An updated Barnes maze protocol for assessing the outcome of controlled cortical impact mouse models of traumatic brain injury.

BACKGROUND: The Barnes Maze (BM) is a common method of testing cognitive deficits in rodents. Adapting BM protocols for specific neurological disorders could potentially aid in more effective testing, reduce research time, and help decrease variability between studies. NEW METHOD: We tested differences an updated, shortened BM consisting of 6 days, 3 trials per day, only covering the equivalent of the spatial acquisition week BM protocol and a probe trial day consisting of one trial (7 total days). RESULTS: Kaplan-Meier plots of escape percentage as a function of total latency showed a significant difference between control and CCI mice in the updated protocol on days 3 through 6. Additionally, probe trial data showed significant differences in primary latency, primary errors, and returns to goal. COMPARISON WITH EXISTING METHODS: We tested differences between a traditional 5 days per week, 2 trials per day, spatial acquisition and reversal weeks BM protocol (12 total days with probe trials) with an updated 6-day BM protocol (7 total days with probe trial). In the probe trial, the updated protocol control mice showed an over 5-fold decrease in primary latency and primary errors and a 4.6-fold increase in returns to goal compared to the traditional protocol. Additionally, mice in both protocols performed similarly on a trial-by-trial basis suggesting that the changes made for the updated protocol increased learning and memory and was not simply an easier task. CONCLUSION: The updated BM protocol showed an improved ability to distinguish between control and CCI mice and promoted improved and more consistent learning for both the control and CCI groups.

Antioxidant theranostic copolymer-mediated reduction in oxidative stress following traumatic brain injury improves outcome in a mouse model.

Following a traumatic brain injury (TBI), excess reactive oxygen species (ROS) and lipid peroxidation products (LPOx) are generated and lead to secondary injury beyond the primary insult. A major limitation of current treatments is poor target engagement, which has prevented success in clinical trials. Thus, nanoparticle-based treatments have received recent attention because of their ability to increase accumulation and retention in damaged brain. Theranostic neuroprotective copolymers (NPC3) containing thiol functional groups can neutralize ROS and LPOx. Immediate administration of NPC3 following injury in a controlled cortical impact (CCI) mouse model provides a therapeutic window in reducing ROS levels at 2.08-20.83 mg/kg in males and 5.52-27.62 mg/kg in females. This NPC3-mediated reduction in oxidative stress improves spatial learning and memory in males, while females show minimal improvement. Notably, NPC3-mediated reduction in oxidative stress prevents the bilateral spread of necrosis in male mice, which was not observed in female mice and likely accounts for the sex-based spatial learning and memory differences. Overall, these findings suggest sex-based differences to oxidative stress scavenger nanoparticle treatments, and a possible upper threshold of antioxidant activity that provides therapeutic benefit in injured brain since female mice benefit from NPC3 treatment to a lesser extent than male mice.

Theranostic Copolymers Neutralize Reactive Oxygen Species and Lipid Peroxidation Products for the Combined Treatment of Traumatic Brain Injury.

Traumatic brain injury (TBI) results in the generation of reactive oxygen species (ROS) and lipid peroxidation product (LPOx), including acrolein and 4-hydroxynonenal (4HNE). The presence of these biochemical derangements results in neurodegeneration during the secondary phase of the injury. The ability to rapidly neutralize multiple species could significantly improve outcomes for TBI patients. However, the difficulty in creating therapies that target multiple biochemical derangements simultaneously has greatly limited therapeutic efficacy. Therefore, our goal was to design a material that could rapidly bind and neutralize both ROS and LPOx following TBI. To do this, a series of thiol-functionalized biocompatible copolymers based on lipoic acid methacrylate and polyethylene glycol monomethyl ether methacrylate (FW ∼ 950 Da) (O950) were prepared. A polymerizable gadolinium-DOTA methacrylate monomer (Gd-MA) was also synthesized starting from cyclen to facilitate direct magnetic resonance imaging and in vivo tracking of accumulation. These neuroprotective copolymers (NPCs) were shown to rapidly and effectively neutralize both ROS and LPOx. Horseradish peroxidase absorbance assays showed that the NPCs efficiently neutralized H2O2, while R-phycoerythrin protection assays demonstrated their ability to protect the fluorescent protein from oxidative damage. 1H NMR studies indicated that the thiol-functional NPCs rapidly form covalent bonds with acrolein, efficiently removing it from solution. In vitro cell studies with SH-SY5Y-differentiated neurons showed that NPCs provide unique protection against toxic concentrations of both H2O2 and acrolein. NPCs rapidly accumulate and are retained in the injured brain in controlled cortical impact mice and reduce post-traumatic oxidative stress. Therefore, these materials show promise for improved target engagement of multiple biochemical derangements in hopes of improving TBI therapeutic outcomes.

