The Mechanisms of Action of Tumor Treating Fields.

Tumor treating fields (TTFields), a new modality of cancer treatment, are electric fields transmitted transdermally to tumors. The FDA has approved TTFields for the treatment of glioblastoma multiforme and mesothelioma, and they are currently under study in many other cancer types. While antimitotic effects were the first recognized biological anticancer activity of TTFields, data have shown that tumor treating fields achieve their anticancer effects through multiple mechanisms of action. TTFields therefore have the ability to be useful for many cancer types in combination with many different treatment modalities. Here, we review the current understanding of TTFields and their mechanisms of action.

Permeabilizing Cell Membranes with Electric Fields.

The biological impact of exogenous, alternating electric fields (AEFs) and direct-current electric fields has a long history of study, ranging from effects on embryonic development to influences on wound healing. In this article, we focus on the application of electric fields for the treatment of cancers. In particular, we outline the clinical impact of tumor treating fields (TTFields), a form of AEFs, on the treatment of cancers such as glioblastoma and mesothelioma. We provide an overview of the standard mechanism of action of TTFields, namely, the capability for AEFs (e.g., TTFields) to disrupt the formation and segregation of the mitotic spindle in actively dividing cells. Though this standard mechanism explains a large part of TTFields' action, it is by no means complete. The standard theory does not account for exogenously applied AEFs' influence directly upon DNA nor upon their capacity to alter the functionality and permeability of cancer cell membranes. This review summarizes the current literature to provide a more comprehensive understanding of AEFs' actions on cell membranes. It gives an overview of three mechanistic models that may explain the more recent observations into AEFs' effects: the voltage-gated ion channel, bioelectrorheological, and electroporation models. Inconsistencies were noted in both effective frequency range and field strength between TTFields versus all three proposed models. We addressed these discrepancies through theoretical investigations into the inhomogeneities of electric fields on cellular membranes as a function of disease state, external microenvironment, and tissue or cellular organization. Lastly, future experimental strategies to validate these findings are outlined. Clinical benefits are inevitably forthcoming.

Fiber Threshold Accommodation as a Mechanism of Burst and High-Frequency Spinal Cord Stimulation.

OBJECTIVES: Burst and high-frequency spinal cord stimulation (SCS), in contrast to low-frequency stimulation (LFS, < 200 Hz), reduce neuropathic pain without the side effect of paresthesia, yet it is unknown whether these methods' mechanisms of action (MoA) overlap. We used empirically based computational models of fiber threshold accommodation to examine the three MoA. MATERIALS AND METHODS: Waveforms used in SCS are composed of cathodic, anodic, and rest phases. Empirical studies of human peripheral sensory nerve fibers show different accommodation effects occurring in each phase. Notably, larger diameter fibers accommodate more than smaller fibers. We augmented our computational axon model to replicate fiber threshold accommodation behavior for diameters from 5 to 15 µm in each phase. We used the model to predict threshold change in variations of burst, high frequency, and LFS. RESULTS: The accommodation model showed that 1) inversion of larger and smaller diameter fiber thresholds produce a therapeutic window in which smaller fibers fire while larger ones do not and 2) the anodic pulses increase accommodation and perpetuate threshold inversion from burst to burst and between cathodic pulses in burst, high frequency, and variations, resulting in an amplitude "window" in which larger fibers are inactivated while smaller fibers fire. No threshold inversion was found for traditional LFS. CONCLUSIONS: The model, based on empirical data, predicts that, at clinical amplitudes, burst and high-frequency SCS do not activate large-diameter fibers that produce paresthesia while driving medium-diameter fibers, likely different from LFS, which produce analgesia via different populations of dorsal horn neural circuits.

Dynamic Computational Model of the Human Spinal Cord Connectome.

Connectomes abound, but few for the human spinal cord. Using anatomical data in the literature, we constructed a draft connectivity map of the human spinal cord connectome, providing a template for the many calibrations of specialized behavior to be overlaid on it and the basis for an initial computational model. A thorough literature review gleaned cell types, connectivity, and connection strength indications. Where human data were not available, we selected species that have been studied. Cadaveric spinal cord measurements, cross-sectional histology images, and cytoarchitectural data regarding cell size and density served as the starting point for estimating numbers of neurons. Simulations were run using neural circuitry simulation software. The model contains the neural circuitry in all ten Rexed laminae with intralaminar, interlaminar, and intersegmental connections, as well as ascending and descending brain connections and estimated neuron counts for various cell types in every lamina of all 31 segments. We noted the presence of highly interconnected complex networks exhibiting several orders of recurrence. The model was used to perform a detailed study of spinal cord stimulation for analgesia. This model is a starting point for workers to develop and test hypotheses across an array of biomedical applications focused on the spinal cord. Each such model requires additional calibrations to constrain its output to verifiable predictions. Future work will include simulating additional segments and expanding the research uses of the model.

