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Previous work in our laboratory has shown a correspondence between the chemosensitivity of C6 rat glioma and that of human glioblastoma (GBM) to a panel of chemotherapeutic agents in vitro, as determined by the MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide] colorimetric assay. In the present study, an in vivo model of intracerebral C6 glioma in Sprague-Dawley rats was used to determine if a correlation exists between in vitro chemosensitivity and in vivo survival of the animals, and post-mortem histopathological changes in the tumor. Cisplatin (CDDP) and methotrexate (MTX), agents previously shown to demonstrate high and low in vitro cytotoxicity, respectively, against C6, were administered by intra-carotid infusion over the course of two days. In a separate series of animals, LTC4 was administered prior to infusion of CDDP or MTX; LTC4 was used in view of its known, selective, vasogenic effect on the permeability of brain tumor capillaries. It was found that survival of animals treated with CDDP alone was increased, although this did not reach statistical significance; histopathologically, CDDP-treated animals showed significant tumor necrosis. However, in CDDP-treated animals, pre-treatment with LTC4 increased survival to a statistically significant degree. When administered alone, LTC4 (not followed by CDDP) had no effect on either survival or histology. The survival-enhancing effect of CDDP, when combined with LTC4, was probably not due to any cytotoxic effect of LTC4; this is based on our finding that, on the in vitro MTT colorimetric assay, LTC4 showed low cytotoxicity for C6 glioma cells. By contrast with CDDP, MTX -- with or without pretreatment with LTC4 -- affected neither survival nor tumor histology. With respect to the question of correspondence between the MTT colorimetric in vitro assay and in vivo effect, MTX showed a clear correlation: low cytotoxicity in vitro and poor in vivo response. In the case of CDDP, the correspondence was not clear-cut: there was a high level of in vitro chemosensitivity of the C6 cell line to CDDP as well as post-mortem tumor necrosis, but in vivo testing showed no significant prolongation of survival. However, pre-treatment with LTC4 did significantly extend survival in animals treated with CDDP.

This study concerns the use of the MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide] colorimetric assay to evaluate the chemosensitivity of the C6 rat glioma cell line to a panel of twelve chemotherapeutic agents. In a previous study of in vitro chemosensitivity of human glioma cell lines, the present authors found a range of sensitivities of the respective cell lines to a panel of chemotherapeutic agents [1]. We then devised an experimental strategy to begin an in vivo evaluation of the correlation between in vitro chemosensitivity and clinical response in an in vivo animal model, such as the model employing the C6 rat glioma cell line. As a step towards utilizing the C6 rat glioma in vivo model, we carried out the present study (Part 1) to determine the correspondence between chemosensitivity to human glioma cell lines and the rat C6 glioma cell line. If a correspondence were to be found, this would enable experimental use of the C6 tumor model for in vivo testing of chemotherapeutic agents. As reported in this paper (Part 1), a correspondence was found, suggesting that the C6 rat glioma represents a suitable model of human glioma for chemotherapeutic studies. This finding served as a basis for proceeding with an in vivo study of chemotherapeutic efficacy which is the subject of a companion report [2].

Chloroquine has been shown to increase the cellular retention and nuclear incorporation of 125I-labeled monoclonal antibody (MAb) 425, a murine anti-epidermal growth factor receptor monoclonal antibody, in human high-grade glioma cells in vitro. The objective of this study was to examine the effect of chloroquine on the biodistribution of 125I-MAb 425 in an intracerebral xenogeneic transplant of glioma cells. Nude rats were stereotaxically implanted in the right hemisphere with A1207 human high-grade glioma cells. After 14 days, animals were injected i.v. with chloroquine (40 mg/kg) followed 2 h later by an 125I-MAb 425 (9 MBq) infusion. Tissue distributions were performed up to 168 h post 125I-MAb 425 injection. From 24 to 168 h, tumor-to-contralateral left brain ratios increased from 9 to 15 for 125I-MAb 425 alone, and 7 to 13 for the 125I-MAb 425/chloroquine combination, respectively. A single administration of chloroquine did not result in any significant difference in radiolabeled MAb accumulation in either the tumor site or other tissues. We conclude that chloroquine did not increase the amount of 125I-MAb 425 into the tumor; however, it is safe to administer i.v. at the 40 mg/kg dose. Under these experimental conditions, the increased radioactive accumulation observed for in vitro data did not translate into similar in vivo results.

