IMAGING NERVE FUNCTION AND PATHOLOGIES
1. A computer-implemented method of detecting nerve activity from a time series of diffusion MRI images, the method comprising:
- obtaining, using an MM scanner, a time-series of diffusion MRI datasets from a nerve, each diffusion MM dataset comprising a plurality of voxels and associated diffusion MM signals;
applying, using a computing device, a diffusion basis spectrum imaging (DBSI) model to each diffusion MM dataset of the time-series to obtain a time-series of at least one DBSI parameter;
detecting, using the computing device, the nerve activity at each voxel of the plurality of voxels based on changes in at least one value within the time series of the at least one DBSI parameter according to a nerve activity detection rule;
transforming, using the computing device, the detected nerve activity into a spatial map of nerve activity at each voxel position of the diffusion MM dataset; and
displaying to a user, using a display device operatively coupled to the computing device, the spatial map of nerve activity.
Repetitive electrical activity produces microstructural alteration in myelinated axons. These transient microstructural changes can be non-invasively visualized via two different magnetic-resonance-based approaches: diffusion fMRI and dynamic T2 spectroscopy in the ex vivo perfused bullfrog sciatic nerves. Non-invasive diffusion fMRI, based on standard diffusion tensor imaging (DTI), clearly localized the sites of axonal conduction blockage as might be encountered in neurotrauma or other lesion types. Diffusion fMRI response was graded in proportion to the total number of electrical impulses carried through a given locus. Diffusion basis spectrum imaging (DBSI) method revealed a reversible shift of tissue water into a restricted isotropic diffusion signal component, consistent with sub-myelinic vacuole formation.
- 1. A computer-implemented method of detecting nerve activity from a time series of diffusion MRI images, the method comprising:
obtaining, using an MM scanner, a time-series of diffusion MRI datasets from a nerve, each diffusion MM dataset comprising a plurality of voxels and associated diffusion MM signals; applying, using a computing device, a diffusion basis spectrum imaging (DBSI) model to each diffusion MM dataset of the time-series to obtain a time-series of at least one DBSI parameter; detecting, using the computing device, the nerve activity at each voxel of the plurality of voxels based on changes in at least one value within the time series of the at least one DBSI parameter according to a nerve activity detection rule; transforming, using the computing device, the detected nerve activity into a spatial map of nerve activity at each voxel position of the diffusion MM dataset; and displaying to a user, using a display device operatively coupled to the computing device, the spatial map of nerve activity.
- View Dependent Claims (2, 3, 4, 5)
- 6. A computer-implemented method of detecting optic nerve activity in a human subject from a time series of diffusion MM images, the method comprising:
obtaining, using an MM scanner, a time-series of diffusion MRI datasets from an optic nerve, each diffusion MM dataset comprising a plurality of voxels and associated diffusion MM signals;
- View Dependent Claims (7, 8)
- 9. A computer-implemented method of detecting optic nerve pathology in a human subject from a time series of diffusion MM images, the method comprising:
obtaining, using an MM scanner, a diffusion MRI dataset from an optic nerve, the diffusion MM dataset comprising a plurality of voxels and associated diffusion MM signals; applying, using a computing device, a diffusion basis spectrum imaging (DBSI) model to the diffusion MRI dataset to obtain at least one DBSI parameter; detecting, using the computing device, the optic nerve pathology at each voxel of the plurality of voxels based on at least one value of the at least one DBSI parameter according to a optic nerve pathology detection rule; transforming, using the computing device, the detected optic nerve pathology into a spatial map of optic nerve pathology at each voxel position of the diffusion MM dataset; and displaying to a user, using a display device operatively coupled to the computing device, the spatial map of optic nerve pathology.
- View Dependent Claims (10, 11)
This application claims the benefit of priority to U.S. Provisional Application No. 62/663,816, filed Apr. 27, 2018, which is hereby incorporated by reference herein in its entirety.
This invention was made with government support under grants U01EY025500, R01NS047592, and P01NS059560 awarded by the National Institutes of Health. The government has certain rights in the invention.
BOLD functional MRI has been extremely successful and forms the basis of the NIH-supported Human Connectome Project. Despite the continued success of resting-state and task-specific fMRI applications, few have demonstrated the ability to image nerve activation using BOLD fMRI. Diffusion-weighted MRI has recently been proposed to image brain function as those seen by BOLD fMRI, without a consensus in its utility or mechanisms.
Blood oxygen-level-dependent functional magnetic resonance imaging (BOLD fMRI) has revolutionized our ability to study human brain function. Because grey matter is relatively well-vascularized, the BOLD effect works exceptionally well as a means to indirectly monitor neuronal activity. By comparison, successful studies with white matter (WM) BOLD fMRI are limited because both blood volume and the tissue'"'"'s capacity for physiologic modulation of blood flow are considerably less than in grey matter.
Diffusion Tensor Imaging (DTI) has been employed extensively to investigate WM structure. DTI models random thermal displacement of water as a single tensor, which can be used to encapsulate the direction-dependent behavior of water diffusion, reflecting fiber orientations in ordered tissues. Using DTI, it has been demonstrated that prolonged periods (weeks, months, years) of training activities, such as piano playing or juggling, result in detectable changes of the diffusion MM properties of the requisite WM pathways. Presumably, the diffusion MRI changes are due to increased myelination of the chronically electrically-active axons in the WM tracts used in these coordinated behaviors.
White matter comprises approximately 50% of the volume of the human brain, and various pathologies (e.g., multiple sclerosis) directly target WM and lead to disability. Thus, non-invasive assessment of WM function, including pinpointing lesions that block conduction of action potentials, could conceivably be very powerful for understanding disease progression and monitoring therapeutic response.
White matter diffusion fMRI investigations have applied DTI, or variants thereof, to monitor short-term structural alterations with WM electrical activity. For example, functional DTI was performed to measure task-related changes in fractional anisotropy (FA) along the thalamocortical tract and optic radiation upon tactile and visual stimulation, respectively. Additional previous studies have used ion-sensitive electrodes to monitor the concentration of the tetramethylammonium ion in the extracellular space of perfused rat optic nerves, and reported a reversible decrease in the volume of the extracellular space in WM tissue with repetitive electrical stimulation.
These studies motivated an in vivo WM diffusion fMRI study, wherein we demonstrated that in healthy mice, a flashing-light visual stimulus led to a reversible decrease in the apparent diffusion coefficient of water perpendicular to the optic nerve fibers (ADC⊥, also denoted as λ⊥) of the stimulated eye. Diffusion changes were absent in the optic nerve of the contralateral, non-stimulated eye. Moreover, the ADC⊥ decrease was found to be attenuated in mice with acute optic neuritis, the magnitude of the WM diffusion fMRI response being correlated with visual acuity in the affected eye
As an extension of DTI, Diffusion Basis Spectrum Imaging (DBSI) models the tissue water diffusion signal as one or more discrete anisotropic tensors (reflecting one or more fiber components) and a spectrum of isotropic diffusion tensors. A pictorial overview of this model is presented in
The isotropic diffusion spectrum can reflect more complex features of the diffusion MRI signal and tissue pathology/structure. The isotropic diffusion spectrum is typically divided into a restricted component (the lowest range of isotropic diffusion coefficients, which can reflect variation in tissue cellularity occurring with inflammation or cancer), a hindered component (the intermediate band of the isotropic diffusion spectrum, which may be elevated in vascular edema or axonal loss), and a free diffusion component (for example, when partial volume effects lead to inclusion of cerebrospinal fluid in an imaging voxel'"'"'s total signal). Further details of the tissue microstructural alterations in electrically-active myelinated axons are observable with DBSI—details that are not accessible in a DTI analysis of the diffusion fMRI data.