Theoretical Effect of DBS on Axonal Fibers of Passage: Firing Rates, Entropy, and Information Content.

BACKGROUND: Deep brain stimulation (DBS) has effects on axons that originate and terminate outside the DBS target area. OBJECTIVE: We hypothesized that DBS generates action potentials (APs) in both directions in "axons of passage," altering their information content and that of all downstream cells and circuits, and sought to quantify the change in fiber information content. METHODS: We incorporated DBS parameters (fiber firing frequency and refractory time, and AP initiation location along the fiber and propagation velocity) in a filtering function determining the AP frequency reaching the postsynaptic cell. Using neural circuitry simulation software, we investigated the ability of the filtering function to predict the firing frequency of APs reaching neurons targeted by axons of passage. We calculated their entropy with and without DBS, and with the electrode applied at various distances from the cell body. RESULTS: The predictability of the filtering function exceeded 98%. Entropy calculations showed that the entropy ratio "without DBS" to "with DBS" was always >1.0, thus DBS reduces fiber entropy. CONCLUSIONS: (1) The results imply that DBS effects are due to entropy reduction within fibers, i.e., a reduction in their information. (2) Where fibers of passage do not terminate in target regions, DBS may have side effects on nontargeted circuitry.

Dodecafluoropentane Improves Neurological Function Following Anterior Ischemic Stroke.

Dodecafluoropentane emulsion (DDFPe), an advanced oxygen transport drug, given IV at 90-min intervals maintains viability in the penumbra during cerebral ischemia in the standard rabbit anterior stroke model (STND). This study investigated shortened dosage schedules of DDFPe in nonstandard posterior (NSTND) strokes following occlusions of the posterior cerebral arteries. DDFPe given at shortened schedules of 30 or 60-min injection intervals will reduce neurological deficits, percent stroke volume (%SV), and serum glutamate levels in NSTND ischemic strokes. New Zealand White rabbits (N = 26) were randomly placed into three groups: A (n = 9) controls given saline injections every 60 min, B (n = 9) 2 % DDFPe given IV every 30 min, and C (n = 8) DDFPe every 60 min. Injections began 1 h after embolization. Groups were subdivided into STND and NSTND based on angiographically verified embolization of the cerebral arteries. Neurological assessments and blood samples were done at 0.5-1-h intervals. Rabbits were euthanized at 7 h following embolization. Stained brain slices were measured for %SV. The 30 and 60-min subgroups did not differ and were combined as DDFPe-STND or DDFPe-NSTND groups. In the DDFPe-STND stroke group, the %SV, neurological assessment scores (NAS), and serum glutamate were decreased vs. STND controls (p = 0.0016, 0.008, and 0.016, respectively). In the DDFPe-NSTND stroke group, %SV, NAS, and serum glutamate did not differ statistically compared to NSTND controls (p = 0.82, 0.097, and 0.06, respectively). More frequent dosage schedules provided no additional improvement. In anterior strokes, DDFPe improves recovery but not in the more severe NSTND strokes.

Investigation of mechanisms of vagus nerve stimulation for seizure using finite element modeling.

OBJECTIVE: While the efficacy of vagus nerve stimulation (VNS) to reduce seizures in pharmaco-resistant patients is clinically proven, its efficacy and side effects mechanisms are not fully understood. Our goals were 1) to use a finite element model (FEM) and axon models to examine different fiber activation and blocking thresholds and 2) examine fiber activation and blocking in three fiber groups likely to be responsible for efficacy and side effects. METHODS: Using FEM, we examined the field potential along axons within a vagus nerve model with fascicles. These data were input to a computational fiber model to estimate numbers of axons activated across all diameters. We estimated numbers of activated and blocked fibers by diameter. RESULTS: 1) At the low end of VNS amplitudes, little efficacy for seizure control is appreciated while large Aß fibers associated with the recurrent laryngeal nerve are recruited. As amplitudes are increased, Aß fibers can produce hoarseness, and next recruited are fast B fibers associated with the aortic fascicle. We hypothesize these B fibers are the source of efficacy in treating seizure. As amplitudes are further increased, coughing may occur, possibly due to recruitment of smaller and deeper pulmonary fibers. 2) Clinical parameters are in a range that could cause inadvertent blocking at the cathode and activation at the anode. Conversely, innovative approaches to field shape and charge-balancing can allow controlled fiber activation at the cathode for maximum activation of the fibers responsible for efficacy, and possibly blocking at the anode to minimize side effects and expand therapeutic range. In design and operation, the cathode and anode can each be approached as a band pass filter. SIGNIFICANCE: The B fiber group is necessary and possibly sufficient to produce VNS efficacy in epilepsy. This group may emanate from aortic baroreceptors that, via synapses in the solitary tract nucleus, stimulate the locus coeruleus, hypothalamus and other influential targets such as the hippocampus. Responder rates may be increased with a lead that fully encircles the nerve. With better identification of the fiber groups involved in VNS, efficacy, side effects, therapeutic range and responder rates can be optimized.

