Studies of the development of congenital anomalies in rats. III. Effects of inhibition of mitochondrial energy systems on embryonic development.

Pregnant rats were treated with various inhibitors of mitochondrial oxidative energy metabolism and with lowered oxygen tension, and the embryo fetuses examined for the occurrence of congenital malformations and for changes in enzymatic activities. Treatment with all agents tested resulted in the production of skeletal anomalies. Sodium phenobarbital was the most teratogenic of the drugs tested and produced a high incidence of malformations which included cleft palate, tail anomalies, spinal retroflexion, domed head, and facial hypoplasia. Diphenylhydantoin produced a low incidence of syndactyly and oligodactyly. In addition to its effects on fetal growth and development chloramphenicol appeared to interfere with implantation. Tissue preparations from embryos exposed to sodium phenobarbital and chloramphenicol showed markedly lowered levels of DPNH oxidase activity. Cytochrome oxidase activity was also markedly lowered in the preparations from chloramphenicol-exposed embryos. Enzyme activities in preparations from embryos exposed to malonate and diphenylhydantoin appeared unaffected, although the drugs are strong inhibitors of electron transport in vitro; the lack of apparent effect may be due to the fact that both drugs do not bind to the enzyme preparations and were diluted 100- to 200-fold during preparation and assay of the tissue homogenates.     

Distinct neural precursors in the developing human spinal cord

Both embryonic and adult central nervous system have been shown to contain multipotent neural stem cells, but it is not yet clear whether they consist of a single or distinct populations of neural precursors. Since embryonic human neural precursors, particularly in the spinal cord, have not been extensively characterized, we have studied their behaviour at different days of gestation and in different culture conditions. Depending on dissociation and culture conditions, neurospheres which contain nestin- and vimentin-positive or only vimentin-positive neural precursors can be isolated. Whereas the former can be isolated only at early developmental stages, the latter appear to be present at all the stages examined, between 45 and 89 days of gestation. Furthermore, comparison of the effect of FGF, EGF and the two factors in combination on colony formation shows an additive effect of the two growth factors, indicating the existence of more than one type of neural precursor. Overall our results suggest that the human spinal cord contains distinct and dynamic populations of neural precursors which are developmentally regulated.     

Differences in performance between trained and untrained subjects during a 30-s sprint test in a wheelchair ergometer

To compare physiological responses and propulsion technique of able bodied subjects with no prior experience of wheelchairs (AB) and wheelchair dependent subjects (WD), ten AB and nine WD performed a 30-s sprint test in a wheelchair ergometer. The WD had spinal cord injuries with a lesion at T8 or lower. The WD and AB did not show significantly different physiological responses. The power values averaged for the right wheel over the 30 s of the test were 50.2 (SD 14.7) W and 48.0 (SD 4.4) W for WD and AB, respectively. No significant differences in torque application could be discerned, although WD subjects seemed to have a more flattened torque curve with a smaller negative deflection at the beginning of the push. The WD applied a significantly higher horizontal propulsive force to the handrims but did not apply force more effectively. The percentages of effective force to total propulsive force were 61 (SD 16)% for WD and 57 (SD 4)% for AB. With regard to the kinematic parameters, AB followed the handrims significantly longer than WD (end angle AB 65°, WD 44°), started the push phase with their arms more in retroflexion and flexed their trunks further forward. The AB did however show a movement pattern comparable to that of wheelchair athletes measured in a comparable experiment. It could not be decided conclusively that inexperience in wheelchair propulsion led to a less effective propulsion technique. Despite the selection of WD with respect to lesion level, interindividual differences in terms of level of training may have been responsible for the absence of significant results.     

Comparative distribution of the mRNAs encoding urotensin I and urotensin II in zebrafish

The neural neurosecretory system of fishes produces two biologically active neuropeptides, i.e. the corticotropin-releasing hormone paralog urotensin I (UI) and the somatostatin-related peptide urotensin II (UII). In zebrafish, we have recently characterized two UII variants termed UIIalpha and UIIbeta. In the present study, we have investigated the distribution of UI, UIIalpha and UIIbeta mRNAs in different organs by quantitative RT-PCR analysis and the cellular localization of the three mRNAs in the spinal cord by in situ hybridization (ISH) histochemistry. The data show that the UI gene is mainly expressed in the caudal portion of the spinal cord and, to a lesser extent, in the brain, while the UIIalpha and the UIIbeta genes are exclusively expressed throughout the spinal cord. Single-ISH labeling revealed that UI, UIIalpha and UIIbeta mRNAs occur in large cells, called Dahlgren cells, located in the ventral part of the caudal spinal cord. Double-ISH staining showed that UI, UIIalpha and UIIbeta mRNAs occur mainly in distinct cells, even though a few cells were found to co-express the UI and UII genes. The differential expression of UI, UIIalpha and UIIbeta genes may contribute to the adaptation of Dahlgren cell activity during development and/or in various physiological conditions.     

