Fluorescent Detection of Vestibular Schwannoma Using Intravenous Sodium Fluorescein In Vivo

Abstract

Background:

Vestibular schwannoma (VS) are intracranial tumors caused by merlin deficiency. Sodium fluorescein (SF) is a fluorescent compound that accumulates in various intracranial tumors, causing tumors to emit green fluorescence after blue light excitation.

Hypothesis:

Intravenous SF preferentially deposits in VS, helping surgeons differentiate tumor from surrounding tissue.

Methods:

Merlin-deficient Schwann cells were grafted onto cochleovestibular nerves of immunodeficient rats. Rats were randomized to receive SF (7.5 mg/kg; n=5) or saline (n=3). Tissues were harvested at 1 hour and photographed in white and blue light. Sixteen surgeons identified and marked the tumor-tissue interfaces on images. Fluorescence was measured on tissue specimens using the IVIS® imaging system and on tissue cross-sections obtained with confocal microscopy. Western blot was performed to measure levels of organic anion transporting polypeptide (OATP), a drug transporter specific for SF.

Results:

Under blue light, tumors from SF rats demonstrated bright green fluorescence under direct visualization, higher fluorescence measurements on tissue specimens (p<0.001), and more SF deposition on tissue cross-sections (p<0.001), when compared to surrounding tissues and placebo rats. Surgeons were better able to distinguish the tumor-tissue interfaces in SF rats. Furthermore, the expression level of OATP1C1 was significantly higher in tumors than in surrounding tissues (p<0.0001).

Conclusion:

In a xenograft model of VS, intravenous SF preferentially deposits in tumors, compared to normal surrounding tissue. Under blue light, tumors emit an intense green fluorescence that can help surgeons differentiate tumor from critical structures nearby, which may improve clinical outcomes in complicated VS surgery.

Introduction

Vestibular schwannoma (VS) is a benign intracranial tumor of the cochleovestibular nerve known to cause facial paralysis, hearing loss, dizziness and life-threatening complications from compression of critical neurological structures. Mutations in the NF2 gene which encodes merlin protein are known to cause VSs. VSs occurs predominantly on a spontaneous basis (~95%) or as part of an inherited syndrome called Neurofibromatosis Type II (NF2; 5%). Approximately 3,300 cases of VS are diagnosed in the United States each year with an incidence of 1.09 cases per 100,000 individuals. [1] The incidence of VS has been rising and 1 in 1,000 individuals may develop VS in their lifetime. [2]

The main treatment options for VS include observation with tumor surveillance, radiation therapy, and microsurgical resection. The optimal treatment modality depends on a number of factors, including age, medical comorbidities, hearing, tumor size, and NF2 status. [3–6] Complete surgical removal of VS may not always be possible as identifying the tumor-nerve or tumor-brain interfaces is challenging in adherent tumors, which can result in incomplete tumor resection or risk injury to normal brain and nerve. Fluorescence-guided surgical resection of intracranial tumors can potentially improve gross total resection while preserving adjacent normal tissues.

Sodium fluorescein (SF) is a fluorescent compound that has preferential uptake in several intracranial tumors, which allows the tumors to emit a green fluorescence when excited by blue light. [7] SF is a low-cost drug with a good safety profile and was first used by George Moore in 1947 to differentiate normal non-fluorescent tissue from tumors of the brain and spinal cord that display intense fluorescence under ultraviolet light. [8,9] Throughout the last decade, neurosurgeons have utilized intravenous (IV) SF to better visualize malignant gliomas, thus improving gross total resection while minimizing morbidity by preserving adjacent normal brain. [7, 10, 11] Our in vitro data using mouse merlin-deficient Schwann cells (MDSC) and primary human VS cells from 6 patients showed a preferential uptake of SF in these tumor cells, compared to normal Schwann cells that ensheath adjacent neuronal axons. [12] MDSCs emitted significantly higher relative fluorescence units (RFUs) that increase with greater drug concentrations and cell densities, in comparison to normal Schwann cells that display only low levels of fluorescence. Using our validated Likert scale for fluorescence visualization, we also demonstrated that blinded observers were able to differentiate SF-treated normal Schwann cells from MD-SCs and primary human VS cells. These findings suggest that SF-guided VS surgery may improve detection of residual tumor and identification of the tumor-nerve interface, thereby improving surgical resection and reducing devastating neurological complications.

