Proximodistal degeneration of C-fibers detached from their perikarya

Degeneration was followed in the garfish olfactory nerve after removal of the mucosa containing the cell bodies. Degeneration, as measured by a decrease in the weight of consecutive 3-mm nerve segments, spreads at constant velocity from the site of injury toward the synaptic area. The proximodistal degeneration is temperature dependent and progresses from 0.3 mm/d at 10 degrees C to 13.0 mm/d at 35 degrees C. Between 14 and 35 degrees C, the velocity increases linearly with temperature. At all the temperatures investigated, these proximodistal degeneration velocities are identical to the rates of slow intraaxonal flow measured in axons detached from their cell bodies, or to the rates measured in regenerating fibers, and, except at 10 degrees C, are 3.3 times faster than the rate of slow flow in intact nerves. These results were confirmed by light and electron microscopy. We hypothesize that the collapse and subsequent degeneration of the axons is the result of a proximodistal depletion of cytoskeletal elements no longer provided by the cell body to the axon by slow intraaxonal flow. A significant number of axons disappeared rapidly from the nerve before the arrival of the slow degenerative wave. From studies by other groups, this rapid degeneration may be the result of a lack of rapidly transported, mainly membranous components.     

Slow transport of the cytoskeleton after axonal injury

The delivery of cytoskeletal proteins to the axon occurs by slow axonal transport. We examined how the rate of slow transport was altered after axonal injury. When retinal ganglion cell (RGC) axons regenerated through peripheral nerve grafts, an increase in the rate of slow transport occurred during regrowth of the injured axons. We compared these results to axonal injury in the optic nerve where no substantial regrowth occurs and found a completely different response. Slow transport was decreased approximately tenfold in rate in the proximal segment of crushed optic nerves. This decreased rate of slow transport was not induced immediately, but occurred about 1 week after injury. To explore whether a decrease in the rate of slow transport was induced when the regeneration of peripheral nerves was physically blocked, we examined slow transport in motor neurons after the sciatic nerve was transected and ligated. In this case, no change in the rate of the comigrating tubulin and neurofilament (NF) radioactive peaks were observed. We discuss how the changes in the rate of slow transport may reflect different neuronal responses to injury and speculate about the possible molecular changes in the expression of tubulin which may contribute to the observed changes. © 1992 John Wiley & Sons, Inc.     

Slow transport of the cytoskeleton after axonal injury.

The delivery of cytoskeletal proteins to the axon occurs by slow axonal transport. We examined how the rate of slow transport was altered after axonal injury. When retinal ganglion cell (RGC) axons regenerated through peripheral nerve grafts, an increase in the rate of slow transport occurred during regrowth of the injured axons. We compared these results to axonal injury in the optic nerve where no substantial regrowth occurs and found a completely different response. Slow transport was decreased approximately tenfold in rate in the proximal segment of crushed optic nerves. This decreased rate of slow transport was not induced immediately, but occurred about 1 week after injury. To explore whether a decrease in the rate of slow transport was induced when the regeneration of peripheral nerves was physically blocked, we examined slow transport in motor neurons after the sciatic nerve was transected and ligated. In this case, no change in the rate of the comigrating tubulin and neurofilament (NF) radioactive peaks were observed. We discuss how the changes in the rate of slow transport may reflect different neuronal responses to injury and speculate about the possible molecular changes in the expression of tubulin which may contribute to the observed changes.     

Clots of beta-fibrin. Viscoelastic properties, temperature dependence of elasticity, and interaction with fibrinogen-binding tetrapeptides.

