Laminin B1 and Collagen Type IV Gene Expression in Transected Peripheral Nerve: Reinnervation Compared to Denervation

The expression of B1 laminin and type IV collagen was followed in the microsurgically isolated endoneurium of transected rat sciatic nerves from 3 days until 8 weeks. Northern hybridizations revealed that after nerve transection the proximal stumps of denervated, as well as freely regenerating, nerves showed a markedly increased expression of laminin and type IV collagen which lasted from 3 days up to 8 weeks. In the distal stumps, close to the site of transection (2-7 mm), the expression of laminin, and to a certain extent that of type IV collagen, seemed to be enhanced if free axonal reinnervation was allowed. Further distally (10-15 mm), the patterns of B1 laminin and type IV collagen expression were similar in both experimental groups, so that an increased expression was noticed during the first 2 weeks. The present results suggest that laminin and type IV collagen gene expression is markedly different in different parts of transected rat sciatic nerve. During peripheral nerve regeneration, there is a long-lasting basement membrane gene expression in the proximal stump. In the distal part of the transected nerve, the axonal reinnervation possibly up-regulates, but is not essential for, the expression of B1 laminin and type IV collagen.     

Estimating Temporal Causal Interaction between Spike Trains with Permutation and Transfer Entropy

Estimating the causal interaction between neurons is very important for better understanding the functional connectivity in neuronal networks. We propose a method called normalized permutation transfer entropy (NPTE) to evaluate the temporal causal interaction between spike trains, which quantifies the fraction of ordinal information in a neuron that has presented in another one. The performance of this method is evaluated with the spike trains generated by an Izhikevich’s neuronal model. Results show that the NPTE method can effectively estimate the causal interaction between two neurons without influence of data length. Considering both the precision of time delay estimated and the robustness of information flow estimated against neuronal firing rate, the NPTE method is superior to other information theoretic method including normalized transfer entropy, symbolic transfer entropy and permutation conditional mutual information. To test the performance of NPTE on analyzing simulated biophysically realistic synapses, an Izhikevich’s cortical network that based on the neuronal model is employed. It is found that the NPTE method is able to characterize mutual interactions and identify spurious causality in a network of three neurons exactly. We conclude that the proposed method can obtain more reliable comparison of interactions between different pairs of neurons and is a promising tool to uncover more details on the neural coding.     

3D Visualization and Measurement of Capillaries Supplying Metabolically Different Fiber Types in the Rat Extensor Digitorum Longus Muscle During Denervation and Reinnervation

The aim of this study was to determine whether capillarity in the denervated and reinnervated rat extensor digitorum longus muscle (EDL) is scaled by muscle fiber oxidative potential. We visualized capillaries adjacent to a metabolically defined fiber type and estimated capillarity of fibers with very high oxidative potential (O) vs fibers with very low oxidative potential (G). Capillaries and muscle fiber types were shown by a combined triple immunofluorescent technique and the histochemical method for NADH-tetrazolium reductase. Stacks of images were captured by a confocal microscope. Applying the Ellipse program, fibers were outlined, and the diameter, perimeter, cross-sectional area, length, surface area, and volume within the stack were calculated for both fiber types. Using the Tracer plug-in module, capillaries were traced within the three-dimensional (3D) volume, the length of capillaries adjacent to individual muscle fibers was measured, and the capillary length per fiber length (Lcap/Lfib), surface area (Lcap/Sfib), and volume (Lcap/Vfib) were calculated. Furthermore, capillaries and fibers of both types were visualized in 3D. In all experimental groups, O and G fibers significantly differed in girth, Lcap/Sfib, and Lcap/Vfib, but not in Lcap/Lfib. We conclude that capillarity in the EDL is scaled by muscle fiber size and not by muscle fiber oxidative potential. (J Histochem Cytochem 57:437–447, 2009)     

Effect of Denervation and Reinnervation on Oxidation of [6-14C]Gucose by Rat Skeletal Muscle Homogenates

