KR101497973B1 - Isolation and Yield Enhancement of Primo Vessels Inside of Rabbit Lymph Vessels by Using Sound Wave Vibration - Google Patents

Abstract

The present invention relates to a method for separating primotypes in a lymphatic vessel of a laboratory animal using sonic vibration, and more particularly, to a method for separating primotypes from an experimental rabbit by providing sound waves prior to separation of the primotypes from experimental rabbits, The present invention relates to a method for isolating a primo tube in a lymph vessel of a laboratory animal using sonic vibration,
Applying music and sound waves to the laboratory animal; Anesthetizing the animal to prepare the experiment; Lancing the anesthetized laboratory animal; Staining lymphatic vessels; Observing the lymphatic vessels and priming the lymphatic vessels from the lymphatic vessels;
The present invention has the effect of providing a sound wave to a laboratory animal, minimizing the tensions of the laboratory animal, and dissolving the animal under anesthesia in a state in which the blood circulation is performed in a satisfactory manner, thereby making it possible to more easily extract the primo tube from the lymphatic vessel.

Description

Technical Field [0001] The present invention relates to a method for isolating a primer tube in a lymph vessel of a laboratory animal using sonic vibration,

The present invention relates to a method for separating primotypes in a lymph vessel of a laboratory animal using sonic vibrations. In particular, prior to separation of primotypes from experimental rabbits, The present invention relates to a method of isolating a primotube in a lymph vessel of a laboratory animal using sonic vibrations.

In the early 1960s, Dr. Kim Bong - Han of Pyongyang University Medical School discovered a new circulatory system distinct from the existing blood vessels, lymph vessels, and nerves, and published a study showing that some of them exist as threadlike structures in blood vessels.

Since Kim Bong-Han's research has been published, many researches have been conducted on Primo vessel, a third circulatory system, in Korea to reproduce Dr. Kim's research.

As a result, it has been found that the various dyes and experimental methods have the presence of primotube in the cardiovascular, organ, and lymphatic vessels. As a follow-up study, many studies have been conducted to visualize a nerve primo vessel present in nerve tissue.

The primo vascular system (PVS; formerly called the Bonghan System) is a structure found in the 1960s as a new circulatory system in acupuncture and acupuncture (Bonghan Kim, "Bonghan theory"; Kim BH, The Kyungrak System, J Jo Sun Med 1965; 108: 1-38.). Bonghan Kim proposed the existence of a network of NPVS consisting of primo nodes (PN) and primo vessels (PV) in cerebrospinal fluid in the brain and vertebrae.

To confirm these proposals, we have studied techniques for visualization of primonal and primo tubes in the brain ventricles and central canal of rabbits. The present inventors have succeeded in developing a technique for visualizing primonal and primo tubes in the internal organs of rats using trypan blue (TB) staining in previous studies (Lee BC, So KS, Visualizing This staining was performed in situ and in vivo in the brain, spinal cord, and sciatic nerve of rats, and in situ and in vivo in the rat brain, spinal cord, and sciatic nerve It is applied to visualization.

However, there is a problem that the primo tube is difficult to be found because it is very thin and thin.

In particular, when the animal becomes strained, the vessels contract and it becomes more difficult to find the primo tube in the lymphatic vessel.

For reference, the primordial tube of the vascular structure is transparent, so it is difficult to distinguish it from the thrombus, and the tube is dense like the root of the plant, and the fluorescent dye put in it moves very slowly (0.3 mm per minute) , So it is not easy to find a primo tube.

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made to solve the above-mentioned problems, and it is an object of the present invention to provide a sound wave to a laboratory animal, minimize anxiety of a laboratory animal, So that it can be extracted.

