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Patients undergoing gynecological procedures suffer from lasting side effects due to intraoperative nerve damage. Small, delicate nerves with complex and nonuniform branching patterns in the female pelvic neuroanatomy make nerve-sparing efforts during standard gynecological procedures such as hysterectomy, cystectomy, and colorectal cancer resection difficult, and thus many patients are left with incontinence and sexual dysfunction. Herein, a near-infrared (NIR) fluorescent nerve-specific contrast agent, LGW08-35, that is spectrally compatible with clinical fluorescence guided surgery (FGS) systems is formulated and characterized for rapid implementation for nerve-sparing gynecologic surgeries. The toxicology, pharmacokinetics (PK), and pharmacodynamics (PD) of micelle formulated LGW08-35 are examined, enabling the determination of the optimal imaging doses and time points, blood and tissue uptake parameters, and maximum tolerated dose (MTD). Application of the formulated fluorophore to imaging of female rat and swine pelvic neuroanatomy validates the continued clinical translation and use for real-time identification of important nerves such as the femoral, sciatic, lumbar, iliac, and hypogastric nerves. Further development of LGW08-35 for clinical use will unlock a valuable tool for surgeons in direct visualization of important nerves and contribute to the ongoing characterization of the female pelvic neuroanatomy to eliminate the debilitating side effects of nerve damage during gynecological procedures., (© 2023 Wiley‐VCH GmbH.)

Fluorescence-guided surgery (FGS) is poised to revolutionize surgical medicine through near-infrared (NIR) fluorophores for tissue- and disease-specific contrast. Clinical open and laparoscopic FGS vision systems operate nearly exclusively at NIR wavelengths. However, tissue-specific NIR contrast agents compatible with clinically available imaging systems are lacking, leaving nerve tissue identification during prostatectomy a persistent challenge. Here, it is shown that combining drug-like molecular design concepts and fluorophore chemistry enabled the production of a library of NIR phenoxazine-based fluorophores for intraoperative nerve-specific imaging. The lead candidate readily delineated prostatic nerves in the canine and iliac plexus in the swine using the clinical da Vinci Surgical System that has been popularized for minimally invasive prostatectomy procedures. These results demonstrate the feasibility of molecular engineering of NIR nerve-binding fluorophores for ready integration into the existing surgical workflow, paving the path for clinical translation to reduce morbidity from nerve injury for prostate cancer patients., (© 2023 The Authors. Advanced Materials published by Wiley‐VCH GmbH.)

Purpose: Reliable and rapid identification of tumor in the margins of breast specimens during breast-conserving surgery to reduce repeat surgery rates is an active area of investigation. Dual-stain difference imaging (DDSI) is one of many approaches under evaluation for this application. This technique aims to topically apply fluorescent stain pairs (one targeted to a receptor-of-interest and the other a spectrally distinct isotype), image both stains, and compute a normalized difference image between the two channels. Prior evaluation and optimization in a variety of preclinical models produced encouraging diagnostic performance. Herein, we report on a pilot clinical study which evaluated HER2-targeted DDSI on 11 human breast specimens., Procedures: Gross sections from 11 freshly excised mastectomy specimens were processed using a HER2-receptor-targeted DDSI protocol shortly after resection. After staining with the dual-probe protocol, specimens were imaged on a fluorescence scanner, followed by tissue fixation for hematoxylin and eosin and anti-HER2 immunohistochemical staining. Receiver operator characteristic curves and area under the curve (AUC) analysis were used to assess diagnostic performance of the resulting images. Performance values were also compared to expression level determined from IHC staining., Results: Eight of the 11 specimens presented with distinguishable invasive ductal carcinoma and/or were not affected by an imaging artifact. In these specimens, the DDSI technique provided an AUC = 0.90 ± 0.07 for tumor-to-adipose tissue and 0.81 ± 0.15 for tumor-to-glandular tissue, which was significantly higher than AUC values recovered from images of the targeted probe alone. DDSI values and diagnostic performance did not correlate with HER2 expression level, and tumors with low HER2 expression often produced high AUC, suggesting that even the low expression levels were enough to help distinguish tumor., Conclusions: The results from this preliminary study of rapid receptor-specific staining in human specimens were consistent with prior preclinical results and demonstrated promising diagnostic potential., (© 2023. The Author(s).)

