Intra-Tumoral Nerve-Tracing in a Novel Syngeneic Model of High-Grade Serous Ovarian Carcinoma.

Dense tumor innervation is associated with enhanced cancer progression and poor prognosis. We observed innervation in breast, prostate, pancreatic, lung, liver, ovarian, and colon cancers. Defining innervation in high-grade serous ovarian carcinoma (HGSOC) was a focus since sensory innervation was observed whereas the normal tissue contains predominantly sympathetic input. The origin, specific nerve type, and the mechanisms promoting innervation and driving nerve-cancer cell communications in ovarian cancer remain largely unknown. The technique of neuro-tracing enhances the study of tumor innervation by offering a means for identification and mapping of nerve sources that may directly and indirectly affect the tumor microenvironment. Here, we establish a murine model of HGSOC and utilize image-guided microinjections of retrograde neuro-tracer to label tumor-infiltrating peripheral neurons, mapping their source and circuitry. We show that regional sensory neurons innervate HGSOC tumors. Interestingly, the axons within the tumor trace back to local dorsal root ganglia as well as jugular-nodose ganglia. Further manipulations of these tumor projecting neurons may define the neuronal contributions in tumor growth, invasion, metastasis, and responses to therapeutics.

Reverse engineering of an anatomically equivalent nerve conduit.

Reconstruction of peripheral nervous tissue remains challenging in critical-sized defects due to the lack of Büngner bands from the proximal to the distal nerve ends. Conventional nerve guides fail to bridge the large-sized defect owing to the formation of a thin fibrin cable. Hence, in the present study, an attempt was made to reverse engineer the intricate epi-, peri- and endo-neurial tissues using Fused Deposition Modeling based 3D printing. Bovine serum albumin protein nanoflowers (NF) exhibiting Viburnum opulus 'Roseum' morphology were ingrained into 3D printed constructs without affecting its secondary structure to enhance the axonal guidance from proximal to distal ends of denuded nerve ends. Scanning electron micrographs confirmed the uniform distribution of protein NF in 3D printed constructs. The PC-12 cells cultured on protein ingrained 3D printed scaffolds demonstrated cytocompatibility, improved cell adhesion and extended neuronal projections with significantly higher intensities of NF-200 and tubulin expressions. Further suture-free fixation designed in the current 3D printed construct aids facile implantation of printed conduits to the transected nerve ends. Hence the protein ingrained 3D printed construct would be a promising substitute to treat longer peripheral nerve defects as its structural equivalence of endo- and perineurial organization along with the ingrained protein NF promote the neuronal extension towards the distal ends by minimizing axonal dispersion.

Sight and switch off: Nerve density visualization for interventions targeting nerves in prostate cancer.

Nerve density is associated with prostate cancer (PCa) aggressiveness and prognosis. Thus far, no visualization methods have been developed to assess nerve density of PCa in vivo. We compounded propranolol-conjugated superparamagnetic iron oxide nerve peptide nanoparticles (PSN NPs), which achieved the nerve density visualization of PCa with high sensitivity and high specificity, and facilitated assessment of nerve density and aggressiveness of PCa using magnetic resonance imaging and magnetic particle imaging. Moreover, PSN NPs facilitated targeted therapy for PCa. PSN NPs increased the survival rate of mice with orthotopic PCa to 83.3% and decreased nerve densities and proliferation indexes by more than twofold compared with the control groups. The present study, thus, developed a technology to visualize the nerve density of PCa and facilitate targeted neural drug delivery to tumors to efficiently inhibit PCa progression. Our study provides a potential basis for clinical imaging and therapeutic interventions targeting nerves in PCa.

Determination of optical properties of human tissues obtained from parotidectomy in the spectral range of 250 to 800 nm.

