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Surgical procedures in small laboratory animals
Journal of Immunological Methods 4 (1974) 213-216. © North-Holland Publishing Company
SURGICAL PROCEDURES IN SMALL LABORATORY ANIMALS J.E. CASTRO* Division of Surgical Science (Transplantation Biology Section) Clinical Research Centre, Northwick Park, Harrow, Middlesex, England
Received 10 September 1973
Accepted 30 October 1973
1. INTRODUCTION Several simple operative procedures in mice will be described. The operations are used in laboratory investigations and criteria of their success are the ability to operate rapidly on large numbers of animals, certainty of effect and minimal mortality and morbidity.
2. ANAESTHESIA Mode of anasthesia depended upon age, species and duration of operation. Hypothermia (East and Parrott 1962) was used for neonates, open ether for operations of short duration and intraperitoneal nembutal (0.1 ml. Nembutal (Abbott) diluted 1:10 with normal saline/10 g body weight) for longer ones. Mice were laid on the operating table, but not restrained during surgery. Operations were performed using a clean, but not sterile, technique and skin was prepared by wiping it with 70% alcohol.
3. ORCHIDECTOMY The peritoneum was entered through a small transverse suprapubic incision (fig. 1). Testes were delivered to the surface of the wound and the vas deferens and spermatic vessels transected distal to the epididymis so that testis, epididymis, surrounding fat and gubernaculum were removed. In most instances haemostasis was not necessary but in special cases spermatic vessels were ligated with catgut. * Present address: Urological Unit, Department of Surgery, Royal Postgraduate Medical School, Hammersmith Hospital, Du Cane Road, London, W12 0HS, England. 213
214
J.E. CASTRO
~
Thyrnectorny Splenectorn "~(~Adrenalectorny ~----Oophorectomy C] Z~ Orchidectorny~ c
~
J
lenectorny
) DORSAL
VENTRAL
Fig. 1. To show the incision used for surgical operations (see text). The wound was closed with a single 3/0 silk suture to include skin and muscle and more than 30 mice were operated upon each hour. Mortality within 24 hr of operating was 1% and was due to wound dehiscence or haemorrhage. Later death did not occur and non fatal complications were also unusual; incisional hernia and fistula occasionally occurring. Neonatal orchidectomy was performed on mice within 24 hr of birth. Animals were sexed by the length of the perineum (which was greater in males) and by increased pigmentation of the perineum (which occurred in males). The surgical technique was similar to that used for adults but the incision was closed with 7/0 silk suture and celloidin was applied to the wound to discourage canibalization. Overall mortality was approximately 40%.
4. OOPHORECTOMY Oophorectomy was through a dorsal transverse cutaneous incision at the level of the second lumbar vertebra (fig. 1). The skin wound was moved to each side so that the peri-ovarian fat was visualized through the body wall lateral to the vertebral column. The b o d y wall was incised at this point and the ovary, peri-ovarian fat and distal fifth of the uterine horn were excised. Haemostasis was not necessary and skin alone was closed with a single suture. Thirty mice were operated on each hour and operative mortality was less than 1%. kate complications did not occur.
5. ADRENALECTOMY Adrenalectomy was performed through a small dorsal transverse cutaneous incision at the level of the 12th thoracic vertebra (fig. 1) using modifications of the methods described by Llaurado 1958. The skin was manipulated so that the lower ribs of one side were visualized and a transmuscular incision made below the 12th
Surgical procedures in small laboratory animals
215
rib. The upper pole of the kidney was visualized and the adrenal gland located. Adrenal vessels were grasped with forceps, the peri-adrenal tissues including the adrenal gland were grasped with another pair of curved forceps and avulsed. The skin wound was then manipulated so that the 12th rib on the other side could be seen and the procedure was repeated. Skin alone was closed with a single suture and 15 mice underwent bilateral adrenalectomy each hour. After removal both glands were examined to confirm completeness of their removal but despite adequate adrenalectomy some strains of mice have functional ectopic adrenal tissue, which may abrogate the effects of operation (Dunn 1970). The routine postoperative management of adrenalectomised mice included weekly, subcutaneous administration of mineral-corticoid supplements in the form of 0.1 mg deoxycortone pivalate B.P. (Percorten M., Ciba Ltd.) or alternatively 0.45% sodium chloride in 5% dextrose in place of normal drinking water. Operative mortality for adrenalectomy was 5%, mainly from haemorrhage. In some strains of mice (Strain A) right adrenalectomy was almost impossible because of the close proximity of the adrenal to the inferior vena cava. Later death occurred in a further 5-10% of mice despite adequate replacement therapy, lf adrenalectomised mice required further procedures, or anaesthesia, pre-treatment with 100/lg hydrocortisone acetate was required to prevent death from stress.
