Improved anti-tumor effect of liposomal doxorubicin after targeted blood-brain barrier disruption by MRI-guided focused ultrasound in rat glioma.

The blood-brain barrier (BBB) inhibits the entry of the majority of chemotherapeutic agents into the brain. Previous studies have illustrated the feasibility of drug delivery across the BBB using focused ultrasound (FUS) and microbubbles. Here, we investigated the effect of FUS-enhanced delivery of doxorubicin on survival in rats with and 9L gliosarcoma cells inoculated in the brain. Each rat received either: (1) no treatment (control; N = 11), (2) FUS only (N = 9), (3) IV liposomal doxorubicin (DOX only; N = 17), or (4) FUS with concurrent IV injections of liposomal doxorubicin (FUS+DOX; N = 20). Post-treatment by magnetic resonance imaging (MRI) showed that FUS+DOX reduced tumor growth compared with DOX only. Further, we observed a modest but significant increase in median survival time after a single treatment FUS+DOX treatment (p = 0.0007), whereas neither DOX nor FUS had any significant impact on survival on its own. These results suggest that combined ultrasound-mediated BBB disruption may significantly increase the antineoplastic efficacy of liposomal doxorubicin in the brain.

The Role of preload and leakage correction in gadolinium-based cerebral blood volume estimation determined by comparison with MION as a criterion standard.

BACKGROUND AND PURPOSE: Contrast extravasation in DSC-MRI potentiates inaccurate and imprecise estimates of glioma rCBV. We tested assertions that preload and postprocessing algorithms minimize this error by comparing Gd-rCBV using permutations of these 2 techniques with criterion standard rCBV using MION, an intravascular agent. MATERIALS AND METHODS: We imaged 7 Fisher rats with 9L gliosarcomas, by using 3T gradient-echo DSC-MRI with MION (2.0 mg Fe/kg) and staged injection of Gd-diethylene triamine pentaacetic acid: a 0.1-mmol/kg bolus provided no preload (P-) data and served as preload (P+) for a subsequent 0.2-mmol/kg bolus. We computed MION-rCBV (steady-state ΔR2*, tumor versus normal brain) and Gd-rCBV ΔR2* [t] integration) without (C-) and with (C+) postprocessing correction, thereby testing 4 correction permutations: P-C-, P-C+, P+C-, and P+C+. We tested whether each permutation reduced bias and variance of the Gd/MION rCBV differences by using generalized estimating equations and Fmax statistics (P < .05 significant). RESULTS: Gd-rCBV progressively better approximated MION-rCBV with increasing leakage correction. There was no statistically significant bias for the mean percentage deviation of Gd-rCBV from MION-rCBV for any correction permutation, but there was significantly reduced variance by using P+C- (22-fold), P-C+ (32-fold), and P+C+ (267-fold) compared with P-C-. P+C+ provided significant additional variance reduction compared with P+C- (12-fold) and P-C+ (8-fold). Linear regression of Gd-rCBV versus MION-rCBV revealed P+C+ to have the closest slope and intercept compared with the ideal, substantially better than P+C-. CONCLUSIONS: Preload and postprocessing correction significantly reduced the variance of Gd-rCBV estimates, and bias reduction approached significance. Postprocessing correction provide significant benefit beyond preload alone.

Gliosarcoma with bone infiltration and extracranial growth: case report and review of literature.

Gliosarcoma is a relatively rare and highly malignant brain tumor consisting of both a glioblastoma and a mesenchymal component. Because of the natural barrier of the dura mater, that prevents intra or extradural neoplasm dissemination, cases of penetration of the dura and cranium by gliosarcomas without previous surgery or radiation are very rarely reported. We report an unusual case of gliosarcoma that involved the temporal skull base and the dura without antecedent radiation or surgery, although the lesion traversed the dura without radiologic or gross interruption of the dura. Remarkable in our case is the initial integrity of cerebral parenchyma. Follow-up revealed a tumorous infiltration of the temporal lobe almost one year after initial diagnosis. Thus the origin of the gliosarcoma in our case seemed to be extradural in the temporal skull base. Furthermore, this report demonstrates that extensive multi-modality treatment might be effective in patients with gliosarcomas and poor prognostic factors, for example unmethylated MGMT status.

