Laser threshold and cell damage mechanism for intravascular photoacoustic imaging.

OBJECTIVES: Intravascular photoacoustic (IVPA) imaging is being developed to image atherosclerotic plaques, a leading cause of morbidity and mortality in the United States. However, the safety of this imaging modality, which requires repeated irradiation with short laser pulses, has not yet been investigated. This study has two objectives. First, determine in vitro the limit of cumulative fluence that can be applied to cells before death at IVPA relevant wavelengths. Second, evaluate if high single pulse fluences are a potential cause of cell death during IVPA imaging. MATERIALS AND METHODS: Experiments were conducted using endothelial cells, macrophages, and smooth muscle cells. The cumulative fluence experiments were conducted at 1064 and 1197 nm, using a high pulse repetition frequency laser. Cells were irradiated with a wide range of cumulative fluences and evaluated for cell death. The thresholds for death were compared to the maximum expected clinical cumulative fluence. To evaluate the effect of single pulse fluences, cells were irradiated at 1064, 1210, and 1720 nm. Light was delivered at a range of pulse energies to emulate the fluences that cells would be exposed to during clinical IVPA imaging. RESULTS: At 1064 nm, all three cell types remained viable at cumulative fluences above the maximum expected clinical cumulative fluence, which is calculated based on common IVPA imaging protocols. At 1197 nm, cells were viable near or just below the maximum expected clinical cumulative fluence, with some cell type to cell type variation. All three cell types remained viable after irradiation with high single pulse fluences at all three wavelengths. CONCLUSION: The cumulative fluence experiments indicate that safety considerations are likely to put constraints on the amount of irradiation that can be used in IVPA imaging protocols. However, this study also indicates that it will be possible to use IVPA imaging safely, since cumulative fluences could be reduced by as much as two orders of magnitude below the maximum expected clinical cumulative fluence by varying the imaging protocol, albeit at the expense of image quality. The single pulse fluence experiments indicate that cell death from single pulse fluence is not likely during IVPA imaging. Thus, future studies should focus on heat accumulation as the likely mechanism of tissue damage. Lasers Surg. Med. 51:466-474, 2019. © 2018 Wiley Periodicals, Inc.

Photoacoustic Image-Guided Delivery of Plasmonic-Nanoparticle-Labeled Mesenchymal Stem Cells to the Spinal Cord.

Regenerative therapies using stem cells have great potential for treating neurodegenerative diseases and traumatic injuries in the spinal cord. In spite of significant research efforts, many therapies fail at the clinical phase. As stem cell technologies advance toward clinical use, there is a need for a minimally invasive, safe, affordable, and real-time imaging technique that allows for the accurate and safe monitoring of stem cell delivery in the operating room. In this work, we present a combined ultrasound and photoacoustic imaging tool to provide image-guided needle placement and monitoring of nanoparticle-labeled stem cell delivery into the spinal cord. We successfully tagged stem cells using gold nanospheres and provided image-guided delivery of stem cells into the spinal cord in real-time, detecting as few as 1000 cells. Ultrasound and photoacoustic imaging was used to guide needle placement for direct stem cell injection to minimize the risk of needle shear and accidental injury and to improve therapeutic outcomes with accurate, localized stem cell delivery. Following injections of various volumes of cells, three-dimensional ultrasound and photoacoustic images allowed the visualization of stem cell distribution along the spinal cord, showing the potential to monitor the migration of the cells in the future. The feasibility of quantitative imaging was also shown by correlating the total photoacoustic signal over the imaging volume to the volume of cells injected. Overall, the presented method may allow clinicians to utilize imaged-guided delivery for more accurate and safer stem cell delivery to the spinal cord.

Characterization of a murine model of SMA.

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disease, which is the leading genetic cause of mortality in children. To date no effective treatment exists for SMA. The genetic basis for SMA has been well documented as a mutation in the gene for survival of motor neuron (SMN). Because there is an understanding of which gene needs to be replaced (SMN) and where it needs to be replaced (spinal motor systems), SMA is an ideal target for gene replacement via gene therapy. While a variety of animal models for SMA exist, they are either too fulminant to realistically test most gene delivery strategies, or too mild to provide a robust read out of the therapeutic effect. The field, therefore, requires a robust model with a slower symptomatic progression. A conditional knockout of SMN in neuronal cell types, giving a phenotype of functional motor defects, weight loss and reduced life expectancy partially satisfies this need (Frugier, Tiziano et al. 2000). This Cre/LoxP mediated neuron specific model presents an attractive alternative. In the present manuscript, we characterize the functional motor deficits of the model. We observed a decline in locomotor ability, as assessed by open field testing. The finer functions of motor skills such as righting reflex and grip strength were also observed to degenerate in the SMA mice. The decline in motor function that we observed here correlates with the anatomical decline in motor neurons and motor axons presented in the literature (Ferri, Melki et al. 2004). This work adds to our understanding and knowledge base of this Cre/LoxP model and provides a basis from which functional recovery, following interventions can be assessed.

