The cryopreservation of Chlorella 1. Interactions of rate of cooling,protective additive and warming rate

The cryoprotective additives glycerol and dimethylsulphoxide were found to be toxic to Chlorella cells at concentrations greater then 2.5% w/v. Polyvinylpyrrolidone, was not damaging up to a concentration of 15% w/v. Chlorella 211/7a had a recovery rate greater than 95% at all rates of cooling studied. With Chlorella 211/8h the survival was lower than 0.1% at all rates examined. The addition of dimethylsulphoxide (5% w/v) to Chlorella 211/8h increased the recovery, particularly at the faster rates of cooling; with polyvinylpyrrolidone (10% w/v) there was an optimum range of cooling rate.Cells of Chlorella 211/7a from the exponential phase of growth were found to be damaged both by a temperature reduction from 25°C to 0°C (thermal shock) and by freezing and thawing. In contrast cells from the stationary phase of growth were resistant to these stresses.Abbreviations DMSO dimethylsulphoxide - HEPES N-2-hydroxyethylpiperazine-N-2-ethansulphonic acid - PVP polyvinylpyrrolidone     

Cold shock injury in Tetrahymena pyriformis

The ciliated protozoan Tetrahymena pyriformis has been used to study the biochemistry of cellular injury induced by rapid cooling (cold shock). Cellular viability was found to depend on the time and temperature of cold exposure, and the rate of cooling. During cooling to −7.5 °C, in the absence of ice, an optimal rate of cooling of 2.5 °C min⁻¹ was observed; at both faster and slower cooling the recovery decreased. Following acclimation at a reduced temprature (10 °C) the viability following rapid cooling was significantly different from that of cultures maintained at 20 °C. Analysis of the phospholipid fatty acids from cells grown at 10 °C demonstrated that, at the reduced temperature, there was an increase in the average degree of fatty acyl unsaturation. Cold-shock injury in Tetrahymena is associated with membrane thermotropic events which are determined by temperature per se, whereas viability is a function of the rate of cooling. A hypothesis of injury is presented in which the presence of gel-phase lipid within the membrane is not the critical event, but it is the pattern of nucleation within the membrane which ultimately determines the extent of cellular injury.     

Improved cryopreservation by diluted vitrification solution with supercooling-facilitating flavonol glycoside

The effect of kaempferol-7-O-glucoside (KF7G), one of the supercooling-facilitating flavonol glycosides which was originally found in deep supercooling xylem parenchyma cells of the katsura tree and was found to exhibit the highest level of supercooling-facilitating activity among reported substances, was examined for successful cryopreservation by vitrification procedures, with the aim of determining the possibility of using diluted vitrification solution (VS) to reduce cryoprotectant toxicity and also to inhibit nucleation at practical cooling and rewarming by the effect of supplemental KF7G. Examination was performed using shoot apices of cranberry and plant vitrification solution 2 (PVS2) with dilution. Vitrification procedures using the original concentration (100%) of PVS2 caused serious injury during treatment with PVS2 and resulted in no regrowth after cooling and rewarming (cryopreservation). Dilution of the concentration of PVS2 to 75% or 50% (with the same proportions of constituents) significantly reduced injury by PVS2 treatment, but regrowth was poor after cryopreservation. It is thought that dilution of PVS2 reduced injury by cryoprotectant toxicity, but such dilution caused nucleation during cooling and/or rewarming, resulting in poor survival. On the other hand, addition of 0.5 mg/ml (0.05% w/v) KF7G to the diluted PVS2 resulted in significantly (p < 0.05) higher regrowth rates after cryopreservation. It is thought that addition of supercooling-facilitating KF7G induced vitrification even in diluted PVS2 probably due to inhibition of ice nucleation during cooling and rewarming and consequently resulted in higher regrowth. The results of the present study indicate the possibility that concentrations of routinely used VSs can be reduced by adding supercooling-facilitating KF7G, by which more successful cryopreservation might be achieved for a wide variety of biological materials.     

