🌱🤝🌍 Anil Achyuta

Electrode arrays for recording and stimulation in the central nervous system have enabled numerous advances in basic science and therapeutic strategies. In particular, micro-fabricated arrays with precision size and spacing offer the benefit of accessing single neurons and permit mapping of neuronal function. Similar advances are envisioned toward understanding the autonomic nervous system and developing therapies based on its modulation, but appropriate electrode arrays are lacking. Here, we… Show more

Electrode arrays for recording and stimulation in the central nervous system have enabled numerous advances in basic science and therapeutic strategies. In particular, micro-fabricated arrays with precision size and spacing offer the benefit of accessing single neurons and permit mapping of neuronal function. Similar advances are envisioned toward understanding the autonomic nervous system and developing therapies based on its modulation, but appropriate electrode arrays are lacking. Here, we present for the first time, a multi-channel electrode array suitable for penetration of peripheral nerves having diameters as small as 0.1mm, and demonstrate performance in vivo. These arrays have the potential to access multiple discrete nerve fibers in small nerves. We fabricated and characterized five-channel arrays and obtained preliminary recordings of activity when penetrating rat carotid sinus nerve. The electrodes were constructed using hybrid microfabrication processes. The individual electrode shafts are as small as 0.01mm in diameter and at its tip each has a defined site that is addressable via a standard electronic connector. In addition to acute in vivo results, we evaluate the device by electrochemical impedance spectroscopy. Having established the fabrication method, our next steps are to incorporate the arrays into an implantable configuration for chronic studies, and here we further describe concepts for such a device. Show less

Synthetic neural scaffolds hold promise to eventually replace nerve autografts for tissue repair following peripheral nerve injury. Despite substantial evidence for the influence of scaffold geometry and dimensions on the rate of axonal growth, systematic evaluation of these parameters remains a challenge due to limitations in materials processing. We have employed fiber drawing to engineer a wide spectrum of polymer-based neural scaffolds with varied geometries and core sizes. Using isolated… Show more

Synthetic neural scaffolds hold promise to eventually replace nerve autografts for tissue repair following peripheral nerve injury. Despite substantial evidence for the influence of scaffold geometry and dimensions on the rate of axonal growth, systematic evaluation of these parameters remains a challenge due to limitations in materials processing. We have employed fiber drawing to engineer a wide spectrum of polymer-based neural scaffolds with varied geometries and core sizes. Using isolated whole dorsal root ganglia as an in vitro model system we have identified key features enhancing nerve growth within these fiber scaffolds. Our approach enabled straightforward integration of microscopic topography at the scale of nerve fascicles within the scaffold cores, which led to accelerated Schwann cell migration, as well as neurite growth and alignment. Our findings indicate that fiber drawing provides a scalable and versatile strategy for producing nerve guidance channels capable of controlling direction and accelerating the rate of axonal growth. Show less

Due to the limited regenerative ability of neural tissue, a diverse set of biochemical and biophysical cues for increasing nerve growth has been investigated, including neurotrophic factors, topography, and electrical stimulation. In this report, we explore optogenetic control of neurite growth as a cell-specific alternative to electrical stimulation. By investigating a broad range of optical stimulation parameters on dorsal root ganglia (DRGs) expressing channelrhodopsin 2 (ChR2), we… Show more

Due to the limited regenerative ability of neural tissue, a diverse set of biochemical and biophysical cues for increasing nerve growth has been investigated, including neurotrophic factors, topography, and electrical stimulation. In this report, we explore optogenetic control of neurite growth as a cell-specific alternative to electrical stimulation. By investigating a broad range of optical stimulation parameters on dorsal root ganglia (DRGs) expressing channelrhodopsin 2 (ChR2), we identified conditions that enhance neurite outgrowth by three-fold as compared to unstimulated or wild-type (WT) controls. Furthermore, optogenetic stimulation of ChR2 expressing DRGs induces directional outgrowth in WT DRGs co-cultured within a 10 mm vicinity of the optically sensitive ganglia. This observed enhancement and polarization of neurite growth was accompanied by an increased expression of neural growth and brain derived neurotrophic factors (NGF, BDNF). This work highlights the potential for implementing optogenetics to drive nerve growth in specific cell populations. Show less

Oral presentation and conference proceedings.

