In vivo imaging of axonal transport of mitochondria in the diseased and aged mammalian CNS

The lack of intravital imaging of axonal transport of mitochondria in the mammalian CNS precludes characterization of the dynamics of axonal transport of mitochondria in the diseased and aged mammalian CNS. Glaucoma, the most common neurodegenerative eye disease, is characterized by axon degeneration and the death of retinal ganglion cells (RGCs) and by an age-related increase in incidence. RGC death is hypothesized to result from disturbances in axonal transport and in mitochondrial function. Here we report minimally invasive intravital multiphoton imaging of anesthetized mouse RGCs through the sclera that provides sequential time-lapse images of mitochondria transported in a single axon with submicrometer resolution. Unlike findings from explants, we show that the axonal transport of mitochondria is highly dynamic in the mammalian CNS in vivo under physiological conditions. Furthermore, in the early stage of glaucoma modeled in adult (4-mo-old) mice, the number of transported mitochondria decreases before RGC death, although transport does not shorten. However, with increasing age up to 23–25 mo, mitochondrial transport (duration, distance, and duty cycle) shortens. In axons, mitochondria-free regions increase and lengths of transported mitochondria decrease with aging, although totally organized transport patterns are preserved in old (23- to 25-mo-old) mice. Moreover, axonal transport of mitochondria is more vulnerable to glaucomatous insults in old mice than in adult mice. These mitochondrial changes with aging may underlie the age-related increase in glaucoma incidence. Our method is useful for characterizing the dynamics of axonal transport of mitochondria and may be applied to other submicrometer structures in the diseased and aged mammalian CNS in vivo.Globally, longevity is increasing, and the cohort aged 60 y or over is the fastest growing portion of the population. These trends place neurodegenerative diseases among the greatest clinical threats. Glaucoma, the most common progressive neurodegenerative eye disease (1), globally affects an estimated 60.5 million people, of whom 8.4 million are bilaterally blind (2). Similar to other neurodegenerative diseases of the CNS, including Alzheimer’s disease (3) and Parkinson’s disease (4), the incidence of glaucoma (5) increases with aging. Glaucoma is characterized by axon degeneration and the death of retinal ganglion cells (RGCs) (6). Histological studies in glaucoma models have suggested disturbances in the axonal transport of mitochondria in RGCs (7, 8).Axonal transport is essential for delivering the organelles and proteins that are required for axonal function and maintenance. Mitochondria are organelles that must be transported in axons and distributed appropriately to function (9, 10), because mitochondria play a pivotal role in the function and survival of neurons by generating ATP, maintaining Ca²⁺ and reduction-oxidation (redox) homeostasis, and signaling in apoptosis. Disturbances in mitochondrial dynamics are suggested to be involved in neurodegenerative diseases and CNS aging (11–14). In vivo imaging of axonal transport of mitochondria has been reported using explant imaging of the Drosophila nervous system (15) and rat cerebellar slice cultures (16) and intravital imaging (direct in vivo imaging of living animals at subcellular resolution) of the mouse peripheral nervous system (17) and zebrafish nervous system (18). However, intravital imaging of axonal transport of mitochondria has not been achieved in the mammalian CNS. Importantly, the lack of intravital imaging of axonal transport of mitochondria in the mammalian CNS under physiological oxygen levels and metabolism and with intact blood flow has precluded the characterization of the dynamics of axonal transport of mitochondria in the CNS of diseased and aged mammals in vivo.To perform intravital imaging of axonal transport of mitochondria in the mammalian CNS, we developed the technique we call “MIMIR” (for “minimally invasive intravital imaging of mitochondrial axonal transport in RGCs”). MIMIR does not involve thinning or opening of the sclera or produce changes in the intraocular humor. MIMIR directly showed, at submicrometer resolution, that axonal transport of mitochondria is highly dynamic in the mammalian CNS in vivo under physiological conditions. It enabled us to characterize disturbances of mitochondrial transport in a mouse model of glaucoma and age-related changes of mitochondrial transport in old (23- to 25-mo-old) mice.     

Prognostic value of cardiovascular magnetic resonance derived right ventricular function in patients with interstitial lung disease

Background

Cardiovascular magnetic resonance (CMR) provides non-invasive and more accurate assessment of right ventricular (RV) function in comparison to echocardiography. Recent study demonstrated that assessment of RV function by echocardiography was an independent predictor for mortality in patients with interstitial lung disease (ILD). The purpose of this study was to determine the prognostic significance of CMR derived RV ejection fraction (RVEF) in ILD patients.

Methods

We enrolled 76 patients with ILD and 24 controls in the current study. By using 1.5 T CMR scanner equipped with 32 channel cardiac coils, we performed steady-state free precession cine CMR to assess the RVEF. RV systolic dysfunction (RVSD) was defined as RVEF ≤45.0% calculated by long axis slices. Pulmonary hypertension (PH) was defined as mean pulmonary artery pressure (mPAP) of more than 25 mmHg at rest in the setting of pulmonary capillary wedge pressure ≤15 mmHg.