In vivo imaging of the mouse spinal cord using two-photon microscopy

In vivo imaging using two-photon microscopy in mice that have been genetically engineered to express fluorescent proteins in specific cell types has significantly broadened our knowledge of physiological and pathological processes in numerous tissues in vivo. In studies of the central nervous system (CNS), there has been a broad application of in vivo imaging in the brain, which has produced a plethora of novel and often unexpected findings about the behavior of cells such as neurons, astrocytes, microglia, under physiological or pathological conditions. However, mostly technical complications have limited the implementation of in vivo imaging in studies of the living mouse spinal cord. In particular, the anatomical proximity of the spinal cord to the lungs and heart generates significant movement artifact that makes imaging the living spinal cord a challenging task. We developed a novel method that overcomes the inherent limitations of spinal cord imaging by stabilizing the spinal column, reducing respiratory-induced movements and thereby facilitating the use of two-photon microscopy to image the mouse spinal cord in vivo. This is achieved by combining a customized spinal stabilization device with a method of deep anesthesia, resulting in a significant reduction of respiratory-induced movements. This video protocol shows how to expose a small area of the living spinal cord that can be maintained under stable physiological conditions over extended periods of time by keeping tissue injury and bleeding to a minimum. Representative raw images acquired in vivo detail in high resolution the close relationship between microglia and the vasculature. A timelapse sequence shows the dynamic behavior of microglial processes in the living mouse spinal cord. Moreover, a continuous scan of the same z-frame demonstrates the outstanding stability that this method can achieve to generate stacks of images and/or timelapse movies that do not require image alignment post-acquisition. Finally, we show how this method can be used to revisit and reimage the same area of the spinal cord at later timepoints, allowing for longitudinal studies of ongoing physiological or pathological processes in vivo.     

Calculation of the strain-deformation condition of the spinal motor segment during loading

A mathematical model is proposed to analyze the spinal strain-deformation condition resulting from axial and lateral g-loads that are generated by changes in the gravity field and/or pilot’s actions during high-performance aircraft maneuvering under flight overload conditions. An algorithm of solution has been developed, which takes into account changes in the intervertebral disk pressure and the fibrous ring shape at the time when loading reaches close-to-critical g-values. Calculation of the spinal-strain deformation condition was implemented using the SPLEN computer system (KOMMEK, Russia). Analysis of the spinal straindeformation condition was made for two types of external loads: normal load and unilateral load with the bending moment. Maximum permissible loads on the spinal segment were evaluated, and a pattern of distribution of strain intensity and mean strains, spinal deformation, and the destruction field was described. The developed computer models can be used as a basis for developing a technique of predicting characteristic spinal injuries due to different extreme loads and pathologies.     

Spearman Correlation Identifies Statistically Significant Gene Expression Clusters in Spinal Cord Development and Injury

An important problem in the analysis of large-scale gene expression data is the validation of gene expression clusters. By examining the temporal expression patterns of 74 genes expressed in rat spinal cord under three different experimental conditions, we have found evidence that some genes cluster together under multiple conditions. Using RT-PCR data from spinal cord development and two sets of microarray data from spinal injury, we applied Spearman correlation to identify clusters and to assign P values to pairs of genes with highly similar temporal expression patterns. We found that 15% of genes occurred in statistically significant pairs in all three experimental conditions, providing both statistical and experimental support for the idea that genes that cluster together are co-regulated. In addition, we demonstrated that DNA microarray and RT-PCR data are comparable, and can be combined to confirm gene expression relationships.     

