Near infrared fluorescence imaging of rabbit thyroid and parathyroid glands

Accepted Manuscript Near Infrared fluorescence imaging of rabbit thyroid and parathyroid glands R. Antakia , P. Gayet , S. Guillermet , T.J. Stephenson , N.J. Brown , B.J. Harrison , S.P. Balasubramanian PII:

S0022-4804(14)00530-7

DOI:

10.1016/j.jss.2014.05.061

Reference:

YJSRE 12763

To appear in:

Journal of Surgical Research

Received Date: 26 March 2014 Revised Date:

1 May 2014

Accepted Date: 19 May 2014

Please cite this article as: Antakia R, Gayet P, Guillermet S, Stephenson T, Brown N, Harrison B, Balasubramanian S, Near Infrared fluorescence imaging of rabbit thyroid and parathyroid glands, Journal of Surgical Research (2014), doi: 10.1016/j.jss.2014.05.061. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

1

ACCEPTED MANUSCRIPT Near Infrared fluorescence imaging of rabbit thyroid and parathyroid glands (revised 28/04/2014)

Department of Oncology, University of Sheffield, UK

2

Fluoptics, Grenoble, FRANCE

3

Department of Histopathology, Sheffield Teaching Hospitals, UK

4

Department of General Surgery, Sheffield Teaching Hospitals, UK

SC

1

RI PT

Antakia R1, Gayet P2, Guillermet S2, Stephenson TJ3, Brown NJ1, Harrison BJ4, Balasubramanian SP1

Antakia R; MBChB (Hons), MRCS; designed the study, undertook experimental work, analysed data and

M AN U

drafted paper.

Gayet P; PhD; participated in experimental work and data analysis.

Guillermet S; PhD; participated in study design, experimental work and data analysis.

and manuscript preparation.

TE D

Stephenson TJ; MBChB (Hons), MA, MD, MBA, FRCPath; histopathology confirmation of excised tissue

Brown NJ; BSc, DipEd, PhD; assisted in study design (in vivo experiments) and manuscript preparation.

EP

Harrison BJ; MS, FRCS; assisted in manuscript preparation.

AC C

Balasubramanian SP; MS, FRCS, PhD; study design, assisted in experimental work & manuscript preparation.

*Corresponding author: [email protected] Department of Oncology, University of Sheffield, FU32, Beech Hill Road, Sheffield, S10 2RX, UK Telephone number: 0798 333 6264 / 0114 271 2510 Funding: Internal funding

Fax number: 0114 271 3314

Category: Original article

No conflicts of interest to declare. I confirm all co-authors have contributed and approved the final report. Presented in the BJS oral prize session in the BAETS annual conference in Rome on the 10/10/2013.

2

ACCEPTED MANUSCRIPT Abstract Background: Near infrared (NIR) fluorescence imaging using intravenous (IV) Methylene Blue (MB) is a novel technique that has potential to aid parathyroid gland localisation during thyroid and parathyroid surgery. The aim of this study was to examine MB fluorescence in the rabbit neck and

RI PT

determine the influence of MB dose and time following administration on fluorescence from thyroid and parathyroid glands.

Methods: Thyroid and external parathyroid glands were exposed in six New Zealand White rabbits

SC

under anaesthesia. Varying doses of MB (0.025 - 3 mg/kg) were injected through the marginal ear vein. NIR fluorescence from exposed tissues was recorded at different time intervals (10 - 74 minutes)

M AN U

using Fluobeam®700. Specimens of identified glands were then resected for histological assessment. Results: Histology confirmed accurate identification of all excised thyroid and parathyroid glands; these were the only neck structures to demonstrate significant fluorescence. The parathyroid demonstrated lower fluorescence intensities and reduced washout times at all MB doses compared to

TE D

the thyroid gland. A dose of 0.1 mg/kg MB was adequate to identify fluorescence; this also delineated the blood supply of the external parathyroid glands. Conclusion: The study demonstrates that near infra-red fluorescence with IV MB helps differentiate

EP

between thyroid and parathyroid glands in the rabbit. This has potential to improve outcomes in thyroid and parathyroid surgery by increasing the accuracy of parathyroid identification; however the

AC C

findings require replication in human surgery. The use of low doses of MB may also avoid the side effects associated with currently used doses in humans (3-7mg/kg). Keywords: Methylene Blue, near-infrared fluorescence, thyroid, parathyroid

3

ACCEPTED MANUSCRIPT Background: Thyroid surgery is the most frequently performed endocrine surgical procedure. Hypocalcaemia is the most common postoperative complication following thyroidectomy, with rates of transient and permanent hypocalcaemia ranging from 2% - 50% (1-3) and 0.4% - 13.8% (4-6) respectively. Symptoms

RI PT

and signs of hypocalcaemia include circumoral paraesthesia, tetany, laryngospasm, ECG changes (7). Although hypocalcaemia can be treated effectively with calcium and/or vitamin-D supplements, there may be persistent long-term morbidity due to adverse effects on bones and kidneys (7-9). Primary

SC

hyperparathyroidism is the main indication for parathyroid surgery and postoperative morbidity can include failure to cure due to under-treatment, and the likelihood of hypocalcaemia with over-

M AN U

treatment (10).

