Impact of freeze-thaw-induced pit aspiration on stem water transport in the subalpine conifer Abies veitchii.

During winter, subalpine conifers experience frequent freeze-thaw cycles in stem xylem that may cause embolism and pit aspiration due to increased water volume during the sap to ice transition. This study examined the occurrence and ecological impacts of a combination of freeze-thaw-induced pit aspiration and embolism triggered by natural and artificial stem freezing. In subalpine Veitch's fir (Abies veitchii) trees, the fraction of closed pits and embolized tracheids as well as conductivity losses were measured to examine pit aspiration and its effects. When trees incurred mild drought stress in February and early March, 70%-80% of stem conductivity was lost. Cryo-scanning electron microscopy indicated <20% embolized tracheids but ∼90% closed pits. Severe drought stress in late March caused 96% ± 1.2% (mean ± standard error) loss of stem conductivity, while the fraction of embolized tracheids increased to 64% ± 6.6%, and aspirated pit fraction decreased to 23% ± 5.6%. Experimental freeze-thaw cycles also increased pit aspiration from 7.1% ± 0.89% to 49% ± 10%, and the fraction of closed pits was positively correlated to the percent loss of stem hydraulic conductivity. The results indicated that freezing-induced pit aspiration is an important factor for stem xylem dysfunction under mild drought, and upon severe drought in winter; stem water transport is predominantly inhibited by xylem embolism.

Xylem embolism and bubble formation during freezing suggest complex dynamics of pressure in Betula pendula stems.

Freeze-thaw-induced embolism, a key limiting factor for perennial plants results from the formation of gas bubbles during freezing and their expansion during thawing. However, the ice volumetric increase generates local pressures, which can affect the formation of bubbles. To characterize local dynamics of pressure tension and the physical state of the sap during freeze-thaw cycles, we simultaneously used ultrasonic acoustic emission analysis and synchrotron-based high-resolution computed tomography on the diffuse-porous species Betula pendula. Visualization of individual air-filled vessels and the distribution of gas bubbles in frozen xylem were performed.. Ultrasonic emissions occurred after ice formation, together with bubble formation, whereas the development of embolism took place after thawing. The pictures of frozen tissues indicated that the positive pressure induced by the volumetric increase of ice can provoke inward flow from the cell wall toward the lumen of the vessels. We found no evidence that wider vessels within a tissue were more prone to embolism, although the occurrence of gas bubbles in larger conduits would make them prone to earlier embolism. These results highlight the need to monitor local pressure as well as ice and air distribution during xylem freezing to understand the mechanism leading to frost-induced embolism.

High potential for foliar water uptake in early stages of leaf development of three woody angiosperms.

Foliar water uptake (FWU) is a widespread mechanism that may help plants cope with drought stress in a wide range of ecosystems. FWU can be affected by various leaf traits, which change during leaf development. We exposed cut and dehydrated leaves to rainwater and measured FWU, changes in leaf water potential after 19 h of FWU (ΔΨ), minimum leaf conductance (gmin ), and leaf wettability (abaxial and adaxial) of leaves of Acer platanoides, Fagus sylvatica, and Sambucus nigra at three developmental stages: unfolding (2-5-day-old), young (1.5-week-old) and mature leaves (8-week-old). FWU and gmin were higher in younger leaves. ΔΨ corresponded to FWU and gmin in all cases but mature leaves of F. sylvatica, where ΔΨ was highest. Most leaves were highly wettable, and at least one leaf surface (adaxial or abaxial) showed a decrease in wettability from unfolding to mature leaves. Young leaves of all studied species showed FWU (unfolding leaves: 14.8 ± 1.1 µmol m-2 s-1 ), which may improve plant water status and thus counterbalance spring transpirational losses due to high gmin . The high wettability of young leaves probably supported FWU. We observed particularly high FWU and respective high ΔΨ in older leaves of F. sylvatica, possibly aided by trichomes.