The cryopreservation ofChlorella

Following growth under sub-optimal concentrations of nutrients, cells ofChlorella emersonii accumulated lipid and became more resistant to the damage caused by freezing and thawing. These results suggest that the factor responsible for the cold hardening of someChlorella spp is not the effect of low temperatures per se but simply that of the reduced metabolic rate. Evidence is given that the post-thaw injury observed following rapid rates of cooling is associated with the vacuole.Abbreviations TLC Thin layer chromatography; fatty acids are noted by two numbers, the first of which give the number of carbon atoms and the second the number of double bonds     

Control of the syntheses of bacteriochlorophyll,c- and b-type cytochromes,and coproporphyrin III in Rhodopseudomonas sphaeroides mutant H5

Rhodopseudomonas sphaerodes mutant H5 lacking 5-aminolevulinic acid synthase was grown phototrophically in chemostat cultures limited by malate. Tetrapyrrole formation was limited by 5-aminolevulinic acid. With variation of dilution rates the cultures exhibited two regions of almost constant cell protein, dry weight and bacteriochlorophyll levels suggesting the formation of two physiological modifications of the strain. These modifications were further characterized by differences in the rates of 5-aminolevulinic acid consumption, the production of reserve material, the stoichiometries of 5-aminolevulinic acid consumption and bacteriochlorophyll or cytochrome production, specific bacteriochlorophyll and cytochrome contents as well as the ratio of bacteriochlorophyll protein complexes. In contrast, cellular levels of coproporphyrin II stayed almost constant over the entire range of dilution rates employed. Bacteriochlorophyll and b-type cytochrome cellular levels exhibited hyperbolic dependencies on the specific rate of 5-aminolevulinic acid consumption, and c-type cytochrome levels a signmoidal dependency. Bacteriochlorophyll cellular levels showed a biphasic dependency with half maximal saturations at 2.6 and 15.4 nmol of 5-aminolevulinic acid consumed per mg of protein and h, and maximal levels of 15.2 and 21 nmol bacteriochlorophyll per mg of protein. Cellular levels of c- and b-type cytochromes were half maximally saturated at 19.5 and 14.5 nmol 5-aminolevulinic acid consumed per mg protein and h while maximal levels were reached at 0.5 and 0.17 nmol of c- and b-type cytochromes, respectively, per mg of protein.The data suggest that within the cell bacteriochlorophyll as well as c- and b-type cytochrome units are assembled according to a defined pattern of kinetics characteristic of each group of compounds. Under otherwise constant external conditions the expression of the pattern is controlled by the rate of 5-aminolevulinic acid supply.     

The effect of cooling rate and warming rate on the packing effect in human erythrocytes frozen and thawed in the presence of 2 M glycerol

The effect of hematocrit (2 versus 75%) has been studied on human red blood cells frozen and thawed in 2 M glycerol at a range of cooling rates (0.8-850 degrees C/min) and warming rates (0.1-200 degrees C/min). The data obtained at a hematocrit of 2% agree well with the data of R. H. Miller and P. Mazur (Cryobiology 13, 404-414, 1976). The results at a hematocrit of 75% show a decrease in recovery with increased cell packing, primarily dependent on warming rate at cooling rates less than 100 degrees C/min and on cooling rate at higher cooling rates. Rapid warming reduced the packing effect, whereas cooling faster than 100 degrees C/min accentuated it. It has been argued that these effects are unlikely to be due to modulation of the generally accepted mechanisms of freezing injury, that is, solution effects and intracellular freezing. It has been suggested that they may be explained by effects of cooling and warming rates on the dimensions of the liquid channels in which the cells are accommodated during freezing and thawing.     

Low cryoprotectant concentrations and fast cooling for nematode cryostorage

Cryopreservation protocols based on slow freezing or vitrification often result in cell injury due to ice formation, cell dehydration and/or toxic concentrations of cryoprotectant (CPA).In this study, we present a cryopreservation technique based on low, non-toxic concentrations of cryoprotectants (≈2–4 M) combined with a rapid cooling rate in the liquid nitrogen phase (−196 °C). Protocols for successfully cryopreserving the plant parasitic nematodes Globodera tabacum tabacum, Heterodera schachtii and Meloidogyne incognita were developed, as demonstrated by the high survival rates and reproducibility of cyst and root-knot nematode species post-cryostorage. This approach for effective cryopreservation of viable plant-parasitic nematodes was developed by inducing an “apparent vitrification” by rapid cooling of the microscopic samples in less than 2 M of cryoprotectant. The extremely thin structure (15–20 μm width, 350–400 μm length) of these nematodes, in combination with a direct and rapid exposure to LN₂, likely prevents the formation of damaging ice crystals. Moreover, this procedure results in viability of both short- and long-cryostorage samples. These techniques could potentially be used for the near-indefinite preservation of thousands of different nematode species. A cryo-nematode collection produced in our lab is available and presented here.     