CTCF regulates ataxin-7 expression through promotion of a convergently transcribed, antisense noncoding RNA

Neural networks in the spinal cord control two basic features of locomotor movements: rhythm generation and pattern generation. Rhythm generation is generally considered to be dependent on glutamatergic excitatory neurons. Pattern generation involves neural circuits controlling left-right alternation, which has been described in great detail, and flexor-extensor alternation, which remains poorly understood. Here, we use a mouse model in which glutamatergic neurotransmission has been ablated in the locomotor region of the spinal cord. The isolated in?vitro spinal cord from these mice produces locomotor-like activity-when stimulated with neuroactive substances-with prominent flexor-extensor alternation. Under these conditions, unlike in control mice, networks of inhibitory interneurons generate the rhythmic activity. In the absence of glutamatergic synaptic transmission, the flexor-extensor alternation appears to be generated by Ia inhibitory interneurons, which mediate reciprocal inhibition from muscle proprioceptors to antagonist motor neurons. Our study defines a minimal inhibitory network that is needed to produce flexor-extensor alternation during locomotion.     

Determination of 5-methyltetrahydrofolic acid in plasma and spinal fluid by high-performance liquid chromatography,using on-column concentration and electrochemical detection

Chromatographic conditions for the determination of 5-methyltetrahydrofolic acid in plasma and spinal fluid are described, involving simple pretreatment of the sample. Electrochemical detection was used. The linear range of the method is more than 10³. Recovery from plasma and spinal fluid is 100%, and the detection limit of the method is 2·10^?9M, sufficient for the detection of endogenous plasma and spinal fluid levels. The detection conditions are discussed. Endogenous concentrations of the compound in plasma and spinal fluid were determined and correlated with a folate bioassay. Plasma concentrations have been shown after the administration of leucovorin which is used in anticancer therapy.     

Manual examination of so-called lumbosacral instability

By the manual test of the lumbosacral instability described by Eder and Tilscher we can never find out for certain whether the fifth lumbar vertebra can be shifted ventrally above the sacrum. Before this test the patient has to be turned over on to his side with hips flexed, which causes a ventral flexion (anteflexion) of the lumbosacral segment. During the test, however, the lumbosacral segment is forced to a dorsal flexion (retroflexion); therefore the spinous process of the fifth lumbar vertebra retires from the back surface simulating a sliding forward of the whole vertebra.     

Origin of new glial cells in intact and injured adult spinal cord

Several distinct cell types in the adult central nervous system have been suggested to act as stem or progenitor cells generating new cells under physiological or pathological conditions. We have assessed the origin of new cells in the adult mouse spinal cord by genetic fate mapping. Oligodendrocyte progenitors self-renew, give rise to new mature oligodendrocytes, and constitute the dominating proliferating cell population in the intact adult spinal cord. In contrast, astrocytes and ependymal cells, which are restricted to limited self-duplication in the intact spinal cord, generate the largest number of cells after spinal cord injury. Only ependymal cells generate progeny of multiple fates, and neural stem cell activity in the intact and injured adult spinal cord is confined to this cell population. We provide an integrated view of how several distinct cell types contribute in complementary ways to cell maintenance and the reaction to injury.     

Assessment of spinal temperature sensitivity in conscious goats by feedback signals

Summary In two conscious goats with chronically implanted spinal thermodes, fifty-six experiments were carried out at two environmental conditions of + 5 °C DB and 30 °C DB. The temperature of the spinal cord was altered by perfusing the thermodes with water whose temperature, as measured at the inlet of the thermodes, varied between 30 °C and 43 °C. Heat production, respiratory evaporative heat loss, rectal and oesophageal temperatures were measured. At the lower air temperature, spinal cord cooling resulted in an elevation of rectal temperature, while spinal cord heating caused a fall in rectal temperature. At the higher air temperature, spinal cord cooling did not result in an increase of rectal temperature. As in the lower air temperature, spinal cord heating caused a fall hi rectal temperature. The experiments suggest that the generation of spinal warm signals is independent of air temperature between +5 °C and 30 °C, while spinal cold signals are not generated in the absence of skin cold signals.     