Assessment of formal proceedings and out-of-court reorganisation: results from a survey among turnaround professionals in Austria.

This study analyses the decision criteria for a specific form of reorganisation in a creditor-friendly bankruptcy system such as that of Austria. From a neoinstitutional perspective, we present different forms of bankruptcy law and the specifics of reorganisation in Austria. Next, we show several distinctive criteria and influencing factors for formal reorganisation and workouts. We group these factors into constitutions and institutional settings, process and handling, and implementation of the reorganisation. Using a sample of 411 survey responses from turnaround professionals, our empirical study analyses the decision criteria for a specific form of reorganisation. We apply a multivariate approach comprising two-sided paired samples Wilcoxon tests to assess the derived hypotheses and a hierarchical cluster analysis. Our results indicate significant differences in the valuation of the two forms: the turnaround professionals rate public perception much higher for out-of-court reorganisation, whereas legal certainty is rated significantly better for formal proceedings. Regarding process and handling, transparency and the handling of blocking positions are arguments for formal reorganisation, whereas flexibility is valuated better for workouts. In terms of implementation, respondents see advantages for out-of-court reorganisation, as it facilitates the implementation of both financial and operational measures. Taxation, the handling of blocking positions, and the improvement of public perception were identified as key development aspects for the legal framework conditions of the various reorganisation forms.

Advances in understanding Norway spruce natural resistance to needle bladder rust infection: transcriptional and secondary metabolites profiling.

BACKGROUND: Needle rust caused by the fungus Chrysomyxa rhododendri causes significant growth decline and increased mortality of young Norway spruce trees in subalpine forests. Extremely rare trees with enhanced resistance represent promising candidates for practice-oriented reproduction approaches. They also enable the investigation of tree molecular defence and resistance mechanisms against this fungal disease. Here, we combined RNA-Seq, RT-qPCR and secondary metabolite analyses during a period of 38 days following natural infection to investigate differences in constitutive and infection-induced defence between the resistant genotype PRA-R and three susceptible genotypes. RESULTS: Gene expression and secondary metabolites significantly differed among genotypes from day 7 on and revealed already known, but also novel candidate genes involved in spruce molecular defence against this pathogen. Several key genes related to (here and previously identified) spruce defence pathways to needle rust were differentially expressed in PRA-R compared to susceptible genotypes, both constitutively (in non-symptomatic needles) and infection-induced (in symptomatic needles). These genes encoded both new and well-known antifungal proteins such as endochitinases and chitinases. Specific genetic characteristics concurred with varying phenolic, terpene, and hormone needle contents in the resistant genotype, among them higher accumulation of several flavonoids (mainly kaempferol and taxifolin), stilbenes, geranyl acetone, α-ionone, abscisic acid and salicylic acid. CONCLUSIONS: Combined transcriptional and metabolic profiling of the Norway spruce defence response to infection by C. rhododendri in adult trees under subalpine conditions confirmed the results previously gained on artificially infected young clones in the greenhouse, both regarding timing and development of infection, and providing new insights into genes and metabolic pathways involved. The comparison of genotypes with different degrees of susceptibility proved that several of the identified key genes are differently regulated in PRA-R, and that the resistant genotype combines a strong constitutive defence with an induced response in infected symptomatic needles following fungal invasion. Genetic and metabolic differences between the resistant and susceptible genotypes indicated a more effective hypersensitive response (HR) in needles of PRA-R that prevents penetration and spread of the rust fungus and leads to a lower proportion of symptomatic needles as well as reduced symptom development on the few affected needles.

Cavitation fatigue in conifers: a study on eight European species.