The blood-brain barrier (BBB) maintains the brain homeostasis and dynamically responds to events associated with systemic and/or rheological impairments (e.g., inflammation, ischemia) including the exposure to harmful xenobiotics. Thus, understanding the BBB physiology is crucial for the resolution of major central nervous system CNS) disorders challenging both health care providers and the pharmaceutical industry. These challenges include drug delivery to the brain, neurological disorders… Show more

The blood-brain barrier (BBB) maintains the brain homeostasis and dynamically responds to events associated with systemic and/or rheological impairments (e.g., inflammation, ischemia) including the exposure to harmful xenobiotics. Thus, understanding the BBB physiology is crucial for the resolution of major central nervous system CNS) disorders challenging both health care providers and the pharmaceutical industry. These challenges include drug delivery to the brain, neurological disorders, toxicological studies, and biodefense. Studies aimed at advancing our understanding of CNS diseases and promoting the development of more effective therapeutics are primarily performed in laboratory animals. However, there are major hindering factors inherent to in vivo studies such as cost, limited throughput and translational significance to humans. These factors promoted the development of alternative in vitro strategies for studying the physiology and pathophysiology of the BBB in relation to brain disorders as well as screening tools to aid in the development of novel CNS drugs. Herein, we provide a detailed review including pros and cons of current and prospective technologies for modelling the BBB in vitro including ex situ, cell based and computational (in silico) models. A special section is dedicated to microfluidic systems including micro-BBB, BBB-on-a-chip, Neurovascular Unit-on-a-Chip and Synthetic Microvasculature Blood-brain Barrier. Show less

While many advanced liver models support hepatic phenotypes necessary for drug and disease studies, these models are characterized by intricate features such as coculture with one of more supporting cell types or advanced media perfusion systems. These systems have helped elucidate
some of the critical biophysical features missing from standard well-plate based hepatocyte culture, but their advanced designs add to their complexity. Additionally, regardless of the culture system, primary… Show more

While many advanced liver models support hepatic phenotypes necessary for drug and disease studies, these models are characterized by intricate features such as coculture with one of more supporting cell types or advanced media perfusion systems. These systems have helped elucidate
some of the critical biophysical features missing from standard well-plate based hepatocyte culture, but their advanced designs add to their complexity. Additionally, regardless of the culture system, primary hepatocyte culture systems suffer from reproducibility issues due to phenotypic variation and expensive, limited supplies of donor lots. Here we describe a microfluidic bilayer device that sustains primary human hepatocyte phenotypes, including albumin production, factor IX production, cytochrome P450 3A4 drug metabolism and bile canaliculi formation for at least 14 days in a simple
monoculture format with static media. Using a variety of channel architectures, we describe how primary cell phenotype is promoted by spatial confinement within the
microfluidic channel, without the need for perfusion or coculture By sourcing human hepatocytes expanded in the Fah, Rag2, and Il2rg-knockout (FRG™-KO) humanized mouse
model, utilizing a few hundred hepatocytes within each channel, and maintaining hepatocyte function for weeks in vitro within a relatively simple model, we demonstrate a basic
primary human hepatocyte culture system that addresses many of the major hurdles in human hepatocyte culture research. Show less

Realizing the vision of a new class of medicines based on modulating the electrical signalling patterns of the peripheral nervous system needs a firm research foundation. Here, an interdisciplinary community puts forward a research roadmap for the next 5 years.

