A device for intraperitoneal injection of quantitative drug delivery in small animals and its method of use

Abstract

This invention relates to a small animal intraperitoneal injection quantitative drug delivery device and its method of use, belonging to the field of experimental animal drug delivery technology. The small animal intraperitoneal injection quantitative drug delivery device and its method of use include: a syringe and a support mechanism, the support mechanism being disposed at the bottom end of the syringe barrel; an injection mechanism, the injection mechanism including a mounting plate disposed on the surface of the syringe barrel, the inner top wall of the mounting plate being fixedly connected to a servo motor; through the structural design of the connecting component's internal toothed locking mechanism combined with spring pressure adjustment, automatic identification and interruption of puncture position errors are achieved, solving the problem that traditional injections cannot promptly identify puncture errors, easily leading to ineffective drug delivery and waste of experimental samples: when an incorrect puncture position causes an abnormally high injection resistance, the locking teeth are compressed by the spring, automatically releasing the lock with the connecting rod, separating the connecting rod from the connecting seat, immediately interrupting the injection, allowing the operator to adjust the puncture position in a timely manner.

Description

Technical Field

[0001] This invention relates to the field of experimental animal drug delivery technology, and in particular to a device for quantitative intraperitoneal injection of drugs for small animals and its method of use. Background Technology

[0002] With the development of perinatal medicine and neonatal intensive care technology, the survival rate of premature infants has significantly improved, but the incidence of premature brain injury has also increased. Premature brain injury is a key challenge in the field of perinatal medicine, and its incidence increases significantly with decreasing gestational age. Hyperoxia exposure is an independent risk factor for premature brain injury, and early hyperoxia exposure is significantly associated with the risk of brain injury in extremely premature infants. Studies have shown that the levels of blood oxygen saturation (SpO2) and inhaled oxygen concentration (FiO2) in extremely premature infants in the first 3 days after birth are closely related to later brain injury and neurodevelopmental outcomes. Even though hyperoxia therapy can save the lives of premature infants in the short term, the risk of long-term neurological sequelae is significantly increased, including cognitive impairment, motor dysfunction, and even death, placing a huge burden on individuals, families, and society. At present, the mechanism of hyperoxia-related premature brain injury is still unclear, and there is a lack of specific treatments. Therefore, elucidating the mechanism of hyperoxia-related premature brain injury and finding effective treatments are urgent hot issues that need to be addressed.

[0003] Pyroptosis is an inflammatory programmed cell death process primarily mediated by inflammasomes, which activates various cysteine-containing aspartate-specific proteinases (Caspases), leading to the cleavage and polymerization of Gasdermin D (GSDMD). The cleaved GSDM protein translocates to the cell membrane, causing cell swelling and death. Recent studies have found that knocking out GSDMD effectively alleviates hyperoxia-induced inflammatory damage, cell survival, and cell death in the hippocampus of preterm infants. Furthermore, the Caspase-1 inhibitor Ac-YVAD-CMK effectively reduces hyperoxia-induced brain injury in preterm infants. Therefore, [the text abruptly ends here, likely due to an incomplete translation or source material.] Increased neuronal pyroptosis may be one of the pathogenic mechanisms of hyperoxia-associated brain injury in preterm infants. In various neurological disease models such as hypoxic-ischemic encephalopathy and traumatic encephalopathy, neuronal pyroptosis is one of the main pathogenic mechanisms. Our previous studies have also confirmed that after hyperoxia, the expression of NOD-like receptor thermal protein domain associated protein 3 (NLRP3) and its downstream inflammatory factor (IL-1β) increases in the hippocampus of newborn mice. Therefore, we believe that pyroptosis may be one of the important mechanisms of hyperoxia-associated brain injury in preterm infants, and targeting neuronal pyroptosis may be an important entry point for the treatment of hyperoxia-associated brain injury in preterm infants.

[0004] Betaine (Bet) is an alkaloid, chemically named N,N,N-trimethylglycine, primarily derived from sugar beets, spinach, and seafood. It is transported to the brain via the Bet-GABA transporter to exert its effects. Numerous studies have shown that Bet supplementation has a protective effect against various neurological diseases, including Alzheimer's disease, Parkinson's disease, and schizophrenia. Previous research by our group further revealed that after Bet intervention, the expression of NLRP3 and its downstream inflammatory factors in the brains of newborn mice after hyperoxia was reduced, and neurobehavioral abnormalities were significantly improved. The study suggests that bet has a protective effect against hyperoxia-related brain injury in newborn mice, and its mechanism warrants further investigation. Bet is an important N6-methyladenosine (m6A) activator. N6-methyladenosine (m6A) is a dynamic and reversible modification of the methyl group at the 6th N atom of base A in eukaryotic mRNA, regulated synergistically by methyltransferases, demethylases, and corresponding readers, thereby affecting mRNA transcription, splicing, transport, and translation. Translation and degradation are among the most common post-transcriptional chemical epigenetic modifications of mRNA. The function of m6A-methylated RNA is mainly mediated by the m6A "reader." Pyroptosis may be one of the important mechanisms of hyperoxia-related brain injury in preterm infants, and NLRP3 expression is regulated by m6A. Betaine can attenuate NLRP3-mediated neuronal pyroptosis in an m6A-dependent manner and alleviate homocysteine-induced cognitive impairment. However, whether betaine exerts a neuroprotective effect by inhibiting neuronal pyroptosis through m6A modification to alleviate hyperoxia-related neonatal rat brain injury remains to be seen. The extent of the damage remains unclear. Therefore, we hypothesize that Bet can protect against hyperoxia-related neonatal brain injury by increasing m6A modification of NLRP3 mRNA and inhibiting neuronal pyroptosis. The specific "reader" involved in this process requires further investigation. This project will be the first to explore the mechanism of targeting neuronal pyroptosis with NLRP3 mRNA m6A modification in hyperoxia-related brain injury. Successful implementation of this project will provide new theoretical basis and targets for the treatment of hyperoxia-related brain injury and lay the foundation for the clinical translation of Bet therapy.

