Activation of NLRP3 inflammasome in a rat model of cerebral small vessel disease.

Cerebral small vessel disease (CSVD) is increasingly being recognized as a leading contributor to cognitive impairment in the elderly. However, there is a lack of effective preventative or therapeutic options for CSVD. In this exploratory study, we investigated the interplay between neuroinflammation and CSVD pathogenesis as well as the cognitive performance, focusing on NLRP3 signaling as a new therapeutic target. Spontaneously hypertensive stroke-prone (SHRSP) rats served as a CSVD model. We found that SHRSP rats showed decline in learning and memory abilities using morris water maze test. Activated NLRP3 signaling and an increased expression of the downstream pro-inflammatory factors, including IL (interleukin)-6 and tumor necrosis factor α were determined. We also observed a remarkable increase in the production of pyroptosis executive protein gasdermin D, and elevated astrocytic and microglial activation. In addition, we identify several neuropathological hallmarks of CSVD, including blood-brain barrier breakdown, white matter damage, and endothelial dysfunction. These results were in correlation with the activation of NLRP3 inflammasome. Thus, our findings reveal that the NLRP3-mediated inflammatory pathway could play a central role in the pathogenesis of CSVD, presenting a novel target for potential CSVD treatment.

GEOMETRIC DETERMINANTS OF CELL VIABILITY FOR 3D-PRINTED HOLLOW MICRONEEDLE ARRAY-MEDIATED DELIVERY.

A wide range of emerging biomedical applications and clinical interventions rely on the ability to deliver living cells via hollow, high-aspect-ratio microneedles. Recently, microneedle arrays (MNA) have gained increasing interest due to inherent benefits for drug delivery; however, studies exploring the potential to harness such advantages for cell delivery have been impeded due to the difficulties in manufacturing high-aspect-ratio MNAs suitable for delivering mammalian cells. To bypass these challenges, here we leverage and extend our previously reported hybrid additive manufacturing (or "three-dimensional (3D) printing) strategy-i.e., the combined the "Vat Photopolymerization (VPP)" technique, "Liquid Crystal Display (LCD)" 3D printing with "Two-Photon Direct Laser Writing (DLW)"-to 3D print hollow MNAs that are suitable for cell delivery investigations. Specifically, we 3D printed four sets of 650 µm-tall MNAs corresponding to needle-specific inner diameters (IDs) of 25 µm, 50 µm, 75 µm, and 100 µm, and then examined the effects of these MNAs on the post-delivery viability of both dendritic cells (DCs) and HEK293 cells. Experimental results revealed that the 25 µm-ID case led to a statistically significant reduction in post-MNA-delivery cell viability for both cell types; however, MNAs with needle-specific IDs ≥ 50 µm were statistically indistinguishable from one another as well as conventional 32G single needles, thereby providing an important benchmark for MNA-mediated cell delivery.

Effect of brackish water irrigation on cadmium migration in a soil-maize system.

Phytoremediation is an effective way to reduce heavy metal content in agricultural soil. The effects of brackish water irrigation on phytoremediation efficiency of plants have not yet been completely understood. In this study, the effects of brackish water irrigation on cadmium (Cd) uptake by maize as the phytoremediator were investigated. In a pot experiment, maize seedlings were grown in soil with exogenously added Cd (0, 5, 10, or 15 mg kg-1) and irrigated with deionized water (T1), natural brackish water (T2), or water with NaCl with salinity equal to that of natural brackish water (T3). Salt stress and cation antagonism caused by brackish water affected maize plant growth and Cd uptake. Under 5, 10, and 15 mg kg-1 Cd, Cd accumulation in maize shoots was 5.55, 7.08, and 5.71 µg plant-1; 4.08, 3.04, and 5.38 µg plant-1; and 2.48, 3.44, and 5.33 µg plant-1 under the T1, T2, and T3 treatments, respectively. Cd accumulation in the shoots was significantly lower under the T2 and T3 treatments than under the T1 treatment at 5 and 10 mg kg-1 Cd; however, no significant differences were observed among all treatments at 15 mg kg-1 Cd. These findings indicated that phytoremediation efficiency decreased in response to both salt stress and cation antagonism caused by brackish water under low soil-Cd concentrations; however, this effect was negligible under high soil-Cd concentration. Therefore, brackish water irrigation can be considered for the phytoremediation of soils contaminated with high Cd levels to save freshwater resources.

Creatine mapping of the brain at 3T by CEST MRI.