In one aspect, a computer-implemented method of detecting nerve activity from a time series of diffusion MRI images is provided. The method includes obtaining, using an MRI scanner, a time-series of diffusion MM datasets from a nerve. Each diffusion MM dataset includes a plurality of voxels and associated diffusion MRI signals. The method further includes applying, using a computing device, a diffusion basis spectrum imaging (DBSI) model to each diffusion MRI dataset of the time-series to obtain a time-series of at least one DBSI parameter. The method further includes detecting, using the computing device, the nerve activity at each voxel of the plurality of voxels based on changes in at least one value within the time series of the at least one DBSI parameter according to a nerve activity detection rule. The method further includes transforming, using the computing device, the detected nerve activity into a spatial map of nerve activity at each voxel position of the diffusion MRI dataset, and displaying to a user, using a display device operatively coupled to the computing device, the spatial map of nerve activity. The nerve activity detection rule further includes detecting a nerve activity for at least one of: an increase of a fiber component (ffiber) value above a first threshold value; an increase of a restricted component (fR) value above a second threshold value; a decrease of an axial diffusivity (λ∥) value below a third threshold value; a decrease of a radial diffusivity (λ⊥) value below a fourth threshold value; a decrease of a hindered isotropic spectral component (DH) value below a fifth threshold value; and an increase of a restricted isotropic spectral component (DR) value above a sixth threshold value. In one aspect, the first threshold value is 1% of a total diffusion signal higher than a baseline ffiber value associated with a non-stimulated nerve; the second threshold value is about 0.3% of the total diffusion signal higher than a baseline fR value associated with a non-stimulated nerve; the third threshold value is about 0.06 μm2/ms less than a baseline λ∥ value associated with a non-stimulated nerve; the fourth threshold value is about 0.02 μm2/ms less than a baseline fR value associated with a non-stimulated nerve; the fifth threshold value is about 0.1% μm2/ms less than a baseline DH value associated with a non-stimulated nerve; and the sixth threshold value is about 0.03 μm2/ms less than a baseline DR value associated with a non-stimulated nerve. In another aspect, the method may further include estimating, using the computing device, an intensity of nerve activation based on changes in at least one value within the time series of the at least one DBSI parameter according to a nerve activity intensity rule. The nerve activity intensity rule includes an empirically-derived relationship between a change in the axial diffusivity (λ∥) value and a nerve activity intensity, wherein further increases of axial diffusivity (λ∥) value below the third threshold value are indicative of increased nerve activity intensity.
In an additional aspect, a computer-implemented method of detecting optic nerve activity in a human subject from a time series of diffusion MRI images is disclosed that includes obtaining, using an MRI scanner, a time-series of diffusion MM datasets from an optic nerve. Each diffusion MRI dataset includes a plurality of voxels and associated diffusion MM signals. The method also includes applying, using a computing device, a diffusion basis spectrum imaging (DBSI) model to each diffusion MM dataset of the time-series to obtain a time-series of at least one DBSI parameter. The method further includes detecting, using the computing device, the nerve activity at each voxel of the plurality of voxels based on changes in at least one value within the time series of the at least one DBSI parameter according to a nerve activity detection rule. The method also includes transforming, using the computing device, the detected nerve activity into a spatial map of nerve activity at each voxel position of the diffusion MRI dataset, and displaying to a user, using a display device operatively coupled to the computing device, the spatial map of nerve activity. In another aspect, the nerve activity detection rule includes detecting a nerve activity for at least one of: a decrease of an axial diffusivity (λ∥) value below a third threshold value; and a decrease of a radial diffusivity (λ⊥) value below a fourth threshold value. In an additional aspect, the third threshold value is about 0.2 μm2/ms less than a baseline λ∥ value associated with a non-stimulated nerve; and the fourth threshold value is about 0.3 μm2/ms less than a baseline λ⊥ value associated with a non-stimulated nerve.
In yet another aspect, a computer-implemented method of detecting optic nerve pathology in a human subject from a time series of diffusion MRI images. The method includes obtaining, using an MRI scanner, a diffusion MM dataset from an optic nerve, the diffusion MRI dataset comprising a plurality of voxels and associated diffusion MRI signals. The method further includes applying, using a computing device, a diffusion basis spectrum imaging (DBSI) model to the diffusion MRI dataset to obtain at least one DBSI parameter. The method also includes detecting, using the computing device, the optic nerve pathology at each voxel of the plurality of voxels based on at least one value of the at least one DBSI parameter according to a optic nerve pathology detection rule. The method also includes transforming, using the computing device, the detected optic nerve pathology into a spatial map of optic nerve pathology at each voxel position of the diffusion MM dataset, and displaying to a user, using a display device operatively coupled to the computing device, the spatial map of optic nerve pathology. In another aspect, the optic nerve pathology detection rule includes detecting an optic nerve pathology for at least one of: a decrease of an axial diffusivity (λ∥) value below a third threshold value; and an increase of a radial diffusivity (λ⊥) value above a fourth threshold value. In another aspect, the third threshold value is about 0.5 μm2/ms less than a baseline λ∥ value associated with a non-stimulated nerve; and the fourth threshold value is about 0.1 μm2/ms greater than a baseline λ⊥ value associated with a non-stimulated nerve.
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Advantages will become more apparent to those skilled in the art from the following description of the preferred aspects which have been shown and described by way of illustration. As will be realized, the present aspects may be capable of other and different aspects, and their details are capable of modification in various respects. Accordingly, the drawings and description are to be regarded as illustrative in nature and not as restrictive.
In various aspects, systems and methods for monitoring electrical activity of myelinated axons using diffusion basis spectrum imaging (DBSI) are disclosed herein. Without being limited to any particular theory, repetitive electrical activity produces microstructural alteration in myelinated axons, which enables noninvasively monitoring of the function of myelinated fibers in peripheral nervous system (PNS)/CNS pathways. In various aspects, microstructural changes in myelinated axons resulting from repetitive electrical activity are assessed using noninvasive diffusion basis spectrum imaging (DBSI) analysis of diffusion functional MRI data. In one aspect, a reversible shift of tissue water into a restricted isotropic diffusion signal component is observed in myelinated axons after exposure to repetitive stimulation. As described in various examples below, this reversible shift of tissue water into the restricted isotropic diffusion signal component is thought to result from the formation of submyelinic vacuoles observed in electron-microscopy images of tissue fixed during electrical stimulation. In various aspects, the disclosed method uses correlations between electrophysiology and DBSI parameters during and immediately after stimulation to determine the electrical activity of myelinated axons non-invasively.
BOLD fMRI has proven to be extremely powerful for studying brain function, but is essentially limited to applications in gray matter. In various aspects, novel MM-based approaches that can non-invasively monitor function of myelinated axonal fibers in PNS/CNS pathways as well as the underlying mechanisms that give rise to the observed functional MRI response are disclosed. The ex vivo perfused bullfrog sciatic nerve is an exceptionally robust model system that allows for well-defined stimulus intensity (duration and frequency of supramaximal voltage stimulus), recording of compound action potentials (CAP), and simultaneous MM data acquisition. Electron microscopy of perfused nerves fixed resting or undergoing stimulation supports microstructural interpretations based upon electrophysiology and magnetic resonance.
Diffusion-weighted MM (DWI) may be used to assess optic nerve response to visual stimulation in various organisms in vivo and ex vivo, including, but not limited to, vertebrates such as mice, frogs, and humans. To better understand the underlying mechanism of diffusion fMRI, a perfused isolated frog nerve system was used in conjunction with DBSI to assess nerve structural changes during repeated electrical stimulations. Through this investigation, DBSI was found to more sensitively detect nerve response than DWI or the more complicated diffusion tensor imaging (DTI) because the multi-tensor modeling offers a unique opportunity to specifically assess nerve structural changes without the confounding effects from surround environment such as CSF, inflammation, or other injuries. DBSI on living subjects undergoing visual stimulation using a flashing checkerboard was also performed. Close to 4-fold stronger optic nerve response using DBSI then conventional DTI was observed (see
In various aspects, “functional DBSI” assesses nerve function through dynamic measurements. The baseline DBSI measurements offer the conventional static DBSI assessment of nerve pathologies. In an aspect, “functional DBSI” may noninvasively and simultaneously assess nerve function and pathologies without the need to inject any tracers. The disclosed methods allow a direct correlation between underlying pathologies on nerve function. For example, one can assess nerve function in the presence of inflammation (likely reversible) or axonal loss (probably irreversible) facilitating stratification of patient management.