An optical-electrical neural ‘Optrode’ is developed for quantitative optical spectroscopic assessment of cortical tissue damage around a neural implant. In contrast to other Optrodes which illuminate tissue for neural activation, this work uses bi-directional waveguides to obtain spectroscopic information from the tissue. An optical assessment has the potential for providing a real-time histological assessment, by quantifying changes in absorption and scattering properties associated with… Show more

An optical-electrical neural ‘Optrode’ is developed for quantitative optical spectroscopic assessment of cortical tissue damage around a neural implant. In contrast to other Optrodes which illuminate tissue for neural activation, this work uses bi-directional waveguides to obtain spectroscopic information from the tissue. An optical assessment has the potential for providing a real-time histological assessment, by quantifying changes in absorption and scattering properties associated with oxygenation and tissue density around the neural probe, in contrast to impedance spectroscopy which provides limited insight. Optrode performance is characterized in optical tissue phantoms and a proof-of-concept chronic rat model is developed. Optrodes were chronically implanted in rat cerebral cortex. The pilot study demonstrates feasibility for chronic optical assessment. Future Optrodes may lead to high throughput and real-time assessments for studying the dynamic nature of the foreign body response in neural tissue. Show less

Our understanding of biological mechanisms and treatment options for traumatic brain injury (TBI) is limited. Here, we employed quantitative real-time PCR (QRT-PCR) and immunohistochemical analyses to determine the dynamic expression of cell proliferation and apoptosis in an effort to provide insights into the therapeutic window for developing regenerative strategies for TBI. For this purpose, young adult Sprague-Dawley rats were subjected to experimental TBI using a controlled cortical… Show more

Our understanding of biological mechanisms and treatment options for traumatic brain injury (TBI) is limited. Here, we employed quantitative real-time PCR (QRT-PCR) and immunohistochemical analyses to determine the dynamic expression of cell proliferation and apoptosis in an effort to provide insights into the therapeutic window for developing regenerative strategies for TBI. For this purpose, young adult Sprague-Dawley rats were subjected to experimental TBI using a controlled cortical impactor, then euthanized 1-48 hours after TBI for QRT-PCR and immunohistochemistry. QRT-PCR revealed that brains from TBI exposed rats initially displayed nestin mRNA expression that modestly increased as early as 1-hour post-TBI, then significantly peaked at 8 hours, but thereafter reverted to pre-TBI levels. On the other hand, caspase-3 mRNA expression was slightly elevated at 8 hours post-TBI, which did not become significantly upregulated until 48 hours. Immunofluorescent microscopy revealed a significant surge in nestin immunoreactive cells in the cortex, corpus callosum, and subventricular zone at 24 hours post-TBI, whereas a significant increase in the number of active caspase-3 immunoreactive cells was only found in the cortex and not until 48 hours. These results suggest that the injured brain attempts to repair itself via cell proliferation immediately after TBI, but that this endogenous regenerative mechanism is not sufficient to abrogate the secondary apoptotic cell death. Treatment strategies designed to amplify cell proliferation and to prevent apoptosis are likely to exert maximal benefits when initiated at the acute phase of TBI. Show less

In this work, we describe the fabrication and working of a modular microsystem that recapitulates the functions of the “Neurovascular Unit”. The microdevice comprised of a vertical stack of poly (dimethylsiloxane) (PDMS) neural parenchymal chamber separated by a vascular channel via a microporous polycarbonate (PC) membrane. The neural chamber housed a mixture of neurons (~4%), astrocytes (~95%), and microglia (~1%). The vascular channel was lined with a layer of rat brain microvascular… Show more

