Gy Rats Research Articles

Abstract A09: Predicting response to whole brain radiotherapy in a murine model of glioma

Abstract Introduction: Glioblastoma is a highly invasive and aggressive brain tumor which accounts for nearly 82% of all gliomas. One promising direction for improving the clinical care of cancer, in general, and glioblastomas, in particular, is the development of accurate and precise predictive mathematical models of tumor growth. Specifically, models that can accurately characterize tumor response to radiotherapy could improve the design and optimization of individual radiotherapy plans. Using non-invasive imaging measurements from magnetic resonance imaging (MRI), a biophysical model of tumor growth has been shown to accurately predict future tumor growth on an individual basis in a murine model of glioma. In this work, we have expanded upon this biophysical model to incorporate the effects of radiotherapy. Furthermore, we evaluate the accuracy of model predictions relative to in vivo measurements of tumor growth. Methods: Untreated tumor growth was modeled using a mechanically-coupled reaction-diffusion model incorporating a voxel-specific carrying capacity, a voxel-specific tumor cell proliferation rate, and a global value for tumor cell diffusion. The effects of radiotherapy were modeled as resulting in either instantaneous cell death (Md model), the reduction in the net proliferation rate (Mp model), or a combination of the two effects (Mdp ). Instantaneous cell death was incorporated into the Md and Mdp models by estimating a death rate that was applied only during treatment. Similarly, decreased proliferation was incorporated into the Mp and Mdp models by estimating a proliferation fraction that was applied during and after treatment. To evaluate these models, rats with C6 gliomas were imaged over twelve days with diffusion-weighted MRI (DW-MRI) and contrast enhanced MRI (CE-MRI). Rats were imaged at three time points prior to receiving whole brain radiation therapy with a dose of either 20 Gy (N = 5) or 40 Gy (N = 7), and were imaged an additional four times following treatment. Tumor cellularity was estimated within enhancing regions in CE-MRI images using DW-MRI data. The pre-treatment measurements of cellularity were used to estimate tumor cell proliferation rate, carrying capacity, and the tumor cell diffusion coefficient. Two post-treatment measurements were used to estimate the post-treatment model parameters. The estimated model parameters were then used in a finite difference simulation to predict tumor growth at the remaining (6th through 7th) imaging time points. Error between the predicted radiotherapy response and the observed radiotherapy response was assessed by calculating the percent error in tumor volume, percent error in voxel cell number, and the normalized root mean square error (nRMSE). Results: For the 20 Gy rats no statistically significant differences were observed for model error in tumor volume (ranging from 7.7 to 21.2%), voxel cell number (ranging from 14.0 to 18.4%), or nRMSE (ranging from 0.14 to 0.16) between models. For the 40 Gy rats, the error in tumor volume prediction was significantly lower (p < 0.05) for the Mp and Mdp models (ranging from 22.2 to 36.3%) compared to the Md model (error greater than 54%). Error in voxel cell number ranged from 17.2 to 23.3% for all three models. The Mp and Mdp models had statistically lower nRMSE (nRMSE < 0.30, p = 0.05) compared to the Md model (nRMSE = 0.64). Conclusion: For 20 Gy whole brain radiotherapy, all three models demonstrated accurate prediction of future bulk tumor growth. However, at 40 Gy the model predictions of future bulk tumor growth were poor, although the Mp and Mdp models provide lower error in predictions than the Md model. Additionally, all three models fail to capture the development of low cell density, potentially necrotic, regions following radiotherapy for both the 20 and 40 Gy groups. Further work is needed to better characterize intra-tumor response at all doses. Citation Format: David A. Hormuth, II, Jared A. Weis, Stephanie B. Eldridge, Michael I. Miga, Erin C. Rericha, Vito Quaranta, Thomas E. Yankeelov. Predicting response to whole brain radiotherapy in a murine model of glioma. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr A09.

New and safe experimental model of radiation-induced neurovascular histological changes for microsurgical research.

