Retinal metabolic changes in an experimental model of optic nerve transection by ex vivo 1H magnetic resonance spectroscopy

This study aims to investigate the retinal metabolic processes in a rat axotomy model. Retinal metabolic changes in optic nerve transection (ONT) rat model were analyzed by ¹H magnetic resonance spectroscopy (¹H-MRS). Retinal ganglion cells (RGCs) densities were assessed from retinal whole mounts. The retina was stained immunohistochemically with glial fibrillary acidic protein (GFAP). The results showed that the retina in ONT rats had significantly decreased concentrations of γ-aminobutyric acid (GABA), N-acetylaspartate (NAA), taurine (Tau), creatine (Cr) and increased concentrations of alanine (Ala) compared with control. Examination of glutamate (Glu), glutamine (Gln) and Glx (Glu + Gln) concentrations disclosed no significant differences. The mean density of RGCs reduced from 2,249 ± 87 cells/mm² in control group to 320 ± 56 cells/mm² in ONT group. GFAP immunoreactivity was markedly higher in ONT group than that in control group. The retinal metabolism after ONT was associated with neurotransmitter recycling/production perturbation, as well as other metabolic disequilibrium.     

A quantitative three-dimensional model of the Drosophila optic lobes

A big step in the neurobiology of Drosophila would be to establish a standard for brain anatomy to which to relate morphological, developmental and genetic data. We propose that only an average brain and its variance would be a biologically meaningful reference and have developed an averaging procedure. Here, we present a brief outline of this method and apply it to the optic lobes of Drosophila melanogaster wild-type Canton S. Whole adult brains are stained with a fluorescent neuropil marker and scanned with the confocal microscope. The resulting three-dimensional data sets are automatically aligned into a common coordinate system and intensity averages calculated. We use effect-size maps for the fast detection of differences between averages. For morphometric analysis, neuropil structures are labelled and superimposed to give a three-dimensional probabilistic map. In the present study, the method was applied to 66 optic lobes. We found their size, shape and position to be highly conserved between animals. Similarity was even higher between left and right optic lobes of the same animal. Sex differences were more pronounced. Female optic lobes were 6% larger than those of males. This value corresponds well with the higher number of ommatidia in females. As females have their additional ommatidia dorsally and ventrally, the additional neuropil in the medulla, lobula and lobula plate, accordingly, was found preferentially at these locations. For males, additional neuropil was found only at the posterior margin of the lobula. This finding supports the notion of male-specific neural processing in the lobula as described for muscid and calliphorid flies.     

A tridomain model for potassium clearance in optic nerve of Necturus

Complex fluids flow in complex ways in complex structures. Transport of water and various organic and inorganic molecules in the central nervous system are important in a wide range of biological and medical processes. However, the exact driving mechanisms are often not known. In this work, we investigate flows induced by action potentials in an optic nerve as a prototype of the central nervous system. Different from traditional fluid dynamics problems, flows in biological tissues such as the central nervous system are coupled with ion transport. They are driven by osmosis created by concentration gradient of ionic solutions, which in turn influence the transport of ions. Our mathematical model is based on the known structural and biophysical properties of the experimental system used by the Harvard group Orkand et al. Asymptotic analysis and numerical computation show the significant role of water in convective ion transport. The full model (including water) and the electrodiffusion model (excluding water) are compared in detail to reveal an interesting interplay between water and ion transport. In the full model, convection due to water flow dominates inside the glial domain. This water flow in the glia contributes significantly to the spatial buffering of potassium in the extracellular space. Convection in the extracellular domain does not contribute significantly to spatial buffering. Electrodiffusion is the dominant mechanism for flows confined to the extracellular domain.     