[0005] This study consists of in vivo and in vitro experiments. In the in vivo experiments, the following objectives were made: 1. To verify the protective effect of Bet on neuronal pyroptosis in animal models; 2. To verify the potential mechanism by which Bet inhibits neuronal pyroptosis by increasing m6A modification of NLRP3 mRNA in animal models. Therefore, P1WTC57BL / 6 mice were used to establish a pyroptosis model by continuous exposure to 80% oxygen concentration for 7 days. The intervention group was injected intraperitoneally with betaine (GN0134, GLPBIO) (20 mg / kg) once a day, while the control group was injected with an equal volume of saline. Mice were randomly divided into NO (noroxic group), HO (hyperoxic group), HO+Bet (hyperoxic + betaine group, i.e., intervention group), HO+solvent group (i.e., control group), and HO+Bet+MCC950 (NLRP3 inhibitor). Pathological changes in the brains of newborn mice before and after the intervention, the number and morphology of neurons, the expression of pyroptosis-related molecules, and neurobehavioral assessments were examined to verify the protective effect of Bet on neuronal pyroptosis in animal models. Current experimental procedures require periodic intraperitoneal injections, but the existing administration methods have the following main problems in practical use: 1. When administering intraperitoneal injections to small animals manually, the dosage relies entirely on the operator's experience. It is difficult to maintain a stable dosage during the injection process, which can easily lead to numerical deviations in the injection dosage. This makes it impossible to achieve precise quantitative injection, and the deviation in dosage will directly interfere with the test sample, thereby affecting the accuracy and reliability of the overall animal test results. 2. In intraperitoneal injection procedures in small animal pharmacology experiments, manual injection relies entirely on the operator's experience to judge the puncture site, which is prone to problems such as injection site deviation and misalignment. If the needle pierces the tissue (instead of the peritoneum), abnormal resistance will automatically stop the injection, resulting in drug administration failure. 3. In intraperitoneal injection procedures in small animal pharmacology experiments, due to the lack of specially adapted external support and positioning structures, the operator relies solely on holding the syringe to complete the puncture and injection. During the injection process, the operator's natural micro-movements can easily affect the position of the injection needle, causing not only puncture positioning errors but also difficulty in maintaining a stable injection depth and position. Summary of the Invention

[0006] Therefore, it is necessary to provide a quantitative drug delivery device for intraperitoneal injection in small animals and its usage method to address the problems of dosage deviation and puncture position deviation that are prone to occur during intraperitoneal injection in small animal experiments, making it difficult to ensure injection accuracy.

[0007] A device for intraperitoneal injection of quantitative drug delivery in small animals includes: a syringe and a support mechanism, wherein the support mechanism is disposed at the bottom end of the syringe barrel; An injection mechanism includes a mounting plate disposed on the surface of a syringe barrel, a servo motor fixedly connected to the inner top wall of the mounting plate, a lead screw fixedly connected to the output shaft of the servo motor, a moving block threadedly connected to the surface of the lead screw, and a connecting assembly disposed between the moving block and the top end of the syringe piston rod. The syringe barrel surface is provided with an adjustable limiting plate, the top of the limiting plate is provided with a pressure sensor, and the connecting assembly is provided with a trigger plate that works in conjunction with the limiting plate. The connecting assembly includes a first spring, a connecting rod fixedly connected to the movable block, and a connecting seat fixedly connected to the syringe piston rod. The trigger plate is fixedly connected to the connecting rod. The top end of the connecting rod is inserted into the bottom end of the connecting seat. The surface of the connecting rod is provided with fixing teeth. The first spring is located inside the connecting seat and one end is fixedly connected with a locking tooth. The locking tooth cooperates with the fixing tooth. The surface of the connecting seat is threaded with an adjusting bolt. The end of the adjusting bolt passes through the connecting seat and is rotatably connected to a movable plate. The end of the movable plate away from the adjusting bolt is fixedly connected to the other end of the first spring.

[0008] In one embodiment, the support mechanism includes a mounting base detachably mounted to the bottom end of the syringe barrel. A round tube is fixedly connected to the bottom end of the mounting base. An adjusting ring is provided on the surface of the round tube. A support rod is fixedly connected to the surface of the adjusting ring. A suction cup is provided at the end of the support rod away from the adjusting ring. An air tube is provided on the surface of the suction cup. A sealing plug is provided at the end of the air tube away from the suction cup.

[0009] In one embodiment, the connecting seat has a cavity, the end of the adjusting bolt, the first spring and the locking teeth are all located in the cavity, and the moving plate slides in the cavity.

[0010] In one embodiment, the syringe barrel handle is provided with two limiting rods, which are slidably connected to a limiting plate.

[0011] In one embodiment, the limiting plate is semi-circular, the inner edge of the limiting plate is provided with a locking plate, the outer wall of the limiting plate is threaded with a locking bolt, and the end of the locking bolt passes through the limiting plate and is rotatably connected to the locking plate.

[0012] In one embodiment, the inner edge of the limiting plate is provided with an arc-shaped cavity, the locking plate is located in the arc-shaped cavity, and a rubber layer is provided at one end of the locking plate near the surface of the syringe barrel.

[0013] In one embodiment, the syringe barrel surface is provided with scale lines, which are used in conjunction with a limiting plate.

[0014] In one embodiment, the movable block is U-shaped and is slidably connected to the mounting plate.

