This manuscript describes the development of an animal model that allows for the direct testing of the effects of tumor hypoxia on metastasis and the deciphering the mechanisms of its action. Although the experiments described here focus on Ewing sarcoma, a similar approach can be applied to other tumor types.
Hypoxia has been implicated in the metastasis of Ewing sarcoma (ES) by clinical observations and in vitro data, yet direct evidence for its pro-metastatic effect is lacking and the exact mechanisms of its action are unclear. Here, we report an animal model that allows for direct testing of the effects of tumor hypoxia on ES dissemination and investigation into the underlying pathways involved. This approach combines two well-established experimental strategies, orthotopic xenografting of ES cells and femoral artery ligation (FAL), which induces hindlimb ischemia. Human ES cells were injected into the gastrocnemius muscles of SCID/beige mice and the primary tumors were allowed to grow to a size of 250 mm3. At this stage either the tumors were excised (control group) or the animals were subjected to FAL to create tumor hypoxia, followed by tumor excision 3 days later. The efficiency of FAL was confirmed by a significant increase in binding of hypoxyprobe-1 in the tumor tissue, severe tumor necrosis and complete inhibition of primary tumor growth. Importantly, despite these direct effects of ischemia, an enhanced dissemination of tumor cells from the hypoxic tumors was observed. This experimental strategy enables comparative analysis of the metastatic properties of primary tumors of the same size, yet significantly different levels of hypoxia. It also provides a new platform to further assess the mechanistic basis for the hypoxia-induced alterations that occur during metastatic tumor progression in vivo. In addition, while this model was established using ES cells, we anticipate that this experimental strategy can be used to test the effect of hypoxia in other sarcomas, as well as tumors orthotopically implanted in sites with a well-defined blood supply route.
Ewing sarcoma (ES) is an aggressive malignancy affecting children and adolescents.1 The tumors develop in soft tissues and bones, commonly in limbs. While the presence of metastases is the single most powerful adverse prognostic factor for ES patients, the mechanisms underlying their development remain unclear.2 Tumor hypoxia is one of the few factors implicated in ES progression. In ES patients, the presence of non-perfused areas within the tumor tissue is associated with poor prognosis.3 In vitro, hypoxia increases invasiveness of ES cells and triggers expression of pro-metastatic genes.4-6 However, despite these lines of evidence, no direct proof for hypoxia-induced ES progression and spread exists. Moreover, the mechanisms by which hypoxia exerts such effects are, at present, unknown. Hence, we have created an in vivo model to fill the gap between existing in vitro data and clinical observations. This model system enables direct testing of the effects of hypoxia on tumors occurring in their natural environment, using magnetic resonance imaging (MRI) to follow tumor progression and metastasis in vivo in combination with ex vivo pathological and molecular analyses (Figure 1).
Since no established transgenic model of ES is currently available, the in vivo studies on metastatic properties of these tumors rely on injections of human cells into immunocompromised mice. While the use of immunologically impaired animals may underestimate the impact of the immune system on the disease progression, the ability to use human cells increases translatability of such studies. Among different xenograft models, systemic injections into tail vein are the easiest to perform, yet they omit the initial steps of tumor cell intravasation and escape from the primary site of growth.7-12 On the other hand, orthotopic xenografting, which involves injections of tumor cells into bones (femur, rib) or muscles, is more technically challenging, but also more biologically relevant to human cancer.13-16 However, in the past, the high morbidity associated with rapid growth of primary tumors has often necessitated animal euthanasia before metastasis development. In this study, we employed a previously established model of cell injections into the gastrocnemius muscle followed by excision of the resulting primary tumor combined with longitudinal monitoring of metastatic progression by MRI.17,18 Such injections into gastrocnemius muscle in close proximity to the tibia allow for tumor growth in two natural ES environments — muscles and bones — and result in distant metastases to locations typically affected in humans.18 Thereby, this model accurately recapitulates the metastatic processes occurring in ES patients during disease progression.
