Regeneration of long spinal axons in the rat

Summary To investigate regeneration of long spinal axons, the right lateral column of the rat spinal cord was cut at high cervical, low cervical, midthoracic or lumbar level, and one end of an autologous sciatic nerve segment was grafted to the spinal cord at the site of incision. Three to six months after operation, the origin of axons in the grafts was traced retrogradely with horseradish peroxidase injected into the grafts and, in some cases, anterogradely with radioautography of tritiated amino acids injected into the brainstem. Axons from each of the major lateral spinal tracts arising in the brainstem as well as axons ascending from the lower spinal cord succeeded in growing into low cervical grafts. However, long descending axons rarely regenerated after midthoracic or lumbar injury; axons ascending from lumbar segments of the spinal cord usually failed to enter high cervical grafts. Differences in axonal regrowth at the four segmental levels were not simply attributable to dwindling of axonal number in fibre tracts. Axonal regeneration from Clarke's column or the red nucleus was observed only with lesions causing atrophy of many neurons.There was no obvious example of a fibre tract in the lateral spinal columns from which axons failed to regenerate nor from which axons regenerated exceptionally well. Under the conditions of these experiments, the distance from cell body to injury appeared to be an important determinant of axonal regeneration.     

Long-fragment DNA of blood plasma as one of the criteria of individual sensitivity to emotional stress and to cerebral ischemia

Intravenous injection ofpolyethylenoxide WSR-301 reducing hydrodynamic blood resistance (Toms effect) improves gas exchange in the lungs and halved lethality of the animals with cerebral ischemia. The aim of the study was to establish whether free plasma DNA influences blood gases and lethality of the animals with brain ischemia. Common carotid arteries were ligated for 15 min in intact stressed and tested in the open field Wistar male rats, then some of the rats received intravenous solution of homologous long-fragment DNA (20x10(-6) g/ml of blood). Cerebral circulation, acid-base equilibrium, paO2, paCO2, asymptotic blood viscosity, plasmic concentration and length of DNA fragments in plasma, lethality and neurological status of the survivors were studied. It was found that long fragments of rat DNA show hydrodynamic Toms effect. In normal passive rats sensitive to cerebral ischemia part of plasm DNA is fragmented, gas composition and blood viscosity of blood is worse (p < 0.05) than in active animals. There is a direct correlation between the level of long-fragment DNA in plasm and paO2 (r = 0.55) and inverse--with paCO2 (r = -0.84). Intravenous injection of long-fragment DNA improved the course and reduced lethality of brain ischemia 2-3-fold. Thus, qualitative and quantitative characteristics of plasma circulating DNA are responsible for differences in blood gases in rats differently tolerable to cerebral ischemia and can serve as one of the criteria of individual sensitivity to it being essential in pathogenesis of ischemic stroke.     

Physiological identification of static β axons in primate muscle

Summary Skeletofusimotor () axons exerting a static action on spindle primary endings were identified in the peroneus brevis and tertius muscles of the cynomolgus monkey.In most of the identified static motor units, the extrafusal portion was of the fast-contracting fatiguable type.Supported by NINCDS grant n 1 R01-NS-14702Dr. Murthy died on June 4, 1982The present paper was edited by L. Jami, Laboratoire de Neurophysiologie, Collège de France, F-75231 Paris Cédex 05, France     

Mechanical properties of rat cerebral arteries as studied by a sensitive device for recording of mechanical activity in isolated small blood vessels*

A sensitive device for recording of mechanical activity in isolated small blood vessels with calibres down to 100 μm is described. This equipment was used to examine the mechanical properties of rat cerebral arteries. The ultrastructure of the preparations was investigated by light-, transmission, and scanning electron-microscopy. In general the walls of the middle cerebral and basilar arteries consisted of 3 layers of smooth muscle cells, which occupied approximately 80% of the total wall thickness. The present technique preserved the integrity of the vessel wall and caused no observable damage to the smooth muscle or endothelial cells. Neither the basilar nor the middle crebral arteries developed spontaneous phasic contractions under standard conditions. Potassium excess (124 mM) induced a biphasic contractile response characterized by a fast and partly transient increase in tension (phase A), followed by a slowly developing sustained contraction (phase B). The responses to K⁺ were strong, highly reproducible and not influenced by pH changes in the range 6.9-7.8, making K⁺-stimulation suitable for testing of vascular contractility. Length-tension measurements were performed on relaxed and K⁺-activated basilar arteries. The mechanical behaviour of the vessels conformed to a sliding-filament model of muscular contraction. Using the “Maxwell model” of a muscle, the length at which the contractile element produced maximum active tension was established. The passive wall tension at this length (? 1 mN/mm) averaged only about 20% of the total wall tension the arteries were capable of producing when activated by K⁺. Under isometric conditions the K⁺-contracted basilar artery developed a maximum active wall stress of approximately 240 mN/mm². In the light of the mechanical data obtained from the length-tension measurements, the optimum resting wall tension for registration of vascular responses is discussed. It appears that the present in vitro system can be of great value in investigations of the smooth muscle function in small blood vessels.     