Role of vagal and spinal sensory pathways on eupneic diaphragmatic activity

The interactions between vagal and spinal afferents in the control of eupneic diaphragmatic activity were studied in two groups of cats anesthetized either with pentobarbital sodium (SPB) or with ethyl carbamate-alpha-chloralose (ECC), which enhanced spinal reflexes. Under both conditions of anesthesia two experimental protocols were performed: 1) bilateral cervical vagotomy followed by spinal section at C8 level or 2) spinal section followed by vagotomy. Changes in integrated diaphragmatic activity (Edi) were studied during eupneic ventilation and tracheal occlusion at end expiration. Vagotomy always significantly increased the amplitude of Edi during eupnea (SPB + 30%; ECC + 15%) and prolonged its duration (Tdi) (SPB + 110%; ECC + 75%) but did not modify the overall shape of the Edi vs. time relationship. Spinal section induced reverse changes in the amplitude of Edi, whether vagal afferents were present or suppressed and modified the shape of the Edi wave, but did not significantly modify Tdi. These results indicate that both vagal and spinal afferents may participate in the control of eupneic inspiration but exert different and interdependent influences on the recruitment and firing time of phrenic motoneurons. In addition, Tdi measured during tracheal occlusion (Todi) was markedly prolonged under ECC anesthesia. In this situation spinal section reduced Todi, which became close to the values obtained in intact or spinal cats under SPB anesthesia. Thus the response to tracheal occlusion at end expiration cannot be interpreted as resulting from the sole suppression of volume related vagal information.     

A kinematic model of the shoulder complex to evaluate the arm-reachable workspace

Upper-arm evaluation including shoulder motion in physiotherapy has no three-dimensional tool for an arm-functioning evaluation, which hampers an uniform, objective comparison. Human shoulder complex models suffer from lack of shoulder girdle kinematic data. A kinematic shoulder-complex model with six degrees of freedom is proposed as the composition of the inner joint representing the shoulder-girdle joints and outer joint representing the glenohumeral joint. The outer shoulder joint has three perpendicular rotations: adduction/abduction, retroflexion/flexion and internal/external rotation of the humerus. The inner shoulder joint has two rotations, depression/elevation and retraction/protraction, and one translation, which are all dependent on the elevation angle of the humerus. The human arm-reachable workspace that represents the area within reach of the wrist is calculated on the basis of the shoulder-complex model and the additional elbow-joint direct kinematics. It was demonstrated that cross-sections of the calculated workspace are in agreement with the measured arm-reachable workspace in all three anatomical planes. The arm-reachable workspace volume and graphics were calculated and a comparison of the arm's workspaces during a patient's shoulder treatment was made. The obtained numerical and graphical arm-reachable workspaces can be used for arm-functioning evaluations in rehabilitation and ergonomics.     

Release in vivo of Met-enkephalin and encrypted forms of Met-enkephalin from brain and spinal cord of the anesthetized cat

Processing of the proenkephalin molecule will result in peptide fragments in which the pentapeptide YGGFM is included. We have employed a molecular sieve (2 kDa) separation, enzyme hydrolysis radioimmunoassay (RIA) treatment sequence which permits concurrent measurement of Met-enkephalin (Enk) and several enkephalin-encrypting (X-Enk) peptides in a single sample. Using this protocol, the release of Enk and X-Enk (total Enk - Enk) greater and less than 2 kDa from spinal cord and the mesencephalic aqueductal grey was assessed under resting conditions and during stimulation of the sciatic nerve in the chloralose-urethane anesthetized cat. Under resting conditions measurable levels of Enk (10.5±4.7; 9.1±2.1 pg/min) and X-Enk (47.8±19.7; 45.7±12.3 pg/min) are found in aqueductal and spinal superfusates, respectively. The X-Enk measured under resting and evoked conditions in aqueductal and spinal perfusates is associated almost exclusively (>90–95%) with fragments >2 kDa in size. These results, showing the relative absence of detectable levels of X-Enk forms <2 kDa, were confirmed by reverse phase chromatography. During sciatic nerve stimulation, the levels of both Enk and X-Enk were mildly elevated in spinal and ventricular perfusates. With the addition of thiorphan (10⁻⁵ M), though there was no effect on the resting release of either Enk or X-Enk, the levels of Enk measured under evoked conditions were significantly augmented in both ventricular and spinal perfusates.     

Effect of Flexible Spine Motion on Energy Efficiency in Quadruped Running

Energy efficiency is important in the performance of quadruped robots and mammals.Flexible spine motion generally exists in quadruped mammals.This paper mainly explores the effect of flexible spinal motion on energy efficiency.Firstly,a planar simplified model of the quadruped robot with flexible spine motion is introduced and two simulation experiments are carried out.The results of simulation experiments demonstrate that both spine motion and spinal flexibility can indeed increase energy efficiency,and the curve of energy efficiency change along with spinal stiffness is acquired.So,in order to obtain higher energy efficiency,quadruped robots should have flexible spine motion.In a certain speed,there is an optimal spinal stiffness which can make energy efficiency to be the best.Secondly,a planar quadruped robot with flexible spine motion is designed and the conclusions drawn in the two simulation experiments are verified.Lastly,the third simulation experiment is carried out to explore the relationship between the optimal spinal stiffness,speed and total mass.The optimal spinal stiffness increases with both speed and total mass,which has important guiding significance for adjusting the spinal stiffness of quadruped robots to make them reach the best energy efficiency.     

