Mapping In Vivo Tumor Oxygenation within Viable Tumor by 19F-MRI and Multispectral Analysis

AbstractQuantifying oxygenation in viable tumor remains a major obstacle toward a better understanding of the tumor microenvironment and improving treatment strategies. Current techniques are often complicated by tumor heterogeneity. Herein, a novel in vivo approach that combines 19F magnetic resonance imaging (19F-MRI)R1 mapping with diffusionbased multispectral (MS) analysis is introduced. This approach restricts the partial pressure of oxygen (pO2) measurements to viable tumor, the tissue of therapeutic interest. The technique exhibited sufficient sensitivity to detect a breathing gas challenge in a xenograft tumor model, and the hypoxic region measured by MS 19F-MRI was strongly correlated with histologic estimates of hypoxia. This approach was then applied to address the effects of antivascular agents on tumor oxygenation, which is a research question that is still under debate. The technique was used to monitor longitudinal pO2 changes in response to an antibody to vascular endothelial growth factor (B20.4.1.1) and a selective dual phosphoinositide 3-kinase/mammalian target of rapamycin inhibitor (GDC-0980). GDC-0980 reduced viable tumor pO2 during a 3-day treatment period, and a significant reduction was also produced by B20.4.1.1. Overall, this method provides an unprecedented view of viable tumor pO2 and contributes to a greater understanding of the effects of antivascular therapies on the tumor's microenvironment

A novel inhibitor of the alternative pathway of complement reverses inflammation and bone destruction in experimental arthritis

Complement is an important component of the innate and adaptive immune response, yet complement split products generated through activation of each of the three complement pathways (classical, alternative, and lectin) can cause inflammation and tissue destruction. Previous studies have shown that complement activation through the alternative, but not classical, pathway is required to initiate antibody-induced arthritis in mice, but it is unclear if the alternative pathway (AP) plays a role in established disease. Previously, we have shown that human complement receptor of the immunoglobulin superfamily (CRIg) is a selective inhibitor of the AP of complement. Here, we present the crystal structure of murine CRIg and, using mutants, provide evidence that the structural requirements for inhibition of the AP are conserved in human and mouse. A soluble form of CRIg reversed inflammation and bone loss in two experimental models of arthritis by inhibiting the AP of complement in the joint. Our data indicate that the AP of complement is not only required for disease induction, but also disease progression. The extracellular domain of CRIg thus provides a novel tool to study the effects of inhibiting the AP of complement in established disease and constitutes a promising therapeutic with selectivity for a single complement pathway

Effect of basic fibroblast growth factor on experimental focal ischemia studied by diffusion-weighted and perfusion imaging

BACKGROUND AND PURPOSE: Basic fibroblast growth factor (bFGF) has documented neuroprotective properties. This study was performed to evaluate the effects of bFGF on infarct size when administered 30 minutes after induction of focal cerebral ischemia in rats. Diffusion-weighted and perfusion MRI were used during the drug infusion. METHODS: We blindly randomized 20 Sprague-Dawley rats to receive either drug (n = 10) or vehicle (n = 10). The animals underwent middle cerebral artery (MCA) occlusion using the suture model. Diffusion-weighted MRI was initiated 30 minutes after induction of ischemia and repeated frequently for 3.5 hours. Drug (45 micrograms/kg per hour) or vehicle (saline) infusion began 30 minutes after MCA occlusion and continued for 3 hours. Perfusion images were made at 25, 90, and 150 minutes after MCA occlusion. The animals were killed after 24 hours of permanent MCA occlusion, and brains were stained with 2,3,5-triphenyltetrazolium chloride (TTC). RESULTS: The TTC-derived, corrected infarct volume postmortem in the bFGF-treated group was significantly smaller than that in controls (126.6 +/- 51.9 versus 180.2 +/- 54.9 mm3, mean +/- SD, P = .038). Diffusion imaging showed essentially equal lesion volumes 3 hours after MCA occlusion (195.4 +/- 61 mm3 in the drug-treated group and 194.4 +/- 65 mm3 in controls). At 4 hours, ischemic lesion size was 182.1 +/- 56.9 mm3 in treated animals and 222.9 +/- 88.7 mm3 in the controls (P = .24, NS). Perfusion imaging did not show a change of cerebral perfusion within ischemic brain regions in the bFGF group during the infusion. No behavioral or physiological side effects were observed. CONCLUSIONS: bFGF is a safe and effective treatment for focal cerebral ischemia in rats. We observed a modest delayed difference of ischemic lesion size in vivo with diffusion MRI. The diffusion-weighted MRI findings suggest a potential delayed therapeutic effect of bFGF, and the perfusion imaging findings imply that the effect is not due to increased blood flow to the ischemic region

