Axons In Spinal Cord Research Articles

In vivo imaging in mouse spinal cord reveals that microglia prevent degeneration of injured axons

Microglia, the primary immune cells in the central nervous system, play a critical role in regulating neuronal function and fate through their interaction with neurons. Despite extensive research, the specific functions and mechanisms of microglia-neuron interactions remain incompletely understood. In this study, we demonstrate that microglia establish direct contact with myelinated axons at Nodes of Ranvier in the spinal cord of mice. The contact associated with neuronal activity occurs in a random scanning pattern. In response to axonal injury, microglia rapidly transform their contact into a robust wrapping form, preventing acute axonal degeneration from extending beyond the nodes. This wrapping behavior is dependent on the function of microglial P2Y12 receptors, which may be activated by ATP released through axonal volume-activated anion channels at the nodes. Additionally, voltage-gated sodium channels (NaV) and two-pore-domain potassium (K2P) channels contribute to the interaction between nodes and glial cells following injury, and inhibition of NaV delays axonal degeneration. Through in vivo imaging, our findings reveal a neuroprotective role of microglia during the acute phase of single spinal cord axon injury, achieved through neuron-glia interaction.

Effects of transcranial magnetic stimulation on axonal regeneration in the corticospinal tract of female rats with spinal cord injury

BackgroundThis study investigates the potential of transcranial magnetic stimulation (TMS) to enhance spinal cord axon regeneration by modulating corticospinal pathways and improving motor nerve function recovery in rats with spinal cord injury (SCI). New methodTMS is a non-invasive neuromodulation technique that generates a magnetic field to activate neurons in the brain, leading to depolarization and modulation of cortical activity. Initially utilized for brain physiology research, TMS has evolved into a diagnostic and prognostic tool in clinical settings, with increasing interest in its therapeutic applications. However, its potential for treating motor dysfunction in SCI has been underexplored. ResultsThe TMS intervention group exhibited significant improvements compared to the control group across behavioral assessments, neurophysiological measurements, pathological analysis, and immunological markers. Comparison with existing methodsUnlike most studies that focus on localized spinal cord injury or muscle treatments, this study leverages the non-invasive, painless, and highly penetrating nature of TMS to focus on the corticospinal tracts, exploring its therapeutic potential for SCI. ConclusionsTMS enhances motor function recovery in rats with SCI by restoring corticospinal pathway integrity and promoting axonal regeneration. These findings highlight TMS as a promising therapeutic option for SCI patients with currently limited treatment alternatives.

Tempol, a Superoxide Dismutase Mimetic, Inhibits Wallerian Degeneration Following Spinal Cord Injury by Preventing Glutathione Depletion and Aldose Reductase Activation.

Spinal cord contusion injury results in Wallerian degeneration of spinal cord axonal tracts, which are necessary for locomotor function. Axonal swelling and loss of axonal density at the contusion site, characteristic of Wallerian degeneration, commence within hours of injury. Tempol, a superoxide dismutase mimetic, was previously shown to reduce the loss of spinal cord white matter and improve locomotor function in an experimental model of spinal cord contusion, suggesting that tempol treatment might inhibit Wallerian degeneration of spinal cord axons. Here, we report that tempol partially inhibits Wallerian degeneration, resulting in improved locomotor recovery. We previously reported that Wallerian degeneration is reduced by inhibitors of aldose reductase (AR), which converts glucose to sorbitol in the polyol pathway. We observed that tempol inhibited sorbitol production in the injured spinal cord to the same extent as the AR inhibitor, sorbinil. Tempol also prevented post-contusion upregulation of AR (AKR1B10) protein expression within degenerating axons, as previously observed for AR inhibitors. Additionally, we hypothesized that tempol inhibits axonal degeneration by preventing loss of the glutathione pool due to polyol pathway activity. Consistent with our hypothesis, tempol treatment resulted in greater glutathione content in the injured spinal cord, which was correlated with increased expression and activity of gamma glutamyl cysteine ligase (γGCL; EC 6.3.2.2), the rate-limiting enzyme for glutathione synthesis. Administration of the γGCL inhibitor buthionine sulfoximine abolished all observed effects of tempol administration. Together, these results support a pathological role for polyol pathway activation in glutathione depletion, resulting in Wallerian degeneration after spinal cord injury (SCI). Interestingly, methylprednisolone, oxandrolone, and clenbuterol, which are known to spare axonal tracts after SCI, were equally effective in inhibiting polyol pathway activation. These results suggest that prevention of AR activation is a common target of many disparate post-SCI interventions.

