Translational control in the spinal cord regulates gene expression and pain hypersensitivity in the chronic phase of neuropathic pain.

Sensitization of spinal nociceptive circuits plays a crucial role in neuropathic pain. This sensitization depends on new gene expression that is primarily regulated via transcriptional and translational control mechanisms. The relative roles of these mechanisms in regulating gene expression in the clinically relevant chronic phase of neuropathic pain are not well understood. Here, we show that changes in gene expression in the spinal cord during the chronic phase of neuropathic pain are substantially regulated at the translational level. Downregulating spinal translation at the chronic phase alleviated pain hypersensitivity. Cell-type-specific profiling revealed that spinal inhibitory neurons exhibited greater changes in translation after peripheral nerve injury compared to excitatory neurons. Notably, increasing translation selectively in all inhibitory neurons or parvalbumin-positive (PV+) interneurons, but not excitatory neurons, promoted mechanical pain hypersensitivity. Furthermore, increasing translation in PV+ neurons decreased their intrinsic excitability and spiking activity, whereas reducing translation in spinal PV+ neurons prevented the nerve injury-induced decrease in excitability. Thus, translational control mechanisms in the spinal cord, particularly in inhibitory neurons, play a role in mediating neuropathic pain hypersensitivity.

Protocol for measuring protein synthesis in specific cell types in the mouse brain using in vivo non-canonical amino acid tagging.

The fluorescent non-canonical amino acid tagging (FUNCAT) technique has been used to visualize newly synthesized proteins in cell lines and tissues. Here, we present a protocol for measuring protein synthesis in specific cell types in the mouse brain using in vivo FUNCAT. We describe steps for metabolically labeling newly synthesized proteins with azidohomoalanine, which introduces an azide group into the polypeptide. We then detail procedures for binding a fluorophore-conjugated alkyne to the azide group to allow its visualization. For complete details on the use and execution of this protocol, please refer to tom Dieck et al. (2012)1 and Hooshmandi et al. (2023).2.

4E-BP1-dependent translation in nociceptors controls mechanical hypersensitivity via TRIM32/type I interferon signaling.

Activation of the mechanistic target of rapamycin complex 1 (mTORC1) contributes to the development of chronic pain. However, the specific mechanisms by which mTORC1 causes hypersensitivity remain elusive. The eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) is a key mTORC1 downstream effector that represses translation initiation. Here, we show that nociceptor-specific deletion of 4E-BP1, mimicking activation of mTORC1-dependent translation, is sufficient to cause mechanical hypersensitivity. Using translating ribosome affinity purification in nociceptors lacking 4E-BP1, we identified a pronounced translational up-regulation of tripartite motif-containing protein 32 (TRIM32), an E3 ubiquitin ligase that promotes interferon signaling. Down-regulation of TRIM32 in nociceptors or blocking type I interferon signaling reversed the mechanical hypersensitivity in mice lacking 4E-BP1. Furthermore, nociceptor-specific ablation of TRIM32 alleviated mechanical hypersensitivity caused by tissue inflammation. These results show that mTORC1 in nociceptors promotes hypersensitivity via 4E-BP1-dependent up-regulation of TRIM32/interferon signaling and identify TRIM32 as a therapeutic target in inflammatory pain.

Excitatory neuron-specific suppression of the integrated stress response contributes to autism-related phenotypes in fragile X syndrome.

Dysregulation of protein synthesis is one of the key mechanisms underlying autism spectrum disorder (ASD). However, the role of a major pathway controlling protein synthesis, the integrated stress response (ISR), in ASD remains poorly understood. Here, we demonstrate that the main arm of the ISR, eIF2α phosphorylation (p-eIF2α), is suppressed in excitatory, but not inhibitory, neurons in a mouse model of fragile X syndrome (FXS; Fmr1-/y). We further show that the decrease in p-eIF2α is mediated via activation of mTORC1. Genetic reduction of p-eIF2α only in excitatory neurons is sufficient to increase general protein synthesis and cause autism-like behavior. In Fmr1-/y mice, restoration of p-eIF2α solely in excitatory neurons reverses elevated protein synthesis and rescues autism-related phenotypes. Thus, we reveal a previously unknown causal relationship between excitatory neuron-specific translational control via the ISR pathway, general protein synthesis, and core phenotypes reminiscent of autism in a mouse model of FXS.

Parvalbumin interneuron loss mediates repeated anesthesia-induced memory deficits in mice.

