Advanced hepatic tissue destruction in ablative cryosurgery: potentials of intermittent freezing and selective vascular inflow occlusion

Recent studies indicate that cryosurgery represents a promising approach to treat non-resectable liver tumors. To improve parenchymal tissue destruction, a variety of modifications of the freeze-thaw procedure have been suggested, including repetitive freezing and portal-triad cross-clamping. The aim of the present study was to analyze whether intermittent freezing by application of a double freeze-thaw procedure or selective vascular inflow occlusion are more effective than a single freeze-thaw cycle to achieve complete hepatic tissue destruction. Using a porcine model, intrahepatic cryolesions were induced by freezing the hepatic tissue for a total of 15 min (n=6, SF). Additional animals (n=6) underwent a double freeze-thaw cycle of 7.5 min each (DF). A third group of animals (n=6) was treated by a single 15-min freeze-thaw cycle during selective vascular inflow occlusion (VO-SF). Seven days after freezing, DF did not change the volume of the cryolesion (25.4+/-1.7 cm(3)) compared to SF (29.9+/-3.7 cm(3)), however, resulted in enhanced destruction of hepatocyte nuclear morphology (DF-score: 2.4+/-0.2 versus SF-score: 1.1+/-0.3; p<0.05) and attenuated leukocyte infiltration within the margin of the cryolesion (DF-score: 1.5+/-0.2 versus SF-score: 2.8+/-0.1; p<0.05). VO-SF was also effective to significantly enhance destruction of hepatocyte nuclear morphology (2.8+/-0.1; p<0.05 versus SF), but, additionally, markedly increased the volume of the cryolesions (43.3+/-5.3 cm(3); p<0.05 versus SF and DF). Interestingly, VO-SF further increased the number of apoptotic cells, while leukocyte infiltration (2.3+/-0.3) was not affected compared to that after SF-treatment. Thus, our data indicate that both DF and VO-SF are effective to enhance parenchymal cell destruction within the margin of the cryolesion. VO-SF additionally increases the volume of the lesion and may therefore be most attractive for successful clinical application.     

Mesophyll freezing and effects of freeze dehydration visualized by simultaneous measurement of IDTA and differential imaging chlorophyll fluorescence

Infrared differential thermal analysis (IDTA) and differential imaging chlorophyll fluorescence (DIF) were employed simultaneously to study the two-dimensional pattern of ice propagation in leaves and mesophyll freeze dehydration as detected by a significant increase of basic chlorophyll fluorescence (F(0)). IDTA and DIF technique gave different insights into the freezing process of leaves that was highly species-specific. IDTA clearly visualized the freezing process consisting of an initial fast spread of ice throughout the vascular system followed by mesophyll freezing. While mesophyll freezing was homogeneously in Poa alpina, Rhododendron ferrugineum and Senecio incanus as determined by IDTA, DIF showed a distinct pattern only in S. incanus, with the leaf tips being affected earlier. In Cinnamomum camphora, a mottled freezing pattern of small mesophyll compartments was observed by both methods. In IDTA images, a random pattern predominated, while in DIF images, compartments closer to lower order veins were affected earlier. The increase of F(0) following mesophyll freezing started after a species-specific time lag of up to 26 min. The start of the F(0) increase and its slope were significantly enhanced at lower temperatures, which suggest a higher strain on mesophyll protoplasts when freezing occurs at lower temperatures.     

Impact of freezing on pH of buffered solutions and consequences for monoclonal antibody aggregation

Freezing of biologic drug substance at large scale is an important unit operation that enables manufacturing flexibility and increased use‐period for the material. Stability of the biologic in frozen solutions is associated with a number of issues including potentially destabilizing pH changes. The pH changes arise from temperature‐associated change in the pK_as, solubility limitations, eutectic crystallization, and cryoconcentration. The pH changes for most of the common protein formulation buffers in the frozen state have not been systematically measured. Sodium phosphate buffer, a well‐studied system, shows the greatest change in pH when going from +25 to ?30°C. Among the other buffers, histidine hydrochloride, sodium acetate, histidine acetate, citrate, and succinate, less than 1 pH unit change (increase) was observed over the temperature range from +25 to ?30°C, whereas Tris‐hydrochloride had an ～1.2 pH unit increase. In general, a steady increase in pH was observed for all these buffers once cooled below 0°C. A formulated IgG2 monoclonal antibody in histidine buffer with added trehalose showed the same pH behavior as the buffer itself. This antibody in various formulations was subject to freeze/thaw cycling representing a wide process (phase transition) time range, reflective of practical situations. Measurement of soluble aggregates after repeated freeze–thaw cycles shows that the change in pH was not a factor for aggregate formation in this case, which instead is governed by the presence or absence of noncrystallizing cryoprotective excipients. In the absence of a cryoprotectant, longer phase transition times lead to higher aggregation. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Farinose fiavonoids are associated with high freezing tolerance in fairy primrose (Primula malacoides) plants

