行政院國家科學委員會專題研究計畫 成果報告
偵測缺氧核醫藥物 Tc-99m HL91 於腦部缺氧模式的離體及活體之研究(3/3)
計畫類別： 個別型計畫
計畫編號： NSC 94-2314-B-006-002-
執行期間： 94 年 08 月 01 日 至 95 年 10 月 31 日 執行單位： 國立成功大學醫學系核子醫學科
計畫主持人： 李碧芳
共同主持人： 簡基憲，邱南津，沈立漢
報告類型： 完整報告
處理方式： 本計畫可公開查詢
中 華 民 國 95 年 10 月 31 日
計畫中文摘要
“缺血性半影”的定義為梗塞周圍的組織，雖然功能已受損，但是結構仍完整，且具有潛在地可 以被搶救回來的可能性。假如存活的半影組織越多，則神經學預後會越好。因此，在中風之後，若能 預防半影梗塞惡化，就必定會改善患者臨床結果。
Tc-99m HL 91 是一個具有潛力成為偵測缺氧存在的核醫葯物，而現已有關於心肌缺血組織及腫 瘤缺氧的研究報告。至於在急性缺血性中風所引起的腦部缺氧之偵測，經由 Ovid Medline 搜尋文獻，
則尚未有研究報告發表。
正常鼠的 Tc-99m HL91 生物分佈實驗之研究結果顯示 Tc-99m HL91 在腦部活性不高。在正常鼠 的針孔閃爍攝影結果顯示 Tc-99m HL91 在左右腦部的放射活性分布均勻。而在腦部缺氧模式老鼠的 針孔閃爍攝影結果顯示 Tc-99m HL91 在患侧腦部的放射活性呈現明顯的增加。
應用自動放射攝影，繼續進行 Tc-99m HL 91 在組織層次的相關性及作用機制之探討。結果顯示 組織切片的缺氧處,確實在〝自動放射攝影〞患處有增加放射活性的表現。在活體上的核醫照影以及 將腦單獨取出來的離体核醫照影中,在患處皆呈現 Tc-99m HL91 增加的情形。
關鍵字: Tc-99m HL 91, 腦部缺氧, 閃爍攝影
I I
計畫英文摘要
Background: Tc-99m HL91 is a new hypoxia imaging agent that demonstrates the presence of tumor hypoxia and ischemic myocardium. The purpose of this study was to determine whether Tc-99m HL91 could detect cerebral ischemia of the mice in vivo by gamma camera imaging.
Materials and methods: Studies were carried out in C57BL/6NCrj mice that were divided into 2 groups,
one sham-control group and the other middle cerebral artery occlusion (MCAO) group. Craniotomy was
performed to expose the right middle cerebral artery transcranially. The MCAO was done by
electrocoagulation using fine bipolar electrodes. Twenty-four hours following MCAO, 100 MBq of
Tc-99m HL91 was injected via the tail vein. Two hours post-injection, in vivo brain scintigraphies were
acquired using a gamma camera. Then, the brains were removed from the skulls to obtain ex vivo images.
Subsequently, the brains were sliced and were stained with 1% 2, 3, 5-triphenyl-tetrazolium chloride
(TTC). Finally, these slices were applied to autoradiography. On the in vivo, ex vivo images and
autoradiography, we analyzed Ratio R/L, i.e. the ratio of mean pixel counts in the manually drawn
regions of interest (ROI) at the right-MCAO area to its contralateral area.
Results: “Hot Spots” were detected in vivo and ex vivo by gamma camera imaging at 2 hours
postinjection. ROI analysis of the in vivo, ex vivo images and autoradiography demonstrated
significantly higher Ratio R/L in the MACO group than in the sham-controls. The autoradiography
revealed the areas with increased Tc-99m HL91 accumulation were larger than the cerebral infarction,
TTC-unstained areas.
Conclusions: Tc-99m HL91 may be a promising agent to detect the presence of cerebral ischemia.
