Translational Orthotopic Models of Glioblastoma Multiforme

Summary

Here, we describe a preclinical orthotopic mouse model for GBM, established by intracranial injection of cells derived from genetically engineered mouse model tumors. This model displays the disease hallmarks of human GBM. For translational studies, the mouse brain tumor is tracked by in vivo MRI and histopathology.

Abstract

Genetically engineered mouse (GEM) models for human glioblastoma multiforme (GBM) are critical to understanding the development and progression of brain tumors. Unlike xenograft tumors, in GEMs, tumors arise in the native microenvironment in an immunocompetent mouse. However, the use of GBM GEMs in preclinical treatment studies is challenging due to long tumor latencies, heterogeneity in neoplasm frequency, and the timing of advanced grade tumor development. Mice induced via intracranial orthotopic injection are more tractable for preclinical studies, and retain features of the GEM tumors. We generated an orthotopic brain tumor model derived from a GEM model with Rb, Kras, and p53 aberrations (TRP), which develops GBM tumors displaying linear foci of necrosis by neoplastic cells, and dense vascularization analogous to human GBM. Cells derived from GEM GBM tumors are injected intracranially into wild-type, strain-matched recipient mice and reproduce grade IV tumors, therefore bypassing the long tumor latency period in GEM mice and allowing for the creation of large and reproducible cohorts for preclinical studies. The highly proliferative, invasive, and vascular features of the TRP GEM model for GBM are recapitulated in the orthotopic tumors, and histopathology markers reflect human GBM subgroups. Tumor growth is monitored by serial MRI scans. Due to the invasive nature of the intracranial tumors in immunocompetent models, carefully following the injection procedure outlined here is essential to prevent extracranial tumor growth.

Introduction

Glioblastoma (GBM; grade IV glioma) is the most common and malignant brain tumor, and current therapies are ineffective, resulting in a median survival of 15 months¹. Reliable and accurate preclinical models that represent the complex signaling pathways involved in brain tumor growth and pathogenesis are essential to expedite the progress in evaluating new therapeutic regimens for GBM. Mouse models in which human brain tumor cell lines are implanted subcutaneously in immunocompromised mice do not reflect the native immune environment of brain tumors, nor can they be used to evaluate the ability of therapeutics to cross the blood-brain barrier². Ideally, preclinical mouse models should also reproduce closely the human GBM histopathology, including the high level of invasiveness into the surrounding parenchyma³. Although genetically engineered mouse (GEM) models develop tumors in the context of an intact immune system, complicated breeding schemes are often required, and tumors may develop slowly and inconsistently⁴. GEM-derived allograft models are better suited for preclinical therapeutic studies, where large cohorts of tumor-bearing mice are needed in a shorter time frame.

In a previous report, we described an orthotopic GBM mouse model derived directly from GEM tumors. Tumorigenesis in the GEM is initiated by genetic events in cell populations (primarily astrocytes) expressing glial fibrillary acidic protein (GFAP), that result in progression to GBM. These TRP GEMs harbor a TgGZT121 transgene (T), which expresses T121 after exposure to the GFAP-driven Cre recombinase. T121 protein expression results in the suppression of Rb (Rb1, p107, and p103) protein activity. Co-expression of a GFAP-driven Cre transgene (GFAP-CreERT2) targets expression to adult astrocytes after induction with tamoxifen. TRP mice also harbor a Cre-dependent mutant Kras (Kras^G12D; R) allele, to represent activation of the receptor tyrosine kinase pathway, and are heterozygous for the loss of Pten (P)^5,6. Concurrent gene aberrations in the receptor tyrosine kinase (RTK), PI3K, and RB networks are implicated in 74% of GBM pathogenesis⁷. Therefore, the primary signaling pathways altered in human GBM are represented by the engineered mutations in TRP mice, in particular GBM tumors, in which shared downstream targets of RTKs are activated⁵.

The GEM-derived syngeneic orthotopic model was validated as a model that recapitulates features of human brain tumors, including invasiveness and the presence of subtype biomarkers, for use as a platform to evaluate cancer therapeutics targeting aberrant pathways in GBM. Cells were cultured from tumors harvested from TRP brains and re-implanted in the brain of strain-matched mice, using stereotactic equipment for intracranial injection in the cortex. This preclinical orthotopic mouse model developed GBM tumors that were highly cellular, invasive, pleomorphic with a high mitotic rate, and displayed linear foci of necrosis by neoplastic cells and dense vascularization, as observed for human GBM. Tumor volumes and growth were measured by in vivo magnetic resonance imaging (MRI).

