Novel AAV variants with improved tropism for human Schwann cells.

Gene therapies and associated technologies are transforming biomedical research and enabling novel therapeutic options for patients living with debilitating and incurable genetic disorders. The vector system based on recombinant adeno-associated viral vectors (AAVs) has shown great promise in recent clinical trials for genetic diseases of multiple organs, such as the liver and the nervous system. Despite recent successes toward the development of novel bioengineered AAV variants for improved transduction of primary human tissues and cells, vectors that can efficiently transduce human Schwann cells (hSCs) have yet to be identified. Here, we report the application of the functional transduction-RNA selection method in primary hSCs for the development of AAV variants for specific and efficient transgene delivery to hSCs. The two identified capsid variants, Pep2hSC1 and Pep2hSC2, show conserved potency for delivery across various in vitro, in vivo, and ex vivo models of hSCs. These novel AAV capsids will serve as valuable research tools, forming the basis for therapeutic solutions for both SC-related disorders or peripheral nervous system injury.

Perineurial Window is Critical for Experimental Reverse End-to-Side Nerve Transfer.

BACKGROUND: The depth of connective tissue window in the side of a recipient nerve in reverse end-to-side transfers (RETS) remains controversial. OBJECTIVE: To test whether the depth of connective tissue disruption influences the efficiency of donor axonal regeneration in the context of RETS. METHODS: Sprague-Dawley rats (n = 24) were assigned to 1 of the 3 groups for obturator nerve to motor femoral nerve RETS: group 1, without epineurium opening; group 2, with epineurium only opening; and group 3, with epineurium and perineurium opening. Triple retrograde labeling was used to assess the number of motor neurons that had regenerated into the recipient motor femoral branch. Thy1-GFP rats (n = 8) were also used to visualize the regeneration pathways in the nerve transfer networks at 2- and 8-week time point using light sheet fluorescence microscopy. RESULTS: The number of retrogradely labeled motor neurons that had regenerated distally toward the target muscle was significantly higher in group 3 than that in groups 1 and 2. Immunohistochemistry validated the degree of connective tissue disruption among the 3 groups, and optical tissue clearing methods demonstrated donor axons traveling outside the fascicles in groups 1 and 2 but mostly within the fascicles in group 3. CONCLUSION: Creating a perineurial window in the side of recipient nerves provides the best chances of robust donor axonal regeneration across the RETS repair site. This finding aids nerve surgeons by confirming that a deep window should be undertaken when doing a RETS procedure.

A Multi-Stage Bioprocess for the Expansion of Rodent Skin-Derived Schwann Cells in Computer-Controlled Bioreactors.

Regenerative therapies for the treatment of peripheral nerve and spinal cord injuries can require hundreds of millions of autologous cells. Current treatments involve the harvest of Schwann cells (SCs) from nerves; however, this is an invasive procedure. Therefore, a promising alternative is using skin-derived Schwann cells (Sk-SCs), in which between 3-5 million cells can be harvested from a standard skin biopsy. However, traditional static planar culture is still inefficient at expanding cells to clinically relevant numbers. As a result, bioreactors can be used to develop reproducible bioprocesses for the large-scale expansion of therapeutic cells. Here, we present a proof-of-concept SC manufacturing bioprocess using rat Sk-SCs. With this integrated process, we were able to simulate a feasible bioprocess, taking into consideration the harvest and shipment of cells to a production facility, the generation of the final cell product, and the cryopreservation and shipment of cells back to the clinic and patient. This process started with 3 million cells and inoculated and expanded them to over 200 million cells in 6 days. Following the harvest and post-harvest cryopreservation and thaw, we were able to maintain 150 million viable cells that exhibited a characteristic Schwann cell phenotype throughout each step of the process. This process led to a 50-fold expansion, producing a clinically relevant number of cells in a 500 mL bioreactor in just 1 week, which is a dramatic improvement over current methods of expansion.

Behavioral and axon histomorphometric analyses after intraneural transplantation of human skin- and nerve-derived Schwann cells in nude rats.

