1.0 OBJECTIVE: To design a Standard Operating Procedure that describes the procedure for Anesthesia.
2.0 RESPONSIBILITY: It is the responsibility of all Researchers and Animals Care Personals.
3.0 TYPES OF ANESTHESIA:
3.1 Ketamine- Ketamine is a dissociative anesthetic, that produces sedation and immobility, increased blood pressure, increased muscle tone, increased salivary secretions, and only slight respiratory depression in most species (severe in rodents), variable analgesia, and may cause apnea. Ketamine should be administered in combination with xylazine or diazepam to induce surgical anesthesia.
3.2 Pentobarbital- Pentobarbital should be used intravenously (IV) to reduce the risk of cardiovascular and respiratory depression. Calculate the required pentobarbital volume based on mg/kg dose and draw this volume into a syringe; administer approximately half of the volume by rapid IV injection to achieve basal narcosis, and then slowly inject additional incremental volume until surgical anesthesia is achieved. The intraperitoneal (IP) route of administration should be used in rodents only.
3.3 Xylazine- Xylazine is a centrally acting alpha-2 adrenergic receptor agonist with analgesic and sedative effects. Xylazine can induce profound bradycardia, decreased cardiac output, emesis, and depressed thermoregulation.
3.4 Isoflurane- Isoflurane is a halogenated ether used for inhalational anesthesia. Isoflurane is always administered in conjunction with air and/or pure oxygen. Often nitrous oxide is also used. Although its physical properties imply that anesthesia can be induced more rapidly than with halothane, its pungency can irritate the respiratory system, negating this theoretical advantage conferred by its physical properties. It is usually used to maintain a state of general anesthesia that has been induced with another drug, such as thiopentone or propofol. It vaporizes readily but is a liquid at room temperature. It is completely non-flammable.
|
NAME OF ANESTHETICS |
DOSE/ROUTE IN MICE |
DOSE/ROUTE IN RAT |
DOSE/ROUTE IN RABBIT |
|
Pentobarbital |
35
mg/kg IV 40
– 70 mg/kg IP |
30
– 40 mg/kg IV 40
– 60 mg/kg IP |
30
– 40 mg/kg IV |
|
Ketamine
+ Xylazine |
100
mg/kg (K) + 10 mg/kg (X) IP |
60
– 90 mg/kg (K)+ 6 – 9 mg/kg (X) IP |
35
– 50 mg/kg (K) + 5 – 10 mg/kg (X) IM |
|
Ketamine |
44
mg/kg SC |
50
– 100 mg/kg IP |
30
mg/kg IM for sedation |
|
Isoflurane |
In
general, 3-4% induction, 1-2% maintenance; inhalation |
In
general, 3-4% induction, 1-2% maintenance; inhalation |
In
general, 3-4% induction, 1-2% maintenance; inhalation |
4.0 PROCEDURE:
For Rats
4.1 Isoflurane anesthesia:
4.1.1 Induction:
4.1.1.1 The animal was placed in the induction chamber.
4.1.1.2 The oxygen flow meter was adjusted to 0.8 to 1.5 L/min.
4.1.1.3 The isoflurane vaporizer was adjusted to 3% to 5%
4.1.2 Maintenance:
4.1.2.1 The mask connected to the Bain circuit was used.
4.1.2.2 The flow meter was adjusted to 400 to 800mL/min.
4.1.2.3 The isoflurane vaporizer was adjusted to 2 to 2.5%.
4.1.2.4 Ophthalmic ointment (natural tears) was applied to both eyes to prevent dryness and damage to the cornea.
4.1.3 Recovery:
4.1.3.1 The isoflurane vaporizer was turned off but the animals were kept on oxygen.
4.1.3.2 The animals were transferred to their cage once they begin to move, and were then allowed to recover fully (sternal position).
4.1.4 Ketamine/Xylazine anesthesia:
4.1.4.1 Anaesthetic dose: Ketamine 50mg/kg, Xylazine 5mg/kg.
4.1.4.2 To prepare cocktail, in a sterile vial or bottle with a rubber stopper, 5mL of ketamine (100mg/mL) + 2.5mL xylazine (20mg/mL) + 1mL acepromazine (10mg/mL) + 1.5mL of sterile isotonic saline or sterile water for injection were mixed.
4.1.4.3 It was labeled as “Rodent Cocktail” and expiry date was indicated on vial or bottle (maximum 6 months).
4.1.4.4 Mixed cocktail was protected from light and stored in a cool place.
4.1.4.5 0.1mL/100g body weight was administered intramuscularly or intraperitoneally.
4.1.4.6 Ophthalmic ointment (natural tears) was applied to both eyes to prevent dryness and damage to the cornea.
4.1.4.7 Duration of anesthesia was approximately 30 minutes.
4.1.4.8 After 30 minutes, half the dose was administered when needed.
For Mice
4.2 Isoflurane anesthesia:
4.2.1 Induction:
4.2.1.1 The animal was placed in the induction chamber.
4.2.1.2 The oxygen flow meter was adjusted to 0.8 to 1.5 L/min.
3.1.1.1 The isoflurane vaporizer was adjusted from 3% to 5%.
3.1.2 Maintenance:
3.1.2.1 The mask connected to the Bain circuit was used.
3.1.2.2 The flow meter was adjusted to 400 to 800mL/min.
3.1.2.3 The isoflurane vaporizer was adjusted to 2 to 2.5%.
3.1.2.4 Ophthalmic ointment (natural tears) was applied to both eyes to prevent dryness and damage to the cornea.
3.1.3 Recovery:
3.1.3.1 The isoflurane vaporizer was turned off but the animals were kept on oxygen.
3.1.3.2 The animal were transferred to their cage once they begin to move, and they were allowed to recover fully (sternal position).