In this report, we describe the optimal technique for the intracranial injection of primary GBM cells or cell lines into the wild-type mouse brain, using TRP tumors as an example. The same protocol may be adapted for immunocompromised mice and other GBM cell lines. Crucial tips are given for avoiding common pitfalls, such as suboptimal cell preparation or cell leakage at the injection site, and for using the stereotactic equipment correctly to ensure model reproducibility and reliability. For translational purposes, we validate the model by MRI detection of brain tumor growth in live animals, histological characterization, and present an example of treatment in tumor-bearing mice.

Protocol

The study protocol described here was approved by the NCI at Frederick Animal Care and Use Committee. NCI-Frederick is accredited by AAALAC International and follows the Public Health Service Policy for the Care and Use of Laboratory Animals. Animal care was provided in accordance with the procedures outlined in the “Guide for Care and Use of Laboratory Animals (National Research Council, 2011; The National Academies Press, Washington D.C.).

1. Preparation of cells for injection

NOTE: Mouse brain tumor primary cells (MBRs) used for this model were originally isolated from tamoxifen induced TRP GEM mice, as described in El Meskini et al.⁵. Details on the cell preparation can be found in this reference.

1. Perform the following steps using sterile technique in a biosafety cabinet.
2. Culture primary brain tumor cells in vitro until they reach the exponential growth phase.
3. Harvest the cells in 0.25% trypsin, and once they have detached, dilute them with growth medium to inactivate the trypsin.
4. Pellet the cells by centrifugation at 400 x g and wash with serum-free media. Repeat once prior to counting.
5. Count the cells manually or with an automated cell counter. Resuspend the cells in 5% sterile methylcellulose in 1x phosphate buffered saline (PBS) at the desired concentration, based on a final injection volume of 2 µL. The desired cell concentration may vary based on the cell line and brain tumor model. For GBM GEM TRP cells, we inject 2 µL of a 25 x 10⁶ cells/ml solution, or 50,000 cells.

2. Mouse strain

1. Breed or purchase an appropriate mouse strain that matches the strain background of the brain tumor cells. For TRP cells, use 9-week-old B6D2F1/J mice (from a cross between C57BL/6J [B6] females and DBA/2J [D2] males) as tumor cell recipients.

3. Setting up the surgical area

NOTE: All surgical steps are conducted using aseptic technique in a clean, sanitized environment. Scrubs and personal protective equipment, including a mask, should be worn by the surgeon. Surgical tools must be heat-sterilized prior to use.

1. Place surface protectors under the stereotaxic apparatus and on the work surface.
2. Attach the vinyl tubing from the anesthesia machine to the IN port of the gas anesthesia platform of the stereotaxic apparatus, and another tube to the OUT port.
3. Connect the digital display to a power source and to the apparatus.
4. Connect the micropump controller (Figure 1A) to a power source and attach the micropump (Figure 1A) to the manipulator arm of the apparatus (also see the manufacturer instructions).
5. Set the micropump to 5.6 nL/s for the 2,000 nL volume injection.
6. Turn on the hot bead sterilizer.
7. Plug the mouse heating pad into the temperature controller and connect to a power source. Place the plate on the stage platform. Set the plate temperature to 37 °C.
8. Backload a 30 G precision syringe, being careful not to introduce bubbles. Insert the plunger and attach the needle. Make sure to grip the needle by the hub only. Depress the plunger until a drop of the methylcellulose cell mixture dispenses through the needle. Clean the needle with a 70% alcohol preparation pad by carefully wiping the sides of the needle, or by placing a sterile gauze pad on the work surface and blotting it. Take care not to bend or break the needle.
9. Attach the precision syringe to the micropump and rotate manipulator arm away from the stage, to prevent syringe-needle displacement prior to placing the mouse on the stage.

