Alzet Pump Recommendations

Implantation of Osmotic Pumps in R= odents

Division of Laboratory Anim= al Resources (DLAR)

University of Kentucky Medi= cal Center

PURPOSE: This document provides recommendat= ions of the DLAR Veterinary Staff for the implantation of ALZET^® osmotic pumps in rodents. A model e= -Sirius 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 an= imal 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 us= ed to deliver test drugs or agents by subcutaneous (SC), intraperitoneal (IP), intravenous (IV), intracerebral (IC), and other routes. The pump manufacturer maintains a website: <= /span>http://alzet.com/#<= /a> t= hat provides extensive technical resources for appropriate selection, preparati= on and use of the pumps, as well as information on special research applicatio= ns 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: http://www.researc= h.uky.edu/dlar/contact_us.htm

PROCEDURE:

NOTES:

1) Before selecting a pump or planning an experimen= t all users should first review the manufacturer’s comprehensive checklist = Checklist for use to assure appropriate selection and application of a pump for an experimental study. The “Technical Tips” <= /span>http://www.alzet.c= om/products/TechnicalTips.php and “Chemical compatibility” http://www.alze= t.com/products/alzaid.php sections should also be reviewed.

2) Implantation of an ALZET^® pump re= quires strict adherence to sterile surgical technique. The pump represents a non-absorbable foreign body that, if contaminated, can persistently harbor bacterial growth. The IACUC Policie= s, Procedures, and Guidelines regarding Rodent Surgery should be referred = to for principals for conducting sterile rodent surgery: http://www.research.uky.edu/ori/univet/resources/sop/IACUC_= 102_Rodent_Surgery.doc

3) 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.

4) It is critical to assure correct matching of pum= p size to animal bodyweight (Table-1)= . 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 monofilam= ent sutures (e.g. 5.0 polypropylene or nylon) can result in less mechanical stress than wound clips.

= Table-1: Esti= mated Minimum Animal Size for Implantation of ALZET^® Pumps

ALZET^{® Pump Model No.}
Mice	1003D, 1007D, 1002, 1004	2001D, 2001, 2002, 2004, 2006	2ML1, 2ML2, 2ML4
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 usi= ng the pumps with other types or genders of rats and mice, or with animals o= ther than rats and mice, these guidelines should be modified accordingly. (N/A= =3D not applicable)

<= o:p>

5)&n= bsp; If an experiment w= ill 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 con= tinues 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).

Table-2: Manufacturers Recommended Pump Explantation Schedule by ALZET^® Model<= /o:p>

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. =

6) Surgical Anesthesia Recommendations for ALZET® Pump Implantation: Animals and occupied animal cages should be placed on a preheated circulating water hea= ting 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/xylazin= e or other injectable agents opioids such as buprenorphine should be administere= d 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 recov= ery 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/k= g 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 postopera= tive 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 (75-1= 00 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 procedur= es (only recommended for subcutaneous pump implantation) and has a more rapid postoperative recovery. Addit= ional acceptable anesthetic regimens can be found on the DLAR web-site (http://www.research.uky.edu/dlar/animal_health.ht= ml) or in consultation with the DLAR veterinarians.

7) Post-Operative Analgesia Recommendations for ALZ= ET^® Pump Implantation: The implanta= tion of Alzet^® pumps is a potentially painful procedure requiring postoperative pain relief unless specifically contraindicated by the experimental design. Long act= ing non-steroidal anti-inflammatory drugs (NSAID) (meloxicam or carprofen) or opioids (buprenorphine) are recommended for systemic post-operative analges= ia. 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 a= s an adjunct to systemic analgesics. The recommended duration of postoperative analgesia is dependent upon the type of implantation procedure:

Table-3: Recommended Postoperative Analgesia Durations

Osmotic Pump Implantation Site	Minimum Recommended Postoperative Analgesia Du= ration
Subcutaneous	6 hours (12-24 hours preferred)
Intraperitoneal	24 hours (48 hours preferred)
Subcutaneous with I= ntravenous Cannula	12 hours (24-48 hou= rs preferred)
Subcutaneous with Intracranial Cannula	48 hours=

