction area of anatomical samples had a significant increase in the normothermia group (P<0.01); similar difference were observed in fMRI images (P<0.01) and in NCT images (P<0.05). The correlation between anatomical samples infarction area and NCT image area for infarction focus werer=0.820 for normothermia group (P<0.05) and r=0.851 for hypothermia group (P<0.05), respectively. The correlation between fMRI and NCT image area for infarction focus were r= 0.874 for normothermia group (P<0.05) and r= 0.782 for hypothermia group (P<0.05), respectively.
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Fig.3 Cortical infarction area measured with NCT,fMRI and anatomical techniques for
(a) photosensitizer induced cortex infarct model of 24 hours group and 2 month group;(b)MCAO model of normothermia group and hypothermia group
Discussion
There are a series of hemodynamic changes such as higher erythrocyte sedimentation rate[12], slower blood flow velocity that result in blood supplement reduction in ischemic focus while ischemic stroke impending in ischemic stroke patient. This kind of CBF changes may induce decrease of [oxy-Hb], [deoxy-Hb] and t-Hb content that differ from the hemodynamic changes caused by other diseases[15]. This is the pathphysiological basis for predicting ischemic stroke by NCT.
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NCT simultaneously measures the oxy-Hb, deoxy-Hb and t-Hb in local tissue and offers another information about blood supply and blood volume in unit tissue. The basic principal makes it possible to measure the cerebral blood supply[9]. The theory about [oxy-Hb], [deoxy-Hb] absorption in near-infrared spectrum has been elucidated by many authors[3,11]. However, no proper animal model has been established to validate the theory. Another barrier for NCT technique is to establish a corresponding relation between the NCT signals and anatomical structure. This is a perquisite for NIR localizing diagnosis[4,10].
, 百拇医药
In the present study, a remarkable finding is that NCT images reliably reflect CBF changes caused by ischemic stroke in the animal model. Our results confirmed the hypothesis that NIRS signals are correspondent to total Hb content changes that reflect blood supply. Specifically, subtle changes of NCT exactly reflect the rehabilitation of neurons in the ischemic focus in the photosensitizer induced intracranial infarct model and had millimeter spatial resolution and piccosecond temporal resolution[6]. And in the MCAO model, NCT can show penumbra that predicts physical and functional rehabilitation ability. The central ischemic focus in the anatomical samples completely corresponded to NCT central low signal core. A well match relationship between measurement point and anatomical structure has been obtained with neutronavigator in both photosensitizer induced intracranial infarct models and MCAO models.
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Although fMRI has a high spatial resolution, the complicated system made it unpractical to be used as a portable and common instrument for tracing patient's hemodynamics changes caused by various factors. More important, fMRI studies on cerebral function may overlook some key information when the studied focus is not associated with reduction of deoxy-hemoglobin[14].
Comparing with MRI, currently available NCT has lower spatial resolution, but it could catch some subtle physiological signals that is important for the disease diagnosis owing to its high temporal resolution. Meanwhile, the outstanding property of compact and reliability of NCT enable it to become a common diagnostic instrument. Moreover, it overcomes the disadvantage of fMRI because NCT simultaneously measures deoxy-Hb and oxy-Hb. Of course, NCT could not replace other techniques, such as MRI, because it has relatively worse spatial resolution. However, it is complementary to other tools because it is compact and can measure with high temporal resolution. So, NCT is thus useful for tracing regional cerebral blood flow and investigating the subtle hemodynamic changes during surface brain activity in real time. Basing on the above results, we can measure more complex functional activities such as semantic, phonological and orthographic processes and explore the associations among those processes in temporal sequence[4]. Although NCT currently available could only reflect the two-dimension area of the infarct focus for the restriction of detection depth, many researchers began to improve the optical entrance model and the imaging technique[5]. Our further study will be focused on the measurement of hematoma in the deep cerebral tissue with 200 MHz frequency phase-differentiation technique and developing NIR tomography technique just as computerized tomography.
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In conclusion, combined with neutronavigator, the NCT could localize the hemodynamic change area causing the stroke. As a compact and reliable instrument, it could be used as real time monitoring instrument at bedside and in critical care ward for warning the patient with an increased risk of stroke. The instrument can also help clinicians to understand a patient's potential for rehabilitation and set realistic plans for treatment during rehabilitation.
