Annexin V imaging of acute doxorubicin cardiotoxicity (apoptosis) in rats.

Anthracyclines are widely used in chemotherapy regimens for several malignancies, with cardiotoxicity being the major limiting factor in high-dose schedules. Recently, it was reported that doxorubicin induces apoptosis in cardiac muscle cells in vivo and, as such, is expected to be involved in the genesis of doxorubicin-induced cardiomyopathy. The aim of this study was to validate an animal model for in vivo monitoring of doxorubicin cardiotoxicity by means of scintigraphic detection of apoptosis. METHODS: Three groups of 5 male Wistar rats each were treated for 3, 4, and 5 times with a weekly intraperitoneal injection of doxorubicin at 2.5 mg/kg. At 24 h before and 24 h after the final treatment, (99m)Tc-annexin pinhole scintigraphy was performed. A control group of 5 rats was scanned without doxorubicin treatment. A cardiac uptake ratio was calculated from planar scintigraphy results with the following formula: (mediastinum - fat)/fat. After scintigraphy, the rats were sacrificed, and the heart was processed for histologic analysis. RESULTS: Incremental general signs of illness were observed with increasing total cumulative doxorubicin dose. Rats treated for 3, 4, and 5 wk with doxorubicin showed significantly higher uptake ratios of, respectively, 4.0 +/- 0.52 (mean +/- SEM), 4.8 +/- 0.46, and 5.2 +/- 0.17 after the final treatment; the ratio for controls was 1.84 +/- 0.05 (P < 0.05). Histologic analysis confirmed cardiac stress in treated groups, with an increasing left ventricular atrial natriuretic factor messenger RNA expression level with increasing cumulative doxorubicin dose. Late apoptosis was confirmed by terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling in the rats treated for 5 wk. CONCLUSION: Acute doxorubicin-induced cardiomyopathy based on early apoptosis can be assessed and imaged with annexin V scintigraphy in rats. This finding makes it possible to use this animal model for repetitive noninvasive evaluation of cardioprotective regimens for anthracycline cardiotoxicity.     

In vivo metabolic studies of thetrans-(R,R) isomer of radioiodinated IQNP: A new ligand with high affinity for the M1 muscarinic-cholinergic receptor

E-(R,R)-IQNP is a new ligand analogue of IQNB, which has high affinity for the cholinergic-muscarinic receptor. Earlier studies have demonstrated high cerebral uptake of activity with selective localization in M₁ receptor subtype areas of the brain. In this paper we describe the results of metabolic studies of E-(R,R)-IQNP directed at determining the metabolic fate of this ligand and the identification of the radioactive species observed in the brain and heart tissue. Tissue Folch extracts demonstrated that the lipid-soluble extracts from brain contained 87.0%±1.65% of the activity up to 24 h. In the heart, 61.9%±7.50% of the activity was extracted in the lipid-soluble extract after 30 min, decreasing to 51.4%± 0.65% by 4 h. In contrast, data from other tissues studied demonstrated large amounts of either aqueous soluble activity or activity which was not extracted from the tissue pellet material; analysis of lipid organic extracts revealed the following results: liver (4 h), 7.43%± 0.96%; serum (4 h), 3.73%±0.87%; urine (24 h), 9.4%; feces (24 h), 16.5%. Thin-layer chromatographic (TLC) and high-performance liquid chromatographic (HPLC) analyses of lipid-soluble brain extracts indicated that only unmetabolized E-(R,R)-IQNP was detected (99.4%± 1.25%). Activity which was extracted into the organic phase from heart tissue was also determined by TLC and HPLC analysis to contain large amounts of unmetabolized ligand after 4 h (88.5%± 0.57%). In addition, however, low levels of two additional radioactive components were detected which increased with time. TLC analysis of the plasma lipid extracts indicated only a small amount of unmetabolized E-(R,R)-IQNP. In comparison, the liver, feces, and urine lipid extracts contained only metabolites. These initial studies clearly indicate that radioactivity present in the brain after intravenous administration of radioiodinated E-(R,R)-IQNP represents only the unmetabolized ligand and that this new ligand shows promise for single-photon emission tomographic imaging of muscarinic receptors in humans.     