Time dependent mechanical properties of bone cement. An in vitro study over one year

Changes in mechanical properties of bone cements over time are of clinical importance, but not well documented. Specifications for testing do not address the time factor. This study recorded changes in compressive properties and microstructure of one bone cement stored under simulated physiological conditions (water at 37 degrees C) from 20 min up to 1 year and in dry air at 37 degrees C for comparison. Compressive strength increased within the first week (p < 0.001), decreased at 1 month (p < 0.001), and remained at that level at 1 year. Elastic modulus showed a similar development. Maximum strain values, indicating plastic deformability, increased continuously over 1 year. Microscopy revealed microcracks between the pre-polymer beads and the matrix in specimens tested after 20 min, whereas there were less cracks in 1 year specimens. Increase in strength during the first week is due to polymerization and formation of interpenetrating molecular networks. The subsequent decrease could be due to the plasticizing effect of water uptake, as supported by higher values for dry specimens. It can be speculated that microcracks which could be initiated while reducing an arthroplasty at 15 min, acting as initiators for fatigue fractures in the cement mantle, contribute to cement failure. It is recommended that testing of bone cements should be performed after extended ageing at simulated physiological conditions, for the present cement at least 5 weeks. Results obtained at less than one week could be influenced by ongoing polymerization, as well as microcracks and lower coherence between the prepolymer beads and the matrix.     

Branching of individual somatosensory cerebropontine axons in rat: evidence of divergence

The cerebral cortex conveys major input to the granule cell layer of the cerebellar hemispheres by way of the pontine nuclei. Cerebrocortical projections terminate in multiple, widely distributed clusters in the pontine nuclei. This clustered organization is thought to provide the transition between the different organizational principles of the cerebrum and cerebellum, and indicates that parallel processing occurs at multiple sites in the pontine nuclei. At a cellular level, however, it is unknown whether individual cerebropontine neurons target pontocerebellar cells located in different clusters or not. We have employed anterograde axonal tracing and 3D computerized reconstruction techniques to characterize the branching pattern and morphology of individual cerebropontine axons from the primary somatosensory cortex (SI). Our findings show that 43% of the cerebrobulbar fibers arising from SI whisker representations provide two or three fibers entering the pontine nuclei, whereas 39% have only one fiber, and the remaining 18% do not project to the pontine nuclei. Thus, it appears that a majority of cerebropontine axons originating in SI whisker representations diverge to contact multiple, separated pontocerebellar cells. Further, 84% of the somatosensory cerebropontine fibers are collateral branches from cerebrobulbar and/or cerebrospinal parent fibers, while 16% are direct cerebropontine projections without a further descending projection. A range of thicknesses of the fibers entering the pontine nuclei were observed, with collaterals of corticobulbar fibers having the smallest diameter. Taken together, these findings may be related to previously described separate cerebropontine transmission lines with different properties.     

Physiological properties of neurons in the mouse superior olive: membrane characteristics and postsynaptic responses studied in vitro