Smooth muscle cells affect differential nanoparticle accumulation in disturbed blood flow-induced murine atherosclerosis.

Atherosclerosis is a lipid-driven chronic inflammatory disease that leads to the formation of plaques in the inner lining of arteries. Plaques form over a range of phenotypes, the most severe of which is vulnerable to rupture and causes most of the clinically significant events. In this study, we evaluated the efficacy of nanoparticles (NPs) to differentiate between two plaque phenotypes based on accumulation kinetics in a mouse model of atherosclerosis. This model uses a perivascular cuff to induce two regions of disturbed wall shear stress (WSS) on the inner lining of the instrumented artery, low (upstream) and multidirectional (downstream), which, in turn, cause the development of an unstable and stable plaque phenotype, respectively. To evaluate the influence of each WSS condition, in addition to the final plaque phenotype, in determining NP uptake, mice were injected with NPs at intermediate and fully developed stages of plaque growth. The kinetics of artery wall uptake were assessed in vivo using dynamic contrast-enhanced magnetic resonance imaging. At the intermediate stage, there was no difference in NP uptake between the two WSS conditions, although both were different from the control arteries. At the fully-developed stage, however, NP uptake was reduced in plaques induced by low WSS, but not multidirectional WSS. Histological evaluation of plaques induced by low WSS revealed a significant inverse correlation between the presence of smooth muscle cells and NP accumulation, particularly at the plaque-lumen interface, which did not exist with other constituents (lipid and collagen) and was not present in plaques induced by multidirectional WSS. These findings demonstrate that NP accumulation can be used to differentiate between unstable and stable murine atherosclerosis, but accumulation kinetics are not directly influenced by the WSS condition. This tool could be used as a diagnostic to evaluate the efficacy of experimental therapeutics for atherosclerosis.

Claudin-1-Targeted Nanoparticles for Delivery to Aging-Induced Alterations in the Blood-Brain Barrier.

Aging-induced alterations to the blood-brain barrier (BBB) are increasingly being seen as a primary event in chronic progressive neurological disorders that lead to cognitive decline. With the goal of increasing delivery into the brain in hopes of effectively treating these diseases, a large focus has been placed on developing BBB permeable materials. However, these strategies have suffered from a lack of specificity toward regions of disease progression. Here, we report on the development of a nanoparticle (C1C2-NP) that targets regions of increased claudin-1 expression that reduces BBB integrity. Using dynamic contrast enhanced magnetic resonance imaging, we find that C1C2-NP accumulation and retention is significantly increased in brains from 12 month-old mice as compared to nontargeted NPs and brains from 2 month-old mice. Furthermore, we find C1C2-NP accumulation in brain endothelial cells with high claudin-1 expression, suggesting target-specific binding of the NPs, which was validated through fluorescence imaging, in vitro testing, and biophysical analyses. Our results further suggest a role of claudin-1 in reducing BBB integrity during aging and show altered expression of claudin-1 can be actively targeted with NPs. These findings could help develop strategies for longitudinal monitoring of tight junction protein expression changes during aging as well as be used as a delivery strategy for site-specific delivery of therapeutics at these early stages of disease development.

Antioxidant thioether core-crosslinked nanoparticles prevent the bilateral spread of secondary injury to protect spatial learning and memory in a controlled cortical impact mouse model of traumatic brain injury.

The secondary phase of traumatic brain injury (TBI) is partly caused by the release of excess reactive oxygen species (ROS) from the primary injury. However, there are currently no therapies that have been shown to reduce the secondary spread of injury beyond the primary insult. Nanoparticles offer the ability to rapidly accumulate and be retained in injured brain for improved target engagement. Here, we utilized systemically administered antioxidant thioether core-cross-linked nanoparticles (NP1) that scavenge and inactivate ROS to reduce this secondary spread of injury in a mild controlled cortical impact (CCI) mouse model of TBI. We found that NP1 treatment protected CCI mice from injury induced learning and memory deficits observed in the Morris water maze (MWM) test at 1-month post-CCI. This protection was likely a result of NP1-mediated reduction in oxidative stress in the ipsilateral hemisphere as determined by immunofluorescence imaging of markers of oxidative stress and the spread of neuroinflammation into the contralateral hippocampus as determined by immunofluorescence imaging of activated microglia and neuron-astrocyte-microglia triad formation. These data suggest NP1-mediated reduction in post-traumatic oxidative stress correlates with the reduction in the spread of injury to the contralateral hippocampus to protect spatial memory and learning in CCI mice. Therefore, these materials may offer an improved treatment strategy to reduce the secondary spread of TBI.

