The unique properties of Big tau in the visual system.

Tau is a microtubule associated protein that plays important roles in regulating the properties of microtubules and axonal transport, as well as tauopathies associated with toxic aggregates leading to neurodegenerative diseases. It is encoded by the MAPT gene forming multiple isoforms (45-60 kDa) by alternative splicing which are developmentally regulated. The high molecular weight (MW) tau isoform of 105 kDa, termed Big tau, was originally discovered in the peripheral nervous system (PNS) but later found in selective CNS areas. It contains an additional large exon 4a generating a long projecting domain of about 250 amino acids. Here we investigated the properties of Big tau in the visual system of rats, its distribution in retinal ganglion cells and the optic nerve as well as its developmental regulation using biochemical, molecular and histological analyses. We discovered that Big tau is expresses as a 95 kDa protein (termed middle MW) containing exons 4a, 6 as well as exon 10 which defines a 4 microtubule-binding repeats (4R). It lacks exons 2/3 but shares the extensive phosphorylation characteristic of other tau isoforms. Importantly, early in development the visual system expresses only the low MW isoform (3R) switching to both the low and middle MW isoforms (4R) in adult retinal ganglion neurons and their corresponding axons. This is a unique structure and expression pattern of Big tau, which we hypothesize is associated with the specific properties of the visual system different from what has been previously described in the PNS and other areas of the nervous system.

Matching mechanical heterogeneity of the native spinal cord augments axon infiltration in 3D-printed scaffolds.

Scaffolds delivered to injured spinal cords to stimulate axon connectivity often match the anisotropy of native tissue using guidance cues along the rostral-caudal axis, but current approaches do not mimic the heterogeneity of host tissue mechanics. Although white and gray matter have different mechanical properties, it remains unclear whether tissue mechanics also vary along the length of the cord. Mechanical testing performed in this study indicates that bulk spinal cord mechanics do differ along anatomical level and that these differences are caused by variations in the ratio of white and gray matter. These results suggest that scaffolds recreating the heterogeneity of spinal cord tissue mechanics must account for the disparity between gray and white matter. Digital light processing (DLP) provides a means to mimic spinal cord topology, but has previously been limited to printing homogeneous mechanical properties. We describe a means to modify DLP to print scaffolds that mimic spinal cord mechanical heterogeneity caused by variation in the ratio of white and gray matter, which improves axon infiltration compared to controls exhibiting homogeneous mechanical properties. These results demonstrate that scaffolds matching the mechanical heterogeneity of white and gray matter improve the effectiveness of biomaterials transplanted within the injured spinal cord.

Regulation of Tau Expression in Superior Cervical Ganglion (SCG) Neurons In Vivo and In Vitro.

The superior cervical ganglion (SCG) is part of the autonomic nervous system providing sympathetic innervation to the head and neck, and has been regularly used to prepare postnatal neuronal cultures for cell biological studies. We found that during development these neurons change tau expression from the low molecular weight (LMW) isoforms to Big tau, with the potential to affect functions associated with tau such as microtubule dynamic and axonal transport. Big tau contains the large 4a exon that transforms tau from LMW isoforms of 45-60 kDa to 110 kDa. We describe tau expression during postnatal development reporting that the transition from LMW tau to Big tau which started at late embryonic stages is completed by about 4-5 weeks postnatally. We confirmed the presence of Big tau in dissociated postnatal SCG neurons making them an ideal system to study the function of Big tau in neurons. We used SCG explants to examine the response of SCG neurons to lesion and found that Big tau expression returned gradually along the regrowing neurites suggesting that it does not drives regeneration, but facilitates the structure/function of mature SCG neurons. The structural/functional roles of Big tau remain unknown, but it is intriguing that neurons that express Big tau appear less vulnerable to tauopathies.

Magnetic alignment of injectable hydrogel scaffolds for spinal cord injury repair.