1. The physiological properties of cells in the superior olivary complex (SOC) were studied in 400-microns brain slices taken through the mouse auditory brain stem. Coronal sections were prepared from fresh brain tissue and were placed fully submerged in an oxygenated saline solution. The boundaries of the medial nucleus of the trapezoid body (MNTB), the lateral superior olive (LSO), and the fibers of the trapezoid body were visualized through a dissecting microscope, and micropipettes filled with 4 M potassium acetate were inserted into the LSO or MNTB. 2. Bipolar stimulating electrodes were placed along the trapezoid body usually at the midline decussation and at a location just lateral to the LSO. This arrangement allowed for stimulation of the trapezoid body both contralateral and ipsilateral to the SOC. Synaptic potentials were elicited by delivering brief (0.1 ms) current pulses to the fibers of the trapezoid body. In some cases the integrity of the fibers was confirmed by transport of horseradish peroxidase (HRP) after extracellular microinjections at various locations along the pathway. The HRP reaction product revealed active transport within the trapezoid body and characteristic synaptic and terminal morphology in the MNTB and LSO. The MNTB contained primarily large-diameter fibers terminating in specialized endings (the calyces of Held), whereas the LSO contained mainly small-diameter fibers and punctate terminal boutons. 3. Membrane characteristics of cells in MNTB and LSO were determined by injecting current into the cell and measuring the corresponding voltage change. Neurons in LSO exhibited a roughly linear relation between voltage and intracellularly injected current. Negative current resulted in a graded hyperpolarization of the cell membrane, and positive current resulted in a graded depolarization that led to the production of action potentials. The number of action potentials was directly related to the strength of the current injected. In contrast, the neurons in MNTB had current-voltage relations that were strongly nonlinear around resting potential. The injection of negative current led to graded hyperpolarization, but injection of positive current produced a limited depolarization that resulted in either a single large action potential or an action potential followed by several spikes with greatly reduced amplitude. 4. Excitatory postsynaptic potentials (EPSPs) could be elicited in LSO by ipsilateral stimulation of the trapezoid body and in MNTB by contralateral stimulation. In response to repeated stimulation, some cells in LSO exhibited temporal summation, that is, a series of slightly subthreshold current pulses produced postsynaptic potentials that combined to elicit action potentials.(ABSTRACT TRUNCATED AT 400 WORDS)     

Physiological properties of mouse skin sensory neurons recorded intracellularly in vivo: temperature effects on somal membrane properties

Recent combined analyses of the structural, functional, and molecular attributes of individual skin sensory neurons using semi-intact in vitro preparations from mice have provided a wealth of novel insights into nociceptor biology. How these findings translate to more natural conditions nevertheless remains unresolved. Toward this end, intracellular recordings were obtained from 362 physiologically identified dorsal root ganglion (DRG) neurons in a new in vivo mouse preparation developed for combined structure/function analyses of individual skin sensory neurons. Recordings were conducted at thoracic levels in adult decorticate mice for comparison with in vitro findings from the same trunk region. In all, 270 neurons were recorded at DRG temperatures tightly regulated at normal core values to establish a baseline and 137 skin sensory neurons were included in detailed analyses of somal properties for comparisons with similar data obtained under reduced temperatures mirroring in vitro conditions. Recovery of Neurobiotin-labeled central projections was crucial for verifying perceived afferent identity of certain neurons. Further, profound temperature dependency was seen across diverse physiological properties. Indeed, the broad, inflected somal spikes normally viewed as diagnostic of myelinated nociceptors were found to be a product of reduced temperatures and were not present at normal core values. Moreover, greater complexity was observed peripherally in the mechanical and thermal sensitivity profile of nociceptive and nonnociceptive populations than that seen under in vitro conditions. This novel in vivo preparation therefore holds considerable promise for future analyses of nociceptor function and plasticity in normal and transgenic models of pain mechanisms.     

Ultrastructural changes of the patellar tendon as a cruciate ligament substitute (one year and two year results)

In four black-faced sheep, the posterior cruciate ligament was replaced with a free autogenous patellar tendon transplant. Tissue samples from the transplants were investigated by light and electron microscopy 1 year and 2 years after surgery. The normal contralateral posterior cruciate ligament and the normal contralateral patellar tendon were used as controls. The structural differences concerned cells, collagen fibrils, elastic tissue and proteoglycans. Most of the cells of the contralateral patellar tendon were spindle-shaped, whereas those of the transplant were frequently chondroid. In the central region of the transplant as well as in the area far from the bone, cell degenerations, and occasionally hypo- or even acellular zones were found. Measurements of the diameter of collagen fibrils in both contralateral patellar tendon and posterior cruciate ligament showed a more or less pronounced bimodal distribution. A unimodal distribution with mainly thin fibrils (20-60 nm) was demonstrated in the transplant tissue which also revealed some morphological alterations of the collagen fibrils. Thin elastic fibers (microfibrils and amorphous material) were randomly scattered among the collagen fibrils of the control samples, bundles of microfibrils (without amorphous material) characterized the transplant. Staining with Alcian blue in the presence of 0.3 M MgCl2 demonstrated a close relationship between proteoglycans and collagen fibrils as well as elastic components in patellar tendon. This arrangement was lost in the transplant where abundant proteoglycans were revealed which, however, composed a tight irregular network between the collagen fibrils. The results serve as a baseline for understanding the impaired biochemical properties of a free autogenous patellar tendon transplant.     