[0015] In one embodiment, the support mechanism further includes a pull ring and a second spring. The surface of the circular tube is provided with evenly distributed locking grooves. The adjusting ring is provided with an installation groove. One end of the second spring is fixedly connected to one end of the inner wall of the installation groove. One end of the second spring is fixedly connected to the pull ring. One end of the pull ring passes through the second spring, the installation groove and the adjusting ring in sequence and is engaged with the locking groove.

[0016] In one embodiment, three evenly distributed sliding rods are fixedly connected to the surface of the circular tube, and the inner wall of the adjusting ring is provided with a sliding groove that cooperates with the sliding rods.

[0017] A method for using a small animal intraperitoneal injection quantitative drug delivery device includes the following steps: Device positioning stage: When administering intraperitoneal injections to animals, first restrain the animal's limbs and expose the animal's abdomen. Then, use the support rod and suction cup in conjunction to contact the working surface. With the suction cup on the working surface, adjust the angle and position of the syringe, and lock the support rod and suction cup. During the puncture procedure, pulling the pull ring outward stretches the second spring, disengaging the pull ring from the locking groove. This pushes the device downward, causing the syringe, mounting base, and cylindrical tube to descend as a whole. The syringe needle then punctures the animal's abdomen until the puncture is complete. Injection volume adjustment phase: When administering intraperitoneal injection to an animal, rotate the locking bolt to disengage the locking plate from the syringe barrel surface, slide the limiting plate to the metering position, and then use the locking bolt and locking plate together to lock the limiting plate. Injection phase: The servo motor needs to be started first. The servo motor drives the lead screw to rotate. The moving block will not rotate under the action of the limit. Under the action of the thread, it will move in the direction of the mounting plate. The moving block will use the connecting component to pull the piston rod of the syringe to achieve a stable intraperitoneal injection operation for the animal until the trigger plate moves down until the trigger plate contacts the limit plate. The pressure sensor will be squeezed and generate a signal to stop the servo motor and complete the intraperitoneal injection operation for the animal. Automatic detachment phase after injection: When the syringe needle tip deviates from the puncture position, the injection pressure increases. The moving block drives the connecting rod downward, and the downward pressure of the connecting seat and the syringe piston rod increases. The moving block and connecting rod move according to the normal pressure value. As the pressure value increases, the fixed teeth move synchronously with the connecting rod and squeeze the locking teeth. The locking teeth squeeze the first spring and move towards the cavity, automatically releasing the lock and separating the connecting rod and the connecting seat, thereby interrupting the injection operation on the animal.

[0018] Debugging phase of the connection components: Rotate the adjusting bolt to adjust the position of the moving plate in the cavity, thereby adjusting the compression distance of the first spring and the pressure value at which the locking tooth disengages from the fixed tooth. This can be adjusted according to the actual situation of intraperitoneal injection in animals. Beneficial effects

[0019] 1. The core innovation of this invention lies in its integrated structural improvement addressing the three major pain points of existing manual intraperitoneal injections. Through a combination of a servo motor, lead screw, limiting plate, and pressure sensor, fully automated and precise control of the drug dosage is achieved. Compared to traditional manual injection, which relies entirely on operator experience, this invention can flexibly adjust the position of the limiting plate according to a preset dosage volume and automatically stop after injection. This eliminates dosage errors inherent in manual injections at the structural source, significantly improving the dosage accuracy of intraperitoneal administration in small animals. It effectively avoids interference from dosage deviations in experimental results, fully ensuring the reliability of animal experimental data. Furthermore, the adjustment operation is simple and convenient, quickly adapting to different dosage requirements in different experiments. Its flexibility and applicability far surpass traditional manual injection. This method, through the structural design of the connecting component's internal toothed locking and spring pressure adjustment, achieves automatic identification and interruption of puncture position errors. It solves the problem that traditional injection cannot identify puncture errors in time, which can easily lead to ineffective drug administration and waste of experimental samples. When the puncture position error causes an abnormal increase in injection resistance, the locking tooth is compressed by the spring, automatically releasing the lock with the connecting rod, separating the connecting rod from the connecting seat, and immediately interrupting the injection. This allows the operator to adjust the puncture position in time. At the same time, the spring preload can be adjusted by adjusting the bolt to change the resistance threshold of the unlocking trigger. It can flexibly adapt to the judgment requirements of different experiments, further improving the accuracy of puncture error identification, reducing the dependence of drug administration on the operator's experience, and effectively improving the consistency and success rate of batch drug administration. 2. This invention solves the problems of unstable positioning and uncontrollable puncture depth in traditional manual puncture by using an adjustable support mechanism with suction cups at the bottom of the syringe. Three support rods with suction cups can stably fix the entire device to the operating surface under negative pressure. After pre-adjusting and locking the puncture angle and direction, it can completely avoid needle deviation caused by slight hand movements of the operator. Moreover, the adjustment ring can slide and lock along the round tube, which can accurately control the needle extension length and puncture depth, adapting to the puncture needs of small animals of different sizes. It avoids the needle from penetrating too deeply and damaging the small animal's organs, and also avoids the needle from penetrating too shallowly and remaining in the subcutaneous tissue, resulting in drug administration failure. It greatly improves the accuracy and stability of puncture positioning and reduces the probability of drug administration failure. At the same time, the suction cups can be released simply by pulling out the sealing plug and venting, which is convenient and adapts to the rapid operation needs of batch injection of small animals. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0021] Figure 1 This is a schematic diagram of the overall structure of the present invention; Figure 2 This is an exploded view of the present invention; Figure 3 This is a schematic diagram of the injection mechanism of the present invention; Figure 4 This is a schematic diagram of the structure of the connection component of the present invention; Figure 5 This is an exploded view of the limiting plate and limiting rod of the present invention; Figure 6 This is a schematic diagram of the support mechanism of the present invention; Figure 7 This is a schematic diagram of the support rod and suction cup of the present invention; Figure 8 This is an exploded view of the circular tube and adjusting ring of the present invention.