After drought-induced embolism and repair, tree xylem may be weakened against future drought events (cavitation fatigue). As there are few data on cavitation fatigue in conifers available, we quantified vulnerability curves (VCs) after embolism/repair cycles on eight European conifer species. We induced 50% and 100% loss of conductivity (LC) with a cavitron, and analyzed VCs. Embolism repair was obtained by vacuum infiltration. All species demonstrated complete embolism repair and a lack of any cavitation fatigue after 50% LC . After 100% LC, European larch (Larix decidua), stone pine (Pinus cembra), Norway spruce (Picea abies), and silver fir (Abies alba) remained unaffected, while mountain pine (Pinus mugo), yew (Taxus baccata), and common juniper (Juniperus communis) exhibited 0.4-0.9 MPa higher vulnerability to embolism. A small cavitation fatigue observed in Scots pine (Pinus sylvestris) was probably biased by incomplete embolism repair, as indicated by a correlation of vulnerability shifts and conductivity restoration. Our data demonstrate that cavitation fatigue in conifers is species-specific and depends on the intensity of preceding LC. The lack of fatigue effects after moderate LC, and relevant effects in only three species after high LC, indicate that conifers are relatively resistant against cavitation fatigue. This is remarkable considering the complex and delicate conifer pit architecture and may be important considering climate change projections.

Amplifying effects of recurrent drought on the dynamics of tree growth and water use in a subalpine forest.

Despite recent advances in our understanding of drought impacts on tree functioning, we lack knowledge about the dynamic responses of mature trees to recurrent drought stress. At a subalpine forest site, we assessed the effects of three years of recurrent experimental summer drought on tree growth and water relations of Larix decidua Mill. and Picea abies (L. Karst.), two common European conifers representative for contrasting water-use strategies. We combined dendrometer and xylem sap flow measurements with analyses of xylem anatomy and non-structural carbohydrates and their carbon-isotope composition. Recurrent drought increased the effects of soil moisture limitation on growth and xylogenesis, and to a lesser extent on xylem sap flow. P. abies showed stronger growth responses to recurrent drought, reduced starch concentrations in branches and increased water-use efficiency when compared to L. decidua. Despite comparatively larger maximum tree water deficits than in P. abies, xylem formation of L. decidua was less affected by drought, suggesting a stronger capacity of rehydration or lower cambial turgor thresholds for growth. Our study shows that recurrent drought progressively increases impacts on mature trees of both species, which suggests that in a future climate increasing drought frequency could impose strong legacies on carbon and water dynamics of treeline species.

Alpine dwarf shrubs show high proportions of nonfunctional xylem: Visualization and quantification of species-specific patterns.

Xylem conductive capacity is a key determinant of plant hydraulic function and intimately linked to photosynthesis and productivity, but can be impeded by temporary or permanent conduit dysfunctions. Here we show that persistent xylem dysfunctions in unstressed plants are frequent in Alpine dwarf shrubs and occur in various but species-specific cross-sectional patterns. Combined synchrotron micro-computed tomography (micro-CT) imaging, xylem staining, and flow measurements in saturated samples of six widespread Ericaceae species evidence a high proportion (19%-50%) of hydraulically nonfunctional xylem areas in the absence of drought stress, with regular distribution of dysfunctions between or within growth rings. Dysfunctions were only partly reversible and reduced the specific hydraulic conductivity to 1.38 to 3.57 ×10-4 m2 s-1 MPa-1 . Decommission of inner growth rings was clearly related to stem age and a higher vulnerability to cavitation of older rings, while the high proportion of nonfunctional conduits in each annual ring needs further investigations. The lower the xylem fraction contributing to the transport function, the higher was the hydraulic efficiency of conducting xylem areas. Improved understanding of the functional lifespan of xylem elements and the prevalence and nature of dysfunctions is critical to correctly assess structure-function relationships and whole-plant hydraulic strategies.

Leaf water potential measurements using the pressure chamber: Synthetic testing of assumptions towards best practices for precision and accuracy.