4. Preparing the mouse for surgery

1. Anesthetize the mouse by placing it in the induction chamber with the isoflurane vaporizer set to 2.5%, or according to institutional guidelines. Turn on the isoflurane flow to the nosecone.
2. Transfer the mouse to the stereotaxic apparatus and secure to the nosecone, with the top teeth positioned onto the nosecone support (Figure 1B, 9). Tighten the knob on the nosecone to secure (Figure 1B). Ensure an appropriate level of anesthesia by performing a toe pinch to check for reflex.
   1. Monitor the ears, feet, and mucous membranes for color (pink) to ensure adequate oxygenation. Also, monitor the respiratory rate (an increase or decrease could indicate the need to adjust isoflurane levels).
3. Insert ear bars (Figure 1B) into both ears and tighten the knob to secure the head.
4. Apply eye ointment to lubricate the eyes while the mouse is under anesthesia.
5. Use curved forceps to pluck the hair on the mouse's head to an area about 150% larger than the planned incision, to ensure adequate aseptic conditions.
6. Inject 0.5-1 mg/kg buprenorphine SR analgesia subcutaneously, or use another protocol-approved analgesic.
7. Position the mouse rectal probe to monitor internal temperature and prevent hypothermia due to anesthesia. Keep the mouse body temperature between 36.5 and 38.5 °C.
8. Sanitize the surgical field using outward circles, alternating between a surgical scrub and ethanol three times.
9. Using forceps to pull the skin taut, make an incision of approximately 1 cm with the scalpel blade, starting between the eyes. The bregma should be visible through the incision.
   NOTE: For proper sterile technique, touch the surgical site only with the tips of sterilized tools, and place tool tips down on a sterile surface only (such as the inside of an autoclaved tool pack).
10. Use the wooden end of cotton-tipped applicator to scrape away excess connective tissue, and then the cotton end of another applicator to dry.

5. Cell injection

1. Return the manipulator arm with the syringe attachment over the mouse, tightening the knob to secure. Use the X and Y knobs in the horizontal plane to move the syringe mount over bregma. Lower the needle using the Z knob to confirm the bregma position. Set the digital readout console to zero.
2. Use the X and Y knobs and the corresponding digital readout to move the needle to the desired position. For the cortical cortex location, the appropriate coordinates are 3 mm posterior, 2 mm lateral right to the bregma, and 2 mm deep from the dura mater. Use the Z knob to move the needle to surface of skull.
3. Puncture a hole in the skull using a 1 mL syringe with an attached 25 G needle. Place the bevel of the needle toward the 30 G precision syringe and needle, and carefully rotate the manipulator arm to the side. Using thumb and finger, roll the needle back and forth slowly with gentle pressure until the needle tip just pierces the skull cap.
4. Use a cotton tipped applicator to dab any blood away from the needle hole. Replace the manipulator arm to the appropriate position with the loaded precision syringe needle above the hole. Align the tip of the needle with the hole, using the Z knob to lower the needle down to the brain dura, and set the digital readout console to zero.
5. Using the Z knob, lower the needle 1 mm, and then wait 1 min. Repeat until the desired depth is reached (2 mm as indicated).
   NOTE: The needle is lowered slowly to prevent additional damage to the surrounding brain tissue and back-flow of the cell solution.
6. Start the micropump, and then monitor to ensure that the pump stops when 2 µL has been injected. This process should take about 6 min at the indicated speed. Then, wait 1 min prior to moving the needle.

6. Removal of the needle and wound closure

1. Raise the needle 1 mm and then wait 1 min. Repeat until the needle is completely free from the skull.
2. If necessary, use a cotton-tipped applicator to dab any blood away from the injection site.
3. Using the wooden end of a cotton-tipped applicator, take a small piece of bone wax (~1 mm) and shape it into a cone. Place it into the opening in the skull, pushing the wax into the hole.
4. Heat forceps using a bead sterilizer and use them to melt remaining wax on the skull and smooth it.
5. Place roughly two drops of bupivacaine (anesthetic) solution into the incision and use forceps to pull the edges of skin together.
6. Pull the skin taut and place one or two wound clips to close the skin.
7. Place the mouse in a clean recovery cage on a heating pad in a draft-free area, and closely observe the mouse. Allow the mouse to wake up fully from the anesthesia, resuming normal activity, before returning it to regular housing.
8. Check on the mouse daily following the surgical procedure and administer pain management, according to institutional guidelines.
   1. If buprenorphine SR (Step 4.6) is used for analgesia, the sustained-release formula lasts for 72 h. Repeat the injection only if visible pain or discomfort is present after 72 h. When performed correctly, the mice recover well from the intracranial injections and additional analgesia is not needed.
   2. Remove the staples 7-10 days post-surgery.
9. Monitor tumor growth by live animal imaging (MRI).
   1. Euthanize the mice when humane endpoints are reached (i.e., if the animal loses 20% body weight or becomes hypothermic).
   2. Due to the invasive nature of these brain tumors, observe the mice for neurological symptoms, such as uneven gait, partial paralysis, spinning, or head tilt. Euthanize a mouse if any of these clinical signs of advanced tumor growth are observed.