Significance: Positive margin status due to incomplete removal of tumor tissue during radical prostatectomy for high-risk localized prostate cancer requires reoperation or adjuvant therapy, which increases morbidity and mortality. Adverse effects of prostate cancer treatments commonly include erectile dysfunction, urinary incontinence, and bowel dysfunction, making successful initial curative prostatectomy imperative., Aim: Current intraoperative tumor margin assessment is largely limited to frozen section analysis, which is a lengthy, labor-intensive process that is obtrusive to the clinical workflow within the operating room (OR). Therefore, a rapid method for prostate cancer margin assessment in the OR could improve outcomes for patients., Approach: Dual probe difference specimen imaging (DDSI), which uses paired antibody-based probes that are labeled with spectrally distinct fluorophores, was shown herein for prostate cancer margin assessment. The paired antibody-based probes consisted of a targeted probe to prostate-specific membrane antigen (PSMA) and an untargeted probe, which were used as a cocktail to stain resected murine tissue specimens including prostate tumor, adipose, muscle, and normal prostate. Ratiometric images (i.e., DDSI) of the difference between targeted and untargeted probe uptake were calculated and evaluated for accuracy using receiver operator characteristic curve analysis with area under the curve values used to evaluate the utility of the DDSI method to detect PSMA positive prostate cancer., Results: Targeted and untargeted probe uptake was similar between the high and low PSMA expressing tumor due to nonspecific probe uptake after topical administration. The ratiometric DDSI approach showed substantial contrast difference between the PSMA positive tumors and their respective normal tissues (prostate, adipose, muscle). Furthermore, DDSI showed substantial contrast difference between the high PSMA expressing tumors and the minimally PSMA expressing tumors due to the ratiometric correction for the nonspecific uptake patterns in resected tissues., Conclusions: Previous work has shown that ratiometic imaging has strong predictive value for breast cancer margin status using topical administration. Translation of the ratiometric DDSI methodology herein from breast to prostate cancers demonstrates it as a robust, ratiometric technique that provides a molecularly specific imaging modality for intraoperative margin detection. Using the validated DDSI protocol on resected prostate cancers permitted rapid and accurate assessment of PSMA status as a surrogate for prostate cancer margin status. Future studies will further evaluate the utility of this technology to quantitatively characterize prostate margin status using PSMA as a biomarker., (© 2023 The Authors.)

Significance: This first-in-kind, perfused, and amputated human limb model allows for the collection of human data in preclinical selection of lead fluorescent agents. The model facilitates more accurate selection and testing of fluorophores with human-specific physiology, such as differential uptake and signal in fat between animal and human models with zero risk to human patients. Preclinical testing using this approach may also allow for the determination of tissue toxicity, clearance time of fluorophores, and the production of harmful metabolites., Aim: This study was conducted to determine the fluorescence intensity values and tissue specificity of a preclinical, nerve tissue targeted fluorophore, as well as the capacity of this first-in-kind model to be used for lead fluorescent agent selection in the future., Approach: Freshly amputated human limbs were perfused for 30 min prior to in situ and ex vivo imaging of nerves with both open-field and closed-field commercial fluorescence imaging systems., Results: In situ , open-field imaging demonstrated a signal-to-background ratio (SBR) of 4.7 when comparing the nerve with adjacent muscle tissue. Closed-field imaging demonstrated an SBR of 3.8 when the nerve was compared with adipose tissue and 4.8 when the nerve was compared with muscle., Conclusions: This model demonstrates an opportunity for preclinical testing, evaluation, and selection of fluorophores for use in clinical trials as well as an opportunity to study peripheral pathologies in a controlled environment., (© 2023 The Authors.)

We have co-developed a first-in-kind model of fluorophore testing in freshly amputated human limbs. Ex vivo human tissue provides a unique opportunity for the testing of pre-clinical fluorescent agents, collection of imaging data, and histopathologic examination in human tissue prior to performing in vivo experiments. Existing pre-clinical fluorescent agent studies rely primarily on animal models, which do not directly predict fluorophore performance in humans and can result in wasted resources and time if an agent proves ineffective in early human trials. Because fluorophores have no desired therapeutic effect, their clinical utility is based solely on their safety and ability to highlight tissues of interest. Advancing to human trials even via the FDA's phase 0/microdose pathway still requires substantial resources, single-species pharmacokinetic testing, and toxicity testing. In a recently concluded study using amputated human lower limbs, we were able to test successfully a nerve-specific fluorophore in pre-clinical development. This study used systemic administration via vascular cannulization and a cardiac perfusion pump. We envision that this model may assist with early lead agent testing selection for fluorophores with various targets and mechanisms.

Iatrogenic nerve injury is a common complication across all surgical specialties. Better nerve visualization and identification during surgery will improve outcomes and reduce nerve injuries. The Gibbs Laboratory at Oregon Health and Science University has developed a library of near-infrared, nerve-specific fluorophores to highlight nerves intraoperatively and aid surgeons in nerve identification and visualization; the current lead agent is LGW16-03. Prior to this study, testing of LGW16-03 was restricted to animal models; therefore, it was unknown how LGW16-03 performs in human tissue. To advance LGW16-03 to clinic, we sought to test this current lead agent in ex vivo human tissues from a cohort of patients and determine if the route of administration affects LGW16-03 fluorescence contrast between nerves and adjacent background tissues (muscle and adipose). LGW16-03 was applied to ex vivo human tissue from lower limb amputations via two strategies: (1) systemic administration of the fluorophore using our first-in-kind model for fluorophore testing, and (2) topical application of the fluorophore. Results showed no statistical difference between topical and systemic administration. However, in vivo human validation of these findings is required.