High-Frequency Stimulation of Dorsal Column Axons: Potential Underlying Mechanism of Paresthesia-Free Neuropathic Pain Relief.

OBJECTIVE: Spinal cord stimulation (SCS) treats neuropathic pain through retrograde stimulation of dorsal column axons and their inhibitory effects on wide dynamic range (WDR) neurons. Typical SCS uses frequencies from 50-100 Hz. Newer stimulation paradigms use high-frequency stimulation (HFS) up to 10 kHz and produce pain relief but without paresthesia. Our hypothesis is that HFS preferentially blocks larger diameter axons (12-15 µm) based on dynamics of ion channel gates and the electric potential gradient seen along the axon, resulting in inhibition of WDR cells without paresthesia. METHODS: We input field potential values from a finite element model of SCS into an active axon model with ion channel subcomponents for fiber diameters 1-20 µm and simulated dynamics on a 0.001 msec time scale. RESULTS: Assuming some degree of wave rectification seen at the axon, action potential (AP) blockade occurs as hypothesized, preferentially in larger over smaller diameters with blockade in most medium and large diameters occurring between 4.5 and 10 kHz. Simulations show both ion channel gate and virtual anode dynamics are necessary. CONCLUSION: At clinical HFS frequencies and pulse widths, HFS preferentially blocks larger-diameter fibers and concomitantly recruits medium and smaller fibers. These effects are a result of interaction between ion gate dynamics and the "activating function" (AF) deriving from current distribution over the axon. The larger fibers that cause paresthesia in low-frequency simulation are blocked, while medium and smaller fibers are recruited, leading to paresthesia-free neuropathic pain relief by inhibiting WDR cells.

Mechanism of dorsal column stimulation to treat neuropathic but not nociceptive pain: analysis with a computational model.

OBJECTIVE: Stimulation of axons within the dorsal columns of the human spinal cord has become a widely used therapy to treat refractory neuropathic pain. The mechanisms have yet to be fully elucidated and may even be contrary to standard "gate control theory." Our hypothesis is that a computational model provides a plausible description of the mechanism by which dorsal column stimulation (DCS) inhibits wide dynamic range (WDR) cell output in a neuropathic model but not in a nociceptive pain model. MATERIALS AND METHODS: We created a computational model of the human spinal cord involving approximately 360,000 individual neurons and dendritic processing of some 60 million synapses--the most elaborate dynamic computational model of the human spinal cord to date. Neuropathic and nociceptive "pain" signals were created by activating topographically isolated regions of excitatory interneurons and high-threshold nociceptive fiber inputs, driving analogous regions of WDR neurons. Dorsal column fiber activity was then added at clinically relevant levels (e.g., Aß firing rate between 0 and 110 Hz by using a 210-µsec pulse width, 50-150 Hz frequency, at 1-3 V amplitude). RESULTS: Analysis of the nociceptive pain, neuropathic pain, and modulated circuits shows that, in contradiction to gate control theory, 1) nociceptive and neuropathic pain signaling must be distinct, and 2) DCS neuromodulation predominantly affects the neuropathic signal only, inhibiting centrally sensitized pathological neuron groups and ultimately the WDR pain transmission cells. CONCLUSION: We offer a different set of necessary premises than gate control theory to explain neuropathic pain inhibition and the relative lack of nociceptive pain inhibition by using retrograde DCS. Hypotheses regarding not only the pain relief mechanisms of DCS were made but also regarding the circuitry of pain itself, both nociceptive and neuropathic. These hypotheses and further use of the model may lead to novel stimulation paradigms.

Design, assembly, and optical bench testing of a high-numerical-aperture miniature injection-molded objective for fiber-optic confocal reflectance microscopy.