Uptake of SF by brain tumors is thought to be from extravasation through a leaky blood-brain barrier into the extracellular space of the tumor. [10] However, a recent study demonstrated that unbound SF can cross the BBB and also deposit in normal brain as well, thereby limiting the tumor-to-normal contrast during fluorescence-guided resection of gliomas. [13] Other investigations suggest that differential expression of organic anion transporting polypeptides (OATPs) may also be important for preferential SF uptake. [14, 15] OATPs are tissue specific transmembrane proteins that facilitate the transport of a variety of endogenous compounds and clinical drugs. [16] There are eleven human OATPs of which only a few have been studied in detail. Several of these human OATPs are naturally expressed in hepatocytes; however, recent studies have demonstrated that various tumors also express high levels of OATP. [17, 18]

We hypothesize that IV SF preferentially deposits in schwannomas, helping surgeons differentiate tumor from normal nervous tissue. In this study, we utilize an animal model of VS to: (1) determine if IV SF preferentially accumulates in tumors compared to normal adjacent tissues by measuring fluorescent intensities in gross tissue specimens and on histology, and (2) assess if surgeons can better identify the tumor-brain interface in blue light compared to white light conditions. Furthermore, we measured the expression levels of OATP1C1 drug transporter to determine if this may be a contributing factor for tumor fluorescence.

Materials and Methods

Animal Model

To create our xenograft model of VS, we implanted mouse MDSCs as previously described in Dinh et al. 2018. [19] The mouse MDSCs (i.e. MTC-10+ luciferase) were obtained from Dr. Fernandez-Valle, who developed this cell line by isolating Schwann cell from neonatal NF2^flox2/flox2 mouse sciatic nerves and transducing the cells with Cre recombinase adenovirus to express luciferase. [20] Tumors that developed in the xenograft model demonstrated S100 reactivity and histologic features of schwannoma (Antoni A/B and early Verocay bodies). The study was performed in accordance with the University of Miami Institutional Animal Care and Use Committee (Protocol 18–149). In brief, Rowett nude rats (Crl:NIH-Foxn1rnu) are t-cell deficient, athymic nude rats commonly used for tumor biology and xenograft research. Eight Rowett Nude rats were anesthetized with isoflurane and ~10⁵ MDSCs were grafted onto the cochleovestibular nerve. All animals were observed daily for adverse clinical signs that would require immediate intervention or euthanasia. Postoperatively, the rats received oral enrofloxacin (0.25 mg/mL in drinking water) for 7 days and triple antibiotic ointment daily to the surgical incision for 3 days. For pain management, animals received meloxicam sustained release (4 mg/kg) every 72 hours for a total of 2 doses.

To ensure that rats developed tumors, they were injected with intraperitoneal D-Luciferin (#122799; Perkin Elmer, Waltham, MA, USA), anesthetized with isoflurane, and underwent bioluminescence live imaging (BLI) using the Caliper/Xenogen IVIS® Spectrum (#12462, Perkin Elmer, Hopkinton, MA, USA) platform at 2 and 4 weeks after surgery. Because the implanted mouse MD-SCs express luciferase, rats that develop intracranial tumors will have detectable luminescence readings [i.e. total flux (photons/sec) and average radiance (photons/sec/cm2/sr)] on bioluminescence live imaging.

At 4 weeks, rats that demonstrated tumor formation were randomized to receive either IV SF (7.5 mg/kg; n=5, #1277004, United States Pharmacopeia, Rockville, MD, USA) or IV saline (n=3, Hospira, Lake Forest, IL, USA) by tail vein injection which circulated systemically for 60 minutes prior to harvesting of tissues.

Tissue Fluorescence

Tissues were harvested using a mid-sagittal craniotomy to expose the implanted tumors as well as the right and left brainstem and cerebellum. Fluorescence imaging using the IVIS® Spectrum platform (Perkins In Vivo Imaging System, Perkin Elmer, #12462, Hopkinton, MA, USA) was performed on gross tissue specimens to determine the total photon flux (photons/second), an objective measurement of tissue fluorescence. Five regions of interest were selected, including the tumor, ipsilateral brainstem, ipsilateral cerebellum, contralateral brainstem and contralateral cerebellum. Fluorescence measurements of the cochleovestibular and facial nerves were not performed because exposure for fluorescence imaging was not adequate to be able to obtain reliable measurements without simultaneously removing the tumor.

