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Circumscribing Laser Cuts Attenuate Seizure Propagation in a Mouse Model of Focal Epilepsy

Seth Lieberman, Daniel A. Rivera, Ryan Morton, Amrit Hingorani, Teresa L. Southard, Lynn Johnson, Jennifer Reukauf, Ryan E. Radwanski, Mingrui Zhao, Nozomi Nishimura, Oliver Bracko, Theodore H. Schwartz, Chris B. Schaffer

Advanced Science (2024)

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In partial onset epilepsy, seizures arise focally in the brain and often propagate. Patients frequently become refractory to medical management, leaving neurosurgery, which can cause neurologic deficits, as a primary treatment. In the cortex, focal seizures spread through horizontal connections in layers II/III, suggesting that severing these connections can block seizures while preserving function. Focal neocortical epilepsy is induced in mice, sub-surface cuts are created surrounding the seizure focus using tightly-focused femtosecond laser pulses, and electrophysiological recordings are acquired at multiple locations for 3–12 months. Cuts reduced seizure frequency in most animals by 87%, and only 5% of remaining seizures propagated to the distant electrodes, compared to 80% in control animals. These cuts produced a modest decrease in cortical blood flow that recovered and left a ~20-µm wide scar with minimal collateral damage. When placed over the motor cortex, cuts do not cause notable deficits in a skilled reaching task, suggesting they hold promise as a novel neurosurgical approach for intractable focal cortical epilepsy.

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Extracellular vesicles incorporating retrovirus-like capsids for the enhanced packaging and systemic delivery of mRNA into neurons

Wenchao Gu, Sijin Luozhong, Simian Cai, Ketaki Londhe, Nadine Elkasri, Robert Hawkins, Zhefan Yuan, Kai Su-Greene, Yujie Yin, Margaret Cruz, Yu-Wei Chang, Patrick McMullen, Chunyan Wu, Changwoo Seo, Akash Guru, Wenting Gao, Tara Sarmiento, Chris Schaffer, Nozomi Nishimura, Richard Cerione, Qiuming Yu, Melissa Warden, Robert Langer, Shaoyi Jiang

Nature Biomedical Engineering (2024)

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The blood–brain barrier (BBB) restricts the systemic delivery of messenger RNAs (mRNAs) into diseased neurons. Although leucocyte-derived extracellular vesicles (EVs) can cross the BBB at inflammatory sites, it is difficult to efficiently load long mRNAs into the EVs and to enhance their neuronal uptake. Here we show that the packaging of mRNA into leucocyte-derived EVs and the endocytosis of the EVs by neurons can be enhanced by engineering leucocytes to produce EVs that incorporate retrovirus-like mRNA-packaging capsids. We transfected immortalized and primary bone-marrow-derived leucocytes with DNA or RNA encoding the capsid-forming activity-regulated cytoskeleton-associated (Arc) protein as well as capsid-stabilizing Arc 5’-untranslated-region RNA elements. These engineered EVs inherit endothelial adhesion molecules from donor leukocytes, recruit endogenous enveloping proteins to their surface, cross the BBB, and enter the neurons in neuro-inflammatory sites. Produced from self-derived donor leukocytes, the EVs are immunologically inert, and enhanced the neuronal uptake of the packaged mRNA in a mouse model of low-grade chronic neuro-inflammation.

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Intravital Multiphoton Microscopy Captures Dynamics of the Beating Heart

Anne Buglione, Nozomi Nishimura

Photonics Spectra (2024)

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Advanced imaging shows the mouse heart as it pumps, leading to insights into cardiac physiology and disease genesis.