Representative Results

Mice injected with brain tumor cells should be monitored daily for signs of tumor growth such as seizures, ataxia, or weight loss. Brain tumor growth may also be monitored by MRI scanning at regular intervals. Weekly MRI scans allow the visualization of increasing tumor burden within the brain and tumor volume measurements (Figure 1C). In particular, TRP tumors exhibit aggressive growth, and 3D tumor volumes are measurable by MRI within 2 to 3 weeks post-intracranial injection (with an average volume of 30 to 40 mm³). In one representative study, a cohort of brain tumor-bearing mice was divided into two groups for control versus radiation therapy; MRI tumor volume measurement revealed a lack of tumor growth suppression after radiation treatment (Figure 2A), resulting in no increase in survival for the treated mice (Figure 2B).

At necropsy, tumors may appear as a dark spot on the brain within a swollen right hemisphere (Figure 3A, middle panel), or in the case of larger tumors, as a raised, darkened region. For histopathology analysis, sagittal sectioning through the injection site region (Figure 3A) allows for optimal assessment of tumor growth and the extent of invasion into non-malignant brain tissue. GBM tumors in syngeneic models may grow aggressively and breach the skull by the terminal endpoint, which can be observed at necropsy. In contrast, extracranial growth soon after cell implantation likely indicates a leakage of cells during the injection, which can be observed on MRI scans (Figure 3B), or in a live mouse by a domed area on the head. The needle may have been removed from the injection site too quickly (see Section 6). Extracranial growth is confirmed as leakage at the injection site by histological assessment. 

In the TRP orthotopic model, histopathology confirmed the presence of grade IV astrocytoma/GBM, including recapitulation of the distinctive features observed in TRP GEM tumors such as pseudopalisading and necrosis (Figure 4). GBM neural stem cell and progenitor markers described for human GBM subclassification were evaluated by immunohistochemistry (Figure 5). Widespread expression of glial fibrillary acidic protein (GFAP) indicates a proliferative tumor of astrocytic progenitor origin, whereas Nestin and Sox-2 are established neuronal progenitor markers, and Olig-2 expression confirms the presence of cells of oligodendrocytic origin^5,7. All four markers are highly expressed in subtypes of human GBM⁸.

Figure 1: Intracranial implant apparatus setup and growth of TRP GBM tumors visualized by serial MRI. (A) Apparatus setup with different elements necessary for intracranial implant (1 to 11 described); specific mouse head placement and inhalation anesthesia unit is magnified in (B). Weekly imaging was performed on an MRI 3.0T clinical scanner with a custom-built four mouse SENSE array surface coil^5,9. Multi-slice T2-weighted turbo spin echo (T2w-TSE) sequence: (TR/TE (4437/100 ms), in plane resolution (0.12 x 0.15 mm), slice thickness (0.5 mm), SENSE acceleration factor (4) images were acquired in the axial plane to cover the entire mouse brain. Shown are coronal sections from tumors at 3, 4, and 5 weeks post-implant. A representative scale bar for MRI images is indicated in the bottom right (C). Please click here to view a larger version of this figure.

Figure 2: Representative efficacy study of an orthotopic GBM model with radiation treatment. (A) Three-dimensional tumor volumes calculated from MRI scans, corresponding to weeks 2, 3, and 4 post-tumor implants. The volumes of the brain tumors were measured through analysis of the MRI images. After uploading an MRI image into the ITK_SNAP program, the image contrast and the image intensity region filter were adjusted. Following the active contour initialization and image segmentation, the tumor volume was measured in cubic milimieters. Individual control (untreated) mice and mice treated by irradiation (3 Gy per day for 5 days for total of 15 Gy) are plotted. (B) Survival of radiation-treated mice compared to control. The survival percent was computed with graphing software (see Table of Materials) from a total of n = 11 untreated mice and n = 12 radiation-treated mice. Study mice were euthanized as humane endpoints were reached, based on our institutional guidelines. Please click here to view a larger version of this figure.