Tumor Brain Interface Identification

High resolution photos of mid-sagittal tissue specimens were taken using a trinocular stereomicroscope (AmScope, Irvine, CA, USA) with attached camera (HD200VP-UM, AmScope, Irvine, CA, USA) in white light and blue light conditions. Blue light conditions were created by using a microscope-mounted 450nm blue ring light (#HY-MG2-128W, Hayear, Futian, Shenzhen China) and photographed through a blue light barrier filter (Electron Microscopy Sciences, Hatfield, PA, USA). Sixteen Otolaryngology surgeons were then asked to view photos of tissue specimens on an iPad® (Apple, Cupertino, CA, USA) using an architecture drawing application (Morpholio Trace®, Raleigh, NC, USA) capable of calculating the surface area of hand drawn objects. Surgeons were asked to mark the tumor-brain interface in each condition by drawing the perceived border with a stylus. This was done using 6 different rat specimens (3 IV SF and 3 IV saline) in random order, first of photographs in white light and then photographs of the identical specimen in blue light.

Two calculations were performed: (1) the percentage of the tumor area correctly detected, and (2) the percentage of tissue area outlined beyond the tumor border. Formulas are described below.

$$\%\text{ of tumor area correctly detected}=[\text{total observed area within the true tumor area}]/[\text{true tumor area}]$$

$$\%\text{ of tissue area outlined beyond tumor border}=[\text{total observed area}–\text{tumor area correctly detected by surgeon}]/\left[\text{tumor area correctly detected by surgeon}\right]$$

Fluorescence Histology

Intracranial tissues (i.e. tumor, brainstem, cerebellum) were fixed in 4% paraformaldehyde for 60 minutes, embedded in optimal cutting temperature media (Tissue-Tek^™, Radnor, PA, USA), flash frozen in liquid nitrogen, sectioned, and placed on slides. Tissues were counterstained with 4’,6-diamidino-2-phenylindole (DAPI, ab104139, Abcam, Cambridge, MA, USA) nuclear stain. Slides were imaged using at 40X magnification with a Zeiss LSM 800 confocal microscope (Carl Zeiss Microscopy, Thornwood, NY, USA) and software. The confocal images were processed using ImageJ software (U. S. National Institutes of Health, Bethesda, Maryland, USA) to quantify the green fluorescence resulting from IV SF deposition in relative fluorescence units (RFU).

Western Blot of Organic Anion Transporting Polypeptide

Protein was extracted from liver, tumor, cerebellum, and brainstem from our experimental xenograft model of VS. Protein was isolated using radioimmunoprecipitation assay buffer with protease and phosphatase inhibitors (Thermo Scientific, Inc., Waltham, MA). This was separated after loading ~8 micrograms of total protein per well and subsequently transferred to a polyvinylidene fluoride membrane (Mini-Protein^™, Biorad, Hercules, CA). The membranes were incubated in 1:500 rabbit anti-OATP1C1 antibody (ABIN635126, Antibodiesonline, Limerick, PA) and 1:5000 mouse anti-beta-actin (NB5600–501, Novus, Centennial, CO) overnight at 4 degrees Celsius. Lastly, the membrane was incubated with goat anti-rabbit antibody conjugated to Alexa 488 (1:1000; Thermo Scientific, Inc., Waltham, MA) and goat anti-mouse antibody conjugated to Alexa 555 (1:1000; Thermo Scientific, Inc., Waltham, MA) then imaged using ImageQuant LAS 4000 Imager (GE Healthcare, Chicago, IL). The mean density of each band was objectively measured using ImageJ software (NIH). Subsequently, we calculated the fold change in expression of OATP1C1 in all tissues, relative to tumor.

Statistical Analysis

Depending on normality, statistical analysis was performed using Kruskal-Wallis test and Friedman’s test with Tukey’s post-hoc testing and Bonferroni correction. Significance was set at a p-value less than 0.05.