Repeated or prolonged, but not short-term, general anesthesia during the early postnatal period causes long-lasting impairments in memory formation in various species. The mechanisms underlying long-lasting impairment in cognitive function are poorly understood. Here, we show that repeated general anesthesia in postnatal mice induces preferential apoptosis and subsequent loss of parvalbumin-positive inhibitory interneurons in the hippocampus. Each parvalbumin interneuron controls the activity of multiple pyramidal excitatory neurons, thereby regulating neuronal circuits and memory consolidation. Preventing the loss of parvalbumin neurons by deleting a proapoptotic protein, mitochondrial anchored protein ligase (MAPL), selectively in parvalbumin neurons rescued anesthesia-induced deficits in pyramidal cell inhibition and hippocampus-dependent long-term memory. Conversely, partial depletion of parvalbumin neurons in neonates was sufficient to engender long-lasting memory impairment. Thus, loss of parvalbumin interneurons in postnatal mice following repeated general anesthesia critically contributes to memory deficits in adulthood.

mTORC2 mediates structural plasticity in distal nociceptive endings that contributes to pain hypersensitivity following inflammation.

The encoding of noxious stimuli into action potential firing is largely mediated by nociceptive free nerve endings. Tissue inflammation, by changing the intrinsic properties of the nociceptive endings, leads to nociceptive hyperexcitability and thus to the development of inflammatory pain. Here, we showed that tissue inflammation-induced activation of the mammalian target of rapamycin complex 2 (mTORC2) triggers changes in the architecture of nociceptive terminals and leads to inflammatory pain. Pharmacological activation of mTORC2 induced elongation and branching of nociceptor peripheral endings and caused long-lasting pain hypersensitivity. Conversely, nociceptor-specific deletion of the mTORC2 regulatory protein rapamycin-insensitive companion of mTOR (Rictor) prevented inflammation-induced elongation and branching of cutaneous nociceptive fibers and attenuated inflammatory pain hypersensitivity. Computational modeling demonstrated that mTORC2-mediated structural changes in the nociceptive terminal tree are sufficient to increase the excitability of nociceptors. Targeting mTORC2 using a single injection of antisense oligonucleotide against Rictor provided long-lasting alleviation of inflammatory pain hypersensitivity. Collectively, we showed that tissue inflammation-induced activation of mTORC2 causes structural plasticity of nociceptive free nerve endings in the epidermis and inflammatory hyperalgesia, representing a therapeutic target for inflammatory pain.

Microglia-mediated degradation of perineuronal nets promotes pain.

Activation of microglia in the spinal cord dorsal horn after peripheral nerve injury contributes to the development of pain hypersensitivity. How activated microglia selectively enhance the activity of spinal nociceptive circuits is not well understood. We discovered that after peripheral nerve injury, microglia degrade extracellular matrix structures, perineuronal nets (PNNs), in lamina I of the spinal cord dorsal horn. Lamina I PNNs selectively enwrap spinoparabrachial projection neurons, which integrate nociceptive information in the spinal cord and convey it to supraspinal brain regions to induce pain sensation. Degradation of PNNs by microglia enhances the activity of projection neurons and induces pain-related behaviors. Thus, nerve injury-induced degradation of PNNs is a mechanism by which microglia selectively augment the output of spinal nociceptive circuits and cause pain hypersensitivity.

Functional Grading of a Transversely Isotropic Hyperelastic Model with Applications in Modeling Tricuspid and Mitral Valve Transition Regions.

Surgical simulators and injury-prediction human models require a combination of representative tissue geometry and accurate tissue material properties to predict realistic tool-tissue interaction forces and injury mechanisms, respectively. While biological tissues have been individually characterized, the transition regions between tissues have received limited research attention, potentially resulting in inaccuracies within simulations. In this work, an approach to characterize the transition regions in transversely isotropic (TI) soft tissues using functionally graded material (FGM) modeling is presented. The effect of nonlinearities and multi-regime nature of the TI model on the functional grading process is discussed. The proposed approach has been implemented to characterize the transition regions in the leaflet (LL), chordae tendinae (CT) and the papillary muscle (PM) of porcine tricuspid valve (TV) and mitral valve (MV). The FGM model is informed using high resolution morphological measurements of the collagen fiber orientation and tissue composition in the transition regions, and deformation characteristics predicted by the FGM model are numerically validated to experimental data using X-ray diffraction imaging. The results indicate feasibility of using the FGM approach in modeling soft-tissue transitions and has implications in improving physical representation of tissue deformation throughout the body using a scalable version of the proposed approach.

Chronic pain produces hypervigilance to predator odor in mice.

