The Impact of Motor Axon Misdirection and Attrition on Behavioral Deficit Following Experimental Nerve Injuries

Peripheral nerve transection and neuroma-in-continuity injuries are associated with permanent functional deficits, often despite successful end-organ reinnervation. Axonal misdirection with non-specific reinnervation, frustrated regeneration and axonal attrition are believed to be among the anatomical substrates that underlie the poor functional recovery associated with these devastating injuries. Yet, functional deficits associated with axonal misdirection in experimental neuroma-in-continuity injuries have not yet been studied. We hypothesized that experimental neuroma-in-continuity injuries would result in motor axon misdirection and attrition with proportional persistent functional deficits. The femoral nerve misdirection model was exploited to assess major motor pathway misdirection and axonal attrition over a spectrum of experimental nerve injuries, with neuroma-in-continuity injuries simulated by the combination of compression and traction forces in 42 male rats. Sciatic nerve injuries were employed in an additional 42 rats, to evaluate the contribution of axonal misdirection to locomotor deficits by a ladder rung task up to 12 weeks. Retrograde motor neuron labeling techniques were utilized to determine the degree of axonal misdirection and attrition. Characteristic histological neuroma-in-continuity features were demonstrated in the neuroma-in-continuity groups and poor functional recovery was seen despite successful nerve regeneration and muscle reinnervation. Good positive and negative correlations were observed respectively between axonal misdirection (p<.0001, r²=.67), motor neuron counts (attrition) (p<.0001, r²=.69) and final functional deficits. We demonstrate prominent motor axon misdirection and attrition in neuroma-in-continuity and transection injuries of mixed motor nerves that contribute to the long-term functional deficits. Although widely accepted in theory, to our knowledge, this is the first experimental evidence to convincingly demonstrate these correlations with data inclusive of the neuroma-in-continuity spectrum. This work emphasizes the need to focus on strategies that promote both robust and accurate nerve regeneration to optimize functional recovery. It also demonstrates that clinically relevant neuroma-in-continuity injuries can now also be subjected to experimental investigation.     

Landscape-Scale Disturbances Modified Bird Community Dynamics in Successional Forest Environment

Ecosystem-based forest management strives to develop silvicultural practices that best emulate natural disturbances such as wildfire to conserve biodiversity representative of natural forest ecosystems. Yet, current logging practices alter forest structure and reduce the proportion of old-growth forest and, consequently, can exert long-term effects on the dynamics of forest biota. The stand- and landscape-scale factors driving bird community dynamics in post-disturbance environment remain poorly understood. In this study, we examined bird community dynamics along successional gradients in boreal ecosystems originating from fire and logging in landscapes dominated by old-growth forest. We tested if bird species richness and community compositions in clear-cutting stands became comparable to those in natural stands after 70 years, and identified the relative contributions of stand- and landscape-scale forest attributes in bird community dynamics. Based on records of bird occurrences at 185 field sites in natural and clearcutting stands, we demonstrate that (1) both forest structures and bird communities underwent evident changes along successional gradients in post-clearcutting environment; (2) bird species richness and community composition in 60- to 70-years-old clearcutting stands still differed from those in 50- to 79-years-old natural stands, in spite of the fact that most forest attributes of clearcutting stands became comparable to those of natural stands after 40 years; and (3) landscape disturbances contributed more than stand characteristics in explaining the lack of convergence of mature forest species, residents, and short-distance migrants in post-clearcutting environment. Our study points out that more regards should be paid to improve the landscape configuration of the managed forests, and implies that old-growth forest retention within logged areas, combined with selection cutting and prolonged logging rotations, can better emulate fire and alleviate forest harvesting effects on bird community assemblages typical of natural boreal ecosystem.     

Tumor versus Stromal Cells in Culture—Survival of the Fittest?

Two of the signature genetic events that occur in human gliomas, EGFR amplification and IDH mutation, are poorly represented in experimental models in vitro. EGFR amplification, for example, occurs in 40 to 50% of GBM, and yet, EGFR amplification is rarely preserved in cell cultures derived from human tumors. To analyze the fate of EGFR amplified and IDH mutated cells in culture, we followed the development over time of cultures derived from human xenografts in nude rats enriched for tumor cells with EGFR amplification and of cultures derived from patient samples with IDH mutations, in serum monolayer and spheroid suspension culture, under serum and serum free conditions. We observed under serum monolayer conditions, that nestin positive or nestin and SMA double positive rat stromal cells outgrew EGFR amplified tumor cells, while serum spheroid cultures preserved tumor cells with EGFR amplification. Serum free suspension culture exhibited a more variable cell composition in that the resultant cell populations were either predominantly nestin/SOX2 co-expressing rat stromal cells or human tumor cells, or a mixture of both. The selection for nestin/SMA positive stromal cells under serum monolayer conditions was also consistently observed in human oligodendrogliomas and oligoastrocytomas with IDH mutations. Our results highlight for the first time that serum monolayer conditions can select for stromal cells instead of tumor cells in certain brain tumor subtypes. This result has an important impact on the establishment of new tumor cell cultures from brain tumors and raises the question of the proper conditions for the growth of the tumor cell populations of interest.     