Delayed treatment with an adenosine kinase inhibitor, GP683, attenuates infarct size in rats with temporary middle cerebral artery occlusion

BACKGROUND AND PURPOSE: Brain ischemia is associated with a marked increase in extracellular adenosine levels. This results in activation of cell surface adenosine receptors and some degree of neuroprotection. Adenosine kinase is a key enzyme controlling adenosine metabolism. Inhibition of this enzyme enhances the levels of endogenous brain adenosine already elevated as a result of the ischemic episode. We studied a novel adenosine kinase inhibitor (AKI), GP683, in a rat focal ischemia model. METHODS: Four groups of 10 adult Sprague-Dawley rats were exposed to 90 minutes of temporary middle cerebral artery (MCA) occlusion. Animals were injected intraperitoneally with vehicle, 0.5 mg/kg, 1.0 mg/kg, or 2.0 mg/kg of GP683 30, 150, and 270 minutes after the induction of ischemia by a researcher blinded to treatment group. The animals were euthanatized 24 hours after MCA occlusion, and brains were stained with 2,3,5-triphenyltetrazolium chloride. We measured brain temperatures in a separate group of 6 rats before and after administration of 1.0 mg/kg GP683. RESULTS: All treated groups showed a reduction in infarct volumes, but a significant effect was observed only in the 1.0 mg/kg-dose group (44% reduction, P=0.0077). Body weight, physiological parameters, neurological scores, and mortality did not differ among the 4 groups. No apparent behavioral side effects were observed. Brain temperatures did not change after drug injection. CONCLUSIONS: Our results indicate that the use of AKIs offers therapeutic potential and may represent a novel approach to the treatment of acute brain ischemia. The therapeutic effect observed was not caused by a decrease in brain temperature

A novel endothelin antagonist, A-127722, attenuates ischemic lesion size in rats with temporary middle cerebral artery occlusion: a diffusion and perfusion MRI study

BACKGROUND AND PURPOSE: Endothelins (ETs) are potent vasoconstrictors. Plasma ET levels increase during acute brain ischemia and may worsen the ischemic damage. Diffusion-weighted MRI (DWI) and perfusion imaging (PI) are powerful tools for evaluation of acute cerebral ischemia. We studied the effects of A-127722, a novel ET(A)-selective ET antagonist, on cerebral ischemic lesion size using 2,3,5-triphenyltetrazolium chloride (TTC) staining postmortem, on acute ischemic lesion development with DWI, and on the cerebral circulation using PI. METHODS: Twenty male Sprague-Dawley rats received either 5 mg/kg of A-127722 or vehicle (n=10 per group) intravenously 30 minutes and subcutaneously 4 hours after middle cerebral artery occlusion (MCAO). Whole-brain DWI and single-slice PI were done before initiation of treatment and repeated frequently thereafter up to 4 hours after MCAO. The animals were reperfused in the MRI scanner 90 minutes after the onset of MCAO. At 24 hours the animals were killed, and the brains were cut into six 2-mm-thick slices and stained with 2% TTC. Percent hemispheric lesion volume (%HLV) was calculated for each animal. RESULTS: Physiological parameters, body weight, neurological scores, and premature mortality (2 versus 2) did not differ between the two groups. No hypotension, abnormal behavior, or other adverse effects were seen. TTC-derived %HLV was 25.3+/-5.6% for controls and 16.2+/-9.6% for treated animals (36% reduction, PCONCLUSIONS: A-127722 significantly reduced ischemic lesion size in rats without observable adverse effects. It is not clear whether the effect was due to vasodilatation of collateral arterioles not detectable by PI or whether A-127722 has neuroprotective properties that are independent of vascular effects