Results

Tissue Fluorescence

Using blue light, tumors from rats treated with IV SF demonstrated intense green fluorescence under direct visualization, when compared to adjacent normal tissues and control rats (Figure 1A and 1B). In animals that received IV SF, green fluorescence was visualized mainly within tumors; minimal to no fluorescence was observed in other neural tissues. Additionally, there was no significant gross tissue auto-fluorescence in control rats. When measured objectively under blue light excitation at five different regions of interest (Figure 2A and 2B), the total photon flux was significantly higher in tumors from rats that received IV SF, when compared to adjacent neural structures and to tissues from rats that received IV saline (p<0.001) (Figure 2C). Under blue light, the mean fold changes in fluorescence between tumor and ipsilateral cerebellum and ipsilateral brainstem were significantly higher in rats that received IV SF, when compared to rats the received IV saline (p<0.001) (Figure 2D).

Figure 1. Photos of Tissue Specimens in White and Blue Light.

Mid-sagittal craniotomies displaying cross-sectional anatomy with tumors labelled with white arrows. (A) Tissue specimens from animal that received intravenous saline demonstrate no green fluorescence when visualized in white and blue light. (B) Tumors from animals that received intravenous sodium fluorescein demonstrated strong green fluorescence under blue excitation, when compared to white light alone.

Figure 2. Tissue Fluorescent Measurements with the IVIS® Imaging System.

(A-B) The IVIS platform was utilized to obtain objective fluorescence measurements on tissue specimens in five different regions of interests from 5 animals treated with intravenous sodium fluorescein (IV SF) and 3 rats that received saline (control). (C) Fluorescent measurements of the tumor were significantly higher in rats that received IV SF, when compared to ipsilateral and contralateral cerebellums and brainstems. Bar=mean, error bar=standard error mean, ***p<0.001. (B) The fold changes in fluorescence between the tumor and its ipsilateral cerebellum and brainstem were significantly higher in rats that received IV SF, compared to the control condition. Box=25-50-75th percentile, error bar=minimum-maximum, circle=mean. Friedman’s test, ***p<0.001.

Tumor Brain Interface Identification

Figure 3A shows an example of a tumor in white light after a mid-sagittal craniotomy was performed. The true tumor area is highlighted in green, while the surgeon’s perspective of the tumor outline is shown with a black dotted line. The tumor area correctly detected by the surgeon is shown highlighted in blue. After surgeons subjectively outlined the tumor-brain interface, the percentage of tumor area correctly detected and the percentage of tissue area outlined beyond the tumor border were calculated. There were no statistically significant differences in the percentage of tumor area detected when comparing treatment condition and type of light (Figure 3B). In contrast, there was a statistically significant decrease in the percentage of tissue area outlined beyond the tumor border in animals that received IV SF with specimens viewed in blue light, when compared to animals that received saline with specimens observed in white light (p<0.05) (Figure 3C). There were no statistically significant differences in the tissue area outlined beyond the actual tumor border when comparing blue light to white light in control animals and animals treated with IV SF.

Figure 3. Percentages of Tumor Area Correctly Detected and Tissue Area Outlined Beyond Tumor Border.

Sixteen blinded surgeons were asked to outline the tumor-brain interface on images of mid-sagittal craniotomies demonstrating the tumor and adjacent neural structures, obtained in white and blue light. (A) Photograph of mid-sagittal craniotomy with tumor in white light. The true tumor area is shown in green, while the total observed area (surgeon’s perspective of tumor) is presented with a dashed line. The tumor area correctly detected by the surgeon is shown in blue. (B) There were no significant differences in the percentage of tumor area detected by treatment condition or type of light. (C) There was a statistically significant decrease in the percentage of tissue area outlined beyond the actual tumor border in rats that received intravenous sodium fluorescein with specimens visualized in blue light. Friedman’s test. Bar=mean, error bar=standard error mean, *p<0.05, NS=not significant.

Fluorescence Histology

Confocal images of tumor sections obtained from rats treated with IV SF showed more green fluorescence than adjacent brainstem or cerebellum (Figure 4A). Green fluorescence was observed around nuclei of tumor cells in rats that received IV SF, suggesting that the pattern of SF deposition was likely intracellular and extranuclear. In addition, there was mild green fluorescence in areas without nuclei, implying that SF deposition may also be in the extracellular space of tumors. When measured objectively, the histologic green fluorescent intensity was significantly higher in tumor sections from animals that received IV SF, when compared to adjacent neural tissues and sections obtained from rats given IV saline (p<0.001) (Figure 4B).

Figure 4. Tumor and Tissue Fluorescence on Confocal Imaging.