As means for achieving the above object,

The present invention relates to a method and apparatus for applying music and sound waves output from MP3 or PC to a laboratory animal (S10); (S20) preparing an experiment by anesthetizing the animal; A step (S30) of raising an anesthetized laboratory animal; Dyeing the lymphatic tissue (S40); Observing the lymphatic vessel and priming the lymphatic vessel (S50); The step (10) of applying the music and the sound wave to the laboratory animal comprises the steps of (S11) placing the laboratory animal in a box and placing it on the sound wave vibrating machine; (S12, S13) of operating the sound wave vibration device to provide music to the laboratory animal and applying the vibration for a period of 30 minutes at a predetermined frequency with a constant intensity; The animal test preparation step (S20) comprises injecting an anesthetic agent into the leg muscles of a laboratory animal, injecting an anesthetic using a 3 ml syringe into a leg of a laboratory animal (S21); Placing an anesthetized animal in a box, placing the animal on a sound wave vibration apparatus, setting frequency and intensity, and applying it for 10 minutes (S23); The dyeing step S40 may include a step S41 of finding a lymph node or a lymph vessel and injecting a dye; (S42) when it is confirmed that the blue liquid flows through the lymphatic vessel after the injection of the Alcian Blue Solution, and waits until the washing is performed; It is characterized in that the sound wave vibration is provided at 7 Hz to 14 Hz.

delete

delete

delete

delete

delete

1 is a block diagram of a vibration mechanism of a sound wave vibration device;
FIG. 2A is a photograph of a state in which a rabbit in a cage (box) is simultaneously subjected to music and sound waves before anesthesia; FIG.
2B is a photograph of an operation panel of a device providing sound waves;
Figure 2c is a photographic view showing providing a sound wave to a rabbit;
FIG. 2d is a photograph showing that an anesthetic and shaving rabbits are contained in a box to provide sound waves of appropriate frequency and intensity. FIG.
3A is a photograph showing a state in which a primo tube near the abdominal vein is separated to find a lymph node and a syringe is inserted.
Figure 3b is another angle photograph of Figure 3a.
FIG. 3c is a photograph showing that the dyeing solution exits through the lymphatic vessel in an instant after injecting the staining liquid into the lymph node.
FIG. 3D is a photographic view showing that only a stain remained in a primo tube in a lymph vessel.
3E is a photograph showing a process of separating the dyed primot tube.
FIG. 3f is another photograph showing the process of separating the dyed primot tube. FIG.
4A is a photograph showing that a primo tube is observed in a lymphatic vessel in a vein of a vein.
4B is a photograph showing various types of primo tubes in the lymphatic system connected to each other and having various shapes.
4C is a photograph showing the morphology of the separated primot tube.
4D is a photograph showing another structure of the separated primot tube.
FIG. 5 is an overall flowchart of a method for isolating a primorum tube in a lymphatic vessel of an animal of the present invention. FIG.
FIG. 6 is a flow chart showing an operation sequence of applying music and sound waves of the present invention to a laboratory animal.
FIG. 7 is a flow chart showing an operation sequence for preparing an experiment by anesthetizing an animal in the present invention. FIG.
8 is a flow chart of the operation of the step of staining lymphatic vessels in the present invention.

The operation principle of the preferred embodiment of the present invention will be described in detail with reference to the accompanying drawings and description. It should be understood, however, that the drawings and the following detailed description are exemplary and explanatory and are intended to provide further explanation of the invention, and are not to be construed as limiting the present invention.

In the following description of the present invention, a detailed description of known functions and configurations incorporated herein will be omitted when it may make the subject matter of the present invention rather unclear. The terms used below are defined in consideration of the functions of the present invention, which may vary depending on the user, intention or custom of the operator. Therefore, the definition should be based on the contents throughout the present invention.

It is to be understood that both the foregoing general description and the following detailed description of the present invention are exemplary and explanatory and are intended to provide further explanation of the invention as claimed. The configuration is omitted as much as possible, and a functional configuration that should be additionally provided for the present invention is mainly described.

Those skilled in the art will readily understand the functions of the components that have been used in the prior art among the functional configurations that are not shown in the following description, The relationship between the elements and the components added for the present invention will also be clearly understood.

In order to efficiently explain the essential technical features of the present invention, the following embodiments properly modify the terms so that those skilled in the art can clearly understand the present invention, It is by no means limited.

As a result, the technical idea of the present invention is determined by the claims, and the following embodiments are merely illustrative of the technical idea of the present invention in order to efficiently explain the technical idea of the present invention to a person having ordinary skill in the art to which the present invention belongs. .

