A Model for Encephalomyosynangiosis Treatment after Middle Cerebral Artery Occlusion-Induced Stroke in Mice

Summary

The protocol aims to provide methods for encephalomyosynangiosis-grafting of a vascular temporalis muscle flap on the pial surface of ischemic brain tissue-for the treatment of non-moyamoya acute ischemic stroke. The approach's efficacy in increasing angiogenesis is evaluated using a transient middle cerebral artery occlusion model in mice.

Abstract

There is no effective treatment available for most patients suffering with ischemic stroke, making development of novel therapeutics imperative. The brain's ability to self-heal after ischemic stroke is limited by inadequate blood supply in the impacted area. Encephalomyosynangiosis (EMS) is a neurosurgical procedure that achieves angiogenesis in patients with moyamoya disease. It involves craniotomy with placement of a vascular temporalis muscle graft on the ischemic brain surface. EMS has never been studied in the setting of acute ischemic stroke in mice. The hypothesis driving this study is that EMS enhances cerebral angiogenesis at the cortical surface surrounding the muscle graft. The protocol shown here describes the procedure and provides initial data supporting the feasibility and efficacy of the EMS approach. In this protocol, after 60 min of transient middle cerebral artery occlusion (MCAo), mice were randomized to either MCAo or MCAo + EMS treatment. The EMS was performed 3-4 h after occlusion. The mice were sacrificed 7 or 21 days after MCAo or MCAo + EMS treatment. Temporalis graft viability was measured using nicotinamide adenine dinucleotide reduced-tetrazolium reductase assay. A mouse angiogenesis array quantified angiogenic and neuromodulating protein expression. Immunohistochemistry was used to visualize graft bonding with brain cortex and change in vessel density. The preliminary data here suggest that grafted muscle remained viable 21 days after EMS. Immunostaining showed successful graft implantation and increase in vessel density near the muscle graft, indicating increased angiogenesis. Data show that EMS increases fibroblast growth factor (FGF) and decreases osteopontin levels after stroke. Additionally, EMS after stroke did not increase mortality suggesting that protocol is safe and reliable. This novel procedure is effective and well-tolerated and has the potential to provide information of novel interventions for enhanced angiogenesis after acute ischemic stroke.

Introduction

Ischemic stroke is an acute neurovascular injury with devastating chronic sequelae. Most of the stroke survivors, 650,000 per year, in the US suffer from permanent functional disability¹. None of the available treatments confer neuroprotection and functional recovery after the acute phase of ischemic stroke. After an acute ischemic stroke, both direct and collateral blood supplies are diminished which leads to dysfunction of brain cells and networks, resulting in sudden neurological deficits^2,3. Restoration of blood supply to the ischemic region remains the foremost goal of stroke therapy. Thus, enhancing angiogenesis to promote blood supply in the ischemic territory is a promising therapeutic approach; however, previously studied methods for promoting post-stroke angiogenesis, including erythropoietin, statins, and growth factors, have been limited by unacceptable levels of toxicity or translatability⁴.

Encephalomyosynangiosis (EMS) is a surgical procedure that enhances cerebral angiogenesis in humans with moyamoya disease, a condition of narrowed cranial arteries that often leads to stroke. EMS involves partial detachment of a vascular section of the patient's temporalis muscle from the skull, followed by craniotomy and grafting of the muscle onto the affected cortex. This procedure is well tolerated and induces cerebral angiogenesis, reducing the risk of ischemic stroke in patients with moyamoya disease^5,6. Thus, the procedure serves largely a preventative role in these patients. The angiogenesis brought about by this procedure may also have a role in promoting neurovascular protection and recovery in the setting of ischemic stroke. This report supports the hypothesis that angiogenesis brought about by EMS has the potential to expand the understanding of and therapeutic options for cerebral ischemia.

Beside EMS, there are several pharmacological and surgical approaches to improve angiogenesis, but they have several limitations. Pharmacological approaches such as vascular endothelial growth factor (VEGF) administration has been found to be insufficient or even detrimental due to several limitations, including the formation of chaotic, disorganized, leaky, and primitive vascular plexuses, which resemble those found in the tumor tissues^7,8 and have no beneficial effects in clinical trials⁹.