Bioactive hydrogels loaded with BMSC-EXOs and GDNF for synergistically spinal cord injury repairing

Spinal cord injury (SCI) is a common lethal injury to the central nervous system (CNS), which severely affects the quality of life of SCI patients. Attempts to enhance the treatment of SCI typically tend to develop functional bioactive hydrogel scaffolds with drug encapsulation. In this work, we proposed novel RGD-functionalized bioactive hydrogel loaded with bone marrow stromal cells-derived exosomes (BMSC-EXOs) and glial cell line-derived neurotrophic factor (GDNF) to synergistically promote SCI repair. The functional hydrogel consisted of methacrylic anhydride-grafted sodium alginate and acrylated RGD, and then combined with BMSC-EXOs and GDNF by photocrosslinking to form a composite bioactive hydrogel. The hydrogel showed good drug-release properties and biocompatibility. When the bioactive hydrogel was implanted into the SCI area of the SD rats, it effectively modulated the adverse inflammatory response in the SCI area. Furthermore, the bioactive hydrogel promoted the regeneration of spinal cord neurons and axons, leading to the improvement in motor function recovery. These results indicate that the bioactive hydrogel loaded with BMSC-EXOs and GDNF has great potential as a therapeutic approach for the repair of spinal cord injuries.

Improvement of diabetes-induced spinal cord axon injury with taurine via nerve growth factor-dependent Akt/mTOR pathway

Diabetic neuropathy (DN) is a common neurological complication caused by diabetes mellitus (DM). Axonal degeneration is generally accepted to be the major pathological change in peripheral DN. Taurine has been evidenced to be neuroprotective in various aspects, but its effect on spinal cord axon injury (SCAI) in DN remains barely reported. This study showed that taurine significantly ameliorated axonal damage of spinal cord (SC), based on morphological and functional analyses, in a rat model of DN induced by streptozotocin (STZ). Taurine was also found to induce neurite outgrowth in cultured cerebral cortex neurons with high glucose exposure. Moreover, taurine up-regulated the expression of nerve growth factor (NGF) and neurite outgrowth relative protein GAP-43 in rat DN model and cultured cortical neurons/VSC4.1 cells. Besides, taurine increased the activating phosphorylation signals of TrkA, Akt, and mTOR. Mechanistically, the neuroprotection by taurine was related to the NGF–pAKT–mTOR axis, because either NGF-neutralizing antibody or Akt or mTOR inhibitors was found to attenuate its beneficial effects. Together, our results demonstrated that taurine promotes spinal cord axon repair in a model of SCAI in STZ-induced diabetic rats, mechanistically associating with the NGF-dependent activation of Akt/mTOR pathway.

Serum response factor promoting axonal regeneration by activating the Ras-Raf-Cofilin signaling pathway after the spinal cord injury.

Serum response factor (SRF) is important in muscle development, tissue repair, and neuronal regulation. This research aims to thoroughly examine the effects of SRF on spinal cord injury (SCI) and its ability to significantly impact the recovery and regeneration of neuronal axons. The researchers created rat models of SCI and scratch injury to primary spinal cord neurons to observe the expression of relevant factors after neuronal injury. We found that the SRF, Ras, Raf, and cofilin levels increased after injury and gradually returned to normal levels. Afterward, researchers gave rats with SCI an SRF inhibitor (CCG1423) and studied the effects with nuclear magnetic resonance and transmission electron microscopy. The SRF inhibitor rodents had worse spinal cord recovery and axon regrowth than the control group. And the apoptosis of primary neurons after scratch injury was significantly higher in the SRF inhibitor group. Additionally, the researchers utilized lentiviral transfection to modify the SRF expression in neurons. SRF overexpression increased neuron migration while silencing SRF decreased it. Finally, Western blotting and RT-PCR were conducted to examine the expression changes of related factors upon altering SRF expression. The results revealed SRF overexpression increased Ras, Raf, and cofilin expression. Silencing SRF decreased Ras, Raf, and Cofilin expression. Based on our research, the SRF promotes axonal regeneration by activating the "Ras-Raf-Cofilin" signaling pathway.