[0022] Figure label: 100. Syringe; 110. Scale line; 200. Injection mechanism; 210. Mounting plate; 211. Servo motor; 212. Lead screw; 213. Moving block; 214. Trigger plate; 215. Limiting plate; 2151. Locking bolt; 2152. Locking plate; 216. Limiting rod; 217. Pressure sensor; 220. Connecting assembly; 221. Connecting seat; 222. Connecting rod; 223. Fixing tooth; 224. Locking tooth; 225. Adjusting bolt; 226. Moving plate; 227. First spring; 300. Support mechanism; 310. Mounting seat; 311. Round tube; 312. Adjusting ring; 313. Support rod; 314. Suction cup; 3141. Air tube; 3142. Sealing plug; 315. Locking groove; 320. Mounting groove; 321. Pulling ring; 322. Second spring. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] It should be noted that when a component is referred to as being "fixed to" or "set on" another component, it can be directly on the other component or there may be an intermediate component. When a component is considered to be "connected to" another component, it can be directly connected to the other component or there may be an intermediate component present. The terms "vertical," "horizontal," "upper," "lower," "left," "right," and similar expressions used in this specification are for illustrative purposes only and do not represent the only possible implementation.

[0025] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0026] In this invention, unless otherwise explicitly specified and limited, "above" or "below" the second feature can mean that the first feature is in direct contact with the second feature, or that the first feature and the second feature are in indirect contact through an intermediate medium. Furthermore, "above," "over," and "on top" the second feature can mean that the first feature is directly above or diagonally above the second feature, or simply indicates that the first feature is at a higher horizontal level than the second feature. "Below," "below," and "under" the second feature can mean that the first feature is directly below or diagonally below the second feature, or simply indicates that the first feature is at a lower horizontal level than the second feature.

[0027] Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used in this specification includes any and all combinations of one or more of the associated listed items.

[0028] The following is combined with Figures 1-8 This invention describes the small animal intraperitoneal injection quantitative drug delivery device and its method of use.

[0029] A small animal intraperitoneal injection quantitative drug delivery device includes: a syringe 100 and a support mechanism 300, wherein the support mechanism 300 is disposed at the bottom end of the syringe barrel of the syringe 100. The injection mechanism 200 includes a mounting plate 210 disposed on the surface of the syringe barrel of the syringe 100. A servo motor 211 is fixedly connected to the inner top wall of the mounting plate 210. A lead screw 212 is fixedly connected to the output shaft of the servo motor 211. A moving block 213 is threadedly connected to the surface of the lead screw 212. A connecting assembly 220 is disposed between the moving block 213 and the top end of the piston rod of the syringe 100. The syringe 100 has an adjustable limiting plate 215 on its surface, a pressure sensor 217 on its top end, and a trigger plate 214 on its connecting assembly 220 that works in conjunction with the limiting plate 215. The connecting assembly 220 includes a first spring 227, a connecting rod 222 fixedly connected to the moving block 213, and a connecting seat 221 fixedly connected to the piston rod of the syringe 100. The trigger plate 214 is fixedly connected to the connecting rod 222. The top end of the connecting rod 222 is inserted into the bottom end of the connecting seat 221. The surface of the connecting rod 222 is provided with fixing teeth 223. The first spring 227 is located inside the connecting seat 221 and one end is fixedly connected with a locking tooth 224. The locking tooth 224 and the fixing tooth 223 are used in conjunction. The surface of the connecting seat 221 is threaded with an adjusting bolt 225. The end of the adjusting bolt 225 passes through the connecting seat 221 and is rotatably connected to a movable plate 226. The end of the movable plate 226 away from the adjusting bolt 225 is fixedly connected to the other end of the first spring 227.

[0030] In this embodiment, the injection phase: First, the servo motor 211 needs to be started. The servo motor 211 drives the lead screw 212 to rotate. The moving block 213 will not rotate under the action of the limit. Under the action of the thread, it will move in the direction of the mounting plate 210. The moving block 213 will cooperate with the connecting component 220 to pull the piston rod of the syringe 100 to achieve a stable intraperitoneal injection operation for animals until the trigger plate 214 moves downward until the trigger plate 214 contacts the limit plate 215. The pressure sensor 217 will be squeezed and generate a signal to stop the servo motor 211 and complete the intraperitoneal injection operation for animals. When the needle tip of syringe 100 deviates from the puncture position, the injection pressure increases. The moving block 213 will drive the connecting rod 222 to move downward. The pressure value of the connecting seat 221 and the piston rod of syringe 100 moving downward will increase. The moving block 213 and the connecting rod 222 will move according to the normal pressure value. As the pressure value increases, the fixed tooth 223 will move synchronously with the connecting rod 222 and squeeze the locking tooth 224. The locking tooth 224 will squeeze the first spring 227 and move towards the cavity, realizing automatic unlocking and separating the connecting rod 222 and the connecting seat 221, thereby interrupting the animal injection operation. It should be noted that rotating the adjusting bolt 225 allows for adjustment of the position of the moving plate 226 within the cavity, thereby adjusting the compression distance of the first spring 227 and the pressure value at which the locking tooth 224 disengages from the fixed tooth 223. This adjustment can be made according to the actual situation of intraperitoneal injection in animals.