The optical properties of human tissues are an important parameter in medical diagnostics and therapy. The knowledge of these parameters can encourage the development of automated, computer-driven optical tissue analysis methods. We determine the absorption coefficient µa and scattering coefficient µs' of different tissue types obtained during parotidectomy in the wavelength range of 250 to 800 nm. These values are determined by high precision integrating sphere measurements in combination with an optimized inverse Monte Carlo simulation. To conserve the optical behavior of living tissues, the optical spectroscopy measurements are performed immediately after tissue removal. Our study includes fresh samples of the ear, nose, and throat (ENT) region, as muscle tissue, nervous tissue, white adipose tissue, stromal tissue, parotid gland, and tumorous tissue of five patients. The measured behavior of adipose corresponds well with the literature, which sustains the applied method. It is shown that muscle is well supplied with blood as it features the same characteristic peaks at 430 and 555 nm in the absorption curve. The parameter µs' decreases for all tissue types above 570 nm. The accuracy is adequate for the purposes of providing µa and µs' of different human tissue types as muscle, fat, nerve, or gland tissue, which are embedded in large complex structures such as in the ENT area. It becomes possible for the first time to present reasonable results for the optical behavior of human soft tissue located in the ENT area and in the near-UV, visual, and near-infrared areas.

Extended periprostatic nerve distributions on the prostate surface confirmed using diffusion tensor imaging.

OBJECTIVE: To perform a descriptive microscopic study of prostatectomy specimens from 19 patients which anatomically characterizes the distributions of periprostatic nerve qualities, and to visualize these using diffusion tensor imaging (DTI). MATERIALS AND METHODS: Serial whole-mounted sections were stained for cholinergic (neuronal nitric oxide synthase), adrenergic (tyrosine hydroxylase) and sensory (calcitonin gene-related peptide) nerves. Extracapsular stained nerves were counted by prostate surface sector, and classified by diameter. Stain-related relative density was calculated, and distribution patterns were evaluated. To better visualize the reported neuronal structures and independently confirm our findings, nerve concordance in five male volunteers was investigated using a 3-Tesla magnetic resonance imaging-DTI system. RESULTS: At the base, cholinergic nerves were distributed from the anterolateral to posterior sectors, continuing posterolaterally (mid-section) into the posterolateral-posterior sector toward the apex. Adrenergic nerves were distributed across the anterolateral-posterior sectors at the base, with the course narrowing to the posterolateral-posterior sectors at the mid- and apical levels. Sensory fibres were found posterolaterally posteriorly at the base, continuing posterolaterally over the mid- and apical levels. Although it was not possible to determine the different nerve qualities, DTI confirmed histological findings from the base to the apex. CONCLUSIONS: Different types of nerve fibres were found to vary in distribution. When linked to possible functional aspects of the different nerve types, this morphological evidence may be of importance to further protect function after radical prostatectomy (RP). To our knowledge, this is the first time that DTI has confirmed reported histological findings in nerve-sparing RPs. DTI could be an important tool with which to correlate nerves to tumour for better preoperative planning and to incorporate imaging into treatment.

Functional magnetic resonance electrical impedance tomography (fMREIT) sensitivity analysis using an active bidomain finite-element model of neural tissue.

PURPOSE: A direct method of imaging neural activity was simulated to determine typical signal sizes. METHODS: An active bidomain finite-element model was used to estimate approximate perturbations in MR phase data as a result of neural tissue activity, and when an external MR electrical impedance tomography imaging current was added to the region containing neural current sources. RESULTS: Modeling-predicted, activity-related conductivity changes should produce measurable differential phase signals in practical MR electrical impedance tomography experiments conducted at moderate resolution at noise levels typical of high field systems. The primary dependence of MR electrical impedance tomography phase contrast on membrane conductivity changes, and not source strength, was demonstrated. CONCLUSION: Because the injected imaging current may also affect the level of activity in the tissue of interest, this technique can be used synergistically with neuromodulation techniques such as deep brain stimulation, to examine mechanisms of action.

Nile Red derivatives enable improved ratiometric imaging for nerve-specific contrast.

