A method for constructing a model of post-amputation secondary pain

Abstract

The application discloses a method for constructing a secondary pain model after amputation, which comprises the following steps: anesthetizing a mouse, performing limb disconnection at the wrist radius junction of the forelimb of the mouse, suturing the amputated limb stump of the mouse, detecting the pain threshold of the ipsilateral hind limb and the contralateral forelimb of the mouse by a von Frey fiber, and constructing a secondary pain model after amputation. The method can better simulate the situation of upper limb amputation in the clinic. Through the method, the model mouse can produce stable and significant secondary pain in the non-injury parts such as the ipsilateral hind limb and the contralateral forelimb. Moreover, the whole process of the construction method is short, the mouse wakes up quickly after the operation, the mouse can be induced to have secondary pain within a few days after the operation, and the success rate is high.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and medicine, and specifically to a method for constructing a model of secondary pain after amputation. Background Technology

[0002] Amputation refers to the removal of the distal end of a limb. Clinically, amputation surgery typically involves the removal of a necrotic, damaged, severely diseased, painful, or life-threatening limb, or a completely disused limb that impairs function. Amputees often experience significant pain. Clinical studies show that, in addition to stump pain and phantom limb pain, more than half of upper or lower limb amputees also experience significant back pain and other secondary pain, such as pain in the non-amputated limb. Therefore, studying the neural mechanisms of secondary pain after amputation is of great significance for alleviating the suffering of amputee patients.

[0003] Currently, stump neuroma animal models are commonly used to simulate amputation, allowing for the detection of stump pain and phantom limb pain. For example, in a mouse model of sciatic nerve transection, animals may exhibit behaviors such as biting off nails, toes, and even the soles of the affected limb 1-3 weeks post-surgery. Autophagy scores quantify the degree of aggression towards the denervated limb, mimicking phantom limb pain symptoms in amputees to some extent. Tibial neuroma transplantation models can be used to simulate stump pain and phantom limb pain in amputated animals. However, it remains unclear whether these common stump neuroma models in animals such as mice can induce stable and easily assessable secondary pain after amputation. Simulating secondary pain in non-injured areas, such as back pain in amputees and pain in non-amputated limbs, remains a challenge in basic research. Summary of the Invention

[0004] To address the uncertainty in inducing post-amputation secondary pain in animals using the aforementioned stump neuroma model, the present invention aims to provide a mouse model capable of inducing stable and easily measurable post-amputation secondary pain, facilitating the study of the mechanism of post-amputation secondary pain.

[0005] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows:

[0006] A method for constructing a model of secondary pain after amputation is provided, which includes the following steps:

[0007] Mice were anesthetized, and the limbs were severed at the carpal-radial junction using scissors. The amputated limb stumps were then sutured together. The pain thresholds of the ipsilateral hindlimb and contralateral forelimb of the mouse stumps were detected using von Frey fibers to construct a model of secondary pain after amputation.

[0008] Furthermore, the specific steps include the following:

[0009] (1) Place the mouse’s head into the inhalation mask of the air anesthesia machine, turn on the air anesthesia machine knob, so that the mouse can continuously inhale isoflurane and be under full anesthesia during the operation.

[0010] (2) Inject 0.5% lidocaine into the left or right forelimb of the mouse for local analgesia at the site of limb amputation;

[0011] (3) Use scissors to sever the limb at the junction of the carpal and radius bones in the forelimb of the mouse;

[0012] (4) The mouse amputation stump was sutured with an absorbable suture needle with suture thread;

[0013] (5) Apply erythromycin eye ointment to the mouse stump for antibacterial purposes, remove its head from the air anesthesia machine mask, place it on a heating pad to maintain its body temperature, and return it to its original breeding environment after the mouse wakes up.

[0014] (6) Carbofen and enrofloxacin were injected into the mice within 48 hours after the operation to relieve their pain, and the mice were observed daily.

[0015] (7) After surgery, the pain threshold of the ipsilateral hindlimb and contralateral forelimb of the mouse residual limb was detected by von Frey fiber to construct a model of secondary pain after amputation.

[0016] The beneficial effects of this invention are as follows:

[0017] (1) This method can better simulate the situation of upper limb amputation in clinical practice.

[0018] (2) By using this method, the model mice were able to generate stable and significant secondary pain in non-injured areas such as the ipsilateral hind limb and the contralateral forelimb.