[0031] It should be noted that the model of servo motor 211 is JGA12-N20 miniature geared servo motor; The pressure sensor 217 is a high-precision thin-film pressure sensor with model FSS1500NST. Its advantages include: small size that can be installed on the top of the limit plate 215; sensing range of 0~10N; high resolution; and the ability to accurately detect the squeezing trigger pressure of the trigger plate 214, meeting the pressure triggering requirements for small animal injections. It should be noted that when the pressure sensor 217 is squeezed, the pressure sensor 217 will convert the received pressure signal into an analog electrical signal output. The electrical signal is transmitted to the control circuit board (preset PLC control program) of the equipment. After the control board receives the signal corresponding to the pressure threshold, it will immediately output a stop command. The command is transmitted to the driver of the servo motor 211 through the terminal block. The driver directly cuts off the power supply output of the servo motor 211, and the servo motor 211 immediately stops running, completing the automatic stop of quantitative injection and accurately controlling the dosage.

[0032] like Figure 1 , Figure 2 , Figure 6 , Figure 7 and Figure 8 As shown, the support mechanism 300 includes a mounting base 310 detachably mounted on the bottom end of the syringe barrel of the syringe 100. A round tube 311 is fixedly connected to the bottom end of the mounting base 310. An adjusting ring 312 is provided on the surface of the round tube 311. A support rod 313 is fixedly connected to the surface of the adjusting ring 312. A suction cup 314 is provided at the end of the support rod 313 away from the adjusting ring 312. An air tube 3141 is provided on the surface of the suction cup 314. A sealing plug 3142 is provided at the end of the air tube 3141 away from the suction cup 314.

[0033] The support mechanism 300 also includes a pull ring 321 and a second spring 322. The surface of the round tube 311 is provided with evenly distributed locking grooves 315. The adjusting ring 312 is provided with an installation groove 320. One end of the second spring 322 is fixedly connected to one end of the inner wall of the installation groove 320. One end of the second spring 322 is fixedly connected to the pull ring 321. One end of the pull ring 321 passes through the second spring 322, the installation groove 320 and the adjusting ring 312 in sequence and is engaged with the locking groove 315.

[0034] Three evenly distributed sliding rods are fixedly connected to the surface of the round tube 311, and the inner wall of the adjusting ring 312 is provided with a sliding groove that cooperates with the sliding rods.

[0035] In this embodiment, when performing intraperitoneal injection on an animal, the animal's limbs are first restrained, and the animal's abdomen is exposed. Then, the support rod 313 and suction cup 314 are used to contact the working surface. The suction cup 314 is on the working surface. The angle of the syringe 100 is adjusted and the position is adjusted. The support rod 313 and suction cup 314 are then locked. During the puncture process, the pull ring 321 is pushed outward, the second spring 322 is stretched by force, the pull ring 321 disengages from the locking groove 315, and the device is pushed downward. The syringe 100, the mounting base 310 and the round tube 311 are pushed downward as a whole, and the injection needle of the syringe 100 punctures the abdomen of the animal until the puncture is in place.

[0036] It should be noted that when it is necessary to release the support mechanism 300, first separate the suction cup 314 from the working surface, remove the sealing plug 3142, and external gas will enter between the suction cup 314 and the working surface through the air pipe 3141. The suction cup 314 will then separate from the working surface, and the locking between the device and the working surface can be quickly released.

[0037] like Figure 1 , Figure 2 , Figure 3 and Figure 4 As shown, a cavity is provided inside the connecting seat 221, the end of the adjusting bolt 225, the first spring 227 and the locking tooth 224 are all located in the cavity, and the moving plate 226 slides in the cavity.

[0038] Two limiting rods 216 are provided at the syringe handle of syringe 100, and the limiting rods 216 are slidably connected to the limiting plate 215.

[0039] like Figure 1 , Figure 2 , Figure 3 , Figure 4 and Figure 5 As shown, the limiting plate 215 is semi-circular, with a locking plate 2152 on its inner edge. A locking bolt 2151 is threaded onto the outer wall of the limiting plate 215, and the end of the locking bolt 2151 passes through the limiting plate 215 and is rotatably connected to the locking plate 2152. Before intraperitoneal injection in an animal, the required injection volume is adjusted in advance. The locking bolt 2151 is rotated to disengage the locking plate 2152 from the syringe barrel surface of the syringe 100. The limiting plate 215 is then slid to the metering position, and the locking bolt 2151 and locking plate 2152 are used to lock the limiting plate 215.

[0040] The inner edge of the limiting plate 215 has an arc-shaped cavity, and the locking plate 2152 is located inside the arc-shaped cavity. The end of the locking plate 2152 near the surface of the syringe 100 has a rubber layer. The arc-shaped cavity can match the locking plate 2152 and limit the locking plate 2152 to prevent the locking plate 2152 from deviating from its path during movement.

[0041] The syringe 100 has a scale line 110 on its syringe barrel surface, which works in conjunction with the limiting plate 215. The scale line 110 provides positional data support for the adjustment of the limiting plate 215, making the adjustment position more precise.

[0042] The movable block 213 is U-shaped and is slidably connected to the mounting plate 210. When the U-shaped movable block 213 moves, it will conform to the mounting plate 210, thereby limiting the path and ensuring that the movable block 213 and the connecting rod 222 will not deviate during the movement.

[0043] It should be noted that the accompanying research project for this device is a study on the role and mechanism of betaine in protecting against hyperoxia-related brain injury in newborn mice by regulating RNA methylation (m6A), including the following steps; I. Main Research Content: This study is divided into in vivo experiments and in vitro experiments.

