A method for preparing a whole-body nerve specimen from a domestic rabbit

Abstract

This invention discloses a method for preparing a whole-body rabbit nerve specimen, comprising the following steps: S1, skinning and preservation: Skinning a rabbit carcass, separating the visceral nerves, removing the viscera, and then performing preservation treatment to obtain sample 1; S2, softening: Immersing sample 1 sequentially in a low-temperature softening enzyme EB solution, a mixed solution of hydrochloric acid and glycerol, and an alkaline solution to obtain sample 2; S3, nerve separation: Taking sample 2, removing muscle, fat, and bone, and separating the whole-body rabbit nerves; S4, defatting: Immersing the whole-body rabbit nerves sequentially in a sodium hydroxide solution and petroleum ether for defatting treatment to obtain sample 3; S5, bleaching and fixation: Bleaching sample 3 in hydrogen peroxide, then washing and drying to obtain sample 4; Fixing sample 4 onto a substrate in the upright posture of the rabbit to obtain the whole-body rabbit nerve specimen. This invention is simple to operate, and the resulting whole-body rabbit nerve specimen is easy to preserve and has a complete and aesthetically pleasing structure.

Description

Technical Field

[0001] This invention relates to the field of specimen preparation technology, and in particular to a method for preparing a whole-body nerve specimen of a rabbit. Background Technology

[0002] Animal specimens are created by processing whole or parts of an animal using physical or chemical methods. Animal specimens play a vital role in teaching and research, primarily serving as a visual teaching tool, providing practical teaching materials, and enhancing students' hands-on skills through specimen preparation. In scientific research, animal specimens provide physical evidence for the publication of new species and offer genetic information for studies on systematics, biodiversity, and the conservation of endangered species.

[0003] The nervous system is intricate and complex. Nerve specimens can visualize the abstract and difficult-to-understand nervous system in actual teaching, which can improve teaching effectiveness. However, the current methods of making nerve specimens often use instruments and tools to forcibly remove the muscles and bones (especially the skull) that cover the nerves. This will cause the nerve ends to break, making it impossible to make complete nerve specimens. The nerve specimens currently used are often only a part of the nerves, and there are few whole-body nerve specimens. It is difficult to see the whole picture of the cranial nerves and spinal nerves. Summary of the Invention

[0004] Based on the technical problems existing in the background art, the present invention proposes a method for preparing a whole-body nerve specimen of a rabbit. The present invention is simple to operate, low in cost, and the prepared whole-body nerve specimen of a rabbit is easy to preserve, with complete and beautiful structure, and clear and natural morphology and position.

[0005] This invention proposes a method for preparing a whole-body nerve specimen from a rabbit, comprising the following steps:

[0006] S1. Skinning and preservation: The rabbit carcass was skinned, the internal organs and nerves were separated and the internal organs were removed. Then, preservation treatment was performed to obtain sample 1.

[0007] S2, softening: Sample 1 was soaked in a low-temperature softening enzyme EB solution, a mixed solution of hydrochloric acid and glycerol, and an alkaline solution in sequence to obtain sample 2;

[0008] S3. Nerve separation: Take sample 2, remove muscle, fat and bone, and separate the whole nerve of the rabbit.

[0009] S4. Degreasing: The whole nerves of the rabbit were sequentially immersed in sodium hydroxide solution and petroleum ether for degreasing treatment to obtain sample 3;

[0010] S5. Bleaching and Fixation: Sample 3 was bleached by soaking in hydrogen peroxide, then washed and dried to obtain Sample 4; Sample 4 was fixed on a substrate in the upright posture of the rabbit to obtain a whole-body nerve specimen of the rabbit.

[0011] Preferably, in S2, the mass fraction of the low-temperature softening enzyme EB solution is 0.4-0.6 wt%, and the solution is ultrasonically soaked at 30-35°C for 2-3 hours.

[0012] Preferably, in S2, the mixed solution of hydrochloric acid and glycerol has a hydrochloric acid mass fraction of 13-18 wt% and a glycerol mass fraction of 13-17 wt%, and is soaked for 2-3 days.

[0013] Preferably, in S2, the alkaline solution is an aqueous solution of sodium carbonate or an aqueous solution of sodium hydroxide.

[0014] Preferably, in S1, the skin of the rabbit's ear is preserved.

[0015] Preferably, in S1, the sample is soaked in formalin solution for 0.8-1.2 months for preservation treatment.

[0016] Preferably, in S4, the mass fraction of the sodium hydroxide solution is 2.5-3.5 wt%.

[0017] Preferably, in S4, the sample is soaked in a sodium hydroxide solution for 1.5-2.5 days.