Spontaneous regeneration of cholecystokinergic reticulospinal axons after a complete spinal cord injury in sea lampreys

In contrast to humans, lampreys spontaneously recover their swimming capacity after a complete spinal cord injury (SCI). This recovery process involves the regeneration of descending axons. Spontaneous axon regeneration in lampreys has been mainly studied in giant descending neurons. However, the regeneration of neurochemically distinct descending neuronal populations with small-caliber axons, as those found in mammals, has been less studied. Cholecystokinin (CCK) is a regulatory neuropeptide found in the brain and spinal cord that modulates several processes such as satiety, or locomotion. CCK shows high evolutionary conservation and is present in all vertebrate species. Work in lampreys has shown that all CCKergic spinal cord axons originate in a single neuronal population located in the caudal rhombencephalon. Here, we investigate the spontaneous regeneration of CCKergic descending axons in larval lampreys following a complete SCI. Using anti-CCK-8 immunofluorescence, confocal microscopy and lightning adaptive deconvolution, we demonstrate the partial regeneration of CCKergic axons (81% of the number of axonal profiles seen in controls) 10 weeks after the injury. Our data also revealed a preference for regeneration of CCKergic axons in lateral spinal cord regions. Regenerated CCKergic axons exhibit colocalization with synaptic vesicle marker SV2, indicative of functional synaptic connections. We also extracted swimming dynamics in injured animals by using DeepLabCut. Interestingly, the degree of CCKergic reinnervation correlated with improved swimming performance in injured animals, suggesting a potential role in locomotor recovery. These findings open avenues for further exploration into the role of specific neuropeptidergic systems in post-SCI spinal locomotor networks.

Morphological analysis of descending tracts in mouse spinal cord using tissue clearing, tissue expansion and tiling light sheet microscopy techniques

Descending tracts carry motor signals from the brain to spinal cord. However, few previous studies show the full view of the long tracts from a 3D perspective. In this study, we have followed five less well-known tracts that project from midbrain, hindbrain, and cerebellum to the mouse spinal cord, using the tissue clearing method in combination with tiling light sheet microscopy. By tracing axons in spinal cord, we found several notable features: among the five tracts the collateral "sister" branches occurred only in the axons originating from the cerebellospinal tracts; the axons from the spinal trigeminal nucleus crossed the midline of spinal cord to the contralateral side; those arising in the medullary reticular formation ventral part gave many branches in both cervical and lumbar segments; the axons from superior colliculus terminated only at upper cervical but with abundant branches in the hindbrain. Furthermore, we investigated the monosynaptic connections between the tracts and motor neurons in the spinal cord through hydrogel-based tissue expansion, and found several monosynaptic connections between the medullary reticular formation ventral part axons and spinal motor neurons. We believe that this is the first study to show the full 3D scope of the projection patterns and axonal morphologies of these five descending tracts to the mouse spinal cord. In addition, we have developed a new method for future study of descending tracts by three-dimensional imaging.

Oligodendrocyte development and myelin sheath formation are regulated by the antagonistic interaction between the Rag-Ragulator complex and TFEB.

Myelination by oligodendrocytes is critical for fast axonal conduction and for the support and survival of neurons in the central nervous system. Recent studies have emphasized that myelination is plastic and that new myelin is formed throughout life. Nonetheless, the mechanisms that regulate the number, length, and location of myelin sheaths formed by individual oligodendrocytes are incompletely understood. Previous work showed that the lysosomal transcription factor TFEB represses myelination by oligodendrocytes and that the RagA GTPase inhibits TFEB, but the step or steps of myelination in which TFEB plays a role have remained unclear. Here, we show that TFEB regulates oligodendrocyte differentiation and also controls the length of myelin sheaths formed by individual oligodendrocytes. In the dorsal spinal cord of tfeb mutants, individual oligodendrocytes produce myelin sheaths that are longer than those produced by wildtype cells. Transmission electron microscopy shows that there are more myelinated axons in the dorsal spinal cord of tfeb mutants than in wildtype animals, but no significant change in axon diameter. In contrast to tfeb mutants, oligodendrocytes in rraga mutants produce shorter myelin sheaths. The sheath length in rraga; tfeb double mutants is not significantly different from wildtype, consistent with the antagonistic interaction between RagA and TFEB. Finally, we find that the GTPase activating protein Flcn and the RagCa and RagCb GTPases are also necessary for myelination by oligodendrocytes. These findings demonstrate that TFEB coordinates myelin sheath length and number during myelin formation in the central nervous system.