The disclosed methods can be used in various clinical applications. Functional assessment of a patient suffering neurological dysfunction is challenging and misdiagnosis is a frequent occurrence. One of the most promising neuroimaging technologies, resting-state fMRI, has shown utility to decipher connections between functional zones. However, resting-state fMRI remains incapable of determining where or how the disconnection occurs. The disclosed methods may be used to detect the connection between functional zones in brain and to provide insights to the function and pathologies of the connection. The disclosed methods offer a guide to treatment stratification allowing a more effective therapy.
For example, visual impairment may be detected by regular visual exams, RS-fMRI, or visual evoked potential. However, all of these existing detection methods reflect overall system response without the ability to pinpoint the breakpoint of the pathway. The disclosed method offers a means to detect the exact point where the signal is disrupted leading to visual impairment. With DBSI'"'"'s ability to assess nerve pathologies the disclosed method will also provide the potential mechanism why the functional impairment occurs.
Conventional functional assessment methods may not be sufficient to detect subtle recovery of injured nerves. The disclosed method may detect subclinical function recovery and subclinical pathologies of a nerve. This would be crucial to allow a treatment to continue avoiding premature discontinuation for not detecting the conventional functional responses. For neurodegenerative disease such as AD, RS-fMRI and other neuroimaging technologies have been employed to predict the disease'"'"'s progression. None of the current neuroimaging technologies assesses function of nerves connecting brain regions. It is likely that brain has the reserve to sustain the function in the presence of neuronal injury. Neurological impairment in AD or other neurodegenerative diseases could result from dysfunction and/or pathologies of connecting nerves.
In various aspects, a method of dynamic T2 spectroscopy to monitor signatures of repetitive electrical stimulation is provided. In T2 spectroscopy, the transverse magnetization signal decay in a multi-echo magnetic resonance measurement is decomposed into a spectrum of T2 decay components. This approach applied in imaging mode has formed the basis of myelin water fraction (MWF) imaging, which is being pursued as a means to assess demyelination/remyelination in human brain diseases. In CNS white matter, two T2 spectral components are observable, the shorter T2 decay component (T2<30 ms) corresponding to myelin water, and the longer component corresponding to intra- and extra-cellular water (T2˜200 ms). In tissue of the peripheral nervous system (PNS), three T2 decay components are observable, which are attributed to myelin water (T2<30 ms), extracellular water (T2˜50-100 ms) and intracellular water (T2˜200 ms). It has been suggested that the shorter extracellular than intracellular water T2 in PNS is the result of efficient relaxation of transverse magnetization facilitated by the ubiquitous presence of collagen fibers. A number of investigators have successfully applied T2 spectroscopy measurements to assess the myelin water fraction in ex vivo nerves.
In various aspects, the methods described herein may be implemented using an MRI system.
Although the present invention is described in connection with an exemplary imaging system environment, embodiments of the invention are operational with numerous other general purpose or special purpose imaging system environments or configurations. The imaging system environment is not intended to suggest any limitation as to the scope of use or functionality of any aspect of the invention. Moreover, the imaging system environment should not be interpreted as having any dependency or requirement relating to any one or combination of components illustrated in the exemplary operating environment. Examples of well-known imaging systems, environments, and/or configurations that may be suitable for use with aspects of the invention include, but are not limited to, personal computers, server computers, hand-held or laptop devices, multiprocessor systems, microprocessor-based systems, set top boxes, programmable consumer electronics, mobile telephones, network PCs, minicomputers, mainframe computers, distributed computing environments that include any of the above systems or devices, and the like.
Computer systems, as described herein, refer to any known computing device and computer system. As described herein, all such computer systems include a processor and a memory. However, any processor in a computer system referred to herein may also refer to one or more processors wherein the processor may be in one computing device or a plurality of computing devices acting in parallel. Additionally, any memory in a computer device referred to herein may also refer to one or more memories wherein the memories may be in one computing device or a plurality of computing devices acting in parallel.
The term processor, as used herein, refers to central processing units, microprocessors, microcontrollers, reduced instruction set circuits (RISC), application specific integrated circuits (ASIC), logic circuits, and any other circuit or processor capable of executing the functions described herein. The above are examples only, and are thus not intended to limit in any way the definition and/or meaning of the term “processor.”
As used herein, the term “database” may refer to either a body of data, a relational database management system (RDBMS), or to both. As used herein, a database may include any collection of data including hierarchical databases, relational databases, flat file databases, object-relational databases, object-oriented databases, and any other structured collection of records or data that is stored in a computer system. The above examples are example only, and thus are not intended to limit in any way the definition and/or meaning of the term database. Examples of RDBMSs include, but are not limited to including, Oracle® Database, MySQL, IBM® DB2, Microsoft® SQL Server, Sybase®, and PostgreSQL. However, any database may be used that enables the systems and methods described herein. (Oracle is a registered trademark of Oracle Corporation, Redwood Shores, Calif.; IBM is a registered trademark of International Business Machines Corporation, Armonk, N.Y.; Microsoft is a registered trademark of Microsoft Corporation, Redmond, Wash.; and Sybase is a registered trademark of Sybase, Dublin, Calif.)
In one embodiment, a computer program is provided to enable the data processing of the method as described herein, and this program is embodied on a computer readable medium. In an example embodiment, the computer system is executed on a single computer system, without requiring a connection to a server computer. In a further embodiment, the computer system is run in a Windows® environment (Windows is a registered trademark of Microsoft Corporation, Redmond, Wash.). In yet another embodiment, the computer system is run on a mainframe environment and a UNIX® server environment (UNIX is a registered trademark of X/Open Company Limited located in Reading, Berkshire, United Kingdom). Alternatively, the computer system is run in any suitable operating system environment. The computer program is flexible and designed to run in different environments without compromising any major functionality. In some embodiments, the computer system includes multiple components distributed among a plurality of computing devices. One or more components may be in the form of computer-executable instructions embodied in a computer-readable medium.
The computer systems and processes are not limited to the specific embodiments described herein. In addition, components of each computer system and each process can be practiced independent and separate from other components and processes described herein. Each component and process also can be used in combination with other assembly packages and processes.
In one embodiment, the computer system may be configured as a server system.
In this aspect, the server system 3001 includes a processor 3005 for executing instructions. Instructions may be stored in a memory area 3010, for example. The processor 3005 may include one or more processing units (e.g., in a multi-core configuration) for executing instructions. The instructions may be executed within a variety of different operating systems on the server system 3001, such as UNIX, LINUX, Microsoft Windows®, etc. It should also be appreciated that upon initiation of a computer-based method, various instructions may be executed during initialization. Some operations may be required in order to perform one or more processes described herein, while other operations may be more general and/or specific to a particular programming language (e.g., C, C#, C++, Java, or any other suitable programming languages).
The processor 3005 is operatively coupled to a communication interface 3015 such that server system 3001 is capable of communicating with a remote device, such as the MRI scanner 1100, a user system, or another server system 301. For example, communication interface 3015 may receive requests (e.g., requests to provide an interactive user interface to receive sensor inputs and to control one or more devices of system 1000 from a client system via the Internet.
