Development of a Longitudinal Imaging System for Murine Brain Injury Models

Following traumatic brain injury (TBI) and stroke, secondary injury cascades can lead to axonal damage and persistent microglia activation, respectively, in subcortical regions of mice. Current techniques, such as diffusion tensor imaging (DTI) and histology are used to observe features related to damage, but DTI lacks cellular resolution and histology is conducted on fixed tissue, preventing longitudinal studies in the same mouse. The combination of cranial windows and multiphoton microscopy (MPM) is used to image cells in the upper layers of the mouse cortex, but resolution rapidly degrades with imaging depth, making it difficult to observe white matter and subcortical regions following TBI and stroke. To circumvent this challenge, a novel imaging system was developed capable of obtaining longitudinal data related to this secondary damage in subcortical regions of mice. An existing technology, gradient refractive index (GRIN) lenses, was used in conjunction with MPM to image mice before and after injury models. GRIN lenses were attached to low profile head plates and surgically implanted into the brain of mice to acquire time-lapse images of white matter for 60 days following midline fluid percussion injury and microglia 24 hours after a MCAo stroke model. Thy1-YFP and Cx3cr1- tdTomato mice were used to compare changes in white matter fiber tracks and microglia dynamics, respectively. In a model of stroke, injured mice exhibited larger soma areas as compared to control treated animals, demonstrating their activated morphology. When the potential therapeutic, Annexin A1, was administered, treated animals had smaller soma areas as compared to the saline treated animals, signifying Annexin A1’s potential to mitigate inflammation. The system was also used in a model of TBI and the results indicated injured animals developed significantly more varicosities and terminal bulbs than uninjured animals. When minocycline, an FDA approved antibiotic, was administered, treated animals had fewer varicosities and terminal bulb development, demonstrating the potential of the therapeutic to protect against axonal degeneration following TBI. Overall, the imaging system was successfully used in preclinical trials to demonstrate the effectiveness of two potential therapeutics in two brain injury models

Longitudinal optical imaging technique to visualize progressive axonal damage after brain injury in mice reveals responses to different minocycline treatments

A high-resolution, three-dimensional, optical imaging technique for the murine brain was developed to identify the effects of different therapeutic windows for preclinical brain research. This technique tracks the same cells over several weeks. We conducted a pilot study of a promising drug to treat diffuse axonal injury (DAI) caused by traumatic brain injury, using two different therapeutic windows, as a means to demonstrate the utility of this novel longitudinal imaging technique. DAI causes immediate, sporadic axon damage followed by progressive secondary axon damage. We administered minocycline for three days commencing one hour after injury in one treatment group and beginning 72 hours after injury in another group to demonstrate the method’s ability to show how and when the therapeutic drug exerts protective and/or healing effects. Fewer varicosities developed in acutely treated mice while more varicosities resolved in mice with delayed treatment. For both treatments, the drug arrested development of new axonal damage by 30 days. In addition to evaluation of therapeutics for traumatic brain injury, this hybrid microlens imaging method should be useful to study other types of brain injury and neurodegeneration and cellular responses to treatment

Self-Assembled Metal–Organic Biohybrids (MOBs) Using Copper and Silver for Cell Studies

The novel synthesis of metal-containing biohybrids using self-assembly methods at physiological temperatures (37 °C) was compared for copper and silver using the amino acid dimer cystine. Once assembled, the copper containing biohybrid is a stable, high-aspect ratio structure, which we call CuHARS. Using the same synthesis conditions, but replacing copper with silver, we have synthesized cystine-capped silver nanoparticles (AgCysNPs), which are shown here to form stable colloid solutions in contrast to the CuHARS, which settle out from a 1 mg/mL solution in 90 min. Both the copper and silver biohybrids, as synthesized, demonstrate very low agglomeration which we have applied for the purpose of applications with cell culture methods, namely, for testing as anti-cancer compounds. AgCysNPs (1000 ng/mL) demonstrated significant toxicity (only 6.8% viability) to glioma and neuroblastoma cells in vitro, with concentrations as low as 20 ng/mL causing some toxicity. In contrast, CuHARS required at least 5 μg/mL. For comparative purposes, silver sulfate at 100 ng/mL decreased viability by 52% and copper sulfate at 100 ng/mL only by 19.5% on glioma cells. Using these methods, the novel materials were tested here as metal–organic biohybrids (MOBs), and it is anticipated that the functionalization and dynamics of MOBs may result in building a foundation of new materials for cellular applications, including cell engineering of both normal and diseased cells and tissue constructs