Surgical nerve damage due to difficulty with identification remains a major risk for postsurgical complications and decreased quality of life. Fluorescence-guided surgery offers a means to specifically highlight tissues of interest such as nerves and a number of fluorescence-guided surgical systems are in clinical trial or are approved for clinical use. However, no clinically approved nerve-specific fluorophores exist. In addition, many preclinical nerve-specific fluorophores tend to accumulate in adipose tissue due to the molecular composition similarities between the two tissues, making it challenging to generate a specific nerve signal. To alleviate this difficulty, we have synthesized a library of oxazine fluorophores based on the Nile Red scaffold, with the goal of strong adipose specificity without nerve uptake to facilitate ratiometric imaging. The library was screened for tissue specificity ex vivo and in vivo, enabling quantification of adipose-, nerve- and muscle-specific uptake as well as selection of the best candidate for adipose selectivity without nerve signal. We showed our selected Nile Red fluorophore improved nerve contrast using ratiometric imaging, especially nerve-to-adipose contrast as compared to the parent Nile Red compound or nerve-specific imaging alone. This adipose-specific Nile Red derivative could be used in future fluorescence-guided surgery applications where adipose- or nerve-specific contrast is required.

Optical signature of nerve tissue-Exploratory ex vivo study comparing optical, histological, and molecular characteristics of different adipose and nerve tissues.

BACKGROUND: During several anesthesiological procedures, needles are inserted through the skin of a patient to target nerves. In most cases, the needle traverses several tissues-skin, subcutaneous adipose tissue, muscles, nerves, and blood vessels-to reach the target nerve. A clear identification of the target nerve can improve the success of the nerve block and reduce the rate of complications. This may be accomplished with diffuse reflectance spectroscopy (DRS) which can provide a quantitative measure of the tissue composition. The goal of the current study was to further explore the morphological, biological, chemical, and optical characteristics of the tissues encountered during needle insertion to improve future DRS classification algorithms. METHODS: To compare characteristics of nerve tissue (sciatic nerve) and adipose tissues, the following techniques were used: histology, DRS, absorption spectrophotometry, high-resolution magic-angle spinning nuclear magnetic resonance (HR-MAS NMR) spectroscopy, and solution 2D 13 C-1 H heteronuclear single-quantum coherence spectroscopy. Tissues from five human freshly frozen cadavers were examined. RESULTS: Histology clearly highlights a higher density of cellular nuclei, collagen, and cytoplasm in fascicular nerve tissue (IFAS). IFAS showed lower absorption of light around 1200 nm and 1750 nm, higher absorption around 1500 nm and 2000 nm, and a shift in the peak observed around 1000 nm. DRS measurements showed a higher water percentage and collagen concentration in IFAS and a lower fat percentage compared to all other tissues. The scattering parameter (b) was highest in IFAS. The HR-MAS NMR data showed three extra chemical peak shifts in IFAS tissue. CONCLUSION: Collagen, water, and cellular nuclei concentration are clearly different between nerve fascicular tissue and other adipose tissue and explain some of the differences observed in the optical absorption, DRS, and HR-NMR spectra of these tissues. Some differences observed between fascicular nerve tissue and adipose tissues cannot yet be explained but may be helpful in improving the discriminatory capabilities of DRS in anesthesiology procedures. Lasers Surg. Med. 50:948-960, 2018. © 2018 The Authors. Lasers in Surgery and Medicine Published by Wiley Periodicals, Inc.

Nerve detection using optical spectroscopy, an evaluation in four different models: In human and swine, in-vivo, and post mortem.