Methylene Blue (MB) is a dye that localises in both thyroid and parathyroid tissue on intravenous administration. Its use in parathyroid surgery was first described in 1971 to aid the ‘naked eye’ visualisation of enlarged parathyroid glands (11, 12) and subsequently used at doses ranging from 3 to

TE D

7.5mg/kg depending on local practice (13). Side effects and serious neurotoxicity have been reported, especially in patients on Selective Serotonin Reuptake Inhibitors (SSRIs) (13-15). MB also exhibits fluorescent properties in the near-infrared (NIR) range (light outside the visible spectrum) at

EP

significantly reduced concentrations than those used in clinical practice (16) and has the potential to reduce the adverse effects of MB, whilst improving the accuracy of parathyroid identification.

AC C

Intra-operative NIR fluorescence imaging is a new technology that allows real-time visualisation of normal and abnormal tissues. In surgical oncology, combined with appropriate fluorescent agents, the technique has been found beneficial in the assessment of cancer resection margins (17, 18), imageguided lymph node mapping (19-22), lymph node mapping in gynaecological cancer surgery (23) and to confirm the patency of biliary anastomoses in pancreatico-duodenal biliary surgery (24). In thyroid and parathyroid surgery, the technique has the potential to differentiate parathyroid glands from thyroid nodules and lymph nodes and subsequently reduce the incidence of inadvertent damage and devascularisation of normal glands, improving patient outcomes.

4

ACCEPTED MANUSCRIPT The aim of the current in vivo experiment was to establish the pattern of MB-induced fluorescence detected by Fluobeam®700 from the soft tissue structures in the rabbit neck. This animal model was chosen as it has an extra-thyroidal 'external' parathyroid gland on each side of the neck (25). The specific objectives were to:

RI PT

1. Determine the lowest systemic MB dose that allows detection of fluorescence in the neck tissues. 2. Identify and study patterns of fluorescence (onset, peak intensity and duration) in thyroid and

AC C

EP

TE D

M AN U

SC

parathyroid glands.

5

ACCEPTED MANUSCRIPT Methodology: Animals Two New Zealand White (NZW) rabbits were obtained from Highgate Farm in Sheffield (Home

RI PT

Office authorised supplier), and held in the animal facility at University of Sheffield field laboratories 3 months before experimentation. Animals were housed in a humidity- and temperature-controlled environment and allowed access to water and food ad libitum. Procedures were performed in

accordance with UK Home Office Animal Procedures Act (1986), under Project License number

SC

40/3531 (NJB). Following terminal anaesthesia, the neck was dissected for the researchers to become

M AN U

familiar with the anatomy of rabbit thyroid and parathyroid glands.

Further experiments were performed under general anaesthesia on six NZW rabbits at INRA (Institute of National Agronomic Research) Animal Institute, Tours, France. Rabbits were obtained from HYPHARM (Roussay, France) and held in the animal facility at the “Platform for Experimental Infectious Diseases” (INRA, Nouzilly, France) for 6 days before experimentation. Experiments were

TE D

conducted in accordance with the European directive 2010/63/EU on the protection of animals used for scientific purposes and approved by the regional ethics committee (CEEA VdL) and in accordance

EP

to the UK animal welfare act 2006 (26). Experimental protocol

AC C

Two rabbits were dissected post mortem in Sheffield to determine the anatomy of the neck central compartment, with specific reference to the thyroid and parathyroid glands (PGs). The tissues were then resected and processed for histological evaluation. At INRA Tours, anaesthesia was induced with intramuscular injection of Ketamine (35 mg/kg), Xylazine (5 mg/kg) and morphine (2 mg). Inhalational isoflurane 1.5% was used for maintenance of anaesthesia. The central neck compartment was exposed via a vertical midline incision over the ventral aspect of the neck. Care was taken to preserve the parathyroid vascular supply during dissection.

