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Delivery of a peptide (APP96-110), derived from amyloid precursor protein (APP), has been shown to elicit neuroprotective effects following cerebral stroke and traumatic brain injury. In this study, the effect of APP96-110 or a mutant version of this peptide (mAPP96-110) was assessed following moderate (200 kdyn, (2 N)) thoracic contusive spinal cord injury (SCI) in adult Nude rats. Animals received a single tail vein injection of APP96-110 or mAPP96-110 at 30 minutes post-SCI and were then assessed for functional improvements over the next 8 weeks. A cohort of animals also received transplants of either viable or non-viable human mesenchymal stromal cells (hMSCs) into the SC lesion site at one week post-injury to assess the effect of combining intravenous APP96-110 delivery with hMSC treatment. Rats were perfused 8 weeks post-SCI and longitudinal sections of spinal cord analyzed for a number of factors including hMSC viability, cyst size, axonal regrowth, glial reactivity and macrophage activation. Analysis of sensorimotor function revealed occasional significant differences between groups using Ladderwalk or Ratwalk tests, however there were no consistent improvements in functional outcome after any of the treatments. mAPP96-110 alone, and APP96-110 in combination with both viable and non-viable hMSCs significantly reduced cyst size compared to SCI alone. Combined treatments with donor hMSCs also significantly increased βIII tubulin+, glial fibrillary acidic protein (GFAP+) and laminin+ expression, and decreased ED1+ expression in tissues. This preliminary study demonstrates that intravenous delivery of APP96-110 peptide has selective, modest neuroprotective effects following SCI, which may be enhanced when combined with hMSC transplantation. However, the effects are less pronounced and less consistent compared to the protective morphological and cognitive impact that this same peptide has on neuronal survival and behaviour after stroke and traumatic brain injury. Thus while the efficacy of a particular therapeutic approach in one CNS injury model may provide justification for its use in other neurotrauma models, similar outcomes may not necessarily occur and more targeted approaches suited to location and severity are required. All animal experiments were approved by The University of Western Australia Animal Ethics Committee (RA3/100/1460) on April 12, 2016.

A unique, biomimetic self-assembling peptide (SAP) hydrogel, Fmoc-DIKVAV, has been shown to be a suitable cell and drug delivery system in the injured brain. In this study, we assessed its utility in adult Fischer 344 (F344) rats as a stabilizing scaffold and vehicle for grafted cells after mild thoracic (thoracic level 10 [T10]) contusion spinal cord injury (SCI). Treatments were as follows: Fmoc-DIKVAV alone, Fmoc-DIKVAV containing viable or nonviable rat mesenchymal precursor cells (rMPCs), and rMPCs alone. The majority of post-SCI treatments were administered at 11-15 days (mean 13.5 days) and the results then compared to SCI-only control (no treatment) rats. Postinjury behavior was quantified using open field locomotion (BBB) and LadderWalk analysis. After perfusion at 8 weeks, longitudinal spinal cord sections were immunostained with a panel of antibodies. Qualitatively, in the SAP-only treatment group, implanted gels contained regenerate axons as well as astrocytic, immune cell, and extracellular matrix (ECM) component profiles. Grafts of Fmoc-DIKVAV plus viable or nonviable rMPCs also contained numerous macrophages/microglia and ECM components, but astrocytes were generally confined to implant margins, and axons were rare. Quantitative analysis showed that, while average cyst size was reduced in all experimental groups, the decrease compared to SCI-only controls was only significant in the SAP and rMPC treatment groups. There was gradual improvement in functionality after SCI, but a consistent trend was only seen between the rMPC treatment group and SCI-only controls. In summary, after contusion SCI, implantation of Fmoc-DIKVAV hydrogel provided a favorable microenvironment for cellular infiltration and axonal regrowth, a supportive role that unexpectedly appeared to be compromised by prior inclusion of rMPCs into the gel matrix. Impact statement The self-assembling peptide hydrogel, Fmoc-DIKVAV, is a biomimetic scaffold that is an effective cell and drug delivery system in the injured brain. We examined whether this hydrogel, alone or combined with mesenchymal precursor cells, was also able to stabilise spinal cord tissue after thoracic contusion injury and improve morphological and behavioral outcomes. While improved functionality was not consistently seen, there was reduced cyst size and increased tissue sparing in some groups. There was regenerative axonal growth into hydrogels, but only in initially cell-free implants. This type of polymer is a suitable candidate for further testing in spinal cord injury models.

Abnormally high level of methylation in the epigenome is a biomarker for various forms of cancer. It has been reported that a methylated nucleoside, 5-methylcytidine, when dissolved in water and then frozen, has a resonance absorption peak around 1.6 THz, which could allow for the determination of the degree of methylation using terahertz spectroscopy. This study attempts to replicate the experiment and reproduce this observation independently. Samples of ice were measured at 173 K to establish a baseline and frozen solutions of 5-methylcytidine were measured. Normal (physiological) 0.15 M saline, which is known to have a peak at 1.6 THz, was also measured. The use of a Gaussian function to fit a baseline to the broad (0.2 to 2.5 THz) absorption spectra consistently produced a peak residual around 1.6 THz for all three types of samples, with saline having the highest peak followed by ice. The peak for 5-methylcytidine was found to be smaller than that for ice, which contradicts previously published findings and illustrates the strong dependence of the result on the method of baseline fitting.