The aim is to create a new and safe experimental model of radiation-induced neurovascular histological changes with reduced morbidity and mortality for use with experimental microsurgical techniques. Seventy-two Sprague-Dawley rats (250-300 g) were divided as follows: Group I: control group, 24 rats clinically evaluated during six weeks; Group II: evaluation of acute side-effects (two-week follow-up period), 24 irradiated (20 Gy) rats; and Group III: evaluation of subacute side-effects (six-week follow-up period), 24 irradiated (20 Gy) rats. Variables included clinical assessments, weight, vascular permeability (arterial and venous), mortality and histological studies. No significant differences were observed between groups with respect to the variables studied. Significant differences were observed between groups I vs II-III regarding survival rates and histological changes to arteries, veins and nerves. Rat body weights showed progressive increases in all groups, and the mortality rate of the present model is 10.4% compared with 30-40% in the previous models. In conclusion, the designed model induces selective changes by radiotherapy in the neurovascular bundle without histological changes affecting the surrounding tissues. This model allows therapeutic experimental studies to be conducted, including the viability of microvascular and microneural sutures post radiotherapy in the cervical neurovascular bundle.

Cellular alterations upon IR-laser (890 nm) exposures, in vivo.

Exposure of cultured cells and small animals to ionizing radiation as well as irradiation of cultured cells with He-Ne laser can cause changes in the functional condition of plasma membranes. The ionizing radiation-induced cell membrane alterations have been determined after either partial or local exposures. The aim of the present study was to reveal whether the local laser treatments cause a general, distant, so called abscopal" effect measured at cellular level, when the laser treatment is intended as a stimulatory procedure. The biological effect of infrared laser (mean power of 5 Watts, 150 Hz frequency, 890 nm wavelength) was demonstrated through 3H-concanavalin A binding by blood cells of daily irradiated (altogether 10 exposures) oncological and non-oncological patients as well as by changes in the proliferation of bone marrow cells of whole body gamma-irradiated (4 Gy) rats, partially laser-treated. The lectin binding of lymphocytes of oncological, as well as ischaemic heart disease patients was increased immediately after the first laser treatment. However, it was decreased after completion of the full course. In cases of inflammatory diseases the test parameters were either unchanged or decreased as compared to their self-control values. The platelets and erythrocytes did not react in any group. Gamma irradiation caused a deep inhibition of proliferation of rat bone marrow cells. The number of fibroblast colony-forming units (CFU-F) could be increased again if the animals were partially exposed to laser. Laser irradiation of one of the femurs led to some recovery of CFU-F values in the exposed as well as unexposed femur. Thus, local infrared laser treatment induces abscopal effects on the cell membrane and cell proliferation characteristics.

Bile secretion of cadmium, silver, zinc and copper in the rat. Involvement of various transport systems

In the present study we compared, in vivo in rats, the hepatobiliary transport of monovalent (silver:Ag) and divalent metals (zinc:Zn; cadmium:Cd) with that of copper (Cu). Cu can have two oxidation states in vivo, i.e. Cu(I) and Cu(II). Studies were performed in normal Wistar (NW) rats and mutant GY Wistar rats. The latter express defective canalicular ATP-dependent glutathione-conjugate transport (cMOAT); reduced glutathione (GSH) is virtually absent in bile of these mutants. Cd (400 nmol/100g body Wt, I.v.) was rapidly secreted into bile in NW rats concommitant with a 4-fold increase in biliary GSH secretion. In contrast, biliary Cd concentrations remained below detection limits in GY rats. Injection of Zn (1500 nmol/100g body wt) did not affect Zn secretion in GY rats and resulted only in a very small increase in NW rats (recovery < 2%). The biliary secretion pattern of Ag (80 nmol/100g body Wt, I.v.) was highly similar to that of Cu (260 nmol/100g body wt). A biphasic pattern composed of a rapid and slow phase was observed in NW rats for both metals with a recovery of 48.5 ± 10.6% and 44.9 ± 8.4% of the dose for Ag and Cu, respectively. In GY rats, the rapid phase of both Ag and Cu secretion was absent and recoveries were 23.2 ± 3.6% and 19.7 ±3.2%, respectively. When Ag and Cu were administered simultaneously, the recoveries of Ag and Cu were decreased in NW and GY rats when compared to single administration. Our data indicate that divalent and monovalent metals are secreted into bile via different transport systems in the rat. The absence of Cd and Zn secretion into bile of GY rats after their i.v. administration suggest a role of cMOAT in their biliary elimination. Cu and Ag probably share common transport systems for hepatobiliary removal, being in part dependent on the presence of either GSH in bile or cMOAT activity or on both. The GSH-independent portion of Transport, I.e. the slow phase, may be mediated by the newly identified Cu transporting P-type ATPase (cCOP).