[0019] (3) The entire construction process is short, the mice recover quickly after surgery, and secondary pain can be induced in mice within a few days after surgery, with a high success rate.

[0020] (4) The construction method is simple to operate, highly secure, and highly repeatable. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of a mouse forelimb amputation surgery provided in an embodiment of the present invention;

[0022] Figure 2 This is a schematic diagram illustrating the detection of mechanical leg lifting in mice using the von Frey fiber test, as an embodiment of the present invention.

[0023] Figure 3 This is a schematic diagram of the pain threshold of the ipsilateral hind limb at different time points after forelimb amputation surgery in mice according to an embodiment of the present invention;

[0024] Figure 4This is a schematic diagram of the pain threshold of the contralateral forelimb at different time points after forelimb amputation surgery in mice according to an embodiment of the present invention. Detailed Implementation

[0025] The specific embodiments of the present invention are described below to enable those skilled in the art to understand the present invention. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various changes are obvious as long as they are within the spirit and scope of the present invention as defined and determined by the appended claims. All inventions utilizing the concept of the present invention are protected.

[0026] Example

[0027] (1) Place the mouse’s head into the inhalation mask of the air anesthesia machine, turn on the air anesthesia machine knob, so that the mouse can continuously inhale isoflurane and be under full anesthesia during the operation.

[0028] (2) Inject 0.5% lidocaine into the left forelimb (or right forelimb, in this example the left forelimb) of the mouse for local analgesia at the limb amputation site;

[0029] (3) Use scissors to sever the limb at the junction of the carpal and radius bones in the forelimb of the mouse;

[0030] (4) The mouse amputation stump was sutured with an absorbable suture needle with suture thread;

[0031] (5) Apply erythromycin eye ointment to the mouse stump for antibacterial purposes, remove its head from the air anesthesia machine mask, place it on a heating pad to maintain its body temperature, and return it to its original breeding environment after the mouse wakes up.

[0032] (6) Carbofen and enrofloxacin were injected into the mice within 48 hours after the operation to relieve their pain, and the mice were observed daily.

[0033] (7) The pain threshold of the ipsilateral hindlimb and contralateral forelimb of the mouse residual limb was detected by von Frey fiber after surgery.

[0034] Use such as Figure 1 The method shown involved amputation of the left forelimb in mice. The mechanical foot-lifting threshold of the ipsilateral hindlimb (left hindlimb) was measured using von Frey fibers at different time points: before surgery (BL) and at 1, 3, 7, 14, 21, 28, 35, 42, 49, and 56 days post-surgery. Statistical analysis showed a significant decrease in the threshold of the ipsilateral hindlimb in the amputation group compared to the control group (unamputated group) (***).

[0035] Use such as Figure 1The method shown involved amputation of the left forelimb of mice. The mechanical foot-lifting threshold of the contralateral forelimb (right forelimb) was measured using von Frey fibers at different time points: before surgery (BL) and at 1, 3, 7, 14, 21, 28, 35, 42, 49, and 56 days post-surgery. Statistical analysis revealed a significant decrease in the threshold of the contralateral forelimb in the surgical group compared to the control group (unsurgery group) (***).

[0036] Combination Figure 3 and Figure 4 It can be seen that amputation of the left forelimb in mice can induce significant secondary pain in non-injured areas such as the ipsilateral hindlimb and the contralateral forelimb, and this pain can persist for 56 days post-surgery. This demonstrates the effectiveness and efficiency of the model.

[0037] In summary, this invention provides a mouse model that can induce stable and easily measurable post-amputation secondary pain, facilitating the study of the mechanism of post-amputation secondary pain.

[0038] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

[0039] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for constructing an animal model of secondary pain after amputation, characterized in that, Includes the following steps: Experimental mice were obtained by limb amputation at the carpal-radial junction of the forelimb; On days 1, 3, 7, 14, 21, 28, 35, 42, 49, and 56 after the limb amputation procedure, the mechanical pain threshold of the ipsilateral hindlimb and contralateral forelimb of the experimental mice was detected using von Frey fibers. The pain thresholds obtained at each time point were compared with the corresponding pain thresholds of normal control mice at the same time point, and the differences were calculated. If the pain threshold detected at the above multiple time points is consistently different from that of the normal control group, then the model is considered to have formed stable post-amputation secondary pain.

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