OBJECTIVE: Identification of peripheral nerve tissue is crucial in both surgery and regional anesthesia. Recently, optical tissue identification methods are presented to facilitate nerve identification in transcutaneous procedures and surgery. Optimization and validation of such techniques require large datasets. The use of alternative models to human in vivo, like human post mortem, or swine may be suitable to test, optimize and validate new optical techniques. However, differences in tissue characteristics and thus optical properties, like oxygen saturation and tissue perfusion are to be expected. This requires a structured comparison between the models. STUDY DESIGN: Comparative observational study. METHODS: Nerve and surrounding tissues in human (in vivo and post mortem) and swine (in vivo and post mortem) were structurally compared macroscopically, histologically, and spectroscopically. Diffuse reflective spectra were acquired (400-1,600 nm) after illumination with a broad band halogen light. An analytical model was used to quantify optical parameters including concentrations of optical absorbers. RESULTS: Several differences were found histologically and in the optical parameters. Histologically nerve and adipose tissue (subcutaneous fat and sliding fat) showed clear similarities between human and swine while human muscle enclosed more adipocytes and endomysial collagen. Optical parameters revealed model dependent differences in concentrations of ß-carotene, water, fat, and oxygen saturation. The similarity between optical parameters is, however, sufficient to yield a strong positive correlation after cross model classification. CONCLUSION: This study shows and discusses similarities and differences in nerve and surrounding tissues between human in vivo and post mortem, and swine in vivo and post mortem; this could support the discussion to use an alternative model to optimize and validate optical techniques for clinical nerve identification. Lasers Surg. Med. 50:253-261, 2018. © 2017 Wiley Periodicals, Inc.

Effect of Electrothermal Treatment on Nerve Tissue Within the Triangular Fibrocartilage Complex, Scapholunate, and Lunotriquetral Interosseous Ligaments.

PURPOSE: To evaluate the effect of thermal treatment on neural tissue in the triangular fibrocartilage complex (TFCC), scapholunate interosseous ligament (SLIL), and lunotriquetral interosseous ligament (LTIL). METHODS: The intact TFCC, SLIL, and LTIL were harvested from cadaveric specimens and treated with a radiofrequency probe as would be performed intraoperatively. Slides were stained using a triple-stain technique for neurotrophin receptor p75, pan-neuronal marker protein gene product 9.5 (PGP 9.5), and 4',6-diamidino-2-phenylindole for neural identification. Five TFCC, 5 SLIL, and 4 LTIL specimens were imaged with fluorescence microscopy. Imaging software was used to measure fluorescence signals and compare thermally treated areas with adjacent untreated areas. A paired t test was used to compare treated versus untreated areas. P < .05 was considered significant. RESULTS: For the TFCC, a mean of 94.9% ± 2.7% of PGP 9.5-positive neural tissue was ablated within a mean area of 11.7 ± 2.5 mm(2) (P = .02). For the SLIL treated from the radiocarpal surface, 97.4% ± 1.0% was ablated to a mean depth of 2.4 ± 0.3 mm from the surface and a mean horizontal spread of 3.4 ± 0.5 mm (P = .01). For the LTIL, 96.0% ± 1.5% was ablated to a mean depth of 1.7 ± 0.7 mm and a mean horizontal spread of 2.6 ± 1.0 mm (P = .02). Differences in the presence of neural tissue between treated areas and adjacent untreated areas were statistically significant for all specimens. CONCLUSIONS: Our study confirms elimination of neuronal markers after thermal treatment of the TFCC, SLIL, and LTIL in cadaveric specimens. This effect penetrates below the surface to innervated collagen tissue that is left structurally intact after treatment. CLINICAL RELEVANCE: Electrothermal treatment as commonly performed to treat symptomatic SLIL, LTIL, and TFCC tears eliminates neuronal tissue in treated areas and may function to relieve pain through a denervation effect.

IMAGING OF NEURO-INFLAMMATION: PAST, PRESENT AND FUTURE.

The investigation of dynamic processes with long time profiles can profit from longitudinal noninvasive imaging modalities. In this contribution the potential and limits of in vivo imaging modalities are discussed as regards monitoring neuro-inflammation. Complementary information derived from magnetic resonance imaging (MRI), positron emission tomography (PET) and bioluminescence imaging (BLI) will be presented.

