Method for determining entry of injection needle into abdominal cavity state of rat or mouse and method for intraperitoneal injection

Abstract

The invention provides a method for rat or mouse intraperitoneal injection and determining that an injection needle enters the rat or mouse intraperitoneal state. The rat intraperitoneal injection method comprises the steps that a rat is fixed; at the injection position, the injection needle penetrates subcutaneously and penetrates through the abdominal muscle of the mouse to pierce into the abdominal cavity; determining that the injection needle enters an abdominal cavity state. Standardized operation procedures are carried out on a rat or mouse fixing method, injection position determination and judgment on whether an injection needle enters the abdominal cavity state, the success rate of intraperitoneal injection is effectively increased, the mechanical stimulation and bleeding risk of abdominal tissue are reduced, the cecum and small intestine puncture risk is reduced, stress response is reduced, and the safety of intraperitoneal injection is improved. And technical support is provided for drug research and development and disease model construction.

Description

Technical Field

[0001] The present invention relates to the field of biology, and in particular to a method for determining the state of an injection needle entering the abdominal cavity of a rat or mouse and performing intraperitoneal injection. Background Art

[0002] Intraperitoneal injection is a common technique for drug delivery through peritoneal absorption. It is characterized by rapid absorption and easy operation, and plays an important role in drug development, disease model construction, pharmacokinetic research, and anesthesia administration. The success rate of this technology directly affects the reliability and repeatability of experimental data. Non-standard technical operations can lead to animal stress and tissue damage. At present, there is no standardized operating procedure for intraperitoneal injection. The main technical bottleneck is misjudgment of the injection site. The misjudged injection site is mainly in the cecum, followed by the small intestine. Inadvertent injection into non-abdominal sites can cause certain damage and infection to mice and rats, such as complications such as peritonitis and tissue adhesions, and at the same time induce stress behaviors in animals, such as screaming, urinary incontinence, tremors, etc., involving animal rights and welfare issues. Therefore, accurately judging whether the injection needle has entered the abdominal cavity is crucial for successful intraperitoneal injection. Summary of the Invention

[0003] This application aims to at least partially resolve the technical problems existing in the prior art. To this end, this application proposes a method for determining the state of an injection needle entering the abdominal cavity of a rat or mouse and a method for mouse intraperitoneal injection. This application implements standardized operating procedures for the mouse fixation method, injection location, and determination of the state of the injection needle entering the abdominal cavity, effectively improving the success rate of intraperitoneal injection, reducing the risks of mechanical irritation and bleeding of abdominal tissue, reducing cecal and small intestinal puncture, and reducing stress reactions. This has promoted the development of experimental animal technology and protected animal welfare, providing technical support for drug development and disease model construction, and is of great significance.

[0004] In one aspect, the present application provides a method for determining whether an injection needle has entered the abdominal cavity of a mouse. According to an embodiment of the present application, the method includes: after the injection needle penetrates the abdominal muscle of a rat or mouse and enters the abdominal cavity, determining whether the injection needle has entered the abdominal cavity based on whether there is a sense of empty space in the following manner: 1) If there is no sense of empty space, determining that the injection needle has not entered the abdominal cavity; 2) If there is a sense of empty space, determining that the injection needle has entered the abdominal cavity, and then proceeding to the following steps: lifting the injection needle toward the abdominal wall until the needle body is parallel to and in contact with the abdominal wall, swinging the needle left and right, and withdrawing the injection needle plunger, and determining the state of the injection needle in the abdominal cavity based on the following manner: a) If there is no sense of obstruction during the swinging of the needle left and right, and no liquid is present in the injection needle cavity after withdrawing the injection needle plunger, determining that the injection needle has not contacted organs and / or blood vessels in the abdominal cavity; b) If there is a sense of obstruction during the swinging of the needle left and right and / or liquid is present in the injection needle cavity after withdrawing the injection needle plunger, determining that the injection needle has contacted organs and / or blood vessels in the abdominal cavity.

[0005] According to the method of the present application, whether the injection needle has entered the abdominal cavity is initially determined based on whether there is a sense of empty space after the injection needle penetrates the abdominal muscle of the mouse and enters the abdominal cavity. If there is an empty space, it can be preliminarily determined that the injection needle has entered the abdominal cavity; otherwise, it has not entered.

[0006] After initially confirming entry into the abdominal cavity, the needle is further assessed for contact with organs and / or blood vessels within the abdominal cavity. Specifically, the needle is lifted toward the abdominal wall until the needle shaft is parallel to and in contact with the abdomen. This prevents intestinal puncture and entanglement, facilitating a stable injection. Next, the needle is swung left and right while withdrawing the syringe. Whether the needle has contact with organs and / or blood vessels within the abdominal cavity is determined based on any obstruction felt during the swinging process and the presence of fluid in the syringe cavity after withdrawing the needle plunger.

[0007] Specifically, if there is no sense of obstruction when the needle is swung left and right, and there is no liquid in the injection needle cavity after the injection needle piston is withdrawn, it is determined that the injection needle has not contacted the organs and / or blood vessels in the abdominal cavity; if there is a sense of obstruction when the needle is swung left and right and / or there is liquid in the injection needle cavity after the injection needle piston is withdrawn, it is determined that the injection needle has contacted the organs and / or blood vessels in the abdominal cavity.

[0008] Therefore, the method of the present application can accurately determine the state of the injection needle entering the mouse's abdominal cavity. It can be used as a standardized operating procedure to effectively improve the success rate of intraperitoneal injection, reduce the risk of mechanical irritation and bleeding of abdominal tissue, reduce the risk of cecal and small intestine puncture, and reduce the changes in acute and chronic stress biochemical indicators such as creatine kinase (CK), alanine aminotransferase (ALT), and serum albumin (ALB). It promotes the development of experimental animal technology and protects animal welfare, provides technical support for drug development and disease model construction, and has reference significance for technological innovation and cross-domain application development.