The Nanotheranostic Researcher's Guide for Use of Animal Models of Traumatic Brain Injury.

Traumatic brain injury (TBI) is currently the leading cause of injury-related morbidity and mortality worldwide, with an estimated global cost of USD 400 billion annually. Both clinical and preclinical behavioral outcomes associated with TBI are heterogeneous in nature and influenced by the mechanism and frequency of injury. Previous literature has investigated this relationship through the development of animal models and behavioral tasks. However, recent advancements in these methods may provide insight into the translation of therapeutics into a clinical setting. In this review, we characterize various animal models and behavioral tasks to provide guidelines for evaluating the therapeutic efficacy of treatment options in TBI. We provide a brief review into the systems utilized in TBI classification and provide comparisons to the animal models that have been developed. In addition, we discuss the role of behavioral tasks in evaluating outcomes associated with TBI. Our goal is to provide those in the nanotheranostic field a guide for selecting an adequate TBI animal model and behavioral task for assessment of outcomes to increase research in this field.

Ultrasmall Mixed Eu-Gd Oxide Nanoparticles for Multimodal Fluorescence and Magnetic Resonance Imaging of Passive Accumulation and Retention in TBI.

Traumatic brain injury (TBI) is a leading cause of death and disability worldwide. TBI can have a long-term impact on the quality of life for survivors of all ages. However, there remains no approved treatment that improves outcomes following TBI, which is partially due to poor delivery of therapies into the brain. Therefore, there is a significant unmet need to develop more effective delivery strategies that increase the accumulation and retention of potentially efficacious treatments in the injured brain. Recent work has revealed that nanoparticles (NPs) may offer a promising approach for site-specific delivery; however, a detailed understanding of the specific NP properties that promote brain accumulation and retention are still being developed. Multimodal imaging plays a vital role in the understanding of physicochemical properties that initiate the uptake and accumulation of NPs in the brain at both high spatial (e.g., fluorescence imaging) and temporal (e.g., magnetic resonance imaging, MRI) frequency. However, many NP systems that are currently used in TBI only provide contrast in a single imaging modality limiting the imaging data that can be obtained, and those that offer multimodal imaging capabilities have complicated multistep synthesis methods. Therefore, the goal of this work was to develop an ultrasmall NP with simple fabrication capable of multimodal imaging. Here, we describe the development, characterization, accumulation, and retention of poly(ethylene glycol) (PEG)-coated europium-gadolinium (Eu-Gd) mixed magnetic NPs (MNPs) in a controlled cortical impact mouse model of TBI. We find that these NPs having an ultrasmall core size of 2 nm and a small hydrodynamic size of 13.5 nm can be detected in both fluorescence and MR imaging modalities and rapidly accumulate and are retained in injured brain parenchyma. These NPs should allow for further testing of NP physicochemical properties that promote accumulation and retention in TBI and other disease models.

Evaluating differential nanoparticle accumulation and retention kinetics in a mouse model of traumatic brain injury via Ktrans mapping with MRI.

Traumatic brain injury (TBI) is a leading cause of injury-related death worldwide, yet there are no approved neuroprotective therapies that improve neurological outcome post-injury. Transient opening of the blood-brain barrier following injury provides an opportunity for passive accumulation of intravenously administered nanoparticles through an enhanced permeation and retention-like effect. However, a thorough understanding of physicochemical properties that promote optimal uptake and retention kinetics in TBI is still needed. In this study, we present a robust method for magnetic resonance imaging of nanoparticle uptake and retention kinetics following intravenous injection in a controlled cortical impact mouse model of TBI. Three contrast-enhancing nanoparticles with different hydrodynamic sizes and relaxivity properties were compared. Accumulation and retention were monitored by modelling the permeability coefficient, Ktrans, for each nanoparticle within the reproducible mouse model. Quantification of Ktrans for different nanoparticles allowed for non-invasive, multi-time point assessment of both accumulation and retention kinetics in the injured tissue. Using this method, we found that 80 nm poly(lactic-co-glycolic acid) nanoparticles had maximal Ktrans in a TBI when injected 3 hours post-injury, showing significantly higher accumulation kinetics than the small molecule, Gd-DTPA. This robust method will enable optimization of administration time and nanoparticle physicochemical properties to achieve maximum delivery.