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1.
Ana Mijovilovich Barbara Leitenmaier Wolfram Meyer-Klaucke Peter M.H. Kroneck Birgit G?tz Hendrik Küpper 《Plant physiology》2009,151(2):715-731
The cadmium/zinc hyperaccumulator Thlaspi caerulescens is sensitive toward copper (Cu) toxicity, which is a problem for phytoremediation of soils with mixed contamination. Cu levels in T. caerulescens grown with 10 μm Cu2+ remained in the nonaccumulator range (<50 ppm), and most individuals were as sensitive toward Cu as the related nonaccumulator Thlaspi fendleri. Obviously, hyperaccumulation and metal resistance are highly metal specific. Cu-induced inhibition of photosynthesis followed the “sun reaction” type of damage, with inhibition of the photosystem II reaction center charge separation and the water-splitting complex. A few individuals of T. caerulescens were more Cu resistant. Compared with Cu-sensitive individuals, they recovered faster from inhibition, at least partially by enhanced repair of chlorophyll-protein complexes but not by exclusion, since the content of Cu in their shoots was increased by about 25%. Extended x-ray absorption fine structure (EXAFS) measurements on frozen-hydrated leaf samples revealed that a large proportion of Cu in T. caerulescens is bound by sulfur ligands. This is in contrast to the known binding environment of cadmium and zinc in the same species, which is dominated by oxygen ligands. Clearly, hyperaccumulators detoxify hyperaccumulated metals differently compared with nonaccumulated metals. Furthermore, strong features in the Cu-EXAFS spectra ascribed to metal-metal contributions were found, in particular in the Cu-resistant specimens. Some of these features may be due to Cu binding to metallothioneins, but a larger proportion seems to result from biomineralization, most likely Cu(II) oxalate and Cu(II) oxides. Additional contributions in the EXAFS spectra indicate complexation of Cu(II) by the nonproteogenic amino acid nicotianamine, which has a very high affinity for Cu(II) as further characterized here.Many heavy metals are well known to be essential microelements for plants, but elevated concentrations of these metals cause toxicity (for review, see Prasad and Hagemeyer, 1999; Küpper and Kroneck, 2005), as explained in more detail in our companion article (Küpper et al., 2009) on copper (Cu) complexation and toxicity in Crassula helmsii. Plants developed a number of strategies to resist this toxicity, including active efflux, sequestration, and binding of heavy metals inside the cells by strong ligands. Among the zinc (Zn) and cadmium (Cd) hyperaccumulators (Brooks, 1998; Lombi et al., 2000), the best known species is Thlaspi caerulescens, which has been proposed as a hyperaccumulator model species by several authors (Assunção et al., 2003; Peer et al., 2003, 2006). An enhanced uptake of metals into the root symplasm was found in T. caerulescens compared with the related nonaccumulator Thlaspi arvense (Lasat et al., 1996, 1998), and a reduced sequestration into the root vacuoles was associated with the higher root-to-shoot translocation efficiency of T. caerulescens (Shen et al., 1997; Lasat et al., 1998). This is likely related to elevated expression of xylem-loading transporters in the roots (Papoyan and Kochian, 2004; Weber et al., 2004). One of these, the Cd/Zn-pumping P1b-type ATPase TcHMA4, was recently purified as a protein, which revealed posttranslational processing, and its biochemical characterization showed Cd and Zn transport affinity in the submicromolar range (Parameswaran et al., 2007). Studies of cellular metal compartmentation have shown that in most hyperaccumulators, the metal is sequestered preferentially into compartments where it does no harm to the metabolism. These are, in most cases studied so far, the epidermal vacuoles (Küpper et al., 1999, 2001; Frey et al., 2000; Bidwell et al., 2004; Broadhurst et al., 2004), where concentrations of several hundred mmol L−1 can be reached in the large metal storage cells (Küpper et al., 1999, 2001). The latter showed that hyperaccumulation must be mediated by active pumping of the heavy metals into their storage sites, which was shown to be achieved by an extremely increased expression of metal transport proteins in leaves of hyperaccumulators compared with nonaccumulators (Pence et al., 2000; Assunção et al., 2001; Becher et al., 2004; Papoyan and Kochian, 2004; Küpper et al., 2007b). Strong sulfur (S) ligands like phytochelatins were shown not to be relevant for Cd detoxification in the Cd hyperaccumulator T. caerulescens. Phytochelatin levels are lower in this plant than in the related nonaccumulator T. arvense (Ebbs et al., 2002), inhibition of phytochelatin synthase in hyperaccumulators does not affect their Cd resistance (Schat et al., 2002), and direct measurements of the Cd ligands by EXAFS (Küpper et al., 2004). Thus, the main detoxification strategy in hyperaccumulators is clearly not binding to strong ligands but sequestration of the hyperaccumulated heavy metals. However, the nonproteogenic amino acid nicotianamine (NA) seems to play an important role in metal homeostasis of plants. According to several studies, it binds iron, Zn, and Cu, mainly for long-distance transport in the vascular bundle (Stephan and Scholz, 1993; Pich et al., 1994; Schmidke and Stephan, 1995; Stephan et al., 1996, Pich and Scholz, 1996; von Wiren et al., 1999; Liao et al., 2000), and NA synthase has been shown to be highly overexpressed in hyperaccumulators compared with nonaccumulator plants (Becher et al., 2004; Weber et al., 2004; van de Mortel et al., 2006, 2008).