Experimental surgery in animal models often requires prolonged periods of general anesthesia. Animals undergoing these procedures may have difficulty maintaining normal ventilation and oxygenation and therefore may require physiologic monitoring and endotracheal intubation to avoid hypoxia that could adversely affect the results of the investigation. Monitoring has traditionally included measurements of PaO2, PCO2, pH, and oxygen saturation calculated from arterial blood. Endotracheal intubation and placement of arterial catheters necessary for the intraoperative collection of specimens for blood gas analysis are labor-intensive and time-consuming. In this study, rats undergoing thoracic laminectomy as part of a study on spinal cord injury were monitored by noninvasive pulse oximetry, with a reflectance transducer placed on the skin/fur of the neck overlying the cervical carotid artery. Comparison of these data with intermittent arterial blood gas measurements in the same animals indicated > 90% concordance with the pulse oximetry values. We also determined the fraction of inspired oxygen (FiO2) that, delivered via facemask, can achieve at least the minimal normal oxygen saturation (SaO2) of 90%. These findings suggest that physiologic monitoring during experimental rodent surgery can be substantially simplified by pulse oximetry and by delivery of oxygen via facemask, eliminating the need for arterial blood gas determinations or endotracheal intubation.

The relationship between the severity of the posttraumatic functional deficit and findings on magnetic resonance imaging (MRI) was investigated in a rat model of experimental spinal cord trauma. Thirty Sprague-Dawley rats were subjected to an identical, moderate, contusion injury of the spinal cord. Control animals underwent laminectomy without cord injury. The severity of the functional deficit was assessed with the Combined Behavioral Score (CBS). Animals were killed at 3, 7, 14, 21, or 28 days after injury, and the fixed, excised spinal cords were studied with MRI at 1.9 T. The lesion length was measured on sagittal spin-echo MRI. The lesion length measured on MRI was highly correlated with the CBS functional score (r = 0.56, P = 0.002). There were significant correlations between lesion length as determined by MRI and by histological morphometry (r = 0.44, P = 0.02), between histological morphometric lesion length and CBS functional deficit (r = 0.76, P < 0.001), and between the area of residual white matter at the lesion epicenter, determined by histological techniques, and the severity of functional deficit (r = -0.59, P = 0.001). A qualitative estimate of the area of preserved white matter, derived from MRI, was significantly correlated with the severity of functional deficit (r = -0.56, P = 0.006). A multiple regression of MRI-determined lesion length and MRI estimate of residual white matter versus CBS explained more than 42% of the variability of the functional deficit among these animals subjected to the same weight drop injury. We conclude that MRI parameters are reliable predictors of the severity of neurological deficit in experimental spinal cord trauma.

Apparent diffusion coefficients (ADC) were measured in a rat spinal cord weight-drop injury model. After sacrifice, the spinal cords were fixed in situ and excised for MR imaging and ADC measurement. Diffusion is anisotropic in normal gray and white matter. There were significant decreases in ADCs measured along the longitudinal axis of the injured cord and increases in ADCs measured transverse to the cord. Injured segments demonstrated reductions in diffusion anisotropy in the white matter. Diffusion was completely isotropic at the epicenter of the weight-drop injury. Significant decreases in longitudinal ADC and increases in transverse ADC were observed in portions of the cord which appeared normal on conventional spin-echo and calculated T2 images. Thus ADC measurement may complement routine imaging for evaluation of spinal cord injury.

In order to obtain high resolution images of fixed excised rat spinal cords we have developed a technique using a 6-mm bore, two-turn saddle coil, with a usable imaging length of approximately 4 cm. MR imaging is performed on a prototype 31-cm bore, 1.9-T system with a 1.5-mm section thickness and 7.6-mm field of view.