Abstract: We studied the effects of denervation and reinnervation of the rat extensor digitorum longus muscle (EDL) on the oxidation of [6-¹⁴C]glucose to ¹⁴CO₂. The rate of ¹⁴CO₂ production decreased dramatically following denervation, and the decrease became significant 20 days after nerve section. Prior to day 20, changes apparently reflected the decline of muscle mass. Decreased ¹⁴CO₂ production was due to reduced capacity of the enzymatic system (apparent V_max); there was no change in apparent affinity for glucose (apparent K _m). Mixing experiments revealed that the loss of oxidative capacity following denervation is not caused by production of soluble inhibitors by degenerating muscle. Oxidative metabolism, as measured by ¹⁴CO₂ evolution, recovered during reinnervation. Surprisingly, the specific activity in reinnervated muscles displayed an "overshoot" of approximately 50%, which returned to control by day 60, possibly reflecting increased energy demand by the growing muscle. The time-course of the denervation-mediated change indicates that altered oxidative capacity is secondary to events that initiate denervation changes in muscle. Nevertheless, diminished oxidative capacity may be of considerable metabolic significance in denervated muscle.     

Muscle Plasticity During Sprouting and Reinnervation

SYNOPSIS. When peripheral nerves are cut, the axotomized nervesand denervated muscles undergo atrophic changes which are reversedonly when functional connections are remade in the periphery.The restored interaction completely reverses the effects ofaxotomy and denervation and leads to rematching of the sizeof the motoneuron, muscle unit force, speed and histochemicalproperties, according to the size principle. Differences inunit force and fatigue characteristics between motor unit typesare not fully restored in reinnervated muscles but do not obscuresize relationships between the motoneurons and their muscleunits. Although intact motoneurons will supply increased numbers ofmuscle fibers after partial nerve injuries, regenerating axonsappear to be limited in their ability to enlarge their muscleunits. Increased motor unit force in reinnervated slow motorunits is accounted for primarily by an increase in fiber diameter;fast motor units do not increase their mean force output. As a result of the rematching of muscle unit properties withthe size of the motoneurons that reinnervate them, motor unitproperties are appropriate for fine control of movement aftercomplete or partial nerve injuries. However, regenerating axonsdo not reinnervate their original muscle fibers and unless thefibers are injured close to the muscles, they often fail toreinnervate their original muscles. The mismatching of motorpools with inappropriate target muscles is probably the mainfactor responsible for poor recovery of motor function aftercomplete nerve injuries.     

Denervation supersensitivity and the delay phenomenon.

We have reviewed the denervation supersensitivity-AV shunt hypothesis (explaining the delay phenomenon) and assessed the contribution of each of the two components of denervation supersensitivy to delay. We concluded that adrenergic denervation supersensitivity contributes little to the delay phenomenon. We propose a new hypothesis, based on the effects of prolonged vascular smooth muscle relaxation in the precapillary arterioles, to explain the delay phenomenon.     

Diaphragmatic fatigue in normoxia and hyperoxia

The effect of choline deficiency on the lung lipids of actively growing male Sprague-Dawley rats was investigated using a washed soy protein diet deficient in choline and methionine (lipotrophic). The livers from deficient animals had a significantly increased total lipid content and decreased phosphatidylcholine (PC) content and PC-to-phosphatidylethanolamine ratio (P less than 0.01). Although lung free choline levels were decreased 40% compared with controls (P less than 0.05), the PC content of the whole lung homogenate was unchanged. However, disaturated phosphatidylcholine from animals receiving the lipotrophic diet was significantly increased in the lavage and proportionally decreased in the lavaged lung tissue compared with controls (P less than 0.01). This study indicates that, despite decreased lung choline levels as a result of ingesting a lipotrophic diet, and unlike the liver, lung PC content is maintained at normal values. Although the lung total PC levels are maintained, there is a change in the partition of this lipid pool between the tissue and the alveolar space.     