INTRODUCTION/AIMS: We have recently isolated and expanded skin-derived Schwann cells (Sk-SCs) from human skin and showed that they are largely similar to nerve-derived Schwann cells (N-SCs). Here, we extend our investigation into functional assessments of the nude rats that received human Sk-SCs and N-SCs after intraneural delivery into crushed and decellularized tibial nerve in adult nude rats. METHODS: Sk-SCs, N-SCs, dermal fibroblasts, or control culture medium was injected into the crushed and decellularized tibial nerve using in situ repeated freeze-thaw cycles. Animals were then subjected to a ladder rung walking test, nociceptive von Frey testing, and walking gait analysis weekly. Animals were euthanized 6 weeks after surgery, gastrocnemius and soleus muscles were weighed, distal nerves were harvested, and whole semithin cross-sections were analyzed using segmentation software. RESULTS: N-SC-injected and dermal fibroblast-injected animals improved significantly at 4 to 6 weeks postinjury in nociceptive assessment compared with medium-injected controls. Sk-SCs recovered more rapidly in tibial functional index at 2 weeks postinjury compared with medium-injected controls. No significant difference was observed for the ladder rung walking test or muscle weight ratio. Histologically, the number of myelinated axons was significantly higher in all cell injection groups compared with medium-injected controls. No significant difference was observed in g ratio, axon diameter, or myelin thickness. DISCUSSION: Cell injection significantly improved axon regeneration across an in situ decellularized nerve segment. However, a more human cell-permissive animal model is required to delineate functional differences between cell types for preclinical transplantation studies.

Successful retrograde regeneration using a sensory branch for motor nerve transfer.

OBJECTIVE: The objective of this study was to test whether regenerating motor axons from a donor nerve can travel in a retrograde fashion using sensory branches to successfully reinnervate a motor nerve end organ. METHODS: This study has two parts. In part I, rats (n = 30) were assigned to one of five groups for obturator nerve (ON)-to-femoral nerve transfer: group 1, ON-to-saphenous nerve (SN) distal stump; group 2, ON-to-SN proximal stump without femoral nerve proper (FNP) injury; group 3, ON-to-SN proximal stump with FNP crush injury; group 4, ON-to-SN proximal stump with FNP transection injury; and group 5, gold standard transfer, ON-to-motor femoral nerve (MFN) branch. At 8 weeks, retrograde labeling was done from the distal MFN, and the spinal cords were examined to assess the degree of obturator motor axon regeneration across the five groups. In part II, only group 4 was examined (n = 8). Through use of immunostaining and optical tissue clearing methods, the nerve transfer networks were cleared and imaged using light-sheet fluorescence microscopy to visualize the regeneration pathways in 2D and 3D models at 2- and 8-week time points. RESULTS: Proximal FNP transection (group 4) enabled a significantly higher number of retrogradely regenerated motor axons compared with control groups 1-3. Moreover, group 4 had modest, but nonsignificant, superiority of motor neuron counts compared with the positive control group, group 5. Optical tissue clearing demonstrated that the axons traveled in a retrograde fashion from the recipient sensory branch to the FNP mixed stump, then through complex turns, down to distal branches. Immunostaining confirmed the tissue clearing findings and suggested perineurium disruption as a means by which axons could traverse across fascicular boundaries. CONCLUSIONS: Sensory branches can transmit regenerating axons from donor nerves back to main mixed recipient nerves, then distally toward target organs. The extent of retrograde regeneration is markedly influenced by the type and severity of injury sustained by the recipient nerve. Using a sensory branch as a bridge for retrogradely regenerating axons can open new potential horizons in nerve repair surgery for severely injured mixed nerves.

Functional Gait Assessment Using Manual, Semi-Automated and Deep Learning Approaches Following Standardized Models of Peripheral Nerve Injury in Mice.