In vitro modeling of liver membrane copper transport

The process of hepatobiliary copper (Cu) secretion is still poorly understood Cu secretion as a complex with glutathione and transport via a lysosomal pathway have been proposed. The recent cloning and sequencing of the gene for Wilson disease indicates that Cu transport in liver cells may be mediated by a Cu transporting Ptype ATPase. Biochemical evidence for ATP-dependent Cu transport in mammalian systems, however, has not been reported so far. We have investigated Cu transport in rat liver plasma membrane vesicles enriched in canalicular or basolateral membranes in the presence and absence of ATP (4 mM) and an ATP-regenerating system. The presence of ATP clearly stimulated uptake of radiolabeled Cu (64Cu, 10 μM) into canalicular plasma membrane vesicles and, to a lesser extent, also into basolateral plasma membrane vesicles. ATP-dependent Cu transport was dose-dependently inhibited by the P-type ATPase inhibitor vanadate, and showed saturation kinetics with an estimated K m of 8.6 μM and a V max of 6.9 nmol/min/mg protein. ATP-stimulated Cu uptake was similar in canalicular membrane vesicles of normal Wistar rats and those of mutant GY rats, expressing a congenital defect in the activity of the ATP-dependent canalicular glutathione-conjugate transporter (cMOAT). These studies demonstrate the presence of an ATP-dependent Cu transporting system in isolated plasma membrane fractions of rat liver distinct from cMOAT. (J. Clin. Invest. 1995.95:412-416.) Key words: copper · bile secretion · P-type ATPase · vesicle transport · Wilson disease. (Reproduced from The Journal of Clinical Investigation, 1995, vol. 95, pp. 412-416 by copyright permission of The Society for Clinical Investigation.).

Adenosine triphosphate-dependent copper transport in isolated rat liver plasma membranes.

The process of hepatobiliary copper (Cu) secretion is still poorly understood: Cu secretion as a complex with glutathione and transport via a lysosomal pathway have been proposed. The recent cloning and sequencing of the gene for Wilson disease indicates that Cu transport in liver cells may be mediated by a Cu transporting P-type ATPase. Biochemical evidence for ATP-dependent Cu transport in mammalian systems, however, has not been reported so far. We have investigated Cu transport in rat liver plasma membrane vesicles enriched in canalicular or basolateral membranes in the presence and absence of ATP (4 mM) and an ATP-regenerating system. The presence of ATP clearly stimulated uptake of radiolabeled Cu (64Cu, 10 microM) into canalicular plasma membrane vesicles and, to a lesser extent, also into basolateral plasma membrane vesicles. ATP-dependent Cu transport was dose-dependently inhibited by the P-type ATPase inhibitor vanadate, and showed saturation kinetics with an estimated Km of 8.6 microM and a Vmax of 6.9 nmol/min/mg protein. ATP-stimulated Cu uptake was similar in canalicular membrane vesicles of normal Wistar rats and those of mutant GY rats, expressing a congenital defect in the activity of the ATP-dependent canalicular glutathione-conjugate transporter (cMOAT). These studies demonstrate the presence of an ATP-dependent Cu transporting system in isolated plasma membrane fractions of rat liver distinct from cMOAT.

Processing of the phospholipid analogue phosphatidyl(N-sulphorhodamine B sulphonyl)ethanolamine by rat hepatocytes in vitro and in vivo.

We have investigated the processing of the non-exchangeable fluorescent phospholipid analogue phosphatidyl(N-sulphorhodamine B sulphonyl)ethanolamine (N-Rh-PE) by rat liver cells. In the hepatocyte couplet system, N-Rh-PE was incorporated into the plasma membrane at 2 degrees C and readily internalized upon warming to 37 degrees C. Fluorescence was initially found to be concentrated in vesicles clustered throughout the cell, but subsequently it started to accumulate in pericanalicular vesicles, tentatively identified as lysosomes, and in the bile canalicular lumen. Analysis of cells and media by t.l.c. revealed the slow formation of at least two metabolites. After intravenous injection into bile-fistula rats of [9,10-3H-oleoyl]N-Rh-PE incorporated in small unilamellar liposomes, the initial rates of elimination from plasma of 3H and rhodamine label were virtually identical. However, biliary secretion of the 3H label (5.5% of dose at 2 h) was much slower than that of the rhodamine label (49.3% at 2 h). The rhodamine label in bile was chloroform-soluble, but not identical to the native molecule, and was resistant to phospholipase A2 and alkaline hydrolysis. To gain insight in the mechanism of the rapid bile secretion of this metabolite, we compared the processing of N-Rh-PE, its deacylated form [glycerophospho(N-sulphorhodamine B sulphonyl)ethanolamine; Gly-N-Rh] and the rhodamine label itself (sulphorhodamine B sulphonyl chloride; SRho). Intravenous injection of chloroform-soluble N-Rh-PE and of methanol/water-soluble Gly-N-Rh complexed with albumin both resulted in rapid bile secretion of chloroform-soluble fluorescent compounds (60.2% and 86.3% respectively at 2 h), which showed behaviour identical to that of the metabolite of liposomal N-Rh-PE on t.l.c. Methanol/water-soluble SRho was also rapidly secreted into bile (89.5% at 2 h) without being metabolized. Bile secretion of the chloroform-soluble metabolite of N-Rh-PE and of SRho was markedly impaired (-31% and -52% respectively) in GY Wistar rats, which express a genetic defect in the hepatobiliary transport of organic anions. Our data show that the rat hepatocyte is capable of modifying the structure of N-Rh-PE, a process which proceeds considerably faster in vivo than in vitro. The chloroform-soluble metabolite is subsequently rapidly removed via the bile. The canalicular organic anion transporting system, which is deficient in GY rats, appears to be involved in the excretion of this apolar product of hepatic metabolism.