The localization of primary tumors in the lower hindlimb also facilitates the precise control of the blood supply to the tumor tissue. Femoral artery ligation (FAL) is a well-established technique utilized in angiogenesis research to block blood flow to distal regions of the leg and investigate tissue vascularization in response to ischemia.19,20 Importantly, the initial drop in blood flow is followed by collateral vessel opening and tissue reperfusion observed approximately 3 days after FAL.20 Thus, when performed in a tumor-bearing limb, this model recreates hypoxia/reperfusion events that occur naturally in rapidly growing tumors and enables the escape of metastatic tumor cells due to restoration of perfusion to the lower hindlimb via newly opened collateral vessels.21 Importantly, this procedure must be performed when the tumor size is small enough to prevent excessive hypoxia in control tumors (typically at the tumor-bearing calf volume of 150 – 250 mm3), ensuring significant differences in levels of tumor hypoxia between control and FAL-treated groups.
In addition to longitudinal monitoring of the effect of hypoxia on ES latency and the frequency of metastases, this model also allows for the collection of tissues and the development of new cell lines from both primary tumors and metastases. Importantly, previous work established that metastases-derived cell lines exhibit enhanced metastatic potential upon reintroduction to animals, indicating that tumor dissemination is associated with permanent changes in the tumor cell phenotype, and thereby validating the use of these cell lines to decipher the metastatic processes.18 Collectively, these models can now be used for the genetic and molecular analyses required for identifying hypoxia-induced metastatic pathways.
As hypoxia is a pro-metastatic factor enhancing the malignancy of various tumors, our model can be used as a platform to investigate the role of hypoxia in other tumor types that naturally develop in limbs, such as osteosarcoma and rhabdomyosarcoma.21-23 Moreover, a similar approach can be applied to malignancies growing in other anatomical locations with a well-defined route of blood supply. Ultimately, the model can be modified and its utility further extended, depending on individual research needs.
All procedures were approved by the Georgetown University Institutional Animal Care and Use Committee.
1. Cell Preparation for Orthotopic Injections
2. Orthotopic Injection of ES cells into Gastrocnemius Muscle
3. Monitoring Primary Tumor Growth
4. Femoral Artery Ligation (FAL) for Inducing Hypoxia in the Tumor-bearing Hindlimb
5. Primary Tumor Excision by Leg Amputation
NOTE: Amputate the tumor-bearing lower hindlimb when the calf size reaches 250 mm3 for the control group or 3 days after FAL for the hypoxic group.
6. Monitoring Mice for the Presence of Metastases
7. Magnetic Resonance Imaging (MRI) for Detecting Metastases
8. Euthanasia and Necropsy
9. Primary Cell Culture
Following injection of ES cells into gastrocnemius muscle, the primary tumors are allowed to grow to a calf size of 250 mm3 (Figure 1, 2). The time necessary for the tumors to reach this volume typically ranges from 10 – 15 days for TC71 to 20-25 days for SK-ES1 xenografts, respectively. Tumors at a calf volume of 250 mm3 exhibit a relatively low level of endogenous hypoxia (approximately 3% of tumor tissue), based on hypoxybrobe-1 (pimonidazole) staining (Figure 4A, C). Importantly, in these control tumors, the positive staining for hypoxyprobe-1 was observed solely in the areas of physiological hypoxia, i.e., in tumor cells that are distant from the vasculature and start to undergo cell death (Figure 4A). Such staining exhibits a characteristic "air-brush" pattern, while the bulk of healthy tumor tissue remains negative for hypoxyprobe-1.6 In contrast, FAL performed at this stage creates profound hypoxia, as evidenced by the positive staining for hypoxyprobe-1 observed in the vast majority of the tumor cells at 4 hr post-FAL (Figure 4B). Notably, the hypoxyprobe-1-positive tumor cells are also observed in close proximity to blood vessels and the characteristic pattern of physiological hypoxia is lost. In these FAL-treated tumors, 73% of tissue consists of hypoxic tumor cells (Figure 4C). The tumor tissue also exhibits the first signs of hypoxia-induced damage, including cell shrinkage and loose structure (Figure 4B).
