The rat as a model for studying noise injury and otoprotection.

A major challenge for those studying noise-induced injury pre-clinically is the selection of an animal model. Noise injury models are particularly relevant in an age when people are constantly bombarded by loud noise due to occupation and/or recreation. The rat has been widely used for noise-related morphological, physiological, biochemical, and molecular assessment. Noise exposure resulting in a temporary (TTS) or permanent threshold shift (PTS) yields trauma in peripheral and central auditory related pathways. While the precise nature of noise-related injuries continues to be delineated, both PTS and TTS (with or without hidden hearing loss) result in homeostatic changes implicated in conditions such as tinnitus and hyperacusis. Compared to mice, rats generally tolerate exposure to loud sounds reasonably well, often without exhibiting other physical non-inner ear related symptoms such as death, loss of consciousness, or seizures [Skradski, Clark, Jiang, White, Fu, and Ptacek (2001). Neuron 31, 537-544; Faingold (2002). Hear. Res. 168, 223-237; Firstova, Abaimov, Surina, Poletaeva, Fedotova, and Kovalev (2012). Bull Exp. Biol. Med. 154, 196-198; De Sarro, Russo, Citraro, and Meldrum (2017). Epilepsy Behav. 71, 165-173]. This ability of the rat to thrive following noise exposure permits study of long-term effects. Like the mouse, the rat also offers a well-characterized genome allowing genetic manipulations (i.e., knock-out, viral-based gene expression modulation, and optogenetics). Rat models of noise-related injury also provide valuable information for understanding mechanistic changes to identify therapeutic targets for treatment. This article provides a framework for selection of the rat as a model for noise injury studies.

MRI compatible MS2 nanoparticles designed to cross the blood-brain-barrier: providing a path towards tinnitus treatment.

Fundamental challenges of targeting specific brain regions for treatment using pharmacotherapeutic nanoparticle (NP) carriers include circumventing the blood-brain-barrier (BBB) and tracking delivery. Angiopep-2 (AP2) has been shown to facilitate the transport of large macromolecules and synthetic nanoparticles across the BBB. Thus, conjugation of AP2 to an MS2 bacteriophage based NP should also permit transport across the BBB. We have fabricated and tested a novel MS2 capsid-based NP conjugated to the ligand AP2. The reaction efficiency was determined to be over 70%, with up to two angiopep-2 conjugated per MS2 capsid protein. When linked with a porphyrin ring, manganese (Mn2+) remained stable within MS2 and was MRI detectable. Nanoparticles were introduced intracerebroventricularly or systemically. Systemic delivery yielded dose dependent, non-toxic accumulation of NPs in the midbrain. Design of a multifunctional MRI compatible NP platform provides a significant step forward for the diagnosis and treatment of intractable brain conditions, such as tinnitus.

Stimulation of otolith irregular fibers produces a rostro-caudal gradient in activity in the vestibular nuclear complex (VNC), but not the vestibulocerebellum (VeCb).

The vestibular system is important for posture, balance, motor control, and spatial orientation. Each of the vestibular end organs have specialized neuroepithelia with both regular and irregular afferents. In otolith organs, the utricle and saccule, afferents most responsive to linear jerk (jerk - derivative of acceleration) are located in the striola and project centrally to the vestibular nuclear complex (VNC) as well as the uvula and nodulus of the vestibulocerebellum (VeCb). The pattern of central neuronal activation attributed to otolith irregular afferents is relatively unknown. To address this gap, c-Fos was used as a marker of neuronal activity to map the distribution of active neurons throughout the rostro-caudal extent of the VNC and VeCb. Immunohistochemistry for c-Fos was performed to assess activation of VNC and VeCb neurons in response to a linear jerk stimulus delivered in the naso-occipital plane. Activated neurons were distributed throughout the VNC, including the lateral vestibular nucleus (LVe), magnocellular medial vestibular nucleus (MVeMC), parvocellular medial vestibular nucleus (MVePC), spinal vestibular nucleus (SpVe), and superior vestibular nucleus (SuVe). Notably, after stimulation, the MVePC exhibited the greatest number of c-Fos labeled nuclei. Significant increases in c-Fos labeling were found in mid-rostrocaudal and caudal regions of the VNC in the LVe, MVe, and SpVe. Additionally, c-Fos labeling was observed across all regions of the VeCb after jerk stimulation. Significant increases in the number of labeled nuclei were found throughout the rostro-caudal extent of the nodulus and uvula. However, jerk stimulated increases in activity for the paraflocculus were restricted to the caudal VeCb. The distribution of neuronal activity suggests that regions receiving the greatest direct otolith input exhibit the most substantial changes in response to otolith derived, irregular fiber stimulation. Highlights: Nuclei with descending projections (LVe, MVePC, and SpVe) demonstrated the greatest change in activity after naso-occipital jerk stimulation.Naso-occipital jerk stimulation preferentially activates caudal VNC neuronsNaso-occipital jerk stimulation activates neurons throughout the VeCbJerk stimulation in the naso-occipital plane has the greatest effects on activity in VNC and VeCb regions with the greatest inputs from afferents originating in gravity receptors.

