Persistence of Hyper-Ramified Microglia in Porcine Cortical Gray Matter after Mild Traumatic Brain Injury.

Traumatic brain injury (TBI) is a major contributor to morbidity and mortality in the United States as several million people visit the emergency department every year due to TBI exposures. Unfortunately, there is still no consensus on the pathology underlying mild TBI, the most common severity sub-type of TBI. Previous preclinical and post-mortem human studies have detailed the presence of diffuse axonal injury following TBI, suggesting that white matter pathology is the predominant pathology of diffuse brain injury. However, the inertial loading produced by TBI results in strain fields in both gray and white matter. In order to further characterize gray matter pathology in mild TBI, our lab used a pig model (n = 25) of closed-head rotational acceleration-induced TBI to evaluate blood-brain barrier disruptions, neurodegeneration, astrogliosis, and microglial reactivity in the cerebral cortex out to 1 year post-injury. Immunohistochemical staining revealed the presence of a hyper-ramified microglial phenotype-more branches, junctions, endpoints, and longer summed process length-at 30 days post injury (DPI) out to 1 year post injury in the cingulate gyrus (p < 0.05), and at acute and subacute timepoints in the inferior temporal gyrus (p < 0.05). Interestingly, we did not find neuronal loss or astroglial reactivity paired with these chronic microglia changes. However, we observed an increase in fibrinogen reactivity-a measure of blood-brain barrier disruption-predominately in the gray matter at 3 DPI (p = 0.0003) which resolved to sham levels by 7 DPI out to chronic timepoints. Future studies should employ gene expression assays, neuroimaging, and behavioral assays to elucidate the effects of these hyper-ramified microglia, particularly related to neuroplasticity and responses to potential subsequent insults. Further understanding of the brain's inflammatory activity after mild TBI will hopefully provide understanding of pathophysiology that translates to clinical treatment for TBI.

Vector Affinity and Receptor Distribution Define Tissue-Specific Targeting in an Engineered AAV Capsid.

Unbiased in vivo selections of diverse capsid libraries can yield engineered capsids that overcome gene therapy delivery challenges like traversing the blood-brain barrier (BBB), but little is known about the parameters of capsid-receptor interactions that govern their improved activity. This hampers broader efforts in precision capsid engineering and is a practical impediment to ensuring the translatability of capsid properties between preclinical animal models and human clinical trials. In this work, we utilize the adeno-associated virus (AAV)-PHP.B-Ly6a model system to better understand the targeted delivery and BBB penetration properties of AAV vectors. This model offers a defined capsid-receptor pair that can be used to systematically define relationships between target receptor affinity and in vivo activity of engineered AAV vectors. Here, we report a high-throughput method for quantifying capsid-receptor affinity and demonstrate that direct binding assays can be used to organize a vector library into families with varied affinity for their target receptor. Our data indicate that efficient central nervous system transduction requires high levels of target receptor expression at the BBB, but it is not a requirement for receptor expression to be limited to the target tissue. We observed that enhanced receptor affinity leads to reduced transduction of off-target tissues but can negatively impact on-target cellular transduction and penetration of endothelial barriers. Together, this work provides a set of tools for defining vector-receptor affinities and demonstrates how receptor expression and affinity interact to impact the performance of engineered AAV vectors in targeting the central nervous system. IMPORTANCE Novel methods for measuring adeno-associated virus (AAV)-receptor affinities, especially in relation to vector performance in vivo, would be useful to capsid engineers as they develop AAV vectors for gene therapy applications and characterize their interactions with native or engineered receptors. Here, we use the AAV-PHP.B-Ly6a model system to assess the impact of receptor affinity on the systemic delivery and endothelial penetration properties of AAV-PHP.B vectors. We discuss how receptor affinity analysis can be used to isolate vectors with optimized properties, improve the interpretation of library selections, and ultimately translate vector activities between preclinical animal models and humans.

Concussion leads to widespread axonal sodium channel loss and disruption of the node of Ranvier.

