Construction and application of empathy analgesia animal model

Abstract

The present invention discloses a method for constructing an empathy analgesia animal model and its application. The present invention is based on the fact that social interaction between pain mice and analgesia mice can induce analgesia in pain mice, thereby successfully constructing an empathy analgesia animal model. This model has translational value in clinical research on pain treatment. As a simple, safe and easy-to-use intervention measure outside the hospital, empathy analgesia has broad application prospects.

Description

Technical Field

[0001] The present invention relates to the field of empathy analgesia, and in particular to a method for constructing an empathy analgesia animal model and its application. Background Art

[0002] Empathy is the ability to share and understand the experiences of other individuals, and it is essential to human social and emotional life. Recent studies have shown that empathy even exists in rodents. For example, in a pair of mice housed together, injecting a pain-inducing drug into one of them (the demonstrator mouse) and then allowing it to socialize with another mouse that did not experience pain (the observer mouse) for one hour can induce a significant decrease in the mechanical pain threshold of the observer mouse that did not experience pain.

[0003] There are reports that oxytocin has effects on pain empathy and drug addiction, such as "Study on the neural mechanism of oxytocin-mediated pain empathy response in rats" and WO2011146726A1. However, the effect of oxytocin on empathy is different, or even completely opposite. There is currently no research on oxytocin and empathic analgesia (relieving pain through social interaction). Empathic analgesia has a high clinical translation value. If it can be used for pain treatment, it will have broad application prospects as a simple, safe, and easy-to-use intervention outside the hospital.

[0004] The information in the background technology is only for illustrating the general background of the present invention and should not be regarded as admitting or suggesting in any form that such information constitutes the prior art known to a person skilled in the art. Summary of the invention

[0005] In order to solve at least some of the technical problems in the prior art, the present invention has conducted in-depth research on empathic analgesia, and found that analgesic behavior exists between familiar mice, but not between unfamiliar mice, and oxytocin treatment can greatly increase the duration of empathic analgesic behavior. The present invention is completed based on at least this discovery. Specifically, the present invention includes the following contents.

[0006] A first aspect of the present invention provides a method for constructing an empathy analgesia animal model, comprising the following steps:

[0007] (1) providing a first group of animals that are familiar with each other and a second group of animals that are unfamiliar with each other, wherein the animals in the first group of animals and the second group of animals are all animals in pain;

[0008] (2) respectively subjecting some of the first group of animals and some of the second group of animals to analgesia, and administering oxytocin to some of the animals via nasal spray, and then allowing the analgesic-treated animals and the oxytocin-treated animals to contact and socialize; and

[0009] (3) Detection of pain parameters in oxytocin-treated animals.

[0010] In certain embodiments, the method for constructing an empathy analgesia animal model according to the present invention further comprises the step of treating some animals in the first group of animals and some animals in the second group of animals with physiological saline respectively.

[0011] In certain embodiments, the method for constructing an empathic analgesia animal model according to the present invention further comprises the step of comparing pain parameters of oxytocin-treated animals in the first group of animals with those of oxytocin-treated animals in the second group of animals.

[0012] In certain embodiments, according to the method for constructing an empathy analgesia animal model of the present invention, the pain parameter is selected from at least one of mechanical pain threshold, analgesia duration, electroencephalogram parameter and NRS score.

[0013] In certain embodiments, according to the method for constructing an empathy analgesia animal model of the present invention, the analgesic treatment in step (2) includes the step of administering analgesic drugs to the animal.

[0014] In certain embodiments, according to the method for constructing an empathy analgesia animal model of the present invention, the mutual familiarity in step (1) refers to multiple animals that are not administered with pain-inducing drugs being housed in the same cage for at least 7 days.

[0015] In certain embodiments, according to the method for constructing an empathy analgesia animal model of the present invention, wherein the step (2) of allowing the analgesic-treated animal and the oxytocin-treated animal to contact and socialize refers to keeping the analgesic-treated animal and the oxytocin-treated animal in the same cage for more than 1 hour.

[0016] In certain embodiments, according to the method for constructing an empathy analgesia animal model of the present invention, the oxytocin treatment dose in step (2) is 0.2-1.5 mg / kg body weight.

