The TrkB agonist, 7,8-dihydroxyflavone, impairs fracture healing in mice.

OBJECTIVES: To study the effects of the selective TrkB agonist, 7,8-dihydroxyflavone (7,8-DHF), on fracture healing in mice and on an osteoprogenitor cell line, Kusa4b10, in vitro. METHODS: Mice received unilateral closed mid-shaft tibial fractures and treated for two weeks with vehicle or 5 mg/kg/day DHF and euthanised at 28 days post-fracture. Calluses were analysed by micro-computed tomography (µCT) and three-point bending biomechanical test. Kusa4b10 cells were cultured with 50nM of 7,8-DHF or vehicle for 3-, 7-, 14-days for RT-PCR, and 21 days for mineralization. RESULTS: µCT found 7,8-DHF calluses had decreased tissue volume (p=0.042), mean polar moment of inertia (p = 0.004), and mean cross-sectional area (p=0.042) compared to controls. At 28 days biomechanical analyses showed 7,8-DHF treatment decreased peak force (p=0.011) and stiffness per unit area (p=0.012). 7,8-DHF treatment did not change Kusa4b10 gene expression of Runx2 and alkaline phosphatase at all time points, nor mineralization. CONCLUSIONS: 7,8-DHF treatment had a negative impact on fracture healing at 28 days post-fracture via an unknown mechanism. 7,8-DHF may have had a central role in impairing fracture healing.

The selective TrkA agonist, gambogic amide, promotes osteoblastic differentiation and improves fracture healing in mice.

OBJECTIVES: To study effects of the selective TrkA agonist, gambogic amide (GA), on fracture healing in mice and on an osteoprogenitor cell line in vitro. METHODS: Mice were given bilateral fibular fractures and treated for two weeks with vehicle or 1 mg/kg/day GA and euthanized at 14-, 21-, and 42-days post-fracture. Calluses were analysed by micro-computed tomography (µCT), three-point bending and histology. For RT-PCR analyses, Kusa O cells were treated with 0.5nM of GA or vehicle for 3, 7, and 14 days, while for mineralization assessment, cells were treated for 21 days. RESULTS: µCT analysis found that 21-day GA-treated calluses had both decreased tissue volume (p<0.05) and bone surface (p<0.05) and increased fractional bone volume (p<0.05) compared to controls. Biomechanical analyses of 42-day calluses revealed that GA treatment increased stiffness per unit area by 53% (p<0.01) and load per unit area by 52% (p<0.01). GA treatment increased Kusa O gene expression of alkaline phosphatase and osteocalcin (p<0.05) by 14 days as well as mineralization at 21 days (p<0.05). CONCLUSIONS: GA treatment appeared to have a beneficial effect on fracture healing at 21- and 42-days post-fracture. The exact mechanism is not yet understood but may involve increased osteoblastic differentiation and matrix mineralization.

Oculomotor Cognitive Control Abnormalities in Australian Rules Football Players with a History of Concussion.

This study used oculomotor, cognitive, and multi-modal magnetic resonance imaging (MRI) measures to assess for neurological abnormalities in current asymptomatic amateur Australian rules footballers (i.e., Australia's most participated collision sport) with a history of sports-related concussion (SRC). Participants were 15 male amateur Australian rules football players with a history of SRC greater than 6 months previously, and 15 sex-, age-, and education-matched athlete control subjects that had no history of neurotrauma or participation in collision sports. Participants completed a clinical interview, neuropsychological measures, and oculomotor measures of cognitive control. MRI investigation involved structural imaging, as well as diffusion tensor imaging and resting-state functional MRI sequences. Despite no group differences on conventional neuropsychological tests and multi-modal MRI measures, Australian rules football players with a history of SRC performed significantly worse on an oculomotor switch task: a measure of cognitive control that interleaves the response of looking towards a target (i.e., a prosaccade) with the response of looking away from a target (i.e., an antisaccade). Specifically, Australian footballers performed significantly shorter latency prosaccades and found changing from an antisaccade trial to a prosaccade trial (switch cost) significantly more difficult than control subjects. Poorer switch cost was related to poorer performance on a number of neuropsychological measures of inhibitory control. Further, when comparing performance on the cognitively more demanding switch task with performance on simpler, antisaccade/prosaccades tasks which require a single response, Australian footballers demonstrated a susceptibility to increased cognitive load, compared to the control group who were unaffected. These initial results suggest that current asymptomatic amateur Australian rules football players with a history of SRC may have persisting, subtle, cognitive changes, which are demonstrable on oculomotor cognitive measures. Future studies are required in order to further elucidate the full nature and clinical relevance of these findings.

Gambogic amide, a selective TrkA agonist, does not improve outcomes from traumatic brain injury in mice.

OBJECTIVES: There is evidence that treatment with nerve growth factor (NGF) may reduce neuroinflammation and apoptosis after a traumatic brain injury (TBI). NGF is thought to exert its effects via binding to either TrkA or p75 neurotrophin receptors. This study aimed to investigate the effects of a selective TrkA agonist, gambogic amide (GA), on TBI pathology and outcomes in mice following lateral fluid percussion injury. METHODS: Male C57BL/6 mice were given either a TBI or sham injury, and then received subcutaneous injections of either 2 mg/kg of GA or vehicle at 1, 24, and 48 h post-injury. Following behavioural studies, mice were euthanized at 72 h post-injury for analysis of neuroinflammatory, apoptotic, and neurite outgrowth markers. RESULTS: Behavioural testing revealed that GA did not mitigate motor deficits after TBI. TBI caused an increase in cortical and hippocampal expression of several markers of neuroinflammation and apoptosis compared to sham groups. GA treatment did not attenuate these increases in expression, possibly contributed to by our finding of TrkA receptor down-regulation post-TBI. CONCLUSIONS: These findings suggest that GA treatment may not be suitable for attenuating TBI pathology and improving outcomes.

Treatment with an interleukin-1 receptor antagonist mitigates neuroinflammation and brain damage after polytrauma.

Traumatic brain injury (TBI) and long bone fracture are common in polytrauma. This injury combination in mice results in elevated levels of the pro-inflammatory cytokine interleukin-1ß (IL-1ß) and exacerbated neuropathology when compared to isolated-TBI. Here we examined the effect of treatment with an IL-1 receptor antagonist (IL-1ra) in mice given a TBI and a concomitant tibial fracture (i.e., polytrauma). Adult male C57BL/6 mice were given sham-injuries or polytrauma and treated with saline-vehicle or IL-1ra (100mg/kg). Treatments were subcutaneously injected at 1, 6, and 24h, and then once daily for one week post-injury. 7-8 mice/group were euthanized at 48h post-injury. 12-16 mice/group underwent behavioral testing at 12weeks post-injury and MRI at 14weeks post-injury before being euthanized at 16weeks post-injury. At 48h post-injury, markers for activated microglia and astrocytes, as well as neutrophils and edema, were decreased in polytrauma mice treated with IL-1ra compared to polytrauma mice treated with vehicle. At 14weeks post-injury, MRI analysis demonstrated that IL-1ra treatment after polytrauma reduced volumetric loss in the injured cortex and mitigated track-weighted MRI markers for axonal injury. As IL-1ra (Anakinra) is approved for human use, it may represent a promising therapy in polytrauma cases involving TBI and fracture.