Anatomy and Neuroscience - Research Publications
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ItemNo Preview AvailableThe effect of TrkB receptor knockdown on mouse retinal ganglion cell function and their response to acute mild intraocular pressure stress(Association for Research in Vision and Ophthalmology, 2019-07-01)Purpose : To examine the effect of tropomyosin receptor kinase B (TrkB) receptor removal on basal retinal ganglion cell (RGC) function and recovery from acute intraocular pressure (IOP) stress following conditional deletion of neuronal-specific TrkB receptors in adult mice. Methods : Conditional TrkB receptor knockout (KO) from Thy-1 positive neurons was induced via daily tamoxifen injections (100 ul i.p. 75mg/kg, 5 days) in 3-month old Thy1-GFP CreERT2+/- TrkBfl/fl transgenic (TrkB KO n=12) and control CreERT2-/- TrkBfl/fl mice (n=12). Four weeks later, one eye was exposed to controlled IOP stress of 50mmHg for 30 minutes, achieved via anterior chamber cannulation (50 μm glass micropipette connected to height-adjustable Hanks balanced salt solution reservoir; 80:10mg/kg ketamine:xylazine). After 7 days of recovery, retinal function (full-field electroretinogram -5.53 – 2.07 log cd.s/m2) and structure (optical coherence tomography) were assessed in sedated mice. Following in vivo assays, eyes were enucleated for immunohistochemical assessment of TrkB receptor KO efficiency using confocal microscopy. Unpaired t-test and two-way ANOVA were used for statistical analysis. Results : TrkB receptor expression was largely confined to the ganglion cell layers and reduced by 81.3±5.8% in TrkB KO retinas compared to controls (P<0.05). Deletion of TrkB receptors significantly reduced RGC-mediated negative scotopic threshold response (nSTR -39.1±13.7% P<0.05, positive STR -38.0±12.1% P=0.05). No changes in photoreceptor (amplitude P>0.05, sensitivity P>0.05) and bipolar cell (amplitude P>0.05, sensitivity P>0.05) function. At day 7 post-IOP stress, photoreceptor and bipolar cell responses recovered back to baseline whilst RGC function did not (pSTR P<0.05; nSTR P<0.05). This effect was similar for both genotypes. TrkB KO did not affect total retinal, retinal nerve fibre, ganglion cell and inner plexiform layer thicknesses compared with control retina (P>0.05). Conclusions : Conditional removal of TrkB receptors in adult mice suggests that TrkB is critical for the ongoing maintenance of ganglion cell function. Specific changes in RGC morphology, synapse expression or intrinsic excitability associated with TrkB deficiency remain to be elucidated. It appears that TrkB receptors do not play an integral role in recovery from a single episode of mild IOP stress.
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ItemPeer Tutoring for Anatomy Workshops in Cambodia(WILEY, 2019-01)Historical loss of staff and teaching resources in Cambodia has resulted in significant challenges to anatomy education. Small group anatomy teaching opportunities are limited. A visit to Cambodia by a teaching team from the University of Melbourne in 2010 demonstrated it was possible to implement well-resourced anatomy workshops for this purpose. However, continuation of the workshop program was inhibited by the limited number of local teaching staff. In 2015, another team from the University of Melbourne returned to Cambodia to implement anatomy workshops that incorporated peer tutoring. The objective was to improve teacher-to-student ratios and to demonstrate that interactive anatomy workshops could be delivered successfully despite low staff numbers. The anatomy workshops were attended by 404 students of Medicine, Dentistry, Nursing, and Midwifery at the University of Puthisastra. Medical students were invited to act as peer tutors for nursing students. A five-point Likert scale questionnaire was used to determine student satisfaction with both the workshops and peer tutoring. The overwhelming majority were positive about the workshops and keen for them to continue. Almost all medical students who acted as peer tutors agreed or strongly agreed that this role increased their anatomical knowledge (98%) and confidence (94%). Most nursing students agreed or strongly agreed with statements that they would like peer tutoring to continue (94%) and that they would like to be peer tutors themselves (88%). This report demonstrates that peer tutoring could be an effective tool in educational settings where poor staff-to-student ratios limit delivery of interactive workshops.
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ItemPeripheral Nerve Regeneration Is Independent From Schwann Cell p75NTR Expression(FRONTIERS MEDIA SA, 2019-05-29)Schwann cell reprogramming and differentiation are crucial prerequisites for neuronal regeneration and re-myelination to occur following injury to peripheral nerves. The neurotrophin receptor p75NTR has been identified as a positive modulator for Schwann cell myelination during development and implicated in promoting nerve regeneration after injury. However, most studies base this conclusion on results obtained from complete p75NTR knockout mouse models and cannot dissect the specific role of p75NTR expressed by Schwann cells. In this present study, a conditional knockout model selectively deleting p75NTR expression in Schwann cells was generated, where p75NTR expression is replaced with that of an mCherry reporter. Silencing of Schwann cell p75NTR expression was confirmed in the sciatic nerve in vivo and in vitro, without altering axonal expression of p75NTR. No difference in sciatic nerve myelination during development or following sciatic nerve crush injury was observed, as determined by quantification of both myelinated and unmyelinated nerve fiber densities, myelinated axonal diameter and myelin thickness. However, the absence of Schwann cell p75NTR reduced motor nerve conduction velocity after crush injury. Our data indicate that the absence of Schwann cell p75NTR expression in vivo is not critical for axonal regrowth or remyelination following sciatic nerve crush injury, but does play a key role in functional recovery. Overall, this represents the first step in redefining the role of p75NTR in the peripheral nervous system, suggesting that the Schwann cell-axon unit functions as a syncytium, with the previous published involvement of p75NTR in remyelination most likely depending on axonal/neuronal p75NTR and/or mutual glial-axonal interactions.