Spinal cord injury regulates circular RNA expression in axons.

Neurons transport mRNA and translational machinery to axons for local translation. After spinal cord injury (SCI), de novo translation is assumed to enable neurorepair. Knowledge of the identity of axonal mRNAs that participate in neurorepair after SCI is limited. We sought to identify and understand how axonal RNAs play a role in axonal regeneration. We obtained preparations enriched in axonal mRNAs from control and SCI rats by digesting spinal cord tissue with cold-active protease (CAP). The digested samples were then centrifuged to obtain a supernatant that was used to identify mRNA expression. We identified differentially expressed genes (DEGS) after SCI and mapped them to various biological processes. We validated the DEGs by RT-qPCR and RNA-scope. The supernatant fraction was highly enriched for mRNA from axons. Using Gene Ontology, the second most significant pathway for all DEGs was axonogenesis. Among the DEGs was Rims2, which is predominately a circular RNA (circRNA) in the CNS. We show that Rims2 RNA within spinal cord axons is circular. We found an additional 200 putative circRNAs in the axonal-enriched fraction. Knockdown in primary rat cortical neurons of the RNA editing enzyme ADAR1, which inhibits formation of circRNAs, significantly increased axonal outgrowth and increased the expression of circRims2. Using Rims2 as a prototype we used Circular RNA Interactome to predict miRNAs that bind to circRims2 also bind to the 3'UTR of GAP-43, PTEN or CREB1, all known regulators of axonal outgrowth. Axonally-translated GAP-43 supports axonal elongation and we detect GAP-43 mRNA in the rat axons by RNAscope. By enriching for axonal RNA, we detect SCI induced DEGs, including circRNA such as Rims2. Ablation of ADAR1, the enzyme that regulates circRNA formation, promotes axonal outgrowth of cortical neurons. We developed a pathway model using Circular RNA Interactome that indicates that Rims2 through miRNAs can regulate the axonal translation GAP-43 to regulate axonal regeneration. We conclude that axonal regulatory pathways will play a role in neurorepair.

Neurogenesis redirects β-catenin from adherens junctions to the nucleus to promote axonal growth.

Here, we show that, in the developing spinal cord, after the early Wnt-mediated Tcf transcription activation that confers dorsal identity to neural stem cells, neurogenesis redirects β-catenin from the adherens junctions to the nucleus to stimulate Tcf-dependent transcription in a Wnt-independent manner. This new β-catenin activity regulates genes implicated in several aspects of contralateral axon growth, including axon guidance and adhesion. Using live imaging of ex-vivo chick neural tube, we showed that the nuclear accumulation of β-catenin and the rise in Tcf-dependent transcription both initiate before the dismantling of the adherens junctions and remain during the axon elongation process. Notably, we demonstrated that β-catenin activity in post-mitotic cells depends on TCF7L2 and is central to spinal commissural axon growth. Together, our results reveal Wnt-independent Tcf/β-catenin regulation of genes that control the growth and guidance of commissural axons in chick spinal cord.

The oncomodulin receptor ArmC10 enables axon regeneration in mice after nerve injury and neurite outgrowth in human iPSC-derived sensory neurons.

Oncomodulin (Ocm) is a myeloid cell-derived growth factor that enables axon regeneration in mice and rats after optic nerve injury or peripheral nerve injury, yet the mechanisms underlying its activity are unknown. Using proximity biotinylation, coimmunoprecipitation, surface plasmon resonance, and ectopic expression, we have identified armadillo-repeat protein C10 (ArmC10) as a high-affinity receptor for Ocm. ArmC10 deletion suppressed inflammation-induced axon regeneration in the injured optic nerves of mice. ArmC10 deletion also suppressed the ability of lesioned sensory neurons to regenerate peripheral axons rapidly after a second injury and to regenerate their central axons after spinal cord injury in mice (the conditioning lesion effect). Conversely, Ocm acted through ArmC10 to accelerate optic nerve and peripheral nerve regeneration and to enable spinal cord axon regeneration in these mouse nerve injury models. We showed that ArmC10 is highly expressed in human-induced pluripotent stem cell-derived sensory neurons and that exposure to Ocm altered gene expression and enhanced neurite outgrowth. ArmC10 was also expressed in human monocytes, and Ocm increased the expression of immune modulatory genes in these cells. These findings suggest that Ocm acting through its receptor ArmC10 may be a useful therapeutic target for nerve repair and immune modulation.