Leaf water potential (ψleaf ), typically measured using the pressure chamber, is the most important metric of plant water status, providing high theoretical value and information content for multiple applications in quantifying critical physiological processes including drought responses. Pressure chamber measurements of ψleaf (ψleafPC ) are most typical, yet, the practical complexity of the technique and of the underlying theory has led to ambiguous understanding of the conditions to optimize measurements. Consequently, specific techniques and precautions diversified across the global research community, raising questions of reliability and repeatability. Here, we surveyed specific methods of ψleafPC from multiple laboratories, and synthesized experiments testing common assumptions and practices in ψleafPC for diverse species: (i) the need for equilibration of previously transpiring leaves; (ii) leaf storage before measurement; (iii) the equilibration of ψleaf for leaves on bagged branches of a range of dehydration; (iv) the equilibration of ψleaf across the lamina for bagged leaves, and the accuracy of measuring leaves with artificially 'elongated petioles'; (v) the need in ψleaf measurements for bagging leaves and high humidity within the chamber; (vi) the need to avoid liquid water on leaf surfaces; (vii) the use of 'pulse' pressurization versus gradual pressurization; and (viii) variation among experimenters in ψleafPC determination. Based on our findings we provide a best practice protocol to maximise accuracy, and provide recommendations for ongoing species-specific tests of important assumptions in future studies.

Hydraulic-stomatal coordination in tree seedlings: tight correlation across environments and ontogeny in Acer pseudoplatanus.

Hydraulic conductance is recognized as a major determinant of gas exchange and productivity. However, whether this also applies to seedlings, a critically important stage for vegetation regeneration, has been largely unknown. We analyzed the hydraulic and stomatal conductance of leaves and shoots for 6-wk-old Acer pseudoplatanus seedlings emerging in different lowland and treeline habitats and under glasshouse conditions, respectively, as well as on 9-, 15- and 18-wk-old plants, and related findings to leaf and xylem anatomical traits. Treeline seedlings had higher leaf area-specific shoot hydraulic conductance (Kshoot-L ), and stomatal conductance (gs ), associated with wider xylem conduits, lower leaf area and higher stomatal density than lowland and glasshouse-grown plants. Across the first 18 wk of development, seedlings increased four-fold in absolute shoot hydraulic conductance (Kshoot ) and declined by half in Kshoot-L , with correlated shifts in xylem and leaf anatomy. Distal leaves had higher leaf hydraulic conductance (Kleaf ) and gs compared to basal leaves. Seedlings show strong variation across growth environments and ontogenetic shifts in hydraulic and anatomical parameters. Across growth sites, ontogenetic stages and leaf orders, gs was tightly correlated with Kshoot-L and Kleaf , balancing hydraulic supply with demand for the earliest stages of seedling establishment.

Shade-induced reduction of stem nonstructural carbohydrates increases xylem vulnerability to embolism and impedes hydraulic recovery in Populus nigra.

Nonstructural carbohydrates (NSCs) have been suggested to affect xylem transport under fluctuating water availability, but conclusive evidence is still lacking. We tested the effect of shade-induced NSC depletion on xylem vulnerability to embolism and hydraulic recovery on Populus nigra saplings. Vulnerability was assessed in light-exposed (L) and shaded (S) plants with the hydraulic method, and in vivo with the optical method and X-ray micro-computed tomography. Plants were stressed to 80% loss of hydraulic conductance (PLC) and re-irrigated to check for possible recovery. We measured PLC, bark and wood NSC content, as well as xylem sap pH, surface tension (γsap ) and sugar concentration, before, during and after drought. Shading induced depletion of stem NSC (mainly starch) reserves. All methods converged in indicating higher xylem vulnerability in S than in L plants. This difference was not explained by xylem vessel and pit anatomy or by γsap . Shading impeded sap acidification and sugar accumulation during drought in S plants and prevented hydraulic recovery, which was observed in L plants. Our results highlight the importance of stem NSCs to sustain xylem hydraulic functioning during drought and suggest that light and/or adequate stem NSC thresholds are required to trigger xylem sap chemical changes involved in embolism recovery.