[0017] The second aspect of the present invention provides a method for studying empathy analgesia, which includes the steps of using the method described in the first aspect as a research step, or using an empathy analgesia animal model obtained by the method described in the first aspect of the present invention.

[0018] The third aspect of the present invention provides an application of an empathy analgesia animal model in drug research or clinical trials, wherein the empathy analgesia animal model is obtained by the method described in the first aspect.

[0019] The present invention successfully constructed an empathy analgesia animal model based on the fact that social interaction between pain mice and analgesic mice can induce analgesia in pain mice. This model has important translational value in clinical research on pain treatment. As a simple, safe and easy-to-use intervention measure outside the hospital, empathy analgesia has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Results of mechanical pain threshold measurement in the unfamiliar mouse group. Figure 1 It can be seen that compared with the blank control group that only underwent pain treatment and social interaction without any analgesic treatment, no significant changes in the mechanical pain threshold were observed after the pain mice socialized with the unfamiliar analgesic mice, regardless of whether they were sprayed with nasal saline or oxytocin.

[0021] Figure 2 The results of mechanical pain threshold measurement in the familiar mice group. Figure 2 It can be seen that compared with the blank control group that only underwent pain treatment and social interaction without any analgesic treatment, the pain threshold of mice that socialized with familiar mice and inhaled normal saline increased significantly within about 4 hours after social interaction, and an analgesic effect of about 4 hours was observed; and the pain threshold of mice that interacted with familiar mice and inhaled oxytocin increased significantly within about 48 hours after social interaction, and an analgesic effect of about 48 hours was observed. DETAILED DESCRIPTION

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that the upper and lower limits of the scope and each intermediate value therebetween are specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0024] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which the invention belongs. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0025] [Construction method]

[0026] A first aspect of the present invention provides a method for constructing an empathy analgesia animal model, which at least comprises the following steps:

[0027] (1) providing a first group of animals that are familiar with each other and a second group of animals that are unfamiliar with each other, wherein the animals in the first group of animals and the second group of animals are all animals in pain;

[0028] (2) respectively subjecting some of the first group of animals and some of the second group of animals to analgesia, and administering oxytocin to some of the animals via nasal spray, and then allowing the analgesic-treated animals and the oxytocin-treated animals to contact and socialize; and

[0029] (3) Detection of pain parameters in oxytocin-treated animals.

[0030] Optionally, the construction method of the present invention further comprises the step of (4) treating some animals in the first group of animals and some animals in the second group of animals with physiological saline, and (5) comparing pain parameters between individuals in different groups, in particular, comparing pain parameters between oxytocin-treated animals in the first group of animals and oxytocin-treated animals in the second group of animals. Each step is described in detail below.

[0031] Step (1):

[0032] Step (1) of the present invention is to provide a first group of animals that are familiar with each other and a second group of animals that are unfamiliar with each other. Generally, the first group of animals is an experimental animal group, and the second group of animals is a control experimental animal group. The animals in the first group of animals and the second group of animals are preferably pain animals. For the first group of animals, pain can be caused to the animals before, when or after they are familiar with each other.

[0033] In the present invention, the pain animal can be prepared by any known method, and its exemplary methods include chemical stimulation method, physical stimulation method or neurogenic injury method, etc. Among them, chemical stimulation method includes inflammatory stimulation pain and drug stimulation pain. Physical stimulation method includes thermal stimulation method, cold stimulation method-cold plate method, electrical stimulation method, etc.

[0034] In certain embodiments, the preparation of the painful animals of the present invention is the inflammatory agent writhing method, which includes injecting 0.6% glacial acetic acid 0.2 ml / mouse or 0.05% potassium antimony tartrate 0.2 ml / mouse into the abdominal cavity of the mouse to induce pain. The number of writhing reactions or the number of animals with writhing reactions is observed for 10-20 minutes, and the analgesic percentage is calculated.