Objective: This study was conducted to determine the association between lesion length measured on MRI and the severity of mechanical injury in a rat model of spinal cord trauma., Materials and Methods: Sprague-Dawley rats were subjected to a modified Allen weight drop injury and killed 4 h after injury. Their fixed, exercised cords were studied with MRI at 1.9 T., Results: There were strong correlations between lesion length on MR images and weight drop height and square root of drop height (r2 = 0.55, p < 0.0001 and r2 = 0.63, p < 0.0001, respectively). The lesion length differences were significant versus controls for all drop heights at p values of < or = 0.002 and for the 2.5 cm animals versus the 5 and 15 cm animals (p = 0.01 and p = 0.0002, respectively), but the lengths of the 5 vs. 15 cm groups only approached significance (p = 0.06)., Conclusion: These results suggest that lesion length determined on MR images is a reliable indicator of the severity of trauma among animals subjected to weight drop injury.

We have developed an implanted radiofrequency coil to obtain high resolution in vivo MR images at 1.9 Tesla of rat spinal cords that have been injured using a standardized weight drop technique. The signal-to-noise ratio and motion artifact suppression of these images is superior to that achieved in earlier attempts at this field strength using an external surface coil. The high quality and spatial resolution provided by this technique afford the possibility for longitudinal studies of experimental spinal cord injury before and after treatment, as well as detailed correlation of in vivo MRI contrast, histopathological findings, and functional deficit, in a controlled setting.

Eleven athymic nude rats had stereotactic intracerebral inoculation of cells from one of three established human glioma cell lines (A172, A1207, and A1235). The implants grew progressively in 9 of 11 instances, which led to spontaneous death of the host in 14 to 37 days. For comparison, two Sprague-Dawley normal albino rats were implanted with the rat C6 glioma cell line. One rat died at 14 days, and the other was killed at Day 16. The human glioma cells developed into partially (A172, A1235) or totally (A1207) circumscribed tumor masses. Invasion, when present, was manifested as infiltrating prongs of cells rather than as individual cell infiltration. The growth of the human glioma cells was accompanied by a small zone of surrounding edema and marked central necrosis. These features were not encountered in the C6 implants. Inflammatory changes were minimal to nonexistent in all cases. All tumor lines produced internal cerebral herniation and neuraxis spread with implants seeded throughout the ventricular system, often associated with ventricular dilation. In situ hybridization, by the use of isotopic and nonisotopic detection methods, was used to study the cellular expression of the acidic fibroblast growth factor and basic fibroblast growth factor genes in A172 glioma xenografts. The expression of these genes was not seen in normal rat brain, but the genes were selectively overexpressed by the glioma cell implants, with especially high signal in the tumor periphery.(ABSTRACT TRUNCATED AT 250 WORDS)

This study was undertaken to evaluate the colorimetric MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide] assay as a means of testing the sensitivity of gliomas to chemotherapeutic agents in vitro. Eight human glioma established cell lines were plated in 96-well tissue culture plates and incubated for 4 days with 10 different anti-cancer agents; 5 different concentrations of each drug were tested. The MTT dye was then added to the wells, and the resulting formazan precipitate was solubilized with dimethylsulfoxide (DMSO). The spectrophotometric absorbance (measured at 570 nm) of control and experimental wells was used to calculate the cytotoxicity index (CI). Values with a CI greater than 50% growth inhibition indicated cytotoxic efficacy (sensitivity to the chemotherapeutic drug). Six of the seven (85.7%) glioma cell lines were highly sensitive at varying concentrations to mitomycin C, cisplatin, and doxorubicin. Four of the seven (57.1%) cell lines demonstrated intermediate sensitivity to mitoxantrone and vinblastine. Five of the seven (71.4%) cell lines exhibited resistance to etoposide, bleomycin, cosmegen, and BCNU. One of the cell lines tested, U-138MG, failed to produce the MTT formazan precipitate, so that the sensitivity of this cell line to the panel chemotherapeutic drugs could not be determined. The variability of the results indicates the need for an in vitro screening method to evaluate the effectiveness of clinical and experimental chemotherapeutic agents. The MTT assay provides a rapid method of screening antineoplastic agents against gliomas for cytotoxicity.