A bipedal mammalian model for spinal cord injury research: The tammar wallaby [version 1; referees: 2 approved]

Background: Most animal studies of spinal cord injury are conducted in quadrupeds, usually rodents. It is unclear to what extent functional results from such studies can be translated to bipedal species such as humans because bipedal and quadrupedal locomotion involve very different patterns of spinal control of muscle coordination. Bipedalism requires upright trunk stability and coordinated postural muscle control; it has been suggested that peripheral sensory input is less important in humans than quadrupeds for recovery of locomotion following spinal injury. Methods: We used an Australian macropod marsupial, the tammar wallaby (Macropus eugenii), because tammars exhibit an upright trunk posture, human-like alternating hindlimb movement when swimming and bipedal over-ground locomotion. Regulation of their muscle movements is more similar to humans than quadrupeds. At different postnatal (P) days (P7–60) tammars received a complete mid-thoracic spinal cord transection. Morphological repair, as well as functional use of hind limbs, was studied up to the time of their pouch exit. Results: Growth of axons across the lesion restored supraspinal innervation in animals injured up to 3 weeks of age but not in animals injured after 6 weeks of age. At initial pouch exit (P180), the young injured at P7-21 were able to hop on their hind limbs similar to age-matched controls and to swim albeit with a different stroke. Those animals injured at P40-45 appeared to be incapable of normal use of hind limbs even while still in the pouch. Conclusions: Data indicate that the characteristic over-ground locomotion of tammars provides a model in which regrowth of supraspinal connections across the site of injury can be studied in a bipedal animal. Forelimb weight-bearing motion and peripheral sensory input appear not to compensate for lack of hindlimb control, as occurs in quadrupeds. Tammars may be a more appropriate model for studies of therapeutic interventions relevant to humans

Combination of Histone Deacetylase Inhibitor Panobinostat (LBH589) with β-Catenin Inhibitor Tegavivint (BC2059) Exerts Significant Anti-Myeloma Activity Both In Vitro and In Vivo

Over the last three decades changes in the treatment paradigm for newly diagnosed multiple myeloma (MM) have led to a significant increase in overall survival. Despite this, the majority of patients relapse after one or more lines of treatment while acquiring resistance to available therapies. Panobinostat, a pan-histone deacetylase inhibitor, was approved by the FDA in 2015 for patients with relapsed MM but how to incorporate panobinostat most effectively into everyday practice remains unclear. Dysregulation of the Wnt canonical pathway, and its key mediator β-catenin, has been shown to be important for the evolution of MM and the acquisition of drug resistance, making it a potentially attractive therapeutic target. Despite concerns regarding the safety of Wnt pathway inhibitors, we have recently shown that the β-catenin inhibitor Tegavivint is deliverable and effective in in vivo models of MM. In this study we show that the combination of low concentrations of panobinostat and Tegavivint have significant in vitro and in vivo anti-MM effects including in the context of proteasome inhibitor resistance, by targeting both aerobic glycolysis and mitochondrial respiration and the down-regulation of down-stream β-catenin targets including myc, cyclinD1, and cyclinD2. The significant anti-MM effect of this novel combination warrants further evaluation for the treatment of MM patients with relapsed and/or refractory MM

Cellular specificity of the bloodCSF barrier for albumin transfer across the choroid plexus epithelium

To maintain the precise internal milieu of the mammalian central nervous system, well-controlled transfer of molecules from periphery into brain is required. Recently the soluble and cell-surface albumin-binding glycoprotein SPARC (secreted protein acidic and rich in cysteine) has been implicated in albumin transport into developing brain, however the exact mechanism remains unknown. We postulate that SPARC is a docking site for albumin, mediating its uptake and transfer by choroid plexus epithelial cells from blood into cerebrospinal fluid (CSF). We used in vivo physiological measurements of transfer of endogenous (mouse) and exogenous (human) albumins, in situ Proximity Ligation Assay (in situ PLA), and qRT-PCR experiments to examine the cellular mechanism mediating protein transfer across the bloodCSF interface. We report that at all developmental stages mouse albumin and SPARC gave positive signals with in situ PLAs in plasma, CSF and within individual plexus cells suggesting a possible molecular interaction. In contrast, in situ PLA experiments in brain sections from mice injected with human albumin showed positive signals for human albumin in the vascular compartment that were only rarely identifiable within choroid plexus cells and only at older ages. Concentrations of both endogenous mouse albumin and exogenous (intraperitoneally injected) human albumin were estimated in plasma and CSF and expressed as CSF/plasma concentration ratios. Human albumin was not transferred through the mouse bloodCSF barrier to the same extent as endogenous mouse albumin, confirming results from in situ PLA. During postnatal development Sparc gene expression was higher in early postnatal ages than in the adult and changed in response to altered levels of albumin in blood plasma in a differential and developmentally regulated manner. Here we propose a possible cellular route and mechanism by which albumin is transferred from blood into CSF across a sub-population of specialised choroid plexus epithelial cells.HEALTH-F2-2009-24177

Weight-Bearing Locomotion in the Developing Opossum, <i>Monodelphis domestica</i> following Spinal Transection: Remodeling of Neuronal Circuits Caudal to Lesion