6

ACCEPTED MANUSCRIPT Details of each experiment were recorded using a standard proforma. MB was administered as bolus injections into the marginal ear vein at doses ranging from 0.025 mg/kg to 3 mg/kg and flushed with an infusion of normal saline. Volumes of MB injections ranged from 0.29 ml to 2.16 ml, with varying dilutions, appropriate for an approximate circulatory volume of 56 ml/kg body weight (27-29). NIR

RI PT

fluorescent images of the exposed neck soft tissues (thyroid, external parathyroid glands, and skeletal muscle) were recorded using Fluobeam® 700 (Fluoptics, Grenoble, France), with black and white images of the operating field before and after each intravenous MB bolus (Figure 1a and 1b).

SC

Oxygen saturation and heart rate were monitored throughout surgery to identify whether the

circulation was compromised, as this could affect MB delivery to the soft tissues of interest. At the

M AN U

end of each experiment, the rabbit was euthanized using IV thiopental (350 mg). Specimens from both external parathyroid glands and the thyroid were retrieved for histopathological confirmation. Image J software, developed by the National Institute (30, 31), was used for quantification of fluorescent intensity emitted from the thyroid, parathyroid glands and the strap muscles. Fluorescent intensities

TE D

were recorded as numerical values on an Excel spreadsheet (Microsoft Office 2007), and displayed in tables and charts to demonstrate the evolution of MB fluorescence. Ratios, median values and ranges are also reported.

EP

Fluobeam Optical Imaging

AC C

Fluobeam® is a 2D NIR portable imaging system composed of the following: an electric panel box (laser emitting at 690 nm with its power supply); light-emitting diodes (LEDs); a highly sensitive charge-coupled device (CCD) camera; and a dedicated optical system to scatter the laser light and white LEDs. The fluorescence signal is collected through a high-pass filter over 700 nm. The optical system allows the laser beam to scatter over a field of about 6 cm in diameter at a distance of 15 cm from the extremity of the optical head (32).

7

ACCEPTED MANUSCRIPT Histology processing Specimens were fixed in 10% buffered formalin solution. Tissues were processed by ‘Leica TP1020 tissue processer’ passing through increasing concentrations of alcohol (70-100%), Xylene and wax

3020 microtome’ at 5 micron thickness onto ‘superfrost plus’ slides.

RI PT

over 22 hours. Specimens were then embedded in paraffin wax blocks and sectioned using ‘Leica

Slides were stained using haematoxylin and eosin (H&E) using the following technique: Sections were dewaxed in Xylene and taken to water through descending concentrations of alcohol. Aqueous

SC

Gill’s haematoxylin stain was applied for 1-2 minutes; followed by 5 minutes wash in tap water.

M AN U

Alcoholic eosin was then applied after increasing alcohol concentrations (70 to 95% ethanol), stained with eosin in 95% ethanol for 1 minute, 100% ethanol (1 minute) and Xylene (2 x 5 minutes).

AC C

EP

TE D

Sections were then mounted in DPX (distyrene, a plasticizer, and Xylene) and coverslip.

8

ACCEPTED MANUSCRIPT Results: The characteristic features of the rabbits and the experimental protocol are presented in Table 1. In all rabbits, thyroid and the external PGs were initially identified by naked-eye examination then confirmed by visualisation of MB fluorescence. A range of MB concentrations were used, with the

RI PT

results from the first experiment informing the dose range used in subsequent experiments (Table 1). The period of observation with each MB bolus ranged from 10 to 74 minutes in all six rabbits. In the first two experiments, longer periods of observation were required to establish the patterns of

SC

fluorescence and decay of fluorescence in the tissues of interest. The periods of observation were reduced in the subsequent four experiments as it was clear that the return to basal fluorescence in the

M AN U

parathyroid glands was rapid (ranging from 3 to 12 minutes) and the thyroid gland fluorescence lasted longer (up to 74 minutes). Neck anatomy

Neck dissection of the two rabbits in Sheffield demonstrated that the thyroid gland consists of 2 fleshy

TE D

reddish lobes lying on either side of the upper trachea connected by a thin isthmus. Each lobe is 1520mm in cephalo-caudal length and 6-12mm in width. The external PGs, 3-4mm in size, were located either adjacent or inferior to the lower pole of the thyroid lobe or in close proximity to the carotid

EP

sheath. This was consistent with anatomy described previously (25). The internal PGs were located

AC C

within the thyroid tissue as demonstrated on histology. In the subsequent rabbits, the positions of external PGs were confirmed as described above (Figure 2) with one exception, where the right external PG was lying over the anterior surface of the right thyroid lobe. Histology All thyroid and parathyroid glands were confirmed by histopathology examination (TJS) in H&E stained sections. Rabbit parathyroid glands demonstrated similar morphology to human glands apart from minor differences in the proportions of the constituent cell types, lobulation of the tissue, and its