6. SPLENECTOMY Splenectomy was through a left anterolateral subcostal, musculocutaneous incision (fig. 1). Splenic vessels were visualized and coagulated by diathermy or ligated. The connective tissues at the splenic hilum were divided, particular care being taken to avoid injury to the tail of the pancreas, and the spleen was removed. The wound was closed with a single suture through skin and muscle and 30 mice were operated upon each hour. Operative mortality was 1%, due to haemorrhage, and late mortality and morbidity did not occur.
7. THYMECTOMY Thymectomy was by modifications of the methods of Miller (1961). Young mice aged 3 - 4 weeks were used, for in older ones the thymus atrophies, undergoes fibrosis and is more difficult to remove. A small vertical suprasternal incision was made (fig. 1). The submandibular gland was visualized and elevated and the cervical strap muscles were separated in the midline using forceps so that the trachea was exposed. A small, angled, finger controlled sucker (fig. 2) was then inserted behind the manubrium in a plane immediately anterior to the trachea and behind the cervical strap muscles. The tip of the sucker was advanced to the posterior mid manubrial level and suction applied so that one half of the thymus was removed.
216
J.E. CASTRO
Fig. 2. To show the sucker end used for thymectomy. It was attached to a top operated suction pump. The tip of the sucker was then angled to the other side so that the remaining thymus tissue was removed. After removal of the sucker strap, muscles were allowed to fall together and the skin was not sutured but apposed by pinching it together. If thymuses are to be collected and used, a small trap can be inserted into the suction set (Boyse et al., 1971). Forty mice were thymectomised each hour, immediate postoperative mortality was 5 - 1 0 % and later death and morbidity did not occur.
REFERENCES Boyse, E.A., L.J. Old and C.A. Iritani, 1971, Transplantation 12, 93. Dunn, Thelma B., 1970, J. Natl. Cancer Inst. 44, 1323. Llaurado J.G., 1958, J. Anim. Technician Association 8, 75. Miller, J.F.A.P., 1961, Lancet 2,748.
SURGICAL PROCEDURES IN SMALL LABORATORY ANIMALS J.E. CASTRO* Division of Surgical Science (Transplantation Biology Section) Clinical Research Centre, Northwick Park, Harrow, Middlesex, England
Received 10 September 1973
Accepted 30 October 1973
1. INTRODUCTION Several simple operative procedures in mice will be described. The operations are used in laboratory investigations and criteria of their success are the ability to operate rapidly on large numbers of animals, certainty of effect and minimal mortality and morbidity.
2. ANAESTHESIA Mode of anasthesia depended upon age, species and duration of operation. Hypothermia (East and Parrott 1962) was used for neonates, open ether for operations of short duration and intraperitoneal nembutal (0.1 ml. Nembutal (Abbott) diluted 1:10 with normal saline/10 g body weight) for longer ones. Mice were laid on the operating table, but not restrained during surgery. Operations were performed using a clean, but not sterile, technique and skin was prepared by wiping it with 70% alcohol.
3. ORCHIDECTOMY The peritoneum was entered through a small transverse suprapubic incision (fig. 1). Testes were delivered to the surface of the wound and the vas deferens and spermatic vessels transected distal to the epididymis so that testis, epididymis, surrounding fat and gubernaculum were removed. In most instances haemostasis was not necessary but in special cases spermatic vessels were ligated with catgut. * Present address: Urological Unit, Department of Surgery, Royal Postgraduate Medical School, Hammersmith Hospital, Du Cane Road, London, W12 0HS, England. 213
214
J.E. CASTRO
~
Thyrnectorny Splenectorn "~(~Adrenalectorny ~----Oophorectomy C] Z~ Orchidectorny~ c
~
J
lenectorny
) DORSAL
VENTRAL
Fig. 1. To show the incision used for surgical operations (see text). The wound was closed with a single 3/0 silk suture to include skin and muscle and more than 30 mice were operated upon each hour. Mortality within 24 hr of operating was 1% and was due to wound dehiscence or haemorrhage. Later death did not occur and non fatal complications were also unusual; incisional hernia and fistula occasionally occurring. Neonatal orchidectomy was performed on mice within 24 hr of birth. Animals were sexed by the length of the perineum (which was greater in males) and by increased pigmentation of the perineum (which occurred in males). The surgical technique was similar to that used for adults but the incision was closed with 7/0 silk suture and celloidin was applied to the wound to discourage canibalization. Overall mortality was approximately 40%.