Key words: Tc-99m-HL91, cerebral ischemia, scintigraphy.
II
報告內容
INTRODUCTION
The ischemic penumbra is defined as peri-infarct tissue that is functionally impaired but structurally
intact and remains potentially salvageable (1). The greater the amount of penumbral tissue that survives, the
better is the neurological outcome. Preventing penumbral infarction should therefore improve clinical
outcome after stroke.
Detection of tissue hypoxia by a radionuclide imaging technique was first proposed by Chapman et al.
(2). Since then, nitroimidazole compounds have received much attention (3-6). Clinical studies using F–18
fluoromisonidazole (FMISO) show imageable differences between normal and hypoxic cerebral tissues
(7-10). Tc-99m is a very convenient isotope for use with such imaging because it is readily available and,
with a 6-h half-life, it is well suited for routine clinical use. Tc-99m labeled ligands containing
2-nitroimidazole, including BMS 181321, and BRU59-21, displayed hypoxia selectivity (11).
Butyleneamine oxime (BnAO, HL91) is another Tc-99m complex and has shown good hypoxia selectivity in
ischemic heart (12), and tumor hypoxia (13-14). HL91 does not contain a 2-nitroimidazole group and its
mechanism of localization is not understood. During the development of this tracer, it was found that the nitroimidazole“hypoxia-localizing moiety” minimally affectsthetargeting oftheseradiometalcomplexes,
since the intrinsic properties of Tc-99m complexes are predominantly responsible for the hypoxia selectivity
(14).
Tc-99m HL 91 is a potential agent for imaging hypoxic tissue in vitro and in vivo. Uses of this drug to
image ischemic tissue in the myocardium (12), and hypoxia in solid tumors have been reported (13-14). Our
preliminary result (15) identified the increased accumulation of Tc-99m HL91 in the region of cerebral
ischemia, in accordance with the preliminary findings of Ningyi J et al (16). Yet, to the best of our
knowledge, there are no published studies on the application of Tc-99m HL91 in detecting hypoxic tissue
after acute ischemic stroke. Therefore, we evaluated the value of Tc-99m HL 91 as a hypoxia marker in the
middle cerebral artery occlusion (MCAO) model of the mice.
MATERIALS AND METHODS
Radiolabeled tracer
The lyophilized HL91 kits were prepared by Institute of Nuclear Energy Research, Lung-Tan, Taiwan.
Each HL91 kit contains 200 µg of HL91 ligand, 20 µg of stannous chloride dihydrate, 40 µg of methylene
diphosphonic acid, and buffer salts, in a 10-ml glass vial sealed under nitrogen atmosphere. Tc- 99m-HL91
was prepared by reconstitution of a HL91 kit with 4.5 ml, 900 MBq (24.3 mCi) of Tc-99m-sodium
pertechnetate solution.
Middle cerebral artery occlusion (MCAO) model in mice
C57BL/6NCrj (B6) mice (n =22, males, body weight 31 ± 2.3 grams) were purchased from Laboratory
Animal Center of National Cheng-Kung University (NCKU). These mice were bred and maintained at
NCKU. This study was performed in accordance with the National Institutes of Health Guide for the Care
and Use of Laboratory Animals. All procedures were approved by the Animal Care Committee at National
Cheng Kung University.
In this MCAO model, mice (n =16) were anaesthetized with an intraperitoneal injection of chloral
hydrate (60 mg/kg). The skin and temporal muscle were cut and craniotomy was performed to expose the
right MCA transcranially. After making a cut in the dura mater, the MCA was lifted off distally and
occluded by electrocoagulation using fine bipolar electrodes. The artery was then transected to avoid
recanalisation. The sham-control group (n =6), in which surgical procedure was performed without MCAO,
were carried out to investigate the effect of surgical manipulation upon tracer distribution.