Iatrogenic nerve injury significantly affects surgical outcomes. Although intraoperative neuromonitoring is utilized, nerve identification remains challenging and the success of nerve sparing is strongly correlated with surgeon experience levels. Fluorescence guided surgery (FGS) offers a potential solution for improved nerve sparing by providing direct visualization of nerve tissue intraoperatively. However, novel probes for FGS face a long regulatory pathway to achieve clinical translation. Herein, we report on the development of a clinically-viable, gel-based formulation that enables direct administration of nerve-specific probes for nerve sparing FGS applications, facilitating clinical translation via the exploratory investigational new drug (eIND) guidance. The developed formulation possesses unique gelling characteristics, allowing it to be easily spread as a liquid followed by rapid gelling for subsequent tissue hold. Optimization of the direct administration protocol with our gel-based formulation enabled a total staining time of 1-2 min for compatibility with surgical procedures and successful clinical translation., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Nerves are extremely difficult to identify and are often accidently damaged during surgery, leaving patients with lasting pain and numbness. Herein, a novel near-infrared (NIR) nerve-specific fluorophore, LGW01-08, was utilized for enhanced nerve identification using fluorescence guided surgery (FGS), formulated using clinical translatable strategies. Formulated LGW01-08 was examined for toxicology, pharmacokinetics (PK), and pharmacodynamics (PD) parameters in preparation for future clinical translation. Optimal LGW01-08 imaging doses were identified in each formulation resulting in a 10x difference between the toxicity to imaging dose window. Laparoscopic swine surgery completed using the da Vinci surgical robot (Intuitive Surgical) demonstrated the efficacy of formulated LGW01-08 for enhanced nerve identification. NIR fluorescence imaging enabled clear identification of nerves buried beneath ~3 mm of tissue that were unidentifiable by white light imaging. These studies provide a strong basis for future clinical translation of NIR nerve-specific fluorophores for utility during FGS to improve patient outcomes., Competing Interests: COMPETING INTERESTS Alexander Antaris, Alwin Klaassen and Jonathan Sorger are employees of Intuitive Surgical. Lei Wang, Connor Barth and Summer Gibbs are inventors on patent application PCT/US19/43739 held by Oregon Health and Science University that cover the composition and methods of use of the nerve-specific oxazine compound (LGW01–08) discussed in this manuscript. Lei Wang, Connor Barth, Vidhi Shah, Adam Alani and Summer Gibbs are inventors on patent application PCT/US19/45347 held jointly by Oregon Health and Science University and Oregon State University that covers the compositions and methods of use of the formulation strategies developed herein. Lei Wang, Connor Barth and Summer Gibbs are co-founders of Inherent Targeting, LLC.

Background: Re-excision rates following breast conserving surgery (BCS) remain as high as ~ 35%, with positive margins detected during follow-up histopathology. Additional breast cancer resection surgery is not only taxing on the patient and health care system, but also delays adjuvant therapies, increasing morbidity and reducing the likelihood of a positive outcome. The ability to precisely resect and visualize tumor margins in real time within the surgical theater would greatly benefit patients, surgeons and the health care system. Current tumor margin assessment technologies utilized during BCS involve relatively lengthy and labor-intensive protocols, which impede the surgical work flow., Methods: In previous work, we have developed and validated a fluorescence imaging method termed dual probe difference specimen imaging (DDSI) to accurately detect benign and malignant tissue with direct correlation to the targeted biomarker expression levels intraoperatively. The DDSI method is currently on par with touch prep cytology in execution time (~ 15-min). In this study, the main goal was to shorten the DDSI protocol by decreasing tissue blocking and washing times to optimize the DDSI protocol to < 10-min whilst maintaining robust benign and malignant tissue differentiation., Results: We evaluated the utility of the shortened DDSI staining methodology using xenografts grown from cell lines with varied epidermal growth factor receptor (EGFR) expression levels, comparing accuracy through receiver operator characteristic (ROC) curve analyses across varied tissue blocking and washing times. An optimized 8-min DDSI methodology was developed for future clinical translation., Conclusions: Successful completion of this work resulted in substantial shortening of the DDSI methodology for use in the operating room, that provided robust, highly receptor specific, sensitive diagnostic capabilities between benign and malignant tissues.

Nerve damage is a major complication of surgery, causing pain and loss of function. We have identified novel near-infrared nerve-specific fluorophores that provide excellent nerve contrast with the ability to identify buried nerve tissue.