Despite being a major health concern, little is known about the pathophysiological changes that underly concussion. Nonetheless, emerging evidence suggests that selective damage to white matter axons, or diffuse axonal injury (DAI), disrupts brain network connectivity and function. While voltage-gated sodium channels (NaChs) and their anchoring proteins at the nodes of Ranvier (NOR) on axons are key elements of the brain's network signaling machinery, changes in their integrity have not been studied in context with DAI. Here, we utilized a clinically relevant swine model of concussion that induces evolving axonal pathology, demonstrated by accumulation of amyloid precursor protein (APP) across the white matter. Over a two-week follow-up post-concussion with this model, we found widespread loss of NaCh isoform 1.6 (Nav1.6), progressive increases in NOR length, the appearance of void and heminodes and loss of ßIV-spectrin, ankyrin G, and neurofascin 186 or their collective diffusion into the paranode. Notably, these changes were in close proximity, yet distinct from APP-immunoreactive swollen axonal profiles, potentially representing a unique, newfound phenotype of axonal pathology in DAI. Since concussion in humans is non-fatal, the clinical relevance of these findings was determined through examination of post-mortem brain tissue from humans with higher levels of acute traumatic brain injury. Here, a similar loss of Nav1.6 and changes in NOR structures in brain white matter were observed as found in the swine model of concussion. Collectively, this widespread and progressive disruption of NaChs and NOR appears to be a form of sodium channelopathy, which may represent an important substrate underlying brain network dysfunction after concussion.

A Preliminary Report: The Hippocampus and Surrounding Temporal Cortex of Patients With Schizophrenia Have Impaired Blood-Brain Barrier.

Though hippocampal volume reduction is a pathological hallmark of schizophrenia, the molecular pathway(s) responsible for this degeneration remains unknown. Recent reports have suggested the potential role of impaired blood-brain barrier (BBB) function in schizophrenia pathogenesis. However, direct evidence demonstrating an impaired BBB function is missing. In this preliminary study, we used immunohistochemistry and serum immunoglobulin G (IgG) antibodies to investigate the state of BBB function in formalin-fixed postmortem samples from the hippocampus and surrounding temporal cortex of patients with schizophrenia (n = 25) and controls without schizophrenia (n = 27) matched for age, sex, and race. Since a functional BBB prevents the extravasation of IgGs, detection of IgGs in the parenchyma is used as direct evidence of BBB breakdown. We also developed a semi-quantitative approach to quantify the extent of IgG leak and therein BBB breach. Analysis of our immunohistochemistry data demonstrated a significantly higher incidence of IgG leak in patients with schizophrenia compared to controls. Further, BBB permeability was significantly higher in advanced-age patients with schizophrenia than both advanced-age controls and middle-aged patients with schizophrenia. Male patients with schizophrenia also demonstrated a significant increase in IgG permeability compared to control males. Interestingly, the extravasated IgGs also demonstrated selective immunoreactivity for neurons. Based on these observations, we suggest that BBB dysfunction and IgG autoantibodies could be two key missing pathoetiological links underwriting schizophrenia hippocampal damage.

Postirradiation Necrosis after Slow Microvascular Breakdown in the Adult Rat Spinal Cord is Delayed by Minocycline Treatment.

To better understand the spatiotemporal course of radiation-induced central nervous system (CNS) vascular necrosis and assess the therapeutic potential of approaches for protecting against radiation-induced necrosis, adult female Sprague Dawley rats received 40 Gy surface dose centered on the T9 thoracic spinal cord segment. Locomotor function, blood-spinal cord barrier (BSCB) integrity and histology were evaluated throughout the study. No functional symptoms were observed for several months postirradiation. However, a sudden onset of paralysis was observed at approximately 5.5 months postirradiation. The progression rapidly led to total paralysis and death within less than 48 h of symptom onset. Open-field locomotor scores and rotarod motor coordination testing showed no evidence of neurological impairment prior to the onset of overt paralysis. Histological examination revealed minimal changes to the vasculature prior to symptom onset. However, Evans blue dye (EvB) extravasation revealed a progressive deterioration of BSCB integrity, beginning at one week postirradiation, affecting regions well outside of the irradiated area. Minocycline treatment significantly delayed the onset of paralysis. The results of this study indicate that extensive asymptomatic disruption of the blood-CNS barrier may precede onset of vascular breakdown by several months and suggests that minocycline treatment has a therapeutic effect by delaying radiation-induced necrosis after CNS irradiation.

Intravenously delivered mesenchymal stem cell-derived exosomes target M2-type macrophages in the injured spinal cord.