Plasma High Sensitivity Troponin T Levels in Adult Survivors of Childhood Leukaemias: Determinants and Associations with Cardiac Function

Background

We sought to quantify plasma high sensitivity cardiac troponin (hs-cTnT) levels, their determinants, and their associations with left ventricular (LV) myocardial deformation in adult survivors of childhood acute leukaemias.

Methods and Results

One hundred adult survivors (57 males) of childhood acute leukaemias, aged 24.1±4.2 years, and 42 age-matched controls (26 males) were studied. Plasma cTnT was determined using a highly sensitive assay. Genotyping of NAD(P)H oxidase and multidrug resistance protein polymorphisms was performed. Left ventricular function was assessed by conventional, three-dimensional, and speckle tracking echocardiography. The medians (interquartile range) of hs-cTnT in male and female survivors were 4.9 (4.2 to 7.2) ng/L and 1.0 (1.0 to 3.5) ng/L, respectively. Nineteen survivors (13 males, 6 females) (19%) had elevated hs-cTnT (>95^th centile of controls). Compared to those without elevated hs-TnT levels, these subjects had received larger cumulative anthracycline dose and were more likely to have leukaemic relapse, stem cell transplant, and cardiac irradiation. Their LV systolic and early diastolic myocardial velocities, isovolumic acceleration, and systolic longitudinal strain rate were significantly lower. Survivors having CT/TT at CYBA rs4673 had higher hs-cTnT levels than those with CC genotype. Functionally, increased hs-cTnT levels were associated with worse LV longitudinal systolic strain and systolic and diastolic strain rates.

Conclusions

Increased hs-cTnT levels occur in a significant proportion of adult survivors of childhood acute leukaemias and are associated with larger cumulative anthracycline dose received, history of leukaemic relapse, stem cell transplant, and cardiac irradiation, genetic variants in free radical metabolism, and worse LV myocardial deformation.     

Involvement of Autonomic Nervous Activity Changes in Gastroesophageal Reflux in Neonates during Sleep and Wakefulness

Background

It has been suggested that disturbed activity of the autonomic nervous system is one of the factors involved in gastroesophageal reflux (GER) in adults. We sought to establish whether transient ANS dysfunction (as assessed by heart rate variability) is associated with the occurrence of GER events in neonates during sleep and wakefulness.

Methods

Nineteen neonates with suspected GER underwent simultaneous, synchronized 12-hour polysomnography and esophageal multichannel impedance-pH monitoring. We compared changes in HRV parameters during three types of periods (control and prior to and during reflux) with respect to the vigilance state.

Results

The vigilance state influenced the distribution of GER events (P<0.001), with 53.4% observed during wakefulness, 37.6% observed during active sleep and only 9% observed during quiet sleep. A significant increase in the sympathovagal ratio (+32%, P=0.013) was observed in the period immediately prior to reflux (due to a 15% reduction in parasympathetic activity (P=0.017)), relative to the control period. This phenomenon was observed during both wakefulness and active sleep.

Conclusion

Our results showed that GER events were preceded by a vigilance-state-independent decrease in parasympathetic tone. This suggests that a pre-reflux change in ANS activity is one of the factors contributing to the mechanism of reflux in neonates.     

Automated analysis of courtship suppression learning and memory in Drosophila melanogaster

Study of the fruit fly, Drosophila melanogaster, has yielded important insights into the underlying molecular mechanisms of learning and memory. Courtship conditioning is a well-established behavioral assay used to study Drosophila learning and memory. Here, we describe the development of software to analyze courtship suppression assay data that correctly identifies normal or abnormal learning and memory traits of individual flies. Development of this automated analysis software will significantly enhance our ability to use this assay in large-scale genetic screens and disease modeling. The software increases the consistency, objectivity, and types of data generated.     

Modulation of distinct isoforms of L-type calcium channels by Gq-coupled receptors in Xenopus oocytes

L-type voltage dependent Ca²⁺ channels (L-VDCCs; Ca_v1.2) are crucial in cardiovascular physiology. In heart and smooth muscle, hormones and transmitters operating via G_q enhance L-VDCC currents via essential protein kinase C (PKC) involvement. Heterologous reconstitution studies in Xenopus oocytes suggested that PKC and G_q-coupled receptors increased L-VDCC currents only in cardiac long N-terminus (NT) isoforms of α_1C, whereas known smooth muscle short-NT isoforms were inhibited by PKC and G_q activators. We report a novel regulation of the long-NT α_1C isoform by Gβγ. Gβγ inhibited whereas a Gβγ scavenger protein augmented the G_q- but not phorbol ester-mediated enhancement of channel activity, suggesting that Gβγ acts upstream from PKC. In vitro binding experiments reveal binding of both Gβγ and PKC to α_1C-NT. However, PKC modulation was not altered by mutations of multiple potential phosphorylation sites in the NT, and was attenuated by a mutation of C-terminally located serine S1928. The insertion of exon 9a in intracellular loop 1 rendered the short-NT α_1C sensitive to PKC stimulation and to Gβγ scavenging. Our results suggest a complex antagonistic interplay between G_q-activated PKC and Gβγ in regulation of L-VDCC, in which multiple cytosolic segments of α_1C are involved.     