4. OOPHORECTOMY Oophorectomy was through a dorsal transverse cutaneous incision at the level of the second lumbar vertebra (fig. 1). The skin wound was moved to each side so that the peri-ovarian fat was visualized through the body wall lateral to the vertebral column. The b o d y wall was incised at this point and the ovary, peri-ovarian fat and distal fifth of the uterine horn were excised. Haemostasis was not necessary and skin alone was closed with a single suture. Thirty mice were operated on each hour and operative mortality was less than 1%. kate complications did not occur.
5. ADRENALECTOMY Adrenalectomy was performed through a small dorsal transverse cutaneous incision at the level of the 12th thoracic vertebra (fig. 1) using modifications of the methods described by Llaurado 1958. The skin was manipulated so that the lower ribs of one side were visualized and a transmuscular incision made below the 12th
Surgical procedures in small laboratory animals
215
rib. The upper pole of the kidney was visualized and the adrenal gland located. Adrenal vessels were grasped with forceps, the peri-adrenal tissues including the adrenal gland were grasped with another pair of curved forceps and avulsed. The skin wound was then manipulated so that the 12th rib on the other side could be seen and the procedure was repeated. Skin alone was closed with a single suture and 15 mice underwent bilateral adrenalectomy each hour. After removal both glands were examined to confirm completeness of their removal but despite adequate adrenalectomy some strains of mice have functional ectopic adrenal tissue, which may abrogate the effects of operation (Dunn 1970). The routine postoperative management of adrenalectomised mice included weekly, subcutaneous administration of mineral-corticoid supplements in the form of 0.1 mg deoxycortone pivalate B.P. (Percorten M., Ciba Ltd.) or alternatively 0.45% sodium chloride in 5% dextrose in place of normal drinking water. Operative mortality for adrenalectomy was 5%, mainly from haemorrhage. In some strains of mice (Strain A) right adrenalectomy was almost impossible because of the close proximity of the adrenal to the inferior vena cava. Later death occurred in a further 5-10% of mice despite adequate replacement therapy, lf adrenalectomised mice required further procedures, or anaesthesia, pre-treatment with 100/lg hydrocortisone acetate was required to prevent death from stress.
6. SPLENECTOMY Splenectomy was through a left anterolateral subcostal, musculocutaneous incision (fig. 1). Splenic vessels were visualized and coagulated by diathermy or ligated. The connective tissues at the splenic hilum were divided, particular care being taken to avoid injury to the tail of the pancreas, and the spleen was removed. The wound was closed with a single suture through skin and muscle and 30 mice were operated upon each hour. Operative mortality was 1%, due to haemorrhage, and late mortality and morbidity did not occur.
7. THYMECTOMY Thymectomy was by modifications of the methods of Miller (1961). Young mice aged 3 - 4 weeks were used, for in older ones the thymus atrophies, undergoes fibrosis and is more difficult to remove. A small vertical suprasternal incision was made (fig. 1). The submandibular gland was visualized and elevated and the cervical strap muscles were separated in the midline using forceps so that the trachea was exposed. A small, angled, finger controlled sucker (fig. 2) was then inserted behind the manubrium in a plane immediately anterior to the trachea and behind the cervical strap muscles. The tip of the sucker was advanced to the posterior mid manubrial level and suction applied so that one half of the thymus was removed.
216
J.E. CASTRO
Fig. 2. To show the sucker end used for thymectomy. It was attached to a top operated suction pump. The tip of the sucker was then angled to the other side so that the remaining thymus tissue was removed. After removal of the sucker strap, muscles were allowed to fall together and the skin was not sutured but apposed by pinching it together. If thymuses are to be collected and used, a small trap can be inserted into the suction set (Boyse et al., 1971). Forty mice were thymectomised each hour, immediate postoperative mortality was 5 - 1 0 % and later death and morbidity did not occur.
REFERENCES Boyse, E.A., L.J. Old and C.A. Iritani, 1971, Transplantation 12, 93. Dunn, Thelma B., 1970, J. Natl. Cancer Inst. 44, 1323. Llaurado J.G., 1958, J. Anim. Technician Association 8, 75. Miller, J.F.A.P., 1961, Lancet 2,748.