Tc-99m HL91 imaging
We used a semiconductor (CdZnTe)-based gamma camera for Tc-99m HL91 imaging. This gamma
camera (eZ-SCOPE, Anzai Medical Co., Ltd., Tokyo, Japan) has a single wafer of 5-mm thick CdZnTd that
is divided into a 16 x 16 array (256 pixels). The field of view is 3.2 cm x 3.2 cm. The full-width at
half-maximum (FWHM) is 2.2 mm when using the low-energy, high-resolution parallel-hole collimator
(LEHR).
For in vivo and ex vivo Tc-99m HL91 planar brain scintigraphy, the gamma camera was equipped
with LEHR parallel-hole collimator and the energy peak was set at 140 kev ± 10%. In vivo images (100K
counts/image) were acquired at 30min, 1 hour, 1 hour30min, and 2 hours post-injection of the radiotracer.
Then the intact brains were removed from the skulls for ex vivo imaging. In addition to visual assessment
of the scintigraphic manifestations, region of interest (ROI) was drawn manually at right-MCAO area and
its contralateral zones to analyze the mean counts and to calculate the ratios of right-MCAO to left-brain
zones (RatioR/L).
TTC staining and autoradiography
After ex vivo imaging, the brains were sectioned into series of 2 mm coronal slices and were stained with
vital dye 1% 2,3,5-triphenyl-tetrazolium chloride (TTC) (Sigma, St Louis, Missouri, USA) for 30 min at 37
℃ in the dark to obtain images of the brain infarction. The TTC stained healthy brain tissue red, whereas
infarct tissue was unstained. For autoradiography, the brain slices were then applied to the BASMP2040
imaging plate (IP) inside the cassette (Fuji Photo Film Co., Tokyo, Japan) for 1 day. After exposure was
completed, the cassette was opened under subdued light and the brain slices were removed. The IP was
measured with a FUji FLA5000 Multifunction Imaging System (Fuji Photo Film Co., Tokyo, Japan).
Captured images were analyzed using Image Gauge, version 4.0 (Fuji Photo Film Co., Tokyo, Japan). The
distribution of radioactivity was estimated by comparing the degree of blackening; darker gray levels
represent higher radioactivity. Furthermore, region of interest (ROI) was drawn manually at right-MCAO
and left-brain zones to calculate the ratios of right-MCAO to left-brain zones (RatioR/L).
RESULTS
There were totally twenty-two male B6 mice enrolled in the study: six in the sham-control group, and
sixteen in the MCAO group. Mortality during the procedure did not exceed 10%.
Tc-99m HL91 imaging: In vivo Tc-99m HL91 planar brain scintigraphy showed no selective accumulation
of the radiotracer over the head in the sham-control group (Figure 1A) while increased radioactivity in the
MCAO area was identified in the MCAO group (Figure 1B). There was significantly higher RatioR/L( p <
0.05) in the MCAO group (1.45±0.11, range: 1.34 to 1.71) than in the sham-control group (1.02±0.02,
range: 0.99 to 1.03) (Figure 1C). Ex vivo Tc-99m HL91 images revealed similar results as the in vivo study
in the sham-control (Figure 2A) and in the MCAO group (Figure 2B). Also, the the RatioR/Lwas higher in
the MCAO group (2.33±0.12, range: 2.25 to 2.61) than in the sham-control group(1.03±0.05, range: 0.96 to
1.09) (Figure 2C).
TTC staining and autoradiography: The brain coronal sections were stained with TTC to identify the
infarct region and the contralateral brain, as shown in Figure 3A. The autoradiographic imaging (Figure 3B)
obtained from coronal sections showed increased radioactivity in the right MCAO area at the slice-1, slice-2
and slice-3. Little radioactivity was identified at the slice-4. The areas with increased Tc-99m HL91
accumulation was larger than the infarct region demonstrated by unstaining of TTC. Figure 3C exhibited the
statistically significant differences of Ratio R/Lbetween the MCAO group and the sham-control group. The
RatioR/Lfor Tc-99m HL91 at the MCAO group ranged from 1.33 to 1.58 (1.48±0.10) at the slice-1, 1.50 to
1.70 (1.61±0.08) at the slice-2, and 1.29 to 1.51 (1.38±0.08) at the slice-3. The figures for the sham-control
group ranged from 1.02 to 1.10 (1.09±0.06) at the slice-1, 0.98 to 1.03 (1.01±0.04) at the slice-2, and 1.03 to
1.09 (1.06±0.02) at the slice-3.