[0035] In certain embodiments, the preparation of the painful animals of the present invention is the bee venom-induced colitis pain method, in which bee venom is injected under visual conditions into the submucosal sigmoid colon of immediately anesthetized rats, and the tension pain behaviors of awake rats due to visceral pain, such as licking and biting the lower abdomen, abdominal contraction, body stretching and whole body contraction, are observed within a certain time range.

[0036] In certain embodiments, the preparation of the pain animal of the present invention is a method of local joint swelling and pain caused by an inflammatory agent, which includes selecting an appropriate inflammatory agent and injecting it subcutaneously at the hind foot or ankle of a rat or mouse. For example, short-acting inflammatory agents such as egg white, 5-HT, histamine, bradykinin, dextran, etc., which reach a peak swelling 0.5-1h after injection, or medium-acting inflammatory agents such as formalin, cross-gum (0.1ml, 0.5%), agar, white clay, yeast, etc., which reach a peak swelling 2-4h after injection; formaldehyde (0.2mg / 50μl), nitrogen mustard, and crystalline sodium urate reach a peak only 24h after injection, lasting about two weeks, and subsiding late, which are long-acting inflammatory agents. After injection, the joint or plantar swelling is caused, and the plantar volume or joint circumference is measured.

[0037] In certain embodiments, the preparation of painful animals is a potassium ion subcutaneous permeation or injection-induced inflammatory pain model, and the animals commonly used are rabbits and rats. Saturated KCl solution is permeated or directly injected into different subcutaneous parts of the animals with direct current of a certain intensity to cause pain.

[0038] In certain embodiments, the preparation of the pain animal of the present invention is a capsaicin intravenous injection-induced migraine model, which includes the method of intravenous stimulation and electrical stimulation with capsaicin to cause plasma protein infiltration in the rat dura mater, thereby establishing a neurogenic inflammation animal migraine model.

[0039] In certain embodiments, the preparation of the pain animal of the present invention is the adjuvant arthritis method, the basic method of which is to inject complete Freund's adjuvant into the rat's tail root skin or the plantar part of the foot. After the injection, the primary disease is an inflammatory reaction of the inflammation local area. Redness, swelling and pain appear in the inflammation local area 8 hours after the inflammation, and the peak can be reached in 18 hours, which lasts for about 6 days, and then gradually subsides, lasting for several weeks. Secondary lesions generally appear about 10 days after the inflammation, showing systemic multiple joint damage. This model is very similar to human rheumatoid arthritis, and has high modeling specificity and low false positive rate, so this type of pain animal model is preferred.

[0040] In the present invention, familiarity preferably refers to multiple animals contacting each other and living together for a period of time to establish a familiar relationship. Preferably, the animals contacting each other are non-pain animals, for example, animals that have not been administered with pain-inducing drugs. Exemplarily, familiarity refers to multiple animals that have not been administered with pain-inducing drugs being housed in the same cage for at least 7 days, for example, 8 days, 9 days, 10 days, 12 days, 14 days, 16 days, 18 days or more than 20 days.

[0041] In the present invention, being strangers to each other preferably means that the multiple animals have never come into contact or interacted with each other. Being strangers to each other can be achieved by raising the multiple animals in different environments, such as cages.

[0042] In the present invention, the first group of animals and the second group of animals are only for the purpose of distinguishing different groups, and do not refer to animals of different species. Usually, the number of animals in the first group of animals and the second group of animals is equal, but not necessarily equal. In some special cases, the number of animals in the first group of animals and the number of animals in the second group are not equal. Exemplary number of animals in the first group of animals or the second group of animals is 2 or more, such as 4 or more, 6 or more, 8 or more, 10 or more, etc.

[0043] Step (2):

[0044] Step (2) of the present invention is to perform analgesia treatment on some animals in the first group and some animals in the second group, respectively, and at the same time administer oxytocin to some animals through nasal spray, and then allow the analgesia-treated animals and the oxytocin-treated animals to contact and socialize.

[0045] In the present invention, analgesia and oxytocin treatment are respectively for different animal individuals. That is, in each group of animals, if an individual is treated with analgesia, then oxytocin treatment cannot be further performed. Similarly, if an individual is treated with oxytocin, then analgesia cannot be further performed.