The design, analysis, assembly methods, and optical-bench test results for a miniature injection-molded plastic objective lens used in a fiber-optic confocal reflectance microscope are presented. The five-lens plastic objective was tested as a stand-alone optical system before its integration into a confocal microscope for in vivo imaging of cells and tissue. Changing the spacing and rotation of the individual optical elements can compensate for fabrication inaccuracies and improve performance. The system performance of the miniature objective lens is measured by use of an industry-accepted slanted-edge modulation transfer function (MTF) metric. An estimated Strehl ratio of 0.61 and a MTF value of 0.66 at the fiber-optic bundle Nyquist frequency have been obtained. The optical bench testing system is configured to permit interactive optical alignment during testing to optimize performance. These results are part of an effort to demonstrate the manufacturability of low-cost, high-performance biomedical optics for high-resolution in vivo imaging. Disposable endoscopic microscope objectives could help in vivo confocal microscopy technology mature to permit wide-scale clinical screening and detection of early cancers and precancerous lesions.

Confocal microscopy: imaging cervical precancerous lesions.

OBJECTIVES: We explore the clinical potential of reflectance and fluorescence confocal microscopy to image the morphologic and biochemical changes associated with precancer, in order to aid in the detection and diagnosis of cervical dysplasia. METHODS: Cervical epithelial tissue samples imaged ex vivo or in vivo were obtained from M. D. Anderson Cancer Center and Lyndon B. Johnson Hospital in Houston, Texas. Confocal reflectance microscopy was used to image ex vivo cervical biopsies and in vivo cervical tissue. Confocal fluorescence microscopy was used to image ex vivo cervical tissue slices. RESULTS: We present reflectance and fluorescence confocal images of cervical tissue demonstrating the ability to differentiate between normal and abnormal cervical tissue. CONCLUSIONS: We believe that there is significant clinical potential for confocal microscopy to provide a sensitive and specific method for cervical precancer detection.

An image model and segmentation algorithm for reflectance confocal images of in vivo cervical tissue.

The automatic segmentation of nuclei in confocal reflectance images of cervical tissue is an important goal toward developing less expensive cervical precancer detection methods. Since in vivo confocal reflectance microscopy is an emerging technology for cancer detection, no prior work has been reported on the automatic segmentation of in vivo confocal reflectance images. However, prior work has shown that nuclear size and nuclear-to-cytoplasmic ratio can determine the presence or extent of cervical precancer. Thus, segmenting nuclei in confocal images will aid in cervical precancer detection. Successful segmentation of images of any type can be significantly enhanced by the introduction of accurate image models. To enable a deeper understanding of confocal reflectance microscopy images of cervical tissue, and to supply a basis for parameter selection in a classification algorithm, we have developed a model that accounts for the properties of the imaging system and of the tissues. Using our model in conjunction with a powerful image enhancement tool (anisotropic median-diffusion), appropriate statistical image modeling of spatial interactions (Gaussian Markov random fields), and a Bayesian framework for classification-segmentation, we have developed an effective algorithm for automatically segmenting nuclei in confocal images of cervical tissue. We have applied our algorithm to an extensive set of cervical images and have found that it detects 90% of hand-segmented nuclei with an average of 6 false positives per frame.

Fluorescent nanocrystals for use in early cervical cancer detection.

BACKGROUND: Quantum dots (qdots) are a promising alternative to organic fluorophores for biological imaging. Advantages of quantum dots over organic fluorophores include broad excitation coupled with narrow, tunable emission, high resistance to chemical and metabolic degradation, a higher photobleaching threshold and finally the ability to be modified with a targeting ligand. These many properties allow quantum dots to be used in conjunction with optical detection methods for imaging. METHODS: We are investigating the use of quantum dots to detect precancerous biomarkers. We have directly targeted epidermal growth factor receptors with quantum dots conjugated to anti-EGFR antibodies. RESULTS: Compared to appropriate controls, we do see specific labeling of EGF receptors. CONCLUSIONS: Quantum dots provide a promising alternative to conventional organic dyes for biological imaging. Combined with optical imaging technologies, quantum dots can help visualize changes in cervical cancer at the molecular level. This ability may alert health care providers to the need for intervention before a cancer can metastasize.

In vivo fiber-optic confocal reflectance microscope with an injection-molded plastic miniature objective lens.

For in vivo optical diagnostic technologies to be distributed to the developed and developing worlds, optical imaging systems must be constructed of inexpensive components. We present a fiber-optic confocal reflectance microscope with a cost-effective injection-molded plastic miniature objective lens for in vivo imaging of human tissues in near real time. The measured lateral resolution is less than 2.2 microm, and the measured axial resolution is 10 microm. Confocal images of ex vivo cervical tissue biopsies and in vivo human lip taken at 15 frames/s demonstrate the microscope's capability of imaging cell morphology and tissue architecture.