[0009] According to an embodiment of the present application, the method for determining whether an injection needle has entered the abdominal cavity of a rat or mouse may also have the following additional technical features: According to an embodiment of the present application, the amplitude of the left and right swing of the needle is 0.8 cm to 1.2 cm.

[0010] According to an embodiment of the present application, the depth of the injection needle inserted into the abdominal cavity is 0.4 cm to 1.2 cm.

[0011] According to an embodiment of the present application, the depth of the injection needle inserted into the mouse abdominal cavity is 0.4 cm to 0.6 cm; or, the depth of the injection needle inserted into the rat abdominal cavity is 0.8 cm to 1.2 cm.

[0012] According to an embodiment of the present application, the needle body of the injection needle is at a 40°~50° angle to the skin surface and penetrates the abdominal muscle into the abdominal cavity.

[0013] In another aspect of the present application, a method for intraperitoneal injection of rats or mice is proposed. According to an embodiment of the present application, the method comprises: (1) fixing the rat or mouse; (2) passing the injection needle along the subcutaneous tissue at the injection site, through the abdominal muscle and into the abdominal cavity; (3) determining the state of the injection needle entering the abdominal cavity of the rat or mouse according to the method for determining the state of the injection needle entering the abdominal cavity of the rat or mouse described above; if it is determined that the injection needle has entered the abdominal cavity and has not touched the organs and blood vessels in the abdominal cavity, injecting the injection solution; if it is determined that the injection needle has not entered the abdominal cavity or has touched the organs and / or blood vessels in the abdominal cavity, repeating steps (2) and (3) at least once until it is determined that the injection needle has entered the abdominal cavity and has not touched the organs and blood vessels in the abdominal cavity, and injecting the injection solution; wherein, each time step (2) is repeated, the injection site is changed. Therefore, the method of the present application can accurately achieve intraperitoneal injection in mice, can be used as a standardized operating procedure, effectively improve the success rate of intraperitoneal injection, reduce the risk of mechanical irritation and bleeding of abdominal tissue, reduce cecal and small intestine puncture, promote the development of experimental animal technology and protect animal welfare, provide technical support for drug development and disease model construction, and have reference significance for technological innovation and cross-field application development.

[0014] According to an embodiment of the present application, the method for fixing a mouse includes: grabbing the skin on the right side of the mouse's neck and back.

[0015] According to an embodiment of the present application, the method of fixing the mouse is a one-handed operation, including: grabbing the skin on the right side of the mouse's neck and back with the thumb and index finger below the mouse's right ear, and pinching the tail with the ring finger and little finger, with the mouse in a head-low tail-high position.

[0016] According to an embodiment of the present application, the method for fixing a mouse is a one-handed operation, including: grabbing the skin on the right side of the mouse's neck and back with the thumb and index finger below the mouse's right ear, pinching the tail with the ring finger and little finger, and simultaneously supporting the back with the middle finger, so that the mouse is in a head-low and tail-high position; or, the method for fixing a rat is a one-handed operation, including: using the index finger to drive the head up, grabbing the skin on the right side of the mouse's neck and back with the thumb and index finger below the mouse's right ear, and pinching the remaining back skin with the other three fingers, and then pinching the tail with the ring finger and little finger, so that the mouse is in a head-low and tail-high position.

[0017] According to an embodiment of the present application, an auxiliary line is defined through the lower end of the ilium, the auxiliary line is perpendicular to the abdominal midline and has an intersection with the abdominal midline, and an injection area is formed with the midpoint of the line connecting the lower end of the ilium and the intersection as the center, and the injection position is located within the injection area.

[0018] According to an embodiment of the present application, for mice, the injection area is a circle with a radius of 0.1 cm to 0.3 cm; or, for rats, the injection area is a circle with a radius of 0.3 cm to 0.5 cm.

[0019] According to an embodiment of the present application, the depth of subcutaneous penetration is 0.4 cm to 0.6 cm.

[0020] According to an embodiment of the present application, after the injection is completed, the injection site is pressed with a cleaning object and the needle is rotated and pulled out.