Multifaceted freezing injury in human polymorphonuclear cells at high subfreezing temperatures

Extracellular freezing injury at high subzero temperatures in human polymorphonuclear cells (PMNs) was studied with a cryomicroscope, electron microscope, and functional assays (phagocytosis, microbicidal activity, and chemotaxis). There are at least four major factors in freezing injury: osmotic stress, chilling, cold shock, and dilution shock. Extracellularly frozen PMNs lose functions when cooled to -2 degrees C without a cryoprotectant. Cells lose volume on freezing to the same degree as in hypertonic exposure. PMNs have a minimum volume to which they can shrink without injury. Greater dehydration produces irreversible injury to cellular functions, and cells eventually collapse under high osmotic stress. Chilling sensitivity is seen in slowly chilled, supercooled PMNs below -5 degrees C; at -7 degrees C, functions are lost in 1 h. This injury can be prevented by the addition of Me2SO but not glycerol. Me2SO does not, however, prevent cold shock (injury due to rapid cooling), which is seen during cooling at 10 degrees C/min to -14 degrees C, but not during slow cooling at 0.5 degrees C/min. One of the problems of using glycerol as a cryoprotectant stems from the high sensitivity of PMNs to dilution shock during the dilution or removal of glycerol.     

Rapidly cooled horse spermatozoa: loss of viability is due to osmotic imbalance during thawing, not intracellular ice formation

The cellular damage that spermatozoa encounter at rapid rates of cooling has often been attributed to the formation of intracellular ice. However, no direct evidence of intracellular ice has been presented. An alternative mechanism has been proposed by Morris (2006) that cell damage is a result of an osmotic imbalance encountered during thawing. This paper examines whether intracellular ice forms during rapid cooling or if an alternative mechanism is present. Horse spermatozoa were cooled at a range of cooling rates from 0.3 to 3,000 degrees C/min in the presence of a cryoprotectant. The ultrastructure of the samples was examined by Cryo Scanning Electron Microscopy (CryoSEM) and freeze substitution, to determine whether intracellular ice formed and to examine alternative mechanisms of cell injury during rapid cooling. No intracellular ice formation was detected at any cooling rate. Differential scanning Calorimetry (DSC) was employed to examine the amount of ice formed at different rate of cooling. It is concluded that cell damage to horse spermatozoa, at cooling rates of up to 3,000 degrees C/min, is not caused by intracellular ice formation. Spermatozoa that have been cooled at high rates are subjected to an osmotic shock when they are thawed.     

A comparative study of the morphology and viability of hyphae of Penicillium expansum and Phytophthora nicotianae during freezing and thawing

The changes in morphology of Penicillium expansum Link and Phytophthora nicotianae Van Breda de Haan during freezing and thawing in a growth medium with and without the cryoprotective additive glycerol were examined with a light microscope fitted with a temperature-controlled stage. Viability of 0.5-1.0 mm diameter colonies of both fungi was determined after equivalent rates of cooling to -196 degrees C in the presence or absence of glycerol. In P. expansum shrinkage occurred in all hyphae at rates of cooling of less than 15 degrees C min-1; at faster rates intracellular ice nucleation occurred. The addition of glycerol increased the rate of cooling at which 50% of the hyphae formed intracellular ice from 18 degrees C min-1 to 55 degrees C min-1. This species was particularly resistant to freezing injury and recovery was greater than 60% at all rates of cooling examined. At rapid rates of cooling recovery occurred in hyphae in which intracellular ice had nucleated. In contrast, during the cooling of Ph. nicotianae in the growth medium, shrinkage occurred and no samples survived on thawing from -196 degrees C. However, on the addition of glycerol, shrinkage during freezing decreased and viable hyphae were recovered upon thawing; at rates of cooling over 10 degrees C min-1 the loss of viability was related to glycerol-induced osmotic shrinkage during cooling rather than to the nucleation of intracellular ice.     