A quantitative electron microscopic analysis of myelination in the optic nerve of suckling rats treated with an inhibitor of cholesterol biosynthesis

Summary The effect of AY-9944, an inhibitory cholesterol biosynthesis, on the myelination of the optic nerve of rats was studied. Suckling rats were injected intraperitoneally with the drug every other day from birth, and were sacrificed at 10, 20 and 30 days of age together with littermate controls. The analysis is based on counting, at the electron-microscope level, the number of unmyelinated axons and the number of myelin lamellae surrounding each myelinating axon. The results indicate that a decrease in endogenous cholesterol by AY 9944, induced an overall retardation of the myelination process in the optic nerve: a larger proportion of myelinated axons and smaller number of myelin lamellae around the myelinating axons, when compared with the littermate controls, was observed. Exogenous cholesterol from the maternal milk did not compensate for a lack in endogenous cholesterol.Degenerating myelin sheaths were frequently seen in the experimental optic nerves at 20 and 30 days of age. Numerous membranous, intracytoplasmic drug-induced inclusions were found at all ages studied. Acknowledgements. The author is particularly indebted to Dr. B. G. Uzman and Dr. G. M. Villegas for their valuable discussion and suggestions. He wishes also to thank Mr. F. Paredes, Mr. J. Aristimuño and Miss Marcia Escala for their technical assistance; Mr. J. Bigorra for the photographic aid, and Miss Sonia Rodríguez for her secretarial help.     

Gliogenesis and myelination in the optic nerve of trisomy 19 mice. A quantitative electron-microscopic study

Trisomy 19 (ts19) of the mouse permits detailed studies on the influence of an extra autosome upon the postnatal development of the central nervous system. To examine gliogenesis and myelinogenesis, the optic nerves of 19 ts19 pugs aged 1-15 days have been examined by light and electron microscopy and compared to those of litter-mate controls. Differentiation of astrocytes and oligodendrocytes, myelinogenesis as well as the opening of the eyes are each delayed by about 2 days. Myelin sheaths are normally structured in ts19. There is a decrease in the percentage of myelinated fibres. The cross-sectional area of the ts19 optic nerve is reduced. The fibre density, which decreases with age both in ts19 and control mice, is higher in ts19 mice. Both with ts19 and control animals, the distribution of fibre diameters of myelinated axons overlaps with that of promyelinated and unmyelinated fibres, but myelinated axons cannot be observed below a diameter of 0.3 micron, and unmyelinated axons are always smaller than 1 micron. The mean diameter of promyelinated axons is identical in ts19 and control animals. Myelination is therefore not severely disturbed in the ts19 optic nerve. As retinal differentiation in ts19 is delayed by 2 days as well, reports on an asynchronous development of neurons and myelin sheaths cannot be confirmed for the visual system.     

Changes in oxygen partial pressure of brain tissue in an animal model of obstructive apnea

Background

Prolonged weaning from mechanical ventilation has a major impact on ICU bed occupancy and patient outcome, and has significant cost implications. There is evidence in patients around the period of extubation that helium-oxygen leads to a reduction in the work of breathing. Therefore breathing helium-oxygen during weaning may be a useful adjunct to facilitate weaning. We hypothesised that breathing helium-oxygen would reduce carbon dioxide production during the weaning phase of mechanical ventilation.

Materials/patients and methods

We performed a prospective randomised controlled single blinded cross-over trial on 19 adult intensive care patients without significant airways disease who fulfilled criteria for weaning with CPAP. Patients were randomised to helium-oxygen and air-oxygen delivered during a 2 hour period of CPAP ventilation. Carbon dioxide production (VCO₂) was measured using a near patient main stream infrared carbon dioxide sensor and fixed orifice pneumotachograph.

Results

Compared to air-oxygen, helium-oxygen significantly decreased VCO₂ production at the end of the 2 hour period of CPAP ventilation; there was a mean difference in CO₂ production of 48.9 ml/min (95% CI 18.7-79.2 p = 0.003) between the groups. There were no significant differences in other respiratory and haemodynamic parameters.

Conclusion

This study shows that breathing a helium-oxygen mixture during weaning reduces carbon dioxide production. This physiological study supports the need for a clinical trial of helium-oxygen mixture during the weaning phase of mechanical ventilation with duration of weaning as the primary outcome.