Objective: To develop a standardized model of stretch−crush sciatic nerve injury in mice, and to compare outcomes of crush and novel stretch−crush injuries using standard manual gait and sensory assays, and compare them to both semi-automated as well as deep-learning gait analysis methods. Methods: Initial studies in C57/Bl6 mice were used to develop crush and stretch−crush injury models followed by histologic analysis. In total, 12 eight-week-old 129S6/SvEvTac mice were used in a six-week behavioural study. Behavioral assessments using the von Frey monofilament test and gait analysis recorded on a DigiGait platform and analyzed through both Visual Gait Lab (VGL) deep learning and standardized sciatic functional index (SFI) measurements were evaluated weekly. At the termination of the study, neurophysiological nerve conduction velocities were recorded, calf muscle weight ratios measured and histological analyses performed. Results: Histological evidence confirmed more severe histomorphological injury in the stretch−crush injured group compared to the crush-only injured group at one week post-injury. Von Frey monofilament paw withdrawal was significant for both groups at week one compared to baseline (p < 0.05), but not between groups with return to baseline at week five. SFI showed hindered gait at week one and two for both groups, compared to baseline (p < 0.0001), with return to baseline at week five. Hind stance width (HSW) showed similar trends as von Frey monofilament test as well as SFI measurements, yet hind paw angle (HPA) peaked at week two. Nerve conduction velocity (NCV), measured six weeks post-injury, at the termination of the study, did not show any significant difference between the two groups; yet, calf muscle weight measurements were significantly different between the two, with the stretch−crush group demonstrating a lower (poorer) weight ratio relative to uninjured contralateral legs (p < 0.05). Conclusion: Stretch−crush injury achieved a more reproducible and constant injury compared to crush-only injuries, with at least a Sunderland grade 3 injury (perineurial interruption) in histological samples one week post-injury in the former. However, serial behavioral outcomes were comparable between the two crush groups, with similar kinetics of recovery by von Frey testing, SFI and certain VGL parameters, the latter reported for the first time in rodent peripheral nerve injury. Semi-automated and deep learning-based approaches for gait analysis are promising, but require further validation for evaluation in murine hind-limb nerve injuries.

Utilization of focused ultrasound for opening of the blood-nerve barrier.

Objective. Focused ultrasound (FUS) use with and without microbubbles (MB) for investigation of the blood-nerve barrier (BNB) within the peripheral nervous system (PNS) has been performed in this study. We evaluate the feasibility of BNB opening in a rodent sciatic nerve model by direct vision FUS treatment and provide preliminary results of magnetic resonance guided FUS (MRgFUS).Approach. Twenty rodent bilateral sciatic nerves were investigated. Rodents were treated using a benchtop FUS system to directly visualize nerve FUS studies. Definity MB, Evans blue dye (EB) and latex micro beads were injected during studies. Selected animals underwent further compound muscle action potential (CMAP) studies. Sonication peak pressure (MPa), width, duty-cycle and duration as well as MB concentration were varied to investigate effective pressure threshold. Further preliminary MRgFUS studies were performed on selected animals. Immunohistochemistry and histological analysis under florescent microscopy were performed at termination of experiments to verify treatment outcomes.Main results. Three ultrasound pressures and three microbubble concentrations at a single sonication frequency (476.5 kHz) were performed under direct open targeting. Histological analysis demonstrated nerve internal architecture disruption at 1.2 MPa with 166.7µl kg-1while 0.3 MPa, with 40µl kg-1MB concentration was the lower threshold for consistently observed disruption of the BNB without anatomical microarchitecture disruption. EB leakage was confirmed at the target region in histological evaluation of nerve following MB injection and FUS sonication. Supra-harmonic emissions were detected during FUS exposures following MB injection but not at baseline reference, indicating effective MB response and stable cavitation. CMAP amplitudes showed delayed onset latency and lower amplitudes in sonicated nerves compared to control nerves without evidence of complete conduction block, suggesting a transient BNB disruption, while at lower limit pressure subtle conduction changes were observed. In MRgFUS, targeted nerves demonstrated further contrast agent leak as well as supra-harmonic frequency detection.Significance. Opening of the BNB in the PNS was achieved using FUS and MB in a rodent model. Ongoing work aims to refine FUS parameters for drug delivery into the nerve after experimental transient BNB disruption.

Comparison of human skin- and nerve-derived Schwann cells reveals many similarities and subtle genomic and functional differences.

