LinBit - Product Instructions
(For Diabetic Mice Only)

The following sections contain helpful information on the proper uses of the LinBit implants in diabetic mice. As the severity of diabetes, health status, and response to insulin may vary, it is advisable to monitor the initial phase of implant therapy until the routines have become familiar.

A) Recommended Implantation Sites:

The LinBit implants should be placed subcutaneously under the mid dorsal skin, or any other convenient site.

B) Anesthesia:

The use of volatile halohydrocarbon liquids or any short-acting anesthetic agents of a few minutes duration is recommended.

C) Insertion:

1) Clip the hair from a spot ~1 cm in diameter using a pair of small scissors with curved tips.
2) Cleanse the spot with Betadine (a 10% povidone-iodine solution for skin disinfection) and then pinch the skin between the thumb and the index fingers.
3) Pierce the pinched skin with a 16G disposable hypodermic needle and withdraw.
4) Briefly immerse the 12-gauge trocar in a diluted (~2%) solution of Betadine, then push it through the skin orifice just created to a length of at least 1.5 cm.
5) Immerse each implant briefly (for 5 sec) in the ~2% solution of Betadine, just before inserting it into the proximal end of the trocar.
6) After immersing in the ~2% Betadine solution, use the stylet to push the implant until it exits from the distal end of the trocar.
7) Repeat steps 5 and 6 when required.
8) Upon completion, place a drop of the 10% Betadine solution over the skin opening.
9) The skin defect will soon contract, and requires no suturing or wound clips for closure.

D) After Care:

Food and water must be available at all times. No other specific care is required, unless otherwise stated. For blood and urine glucose tests see Section F.

E) Dosages:

1) The LinBit implants are intended to treat diabetic mice only. For other diabetic animals, use Linplant which has a much higher insulin release rate.
2) Do not equate or correlate the daily injection dose directly with the sustained release dose. The sustained release formulation may potentiate the insulin action manifold.
3) Use 2 LinBit implants for the first 20 g in body-weight of a streptozotocin-induced or naturally diabetic mouse. For each additional 5 g or fraction thereof, add another LinBit implant.
For example, a 28.4 g diabetic mouse = 20 g + 5 g + 3.4 g in body-weight. The recommended dose is 2 Bits + 1 Bit + 1 Bit = 4 LinBit implants.
4) By using a different number of LinBit implants, various degrees of severity in sustained hyperglycemia may be obtained for physiopathological or nutritional studies. However, a wider fluctuation range in blood glucose levels should be expected.

F) Monitoring (Blood and Urine Glucose Tests):

1) Diabetic mice consume a substantial amount of feed frequently in the day time as well. Therefore, even with adequate insulin therapy, random tests still often show hyperglycemia and glucosuria. Hence, meaningful results should be obtained from a standardized schedule as suggested below.
2) In the morning, the feed is removed and the required tests are then performed. If urinary glucose is negligible or blood glucose is <8 mmol/L (<144 mg/dL), the feed is restored at once.
3) If there is glucose in the urine or blood glucose is >8 mmol/L, repeat the test after every 3 hours. When the results are close to the criteria stated in Step F.2 above after the first 3-hr interval, the feed should be restored.
4) When there is an adequate amount of insulin being released from the LinBit implants, the criteria in Step F.3 are usually realized before or not later than the test conducted after the second 3-hr. interval. Diabetic mice (especially with natural diabetes) without sufficient exogenous insulin rarely meet the above stated criteria when tested, even after the second 3-hr interval.

G) Monitoring (Feed and Water Consumption):

1) A diabetic mouse receiving an adequate dose from LinBit implants has a relatively consistent consumption rate of ~0.25 g of feed and ~0.7 mL of water per hour nocturnally. Without the sustained release insulin, the consumption rate is about double for feed and fourfold for water. Therefore, noting the change in the drinking water supply overnight (e.g., 5 p.m. to 9 a.m. the next day) is a convenient alternative to the glucose tests in monitoring the efficacy of implant therapy.
2) If the water consumption rate becomes inconsistent or threefold higher than before, especially when it occurs >30 days post-implantation, the scheduled glucose test procedure suggested in Section F should be considered.