Repetitive transcranial magnetic stimulation (rTMS) is a form of non-invasive brain stimulation frequently used to induce neuroplasticity in the brain. Even at low intensities, rTMS has been shown to modulate aspects of neuronal plasticity such as motor learning and structural reorganisation of neural tissue. However, the impact of low intensity rTMS on glial cells such as astrocytes remains largely unknown. This study investigated changes in RNA (qPCR array: 125 selected genes) and protein levels (immunofluorescence) in cultured mouse astrocytes following a single session of low intensity repetitive magnetic stimulation (LI-rMS – 18 mT). Purified neonatal cortical astrocyte cultures were stimulated with either 1Hz (600 pulses), 10Hz (600 or 6000 pulses) or sham (0 pulses) LI-rMS, followed by RNA extraction at 5 h post-stimulation, or fixation at either 5 or 24-h post-stimulation. LI-rMS resulted in a two-to-four-fold downregulation of mRNA transcripts related to calcium signalling (Stim1 and Orai3), inflammatory molecules (Icam1) and neural plasticity (Ncam1). 10Hz reduced expression of Stim1, Orai3, Kcnmb4, and Ncam1 mRNA, whereas 1Hz reduced expression of Icam1 mRNA and signalling-related genes. Protein levels followed a similar pattern for 10Hz rMS, with a significant reduction of STIM1, ORAI3, KCNMB4, and NCAM1 protein compared to sham, but 1Hz increased STIM1 and ORAI3 protein levels relative to sham. These findings demonstrate the ability of 1Hz and 10Hz LI-rMS to modulate specific aspects of astrocytic phenotype, potentially contributing to the known effects of low intensity rTMS on excitability and neuroplasticity.

The incorporation of nanotechnology in regenerative medicine is at the nexus of fundamental innovations and early-stage breakthroughs, enabling exciting biomedical advances. One of the most exciting recent developments is the use of nanoscale constructs to influence the fate of cells, which are the basic building blocks of healthy function. Appropriate cell types can be effectively manipulated by direct cell reprogramming; a robust technique to manipulate cellular function and fate, underpinning burgeoning advances in drug delivery systems, regenerative medicine, and disease remodeling. Individual transcription factors, or combinations thereof, can be introduced into cells using both viral and nonviral delivery systems. Existing approaches have inherent limitations. Viral-based tools include issues of viral integration into the genome of the cells, the propensity for uncontrollable silencing, reduced copy potential and cell specificity, and neutralization via the immune response. Current nonviral cell reprogramming tools generally suffer from inferior expression efficiency. Nanomaterials are increasingly being explored to address these challenges and improve the efficacy of both viral and nonviral delivery because of their unique properties such as small size and high surface area. This review presents the state-of-the-art research in cell reprogramming, focused on recent breakthroughs in the deployment of nanomaterials as cell reprogramming delivery tools.

Repetitive transcranial magnetic stimulation (rTMS) is a non-invasive brain stimulation technique that has the potential to treat a variety of neurologic and psychiatric disorders. The extent of rTMS-induced neuroplasticity may be dependent on a subject’s brain state at the time of stimulation. Chronic low intensity rTMS (LI-rTMS) has previously been shown to induce beneficial structural and functional reorganisation within the abnormal visual circuits of ephrin-A2A5-/- mice in ambient lighting. Here, we administered chronic LI-rTMS in adult ephrin-A2A5-/- mice either in a dark environment or concurrently with voluntary locomotion. One day after the last stimulation session, optokinetic responses were assessed and fluorescent tracers were injected to map corticotectal and geniculocortical projections. We found that LI-rTMS in either treatment condition refined the geniculocortical map. Corticotectal projections were improved in locomotion+LI-rTMS subjects, but not in dark + LI-rTMS and sham groups. Visuomotor behaviour was not improved in any condition. Our results suggest that the beneficial reorganisation of abnormal visual circuits by rTMS can be significantly influenced by simultaneous, ambient visual input and is enhanced by concomitant physical exercise. Furthermore, the observed pathway-specific effects suggest that regional molecular changes and/or the relative proximity of terminals to the induced electric fields influence the outcomes of LI-rTMS on abnormal circuitry.

In an attempt to reproduce a previously-reported absorption peak in the spectrum of a frozen aqueous solution of 5-methylcytidine (5-mc), we observed a resonant feature around 1.6 THz in all samples containing ice, including pure ice itself. Thus, simply fitting the left-hand side of a Gaussian function to the spectrum of ice does not fully describe it. The previously reported peak feature depends on the subtraction of the Gaussian fit from the 5-mc sample spectrum. Through direct subtraction of the ice spectrum from that of 5-mc, our data did not reveal any statistically distinct spectral features that would allow differentiation between 5-mc and ice, however, we observed a variation in the overall absorption over the entire frequency range measured. X-ray diffraction confirmed the formation of complex hydrates, the structure of which may have a strong influence on the overall absorption based on the theory of hydration shells. The difference in the overall absorption may allow for the detection of methylated cytidine at high concentrations.