Results

The median RVEF was 59.2% in controls (n = 24), 53.8% in ILD patients without PH (n = 42) and 43.1% in ILD patients with PH (n = 13) (p < 0.001 by one-way ANOVA). During a mean follow-up of 386 days, 18 patients with RVSD had 11 severe events (3 deaths, 3 right heart failure, 3 exacerbation of dyspnea requiring oxygen, 2 pneumonia requiring hospitalization). In contrast, only 2 exacerbation of dyspnea requiring oxygen were observed in 58 patients without RVSD. Multivariate Cox regression analysis showed that RVEF independently predicted future events, after adjusting for age, sex and RVFAC by echocardiography (hazard ratio: 0.889, 95% confidence interval: 0.809 – 0.976, p = 0.014).

Conclusions

The current study demonstrated that RVSD in ILD patients can be clearly detected by cine CMR. Importantly, low prevalence of PH (17%) indicated that population included many mild ILD patients. CMR derived RVEF might be useful for the risk stratification and clinical management of ILD patients.     

Raloxifene prevents cardiac hypertrophy and dysfunction in pressure-overloaded mice

17beta-estradiol reduces myocardial hypertrophy and left ventricular mass, suggesting that the selective estrogen receptor modulator raloxifene may have similar effects. However, it is not clear whether raloxifene inhibits both cardiac hypertrophy and dysfunction. We used transverse aortic-banded mice to produce pressure-overload cardiac hypertrophy and used neonatal rat ventricular cardiomyocytes to investigate the cellular mechanisms of raloxifene on cardiac hypertrophy. Left ventricular mass and fractional shortening of mice hearts were measured by transthoracic echocardiography. Protein synthesis of cardiomyocytes was evaluated by incorporation of [3H]leucine into cardiomyocytes exposed to angiotensin II. Phosphorylation of mitogen-activated protein (MAP) kinase was also observed in cardiomyocytes. Raloxifene prevented increases in left ventricular mass and decreases of fractional shortening at 4 weeks after aortic banding. Pretreatment with raloxifene before angiotensin II stimulation inhibited the increase in [3H]leucine incorporation into neonatal rat cardiomyocytes in a concentration-dependent manner. This inhibition was partially but not significantly attenuated by N(G)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase, and completely abolished by ICI182780, an estrogen receptor antagonist. Although the phosphorylation of p38 MAP kinase, c-Jun N-terminal kinase (JNK), or extracellular signal-regulated protein kinase (ERK) in cardiomyocytes was significantly increased by angiotensin II stimulation as compared with the control, pretreatment with raloxifene attenuated p38 MAP kinase phosphorylation, but neither JNK nor ERK phosphorylation. We conclude that raloxifene inhibits cardiac hypertrophy and dysfunction and that the inhibition of p38 MAP kinase phosphorylation after the stimulation of estrogen receptors may be involved in the cellular mechanisms of this agent.     

PKC/MEK inhibitors suppress oxaliplatin‐induced neuropathy and potentiate the antitumor effects

Oxaliplatin is a key drug commonly used in colorectal cancer treatment. Despite high clinical efficacy, its therapeutic application is limited by common, dose‐limiting occurrence of neuropathy. As usual symptomatic neuropathy treatments fail to improve the patients' condition, there is an urgent need to advance our understanding of the pathogenesis of neuropathy to propose effective therapy and ensure adequate pain management. Oxaliplatin‐induced neuropathy was recently reported to be associated with protein kinase C (PKC) activation. It is unclear, however, whether PKC inhibition can prevent neuropathy. In our current studies, we found that a PKC inhibitor, tamoxifen, inhibited oxaliplatin‐induced neuropathy via the PKC/extracellular signal‐regulated kinase (ERK)/c‐Fos pathway in lumbar spinal cords (lumbar segments 4–6). Additionally, tamoxifen was shown to act in synergy with oxaliplatin to inhibit growth in tumor cells‐implanted mice. Moreover, mitogen‐activated protein kinase kinase (MEK) 1/2 inhibitor, PD0325901, suppressed oxaliplatin‐induced neuropathy and enhanced oxaliplatin efficacy. Our results indicate that oxaliplatin‐induced neuropathy is associated with PKC/ERK/c‐Fos pathway in lumbar spinal cord. Additionally, we demonstrate that disruption of this pathway by PKC and MEK inhibitors suppresses oxaliplatin‐induced neuropathy, thereby suggesting that PKC and MEK inhibitors may be therapeutically useful in preventing oxaliplatin‐induced neuropathy and could aid in combination antitumor pharmacotherapy.     

Oxidation and hydrogenation of Pd: suppression of oxidation by prolonged H2 exposure

We investigate the phase transition of a Pd surface in both oxidizing and reducing environments by environmental transmission electron microscopy (ETEM). ETEM allows us to study sequential exposure of Pd to O₂ and H₂ in the same TEM conditions. First, under ETEM observation, oxidation occurs at step edges but it can also occur at terraces. Second, as the most important result, we observed a novel process where previous exposure to H₂ suppresses new oxidation of the Pd surface. Third, we show by electron energy loss spectroscopy (EELS) that this process, suppression of oxidation by previous exposure to H₂, is not due to the formation of bulk β-phase Pd hydride. We also demonstrate that this process is not present in Pt. Finally, we discuss the hypothesis to explain this phenomenon: formation of surface–Pd–hydride suppresses the new oxidation. This observation, suppression of oxidation by H₂ exposure, may eventually lead to new breakthroughs.

Oxidation of the Pd surface exhibits dependence on the history of pre-exposure to H₂.