Role of cytoplasmic proteins in cold shock injury of Amoeba

Strains of Amoeba have been used to study the mechanisms of cellular injury induced by rapid cooling (cold shock). Cell viability was found to depend on the time and temperature of cold exposure, on the rate of cooling and on the morphology of the cells prior to chilling. All strains underwent a granuloplasmic contraction following undercooling to ?10 °C, although its extent varied; strains most damaged by cold shock exhibited the most violent cytoplasmic contractions. Cryomicroscopy demonstrated that the cellular contraction occurred upon rewarming, not during cooling. Cells damaged by cold shock were osmotically responsive, demonstrating that irreversible damage to the plasmalemma does not account for the phenomenon.Several compounds protected Amoeba against cold shock injury, glycerol and glucose being the most effective. With glycerol an optimum rate of cooling was observed upon cooling to ?10 °C, at both faster and slower cooling rates damage increased.The state of cellular actin in control cells and following cold shock was monitored by the DNase 1 inhibition assay and by electron microscopy. A comparatively “cold shock resistant” strain of A. proteus was found to contain less total actin per unit cellular protein than the more “sensitive” Amoeba sp. strain Bor. In the Bor strain a cold-induced aggregation of cytoplasmic filaments was evident in electron micrographs, presumably a crosslinking of preexisting F-actin.     

State and Phase Transition Behaviors ofQuercus rubraSeed Axes and Cotyledonary Tissues: Relevance to the Desiccation Sensitivity and Cryopreservation of Recalcitrant Seeds

Freezing and melting transitions of cellular water in embryonic axes and cotyledonary tissues of recalcitrantQuercus rubra(red oak) seeds were compared under slow and rapid cooling conditions. The relevance of desiccation sensitivity (critical water content) and state/phase transition behaviors to cryopreservation was examined. Under a slow to intermediate cooling condition (≤10°C min⁻¹), unfrozen water content in the tissues decreased to less than the critical water content, resulting in a dehydration damage. Under a rapid cooling condition (>100°C min⁻¹) using liquid nitrogen (LN₂), freeze-induced dehydration damage could be avoided if the initial water content was >0.50 g g⁻¹dry wt. However, at water content >0.50 g g⁻¹dry wt, the vitrified cellular matrix was highly unstable upon warming at 10°C min⁻¹. These results offered a theoretical explanation on the difficulty for successful cryopreservation of recalcitrant red oak embryonic axes. A complete state/phase transition diagram for red oak axes was constructed, and a vitrification-based cryopreservation protocol that employed predehydration and rapid cooling was examined. State/phase transition behaviors of cellular water are important parameters for cryopreservation; however, vitrification alone was not sufficient for seed tissues to survive the cryopreservation condition.     

Effects of temperature on silkmoth olfactory responses to pheromone can be simulated by modulation of resting cell membrane resistances

Electrophysiological parameters were measured at different temperatures in resting and pheromone-stimulated olfactory sensilla trichodea of male Antheraea polyphemus (Saturniidae). A method for selective cooling of either the olfactory hair or the antennal branch was developed.The resting preparation resistance increased with lower temperatures, the transepithelial potential decreased. These effects were also observed when the antennal branch was cooled, but were absent during cooling the hair, suggesting a major influence of auxiliary cells on the transepithelial potential and resistance. Together with the preparation resistance, the responses to pheromone stimuli increased with lower temperatures.Computer simulation of the current flow in the sensillum showed that the temperature dependence of responses to pheromone can be explained by modulation of resting resistances of cell membranes alone, without effects of temperature on stimulus transduction. The weak temperature dependence of transepithelial potential might be due to temperature dependence of the electrogenic pump producing the transepithelial potential.Selective cooling of the olfactory hair had no effect on the shape of nerve impulses, cooling of the antennal branch caused changes similar to that obtained by cooling the entire sensillum. This supports the idea that the nerve impulses are generated in the soma of the receptor cell.Abbreviations R _prep preparation resistance - R _prep reduction of R _prep during chemical stimulation - TEP transepithelial potential - TEP receptor-potential amplitude - t _hd half-time of decline of the receptor potential - t _hr half-time of rise of the receptor potential     

Formation of domoic acid and fatty acids inPseudonitzschia pungens f.multiseries with scale of culture

