Implantation of Osmotic Pumps in Rodents
PURPOSE: This document provides recommendations of the DLAR Veterinary Staff for the implantation of ALZET® osmotic pumps in rodents. A model Cayuse Animal Oversight surgical procedure description is provided in the PROCEDURE section to assist researchers in adding ALZET® pump implantation to a research protocol. This document is intended to serve as a guide to help standardize the procedure by which an osmotic pump system may be implanted in rodent models. It is also meant to serve as a resource when preparing an animal use protocol. Information may be copied and pasted into your protocol document(s) as needed.
APPLICATION: ALZET® pumps are miniature, osmotic infusion pumps for the continuous and controlled dosing of laboratory animals in vivo. The pumps can be used to deliver test drugs or agents by subcutaneous (SC), intraperitoneal (IP), intravenous (IV), intracerebral (IC), and other routes. The pump manufacturer maintains a website: http://alzet.com that provides extensive technical resources for appropriate selection, preparation and use of the pumps, as well as information on special research applications and accessory components. It should be reviewed by all users. For special infusion applications or assistance with surgical, anesthetic, or analgesic recommendations please contact a DLAR clinical veterinarian.
procedure
NOTES:
- Before selecting a pump or planning an experiment all users should first review the manufacturer’s comprehensive checklist for use to assure appropriate selection and application of a pump for an experimental study. The “Technical Tips” and “Chemical compatibility” sections should also be reviewed.
- Implantation of an ALZET® pump requires strict adherence to sterile surgical technique. The pump represents a non-absorbable foreign body that, if contaminated, can persistently harbor bacterial growth. The IACUC Policies, Procedures, and Guidelines regarding Rodent Surgery should be referred to for principals for conducting sterile rodent surgery.
- Sterile technique is also essential when filling and handling pumps. Contamination of the infused solution or pump flow moderator during filling may result in the growth of potentially activity-destroying microorganisms, tissue irritation, and erratic results. If the sterility of your solution is a concern fill the pumps using a 0.22 µM syringe-end filter.
- It is critical to assure correct matching of pump size to animal bodyweight (Table-1 below). A primary cause of implant failure/problems is excessive skin tension due to inappropriate pump size. When using a mouse strain known to have thin or fragile skin (e.g. nude mice) closing the skin incision with fine monofilament sutures (e.g. 5.0 polypropylene or nylon) can result in less mechanical stress than wound clips.
| ALZET® Pump Model No. | |||
|---|---|---|---|
| 1003D, 1007D, 1002, 1004 | 2001D, 2001, 2002, 2004, 2006 | 2ML1, 2ML2, 2ML4 | |
| Mice | |||
| Subcutaneous | 10g | 20g | n/a |
| Intraperitoneal | 20g | n/a | n/a |
| Rats | |||
| Subcutaneous | 10g | 20g | 150g |
| Intraperitoneal | 20g | 150g | 300g |
| Note: The minimum animal size estimates are based on experience with male Sprague Dawley rats and Swiss Webster mice. When using the pumps with other types of genders of rats and mice, or with animals other than rats and mice, these guidelines should be modified accordingly. | |||
- If an experiment will extend beyond the designed infusion period for the osmotic pump it may be necessary to explant the pump. After its pumping lifetime an ALZET® osmotic pump continues to attract water that can cause it to swell and leak a concentrated salt solution, resulting in local irritation of tissues around the pump. The manufacturer recommends pump removal according to the following schedule (Table 2).