Open in a separate windowUnder metal-induced stress, the heavy metal accumulation pattern changes. Under such conditions, heavy metal (Cd, nickel) accumulation was enhanced in a few cells of the mesophyll (Küpper et al., 2000a, 2001). The same cells contained elevated levels of magnesium, which was interpreted as a defense against substitution of Mg2+ in chlorophyll (Chl; Küpper et al., 1996, 1998, 2002) by heavy metals. Recently, it was found that this heterogeneity of Cd accumulation is a transient phenomenon in T. caerulescens under Cd-toxicity stress, correlating with a heterogeneity in photosynthesis and disappearing when the plants acclimate to the stress (Küpper et al., 2007a). It was postulated that this transient heterogeneity constitutes an emergency defense against Cd toxicity by sacrificing a few mesophyll cells as additional storage sites until the metal sequestration in the epidermis is sufficiently up-regulated. Furthermore, this acclimation response showed that at least part of the Cd resistance of T. caerulescens is inducible (Küpper et al., 2007a).While Cd resistance of T. caerulescens is strongly enhanced compared with related nonaccumulator plants, T. caerulescens is known to be sensitive to Cu to a similar extent as nonaccumulator plants, limiting its application for phytoremediating soils with mixed contamination (Walker and Bernal, 2004; Benzarti et al., 2008).In contrast to other metals, the speciation (redox state and coordination) of Cu in plant tissues is poorly understood, despite the detailed structural and functional knowledge about numerous Cu-dependent enzymes and Cu chaperones (for review, see Pilon et al., 2006). This is most likely due to the difficulty of measuring Cu speciation. The concentrations of Cu in plant tissue are too small for NMR and were too small for EXAFS for a long time. Methods relying on fractionation or homogenization of fresh tissues (e.g. for chromatography) cause breaking of intracellular membranes. This brings weakly bound metal that was localized in the vacuole into contact with the various strong ligands of the cytoplasm, causing artifactual changes of speciation. To our knowledge, so far only one EXAFS study on Cu speciation in plant tissues used environmental Cu concentrations: Polette et al. (2000) studied Cu speciation in the Cu-resistant Cu indicator plant Larrea tridentata. In their EXAFS measurements on samples frozen in liquid nitrogen, they found evidence for a Cu(II)-phytochelatin complex involved in transport and an unknown Cu complex involved in Cu storage. A second study using the same sample preparation method (that due to the formation of a gas layer around the sample slows freezing, resulting in ice formation with the risk of membrane damage) investigated the Cu-tolerant Cu-excluder plant Elsholtzia splendens (Shi et al., 2008). Unfortunately, these authors applied very high, physiologically not relevant Cu levels (300 μm) in their nutrient solution in order to force enough Cu into the tissues to get an acceptable signal-noise level in their EXAFS data within the limited synchrotron beam time. In two further Cu-EXAFS studies on plant tissues (Gardea-Torresdey et al., 2001, on L. tridentata; Sahi et al., 2007, on Sesbania drummondii), strong artifacts were most likely introduced by drying of tissues. Drying disrupts membranes as discussed above and can remove aquo ligands from metal ions (Schünemann et al., 1999). Additionally, extremely high Cu concentrations in the nutrient solution were applied to the plants (1,000 and 10,000 μm by Gardea-Torresdey et al., 2001; 400–5,000 μm by Sahi et al., 2007). Thus, the biological relevance of these studies has to be questioned.In this study, we addressed two related questions, both in comparison with the Cu accumulator C. helmsii described by Küpper et al. (2009). How do environmentally relevant (10 μm) toxic levels of Cu affect T. caerulescens? And how does the plant try to defend itself by binding the Cu ions? This was done by investigating mechanisms of Cu-induced inhibition of photosynthesis and analyzing the complexation of Cu in leaves. The latter was done in order to find out whether T. caerulescens utilizes different mechanisms for detoxification of the nonaccumulated Cu in comparison with the hyperaccumulated Cd. The speciation of Cu was analyzed by EXAFS, which is an element-specific method and therefore particularly suited for analyzing the in vivo