Photopic visual input is necessary for emmetropization in mice.

It was recently demonstrated that refractive errors in mice stabilize around emmetropic values during early postnatal development, and that they develop experimental myopia in response to both visual form deprivation and imposed optical defocus similar to other vertebrate species. Animal studies also suggest that photopic vision plays critical role in emmetropization in diurnal species; however, it is unknown whether refractive eye development is guided by photopic vision in the mouse, which is a nocturnal species. We used an infrared mouse photorefractor and a high-resolution MRI to clarify the role of photopic visual input in refractive eye development in the mouse. Refractive eye development and form-deprivation myopia in P21-P89 C57BL/6J mice were analyzed under 12:12 h light-dark cycle, constant light and constant darkness regimens. Animals in all experimental groups were myopic at P21 (-13.2 ± 1.6 D, light-dark cycle; -12.5 ± 0.9 D, constant light; -12.5 ± 2.0 D, constant dark). The mean refractive error in the light-dark-cycle-reared animals was -0.5 ± 1.3 D at P32 and, and did not change significantly until P40 (+0.3 ± 0.6 D, P40). Animals in this group became progressively hyperopic between P40 and P89 (+2.2 ± 0.6 D, P67; +3.7 ± 2.0 D, P89). The mean refractive error in the constant-light-reared mice was -1.0 ± 0.7 D at P32 and remained stable until P89 (+0.1 ± 0.6 D, P40; +0.3 ± 0.6 D, P67; 0.0 ± 0.4 D, P89). Dark-reared animals exhibited highly hyperopic refractive errors at P32 (+5.2 ± 1.8 D) and became progressively more hyperopic with age (+8.7 ± 1.9 D, P40; +11.2 ± 1.4 D, P67). MRI analysis revealed that emmetropization in the P40-P89 constant-light-reared animals was associated with larger eyes, a longer axial length and a larger vitreous chamber compared to the light-dark-cycle-reared mice. Constant-light-reared mice also developed 4 times higher degrees of form-deprivation myopia on average compared to light-dark-cycle-reared animals (-12.0 ± 1.4 D, constant light; -2.7 ± 0.7 D, light-dark cycle). Dark-rearing completely prevented the development of form-deprivation myopia (-0.3 ± 0.5 D). Thus, photopic vision plays important role in normal refractive eye development and ocular response to visual form deprivation in the mouse.

Postmortem neuroimaging: Temporal and spatial sensitivity of manganese-enhanced magnetic resonance imaging (MEMRI) and impact of Mn2+ uptake.

Magnetic resonance imaging data collection and analysis have been challenges in the field of auditory neuroscience. Recent studies have addressed these concerns by using manganese-enhanced magnetic resonance imaging (MEMRI). Basic challenges for in vivo application of MEMRI in rodents includes how to set inclusion criteria for adequate Mn2+ uptake and whether valid data can be collected from brains postmortem. Since brain Mn2+ uptake is complete within 2-4 h and clearance can take 2-4 weeks, one assumption has been that Mn2+-enhanced R1 values continue to reliably reflect the degree of Mn2+-uptake for some indeterminate time after death. To address these issues, the impact of death on R1 values was determined in rats administered Mn2+ and rats that were not. Images of auditory nuclei were collected at fixed intervals from rats before and after death for up to 10 h postmortem. By taking a ratio of pituitary and muscle T1-W intensities (P/M), a reliable quantitative method for assessing adequate brain Mn2+ uptake was created and suggest that P/M ratios should be adopted to objectively measure the quality of the Mn2+ injection. Postmortem R1 values decreased in all brain regions in both the After Mn2+ and No Mn2+ groups. However, the time-course of postmortem changes in R1 was dependent on brain region and degree of Mn2+ uptake. Thus, postmortem R1 values not only differ after death, but vary with time and across brain regions. Postmortem R1 values in unfixed brain tissue, including the auditory nuclei, should be interpreted with caution.