H) Next Implantation:

1) The implant dose, as suggested in Section E, has enough insulin to last for >40 days at the specified release rate. However, insulin is also an anabolic hormone, and body weight gain often still occurs even for fully matured adult mice. There is also the possibility of tolerance to the exogenous insulin. Therefore, at ~30 days, the suggested dose may appear inadequate. If glucosuria or hyperglycemia >16 mmo1/L (>288 mg/dL) is found to persist at the test scheduled for the second 3-hr interval as referred to in Section F, repeat the insertion as outlined in Section C, and at the dose suggested in Section E. The remnants of the insulin-depleted implants will be totally absorbed in the next few weeks, and removal is not necessary.
2. If the implants are removed in <30 days due to experimental design, new implants may be inserted at any time by following the procedure outlined in Section C.

I) Complications:

1) Occasionally, (<3% occurrence), skin tissue debris or hair remnants are carried into the implant site by the needle or trocar. An abscess, which is usually palpable, nodular, and soft, may develop in 1 to 3 weeks, and when monitored (see Section F), the response to the action of the implant may appear to cease abruptly. It is recommended that the abscess be left undisturbed, as it will soon recede gradually. If desired, new implants should be inserted at another site.
2) For some mice with induced diabetes, continuous implant therapy for >6 months may lead to a reduction in the severity of hyperglycemia or result in remission.
3) Do not attempt RIA of serum insulin because antibodies against the exogenous insulin will be present. Use athymic mice with induced diabetes for assays of circulating insulin level derived from the sustained release implant.
4) Hypoglycemia may occur even with the recommended doses if the usual food intake is greatly reduced due to whatever causes (i.e. supplemental medications, brand of chow, pregnancy, lactation, etc.).

J) Storage:

Store at room temperature. No refrigeration required.

K) Product Information:

Composition: Insulin & Micro-Recrystallized Palmitic Acid
Weight: 13 ± 2 mg per implant
Dimension: 2 mm in diameter, 3 mm in length. Insertable by a 12G Needle
Release Rate: ~0.1 U/24 hr/implant for >30 days, subcutaneously (erodible in vivo)
Indication: For laboratory research with diabetic mice

Note: Implants have not been sterilized, they may be immersed briefly (for 5 sec) in dilute (~2%) povidone-iodine solution, or exposed to UV. Do not immerse in alcohol.

WARNING: NOT FOR DRUG USE

L) References:

1. Ohzato, H., Porter, J., Monaco, A.P., Montanya, E. and Maki, T. "Improved outcome of islet transplantation in insulin-treated diabetic mice: effects on beta-cell mass and function." Transplantation 56 (2), 270-274 (1993).

2. Davalli, A.M., Sagalia, L., Zangen, D.H., Hollister, J., Bonner-Weir, S., Weir, G.C. "Vulnerability of islets in immediate post-transplantation period: Dynamic changes in structure and function." Diabetes 19, 1161-1167 (1996).

3. Merino, J.F., Nacher, V., Raurell, M., Aranda, O., Soler, J., & Montanya, E., "Improved outcome of islet transplantation in insulin treated diabetic mice: effects on beta cell mass and function." Diabetalogica 40, 1004-1010 (1997).

4. Nacher, V., Marino, J.F., Raurell, M., Soler, J., & Montanya, E., "Normoglycemia restores B-cell replicative response to glucose in transplanted islets exposed to chronic hyperglycemia." Diabetes 47, 192-196 (1998).

5. Markees, T.G., Serreze, D.V., Phillips, N.E., Sorli, C.H., Gordon, E.J., Shultz, L.D., Noelle, R.J., Woda, B.A., Greiner, D.L., Mordes, J.P., & Rossini, A.A., "NOD mice have a generalized defect in their response to transplantation tolerance induction." Diabetes 48, 967-974 (1999).

6. Atkinson et al., "Adeno-associated virus-mediated IL-10 gene therapy inhibits diabetes recurrence in syngeneic islet cell transplantation of NOD mice." Diabetes 52, 708-716 (2003).

MADE IN CANADA
CANADIAN PATENT 1 257 199
U.S. PATENT 5 110 595 and others