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MousePZT: A simple, reliable, low-cost device for vital sign monitoring and respiratory gating in mice under anesthesia

Daniel A. Rivera, Anne E. Buglione, Sadie E. Ray, Chris B. Schaffer

PLOS ONE (2024)

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Small animal studies in biomedical research often require anesthesia to reduce pain or stress experienced by research animals and to minimize motion artifact during imaging or other measurements. Anesthetized animals must be closely monitored for the safety of the animals and to prevent unintended effects of altered physiology on experimental outcomes. Many currently available monitoring devices are expensive, invasive, or interfere with experimental design. Here, we present MousePZT, a low-cost device based on a simple piezoelectric sensor, with a custom circuit and computer software that allows for measurements of both respiratory rate and heart rate in a non-invasive, minimal contact manner. We find the accuracy of the MousePZT device in measuring respiratory and heart rate matches those of commercial systems. Using the widely-used gas isoflurane and injectable ketamine/xylazine combination, we also demonstrate that changes in respiratory rate are more easily detected and can precede changes in heart rate associated with variations in anesthetic depth. Additional circuitry on the device outputs a respiration-locked trigger signal for respiratory-gating of imaging or other data acquisition and has high sensitivity and specificity for detecting respiratory cycles. We provide detailed instruction documents and all necessary microcontroller and computer software, enabling straightforward construction and utilization of this device.

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Cutting Edge: CCR9 Promotes CD81 T Cell Recruitment to the Brain during Congenital Cytomegalovirus Infection

Zachary T. Hilt, Wisler Charles, Katarina E. Cheng, Cybelle Tabilas, Megan Steinhilber, Samantha P. Wesnak, Norah L. Smith, Chris B. Schaffer, Brian D. Rudd

Journal of Immunology (2023)

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CD81 T lymphocytes infiltrate the brain during congenital CMV infection and promote viral clearance. However, the mechanisms by which CD81 T cells are recruited to the brain remain unclear. Using a mouse model of congenital CMV, we found a gut-homing chemokine receptor (CCR9) was preferentially expressed in CD81 T cells localized in the brain postinfection. In the absence of CCR9 or CCL25 (CCR9’s ligand) expression, CD81 T cells failed to migrate to key sites of infection in the brain and protect the host from severe forms of disease. Interestingly, we found that expression of CCR9 on CD81 T cells was also responsible for spatial temporal positioning of T cells in the brain. Collectively, our data demonstrate that the CMVinfected brain uses a similar mechanism for CD81 T cell homing as the small intestine.

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Evaluation of a gain-managed nonlinear fiber amplifier for multiphoton microscopy

Pavel Sidorenko*, Michael Buttolph*, Menansili Mejooli*, Chi-Yong Eom, Chris B. Schaffer, Frank Wise

Biomedical Optics Express (2023)

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Two-photon excited fluorescence microscopy is a widely-employed imaging technique that enables the noninvasive study of biological specimens in three dimensions with submicrometer resolution. Here, we report an assessment of a gain-managed nonlinear (GMN) fiber amplifier for multiphoton microscopy. This recently-developed source delivers 58-nJ and 33-fs pulses at 31-MHz repetition rate. We show that the GMN amplifier enables high-quality deep-tissue imaging, and furthermore that the broad spectral bandwidth of the GMN amplifier can be exploited for superior spectral resolution when imaging multiple distinct fluorophores.

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Quasi-analytic solution for real-time multi-exposure speckle imaging of tissue perfusion

Daniel A. Rivera, Chris B. Schaffer

Biomedical Optics Express (2023)

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Laser speckle contrast imaging (LSCI) is a widefield imaging technique that enables high spatiotemporal resolution measurement of blood flow. Laser coherence, optical aberrations, and static scattering effects restrict LSCI to relative and qualitative measurements. Multi-exposure speckle imaging (MESI) is a quantitative extension of LSCI that accounts for these factors but has been limited to post-acquisition analysis due to long data processing times. Here we propose and test a real-time quasi-analytic solution to fitting MESI data, using both simulated and real-world data from a mouse model of photothrombotic stroke. This rapid estimation of multi-exposure imaging (REMI) enables processing of full-frame MESI images at up to 8 Hz with negligible errors relative to time-intensive least-squares methods. REMI opens the door to real-time, quantitative measures of perfusion change using simple optical systems.