Radiation Hepatology of the Rat: Microvascular Fibrosis and Enhancement of Liver Dysfunction by Diet and Drugs

Prior to whole-liver irradiation (0, 15, 25 Gy) rats were not treated, placed on a protein-deficient diet for 2 weeks, administered cyclophosphamide, and/or depleted of intracellular glutathione by injection with buthionine sulfoximide and diethyl maleate. At various times (0 to 84 days) after 0- and 25-Gy liver irradiation, liver function, liver mass, and hydroxyproline content were measured in nontreated animals. Liver function was measured in all other preirradiation regimens 2 to 3 months postirradiation. Histology and/or India ink perfusion of the liver were done on ascitic and jaundiced animals from all treatment groups. Approximately 20% of the 25-Gy whole-liver-irradiated animals in each preirradiation treatment group developed clinical signs of acute hepatitis (ascites, jaundice, and elevated liver enzymes in the plasma) 70 to 100 days after irradiation. Twenty-five-gray whole-liver irradiation also resulted in significant liver fibrosis that preceded the onset of liver dysfunction. Fibrotic changes were most dramatic in and around hepatic veins, sometimes resulting in complete or partial occlusion that disrupted intrahepatic blood flow and diminished liver function. In addition, a substantial accumulation of fluid in the liver of 25-Gy-irradiated livers occurred, resulting in a lower dry/wet liver weight ratio. Functional hepatic injury was enhanced by preirradiation treatment with a protein-deficient diet, cyclophosphamide, and/or depletion of intracellular glutathione. This enhancement of functional injury was pronounced after 15-Gy whole-liver irradiation when injury from radiation alone was minimal.

Two pathways for biliary copper excretion in the rat: The role of glutathione

To evaluate the role of glutathione in biliary copper excretion, we studied this process in control Wistar rats and in mutant Wistar rats (GY rats), in which the secretion of glutathione into bile is deficient. For comparison, biliary zinc excretion was determined simultaneously. In spite of the markedly reduced bile flow (−45%) in GY rats, biliary output rates of endogenous copper were virtually identical in GY and control rats. In contrast, zinc output was drastically reduced in GY rats compared to controls (−80%). Biliary excretion patterns after intravenous administration of copper, in doses ranging from 65 to 2265 nmol/100 g/body wt, showed a distinct rapid and slow phase in control rats. In GY rats, on the other hand, the rapid phase in copper excretion was absent but the slow phase appeared to be unaffected. Pretreatment of rats with diethylmaleate to deplete hepatic and biliary glutathione abolished the rapid phase of copper excretion in control rats, while the slow phase remained unaffected. No significant effect of diethylmaleate on the hepatic handling of exogenous copper was observed in GY rats. The maximal capacity of the slow copper excretion pathway was 40–45 nmol/hr/100 g body wt, both in control and GY rats; the capacity of rapid excretion pathway depended on the administered copper load. Intravenous injection of copper induced the biliary excretion of a substantial amount of zinc in control rats; but not in GY rats. These results indicate the existence of at least two distinct biliary excretory pathways for copper in the rat. i.e. a slow and a rapid pathway, with a glutathione dependency of the latter only. The basal excretion of (endogenous) copper, in contrast to that of zinc, can proceed independently of glutathione excretion. However, glutathione appears to be involved in the rapid secretion of excess copper.