There is evidence that millimeter waves (MMWs) can have an impact on cellular function, including neurons. Earlier in vitro studies have shown that exposure levels well below the recommended safe limit of 1 mW/cm(2) cause changes in the action potential (AP) firing rate, resting potential, and AP pulse shape of sensory neurons in leech preparations as well as alter neuronal properties in rat cortical brain slices; these effects differ from changes induced by direct heating. In this article, we compare the responses of thermosensitive primary nociceptors of the medicinal leech under thermal heating and MMW irradiation (80–170 mW/cm(2) at 60 GHz). The results show that MMW exposure causes an almost twofold decrease in the threshold for activation of the AP compared with thermal heating (3.9 ± 0.4 vs. 8.3 ± 0.4 mV, respectively). Our analysis suggests that MMWs-mediated threshold alterations are not caused by the enhancement of voltage-gated sodium and potassium conductance. We propose that the reduction in AP threshold can be attributed to the sensitization of the transient receptor potential vanilloid 1-like receptor in the leech nociceptor. In silico modeling supported our experimental findings. Our results provide evidence that MMW exposure stimulates specific receptor responses that differ from direct thermal heating, fostering the need for additional studies.

PTEN expression is lost in many cancers, and even small changes in PTEN activity affect susceptibility and prognosis in a range of highly aggressive malignancies, such as melanoma and triple-negative breast cancer (TNBC). Loss of PTEN expression occurs via multiple mechanisms, including mutation, transcriptional repression and epigenetic silencing. Transcriptional repression of PTEN contributes to resistance to inhibitors used in the clinic, such as B-Raf inhibitors in BRAF mutant melanoma. We aimed to activate PTEN expression using the CRISPR system, specifically dead (d) Cas9 fused to the transactivator VP64-p65-Rta (VPR). dCas9-VPR was directed to the PTEN proximal promoter by single-guide RNAs (sgRNAs), in cancer cells that exhibited low levels of PTEN expression. The dCas9-VPR system increased PTEN expression in melanoma and TNBC cell lines, without transcriptional regulation at predicted off-target sgRNA binding sites. PTEN activation significantly repressed downstream oncogenic pathways, including AKT, mTOR, and MAPK signaling. BRAF V600E mutant melanoma cells transduced with dCas9-VPR displayed reduced migration, as well as diminished colony formation in the presence of B-Raf inhibitors, PI3K/mTOR inhibitors, and with combined PI3K/mTOR and B-Raf inhibition. CRISPR-mediated targeted activation of PTEN may provide an alternative therapeutic approach for highly aggressive cancers that are refractory to current treatments. Keywords: human PTEN protein, CRISPR/Cas systems, tumor suppressor genes, melanoma, breast neoplasms

During development of retinofugal pathways there is naturally occurring cell death of at least 50% of retinal ganglion cells (RGCs). In rats, RGC death occurs over a protracted pre- and early postnatal period, the timing linked to the onset of axonal ingrowth into central visual targets. Gene expression studies suggest that developing RGCs switch from local to target-derived neurotrophic support during this innervation phase. Here we investigated, in vitro and in vivo, how RGC birthdate affects the timing of the transition from intra-retinal to target-derived neurotrophin dependence. RGCs were pre-labeled with 5-Bromo-2′-Deoxyuridine (BrdU) at embryonic (E) day 15 or 18. For in vitro studies, RGCs were purified from postnatal day 1 (P1) rat pups and cultured with or without: (i) brain derived neurotrophic factor (BDNF), (ii) blocking antibodies to BDNF and neurotrophin 4/5 (NT-4/5), or (iii) a tropomyosin receptor kinase B fusion protein (TrkB-Fc). RGC viability was quantified 24 and 48 h after plating. By 48 h, the survival of purified βIII-tubulin immunopositive E15 but not E18 RGCs was dependent on addition of BDNF to the culture medium. For E18 RGCs, in the absence of exogenous BDNF, addition of blocking antibodies or TrkB-Fc reduced RGC viability at both 24 and 48 h by 25–40%. While this decrease was not significant due to high variance, importantly, each blocking method also consistently reduced complex process expression in surviving RGCs. In vivo, survival of BrdU and Brn3a co-labeled E15 or E18 RGCs was quantified in rats 24 h after P1 or P5 injection into the eye or contralateral superior colliculus (SC) of BDNF and NT-4/5 antibodies, or serum vehicle. The density of E15 RGCs 24 h after P1 or P5 injection of blocking antibodies was reduced after SC but not intraretinal injection. Antibody injections into either site had little obvious impact on viability of the substantially smaller population of E18 RGCs. In summary, most early postnatal RGC death in the rat involves the elimination of early-born RGCs with their survival primarily dependent upon the availability of target derived BDNF during this time. In contrast, late-born RGC survival may be influenced by additional factors, suggesting an association between RGC birthdate and developmental death mechanisms.

Recently, it was demonstrated that Terahertz (THz) radiation facilitates elongation of tubulin microtubules and actin microfilaments. The phenomenon could have a dramatic impact on living cells, especially in the phase of cell development when dramatic structural changes in cytoskeleton occur. In this study, the THz/Far-IR Beamline of Australian Synchrotron was used as a broadband source of THz radiation to stimulate perturbation in cytoskeleton structure in neuronal culture. Preliminary results indicate alterations of neurite shape and length in THz exposed cell cultures.