Lentiviral vector delivery of short hairpin RNA to NG2 and neurotrophin-3 promotes locomotor recovery in injured rat spinal cord.

BACKGROUND AIMS: In this study we investigated the effect of neurotrophin-3 (NT-3) and knockdown of NG2, one of the main inhibitory chondroitin sulfate proteoglycans (CSPG), in the glial scar following spinal cord injury (SCI). METHODS: Short hairpin (sh) RNA were designed to target NG2 and were cloned into a lentiviral vector (LV). A LV was also constructed containing NT-3. LV expressing NT-3, shRNA to NG2 or combinations of both vectors were injected directly into contused rat spinal cords 1 week post-injury. Six weeks post-injection of LV, spinal cords were examined by histology for changes in scar size and by immunohistochemistry for changes in expression of CSPG, NT-3, astrocytes, neurons and microglia/macrophages. Motor function was assessed using the Basso, Beattie and Bresnahan (BBB) locomotor scale. RESULTS: Animals that received the combination treatment of LV shNG2 and LV NT-3 showed reduced scar size. These animals also showed an increase in levels of neurons and NG2, a decrease in levels of astrocytes and a significant functional recovery as assessed using the BBB locomotor scale at 2 weeks post-treatment. CONCLUSIONS: The improvement in locomotor recovery and decrease in scar size shows the potential of this gene therapy approach as a therapeutic treatment for SCI.

Lentiviral vector-mediated knockdown of the NG2 [corrected] proteoglycan or expression of neurotrophin-3 promotes neurite outgrowth in a cell culture model of the glial scar.

BACKGROUND: Following spinal cord injury, a highly inhibitory environment for axonal regeneration develops. One of the main sources of this inhibition is the glial scar that is formed after injury by reactive astrocytes. The inhibitory environment is mainly a result of chondroitin sulphate proteoglycans (CSPGs). NG2, [corrected] one of the main inhibitory CSPGs, is up-regulated following spinal cord injury. METHODS: Small interfering RNA (siRNA) was designed to target NG2 and this short hairpin RNA (shRNA) was cloned into a lentiviral vector (LV). The neurotrophic factor neurotrophin-3 (NT-3) promotes the growth and survival of developing neurites and has also been shown to aid regeneration. NT-3 was also cloned into a LV. In vitro assessment of these vectors using a coculture system of dorsal root ganglia (DRG) neurones and Neu7 astrocytes was carried out. The Neu7 cell line is a rat astrocyte cell line that overexpresses NG2, thereby mimicking the inhibitory environment following spinal cord injury. RESULTS AND DISCUSSION: These experiments show that both the knockdown of NG2 via shRNA and over-expression of NT-3 can significantly increase neurite growth, although a combination of both vectors did not confer any additional benefit over the vectors used individually. These LVs show promising potential for growth and survival of neurites in injured central nervous system tissue (CNS).

Effects of Freezing on Mesenchymal Stem Cells Labeled with Gold Nanoparticles.