Clots of human beta-fibrin, in which only (or predominantly) the B fibrinopeptide is released, were formed at 14 degrees C by copperhead venom procoagulant enzyme (CVE or venzyme), at pH 8.5, ionic strength 0.45. The shear modulus of elasticity increased slowly and after several days attained a constant value, which was lower than those of alpha-fibrin or alpha beta-fibrin under the same conditions. Before studying the temperature dependence of elasticity, the CVE was then inhibited by introducing phenyl methyl sulfonyl chloride (PMSF) by diffusion. With increasing temperature, the modulus decreased progressively from 5 degrees C to nearly zero at 35 degrees and was essentially reversible with temperature change; recovery of elasticity after change from 34.5 degrees to 14 degrees required approximately 2 d but was considerably faster than the initial buildup of elasticity by CVE at 14 degrees. Creep and creep recovery measurements on unligated clots showed creep rates and irrecoverable deformation that were similar in magnitude to those of alpha-fibrin clots formed with batroxobin and much larger than those of alpha beta-fibrin clots formed with thrombin, under the same conditions. During creep and creep recovery, the differential modulus or compliance remained constant, showing that there was no permanent structural damage, and if network strands are severed in slow flow, they must rejoin in new configurations.(ABSTRACT TRUNCATED AT 250 WORDS)     

An isolated perfused rat mesentery model for direct observation of the vasculature during cryopreservation

An intact vasculature is essential for successful hypothermic perfusion and cryopreservation of solid organs, but few studies have specifically assessed the vascular effects of these procedures. A technique was therefore developed for continuous, direct observation of an isolated vascular bed during hypothermic perfusion with cryoprotectants, and during freezing and thawing. The isolated rat mesentery was spread across a controlled low temperature microscope stage and perfused with solutions containing fluorescein isothiocyanate (FITC)-Dextran 70 as an indicator of macromolecular permeability of the vessels. Hypertonic citrate washout, HP-5 perfusion (23), rapid and slow addition and removal of glycerol, and freezing/thawing were studied. Control perfusion with HP-5 produced slow FITC-Dextran leakage, reflecting normal physiological macromolecular permeability of vessels. Rapid addition of glycerol dramatically increased vascular permeability, consistent with osmotic damage to vessels. Rapid removal stopped flow through capillaries and decreased vascular dimensions, suggesting overhydration of endothelial cells and extravascular tissue. Venules and capillaries were the most susceptible vessels to osmotic stress. Slow addition and removal of glycerol (80 mmol/liter/min) produced results similar to control perfusions. During slow freezing (0.5 degree C/min to -5 degrees C) extravascular ice compressing vessels was more obvious than intravascular ice. Glycerol afforded some protection to the microvasculature during freeze/thaw cycles since flow was reestablished in venules and arterioles after thawing, although FITC-Dextran leakage indicated that damage had occurred.     

Multipotent hair follicle stem cells promote repair of spinal cord injury and recovery of walking function

The mouse hair follicle is an easily accessible source of actively growing, pluripotent adult stem cells. C57BL transgenic mice, labeled with the fluorescent protein GFP, afforded follicle stem cells whose fate could be followed when transferred to recipient animals. These cells appear to be relatively undifferentiated since they are positive for the stem cell markers nestin and CD34 but negative for the keratinocyte marker keratin 15. These hair follicle stem cells can differentiate into neurons, glia, keratinocytes, smooth muscle cells, and melanocytes in vitro. Implanting hair follicle stem cells into the gap region of severed sciatic or tibial nerves greatly enhanced the rate of nerve regeneration and restoration of nerve function. The transplanted follicle cells transdifferentiated mostly into Schwann cells, which are known to support neuron regrowth. The treated mice regained the ability to walk essentially normally. In the present study, we severed the thoracic spinal chord of C57BL/6 immunocompetent mice and transplanted GFP-expressing hair follicle stem cells to the injury site. Most of the transplanted cells also differentiated into Schwann cells that apparently facilitated repair of the severed spinal cord. The rejoined spinal cord reestablished extensive hind-limb locomotor performance. These results suggest that hair follicle stem cells can promote the recovery of spinal cord injury. Thus, hair follicle stem cells provide an effective accessible, autologous source of stem cells for the promising treatment of peripheral nerve and spinal cord injury.     