Synthesis, biodistribution and effects of farnesyltransferase inhibitor therapy on tumour uptake in mice of 99mTc labelled epidermal growth factor

OBJECTIVE: The goal of this study was to develop a 99mTc labelled human epidermal growth factor (hEGF) for the in-vivo prediction of cancer cell response to farnesyltransferase inhibitor (FTI) therapy. This is based on the observation that internalization of EGF receptors is inhibited by FTIs. METHODS: We describe the radiolabelling of 99mTc-hEGF using the hydrazinonicotinamide (HYNIC) linker. Binding characteristics of 99mTc-HYNIC-hEGF to the EGF receptor are explored using an in-vitro binding assay. Biodistribution data of the compound in mice and tumour uptake in LoVo tumour bearing athymic mice before and after farnesyltransferase inhibitor therapy are presented. RESULTS: No colloid formation was observed. Binding parameters and LoVo tumour uptake of 99mTc-HYNIC-hEGF did not differ significantly from directly labelled 123I-hEGF values. However, the biodistribution data of the 99mTc-HYNIC-hEGF showed higher uptake in liver and intestines and decreased stomach uptake compared to its 123I analogue. Eight hours after farnesyltransferase inhibitor therapy with R115777, LoVo tumour uptake of 99mTc-HYNIC-hEGF decreased significantly, as shown using planar gamma scintigraphy (the ratio tumour vs. thigh dropped from 2.54+/-0.83 to 0.99+/-0.18). These data confirm the results obtained using 123I-hEGF. CONCLUSION: These data suggest that 99mTc-HYNIC-hEGF is a promising and selective new radiotracer for in-vivo monitoring of the EGF receptor with SPECT. Moreover, 99mTc-HYNIC-hEGF is a possible tool for early therapy response prediction of farnesyltransferase inhibitors.     

In vivo imaging of the neutron capture therapy agent BSH in mice using (10)B MRI.

Boron neutron capture therapy (BNCT) is an experimental cancer treatment modality requiring the targeting of (10)B-enriched compounds to the tumor, which is then irradiated by low-energy neutrons. One of the boron-containing compounds used for this purpose is the mercaptoborane Na(2)B(12)H(11)SH (BSH). The first in vivo MR images of (10)B-enriched BSH are presented here. BSH, injected into the tail vein of mice with implanted M2R melanoma xenografts, was imaged using 3D gradient echo (10)B MRI. (10)B NMR spectroscopy, localized mainly to the tumor by virtue of the use of a small surface coil, was applied to measure the T(1) (2.9 +/- 0.3 ms) and T(2) (1.75 +/- 0.25 ms) values of the (10)B signal. The MRI experiments detected levels of about 20 ppm (microg boron / g tissue) at 6 x 6 x 6 mm spatial resolution in a total scan time of 16 min. Magn Reson Med 46:13-17, 2001.     

Kinetics of radioiodinated species in subcellular fractions from rat hearts following administration of iodine-123-labelled 15-(p-iodophenyl)-3-(R,S)-methylpentadecanoic acid (123I-BMIPP)

It is recognized that iodine-123-labelled 15-(p-iodophenyl)-3-(R,S)-methylpentadecanoic acid (¹²³IBMIPP) slowly washes out of the myocardium. The mechanism for the washout was investigated in normal rat hearts by analyses of the subcellular distribution and lipid classes based on the BMIPP metabolism. Rat hearts were excised at 1–120 min after intravenous injection of¹²³I-BMIPP. After counting the radioactivity, the hearts were digested with Nagarse and homogenized, and then fractionated into the cytosolic, mitochondrial, microsomal and crude nuclear fractions by centrifugations. The radioactivity of each fraction was counted, and the lipid classes were analysed by radio-thin-layer chromatographic and high-performance liquid chromatographic methods. The heart uptake of 1231-BMIPP was maximal at 5 min (6.81%±0.36% ID/g), and 41% of the radioactivity disappeared within 120 min. The myocardial radioactivity was immediately distributed into the cytosolic, mitochondrial, microsomal and crude nuclear fractions. The distribution (%) of each fraction was almost identical from 5 min through 120 min. The cytosolic fraction was always the major site of radioactivity deposition (60%), and the time-activity curve of the cytosolic fraction paralleled that of the whole heart throughout the 120-min study period. In the cytosolic fraction, most of the radioactivity was incorporated into the triglyceride class, and the rest was present in the free fatty acid, phospholipid (phosphatidylcholine) and diglyceride classes. In the mitochondrial fraction, the radioactivity was mostly incorporated into the phospholipid class (phosphatidylethanolamine), followed by free fatty acids. The final metabolite of¹²³I-BMIPP,¹²³I-p-iodophenylacetic acid (¹²³I-PIPA), initially appeared in the mitochondrial fraction as early as 1 min, and subsequently in the cytosolic fraction at 5 min. Another intermediary metabolite,¹²³I-p-iodophenyldodecanoic acid (¹²³I-PIPC₁₂), was found only in the mitochondrial fraction after 5 min. In conclusion, the slow washout kinetics of¹²³I-BMIPP from the myocardium mainly reflects the turnover rate of the triglyceride pool in the cytosol. The BMIPP metabolism, i.e. initial -oxidation followed by subsequent cycles of -oxidation, was confirmed in vivo. The participation of the mitochondria in the metabolism was also proven.     