Injectable hydrogels for cell delivery and tissue regeneration have several advantages over pre-fabricated scaffolds that require more invasive transplantation procedures, but lack the ability to implement tunable topologies. Here, we describe an approach to create patternable and injectable scaffolds using magnetically-responsive (MR) self-assembling peptide hydrogels, and validate their efficacy to promote and align axon infiltration at the site of a spinal cord injury. In vitro experiments reveal the parameters needed to align the fibers using the application of an external magnetic field. These results indicate that applying a 100-Gauss (G) field to the peptide hydrogels during polymerization causes fiber alignment as measured by electron microscopy, even in the presence of cells. In order to mimic infiltrating axons, neural progenitor cells (NPCs) are seeded on the surface of peptide hydrogels to interrogate the effects of both magnetic alignment and embedding human mesenchymal stem cells (hMSCs) in the scaffold. NPCs infiltrate peptide hydrogels seeded with hMSCs, and exhibit increased alignment and elongation in aligned gels. In order to evaluate these injectable and patternable scaffolds in vivo, hMSC-seeded peptide hydrogels are injected at the site of a contusion spinal cord injury with and without the presence of a magnetic field to align the resulting fibrous network. Measurements of axon growth and orientation as well as inflammation and glial scar formation indicate that these metrics are improved in magnetically aligned hMSC-seeded hydrogels. The results verify that MR hydrogels can dictate the orientation of infiltrating axons, providing a viable means to control the topology of injectable scaffolds.

Transplanting Neural Progenitor Cells into a Chronic Dorsal Column Lesion Model.

Cell transplantation therapy is a promising strategy for spinal cord injury (SCI) repair. Despite advancements in the development of therapeutic strategies in acute and subacute SCI, much less is known about effective strategies for chronic SCI. In previous studies we demonstrated that transplants of neural progenitor cells (NPC) created a permissive environment for axon regeneration and formed a neuronal relay across the injury following an acute dorsal column injury. Here we explored the efficacy of such a strategy in a chronic injury. We tested two preparations of NPCs derived from rat spinal cord at embryonic day 13.5: one prepared using stocks of cultured cells and the other of dissociated cells transplanted without culturing. Transplantation was delayed for 4-, 6- and 12-weeks post injury for a chronic injury model. We found that the dissociated NPC transplants survived and proliferated for at least 5 weeks post transplantation, in contrast to the poor survival of transplants prepared from cultured NPC stocks. The dissociated NPC transplants differentiated into neurons expressing excitatory markers, promoted axon regeneration into the injury/transplant site and extended axons from graft-derived neurons into the host. These results support the potential of NPC transplants to form neuronal relays across a chronic SCI, but they also underscore the challenges of achieving efficient cell survival in the environment of a chronic injury.

Locomotor deficits induced by lumbar muscle inflammation involve spinal microglia and are independent of KCC2 expression in a mouse model of complete spinal transection.

Spinal cord injury (SCI) is associated with damage to musculoskeletal tissues of the spine. Recent findings show that pain and inflammatory processes caused by musculoskeletal injury mediate plastic changes in the spinal cord. These changes could impede the adaptive plastic changes responsible for functional recovery. The underlying mechanism remains unclear, but may involve the microglia-BDNF-KCC2 pathway, which is implicated in sensitization of dorsal horn neurons in neuropathic pain and in the regulation of spinal excitability by step-training. In the present study, we examined the effects of step-training and lumbar muscle inflammation induced by complete Freund's adjuvant (CFA) on treadmill locomotion in a mouse model of complete spinal transection. The impact on locomotor recovery of each of these interventions alone or in combination were examined in addition to changes in microglia and KCC2 expression in the dorsal and ventral horns of the sublesional spinal cord. Results show that angular motion at the hip, knee and ankle joint during locomotion were decreased by CFA injection and improved by step-training. Moreover, CFA injection enhanced the expression of the microglial marker Iba1 in both ventral and dorsal horns, with or without step-training. However, this change was not associated with a modulation of KCC2 expression, suggesting that locomotor deficits induced by inflammation are independent of KCC2 expression in the sublesional spinal cord. These results indicate that musculoskeletal injury hinders locomotor recovery after SCI and that microglia is involved in this effect.