9

ACCEPTED MANUSCRIPT interface with surrounding adipose connective tissue. MB administration had no discernible or detrimental effects on the thyroid and parathyroid specimens. Fluorescence intensity

RI PT

The time to peak fluorescence following MB administration and peak fluorescence ratios (ratio of peak fluorescence in the tissue of interest compared to fluorescence from the strap muscle) in both thyroid lobes and external PGs was determined. In rabbit two, the right external PG was lying over the anterior surface of the right thyroid lobe which interfered with fluorescence measurements; especially

SC

after the third MB dose. In rabbit three, the right external PG was partially devascularised during neck

M AN U

dissection; this was reflected by the lower fluorescent intensities compared to the left external PG. Following anaesthetic induction, all rabbits remained physiologically stable throughout the experiments and MB-related toxicity was not encountered. Transient hypoxia and bradycardia occurred during the first MB bolus in rabbit three, this resolved following repositioning of the endotracheal tube.

TE D

Thyroid and external PGs were the only soft tissues to fluoresce in the neck. Onset of fluorescence following administration of MB was similar in both thyroid and parathyroid glands (Figure 3). Peak fluorescence intensities in the external PGs were reached either at the same time or earlier when

EP

compared to thyroid with quicker decay in the parathyroids. However, thyroid peak fluorescence was consistently higher (Table 2) and persisted for a longer duration (Figure 3) at all concentrations. Large

AC C

differences in peak fluorescence ratio were observed between both thyroid lobes only on four occasions. This was attributed to partial rotation of the rabbit neck, which altered the measurements from the area exposed during image capture. The changes in fluorescent intensities following administration of various doses of MB from thyroid, external PGs and muscle are demonstrated graphically in Figure 3. Very low doses of MB (0.025 and 0.05 mg/kg) were associated with minimal differences in both peak fluorescence and persistence of fluorescence between thyroid and parathyroid tissue (data not shown). In contrast, following administration of 0.1, 0.3 and 0.6 mg/kg of MB, large differences in fluorescence intensity were

10

ACCEPTED MANUSCRIPT observed between thyroid and parathyroid tissue. This was more pronounced at the two higher concentrations (0.3 and 0.6 mg/kg; Table 2 and Figure 3). The time to peak fluorescence was increased for the thyroid (median 3.28 minutes, range 0.6-7.45, n=30) compared to the PGs (median 0.9 minute, range 0.6-2.5, n=29) when amalgamating data from the three MB concentrations (0.1, 0.3

RI PT

and 0.6 mg/kg). The evolution of the fluorescence signal in thyroid, parathyroid glands and muscle of one rabbit following IV injection of 0.1 mg/kg MB, is represented in Figure 4. This shows an obvious difference

SC

between thyroid, parathyroid and muscle fluorescence, the rapid reduction in parathyroid

AC C

EP

TE D

not achieved with higher MB doses.

M AN U

fluorescence, and the persistence of fluorescence in the thyroid. Further differential enhancement was

11

ACCEPTED MANUSCRIPT Discussion: To our knowledge, this is the first study to examine the patterns of near infra-red (NIR) fluorescence induced by administration of systemic MB in normal thyroid and parathyroid tissue in an in vivo model. NIR wavelength (700-900 nm) is considered the most appropriate optical range for biomedical

RI PT

research due to increased penetration depth, decreased scattering and tissue absorption compared to UV-visible light range (200-700 nm) (19). MB administered at 0.1 mg/kg (1/30th of the dose currently used in clinical practice) was the lowest concentration that clearly demonstrated differences in

SC

fluorescence between the soft tissues of interest in the rabbit neck.

M AN U

Thyroid and PGs were the only soft tissues to fluoresce in the neck central compartment suggesting that only these tissues retain and subsequently emit fluorescence from MB, in contrast to other well vascularised tissues such as muscle. The mechanism underlying the increased uptake of MB by thyroid/parathyroid glands is unclear. It may be due to the relatively increased blood supply of these glands or the higher uptake and retention of MB by these tissues.

TE D

The aim of the study was to evaluate the onset, time to peak and duration of fluorescence from thyroid and parathyroid glands using a range of MB concentrations. Understanding these fluorescence patterns in the rabbit model will guide further clinical studies to determine whether lower MB

EP

concentrations differentiate between the structures of interest during thyroid and parathyroid surgery. This has the potential to decrease the morbidity associated with MB administration and improve

AC C

patient outcomes.