The adaptive significance of acute pain (to withdraw from tissue-damaging or potentially tissue-damaging external stimuli, and to enhance the salience of the stimulus resulting in escape and avoidance learning) and tonic pain (to enforce recuperation by punishing movement) are well-accepted [1]. Pain researchers, however, generally assert that chronic pain has no adaptive significance, representing instead a pathophysiological state. This belief was recently challenged by the observation [2] that nociceptive sensitization caused by a chronic pain-producing injury reduced predation risk in squid (Doryteuthis pealeii). In that study, injury to an arm (removal of the tip with a scalpel) 6 hours prior led to increased targeting by black sea bass, resulting in decreased survival of the squid in a 30-minute trial featuring free interaction between predator and prey. The surprising finding was that anesthesia during surgery, preventing the chronic nociceptor sensitization associated with such injuries, led to even lower probability of survival. That is, the likely presence of pain increased apparent fitness, and the authors concluded that the chronic pain state and its associated nociceptive sensitization represented an adaptive function. Pain-induced defensive behaviors affecting fitness have also been reported in crustaceans (Gammarus fossarum) [3]. It is, however, currently unknown whether this may also be true in any other species, including in Mammalia.

Advanced Methodology and Preliminary Measurements of Molecular and Mechanical Properties of Heart Valves under Dynamic Strain.

Mammalian heart valves are soft tissue assemblies with multi-scale material properties. This is because they are constructs comprising both muscle and non-contractile extracellular matrix proteins (such as collagens and proteoglycans) and transition regions where one form of tissue structure becomes another, significantly different form. The leaflets of the mitral and tricuspid valves are connected to chordae tendinae which, in turn, bind through papillary muscles to the cardiac wall of the ventricle. The transition regions between these tissue subsets are complex and diffuse. Their material composition and mechanical properties have not been previously described with both micro and nanoscopic data recorded simultaneously, as reported here. Annotating the mechanical characteristics of these tissue transitions will be of great value in developing novel implants, improving the state of the surgical simulators and advancing robot-assisted surgery. We present here developments in multi-scale methodology that produce data that can relate mechanical properties to molecular structure using scanning X-ray diffraction. We correlate these data to corresponding tissue level (macro and microscopic) stress and strain, with particular emphasis on the transition regions and present analyses to indicate points of possible failure in these tissues.

Development of the CAVEMAN Human Body Model: Validation of Lower Extremity Sub-Injurious Response to Vertical Accelerative Loading.

Improving injury prediction accuracy and fidelity for mounted Warfighters has become an area of focus for the U.S. military in response to improvised explosive device (IED) use in both Iraq and Afghanistan. Although the Hybrid III anthropomorphic test device (ATD) has historically been used for crew injury analysis, it is only capable of predicting a few select skeletal injuries. The Computational Anthropomorphic Virtual Experiment Man (CAVEMAN) human body model is being developed to expand the injury analysis capability to both skeletal and soft tissues. The CAVEMAN model is built upon the Zygote 50th percentile male human CAD model and uses a finite element modeling approach developed for high performance computing (HPC). The lower extremity subset of the CAVEMAN human body model presented herein includes: 28 bones, 26 muscles, 40 ligaments, fascia, cartilage and skin. Sensitivity studies have been conducted with the CAVEMAN lower extremity model to determine the structures critical for load transmission through the leg in the underbody blast (UBB) environment. An evaluation of the CAVEMAN lower extremity biofidelity was also carried out using 14 unique data sets derived by the Warrior Injury Assessment Manikin (WIAMan) program cadaveric lower leg testing. Extension of the CAVEMAN lower extremity model into anatomical tissue failure will provide additional injury prediction capabilities, beyond what is currently achievable using ATDs, to improve occupant survivability analyses within military vehicles.

Slotted photonic crystal nanobeam cavity with an ultrahigh quality factor-to-mode volume ratio.

We describe the design, fabrication, and characterization of a 1-dimensional silicon photonic crystal cavity with a quality factor-to-mode volume ratio greater than 10(7), which exceeds the highest previous values by an order of magnitude. The maximum of the electric field is outside the silicon in a void formed by a central slot. An extremely small calculated mode volume of 0.0096 (λvac/n)(3) is achieved through the abrupt change of the electric field in the slot, despite which a high quality factor of 8.2 × 10(5) is predicted by simulation. Quality factors up to 1.4 × 10(5) are measured in actual devices. The observation of pronounced thermo-optic bistability is consistent with the strong confinement of light in these cavities.

Development of in vivo constitutive models for liver: application to surgical simulation.

Advancements in real-time surgical simulation techniques have provided the ability to utilize more complex nonlinear constitutive models for biological tissues which result in increased haptic and graphic accuracy. When developing such a model, verification is necessary to determine the accuracy of the force response as well as the magnitude of tissue deformation for tool-tissue interactions. In this study, we present an experimental device which provides the ability to obtain force-displacement information as well as surface deformation of porcine liver for in vivo probing tasks. In addition, the system is capable of accurately determining the geometry of the liver specimen. These combined attributes provide the context required to simulate the experiment with accurate boundary conditions, whereby the only variable in the analysis is the material properties of the liver specimen. During the simulation, effects of settling due to gravity have been taken into account by a technique which incorporates the proper internal stress conditions in the model without altering the geometry. Initially, an Ogden model developed from ex vivo tension and compression experimentation is run through the simulation to determine the efficacy of utilizing an ex vivo model for simulation of in vivo probing tasks on porcine liver. Subsequently, a method for improving upon the ex vivo model was developed using different hyperelastic models such that increased accuracy could be achieved for the force characteristics compared to the displacement characteristics, since changes in the force variation would be more perceptible to a user in the simulation environment, while maintaining a high correlation with the surface displacement data. Furthermore, this study also presents the probing simulation which includes the capsule surrounding the liver.