A comparative study of 188Re(V)-meso-DMSA and 188Re(V)-rac-DMSA: preparation and in vivo evaluation in nude mice xenografted with a neuroendocrine tumor

Dimercaptosuccinic acid (DMSA) exists in meso and racemic (rac) forms. Unlike a meso isomer, rac-2,3-DMSA is very soluble in water, strongly acidic solutions and organic solvents. Despite these differences, rac-2,3-DMSA has not been studied as a radiopharmaceutical. In this study, ¹⁸⁸Re complexes with diastereomeric DMSA were prepared to compare the properties of ¹⁸⁸Re(V)-rac-DMSA with those of ¹⁸⁸Re(V)-meso-DMSA in in vitro and in vivo models.

Methods

rac-2,3-DMSA was synthesized and radiolabeled with ¹⁸⁸Re. The biodistribution and gamma camera imaging of ¹⁸⁸Re(V)-meso-DMSA and ¹⁸⁸Re(V)-rac-DMSA were performed in nude mice subcutaneously implanted with PC-12 cell lines.

Results and conclusions

Both ¹⁸⁸Re(V)-meso-DMSA and ¹⁸⁸Re(V)-rac-DMSA showed excellent radiochemical purity and stability at room temperature. Compared with ¹⁸⁸Re(V)-meso-DMSA, ¹⁸⁸Re(V)-rac-DMSA needed a higher concentration of rac-DMSA and metabisulfite for maximum yields. ¹⁸⁸Re(V)-meso-DMSA showed high labeling efficiency at pH 2, whereas ¹⁸⁸Re(V)-rac-DMSA showed maximum yields at pH 5. The tumor uptake of ¹⁸⁸Re(V)-rac-DMSA was 3.5 times higher than that of ¹⁸⁸Re(V)-meso-DMSA at 1 h (P<.01). Gamma camera images showed that ¹⁸⁸Re(V)-rac-DMSA was more selectively localized than ¹⁸⁸Re(V)-meso-DMSA at the tumor region in a xenograft model. These results demonstrate that ¹⁸⁸Re(V)-rac-DMSA may have better potential than ¹⁸⁸Re(V)-meso-DMSA as a therapeutic agent against neuroendocrine tumors.     

In vivo R1-enhancement mapping of canine myocardium using ceMRI with Gd(ABE-DTTA) in an acute ischemia-reperfusion model

PURPOSE: To demonstrate the usefulness of normalized DeltaR1 (DeltaR1(n)) mapping in myocardial tissue following the administration of the contrast agent (CA) Gd(ABE-DTTA). MATERIALS AND METHODS: Ischemia-reperfusion experiments were carried out in 11 dogs. The method exploited the relatively long tissue lifetime of Gd(ABE-DTTA), and thus no fast R1 measurement technique was needed. Myocardial perfusion was determined with colored microspheres (MP). RESULTS: With varying extent of ischemia, impaired wall motion (WM) and lower DeltaR1(n) values were detected in the ischemic sectors, as opposed to the nonischemic sectors where normal WM and higher DeltaR1(n) were observed. Based on the DeltaR1(n), data from the myocardial perfusion assay and the DeltaR1(n) maps were compared in the ischemic sectors. A correlation analysis of these two parameters demonstrated a significant correlation (R = 0.694, P < 0.005), validating the DeltaR1(n)-mapping method for the quantitation of ischemia. Similarly, pairwise correlations were found for the MP, DeltaR1(n), and wall thickening (WT) values in the same areas. Based on the correlation between DeltaR1(n) and MP, DeltaR1(n) maps calculated with a pixel-by-pixel resolution can be converted to similarly high-resolution myocardial perfusion maps. CONCLUSION: These results suggest that the extent of the severity of ischemia can be quantitatively represented by DeltaR1(n) maps obtained in the presence of our CA.     