Dopamine in Auditory Nuclei and Lemniscal Projections is Poised to Influence Acoustic Integration in the Inferior Colliculus.

Dopamine (DA) modulates the activity of nuclei within the ascending and descending auditory pathway. Previous studies have identified neurons and fibers in the inferior colliculus (IC) which are positively labeled for tyrosine hydroxylase (TH), a key enzyme in the synthesis of dopamine. However, the origins of the tyrosine hydroxylase positive projections to the inferior colliculus have not been fully explored. The lateral lemniscus (LL) provides a robust inhibitory projection to the inferior colliculus and plays a role in the temporal processing of sound. In the present study, immunoreactivity for tyrosine hydroxylase was examined in animals with and without 6-hydroxydopamine (6-OHDA) lesions. Lesioning, with 6-OHDA placed in the inferior colliculus, led to a significant reduction in tyrosine hydroxylase immuno-positive labeling in the lateral lemniscus and inferior colliculus. Immunolabeling for dopamine beta-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT), enzymes responsible for the synthesis of norepinephrine (NE) and epinephrine (E), respectively, were evaluated. Very little immunoreactivity for DBH and no immunoreactivity for PNMT was found within the cell bodies of the dorsal, intermediate, or ventral nucleus of the lateral lemniscus. The results indicate that catecholaminergic neurons of the lateral lemniscus are likely dopaminergic and not noradrenergic or adrenergic. Next, high-pressure liquid chromatography (HPLC) analysis was used to confirm that dopamine is present in the inferior colliculus and nuclei that send projections to the inferior colliculus, including the cochlear nucleus (CN), superior olivary complex (SOC), lateral lemniscus, and auditory cortex (AC). Finally, fluorogold, a retrograde tracer, was injected into the inferior colliculus of adult rats. Each subdivision of the lateral lemniscus contained fluorogold within the somata, with the dorsal nucleus of the lateral lemniscus showing the most robust projections to the inferior colliculus. Fluorogold-tyrosine hydroxylase colocalization within the lateral lemniscus was assessed. The dorsal and intermediate nuclei neurons exhibiting similar degrees of colocalization, while neurons of the ventral nucleus had significantly fewer colocalized fluorogold-tyrosine hydroxylase labeled neurons. These results suggest that several auditory nuclei that project to the inferior colliculus contain dopamine, dopaminergic neurons in the lateral lemniscus project to the inferior colliculus and that dopaminergic neurotransmission is poised to play a pivotal role in the function of the inferior colliculus.

Erythrocyte flow in choriocapillaris of normal and diabetic rats.

The choriocapillaris is a unique capillary bed that provides nutrients to the retinal photoreceptors. It changes anatomically in diabetes, but the impact of these changes on blood flow is unknown. In this study hemodynamic parameters in individual choriocapillaris vessels were compared in normal and diabetic rats. Three groups were studied: normal buffer-injected control rats, streptozotocin (STZ)-injected mildly hyperglycemic (STZ-MH) rats, and STZ-injected diabetic (STZ-D) rats. 7-8 weeks after STZ injection, the rats were anesthetized, and epifluorescent, intravital microscopy was used to record the flow of fluorescent red blood cells (RBC) in the choriocapillaris. Diameter, RBC flux, and RBC velocity were measured in 153 capillary pathways in five control rats, 98 pathways in four STZ-MH rats, and 153 pathways in seven STZ-D rats. There was no difference in capillary diameter among the groups. RBC flux and velocity were lower in the STZ-injected rats compared to the controls (p

Novel QUEST MRI In Vivo Measurement of Noise-induced Oxidative Stress in the Cochlea.