Defective biliary secretion of bile acid 3-O-glucuronides in rats with hereditary conjugated hyperbilirubinemia.

Biliary secretion of bile acid glucuronides was studied in control rats and in rats with a congenital defect in hepatobiliary transport of organic anions (GY rats). In control animals, hepatobiliary transport of [3H]lithocholic acid 3-O-glucuronide and [3H]cholic acid 3-O-glucuronide was efficient (greater than 95% in 1 h) and comparable to that of [14C]taurocholic acid. Secretion of both glucuronides was impaired in GY rats (24% and 71% at 1 h), whereas that of taurocholate was similar to control values. However, recovery of the glucuronides in bile was nearly complete within 24 h; virtually no radioactivity was found in urine. In control rats, biliary secretion of lithocholic acid 3-O-glucuronide, but not that of cholic acid 3-O-glucuronide or taurocholate, could be delayed by simultaneous infusion of dibromosulphthalein. In mutant rats, dibromosulphthalein infusion was also able to inhibit secretion of cholic acid 3-O-glucuronide. [3H]Hydroxyetianic acid, a C20 short-chain bile acid, was secreted by control rats as a mixture of 20% carboxyl-linked and 80% hydroxyl-linked (3-O-)glucuronide; secretion was very efficient (99% in 1 h). In GY rats, secretion was drastically impaired (16% at 1 h and 74% over a 24-h period). Initially, the mutant secreted more carboxyl- than hydroxyl-linked glucuronide, but the ratio reached that of control animals after 24 h. The rates of formation of both types of hydroxyetianic acid glucuronide by hepatic microsomes from mutant rats were similar or even slightly higher than those of control microsomes. These findings indicate that bile acid 3-O-glucuronides, but probably not carboxyl-linked glucuronides, are secreted into bile by a transport system shared with organic anions such as conjugated bilirubin and dibromosulphthalein, but different from that for amino acid-conjugated bile acids.

Hypertension after Bilateral Kidney Irradiation in Young and Adult Rats

The mechanism of a rise in blood pressure after kidney irradiation is unclear but most likely of renal origin. We have investigated the role of the renin-angiotensin system and dietary salt restriction in the development of systolic hypertension after bilateral kidney irradiation in young and adult rats. Three to 12 months after a single X-ray dose of 7.5 or 12.5 Gy to both kidneys of young and adult rats, the systolic blood pressure (SBP) and plasma renin concentration (PRC) were measured regularly. A single X-ray dose of 12.5 Gy caused a moderate rise in SBP and a slight reduction in PRC in both young and adult rats. A dose of 7.5 Gy did not significantly alter the SBP or PRC during the follow-up period of 1 year. In a second experiment, the kidneys of young rats received an X-ray dose of 20 Gy. Subsequently, rats were kept on a standard diet (110 mmol sodium/kg) or a sodium-poor diet (10 mmol sodium/kg). On both diets, SBP started to rise rapidly 3 months after kidney irradiation. Sodium balance studies carried out at that time revealed an increased sodium retention in the irradiated rats compared to controls on the same diet. In rats on a low sodium intake, there was neither a delay nor an alleviation in the development of hypertension. Compared to controls, the PRC tended to be lower in irradiated rats up to 4 months after irradiation. Subsequently, malignant hypertension developed in all 20 Gy rats, resulting in pressure natriuresis, stimulating the renin-angiotensin system. Our findings indicated that hypertension after bilateral kidney irradiation was not primarily the result of an activation of the renin-angiotensin system. Although there were some indications that sodium retention played a role, dietary sodium restriction did not influence the development of hypertension.

The late effects of radiation on the blood brain barrier

Rats were irradiated with 60 to 20 Gy in a single dose focussed to a volume of 0.5 cc in the center of the left hemisphere. Breakdown of the BBB was detected by (a) the presence of spikes on EEG after subcutaneous injection of Bicuculline methiodide, and (b) the presence of staining following Evans Blue dye injection. Breakdown of the BBB (mean and standard deviation) occurred in 38 46 of the 60 Gy rats at 98 ± 16 days, 7 11 of the 50 Gy rats at 128 ± 25 days, 11 18 of the 40 Gy rats at 162 ± 3 days, 5 11 of the 30 Gy rats at 178 ± 5 days and, 7 12 of the 20 Gy rats at 217 ± 7 days post irradiation. This study suggests that endothelial damage could be the principle mechanism mediating the late radiation syndrome.