PURPOSE: To assess the feasibility of CEST-based creatine (Cr) mapping in brain at 3T using the guanidino (Guan) proton resonance. METHODS: Wild type and knockout mice with guanidinoacetate N-methyltransferase deficiency and low Cr and phosphocreatine (PCr) concentrations in the brain were used to assign the Cr and protein-based arginine contributions to the GuanCEST signal at 2.0 ppm. To quantify the Cr proton exchange rate, two-step Bloch-McConnell fitting was used to fit the extracted CrCEST line-shape and multi-B1 Z-spectral data. The pH response of GuanCEST was simulated to demonstrate its potential for pH mapping. RESULTS: Brain Z-spectra of wild type and guanidinoacetate N-methyltransferase deficiency mice show a clear Guan proton peak at 2.0 ppm at 3T. The CrCEST signal contributes ∼23% to the GuanCEST signal at B1 = 0.8 µT, where a maximum CrCEST effect of 0.007 was detected. An exchange rate range of 200-300 s-1 was estimated for the Cr Guan protons. As revealed by the simulation, an elevated GuanCEST in the brain is observed when B1 is less than 0.4 µT at 3T, when intracellular pH reduces by 0.2. Conversely, the GuanCEST decreases when B1 is greater than 0.4 µT with the same pH drop. CONCLUSIONS: CrCEST mapping is possible at 3T, which has potential for detecting intracellular pH and Cr concentration in brain.

3D printing-based frugal manufacturing of glass pipettes for minimally invasive delivery of therapeutics to the brain.

Objective: Intracerebral delivery of agents in liquid form is usually achieved through commercially available and durable metal needles. However, their size and texture may contribute to mechanical brain damage. Glass pipettes with a thin tip may significantly reduce injection-associated brain damage but require access to prohibitively expensive programmable pipette pullers. This study is to remove the economic barrier to the application of minimally invasive delivery of therapeutics to the brain, such as chemical compounds, viral vectors, and cells. Methods: We took advantage of the rapid development of free educational online resources and emerging low-cost 3D printers by designing an affordable pipette puller (APP) to remove the cost obstacle. Results: We showed that our APP could produce glass pipettes with a sharp tip opening down to 20 µm or less, which is sufficiently thin for the delivery of therapeutics into the brain. A pipeline from pipette pulling to brain injection using low-cost and open-source equipment was established to facilitate the application of the APP. Conclusion: In the spirit of frugal science, our device may democratize glass pipette-puling and substantially promote the application of minimally invasive and precisely controlled delivery of therapeutics to the brain for finding more effective therapies of brain diseases.

Stimulus edges induce orientation tuning in superior colliculus.

Orientation columns exist in the primary visual cortex (V1) of cat and primates but not mouse. Intriguingly, some recent studies reported the presence of orientation and direction columns in the mouse superficial superior colliculus (sSC), while others reported a lack of columnar organization therein. Using in vivo calcium imaging of sSC in the awake mouse brain, we found that the presence of columns is highly stimulus dependent. Specifically, we observed orientation and direction columns formed by sSC neurons retinotopically mapped to the edge of grating stimuli. For both excitatory and inhibitory neurons in sSC, orientation selectivity can be induced by the edge with their preferred orientation perpendicular to the edge orientation. Furthermore, we found that this edge-induced orientation selectivity is associated with saliency encoding. These findings indicate that the tuning properties of sSC neurons are not fixed by circuit architecture but rather dependent on the spatiotemporal properties of the stimulus.

A modular chemigenetic calcium indicator enables in vivo functional imaging with near-infrared light.

Genetically encoded fluorescent calcium indicators have revolutionized neuroscience and other biological fields by allowing cellular-resolution recording of physiology during behavior. However, we currently lack bright, genetically targetable indicators in the near infrared that can be used in animals. Here, we describe WHaloCaMP, a modular chemigenetic calcium indicator built from bright dye-ligands and protein sensor domains that can be genetically targeted to specific cell populations. Fluorescence change in WHaloCaMP results from reversible quenching of the bound dye via a strategically placed tryptophan. WHaloCaMP is compatible with rhodamine dye-ligands that fluoresce from green to near-infrared, including several dye-ligands that efficiently label the central nervous system in animals. When bound to a near-infrared dye-ligand, WHaloCaMP1a is more than twice as bright as jGCaMP8s, and shows a 7× increase in fluorescence intensity and a 2.1 ns increase in fluorescence lifetime upon calcium binding. We use WHaloCaMP1a with near-infrared fluorescence emission to image Ca2+ responses in flies and mice, to perform three-color multiplexed functional imaging of hundreds of neurons and astrocytes in zebrafish larvae, and to quantitate calcium concentration using fluorescence lifetime imaging microscopy (FLIM).