Effective personalized therapeutic treatment for hearing loss is currently not available. Cochlear oxidative stress is commonly identified in the pathogenesis of hearing loss based upon findings from excised tissue, thus suggesting a promising druggable etiology. However, the timing and site(s) to target for anti-oxidant treatment in vivo are not clear. Here, we address this long-standing problem with QUEnch-assiSTed Magnetic Resonance Imaging (QUEST MRI), which non-invasively measures excessive production of free radicals without an exogenous contrast agent. QUEST MRI is hypothesized to be sensitive to noise-evoked cochlear oxidative stress in vivo. Rats exposed to a loud noise event that resulted in hair cell loss and reduced hearing capability had a supra-normal MRI R1 value in their cochleae that could be corrected with anti-oxidants, thus non-invasively indicating cochlear oxidative stress. A gold-standard oxidative damage biomarker [heme oxidase 1 (HO-1)] supported the QUEST MRI result. The results from this study highlight QUEST MRI as a potentially transformative measurement of cochlear oxidative stress in vivo that can be used as a biomarker for improving individual evaluation of anti-oxidant treatment efficacy in currently incurable oxidative stress-based forms of hearing loss.

Publisher Correction: Novel QUEST MRI In Vivo Measurement of Noise-induced Oxidative Stress in the Cochlea.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Measurement of human choroidal melanoma xenograft volume in rats using high-frequency ultrasound.

PURPOSE: The purpose of this study was to test the hypothesis that the volume of primary orthotopic human choroidal melanoma xenografts can be quantified noninvasively in the same nude rat over time using a portable, high-resolution, high-frequency ultrasound (HF-US) system. METHODS: C918 human choroidal melanoma spheroids were implanted in the superior suprachoroidal space of 26 WAG/RijHsd-rnu nude rats. Fourteen rats were anesthetized 14 days after tumor implantation, and HF-US B-scan images of the tumor-bearing eye were captured at 250-mum intervals. Tumor areas were measured on each image and numerically integrated to calculate volume. Tumor volumes were also estimated from serial histologic sections in six rats. Twelve other rats were anesthetized and weighed every 4 to 5 days after implantation for 2 weeks, and HF-US B-scan image series were acquired for subsequent measurement of tumor volume. RESULTS: Tumors could be visualized as heterogeneous, relatively hyperechoic regions in the superior portion of the eye. These regions were verified as tumor by comparison with histologic sections, and histologic and HF-US volumes were highly correlated (r = 0.961; P = 0.002). For the determination of HF-US volume, the intraobserver variability was 9.7% +/- 5.1% (n = 8), and the coefficient of variation for multiple measurements was 12.1% +/- 6.8% (n = 12). Tumor volume could be repeatedly measured in the same rat every 4 to 5 days for 2 weeks without significant weight loss. CONCLUSIONS: HF-US is a safe, practical method to measure tumor volume in the same nude rat over time in this orthotopic xenograft model of human choroidal melanoma.

Direct demonstration of instabilities in oxygen concentrations within the extravascular compartment of an experimental tumor.

To test the hypothesis that temporal variations in microvessel red cell flux cause unstable oxygen levels in tumor interstitium, extravascular oxygenation of R3230Ac mammary tumors grown in skin-fold window chambers was measured using recessed tip polarographic microelectrodes. Red cell fluxes in microvessels surrounding pO2 measurement locations were measured using fluorescently labeled red cells. Temporal pO2 instability was observed in all experiments. Median pO2 was inversely related to radial distance from microvessels. Transient fluctuations above and below 10 mm Hg were consistently seen, except in one experiment near the oxygen diffusion distance limit (140 microm) where pO2 fluctuations were <2 mm Hg and median pO2 was <5 mm Hg. Vascular stasis was not seen in these experiments. These results show that fluctuations in red cell flux, as opposed to vascular stasis, can cause temporal variations in pO2 that extend from perivascular regions to the maximum oxygen diffusion distance.

Manganese-enhanced MRI of human choroidal melanoma xenografts.