Nerve-binding fluorophores with near-infrared (NIR; 650 to 900 nm) emission could reduce iatrogenic nerve injury rates by providing surgeons precise, real-time visualization of the peripheral nervous system. Unfortunately, current systemically administered nerve contrast agents predominantly emit at visible wavelengths and show nonspecific uptake in surrounding tissues such as adipose, muscle, and facia, thus limiting detection to surgically exposed surface-level nerves. Here, a focused NIR fluorophore library was synthesized and screened through multi-tiered optical and pharmacological assays to identify nerve-binding fluorophore candidates for clinical translation. NIR nerve probes enabled micrometer-scale nerve visualization at the greatest reported tissue depths (~2 to 3 mm), a feat unachievable with previous visibly emissive contrast agents. Laparoscopic fluorescent surgical navigation delineated deep lumbar and iliac nerves in swine, most of which were invisible in conventional white-light endoscopy. Critically, NIR oxazines generated contrast against all key surgical tissue classes (muscle, adipose, vasculature, and fascia) with nerve signal-to-background ratios ranging from ~2 (2- to 3-mm depth) to 25 (exposed nerve). Clinical translation of NIR nerve-specific agents will substantially reduce comorbidities associated with surgical nerve damage., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

In the past several decades, a number of novel fluorescence image-guided surgery (FGS) contrast agents have been under development, with many in clinical translation and undergoing clinical trials. In this review, we have identified and summarized the contrast agents currently undergoing clinical translation. In total, 39 novel FGS contrast agents are being studied in 85 clinical trials. Four FGS contrast agents are currently being studied in phase III clinical trials and are poised to reach FDA approval within the next two to three years. Among all novel FGS contrast agents, a wide variety of probe types, targeting mechanisms, and fluorescence properties exists. Clinically available FGS imaging systems have been developed for FDA approved FGS contrast agents, and thus further clinical development is required to yield FGS imaging systems tuned for the variety of contrast agents in the clinical pipeline. Additionally, study of current FGS contrast agents for additional disease types and development of anatomy specific contrast agents is required to provide surgeons FGS tools for all surgical specialties and associated comorbidities. The work reviewed here represents a significant effort from many groups and further development of this promising technology will have an enormous impact on surgical outcomes across all specialties.

Accidental nerve damage or transection of vital nerve structures remains an unfortunate reality that is often associated with surgery. Despite the existence of nerve-sparing techniques, the success of such procedures is not only complicated by anatomical variance across patients but is also highly dependent on a surgeon's first-hand experience that is acquired over numerous procedures through trial and error, often with highly variable success rates. Fluorescent small molecules, such as rhodamines and fluoresceins have proven incredibly useful for biological imaging in the life sciences, and they appeared to have potential in illuminating vital nerve structures during surgical procedures. In order to make use of the current clinically relevant imaging systems and to provide surgeons with fluorescent contrast largely free from the interference of hemoglobin and water, it was first necessary to spectrally tune known fluorescent scaffolds towards near infrared (NIR) wavelengths. To determine whether the well-documented Si-substitution strategy could be applied towards developing a NIR fluorophore that retained nerve-specific properties of candidate molecules, an in vivo comparison was made between two compounds previously shown to highlight nervous structures - TMR and Rhodamine B - and their Si-substituted derivatives.

The current five-year survival rate of <5% for pancreatic ductal adenocarcinoma (PDAC) is compounded by late diagnosis, a lack of PDAC-specific intraoperative guidance to ensure complete resection, and the ineffectiveness of current therapies. Previously, utilizing compound 1, a fluorophore with inherent PDAC selectivity, PDAC was visualized both in vivo and ex vivo in a murine model. In the current study, human PDAC tissue is targeted. Compound 1 selectively stains ducts of the adenocarcinoma versus the surrounding stroma, enabling the imaging of PDAC in frozen tissue sections with high contrast. To enhance the potential of 1 for intraoperative applications, the ex vivo staining protocol was optimized for rapid margin assessment, with a final staining time of ~15 min. To measure diagnostic performance, the area under a receiver operating characteristic (ROC) curve was measured for the identification of ductal adenocarcinoma vs. stroma. The bright fluorescence contrast enabled quantitative determination of PDAC (or precancerous PanIN lesions) versus healthy pancreas tissue in human tissue array samples., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Accidental nerve transection or injury is a significant morbidity associated with many surgical interventions, resulting in persistent postsurgical numbness, chronic pain, and/or paralysis. Nerve-sparing can be a difficult task due to patient-to-patient variability and the difficulty of nerve visualization in the operating room. Fluorescence image-guided surgery to aid in the precise visualization of vital nerve structures in real time during surgery could greatly improve patient outcomes. To date, all nerve-specific contrast agents emit in the visible range. Developing a near-infrared (NIR) nerve-specific fluorophore is poised to be a challenging task, as a NIR fluorophore must have enough "double-bonds" to reach the NIR imaging window, contradicting the requirement that a nerve-specific agent must have a relatively low molecular weight to cross the blood-nerve-barrier (BNB). Herein we report our efforts to investigate the molecular characteristics for the nerve-specific oxazine fluorophores, as well as their structurally analogous rhodamine fluorophores. Specifically, optical properties, physicochemical properties and their in vivo nerve specificity were evaluated herein.