ligand environment of metals in plants. Furthermore, in contrast to most other methods, it is applicable to intact frozen-hydrated plant tissues, which were first used by Salt et al. (1995). Moreover, EXAFS beamlines can now reach much lower metal concentrations than heteronuclear NMR (Ascone et al., 2003). So far, metal NMR was applied only in the case of Cd in T. caerulescens (Ueno et al., 2005), but although the concentration of this hyperaccumulated metal in the tissue was about 100 times higher than that of Cu, the NMR signal obtained was so weak that only the most abundant ligand type (organic acids, confirming previous EXAFS work [Küpper et al. 2004]) could be detected; everything else was drowned in noise. Thus, EXAFS is the method of choice for identifying ligands of both Cu(I) and Cu(II) in intact tissues in the absence of artifacts introduced by sample preparation (see above). EXAFS model complexes were prepared as references, and among these, Cu(II)-NA was characterized in more detail by UV/VIS and electron paramagnetic resonance (EPR) spectroscopy in view of its importance for Cu(II) binding in T. caerulescens. Inhibition of photosynthesis was investigated, as in our C. helmsii study, by the two-dimensional (imaging) microscopic in vivo measurement of the transients of Chl variable fluorescence with a recently described instrument, the fluorescence kinetic microscope (FKM; Küpper et al., 2007a). This was complemented with data on Cu accumulation, changes in pigment composition, and plant growth. 相似文献
Table I.
Explanation of technical termsTechnical Term | Explanation/Definition |
---|---|
Antenna connectivity | The likelihood of energy transfer between antennae of different photosystems (PSII and/or PSI) |
DW | Debye-Waller factor of the EXAFS refinements, accounts for both structural and thermal disorder of the metal ion coordination shells (Binstead et al., 1992) |
EF | Fermi energy of the EXAFS refinements, defines the threshold for the EXAFS spectra (Rehr and Albers, 2000) |
EXAFS | Extended x-ray absorption fine structure |
FI | Fit index of the EXAFS refinements = sum of the square of the residuals (this is what is minimized in the refinement) |
F0 | Minimal fluorescence yield of a dark-adapted sample, fluorescence in nonactinic measuring light |
Fm | Maximum fluorescence yield of a dark-adapted sample after supersaturating irradiation pulse |
Fm′ | Maximum fluorescence yield of a light-adapted sample after supersaturating irradiation pulse |
Fp | Fluorescence yield at the P level of the induction curve after the onset of actinic light exposure |
Fv = Fm − F0 | Variable fluorescence |
Fv/Fm = (Fm − F0)/Fm | Maximal dark-adapted quantum yield of PSII photochemistry |
ΦPSII = Φe = (Fm′ − Ft′)/Fm′ | The light-acclimated efficiency of PSII (Genty et al., 1989). In this article, the use of this parameter is extended to the relaxation period after the end of actinic light to analyze the return of the system to its dark-acclimated state as measured by Fv/Fm. |
k | Wave number of the photoelectron, proportional to the square of the energy difference from the threshold energy (Efermi) |
k-range | Energy range above the x-ray absorption edge of the metal where EXAFS was analyzed. |
Light saturation | Measured by the increased amplitude of Fp relative to Fm after subtraction of F0. (Fp − F0)/(Fm − F0) is mostly dependent on the ratio of functional antenna molecules to functional reaction centers and electron transport chains. Under constant actinic irradiance for measuring Fp, a large antenna capturing photons and delivering them to its reaction center will cause more of the “electron traffic jam” that leads to Fp than a small antenna. |
Multiple scattering | Contributions in EXAFS that originate not from direct interaction between the central ion and another atom, but from interactions between the different atoms of a ligand molecule. This can only be observed in very rigid (parts of) ligand molecules, e.g. the imidazole ring of His. Further, the contribution is most significant for forward scattering at angles close to 180°, which is the case for the atoms in an imidazole ring. |
NPQ = (Fm − Fm′)/Fm | Nonphotochemical quenching, in this article used as an acronym for the name of this phenomenon. In this article, we measure nonphotochemical quenching as qCN = “complete nonphotochemical quenching of Chl fluorescence”, i.e. with normalization to Fm. |
Z | Atomic number |
2.