Inherited retinal diseases (IRDs) comprise a clinically and genetically heterogeneous group of disorders that can ultimately result in photoreceptor dysfunction/death and vision loss. With over 270 genes known to be involved in IRDs, translation of treatment strategies into clinical applications has been historically difficult. However, in recent years there have been significant advances in basic research findings as well as translational studies, culminating in an increasing number of clinical trials with the ultimate goal of reducing vision loss and associated morbidities. The recent approval of Luxturna® (voretigene neparvovec-rzyl) for Leber congenital amaurosis type 2 (LCA2) prompts a review of the current clinical trials for IRDs, with a particular focus on the importance of adeno-associated virus (AAV)-based gene therapies. The present article reviews the current state of AAV use in gene therapy clinical trials for IRDs, with a brief background on AAV and the reasons behind its dominance in ocular gene therapy. It will also discuss pre-clinical progress in AAV-based therapies aimed at treating other ocular conditions that can have hereditable links, and what alternative technologies are progressing in the same therapeutic space.

Multiple studies showed various effects of millimeter wave (MMW) radiation in biological objects [1]. The most common subjects to observe are genetic apparatus, shifts in activity and structure of macromolecules and cell function. Reasonably, biological effects of MMW radiation on living tissues are associated with inevitable heating of the sample due to high water content. Nevertheless, opposite to conductive heating alterations in neuronal electrophysiological activity was reported. This induces the question regarding the intracellular calcium homeostasis in neurons, due to critical importance of intracellular level of free calcium for neuron’s activity. This study indicated upregulation of free intracellular calcium in ex vivo experiment along with corelating morphological changes in the leech ganglia neuron.

Gene delivery has been extensively investigated for introducing foreign genetic material into cells to promote expression of therapeutic proteins or to silence relevant genes. This approach can regulate genetic or epigenetic disorders, offering an attractive alternative to pharmacological therapy or invasive protein delivery options. However, the exciting potential of viral gene therapy has yet to be fully realized, with a number of clinical trials failing to deliver optimal therapeutic outcomes. Reasons for this include difficulty in achieving localized delivery, and subsequently lower efficacy at the target site, as well as poor or inconsistent transduction efficiency. Thus, ongoing efforts are focused on improving local viral delivery and enhancing its efficiency. Recently, biomaterials have been exploited as an option for more controlled, targeted and programmable gene delivery. There is a growing body of literature demonstrating the efficacy of biomaterials and their potential advantages over other delivery strategies. This review explores current limitations of gene delivery and the progress of biomaterial-mediated gene delivery. The combination of biomaterials and gene vectors holds the potential to surmount major challenges, including the uncontrolled release of viral vectors with random delivery duration, poorly localized viral delivery with associated off-target effects, limited viral tropism, and immune safety concerns.

After damage to the adult mammalian central nervous system (CNS), surviving neurons have limited capacity to regenerate and restore functional connectivity. Conditional genetic deletion of PTEN results in robust CNS axon regrowth, while PTEN repression with short hairpin RNA (shRNA) improves regeneration but to a lesser extent, likely due to suboptimal PTEN mRNA knockdown using this approach. Here we employed the CRISPR/dCas9 system to repress PTEN transcription in neural cells. We targeted the PTEN proximal promoter and 5′ untranslated region with dCas9 fused to the repressor protein Krüppel-associated box (KRAB). dCas9-KRAB delivered in a lentiviral vector with one CRISPR guide RNA (gRNA) achieved potent and specific PTEN repression in human cell line models and neural cells derived from human iPSCs, and induced histone (H)3 methylation and deacetylation at the PTEN promoter. The dCas9-KRAB system outperformed a combination of four shRNAs targeting the PTEN transcript, a construct previously used in CNS injury models. The CRISPR system also worked more effectively than shRNAs for Pten repression in rat neural crest-derived PC-12 cells, and enhanced neurite outgrowth after nerve growth factor stimulation. PTEN silencing with CRISPR/dCas9 epigenetic editing may provide a new option for promoting axon regeneration and functional recovery after CNS trauma.