Figure 3: Tumor location in the brain at necropsy. (A) Correct location of tumor growth in the right mouse hemisphere. The left panel shows an example of a tumor within the brain from an MRI scan at study termination; the red arrow indicates the tumor location. The dissected mouse brain is shown within the skull (middle panel) and removed from the cranial cavity (right panel). In the middle panel, blue arrows indicate the needle impact of the injection site, and double-headed arrows indicate the recommended location of the brain sagittal cut for histology, splitting the right hemisphere into two parts. Both brain parts are embedded in paraffin in an "open book" fashion. The dashed line indicates the median longitudinal fissure separating the two brain hemispheres as a reference. In the right panel, the area enclosed by the dashed line represents the visible tumor region at necropsy. (B) An example of a brain tumor growing outside the mouse skull is shown in the MRI scan. A representative scale bar for both MRI images is shown in Figure 3A. Please click here to view a larger version of this figure.

Figure 4: Histopathology of orthotopic TRP GBM tumors recapitulates the hallmarks of human GBM. Hematoxylin and eosin (H&E) stained, sagittal sections of the entire brain were evaluated by an ACVP-board-certified veterinary pathologist, and the tumors were graded based upon the current World Health Organization (WHO) classification for human astrocytomas^10,11. Orthotopic GBM tumors are highly proliferative, invasive (I) and vascularized (V), and exhibit hallmarks of human GBM histopathology including necrosis (N) and pseudopalisading (P) by neoplastic cells¹². Dashed lines show magnification of the areas within the brain tumor and at the periphery, adjacent to normal brain tissue (top and bottom right, respectively). Antibodies and methods for immunohistochemistry are described in El Meskini et al.⁵. A scale bar for H&E images is shown for both low and high magnifications. Please click here to view a larger version of this figure.

Figure 5: Biomarkers of GBM expressed in the orthotopic model. Expression of the multipotent progenitor marker SOX-2 (SRY-Box transcription factor 2), neural progenitors Nestin and Olig-2, and the glial fibrillary acidic protein (GFAP) astrocytic marker indicate cellular heterogeneity, a common feature of human GBM^7,8. A representative scale bar for all IHC images is shown in Olig2 (Oligodendrocyte transcription factor 2;bottom right). Please click here to view a larger version of this figure.

Discussion

Preclinical models are essential for the evaluation of new therapeutic targets and novel treatment strategies in GBM. Genetically engineered mouse models for GBM have the advantage of tumor occurrence in the autochthonous site, but often with a long latency and unpredictable tumor growth¹³. The GEM model tumors exhibit a latency of 4-5 months, and the ideal time window for imaging, recruitment, and treatment is variable among individual mice. The orthotopic model has a well-established and tractable growth and treatment timeline of 4-5 weeks, and tumors are reliably detected by MRI after implant. The use of GEM-derived orthotopic models allows for placement of the tumor in the exact location in each mouse, as well as temporal control of tumor growth initiation, while maintaining the advantage of the surrounding brain microenvironment. In the TRP orthotopic model, activated RTK, PTEN, and RB pathways produce a tumor model that recapitulates the histological features of human GBM. Analogous to human GBM, orthotopic TRP GBM tumors display features of aggressive growth, such as necrotic foci surrounded by pseudopalisading neoplastic cells and neovascularization. Tumors are also invasive into the surrounding brain parenchyma. Therefore, agents that may suppress tumor infiltration and migration in patients can be evaluated preclinically in TRP orthotopic tumors. The process of injecting cells from invasive GEM tumors into syngeneic mice requires particular caution, to avoid the unintended leakage and growth of cells outside of the targeted region. The method described here, when routinely practiced, can be used to generate large cohorts of tumor-bearing mice with consistent tumor growth.

Critical steps in the protocol that may impact successful tumor injection include removal of the needle from the brain post-cell injection and the use of bone wax. The needle must be inserted and removed 1 mm at a time (as described in steps 5 and 6), to prevent additional damage to the surrounding brain tissue and the leakage of residual cells or backflow of the injected suspension¹⁴. The use of melted bone wax to plug and seal the needle hole creates a barrier to prevent cell leakage, as does implanting cells in a methylcellulose suspension. Importantly, methylcellulose must be mixed in a buffered solution such as PBS (or alternatively, serum-free media) prior to resuspending the brain cells. Additionally, the use of inhalation anesthesia is preferred over injectable anesthesia, to shorten the recovery time.

Note that head size may differ slightly between mouse strains, or by age and sex; thus, the stereotactic equipment may need to be re-calibrated when changing from one mouse cohort type to another. We also recommend optimization of the cell number for injection for each cell line of interest in a small number of mice before proceeding with a large cohort. Compared to human xenografts, murine tumors may grow aggressively when re-implanted in strain-matched mice and require fewer cells for injection, as we observed with TRP lines.