Surgical approaches include direct anastomosis such as superficial temporal artery-middle cerebral artery anastomosis, indirect anastomosis such as encephalo-duro arterio-synangiosis (EDAS), encephalomyosynangiosis (EMS), and combinations of direct and indirect anastomosis¹⁰. All these procedures are very technically challenging and demanding in small animals, except for EMS. Whereas the other procedures require complex vascular anastomosis, EMS requires a relatively simple muscle graft. Moreover, the proximity of the temporalis muscle to the cortex makes it a natural choice for grafting, as it does not need to be completely excised or disconnected from its blood supply, as would be necessary if a more distant muscle were used for grafting.

EMS has been studied in chronic cerebral hypoperfusion models in rats^7,11. However, EMS using a temporalis muscle graft has never been studied in acute ischemic stroke in rodents. Here, we describe a novel protocol of EMS in mice after an ischemic stroke via middle cerebral artery occlusion model (MCAo). This manuscript serves as a description of methods and early data for this novel approach of EMS in mice after MCAo.

Protocol

All experiments were approved by the Institutional Animal Care and Use Committee of UConn Health and conducted in accordance with US guidelines. The following protocol should work in any species or strain of rodent. Here, 8- to 12-week-old, age- and weight-matched C57BL/6 wild-type male mice were used. Mice were fed standard chow diet and water ad libitum. Standard housing conditions were maintained at 72.3 °F and 30%-70% relative humidity with a 12 h light/dark cycle.

1. Pre-surgery preparation

1. Sterilize all instruments by autoclaving prior to surgery. Sanitize the operating surface with 70% ethanol and warm the operating surface to 37 °C with an electric heating pad.
2. Use an induction chamber to anesthetize the mouse with 4%-5% isoflurane for induction. Deliver 1.5%-2.0% isoflurane via nose cone for maintenance until the end of surgery. Ensure prior to surgery that the mouse is properly anesthetized by assessing the lack of a response to a firm hind foot pinch and loss of the postural reaction and righting reflex.
3. Place the mouse on its left side on the operating surface and apply eye ointment to protect both eyes.
4. Shave hair over the surgical field (i.e., right lateral cranium between eye and ear) with electric clippers. Clean the surgical field in concentric circles outward from the middle of the surgical site, with 70% ethanol followed by povidone solution, and repeat these steps 2x.
   NOTE: Due to the surgery site being close to the eye, removing 150% of the area surrounding a surgical site may not be possible to avoid irritation or accidental injury to the eye.
5. Administer a single dose of 0.25% bupivacaine (up to 8 mg/kg body weight) by subcutaneous injection as pre-operative analgesia at the site of surgery.
6. Set up a surgical microscope at 4x magnification. The microscope is used for all surgical steps.

2. Surgery procedure

NOTE: The surgery steps are presented in Figure 1. For this protocol, three mice were allocated to sham group, three mice for EMS alone, 12 mice for MCAo, and 23 mice for MCAo + EMS group.

1. MCAo surgery
   NOTE: MCAo is a well-characterized model of ischemic stroke in rodents, as described by us and others^12,13,14. The surgery steps are described in brief here. Focal transient cerebral ischemia was induced by a 60 min right MCAo under isoflurane anesthesia followed by reperfusion for 7 or 21 days.
   1. Make a midline ventral neck incision followed by unilateral right MCAo by advancing a 10-11 mm long 6.0 silicone rubber-coated monofilament from the internal carotid artery bifurcation via an external carotid artery stump. In sham mice, perform identical surgeries except for the advancement of the suture into the internal carotid artery.
   2. Measure rectal temperatures using a temperature control system, maintaining the temperature at ~37 °C during surgery with an automatic heating pad.
   3. Use laser Doppler flowmetry to measure cerebral blood flow prior to suture insertion by placing the Doppler probe against the lateral skull (corresponding to the MCA territory) and recording the value⁸. To confirm occlusion reduction to 15% of baseline cerebral blood flow, use the same procedure after the suture is advanced. To confirm reperfusion, use the same procedure after the suture is removed.
   4. Feed all animals with wet mash until sacrifice and/or 1 week after surgery to ensure adequate nutrition for chronic endpoints, as animals have rearing deficits after stroke.
2. EMS surgery
   1. After 60 min of MCAo, randomize mice into MCAo-only or MCAo + EMS groups. Perform EMS 4 h after MCAo (MCAo + EMS group) or sham surgery for select experiments (EMS-only group). Change into a new pair of sterile surgical gloves before the surgery.
      NOTE: The mice recovered from anesthesia after 60 minutes of MCAo and were re-anesthetized before the EMS surgery.
   2. For groups that receive EMS (MCAo + EMS or EMS-only groups), make a 10-15 mm skin incision with scissors, extending from 1-2 mm rostral to the right ear to 1-2 mm caudal to the right eye.
      NOTE: Sterile scissors were used to prevent accidental damage to the temporalis muscles underneath.
   3. Retract skin flaps using clamps and visually identify the temporalis muscle and the skull.
   4. Bluntly dissect the temporalis muscle away from the skull using scissors with a spreading technique. Perform a 2-3 mm myotomy directed ventrally along the caudal border of the muscle to facilitate ventral reflection.
   5. Perform a craniotomy ~5 mm in diameter at the skull underneath the reflected temporalis muscle using a micro drill.
   6. Remove the dura mater with tweezers to expose the pial surface of the brain. Use extreme caution to avoid accidental injury to the brain.
   7. Suture the dorsal border of the temporalis muscle to the subcutaneous tissue of the dorsal skin flap with 6-0 monocryl filaments, making it flush to the exposed cerebral cortex.
   8. Close the skin incision with 6-0 monofilament suture. Place the mouse back into its cage and monitor until recovery from anesthesia. Return the mouse to its housing facility.