Sabine Drevet Bertrand Favier Emmanuel Brun Gaëtan Gavazzi Bernard Lardy 《Comparative medicine》2022,72(1):3
Osteoarthritis (OA) is a multidimensional health problem and a common chronic disease. It has a substantial impact on patient quality of life and is a common cause of pain and mobility issues in older adults. The functional limitations, lack of curative treatments, and cost to society all demonstrate the need for translational and clinical research. The use of OA models in mice is important for achieving a better understanding of the disease. Models with clinical relevance are needed to achieve 2 main goals: to assess the impact of the OA disease (pain and function) and to study the efficacy of potential treatments. However, few OA models include practical strategies for functional assessment of the mice. OA signs in mice incorporate complex interrelations between pain and dysfunction. The current review provides a comprehensive compilation of mouse models of OA and animal evaluations that include static and dynamic clinical assessment of the mice, merging evaluation of pain and function by using automatic and noninvasive techniques. These new techniques allow simultaneous recording of spontaneous activity from thousands of home cages and also monitor environment conditions. Technologies such as videography and computational approaches can also be used to improve pain assessment in rodents but these new tools must first be validated experimentally. An example of a new tool is the digital ventilated cage, which is an automated home-cage monitor that records spontaneous activity in the cages.Osteoarthritis (OA) is a multidimensional health problem and a common chronic disease.36 Functional limitations, the absence of curative treatments, and the considerable cost to society result in a substantial impact on quality of life.76 Historically, OA has been described as whole joint and whole peri-articular diseases and as a systemic comorbidity.9,111 OA consists of a disruption of articular joint cartilage homeostasis leading to a catabolic pathway characterized by chondrocyte degeneration and destruction of the extracellular matrix (ECM). Low-grade chronic systemic inflammation is also actively involved in the process.42,92 In clinical practice, mechanical pain, often accompanied by a functional decline, is the main reason for consultations. Recommendations to patients provide guidance for OA management.22, 33,49,86 Evidence-based consensus has led to a variety of pharmacologic and nonpharmacologic modalities that are intended to guide health care providers in managing symptomatic patients. Animal-based research is of tremendous importance for the study of early diagnosis and treatment, which are crucial to prevent the disease progression and provide better care to patients.The purpose of animal-based OA research is 2-fold: to assess the impact of the OA disease (pain and function) and to study the efficacy of a potential treatment.18,67 OA model species include large animals such as the horse, goat, sheep, and dog, whose size and anatomy are expected to better reflect human joint conditions. However, small animals such as guinea pig, rabbit, mouse, and rat represent 77% of the species used.1,87 In recent years, mice have become the most commonly used model for studying OA. Mice have several advantageous characteristics: a short development and life span, easy and low-cost breeding and maintenance, easy handling, small joints that allow histologic analysis of the whole joint,32 and the availability of genetically modified lines.108 Standardized housing, genetically defined strains and SPF animals reduce the genetic and interindividual acquired variability. Mice are considered the best vertebrate model in terms of monitoring and controlling environmental conditions.7,14,15,87 Mouse skeletal maturation is reached at 10 wk, which theoretically constitutes the minimal age at which mice should be entered into an OA study.64,87,102 However, many studies violate this limit by testing mice at 8 wk of age.Available models for OA include the following (32,111 physical activity and exercise induced OA; noninvasive mechanical loading (repetitive mild loading and single-impact injury); and surgically induced (meniscectomy models or anterior cruciate ligament transection). The specific model used would be based on the goal of the study.7 For example, OA pathophysiology, OA progression, and OA therapies studies could use spontaneous, genetic, surgical, or noninvasive models. In addition, pain studies could use chemical models. Lastly, post-traumatic studies would use surgical or noninvasive models; the most frequently used method is currently destabilization of the medial meniscus,32 which involves transection of the medial meniscotibial ligament, thereby destabilizing the joint and causing instability-driven OA. An important caveat for mouse models is that the mouse and human knee differ in terms of joint size, joint biomechanics, and histologic characteristics (layers, cellularity),32,64 and joint differences could confound clinical translation.10 Table 1. Mouse models of osteoarthritis.