In this work, we describe the fabrication and working of a modular microsystem that recapitulates the functions of the “Neurovascular Unit”. The microdevice comprised of a vertical stack of poly (dimethylsiloxane) (PDMS) neural parenchymal chamber separated by a vascular channel via a microporous polycarbonate (PC) membrane. The neural chamber housed a mixture of neurons (~4%), astrocytes (~95%), and microglia (~1%). The vascular channel was lined with a layer of rat brain microvascular endothelial cell line (RBE4). Cellular components in neural chamber and vascular channel showed viability (> 90%). The neural cells fired inhibitory as well as excitatory potentials following 10 days of culture. The endothelial cells showed diluted-acetylated low density lipoprotein (dil-a-LDL) uptake, expressed von Willebrand factor (vWF) and zonula occludens (ZO-1) tight junctions, and showed decreased Alexafluor™-conjugated dextran leakage across their barrier significantly compared with controls (p < 0.05). When the vascular layer was stimulated with TNF-a for 6h, about 75% of resident microglia and astrocytes on the neural side were activated significantly (p < 0.05 compared to controls) recapitulating tissue-mimetic responses resembling neuroinflammation. The impact of this microsystem lies in the fact that this biomimetic neurovascular platform might not only be harnessed for obtaining mechanistic insights for neurodegenerative disorders, but could also serve as a potential screening tool for central nervous system (CNS) therapeutics in toxicology and neuroinfectious diseases. Show less

Over the past few decades, tremendous advancements have been made in the neural microelectrode interfaces realm lending hope for patients suffering from harrowing neurological deficits. The electrode designing, fabrication, implantation, and operation of these microdevices are all highly complex and established areas in their own right, described in the earlier sections of this chapter. However, the full potential of these devices are unrealized due to existing challenges to reliably record… Show more

Over the past few decades, tremendous advancements have been made in the neural microelectrode interfaces realm lending hope for patients suffering from harrowing neurological deficits. The electrode designing, fabrication, implantation, and operation of these microdevices are all highly complex and established areas in their own right, described in the earlier sections of this chapter. However, the full potential of these devices are unrealized due to existing challenges to reliably record in clinically relevant settings. This recording inconsistency stems from a complex biological response triggered from the neural tissue against the implanted electrodes-traditionally referred as “biocompatibility” of the implant. In order to design more robust next generation electrode interfaces, it is essential that the tissue reaction be mitigated and hence, a thorough knowledge of such a biological response is absolutely fundamental. Show less

Vapor-deposited silicone coatings are attractive candidates for providing insulation in neuroprosthetic devices owing to their excellent resistivity, adhesion, chemical inertness and flexibility. A biocompatibility assessment of these coatings is an essential part of the materials design process, but current techniques are limited to rudimentary cell viability assays or animal muscle implantation tests. This article describes how a recently developed in vitro model of glial scar formation can… Show more

Vapor-deposited silicone coatings are attractive candidates for providing insulation in neuroprosthetic devices owing to their excellent resistivity, adhesion, chemical inertness and flexibility. A biocompatibility assessment of these coatings is an essential part of the materials design process, but current techniques are limited to rudimentary cell viability assays or animal muscle implantation tests. This article describes how a recently developed in vitro model of glial scar formation can be utilized to assess the biocompatibility of vapor-deposited silicone coatings on micron-scale wires. A multi-cellular monolayer comprising mixed glial cells was obtained by culturing primary rat midbrain cells on poly(D-lysine)-coated well plates. Stainless steel microwires were coated with two novel insulating thin film silicone polymers, namely poly(trivinyltrimethylcyclotrisiloxane) (polyV3D3) and poly(trivinyltrimethylcyclotrisiloxane–hexavinyldisiloxane) (polyV3D3–HVDS) by initiated chemical vapor deposition (iCVD). The monolayer of midbrain cells was disrupted by placing segments of coated microwires into the culture followed by immunocytochemical analysis after 7 d of implantation. Microglial proximity to the microwires was observed to correlate with the amount of fibronectin adsorbed on the coating surface; polyV3D3–HVDS adsorbed the least amount of fibronectin compared to both stainless steel and polyV3D3. Consequently, the relative number of microglia within 100 µm of the microwires was least on polyV3D3–HVDS coatings compared to steel and polyV3D3. In addition, the astrocyte reactivity on polyV3D3–HVDS coatings was lower compared to stainless steel and polyV3D3. The polyV3D3–HVDS coating was therefore deemed to be most biocompatible, least reactive and most preferable insulating coating for neural prosthetic devices. Show less