Drought-Induced Xylem Embolism Limits the Recovery of Leaf Gas Exchange in Scots Pine.

Climate change increases the occurrence of prolonged drought periods with large implications for forest functioning. Scots pine (Pinus sylvestris) is one of the most abundant conifers worldwide, and evidence is rising that its resilience to severe drought is limited. However, we know little about its ability to recover from drought-induced embolism. To analyze postdrought hydraulic recovery, we investigated stress and recovery dynamics of leaf gas exchange, nonstructural carbohydrates, and hydraulic properties in 2.5-year-old Scots pine seedlings. We quantified the degree of xylem embolism by combining in vivo x-ray microtomography with intrusive techniques including measurements of hydraulic conductivity and dye staining during drought progression and short-term (2 d) and long-term (4 weeks) recovery. Seedlings were grown under controlled conditions, and irrigation was withheld until stomata closed and xylem water potential declined to -3.2 MPa on average, causing a 46% loss of stem hydraulic conductivity. Following drought release, we found a gradual recovery of leaf gas exchange to 50% to 60% of control values. This partial recovery indicates hydraulic limitations due to drought-induced damage. Whereas xylem water potential recovered close to control values within 2 d, both x-ray microtomography and intrusive measurements revealed no recovery of stem hydraulic conductivity. Moreover, we did not find indications for nonstructural carbohydrate reserves limiting hydraulic recovery. Our findings demonstrate that Scots pine is able to survive severe drought and to partially recover, although we assume that xylem development during the next growing season might compensate for some of the hydraulic impairment. Such drought-induced legacy effects are important when considering vegetation responses to extreme events.

When the heat is on: High temperature resistance of buds from European tree species.

The heat resistance of meristematic tissues is crucial for the survival of plants exposed to high temperatures, as experienced during a forest fire. Although the risk and frequency of forest fires are increasing due to climate change, knowledge about the heat susceptibility of buds, which enclose apical meristems and thus enable resprouting and apical growth, is scarce. In this study, the heat resistance of buds in two different phenological stages was experimentally assessed for 10 European tree species. Cellular heat tolerance of buds was analyzed by determining the electrolyte leakage following heat exposure. Further, the heat insulation capability was tested by measuring the time required to reach lethal internal temperatures linked to bud traits. Our results highlighted differences in cellular heat tolerance and insulation capability among the study species. The phenological stage was found to affect both the thermal stability of cells and the buds' insulation. Further, a good relationship between size-related bud traits and insulation capability was established. Species-specific data on the heat resistance of buds give a more accurate picture of the fire susceptibility of European tree species and provide useful information for estimating tree post-fire responses more precisely.

Subalpine dwarf shrubs differ in vulnerability to xylem cavitation: An innovative staining approach enables new insights.

Dwarf shrubs are a key functional group of the high-elevation vegetation belt. Despite their ecological relevance and high sensitivity to environmental changes, the hydraulic adaptations and species-specific variations in drought tolerance within this growth form are mostly unknown. Here, we assess the xylem vulnerability to cavitation of important character species of the Alpine dwarf shrub heaths in Central Europe. Due to the high percentage of nonfunctional xylem areas in these species, vulnerability curves were gained by an innovative staining approach with safranin, validated by hydraulic and xylem anatomical measurements. The loss of 50% conducting xylem area appeared in the range of -1.78 to -2.91 MPa. Midday plant water potential during an intense summer drought remained above these critical thresholds but was correlated with cavitation resistance. No trade-off between hydraulic safety and efficiency was detected across analyzed species. We conclude that the characteristic clustered occurrence of species in the heterogeneous mountain terrain (mainly interpreted as a consequence of varying snow cover dynamics) is also reflected in species-specific adjustments in xylem hydraulics. The interspecific variety in vulnerability thresholds and plant water potentials during summer drought indicates different hydraulic strategies and adjustments in water relations between these co-occurring shrubs.