A key advantage of orthotopic brain injection compared to the initiation and growth of glioblastoma in a GEM model is that the tissue surrounding the injected tumor is normal, and local invasiveness may be assessed by histopathology. However, we cannot rule out changes in the microenvironment due to the injection wound itself, which possibly result in a local inflammatory response or perturbation of the blood-brain barrier.

In conclusion, orthotopic models allow for the study of GBM, with a reasonable timeline for treatment and within the context of the native tumor location. Our model recapitulates human GBM features but in an immunocompetent mouse; therefore, the immune microenvironment may be analyzed relative to tumor growth, or in response to immunotherapy. Tumor growth is aggressive and invasive, unlike many cell-line xenograft GBM models in which the tumor remains encapsulated and does not exhibit the histological features of human GBM^13,15,16. When performed correctly, the procedure described here results in reliable tumor growth without cell leakage or extracranial spread.

Lastly, although this protocol has been optimized in the context of GBM as an orthotopic mouse model, its accuracy and reliable application in preclinical translation can be adapted to models of other brain tumor subclasses. Methods and reagents in this protocol can be utilized for different cell type implants carrying mutations for the brain tumor model of interest (e.g., diffuse midline glioma¹⁷), with the adjustment of stereotaxic coordinates.

Materials

Name	Company	Catalog Number	Comments
5% methylcellulose in 1X PBS, autoclaved	Millipore Sigma	M7027
1mL Tuberculin Syringe, slip tip	BD	309659
6" Cotton Tipped Applicators	Puritan	S-18991
Adjustable stage platform	David Kopf Instruments	Model 901
Aerosol Barrier Tips	Fisher Scientific	02-707-33
Alcohol Prep Pads Sterile, Large - 2.5 x 3 Inch	PDI	C69900
B6D2  mouse strain (C57Bl/6J x DBA/2J)	Jackson Laboratory	Jax #10006
Bone Wax	Surgical Specialties	901
Bupivacaine 0.25%	Henry Schein	6023287
BuprenorphineSR	ZooPharm	n/a
Clear Vinyl Tubing 1/8ID X 3/16OD	UDP	T10004001
CVS Lubricant Eye Ointment	CVS Pharmacy	247881
Disposable Scalpels, #10 blade	Scalpel Miltex	16-63810
Gas anesthesia machine with oxygen hook-up and anesthesia box	Somni Scientific	n/a	Investigator may use facility standard equipment
Gas anesthesia platform for mice	David Kopf Instruments	Model 923-B
GraphPad Prism	Graphpad	Prism      9      version 9.4.1
Hamilton 30 g needle, ½ “, small hub, point pst 3	Hamilton	Special Order
Hamilton precision microliter syringe, 1701 RN, no needle 10 µL	Hamilton	7653-01
Hot bead sterilizer with beads	Fine Science Tools	18000-45
Invitrogen Countess 3 Automated Cell Counter	Fisher Scientific	AMQAX2000
IsoFlurane	Piramal Critical Care	29404
Isopropyl Alcohol Prep Pads	PDI	C69900
ITK_SNAP (Version 36.X, 2011-present)	Penn Image Computing and Science Laboratory (PICSL) at the University of Pennsylvania, and the Scientific Computing and Imaging Institute (SCI) at the University of Utah
KOPF Small Animal Stereotaxic Instrument with digital readout console	David Kopf Instruments	Model 940
Masterflex Fitting, PVDF, Straight, Hose Barb Reducer, 1/4" ID x 1/8" ID	Masterflex	HV-30616-16
Mouse Heating Plate	David Kopf Instruments	PH HP-4M
Mouse Rectal Probe	David Kopf Instruments	PH RET-3-ISO
Nalgene Super Versi-Dry Surface Protectors	ThermoFisher Scientific	74000-00
P20 pipette	Gilson	F123600
Povidone Iodine Surgical Scrub	Dynarex	1415
Reflex 9 mm Wound Clip Applicator	Fine Science Tools	12031-09
Reflex 9 mm Wound Clip Remover	Fine Science Tools	12033-00
Reflex 9 mm Wound Clips	Fine Science Tools	12032-09
Semken forceps, curved	Fine Science Tools	11009-13
Temperature Controller	David Kopf Instruments	PH TCAT-2LV
Trypsin-EDTA (0.25%)	ThermoFisher Scientific	25200056
Tuberculin Syringe with 25g needle, slip tip	BD	309626
UltraMicroPump 3 with Micro2T Controller	World Precision Instruments	Model UMP3T