Towards a soft-tissue cutting simulator using the cohesive zone approach.

Advancements in computational techniques have provided the ability to utilize detailed models in surgical training systems. The development of more complicated procedures coupled with reduced training time for medical residents has driven the need for accurate reality-based medical simulators. Extensive research has been conducted in the area of modeling general deformation of biological tissue; however few studies have focused on the physical properties of specific tool tissue interactions such as cutting. This paper presents a fracture mechanics based method to model the scalpel cutting of porcine liver by implementing a cohesive zone approach. Using in vivo cutting data, the parameters of the cohesive zone model are defined for the scalpel cutting process of soft biological tissues.

Constitutive modeling of liver tissue: experiment and theory.

Realistic surgical simulation requires incorporation of the mechanical properties of soft tissue in mathematical models. In actual deformation of soft-tissue during surgical intervention, the tissue is subject to tension, compression, and shear. Therefore, characterization and modeling of soft-tissue in all these three deformation modes are necessary. In this paper we applied two types of pure shear test, un-confined compression and uniaxial tension test to characterize porcine liver tissue. Digital image correlation technique was used to accurately measure the tissue deformation field. Due to gravity and its effect on the soft tissue, a maximum stretching band was observed from the relative strain field on sample undergoing tension and pure shear test. The zero strain state was identified according to the position of this maximum stretching band. Two new constitutive models based on combined exponential/logarithmic and Ogden strain energy were proposed. The models are capable to represent the observed non-linear stress-strain relation of liver tissue for full range of tension and compression and also the general response of pure shear.

Real-time, haptics-enabled simulator for probing ex vivo liver tissue.

The advent of complex surgical procedures has driven the need for realistic surgical training simulators. Comprehensive simulators that provide realistic visual and haptic feedback during surgical tasks are required to familiarize surgeons with the procedures they are to perform. Complex organ geometry inherent to biological tissues and intricate material properties drive the need for finite element methods to assure accurate tissue displacement and force calculations. Advances in real-time finite element methods have not reached the state where they are applicable to soft tissue surgical simulation. Therefore a real-time, haptics-enabled simulator for probing of soft tissue has been developed which utilizes preprocessed finite element data (derived from accurate constitutive model of the soft-tissue obtained from carefully collected experimental data) to accurately replicate the probing task in real-time.

Towards a teleoperated needle driver robot with haptic feedback for RFA of breast tumors under continuous MRI.

OBJECTIVE: The purpose of this paper is to explore the feasibility of developing a MRI-compatible needle driver system for radiofrequency ablation (RFA) of breast tumors under continuous MRI imaging while being teleoperated by a haptic feedback device from outside the scanning room. The developed needle driver prototype was designed and tested for both tumor targeting capability as well as RFA. METHODS: The single degree-of-freedom (DOF) prototype was interfaced with a PHANToM haptic device controlled from outside the scanning room. Experiments were performed to demonstrate MRI-compatibility and position control accuracy with hydraulic actuation, along with an experiment to determine the PHANToM's ability to guide the RFA tool to a tumor nodule within a phantom breast tissue model while continuously imaging within the MRI and receiving force feedback from the RFA tool. RESULTS: Hydraulic actuation is shown to be a feasible actuation technique for operation in an MRI environment. The design is MRI-compatible in all aspects except for force sensing in the directions perpendicular to the direction of motion. Experiments confirm that the user is able to detect healthy vs. cancerous tissue in a phantom model when provided with both visual (imaging) feedback and haptic feedback. CONCLUSION: The teleoperated 1-DOF needle driver system presented in this paper demonstrates the feasibility of implementing a MRI-compatible robot for RFA of breast tumors with haptic feedback capability.

Soft-tissue characterization during monopolar electrocautery procedures.

Advances in electrosurgical technology have promoted the use of electrocautery in many surgical procedures. Precise modeling of soft tissue deformation during electrocautery with electrosurgical generators can be a valuable tool in training simulators for surgical procedures. Coupling the visualization of electrocautery with the force feedback during an electrocautery process (to maintain optimal current) without causing necrosis is an important learning tool. Realistic simulation will provide surgeon trainees a method to practice electrocautery techniques prior to experimenting on live tissue as well as allow surgeons to gain a feel for electrocautery procedures.