3.2 Ketamine/Xylazine/Acepromazine anesthesia:
3.2.1 Anesthetic dose: Ketamine 100mg/kg, Xylazine 10mg/kg, Acepromazine 3mg/kg.
3.2.2 The solution was prepared a day before or shaken thoroughly before use.
3.2.3 The cocktail was prepared in a sterile vial or bottle with a rubber stopper, by mixing: 1mL of Ketamine (100mg/mL) + 0.5mL Xylazine (20mg/mL) + 0.3mL Acepromazine (10mg/mL) + 8.2mL of sterile isotonic saline or sterile water for injection.
3.2.4 It was labelled as “Mouse Cocktail” and the expiry date on vial or bottle (maximum 6 months) was indicated.
3.2.5 Mixed cocktail was protected from light and stored in a cool place.
3.2.6 0.05-0.1mL/10g body weight was administered intraperitoneally.
3.1.1 Ophthalmic ointment (natural tears) was applied to both eyes to prevent dryness and damage to the cornea.
3.1.2 Duration of anaesthesia was approximately 20 minutes.
3.1.3 After 20 minutes, half the dose was administered.
3.1.4 Atipamezole 1-2 mg/kg SC or IP was administered to improve respiration or speed up the recovery if needed. Atipamezole is the antidote for xylazine.
For Rabbits
3.2 Sedation:
3.2.1 Used for short periods of restraint for non-painful procedures (e.g. blood collection).
3.2.2 Dose: Buprenorphine 0.2 mg/kg, Acepromazine 1mg/kg.
3.2.3 Both drugs can be mixed in the same syringe.
3.2.4 Was injected intramuscularly.
3.2.5 Animal was adequately sedated after 15 minutes.
3.2.6 Duration of sedation is approximately 1 hour.
3.3 Injectable anesthesia:
3.3.1 Can be used alone for short, non-invasive procedures.
3.3.2 Used for induction prior to use of Isoflurane anesthesia for smooth and rapid induction and to facilitate intubation.
3.3.3 Ketamine-Xylazine-Acepromazine anesthesia
3.3.3.1 Acepromazine and xylazine were injected intramuscularly. Both drugs were mixed in the same syringe. Dose of acepromazine: 0.75mg/kg. Dose of xylazine: 5mg/kg.
3.3.3.2 Ketamine was injected intramuscularly in a different muscle. Dose: 20-35mg/kg.
3.3.3.3 Ophthalmic ointment (natural tears) was applied to both eyes to prevent dryness and damage to the cornea.
3.4 Isoflurane anaesthesia:
3.4.1 Induction (if injectable anaesthetics not previously administered).
3.4.2 The animal was placed in the induction chamber.
3.4.3 The oxygen flow meter was adjusted to 0.8 to 1.5 L/min.
3.4.4 The isoflurane vaporizer was adjusted to 3% to 3%.
3.4.5 Maintenance:
3.4.5.1 A tight-fitting mask was used and was connected to the Bain circuit to incubate the rabbit.
3.4.5.2 The flow meter was adjusted to 400 to 800mL/min.
3.4.5.3 The isoflurane vaporizer was adjusted to 1.5 to 3%.
3.4.5.4 Ophthalmic ointment (natural tears) was applied to both eyes to prevent dryness and damage to the cornea.
3.4.6 Recovery:
3.4.6.1 The isoflurane vaporizer was turned off but the animals were kept on oxygen.
3.4.6.2 The animals were transferred to their cage and once they begin to move and were allowed to recover fully.
4.0 PRECAUTIONS:
4.1 The animals should be checked carefully for its health status
4.2 The animals should be handled carefully.
4.3 All the checkups of the animals should be done in the presence of Veterinary Personal.
5.0 REFERENCES:
5.1 Brown MJ, Pearson PT, Tomson FN. Guidelines for animal surgery in research and teaching. Am J Vet Res.1993;54:1544–1559. [PubMed]
5.2 Butler TM, Brown BG, Dysko RC, Ford EW, Hoskins DE, Klein HJ, Levin JL, Murray KA, Rosenberg DP, Southers JL, Swenson RB. Medical management. Nonhuman Primates in Biomedical Research: Biology and Management. Bennett BT, Abee CR, Hendrickson R, editors. San Diego: Academic Press; 1995. pp. 255–334.
5.3 Callahan BM, Hutchinson KA, Armstrong AL, Keller LSF. A comparison of four methods for sterilizing surgical instruments for rodent surgery. Contemp Top Lab Anim Sci. 1995;34:57–60. [PubMed]
5.4 Capitanio JP, Kyes RC, Fairbanks LA. Considerations in the selection and conditioning of Old World monkeys for laboratory research: Animals from domestic sources. ILAR J. 2006;47:294–306. [PubMed]
5.5 Carstens E, Moberg GP. Recognizing pain and distress in laboratory animals. ILAR J. 2000;41:62–71.[PubMed]
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