(Acknowledgments：Special thanks are expressed to Professor Liyun Li at State Key Laboratory for Atom Physics and Spectroscopy, Institute for Physics and Mathematics, Chinese Academy of Science, for her MRI data analysis.We appreciate Dr.Steven L.Jacques for revise of the paper.)
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Foundation item:Supported by the National Nature Science Foundation of China(39770228)
[References]
[1] Dennis M,Bamford J,Sandercock P et al.Prediction of transient ischemic attacks in the Oxfordshire Community Stroke Project[J].Stroke,1990,21:848～853
[2] Honig CR,Lammers C,Buttner T et al. Long-term prediction of infratentorial transient ischemic attacks and minor stroke[J].Stroke,1992,23 (2): 199～204
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[3] Chance B,Zhuang C,Unan C et al.Cognition-activated low-frequency modulation of light absorption in human brain[J].Proc Natl Acad Sci USA,1993,90: 3370～3374
[4] Maki A,Yamshita Y,Ito Y et al.Spatial and temporal analysis of motor activity using noninvasive NIR topography[J].Med Phys,1995,22(12):1997-2005
[5] Luo QM,Zeng SQ,Nioka S et al.Real time functional near-infrared imager[C].Proceedings of SPIE,1998,Vol.3548: 109～113
, 百拇医药
[6] Zea LE,Weinstein PR,Carlson S et al.Reversible middle cerebral artery occlusion without crainiectomy in rats[J].Stroke,1989,20: 84～91
[7] Riddenour TR,Warner DS,Todd MM et al.Mild hypothermia reduces infract size resulting from temporary but not permanent focal ischemia in rats[J].Stroke,1992,23(5): 733～738
[8] Waston BD,Dtrich WD,Prade R et al.Argon laser induced arterial photothrombosis[J].J Neurosurg,1987,66:748～754
, 百拇医药
[9] Villlringer A,Planck J,Hock C et al.Near-infrared spectroscopy (NIRS)-A new tool to study hemodynamics changes during activation of brain function in human adults[J].Neurosci Lett,1993,154:101～104
[10] Li JY,Ueda H,Seiyama A et al.A near-infrared spectroscopy study of cerebral ischemia and ischemic tolerance in gerbils[J].Stroke,1997,28(7): 1451～1457
[11] Cope M,Delpy DT,Reynolds EO et al.Methods of quantitating cerebral near infrared spectroscopy data[J].Adv Exp Med Biol,1988,222:183～189
, 百拇医药
[12] Chamorro A,Vila N,Ascaso C et al.Early prediction of stroke severity Role of the erythrocyte sedimentation rate[J].Stroke,1995,26: 573～576
[13] Rijen PC,Verheul HB,Echteld CJA et al. Effect of dextromethorphan on rat brain during ischemia and reperfusion assessed by magnetic resonance spectroscopy[J].Stroke,1991,22: 343～350
[14] Sakatni K,Xie YX,Lichty W et al.Language-activated cerebral blood oxygenation and hemodynamic changes of left prefrontal cortex in poststroke aphastic patients a near-infrared spectroscopy study[J].Stroke,1998,29(7): 1299～1304
Recieved date:1999-07-13, http://www.100md.com
单位：陈卫国.华中理工大学生物医学光子学研究所，武汉 430074
关键词：近红外光谱；脑血流; 脑梗塞；实时；预测
航天医学与医学工程000202
摘要：目的 在标准的动物脑梗塞模型上，对近红外光大脑地形图技术反映梗塞灶血流动力学变化进行评价。 方法 建立大鼠线栓脑中动脉梗塞模型和光敏剂诱导皮层梗塞模型，用近红外光大脑地形图(NCT),功能核磁共振成像(fMRI)和TTC染色成像技术分别对皮层进行定位测量和成像检测，并进行结果比较。 结果 光敏剂诱导模型，24 h组，NCT对梗塞灶成像面积和TTC染色样本的相关系数为0.897(P<0.05)；对2月组，相关系数为0.906(P<0.01)。大脑中动脉梗塞模型，常温组，NCT梗塞面积和染色样本相关系数为0.820(P<0.05)，NCT和fMRI对梗塞灶成像面积的相关系数为0.874(P<0.05)；亚低温组，NCT梗塞灶成像面积和样本梗塞灶染色面积的相关系数为0.851(P<0.05)，NCT、fMRI对梗塞灶成像面积的相关系数为0.782(P<0.05)。 结论 NCT技术能较好地反映局部脑血流的变化，该技术可望成为脑卒中趋向病人的实时预测及康复监测。
, 百拇医药
中图分类号：R319；R743 文献标识码：A 文章编号：1002-0832(2000)02-0084-06
Hemodynamic Assessment of Ischemic Stroke with Near-Infrared Spectroscopy
CHEN Wei-guo1，LI Peng-cheng1, LUO Qing-ming1, ZENG Shao-qun1,HU Bo2
(1.Institute of Biomedical Photonics, Huazhong University of Science & Technology, Wuhan, Hubei 430030, China；2.Xiehe Hospital, Tongji Medical University, Wuhan, Hubei 430074, China)
, 百拇医药