Identification of tumor margins in the operating room in real time is a critical challenge for surgical procedures that serve as cancer cure. Breast conserving surgery (BCS) is particularly affected by this problem, with current re-excision rates above 25%. Due to a lack of clinically available methodologies for detection of involved or close tumor margins, much effort is focused on developing intraoperative margin assessment modalities that can aid in addressing this unmet clinical need. BCS provides a unique opportunity to design contrast-based technologies that are able to assess tumor margins independent from the patient, providing a rapid pathway from bench to bedside at a much lower cost. Since resected tissue is removed from the patient's blood supply, non-specific contrast agent uptake becomes a challenge due to the lack of clearance. Therefore, a dual probe, ratiometric fluorescence imaging approach was taken in an effort to reduce non-specific signal, and provide a modality that could demonstrate rapid, robust margin assessment on resected patient samples. Termed, dual-stain difference specimen imaging (DDSI), DDSI includes the use of spectrally unique, and fluorescently labeled target-specific, as well as non-specific biomarkers. In the present study, we have applied epidermal growth factor receptor (EGFR) targeted DDSI to tumor xenografts with variable EGFR expression levels using a previously optimized staining protocol, allowing for a quantitative assessment of the predictive power of the technique under biologically relevant conditions. Due to the presence of necrosis in the model tumors, ring analysis was employed to characterize diagnostic accuracy as measured by receiver operator characteristic (ROC) curve analysis. Our findings demonstrate the robust nature of the DDSI technique even in the presence of variable biomarker expression and spatial patterns. These results support the continued development of this technology as a robust diagnostic tool for tumor margin assessment in resected specimens during BCS.

Intraoperative margin assessment is imperative to cancer cure but is a continued challenge to successful surgery. Breast conserving surgery is a relevant example, where a cosmetically improved outcome is gained over mastectomy, but re-excision is required in >25  %   of cases due to positive or closely involved margins. Clinical translation of margin assessment modalities that must directly contact the patient or required administered contrast agents are time consuming and costly to move from bench to bedside. Tumor resections provide a unique surgical opportunity to deploy margin assessment technologies including contrast agents on the resected tissues, substantially shortening the path to the clinic. However, staining of resected tissues is plagued by nonspecific uptake. A ratiometric imaging approach where matched targeted and untargeted probes are used for staining has demonstrated substantially improved biomarker quantification over staining with conventional targeted contrast agents alone. Our group has developed an antibody-based ratiometric imaging technology using fluorescently labeled, spectrally distinct targeted and untargeted antibody probes termed dual-stain difference specimen imaging (DDSI). Herein, the targeted biomarker expression level and pattern are evaluated for their effects on DDSI diagnostic potential. Epidermal growth factor receptor expression level was correlated to DDSI diagnostic potential, which was found to be robust to spatial pattern expression variation. These results highlight the utility of DDSI for accurate margin assessment of freshly resected tumor specimens.

Rationale: Positive margin status due to incomplete removal of tumor tissue during breast conserving surgery (BCS) is a prevalent diagnosis usually requiring a second surgical procedure. These follow-up procedures increase the risk of morbidity and delay the use of adjuvant therapy; thus, significant efforts are underway to develop new intraoperative strategies for margin assessment to eliminate re-excision procedures. One strategy under development uses topical application of dual probe staining and a fluorescence imaging strategy termed dual probe difference specimen imaging (DDSI). DDSI uses a receptor-targeted fluorescent probe and an untargeted, spectrally-distinct fluorescent companion imaging agent topically applied to fresh resected specimens, where the fluorescence from each probe is imaged and a normalized difference image is computed to identify tumor-target distribution in the specimen margins. While previous reports suggested this approach is a promising new tool for surgical guidance, advancing the approach into the clinic requires methodical protocol optimization and further validation., Methods: In the present study, we used breast cancer xenografts and receiver operator characteristic (ROC) curve analysis to evaluate a wide range of staining and imaging parameters, and completed a prospective validation study on multiple tumor phenotypes with different target expression. Imaging fluorophore-probe pair, concentration, and incubation times were systematically optimized using n=6 tissue specimen replicates per staining condition. Resulting tumor vs. normal adipose tissue diagnostic performance were reported and staining patterns were validated via receptor specific immunohistochemistry colocalization. Optimal staining conditions were tested in receptor positive and receptor negative cohorts to confirm specificity., Results: The optimal staining conditions were found to be a one minute stain in a 200 nM probe solution (area under the curve (AUC) = 0.97), where the choice of fluorescent label combination did not significantly affect the diagnostic performance. Using an optimal threshold value determined from ROC curve analysis on a training data set, a prospective study on xenografts resulted in an AUC=0.95 for receptor positive tumors and an AUC = 0.50 for receptor negative (control) tumors, confirming the diagnostic performance of this novel imaging technique., Conclusions: DDSI provides a robust, molecularly specific imaging methodology for identifying tumor tissue over benign mammary adipose tissue. Using a dual probe imaging strategy, nonspecific accumulation of targeted probe was corrected for and tumor vs. normal tissue diagnostic potential was improved, circumventing difficulties with ex vivo tissue specimen staining and allowing for rapid clinical translation of this promising technology for tumor margin detection during BCS procedures., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

