Intraperitoneal (IP) injection is a foundational technique in biomedical research, involving the administration of a substance directly into the mouse’s peritoneal cavity. This route is favored because the peritoneal cavity is highly vascularized, allowing for rapid absorption and systemic drug delivery. Successful IP injection requires meticulous attention to detail and precise technique to ensure animal welfare and the integrity of research data.
Essential Preparation and Safety Measures
The preparatory phase for an intraperitoneal injection focuses on maintaining sterility and selecting appropriate equipment to minimize tissue trauma. Fine-gauge needles, typically 25 to 27 gauge, are recommended for mice. These smaller diameters help reduce the size of the puncture wound in the abdominal wall and lessen the animal’s discomfort.
Needle length should be short, often between one-half to three-quarters of an inch, which is sufficient to penetrate the abdominal wall without excessively entering the cavity. All substances intended for injection must be sterile, as contamination can lead to peritonitis, compromising animal health and experimental results. Preparing the substance also includes warming it to room or body temperature, ideally not exceeding 37°C, to prevent discomfort or a drop in the mouse’s body temperature.
Aseptic technique must be maintained throughout the procedure. This includes disinfecting the top of the substance vial with 70% alcohol before drawing up the solution. Furthermore, all personnel involved must wear appropriate personal protective equipment (PPE), such as gloves and eye protection, to ensure handler safety and procedural cleanliness.
The entire procedure must align with institutional protocols, often dictated by an Institutional Animal Care and Use Committee (IACUC). These protocols outline acceptable volumes, needle sizes, and the maximum number of attempts permitted per injection site. Adherence to these guidelines is necessary for ethical and scientifically sound research.
Principles of Mouse Restraint and Handling
Proper and secure manual restraint is paramount to the success and safety of the intraperitoneal injection procedure. The standard method for immobilizing a mouse is the “scruff” hold, which involves grasping the loose skin over the neck and shoulders. This technique effectively immobilizes the mouse’s head and upper body, preventing sudden movements that could lead to an incorrect or injurious needle insertion.
The handler uses the thumb and forefinger to gather a substantial amount of skin, pulling it gently but firmly upward until the skin is taut. This firm grip is essential for maintaining control, but it must not restrict the animal’s breathing. The mouse is then held in dorsal recumbency, lying on its back with the abdomen exposed.
For IP injection, the mouse should be tilted downward, positioning its head slightly lower than its hindquarters. This downward tilt uses gravity to cause the internal abdominal organs (viscera) to shift cranially, away from the lower injection site. Moving the organs out of the target area minimizes the risk of inadvertently puncturing the intestines or other vital structures.
Acclimating mice to handling before the procedure helps reduce their stress and resistance. Mice that are calm are less likely to struggle, allowing for a quicker, more accurate injection, which improves animal welfare. The entire restraint process should be conducted with confidence and speed, keeping the duration of the stressor as short as possible.
Executing the Intraperitoneal Injection
The execution of the injection requires precise anatomical knowledge and a controlled hand motion to ensure the substance is delivered correctly into the peritoneal space. The target site is located in the lower abdominal quadrant, lateral to the midline. Injecting into the animal’s lower right quadrant is preferred, as this location helps avoid the cecum, which is typically situated on the left side of the mouse’s abdomen.
The needle must be inserted at a shallow angle, typically between 10 and 30 degrees relative to the abdominal wall. This low angle ensures the needle passes through the muscle layer and into the peritoneal cavity without excessive deep penetration. Inserting the needle with the bevel facing upward facilitates a smoother penetration of the tissue layers.
Before injection, the technician must perform aspiration by gently pulling back on the syringe plunger. If the needle is correctly placed in the peritoneal space, no fluid or blood should be drawn back. The appearance of blood indicates a punctured blood vessel; yellowish fluid suggests the bladder has been hit; and green matter indicates the bowel has been entered.
If any fluid is aspirated, the needle must be withdrawn, and the syringe and needle replaced with a fresh, sterile set. If aspiration yields negative pressure, the substance can be administered by depressing the plunger smoothly and steadily. The needle should be withdrawn along the same angle of entry immediately after delivery, and the injection site observed for any leakage.
Critical Volume and Post-Procedure Considerations
The volume of fluid injected is a critical parameter that directly affects the safety and well-being of the mouse. The maximum safe volume for a single intraperitoneal injection is commonly set at 10 to 20 milliliters per kilogram of body weight. For an average adult mouse, this translates to an absolute volume typically ranging from 0.2 to 0.5 milliliters, though the lower end of this range is often recommended to maximize safety.
Exceeding the maximum safe volume can lead to complications such as organ compression or respiratory distress due to the pressure exerted on the diaphragm. When administering multiple injections or higher volumes, the protocol should specify the use of the lowest possible volume that achieves the therapeutic goal. If repeated injections are necessary, alternating the injection site between the right and left lower quadrants helps minimize tissue irritation and potential scarring at one location.
After the needle is removed, the mouse should be returned to its cage and closely monitored for any immediate signs of discomfort or complications. Technicians observe for signs like labored breathing, lethargy, or leakage of the injected fluid from the site. Leakage often indicates the injection was too shallow, resulting in the substance being delivered subcutaneously or into the muscle layer rather than the peritoneal cavity.
Recognizing signs of technical errors is necessary for immediate intervention and protocol refinement. Injection into the bladder is suspected if the mouse exhibits immediate urination after the procedure. Any signs of abdominal pain, such as hunched posture or guarding the abdomen, warrant immediate attention and may require consultation with a veterinary professional to ensure appropriate care and administration of pain relief.