Automatic segmentation of nerve structures in ultrasound images using Graph Cuts and Gaussian processes.

Peripheral Nerve Blocking (PNB), is a procedure used for performing regional anesthesia, that comprises the administration of anesthetic in the proximity of a nerve. Several techniques have been used with the purpose of locating nerve structures when the PNB procedure is performed: anatomical surface landmarks, elicitation of paresthesia, nerve stimulation and ultrasound imaging. Among those, ultrasound imaging has gained great attention because it is not invasive and offers an accurate location of the nerve and the structures around it. However, the segmentation of nerve structures in ultrasound images is a difficult task for the specialist, since such images are affected by echo perturbations and speckle noise. The development of systems for the automatic segmentation of nerve structures can aid the specialist for locating nerve structures accurately. In this paper we present a methodology for the automatic segmentation of nerve structures in ultrasound images. An initial step is carried out using Graph Cut segmentation in order to generate regions of interest; we then use machine learning techniques with the aim of segmenting the nerve structure; here, a specific non-linear Wavelet transform is used for the feature extraction stage, and Gaussian processes for the classification step. The methodology performance is measured in terms of accuracy and the dice coefficient. Results show that the implemented methodology can be used for automatically segmenting nerve structures.

Scanning electron microscopy of fetal murine myelomeningocele reveals growth and development of the spinal cord in early gestation and neural tissue destruction around birth.

BACKGROUND: Previous studies demonstrated that the spinal cord within a fetal myelomeningocele (MMC) lesion suffers progressive destruction during gestation. This study aims at elucidating this pathophysiologic feature on a cellular and ultrastructural level in a model of genetically determined MMC. METHODS: Curly tail/loop tail mouse fetuses at various gestational stages and neonates were analyzed electron-microscopically to document time-point and nature of neural tissue development and pathologic alterations within the MMC. RESULTS: At embryonic day (E) 8.5 and E9.5, round cells displaying multiple microvilli covered the entire region of interest, and some specimens showed initial stages of neurulation. At E10.5, neurulation was terminated in normal animals, whereas the neural placode remained unfolded in MMC fetuses and became distinguishable from adjacent epidermal layers. At E15.5, an apparently normal differentiation was found. Until this time-point, there was no tissue damage or inflammation. Thereafter, increasingly severe tissue alterations were identified with ongoing gestation leading to almost complete loss of neural tissue at birth. CONCLUSION: We show here in fetal mice with MMC that, apart from absent neurulation, growth and development of the otherwise perfectly intact exposed spinal cord appear normal in early gestation, whereas later, the unprotected neural tissue is progressively destroyed.

Ultrasound of nerve and muscle.

Over the last two decades, ultrasound has developed into a useful technology for the evaluation of diseases of nerve and muscle. Since it is currently not used at by the majority of clinicians involved in diagnosis or care of patients with neuromuscular disorders, this review briefly describes the technical aspects of ultrasound and its physical principles. It relates normal muscle anatomy and movement to ultrasound images in the axial and sagittal planes and follows with a discussion of ultrasound findings in chronic muscle disease. These include evident atrophy and the loss of the hypoechoic architecture of normal muscle tissue. It highlights evolving uses of the technique to measure other pathologic changes in disease including altered muscle dynamics. With high-resolution instruments nerve imaging has now become standard, and the relationships of median nerve anatomy and observations of static and dynamic images from ultrasound are reviewed. Changes seen in carpal tunnel syndrome include significant increases in the cross-sectional area of the nerve just proximal to the site of compression, loss of hyperechoic intensities within nerve, and reduced mobility. Preliminary use of the technique for the study of other nerves is reviewed as well. Ultrasound is an ideal tool for the clinical and research investigation of normal and diseased nerve and muscle complementary to existing diagnostic techniques. As the technology continues to evolve, it will likely assume a more significant role in these areas as those most able to exploit its potential, clinical neurophysiologists and neuromuscular clinicians, incorporate its use at the bedside.