Trial registration

ISRCTN56470948     

Decline of IXth nerve taste responses following nerve transection

Summated impulse discharges to taste solutions were recordedfrom intact and transected IXth nerves in the Mongolian gerbil(Meriones unguiculatus). Five taste stimuli were used: 0.3 MNH₄Cl, 0.3 M NaCl, 0.01 M HCl, 0.01 M quinine hydrochloride,and 0.5 M sucrose. 0.3 M NH₄Cl was the most effective stimulus.Taste responses from intact nerves were stable for more than10 hours. Following IXth nerve transection, the peak summatedresponse to 0.3 M NH₄Cl declined by 50% in a mean of 119 min.(Some animals failed to show this taste response decline inthe winter months.) The transected IXth nerve's spontaneousactivity and responses to other taste solutions also typicallydeclined. The continued presence of normal compound action potentialsindicated that the transection-induced decline in taste responsesdid not result from a failure of impulse propagation mechanismsin the nerve trunk. The results are consistent with the propositionthat transection interferes with axonal transport of materialsvital to the short-term maintenance of taste responses.     

A novel strategy for quantitative proteomics using isotope-coded protein labels

Stable isotope labelling in combination with mass spectrometry has emerged as a powerful tool to identify and relatively quantify thousands of proteins within complex protein mixtures. Here we describe a novel method, termed isotope-coded protein label (ICPL), which is capable of high-throughput quantitative proteome profiling on a global scale. Since ICPL is based on stable isotope tagging at the frequent free amino groups of isolated intact proteins, it is applicable to any protein sample, including extracts from tissues or body fluids, and compatible to all separation methods currently employed in proteome studies. The method showed highly accurate and reproducible quantification of proteins and yielded high sequence coverage, indispensable for the detection of post-translational modifications and protein isoforms. The efficiency (e.g. accuracy, dynamic range, sensitivity, speed) of the approach is demonstrated by comparative analysis of two differentially spiked proteomes.     

The conduction of optic nerve fibers using different visual patterns

The Authors have studied the behaviour of checkerboard pattern visual evoked potential (VEP) latencies by using different spatial frequency stimuli and different stimulating visual fields in order to demonstrate whether spatial frequency might constitute a parameter capable of exciting different retinal regions like different stimulus fields. According to the recent literature low spatial frequency stimuli generate VEP with latencies which are significantly shorter than high spatial frequency stimuli, making this method more reliable for the differentiation of macular and peripheral retinal fields.     

Alterations in the morphology of ganglion cell dendrites in the adult rat retina after optic nerve transection and grafting of peripheral nerve segments

Summary Transected ganglion cell axons from the adult retina are capable of reinnervating their central targets by growing into transplanted peripheral nerve (PN) segments. Injury of the optic nerve causes various metabolic and morphological changes in the retinal ganglion cell (RGC) perikarya and in the dendrites. The present work examined the dendritic trees of those ganglion cells surviving axotomy and of those whose severed axons re-elongated in PN grafts to reach either the superior colliculus (SC), transplanted SC, or transplanted autologous thigh muscle. The elaboration of the dendritic trees was visualized by means of the strongly fluorescent carbocyanine dye DiI, which is taken up by axons and transported to the cell bodies and from there to the dendritic branches. Alternatively, retinofugal axons regrowing through PN grafts were anterogradely filled from the eye cup with rhodamine B-isothiocyanate. The transection of the optic nerve resulted in characteristic changes in the ganglion cell dendrites, particularly in the degeneration of most of the terminal and preterminal dendritic branches. This occurred within the first 1 to 2 weeks following axotomy. The different types of ganglion cells appear to vary in their sensitivity to axotomy, as reflected by a rapid degeneration of certain cell dendrites after severance of the optic nerve. The most vulnerable cells were those with small perikarya and small dendritic fields (type II), whereas larger cells with larger dendritic fields (type I and III) were slower to respond and less dramatically affected. Regrowth of the lesioned axons in peripheral nerve grafts and reconnection of the retina with various tissues did not result in a significant immediate recovery of ganglion cell dendrites, although it did prevent some axotomized cells from further progression toward posttraumatic cell death.     