PURPOSE: To test the hypothesis that the structure and function of an experimental human choroidal melanoma xenograft and neighboring non-tumor-bearing retina can be simultaneously assessed by using manganese-enhanced MRI (MEMRI). METHODS: Spheroids grown from the human choroidal melanoma cell line C918 were implanted in the superior suprachoroidal space of 11 WAG/Nij-rnu nude rats. Two weeks later, MRI data were collected 4 hours after intraperitoneal injection of saline or MnCl(2), an MRI contrast agent that can act as a biomarker of cellular demand for ions, such as calcium. The following parameters were measured: (1) tumor signal intensity, (2) inner and outer retinal signal intensity in non-tumor-bearing inferior retina, and (3) whole and inner retinal thickness of inferior retina. Separate MEMRI experiments were performed on spheroids in vitro after MnCl(2) exposure and washing. RESULTS: In vitro, spheroids exposed to MnCl(2) retained sufficient Mn(2+) to demonstrate contrast enhancement during MEMRI. In vivo, injection of MnCl(2) resulted in a 30% increase in tumor signal intensity compared with tumors in rats injected with saline (P < 0.05). In inferior retina of tumor-bearing eyes, outer retinal signal intensity increased by 17% relative to a similar region in control eyes (P < 0.05), but there was no change in the inferior inner retinal intensity. Total retinal thickness of the inferior retina in the tumor-bearing eyes increased by 8%, compared with that in the non-tumor-bearing eyes (P < 0.05). CONCLUSIONS: The present identification of regions of enhanced Mn(2+) uptake in choroidal melanoma and a somewhat unexpected edema and increased outer retinal ion demand in neighboring non-tumor-bearing retina highlights MEMRI as a potentially powerful method for noninvasively monitoring tumor progression and treatment response and efficacy.

Orthotopic human choroidal melanoma xenografts in nude rats with aggressive and nonaggressive PAS staining patterns.

PURPOSE: Choroidal melanoma is the most common primary ocular cancer among the adult population. Patient survival has been linked to the periodic acid-Schiff base (PAS)-positive vascular patterns in the tumors. The presence of PAS-positive loops or cross-linking parallel channels is a marker of an aggressive tumor. The purpose of this study was to develop new xenograft models of human choroidal melanoma that predictably demonstrate the PAS staining patterns associated with nonaggressive and aggressive tumors in humans. METHODS: Three human choroidal melanoma cell lines (C918, M619, and OCM-1) were used. C918 and M619 are considered aggressive, based on their ability to form PAS-positive channels in vitro. The nonaggressive OCM-1 cells do not form these channels. C918, M619, and OCM-1 spheroids were grown and implanted in the suprachoroidal space of 20, 17, and 16 WAG/RijHs-rnu nude rats, respectively. Tumors were grown for 1 to >4 weeks, and histology was performed to evaluate tumor growth and determine PAS labeling patterns. RESULTS: Growth of C918, M619, and OCM-1 xenografts were histologically verified in 20/20, 15/17, and 16/16 rats, respectively. PAS staining revealed loops and cross-linking parallel channels, typical of aggressive tumors in patients, in 90% of C918 and 100% of M619 xenografts. Only 4 of 16 OCM-1 xenografts showed PAS-positive loops. The rest showed no PAS staining or only perivascular staining, indicative of nonaggressive tumors. CONCLUSIONS: It is possible to grow human choroidal melanoma orthotopic xenografts in nude rats that reproduce the PAS staining patterns associated with aggressive and nonaggressive choroidal melanomas in patients.

Effect of longitudinal oxygen gradients on effectiveness of manipulation of tumor oxygenation.

The purpose of this study was to test the hypothesis that longitudinal O(2) gradients in tumor affect response to manipulation of oxygenation. Previously we showed that pO(2) is higher on the fascial than the tumor surface of the R3230Ac rat mammary carcinoma when growing in a dorsal skin-fold window chamber, reflecting a longitudinal oxygen gradient. Magnetic resonance angiography verified prior results: the fascial surface has arterioles and higher vascular density than tumor; and the tumor surface has no arterioles. Phosphorescence lifetime imaging was used to measure each surface hypoxic percentage (HP; percentage of pixels < 10 mm Hg) before and after administration of mannitol or glucose (1 g/kg, i.v.) followed by O(2) breathing. The fascial surface had a smaller HP (median = 2.72%) than tumor (median = 27.94%; P = 0.0002) at baseline. HP on the fascial surface was positively correlated with HP on the tumor surface (P = 0.0067). HP decreased on the fascial surface after either sugar + O(2) (mannitol P = 0.03; glucose P = 0.06; combined P = 0.002), but HP did not change on the tumor surface. Therefore, the tumor surface is refractory to improvement in pO(2) with this method. Additional refinements may be needed to improve pO(2) of analogous regions in larger tumors; mechanism-driven suggestions are provided.