DISCUSSION
To our knowledge, this is the first study to explore the use of in vivo and ex vivo Tc-99m HL91 planar
brain scintigraphy in assessing cerebral hypoxia by using the animal model of permanent MCAO. We found
that cerebral ischemia can be detected, in vivo and ex vivo, by Tc-99m HL91 imaging at 24 hours following
permanent MCAO (Fig 1 and Fig 2). Furthermore, by comparing the autoradiography (Fig 3B) and TTC
staining (Fig 3A), we observed that the accumulation of Tc-99m HL91 in the areas of cerebral ischemia
were larger than the TTC unstained areas, i.e. infarct areas.
In this study, there were statistically significant differences of Ratio R/Lbetween the MCAO group and
the sham-control group at the Tc-99m HL91 planar brain scintigraphy in vivo (Fig 1), ex vivo (Fig 2) and the
autoradiography of the brain coronal sections (Fig 3). Figures 1 and 2 of the mouse brains supported our
previous findings of Tc-99m HL91 planar brain scintigraphy in the rats following MCAO that cerebral
ischemia appeared as “hot spots”
in vivo by gamma camera imaging (15). In addition, our findings of the
autoradiography were in accordance with that of Ningyi et al. (16). They found that the ischemic territory
accumulated more Tc-99m HL91 than the opposite site in the autoradiogram, and Tc-99m HL91 can be
avidly taken up by ischemic penumbra.
The concept of penumbra during focal cerebral ischemia refers to the regions of brain tissue, usually
peripheral in location, where blood flow is sufficiently reduced to result in hypoxia severe enough to arrest
physiological function, but not so complete as to cause irreversible failure of energy metabolism and cellular
necrosis (1). For detecting penumbra, a variety of imaging techniques have been developed. ATP,
phosphocreatine, pH, lactate, and n-acetyl aspartate (NAA) concentrations can be estimated experimentally
and clinically with magnetic resonance spectroscopy (MRS) to distinguish infarct core from penumbra (17).
Clinical application of MRS imaging has been limited in acute stroke patients by complexity and duration of
data acquisition and processing required and subsequent availability of more rapid diffusion and perfusion
imaging methodology. Diffusion-weighted magnetic resonance images (DMRI)-derived autordiographic
maps distinguished penumbra from core at 36 minutes but not at 48 or more minutes after 1-hour temporary
MCA occlusion in rats (18). Another example of ischemic molecular penumbra that can be detected with in
vivo early T2 MRI and late T1 MRI and appears to play a role in delayed cell damage is manganese
(Mn)-mediated striatal neurodegeneration, which developed between 5 and 28 days after 15-minute
temporary MCA occlusion in rats (19).
Scintigraphic techniques are frequently used for the evaluation of penumbra. The penumbra is
characterized by PET as a region with reduced regional cerebral blood flow (rCBF), an increased oxygen
extraction fraction, and relatively preserved oxygen consumption (CMRO2) (20). Relative or absolute rCBF
maps fail to distinguish between the infarct core and ischemic penumbra and have limited value in the late
diagnosis or prognosis of recovery in patients with ischemic stroke. However, when rCBF is measured in
combination with CMRO2 or DMRI, it can reveal areas of misery perfusion that may proceed to infarction.
Therefore, this may prove useful in classifying areas of recoverable tissue (21).