A new method to improve in-bore middle cerebral artery occlusion in rats: demonstration with diffusion- and perfusion-weighted imaging

BACKGROUND AND PURPOSE: In-bore middle cerebral artery occlusion (MCAO) enables investigators to acquire preischemic MRI data and to image ischemic changes immediately after occlusion. We have developed a highly successful in-bore MCAO method. This study describes the methods and pertinent techniques. METHODS: Sixty-seven Sprague-Dawley rats were subjected to temporary (n=36) or permanent (n=31) MCAO. The occluding device consisted of a supporting tubing, a driving line, and a silicone-coated 4-0 nylon suture occluder. Outside the magnet, the occluder was positioned in the carotid canal. MCAO was achieved in the magnet bore by remotely advancing the driving line until resistance was felt. Diffusion-weighted imaging (DWI) and perfusion-weighted imaging (PWI) were acquired before and immediately after occlusion and were used to document the presence of MCAO. RESULTS: Fifty-nine (88.1%) rats were successfully occluded, demonstrating hyperintensity on DWI, perfusion deficits on PWI, and no subarachnoid hemorrhage at postmortem examination. The average values of the apparent diffusion coefficient in both the frontoparietal cortex and the lateral caudoputamen significantly decreased as early as 3 minutes after the onset of ischemia. The failures included preocclusion damage (1/67), sliding out of the occluder during occlusion (1/67), no occlusion (2/67), and arterial perforation (4/67). CONCLUSIONS: Our in-bore MCAO method is easily performed and is as successful as MCAO induced outside the magnet

Efficacy of intra-arterial and intravenous prourokinase in an embolic stroke model evaluated by diffusion-perfusion magnetic resonance imaging

OBJECTIVE: To explore the utility of intravenous (i.v.) prourokinase treatment, we compared intra-arterial (i.a.) and i.v. delivery in a rat embolic stroke model, using diffusion (DWI) and perfusion (PI) magnetic resonance imaging to assess in vivo effects on ischemic lesion evolution and reperfusion. BACKGROUND: Thrombolytic therapy with recombinant tissue-type plasminogen activator (rt-PA) for acute ischemic stroke is useful during the initial hours after onset. Prourokinase is a novel thrombolytic agent with potential safety advantages in comparison to rt-PA. METHODS: Twenty-four male Sprague-Dawley rats were embolized with autologous blood clots into the middle cerebral artery territory and then randomly assigned at 30 minutes after embolization to a 2-hour bolus infusion with i.a. prourokinase, i.v. prourokinase, or vehicle. DWI and PI were performed before treatment and repeated during and at the end of the treatment. RESULTS: PI demonstrated that both i.a. and i.v. significantly improved the percentage of the ischemic hemisphere that was normally perfused when the 20-minute, pretreatment, and 145-minute after embolization time points were compared; in the control group, the hypoperfused volume increased over time. DWI disclosed that the ischemic lesion evolution slightly decreased in the i.a. group, remained stable in the i.v. group, and increased over time in the control group. Infarct volume by triphenyltetrazolium chloride (TTC) staining was significantly smaller in both treatment groups than controls. CONCLUSIONS: These results demonstrate that i.a. and i.v. therapy with prourokinase are equally effective in promoting reperfusion and inhibiting the development of focal ischemic injury in this rat embolic stroke model