Abstract: Objective To validate near-infrared cerebral topography (NCT) as a practical toolin tracing the regional hemodynamic changes during normal ischemic stroke model of rat. Method Middle cerebral artery occlusion (MCAO) and photosensitizer induced intracranial infarction model of rat were established. The geometric shape and infarction area were measured by NCT, functional magnetic resonance imaging (fMRI), and TTC stained anatomical imaging techniques. Result In photosensitizer induced infarction model, the correlation between anatomical infarct area and NCT image area for infarct focus were r= 0.897 for 24 h group (P<0.05) and r=0.906 for 2 months group (P<0.01),respectively. The correlation between anatomical infarction area and NCT image area for infarct focus were r=0.820 for normothermia group (P<0.05) and r= 0.851 for hypothermia group (P<0.05), respectively. The correlation between fMRI and NCT image area for infarction focus were r= 0.874 for normothermia group (P<0.05) and r= 0.782 for hypothermia group (P<0.05),respectively. Conclusion Measurement with NCT for infarction focus matched well with fMRI and anatomic sample in rats. NCT technique might be a practical tool for short-term prediction of stroke and the rehabilitation after stroke in real time.
, http://www.100md.com
Key words: near-infrared spectroscopy; cerebral blood flow; cerebral infarction; real time; prediction
Ischemic stroke is a dangerous cerebrovascular disease, which has high mortality and disability rate. The prediction and prevention are more important than treatment because the recovery of nervous system is very difficult[1].The prediction of stroke includes long-term and short-term prediction. The long-term prediction could be acquired from analysis of hemodynamics, cerebrovascular structure changes etc. Infact, several cerebral functional mapping techniques such as fMRI, CT, transcranial Doppler ultrasound (TCD)[2] have been used in prognosis of ischemic stroke. However, CT and brain MRI information concerning the extent of cerebral infarction usually come after the clinical onset, it may be helpful for the treatment,but is of little value in predicting the attack.
, 百拇医药
The short-term prediction or real-time prediction (e.g. in 5 min) could warn the patient on time to take action to protect the brain from stroke attack. But the major requirement for short-term prediction instruments is that it should be small enough to wear. It is obvious that current instruments could not meet these requirements. The short-term prediction and tracing of stroke hawe not got come true.
Near-infrared spectroscopy (NIRS), as a novel noninvasive optical technique can measure changes in concentration of oxygenated and deoxygenated hemoglobin ([oxy-Hb], [deoxy-Hb]) in cerebral blood. The changes in total hemoglobin concentration [t-Hb] reflect those in blood volume which in the optical field, are closely related to those in cerebral blood flow. Many researchers have elucidated the optical mechanism of hemoglobin state changes, which reflect the CBF and have set up non-animal models to verify the hypothesis[3]. Even more, some mapping and spectroscopy studies on human nervous diseases and activities have been made[4]. However, no proper animal model has been established to confirm the theory. Meanwhile, these were no consideration of the brain structure, thus localization of the focus was impossible. In fact, a certain cerebral function involves many functional areas. Without cerebral structure mapping, it is difficult to localize the functional areas involved in cerebral diseases or psychic activities. The main purpose of this study was to establish a localizing diagnostic technique of ischemic stroke and to analyze the spatial characteristics of the Near-infrared cerebral topography(NCT) images corresponding to the hemodynamic alternations during ischemia.