SARM1 deletion delays cerebellar but not spinal cord degeneration in an enhanced mouse model of SPG7 deficiency.

Hereditary spastic paraplegia is a neurological condition characterized by predominant axonal degeneration in long spinal tracts, leading to weakness and spasticity in the lower limbs. The nicotinamide adenine dinucleotide (NAD+)-consuming enzyme SARM1 has emerged as a key executioner of axonal degeneration upon nerve transection and in some neuropathies. An increase in the nicotinamide mononucleotide/NAD+ ratio activates SARM1, causing catastrophic NAD+ depletion and axonal degeneration. However, the role of SARM1 in the pathogenesis of hereditary spastic paraplegia has not been investigated. Here, we report an enhanced mouse model for hereditary spastic paraplegia caused by mutations in SPG7. The eSpg7 knockout mouse carries a deletion in both Spg7 and Afg3l1, a redundant homologue expressed in mice but not in humans. The eSpg7 knockout mice recapitulate the phenotypic features of human patients, showing progressive symptoms of spastic-ataxia and degeneration of axons in the spinal cord as well as the cerebellum. We show that the lack of SPG7 rewires the mitochondrial proteome in both tissues, leading to an early onset decrease in mito-ribosomal subunits and a remodelling of mitochondrial solute carriers and transporters. To interrogate mechanisms leading to axonal degeneration in this mouse model, we explored the involvement of SARM1. Deletion of SARM1 delays the appearance of ataxic signs, rescues mitochondrial swelling and axonal degeneration of cerebellar granule cells and dampens neuroinflammation in the cerebellum. The loss of SARM1 also prevents endoplasmic reticulum abnormalities in long spinal cord axons, but does not halt the degeneration of these axons. Our data thus reveal a neuron-specific interplay between SARM1 and mitochondrial dysfunction caused by lack of SPG7 in hereditary spastic paraplegia.

Ca2+-induced myelin pathology precedes axonal spheroid formation and is mediated in part by store-operated Ca2+ entry after spinal cord injury.

The formation of axonal spheroid is a common feature following spinal cord injury. To further understand the source of Ca2+ that mediates axonal spheroid formation, we used our previously characterized ex vivo mouse spinal cord model that allows precise perturbation of extracellular Ca2+. We performed two-photon excitation imaging of spinal cords isolated from Thy1YFP+ transgenic mice and applied the lipophilic dye, Nile red, to record dynamic changes in dorsal column axons and their myelin sheaths respectively. We selectively released Ca2+ from internal stores using the Ca2+ ionophore ionomycin in the presence or absence of external Ca2+. We reported that ionomycin dose-dependently induces pathological changes in myelin and pronounced axonal spheroid formation in the presence of normal 2 mM Ca2+ artificial cerebrospinal fluid. In contrast, removal of external Ca2+ significantly decreased ionomycin-induced myelin and axonal spheroid formation at 2 hours but not at 1 hour after treatment. Using mice that express a neuron-specific Ca2+ indicator in spinal cord axons, we confirmed that ionomycin induced significant increases in intra-axonal Ca2+, but not in the absence of external Ca2+. Periaxonal swelling and the resultant disruption in the axo-myelinic interface often precedes and is negatively correlated with axonal spheroid formation. Pretreatment with YM58483 (500 nM), a well-established blocker of store-operated Ca2+ entry, significantly decreased myelin injury and axonal spheroid formation. Collectively, these data reveal that ionomycin-induced depletion of internal Ca2+ stores and subsequent external Ca2+ entry through store-operated Ca2+ entry contributes to pathological changes in myelin and axonal spheroid formation, providing new targets to protect central myelinated fibers.

AAV Vector Mediated Delivery of NG2 Function Neutralizing Antibody and Neurotrophin NT-3 Improves Synaptic Transmission, Locomotion, and Urinary Tract Function after Spinal Cord Contusion Injury in Adult Rats.