Optimized speckle variance OCT imaging of microvasculature.

We optimize speckle variance optical coherence tomography (svOCT) imaging of microvasculature in high and low bulk tissue motion scenarios. To achieve a significant level of image contrast, frame rates must be optimized such that tissue displacement between frames is less than the beam radius. We demonstrate that higher accuracy estimates of speckle variance can enhance the detection of capillaries. These findings are illustrated in vivo by imaging the dorsal window chamber model (low bulk motion). We also show svOCT imaging of the nonstabilized finger (high bulk motion), using optimized imaging parameters, demonstrating better vessel detection than Doppler OCT.

A novel brainstem tumor model: functional and histopathological characterization.

INTRODUCTION: Diffuse pontine gliomas remain a challenging and frustrating disease to treat. The survival rates for these high-grade brainstem tumors (BSTs) is dismal and optimal therapy has yet to be determined. The development of a satisfactory brainstem tumor model is necessary for testing new therapeutic paradigms that may prolong survival. MATERIALS AND METHODS: We report the surgical technique, functional testing, and histopathological features of a novel brainstem tumor model in rats. Female Fischer 344 rats (n=45) were randomized to receive an injection of either 3 microl of 9L gliosarcoma cells (100,000 cells, n=), 3 microl of F98 glioma cells (100,000 cells, n=10), or 3 microl of medium (Dulbecco's modified eagle medium) into the pontine tegmentum. Using a cannulated guide screw system, implanted in the skull of the animal, we injected each group at coordinates 1.4 mm right of the sagittal and 1.0 mm anterior of the lambdoid sutures, at a depth of 7.0 mm from the dura. The head was positioned 5 degrees from horizontal before injection. The rats were post-operatively evaluated for neurological deficits using an automated test. Kaplan-Meier curves were generated for survival and disease progression, and brains were processed postmortem for histopathology. RESULTS AND DISCUSSION: 9L and F98 tumor cells grew in 100% of animals injected and resulted in a statistically significant mean onset of hemiparesis of 16.5+/-0.56 days (P=0.001, log-rank test), compared to animals in the control group which lacked neurological deficits by day 60. The animals with tumor cells implanted demonstrated significant deterioration of function on the automated rod testing. Animals in the control group showed no functional or pathological signs of tumor. Progression to hemiparesis was consistent in all tumor-injected animals, with predictable onset of symptoms occurring approximately 17 days post-surgery. The histopathological characteristics of the 9L and F98 BSTs were comparable to those of aggressive human BSTs. CONCLUSION: The establishment of this animal tumor model will facilitate the testing of new therapeutic paradigms for the treatment of BSTs.

A novel model of intramedullary spinal cord tumors in rats: functional progression and histopathological characterization.

OBJECTIVE: Intramedullary spinal cord tumors are difficult lesions to treat given their recurrence rate and limited treatment options. The absence of an adequate animal model, however, has hindered the development of new treatment paradigms. In this study, we describe the technique for intramedullary injection of two experimental rodent gliomas (9L and F98) and present the methodology for functional and histopathological analysis of tumor progression. METHODS: F344 rats (n = 24) were randomized into three groups. Group 1 (n = 8) received a 5 microl intramedullary injection of Dulbecco's modified Eagle medium, Group 2 received a 5 microl intramedullary injection of 9L gliosarcoma (100,000) cells, and Group 3 received a 5 microl intramedullary injection of F98 glioma (100,000) cells. The animals were anesthetized, a 2 cm incision was made in the dorsal mid-thoracic region, and the spinous process of the T5 vertebrae was removed to expose the intervertebral space. The ligamentum flavum was removed, and an intramedullary injection was made into the spinal cord. The animals were evaluated daily for signs of paralysis using the Basso, Beattie, and Bresnahan scale and sacrificed after the onset of deficits for histopathological analysis. RESULTS: Animals injected with 9L-gliosarcoma had a median onset of hind limb paresis at 12 +/- 2.9 days. Animals injected with F98 glioma had a median onset of hind limb paresis at 19 +/- 3 days. Animals injected with Dulbecco's modified Eagle medium did not show neurological deficits. Hematoxylin-eosin cross sections confirmed the presence of intramedullary 9L and F98 tumor invading the spinal cord. Control animals had no significant histopathological findings. CONCLUSION: Animals injected with 9L or F98 consistently developed hind limb paresis in a reliable and reproducible manner. The progression of neurological deficits is similar to that seen in patients with intramedullary spinal cord tumors. These findings suggest that this model mimics the behavior of intramedullary spinal cord tumors in humans and may be used to examine the efficacy of new treatment options for both low- and high-grade intramedullary tumors.