Tumor-dependent kinetics of partial pressure of oxygen fluctuations during air and oxygen breathing.

The primary purpose of this study was to examine the kinetics of partial pressure of oxygen (pO2) fluctuations in fibrosarcoma (FSA) and 9L tumors under air and O2 breathing conditions. The overall hypothesis was that key factors relating to oxygen tension fluctuations would vary between the two tumor types and as a function of the oxygen content of the breathing gas. To assist in the interpretation of the temporal data, spatial pO2 distributions were measured in 10 FSA and 8 9L tumors transplanted into the subcutis of the hind leg of Nembutal-anesthetized (50 mg/kg) Fischer 344 rats. Recessed-tip oxygen microelectrodes were inserted into the tumor, and linear pO2 measurements were recorded in 50-microm steps along a 3-mm path, and blood pressure was simultaneously measured via femoral arterial access. Additionally, pO2 was measured at a single location for 90 to 120 minutes in FSA (n=11) or 9L tumors (n=12). Rats were switched from air to 100% O2 breathing after 45 minutes. Temporal pO2 records were evaluated for their potential radiobiological significance by assessing the number of times they crossed a 10-mm-Hg threshold. In addition, the data were subjected to Fourier analysis for air and O2 breathing. FSA and 9L tumors had spatial median pO2 measurements of 4 and 1 mm Hg, respectively. 9L had more low pO2 measurements < or =2.5 mm Hg than did FSA, whereas between 2.5 and 10 mm Hg this pattern was reversed. Pimonidazole staining patterns in FSA and 9L tumors supported these results. Temporal pO2 instability was observed in all experiments during air and O2 breathing. Threshold analyses indicated that the 10 mm Hg threshold was crossed 2 to 5 times per hour, independent of tumor type. However, the magnitude of 9L pO2 fluctuations was approximately eight times greater than FSA fluctuations, as assessed with Fourier transform analysis (Wilcoxon, P < 0.005). O2 breathing significantly increased median pO2 in FSA from 3 to 8 mm Hg (P < 0.005) and caused a significant increase in frequency and magnitude of pO2 fluctuations. One hundred percent O2 breathing had no effect on 9L tumor pO2, and it decreased the magnitude of pO2 fluctuations with borderline significance. These results show that these two tumors differ significantly with respect to spatial and temporal oxygenation conditions under air and O2 breathing. Fluctuations of pO2 of the type reported herein are predicted to significantly affect radiotherapy response and could be a source for genetic instability, increased angiogenesis, and metastases.

Reduction of wound angiogenesis in patients treated with BMS-275291, a broad spectrum matrix metalloproteinase inhibitor.

PURPOSE: The purpose of this study was to evaluate the feasibility of incorporating a novel wound angiogenesis assay into a Phase I study of BMS-275291, a broad-spectrum matrix metalloproteinase inhibitor, and to determine whether the wound angiogenesis assay was able to detect the inhibition of angiogenesis in patients treated with BMS-275291. EXPERIMENTAL DESIGN: Before treatment began, a 4-mm skin biopsy was performed. The wound was imaged for 14 days. Treatment was started on day 0, and a separate 4-mm biopsy was performed 14 days later. The second wound was also imaged for 14 days. Wound angiogenesis was scored by two independent observers who were blinded to treatment status. RESULTS: The median times in days (95% confidence interval) to reach the target average vascular score (AVS) of 1.5 and 2.0 based on the data of Observer 1 were 3.7 (2.2-6.9) and 8.0 (5.0-10.0) pretreatment whereas on-treatment the values were 4.9 (3.7-8.0) and 9.3 (7.0-11.5), respectively. The delay in the median time to reach an AVS of 1.5 was 1.2 days or a 32% reduction when comparing pretreatment with on-treatment (P = 0.06). For the target AVS of 2.0 the delay in the median time pretreatment versus on-treatment was 1.3 days or a 16% reduction (P = 0.04). CONCLUSIONS: The wound angiogenesis assay used in this study was practical, well tolerated, and reproducible. Delays in wound angiogenesis because of BMS-275291 were detectable with this assay. This technique warrants additional investigation in clinical trials of other antiangiogenic agents.