FIG. 5 is an overall flowchart of a method for isolating a primorum tube in a lymphatic vessel of an animal of the present invention. FIG.

FIG. 6 is a flow chart showing an operation sequence of applying music and sound waves of the present invention to a laboratory animal.

FIG. 7 is a flow chart showing an operation sequence for preparing an experiment by anesthetizing an animal in the present invention. FIG.

FIG. 8 is a flow chart illustrating a procedure of dyeing lymphatic vessels in the present invention,

The present invention extracts the primo tube from the rabbit through the following steps.

The rabbit is a New Zealand mountain, supplied by the Korea Bio Link Company, located in Chungcheongbuk-do province. The gender is female. The age is 10th and the weight is 1.5kg ~ 1.8kg.

The following three points were noted during dissection. ① Saline (saline) at the same temperature (about 40 degrees) as the rabbit's body temperature is sprayed to the body for a long time, so that circulation of body fluids is smooth. ② If you have bleeding, gauze bleeding is done. ③ By twisting the membrane with tweezers, be careful not to touch the blood vessels when observing.

The entire experimental procedure of rabbit anatomy is through the following six major steps.

1. Music and sonic steps

① Place the rabbit in a box and place it on a sound wave vibration device.

② It usually plays the music that the rabbit likes (meeting rabbits, looking for music that rabbit likes through cafe).

③ Sound wave Set the frequency and intensity of the vibration device and apply it for 30 minutes.

2. Preparation stage of animal experiment

① Anesthetize the leg muscles of the rabbit. That is, an anesthetic agent (Zoletil 0.5 ml + Rompun 2.5 ml) is injected into a muscle of a rabbit leg using a 3 ml syringe.

② Shaves the abdomen hair. In other words, the hair around the abdomen is trimmed with an epilator in preparation for raising anesthetized rabbits. It is necessary to be careful not to hurt the rabbit.

③ Anesthetized rabbits are placed in a box, placed on a sound wave vibration apparatus, and frequency and intensity are set for 10 minutes.

④ Lay the rabbit on a large scale on a bench and fasten the arms and legs with a bandage.

3. Opening phase

① Sprinkle the whole abdomen with alcohol to disinfect.

② Use the scissors to cut the center of the abdominal skin layer.

③ When the peritoneum is exposed, it extends from the bladder to the center of the stomach along the white line at the center.

④ Take the organs out and put them in one side and cover the gauze. Spray the saline gaps to keep the device dry.

⑤ If there is urine in the bladder, remove the urine with a syringe to secure the search field.

4. Dyeing Step

Dye: Alcian Blue Powder 0.05 g + PBS 1 × 5 ml (1: 100 ratio)

① Locate the lymph node (lymph node) or lymphatic vessel (Lymph Vessel) and inject the Alcian Blue Solution.

② Alcian Blue Solution is injected. When it is confirmed that the blue liquid flows through the lymphatic vessel, wait for washing.

5. Observation and primo extraction steps

① Observe with a microscope and look for primo dyed in the lymphatic duct. In other words, a thin, threadlike primo is observed in the washed lymph. If Primo is not visible, tear it with tweezers and look for tissue near the blood vessels. Primo is not always visible, depending on the condition of the rabbit and the various complex factors.

② Use the tweezers to carefully remove the lymph vessels to find the primo canal and collect the primo canal inside the lymph. When the lymphatic tube is removed, the primot tube may be contracted. In addition, the necessary parts of the lymphatic tubes are extracted with a micro-forceps and stored in refrigerator in 5 ml of Eppendorf Tubes containing 1: 100 ratio of PBS (phosphate buffered saline).

1 shows a vibration mechanism of a sound wave vibration device. Sound wave vibration equipment is a device that transmits sound energy to the human body organs of the person who climbs up the plate by adjusting the intensity of the frequency and intensity by generating the sound wave with the principle of the speaker. As shown in FIG. 1, music (MP) 3 or music from a PC is connected to be designed so that the musical frequency and the intensity of the sound source can be applied. Sound wave vibration is applied to various parts of the body such as bones, fractures, auditory nerves and muscles at various frequencies. In recent years, research has been reported that the circulatory system, endocrine system, and musculoskeletal system are affected evenly, and it is widely supplied as a device capable of exercising the human body in the room.