Large peripheral nerve (PN) defects require bridging substrates to restore tissue continuity and permit the regrowth of sensory and motor axons. We previously showed that cell-free PN segments repopulated ex vivo with Schwann cells (SCs) transduced with lentiviral vectors (LV) to express different growth factors (BDNF, CNTF or NT-3) supported the regeneration of axons across a 1 cm peroneal nerve defect (Godinho et al., 2013). Graft morphology, the number of regrown axons, the ratio of myelinated to unmyelinated axons, and hindlimb locomotor function differed depending on the growth factor engineered into SCs. Here we extend these observations, adding more LVs (expressing GDNF or NGF) and characterising regenerating sensory and motor neurons after injection of the retrograde tracer Fluorogold (FG) into peroneal nerve distal to grafts, 10 weeks after surgery. Counts were also made in rats with intact nerves and in animals receiving autografts, acellular grafts, or grafts containing LV-GFP transduced SCs. Counts and analysis of FG positive (+) DRG neurons were made from lumbar (L5) ganglia. Graft groups contained fewer labeled sensory neurons than non-operated controls, but this decrease was only significant in the LV-GDNF group. These grafts had a complex fascicular morphology that may have resulted in axon trapping. The proportion of FG+ sensory neurons immunopositive for calcitonin-gene related peptide (CGRP) varied between groups, there being a significantly higher percentage in autografts and most neurotrophic factor groups compared to the LV-CNTF, LV-GFP and acellular groups. Furthermore, the proportion of regenerating isolectin B4+ neurons was significantly greater in the LV-NT-3 group compared to other groups, including autografts and non-lesion controls. Immunohistochemical analysis of longitudinal graft sections revealed that all grafts contained a reduced number of choline acetyltransferase (ChAT) positive axons, but this decrease was significant only in the GDNF and NT-3 graft groups. We also assessed the number and phenotype of regrowing lumbar FG+ motor neurons in non-lesioned animals, and in rats with autografts, acellular grafts, or in grafts containing SCs expressing GFP, CNTF, NGF or NT-3. The overall number of FG+ motor neurons per section was similar in all groups; however in tissue immunostained for NeuN (expressed in α- but not γ-motor neurons) the proportion of NeuN negative FG+ neurons ranged from about 40–50% in all groups except the NT-3 group, where the percentage was 82%, significantly more than the SC-GFP group. Immunostaining for the vesicular glutamate transporter VGLUT-1 revealed occasional proprioceptive terminals in ‘contact’ with regenerating FG+ α-motor neurons in PN grafted animals, the acellular group having the lowest counts. In sum, while all graft types supported sensory and motor axon regrowth, there appeared to be axon trapping in SC-GDNF grafts, and data from the SC-NT-3 group revealed greater regeneration of sensory CGRP+ and IB4+ neurons, preferential regeneration of γ-motor neurons and perhaps partial restoration of monosynaptic sensorimotor relays.

Inherited retinal diseases (IRDs) are a leading cause of blindness. To date, 260 disease-causing genes have been identified, but there is currently a lack of available and effective treatment options. Cone photoreceptors are responsible for daylight vision but are highly susceptible to disease progression, the loss of cone-mediated vision having the highest impact on the quality of life of IRD patients. Cone degeneration can occur either directly via mutations in cone-specific genes (primary cone death), or indirectly via the primary degeneration of rods followed by subsequent degeneration of cones (secondary cone death). How cones degenerate as a result of pathological mutations remains unclear, hindering the development of effective therapies for IRDs. This review aims to highlight similarities and differences between primary and secondary cone cell death in inherited retinal diseases in order to better define cone death mechanisms and further identify potential treatment options.

Age-related changes in ventral lumbar spinal cord (L3-L5) were compared in young [3 month, (M)] and old (27 M) C57BL/6J male mice. The aged mice had previously been shown to exhibit sarcopenia and changes to peripheral nerve morphology. The putative connectivity of β-III tubulin positive α-motor neurons was compared in immunostained transverse sections using excitatory and inhibitory terminal markers vesicular glutamate transporter-1 (VGLUT1) and vesicular GABA transporter (VGAT). Glial fibrillary acidic protein (GFAP) and ionized calcium binding adaptor molecule 1 (Iba1) immunostaining was used to monitor changes in astrocyte and microglial phenotype respectively. For a given motor neuron, the neuronal perimeter was outlined and terminals immunoreactive for VGLUT1 or VGAT in close apposition to the soma were identified. By 27 M, the percentage coverage and total number of VGLUT1 immunoreactive terminals immediately adjacent to the soma of α-motor neurons was significantly decreased compared with young mice. However, percentage coverage of immunoreactive VGAT inhibitory terminals did not change significantly with age. The gray matter of 27 M spinal cords showed increased astrocytic and microglial activity. The loss of VGLUT1 terminals on α-motor neurons, terminals known to be derived from proprioceptive muscle afferents, may further impair sensorimotor control of hind limb skeletal muscle function in old mice.

Background Repetitive transcranial magnetic stimulation (rTMS) is a non-invasive technique that uses magnetic pulses over the cranium to induce electrical currents in underlying cortical tissue. Although rTMS has shown clinical utility for a number of neurological conditions, we have only limited understanding of how rTMS influences cellular function and cell-cell interactions. Objective In this study, we sought to investigate whether repeated magnetic stimulation (rMS) can influence astrocyte biology in vitro. Method We tested four different rMS frequencies and measured the calcium response in primary neonatal astrocyte cultures. We also tested the effect of rMS on astrocyte migration and proliferation in vitro. We tested 3 to 4 culture replicates and 17 to 34 cells for each rMS frequency (sham, 1 Hz, cTBS, 10 Hz and biomemetic high frequency stimulation - BHFS). Results Of all frequencies tested, 1 Hz stimulation resulted in a statistically significant rise in intracellular calcium in the cytoplasmic and nuclear compartments of the cultured astrocytes. This calcium rise did not affect migration or proliferation in the scratch assay, though astrocyte hypertrophy was reduced in response to 1 Hz rMS, 24 hours post scratch injury. Conclusion Our results provide preliminary evidence that rMS can influence astrocyte physiology, indicating the potential for a novel mechanism by which rTMS can influence brain activity.