The effectiveness of FAL was further supported by the complete inhibition of primary tumor growth. During the 3-day period between FAL and amputation, the size of primary tumors did not increase (Figure 5A). In contrast, primary tumors from mice subjected to sham surgery continued to grow rapidly, reaching a volume of approximately 650 mm3, thus mimicking the growth rate of tumors in control mice not subjected to surgery (Figure 5A).18 In line with these data, histopathological analysis revealed extensive areas of necrosis in FAL-treated primary tumors harvested 3 days post-surgery (Figure 5B). Nevertheless, there were groups of viable tumor cells present within the tumor tissue, usually located at the edges of the tumor, close to the vasculature (Figure 5B). To investigate the oxygenation status of these cells we used hypoxyprobes, which are activated in live hypoxic cells, subsequently gaining the ability to covalently bind to proteins within these cells.29 As such, these probes serve as a permanent marker of the cells that experienced hypoxia at any point in time. Thus, to detect changes in the oxygen levels of the tumor cells throughout the duration of the experiment, we used two different hypoxyprobes that can be detected independently by immunohistochemistry. The probes were administered at two time points — immediately before FAL (hypoxyprobe-1) and prior to amputation (hypoxyprobe-2, CCI-103F) — and their localization was detected in tumors harvested 3 days post-FAL (Figure 1). Hypoxyprobe-1, which was present in the system at the time of FAL, marked a majority of the tumor cells, as the tissue became severely hypoxic (Figure 5C). This labelling is also evident in areas of necrosis/apoptosis, since these cells were still alive at the time of FAL and therefore capable of permanently binding hypoxyprobe-1, as shown in Figure 4B. In contrast, hypoxyprobe-2 administered 2 days post-FAL only stained viable cells, as the cells in necrotic areas were already dead and unable to activate the probe at the time of its administration (Figure 5D). Interestingly, we also observed viable tumor tissue adjacent to the vasculature that was initially hypoxic (hypoxyprobe-1-positive), but adequately oxygenated at the later time point (hypoxyprobe-2-negative) (Figure 5C, D). This finding is in agreement with previous reports indicating that collateral vessel-dependent tissue reperfusion restores blood flow in the hindlimb 3 days post-FAL.20 The viable tumor cells remaining in the tumor tissue 3 days post-FAL were highly invasive, as evidenced by their massive intravasation on vascularized edges of the tumor (Figure 5E). Some of the cells within the vessel lumen were positive for hypoxyprobe-1, indicating that they had become hypoxic upon FAL, yet remained viable. Collectively, these data suggest that tissue reperfusion following severe hypoxia may facilitate the escape of cells from the primary tumors.