A novel brainstem tumor model: guide screw technology with functional, radiological, and histopathological characterization.

OBJECT: Survival rates for high-grade brainstem tumors are approximately 10% and optimal therapy has yet to be determined. Development of a satisfactory brainstem tumor model is necessary for testing new therapeutic paradigms that may prolong survival. The authors report the technique, functional progression, radiological appearance, and histopathological features of a novel brainstem tumor model in rats. METHODS: Thirty female Fischer 344 rats were randomized (10 animals/group) to receive an injection of either 3 ml of 9L gliosarcoma cells (100,000 cells), 3 ml of F98 glioma cells (100,000 cells), or 3 ml of medium (Dulbecco modified Eagle medium) into the pontine tegmentum of the brainstem. Using a cannulated guide screw system implanted in the skull of the animal, rats in each group were injected at coordinates 1.4 mm to the right of the sagittal and 1 mm anterior to the lambdoid sutures, at a depth of 7 mm from the dura mater. The angle of the syringe during injection was anteflexed 5 degrees from the vertical. Postoperatively, the rats were evaluated for neurological deficits by using an automated rotarod test. High-resolution [18F]fluorodeoxyglucose-positron emission tomography (FDG-PET) fused with computerized tomography (CT) scans were acquired pre- and postoperatively through the onset of hemiparesis and correlated accordingly. Kaplan-Meier curves were generated for survival and disease progression, and brains were processed postmortem for histopathological investigation. The 9L and F98 tumor cells grew in 95% of the animals in which they were injected and resulted in a statistically significant mean onset of hemiparesis of 16.5 6 0.56 days (p = 0.001, log-rank test), compared with animals in the control group, which had no neurological deficits by Day 45. The FDG-PET studies coregistered with CT scans demonstrated space-occupying brainstem lesions, and this finding was confirmed by histological studies. Animals in the control group showed no functional, radiological, or pathological signs of tumor. CONCLUSIONS: Progression to hemiparesis was consistent in all tumor-injected animals, with predictable onset of symptoms occurring approximately 17 days postsurgery. The histopathological and radiological characteristics of the 9L and F98 brainstem tumors were comparable to those of aggressive primary human brainstem tumors. Establishment of this animal tumor model will facilitate the testing of new therapeutic paradigms for the treatment of these lesions.

Detection of experimentally induced brain tumors in rats using high resolution computed tomography.

OBJECTIVE: The feasibility of high resolution CT imaging for evaluating experimentally-induced brain tumors in rats was assessed. METHODS: The gliosarcoma cell line (9 L/lacZ) was inoculated in 34 male Fischer 344 rats and CT studies were performed prior to and at 4, 7, 9, 12 and 14 days post-tumor cell implantation. Brain imaging pre- and post-contrast was performed and correlated with autopsy findings. RESULTS: Tumors were identified by CT in 19 of the 34 animals after contrast administration and their presence was confirmed at autopsy. Tumors were present at autopsy and not identified by CT scanning in eight additional animals and in the remaining seven animals, the CT scan was normal and no tumors were present at autopsy. The sensitivity and specificity of CT scanning with contrast in detecting tumors in this rat model of gliosarcoma was 70 and 100%, respectively. CONCLUSION: The improved CT technology currently available can be used to identify and follow tumor burden in a rat model of gliosarcoma, and be a good tool to utilize in determining treatment outcomes experimentally, especially when MR imaging is not available.