3D-Printed Microinjection Needle Arrays via a Hybrid DLP-Direct Laser Writing Strategy.

Microinjection protocols are ubiquitous throughout biomedical fields, with hollow microneedle arrays (MNAs) offering distinctive benefits in both research and clinical settings. Unfortunately, manufacturing-associated barriers remain a critical impediment to emerging applications that demand high-density arrays of hollow, high-aspect-ratio microneedles. To address such challenges, here, a hybrid additive manufacturing approach that combines digital light processing (DLP) 3D printing with "ex situ direct laser writing (esDLW)" is presented to enable new classes of MNAs for fluidic microinjections. Experimental results for esDLW-based 3D printing of arrays of high-aspect-ratio microneedles-with 30 µm inner diameters, 50 µm outer diameters, and 550 µm heights, and arrayed with 100 µm needle-to-needle spacing-directly onto DLP-printed capillaries reveal uncompromised fluidic integrity at the MNA-capillary interface during microfluidic cyclic burst-pressure testing for input pressures in excess of 250 kPa (n = 100 cycles). Ex vivo experiments perform using excised mouse brains reveal that the MNAs not only physically withstand penetration into and retraction from brain tissue but also yield effective and distributed microinjection of surrogate fluids and nanoparticle suspensions directly into the brains. In combination, the results suggest that the presented strategy for fabricating high-aspect-ratio, high-density, hollow MNAs could hold unique promise for biomedical microinjection applications.

Fast and sensitive GCaMP calcium indicators for imaging neural populations.

Calcium imaging with protein-based indicators1,2 is widely used to follow neural activity in intact nervous systems, but current protein sensors report neural activity at timescales much slower than electrical signalling and are limited by trade-offs between sensitivity and kinetics. Here we used large-scale screening and structure-guided mutagenesis to develop and optimize several fast and sensitive GCaMP-type indicators3-8. The resulting 'jGCaMP8' sensors, based on the calcium-binding protein calmodulin and a fragment of endothelial nitric oxide synthase, have ultra-fast kinetics (half-rise times of 2 ms) and the highest sensitivity for neural activity reported for a protein-based calcium sensor. jGCaMP8 sensors will allow tracking of large populations of neurons on timescales relevant to neural computation.

Preserving cognitive function in patients with Alzheimer's disease: The Alzheimer's disease neuroprotection research initiative (ADNRI).

The global trend toward aging populations has resulted in an increase in the occurrence of Alzheimer's disease (AD) and associated socioeconomic burdens. Abnormal metabolism of amyloid-ß (Aß) has been proposed as a significant pathomechanism in AD, supported by results of recent clinical trials using anti-Aß antibodies. Nonetheless, the cognitive benefits of the current treatments are limited. The etiology of AD is multifactorial, encompassing Aß and tau accumulation, neuroinflammation, demyelination, vascular dysfunction, and comorbidities, which collectively lead to widespread neurodegeneration in the brain and cognitive impairment. Hence, solely removing Aß from the brain may be insufficient to combat neurodegeneration and preserve cognition. To attain effective treatment for AD, it is necessary to (1) conduct extensive research on various mechanisms that cause neurodegeneration, including advances in neuroimaging techniques for earlier detection and a more precise characterization of molecular events at scales ranging from cellular to the full system level; (2) identify neuroprotective intervention targets against different neurodegeneration mechanisms; and (3) discover novel and optimal combinations of neuroprotective intervention strategies to maintain cognitive function in AD patients. The Alzheimer's Disease Neuroprotection Research Initiative's objective is to facilitate coordinated, multidisciplinary efforts to develop systemic neuroprotective strategies to combat AD. The aim is to achieve mitigation of the full spectrum of pathological processes underlying AD, with the goal of halting or even reversing cognitive decline.

Hyperosmolar blood-brain barrier opening using intra-arterial injection of hyperosmotic mannitol in mice under real-time MRI guidance.