If the use of systemic analgesics is likely to interfere with experimental dat= a, the use of postoperative local anesthetics may be considered as an alternative regimen for subcutaneous pump implantations. The application of 0.1 ml (mic= e) to 0.5 ml (rats) of 0.08% bupivicaine solution to the incision area prior to surgical closure will provide 6-8 hours of postoperative pain relief. =

&nbs= p; Table-4: Post-operative Analgesia Recommendations for AL= ZET^® Pump Implantation^A

An= algesic Regimen

Mi= ce

Ra= ts

Meloxicam

2-5 mg/kg IP q 12-2= 4 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<= /o:p>

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% sol= ution at site

0.5 ml of 0.08% sol= ution at site

EMLA^=
® Cream (local anesthetic)^C

At closure, repeat = at 3-4 hrs

At closure, repeat = at 3-4 hrs

^A<= span style=3D'font-size:9.0pt'>See: http://www.res= earch.uky.edu/dlar/animal_health.html DLAR Anesthesia and Analgesia Drug Recommendations for additional ag= ents and species-specific dose information.

^BFor 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. =

^CBupivicaine and EMLA^® Cream are acceptable as the sole source of postoperative analgesia only for subcutaneous osmotic pump implantation.

Sample: E-= Sirius Surgical Description for Subcutaneous ALZET^® Pump Implantati= on

Select location(s) in which surger= ies are performed.

Selec= t

Surgi= cal Location

Room = Number

= ;

=

=

&n= bsp;

Type of surgery to be performed

Surgical Implantation: Subcutane= ous ALZET® Osmotic Pump in Rodents

Is this a Recover= y or Non-recovery Type of Surgery

Recovery<= /span>

Duration of anesthesia/surgery

15-30 min=

Describe pre-op heal= th assessment

Animals will be visu= ally examined to ensure they are healthy and normally active. Individual animal body weights will be determ= ined and noted on the pink DLAR Surgery Cards.

Describe use of pre-anesthetic medications

Mice: Meloxicam 2-5mg/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) I= P

=

Refer= to Table 3 (above) for other suggested surgical anesthetic regiments.

Note= : If experimentally acceptable, more than one anesthetic option can be liste= d 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 an= esthesia will be assessed by presence of regular steady respirations and absence= of a withdrawal reflex (toe or tail pinch) or corneal reflexes.=

Surgical table, inci= sion 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 w= ill be performed in a designated area of the laboratory specifically prepar= ed 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 w= ear 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 instrum= ents from the hot bead sterilizer are normalized before use. The pump will be prepar= ed aseptically.

Support equipment

I= soflurane 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 implantati= on of ALZET® pumps in mice and rats is on the back, slightly posterior to the scapul= ae. Other regions may be used, provided that the pump does not put pressure= on vital organs or impede respiration. If the pump is implanted subcutaneo= usly without a catheter attachment, the contents of the pump will be deliver= ed into the local subcutaneous space. Absorption of the compound by local capillaries results in systemic administration.

Subcutaneous Implantation Procedure (adapted from = www.alzet.com)

1.       An incision is made adjacent to the site chosen for pump placement. If the back of the animal is the site of choice, make a mid-scapular incision.=

2.       A sterile hemostat is inserted into the incision and, by opening and clos= ing the jaws, a subcutaneous pocket is made for the pump. The pocket is made large enough to allow some free movement of the pump (e.g. 1 cm longer = than the pump) but not too large, as this will allow the pump to turn around= or slip down on the flank of the animal.

3.       The pump is inserted into the pocket, delivery portal first, which minimizes interaction between the compound delivered and the healing of the incis= ion. The pump location is checked to assure that it does not rest immediately beneath the incision, which could interfere with the healing of the incision.

4.       The wound is closed with wound clips or non-absorbable monofilament sutures. Two clips will normally suffice.

5.       An anal= gesic is given post-operatively as described belo= w.

See http://www.alzet.com/n= ews/downloads.php for a video demonstrating the subcutaneous pump implantation procedure.=

=

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 ty= pe as indicated/needed.