Continuous arterial spin labeling using a train of adiabatic inversion pulses.

PURPOSE: To develop a simple and robust magnetic resonance imaging (MRI) pulse sequence for the quantitative measurement of blood flow in the brain and cerebral tumors that has practical implementation advantages over currently used continuous arterial spin labeling (CASL) schemes. MATERIALS AND METHODS: Presented here is a single-coil protocol that uses a train of hyperbolic secant inversion pulses to produce continuous arterial spin inversion for perfusion weighting of fast spin echo images. Flow maps of normal rat brains and those containing a 9L gliosarcoma orthotopic tumor model conditions were acquired with and without carbogen. RESULTS: The perfusion-weighted images have reduced magnetization transfer signal degradation as compared to the traditional single-coil CASL while avoiding the use of a more complex two-coil CASL technique. Blood flow measurements in tumor and normal brain tissue were consistent with those previously reported by other CASL techniques. Contralateral and normal brain showed increased blood flow with carbogen breathing, while tumor tissue lacked the same CO(2) reactivity. CONCLUSION: This variation of the CASL technique is a quantitative, robust, and practical single-coil method for measuring blood flow. This CASL method does not require specialized radiofrequency coils or amplifiers that are not routinely used for anatomic imaging of the brain, therefore allowing these flow measurements to be easily incorporated into traditional rodent neuroimaging protocols.

Antiangiogenic effects of dexamethasone in 9L gliosarcoma assessed by MRI cerebral blood volume maps.

Depending on dose, dexamethasone has been shown to inhibit or stimulate growth of rat 9L gliosarcoma and decrease the expression of vascular endothelial growth factor (VEGF), an important mediator of tumor-associated angiogenesis. We demonstrate, by constructing relative cerebral blood volume (rCBV) maps with MRI, that dexamethasone also decreases total blood volume while increasing microvascular blood volume in Fischer rats bearing intracranial 9L gliosarcoma. Animals were inoculated with 1 x 10(5) 9L gliosarcoma tumor cells. On days 10-14 after tumor cell inoculation, animals were intra-peritoneally injected with dexamethasone (3 mg/kg) over 5 days. MRI-derived gradient echo (GE) and spin-echo (SE) rCBV maps were created to demonstrate total vasculature (GE) and microvasculature (SE). After MRI studies were performed, the rat's vasculature was perfused with a latex compound. Total vessel volume and diameters were assessed by microscopy. Dexamethasone decreased the tumor-enhancing area of postcontrast T1-weighted images (P < 0.0001) and total tumor volume(P = 0.0085). In addition, there was a greater than 50% decrease in GE rCBV (total vasculature) (P = 0.007) as well as a significant decrease in total fractional blood volume, as validated by histology (P = 0.0007). Conversely, there was an increase in SE rCBV signal (microvasculature) in animals treated with dexamethasone (P = 0.05), which was consistent with microscopy (P < 0.0001). These data demonstrate that (1) dexamethasone selectively treats tumor vasculature, suggesting a vessel-size selective effect and (2) MRI-derived rCBV is a noninvasive technique that can be used to evaluate changes in blood volume and vascular morphology.

Steady-state blood volume measurements in experimental tumors with different angiogenic burdens a study in mice.

PURPOSE: To experimentally validate the effectiveness of magnetic resonance (MR) imaging enhanced with long-circulating iron oxide for measurement of vascular volume fractions (VVFs) as indicators of angiogenesis in different experimental tumor models. MATERIALS AND METHODS: Tumors with differing degrees of angiogenesis-9L rodent gliosarcoma, DU4475 human mammary adenocarcinoma, HT1080 human fibrosarcoma, and EOMA hemangioendothelioma--were implanted in nude mice. Tumoral VVFs were measured at submillimeter voxel resolutions by using 1.5-T MR imaging. A technetium-labeled intravascular radiotracer was injected into a subset of the animals to validate the MR imaging measurements. Microvessel density and vascular endothelial growth factor (VEGF) also were measured. Statistical analysis was performed with analysis of variance. RESULTS: High-resolution multisection MR maps of tumor blood volume were obtained in all tumor models. Mean tumoral VVF differed significantly among the different tumors: 2.1% +/- 0.3 (standard error of mean) for 9L gliosarcoma, 3.1% +/- 0.4 for DU4475 mammary adenocarcinoma, 5.5% +/- 0.8 for HT1080 fibrosarcoma, and 6.6% +/- 0.9 for EOMA hemangioendothelioma (P <.01). There was a strong correlation between the MR imaging and radiotracer measurements. There was considerable intra- and intertumoral heterogeneity among the VVFs. MR imaging measurements were in accordance with conventional measurements of angiogenesis, such as microvessel density count and VEGF. CONCLUSION: Measurements of tumoral VVF at high-resolution MR imaging with long-circulating iron oxide are feasible and correlate with angiogenic burden in experimental tumor models.