[0021] Additional aspects and advantages of the present application will be given in part in the description below, and in part will become obvious from the description below, or will be learned through practice of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The above and / or additional aspects and advantages of the present application will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which: Figure 1 Figure 1 shows the intraperitoneal injection techniques for mice and rats; (A) the intraperitoneal injection technique for mice of this application; (B) the intraperitoneal injection technique for rats of this application; (C) the conventional intraperitoneal injection technique for mice; (D) the conventional intraperitoneal injection technique for rats; the intraperitoneal injection procedure for mice and rats shows: (a) grasping, (b) fixation, (c) head-low-tail-high position, (d) injection location, (e) subcutaneous passage, (f) intraperitoneal puncture, (g) injection judgment, and (h) completion of injection; white or black crosshatching in the figure indicates the intraperitoneal injection reference line, red circles indicate the injection area, and arrows or angle symbols indicate the corresponding area of ​​instruction; Figure 2 Shows H&E and S&R sections of mouse abdominal skin tissue; (A) 40× and 200× H&E and S&R cross-sections of skin; (B) 40× and 200× H&E longitudinal sections of the muscular layer of subcutaneous tissue; white boxes indicate magnified areas, and red dotted lines indicate blood vessels or bleeding sites; Figure 3 Figures show H&E and S&R sections of rat abdominal skin tissue; (A) 40× and 200× H&E transverse sections, the upper section is the skin tissue area, and the lower section is mainly the subcutaneous tissue area; (B) 40× and 200× S&R transverse sections, the upper section is the skin tissue area, and the lower section is mainly the subcutaneous tissue area; (C) 40× and 200× H&E longitudinal sections; white boxes in the figures indicate magnified sections, and red dotted lines indicate blood vessels or bleeding sites; Figure 4 The effects of intraperitoneal injection on stress biochemical indicators in mice are shown; (A) Observation of the abdominal area of ​​mice on day 14, the red dotted circle indicates the trauma point at the intraperitoneal injection site; (B) Changes in CK indicators in mice on day 1 and day 14; (C) Changes in ALT indicators in mice on day 1 and day 14; (D) Changes in ALB indicators in mice on day 1 and day 14; Data are represented as: Mean ± SD (n = 4); #p<0.05, ## p < 0.01 indicates comparison with the control group; α p < 0.05 indicates comparison with the traditional method group; Figure 5 Figure 3. Effects of intraperitoneal injection on stress biochemical indices in rats. (A) Observation of the abdominal area of ​​rats on day 14. The red dotted circle indicates the trauma point at the intraperitoneal injection site. (B) Changes in CK in rats on day 1 and day 14. (C) Changes in ALT in rats on day 1 and day 14. (D) Changes in ALB in rats on day 1 and day 14. Data are expressed as mean ± SD (n = 3). # p<0.05, ## p < 0.01 indicates comparison with the control group; α p<0.05 indicates comparison with the traditional method group. DETAILED DESCRIPTION

[0023] The embodiments of the present application are described in detail below. The embodiments described below are exemplary and are only used to explain the present application, and should not be understood as limiting the present application.

[0024] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, features defined as "first" or "second" may explicitly or implicitly include one or more of such features. Furthermore, in the description of this application, unless otherwise specified, "plurality" means two or more.

[0025] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0026] In this document, the terms "include" or "comprising" are open expressions, that is, including the contents specified in this application, but not excluding other contents.

[0027] The present application proposes a method for determining the state of an injection needle entering the abdominal cavity of a rat or mouse and a method for intraperitoneal injection of a rat or mouse, which will be described in detail below.

[0028] Method for determining the status of injection needle entering the abdominal cavity of rats or mice In one aspect, the present application provides a method for determining whether an injection needle has entered the abdominal cavity of a rat or mouse. According to an embodiment of the present application, the method comprises: after the injection needle penetrates the abdominal muscle of the mouse and enters the abdominal cavity, determining whether the injection needle has entered the abdominal cavity based on whether there is a sense of missing air, in the following manner: 1) If there is no empty feeling, the injection needle has not entered the abdominal cavity; 2) If there is a sense of missing the needle, the needle has entered the abdominal cavity and the following steps should be performed: Lift the needle toward the abdominal wall until the needle is parallel to the abdomen and in contact with the abdominal wall. Swing the needle left and right while withdrawing the needle plunger. Determine the position of the needle in the abdominal cavity based on the following: a) If there is no resistance when the needle is swung left and right, and there is no liquid in the needle cavity after the needle plunger is withdrawn, it is determined that the needle has not contacted organs and / or blood vessels in the abdominal cavity; b) If there is a sense of resistance when swinging the needle left or right and / or liquid is found in the needle cavity after withdrawing the needle piston, it is determined that the needle is in contact with organs and / or blood vessels in the abdominal cavity.

[0029] Therefore, the method of the present application can be used to accurately judge the state of the injection needle entering the abdominal cavity of rats and mice. It can be used as a standardized operating procedure to effectively improve the success rate of intraperitoneal injection, reduce the risk of mechanical irritation and bleeding of abdominal tissue, reduce the risk of cecal and small intestine puncture, and reduce the changes in acute and chronic stress biochemical indicators such as creatine kinase (CK), alanine aminotransferase (ALT), and serum albumin (ALB). It promotes the development of experimental animal technology and protects animal welfare, provides technical support for drug development and disease model construction, and has reference significance for technological innovation and cross-domain application development.

[0030] It should be noted that when the needle passes through the abdominal muscles and enters the abdominal cavity, the resistance encountered by the needle suddenly disappears due to the relative spaciousness of the abdominal cavity, resulting in a feeling of unobstructed movement, a feeling known as "falling empty." If there is resistance while swinging the needle left and right, this is referred to as a "sense of resistance," which may be caused by touching the wall of the cecum or small intestine, or other intra-abdominal organs. The "liquid" in "whether there is liquid in the needle cavity after withdrawing the needle piston" includes blood, intestinal contents, and the like. "Organs and / or blood vessels in the abdominal cavity" may include the cecum, small intestine, stomach, liver, gallbladder, spleen, pancreas, and the like.

[0031] According to an embodiment of the present application, the amplitude of the needle's left-right swing is 0.8 cm to 1.2 cm. In some embodiments, the amplitude of the needle's left-right swing is 0.8 cm, 0.9 cm, 1.0 cm, 1.1 cm, or 1.2 cm. This allows accurate determination of intestinal puncture or entanglement, avoiding areas with thicker or denser capillaries in the abdomen, thereby reducing the animal's stress behavior and the likelihood of complications such as peritonitis and tissue adhesions.