In a previous report we showed that intravenous infusion of bone marrow-derived mesenchymal stem cells (MSCs) improved functional recovery after contusive spinal cord injury (SCI) in the non-immunosuppressed rat, although the MSCs themselves were not detected at the spinal cord injury (SCI) site [1]. Rather, the MSCs lodged transiently in the lungs for about two days post-infusion. Preliminary studies and a recent report [2] suggest that the effects of intravenous (IV) infusion of MSCs could be mimicked by IV infusion of exosomes isolated from conditioned media of MSC cultures (MSCexos). In this study, we assessed the possible mechanism of MSCexos action on SCI by investigating the tissue distribution and cellular targeting of DiR fluorescent labeled MSCexos at 3 hours and 24 hours after IV infusion in rats with SCI. The IV delivered MSCexos were detected in contused regions of the spinal cord, but not in the noninjured region of the spinal cord, and were also detected in the spleen, which was notably reduced in weight in the SCI rat, compared to control animals. DiR "hotspots" were specifically associated with CD206-expressing M2 macrophages in the spinal cord and this was confirmed by co-localization with anti-CD63 antibodies labeling a tetraspanin characteristically expressed on exosomes. Our findings that MSCexos specifically target M2-type macrophages at the site of SCI, support the idea that extracellular vesicles, released by MSCs, may mediate at least some of the therapeutic effects of IV MSC administration.

Chronic TNFα Exposure Induces Robust Proliferation of Olfactory Ensheathing Cells, but not Schwann Cells.

TNFα is persistently elevated in many injury and disease conditions. Previous reports of cytotoxicity of TNFα for oligodendrocytes and their progenitors suggest that the poor endogenous remyelination in patients with traumatic injury or multiple sclerosis may be due in part to persistent inflammation. Understanding the effects of inflammatory cytokines on potential cell therapy candidates is therefore important for evaluating the feasibility of their use. In this study, we assessed the effects of long term exposure to TNFα on viability, proliferation, migration and TNFα receptor expression of cultured rat olfactory ensheathing cells (OECs) and Schwann cells (SCs). Although OECs and SCs transplanted into the CNS produce similar myelinating phenotypes, and might be expected to have similar therapeutic uses, we report that they have very different sensitivities to TNFα. OECs exhibited positive proliferative responses to TNFα over a much broader range of concentrations than SCs. Low TNFα concentrations increased proliferation and migration of both OECs and SCs, but SC number declined in the presence of 100 ng/ml or higher concentrations of TNFα. In contrast, OECs exhibited enhanced proliferation even at high TNFα concentrations (up to 1 µg/ml) and showed no evidence of TNF cytotoxicity even at 4 weeks post-treatment. Furthermore, while both OECs and SCs expressed TNFαR1 and TNFαR2, TNFα receptor levels were downregulated in OECs after exposure to100 ng/ml TNFα for 5-7 days, but were either elevated or unchanged in SCs. These results imply that OECs may be a more suitable cell therapy candidate if transplanted into areas with persistent inflammation.

Diffuse and persistent blood-spinal cord barrier disruption after contusive spinal cord injury rapidly recovers following intravenous infusion of bone marrow mesenchymal stem cells.

Intravenous infusion of mesenchymal stem cells (MSCs) has been shown to reduce the severity of experimental spinal cord injury (SCI), but mechanisms are not fully understood. One important consequence of SCI is damage to the microvasculature and disruption of the blood spinal cord barrier (BSCB). In the present study we induced a contusive SCI at T9 in the rat and studied the effects of intravenous MSC infusion on BSCB permeability, microvascular architecture and locomotor recovery over a 10week period. Intravenously delivered MSCs could not be identified in the spinal cord, but distributed primarily to the lungs where they survived for a couple of days. Spatial and temporal changes in BSCB integrity were assessed by intravenous infusions of Evans blue (EvB) with in vivo and ex vivo optical imaging and spectrophotometric quantitation of EvB leakage into the parenchyma. SCI resulted in prolonged BSCB leakage that was most severe at the impact site but disseminated extensively rostral and caudal to the lesion over 6weeks. Contused spinal cords also showed an increase in vessel size, reduced vessel number, dissociation of pericytes from microvessels and decreases in von Willebrand factor (vWF) and endothelial barrier antigen (EBA) expression. In MSC-treated rats, BSCB leakage was reduced, vWF expression was increased and locomotor function improved beginning 1 week post-MSC infusion, i.e., 2weeks post-SCI. These results suggest that intravenously delivered MSCs have important effects on reducing BSCB leakage which could contribute to their therapeutic efficacy.

Acute demyelination disrupts the molecular organization of peripheral nervous system nodes.