Processor 3005 may also be operatively coupled to a storage device 3134. Storage device 3134 is any computer-operated hardware suitable for storing and/or retrieving data. In some embodiments, storage device 3134 is integrated in server system 3001. For example, server system 3001 may include one or more hard disk drives as storage device 3134. In other embodiments, storage device 3134 is external to server system 3001 and may be accessed by a plurality of server systems 3001. For example, storage device 3134 may include multiple storage units such as hard disks or solid-state disks in a redundant array of inexpensive disks (RAID) configuration. Storage device 3134 may include a storage area network (SAN) and/or a network attached storage (NAS) system.
In some embodiments, processor 3005 is operatively coupled to storage device 3134 via a storage interface 3020. Storage interface 3020 is any component capable of providing processor 3005 with access to storage device 3134. Storage interface 3020 may include, for example, an Advanced Technology Attachment (ATA) adapter, a Serial ATA (SATA) adapter, a Small Computer System Interface (SCSI) adapter, a RAID controller, a SAN adapter, a network adapter, and/or any component providing processor 3005 with access to storage device 3134.
Memory area 3010 may include, but are not limited to, random access memory (RAM) such as dynamic RAM (DRAM) or static RAM (SRAM), read-only memory (ROM), erasable programmable read-only memory (EPROM), electrically erasable programmable read-only memory (EEPROM), non-volatile RAM (NVRAM), registers, hard disk memory, a removable disk, a CD-ROM, or any other form of computer-readable storage medium known in the art. The above memory types are exemplary only, and are thus not limiting as to the types of memory usable for storage of a computer program.
In another embodiment, the computer system may be provided in the form of a computing device, such as a computing device 402 (shown in
In another embodiment, the memory included in the computing device 402 may include a plurality of modules. Each module may include instructions configured to execute using at least one processor. The instructions contained in the plurality of modules may implement at least part of the method for simultaneously regulating a plurality of process parameters as described herein when executed by the one or more processors of the computing device. Non-limiting examples of modules stored in the memory of the computing device include: a first module to receive measurements from one or more sensors and a second module to control one or more devices of the MM imaging system 1000.
Computing device 402 also includes one media output component 408 for presenting information to a user 400. Media output component 408 is any component capable of conveying information to user 400. In some embodiments, media output component 408 includes an output adapter such as a video adapter and/or an audio adapter. An output adapter is operatively coupled to processor 404 and is further configured to be operatively coupled to an output device such as a display device (e.g., a liquid crystal display (LCD), organic light emitting diode (OLED) display, cathode ray tube (CRT), or “electronic ink” display) or an audio output device (e.g., a speaker or headphones).
In some embodiments, client computing device 402 includes an input device 410 for receiving input from user 400. Input device 410 may include, for example, a keyboard, a pointing device, a mouse, a stylus, a touch sensitive panel (e.g., a touch pad or a touch screen), a camera, a gyroscope, an accelerometer, a position detector, and/or an audio input device. A single component such as a touch screen may function as both an output device of media output component 408 and input device 410.
Computing device 402 may also include a communication interface 412, which is configured to communicatively couple to a remote device such as server system 302 or a web server. Communication interface 412 may include, for example, a wired or wireless network adapter or a wireless data transceiver for use with a mobile phone network (e.g., Global System for Mobile communications (GSM), 3G, 4G or Bluetooth) or other mobile data network (e.g., Worldwide Interoperability for Microwave Access (WIMAX)).
Stored in memory 406 are, for example, computer-readable instructions for providing a user interface to user 400 via media output component 408 and, optionally, receiving and processing input from input device 410. A user interface may include, among other possibilities, a web browser and an application. Web browsers enable users 400 to display and interact with media and other information typically embedded on a web page or a website from a web server. An application allows users 400 to interact with a server application.
Exemplary embodiments of methods, systems, and apparatus for use in diffusion basis spectrum imaging are described herein. The methods, systems, and apparatus are not limited to the specific embodiments described herein but, rather, operations of the methods and/or components of the systems and/or apparatus may be utilized independently and separately from other operations and/or components described herein. Further, the described operations and/or components may also be defined in, or used in combination with, other systems, methods, and/or apparatus, and are not limited to practice with only the systems, methods, and apparatus described herein.
The order of execution or performance of the operations in the embodiments of the invention illustrated and described herein is not essential, unless otherwise specified. That is, the operations may be performed in any order, unless otherwise specified, and embodiments of the invention may include additional or fewer operations than those disclosed herein. For example, it is contemplated that executing or performing a particular operation before, contemporaneously with, or after another operation is within the scope of aspects of the disclosure.
It will be understood by those of skill in the art that information and signals may be represented using any of a variety of different technologies and techniques. For example, data, instructions, commands, information, signals, bits, symbols, and/or chips may be represented by voltages, currents, electromagnetic waves, magnetic fields or particles, optical fields or particles, or any combination thereof. Similarly, the various illustrative logical blocks, modules, circuits, and algorithm operations described herein may be implemented as electronic hardware, computer software, or a combination of both, depending on the application and the functionality. Moreover, the various logical blocks, modules, and circuits described herein may be implemented or performed with a general purpose computer, a digital signal processor (DSP), an application specific integrated circuit (ASIC), a field programmable gate array (FPGA), or other programmable logic device, discrete gate or transistor logic, discrete hardware components, or any combination thereof designed to perform the functions described herein. Exemplary general-purpose processors include, but are not limited to only including, microprocessors, conventional processors, controllers, microcontrollers, state machines, or a combination of computing devices.
When introducing elements of aspects of the invention or embodiments thereof, the articles “a,” “an,” “the,” and “said” are intended to mean that there are one or more of the elements. The terms “comprising,” “including,” and “having” are intended to be inclusive and mean that there may be additional elements other than the listed elements.
In the examples described below, it was demonstrated that short-term dynamic changes in diffusion MRI and T2 relaxation properties accompany repetitive electrical activity in myelinated nerve fibers. These changes were observable on the timescale of minutes. From the MRI electrophysiology, and microscopy data (post-stimulation-fixed or quiescent-fixed tissues), a consistent picture emerges—repetitive electrical activity leads to osmotically-driven microstructural modification of the myelinated fibers. The dynamic changes in water T2 spectral components with electrical activity in WM observed using the disclosed methods may form the basis of an additional, complementary MM-based approach to non-invasively assess WM function in vivo. Further, it was demonstrated that WM diffusion fMRI can non-invasively localize foci of electrical conduction blockage in axonal pathways, thereby providing an important clinical tool for use in management of WM neurodegenerative diseases and/or neurotrauma.
To compare the effectiveness of the DBSI and DTI analysis models for the detection of optic nerve pathology in mice, the following experiments were conducted.
Eight 10-week-old female C57BL/6 mice underwent optic nerve DBSI, followed by a week-long recuperation prior to active immunization for experimental autoimmune encephalomyelitis (EAE). Visual acuity of all mice was assessed daily. Longitudinal DBSI was performed in mouse optic nerves at baseline (naïve, before immunization), before, during, and after the onset of optic neuritis.
To compare the effectiveness of the DBSI and DTI analysis models for the detection of optic nerve function non-invasively in human subjects, the following experiments were conducted.
Single-direction diffusion functional MM (fMRI) has been previously employed to assess activation of mouse optic nerve via decreased apparent diffusion coefficient perpendicular to axonal fibers (ΔADC⊥) during flashing-light visual or direct electrical stimulation. Unlike blood-oxygen level dependent (BOLD) fMRI, the stimulation-induced ΔADC⊥ was found to be independent of vascular effects using diffusion functional MRI. This functional fMRI response (ΔADC⊥) was found to be attenuated in optic nerves from mice with optic neuritis.
In this experiment, diffusion-weighted imaging (DWI) measurements were performed in human optic nerves with visual stimulation and the data were analyzed using diffusion basis spectrum imaging (DBSI) and diffusion tensor imaging (DTI). It was hypothesized that DBSI-estimated Δλ⊥ was more sensitive for detection of axonal activation than DTI-Δλ⊥ or DWI-ΔADC⊥ since DBSI enabled the exclusion of confounding effects of surrounding CSF to a relatively higher extent as compared to conventional DTI.