[0032] According to the embodiments of the present application, the depth of the injection needle's insertion into the abdominal cavity is 0.4 cm to 1.2 cm. In some embodiments, the depth of the injection needle's insertion into the abdominal cavity is 0.4 cm, 0.5 cm, 0.6 cm, 0.7 cm, 0.8 cm, 0.9 cm, 1.0 cm, 1.1 cm, or 1.2 cm. For mice, the depth of the injection into the abdominal cavity is 0.4 cm to 0.6 cm, and for rats, the depth of the injection into the abdominal cavity is 0.8 cm to 1.2 cm. This can effectively avoid puncture and entanglement of the intestines, reduce the risk of puncture of the cecum or small intestine, and avoid areas with thicker or dense capillaries in the abdomen, thereby reducing the animal's stress behavior and the probability of complications such as peritonitis and tissue adhesions. In addition, the depth of the injection into the abdominal cavity is combined with specific needle insertion criteria, such as lifting the needle after acupuncture, and gently swinging the needle parallel to the abdomen without any obstruction, which helps to accurately determine whether the injection needle has entered the abdominal cavity, and can stably inject the drug into the abdominal cavity, thereby improving the success rate and accuracy of the injection.

[0033] As used herein, the terms "rat" and "mouse" are well known in the art. Rats belong to the genus Rattus and are relatively large. Mice belong to the genus Mus and are relatively small. The methods of this application are applicable to both rats and mice and are therefore suitable for a wide range of applications.

[0034] According to an embodiment of the present application, the needle body of the injection needle is oriented at a 40° to 50° angle to the skin surface, penetrating the mouse abdominal muscle into the abdominal cavity. In some embodiments, the needle body of the injection needle is oriented at a 40°, 42°, 44°, 45°, 46°, 48°, or 50° angle to the skin surface, penetrating the mouse abdominal muscle into the abdominal cavity. This helps the needle tip smoothly penetrate the skin and abdominal muscle layer and enter the abdominal cavity, while avoiding excessive penetration and damaging internal organs (such as the intestines, liver, or bladder). The needle tip's path into the abdominal cavity is shorter and its direction is controllable, reducing the risk of puncturing the bladder, intestines, or blood vessels, ensuring consistency between different operators or experimental batches and reducing human error.

[0035] Intraperitoneal injection in rats or mice In another aspect of the present application, the present application provides a method for intraperitoneal injection of rats or mice. According to an embodiment of the present application, the method comprises: (1) Immobilize rats or mice; (2) Insert the injection needle subcutaneously at the injection site, through the abdominal muscle of the mouse and into the abdominal cavity; (3) Determine the state of the injection needle entering the mouse's abdominal cavity using the method described above for determining the state of the injection needle entering the rat or mouse's abdominal cavity; If it is determined that the injection needle has entered the abdominal cavity and has not come into contact with organs and blood vessels within the abdominal cavity, the injection solution is administered; If it is determined that the injection needle has not entered the abdominal cavity or has contacted the organs and / or blood vessels in the abdominal cavity, repeat steps (2) and (3) at least once until it is determined that the injection needle has entered the abdominal cavity and has not contacted the organs and blood vessels in the abdominal cavity, and the injection solution is injected; wherein, each time step (2) is repeated, the injection position is changed.

[0036] Therefore, the method of the present application can accurately achieve intraperitoneal injection in rats and mice, and can be used as a standardized operating procedure to effectively improve the success rate of intraperitoneal injection, reduce the risk of mechanical irritation and bleeding of abdominal tissue, and reduce cecal and small intestinal puncture, thereby promoting the development of experimental animal technology and protecting animal welfare. It provides technical support for drug development and disease model construction, and has reference significance for technological innovation and cross-domain application development. The characteristics and advantages described above for the method of determining the state of the injection needle entering the abdominal cavity of rats or mice are also applicable to the method of intraperitoneal injection of rats or mice, and will not be repeated here.

[0037] According to an embodiment of the present application, a method for immobilizing a rat or mouse includes: grasping the skin on the right side of the neck and back of the mouse, thereby securely immobilizing the mouse, keeping the head and body in the same plane, facilitating breathing, and reducing stress behaviors.

[0038] According to an embodiment of the present application, the method for fixing a rat or mouse is a one-handed operation, comprising: below the right ear of the mouse, grasping the skin on the right side of the neck and back of the mouse with the thumb and index finger, pinching the tail with the ring finger and little finger, and simultaneously supporting the back with the middle finger, so that the mouse is in a head-low tail-high position. In some embodiments, for mice, the method for fixing the mouse is a one-handed operation, comprising: below the right ear of the mouse, grasping the skin on the right side of the neck and back of the mouse with the thumb and index finger, pinching the tail with the ring finger and little finger, and simultaneously supporting the back with the middle finger, so that the mouse is in a head-low tail-high position; for rats, the method for fixing the mouse is a one-handed operation, comprising: using the index finger to drive the head up, below the right ear of the mouse, grasping the skin on the right side of the neck and back of the mouse with the thumb and index finger, and grasping the rest of the back skin with the other three fingers, and then pinching the tail with the ring finger and little finger, so that the mouse is in a head-low tail-high position.

[0039] One-handed operation can more flexibly control the animal fixation and injection process, allowing the operator to more accurately adjust the animal's position and angle to ensure the accuracy and stability of the injection. The mouse is in a head-low and tail-high position, which optimizes the operating space and safety of intraperitoneal injection through gravity. It is an important measure to reduce technical errors, improve experimental repeatability and protect animal welfare. Generally speaking, technicians in this field may have the ability to change the fixation method. The operation method is subjective and variable, and there is no standardized fixation method. The above fixation method can securely fix the mouse, reduce stress behavior, has practical operability, and can form a standardized process.

[0040] According to an embodiment of the present application, an auxiliary line is defined through the lower end of the ilium, the auxiliary line is perpendicular to the abdominal midline and has an intersection with the abdominal midline, and an injection area is formed with the midpoint of the line connecting the lower end of the ilium and the intersection as the center, and the injection position is located within the injection area.