Van der Vorst el al., (2013) used an imaging system (Mini-FLARE 700 nm wavelength) similar to the one in the current study and demonstrated the feasibility of MB NIR in guiding intra-operative identification of parathyroid adenomas in human surgery (16). A single concentration of MB (0.5 mg/kg) was used in 12 patients with parathyroid disease (benign and malignant), however the fluorescence emission from ‘normal’ parathyroid and thyroid tissue was not reported in detail. The study concluded that NIR fluorescence from low-dose MB can be used to identify parathyroid adenomas. Another study by McWade et al evaluated the auto-fluorescence emission spectra without

12

ACCEPTED MANUSCRIPT using any extrinsic fluorophores in 45 patients with mixed thyroid and parathyroid disease (33). They reported that parathyroid auto-fluorescence was 1.2-18 times higher than thyroid at 822 nm wavelength regardless of disease, although parathyroid histology confirmation was only available in 22 cases.

RI PT

The current 'proof of concept' study only used six rabbits. Given that specific fluorescence patterns from the neck soft tissues have been consistently obtained at MB doses much lower than currently used in clinical practice, this number was deemed sufficient. Although several concentrations of MB

SC

were utilised in each animal, sufficient time was allowed to elapse between consecutive doses. This allowed fluorescence to decay to near baseline values, thus limiting the impact of cumulative

M AN U

fluorescence. Regardless of the first MB bolus, clear differentiation between the pattern of parathyroid and thyroid fluorescence were noted with doses of 0.1, 0.3 and 0.6 mg/kg body weight. Although differentiating between thyroid and parathyroid gland fluorescence was difficult during image analysis done after the experiments; during the surgery, we could distinguish thyroid from

limitation in clinical studies.

TE D

parathyroid fluorescence even when the glands were in close proximity to each other. This may be a

In summary, this study clearly demonstrates that normal thyroid and PGs demonstrate intense

EP

fluorescence in the NIR range with intravenous low dose MB in an in vivo model. PG fluorescence decays rapidly compared to the thyroid gland. These results will inform the design of a human study

AC C

of NIR fluorescence from MB administered during thyroid and parathyroid surgery. The ability of MB at doses of 0.1 and 0.3 mg/kg (which are lower than that used by Van der Vorst et al (16)) to distinguish between thyroid and parathyroid tissue (both normal and diseased) will be evaluated. If the results are replicated in human tissues, the technology has the potential to facilitate early identification and preservation of parathyroid glands during thyroid surgery. In addition, there is potential for clinical benefit in other scenarios such as the identification of ectopic parathyroid glands at parathyroid surgery; assessing the integrity of parathyroid vascular supply after thyroidectomy i.e.

13

ACCEPTED MANUSCRIPT gland viability; and the potential value of thyroid fluorescence in guiding re-operative surgery in patients with residual and recurrent thyroid cancer. Acknowledgment:

RI PT

We gratefully acknowledge the staff at “INRA Tours” (INRA Centre de recherche Val de Loire, 37380 Nouzilly, France) for arranging the animal dissections and assisting with the experimentation, Mrs Jenny Globe for processing the histology specimens, and the funding from the University of

AC C

EP

TE D

M AN U

SC

Sheffield and BMI Healthcare.

14

ACCEPTED MANUSCRIPT References:

AC C

EP

TE D

M AN U

SC

RI PT

1. Lang BH, Ng SH, Lau L, Cowling B, Wong KP, Wan KY. A systematic review and meta-analysis of prophylactic central neck dissection on short-term locoregional recurrence in papillary thyroid carcinoma after total thyroidectomy. Thyroid. 2013. Epub 2013/02/14. doi: 10.1089/thy.2012.0608. PubMed PMID: 23402640. 2. Sanabria A, Dominguez LC, Vega V, Osorio C, Duarte D. Cost-effectiveness analysis regarding postoperative administration of vitamin-D and calcium after thyroidectomy to prevent hypocalcaemia. Revista de salud publica (Bogota, Colombia). 2011;13(5):804-13. Epub 2012/05/29. PubMed PMID: 22634947. 3. Dionigi G, Bacuzzi A, Bertocchi V, Carrafiello G, Boni L, Rovera F, et al. Prospectives and surgical usefulness of perioperative parathyroid hormone assay in thyroid surgery. Expert Rev Med Devices. 2008;5(6):699-704. Epub 2008/11/26. doi: 10.1586/17434440.5.6.699. PubMed PMID: 19025346. 4. Yang W, Shao T, Ding J, Jin X, Li Q, Chu PG, et al. The feasibility of total or near-total bilateral thyroidectomy for the treatment of bilateral multinodular goiter. Journal of Investigative Surgery. 2009;22(3):195-200. PubMed PMID: 2010053500. 5. Bergenfelz A, Jansson S, Kristoffersson A, Martensson H, Reihner E, Wallin G, et al. Complications to thyroid surgery: results as reported in a database from a multicenter audit comprising 3,660 patients. Langenbeck's archives of surgery / Deutsche Gesellschaft fur Chirurgie. 2008;393(5):667-73. Epub 2008/07/18. doi: 10.1007/s00423-008-0366-7. PubMed PMID: 18633639. 6. Sousa Ade A, Salles JM, Soares JM, Moraes GM, Carvalho JR, Savassi-Rocha PR. Predictors factors for post-thyroidectomy hypocalcaemia. Revista do Colegio Brasileiro de Cirurgioes. 2012;39(6):476-82. Epub 2013/01/26. PubMed PMID: 23348643. 7. Fong J, Khan A. Hypocalcemia: updates in diagnosis and management for primary care. Canadian family physician Medecin de famille canadien. 2012;58(2):158-62. Epub 2012/03/24. PubMed PMID: 22439169; PubMed Central PMCID: PMCPMC3279267. 8. Schaffler A. Hormone replacement after thyroid and parathyroid surgery. Dtsch Arztebl Int. 2010;107(47):827-34. Epub 2010/12/22. doi: 10.3238/arztebl.2010.0827. PubMed PMID: 21173898; PubMed Central PMCID: PMCPmc3003466. 9. Mitchell DM, Regan S, Cooley MR, Lauter KB, Vrla MC, Becker CB, et al. Long-term follow-up of patients with hypoparathyroidism. The Journal of clinical endocrinology and metabolism. 2012;97(12):4507-14. Epub 2012/10/09. doi: 10.1210/jc.2012-1808. PubMed PMID: 23043192; PubMed Central PMCID: PMCPmc3513540. 10. Bergenfelz AO, Jansson SK, Wallin GK, Martensson HG, Rasmussen L, Eriksson HL, et al. Impact of modern techniques on short-term outcome after surgery for primary hyperparathyroidism: a multicenter study comprising 2,708 patients. Langenbeck's archives of surgery / Deutsche Gesellschaft fur Chirurgie. 2009;394(5):851-60. Epub 2009/07/21. doi: 10.1007/s00423-009-0540-6. PubMed PMID: 19618204. 11. Dudley NE. Methylene blue for rapid identification of the parathyroids. British medical journal. 1971;3(5776):680-1. Epub 1971/09/18. PubMed PMID: 5569552; PubMed Central PMCID: PMCPMC1798947. 12. Meekin GK. Intraoperative use of methylene blue to localize parathyroid adenoma. Laryngoscope. 1998;108(5):772-3. Epub 1998/05/20. doi: 10.1097/00005537-199805000-00027. PubMed PMID: 9591562. 13. Patel HP, Chadwick DR, Harrison BJ, Balasubramanian SP. Systematic review of intravenous methylene blue in parathyroid surgery. The British journal of surgery. 2012;99(10):1345-51. Epub 2012/09/11. doi: 10.1002/bjs.8814. PubMed PMID: 22961511.