The deposition of surface(farinose)fiavonoids on aerial parts of some Primula species is a well-documented but poorly understood phenomenon.Here,we show thatfiavonoid deposition on the leaves and winter buds may contribute strongly to preventing freezing damage in these plants.The ice nucleation temperature of fairy primrose(Primula malacoides)leaves covered with naturalfiavone was approximately 6°C lower compared to those that had theirfiavone artificially removed.Additionally,farinosefiavonoids on the leaves reduced subsequent electrolyte leakage(EL)from the cells exposed to freezing temperatures.Interestingly,exogenous application offiavone at4 mg/g fresh weight to P.malacoides leaves,which had the originalfiavone mechanically removed,restored freezing tolerance,and diminished EL from the cells to pretreatment values.Our results suggest that farinosefiavonoids may function as mediators of freezing tolerance in P.malacoides,and exogenous application offiavone could be used to reduce freezing damage during sudden but predictable frost events in other plant species.     

Farinose freezing （Primula flavonoids,are associated with high tolerance in fairy primrose malacoides） plants

The deposition of surface （farinose） flavonoids on aerial parts of some Primula species is a well-documented but poorly understood phenomenon. Here, we show that flavonoid deposition on the leaves and winter buds may contribute strongly to preventing freezing damage in these plants. The ice nucleation temperature of fairy primrose （Primula malacoides） leaves covered with natural flavone was approximately 6~C lower compared to those that had their flavone artificially removed. Additionally, farinose flavonoids on the leaves reduced subse- quent electrolyte leakage （EL） from the cells exposed to freezing temperatures. Interestingly, exogenous application of flavone at 4 mg/g fresh weight to P. malacoides leaves, which had the original flavone mechanically removed, restored freezing tolerance, and diminished EL from the cells to pretreatment values. Our results suggest that farinose flavonoids may function as mediators of freezing tolerance in P. malacoides, and exogenous application of flavone could be used to reduce freezing damage during sudden but predictable frost events in other plant species.     

Proteins accumulate in the apoplast of winter rye leaves during cold acclimation

During cold acclimation, winter rye ( Secale cereale L.) plants develop the ability to tolerate freezing temperatures by forming ice in intercellular spaces and xylem vessels. In this study, proteins were extracted from the apoplast of rye leaves to determine their role in controlling extracellular ice formation. Several polypeptides in the 15 to 32 kDa range accumulated in the leaf apoplast during cold acclimation at 5°C and decreased during deacclimation at 20°C. A second group of polypeptides (63, 65 and 68 kDa) appeared only when the leaves were maximally frost tolerant. Ice nucleation activity, as well as the previously reported antifreeze activity, was higher in apoplastic extracts from cold-acclimated than from nonacclimated rye leaves. These results indicate that apoplastic proteins exert a direct influence on the growth of ice. In addition, freezing injury was greater in extracted cold-acclimated leaves than in unextracted cold-acclimated leaves, which suggests that the proteins present in the apoplast are an important component of the mechanism by which winter rye leaves tolerate ice formation     

Climate change and perishable food hoards of an avian predator: Is the freezer still working?

Changing climate can modify predator–prey interactions and induce declines or local extinctions of species due to reductions in food availability. Species hoarding perishable food for overwinter survival, like predators, are predicted to be particularly susceptible to increasing temperatures. We analysed the influence of autumn and winter weather, and abundance of main prey (voles), on the food‐hoarding behaviour of a generalist predator, the Eurasian pygmy owl (Glaucidium passerinum), across 16 years in Finland. Fewer freeze–thaw events in early autumn delayed the initiation of food hoarding. Pygmy owls consumed more hoarded food with more frequent freeze–thaw events and deeper snow cover in autumn and in winter, and lower precipitation in winter. In autumn, the rotting of food hoards increased with precipitation. Hoards already present in early autumn were much more likely to rot than the ones initiated in late autumn. Rotten food hoards were used more in years of low food abundance than in years of high food abundance. Having rotten food hoards in autumn resulted in a lower future recapture probability of female owls. These results indicate that pygmy owls might be partly able to adapt to climate change by delaying food hoarding, but changes in the snow cover, precipitation and frequency of freeze–thaw events might impair their foraging and ultimately decrease local overwinter survival. Long‐term trends and future predictions, therefore, suggest that impacts of climate change on wintering food‐hoarding species could be substantial, because their ‘freezers’ may no longer work properly. Altered usability and poorer quality of hoarded food may further modify the foraging needs of food‐hoarding predators and thus their overall predation pressure on prey species. This raises concerns about the impacts of climate change on boreal food webs, in which ecological interactions have evolved under cold winter conditions.     