3. Post-operative considerations

1. Monitor the mice for illness and the surgical site for infection daily. Give subcutaneous normal saline (1% volume by body weight) daily to support hydration.
2. Monitor for severe dehydration (loss of body weight >20%) until 7 days after surgery. Administer an additional bolus of subcutaneous normal saline 1% volume by body weight if >20% weight loss.
3. Proceed with injections, physiologic monitoring, and other testing without special considerations.
   NOTE: In this procedure, the use of opioids or non-steroidal anti-inflammatory drugs (NSAIDs) to treat post-surgery was avoided due to the known effects of these agents on stroke outcome or infarct size in consultation with in-house institutional animal care and use committee^15,16,17,18. However, the use of post-operative analgesia is highly encouraged for EMS surgery with other models. Please consult the Institutional Animal Care and Use Committee (IACUC) for this.

Representative Results

A total of 41 mice were used for this study. After three mortalities, one in MCAo and two in MCAo + EMS, a total of 38 mice were used for obtaining the results shown.

Statistics
Data from each experiment are presented as mean ± standard deviation (S.D.). Significance was determined using either unpaired student's t-test for comparing two groups or one-way ANOVA for more than two groups, with a Newman−Keuls post-hoc test to correct for multiple comparisons.

Nicotinamide adenine dinucleotide (reduced)-tetrazolium reductase (NADH-TR) staining
This staining was done to assess the long-term viability of the grafted muscle as in Turoczi et al.¹⁹. Briefly, at the time of sacrifice, the grafted muscle flap was carefully excised, fixed with 4% paraformaldehyde for 30 min, and cryopreserved in optimum cutting temperature (OCT) medium at -80 °C. Several 12 µm thick cryosections of temporalis muscle tissue were stained for NADH-TR enzyme-histochemical reaction. Slides were incubated for 30 min at 37 °C in a solution of nitroblue tetrazolium (1.8 mg/dL) and NADH (15 mg/dL) in 0.05 M Tris buffer (pH 7.6). Unused tetrazolium reagent was removed using increasing followed by decreasing concentrations of acetone. Quantitative assessment of NADH-tetrazolium-stained muscle was performed on muscle images taken at 40x magnification.

Immunostaining studies
Immunostaining was used to visualize muscle graft bonding with cortex and blood vessel density at the junction of muscle and cortex^20,21. For visualization of muscle bonding with brain tissue, mice that had undergone EMS surgery were used here. At the end of each respective time point, mice were anesthetized with an avertin injection (50 mg/kg body weight), followed by perfusion with 1x PBS containing 5 mM Ethylenediaminetetraacetic acid (EDTA) and fixation with 4% paraformaldehyde. The skull was carefully cut to prevent accidental detachment of temporalis muscle (TM) graft from brain cortex. TM graft above the brain cortex was then separated from the remaining temporalis muscle. The brain was carefully removed and post-fixed in 4% paraformaldehyde overnight. The fixed brain was then dehydrated with 30% sucrose in 1x PBSuntil the brain sunk to the bottom of the vial (approximately 1-3 days). Tissue sections of 30 µm size were cut with freezing microtome and mounted on slides.