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ItemInhibiting Bone Morphogenetic Protein 4 Type I Receptor Signaling Promotes Remyelination by Potentiating Oligodendrocyte Differentiation.(Society for Neuroscience, 2019)Blocking inhibitory factors within CNS demyelinating lesions is regarded as a promising strategy to promote remyelination. Bone morphogenetic protein 4 (BMP4) is an inhibitory factor present in demyelinating lesions. Noggin, an endogenous antagonist to BMP, has previously been shown to increase the number of oligodendrocytes and promote remyelination in vivo. However, it remains unclear how BMP4 signaling inhibits remyelination. Here we investigated the downstream signaling pathway that mediates the inhibitory effect that BMP4 exerts upon remyelination through pharmacological and transgenic approaches. Using the cuprizone mouse model of central demyelination, we demonstrate that selectively blocking BMP4 signaling via the pharmacological inhibitor LDN-193189 significantly promotes oligodendroglial differentiation and the extent of remyelination in vivo This was accompanied by the downregulation of transcriptional targets that suppress oligodendrocyte differentiation. Further, selective deletion of BMP receptor type IA (BMPRIA) within primary mouse oligodendrocyte progenitor cells (OPCs) significantly enhanced their differentiation and subsequent myelination in vitro Together, the results of this study identify that BMP4 signals via BMPRIA within OPCs to inhibit oligodendroglial differentiation and their capacity to myelinate axons, and suggest that blocking the BMP4/BMPRIA pathway in OPCs is a promising strategy to promote CNS remyelination.
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ItemActivity-dependent central nervous system myelination throughout life(WILEY, 2019-02)Myelin, the multilayered membrane surrounding many axons in the nervous system, increases the speed by which electrical signals travel along axons and facilitates neuronal communication between distant regions of the nervous system. However, how neuronal signals influence the myelinating process in the CNS is still largely unclear. Recent studies have significantly advanced this understanding, identifying important roles for neuronal activity in controlling oligodendrocyte development and their capacity of producing myelin in both developing and mature CNS. Here, we review these recent advances, and discuss potential mechanisms underpinning activity-dependent myelination and how remyelination may be stimulated via manipulating axonal activity, raising new questions for future research.
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ItemImaging and Quantification of Myelin Integrity After Injury With Spectral Confocal Reflectance Microscopy(FRONTIERS MEDIA SA, 2019-11-19)Developing a high-throughput approach to quantify the extent of myelin integrity in preclinical models of demyelinating diseases will enhance our capacity to identify novel therapies for myelin repair. In light of the technical limitations of electron microscopy and immunohistochemical analyses of myelination, we have utilized a novel imaging technique, spectral confocal reflectance (SCoRe) microscopy. SCoRe takes advantage of the optically reflective properties of compact myelin, allowing the integrity of compact myelin to be quantified over the course of the cuprizone-induced model of central demyelination. We applied SCoRe imaging on fixed frozen brain sections. SCoRe analysis of control mice identified an increase in corpus callosum myelination during the period of cuprizone administration and recovery, suggesting that the normal developmental processes of myelination are ongoing at this time. Importantly, analysis of mice subjected to cuprizone identified a significant reduction in compact myelin in both rostral and caudal corpus callosum compared to age-matched control mice. SCoRe microscopy also allowed the visualization and quantification of the amount of myelin debris in demyelinating lesions. Combining SCoRe imaging with immunohistochemistry, we quantified the amount of myelin debris within IBA-1+ microglia and found that 11% of myelin debris colocalized in microglia irrespective of the callosal regions, with the vast majority of debris outside of microglia. In summary, we have demonstrated that SCoRe microscopy is an effective and powerful tool to perform both quantitative and qualitative analyses of compact myelin integrity in health or after injury in vivo, demonstrating its future application in high-throughput assessments and screening of the therapeutic efficacy of myelin repair therapies in preclinical animal models of demyelinating diseases.
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ItemTrkB Agonist LM22A-4 Increases Oligodendroglial Populations During Myelin Repair in the Corpus Callosum(FRONTIERS MEDIA SA, 2019-08-27)The neurotrophin, brain-derived neurotrophic factor (BDNF) promotes central nervous system (CNS) myelination during development and after injury. This is achieved via activation of oligodendrocyte-expressed tropomyosin-related kinase (Trk) B receptors. However, while administration of BDNF has shown beneficial effects, BDNF itself has a poor pharmacokinetic profile. Here, we compare two TrkB-targeted BDNF-mimetics, the structural-mimetic, tricyclic dimeric peptide-6 (TDP6) and the non-peptide small molecule TrkB agonist LM22A-4 in a cuprizone model of central demyelination in female mice. Both mimetics promoted remyelination, increasing myelin sheath thickness and oligodendrocyte densities after 1-week recovery. Importantly, LM22A-4 exerts these effects in an oligodendroglial TrkB-dependent manner. However, analysis of TrkB signaling by LM22A-4 suggests rather than direct activation of TrkB, LM22A-4 exerts its effects via indirect transactivation of Trk receptors. Overall, these studies support the therapeutic strategy to selectively targeting TrkB activation to promote remyelination in the brain.