In vivo detection of brain glycine with echo-time-averaged (1)H magnetic resonance spectroscopy at 4.0 T.

A single-voxel proton magnetic resonance spectroscopy ((1)H-MRS) method is described that enables the in vivo measurement of endogenous brain glycine (Gly) levels in human subjects. At 4.0 T, TE-averaging (1)H-MRS dramatically attenuates the overlapping myo-inositol (mI) resonances at 3.52 ppm, permitting a more reliable measure of the Gly singlet peak. This methodology initially is described and tested in phantoms. The phantom data infers that the 3.55-ppm peak predominantly is Gly with a smaller contribution from mI. The composite resonance thus is differentiated from pure Gly and mI and is labeled Gly*. The mI contribution was calculated as <2% of the total Gly* signal for a 1:1 mI/Gly mixture. The technique subsequently was used to acquire TE-averaged (1)H-MRS data from the occipital cortex of healthy control subjects. The resultant spectra closely resembled experimental phantom data. LC-model analysis provided a means for quantifying TE-averaged (1)H-MRS spectra and a mean test-retest variability measure of 15% was established for brain Gly* levels in studies of six healthy subjects.     

(123)I-Labeled chitinase as specific radioligand for in vivo detection of fungal infections in mice.

Given the scarcity of diagnostic tools for invasive fungal infections, the aim of this project was to develop new, specific radiopharmaceuticals for diagnosis of fungal infections. Chitin, which is expressed in the fungal cell wall but is absent in mammalian and bacterial cells, represents a potentially selective target for development of tracers for fungal infections. ChiB_E144Q (ChiB = chitinase B) from Serratia marcescens was labeled with (123)I, and in vitro and in vivo studies were assessed. METHODS: (123)I labeling of ChiB_E144Q from S. marcescens by direct iodination was characterized by high-pressure liquid chromatography (HPLC), and stability was evaluated. The in vitro binding properties of the compound to living bacteria, Candida albicans, and Aspergillus fumigatus were examined. Scintigraphy was performed and in vivo characteristics were studied in mice with infected thigh muscles. RESULTS: An average radiochemical yield of 35% was obtained. Radiochemical purity was >97% with a stability of >24 h as determined by HPLC and instant thin-layer chromatography. The average specific activity of the noncarrier-free (123)I-chitinase was 9.25 MBq/ micro g of enzyme. Binding assays showed virtually no binding to Eschericha coli and Staphylococcus aureus, and 2.4 x 10(3) Bq per 1 x 10(7) cells for A. fumigatus and 3.0 x 10(3) Bq per 1 x 10(7) cells for C. albicans (P < 0.05). Binding of the tracer dropped to almost zero for organisms previously incubated with a 50-fold excess of unlabeled enzyme. At 24 h after injection, target-to-nontarget (T/NT) ratios in mice were 20.6 +/- 3.6 for C. albicans and 15.2 +/- 3.7 for A. fumigatus infections, respectively, whereas T/NT ratios for S. aureus -and E. coli-infected thigh muscles or thigh muscles with a sterile inflammation did not exceed 4.9 +/- 2.6, 3.0 +/- 2.3, and 5.3 +/- 2.8, respectively (P < 0.05). Target-to-blood ratios for fungus-infected thighs were always >1. CONCLUSION: Our results show that (123)I-ChiB_E144Q has affinity in vitro for fungi. In vivo, the tracer accumulates in tissue infected with C. albicans and A. fumigatus but not in tissue infected with gram-positive or gram-negative bacteria, or in sterile inflammations, proving it to be a valuable SPECT diagnostic.     