The blood-brain barrier (BBB) is the main obstacle to the effective delivery of therapeutic agents to the brain, compromising treatment efficacy for a variety of neurological disorders. Intra-arterial (IA) injection of hyperosmotic mannitol has been used to permeabilize the BBB and improve parenchymal entry of therapeutic agents following IA delivery in preclinical and clinical studies. However, the reproducibility of IA BBB manipulation is low and therapeutic outcomes are variable. We demonstrated that this variability could be highly reduced or eliminated when the procedure of osmotic BBB opening is performed under the guidance of interventional MRI. Studies have reported the utility and applicability of this technique in several species. Here we describe a protocol to open the BBB by IA injection of hyperosmotic mannitol under the guidance of MRI in mice. The procedures (from preoperative preparation to postoperative care) can be completed within ~1.5 h, and the skill level required is on par with the induction of middle cerebral artery occlusion in small animals. This MRI-guided BBB opening technique in mice can be utilized to study the biology of the BBB and improve the delivery of various therapeutic agents to the brain.

An adaptive optics module for deep tissue multiphoton imaging in vivo.

Understanding complex biological systems requires visualizing structures and processes deep within living organisms. We developed a compact adaptive optics module and incorporated it into two- and three-photon fluorescence microscopes, to measure and correct tissue-induced aberrations. We resolved synaptic structures in deep cortical and subcortical areas of the mouse brain, and demonstrated high-resolution imaging of neuronal structures and somatosensory-evoked calcium responses in the mouse spinal cord at great depths in vivo.

Traumatic brain injury does not disrupt costimulatory blockade-induced immunological tolerance to glial-restricted progenitor allografts.

BACKGROUND: Cell transplantation-based treatments for neurological disease are promising, yet graft rejection remains a major barrier to successful regenerative therapies. Our group and others have shown that long-lasting tolerance of transplanted stem cells can be achieved in the brain with systemic application of monoclonal antibodies blocking co-stimulation signaling. However, it is unknown if subsequent injury and the blood-brain barrier breach could expose the transplanted cells to systemic immune system spurring fulminant rejection and fatal encephalitis. Therefore, we investigated whether delayed traumatic brain injury (TBI) could trigger graft rejection. METHODS: Glial-restricted precursor cells (GRPs) were intracerebroventricularly transplanted in immunocompetent neonatal mice and co-stimulation blockade (CoB) was applied 0, 2, 4, and 6 days post-grafting. Bioluminescence imaging (BLI) was performed to monitor the grafted cell survival. Mice were subjected to TBI 12 weeks post-transplantation. MRI and open-field test were performed to assess the brain damage and behavioral change, respectively. The animals were decapitated at week 16 post-transplantation, and the brains were harvested. The survival and distribution of grafted cells were verified from brain sections. Hematoxylin and eosin staining (HE) was performed to observe TBI-induced brain legion, and neuroinflammation was evaluated immunohistochemically. RESULTS: BLI showed that grafted GRPs were rejected within 4 weeks after transplantation without CoB, while CoB administration resulted in long-term survival of allografts. BLI signal had a steep rise following TBI and subsequently declined but remained higher than the preinjury level. Open-field test showed TBI-induced anxiety for all animals but neither CoB nor GRP transplantation intensified the symptom. HE and MRI demonstrated a reduction in TBI-induced lesion volume in GRP-transplanted mice compared with non-transplanted mice. Brain sections further validated the survival of grafted GRPs and showed more GRPs surrounding the injured tissue. Furthermore, the brains of post-TBI shiverer mice had increased activation of microglia and astrocytes compared to post-TBI wildtype mice, but infiltration of CD45+ leukocytes remained low. CONCLUSIONS: CoB induces sustained immunological tolerance towards allografted cerebral GRPs which is not disrupted following TBI, and unexpectedly TBI may enhance GRPs engraftment and contribute to post-injury brain tissue repair.

A Distinct Population of L6 Neurons in Mouse V1 Mediate Cross-Callosal Communication.

Through the corpus callosum, interhemispheric communication is mediated by callosal projection (CP) neurons. Using retrograde labeling, we identified a population of layer 6 (L6) excitatory neurons as the main conveyer of transcallosal information in the monocular zone of the mouse primary visual cortex (V1). Distinct from L6 corticothalamic (CT) population, V1 L6 CP neurons contribute to an extensive reciprocal network across multiple sensory cortices over two hemispheres. Receiving both local and long-range cortical inputs, they encode orientation, direction, and receptive field information, while are also highly spontaneous active. The spontaneous activity of L6 CP neurons exhibits complex relationships with brain states and stimulus presentation, distinct from the spontaneous activity patterns of the CT population. The anatomical and functional properties of these L6 CP neurons enable them to broadcast visual and nonvisual information across two hemispheres, and thus may play a role in regulating and coordinating brain-wide activity events.