Skin is an easily accessible tissue and a rich source of Schwann cells (SCs). Toward potential clinical application of autologous SC therapies, we aim to improve the reliability and specificity of our protocol to obtain SCs from small skin samples. As well, to explore potential functional distinctions between skin-derived SCs (Sk-SCs) and nerve-derived SCs (N-SCs), we used single-cell RNA-sequencing and a series of in vitro and in vivo assays. Our results showed that Sk-SCs expressed typical SC markers. Single-cell sequencing of Sk- and N-SCs revealed an overwhelming overlap in gene expression with the exception of HLA genes which were preferentially up-regulated in Sk-SCs. In vitro, both cell types exhibited similar levels of proliferation, migration, uptake of myelin debris and readily associated with neurites when co-cultured with human iPSC-induced motor neurons. Both exhibited ensheathment of multiple neurites and early phase of myelination, especially in N-SCs. Interestingly, dorsal root ganglion (DRG) neurite outgrowth assay showed substantially more complexed neurite outgrowth in DRGs exposed to Sk-SC conditioned media compared to those from N-SCs. Multiplex ELISA array revealed shared growth factor profiles, but Sk-SCs expressed a higher level of VEGF. Transplantation of Sk- and N-SCs into injured peripheral nerve in nude rats and NOD-SCID mice showed close association of both SCs to regenerating axons. Myelination of rodent axons was observed infrequently by N-SCs, but absent in Sk-SC xenografts. Overall, our results showed that Sk-SCs share near-identical properties to N-SCs but with subtle differences that could potentially enhance their therapeutic utility.

Simultaneous Localized Brain Mild Hyperthermia and Blood-Brain Barrier Opening via Feedback-Controlled Transcranial MR-Guided Focused Ultrasound and Microbubbles.

OBJECTIVE: Non-invasive methods to enhance drug delivery and efficacy in the brain have been pursued for decades. Focused ultrasound hyperthermia (HT) combined with thermosensitive therapeutics have been demonstrated promising in enhancing local drug delivery to solid tumors. We hypothesized that the presence of microbubbles (MBs) combined with transcranial MR-guided focused ultrasound (MRgFUS) could be used to reduce the ultrasound power required for HT while simultaneously increasing drug delivery by locally opening the blood-brain barrier (BBB). METHODS: Transcranial HT (42 °C, 10 min) was performed in wild-type mice using a small animal MRgFUS system incorporated into a 9.4T Bruker MR scanner, with infusions of saline or Definity MBs with doses of 20 or 100 µl/kg/min (denoted as MB-20 and MB-100). MR thermometry data was continuously acquired as feedback for the ultrasound controller during the procedure. RESULTS: Spatiotemporally precise transcranial HT was achieved in both saline and MB groups. A significant ultrasound power reduction (-45.7%, p = 0.006) was observed in the MB-20 group compared to saline. Localized BBB opening was achieved in MB groups confirmed by CE-T1w MR images. There were no structural abnormalities, edema, hemorrhage, or acutemicroglial activation in all groups, confirmed by T2w MR imaging and histology. CONCLUSION: Our investigations showed that it is feasible and safe to achieve spatiotemporally precise brain HT at significantly reduced power and simultaneous localized BBB opening via transcranial MRgFUS and MBs. SIGNIFICANCE: This study provides a new synergistic brain drug delivery method with clinical translation potential.

Traumatic Injury Reduces Amyloid Plaque Burden in the Transgenic 5xFAD Alzheimer's Mouse Spinal Cord.

BACKGROUND: Axonal injury has been implicated in the development of amyloid-ß in experimental brain injuries and clinical cases. The anatomy of the spinal cord provides a tractable model for examining the effects of trauma on amyloid deposition. OBJECTIVE: Our goal was to examine the effects of axonal injury on plaque formation and clearance using wild type and 5xFAD transgenic Alzheimer's disease mice. METHODS: We contused the spinal cord at the T12 spinal level at 10 weeks, an age at which no amyloid plaques spontaneously accumulate in 5xFAD mice. We then explored plaque clearance by impacting spinal cords in 27-week-old 5xFAD mice where amyloid deposition is already well established. We also examined the cellular expression of one of the most prominent amyloid-ß degradation enzymes, neprilysin, at the lesion site. RESULTS: No plaques were found in wild type animals at any time points examined. Injury in 5xFAD prevented plaque deposition rostral and caudal to the lesion when the cords were examined at 2 and 4 months after the impact, whereas age-matched naïve 5xFAD mice showed extensive amyloid plaque deposition. A massive reduction in the number of plaques around the lesion was found as early as 7 days after the impact, preceded by neprilysin upregulation in astrocytes at 3 days after injury. At 7 days after injury, the majority of amyloid was found inside microglia/macrophages. CONCLUSION: These observations suggest that the efficient amyloid clearance after injury in the cord may be driven by the orchestrated efforts of astroglial and immune cells.