For immunostaining of blood vessels in the ipsilateral brain cortex, MCAo and MCAo + EMS mice were sacrificed, perfused, fixed, and processed as above. Brain slices of 30 µm size were sectioned on a freezing microtome and mounted on a glass side. Antigen retrieval was done using citrate buffer (pH 6.0) and sections were incubated with blocking buffer followed by incubation overnight with primary antibodies, anti-alpha skeletal muscle actin 1:200, and Lectin-Dy594^21,22. Three coronal brain sections per mouse (n = 5 mice/group; total = 15 sections) were taken between 0.45 mm and 0.98 mm from bregma, stained, and visualized for quantification at 20x magnification at the junction of the ischemic core and penumbra regions. A blinded observer quantified lectin positive vessel density in the brain parenchyma using ImageJ software.

Muscle graft remains viable at 21 days after EMS
One prerequisite for success of this surgery is long-term viability of the grafted temporalis muscle. The TM graft showed transient damage of muscle cells at 7 days after surgery in grafted muscle vs. control muscle (71.32% muscle cell survival ± 16.64% vs. 97.19% ± 3.81%). However, this difference between grafted and the control muscle vanished, and muscles recovered completely 21 days after surgery (98.22% ± 3.965 vs. 96.87% ± 2.27%; Figure 2A).

Muscle grafts make loose bonds with brain tissue
Successful grafting of the temporalis muscle onto the brain cortex surface is a foremost requirement for the success of this model. In both the EMS + MCAo and EMS-only model, the temporalis muscle grafts adhered to the cortical surface 21 days after EMS, suggesting successful surgery, graft implantation, and bonding (Figure 1B and Figure 2B).

Blood vessel density increases in the perilesional cortex after EMS
Acute stroke leads to acute reduction in cerebral blood flow, impeded recruitment of collateral vessels, abnormal vascular sprouting, and dysfunctional angiogenesis, which contribute to poor stroke outcomes²³. EMS significantly increases blood vessel surface area and integrated density in perilesional cortex after stroke (p < 0.05 vs. MCAo-only; Figure 3).

Analysis of angiogenic and neuromodulating proteins
A mouse angiogenesis array was used to compare expression of angiogenic and neuromodulating proteins 7 days and 21 days after MCAo in MCAo-only vs. MCAo + EMS mice as per manufacturer's instructions²⁴. ImageJ software was used to quantify pixel density for each data point of the protein dot blot. Data were recorded as the ratio of the density of each analyzed protein to the averaged density of the standards for each blot.

Fibroblast growth factor (FGF)-acidic is upregulated and osteopontin is downregulated after EMS
Protein array results showed a significant increase in protein levels of FGF-acidic (0.677 ± 0.007 vs. 0.585 ± 0.014, p = 0.045), a potent angiogenic factor, and decrease in osteopontin levels, a multifunctional molecule expressed in inflammatory conditions (0.692 ± 0.007 vs. 0.758 ± 0.014, p = 0.048) in the MCAo + EMS group 21 days after stroke, suggesting improved angiogenesis and neuroprotection (Figure 4A).

Mortality outcomes for EMS after stroke
Both MCAo and EMS are invasive surgical techniques that may cause some mortality in mice. In this experiment, there was between 10%-11% mortality in mice 21 days after MCAo surgery, which is an accepted death rate for mice subjected to 60 min of MCAo¹⁴. Performing EMS on mice after MCAo did not increase mortality (Figure 4B) suggesting tolerance of EMS surgery even after MCAo.

Figure 1. Stepwise EMS procedure after middle cerebral artery occlusion (MCAo): (A) Step 1. A skin incision is made over the right middle cerebral artery territory. The skin and subcutaneous tissues are reflected, exposing the skull and temporalis muscle. Step 2. The temporalis muscle is dissected away from the skull and reflected ventrally. Step 3. A craniotomy is performed (4-5 mm) and the dura is gently removed. Step 4. The temporalis muscle is placed directly on brain surface to cover the exposed cortex. Step 5. The dorsal edge of the temporalis muscle is sutured to the subcutaneous tissue of the dorsal skin flap, flush with the brain surface. Step 6. The incision is closed, and the mouse is removed from anesthesia and returned to its cage.This part of figure has been modified from²⁵. (B) Conceptual schematic for encephalomyosynangiosis (EMS) treatment of MCAo-induced stroke. Abbreviations: FGF = Fibroblast growth factor. Please click here to view a larger version of this figure.