In vitro and in vivo characteristics of [iodine-125] 3-(R)-quinuclidinyl (S)-4-iodobenzilate

The radioiodinated muscarinic acetylcholine receptor antagonist, [125I] 3-quinuclidinyl 4-iodobenzilate, has two high affinity diastereomeric forms, the (R,R) and (R,S)-isomers. The (R,S)-diastereomer is only threefold lower in affinity than the (R,R)-isomer, but the kinetic properties are considerably different--the dissociation rate constant is 13-fold faster for the (R,S)-isomer and the association rate constant is two to threefold faster. The calculated affinity is therefore only fourfold lower. In vivo, the clearance of (R,S)-4IQNB from receptor-rich tissue is also more rapid than that of the (R,R)-isomer, that is a reflection of the more rapid in vitro kinetic properties since the physicochemical properties and the metabolic clearance of the diastereomers is the same.     

In vivo imaging of nicotinic receptor upregulation following chronic (-)-nicotine treatment in baboon using SPECT

To quantify changes in neuronal nAChR binding in vivo, quantitative dynamic SPECT studies were performed with 5-[(123)I]-iodo-A-85380 in baboons pre and post chronic treatment with (-)-nicotine or saline control. Infusion of (-)-nicotine at a dose of 2.0 mg/kg/24h for 14 days resulted in plasma (-)-nicotine levels of 27.3 ng/mL. This is equivalent to that found in an average human smoker (20 cigarettes a day). In the baboon brain the regional distribution of 5-[(123)I]-iodo-A-85380 was consistent with the known densities of nAChRs (thalamus > frontal cortex > cerebellum). Changes in nAChR binding were estimated from the volume of distribution (V(d) ) and binding potential (BP) derived from 3-compartment model fits. In the (-)-nicotine treated animal V(d) was significantly increased in the thalamus (52%) and cerebellum (50%) seven days post cessation of (-)-nicotine treatment, suggesting upregulation of nAChRs. The observed 33% increase in the frontal cortex failed to reach significance. A significant increase in BP was seen in the thalamus. In the saline control animal no changes were observed in V(d) or BP under any experimental conditions. In this preliminary study, we have demonstrated for the first time in vivo upregulation of neuronal nAChR binding following chronic (-)-nicotine treatment.     

In vivo comparative imaging of dopamine D2 knockout and wild-type mice with (11)C-raclopride and microPET.

The use of mice with targeted gene deletions (knockouts [KOs]) provides an important tool to investigate the mechanisms underlying behavior, neuronal development, and the sequella of neuropsychiatric diseases. MRI has been used to image brain structural changes in KO mice but, to our knowledge, the feasibility of using PET to investigate brain neurochemistry in KO mice has not been demonstrated. METHODS: We have evaluated the sensitivity of the microPET to image dopamine D2 receptor (DRD2) KO mice (D2-/-). PET measurements were performed in wild-type (D2+/+) mice and KO (D2-/-) mice using a microPET scanner. Briefly, each animal was anesthetized and injected intravenously with (11)C-raclopride, a DRD2-specific ligand, and dynamic PET scanning was performed for 60 min. RESULTS: The (11)C-raclopride images of the KO mice showed significantly lower binding in the striatum (ST) than those of the wild-type (WT) mice, which was confirmed by the time-activity curves that revealed equivalent binding in the ST and cerebellum (CB) in KO mice, whereas the WT mice had significantly higher binding in the ST than in the CB. The ST/CB ratio was significantly higher in WT mice than in KO mice (ST/CB = 1.33 +/- 0.13 and 1.05 +/- 0.03, respectively; P < 0.002; n = 10). The microPET images were compared qualitatively with conventional autoradiography images. CONCLUSION: These data support the use of microPET as an effective in vivo imaging tool for studying noninvasively KO mice. These same tools can be extended to investigate other genetically engineered murine models of disease. Future studies will seek to use microPET to investigate the relationships between genes, neuronal activity, and behavior.     

Noninvasive in vivo MRI detection of neuritic plaques associated with iron in APP[V717I] transgenic mice, a model for Alzheimer's disease.

Transgenic mice overexpressing the London mutant of human amyloid precursor protein (APP[V717I]) in neurons develop amyloid plaques in the brain, thus demonstrating the most prominent neuropathological hallmark of Alzheimer's disease. In vivo 3D T2*-weighted MRI on these mice (24 months of age) revealed hypointense brain inclusions that affected the thalamus almost exclusively. Upon correlating these MRI observations with a panel of different histologic staining techniques, it appeared that only plaques that were positive for both thioflavin-S and iron were visible on the MR images. Numerous thioflavin-S-positive plaques in the cortex that did not display iron staining remained invisible to MRI. The in vivo detection of amyloid plaques in this mouse model, using the intrinsic MRI contrast arising from the iron associated with the plaques, creates an unexpected opportunity for the noninvasive investigation of the longitudinal development of the plaques in the same animal. Thus, this work provides further research opportunities for analyzing younger APP[V717I] mouse models with the knowledge of the final outcome at 24 months of age.     