Local delivery of mitoxantrone for the treatment of malignant brain tumors in rats.

OBJECT: Mitoxantrone is a drug with potent in vitro activity against malignant brain tumor cell lines; however, its effectiveness as a systemic agent has been hampered by poor central nervous system penetration and dose-limiting myelosuppression. To avoid these problems, we incorporated mitoxantrone into biodegradable polymeric wafers to be used for intracranial implantation, a strategy that has been shown to be safe and successful in the treatment of malignant gliomas. The authors investigated the release kinetics, toxicity, distribution, and efficacy of mitoxantrone delivered from intracranially implanted biodegradable wafers in the treatment of 9L gliosarcoma in Fischer 344 rats. METHODS: Mitoxantrone released from the biodegradable wafer matrix reached therapeutic drug concentrations in the brain for at least 35 days. Only animals with implanted wafers of the highest drug loading dose (20% mitoxantrone by weight) showed signs of significant toxicity. In three separate efficacy experiments, animals treated with mitoxantrone-loaded biodegradable wafers had significantly improved survival compared with control animals. The combined median survival for each treatment group was the following: 0% mitoxantrone wafers, 19 days; 1%, 30 days, p < 0.0001; 5%, 34 days, p < 0.0001; and 10%, 50 days, p < 0.0001. CONCLUSIONS: These findings establish that mitoxantrone delivered from intracranially implanted biodegradable wafers is effective in the treatment of malignant gliomas in rodents and should be considered for future clinical application in humans.

Endostatin reduces vascularization, blood flow, and growth in a rat gliosarcoma.

Endostatin, the 20-kDa C-terminal fragment of collagen XVIII, has previously been shown to inhibit growth and induce regression of different experimental tumors in rodents. In this study, we show that recombinant murine and human endostatin, produced in 293 EBNA cells and yeast, respectively, inhibit ectotopic as well as orthotopic growing BT4Cn gliosarcomas in BD-IX rats. In rats in which s.c. gliomas were grown for a total of 29 days, systemic treatment with recombinant murine endostatin induced about 50% reduction of intratumoral blood flow and tumor size after only 10 days of therapy. In contrast, the blood flow to irrelevant organs was unaffected by endostatin, indicating its specificity of action. Tumors were not observed to increase in size or regrow after cessation of therapy. Furthermore, endostatin-treated rats with i.c. tumors had significantly longer survival time than did untreated controls. In the treated rats, endostatin therapy resulted in a reduced tumor blood vessel volume and an increased tumor cell density with an increased apoptotic index within a given tumor volume, as verified by flow cytometry and by staining with deoxynucleotidyltransferase-mediated dUTP nick-end labeling. This work verifies the general anti-angiogenic and antitumor effects of endostatin and indicates that the protein may also be considered as a treatment strategy for malignant brain tumors.

TmDOTP(5-) as a (23)Na shift reagent for the subcutaneously implanted 9L gliosarcoma in rats.