Figure 2. Immunostaining studies. (A) Temporalis muscle grafts maintain viability. Temporalis muscle grafts (EMS) on ischemic cortex tissue maintain high viability. (Left) Representative image of nicotinamide adenine dinucleotide (reduced)-tetrazolium reductase-stained muscle tissue cells from control (naïve muscle from contralateral side) and grafted muscle at 7 days after middle cerebral artery occlusion (MCAo) + encephalomyosynangiosis (EMS) surgery. Black arrow () shows damaged cells. (Right) Quantification of live/dead muscle cells. Muscle cells at 7 days after EMS show some mild damage (p < 0.1; t-test) that completely recovered at 21 days. (n = 5 mice/time points = total 10 mice in this group) Data are mean ± S.D. Scale bar = 20 µm. (B) Bonding of grafted temporalis muscle with brain cortex 21 days after EMS surgery. EMS tissues stained with anti-alpha skeletal muscle actin (green) and Lectin-Dy594 (red; blood vessel marker) antibody (n = 3 mice). Scale bar = 100 µm. Please click here to view a larger version of this figure.

Figure 3: Encephalomyosynangiosis (EMS) surgery increases blood vessel density in ischemic lesions 21 days after stroke. (A) Representative images of coronal brain sections from mice subjected to (left) middle cerebral artery occlusion (MCAo) or (Right) MCAo + EMS and stained with L. esculentum (Tomato) Lectin-Dy594, which binds to glycoproteins in the basal membrane of endothelial cells. Graphs are quantified areas. MCAo + EMS mice showed higher endothelium network using parameters viz. vascular fraction area (B) and integrated density (C). **p < 0.01 (unpaired t-test), while MCAo-only mice showed damage close to the ischemic lesion (dashed line). N = 5 mice/group= 10 mice total. Data are mean ± S.D. Scale bar = 100 µm. Abbreviations: Contra = contralateral side; Ipsi =ipsilateral side. Please click here to view a larger version of this figure.

Figure 4: Encephalomyosynangiosis modulates angiogenic proteins after stroke. (A) A mouse angiogenesis array (ARY015) was used to simultaneously assess the relative levels of 53 mouse angiogenesis-related proteins after middle cerebral artery occlusion (MCAo) and MCAo + EMS (day 21 after MCAo) in brain tissue lysates from the perilesional cortex. Quantitative analysis shows that EMS surgery significantly reduced osteopontin and increased fibroblast growth factor (FGF)-acidic protein after stroke (*p < 0.05 or **p < 0.01) vs ipsilateral MCAo. Data are mean ± S.D.; n = 3 mice/group/time point = total 15 mice. (B) EMS did not increase mortality after stroke (MCAo). Kaplan Meier survival curve shows that EMS + MCAO did not change post-stroke mortality vs. MCAO alone (p = 0.54). For EMS n = 3; for MCAo n = 11; and for MCAo + EMS n = 21. Please click here to view a larger version of this figure.

Discussion

This protocol describes a successful EMS procedure in a mouse model of MCAo-induced stroke. The data show that grafted tissue remains viable and can form bonds with brain cortex long after EMS surgery. These findings support the rationale for using a cerebral muscle graft to gradually develop a richly vascular trophic environment at the site of stroke. EMS is a promising therapy for potentially repairing infarcted cerebral tissue in the same environment.

The critical steps of the protocol include step 2.2.4: this step causes unavoidable trauma to the TM, which may reduce its ability to bond with the cortex and release trophic factors. Take care to limit TM trauma to the extent possible. An alternative strategy to reduce tissue trauma is to bluntly dissect the TM from the skull only at its dorsal border and forego the myotomy. In this case, the TM would be lifted away from the skull (rather than fully reflected), and the craniotomy would be performed with the craniotomy drill underneath the muscle. This reduces the amount of space available to perform this step but again may reduce TM trauma. Further, extreme care and practice is necessary in steps 2.2.5 and 2.2.6, for preventing injury to the underlying brain cortex during craniotomy and manipulation of the dura.

This EMS model is a natural adjunct to the well-established MCAo model. Because the MCAo model closely simulates the pathophysiology of ischemia and vascular network damage, that is common in human patients, the MCAo + EMS model is likely to have a high level of translatability to humans. The EMS model presented here is the first therapeutic intervention that has been studied for ischemic stroke in the preclinical setting that relies only on autologous tissue. Moreover, because the TM graft is organic and autologous, it may demonstrate paracrine signaling interactions with the adjacent injured brain that serve to regulate the release of trophic factors to optimum levels at various time points.