Molecular probes that selectively highlight pancreatic cancer (PC) tissue have the potential to improve pancreatic ductal adenocarcinoma (PDAC) margin assessment through the selective highlighting of individual PC cells. Herein, we report a simple and unique family of systematically modified red and near-infrared fluorescent probes that exhibit a field-effect-derived redshift. Two of thirteen probes distributed to the normal mouse pancreas following systemic administration. One selectively accumulated in genetically modified mouse models of PDAC. The probe exhibited intracellular accumulation and enabled visualization of four levels of the structure, including the whole organ, resected tissue, individual cells, and subcellular organelles. In contrast to the small-molecule probes reported previously, it possesses an inherent affinity toward PDAC cells and thus does not require conjugation to any targeting agent. The fluorescent probe can thus promote new strategies not only for precision image-guided surgery, but also for PC detection, monitoring of therapeutic outcomes, and basic research., Competing Interests: The authors declare no competing financial interest.

Nerve damage remains a major morbidity following nerve sparing radical prostatectomy, significantly affecting quality of life post-surgery. Nerve-specific fluorescence guided surgery offers a potential solution by enhancing nerve visualization intraoperatively. However, the prostate is highly innervated and only the cavernous nerve structures require preservation to maintain continence and potency. Systemic administration of a nerve-specific fluorophore would lower nerve signal to background ratio (SBR) in vital nerve structures, making them difficult to distinguish from all nervous tissue in the pelvic region. A direct administration methodology to enable selective nerve highlighting for enhanced nerve SBR in a specific nerve structure has been developed herein. The direct administration methodology demonstrated equivalent nerve-specific contrast to systemic administration at optimal exposure times. However, the direct administration methodology provided a brighter fluorescent nerve signal, facilitating nerve-specific fluorescence imaging at video rate, which was not possible following systemic administration. Additionally, the direct administration methodology required a significantly lower fluorophore dose than systemic administration, that when scaled to a human dose falls within the microdosing range. Furthermore, a dual fluorophore tissue staining method was developed that alleviates fluorescence background signal from adipose tissue accumulation using a spectrally distinct adipose tissue specific fluorophore. These results validate the use of the direct administration methodology for specific nerve visualization with fluorescence image-guided surgery, which would improve vital nerve structure identification and visualization during nerve sparing radical prostatectomy., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

Nerve damage plagues surgical outcomes and remains a major burden for patients, surgeons, and the healthcare system. Fluorescence image-guided surgery using nerve specific small molecule fluorophores offers a solution to diminish surgical nerve damage through improved intraoperative nerve identification and visualization. Oxazine 4 has shown superior nerve specificity in initial testing in vivo , while exhibiting a red shifted excitation and emission spectra compared to other nerve-specific fluorophores. However, Oxazine 4 does not exhibit near-infrared (NIR) excitation and emission, which would be ideal to improve penetration depth and nerve signal to background ratios for in vivo imaging. Successful development of a NIR nerve-specific fluorophore will require understanding of the molecular target of fluorophore nerve specificity. While previous small molecule nerve-specific fluorophores have demonstrated excellent ex vivo nerve specificity, Oxazine 4 ex vivo nerve specific fluorescence has been difficult to visualize. In the present study, we examined each step of the ex vivo fluorescence microscopy sample preparation procedure to discover how in vivo nerve-specific fluorescence is changed during ex vivo tissue sample preparation. Through step-by-step examination we found that Oxazine 4 fluorescence was significantly diminished by washing and mounting tissue sections for microscopy. A method to preserve Oxazine 4 nerve specific fluorescence ex vivo was determined, which can be utilized for visualization by fluorescence microscopy.