A novel Drosophila model of nerve injury reveals an essential role of Nmnat in maintaining axonal integrity

Axons damaged by acute injury, toxic insults, or during neurodegenerative diseases undergo Wallerian or Wallerian-like degeneration, which is an active and orderly cellular process, but the underlying mechanisms are poorly understood. Drosophila has been proven to be a successful system for modeling human neurodegenerative diseases. In this study, we established a novel in vivo model of axon injury using the adult fly wing. The wing nerve highlighted by fluorescent protein markers can be directly visualized in living animals and be precisely severed by a simple wing cut, making it highly suitable for large-scale screening. Using this model, we confirmed an axonal protective function of Wld(S) and nicotinamide mononucleotide adenylyltransferase (Nmnat). We further revealed that knockdown of endogenous Nmnat triggered spontaneous, dying-back axon degeneration in vivo. Intriguingly, axonal mitochondria were rapidly depleted upon axotomy or downregulation of Nmnat. The injury-induced mitochondrial loss was dramatically suppressed by upregulation of Nmnat, which also protected severed axons from degeneration. However, when mitochondria were genetically eliminated from axons, upregulation of Nmnat was no longer effective to suppress axon degeneration. Together, these findings demonstrate an essential role of endogenous Nmnat in maintaining axonal integrity that may rely on and function by stabilizing mitochondria.     

A quantitative model for metabolic intervention using gut microbes

As medicine shifts toward precision-based and personalized therapeutics, utilizing more complex biomolecules to treat increasingly difficult and rare conditions, microorganisms provide an avenue for realizing the production and processing necessary for novel drug pipelines. More so, probiotic microbes can be co-opted to deliver therapeutics by oral administration as living drugs, able to survive and safely transit the digestive tract. As living therapeutics are in their nascency, traditional pharmacokinetic–pharmacodynamic (PK–PD) models for evaluating drug candidates are not appropriate for this novel platform. Using a living therapeutic in late-stage clinical development for phenylketonuria (PKU) as a case study, we adapt traditional oral drug delivery models to properly evaluate and inform the engineering of living therapeutics. We develop the adapted for living therapeutics compartmental absorption and transit (ALT-CAT) model to provide metrics for drug efficacy across nine age groups of PKU patients and evaluate model parameters that are influenced by patient physiology, microbe selection and therapeutic production, and dosing formulations. In particular, the ALT-CAT model describes the mathematical framework to model the behavior of orally delivered engineered bacteria that act as living therapeutics by adapting similar methods that have been developed and widely-used for small molecular drug delivery and absorption.     

A statistical analyzer for optic nerve messages

A statistical mathematical model of the discharge in a single optic nerve fiber is proposed, based on a discharge with intervals between impulses distributed independently according to a gamma distribution ("gamma discharge"). A light stimulus distorts the time axis of this discharge according to a "frequency function" which is characteristic of the stimulus. A linear filter is described which calculates the likelihood of a certain stimulus when the nerve fiber message is fed into it. This filter forms the basis of theoretical nerve message analyzers for three visual experiments: (a) The detection of the occurrence of a flash of light of known intensity and time of occurrence, (b) the detection of the time of occurrence of a flash of known intensity, and (c) The estimation of the intensity of a flash occurring at a known time. Possible neural mechanisms in the brain for analyzing optic nerve messages, based on the above mathematical models, are suggested. Changes of excitability or discharge frequency correspond to the output of the likelihood filter. Any such mechanism must be sufficiently plastic to have a response matched to each expected stimuus for most efficient vision near threshold.     

Rapid expression of novel proteins in goldfish retina following optic nerve crush

Polyadenylated messenger RNA was isolated from goldfish retinas at various times following unilateral crush of the optic nerve. RNA was translated in a cell-free system and product proteins analyzed by two-dimensional gel electrophoresis and autofluorography. Poly(A)+ mRNA-directed protein synthesis revealed an 8-fold increase in the labeling of polypeptides of about 30 kd Mr and a pI of 5.5 in retinas 2 d following optic nerve crush, compared with control retina mRNA translation products. In vitro labeling of retinal proteins revealed the enhanced synthesis of comparable 30 kd proteins in 2 d post-crush retinas. Evidence presented suggests that this 30 kd protein cluster may correspond to fish 30 kd stress or heat-shock proteins (hsp-30).