[0018] Preferably, in S4, the sample is soaked in petroleum ether for 6-8 days.

[0019] Preferably, in S5, the mass fraction of hydrogen peroxide in the hydrogen peroxide solution is 2.5-3.5 wt%.

[0020] Preferably, in S5, the sample is soaked in hydrogen peroxide for 2-3 days.

[0021] The aforementioned low-temperature softening enzyme EB is available commercially.

[0022] Beneficial effects:

[0023] This invention first uses a low-temperature softening enzyme EB to soak the sample, which can break down muscles and fat, making the muscles and fat in sample 1 loose and easier to separate from the bone. Additionally, the low-temperature softening enzyme EB can also soften the bone appropriately. Furthermore, by selecting an appropriate concentration and ultrasonic soaking time, damage to small nerves can be avoided. Then, a suitable concentration of hydrochloric acid and glycerol is used in combination. Hydrochloric acid can easily penetrate through the muscles and fat to soften the bone, while the addition of glycerol prevents the hydrochloric acid from corroding the nerves, thus protecting their integrity. An alkaline solution can neutralize the hydrochloric acid. After softening treatment by this invention, the nerves can be easily separated from the rabbit's bones, muscles, fat, fascia, etc., avoiding the problem of nerve breakage during dissection.

[0024] The rabbit's entire nervous system was gradually degreased using sodium hydroxide solution and petroleum ether in sequence. This process ensured thorough degreasing and prevented the slow seepage of oil from the nerves during long-term use and storage, which could lead to contamination, mold growth, and damage to the specimen. Hydrogen peroxide was then used for bleaching, which improved the appearance of the rabbit's nervous system without damaging the nerve specimen.

[0025] The rabbit whole nervous system specimen prepared by this invention is easy to preserve, has a complete and beautiful structure, and a clear and natural morphology and position. Each nerve trunk of the peripheral nerves is well displayed and fixed in the natural position of the rabbit. The positional relationship between the brain, cranial nerves, spinal cord, spinal nerves and visceral nerves is clear and natural. Moreover, the preparation method of this invention is simple, easy to implement and low in cost. Attached Figure Description

[0026] Figure 1 This is a photograph of a whole-body nerve specimen of a rabbit obtained in Example 1.

[0027] Figure 2 A photograph of the family nerves obtained for Comparative Example 1. Detailed Implementation

[0028] The technical solution of the present invention will be described in detail below through specific embodiments. However, it should be clearly stated that these embodiments are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0029] Example 1

[0030] A method for preparing a whole-body nerve specimen from a rabbit includes the following steps:

[0031] S1. Skinning and preservation: The rabbit carcass was placed supine on the dissection table. An incision was made in the skin of the rabbit's abdomen using a scalpel. The skin was peeled off from the abdomen to the head and tail. Circular incisions were made on both sides of the rabbit's ears to preserve the ear skin and avoid damaging the auditory nerves. The skin of the eyes and lips was separated, and the rabbit skin was completely peeled off. The superficial fascia and fat attached to the muscles were removed, and the visceral nerves were separated and the internal organs were removed. The carcass was then fixed in a 37wt% formalin solution for preservation for 1 month to obtain Sample 1.

[0032] S2. Softening: Sample 1 was immersed in a 0.5 wt% low-temperature softening enzyme EB aqueous solution and ultrasonically soaked at 32°C for 2.5 h. After being removed and washed with water, it was soaked in a mixed solution of hydrochloric acid and glycerol for 3 days. After being removed, it was soaked in a 50 wt% sodium carbonate aqueous solution for 8 h to neutralize the hydrochloric acid and obtain Sample 2. The mixed solution of hydrochloric acid and glycerol contained 15 wt% hydrochloric acid and 15 wt% glycerol.

[0033] S3. Nerve Separation: Take sample 2 and sequentially separate the cranial nerves, spinal cord, upper limb nerves, lower limb nerves, thoracic nerves, lumbar plexus nerves, and sacral plexus nerves. The specific steps are as follows:

[0034] a. Separate the cranial nerves, peel off the superficial fascia on the surface of the muscles, free the facial nerve and its branches to the root of the ear, and remove the adipose tissue around the nerves; incise and remove the masseter muscle at the mandible, clamp the decalcified condyle and zygomatic arch with hemostatic forceps to expose the eyeball, dissect the eyeball and locate the optic nerve; carefully separate the mandible, gently remove the medial pterygoid, lateral pterygoid, and pectineal muscles to locate the mandibular nerve, and through the main trunk of the mandibular nerve, locate the inferior alveolar nerve, lingual nerve, and buccal nerve, carefully separate the inferior alveolar nerve to the mandibular foramen, and carefully... Separate the lingual nerve on the medial side of the mandibular ramus and the buccal nerve on the pterygoid mandible; locate the trigeminal ganglion through the foramen ovale, the ophthalmic nerve through the superior orbital fissure, and the maxillary nerve through the foramen rotundum; remove the tympanic tegmentum at the internal auditory meatus to locate the origin of the facial nerve; remove the vertebrae, connective tissue, and blood vessels, and separate the spinal cord; while dissecting the nerve along its course and distribution, cut away the muscles, remove the connective tissue and blood vessels along the intermuscular space, separate the serosa covering the nerve, expose the rabbit's nerve, and completely separate it;

[0035] b. Separate the spinal cord, use a scalpel and surgical scissors to remove the skull and muscles around the spine, lift the skull upward from the external occipital protuberance to expose the brain, and lift the vertebral arch downward to expose the spinal cord up to the cauda equina.

[0036] c. Separate the nerves of the upper limb. Incise the platysma and sternocleidomastoid muscles along the midline of the trachea to find the brachial plexus. Incise the pectoralis major and biceps brachii muscles along the lateral bundle of the brachial plexus to separate the musculocutaneous nerve and lateral thoracic nerve. Incise the brachialis and pronator teres muscles along the medial bundle of the brachial plexus to separate the medial cutaneous nerve of the arm, the medial cutaneous nerve of the forearm, the medial thoracic nerve, and the ulnar nerve. Separate the subscapularis nerve, radial nerve, axillary nerve, and thoracodorsal nerve along the posterior bundle of the brachial plexus using the triceps brachii and subscapularis muscles.

[0037] d. Separate the lower limb nerves. In the posterior thigh region, cut the gluteus maximus, gluteus medius, biceps femoris, and semitendinosus muscles to separate the sciatic nerve. Proceed to the upper corner of the popliteal fossa and cut the posterior triceps surae muscle to separate the tibial nerve and common peroneal nerve. In the anteromedial thigh region, cut the five muscles of the anterior thigh group (sartorius, quadriceps femoris, and medial thigh group) to separate the femoral nerve.

[0038] e. Separate the thoracic nerves. Locate the intercostal nerves in the intercostal spaces on the inner side of the chest, close to the lower edge of the upper rib. Separate the lateral cutaneous branches of the intercostal nerves in the middle of the rib. Separate the intercostal nerves gradually from the costal cartilage toward the spine. This can avoid the intercostal nerves from being broken and can separate the intercostal nerves more completely. Separate the intervertebral foramen upwards to fully expose the intercostal nerves and spinal cord. Separate other intercostal nerves downwards along the spine.

[0039] f. Separate the lumbar plexus, cut the transverse process muscles and psoas major muscles of the lumbar vertebrae, and separate the iliohypogastric nerve, ilioinguinal nerve, lateral femoral cutaneous nerve, femoral nerve, obturator nerve and genitofemoral nerve from the intervertebral foramen downwards in sequence to fully expose them;

[0040] g. Separate the sacral plexus, find its origin by ascending along the separated sciatic nerve, separate the intervertebral foramen, at which point the spinal nerves have been fully freed and the spinal cord has been fully exposed; finally, obtain the whole nervous system of the rabbit;

[0041] S4. Degreasing: Rinse the entire nervous system of the rabbit with water, place it in an immersion container, and then add a 3wt% sodium hydroxide aqueous solution to completely submerge the rabbit's nervous system. After soaking for 2 days, remove the rabbit, rinse it with water, and remove any remaining muscle with a scalpel. Then, put the rabbit back into a sufficient amount of petroleum ether for 7 days (to prevent the petroleum ether from evaporating completely, it should be replenished every 2 days. During the degreasing process, the immersion container should be sealed tightly with plastic wrap to prevent the solution from evaporating and causing harm to the operators). Sample 3 was obtained.

[0042] S5. Bleaching and Fixation: Sample 3 was soaked in hydrogen peroxide with a mass fraction of 3wt% for 3 days to make sample 3 white. Then it was rinsed with clean water and dried to obtain sample 4. Sample 4 was fixed to an acrylic plate with glue to keep the nerve in the upright posture of the rabbit. Then it was placed in a display box to obtain a whole-body nervous system specimen of the rabbit.