DWI (25-direction repeated with polarity reversal, max b-value=1,000 s/mm2, 3T Siemens Prisma) was performed with inner-volume single-shot EPI using a 64-channel head coil: TR=2.5 s, TE=53.8 ms, Δ=18 ms, d=6 ms, in-plane resolution=1.1×1.1 mm, slice thickness=4 mm, echo-train length=30, and acquisition time=2.4 minutes. Imaging slices were perpendicular to optic nerve (
As illustrated in
In another experiment, seven healthy subjects were recruited. Three had normal vision uncorrected, and four wore contact lenses. Scans were performed on a 3T Siemens Prisma scanner. A 64-channel head coil was used with a mirror to allow the subject to see the flashing checkerboard while in the scanner. One eye was covered by gauze and taped shut with medical tape.
Whole brain MPRAGE was acquired to precisely locate the optic nerves (
Raw DWIs were post-processed and coregistered before DBSI-λ⊥ and DTI-λ⊥ were derived using lab-developed software. Measurements from both image slices were averaged for each subject.
Baseline DBSI-λ⊥ was lower and λ| was higher than baseline DTI-λ⊥ and λ|, respectively (
The results of these experiments demonstrated that DBSI-Δλ⊥ detected greater axonal activation-induced changes than DTI-Δλ⊥. These experiments are the first to detect axonal function-associated diffusion changes in a living human subject.
To demonstrate the ability to perform function imaging of neuronal activity using DBSI and T2 spectral methods, the following experiments were conducted. The experiments were carried out on perfused, ex vivo frog sciatic nerves, which due to their extreme robustness have been the subject of numerous electrophysiology and MM studies.
Frog Nerve Preparation
Jumbo bullfrogs (Rana catesbeiana) were purchased from Rana Ranch (Twin Falls, Id.) and housed in a climate-controlled environment with a 12/12 hr-light/dark cycle. The animal housing consisted of a standard clear plastic container [18″(1)×10″(w)×10″(h)] holding 1.5 L of water and tipped at an angle to provide a dry area within the cage. Frogs were fed pelleted chow (5LP3, Lab Diet, St. Louis, Mo.) three times per week.
Frogs were anesthetized in a solution of tricaine methanesulfonate (Sigma-Aldrich, St. Louis, Mo.), 0.5 g/L in dechlorinated tap water with pH adjusted to 7.4. Once immobilized, frogs were decapitated and pithed. After removal of the skin, the body was pinned into a dissecting tray and a 4-5 cm segment of the sciatic nerve was dissected from the lower back to just proximal to its branching into peroneal and tibial nerves. During dissection, the surgical field was irrigated frequently with frog Ringer'"'"'s solution without glucose (see below) to maintain tissue moisture content. After gently debriding fat and surrounding connective tissue from the sciatic nerve, the nerve segment was tied off first at the proximal and then at the distal end with 2-0 silk suture and cut out. Twitching of the gastrocnemius muscle upon tying off the nerve at the proximal end served as verification that the sciatic nerve has not been damaged during the dissection procedure. Once dissected, the tied-off nerve was placed in a Petri dish of oxygenated frog Ringer'"'"'s solution containing glucose at room temperature.
The frog Ringer'"'"'s solution recipe used in these experiments was intended to match the ionic composition and osmolarity of Rana catesbeiana blood as nearly as possible. Frog Ringer'"'"'s solution with glucose contained: 100 mM Na+, 2.6 mM K+, 99.6 mM Cl−, 1 mM Mg2+, 1 mM Ca2+, 1 mM SO42−, 2.5 mM HPO42−, and 2 mM glucose, pH adjusted to 7.4. The glucose-free Ringer'"'"'s solution used for tissue irrigation during dissection was the same, except for the omission of glucose and the addition of one extra mmol/L of NaCl to maintain the same solution osmolarity. The frog Ringer'"'"'s solution used in perfused nerve experiments was oxygenated by heating the solution to 55° C. to displace any inert dissolved gases, and then the solution was bubbled with O2 via a submerged gas dispersion tube at a rate of 2 L/min during the time required for the solution temperature to drop to 30° C. (>1 hr.). Further thermal equilibration to room temperature occurred without bubbling of the solution. The solution, thus oxygen-saturated at 30° C., contained ˜1.15 μmol/mL of dissolved O2. In the case of perfused Rana pipiens sciatic nerves undergoing 100 Hz electrical stimulation at 20° C., oxygen consumption by the nerve tissue was previously reported to increase to 1.15 μmol O2/hr.·g wet weight. The typical fresh jumbo bullfrog sciatic nerve weighed ˜15 mg/cm length. Assuming roughly similar O2 consumption in Rana catesbeiana sciatic nerves with electrical activity, at a perfusate flow rate of 3 mL/hr., the oxygen supply (3.5 μmol O2/hr.) would well exceed the tissue oxygen requirement of the perfused nerve (˜0.07 μmol O2/hr.) with the most vigorous electrical stimulation frequency employed in these experiments.
Electrophysiology Flow Cell for MRI
A perfusion flow cell was constructed from readily-available materials, holding the excised nerve immobilized at the two ends with retractable suction electrodes used for electrical stimulation and recording of the compound action potential. As illustrated in
Electrical stimulation was delivered as 100 μs voltage pulses from a Pulse/Function Generator (model 33210A, Agilent Technologies, Santa Clara, Calif.) into an Analog Stimulus Isolation Unit (model 2200, A-M Systems, Sequim, Wash.). The DC stimulus/compound action potential (CAP) signals were carried to/from the magnet via shielded triax-cables. Suction electrodes were used for nerve stimulation and CAP recording. Placement of the recording amplifier grounding electrode in close proximity to the tip of the stimulating electrode was found to significantly reduce interference artifacts. The compound action potential signal was amplified (1000×) and filtered (high-pass cutoff: 160 Hz, low-pass cutoff: 20 kHz) via a home-built two-stage amplifier before passing to a digital oscilloscope (model TBS1052B, Tektronix, Beaverton, Oreg.) for signal averaging (128 averages). The signal-averaged CAP waveform was ported to a laptop pc and saved for offline processing and analysis.
For one set of the nerves studied (the diffusion fMRI studies carried out with 40-minute temporal resolution, described below), the electrical stimulation consisted of super-maximal stimulation delivered at 100 Hz frequency for a period of 40 minutes. In the other set of nerves, imaged with 20-minute temporal resolution, the nerves were stimulated first at 50 Hz×24 minutes, followed by a resting period of 3 hours, before undergoing a second round of electrical stimulus 100 Hz×24 minutes.
Magnetic Resonance Imaging
MRI measurements were carried out in a 4.7 Tesla Agilent small-animal imaging system using home-built RF coils for signal transmit/receive.
Diffusion fMRI data was acquired with a multi-echo spin-echo diffusion-weighted imaging sequence. Acquisition parameters included TR=1.5 s, 3-echoes (TE1=28.8 ms, TE2=8.3 ms). A maximum diffusion-weighting b value of 3 ms/μm2, was employed with a 25-direction diffusion-encoding scheme, and diffusion timings Δ=18 ms, δ=5 ms. A 1.5-mm slice thickness with a 0.5 mm gap between slices was used. The imaging field-of-view was 7×7 mm2 with an acquisition matrix size of 32×32 (20 minutes per image) or 64×64 (40 minutes per image).