(A) Fluorescent images of tumor, brainstem and cerebellum were obtained after tissues were fixed, sectioned, and treated with 4’,6-diamidino-2-phenylindole (DAPI) nuclear stain. Green fluorescence was observed primarily in tumor sections obtained from rats that received intravenous (IV) sodium fluorescein (SF). The pattern of green fluorescence suggests that SF deposition in tumor is cytoplasmic and extranuclear as well as extracellular. White line = 20 μm. (B) The green fluorescence was quantified on tissue sections. The fluorescent measurements were significantly higher in tumor sections from rats that received IV SF, when compared to adjacent neural structures and tissues obtained from control animals (p<0.001). N=6 sections per tissue location and treatment. Friedman’s test. Bar=mean, error bar=standard error mean, ***p<0.001.

Western Blot of Organic Anion Transporting Polypeptide

Western blots were performed to analyze expression of OATP1C1 in rat liver, tumor, brainstem and cerebellum. We found OATP1C1 expression in all tissues of interest, including the rat liver (positive control) (Figure 5). The tumor demonstrated significantly higher OATP1C1 expression, when compared to cerebellum and brainstem (p<0.0001), suggesting that the tumor may have preferential uptake of SF by expressing more OATP1C1.

Figure 5. Fold Change in Organic Anion Transporting Polypeptide 1C1 (OATP1C1) Expression by Tissue Type.

(A) Western blot of OATP1C1 in liver (positive control), tumor, cerebellum and brainstem. (B) Box plot of fold changes in OATP1C1 expression (relative to tumor). The expression of OATP1C1 was significantly higher in the tumor, compared to the cerebellum and brainstem. Box=25-50-75th percentile, error bar=minimum-maximum. Kruskal-wallis test, ***p<0.0001.

Discussion

Microsurgical resection of VS can be challenging particularly when tumors are large, distort normal anatomical structures, or adherent to critical intracranial structures naturally or as a result of radiosurgery. Difficulty identifying the tumor-brain and tumor-nerve interfaces during VS surgery can lead to cranial nerve, brainstem and cerebellar injuries that can result in devastating clinical outcomes, such as hearing loss, facial paralysis, and even stroke.

Because VS tumors are being diagnosed earlier when patients have normal or near-normal hearing, hearing preservation has become an important outcome measure in the management. [21] There is ongoing debate as to the best treatment modality (tumor surveillance, radiation therapy, and microsurgical resection) for VS, particularly for small tumors less than 2 cm. [21, 22–23] For cases in which microsurgical resection is selected for hearing preservation, either the middle cranial fossa approach or the retrosigmoid approach is utilized. In 125 patients with tumors less than 1.5 cm, Sameshema et al. demonstrated approximately 75% hearing preservation rate (American Academy of Otolaryngology-Head and Neck Surgery Hearing Grade A/B) with the middle cranial fossa (76.7%, n=43) and retrosigmoid (73.2%, n=82) approaches for resection. [23] In patients with tumors larger than 3 cm and preoperative serviceable hearing, the rate of hearing preservation after microsurgical resection is much less at approximately 50%. [24,25] This drop in hearing preservation rates with larger tumors is likely in part from tumor compression, adhesion, distortion and displacement of adjacent neural structures that makes cochlear nerve dissection exceedingly difficult.

Facial nerve function is a critical determinant of post-operative quality of life following surgery. [26] Preservation of facial function (House Brackman Grade 1 and 2) ranges from 35–84% in patients undergoing VS surgery; however, it is well known that the preservation of facial function is more challenging with larger tumors. [27–29] Moreover, revision surgery negatively affects facial nerve outcomes. [30] When VS tumors grow, they can compress the facial nerve over time, causing a thick nerve bundle to flatten and become a thin layer of facial nerve fibers that are difficult to discern from tumor and tumor capsule. In difficult cases, being able to clearly identify the tumor-nerve interface during microsurgical resection can potentially improve surgical outcomes and ultimately quality of life.

Because large VS tumors can compress and distort the cerebellum, brainstem, and their blood supply, another devastating complication of VS surgery is stroke. Stroke occurs in approximately 2% of VS patients undergoing surgery; however, in low volume hospitals, the rate of stroke is slightly higher at 3.2%. [31, 32] Therefore, there is a need for new techniques that can improve surgical outcomes by helping surgeons better define the tumor-tissue interface. Furthermore, by improving visualization of the tumor-tissue interface, we can potentially increase gross tumor resection rates while also minimizing surgical morbidity.