Markers of hypoxic tissue have been tested for their ability to identify penumbral tissues. PET with
F-18 fluoromisonidazole (FMISO), a nitroimidazole derivative, was studied as an alternative, simple method
for identifying penumbral tissues in patients with acute ischemic stroke. This method identified hypoxic
tissues during the first 48 hours after stroke (7-10). Furthermore, autoradiography with labeled
nitroimidazole derivatives revealed increased tracer uptake that was associated with histologically damaged
areas and adjacent areas that appeared intact in animal models of cerebral ischemia (3-6). The uptake of
Tc-99m ethylene dicysteine metronidazole (EC-MN) in peri-infarcted areas suggesting the presence of
actual viable neurons was observed during the subacute stage of cerebral infarction (22). Moreover, Tc-99m
HL91 has been used to image hypoxic tissue in animal models of myocardial ischemia (12), tumor hypoxia
(13-14), and cerebral ischemia (15-16).
In comparison with other modalities, Tc-99m HL91 scintigraphy is ideally suited to obtain a precise
snapshot of the distribution and intensity of tissue hypoxia (12-16). Tc-99m has favorable physical
characteristics, has a low price, and is readily available. In this mouse MCAO model, our results
demonstrated that gamma camera imaging using Tc-99m HL91 can identify the presence of cerebral
ischemia.
Di Rocco RJ et al. (4) revealed that the Tc-99m complex of a 2-nitroimidazole–derivatized propylene
amine oxime is selectively retained in acutely ischemic brain but not in the ischemic infarct before
disruption of the blood-brain barrier (BBB). However, Jingtao J et al. (23) identified a change in
permeability of the BBB occurs between the first and the third day after ischemia. Because our study was
performed at 24 hours following MCAO, we could not exclude that trapping of Tc-99m HL91 might be
influenced by disruption of the BBB in ischemic tissue. Although no direct data concerning this issue are
available, trapping of 2-nitronidazole derivatives is dependent on tissue hypoxia and is not associated with
blood flow (5, 8, 9). Lythgoe et al (5) showed that I-125 labeled iodoazomycin arabinoside was trapped in
regions of moderately reduced perfusion but not in the infarct core, where perfusion was severely reduced,
supporting the hypothesis that nitroimidazole labels penumbral peri-infarct tissues. Further study will be
performed to investigate the relationship between the disruption of the BBB and Tc-99m HL91 uptake in
ischemic tissue at the animal model of MCAO.
CONCLUSION
The present studies showing increased Tc-99m HL91 uptake in experimental stroke and prior work
showing increased uptake in myocardial ischemia (12), tumor hypoxia (13-14), and cerebral ischemia (15-16)
provide evidence that the penumbra can be imaged in vivo with Tc-99m HL91. Furthermore, Tc-99m HL91
uptake in peri-infarct areas suggests that the presence of actual viable neurons was observed at 24h
following MCAO. Tc-99m HL 91 is a potential agent for imaging the penumbral peri-infarct tissues at 24h
following MCAO.
ACKNOWLEDGEMENTS
This work was supported in part by grants NSC from the Taiwan National Science Council
(NSC94-2314-B-006-002, NSC93-2314-B-006-012, and NSC 92-2314-B-006-056). We thank Chin-Ling
Chu for statistical advice, Jin-Wen Hung, and Ji-Ing Huang for technical assistance, Szu-Fu Chen, and
Kung-Ju Lin for helpful conversations on experimental design. We also thank Hui-Ling Lee for her
secretarial help.
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Figure 1 A.
B.
C.
Figure 1
A. Tc-99m HL91 in vivo planar brain scintigraphy of the mouse head in the sham-control group reveals the normal distribution of HL-91 over the brain. The mouse head shown on the left is digitized photographic images of the mouse head. On the right is the distribution of Tc-99m HL91 over the mouse head by in vivo planar brain scintigraphy of the mouse head.
B. Tc-99m HL91 in vivo planar brain scintigraphy of the mouse head in the MCAO group shows increased radiotracer accumulation of HL91 over the right MCAO lesion.