Stem cell therapies are a promising treatment for many patients suffering from diseases with poor prognosis. However, clinical translation is inhibited by a lack of in vivo monitoring techniques to track stem cells throughout the course of treatment. Ultrasound-guided photoacoustic (PA) imaging of nanoparticle-labeled stem cells may be a solution. To allow PA tracking, stem cells must be labeled with an optically absorbing contrast agent. Gold nanoparticles are one option due to their cytocompatibility and strong optical absorption in the near-infrared region. However, stem cell labeling can require up to 24-h incubation with nanoparticles in culture before use. Although stem cell monitoring is critically needed, the additional preparation time may not be feasible-it is cost prohibitive and stem cell treatments should be readily available in emergency situations as well as scheduled procedures. To remedy this, stem cells can be labeled before freezing and long-term storage. While it is well known that stem cells retain their cellular function after freezing, storage, and thawing, the impact of gold nanoparticles on this process has yet to be investigated. Therefore, we assessed the viability, multipotency, and PA activity of gold nanosphere-labeled mesenchymal stem cells (MSCs) after freezing, storing, and thawing for 1 week, 1 month, or 2 months and compared to unlabeled, naive MSCs which were frozen, stored, and thawed at the same time points. Results indicated no substantial change in viability as assessed by the MTT assay. Differentiation, observed through adipogenesis and osteogenesis, was also comparable to controls. Finally, strong PA signals and similar PA spectral signatures remained. Further studies involving more diverse stem cell types and nanoparticles are required, but our data suggest that function and imaging properties of nanoparticle-labeled stem cells are maintained after freezing and storage, which improve translation of stem cell monitoring techniques by simplifying integration with clinical protocols. Impact statement Although stem cell tracking techniques are critically needed, stem cells must be labeled with contrast agents in advance of procedures, which is not clinically feasible due to increased procedure time. As a solution, a stock of labeled stem cells could be frozen and stored, ready for immediate use. Results showed that gold nanosphere-labeled stem cells can be frozen and stored long-term without impacting cellular function or photoacoustic imaging contrast, supporting further investigation of other contrast agents and cell types. Creating a bank of nanoparticle-labeled stem cells advances translation and scalability of stem cell tracking methods by improving integration with clinical protocols.

Photoacoustic imaging of gold nanorods in the brain delivered via microbubble-assisted focused ultrasound: a tool for in vivo molecular neuroimaging.

The protective barriers of the CNS present challenges during the treatment and monitoring of diseases. In particular, the blood brain barrier is a major hindrance to the delivery of imaging contrast agents and therapeutics to the brain. In this work, we use gas microbubble-assisted focused ultrasound to transiently open the blood brain barrier and locally deliver silica coated gold nanorods across the barrier. This particular nanoagent possesses a strong optical absorption which enables in vivo and ex vivo visualization of the delivered particles using ultrasound-guided photoacoustic imaging. The results of these studies demonstrate the potential of ultrasound-guided photoacoustics to image contrast agents delivered via microbubble-assisted focused ultrasound for longitudinal diagnostic imaging and for therapeutic monitoring of neurological diseases.

Laser-activated perfluorocarbon nanodroplets: a new tool for blood brain barrier opening.

A major obstacle in the monitoring and treatment of neurological diseases is the blood brain barrier (BBB), a semipermeable barrier that prevents the delivery of many therapeutics and imaging contrast agents to the brain. In this work, we explored the possibility of laser-activated perfluorocarbon nanodroplets (PFCnDs) to open the BBB and deliver agents to the brain tissue. Specifically, near infrared (NIR) dye-loaded PFCnDs comprised of a perfluorocarbon (PFC) core with a boiling point above physiological temperature were repeatedly vaporized and recondensed from liquid droplet to gas bubble under pulsed laser excitation. As a result, this pulse-to-pulse repeated behavior enabled the recurring interaction of PFCnDs with the endothelial lining of the BBB, allowing for a BBB opening and extravasation of dye into the brain tissue. The blood brain barrier opening and delivery of agents to tissue was confirmed on the macro and the molecular level by evaluating Evans Blue staining, ultrasound-guided photoacoustic (USPA) imaging, and histological tissue analysis. The demonstrated PFCnD-assisted pulsed laser method for BBB opening, therefore, represents a tool that has the potential to enable non-invasive, cost-effective, and efficient image-guided delivery of contrast and therapeutic agents to the brain.

Release and hemodynamic influence of nitro-glycerine-derived nitric oxide in endotoxemic rats.

BACKGROUND AND AIM: Nitric oxide released from nitro-glycerine (NG) has been considered to improve the microcirculation. Septic conditions are, however, associated with excessive formation of nitric oxide (NO), which is formed from l-arginine by the inducible NO synthase (iNOS) activity. Since the characteristics and influence of NG-derived NO in sepsis remains unclear, the major aims of the present study were to quantify the release and to determine the effects of NO formed from NG on systemic blood pressure under endotoxemic conditions. MATERIAL AND METHODS: Four hours following endotoxin challenge (10 mg/kg intraperitoneally), rats received an infusion of NG (0.5 or 5.0 micromol/kg/h) over 45 min. We determined the changes in blood pressure and the NO concentrations generated in brain, heart, intestine, kidney, liver, and lung by means of NO trapping and EPR technique. RESULTS: NG infusion in control rats and endotoxin challenge decreased systemic blood pressure to the same extent. However, in rats subjected to endotoxin challenge NG infusion did not affect the blood pressure. The endotoxin-induced increase in tissue NO concentrations were found to be 15-folds higher than tissue levels of NO following NG infusion. CONCLUSION: Our results suggest that under endotoxic shock conditions in rats NG may not additionally affect the systemic blood pressure. This may relate to the excessive tissue NO levels induced by endotoxin that are not further increased by NG.