NG2 is a structurally unique transmembrane chondroitin sulfate proteoglycan (CSPG). Its role in damaged spinal cord is dual. NG2 is considered one of key inhibitory factors restricting axonal growth following spinal injury. Additionally, we have recently detected its novel function as a blocker of axonal conduction. Some studies, however, indicate the importance of NG2 presence in the formation of synaptic contacts. We hypothesized that the optimal treatment would be neutralization of inhibitory functions of NG2 without its physical removal. Acute intraspinal injections of anti-NG2 monoclonal antibodies reportedly prevented an acute block of axonal conduction by exogenous NG2. For prolonged delivery of NG2 function neutralizing antibody, we have developed a novel gene therapy: adeno-associated vector (AAV) construct expressing recombinant single-chain variable fragment anti-NG2 antibody (AAV-NG2Ab). We examined effects of AAV-NG2Ab alone or in combination with neurotrophin NT-3 in adult female rats with thoracic T10 contusion injuries. A battery of behavioral tests was used to evaluate locomotor function. In vivo single-cell electrophysiology was used to evaluate synaptic transmission. Lower urinary tract function was assessed during the survival period using metabolic chambers. Terminal cystometry, with acquisition of external urethral sphincter activity and bladder pressure, was used to evaluate bladder function. Both the AAV-NG2Ab and AAV-NG2Ab combined with AAV-NT3 treatment groups demonstrated significant improvements in transmission, locomotion, and bladder function compared with the control (AAV-GFP) group. These functional improvements associated with improved remyelination and plasticity of 5-HT fibers. The best results were observed in the group that received combinational AAV-NG2Ab+AAV-NT3 treatment.SIGNIFICANCE STATEMENT We recently demonstrated beneficial, but transient, effects of neutralization of the NG2 proteoglycan using monoclonal antibodies delivered intrathecally via osmotic mini-pumps after spinal cord injury. Currently, we have developed a novel gene therapy tool for prolonged and clinically relevant delivery of a recombinant single-chain variable fragment anti-NG2 antibody: AAV-rh10 serotype expressing scFv-NG2 (AAV-NG2Ab). Here, we examined effects of AAV-NG2Ab combined with transgene delivery of Neurotrophin-3 (AAV-NT3) in adult rats with thoracic contusion injuries. The AAV-NG2Ab and AAV-NG2Ab+AAV-NT3 treatment groups demonstrated significant improvements of locomotor function and lower urinary tract function. Beneficial effects of this novel gene therapy on locomotion and bladder function associated with improved transmission to motoneurons and plasticity of axons in damaged spinal cord.

转录因子Nkx6.1对脊髓损伤后中间神经元和星形胶质细胞发生的影响

<p indent="0mm">Interneurons are the main local loop connecting sensory and motor neurons and play an important role in the pathological changes after spinal cord injury and the corresponding repair process. After spinal cord injury, astrocyte proliferation and glial scar formation are the main factors hindering the effective regeneration and growth of injured spinal cord axons. In the early stage of neurogenesis, neuroepithelial cells produce astrocytes and intermediate neurons. Therefore, it is likely that astrocytes and intermediate neurons arise from common progenitor cells. In the spinal cord, neuron and glial cell differentiation is regulated by a transcription factor network. In particular, Nkx6.1 is critical for the fate determination of progenitor cells into ventral interneurons. However, the role of Nkx6.1 in the birth of spinal astrocytes after spinal cord injury is still unclear. Herein, we first used the Cre-LoxP system and Rosa26 gene knock-in technology to specifically induce an <italic>Nkx6</italic>.<italic>1</italic> knockout and introduce a lycopene marker gene in different neural precursor cells of adult mice’s spinal cord. Next, we constructed a knockout spinal cord injury model and performed immunofluorescence staining. The staining results showed a substantial number of Nkx6.1⁺ cells in the injured area after spinal cord injury, while <italic>Nkx6</italic>.<italic>1</italic> ablation one week after spinal cord injury would hinder the formation of the glial scar and inhibit the production of En1⁺ intermediate neurons by regulating the expression pattern of Pax6<italic> </italic>in the central neural tube. In conclusion, our study shows that <italic>Nkx6</italic>.<italic>1</italic> plays a crucial role in spinal cord injury, being responsible for effective axon regeneration and motor function recovery.

Downstream projection of Barrington's nucleus to the spinal cord in mice.