During mammalian visual system development, retinal ganglion cells (RGCs) undergo extensive apoptotic death. In mouse retina, approximately 50% of RGCs present at birth (postnatal day 0; P0) die by P5, at a time when axons innervate central targets such as the superior colliculus (SC). We examined whether RGCs that make short-range axonal targeting errors within the contralateral SC are more likely to be eliminated during the peak period of RGC death (P1-P5), compared with RGCs initially making more accurate retinotopic connections. A small volume (2.3nl) of the retrograde nucleophilic dye Hoechst 33342 was injected into the superficial left SC of anaesthetised neonatal C57Bl/6J mice at P1 (n=5) or P4 (n=8), and the contralateral retina wholemounted 12 hours later. Retrogradely labelled healthy and dying (pyknotic) RGCs were identified by morphological criteria and counted. The percentage of pyknotic RGCs was analysed in relation to distance from the area of highest density RGC labelling, presumed to represent the most topographically accurate population. As expected, pyknotic RGC density at P1 was significantly greater than P4 (p

Intraorbital optic nerve crush in rodents is widely used as a model to study axon regeneration in the adult mammalian central nervous system. Recent studies using appropriate genetic manipulations have revealed remarkable abilities of mature retinal ganglion cell (RGC) axons to regenerate after optic nerve injury, with some studies demonstrating that axons can then go on to re-innervate a number of central visual targets with partial functional restoration. However, one confounding factor inherent to optic nerve crush injury is the potential incompleteness of the initial lesion, leaving spared axons that later on could erroneously be interpreted as regenerating distal to the injury site. Careful examination of axonal projection pattern and morphology may facilitate separating spared from regenerating RGC axons. Here we discuss morphological criteria and strategies that may be used to differentiate spared versus regenerated axons in the injured mammalian optic nerve.

Repetitive transcranial magnetic stimulation (rTMS), a non-invasive form of brain stimulation, has shown experimental and clinical efficacy in a range of neuromodulatory models, even when delivered at low intensity (i.e. subthreshold for action potential generation). After central nervous system (CNS) injury, studies suggest that reactive astrocytes and microglia can have detrimental but also beneficial effects; thus modulating glial activity, for example through application of rTMS, could potentially be a useful therapeutic tool following neurotrauma. Immunohistochemistry was used to measure the effect of low intensity rTMS (LI-rTMS) on GFAP (astrocyte), IBA1 (microglial), and CS56 (proteoglycan) expression in a unilateral penetrating cortical stab injury model of glial scarring in young adult and aged male and female C57BL6/J mice. Mice received contralateral low frequency, ipsilateral low frequency, ipsilateral high frequency or sham LI-rTMS (4-5mT intensity), for two weeks following injury. There was no significant difference in the overall volume of tissue containing GFAP positive (+) astrocytes, IBA1+ microglia, or proteoglycan expression, between sham and LI-rTMS-treated mice of all ages and sex. Importantly however, the density of GFAP+ astrocytes and IBA1+ microglia immediately adjacent to the injury was significantly reduced following ipsilateral low and high frequency stimulation in adult and aged females (p≤0.05), but was significantly increased in adult and aged males (p≤0.05). LI-rTMS effects were generally of greater magnitude in aged mice compared to young adult mice. These results suggest that sex differences need to be factored into therapeutic rTMS protocols. In particular, more work analyzing frequency and intensity specific effects, especially in relation to age and sex, is required to determine how rTMS can best be used to modify glial reactivity and phenotype following neurotrauma.

© 2017 The Author(s). Background: Following partial injury to the central nervous system, cells beyond the initial injury site undergo secondary degeneration, exacerbating loss of neurons, compact myelin and function. Changes in Ca2+ flux are associated with metabolic and structural changes, but it is not yet clear how flux through specific ion channels contributes to the various pathologies. Here, partial optic nerve transection in adult female rats was used to model secondary degeneration. Treatment with combinations of three ion channel inhibitors was used as a tool to investigate which elements of oxidative and structural damage related to long term functional outcomes. The inhibitors employed were the voltage gated Ca2+ channel inhibitor Lomerizine (Lom), the Ca2+ permeable AMPA receptor inhibitor YM872 and the P2X7 receptor inhibitor oxATP. Results: Following partial optic nerve transection, hyper-phosphorylation of Tau and acetylated tubulin immunoreactivity were increased, and Nogo-A immunoreactivity was decreased, indicating that axonal changes occurred acutely. All combinations of ion channel inhibitors reduced hyper-phosphorylation of Tau and increased Nogo-A immunoreactivity at day 3 after injury. However, only Lom/oxATP or all three inhibitors in combination significantly reduced acetylated tubulin immunoreactivity. Most combinations of ion channel inhibitors were effective in restoring the lengths of the paranode and the paranodal gap, indicative of the length of the node of Ranvier, following injury. However, only all three inhibitors in combination restored to normal Ankyrin G length at the node of Ranvier. Similarly, HNE immunoreactivity and loss of oligodendrocyte precursor cells were only limited by treatment with all three ion channel inhibitors in combination. Conclusions: Data indicate that inhibiting any of a range of ion channels preserves certain elements of axon and node structure and limits some oxidative damage following injury, whereas ionic flux through all three channels must be inhibited to prevent lipid peroxidation and preserve Ankyrin G distribution and OPCs.