Open in a separate windowSince all animal models have strengths and weaknesses, it is often best to plan using a number of models and techniques together to combine the results.In humans, the lack of correlation between OA imaging assessment and clinical signs highlights the need to consider the functional data and the quality of life to personalize OA management. Clinical outcomes are needed to achieve 2 main goals: to assess the impact of the OA in terms of pain and function and to study the efficacy of treatments.65 Recent reviews offer few practical approaches to mouse functional assessment and novel approaches to OA models in mice.7,32,67,75,79,83,87, 100,120 This review will focus on static and dynamic clinical assessment of OA using automatic and noninvasive emerging techniques (Test name Techniques Kind of assessment Output Specific equipment required Static measurement Von Frey filament testing Calibrated nylon filaments of various thickness (and applied force) are pressed against the skin of the plantar surface of the paw in ascending order of force Stimulus- evoked pain-like behavior
Mechanical stimuli - Tactile allodynia
The most commonly used test Latency to paw withdrawal
and
Force exerted are recorded Yes Knee extension test Apply a knee extension on both the intact and affected knee
or
Passive extension range of the operated knee joint under anesthesia Stimulus-evoked pain-like behavior Number of vocalizations evoked in 5 extensions None Hotplate Mouse placed on hotplate. A cutoff latency has been determined to avoid lesions Stimulus-evoked pain-like behavior
Heat stimuli- thermal sensitivity Latency of paw withdrawal Yes Righting ability Mouse placed on its back Neuromuscular screening Latency to regain its footing None Cotton swab test Bringing a cotton swab into contact with eyelashes, pinna, and whiskers Stimulus-evoked pain-like behavior
Neuromuscular screening Withdrawal or twitching response None Spontaneous activity Spontaneous cage activity One by one the cages must be laid out in a specific platform Spontaneous pain behavior
Nonstimulus evoked pain
Activity Vibrations evoked by animal movements Yes Open field analysis Experiment is performed in a clear chamber and mice can freely explore Spontaneous pain behavior
Nonstimulus evoked pain
Locomotor analysis Paw print assessment
Distance traveled, average walking speed, rest time, rearing Yes Gait analysis Mouse is placed in a specific cage equipped with a fluorescent tube and a glass plate allowing an automated quantitative gait analysis Nonstimulus evoked pain
Gait analysis
Indirect nociception Intensity of the paw contact area, velocity, stride frequency, length, symmetry, step width Yes Dynamic weight bearing system Mouse placed is a specific cage. This method is a computerized capacitance meter (similar to gait analysis) Nonstimulus evoked pain
Weight-bearing deficits
Indirect nociception Body weight redistribution to a portion of the paw surface Yes Voluntary wheel running Mouse placed is a specific cage with free access to stainless steel activity wheels. The wheel is connected to a computer that automatically record data Nonstimulus evoked pain
Activity Distance traveled in the wheel Yes Burrowing analysis Mouse placed is a specific cage equipped with steel tubes (32 cm in length and 10 cm in diameter) and quartz sand in Plexiglas cages (600 · 340x200 mm) Nonstimulus evoked pain
Activity Amount of sand burrowed Yes Digital video recordings Mouse placed is a specific cage according to the tool Nonstimulus evoked pain
Or
Evoked pain Scale of pain or specific outcome Yes Digital ventilated cage system Nondisrupting capacitive-based technique: records spontaneous activity 24/7, during both light and dark phases directly from the home cage rack Spontaneous pain behavior
Nonstimulus evoked pain
Activity-behavior Distance walked, average speed, occupation front, occupation rear, activation density.