[0041] In this article, the term "lower end of the ilium" is an anatomical position known in the art, which is where the knee joint can be fixed for easy observation during actual operation. Figure 1 Taking d in B as an example, the vertical line is the midline of the abdomen, the left end of the horizontal line is the lower part of the ilium, and the red circle area is the injection area.

[0042] Determining the above-mentioned injection location through morphological observation and anatomy can reduce the risk of cecal and small intestine puncture and avoid areas with thicker or dense capillaries in the abdomen.

[0043] According to the embodiments of the present application, for mice, the injection area is a circle with a radius of 0.1 cm to 0.3 cm, exemplified by 0.1 cm, 0.15 cm, 0.2 cm, 0.25 cm, and 0.3 cm. For rats, the injection area is a circle with a radius of 0.3 cm to 0.5 cm, exemplified by 0.3 cm, 0.35 cm, 0.4 cm, 0.45 cm, and 0.5 cm. This reduces the risk of puncturing the cecum and small intestine and avoids areas of the abdomen with thicker or denser capillaries.

[0044] According to an embodiment of the present application, after the injection is completed, a clean object is used to press the injection site and the needle is removed by rotation. For example, the clean object can be a cotton ball or cotton swab. By pressing the injection site and removing the needle by rotation, the injection solution is ensured to fully and accurately enter the abdominal cavity, while also minimizing extravasation and tissue damage.

[0045] Below in conjunction with embodiment, the scheme of the application will be explained. Those skilled in the art will appreciate that the following examples are merely for illustration of the application and should not be considered as limiting the scope of the application. Where specific techniques or conditions are not indicated in the examples, they are carried out according to the techniques or conditions described in the literature in this area or according to the product specifications. Reagents used or instruments not indicated by the manufacturer are conventional products that can be obtained commercially.

[0046] Example 1 Figure 1 A and Figure 1 B describes the intraperitoneal injection operation process of this application, which is as follows: 1. Fix the mouse with one hand, and use your thumb and index finger to grab the skin on the right side of the mouse's neck and back below the mouse's right ear and turn it over ( Figure 1 A (a) , pinch the tail with the ring finger and little finger, and gently lift the back with the middle finger. The mouse is now firmly fixed ( Figure 1 A (b)); The rat is fixed and its head is lifted by the index finger ( Figure 1 B (a)), the thumb and index finger grasped the right side of the rat's neck, and the other three fingers grasped the rest of the back skin, and then the tail was fixed between the ring finger and the little finger ( Figure 1 B (b)); The animal is in a head-low tail-high posture (c).

[0047] 2. Disinfect with 75% alcohol. The injection site is the midpoint between the lower end of the ilium and the midline of the abdomen (d). The radius of the injection area is 0.2 cm for mice and 0.4 cm for rats. The needle is inserted subcutaneously for about 0.5 cm (e). The needle is placed at a 45° angle to the skin surface, passing through the abdominal muscle and into the abdominal cavity (f). At this point, there is a sense of empty space. The depth of insertion into the abdominal cavity is controlled to be about 0.5 cm for mice and about 1 cm for rats. The needle is gently lifted toward the abdominal wall until it is parallel to the abdomen (g). The needle body is in contact with the abdominal wall, and the abdomen is visible. The needle is gently swung left and right with an amplitude of about 1 cm without any obstruction. After withdrawing the piston, the injection is performed. After the injection is completed, lightly press the injection site with a sterile dry cotton ball and rotate to remove the needle (h).

[0048] Comparative Example 1 Figure 1 C and Figure 1 D describes the traditional intraperitoneal injection procedure as follows: 1. Grab the skin on the back of the mouse's neck (a), and fix the mouse's tail. The mouse presses the tail with its ring finger ( Figure 1 C(b)), rats were fixed with the ring and little fingers ( Figure 1 D (b)), the animal is in a head-low tail-high posture (c).

[0049] 2. Wipe the abdominal injection area with a 75% alcohol cotton ball. Select the midpoint of the abdominal midline of the mouse or rat as the injection site (d). Pass the needle subcutaneously for about 0.5 cm (e). Make sure the needle is at a 45° angle to the skin surface, penetrate the abdominal muscle, and pierce the abdominal cavity (f). At this point, there will be a sense of empty space. Draw back the piston until there is no liquid and inject (g). After the injection is completed, gently press the injection site with a sterile dry cotton ball and rotate to remove the needle (h).

[0050] Comparative Example 2 The mouse intraperitoneal injection was performed according to the method of Example 1, except that the operation of step 1 was as follows: Grab the skin on the back of the mouse's neck (a), fix the mouse's tail, and press the tail with the mouse's ring finger ( Figure 1 C(b)), rats were fixed with the ring and little fingers ( Figure 1 D (b)), the animal is in a head-low tail-high posture (c).

[0051] Comparative Example 3 The mouse intraperitoneal injection was performed according to the method of Example 1, except that the operation of step 2 was as follows: Disinfect with 75% alcohol. The injection site is the midpoint of the abdominal midline (d). Pass the needle subcutaneously for about 0.5 cm (e). The needle is inserted through the abdominal muscle at a 45° angle to the skin surface into the abdominal cavity (f). At this time, there is a sense of empty space. The depth of insertion into the abdominal cavity is controlled at about 0.5 cm for mice and about 1 cm for rats. After withdrawing the piston and removing the liquid, the injection is performed. After the injection is completed, lightly press the injection site with a sterile dry cotton ball and rotate to remove the needle (h).