The aim of the present study was to determine whether adeno-associated viral vector (AAV) mediated transfer of ciliary neurotrophic factor (CNTF) and RhoA shRNA has additive effects on promoting the survival and axon regeneration of retinal ganglion cells (RGCs) after optic nerve crush (ONC). Silencing effects of AAV-RhoA shRNA were confirmed by examining neurite outgrowth in PC12 cells, and by quantifying RhoA expression levels with western blotting. Young adult Fischer rats received an intravitreal injection of (i) saline, (ii) AAV green fluorescent protein (GFP), (iii) AAV-CNTF, (iv) AAV-RhoA shRNA, or (v) a combination of both AAV-CNTF and AAV-RhoA shRNA. Two weeks later, the ON was completely crushed. Three weeks after ONC, RGC survival was estimated by counting βIII-tubulin-positive neurons in retinal whole mounts. Axon regeneration was evaluated by counting GAP-43-positive axons in the crushed ON. It was found that AAV-RhoA shRNA decreased RhoA expression levels and promoted neurite outgrowth in vitro. In the ONC model, AAV-RhoA shRNA by itself had only weak beneficial effects on RGC axon regeneration. However, when combined with AAV-CNTF, AAV-RhoA shRNA significantly improved the therapeutic effect of AAV-CNTF on axon regeneration by nearly two fold, even though there was no significant change in RGC viability. In sum, this combination of vectors increases the regenerative response and can lead to more successful therapeutic outcomes following neurotrauma.

MMW radiation can be used to focally deposit energy into biological tissue, which converts in it into thermal energy due to high absorption properties, specifically of water. As a result, it can affect the functionality of some biological tissues. Neuronal tissue is one of the most susceptible types due to its high sensitivity to environmental changes, such as electric field and temperature. Here we investigate the effect of MMW mediated heating and conventional thermal heating on action potential voltage initiation in spontaneously active interneuron of leech. The results demonstrate that MMW radiation has a different effect to thermal heating but only at initial stages of sample heating.

We previously identified that ngr1 allele deletion limits the severity of experimental autoimmune encephalomyelitis (EAE) by preserving axonal integrity. However, whether this favorable outcome observed in EAE is a consequence of an abrogated neuronal-specific pathophysiological mechanism, is yet to be defined. Here we show that, Cre-loxP-mediated neuron-specific deletion of ngr1 preserved axonal integrity, whereas its re-expression in ngr1(−/−) female mice potentiated EAE-axonopathy. As a corollary, myelin integrity was preserved under Cre deletion in ngr1(flx/flx), retinal ganglion cell axons whereas, significant demyelination occurred in the ngr1(−/−) optic nerves following the re-introduction of NgR1. Moreover, Cre-loxP-mediated axon-specific deletion of ngr1 in ngr1(flx/flx) mice also demonstrated efficient anterograde transport of fluorescently-labeled ChTxβ in the optic nerves of EAE-induced mice. However, the anterograde transport of ChTxβ displayed accumulation in optic nerve degenerative axons of EAE-induced ngr1(−/−) mice, when NgR1 was reintroduced but was shown to be transported efficiently in the contralateral non- recombinant adeno-associated virus serotype 2-transduced optic nerves of these mutant mice. We further identified that the interaction between the axonal motor protein, Kinesin-1 and collapsin response mediator protein 2 (CRMP2) was unchanged upon Cre deletion of ngr1. Whereas, this Kinesin-1/CRMP2 association was reduced when NgR1 was re-expressed in the ngr1(−/−) optic nerves. Our data suggest that NgR1 governs axonal degeneration in the context of inflammatory-mediated demyelination through the phosphorylation of CRMP2 by stalling axonal vesicular transport. Moreover, axon-specific deletion of ngr1 preserves axonal transport mechanisms, blunting the induction of inflammatory demyelination and limiting the severity of EAE. SIGNIFICANCE STATEMENT Multiple sclerosis (MS) is commonly induced by aberrant immune-mediated destruction of the protective sheath of nerve fibers (known as myelin). However, it has been shown that MS lesions do not only consist of this disease pattern, exhibiting heterogeneity with continual destruction of axons. Here we investigate how neuronal NgR1 can drive inflammatory-mediated axonal degeneration and demyelination within the optic nerve by analyzing its downstream signaling events that govern axonal vesicular transport. We identify that abrogating the NgR1/pCRMP2 signaling cascade can maintain Kinesin-1-dependent anterograde axonal transport to limit inflammatory-mediated axonopathy and demyelination. The ability to differentiate between primary and secondary mechanisms of axonal degeneration may uncover therapeutic strategies to limit axonal damage and progressive MS.

