|Surgical organ perfusion method for somatic gene transfer: An experimental study on gene transfer into the kidney, spleen, lung and mammary gland|
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The kidneys of thirteen animals were perfused in vivo via laparotomy. Three animals died on the table from cardiac arrhythmias of unknown reason. One of the ten successful in vivo perfusion experiments was made without the viral preparation as a control. The animals were given prophylactic antibiotic, 750 mg of cefuroxime sodium (Lifurox®, Eli Lilly, Italy) intravenously in the induction of anesthesia, and 250 mg of cefuroxime sodium was added to the perfusate. While the kidney was connected to the perfusion system, it was isolated from the systemic circulation by vascular clamps placed on the renal artery proximally and vein distally. Before the clamping of the vessels, 2500 IU of heparin was administered intravenously to the animal. The ureter was cannulated to monitor diuresis and to collect the urine into the reservoir during the perfusion. For perfusion, the cannulas were attached to the vessels by a silk ligature. Perfusion pressure was measured directly from the arterial inlet, and the flow rate was adjusted to the level of 75/67 mmHg. Seven in vivo viral perfusions and the control perfusion experiments were continued for 60 minutes, and two viral perfusions for 120 minutes. At the end of the perfusion, 50 ml of physiological 0.9% saline was infused into the renal artery to flush out the excess adenoviral solution. The ligatures and cannulas were removed, and the puncture sites in the artery and vein were sutured with 7–0 monofilament sutures (Prolene®,Ethicon, USA). Hydrocortison 50 mg (Solucortef®, Pharmacia & Upjohn, Belgium) was administered intramuscularly after the operation.
The serum creatinine levels were measured before and 4 and 28 days after the perfusion. An intravenous excretory urogram was taken four weeks after the perfusion to evaluate the viability of the perfused kidney. Four animals were sacrificed on the fourth postoperative day, and the whole perfused kidney was removed for histological analysis. After the perfusion, five animals perfused with adenovirus and the control animal underwent open biopsy of the perfused kidney on the seventh day for a determination of transduction efficiency. Three perfused animals and the control were maintained for up to four weeks after the operation, after which they were sacrificed and both the left kidney and the untreated right kidney were taken for histological analysis. Serum anti‐β -galactosidase antibodies and anti‐AdV antibodies were examined by ELISA and IF, respectively, before the perfusion and two and four weeks thereafter, to find out the possible immunological reactions towards the adenoviral vector and the lacZ transgene. Two animals were maintained for up to 12 weeks after the viral kidney perfusion, after which the kidneys were removed for histological analysis.
A total of ten in vivo spleen perfusion experiments were made. Initially, an attempt was made to cannulate the hilar artery and vein of the spleen, but that was technically difficult due to the posterior location of the porcine spleen. The porcine spleen is elongated in shape, and this allowed vascular clamps to be placed on the splenic artery and vein 5 cm distal to the hilum. The proximal part of the spleen thus remained intact during the procedure, and this part served as a control in the later histological examination. The spleen was attached to the perfusion system by cannulating the splenic artery with an 18 G cannula and the vein with a 16 G cannula, and the perfusion procedure was carried out in exactly the same way as in the kidney perfusions, except that the amount of perfusate remained stable in spleen perfusions because of the lack of excretory functions in the spleen. I.v. heparin was administered similarly as in the kidney experiments before clamping the vessels. Before connecting the spleen to the perfusion system, 10 ml of lidocain‐heparin solution at the same doses as in the kidney experiments was infused through the splenic artery. The adenoviral preparation, 1 x 1011 pfu in 20 ml of Krebs‐Ringer solution, was then infused into the arterial inlet, and the perfusion was initiated immediately. The perfusion pressure was kept at 82.5/75 mmHg during the perfusion procedure. The viral perfusions were continued for 30 (N3) and 60 (N5) minutes. Oxygen saturation in the perfusate was measured during the perfusion, and it was 99%. Hydrocortison 50 mg (Solucortef®, Pharmacia & Upjohn, Belgium) was administered intramuscularly after the operation. The laparotomy incision was closed, and the animal was allowed to recover.
The animals were sacrificed on the fourth postoperative day and splenectomy was made. Samples from the perfused part and the proximal control part of the spleen were taken for histological analysis and the expression of the lacZ gene was examined visually after staining with X‐gal and PAS. Samples from the non-perfused part of the spleen were taken for histological analysis to serve as a control.
A closed‐circuit perfusion system for continuous circulation of the adenoviral vector solution in the right middle lobe of the lung in vivo was used. The perfusions were carried out into the right middle lobe of the lung in eleven farm pigs. This lobe is triangular in shape, being narrowest near the hilum and being separated from the other lobes by a thin fissure. The animal was intubated into the left main bronchus to obtain better exposure of the right lung hilum.
A right intercostal thoracotomy incision was made. After dissection of the lung hilum and administration of i.v. heparin 2500 IU, vascular clamps were placed on the right middle lobe branch of the pulmonary artery and vein at the hilum. The lung was attached to the perfusion system by cannulating the artery of the middle lobe with an 18 G cannula and the vein with a 16 G cannula, as in the previous experiments with the kidney and spleen. The venous effluent was collected directly into the reservoir. The whole perfusion experiment was carried out in the same way as the previous experiments with the kidney and spleen. The perfusion pressure was kept at 20/15 mmHg throughout the perfusion procedure. The viral perfusions were continued for 60 minutes. Thereafter the cannulas were removed and the puncture sites in the lobar artery and vein were sutured with 7–0 nonresorbable monofilament (Prolene®, Ethicon, USA) sutures. Circulation was restored immediately. The chest wall was closed with a suction chest tube in situ, which was removed during the recovery from anesthesia. Hydrocortison 50 mg (Solucortef®, Pharmacia & Upjohn, Belgium) was administered intramuscularly after the operation.
The animals were sacrificed on the seventh postoperative day and the treated right middle lung lobe was taken for examination. Samples from the lung were prepared for histological analysis, and the expression of the lacZ gene was visually examined after staining with X‐gal and PAS. Samples from the untreated left lung were prepared for analysis as a control.
The organ perfusion method was tested for in vivo gene transfer into the mammary gland by introducing retroviral vectors carrying the growth hormone gene as a reporter gene into goat mammary gland. The purpose was to evaluate the possibility of producing therapeutic proteins in goat´s milk by closed-circuit organ perfusion gene transfer. Three farm goats were operated. The glands were induced by single intravaginal natural progesterone device administration (Eazi-Breed Cidr. G“, Carter Holt Harwey Plastic Products, USA) into the lactating phase two weeks before the operation. Estrogen 0.3 mg kg–1 (Estrogen vet.®, Orion, Finland) was administered as a single intramuscular injection two days before the operation.
The incision was made caudal to the right mammary gland, and the main mammary artery and vein were prepared and clamped proximally and distally with vascular clamps, respectively. The artery and vein were cannulated with 16 G cannules. The retroviral vectors carrying PA 317 1.5 x 108 packaging cells in a volume of 300 ml were infused at the beginning of the perfusion into the arterial cannula, and the perfusion was continued, as in the previous experiments, for 60 minutes. The flow rate was set at 40 ml min–1. The cannulas were removed and the puncture site closed with one 7–0 monofilament (Prolene“, Ethicon, USA) suture. The goats were milked twice a day and human growth hormone secretion into the milk was measured by fluoroimmunologic assay (DELFIA, hGH Kit Wallac“, Orion, Finland) daily for one week and after that for as long as expression was detected, i.e. up to five months.
|Ex vivo gene transfer into kidney by the closed-circuit organ perfusion method||Up||Histochemical analysis|