[0052] Test Example 1 1. Experimental Animals Male or female Sprague-Dawley rats (weighing 200–400 g) were obtained from the Guangdong Medical Laboratory Animal Center (license number: SCXK (Yue) 2022-0002). Male or female C57BL / 6 mice (20–30 g) were purchased from Hunan Slake Jingda Laboratory Animal Co., Ltd. (Hunan, China, license number: SCXK (Xiang) 2021-0002). This experiment was conducted at Guangdong East Sunshine Pharmaceutical Co., Ltd. (license number: SYXK (Yue) 2023-0135) and was approved by the Institutional Animal Care and Use Committee (no.: IAEC-K-240615-01).

[0053] 2. Experimental Methods India ink is often used as a marker or staining tool in experimental research. India ink is used as a tracer to visually evaluate the success rate and injection site of intraperitoneal injection, and to take photos and record the data.

[0054] The experiment was divided into four groups (using the methods of Comparative Examples 1-3 and Example 1), with 20 mice in each group. Seven batches of mice were performed, and each batch was operated simultaneously according to the injection method described for both groups. Seven operators were trained and qualified in standardized intraperitoneal injection procedures. The injection solution was India ink (Biotech Grade For Biological stain) diluted fivefold with normal saline (Chifeng Yuansheng Pharmaceutical Co., Ltd, Inner Mongolia, China). 0.3 mL was injected per mouse using a 1 mL syringe (0.3 mm; Jiangnan Medical Equipment Co., Ltd, Anhui, China). Following injection, mice were euthanized using a CO2 euthanasia device (flow range: 0-25 L / min; Siargo, Ltd, Sichuan, China). Dissections were performed by specialized personnel who were unaware of the injection method and operator information. The abdominal condition of the animals was observed, and the success rate, failure rate, and location of injection failure were recorded.

[0055] Data were analyzed using SPSS 25.0 software (SPSS Inc., Chicago, USA). Qualitative data were analyzed using the chi-square test. A p value < 0.05 was considered statistically significant. 3. Experimental Results Compared to the conventional method of Comparative Example 1, the method of Example 1 significantly improved the injection success rate in mice and rats (p < 0.01), increasing the success rate of intraperitoneal injection in mice from 83.57% to 100% (N = 140) and in rats from 83.57% to 99.29% (N = 140). The injection success rates of the methods of Comparative Examples 1-3 were all significantly lower than those of Example 1, while the results of Comparative Examples 1-3 were similar. The results and analysis are provided below using the conventional method of Comparative Example 1 as an example.

[0056] As shown in Tables 1 and 2, the failure rate of the method in Comparative Example 1, used in mice, was as high as 16.43%, primarily due to misinjection into the cecum (12.86%), followed by injection into the small intestine (3.57%). In contrast, the success rate of the method in Example 1 was as high as 100%. Compared with the method in Comparative Example 1, the success rate of the method in Example 1 was significantly improved (p < 0.01), while the incidence of injection into the cecum and small intestine was significantly reduced (p < 0.05).

[0057] In rat intraperitoneal injections (Tables 3 and 4), the method of Example 1 significantly improved the success rate (p < 0.01), reducing the failure rate to only 0.71% and reducing the probability of injection into the cecum (p < 0.05) and small intestine (p < 0.05). In contrast, the method of Comparative Example 1 resulted in a high failure rate of 16.43% in rats, primarily into the cecum (8.57%) and small intestine (7.86%).

[0058] Table 1 Success rate and injection failure sites of mice intraperitoneal injection

[0059] Table 2 Statistical analysis of intraperitoneal injection in mice

[0060] Note: superscript a or b Indicates that there are differences compared with traditional methods ( p<0.05 ), the same letters indicate no difference.

[0061] Table 3 Success rate and injection failure sites of rat intraperitoneal injection

[0062] Table 4 Statistical analysis of rat intraperitoneal injection

[0063] Note: superscript a or b Indicates that there are differences compared with traditional methods ( p<0.05 ), the same letters indicate no difference.

[0064] Test Example 2 1. Experimental Animals Male or female Sprague-Dawley rats (weighing 200–220 g) were purchased from the Guangdong Medical Laboratory Animal Center (license number: SCXK (Yue) 2022-0002). Male or female C57BL / 6 mice (20–22 g) were purchased from Hunan Slake Jingda Laboratory Animal Co., Ltd. (Hunan, China, license number: SCXK (Xiang) 2021-0002). This experiment was conducted at Guangdong East Sunshine Pharmaceutical Co., Ltd. (license number: SYXK (Yue) 2023-0135) and was approved by the Institutional Animal Care and Use Committee (No.: IAEC-K-240615-01).

[0065] 2. Experimental Methods H&E sections are often used to evaluate the structural morphology of microscopic histopathology. S&R staining can specifically display collagen fibers and further distinguish blood vessels from other tissues. These staining methods were used to evaluate the microscopic effects of injection procedures on abdominal skin tissue in mice and rats.

[0066] Three experimental groups were set up: a control group, a group using the conventional method of Comparative Example 1, and a group using the method of Example 1. The control group underwent no manipulation. The other two groups received intraperitoneal injections as described, with two injections at the same site. The animals were then euthanized with CO2. The abdominal area was hairless using a hair shaver (Ningbo Aiklipu Electric Appliance Co., Ltd, Zhejiang, China) and Veetin hair removal cream (ReckittBenckiser, France). 5 mm × 5 mm abdominal skin tissue was collected, centered at the injection site, and fixed in 10% formalin solution (Kangnaixin Biomedical Technology Co., Ltd, Guangdong). Sections were observed using transverse and longitudinal sections with a thickness of 3 μm. The skin tissue was stained with hematoxylin and eosin (H&E) and picrosirius red (S&R), respectively. Sections were observed and photographed under a fluorescence microscope (Nikon, Japan).