Comparative Behavioral Assessment of Lewis and Nude Rats after Peripheral Nerve Injury.

Cell therapy has shown potential in the field of peripheral nerve repair, and research using rodents is a critical and essential step toward clinical development of this approach. Traditionally, most experimental peripheral nerve injuries are conducted in inbred Lewis or outbred Sprague-Dawley strains. However, transplantation of xenogeneic cells such as human-derived cells typically triggers rejection in these animals. An alternative approach is to use immunodeficient animals, such as athymic nude rats. The lack of functional T cells in these animals renders them more accommodating to foreign cells from a different host. Currently, no literature exists regarding sensorimotor behavioral assessment of nude rats after peripheral nerve injury. To this end, we compared the functional recovery during a 6-wk period of behavioral testing of Lewis and nude rats after unilateral sciatic nerve crushing injury. Three sensorimotor behavioral assessments were performed weekly: a ladder rungwalking task to assess slip ratio and cross duration, von Frey nociception testing to determine the paw withdrawal threshold thus monitoring the regaining of sensory function, and sciatic functional index evaluation to monitor the recovery of integrated motor function. Both strains demonstrated significant sensory and motor deficits in the first week after injury, with a slight regain of sensory function, reduced slip ratio, and increased sciatic functional index starting at 2 wk. No significance difference existed between nude and Lewis rats in their recovery courses. We conclude that nude rats are a suitable model for behavioral training and assessment for cell transplantation studies in peripheral nerve injury and repair.

Insights Into the Role and Potential of Schwann Cells for Peripheral Nerve Repair From Studies of Development and Injury.

Peripheral nerve injuries arising from trauma or disease can lead to sensory and motor deficits and neuropathic pain. Despite the purported ability of the peripheral nerve to self-repair, lifelong disability is common. New molecular and cellular insights have begun to reveal why the peripheral nerve has limited repair capacity. The peripheral nerve is primarily comprised of axons and Schwann cells, the supporting glial cells that produce myelin to facilitate the rapid conduction of electrical impulses. Schwann cells are required for successful nerve regeneration; they partially "de-differentiate" in response to injury, re-initiating the expression of developmental genes that support nerve repair. However, Schwann cell dysfunction, which occurs in chronic nerve injury, disease, and aging, limits their capacity to support endogenous repair, worsening patient outcomes. Cell replacement-based therapeutic approaches using exogenous Schwann cells could be curative, but not all Schwann cells have a "repair" phenotype, defined as the ability to promote axonal growth, maintain a proliferative phenotype, and remyelinate axons. Two cell replacement strategies are being championed for peripheral nerve repair: prospective isolation of "repair" Schwann cells for autologous cell transplants, which is hampered by supply challenges, and directed differentiation of pluripotent stem cells or lineage conversion of accessible somatic cells to induced Schwann cells, with the potential of "unlimited" supply. All approaches require a solid understanding of the molecular mechanisms guiding Schwann cell development and the repair phenotype, which we review herein. Together these studies provide essential context for current efforts to design glial cell-based therapies for peripheral nerve regeneration.

Droplet Barcoding-Based Single Cell Transcriptomics of Adult Mammalian Tissues.

The analysis of single cell gene expression across thousands of individual cells within a tissue or microenvironment is a valuable tool for identifying cell composition, discrimination of functional states, and molecular pathways underlying observed tissue functions and animal behaviors. However, the isolation of intact, healthy single cells from adult mammalian tissues for subsequent downstream single cell molecular analysis can be challenging. This protocol describes the general processes and quality control checks necessary to obtain high-quality adult single cell preparations from the nervous system or skin that enabled subsequent unbiased single cell RNA sequencing and analysis. Guidelines for downstream bioinformatic analysis are also provided.

The triple monoamine re-uptake inhibitor DOV 216,303 promotes functional recovery after spinal cord contusion injury in mice.