Hemodynamic parameters in blood vessels in choroidal melanoma xenografts and rat choroid.

PURPOSE: Choroidal melanoma is the most common primary ocular cancer among the adult population. To avoid enucleation, there has been a concerted effort to develop therapies that spare the affected eye and the patient's vision. Blood flow helps shape the tumor's microenvironment, plays a key role in the tumor's response to many different types of therapy, and is necessary for delivery of chemotherapeutic agents. To rationally design new therapies and optimize existing treatments, it is essential to learn as much as possible about blood flow and the microcirculation in this tumor. In recent years, much has been discovered about the anatomy of the microvasculature and the dynamics of overall blood flow in choroidal melanoma, but little is known about the factors that determine microvascular blood flow. In this study hemodynamic parameters in individual microvessels of a human choroidal melanoma xenograft were compared with those same parameters in a normal rat choroid. METHODS: Nude, athymic WAG/RijHs-rnu rats were used in this study. The human choroidal melanoma cell line OCM-1 was used to grow solid tumors subcutaneously in the flanks of donor rats. Small pieces of these tumors were then implanted into the choroidal space of recipient rats. After 6 to 8 weeks, the rats were anesthetized with a subcutaneous injection of urethane, and the sclera was exposed. Rhodamine-labeled liposomes and red blood cells (RBCs) labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) were injected intravenously. Epifluorescent, intravital microscopy was used to visualize the flow of fluorescent RBCs through individual vessels in the choroid or tumor. Flow through multiple vessels was recorded on videotape for later analysis. From the recordings, RBC flux, RBC velocity (V(c)), and microvascular hematocrit (HCT(m)) were determined. Similar experiments were performed in rats with no tumor growth, and these same parameters were calculated in normal choroidal vessels. RBC flow was characterized in 55 vessels in six OCM-1 tumors and in 153 choroidal vessels in five non-tumor-bearing rats. RESULTS: RBC flux was higher in larger tumor vessels (>30 micro m in diameter) compared with similarly sized choroidal vessels. There were no differences in the velocities of RBCs through the two types of vessels. HCT(m) was significantly higher in medium-sized (>20 micro m in diameter) and larger tumor vessels compared with normal choroidal vessels. CONCLUSIONS: These experiments demonstrate differences between hemodynamic parameters in normal choroidal microvessels and microvessels in choroidal melanoma in this animal model. Because HCT(m) is a key determinant of apparent viscosity, abnormally high HCT(m) in the tumor vessels would increase vascular resistance and decrease flow. This could have a negative impact on the tumor oxygen levels and on the ability to deliver drugs effectively. On the contrary, higher local HCT(m) has also been shown to increase oxygen delivery. The impact and interplay of these two effects on tumor oxygenation remain to be determined.

Modeling human choroidal melanoma xenograft growth in immunocompromised rodents to assess treatment efficacy.

PURPOSE: To evaluate potential treatments of primary uveal melanoma in rodent xenograft models, it is necessary to track individual tumor growth during treatment. Previously, high-frequency ultrasound (HF-US) was used to measure tumor volume in nude rats for up to 2 weeks. This study tests the hypothesis that HF-US can be used to repeatedly measure tumor volume for at least a month in both nude rat and severe combined immunodeficiency (SCID) mouse xenograft models of human uveal melanoma, with the goal of modeling tumor growth to evaluate treatment efficacy. METHODS: C918 human uveal melanoma spheroids were implanted in the choroids of six nude rats and six severe combined immunodeficiency mice. OCM-1 human uveal melanoma spheroids were implanted in six nude rats. Every 4-7 days thereafter for up to 5 weeks, HF-US images of the tumor-bearing eye were captured every 100 or 250 µm. Tumor areas were measured on each image and integrated to calculate volume. Tumor growth was modeled using a logistic curve, and parameters characterizing growth, including the time to reach a target volume (t(T)), were evaluated as potential measures of treatment efficacy. RESULTS: Tumor volume could be measured for up to 5 weeks in all models, and the logistic curve described the growth well. The parameter t(T) was shown to be a suitable endpoint to evaluate treatments. CONCLUSIONS: HF-US is a practical method to track uveal melanoma growth in the same nude rat or SCID mouse for up to a month. Such growth data can be used to evaluate treatments in these xenograft models.