[0067] 3. Experimental Results Histopathological evaluation showed that the injection method of Example 1 effectively reduced mechanical irritation and bleeding risk in abdominal tissue compared to the traditional method of Comparative Example 1. H&E and S&R staining analysis showed that the skin integrity of animals in the Example 1 group was higher and bleeding was less.

[0068] like Figure 2 As shown in A and 2B, the skin morphology and structure of the mice in the control group were clear, with hair follicles, sebaceous glands, and sweat glands visible in the dermis, and fat lobules, blood vessels, loose connective tissue, and muscle layer arrangement visible in the subcutaneous tissue. The group using the method of Comparative Example 1 (traditional method) showed typical mechanical damage characteristics: cross-sections showed damage to the dermis and subcutaneous tissue, and rupture of subcutaneous blood vessels resulted in bleeding foci (red dotted area). The group using the method of Example 1 (improved method) showed significantly reduced vascular rupture and bleeding, with only local bleeding observed, and no accumulation of muscle layer tissue. The effects of Comparative Examples 2-3 were similar to those of Comparative Example 1. Taking Comparative Example 1 as an example, the skin morphology and structure ( Figure 2 and Figure 3 ).

[0069] Example 1 The method improves abdominal skin tissue bleeding and damage in rats ( Figure 3A, 3B, 3C). The abdominal skin tissue structure of the rats in the control group was clear; after injection, the rats in the conventional method group of Comparative Example 1 had a large number of ruptured blood vessels in the subcutaneous tissue. Blood cells were clearly visible in the red dotted area in the surrounding tissues, spreading to the epidermis and dermis, causing damage to the surrounding tissues. The red dotted area in the rats in the Example 1 method group was significantly reduced, causing less damage to the subcutaneous tissue area of ​​the rats.

[0070] Test Example 3 1. Experimental Animals Male or female Sprague-Dawley rats (weighing 200–220 g) were purchased from the Guangdong Medical Laboratory Animal Center (license number: SCXK (Yue) 2022-0002). Male or female C57BL / 6 mice (20–22 g) were purchased from Hunan Slake Jingda Laboratory Animal Co., Ltd. (Hunan, China, license number: SCXK (Xiang) 2021-0002). This experiment was conducted at Guangdong East Sunshine Pharmaceutical Co., Ltd. (license number: SYXK (Yue) 2023-0135) and was approved by the Institutional Animal Care and Use Committee (No.: IAEC-K-240615-01).

[0071] 2. Experimental Methods Animal stress is often used to assess the biological impact of experimental techniques. Acute (2-24 hours) and chronic stress (3-21 days) monitoring can reveal short-term effects and long-term cumulative damage, respectively. Blood biochemical indices directly reflect global or local metabolic changes in organs and tissues during animal stress. Studies have shown that stress can affect serum enzymes, such as alanine aminotransferase (ALT), which can undergo changes. When tissues are damaged or fatigued, creatine kinase (CK) levels in the blood increase, accompanied by imbalances in protein metabolism, such as changes in ALB (serum albumin). To evaluate the advantages of the proposed method, this study designed a 14-day intraperitoneal injection stress experiment and used a biochemical analyzer to quantitatively compare the effects of the two methods on blood biochemical stress indices.

[0072] The experiment was divided into five groups: a control group, groups treated with the methods of Comparative Examples 1-3, and a group treated with the method of Example 1. Each group consisted of four mice and three rats, and the experimental period lasted 14 days. The blank control group received no treatment. The groups treated with the methods of Comparative Examples 1-3 and Example 1 received intraperitoneal injections of normal saline (10 mL / kg) twice daily, and the animals were observed. Blood was collected on days 1 and 14 of the experiment, and creatine kinase (CK), alanine aminotransferase (ALT), and serum albumin (ALB) were measured using a Cobas c311 biochemical analyzer (Roche, Switzerland).

[0073] Data were analyzed using SPSS 25.0 software (SPSS Inc., Chicago, USA). Continuous data are expressed as mean ± standard deviation. One-way analysis of variance was performed, with homogeneity of variance tested using the LSD test; otherwise, the Dunnett T3 test was used. Nonparametric data were compared using the Mann-Whitney rank-sum test. P < 0.05 was considered statistically significant.

[0074] 3. Experimental Results After 14 consecutive days of intraperitoneal injection, the mice in the comparative example 1 conventional method had trauma points on their abdomens ( Figure 4 A), while no obvious damage was observed in mice using the method of Example 1 (improved method). In the control group of the traditional method of Example 1, the CK value increased significantly on the 1st and 14th days (p < 0.01), while that of the group using the method of Example 1 remained at a normal level ( Figure 4 B). Compared with the control group, the ALT level of the conventional method group of comparative example 1 was significantly reduced (p < 0.01), while the ALT level of the method group of example 1 was significantly restored to normal level compared with the conventional method group (p < 0.05) ( Figure 4 C). Compared with the control group, the ALB level of the traditional method in comparative example 1 was significantly reduced (p < 0.01), and the method in example 1 was consistent with that of the control group ( Figure 4 D), it can be seen that the method of Example 1 significantly reduced the impact of the traditional method on ALB levels (p < 0.05). The results of Comparative Examples 2-3 are similar to those of Comparative Example 1 and are not repeated here.