Determination of focal ischemic lesion volume in the rat brain using multispectral analysis

Multispectral (MS) analysis was used to determine the ischemic lesion volume in the rat brain after permanent middle cerebral arterial occlusion. MS analysis used a four-dimensional MS model consisting of an estimate of the average apparent diffusion coefficient of water (ADC(av)), T2, proton density, and perfusion. Four classification methods were investigated: (a) multivariate gaussian (MVG); (b) k-nearest neighbor (k-NN); (c) k-means (KM); and (d) fuzzy c-means (FCM). MVG and k-NN classifiers are supervised methods requiring labeled training data to characterize the stroke lesion. Unsupervised classifiers (KM, FCM) do not require previous statistics or labeled training data, resulting in potentially greater clinical usefulness. All MS methods provided significant correlation with postmortem findings beyond the use of ADC(av) alone (partial correlation given the ADC(av) estimate: MVG, .66; k-NN, .75; KM, .68; FCM, .70). This study demonstrates that MS analysis provides an improved estimate of ischemic lesion volume over that obtained from ADC alone

Broad-spectrum cation channel inhibition by LOE 908 MS reduces infarct volume in vivo and postmortem in focal cerebral ischemia in the rat

Cation channels conduct calcium, sodium and potassium, cations that are likely deleterious in the evolution of focal ischemic injury. We studied the effects of a novel, broad-spectrum inhibitor of several cation channels, LOE 908 MS, on acute ischemic lesion development with diffusion-weighted magnetic resonance imaging (DWI) and on cerebral perfusion with perfusion imaging (PI) in vivo and on cerebral infarct size using 2,3,5-triphenyltetrazolium chloride (TTC) staining postmortem. A total of 18 male Sprague-Dawley rats underwent 90 min of middle cerebral artery occlusion (MCAO) and were randomly and blindly assigned to either LOE 908 MS or vehicle starting 30 min after inducing focal ischemia and continuing for 4 h. Whole-brain DWI and multislice PI were done before initiation of treatment and repeated frequently for the next 3.5 h. DWI-derived lesion volume at 4 h showed a significant difference in favor of the drug treated group (P=0.03), whereas PI-derived perfusion deficit volumes did not significantly differ between the groups. The postmortem infarct volume at 24 h was significantly attenuated in the treated group in comparison to controls (P=0.0001) and neurological score was significantly better in the treated group (P\u3c0.02). Blocking several distinct cation channels with LOE 908 MS significantly reduced infarct size and improved neurological outcome without observable adverse effects in this focal ischemia model

The role of spreading depression in focal ischemia evaluated by diffusion mapping

This study investigated the role of spontaneous and induced spreading depression (SD) on the evolution of focal ischemia in vivo. We induced focal ischemia in 12 rats using the middle cerebral artery suture occlusion (MCAO) method. Chemical stimulation of nonischemic ipsilateral cortex by potassium chloride application (KCl group; n = 7) and saline (NaCl group; n = 5) was performed at 15, 30, 45, and 60 minutes following MCAO, and SD was detected electrophysiologically. Ischemic lesion volumes assessed over 15-minute intervals, evaluated by continuous apparent diffusion coefficient (ADC) of water mapping, demonstrated that the ischemic region increased significantly during 15-minute time epochs with a single SD episode (36.5 +/- 12.9 mm3, mean +/- SD) or multiple SD episodes (39.8 +/- 22.3) compared with those without SD (13.9 +/- 11.5) (p = 0.0009). Infarct volume at postmortem 24 hours after MCAO was significantly larger in the KCl group, with more total SDs (237.8 +/- 13.8) than the NaCl group (190.5 +/- 12.6) (p = 0.0001). This study demonstrates that ischemia-related and induced SDs increase significantly ischemic lesion volume in vivo, supporting the hypothesis for a causative role of SD in extending focal ischemic injury