Human tumor cell proliferation evaluated using manganese-enhanced MRI.

BACKGROUND: Tumor cell proliferation can depend on calcium entry across the cell membrane. As a first step toward the development of a non-invasive test of the extent of tumor cell proliferation in vivo, we tested the hypothesis that tumor cell uptake of a calcium surrogate, Mn(2+) [measured with manganese-enhanced MRI (MEMRI)], is linked to proliferation rate in vitro. METHODOLOGY/PRINCIPAL FINDINGS: Proliferation rates were determined in vitro in three different human tumor cell lines: C918 and OCM-1 human uveal melanomas and PC-3 prostate carcinoma. Cells growing at different average proliferation rates were exposed to 1 mM MnCl(2) for one hour and then thoroughly washed. MEMRI R(1) values (longitudinal relaxation rates), which have a positive linear relationship with Mn(2+) concentration, were then determined from cell pellets. Cell cycle distributions were determined using propidium iodide staining and flow cytometry. All three lines showed Mn(2+)-induced increases in R(1) compared to cells not exposed to Mn(2+). C918 and PC-3 cells each showed a significant, positive correlation between MEMRI R(1) values and proliferation rate (p≤0.005), while OCM-1 cells showed no significant correlation. Preliminary, general modeling of these positive relationships suggested that pellet R(1) for the PC-3 cells, but not for the C918 cells, could be adequately described by simply accounting for changes in the distribution of the cell cycle-dependent subpopulations in the pellet. CONCLUSIONS/SIGNIFICANCE: These data clearly demonstrate the tumor-cell dependent nature of the relationship between proliferation and calcium influx, and underscore the usefulness of MEMRI as a non-invasive method for investigating this link. MEMRI is applicable to study tumors in vivo, and the present results raise the possibility of evaluating proliferation parameters of some tumor types in vivo using MEMRI.

Uptake rate of cationic mitochondrial inhibitor MKT-077 determines cellular oxygen consumption change in carcinoma cells.

OBJECTIVE: Since tumor radiation response is oxygen-dependent, radiosensitivity can be enhanced by increasing tumor oxygenation. Theoretically, inhibiting cellular oxygen consumption is the most efficient way to increase oxygen levels. The cationic, rhodacyanine dye-analog MKT-077 inhibits mitochondrial respiration and could be an effective metabolic inhibitor. However, the relationship between cellular MKT-077 uptake and metabolic inhibition is unknown. We hypothesized that rat and human mammary carcinoma cells would take up MKT-077, causing a decrease in oxygen metabolism related to drug uptake. METHODS: R3230Ac rat breast adenocarcinoma cells were exposed to MKT-077. Cellular MKT-077 concentration was quantified using spectroscopy, and oxygen consumption was measured using polarographic electrodes. MKT-077 uptake kinetics were modeled by accounting for uptake due to both the concentration and potential gradients across the plasma and mitochondrial membranes. These kinetic parameters were used to model the relationship between MKT-077 uptake and metabolic inhibition. MKT-077-induced changes in oxygen consumption were also characterized in MDA-MB231 human breast carcinoma cells. RESULTS: Cells took up MKT-077 with a time constant of ∼1 hr, and modeling showed that over 90% of intracellular MKT-077 was bound or sequestered, likely by the mitochondria. The uptake resulted in a rapid decrease in oxygen consumption, with a time constant of ∼30 minutes. Surprisingly the change in oxygen consumption was proportional to uptake rate, not cellular concentration. MKT-077 proved a potent metabolic inhibitor, with dose-dependent decreases of 45-73% (p = 0.003). CONCLUSIONS: MKT-077 caused an uptake rate-dependent decrease in cellular metabolism, suggesting potential efficacy for increasing tumor oxygen levels and radiosensitivity in vivo.