[0075] like Figure 5 As shown in A, trauma points appeared on the abdomen of rats in the comparative example 1 method, while no obvious damage was observed in the example 1 method (improved method). The CK values ​​of rats in the comparative example 1 traditional method group increased on the 1st and 14th days, while the CK values ​​of the example 1 method group were close to the control group level ( Figure 5 B). Compared with the control group, the ALT level of rats in the traditional method group of comparative example 1 was significantly reduced on the 14th day (p < 0.05), while the ALT level of rats in the method group of example 1 was significantly restored to normal level (p < 0.05) ( Figure 5 C). Compared with the control group, the ALB level of the rats in the conventional method group of comparative example 1 was significantly reduced on the 14th day (p < 0.01), while the ALB level of the rats in the method group of example 1 remained the same as that of the control group ( Figure 5 D). The results of Comparative Examples 2-3 are similar to those of Comparative Example 1 and are not described in detail.

[0076] This shows that the method of Example 1 of the present invention significantly reduces the changes in the biochemical indicators CK, ALT, and ALB of acute and chronic stress, and is consistent with the normal levels of the control group. Its effect is significantly better than the traditional method of Comparative Example 1 and the methods of Comparative Examples 2-4.

[0077] Although the embodiments of the present application have been shown and described above, it can be understood that the above embodiments are exemplary and cannot be understood as limitations on the present application. Ordinary technicians in this field can change, modify, replace and modify the above embodiments within the scope of the present application.

Claims

1. A method for determining the state of an injection needle entering the abdominal cavity of a rat or mouse, characterized in that: include: After the needle penetrates the abdominal muscle and enters the abdominal cavity, determine whether the needle has entered the abdominal cavity based on whether there is a feeling of empty space as follows: 1) If there is no empty feeling, the injection needle has not entered the abdominal cavity; 2) If there is a sense of missing the needle, the needle has entered the abdominal cavity and the following steps should be performed: Lift the injection needle toward the abdominal wall until the needle is parallel to the abdomen and in contact with the abdominal wall. Swing the needle left and right and withdraw the injection needle plunger. Determine the position of the injection needle in the abdominal cavity based on the following method: a) If there is no resistance when the needle is swung left and right, and there is no liquid in the needle cavity after the needle piston is withdrawn, it is determined that the needle has not contacted organs and / or blood vessels in the abdominal cavity; b) If there is a sense of resistance when the needle is swung left and right and / or liquid is present in the needle cavity after the needle piston is withdrawn, it is determined that the needle is in contact with organs and / or blood vessels in the abdominal cavity.

2. The method according to claim 1, wherein The amplitude of the left-right swing of the needle is 0.8 cm to 1.2 cm.

3. The method according to claim 1, wherein The injection needle penetrates the abdominal cavity to a depth of 0.4 cm to 1.2 cm.

4. The method according to claim 3, wherein The injection needle was inserted into the mouse abdominal cavity to a depth of 0.4 cm to 0.6 cm; Alternatively, the injection needle is inserted into the rat's abdominal cavity to a depth of 0.8 cm to 1.2 cm.

5. The method according to claim 1, wherein The needle body of the injection needle is 40° to 50° with the skin surface and penetrates the abdominal muscle into the abdominal cavity.

6. A method for intraperitoneal injection in rats or mice, characterized in that: include: (1) Immobilize rats or mice; (2) Insert the needle under the skin at the injection site, through the abdominal muscles and into the abdominal cavity; (3) Determining the state of the injection needle entering the abdominal cavity of a rat or mouse by the method for determining the state of the injection needle entering the abdominal cavity of a rat or mouse according to any one of claims 1 to 5; If it is determined that the injection needle has entered the abdominal cavity and has not come into contact with organs and blood vessels within the abdominal cavity, the injection solution is administered; If it is determined that the injection needle has not entered the abdominal cavity or has contacted the organs and / or blood vessels in the abdominal cavity, repeat steps (2) and (3) at least once until it is determined that the injection needle has entered the abdominal cavity and has not contacted the organs and blood vessels in the abdominal cavity, and the injection solution is injected; wherein, each time step (2) is repeated, the injection position is changed.

7. The method according to claim 6, characterized in that The method for fixing a rat or mouse comprises: grabbing the skin on the right side of the neck and back of the mouse; Optionally, the method for fixing the rat or mouse is a one-handed operation, comprising: grasping the skin on the right side of the mouse's neck and back with the thumb and index finger below the mouse's right ear, and pinching the tail with the ring finger and little finger, with the mouse in a head-down and tail-up position.

8. The method according to claim 7, characterized in that The method for fixing the mouse is a one-handed operation, comprising: grasping the skin on the right side of the mouse's neck and back with the thumb and index finger below the mouse's right ear, pinching the tail with the ring finger and little finger, and simultaneously propping up the back with the middle finger, with the mouse in a head-low tail-high position; Alternatively, the method for fixing the rat is a one-handed operation, including: using the index finger to drive the head up, grabbing the skin on the right side of the rat's neck and back with the thumb and index finger below the right ear of the rat, and grabbing the remaining back skin with the other three fingers, and then pinching the tail with the ring finger and little finger, so that the rat is in a head-low tail-high position.

9. The method according to claim 6, characterized in that An auxiliary line is defined through the lower end of the ilium, the auxiliary line being perpendicular to the midline of the abdomen and having an intersection with the midline of the abdomen, an injection area is formed with the midpoint of the line connecting the lower end of the ilium and the intersection as the center, and the injection position is located within the injection area; Optionally, for mice, the injection area is a circle with a radius of 0.1 cm to 0.3 cm; Alternatively, for rats, the injection area is a circle with a radius of 0.3 cm to 0.5 cm.

10. The method according to claim 6, wherein The depth of the subcutaneous passage is 0.4 cm to 0.6 cm; Optionally, after the injection is completed, press the injection site with a cleanser and twist to remove the needle.

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