Fig. 2 is a photograph showing the application of a music and sound wave vibration device to a rabbit. FIG. 2 (a) shows a case where a rabbit in a cage (box) applies music and sound waves simultaneously before anesthesia. FIGS. 2 (b) and 2 (c) are views showing a portion for adjusting the intensity and frequency of the sound wave vibration device, and music for the rabbit. Fig. 2 (d) shows that rabbits that underwent anesthesia and shaving were subjected to sonic fluctuations at appropriate frequency and intensity within the cage. In the case of sound wave vibration equipment, it transmits precise sound wave vibration of 3 Hz to 50 Hz to the body, and has the function of strengthening muscle strength, increasing bone density, and improving blood circulation and lymph circulation without imposing on joints or ligaments

FIG. 3 is a photograph showing the representative six steps required for separating the primo tube near the abdominal vein. First, locate the largest lymph node near the abdominal vein and carefully insert the syringe as shown in Figures 3 (a) and 3 (b). FIG. 3 (c) shows the staining solution spreading through the lymphatic vessels in an instant after injecting the Alcian blue staining solution into the lymph nodes. FIG. 3 (d) shows that the lymphatic fluid stained only in the primordium tube in the lymphatic vessel after the lymphatic fluid flowed along the tube, mostly after the passage of a few minutes, while the staining solution gradually became thinner. With the staining solution attached to the wall of the primo tube having the vascular structure characteristic, while maintaining the shape of the primo tube while the lymph flow is flowing, it remains. Thus, it can be easily observed that the presence of the primotube in the floating state in the lymphatic vessel is stained. Figs. 3 (e) and 3 (f) show the process of carefully collecting and separating the stained primot tube. Through these experimental procedures, the separated primo tubes are stuck together with the lymphatic tubes and stored in a sample box in PBS. This is followed by separation of the primo-tubes in the lymphatic vessels using ultrasound or micro-pincette to provide the primo-tubes for electrophysiological or histological functional studies.

   In this study, to characterize the primordial tube with the nucleus of the vascular structure, it was washed well in PBS solution, and the primordial tube was placed on a slide. Acridine orange dye and DAPI (4 ', 6-diamidino-2-phenylindole, dihydrochloride) The coloring phenomenon was observed with a confocal laser scanning microscope. Nuclear staining images were obtained and analyzed to confirm that they have the typical size and shape of the primot tube

Also, we could obtain the morphological data of the primordial tube connected with the thickness and length of the primordial tube and the primonal before applying the sonodynamic vibration device. Fig. 4 shows the appearance of various representative primo tubes. FIG. 4 (a) shows a large-sized lymphatic tube having a diameter of several millimeters and a small-sized lymphatic tube having a size of about several hundreds of micrometers, which are adjacent to the abdominal vein, and a primo tube can be observed in a small lymphatic vessel. FIG. 4 (b) shows various types of primo tubes connected to the primordodes of the various primo tubes in the lymphatic system. 4 (c) and 4 (d) are photographs showing the morphology of the separated primot tube. Table 1 summarizes the morphological and structural data of these primo-tubes. We analyzed the results and explained improvement of sound wave vibration and primo separation method.

Table 1 shows that the intensity is 20, which is the same for all three, four, five, and six experiments except for the first and second experiments. Here, the intensity, which is the intensity of the sound wave vibration device, represents a given frequency step, and the unit is a step.

The size of the intensity 50 step at 7 Hz obtained with a compact type vibration measuring instrument (TYPE 3116, Aco Co., Ltd., Japan) as an acceleration sensor was 1.04 m / s ² . Among these four, the primo tube was found at frequencies of 11 Hz and 14 Hz, but not at frequencies of 4 Hz and 30 Hz. Therefore, it was not easy to find a primo tube at a relatively low frequency (4 Hz) or a very high frequency (30 Hz) based on 7 Hz. In the experiments 1 and 2, the intensity and frequency were the same at 50 Hz and 7 Hz. In addition, Primo can be found. Table 1 summarizes these results. Considering the frequencies of 7 Hz, 11 Hz, and 14 Hz of sonic interac- tions, it can be seen that a rabbit primo tube was found at frequencies between 7 Hz and 14 Hz. The thickness and the length of the found primotube were about 42.5 ㎛ and 23.5 ㎜, respectively.