Animal locomotion index, animal tracking distance, animal tracking speed, animal running wheel distance and speed or rotation Yes Challenged activity Rotarod test Gradual and continued acceleration of a rotating rod onto which mice are placed Motor coordination
Indirect nociception Rotarod latency: riding time and speed with a maximum cut off. Yes Hind limb and fore grip strength Mouse placed over a base plate in front of a connected grasping tool Muscle strength of limbs Peak force, time resistance Yes Wire hang analysis Suspension of the mouse on the wire and start the time Muscle strength of limbs: muscle function and coordination Latency to fall gripping None
(self -constructed)
Models | Pros | Cons | |
---|---|---|---|
Spontaneous | Wild type mice7,9,59,67,68,70,72,74,80,85,87,115,118,119,120 | - Model of aging phenotype - The less invasive model - Physiological relevance: mimics human pathogenesis - No need for technical expertise - No need for specific equipment | - Variability in incidence - Large number of animals at baseline - Long-term study: Time consuming (time of onset: 4 -15 mo) - Expensive (husbandry) |
Genetically modified mice2,7,25,40,50,52,67,72,79,80, 89,120 | - High incidence - Earlier time of onset: 18 wk - No need for specific equipment - Combination with other models | - Time consuming for the strain development - Expensive | |
Chemical- induced | Mono-iodoacetate injection7,11,46,47,60,66,90,91,101,128 | - Model of pain-like phenotype - To study mechanism of pain and antalgic drugs - Short-term study: Rapid progression (2-7 wk) - Reproducible - Low cost | - Need for technical expertise - Need for specific equipment - Systemic injection is lethal - Destructive effect: does not allow to study the early phase of pathogenesis |
Papain injection66,67,120 | - Short-term study: rapid progression - Low cost | - Need for technical expertise - Need for specific equipment - Does not mimic natural pathogenesis | |
Collagenase injection7,65,67,98 | - Short-term study: rapid progression (3 wk) - Low cost | - Need for technical expertise - Need for specific equipment - Does not mimic natural pathogenesis | |
Non-invasive | High-fat diet (Alimentary induced obesity model)5,8,43,45,57,96,124 | Model of metabolic phenotype No need for technical expertise No need for specific equipment Reproducible | Long-term study: Time consuming (8 wk–9 mo delay) Expensive |
Physical activity and exercise model45,73 | Model of post traumatic phenotype No need for technical expertise | Long-term study: time consuming (18 mo delay) Expensive Disparity of results | |
Mechanical loading models Repetitive mild loading models Single-impact injury model7,16,23,24, 32,35,104,105,106 | Model of post traumatic phenotype Allow to study OA development Time of onset: 8-10 wk post injury Noninvasive | Need for technical expertise Need for specific equipment Heterogeneity in protocol practices Repetitive anesthesia required or ethical issues | |
Surgical | Ovariectomy114 | Contested. | |
Meniscectomy model7,32,63,67,87 | Model of post traumatic phenotype High incidence Short-term study: early time of onset (4 wk from surgery) To study therapies | Need for technical expertise Need for specific equipment Surgical risks Rapid progression compared to human | |
Anterior cruciate ligament transection (ACLT)7,39,40,61,48,67,70,87,126 | Model of posttraumatic phenotype High incidence Short-term study: early time of onset (3-10 wk from surgery) Reproducible To study therapies | Need for technical expertise Need for specific equipment Surgical risks Rapid progression compared to human | |
Destabilization of medial meniscus (DMM)7,32,39,40 | Model of post traumatic phenotype High incidence Short-term study: early time of onset (4 wk from surgery) To study therapies The most frequently used method | Need for technical expertise Need for specific equipment Surgical risks Rapid progression compared to human |
Mechanical stimuli - Tactile allodynia
The most commonly used test
and
Force exerted are recorded
or
Passive extension range of the operated knee joint under anesthesia
Heat stimuli- thermal sensitivity
Neuromuscular screening
Nonstimulus evoked pain
Activity
Nonstimulus evoked pain
Locomotor analysis
Distance traveled, average walking speed, rest time, rearing
Gait analysis
Indirect nociception
Weight-bearing deficits
Indirect nociception
Activity
Activity
Or
Evoked pain
Nonstimulus evoked pain
Activity-behavior
Animal locomotion index, animal tracking distance, animal tracking speed, animal running wheel distance and speed or rotation
Indirect nociception
(self -constructed)