Controlled-rate freezing of human ES cells

A significant obstacle to using human embryonic stem cells (hESCs) arises from extremely poor survival associated with freezing, typically in the range of 1%. This report describes a slow controlled-rate freezing technique commonly used for mammalian embryo cryopreservation. Using a combination of surviving colony number and colony diameter; survival was determined relative to untreated hESCs. Using a dimethyl sulfoxide (DMSO) cryoprotectant and either a homemade controlled-rate freezing device or a commercial freezing device, survival rates of 20%-80% were obtained. To achieve the highest levels of survival, the critical factors were an ice crystal seed (at -7 degrees to -10 degrees C), a freeze rate between 0.3 degrees and 1.8 degrees C/min, and a rapid thaw rate using room temperature water. Slow controlled-rate cooling allows a rapid, simple, and reproducible means of cryopreserving hESCs.     

INFLUENCE OF TEMPERATURE ON THE VELOCITY AND ON THE ISOTOPE PROFILE OF SLOWLY TRANSPORTED LABELED PROTEINS

Abstract— Slow intra-axonal flow of [³H]leucine labeled proteins has been studied in the garfish olfactory nerve. Because of the homogeneity of the nerve a very well defined peak of slowly transported radioactivity is observed. The velocity of slow flow increases linearly with temperature. Between 14 and 28°C, the rate of the peak apex increases from 0.26 to 1.57 mm/day and the rate of the leading edge of the wavefront from 0.54 to 2.75 mm/day. Extrapolation of the rate-temperature function indicates that slow flow should stop at 11°C. However, a velocity of 0.1 mm/day was determined for experiments conducted at 10°C. Between 15 and 25°C a Q ₁₀ of 3.7 was determined for the peak apex and of 3.3 for the leading edge of the wavefront. The Q₁₀'s are significantly larger than the value of 2.2 found for fast transport (G ross & B eidler , 1975) and support the possibility of at least partial differences between the mechanisms of fast and slow transport. A very small peak was found to migrate in front of the main peak. The positioning of this peak seems to be similar to one found by L asek & H offman (1976) in rat ventral motor neurons.
A temperature dependent exponential decrease of the slow moving peak height was measured and it can be estimated that only 1% of the slowly transported radioactivity reaches the synapses. Most of the slow radioactivity appears to remain in the axon behind the peak. The plateau height was also found to decrease exponentially with time. The rate of disappearance greatly affects the profile determined by the slowly transported labeled proteins along the nerve.     

Evaluation of dog semen quality after slow (biological freezer) or rapid (nitrogen vapours) freezing

Three ejaculates were collected from each of five dogs. After initial evaluation, the sperm-rich fractions were diluted to 100 x 10(6) spermatozoa x mL(-1) in two steps with an egg yolk-TRIS extender containing a final concentration of 5% glycerol and 0.5% Equex STM paste. Half of the 0.5 mL straws obtained from each ejaculate were frozen on nitrogen vapours (4 cm above the liquid surface) ("rapid freezing"), while the other half was frozen in a biological freezer at a rate of 0.5 degrees C x min(-1) between 5 degrees C and -10 degrees C and of 8 degrees C x min(-1) between -10 degrees C and -60 degrees C, followed by immersion in liquid nitrogen ("slow freezing"). After an average storage of 30 days, the straws were thawed in a water-bath at 37 degrees C for 1 min. Progressive motility was subjectively estimated hourly for 8 h on semen incubated at 38 degrees C. Immediately after thawing and after 2 h of incubation, motility parameters were also measured by a motility analyser. Sperm membrane function and chromatin stability were assessed immediately post-thaw, using the hypo-osmotic swelling test and acridine orange staining, respectively. Slow freezing significantly improved total post-thaw motility, which showed a slower decline over time, although spermatozoal average path and straight line velocity were lower compared to the fast rate. Also the number of intact membrane spermatozoa was significantly higher in slow-frozen samples while the proportion of spermatozoa with single-stranded DNA was minimal after both freezing procedures.     