Based on the pilot experiment, widespread metastases were detectable by MRI approximately 35 days post-amputation for SK-ES1 xenografts, while TC71 cells typically metastasized within 15 days. Consequently, for comparative analysis of metastasis latency and frequency, MRI was performed at day 15 and 35 post-amputation for the animals bearing SK-ES1 tumors, while one imaging at day 15 was sufficient for mice with TC71 tumors. Euthanasia was performed at day 50 and day 25 for the animals with SK-ES1 and TC71 xenografts, respectively. As previously reported, the most common types of metastases included soft tissue masses in the shoulder area, as well as bone metastases to contralateral legs, maxillary area and spine for SK-ES1 cells, and lung and pelvic tumors for TC71.18 In addition, frequent metastases to internal organs, including adrenal glands, lungs and liver, as well as bone marrow infiltration, were observed in mice bearing FAL-treated SK-ES1 xenografts (Figure 6). In general, hypoxia significantly increased the overall (SK-ES1) or site-specific (TC71) frequency and multiplicity of metastases in both cell lines. However, TC71 xenografts also developed frequent recurrent masses at the site of amputation (25% for control and 40% for FAL-treated tumors), as previously described for the large TC71 primary tumors.18
The tissues from control and FAL-treated primary tumors and metastases can also be a source of material for comprehensive molecular analyses aimed at the identification of hypoxia-activated pathways involved in ES dissemination. For example, we observed significant differences in metabolomic profiles of control and hypoxic primary tumors (data not shown). We were also able to successfully isolate multiple cell lines from all of the above tissues. In many cases, the cells derived from FAL-treated tumors exhibited noticeable changes in morphology, as compared to the original cells and those derived from control tumors (Figure 7A). The purity of these cell cultures was confirmed by positive immunostaining for the Ewing sarcoma marker, CD99 (Figure 7B). Moreover, 100% of cells in the established cell lines had a positive signal in fluorescence in situ hybridization (FISH) with human centromeric probes, often presenting with increased chromosome numbers (Figure 8A). The human origin of these cells was further confirmed by analysis of the metaphase chromosomes (Figure 8B) that exhibited cytogenetic rearrangements previously described for SK-ES1 cells in both the original cell line and cells derived from FAL-treated tumors (Figure 8B).30
Figure 1. Outline of the ES Hypoxia Model. ES cells (1 – 2 x 106) were injected into gastrocnemius muscles of SCID/beige mice. Once the resulting primary tumors reached a calf volume of 250 mm3, the mice were divided into two experimental groups. Mice from the control group were injected with hypoxyprobe-1 (HP-1), and the primary tumors were excised by limb amputation. Mice from the hypoxic group were injected with hypoxyprobe-1 and then subjected to femoral artery ligation (FAL). Two days later the mice were injected with hypoxyprobe-2 (HP-2), and then the tumor-bearing limbs were amputated three days post-FAL. Animals from both groups were monitored by periodic magnetic resonance imaging (MRI). Once metastases became apparent based on imaging and clinical signs, the mice were euthanized and the presence of metastases was confirmed by necropsy and histopathological analyses. The time of tumor growth, imaging and euthanasia is based on the SK-ES1 cell rate of growth and metastasis. Note that while hypoxyprobe-2 was necessary to establish the method and visualize changes in hypoxia levels during the period between FAL and limb amputation, its use is not essential for the actual experiments. Therefore, this step is not included in the protocol. Please click here to view a larger version of this figure.
Figure 2. Primary Tumor Growth. A. Magnetic resonance image (MRI) of an intact hindlimb. The red arrow indicates the site and direction of the tumor cell orthotopic injection. B. Primary tumor at a calf volume of approximately 250 mm3 growing in the gastrocnemius muscle (red outline). Red arrowheads indicate sites of bone destruction. T – tibia. Please click here to view a larger version of this figure.
Figure 3. The Site of Femoral Artery Ligation. A representative postmortem x-ray arteriogram of a 129S1/SvJ mouse perfused with barium sulfate immediately after ligation is shown to highlight the presence of pre-existing collateral vessels. Note that while the image depicts two ligations (Xs), one distal to the lateral circumflex femoral artery (LCFA) (red X) and another proximal to the genu artery (white X), the tumor hypoxia model only used the proximal ligation, near the inguinal ligament and distal to the LCFA (red X). The region of collateral vessel development is shown as described (faint vessels), and they are identified by their tortuous, cork-screw shape. Please click here to view a larger version of this figure.
Figure 4. Tumor Hypoxia Induced by Femoral Artery Ligation (FAL). A. A control tumor at a calf volume of 250 mm3 stained by hematoxylin & eosin (H&E) and immunostained for hypoxyprobe-1 (brown). Hypoxyprobe-1 immunostaining is solely observed in the areas of physiological tumor hypoxia, where the tumor cells distant from vasculature are deprived of oxygen and start to undergo cell death. B. FAL-treated primary tumor at a similar size harvested 4 hr post-ligation and stained with H&E and immunostained for hypoxyprobe-1. The majority of tumor cells are highly positive for hypoxyprobe-1, including those in close proximity to the vasculature. V – blood vessel. C. The area of positive staining for hypoxyprobe-1 was quantified in control and FAL-treated tumors using ImageJ software. Error bars represent SEM. *** – p < 0.001 Please click here to view a larger version of this figure.