Do angiosperm tree species adjust intervessel lateral contact in response to soil drought?

During soil drought (i.e. limited soil water availability to plants), woody species may adjust the structure of their vessel network to improve their resistance against future soil drought stress. Impacts of soil drought on intervessel lateral contact remain poorly understood despite of its significance to xylem transport efficiency and safety. Here, we analysed drought-induced modifications in xylem structures of temperate angiosperm trees with a focus on intervessel lateral contact. Anatomical analyses were performed both in stems of seedlings cultivated under different substrate water availability and annual rings of mature individuals developed during years of low and high soil drought intensities. In response to limited water availability, a decrease in vessel diameter (up to -20%) and simultaneous increase in vessel density (up to +60%) were observed both in seedlings and mature trees. Conversely, there were only small and inconsistent drought-induced changes in intervessel contact frequency and intervessel contact fraction (typically up to ±15%) observed across species, indicating that intervessel lateral contact is a conservative trait. The small adjustments in intervessel lateral contacts were primarily driven by changes in the contact frequencies between neighbouring vessels (i.e. vessel grouping) rather than by changes in proportions of shared cell walls. Our results demonstrate that angiosperm tree species, despite remarkable adjustments in vessel dimensions and densities upon soil drought, exhibit surprisingly invariant intervessel lateral contact architecture.

RNA-Seq and secondary metabolite analyses reveal a putative defence-transcriptome in Norway spruce (Picea abies) against needle bladder rust (Chrysomyxa rhododendri) infection.

BACKGROUND: Norway spruce trees in subalpine forests frequently face infections by the needle rust fungus Chrysomyxa rhododendri, which causes significant growth decline and increased mortality of young trees. Yet, it is unknown whether trees actively respond to fungal attack by activating molecular defence responses and/or respective gene expression. RESULTS: Here, we report results from an infection experiment, in which the transcriptomes (via RNA-Seq analysis) and phenolic profiles (via UHPLC-MS) of control and infected trees were compared over a period of 39 days. Gene expression between infected and uninfected ramets significantly differed after 21 days of infection and revealed already known, but also novel candidate genes involved in spruce molecular defence against pathogens. CONCLUSIONS: Combined RNA-Seq and biochemical data suggest that Norway spruce response to infection by C. rhododendri is restricted locally and primarily activated between 9 and 21 days after infestation, involving a potential isolation of the fungus by a hypersensitive response (HR) associated with an activation of phenolic pathways. Identified key regulatory genes represent a solid basis for further specific analyses in spruce varieties with varying susceptibility, to better characterise resistant clones and to elucidate the resistance mechanism.

Die hard: timberline conifers survive annual winter embolism.

During winter, timberline trees are exposed to drought and frost, factors known to induce embolism. Studies indicated that conifers cope with winter embolism by xylem refilling. We analysed the loss of hydraulic conductivity (LC) in Picea abies branch xylem over 10 years, and correlated winter embolism to climate parameters. LC was investigated by direct X-ray micro-computer tomography (micro-CT) observations and potential cavitation fatigue by Cavitron measurements. Trees showed up to 100% winter embolism, whereby LC was highest, when climate variables indicated frost drought and likely freeze-thaw stress further increased LC. During summer, LC never exceeded 16%, due to hydraulic recovery. Micro-CT revealed homogenous embolism during winter and that recovery was based on xylem refilling. Summer samples exhibited lower LC in outermost compared to older tree rings, although no cavitation fatigue was detected. Long-term data and micro-CT observations demonstrate that timberline trees can survive annual cycles of pronounced winter-embolism followed by xylem refilling. Only a small portion of the xylem conductivity cannot be restored during the first year, while remaining conduits are refilled without fatigue during consecutive years. We identify important research topics to better understand the complex induction and repair of embolism at the timberline and its relevance to general plant hydraulics.

Insights into trunks of Pinus cembra L.: analyses of hydraulics via electrical resistivity tomography.