Vascularization of self-assembled peptide scaffolds for spinal cord injury repair.

The disruption of the blood-spinal cord barrier (BSCB) following spinal cord injury contributes to inflammation and glial scarring that inhibits axon growth and diminishes the effectiveness of conduits transplanted to the injury site to promote this growth. The purpose of this study is to evaluate whether scaffolds containing microvessels that exhibit BSCB integrity reduce inflammation and scar formation at the injury site and lead to increased axon growth. For these studies, a self-assembling peptide scaffold, RADA-16I, is used due to its established permissiveness to axon growth and ability to support vascularization. Immunocytochemistry and permeability transport assays verify the formation of tight-junction containing microvessels within the scaffold. Peptide scaffolds seeded with different concentrations of microvascular cells are then injected into a spinal contusion injury in rats to evaluate how microvessels affect axon growth and neurovascular interaction. The effect of the vascularized scaffold on inflammation and scar formation is evaluated by quantifying histological sections stained with ED-1 and GFAP, respectively. Our results indicate that the peptide scaffolds containing microvessels reduce inflammation and glial scar formation and increase the density of axons growing into the injury/transplant site. These results demonstrate the potential benefit of scaffold vascularization to treat spinal cord injury. STATEMENT OF SIGNIFICANCE: This study evaluates the benefit of transplanting microvascular cells within a self-assembling peptide scaffold, RADA-16I, that has shown promise for facilitating regeneration in the central nervous system in previous studies. Our results indicate that vasculature featuring tight junctions that give rise to the blood-spinal cord barrier can be formed within the peptide scaffold both in vitro and in a rat model of a subacute contusion spinal cord injury. Histological analysis indicates that the presence of the microvessels encourages axon infiltration into the site of injury and reduces the area of astrocyte activation and inflammation. Overall, these results demonstrate the potential of vascularizing scaffolds for the repair of spinal cord injury.

Harnessing neurovascular interaction to guide axon growth.

Regulating the intrinsic interactions between blood vessels and nerve cells has the potential to enhance repair and regeneration of the central nervous system. Here, we evaluate the efficacy of aligned microvessels to induce and control directional axon growth from neural progenitor cells in vitro and host axons in a rat spinal cord injury model. Interstitial fluid flow aligned microvessels generated from co-cultures of cerebral-derived endothelial cells and pericytes in a three-dimensional scaffold. The endothelial barrier function was evaluated by immunostaining for tight junction proteins and quantifying the permeability coefficient (~10-7 cm/s). Addition of neural progenitor cells to the co-culture resulted in the extension of Tuj-positive axons in the direction of the microvessels. To validate these findings in vivo, scaffolds were transplanted into an acute spinal cord hemisection injury with microvessels aligned with the rostral-caudal direction. At three weeks post-surgery, sagittal sections indicated close alignment between the host axons and the transplanted microvessels. Overall, this work demonstrates the efficacy of exploiting neurovascular interaction to direct axon growth in the injured spinal cord and the potential to use this strategy to facilitate central nervous system regeneration.

Glial restricted precursors maintain their permissive properties after long-term expansion but not following exposure to pro-inflammatory factors.