The RF coil setup was modified during the course of these experiments. An actively-decoupled transmit/receive coil pair was employed in earlier experiments (32×32) with a Helmholtz transmit coil and hemi-cylindrical surface receive coil for signal detection. For later experiments, with higher spatial resolution, a single transmit/receive coil was used (see
Carr-Purcell-Meiboom-Gill Multi-Echo Acquisition (T2 Relaxation Spectra) was used to conduct a non-localized spectroscopy experiment. Trains of 4,096 echoes were acquired with a 1 ms inter-echo spacing. A repetition time of 20 s was used between the start of consecutive echo trains, and data from sixteen echo trains were averaged, thus the total measurement time was 5.3 minutes.
After setup in the magnet and verifying electrical activity of the perfused nerve, two baseline diffusion MRI scans were acquired, followed by a series of interleaved T2 spectroscopy (5.3 min) and diffusion MRI (40 min) acquisitions. The second T2 spectroscopy dataset was acquired just prior to commencement of repetitive electrical stimulation (100 Hz×40 min). One diffusion dataset was acquired during application of repetitive electrical stimulation. Upon completion of electrical stimulation, the interleaved T2 and diffusion MM acquisitions continued. Measurements terminated at 5 hours after the start of electrical stimulation.
MRI Data Analysis
For DBSI analysis, the raw k-space image data was multiplied by a Hamming window filter prior to zero-filling to a 128×128 matrix and denoising, with signal-averaging of the three acquired echoes. The resulting image data was then analyzed via the diffusion basis spectrum imaging (DBSI) package developed in-house and running in Matlab 2015b (MathWorks, Natick, Mass.). The 25-direction diffusion-weighted signal (k: 1-25) was modeled as contributions from a basis set of anisotropic, cylindrically-symmetric diffusion tensors (i: 1-NAniso) with axial and radial diffusivities (λ∥,i and λ⊥,i, respectively) and a spectrum of isotropic diffusion with amplitude f(D) at diffusivity D, as summarized in Equation .
Since the frog sciatic nerve was composed of a single, coherent axonal fiber bundle (i.e., no crossing fibers), the anisotropic modeling was limited to a single anisotropic tensor (NAniso=1), referred to as the fiber component (ffiber). The isotropic spectral components were differentiated based upon diffusivity and denoted as restricted, (0<DR≤0.3 μm2/ms), hindered, (0.3<DH≤2.0 μm2/ms), or free (D>2.0 μm2/ms).
Voxel-wise prescriptions of regions of interest (ROIs) for inclusion in diffusion MRI metric measurement for a given nerve at a given imaging time-point were based solely on the DBSI ffiber parameter map. Contiguous imaging voxels within each slice that satisfied the cutoff ffiber ≥0.67 were included in the nerve ROI. The nerve ROIs, prescribed in this manner and outlined using ITK-Snap software [ITK-SNAP, version 3.6.0, University of Pennsylvania] were saved and the average diffusion parameter values in the nerve ROI for each slice were extracted from the DTI and DBSI parameter image maps using an in-house script running in Python 2.7 (www.python.org).
DTI analysis of the same 25-direction diffusion-weighted imaging data sets was performed using existing methods known in the art.
To perform the T2 spectral analysis, the CPMG echo data was decomposed into T2 spectral components via non-negative least square fitting (NNLS) with a first-derivative smoothing term. In this approach, echo-decay data was assumed to be described by the form Equation :
For the T2 spectral analysis, N=4,068 echoes were used (ti: 8-4,076 ms), and the spectrum was calculated as the set of amplitudes (sj) at M=1,001 log-spaced T2 grid points (T2j: 2-5000 ms), minimizing the least-squares misfit subject to the first-derivative smoothing constraint. The extent of smoothing employed for each dataset [i.e., the coefficient μ] was determined based upon the F-statistic for the sum of squares of the data/model misfit as compared to the misfit with no smoothing term applied (μ=0). The value of μ employed for the final accepted T2 spectral modeling was the minimum value which resulted in p<0.10. This statistical-threshold was found to produce consistent results for estimated T2 and component amplitudes amongst independent data sets, and values similar to those reported previously in the literature for ex vivo sciatic nerve of various frog species.
Diffusion fMRI DTI Time-Course
As illustrated in
The principal eigenvalue of the DTI diffusion tensor represented axial diffusivity (λ∥) of the diffusion ellipsoid. With the assumption of cylindrical symmetry, the radial diffusivity is taken as the average of the other two eigenvectors. Time-points which were statistically-significantly different than the pre-stimulus measurement, as determined via repeated-measures ANOVA/Tukey post-hoc testing, are indicated by an asterisk.
As illustrated in
As illustrated in
While the decrease in λ∥, appeared to be transient and recovered to baseline values, decreases in λ⊥, and ADCDTI were more sustained over ˜5 h post stimulation.
Similar DTI measurements of the perfused frog axon were derived from image data acquired with a 32×32 image matrix (20-minute temporal resolution).
Diffusion Functional DBSI Time-Course
The readouts on fiber-component axial (λ∥,fiber) and radial (λ⊥,fiber) diffusivities from DBSI were similar to those provided by DTI modeling (
As illustrated in
As illustrated in
As illustrated in
As illustrated in
As illustrated in
While some of the DBSI signal components exhibited only transient changes (fR, DH, DR, and λ∥,1), the induced change in DBSI fiber radial diffusivity (λ⊥,1) was sustained over the post stimulus observation period.
T2 Spectral Changes
To determine whether dynamic T2 spectroscopy could non-invasively detect structural changes accompanying repetitive electrical activity, dynamic T2 spectra were successfully acquired from five perfused nerves.
For the T2 spectral analysis, N=4,068 echoes were used (ti: 8-4,076 ms), and the spectrum was calculated as the set of amplitudes (sj) at M=1,001 log-spaced T2 grid points (T2,j: 2-5000 ms), minimizing the least-squares misfit subject to the first-derivative smoothing constraint. The extent of smoothing employed for each dataset [the coefficient μ] was determined based upon the F-statistic for the sum of squares of the data/model misfit as compared to the misfit with no smoothing term applied (μ=0). The value of μ employed for the final accepted T2 spectral modeling was the minimum value which resulted in p<0.10. This statistical-threshold was found to produce consistent results for estimated T2 and component amplitudes amongst independent data sets, and values similar to those reported previously in the literature for ex vivo sciatic nerve of various frog species.
The pre-stimulus T2-spectroscopy-based signal fractions [fM=0.173±0.009 (myelin), fEx=0.568±0.011 (extracellular), fAx=0.259±0.011 (axonal), mean±SEM] were in reasonably good agreement with existing literature reports for in vivo and ex vivo sciatic nerves of various frog species.
Diffusion fMRI Dose-Response
To assess diffusion fMRI responses to stimuli of reduced duration and/or frequency of applied super-maximal electrical stimulation, the following experiments were conducted.
Starting from the DTI results, the simplest interpretation, considering differences between pre-stimulus and post-stimulus values of λ⊥ and assuming the diffusion behavior is dominated by the diffusivity of the extracellular volume fraction, would be that the bout of sustained electrical stimulus leads to swelling of the axonal fibers and shrinkage of the extracellular space. This presumed shift of water out of the extracellular space did not appear to be fully reversible within the time-frame of the current measurements. With this rationale, however, the observed dynamics of λ∥ were not as obvious. Possibilities included changes of the axonal microstructure and/or differences in the free diffusivities/viscosities of intra-axonal and extracellular water. This may be understood more fully in light of the DBSI and microscopy results (see below).
In DBSI modelling, the behavior of fR suggested the formation of a transient membrane-bounded vesicular structure, induced by repetitive electrical stimulation, within the perfused frog sciatic nerve. While this phenomenon cannot be revealed by DTI modeling of the diffusion fMRI data, it may account for some of the observed decrease in λ∥ from DTI.