Denervation and age modify neuromuscular positional selectivity

The rostrocaudal position of neurons within the spinal motor pool maps systematically onto the surface of several muscles in mammals. In an effort to understand the mechanisms that generate such maps, we have been studying choices made by embryonic spinal cord neurons on muscle membrane substrates in the in vitro stripe assay. In this report we explore the effects of postnatal age of the muscle on neurite choice, and how prior denervation modifies this choice. Our results further differentiate rostral from caudal motor neurons in preferring one substrate to another. First, caudal neurites prefer to grow on P6 neonatal caudal over rostral membranes, but lose this ability to distinguish axial position of origin in older muscles. Rostral neurites prefer growth on rostral membranes, but this preference also diminishes with age. Second, when adult muscles have been denervated, both rostral and caudal neurites regain their positional growth selectivity. Third, caudal neurites are particularly sensitive to substrate choice. When growing on a preferred substrate (gluteus) caudal neurites prefer neonatal over adult membranes. These results support the concept of fundamental differences in the growth preferences of rostral and caudal spinal neurites. These differences will assist in the identification of molecular guidance cues that determine the formation of neuromuscular positional maps.     

A Study of the Reinnervation of Fast and Slow Mammalian Muscles

Miniature end plate potential (mepp) frequency in innervated extensor muscle is significantly higher than in soleus muscle. 9 days after nerve crush mepps of low amplitude and prolonged duration reappeared at a frequency of 2% of control and were similar to normal muscles after 35 days. Membrane potential began to increase 9–10 days after nerve crush and at 30 days was similar to controls. The region most sensitive to ACh in denervated and reinnervated muscles was the end plate. Caffeine (20 mM, 23°C) induced contracture in innervated soleus but not in extensor muscles. After denervation the extensor became sensitive to caffeine while the soleus muscles decreased in sensitivity to the drug; 4–5 days after reinnervation the effect of caffeine on these muscles was similar to control. The events during reinnervation are: (a) reappearance of mepps at the same time as end plate potential and muscle twitch; (b) partial restoration of the membrane potential; (c) return of caffeine-induced contracture to normal levels in the soleus and its absence in the extensor muscles; (d) return of membrane resistance to normal values in both muscles at about 25 days; and (e) return of ACh-sensitivity to control levels at about 30 days in both muscles. Although these results suggest that the membrane potential and sarcoplasmic reticulum are under neural influence, it remains to be established whether or not separate neurotrophic factors are involved.     

EpiProfile Quantifies Histone Peptides With Modifications by Extracting Retention Time and Intensity in High-resolution Mass Spectra