IV SF is well described in the neurosurgical literature for its intraoperative use to improve visualization of gliomas. Several of these groups have demonstrated improved gross total resection and decreased morbidity by preserving surrounding normal brain tissue. [7, 10, 11] To date, there is only one case series describing the use of IV SF for improved visualization of VS. [33] In this study, the authors administered IV SF to six patients, one of which had a VS. They photographed the tumors using an operating microscope and observed tumor discoloration in white light. Aside from a small sample size, this study did not evaluate the signal intensity before and after SF administration. In addition, the appropriate filters for visualizing tissue fluorescence were not utilized, which is the current standard for intraoperative use of IV SF.

We recently showed that MDSCs and primary VS cells demonstrated preferential uptake of SF in vitro, when compared to normal Schwann cells. [12] This finding led us to evaluate the potential of IV SF in the detection of VS in a preclinical animal model. [19] After administering IV SF in our xenograft model of VS, we were able to directly visualize tumor fluorescence under blue light excitation (Figure 1), objectively quantify the tumor fluorescence (Figure 2), and demonstrate that SF accumulates within VS tumors on histologic tissue sections (Figure 4). We also established that this technique can improve a surgeon’s ability to differentiate between tumor and normal cerebellar and brainstem tissue and detect the tumor-brain interface with blue light excitation (Figure 3). In particular, our findings suggest that with IV SF and blue light excitation, surgeons can potentially minimize the resection of normal cerebellar and brainstem adjacent to the tumor and reduce surgical morbidity.

The exact mechanism of IV SF uptake in VS remains unknown. In gliomas, a recent study suggests that preferential uptake by tumors occurs through SF extravasation through a leaky BBB. [10] Our study demonstrates that VSs in our xenograft model preferentially uptake IV SF, and the deposition of SF is both extracellular and intracellular. Although the extravasation of SF into the tumor through a leaky BBB can explain the extracellular accumulation of SF in the tumor, it does not entirely account for the tumor’s intracellular uptake of SF. OATPs are transmembrane proteins that play an important role in the uptake of several endogenous compounds, including hormones and bile acids. They are also responsible for the cellular uptake of several drugs, including chemotherapeutic agents and SF. [14, 15, 34, 35] In our xenograft model of VS, tumors demonstrated higher levels of OATP1C1 (Figure 5), when compared to normal brain tissue, suggesting that increased OATP expression may also be a contributing factor to the intracellular uptake of SF in our tumors. Further studies are warranted to understand the true mechanisms of preferential SF uptake in VS.

The limitations of our study include our preclinical xenograft model and a small sample size. Additionally, IV SF uptake was studied at only one time point and one dosage. We were unable to photograph and reliably measure fluorescence in the facial and cochleovestibular nerves because of the small size of these cranial nerves and their anatomical location with respect to the craniotomy. Although we were not able to quantify the tumor-nerve interface, we observed subjectively that several tumors demonstrated green fluorescence under blue light, when compared to nerve, in rats that received IV SF (Figure 6). Future investigations should focus on understanding the exact mechanisms of preferential SF uptake by VS and testing the utility of IV SF in tumor detection and microsurgical resection of VS in NF2 animal models and clinical trials.

Figure 6. Photo of Cochleovestibular and Facial Nerves in White + Blue Light.

Mid-sagittal craniotomy displaying cross-sectional anatomy in an animal that received intravenous sodium fluorescein. The tumor is labelled with a white block arrow. On the right, the cerebellum and brainstem were removed and the tumor was debulked in order to visualize the cochleovestibular and facial nerves (double white arrow) entering into the internal auditory canal. The tumor demonstrated bright green fluorescence on white + blue light excitation, while nerves did not show obvious fluorescence.

In conclusion, tumor from our xenograft model of VS demonstrated preferential uptake of IV SF and emitted an intense green fluorescence under blue light that helped surgeons differentiate tumor from normal cerebellum and brainstem nearby. These findings support further investigations into the effectiveness of IV SF in helping surgeons distinguish the tumor-nerve interface as well as the clinical utility of IV SF for improving tumor resection and surgical outcomes in VS patients.