C. The ROI analysis of in vivo planar brain scintigraphy demonstrates significantly higher ratio of right MCAO lesion to left ipsilateral brain (Ratio R/L) at the MCAO group (group A) than in the sham-controls (group B). (*p<0.05)
Figure 2.
A.
B.
C.
Figure 2.
A. Tc-99m HL91 ex vivo planar brain scintigraphy of the isolated brain in the sham-control group exhibits the normal distribution of Tc-99m HL-91 over the brain. The isolated brain shown on the left is digitized photographic images of the isolated brain. On the right is the distribution of Tc-99m HL91 over the isolated brain by ex vivo planar brain scintigraphy of the isolated brain.
B. Tc-99m HL91 ex vivo planar brain scintigraphy in the MCAO group shows increased radiotracer accumulation of Tc-99m HL91 over the right MCAO lesion.
C. The ROI analysis of ex vivo planar brain scintigraphy demonstrates significantly higher ratio of right MCAO lesion to left brain (RatioR/L) at the MCAO group (group A) as compared with the sham-controls (group B). (*p<0.05)
Figure 3.
A.
slice-1 slice-2 slice-3 slice-4
B.
C
Figure 3.
A. The histological finding of staining with 1% 2, 3, 5-triphenyl-tetrazolium chloride (TTC) of serial coronal sections of mouse brain was examined at 24 hr following the middle cerebral artery occlusion (MCAO). The TTC dye stains healthy tissue red, and leaves infarct tissue unstained.
B. The autoradiograms of the coronal sections were taken from the same blocks used for the TTC in panel A.
The mice were subjected to MCAO. The animals were then reanesthetized and injected intravenously with Tc-99m HL91 24 hr following MCAO, and euthanized 2 hr after isotope injection. The autoradiography using Tc-99m HL91 demonstrates increased radioactivity of Tc-99m HL91 at the corresponding cerebral ischemia on the TTC staining.
C. The ROI analysis of the autoradiography finds significantly higher RatioR/Lin the MCAO group (group A) as compared with the sham-controls (group B) at the slice-1, slice-2, and slice-3. (*p<0.05)
計畫成果自評
本三年期計畫成果，已發表於”2003 年日本核醫學會年會”，”2004 年日本核醫學會年會”
及”2006 年英國放射學會年會” (註)，預計將寫成論文而在學術期刊發表。因此，吾人預計此三年
期計劃，除了可將 Tc-99m HL91 的組織、器官、細胞及次細胞分佈特性加以闡明，使其作用機轉更加
明瞭之外；對於未來核醫診斷藥劑的研究發展，更是具有不可限量的發展潛力。期望核醫基礎研究向
下紮根，然後能夠運用到臨床疾病的診斷及治療，以做到”早期診斷，早期治療”如此一來對人類社
會的健康福祉能有那麼一些些的貢獻；這些研究，是值得吾人去辛勤努力加以探討的。
註:
1. Nan-Tsing Chiu, Bi-Fang Lee, Hsin-Hung Chen, Chi-Hsien Chien. Hsia CC, Shen LH, Fu YK. Oct 27-29, 2003. Tc-99m HL91:“HotSpot”Detection ofCerebralIschemiain Vivo by GammaCameraImaging after acute middle cerebral artery occlusion in rats. The 43st Annual Meeting of the Japanese Society of Nuclear Medicine. Tokyo, Japan.
2. N.T. Chiu, B.F. Lee, Hsia CC, Shen LH, Fu YK. November 4-6. 2004. Accumulation of Tc-99m HL91:
in vitro Cell Culture and in vivo Tumor Model. The 44th Annual Meeting of the Japanese Society of Nuclear
Medicine. Kyoto, Japan.3. Lee BF, Chiu NT, Fu WM, Wu PS, Hsia CC, Shen LH. May 15-17. 2006. The value of Tc-99m HL91 in a cerebral ischemic model in vivo. UK radiological congress 2006, Birminhan, UK.
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