From the perspective of the dynamic T2 data, considering the nature of the sub-myelinic vacuoles (
The results from dynamic T2 spectroscopy suggested that an increase in myelin water, which was uniformly observed for the electrically-stimulated nerves, may merit future investigation as a complementary approach to non-invasively visualize rapid structural changes associated with electrical activity in white-matter or PNS. The measurements obtained in these experiments, which used non-localized CPMG echoes, were less demanding than in vivo imaging-mode implementation of dynamic myelin-water assessment. Further, the correlations between the T2 axonal water fraction and DBSI restricted fraction may be challenging to study in the context of the CNS, since typically T2 axonal and extracellular fractions are distinctly separated only in PNS tissue. However, application of exogenous T2 relaxation agents may enable additional study of the observed correlations between the T2 axonal water fraction and DBSI restricted fraction.
The diffusion fMRI results of this experiment further highlight differences between the perfused frog sciatic nerve and previously published in vivo mouse optic nerve results. For example, in the optic nerve diffusion fMRI studies, there was no observable change in the axial diffusivity with repetitive axonal activation. Further, although the mouse optic nerve the observable diffusion fMRI response in ΔADC1 was rapidly reversible, the perfused frog sciatic nerve, Δλ1 was not rapidly reversible. It is to be note, however, that the perfused sciatic nerve required vigorous electrical stimulation to achieve a smaller relative Δλ1 than was seen in ΔADC⊥ for the in vivo mouse optic nerve after more modest stimulation.
Further, some of these differences likely arise from the markedly different tissue geometry in the two nerves. For instance, the extracellular space in the frog sciatic nerve tissue accounts for at least 50% of the total water content of the frog sciatic nerve, based upon T2 spectroscopy measurements, hence the axonal packing density is reduced in the frog PNS tissue as compared with mouse CNS. Without being limited to any particular theory, the extracellular space typically accounts for 20% of tissue volume in normal CNS. Thus, a shift of water out of the extracellular space would be expected to have a larger impact on the overall diffusion characteristics in mouse CNS. The demand for highly-interconnected neurons in CNS of higher organisms is often cited as the selective pressure that spurred the evolutionary development of dense packing and smaller gauge axons in CNS tissue. Typical mouse optic nerve axons are on the order of 1 μm in diameter and are uniformly myelinated in healthy tissue. In addition to very small unmyelinated axons, the frog PNS contains myelinated axons with a distribution of outer diameters whose upper limit is ˜16 μm.
The mouse optic nerve and the frog sciatic nerve further differ with respect to the myelin itself. The larger PNS axons tend to be blanketed by very thick myelin wrapping, sometimes over 100 layers deep, whereas for the smaller axons in the CNS, fewer total layers of myelin are the rule. Additionally, the internodal spacing in mouse optic nerve is much shorter [138 μm, on average] than that in frog sciatic nerve axons [wherein, it ranges up to 1.5 mm].
The experiments described above performed using perfused frog sciatic nerves were relatively large and robust compared to corresponding CNS nerves, such as perfused mouse optic nerves. This may pose a challenge for any additional experiments performed using perfused mouse optic nerves, including in animal models of optic neuritis and/or trauma, which represent a natural next step for mechanistic exploration in mammalian CNS.
To assess the structures involved in activation and deactivation of the frog sciatic nerves visualized using DBSI and T2 spectral methods as described above, the following experiments were conducted. The frog sciatic nerves obtained as described above in Example 2 were fixed and subjected to imaging using electron microscopy as described below.
Tissue Fixation and Electron Microscopy
The fixative used for perfusion fixation contained 2% glutaraldehyde, 2 mM calcium acetate and 20 mM sodium cacodylate. After 1 hour in fixative at room temperature, the nerve-containing fixative solution was transferred to a refrigerator and stored at 4° C. for a total of 12 hours. After fixation, the nerve tissue was diced into 3-mm segments and rinsed in 0.10 M cacodylate buffer at pH 7.4 with 2 mM calcium chloride 3 times for 10 minutes each time. Samples were then subjected to a secondary fixation step for one hour in 1% osmium textroxide/1.5% potassium ferrocyanide in cacodylate buffer. Subsequently, samples were rinsed in 0.10 M maleate buffer at pH 6.0 three times for 10 minutes per rinse and en bloc stained for 1 hour with 1% uranyl acetate in maleate buffer. At the completion of staining, samples were briefly washed in ultrapure water, dehydrated in a graded acetone series (50%, 70%, 90%, 100%×2) for 10 minutes each step, infiltrated with microwave assistance (Pelco BioWave Pro, Redding, Calif.) into LX112 resin, and flat embedded in a silicone mold to orient the nerve segment for cross-sectioning. Samples were cured in an oven at 60° C. for 48 hours. Once the resin was cured, 70 nm thin sections were taken. TEM imaging was performed at 80 keV using a JEOL JEM-1400 Plus microscope (JEOL, Tokyo, Japan).
EM images of individual axons were segmented using a semi-automatic image segmentation method to define the cross-sectional area occupied by myelin, axoplasm and periaxonal vacuole for each axon. The Axon/Myelin/Vacuole segmentation method was a region or pixel-based image segmentation method that involved the selection of initial seed points. A seed pixel marker was manually placed in myelin, axon, and possibly vacuole regions (depending upon whether or not they were seen in a given image). In practice, the segmented areas of vacuole typically overlapped with axon.
The images were first pre-processed to enhance the contrast between the low intensity myelin and relatively higher intensity axonal regions, with default parameters of the built-in MATLAB (2015b) function, imadjust. Images were then filtered to smooth the intensities as well as sharpened boundaries/edges of the axon and myelin regions. Filtering was done in the frequency domain. Smoothing the intensities was implemented using a Gaussian low pass filter (˜1.3% of the width of the Fourier transform of the image) and the edges were sharpened using a Gaussian high pass filter (−0.6% of the width of the Fourier transform of the image). This preprocessing step was thought to improve segmentation and demarcate their edges from surrounding regions.
A region growing segmentation approach was then used to examine neighboring pixels of initial seed points and determine whether the pixel neighbors should be added to a region based on pixel intensity. The region growing segmentation was based on MATLAB (2015b) built-in function grayconnected, which finds connected regions of similar intensity in the grayscale image, within a range of intensity values determined by a threshold or tolerance that can be user-controlled. The user specifies the intensity value to use as a starting point, the seed pixel, and the function grayconnected generates a binary mask image, where all of the foreground pixels are 8-connected to the seed pixel by pixels of similar intensity.
The area of the binary mask image, representing a segmented region corresponding to axon, vacuole or myelin area, was measured using the MATLAB (2015b) built-in function regionprops. The diameter of each segmented region was also obtained from the area using the formula for area of an equivalent circle, A=(π/4)2, where A=area of segmented region and D=equivalent diameter of segmented region. The measurements (area and diameter) for each axon and myelin segmented were recorded or saved into a table/spreadsheet in .csv format. Areas were reported in pixel units.
The myelin area reported in the .csv output was representative of the total area bounded by the myelin, including the enclosed axon and vacuole roi'"'"'s. Conversion from image pixels to areas in μm2 were performed offline based upon the known magnification/scale of the acquired EM images.
Statistical tests of the electron microscopy images were performed using Statistica 13.2 (Tibco Software, Inc., Palo Alto, Calif.). The distributions of sub-myelinic vacuole radii in stimulation-fixed versus rested-fixed frog nerve axons, which followed roughly exponential distributions, were compared via the Cox-Mantel test within the Survival and Failure Time Analysis Module of Statistica.
The two sciatic nerves of a single bullfrog were excised and assembled in separate perfusion flow cells on the benchtop. One of the nerves was stimulated for 40 min×100 Hz (as in the in-magnet diffusion fMRI studies) and perfusion-fixed immediately at the end of the stimulation period. The other nerve was rested and perfusion fixed at the same time as the stimulation-fixed nerve.