Imaging nerve and muscle with ultrasound.

Over the last two decades significant advances in ultrasound have made it possible for investigators to image and interpret pathologic changes in muscle and nerve. In addition to being able to assess the pathologic changes in these structures themselves, ultrasound also provides the unique ability to identify anatomic lesions responsible for nerve or muscle injury. They can be correlated with changes in neural structures or affected muscles. Like electrodiagnostic studies, ultrasound is portable and inexpensive, but it is even less invasive, and surprisingly sensitive in detecting a variety of unusual and common causes of neuromuscular dysfunction. Given recent developments in the field, ultrasound shows promise as the technique most suitable for clinical neurophysiologists and neuromuscular clinicians in the growing field of imaging. Such involvement is required to best adopt and exploit the potential of imaging for the research and clinical evaluation of neuromuscular disorders.

Positron emission tomography imaging of transplant function.

In this article, the role of functional imaging for providing objective evidence that grafts of fetal tissue can survive and form connections in Parkinson's and Huntington's disease patients is reviewed. The dissociation between dopamine storage capacity, clinical improvement, and normalization of brain metabolism in PD is discussed, and possible mechanisms underlying the phenomenon of dyskinesias off medication are presented. It is concluded the positron emission tomography and single photon emission computed tomography can provide valuable ancillary information alongside clinical observations but are not currently appropriate modalities for use as surrogate endpoints.

Pyridoxine-dependent seizures associated with white matter abnormalities.

Pyridoxine-dependent seizures are a disorder of GABA metabolism probably due to a defective binding of pyridoxal phosphate coenzyme (PALP) with glutamate decarboxylase (GAD), the rate-limiting enzyme in GABA synthesis. The resulting GABA deficiency causes severe epilepsy in infancy. We report on a boy with seizures starting soon after birth, and only controlled by pyridoxine at pharmacological dosages. After two months without seizures, a CT scan showed hypodense white matter in frontal and occipital lobes suggestive of a retarded or defective myelination. We are not aware of other descriptions of such morphological abnormalities in a patient with this disorder.

[Multiple hemorrhagic cerebral amyloid angiopathy associated with leukoencephalopathy].

The authors present a case of cerebral amyloid angiopathy (CAA) which caused multiple recurrent subcortical hemorrhages and leukoencephalopathy similar to Binswanger's disease. CT scan revealed bilateral hypodensity of the hemispheric white matter in addition to multiple subcortical hemorrhages. The difference between white and gray matter densities was 14 approximately 15 HU. T2-weighted MRI showed the hemispheric white matter as high intensity. These findings on CT and MRI are compatible with those observed in leukoencephalopathy like Binswanger's disease. A biopsy of the cortex was performed and histological examination revealed amyloid deposition in the arterial wall. In addition to amyloid deposition, obliterative intimal proliferation and hyaline degeneration of the vessel were observed. It is thought that the occlusive vascular change due to CAA may induce chronic hypoperfusion of the hemispheric white matter and consequently the leukoencephalopathy may develop. In the aged population, CAA is noteworthy as a cause of both hemorrhagic and ischemic lesions, because the incidence of CAA is increasing with age.

[Computed tomography of Creutzfeldt-Jakob disease].

Creutzfeldt-Jakob disease (CJD), one of the transmissible spongiform encephalopathy, affects gray matter primarily. Degeneration of the white matter is uncommon and when it occurs is usually mild, and limited in distribution. Mizutani separated the "panencephalopathic type" of CJD from the other type, because this panencephalopathic type has extensive white matter changes, that could not be explained simply as secondary to cortical degeneration. This communication discusses six necropsy cases of CJD. In four cases of panencephalopathic type, out of our cases, there were extensive changes of cerebral white matter in addition to severe loss of neurons and hypertrophic astrogliosis in cortex and basal ganglia. Computed tomography demonstrated diffuse low attenuation areas in the white matter with marked atrophy of the cortex, brainstem and cerebellum.