[0043] Example 2

[0044] A method for preparing a whole-body nerve specimen from a rabbit includes the following steps:

[0045] S1. Skinning and preservation: The rabbit carcass was placed supine on the dissection table. An incision was made in the skin of the rabbit's abdomen using a scalpel. The skin was peeled off from the abdomen to the head and tail. Circular incisions were made on both sides of the rabbit's ears to preserve the ear skin and avoid damaging the auditory nerves. The skin of the eyes and lips was separated, and the rabbit skin was completely peeled off. The superficial fascia and fat attached to the muscles were removed, and the visceral nerves were separated and the internal organs were removed. The carcass was then fixed in a 37wt% formalin solution for preservation for 1 month to obtain Sample 1.

[0046] S2. Softening: Sample 1 was immersed in a 0.4 wt% low-temperature softening enzyme EB aqueous solution and ultrasonically soaked at 30°C for 3 hours. After being removed and washed with water, it was immersed in a mixed solution of hydrochloric acid and glycerol for 2 days. After being removed, it was immersed in a 50 wt% sodium carbonate aqueous solution for 8 hours to neutralize the hydrochloric acid and obtain Sample 2. The mixed solution of hydrochloric acid and glycerol contained 13 wt% hydrochloric acid and 13 wt% glycerol.

[0047] S3, the same as S3 in Example 1, finally obtained the whole-body nerve of the rabbit;

[0048] S4. Degreasing: Rinse the entire nervous system of the rabbit with water, place it in an immersion container, and then add a 2.5 wt% sodium hydroxide aqueous solution to completely submerge the rabbit's nervous system. After soaking for 2.5 days, remove the rabbit, rinse it with water, and remove any remaining muscle with a scalpel. Then, put the rabbit back into a sufficient amount of petroleum ether for 8 days (to prevent the petroleum ether from evaporating completely, it should be replenished every 2 days. During the degreasing process, the immersion container should be sealed tightly with plastic wrap to prevent the solution from evaporating and causing injury to the operators). Sample 3 was obtained.

[0049] S5. Bleaching and Fixation: Sample 3 was soaked in hydrogen peroxide with a mass fraction of 3.5 wt% for 2 days to make sample 3 white. Then it was rinsed with clean water and dried to obtain sample 4. Sample 4 was fixed to an acrylic plate with glue to keep the nerve in the upright posture of the rabbit. Then it was placed in a display box to obtain a whole-body nervous system specimen of the rabbit.

[0050] Example 3

[0051] A method for preparing a whole-body nerve specimen from a rabbit includes the following steps:

[0052] S1. Skinning and preservation: The rabbit carcass was placed supine on the dissection table. An incision was made in the skin of the rabbit's abdomen using a scalpel. The skin was peeled off from the abdomen to the head and tail. Circular incisions were made on both sides of the rabbit's ears to preserve the ear skin and avoid damaging the auditory nerves. The skin of the eyes and lips was separated, and the rabbit skin was completely peeled off. The superficial fascia and fat attached to the muscles were removed, and the visceral nerves were separated and the internal organs were removed. The carcass was then fixed in a 37wt% formalin solution for preservation for 1 month to obtain Sample 1.

[0053] S2. Softening: Sample 1 was immersed in a 0.6 wt% low-temperature softening enzyme EB aqueous solution and ultrasonically soaked at 35°C for 2 hours. After rinsing with water, it was immersed in a mixed solution of hydrochloric acid and glycerol for 2.5 days. After that, it was immersed in a 50 wt% sodium carbonate aqueous solution for 8 hours to neutralize the hydrochloric acid and obtain Sample 2. The mixed solution of hydrochloric acid and glycerol contained 18 wt% hydrochloric acid and 17 wt% glycerol.

[0054] S3, the same as S3 in Example 1, finally obtained the whole-body nerve of the rabbit;

[0055] S4. Degreasing: Rinse the entire nervous system of the rabbit with water, place it in an immersion container, and then add a 3.5 wt% sodium hydroxide aqueous solution to completely submerge the rabbit's nervous system. After soaking for 1.5 days, remove the rabbit, rinse it with water, and remove any remaining muscle with a scalpel. Then, put the rabbit back into a sufficient amount of petroleum ether for 6 days (to prevent the petroleum ether from evaporating completely, it should be replenished every 2 days. During the degreasing process, the immersion container should be sealed tightly with plastic wrap to prevent the solution from evaporating and causing injury to the operators). Sample 3 was obtained.

[0056] S5. Bleaching and Fixation: Sample 3 was soaked in hydrogen peroxide with a mass fraction of 2.5 wt% for 3 days to make sample 3 white. Then it was rinsed with clean water and dried to obtain sample 4. Sample 4 was fixed to an acrylic plate with glue to keep the nerve in the upright posture of the rabbit. Then it was placed in a display box to obtain a whole-body nervous system specimen of the rabbit.