三叉神经对去运动神经支配面肌肌萎缩的影响

本文用组织化学、电镜以及肌球蛋白和肌动蛋白电泳分析了在单纯性面神经切断和三叉神经、面神经同时切断后面肌萎缩的病理改变--肌纤维的显微结构改变以及面肌收缩蛋白质在手术后不同时期的降解变化。实验证明,面部肌肉在不同的神经切断情况下其病理改变不同,正常的三叉神经支配可以延缓面瘫后肌肉蛋白质的降解,减少胶原纤维结缔组织的增生,较好地保护肌纤维的显微结构,延缓和减轻去运动神经支配面肌肌萎缩。本研究结果可以为临床治疗面瘫提供一定的理论指导。     

Modification of nerve growth factor receptor properties by wheat germ agglutinin

PC12 is a nerve growth factor (NGF) responsive cell line which exhibits two classes of NGF receptors distinguishable by different kinetic rate constants, sensitivity to trypsin and resistance to Triton detergent solubilization. Whereas incubation of PC12 cells with wheat germ agglutinin (WGA) prior to addition of 125I-NGF inhibits binding of NGF to both classes of receptors, treatment with WGA subsequent to incubation with NGF does not inhibit NGF binding but causes the class of NGF receptors which exhibit rapid or "Fast" dissociation kinetics prior to lectin treatment to be converted to the form which exhibits "Slow" dissociation kinetics. This WGA-mediated receptor conversion is lectin specific, blocked by N-acetyl-D-glucosamine, occurs at similar rates at 4 and 37 degrees C, and is not impaired by a metabolic poison. NGF receptors converted by WGA, like pre-existing Slow receptors, are resistant to trypsinization and remain associated to Triton X-100 extracted "cytoskeletons." Very similar results were obtained for NGF receptors on a human melanoma cell line A875. These results suggest that Fast and Slow receptors are two interconvertible forms of a single protein, rather than distinct proteins. The significance of the generality of these properties for NGF receptors from diverse species and cell types is discussed.     

Cryopreservation of mouse oocytes using a medium with low sodium content: effect of plunge temperature

The effect of various combinations of plunge temperature and thawing protocol on the survival and viability of mouse oocytes was examined. The oocytes were frozen either in a standard freezing medium (ETFM, embryo transfer freezing medium) or in a low-sodium, choline-based freezing medium (CJ2), with 1.5 M 1,2-propanediol and 0.1 M sucrose, and using a conventional slow cooling method. The criteria used to assess survival were morphological state after thawing (intact or lysed), ability to become fertilized, and ability to develop to the two-cell, morula, and blastocyst stage in vitro. Oocytes frozen in CJ2 and plunged into liquid nitrogen (LN(2)) from -10, -20, or -33 degrees C remained intact and developed to the blastocyst stage at significantly higher rates than oocytes frozen in ETFM. For oocytes plunged into LN(2) from -33 degrees C, very rapid thawing (10 s in 30 degrees C water) was more detrimental than rapid or slow thawing (holding in air at room temperature for 10 or 30 s, respectively, prior to submersion in water at 30 degrees C for 10 s). By contrast, oocytes plunged into LN(2) from -10 or -20 degrees C survived better when thawing was very rapid or rapid than when thawing was slow. With the current protocol CJ2 was very effective over a wide range of plunge temperatures (-20 to -33 degrees C), although the optimal thawing protocol depended on the particular plunge temperature. Over 90% of oocytes surviving after slow cooling in CJ2 to -33 degrees C could be plunged to -196 degrees C with little or no further damage.     

Axon regeneration across the site of injury in the optic nerve of the newt Triturus pyrrhogaster

Summary The process by which axons regenerate following a freeze injury to the optic nerve of the newt was analyzed by light and electron microscopy. Freezing destroys cellular constituents in a one millimeter segment of the nerve, leaving intact the basal lamina and the blood supply to the eye. No axons are seen at the site of injury one to seven days post lesion. This contrasts with the persistence of normal-appearing but severed unmyelinated axons within the cranial stump which thus give a false appearance of early regeneration. The first axon sprouts traverse the lesion and enter the cranial stump by ten days. The number of regenerating axons increases rapidly thereafter with no signs of random growth at the site of injury. These axon sprouts tend to be somewhat larger than normal unmyelinated axons and contain dense core vesicles and abnormal organelles similar to those in growing axons in tissue culture. The persisting basal lamina inside the optic sheath appears to provide continuity across the site of injury, to orient axon sprouts, and to favor an orderly process of axon regeneration without neuroma formation.The authors wish to express their gratitude to Barbara Heindel and Jill Jones for extremely helpful technical assistance. This work was supported by grants NS 10864 and NS 05666 from the U.S. Public Health Service and by the Medical Research Service of the Veterans Administration     