Serotonin, noradrenaline and dopamine are important neuromodulators for locomotion in the spinal cord. Disruption of descending axons after spinal cord injury resulted in reduction of excitatory and neuromodulatory inputs to spinal neurons for locomotion. Receptor agonists or reuptake inhibitors for these neuromodulators have been shown to be beneficial in incomplete spinal cord injury. In this study, we tested a triple re-uptake inhibitor, DOV 216,303, for its ability to affect motor function recovery after spinal cord injury in mice. We impacted C57 mouse spinal cord at the T11 vertebral level and administered vehicle or DOV 216,303 at 10 mg/kg, b.i.d via intraperitoneal injections for 7 days. We monitored motor function with the Basso Mouse Scale for locomotion for 4 weeks. Spinal cords were harvested and histological examinations were performed to assess tissue sparing and lesion severity. Results showed that DOV 216,303-treated mice recovered significantly better than vehicle treated mice starting at 14 days post injury until the end of the survival period. Lesion size of the DOV 216,303 treated mice was also smaller compared to that of vehicle treated mice. This study suggests DOV 216,303 as a potential therapeutic after spinal cord injury warrants further investigation.

Axonal and myelinic pathology in 5xFAD Alzheimer's mouse spinal cord.

As an extension of the brain, the spinal cord has unique properties which could allow us to gain a better understanding of CNS pathology. The brain and cord share the same cellular components, yet the latter is simpler in cytoarchitecture and connectivity. In Alzheimer's research, virtually all focus is on brain pathology, however it has been shown that transgenic Alzheimer's mouse models accumulate beta amyloid plaques in spinal cord, suggesting that the cord possesses the same molecular machinery and conditions for plaque formation. Here we report a spatial-temporal map of plaque load in 5xFAD mouse spinal cord. We found that plaques started to appear at 11 weeks, then exhibited a time dependent increase and differential distribution along the cord. More plaques were found in cervical than other spinal levels at all time points examined. Despite heavy plaque load at 6 months, the number of cervical motor neurons in 5xFAD mice is comparable to wild type littermates. On detailed microscopic examination, fine beta amyloid-containing and beta sheet-rich thread-like structures were found in the peri-axonal space of many axons. Importantly, these novel structures appear before any plaque deposits are visible in young mice spinal cord and they co-localize with axonal swellings at later stages, suggesting that these thread-like structures might represent the initial stages of plaque formation, and could play a role in axonal damage. Additionally, we were able to demonstrate increasing myelinopathy in aged 5xFAD mouse spinal cord using the lipid probe Nile Red with high resolution. Collectively, we found significant amyloid pathology in grey and white matter of the 5xFAD mouse spinal cord which indicates that this structure maybe a useful platform to study mechanisms of Alzheimer's pathology and disease progression.

Fluorescent Phosphorus Dendrimer as a Spectral Nanosensor for Macrophage Polarization and Fate Tracking in Spinal Cord Injury.

Dendrimers and dendriplexes, highly branched synthetic macromolecules, have gained popularity as new tools for a variety of nanomedicine strategies due to their unique structure and properties. We show that fluorescent phosphorus dendrimers are well retained by bone marrow-derived macrophages and exhibit robust spectral shift in its emission in response to polarization conditions. Fluorescence properties of this marker can also assist in identifying macrophage presence and phenotype status at different time points after spinal cord injury. Potential use of a single dendrimer compound as a drug/siRNA carrier and phenotype-specific cell tracer offers new avenues for enhanced cell therapies combined with monitoring of cell fate and function in spinal cord injury.

Down-regulation of apurinic/apyrimidinic endonuclease 1 (APE1) in spinal motor neurones under oxidative stress.