The morphology and distribution of 'Kolmer-Agduhr cells', a class of cerebrospinal-fluid-contacting neurons revealed in the frog embryo spinal cord by GABA immunocytochemistry

An immunocytochemical method that localizes GABA in glutaraldehyde-fixed tissue has been applied to the study of the Xenopus embryo spinal cord. This procedure stained an anatomical class of neuron, which had somata forming two more or less continuous rows, one on either side of the central canal, in the ventral part of the spinal cord. The total number of stained neurons in the stage 37-38 embryo spinal cord was about 300. The medial surface on the soma protruded into the central canal and had a brush border which electron microscope studies showed to consist of many microvilli or stereocilia and one or two cilia. The external end of the neuron soma had an ipsilateral ascending axon. The axon of many of these neurons had a growth cone which was also clearly stained. We propose calling these neurons 'Kolmer-Agduhr cells' after W. Kolmer and E. Agduhr who described them in the spinal cords of many vertebrate classes. Their early embryonic origin, GABA-like immunoreactivity, axonal projections and distribution as a whole population have not previously been known.     

Patient-specific finite element model of the spine and spinal cord to assess the neurological impact of scoliosis correction: preliminary application on two cases with and without intraoperative neurological complications

Scoliosis is a 3D deformation of the spine and rib cage. For severe cases, surgery with spine instrumentation is required to restore a balanced spine curvature. This surgical procedure may represent a neurological risk for the patient, especially during corrective maneuvers. This study aimed to computationally simulate the surgical instrumentation maneuvers on a patient-specific biomechanical model of the spine and spinal cord to assess and predict potential damage to the spinal cord and spinal nerves. A detailed finite element model (FEM) of the spine and spinal cord of a healthy subject was used as reference geometry. The FEM was personalized to the geometry of the patient using a 3D biplanar radiographic reconstruction technique and 3D dual kriging. Step by step surgical instrumentation maneuvers were simulated in order to assess the neurological risk associated to each maneuver. The surgical simulation methodology implemented was divided into two parts. First, a global multi-body simulation was used to extract the 3D displacement of six vertebral landmarks, which were then introduced as boundary conditions into the personalized FEM in order to reproduce the surgical procedure. The results of the FEM simulation for two cases were compared to published values on spinal cord neurological functional threshold. The efficiency of the reported method was checked considering one patient with neurological complications detected during surgery and one control patient. This comparison study showed that the patient-specific hybrid model reproduced successfully the biomechanics of neurological injury during scoliosis correction maneuvers.     

Immature spinal cord neurons are dynamic regulators of adult nociceptive sensitivity

Chronic pain is a debilitating condition with unknown mechanism. Nociceptive sensitivity may be regulated by genetic factors, some of which have been separately linked to neuronal progenitor cells and neuronal differentiation. This suggests that genetic factors that interfere with neuronal differentiation may contribute to a chronic increase in nociceptive sensitivity, by extending the immature, hyperexcitable stage of spinal cord neurons. Although adult rodent spinal cord neurogenesis was previously demonstrated, the fate of these progenitor cells is unknown. Here, we show that peripheral nerve injury in adult rats induces extensive spinal cord neurogenesis and a long‐term increase in the number of spinal cord laminae I–II neurons ipsilateral to injury. The production and maturation of these new neurons correlates with the time course and modulation of nociceptive behaviour, and transiently mimics the cellular and behavioural conditions present in genetically modified animal models of chronic pain. This suggests that the number of immature neurons present at any time in the spinal cord dorsal horns contributes to the regulation of nociceptive sensitivity. The continuous turnover of these neurons, which can fluctuate between normal and injured states, is a dynamic regulator of nociceptive sensitivity. In support of this hypothesis, we find that promoters of neuronal differentiation inhibit, while promoters of neurogenesis increase long‐term nociception. TrkB agonists, well‐known promoters of nociception in the short‐term, significantly inhibit long‐term nociception by promoting the differentiation of newly produced immature neurons. These findings suggest that promoters of neuronal differentiation may be used to alleviate chronic pain.