Factors affecting the cryosurvival of mouse two-cell embryos

A series of 4 experiments was conducted to examine factors affecting the survival of frozen-thawed 2-cell mouse embryos. Rapid addition of 1.5 M-DMSO (20 min equilibration at 25 degrees C) and immediate, rapid removal using 0.5 M-sucrose did not alter the frequency (mean +/- s.e.m.) of blastocyst development in vitro when compared to untreated controls (90.5 +/- 2.7% vs 95.3 +/- 2.8%). There was an interaction between the temperature at which slow cooling was terminated and thawing rate. Termination of slow cooling (-0.3 degrees C/min) at -40 degrees C with subsequent rapid thawing (approximately 1500 degrees C/min) resulted in a lower frequency of blastocyst development than did termination of slow cooling at -80 degrees C with subsequent slow thawing (+8 degrees C/min) (36.8 +/- 5.6% vs 63.9 +/- 5.7%). When slow cooling was terminated between -40 and -60 degrees C, higher survival rates were achieved with rapid thawing. When slow cooling was terminated below -60 degrees C, higher survival rates were obtained with slow thawing rates. In these comparisons absolute survival rates were highest among embryos cooled below -60 degrees C and thawed slowly. However, when slow cooling was terminated at -32 degrees C, with subsequent rapid warming, survival rates were not different from those obtained when embryos were cooled to -80 degrees C and thawed slowly (52.4 +/- 9.5%, 59.5 +/- 8.6%). These results suggest that optimal cryosurvival rates may be obtained from 2-cell mouse embryos by a rapid or slow thawing procedure, as has been found for mouse preimplantation embryos at later stages.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fast and slow milk-coagulating variants of Lactobacillus helveticus HLM 1

Slow milk-coagulating variants were isolated from Lactobacillus helveticus HLM 1, a fast strain which coagulates milk in 16 h at 42 degrees C. Variants were isolated after subculturing in reconstituted skim milk or complex broth media. Analysis of plasmid content revealed that in slow variants a 3.5-megadalton plasmid was missing.     

Procedures for rat in situ skeletal muscle contractile properties

There are many circumstances where it is desirable to obtain the contractile response of skeletal muscle under physiological circumstances: normal circulation, intact whole muscle, at body temperature. This includes the study of contractile responses like posttetanic potentiation, staircase and fatigue. Furthermore, the consequences of disease, disuse, injury, training and drug treatment can be of interest. This video demonstrates appropriate procedures to set up and use this valuable muscle preparation. To set up this preparation, the animal must be anesthetized, and the medial gastrocnemius muscle is surgically isolated, with the origin intact. Care must be taken to maintain the blood and nerve supplies. A long section of the sciatic nerve is cleared of connective tissue, and severed proximally. All branches of the distal stump that do not innervate the medial gastrocnemius muscle are severed. The distal nerve stump is inserted into a cuff lined with stainless steel stimulating wires. The calcaneus is severed, leaving a small piece of bone still attached to the Achilles tendon. Sonometric crystals and/or electrodes for electromyography can be inserted. Immobilization by metal probes in the femur and tibia prevents movement of the muscle origin. The Achilles tendon is attached to the force transducer and the loosened skin is pulled up at the sides to form a container that is filled with warmed paraffin oil. The oil distributes heat evenly and minimizes evaporative heat loss. A heat lamp is directed on the muscle, and the muscle and rat are allowed to warm up to 37°C. While it is warming, maximal voltage and optimal length can be determined. These are important initial conditions for any experiment on intact whole muscle. The experiment may include determination of standard contractile properties, like the force-frequency relationship, force-length relationship, and force-velocity relationship. With care in surgical isolation, immobilization of the origin of the muscle and alignment of the muscle-tendon unit with the force transducer, and proper data analysis, high quality measurements can be obtained with this muscle preparation.     