Pseudonitzschia pungens f.multiseries was cultured in 20-L polycarbonate carboys, 350-L fibreglass columns and 500-L plastic bags to determine the effects of medium enrichment and scale of culture on cell yield, production of cellular domoic acid and formation of fatty acids, particularly the potential tracer acid 16:4n-1. Cell concentrations were highest in seawater enriched with stock levels of nitrate and phosphate, but with double the stock level of silicate, at all scales of culture. Cellular toxin in 20, 350 and 500-L cultures averaged 0.32, 0.04 and 2.56 pg cell^-1 and was independent of medium used. The order of magnitude difference in levels of cellular toxin was considered to reflect the varying levels of irradiance within the culture vessels. Support was given to this by the significant difference in content of total cellular fatty acids, due principally to the algal storage acid 16: 1n-7, which is known to be influenced by irradiance. Levels of cellular domoic acid correlated significantly with total fatty acids in 350 and 500-L cultures. Bag cultures producing significantly higher levels of cellular domoic acid provided lower relative proportions of 16:4n-1, which limited its use as a tracer for food-web studies.     

The use of a temperature-profiled position transducer for the study of low-temperature growth in Gramineae

A device is described for measuring linear extension of grass leaves during controlled cooling and heating of the growing region. The instrument was employed to investigate the sensitivity to temperature of the expanding third and fourth leaves of Lolium temulentum L. seedlings. Using a stepped temperature profile it was established that there was no lag in the response of growth rate to rapid changes in temperature below 16°C. If cooling was continued to the point where growth ceased (1°C) but no further, then rates of growth on rewarming were enhanced over the chilling range and reverted to the original rate at 20°C. Cooling to successively lower subzero temperatures before rewarming abolished the hysteretic enhancement, progressively raised the temperature at which growth resumed and decreased the rate of extension until, at-5.3°C, no recovery occurred. The temperature sensitivity of growth, measured as Q₁₀, was essentially constant when cooling from 20°C to 5°C, with 5°C-grown leaf tissue exhibiting a higher mean Q₁₀ than tissue developed at 20°C. The possible physiological significance of these data is discussed.Abbreviations LVDT linear variable displacement transformer - Pe, Fx temperatures at which growth ceases during cooling and resumes during rewarming     

Cryopreservation of heart cells from the eastern oyster

Summary Conditions were developed to cryopreserve cells from pronase-dissociated atria and ventricles of eastern oysters (Crassostrea virginica). The effect of three concentrations (5, 10, 15%) of the cryoprotectants (dimethyl sulfoxide, glycerol, and propylene glycol), three thawing temperatures (25, 45, 75°C), and three cooling rates (slow, medium, fast) were compared. Cells were frozen at −80°C and plunged in liquid nitrogen. Thawed cells were seeded in 96-well plates and primary cultures were evaluated after 3 d by measuring the metabolic activity using a tetrazolium compound, 3-(4,5-dimethylthiazol-2-yl)-5-( 3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, and by comparing the relative spreading of cells between treatments. The best conditions for freezing and thawing of cells for each cryoprotectant were selected and a final study was performed to compare cryoprotectants. For this final study, we measured the number of cells and their viability 3 d after thawing, in addition to determining cell metabolic activity and cell spreading. Primary cultures of cells frozen without cryoprotectant and of nonfrozen cells were used as controls in all studies. Atrial cells were best cryopreserved with glycerol at a concentration of 10%, a medium cooling rate, and thawing at 45°C. After thawing, atrial cells showed 53±5% of the metabolic activity, 84±5% of the number, and 92±2% of the viability of nonfrozen cells. For ventricular cells, 10% glycerol, a medium cooling rate, and thawing at 25°C yielded the best results. The thawed ventricular cells showed 83±5% of the metabolic activity, 91±5% of the number, and 96±2% of the viability of nonfrozen cells.     