, 百拇医药
Method
The principle and system description of NCT The theory of [oxy-Hb], [deoxy-Hb] absorption in near-infrared spectrum has been elucidated by many authors[3,4]. NCT is an optical method for measuring concentration changes of oxy-Hb and deoxy-Hb in cerebral vessels by means of characteristic absorption spectra of hemoglobin in the near-infrared range. Changes in total-Hb (sum of oxy-Hb and deoxy-Hb) indicate blood volume changes and are correlated with CBF changes under condition with constant hematocrit and perfusion. Thus, NCT can measure both blood oxygenation and hemodynamics in the brain noninvasively. In our system, hemodynamic changes in cerebral cortex could be localized by the combination of the NCT signals with relative cortex points by neutrona-vigator in rats or humans.
, http://www.100md.com
The NCT developed by us is shown in figure 1. Two laser diodes were used as the continuous light sources; their wavelengths are 760 nm and 850 nm respectively and their intensities were modulated at 1 kHz. The distance between the end surface of incident and receiving (for reflected light) fiberoptic bundles on the rat's scalp is about 4 mm which is just the length of the path of the reflected light from the cerebral cortex, passing through the skin and the skull. We then will refer to the middle point from the incident and the received points as the measurement position. The reflected light was collected in the receiving fiberoptic bundle and transmitted to a photomultiplier tube(PMT). The PMT output was separated into two modulated signals for the corresponding wavelengths with two lock-in amplifiers. After analog-to-digital conversion, the signals were fed to a computer at a sampling rate of 1 s.
, http://www.100md.com
Fig.1 Experimental setup of NCT
Nylon monofilament for intraluminal middle cerebral artery occlusion 20 male sprague-dawley rats weighing 200～250 g were used. All processes were performing in according with institutional guidelines. Intraluminal ligature was made of 4-0 surgical nylon monofilament (Nitcho Kogyo Co,Inc).The tips of the ligatures were rounded by flame heating and coated with silicone.
Animals were initially anesthetized with 400 mg/kg IP chloral hydrate, then the anesthetic state was maintained with 0.5% to 2% halothane delivered in an air oxygen mixture gas through a facemask. The ostium of the left MCAO in each rat was occluded intraluminally[6].A ligature was introduced through the left CCA into the internal carotid artery, then advanced approximately 17mm intracranially from the CCA bifurcation. After the model was set up, the rats were randomly assigned to either normothermia (37℃,n=10) or hypothermia (33℃,n=10) condition created by servoregulation of pericranial temperature to the indicated values. The MCA was occluded for 1 h. After drawing out the ligature, the wound was closed, and normothermia (37℃) or hypothermia (33℃) was continued for 1 h[7].
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Photosensitizer induced intracranial infarct model 20 male spargue-dawley rats weighing 200～250g were anesthetized according to the methods described above. The rats were pronely positioned on an operation table. A YAG:Nd pumped dye laser (wavelength, 562 nm, peak power, 325 mw) was focused onto the right prefrontal lobe for 30 min.Simultaneously,the photosensitizer dye rose Bengal [2%(v/v) in 0.9%saline, 0.125 ml/100g weight] was injected intravenously into the circulation over 90 s. This irradiation procedure has been shown to produce cortical infarction. After the irradiation, wounds were sutured, and the rats were randomly assigned to either chronic (2months,n=10) or acute (24 h, n=10) group, and then were allowed to recover in their cages[8].
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Magnetic resonance spectroscopy protocols All MRI images were obtained on a Bruker Biospec 47/30 spectrometer. The probe was inserted into a Bruker Biospec200 spectrometer with a vertical bore and a magnetic field strength of 4.7T. The magnetic field homogeneity was optimized using the water signal. Homogeneity of 0.5 parts per million (PPM) was obtained in all experiments, leading to a constant line width. The excitation pulse was optimized by maximizing the water signal in the 1H spectra[13]. A T2-weighted MRI (Slice thickness, 1 mm; interlice separation, 1 mm;repeat time [TR], 2.05 s; echo time [TE], 25 ms;) was used to identify the location of the stroke and for lesion volume estimation.