(Table 1) Subject
Number Weight
(kg) Intensity Frequency
(Hz) Music After Anesthesia
(10 min) Primo
Vessel P _D
(탆) P _L
(cm) One 1.6 50 7 × Frequency ○ 35 2.3 2 1.7 50 7 × Frequency ○ 25 2.5 3 1.5 20 14 ○ Music On ○ 45 2.0 4 1.6 20 4 ○ Music On × - - 5 1.7 20 30 ○ Music On × - - 6 1.8 20 11 ○ Music On ○ 60 2.6

In the second experiment, the frequency of 7 Hz to 14 Hz and the intensity of 60 were applied first, as in the first experiment, and then the sound wave was applied again for 10 minutes after shaving. The results are shown in Table 2. The thickness and length of the found primotube were about 26.8 ㎛ and 27 ㎜, respectively.

(Table 2) Subject
Number Weight
(kg) Intensity Frequency
(Hz) Music After Anesthesia
(10 min) Primo
Vessel P _D
(탆) P _L
(cm) One 1.6 60 7 × Frequency ○ 27 2.4 2 1.7 60 7 × Frequency ○ 28 2.0 3 1.5 60 7 × Frequency ○ 26 3.9 4 1.6 60 14 × Frequency ○ 23 2.8 5 1.7 60 8 × Frequency ○ 30 2.4 6 1.8 60 8 × Frequency × - -

As shown in Table 2, no secondary primer was found. However, it was found that the probability of finding a primo tube at a frequency of 7 Hz to 14 Hz was high.

Table 3 shows the comparison with the general experiment in which the sound wave vibration was not performed. From this we could see that the probability of finding primo at 7 Hz to 14 Hz was more pronounced. In case of general experiment without excitation vibration, 5 times of 12 trials were successful and showed about 42% probability. On the other hand, when a sound wave is applied, a primo tube is found at a frequency of 7 Hz to 14 Hz except for extreme frequencies of 4 Hz and 30 Hz with a 90% probability.

(Table 3) Subject
Number Normal Experiment Sound Wave Experiment Frequency
(Hz) Intensity Primo
Vessel Frequency
(Hz) Intensity Primo
Vessel One - - ○ 7 50 ○ 2 - - × 7 50 ○ 3 - - × 14 20 ○ 4 - - ○ 4 20 × 5 - - × 30 20 × 6 - - ○ 11 20 ○ 7 - - × 7 60 ○ 8 - - ○ 7 60 ○ 9 - - ○ 7 60 ○ 10 - - × 14 60 ○ 11 - - × 8 60 ○ 12 - - × 8 60 ×

Claims (5)

(S10) applying music and sound waves output from MP3 or PC to a laboratory animal; (S20) preparing an experiment by anesthetizing the animal; A step (S30) of raising an anesthetized laboratory animal; Dyeing the lymphatic tissue (S40); Observing the lymphatic vessel and priming the lymphatic vessel (S50);
The step (10) of applying the music and sound waves to a laboratory animal comprises the steps of (S11) placing a laboratory animal in a box and placing it on a sound wave vibration apparatus; (S12, S13) of operating the sound wave vibration device to provide music to the laboratory animal and applying the vibration for a period of 30 minutes at a predetermined frequency with a constant intensity;
The animal test preparation step (S20) comprises injecting an anesthetic agent into leg muscles of a laboratory animal, injecting the anesthetic agent into a leg of a laboratory animal using a 3 ml syringe (S21), and Placing an anesthetized animal in a box, placing the animal on a sound wave vibration apparatus, setting frequency and intensity, and applying it for 10 minutes (S23);
The dyeing step S40 may include a step S41 of finding a lymph node or a lymph vessel and injecting a dye; (S42) when it is confirmed that the blue liquid flows through the lymphatic vessel after the injection of the Alcian Blue Solution, and waits until the washing is performed;
Wherein the sonic vibrations are provided in a range of 7 Hz to 14 Hz. delete delete delete delete

Images