KEY MESSAGE: The lack of elevational changes in electrical resistivity in Pinus cembra trunks indicated consistent growth and hydraulics across elevations. Though, electrical resistivity tomograms exhibited pronounced temperature-driven seasonal changes. ABSTRACT: Alpine conifers growing at high elevation are exposed to low temperatures, which may limit xylogenesis and cause pronounced seasonal changes in tree hydraulics. Electrical resistivity (ER) tomography enables minimal invasive monitoring of stems in situ. We used this technique to analyze Pinus cembra trunks along a 400 m elevational gradient up to the timberline and over seasons. Furthermore, ER data of earlywood across tree rings were compared with the respective specific hydraulic conductivity (K S), measured on extracted wood cores. ER tomograms revealed pronounced changes over the year and a strong correlation between average ER (ERmean) and air and xylem temperatures. Surprisingly, no elevational changes in ERmean, earlywood ER or K S were observed. ER data corresponded to variation in earlywood K S, which decreased from the youngest (ca. 4-5 cm2s-1 MPa-1) to the oldest tree rings (0.63 ± 0.22 cm2s-1 MPa-1). The lack of changes in ER data and earlywood K S along the study transect indicated consistent growth patterns and no major changes in structural and functional hydraulic traits across elevation. The constant decrease in earlywood K S with tree ring age throughout all elevations highlights the hydraulic relevance of the outermost tree rings in P. cembra. Seasonal measurements demonstrated pronounced temperature effects on ER, and we thus recommend a detailed monitoring of trunk temperatures for ER tomography.

Employing Nanostructured Scaffolds to Investigate the Mechanical Properties of Adult Mammalian Retinae Under Tension.

Numerous eye diseases are linked to biomechanical dysfunction of the retina. However, the underlying forces are almost impossible to quantify experimentally. Here, we show how biomechanical properties of adult neuronal tissues such as porcine retinae can be investigated under tension in a home-built tissue stretcher composed of nanostructured TiO2 scaffolds coupled to a self-designed force sensor. The employed TiO2 nanotube scaffolds allow for organotypic long-term preservation of adult tissues ex vivo and support strong tissue adhesion without the application of glues, a prerequisite for tissue investigations under tension. In combination with finite element calculations we found that the deformation behavior is highly dependent on the displacement rate which results in Young's moduli of (760-1270) Pa. Image analysis revealed that the elastic regime is characterized by a reversible shear deformation of retinal layers. For larger deformations, tissue destruction and sliding of retinal layers occurred with an equilibration between slip and stick at the interface of ruptured layers, resulting in a constant force during stretching. Since our study demonstrates how porcine eyes collected from slaughterhouses can be employed for ex vivo experiments, our study also offers new perspectives to investigate tissue biomechanics without excessive animal experiments.

Regeneration of TiO2 Nanotube Arrays after Long-Term Cell and Tissue Culture for Multiple Use - an Environmental Scanning Electron Microscopy (ESEM) Survey of Adult Pig Retina and beyond.

Long-term organotypic culture of adult tissues not only open up possibilities for studying complex structures of explants in vitro, but also can be employed e.g. to investigate pathological changes, their fingerprints on tissue mechanics, as well as the effectiveness of drugs. While conventional culture methods do not allow for survival times of more than a few days, we have demonstrated recently that TiO2 nanotube arrays allow to maintain integrity of numerous tissues, including retina, brain, spline and tonsils, for as long as 2 weeks in vitro. A mystery in culturing has been the interaction of tissue with these substrates, which is also reflected by tissue debris after liftoff. As the latter reveals fingerprints of tissue adhesion and impedes with nanotube array reuse, we address within the present environmental scanning electron study debris nature and the effectiveness of cleaning approaches of distinct physical and chemical methods, including UV-light irradiation, O2 plasma treatment and application of an enzyme-based buffer. This will lays the foundation for large-scale regeneration and reuse of nanotube arrays in science and clinical research.