The peripheral sensory nerves that innervate the cornea can be easily damaged by trauma, surgery, infection or diabetes. Several growth factors and axon guidance molecules, such as Semaphorin3A (Sema3A) are upregulated upon cornea injury. Nerves can regenerate after injury but do not recover their original density and patterning. Sema3A is a well known axon guidance and growth cone repellent protein during development, however its role in adult cornea nerve regeneration remains undetermined. Here we investigated the neuro-regenerative potential of Sema3A on adult peripheral nervous system neurons such as those that innervate the cornea. First, we examined the gene expression profile of the Semaphorin class 3 family members and found that all are expressed in the cornea. However, upon cornea injury there is a fast increase in Sema3A expression. We then corroborated that Sema3A totally abolished the growth promoting effect of nerve growth factor (NGF) on embryonic neurons and observed signs of growth cone collapse and axonal retraction after 30 min of Sema3A addition. However, in adult isolated trigeminal ganglia or dorsal root ganglia neurons, Sema3A did not inhibited the NGF-induced neuronal growth. Furthermore, adult neurons treated with Sema3A alone produced similar neuronal growth to cells treated with NGF and the length of the neurites and branching was comparable between both treatments. These effects were replicated in vivo, where thy1-YFP neurofluorescent mice subjected to cornea epithelium debridement and receiving intrastromal pellet implantation containing Sema3A showed increased corneal nerve regeneration than those receiving pellets with vehicle. In adult PNS neurons, Sema3A is a potent inducer of neuronal growth in vitro and cornea nerve regeneration in vivo. Our data indicates a functional switch for the role of Sema3A in PNS neurons where the well-described repulsive role during development changes to a growth promoting effect during adulthood. The high expression of Sema3A in the normal and injured adult corneas could be related to its role as a growth factor.

Semaphorin3A induces nerve regeneration in the adult cornea-a switch from its repulsive role in development.

Electrical brain stimulation is a proven therapy for epilepsy, but long-term seizure free outcomes are rare. Early implantable devices were developed for open-loop stimulation without sensing, embedded computing or adaptive therapy. Recent device advances include sensing and closed-loop responsive stimulation, but these clinically available devices lack adequate computing, data storage and patient interface to concisely catalog behavior, seizures, and brain electrophysiology, despite the critical importance of these details for epilepsy management. Here we describe the first application of a distributed brain co-processor providing an intuitive, bi-directional interface between device implant, patient & physician, and implement it in human and canine patients with epilepsy living in their natural environments. Automated behavioral state tracking (awake and sleep) and electrophysiologic classifiers for interictal epileptiform discharges and electrographic seizures are run on local hand-held and distributed cloud computing resources to guide adaptive electrical stimulation. These algorithms were first developed and parameterized using long-term retrospective data from 10 humans and 11 canines with epilepsy and then implemented prospectively in two pet canines and one human with drug resistant epilepsy as they naturally navigate their lives in society. One Sentence SummaryWe created a distributed brain co-processor for continuous neurophysiologic tracking and controlling adaptive brain stimulation to treat epilepsy.

https://www.biorxiv.org/content/biorxiv/early/2021/03/09/2021.03.08.434476.full.pdf

Distributed Brain Co-Processor for Neurophysiologic Tracking and Adaptive Stimulation: Application to Drug Resistant Epilepsy

Drug-resistant epilepsy, characterized by ongoing seizures despite appropriate trials of anti-seizure medications, affects approximately one-third of people with epilepsy. Brain stimulation has recently become available as an alternative treatment option to reduce symptomatic seizures in short and long-term follow-up studies. Several questions remain on how to optimally develop patient-specific treatments and manage therapy over the long term. This review aims to discuss the clinical use and mechanisms of action of Responsive Neural Stimulation and Deep Brain Stimulation in the treatment of epilepsy and highlight recent advances that may both improve outcomes and present new challenges. Finally, a rational approach to device selection is presented based on current mechanistic understanding, clinical evidence, and device features.

Brain Stimulation Treatments in Epilepsy: Basic Mechanisms and Clinical Advances.