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RNA structures and dynamics with Å resolution revealed by x-ray free-electron lasers

Kara A. Zielinski, Shuo Sui, Suzette A. Pabit, Daniel A. Rivera, Tong Wang, Qingyue Hu, Maithri M. Kashipathy, Stella Lisova, Chris B. Schaffer, Valerio Mariani, Mark S. Hunter, Christopher Kupitz, Frank R. Moss III, Frédéric P. Poitevin, Thomas D. Grant, Lois Pollack

Science Advances (2023)

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RNA macromolecules, like proteins, fold to assume shapes that are intimately connected to their broadly recognized biological functions; however, because of their high charge and dynamic nature, RNA structures are far more challenging to determine.We introduce an approach that exploits the high brilliance of x-ray free-electron laser sources to reveal the formation and ready identification of angstrom-scale features in structured and unstructured RNAs. Previously unrecognized structural signatures of RNA secondary and tertiary structures are identified through wide-angle solution scattering experiments. With millisecond time resolution, we observe an RNA fold from a dynamically varying single strand through a base-paired intermediate to assume a triplehelix conformation. While the backbone orchestrates the folding, the final structure is locked in by base stacking. This method may help to rapidly characterize and identify structural elements in nucleic acids in both equilibrium and time-resolved experiments.

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Femtosecond optical parametric chirped-pulse amplification in birefringent step-index fiber

Michael L. Buttolph, Pavel Sidorenko, Chris B. Schaffer, Frank W. Wise

Optics Letters (2022)

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We demonstrate an optical parametric chirped-pulse amplifier (OPCPA) that uses birefringence phase matching in a step-index single-mode optical fiber. The OPCPA is pumped with chirped pulses that can be compressed to sub-30-fs duration. The signal (idler) pulses are generated at 905 nm (1270 nm), have 26 nJ (20 nJ) pulse energy, and are compressible to 70 fs duration. The short compressed signal and idler pulse durations are enabled by the broad bandwidth of the pump pulses. Numerical simulations guiding the design are consistent with the experimental results and predict that scaling to higher pulse energies will be possible. Forgoing a photonic crystal fiber for phase-matching offers practical advantages, including allowing energy scaling with mode area.

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Hypoxia-induced carbonic anhydrase mediated dorsal horn neuron activation and induction of neuropathic pain

Marlene E. Da Vitoria Lobo, Nick Weir, Lydia Hardowar, Yara Al Ojaimi, Ryan Madden, Alex Gibson, Samuel M. Bestall, Masanori Hirashima, Chris B. Schaffer, Lucy F. Donaldson, David O. Bates, Richard Philip Hulse

Pain (2022)

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Neuropathic pain, such as that seen in diabetes mellitus, results in part from central sensitisation in the dorsal horn. However, the mechanisms responsible for such sensitisation remain unclear. There is evidence that disturbances in the integrity of the spinal vascular network can be causative factors in the development of neuropathic pain. Here we show that reduced blood flow and vascularity of the dorsal horn leads to the onset of neuropathic pain. Using rodent models (type 1 diabetes and an inducible endothelial-specific vascular endothelial growth factor receptor 2 knockout mouse) that result in degeneration of the endothelium in the dorsal horn, we show that spinal cord vasculopathy results in nociceptive behavioural hypersensitivity. This also results in increased hypoxia in dorsal horn neurons, depicted by increased expression of hypoxia markers such as hypoxia inducible factor 1a, glucose transporter 3, and carbonic anhydrase 7. Furthermore, inducing hypoxia through intrathecal delivery of dimethyloxalylglycine leads to the activation of dorsal horn neurons as well as mechanical and thermal hypersensitivity. This shows that hypoxic signalling induced by reduced vascularity results in increased hypersensitivity and pain. Inhibition of carbonic anhydrase activity, through intraperitoneal injection of acetazolamide, inhibited hypoxia-induced pain behaviours. This investigation demonstrates that induction of a hypoxic microenvironment in the dorsal horn, as occurs in diabetes, is an integral process by which neurons are activated to initiate neuropathic pain states. This leads to the conjecture that reversing hypoxia by improving spinal cord microvascular blood flow could reverse or prevent neuropathic pain.

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