Nerve damage during surgery is a common morbidity experienced by patients that leaves them with chronic pain and/or loss of function. Currently, no clinically approved imaging technique exists to enhance nerve visualization in the operating room. Fluorescence image-guided surgery has gained in popularity and clinical acceptance over the past decade with a handful of imaging systems approved for clinical use. However, contrast agent development to complement these fluorescence-imaging systems has lagged behind with all currently approved fluorescent agents providing untargeted blood pool information. Nerve-specific fluorophores are known, however translations of these agents to the clinic has been complicated by their lipophilic nature, which necessitates specialized formulation strategies for successful systemic administration. To date the known nerve-specific fluorophores have only been demonstrated preclinically due to the necessity of a dimethyl sulfoxide containing formulation for solubilization. In the current study, a polymeric micellar (PM) formulation strategy was developed for a representative nerve-specific fluorophore from the distyrylbenzene family, BMB. The PM formulation strategy was able to solubilize BMB and demonstrated improved nerve-specific accumulation and fluorescence intensity when the same fluorophore dose was administered to mice utilizing the previous formulation strategy. The success of the PM formulation strategy will be important for moving toward clinical translation of these novel nerve-specific probes as it is nontoxic and biodegradable and has the potential to decrease the necessary dose for imaging while also improving the safety profile.

Population-based threshold models using hydrothermal time (HTT) have been widely used to model seed germination. We used HTT to model conidial germination and mycelial growth for the seed pathogen Pyrenophora semeniperda in a novel approach to understanding its interactions with host seeds. Germination time courses and mycelial growth rates for P.semeniperda were measured on PDA amended to achieve a series of five water potentials (ca. 0 to -6 MPa) at six constant temperatures (5-30 °C). Conidial germination was described with alternative population-based models using constant or variable base and maximum temperature and water potential parameters. Mycelial growth was modeled as a continuous, linear process with constant base temperature and base water potential. Models based on HTT showed reasonable fit to germination and growth rate data sets. The best-fit conidial germination model (R(2) = 0.859) was based on variable base and maximum temperature as a function of water potential. The good fit of the linear mycelial growth model (R(2) = 0.916) demonstrated the utility of HTT for modeling continuous as well as population-based processes. HTT modeling may be a useful approach to the quantification of germination and growth processes in a wide range of filamentous fungi., (Published by Elsevier Ltd.)

We evaluated the efficacy and safety of elective percutaneous coronary intervention (PCI) at a hospital without onsite cardiac surgery. A growing number of hospitals without onsite cardiac surgery perform elective PCI. Few hospitals have reported outcomes, despite controversy surrounding this practice. From August 2003 to December 2005, 1,090 elective PCI were performed at Saint Luke's South Hospital (SLS), a hospital without onsite cardiac surgery, for which the referral center is the Mid America Heart Institute (MAHI). The elective PCI program used experienced interventionalists, technicians, and nurses; a tested helicopter transport protocol; a well-equipped catheterization laboratory; and a quality assurance process. Baseline characteristics, procedural success, and adverse clinical outcomes were compared. Observed frequencies of in-hospital death, a combined end point of Q-wave myocardial infarction (MI)/emergency coronary artery bypass grafting (CABG) surgery, and vascular complications were compared with prediction models. SLS, with lower risk characteristics than MAHI, had unadjusted frequencies of procedural success (93% vs 94%, p = NS), Q-wave MI (0.3% vs 0.3%, p = NS), emergency CABG surgery (0.2% vs 0.03%, p = 0.09), vascular complications (0.6% vs 0.6%, p = NS), and in-hospital death (0.1% vs 0.8%, p = 0.002) that compared favorably with MAHI. Two patients transferred from SLS to MAHI for emergency CABG surgery without adverse effects. Fewer in-hospital deaths and vascular complications were observed at SLS than predicted by models. In conclusion, favorable clinical outcomes were achieved for elective PCI at a hospital without onsite cardiac surgery that used strict program requirements.

Background: The optimal percutaneous interventional strategy for dealing with significant non-culprit lesions in patients with multivessel disease (MVD) with acute myocardial infarction (AMI) at presentation remains controversial., Methods: A total of 820 patients treated with primary angioplasty for AMI between 1998 and 2002 were classified in groups of patients with single vessel disease (SVD) or MVD (> or =70% stenosis of > or =2 coronary arteries). Patients with MVD were subdivided in 3 groups on the basis of the revascularization strategy: 1) patients undergoing percutaneous coronary intervention (PCI) of the infarct-related artery (IRA) only; 2) patients undergoing PCI of both the IRA and non-IRA(s) during the initial procedure; and 3) patients undergoing PCI of the IRA followed by staged, in-hospital PCI of the non-IRA(s). Procedural, 30-day, and 1-year outcomes are reported., Results: At 1 year, compared with patients with SVD, patients with MVD had a higher incidence of re-infarction (5.9% vs 1.6%, P =.003), revascularization (18% vs 9.6%, P <.001), mortality (12% vs 3.2%, P <.001), and major adverse cardiac events (MACEs; 31% vs 13%, P <.001). In patients with MVD, compared with PCI restricted to the IRA only, multivessel PCI was associated with higher rates of re-infarction (13.0% vs 2.8%, P <.001), revascularization (25% vs 15%, P =.007), and MACEs (40% vs 28%, P =.006). Multivessel PCI was an independent predictor of MACEs at 1 year (odds ratio = 1.67, P =.01)., Conclusions: These data suggest that in patients with MVD, PCI should be directed at the IRA only, with decisions about PCI of non-culprit lesions guided by objective evidence of residual ischemia at late follow-up. Further studies are needed to confirm these findings.