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The defined 25 measurement points on the rat's scalp corresponding to relevant cortex points are shown in figure 2[5]. Pre-and post-MCAO, changes in the concentration of oxy-and deoxy-and total hemoglobin in occluded and controlled hemisphere were recorded, and a color map was reconstructed with these data. According to the rat's cerebral cortex map, the NCT overlapped on the rat's cerebral cortex. An NCT, which mapped total-Hb concentration distribution, was acquired and compared with a MRI image and anatomical sample from the same rat. We were thereby able to validate the NCT images with the corresponding ischemic area and to analyze the spatial characteristics of the hemodynamic alternations caused by the ischemia.
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Fig.2 With neutronavigator, 25 white circles on the scalp representing different cortex points were selected to measure with NCT
R—rat head right side, L—rat head left side
For each rat with acute photosensitizer induced intracranial infarction model and MCAO model (including normothermia group and hypothermia group), NCT and fMRI were obtained at preoperation and at 24 h postoperation. After recovering for 2 months, NCT and MRI images for another 10 rats with chronic photosensitizer induced intracranial infarcton model were made.
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Neurological evaluation was performed 24 h after the induction of ischemia and scored on a 6-point scale, as described previously by Zea LE et al[6]. For the acute photosensitizer induced intracranial infarction model and MCAO model, animals were anesthetized with 300 mg/kg IP chloral hydrate and decapitated. The brains were quickly removed and inspected to confirm the position of the infarction focus, the absence of subarachnoid hemorrhage and/or arterial penetration with the ligature. The brains were sectioned coronarily with for 30 min in 2% solution of TTC at 37℃ for vital staining. The occlusion focus areas were calculated with the following formula:
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Infarction focus area (%) = infarction cortex lattice work number/ whole cortex latticework number
For the chronic photosensitizer induced intracranial infarct model (2 months), same procedures were performed for obtaining brain samples as described above.
Physiological values and infarction area ratio were compared between groups with Chi test analysis of variance and with one way ANOVA analysis. The association between infarction size and neurological deficit score was analyzed with correlation coefficient. Parametric values are represented as mean(
±s). Significance was assumed at the value P<0.05., 百拇医药
Results
To evaluate the spatial resolution of NCT, we established the photosensitizer introduced cortex infarction model that had a small infarction focus. Now, the spatial characteristics of changes in the concentration of Hb caused by infarction are described as follows. To visualize these characteristics, we mapped the static topograms of the changes in concentration of Hb during pre-and post-ischemic models. Meanwhile, a series of fMRI images were obtained on the same rats for identifying the reliability of the NCT images.
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The correlation of geometric shape with image areas of different techniques was compared with. In the photosensitizer induced infarction model (Fig.3.a), one way ANONAs analysis showed that there were no significant difference between anatomical samples, fMRI images and NCT images for infarction focus [F(2,27)=3.10, P>0.05 for 24 h group; F(2,27)= 2.68, P>0.05 for 2 months group]. Comparing with 24 h group, fMRI images for infarction area showed a significant decrease in the 2 months group (P<0.01); Similar phenomenon was observed in NCT images and in anatomical samples (P<0.01;P<0.01, respectively). The correlation in infarction area between anatomical samples and NCT image area for infarction focus were r=0.897 for 24 h group (P<0.05) and r=0.906 for 2 months group (P<0.01), respectively.
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For the hypothermia MCAO group (Fig.3.b), the fMRI mean image area of infarction focus was 16.03%±2.1%;the NCT images mean image area for infraction focus was 19.36%±2.82%;the infarction focus mean area of anatomical samples was 16.93%±1.57%. There were no significant difference among NCT images, fMRI mean image and anatomical samples [F(2,27)=2.47, P>0.05]; In the normothermia MCAO group, the fMRI mean image area of infarction focus was 40.32%±2.16%;the NCT images mean image area of infraction focus was 42.48%±5.23%; The infract focus mean area of anatomical samples was 36.65%±4.35%. There were no significant difference among NCT images, fMRI mean image and anatomical samples [F(2,27)= 2.64,P>0.05], similarly.
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Comparing with hypothermia group, infraction area of anatomical samples had a significant increase in the normothermia group (P<0.01); similar difference were observed in fMRI images (P<0.01) and in NCT images (P<0.05). The correlation between anatomical samples infarction area and NCT image area for infarction focus werer=0.820 for normothermia group (P<0.05) and r=0.851 for hypothermia group (P<0.05), respectively. The correlation between fMRI and NCT image area for infarction focus were r= 0.874 for normothermia group (P<0.05) and r= 0.782 for hypothermia group (P<0.05), respectively.