AIM: Apurinic/apyrimidinic endonuclease 1 (APE1) is an intermediate enzyme in base excision repair which is important for removing damaged nucleotides under normal and pathological conditions. Accumulation of damaged bases causes genome instability and jeopardizes cell survival. Our study is to examine APE1 regulation under oxidative stress in spinal motor neurones which are vulnerable to oxidative insult. METHODS: We challenged the motor neurone-like cell line NSC-34 with hydrogen peroxide and delineated APE1 function by applying various inhibitors. We also examined the expression of APE1 in spinal motor neurones after spinal root avulsion in adult rats. RESULTS: We showed that hydrogen peroxide induced APE1 down-regulation and cell death in a differentiated motor neurone-like cell line. Inhibiting the two functional domains of APE1, namely, DNA repair and redox domains potentiated hydrogen peroxide induced cell death. We further showed that p53 phosphorylation early after hydrogen peroxide treatment might contribute to the down-regulation of APE1. Our in vivo results similarly showed that APE1 was down-regulated after root avulsion injury in spinal motor neurones. Delay of motor neurone death suggested that APE1 might not cause immediate cell death but render motor neurones vulnerable to further oxidative insults. CONCLUSION: We conclude that spinal motor neurones down-regulate APE1 upon oxidative stress. This property renders motor neurones susceptible to continuous challenge of oxidative stress in pathological conditions.

Neural progenitor cell apoptosis and differentiation were affected by activated microglia in spinal cord slice culture.

Neural progenitor cell (NPC) transplantation offers great potential to treat spinal cord injury (SCI). NPCs may replace lost neurons or oligodendrocytes and act as a source of neurotrophic factors to support survival of remaining cells. However, their efficiency was limited by poor survival after transplantation, and they tended more to differentiate into astrocytes, but not neurons and oligodendrocytes. This study investigated whether activated microglia is a factor that contributes to this phenomenon. Organotypic spinal cord slice (SCS) culture was used to mimic the local environment after SCI, and NPCs were co-cultured with them to share the culture medium. After specific depletion of microglia in the SCSs with clodronate loaded liposome, the apoptotic rate of NPCs decreased, more NPCs differentiated into neurons, and glial differentiation was impaired. This suggested that microglia may impair NPC survival, and neuronal differentiation, but improve astrocyte differentiation. In NPC transplantation strategy for SCI, microglia would be manipulated to improve the survival and neuronal differentiation of NPCs.

Minocycline inhibited the pro-apoptotic effect of microglia on neural progenitor cells and protected their neuronal differentiation in vitro.

Neural progenitor cell (NPC) transplantation offers great potential to treat spinal cord injury (SCI), but their efficiency is limited by poor survival and neuronal differentiation after transplantation. In the injury site, microglia may become activated and participate in the inflammation reaction. In vitro studies indicated that activated microglia might impair NPC survival and neuronal differentiation, but resting microglia did not. This study investigated the potential of minocycline to modify the negative effects of activated microglia on NPCs in vitro. First, the direct effects of minocycline on NPCs were tested. The results showed that at the concentration of 10µg/ml or lower, minocycline did not affect NPC survival and proliferation, but impaired neuronal differentiation. Then microglia were activated with lipopolysaccharide (LPS) or treated with LPS plus minocycline (LPSMC), and the effects of conditioned media on NPC apoptosis and differentiation were studied. The results showed that, compared with LPS treatment group, the microglia conditioned media of LPSMC treatment group resulted in a significantly lower apoptotic rate of NPCs, and increased the neuronal differentiation of NPCs. This suggested that minocycline might inhibit the negative effects of microglia on NPCs, and have the potential to support the survival and neuronal differentiation of transplanted NPCs for SCI.

Co-expression of GAP-43 and nNOS in avulsed motoneurons and their potential role for motoneuron regeneration.

Neuronal nitric oxide synthase (nNOS) is induced after axonal injury. The role of induced nNOS in injured neurons is not well established. In the present study, we investigated the co-expression of nNOS with GAP-43 in spinal motoneurons following axonal injury. The role of induced nNOS was discussed and evaluated. In normal rats, spinal motoneurons do not express nNOS or GAP-43. Following spinal root avulsion, expression of nNOS and GAP-43 were induced and colocalized in avulsed motoneurons. Reimplantation of avulsed roots resulted in a remarkable decrease of GAP-43- and nNOS-IR in the soma of the injured motoneurons. A number of GAP-43-IR regenerating motor axons were found in the reimplanted nerve. In contrast, the nNOS-IR was absent in reimplanted nerve. These results suggest that expression of GAP-43 in avulsed motoneurons is related to axonal regeneration whereas nNOS is not.