Histone post-translational modifications contribute to chromatin function through their chemical properties which influence chromatin structure and their ability to recruit chromatin interacting proteins. Nanoflow liquid chromatography coupled with high resolution tandem mass spectrometry (nanoLC-MS/MS) has emerged as the most suitable technology for global histone modification analysis because of the high sensitivity and the high mass accuracy of this approach that provides confident identification. However, analysis of histones with this method is even more challenging because of the large number and variety of isobaric histone peptides and the high dynamic range of histone peptide abundances. Here, we introduce EpiProfile, a software tool that discriminates isobaric histone peptides using the distinguishing fragment ions in their tandem mass spectra and extracts the chromatographic area under the curve using previous knowledge about peptide retention time. The accuracy of EpiProfile was evaluated by analysis of mixtures containing different ratios of synthetic histone peptides. In addition to label-free quantification of histone peptides, EpiProfile is flexible and can quantify different types of isotopically labeled histone peptides. EpiProfile is unique in generating layouts (i.e. relative retention time) of histone peptides when compared with manual quantification of the data and other programs (such as Skyline), filling the need of an automatic and freely available tool to quantify labeled and non-labeled modified histone peptides. In summary, EpiProfile is a valuable nanoflow liquid chromatography coupled with high resolution tandem mass spectrometry-based quantification tool for histone peptides, which can also be adapted to analyze nonhistone protein samples.The nucleosome, the basic unit of chromatin, consists of 147 base pairs of DNA wrapped around histone proteins (H2A, H2B, H3, and H4). Histones play vital roles in chromatin, interacting with many signaling proteins and chromatin-structural proteins through various post-translational modifications (PTMs)¹ (1–3). There are numerous PTMs on histones, including methylation (mono - me1, di - me2, tri - me3), acetylation (ac), phosphorylation (ph), ubiquitination, and SUMOylation (4). Histone PTMs can affect chromatin function, and therefore influence processes such as gene accessibility, DNA repair and chromosome condensation. Moreover, histone PTMs cross-talk in a synergistic manner to fine-tune gene expression (5). Therefore, quantification of histone PTMs has become a high priority to investigate cell regulation and epigenetics (6).Traditionally, antibody-based methods (e.g. Western blot) have been used to analyze histone modifications (7), which have multiple disadvantages. First, antibodies are not available for every new PTM discovered. Second, PTMs on neighboring amino acids (e.g. H3K9me1–3 and H3S10ph) may prevent antibody binding, a phenomenon called epitope occlusion. Third, the quantification of PTMs via antibody-based methods is not sensitive to small differences (e.g. <twofold). Mass spectrometry (MS) has emerged as a sensitive and efficient technique to detect known and novel PTMs (8). The high mass accuracy and the high speed of modern mass spectrometers allow for sensitive, confident, and accurate peptide quantification when coupled with nanoflow liquid chromatography (nanoLC).NanoLC-MS/MS analysis of protein digests (i.e. bottom-up MS) is nowadays a mature and widely applied technology. Data-dependent acquisition is the most commonly adopted MS acquisition method to identify peptides via bottom-up MS (9–12), generating MS1 and MS2 spectra. Nevertheless, histone proteins are particularly challenging to analyze by using the generalized bottom-up workflow. As histones are rich with lysines and arginines, tryptic digest of histones generates short peptides that are difficult to be retained on C18 columns. To improve histone peptide retention, the unmodified and mono-methylated lysines and peptide N terminus can be selectively chemically propionylated (13–16), preventing tryptic digest after lysine to generate longer peptides. Moreover, peptide identification through traditional database searches leads to a large number of false positives, as allowing several dynamic modifications (e.g. me1/me2/me3, ac, ph) dramatically increases the number of molecular candidates and thus the possibility to achieve a false hit (12). Therefore, software tools that quantify histone peptides require additional data to correctly map a given peptide, such as previous knowledge of peptide retention time.Quantification of histone peptides is particularly challenging because of presence of isobaric peptides, near isobaric PTMs such as tri-methylation (42.047 Da) and acetylation (42.011 Da), and low abundant species. Previous knowledge about relative peptide retention time (RT) enables differentiation between species close in mass and therefore selection of the correct peak for integration of the area of the chromatographic peak (i.e. area under curve or AUC). However, determination of peptide RT might be difficult because of their low abundance though acid extraction was performed to purify histones. This problem can be solved by using isotopically labeled synthetic histone peptides (17), or data independent approaches (18). When using relative retention time information to assign peak identities, reproducible nanoLC is crucial, especially because some isobaric peptides co-elute. In this case, the MS acquisition method must perform targeted MS2 for the co-eluting isobaric peptides at the specific time that they elute. These species can be discriminated and quantified based on the intensity of fragment ions unique to each species. For instance, the peptides KacSTGGKAPR (H3K9ac) and KSTGGKacAPR (H3K14ac) have the same mass and overlap at the nanoLC elution (the full protein sequence of human canonical histone H3 and H4 are shown in Fig. 1A). Thus, the co-eluting isobaric peptides could not be quantified separately based on the MS1 signal, but the unique fragment ions present in MS2 spectra allow them to be quantified individually.Open in a separate windowFig. 1.Histones are a challenge for quantitative mass spectrometry analyses. A, Human histone H3.1 and H4 protein sequences. B, Spline fitting to calculate AUC: blue lines are the original peaks and pink lines are the fitted peaks. C, An example of isobaric PTM modified peptides. The above MS2 is matched with H3K18ac, and the same MS2 is also matched with H3K23ac below. D, The workflow of EpiProfile: inputting precursor m/z and charge state, extracting elution profiles, selecting the correct chromatographic peak, calculating AUC, and outputting quantification tables and figures.There have been few computational investigations attempting to solve the problem of quantifying co-eluting isobaric peptides. DiMaggio et al. used a mixed integer linear optimization (MILP) framework to quantify partially co-eluting isobaric histone peptides from electron transfer dissociation (ETD) spectra (19). The framework is comprised of two MILP models: (1) enumerating the entire space of the modified forms that satisfy a given peptide mass and (2) determining the relative composition of the modified forms in the spectrum. Another study by Guan et al. identified isobaric peptides by searching ETD MS/MS spectra for ions representing all possible configurations of modified peptides using a visual assistance program. The relative abundances of these species were estimated by using a nonnegative least squares procedure (20). Other quantification programs can also perform accurate peak picking, but are commonly not as suitable for heavily modified and isobaric histone peptides (e.g. Skyline) (21). These software programs are unable to provide the layouts of histone peptides (i.e. relative RTs) or discriminate all isobaric modified peptides, two tasks that are vital for full characterization of a histone sample.In this study, we developed a new quantification program named EpiProfile. EpiProfile extracts ion chromatography for known histone peptides by using previous knowledge about their elution profiles. Moreover, it discriminates and quantifies the isobaric histone peptides by resolving the linear equations listed with the peak heights of unique fragment ions between the two modification sites in the MS2 spectra (e.g. ions between H3K9ac and H3K14ac). We evaluated the accuracy of EpiProfile by mixing different ratios of synthetic histone peptides, and then tested EpiProfile by analyzing nanoLC-MS/MS data sets of the following samples: purified histones from HeLa cells, a synthetic histone peptide library, and histone peptides labeled during cell growth with ¹³C-labeled glucose media or stable isotope labeling by amino acids in cell culture (SILAC) (22). We compared EpiProfile to manual quantification of the data, and also with the openly available program Skyline. We found that manual quantification is obviously time-consuming and that Skyline cannot generate the layouts of histone peptides and cannot discriminate four or six-component isobaric peptides, a common occurrence in histone data. Moreover, EpiProfile is highly flexible, and thus it can be used to analyze various protein samples, including isotopically labeled peptides and nonhistone data sets.     