| ALZET® Model | Explant by* | ALZET® Model | Explant by* |
|---|---|---|---|
| 1003D | Day 5 | 2002 | Day 21 |
| 1007D | Day 10 | 2004 | Day 42 |
| 1002 | Day 21 | 2006 | Day 63 |
| 1004 | Day 42 | 2ML1 | Day 10 |
| 2001D | Day 1.5 | 2ML2 | Day 21 |
| 2001 | Day 10 | 2ML4 | Day 42 |
| *Data shown correspond to the nominal duration. Actual explant date should be calculated using the exact specifications for each lot of pumps. | |||
- Surgical Anesthesia Recommendations for ALZET® Pump Implantation: Animals and occupied animal cages should be placed on a preheated circulating water heating pads set to 90-100F to prevent hypothermia which can result in slowed metabolism, prolonged induction, and extended postsurgical recovery times. A “Balanced Anesthetic Regimen” is recommended, which includes pre-emptive analgesia administration with an opioid or a non-steroidal anti-inflammatory drug (NSAID) given 15-60 minutes before induction (Note: when using ketamine/xylazine or other injectable agents opioids such as buprenorphine should be administered as the animal begins to recover from anesthesia and not pre-emptively as it can increase adversely the depth of anesthesia).
Isoflurane inhalation anesthesia (1-4% in O2) is preferred and recommended as the anesthesia depth is more readily controlled and the postoperative recovery time is rapid (minutes). The use of isoflurane anesthesia requires the use of a precision vaporizer and the provision of a system to exhaust waste anesthetic gases. Ketamine and xylazine (90-120 mg/kg and 10 mg/kg respectively IP in the mouse and 40-80 mg/kg and 5-10 mg/kg respectively IP in the rat) is the most commonly used injectable anesthetic for osmotic pump implantation. The ketamine/xylazine combination produces an adequate depth of anesthesia for major surgeries of 15-20 minutes and can be extended by additional ketamine administration. The postoperative recovery period can extend up to 30-40 minutes after the surgery requiring extended observation of the animals. An alternative anesthetic regimen of ketamine and medetomidine (75-100 mg/kg and 1 mg/kg respectively IP in the mouse and 75 mg/kg and 0.5 mg/kg respectively IP in the rat) provides anesthesia adequate for minor procedures (only recommended for subcutaneous pump implantation) and has a more rapid postoperative recovery. Additional acceptable anesthetic regimens can be found on the DLAR Animal Health page or in consultation with the DLAR veterinarians. - Post-Operative Analgesia Recommendations for ALZET® Pump Implantation: The implantation of Alzet® pumps is a potentially painful procedure requiring postoperative pain relief unless specifically contraindicated by the experimental design. Long acting non-steroidal anti-inflammatory drugs (NSAID) (meloxicam or carprofen) or opioids (buprenorphine) are recommended for systemic post-operative analgesia. Additionally in the case of intracranial cannula, the local application of bupivicaine with epinephrine [0.1(mouse)-0.5(rat) ml of a 0.08% solution of bupivicaine] to the craniotomy site at the time of surgery is recommended as an adjunct to systemic analgesics. The recommended duration of postoperative analgesia is dependent upon the type of implantation procedure:
| Osmotic Pump Implantation Site | Minimum Recommended Postoperative Analgesia Duration |
|---|---|
| Subcutaneous | 6 hours (12-24 hours preferred) |
| Intraperitoneal | 24 hours (48 hours preferred) |
| Subcutaneous with Intravenous Cannula | 12 hours (24-48 hours preferred) |
| Subcutaneous with Intracranial Cannual | 48 hours |
If the use of systemic analgesics is likely to interfere with experimental data, the use of postoperative local anesthetics may be considered as an alternative regimen for subcutaneous pump implantations. The application of 0.1 ml (mice) to 0.5 ml (rats) of 0.08% bupivicaine solution to the incision area prior to surgical closure with provide 6-8 hours of postoperative pain relief.
| Analgesic Regimen | Mice | Rats |
|---|---|---|
| Meloxicam | 2-5 mg/kg IP q 12-24 hours | 1-4 mg/kg SC or IP q 24 hours |
| Carprofen | 10.0 mg/kg SC or PO q 12-24 hours | 5-7 mg/kg SC or PO q 12-24 hours |
| Buprenorphine | 0.05-0.1 mg/kg SC q 6-12 hours | 0.02-0.05 mg/kg SC q 6-12 hours |
| Bupivicaine (local anesthetic)B,C | 0.1 ml of 0.08% solution at site | 0.5 ml of 0.08% solution at site |
| EMLA® Cream (local anesthetic)C | At closure, repeat at 3-4 hours | At closure, repeat at 3-4 hours |
A See DLAR Anesthesia and Analgesia Drug Recommendations for additional agents and species-specific dose information.