[0044] In vivo experiments (1) Verify the protective effect of Bet on neuronal pyroptosis in animal models. P1WTC57BL / 6 mice were used to establish a pyroptosis model by continuous exposure to 80% oxygen concentration for 7 days. The intervention group received intraperitoneal injections of betaine (GN0134, GLPBIO) (20 mg / kg) once a day, while the control group received an equal volume of saline. Mice were randomly divided into NO (noroxic group), HO (hyperoxic group), HO+Bet (hyperoxic + betaine group, i.e., intervention group), HO+solvent group (i.e., control group), and HO+Bet+MCC950 (NLRP3 inhibitor). Pathological changes in the brains of newborn mice before and after intervention, the number and morphology of neurons, the expression of pyroptosis-related molecules, and neurobehavioral assessments were examined to verify the protective effect of betaine on neuronal pyroptosis in animal models.

[0045] (2) To verify the potential mechanism by which Bet inhibits neuronal pyroptosis by increasing m6A modification of NLRP3 mRNA in animal models. Modeling and betaine intervention were the same as before. Mice were randomly divided into NO (noroxic group), HO (hyperoxic group), HO+Bet+blank control vector group, HO+Bet+overexpression group, HO+Bet+silence group, and HO+Bet+overexpression+MCC950 group. EpiQuik m6A RNA methylation quantitative detection kit was used to compare the total RNA m6A level in tissues, and RNA methylation m6A immunoprecipitation (MeRIP-qPCR) was used to compare the NLRP3 mRNA m6A level in tissues. SRAMP, CVm6A, and other websites were used to predict the m6A-modified regions in the NLRP3 mRNA sequence, and the corresponding reading frames were screened experimentally. Lentiviral vectors for overexpression and silencing of the corresponding reading frames were constructed. The overexpression and silencing lentiviral vectors and their blank control vectors were used to achieve local brain transfection via lateral ventricle injection of lentivirus. The expression of NLRP3 and IL-1β was detected to explore the potential mechanism by which Bet inhibits neuronal pyroptosis by increasing m6A modification of NLRP3 mRNA.

[0046] In vitro experiments (1) Verify the protective effect of Bet on neuronal pyroptosis in a cell model. SH-SY5Y cells were cultured and subjected to 80% oxygen for 24 hours to induce a pyroptosis model. Cells were then co-cultured with Bet (10 mM) or MCC950 (10 μM) for 4 hours. Cells were randomly divided into NO (noroxic group), HO (hyperoxic group), HO+Bet (hyperoxic + betaine group), HO+DMSO group, and HO+Bet+MCC950 (NLRP3 inhibitor). Cell viability, apoptosis rate, number and morphology of neurons, and expression of pyroptosis-related molecules were measured to verify the protective effect of Bet on neuronal pyroptosis in the cell model.

[0047] (2) Explore the potential mechanism by which Bet inhibits neuronal pyroptosis by increasing m6A modification of NLRP3 mRNA in cell models. Plasmids containing the corresponding reading frames obtained from overexpression or silencing of m6A were used for modeling and betaine intervention as before. Patients were randomly divided into four groups: NO (noroxic group), HO (hyperoxic group), HO+Bet+blank control vector group, HO+Bet+overexpression group, HO+Bet+silencing group, and HO+Bet+overexpression+MCC950 group. Cell viability, apoptosis rate, number and morphology of neurons, and expression of pyroptosis-related molecules were measured to explore the potential mechanism by which Bet inhibits neuronal pyroptosis by increasing m6A modification of NLRP3 mRNA.

[0048] II. Research Methods, Techniques, and Technical Approaches: Research Methods and Techniques Validating the protective effect of Bet against neuronal pyroptosis in animal models. P1WTC57BL / 6 mice were used to establish the hyperoxia model by continuous exposure to 80% oxygen concentration for 7 days. The oxygen concentration was set at 80%, and the oxygen concentration in the hyperoxia chamber was monitored during exposure, with actual measured values ​​controlled to fluctuate between 80-85%. Sodium lime was placed in the hyperoxia chamber to adsorb carbon dioxide, and anhydrous calcium chloride was used to adsorb moisture, with carbon dioxide concentration monitored to be <0.5% and humidity maintained at 55-65%. Newborn mice were placed in the hyperoxia chamber with their mothers, and the mothers were rotated daily to prevent hyperoxia injury in the mothers. After model establishment, the mice were randomly divided into NO (noroxic group), HO (hyperoxia group), HO+Bet (hyperoxia + betaine group, i.e., intervention group), HO+solvent group (i.e., control group), and HO+Bet+MCC950 (NLRP3 inhibitor). The intervention group received intraperitoneal injections of betaine (GN0134, GLPBIO) (20 mg / kg) once daily, while the control group received an equal volume of physiological saline. The mice were then housed in the air for 28 days. At P8, partial neonatal mouse brain tissue sections were dehydrated, trimmed, embedded, sectioned, stained, and mounted. Images were acquired using an automated confocal scanner, and Nissl and HE staining were performed to observe pathological changes in the neonatal mouse brain before and after intervention. After frozen section preparation, immunofluorescence or immunohistochemistry was performed to detect the number and morphology of neurons and the expression of pyroptosis-related molecules in the brains of mice in the above groups, following steps including rewarming, permeabilization, blocking, primary antibody incubation, secondary antibody incubation, and DAPI nuclear staining. Western blotting was used to detect neurons and pyroptosis-related molecules. Finally, at P28, the Morris test was performed on neonatal mice to conduct neurobehavioral assessments to observe their learning and memory.