Quick freezing of the murine CNS: comparison of regional cooling rates and metabolite levels when using liquid nitrogen of Freon-12

Abstract— Murine brains were frozen in situ, either in liquid N₂ or in Freon-12 cooled to its freezing point. The effect of these coolants on cooling rates and times in various CNS regions was determined. In addition, levels of ATP, P-creatine and lactate were measured in selected regions of brains from both intact animals and severed heads frozen in either coolant. For both the intact animals and severed heads, superficial regions of brain cooled to 0°C and deeper regions to 25°C, at the same rate in either liquid N2 or Freon. Subsequent cooling was more rapid in liquid N₂ in both regions. Levels of ATP, P-creatine and lactate were similar in brains frozen in either coolant, probably because CNS utilization of highenergy phosphates decreased markedly as body temperature fell. In brains of animals frozen intact, levels of ATP and P-creatine were higher and levels of lactate were lower than those in brains from heads which were severed prior to freezing. This difference may be a result of the marked stimulation which accompanies decapitation and may also reflect continued cerebral circulation in the intact animal for a brief time after immersing the animal in the coolant.     

Temperature dependence of speed of actin filaments propelled by slow and fast skeletal myosin isoforms.

It was shown that the temperature sensitivity of shortening velocity of skeletal muscles is higher at temperatures below physiological (10-25 degrees C) than at temperatures closer to physiological (25-35 degrees C) and is higher in slow than fast muscles. However, because intact muscles invariably express several myosin isoforms, they are not the ideal model to compare the temperature sensitivity of slow and fast myosin isoforms. Moreover, temperature sensitivity of intact muscles and single muscle fibers cannot be unequivocally attributed to a modulation of myosin function itself, as in such specimen myosin works in the structure of the sarcomere together with other myofibrillar proteins. We have used an in vitro motility assay approach in which the impact of temperature on velocity can be studied at a molecular level, as in such assays acto-myosin interaction occurs in the absence of sarcomere structure and of the other myofibrillar proteins. Moreover, the temperature modulation of velocity could be studied in pure myosin isoforms (rat type 1, 2A, and 2B and rabbit type 1 and 2X) that could be extracted from single fibers and in a wide range of temperatures (10-35 degrees C) because isolated myosin is stable up to physiological temperature. The data show that, at the molecular level, the temperature sensitivity is higher at lower (10-25 degrees C) than at higher (25-35 degrees C) temperatures, consistent with experiments on isolated muscles. However, slow myosin isoforms did not show a higher temperature sensitivity than fast isoforms, contrary to what was observed in intact slow and fast muscles.     

Influence of cooling rate on Saccharomyces cerevisiae destruction during freezing: unexpected viability at ultra-rapid cooling rates

The purpose of this work was to study cell viability as a function of cooling rate during freezing. Cooling rate strongly influences the viability of cells during cold thermal stress. One of the particularities of this study was to investigate a large range of cooling rates and particularly very rapid cooling rates (i.e., faster than 20000 degrees C min (-1)). Four distinct ranges of cooling rates were identified. The first range (A(')) corresponds to very slow cooling rates (less than 5 degrees C min (-1)), and results in high cell mortality. The second range (A) corresponds to low cooling rates (5-100 degrees C min (-1)), at which cell water outflow occurs slowly and does not damage the cells. The third range (B) corresponds to rapid cooling rates (100-2000 degrees C min (-1)), at which there is competition between heat flow and water flow. In this case, massive water outflow, which is related to the increase in extracellular osmotic pressure and the membrane-lipid phase transition, can cause cell death. The fourth range (C) corresponds to very high cooling rates (more than 5000 degrees C min (-1)), at which the heat flow is very rapid and partially prevents water exit, which seems to preserve cell viability.