Regulated neuronal neuromodulation via spinal cord expression of the gene for the inwardly rectifying potassium channel 2.1 (Kir2.1).

BACKGROUND: Neuromodulation is used to restore neural function in disorders that stem from an imbalance in the activity of specific neural networks when they prove refractory to pharmacological therapy. The Kir2.1 gene contributes to stabilizing the resting potential below the threshold of activation of voltage-gated sodium channels and action potentials. Therefore, the delivery of the Kir2.1 gene to neuronal cells could reduce the probability of action potential generation, inhibiting excessive neural activity. OBJECTIVE: To address the hypothesis that overexpression of the inwardly rectifying potassium channel 2.1 (Kir2.1) gene could inhibit motor neuron activity and therefore be therapeutically used in gene-based neuromodulation. METHODS: To induce expression of Kir2.1, the inducible RheoSwitch promoter was used and controlled by ligand. In vivo gene expression was accomplished by an adenoviral vector to deliver unilaterally into the lumbar spinal cord of rats. RESULTS: Behavioral assays demonstrated that neuromuscular inhibition was exclusive to rats that received the ligand. Histological analysis also showed evidence of some motor neuron loss in these animals. Behavioral effects of Kir2.1 expression were completely reversible, arguing that the behavioral effect did not result from motor neuron death. CONCLUSION: Delivery of the gene for Kir2.1 inhibits neurons by resisting depolarization to the action potential threshold. Regulated neuronal expression of Kir2.1 may provide an elegant means for neuromodulation in a selected neuronal population.

Update on gene and stem cell therapy approaches for spinal muscular atrophy.

INTRODUCTION: Spinal muscular atrophy (SMA) is the leading genetic cause of pediatric death to which at present there is no effective therapeutic. The genetic defect is well characterized as a mutation in exon 7 of the survival of motor neuron (SMN) gene. The current gene therapy approach focuses on two main methodologies, the replacement of SMN1 or augmentation of SMN2 readthrough. The most promising of the current work focuses on the delivery of SMN via AAV9 vectors via intravenous delivery. AREAS COVERED: In the review the authors examine the current research in the field of stem cell and gene therapy approaches for SMA. Also focusing on delivery methods, timing of administration and general caveats that must be considered with translational work for SMA. EXPERT OPINION: Gene therapy currently offers the most promising avenue of research for a successful therapeutic for SMA. There are many important practical and ethical considerations which must be carefully considered when dealing with clinical trial in infants such as the invasiveness of the surgery, the correct patient cohort and the potential risks.

Stem cell therapy for the spinal cord.

Injury and disease of the spinal cord are generally met with a poor prognosis. This poor prognosis is due not only to the characteristics of the diseases but also to our poor ability to deliver therapeutics to the spinal cord. The spinal cord is extremely sensitive to direct manipulation, and delivery of therapeutics has proven a challenge for both scientists and physicians. Recent advances in stem cell technologies have opened up a new avenue for the treatment of spinal cord disease and injury. Stem cells have proven beneficial in rodent models of spinal cord disease and injury. In these animal models, stem cells have been shown to produce their effect by the dual action of cell replacement and the trophic support of the factors secreted by these cells. In this review we look at the main clinical trials involving stem cell transplant into the spinal cord, focusing on motor neuron diseases and spinal cord injury. We will also discuss the major hurdles in optimizing stem cell delivery methods into the spinal cord. We shall examine current techniques such as functional magnetic resonance imaging guidance and cell labeling and will look at the current research striving to improve these techniques. With all caveats and future research taken into account, this is a very exciting time for stem cell transplant into the spinal cord. We are only beginning to realize the huge potential of stem cells in a central nervous system setting to provide cell replacement and trophic support. Many more trials will need to be undertaken before we can fully exploit the attributes of stem cells.