[0049] The potential mechanism by which Bet inhibits neuronal pyroptosis by increasing m6A modification of NLRP3 mRNA was validated in animal models. Modeling and betaine intervention were the same as before. Mice were randomly divided into NO (noroxic group), HO (hyperoxic group), HO+Bet+blank control vector group, HO+Bet+overexpression group, HO+Bet+silence group, and HO+Bet+overexpression+MCC950 group. EpiQuik m6A RNA methylation quantitative detection kit (colorimetric method) was used to compare the total RNA m6A level in tissues, and MeRIP-qPCR was used to compare the NLRP3 mRNA m6A level in tissues. qRT-PCR and WB were used to compare the expression of GSDMD and IL-1β. SRAMP, CVm6A, and other websites were used to predict the m6A-modified regions in the NLRP3 mRNA sequence. After experimental verification, the corresponding reading frames obtained from m6A were screened, and lentiviral vectors for overexpression and silencing of these reading frames were constructed. The overexpression and silencing lentiviral vectors and their blank control vectors were used to achieve local brain transfection via lateral ventricle injection of lentivirus. The levels of pyroptosis-related molecules were detected to explore the potential mechanism by which Bet inhibits neuronal pyroptosis by increasing m6A modification of NLRP3 mRNA.

[0050] Validating the protective effect of Bet against neuronal pyroptosis in cell models. SH-SY5Y cells were cultured and subjected to 80% oxygen for 24 hours to establish a cell model. They were then co-cultured with either Bet or MCC950 (10 μM) for 4 hours. Cells were randomly assigned to NO (noroxic group), HO (hyperoxic group), HO+Bet (hyperoxic + betaine group), HO+DMSO group, and HO+Bet+MCC950 (NLRP3 inhibitor). Cell viability was assessed using CCK8 assay, apoptosis rate was detected by flow cytometry, the number and morphology of neurons were detected by immunofluorescence or immunohistochemistry, and the expression of pyroptosis-related molecules was detected by Western blotting.

[0051] Exploring the potential mechanism by which Bet inhibits neuronal pyroptosis by increasing m6A modification of NLRP3 mRNA in cellular models. Modeling and betaine intervention were the same as before. Cells were randomly divided into four groups: NO (noroxic group), HO (hyperoxic group), HO+Bet+ blank control vector group, HO+Bet+ overexpression group, HO+Bet+ silencing group, and HO+Bet+ overexpression + MCC950 group. Total RNA m6A and NLRP3 mRNA m6A were compared in each group using colorimetric assays and MeRIP-qPCR. RNA-pulldown and RIP-qPCR (RNA-binding protein immunoprecipitation) assays were performed on each group. RNA-pulldown: NLRP3 mRNA was labeled with a biotinylated probe and bound to magnetic beads to form a magnetic bead-mRNA mixture. The mixture was incubated with cell lysis buffer to form a magnetic bead-mRNA-protein complex. After separation, purification, and elution of the complex, Western blotting was used to detect the expression of the selected reading frames to verify whether the reading frame bound to NLRP3 mRNA. RIP-qPCR: Cells were lysed, and co-precipitated mRNA was isolated and purified. qRT-PCR was used to detect the enrichment of NLRP3 in the immunoprecipitate of the selected reading frames, verifying whether the reading frames directly bind to NLRP3 mRNA. Cell viability, apoptosis rate, and the number and morphology of neurons were further measured to explore the potential mechanism by which Bet inhibits neuronal pyroptosis by increasing m6A modification of NLRP3 mRNA.

[0052] III. Key issues to be addressed: Hyperoxia-associated brain injury (HBI) can lead to disability and death in children, and its mechanisms remain unclear and specific treatments are lacking. This study aims to address the following scientific questions: whether betaine has a protective effect against hyperoxia-associated brain injury in newborn mice, and to explore its mechanism by targeting neuronal pyroptosis through m6A modification of NLRP3 mRNA. This will further elucidate the potential mechanism by which betaine inhibits neuronal pyroptosis by increasing m6A modification of NLRP3 mRNA, providing new theoretical evidence and therapeutic targets for hyperoxia-associated brain injury in newborn mice, and laying the foundation for the clinical translation of betaine treatment for hyperoxia-associated brain injury in preterm infants.

[0053] This device is a quantitative drug delivery system for intraperitoneal injection in small animals, along with its usage method. It enables quantitative injection operations in the aforementioned animal experiments, thereby facilitating the normal progress of the research project.

[0054] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0055] The above-described embodiments are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. A device for intraperitoneal injection of quantitative drug delivery in small animals, characterized in that, include: A syringe (100) and a support mechanism (300), wherein the support mechanism (300) is disposed at the bottom end of the syringe (100) syringe barrel; An injection mechanism (200) includes a mounting plate (210) disposed on the surface of the syringe (100) syringe barrel. A servo motor (211) is fixedly connected to the inner top wall of the mounting plate (210). A lead screw (212) is fixedly connected to the output shaft of the servo motor (211). A moving block (213) is threadedly connected to the surface of the lead screw (212). A connecting assembly (220) is disposed between the moving block (213) and the top end of the piston rod of the syringe (100). The syringe (100) has an adjustable limiting plate (215) on its syringe surface. A pressure sensor (217) is provided at the top of the limiting plate (215). A trigger plate (214) is provided on the connecting assembly (220) to cooperate with the limiting plate (215). The connecting assembly (220) includes a first spring (227), a connecting rod (222) fixedly connected to the moving block (213), and a connecting seat (221) fixedly connected to the piston rod of the syringe (100). The trigger plate (214) is fixedly connected to the connecting rod (222). The top end of the connecting rod (222) is inserted into the bottom end of the connecting seat (221). The surface of the connecting rod (222) is provided with fixing teeth (223). The first spring (227) is located inside the connecting seat (221) and one end is fixedly connected with a locking tooth (224). The locking tooth (224) and the fixing tooth (223) are used in cooperation. The surface of the connecting seat (221) is threaded with an adjusting bolt (225). The end of the adjusting bolt (225) passes through the connecting seat (221) and is rotatably connected to a moving plate (226). The end of the moving plate (226) away from the adjusting bolt (225) is fixedly connected to the other end of the first spring (227).