Transverse EM images of the two sciatic nerves showed clear microstructural differences. At relatively low magnification, the myelin in both nerves was observed to be well-preserved and intact. A low-magnification image of the stimulation-fixed nerve is shown in
In the stimulation-fixed axons, the sub-myelinic vacuoles were both more prevalent and larger in size than in the rest-fixed axons (
Transient paranodal intramyelinic vacuoles induced by repetitive in situ electrical stimulation of the frog sciatic nerve have been reported previously, although previous studies have specifically focused on microstructural alterations of the paranodal myelin by examination of electron microscopy (EM) images of longitudinally-sectioned nodes of Ranvier. Without being limited to any particular theory, the spacings between nodes of Ranvier in frog sciatic nerves are known to be proportional to axonal diameter and may be as large as 1.5 mm. Thus, successfully sampling enough nodes of Ranvier via EM for statistical analysis may be exceptionally time-consuming in longitudinally-sectioned specimens.
Examining transverse-sectioned EM images of fixed frog sciatic nerves enabled more time-efficient sampling of axons. However, this sampling is not restricted to any unique axial position (nodal, paranodal, internodal, etc.). Even with the introduction of variation due to differences in axial position of various samples, the distribution of vacuole radii in the sampling of stimulation-fixed and rest-fixed axons were statistically-significantly different.
The amalgamated results from diffusion fMRI described above and electron microscopy results of this experiment suggested that sub-myelinic vacuole formation was prominent in the microstructural rearrangements that occurred with repetitive electrical activity in the frog sciatic nerve model system.
Without being limited to any particular theory, a biophysical mechanism that likely accounts for the observed microstructural changes may include localization of K+ voltage-gated channels, which open up to allow egress of potassium ions from the axon during the re-polarization step of action potential conduction, to the sub-myelinic axonal membrane when the nerve is activated. In the bullfrog sciatic nerve, these Kv channels are known to be distributed along the length of the internodal axonal membrane. According to this biophysical mechanism, repetitive electrical activity in myelinated axons results in an accumulation of K+ and its osmotically-associated water in the confined space. The stimulation-induced sub-myelinic vacuoles observed via electron microscopy and transient post-stimulus DBSI restricted diffusion signal component supported this biophysical model for the perfused frog sciatic nerve model system.
To assess the physiological mechanisms involved in activation and deactivation of the frog sciatic nerves visualized using DBSI and T2 spectral methods as described above, the following experiments were conducted. The CAP recordings of the frog sciatic nerves obtained as described in Ex. 2 were analyzed and correlated against T2 and diffusion MM measurements as described below.
Electrophysiology of the Frog Sciatic Nerve
A universal observation in the perfused nerve electrophysiology experiments was that sustained, repetitive electrical stimulation produced a decrease in the compound action potential (CAP) conduction velocity during the stimulation period. In concert with the conduction velocity decrease, the CAP waveform was observed to broaden with a concomitant decrease in peak-to-peak amplitude (
Correlations Amongst Electrophysiology, T2, and Diffusion fMRI Changes
For linear correlations amongst dynamic T2 spectroscopy, diffusion fMRI, and electrophysiology measures of nerve response, p<0.05 was taken as the threshold for statistical significance. Repeated-measures ANOVA with Tukey post-hoc testing was used to identify the time-point at which diffusion MRI parameters differed from pre-stimulus values.
Statistically-significant correlations were observed between the normalized axonal CAP conduction velocity measured at the end of the 40 min×100 Hz stimulation period (vCAP,40 min/vCAP,0) and DTI-based diffusion fMRI response measures (
As described above, sub-myelinic vacuole formation was prominent in the microstructural rearrangements that occurred with repetitive electrical activity in the frog sciatic nerve model system. This phenomenon was consistent with observed changes in nerve electrophysiology and dynamic T2 spectroscopy described above.
Without being limited to any particular theory, from a mechanistic standpoint, the electrical disturbance of the action potential (AP) wave-front charges up the next downstream node of Ranvier to its threshold potential as it propagates down a given axon. As each node of Ranvier reaches threshold potential, sodium voltage-gated channels node open, depolarizing the axon and sustaining the AP propagation. The physical parameters that dictate τ, the exponential time-constant describing the charging at a node of Ranvier, are ri (the longitudinal ohmic resistance to current flow in the axoplasm) and cn (the nodal membrane capacitance), such that τ=ri·cn.
Without being limited to any particular theory, the CAP waveforms observed in these experiments are summations of action potentials arising from the entire ensemble of conducting axons within the nerve, with the larger-gauge axons conducting more rapidly than the finer-gauge axons. As a result, a proportional decrease in action potential conduction velocity across the ensemble of axons accounts for the broadening of the CAP waveform.
Without being limited to any particular theory, the ballooning sub-myelinic vacuoles of the activated sciatic nerve, observed via EM (
To demonstrate the visualization of axonal conduction blockage using the DBSI and T2 spectrum methods described above the following experiments were conducted.
A perfused frog sciatic nerve tied off in the center with three sutures (
Two types of diffusion MR images of the perfused nerve were acquired before and after applying electrical stimulation to the proximal end of the nerve (100 Hz×60 min). One image set consisted of six axial slices through the nerve (3 proximal to the conduction blockage and 3 distal to the sutures/conduction blockage,
To demonstrate the diagnosis of an optic neuropathy using the DBSI methods described above, the following experiments were conducted.
Axon damage is frequently seen in optic neuropathies such as glaucoma and optic neuritis (ON). Glaucoma is a group of optic neuropathies characterized by progressive degeneration of retinal ganglion cells (RGCs) and their axons and is the leading cause of irreversible blindness worldwide. Although pathogenesis of glaucoma remains incompletely understood, optic nerve axon injury appears to play a crucial role in RGC axon and cell body degeneration. Optic neuritis is an autoimmune optic neuropathy, frequently the presenting feature of multiple sclerosis (MS). Although inflammatory demyelination is the hallmark pathology of ON, the axonal and neuronal injury has been widely observed. Despite differing etiologies, axonal injury in the optic nerves of both glaucoma and ON patients could potentially advance and cause neuronal damage. With the simultaneous and noninvasive assessment of optic nerve pathology and function afforded by the DBSI methods described above, an enhanced understanding of the underlying pathophysiology in glaucoma and ON may lead to the improved patient care and a surrogate end point for future clinical trials.
Four glaucoma patients (1 male, 3 female, ages ranging 58-76) were subjected to DBSI assessment of optic nerves using the DBSI methods described above. Among the glaucoma patient population, two patients were low tension glaucoma and two patients were primary open angle glaucoma.
To compare the effectiveness of the DBSI and DTI analysis models for the detection of nerve activation and deactivation using of fMRI, the following experiments were conducted.
A cross-validation (CV) of the DTI and DBSI model fits of the data was carried out on data from two pre-stimulus MRI measurements previously analyzed in Example 2 ((the second and third time points in
Signal intensities (in scanner signal intensity units) of each of the 25 different diffusion weightings/directions from each of the six nerves were compared and analyzed using the DBSI and DTI models. For each data point in
To compare the effectiveness of the DBSI and DTI analysis models for the detection of optic nerve function non-invasively in human subjects, the following experiments were conducted. DTI and diffusion basis spectrum imaging (DBSI) were used to assess human optic nerve activation during exposure to a flashing checkerboard stimulation.
Previously, single-direction diffusion-weighted MRI of optic nerves in mice demonstrated that the mouse optic nerves exhibited decreased apparent diffusion coefficient perpendicular to axonal fibers (ADC⊥) during flashing-light stimulation. Unlike blood-oxygen level dependent (BOLD) fMRI, the activation induced ADC⊥ decrease was independent of alterations in blood flow and more directly associated with axonal activation. In a subsequent study, we observed that the decrease in ADC⊥ was attenuated in optic nerves affected by optic neuritis in mice.2 We speculate this is due, in part, to the presence of inflammation confounding ADC⊥ measurements which may have masked the effects of actual axonal activation.