[0046] In the present invention, before analgesia and oxytocin treatment, the painful individuals in the first group of animals can be contacted to enhance familiarity, or analgesia and oxytocin treatment can be directly performed without contact after the individuals are in pain, and then the animals with different treatments can be contacted and interacted.

[0047] In the present invention, analgesic treatment includes the use of drugs, and exemplary drugs include narcotic analgesics and antipyretic analgesics and anti-inflammatory drugs. Examples of narcotic analgesics include but are not limited to morphine, and antipyretic analgesics and anti-inflammatory drugs include but are not limited to aspirin and the like.

[0048] In the present invention, oxytocin treatment includes administering oxytocin drugs to individuals to enhance empathic behavior. The gender of the animal treated with oxytocin is not limited, and it can be a female animal or a male animal. The dosage of oxytocin is generally 0.2-1.5 mg / kg body weight, such as 0.3 mg / kg body weight, 0.4 mg / kg body weight, 0.5 mg / kg body weight, 0.6 mg / kg body weight, 0.7 mg / kg body weight, 0.8 mg / kg body weight, 0.9 mg / kg body weight, 1.0 mg / kg body weight, 1.1 mg / kg body weight, 1.2 mg / kg body weight, 1.3 mg / kg body weight, 1.4 mg / kg body weight, etc.

[0049] In the present invention, further contact and interaction is required between different individuals after analgesia treatment and oxytocin treatment. Exemplarily, analgesia-treated animals and oxytocin-treated animals are kept in the same cage for more than 1 hour, such as 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, etc.

[0050] Step (3):

[0051] Step (3) of the present invention is to detect pain parameters of oxytocin-treated animals. In the present invention, pain parameters refer to parameters related to the degree of pain. For example, pain parameters include but are not limited to mechanical pain threshold, analgesia duration, electroencephalogram parameters and NRS scores, etc. The present invention can use a combination of one or more of the above parameters. In the present invention, the detection of pain parameters can be carried out using known methods, which are not particularly limited.

[0052] In an optional embodiment, step (3) of the present invention further comprises the step of detecting pain parameters of other groups of individuals. The other groups include analgesic-treated individuals and / or saline-treated individuals in the first group; or analgesic-treated individuals and / or saline-treated individuals in the second group.

[0053] Step (4):

[0054] In the present invention, step (4) is an optional step, which includes the step of treating some animals in the first group of animals and some animals in the second group of animals with saline. In the present invention, animals that are nasally sprayed with saline and subjected to pain treatment are usually used as controls, and they are different individuals from the analgesic individuals or oxytocin-treated individuals. In the case of including step (4), the first group and the second group of animals respectively contain three types of differently treated individuals, namely analgesic individuals, oxytocin-treated individuals and saline-treated individuals.

[0055] Step (5):

[0056] Step (5) of the present invention is a step of comparing pain parameters of individuals in different groups, in particular, a step of comparing pain parameters of oxytocin-treated animals in the first group of animals with those of oxytocin-treated animals in the second group of animals.

[0057] Step (5) of the present invention preferably further comprises a step of contacting different individuals in groups. Exemplarily, the grouping is two-by-two groups, that is, two individuals form a group, and the individuals in a group are contacted and interacted, such as being caged for a specific period of time.

[0058] In certain embodiments, the groupings of the present invention are as follows:

[0059] Group I: saline-treated individuals + saline-treated individuals;

[0060] Group II: analgesic individuals + saline-treated individuals;

[0061] Group III: analgesic individuals + oxytocin-treated individuals;

[0062] Among them, the two saline-treated individuals in Group I can be the same.

[0063] In the above case, the comparison in step (5) of the present invention comprises a comparison of pain parameters between the saline-treated individuals in group I, the saline-treated individuals in group II, and the oxytocin-treated individuals in group III.

[0064] [Research Methods of Empathic Analgesia]

[0065] The second aspect of the present invention provides a method for studying empathy analgesia, which includes the step of constructing an empathy analgesia animal model using the method described in the first aspect, or the step of using an empathy analgesia animal obtained using the method described in the first aspect.

[0066] The research method of the present invention includes animal empathy behavior research, especially analgesia behavior research, or further as a simulation study of human empathy behavior.