15

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

14. Patel AS, Singh-Ranger D, Lowery KA, Crinnion JN. Adverse neurologic effect of methylene blue used during parathyroidectomy. Head & neck. 2006;28(6):567-8. Epub 2006/05/09. doi: 10.1002/hed.20416. PubMed PMID: 16680760. 15. Khavandi A, Whitaker J, Gonna H. Serotonin toxicity precipitated by concomitant use of citalopram and methylene blue. Med J Aust. 189. Australia2008. p. 534-5. 16. van der Vorst JR, Schaafsma BE, Verbeek FP, Swijnenburg RJ, Tummers Q, Hutteman M, et al. Intraoperative Near-Infrared Fluorescence Imaging of Parathyroid Adenomas using Low-Dose Methylene Blue. Head & neck. 2013. Epub 2013/05/31. doi: 10.1002/hed.23384 [Epub ahead of print]. PubMed PMID: 23720199. 17. Gotoh K, Yamada T, Ishikawa O, Takahashi H, Eguchi H, Yano M, et al. A novel image-guided surgery of hepatocellular carcinoma by indocyanine green fluorescence imaging navigation. J Surg Oncol. 2009;100(1):75-9. Epub 2009/03/21. doi: 10.1002/jso.21272. PubMed PMID: 19301311. 18. Ishizawa T, Fukushima N, Shibahara J, Masuda K, Tamura S, Aoki T, et al. Real-time identification of liver cancers by using indocyanine green fluorescent imaging. Cancer. 2009;115(11):2491-504. Epub 2009/03/28. doi: 10.1002/cncr.24291. PubMed PMID: 19326450. 19. Tanaka E, Choi HS, Fujii H, Bawendi MG, Frangioni JV. Image-guided oncologic surgery using invisible light: completed pre-clinical development for sentinel lymph node mapping. Ann Surg Oncol. 2006;13(12):1671-81. Epub 2006/09/30. doi: 10.1245/s10434-006-9194-6. PubMed PMID: 17009138; PubMed Central PMCID: PMCPmc2474791. 20. van der Vorst JR, Schaafsma BE, Verbeek FP, Keereweer S, Jansen JC, van der Velden LA, et al. Near-infrared fluorescence sentinel lymph node mapping of the oral cavity in head and neck cancer patients. Oral oncology. 2013;49(1):15-9. Epub 2012/09/04. doi: 10.1016/j.oraloncology.2012.07.017. PubMed PMID: 22939692; PubMed Central PMCID: PMCPmc3608510. 21. van der Vorst JR, Schaafsma BE, Verbeek FP, Swijnenburg RJ, Hutteman M, Liefers GJ, et al. Dose optimization for near-infrared fluorescence sentinel lymph node mapping in patients with melanoma. The British journal of dermatology. 2013;168(1):93-8. Epub 2012/10/20. doi: 10.1111/bjd.12059. PubMed PMID: 23078649; PubMed Central PMCID: PMCPmc3607940. 22. Crane LM, Themelis G, Arts HJ, Buddingh KT, Brouwers AH, Ntziachristos V, et al. Intraoperative near-infrared fluorescence imaging for sentinel lymph node detection in vulvar cancer: first clinical results. Gynecologic oncology. 2011;120(2):291-5. Epub 2010/11/09. doi: 10.1016/j.ygyno.2010.10.009. PubMed PMID: 21056907. 23. Hutteman M, van der Vorst JR, Gaarenstroom KN, Peters AA, Mieog JS, Schaafsma BE, et al. Optimization of near-infrared fluorescent sentinel lymph node mapping for vulvar cancer. American journal of obstetrics and gynecology. 206. United States: Inc; 2012. p. 89 e1-5. 24. Hutteman M, van der Vorst JR, Mieog JS, Bonsing BA, Hartgrink HH, Kuppen PJ, et al. Nearinfrared fluorescence imaging in patients undergoing pancreaticoduodenectomy. European surgical research Europaische chirurgische Forschung Recherches chirurgicales europeennes. 47. Switzerland: Basel.; 2011. p. 90-7. 25. Tan SQ, Thomas D, Jao W, Bourdeau JE, Lau K. Surgical thyroparathyroidectomy of the rabbit. The American journal of physiology. 1987;252(4 Pt 2):F761-7. Epub 1987/04/01. PubMed PMID: 3565584. 26. Animal Welfare Act 2006 - Codes of Practice 2007. Available from: http://www.legislation.gov.uk/ukpga/2006/45/pdfs/ukpga_20060045_en.pdf. 27. Gonzalez-Fajardo J, Beatriz A, Perez-Burkhardt JL, Alvarez T, Fernandez L, Ramos G, et al. Epidural regional hypothermia for prevention of paraplegia after aortic occlusion: experimental evaluation in a rabbit model. J Vasc Surg. 23. United States1996. p. 446-52. 28. Chang YS, Tseng SY, Tseng SH, Chen YT, Hsiao JH. Comparison of dyes for cataract surgery. Part 1: cytotoxicity to corneal endothelial cells in a rabbit model. J Cataract Refract Surg. 31. United States2005. p. 792-8.

16

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

29. Buzato MA, Viaro F, Piccinato CE, Evora PR. The use of methylene blue in the treatment of anaphylactic shock induced by compound 48/80: experimental studies in rabbits. Shock. 23. United States2005. p. 582-7. 30. Barboriak DP, Padua AO, York GE, Macfall JR. Creation of DICOM--aware applications using ImageJ. Journal of digital imaging. 2005;18(2):91-9. Epub 2005/04/14. doi: 10.1007/s10278-0041879-4. PubMed PMID: 15827831; PubMed Central PMCID: PMCPmc3046706. 31. Collins TJ. ImageJ for microscopy. BioTechniques. 2007;43(1 Suppl):25-30. Epub 2007/10/16. PubMed PMID: 17936939. 32. Fluoptics Fluorescence imaging for surgery 2014. Available from: http://fluoptics.com/index.php. 33. McWade MA, Paras C, White LM, Phay JE, Mahadevan-Jansen A, Broome JT. A novel optical approach to intraoperative detection of parathyroid glands. Surgery. 2013;154(6):1371-7; discussion 7. Epub 2013/11/19. doi: 10.1016/j.surg.2013.06.046. PubMed PMID: 24238054; PubMed Central PMCID: PMCPmc3898879.