Mycobacterium ulcerans Disease: Experience with Primary Oral Medical Therapy in an Australian Cohort

Background

Mycobacterium ulcerans (MU) is responsible for disfiguring skin lesions and is endemic on the Bellarine peninsula of southeastern Australia. Antibiotics have been shown to be highly effective in sterilizing lesions and preventing disease recurrences when used alone or in combination with surgery. Our practice has evolved to using primarily oral medical therapy.

Methods

From a prospective cohort of MU patients managed at Barwon Health, we describe those treated with primary medical therapy defined as treatment of a M. ulcerans lesion with antimicrobials either alone or in conjunction with limited surgical debridement.

Results

From 1/10/2010 through 31/12/11, 43 patients were treated with exclusive medical therapy, of which 5 (12%) also underwent limited surgical debridement. The median patient age was 50.2 years, and 86% had WHO category 1 and 91% ulcerative lesions. Rifampicin was combined with ciprofloxacin in 30 (70%) and clarithromycin in 12 (28%) patients. The median duration of antibiotic therapy was 56 days, with 7 (16%) receiving less than 56 days. Medication side effects requiring cessation of one or more antibiotics occurred in 7 (16%) patients. Forty-two (98%) patients healed without recurrence within 12 months, and 1 patient (2%) experienced a relapse 4 months after completion of 8 weeks of antimicrobial therapy.

Conclusion

Our experience demonstrates the efficacy and safety of primary oral medical management of MU infection with oral rifampicin-based regimens. Further research is required to determine the optimal and minimum durations of antibiotic therapy, and the most effective antibiotic dosages and formulations for young children.     

Validation of a Remote Sensing Model to Identify Simulium damnosum s.l. Breeding Sites in Sub-Saharan Africa

Background

Recently, most onchocerciasis control programs have begun to focus on elimination. Developing an effective elimination strategy relies upon accurately mapping the extent of endemic foci. In areas of Africa that suffer from a lack of infrastructure and/or political instability, developing such accurate maps has been difficult. Onchocerciasis foci are localized near breeding sites for the black fly vectors of the infection. The goal of this study was to conduct ground validation studies to evaluate the sensitivity and specificity of a remote sensing model developed to predict S. damnosum s.l. breeding sites.

Methodology/Principal Findings

Remote sensing images from Togo were analyzed to identify areas containing signature characteristics of S. damnosum s.l. breeding habitat. All 30 sites with the spectral signature were found to contain S. damnosum larvae, while 0/52 other sites judged as likely to contain larvae were found to contain larvae. The model was then used to predict breeding sites in Northern Uganda. This area is hyper-endemic for onchocerciasis, but political instability had precluded mass distribution of ivermectin until 2009. Ground validation revealed that 23/25 sites with the signature contained S. damnosum larvae, while 8/10 sites examined lacking the signature were larvae free. Sites predicted to have larvae contained significantly more larvae than those that lacked the signature.

Conclusions/Significance

This study suggests that a signature extracted from remote sensing images may be used to predict the location of S. damnosum s.l. breeding sites with a high degree of accuracy. This method should be of assistance in predicting communities at risk for onchocerciasis in areas of Africa where ground-based epidemiological surveys are difficult to implement.     

Chikungunya Virus Infection Results in Higher and Persistent Viral Replication in Aged Rhesus Macaques Due to Defects in Anti-Viral Immunity

Chikungunya virus (CHIKV) is a re-emerging mosquito-borne Alphavirus that causes a clinical disease involving fever, myalgia, nausea and rash. The distinguishing feature of CHIKV infection is the severe debilitating poly-arthralgia that may persist for several months after viral clearance. Since its re-emergence in 2004, CHIKV has spread from the Indian Ocean region to new locations including metropolitan Europe, Japan, and even the United States. The risk of importing CHIKV to new areas of the world is increasing due to high levels of viremia in infected individuals as well as the recent adaptation of the virus to the mosquito species Aedes albopictus. CHIKV re-emergence is also associated with new clinical complications including severe morbidity and, for the first time, mortality. In this study, we characterized disease progression and host immune responses in adult and aged Rhesus macaques infected with either the recent CHIKV outbreak strain La Reunion (LR) or the West African strain 37997. Our results indicate that following intravenous infection and regardless of the virus used, Rhesus macaques become viremic between days 1–5 post infection. While adult animals are able to control viral infection, aged animals show persistent virus in the spleen. Virus-specific T cell responses in the aged animals were reduced compared to adult animals and the B cell responses were also delayed and reduced in aged animals. Interestingly, regardless of age, T cell and antibody responses were more robust in animals infected with LR compared to 37997 CHIKV strain. Taken together these data suggest that the reduced immune responses in the aged animals promotes long-term virus persistence in CHIKV-LR infected Rhesus monkeys.