[0067] [application]

[0068] The third aspect of the present invention provides the use of an empathy analgesia animal model in drug research or clinical trials. As long as the method described in the first aspect of the present invention is used or the animal model is obtained by the method, the specific application is not particularly limited. In the future, it may be used in the clinical treatment of pain diseases to reduce the use of analgesics, thereby reducing drug side effects and alleviating the burden on patients.

[0069] Example

[0070] 1. Implementation Methods

[0071] 1. Animals

[0072] C57BL / 6N male mice were purchased from the Department of Laboratory Animal Science, Peking University Health Science Center for use in the experiments. All mice were housed in a pathogen-free environment with a 12-hour artificial alternating light / dark cycle and unlimited access to food and water. All behavioral tests were performed during the dark cycle of mice.

[0073] 2. Empathy Task

[0074] Social interactions were performed by pairs of mice living in the same cage (cage mates) or by unfamiliar mice (strangers). One mouse in the pair was randomly selected to act as the demonstrator, while the other served as the spectator.

[0075] Half an hour before the social interaction, bystanders received 12 μl of oxytocin (1 μg / μl) or inhaled an equal amount of saline. The solution was applied bilaterally to the nasal cavity of the mouse using a pipette and inhaled via the mouse's respiratory tract, i.e. the hairless skin area around the nostrils.

[0076] 3. Construction of analgesia model

[0077] On the test day, mice were lightly restrained and 20 μl of complete Freund's adjuvant was injected subcutaneously into the plantar surface of the left hind paw.

[0078] Immediately thereafter, 10 mg / kg of morphine (demonstrators receiving analgesics) or saline (bystanders not receiving analgesics) was subcutaneously injected. Pairs of mice were then placed in clean cages for 1 hour for social interaction. The bystanders were then tested for mechanical pain thresholds. After the initial pain threshold test, the mice were separated and kept in single cages for 3 days for subsequent pain threshold determination. In addition, 12 pairs of mice housed in the same cage that were only injected with complete Freund's adjuvant in the left hind foot and were socially interacted without any analgesic treatment were set as blank controls.

[0079] 4. Mechanical pain threshold measurement

[0080] The up-down method was used to detect mechanical pain in mice. Specifically, von Frey fibers (0.008 to 6 g plastic fibers) were used to mechanically stimulate the plantar surface of the left hind paw, and the mouse's response (withdrawal, shaking or licking the paw) was observed to determine the mouse's response threshold to mechanical pain stimulation.

[0081] This method repeatedly tests 6 times near the pain response threshold to determine the median 50% threshold of the response. Two days before the start of the experiment, the mice were acclimated to the pain test venue for 30 minutes in advance, and were placed in the test venue 30 minutes before each pain test to allow them to obtain a quiet state to provide an accurate pain threshold.

[0082] 2. Experimental Results

[0083] The mechanical pain threshold of the blank control group before pain modeling was 0.72±0.14g; within two days after pain modeling without any analgesia and socialization, the mechanical pain threshold was stable at 0.11±0.12g. The continued sensitivity to mechanical stimulation indicated that the pain modeling was successful and the effect was definite.

[0084] The pain threshold of mice that socialized with unfamiliar analgesic mice before modeling was 0.73±0.06g, similar to that of the blank control group; within two days after pain modeling and intranasal oxytocin spraying and socializing with analgesic mice, their pain threshold was stable at 0.13±0.12g, similar to that of the blank control group, and no significant analgesic effect was found ( P=0.99). Within two days after pain modeling, nasal saline spraying and socializing with analgesic mice, the pain threshold was also stable at 0.14±0.14g, similar to the blank control group, and no significant analgesic effect was found ( P =0.98). In summary, social interaction between stranger mice could not produce effective analgesia regardless of whether oxytocin was sprayed intranasally.