2. The small animal intraperitoneal injection quantitative drug delivery device according to claim 1, characterized in that, The support mechanism (300) includes a mounting base (310) detachably mounted on the bottom end of the syringe (100) syringe barrel. A round tube (311) is fixedly connected to the bottom end of the mounting base (310). An adjusting ring (312) is provided on the surface of the round tube (311). A support rod (313) is fixedly connected to the surface of the adjusting ring (312). A suction cup (314) is provided at the end of the support rod (313) away from the adjusting ring (312). An air tube (3141) is provided on the surface of the suction cup (314). A sealing plug (3142) is provided at the end of the air tube (3141) away from the suction cup (314).

3. The small animal intraperitoneal injection quantitative drug delivery device according to claim 1, characterized in that, The connecting seat (221) has a cavity, the end of the adjusting bolt (225), the first spring (227) and the locking tooth (224) are all located in the cavity, and the moving plate (226) slides in the cavity.

4. The small animal intraperitoneal injection quantitative drug delivery device according to claim 1, characterized in that, The syringe (100) has two limiting rods (216) at the syringe handle, and the limiting rods (216) are slidably connected to the limiting plate (215).

5. The small animal intraperitoneal injection quantitative drug delivery device according to claim 1, characterized in that, The limiting plate (215) is semi-circular. A locking plate (2152) is provided on the inner edge of the limiting plate (215). A locking bolt (2151) is threadedly connected to the outer wall of the limiting plate (215). The end of the locking bolt (2151) passes through the limiting plate (215) and is rotatably connected to the locking plate (2152).

6. The small animal intraperitoneal injection quantitative drug delivery device according to claim 5, characterized in that, The inner edge of the limiting plate (215) is provided with an arc-shaped cavity, and the locking plate (2152) is located in the arc-shaped cavity. The end of the locking plate (2152) near the surface of the syringe (100) is provided with a rubber layer.

7. The small animal intraperitoneal injection quantitative drug delivery device according to claim 1, characterized in that, The syringe (100) has a scale line (110) on its syringe surface, which is used in conjunction with the limiting plate (215).

8. The small animal intraperitoneal injection quantitative drug delivery device according to claim 1, characterized in that, The movable block (213) is U-shaped and is slidably connected to the mounting plate (210).

9. The small animal intraperitoneal injection quantitative drug delivery device according to claim 2, characterized in that, The support mechanism (300) further includes a pull ring (321) and a second spring (322). The surface of the round tube (311) is provided with evenly distributed locking grooves (315). The adjusting ring (312) is provided with an installation groove (320). One end of the second spring (322) is fixedly connected to one end of the inner wall of the installation groove (320). One end of the second spring (322) is fixedly connected to the pull ring (321). One end of the pull ring (321) passes through the second spring (322), the installation groove (320) and the adjusting ring (312) in sequence and is engaged with the locking groove (315). Three evenly distributed sliding rods are fixedly connected to the surface of the round tube (311). The inner wall of the adjusting ring (312) is provided with a sliding groove that cooperates with the sliding rods.

10. A method of using a small animal intraperitoneal injection quantitative drug delivery device, characterized in that, Includes the following steps: Device positioning stage: When performing intraperitoneal injection on an animal, first restrain the animal's limbs and expose the animal's abdomen. Then, use the support rod (313) and suction cup (314) to contact the working surface. With the suction cup (314) on the working surface, adjust the angle and position of the syringe (100), and lock the support rod (313) and suction cup (314). During the puncture process, the pull ring (321) is pushed outward, the second spring (322) is stretched by force, the pull ring (321) disengages from the locking groove (315), and the device is pushed downward. The syringe (100), the mounting base (310) and the round tube (311) are pushed downward as a whole, and the injection needle of the syringe (100) punctures the abdomen of the animal until the puncture is in place; Injection volume adjustment phase: When administering an intraperitoneal injection to an animal, rotate the locking bolt (2151) to disengage the locking plate (2152) from the syringe (100) surface, slide the limiting plate (215) to the quantitative position, and then use the locking bolt (2151) and the locking plate (2152) to lock the limiting plate (215). Injection phase: The servo motor (211) needs to be started first. The servo motor (211) drives the lead screw (212) to rotate. The moving block (213) will not rotate under the action of the limit. Under the action of the thread, it will move in the direction of the mounting plate (210). The moving block (213) will use the connecting component (220) to pull the piston rod of the syringe (100) to achieve a stable intraperitoneal injection operation for animals until the trigger plate (214) moves down until the trigger plate (214) contacts the limit plate (215). The pressure sensor (217) will be squeezed and generate a signal to stop the servo motor (211) and complete the intraperitoneal injection operation for animals. Automatic detachment phase after injection: When the needle tip of the syringe (100) deviates from the puncture position, the injection pressure will increase. The moving block (213) will drive the connecting rod (222) to move downward. The pressure value of the connecting seat (221) and the piston rod of the syringe (100) moving downward will increase. The moving block (213) and the connecting rod (222) will move according to the normal pressure value. As the pressure value increases, the fixed tooth (223) will move synchronously with the connecting rod (222) and squeeze the locking tooth (224). The locking tooth (224) will squeeze the first spring (227). The locking tooth (224) will move towards the cavity to automatically unlock and separate the connecting rod (222) and the connecting seat (221), thereby interrupting the animal injection operation. Commissioning phase of the connection component (220): Rotate the adjusting bolt (225) to adjust the position of the moving plate (226) in the cavity, thereby adjusting the compression distance of the first spring (227) and the pressure value of the locking tooth (224) disengaging from the fixed tooth (223). This can be adjusted according to the actual situation of intraperitoneal injection in animals.

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