B For bupivicaine (Marcaine®): To avoid potential toxicity in rodents dilute stock 0.25% bupivicaine (2.5mg/ml) to a 0.08% solution (Buy 0.25% Marcaine® and dilute it 1:3 with sterile saline). Apply approximately 100 ul (0.1 ml) of the 0.08% solution to the incision area for a 25 gram mouse. Prepare fresh before use, assure sterility.
C Bupivicaine and EMLA® Cream are acceptable as the sole source of postoperative analgesia only for subcutaneous osmotic pump implantation.
sample
Sample: Cayuse AO Surgical Description for Subcutaneous ALZET® Pump Implantation
Select location(s) in which surgeries are performed.
| Select | Surgical Location | Room Number |
|---|---|---|
| Type of surgery to be performed | Surgical Implantation: Subcutaneous ALZET® Osmotic Pump in Rodents |
| Is this a Recovery or Non-recovery type of surgery? | Recovery |
| Duration of anesthesia/surgery | 15-30 min |
| Describe pre-op health assessment | Animals will be visually examined to ensure they are healthy and normally active. Individual animal body weights will be determined and noted on the pink DLAR surgery cards. |
| Describe use of pre-anesthetic medications | Mice: Meloxicam 2-5 mg/kg IP OR Buprenorphine 0.05-0.1 mg/kg SC (only when using Isoflurane anesthesia) |
| Rats: Meloxicam 1-4 mg/kg SC or IP OR Buprenorphine 0.01-0.05 mg/kg SC (only when using Isoflurane anesthesia) | |
| Operative Anesthesia | Mice: Isoflurane (1-4% in O2, inhaled-nosecone) OR Ketamine (90-120 mg/kg) and Xylazine (10 mg/kg) IP |
| Rats: Isoflurane (1-4% in O2, inhaled-nosecone) OR Ketamine (40-80 mg/kg) and Xylazine (5-10 mg/kg) IP | |
| Refer to Table 3 (above) for other suggested surgical anesthetic regiments. Note: If experimentally acceptable, more than one anesthetic option can be listed to enhance protocol versatility and accommodate individual researcher(s)' preference. |
|
| Supplemental doses of anesthetic | When using injectable Ketamine-mixtures prolongation of surgical time is accomplished by re-dosing with 25-35% of the original dose of Ketamine, alone. |
| How will anesthetic depth be measured? | Adequate depth of anesthesia will be assessed by presence of regular steady respirations and absence of a withdrawal reflex (toe or tail pinch) or corneal reflexes. |
| Surgical table, incision site and surgeon preparation | Standard procedures for rodent survival surgery will be followed as described in the IACUC Policies, Procedures and Guidelines on Rodent Surgery. Procedures will be performed in a designated area of the laboratory specifically prepared in advance for that purpose. Following induction of anesthesia an artificial tear or ophthalmic ointment is applied to both eyes. The skin will then be shaved and scrubbed thoroughly with Povidone iodine or Chlorhexidine solution. The animal’s body temperature will be maintained with heating pads or lamps, as necessary. The surgeon will wear a mask and sterile gloves. Instruments will be steam autoclaved prior to use and sterilized with a glass bead sterilizer between animals. Assurance is made that the temperature of instruments from the hot bead sterilizer are normalized before use. The pump will be prepared aseptically. |
| Support Equipment | Isoflurane vaporizer with gas scavenging (if using isoflurane), recirculating water heating pad(s), and glass bead sterilizer. |
| Describe surgical procedure | Subcutaneous Pump Implantation: The usual site for subcutaneous implantation of ALZET® pumps in mice and rats is on the back, slightly posterior to the scapulae. Other regions may be used, provided that the pump does not put pressure on vital organs or impede respiration. If the pump is implanted subcutaneously without a catheter attachment, the contents of the pump will be delivered into the local subcutaneous space. Absorption of the compound by local capillaries results in systemic administration. |