[0085] The pain threshold of mice that socialized with the familiar analgesic mice before modeling was 0.7±0.1g, which was similar to that of the blank control group. The pain threshold of mice that socialized with the analgesic mice after pain modeling and nasal saline spraying was about 0.34±0.05g immediately after socializing, showing a significant and strong analgesic effect ( P <0.01); its pain threshold was about 0.52±0.05g 4 hours after social interaction, showing a significant and strong analgesic effect ( P <0.05); 24 hours after social interaction, it was about 0.29±0.1g, and the analgesic effect was weak and not significant ( P =0.15), while at 48 hours of social interaction, it stabilized at 0.18±0.12g, with almost no difference from the blank control group ( P =0.87). After pain modeling and intranasal oxytocin injection in mice, the pain threshold of mice was about 0.34±0.07g immediately after socialization, showing a significant and strong analgesic effect ( P <0.01); its pain threshold was about 0.38±0.1g 4 hours after social interaction, showing a significant and strong analgesic effect ( P <0.01); its pain threshold was about 0.42±0.1g 24 hours after social interaction, showing a significant and strong analgesic effect ( P <0.01); its pain threshold was about 0.37±0.07g after 48 hours of social interaction, showing a significant and strong analgesic effect ( P <0.01).

[0086] In summary, nasal spraying of normal saline and socializing with familiar mice can induce significant analgesic effects immediately and 4 hours after socializing, while nasal spraying of oxytocin and socializing with familiar mice can induce significant analgesic effects immediately, 4 hours, 24 hours and 48 hours after socializing. Compared with normal saline, nasal spraying of oxytocin can extend the analgesic time after socializing from 4 hours to 48 hours.

[0087] Although the present invention has been described with reference to exemplary embodiments, it should be understood that the present invention is not limited to the disclosed exemplary embodiments. Various adjustments or changes may be made to the exemplary embodiments of the present specification without departing from the scope or spirit of the present invention. The scope of the claims should be based on the broadest interpretation to cover all modifications and equivalent structures and functions.

Claims

1. A method for constructing an empathy analgesia animal model, characterized in that: The animal is a mouse, and the construction method comprises the following steps: (1) providing a first group of animals that are familiar with each other and a second group of animals that are unfamiliar with each other, wherein the animals in the first group of animals and the second group of animals are all pain animals, the familiarity means that the multiple animals are housed in the same cage without the administration of pain-inducing drugs for at least 7 days, and the unfamiliarity means that the multiple animals have never come into contact or interacted with each other; (2) performing analgesic treatment on some of the animals in the first group and some of the animals in the second group, and administering oxytocin to some of the animals through nasal spray, and then bringing the analgesic-treated animals into contact with and socializing with the oxytocin-treated animals, wherein the analgesic treatment includes the step of administering analgesics to the animals, bringing the analgesic-treated animals into contact with and socializing with the oxytocin-treated animals means that the analgesic-treated animals and the oxytocin-treated animals are housed in the same cage for more than 1 hour, and the analgesic treatment and the oxytocin treatment are respectively for different individual animals, and if an individual is subjected to analgesic treatment, the oxytocin treatment cannot be further performed; and (3) Detection of pain parameters in oxytocin-treated animals.

2. The method for constructing an empathy analgesia animal model according to claim 1, characterized in that: The method further comprises the step of treating some animals in the first group of animals and some animals in the second group of animals with physiological saline respectively.

3. The method for constructing an empathy analgesia animal model according to claim 1, characterized in that: Further comprising the step of comparing pain parameters of the oxytocin-treated animals in the first group of animals with the oxytocin-treated animals in the second group of animals.

4. The method for constructing an empathy analgesia animal model according to claim 1, characterized in that: The pain parameter is selected from at least one of mechanical pain threshold, analgesia duration, electroencephalogram parameter and NRS score.

5. The method for constructing an empathy analgesia animal model according to claim 1, characterized in that: The oxytocin treatment dose in step (2) is 0.2-1.5 mg / kg body weight.

6. A method for studying empathy analgesia, characterized in that: The method comprises the steps of using the method according to any one of claims 1 to 5 as a research step, or using the empathy analgesia animal model obtained by the method according to any one of claims 1 to 5.

7. Application of empathic analgesia animal model in drug research or clinical trials, characterized in that: The empathy analgesia animal model is obtained according to the method according to any one of claims 1-5.

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