Diaphragmatic blood flow in hypoxia and hypercapnia]

By means of ultrasonic method, used in acute experiments on cats with closed abdominal cavity under nembutal narcosis, we studied the linear and volumetric blood flow velocity in the left phrenic artery, vascular resistance, systemic blood pressure, lung ventilation, arterial blood gases during different degrees of hypoxia and hypercapnia. It was shown that hypoxia and hypercapnia resulted in a decrease of the phrenic artery vascular resistance and an increase of the blood flow in the phrenic artery, not always proportional to hypoxia and hypercapnia degree. The correlation of an increase of the lung ventilation with an increase of the blood flow in the phrenic artery depends on the factor causing activation of the diaphragm performance. Some extreme conditions (prolonged asphyxia, blood loss, the exposure to 3% O2) lower phrenic vascular resistance, providing maximal blood supply of the diaphragm.     

Entropy and diversity

Entropies such as the Shannon–Wiener and Gini–Simpson indices are not themselves diversities. Conversion of these to effective number of species is the key to a unified and intuitive interpretation of diversity. Effective numbers of species derived from standard diversity indices share a common set of intuitive mathematical properties and behave as one would expect of a diversity, while raw indices do not. Contrary to Keylock, the lack of concavity of effective numbers of species is irrelevant as long as they are used as transformations of concave alpha, beta, and gamma entropies. The practical importance of this transformation is demonstrated by applying it to a popular community similarity measure based on raw diversity indices or entropies. The standard similarity measure based on untransformed indices is shown to give misleading results, but transforming the indices or entropies to effective numbers of species produces a stable, easily interpreted, sensitive general similarity measure. General overlap measures derived from this transformed similarity measure yield the Jaccard index, Sørensen index, Horn index of overlap, and the Morisita–Horn index as special cases.