Xylem dysfunction caused by water stress and freezing in two species of co-occurring chaparral shrubs

Water transport from the roots to leaves in chaparral shrubs of California occurs through xylem vessels and tracheids. The formation of gas bubbles in xylem can block water transport (gas embolism), leading to shoot dieback. Two environmental factors that cause gas embolism formation in xylem conduits are drought and freezing air temperatures. We compared the differential vulnerabilities of Rhus laurina and Ceanothus megacarpus, co-dominant shrub species in the coastal regions of the Santa Monica Mountains of southern California, to both water stress-induced and freezing-induced embolism of their xylem. Rhus laurina has relatively large xylem vessel diameters, a deep root system, and a large basal burl from which it vigorously resprouts after wildfire or freezing injury. In contrast, Ceanothus megacarpus has small-diameter vessels, a shallow root system, no basal burl and is a non-sprouter after shoot removal by wildfire. We found that R. laurina became 50% embolized at a water stress of –3 MPa and 100% embolized by a freeze–thaw cycle at all hydration levels. In contrast, C. megacarpus became 50% embolized at a water stress of –9 MPa and 100% embolized by freeze–thaw events only at water potentials lower than –3 MPa. Reducing thaw rates from 0·8 °C min^?1 to 0·08 °C min^?1 (the normal thaw rate measured in situ) had no effect on embolism formation in R. laurina but significantly reduced embolism occurrence in well-hydrated C. megacarpus (embolism reduced from 74 to 35%). These results were consistent with the theory of gas bubble formation and dissolution in xylem sap. They also agree with field observations of differential shoot dieback in these two species after a natural freeze–thaw event in the Santa Monica Mountains.     

Ice Nucleation Activity in the Freezing-Tolerant Antarctic NematodePanagrolaimus davidi

Ice nucleation spectrometry was used to look for the presence of ice nucleating agents (INAs), and their inhibitors, in cultures ofPanagrolaimus davidi, an Antarctic nematode which survives intracellular freezing. INA activity was absent in both nematode suspensions and homogenates. The nematodes produce a substance which inhibits the nucleation activity of organic INAs but not of an inorganic INA (AgI). The nucleation inhibitor is both released from the nematode by homogenization and excreted by them into the medium, but the former was more effective at inhibiting nucleation. The inhibitory activity was destroyed by heating. A thermal hysteresis protein, or a similar ice-active substance, may be responsible for the nucleation inhibition.     

Separate signal pathways regulate the expression of a low-temperature-induced gene in Arabidopsis thaliana (L.) Heynh.

A cDNA clone corresponding to a novel low-temperature-induced Arabidopsis thaliana gene, named lti140, was employed for studies of the environmental signals and the signal pathways involved in cold-induced gene expression. The single-copy lti140 gene encodes a 140 kDa cold acclimation-related polypeptide. The lti140 mRNA accumulates rapidly in both leaves and roots when plants are subject to low temperature or water stress or are treated with the plant hormone abscisic acid (ABA), but not by heat-shock treatment. The low-temperature induction of lti140 is not mediated by ABA, as shown by normal induction of the lti140 mRNA in both ABA-deficient and ABA-insensitive mutants and after treatment with the ABA biosynthesis inhibitor fluridone. The effects of low temperature and exogenously added ABA are not cumulative suggesting that these two pathways converge. The induction by ABA is abolished in the ABA-insensitive mutant abi-1 indicating that the abi-1 mutation defines a component in the ABA response pathway. Accumulation of the lti140 mRNA in plants exposed to water stress was somewhat reduced by treatment with fluridone and in the ABA-insensitive mutant abi-1 suggesting that the water stress induction of lti140 could be partly mediated by ABA. It is concluded that three separate but converging signal pathways regulate the expression of the lti140 gene.     

The expression of a rab-related gene,rab18, is induced by abscisic acid during the cold acclimation process of Arabidopsis thaliana (L.) Heynh

We have isolated a rab-related (responsive to ABA) gene, rab18 from Arabidopsis thaliana. The gene encodes a hydrophilic, glycine-rich protein (18.5 kDa), which contains the conserved serine- and lysine-rich domains characteristic of similar RAB proteins in other plant species. The rab18 mRNA accumulates in plants exposed to low temperature, water stress or exogenous ABA but not in plants subjected to heat shock. This stress-related accumulation of the rab18 mRNA is markedly decreased in the ABA-synthesis mutant aba-1, the ABA-response mutant abi-1 or in wild-type plants treated with the carotenoid synthesis inhibitor, fluridone. Exogenous ABA treatment can induce the rab18 mRNA in the aba-1 mutant but not in the abi-1 mutant. These results provide direct genetic evidence for the ABA-dependent regulation of the rab18 gene in A. thaliana.