Repetitive Transcranial Magnetic Stimulation (rTMS) is a form of non-invasive brain stimulation that has shown therapeutic potential for various nervous system disorders. In addition to its modulatory effects on neuronal excitability, rTMS is capable of altering neurotransmitter (e.g., glutamate, GABA, dopamine and serotonin) concentrations in cortical and subcortical brain regions. Here we used a modified liquid chromatography coupled tandem mass spectrometry (LC-MS/MS) to quantify changes in 27 free amino acids and the monoamines: dopamine (DA), serotonin (5HT) and their metabolites (DOPAC, HVA; 5HIAA) in the mouse brain. Awake C57BL/6 J mice (either sex, 8-12 weeks old) received 10 Hz rTMS using two devices that can deliver low (LI-; 12 mT; custom built) or high (Fo8-; 1.2 T; MagVenture) intensity rTMS. Sham (unstimulated) mice were used as controls. Samples were collected immediately following a single session of rTMS or sham and processed for LC-MS/MS. The modified LC-MS/MS method used to detect DA, 5-HT and their metabolites showed good accuracy and precision with regression coefficients greater than 0.999, and an intra- and inter-day reproducibility with values 13%. Fo8-rTMS induced a significant reduction in cortical 5-HT turnover rates, hippocampal DOPAC and an increase in striatal DOPAC concentrations. Fo8-rTMS also reduced concentrations of hippocampal α-aminoadipic acid, and striatal serine, threonine, sarcosine, aspartate and glutamate. There were no changes in the level of any compounds following LI-rTMS as compared to sham. The rapid change in monoamine turnover and amino acid concentrations following Fo8-rTMS but not LI-rTMS suggests that different stimulation parameters recruit different cellular mechanisms related to rTMS-induced plasticity. The described method can be used for the characterisation of trace levels of neurotransmitters and amino acids in brain tissue homogenates, providing a useful and precise tool to investigate localised neurotransmitter changes in animal models of health and disease.

Neuroethics is central to the Australian Brain Initiative’s aim to sustain a thriving and responsible neurotechnology industry. Diverse and inclusive community and stakeholder engagement and a trans-disciplinary approach to neuroethics will be key to the success of the Australian Brain Initiative. Refereed/Peer-reviewed

Changes within the dopaminergic system induced by repetitive transcranial magnetic stimulation (rTMS) may contribute to its therapeutic effects; however, dopamine-related behavioral effects of rTMS have not been widely investigated. We recently showed that ephrin-A2A5-/- mice completed significantly fewer trials in a visual task than wildtype mice, and that concurrent low-intensity (LI-) rTMS during the task could partially rescue the abnormal behavior [Poh et al. 2018, eNeuro, vol. 5]. Here, we investigated whether the behavioral differences in ephrin-A2A5-/- mice are due to abnormal motivation, primarily a dopamine-modulated behavior, and whether LI-rTMS would increase motivation. Ephrin-A2A5-/- and wildtype mice underwent 14 daily sessions of progressive ratio (PR) tasks and received either sham or LI-rTMS during the first 10 min. Ephrin-A2A5-/- mice responded more than wildtype comparisons, and LI-rTMS did not influence task performance for either strain. Therefore concurrent stimulation does not influence motivation in a PR task. However, ephrin-A2A5-/- mice did have abnormal performance in the PR tasks after a change in the PR schedule which suggests perseverative behavior. We stained for c-Fos in the prelimbic area (PrL), ventral tegmental area and nucleus accumbens (NAc) core and shell to examine neuronal activity from the final PR session. Sham ephrin-A2A5-/- mice had lower c-Fos expression in the PrL and NAc vs. wildtype mice. Ephrin-A2A5-/- mice that received LI-rTMS showed c-Fos expression closer to wildtype levels in the NAc. Combined with high PR performance, ephrin-A2A5-/- mice show an abnormal shift to habitual responding and LI-rTMS may attenuate this shift.

Cellular transplantation strategies utilizing intraspinal injection of mesenchymal progenitor cells (MPCs) have been reported as beneficial for spinal cord injuries. However, intraspinal injection is not only technically challenging, but requires invasive surgical procedures for patients. Therefore, we investigated the feasibility and potential benefits of noninvasive intravenous injection of MPCs in two models of cervical spinal cord injury, unilateral C5 contusion and complete unilateral C5 hemisection. MPCs isolated from green fluorescence protein (GFP)-luciferase transgenic mice compact bone (1 × 106 cells), or vehicle Hank's Buffered Saline Solution (HBSS), were intravenously injected via the tail vein at D1, D3, D7, D10, or D14. Transplanted MPCs were tracked via bioluminescence imaging. Live in vivo imaging data showed that intravenously injected MPCs accumulate in the lungs, confirmed by postmortem bioluminescence signal—irrespective of the time of injection or injury model. The results showed a rapid, positive modulation of the inflammatory response providing protection to the injured spinal cord tissue. Histological processing of the lungs showed GFP+ cells evenly distributed around the alveoli. We propose that injected cells can act as cellular target decoys to an immune system primed by injury, thereby lessening the inflammatory response at the injury site. We also propose that intravenous injected MPCs modulate the immune system via the lungs through secreted immune mediators or contact interaction with peripheral organs. In conclusion, the timing of intravenous injection of MPCs is key to the success for improving function and tissue preservation following cervical spinal cord injury.