The use of TmDOTP(5-) as an in vivo (23)Na NMR shift reagent (SR) for subcutaneously implanted 9L gliosarcoma was evaluated. TmDOTP(5-) produced a single sharp extracellular peak after about 50-60 min of infusion, and did not cause any changes in the (31)P resonance areas or chemical shifts, suggesting that the SR is homogeneously distributed in the extracellular space and does not alter tumor bioenergetic status. TmDOTP(5-) and CoEDTA(-) as extracellular space markers gave identical results for relative extracellular space (0.25 +/- 0.03 and 0.25 +/- 0.04, respectively) and intracellular Na(+) concentration (19.3 +/- 4.0 mM and 18.6 +/- 3.9 mM, respectively), indicating that the biodistribution of the SR is the same as the well-accepted extracellular space marker. The in vivo T(1) and T(2) relaxation times of intra- and extracellular Na(+) were also measured. Our results indicate that TmDOTP(5-) promises to be an effective shift reagent and extracellular space marker in the 9L gliosarcoma and perhaps other tumors. Magn Reson Med 45:436-442, 2001.

Experimental gliosarcoma induces chemokine receptor expression in rat brain.

Macrophage/microglial infiltration is a characteristic feature of brain tumors. The functional role(s) of these cells is complex and could include both trophic and suppressive effects on tumors. Information has recently emerged about the molecular signals that regulate the accumulation and function of monocytes in pathological disorders. Recent data indicate that the chemokine, monocyte chemoattractant protein-1 (MCP-1), a potent monocyte activating and chemotactic factor, is a primary regulator of the macrophage response in brain tumors. We hypothesized that if MCP-1 regulates macrophage/microglial infiltration, then expression of the specific MCP-1 receptor, CCR2, will be induced in peritumoral tissue and/or within brain tumors. Identification of a specific receptor that is preferentially expressed in brain tumors could be important both in terms of tumor biology and as a potential therapeutic target. We used an established experimental gliosarcoma model, induced by intracranial transplantation of cultured 9L cells into adult rat brain, to test this hypothesis. RT-PCR analysis showed high levels of both MCP-1 and CCR2 mRNA and Western blot analysis demonstrated increased CCR2 protein in tumor extracts. Immunocytochemistry showed CCR2 immunoreactive microglia in peritumoral tissue and, unexpectedly, that intrinsic tumor cells, rather than monocytes, were the predominant source of CCR2. These results demonstrate that CCR2 expression is markedly upregulated in this brain tumor model.

Unusual evolution and computerized tomographic appearance of a gliosarcoma.

A patient with a remote infarct, seizures, mild hemiparesis, and dysphasia became obtunded over four months and died. Computerized tomography (CT) over 5 years showed a consistent, large, wedge-shaped left hemisphere hypodensity with a central calcification, but without signs of mass effect. This was interpreted as an infarct of the left middle cerebral artery territory. Post-mortem examination of the brain revealed the entire area appearing as infarct on CT was a gliosarcoma. We suspect that the unusual CT appearance of the lesion was likely caused by multiple pathologies: a low grade glioma transforming into a gliosarcoma that was able to spread throughout the area of infarct encephalomalacia without revealing a typical CT appearance of mass effect. The patient's brief period of deterioration probably coincided with transformation of the tumor into a gliosarcoma. The variable CT characteristics of gliosarcomas are reviewed.

Relationship of perfusion to edema in the 9L gliosarcoma.

The relationship between tumor perfusion and edema was analyzed, with edema characterized as tumor wet/dry weight ratio. Perfusion of subcutaneous 9L gliosarcoma was measured by injection of 133Xe in saline into the tumor core, followed by gamma camera imaging of 133Xe washout kinetics. A significant inverse correlation was found between edema and tumor perfusion (p < 0.0002), suggesting that edema can limit tumor perfusion, perhaps through a mechanism of increased interstitial fluid pressure. The perfusion rate of highly edematous tumors was reduced to less than 10% of the perfusion rate of less edematous tumors (p < 0.001). It was also found that tumor edema increased significantly with increasing tumor volume (p < 0.001), which could account for the finding that perfusion declined significantly with increasing tumor volume (p < 0.02). These findings are potentially important because it is possible to quantify tumor edema in vivo, with millimeter resolution, using 1H magnetic resonance imaging (MRI). Thus MRI may provide a non-invasive technique for characterizing tumor perfusion or tumor drug delivery.