Figure 5. Effect of Femoral Artery Ligation (FAL) on Primary Tumor Growth. A. Primary tumor growth during the 3-day period between surgery and amputation in FAL- (n = 4) and sham surgery-treated (n = 4) animals, as compared to intact control tumors (n= 6). Error bars represent SD. *** – p < 0.001, as compared to both control and sham surgery-treated tumors. B. Hematoxylin and eosin (H&E) staining of a FAL-treated tumor harvested 3 days post-ligation reveals widespread necrosis within tumor. The red outline indicates an area of viable tumor cells in close proximity to a highly vascularized region perfused 3 days post-FAL. C. FAL-treated tumor tissue harvested 3 days post-surgery immunostained for hypoxyprobe-1. Hypoxyprobe-1 was injected before FAL. Therefore, the positive immunostaining indicates the existence of extensive tissue hypoxia immediately post-surgery. D. Hypoxyprobe-2 was injected 2 days post-FAL, and the tissue collected 24h later, at amputation. Positive hypoxyprobe-2 staining identifies tissue hypoxia at the time of amputation. The red arrow indicates tissue that is hypoxic at both time points while the yellow arrow indicates an area adjacent to the vasculature that was hypoxic immediately after FAL (hypoxyprobe-1-positive), but negative for hypoxyprobe-2 at the time of excision, which is indicative of tissue reperfusion. The lack of hypoxyprobe-2 staining in necrotic areas suggests that the cells in this region were not viable at the time of its administration and therefore unable to actively bind the probe. The staining presented in panels B-D was performed on serial tissue sections. E. Intravasation in FAL-treated tumor 3 days post-FAL. The immunostaining identifies viable cells positive for hypoxyprobe-1 within the vessel lumen (red arrow). Please click here to view a larger version of this figure.
Figure 6. Examples of Metastases in Mice Bearing FAL-treated SK-ES1 Tumors. The metastases were detected by magnetic resonance imaging (MRI) and confirmed by necropsy and histopathological analyses (hematoxylin & eosin staining, H&E). M – metastasis. Please click here to view a larger version of this figure.
Figure 7. Characteristics of Cells Derived from FAL-treated Primary Tumors. A. Viable cell cultures were established from tissues harvested from control and hypoxic primary tumors and their morphology was compared to the original cell lines used for initial orthotopic injections. The cells derived from FAL-treated primary tumors exhibit dramatic changes in their sizes and morphology. Representative images of the original and primary tumor-derived SK-ES1 cells are shown. B. Representative images of cells derived from FAL-treated tumors immunostained for the ES marker, CD99, and counterstained with the DNA-binding dye, DAPI. All cells are CD99-positive, including hypertrophic cells containing enlarged nuclei (white arrow). Please click here to view a larger version of this figure.
Figure 8. Chromosomal Analysis of the Cells Derived from FAL-treated Tumors. A. Fluorescence in situ hybridization (FISH) analysis in interphase cells showing two nuclei with signals for human centromeric 4 probe. The large nucleus with four signals indicates a tetraploid (4n) cell (white arrow). B. FISH analysis in representative metaphases of the SK-ES1 original cell line and of the cell derived from FAL-treated primary tumor. Red arrows indicate inv(1)(p13.1q21), a non-random cytogenetic rearrangement characteristic of the SK-ES1 cells. Red signals: human centromeric 4 probe; Green signals in the original SK-ES1 cells: NPY5R probe on chromosome 4q32.2. Please click here to view a larger version of this figure.