Study on the outcome of pregnancies for one year in Dehradun city

Data on the outcome of pregnancies and birth weight of neonates for one year from the government women hospital, Dehradun (U.P.) are analysed. The frequency of live births, stillbirths, twin births and abortions are found to occur higher in autumn. This may be due to higher number of marriages in winter months and higher conception rate in cooler months. The live birth sex ratio is found to be at par with the other world populations. The sex ratio for single live births as well as stillbirths is lowest in winter and for twin births, it is lowest in summer. Single live born neonates are heavier than the stillborn and twin born neonates. Summer live born and winter stillborn single neonates are heavier than those born in other seasons.     

The arborization of single callosal axons in the mouse cerebral cortex

After several large cortical injections of horseradish peroxidase, individual callosal axons could be observed in most cortical areas contralateral to the injected hemisphere. They left the white matter and travelled for various distances (up to 2 mm) deep in layer VI, then turned to penetrate the cortex radially or obliquely, giving collaterals to several layers and forming narrow terminal arborisations in supragranular layers. In addition, callosal fibers were seen predominantly in deep cortical layers, which fibers could be interpreted either as collaterals of the thick fibers or as a distinct class of callosal afferents.     

Towing of sensory axons by their migrating target cells in vivo

Many pathfinding axons must locate target fields that are themselves positioned by active migration. A hypothetical method for ensuring that these migrations are coordinated is towing, whereby the extension of axons is entirely dependent on the migration of their target cells. Here we combine genetics and time-lapse imaging in the zebrafish to show that towing by migrating cells is a bona fide mechanism for guiding pathfinding axons in vivo.     

Resealing of transected myelinated mammalian axons in vivo: evidence for involvement of calpain.

The mechanisms underlying resealing of transected myelinated rat dorsal root axons were investigated in vivo using an assay based on exclusion of a hydrophilic dye (Lucifer Yellow-biocytin conjugate). Smaller caliber axons (<5 microm outer diameter) resealed faster than larger axons. Resealing was Ca2+ dependent, requiring micromolar levels of extracellular [Ca2+] to proceed, and further accelerated in 1 mM Ca2+. Two hours after transection, 84% of axons had resealed in saline containing 2 mM Ca2+, 28% had resealed in saline containing no added Ca2+ and only 3% had resealed in the Ca2+ buffer BAPTA (3 mM). The enhancing effect of Ca2+ could be overcome by both non-specific cysteine protease inhibitors (e.g., leupeptin) and inhibitors specific for the calpain family of Ca2+ -activated proteases. Resealing in 2 mM Ca2+ was not inhibited by an inhibitor of phospholipase A2. Resealing in low [Ca2+] was not enhanced by agents which disrupt microtubules, but was enhanced by dimethylsulfoxide (0.5-5%). These results suggest that activation of endogenous calpain-like proteases by elevated intra-axonal [Ca2+] contributes importantly to membrane resealing in transected myelinated mammalian axons in vivo.     

The long internal loop of the alpha 3 subunit targets nAChRs to subdomains within individual synapses on neurons in vivo

Different types of neurotransmitter receptors coexist within single neurons and must be targeted to discrete synaptic regions for proper function. In chick ciliary ganglion neurons, nicotinic acetylcholine receptors (nAChRs) containing alpha 3 and alpha 5 subunits are concentrated in the postsynaptic membrane, whereas alpha-bungarotoxin receptors composed of alpha 7 subunits are localized perisynaptically and excluded from the synapse. Using retroviral vector-mediated gene transfer in vivo, we show that the long cytoplasmic loop of alpha 3 targets chimeric alpha 7 subunits to the synapse and reduces endogenous nAChR surface levels, whereas the alpha 5 loop does neither. These results show that a particular domain of one subunit targets specific receptor subtypes to the interneuronal synapse in vivo. Moreover, our findings suggest a difference in the mechanisms that govern assembly of interneuronal synapses as compared to the neuromuscular junction in vertebrates.     

Divergent projection of individual corticospinal axons to motoneurons of multiple muscles in the monkey

Intracellular staining with horseradish peroxidase (HRP) of physiologically identified corticospinal (CS) axons originating from the monkey motor cortex revealed the intraspinal morphology of their branching patterns. CS collaterals spread in a delta-like fashion in the intermediate zone and lamina IX. Virtually all CS axons examined terminated in lamina IX, and it was shown by labeling motoneurons with retrograde transport of HRP that individual CS axons made direct contacts with dendrites of motoneurons of different muscle species.