Post-surgery/anesthe= tic recovery monitoring plan

Animals will be observed every f= ew minutes until they are recovered (i.e., awake) from anesthesia (sternal= ly recumbent, purposeful movement). They will be returned to their home ca= ge when fully conscious. Food and water will be offered once the animals a= re 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 D= LAR 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 a= re 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 Operat= ive Medications and Describe Postop Care

1)     &n= bsp; Mice: Meloxicam (OR buprenorphine), administered as a preanesthetic medication, will provide 12-24 hours of postoperative pain relief OR buprenorphine (0.05-0.1 mg/kg SC) will be administered for postoperative analgesia when the mi= ce have recovered from anesthesia (sternally recumbent, purposeful movement).

Rats: Meloxicam (OR buprenorphine), administered as a preanesthetic medication, will provide 12-24 hours of postoperative pain relief OR buprenorphine (0.05 mg/kg SC) will be administered for postoperative analgesia when the rats have recovered from anesthesia (sternally recumbent, purposeful movement).

2)     &n= bsp; A pink DLAR Surgery Card with the date the procedure was performed will be used to mark cages after surgery. Animals that have had surgery w= ill be observed daily (5-7 days) by the PI, or a member of the research team, = and the results of observations recorded (e.g. back of pink card). The anim= als will be observed with regard to normal activity, inquisitiveness, condi= tion of hair coat, eating, drinking, defecation, urination, appearance of ey= es, breathing rate, gait, and body condition. The animals will also be eval= uated for vocalization, dehydration, and appearance of the surgical wound (e.= g. red edges, swelling, or exudates), and suture/wound clip displacement. The cards will be removed after suture/staple removal.

3)     &n= bsp; If abnormal conditions are observed, the PI = or a co-investigator will consult with a DLAR veterinarian.

When will sutures/st= aples be removed?

10-14 days following= surgery

        &= nbsp;           &nbs= p;            &= nbsp;

Surgical Description for Intraperitoneal Pump implantation and References for Additi= onal Pump application procedures by ALZET®

Intraperitoneal Implantation Procedure: ALZET® pumps can be implant= ed intraperitoneally in animals with sufficiently large peritoneal cavities. Depending on the size of the animal relative to the pump, intraperitoneal implantation can disrupt normal feedi= ng 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 majo= rity of the dose may be absorbed via the hepatic portal circulation rather than = by the capillaries. For substances that are extensively metabolized by the liv= er (i.e., have a high “first pass effect”), the intraperitoneal ro= ute of administration may produce highly variable concentrations of agent in pl= asma and consequently highly variable effects. Therefore, the intraperitoneal ro= ute should probably be avoided with agents that have a significant first-pass effect. =

        =             &nb= sp; Intraperitoneal implantation Procedure (adapted from www.= alzet.com)

1.       Once the animal is anesthetized, shave and wash the skin over the implantation s= ite.

2.       A midline skin incision, approximately 1.0 cm long, is made in the lower abdo= men caudal to the rib cage.

3.       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.=

4.       A sterile filled pump is inserted, delivery portal first, into the peritoneal cavity.

5.       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.

6.       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 adhes= ive, or subcuticular closure using 4.0 or 5.0 absorbable suture.

<= span style=3D'font-size:10.0pt'>See: http://www.alzet.com/news/downloads.php for a video demonstrating the intraperitoneal pump implantation procedure.

<= span style=3D'font-size:10.0pt;color:black'>

Additi= onal surgical procedure descriptions= : 1) Intravenous Infusion (via = the External Jugular Vein) in Rats, 2) IV Cannulation in Mice, 3) CNS Infusion (via Brain Cannulation) in Rats, and 4) CNS Infusion in Mice can be obtained from ALZE= T® at the following link: http://www.alzet.com/news/link= s.php (See ALZET® Surgical Implantation Training & Resources-IACUC Approval Form Guide).=   &nb= sp;

Subcutaneous Implantation Procedure (adapted from = www.alzet.com)

= &nb= sp; Intraperitoneal implantation Procedure (adapted from www.= alzet.com)

An= algesic Regimen	Mi= ce	Ra= ts
Meloxicam	2-5 mg/kg IP q 12-2= 4 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<= /o:p>	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% sol= ution at site	0.5 ml of 0.08% sol= ution at site
EMLA^= ® Cream (local anesthetic)^C	At closure, repeat = at 3-4 hrs	At closure, repeat = at 3-4 hrs