Barrington's nucleus (Bar), which controls micturition behavior through downstream projections to the spinal cord, contains two types of projection neurons, BarCRH and BarESR1, that have different functions and target different spinal circuitry. Both types of neurons project to the L6-S1 spinal intermediolateral (IML) nucleus, whereas BarESR1 neurons also project to the dorsal commissural nucleus (DCN). To obtain more information about the spinal circuits targeted by Bar, we used patch-clamp recording in spinal slices from adult mice in combination with optogenetic stimulation of Bar terminals. Recording of opto-evoked excitatory postsynaptic currents (oEPSCs) in 1,1'-dilinoleyl-3,3,3',3'-tetramethylindocarbocyanine, 4-chlorobenzenesulfonate (DiI)-labeled lumbosacral preganglionic neurons (LS-PGNs) revealed that both Bar neuronal populations make strong glutamatergic monosynaptic connections with LS-PGNs, whereas BarESR1 neurons also elicited smaller-amplitude glutamatergic polysynaptic oEPSCs or polysynaptic opto-evoked inhibitory postsynaptic currents (oIPSCs) in some LS-PGNs. Optical stimulation of BarCRH and BarESR1 terminals also elicited monosynaptic oEPSCs and polysynaptic oIPSCs in sacral DCN neurons, some of which must include interneurons projecting to either the IML or ventral horn. Application of capsaicin increased opto-evoked firing during repetitive stimulation of Bar terminals through the modulation of spontaneous postsynaptic currents in LS-PGNs. In conclusion, our experiments have provided insights into the synaptic mechanisms underlying the integration of inputs from Bar to autonomic circuitry in the lumbosacral spinal cord that may control micturition.NEW & NOTEWORTHY Photostimulation of BarCRH or BarESR1 axons in the adult mouse spinal cord elicits excitatory or inhibitory postsynaptic responses in multiple cell types related to the autonomic nervous system including preganglionic neurons (PGNs) in the lumbosacral intermediolateral nucleus and interneurons in the lumbosacral dorsal commissure nucleus. Integration of excitatory inputs from Bar and from visceral primary afferents in PGNs may be important in the regulation of micturition behavior.

Influence of Lysine and TRITC Conjugation on the Size and Structure of Dextran Nanoconjugates with Potential for Biomolecule Delivery to Neurons.

As a potent nonviral system for biomolecular delivery to neurons via their axons, we have studied molecular characteristics of lysinated fluorescent dextran nanoconjugates with degrees of conjugation of 0.54-15.2 mol lysine and 0.25-7.27 mol tetramethyl rhodamine isothiocyanate (TRITC) per mol dextran. We studied the influence of conjugation with lysine and TRITC on the size and structure of different molecular weight dextrans and their mobility within axons. Dynamic light scattering (DLS) and small-angle neutron scattering (SANS) experiments revealed significant differences in the size and structure of unmodified and modified dextrans. Unexpectedly, lower-molecular-weight conjugated dextrans exhibited higher molecular volumes, which we propose is due to fewer intramolecular interactions than in higher-molecular-weight conjugated dextrans. Assessment of retrograde and anterograde movement of lysine- and TRITC-conjugated dextrans in axons in the lumbar spinal cord of chicken embryos showed that lower-molecular-weight dextrans translocate more efficiently than higher-molecular-weight dextrans, despite having larger molecular volumes. This comparative characterization of different molecular weight dextrans will help define optimal features for intracellular delivery.

Defining Spatial Relationships Between Spinal Cord Axons and Blood Vessels in Hydrogel Scaffolds.