Our model involves the comparison of metastasis in two experimental groups — a control group, where tumors are allowed to develop in the hindlimb followed by amputation upon reaching a calf volume of 250 mm3, and a hypoxia-exposed group, in which the tumor-bearing hindlimb is subjected to FAL at the same volume, followed by amputation 3 days later. Even though in these experiments the FAL-treated tumors are amputated with a slight delay, as compared to the control tumors, their size does not increase during the 3-day period between ligation and amputation. Therefore, this approach enables the comparison of metastatic potential between tumors of comparable sizes but with dramatically different levels of hypoxia. In contrast, using sham surgery, a commonly utilized control for surgical interventions, followed by 3 days of tumor growth would result in significant differences in tumor size between the experimental groups, with the sham surgery-treated primary tumors possessing significant levels of endogenous hypoxia. Based on the pilot experiments, sham surgery does not significantly affect tumor growth or its hypoxia levels and can thus be eliminated from the experimental design. Instead, the strategy of comparing tumors of similar sizes and different hypoxia levels is a highly relevant model for elucidating the effects of hypoxia on dissemination of the disease. Importantly, unlike previous in vitro studies, this approach allows for comprehensive evaluation of the pro-metastatic actions of hypoxia in a natural tumor microenvironment.4-6
The goal of the experimental design was to achieve a low basal level of metastases that would allow for the detection of a hypoxia-induced stimulatory effect on their formation. We established that a calf size of 250 mm3 at FAL and amputation was optimal for SK-ES1 primary tumors. However, this parameter will need to be optimized for each cell line, depending on their metastatic potential and local invasiveness. For tumors with higher metastatic potential, the primary tumor size at amputation may need to be decreased. For example, TC71 tumors exhibit a high local invasiveness manifested by frequent formation of recurrent tumors at the site of amputation.18 Once such masses develop, the animals have to be excluded from analyses, as it would not be clear if subsequent distant metastases originated from the primary tumors or from recurrent tumors that often grow rapidly and exhibit high levels of endogenous hypoxia. From previous experience, independent of the cell line used, 150 mm3 is the minimal tumor-bearing calf size that can be reliably measured and normalized. If decreasing the tumor size at FAL and amputation does not eliminate recurrent tumors or decrease the basal metastasis rate sufficiently, the particular cell line in question may not be suitable for these experiments.
Likewise, it is important to establish the timeline of MRI and euthanasia for each individual cell line, depending on the latency of its metastasis. Notably, the surgical wound clips that interfere with MRI can be removed no earlier than 10 days post-surgery, and this period must sometimes be extended due to complications with healing. Thus, in this study we established day 15 as the time of the first MRI.
Another important consideration in this model is maintaining a level of hypoxia capable of inducing metastatic processes without causing complete tumor necrosis, which would prevent tumor cell dissemination. Although we have not encountered this problem in this study, if needed, the severity of hypoxia may be regulated by altering the exact site and number of ligations in relation to the branches of the femoral artery, such as the LCFA and genu artery.20 Thus, a ligation proximal to the LCFA will create more severe ischemia in the hindlimb circulation, thereby increasing the hypoxic conditions in the distal leg. In contrast, ligating distal to subsequent branches of the femoral artery will decrease the resulting hypoxia in the lower hindlimb.
Importantly, there were no severe adverse effects of combined FAL and amputation procedures and no deaths related to the surgeries, as previously reported for these techniques performed separately.18-20 Thus, the success of the method is limited mainly by the rate of tumor take at the primary site and the frequency of recurrent tumors, both of which are cell line-dependent.
As hypoxia has been implicated as a pro-metastatic factor in many tumor types, this approach can also be suitable for directly testing its effects and defining its mechanisms of actions in other sarcomas that develop in limbs.23 Moreover, a similar strategy can be applied to other malignancies growing in orthotopic environments with a well-defined blood supply route. Thus, with appropriate modifications, this model can be a useful tool in research beyond the field of ES biology.
The authors have nothing to disclose.