Neuroprosthetic devices have made a major impact in the treatment of a variety of disorders such as paralysis and stroke. However, a major impediment in the advancement of this technology is the challenge of maintaining device performance during chronic implantation (months to years) due to complex intrinsic host responses such as gliosis or glial scarring. The objective of this review is to bring together research communities in neurobiology, tissue engineering, and neuroprosthetics to address… Show more

Neuroprosthetic devices have made a major impact in the treatment of a variety of disorders such as paralysis and stroke. However, a major impediment in the advancement of this technology is the challenge of maintaining device performance during chronic implantation (months to years) due to complex intrinsic host responses such as gliosis or glial scarring. The objective of this review is to bring together research communities in neurobiology, tissue engineering, and neuroprosthetics to address the major obstacles encountered in the translation of neuroprosthetics technology into long-term clinical use. This article draws connections between specific challenges faced by current neuroprosthetics technology and recent advances in the areas of nerve tissue engineering and neurobiology. Within the context of the device–nervous system interface and central nervous system implants, areas of synergistic opportunity are discussed, including platforms to present cells with multiple cues, controlled delivery of bioactive factors, three-dimensional constructs and in vitro models of gliosis and brain injury, nerve regeneration strategies, and neural stem/progenitor cell biology. Finally, recent insights gained from the fields of developmental neurobiology and cancer biology are discussed as examples of exciting new biological knowledge that may provide fresh inspiration toward novel technologies to address the complexities associated with long-term neuroprosthetic device performance. Show less

Occlusion or blockage of silicone shunts utilized in the treatment of hydrocephalus is a major challenge that is currently addressed by multiple shunt replacements. Shunt occlusion is caused by the adhesion and proliferation of reactive cells, such as glial and vascular cells, into the lumen of the catheter and on valve components. This in vitro study describes how the adhesive behavior of four human cell types on poly(dimethylsiloxane) (PDMS) surfaces can be suppressed by functionalization… Show more

Occlusion or blockage of silicone shunts utilized in the treatment of hydrocephalus is a major challenge that is currently addressed by multiple shunt replacements. Shunt occlusion is caused by the adhesion and proliferation of reactive cells, such as glial and vascular cells, into the lumen of the catheter and on valve components. This in vitro study describes how the adhesive behavior of four human cell types on poly(dimethylsiloxane) (PDMS) surfaces can be suppressed by functionalization with trypsin, a proteolytic enzyme. The impact of this work lies in the recognition that the well-known proteolytic characteristics of trypsin can be harnessed by covalent surface immobilization to suppress cell adhesion and protein adsorption Show less

Poly(trivinyltrimethylcyclotrisiloxane) or polyV3D3 is a promising insulating thin film known for its potential application in neural probe fabrication. However, its time-consuming synthesis rate renders it impractical by manufacturing standards. Previously, the growth mechanism of polyV3D3 was shown to be affected by significant steric barriers. This article describes the synthesis of a copolymer of polyV3D3 via initiated chemical vapor
deposition (iCVD) using V3D3 as the monomer… Show more

Poly(trivinyltrimethylcyclotrisiloxane) or polyV3D3 is a promising insulating thin film known for its potential application in neural probe fabrication. However, its time-consuming synthesis rate renders it impractical by manufacturing standards. Previously, the growth mechanism of polyV3D3 was shown to be affected by significant steric barriers. This article describes the synthesis of a copolymer of polyV3D3 via initiated chemical vapor
deposition (iCVD) using V3D3 as the monomer, hexavinyldisiloxane (HVDS) as a spacer, and tert-butyl peroxide
(TBP) as the initiator to obtain nearly a 4-fold increase in deposition rate. Show less

Immobilized ECM proteins and neurotrophins have been extensively studied to enhance neuronal adhesion and proliferation on surfaces for applications in nerve tissue engineering and neuroprosthetic devices. This paper describes how the co-immobilization of laminin, an extracellular matrix (ECM) protein and nerve growth factor (NGF), a neurotrophin, can enhance neurite outgrowth observed separately with each type of molecule.