Drug-resistant epilepsy, characterized by ongoing seizures despite appropriate trials of anti-seizure medications, affects approximately one-third of people with epilepsy. Brain stimulation has recently become available as an alternative treatment option to reduce symptomatic seizures in short and long-term follow-up studies. Several questions remain on how to optimally develop patient-specific treatments and manage therapy over the long term. This review aims to discuss the clinical use and mechanisms of action of Responsive Neural Stimulation and Deep Brain Stimulation in the treatment of epilepsy and highlight recent advances that may both improve outcomes and present new challenges. Finally, a rational approach to device selection is presented based on current mechanistic understanding, clinical evidence, and device features. 

Brain stimulation treatments in epilepsy: Basic mechanisms and clinical advances

Poly(3,4‐ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS) is a promising material because of its favorable electrical and mechanical properties, stability in ambient environments, and biocompatibility. It finds broad application in energy storage, flexible electronics, and bioelectronics. Additive manufacturing opens a plethora of new avenues to form and shape PEDOT:PSS, allowing for the rapid construction of customized geometries. However, there are difficulties in printing PEDOT:PSS while maintaining its attractive properties. A 3D printing method for PEDOT:PSS using a room‐temperature coagulation bath‐based direct ink writing technique is reported. This technique enables fabrication of PEDOT:PSS into parts that are of high resolution and high conductivity, while maintaining stable electrochemical properties. The coagulation bath can be further modified to improve the mechanical properties of the resultant printed part via a one‐step reaction. Furthermore, it is demonstrated that a simple post‐processing step allows the printed electrodes to strongly adhere to several substrates under aqueous conditions, broadening their use in bioelectronics. Employing 3D printing of PEDOT:PSS, a cortex‐wide neural interface is fabricated, and intracranial electrical stimulation and simultaneous optical monitoring of mice brain activity with wide field calcium imaging are demonstrated. This reported 3D‐printing technique eliminates the need for cumbersome experimental setups while offering desired material properties.

Coagulation Bath‐Assisted 3D Printing of PEDOT:PSS with High Resolution and Strong Substrate Adhesion for Bioelectronic Devices

Mesial temporal lobe epilepsy (MTLE) is the most common form of focal, pharmacoresistant epilepsy in adults and is often associated with hippocampal sclerosis. Here, we established the efficacy of optogenetic and electrical low-frequency stimulation (LFS) in interfering with seizure generation in a mouse model of MTLE. Specifically, we applied LFS in the sclerotic hippocampus to study the effects on spontaneous subclinical and evoked generalized seizures. We found that stimulation at 1 Hz for 1 hr resulted in an almost complete suppression of spontaneous seizures in both hippocampi. This seizure-suppressive action during daily stimulation remained stable over several weeks. Furthermore, LFS for 30 min before a pro-convulsive stimulus successfully prevented seizure generalization. Finally, acute slice experiments revealed a reduced efficacy of perforant path transmission onto granule cells upon LFS. Taken together, our results suggest that hippocampal LFS constitutes a promising approach for seizure control in MTLE.

Hippocampal low-frequency stimulation prevents seizure generation in a mouse model of mesial temporal lobe epilepsy.

One characteristic feature of mesial temporal lobe epilepsy is granule cell dispersion (GCD), a pathological widening of the granule cell layer in the dentate gyrus. The loss of the extracellular matrix protein Reelin, an important positional cue for neurons, correlates with GCD formation in MTLE patients and in rodent epilepsy models. Here, we used organotypic hippocampal slice cultures (OHSC) from transgenic mice expressing enhanced green fluorescent protein (eGFP) in differentiated granule cells (GCs) to monitor GCD formation dynamically by live cell video microscopy and to investigate the role of Reelin in this process. We present evidence that following treatment with the glutamate receptor agonist kainate (KA), eGFP-positive GCs migrated mainly toward the hilar region. In the hilus, Reelin-producing neurons were rapidly lost following KA treatment as shown in a detailed time series. Addition of recombinant Reelin fragments to the medium effectively prevented the KA-triggered movement of eGFP-positive GCs. Placement of Reelin-coated beads into the hilus of KA-treated cultures stopped the migration of GCs in a distance-dependent manner. In addition, quantitative Western blot analysis revealed that KA treatment affects the Reelin signal transduction pathway by increasing intracellular adaptor protein Disabled-1 synthesis and reducing the phosphorylation of cofilin, a downstream target of the Reelin pathway. Both events were normalized by addition of recombinant Reelin fragments. Finally, following neutralization of Reelin in healthy OHSC by incubation with the function-blocking CR-50 Reelin antibody, GCs started to migrate without any direction preference. Together, our findings demonstrate that normotopic position of Reelin is essential for the maintenance of GC lamination in the dentate gyrus and that GCD is the result of a local Reelin deficiency.