This work was supported by National Institutes of Health (NIH) grants: UL1TR000101 (previously UL1RR031975) through the Clinical and Translational Science Awards Program, 1RO1CA123211, 1R03CA178809, R01CA197964 and 1R21CA198698 to JK. MRI was performed in the Georgetown-Lombardi Comprehensive Cancer Center’s Preclinical Imaging Research Laboratory (PIRL) and tissue processing in the Georgetown-Lombardi Comprehensive Cancer Center’s Histopathology & Tissue Shared Resource, both supported by NIH/NCI grant P30-CA051008. The authors thank Dan Chalothorn and James E. Faber, Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, for their assistance with postmortem x-ray angiography, and providing insight and expertise on collaterogenesis.
SK-ES1 Human Ewing sarcoma (ES) cells | ATCC | ||
TC71 Human ES cells | Kindly provided from Dr. Toretsky | ||
McCoy's 5A (modified) Medium | Gibco by Life Technologies | 12330-031 | |
RPMI-1640 | ATCC | 30-2001 | |
PBS | Corning Cellgro | 21-040-CV | |
FBS | Sigma-Aldrich | F2442-500mL | |
0.25% Trypsin-EDTA (1X) | Gibco by Life Technologies | 25200-056 | |
Penicillin-Streptomycin | Gibco by Life Technologies | 15140-122 | |
Fungizone® Antimycotic | Gibco by Life Technologies | 15290-018 | |
MycoZap™ Prophylactic | Lonza | VZA-2032 | |
Collagen Type I Rat tail high concetration | BD Biosciences | 354249 | |
SCID/beige mice | Harlan or Charles River | 250 (Charles River) or 18602F (Harlan) | |
1 mL Insulin syringes with permanently attached 28G½ needle | BD | 329424 | |
Saline (0.9% Sodium Chloride Injection, USP) | Hospira, INC | NDC 0409-7984-37 | |
Digital calipers | World Precision Instruments, Inc | 501601 | |
Surgical Tools | Fine Science Tools | ||
Rimadyl (Carprofen) Injectable | Zoetis | ||
Hypoxyprobe-1 (Pimonidazole Hydrochloride solid) | HPI, Inc | HP-100mg | |
hypoxyprobe-2 (CCI-103F-250mg) | HPI, Inc | CCI-103F-250mg | |
Povidone-iodine Swabstick | PDI | S41350 | |
Sterile alcohol prep pad | Fisher HealthCare | 22-363-750 | |
LubriFresh P.M. (eye lubricant ointment) | Major Pharaceuticals | NDC 0904-5168-38 | |
VWR Absorbent Underpads with Waterproof Moisture Barrier | VWR | 56617-014 | |
Oster Golden A5 Single Speed Vet Clipper with size 50 blade | Oster | 078005-050-002 (clipper), 078919-006-005 (blade) | |
Nair Lotion with baby oil | Church & Dwight Co., Inc. | ||
Silk 6-0 | Surgical Specialties Corp | 752B | |
Prolene (polypropylene) suture 6-0 | Ethicon | 8680G | |
Vicryl (Polyglactin 910) suture 4-0 | Ethicon | J386H | |
Fisherbrand Applicators (Purified cotton) | Fisher Scientific | 23-400-115 | |
GelFoam Absorbable Dental Sponges – Size 4 | Pfizer Pharmaceutical | 9039605 | |
Autoclip Wound Clip Applier | BD | 427630 | |
Stereo Microscope | Olympus | SZ61 | |
Autoclip remover | BD | 427637 | |
Aound clip | BD | 427631 | |
MRI 7 Tesla | Bruker Corporation | ||
Paravision 5.0 software | Bruker Corporation | ||
CO2 Euthanasia system | VetEquip | ||
25G 5/8 Needle (for heart-puncture) | BD | 305122 | |
0.1 mL syringe (for heart-puncture) | Terumo | SS-01T | |
K3 EDTA Micro tube 1.3ml | Sarstedt | 41.1395.105 | |
10% Neutral Buttered Formalin | Fisher Scientific | SF100-4 |