<div><p>Complete spinal transection in the mature nervous system is typically followed by minimal axonal repair, extensive motor paralysis and loss of sensory functions caudal to the injury. In contrast, the immature nervous system has greater capacity for repair, a phenomenon sometimes called the infant lesion effect. This study investigates spinal injuries early in development using the marsupial opossum <i>Monodelphis domestica</i> whose young are born very immature, allowing access to developmental stages only accessible <i>in utero</i> in eutherian mammals. Spinal cords of <i>Monodelphis</i> pups were completely transected in the lower thoracic region, T10, on postnatal-day (P)7 or P28 and the animals grew to adulthood. In P7-injured animals regrown supraspinal and propriospinal axons through the injury site were demonstrated using retrograde axonal labelling. These animals recovered near-normal coordinated overground locomotion, but with altered gait characteristics including foot placement phase lags. In P28-injured animals no axonal regrowth through the injury site could be demonstrated yet they were able to perform weight-supporting hindlimb stepping overground and on the treadmill. When placed in an environment of reduced sensory feedback (swimming) P7-injured animals swam using their hindlimbs, suggesting that the axons that grew across the lesion made functional connections; P28-injured animals swam using their forelimbs only, suggesting that their overground hindlimb movements were reflex-dependent and thus likely to be generated locally in the lumbar spinal cord. Modifications to propriospinal circuitry in P7- and P28-injured opossums were demonstrated by changes in the number of fluorescently labelled neurons detected in the lumbar cord following tracer studies and changes in the balance of excitatory, inhibitory and neuromodulatory neurotransmitter receptors’ gene expression shown by qRT-PCR. These results are discussed in the context of studies indicating that although following injury the isolated segment of the spinal cord retains some capability of rhythmic movement the mechanisms involved in weight-bearing locomotion are distinct.</p></div

Behavioural assessments.

<p>Control (n = 11, white bars), P7-injured (n = 7, black bars) and P28-injured (n = 9, grey bars) opossums. <b><i>A</i></b>: BBB Locomotor rating scores. <b><i>B</i></b>: Representative gait traces from treadmill locomotion. <b><i>C</i></b>: Regularity index. <i>D</i>: Step duration. <i>E</i>: Stance duration. <i>F</i>: Swing duration. Mean ± sem; *<i>P</i>≤0.05 vs control; ^#<i>P</i>≤0.05 vs P7-inj, by One-way ANOVA.</p

Limb placement phase lags.

<p>Control (n = 11, white bars), P7-injured (n = 7, black bars) and P28-injured (n = 9,grey bars) opossums. <b><i>A</i></b>: Schematic examples showing calculation method for girdle, diagonal and ipsilateral phase lags. These examples are taken from the control animal in Fig. 1B. <b><i>B</i></b>: Forelimb and hindlimb girdle phase lags <b><i>C</i></b>: Diagonal phase lags. <b><i>D</i></b>: Ipsilateral phase lags. Mean ± sem; *<i>P</i>≤0.05 vs control; ^#<i>P</i>≤0.05 vs P7-inj, by One-way ANOVA.</p

Morphometric measurements of <i>Monodelphis</i> spinal cords.

<p>Control (n = 5, white circles), P7-injured (n = 3, black circles) and P28-injured (n = 4, grey circles) opossum spinal cords. <b><i>A</i></b>: Representative whole mounts of lower cervical and thoracic spinal cords from control (top), P7-injured (middle) and P28-injured (bottom) <i>Monodelphis</i>. <b><i>B</i></b>: Whole spinal cord cross-sectional area along the length of the spinal cord. <b><i>C</i></b>: Grey matter area. <b><i>D</i></b>: White matter area. Mean ± sem. *<i>P</i>≤0.05 vs control. Stars denoting significance appear immediately <i>above</i> data for P7-injured animals or immediately <i>below</i> data for P28-injured animals.</p

Primers used in this study.

<p>Designed with reference to <i>Monodelphis domestica</i> database <b>(Ensembl database MonDom5</b><a href="http://www.ensembl.org/Monodelphis_domestica" target="_blank">http://www.ensembl.org/Monodelphis_domestica</a><b>)</b>.</p

Gene expression quantitation by qRT-PCR for L1–2 and L3–5 spinal segments.

<p>Gene expression changes in the spinal cords of P7- (solid bars) and P28-injured (hatched bars) opossums are shown relative to control expression. <b><i>A</i></b>: Gene expression ratios for the L1–2 spinal segments. <b><i>B</i></b>: Gene expression ratios for the L3–5 spinal segments. All data are mean ± sem. *<i>P≤0.05</i>; <i>P</i> values ≤0.1 are indicated in parentheses.</p

Re-transection of P28-injured opossum spinal cord.

<p>In adulthood the spinal cord of one P28-injured <i>Monodelphis</i> was re-transected through the centre of the injury site and allowed to recover for 3 weeks before behavioural and labelling studies were performed. <b><i>A</i></b><i>:</i> Representative gait trace from P28-injured opossum in adulthood before (left panel) and after (right panel) re-transection. <b><i>B</i></b><i>:</i> Image of section through the medulla of re-transected P28-injured animal following retrograde tracing. <b><i>C</i></b><i>:</i> Images of sections through the spinal cord above (left panel) and below (right panel) the level of the re-transection. Re-transection was performed at the same level as the initial transection during the neonatal period and did not result in a second distinct transection site (data not shown).</p