In vivo monitoring of intranuclear p27 protein expression in breast cancer cells during trastuzumab (Herceptin) therapy

IntroductionTrastuzumab, a humanized antibody directed against the Her2 receptor, induces the expression of p27^kip1, an intranuclear cyclin-dependent kinase inhibitor in some breast cancer cells. The aim of this study was to develop a radioimmunoconjugate (RIC) to monitor trastuzumab-induced p27^kip1 protein up-regulation in vivo.Materials and MethodsAnti-p27^kip1 IgG was purified, and conjugated to diethylenetriaminopentaacetate, to allow radiolabeling with ¹¹¹In for in vivo detection. Then tat peptide (GRKKRRQRRRPPQGYG), containing a nuclear localization sequence (underlined), was conjugated to the Fc-domain of IgG, using NaIO₄ oxidation of carbohydrates and the resulting Schiff base stabilized with NaCNBH₃. The conjugate was radiolabeled with ¹¹¹In, yielding [¹¹¹In]-anti-p27^kip1-tat. ¹¹¹In labeling efficiency, purity and p27^kip1 binding were measured. Trastuzumab-induced p27^kip1 up-regulation was assessed in a panel of breast cancer cell lines by Western blot analysis. Uptake and retention of [¹¹¹In]-anti-p27^kip1-tat were measured in MDA-MB-361 and SKBr3 cells after exposure to trastuzumab. Uptake of [¹¹¹In]-anti-p27^kip1-tat was determined at 72 h postintravenous injection in MDA-MB-361 xenografts in athymic mice treated with trastuzumab or saline.Results[¹¹¹In]-anti-p27^kip1-tat was synthesized to 97% purity. The RIC was able to bind to p27^kip1 protein and internalized in the cells and was transported to the nuclei of MDA-MB-361 cells. The level of p27^kip1 protein in MDA-MB-361 cells was increased after exposure to clinically relevant doses of trastuzumab for 3 days. Trastuzumab-mediated induction of p27^kip1 was not associated with increased cellular uptake or nuclear localization of [¹¹¹In]-anti-p27^kip1-tat (6.53±0.61% vs. 6.98±1.36% internalized into trastuzumab-treated vs. control cells, respectively). However, retention of [¹¹¹In]-anti-p27^kip1-tat at 72 h was increased approximately twofold (13.5±1.3% vs. 6.6±0.6% of internalized [¹¹¹In]-anti-p27^kip1-tat was retained in trastuzumab-treated vs. control cells, respectively; P=.016). Immunohistochemistry showed up-regulation of p27^kip1 in trastuzumab-treated xenografts. Tumour uptake of [¹¹¹In]-anti-p27^kip1-tat was significantly higher in trastuzumab-treated compared to control animals (6.5±0.9 vs. 4.8±0.1 %ID/g at 72 h postinjection, respectively; P=.0065).Conclusion[¹¹¹In]-Anti-p27^kip1-tat may be useful for monitoring changes in the expression of the intranuclear protein, p27^kip1. Up-regulation of p27^kip1 resulted in increased retention of [¹¹¹In]-anti-p27^kip1-tat in cells treated with trastuzumab. Modest, but statistically significantly higher, retention was also observed in tumours in mice treated with trastuzumab. Since responsiveness to trastuzumab correlated to up-regulation of p27^kip1, it may be possible to use [¹¹¹In]-anti-p27^kip1-tat to guide treatment with Herceptin and other drugs which alter p27^kip1 expression.     

In vivo dosimetry with semiconductors in medium dose rate (MDR) brachytherapy for cervical cancer

Purpose

This study was performed to evaluate the role of in vivo dosimetry with semiconductor detectors in gynaecological medium dose rate brachytherapy, and to compare the actual doses delivered to organs at risk (as measured using in vivo dosimetry) with those calculated during treatment planning.