ACCEPTED MANUSCRIPT Table 1. Demographics and characteristics of rabbits used in fluorescent imaging experiments Rabbit

Species

Gender / Age

Weight (kg)

MB dose – period of observation

Comments

R1

NZ White

Male / 16 weeks

2.9

None

R2

NZ White

Male / 16 weeks

2.9

R3

NZ White

Male / 16 weeks

2.8

0.05 mg/kg for 36 minutes 0.1 mg/kg for 74 minutes 0.3 mg/kg for 18 minutes 3 mg/kg for 11 minutes 0.025 mg/kg for 16 minutes 0.05 mg/kg for 10 minutes 0.1 mg/kg for 13 minutes 0.3 mg/kg for 22 minutes 1 mg/kg for 26 minutes 0.05 mg/kg for 16 minutes 0.1 mg/kg for 20 minutes 0.3 mg/kg for 16 minutes 0.6 mg/kg for 19 minutes

R4

NZ White

Male / 16 weeks

2.8

R5

NZ White

Male / 16 weeks

2.8

R6

NZ WhiteCalifornian

Female / 16 weeks

3.6

RI PT

0.1 mg/kg for 30 minutes 0.3 mg/kg for 19 minutes 0.6 mg/kg for 17 minutes

None

SC

None

M AN U

TE D EP

Right PG partially devascularised during dissection + bradycardia, hypoxia & intermittent cannula obstruction during first bolus

0.1 mg/kg for 28 minutes 0.3 mg/kg for 23 minutes 0.6 mg/kg for 14 minutes 0.3 mg/kg for 26 minutes 0.6 mg/kg for 25 minutes

Note: MB – Methylene Blue; PG – Parathyroid Gland.

AC C

Right external PG lying over the Right thyroid lobe

None

ACCEPTED MANUSCRIPT Table 2. Peak fluorescence ratio of the rabbit thyroid and external parathyroid glands compared to strap muscle (control tissue) at Methylene Blue doses of 0.1, 0.3 and 0.6 mg/kg

3.5 (1.9 - 4.5)

1.85 (1.3 – 2.1)

11.575 (9.7 – 14.95)

2.125 (1.1 – 2.7)

TE D

M AN U

SC

7.475 (4.4 – 10.85)

RI PT

Peak parathyroid fluorescence ratio (compared to muscle) 0.1 mg/kg 0.3 mg/kg 0.6 mg/kg 1.45 1.65 n/a 1.3 2.7 n/a 1.85 1.1 1.25 2.1 2.5 2.5 n/a 2.6 2.55 1.95 1.75 1.9

EP

1 2 3 4 5 6 Median (range)

Peak thyroid fluorescence ratio (compared to muscle) 0.1 mg/kg 0.3 mg/kg 0.6 mg/kg 4.3 7.95 n/a 1.9 4.4 n/a 3.5 4.65 10.35 4.5 10.85 14.95 n/a 7.05 9.7 3.45 7.9 12.8

AC C

Rabbit

2.2 (1.25 – 2.55)

ACCEPTED MANUSCRIPT Figures Figure 1. Images of soft tissues in the central compartment of the rabbit neck (a) before and (b) after Methylene Blue and infrared exposure.

AC C

EP

TE D

M AN U

SC

RI PT

1a.

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

1b. Image taken approximately 20-25 seconds following MB bolus injection

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

Figure 2. Central neck compartment exposed following dissection

ACCEPTED MANUSCRIPT Figure 3. Chart illustrating changes in fluorescence intensities in rabbit thyroid, parathyroid and muscle (rabbit four). X-axis plots time following exposure of the central neck compartment to MB. Y-axis shows fluorescent intensity. The times described adjacent to the peaks refer to the 'time to peak fluorescence' in thyroid (purple) and external parathyroid glands (black). The

AC C

EP

TE D

M AN U

SC

RI PT

doses of MB used are indicated by arrows.

ACCEPTED MANUSCRIPT Figure 4. Graph showing the evolution of the fluorescence signal in thyroid and parathyroid

AC C

EP

TE D

M AN U

SC

RI PT

glands and in muscle following the IV injection of 0.1 mg/ kg of MB in rabbit 6.