Intraneurally injected lysolecithin causes both segmental and paranodal demyelination. In demyelinated internodes, axonal components of nodes fragment and disappear, glial and axonal paranodal and juxtaparanodal proteins no longer cluster, and axonal Kv1.1/Kv1.2 K+ channels move from the juxtaparanodal region to appose the remaining heminodes. In paranodal demyelination, a gap separates two distinct heminodes, each of which contains the molecular components of normal nodes; paranodal and juxtaparanodal proteins are properly localized. As in normal nodes, widened nodal regions contain little or no band 4.1B. Lysolecithin also causes "unwinding" of paranodes: The spiral of Schwann cell membrane moves away from the paranodes, but the glial and axonal components of septate-like junctions remain colocalized. Thus, acute demyelination has distinct effects on the molecular organization of the nodal, paranodal, and juxtaparanodal region, reflecting altered axon-Schwann cell interactions.

Recent progress on the molecular organization of myelinated axons.

The structure of myelinated axons was well described 100 years ago by Ramón y Cajal, and now their molecular organization is being revealed. The basal lamina of myelinating Schwann cells contains laminin-2, and their abaxonal/outer membrane contains two laminin-2 receptors, alpha6beta4 integrin and dystroglycan. Dystroglycan binds utrophin, a short dystrophin isoform (Dp116), and dystroglycan-related protein 2 (DRP2), all of which are part of a macromolecular complex. Utrophin is linked to the actin cytoskeleton, and DRP2 binds to periaxin, a PDZ domain protein associated with the cell membrane. Non-compact myelin--found at incisures and paranodes--contains adherens junctions, tight junctions, and gap junctions. Nodal microvilli contain F-actin, ERM proteins, and cell adhesion molecules that may govern the clustering of voltage-gated Na+ channels in the nodal axolemma. Na(v)1.6 is the predominant voltage-gated Na+ channel in mature nerves, and is linked to the spectrin cytoskeleton by ankyrinG. The paranodal glial loops contain neurofascin 155, which likely interacts with heterodimers composed of contactin and Caspr/paranodin to form septate-like junctions. The juxtaparanodal axonal membrane contains the potassium channels Kv1.1 and Kv1.2, their associated beta2 subunit, as well as Caspr2. Kv1.1, Kv1.2, and Caspr2 all have PDZ binding sites and likely interact with the same PDZ binding protein. Like Caspr, Caspr2 has a band 4.1 binding domain, and both Caspr and Caspr2 probably bind to the band 4.1 B isoform that is specifically found associated with the paranodal and juxtaparanodal axolemma. When the paranode is disrupted by mutations (in cgt-, contactin-, and Caspr-null mice), the localization of these paranodal and juxtaparanodal proteins is altered: Kv1.1, Kv1.2, and Caspr2 are juxtaposed to the nodal axolemma, and this reorganization is associated with altered conduction of myelinated fibers. Understanding how axon-Schwann interactions create the molecular architecture of myelinated axons is fundamental and almost certainly involved in the pathogenesis of peripheral neuropathies.

Genetic dysmyelination alters the molecular architecture of the nodal region.

We have examined the molecular organization of axons in the spinal cords of myelin-deficient (md) rats, which have profound CNS dysmyelination associated with oligodendrocyte cell death. Although myelin sheaths are rare, most large axons are at least partially surrounded by oligodendrocyte processes. At postnatal day 7 (P7), almost all node-like clusters of voltage-gated Na+ channels and ankyrinG are adjacent to axonal segments ensheathed by oligodendrocytes, but at P21, many node-like clusters are found in axonal segments that lack oligodendrocyte ensheathment. In P21 wild-type (WT) rats, the voltage-gated Na+ channels Na(v)1.2, Na(v)1.6, and Na(v)1.8, are found in different subpopulations of myelinated axons, and md rats have a similar distribution. The known molecular components of paranodes--contactin, Caspr, and neurofascin 155--are not clustered in md spinal cords, and no septate-like junctions between oligodendrocyte processes and axons are found by electron microscopy. Furthermore, Kv1.1 and Kv1.2 K+ channels are not spatially segregated from the node-like clusters of Na+ channels in md rats, in contrast to their WT littermates. These results suggest the following: node-like clusters of voltage-gated Na+ channels and ankyrinG form adjacent to ensheathed axonal segments even in the absence of a myelin sheath; these clusters persist after oligodendrocyte cell death; dysmyelination does not alter the expression of different nodal of voltage-gated Na+ channels; the absence of paranodes results in the mislocalization of neurofascin155, contactin, and Caspr, and the aberrant localization of Kv1.1 and Kv1.2.