White matter demyelination predates axonal injury after ischemic stroke in cynomolgus monkeys.

Unraveling the pathology of stroke is a prerequisite for designing therapeutic strategies. It was reported that myelin injury exceeded axonal loss in the peri-infarct region of rodent white matter stroke. An in-depth investigation of the post-stroke white matter damage in higher-order species might innovate stroke intervention. In this study, adult male cynomolgus monkeys received surgical middle cerebral artery occlusion (MCAO), and serial magnetic resonance scans to non-invasively assess brain damage. Spontaneous movements were recorded to evaluate post-stroke behavior. The axon and myelin loss, as well as immune cell infiltration were examined using immunohistochemistry. Magnetic resonance imaging revealed cerebral infarcts and white matter injury after MCAO in monkeys, which were confirmed by neurological deficits. Immunostaining of white matter fibers showed substantial demyelination whilst retention of axons in the infarcts 8 days post MCAO, while a progressive loss of myelin and axons was observed after one month. Gliosis, microglia activation, and leukocyte infiltration were identified in the lesions. These results demonstrate that demyelination predates axonal injury in non-human primate ischemic stroke, which provides a time window for stroke intervention focusing on prevention of progressive axonal loss through myelin regeneration.

Long term intravital single cell tracking under multiphoton microscopy.

Visualizing and tracking cells over time in a living organism has been a much-coveted dream before the invention of intravital microscopy. The opaque nature of tissue was a major hurdle that was remedied by the multiphoton microscopy. With the advancement of optical imaging and fluorescent labeling tools, intravital high resolution imaging has become increasingly accessible over the past few years. Long-term intravital tracking of single cells (LIST) under multiphoton microscopy provides a unique opportunity to gain insight into the longitudinal changes in the morphology, migration, or function of cells or subcellular structures. It is particularly suitable for studying slow-evolving cellular and molecular events during normal development or disease progression, without losing the opportunity of catching fast events such as calcium signals. Here, we review the application of LIST under 2-photon microscopy in various fields of neurobiology and discuss challenges and new directions in labeling and imaging methods for LIST. Overall, this review provides an overview of current applications of LIST in mammals, which is an emerging field that will contribute to a better understanding of essential molecular and cellular events in health and disease.

Modeling human pediatric and adult gliomas in immunocompetent mice through costimulatory blockade.

Currently, human glioma tumors are mostly modeled in immunodeficient recipients; however, lack of interactions with adaptive immune system is a serious flaw, particularly in the era when immunotherapies dominate treatment strategies. Our group was the first to successfully establish the orthotopic transplantation of human glioblastoma (GBM) in immunocompetent mice by inducing immunological tolerance using a short-term, systemic costimulation blockade strategy (CTLA-4-Ig and MR1). In this study, we further validated the feasibility of this method by modeling pediatric diffuse intrinsic pontine glioma (DIPG) and two types of adult GBM (GBM1, GBM551), in mice with intact immune systems and immunodeficient mice. We found that all three glioma models were successfully established, with distinct difference in tumor growth patterns and morphologies, after orthotopic xenotransplantation in tolerance-induced immunocompetent mice. Long-lasting tolerance that is maintained for up to nearly 200 d in GBM551 confirmed the robustness of this model. Moreover, we found that tumors in immunocompetent mice displayed features more similar to the clinical pathophysiology found in glioma patients, characterized by inflammatory infiltration and strong neovascularization, as compared with tumors in immunodeficient mice. In summary, we have validated the robustness of the costimulatory blockade strategy for tumor modeling and successfully established three human glioma models including the pediatric DIPG whose preclinical study is particularly thwarted by the lack of proper animal models.

jYCaMP: an optimized calcium indicator for two-photon imaging at fiber laser wavelengths.

Femtosecond lasers at fixed wavelengths above 1,000 nm are powerful, stable and inexpensive, making them promising sources for two-photon microscopy. Biosensors optimized for these wavelengths are needed for both next-generation microscopes and affordable turn-key systems. Here we report jYCaMP1, a yellow variant of the calcium indicator jGCaMP7 that outperforms its parent in mice and flies at excitation wavelengths above 1,000 nm and enables improved two-color calcium imaging with red fluorescent protein-based indicators.