[0057] Comparative Example 1

[0058] In S2, sample 1 was soaked in a 15 wt% hydrochloric acid solution for 3 days, and then taken out and soaked in a 50 wt% sodium carbonate aqueous solution for 8 hours to neutralize the hydrochloric acid to obtain sample 2; the rest is the same as in Example 1.

[0059] Figure 1 Photographs of a whole-body nerve specimen from a rabbit obtained in Example 1, wherein 1 is the ophthalmic nerve, 2 is the olfactory nerve, 3 is the maxillary nerve, 4 is the mandibular nerve, 5-6 are the facial nerves, 7 is the first pair of cervical nerves, 8 is the second pair of cervical nerves, 9 is the third pair of cervical nerves, 10 is the fourth pair of cervical nerves, 11-12 are the cephalic trunks of the brachial plexus, 13 is the middle trunk of the brachial plexus, 14-15 are the caudal trunks of the brachial plexus, 16 is the second pair of thoracic nerves, 17 is the third pair of thoracic nerves, 18 is the fourth pair of thoracic nerves, 19 is the fifth pair of thoracic nerves, and 20 is the sixth pair of thoracic nerves. Nerves: 21 is the seventh thoracic nerve, 22 is the eighth thoracic nerve, 23 is the ninth thoracic nerve, 24 is the tenth thoracic nerve, 25 is the eleventh thoracic nerve, 26 is the twelfth thoracic nerve, 27 is the first lumbar nerve, 28-29 are the second lumbar nerve, 30 is the third lumbar nerve, 31 is the fourth lumbar nerve, 32 is the fifth lumbar nerve, 33 is the sixth lumbar nerve, 34 is the seventh lumbar nerve, 35 is the first sacral nerve, 36 is the sciatic nerve, 37 is the tibial nerve, 38 is the common peroneal nerve, and 39 is the cauda equina.

[0060] Figure 2 This is a photograph of the rabbit's nervous system obtained for Comparative Example 1.

[0061] Depend on Figure 1-2It can be seen that the specimen prepared by the present invention has a complete and beautiful structure, clear and natural morphology and position, and each nerve trunk of the peripheral nerves is well displayed and fixed in the natural position of the rabbit. The positional relationship of the brain, cranial nerves, spinal cord, spinal nerves and visceral nerves is clear and natural; while in Comparative Example 1, some nerves were broken during dissection.

[0062] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for preparing a whole-body nerve specimen from a rabbit, characterized in that, Includes the following steps: S1. Skinning and preservation: The rabbit carcass was skinned, the internal organs and nerves were separated, the internal organs were removed, and then preservation treatment was performed to obtain sample 1. S2, softening: Sample 1 was soaked in a low-temperature softening enzyme EB solution, a mixed solution of hydrochloric acid and glycerol, and an alkaline solution in sequence to obtain sample 2; S3. Nerve separation: Take sample 2, remove muscle, fat and bone, and separate the whole nerve of the rabbit. S4. Degreasing: The entire nervous system of the rabbit was sequentially immersed in sodium hydroxide solution and petroleum ether for degreasing treatment to obtain sample 3; S5. Bleaching and Fixation: Sample 3 was bleached by soaking in hydrogen peroxide, then washed and dried to obtain Sample 4; Sample 4 was fixed on the substrate according to the upright posture of the rabbit to obtain the whole-body nerve specimen of the rabbit. In S2, the mass fraction of the low-temperature softening enzyme EB solution is 0.4-0.6 wt%, and it is ultrasonically soaked at 30-35℃ for 2-3 hours; In S2, a mixed solution of hydrochloric acid and glycerol, with hydrochloric acid having a mass fraction of 13-18 wt% and glycerol having a mass fraction of 13-17 wt%, is soaked for 2-3 days. In S2, the alkaline solution is an aqueous solution of sodium carbonate and an aqueous solution of sodium hydroxide. In S4, the mass fraction of sodium hydroxide solution is 2.5-3.5 wt%. In S4, soak in sodium hydroxide solution for 1.5-2.5 days; In S4, soak in petroleum ether for 6-8 days; In S5, the mass fraction of hydrogen peroxide in the hydrogen peroxide solution is 2.5-3.5 wt%. In S5, soak in hydrogen peroxide for 2-3 days.

2. The method for preparing a whole-body rabbit nerve specimen according to claim 1, characterized in that, In S1, the skin of the rabbit's ears is preserved.

3. The method for preparing a whole-body nerve specimen from a rabbit according to claim 1, characterized in that, In S1, it is soaked in formalin solution for 0.8-1.2 months for preservation treatment.

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