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Fig.3 Cortical infarction area measured with NCT,fMRI and anatomical techniques for
(a) photosensitizer induced cortex infarct model of 24 hours group and 2 month group;(b)MCAO model of normothermia group and hypothermia group
Discussion
There are a series of hemodynamic changes such as higher erythrocyte sedimentation rate[12], slower blood flow velocity that result in blood supplement reduction in ischemic focus while ischemic stroke impending in ischemic stroke patient. This kind of CBF changes may induce decrease of [oxy-Hb], [deoxy-Hb] and t-Hb content that differ from the hemodynamic changes caused by other diseases[15]. This is the pathphysiological basis for predicting ischemic stroke by NCT.
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NCT simultaneously measures the oxy-Hb, deoxy-Hb and t-Hb in local tissue and offers another information about blood supply and blood volume in unit tissue. The basic principal makes it possible to measure the cerebral blood supply[9]. The theory about [oxy-Hb], [deoxy-Hb] absorption in near-infrared spectrum has been elucidated by many authors[3,11]. However, no proper animal model has been established to validate the theory. Another barrier for NCT technique is to establish a corresponding relation between the NCT signals and anatomical structure. This is a perquisite for NIR localizing diagnosis[4,10].
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In the present study, a remarkable finding is that NCT images reliably reflect CBF changes caused by ischemic stroke in the animal model. Our results confirmed the hypothesis that NIRS signals are correspondent to total Hb content changes that reflect blood supply. Specifically, subtle changes of NCT exactly reflect the rehabilitation of neurons in the ischemic focus in the photosensitizer induced intracranial infarct model and had millimeter spatial resolution and piccosecond temporal resolution[6]. And in the MCAO model, NCT can show penumbra that predicts physical and functional rehabilitation ability. The central ischemic focus in the anatomical samples completely corresponded to NCT central low signal core. A well match relationship between measurement point and anatomical structure has been obtained with neutronavigator in both photosensitizer induced intracranial infarct models and MCAO models.
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Although fMRI has a high spatial resolution, the complicated system made it unpractical to be used as a portable and common instrument for tracing patient's hemodynamics changes caused by various factors. More important, fMRI studies on cerebral function may overlook some key information when the studied focus is not associated with reduction of deoxy-hemoglobin[14].
Comparing with MRI, currently available NCT has lower spatial resolution, but it could catch some subtle physiological signals that is important for the disease diagnosis owing to its high temporal resolution. Meanwhile, the outstanding property of compact and reliability of NCT enable it to become a common diagnostic instrument. Moreover, it overcomes the disadvantage of fMRI because NCT simultaneously measures deoxy-Hb and oxy-Hb. Of course, NCT could not replace other techniques, such as MRI, because it has relatively worse spatial resolution. However, it is complementary to other tools because it is compact and can measure with high temporal resolution. So, NCT is thus useful for tracing regional cerebral blood flow and investigating the subtle hemodynamic changes during surface brain activity in real time. Basing on the above results, we can measure more complex functional activities such as semantic, phonological and orthographic processes and explore the associations among those processes in temporal sequence[4]. Although NCT currently available could only reflect the two-dimension area of the infarct focus for the restriction of detection depth, many researchers began to improve the optical entrance model and the imaging technique[5]. Our further study will be focused on the measurement of hematoma in the deep cerebral tissue with 200 MHz frequency phase-differentiation technique and developing NIR tomography technique just as computerized tomography.
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In conclusion, combined with neutronavigator, the NCT could localize the hemodynamic change area causing the stroke. As a compact and reliable instrument, it could be used as real time monitoring instrument at bedside and in critical care ward for warning the patient with an increased risk of stroke. The instrument can also help clinicians to understand a patient's potential for rehabilitation and set realistic plans for treatment during rehabilitation.
(Acknowledgments：Special thanks are expressed to Professor Liyun Li at State Key Laboratory for Atom Physics and Spectroscopy, Institute for Physics and Mathematics, Chinese Academy of Science, for her MRI data analysis.We appreciate Dr.Steven L.Jacques for revise of the paper.)
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Foundation item:Supported by the National Nature Science Foundation of China(39770228)
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Recieved date:1999-07-13, http://www.100md.com