Materials and methods

Doses to the rectum and bladder were measured in a group of patients with cervical carcinoma using semiconductor detectors and compared to the doses calculated using a treatment planning system. 36 applications of brachytherapy at dose rates of 1.8–2.3 Gy/h were performed in the patients.

Results

The mean differences between the measured and calculated doses were 3 % for the rectum and 11 % for the bladder.

Conclusions

The main reason for the differences between the measured and calculated doses was patient movement. To reduce the risk of large errors in the dose delivered, in vivo dosimetry should be performed in addition to treatment planning system computations.     

In vivo detection of multidrug-resistant (MDR1) phenotype by technetium-99m sestamibi scan in untreated breast cancer patients

Technetium-99m sestamibi is a transport substrate recognised by the multidrug-resistant P-glycoprotein (Pgp). To test whether^99mTc-sestamibi efflux is enhanced in breast carcinomas overexpressing Pgp, we determined the efflux rates of^99mTc-sestamibi and Pgp levels in tumours from 30 patients with untreated breast carcinoma. Patients were intravenously injected with 740 MBq of^99mTc-sestamibi and underwent a 15-min dynamic study followed by the acquisition of static planar images at 0.5, 1, 2 and 4 h. Tumour specimens were obtained from each patient 24 h after^99mTc-sestamibi scan and Pgp levels were determined using¹²⁵I-MRK16 monoclonal antibody and in vitro quantitative autoradiography. All breast carcinomas showed high uptake of^99mTc-sestamibi and data from region of interest analysis on sequential images were fitted with a monoexponential function. The efflux rates of^99mTc-sestamibi, calculated from decay-corrected time-activity curves, ranged between 0.00121 and 0.01690 min⁻¹ and were directly correlated with Pgp levels measured in the same tumours (r=0.62;P<0.001). Ten out of 30 breast carcinomas (33%) contained 5 times more Pgp than benign breast lesions and showed a mean concentration of 5.73±1.63 pmol/g of tumour (group A). The remaining 20 breast carcinomas had a mean Pgp concentration of 1.29±0.64 pmol/g (group B), equivalent to that found in benign breast lesions.^99mTc-sestamibi efflux from tumours of group A was 2.7 times higher than that observed in tumours of group B (0.00686±0.00390 min⁻¹ vs 0.00250±0.00090 min⁻¹,P<0.001). The in vivo functional test with^99mTc-sestamibi showed a sensitivity and a specificity of 80% and 95%, respectively. In conclusion, the efflux rate of^99mTc-sestamibi may be used for the in vivo identification of the multidrug resistant (MDR1) phenotype in untreated breast cancer patients.     

In vivo imaging of induction of heat-shock protein-70 gene expression with fluorescence reflectance imaging and intravital confocal microscopy following brain ischaemia in reporter mice

Purpose

Stroke induces strong expression of the 72-kDa heat-shock protein (HSP-70) in the ischaemic brain, and neuronal expression of HSP-70 is associated with the ischaemic penumbra. The aim of this study was to image induction of Hsp-70 gene expression in vivo after brain ischaemia using reporter mice.

Methods

A genomic DNA sequence of the Hspa1b promoter was used to generate an Hsp70-mPlum far-red fluorescence reporter vector. The construct was tested in cellular systems (NIH3T3 mouse fibroblast cell line) by transient transfection and examining mPlum and Hsp-70 induction under a challenge. After construct validation, mPlum transgenic mice were generated. Focal brain ischaemia was induced by transient intraluminal occlusion of the middle cerebral artery and the mice were imaged in vivo with fluorescence reflectance imaging (FRI) with an intact skull, and with confocal microscopy after opening a cranial window.

Results

Cells transfected with the Hsp70-mPlum construct showed mPlum fluorescence after stimulation. One day after induction of ischaemia, reporter mice showed a FRI signal located in the HSP-70-positive zone within the ipsilateral hemisphere, as validated by immunohistochemistry. Live confocal microscopy allowed brain tissue to be visualized at the cellular level. mPlum fluorescence was observed in vivo in the ipsilateral cortex 1 day after induction of ischaemia in neurons, where it is compatible with penumbra and neuronal viability, and in blood vessels in the core of the infarction.

Conclusion

This study showed in vivo induction of Hsp-70 gene expression in ischaemic brain using reporter mice. The fluorescence signal showed in vivo the induction of Hsp-70 in penumbra neurons and in the vasculature within the ischaemic core.