Glial restricted precursors (GRP) are a promising cellular source for transplantation therapy of spinal cord injury (SCI), capable of creating a permissive environment for axonal growth and regeneration. However, there are several issues regarding the nature of their permissive properties that remain unexplored. For example, cellular transplantation strategies for spinal cord repair require the preparation of a large number of cells, but it is unknown whether the permissive properties of GRP are maintained following the process of in vitro expansion. We used rat GRP isolated from the embryonic day 13.5 spinal cord to compare the properties of early (10-20 days) and late (120-140 days) passage GRP. We found that late passage GRP showed comparable effects on neurite outgrowth of adult rat DRG to early passage GRP in both in vitro co-culture and conditioned medium experiments. In addition, to further examine the effects of the inflammatory cascade activated in the aftermath of SCI on the microenvironment, we studied the direct effects of strong inflammatory mediators, Lipopolysaccharide and interferon gamma (LPS and IFNɤ, respectively), on the properties of GRP. We showed that exposure to these pro-inflammatory mediators altered GRP phenotype and attenuated their growth-promoting effects on neurite outgrowth in a dose dependent manner. Taken together, our data suggest that GRP maintain their growth-promoting properties following extensive in vitro passaging and underscore the importance of modulating the inflammatory environment at the injured spinal cord.

Evaluation of the anatomical and functional consequences of repetitive mild cervical contusion using a model of spinal concussion.

Spinal cord concussion is characterized by a transient loss of motor and sensory function that generally resolves without permanent deficits. Spinal cord concussions usually occur during vehicular accidents, falls, and sport activity, but unlike brain concussions, have received much less attention despite the potential for repeated injury leading to permanent neurological sequelae. Consequently, there is no consensus regarding decisions related to return to play following an episode of spinal concussion, nor an understanding of the short- and long-term consequences of repeated injury. Importantly, there are no models of spinal concussion to study the anatomical and functional sequelae of single or repeated injury. We have developed a new model of spinal cord concussion focusing on the anatomical and behavioral outcomes of single and repeated injury. Rats received a very mild (50 kdyn, IH impactor) spinal contusion at C5 and were separated into two groups three weeks after the initial injury--C1, which received a second, sham surgery, and C2, which received a second contusion at the same site. To track motor function and recovery, animals received weekly behavioral tests--BBB, CatWalk™, cylinder, and Von Frey. Analysis of locomotor activity by BBB demonstrated that rats rapidly recovered, regaining near-normal function by one week after the first and second injury, which was confirmed using the more detailed CatWalk™ analysis. The cylinder test showed that a single contusion did not induce significant deficits of the affected limb, but that repeated injury resulted in significant alteration in paw preference, with animals favoring the unaffected limb. Intriguingly, Von Frey analysis demonstrated an increased sensitivity in the contralateral hindlimb in the C2 group vs. the C1 group. Anatomical analyses revealed that while the lesion volume of both groups was minimal, the area of spared white matter in the C2 group was significantly reduced 1 and 2mm rostral to the lesion epicenter. Reactive astrocytes were present in both groups, with the majority found at the lesion epicenter in the C1 group, whereas the C2 group demonstrated increased reactive astrocytes extending 1mm caudal to the lesion epicenter. Macrophages accumulated within the injured, dorsal and ipsilateral spinal cord, with significant increases at 2 and 3mm rostral to the epicenter in the C2 group. Our model is designed to represent the clinical presentation of spinal cord concussion, and highlight the susceptibility and functional sequelae of repeated injury. Future experiments will examine the temporal and spatial windows of vulnerability for repeated injuries.

Behavioral and anatomical consequences of repetitive mild thoracic spinal cord contusion injury in the rat.

Moderate and severe spinal cord contusion injuries have been extensively studied, yet much less is known about mild injuries. Mild contusions result in transient functional deficits, proceeding to near-complete recovery, but they may render the spinal cord vulnerable to future injuries. However, to date there have been no appropriate models to study the behavioral consequences, anatomical changes, and susceptibility of a mild contusion to repeated injuries, which may occur in children as well as adults during competitive sport activities. We have developed a novel mild spinal cord contusion injury model characterized by a sequence of transient functional deficits after the first injury and restoration to near-complete motor and sensory function, which is then followed up by a second injury. This model can serve not only to study the effects of repeated injuries on behavioral and anatomical changes, but also to examine the relationship between successive tissue damage and recovery of function. In the present study, we confirmed that mild thoracic spinal cord contusion, utilizing the NYU impactor device, resulted in localized tissue damage, characterized by a cystic cavity and peripheral rim of spared white matter at the injury epicenter, and rapid functional recovery to near-normal levels utilizing several behavioral tests. Repeated injury after 3weeks, when functional recovery has been completed, resulted in worsening of both motor and sensory function, which did not recover to prior levels. Anatomical analyses showed no differences in the volumes of spared white matter, lesion, or cyst, but revealed modest extension of lesion area rostral to the injury epicenter as well as an increase in inflammation and apoptosis. These studies demonstrate that a mild injury model can be used to test efficacy of treatments for repeated injuries and may serve to assist in the formulation of policies and clinical practice regarding mild SCI injury and spinal concussion.