While stroke creates a proangiogenic environment and stimulates angiogenesis itself²⁶, the intrinsic post-stroke response is not sufficient to improve vascular supply in the damaged region due to subthreshold levels of angiogenic factors. Here, EMS further enhanced FGF-acidic protein expression compared to stroke-only animals. This protein indirectly controls neovascularization in concert with other growth factors. FGF-acidic also works as a neurotrophic factor, promoting neuroprotection and neurogenesis^27,28. Some of the neuroprotective effects of FGF-acidic are mediated by activation of AKT and MAPK/EPK pathways²⁹. In addition to FGF, there was also reduced expression of the protein osteopontin. Osteopontin is a pro-inflammatory, pleotropic cytokine that is increasingly being recognized for its role in multiple neuropathologies and tissue remodeling processes, among other functions. The role of osteopontin in stroke is still uncertain³⁰. However, recent studies in humans point to osteopontin as a poor prognostic factor after stroke. A decrease in serum osteopontin levels after stroke was shown in one study to predict favorable outcomes (modified Rankin scale score < 2 at 90 days) in human patients with stroke³¹. Another study showed a dose-dependent relationship between higher levels of plasma osteopontin and outcomes of death and disability in human patients after stroke³². In line with these clinical studies, the data here suggest that reduced osteopontin after EMS may promote an anti-inflammatory environment to increase neo-vessel formation. Overall, the differential expression of FGF-acidic and osteopontin points toward mechanisms governing the angiogenesis following EMS in this mouse model and increases the likelihood that the procedure that can also bring about neuroprotection and neuro-regeneration in addition to angiogenesis.

There are some potential limitations of this procedure. Measuring cerebral flow due to increased blood vessel density is challenging in this procedure, as commonly used procedures of laser Doppler or laser speckle flowmeter are affected by the presence of temporalis muscle on the top of cortex which prevent true blood measurement on cortical surface. Thus, this procedure may need more sophisticated, but rarely available, small rodent MRI scan if real time flow measurement is required. However, the use of blood vessel density measurement indirectly supports the success of EMS procedure in improving angiogenesis as suggested by our data. Another limitation is the invasive nature of EMS interventions on the top of MCAo, which itself is an invasive procedure. While there was no increased mortality with EMS in this study compared to MCAo only, the requirement for hemicraniectomy may limit its future translatability for all type of stroke. However, in clinical practice, >10% of patients with large ischemic stroke require hemicraniectomy to manage increased intracranial pressure²³, and this EMS model may have translational value for this subgroup of stroke patients in particular. Finally, the time point of 4 h post-MCAo for performance of EMS was chosen to fall within the standard treatment window of rT-PA for most human patients, though future studies will use later time points to evaluate the therapeutic window for EMS.

Overall, the EMS model provides a well-tolerated option for inducing angiogenesis after ischemic stroke, and in addition to its potential clinical translation, may be used in future studies examining the pathophysiology of stroke and angiogenesis.

The EMS model described here offers a safe method of achieving cerebral angiogenesis for preclinical study, obviating the need for pharmacologic interventions, which often lead to unwanted side effects or uncontrolled angiogenesis. Many patients with large ischemic strokes require a hemicraniectomy during their clinical course to manage increasing intracranial pressure. This EMS procedure, which also includes hemicraniectomy in mice for muscle grafting, may provide preclinical proof of concept for translational application of EMS in ischemic stroke. Therefore, this model has the potential to expand the knowledge of neurovascular recovery after an ischemic stroke and to facilitate the development of innovation, which are the need of the hour, in therapeutics for stroke survivors.

Materials

Name	Company	Catalog Number	Comments
6-0 monocryl suture	Ethilon	697G
70% ethanol to sanitize operating surface	Walgreens
Bupivacaine 0.25% solution	Midwest Vet
Clamps for tissue retraction	Roboz
Doccal suture with silicone coating	Doccal Corporation	602145PK10Re
Electric heating pad for operating surface
Isoflurane anesthesia	Piramal Critical Care Inc
Isoflurane delivery apparatus	B6Surgivet (Isotech 4)
Micro drill	Harvard Apparatus
Microdissecting tweezers, curved x2	Piramal Critical Care Inc
mouse angiogenesis panel arrat	R& D biotech	ARY015
Needle driver	Ethilon
Ointment for eye protection	Walgreens
Operating microscope	Olympus
Operating surface	Olympus
Povidone iodine solution	Walgreens
Rectal thermometer	world precison instrument
Saline or 70% ethanol for irrigation	Walgreens
Small electric razor to shave operative site	Generic
Surgical scissors	Roboz