Growth characteristics of flagellated cells of Phaeocystis pouchetii revealed by diel changes in cellular DNA content

The present study reports on effects of different light:dark periods, light intensities, N:P ratios and temperature on the specific growth rate of flagellated cells of Phaeocystis pouchetii in culture. The specific growth rate was estimated by diel changes in cellular DNA content. The cellular DNA content and cell cycle of flagellated cells of P. pouchetii are shown, and the importance of light:dark period in cell division is demonstrated. Diel patterns of the cellular DNA content showed that cell division was confined to the dark period. The cells dealt with more than one division per day by rapid divisions shortly after each other.The specific growth rates (μ_DNA) based on the DNA cell cycle model were in close agreement with specific growth rates (μ_Cell) determined from cell counts. The temperature affected the specific growth rates (multiple regression, p < 0.01) and were higher at 5 °C (μ ≤ 2.2 d⁻¹) than at 10 °C (μ ≤1.6 d⁻¹). Increasing the light:dark period from 12:12 h to 20:4 h affected the specific growth rate of P. pouchetii at the lower temperature tested (5 °C) (multiple regression, p < 0.01), resulting in higher specific growth rates than at 10 °C. At 10 °C, the effect of light:dark period was severely reduced. Neither light nor nutrients could compensate the reduction in specific growth rates caused by elevated temperature. The specific growth rates was not affected by the N:P ratios tested (multiple regression, p = 0.21). The experiments strongly suggest that the flagellated cells have a great growth potential and could play a dominating role in northern areas at increased day length.     

Direct uptake of nitrogen by Pfiesteria piscicida and Pfiesteria shumwayae, and nitrogen nutritional preferences

The rates of uptake of a range of forms of nitrogenous nutrients were measured in cultures of Pfiesteria piscicida and Pfiesteria shumwayae maintained at varying physiological states. The measured rates of dissolved N uptake under some conditions approached the rates of N uptake that are achieved through phagotrophy. Rates of dissolved N uptake by P. piscicida contributed <10% of the cellular N of flagellated cells feeding on algae, but were equal to or greater than phagotrophic N acquisition in cells recently removed from fish cultures. Specific N uptake rates (V, h⁻¹) were higher for cells that were maintained on algal prey for long periods (months) than those that were grown with live fish. However, rates of N uptake on a cellular basis for cells grown on or recently removed from fish were comparable to those maintained on algal prey, likely reflecting differences in the sizes of cells of different physiological condition. Preferences for form of N generally followed a decreasing trend of amino acids > urea > NH₄⁺ > NO₃⁻. Nitrate consistently was not a preferred form of N. Although Pfiesteria spp. are often found in eutrophic environments, the relationship between Pfiesteria spp. and nutrient availability is likely to be primarily indirect, mediated through the production of various prey on which Pfiesteria spp. feed. These findings also confirm, however, that when dissolved N concentrations are elevated, they can contribute to the supplemental nutrition of these cells, and thus may provide a significant source of N to Pfiesteria spp. in nature.     

Freezing tolerance in relation to cooling rate in an adult insect

In the adult tenebrionid beetle Upis ceramboides unusually low cooling rates are required to demonstrate maximum freezing tolerance, and a very slight change in rate can reduce survival from 100 to 0%. Freezing to ?50 °C results in 100% mortality at rates above 0.35 °C/min, but no injury is apparent if the cooling rate is 0.28 °C/min. The lower lethal temperature, determined with a cooling rate of 0.17 °C/min, is about ?60 °C. The maximum cooling rate which allows full survival is nearly identical to optimal cooling rates previously found for mouse embryos and some lymphocytes, but the striking sensitivity to very slight changes in rate is unique to Upis. Most studies dealing with insect freezing tolerance have utilized rates of 1 °C/min or faster, and the failure of some of these laboratory studies to observe freezing survival may be due to the use of lethal cooling rates.     

Rapid cold-hardening increases membrane fluidity and cold tolerance of insect cells

The rapid cold-hardening (RCH) response not only confers dramatic protection against cold-shock (non-freezing) injury, but also "instantaneously" enhances organismal performance. Since cold-shock injury is associated with damage to the cell membrane, we investigated the relationship between RCH and changes in cold tolerance and membrane fluidity at the cellular level. None of the adult flies (Sarcophaga bullata) in the cold-shocked treatment group survived direct transfer to -8 degrees C for 2 h; in contrast, 64.5% of flies in the RCH group survived exposure to -8 degrees C. Differences between the treatment groups also were reflected at the cellular level; only 21.3% of fat body cells in the cold-shocked group survived compared to 68.5% in the RCH group. Using 31P solid-state NMR spectroscopy, we determined that membrane fluidity increased concurrently with rapid cold-hardening of fat body cells. This result suggests that membrane characteristics may be modified very rapidly to protect cells against cold-shock injury.