Positively charged oligo(poly(ethylene glycol) fumarate) (OPF+) hydrogel scaffolds, implanted into a complete transection spinal cord injury (SCI), facilitate a permissive regenerative environment and provide a platform for controlled observation of repair mechanisms. Axonal regeneration after SCI is critically dependent upon nutrients and oxygen from a newly formed blood supply. Our objective was to investigate fundamental characteristics of revascularization in association with the ingrowth of axons into hydrogel scaffolds, thereby defining spatial relationships between axons and the neovasculature. A novel combination of stereologic estimates and precision image analysis techniques quantitate neurovascular regeneration in rats. Multichannel hydrogel scaffolds containing Matrigel-only (MG), Schwann cells (SCs), or SCs with rapamycin-eluting poly(lactic co-glycolic acid) microspheres (RAPA) were implanted for 6 weeks following complete spinal cord transection. Image analysis of 72 scaffold channels identified a total of 2494 myelinated and 4173 unmyelinated axons at 10 μm circumferential intervals centered around 708 individual blood vessel profiles. Blood vessel number, density, volume, diameter, intervessel distances, total vessel surface and cross-sectional areas, and radial diffusion distances were compared. Axon number and density, blood vessel surface area, and vessel cross-sectional areas in the SC group exceeded that in the MG and RAPA groups. Individual axons were concentrated within a concentric radius of 200-250 μm from blood vessel walls, in Gaussian distributions, which identified a peak axonal number (Mean Peak Amplitude) corresponding to defined distances (Mean Peak Distance) from each vessel, the highest concentrations of axons were relatively excluded from a 25-30 μm zone immediately adjacent to the vessel, and from vessel distances >150 μm. Higher axonal densities correlated with smaller vessel cross-sectional areas. A statistical spatial algorithm was used to generate cumulative distribution F- and G-functions of axonal distribution in the reference channel space. Axons located around blood vessels were definitively organized as clusters and were not randomly distributed. A scoring system stratifies 5 direct measurements and 12 derivative parameters influencing regeneration outcomes. By providing methods to quantify the axonal-vessel relationships, these results may refine spinal cord tissue engineering strategies to optimize the regeneration of complete neurovascular bundles in their relevant spatial relationships after SCI. Impact statement Vascular disruption and impaired neovascularization contribute critically to the poor regenerative capacity of the spinal cord after injury. In this study, hydrogel scaffolds provide a detailed model system to investigate the regeneration of spinal cord axons as they directly associate with individual blood vessels, using novel methods to define their spatial relationships and the physiologic implications of that organization. These results refine future tissue engineering strategies for spinal cord repair to optimize the re-development of complete neurovascular bundles in their relevant spatial architectures.

Implantation of Engineered Axon Tracts to Bridge Spinal Cord Injury Beyond the Glial Scar in Rats.

Regeneration after spinal cord injury (SCI) is limited by the presence of a glial scar and inhibitory cell signaling pathways that favor scar formation over regrowth of endogenous neurons. Tissue engineering techniques, including the use of allografted neural networks, have shown promise for nervous system repair in prior studies. Through the use of a minimally invasive injury model in rats, we describe the implantation of micro-tissue engineered neural networks (micro-TENNs) across a region of SCI, spanning the glial scar to promote axonal regeneration. Forty-three female Sprague-Dawley rats were included in this study. Micro-TENNs were preformed in vitro before implant, and comprised rat sensory dorsal root ganglion (DRG) neurons projecting long bundled axonal tracts within the lumen of a biocompatible hydrogel columnar encasement (1.2 cm long; 701 μm outer diameter × 300 μm inner diameter). Animals were injured using a 2F embolectomy catheter inflated within the epidural space. After a 2-week recovery period, micro-TENNs were stereotactically implanted across the injury. Animals were euthanized at 1 week and 1 month after implantation, and the tissue was interrogated for the survival of graft DRG neurons and outgrowth of axons. No intraoperative deaths were noted with implantation of the micro-TENNs to span the injury cavity. Graft DRG axons were found to survive at 1 week postimplant within the hydrogel encasement. Graft-derived axonal outgrowth was observed within the spinal cord up to 4.5 mm from the implant site at 1 month postinjury. Limited astroglial response was noted within the host, suggesting minimal trauma and scar formation in response to the graft. Micro-TENN sensory neurons survive and extend axons into the host spinal cord following a minimally invasive SCI in rats. This work serves as the foundation for future studies investigating the use of micro-TENNs as a living bridge to promote recovery following SCI. Impact statement As spinal cord injury pathology develops, the establishment of a glial scar puts an end to the hope of regeneration and recovery from the consequent neurological deficits. Therefore, growing attention is given to bioengineered scaffolds that can bridge the lesions bordered by this scar tissue. The utilization of longitudinally aligned preformed neural networks-referred to as micro-tissue engineered neural networks (TENNs)-presents a promising opportunity to provide a multipurpose bridging strategy that may take advantage of several potential mechanisms of host regeneration. In addition to providing physical support for regenerating spinal cord axons, micro-TENNs may serve as a functional "cable" that restores lost connections within the spinal cord.