/pdf/reelin-is-required-for-maintenance-of-granule-cell-24y2q06j4k.pdf

Reelin Is Required for Maintenance of Granule Cell Lamination in the Healthy and Epileptic Hippocampus

Optogenetic tools allow precise manipulation of neuronal activity via genetically encoded light-sensitive proteins. Currently available optogenetic inhibitors are not suitable for prolonged use due to short-lasting photocurrents, tissue heating, and unintended changes in ion distributions, which may interfere with normal neuron physiology. To overcome these limitations, a novel potassium channel-based optogenetic silencer, named PACK, was recently developed. The PACK tool has two components: a photoactivated adenylyl cyclase from Beggiatoa (bPAC) and a cAMP-dependent potassium channel, SthK, which carries a large, long-lasting potassium current in mammalian cells. Previously, it has been shown that activating the PACK silencer with short light pulses led to a significant reduction of neuronal firing in various in vitro and acute in vivo settings. Here, we examined the viability of performing long-term studies in vivo by looking at the inhibitory action and side effects of PACK and its components in healthy and epileptic adult male mice.We targeted hippocampal cornu ammonis (CA1) pyramidal cells using a viral vector and enabled illumination of these neurons via an implanted optic fiber. Local field potential (LFP) recordings from CA1 of freely moving mice revealed significantly reduced neuronal activity during 50-min intermittent (0.1 Hz) illumination, especially in the gamma frequency range. Adversely, PACK expression in healthy mice induced chronic astrogliosis, dispersion of pyramidal cells, and generalized seizures. These side effects were independent of the light application and were also present in mice expressing bPAC without the potassium channel. Light activation of bPAC alone increased neuronal activity, presumably via enhanced cAMP signaling. Furthermore, we applied bPAC and PACK in the contralateral hippocampus of chronically epileptic mice following a unilateral injection of intrahippocampal kainate. Unexpectedly, the expression of bPAC in the contralateral CA1 area was sufficient to prevent the spread of spontaneous epileptiform activity from the seizure focus to the contralateral hippocampus.Our study highlights the PACK tool as a potent optogenetic inhibitor in vivo. However, further refinement of its light-sensitive domain is required to avoid unexpected physiological changes. 

/pdf/long-term-in-vivo-application-of-a-potassium-channel-based-166dqrof.pdf

Long-term in vivo application of a potassium channel-based optogenetic silencer in the healthy and epileptic mouse hippocampus

/pdf/long-term-in-vivo-application-of-a-potassium-channel-based-199vq7vp.pdf

Mesial temporal lobe epilepsy (MTLE) is the most common form of focal epilepsy in adults and is typically associated with hippocampal sclerosis and drug-resistant seizures. As an alternative to curative epilepsy surgery, brain stimulation evolves as a promising approach for seizure-interference. However, particularly in MTLE with severe hippocampal sclerosis, current stimulation protocols are often not effective. Here, we show that optogenetic low-frequency stimulation (oLFS) of entorhinal afferents exhibits unprecedented anti-ictogenic effects in chronically epileptic mice. Photostimulation at 1 Hz resulted in an almost complete suppression of focal seizures, independent of the degree of hippocampal sclerosis. Furthermore, by performing oLFS for 30 min before a pro-convulsive stimulus, seizure generalization was successfully prevented. Finally, acute slice experiments revealed a decreased excitability upon oLFS, which may partially explain the observed anti-epileptic effects. Taken together, our results suggest that oLFS of entorhinal afferents constitutes a promising approach for seizure control in MTLE.

https://biorxiv.org/content/biorxiv/early/2020/01/10/2020.01.09.900084.full-text.pdf

Enya Paschen

Papers

Hippocampal low-frequency stimulation prevents seizure generation in a mouse model of mesial temporal lobe epilepsy.

Reelin Is Required for Maintenance of Granule Cell Lamination in the Healthy and Epileptic Hippocampus

Long-term in vivo application of a potassium channel-based optogenetic silencer in the healthy and epileptic mouse hippocampus

Long-term in vivo application of a potassium channel-based optogenetic silencer in the healthy and epileptic mouse hippocampus

Optogenetic low-frequency stimulation of dentate granule cells prevents seizure generation in experimental epilepsy