Distal embolization of atheroma and thrombus is a major concern when performing balloon angioplasty in coronary saphenous vein grafts (SVGs). The transluminal extraction catheter (TEC) is designed to remove this material and may improve the safety of percutaneous treatment of SVG disease. We assessed the acute results and long-term outcome of 67 patients (mean age 65.6 +/- 8.1 years; range 47-83 years) who underwent 73 separate TEC atherectomy procedures. Eighty-eight SVG lesions were treated (mean age 8.7 +/- 3.8 years from bypass surgery). Procedural success (< 50% final diameter stenosis and absence of major complications) was obtained in 63 patients (86%). Adjunctive balloon angioplasty and/or directional coronary atherectomy was required in 69 of the procedures (95%). Major complications, occurring in 8 patients (11%), were acute closure in 4 (5%), resulting in Q-wave myocardial infarction in 3 and urgent bypass surgery in 1, and distal embolization in 4 (5%; 1 associated with Q-wave myocardial infarction). Angiographic follow-up was available for 50 patients and restenosis was present in 26 (52%). These data suggest TEC atherectomy can be performed in SVGs with an acceptable procedural risk, but restenosis remains a significant limitation which will require other strategies to overcome.

Blunt chest trauma can result in significant cardiothoracic injury, which can include cardiac contusion, aortic injury, and myocardial valvular injury. Nineteen patients with no prior history of cardiac abnormalities who sustained severe blunt chest trauma and had widening of the mediastinum on chest radiographs were prospectively evaluated using transesophageal echocardiography (TEE). In each instance TEE was performed without difficulty, excellent images were obtained of the aorta and heart, and no complications were noted. Abnormalities were seen in 12 (63%) patients, with hypokinetic regional wall motion consistent with cardiac contusion demonstrated in five (26%) patients. Tricuspid regurgitation was found in three (16%) patients, and aortic and mitral regurgitation in one (5%) patient each. Aortic wall hematomas were seen in two patients, one of whom had an intimal tear on aortography, and a pericardial effusion was seen in one patient with an aortic intimal tear confirmed angiographically. Thus TEE can be performed safely in the acute setting of patients sustaining severe blunt chest trauma and yield useful information with respect to cardiovascular function and the aorta.

It is often difficult to delineate the true course of anomalous coronary arteries by angiography because it only provides a 2-dimensional view of a complex 3-dimensional structure. The purpose of this study was to confirm morphologically the radiographic appearance of anomalous coronary arteries and to construct a protocol for rapid determination of their true course. Twenty-one adults who had anomalous origin of coronary arteries without other evidence of congenital heart disease were reviewed. Using an anatomically correct model of the heart, solder wire was placed in the pathologically described anomalous positions and radiographed. With this model the pathologically described courses could be easily recognized and separated radiographically. These courses were confirmed in the operating room in 2 patients and a rare anomaly of posterior origin of a coronary artery was also confirmed by autopsy.

Findings are described in five patients who at necropsy were found to have origin of the left main coronary artery from the right sinus of Valsalva and coursing of the anomalously arising artery between aorta and pulmonary trunk to reach the left side of the heart. Three of the five patients were boys and died suddenly at ages 13, 14 and 19 years, respectively: two of them had had one or more episodes of syncope and the third had an abnormal electrocardiogram. The fourth patient, a 64 year old woman, died of chronic congestive heart failure 1 year after an acute myocardial infarction. She had insignificant coronary atherosclerosis. The fifth patient, an 81 year old man, died of chronic alcoholism, having been free of symptoms of cardiac dysfunction during life. Additionally, clinical and necropsy findings are summarized in 38 previously reported necropsy patients with the coronary anomaly. Of these 38 (34 male [89%]), 23 (61%) died suddenly in the first two decades of life; death in 6 others (16%) appears to have been related to coronary atherosclerosis and 9 patients (24%) died from noncoronary causes. Thus, this anomaly is life-threatening. Why it frequently causes fatal cardiac arrest in some young individuals and allows a normal life span in others remains unclear.