Subcutaneous Implantation Procedure (adapted from www.alzet.com)
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| Wound Closure | Surgical wounds will be closed with: 1)wound clips, or 2) 5.0 monofilament non-absorbable sutures. Note: For flexibility, it is good to add several methods. Add additional closure methods (subcuticular sutures, tissue adhesive, etc); revise suture size or type as indicated/needed. |
| Post-surgery/anesthetic recovery monitoring plan | Animals will be observed every few minutes until they are recovered (i.e., awake) from anesthesia (sternally recumbent, purposeful movement). They will be returned to their home cage when fully conscious. Food and water will be offered once the animals are alert. Failure to eat and/or to move normally, unusual vocalizations, or failure of the wound to heal normally will be taken as suggestive of potential post-operative complications or discomfort. In such cases a DLAR veterinarian will be consulted. |
| Will post-operative analgesics be administered? | Yes |
| If Recovery Surgery and post-operative analgesics are not used, provide scientific justification for withholding analgesics | Note: If post-operative analgesics are not being given, complete this section, with scientific rational indicating why the administration of analgesics would interfere with the scientific objectives of the study. |
| Indicate Post Operative Medications and Describe Post-Op Care |
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| When will sutures/staples be removed? | 10-14 days following surgery |
Intraperitoneal Implantation Procedure: ALZET® pumps can be implanted intraperitoneally in animals with sufficiently large peritoneal cavities. Depending on the size of the animal relative to the pump, intraperitoneal implantation can disrupt normal feeding and weight gain for a day or two thereafter. Allow 24 to 48 hours for the animal to recover after intraperitoneal implantation. With any substance administered intraperitoneally, whether by injection or by infusion, a majority of the dose may be absorbed via the hepatic portal circulation rather than by the capillaries. For substances that are extensively metabolized by the liver (i.e., have a high “first pass effect”), the intraperitoneal route of administration may produce highly variable concentrations of agent in plasma and consequently highly variable effects. Therefore, the intraperitoneal route should probably be avoided with agents that have a significant first-pass effect.
Intraperitoneal implantation Procedure (adapted from www.alzet.com)
- Once the animal is anesthetized, shave and wash the skin over the implantation site.
- A midline skin incision, approximately 1.0 cm long, is made in the lower abdomen caudal to the rib cage.
- The musculo-peritoneal layers are grasped by the linea alba with forceps and carefully tented up and incised being careful to avoid damage to the bowel.
- A sterile filled pump is inserted, delivery portal first, into the peritoneal cavity.
- The musculo-peritoneal layer is carefully closed with 4.0 (rats) or 5.0 (mice) absorbable sutures in an interrupted or continuous pattern, taking care to avoid perforation of the underlying bowel.
- The skin incision is closed with 2 or 3 wound clips, simple interrupted monofilament sutures (e.g. 4.0 or 5.0 polypropylene or nylon), tissue adhesive, or subcuticular closure using 4.0 or 5.0 absorbable suture.
See: http://www.alzet.com/news/downloads.php for a video demonstrating the intraperitoneal pump implantation procedure.
Additional surgical procedure descriptions:
- Intravenous Infusion (via the External Jugular Vein) in Rats,
- IV Cannulation in Mice,
- CNS Infusion (via Brain Cannulation) in Rats, and
- CNS Infusion in Mice can be obtained from ALZET® at the following link: http://www.alzet.com/news/links.php (See ALZET® Surgical Implantation Training & Resources-IACUC Approval Form Guide).