Exogenous BDNF enhances the integration of chronically injured axons that regenerate through a peripheral nerve grafted into a chondroitinase-treated spinal cord injury site.

Although axons lose some of their intrinsic capacity for growth after their developmental period, some axons retain the potential for regrowth after injury. When provided with a growth-promoting substrate such as a peripheral nerve graft (PNG), severed axons regenerate into and through the graft; however, they stop when they reach the glial scar at the distal graft-host interface that is rich with inhibitory chondroitin sulfate proteoglycans. We previously showed that treatment of a spinal cord injury site with chondroitinase (ChABC) allows axons within the graft to traverse the scar and reinnervate spinal cord, where they form functional synapses. While this improvement in outgrowth was significant, it still represented only a small percentage (<20%) of axons compared to the total number of axons that regenerated into the PNG. Here we tested whether providing exogenous brain-derived neurotrophic factor (BDNF) via lentivirus in tissue distal to the PNG would augment regeneration beyond a ChABC-treated glial interface. We found that ChABC treatment alone promoted axonal regeneration but combining ChABC with BDNF-lentivirus did not increase the number of axons that regenerated back into spinal cord. Combining BDNF with ChABC did increase the number of spinal cord neurons that were trans-synaptically activated during electrical stimulation of the graft, as indicated by c-Fos expression, suggesting that BDNF overexpression improved the functional significance of axons that did reinnervate distal spinal cord tissue.

Transplantation of human glial restricted progenitors and derived astrocytes into a contusion model of spinal cord injury.

Transplantation of neural progenitors remains a promising therapeutic approach to spinal cord injury (SCI), but the anatomical and functional evaluation of their effects is complex, particularly when using human cells. We investigated the outcome of transplanting human glial-restricted progenitors (hGRP) and astrocytes derived from hGRP (hGDA) in spinal cord contusion with respect to cell fate and host response using athymic rats to circumvent xenograft immune issues. Nine days after injury hGRP, hGDA, or medium were injected into the lesion center and rostral and caudal to the lesion, followed by behavioral testing for 8 weeks. Both hGRP and hGDA showed robust graft survival and extensive migration. The total number of cells increased 3.5-fold for hGRP, and twofold for hGDA, indicating graft expansion, but few proliferating cells remained by 8 weeks. Grafted cells differentiated into glia, predominantly astrocytes, and few remained at progenitor state. About 80% of grafted cells around the injury were glial fibrillary acidic protein (GFAP)-positive, gradually decreasing to 40-50% at a distance of 6 mm. Conversely, there were few graft-derived oligodendrocytes at the lesion, but their numbers increased away from the injury to 30-40%. Both cell grafts reduced cyst and scar formation at the injury site compared to controls. Microglia/macrophages were present at and around the lesion area, and axons grew along the spared tissue with no differences among groups. There were no significant improvements in motor function recovery as measured by the Basso, Beattie, and Bresnahan (BBB) scale and grid tests in all experimental groups. Cystometry revealed that hGRP grafts attenuated hyperactive bladder reflexes. Importantly, there was no increased sensory or tactile sensitivity associated with pain, and the hGDA group showed sensory function returning to normal. Although the improved lesion environment was not sufficient for robust functional recovery, the permissive properties and lack of sensory hypersensitivity indicate that human GRP and astrocytes remain promising candidates for therapy after SCI.